key: cord-270458-7imgvale authors: franchini, massimo; farrugia, albert; velati, claudio; zanetti, alessandro; romanò, luisa; grazzini, giuliano; lopez, nadia; pati, ilaria; marano, giuseppe; pupella, simonetta; liumbruno, giancarlo maria title: the impact of the sars‐cov‐2 outbreak on the safety and availability of blood transfusions in italy date: 2020-04-13 journal: vox sang doi: 10.1111/vox.12928 sha: doc_id: 270458 cord_uid: 7imgvale coronaviruses are enveloped single-stranded rna viruses belonging to the family of coronaviridae. while initial research focused on their ability to cause enzootic infections, infections which have emerged in the past two decades demonstrate their ability to cross the species barrier and infect humans [1,2]. the ensuing epidemics have included severe acute respiratory syndrome (sars) in 2002 and the more recent middle east respiratory syndrome (mers) in 2012, and have resulted in severe disease burden, mortality and economic disruption [3]. a novel flu-like coronavirus, emerging towards the end of 2019 and subsequently named sars-cov-2, has been associated with an epidemic initially focused in wuhan, china. coronaviruses are enveloped single-stranded rna viruses belonging to the family of coronaviridae. while initial research focused on their ability to cause enzootic infections, infections that have emerged in the past two decades demonstrate their ability to cross the species barrier and infect humans [1, 2] . the ensuing epidemics have included severe acute respiratory syndrome (sars) in 2002 and the more recent middle east respiratory syndrome (mers) in 2012 and have resulted in severe disease burden, mortality and economic disruption [3] . a novel flu-like coronavirus, emerging towards the end of 2019 and subsequently named sars-cov-2, has been associated with an epidemic initially focused in wuhan, china. sars-cov-2 belongs to the same group (b-coronavirus) of coronaviruses responsible of sars and mers [2] . over december 2019 and february, sars-cov-2 spread rapidly throughout china, infecting many thousands of people and causing more than 3000 deaths [4, 5] . as a consequence, on 30 january the world health organization (who) announced that the outbreak of sars-cov-2 in china was a public health emergency of international concern (pheic) [6] . the influence of globalization, particularly on travel, rapidly caused the infection to spread to countries outside china, initially to neighbouring asian countries and then to more than 120 countries around the world. as of the time of writing this commentary, more than 200 000 individuals have contracted sars-cov-2 (data updated on 20 march 2020) [7] . on 11 march, the who declared the rapidly spreading coronavirus outbreak to be a pandemic [6] . currently, italy is the country with the highest number of cases of sars-cov-2 infection after china, a situation which has precipitated a health and social emergency. focusing on our role within the italian blood system, we note that there has been no scientifically documented evidence of the transmission of coronavirus infection through the transfusion of blood components during previous epidemics. the current sars-cov-2 outbreak has stimulated further discussion on this issue, particularly on the safety of blood donations in endemic countries [8] . in italy, the transfusion network is overseen, through regional blood coordinating centres (rbccs), by the italian national blood centre (cns), which is the health ministry's technical and scientific advisory body on matters related to blood and blood products. the cns acts also as the national competent authority, as established by the european commission, on blood and related issues on behalf of the same ministry. to guarantee high standards of blood safety and sufficiency, blood donation is sourced both from public hospital-based blood establishments (bes) and licensed and accredited blood donor associations and federations' blood collection sites in a 2:1 split and is restricted to voluntary unpaid donors. the first sars-cov-2 local transmitted cases in italy were observed on 18 february 2020 and were initially clustered in a few cities in northern italy. the italian government, starting on 23 february and up to the date of writing this manuscript, has released eight decrees aimed at limiting the spread of sars-cov-2. the cns has already released since 22 january 2020 a recommendation outlining preventative measures for the transmission of sars-cov-2 by transfusion of labile blood components related to travels from the people's republic of china. on 24 february 2020, a new document was released with reference also to italian territories. this document included the following recommendations: • request all the personnel working at bes and collection sites to comply scrupulously with behavioural protocols designed to prevent the spread of respiratory infections, including sars-cov-2 infection. in addition, the cns advised the associations and federations of voluntary blood donors to inform adequately all their staff and donors about the epidemiology, symptoms and preventative measures of sars-cov-2 infection. the cns recommended that the rbccs further implement regional patient blood management programmes, in order to conserve the blood supply, and enhance and monitor closely strategic regional blood component stocks (using information systems), in order to ensure balance and adjustment, thus minimizing any intra-or inter-regional blood component shortages. several updates of this document were released by the cns as the epidemiology of sars-cov-2 infection rapidly evolved. on 2 march 2020, following the recommendations of the european centre for disease prevention and control (ecdc) [9] and reflecting the decrees of the italian government, the cns updated the prevention measures, reducing the period of temporary deferral of donors from the previous 28 to 14 days. along with appeals to donate in order to avoid shortages in the blood supply, the same document also included additional recommendations for the associations and federations of voluntary blood donors. these included the maintenance of safe inter-individual distance (a minimum of 1 m) in waiting rooms, a careful planning of the schedule for calling up donors in order to regulate the volume of human traffic within centres, a meticulous disinfection of hands of the be and collection sites' personnel, and similarly rigorous protocols for the sanitation and disinfection of the collection rooms. the use of masks for both be personnel and donors was not required following the recommendations of the who and national health authorities. these measures were progressively extended to most of the cities of northern italy (9 march 2020). following the declaration of the government of the widespread dissemination of the sars-cov-2 infection in italy, the last update of the cns on 10 march 2020 extended these measures to the whole national territory of italy. on 18 march, the cns defined specific criteria for collection of plasma from convalescent subjects. the cns continues to reinforce its appeal to donate blood components in both public hospital bes and associated collection sites. given the limitations on movement imposed by the government, the cns has been pivotal in communicating and managing the necessary deviations from these measures of donors and the healthcare personnel involved in blood donation, in order to maintain the smooth collection of blood. in the first week of march, a reduction of 10% whole blood donations per week was experienced by the italian blood system (from 48 000as a standard number of blood donations per week in this periodto 43 000 nationwide), but the inventory was maintained by a contemporary reduction of elective surgery using mediumhigh amounts of blood. the strategic reserve of blood kept for emergencies was not accessed. during the second week of march, after the campaign of the cns and italian government encouraging blood donation, we observed a 12% increase in the collection of whole blood (from 48 000 to 53 600 nationwide). in fig. 1 , the trend of positive cases for sars-cov-2 infection in italy, updated on 20 march 2020, is reported with a concise chronology of the main documents released and the trend of blood donations in the same period. these measures have been able to guarantee blood safety and supply in italy. we hope that our ongoing experience, as briefly reported in this commentary, will help transfusion medicine specialists from other countries, who are facing the effect of this epidemic on the blood supply. we propose that it is possible to manage a transfusion system during this critical period, using pragmatic, prudent and evidence-based measures. in our country, so far, blood donation, after an initial fall (-10%), has been maintained during sars-cov-2 emergency, having been recognized by the government as a priority in order to maintain basic healthcare services. with the sars-cov-2 outbreak spreading in all european countries, we recommend defining common european guidelines that establish screening criteria for blood donors. such criteria need to take into account the different epidemiological situations in different countries, while establishing common principles ensuring blood safety throughout europe. coronavirus envelope protein: current knowledge coronaviruses: genome structure, replication, andpathogenesis middle east respiratory syndrome covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? the novel chinese coronavirus (2019-ncov) infections: challenges for fighting the storm world health organization: coronavirus disease (covid-19) outbreak world health organization. coronavirus disease 2019 (covid-19) situation report -59. data as reported by national authorities by 00 coronavirus disease 2019: coronaviruses and blood safety european centre for disease prevention and control. outbreak of novel coronavirus disease 2019 (covid-19): increased transmission globally -fifth update key: cord-271176-wdc4p4bc authors: gonzález-scarano, francisco; rima, bert title: infectious etiology in multiple sclerosis: the debate continues date: 1999-12-01 journal: trends microbiol doi: 10.1016/s0966-842x(99)01634-0 sha: doc_id: 271176 cord_uid: wdc4p4bc ‘demonstrating infectious cause: viral and bacterial infections in ms and related disorders’ was held in brighton, uk, 23–25 august 1999. m ultiple sclerosis (ms) affects over 300 000 americans, 85 000 britons and perhaps in excess of two million people worldwide, and has long been suspected to have an infectious etiology. however, despite intensive investigation, no single infectious agent or group of agents has been identified. 'demonstrating infectious cause: viral and bacterial infections in ms and related disorders' brought together immunologists, virologists, epidemiologists and clinicians to review the current knowledge about the role of infectious agents in ms, and to identify key concepts and questions for research. nineteenth-century descriptions of the neuropathology of ms emphasized the prominent infiltration of leukocytes and other inflammatory cells into its characteristic central nervous system (cns) lesion, which is now called a plaque. although it is now well accepted that inflammatory responses often have non-infectious causes, there are a number of clues that point towards an exogenous, possibly infectious, factor or factors as an important component of ms. for example, studies of twins with ms indicate that monozygosity is associated with a disease concordance of ~35% which, although suggesting a strong genetic component (the concordance for dizygotic twins is in the order of 4%), also indicates that the disease is influenced by additional, possibly environmental, factors. furthermore, there is a strong geographical influence on ms prevalence: the rates range from 150-250 per 100 000 in northern europe and in the northernmost usa, whereas in southern europe the prevalence drops to ~40 per 100 000, similar to the rates in the southern usa. remarkably, a large number of epidemiological studies have shown that individuals maintain the 'signature' incidence associated with the geographical area in which they spent their youth, even after migrating from an area of high incidence to one of low incidence, or vice versa. this has been interpreted as evidence that exposure to a microorganism prior to adulthood predisposes people to the development of ms (ref. 1). the most persuasive evidence that foreign antigens are associated with ms, however, is the indication of a humoral immune response within the cns. detectable as 'oligoclonal' bands in electrophoresed samples of concentrated cerebrospinal fluid (csf) proteins, antibodies produced by plasma cells within the cns are usually seen in infectious diseases such as syphilis or aids, where they are directed against antigenic components of the invading microorganism. their presence and persistence in ms (ref. 2) suggests that a chronic infection underlies the clinical syndrome, although it is also possible that these antibodies are directed against as-yet-unidentified self-antigens. additionally, analysis of tissue samples from around the plaques themselves also indicates antibody production and deposition. most investigators believe that identification of the antigens against which these antibodies are directed will provide important information about ms, perhaps even identifying its cause. although there were calls from conference 'break-out' sessions for better stratification of ms patients in epidemiological studies, the consensus of studies pointing in the direction of an environmental cause, as well as the evidence from the oligoclonal immune responses, convinced the majority of delegates that ms is likely to be caused or triggered by an infectious agent. the infectious agent: known or unknown? the majority of studies concerning specific agents in ms are based on the assumption that the microorganisms persist in the patient, probably in the cns but perhaps in other organs. a number of new technologies such as representational difference analysis (rda), differential display, consensus pcr and phage display are now available for identification of unknown or difficult-to-culture organisms. with that in mind, donald gilden (university of colorado, denver, co, usa) has adapted phage-display technology to analyse the antigen specificity of oligoclonal bands by extracting the rna surrounding an ms plaque and amplifying the immunoglobulin heavy and light chains using consensus primers. after cloning into an expression vector, the immunoglobulins are displayed on the coat of a bacteriophage. these immunoglobulins can then be specifically amplified, and the antigen against which they are directed can subsequently be identified. gilden has already achieved success using this approach for at least one chronic infection caused by measles virus (subacute sclerosing panencephalitis; sspe). his laboratory is now directing its attention to the ms lesions and the cns areas where the csf is produced. other presentations concerning molecular technologies gave the overall impression that the new technologies have not yet been applied in a systematic fashion to pathologically well defined ms tissue, and that there was room for more work using these approaches. some investigators felt that the agent that induces ms has already been identified. subramamiam sriram (vanderbilt stallworth rehabilitation hospital, memphis, tn, usa) presented data indicating that the csf of 97% of ms patients (compared with 18% of patients with other neurological diseases) contains genomic material from the bacterium chlamydia pneumoniae, as shown by pcr amplification. sriram was also able to culture c. pneumoniae from the csf of ms patients. furthermore, oligoclonal bands from ms csf were adsorbed by incubation of the fluid with a c. pneumoniae antigen, but not with antigens from herpes simplex or measles viruses. these data support his view that the immune response in those patients was directed against c. pneumoniae. sriram also suggested that a therapeutic trial with antibiotics directed against c. pneumoniae might be required to demonstrate a causal association between this microorganism and ms. konstance knox (institute for viral pathogenesis, milwaukee, wi, usa) presented equally compelling data supporting earlier suggestions 3 that human herpesvirus six (hhv-6) is associated with ms. immunohistochemical analysis of ms plaques has revealed a concentration of an hhv-6 antigen in oligodendrocytes, the cns cells responsible for the formation and maintenance of myelin (myelin destruction is a key component of the ms lesion). knox and collaborators found hhv-6 expression in 17 out of 19 patients with 'active' disease, compared with only three out of 23 patients with 'inactive' ms. there were also dramatic differences in viremia. twenty seven out of 41 patients with ms had active hhv-6 viremia, compared with none of the 61 controls. other investigators presented data supporting potential roles for human endogenous retroviruses (herve perron, biomerieux sa, lyon, france), and delayed exposure to epstein-barr virus (tove christensen and sven haar, aarhus university, aarhus, denmark) as potential triggers 4, 5 . however, as with many such reports, in most individuals there is evidence of prior or current infection with these agents whether or not they have ms, and there are no moredefinitive tests such as viremia or analysis of oligoclonal bands to differentiate patients with ms from those with other diseases. further research will be needed in order to clarify and reconcile these potentially conflicting findings, as several presentations outlined an association with a specific agent in over half of the cases. it is entirely possible that multiple microorganisms are each responsible for a small proportion of this disease, perhaps indirectly, by inducing immune responses that result in an immune attack on the cns, without persisting in the affected patient. several animal models of virally induced demyelination, particularly those associated with murine coronaviruses or with theiler's murine encephalomyelitis virus (tmev), support such an idea. in these models, demyelination is related to the presence of an identified virus within the cns; in tmev-mediated disease, the extent of demyelination depends not only on viral load but also on a locus on mouse chromosome 11 (michel brahic, institut pasteur, paris, france). although an intact immune system is necessary for the disease to manifest in its full form, which arm of the immune response is most critical varies from model to model, and even within models, apparently depending on the viral strain. demyelination after tmev infection of mice transgenic for a human leukocyte antigen (hla) was shown to be modulated by the hla transgene (moses rodriguez, mayo clinic, rochester, mn, usa), a finding that might relate to the preponderance of certain hla subtypes in ms. interestingly, by careful injection, foreign antigens can be introduced into the brain of mice and not be 'seen' by the immune system until after a subsequent peripheral challenge, which leads to detection and commencement of an immunopathological process (hugh perry, university of southampton, southampton, uk). whether or not a single agent is indeed responsible for this disease, there are multiple ways in which an infectious agent could be associated with, or trigger, ms. in some of these, the microorganism might no longer be present by the time the disease manifests but, for example, it affects the cns by changing the intrathecal cytokine environment and promoting pro-inflammatory reactions or by stimulating autoreactive t cells. matthias von herrath (scripps clinic, san diego, ca, usa) indicated that, in an autoimmune diabetes model, the growth kinetics of different strains of lymphocytic choriomeningitis virus determined whether the disease was exacerbated by a second peripheral challenge or whether this was, in fact, protective. balances between immune reactions also produce different responses in borna virus infection in rats (luther stitz, institute of animal health, tüebingen, germany). one of the more popular arguments has been that a microorganism could stimulate an autoimmune response by mimicking an endogenous antigen (antigenic mimicry) (uwe liebert, university of leipzig, leipzig, germany; henry mcfarland, national institutes of health, bethesda, md, usa); circumstantial evidence indicates that such a mechanism could lead to the development of autoimmune, reactive t-cell clones 6, 7 . alternatively, an exogenous agent could act as an mycobacterium tuberculosis infection is complex and multifaceted. as indicated in the review by murray 1 , both cd4 ϩ and cd8 ϩ t cells are essential to control tuberculosis (tb) in mice [2] [3] [4] [5] , and recent data support a role for both types of t cells in human tb resistance [6] [7] [8] . macrophage activation, which is likely to occur within a granuloma, is the key to controlling tb. granulomas consist of macrophages and lymphocytes, but the role(s) of each cell type, the types and kinetics of the cytokines produced by different cell populations, and the evolution of the granuloma remain poorly understood. murine studies have revealed essential roles for certain cytokines, including interferon ␥ (ifn-␥), interleukin 12 (il-12) and tumour necrosis factor ␣ (tnf-␣). however, there is little data regarding the importance of these cytokines in human tb. humans with mutations in the genes encoding ifn-␥ or il-12, or their receptors, often succumb to mycobacterial infections other than tb (ref. 9). although it is probable that these cytokines are significant in human tb, their relative importance, specific roles and cellular targets remain less clear. most patients infected with m. tuberculosis have cd4 ϩ t-cell responses that are expected to be effective in activating macrophages to kill intracellular mycobacteria (e.g. ifn-␥ production). however, because most infected individuals harbor the organism for many years, perhaps for a lifetime, this response is apparently not completely effective in eliminating virulent m. tuberculosis. in fact, in vitro, it has been difficult to demonstrate ifn-␥-induced killing of m. tuberculosis within human macrophages, although similarly activated macrophages eliminate other intracellular pathogens. a recent study has shed some light on this phenomenon 10 however, the infection dramatically inhibits the association of the transcription factor stat1 (signal transducers and activators of transcription 1) with the transcriptional coactivators cbp [camp-response-element-binding protein (creb)-binding protein] and p300, an association that is essential for ifn-␥-induced transcription. live bacteria are not necessary for this effect, as it can be reproduced using cell walls and it does not appear to be mediated by lipoarabinomannan. neutralization of transforming growth factor ␤ (tgf-␤) or il-10 did not reverse the effects of m. tuberculosis infection, suggesting that these cytokines are not the only contributors to macrophage deactivation. this phenomenon might also occur in mice, because mice do not eliminate m. tuberculosis, even though a strong immune response involving both cd4 ϩ and cd8 ϩ t cells and production of ifn-␥, tnf-␣ and other cytokines is present. m. tuberculosis is an enormously successful pathogen that has evolved numerous mechanisms for evading elimination by the host immune response. we are still in the process of learning which immune responses are important in controlling m. tuberculosis during the acute and latent phases of the infection. in most cases, the host maintains control of the infection, but occasionally the bacterium gains the upper hand. dissecting these interactions at an immunological level in both animal models and human systems remains the challenge for tb research in the future. the development of better vaccines ultimately depends on our increased understanding of the host-pathogen interaction. dept of molecular genetics and biochemistry, university of pittsburgh school of medicine, pittsburgh, pa 15261, usa adjuvant, perhaps by incorporating a self-antigen into its structure, much like retroviruses can incorporate cellular proteins into their envelopes 8 . steve miller (northwestern university medical school, chicago, il, usa) presented data to indicate that only antigenpresenting cells from tmevinfected mice with pre-existing myelin damage and not from acutely infected or control mice were able to present proteolipid apoprotein (plp) epitopes to specific t-helper type 1 cell lines. taken together, the presentations and discussion at the brighton conference summarized an impressive amount of information, leading to the conclusion that there are indeed exogenous agents involved in the development of ms. however, it is also clear that at this point there are insufficient data to identify a single culprit, and future research will be directed at replicating some of the most promising findings to date, and at understanding the possible pathogenic mechanisms by which foreign antigens could trigger an immune attack on the cns. why is ifn-␥ insufficient to control tuberculosis? multiple sclerosis key: cord-017917-7aeh6quc authors: marten, norman w.; zhou, jiehao title: the role of metalloproteinases in corona virus infection date: 2005 journal: experimental models of multiple sclerosis doi: 10.1007/0-387-25518-4_48 sha: doc_id: 17917 cord_uid: 7aeh6quc infection with neurotropic strains of mouse hepatitis virus (mhv) results in rapid leukocyte infiltration into the central nervous system (cns). the inflammatory response controls virus replication but fails to mediate sterile clearance. the persistence of viral rna and inflammatory cells within the cns is associated with the development of ongoing demyelination. matrix metalloproteinases (mmps) are a family of proteases involved in degradation of the extracellular matrix (ecm). during inflammatory responses mmps are thought to play a significant role in breaking down the basement membrane surrounding blood vessels as well as parenchymal ecm thereby facilitating leukocyte infiltration. mmps have also been associated with activation of chemokines and perhaps more significantly the degradation of myelin proteins and generation of autoantigens. recent examination of mmp expression during mhv infection suggests that mmp-3, -9 and -12 are involved in the inflammatory response. the proinflammatory effects of these mmps are likely tempered by induction of tissue inhibiter of metalloproteinase-1 expression. mouse hepatitis virus (mhv) can cause respiratory, enteric, hepatic or neurologic infections depending on the viral strain, age of the host and route of infection (33) . mhv infection of the central nervous system (cns) as a model of demyelinating disease has been studied extensively using strains derived from either the neurotropic jhm strain (mhv-jhm; mhv-4 serotype) or the mhv-a59 strain, which is both hepatotropic and neurotropic (33, 43) . as described by others, and ourselves acute mhv infection of the cns induces a potent inflammatory response involving neutrophils, macrophages, nk, b and t cells (33, 43, 52) . in order to reach the site of infection, however, these cells must overcome physical barriers represented by the blood brain barrier as well as the dense extracellular matrix (ecm) of the cns parenchyma. the ability of leukocytes to extravasate from blood vessels and cross the parenchymal tissues is presumably dependent on the activity of extracellular proteases. one family of proteases, which are often associated with inflammatory responses are the matrix metalloproteinases (mmps). the descriptions of mmp regulation and function during normal physiological as well as pathological conditions have been described in numerous publications (32, 23, 41) , thus only a brief summary is provided herein. mmps comprise a large group of endoproteinases that, in conjunction with other proteases, mediate degradation during remodeling of the extracellular matrix (ecm). twenty four different mmps have been identified and classified as collagenases, gelatinases, stromelysins, matrilysin and membrane type mmps (mt-mmps) based on their substrate specificities and protein domain structures (24,34). these proteases are produced by a wide range of cell types, including endothelial, inflammatory and stromal cells (18, 23, 32) . regulation of mmp activity has been classically defined as occurring at three distinct levels. primary regulation of mmp activity occurs at the transcriptional level (5, 12, 22, 30, 40) . mmp gene expression can be induced by a wide range of signals including acute phase cytokines such as il-lc~ and -13, il-6 and tnf-c~, and chemokines such as mip113, and rantes (6, 12, 22) . a second level of regulation for mmp activity is post-translational, as mmps require proteolytic cleavage of their proenzyme form to become activated (4,34). following translation, most prommps are released into the extracellular space. by contrast, a subgroup of membrane associated mmps, the mt-mmps, may play a role in the processing of secreted prommps to their active form (20). the final level of control for mmp activity resides in their regulation by a small group of specific inhibitors, the tissue inhibitors of metalloproteinases (timps) (7) . timps function primarily as specific inhibiters, binding to mmps in a 1:1 stoicheometry. interestingly, there is evidence that timps may also act as chaperone proteins during the processing of mmps from proenzyme to active form, suggesting that they can promote as well as inhibit mmp enzymatic activity, at least for 44) . mmps and their inhibitors are involved in remodeling of the ecm during normal physiological conditions. however they have also been associated with diverse pathological processes from rheumatoid arthritis to tumor invasion and metastasis (16, 25) . a number of mmps and timps have also been associated with the cns inflammatory/demyelinating disease multiple sclerosis (ms) in humans (2,32) as well as its rodent model experimental autoimmune encephalitis (eae) (10,38). in the case of ms, these include mmp-1, -2, -3 -7 and -9 and tlmp-1 (2,11,32). these mmps and timps have been detected by immunohistochemistry in both inflammatory infiltrates and activated glia within acute ms lesions as well as perivascular infiltrates. following eae induction in mice, mmp-3, -9, -12 and -14 as well as timp-1 gene expression were induced within the cns (38). mmp-1 and -10 were also induced although to a lesser degree than the aforementioned genes (38). the induction of mmp-7 mrna expression was not detected but the presence of mmp-7 protein has also been reported during eae in rats (10). the consistent association of mmp and timp expression with inflammatory demyelinating disease suggests that mmps play either a direct or indirect role in promoting cns pathology (2,27,32). however, as discussed below, identifying specific mmps and their exact role in promoting inflammatory/demyelinating has proven to be difficult. in vitro infection of human astrocytic and microglial cell lines with human coronavirus induced up-regulation of mmp-2 and -9 protein suggesting that virus infection can up-regulate mmp expression within the cns (15). the mechanism by which mmps were up-regulated by infection where not known, however, virus infection of the glial cell lines induced expression of il-6, tnf-c~ and mcp-1, which are all inducers of mmp expression (15). transfer of non-infectious supernatants from infected cultures to uninfected cells was sufficient to induce expression of mmp-2 and -9. mmp expression, including mmp-2 and -9, is also elevated in na'fve transgenic mice, which express il-6 and tnf-c~ within the cns (38). these observations suggest that viral infections induce expression of chemokines/cytokines, which in turn induce mmp expression in neighboring cells (15, 38) . recent examination of mmp gene expression within the brains of mice infected with lethal and attenuated mhv-jhm variants showed that mmp-3 and -12 mrna were rapidly up regulated during acute infections (51, 53). by contrast, infection did not alter transcription of the genes encoding mmp-2, -9, -11 or -14, which were constitutively expressed in the cns of na'fve balb/c mice (51). these data are summarized in table 1 . kinetic analysis of mmp expression indicated that mmp-3 and mmp-12 expression peaked by 6 days post infection (p.i.) in accord with viral load, however, expression was significantly higher in lethally infected mice (51,52). comparisons of mmp/timp expression in infected irradiated and control mice revealed relatively little loss in mmp or timp expression in immunosuppressed mice (51). these results indicated that cns resident cells were responsible for the majority of mmp-3, and -12 gene expression detected within the brain and that this gene induction was relatively independent of the presence of inflammatory cell infiltration. expression of mmps constitutively expressed in naive mice (mmp-2, -9, -11, -14) were unaffected by immunosuppression. timp-2 timp-3 rna was prepared from brains of mhv-jhm infected mice. relative gene expression was determined by rnase protection assay. b analysis of mmp-9 expression by rnase protection assay did not reveal an increase in mmp-9 expression in rna prepared from total brain. however, analysis of mmp expression from cd45 hi inflammatory cells isolated from the cns indicated that mmp-9 expression was increased during mhv infection. four timps have been characterized and all are capable of inhibiting a range of mmps (7, 23) . basal expression of timp-1 is low but readily induced in response to a multitude of inflammatory signals (5,7). by comparison, timp-2, -3 and -4 are expressed constitutively and alterations in their expression are less frequently associated with inflammatory responses (26,31,38). examination of naive and mhv-jhm infected brains revealed that timp-2 and -3 were unaffected by the infection (51) ( table 1) . by contrast, mhv infection induced rapid expression of t1mp-1 (51) ( table 1) . timp-1 expression peaked by 6 p.i. in conjunction with mmp-3 and -12 expression (51,52). timp-1 expression was higher in lethally infected mice compared to sublethally infected mice suggesting that expression is dependent on viral load and/or virulence. t1mp-1 expression has been reported to be associated with both tissue resident cells as well as most inflammatory cells (7) . expression levels of timp-1 were higher in irradiated, immuncompromised mice compared to control animals, indicating that most timp-1 is produced by cns resident cells (51). analysis of whole brain samples for mmp rna and protein failed to detect induction of mmp-9 expression during mhv infection (51), which was in sharp contrast to its prominence in ms. however, subsequent analysis of individual cell populations sorted from infected brains indicated that mmp-9 gene expression is up regulated several fold among infiltrating inflammatory cells (zhou, unpublished data) (table 1) . similarly, a strong induction in timp-1 expression within the inflammatory cell populations (zhou, unpublished data) suggests that t cells are, at least in part, a source of both mmp-9 and timp-1 as both of these genes are co-expressed by t cells (35). furthermore, analysis of cell extracts prepared from infiltrating cells revealed that mhv infection also induced a sharp rise in the level of intracellular mmp-9 protein (51, 53). the increase in intracellular mmp-9 protein was attributed to the presence of polymorphonuclear cell infiltrates in response to infection (51, 53). neutrophil synthesis of mmp-9 differs from other cell types in two key ways. first, in contrast to mononuclear cells, which release mmp-9 directly into the extracellular space, neutrophils store mmp-9 proenzyme in granules for release during degranulation (35), therefore permitting neutrophils to rapidly release large quantities of mmp-9 in the absence of de novo synthesis. second, expression of mmp-9 in neutrophils is not linked to co-expression of timp-1 as is the case for t cells (35). thus, mmp-9 from neutrophils is regulated at the level of degranulation as opposed to gene transcription and co-expression of timps. these data suggest mmp-9 is likely released from both mononuclear and polymorphonuclear infiltrates during jhmv infection. thus, with the exception that mmp-7 was not detected during mhv infection (51), the overall expression patterns for both mmps (-3, -9 and -12) and timp-1 during mhv infection, eae and ms are extremely similar. linkage between mmp/timp expression during demyelinating disease has been well chronicled (32,38,51). by contrast, determining a definitive role for mmps during demyelinating disease has proven to be far more elusive. during inflammatory processes within the cns, mmps most likely play two key roles: 1) breaking down the basal lamina surrounding the blood brain barrier, thereby allowing inflammatory cells to enter the cns (35) ; and 2) breaking down connective tissues linking tightly packed cns resident cells, thereby allowing inflammatory cells to migrate along chemokine gradients towards the sites of infection/injury (18, 50) . other key mechanisms in the genesis of inflammatory demyelination have been associated with mmps. these include activation of cytokines and chemokines, such as the conversion of tnf-a from pro to active form (8) and increasing the potency of the chemokine . furthermore, activated mmp-3 can convert pro-mmp-9 into it active form (13). thus mmp-3 produced by cns resident cells may be involved in activating pro-mmp-9 synthesized by inflammatory cells. activation of these proinflammatory proteins by mmps increases the potential for immune mediated damage to the cns. a number of mmps including -3, -9, and -12, which are common to many demyelinating diseases, are capable of breaking down proteins of the myelin sheath (8, 9, 39) . the consequences of this are two fold. first, induction of an inflammatory response to a foreign antigen within the cns may nonspecifically initiate destruction of the myelin sheath through the release of mmps. second, generation of potential autoreactive self-antigens by mmp mediated breakdown of myelin proteins may initiate specific responses against oligodendroglia resulting in additional demyelination (36, 39) . of the mmps associated with mhv infection of the cns, mmp-9 and its specific inhibitor, timp-i, are of particular interest for several reasons. mmp-9 and timp-1 are produced by both activated inflammatory as well as cns resident cells (19, 35, 47) . mmp-9 is specific for type iv collagens, which make up the basement membrane surrounding the blood brain barrier (49). this suggests that mmp-9 contributes to the ability of activated inflammatory cells to cross this barrier. support for the role of mmp-9 in t cell trafficking comes from ms patients undergoing ifn-13 treatment, one of the few therapeutics shown to have a positive effect. although no single conclusive mechanism for the anti-inflammatory activity of ifn-[3 has been demonstrated, mmp-9 gene expression is down regulated by 45, 46) . conversely, timp-i, which inhibits mmp-9, is up regulated by . this suggests that one of the mechanisms by which ifn-13 works is through inhibition of mmp-9 mediated inflammatory cell trafficking. these data are supported by treatment of eae mice with mmp inhibitors, which have generally prevented disease induction or reduced clinical symptoms when treatment was initiated after active disease was apparent ( 17,21,28). mmp and timp expression are closely associated with the human demyelinating disease ms as well as experimental animal models of demyelinating disease. mmp expression within the cns is most likely up regulated by the presence of inflammatory cytokines. however, as stated previously, confirmation of a specific role for mmps in the inflammatory process is lacking. the use of knockout mice to determine the role of specific mmps in inflammatory responses have generated mixed results, particularly in regard to blood brain barrier breakdown and inflammatory cell infiltration across endothelial cell barriers (1, 3, 14, 29) . this may in part be explained by the large number of mmps with overlapping specificities as well as the presence of other proteases. thus, although mmp expression is clearly associated with inflammatory responses during demyelinating disease, the role of individual mmps as active participants or simply as markers remains to be determined. neutrophils from mmp-9-or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro differential matrix metalloproteinase expression in cases of multiple sclerosis and stroke effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia matrix metalloproteinases: a review synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor nf-kb nuclear factor kappab activity is essential for matrix metalloproteinase-1 and -3 upregulation in rabbit dermal fibroblasts macrophage metalloelastase degrades matrix and myelin proteins and processes a tumour necrosis factor-alpha fusion protein matrix metalloproteinases degrade myelin basic protein matrix metalloproteinase expression during experimental autoimmune encephalomyelitis and effects of a combined matrix metalloproteinase and tumour necrosis factoralpha inhibitor plasminogen activators and matrix metalloproteases, mediators of extracellular proteolysis in inflammatory demyelination of the central nervous system resistance of young gelatinase b-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions activation of glial cells by human coronavirus oc43 infection induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5 reversal of experimental autoimmune encephalomyelitis with a hydroxamate inhibitor of matrix metalloproteases matrix metalloproteinases in immunity cytokines regulate gelatinase a and b (matrix metalloproteinase 2 and 9) activity in cultured rat astrocytes the role of matrix metalloproteinases in autoimmune damage to the central and peripheral nervous system suppression of experimental allergic encephalomyelitis in the lewis rat by the matrix metalloproteinase inhibitor ro31-9790 cytokine inducible matrix metalloproteinase expression in immortalized rat chondrocytes is independent of nitric oxide stimulation matrix metalloproteinases in tumor invasion structural biochemistry and activation of matrix metalloproteases analysis of 16 different matrix metalloproteinases (mmp-1 to mmp-20) in the synovial membrane: different profiles in trauma and rheumatoid arthritis differential regulation of timp-1 and timp-2 mrna expression in normal and ha-ras-transformed murine fibroblasts expression of matrix metalloproteinase-9 and its inhibitors in mononuclear blood cells of patients with multiple sclerosis effective treatment of models of multiple sclerosis by matrix metalloproteinase inhibitors gelatinase b-deficient mice are resistant to experimental bullous pemphigoid transcriptional suppression of matrix metalloproteinase-9 gene expression by ifn-gamma and ifn-beta: critical role of stat-lalpha selective induction of tissue inhibitor of metalloproteinase-1 in bleomycin-induced pulmonary fibrosis matrix metalloproteinases in the normal human central nervous system, microglial nodules, and multiple sclerosis lesions mhv infection of the cns: mechanisms of immune-mediated control matrix metalloproteinases gelatinase b functions as regulator and effector in leukocyte biology gelatinase b: a tuner and amplifier of immune functions multiple sclerosis: pro-and anti-inflammatory cytokines and metalloproteinases are affected differentially by treatment with ifn-beta differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states leukocyte gelatinase b cleavage releases encephalitogens from human myelin basic protein cytokine regulation of matrix metalloproteinase activity and its regulatory dysfunction in disease coordinate expression of matrix metalloproteinase family members in the uterus of normal, matrilysin-deficient, and stromelysin-l-deficient mice cell surface binding and activation of gelatinase a induced by expression of membrane-type-l-matrix metalloproteinase (mt1-mmp) mouse hepatitis virus mechanism of cell surface activation of 72-kda type iv collagenase. isolation of the activated form of the membrane metalloprotease interferon beta-lb decreases the migration of t lymphocytes in vitro: effects on matrix metalloproteinase-9 changes of serum sicam-1 and mmp-9 induced by rifnbeta-lb treatment in relapsingremitting ms neutrophil gelatinase b potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades ctap-iii, pf-4, and gro-alpha and leaves rantes and mcp-2 intact gelatinase contributes to the degradation of glomerular basement membrane collagen by human neutrophils matrix metalloproteinases and diseases of the cns matrix metalloproteinase expression correlates with virulence following neurotropic mouse hepatitis virus infection neutrophils modulate inflammation during viral induced encephalitis regulation of matrix metalloproteinase (mmp) and tissue inhibitor of matrix metalloproteinase (timp) genes during jhmv infection of the central nervous system key: cord-021069-v9f9874x authors: morrison, lynda a.; fields, bernard n. title: viral pathogenesis and central nervous system infection date: 2004-11-23 journal: nan doi: 10.1016/1044-5765(91)90002-6 sha: doc_id: 21069 cord_uid: v9f9874x both host defense and viral genetic factors influence the development of viral infection and disease. due to the presence of the blood-brain barrier, infection of the central nervous system creates additional complexities in interactions between a virus and its host. stages in viral pathogenesis defined as (1) virus entry, (2) spread, (3) tropism, (4) virulence and injury to the host, and (5) the outcome of infection are discussed for viral infections in general and those aspects unique to infections of the central nervous system. information about neuronal physiology and function has also been revealed through studying virus infection. an increased understanding of viral pathogenetic mechanisms and host response to infection raises interesting possibilities for vaccine development and for basic studies in neurology and neurobiology. seminars in the neurosciences, vol 3, 1991 : pp 8 3 -91 viral pathogenesis and central nervous system infection lynda a . morrison and bernard n. fields both host defense and viral genetic factors influence the development of viral infection and disease. due to the presence of the blood-brain barrier, infection of the central nervous system creates additional complexities in interactions between a virus and its host. stages in viral pathogenesis defined as (1) virus entry, (2) spread, (3) tropism, (4) virulence and injury to the host, and (5) the outcome of infection are discussed for viral infections in general and those aspects unique to infections of the central nervous system . information about neuronal physiology and function has also been revealed through studying virus infection . an increased understanding of viral pathogenetic mechanisms and host response to infection raises interesting possibilities for vaccine development and for basic studies in neurology and neurobiology . key words : virus / infection / pathogenesis / central nervous system viral pathogenesis has been defined as the process by which a virus causes disease in a susceptible host .' several stages in pathogenesis can be identified that represent unifying themes in this process . all viruses must successfully penetrate the host's barriers to entry, undergo primary replication and then spread to their ultimate target tissue . any virus able to cause disseminated infection has the potential to infect the cns . viruses infecting the cns can either circumvent the blood-brain barrier by entry into peripheral nerve endings and axonal transport into the cns, or they can invade from the blood stream by infection of or transport across the vascular endothelium in the cns, in regions of greater permeability, or by passive transport in infected monocytes . but with all potentially neurotropic viruses successful invasion of the cns is rare, limited by host defenses, the mechanics of the blood-brain from the department of microbiology and molecular genetics, and the shipley institute of medicine, harvard medical school, 25 activity and are thought to gain entry through small immune host defenses . breaks in the integrity of the mucosa, or possibly by uptake at the intact mucosal surface . infection of the conjunctiva, presumably by direct inoculation from contaminated fingers, can occur with enterovirus 70, hsv and some coxsackie viruses . 3 regardless of the route of entry, viruses must evade local phagocytic cells that make an important contribution to early host defense . alveolar macrophages and histiocytes of the reticuloendothelial system efficiently phagocytose and eliminate invading viruses, but some (lymphocytic choriomeningitis virus, cytomegalovirus and the lentiviruses visna and hiv) escape destruction by productively infecting these cells . 1,2,5 elements of the host immune system, immunoglobulin a in mucous secretions and intraepithelial lymphocytes in the intestine, must also spread tissue invasion a period of replication in epithelial or subepithelial tissue at the site of inoculation typically occurs, except when virus is directly inoculated into the blood . whether a virus infection will remain localized at the site of entry or become disseminated is influenced by the temperature of the epithelial surface (e .g . rhinovirus), the propensity of a virus to bud from the apical (influenza) or basolateral (hsv) surface of epithelial cells, 3 following replication at the site of inoculation, virus enters the subepithelial lymphatic capillaries and is carried into the blood stream 3 where it must survive encounters with phagocytic cells and elements of the immune system . primary viremia permits the rapid dissemination either of free virus or of bloodcell associated virus to other susceptible tissues :' successful penetration of the vascular endothelium and replication in these organs amplifies the infection and augments viremia . viremia must be of a magnitude and duration adequate to sustain infection and, for some viruses, to permit an assault on the blood-brain barrier . replication of some arboviruses in cerebral capillary endothelium precedes viral invasion of the neural parenchyma, 6 and replication in or passive transport across the endothelium has been postulated for several other viruses, including enteroviruses, arboviruses and retroviruses .6' 7 because of its fenestrated endothelium and sparse basement membrane, the choroid plexus is also a target for replication or passive transport of mumps and arboviruses ; 1,3 through it, virus enters the cerebral spinal fluid to infect ependymal cells lining the ventricles and subseqently the underlying brain tissue . 3,5 migrating infected monocytes, lymphocytes or leukocytes may be an important vehicle for virus transport into the cns, especially in lentivirus infection (see zink and narayan, this issue,$ and refs 9,10) . neural spread from the periphery to the cns can be considered as two processes, penetration and propagation . penetration probably occurs at nerve terminals, either at the site of initial entry into and replication in the host or at a secondary site of replication in extraneural tissues to which virus is disseminated by the blood . electron microscopic observations have documented accumulation of neuroinvasive rabies virus particles in infected myocytes at neuromuscular junctions and neuromuscular 85 spindles, followed by entry into motor or sensory nerve terminals . 3 receptors used by viruses may be concentrated at nerve terminals and synapses, facilitating uptake and possibly transport of virus (see below, tropism) . once within nerves, propagation is mediated by axonal transport, replication and transsynaptic transfer of virus . hsv, rabies, poliovirus and reovirus are known to spread along nerves by the system of fast axonal transport ." transport has been demonstrated both towards and away from the cell body in somatic motor, somatic sensory and autonomic nerves, under various experimental conditions . bidirectional transport is important in the establishment of, and reactivation from, latency seen with hsv and varicella zoster . 12,13 viruses may have a predilection for a subset of nerve types, as evidenced by the more rapid uptake of reoviruses in motor than sensory fibers innervating the footpad 14 and by the restriction of rabies mutants to transport by sensory but not autonomic paths . 15 in contrast to the fast axonal transport of most viruses, the scrapie agent has been postulated to use the system of slow axonal transport . 3,6,16 disruption of the neurofilament cytoskeleton that supports slow axonal transport has been observed in scrapie-infected neurons and has been implicated in generation of the spongiform changes that characterize this degenerative neurological disease (see hope, this issue, 17 and refs 16, 18) . viral replication occurs once virus particles reach the neuronal cell body. newly synthesized virion components and virus particles then accumulate at post-synaptic sites, probably reflecting directed transport of virus components analogous to directed budding in polarized epithelial cells (see entry into the host, above ; ref 11) . both rabies and vesicular stomatitis virus accumulate at synaptic terminals and beneath the nodes of ranvier . when antiviral antibody is added to infected neuronal cultures, vesicular stomatitis virus buds preferentially from the synapses, indicating that exocytosis by neurons may be facilitated at this point .3 neurons thus transfer virus at synaptic junctions in the periphery . in the cns, the high density of neurons and glia permits many viruses to spread from cell to cell with less fidelity . the site of virus inoculation can influence the ultimate distribution of lesions within the cns, especially for viruses that spread along neural routes . neurons can transport viruses great distances intraaxonally both to and within the cns, and the final destinations of a virus will be determined in part by the particular synaptic connections made with individual neurons whose processes took up virus in the periphery . 19 hsv gaining entry through the genitourinary tract infects and becomes latent in sacral ganglia, whereas hsv entering in the oral mucosa becomes latent in trigeminal sensory neurons . though cases of strictly blood-borne or neural spread of viruses to the nervous system have been documented, a combination of the two routes may be used by certain viruses, or under certain conditions . for example, flaviviruses can spread through the blood to exposed neurons of the olfactory bulb which acetylcholine receptor, localized on muscle cells in the region of neuromuscular junctions, has been identified as a receptor for rabies virus . phosphatidyl serine may function as an alternative receptor for rhabdoviruses, 5,26 indicating that lipid molecules (and carbohydrate residues) as well as proteins can serve as neurotropic virus receptors . other molecules acting as receptors for neurotropic viruses are listed in table 2 . specific interactions with receptors may regulate binding of a particular virus at epithelial surfaces and vascular endothelium as well as nerve terminals, and may explain to some degree tissue specificity and differential susceptibility to infection . numerous viral cell-attachment proteins have also been identified ( gp350, gp220 heparin sulfate proteoglycana gb, gc, basic fibroblast growth gd/gh1 factor receptorb 87 selected with antibody against the e2 envelope protein revealed that a single amino-acid substitution in the e2 molecule determines loss of tropism for neurons without change in tropism for oligodendrocytes . 6,26 little is known about the murine coronavirus receptor protein on neurons or its similarity to the receptor on oligodendrocytes . x-ray crystallographic analysis of the poliovirus virion has provided a detailed molecular view of a ligand in virus-receptor molecular interactions . the analysis has revealed a series of peaks in the vp1 protein surrounded by a broad valley composed of vpi and vp3 that forms the receptor binding pocket (see almond, this issue 29 ; ref 13) . other members of the picornavirus family are known or postulated to have cell-attachment proteins of similar topology . transcriptional regulatory elements are an important post-receptor-binding determinant of viral tropism . 5,30 an enhancer element confers tissuespecific expression for jc papovavirus in human oligodendrocytes . similarly, promoter elements of some neurotropic murine retroviruses determine their species-specificity . on the other hand, host cells that lack enzymatic activities required for maturational cleavage of viral proteins or completion of the virus replicative cycle (as observed for poliovirus) will not be productively infected, thereby limiting tropism .5,25 a number of host factors modulate viral tropism and virulence, influencing viral pathogenetic potential and ultimately the potential for cns infection . 3,31 many neurotropic viruses more readily infect the young. this has been shown to variously result from the immaturity of the immune response, reduced capacity to produce interferon, increased susceptibility to infection of some cell types, and age-specific nature and distribution of receptor proteins2 '3 (see coyle, this issue 32) . genetic determinants of disease susceptibility have been found for infection of mice with strains of most neurotropic viruses, in at least one case of coronavirus reflecting lack of a gene encoding a virus receptor protein . determinants have been linked to histocompatibility genes that regulate immune responses only in chronic diseases of immunopathological etiology . last, sex of the host and nutritional status can influence resistance to infection . the selective vulnerability of different species or one species at different ages, coupled with viral determinants of neurotropism, may explain differences in clinical manifestations of cns disease . the efficiency with which a virus can multiply and extend infection after it has invaded the nervous system, its neurovirulence, determines in part the extent of injury suffered by the host . the effectiveness of the host response and the resulting immunopathology also play a role . although each virus has evolved its own genetic determinants of virulence, some generalizations can be drawn . outer capsid and envelope glycoproteins are frequently implicated as virulence factors because changes in their amino-acid sequence usually weaken their infectivity (attenuation), although this may be a subtle reflection of altered tropism . genetic reassortants between virulent and avirulent strains of bunyaviruses and reoviruses have also been used to identify determinants of neurovirulence by segregation of phenotype with a particular viral gene . 3 genetic recombination between strains of poliovirus and of hsv have similarly been used to identify genes critical for neurovirulence . 5 more than one region of a viral protein identified as a virulence factor may be important3 and, when neurotropism and neurovirulence are determined by the same protein, changes in that protein can have multiple and dramatic effects on the resulting infection . in studies of theiler's murine encephalomyelitis virus, a model of human demyelinating disease (see nash, this issue 33), variants selected with l .a . morrison and b .n. fields antibody to the vp1 protein possess tissue-specific alterations in extent and duration of infection and in capacity to induce demyelination . 13 but not all changes in viral genes are attenuating : mutations causing reversion to neurovirulence have been identified in poliovirus isolates from the feces of recipients of the attenuated trivalent oral vaccine . 34 mutations in genes encoding proteins other than those of the capsid or envelope can also result in attenuation, for example, sequences encoding a retroviral core protein or in the 5' noncoding region of poliovirus rna . 34 transacting transcriptional activators may play a role in determining virulence, exemplified by a mutant of hiv with a defective tatiii gene that shows reduced replication and cytopathic effect .3,6 importantly, neurovirulence may be under multigenic control and determinants of virulence may differ depending on the host species infected . 3 cns injury resulting from viral infections viral infections of the nervous system can have very severe consequences because the neurons vital to host function and survival cannot be replaced once injured or destroyed ; the effect of even minor damage may be catastrophic .2 furthermore, once infection in the cns has begun, the reduced permeability and dense structure of the blood-brain barrier which often impedes the initial spread of virus to the cns may reduce the capacity of the host to resolve infection and limit injury. 9 injury at a cellular level may be directly due to virus infection, occurring when cytopathic effect is significant enough to damage irreparably the plasma membrane (lysis) or to arrest metabolic activity by inhibition of protein, rna or dna synthesis, e .g . poliovirus, vesicular stomatitis virus and herpesvirus, respectively . 35,36 viruses such as rabies may alter nerve impulse conductance without histologically perturbing infected neurons . 19 accumulations of virus capsids or even individual viral proteins may in themselves be toxic . the sequence homology between hiv gp120 envelope protein and certain essential neurotransmitters and neuropeptides has evoked the hypothesis that competitive binding of gp120 may block normal neuronal function (see tillman and wigdahl, this issue 37). gp120 also mediates another potential form of direct injury by virus, syncytium formation . indirect injury to cells has been postulated to occur by release of toxic`factors' from neighboring cells damaged by infection . 36 systemic injury occurs by both direct and indirect mechanisms as well . the consequences of infection vary with the location and function of tissue injured by the virus : for example, motor neuron destruction in poliomyelitis results in paralysis ; demyelinating reactions to virus infection cause incoordination ; and virus infections of cells in the developing nervous system produce a variety of congenital abnormalities and neurological diseases (see coyle, this issue, 32 and refs 19, 38) . indirect mechanisms of viral injury to the host cns include the actions of interferons, 39-41 and the virus-specific cellular and humoral immune response (see sedgwick and dã¶rries, this issue, 42 and refs 2, 40, 43) . paradoxically, the elaborate system of host response to virus infection that may be protective outside the cns can be destructive when generated within this normally isolated compartment, 1,43 where cell lysis and complement activation may injure the host while helping to clear virus . virus replication in macrophages or immunocompetent t or b cells can also induce immune disregulation . 41,43 last, autoimmune reactions may be provoked by viral infections, resulting in indirect injury to the host even without continued presence of virus. 41,44 autoimmunity can be triggered in genetically susceptible individuals by non-specific inflammatory stimuli that alter immune regulation or by cross-reactivity between the viral inducing agent and normal host proteins (molecular mimicry) . 44,45 exposure of normally sequestered antigen due to virus-induced cytolysis and destruction or dysfunction of suppressor t cells have also been postulated as mechanisms in the development of autoimmune reactions . 41 autoimmune demyelination reactions, initiated in the cns against nervous system antigens, are especially prevalent . 46 several outcomes are possible in viral infections of the cns that do not acutely result in death . virus may be effectively cleared from the cns, may remain latent in the nervous system and cause recurrent disease, or may produce persistent disease . clearance is thought to be highly dependent on the immune response, though limitation of viral replication and the type of disease produced can be influenced by nonspecific factors . failure to clear varicella zoster, cytomegalovirus, adenovirus and 89 measles infections in natural cell-mediated immunodeficiency states suggest that t cell responses may be more important than antibody in eventual clearance of many infections . ) successful clearance may still leave the host impaired, depending on the nature and severity of the injury sustained .9" 10 a chronic autoimmune reaction may continue even after virus clearance . when latency is established by some viruses, the viral genome remains within the nervous system after acute infection ceases . no infectious virus is produced until reactivation occurs and the absence of viral protein products transcribed during latency may permit the infected cell to escape immune surveillance . capacity for latent infection in the nervous system is characteristic of the herpesviruses, hsv and varicella zoster (see stevens, this issue, 47 and refs 12,13) . persistent viral infections, in contrast to latency, are characterized by continued production and shedding of virions from infected cells . they are especially prevalent in cns tissues, 9 possibly because these contain non-dividing cells, are isolated behind the blood-brain barrier, have low expression of major histocompatibility complex proteins and may permit increased generation of defective viruses . 19 immune responses are usually generated and maintained but are ineffectual in clearing virus .48 as a result, chronic immune reactions may contribute much of the tissue injury .43 possible mechanisms of establishment and maintenance of persistent infection are listed in table 3 . persistent infection takes many forms . in lymphocytic choriomeningitis virus infection of newborn mice, chronic production of virus occurs without serious detriment to cells . 43 a more thorough understanding of these stages in pathogenesis of viral cns infections may engender new or more effective measures for prevention of disease . development of more efficacious vaccines is a primary objective but requirements for protection from individual viruses differ . 40,50 knowledge of pathogenetic stages of each virus may reveal points of greatest vulnerability and will help to determine requirements for stimulating immunity with a minimum of immunopathological side effects . ideally, infection by any virus with potential to invade the cns should be stopped before entering the nervous system . characterization of neurotropic virus infections also offers interesting possibilities for studying structure and function of the nervous system . basic neurological and neuroscience research has begun to benefit from using the strongly neurotropic rabies, hsv and pseudorabies viruses for tracing neural pathways . 51 more speculative is the use of attenuated neurotropic viruses for delivering foreign proteins into regions of the cns defined through studies of viral pathogenesis . foreign genes can be packaged by many viruses and may be selectively expressed, permitting intracerebral production of pharmacological agents or neurotransmitters . thus studies of viral pathogenesis of the cns may offer tools for the future as well as an understanding of disease states . viral infections of the nervous system viral 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neurovirulence and latency viral persistence pathogenetic aspects of persistent measles virus infections in brain tissue modern approaches to vaccines viruses as transneuronal tracers the authors are grateful to dr k tyler for much helpful advice and discussion, and for reviewing this manuscript . work on the manuscript was supported by public health service grants a108207-02 and program project 5 p50 ns16998 from the national institutes of health . key: cord-259347-3acsko74 authors: cheng, qi; yang, yue; gao, jianqun title: infectivity of human coronavirus in the brain date: 2020-05-28 journal: ebiomedicine doi: 10.1016/j.ebiom.2020.102799 sha: doc_id: 259347 cord_uid: 3acsko74 a new strain of human coronaviruses (hcovs), severe acute respiratory syndrome coronavirus-2 (sars-cov-2), has been identified to be responsible for the current outbreak of the coronavirus disease 2019 (covid-19). though major symptoms are primarily generated from the respiratory system, neurological symptoms are being reported in some of the confirmed cases, raising concerns of its potential for intracranial invasion and neurological manifestations, both in the acute phase and in the long-term. at present, it remains unclear the extent to which sars-cov-2 is present in the brain, and if so, its pathogenic role in the central nervous system (cns). evidence for neuroinvasion and neurovirulence of hcovs has been recognised in animal and human studies. given that sars-cov-2 belongs to the same family and shares characteristics in terms of receptor binding properties, it is worthwhile exploring its potential cns manifestations. this review summarises previous findings from hcovs in relation to the cns, and compares these with the new strain, aiming to provide a better understanding of the effects of sars-cov-2 on the cns. human coronaviruses (hcovs) are a group of enveloped rna viruses that are composed of a single stranded positive sense rna genome (~30 kb). the entire genome of hcovs encodes spike (s) glycoprotein, membrane (m) glycoprotein, envelope (e) glycoprotein, nucleocapsid (n) protein, rna polymerase and genes for numerous accessory proteins that modulate pathogenesis, among which the n protein is highly conserved in most coronaviruses and abundantly expressed during infection [1] , thus acting as a critical antigen for viral detection. the s protein is biologically critical for tropism and modulation [2] , and is considered to be integral in the capability of hcovs to reach the brain [3, 4] . the hcovs include seven strains, three of which, namely sars-cov-2, sars-cov, and middle east respiratory syndrome-related coronavirus (mers-cov), may cause severe respiratory disease with a relatively high morbidity and mortality. in contrast, the other four strains oc43, 229e, nl63 and hku1, are often associated with symptoms akin to the 'common cold' and hence, are relatively harmless. based on genome analysis, the oc43 strain shares a 531% and a 512% identity with sars-cov and hcov-229e, respectively [5] . the new strain, sars-cov-2 (previously called 2019-cov), shares a 79% and 50% identity with sars-cov and mers-cov, respectively [6] . mainly considered as respiratory viruses, a common manifestation from hcovs related acute respiratory disease is hypoxia, which predisposes the brain to edema, disturbed metabolism, and subsequent neurological manifestation. however, hypoxia is not the only way hcovs were related to central nervous system. in fact, hcovs have also been shown to be capable of invading neural cells, and hence manifesting with neurological symptoms and signs ( table 1 ). the potential for significant neurological deficits recently became a concern following the report of a covid-19 patient who demonstrated loss of involuntary control of breathing due to presumed involvement of the inspiratory area in the brainstem, as well as reports of sars-cov-2 infected patients developing ataxia, loss of smell and convulsions [7] . in vitro studies have shown that 229e and oc43 hcov strains can infect a wide range of human neural cell cultures, including neuroblastoma, neuroglioma, astrocytoma, microglial and oligodendrocytic cell lines [8à10]. in addition, animal studies have also revealed the neuroinvasion and neurovirulence of hcov-oc43 [4,5,11à13] . importantly, a significantly higher prevalence of the oc43 strain in terms of viral rna detection has been shown in human brain autopsy samples from multiple sclerosis (ms) patients when compared to other neurological diseases and normal controls, which is consistent with the left lower lobe infiltrates (11) →obvious resolution (28) →progressive bibasilar infiltrations (35) ct: broad encephalic pathological changes of probably ischemia and necrosis and brain edema (33) none eye-ground exam: an exudation around the visual yellow zone (26) death ( recovery [15] all cases had fever and/or flu-like symptoms at the onset. half of the patients (5/10) had headache, dizziness or vomiting as an early sign of neurological manifestation. 2/3 sars patients had seizures, whereas 3/5 mers patients and both oc43-infected cases had facial/limb paralysis. unfortunately, mers-cov has not been detected in csf samples of any of the reported cases. ischaemic changes have been detected in images of half of the patients, while haemorrhagic pathology has been identified on one mers patient. the oc43-related cns-infected patients were younger compared to sars and mers patients. gcs: glasgow coma scale; ncv: nerve conduct velocity; emg: electroneuromyography. capability for neuroinvasion of this hcov [3] . furthermore, oc43 has also been identified in a teenager patient with demyelinating disease, in whom the virus was detected in both csf and nasopharyngeal secretions by pcr technology [14] . the hcov-oc43 has also been associated with a fatal encephalitis in an infant although the underlying circumstances are still unclear [15] . additionally, co-infection with the 229e and oc43 strains has been reported in a young girl who developed an acute flaccid paralysis [16] . sars-cov and mers-cov have also been linked with neurological manifestations. sars-cov has been shown to be capable of infecting human neural cells [17] , and neuroinvasion and neurovirulence have been found in studies involving both sars-cov [18à23] and mers-cov [24, 25] . an association of these two more highly pathogenic viruses with neurological manifestations have also been reported. for instance, sars-cov particles and genomic sequences have been detected from post-mortem brain tissues of sars patients [26à28]. they have also been detected using rt-pcr in csf samples from a 32-year-old pregnant female patient who presented with a brief duration generalized convulsion and accompanying loss of consciousness [29] and within 24 h of a first seizure in a 59-year-old female patient [30] . although there is less of direct evidence of viral presence in the cns, mers patients have also presented with neurological findings, such as altered consciousness, as well as manifested with a wide range of abnormalities on brain mri [31, 32] . regarding the regional distribution of the virus in the cns, data from the post-mortem studies have shown that infection from sars-cov was confined to neurons within selected areas of the brain, including thalamus, cerebrum, brainstem, hypothalamus and cortex [22, 27] . intriguingly, sars-cov has been detected in cerebrum, but not in cerebellum, in both animal [22] and human [28] studies. in animals infected in the cns with mers-cov, the thalamus and brain stem were found to be the highest infected sites [25] . data from multiple hace2 transgenic mouse models has revealed that sars-cov detection in the brain is significantly delayed compared to that within the lung, consistent with the initial establishment of infection within the respiratory system before dissemination to the cns [21à23]. several dissemination routes have been proposed for coronaviruses to gain access to the cns (fig. 1) . the mouse hepatitis virus (mhv), a neurotropic coronavirus, has been shown to enter the cns via a transneuronal route and disseminate within the brain via neuroanatomic pathways [33] . in mice transgenic for hace2, sars-cov seems to enter the brain via the olfactory bulb post intranasal inoculation before disseminating transneuronally to distal connected neurons and causing the dysfunction/ death of these infected neurons [18] . a similar dissemination pattern has also been detected in oc43-inoculated mice [5, 13] . however, different observations were found in another hace2 transgenic mouse model, in which no sars-cov was detected in the olfactory bulb, or preferentially in sites transneuronally connected to the olfactory bulb [22] . in addition, a recent study has revealed that ace2 (angiotensinconverting enzyme 2) and tmprss2 (transmembrane protease, serine 2), two key genes involved in sars-cov-2 entry, are not expressed in olfactory sensory or bulb neurons, but rather in olfactory epithelial cells [63] , suggesting the involvement of other dissemination mechanisms independent of axonal transport. it has also been proposed that hcovs can gain access to the cns via the certain neurotransmitter pathways such as serotoninergic dorsal raphe system [18] , which primarily projects to cerebral cortex, neostriatum, amygdala, substantia nigra, pons, hippocampus, entorhinal cortex and locus coeruleus. however, this hypothesis is speculative and requires to be further tested in vivo models. viremia has been observed following intranasal inoculation with mhv [34] and sars-cov [21] in transgenic mice. in addition, viral rna has been measured in the blood of patients infected with sars-cov and mers-cov [35] and thus, the dissemination of these two viruses to the cns may be mediated by the hematogenous route through a damaged blood-brain barrier (bbb). once crossing the bbb, hcovs-infected cells could potentially invade the brain through perivascular spaces, also known as virchow-robin spaces. these fluid-filled spaces are implicated as sites in which macrophages and lymphocytes interact to initiate immune response in patients with viral encephalitis [36] . for instance, the cryptococcal organisms in aids patients have been shown to disseminate through the virchow-robin spaces before further propagation into other brain regions [37] . whether hcovs could disseminate via the same route remains unclear. the intranasal inoculation of rodents with mhv has also revealed a lymphatic dissemination of coronavirus via cervical and mesenteric lymph nodes in addition to viremia [34] . additional studies are required to determine the role of this potential dissemination route. the infection of hcovs may also result in long-term neurological deficits. the pathologic examination of brain tissue collected from a sars patient revealed necrosis of neurons and broad hyperplasia of gliocytes, indicative of the presence of a chronic progressive encephalitis during the course of viral infection [26] . in addition, scattered red degeneration of neurons, a sign of neuronal hypoxia or ischaemia [27] , as well as oedema around small veins with demyelination of nerve fibres [38] , have been detected in brains of sars patients, which may provide an explanation for an incidence of long-term neurological [39] and/or psychological [40] abnormalities observed in sars survivors. in surviving mice post hcov-oc43 infection, spongiform-like degeneration was a residual finding post acute infection, indicating the possibility of neurodegenerative neuropathological sequelae [4] . further, a decline of locomotor activity and an abnormality in the limb clasping reflex were observed starting several months after viral infection, and neuronal loss especially in the hippocampal region was still detectable one year post infection [41] . although the majority of infectious virus (80%) was cleared in surviving animals, viral rna could persist for months [41] . even for cells that have survived the initial viral infection, little is known about their long-term functional status. to better understand this, cov-infected models that allow the identification and isolation of survival cells, such as the cre reporter mouse model of mhv infection, may be helpful [42] . recovery from acute infection does not necessarily promise complete clearance of the virus, and if an infection were to becomes chronic, it may result in long-term sequelae including chronic neurological diseases. however, given the limited clinical data, the long-term effects of hcovs on the cns post acute infection are unknown and thus, more studies involving the long-term follow-up of survivors are needed. ace2 was reported as a functional cellular receptor for sars-cov due to the ability of the s1 domain of the spike (s) protein to efficiently bind to ace2 for subsequent viral replication in the cytoplasm of sensitive cells [43] . in addition, previous autopsy studies have inferred that only ace2-postive cells were susceptible to sars-cov infection because the s protein and its rna could not be detected in ace2-negative cells [44] . it has been reported that sars-cov-2 might also use ace2 as a cellular receptor, despite a variation at some key residues of the receptor-binding domain compared to that of sars-cov [6] . the lack of ace2 expression in neuronal cells at both transcription and protein level contrasts with the viral susceptibility of these cells and cytopathogenicity during the acute phase of infection in neural cell lines of human and rat origins [17] as well as in humans brains [45] . even in hace2 transgenic mice, when specific brain regions were heavily infected by sars-cov, the expression levels of hace2 in the brain were no more than 01 to 1% of those found in the lungs [18] . furthermore, even in cells highly expressing hace2 in the transgenic animal model, sars-cov infection could be absent, indicating that the expression of hace2 alone might not be sufficient for maintaining viral infection [46] . these findings suggest that other receptor (s) or co-receptor(s) are involved in the viral entry into neural cells. dpp-4 (also known as cd26) was identified as a cellular receptor for mers-cov [47] . two lines of human dpp-4 transgenic mice were found to have high affinity to infection by mers-cov [24, 25] . dpp-4 is expressed in human brains [48] , therefore, when the virus gains access to the brain, cells expressing dpp-4 are potentially available for viral replication within the cns. aminopeptidase n (apn, also known as cd13), a cell-surface metalloprotease, was proposed as a receptor for hcov 229e [49] , as well as sars-cov [50] . human aminopeptidase n was shown, in vitro, to be a cellular receptor for hcov 229e infection of human neuronal, astrocytic and oligodendrocytic cell lines [51] . another glycoprotein, namely cd209l (also called l-sign) has also been identified as an alternative cellular receptor for sars-cov [52] . in addition, the infection of sars-cov on ace2-expressing cells seemed to be dependent on a proteolytic enzyme cathepsin l (ctsl1) in a ph sensitive manner [53, 54] . the expression levels of these receptors in human brain, however, require further elucidation to determine the extent to which they may be responsible for the hcovs infection in the cns. in addition to known and putative receptors, antibody-dependent enhancement (ade) of viral entry using the fusogenic spike protein has also been reported as a pathway to transfer coronavirus (mhv4) from infected cells to non-infected cells [55] . in vitro studies have shown that coronavirus entry could be mediated by special antibodies that bind the virus surface spike protein and facilitate subsequence viral entry of mers-cov [56] and sars-cov [57] in a receptor-like manner. the ade pathway is particularly important for vaccine design and the development of antibody-based drug therapies. 6. is sars-cov-2 likely to be present in the central nervous system? at present, it remains unknown whether sars-cov-2 is present in the cns, possibly due to limited access to brain tissue and csf from patients infected with the virus. autopsies are being increasingly carried out, however, initial biopsies have been taken only from lung, liver and heart for histopathology [58] . given that several strains of hcovs have been shown to be present in the brain, as well as csf [14à16,26à30], with neuroinvasion and neurovirulence shown in cell culture and animal models [4,5,11à13,17à25] , it is reasonable to consider that the sars-cov-2 may also be present in the cns. this is further supported by the fact that some covid-19 patients present with headache, nausea and vomiting, symptoms that potentially indicates neurological involvement [59] . the frequency of neurological manifestations including changes in consciousness and acute cerebrovascular disease increase in parallel with the severity of the disease [60] . one study on nasal epithelium samples taken from a subset of covid-19 patients exhibiting olfactory dysfunction has suggested that the disturbance of smell in these patients is more possibly due to sars-cov-2 infection of non-neuronal cells rather than olfactory neurons [63] . more recently, the report of a cohort of 58 covid-19 patients showed a correlation between acute respiratory distress syndrome (ards) and encephalopathy, mainly agitation, confusion and corticospinal tract signs [61] . however, these findings need to be confirmed by studies on brain samples or csf. the most efficient way to identify involvement of neurological systems remains to be determined. the presence of sars-cov-2 in csf was confirmed by gene sequencing in a 56-year-old patient with covid-19, which was the first direct evidence for the neuroinvasion of this novel coronavirus [62] . brain samples remain the gold standard of confirmation, however, access to such samples will almost certainly remain very limited and it is not a practical or ethical consideration in larger cohorts, especially in mild to moderate cases. several pathways of invasion for hcovs have been proposed, including via the olfactory nerve, neurotransmitter pathways, hematogenous route, virchow-robin space surrounding arterioles and venules, lymphatic systems and receptors, from which alternative parameters and types of samples may offer opportunity for proof of cns involvement. more studies are needed to evaluate and compare these alternative pathways and parameters. in addition, the timing of sampling needs further investigation, as the change of viral load in the human body at different stages of covid-19-related infection and recovery remains unclear. though considered mainly as respiratory viruses, several strains of hcovs have been detected in brains from patients with encephalitis and multiple sclerosis. in addition, the detection of sars-cov in csf of patients with neurological manifestation has also provided direct evidence for the neuroinvasion and neurovirulence of hcovs. however, the role of the virus in the process of the disease in acute phase as well as in the long term still remains elusive. the current strain sars-cov-2 can also potentially infect neuronal cells in the brain. therefore, a better understanding of dissemination characteristics and neuropathogenesis of these hcovs in the cns is urgently needed. catching a viral infection with ensuing cytokine release and tissue lesions may lead to a variety of cns-related manifestations (delirium, headache, vomiting, etc.), which are recognizable for many viruses including hcovs, however, these non-specific clinical symptoms and signs may be constitutional and represent systemic involvement rather than direct cns infection. therefore, we should be cautious when reporting cases with neurological symptoms and/or signs that lack solid evidence for cns infection (e.g. absence of viral detection in csf samples or abnormalities supportive of infection on brain imaging). it is particularly challenging when evaluate the neurological deficits during the advanced stage of covid-19-related disease. the clinical diagnosis and treatment strategy has to be made on the basis of a differential diagnosis which includes hypoxia, respiratory, metabolic acidosis, ards-related encephalopathy, the effect or withdrawal of medications, and viral infection of the cns. pubmed search of articles: "human coronavirus", "sars", "mers", "oc43", "229e", "neurological symptom", "neurological sign", "neurological manifestation", "neuroinvasion", and "neurovirulence". additional articles were selected based on articles in these searches and as suggested by reviewers. we declare no conflicts of interest. identification of an epitope of sars-coronavirus nucleocapsid protein engineering the largest rna virus genome as an infectious bacterial artificial chromosome neuroinvasion by human respiratory coronaviruses vacuolating encephalitis in mice infected by human coronavirus oc43 human respiratory coronavirus oc43: genetic stability and neuroinvasion genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding neurological manifestations in covid-19 caused by sars-cov-2 persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229e acute and persistent infection of human neural cell lines by human coronavirus oc43 infection of primary cultures of human neural cells by human coronaviruses 229e and oc43 murine encephalitis caused by hcov-oc43, a human coronavirus with broad species specificity, is partly immunemediated susceptibility of murine cns to oc43 infection axonal transport enables neuron-to-neuron propagation of human coronavirus oc43 detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis human coronavirus oc43 associated with fatal encephalitis a rare cause of acute flaccid paralysis: human coronaviruses susceptibility of human and rat neural cell lines to infection by sars-coronavirus severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus differential virological and immunological outcome of severe acute respiratory syndrome coronavirus infection in susceptible and resistant transgenic mice expressing human angiotensin-converting enzyme 2 generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis multiple organ infection and the pathogenesis of sars organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways possible central nervous system infection by sars coronavirus detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) neurological complications of middle east respiratory syndrome coronavirus: a report of two cases and review of the literature effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain viremic dissemination of mouse hepatitis virus-jhm following intranasal inoculation of mice middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease immunological and neuropathological significance of the virchow-robin space dilated virchow-robin spaces in cryptococcal meningitis associated with aids: ct and mr findings the clinical pathology of severe acute respiratory syndrome (sars): a report from china persistence of physical symptoms in and abnormal laboratory findings for survivors of severe acute respiratory syndrome posttraumatic stress, anxiety, and depression in survivors of severe acute respiratory syndrome (sars) human coronavirus oc43 infection induces chronic encephalitis leading to disabilities in balb/c mice murine olfactory bulb interneurons survive infection with a neurotropic coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace2+ cells in sars patients: relation to the acute lung injury and pathogenesis of sars tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv human aminopeptidase n is a receptor for human coronavirus 229e aminopeptidase n inhibitors and sars involvement of aminopeptidase n (cd13) in infection of human neural cells by human coronavirus 229e cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells dissemination of mhv4 (strain jhm) infection does not require specific coronavirus receptors molecular mechanism for antibody-dependent enhancement of coronavirus entry antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro pathological findings of covid-19 associated with acute respiratory distress syndrome development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients neurologic features in severe sars-cov-2 infection sars-cov-2: underestimated damage to nervous system non-neuronal expression of sars-cov-2 entry genes in the olfactory system suggests mechanisms underlying covid-19-associated anosmia we thank a/prof dennis cordato, senior specialist/neurologist, liverpool hospital, sydney, australia, for critical review and grammar check of the manuscript. we received no funding for this review. key: cord-276533-cxyndepl authors: mcfarland, henry f.; dhib-jalbut, suhayl title: multiple sclerosis: possible immunological mechanisms date: 1989-01-31 journal: clinical immunology and immunopathology doi: 10.1016/0090-1229(89)90116-5 sha: doc_id: 276533 cord_uid: cxyndepl abstract multiple sclerosis is the principal demyelinating disease of the central nervous system. although the prevalence of the disease is moderately low, averaging about 40 cases per 100,000 people in high risk areas, it is a particularly devastating disease. it primarily affects young adults, is chronic, and has an unpredictable course. most discouraging, the cause of the disease is not known and an effective treatment has not been identified. recently, however, research has yielded some important findings concerning the etiology of ms. much evidence now points to an immunological process as one of the major elements in the disease. it is also likely that an environmental influence, possibly an infectious process, may contribute to the disease. finally, it is now certain that genetic makeup influences susceptibility to the disease. at present, the strongest evidence is for a polygenic effect, not the effect of a single gene or gene locus. this review will examine some of the possible immunologically mediated disease processes that could be involved in ms, especially those that could account for a role for infectious and genetic factors in the disease. multiple sclerosis (ms) is the principal demyelinating disease of the central nervous system (cns). although the prevalence of the disease is moderately low averaging about 40 cases per 100,000 people in high risk areas, it is a particularly devastating disease. it primarily affects young adults, is chronic, and has an unpredictable course. most discouraging, the cause of the disease is not known and an effective treatment has not been identified. recently, however, research has yielded some important findings concerning the etiology of ms. much evidence now points to an immunological process as one of the major elements in the disease (1) . it is also likely that an environmental influence, possibly an infectious process, may contribute to the disease. finally, it is now certain that genetic makeup influences susceptibility to the disease. presently, the strongest evidence is for a polygenic effect, not the effect of a single gene or gene locus. since an excellent review of the various immunological studies relevant to ms has recently been published (11, this review will examine some of the possible immunologically mediated disease processes that could be involved in ms, especially those that could account for a role for infectious and genetic factors in the disease. despite the focus on an immunological process in ms, the evidence for this is largely circumstantial. supporting an immunological mechanism in ms are the s97 abnormalities of immunoglobulin (ig) found in the cerebrospinal fluid (csf) of patients with the disease (2, 3) . the alterations in csf ig represent the most consistent immunological changes found in the disease and this is reflected in their diagnostic importance. these changes include elevations in the levels of igg. igm, and possibly iga in a majority of ms patients (2) . this ig is largely synthesized locally in the csf and is not merely a reflection of a breakdown of the blood-brain barrier (3) . the ig is usually oligoclonal as demonstrated by electrophoresis or isoelectric focusing (4) . the oligoclonal nature of the ig has led to the supposition that the antibody specificity of this ig may be directly related to the disease process: the antibody may be directed to an infectious agent in the brain or the disease may be caused by antibody to myelin or oligodendrocytes. despite extensive study, a clear association between csf ig reactivity and the disease has not been established. increased antibody levels to numerous viruses have been found, and in almost every case, they represent only a small fraction of the ig present. an exception is the recent report demonstrating that a significant portion of csf ig from some ms patients is directed to svs, a paramyxovirus (5) . this finding awaits confirmation. small amounts of antibody to myelin components have been described and it has generally been thought that these antibodies are unlikely to be directly or singularly responsible for demyelination (6) . importantly, evidence is now emerging from experimental models of cell-mediated demyelination that the presence of antibody to components of myelin may augment disease and demyelination. this will be discussed at greater length later in this review. if most or all of the csf ig is not directly related to the cause of the disease, why do elevated levels occur in the csf? a likely explanation is that the csf ig reflects the specificity of b cells that are found in greatest number in the blood, and therefore, have the greatest chance of migrating into an inflammatory site in the cns. the antibody specificities found in the csf of ms patients are generally those with high titers in the blood such as antibodies to viruses that are associated with lifelong immunity. although the accumulation of these b cells may be random, the conditions permitting their differentiation into ig-secreting cells must be present within the cns. generally, differentiation of and ig production by b cells requires two signals, one of which is the perturbation of the ig antigen receptor. if the antigens to which these antibodies react are not in the cns, the continued differentiation of b cells must be due to factors in the cns milieu that are capable of overriding the need for antigen binding to the ig receptor. this could include lymphokines produced by the ongoing immune response or to the effect of neurotransmitters with immunostimulatory activity. obviously, an alternative explanation is that the antigen necessary to drive these b cells is in the cns. this would suggest that multiple viruses could contribute to the disease process in some manner. direct evidence for this is lacking. probably the most consistent antiviral antibody found in csf of ms patients is that to measles virus (7) . although measles virus genome has been demonstrated in brain material from some ms patients (8) , the finding is not specific for ms and efforts to demonstrate the virus by other means have been unsuccessful. it is unlikely that the elevation in antimeasles antibody can be explained by cross-s9x mcfarland and dhib-j.ai.bu i reactivity between the virus and antigens in the nervous system since antibodies to at least four of the five protein components of the virus are elevated (dhib-jalbut and mcfarland, unpublished data). with the exception of the csf ig abnormalities. other evidence for an immunological process in the pathogenesis of ms points to a cellular immune mechanism. the perivenular inflammatory response comprised of lymphocytes and monocytes is certainly consistent with an immunologically mediated disease and resembles the pathological changes seen in postvaccinal encephalomyelitis, a disease of certain immunological cause. similarities between ms and a model of cell-mediated immunopathological disease of the cns. experimental allergic encephalomyelitis (eae). support a similar mechanism in ms. the objections to the appropriateness of eae as a model for ms have been partially overcome by the demonstration of an experimentally produced relapsing remitting disease with close pathological similarities to ms (9) . although the relationship between ms and eae remains uncertain, it is clear that the initiating events in ms are far more complex. finally, a number of abnormalities in lymphocyte distribution and function have been described in ms. these have been reviewed in detail elsewhere (i). these changes tend to be nonspecitic and, in some cases, inconsistent, but in general they support the supposition of an abnormality in cellular immunity in ms. there remain, however, fundamental unanswered questions regarding the nature of a putitive immune-mediated process in ms. these include: what is the actual mechanism of myelin destruction; what is the antigen or antigens responsible for initiating the disease process; how is antigen presented to immune t cells in the cns and is the genetic influence on susceptibility involved at this stage; what is the role of an environmental or infectious agent in the disease; and finally, what accounts for the fluctuating nature of the disease? although there are no definitive answers to these questions, recent studies have begun to provide some insight into these problems. several components of the immune system have been suggested as the effector mechanism producing demyelination. these have included antibody or other poorly characterized serum factors, lymphocytes, particularly cytotoxic t cells, and macrophages. the eloquent ultrastructural studies of acute lesions in ms by prineas provide strong evidence that demyelination is macrophage mediated (io). these studies, as well as similar studies of demyelination in eae, indicate that myelin disruption follows an interaction between myelin and macrophages and that characteristic coated pits are found at the point of myelin-macrophage contact. these coated pits most likely represent the migration of receptors within the macrophage membrane. a critical question is what are the receptors that are concentrated in the coated pit? included among the receptors expressed on macrophages are those for the fc portion of the ig molecule and for complement. either of these receptors could contribute to the myelin-macrophage interaction seen in ms. as pointed out previously, antibodies to various components of myelin occur in ms and those antibodies recognizing components of myelin ex-posed to the outer lamelle could form a means for macrophage attachment via fc receptors. antibody to glactocerebroside (11) and possibly other myelin components have been shown to contribute to the induction of eae in the guinea pig. also, transfer of antibody against components of myelin to mice with a relapsing form of eae seem to augment demyelination and enhance progression of the disease (12) . these findings suggest that antibody to myelin if not necessary for disease may at least contribute to the severity or progression of the process. complement may also serve as a ligand between myelin and macrophages. this could involve complement fixed to antibody bound to myelin or complement bound directly to myelin, since myelin has been shown to bind or fix complement in the absence of antibody. macrophages express two types of complement receptors, cr3 and crl. in addition to being up-regulated on activated macrophages, the cr1 receptor becomes mobile in the macrophage membrane and associates with clathin-coated pits (13). a final consideration in the demyelinating process concerns a more direct role of viruses. demyelination occurs in association with infections with several viruses in the retrovirus family. infection of sheep with visna virus produces an encephalitis that is characterized by inflammation and demyelination (14) . demyelination can also be a prominent finding in aids-related dementia caused by infection with hiv (15) . finally, abnormalities of white matter have been demonstrated by mri in tropical spastic paraparesis (tsp) due to infection with htlv-i (16) . in visna and aids, virus is found in the cns in macrophages and this may form the basis for entry into the nervous system (17, 18) . htlv-i has also been isolated from lymphocytes from patients with tsp and this may provide a similar basis for entry into the cns in this disease (19) . the cause of demyelination in these diseases is uncertain and may be due to infection of oligodendrocytes. the possibility that macrophages infected with viruses, particularly retroviruses, can cause destruction or damage to myelin directly must be considered as well. a possible mechanism could be production of cytokines toxic to myelin. at least in visna, this seems unlikely, since disease is reduced by treatment with immunosuppressive drugs indicating that demyelination may be immune mediated (42) . although a similiar process would seem unlikely in aids which is associated with profound immunosuppression, a role for an immunopathological process in this disease must still be considered. if activated macrophages serve as the final effector arm in demyelination and if macrophage activation is immune mediated, what is the antigen specificity of the immune response producing the activation and why doesn't demyelination follow every inflammatory response in the brain such as those associated with viral infections? the answer to the second question may be that a true delayed-type hypersensitivity (dth) reaction is necessary for macrophage activation. as mentioned previously, the presence of antibody to myelin may also contribute to the potential for demyelination. the antigen specificity or specificities of t cell initiating the local immune reaction in the ms lesion are not known but either neural or viral antigens would seem to be reasonable possibilities. eae, a disease characterized by inflammation, and in some species. demyelination, can be induced by inoculation of experimental animals with myelin basic protein (mbp). the most compelling objections to viewing eae as a model for ms have been based on the relatively acute and monophasic form of the experimental disease as compared to the relapsing course frequently found in ms. recent studies of eae have now demonstrated fluctuating courses under several experimental conditions. importantly, a relapsing form of eae has been produced in mice following the transfer of t cells from mice sensitized to mbp (9) . further, the pathological appearance of the cns of these mice closely resembles that found in patients with ms. that mbp can be the relevant antigen in relapsing eae is supported by the finding that this form of disease can be produced following transfer of t cell clones specific for the amino terminal portion of the mbp molecule (20). as expected, the demonstration that mbp induces an experimental disease with similarities to ms has stimulated extensive studies of mbp reactive t cells in patients with ms. evidence indicating an enhanced cellular immune response to mbp in ms, however, is meager. a recent study of a large number of patients using lymphoproliferation to measure reactivity to mbp found the response to be only slightly greater than that of a control group (21) . the response to two other immunogenic components of myelin, myelin-associated glycoprotein (mag) and proteolipid protein (plp), were not significantly different between the patients and the controls. in a separate study using t cell clones generated from the csf and blood of ms patients, mbp reactive clones could not be recovered from the csf of ms patients but were generated from csf lymphocytes obtained from patients with postinfectious encephalomyelitis (22) . in the same study, t cell clones were generated from lymphocytes obtained from the brain of a patient with ms who had died. none of the clones reacted with mbp. these findings suggest that, even at the site of demyelination, mbp reactive t cells cannot be readily demonstrated. this study probably does not provide a definitive answer to this problem since it can be argued that if lymphocytes were obtained at the very earliest stages of disease, t cells with more relevant specificities might be recovered. also, in studies of viral infections in the brain in which the inflammatory response is known to be generated by viral reactive t cells. only very small numbers of cells with the relevant specificity have been recovered from the inflammatory site (43) . this indicates that a very small number of antigen reactive t cells are capable of initiating an inflammatory response and that the recovery of these cells from the site may be extremely difficult. the evidence for a role for mbp reactive t cells in ms is unconvincing but unfortunately the question is not completely resolved. the other major category of antigens that has been considered in postulating an immunopathological process in ms is viral antigens. a relationship between a virus and ms has been supported by epidemiological studies showing a variation between incidence of disease and geographic region and by the apparent occurrence of small focal epidemic phase of disease (23) . in addition, elevations in antibody to various viruses have been found in the csf of patients with ms (44) . additional support for a relationship between ms and a virus comes from studies of slol various experimental models of virus-induced demyelination. one of the most important of these models is theiler's murine encephalomyelitis virus (tmev) infection in mice (24) . infection of some strains of mice with tmev, a picomavirus, produces, after one to several months, a neurological disease that is characterized pathologically by inflammation and demyelination. the disease appears to be immunologically mediated and susceptibility is influenced by the genetic makeup of the mouse (25) . the genetic factors include genes in the mhc and probably another genetic locus, possibly genes coding for the b chain of the t cell receptor (25) . susceptibility to disease correlates closely with the ability to generate a dth response to the virus. consequently, it has been proposed that t cells recognize virus in the cns and trigger a dth response. the subsequent recruitment and activation of macrophages then leads to demyelination. a process of this nature would not require persistence of infectious or complete virus. persistence of the viral genome and production of a single polypeptide that contains an epitope recognized by t cells would be sufficient to elicit a response. if the polypeptide was incomplete or present in small amounts, it might escape detection by conventional antibody staining. lymphocytes from ms patients have been studied extensively for abnormal reactivity to a wide variety of viruses. most of these studies have used lymphoproliferation to measure reactivity and the results have generally been inconsistent (45) . because of the substantially elevated antibody titers to measles virus in the csf of ms patients, measles virus has been the focus of numerous studies. recently, the generation of virus specific cytotoxic t cells (ctl) was examined in patients with ms and in appropriate control groups (26). the findings demonstrated that a substantial number of patients had a significant reduction in their ability to generate measles virus-specific ctl. in contrast, their ability to generate influenza virus-specific ctl was normal. the abnormality in measles virus ctl is due to a s-to io-fold reduction in the precursor frequency of the population and not due to their complete absence (27) . importantly, measles virus ctl are cd4+ lymphocytes and recognize measles virus antigens in conjunction with class ii hla molecules. this is in distinction to most other well-characterized virus-specific ctl which are predominantly cd8' and hla class i restricted. it remains uncertain if the abnormality seen in the generation of measles virus ctl is specific for measles virus or if it reflects a more general abnormality in a population of cd4+ lymphocytes. if the abnormality is virus specific, it may reflect a defect occurring at the time of initial exposure to the virus or to an abnormality in maintaining normal long-term immunity. it is also tempting to speculate that these cells are reduced in the peripheral blood because they are sequestered within the cns following recognition of viral antigens. recognition of a brain antigen that cross-reacts with measles virus is unlikely, since there is a reduction in the ability to generate ctl using several of the measles virus proteins: the reduction is not specific for a particular viral polypeptide (dhib-jalbut and mcfarland, in press). although the possibility of t cell recognition of viral antigens in the cns triggering an immune response with subsequent demyelination remains an attractive hypothesis, proof is lacking. an alternative means by which a virus could elicit a disease such as ms is by causing sensitization to a neural antigen either by altering tolerance to the antigen during infection or through molecular mimicry. the coronavirus, jhm virus, when inoculated into rodents produces a subacute demyelinating disease. this disease is probably due to infection of oligodendrocytes with virus. an important finding, however, has been that lymphocytes taken from rats with this subacute disease and transferred into normal rats can elicit an eae-like disease that does not seem to be due to transfer of virus (28) . these animals can subsequently be shown to have a cellular immune response to mbp. these findings indicate that infection with jhm virus leads to sensitization to mbp. a similiar observation has been made in the same laboratory using rats infected with a neurotrophic strain of measles virus (29) . although this virus also produces encephalitis. it replicates mostly in neurons and does not cause demyelination. this suggests that the inflammatory response in the brain may be sufficient for sensitization to mbp to occur. sensitization to mbp following viral infections has also been reported in humans. studies of children with complicated or uncomplicated measles virus infections have demonstrated that many of the children acquire a lymphoproliferative response to mbp greater than that found in controls (30) . it is not known if this sensitization follows infection of the nervous system with measles virus or if it is due to some form of cross-reactivity. some minor degrees of sequence homology have been reported between mbp and several viruses that infect humans (31). despite extensive study. however. immunological cross-reactivity between measles virus and mpb has not been demonstrated. regardless of the mechanism, some viral infections apparently can produce enhanced t cell reactivity to neural antigens. if ms is due to a local dth response, the relevant antigen(s) is most likely presented to t cells in the nervous system. the essential requirements for cells to serve as antigen-presenting cells are that they are capable of processing antigens and that they can express hla class ii molecules on their surface. two cell populations, astrocytes and brain endothelial cells, have been shown to have these qualities (32, 331 . it also seems reasonable to assume that microglia may also be able to serve as antigen-presenting cells (46) . neither astrocytes nor cns endothelial cells normally express hla class ii molecules but both can be induced to do so using interferon-y. also endothelial cells from mice with eae express hla class ii molecules and studies of astrocytes from various strains of rats or mice have found a correlation between the ability to induce hla class ii molecules and susceptibility to eae (34) . mhc class ii antigens can also be induced on rat astrocytes with viruses (35). even inactivated virus seems to be capable of inducing mhc class ii antigen on astrocytes and this could be achieved more easily on astrocytes derived from strains of rats susceptible to eae. these findings indicate that persistent infection could contribute to expression of mhc molecules and that this, in turn, could lead to presentation of either viral or neural antigens. this provides an alternative mechanism by which a viral infection could lead to sensitization to cns antigens. importantly, this suggests that a persistent infection could be instrumental to s103 disease production triggered by t cell reactivity to nonviral antigens. a diminished cellular immune response to the virus might increase the likelihood of this since there would be a reduced ability to eliminate virus. differences in the ability to induce hla molecules in humans similar to those demonstrated in those experimental animals could represent one of the components of the genetic susceptibility to ms. clearly, the mechanisms regulating expression of the molecules may be critical to understanding ms. regulation of the immune response one of the striking characteristics of ms is its frequent relapsing remitting course. this has led to speculation that the disease may be related to an abnormality of suppressor cell function with reduced suppression resulting in episodes of disease activity followed by return of suppressor function or local regulation of the response. in fact, deficiencies of suppressor cells have been demonstrated in several experimental, in vitro, systems (1) . the finding that a reduced number of cds+ t cells, which includes suppressor cells, occurs during periods of worsening has been inconsistent. functional studies have shown, however, that there is reduced suppression during progression or exacerbation. these studies have measured suppression as generated by mitogens such as concanavalin a or okt3 which binds to the t cell receptor (36) . similarly, the ability of cd8+ t cells to suppress ig production following pwm stimulation is reduced while other functional parameters of the cd8+ t cells such as ctl activity are normal (37) . a subset of cd4+ cells, the 2h4 cells, which acts to induce suppression through cd8+ t cells, has been shown to be reduced in ms patients with active disease (38) . the autologous mixed lymphocyte reaction (amlr) has been considered an important technique for studying immunoregulatory processes and several investigators have examined the amlr in patients with ms (39, 40, 41) . generally, the response is found to be increased during worsening. it has now been demonstrated that this is due to a reduction in the generation of suppression which normally occurs in the amlr (40) . the significance of the various abnormalities of suppressor cell function is uncertain. ms is not associated with multiple immunological abnormalities which casts some doubt on generalized suppressor cell deficiency but episodic fluctuations in regulatory mechanisms could be involved in exacerbation. alternatively, these changes in immunoregulation could occur as a result of the same process that triggers the immune response occurring within the cns. for example, upregulation in hla class ii antigen expression could lead to increased reactivity in all of these in vitro tests and account for the apparent reduction in suppression. studies of immune regulation are critical to an understanding of the immune process in ms and future studies hopefully will establish if these changes represent reduced suppression or enhanced reactivity secondary to the disease process. conclusion the evidence linking the pathogenesis of ms to an immunological process remains indirect and tentative. it seems increasingly certain that the actual de-struction of myelin is caused by activated macrophages. what is uncertain is the mechanism for activation of these macrophages. although an immunological mechanism would seem most likely, it is still possible that this activation could occur as a direct result of a viral infection. by extrapolating from observations made in various experimental models, it seems that the most likely immunological process would be the induction of a dth type response with subsequent macrophage activation. either a neural or viral antigen could trigger this response and the genetic influence on susceptibility may reflect the genes coding for the hla class ii and t cell receptor makeup necessary for recognition of the relevant antigen. the failure to demonstrate a unique or enhanced t cell reactivity is disturbing, but could be due to the techniques used or to a sequestration of this population within the cns. this latter possibility seems the least likely and has little support from experimental models. it is now clear that both astrocytes and endothelial cells can function as antigenpresenting cells in vitro. it is also likely that microglia can also express class ii hla molecules and function as antigen-presenting cells. the nonspecific induction of hla class ii antigens on these populations by lymphokines such as interferon-y provides an attractive mechanism for presenting antigen to circulating cells and triggering a local immune response which leads to demyelination. periodic increases in hla class ii expression due to nonspecific processes such as viral infections could provide a reasonable explanation for the fluctuating course of the disease. with continued disease progression, the local production of lymphokines may become sufficient to maintain an ongoing immune response. demyelination may be enhanced by local production of antibody capable of binding myelin. b cells with appropriate specificities migrating into the inflammatory site would find both antigen and lymphokines necessary for b cell differentiation. the continued differentiation of b cells with production of antimyelin antibody could contribute to the conversion of the disease from a relapsingremitting course into one of continued progression. the major unanswered questions concern the antigen and the role for the genetic influence on the immune response. the ability of t cells to recognize epitopes consisting of only several amino acids and which are not recognized by antibody may contribute to the difficulty of answering these questions. it is hoped that the techniques that allow manipulation of hla genes and identification of characteristics of the t cell receptor makeup along with more specific immunological methods will contribute a greater understanding to these questions. immunology of multiple sclerosis cellular and humoral components of cerebrospinal fluid proc. natl. acad. sci. usa key: cord-005146-o3roa7br authors: sasaki, makoto; ide, chizuka title: demyelination and remyelination in the dorsal funiculus of the rat spinal cord after heat injury date: 1989 journal: j neurocytol doi: 10.1007/bf01206664 sha: doc_id: 5146 cord_uid: o3roa7br part of the dorsal funiculus of the adult male rat (wistar) spinal cord was treated for l h at the thoracolumbar level by running hot water, at approximately 48–50 ° c, through a polyethylene tube 2 mm in diameter in contact with the dura. animals were fixed 1 day to 4 weeks later and the spinal cords were examined by light and electron microscopy. the affected area in the dorsal funiculus was approximately 1 mm long and less than 1 mm wide at the dorsal surface, and varied from 0.4 to 0.7 mm in depth. within 3 days after treatment, almost all the myelin sheaths in the affected area were degraded, leaving the axons denuded, and at the same time astrocyte endfeet at the glial limiting membrane were swollen and partly destroyed. almost all the denuded axons remained intact, exhibiting no noticeable morphological changes. there was evidence of a moderate vasogenic oedema, but minimal signs of haemorrhage in the lesion. seven days after treatment, many immature schwann cells but no oligodendrocytes were found between the denuded axons. by 2 weeks many of the denuded axons were remyelinated, and by 4 weeks almost all of those axons located near the pial and perivascular surfaces had been remyelinated by schwann cells, while most of those located in the deep and marginal zones bordering the adjoining intact areas were remyelinated by oligodendrocytes. longitudinal sections revealed that at nodes of ranvier pns-type myelin sheaths were apposed by either intact or newly formed cns-type myelin sheaths. a typical glial limiting membrane was not reformed beneath the pial surface, but an inconspicuous one was found between the pnsand cns-type fibre areas. recently, therapeutic hyperthermia in localized or whole-body applications has been used as a new method of cancer therapy. in focal hyperthermia, the tissue is heated with microwave or ultrasound transducers to about 40 ~ c, a temperature which damages tumour cells but does not affect the surrounding normal tissue (hahn, 1982) . changes in the normal brain and spinal cord tissues resulting as an adverse effect of clinical or experimental hyperthermia have been studied only sporadically in the past three decades (barnard et al., 1956; fajardo, 1984; lyons et al., 1986) . in a clinical study (douglas et al., 1981) on the myelopathy induced by whole-body hyperthermia at 41.8~ for 6h, severe demyelination, axonal and gliai cell degeneration, and vascular congestion were noted to occur in the white matter; and swelling and chromatolysis of the neurons were seen in the gray matter. several experimental studies of hyperthermia (barnard et al., 1956; britt et al., 1983; lyons et ai., 1984) have also demonstrated that acute changes, including myelin sheath breakdown, vasogenic oedema and * to whom correspondence should be addressed. haemorrhage, were found mainly in the white matter after heat treatment at a temperature of over 42 ~ c for about 60 min. these studies were done by light microscopy only, and no ultrastructural study has so far been reported with regard to the effects of hyperthermia on normal cns tissues. to investigate more precisely the histological changes induced in cns tissues after high temperature treatment, heat injury was induced in the rat spinal cord by transdural heat application, and early degradation and subsequent repair of the affected tissues were examined by light and electron microscopy. thirty male rats (wistar strain), weighing 200-300 g, were used. the animals were anesthetized by intraperitoneal injection of nembutal (sodium pentobarbital, 100 mg/kg body weight), and the thoracolumbar region of the spinal cord was exposed through a rectangular window made by partial laminectomy. a polyethylene tube, 2 mm in diameter, was bent and the tip of the bent end was placed on the 0300m864/89 $03.00 +. 12 9 chapman and hall ltd. water was circulated by a flow roller pump through a 2 ram-diameter polyethylene tube. the water was warmed to approximately 60 ~ c in the water bath so that the temperature of the thermocouple at the bent tube could be kept at 48-50 ~ c. the tube was wrapped with adiabatic material, and the tip of the bent tube was in contact with the dural surface of the spinal cord. dura mater using a stereotaxic device. hot water, heated in a water bath (yamato bk33), was circulated through this tube. tube. the temperature was maintained at 48-50 ~ c for 1 h, as monitored with a thermocouple (1.0 mm in diameter) inserted between the proximal and distal limbs of the bent tube, 10 mm away from its tip (fig. 1 ). room temperature was kept at 28-30 ~ c, and the tube, except the bent tip, was shielded by adiabatic material to prevent cooling by air. the pressure on the dura exerted by the tube was not so great as to interrupt the blood flow in the dorsal vein of the spinal cord, as checked under a dissecting microscope. control animals were operated on and treated in the same manner, except that hot water was not circulated through the tuba in two animals, and hot water at 40m5 ~ c was circulated in five animals. the animals were killed 1 (n-4), 3 (n=4), 5 (n~3) and 7 (n-4) days, and 2 (n=4) and 4 weeks (n=4) after the operation by perfusion through the heart with a fixative containing 2.0% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m cacodylate buffer. the treated parts of the cord were excised, together with the adjacent cranial and caudal segments and stored overnight in the same fixative. specimens were cut into small blocks, and were postfixed in 1.0% osmium tetroxide solution in 0.1 m cacodylate buffer for 2 h, dehydrated through a graded series of ethanol, and embedded in epon 812. ultrathin sections were cut on a lkb ultrotome and observed in a jeol-100b electron microscope after double staining with uranyl acetate and lead citrate. semithin sections i p~m thick were stained with 1% toluidine blue and observed by light microscopy. the rats did not show any gait or other behaviour disturbance after the operation. the lesion resulting from the heat treatment was confined within the dorsal funiculus of the spinal cord. the affected area was approximately i mm long, and less than 1ram wide at the dorsal surface of the spinal cord. the depth of the lesion varied from 0.4 to 0.7 mm. at every post-treatment stage only a few degenerated axons were observed in the dorsal funiculus at levels cranial to the lesion, indicating that the axons themselves were not damaged by the heat treatment. in the control cases the dorsal surface of the spinal cord was deformed to some extent, presumably due to compression by the tube'. however, no focus of demyelination was found in the dorsal funiculus. a few fibres whose myelin sheaths were degraded in the treated part of the dorsal funiculus were noted by light microscopy. myelinated fibres located near the pial surface appeared loosely packed owing to the increase of extracellular spaces ( fig. 2a, b) . electron microscopy showed that myelin sheath lamellae of some myelinated fibres were loosened or even broken up into irregular structures (fig. 3) . in some fibres the internal loops of myelin sheaths were markedly swollen. these were features of the early stages of myelin sheath degradation which finally led to the complete demyelination of the affected fibres. the extracellular spaces between the axons were enlarged and contained proteinaceous oedema fluid and electron-dense deposits of fibrin. astrocyte processes, including endfeet of the subpial glial limiting membrane (glm), were partly swollen and disrupted, but the basal lamina was preserved as a continuous sheet covering the entire surface of the spinal cord. oligodendrocyte processes forming the myelin sheath outer loop disappeared from the degenerating myelin sheaths. in the affected area astrocytes and oligodendrocytes contained many vacuoles in their cytoplasm suggesting early degenerative changes. the vessel walls appeared undamaged and no haemorrhage occurred in the lesion. light microscopy showed that most fibres located near to the pial surface had myelin sheaths with much less opacity, reflecting advanced myelin lamella disintegration and breakdown. extracellular spaces between the axons were further enlarged, and some macrophages were found in such interaxonal spaces. no haemorrhage was noted (fig. 2c) . electron microscopy showed that many myelin sheath lamellae were split extensively into irregularly arranged sheets and had partially disintegrated into vesicular structures, losing their original electron fig. 4 . three days after treatment. this electron micrograph shows a later stage in myelin sheath degradation with interlamellar splitting of all the fibres. some axons (asterisk) appear to have been damaged by heat injury, but most seem to be intact (ax). astrocytes at the glm beneath the pia are totally destroyed, but the basal laminae are largely preserved (arrowheads). extracellular spaces of the affected area have become much more conspicuous than in fig. 3 . x 8600. opacity (fig. 4) . other myelin sheaths, while retaining densely packed lamellae, exhibited a greatly decreased electron opacity. axons whose myelin sheaths had become degraded appeared normal. astrocyte processes including endfeet of the glm were entirely disrupted and were not visible. however, the basal laminae of the glm remained intact separating the cns from the s u r r o u n d i n g c o n n e c t i v e tissue. no oligodendrocyte cell bodies or processes were found in the areas near the surface or in the centre of the lesion. fragments of degraded myelin sheaths, as well as fibrin, were scattered in wide interaxonal spaces, and macrophages were phagocytosing cellular debris. n u m e r o u s d e n u d e d axons were identified by light microscopy as various-sized bundles within the heat-injured areas. macrophages containing myelin debris were scattered between such d e n u d e d axons. spindle-shaped cells proliferated and made a dense cell layer above the affected area (fig. 2d) . by electron microscopy almost all axons were completely demyelinated and closely packed (fig. 5a) . a notable finding was the presence, between the demyelinated axons, of cells with round nuclei which contained no myelin debris. these cells often extended cytoplasmic processes around axons in the m a n n e r of developing schwann and glial cells, suggesting that they were immature schwann or glial cells migrating through the interaxonal spaces (fig. 5b ). cells identifiable as astrocytes and oligodendrocytes were not observed within the affected area at this time. the glm basal lamina at the pial surface was largely destroyed. thus naked axons and associated imma-ture schwann cells were in direct contact with the pial connective tissues at the pial surface. spindle-shaped non-neural cells were numerous throughout the pial layer. most axons near the pial surface appeared thinly myelinated by light microscopy, while most axons located toward the deep or marginal zones of the lesion remained denuded. also noted were many round cells with relatively dark cytoplasm, intimately associated with the thinly myelinated axons. spindleshaped non-neuronal cells and bundles of collagen fibres formed a thick pial layer on the surface of the injured cord. macrophages containing myelin debris were still present in the lesion (fig. 2e) . by electron microscopy, many axons were noted near the pial surface covered mostly by very thin, but occasionally by relatively thick myelin sheaths (fig. 6) . it was evident that the round cells were associated 1 : 1 with the axons, and that they were covered by basal lamina, features typical of myelinating schwann cells. in the marginal zone bordering the intact area, some axons were thinly myelinated by oligodendrocyte processes (fig. 7) : the myelin sheath had an interlamellar periodicity of about 130a. at this stage oligodendrocytes tended to be in contact with or near these cns-type fibres. astrocytes also appeared in the marginal zone bordering the intact area. by light microscopy it was determined that almost all the axons located near the pial and perivascular surfaces were covered by thick myelin sheaths. schwann cells were associated with axons in a 1:1 relationship. in the deep or marginal zones bordering the intact tissue, most axons had thin myelin, which, unlike schwann cell myelin, had no cytoplasmic layer or basal lamina on the myelin sheath surface, indicating that oligodendrocytes had made the myelin sheaths around them. blood vessels were abundant throughout the lesion but macrophages were no longer found in the injured area (fig. 2f) . by electron microscopy it was determined that almost all the axons near the pia! and perivascular surfaces were myelinated by schwann cells (fig. 8) . the myelin sheaths were 0.5-1 #,m thick and had an interlamellar periodicity of about 150a. unmye-231 linated axons were wrapped in a bundle of 2-3 fibres by a common schwann cell, as in unmyelinated fibres of the pns. schwann cells were invested with basal laminae associated with a few collagen fibrils on their interstitial surfaces. on the other hand, in the deep and marginal zones bordering the intact area almost all the axons were myelinated by oligodendrocytes (fig. 9) . such cns-type myelin sheaths were extremely thin (about 0.1 ~m thick), and had an interlamellar periodicity of about 130 a. at the oligodendrocyte-and schwann cellmyelination transition areas, pns-and cns-type fibres intermingled with one another with no clear line of demarcation (fig. 10) . in such transitional zones, the oligodendrocyte-myelinated fibres were incompletely covered by the astrocyte processes; therefore cns-type myelin sheaths and oligodendrocyte processes were directly contiguous to schwann cell basal laminae or collagen fibrils. peculiar dark cells, which had previously appeared as spindle-shaped cells, proliferated to form a thick layer on the pial surface of the lesion. no astrocytic layer had developed between this 'pial' cell layer and the underlying neural tissue. blood vessels within the schwann cell myelinated areas lacked an astrocytic covering; these vessels were thus of similar appearance to those found in the pns. a few macrophages containing myelin debris were still observed in the affected area. in longitudinal sections, internodes of cns-type remyelinated axons were so short that the entire internode could be followed in one section, whereas those of pns-type internodes were longer and only part of an internode was traceable in one section. nodes of ranvier of the pns-type had a normal structure, while those of a cns-type occasionally exhibited abnormally wide gaps. oligodendrocytes sometimes formed a thin myelin sheath on their own cell body. newly formed pns-or cns-type myelin sheaths were apposed to the adjacent intact cns myelin sheaths at nodes of ranvier. sometimes a single axon was remyelinated by oligodendrocytes and by schwann cells forming alternate pns-and cns-type myelin sheaths (fig. 11 ). usually only a few astrocyte processes covered these newly formed nodes of ranvier. unlike the normal pns-cns transition in the nerve root, lateral loops of oligodendrocytes were directly contiguous with schwann cell processes at the node (fig. 11, inset. ). the present study has documented the morphological changes in the spinal cord injured by heat treatment, as observed by light and electron microscopy. using hot water circulating in a tube, we made a small (less than i mm~), well-confined lesion in the dorsal funiculus of the rat spinal cord. according to previous reports (britt et al., 1983; lyons et al., 1984) , the temperature threshold for cell damage in the cns is 42-43~ c for a duration of 50-60 min. in the present study the tube was kept at 48-50 ~ c on the surface of the dura: this elevated the temperature in the tissue high enough to cause damage to glial cells but not axons. the spinal cord is susceptible to compression: demyelination as well as axonal degeneration can occur following mild compression (gledhill et al., 1973; harrison et al., 1975) . in the present study, demyelination was not found in the dorsal funiculus of any control animals, including those exposed to a temperature o f 40-45 ~ c. even in cases in which spinal cords were accidentally deformed by compression, no demyelination nor axonal degeneration was found in the spinal cord indicating that demyelination was not the result of compression but the direct result of heating. in the present study the widespread demyelination occurred without any noticeable damage to the axons. most myelin sheaths were degraded by splitting of the myelin lamellae as was reported by hirano (1972) , fig. 7 . two weeks after treatment. this electron micrograph was taken from the deep zone bordering the intact area of the affected dorsal funiculus. almost all the axons (ax) in the area are thinly myelinated by oligodendrocytes (o1), while a few axons remain unmyelinated. proliferation of astrocyte processes (as) has occurred in the spaces between these axons, x 7200. while others initially only lost their electron opacity while retaining their original compact lamellae. myelin sheath changes of the latter type have also been found in the cryo-treated spinal cord (unpublished observations). vulnerability of the myelin sheath to heating can probably be related to the high lipid content and structural characteristics of the membrane. raison et al. (1971) utilizing electron spin resonance demonstrated that heating gives rise to changes in the lipid components of the plasma membrane. similarly, the organizations of membraneincorporated proteins are considered to be easily altered by heating (hahn, 1982) . the finding that the glial cells and associated myelin sheaths were more vulnerable to heat treatment than axons is in contrast to previous studies using ultrasound for heating (fry et al., 1955; barnard et al., 1956) which claimed that axons were more vulnerable to heat injury than glial ceils. the discrepancy in the vulnerability between axons and glial cells between their studies and our own may be due to the different methods employed. the conductivity and absorption rate of the heat in the cns tissues would be different in the two methods which could produce different effects on axons and on glial cells. primary demyelination in the cns occurs in multiple sclerosis and experimental infection of theiler's murine encephalomyelitis (tme) virus (dal canto & lipton, 1980; dal canto & barbano, 1984) as well as in other experimental injuries using lysophosphatidylcholine (lcp) (blakemore, 1976) , 6-aminonicotinamide (6-an) (blakemore, 1975) , cuprizone (ludwin, 1978) and csf barbotage (bunge et ai., 1960) , whereas in other experimental injuries such as allergic encefig. 8 . one month after treatment. this electron micrograph shows the subpial area of the lesion. all the axons (ax)are thickly myelinated by schwann cells (s). there is no typical glm at the surface which is covered by a thick cell layer (p). x 7400. phalomyelitis (eae) (lampert, 1967) , diphtheria toxin injection (harrison et ai., 1972) , and impact injury (griffith & mcculloch, 1983) , axons also undergo degenerative changes. in these latter disorders severe vasogenic oedema and local haemorrhage occur, which are considered to be harmful to axons, and thus may be partly responsible for axonal degeneration. it seems that heat treatment does not produce damage of sufficient severity to the blood-brain barrier (bbb) so as to fatally affect the axons, although permeability of damaged blood vessels seemed to increase to a certain degree (muel!er, 1979) . it was shown in the present study that demyelinated axons are effectively remyelinated by either oligo-dendrocytes or schwann cells. the same pattern of remyelination by oligodendrocytes and schwann cells has also been noted in cns lesions induced by the murine corona (jhm) virus (nagashima et al., 1979) , the tme virus (dal canto & barbano, 1984) , and cryo-treatment (collins et al., 1986 ; our unpublished observations) as well as by x-ray irradiation of the infant spinal cord (gilmore & duncan, 1968; blakemore & patterson, 1975; gilmore & sims, 1986) . remyelination by schwann cells is predominant in 6-an (blakemore, 1975) and ethidium bromide injected cns (blakemore, 1982; gra#a & blakemore, 1986) , whereas remyelination by oligodendrocytes is prominent in ordinary eae (lampert, 1965 (lampert, , 1967 and cuprizone treatment (ludwin, 1978) . it should be noted that schwann cell-remyelination was dominant in the subpial and perivascular areas, fig. 9 . one month after treatment. this electron micrograph shows the deep zone bordering the intact areas. most axons (ax) have cns-type myelin sheaths, that is, they have been myelinated by oligodendrocytes, but their myelin sheaths are still very thin. the axons with thick myelin sheaths (c) are those that were unaffected by heat injury. as: astrocyte, x 7800. whereas oligodendrocyte-remyelination was conspicuous in the deep and border areas of a lesion. the appearance o f schwann cells in the cns has been controversial and various prerequisites for ectopic schwann cells in the cns have been proposed which include (1) breakdown of the glm, (2) absence of astrocytes, and (3) presence of denuded axons (blakemore, 1983; sims & gilmore, 1983; harrison, 1985; gilmore & sims, 1986) . as described by previous authors (blakemore, 1983; sims & gilmore 1983; harrison, 1985) , the breakdown of the subpial and perivascular glm might be the most critical factor for the appearance of schwann cells. thus in cuprizone (ludwin, 1978) and ordinary eae (lampert, 1965) injuries in which the glm is retained intact, few schwann cells appear in the lesion. harrison (1985) thinks that schwann cells invade the cns tissue through damaged glm at the perivascular and subpial surfaces. astrocytes therefore seem to block the invasion of schwann cells. no astrocytes were found in cns lesions where axons were exclusively myelinated by schwann cells (hammang et al., 1986) . in contrast, schwann cells are rarely seen at the sites where astrocyte processes have proliferated throughout the cns lesions as a result of lpc and 6-an treatment, and jhm virus and tme virus infection (blakemore, 1975 (blakemore, , 1976 nagashima et al., 1979; dal canto & lipton, 1980) , as well as schwann cell implantation into normal nervous tissue (blakemore et al., 1986) . denuded axons seem necessary for schwann cells to invade the cns and proliferate. it was reported that naked axons have a mitogenic influence on schwann cells in vitro (wood & bunge, 1975) . harrison (1987) fig. 10. one month after treatment. this micrograph was taken at the transitional zone between the schwann cell-myelinated area and the oligodendrocyte-myelinated area. oligodendrocyte-myelinated axons (ax) and oligodendrocyte cytoplasm (o1) both directly abut (arrows) on the basal laminae of the schwann cells (s) without any intervening astrocyte processes. x 23 000. demonstrated by autoradiography that schwann cell division is only initiated and promoted by direct contact with denuded axons. it has also been suggested that ensheathment of denuded axons by oligodendrocytes or astrocytes eliminated mitogenic influences of naked axons on schwann cells (sims & gilmore, 1983) . where are the sources of ectopic schwann cells? since many ectopic schwann cells are found near the nerve root entry, it is supposed that schwann cells in the root could move along the denuded axons into the injured cns (gilmore & duncan, 1968) . however, even when the roots and their entry zones are preserved undamaged, many schwann cells have been seen in the midline of the dorsal funiculus, far from the root entry (gilmore & sims, 1986 ). on the other hand, schwann cells of the perivascular autonomic nerves (ghatak et ai., 1973) and of small nerves located in a pial layer (blakemore, 1983 ) could proliferate and migrate into the lesion. though the sources of ectopic schwann cells could not be clearly identified, the present study indicates that damage to glial cells as well as the glm at the pial and perivascular surfaces might be the most important prerequisite (blakemore et ai., 1986) . oligodendrocytes remyelinate denuded axons after cuprizone intoxication (ludwin, 1987 (ludwin, , 1988 and other cns lesions including chronic relapsing eae (raine & traugott, 1985) and tme virus infection (dal canto & barbano, 1984) . in these studies, as in the present study, remyelination by oligodendrocytes occurred in deep areas of the lesion, where there might be no influence of the damaged glm. on the other hand, there is evidence that astrocytes can co-operate to some extent with oligodendrocytes in the facilitation of myelination (blakemore, 1975 (blakemore, , 1984 fig. 11 . one month after treatment. this electron micrograph shows a longitudinal section from the caudal part of the lesion. a myelin sheath newly formed by schwann cells (s) makes a node of ranvier (arrow) at the junction with a thick cns-type myelin sheath (c) which presumably survived the heat treatment. there are two fibres that are partly myelinated by schwann cells (s) and partly by otigodendrocytes (o1), forming new nodes of ranvier between them (arrowheads). x 7600. inset: a high-powered micrograpl~ showing a similar node of ranvier taken from other section. a lateral loop (asterisk) of the oligodendrocyte is contiguous with a schwann cell process (arrowhead). no astrocyte process is interposed between them. x 28 000. harrison, 1985) . in the present study, however, there were relatively greater numbers of oligodendrocytes in the cns-pns border zone in which astrocytes and their processes were sparse. this finding indicates that oligodendrocytes can myelinate d e n u d e d axons in the presence of even only a few astrocytes. small localized ultrasonic lesions in the white and gray matter of the cat brain remyelination by schwann cells of axons demyelinated by intraspinal injection of 6-aminonicotinamide in the rat remyelination inmultiple sclerosis with peripheral type myelin on the presence of peripheral-like nervous and connective tissue within irradiated spinal cord the role of schwann cells in the repair of glial cell deficits in the spinal cord demyelination and remyelination after acute spinal cord compression delayed remyelination in rat spinal cord following ethidium bromide injection nerve fibres in spinal cord impact injuries part 1. changes in the myelin sheath during the initial 5 weeks hyperthermia and cancer degenerative changes in rat intraspinal schwann cells following tellurium intoxication schwann cell and oligodendrocyte remyelination in lysolecithin-induced lesions in irradiated rat spinal cord schwann cells divide in a demyelinating lesion of the central nervous system remyelination after transient compression of the spinal cord central demyelination produced by diphtheria toxin: an electron microscopic study the pathology of the central myelinated axon demyelination and remyelination in experimental allergic encepahlomyelitis electron microscopic studies on ordinary and hyperacute experimental allergic encephalomyelitis central nervous system demyelination and remyelination in the mouse. an ultrastructural study of cuprizone toxicity regeneration of myelin and oligodendrocytes in the central nervous system remyelination in the central nervous system and the peripheral nervous system localized hyperthermia in the treatment of malignant brain tumors using an interstitial microwave antenna array chronic histological effects of ultrasonic hyperthermia on normal feline brain tissue increased blood-brain barrier permeability during hyperthermia in the awake rat demyelinating encephalomyelitis induced by a long-term corona virus infection in rats remyelination in chronic experimental allergic encephalomyelitis and multiple sclerosis temperature-induced phase changes in mitochondrial membranes detected by spin labeling interactions between intraspinal schwann cells and the cellular constituents normally occurring in the spinal cord: an ultrastructural ,~study in the irradiated rat p~ (1975) evidence that sensory axons are mitogenic for schwann cells the authors wish to thank dr t. ushiki and dr k. tohyama for their kind suggestions in th e preparation of manuscript. the authors also thank professor paul langman for his suggestions concerning english usage. key: cord-011533-im78xwl8 authors: gloude, nicholas j.; dandoy, christopher e.; davies, stella m.; myers, kasiani c.; jordan, michael b.; marsh, rebecca a.; kumar, ashish; bleesing, jack; teusink-cross, ashley; jodele, sonata title: thinking beyond hlh: clinical features of patients with concurrent presentation of hemophagocytic lymphohistiocytosis and thrombotic microangiopathy date: 2020-05-23 journal: j clin immunol doi: 10.1007/s10875-020-00789-4 sha: doc_id: 11533 cord_uid: im78xwl8 hemophagocytic lymphohistiocytosis (hlh) is a syndrome of excessive immune system activation driven mainly by high levels of interferon gamma. the clinical presentation of hlh can have considerable overlap with other inflammatory conditions. we present a cohort of patients with therapy refractory hlh referred to our center who were found to have a simultaneous presentation of complement-mediated thrombotic microangiopathy (tma). twenty-three patients had therapy refractory hlh (13 primary, 4 evb-hlh, 6 hlh without known trigger). sixteen (69.6%) met high-risk tma criteria. renal failure requiring renal replacement therapy, severe hypertension, serositis, and gastrointestinal bleeding were documented only in patients with hlh who had concomitant complement-mediated tma. patients with hlh and without tma required ventilator support mainly due to cns symptoms, while those with hlh and tma had respiratory failure predominantly associated with pulmonary hypertension, a known presentation of pulmonary tma. ten patients received eculizumab for complement-mediated tma management while being treated for hlh. all patients who received the complement blocker eculizumab in addition to the interferon gamma blocker emapalumab had complete resolution of their tma and survived. our observations suggest co-activation of both interferon and complement pathways as a potential culprit in the evolution of thrombotic microangiopathy in patients with inflammatory disorders like refractory hlh and may offer novel therapeutic approaches for these critically ill patients. tma should be considered in children with hlh and multi-organ failure, as an early institution of a brief course of complement blocking therapy in addition to hlh-targeted therapy may improve clinical outcomes in these patients. electronic supplementary material: the online version of this article (10.1007/s10875-020-00789-4) contains supplementary material, which is available to authorized users. hemophagocytic lymphohistiocytosis (hlh) is a rare clinical syndrome of excessive immune activation, characterized by signs and symptoms of extreme inflammation, driven mainly by interferon gamma (ifnγ) and other pro-inflammatory cytokines. our understanding of hlh pathogenesis has significantly evolved in the past two decades. though there are varied molecular etiologies, genetic hlh disorders most often develop due to either defective lymphocyte cytotoxicity or abnormal inflammasome activity. defects in lymphocyte cytotoxicity due to perforin deficiency or degranulation defects lead to prolonged synapse times, increased production of inflammatory cytokines, and abnormal reciprocal activation of mononuclear phagocytes and cytotoxic nk cells and t cells. a c t i v a t e d m o n o n u c l e a r p h a g o c y t e s p r o m o t e hemophagocytosis and release of hlh biomarkers like ferritin, cxcl9, and il-18bp. sap deficiency uniquely impairs restimulation-induced cell death and, like cd27 and cd70 deficiency, prevents normal killing of ebv-infected b cells. the absence of xiap permits pathogenic nlrp3 inflammasome activity and abnormal lymphocyte apoptosis, while activating mutations in nlrc4 drive pathologic nlrc4 inflammasome activity [1] [2] [3] [4] [5] [6] . the current diagnostic criteria for hlh include either a genetic hlh diagnosis or five of the following eight clinical and/or laboratory criteria: fever, splenomegaly, cytopenias, hypertriglyceridemia and/or hypofibrinogenemia, hemophagocytosis, low or absent nk cell activity, elevated ferritin, and elevated sil-2 receptor (sil2r) ( table 1 ) [7] . the most commonly affected organs are the liver, spleen, central nervous system (cns), and bone marrow. hlh is a syndrome with a variable clinical presentation, requiring comprehensive clinical evaluation to rule out other disorders and clinical syndromes, as clinical diagnostic criteria are not specific and there is considerable overlap with other conditions [8] [9] [10] [11] . we present a group of patients referred to our center for the management of hlh, who also had a simultaneous presentation of clinically significant thrombotic microangiopathy (tma). tma is an increasingly recognized clinical syndrome that results from vascular endothelial injury and may occur in the setting of excessive inflammation such as that seen following a toxic injury (spider or snake bite, radiation, or chemotherapy), in highly inflammatory diseases like systemic lupus erythematosus (sle), and after stem cell transplantation (ta-tma) or solid organ transplant [12] . tma presents with a constellation of laboratory features and a specific pattern of organ injury [13] . tma diagnostic criteria include either a histologic diagnosis on tissue biopsy of microangiopathic changes or at least five of the seven laboratory and clinical markers listed in table 1 . if not recognized, tma can progress to multi-organ failure. complement system dysregulation is a known pathogenic pathway leading to tma, and complement blocking agents are used therapeutically with some success [14] . however, other inflammatory pathways may contribute to the endothelial injury that initiate and sustain tma, and can likely be targeted to improve clinical outcomes further [15, 16] . our observations suggest that co-activation of both interferon and complement pathways is important in the evolution of tma and offers novel therapeutic approaches for these critically ill patients. consecutive patients of any age with hemophagocytic lymphohistiocytosis (hlh) refractory to first-line therapy who were treated at our institution from january 2012 through december 2018 were identified by retrospective chart review after institutional review board (irb) approval. all patients were prospectively screened for tma as part of clinical care as previously described [17] . patient demographics, disease characteristics, hlh and tma-targeted therapy, low or absent nk cell activity 6. proteinuria ≥ 30 mg/dl on random urinalysis ×2 or random urine protein creatinine ratio ≥ 2 mg/mg 7. elevated ferritin > 500 ng/ml 7. terminal complement activation: elevated plasma sc5b-9 above normal limit of ≥ 244 ng/ml, or elevated above defined normal laboratory value 8. elevated sil-2 receptor (sil2r) > 2400 u/ml or elevated above defined normal laboratory value note: 6 and 7 are high-risk tma features complications, intervention, and laboratory assessment were captured from the electronic medical record. hlh and tma diagnoses were made prospectively using published diagnostic criteria (table 1) [7, 17] . laboratory tests were performed as part of routine clinical care in a cliacertified laboratory. routine institutional tma monitoring included daily complete blood count (cbc) with attention to schistocytes; twice a week lactate dehydrogenase (ldh); and weekly haptoglobin, free plasma hemoglobin, and urinalysis with a random urine protein/creatinine ratio. for subjects < 18 years of age, hypertension was defined as a blood pressure > 99% for age, sex, and height [18] . hypertension for subjects ≥ 18 years of age was defined as a blood pressure ≥ 140/ 90 mmhg. the number of antihypertensive medications, not including diuretics, required to maintain the blood pressure below the targeted hypertension threshold (99th centile for age) were counted for each patient, and hypertension was classified as severe if management included > 2 antihypertensive medications or a continuous antihypertensive medication infusion for > 12 h. terminal complement activation was tested by measuring the level of sc5b-9 in blood and was checked in patients with elevated ldh and nephrotic range proteinuria (random urine protein/creatinine ratio ≥ 2 mg/mg). sc5b-9 was reported as elevated if the measurement in the blood was > 244 ng/ml by enzyme immunoassay (eia). patients with hematologic signs of tma who had both nephrotic range proteinuria and complement activation as measured by elevated sc5b-9, or evidence of multi-organ injury syndrome (mods), were classified as high-risk tma and were offered therapy with the terminal complement blocker, eculizumab. eculizumab dosing was based on drug pharmacokinetics and pharmacodynamics (pk/pd) as previously published [17, 19] . all patients on eculizumab therapy received antimicrobial prophylaxis against meningococcus until the drug was cleared after therapy completion and complement function was restored. mods was diagnosed when a patient had symptoms of hlh/tma and dysfunction of two or more organ systems: renal failure requiring renal replacement therapy (rrt) or cystatin c glomerular filtration rate (gfr) < 50 ml/min, invasive or non-invasive positive pressure ventilator support for > 24 h, diagnosis of pulmonary hypertension (as determined by echocardiogram and cardiology consultation), serositis (pleural or pericardial effusions), severe hypertension requiring either ≥ 2 medications or continuous infusion of an antihypertensive for > 12 h to maintain blood pressure < 99% for age, cns symptoms (seizures, bleeding, posterior reversible encephalopathy syndrome (pres), or altered mental status), or gastrointestinal symptoms (ileus and/or bleeding) [20] [21] [22] [23] [24] . twenty-three patients with frontline therapy refractory hlh were treated at our institution during the study period. all patients met clinical hlh criteria, as listed in table 1 . twelve (52%) had confirmed genetic hlh, 4 (17.4%) had ebv-associated secondary hlh, and 7 (30.4%) met hlh clinical diagnostic criteria but did not have any identified genetic abnormality, nor ebv infection. sixteen of 23 patients (70%) had cns symptomatology such as altered mental status, encephalopathy, or seizures, and seven of these patients also had a documented cns bleed, as well as radiographic evidence of posterior reversible encephalopathy syndrome (pres). all patients were initially treated with etoposide and dexamethasone except for one patient who received cytarabine and methylprednisolone. all patients had either disease progression on therapy or had an hlh disease relapse after achieving brief remission with additional agents and high dose dexamethasone (≥ 10 mg/m 2 ) (supplemental table 1a) . two patients had hlh relapse after allogenic hct. seventeen patients received emapalumab a monoclonal antibody directed against interferon gamma (ifnγ), as a targeted hlh therapy for refractory disease [25] . fourteen of them received emapalumab as part of the clinical study and three for compassionate use. all patients had active hlh signs including elevated sil2r at the start of emapalumab. all patients initiated prospective monitoring for thrombotic microangiopathy (tma) as part of clinical care on arrival to our institution as listed in "methods" section. sixteen of 23 patients (70%) met clinical and laboratory criteria for highrisk tma in addition to their diagnosis of hlh, including elevated lactate dehydrogenase (ldh), presence of schistocytes in the blood, transfusion-dependent coombs negative hemolytic anemia, transfusion-dependent thrombocytopenia, nephrotic range proteinuria, and severe hypertension. high-risk tma features included nephrotic range proteinuria and complement activation (table 1 ) and/or multi-organ injury. nine patients were tested for complement gene abnormalities using an institutional ahus genetic susceptibility panel (c3, cfb, cfh, cfhr1, cfhr3, cfhr5, cfi, and mlpa analysis for chfr1/chfr3 deletion). six patients had at least one complement gene variant identified. three subjects were heterozygous for cfhr3-cfhr1 by multiple ligationdependent probe amplification (mlpa) analysis that has been reported in heterozygous state in stem cell transplant recipients with tma. one subject had cfhr3 and cfhr1 partial duplication, which is of uncertain clinical significance (vucs). three subjects had more than 1 gene variant identified, including a likely pathogenic variant in cfi (c.1246a>c(p.i416l, het), cfi (c.1217g>a(p.r406h),het) variant reported in macular degeneration and cfhr5 (c.486_487insaa(p.e163fs) heterozygous for a frameshift mutation that has been associated with ahus (supplemental table 1a , b). fourteen patients were tested for the evidence of terminal complement pathway activation by measuring blood sc5b-9 (or soluble membrane attack complex), and thirteen of them had elevated sc5b-9 level (sc5b-9 range was 262 to > 1890 ng/ml, normal is < 244 ng/ml). thirteen patients had nephrotic range proteinuria (random urine protein/creatinine ratio > 2 mg/mg). fifteen patients had multi-organ dysfunction syndrome (mods) with two or more organ systems involved, and 12 of them required intensive care support. twelve patients required positive pressure ventilation for respiratory failure, and four of them had pulmonary hypertension. one patient required ecmo support who did not survive and was found on autopsy to have diffuse alveolar hemorrhage. this patient had cmv viremia that could be a possible trigger for inflammatory process. eight patients had renal failure; seven received renal replacement therapy (rrt), and one patient's family elected not to proceed with rrt. two patients had histologic evidence of tma in kidney on autopsy (supplemental table 1a ). eight additional patients had a > 50% decline in cystatin c gfr from their pre-transplant baseline. all patients with tma had severe hypertension requiring more than two antihypertensive medications or a continuous antihypertensive medication infusion to maintain blood pressure below 99th percentile for age. six had clinically significant serositis requiring pericardial or pleural drain placement. eleven patients in this group had cns symptoms such as seizure or encephalopathy attributed to hlh diagnosis, but six of these patients also had a documented cns bleed clinically attributed to pres ( table 2) . ten of 16 patients in the hlh plus tma group received complement blockade with eculizumab as a targeted therapy for thrombotic microangiopathy using personalized pk/pd dosing (fig. 1) . all of these patients had documented activation of terminal complement (elevated sc5b9). four of these 10 treated patients received eculizumab together with primary hlh therapy with corticosteroid with or without etoposide. none of these four patients proceeded to hsct, and two died with active tma. the other six patients received eculizumab after starting treatment with the ifnγ blocker emapalumab for refractory hlh. one patient started eculizumab 8 days after initiation of emapalumab and had resolution of tma. the patient did not proceed to hcst due to a lack of suitable donor and is alive and well for more than 3 years after therapy. the other five patients in this group who proceeded to hsct after hlh-targeted therapy had active tma at the start of hematopoietic stem cell transplantation (hsct). they continued complement blockade with eculizumab during hsct with successful resolution of their tma. all patients were alive, with only one developing liver gvhd after hsct. the median number of eculizumab doses given to patients with tma was 9 (range 2-11). the median time to complete tma resolution and organ function recovery was 16 weeks (9-21 weeks). eculizumab therapy was well tolerated without any adverse events in this cohort. six patients with hlh and tma did not receive eculizumab (fig. 1) . one patient had resolution of tma with plasma exchange therapy before eculizumab was available and successfully underwent allogeneic hsct. in two other patients, tma resolved with emapalumab given for hlh. of these two, one patient's family opted not to proceed to hsct and one patient subsequently underwent hsct. two patients died shortly after transfer to our institution with both hlh and tma after initiation of emapalumab, but prior to hsct. one patient died prior to the use of either eculizumab or emapalumab. seven of 23 patients (30%) with hlh did not meet criteria for tma and did not have evidence of complement activation in the blood (fig. 1) . four of these seven patients required intensive care admission for respiratory support, mostly due to cns symptoms such as status epilepticus. five patients developed cns symptoms, all attributed to hlh. one patient who was diagnosed with hlh 3 days after birth had evidence of a perinatal intraventricular bleed (ivh). none of the patients without tma required renal replacement therapy, and none had severe hypertension. mods was present in four of these patients. we observed a high incidence of clinically significant complement-mediated thrombotic microangiopathy (tma) associated with multi-organ injury in children and young adults with a diagnosis of hlh. tma has not been previously associated with interferon gamma-driven diseases like hlh [26] , but tma has been reported as an iatrogenic therapylimiting side effect in patients receiving interferon alpha and beta therapy [27] [28] [29] [30] [31] . we hypothesized that high levels of interferon gamma in hlh might contribute directly to endothelial damage or injure endothelium through complement system activation. our previous gene expression analysis in autologous stem cell recipients showed upregulation of interferon-responsive complement genes at the onset of ta-tma that resolved after therapy with complement blocking [32] . high levels of ifnγ promote the formation of neutrophil extracellular traps (nets), leading to complement activation via complement factor p (cfp) [33] . terminal complement activation resulting in vascular endothelial injury is one of the important pathogenic pathways leading to high-risk tma with multi-organ dysfunction syndrome. uncontrolled terminal complement activation can maintain ifnγ secretion via intracellular c5a receptor production resulting in a cycle of continuing tissue injury [34] . vascular endothelium injured by terminal complement can secrete interferons in addition to the robust production of ifnγ by activated t cells, nk cells, and macrophages in patients with immune over-activation syndromes like hlh. while both interferons and complement are essential components of our immune response, over-activation of these pathways results in organ injury as "collateral damage" during high inflammatory states as seen in hlh and tma. in our prior prospective study examining complement genes in hsct, we observed a significant association between tma severity and outcomes in hlh patients undergoing hsct who had complement gene variants identified. in this study, complement gene variants were more common in hlh subjects with tma then those without tma (7/9, 78% vs 0/8, 0%, p = 0.0023). the median number of complement variants detected in these 7 hsct recipients with hlh was 2 (range 1-5), and all patients with complement variants identified had severe tma and multi-organ injury with an elevated blood sc5b-9 level indicating terminal complement over-activation. transplant-related mortality was also significantly higher in hlh patients with tma with six patients of the 9 patients dying from tma-associated complications, while all hlh patients without tma survived after transplant (6/9 67% vs 0/8, 0%, p = 0.09) [35] . based on this prior observation, it is possible that patients with hlh who have complement gene variants in addition to the genetic defects of their primary disease may be more prone to rapid complement activation during active hlh, and may have more severe organ damage resulting from both interferon and complement co-injury of endothelial cells. prompt control of overactivation of the interferon and complement systems is likely needed for successful resolution of both clinical conditions, hlh and tma. our study was limited by small sample size, so we were unable to perform any statistical analysis confirming benefit of eculizumab therapy in subjects with tma. however, all patients with hlh and tma who received the complement blocker eculizumab with the ifnγ blocker emapalumab recovered and are doing well, supporting further investigation of this approach. such targeted therapies can only be successfully applied if these co-existent hyperinflammatory conditions are appropriately recognized. h l h o f t en p r e s en t s w i t h l i v e r f a i l u r e a n d a hyperinflammatory syndrome mimicking sepsis that can ultimately lead to mods due to coagulopathy, hypotension, and capillary leak. however, hlh rarely causes direct renal and pulmonary failure, or gastrointestinal bleeding in the absence of sepsis [36] . hlh commonly causes neutropenia, but the white blood cell counts are rarely affected in tma. patients presenting with a diagnosis of hlh and mods (in particular renal failure, pulmonary failure, and uncontrolled hypertension that is more severe than expected with dexamethasone use) should be evaluated for co-existing tma. these patients can benefit from evaluation for terminal complement activation by measuring sc5b-9 blood levels in addition to routine hematologic and renal tma markers and organ function assessment (fig. 2) . in our patient cohort, ventilator support in patients who had hlh but not tma was generally required because of cns symptoms, while patients with tma were more likely to have respiratory failure due to cardiorespiratory pathology, often pulmonary hypertension. hlh patients with hypoxemic acute respiratory failure should undergo diagnostic evaluation for pulmonary hypertension, a known presentation of tma [21] . in our cohort of patients, renal failure associated with severe hypertension was exclusively seen in patients with hlh and tma. while hypertension in a patient with hlh can be due to the use of dexamethasone, severe hypertension requiring multiple medications or a continuous infusion of antihypertensive medication should trigger evaluation for tma. in contrast, the hyperinflammatory state seen in patients with hlh without tma usually mimics sepsis and is more likely to present with hypotension. nephrotic range proteinuria measured by random urine protein/creatinine ratio of ≥ 2 mg/mg in addition to severe hypertension should also prompt consideration of tma in the differential diagnosis of patients with hlh and renal insufficiency. patients with hlh are more likely to have fluid overload from liver failure leading to hepato-renal syndrome, whereas tma primarily affects the renal vasculature resulting in direct kidney injury requiring renal replacement therapy, especially associated with hypertension. cns involvement in hlh is quite common at clinical presentation, and cns bleeding can also occur, especially in thrombocytopenic patients with coagulopathy. cns injury in hlh and tma can be similar in clinical presentation, but certain features help distinguish cns hlh, such as csf pleocytosis, elevated csf protein, and neopterin, along with radiologic abnormalities such as meningeal enhancement, polymorphic white matter changes, often with non-specific periventricular distribution. cns injury in tma most commonly occurs due to uncontrolled hypertension resulting in altered mental status, pres, seizures, and/or cns bleeding [37] . intestinal bleeding has been reported in patients with hlh, but is not common, while tma is known to cause bowel injury quite often resulting in severe abdominal pain and bleeding, as was seen in our study population [24, 38] . if available, histologic tissue evaluation for tma should be considered in patients with hlh and gi bleeding. patients with clinically significant intestinal bleeding may require higher doses of therapeutic monoclonal antibodies due to increased drug clearance [22] . our clinical observations suggest that hlh patients copresenting with complement-mediated tma often suffer multi-organ injury and likely can benefit from a brief course of therapy with the complement blocking agent eculizumab while receiving therapy for hlh. coadministration of both an interferon blocker and a complement blocker targeting both pathways in tma might provide faster control. while complement activation in our patients with hlh and tma was quite prominent, so pk/pd-based eculizumab dosing should be considered as drug clearance can be rapid, especially during a high inflammatory state. based on our data, we anticipate that a brief course of eculizumab should be sufficient to control complement over-activation while hlh treatment is ongoing. our patients with hlh and tma received a median of six doses of eculizumab. some of our patients started eculizumab late in disease presentation with severe organ injury and were not able to recover. those who survived were able to discontinue eculizumab without recurrence of tma successfully. six patients who received both drugs, an interferon gamma blocker and a complement blocker, resolved both hlh and tma, and are alive and well. primary injury is not common, serositis due to third spacing, liver failure right heart failure as a result of pulmonary hypertension due to pulmonary tma. pericardial effusion vascular system hypotension mimicking sepsis due to capillary leak hypertension more severe that can be attributed to dexamethasone use lungs primary injury is not common, respiratory failure often related to cns symptomatology (airway protection), or capillary leak ("wet lungs") hypoxemia often with clear lungs. pulmonary hypertension, interstitial pulmonary bleeding leading to ards gi primary injury is not common intestinal bleeding due to ischemic colitis hemato-poietic neutropenia, anemia, thrombocytopenia anemia, thrombocytopenia, schistocytosis aki acute kidney injury, pres posterior reversable encephalopathy syndrome, ards acute respiratory distress syndrome our study is limited by retrospective review of the data but provides preliminary insights into additional pathogenetic pathways of tma that could be monitored and targeted, and brings awareness of the co-existence of hlh and tma, especially in patients with multi-organ injury. while both hlh and tma can have overlapping clinical presentation, they also have some distinctive clinical features and organ injury patterns that can be recognized using prospective monitoring (table 3) . emerging new therapies like chimeric antigen receptor t cells (cart) cause cytokine release syndrome (crs) due to high ifnγ production, and disorders like systemic lupus erythematosus and dego's syndrome present with high ifnα along with complement activation. the resolution of tma without tma-directed therapy has been observed in mild to moderate cases of tma triggered by infection or engraftment after hsct. however, severe tma, especially presenting with significant complement activation, leads to high mortality due to multi-organ failure. early institution of complement blocking therapy along with treatment of the primary disease may improve clinical outcomes in these patients. our observations suggest co-activation of both interferon and complement pathways as a potential culprit in the evolution of thrombotic microangiopathy in patients with inflammatory disorders like hlh and may offer novel therapeutic approaches for these critically ill patients. tma should be considered in children with hlh and multi-organ failure, as an early institution of a brief course of complement blocking therapy in addition to hlh-targeted therapy may improve clinical outcomes in these 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hemophagocytic lymphohistiocytosis clinical utility of computed tomography and magnetic resonance imaging for diagnosis of posterior reversible encephalopathy syndrome after stem cell transplantation in children and adolescents hemophagocytic lymphohistiocytosis and gastrointestinal bleeding: what a surgeon should know publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments part of the research reported in this publication was supported by the eunice kennedy shriver national institute of child health and human development of the national institute of health (nih) under award number r01hd093773 (s.jodele). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. samples used in this study were collected and provided by the staff of the cincinnati children's hospital bone marrow tissue repository who we thank for outstanding ongoing technical assistance. the authors would like to thank the clinical staff, patients, and families at cincinnati children's hospital medical center.author contributions njg and sj conceived the project, collected, analyzed, and interpreted the data, and wrote the manuscript. ced, smd, kcm, mbj, ram, ak, jb, and atc analyzed and interpreted the data and edited the manuscript. conflict of interest njg, ced, ram, and jb have no interests to disclose. sj and smd have us patents pending. mbj and ak are consultants for sobi. atc is on the speaker's bureau for sobi. none of these funding sources had any input in the study design, analysis, manuscript preparation, or decision to submit for publication. key: cord-271396-bol1zpji authors: manglani, monica; mcgavern, dorian b title: new advances in cns immunity against viral infection date: 2018-02-28 journal: current opinion in virology doi: 10.1016/j.coviro.2017.12.003 sha: doc_id: 271396 cord_uid: bol1zpji the central nervous system (cns) is an immunologically specialized organ where restrictive barrier structures protect the parenchyma from inflammation and infection. this protection is important in preventing damage to non-renewable resident cell populations, such as neurons, responsible for functions ranging from executive to autonomic. despite these barriers, the cns can be infected through several entry portals, giving rise to meningitis and encephalitis. following infection, resident cells recruit peripherally derived immune cells to sites of viral infection. in this review, we discuss recent advances in immune recruitment and entry at barrier structures as well as current immunotherapeutic strategies for the treatment of persistent viral infections. monica manglani 1,2 and dorian b mcgavern 1 the central nervous system (cns) is an immunologically specialized organ where restrictive barrier structures protect the parenchyma from inflammation and infection. this protection is important in preventing damage to non-renewable resident cell populations, such as neurons, responsible for functions ranging from executive to autonomic. despite these barriers, the cns can be infected through several entry portals, giving rise to meningitis and encephalitis. following infection, resident cells recruit peripherally derived immune cells to sites of viral infection. in this review, we discuss recent advances in immune recruitment and entry at barrier structures as well as current immunotherapeutic strategies for the treatment of persistent viral infections. introduction cns viral infections are a major cause of death and disability globally. furthermore, the socio-economic burden of cns infections is growing rapidly with the (re) emergence of highly pathogenic neurotropic viruses [1] [2] [3] . despite this growing patient population, specific therapies for cns infections are largely unavailable. the current standard-of-care for viral infection is antiviral therapy [4, 5] . however, these drugs are often ineffective in depleting viral reservoirs because they are either nonspecific or fail to cross the specialized cns barrier structures into areas of viral invasion [2] . consequently, there remains an outstanding and growing need for targeted treatment of these viral diseases. inextricably intertwined in our ability to treat cns viral infections is our knowledge of brain anatomy, drainage, and immunity, as reviewed elsewhere [6] [7] [8] [9] . application of these principles can assist in creating targeted viral and immune-mediated therapeutics. over the past decade, several instrumental findings have altered our perspective of cns immunity. once thought to be immune-privileged, the cns is immune competent, dynamic, and in direct contact with the peripheral immune system [10, 11] . within this review, we discuss recent advances in immune recruitment and entry at barrier structures as well as current immunotherapeutic strategies for the treatment of persistent viral infection. the brain parenchyma, enveloped by the meninges, is suspended in cerebrospinal fluid (csf) within the cranial vault ( figure 1 ). the csf, produced by the choroid plexus ( figure 2 ) within the ventricular spaces, and the meninges (figure 3) , comprised of the dura, pia and arachnoid mater, protect the cns from injury and infection. immediately beneath the skull lies the dura mater ( figure 3) , a dense connective tissue layer consisting of fenestrated vessels (without tight junctions) derived from the carotid artery, sensory nerve fibers of the cranial nerves, and an abundant repertoire of immune cells, including meningeal macrophages [9, 12 ] . inferior to the dural tissue lies the arachnoid mater -a trabecular network that creates a subarachnoid space for csf flow. within the arachnoid mater lie granulations responsible for csf absorption into the superior sagittal sinus, the venous drainage system. lastly, the pia mater above the parenchyma gives rise to perivascular (virchow-robin) spaces where penetrating arteries enter the parenchyma. these spaces are known to contain csf and perivascular macrophages [9] . between cells of the cns parenchyma resides interstitial fluid that maintains neuronal and glial homeostasis through solvent exchange between the capillary vasculature and csf [13] . recently, iliff et al. described a system known as the 'glymphatics', where the disparate systems of csf influx and efflux drive clearance of interstitial fluid and its suspended solutes [14, 15] . specifically, csf moves from the meningeal space along penetrating arteries and their periarterial spaces into the cns parenchyma. mediated in part by astrocytic endfeet, interstitial fluid is driven through the parenchyma into perivenous spaces where, ultimately, fluid residing in the perivascular spaces can be resorbed. the csf and interstitial fluid is drained in part by lymphatics into the deep cervical lymph nodes. the cns lymphatics consists of the nasal lymphatics, which travel from olfactory bulb (ob), through the cribriform plate and below the skull, and the dural lymphatics, which travel alongside the superior sagittal sinus and across the transverse sinus [16] [17] [18] . although studies have shown the connection between glymphatics and lymphatics, the exact mechanism of csf drainage into the lymphatics remains unknown [15] . in addition to the cns clearance and drainage systems, the brain and spinal cord are well perfused by an extensive vascular network. unlike choroid plexus or dural vasculature, blood vessels that traverse the cns pia and parenchyma are tightly regulated (figure 4 ) [19, 20] . endothelial cells comprising these vessels form tight junctions. in addition, parenchymal blood vessels are further fortified by pericytes, basement membrane, and astrocytes. these structures decrease permeability, making it difficult for small molecules and cells to enter the cns parenchyma. while the aforementioned anatomy protects the cns, it can still be infected by viruses via the meninges or blood-brain barrier (bbb), retrograde transport by peripheral nerves, or by circulating/infiltrating peripheral immune cells. invasion of neural tissue can initiate serious neurological disorders like meningitis and encephalitis, as reviewed previously [21, 22] . these infections can cause significant and irreversible damage to the cns. understanding how cns resident innate immune cells respond to these infections as well as their role in guiding peripheral immune cell recruitment to sites of viral invasion should aid the development of interventions to treat cns infections. upon viral entry into the body, an immune response is generated in secondary lymphoid organs. primed immune cells are then recruited to the cns by local inflammatory signaling. activated by conserved pathogen-associated molecular patterns (pamps), pattern recognition receptors (prrs), retinoic acid-inducible gene (rig)-i-like receptors, and toll-like receptors (tlrs) initiate innate immune reactions [23, 24] . viral nucleic acids bind to these receptors expressed by innate immune sentinels (e.g. microglia, macrophages, dendritic cells, astrocytes) and cause release of type i interferon (ifn-i) as well as the production of interferon stimulated genes (isgs). ifn-i limits viral spread by upregulating antiviral proteins, recruiting peripheral immune cells, and altering endothelial tight junction proteins to decrease bbb permeability [23,25,26 ,27,28] . a recent intravital imaging study performed in the brains of lymphocytic choriomeningitis virus (lcmv) infected mice revealed that the absence of ifn-i signaling prevented microglia differentiation from a ramified state, decreased peripheral myeloid cell patrolling of cerebrovasculature, and significantly increased the number of infected brain-resident myeloid cells [28] . within this viral model system, ifn-i signaling was responsible for the entire innate immune program within the cns. following infection, ifn-i responses within the cns can be global or region-specific. for example, long-distance ifn-i signaling can prevent viral spread from one brain region to another. this has been observed following vesicular stomatitis virus (vsv) infection, where microglial ifn-i production in the olfactory bulb is thought to limit the anterograde spread of virus into the hindbrain regions [29, 30] . ifn-i responses can also tighten the bbb and limit the extent of viral entry from the blood. astrocytic interferon-a receptor signaling was shown to decrease bbb permeability and protect the hindbrain from west nile virus (wnv) infection [26 ] . these data demonstrate that with certain infections ifn-i can provide a region-specific antiviral defense. while the local ifn-i response plays an important role in cns protection, the activation and recruitment of the adaptive immune cells is usually necessary for viral clearance. lymphoid cells can gain access to the cns via multiple entry portals as shown in figure 1 . these areas are also sites of pathogen entry into the cns, which is likely why the immune system invests so much into surveying these structures. the presence of adhesion and antigen presenting molecules facilitates extravasation and routine immune surveillance of these entry portals despite the presence of specialized barrier proteins [31] . each barrier structure has its own anatomical specializations that result in a tailored immune defense against invading pathogens. some of the recent insights into pathogen-specific immunity within these distinct anatomical regions are described in more detail below. although viral meningitis has been studied extensively, little is known about how resident meningeal cells recruit and direct peripheral immune cell traffic. peripheral immune cells can enter the cns through the blood meningeal barrier [12 ,32] , as depicted in figure 3 . the meningeal stromal cell network consists of blood endothelial cells, pericytes, fibroblasts, and smooth muscle cells [19] . blood vessels within the dura mater are fenestrated and do not express tight junctions [33] , permitting easier access to peripheral immune cells that must still traverse the arachnoid mater before entering the subarachnoid space. by contrast, meningeal vessels beneath the arachnoid mater are non-fenestrated, express tight junctions, and are surrounded by a network of fibroblastic reticular cells that can modulate peripheral immune cells. following coronavirus infection, meningeal endothelial cells and fibroblastic reticular cells release ccl19 and ccl21 to recruit/reactivate antiviral ccr7 + cd4 + and cd8 + t cells in response to resident myeloid and neural cell infection [34 ] . the entry of ccr7 + t cells is likely facilitated by stromal cell reorganization after infection [35] . without ccr7 + lymphoid 118 viral immunology choroid plexus. the choroid plexus resides in ventricular spaces of the brain and is responsible for creating cerebral spinal fluid (csf). the choroid plexus is a dynamic barrier structure home to many immune cells, including choroid plexus macrophages. unlike the bbb, but similar to the dura mater, choroid plexus blood vessels are fenestrated and do not express tight junctions, which provides easier access to pathogens and immune cells into the stromal space. choroid plexus epithelia (ependyma) serve as a barrier between fenestrated blood vessels and the csf. expression of tight junctional proteins between individual epithelial cells protects the csf by restricting solute and cellular movement. during infection, the choroid plexus can serve as a gateway for immune recruitment and entry into the csf. immune cells must first enter the choroid plexus stroma before traversing the epithelial barrier. cell recruitment, animals cannot control viral replication and die of infection [34 ] . however, it is unclear exactly how these immune cells enter the virally infected cns parenchyma from the meninges [36] , although the process presumably involves migration across the glial limitans. non-infectious animal models suggest that meningeal and/or perivascular macrophages 'license' t cells for cns entry through chemokines (e.g. ccl5, cxcl9-11, cxcl12), adhesion molecules, and cognate antigen presentation [37 ] . upon entering the meninges, activated t cells utilize these cues to interact with local macrophages, reactivate, and, ultimately, gain access to the cns parenchyma. however, it is presently not known whether similar steps are required for antiviral t cells to enter the infected brain after migrating into the meninges. understanding how the meninges regulate immune recruitment and traffic into the infected brain should improve our ability to stimulate productive immune responses against parenchymal infections. the choroid plexus consists of fenestrated vasculature without tight junctions that is surrounded by an epithelial network (sealed by tight junctions) capable of hosting a diverse immune repertoire, including t and b cells (figure 2) [12 ,19] . although the choroid plexus is directly infected by some viruses (e.g. coxsackie b3, echovirus 30, lcmv), it is likely that this structure also plays a primary role in the antiviral defense against other cns viruses because of its anatomical localization within the ventricular system, ability to globally activate the cns through release of inflammatory mediators into meninges. the meninges consist of three layers that envelope the brain and spinal cord. the dura mater, the outermost layer, is vascularized by fenestrated vessels and is home to a repertoire of immune cells, including meningeal macrophages that line the vessels. interior to the dura lies the arachnoid mater. the arachnoid, its trabecula and the pia mater form the subarachnoid space, a compartment where cerebrospinal fluid (csf) freely flows. the arachnoid mater also contains tight junctions that help keep materials in the dura mater separate from the subarchnoid space. within the subarachnoid space resides pial vessels that dive into the cns parenchyma. pial vessels are non-fenestrated and express tight junctions. the spaces between these vessels and the parenchyma (referred to as perivascular spaces) are inhabited by perivascular macrophages. the final layer, which lies beneath pia mater, is referred to as the glia limitans -a layer of surface-associated astrocytes that protect the brain and prevent migration of solutes and cells from the csf into the parenchyma. during states of infection, the meninges can serve as an entry point for extravasating immune cells. dural vessels are especially susceptible to immune cell and pathogen entry because they are fenestrated and do not express tight junctions. the csf, and role as a gateway for immune cell entry [38, 39] . under steady state conditions, immune cells travel from the blood across fenestrated endothelium into stromal spaces (connective tissue) within the choroid plexus. more recently, it was observed that cd4 + t cells use ifng to help maintain immune populations within the choroid plexus during homeostasis by promoting expression of adhesion, chemokine and antigen presenting molecules on the epithelium. interestingly, deletion of the ifng receptor was shown to reduce steady state immune cell numbers within the choroid plexus as well as immune trafficking into the csf [40] -a process that depends in part on nfkb/p65 signaling [41] . while immune cell traffic through the choroid plexus has been studied in the context of cns injury and autoimmune disease [42, 43] , less is known in vivo about how this structure functions immunologically following cns viral infection [38] . however, data obtained in other models of cns inflammation might provide clues into the role of the choroid plexus in antiviral immunity. unlike the meninges, the cns parenchyma is home to few immune cells [12 ] . resident microglia survey and respond rapidly to cns infections [44] . after herpes simplex virus-1 (hsv-1) infection, microglia utilize the cgas-sting cytosolic dna sensing pathway to induce release ifn-i, which abrogates viral spread and lethality by activating a neuronal antiviral program [45] [46] [47] . microglia-mediated protection was also observed following dengue virus (denv) infection, where depletion of cns myeloid cells with clodronate increased viral 120 viral immunology blood-brain barrier (bbb). the blood brain barrier creates a selective interface between the blood and cns parenchyma. it consists of endothelial cells and their tight junction proteins, basement membrane, pericytes and astrocytic end feet. in conjunction with barrier function, the bbb can also modulate cerebral blood flow through the neuro-vascular unit, a connection involving neurons, pericytes, astrocytes, and the blood-brain barrier. this allows neural and astrocytic activity to modulate blood vessel tone, resulting in an increase or decrease of regional perfusion. during infection, the bbb is permissive to immune extravasation from the blood into the perivascular spaces. immune cells usually enter the perivascular spaces before gaining access to the parenchyma. these spaces are inhabited by perivascular macrophages. replication and mortality [48] . loss of cns myeloid cells abolished antiviral cd8 + t cell recruitment to the denv-infected brain, suggesting that microglia play a critical role in the antiviral defense against this pathogen. although innate activation and recruitment can be initiated by microglia, neurons can also contribute to protective immunity during infection. following hsv-1 infection, deletion of stat1 signaling specifically in neurons markedly increased viral titer in the brain and trigeminal ganglia as well as viral spread into nonneuronal tissues, resulting in increased mortality [47] . neuronal antiviral protection also occurs following wnv infection. cd8 + t cells are required to clear wnv [49, 50] , and neurons can participate in this clearance by releasing t cell recruitment chemoattractants like cxcl10, which has also been observed following rabies virus infection [51, 52] . interestingly, it was recently demonstrated that wnv infection promotes release of ccl2 and cxcl10 from neurons that depends on activation of receptor-interacting protein-kinase 3 (ripk3), which in turn promotes recruitment of leukocytes to the brain without causing neuronal cell death [53 ] . use of this particular pathway in neurons reflects a nontraditional, neuroprotective role for ripk3 that usually induces necroptotic cell death in other cell types [54] . induction of necroptosis within virally infected neurons would have a profoundly negative effect on the cns. it is very important after infection that the cns parenchyma inhibits viral spread and attracts adaptive immune cells without injuring neurons, which are largely considered a non-replicative cell population. olfactory sensory neurons (osn) are located in the olfactory epithelium of the nose and provide a bridge between the periphery and cns. hair-like cilia expressing olfactory receptors extend from osn dendrites into the mucus layer of the epithelium, and their axons project through the cribriform plate onto mitral cells located in the olfactory bulb ( figure 5 ). this system is responsible for our sense of smell, but provides a direct conduit for olfactory epithelium and bulb. the olfactory epithelium forms part of the nasal airway and serves as a direct interface between the peripheral nervous system (pns) and cns. olfactory sensory neurons within the olfactory epithelium are surrounded by support cells including basal, sustentacular, and microvillar cells. these neurons extend axon fibers that cross the cribriform plate and synapse in the olfactory bulb of the cns. because the dendrites of olfactory sensory neurons extend directly into the airways, these cells can be infected by viruses. viruses that infect olfactory sensory neurons often hijack axonal transport machinery to invade the cns. other pathogens, such as bacteria and amoeba, can also access the cns by traversing the olfactory epithelium and entering holes in the cribriform plate. viruses and other pathogens to enter the cns via the nose. there are many viruses (e.g. vsv, hsv-1, rabv, nipah virus, hendra virus, influenza a virus, etc.) that enter the cns via this route [22, 55] . viral invasion of osns can rapidly lead to encephalitis and host death, as a virus travels from the olfactory bulb to the hindbrain [56] . little is known about the immunological defense mounted by the olfactory epithelium following infection. olfactory ensheathing cells (oecs) are glial cells that insulate osns, have phagocytic capabilities, and can clear neuronal debris during development [57] . these cells can respond to bacterial pamps via tlr2 and tlr4, inducing release of ifn-i [58] [59] [60] ; however, it is unclear how oecs contribute to the immune response against neurotropic nasal viruses. another strategy used by the olfactory epithelium to defend against viral infection is apoptosis. osns are a renewable neuronal population that will sometimes undergo apoptosis following infection to limit the degree of viral dissemination into the cns [61] . despite this ability to rebuild, infection and inflammation within the olfactory epithelium can be detrimental to olfactory function, giving rise to a temporary or permanent loss of smell [62, 63] . nasal associated lymphoid tissue (nalt) is also housed within the nose and is thought to play a role in antimicrobial immunity as well as the efficacy of vaccines delivery nasally [64] . nalt is home to a diverse population of apcs, including macrophages, b cells and dendritic cells. these apcs facilitate adaptive immune responses to infection [65] . the nalt can participate in the initiation of nasal immune responses; however, a recent study demonstrated that following influenza virus infection, t resident memory (trm) cells localized primarily in the nasal tissue (e.g. olfactory epithelium) outside of the nalt [66 ] . interestingly, these trm's contributed to the primary defense against re-challenge by a heterologous strain of influenza virus, which prevented spread of the pathogen into the lower respiratory tract [66 ] . these data indicate that antiviral immunity within the olfactory epithelium can limit viral dissemination into the cns as well as the lower respiratory tract. additional studies are required in this important barrier tissue to determine all the cellular participants within the nasal cavity that coordinate such a formidable defense against continuous microbial challenges. region-specific and/or cell-specific manner, undergoing significant genomic evolution compared to the periphery. reactivation of cns viral reservoirs can result in profound neurological disorders, including epilepsy, cognitive impairment, and motor dysfunction despite antiviral treatment [67] . over the past decade, immunotherapy has revolutionized medicine. by utilizing immune-specific therapies, refractory diseases have become responsive to treatment. the application of these techniques to persistent viral infection could aid in the clearance of cns reservoirs. however, use of immunotherapy to treat persistent viral infections offers two unique challenges. first, neurotropic viruses often take residence in the cns behind selective barriers to evade immune cell traffic and detection. to target these viruses, immunotherapies need to gain access to the anatomical niches despite low levels of neuroinflammation -a scenario often encountered during states of viral persistence. second, immunotherapeutic regimes have the potential to cause a great deal of tissue pathology [68] , which must be considered when attempting to purge a virus from a sensitive compartment like the cns. using adoptive t cell therapies, it is possible to eradicate a virus from the cns parenchyma without causing immunopathology, although this might not be the case with every pathogen [69 ] . tailored immunotherapy for treatment of persistent viral infections can consist of immunomodulators or adoptive cell transfer. during peripheral viral infection, immunomodulatory therapy has focused primarily on cytokine or interferon-based treatments. however, these treatments alone often fail to clear viral reservoirs but instead reactivate or facilitate adaptive immune clearance [70] . for example, tnfa blockade paradoxically reactivates cd4 + and cd8 + t cell effector function to stimulate viral control during a persistent lcmv infection [71] . accordingly, immunomodulatory treatment has immense potential to resolve infectious diseases where adaptive immune function remains intact and the nature of the infection is understood. for example, during wnv infection, cxcr4 antagonism increases cd8 + t cells entry into the brain from the perivascular spaces, thereby decreasing viral load and mortality [49] . similarly, administration of cxcl9 into the cns during hsv-1 infection increases the recruitment of antiviral cd8 + t cells and promotes survival by bypassing the role of the ul13 kinase -a viral protein that downregulates neuronal cxcl9 release during infection [72] . alongside chemokine-based and cytokine-based treatments, checkpoint inhibitors, such as pd-1/pdl-1 blockade, have also been used to modulate immune responses during persistent viral infection [73, 74 ] ; however, their efficacy in treating cns infections is currently unknown. to address this question, a current clinical trial (nct03239899) is underway to determine whether pd-1 blockade is safe in hiv-1 patients and can be used to improve antiviral immunity in the cns, which is thought to be a reservoir for the pathogen. immunocytotherapy or adoptive immunotherapy is another potentially effective strategy to purge a persistent cns viral infection when the host immune system is incapable of doing so [75, 76] . during a persistent lcmv infection, adoptive transfer of virus-specific memory cd8 + and cd4 + t cells can eradicate virus from the cns parenchyma, meninges, and choroid plexus without causing cytopathology or bbb breakdown [69 ,77] . during this clearance process, antiviral t cells convert microglia into cd11c + antigen presenting cells that they purge of virus non-cytopathically [69 ] . these data demonstrate that antiviral t cells are not inherently pathogenic to the cns and have the potential to clear a virus without causing damage [78] . one of the suggested etiologies of multiple sclerosis (ms) includes epstein-barr virus (ebv) infection [79] . because of incomplete ebv clearance and t cell exhaustion, infected b cells persist in the meninges as well as perivascular spaces and associate with ms lesions [80, 81] . therefore, removal of these inflammatory b cells may ameliorate some of the cns dysfunction associated with ms [82] . a recent case report revealed that adoptive transfer of autologous ebv-specific cd8 + t cells improved cognition and motor function in a patient with secondary progressive ms [83] . following treatment, the patient showed expansion of ebv-specific cd8 + t cells in the blood, decreased intrathecal immunoglobulin production, and reduced cns lesions by mri. these promising preliminary data suggest that adoptive immunotherapy directed against ebv might be efficacious in treating ms patients, and an expanded clinical trial will evaluate this possibility more definitively. another virus-induced cns disease that might benefit from adoptive immunotherapy is progressive multifocal leukoencephalopathy (pml) -a progressive demyelinating disease caused by jc virus. jcv has a high seroprevalence within the general population; most carriers remain asymptomatic unless immunocompromised. in a recent study, a patient with jcv-induced pml was successfully treated by adoptively transferring jcv-specific cd8 + t cells directed against the vp1 and large t viral proteins [84] . this treatment cleared virus from the csf and improved neurological function. a clinical trial (nct02694783) is currently underway to test this approach in a larger cohort of pml patients. it will be interesting to find out whether jcv-specific cd8 + t cells alone can provide lasting viral control in all patients. similar to adoptive immunotherapy against lcmv [85] , the antiviral defense against jcv might require cd4 + t cell support of cd8 + t cells to provide durable control [86] . understanding how peripheral immune cells are recruited to the cns and interact with resident cells to fight viral infections can greatly improve the development of immunotherapeutics regimens. the need for treatments has become more relevant than ever with the (re)emergence of pathogens like zika virus, which can cause neonatal microcephaly, adult encephalitis, and neural precursor cell death [87, 88] , and ebola virus, a pathogen that causes acute hemorrhagic fever 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of epstein-barr virus infection in multiple sclerosis dysregulated epstein-barr virus infection in the multiple sclerosis brain epstein-barr virus-specific adoptive immunotherapy: a new horizon for multiple sclerosis treatment epstein-barr virus-specific adoptive immunotherapy for progressive multiple sclerosis this paper showed that adoptive transfer of ebv-specific cd8 + t cells into a patient with secondary progressive multiple sclerosis decreased mri lesion size, reduced intrathecal immunoglobulin production, and improved cognition/motor control polyomavirus jc-targeted t-cell therapy for progressive multiple leukoencephalopathy in a hematopoietic cell transplantation recipient defining parameters for successful immunocytotherapy of persistent viral infection mechanisms of immune escape in central nervous system infection with neurotropic jc virus variant zika virus infects neural progenitors in the adult mouse brain and alters proliferation fatal encephalitis associated with zika virus infection in an adult group pfroeviliiis: cerebrospinal fluid examination in survivors of ebola virus disease late ebola virus relapse causing meningoencephalitis: a case report efficacy and effectiveness of an rvsvvectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination clusterrandomised trial zika virus: immunity and vaccine development this work was supported by the intramural program at the national institute of neurological disorders & stroke (ninds), national institutes of health (nih). we thank alan hoofring and ethan tyler in the nih medical arts design section for their help with the illustrations. key: cord-252389-xrdbmosj authors: kumar, mukesh; thakur, ajit kumar title: neurological manifestations and comorbidity associated with covid-19: an overview date: 2020-10-14 journal: neurol sci doi: 10.1007/s10072-020-04823-6 sha: doc_id: 252389 cord_uid: xrdbmosj first in 2002, severe acute respiratory syndrome coronavirus (sars-cov), second in 2012, middle east respiratory syndrome coronavirus (mers-cov), and now the third in the december 2019, emergence of tremendously pathogenic and large-scale epidemic novel coronavirus (sars-cov-2) has brought the worst conditions into the human inhabitants of the twenty-first century. the sars-cov-2 uses the resembling receptor, angiotensin-converting enzyme 2 (ace2) as that for sars-cov, and mainly feasts through the respiratory tract. the ace2 receptor appearances have been also detected upon glial cells and neurons, which makes them a potential target of sars-cov-2 disease (covid-19). consequently, cells expressing ace2, apart from lung and cardiovascular tissue, neurons and glial cells may act as targets and are thus vulnerable to sars-cov-2 systemic infection as well as its central nervous system (cns) comorbidities. investigation of the neurological manifestations of covid-19 is a step towards better understanding the sars-cov-2 infections, inhibiting the additional spread and treating patients affected by this pandemic. in this concern, more clinical examinations for cns involvement of sars-cov-2 are warranted. in this article, we have reviewed the neurological characteristic features of covid-19 patients, latent neurotropic mechanisms of sars-cov-2 involvement in the comorbidity associated with cns disorders, and neurological manifestations associated with covid-19. therefore, in the perspective of covid-19 pandemic, clinicians and healthcare workers should be aware of a wide spectrum of neurological manifestations associated with covid-19 along with their signs and symptoms for initial diagnosis and isolation of the patients. one of the most contagious viruses is coronavirus (family: coronaviridae), primarily targeting the human respiratory system and infecting the epithelial cells of the respiratory tract, but it also has neuroinvasive capabilities to spread from the respiratory tract to the central nervous system (cns) [1, 2] . earlier epidemics or pandemics of coronaviruses comprise the severe acute respiratory syndrome (sars-cov) during 2002 and middle east respiratory syndrome (mers-cov) during 2012 [3, 4] . the present pandemic is an acute respiratory disease, caused by a novel coronavirus (sars-cov-2, earlier known as 2019-ncov); the sars-cov-2 disease 2019 (covid-19) was spread throughout china and rapidly acknowledged the global attention. on 30 january 2020, the world health organization (who) officially stated the covid-19 epidemic as a public health emergency of global concern. the symptoms of covid-19 infection habitually appear after an incubation period most commonly around 5 days (range from 1 to 14 days) [5] . the most common symptoms of covid-19 illness are fever, cough, and fatigue; other symptoms include headache, hemoptysis, and dyspnea, among others. in the most severe cases, patients may develop pneumonia, acute respiratory distress syndrome, acute cardiac problems, and multi-organ failure [2, 6] . the first cases of covid-19 were reported in december 2019 [7] . the appearance of sars-cov-2 marked the third introduction of extremely pathogenic and large-scale epidemic coronavirus into the human inhabitants after sars-cov and mers-cov [8] . the elderly and people with underlying diseases are more susceptible to infection and prone to serious outcomes, which may be related with acute respiratory distress syndrome (ards) and cytokine storm [9, 10] . the covid-19 pandemic is of great global public health concern in the twenty-first century. special attention and efforts should be applied to prevent and reduce the transmission in susceptible populations, including children, elderly people, and healthcare providers [6] . recently, several reports on meningitis, encephalitis, myelitis, and peripheral nerve affection in the context of covid-19 were published, suggesting that sars-cov-2 can directly or indirectly infect structures and functions of the nervous system [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . coronavirus infections have been associated with the neurological manifestations, viz., febrile seizures, convulsions, change in mental status, and encephalitis [21] . in a recently published systemic review, the neurotropic and neuroinvasive abilities of coronaviruses in humans have been described [2] . a growing body of evidence shows that neuroinvasion and neurotropism is a common feature of human coronaviruses. the neuroinvasive human viruses, respiratory viruses, may damage the cns as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to cns cells (virus-induced neuropathology) [1, 2] . coronavirus enters through nasal infection and reached to the cns through the olfactory bulb, causing neuro-inflammation and demyelination of neuronal cells [9] . it has also been reported and correlated that the patients infected with sars-cov-2 show encephalopathy and neuro-inflammation resulting from a cytokine storm [22] . therefore, exploring the neurologic manifestations associated with covid-19 is urgently required for better understanding the sars-cov-2 brain infections, inhibiting the additional spread and treating patients affected by this pandemic. in this review, we have explored the epidemiology and pathophysiology of covid-19, their comorbidity in brain disorders, and neurological manifestations reported. coronaviruses are enveloped, pleomorphic, spherical particles, 150 to 160 nm in size, positive-sense single-stranded viruses ((+) ssrna virus) belonging to the family coronaviridae, and containing 8-10 open reading frames (orfs) [23] . orf1a and orf1b are translated into polyprotein 1a and polyprotein 1ab, which are processed by viral proteases to produce 16 nonstructural proteins containing rna-dependent rna polymerase enzyme (rdrp). the viral rna is replicated via transcription of a minus-strand template by rdrp. coronaviruses generate 6-9 subgenomic mrnas (sgmrnas) during replication, which lead to translation of accessory and physical proteins from downstream orfs. spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins are required for completion of a viral replication cycle, and these proteins translated from sgmrnas [24] [25] [26] [27] . the antibodies generated against the n protein of sars-cov may cross counter with covid-19; however, the heterophilic antibodies of sars-cov may not afford cross protection to covid-19, though it can be used for diagnostic purposes [23] . an additional possible role of sars-cov-n protein is its capability to counter host immune response as a viral suppressor protein of rnai (vsr) [28] . the vsrs suppress the rnai at the pre-dicer or post-dicer level to overcome the host defense to establish infection [23] . in a clustal w analysis of n-protein of sars-cov and covid-19 by ncbi amino acid blast, it is demonstrated and reported that more than 90% sequence identity were similar in sars-cov and covid-19. so, the n-protein of covid-19 may act in an analogous fashion to sars-cov as a vsr to counter the host defense mechanism [23] . covid-19 has significant consequences for morbidity and mortality worldwide since december 2019. to explore the effects of comorbid chronic diseases on clinical outcomes of covid-19, it was found that diabetes present in 10%, coronary artery disease/cardiovascular disease (cad/cvd) was in 8%, and hypertension was in 20%, which were much higher than that of 3% chronic pulmonary disease [29] . the occurrence of a core and receptor-binding domain (rbd) from the crystal structure of sars-cov-2 has exposed that is more closely involved in recognizing the hace2. although sars-cov and sars-cov-2 both exploit the same receptor hace2 in humans to gain entry into the host, the sars-cov-2 binding is more compacted by a four-residue motif from 482 to 485 in the hace2 ridge, thus improving the binding affinity of sars-cov-2 over sars-cov for hace [30] . moreover, the two viral hot spots, namely, hot spot-31 and hot spot-353 on hace2, are stabilized more by the sars-cov-2 rbd as compared to sars-cov. all this clearly indicates why sars-cov-2 has a selective advantage over sars-cov in causing infection and is a more evolved and lethal strain [22] . the key clinical manifestation of sars-cov-2 is severe pneumonia causing immense respiratory distress in the patients, especially the aged or those with already pre-existing conditions. sars-cov-2 leads to chronic inflammation of the lungs, severe dyspnea, fever, dry cough, and cyanosis and in more vulnerable patients a complete lung failure. ace2, which is the entry point for sars-cov-2, has almost an ubiquitous presence in human organs including lung parenchyma, gastrointestinal tract, nasal mucosa, renal and urinary tract, human airway epithelia, vascular endothelium, lymphoid tissues, reproductive organs, as well as in brain [31] . the virus is expected to enter chiefly through the nasal mucosa or the gastrointestinal tract due to their higher expression of protein hace2. the exciting part though is that recently reported studies have noted altered mental health in some covid-19 patients (n = 214) showing symptoms like anosmia (5.1%) and ageusia (5.6%) without nasal obstruction or other rhinitis symptoms, thereby indicating a neuroinvasive nature of the virus [16, [32] [33] [34] . specifically, pre-existing chronic conditions are strongly correlated with disease severity and being admitted to intensive care unit also, compared to covid-19 patients with no pre-existing chronic diseases. covid-19 patients who present with diabetes, hypertension, cad/cvd, or chronic pulmonary disease have a higher risk of developing severe disease, respectively. surprisingly, the authors found no correlation between chronic conditions and increased risk of mortality (or 2.09, 95% ci 0.26 to 16.67) [29] . engaged together, cardio-metabolic diseases, such as diabetes, hypertension, and cad/cvd, were more common than chronic pulmonary disease in covid-19 patients. however, each comorbid disease was correlated with increased disease severity, and after an active treatment, increased risk of mortality in patients with pre-existing chronic diseases may reduce [29] . the ace2 receptor expression in the brain has been also reported and detected over glial cells and neurons, which makes them a potential target of covid-19 systemic infection as well as its cns comorbidities. thus, cells exposing ace2, such as neurons and glial cells, may act as targets and are thus susceptible to sars-cov-2 infection [35] . it is possible that sars-cov-2 could infect the brain through olfactory nerves located in the nasal cavity, like other neurotropic coronaviruses. moreover, it is also suggested that respiratory failure in severe covid-19 cases might be treated from the perspective of the cns [36] . in an independent prospective clinical study, it was concluded that neurological symptoms are often seen in patients with covid-19. moreover, the headache was the most commonly seen neurological symptom in this disease along with dizziness, impaired consciousness, smell and gustation impairments, cerebrovascular disorders, epileptic seizures, and myalgia [37] . the psychiatric disorders are common in several neurological disorders, e.g., alzheimer's disease, parkinson's disease, epilepsy, migraine, essential tremor, and stroke. these comorbidities increase disease load and may complicate the treatment of mutual disorders. early studies of the comorbidity of psychiatric and neurological disorders were cross-sectional, and the time order of the relatives was not possible to elucidate [38] . the latest work has clarified time associations between psychiatric disorders and neurological disorders, particularly in epilepsy and stroke where epidemiological evidence recommends that there is a bidirectional relationship [39] . though these associations are understood in many neurological disorders, repetitive screening for psychiatric disorders in such cases is infrequent, mostly due to the lack of partnerships between psychiatrists and neurologists and the paucity of neuropsychiatrists [38] . various evidences have been reported and support the possible cns comorbidities associated with covid-19 pathophysiology [1, 2, [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . therefore, the healthcare provider should be aware of the wide spectrum of neurological sign and symptoms associated with covid-19 for early diagnosis and isolation of the patients [40] . apart from these, much more prerequisites should be done to improve the finding and treatment of covid-19 patients suffering from neurological and psychiatric disorders and their comorbidities after sars-cov-2 infection. there is also a need to understand the prospect of these comorbidities which may motivate alliances to improve the lives of the publics affected by neurological and psychiatric disorders associated with covid-19. the neurological manifestations of sars-cov-2 have been recently recognized from computed tomography (ct) and magnetic resonance imaging (mri) images of the brain of a patient who contracted covid-19 and showed symptoms of necrotizing hemorrhagic encephalopathy [13] . a rare disorder such as acute necrotizing encephalopathy (ane) is leading to brain dysfunction generally caused by viruses, which outcomes in liver problems, seizures, mental disorientation, and subsequent infection. this sickness is categorized by multifocal symmetric lesions in the brain that influence the brain stem, thalami, cerebellum, and cerebral white matter. the causes of ane is neuro-inflammation resulting from a cytokine storm characterized primarily by the creation of the interleukin-6 (il-6), secreted by the macrophages, which have been activated by the granulocyte-macrophage colony-stimulating factor (gm-csf) produced by the helper t cells [22] . the resultant cytokine storm may also cause a surge in interleukin (il)-2, il-7, interferon-γ, inducible protein 10, monocyte chemo attractant protein 1, macrophage inflammatory protein 1-α, and tumor necrosis factor-α leading to hyperinflammation [10] . the systemic inflammation causes severe encephalopathy in the patient, and that may lead even to stroke. in the patient, specifically infected with sars-cov-2 showing ane, the mri images displayed clear evidence of hemorrhage through hypointense signal intensity in the susceptibility-weighted images and increase in the rim on the post contrast images [22] . as a pandemic virus, no effective treatment has been established until date for this disease resulting from sars-cov-2. thus, awareness of the possible entry and pathogenicity of sars-cov-2 into the cns will have important guiding significance for the prevention and treatment of covid-19. to identify the possible routes by which this virus invades the cns, several studies have been postulated for the olfactoryhematogenous pathway, trans-neuronal machinery, and lymphatic pathway, as putative routes of coronavirus entry into the cns [1, 19] . if the neuroinvasion of sars-cov-2 gets a part in the extension of respiratory failure in covid-19 patients, the precaution with masks will absolutely be the most efficient measure to defend against the possible entry of the virus into the cns [41] . it might be estimated that the indication of the patients infected via conjunctiva will be lighter than those infected intranasally. the probable neuroinvasion of sars-cov-2 may also partially explain why some patients developed respiratory failure, while others not. as sars-cov-2 may conceal itself in the neurons from the immune recognition, complete clearance of the virus may not be sure even the patients have recovered from the acute infection [41] . in sustain of this, there is evidence that sars-cov-2 is still detectable in some patients during the convalescent period. so, given the probable neuroinvasion, the risk of sars-cov-2 infection may be currently underestimated [31] . it is also a prerequisite to find effective antiviral drug therapies that can cross the blood-brain barrier to prevent and treat cns infections from sars-cov-2. currently, as encephalopathy also has been identified as one of the symptoms of covid-19 [13] , therefore, neuroinvasiveness of sars-cov-2 needs to be evaluated to fully understand the neurological implications of covid-19. the brain reportedly, like most other organs, expresses the hace2 considered to be the entry point of the sars-cov-2 viruses in humans and is therefore not immune to viral infection [22] . though sars-cov-2 is yet to be detected in cerebrospinal fluid, sars-cov with similar structural and functional features has been detected in the cerebrospinal fluid of patients, indicating the ability of the virus to breach the extremely rigid blood-brain barrier [31] . if prior studies with other covs are taken into consideration, then sars-cov-2 like its other family members will first infect the peripheral nerve terminals and then slowly crawl its way through the synapse-connected route into the cns [42] . the associated field with sars-cov and mers-cov earlier studies observed that they infiltrate the brains of transgenic mice when administered intranasally. the virus through infiltrates into the brain took place via the olfactory nerves, ultimately affecting the thalamus and the brain stem [31] . the brain stem has been observed and reported as worst infected in covid-19 patients. several reports warranted for subsequent investigational studies along with the hematologic spread of sars-cov-2 in cns as well as retrograde neuronal transport from the lungs into the cns through vagal nerve afferents must be taken into consideration [31, [42] [43] [44] . also, with reports appealing infection of the gastrointestinal tract by sars-cov-2, the virus could even use the enteric nervous system and its sympathetic afferent neurons to reach the cns. now, with the reports of spontaneous breathing, hyposmia, and ageusia in covid-19 patients, scientists have started to speculate that not only does sars-cov-2 infects lungs but it has severe implications in neurons, specifically those in the medulla oblongata, which regulates breathing, lung, and heart functions, and any damage to it can result in chronic respiratory distress as reported in covid-19 patients [22] . it has been put forward that the latency period of the virus may be enough to destroy the neurons in the medullary region of the brain and can lead to coma or death. as reports of sars-cov-2 reaching the blood-brain barrier through the circulating blood and breaching it by attacking the endothelial layer to gain access to cns emerge, the virus might just be using an alternating route in the form of the olfactory bulb instead of the common hematological route [1, 19] . the neuronal cells infected with the virus, immune systems (microphase, t cells, and monocytes) triggered, and inflammatory system activated leads to cytokine storm and oxidative stress (fig. 1 ). if this is to be measured, the virus might just be making its way into olfactory mucosa, mostly consisting of olfactory neurons along with blood vessels and epithelial cells [22] . the olfactory mucosa relates to the olfactory bulb through the cribriform plate, which is found at the very base of the frontal lobes of the brain [16] . this very much elucidates the hyposmia and other neurological symptoms that are being increasingly observed in covid-19 patients. the fact to note here is that the long-term effects of the neuroinvasive nature of the virus may result in an increased risk of neurodegenerative diseases with involvement in the pathogenesis of neurological disorders like parkinson's disease and multiple sclerosis [42] . further, the conditions are more likely to be worsened in covid-19 patients with pre-existing neurological disorders [22] . neurological manifestations in covid-19 patients generally arise between 1 and 14 days after the beginning of sars-cov-2 infectious symptoms, and it has been predicted on average of 5-day incubation period (time between infection and symptoms onset) [45] . the sars-cov-2 is a singlestranded rna coronavirus, the genome of which has 89.1% similarity in its nucleotide sequence to a group of sars-like coronaviruses. it has been classified as the genus betacoronavirus, subgenus sarbecovirus, and is the seventh member of the coronavirus family that can infect humans [35] . naturally, common cold symptoms are caused by these four human coronaviruses, viz., nl63, hku1, 229e, and oc43, whereas the three others are accountable for several pandemic infectious diseases, such as sars in 2002 (sars-cov), mers in 2012 (mers-cov), and covid-19 (sars-cov-2) in 2019 [7, 46] . the neurological manifestations reported by sars patients, sars-cov was detected in samples/specimens of sars patients. csf samples of a sars patient who presented generalized tonic-clonic convulsion tested positive for sars-cov, suggesting possible infection of the cns by sars-cov [18] . the sars-cov was isolated from a sample of brain tissue of one patient with sars that had presented severe cns symptoms, and pathological examination of the brain tissue showed neuronal necrosis and glial cell hyperplasia [15, 35] . furthermore, sars viral particles and its genomic sequence were detected in the neurons in the brains of all eight confirmed cases of sars autopsies. of these, six confirmed cases presented edema and scattered red degeneration of the neurons [47] . the reported prevalence of neurologic manifestations associated with covid-19 patient is 36.4% in china and 57.4% in europe [32, 48] . in a recent published report from hospital network in chicago, illinois, out of total 509 patients, neurologic manifestations were present at covid-19 onset in 215 (42.2%), at hospitalization in 319 (62.7%), and at any time during the disease course in 419 patients (82.3%). furthermore, it was explained and reported in an independent study that the most frequent neurologic manifestations in covid-19 patients were myalgias (44.8%), headaches (37.7%), encephalopathy (31.8%), dizziness (29.7%), dysgeusia (15.9%), and anosmia (11.4%) [49] . the aspect for these differences in frequencies of neurologic manifestations might be due to genetic factors including polymorphism in expression of the viral receptor ace2 in the nervous system and, possibly, sars-cov-2 strain variations [50] . the first case of meningitis/encephalitis associated with sars-cov-2 was reported from the yamanashi university hospital, japan [11] . the sars-cov-2 rna was detected in the csf specimen that confirms direct evidence of the fig. 1 neurodegenerations and cns comorbidities associated with covid-19. sars-cov-2 reaching the blood-brain barrier through the circulating blood and breaching it by attacking the endothelial layer to gain access to cns emerges. the neuronal cells infected with virus, immune systems (microphase, t cells, and monocytes) triggered, and inflammatory system activated leads to cytokine storm, oxidative stress, and associated neurological manifestations neuroinvasiveness of sars-cov-2 [11, 35] . in addition to this, the pathological findings in brain tissue of covid-19 patients, diagnosed on the 13th day of hospital admission, have been reported in the gross anatomy of a patient admitted because of cerebral infarction. however, only non-specific brain edema and atrophy without further histopathological observations were found by gross anatomical examination of this patient and without any signs of infection. thus, detailed neurological examination and attempts to separate sars-cov-2 from the neuronal tissue are required to provide direct neurotropic evidence of sars-cov-2 [16] . in a recent review [51] , authors have categorized the reported neurological findings related to covid-19 into three categories: a) central (headache, dizziness, impaired consciousness, acute cerebrovascular disease, ataxia, seizures, and special senses) b) peripheral (hypogeusia, hyposmia) c) musculoskeletal (ischemic or hemorrhagic) apart from the above, increasing evidence indicated that coronaviruses may invade the cns, causing neurological disorders. though headache has been the most common neurological manifestations reported by covid-19 patients [52, 53] , a systematic review of covid-19 patients concluded that the mean prevalence of headache was only 8% (95% ci 5.7-10.2%) [54] . apart from headache, dizziness is also a common neurological manifestations of covid-19, with a reported prevalence of between 7 and 9.4% [55, 56] . in an independent study, authors have reported that out of total 138 admitted patients in hospital, those who required icu care (n = 36) were more likely to report dizziness than those who did not (n = 102) (icu 8/36 [22.2%] vs. non-icu 5/102 [4.9%], p = 0.007). confusion and headache were the fifth (9/99 [9%]) and sixth (8/99 [8%] ) most common symptoms of the covid-19 patients (n = 99) at admission, respectively [57] . furthermore, it has recently been reported that several cases of covid-19 have concurrent severe neurological symptoms, such as acute cvd (acute ischemic stroke, cerebral venous sinus thrombosis, cerebral hemorrhage, subarachnoid hemorrhage), meningitis/encephalitis, acute necrotizing hemorrhagic encephalopathy, and acute guillain-barré syndrome [11, 58] . in [32, 35] . an observational study was done with 6147 covid-19 patients in fars province, iran, for the occurrence of seizures in patients with covid-19 and to clarify the circumstances of the occurrence of seizures in these patients. although authors have found seizure rate 0.08% ( as recognition of the disease has developed, the reported incidence of chemo-sensitive impairment (loss of taste and smell) in covid-19 has been considerably higher than in the initial stages, ranging from 19.4 to 88% [33, 59] ; also, as stated by bookstaver et al., the clinical symptoms of viral infection are usually nonspecific and sometimes may be mild or symptomless [17] . therefore, the prevalence of neurological manifestations was very likely underestimated in the early stages of the covid-19 outbreak [17, 33, 60] . however, cvd is among the most prevalent comorbidities of covid-19 patients, especially in severe cases. chen et al. reported that cardiovascular and cerebrovascular diseases were the most prevalent (40/99; 40%) chronic underlying conditions [57] . they have reported that out of total 138 hospitalized patients, cvd (7/138, 5.1%) ranked fifth among the most common comorbidities, while the first to fourth comorbidities were hypertension ( [55] . furthermore, coronary heart disease as well as cvd has been confirmed to be independent risk factors associated with fatal outcomes [61] . most of the reported covid-19 patients from these observations are explainable as have been elderly (age ≥ 50 years old, 70%) who are statistically more likely to have comorbidities and thus more likely to progress into severe cases [62, 63] . additionally, elderly patients' cns comorbidities might not be underestimated rather more studies are warranted through clinical examinations for cns involvement of sars-cov-2. it has been reported that sars-cov-2-infected children tend to have a milder covid-19 disease with lower mortality [64] . however, the occurrence of underlying neurological disorder or cns comorbid condition may predispose them for severe covid-19 manifestations [65] . furthermore, children with neuromuscular disorders are high-risk patients for severe covid-19 disease. neuromuscular disorders exuberated the conditions related to respiratory muscle weakness, tracheostomy, non-invasive or invasive ventilation, weak cough, and compromised airway clearance and predispose for severe covid-19 conditions [66] . in a case-series study, abdel-mannan et al. found that out of the 27 children with covid-19 pediatric multisystem inflammatory syndrome, 4 patients (14.8%) who were previously healthy had new-onset neurological symptoms. symptoms included encephalopathy, headaches, brainstem and cerebellar signs, muscle weakness, and reduced reflexes. authors have concluded that children with covid-19 presented with new neurological symptoms involving both the central and peripheral nervous systems and splenial changes on imaging, in the absence of respiratory symptoms. however, they have warranted additional research to assess the association of neurological symptoms with immune-mediated changes among children with covid-19 as well as close neurodevelopmental surveillance is required to assess the neurological and cognitive outcomes in these patients [67] . therefore, in view of the above reported neurological manifestations associated with covid-19, further studies should be carried out to gather evidences on the best therapeutic and management options of covid-19 and associated comorbidities. however, at present, prevention aimed on reducing transmission in the community remains the only proven efficient option to combat covid-19, until the pharmacotherapies and/or vaccines are developed. during the current context of the covid-19 pandemic, clinicians and healthcare workers should be aware of a wide spectrum of neurological manifestation associated with covid-19 along with their signs and symptoms for initial diagnosis and isolation of the patients. there are increasing evidences that indicated that coronaviruses may invade the cns and exuberated neurological disorders. an increasing number of reports and inclusive clinical data have been emerged out for covid-19 patients of different age groups with severe neurological symptoms. moreover, further clinical studies still needed to bring a reasonable concern of sars-cov-2, being a new neuropathogenic agent that remains underdiagnosed. therefore, exploring the neurologic manifestations of covid-19 is a stage towards better understanding the sars-cov-2 infections, avoiding further spread, and treating patients affected by this pandemic. in this concern, more clinical studies are warranted to elucidate a possible impact of sars-cov-2 infection on common neurodegenerative diseases, such as alzheimer's, parkinson's disease, or multiple sclerosis. human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? central nervous system manifestations of covid-19: a systematic review sars and mers: recent insights into emerging coronaviruses olfactory transmission of neurotropic viruses the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak china 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hospitalized subjects with coronavirus disease 2019 from a cationwide analysis in china clinical characteristics of 140 patients infected with sars-cov-2 in wuhan covid-19: risk factors for severe disease and death systematic review of covid-19 in children shows milder cases and a better prognosis than adults neurologic manifestations in an infant with covid-19 covid-19 pandemic: the concerns of pediatric neurologists neurologic and radiographic findings associated with covid-19 infection in children publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments authors would like to acknowledge the delhi pharmaceutical sciences and research university, new delhi, for providing needful infrastructure and facilities in writing this article. conflict of interest the authors declare that they have no conflict of interest.ethical approval this is a review article, no human or animals ethical approval required.informed consent not applicable. this is not a study. key: cord-271011-5stsx5je authors: singh, m.; foster, d.j.; child, g.; lamb, w.a. title: inflammatory cerebrospinal fluid analysis in cats: clinical diagnosis and outcome date: 2005-03-09 journal: j feline med surg doi: 10.1016/j.jfms.2004.07.001 sha: doc_id: 271011 cord_uid: 5stsx5je the medical records of 62 cats with clinical signs of central nervous system disease and accompanying inflammatory cerebrospinal fluid (csf) analysis were examined retrospectively to determine if signalment, clinical signs, csf analysis and ancillary testing could accurately predict the type of central nervous system disease that was present. an inflammatory csf was defined as one in which a total nucleated cell count was greater than 5 cells/μl or one in which the total nucleated cell count was normal but the nucleated cell differential count was abnormal. sex, degree of csf inflammation, neuroanatomical location and systemic signs provided little contributory information to the final diagnosis. in 63% of the cases a presumptive diagnosis could be made based on a combination of clinical signs, clinicopathological data and ancillary diagnostic tests. csf analysis alone was useful only in the diagnosis of cats with feline infectious peritonitis, cryptococcus species infection, lymphoma and trauma. overall, despite extensive diagnostic evaluation, a specific diagnosis could not be made in 37% of cats. the prognosis for cats with inflammatory csf was poor with 77% of cats surviving less than 1 year. the medical records of 62 cats with clinical signs of central nervous system disease and accompanying inflammatory cerebrospinal fluid (csf) analysis were examined retrospectively to determine if signalment, clinical signs, csf analysis and ancillary testing could accurately predict the type of central nervous system disease that was present. an inflammatory csf was defined as one in which a total nucleated cell count was greater than 5 cells/ml or one in which the total nucleated cell count was normal but the nucleated cell differential count was abnormal. sex, degree of csf inflammation, neuroanatomical location and systemic signs provided little contributory information to the final diagnosis. in 63% of the cases a presumptive diagnosis could be made based on a combination of clinical signs, clinicopathological data and ancillary diagnostic tests. csf analysis alone was useful only in the diagnosis of cats with feline infectious peritonitis, cryptococcus species infection, lymphoma and trauma. overall, despite extensive diagnostic evaluation, a specific diagnosis could not be made in 37% of cats. the prognosis for cats with inflammatory csf was poor with 77% of cats surviving less than 1 year. i nflammation of the central nervous system (cns) is common in cats of all ages. despite this, few attempts have been made to evaluate how well cerebrospinal fluid (csf) analysis, clinical signs and other clinicopathological data correlate with the disease occurring in the cns. causes of cns inflammation in cats include viral, protozoal, fungal, parasitic and bacterial infection, 'immune mediated' and 'idiopathic' disorders of uncertain aetiology (eg, non-suppurative and eosinophilic meningoencephalitis) (munana 2001) . in addition to primary inflammatory cns disorders, there are diseases that can induce cns inflammation secondary to tissue damage and necrosis. such diseases include neoplasia, trauma, intervertebral disc disease, cerebrovascular disease and nutritional disorders (eg, thiamine deficiency). the purpose of this study was to determine if signalment, clinical signs, csf analysis, additional clinicopathological data and diagnostic imaging could be used to determine the specific aetiology of the cns disease in cats with inflammatory csf. prognosis for the various disease classifications was also evaluated. records were searched at three referral centres in sydney, nsw from january 1995 to december 2002 for cats that had csf analysis. csf collection was performed in 111 cats. of these, 62 cats could be classified as having inflammatory cns disease. csf was collected by percutaneous puncture from either the cerebello-medullary cistern or via lumbar puncture from either the l5el6 or l4el5 intervertebral space. csf analysis included total white cell count and red cell count by haemocytometer, cytomorphology by stained sediment (idexx laboratories pty ltd) or stained cytocentrifuge smears (the university of sydney department of clinical pathology) and total protein by evaluation of microprotein on a commercial biochemistry autoanalyser (olympus au 400, idexx laboratories; cobas mira, the university of sydney department of clinical pathology). the reference ranges used by our laboratories for feline csf were: white cell count %5 cells/ml, red cell count 0 cells/ml and total protein !0.3 g/l (canfield and martin 1998, raskin and myer 2001 ). an inflammatory csf analysis was defined as one in which a total nucleated cell count was greater than 5 cells/ml or one in which the total nucleated cell count was normal but the nucleated cell differential count was abnormal (o9% neutrophils, o1% eosinophils) (canfield and martin 1998, raskin and myer 2001) . csf nucleated cell counts were classified as: 1, normal: %5 cells/ml; 2, mildly elevated: 6e50 cells/ml; 3, moderately elevated: 51e1000 cells/ml and 4, markedly elevated: o1000 cells/ml. the nucleated cell population was classified as: 1, suppurative: o50% neutrophils; 2, non-suppurative: o80% mononuclear cells; 3, eosinophilic: o50% eosinophils and 4, mixed: no predominance of any one cell type (canfield and martin 1998, raskin and myer 2001) . csf total protein was classified as 1, normal: %0.3 g/l; 2, mildly elevated: 0.31e10 g/ l; 3, moderately elevated: 11e20 g/l and markedly elevated: o20 g/l. csf erythrocyte counts were classified as: 1, normal: 0 cells/ml; 2, mildly elevated: 1e49 cells/ml; 3, moderately elevated 50e1000 cells/ml and 4, markedly elevated: o1000 cells/ml (table 1 ). the contribution of csf red cell contamination to the total csf white cell count was corrected for by subtracting 1 nucleated cell for every 100 red cells (rand et al 1990) . cats with csf nucleated cell count that was less than or equal to that predicted from blood contamination were excluded from the study. haematology was performed on standard automated counters (celldyn, idexx laboratories; sysmex k-4500, university of sydney clinical pathology). differential cell counts were performed via stained (diff quick) blood smear examination. biochemistry was performed using standard automated chemistry analysers described previously. cryptococcal antigen titres were measured by latex agglutination (negative titre z 0), toxoplasma gondii igg antibody titres were measured by indirect haemagglutination (negative z !1:80), t gondii igm titres were measured by indirect fluorescent antibody (negative ! 1:80), feline leukaemia virus (felv) antigen was measured by elisa, feline immunodeficiency virus (fiv) specific antibody was measured by elisa and feline coronavirus (fecov) antibody was measured by indirect fluorescent antibody (negative titre z !1:100). owners and referring veterinarians were contacted to determine the outcome after discharge from hospital. the evaluation period ranged from 0.4 months to 7 years. the following parameters were evaluated for each cat: 1, signalment; 2, duration of clinical signs; 3, neurological findings and localisation of lesion; 4, systemic signs; 5, cerebrospinal fluid testing; 6, ancillary testing including results of culture, diagnostic imaging, blood evaluation and 7, outcome. sixty-two cases had sufficient data and met the criteria of inflammatory changes within the cns. there was no age or sex distribution pattern. the majority of cats were domestic shorthair (56%), while there were four burmese, four persians and three ragdolls. the remainder represented many different breeds including himalayan, chinchilla, birman, burmilla, siamese, cornish rex, devon rex, russian blue, oriental shorthair, abyssinian and domestic longhair. there were no obvious breed risks associated with the inflammatory changes within the cns. the age range was 4 months to 17 years, with a median age of 8 years. thirteen cats (21%) presented with acute clinical signs of less than 2 weeks' duration. forty-nine cats (79%) presented with clinical signs longer than 2 weeks duration. fifty-two cats (83.9%) presented with gait abnormalities that included ataxia, hypermetria, paresis/paralysis or unilateral lameness. fifteen cats (24.2%) presented with seizures, partial or generalised. twelve cats (19.4%) exhibited nystagmus, 12 cats (19.4%) had a head tilt, 11 cats (17.7%) had other cranial nerve deficits (facial nerve or trigeminal nerve paralysis), eight cats (13%) had reduced mentation and eight (13%) had abnormal postural reactions. other signs encountered included circling, spinal pain, anisocoria and behavioural abnormalities. in 23 cats (37%) the presentation suggested multifocal cns disease while in 39 cats (63%) the lesions appeared focal (cerebral cortex in nine cats, cerebellum in two cats, central vestibular in five 2 3 5 3 6 2 3 3 0 3 11 cryptococcus 2 0 0 1 0 2 1 1 2 2 0 1 1 2 0 4 toxoplasma 0 1 0 0 0 2 0 0 1 1 0 0 1 1 0 2 other meningoencephalitis 2 1 1 0 0 2 2 1 4 0 1 1 1 3 0 5 thiamine deficiency 1 1 0 0 1 1 0 0 0 2 0 0 1 1 0 2 lymphoma 1 4 0 0 0 4 1 1 6 0 0 0 3 2 1 6 other had a total protein of 0.31e10 g/l, two cats had a total protein of 11e20 g/l and three cats had a total protein of o20 g/l. eleven cats (17.7%) did not have a total protein measured because of insufficient sample. twelve cats had more than 1000 red cells/ml. three cats had csf characteristics indicative of haemorrhage characterised by activated macrophages and large numbers of haemosiderophages with erythrophagocytosis. in the remaining cases the increased csf erythrocyte count was consistent with blood contamination during collection. the most common ancillary testing procedures were haematology in 47 cats (76%), biochemistry in 46 cats (74%) and cryptococcal antigen titres in 26 cats (42%). fifteen cats (24.2%) had a myelogram performed, 14 cats (22.5%) had t gondii igg antibody titres measured and 10 cats (16.1%) had felv antigen tested. fiv antibody, thoracic radiographs, skull radiographs and abdominal ultrasound were performed in nine cats each. surgical biopsy was performed in five cats. fecov antibody titre measurement and computed tomography (ct) scan were performed in four cats each. urinalysis and fine needle aspirate biopsy were performed in four cats each. blood pressure, t gondii igm antibody titre, magnetic resonance imaging (mri), thyroid hormone concentration and cholinesterase concentration were performed in two cats each. ammonia tolerance testing, blood lead concentrations and bone marrow aspiration were performed in one cat each. the most frequent abnormalities seen were increased serum globulins in 16 cats (26%), anaemia in nine cats (14.5%), an abnormal myelogram in 11 cats (18%) and a peripheral neutrophilia in five cats (8%). thirty-five cats (53.2%) survived less than 1 month after presentation. three died or were euthanased immediately after cerebrospinal fluid collection. of these, one died due to uncontrollable seizures and two were euthanased because of persistent apnoea. ten cats (16.1%) survived 1e6 months, three cats (4.8%) survived 7e12 months and 12 cats (19.4%) survived greater than 12 months and were still alive at the time of writing. of the cats that died or were euthanased, 18 (37%) had a post mortem performed, 30 cats (62%) did not. two cats were lost to follow-up. based on the results, clinical information, clinical pathology and ancillary testing procedures the following classifications of disease could be made: 1, feline infectious peritonitis (fip); 2, cryptococcus species infection; 3, toxoplasma species infection; 4, other meningoencephalitis; 5, thiamine deficiency; 6, lymphoma; 7, other neoplasia; 8, trauma; 9, intervertebral disc disease; 10, spinal cord granuloma and 11, undiagnosed (table 2) . eleven cats (17%) were diagnosed with fip. a presumptive diagnosis of fip was made based on age, a suppurative or mixed inflammatory csf, poor response to treatment, elevated serum or body cavity effusion fecov antibody titre or a reduced albumin:globulin ratio (!0.5) of serum or body cavity effusions. the diagnosis was confirmed at necropsy in eight cats and on surgical biopsy in one other (83% of the cases). the remaining two cats had a diagnosis of fip made based on ancillary diagnostic tests (serum and body cavity effusion albumin:globulin ratio !0.5, and elevated fecov antibody titre in serum and body cavity effusions). the ages ranged from 4 months to 11 years of age with a median of 12 months. breed distribution did not differ from the total population of cats studied. the most common neurological signs were gait abnormalities in seven cats and reduced mentation in three cats. systemic signs included pyrexia in six cats, lethargy in three cats, ocular abnormalities in six cats and weight loss in four cats. four cats had an abdominal effusion and one cat a pleural effusion. ten cats had clinical signs consistent with multifocal lesions of the central nervous system. these were referrable to a combination of cerebral cortex and brainstem (often central vestibular) signs. one cat had cerebellar signs only. csf analysis was characterised as suppurative in seven cats, mixed in one and mononuclear in three. five cats had a marked elevation in the csf white cell count (o1000 cells/ml), three cats had moderate elevations (51e1000 cells/ml), two cats had mild elevations (6e50 cells/ml) and one cat had insufficient sample for a white cell count. seven cats had increased csf protein concentrations. four cats had a mild elevation (0.31e10 g/l), one cat had a moderate elevation (11e20 g/l) and two cats had marked elevations (o20 g/l). two cats had normal csf protein concentrations and two cats did not have csf protein concentrations measured because of insufficient sample. all 11 cats had haematology and serum biochemistry preformed. six cats had a mild, non-regenerative anaemia and three cats had a mild-moderate mature neutrophilia. no cat was lymphopenic. ten cats had elevated serum globulin concentrations, two had elevated alanine aminotransferase (alt) and one cat had an elevated bilirubin concentration. of the two cats without a histopathological diagnosis, one had a serum fecov antibody titre of 1:800 and an fecov antibody titre in ascitic fluid of 1:800. this cat had an albumin:globulin ratio of 0.4 in the ascitic fluid and a csf albumin:globulin ratio of 0.39. the second cat had a serum fecov antibody titre of 1:1600 and serum albumin:globulin ratio of 0.3. no cat survived longer than 10 days after presentation. one cat has a seizure and died immediately after csf collection. four cats were diagnosed with central nervous system cryptococcosis based on a positive serum antigen titre and/or the presence of cryptococcal organisms in the csf. all were spayed female. the age range was 2e11 years with median age of 9 years. three cats were domestic shorthair and one was a burmese. all four cats had gait abnormalities. other clinical signs observed were reduced mentation, spinal pain, seizures, circling, reduced postural reactions and nystagmus. systemic signs observed included bradycardia, inappetence and ocular abnormalities. the location of the neurological signs was variable, encompassing all central regions except the cerebellum. two cats had suppurative csf and two had mononuclear csf. three cats had large numbers of cryptococcal organisms in the csf. one had no organisms detected in the csf and was initially diagnosed with cns toxoplasmosis (based on a moderate mixed inflammatory csf, t gondii igg of 1:60 and igm of o1:160). at necropsy 8 months later, a large cryptococcal granuloma was detected in the cerebrum. this cat had a premortem cryptococcal antigen titre of 1:32. csf nucleated cell numbers ranged from 15 to 2098 cells/ml. the protein concentration was normal in two cats, markedly elevated in one cat (26 g/l) and not measured in the other. three cats had positive serum cryptococcal antigen titres, although in one cat it was very low (1:32). an antigen titre was not measured in one case. two cats had elevated serum globulins and one cat had a moderate mature neutrophilia. two cats recovered and survived more than 5 years after diagnosis. one cat was euthanased 4 days after presentation and did not undergo a post mortem examination. two cats were diagnosed with cns toxoplasmosis. a presumptive diagnosis was made based on a mild suppurative-mixed csf inflammation, normal-mildly increased csf protein levels and positive igg antibody titre. both cats had the clinical diagnosis confirmed at necropsy (t gondii tachyzoites observed in the cns). one of the cats was diagnosed with suppurative meningitis of unknown cause. it was euthanased 3 months after presentation and necropsy revealed cns toxoplasmosis. both cats presented with gait abnormalities, head tilt and anisocoria. one cat had mild neutrophilic csf inflammation with a mildly elevated protein (0.58 g/l) and did not have t gondii antibody titres measured. the other cat had mild mononuclear csf inflammation. the csf protein levels were not measured in this cat because of insufficient sample. one had a positive t gondii igg antibody titre (1:1024). both cats presented with a chronic course of disease and multifocal neurological signs. both cats had elevated serum globulins and abnormal thoracic radiographs (a single nodular opacity present in both). survival times were 2 and 12 weeks. five cats were diagnosed with meningoencephalitis of unknown cause. they were subclassified, according to response to treatment, into nonsuppurative meningoencephalitis and steroid responsive meningoencephalitis. three cats were diagnosed with non-suppurative meningoencephalitis on the basis of a mononuclear pleocytosis in the csf or histopathology indicating a perivascular mononuclear infiltrate. one cat recovered from the disease over 10 days and was clinically normal 2 years later. the other two cats were euthanased within 4 weeks because of persistent neurological signs. both cats received a post mortem examination that revealed a mononuclear perivascular cellular infiltrate in the cerebral cortex of one cat and in the cerebral cortex and brainstem of the other cat. the ages of these cats ranged from 3 to 8 years with a median age of 5 years. the main neurological signs were seizures in two, and one each of nystagmus, gait abnormalities, behavioural changes and reduced mentation. extra-neural signs included one each of weight loss, inappetence, pyrexia and ocular abnormalities. all three cats presented with chronic signs of illness. two cats had multifocal signs and one had focal cerebral signs. all three cats had mononuclear csf inflammation with mild-moderate elevations in the csf leukocyte counts. csf protein concentrations were normal in two cats and not measured in one because of insufficient sample. no cat had serological testing for infectious diseases. two cats had a marked inflammatory pleocytosis on csf analysis but made a complete recovery with corticosteroid therapy and were diagnosed as a steroid responsive meningoencephalitis. one cat was a 5-year-old female spayed ragdoll, the other a 12-year-old male castrate russian blue. the ragdoll presented with acute signs of seizures, circling, proprioceptive deficits and ataxia. the csf nucleated cell count was markedly elevated (2960 cells/ml) with a mixed inflammatory pattern and moderate blood contamination (90 cells/ml). the csf protein concentration was mildly elevated (4.3 g/l). fip was the initial clinical diagnosis and prednisolone (macrolone; mavab) was prescribed. the cat made a dramatic improvement within 2 days of prednisolone therapy and was gradually weaned off prednisolone over 6 months. twelve months later the cat was clinically normal. the russian blue presented with a chronic history of hindlimb paresis with localisation of signs to the thoracolumbar spinal cord. the nucleated cell count was moderately elevated (623.7 cells/ml) with moderate blood contamination (53 cells/ml). the csf protein concentration was moderately elevated (11.6 g/l). the inflammation was mononuclear. complete blood count, biochemical profile and spinal radiographs did not reveal any abnormalities. the cat was treated with prednisolone (macrolone; mavlab) and gradually improved over the following 6 days. this cat was also gradually weaned off prednisolone over 6 months and was clinically normal 2 years later. two cats were diagnosed with thiamine deficiency on the basis of a clinical history of a thiamine deficient diet, response to thiamine supplementation and a mild suppurative inflammatory csf. a 6-year-old female spayed himalayan had an acute onset of ataxia, behavioural abnormalities, absent menace and absence of physiological nystagmus. there was a mildly elevated csf nucleated cell count (10.1 cells/ml) that was suppurative. the cat had been fed a diet of sulphur dioxide preserved kangaroo meat exclusively for 8 weeks. treatment included thiamine (thiamine hydrochloride; natural health products), 20 mg po q12 h and a diet change to a balanced cat food. the cat showed a dramatic improvement within 24 h of therapy and was clinically normal at long term follow-up 2 years later. the other was a 9-year-old male castrated domestic shorthair that also had with an acute onset of ataxia, depression, nystagmus, postural reaction deficits and weight loss. the csf leukocyte count was normal (2 cells/ml) but consisted entirely of non-degenerate neutrophils. the cat was treated with clindamycin (antirobe; pharmacia and upjohn) as blood tests revealed elevated bilirubin, alanine aminotransferase (alt), alkaline phosphatase (alp), and a toxoplasma species igg of o2560. the cat was euthanased 3 days later because of marked deterioration. necropsy revealed changes consistent with thiamine deficiency and hepatic lipidosis. fourteen cats (22.6%) with inflammatory cerebrospinal fluid were diagnosed with neoplasia. of these 14 cats, six (43%) had a confirmed diagnosis of lymphoma based on a mononuclear inflammatory csf with abnormal lymphocytes, or when needle aspiration/biopsy of extracranial sites confirmed malignant lymphoid cells. eight (57%) were diagnosed with other neoplasms on the basis of chronic progressive clinical signs, mild mixed inflammatory csf and ancillary laboratory tests (radiographs, myelogram, mri, ct, surgery and biopsy, table 3 ). only three of these cases had a confirmed histopathological diagnosis of the type of neoplasia. six cats were diagnosed with cns lymphoma. the ages ranged from 4 to 17 years with a median of 13.5 years. the main clinical signs were gait abnormalities in six cats, reduced conscious proprioception in three cats and a head tilt in two cats. one cat had multiple cranial nerve deficits (nystagmus, anisocoria and facial nerve paralysis). systemic signs included anorexia in one cat and ocular abnormalities in one cat. five cats had a chronic course of disease while one was presented with acute clinical signs. in three cats the clinical signs localised the disease to the spinal cord. two cats had multifocal disease and one cat focal cerebral disease. all six cats had mononuclear inflammation and in five cats, large atypical lymphoid cells were observed in the csf. in the sixth cat, lymphoma was diagnosed via fine needle aspirate biopsy of the kidneys. four cats had mildly elevated csf leukocyte counts (6e50 cells/ml). the remaining two cats had moderate (218 cells/ml) and marked (1144 cells/ml) cns inflammation. one cat had a normal csf protein level and four had mild elevations (0.31e10 g/l). in one cat the protein was not measured due to insufficient sample. the most common abnormal ancillary tests were non-regenerative anaemia in three and leukocyte abnormalities (varying combinations of eosinophilia, neutrophilia, monocytosis and abnormal peripheral lymphocytes) in three. one cat had abnormal thoracic and abdominal radiographs (sternal lymphadenopathy and hepatomegaly), one had an abnormal myelogram (spinal cord compression), one had lymphoma revealed by fine needle aspirate biopsy of the kidneys and one had a computed tomography scan which revealed a mass in the frontal lobe. the cat with hepatomegaly had lymphoma confirmed by transabdominal hepatic fine needle aspirate biopsy. the cat with the abnormal myelogram had lymphoma of the spinal cord confirmed by surgical fine needle aspirate biopsy. three cats survived less than 1 month, two survived 1e6 months and one cat was lost to follow-up. no cat hct z haematocrit, tpp z total protein, bun z blood urea nitrogen, fna z fine needle aspirate, lcat z latex cryptococcal antigen titre, ct z computed tomography, mri z magnetic resonance imaging, other me z other meningoencephalitis (non-suppurative meningoencephalitis and steroid responsive meningoencephalitis), ivdd z intervertebral disc disease, def z deficiency, e z post mortem not undertaken. was recorded to have survived more than 3 months after presentation. no cat had a necropsy performed. of these eight cats, one cat was still alive at the time of the study (11 months after presentation), six were euthanased and one died. the ages ranged from 8 to 14 years with a median age of 9.5 years. the main clinical signs were gait abnormalities in seven, reduced proprioception in three and other cranial nerve deficits (facial nerve paresis, reduced pupillary light response, absent gag reflex) in three. a variety of systemic signs were observed (upper respiratory tract infection, heart murmur, pale mucous membranes). seven cats had a chronic course of disease while one cat presented acutely. the clinical signs localised the disease to the spinal cord in four cats, brainstem in two cats, and central vestibular system in one cat. in one cat the lesions appeared multifocal (vestibular and spinal). in six cats the inflammation in the csf was mixed and in two it was mononuclear. in one cat the csf nucleated cell count was normal (2 cells/ml), in six cats it was mildly elevated (6e50 cells/ml) and in one cat it was moderately elevated (82 cells/ml). six cats had mildly elevated csf protein concentrations (0.31e10 g/l). in two cats the csf protein was not measured because of insufficient sample. five cats had abnormalities on myelogram (widening of spinal cord, intradural mass lesions). one cat had abnormalities on skull radiographs (bilateral radio density in tympanic bullae). one cat had abnormal ct scan (soft tissue density in ethmoid turbinates invading cribiform plate and rostral brain). one cat had an abnormal magnetic resonance image (mass in cerebello-pontine angle, invading petrous temporal bone). other abnormalities seen were one each of leukocytosis, elevated globulins, elevated liver enzymes, elevated creatinine kinase and an abnormal biopsy (nasal carcinoma). four cats survived less than 1 month, one survived 2 months and two cats survived 6e12 months. one cat was still alive at the time of the study (11 months later). this cat was a 10-year-old female, spayed chinchilla with neurological signs of a thoracolumbar spinal lesion. myelogram revealed an extramedullary-intradural lesion. surgical removal was performed which revealed an osteosarcoma of the articular facets. the cat was well but mildly paretic on its hindlimbs 11 months after surgery. two cats had a post mortem examination that revealed a cerebral meningioma in one cat and an undifferentiated nerve root tumour in the other. three cats were diagnosed with cns trauma based on history, myelogram and a marked haemorrhagic csf. all had acute onset of disease (24e48 h). one cat was 7 years of age and two were 8 years of age. the main clinical signs were gait abnormalities in two cats, and one each of increased reflexes, spinal pain, reduced conscious proprioception, urinary and faecal incontinence. no systemic clinical signs were seen. in all three cats the neuroanatomical location was the spinal cord (thoracolumbar in two cats and lumbosacral in one cat). the csf inflammation was mixed in two cats and suppurative in one cat. all cats had a mild elevation in csf protein concentration. myelography was performed in all three cats. this revealed intramedullary swelling in one, loss of spinal cord opacity in another and under opacification with compression of the spinal cord in the third cat. csf haemorrhage was marked in all three cats with evidence of erythrophagia and xanthochromia, however the total nucleated cell counts were higher than could be accounted for with blood contamination alone. all three cats made a full recovery. one cat was diagnosed with intervertebral disc disease. it was a 12-year-old male castrate domestic shorthair with chronic signs of hindlimb paresis localised to the thoracolumbar spinal cord. the cat had a mild mixed inflammatory csf (19.8 cells/ml) with a mildly elevated total protein (2.34 g/l). myelogram revealed spinal cord compression. hemilaminectomy was performed and the cat recovered. two cats were diagnosed with a spinal cord granuloma of unknown cause. both cats had a chronic history of hindlimb paresis and the lesions were localised to the spinal cord in both cats. one cat had a mild mixed inflammatory csf (16 cells/ml). the other cat had a moderate suppurative inflammatory csf (167 cells/ml). the first cat did not have csf protein measured while the second had a mild elevation in csf protein (1.43 g/l). myelography revealed extradural spinal cord compression in both cats. surgery and biopsy in both cats revealed granulomatous spinal cord lesions but no underlying cause was found. the first cat was euthanased 4 months later because of a recurrence of clinical signs. a post mortem examination was not performed. the second cat recovered and was still alive 6 years later. eighteen cats (29%) remained without a diagnosis often despite extensive clinicopathological testing (table 3) . of these, one cat made a complete recovery and was euthanased 3 years later for renal failure. one cat was lost to follow-up. the remaining 16 cats died or were euthanased. the age range was 4 monthse15 years with a median age of 12 years. the predominant neurological signs were gait abnormalities (13 cats), seizures (nine cats), reduced proprioception (six cats), head tilt (four cats), postural reaction deficits (three cats) and other cranial nerve deficits (facial nerve, trigeminal nerve paralysis) (three cats). the main systemic signs were inappetence, weight loss and pyrexia in two cats each. sixteen cats had a chronic course of disease and two cats had acute clinical signs. the neuroanatomical location was multifocal in eight cats, cerebral in four cats, brainstem in five cats, central vestibular in two cats and spinal cord in three cats. the type of inflammation was mixed in six cats, suppurative in eight, mononuclear in three and eosinophilic in one cat. three cats had a normal csf white cell count, 12 had mild elevations in the csf nucleated cell counts (6e50 cells/ml) while three cats had moderate elevations (51e1000 cells/ml). the csf protein concentration was normal in four cats and mildly elevated in 12 cats (0.31e10 g/l). in two cats the csf protein concentration was not measured due to insufficient sample. three cats had blood leukocyte abnormalities (neutropenia, lymphopenia and monocytosis). three cats had elevated globulins, two had azotaemia, one cat had elevated t4 and one had elevated liver enzymes. eleven cats survived less than 1 month, five cats 1e6 months, one cat was lost to follow-up and one cat recovered. two cats underwent a post mortem examination, in which a cause for chronic partial seizures, behavioural changes and hemiparesis in one cat and seizures with cranial nerve deficits in the other cat was not found. the purpose of this study was to establish if an accurate diagnosis in cats with inflammatory csf could be established by clinical signs, csf analysis and ancillary diagnostic tests. a previous study (rand et al 1994b) failed to demonstrate any significant difference in csf parameters between cats diagnosed with primary inflammatory, degenerative, or neoplastic disease of the cns. this may reflect the presence of secondary inflammation and degeneration associated with many cns disorders. categorising csf as 'inflammatory' depends on a number of factors such as the method of collection (particularly peripheral blood contamination), laboratory reference ranges, loss of cells in csf due to delays between sample collection and analysis and experience of the technician interpreting the sample. to control these variables, all the reference ranges chosen were those used by the laboratories involved in processing the sample. all samples were evaluated by an experienced veterinary pathologist within 1 h of collection. peripheral blood contamination is a common confounding problem in csf collection despite good technique. normal csf should not contain any erythrocytes, however up to 30 red blood cells (rbcs/ml) is considered 'normal' contamination as it does not have a significant effect on the csf white cell count (rand et al 1990) . correcting for peripheral blood contamination using the ratio of red cells to white cells in the peripheral blood may not be an accurate indicator of the effects of blood contamination on csf fluid (wilson and stevens 1977) . rand et al (1990) suggested a formula for the correction of csf white blood cell counts based on the csf red blood cell number. this study also showed that the formula was likely to overestimate the degree that blood contamination elevated csf white cell counts in approximately 50% of cases. the maximum expected increase in white cells is calculated based on the assumption that there is one additional white cell/ml of csf per 100 red cells/ml of csf. in this study, cases were excluded if blood contamination was significant enough to explain the elevated white cell count. thus, all cats with true cns inflammation would be included but is possible that some cats with true inflammation were excluded. location of csf collection may alter protein concentration and cell counts. lumbar csf white cell counts can be less and protein concentrations higher than that of cisternally collected csf in dogs. the reason for the reduced white cell counts in the lumbar csf is not known. possible causes include cell lysis, less cells entering the lumbar csf or migration of white cells from lumbar csf to blood (bailey and higgins 1985) . increased protein concentration in the lumbar csf may be caused by sluggish spinal csf circulation resulting in protein accumulation. similar differences have been observed in feline csf protein levels (hochwold et al 1969) . changes in white blood cell counts between lumbar and cisternal punctures have not been compared in cats. csf was collected from both sites as this study encompassed cases from three different referral hospitals and the method of collection was chosen according to the preference of the veterinarian involved. often, the area of collection was not specified in the data. differences may have occurred but this could not be accounted for in the present study. fip was the most common cause of inflammatory csf and accounted for 18% of the cases. this is similar to a previous study (rand et al 1994a (rand et al , 1994b where 18% of cats with primary inflammatory and primary non-inflammatory cns disease were diagnosed with fip. fip is a difficult disease to diagnosis antemortem. the 'gold standard' for diagnosis is histopathology which reveals a perivascular proliferation of macrophages, lymphocytes plasma cells and neutrophils with a central area of necrosis (hartmann et al 2003) . it has been shown that evaluation of albumin:globulin ratio (!0.5) and anti-coronavirus antibody in body cavity effusions (any titre) had a good positive predictive value for the diagnosis of fip. in addition, the highest serum anti-coronavirus antibody titre (1:1600) also had a good positive predictive value however, any serum antibody titre less than this was not valuable (hartmann et al 2003) . while nine cases of fip in this study were diagnosed via histopathology, two cases were diagnosed via the clinical and serological criteria. the next most common disease category in our study was neoplasia, with both confirmed lymphoma and undetermined forms accounting for 22.6% of the cases. in contrast, rand et al (1994a rand et al ( , 1994b found that 16% of cases (combined primary inflammatory and non-inflamma-tory cns disease) were diagnosed with nonsuppurative meningoencephalitis of suspected viral origin. quesnel et al (1997) found 47% of cats presented for seizure disorders to have nonsuppurative meningoencephalitis of possible viral cause. our study found that only three cats had findings that were supportive of non-fip viral infection. the diagnosis is suggested by mononuclear meningitis without visceral lesions of fip and perivascular cuffing as the only histologic lesion (lundgren 1992) . it has been postulated that certain viruses, eg, parvovirus, feline herpesvirus, calicivirus, fiv and felv could all cause these histopathological lesions. the arboviruses have received special interest in canada. there are six strains of arbovirus known to cause encephalitis in humans in canada (artsob and spencer 1979) . some of these are also known to be endemic in the wild animal population and experimental infections in cats (powassan virus) have caused non-suppurative meningoencephalitis (keane et al 1987 , rand et al 1994a . there are more than 70 strains of arbovirus in australia, only two of which are known to cause encephalitis in humans (murray river and kunjiin virus) (boughton 1994) . the cause of feline non-suppurative meningoencephalitis is not known but the apparent variation in the diagnosis of this condition may reflect differences in causative agent(s) seen in the two geographical locations. the median age for cats in this study was 8 years and was reflected in all disease categories except fip, which was markedly lower (median age 1 year). overall, the most common systemic clinical signs were weight loss (18%) and ocular abnormalities (16%), apart from fip where pyrexia was the most common clinical finding (75%). in all groups, the majority of cats had gait abnormalities as the major neurological sign (84%). neuroanatomical location did not help in differentiating between disease categories as many cats were diagnosed with multifocal/ diffuse disease (37%). exceptions to this were cats with lymphoma, other neoplasia, spinal cord granuloma and trauma in which the spinal cord was the most frequent neuroanatomical location. csf protein concentration may provide some help in categorising disease groups. the only cats with moderate or marked csf protein elevations (o10 g/l) were cats with fip (23%), one cat with cryptococcus (26.0 g/l) and one case of other meningoencephalitis (steroid responsive meningitis) (11.6 g/l). cats with fip had, by far, the highest csf protein levels (53 and 62 g/l). hence, markedly elevated protein concentrations should increase the index of suspicion for fip. the lack of protein measurements in 18% of the cases as a consequence of insufficient sample, may have skewed the interpretation. the degree of cns inflammation was inconsistent within groups. severe inflammatory csf was observed more commonly in fip cases (38%). other categories had variable degrees of inflammation. the type of inflammatory pattern was helpful only in cats with lymphoma as all had a mononuclear inflammation. mixed inflammation of the csf was the most common type of inflammatory pattern observed (48.4%) and when observed did little in aiding the diagnosis. interestingly, both cats with thiamine deficiency had a mild suppurative inflammation. to the author's knowledge, there are no reports in veterinary or human literature on the csf findings with thiamine deficiency. as csf collection is a diagnostic test, it is not required if the diagnosis is known. a diagnosis of thiamine deficiency can often be suspected from dietary history and clinical signs. thiamine deficiency results in cerebrocortical necrosis (read and harrington 1986, steel 1997) that could result in a mild suppurative csf infiltrate. seventy-seven percent of cats survived less than 1 year. three cats died or were euthanased immediately after csf collection. this represents almost 5% of cases and serves to illustrate that cats with central nervous system disease can be poor anaesthetic risks and that csf collection can be associated with significant morbidity and mortality. cats diagnosed with cryptococcus had a better prognosis (50% still alive more than 5 years after presentation), as did those diagnosed with trauma and steroid responsive meningitis (100% still alive more than 12 months after presentation). survival times were variable in cats with toxoplasmosis, non-suppurative meningitis and thiamine deficiency (table 2 ). this study revealed that csf analysis alone was helpful only in cns fip, cryptococcus, lymphoma and trauma. in other conditions a presumptive diagnosis could be made using a combination of antemortem findings (age, csf cellular and protein levels, serum and effusion antibody titres, biopsy and outcome). neuroanatomical location provided some indication of the diagnosis in cats with lymphoma, other neoplasia, spinal cord granuloma or trauma (spinal cord). the degree of csf inflammation, csf protein concentration and systemic signs provided little contributory information to the final diagnosis other than in cats with fip. despite often extensive diagnostic evaluation, 37% of cats was left without a premortem aetiological diagnosis. future prospective studies with thorough post mortem examination are warranted as post mortem evaluation was only performed in this retrospective study in 18 of the cases. the increased use of advanced imaging (ct/mri), csf antibody, molecular biological methods, and cns biopsy may aid in the diagnosis of cns disease in the living patient. arctic and tropical arboviruses comparison of total white blood cell count and total protein content of lumbar and cisternal cerebrospinal fluid of healthy dogs arboviruses and disease in australia veterinary cytology: a bench manual for the canine and feline practitioner comparison of different tests to diagnose feline infectious peritonitis california serogroup: powassan virus infection of cats feline non-suppurative meningoencephalomyelitis. a clinical and pathological study consultations in feline internal medicine diagnostic evaluation of cats with seizure disorders: 30 cases (1991e1993) reference intervals for feline cerebrospinal fluid: cell counts and cytological features clinical, cerebrospinal fluid, and histological data from twenty-seven cats with primary inflammatory disease of the central nervous system clinical, cerebrospinal fluid, and histological data from thirty-four cats with primary non-inflammatory disease of the central nervous system experimentally induced thiamine deficiency in beagle dogs: pathologic changes of the central nervous system thiamine deficiency in a cat associated with the preservation of 'pet meat' with sulphur dioxide effects of blood contamination on cerebrospinal fluid analysis the authors would like to thank dr jody braddock, dr carolyn o'brien, the reception staff at the university veterinary centre sydney and dr elizabeth dill-macky, the animal referral hospital, sydney for their assistance in providing cases for this study. we would like to thank the staff of central sydney imaging, royal prince alfred hospital medical centre for providing computed tomography, magnetic resonance imaging equipment and assistance in interpretation. we would also like to thank dr colin dunlop, advanced anaesthesia specialists for anaesthesia of the above cases, idexx laboratories and the clinical pathology department of the university of sydney for laboratory testing. key: cord-009577-29u7pdpk authors: gonzalez‐scarano, f.; tyler, kenneth l. title: molecular pathogenesis of neurotropic viral infections date: 2004-10-08 journal: ann neurol doi: 10.1002/ana.410220502 sha: doc_id: 9577 cord_uid: 29u7pdpk classical virologists defined a number of viruses that affect the nervous system and identified tissue tropism, extraneural replication, and viremia as important parameters that determine whether viral infections will affect the central nervous system. molecular techniques are expanding this knowledge by permitting us to relate specific genes and gene products to two defined phenotypes: neuroinvasion and neurovirulence. two converging situations make this knowledge particularly useful: (1) the development of antiviral drugs and subunit vaccines, which mandate that pathogenesis be related to specific regions of the viral genome; and (2) the expanding problem of central nervous system infections in immunodeficient states. the recent revolution in the biological sciences has had a tremendous impact on virology, which benefited early from the technological advances in molecular biology. viruses are now used to study gene expression and receptor-ligand interactions and to express cloned genes in eukaryotic systems. it has nevertheless remained easier to study viral infections in vitro rather than viral-host interactions in whole organisms. recently a number of significant insights into the molecular and genetic basis for the pathogenesis of viral infections of the nervous system have been reported by different groups. in this review we will highlight some of the major findings in the field. viral pathogenesis is the process by which a virus causes disease in a susceptible host. pathogenesis can be analyzed in terms of a series of stages (see 132) and 154) for reviews). to cause systemic illness, a virus must first enter the host animal, undergo primary replication at a site near its portal of entry, and then ultimately spread to distant target tissues, such as the central nervous system (cns). by definition, all animal viruses are intracellular pathogens, and the process of replication must commence with entry into a susceptible cell. an infecting animal virus faces two main blocks to penetration of the cns or any other specific target organ: (1) a variety of barriers prevent the free access of viruses to target cells, and (2) even when these barriers are ineffective, only certain cell types will support the internalization and replication of a particular virus. it is useful to think of the capacity of a virus to establish a lethal infection within the cns as the property of neumirulence and the ability to penetrate the cns after inoculation and growth at a peripheral site as the property of neuroinvasiveness. experimentally, it is easy to bypass the barriers to cns infection (by intracerebral inoculation, for example), and therefore each of these properties can be tested for independently. a major goal of much of the current work in virology has been the correlation of viral properties such as neurovirulence and neuroinvasiveness with specific viral genes or proteins or, when the systems have been more advanced, with specific regions of these genes andor proteins. each of the potential entry routes into the organismskin, mucosal membranes, gastrointestinal (gi) tractis utilized by viruses capable of eventually infecting the cns. the specific entry point will be determined by the biology and physicochemistry of the virus as well as by the need for vectors like mosquitoes, ticks (arboviruses), or mammals (rabies). for a number of neurotropic viruses, the physical barrier provided by the skin is breached by the bite of an animal or arthropod vector, or through the use of contaminated needles or other foreign bodies. many other neurotropic viruses enter the host via natural portals such as the respiratory and gi tracts. entry via these routes may be direct (via contaminated saliva, for example) or through aerosols. the molecular mechanisms that influence the capacity of viruses to become aerosolized and their subsequent stability are unknown. most nonenveloped viruses (poliovirus, reovirus) appear to lose infectivity in conditions of low (< 50%,) relative humidity. for example, aerosolized poliovirus shows a lo3-fold drop in infectivity when kept for 1 hour at 15 % relative humidity (when compared with 72% humidity) {58]. conversely, the infectivity of aerosols of most enveloped viruses is not notably altered by changes in the relative humidity, although in general enveloped viruses are more sensitive to environmental inactivation. the g i tract presents formidable chemical barriers to the entry of viruses. the mechanisms by which agents such as those present in the gi tract (e.g., acids, bile salts, proteolytic enzymes) affect viral structure, and hence determine the ability of a virus to survive transit through the local environments present at entry sites such as the gi tract, are gradually becoming understood. in the case of nonenveloped viruses, the outer capsid proteins appear to be the major determinants of viral stability to a wide variety of physicochemical agents. for example, rhinoviruses are rapidly inactivated at acidic p h levels, whereas other picornaviruses, such as polio, are not. one practical consequence of this is that rhinoviruses do not survive transit through the stomach and hence rarely produce disease outside the limited confines of the respiratory tract. exposure of rhinoviruses to an acidic p h results in the loss of the viral capsid protein v p 4 . this in turn produces a "hole" in the virus particle through which the viral nucleic acid leaks out, leaving a noninfectious "empty" viral capsid. interestingly, the same type of structural alteration can occur in polioviruses exposed to certain adverse physical conditions, which again emphasizes the role of the viral capsid in determining virion stability [45, 731. another example of the role of outer capsid proteins in determining viral stability can be seen in the reoviruses. these viruses, like the enteroviruses, are neurotropic viruses whose natural portal of entry is via the gi tract. the three serotypes of reovirus show profound variations in their in vitro sensitivity to a number of physicochemical agents, including temperature, ph, alcohol, and detergent solutions. studies using reassortant viruses containing genes derived from "sensitive" and "resistant" serotypes of virus (see fig. 2 for an analogous experiment) allowed specific genes to be identified and associated with particular phenotypes. sensitivity to alkahne p h and guanidine mapped to the viral s 1 gene, sensitivity to high temperature and the detergent sodium dodecyl sulfate mapped to the s4 gene, and sensitivity to phenol and ethanol mapped to the m2 gene. in each case these genes encode proteins that are components of the outer cap-sid of the virus, indiccating again the importance of these proteins in determining susceptibility to environmental conditions [17., 181. just as they differ with respect to sensitivity to p h changes, viruses vary in their response to proteolytic enzymes. some viruses are extremely sensitive to trypsin-foot and mouth disease virus demonstrates a 103 drop in infectious titer upon treatment with it-and others, like influenza, need the effects of a trypsin-like protease to become infectious e23, 411. the human rotaviruses, which are enteric pathogens, show dramatic enhancement of infectivity in the presence of trypsin, which cleaves the v p 3 outer capsid protein 1691. reoviruses also differ in their sensitivity to proteolytic digestion with enzymes such as chymotrypsin [4, 343. genetic studies have shown that the viral m 2 gene, which encodes an outer capsid polypeptide (mlc), is responsible for determining sensitivity or resistance to proteolysiij by chymotrypsin [66] . the effects of proteolytic enzymes on the infectivity of ortho-and paramyxoviruses have been investigated extensively. in the case of influenza virus, a trypsin-like protease present in host cells cleaves the hemagglutinin protein into two smaller peptides, ha1 and ha2, held together by a tlisulfide bond. the cleavage and subsequent removal (of an arginine residue exposes the hydrophobic n h 2 terminal of ha2, which is essential for the fusion function of this virus (see below) [23}. if influenza virus is grown in cells that lack this peptidase activity, h a cleavage does not occur, and, though the virus still binds to cellular receptors, it is not infectious [20, 41, 67, 681. enveloped viruses-those that incorporate a lipid bilayer on their out'er surface-are particularly sensitive to inactivation b y bile salts, which dissociate the lipoprotein components of the viral envelope. this may explain why enveloped neurotropic viruses only rarely enter the h0:jt via the gi tract (the coronaviruses, which include the neurotropic mouse hepatitis virus, are a known exception to this rule). the neurotropic viruses that commonly use the gi portal of entry are all nonenvel.oped and therefore insensitive to the action of bile salts. many viruses, such as those that infect the gi and upper respiratory tracts, attack target cells that are near the point of entry. for those agents, infection of contiguous cells satisfies the requirement of their life cycle. except in special circumstances 131, viruses that infect the cns rnwt reach it after entering the body at a distant site. in general, this means primary replication must occur in a target cell outside the cns, and then the virus must reach the cns by either the bloodstream (hematogenous spread) or via nerves (neural spread). it is (1) cns target cell, (2) the efficiency of spread after the primary infection of these target cells, and (3) the success of the host's immune response that determine whether a virus will be neuroinvasive. incidentally, under most circumstances this combination of factors reduces the incidence of successful cns infections, making these a small proportion of the total number of infections caused by viruses, even those that are traditionally considered neurotropic. most neurotropic viruses reach their target tissue through the bloodstream. arboviruses, for example, are inoculated directly into the subcutaneous tissue or bloodstream, then replicate in extraneural tissues such as muscle or regional lymph nodes 148, 52). figure 1 is a schematic diagram of the spread of an arbovirus from the blood to the nervous system. following primary replication, the virus is released into the bloodstream, where it is transported in the plasma. for a viremia to serve as a source of dissemination of virus to target organs, it must be of adequate magnitude and duration. studies with a variety of arboviruses have shown that the degree and persistence of viremia is directly related to the ability to penetrate the cns c30, 52). the viruses of the california encephalitis group, which are transmitted by mosquito species endemic to the midwestern united states and to upper new york state, are responsible for the majority of the cases of arboviral encephalitis in this country. their rna genome is segmented (a quality they share with the influenza viruses, arenaviruses, and rotaviruses and reoviruses), and different "pieces" can be recombined artificially in the laboratory. by taking genomic segments from neuroinvasive and noninvasive strains, one can ask the question: which genome segment is responsible for the ability to penetrate the cns? figure 2 summarizes such an experiment, in which viruses representing neuroinvasive and noninvasive strains were used to coinfect cells. "reassortant" viruses, containing genomic elements from two different strains, were thus generated and then tested for their ability to penetrate the murine cns. using this approach, it has been determined that the gene coding for the envelope glycoproteins cosegregates with the phenotypic property of neuroinvasiveness, although the genes coding for the other structural proteins of the virus can modulate this effect 13 1). the same line of reasoning can be extended by using viral mutants that have changes in a single protein product. one frequently used approach, primarily with rna viruses, is the selection of antigenic variants with monoclonal antibodies c26, 39, 71, 72) . in this approach, a virus mutant present in the initial stock at low concentration is amplified by neutralizing the wildtype virus with a monoclonal antibody (fig 3) . the resultant virus variant contains a mutation in the protein against which the neutralizing antibody is directed, which in most instances is the protein responsible for attachment to cellular receptors. that mutation can usually be mapped to one or two amino acids which, being altered, prevent binding of the mutant virus by the monoclonal antibody. monoclonal antibody variants have been used to map the antigenic sites of the influenza hemagglutinin 122, 76, 771 and have been used successfully to define important regions of the cellular binding proteins of rabies virus, reovirus, coronaviruses, and the california serogroup-all cns pathogens. antigenic variants of the california serogroup obtained with a single monoclonal antibody have altered neuroinvasiveness; if injected directly into the cns their pathogenesis is not altered. thus, the property of neuroinvasiveness can be specifically associated with a single protein in this system 1261. by sequencing the genome of the variant virus and comparing this sequence with that of the wild-type parent, it may be possible to show that the property of neuroinvasiveness is associated with specific regions within the glycoprotein molecule, as has been done in other systems f34). besides arboviruses, other agents, like poliovirus, also use the bloodstream as their primary route to the nervous system. in polio the organ of primary repli-cation is the gut, yet it is release into the bloodstream that determines whether the characteristic myelitis will occur. in addition to plasma viremia, some viruses, including human irnrnunodeficiency virus (hiv), are transported in the bloodstream in association with lymphocytes or macrophages f2, 371. following hemsttogenous spread, neuroinvasive agents penetrate the cns through the choroid plexus or through the endothelial cells. the lack of endothelial or choroid plexus tissue culture systems has made it difficult to analyze .in detail the molecular mechanisms of penetration of these tissues. classic investigations by pasteur and colleagues on rabies virus, and b,y goodpasture and colleagues on herpesviruses clearly established that neurotropic viruses could spread to the cns via nerves [32] . more recent investigations with a number of viruses including rabies, polio, and herpes have provided abundant confirmation and more detailed insights into the process of neural spread 112, 48, 50, 51, 751 . however, until recently it wm not possible to identify the role of specific viral genes in influencing the capacity of viruses to spread through nerves. in the case of rabies virus, after an initial period of replication in skeletal muscle, the virus concentrates at the neuromuscular junction in close proximity to neuromuscular and neurotendinous spindles. the virus then enters nerve terminals in proximity to these sites and is transported to the dorsal root ganglia and spinal cord [so, 511. rabies virus mutants with alterations in the virion glycoprotein (selected with monoclonal antibodies, as described for california encephalitis virus) appear to have altered capacity to enter certain types of nerve fibers 113-161. these findings suggest that the rabies glycoprotein may play an important role in viral entry into and transport within cells. reovirus type 3 has also been shown to spread from peripheral tissue tc) the cns via nerve cells. furthermore, the microtubule-associated system of fast axonal transport has been implicated in this spread, because experimental infection of the cns following footpad inoculation of mice is inhibited by low concentrations of colchicine. selective inhibitors of slow axonal transport had no effect on spread to the cns {751. like the california encephalitis viruses, reoviruses have a segmented genome (double-stranded rna in this case). experiments similar to the one outlined in figure 2 can be used to generate reassortant viruses. unlike type 3 reovirus, type 1 reoviruses spread to the cns via a hematogenous route. tyler and collaborators exploited this fact to test reassortants of type 1 and type 3 viruses and again ask the question: which gene segmeiit accounts for the different patterns of spread in these two strains? these reassortant viism, because they confer resistance to proteolysis, dehydration, and p h changes. in most instances, this has been determined through a combination of genetic methods, usually involving either reassortant viruses or selected antigenic variants. in the next section we discuss the role of these proteins in determining tropism at the cellular level. ruses, containing gene segments from both type 1 and type 3 viruses, have clearly shown that the property of spread can be associated with the gene that codes for the sigma 1 polypeptide-an outer protein that functions as the viral hemagglutinin and cell attachment protein. additional insights into the molecular basis for neural spread have recently come from the study of herpes simplex virus recombinants. for these experiments, two strains (hsv-1, 17; hsv-2, 186), which differ in their ability to spread to the mouse cns after corneal inoculation, were utilized to generate recombinant viruses [55] . the region of the hsv-1 genome which codes for a number of proteins including the gb glycoprotein, a nucleocapsid protein (p40), and a dna-binding protein (icp-8), as well as the dna polymerase, was found to be important in the spread of viruses from cornea to cns. in summary, the available evidence all points to the conclusion that, in several different viral systems, the envelope proteins (in the case of enveloped viruses) or the capsid proteins (in the nonenveloped agents) are the major determinants of spread to the nervous system. the outside proteins naturally play a very important part in other aspects of penetration into the organthe process of entry of viruses into typical mammalian cells is outlined in figure 4 . experimentally and biologically it can be divided into two main steps: (1) binding to the plasma membrane, and ( 2 ) penetration and uncoating. many of the concepts regarding virus binding to plasma membranes have developed from the pioneering work of brown and goldstein on the low-density lipoprotein receptor {zs}. in this model, macromolecules destined for the cytoplasm first interact with cellular receptors. the interaction with these receptors is quite specific and accounts for the restriction of unwanted macromolecules from the cells. viruses bind to the plasma membrane of susceptible target cells through specific receptors which may be proteins (hiv), lipids (vesicular stomatitis virus), or contain sialic acid (reovirus, influenza) [21, 641. in some instances, neurotropic viruses have been thought to bind to pharmacological receptors. this has been best demonstrated for reovirus type 3, which in some tissues appears to bind to the beta-adrenergic receptor [9, lo] , but it has also been suggested for rabies virus, which may use the acetylcholine receptor as its entry point [43, 5 11 . to a large extent, the specificity of this virus-receptor interaction can dictate the pathogenesis of a particular agent. a topical example is the virus that causes the acquired immunodeficiency syndrome (aids), now termed hiv c21. this virus binds to the t4 molecule, a membrane protein of unknown function which defines a particular subclass of lymphocytes many viruses can infect cells if their nucleic acid genome is introduced directly into them-in the absence of any viral polypeptides. introduction of infectious dna clones has been used to bypass cellular receptors in infections with hiv. in such an experiment a variety of cell types can be infected, not necessarily only those containing the appropriate membrane receptor [ 17. another well-known receptor-virus interaction involves the influenza hemagglutinin, which binds to receptors containing sialic acid. unlike the hiv system, where knowlege of the interaction between the receptor and the virus is still at a descriptive stage, the relationship of the influenza hemagglutinin to its receptor has been characterized to the single amino acid level, because the molecular structure of the influenza hemagglutinin has been determined through x-ray crystallography c76, 77) . the hemagglutinin molecule is present as a trimer on the virion surfaces. this trimer contains a "pocket" that appears to function as the site of attachment of the receptors present on susceptible cells. viruses selected from different hosts (avian and equine, for example), which have naturally different enzymatic activities and slightly different sugar residues on their sialic acid molecules, have variations in the amino acids surrounding this pocket [6, 641. thus, even though the target cells are presumably the same, small changes in the receptor-binding site of influenza virus may account for species specificity. different technology has been used to identify the receptor-binding site of rhinoviruses. the large number of rhinovirus serotypes has made production of a general vaccine impractical. the majority of serotypes use a single specific receptor present on the surface of infectable cells. this receptor was initially identified through the use of a polyclonal antibody, then by monoclonal antibodies ell]. the virus itself has subsequently been crystallized c651, and a large cleft or "canyon" present on each icosahedral surface is thought to be the receptor-binding site, by virtue of its relationship to antibodies capable of neutralizing the virus. drugs are now being designed to block this cleft. although rhinoviruses are not neurotropic, they form part of the same family (picornaviridae) as poliovirus, and many of the features of the viral topology are being extended to that group 1471 as well as to footand-mouth disease c71 and theiler's virus, other members of the picornaviridae. in summary, a necessary condition for the entry of viruses into all cells, and nerve cells in particular, is the availability of specific receptor molecules on the sur-face of such cells. some receptors are undoubtedly present in all cns cells, and viruses that utilize such may cause a generalized infection, or panencephalitis. in the cns, the pattern of illness a virus produces is determined in large part by the specific regions of the brain that are infected and by the population of cells in the affected regions that are injured or destroyed. some viruses, like jc papovavirus, which is responsible for progressive multifocal leukoencephalopathy, infect astrocytes an'd oligodendroglia while largely sparing neurons, whereas herpes simplex infects all cell populations. striking differences also occur in the topographical distribution of viral injury to the cns. rabies virus causes pathology in the cerebellum, hippocampus, and limbic areas, whereas polio affects the motor nuclei in the cortex, brainstem, and anterior horns of the spinal cord c32, 501. many of these specific topographic ;associations have been postulated to be the result of specific receptor-binding interactions. after a virus has bound the appropriate receptors, the process of internalization can proceed in one of two ways: (1) viruses can fuse with the plasma membrane, discharging their contents directly into the cytosol, or (2) viruses may be internalized through the endocytotic pathway. fusion at the plasma membrane has been observed for viruses like herpes simplex and coronaviruses, both of which are capable of forming giant syncytia during routine infection. presumably there are no special requirements for internalization under these circumsi:ances, and if the appropriate receptors are present the virus will appear within the cell. other viruses are not capable of fusing directly with the cell membrane, and for those the endocytotic pathway (outlined in figure 4 ) is the method of internalization. this pathway, which again resembles the mechanism of interrlalization of low-density lipoproteins [2 51, involves ithe collection of receptor-ligand complexes at regions of the membrane characterized by electron-dense material at their cytoplasmic side. the viruses are then transported within endocytotic vesicles, which have been determined to have a mildly acidic ph 142, 461. for many enveloped virusesthose viruses that inc:orporate a lipid bilayer into their structure-it has been shown that exposure to such a low p h leads to alterations in the conformation of the envelope proteins c681. again using the influenza hemagglutinin as a model, such a change probably brings to the surface stretches of hydrophobic amino acids normally buriecl within the viral protein. the hydrophobic residues interact with the membrane of the endosome, fusing the viral envelope to it and releasing the contents of the virus into the cytoplasm, where the process of replication can then begin. in influenza and related viruses, a host protease is responsible for the cleavage step that prepares this portion of the hemagglutin for this exposure (from a precursor protein). this proteolysis is essential for replication and accounts for some of the host specificity of the viruses, as noted above. in fact, epidemics of influenza in avian rpecies have been ascribed to the ease of proteolysis in different strains. for nonenveloped viruses (poliovirus, rhinovirus), there is also evidence that a ph-dependent step may be important in infection, but the process has not been observed clearly, as it has been in the enveloped agents. the degree of acidity that can elicit the changes in the viral proteins necessary for fl5ion to occur varies for different viruses. because of the inherent difficulties of such measurements, the endosomal p h has not been determined for a large number of cell lines (and for none of the cns cell lines). however, one can easily theorize that the relationship between the viral requirements and the acidity of these cellular organelles must influence the potential for infection of a particular cell type. in fact, the ability to fuse has been correlated with the encephalitogenic potential of bunyaviruses and mumps virus strains 126, 78) . once the contents of the viral envelope (genome and proteins necessary for replication) have been discharged into the cytosol, the process of viral replication can begin. it is likely that for most nononcogenic rna viruses the specificity of the virus-target interaction is determined prior to this step. an efficiently replicating virus, like vesicular stomatitis virus, for example, is probably capable of initiating replication of its genome in any cell line that it can penetrate. however, not all proteins or genome segments may be reproducible in any given system, and for lymphocytic choriomeningitis virus it appears that restriction at the segment level may be responsible for the outcome of infection {621. for many dna viruses like herpes simplex and varicella-zoster, persistence is clearly associated with the arrest of replication. such mechanisms are highly specific for different families. recently the role of regulatory signals in the enhancement of replication has become a prominent area of investigation. signals within the viral genome that stimulate gene transcription or translation appear to play an important role in determining viral host range and tissue tropism in certain systems 1191. these signals, variously termed enhancers, promoters, and transactivators depending on their position and orientation, are capable of stimulating the transcription of genes. some of these enhancers determine tissue tropism (for example, in polyoma virus), whereas others are active in all tissues (simian virus {svi-40, herpes simplex) {38, 40, 61, 701. transcriptional activators are genes whose products can activate the transcription of other genes. the viruses associated with aids have prominent transactivating activity, and they may also have regulatory activities at posttranscriptional events. in any case this kind of "tat" activity can greatly amplify the production of viral gene products and may be responsible for increased cytopathological changes. in some instances it may be responsible for host specificity. tat activity had previously been shown to be an important feature of sv-40 1357, an experimentally important virus that is related to jc virus, the etiological agent for progressive multifocal leukoencephalopathy. this kind of activity may be important in the selectivity that this virus shows for glial cells and could play a role in a relationship between hiv and jc. in several experimental models, the relative contribution of jc virus regulatory sequences in conferring tissue specificity has been studied (8, 361. it has been argued that the jc virus promoter may contribute substantially to neurovirulence. outcome of infection the final determinant of pathogenesis is the outcome of infection of the host cell. four major pathways exist: (1) death of the cell; ( 2 ) alteration of its growth pattern and change into a cancerous phenotype (transformation); (3) persistence of infection without obvious cellular change; and (4) persistence of infection with alteration of specialized cellular functions (luxury functions). neurotropic viruses can be responsible for any of these final results. transformation is a highly intricate subject and will not be discussed further. interested readers should refer to general reviews (27, 441. cytopathic effect significant enough to stop cellular metabolic activity is obviously the most common outcome of viral infection in all acute and some subacute viral infections. for the most part, morphological evidence has been utilized as the primary way of determining cell death. at a molecular level, mechanisms of viral cytopathology include the inhibition of production of host cell dna, rna, or protein. for some rna viruses it is known that interference with the transfer of a methylguanosine "cap"-m'gpppn-to host messenger rna (mrna) occurs in order to direct the host machinery to translate viral proteins preferentially. for influenza viruses and bunyaviruses (california encephalitis), the stolen cap is essential for transcription of the viral rna to proceed efficiently [57, 59}. except for these and a few other examples, the specific virally mediated mechanism for inhibition of cellular metabolism is unknown. much has been written about the role of the host immune response in mediating cell death. the immune response is frequently capable of harming the host instead of protecting it from viral infection as has been clearly demonstrated for a variety of neurotropic agents, including rabies, lymphocytic choriomeningitis virus, coronaviruses, and others (for review, see 153)). there has been a recent surge of interest in the role of viruses in inducing cell membrane molecules involved in the recognition of foreign antigens. these h-2 antigens are generally expressed at a low level in neural cells, but they increase in the presence of certain viral infections and interferons 1741. it has been postulated that the presence of these antigens on neural cells may make them a better target for cellular immunity directed at viral components and thus increase the possibility of virally triggered immunopathology. although this work is clearly just beginning, the availability of nucleic acid probes for the mrna's coding for these antigens should make it possible to study potential immunopathological mechanisms at a molecular level. similarly, the wealth of information now available about the t-cell receptor molecules {27) should allow exploration of the genetic correlation with immunopathological diseases. because of the complexity of the work involving virally induced immunopathology, we will not discuss it further here. particularly when dealing with clinical specimens, it can be very useful in terms of analyzing the state of the latent viral dna in experimental infections. although considerable effort has been spent on this question, to date there is no clear indication whether herpes simplex virus is integrated into host dna or is present as an "episome" in latently infected ganglia [51. work by fraser and co-workers indicates that the dna is present in a form distinct from that of the isolated virions 147, and personal communication). similar techniques can also be used to demonstrate that varicella-zoster virus is present latently in human dorsal root ganglia, and probably reactivated to cause zoster dermatitis 1241. this particular herpesvirus has not been cultured in explants of such ganglia. when the histological features so allow, the particular cell type that is latently infected may be identifiable-in varicella-zoster virus the genome is thought to be harbored exclusively by neurons of the dorsal root ganglia. because of the lack of a suitable experimental animal, the state of the varicella-zoster virus latent genome is even less known than that of herpes simplex virus. persistent infection with alteration of lux-ury functions. oldstone and co-workers have recently introduced the concept that some persistent viral infections may lead to the alteration of cellular function without any morphological change. in young mice infected with lymphocytic choriomeningitis virus, changes of growth and glucose metabolism are associated with persistent infection of the pituitary 156). such effects are subjelct to significant variation, depending on the age and strain of the mice as well as the viral strain. however, when present, this persistent infection of the mouse cns is associated with the absent expression of the viral glycoproteins. the synthesis of these glycoproteins may be decreased, allowing the virus to escape detection by the cellular immune mechanisms. summary a variety of factors affect the ability of viruses to infect cells within the cns. neurotropic viruses must penetrate the external barriers that protect the organism, replicate in an appropriate peripheral site, and then spread through either the bloodstream or through nerves to the cns itself. genetic analysis in several systems has pointed to lrhe external proteins as the main determinants of neuroinvasiveness and neurovirulence. however, other factors, including host enzymes, activating factors, and the replicative machinery of the virus itself, must come into play. ultimately, the balance between the viral machinery and the host immune system will determine the outcome of any infection. supported by tida ns007 17 (to f.g-s.) and physician-scientist award a100610 (to k. l. t.) and by the william p. anderson foundation. f.g.4. is a harry weaver neuroscience scholar of the national multiple sclerosis society, and k. l. t. is an alfred p. sloan research fellow. we thank neal nathanson for his helpful comments. presented in part at a conference sponsored by the american society for neurologic investigation, chicago, il, october 1, 1985. production of acquired immunodeficiency syndrome associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone isolation of 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influenza hemagglutinin and their involvement in antigenic variation structure of the hemagglutinin membrane glycoprotein of influenza virus at 3a resolution. nature 289366-373 virulence and persistence of three prototype strains of mumps virus in newborn hamsters key: cord-001017-4qfhltg4 authors: chatterjee, dhriti; biswas, kaushiki; nag, soma; ramachandra, s. g.; das sarma, jayasri title: microglia play a major role in direct viral-induced demyelination date: 2013-06-20 journal: clin dev immunol doi: 10.1155/2013/510396 sha: doc_id: 1017 cord_uid: 4qfhltg4 microglia are the resident macrophage-like populations in the central nervous system (cns). microglia remain quiescent, unable to perform effector and antigen presentation (apc) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the cns. previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (mhv) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (ms). current studies revealed that mhv infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, iba1 (ionized calcium-binding adaptor molecule 1), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. during chronic inflammation (day 30 postinfection), microglia were still present within areas of demyelination. experiments performed in ex vivo spinal cord slice culture and in vitro neonatal microglial culture confirmed direct microglial infection. our results suggest that mhv can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination. microglia are specialized macrophages of the cns that constitute 5-20% of total glial cells in rodents, depending on the specific neuroanatomical region of the cns. microglia are distinguished from neuron as well as glial cells, such as astrocytes and oligodendrocytes, by their origin, morphology, gene expression pattern, and function. while neuron and conventional glial cells are neuroectodermal in origin, microglia are of haematopoietic origin and act as primary responding cells for pathogen infection and injury like monocytes/macrophages in peripheral tissues. microglia exhibit several features that distinguish them from other populations of macrophages, such as their "ramified" branches that emerge from the cell body and communicate with surrounding neurons and other glial cells. microglia can rapidly respond to infectious and traumatic stimuli and adopt a "phagocytotic" nature. activated microglia are known to produce many proinflammatory mediators including cytokines, chemokines, reactive oxygen species (ros), and nitric oxide which mainly contribute to the clearance of pathogens or infections. however, prolonged or unwarranted microglial cell activation may result in pathological forms of inflammation which can lead to several neuroinflammatory conditions of the nervous system. microglia-mediated innate immune response in the cns is now considered to be potentially one of the major pathogenic factors in a number of cns neuroinflammatory diseases that lack 2 clinical and developmental immunology the prominent leukocytic infiltrates of adaptive immune responses [1] . neuroinflammation is associated with many neurodegenerative diseases, including alzheimer's disease (ad), parkinson's disease (pd), amyotrophic lateral sclerosis (als), and multiple sclerosis (ms) [2] . while ad, pd, and als are commonly known to be neurodegenerative disease with underlying neuroinflammatory mechanisms, ms is one of the major chronic inflammatory cns diseases in humans with heterogeneous (chronic/remitting) clinical presentations and course [3, 4] . ms is believed to be an autoimmune inflammatory demyelinating disease in which exposure of genetically predisposed people to environmental factors triggers a breakdown in t-cell tolerance to myelin antigens. demyelination is a complex process, and while the precise mechanisms of this pathology are unclear, inflammatory demyelination is thought to be the result of adaptive immune-mediated responses to myelin antigens in the myelin sheaths of axons and/or in the myelin-forming oligodendrocytes. most studies have focused on the pathogenic role of myelin-specific cd4 + t cells because of the relatively strong association of susceptibility to ms with major histocompatibility complex (mhc) class ii alleles [5, 6] . there is also increasing recognition of the potential importance of cd8 + t cells in the pathogenesis of demyelination [7, 8] . however, the contribution of innate immune cells in mediating ms pathogenesis has been recently gained attention, as several studies demonstrated the role of various innate immune cells in mediating ms pathogenesis, in particular, the potential anti-inflammatory or proinflammatory function of microglial cells along with its physical interaction with myelin [9] [10] [11] . for long time, microglia were known to be present in the chronic inflammatory demyelinating plaque to remove myelin from the dead sick neuron in ms patients but the emerging recognition of microglia as cns resident immune cells and their role in cns health and diseases stimulated substantial efforts to redefine the role and function of microglia in the regulatory mechanisms of demyelination. ms is best studied in some experimental models such as experimental autoimmune encephalitis (eae), theiler's murine encephalomyelitis (tmev), and mouse hepatitis virus-(mhv-) induced neuroinflammation. virtually, all types of adaptive immune response have been proposed to play important roles in the pathogenesis of eae [4, 12] , tmev [13] , and a neurotropic strain of mouse hepatitis virus (mhv); mhv-jhm [14, 15] , mimicking the pathogenesis of the ms. upon intracranial (i.c.) infection of neurotropic mhvs, acute meningoencephalitis (with or without hepatitis) is the major pathologic process (see supplementary figure 1 available online at http://dx.doi.org/10.1155/2013/510396) [16] . natural and genetically constructed recombinant mhv strains (generated by targeted rna recombination) with differential pathological properties were used in several studies to understand the mechanisms of demyelination and concomitant axonal loss [17] [18] [19] [20] . the outcome and degree of mhv-induced disease are dependent on several factors, including the age and strain of the mouse, the strain of mhv, and the route of virus inoculation. even very closely related strains of mhv differ in pathogenic properties. some strains of mhv are purely hepatotropic (e.g., mhv-2) [21] ; some are primarily neurotropic (e.g., jhm, mhv-4, an isolate of jhm) [15, 22] ; while others (e.g., mhv-a59 and mhv3) [16, 23] are both hepatotropic and neurotropic. viral titer reaches its peak at days 3 and 5 postinfection (p.i.) [21] . infectious virus is cleared within the first 10-14 days; however, at this time mice begin to develop demyelination, either clinical or accompanied by chronic hind limb paralysis [16, 24] . both mhv-jhm and mhv-a59 cause inflammatory demyelination in the brain and spinal cord whereas mhv3 only causes vasculitis [23, 25] . it was formerly believed that in primary mhv-induced demyelination neuronal axons remain relatively preserved. recently, it has been shown that axonal damage is, in large part, immune mediated in mhvinfected mice and occurs concomitantly with demyelination. concurrent axonal loss and demyelination have recently also been observed with s protein recombinant demyelinating strain-infected mouse spinal cord [20] . evidence from highly neurovirulent jhm strains of mhv suggests that mhv-induced demyelination is primarily immune mediated [26, 27] . clearance of infectious virus is mediated by both cytolytic and cytokine-mediated mechanisms and microglia, and t cells modulate pathologic changes. demyelination can be prevented in jhminfected lymphocyte-deficient (rag −/− ) mice [28] . however, demyelination will occur upon transfer of splenocytes from immunocompetent mice to rag −/− mice [28] . it has also been shown by depletion and transfer studies in the jhm model that cd8 + t cells can induce demyelination. these studies suggest that an intact adaptive immune system is required to promote demyelination in jhm-mhv infection. contrary to these findings, demyelination, induced by mhv-a59, has been shown to develop in adult immunocompromised mice lacking b and t cells [29] . it has also been demonstrated that the depletion of cd4 + or cd8 + t cells after the acute stage of infection does not reduce demyelination [28, 30] . indeed, mhv-a59 or its isogenic spike protein (hostattachment protein) recombinant strain, rsa59 [19, 20, 31] , induces a ms-like disease in mice mediated by microglia, along with a small population of t cells. the mechanism of demyelination is at least, in part, due to macrophagemediated myelin stripping, with some direct axonal injury as well as without involving the conventional t cells. in our current studies, we have used rsa59 infection in vivo, in vitro, and ex vivo as a model to understand whether mhv can directly infect cns resident microglia and the mechanism of microglial activation in the induction of chronic demyelination. use of animals and all experimental procedures were reviewed and approved by the institutional animal care and use committee at the indian institute of science education and research kolkata and indian institute of science, bangalore india. animal protocols adhered to the guidelines of the cpcsea, india. rsa59 an isogenic recombinant demyelinating strain of mhv-a59, where the spike gene (encodes virus host-attachment protein), was exchanged by mhv-a59 spike gene only in the background of mhv-a59 gene by targeted rna recombination as described in our previous studies [18, 19] . this recombinant strain also expresses enhanced green fluorescence protein (egfp) [32] for easy detection of viral particle by egfp fluorescence. to engineer the targeted recombinant strains, molecularly cloned vector pmh54 [33, 34] , which contains the entire 3 end of the genome from mhv-a59, was used for construction of the recombinant viruses. rsa59 and rsmhv2 are isogenic except the spike protein. rsa59 strain is expressing the mhv-a59 spike in the mhv-a59 background, whereas rsmhv2 strain is expressing the mhv-2 spike in the mhv-a59 background [18] . furthermore, egfp gene was inserted into the mhv genome in place of the nonessential gene 4 in both rsa59 and rsmhv2 [19] . in order to replace gene 4 with the egfp gene, pmh54 was modified by the introduction of a sali site 42 nucleotides downstream of the intergenic sequence for gene 4a and a noti site 102 bp upstream of the stop codon for gene 4b, using the quick change site-directed mutagenesis kit (stratagene, la jolla, ca, usa). (these are coding-silent nucleotide changes.) the coding sequence of egfp was cleaved from the pegfp-n1 vector (clontech, palo alto, ca, usa) using sali and noti and inserted in the place of the sali/noti fragment of pmh54. the resulting plasmid contains 760 bp of non-mhv sequence, including the 722-bp egfp open-reading frame, replacing the entire gene 4a and the rest 213 bp of gene 4b. previous studies reported with jhm strain revealed that the interruption of the orf 4 did not alter the neurovirulence neither in vivo nor the replication in vitro [35] . targeted recombination was used to select mhv isolates with stable and efficient expression of the gene encoding egfp to facilitate the in vivo detection of virus in the mouse cns as well as to trace the viral entry and spread in tissue culture. the viruses replicated with similar kinetics as wild-type virus both in tissue culture and in the mouse cns. they caused similar encephalitis and demyelination in animals as the wildtype virus or their recombinant strains; however, they were somewhat attenuated in virulence [19] . four-week-old, ten mhv-free, c57bl/6 (b6) mice (jackson laboratory, obtained from iisc, bangalore, india) were inoculated intracranially with 50% ld 50 dose of rsa59 strain (20,000 pfu) as described previously [19, 32] . mice were monitored daily for signs of disease. three mock-infected controls were inoculated similarly but with an uninfected cell lysate at a comparable dilution. three mice were sacrificed in between days 5, 6, or 7 (period for peak of inflammation postinfection for routine paraffinbased histopathological analysis), and the other three were used for frozen sections. the rest of the infected mice were sacrificed at day 30 postinfection for routine paraffin-based histopathological analysis. cervical, thoracic, and lumbar regions of spinal cord were successively processed, and 4 quadrants (dorsal/posterior column, anterior column, and two anterior horns) from two separate sections of each spinal cord level were examined. histopathology. at 5, 6, or 7 and 30 days postinfection, brain and spinal cord tissues were harvested from both mock infected and rsa59-infected mice. for routine paraffin sectioning, brain and spinal cord tissues were postfixed in 4% pfa for overnight. fixed tissues were processed and 5 micron thin sections were prepared for routine cns pathology, whereas frozen sections tissues were postfixed with 4% pfa for 4−6 hours and then transferred in 4% sucrose solution for 4-6 hours and in 20% sucrose solution for 16-24 hours and mounted in cryomatrix (thermo shandon). ten micron thin sections were prepared for frozen tissue immunofluorescence. the paraffin-embedded tissue sections were stained with hematoxylin and eosin (h&e) to determine the inflammation, whereas luxol fast blue (lfb) staining was used to detect the loss of myelin sheath. all slides are coded and read in blind manner. analysis. serial sections from brain and spinal cord were stained by the avidin-biotinimmunoperoxidase technique (vector laboratories) using 3,3-diaminobenzidine as substrate and a 1 : 100 dilution of anti-iba1 (wako, richmond, va, usa), 1 : 100 dilution of anti-cd45 (lca; leukocyte common antigen, ly-5, bd pharmingen), anti-iba1 (wako, richmond, va, usa), or cd3 (dako; carpinteria, ca, usa), and 1 : 20 dilution of monoclonal antibody directed against the nucleocapsid protein (n) of mhv-jhm (monoclonal antibody clone 1-16-1 (kindly provided by julian leibowitz)) as primary antibodies. control slides from mock-infected mice were incubated in parallel. cryosections from the spinal cord tissues were washed with pbs at room temperature in a humidified chamber, incubated for 10 min at room temperature with 1 mg/ml nabh 4 in pbs to reduce autofluorescence, washed, incubated for 1 h at room temperature with 1 m glycine in pbs to reduce nonspecific cross-linking, and then washed subsequently with pbs, pbs with 0.5% triton x-100 (tx), and pbs with tx and 2% goat serum (gs). the sections were incubated overnight at 4 ∘ c with a 1 : 100 dilution of a rabbit anti-iba1 antibody diluted in pbs with tx and gs, washed, and then incubated with a secondary antiserum diluted into pbs with gs for 2 hrs at 37 ∘ c. all incubations were carried out in a humidified chamber. viral antigen was detected by egfp in a fluorescein isothiocyanate channel [19] . control slides were incubated in parallel with preimmune rabbit sera, and sections from mock-infected mice were incubated with secondary antibodies only. tissue sections were sequentially washed with pbs plus tx and with pbs and mounted and visualized by ix-81 fluorescence microscopy with a 40x uplanapo objective, with the iris diaphragm partially closed to limit the contribution of out-of-plane fluorescence, and with filter packs suitable for green fluorescence and red fluorescence. images were acquired with a hamamatsu orca-1 charge-coupled device camera and image-pro image analysis software (media cybernetics, silver spring, md, usa). chronic inflammatory stage of rsa59 infection. to further characterize the presence of microglia in the chronic demyelinating plaque at the ultrastructural level, mice were anesthetized, perfused with 4% pfa, and spinal cords from mock-infected and rsa59-infected were harvested and fixed overnight in 2% glutaraldehyde as described earlier [20] . samples for transmission electron microscopy (tem) were postfixed with 1% osmium tetroxide, dehydrated, and flatembedded in poly-bed 812 epoxy resin (polysciences). half micrometer thick sections were cut from the lesional epicenter, stained with toluidine blue, and examined by light microscopy. ultrathin tem sections (600å) were cut from representative foci of demyelination from the toluidine bluestained semithin sections and mounted on 200 mesh copper grids, stained with uranyl acetate and bismuth subnitrate, and viewed under a jeol jem 1010. culture. four-week-old, mhv-free, c57 bl/6 were perfused transcardially with sterile pbs. spinal cord was harvested and washed with pbs containing 1% penicillin/streptomycin (pen/strep). the spinal cord was then embedded in 2% agarose mould, and 200 micron thick crosssections were prepared by vibratome (leica vibrating blade microtome; vt1200s). the slices were washed twice with pbs containing 1% pen/strep. the slices were then transferred to a 24-well plate with one slice in each well. 500 l of dmem containing 10% fbs, 1% pen/strep, and 1% l-glutamine were added in each well and incubated overnight with 5% co 2 . after 24 hrs of explantation, slices were infected with rsa59 at 20,000 pfu (half of the ld 50 dose) in low serum (2%) containing medium for 1 hr and then washed with pbs to remove the unbound viruses, and 10% serum containing medium were added to the infected culture and the cultures were maintained for 72 hrs. at 24 hrs, 48 hrs, and 72 hrs of postinfection, slices were processed for immunostaining with anti-iba1 antibody, and egfp fluorescence was used to detect viral antigen. briefly, at different times postinfection slices were washed gently with pbs and fixed with 4% pfa for 2 hours. postfixed slices were washed with pbs, permeabilized with 0.5% triton x-100 for 5 mins, and blocked with 1% goat serum for 1 hr at rt followed by overnight incubation with anti-iba1 antibody. for better staining, next day antibody solution was replaced with fresh antibody and incubated at 4 ∘ c for additional 16-20 hrs. slices were washed to remove any unbound antibody and then labelled with tritc conjugated goat anti-rabbit igg for 16 hrs. labelled slices were then washed to remove any unbound fluorescent tagged antibody and then mounted in vectashield (vector laboratories, ca, usa with dapi and observed in zeiss confocal microscope (lsm710). images were acquired and processed by using zen2010 software (carl zeiss). brain. primary cultures of mixed glia from day 0 to day 3 newborn mice were prepared as described previously [36] . briefly, following the removal of meninges, brain tissues were minced and incubated in a rocking water bath at 37 ∘ c for 30 min in hanks balanced salt solution (hbss, gibco) in the presence of 300 g/ml of dnasei (sigma) and 0.25% trypsin (sigma). enzyme-digested-dissociated cells were triturated with 0.25% of fetal calf serum (fcs), followed by a wash and centrifugation (300 ×g for 10 min). the pellet was resuspended in hbss, passed through a 70 micron nylon mesh, followed by a second wash and centrifugation (300 ×g for 10 min). following dilutions with astrocytespecific medium (dulbecco's essential medium containing 1% penicillin-streptomycin, 0.2 mm l-glutamine, and 10% fcs), cells were plated and allowed to adhere for 1 day in a humidified co 2 incubator at 37 ∘ c. after 24 hrs, any nonadherent cells were removed and fresh astrocyte-specific medium was added. adherent cells were maintained in astrocyte-specific medium for 10 days. culture. after establishment of the mixed glia culture, feeding was stopped for 10 days to allow for significant microglial growth on top of the astrocyte monolayer. the microglia population peaked at 12-14 days in these cultures. to remove any cells adherent to the astrocyte monolayer, microgliaenriched cultures were thoroughly agitated in an orbital incubator shaker (200 rpm for 40 min at 37 ∘ c). immediately following agitation, all cells suspended in the culture medium were collected and centrifuged at 300 ×g for 5 min at 4 ∘ c. the cell pellet was resuspended and diluted with fresh astrocytespecific medium bringing the cells to a final concentration of 8 × 10 5 cells/ml; 1 ml was added to each well of a two-well cc2-treated chamber slide (specifically made for primary cell culture; nunc) or 2 ml/well of a six-well plate. after 30 min, any non-adherent cells were discarded and adherent cells were maintained in fresh astrocyte-specific medium until infected with a medium change every 3-4 days. to examine different cell types in a given culture, primary antibodies directed against cell-specific antigens were used to determine the presence and/or purity of each of the major glial cell types as described previously. microglia were labelled with biotinylated anti-mouse cd11b (chemicon, diluted 1 : 100 in f-12 nutrient medium) followed by cy3streptavidin (jackson immunoresearch, diluted 1 : 200 in f-12). astrocytes were labelled with polyclonal rabbit antiglial fibrillary acidic protein (anti-gfap; dako) followed by either goat anti-rabbit alexa488 (molecular probes), cy2, or fitc (jackson immunoresearch) secondary antibodies. before processing for double-label immunofluorescence microscopy, cells were washed in f-12 nutrient medium. cells were incubated with primary antibodies to the surface clinical and developmental immunology 5 markers cd11b at room temperature followed by three 2 min washes with f-12. cells were then incubated with fluorescently coupled secondary antibodies for 30 min followed by three washes with pbs containing ca ++ /mg ++ . surfacelabelled cells were fixed for 10 min in 4% paraformaldehyde followed by pbs washes, permeabilized with pbs/tx (pbs with ca ++ /mg ++ , 0.5% triton-x) for 5 min, and successively washed with pbs/tx/gs (pbs with ca ++ /mg ++ , 0.5% triton-x, 2.0% normal goat serum) three times for 5 min each. cells were incubated for 30 min with the astrocytic marker gfap, washed three times with pbs/tx, labelled with an appropriate secondary antibody, and stained with dapi (1 : 500 diluted in pbs without ca ++ /mg ++ from 5 g/ml stock solutions) for 5 min. cells were then washed, mounted using vectashield (vector laboratories), and visualized by fluorescence microscopy (olympus i x-80) with a 20 planapo objective (1.0 numerical aperture). images were acquired with a hamamatsu orca ccd camera and data were analyzed by using image-pro software. on day 2 after seeding, neonatal microglial cultures were infected at a multiplicity of infection (moi) of 2 : 1 with rsa59 or mock-infected with noninfected cell lysate. after allowing viral adsorption for 1 hr, cells were washed and placed in fresh media without virus. at 6, 12, and 24 hrs after infection, cultures were examined by microscopy for egfp fluorescence. 2.14. rsa59 growth curve. confluent monolayers of l2 cells were infected with undiluted and 1 : 2 diluted culture supernatant collected from the in vitro infected microglia and incubated for 1 hr at 37 ∘ c. following adsorption, the cells were washed with tris-buffer saline 3 times and then fed with dmem with 20% fbs mixed with 0.4% agarose for overlaying. 48 hours postinfection, culture was subjected for plaque count [32] . to confirm the rsa59-induced cns inflammation, brain and spinal cord sections from day 7 (peak of inflammation) and day 30 (peak of demyelination) postinfected mice were stained with h&e or lfb and examined. rsa59-induced meningitis (supplementary figure 1(a) ), and encephalomyelitis (accumulation of inflammatory cell and perivascular cuffing) ( supplementary figures 1(b) and 1(c) ) were observed as shown previously [18, 19] (supplementary figure 1 ; these data are partly published but for the ready information compiled in one figure. ). to characterize inflammatory cell types, brain sections from day 7 postinfection were stained immunohistochemically with anti-cd45 (leukocyte common antigen (lca)), anti-cd11b and/or anti-iba1 (macrophage/microglial marker), or anti-cd3 (pan t-cell marker) (data not shown). the majority of inflammatory cells in rsa59-infected brains were immunoreactive for both lca (supplementary figure 1(d) ) and cd11b (supplementary figure 1(e) ) and iba1 (supplementary figure 1(f) ). some cd3-stained infiltrating t cells were also found (data not shown), although nonspecific background staining of neurons with available anti-cd3 antibodies made quantification difficult. no cd4-and cd19positive cells but few cd8-positive cells were observed in the inflamed brain and spinal cord sections in rsa59-infected mice (data not shown). demyelination was observed by lfb staining as early as day 7 as examined (supplementary figure 1(h) ) and it reaches its peak at day 30 postinfection (supplementary figure 1(i) ) as observed earlier [20, 31] . lfb-stained spinal cord section showed no myelin loss (supplementary figure 1(g) ). together, the data indicate that rsa59 causes meningoencephalitis and demyelination. cns inflammation consists of a mixed population of inflammatory cells, predominantly macrophages/microglia as well as a smaller population of t lymphocytes as shown previously [17, 20, 37] . infection during acute inflammation. previously, it has been demonstrated that neurotropic strains of mhv can directly infect different neural cell types [16, 19, 38, 39] but there is no evidence whether neurotropic strain can directly infect microglia or only acquire activity indirectly due to the infection of other neural cell types. in order to determine the tropism of rsa59 in cns resident microglia, fourweek-old, mhv-free, c57bl/6 (b6) mice (jackson laboratory) were inoculated intracranially with rsa59. mice were sacrificed at the peak of inflammation (day 6), and the spinal cord sections were prepared for cryostat sectioning. since rsa59 expresses egfp, viral antigen was viewed directly by fluorescence microscopy. identification of cns resident microglia was performed by using iba1 as a specific marker for microglia/macrophages [40] . while iba1 immunofluorescence was observed in both gray and white matter, double fluorescence/immunofluorescence demonstrated dual labelling of egfp (viral antigen) positive iba1 positive microglia/macrophages were present only in the white matter of rsa59 infected mice (figure 1 ). in the white matter, all the microglia (iba1-positive) were not infected as shown by arrowheads (figures 1(e) and 1(f) ). in the control mock infected spinal cord section, no double fluorescent labelled cells were observed as expected (data not shown). previous studies demonstrated that with time of postinfection viral antigen spread from gray matter to white matter [19] in the infected mice. this phenomenon is more prevalent in the spinal cord of infected mice as gray and white matter is clearly separated from white matter. immunostained section demonstrated that the viral antigen is localized both in gray and white matter at day 7 postinfection (figure 2(a) ). at day 30 viral antigen is below the detection level, more specifically after day 10 postinfection (as observed) viral antigen is below the detection limit (data not shown) as discussed previously [18, 19] . to determine whether microglia also follow the trajectory of the viral spread at days 7 and 30 postinfection, debris in demyelinating plaque. previously microglial accumulation was observed in the demyelinating plaque of rsa59 with an emphasis on the stripping of the myelin sheath [20] . to reemphasize on the accumulation of microglia in the demyelination plaque during chronic phase of the inflammation at ultrastructural level, semithin sections were cut at 1 micron intervals from five infected mice at day 30 post infection. semithin sections were stained with toluidine blue. control mock-infected mouse spinal cord was used to evaluate for background fixation and/or postfixation artefacts ( supplementary figure 2(a) ). rsa59-infected spinal cords showed significant myelin loss and accumulation of phagocytotic microglia within plaques as observed earlier ( supplementary figures 2(b) and 2(c)). representative foci of demyelination were selected from semithin sections, and 600å ultrathin sections from poly-bed embedded blocks were processed for tem. high-resolution tem images show accumulation of large number of microglia with no basement membrane which is the characteristic features of microglia/macrophages (supplementary figure 2(e) ). multiple vacuoles with myelin fragments were seen within the cytoplasm of the microglia in the plaque (supplementary figure 2(f) ). no such microglial accumulation was observed in the control mock infected mice at high-resolution tem images (supple figure 2(d) ). cells. in vivo colocalization of iba1 with egfp-(viral antigen) positive cells demonstrated that rsa59 can directly infect microglia but that does not confirm that infected microglia were resident microglia because in intracranial (ic) inoculation blood brain barrier can be disrupted and blood monocytes/macrophages can migrate and acquire infection. (figures 3(g) and 3 (k)) which demonstrated that cns resident microglia can directly acquire infection and become activated (by morphological analysis as number of processes increased and enlarged). arrowheads in figures 3(b) , 3(c), 3(d), 3(f), 3(g), 3(h), 3(j), 3(k), and 3(l) showed that some of the resident microglia did not get infection. control noninfected explant cultures were also immunolabeled with anti-iba1 antibody (figures 3(a) , 3(e), and 3(i)) as microglia in vitro in culture system behave like activated macrophages due to perturbation of the culture system. figures 3(d) , 3(h), and 3(l) show a merged image of egfp (viral antigen; green), iba1 (microglia; red), and dapi (nucleus; blue) and demonstrate the presence of viral antigen in the cell cytoplasm of microglia. due to the thickness of the slices, clarity of the images was slightly compromised. rsa59 infection in ex vivo explant cultures demonstrated that in the absence of peripheral immune cells cns resident microglia can directly be infected. syncytia. in order to determine whether rsa59 can infect microglial cells in vitro in absence of any neural cells, 2-dayold neonatal microglial cultures were infected at a multiplicity of infection (moi) of 2 : 1 with rsa59 or mock infected with noninfected cell lysate. microglia harvested in the cell suspension by the conventional shake-off method as described earlier [36] were 99 ± 0.5% positive for cd11b staining (figure 4(a) ). very few gfap (astrocyte marker) positive cells were observed in the isolated microglia culture (data not shown). at 0, 6, 12, and 24 hrs after infection, cultures were examined by microscopy for egfp fluorescence. at 0 and 6 hrs, no fluorescence was observed in the infected culture but at 12 hrs bright fluorescence started to appear denoting the presence of viral antigen in the microglia. at 12 hrs postinfection, infected microglia demonstrated stressed morphology and started to fuse with the neighbouring cells, and at 24 hrs postinfection, most of the infected cells were involved into large syncytia formation (figure 4(c) ) which is a characteristic of some enveloped rna viruses and more specifically characteristic of mhv-a59 (parental strain of rsa59), an enveloped demyelinating strain of mhv [41] . nucleus of the syncytia was very obvious as shown in figure 4 staining. in vitro experiment demonstrated that rsa59 can infect primary microglia in isolated culture and can also induce syncytia in primary microglia. to demonstrate that the virus is replicating in the microglia, culture supernatant of infected microglia was assessed by routine plaque assay [32] . routine plaque assay found very few plaques which were below the detection limit. but there, discrete syncytia was observed in the infected plates which denoted that the titer could be 30-50 pfu/ml. to understand the cellular mechanism of demyelination of neurotropic strain of mhv, prior studies in our laboratory have analyzed the detailed pathogenesis of recombinant mhv strain, rsa59 (demyelinating strain (dm)) and compared it with rsmhv2 (nondemyelinating strain (ndm)) that is isogenic except for the spike gene that encodes the virus-host-attachment spike glycoprotein [18, 20] . both strains are capable of causing hepatitis, encephalitis, and meningitis. however, the two strains differ in their ability to induce subsequent demyelination and axonal loss [20] . seven days post infection, rsa59 produces demyelination that is best observed in the spinal cord at day 30 postinfection (peak of inflammation). in contrast, rsmhv2 does not produce demyelination and only rarely demonstrates axonopathic changes in spinal cord white matter [20] . the inability of rsmhv2 to induce demyelination is due in part to a lack of transport of viral antigen (and the subsequent inflammatory reaction) to the white matter. furthermore, in vivo and in vitro experiments demonstrate deficits in the ability of rsmhv2 to spread between neurons when compared to interneuronal spread by rsa59 [20] . rsa59-induced demyelination occurs in the setting of both axonal degeneration and macrophage mediated myelin stripping along intact axons [20] . while spike glycoprotein mediates spread of viral antigen to white matter through axonal transport, specific mechanisms leading to subsequent demyelination are not known. one plausible explanation is that mhv spreads intra-axonally within gray matter and when it reaches the white matter, viral particles may need to spread directly into oligodendrocytes, astrocyte, and microglia, using the spike protein, and can induce local oligodendroglial dystrophy and inflammation. viral antigen in white matter axons may be sufficient to trigger an inflammatory response via microglial activation. infected and activated microglia due to its intrinsic nature of chemotaxis can recruit more microglia to the site of inflammation and strip myelin from the damaged axon and successively cause demyelination. our current in vivo studies support this hypothesis that rsa59 can infect cns resident microglia. the migration and activation of numerous microglia to the white matter during acute inflammation and the retention of microglia in the chronic inflammatory plaque reinforce the hypothesis that cns resident microglia can be recruited to the region of local cns injury. ultrastructural morphology of microglia containing multiple vacuoles with myelin fragments in the cytoplasm in the demyelinating plaque further substantiate that cns resident activated microglia can mediate myelin stripping and can successively mediate demyelination. as rsa59 spread intra-axonally, no colocalization was observed within the gray matter cns resident microglia. if haematogenous propagation of peripheral monocytes/macrophages occurred to the cns, one would expect more widespread distribution of activated microglia throughout the spinal cord which may not discriminate gray/white matter track. furthermore, the delay in complete development of demyelination following partial resolution of encephalitis (up to 30 days after peak inflammation) documented in previous studies would not be expected [16, 18, 31] . moreover, ex vivo colocalization of egfp-positive cells with microglia confirmed that rsa59 can directly infect cns resident microglia in absence of peripheral immune cells. in vitro infections of neonatal microglia demonstrate that rsa59 not only infects, but microglia can also forms syncytia which suggests that microglia supports rsa59 infection via cell-to-cell contact. current combined in vivo, in vitro, and ex vivo explants culture studies established that the recruitment of microglia occurred from the cns resident microglial pool rather than peripheral monocyte/macrophages. our current studies are focused on the understanding of the innate immune mechanism of cns resident microglia activation and maturation to perform phagocytotic activity. affymetrix microarray analyses for mrna expression have revealed that expression of inflammatory mediators by mhv infected microglia, including chemokine and inflammatory cytokines. mhv infection of the mouse spinal cord was also associated with increased expression of genes involved in ifn signalling compared to mock-infected controls in the cns. during chronic infection (day 30 postinfection), microglia are still present within areas of demyelination and microgliaassociated inflammatory mediators are still produced which indicates that microglia are still active. our results suggest that putative activated microglia and inflammatory mediators contribute to a local cns microenvironment that eventually regulates viral replication and ifn-gamma production during acute phase of infection. sequentially, ifn-can activate microglia by promoting phagolysosomes maturation and activation (engulfment of the myelin sheath) leading to demyelination. affymetrix microarray data warrants further confirmation. viral infection in the cns is classically recognized as inflammatory in nature, with meningeal perivascular and parenchymal infiltrates of peripheral leukocytes but rsa59 infection could be an exception where inflammation can proceed with cns resident glial activation without involving the peripheral immune responses like rabies virus infection [42] , hiv infection [43] , and prion diseases [44, 45] . in this perspective, it is tempting to speculate that the underlying mechanism of chronic myelin loss in ms could be a combination of persistence of myelin-related autoimmunogens that has escaped self-tolerance with persistence of activated cns resident microglia which can mediate demyelination by phagocytised myelin. microglia are known for their innate immune function for long time but the role of microglia in chronic inflammation opens a new episode in the field of glial biology of neuroinflammatory diseases. the concept of chronic inflammation as opposed to acute inflammation is more relevant in the context of understanding other cns diseases, more specifically neurodegenerative diseases like alzheimer's disease, amyotrophic lateral sclerosis, parkinson's disease, and huntington's disease. these neurodegenerative diseases lack the prominent infiltrates of mononuclear cells but the underlying mechanism of inflammation could be through activation of cns resident microglia. activation of cns resident microglia in the context of chronic neuroinflammation as one of the underlying mechanism of neurodegeneration warrants further study. microglia as the prime components of an intrinsic cns resident immune system become a major focus in cellular neuroimmunology and, therefore, in neuroinflammation. it has been known for long time that in absence of conventional t cells microglia play a major role in neurotropic mhv-induced demyelination but the mechanism of infection and route of infection were not very clearly known for long time. our current microglial tropism studies revealed that rsa59, an isogenic demyelinating strain of mhv, can infect and activate cns resident microglia, and microglia can help to mediate demyelination by engulfing myelin debris. rsa59-induced neuroinflammatory models are helpful in understanding direct cns cellular injury and demyelination that does not require an intact adaptive immune system. understanding the role of direct cns resident microglial infection and activation will shed some light on the pathogenesis of cns inflammatory disease, not only infectious diseases but also chronic cns disorders. the vision of cnsresident-microglia-driven neuroinflammatory responses in rsa59 with neuropathological consequences has extended the avenue to explore the contribution of microglia in chronic neuroinflammatory cns diseases. microglia and neuroinflammation: a pathological perspective mechanisms underlying inflammation in neurodegeneration multiple sclerosis immunology of multiple sclerosis a full genome search in multiple sclerosis a complete genomic screen for multiple sclerosis underscores a role for the major histocompatibility complex a pathogenic role for myelin-specific cd8 + t cells in a model for multiple sclerosis autoreactive cd8 + tcell responses to human myelin protein-derived peptides role of the innate immune system in the pathogenesis of multiple sclerosis t cells-innate immune lymphocytes? multiple sclerosis: a complicated picture of autoimmunity myelin-specific cd8 t cells in the pathogenesis of experimental allergic encephalitis and multiple sclerosis persistent infection with theiler's virus leads to cns autoimmunity via epitope spreading pathogenesis of mouse hepatitis virus-induced demyelination chronic central nervous system demyelination in mice after jhm virus infection experimental demyelination produced by the a59 strain of mouse hepatitis virus a mechanism of virus-induced demyelination demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism mechanisms of primary axonal damage in a viral model of multiple sclerosis mouse hepatitis virus type-2 infection in mice: an experimental model system of acute meningitis and hepatitis pathogenesis of virusinduced demyelination selective tropism of a neurotropic coronavirus for ependymal cells, neurons, and meningeal cells limbic encephalitis after inhalation of a murine coronavirus ependymitis, leukoencephalitis, hydrocephalus, and thrombotic vasculitis following chronic infection by mouse hepatitis virus 3 (mhv 3) bystander cd8 t cell-mediated demyelination after viral infection of the central nervous system demyelination induced by murine hepatitis virus jhm strian (mhv-4) is immunologically mediated cd4 and cd8 t cells have redundant but not identical roles in virusinduced demyelination neither b cells nor t cells are required for cns demyelination in mice clinical and developmental immunology persistently infected with mhv-a59 cd4 + and cd8 + t cells are not major effectors of mouse hepatitis virus a59-induced demyelinating disease experimental optic neuritis induced by a demyelinating strain of mouse hepatitis virus enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system the internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription inactivation of expression of gene 4 of mouse hepatitis virus strain jhm does not affect virulence in the murine cns magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue macrophagemediated optic neuritis induced by retrograde axonal transport of spike gene recombinant mouse hepatitis virus selective localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy viral infections and demyelinating diseases neurofibromatosis-1 heterozygosity increases microglia in a spatially and temporally restricted pattern relevant to mouse optic glioma formation and growth syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus rabies virusinduced activation of mitogen-activated protein kinase and nf-b signaling pathways regulates expression of cxc and cc chemokine ligands in microglia microglia in human immunodeficiency virusassociated neurodegeneration neuroinflammation in alzheimer's disease and prion disease atypical inflammation in the central nervous system in prion disease key: cord-266441-sd117tzs authors: almutrafi, amna; bashawry, yara; alshakweer, wafaa; al-harbi, musa; altwairgi, abdullah; al-dandan, sadeq title: the epidemiology of primary central nervous system tumors at the national neurologic institute in saudi arabia: a ten-year single-institution study date: 2020-02-15 journal: j cancer epidemiol doi: 10.1155/2020/1429615 sha: doc_id: 266441 cord_uid: sd117tzs objectives: this study is aimed at describing the epidemiological trends of primary cns tumors in children and adults at the national neurologic institute in saudi arabia. methods: a retrospective epidemiological approach was used where data was obtained from the department of pathology registry files and pathology reports. the records of all patients registered from january 2005 to december 2014 with a diagnosis of primary cns tumor (brain and spinal cord) were selected. data about sex, age, tumor location, and histologic type were collected. the classification was based on the international classification of diseases for oncology, 3rd edition (icd-o-3). results: nine hundred and ninety-two (992) cases of primary cns tumors throughout the ten years (2005 to 2014) were reviewed. there were 714 (71.97%) adults and 278 (28.02%) in the pediatric age group. nonmalignant tumors dominated the adult population (60.08%) while malignant tumors were more frequent in the pediatric population. gliomas constituted the most common neoplastic category in children and adults. the most common single tumor entity was meningioma (26.99%, icd-o-3 histology codes 9530/0, 9539/1, and 9530/3). medulloblastomas (icd-o-3 histology codes 9470, 9471, and 9474) were the most common single tumor entity in the pediatric age group (26.62%). conclusions: this is an institution-based, detailed, and descriptive epidemiological study of patients with primary cns tumors in saudi arabia. in contrast to other regional and international studies, the medulloblastomas in our institution are more frequent than pilocytic astrocytomas. limitations to our study included the referral bias and histology-based methodology. the worldwide incidence age-standardized rates (asr) of brain and nervous system cancer in high/very-high hdi (human development index) regions versus low/medium hdi regions was 5.0 and 2.4 for men and 4.0 and 1.7 for women (saudi arabia is classified as very-high hdi according to the united nations development program 4-tier system), respectively. these incidence rates were approximately two-fold higher in high/very-high hdi countries compared with low/medium hdi countries and slightly higher for males compared to females [1] . even though about half of these tumors are benign, they may cause substantial morbidity. brain tumors are the leading cause of cancer death in children and the third cause of death related to cancer in adolescents and adults [2] . in the gulf cooperation council (gcc) countries, brain cancer is the tenth most common cancer (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) [2] . the overall age-standardized rate (asr) of brain cancer between1998 and 2007 was 2.4 for males and 1.6 for females per 100,000 populations [2] . the incidence asr for brain and cns cancer in saudi arabia in 2008-2012 was 2.2 for males and 1.5 for females (per 100,000 populations). brain and cns cancers comprised 3.70% of all cancers in males and 2.30% of all cancers in females [3] . in saudi arabia, asr in 2014 was 2.2 for males and 1.7 for females per 100,000 populations [4] . according to the saudi cancer registry, there were 329 cases of brain cancer, accounting for 2.8% of all reported cancer cases in 2014. brain cancer is ranked 11 th among males and 10 th among females in 2014 [4] . according to the globocan 2018, there were 569 cases of brain and nervous system cancers, accounting for 2.3% of all reported cancer cases in 2018 and ranking 14 th among all cancer cases [5] . the national neuroscience institute (nni) at king fahad medical city (kfmc) is dedicated to the provision of comprehensive medical care to patients with neurologic diseases in saudi arabia. the practice of neurooncology lies at the heart of the nni objective, and the team of health care providers amalgamate clinical experience with modern technologies to offer the best care to patients. over the last ten years, the capacity of nni had progressively increased and so did the number of patients treated for cns tumors. the rates, trends, and epidemiology of primary cns tumors at the nni remain mostly unknown. to understand the current epidemiology of primary cns tumors in saudi arabia, reports that describe the disease according to international reporting standards are needed. there are only a few to define an institution-based frequency or incidence rate of primary cns in different saudi arabian regions [6] [7] [8] [9] . thus, the objective of this study is to outline the epidemiology of primary cns tumors at king fahad medical city, over a ten-year period (2005-2014). our work will enable us to observe any unusual trend in primary cns tumor epidemiology and compare our results with local and regional data. this is a retrospective study carried out using the laboratory information system (corttex), departmental diagnostic registry, and pathology reports at king fahad medical city (kfmc), riyadh, saudi arabia. institutional review board approval was granted before the start of the study. the study evaluated the distribution of primary cns tumors (brain and spinal cord) for ten years from 2005 to 2014 (kfmc opened in 2004 and the study start date was 2005). inclusion criteria included a histopathologic diagnosis of primary brain tumor of any age and sex, availability of clinical data, and histologic slides for confirmation of diagnosis. exclusion criteria included absence of histologic slides and insufficient clinical data. nonneoplastic brain lesions, secondary brain tumors (metastases), and scalp and primary bone tumors with intracranial extension were excluded. diagnosis and grading of tumors were established according to the 2016 who classification of tumors of the cns. review of the histologic slides was carried out whenever there was an ambiguity in the diagnosis or tumor grade in the pathology reports. tumors were divided into nonmalignant (who grades i and ii) and malignant (who grades iii and iv) categories. the icd-o-3 coding system was used. the epidemiology profile of the tumors, including the anatomical location, histologic diagnosis, and world health organization (who) grade in both adult (>18 years) and pediatric populations (0-18 years), was collected. the pediatric population was further divided into infants (0-<1 year), children (1-14 years) , and adolescents (15) (16) (17) (18) . moreover, age and gender were also recorded. all statistical analyses, including counts, means, rates, ratios, and proportions, were performed using the spss 22.0 software package (spss inc., chicago, il, usa). the proportions of malignant to nonmalignant tumors and supratentorial to infratentorial tumors were calculated. nine hundred and ninety-two cases of primary cns tumors were reviewed. there were 714 (71.97%) adults and 278 (28.02%) in the pediatric age group. nonmalignant tumors dominated the adult population (60.08%), while malignant tumors were more frequent (61.37%) in the pediatric population ( figure 1 ). there were 892 saudi citizens and 100 non-saudi residents distributed among all age groups ( figure 2 ). the trends of primary cns tumors over the ten-year period were rising. years 2012 and 2013 had the highest numbers of brain tumors for pediatric as well as for adult patients ( figure 4 ). among all meningiomas, 82.75% were who grade i, 16 .7% were grade ii, and 0.49% were grade iii. nonmalignant tumors included who grade i meningiomas (23.5%), pituitary adenoma (7.14%), neurilemmomas (3.6%), neuronal and mixed glioneuronal tumors (3.5%), craniopharyngiomas (2.1%), and low-grade gliomas (who grades i and ii). adult tumors were more frequently distributed in the supratentorial compartments, with cerebral meninges (icd-o-3 site code c70.0) being the most common location followed by intraparenchymal frontal lobe (icd-o-3 site code c71.1) ( figure 5 ). adult gliomas (glioblastomas, astrocytomas, oligodendrogliomas, and ependymomas) were the most frequent (46.49%) neoplastic category, and glioblastoma was the commonest of all (54.52%) ( figure 6 ). (figure 9 ). the rising trends of primary cns tumors over the ten-year period reflect the expansion in the capacity of the national neurologic institute and king fahad medical city. the reduction of number of tumors in 2014 could be attributable to cancellations of neurosurgical procedures due to the unavailability of intensive care unit (icu) beds. this was largely caused by the influx of critical patients infected in the outbreaks of the middle east respiratory syndrome coronavirus (mers-cov). our results showed that gliomas and especially astrocytomas were the most common pathologic categorical entity similar to a global study conducted by leece et al. [10] . the most common single tumor entity in adults was meningioma. these findings were similar to other studies in different [9] , the western province (meningioma 17.8%) [11] , and the eastern province [12] . our findings are also similar to other countries in the middle east, such as jordan (meningioma 26.2%) and iran (meningioma 27.8%) [13, 14] . meningioma was also the leading histologic tumor type worldwide. in the united states, it was 36.1%; france, 30.9%; and korea, 31.1% [13] . taha et al. found glioblastoma to be the most common pathological type (32%), but their study was based on neuroepithelial tumors and excluded meningeal-based tumors [15] . several studies reported the frontal lobe to be the most common site for primary brain tumors in adults [9, 11] . in our study, we found that the cerebral meninges are the most common site followed by the frontal lobe. this could be attributed to the predominance of meningioma over other neoplasms. our findings are similar to the center of brain tumor registry of the united states (cbtrus) where the most common tumor site in adults was the meninges, representing 36.1% [16, 17] . medulloblastomas were the most commonly reported histology type in the pediatric age group followed by low5 journal of cancer epidemiology grade gliomas with a predominance of pilocytic astrocytoma. similar to our finding, medulloblastoma was the most common morphological type reported nationally among children by the saudi cancer registry [4] . previous two studies carried out at king abdulaziz university hospital (kauh) revealed that astrocytoma was more prevalent in the western region [6, 11] . in the egyptian pediatric population, the most common intracranial tumors were astrocytomas (35%) followed by medulloblastomas (18.8%). pilocytic astrocytomas constituted 55% of all astrocytomas and 19.3% of all brain tumors, only slightly ahead of medulloblastomas [18] . the most common tumor found in a syrian childhood population was medulloblastoma (27.5%), followed by astrocytoma (25.8%) [19] . the lack of icd-o-3 histology coding in most of these previous works could have resulted in the discrepancy between different studies in different populations. additionally, the 2015 cbtrus report for infant and childhood primary brain and cns tumors showed pilocytic astrocytoma to be the leading histological type [16] . the saudi cancer registry used a malignancy-based statistical approach where pilocytic astrocytomas (who grade i) were omitted, which explains the higher frequency of medulloblastoma in their registry. our institution had relatively lower rates of glial neoplasms in general (42.97%) and particularly pilocytic astrocytomas (15.3%). many of the pilocytic astrocytomas were likely decompressed or excised in their original secondary hospitals while most of the medulloblastomas require gross total resection and referral to a tertiary center such as the nni. the high-grade gliomas are likely 9 .0% were who grade iv, and 28.5% were unknown). we had lower percentages of tumors with unknown grades (9.55%) compared to the qaddoumi et al. review (28.5%) because our study is pathology-driven and the histologic slides were reviewed whenever the pathology reports lacked who grades [20] . percentages for pediatric tumors were highest in infratentorial sites (55.39%). these findings are following those of several studies that stated how the infratentorial compartment was reported to be the most common site of brain tumors in the pediatric age group [18, 19] . a systematic review by khan et al. showed that inadequate reporting of cns tumor subtypes was observed in registries of developing countries [21] . they suggested establishing a unified reporting system to help improve health management for cns tumors [16] . unstandardized histology groupings and reporting can lead to different interpretations and incomparable results between different populations. chan et al. reviewed the challenges and opportunities of creating cancer registries in developing nations and suggested investing in quality hospital-based and population-based cancer registries with dedicated financial resources and manpower (health care professionals and information technologists) [22] . this study contains the largest institution-based icd-o and who-classified epidemiological analysis of malignant and nonmalignant primary brain tumors in saudi arabia in adult and pediatric groups. our study is limited by the referral bias and histology-based methodology with the exclusion of radiologically diagnosed tumors. this may potentially lead to underestimating the incidence of some gliomas such as diffuse intrinsic pontine gliomas and optic nerve gliomas. the findings in this study are generalizable to other tertiary centers in saudi arabia but not to community hospitals. most of the reviewed studies of brain tumors in saudi arabia are institution-based. the saudi cancer registry reports malignant brain tumors only. we suggest the establishment of a national brain tumor registry in saudi arabia similar to the central brain tumor registry of the united states (cbtrus). this will help in standardization of diagnostic nomenclature (icd-o coding) of brain tumors and accurate description of their incidence and survival trends. glioma as a broad diagnostic category was most common in both adult and pediatric age groups. with regard to a single tumor entity, meningioma was the most common primary brain tumor in adults while in the pediatric age group, medulloblastoma was the leading histology. the data used to support the findings of this study are restricted by the institutional review board of king fahad medical city in order to protect patient privacy. data are available from dr. wafaa al-shakweer (department of pathology, king fahad medical city) for researchers who meet the criteria for access to confidential data. the authors declare that they have no conflicts of interest. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries ten-year cancer incidence among nationals of the gcc states 15-year cancer incidence among nationals of gcc states saudi arabia, saudi cancer registry international agency for research on cancer and world health organization, the global cancer observatory childhood brain lesions: 15 years experience of king abdulaziz university hospital pattern of intracranial space-occupying lesions: the experience of the king khalid university hospital c.n.s. tumors in eastern saudi arabia does brain tumor epidemiology differ from place to another? saudi single tertiary care center experience global incidence of malignant brain and other central nervous system tumors by histology incidence of brain tumours at an academic centre in western saudi arabia experience with brain tumors in the eastern province of saudi arabia epidemiology of malignant and non-malignant primary brain tumors in jordan epidemiology of primary cns tumors in iran: a systematic review demographic and histopathological patterns of neuro-epithelial brain tumors in eastern province of saudi arabia cbtrus statistical report: primary brain and central nervous system tumors diagnosed in the united states the worldwide incidence and prevalence of primary brain tumors: a systematic review and meta-analysis descriptive epidemiology of pediatric intracranial neoplasms in egypt incidence of childhood brain tumors in syria outcome and prognostic features in pediatric gliomas: a review of 6212 cases from the surveillance, epidemiology, and end results database epidemiological trends of histopathologically who classified cns tumors in developing countries: systematic review challenges and opportunities to advance pediatric neuro-oncology care in the developing world this research was generously supported by a grant from the research center at king fahad medical city. key: cord-008586-5jinvuyn authors: loihl, angela k.; murphy, sean title: chapter 18 expression of nitric oxide synthase-2 in glia associated with cns pathology date: 2008-04-10 journal: prog brain res doi: 10.1016/s0079-6123(08)63213-6 sha: doc_id: 8586 cord_uid: 5jinvuyn this chapter discusses the expression of nitric oxide synthase-2 (nos-2) in glia associated with central nervous system (cns) pathology. the production of nitric oxide (no) in the nervous system is catalyzed by three, highly homologous isoforms of no synthase (nos). nos-2, the dimeric, heme-containing, soluble protein whose activity is independent of a rise in intracellular calcium, is variously termed ‘inducible,’ ‘immunologic,’ and ‘macrophage nos (macnos).’ nitric oxide inhibits not only nos-2 activity but also regulates the level of nos-2 messenger rna (mrna) expression through a mechanism involving nf-(k) b. there is specific evidence for the glial expression of nos-2 associated with neuronal injury and infection of the cns and in neurodegenerative and demyelinating diseases. direct injury in the cns results in a reactive gliosis, characterized by the induction of the glial fibrillary acidic protein gene and changes in astrocyte morphology. while the production of nitric oxide (no) in the nervous system is catalyzed by three, highly homologous isoforms of no synthase (nos) (forstermann et al., 1995) , in this chapter we focus exclusively on nos-2. this dimeric, hemecontaining, soluble protein whose activity is independent of a rise in intracellular calcium, is variously termed 'inducible ', 'immunologic', and 'macnos' (griffith and stuehr, 1995) . these terms are somewhat misleading because nos-2 has been reported in a wide variety of normal and neoplastic cell types, in some cases constitutively (xie and nathan 1994) . all cns parenchymal cells, together with the smooth muscle and endothelium of the vascular wall, can be transcriptionally activated by a variety of agents to express nos-2, as well as cells (neutrophils and monocytes) infiltrating the parenchyma following changes in blood-brain barrier properties (grzybicki et al., 1997 (grzybicki et al., , 1998 . in addition, there are reports of developmentally related nos-2 expression in particular brain regions (galea et al., 1995; arnhold et al., 1997) . initially from studies with cultured cells (murphy et al., 1993) , but now exemplified by a host of *corresponding author. tel.: 319 335 7958; fax: 319 335 8930; uiowa.edu in vivo conditions (see later), identified astrocytes have been shown to express nos-2 upon activation. induction can be triggered in vitro (see table 1 ) by endotoxin and/or combinations of such cytokines as interleukin (1l)-1 p, tumor necrosis factor (tnf)-a and interferon (ifn)-y; hiv envelope proteins also activate the astrocyte gene (koka et al., 1995) . the promoter region of the glial nos-2 gene displays a wide variety of potential response elements for transcription factors, such as ap-i, hif, nfkb, nf-il6, tnf, irf, and gas (spitsin et al., 1997) . in cultured astrocytes the transcript can be detected within a few hours of exposure to cytokines, is maximally transcribed by 4 h and then declines rapidly, such that it is not detectable at 12 h . in contrast, expression of nos-2 in the cns in vivo is an unexpectedly delayed event, which may, as we discuss later, have functional implications. the observation that hiv envelope proteins can induce nos-2 is important as it demonstrates that priming cells with ifn-y (a cytokine not produced by resident cns cells) is not obligatory for gene activation. in addition, cytokine-activated astrocytes, macrophages and cerebrovascular cells release tnfa which, in turn, induces nos-2 in naive cells (borgerding and murphy, 1995; shafer and murphy, 1997) . this implies that induction of nos-2 in cns parenchymal cells could occur 254 feinstein et al., 1996; stewart et al., 1997) . activation of glutamate, atp, angiotensin-i1 and p-adrenergic receptors on astrocytes blunt the nos-2 response to endotoxin and pro-inflammatory cytokines (kopnisky et al., 1997; lin and murphy, 1997; feinstein 1998) . it is clear that some of these modulatory effects result from interference with the subsequent activation of a key transcription factor, nfkb, and/or with its binding to the promoter. nitric oxide inhibits not only nos-2 activity but also regulates the level of nos-2 mrna expression through a mechanism involving nf-kb. some reports suggest a blockade of nf-kb activation (colasanti et al., 1995; peng et al., 1995) , whereas others propose inhibition of binding to its response element on the nos-2 promoter via nitrosylation of a key cysteine residue on one of the nf-kb subunits (delatorre et al., 1997; park et al., 1997) . this regulation might represent a means to limit or focus no production in discrete groups of cells in the cns (togashi et al., 1997) . such a feedback effect of no has been confirmed in vivo (luss et al., 1994) , pointing to potential rebound effects with the therapeutic use of nos-2 inhi bit ors. the nos-2 transcript is rapidly degraded, implying there may be factors co-induced in cells which are responsible for causing this instability (park and murphy, 1996) . the 3'-untranslated region of nos-2 has auuua repeats (galea et al., 1994) which are suggested to be determinants of mrna stability and this could represent yet another mechanism by which the amount and duration of no production is regulated. with all of these potential regulatory mechanisms for nos-2 expression in mind, what is the evidence for expression in vivo? what are the roles that no from this source play in cns function? there are many potential targets for no and its oxidation products (such as peroxynitrite). these include heme and non-heme iron, thiols and dna. such reactions account for the reported biological effects of nos activation, such as mitochondria1 dysfunction (knowles, 1997) , neuromodulation (christopherson and bredt, 1997) , and cell and organism killing (nathan, 1997) . in addition, it is evident that no has subtle effects on the regulation of a number of cytokine and chemokine genes (merrill and murphy, 1997) . however, it is important to distinguish between effects due to n o and those resulting from peroxynitrite exposure. the latter is a strong oxidant with effects that, in general, are biologically deleterious; the production of no in the absence of superoxide anion produces different effects, many of which may be beneficial (butler et al., 1995; nathan, 1997) . since last we reviewed this literature (murphy and grzybicki, 1996) there have been considerable efforts to identify specific neuropathological con-255 ditions that result in the induction of nos-2. the earlier, suggestive reports of nos-2 mrna and/ or protein expression associated with certain types of cns injury have now been confirmed by in situ hybridization and immunohistochemistry and provide evidence for the cellular source. there is specific evidence for glial expression of nos-2 associated with neuronal injury and infection of the cns, and in neurodegenerative and demyelinating diseases (see table 2 ). we refer to these most recent reports for completeness, and then focus on ischemia and the evidence for glial nos-2 expression. finally, we speculate on the possible roles for no in the development of pathology, and in recovery from injury. direct injury in the cns results in a reactive gliosis, characterized by induction of the glial fibrillary acidic protein gene and changes in astrocyte morphology (eddleston and mucke, 1993) . in a freeze-lesion model, we found that nos-2 was induced within a few hours in cells infiltrating the lesion (grzybicki et al., 1998) . only after 24 h could we find nos-2 expression in astrocytes delimiting the lesion edge. in a compression injury to the spinal cord, hamada et al. (1 996) report nos-2 mrna, which peaks at 24 h and persists for three days. the expression was associated with motor dysfunction and application there is also suggestive evidence for nos-2 expression in glia after indirect neuronal injury, which occurs with axotomy or chemical toxicity, and results in a marked microglial, but lesser astroglial cell response. following a local injection of kainic acid into the cortex, lei et al. (1996) describe neuronal degeneration, followed 2-3 days later by expression of nos-2 in reactive astrocytes and in endothelial cells at the edge of the lesion. how viruses cause lesions, or dysfunction without direct damage to the cns, is unclear. diffusible cytokines, together with reactive oxygen and nitrogen species produced as a component of the immune response presumably contribute to the pathology. in many instances, nos-2 expression is associated with viral infection of the cns and, in some, the resulting no appears to be beneficial. intranasal inoculation of mice with the neurotropic hepatitis virus leads to expression of nos-2 in the brain (grzybicki et al., 1997) . the cells responsible for its expression appear to be entirely infiltrating, with no evidence for parenchymal cell expression. however, this infection proves fatal within seven days. lane et al. (1997) used a neuroattenuated strain of this virus which is cleared and the animals thus survive. they report nos-2 in astrocytes and macrophages in the olfactory bulb. we have looked also at expression of nos-2 in persistently infected animals, generated by raising inoculated pups with immunized dams. in these animals, which display marked demyelinating lesions and associated hindlimb paralysis, nos-2 is expressed very prominently in astrocytes surrounding such lesions (sun et al., 1995; grzybicki et al., 1997) . a similar expression was reported by oleszak et al. (1997) in astrocytes surrounding necrotizing lesions in mice infected with theiler's virus. there is also suggestive evidence that nos-2 expression in the cns is 256 associated with hiv-1 (adamson et al., 1996) and siv infection (lane et al., 1996) . the study by lane et al. (1997) in wild-type mice early in the course of systemic inflammation, resulting from an injection of endotoxin, there is a marked expression of nos-2 in vascular, glial and neuronal structures adjacent to areas devoid of a blood-brain barrier (wong et al., 1996; htain et al., 1997) . boje (1996) found that intracisternal administration of endotoxin resulted in no production from the meninges and alteration in blood-brain barrier properties, which could be reversed by a nos inhibitor. it is of interest to note that mice with deletions in the nos-2 gene are protected from death after high-doses of endotoxin (macmicking et al., 1995) . inherited retinal dystrophy results from accumulation of cellular debris in the subretinal space and visual cell degeneration. cotinet et al. (1997) looked at the retinal muller (astrocyte-like) cell from dystrophic rats and found that they produce abnormal amounts of tnfa and nos-2 derived no. as no can decrease phagocytosis of photoreceptor segments, which underlies this dystrophy, they suggest that no may disrupt transmission in retinal neurons. alzheimer's disease (ad) occurs in 10-15% of individuals over 65 and is characterized by the loss of pyramidal neurons in the hippocampus and neocortex, and of cholinergic neurons in the median forebrain. the hallmark of ad is the deposition of p-amyloid aggregates which can be toxic to neurons and also leads to no production from glial cells (rossi and bianchini, 1996; barger and harmon, 1997) and neurons (vodovotz et al., 1996) . weldon et al. (1998) injected soluble and fibrillar p-amyloid into rat striatum. they noted that microglia and astrocytes nearby showed a marked increase in nos-2 expression and speculate that the resultant no may be involved in neuronal killing. characteristic of such diseases is the progressive loss of a subset of neurons and there is evidence to implicate no as causal or, at least, contributory. in a proportion (20%) of cases of familial amyotrophic lateral sclerosis (als) there are multiple missense mutations in cytosolic cu/zn superoxide dismutase (sod) and it is proposed that these result in either a gain of function, or increase an injurious reaction catalyzed by sod. for example, crow and beckman (1995) suggest that there is enhanced nitration by no of neurofilament protein. evidence for nitration being causal in als comes from the observation that nitration blocks phosphorylation of tyrosine residues and that nitrotyrosine resembles the strongly antigenic dinitrophenol, perhaps provoking an autoimmune reaction. demyelinating diseases are characterized by preferential damage to myelin, with relative preservation of axons. multiple sclerosis (ms) and animal models such as experimental autoimmune encephalomyelitis (eae) are inflammatory demyelinating diseases characterized by early damage to the blood-brain barrier. lymphocyte and monocyte infiltration occurs, as well as activation of indigenous glia. what produces the resulting myelin damage is unknown and both indigenous as well as infiltrating cell types may contribute to myelin destruction, either directly or indirectly by secretion of proinflammatory cytokines or no (merrill and benveniste, 1996) . a number of recent reports confirm a suggested role for nos-2 in demyelinating disease. using a spin trap method in vivo hooper et al. (1995) could detect no in the cns of eae animals, 257 which correlated with the onset of neurological symptoms. increased no production occurs in ms as illustrated by elevated levels of nitrite and nitrate levels in csf (johnson et al., 1995) . using immunocytochemistry, nos-2 and nitrotyrosine have been found in diseased brain (okuda et al., 1995; hooper et al., 1995; bagasra et al., 1995; degroot et al., 1997; merrill et al., 1998) . both the protein and the transcript for nos-2 colocalize with markers for macrophages/microglia (bagasra et al., 1995; hooper et al., 1997; van der veen, 1997) and/or astrocytes (okuda et al., 1997; tran et al., 1997; merrill et al.. 1998) . cross et al. (1996) showed that nos-2 transcript and enzyme activity correlate with active eae. it is of interest that ms patients have elevated circulating antibodies to snitrosocysteine, suggesting nitrosylation of proteins in vivo (boullerne et al., 1995) . akin to the human situation with ms, ding et al. (1997) find gender-relatedness in nos-2 expression and no production in mice with eae. based on the evidence for no production in ms and eae, several studies have assessed roles for no using nos inhibitors. the results have been mixed. in one study of acute eae inhibitors of nos provided no benefit (zielasek et al., 1995) . other eae studies showed a small rise in clinical score (ruuls et al., 1996) or a decrease in disease incidence, severity and duration (cross et al., 1994; zhao et al., 1996) . most recently, hooper et al. (1997) report significant therapeutic effects on eae with nos inhibitors, and scavengers of no and peroxynitrite. the current therapeutic available for the treatment of ms is ifnfi, demonstrated to be a blocker of nos-2 expression (stewart et al., 1997) . hall et al. (1997) suggest that it is beneficial through its antagonism of endogenously produced ifny, hence impairing the activation of macrophages and microglia. treatment of eae animals with anti-inflammatory cytokines such as il10, another nos-2 modulator, inhibits disease (willenborg et al., 1995) . however, using the eae model in nos-2 gene deficient mice, fenyk-melody et al. (1998) report that disease is more severe and prolonged. this might suggest either that no is immunosup-pressive at the time of immunization or that, without no, there is a loss ofthe normal regulation of infiltration of inflammatory cells into the cns (de caterina et al., 1995; hickey et al., 1997) . elucidating the sequence of physiological and biochemical events following cerebral ischemia, in an attempt to identify means to reduce the amount of damage that develops, has been the focus of extensive investigation. the early events that occur following blood flow reduction, leading to cell death, have been well characterized in animal models of cerebral ischemia (siesjo, 1992) . and glial cell death such as inflammation, gene activation, and free radical production (for a review see iadecola, 1997) . while the pathogenesis of ischemic brain injury is probably the result of the activation of multiple biochemical pathways, it is evident, given the chain of events that occur, that no has the potential to participate in the mechanisms of cerebral ischemia. there is sufficient evidence that no is produced following both global and focal cerebral ischemia. using techniques such as in vivo spin trapping (sato et al., 1994) , porphyrinic microsensor measurement (malinski et al., 1993) , and measurements of brain nitrite and cgmp levels as a reflection of increased no production (kader et al., 1993; shibata et al., 1996) , increased generation of no in the brain following cerebral ischemia has been demonstrated. transient global ischemia is seen clinically associated with disorders of systemic circulation, such as 258 heart failure or other cardiac dysfunction. blood flow to the brain may be reduced below levels of autoregulation or may be completely interrupted for a period of time. global ischemia induces a gliotic reaction, and delayed neuronal necrosis occurs in selectively vulnerable cells such as pyramidal neurons in the ca1 subfield of the hippocampus. nos-2 protein expression has been shown to occur in a rat model of transient global ischemia (endoh et al., 1994) . three days after a 10 min ischemic episode, nos-2 protein was detected in the cai region of the hippocampus by immunohistochemistry, with immunoreactivity progressively increasing to day 30. double-labeling experiments identified these nos-2-expressing cells as reactive astrocytes, and ruled out microglial expression. in our own studies with spontaneously hypertensive rats, we can detect nos-2 protein by 72 h of reperfusion following 30 min of global ischemia. these cells morphologically resemble astrocytes in the corpus callosum, basal ganglia, and the hippocampus (fig. 1) . in response to global ischemia, nos-2 induction parallels, spatially and temporally, the gliotic reaction. the induction of nos-2 appears to be restricted to astrocytes in regions of neuronal damage, as there is no evidence for nos-2 expression in other cell types or other regions. the mechanism of delayed nos-2 induction, and the role of no generated by this isoform following global ischemia remains to be determined. to date, no studies have been performed investigating global ischemic damage in mice deficient in nos-2 expression. this may be due to the difficulties associated with mice not surviving the ischemic episode (yang et al., 1997) , or it may be due to the complications of strain variability in global ischemic outcome (fujii et al., 1997) . focal cerebral ischemia, or stroke, occurs as a regional injury and results from the prolonged interruption of blood flow due to the occlusion of a single cns artery. a focal vascular occlusion fig. 1 . expression of nos-2 in astrocytes following global isrhrmiu. in tissue sections stained with hematoxylin and eosin. neuronal cell death can be seen in the cai region of the rat hippocampus three days following 30 min of transient global ischemia (a, b) . astrogliosis, indicated by an increase in gfap immunoreactivity. is also evident three days after the insult (c, d) . immunohistochemistry reveals nos-2 protein expressed after three days in cells morphologically resembling astrocytes (e, f) . monoclonal gfap antibody (sigma, 1:200) and polyclonal n o s 2 antibody (transduction labs., 1:loo) labelling was detected using standard immunoperoxidase techniques with 3, 3'diaminobezidine as substrate. all sections were counterstained with hematoxylin and eosin. magnfication: a, 4 0~; b. 2 0 0~; c, 4 0 0~; d-f, 6 0 0~. several studies have provided biochemical, molecular, and immuno-histochemical evidence of nos-2 induction following focal cerebral ischemia. two separate reports demonstrated the induction of nos-2 activity following permanent mca occlusion in rats (yoshida et al., 1995; iadecola 1995a) . both studies found an increase in nos-2 activity at 2-3 days post-occlusion, with a return to baseline by seven days. the time course of nos-2 induction suggests that no generated from this isoform does not contribute to the early stages of ischemic injury. iadecola et al. (1995~; have also investigated nos-2 mrna and protein expression in response to focal ischemia. following permanent mca occlusion in rats, nos-2 mrna was detected by 12 h, peaked at 48 h and returned to baseline by seven days post-occlusion. immunoreactivity for nos-2, observed at 48-96 hours after ischemia, was detected only in neutrophils invading the infarct and was not seen in resident cells (iadecola et al., 1996) . transient 260 focal ischemia in rats also leads to nos-2 induction, though with different spatial and temporal patterns of expression than seen following permanent occlusion. two hours of mca occlusion, followed by reperfusion, resulted in maximal expression of nos2 mrna at an earlier time point, and detection of protein predominantly in vascular cells, rather than in neutrophils (iadecola et al., 1996) . in mice, permanent mca occlusion using the filament model of zealonga et al. (1989) leads to the induction of nos-2 mrna and protein expression. using in situ hybridization, we were able to detect nos-2 message at 24, 48 and 72 h (latest point studied), but not at 6 h post-occlusion ( fig. 2) . at 24 hours after ischemia, nos-2 immunoreactivity was observed primarily in what appeared, morphologically, to be inflammatory cells within the infarct (figure 2) . however, at 48 and 72 h nos-2 immunoreactivity was also observed in cells morphologically resembling astrocytes, as well as by gfap staining on adjacent sections (fig. 2) . these nos-2 positive cells were located primarily in regions surrounding the infarct. in contrast to previously published reports, these data indicate that resident glial cells, in addition to infiltrating cells, express nos-2 in the ischemic brain. the differences in our findings compared to previous reports may reflect the differences in total infarct volumes produced as a result of using different surgical procedures (cauterization versus filament occlusion). the infarct volume generated with the filament model of mca occlusion was typically greater than 80 mm3 (loihl and while infarcts produced by cauterization were generally smaller ( < 40 mm3), and predominantly cortical (iadecola et al., 1997) . while it is clear that no participates in the mechanisms of ischemia, much of the early work investigating the precise role of no yielded contradictory results. this controversy stemmed largely from the fact that much of the research being b fig. 2 . expression of nos-2 revealed by in situ hybridization and imniunohistochemistry at 48 aitd 72 h following middle cerebral artery occlusion in mice. nos-2 mrnas, detected with a 35slabelled crna probe (grzybicki et al., 1997) , was observed at 48 and 72 h following permanent mca occlusion (a, b) . in what appear to be infiltrating inflammatory cells within the infarct (c). nos-2 immunoreactivity at 72 h post-occlusion was also seen in cells morphologically resembling astrocytes located at the border of the infarct (d). gfap immunoreactivity reveals astrocytes located at the infarct border at 48 h post-occlusion (e). nos-2 immunoreactivity at 48 h post-occlusion, in an adjeacent section to that shown in e, in cells resembling astrocytes (f) . see legend to fig. 1 for details of methods. magnification: a, b, 200x; 400x. done involved the use of pharmacological inhibitors that, while somewhat selective, are not actually specific for a particular isoform of nos. experiments in vivo have shown that inhibition of no synthesis ameliorates (nowicki et al., 1991; buisson et al., 1992; nagafugi et al., 1992; ashwal et al., 1993; iadecola et al., 1995b iadecola et al., , 1996 zhang et al., 1996) and worsens (yamamoto et al., 1992; dawson et al., 1992; kuluz et al., 1993) tissue damage in animal models of cerebral ischemia. given the scope of potential biological actions of no, and the different spatial and temporal patterns of expression of the nos isoforms, it is likely that no generated by the different isoforms is subserving different roles at different times following ischemic insult. therefore, non-specifically blocking nos activity overshadows the potential for multiple roles of no in ischemia. to circumvent the limitations of the currently available pharmacological inhibitors, and to elucidate the contributions of the different isoforms of nos, ischemic brain damage has been investigated in genetically altered mice, which are deficient in a particular isoform of nos. evidence provided in the last few years using gene targeting strategies has resolved the debate over the roles of the constitutive isoforms of nos in the pathophysiology of focal cerebral ischemia (iadecola, 1997) . infarct volume, resulting from mca occlusion, in mice deficient for nos-1 is reduced compared to wild type mice. infarct 26 i 262 volume, however, in these mutant mice increased following administration of nitro-l-arginine, a nos inhibitor, presumably due to inhibition of nos-3 (huang et al., 1994) . in mice deficient for nos-3, infarct volumes were larger than in wild type littermates. these results indicate that no derived from nos-1 acts early following cerebral ischemia and contributes to the development of tissue damage, while no generated from nos-3 serves a protective function, most likely by regulating blood flow to the ischemic penumbra. more recently, evidence has been provided that begins to elucidate the role of the nos-2 isoform in focal cerebral ischemia. a report by iadecola et al. (1997) demonstrated that 24 h following mca occlusion, infarct volumes in mice deficient for nos-2 did not vary from infarct volumes in wildtype animals. we have found similar results in our own studies (loihl and . this is consistent with the idea, suggested by enzyme activity studies, that nos-2 does not contribute to the early stages of ischemic damage, and is not particularly surprising given the time course of nos-2 induction. iadecola et al. (1997) (wang et al., 1995; krupinski et al., 1996) . extracellular glutamate and atp are elevated and persist for a few hours following cns injury and the activation of selective chemokine genes is an early event. specific chemokines are elevated following trauma (ghirnikar et al., 1996; grzybicki et al., 1998) , exposure to p-amyloid (meda et al., 1996) , with bacterial infection (spanaus et al., 1997) , in eae and ms (hulkower et al., 1993; karpus et al., 1995; miyagishi et al., 1995; ransohoff et al., 1996) and in ischemia (kim et al., 1995; yamasaki et al., 1995 yamasaki et al., , 1997 takami et al., 1997; yoshimoto et al., 1997) . not only will chemokines such as il-8 and mcp-1 block nos-2 induction (mccall et al., 1992; rojas et al., 1993) but no can also inhibit the expression of il-8 and mcp-1 (andrew et al., 1995; zeiher et al, 1995; de caterina et al., 1995) . a delayed appearance of nos-2 might therefore ensure an environment in which chemokine gene expression can proceed and the entry of hematogenous cells is unhindered by no effects on endothelial adhesion molecule expression (khan et al., 1996; tsao et al., 1996; hickey et al., 1997) . that the later production of no might then terminate infiltration of inflammatory cells into the parenchyma, or downregulate expression of pro-inflammatory cytokines, are intriguing possibilities. together with its general role as a vasodilator, thus improving perfusion in compromised tissue and leading to remodelling, no may therefore prove beneficial by limiting the inflammatory response. from functional and genetic studies, it seems that the effects of no from nos-2 can either worsen or improve recovery from damage. evidence from the various enzyme inhibition studies is mixed, fraught with problems regarding specificity. evidence from studies with nos-2 gene-deficient mice is emerging to suggest that the presence of the enzyme can be beneficial, deleterious or without consequence (nathan 1997) . in cerebral ischemia, while nos-2 clearly does not contribute to pathology within the first 24 h (iadecola et al., 1997; loihl and murphy, 1997) , gene-deficient mice are reported to have smaller infarcts at 4 days (iadecola et al., 1997) . such nos-2 deficient animals have a greater leukocyte response in endotoxemia (hickey et al., 1997) which might explain why the condition of such mice with eae is made worse (fenyk-melody et al., 1998) . the interpretation of results from gene-deficient mice is itself not without problems because of the possibility of developmental compensation and the real potential for expression of a truncated protein, perhaps with novel or unregulated functions. however, and in the absence of truly selective enzyme inhibitors, observing neuropathological outcome in experimental models employing such nos-2 deficient mice is a powerful indicator of the roles of nos-2. verification could then be sought by virally re-introducing the gene in deficient animals. it would also be informative to observe pathological outcomes in transgenic mice in which nos-2 can be overexpressed. however, there are no reports of the generation of such animals to date. immunologic no synthase: elevation in severe aids dementia and induction by hiv-i gp41 nitric oxide regulates 1l-8 expression in melanoma cells at the transcriptional level no synthase i1 is transiently expressed in embryonic mouse olfactroy receptor neurions low dose l-name reduces infarct volume in the rat mca/ 0 reperfusion model activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis microglial activation by alzheiiner aniyloid precursor protein and modulation by apolipoprotein e inhibition of nitric oxide synthase attenuates blood-brain barrier disruption during experimental meningitis expression of no synthase in cerebral endothelial cells is regulated by cytokineactivated astrocytes indirect evidence for nitric oxide involvement in multiple sclerosis by characterization of circulating antibodies directed against conjugated s-nitrosocysteine the neuroprotective effect of a nitric oxide inhibitor in a rat model of focal cerebral ischemia no, nitrosonium ions, nitroxide ions, nitrosothiols and irronitrosyls in biology exacerbation of lymphocytic choriomeningitis in mice treated with the inducible nitric oxide synthase inhibitor aminoguanidine nitric oxide in excitable tissues induction of nitric oxide synthase mrna expression tnf and no production by retinal muller glial cells from rats exhibiting inherited retinal dystrophy nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion interleukin-8 inhibits the induction of nitric oxide synthase in rat peritoneal neutrophils p-amyloid (25-35) peptide and ifn-y synergistically induce the production of the chemotactic cytokine mcp-i/ je in monocytes and microglial cells ) inos and nitrotyrosine in macrophages and glia in demyelinating lesions regulation of gene expression in the nervous system by reactive oxygen and nitrogen species cytokines in inflammatory brain lesions: helpful and harmful macrophage inflammatory protein-la in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological diseases glial no: normal and pathological roles synthesis of nitric oxide in cns glial cells blockade of nitric oxide formation by nw-nitro-l-arginine mitigates ischemic brain edema and subsequent cerebral infarction in rats inducible nitric oxide synthase nitric oxide mediates neuronal death after focal cerebral ischemia in the mouse expression of the inducible isoform of nitric oxide synthase in the central nervous system of mice correlates with the severity of actively induced experimental allergic encephalomyelitis nitric oxide via an inducible isoform of nitirc oxide synthase is a spossible factor to eliminate inflammatory cells from the cns of mice with eae inducible nitric oxide synthase in theiler's murine encephalomyelitis virus infection duration of expression of inducible nitric oxide synthase in glial cells nitric oxide synthase type i1 mrna stability is translation-and transcription-dependent nitric oxide limits transcriptional induction of nitric oxide synthase in cns glial cells nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting nf-kb binding to dna induction and stabilization of ikbn by nitric oxide mediates inhbition of nf-kb chemokines in immune-mediated inflammation of the central nervous system mcp-1 inhibits the induction of nitric oxide synthase in 5774 cells synetgistic induction of nitric oxide by /l-amyloid and cytokines in astrocytes aggravation of experimental allergic encephalomyelitis by administration of nitric oxide synthase inhibitors role of nitric oxide in brain ischemia activated astrocytes induce nitric oxide synthase-2 in cerebral endothelium via tnfa brain nitrite production during global ischemia and reperfusion pathophysiology and treatment of focal cerebral ischemia c-x-c and c-c chemoklines are expressed in the csf in bacterial meningitis and mediate chemotactic activity on peripheral blood-derived polymorphonclear and mononuclear cells in vitro characterization and functional analysis of the human inducible nitric oxide synthaser gene promoter pretreatment of astrocytes with interferon c t / j impairs interferon y induction of nitric oxide synthase activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus induction of mip-la mrna on glial cells after focal ischemia in the rat neuronal (type i) nitric oxide synthase regulates nuclear factor kb activity and immunologic (type 11) nitric oxide synthase expression astrocytes and microglia exporess inducible nitric oxide synthase in mice with eae extensive peroxynitrite actvivity during progrcssive stages of cns inflammation inducible nitric oxide synthase in tangle-hearing neurons of patients with alzheimer's disease transforming growth factor j l exhibits delayed gene expression following focal cerebral ischemia fibrillar j-amyloid induces microglial phagocytosis expression of inducible nitric oxide synthase and loss of a select population of neurons in the rat cns in vivo cytokines and murine autoimmune encephalomyelitis: inhibition or enhancement of disease with antibodies to select cytokines, or by delivery of exogenous cytokines using a recombinant vaccinia virus system inducible nitric oxide synthase gene expression in the lett brain during systemic inflammation the high output nitric oxide pathway: role and regulation transient increase of cytokine-induced neutrophil chemoattractant, a member of the interleukin-s family new therapeutic possibility of blocking cytokine-induced neurtro-phi1 chenioattrdctant on tranisent ischemic brain damage in rats c57bl/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains 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(1995) altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. cell, key: cord-303741-1ou0cy5k authors: stafstrom, carl e.; jantzie, lauren l. title: covid-19: neurological considerations in neonates and children date: 2020-09-10 journal: children (basel) doi: 10.3390/children7090133 sha: doc_id: 303741 cord_uid: 1ou0cy5k the ongoing worldwide pandemic of the novel human coronavirus sars-cov-2 and the ensuing disease, covid-19, has presented enormous and unprecedented challenges for all medical specialists. however, to date, children, especially neonates, have been relatively spared from the devastating consequences of this infection. neurologic involvement is being increasingly recognized among adults with covid-19, who can develop sensory deficits in smell and taste, delirium, encephalopathy, headaches, strokes, and peripheral nervous system disorders. among neonates and children, covid-19-associated neurological manifestations have been relatively rare, yet reports involving neurologic dysfunction in this age range are increasing. as discussed in this review, pediatric neurologists and other pediatric specialists should be alert to potential neurological involvement by this virus, which might have neuroinvasive capability and carry long-term neuropsychiatric and medical consequences. severe and at times fatal symptoms caused by the novel human coronavirus, severe acute respiratory syndrome (sars)-cov-2, and the associated coronavirus disease 2019 (covid19) , are ravaging the world. while symptoms of covid-19 are primarily pulmonary (fever, dry cough, fatigue, pneumonia), it is becoming increasingly recognized that multiple organ systems can be affected, including the brain, with neurological involvement affecting up to~36% of patients [1] [2] [3] [4] [5] . information gained from studies of related coronaviruses in recent epidemics of severe acute respiratory syndrome (sars, 2002) and middle east respiratory syndrome (mers, 2012) suggests that all three coronaviruses might have neurologic consequences [6, 7] , though the relative severity and frequency of neurologic involvement caused by coronaviruses varies and thus the extent to which sars and mers epidemics inform our understanding of covid-19 remains unclear [5] . nevertheless, the possibility has been raised that sars-cov-2 could invade the brain and cause neurological disease [2, 8] . while appealing conceptually, data supporting the idea that the sars-cov-2 virus can infect the peripheral and central nervous systems (pns, cns) are limited, as discussed below. table 1 lists definitions of relevant terms that are often used in the literature. neurotropic viruses vary in their invasiveness, virulence, and propensity to cause inflammation [9] . the purpose of this review is twofold: (1) to discuss the available data about covid-19 infections in neonates and children, and (2) to provide a perspective about potential neurologic involvement in neonates and children with covid-19 infections, in view of neurobiological development. a few points need clarification up front. first, data about the virus and its effects are accumulating rapidly and our understanding of its consequences will evolve over time. second, much of the literature about covid-19 currently exists as case reports or small series; obviously, the impact of such publications is limited, and greater understanding will emerge only as large, rigorous studies are published. third, we must be mindful that a positive test for sars-cov-2 in a patient with a neurological symptom does not necessarily imply that the virus caused the symptom. the covid-19 epidemic has escalated rapidly and spread widely across the globe, with cases continuing to accrue at an alarming rate. the first case was reported from wuhan, china in mid-december 2019 and three months later, in march 2020, the world health organization declared covid-19 a pandemic. as of this writing (late august, 2020), more than 23 million cases of covid19 have been documented worldwide (with many more mildly symptomatic cases likely not reported), with over 800,000 deaths, and more than 175,000 deaths in the united states alone (www.cdc.gov, accessed 24 august 2020). documentation of the numerous clinical presentations, manifestations, and disease course have proliferated in the medical literature. a pubmed search (23 july 2020) using the keyword covid-19 revealed an astounding number of published reports already (34, 310) , over the course of only a few months. of those citations, only 501 reports (1.5%) also included the keyword neonate, attesting to the low published incidence in newborns. when searching pubmed with the key terms covid-19, neonate and neurological or brain, fewer than 10 articles emerged. therefore, at least so far, covid-19 does not seem to be affecting neonates very often from a neurological point of view; pubmed counts probably underestimate the occurrence of neurological involvement in children, being biased toward areas of the world with greater medical resources. these observations do not preclude the potential for neonatal brain involvement in covid-19 nor exclude the possibility of long-term medical, neurodevelopmental, and psychosocial consequences of the disease. indeed, as time goes on, a wider spectrum of neurologic manifestations will likely emerge with as yet undetermined long-term sequelae. as the covid-19 pandemic continues, certain trends are becoming evident. first, while the number of cases and deaths continue to rise, the disease does not affect infants and children nearly as frequently as adults [9, 10] . to date, approximately 2-5% of cases of covid-19 involve children, who appear to be less severely affected than adults, mainly with pulmonary symptoms [11] [12] [13] . second, disease severity in children who develop covid-19 is usually milder than in adults, and children with severe disease often have an underlying co-morbidity such as immunosuppression [10, 14] . indeed, there is accumulating evidence that adults with covid-19 infection manifest with multiple organ system involvement, including the cns and pns, and that older and sicker individuals carry a higher risk for neurologic problems [1, 4, 15] . these age-dependent differences in disease expression and severity have clear implications for healthcare professionals who deal with the pediatric population because children remain at risk for incurring and spreading the virus, yet many remain asymptomatic. several hypotheses have been posited as to why children are less affected by covid-19, including age-related differences in immune responses [16] , a neutralizing antibody response due to prior exposure to coronaviruses [17] , lower prevalence of co-morbidities in children, and age-specific differences in sars-cov-2 receptor function [18] , neurovirulence, intrinsic biological protective mechanisms, and other host factors [19] . none of these hypotheses is supported compellingly by extant data at present. while the number of affected neonates and children remains small, pediatric practitioners cannot become complacent about the potential for neurologic involvement in covid-19. furthermore, the recently described multisystem inflammatory syndrome-children (mis-c), raises the specter that covid-19 or its after-effects also target children (see section 4) [20, 21] . as data from china, europe and other areas affected early by the covid-19 pandemic are reported, some patterns are emerging regarding pregnant women and neonates. first, it is clear that vertical transmission covid-19 from a pregnant mother to her fetus occurs quite rarely. more than a dozen publications attest to this observation, together encompassing over 100 patients (table 2 lists a few relevant publications, selected from the largest available series and omitting small series and single case reports). none of these reports documents unequivocal vertical transmission. in many of the babies, onset of symptoms occurred in the neonatal period but not immediately at birth, so the exact timing of infection remains uncertain. overall, the data does not support robust transplacental transfer of sars-cov-2, but recent case reports are providing proof-of-principle that the virus can be transmitted intrauterine from infected mother to fetus [22, 23] . an especially apropos case demonstrated maternal viremia, placental infection shown by immunohistochemistry, and high placental viral load with subsequent neonatal viremia, implying transplacental transfer of sars-cov-2 from pregnant mother to fetus [24] ; this newborn presented with neurological symptoms as discussed in section 3. of all the infants reported during the first month of life, most had documented exposure to affected family members [13, 32] which emphasizes the importance of controlling horizontal virus transmission from affected family members to the neonate [33] . while this trend may need revision [27, 34] , so far, it is encouraging that most covid-19-positive pregnant women do not transmit the disease to their unborn children. similarly, there is no evidence that covid-19-positive pregnant women incur covid-19 or develop more severe disease than similar-aged women who are not pregnant. however, the impact of chronic inflammation caused by maternal viral infection on fetal development, pregnancy outcomes and long-term neurodevelopment is unknown. similarly, the timing of maternal viral infection with respect to major milestones of in utero neurodevelopment (i.e., second trimester vs. late third trimester) is an unknown and critical consideration for further research and long-term neurodevelopmental followup. there is legitimate concern about the impact of acute and chronic stress during the pandemic (i.e., worry about the medical complications of sars-cov-2 infection, family disruption, job loss, economic pressures, educational uncertainties, food availability, etc.) on the pregnant patient and developing fetus [35] [36] [37] [38] . furthermore, it is reassuring that there is no definitive evidence that the virus is present or can be transmitted in the breast milk of covid-19-positive women [28, 39, 40] . the current american academy of pediatrics recommendation is that covid-19-positive mothers can breast feed directly while wearing a mask or feed expressed breast milk, using appropriate breast and hand hygiene [41] . extensive guidelines are available regarding principles of management of pregnant women with covid-19 and their newborns [40] [41] [42] . again, all of these observations are preliminary and subject to modification over time. although covid-19 primarily affects the pulmonary system, it is a multisystem infection (e.g., gastrointestinal tract, kidneys, liver, heart) and involvement of the pns and cns are increasingly recognized [43] [44] [45] [46] . data on neurological signs and symptoms are limited but increasing, with a wide spectrum of acute and chronic manifestations becoming apparent [47] . in a series of 214 hospitalized adults with covid-19, 88 of whom had "severe" infections, 36.4% of the entire group was reported to manifest some neurologic involvement, including alteration of consciousness, encephalopathy, headache, cerebrovascular disease, and skeletal muscle injury (myalgia, weakness) [1] . ischemic strokes, many affecting young adults with large vessel occlusions, have garnered considerable concern that the etiology may be a prothrombotic state caused by virus-induced inflammation of the vascular epithelium [48, 49] . many of the young adult stroke victims had other vascular risk factors such as diabetes or hypertension, which emphasizes the importance of comorbidities with systemic inflammatory conditions in disease manifestations and severity. stroke has not been reported in children with covid-19 [50] . the occurrence of encephalitis remains controversial, as virus has not been recovered from cerebrospinal fluid (csf) [51] and overall, a surprisingly small number of covid-19 patients develop classic encephalitic symptoms. autopsy studies are beginning to be published. the wide spectrum of postmortem findings include mostly secondary changes to the cns such as hypoxemia and ischemia, rarely localized perivascular and interstitial neuroglial activation with neuronal loss and axonal degeneration [52, 53] , and no other major cns abnormalities [54] . no pediatric autopsy cases have reported neuropathological involvement. clearly, more data are needed before this issue can be clarified. in the chinese series [1] , only 2 out of 214 patients had seizures (1%), which is not greater than the general population, so it is uncertain whether infected patients are at higher risk. the few patients with seizures are reported mainly in case reports [55, 56] . the lack of frequent seizures is rather curious, especially if encephalopathy is indeed a frequent complication of covid-19; this data may be related to sampling bias rather than actual non-occurrence. a few reports of seizures have appeared in adults using electroencephalography (eeg) [57, 58] , but a more concerted effort to evaluate the brain electrophysiology of children with covid-19 would be informative. the examples of seizures in children with covid-19 described in section 3.6 appear to be largely anecdotal. many additional cases are necessary to conclude whether there is increased seizure susceptibility in the pediatric population. other cns symptoms include headache, dizziness and delirium, all of which can occur as a nonspecific consequence of systemic infection or inflammation of the respiratory tract as well as via a cns mechanism. although headaches are reported frequently, the pain often appears to be nonspecific or associated with inflammation or migraine exacerbation rather than meningeal irritation [59] . the most commonly reported symptoms related to the pns are decreased taste (hypogeusia or ageusia) and smell (hyposmia or anosmia) [60, 61] . a neural mechanism is suspected for hyposmia in covid-19, because decreased smell is often the first symptom experienced and occurs in mild disease in the absence of significant local inflammation or mucosal congestion that are typical of the more benign coronavirus or non-coronavirus nasal infections [62] . a few adolescents with covid-19 have been reported with decreased taste or smell [63] ; these symptoms appear to be very uncommon in children but deserve a more concerted ascertainment effort. several cases of guillain-barré syndrome (gbs) have been reported in adults with covid-19, raising the possibility of post-infectious autoimmune responses against the pns [64, 65] . two case reports of children with gbs who developed covid-19 symptoms about 3 weeks later confirms that gbs can occur with covid-19, though this association remains quite rare given the widespread prevalence of covid-19 [66, 67] . finally, the possibility of central demyelination has been raised, e.g., in the form of multiple sclerosis (ms), among patients with covid-19 [68] ; this concern is relevant in that various disease-modifying agents used to treat ms could theoretically exacerbate ms symptoms [69] . fortunately, there is no evidence that covid-19 triggers central demyelinating disease in children [50] . the lack of unequivocal reports of sars-cov-2 being recovered from the csf of individuals affected with presumed neurological involvement nor in brain tissue from the limited number of autopsied cases strengthens the possibility that the virus does not often directly cause the symptoms but rather, that the neurological sequelae are secondary to hypoxia, cytokine involvement, or some other non-direct mechanism (see section 6). it is appropriately concerning that chronic neurologic diseases such as epilepsy, amyotrophic lateral sclerosis, multiple sclerosis, etc., might be exacerbated during concurrent covid-19 infection or that covid-19 may unmask preexisting cns pathology that might have been unrecognized or asymptomatic. as just discussed, neurologic involvement in children, and in particular neonates, with covid-19 appears to be scarce but may be under reported [70] . a few selected examples of case reports of neurologic involvement in neonates and children are presented in table 3 ; such case reports have limited generalizability, and many lack sufficient details to ascribe causality between sars-cov-2 and neurologic symptoms. most children were assumed to have contracted covid-19 from a family member and some children had concurrent infection with other viruses, confounding any argument for causality. importantly, csf was negative for sars-cov-2 in all children on whom spinal fluid was obtained. all children recovered within a few days or weeks, contrasting with the severe and prolonged courses in many adults. available evidence does not allow distinction between a direct effect of sars-cov-2 causing neurologic dysfunction, versus the symptoms instead being secondary to an over activated immune response (see section 5) . in summary, case reports of neurological involvement in babies and children are rare but accumulating, and the recovery of most infants with early neurologic symptoms implicates some virus-or host-related factors that minimize massive neurological devastation. this newly recognized kawasaki syndrome-like hyperinflammatory disorder presents with acute hypotension and cardiogenic shock and is proliferating across the globe. it is likely a post-infectious syndrome or inflammatory reaction following asymptomatic or mildly symptomatic covid-19 [76] . children can develop toxic shock-like symptoms, hypoxia-ischemia, and significant end organ damage to the heart, kidneys, and other organs. while definitive data is not available, there is concern that the inflammation that hallmarks mis-c may have adverse consequences on the developing brain. while no consistent neurologic picture has emerged, several mis-c patients have had cns involvement as part of their course. in a small series of 6 children with mis-c, 4 patients had neurologic symptoms, including headache, altered mental status, and aseptic meningitis [77] . headache was the most common symptom in a series of 58 children with mis-c associated with sars-cov-2, affecting 26% of patients [78] . a series 21 children from france notes that 57% of patients were "irritable" and another 29% had "other neurological features", though these were not specified [79] . in a larger survey of 186 children, 5-11% had neurologic involvement, depending on age, including encephalitis, seizures or mental status alteration, but details are not provided [80] . finally, 4 of 27 children with covid-19 associated mis-c developed new neurologic symptoms including encephalopathy, headache, weakness, ataxia, and dysarthria [81] ; two patients had lumbar punctures and csf was negative for sars-cov-2 in both. three of the four patients had an eeg; each showed diffuse slowing. brain mri scans of all four children showed abnormal signal intensities of the splenium of the corpus callosum (a finding seen in previous cases of encephalopathy and thought to indicate inflammation-induced focal myelin edema [81, 82] . a recent systematic review of eight studies notes neurological symptoms in~25-50% of children with mis-c, depending upon inclusion criteria [83] . a putative impact on the immature cns and developing immune system, including neural-immune maturation, cannot be overlooked, and the long-term neurologic impact of both covid-19 and mis-c on the developing brain need urgent elucidation. sars-cov-2 is a highly contagious and pathogenic rna virus that is transmitted via droplet, contact, or aerosol routes. the virus gains entry into epithelia of the pulmonary system (upper or lower respiratory tracts), digestive tract, or nasopharynx. the virus is composed of single-stranded rna enveloped by surface proteins (s, e, m, n). the spike (s) glycoprotein serves as the attachment site onto angiotensin converting enzyme type 2 (ace-2) receptors on epithelial membranes. the normal function of ace-2 receptors is to provide protection against pulmonary and endothelial injury [84] . sars and sars-cov-2 share~79% genome sequence identity and both viruses infect epithelium by the binding of spike proteins to ace-2 receptors. while most prevalent on airway and pulmonary epithelium [85] , ace-2 receptors are also reportedly present to a lesser degree on neurons and perhaps glia [2] . binding of the s protein to ace-2 receptors leads to proteolytic cleavage of s by the transmembrane protease tmprss2 [5] . viral rna then enters the epithelial cell and replicates rapidly, translating viral proteins and inducing a host immune response. this immune response can be adaptive, attacking and inactivating the virus. by contrast, the immune response can be maladaptive and induce a massive immune reaction, accompanied by a hyper-inflammatory response hallmarked by excessive cytokine secretion and signal transduction (cytokine storm), and robust cellular immune activation and recruitment [86] . the large-scale cytokine storm consists of a massive release of pro-inflammatory humoral agents such as interleukin-6 (il-6), interferon gamma (ifn-y), mcp-1/ccl2 (monocyte chemoattractant protein 1/chemokine ligand 2), il-1, il-12, il-8, tnfα (tumor necrosis factor alpha), and cxcl 10 (c-x-c motif chemokine ligand 10) that exacerbate the underlying pathophysiology [87, 88] . this cytokine release subsequently feeds forward an overactive and dysregulated cellular immune response defined by macrophage, monocyte, neutrophil, and t-cell hyperactivation and recruitment [89] . the impact of this systemic cytokine storm on neurodevelopment is under investigation in preclinical models [90] and should be the focus of future prospective studies. subsequently, the replicated viruses exit the cell, leading to further infection. it is unknown why children, and neonates in particular, seem to be relatively resistant to covid-19 and its severe symptoms, including neurological manifestations. the cytokine response to coronavirus infection appears to be less robust in young children although the recognition of mis-c may suggest host-dependent genetic susceptibility to enhanced cytokine and/or inflammatory responses [91] , but other mechanisms are also plausible [19] . it remains controversial whether ace-2 inhibitors would provide symptomatic relief or prevent the covid-19 disease, and evidence for the effectiveness of these agents in children and neonates is not yet available [92] . the cellular and molecular basis of sars-cov-2 neurotropism, neuroinvasiveness, and neurovirulence are poorly understood [9] : does the virus get into the brain and if so how, and what does it do in the cns once there (e.g., infects neural cells? causes disease?). neurological involvement in covid-19 might be associated with at least four potential mechanisms: 1. a direct neurotropic or neuroinvasive effect of sars-cov-2 (e.g., anosmia, encephalopathy), 2. a secondary effect of the systemic inflammatory responses triggered by the viral infection (e.g., encephalopathy), 3. a secondary effect associated with the vascular and prothrombotic effect of the viral infection on the cns or pns vasculature (e.g., strokes, necrotizing leukoencephalopathy), 4. an immune-mediated para-infectious or post-infectious autoimmune effect in response to the viral infection (e.g., gbs, acute disseminated encephalomyelitis). figure 1 summarizes, in schematic fashion, some hypothetical possibilities about how the virus may infect the brain directly, whether the neurological symptoms and signs may be related to systemic or hyperactivation of immune responses, or both [87] . it is important to consider the mechanisms associated with neurological manifestations of covid-19, with an aim toward developing therapeutic options. the possibilities of direct neurotropism and hyper-responsiveness to immune activation (cytokine storm) are considered separately below, though these mechanisms might work synergistically. the sars-cov-2 virus attaches to olfactory epithelium using the ace-2 receptor. after cell entry, the virus replicates and induces a massive immune response leading to excessive cytokine release, comprising a maladaptive immune response. theoretically, virus particles may reach the cns retrogradely via cranial nerve pathways: v from corneal epithelium or oropharyngeal cutaneous sensory receptors; i via the cribiform plate, infecting olfactory sensory neurons; vii and ix from tongue chemoreceptors; x via pulmonary mechanoreceptors. once reaching cns nuclei including brainstem and cortex, a variety of neurologic signs and symptoms are possible. however, it must be noted that the virus has not been recovered from csf or brain tissue, making all of these pathways hypothetical at this point. abbreviations: np, nasopharynx; gi, gastrointestinal; ace-2, angiotensin converting enzyme type 2 receptor; pns, peripheral nervous system; cns, central nervous system; ich, intracranial hemorrhage; gbs, guillain-barre syndrome; bbb, blood-brain barrier. definitive demonstration of direct viral invasion would require a positive csf reverse transcriptase-polymerase chain reaction (rt-pcr) for sars-cov-2, recovery of infective virus from the csf as demonstrated by viral cultures or "plaque assay" [93] , intrathecal synthesis of antibodies to sars-cov-2, or autopsy-demonstrated sars-cov-2 antigen or rna in brain tissue [5] . current published evidence meeting these strict criteria is minimal. while it is plausible that the virus infects the brain through one of the anatomical pathways discussed below, the lack of viral recovery from the cns gives pause to that notion. neuroinvasion has been demonstrated for the related sars and mers viruses [94] , but sars-cov-2 has not been recovered from the csf or brain tissue. animal models of sars and mers have shown that the virus can enter through epithelium of the nasopharynx and travel retrogradely to the cns [95, 96] . interestingly, wild type mice are not vulnerable to infection and disease by human coronaviruses, but transgenic mice with human ace-2 receptors do develop respiratory and neurological symptoms when infected [95, 97] . in such transgenic mice, intranasal exposure to sars or mers leads to brain infection. one of the proposed portals of entry is via olfactory sensory neurons, crossing the cribiform plate into the olfactory bulb, with subsequent retrograde travel along the olfactory nerve (cranial nerve i) to the brainstem, thalamus, and basal ganglia, all areas that are connected to the olfactory cortex. please note that it has yet to be proven that sars-cov-2 infects olfactory sensory neurons. emerging animal models may clarify whether sars-cov-2 is similarly neuroinvasive as sars and whether this isage dependent the sars-cov-2 virus attaches to olfactory epithelium using the ace-2 receptor. after cell entry, the virus replicates and induces a massive immune response leading to excessive cytokine release, comprising a maladaptive immune response. theoretically, virus particles may reach the cns retrogradely via cranial nerve pathways: v from corneal epithelium or oropharyngeal cutaneous sensory receptors; i via the cribiform plate, infecting olfactory sensory neurons; vii and ix from tongue chemoreceptors; x via pulmonary mechanoreceptors. once reaching cns nuclei including brainstem and cortex, a variety of neurologic signs and symptoms are possible. however, it must be noted that the virus has not been recovered from csf or brain tissue, making all of these pathways hypothetical at this point. abbreviations: np, nasopharynx; gi, gastrointestinal; ace-2, angiotensin converting enzyme type 2 receptor; pns, peripheral nervous system; cns, central nervous system; ich, intracranial hemorrhage; gbs, guillain-barre syndrome; bbb, blood-brain barrier. definitive demonstration of direct viral invasion would require a positive csf reverse transcriptase-polymerase chain reaction (rt-pcr) for sars-cov-2, recovery of infective virus from the csf as demonstrated by viral cultures or "plaque assay" [93] , intrathecal synthesis of antibodies to sars-cov-2, or autopsy-demonstrated sars-cov-2 antigen or rna in brain tissue [5] . current published evidence meeting these strict criteria is minimal. while it is plausible that the virus infects the brain through one of the anatomical pathways discussed below, the lack of viral recovery from the cns gives pause to that notion. neuroinvasion has been demonstrated for the related sars and mers viruses [94] , but sars-cov-2 has not been recovered from the csf or brain tissue. animal models of sars and mers have shown that the virus can enter through epithelium of the nasopharynx and travel retrogradely to the cns [95, 96] . interestingly, wild type mice are not vulnerable to infection and disease by human coronaviruses, but transgenic mice with human ace-2 receptors do develop respiratory and neurological symptoms when infected [95, 97] . in such transgenic mice, intranasal exposure to sars or mers leads to brain infection. one of the proposed portals of entry is via olfactory sensory neurons, crossing the cribiform plate into the olfactory bulb, with subsequent retrograde travel along the olfactory nerve (cranial nerve i) to the brainstem, thalamus, and basal ganglia, all areas that are connected to the olfactory cortex. please note that it has yet to be proven that sars-cov-2 infects olfactory sensory neurons. emerging animal models may clarify whether sars-cov-2 is similarly neuroinvasive as sars and whether this isage dependent [97, 98] . however, since mice are not naturally susceptible to the clinical and immunopathological manifestations of coronaviruses affecting humans, translational studies of pathogenic mechanisms and vaccine development become complicated. extensive efforts to modify mice with transgenic approaches have begun to provide informative models. as mentioned, the olfactory epithelium has been touted as a potential site of viral entry into the brain, and hence explain hyposmia [99] .detailed genetic and immunohistochemical examinations of cell types of the olfactory system reveal that ace-2 and tmprss2 are present on olfactory epithelial cells (especially supporting or "sustenacular" cells) but not on olfactory sensory neurons themselves [54, 85, 100] . moreover, there is some evidence of virus-induced cell death in other coronavirus infections but not yet for sars-cov-2 [84, 101] . likewise, the virus might enter via the sensory system of the tongue that mediates taste, with transmission via cranial nerves vii, ix, and x to the nucleus tractus solitarius, thalamus and eventually, brain. finally, trigeminal nociceptors via cranial nerve v from either the corneal epithelium or buccal epithelium could theoretically reach the cns. these potential pathways could explain the symptoms of hypogeusia and altered vision. however, sars-cov-2 has not been recovered from the brain. transynaptic transport from lower respiratory tract mechano-and chemoreceptors to the brainstem medullary cardiorespiratory centers has been proposed as a hypothetical mechanism that could exacerbate brainstem dysfunction and perhaps even worsen respiratory effort [102] ; however, this hypothesis lacks objective validation and remains controversial. other potential routes for virus to enter the cns are through the bloodstream (hematogenous) or via disruption of the blood brain barrier (bbb). from the systemic circulation, the virus might travel to the cerebral circulation where it can damage capillary epithelium and access the brain. interestingly, there is scant evidence that sars-cov-2 produces a significant or sustained viremia [103] . the bbb is essential for transport of molecules into the brain and exclusion of pathogens and overall maintenance of cerebral homeostasis [104] . the bbb is a dynamic structure, consisting of several cell types and proteins, each with its own maturational profile-astrocyte foot processes, pericytes, tight junction proteins, and extracellular matrix, providing structural and functional support. virus attachment to ace-2 receptors at the bbb might facilitate trafficking of the virus into the cns, facilitating endothelial damage and edema [89] . notably, while the bbb is structurally complete at birth and is sufficiently functional in the neonate to provide protection against many pathogens, its full physiological maturation may take several months [105] . in the context of covid-19 infection, the bbb may be dysfunctional, disrupted either by inflammatory response or the virus itself, allowing transmission of the virus or activated immune cells from the circulation into the cns [8, 84] . the release of inflammatory cytokines by activated glia and neural mast cells exacerbate the inflammation [89] . similarly, flow of the virus through lymphatic channels of the interstitial space of the brain could breach the blood-csf barrier and permit virus entry [106] . to date, there is no evidence for the presence of the virus in pathological specimens of the pns or cns, in part due to the dearth of comprehensive autopsies [52] [53] [54] . obviously, patient care has focused on critical pulmonary and life-support measures so neuropathologically-focused autopsy studies have been uncommon. animal studies of covid-19 will be crucial to complement information gained from prior studies of the other coronaviruses. such animal models will provide more information about mechanisms of virus entry into the nervous system and how the virus affects neural function, neural-immune maturation and neurodevelopment, as well as the critical and yet unanswered question of long-term neurological sequelae of covid-19 [107] . that is, if there is predilection of the virus for certain neural structures or chronic neuroinflammation, long-term consequences may arise in various neural functions such as learning, memory, cognition, seizure predisposition, and other functions. all of this is speculative at present. another essential question, alluded to above, is whether cns disease contributes to the respiratory failure seen in covid-19 patients. ongoing or severe hypoxia can exacerbate ongoing symptoms in other organs. in particular, cns respiratory control centers in the brain stem, nearby the vagus nerve, has been speculated to play a role in respiratory failure [102, 108] . at present, there is some reason for guarded optimism for young patients within the devastating covid-19 pandemic. children, particularly neonates, are less likely to become infected and develop severe symptoms, and their propensity to spread the virus is controversial [109] . there is at best slight evidence for vertical transmission of sars-cov-2 or covid-19 disease from pregnant mother to fetus; rather, neonates are more likely incur the disease by exposure to affected individuals postnatally, and breast milk transmission has not been shown (table 4) . a variety of practical guidelines have been developed for the care of pregnant women who have or are suspected to have covid-19 positivity. analogous guidelines for the care of adult covid-19 patients with neurologic problems are also available and need to be developed for children [110] . table 4 . summary of covid-19 infections in children. severe infection caused by the novel coronavirus, sars-cov-2, has predominant pulmonary involvement but can also affect multiple other organ systems, including the cns and pns. symptoms are less frequent and usually less severe in children and particularly in neonates. vertical transmission of sars-cov-2 from pregnant mother to fetus is rare but anecdotal case reports support this possibility. most cases of covid-19 in early life are due to exposures to infected patients (horizontal transmission). there is no reported transmission of sars-cov-2 via breast milk. regarding neurologic involvement in covid-19, there are plausible mechanisms by which the virus can gain entry into the cns and subsequently incur acute neurologic symptoms, either directly or through immune dysfunction ( table 5 ). the occurrence of long-term medical and neuropsychiatric sequelae is unknown. children can be resilient and yet remain vulnerable to coping with the challenges of covid-19 in the context of other acute and chronic diseases. youngsters may not understand the need for social distancing, prolonged quarantine, and other preventative measures, and it is anticipated that stress-related post-traumatic symptoms will develop in some young people, whether or not they actually acquire symptoms. in children with comorbid chronic conditions and developmental disabilities, the challenges are even more profound. therefore, neuropsychological surveillance and studies of the long-lasting effects of this pandemic on neurodevelopment are critical [111] . finally, the emergence of the hyper-inflammatory multisystem syndrome (mis-c) supplants any conclusion that covid-19 is benign or negligible in the pediatric age range. therefore, it behooves neurologists and other pediatric specialists who deal with neonates and young children to be aware of the potential neurologic involvement of this novel, potentially devastating virus. future animal models should evaluate the impact of sars-cov-2 on maternal infection, inflammatory signal transduction through the maternal-placental-fetal axis, and brain development. the importance of large-scale immunization should a vaccine become available, cannot be over emphasized as should the role of systemic inflammation, neuroinflammation, and neural-immune interactions in novel pathophysiology and symptomology. additionally, mechanism-specific 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respiratory breakdown in covid-19 patients covid-19 super-spreaders: definitional quandaries and implications child neurology society; in collaboration with the neurocritical care society ethics committee aan position statement neurotropic mechanisms in covid-19 and their potential influence on neuropsychological outcomes in children this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license research in the laboratory of stafstrom is supported by the mathias koch memorial fund, the sandra and malcolm berman foundation, and the paine foundation. research in the laboratory of jantzie is supported by the national institutes of health (1r01hl139492) and the department of defense (w81xwh-18-1-0167) . we thank carlos pardo-villamizar for his insightful comments and critique of the manuscript. the authors declare no conflict of interest. key: cord-005734-14ba78cz authors: bennett, jami l.; elhofy, adam; dal canto, mauro c.; tani, mari; ransohoff, richard m.; karpus, william j. title: ccl2 transgene expression in the central nervous system directs diffuse infiltration of cd45(high)cd11b(+) monocytes and enhanced theiler’s murine encephalomyelitis virus-induced demyelinating disease date: 2003 journal: j neurovirol doi: 10.1080/13550280390247551 sha: doc_id: 5734 cord_uid: 14ba78cz ccl2 is a member of the cc chemokine family that mediates the migration and recruitment of monocytes and t cells and has been identified in the central nervous system (cns) during several neuroinflammatory diseases. in order to examine the biological effect of constitutive ccl2 expression in the cns, the authors engineered a mouse that expressed ccl2 in the cns under control of the human glial fibrillary acidic protein (hgfap) promoter. the results demonstrated that transgenic expression of ccl2 in the cns resulted in diffuse cns monocyte infiltration and accumulation. transgenic ccl2 expression did not alter normal development, differentiation, or function of t cells. there was no evidence of overt cns disease or other pathologic phenotype when mice were left unchallenged with antigen or uninfected. however, when ccl2 transgenic mice were given a peripheral challenge of lipopolysaccharide (lps), an inflammatory infiltrate with organized perivascular lesions developed. infection of the transgenic mice with theiler’s murine encephalomyelitis virus (tmev) resulted in accelerated onset and increased severity of clinical and histological disease. these results suggest that ccl2 expression in the cns is a major pathogenic factor that drives macrophage accumulation in the development of cns inflammatory disease. chemokines are small chemotactic molecules that play an important role in immunology, including regulation of leukocyte migration, t-cell differentiation (huang et al, 2001; , organ system development and vascularization, and numerous tissue-specific inflammatory responses (murphy et al, 2000; gu et al, 2000) . these molecules represent a superfamily that can be classified into four groups based on the position of the conserved cysteine residues at the amino terminus of the molecule. the chemokine subfamilies include ccl, cxcl, cl, and cx 3 cl, with the x representing a nonconserved amino acid residue (zlotnik and yoshie, 2000) . corresponding receptors are appropriately named ccr, cxcr, cr, and cx 3 cr based on the family of chemokines to which they bind (murphy et al, 2000) . the binding pattern of chemokine ligands and their receptors suggests an intricate system for directing cell localization throughout the body. multiple ligands may bind the same receptor or one ligand may bind multiple receptors, providing redundancy and complexity for cellular recruitment in different physiological processes (rollins, 1997) . although there is considerable overlap in ligand-receptor binding, ligands are generally restricted to binding receptors of the same family. chemokine receptors signal through seven-transmembrane spanning, g-proteincoupled receptors expressed on the surface of numerous cell populations (murphy et al, 2000) . regulation and expression of corresponding receptors is critical for cells to respond to chemokine gradients produced at sites of tissue inflammation and remodeling (baggiolini, 1998) . murine ccl2 (monocyte chemoattractant protein [mcp]-1) is a member of the cc family of chemokines. this ligand binds ccr2, which is expressed by monocytes, t cells, b cells, natural killer (nk) cells, fibroblasts, and endothelial cells (boring et al, 1996) . ccl2 was first described in mice as a platelet-derived growth factor (pdgf)-inducible gene in fibroblasts, named je (cochran et al, 1983) , and later identified as a recruitment factor for monocytes (matsushima et al, 1989) . ccl2 was also shown to have a role in recruitment of t cells (carr et al, 1994) , polarization of a th2-type t-cell response , regulation of interleukin (il)-4 expression , and down-regulation of il-12 . in the absence of ccl2 expression, th2 responses are impaired in vivo (gu et al, 2000) . intracerebral infection of susceptible inbred strains of mice with theiler's murine encephalomyelitis virus (tmev) leads to a chronic, immune-mediated demyelinating disease . because of the suspected viral etiology, chronic pathology, and primary demyelination accompanied by mononuclear cell infiltration seen in multiple sclerosis (ms), tmev-induced demyelinating disease (tmev-idd) is considered to be an excellent experimental model of ms . virus is rapidly cleared from the peripheral circulation; however, tmev persists within the central nervous system (cns) of susceptible mice for their lifetime (clatch et al, 1990) , serving as a reservoir for chronic activation of virus-specific t cells (lipton et al, 1995) . although tmev has been shown to preferentially reside in cns macrophages, the role of tmev infection in the chronic activation of macrophages to secrete soluble proinflammatory cytokines is unknown. as a result of cns tmev infection, increasing virus-specific (dth-delayed type hypersensitivity) has been shown to correlate with development of increasing disease severity, supporting a role for th1-mediated cns inflammation (clatch et al, 1986) . virus replication occurs in the spinal cord (yamada et al, 1990; ozden et al, 1991) and focal inflammation, consisting primarily of t cells and macrophages, is limited to the spinal cord white matter (lipton, 1975) . from 2 to 5 months following infection, extensive demyelination occurs, leading to clinical paralysis (dal canto and lipton, 1975) . one of the major characteristics of tmev-idd includes cns mononuclear cell infiltration over the disease course. immune responses originate in peripheral lymphoid tissue and once activated, the specific lymphocytes leave the organized lymphoid tissue and accumulate in the cns. in order to further understand the biological role of ccl2 expression in a cns inflammatory disease such as tmev-idd, we generated a transgenic mouse line where astrocytes constitutively expressed ccl2 at low levels (huang et al, 2002) under control of the human glial fibrillary acidic protein (hgfap) promoter (brenner et al, 1994) . astrocytes have been reported as a principle source of ccl2 production in vivo during several cns inflammatory responses (glabinski et al, 1997; berman et al, 1996) . therefore, transgenic ccl2 expression controlled by the hgfap promoter provides the appropriate spatial chemokine distribution for investigation of its role in several neuroinflammatory disease models, including tmev-idd. in order to study the regulation of cns demyelinating diseases by specific chemokines, we constructed a ccl2 transgenic mouse. the hgfap-ccl2 transgene construct ( figure 1a ) was used to produce six transgenic founders, of which four transmitted the transgene to offspring. in the present report, we used the je32 (mcp-1 low ) mouse line for our studies. expression of the hgfap promoter is known to target ccl2 production to astrocytes (huang et al, 2002) . the presence of hgfap-ccl2 was confirmed by southern blotting and polymerase chain reaction (pcr) analysis of tail dna samples (data not shown). to demonstrate tissue-specific expression levels of ccl2 mrna, rnase protection assay (rpa) was performed on cns tissue recovered from mice at 6 to 9 weeks of age. as shown in figure 1b , there was a dramatic increase in ccl2 levels in the forebrain, hindbrain, and spinal cord of je32 transgenic mice compared to minimal expression in wild-type controls. all other tissues were negative (data not shown). astrocytespecific expression of ccl2 was confirmed by in situ hybridization (data not shown). additionally, there was an increase in cxcl10 (crg-2) mrna expression in the je32 transgenic mice ( figure 1b ). because rpa analysis indicated high levels of ccl2 mrna expression in the cns, we wanted to know if the level of chemokine protein was also increased in the periphery. to assess this possibility, serum was recovered from control mice at 32 weeks of age and from je32 mice at both 9 and 36 weeks of age and analyzed by enzyme-linked immunosorbent assay (elisa) for the presence of ccl2. in wild-type mice at 32 weeks of age, very little ccl2 could be detected in the serum ( figure 1c ). however, there was a significant increase in serum ccl2 levels in je32 mice at 9 weeks of age ( figure 1c ). je32 mice that were 36 weeks of age showed a more pronounced level of ccl2 in the serum when compared to either wild-type control or 9-week-old transgenic mice. these data indicate that the ccl2 transgene was expressed in the cns, specifically by astrocytes, and resulted in a translated protein product that could be detected in the serum. given the role of chemokines in regulating lymphocyte migration and accumulation throughout the body (baggiolini, 1998) , it was important to determine if overexpression of ccl2 in the cns affected the number or distribution of lymphocytes in other organs as a result of a developmental abnormality. therefore, we examined spleen, peripheral lymph node, thymus, and peripheral blood of je32 mice for relative numbers of lymphocytes and monocytes. there were increases noted in the size of individual spleen and lymph nodes obtained from je32 mice, but the average weight of organs and average number of mononuclear leukocytes per milligram of tissue was not remarkably different (data not shown). this indicated that there were no apparent developmental defects in the je32 transgenic mice when compared to normal controls. we next analyzed the effect of ccl2 transgene expression in the cns on mononuclear cell infiltration in je32 mice that had not been immunized with antigen or infected with tmev. the cns of wild-type control (sjl×swr) and je32 mice was analyzed by flow cytometry for the presence of microglia (cd45 low cd11b + ), monocytes/macrophages (cd45 high cd11b + ), and lymphocytes (cd45 high cd11b − ). figure 2 shows that the cns of je32 mice that have not been immunized or the je32 ccl2transgenic mouse line was made using the construct shown, consisting of a human gfap (glial fibrilary acidic protein) promoter directing expression of murine ccl2. (b) cns chemokine expression was determined for both control and je32 ccl2-transgenic mice using rpa. the data indicate densitometric evaluation of the rpa analysis. (c) serum levels were determined for normal control mice (32 weeks of age) and normal je32 (9 and 36 weeks of age) using specific elisa. the data represent serum ccl2 concentration + sd of three individual mice per group. cells isolated from the spinal cord of normal control or ccl2transgenic animals were separated by percoll density-gradient centrifugation and analyzed by flow cytometry. antibodies to cd4, cd8, cd45, and cd11b were used to identify infiltrating t cells (cd4 + and cd8 + ), monocytes/macrophages (cd45 high cd11b + ), and resident cns microglia (cd45 low cd11b + ). data are expressed as percent of cd45 + -gated cell population and are representative of at least three similar experiments. infected contains microglia, monocytes/macrophages, and a small population of lymphocytes compared to normal control mice, which only contain microglia. the low-level cns lymphocyte infiltrate in the je32 mice consisted primarily of cd4 + t cells ( figure 2 ). this small population of cd4 + t cells localized to the cns of naïve ccl2-transgenic mice have the phenotype cd62l high , cd25 − , and cd44 + as determined by flow cytometry ( figure 3a ). these cells are also negative for the t-cell activation marker cd69 (data not shown). we were also interested in determining the activation state of the cd45 high cd11b + cells observed in the cns of naïve ccl2-transgenic mice. an absence of disease symptoms suggested that these cells were likely not activated. cells isolated from the cns were further separated by flow cytometric sorting into cd45 low cd11b + and cd45 high cd11b + populations. cdna prepared from the purified cell populations was analyzed by pcr for expression of inducible nitric oxide synthase (inos), a welldocumented marker of activated macrophages (lyons et al, 1992) . controls for inos expression included purified macrophages treated with 75 μg lipopolysaccharide (lps) or phosphate-buffered saline (pbs) for 24 h. the results in figure 3b show that both resident microglia (cd45 low cd11b + ) and infiltrating monocyte/macrophage (cd45 high cd11b + ) populations in ccl2-transgenic mice are not activated, similar to transgene negative controls. this result is consistent with the absence of clinical disease in untreated ccl2-transgenic mice. these data demonstrate that transgenic expression of ccl2 in the murine cns mediates accumulation of mononuclear cells, predominantly monocytes/macrophages, in the absence of inflammatory stimuli, and that these cells have a naïve, unactivated phenotype. ccl2 has been previously shown to induce in vitro th2 polarization and in vivo th2 differentiation (gu et al, 2000) . therefore, we wanted to assess t-cell proliferative and cytokine responsiveness from je32 and control mice. purified t cells were isolated from spleen and lymph node of control and je32 mice and stimulated by immobilized anti-cd3 and anti-cd28 as described in materials and methods. t cells from either the spleen or peripheral lymph nodes of je32 mice proliferated equally as well as control cells (data not shown). in addition, these cells are also capable of producing both interferon (ifn)-γ and il-2 and do not produce detectable levels of il-4 in response to t-cell receptor (tcr) stimulation (data not shown). this suggests that transgenic expression of ccl2 in the cns does not change the fundamental ability of t cells to proliferate or diminish their ability to respond to tcr polyclonal stimulation by production of th1-specific cytokines. to this point, our data suggested that transgenic expression of ccl2 in the cns resulted in an accumulation of cd45 high cd11b + monocytes/macrophages. to assess the ability of recruited monocytes in the cns to initiate inflammation, je32 and control animals were challenged by intraperitoneal (ip) lps administration. three days later, spinal cord tissue was examined by standard hematoxylin and eosin (h&e) histology to determine the level of inflammation. mononuclear cell accumulation and inflammatory lesions were not detected in pbs-treated ( figure 4a ) or lps-treated ( figure 4b ) control animals. in pbstreated je32 animals, there was a diffuse mononuclear cell infiltrate and few small, organized lesions ( figure 4c ). however, following lps administration, distinct perivascular lesions were observed in the je32 spinal cord tissue, primarily in the white matter ( figure 4d ). tracts of mononuclear cell invasion from the meninges into the white matter was also visible. collectively, these results demonstrate that cns transgenic expression of ccl2 results in an accumulation of monocytes/macrophages that have the capacity to induce cns inflammation when challenged with an activating stimulus. activation state of cns-infiltrating cells in ccl2transgenic mice. cells isolated from pooled spinal cords of naïve sjl×swr and ccl2-transgenic mice were purified by percoll density-gradient centrifugation. magnetic antibody coated beads (anti-cd4) were used to further purify cd4 + t cells followed by to test the hypothesis that the presence of ccl2 in the cns could regulate an immune-mediated, macrophage-dependent demyelinating disease, we infected either control, je32, or je95 (described previously; huang et al [2002] ) transgenic mice with tmev and assessed the animals for resulting clinical paralysis. je32 (mcp-1 low ) transgenic mice express low levels of ccl2 in the cns whereas je95 (mcp-1 high ) mice express high levels as measured by rna analysis (huang et al, 2002) . the data shown in figure 5 demonstrate that transgenic overexpression of ccl2 in the cns results in earlier onset of paralytic disease and more severe paralysis during the first 35 days of the disease course. eventually, the severity of disease in either je32 or je95 mice becomes equal to that of the control mice. these data suggest that ccl2-mediated accumulation of monocytes/macrophages in the cns prior to viral infection can result in a more severe disease phenotype once the mice are infected with tmev. the effect of ccl2 overexpression on cns histologic disease following tmev infection was tested by examining spinal cord tissue from control and ccl2transgenic mice. tmev-infected (sjl×swr) control mice develop scattered perivascular mononuclear cell lesions, primarily in the white matter ( figure 6c and d). spinal cord tissue from tmev-infected ccl2transgenic mouse strains (je32 and je95) demonstrates more severe mononuclear cell infiltration and development of numerous perivascular lesions as well as meningial accumulation (je32 shown, figure 6a and b). cns tissue from control and je32 mice was also analyzed for degree of demyelination by epon-embedded, toluidine blue-stained thin sections (data not shown). the results of the demyelination studies were consistent with the h&e histology results, showing slightly higher, though not significantly different, demyelination scores at 2 to 3 weeks post tmev infection compared to controls. these data support the clinical results ( figure 4) where disease onset is more rapid in the ccl2-transgenic mice compared to control mice. incubation with anti-cd45 and anti-cd11b antibodies and flow cytometric sorting in to cd45 low cd11b + and cd45 high cd11b + pools. (a) purified t cells were incubated with antibodies to cd62l, cd25, cd45, and tcrα/β and analyzed by flow cytometry. histogram plots show cd62l and cd25 peaks (shaded) with isotype controls (unshaded). cells are gated out of forward scatter versus side scatter, cd45 high , and tcrα/β + . cd62l high cd25 − staining correlates with a naïve t cell phenotype. (b) cdna isolated from resident microglia (cd45 low cd11b + ) and monocytes/macrophages (cd45 high cd11b + ) was analyzed by pcr for inos expression. controls for inos up-regulation are purified macrophages treated with pbs or lps for 24 h. the results indicate that microglia and monocytes in both sjl×swr and ccl2-transgenic spinal cord tissue do not express inos. the data are representative of at least two experiments. to determine the constituents of the inflammatory infiltrate in both control and je32 mice, we performed flow cytometric analysis of cells recovered from spinal cords of mice 4 weeks post infection, at a time when there was evidence of clinical disease. figure 7 shows that the tmev-infected je32 mice showed a greater percentage of both lymphocytes (cd45 high cd11b − ) and monocytes (cd45 high cd11b + ) than the control mice. in both control and je32 mice, the lymphocyte infiltrate consisted of a mixture of cd4 and cd8 t cells. the ccl2 transgenic mice appeared to have a lower percentage of microglia (cd45 low cd11b + ); however, this was simply due to the increased fraction of lymphocytes and monocytes in the sample. these data support the idea of ccl2mediated enhancement of clinical and histological disease at the cellular level. two distinct possibilities exist to explain the enhanced clinical and histological disease in the ccl2 transgenic mice. one possibility is enhanced tissue damage mediated by an increased mononuclear cell infiltrate consisting of t cells and effector macrophages. the second is augmented tmev replication and viral presence in the cns, possibly inducing direct cytolytic events in oligodendrocytes. to determine between these two possibilities, we infected both control (sjl×swr) and je32 mice with tmev, allowed disease to develop, and harvested spinal cord tissue for analysis of the presence of virus by plaque forming assay. the results in figure 8 demonstrate no significant difference in the viral titers among control and transgenic mice. these data suggests that cns ccl2 transgenic expression does not enhance or diminish the replication of tmev. rather, the data support the idea that cns ccl2 transgenic expression enhances mononuclear cell infiltration, which leads to enhanced clinical and histologic demyelinating disease. our data support the idea that accelerated disease after tmev infection is due to ccl2-mediated monocyte/macrophage recruitment and not altered tmev levels. however, it could be possible that the presence of any virus in the cns of ccl2-transgenic mice would be a sufficient stimulus to activate the accumulated macrophage pool, initiating inflammation and development of demyelinating disease. to test this possibility, sjl×swr control and ccl2transgenic mice were infected intracranially with mcmv (smith strain) or tmev and monitored for disease development. mcmv is a double-stranded dna β-herpesvirus that normally replicates in the liver and spleen of susceptible strains of mice dur-ing the replicative phase (approximately 14 days) before entering a latent phase. mcmv can infect astrocytes, neurons, endothelial cells, radial glia, ependymal cells, microglia, and cells from the meninges and choroid in the mouse cns (tsutsui et al, 1989; shinmura et al, 1997; van den pol et al, 1999) as well as macrophages (brautigam et al, 1979) . the results shown in figure 9 demonstrate that tmev-infected ccl2-transgenic mice developed an earlier-onset disease compared to mcmv-infected ccl2-transgenic mice and and tmev-infected wild-type control mice. following onset of disease in tmev-infected ccl2transgenic mice and the appearance of clinical symptoms in tmev-infected sjl×swr mice, all groups were analyzed by histology ( figure 9b ) and flow cytometry ( figure 9c ) to determine whether infection with mcmv resulted in altered pathology and cellular infiltrate. spinal cords of mcmv-infected sjl×swr mice showed no focal lesions ( figure 9b , a), whereas tmev-infected sjl×swr showed scattered small perivascular lesions and meningial accumulation ( figure 9b, b) . spinal cords of mcmvinfected ccl2-transgenic mice had a diffuse infiltrate with few scattered focal lesions and meningial accumulation ( figure 9b , c) but did not demonstrate the level of pathology associated with ongoing demyelinating disease, as in tmev-infected ccl2-transgenic mice ( figure 9b, d) . finally, flow cytometric analysis of cns-infiltrating cells shows that mcmv infection results in far fewer cd4 + t cells and monocytes/ macrophages in sjl×swr control mice compared to tmev-infected controls ( figure 9c ). this correlates with the absence of demyelinating disease in these animals. surprisingly, mcmv-infected ccl2transgenic mice showed no clinical disease despite the presence of both cd4 + and cd8 + t cells and a similar ratio of cd11b + cd45 high cells in the spinal cord tissue compared to tmev-infected transgenic mice ( figure 9c ). these data demonstrate that the effect of ccl2 on demyelinating disease initiated by tmev is not only due to the enhanced accumulation of macrophages, but is also specific to tmev infection. in the present report, we studied the role of ccl2 in the pathogenesis of cns inflammatory demyelinating disease by creating a transgenic mouse where the astrocytes overexpress our chemokine of interest. the results demonstrated that cns transgenic expression of ccl2 induces a monocyte/macrophage accumulation in the cns (figure 2 ). this cellular accumulation was benign in that the accumulating cells were not activated and no overt histological or clinical inflammatory disease was induced in the absence of an inciting stimulus (figures 3 and 4) . however, when the transgenic mice were challenged with lps, there was evidence of organized cns mononuclear cell lesions (figure 4) . moreover, when the transgenic mice were infected with tmev, clinical disease onset ( figure 5 ) was more rapid and histologic disease was more severe (figure 6 ). ccl2 has been previously overexpressed in the cns using the myelin basic protein promoter (fuentes et al, 1995) where the cellular distribution of the gene product was limited to oligodendrocytes rather than astrocytes. administration of lps to these mice initiated inflammation, presumably by inducing macrophage accumulation the cns, similar to what we have observed in the present report. our results support the idea that ccl2-mediated monocyte/macrophage accumulation in the cns is a pathogenic factor in the development of cns inflammatory demyelinating disease. there are two major possibilities to explain the effect of ccl2 on tmev-induced disease development. the first is that overexpression of ccl2 by astrocytes results in an influx and accumulation of normal monocytes/macrophages into the cns. these cells are not activated and there is no evidence of inflammatory lesions or spontaneous inflammatory disease or histologic disease. these monocytes/macrophages have the capacity to be activated as a peripheral challenge of the je32 transgenic mice with lps-induced organized cns mononuclear cell lesions. these lesions are presumably due to the activation of monocytes/macrophages previously recruited to the cns as a result of ccl2 overexpression. during the normal tmev disease process, ccl2 is expressed in the cns of infected mice (hoffman et al, 1999) and anti-ccl2 treatment results in decreased monocyte accumulation in the cns (manuscript submitted). these findings support the idea that enhanced disease as a result of transgenic overexpression of ccl2 is due to enhanced monocyte accumulation. however, from the histology data, it cannot be ruled out that either lps challenge or tmev infection induced the activation of microglia to form inflammatory foci resembling an accumulation of monocytes/macrophages. the second possibility is that overexpression of ccl2 in the cns results in enhanced t-cell accumulation. because ccl2 can function as a chemoattractant for t cells (carr et al, 1994) and t cells figure 7 flow cytometric analysis of cns infiltrate in tmevinfected control and ccl2-transgenic mice. cells isolated from the spinal cord of tmev-infected control or je32 transgenic animals were separated by percoll density-gradient centrifugation and analyzed by flow cytometry. antibodies to cd4, cd8, cd45, and cd11b were used to identify infiltrating t cells (cd4 + and cd8 + ), monocytes/macrophages (cd45 high cd11b + ) and resident cns microglia (cd45 low cd11b + ). data are expressed as percent of cd45 +gated cell population (t cells) or cd11b + cd45 + (microglia and monocytes/macrophages) and are representative of at least three similar experiments. are required for development of tmev-idd (clatch et al, 1986) , overexpression of ccl2 in both the je32 and je95 mice could promote the accumulation of effector th1 cells that drive the demyelinating disease process. however, our present data ( figure 2 does not support this idea. in unchallenged, uninfected je32 transgenic mice, there is little t-cell (cd4 + or cd8 + ) accumulation but there is a significant monocyte/macrophage (cd45 high cd11b + ) accumulation. this low level of t-cell accumulation could be the result of upregulation of cxcl10 (crg-2, figure 1b ). cd44 expression is usually associated with memory t-cells, but there is no indication of previous activation by other surface markers. cd44 upregulation on unactivated cells with no cd25 or cd69 expression and unchanged cd62l have been previously reported (viret et al, 2000) . further, cd44 expression is thought to play a role in leukocyte migration into tissue, as up-regulation of the molecule has been associated with binding to its ligand, hyaluronan, and rolling or adhesion before extravasation (degrendele et al, 1997) . cd44 has been correlated with cns infiltration by cd4 + t cells (brocke et al, 1999) , suggesting that up-regulation may be required for entry and retention of the small number of t cells observed in ccl-transgenic mice. these cells do not appear to be activated except for cd44 expression. significant accumulation of t cells in the cns occurs only after tmev infection (figure 7) . further support that the murine ccl2 and ccr2 axis primarily controls cns monocyte/macrophage and has little regulation over t-cell accumulation has been demonstrated in murine experimental autoimmune encephalomyelitis (eae) (fife et al, 2000; huang et al, 2001) . it was also possible that transgenic overexpression of ccl2 in the cns promoted enhanced viral replication by inducing accumulation of the macrophage, which is the reservoir for viral persistence (clatch et al, 1990) . the presence of more host cells could potentially result in an increased production of infective virus that could lead to direct infection and cytolysis of oligodrendrocytes (aubert and brahic, 1995) , culminating in enhanced clinical disease ( figure 5 ). we do not believe that this was the case as there is no apparent difference in cns viral titer between control and transgenic mice (figure 8) . therefore, the data argue in favor of the hypothesis that expression of ccl2 promotes cns accumulation of monocytes/macrophages that are not activated. upon infection with tmev, transgenic mice develop disease more rapidly than controls due to the presence of the end-stage effector cells in the cns. eventually, the control mice reach the same level of clinical disease as the transgenic mice as a result of normal accumulation of monocytes/macrophages in this model. further, the mcmv studies demonstrate that the accelerated disease observed in ccl2-transgenic mice is specific to tmev infection and not due to a nonspecific activation of localized monocytes/macrophages by any virus introduced into the cns. this is consistent with our conclusion that the role of ccl2 in tmev-induced demyelinating disease is directing macrophage accumulation. figure 9 mcmv infection of sjl×swr and ccl2-transgenic mice. (a) control sjl×swr and ccl2-transgenic mice were infected intracranially with mcmv or tmev and monitored for development of demyelinating disease symptoms. (b) after 28 days, after disease onset in the ccl2-transgenic tmev-infected mice, spinal cord tissue was recovered by dissection and analyzed by h&e histology. spinal cords of sjl×swr mice infected with mcmv showed no focal lesions (a), whereas sjl×swr infected with tmev showed scattered small perivascular lesions and meningial accumulation (b). spinal cords of ccl2-transgenic mice infected with mcmv had diffuse infiltrate with a few scattered focal lesions and meningial accumulation (c). tmev-infected ccl2-transgenic mice (d) showed multifocal mononuclear cell infiltration and extensive multilayer meningial accumulation associated with ongoing demyelinating disease. (c) cells were also isolated from spinal cord tissue at day 28 after infection and analyzed for t-cell infiltrate (cd4 + and cd8 + ) and microglia or monocyte/macrophage accumulation (cd45 low cd11b + and cd45 high cd11b + ) by flow cytometry. data are derived from the forward scatter versus side scatter and cd45 + gates for all cells, as well as the cd11b + gate for migroglia for monocyte/macrophages. the chemokine regulation of demyelinating disease has been studied in other virus-induced models. in a separate study, tmev-idd was induced using different strains of virus: da, gdvii, and h101. these are neurovirulent strains of tmev that cause different neuropathology and disease when inoculated into sjl/j mice. da virus causes a chronic demyelinating disease, gdvii virus causes an acute fatal encephalomyelitis, and h101 virus causes an acute pachymeningitis with hydrocephalus. ccl2, ccl3, ccl4, ccl5, cxcl10, and mip-2 mrna expression was detected in both the brain and spinal cord during all three infections (theil et al, 2000) . murray et al (2000) examined spinal cords during the acute and chronic phases of tmev infection in mice sus-ceptible (b10.m, h-2f) and resistant (b10, h-2b) to virus-induced demyelination. in this model, tmev infection resulted in robust expression of mrnas for ccl2, ccl5, and, cxcl10, but not cxcl1, in brains and spinal cords of both strains of mice within 5 days. during the chronic, demyelinating phase of infection, there was a resurgence in ccl2, ccl5, and cxcl10 mrnas in spinal cords of susceptible b10.m mice. in both of these studies, the direct function of the chemokines identified as being expressed during the disease process was not directly determined, including whether ccl2 regulated monocyte/macrophage accumulation. lane et al (1998) demonstrated the presence of chemokines in the cns of mice infected with mouse hepatitis virus. by analyzing chemokine mrna, they found that ccl4, ccl5, and cxcl10 expression was evident at the time of chronic demyelinating disease. they went on to demonstrate that ccl5 was required for the development of demyelinating disease , whereas cxcl9 and cxcl10 expression was required for control of viral replication (liu et al, , 2001 . finally, this same group of investigators demonstrated that ccl2 regulation of monocyte/macrophage accumulation was essential for development of demyelinating disease (chen et al, 2001) . these examples point to the essential functions of chemokines during the pathogenesis of virus-induced demyelinating disease and support our contention that ccl2 regulates the accumulation of monocytes/macrophages during the development of tmev-idd. we would speculate that ccl2 is a desirable target for intervention in the demyelinating disease process as it regulates the accumulation of a key mononuclear cell subtype in the cns. development of antagonists to ccr2, the only known receptor for ccl2, could lead to regimens that can interdict during ongoing cns demyelinating disease. indeed, antagonists to ccr1 can treat ongoing eae (liang et al, 2000) by presumably interfering with the ability of ccl3 and/or ccl5 from inducing accumulation of t cells in the cns (fife et al, 2001) . this example points to the possibility of interfering with the functions of ccl2 in a similar manner using receptor antagonists to interfere with ongoing disease. mice ccl2-transgenic mice (je32) were made using standard techniques (huang et al, 2002) . je32 transgenic mice expressed low levels of ccl2 (mcp-1 low ) whereas je95 transgenic mice expressed high levels of ccl2 (mcp-1 high ) (huang et al, 2002) . the modified murine ccl2 gene, with replacement of the mrna cap site, was obtained from dr. barrett rollins (dana farber cancer institute, boston, ma) and was placed under the control of the hgfap promoter to produce the hgfap-ccl2 transgene. initial microinjections of (sjl×swr)f1 mice resulted in six transgene positive founders identified by southern blotting of tail dna for elements of the hgfap gene. four founders transmitted the hgfap-ccl2 transgene to progeny. normal, nontransgenic (sjl×swr)f2 mice were used as controls. sjl×swr controls were established by crossing female sjl/j with male swr mice. the offspring of this cross were then maintained as a repeatedly intercrossed control sjl×swr line in parallel with the transgenic lines. the biological properties of je95 mice were previously described (huang et al, 2002) . animal care and use at northwestern university and the cleveland clinic were approved by the respective institutional animal care and use committees and performed according to public health sevice guidelines. virus was prepared as described previously . briefly, confluent monolayers of bhk-21 cells (atcc) were infected with the bean 8386 strain of tmev for 72 h. virus was precipitated with nacl and polyethylene glycol (peg) and then pelleted by centrifugation. virus was further purified by ultracentrifugation on discontinuous 20% to 70% sucrose and cs 2 so 4 gradients. finally, virus was pelleted, resuspended in pbs, and measured for optical absorbance at 260/280 nm. plaque assays of the supernatant were performed on bhk-21 cells. mice were anesthetized with sodium pentobarbital and injected in the right cerebral hemisphere with 3 × 10 6 plaque-forming units (pfu) of tmev (bean strain) in 30 μl of sterile dmem. mice were examined two to three times per week for the first 3 weeks until all infected animals were exhibiting neurological signs of tmev-idd. after signs of clinical disease, mice were examined biweekly. clinical symptoms were scored as 1 = waddling gait; 2 = severe waddling gait and righting reflex impairment; 3 = hind limb paralysis with/without incontinence. clinical data have been expressed as the mean clinical score at a particular timepoint. chemokine analysis cns chemokine mrna expression was analyzed by rpa as previously described (glabinski et al, 1998) . serum ccl2 concentrations were determined from je32 and control mice of different ages by specific elisa as previously described . histologic evaluation of cns inflammation was performed using standard h&e methodology as previously described (karpus et al, 1995) . transgenic and control animals, aged 6 to 8 weeks, were given 50 μg of lps (escherichia coli serotype 0111:b4; sigma, st. louis, mo) in 100 μl of sterile pbs by ip injection. animals were monitored for 72 h and sacrificed by total body perfusion with pbs through the left ventricle for examination of cns tissue by standard h&e histology. control treatment consisted of ip administration of 100 μl sterile pbs. spinal cord tissue for histology was recovered by microdissection of the dorsal vertebrae to maintain intact meninges. organ harvest and cell isolation spleen, peripheral lymph nodes (inguinal, brachial, axillary), thymus, peripheral blood, and spinal cord ccl2 mediates enhanced tmev-induced demyelination 634 jl bennett et al tissue were harvested from two to four mice per group, aged 6 to 8 weeks. sample weights were determined before processing. splenocytes were isolated by homogenization through 100-mesh stainless steel screens and red blood cells lysed by incubation with 2 ml/organ of tris-nh 4 cl (ph 7.2) at 37 • c for 10 min. lymph nodes and thymi were similarly isolated by homogenization through 100-mesh screens. all samples were washed with hanks buffered salt solution (hbss) (biowhittaker, walkersville, md) and resuspended in 3 ml dulbecco's modified eagle medium containing 5% fetal calf serum (fcs), 1 mm glutamine, 100 u/ml penicillin, 100 μg/ml streptomycin, 1 μm nonessential amino acids, and 5 × 10 −5 m 2-me (complete dmem-5; all components from gibco brl, grand island, ny) for culture. peripheral blood was harvested by cardiac puncture into syringes containing 100 μl heparin sulfate (100 u/ml) per mouse. samples were pooled by experimental group and the total volume was determined before overlay of sample on to 3 ml ficoll (nycomed pharma, oslo, norway) in polyethylene tubes. gradients were produced by spinning at 22 • c for 15 min, 1200 rpm. buffy coat interface was recovered and washed with 10 ml hbss. samples were resuspended in dmem-5 for culture. spinal cords were isolated by flushing the vertebral column with 1 × pbs through a blunted 18-gauge needle and homogenized by passage through 100mesh stainless steel screens. single-cell suspensions were washed with hbss and pelleted by centrifugation at 1200 rpm for 10 min at 4 • c. cells were then resuspended in 5 ml 30% percoll solution (amersham pharmacia biotech ab, uppsala, sweden). percoll solution, 70%, was added by underlay and leukocytes isolated by 10-min spin at 22 • c, 1200 rpm (328 × g). leukocytes were recovered from the interface and washed with 1 ml hbss. cells were pelleted by quick spin (up to 7000 rpm), resuspended in 1 ml flow buffer (pbs containing 0.05% bovine serum albumin [bsa], 0.01% sodium azide), and counted. samples were stored at 4 • c until antibody staining and flow cytometric analysis. purified t cells were recovered from spleen and lymph nodes as described above and isolated using an automacs machine (miltenyi biotech, auburn, ca). briefly, cell samples were washed with macs buffer (1 × pbs, 0.5% bsa, 2 mm edta, ph 6.5), then resuspended in 2 ml buffer containing 50 μg biotinylated anti-cd4 antibody (l3t4) (pharmingen, san diego, ca) and stored on ice for 10 min. following a wash with buffer, automacs streptavidin microbeads were added for 10 min on ice. after a final wash, cd4 + t cells were sorted by positive selection in 2-ml samples and analyzed for purity by staining with cd3-fitc (fluorescein isothiocyanate), cd19-pe (phycoerythrin), cd8-percp (peridinin chlorophyll protein), and cd11b-apc (allophycocyanin). purity was determined to be at least 97%. cellular composition of lymphoid organs was determined by staining with lymphocyte specific antibodies and analysis on a facs-calibur cytometer (becton dickinson, san jose, ca). one million cells were incubated with anti-fc blocking antibody (anti-mouse fcrεii/iii, 24g2) for 15 min at 4 • c and incubated with titered dilutions of the following antibodies: cd4-fitc (rma4-5), cd8-pe (53-6.7), cd45-percp (ly-5), and cd11b-apc (m1/70), cd19-pe (1d3), and cd3-apc (145-2c11) (all antibodies from pharmingen, san diego, ca). data were acquired and analyzed using cellquest pro software (becton dickinson, san jose, ca). data was gated as a function of cd45 expression and side light scatter characteristics. relative cell composition of organs was estimated by multiplying the percent positive by the total cell count for each individual organ. to examine the activation status of t cells and monocytes, cd4 + t cells were depleted from cnsisolated mononuclear cell pools using cd4 dynabeads according to the manufacturer's instructions (dynal biotech, oslo, norway). cd4 + t cells were washed with flow buffer, filtered, and examine by flow cytometry for expression of cd44-fitc (ly-24), cd25-pe (3c7), cd62l-fitc (ly-22), cd69-pe (h1.2f3), cd45 percp (ly-5), and tcr-α/β-apc (h57-597). the remaining cell pool was stained with cd11b-apc (m1/70) and cd45-percp (ly-5), sorted in to cd45 high cd11b + and cd45 low cd11b + populations, and reverse transcriptase (rt)-pcr performed for analysis of inos expression. proliferative capacity of cd4 + t cells isolated from sjl×swr and je32 animals was assayed by conventional mitogenic stimulation. purified t cells were resuspended at a concentration of 10 6 cells/ml in dmem-5. 96-and 24-well plates were coated with 2 μg/well anti-cd3 (145-2c11) and anti-cd28 (37.51) antibodies (pharmingen), or pbs (ph 7.2) as a control, for 90 min at 37 • c and then washed 3 times with pbs before addition of samples. 5 × 10 6 cells/ml were added to the plates in triplicate wells and incubated at 37 • c for 24 h. cells were pulsed with 1 μci 3 h-tdr (icn biochemicals, irvine, ca), incubated an additional 12 h, and harvested onto glass filters (packard, meriden, ct). microscint (packard, meriden, ct) fluid was added to the filter and the 3 h-tdr incorporation determined on a top count liquid scintillation counter. counts per minute (cpm) was calculated by subtracting average cpm values of unstimulated wells from average cpm values of anti-cd3-and cd28-stimulated wells. cytokine production t cells were analyzed for cytokine response to mitogenic stimulation by elisa. purified t cells were added at a concentration of 1 × 10 6 cells/ml to anti-cd3-and anti-cd28-coated plates as described above. supernatants were recovered at 24 and 48 h and stored at −20 • c until assayed. cytokine analysis was performed by standard techniques using interferon (ifn)-γ and il-2 elisa kits according to manufacturer instructions (endogen, cambridge, ma). plates were developed by addition of tmb substrate (dako, carpinteria, ca) and 0.18 m h2so4. all samples were analyzed in triplicate. results were early infection of the central nervous system by the gdvii and da strains of theiler's virus chemokines and leukocyte traffic localization of monocyte chemoattractant peptide-1 expression in the central nervous system in experimental autoimmune encephalomyelitis and trauma in the rat molecular cloning and functional expression of murine je (monocyte chemoattractant protein 1) and murine macrophage inflammatory protein la receptors-evidence for two closely linked c-c chemokine receptors on chromosome 9 pathogenesis of murine cytomegalovirus infection: the macrophage as a permissive cell for cytomegalovirus infection, replication and latency gfap promoter directs astrocyte-specific expression in transgenic mice antibodies to cd44 and integrin α4, but not l-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment monocyte chemoattractant protein 1 acts as a t-lymphocyte chemoattractant lack of ccr2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus characterization of theiler's murine encephalomyelitis virus (tmev)-specific delayed-type hypersensitivity responses in tmev-induced demyelinating disease: correlation with clinical signs ) reading at an absorbance of 450 nm. statistical analysis statistical significance of thymidine incorporation and cytokine levels was determined using student's t test for comparisons of two means molecular cloning of gene sequences regulated by platelet-derived growth factor primary demyelination in theiler's virus infection. an ultrastructural study cd44 activation and associated primary adhesion is inducible via t cell receptor stimulation cc chemokine receptor 2 is critical for induction of experimental autoimmune encephalomyelitis selective cc chemokine receptor expression by central nervous system-infiltrating encephalitogenic t cells during experimental autoimmune encephalomyelitis controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1 synchronous synthesis of α-and βchemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis expression of chemokines rantes, mip-1alpha and gro-alpha correlates with inflammation in acute experimental autoimmune encephalomyelitis control of th2 polarization by the chemokine monocyte chemoattractant protein-1 central nervous system chemokine expression during theiler's virus-induced demyelinating disease pertussis toxin-induced reversible encephalopathy dependent on monocyte chemoattractant protein-1 overexpression in mice absence of monocyte chemoattractant autoimmune encephalomyelitis mip-1alpha and mcp-1 differentially regulate acute and relapsing autoimmune encephalomyelitis as well as th1/th2 lymphocyte differentiation monocyte chemotactic protein 1 regulates oral tolerance induction by inhibition of t helper cell 1-related cytokines differential cc chemokine-induced enhancement of t helper cell cytokine production an important role for the chemokine macrophage inflammatory protein-1α in the pathogenesis of the t cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the cc chemokines macrophage inflammatory protein-1α and monocyte chemotactic protein-1 dynamic regulation of alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease a central role for cd4(+) t cells and rantes in virus-induced central nervous system inflammation and demyelination identification and characterization of a potent, selective, and orally active antagonist of the cc chemokine receptor-1 theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination the predominant virus antigen burden is present in macrophages in theiler's murine encephalomyelitis virus-induced demyelinating disease expression of mig (monokine induced by interferongamma) is important in t lymphocyte recruitment and host defense following viral infection of the central nervous system the t cell chemoattractant ifn-inducible protein 10 is essential in host defense against viral-induced neurologic disease c-c chemokines differentially alter interleukin-4 production from lymphocytes molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line the immunopathogenesis and regulation of t-cellmediated demyelinating diseases theiler's virus-induced demyelinating disease international union of pharmacology. xxii. nomenclature for chemokine receptors biphasic and regionally-restricted chemokine expression in the central nervous system in the theiler's virus model of multiple sclerosis in situ analysis of proteolipid protein gene transcripts during persistent theiler's virus infection differential expression of the immediate-early and early antigens in neuronal and glial cells of developing mouse brains infected with murine cytomegalovirus. y alterations in cytokine but not chemokine mrna expression during three distinct theiler's virus infections susceptibility of brain cells to murine cytomegalovirus infection in the developing mouse brain cytomegalovirus cell tropism, replication, and gene transfer in brain on the self-referential nature of naïve mhc class ii-restricted t cells the relationship between viral rna, myelin-specific mrnas, and demyelination in central nervous system disease during theiler's virus infection chemokines: a new classification system and their role in immunity key: cord-006824-btcdjmfp authors: nan title: key note and state of the art lectures date: 2002-09-07 journal: ann hematol doi: 10.1007/s00277-002-0513-0 sha: doc_id: 6824 cord_uid: btcdjmfp nan non-hodgkin's lymphoma (nhl) is a hematologic malignancy for which a rather dramatic rise in incidence has been noted during the past decade. nhl is a heterogeneous condition in every respect. there is a wide range of histologic, immunologic, molecular and clinical expression of this condition with marked differences in response to therapy and survival. various forms of combination chemotherapy and radiation have been proven to be successful for the different types of nhl, however, on average only one-half of the patient population with disseminated disease enters a durable remission [1] . during the past 25 years, many investigators have explored various forms of high dose therapy followed by either autologous or allogeneic hematopoietic cell transplantation. this review will present some of the original data and provide new information from more recently published clinical trials. the first group of patients treated with high dose combination therapy followed by autologous hct dates back to 1978. in a report from the national cancer institute, 12 nhl patients were described of whom four currently are alive and well more than 25 years after hct [2] . this encouraging observation was followed by a large number of clinical trials, mostly designed as feasibility trials or phase ii studies. a comparison between high dose therapy and autologous hct versus standard dose chemotherapy was reported in 1995 and demonstrated a significant advantage with respect to overall survival and disease-free survival in favor of transplantation [3] . during the past decade, patients with nhl who had high risk features have been transplanted electively during their first clinical remission in order to avoid an early relapse. these extended phase ii trials have yielded promising results. a prospective comparison showing the advantage of high dose sequential therapy versus continued standard dose therapy was reported five years ago [4] . the leading cause for treatment failure after autologous hct for nhl is the relapse of the underlying disease. a number of strategies to improve the outcome of autologous hct can be pursued (see table 1 ). in this presentation, i will show several examples which indicate the importance of some of the treatment strategies mentioned in table 1 [5] . table 1 . opportunities to improve the outcome of autologous hematopoietic cell transplantation • timing of autologous hct with respect to remission status • selection of myeloablative doses and combinations of drugs with or without irradiation • optimizing the graft, i.e., removal of clonogenic tumors without loss of activity for hematopoietic reconstitution • post-autologous hct to consolidate remission (cytokines, monoclonal antibodies, vaccines, cells, involved-field radiation) the timing of high dose therapy and autologous hct can have a major influence upon the treatment result. in a prospective phase ii trial, we treated 37 patients with high dose therapy and autologous bone marrow transplantation for follicular lymphoma in first complete or partial remission [6] . seventeen patients were in first complete remission and 20 patients were in partial remission. the median age of this patient population was 37 years ranging from 24 to 49 years. the preparatory regimen consisted of fractionated total body irradiation (1,200 cgy), etoposide (60 mg/kg) and cy-clophosphamide (100 mg/kg). all patients received autologous bone marrow cells which were purged with a set of monoclonal antibodies directed against cd9, cd10, cd19 and cd20 with complement lysis. an intent-to-treat analysis is shown in figure 1 . at the time of analysis, the 31 surviving patients had been followed for 8-14 years. overall survival was 84%, event-free survival 62% and the relapse rate was 29%. the causes for treatment failure included one early death due to septicemia, two patients died from acute leukemia and three patients succumbed to lymphoma. the excellent long-term survival demonstrated in this study indicates that the natural course of the underlying disease has been markedly altered by the autologous hct approach. a case-matched comparison from the stanford nhl data base indicates a significant survival advantage compared to patients not undergoing transplantation. conversely, patients with follicular lymphoma who proceed to transplantation have a reduced chance for long-term survival. four groups of investigators at boston, london, omaha and stanford have evaluated 360 autologous hct recipients in four independent trials. four to eight years following autologous hct, overall survival ranged from 60-69% and freedom from relapse ranged from 42-63%. the event of a relapse of follicular nhl clearly reduces the chance for disease-free survival. a prospective clinical trial in follicular lymphoma has been completed recently in europe. in this study, patients with this type of lymphoma were treated with cytoreductive therapy to minimal disease and were then randomized to receive either high dose radiochemotherapy and autologous hct or interferon. the data of this trial are currently being prepared for publication. a preliminary review indicates a statistical advantage in progression-free survival in favor of transplantation (w. hiddemann, personal communication, 2002). other efforts to overcome the relapse problem include the use of maximum tolerated regimens with or without total body irradiation. more recently, radiolabeled monoclonal antibodies have been introduced and these interesting efforts have yielded promising results [7] . another approach to reduce the post-transplant relapse rate is the employment of monoclonal antibodies directed against epitopes expressed on lymphoma cells. in an exploratory trial, we have administered the antibody directed against the cd20 epitope to 35 autologous hct recipients whose lymphoma cells expressed cd20 [8] . these 35 patients had a median age of 51 years ranging from 28 to 70 years. following our standardized transplant approach, an intent-to-treat analysis shows that 31 of 35 patients are alive and 29 are in continued complete remission with a follow-up period ranging from 1.3 to 4.1 years. a comparison with similar autologous hct recipients with nhl who had not been treated with the monoclonal antibody after transplantation indicates that figure 1 : overall survival, event-free survival and relapse following autologous bone marrow transplantation in 37 patients with follicular lymphoma during their first complete or first partial chemotherapy-induced remissions this new post-transplant therapy has contributed positively to the treatment outcome. clearly, a prospective randomized study is needed to confirm our promising observation. such a trial is being performed under the auspices of the eastern oncology group (ecog trial 2199). in 1986, a first group of 17 patients (14 with nhl and three with hodgkin's disease) was described indicating that extended disease-free long-term survival can be attained with high dose therapy followed by allogeneic transplantation utilizing hematopoietic cells obtained either from fully or closely matched related donors [9] . the treatment outcome was greatly influenced by the remission status of the transplant recipient at the time when preparation for transplantation was begun, age of the recipient, compatibility with the respective donor and incidence of post-transplant complications. the leading cause for treatment failure remains graft-versus-host disease (gvhd) with associated infectious complications, other toxicities or relapse of the underlying disease. figure 2 illustrates our institutional experience with 36 patients (seven children/adolescents and 29 adults). the median age of the patient population was 35 years ranging from 5 to 60 years. median follow-up in continued complete remission of survivors is in excess of six years ranging from six months to more than 10 years. overall survival is 50% with relapse causing treatment failure in 30% of allogeneic hct recipients. the remaining 20% of patients succumbed to complications such as graft-versus-host disease, infections or veno-occlusive disease. this limited experience is nevertheless representative for the observations made in other trials including results reported from the international bone marrow transplant registry. without question, non-relapse mortality (mostly due to gvhd) is a major obstacle to success and alternative technologies need to be explored. very promising data have recently been reported related to the use of intensity-reduced regimens followed by allogeneic hct [10] . it can be expected that this novel concept which relies mainly on a graft-versus-lymphoma effect will lead to an increase in the successful use of allogeneic hct while the procedure-related mortality is dramatically decreased as compared to high dose therapy regimens. the transplant physician who evaluates and counsels a patient with nhl concerning the type of transplantation is faced with a serious dilemma. because of the heterogeneity of the underlying disease, it has so far not been possible to come to a clear recommendation which of the types of hct should be pursued for any given patient, autologous or allogeneic hct. the earliest comparison was reported 15 years ago and indicated equivalence in outcome [11] . since then, another 10 comparative studies have been described in the peer reviewed literature. in these trials, 1,183 al-lograft recipients were compared to 10,087 autograft recipients [12] . differences in histology, remission status, source of autologous or allogeneic cells (marrow versus peripheral blood, related versus unrelated donor) and post-transplant management make even a meta-analysis relatively meaningless. one can only conclude that autologous hct is associated with a post-transplant relapse rate in the order of 40-50% while allogeneic hct is complicated mostly by gvhd and related toxicities resulting in equivalent outcomes for patients with nhl. a collaborative trial is currently planned by the bone marrow transplant clinical trials network in the united states to address this important question which should help to facilitate the proper choice for transplant candidates. for the time being, most centers would utilize hct if a suitably matched donor is available and to offer autologous hct in the absence of a compatible donor. mortality after hematopoietic stem cell transplantation (hsct) could be reduced to approximately 10% in hla-identical intrafamiliary pediatric transplantations and ranges between 20 and 40% in hla-different, intrafamiliary as well as hla-unrelated transplantations. in contrast, pulmonary complications are a leading cause of mortality after hsct and contribute markedly to the morbidity [33] . the pathophysiology of these diseases is poorly understood and their classification is highly descriptive. this overview is focussed on the pediatric age group and (1) elucidates the circumstances which render recipients at increased risk of non-infectious pulmonary complications, (2) describes the diseases and alterations which are encountered, and (3) discusses diagnostic and therapeutic consequences. the number of hematopoetic stem cell transplantations has increased in recent years [53] . statistically, pulmonary complications with respiratory insufficiency account for as much as 60% of the mortality, depending on the patients' age [12, 19, 36, 70, 64] . furthermore, respiratory insufficiency is included under diagnoses such as graft-versus-host-disease (gvhd) or multi organ insufficiency. the largest retrospective analysis of italian children after allogeneic stem cell transplantation demonstrated a pulmonary mortality of 9% [28] . case reports as well as retrospective or prospective analyses of different patient cohorts demonstrate between 10% and 40% incidence of irreversible pulmonary function loss after hsct [68, 14, 48, 65, 43, 11] . the incidence of late-onset non infectious pulmonary complications (lonipc) is estimated to be approximately 10%. in one study 18 out of 179 patients suffered such complications three months after allogeneic hsct [60, 10] . there has been no improvement over the past decade (11) . risk factors are intrinsic to the procedure (autologous vs. allogeneic transplantation with rejection by residual host t-cells, gvhd), to the immunosuppression (total body irradiation, cyclophosphamide and busulfan, both leading to interstitial pulmonary fibrosis), to the underlying disease (sickle-cell anemia with recurrent episodes of chest syndrome and pulmonary infarction, severe combined immunodeficiency with recurrent episodes of pneumonia), to the treatment of the underlying disease (irradiation, resection) and the hyperimmune status (influenced by the histocompatibility between donor and host, e.g. donor t-cells reacting to hlaantigens). in one series of children undergoing transplantation for hematologic diseases, 44% had pulmonary function abnormalities before the procedure (11) . although the topic of this review is non-infectious pulmonary complications, it is impossible to completely distinguish these from infectious ones, as the latter may trigger hyperimmune reactions, lead to vascular alterations, enhance antigen presentation and fibroblast activity, attract neutrophils and eosinophils with release of their toxic products and disturb the level of immunotolerance. in contrast, augmented immunosuppression in the course of interstitial lung disease enhances the susceptibility for infections leading to additional damage. the extent of pulmonary mortality and morbidity, the degree and the spectrum of pulmonary diseases in children as well as the relevance of possible monocausal or multifactorial etiologic risk factors (gvh-disease, irradiation, chemotherapy especially with cyclophosphamide, bleomycin, busulfane, melphalane) is still classified insufficiently [26, 52, 8, 17, 3] . the introduction of fractional irradiation schedules has led to a reduction of the incidence of interstitial pneumonia [17] . latent viral infections, gvhd and cytotoxic factors are not well defined in their pathogenetic role and s11 data about the influence of fungi is limited [5, 20, 13] . histologic evaluations of lung tissue in gvhd-patients show epithelial and interstitial lesions typical for gvhd of the skin and the intestine [83]. there is some evidence for an active role of cmv in bronchiolitis obliterans (bo) as well as in interstitial pneumonias. since the detection of cmv in bronchoalveolar lavage (bal) can be taken as an indicator of invasive infection [9, 69, 63] , screening for cmv in bal and blood has been suggested as a basis for rigorous treatment [38, 1, 30, 39, 25] . the same may hold true for other viruses such as adeno-, ebv, influenza, parainfluenza virus, hhv-6 and rsv (75, 34) . it is usual to separate pulmonary complications up to day 100 (early) from those after day 100 (late), although for some risks the division is arbitrary. if one follows lung function parameters over time in clinically asyptomatic patients after hsct for childhood leukemia [11] , abnormalities can be observed in more than 80% of patients with predominance of a restrictive pattern 6 months after transplantation (possibly due to conditioning). lung function abnormalities tend to persist in patients with advanced disease states before transplantation but decrease by 50% in those with transplantation at early stages of childhood leukemia, suggesting a summation of adverse effects. toxic pulmonary reactions may occur within 90 days after irradiation. clinically overt non-infectious pulmonary complications in the neutropenic (early) phase after transplantation occur in the form of engraftment-syndrome, idiopathic pneumonia syndrome (ips) or diffuse alveolar hemorrhagesyndrome (dah). furthermore the lung can be part of a systemic vascular process (capillary-leak-syndrome, thrombotic-thrombocytopenic purpura or cytokine-syndrome). late pulmonary sequelae manifest as bronchiolitis obliterans (bo), bronchiolitis obliterans organizing pneumonia (boop) or fibrosis. there are several distinct cellular systems involved in the maintenance of pulmonary integrity and, accordingly, in the disease process: (a) alveolar cells or pneumocytes are a source of surfactant and contribute to metabolism within the lung as well as to lung defence. (b) bronchial epithelial cells contribute to the clearance of bacteria and are important barriers against invasion. (c) vascular endothelial cells are essential for regulating gas exchange, fluid homeostasis (selectins, endothelin), and the migration of blood cells. (d) fibroblasts are sources of metalloproteinases and contribute to the repair (and fibrotic changes) of lung tissue. (e) dendritic cells act as antigen-presenting-cells. (f) t-cells target pulmonary structures in gvhd or autoimmunity. (g) neutrophils are the leading cells in bacterial defense but are also sources of proteases with strong elastolytic activity. (h) recently a role has been proposed for alveolar macrophages in disease processes after hsct (76) . it is beyond the scope of this review to evalute the literature on the spectrum of immune mechanisms which include direct and indirect allorecognition as well as the activation of chemokines and lymphokines. pulmonary diagnostics include lung function testing (bedside with portable spirometers, bodyplethysmography), capillary blood gas analysis, non-invasive transcutaneous assessment of oxygen saturation, exercise tests, bronchoalveolar lavage (bal), transbronchial (tbb) or open lung biopsy, ultrafast or thin section computed tomography (ct) (45, 49, 27, 21, 50, 19, 54) . material obtained should be evaluated by the most sensitive and specific microbiological and pathological techniques (7) . in the early phase, infectious complications are the leading differential diagnoses, predominantly by gram-positive and gram-negative bacteria of the endogenous flora. in prolonged neutropenia, invasive fungal infections pose considerable diagnostic and therapeutic problems. secondly, infections by cytomegaly, ebv, adeno-, respiratory-syncytial, parainfluenza and influenza virus, hhv-6, as well as by chlamydia, mycoplasms, legionella, mycobacteria, toxo-plasma, and pneumocystis-carinii should be considered. infections by herpes virus have reached their nadir 90 days post hsct. however, all viral, bacterial and fungal infections can also occur at a later time point depending on the state of immunity, donor selection, and preparation of the transplant. furthermore, gvhd and its therapy play a pathogenetic role in the development of so-called noninfectious pulmonary complications. the treatment or prophylaxis of infectious diseases may have equally profound impact on the pattern of non-infectious lung diseases observed (4). among non-infectious pulmonary complications in the early phase, a group of diffuse interstitial pneumonias is responsible for high mortality. some of these alterations were defined at the 1991 nihlbi workshop in bethesda as "idiopathic pneumonia syndrome" (ips) [15] . immediately after transplantation the so-called "engraftment syndrome" or "capillary-leak syndrome" play an important role (47) , which has to be distiguished from diffuse alveolar damage (dad) or the idiopathic pneumonia syndrome. the latter ones are considered primarily as forms of ards because their time course, their histopathologic morphological criteria and their clinical appearance closely resemble this complicationwhich, despite various causes, generally has an unfavourable prognosis [12, 13] . the incidence of ips after allogeneic hsct has been reported as 12% [15] or 8% [41] for all ages, and is certainly lower for all donors among children. the mortality of this complication is high, despite intensive care treatment including mechanical ventilation (36) . according to a retrospective analysis of 1165 hsct patients, mortality is estimated to be around 70% [41] . essential diagnostic criteria are (1) indicators of diffuse alveolar damage (multilobular infiltrates in thorax x-ray or disturbance of gas transfer, a restrictive ventilatory defect, clinical signs of pneumonia) and (2) the exclusion of active infection of lower airways by bronchoalveolar lavage (bal) or transbronchial biopsy (tbb). patients with severe acute graft versus host disease (gvhd grade iii-iv) have a largely increased risk of developing ips. it has not been clear whether ips after transplantation represents t-cell-mediated lung damage (e.g. "acute gvhd of the lung") or is triggered by occult viral infections (e.g. adeno or hhv-6), or can be considered as a consequence of a multiple organ failure during generalised activation of inflammatory mediators (analogous to the multifactorial ards of non-transplanted patients) in acute gvhd, in the course of viral infections or sepsis. there is no specific therapy for the ips to date. therefore diffuse infiltrates on x-ray after transplantation have to be subject to a thorough work-up. if they do not respond to diuretics (to exclude pulmonary edema), they have to be evaluated radialogically, treated empirically for a short time and/or evaluated invasively depending upon the radiological appearance before or shortly after the start of antibiotics. diagnostic criteria for "capillary leak syndrome", which also occurs in the early phase after transplantation, are inadequate weight gain and disturbance of gas transfer. therapy with c1-esterase inhibitor is currently under evaluation (56) . diffuse alveolar hemorrhage syndrome (dah) is another early "non-infectious" pulmonary complication (day +7 until +40). its occurrence has been described predominantly after autologous transplantation [78, 2] . mortality of this disorder ranges from 50-80%. nonspecific symptoms such as progredient dyspnoea, dry cough, fever, hypoxia and diffuse lobular consolidation in xray are considered typical. hemoptoe is rare. early diagnosis (bal) is important since high-dose corticosteroids can then be applied efficiently [70] . because of its high mortality, the late complication "bronchiolitis obliterans" (bo) stands -together with non-classifiable interstitial pneumonia, -in the first line of differential diagnoses for non-infectious pulmonary complications after hsct. not unlike bo after lung transplantation, the etiology remains unclear. lymphocyt-ic bronchitis, alloreactivity after pathologic expression of antigens due to stress (viral, radiation, medications), malnutrition of the bronchial wall, vascular changes, occult aspirations -are all considered to be causative events (71) . histologically the disease resembles bo in patients after heart-lung and lung transplantation; it is interpreted as chronic rejection (prevalence >35%) with high mortality (25%) [79] . the first description of bo after allogeneic hematopoetic stem cell transplantation dates from 1982 [66] . since then this complication has been described after all types of transplantation, including cord blood cell transplantation [57] . in adults one assumes a prevalence of bronchiolitis obliterans after hematopoetic stem cell transplantation in a range between 5-10% [62, 81] . in children, incidences of up to 20% have been reported [68] . if unrecognised, this late complication tends to be lethal, since the therapeutic effect of corticosteroids in advanced stages is poor. the speed of progression for bo and the ideal time for therapeutic intervention are unknown. bo is frequently associated with chronic gvhd [81, 60] and they occur in combination in up to 10% of adult patients [42] and up to 30% in children with gvhd [68] . the clinical symptoms are nonspecific with expiratory wheeze, cough, dyspnoea and hypoxia upon exertion. typical hr-ct-signs of the thorax for bo are peripheral hypovascularity, enlargement of segmental-and subsegmental bronchi, fixed hyperinflation ("mosaic pattern"), and deformed peripheral vessels [31, 67, 6, 80, 35, 58] . it is debatable, however, whether these signs are also typical for bo after hematopoietic stem cell transplantation. some of these signs can only be interpreted correctly if sequential examinations have been performed. furthermore, ct studies in children are rare. in bal differential cytology the relative number of neutrophils is elevated [70] . the definite diagnosis of bronchiolitis obliterans can frequently only be achieved by open lung biopsy. the histopathological finding is bronchiolar inflammation with luminar scarring, fibrous tissue and obliteration of the small bronchioli without occluding the alveolar ducts. in lung function the small airways are affected first (hyperinflation) and there is exercise limitation without hypoxia. the involvement of vessels can vary. therapeutically, systemic glucocorticosteroids, cyclophosphamide, azathioprine, chloroquine, glucocorticosteroid pulses, cyclosporine, anti-thymocyte globulin, tacrolimus, methotrexate, total lymphoid irradiation, thalidomide, clofazimine, extracorporal photopheresis, humanized monoclonal anti-il-2 receptor antibodies are applied in bo without convincing success. furthermore, there have been no controlled studies of any of the above-mentioned medications. as a number of interstitial diseases in childhood have shown a favourable response [22, 61, 59] , our approach is to try to detect the disease at an incipient stage and use methylprednisolone pulse therapy as a first-line treatment (32) . in about 30-50% of cases, at least a temporary response can be seen. unfortunately, the time window for intervention is small in most cases and the speed of progression extremely variable, rendering evaluation of therapeutic success difficult. complications of bo are pneumothorax, pneumomediastinum, bronchiectases, hypercapnia and pulmonary hypertension with cardiac failure. the "bronchiolitis obliterans organizing peumonia" (boop), which occurs more rarely, was defined 1985 by epler [24] . the etiology remains unclear. in the literature boop has been described in autoimmune diseases, after medication,as well as with viral and bacterial infection and as idiopathic boop [18] . lately a number of cases have been published after hsct with gvhd [44, 48, 74, 40] . there are also reports on early and fulminant courses [55, 16, 82] . the clinical picture is non-specific, with cough, dyspnea, and hypoxia. lung function shows a restrictive pattern and impairied diffusion capacity. histopathology demonstrates granulation -and fibrous tissue in terminal bronchioles reaching into alveoli -as well as interstitial lymphocytic inflammation. cellular profiles of bal show high lymphocyte counts, with low cd4/cd8-ratio (results from adults) [64] . characteristic hr-ct-patterns in boop are ground-glass opacification and nodular shadows [29] . typical bilateral, focal, predominantly peripheral infiltrates can be detected earlier using hr-ct rather than by x-ray of the thorax [51, 46] . in the literature (including patients after hsct) the beneficial effect of treatment with glucocorticosteroids is emphasized beginning at an early stage and continuing for 6-12 months (18, 40, 44, 48, 50, 75) . bronchiolitis obliterans organizing pneumonia (boop) must be separated diagnostically from "lymphocytic interstitial pneumonia (lip)", "diffuse alveolar damage" (dad) and "lung fibrosis" as well as a nsid (nonspecific interstitial disease) and pulmonary veno-occlusive disease (42, 77, 3, 4) . these diseases tend to occur after hematologic diseases, infections and therapeutical interventions (radiation, chemotherapy). some of them are termed as late onset non specific interstitial lung disease (lonsild). prognosis varies greatly. treatment modalities range from antioxidants to supportive care (oxygen) (72) . with severe progression, the only chance for survival may be lung transplantation. post transplant lymphoproliferative disease (ptlpd) can manifest itself in the lung and can occur any time after transplantation. frequency varies depending on the kind of transplantation, intensity and mode of immunosuppression, ebv-status of donor and recipient, and bone marrow reconstitution. although rarely confined to the lung, the occurrence of this lesion together with other infections (like aspergillus) or gvhd makes diagnosis difficult, usually requiring an invasive approach. the who classification is not applied uniquely. treatment modalities range from lowering immunosuppression to chemotherapy, using anti cd 20 antibodies to antiviral therapy, depending, among other factors, on histology, ebv-status, extent of the disease, and clinical course. lung transplantation has been applied as ultima ratio in patients after an interval of up to 14 years after hsct, mostly after pulmonary complications involving gvhd. the preferred surgical technique in most cases has been double lung transplant. reported survival is similar to transplantation for other causes -with one patient surviving more than seven years [37] . out of this series, one patient developed boop, a condition rarely seen after lung transplantation. a girl with a lobar transplant from the same living donor as her hematopoetic stem cells is the only known person without immunosuppression after lung transplantation [73] . generally, prognosis after lung transplantation is a 45% survival rate after 5 years. pulmonary complications after hematopoetic stem cell transplantation are poorly understood and treatment is unsatisfactory. there is no standardised pulmonary follow-up for children after hematopoetic stem cell transplantation with disappointing rates of pulmonary function measurements [23] . on the other hand, the spectrum of pulmonary complications is limited and the time sequence more or less known. diagnostically it would seem essential to follow a scheduled plan of pulmonary surveillance measures including body plethysmography, assessment of gas exchange and exercise capacity as well as screening for infections. this should make it possible to gain insight into the pathogenesis of the diseases and examine whether bronchiolitis obliterans can be prevented by therapeutical measures at an early stage. as therapeutical options are limited in advanced bo, prophylaxis and preemptive therapy may be crucial. as a prerequisite, risk factors should be carefully studied and causal strategies developed in a multicenter approach. (7) the treatment of inborn metabolic diseases such as lysosomal hydrolase deficiencies [21, 32] and peroxisomal disorders such as adrenoleukodystrophy (ald) [1, 28] has been limited to symptomatic supportive care. intravenous enzyme replacement has been largely ineffective, especially in storage diseases with central nervous system (cns) manifestations. allogeneic hematopoietic cell transplantation (hct) with related or unrelated bone marrow cells, peripheral blood progenitor cells, or placental (umbilical cord) blood cells in these conditions repopulates recipient hematopoietic and lymphoid cells with metabolically normal donor-derived cells and thus provides a self-renewing source of hydrolase. in addition, hct effectively repopulates mononuclear phagocytic cells, including macrophages, hepatic kupffer cells, and pulmonary alveolar macrophages, in which substrate often accumulates in storage diseases. as discussed below, the repopulation of microglia with donor-derived cells after allogeneic hct is a potentially important therapeutic mechanism of allogeneic hct in patients with cns manifestations of metabolic diseases. intercellular transport of lysosomal hydrolase from normal donor cells occurs by both receptor-mediated endocytosis and direct transfer of enzyme from adjacent cells [2] . the heterogeneity of receptor-mediated endocytosis systems limits the extent of hydrolase uptake among various cell types and tissues [38, 40] . direct intercellular transfer of hydrolase occurs independent of specific receptors but requires cell-to-cell contact and participation of adhesion molecules such as lfa-1 and -3, icam-1, -2, and -3, and cd2 (the sheep erythrocyte receptor on t lymphocytes) [2, 34] . unlike lysosomal storage diseases, ald is due to a defective protein component of the peroxisomal membrane that is neither secreted from normal cells nor transferred between cells, leading to defective oxidation and elevated plasma levels of very long chain fatty acids (vlcfas) [30] . co-culture with normal cells does not improve vlcfa oxidation by ald fibroblasts, indicating that improvement of ald after hct is likely due to repopulation by metabolically normal cells instead of intracellular transfer of molecules from donor cells. other beneficial effects of allogeneic hct in ald include normalization of plasma vlcfa levels and decreased perivascular inflammation. microglia are the mononuclear phagocytic cells in the cns [11, 24] and account for approximately 5% to 10% of non-neuronal cells in the brain. activated microglia, also referred to as cns macrophages, are involved in antigen presentation and responses to inflammation, infection, or cns injury [11] . microglia are derived from hematopoietic precursors that normally enter the brain from the peripheral circulation during embryonic and early postnatal life but not in adulthood [35] . in rodent hct recipients, donor-derived cells can be identified throughout the cns [12] and over time completely repopulate the microglial compartment [18, 22, 59] . in feline β-mannosidosis, these donor cells effectively transfer lysosomal hydrolase to recipient neurons in vivo [52] . after hct, donor-derived cells also differentiate into other non-neuronal cns cell populations such as astrocytes (also referred to as macroglia) [5, 31] . postmortem studies confirm that donor mononuclear cells are also present throughout the brains of human allogeneic hct recipients [51] . the kinetics of repopulation of microglia after hct is slower than that observed with other mononuclear phagocytes like pulmonary alveolar macrophages and hepatic kupffer cells [3, 25] . in humans, post-hct repopulation with donor-derived microglia requires approximately 1 year, which may explain the ineffectiveness of allogeneic hct in stabilizing or pre-venting neurological deterioration in some rapidly progressive storage diseases [24, 25] . the availability of spontaneously occurring heritable animal models and the development of transgenic "knockout" lysosomal hydrolase deficient animals allows preclinical evaluation of the biochemical, physiological and clinical effects of allogeneic hct in storage diseases. the most extensive studies of hct in preclinical storage disease models have been in canine α-l-iduronidase deficiency (a model of mucopolysaccharidosis [mps] ih, or hurler syndrome) [3, 8, 45] , murine β-galactosidase deficiency (a model of mps vii, or sly syndrome) [4, 42, 55] , and murine galactosylceramidase deficiency (the "twitcher" mouse, a model of the sphingolipidosis globoid cell leukodystrophy [gcl]). we have extensively studied hct in the twitcher murine model [49] , which most closely resembles krabbe disease, the early infantile form of human gcl. hct leads to prolonged survival, clinical improvement, attenuation of hindlimb paralysis [57, 58] , remyelination in peripheral nerves and cns [58] , and stabilization of motor nerve conduction velocities [50] in presymptomatic twitcher mice but is of no benefit in symptomatic animals. after hct, galactosylceramidase is present in the cns and non-neural tissues [14, 60] , and levels of the toxic metabolite psychosine (galactosylsphingosine) [15] , are significantly decreased in the cns [14] . postnatal hct is not curative in murine gcl, most likely because repopulation of the cns and peripheral nervous system with donor cells does not occur rapidly enough to stabilize the progressive demyelination. murine gcl may be an important model for critical evaluation of the effects of intrauterine cellular transplantation for infantile-onset sphingolipidosis. several recent reports have summarized the results of allogeneic hct for storage diseases and ald [27, 44, 56] . this review will focus on current concepts of allogeneic hct for mps ih (hurler syndrome), globoid cell leukodystrophy and ceramidase deficiency (farber disease). the prognosis of untreated mps ih (α-l-iduronidase deficiency) is poor, with a median survival of 4.5 years and with very few children surviving into the second decade of life [23] . in contrast, survival after hct in children with mps ih is 75% after bone marrow transplantation (bmt) from hla-identical siblings, 53% after bmt from hla-mismatched relatives, and 49% after bmt from unrelated donors [36, 37] . hepatosplenomegaly, joint mobility, and upper airway obstruction in mps ih resolve within months to a year after hct. corneal clouding stabilizes or slowly resolves [9] , and visual acuity may improve even without regression of corneal clouding. unfortunately hct does not correct the skeletal manifestations (dysostosis multiplex) of mps ih, which are due to metabolic dysfunction in chondrocytes and osteoblasts that arise from mesenchymal precursors. the orthopaedic complications of mps ih require ongoing evaluation and surgical management even after hct [7] . mental retardation is a hallmark of untreated mps ih [53] , and both age and neurodevelopmental status are important determinants of outcome after hct. when carried out in patients under age 2 years, hct preserves neurocognitive function and prevents or reverses increased intracranial pressure. this favorable outcome is in part due to the fact that the developmental quotient (dq) falls below 70 in most children with mps ih after age 2 years [43] . in general, children with mps ih with normal intelligence before hct maintain that level of cognitive functioning after transplant [36, 43, 54] , but those with significant neurocognitive impairment (e.g., dq below 70) at hct have progressive deterioration and do not benefit from the procedure. globoid cell leukodystrophy (galactosylceramidase deficiency) is one of the sphingolipidoses, which are characterized by demyelin-ation of the cns and/or peripheral nervous system because of deficiencies in specific acid hydrolases involved in the metabolism of sphingomyelin, gangliosides and cerebrosides [48] . characteristic globoid cells, derived from microglia or cns macrophages and containing periodic acid-schiff (pas)-positive myelin breakdown material, are present in the brains, spinal cord and peripheral nerves. interestingly, the widespread demyelination in gcl is due to accumulation of psychosine (galactosylsphingosine), which is derived from the substrate galactosylceramide and is toxic to both oligodendroglia and schwann cells [15] . the four clinical phenotypes of human gcl vary in clinical manifestations and tempo of disease progression. patients with early (age of onset, 3 to 6 months) or late (age of onset, 6 months to 3 years) infantile gcl have profound psychomotor retardation, failure to thrive, spasticity, seizures, optic atrophy and cortical blindness [10] . juvenile gcl affects children from 3 to 10 years of age, with insidious onset and progression of visual loss, lower extremity spasticity, and in some patients dementia. adult gcl occurs in patients over age 10 years and is characterized by slowly progressive difficulty in walking, long-tract signs, asymmetric weakness of the extremities, and difficulties with coordination and balance, but intellect is generally unaffected [20] . allogeneic hct is not curative in symptomatic early infantile gcl (krabbe disease), in which neurodegeneration persists despite post-transplant biochemical improvement [23, 26] , analogous to observations in the preclinical studies of hct in murine gcl. although very limited experience suggests that presymptomatic infants with infantile gcl may benefit from hct [26] , early identification of the affected infant and of a suitable hct donor are obvious practical challenges to this therapeutic strategy. the role for allogeneic hct is more firmly established in patients with presymptomatic or minimally symptomatic juvenile or adult gcl, in whom hct leads to stabilization and gradual improvement in clinical, neurological and neurocognitive status [13, 26, 43] . of the six phenotypes of ceramidase deficiency [29] , at least 50% of patients have the classic infantile form, or farber disease phenotype. affected infants have painful joint swelling, multiple painful subcutaneous nodules, hoarseness, swallowing difficulties, and failure to thrive [6] . microscopic examination of the nodules shows granulomata containing ceramide-laden macrophages. granulomata in the aerodigestive tract cause hoarseness and deglutition problems. hepatomegaly, recurrent pulmonary infections and respiratory difficulties are common and due in large part to organ infiltration by ceramide-laden macrophages. infants with farber disease die with progressive and profound psychomotor retardation at a mean age of 18 to 20 months [29] . two infants with farber disease received allogeneic bmt at 18 months [47] and 9 months [61] of age, respectively. subcutaneous nodules, joint pain and hoarseness regressed within 2 months after transplant in both patients. the older patient had progressive neurodegeneration and died 6 months after bmt. the younger patient, who had mild developmental delay and slight hypotonia at bmt, had progressive psychomotor retardation and graft failure, with a developmental age of 4 months at chronological age 32 months. despite loss of donor cell engraftment, levels of ceramidase in the peripheral blood leukocytes remained in the donor heterozygous range, suggesting ongoing production of ceramidase by non-circulating donor cells and hydrolase uptake by recipient blood cells [61] . these limited observations indicate that hct does not stabilize the rapid neurological deterioration in classic farber disease. among new approaches for treatment of metabolic disorders by cellular transplantation, intrauterine hct may be worthy of exploration, especially in the rapidly progressive infantile forms in which postnatal hct is not effective. the major limitation to this approach is the low levels of donor cell engraftment, which may not provide sufficient hydrolase or metabolically active cells to correct the underlying biochemical defect [17] . allogeneic hct using reduced-intensity nonmyeloablative preparative regimens may be considered in some indolent forms of storage diseases, providing gradual donor cell engraftment without the risk of aplasia and other short-and long-term toxicities associated with intensive marrow-lethal preparative regimens [19, 46] . the use of these regimens is not feasible in more aggressive forms of storage diseases. insertion of genes for specific lysosomal hydrolases or for the ald protein into autologous hematopoietic stem cells (hscs) and transplantation of these cells is theoretically very attractive but remains at the level of preclinical investigation at this time because of ongoing challenges such as efficient introduction and integration of the exogenous gene in truly primitive hscs and sustained, consistent high-level expression of the hydrolase in these cells and their progeny [41] . a potentially exciting approach for storage diseases with skeletal manifestations (dysostosis multiplex) is the co-transplantation of mesenchymal stem cells (mscs), which may differentiate into chondrocytes and osteoblasts [33, 39] . although clearly not a clinical therapeutic option at this time, intracerebral injection of mscs may favorably affect the neuropathological and biochemical abnormalities in a murine model of acid sphingomyelinase deficiency (niemann-pick disease types a and b) [16] . clinical experience for more than two decades has shown that allogeneic hct may benefit some but not all patients with inherited metabolic diseases. the hct procedure is most effective in presymptomatic patients and those with indolent forms of storage diseases but is ineffective in those with overt neurological symptoms or aggressive neonatal or infantile forms. hct alone does not correct skeletal dysplasia in mpss and may not prevent development or progression of the peripheral neuropathy in sphingolipidoses and ald. decisions regarding hct in 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cord_uid: ecmayzr6 multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns) characterized by demyelination and axonal loss. demyelinating lesions are associated with infiltrating t lymphocytes, bone marrow-derived macrophages (bmdm), and activated resident microglia. tissue damage is thought to be mediated by t cell produced cytokines and chemokines, which activate microglia and/or bmdm to both strip myelin and produce toxic factors, ultimately damaging axons and promoting disability. however, the relative contributions of bmdm and microglia to demyelinating pathology are unclear, as their identification in ms tissue is difficult due to similar morphology and indistinguishable surface markers when activated. the cd4 t cell-induced autoimmune murine model of ms, experimental autoimmune encephalitis (eae), in which bmdm are essential for demyelination, has revealed pathogenic and repair-promoting phenotypes associated with bmdm and microglia, respectively. using a murine model of demyelination induced by a gliatropic coronavirus, in which bmdm are redundant for demyelination, we herein characterize gene expression profiles of bmdm versus microglia associated with demyelination. while gene expression in cns infiltrating bmdm was upregulated early following infection and subsequently sustained, microglia expressed a more dynamic gene profile with extensive mrna upregulation coinciding with peak demyelination after viral control. this delayed microglia response comprised a highly pro-inflammatory and phagocytic profile. furthermore, while bmdm exhibited a mixed phenotype of m1 and m2 markers, microglia repressed the vast majority of m2-markers. overall, these data support a pro-inflammatory and pathogenic role of microglia temporally remote from viral control, whereas bmdm retained their gene expression profile independent of the changing environment. as demyelination is caused by multifactorial insults, our results highlight the plasticity of microglia in responding to distinct inflammatory settings, which may be relevant for ms pathogenesis. introduction multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns), characterized by demyelination and axonal damage. active demyelinating lesions are characterized by cd8 t cells, cd4 t cells expressing both th1 and th17 cytokines, bone marrow-derived macrophages (bmdm) and activated cns resident microglia (1, 2) . myeloid cells activated by t cell effector functions are thought to participate in tissue damage by removing or "stripping" myelin (3) , and secreting toxic factors, such as reactive oxygen species, nitric oxide and the pro-inflammatory cytokines, tumor necrosis factor (tnf), and il-1β (4, 5) . activated microglia also secrete chemokines, which recruit innate and adaptive immune cells into the parenchyma, further amplifying the destructive inflammatory response (5) . however, both bmdm and microglia effector functions are highly heterogeneous depending on the environment and may not only contribute to disease progression but also to resolution (6, 7) . for example, by removing apoptotic cells and debris, their phagocytic activity favors tissue repair and is essential for disease resolution (3) . in addition, both cell populations secrete anti-inflammatory cytokines, such as il-10 and tgf-β, as well as trophic factors, which provide an environment that promotes tissue repair and neuronal protection (8) . the heterogeneity of the inflammatory response associated with ms lesions at the cellular and functional levels, thus makes it difficult to establish detrimental versus disease resolving functions of bmdm and microglia in ms pathogenesis. in addition to the inherent limitations associated with sampling cns tissues for longitudinal studies, the individual role of bmdm versus microglia as pathological mediators remains ambiguous due to morphological similarities and lack of reagents uniquely identifying each population. however, increasing evidence from animal models supports the concept that microglia and bmdm comprise two effector populations with distinct origins (derived from progenitors in the embryonic yolk sac and circulating monocytes respectively) and functions during ms and other neuroinflammatory disorders (9) . a variety of murine models, including autoimmune-and viral-induced demyelination, have been developed to study pathogenic features of ms (10) . the most common is the experimental autoimmune encephalomyelitis (eae), an autoreactive cd4 t cell-induced autoimmune demyelination characterized by infiltration of myelin-specific th1 and th17 cells, bmdm and microglial activation (11, 12) . pathogenesis during eae is associated with temporally distinct microglial activation and bmdm cns infiltration. early microglia activation is insufficient to trigger clinical disease, whereas delayed cns recruitment of bmdm directly correlates with disease progression. importantly, depletion of bmdm but not microglia inhibits eae (13, 14) . similarly, mice deficient in ccl2 (ccl2 −/− ), a chemokine essential for inflammatory monocyte recruitment into the cns (15) , are resistant to eae (16) . in support of detrimental bmdm functions, a combined histological and gene profiling study showed that demyelination is mediated by bmdm associated with nodes of ranvier, whereas debris clearance is achieved by microglia (17) . altogether, studies in the eae model demonstrate that bmdm recruitment into the cns is essential for the process of myelin loss and clinical manifestation. inflammatory demyelination is also induced following infection with two natural viral mouse pathogens, theiler's murine encephalomyelitis virus (tmev) and members of the neurotropic mouse hepatitis viruses (mhv). tmev infection induces an autoimmune disease in which bmdm are essential for both viral persistence and demyelination (18, 19) . however, the function of bmdm as a main reservoir of active viral replication during chronic tmev infection, limits efforts to assess their role in demyelination independent of virus load (20) . in contrast, infection with the non-lethal glia tropic mhv strain designated jhmv predominantly targets oligodendrocytes (olg) and to a lesser extent microglia and astrocytes. viral replication peaks at day 5 post infection (p.i.), but infectious virus is reduced below detection by day 14 p.i. acute infection initiates rapid cns recruitment of predominantly bmdm, but also neutrophils and nk cells, followed by infiltration of both cd8 and cd4 t cells, as observed in active ms lesions. the t cell response, which is essential to reduce viral replication, is highly th1 polarized with no evidence of il-17 or gm-csf production (21) (22) (23) . importantly, t cell-mediated virus control coincides with initiation of demyelination, which peaks between days 14-21 p.i. after infectious virus is cleared (24, 25) . although olg tropism is a requirement for demyelination, immunodeficient mice demonstrated that infection of olg in the absence of adaptive immunity is insufficient to cause demyelination. however, transfer of either virus-specific cd4 or cd8 t cells into virus infected immunodeficient mice leads to demyelination (26, 27) . furthermore, ifn-γ dependent control of infectious virus within olg and no evidence for olg apoptosis, suggested that direct t cell-mediated cytolysis of olg does not play a major role in myelin loss (28) . this implicates t cell activated bmdm and microglia as the most probable mediators of myelin destruction. moreover, both myeloid populations are abundant in lesions and occasionally associated with damaged axons (29) . however, in contrast to eae, genetic or chemical depletion of monocytes during jhmv infection does not alter disease severity, virus replication or myelin loss (30, 31) , suggesting that bmdm are dispensable for jhmv-induced demyelination. this study takes advantage of the distinct tissue environments established during eae and jhmv infection to characterize temporal alterations in gene expression profiles of bmdm versus microglia in a th1 biased demyelination model. to date, we are not aware of any reports evaluating the signature profile of microglia associated with pathogenic functions during demyelination. the results reveal that cns infiltrating bmdm rapidly establish a characteristic profile including m1 and m2 markers, which prevails throughout infection as the population declines. by contrast, gene expression in microglia is only prominently altered remote from viral control concomitant with demyelination; distinct from bmdm, the gene expression pattern is skewed to a highly pro-inflammatory and phagocytic profile. the results overall highlight the plasticity of microglia responses in distinct inflammatory settings, which may be relevant for ms pathogenesis at distinct stages of disease. wild-type (wt) c57bl/6 mice were purchased from the national cancer institute (frederick, md, usa). homozygous ccl2 deficient (ccl2 −/− ) mice were originally obtained from b. j. rollins (dana-farber cancer institute, boston, ma, usa). cx3cr1 gfp/gfp (b6.129p-cx3cr1 tm1litt /j) and ccr2 rfp/rfp (b6.129 (cg)-ccr2 tm2.1ifc /j) mice were purchased from the jackson laboratory (bar harbor, me, usa) and crossed to generate cx3cr1 gfp/+ ccr2 rfp/+ mice. transgenic mice were bred and maintained at the biological research institute under sterile conditions. all procedures were preformed in compliance with the cleveland clinic institutional animal care and use committee approved protocols. the glia tropic jhmv neutralizing monoclonal antibody (mab)derived 2.2v-1 variant was used for all infections (32) . mice of both sexes between 6 and 7 weeks of age were infected in the left hemisphere with 1,000 pfu of jhmv diluted in endotoxin-free dulbecco's phosphate-buffered saline (pbs) in a final volume of 30 µl. mice were monitored daily for clinical disease severity according to the following scale: 0, healthy; 1, hunched back and ruffled fur; 2, partial hind limb paralysis or inability to maintain the upright position; 3, complete hind limb paralysis; 4, moribund or dead. for analytical flow cytometry, anesthetized mice were perfused with ice-cold pbs, and resected brains and spinal cords homogenized using a ten-broeck tissue grinder as described (33) . tissue homogenates were adjusted to 30% percoll (pharmacia, uppsala, sweden) and underlaid with 1 ml 70% percoll prior to centrifugation at 850 g for 30 min at 4°c. cns mononuclear cell were recovered from the 30/70% interface, washed and resuspended in facs buffer (pbs + 1% bovine serum albumin). cells were blocked with anti-mouse cd16/cd32 (clone 2.4g2) mab for 15 min on ice prior to staining. staining was performed for 30 min on ice using fluorescein isothiocyanate, phycoerythrin, peridin chlorophyll protein complex (percp), or allophycocyanin (apc) conjugated mab (all from bd biosciences except where indicated) specific for cd45 (clone ly-5), cd11b (clone m1/70), f4/80 (serotec, raleigh, nc, usa) and major histocompatibility complex (mhc) class ii (clone 2g9). cells were then washed twice in facs buffer prior to analysis using a bd accuri flow cytometer (bd biosciences) and flowjo software (tree star inc., ashland, or, usa). for cell purification, spinal cords from pbs-perfused cx3cr1 gfp/+ ccr2 rfp/+ mice were finely minced with a razor blade. minced tissues were enzymatically digested in rpmi 1640 medium containing 10% fetal calf serum, 0.5% collagenase d (100 mg/ml) roche, basel, switzerland and 1% dnase i (1 mg/ml) (sigma aldrich, st. louis, mo, usa) for 40 min at 37°c. collagenase was then inactivated by addition of 1% 0.1 m edta for 5 min at 37°c prior centrifugation at 400 g for 7 min at 4°c. spinal cord-derived cells from seven mice were pooled and isolated using percoll gradients as described above and then stained with cd11b-percp and cd45-apc for 30 min on ice. spinal cord-derived bmdm (cd45 hi cd1 1b + ccr2 rfp+ ) and microglia (cd45 low cd11b + cx3cr1 gfp+ ) were purified using a facsaria cell sorter (bd biosciences) and resuspended in trizol. yields from 7-pooled mice ranged between 5.4-20 × 10 5 cells for bmdm and 0.5-1.2 × 10 5 cells for microglia depending on the time p.i. microglia from naïve mice were used to assess baseline expression, whereas circulating monocytes were used as controls for cns infiltrated bmdm after infection. monocytes were isolated from blood treated with gey's solution to lyse red blood cells prior to staining and cell sorting. gene expression profiling using ncounter analysis rna was prepared by extraction with trizol reagent (invitrogen, carlsbad, ca, usa) and direct-zol rna mini prep (zymo research, irvine, ca, usa) according to the manufacturer's instructions. gene expression profiles were analyzed using the ncounter mouse myeloid innate immune panel comprising 754 targets representing all major myeloid cell types and generated according to the manufacturer's protocol (nanostring technologies, seattle, wa, usa). the nanostring ncounter system directly captures and counts individual mrna transcripts using a multiplexed measurement system thereby omitting cdna based amplification (34) . analysis was performed using nsolver analysis software v3.0 and ingenuity pathway analysis (qiagen, hilden, germany). venn diagrams from individual gene lists and protein-protein interaction networks were constructed using genespring (agilent, inc.) and string software (http://www. string-db.org). to confirm validity of nanostring ncounter analysis, a small set of selected genes were analyzed by real-time pcr ( figure s1 in supplementary material). following rna extraction as described above, first-strand cdna was synthesized using reverse transcriptase (invitrogen) with oligo-dt and random primers (promega, madison, wi, usa) as described (35) . gene expression analysis was performed using a 7500 fast real-time pcr system (applied biosystems, foster city, ca, usa), sybr green master mix (applied biosystems) and the following primers: gapdh, 5′-catggccttccgtgttccta-3′ (forward) and 5′-atgcctgcttcaccaccttct-3′ (reverse); il15, 5′-tg aggctggcattcatgtctt-3′ (forward) and 5′-tccagtt ggcctctgttttagg-3′ (reverse); il1rn 5′-agatagacatg gtgcctattgacctt-3′ (forward) and 5′-catctccagac ttggcacaaga-3′ (reverse) and arg1 5′-tgggtggatgct cacactga-3′ (forward) and 5′-caggttgcccatgcaga tt-3′ (reverse). transcripts levels were normalized to the housekeeping gene gapdh and converted to a linearized value using the following formula: 2 (c t gapdh-c t gene) × 1,000, where ct represents the threshold cycle value. following pbs perfusion, spinal cords were fixed in 10% neutral buffered formalin, embedded in paraffin and sections stained with luxol fast blue as described to visualize demyelination (36) . for analysis of iba1 + cells spinal cords from ice-cold pbs-perfused mice were quickly embedded in oct and kept at −80°c until 10 µm sections were prepared. sections were fixed with paraformaldehyde for 20 min, treated with blocking solution for 30 min and then stained with rabbit anti-iba1 mab (wako, osaka, japan) overnight at 4°c. goat anti-rabbit secondary ab (invitrogen) was added for 1 h at room temperature and sections mounted with vectashield mounting medium (vector laboratories, burlingame, ca, usa). sections were analyzed using a leica tcs confocal microscope. to better characterize reactivity of microglia and infiltrating bmdm following jhmv infection, we initially monitored cns infiltration of bmdm, as well as upregulation of mhc class ii as an activation marker on both cns bmdm and microglia by flow cytometry. bmdm with a typical cd45 hi cd11b + f4/80 + phenotype comprised the majority of inflammatory leukocytes as early as day 3 p.i. and then progressively decreased as virus replication is controlled by t cells. at the onset of demyelination at day 10 p.i., the bmdm population stabilized at ~10% of the infiltrating leukocytes ( figure 1a ). bmdm initially infiltrated as mhc class ii lo expressing cells, but the vast majority upregulated mhc class ii by day 7 p.i. mhc class ii expression on microglia was sparse at days 3 and 5 p.i., but rapidly increased by day 7 p.i. and then gradually declined by day 14 p.i. (figure 1a) . these kinetics supported that microglia and bmdm activation peaks delayed relative to peak bmdm accumulation and coincides with peak t cell ifn-γ production (36, 37) . enhanced activation of microglia at day 7 p.i., compared to earlier times p.i., was also supported by progression of morphological changes, evidenced by enlarged cell bodies and retracted and thickened processes ( figure 1b) . the decline of bmdm, but an ongoing activation phenotype of microglia at the time of evident demyelination implicated microglia as mediators of tissue damage during jhmv encephalomyelitis. biochemical depletion of peripheral monocytes indeed supported that bmdm are not essential to tissue destruction in jhmv-infected mice (30) . data from our own laboratory further demonstrated that the chemokine ccl2 is essential for bmdm accumulation within the cns (31) . the absence of ccl2 resulted in an ~80% reduction of bmdm at all time points, including day 14 p.i. (31) and ( figure 1c ) when demyelination is prominently evident in wt mice. nevertheless, microglia activation, as monitored by mhc class ii expression, was independent of ccl2 ( figure 1d ). most importantly, the absence of ccl2-dependent bmdm within the cns did not alter demyelination ( figure 1e ). similar myelin loss at day 21 p.i. comparing wt and ccl2 −/− infected mice supported the concept that microglia mediate demyelination during jhmv infection. we next evaluated effector functions of bmdm versus microglia associated with jhmv-induced demyelination by comparing gene expression profiles using ncounter analysis of mrna isolated from purified bmdm and microglia of infected cx3cr1 gfp/+ ccr2 rfp/+ mice. characteristic expression of cx3cr1 gfp and ccr2 rfp on cd45 high cd11b + bmdm (population #1) and cd45 low cd11b + microglia (population #2) is shown in figure 2 throughout days 5-14 p.i. microglia were characterized by high expression of cx3cr1 and undetectable ccr2 expression (figures 2b,c) similar to other inflammatory models (17, 38) . in contrast, cns infiltrating bmdm expressed ccr2 and low levels of cx3cr1 compared to microglia ( figure 2b ). co-expression of ccr2 and cx3cr1 was maintained on bmdm at all time points p.i. and no cx3cr1 − cells were detectable ( figure 2c ). as both microglia and infiltrating bmdm retained their phenotype throughout infection, cd45 low cd11b + cx3cr1 gfphi ccr2 − and cd45 hi cd11b + cx3cr1 gfplow ccr2 + populations were isolated by facs from spinal cords at days 5, 7, 10, and 14 p.i. for subsequent mrna expression analysis. age-matched naïve animals were used to isolate microglia and blood circulating monocytes as precursors of cns-infiltrating bmdm. gene expression profiles for all purified populations were obtained using ncounter analysis and the innate myeloid immune panel. the respective naïve populations were used to assess signature gene expression profiles under homeostatic conditions (figure 3) . figure 3a shows the top 50 highly expressed genes within each population relative to three ncounter platform housekeeping genes, namely g6pdx, polr1b, and tbp, selected for three high, medium, and low expression, respectively, in this part of analysis platform. figure 3b lists the top 50 enriched genes specific for microglia compared to monocytes, or monocytes versus microglia, respectively. among the top 50 genes highly expressed in microglia, 15 were also specific and included genes of the complement cascade (c1qa, c1qb, c1qc, and c3ar1) and trem2, encoding a cell surface receptor involved in phagocytic functions and known to be expressed by microglia (39) . other genes, such as adamts1, f11r or hpgds, found within the top 50 enriched genes expressed by microglia ( figure 3b ) were also previously described as microglia specific (17, 40) . cx3cr1 mrna encoding the fractalkine receptor and used as a marker for microglia (41) , was also among the top 50 highly expressed genes ( figure 3a) , but not unique, consistent with the cx3cr1 lo phenotype on circulating monocytes. similarly, ccr2 expression characteristic of monocytes was confirmed by ccr2 mrna as the second in place of the top 50 expressed genes specific for circulating monocytes (figure 3b) . other specific signature genes of monocytes are related to motility and migration/tissue invasion, e.g., s100a4, s100a8, s100a9, fn1, sema4, mmp8, and to a lesser extent mhc class ii antigen presentation, e.g., h2-ab1, cd74, and fas. microglia and circulating monocytes also shared 17 highly expressed genes, including genes characteristic of the myeloid lineage such as csf1r, a gene coding for a cell surface receptor essential for hematopoietic figure 4a ). interestingly however, three genes among the common and highly expressed genes were regulated differently. ctss mrna, encoding for cathepsin s, a lysosomal cysteine proteinase participating in the mhc class ii molecule antigen presentation pathway as well as nociception (42, 43) , was specifically upregulated within bmdm, with highest levels observed at days 10 and 14 p.i. (figure 4a) , when demyelination increases. by contrast, microglia transiently downregulated ctss mrna at day 7 p.i. opposite regulation was also observed for dusp1 mrna (figure 4a ). dusp1 mrna encodes the dual specificity protein phosphatase 1, an enzyme involved in the cellular stress response and a negative regulator of cell proliferation (44) . while dusp1 mrna levels were vastly upregulated early following bmdm accumulation, but declined by day 7 p.i. and thereafter, levels were rapidly downregulated within microglia ( figure 4b) . finally, tyrobp mrna encoding for the trem2 adaptor dap12 and known to regulate microglia phagocytic functions (39) , was downregulated in bmdm throughout infection, but specifically upregulated within microglia at days 10 and 14 p.i. (figure 4b ). this expression pattern on microglia correlated with the onset of myelin loss and supported trem2 signaling specifically by microglia in response to tissue damage. to determine whether apparently differential functions of bmdm and microglia associated with jhmv-induced demyelination are reflected in distinct gene profiles, we monitored overall up-and downregulation of gene expression relative to basal levels in each population. analysis times were chosen to correlate with innate responses (d5 p.i.), peak t cell effector function (d7 p.i.), resolution of infection and initiation of demyelination (d10 p.i.), and finally viral clearance and overt demyelination (d14 p.i.). at day 5 p.i. a higher number of genes were differentially regulated within infiltrating bmdm compared to microglia (231 versus 76; figure 5a ). moreover, almost 80% of the genes showing altered expression in early infiltrated bmdm were increased compared to basal levels, while only 54% were increased in microglia; the remaining differentially expressed mrnas were decreased ( figure 5b) . the overall number of differentially expressed genes slightly declined in bmdm by day 7 p.i., when t cells exert maximal effector function (37) , and remained fairly constant throughout day 14 p.i. (figure 5a) . furthermore, the relative decline in the proportion of upregulated mrnas coincided with an increased proportion of downregulated genes, reaching a roughly equal distribution at days 10-14 p.i., when virus is largely controlled (figure 5b ). in contrast, microglia altered their gene expression pattern extensively at day 7 p.i. (97 genes, figure 5a ) with 67% of differentially regulated genes showing increases ( figure 5b ). by day 10 p.i., overall altered gene expression remained stable relative to day 7 p.i., with equal proportions showing increases and decreases. however, as myelin loss progresses by day 14 p.i., differentially regulated genes increased again in numbers, with the proportion of upregulated mrnas reaching 95% (figure 5a ). altogether these data show unique regulation of gene profiles in bmdm compared to microglia throughout the course of infection. while most changes were evident in bmdm following initial cns accumulation, microglia revealed most pronounced changes at the time of myelin loss. we further analyzed differential gene expression across time points focusing on upregulated genes using venn diagrams to reveal the relative proportion of genes that were commonly increased at all time points (figure 5c ). of the 183 upregulated genes in bmdm at day 5 p.i., 62 were unique to day 5. on the other hand, 77 genes (representing 42% of all upregulated genes) remained highly expressed at all other time points (figure 5c ). of note, not a single gene transcript was specifically upregulated at day 7 p.i., and only three overlapped with sustained upregulation at days 10 and 14 p.i. similarly, only 4 gene transcripts were specifically elevated at day 10 p.i., 7 were unique to both days 10 and 14, and only one was unique to day 14 p.i. these results indicate that the gene expression profile characterizing bmdm is established early following infection, with sparse unique alterations as bmdm decline during infection. in stark contrast, only 3 of 41 gene transcripts upregulated in microglia at day 5 p.i. were unique to day 5, and no gene transcript was commonly upregulated across all time points analyzed. furthermore, distinct from bmdm, 59 gene transcripts were figure 3 | gene expression characterizing microglia and circulating monocytes under homeostatic conditions. spinal cord-derived microglia (cd45 low cd11b + cx3cr1 gfp+ ) and circulating blood monocytes (cd11b + ccr2 rfp+ ) were purified from naïve cx3cr1 gfp/+ ccr2 rfp/+ mice by facs and rna subjected to ncounter analysis using the myeloid cell probe panel. panel (a) depicts the top 50 highly expressed genes and (b) the enriched genes uniquely characterizing each population. in (a) * highlights genes that are both highly expressed and enriched in each population, while # highlights genes highly expressed and common to both microglia and circulating monocytes. uniquely upregulated by day 7 p.i., with none common to day 10 p.i., and only six overlapping with those upregulated at day 14 p.i. although no gene transcripts were upregulated uniquely at day 10 p.i., 22 overlapped with those upregulated at day 14 p.i. a further 76 genes, comprising 53% of all upregulated genes at day 14 p.i., were specifically expressed at elevated levels at day 14 p.i. coinciding with overt myelin loss ( figure 5c) . these profiles reveal a dynamic range of responses and extensive plasticity of gene expression profiles in microglia throughout jhmv infection ( figure 5c ). we next used a protein-protein network connection constructed based on differential gene expression to specifically examine upregulation of gene transcripts within bmdm and microglia correlating with demyelination at day 14 p.i. for comparison, we also analyzed the network connection at day 5 p.i., when expression profiles were most prominently altered in bmdm, but more modestly in microglia. this comparative analysis was chosen to provide clues about specific functions and involvement of microglia relative to bmdm in tissue destruction (figures 6 and 7) . our initial focus was on temporally altered networks in bmdm (figure 6) . at day 5 p.i., early infiltrated bmdm expressed a wide array of chemokines regulating cns infiltration of both innate (cxcl2, ccl2, ccl4, ccl5, ccl7, and ccl12) and adaptive (cxcl9, cxcl10) immune cells (figure 6a) . a large cluster of molecules regulating the innate immune response, essential to limit early viral replication (45, 46) , was also expressed by bmdm (figure 6a) . these include pathogen recognition receptors such as tlrs (tlr1-4 and tlr9) and molecules linked to the tnf pathway (tnf, traf6, etc.). finally, bmdm expressed molecules involved in antigen presentation, including tap1 and tap2, as well as t cell activating co-stimulatory molecules (cd80 and cd86) or il-12, which induce th1 differentiation (figure 6a) . these results indicate that early infiltrating bmdm orchestrate the acute innate immune response crucial for limiting cns viral spread, as well as initiating the adaptive immune response by recruiting and activating t cells. at day 14 p.i., correlating with peak demyelination, a more restrained number of mrna transcripts were upregulated in bmdm (figure 6b) . the cluster of chemokines mobilizing immune cells was sustained ( figure 6b) . in contrast, the molecular network extended from tnf was more limited at d14 p.i. compared to d5 p.i. (figures 6a,b) . molecules regulating primarily the cd4 t cell response were expressed at day 14 p.i. and comprised gene transcripts involved in antigen presentation including mhc class ii (h2-ab1) and co-stimulatory molecules such as cd80 and cd86 (figure 6b) . interestingly, among the more restricted number of gene transcripts upregulated at day 14 p.i. in bmdm, several were transcripts encoding m2 molecules, which included arg1, il1rn and tgm2 (figure 6b) . in contrast to the vast number of genes upregulated early following infection in bmdm, a significantly lower number of upregulated genes characterized microglia at day 5 p.i. (figure 7a) . transcripts for chemokines regulating migration of both innate and adaptive immune cells, such as ccl2, ccl3, ccl4, ccl12, cxcl10, and cxcl16, were expressed by microglia at day 5 p.i. (figure 7a) . transcripts for tnf and other inflammatory cytokines generally associated with the innate responses, e.g., il1a, il1b, and il18 were also upregulated early in microglia ( figure 7a) . another extended network of tnf comprised psmb8 and psmb9, subunits of the immunoproteasome, essential for antigen presentation by mhc class i molecules. by day 14 p.i., the number of upregulated transcripts extensively increased in microglia (figure 7b) . the most clustered network comprised proteins like ccl5, cxcl9, cxcl10, cxcl13, cxcl16, and cxcr4, all chemokines and chemokine receptors regulating migration and arrest of adaptive immune cells within the cns during inflammation (47) . this chemokine cluster was linked to tnf and inflammatory cytokines previously detected at d5 p.i., e.g., il1a, il1b, and il18. another extended network of tnf comprised psmb8 and psm9, also present at d5 p.i., ctnnb1 encoding b-catenin, a cellular adhesion molecule, and cdkn1a, a cyclin inhibitor. other upregulated gene transcripts associated with class i antigen presentation, e.g., tap1 and tap2, were also linked through a network associated with complement component genes (c3, c3ar1, c4b, c1qa, c1qb, c1qc), which are highly expressed within microglia under homeostatic conditions (figure 3) . similarly, tyrobp and trem2, which formed phagocytic synapses (48), are both highly expressed in microglia during myelin loss. finally, a wide variety of upregulated gene transcripts are associated with mhc class ii antigen presentation and modulation of t cell function. this includes h2-ab1, encoding for the mhc class ii molecules and h2-dm, encoding for a second accessory protein, which facilitates peptide loading. similarly, genes associated with the invariant chain of mhc class ii were increasingly expressed within microglia, such as cd74 and ctss (cathepsin s, which cleaves invariant chain thereby promoting loading on mhc class ii). in addition, genes encoding for modulators of the cd4 t cell response, such as itgax (cd11c), cd86 and cd83 were also expressed by microglia ( figure 7b) . overall, the upregulated networks are related to complement activation, enhanced class i and class 2 antigen processing and presentation (potentially related to ifn-γ responses) as well as migration and phagocytic activity. however, there does not appear to be a bias toward phagocytic receptors over other components activated by pro-inflammatory mediators. pathogenic versus protective functions of myeloid cells following activation have also been correlated to expression of key molecules defined as m1 versus m2 markers. while the strict classification of myeloid cells into the m1 or m2 category has been tempered based on a more dynamic and mixed phenotype during inflammatory responses (49, 50) , the m1 and m2 markers remain helpful to gage overall effector functions. among the 89 analyzed gene transcripts in the nanostring myeloid panel related to m1/m2 polarization (51 m1 and 38 m2 genes), between 50 and 59% were upregulated across the course of jhmv infection with no difference comparing m1 versus m2 genes ( figure 8a) . among the total upregulated m1 markers, about 50% were commonly increased within both infiltrating bmdm and microglia; representative genes were il12b and il15ra (figures 8a,b) . however, while high levels of il12b mrna were observed in both bmdm and microglia at d5 p.i., expression was only sustained in microglia at day 7 p.i. and thereafter (figure 8b ). by contrast, il15ra was increased in both bmdm and to a lesser extent in microglia at d5 p.i., but was decreased in both populations at later time points p.i. (figure 8b ). in addition, between 35 and 45% of m1 markers were specifically expressed by infiltrating bmdm during the course of jhmv infection (figure 8a) , including cd86, atf3, ifng, ptgs2, ccr7, cxcl11, and cxcl12 transcripts ( figure 8b and data not shown). however, only 5 m1 related gene transcripts were specifically upregulated in microglia at the time of demyelination, including ccl5, fas, cxcl13, tnfsf10, and psmb9. (figures 8a,b and data not shown) . importantly, the most prominent difference between microglia and bmdm was noted in m2 marker regulation. among the 50-58% m2 gene transcripts upregulated following jhmv infection, only a small proportion (21-32%) was expressed by microglia ( figure 8a) . fn1 was the only m2 marker specifically expressed by microglia at the time of demyelination ( figure 8a) . although il1rn transcript expression was elevated in both bmdm and microglia, the increase was at best modest in microglia ( figure 8c) . the majority (86-95%) of m2 markers upregulated during jhmv infection were rather expressed by infiltrating bmdm, including arg1, erg2, il-10, and ccl22 (figures 8a,c and data not shown). increased transcript levels were most pronounces at days 5 and 7 p.i., but dropped off thereafter. altogether, these data showed that while infiltrating bmdm express a mixed phenotype of m1 and m2 markers during jhmv infection, microglia expressed primarily pro-inflammatory genes while not expressing m2 markers. microglia and infiltrating macrophages are major components of ms active lesions (51) . their effector functions are highly heterogeneous as evidenced by both pathogenic and protective functions during the course of ms (7) . they can promote tissue damage by releasing toxic and pro-inflammatory molecules, mediate demyelinated axons through phagocytosis as well as propagate inflammation by recruiting and activating adaptive immune cells. on the other hand, both populations also display protective functions by clearing myelin debris, which facilitates remyelination, as well as releasing trophic and anti-inflammatory factors, which promote tissue repair. while it remains a challenge to distinguish infiltrating macrophages from microglia in ms lesions due to morphological and phenotypic similarities, they are disparate effector cells based on animal ms models (17, 52) . questions relating to the pathogenicity of infiltrating macrophages and/or microglia in ms remain unanswered. can both populations display protective functions? do they display dynamic functions throughout the evolution of ms lesions? deciphering the respective roles of macrophages versus microglia in facilitating tissue damage and/or repair is essential to our understanding of ms pathogenesis and development of effective therapeutic strategies. in the murine eae model of ms, infiltrating bmdm are essential in mediating demyelination (53) . gene expression profiles demonstrated that bmdm are indeed highly phagocytic and inflammatory at disease onset, while microglia display a repressed phenotype (17) . by contrast, during jhmv-induced demyelination, recruited bmdm are dispensable for the demyelinating process (30) . distinct from eae, where microglia activation precedes cns infiltration of bmdm (52) , jhmv infection elicits early bmdm infiltration, prior to microglia activation. these distinct kinetics of bmdm recruitment relative to microglia activation thus appear to correlate with the apparently opposing roles of microglia as demyelinating populations. these data further suggest that early responses set the stage or imprint subsequent effector functions of bmdm and microglia. using a similar approach with nanostring analysis as in eae, the present study used gene expression profiling to characterize both bmdm and microglia myeloid functions at various times post jhmv infection. analysis of overall gene expression patterns revealed that the most extensive changes in bmdm were evident early after infection, while microglia showed a more dynamic profile throughout the course of viral encephalomyelitis. importantly, the most drastic gene upregulation in microglia was observed coincident with demyelination, at which time peak viral load and t cell effector function have substantially subsided (54) . our data contrast with eae (17) , where bmdm upregulated far more genes compared to microglia at disease onset, supporting opposing functions of bmdm and microglia in mediating demyelination in these two models. further, while bmdm exhibited a mixed expression profile of both pro-and anti-inflammatory markers, microglia expressed a highly pro-inflammatory profile and repressed most of the m2 markers across the entire time course of jhmv encephalomyelitis. analysis of protein-interacting networks within genes upregulated in microglia at the time of myelin loss revealed several key functions linked to promoting tissue damage. genes associated with complement activation were notably increased, although they were already highly expressed by microglia under homeostatic conditions. complement activation as a pathogenic component in ms has been reported following detection of deposits of the activated products of the complement component c3 in ms lesions (55) . the classical complement pathway has also been shown to mediate olg death thus promoting demyelination (56) . microglia phagocytic activity may also initiate tissue damage by directly removing myelin from axons, especially at the node of ranvier (17) . genes associated with trem2/dap12 signaling were also highly expressed by microglia at time of demyelination. trem2 modulates phagocytic capacity of myeloid cells via dap12 signaling (57) and is expressed on myelin-loaded myeloid cells in ms lesions (58) , supporting a role in ms pathogenesis. similar to jhmv infection, trem2 is predominantly expressed by microglia during eae and cuprizone-induced demyelination (59) (60) (61) . however, trem2-modulated phagocytic functions are essential for removal of myelin debris and remyelination implicating repair-promoting functions of microglia in the specific tissue environments defining these two demyelination models (17, 62) . preferential trem2 expression within microglia compared to bmdm following jhmv infection support a more pathogenic role of trem-2 in jhmv-induced demyelination, potentially by promoting myelin stripping after recognition of glycolipids and phospholipids exposed on damaged myelin. in this context, it is critical to note that jhmv infection is associated with extensive transient production of ifn-γ and its inducible genes, i.e., inos, and cxcr3 ligands, which may drive a more phagocytic pathway in microglia in efforts to remove damaged proteins and lipids (54) . further investigation is required to define inflammatory conditions under which trem-2 modulated or other phagocytic pathways promote tissue damage or repair and whether these are transient and reversible. microglia may also induce demyelination by secreting toxic factors including inflammatory cytokines that are highly expressed by microglia at time of demyelination, including tnf. tnf can induce olg death (63, 64) and is expressed in active ms lesions, as well as elevated tnf in serum and cerebral spinal fluid correlates with enhanced ms pathology (65, 66) . finally, microglia functions during jhmv infection were also associated with promoting adaptive immune response. an extensive network of chemokines and chemokines receptors relating to the recruitment and arrest of t and b cells within the inflamed cns were highly expressed by microglia. similarly, several genes associated with antigen processing and presentation by mhc class i and ii molecules were upregulated within microglia, suggesting that microglia promote t cell reactivation upon cns entry. however, microglia explanted during eae, tmev as well as jhmv infections failed to support myelin-specific cd4 t cell responses ex vivo, despite detection of internalized myelin (67) (68) (69) . a potential deficit in antigen processing was supported by the ability of exogenous peptide to overcome the inability of microglia to prime myelinspecific cd4 t cells (69) . nevertheless, this notion is opposed by our microglia profiling showing upregulation of genes involved in protein degradation and class ii peptide loading, e.g. cd74 (invariant chain), h2-dma (peptide loading), ctss (cathepsin s), which cleaves invariant chain. the apparent inability of microglia to elicit cd4 t cell effector function ex vivo thus remains intriguing. our present study reveals new insights into the plasticity of microglia in adapting to inflammation and expressing pathogenic functions associated with demyelination, characteristics which have previously been ascribed to bmdm (17) . moreover, altered bmdm expression profiles coincided with their early infiltration into the cns and remained largely similar throughout infection. while altered microglia gene expression coincided with the time of early, yet robust demyelination, it remains to be determined whether these changes are sustained at later time points during jhmv persistence, when clinical disease improves and remyelination occurs. it will be of specific interest to assess whether the microglia pro-inflammatory phenotype evolves to an anti-inflammatory, repair-promoting phenotype, as evidenced by the plasticity of myeloid cells in cns autoimmunity (70) . furthermore, our study emphasizes that the distinct tissue environments during eae and jhmv infection drive opposite effector functions of microglia versus infiltrating macrophages. the interplay between t cells, infiltrating macrophages and microglia, as well as astrocytes drives ms pathogenesis, yet mechanisms ultimately leading to loss of repair remain unclear. taking advantage of demyelinating models characterized by distinct inflammatory factors such as both th1 and th17 cells in eae (11) , strong th1 polarized responses during jhmv infection, distinct kinetics of bmdm recruitment versus glia activation promises to reveal essential new insights into the interplay of microglia and bmdm functions in debris clearance versus active myelin stripping and ongoing axonal damage. longitudinal studies will aid in developing efficient future therapies to combat ms pathogenesis. all procedures were performed in compliance with protocols approved by the cleveland clinic institutional animal care and use committee. author contributions cs designed and performed experiments, collected and interpreted the data, and wrote the manuscript. rd analyzed data and edited manuscript. cb designed the research, provided materials, interpreted the data, and wrote the manuscript. all authors approved the final manuscript. the authors would like to thank natasha towne for her exceptional technical assistance, as well as jennifer powers for performing facs purification. this work was supported by the national institutes of health grant ns091183 and national ms society research grant nmss rg4450. rd is supported by the national institutes of health grant ns096148. the supplementary material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.01325/ full#supplementary-material. figure s1 | validation of nanostring ncounter gene expression analysis by real-time pcr. to validate nanostring ncounter data, expression of il-15, il1rn, and arg1 was analyzed by q-pcr in naïve circulating monocytes and microglia, as well as in bone marrow-derived macrophage (bmdm) and microglia isolated from the spinal cord of jhmv-infected mice at days 5, 7, 10, and 14 p.i. levels of mrna expression are normalized to gapdh mrna levels. pathogenesis of tissue injury in ms lesions multiple sclerosis -the plaque and its pathogenesis janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis microglia: active sensor and versatile effector cells in the normal and pathologic brain role of microglia in cns autoimmunity microglial physiology: unique stimuli, specialized responses the benefits and detriments of macrophages/microglia in models of multiple sclerosis microglia as neuroprotective, immunocompetent cells of the cns non-identical twins -microglia and monocyte-derived macrophages in acute injury and autoimmune inflammation modeling the heterogeneity of multiple sclerosis in animals communication 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sclerosis self-reactive cd4(+) t cells activated during viral-induced demyelination do not prevent clinical recovery myeloid cell plasticity in the evolution of central nervous system autoimmunity the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-005393-rhji4io9 authors: popko, brian; corbin, joshua g.; baerwald, kristine d.; dupree, jeffrey; garcia, annie m. title: the effects of interferon-γ on the central nervous system date: 1997 journal: mol neurobiol doi: 10.1007/bf02740619 sha: doc_id: 5393 cord_uid: rhji4io9 interferon-gamma (ifn-γ) is a pleotropic cytokine released by t-lymphocytes and natural killer cells. normally, these cells do not traverse the blood-brain barrier at appreciable levels and, as such, ifn-γ is generally undetectable within the central nervous system (cns). nevertheless, in response to cns infections, as well as during certain disorders in which the cns is affected, t-cell traffic across the blood-brain barrier increases considerably, thereby exposing neuronal and glial cells to the potent effects of ifn-γ. a large portion of this article is devoted to the substantial circumstantial and experimental evidence that suggests that ifn-γ plays an important role in the pathogenesis of the demyelinating disorder multiple sclerosis (ms) and its animal model experimental allergic encephalomyelitis (eae). moreover, the biochemical and physiological effects of ifn-γ are discussed in the context of the potential consequences of such activities on the developing and mature nervous systems. interferon-gamma (ifn-?) was originally discovered by wheelock (1965) as an activity that interfered with viral replication. tlymphocytes and natural killer cells are the only cells known to be capable of expressing this cytokine (reviewed in trinchieri and perussia, 1985) . because under normal condi-under these conditions, neuronal and glial cells that normally do not encounter ifn-',v are exposed to the potent, pleotropic effects of this cytokine. there is considerable evidence that suggests that the effects of ifn-?~ are important in a variety of cns disorders (reviewed in ransohoff and benveniste, 1996) . in this article, we discuss various molecular, biochemical, cellular, and physiological properties of ifn-~, and its evoked response. moreover, the potential role this cytokine plays in cns disorders is discussed. a considerable amount of information has been learned concerning the molecular biology and biochemistry of ifn-~, since the human and mouse cdnas were cloned over a decade ago (gray et al., 1982; goeddel, 1982, 1983) . the human and mouse proteins are 143 and 134 amino acids in length, respectively, and are encoded for by four exons that reside on 6 kb of dna. interestingly; the human and mouse proteins share only limited homology, approx 40% at the amino acid level, and do not bind to the other's receptor to an appreciable extent (reviewed by farrar and schreiber, 1993) . both proteins contain two glycosylation sites (gray and goeddel, 1982; ealick et al., 1991) , although glycosylation is not essential for ifn-'i activity (kelker et al., 1983) . one factor that may play a role in regulating the biological activity of ifn-y is message stability, since the ifn-y mrna contains an au rich sequence in the 3'-untranslated region that has been shown to reduce [he mr_na halfqife of other cytokines dramatically (shaw and kamen, 1986) . biologically active ifn-~/from both human and mouse is a noncovalently bound homodimer that contains two receptor binding sites . the ifn-1 receptor is a multimeric receptor complex composed of a ligand binding subunit (a-chain) (aguet et al., 1988 ) and a transmembrane, accessory factor (~-chain) (soh et al., 1994; hemmi et al., 1994) . the c~-chain is a ubiq-uitously expressed, glycosylated, cell-surface protein that binds ifn-~ , with high affinity (reviewed by farrar and schreiber, 1993) . the extent of glycosylation is variable depending on cell type and accounts for the range of sizes of the a-chain, between 80 and 95 kda. on binding, ifn-7 induces (r-chain dimerization, which is believed to be critical to the initiation of the signal transduction cascade (greenlund et al., 1993; fountoulakis et al., 1992) . this feature of the ifn-7 receptor has been exploited to generate mutant forms that confer ifn-3, unresponsiveness to cells in a dominant manner (dighe et al., 1993 (dighe et al., , 1995 . the crystal structure of the complex between ifn-y and the a-chain of its receptor has recently been reported (walter et al., 1995) . the ~-chain of the receptor, which associates with the or-chain in an ifn-,f-dependent manner, is essential for the induction of the ifn-7 signaling cascade (bach et al., 1996) . recently, considerable progress has been made in the elucidation of the ifn-,~-induced signal transduction pathway (sadowski et al., 1993; darnell et al., 1994; shuai, 1994; vilcek and oliveira, 1994; david, 1995; schindler, 1995) . the janus kinases jak1 and jak2 become phosphorylated following binding of ifn-7 to its receptor, with which the jak kinases associate (muller et al., 1993; wafting et al., 1993) . the jak1 kinase appears to associate with the a-chain, and the jak2 kinase associates with the [3-chain (sakatsume et al., 1995) . following activation, the jak kinases phosphorytate the transcriptional factor known as signal transducer and activator of transcription (stat-i~z), w~hich binds to a specific dna element referred to as ~,-activation site (gas) . stat-la binding results in the rapid transcriptional induction of genes containing the gas element. for example, the genes encoding the gaunylate binding protein and the igg fc receptor are induced in this way: other genes that are stimulated by ifn-~/, such as the class i and class ii molecules of the major histocompatibility complex (mhc), require a longer period (several hours) before transcriptional induction is detected. these genes lack the gas element and are likely activated through ifn-?-induced intermediates (benveniste and benos, 1995) . (summarized in table 1) ifn-? plays a critical role in the regulation of the immune response (young and hardy, 1995) . for this reason, it is often referred to as immune interferon. ifn-y facilitates the stimulation of the ~1 subpopulation of t-lymphocytes, which control cell-mediated immunity and which, in fact, express ifn-y; whereas it impedes the stimulation and activity of the th2 subpopulation of t-cells, :which express il-4 and are involved in regulating humoral immu-ni~ (gajewski et al., 1989; swain et al., 1991; paul and seder, 1994; seder and paul, 1994; reiner and seder, 1995) . cytotoxic t-cells and natural killer cells are also activated by ifn-7 (trinchieri and perussia, 1985) . ifn-? also plays a role in regulating antibody production, promoting an igg2a response through a direct effect on b-cells (snapper and paul, 1987) . importantly, ifn-? is a potent stimulator of expression of the antigen-presenting components of the immune system. mhc class i and ii molecules are dramatically upregulated in the presence of ifn-'y on a variety of cell types (reviewed in vilcek et al., 1985; benveniste and benos, 1995) . ifn-? is also a powerful effector molecule of the inflammatory response (reviewed in schreiber and celada, 1985) . ifnq,-stimulated macrophages release a number of inflammatory cytokines, including tumor necrosis factor-alpha (tnf-a), il-1, il-6, and il-8. in addition, ifn-? increases the microbicidal activities of macrophages through the induction of the synthesis of hydrogen peroxide and nitric oxide . ifn-? is also a potent stimulator of the expression of the fc receptors for igg immunoglobins on macrophages (erbe et al., 1990) . all of these actions of ifn-? on macrophages serve to stimulate the cytocidal activity of these cells. multiple sclerosis (ms) is the most common human demyelinating disease that affects the cns. although the etiology of ms has not been established, it is widely believed that immunological mechanisms are involved (reviewed in martin et al., 1992; raine, 1994a,b) . re cns is immunologically distinct owing to the presence of the blood~brain barrier (crone, 1986) , which inhibits entry of both immune cells and humoral factors. in ms patients, this barrier is disrupted (martin et al., 1992) allowing increased access of blood components (cellular and noncellular) to the cns. the enhanced permeability of the blood-brain barrier is thought to be a contributing factor in immunemediated demyelinating disorders (reviewed in poser, 1987 poser, , 1993 . importantly, there is an increased infiltration of t-lymphocytes (cd4+ and cd8+) into the cns of ms patients (martin et al., 1992; utz and mcfarland, 1994) , with cd4+ (t-helper) t-cells being predominant at the sites of active demyellnation (booss et al., 1983; traugott et al., 1983) . t-cell responses to a variety of myelin antigens, including myelin basic protein (mbp) and proteolipid protein (plp), have been identified in ms patients (reviewed in miller et al., 1995) . experimental allergic encephalomyelitis (eae) is the primary animal model used in the study of ms (reviewed in alvord et al., 1984; zamvil and steinman, 1990; martin and mcfarland, 1995) . eae can be induced in a variety of laboratory animal species by immunization with either cns tissue, myelin, or volume i4, 1997 myelin components. as demonstrated through passive transfer studies, eae is a t-cell (cd4+, thl) mediated disease model (zamvil and steinman, 1990; voskuhi et al., 1993) . a relapsing/remitting form of eae is induced at high incidence in sjl mice immunized with an encephalitogenic epitope of plp (tuohy et al., 1988a (tuohy et al., ,b, 1989 (tuohy et al., , 1992 mcrae et al., 1992) . this model of eae exhibits many of the clinical, pathological, and immunological feat~ares of ms. the t-cells that infiltrate into the cns in ms and eae are activated and produce ifn-i, which is believed to play a role in the pathogenesis of irnmune-rnediated demyelinating disorders (reviewed in hartung et al., 1992; olsson, 1992; panitch, 1992 ). there is considerable circumstantial evidence that suggests that ifn-~, is a key mediator of inflammation in ms and eae. many of the pathological events observed in ms and eae, such as increased mhc expression, macrophage activation, increased expression of leukocyte adhesion molecules on blood-brain barrier endothelial cells (olsson, 1992) , and reactive gliosis (balasingam et al., 1994) , are consistent with the known effects of ifn-~,. ifn-7 is detected in active ms lesions (traugott and lebon, 1988) , and increased numbers of ifn-~,-secreting tlymphocytes are present in the cerebrospinal fluid of ms patients (olsson et al., 1990) . beck et al. (198ff) reported a cor~iation between the onset of clinical symptoms of ms and mitogenstimulated ifn-y production. moreover, eae can be transferred to naive animals using ifn-~,-secreting tqymphocytes isolated from animals experiencing eae (zamvil and stelnman, 1990) , and a significant increase in the level of ifn-?, mrna is observed in the cns of animals with severe eae compared to animals with mild eae (renno et al., 1994) . perhaps the best direct evidence for the deleterious effects of ifn-y in ms came inadvertently from a small clinical trial in the early 1980s. of 18 ms patients receiving ifn-~/, seven experienced exacerbations during the 4-wk treatment period, which was significantly higher than the pretreatment exacerbation rate (panitch et at., 1987; panitch, 1992) . not only did this study clearly eliminate ifn-~/as a potential therapeutic agent for ms, but it also demonstrated that ifn-~, activity can lead to a wo~,ens of tlne disease course, suggesting a role for this cytokine in normal disease progression. there are, however, contradictory experimental data concerning the role of ifn-~, in eae. the administration of antibodies to ifn-~, e~anced the severity of eae in mildly susceptible mice (billiau et al., 1988; voorthuis et al., 1990; duong et al., 1992) , and resulted in eae susceptibility in resistant strains (duong et al., 1994) . moreover, treatment of sjl/j mice, which are very sensitive to eae, with ifn-~, results in enhanced survival (billiau et al., 1988) . nevertheless, the protective effect of ifn-~, is not observed when eae is passively transferred. voorthuis et al. (1990) reported that intraventricular administration of ifn-~/ into eae-induced rats resulted in complete suppression of clinical signs, and willenborg et al. (1995) , using a viral delivery system, suggested that ifn-y had no effect on disease. recently, ferber et al. (1996) examined the eae susceptibility of b10.pl mice that contained an experimentally inactivated ifn-~, gene (dalton et al., 1993) . b10.pl mice are normally very susceptible to mbp-induced eae, and the ifn-?, ka~ockout mice appear equally sensitive to disease induction. although morphological data were not presented, this work dearly demonstrates that ifn-~, is not essential for eae induction, at least not in b10.pl mice. to understand better the effects of ifn-u on cns cells in vivo, in the absence of other t-cellderived factors, a variety of approaches have been used to deliver this cytokine to the cns. the direct injection of ifn-~, into various animal models has been shown to upregulate the volume 14, 1997 expression of both mhc class i and class ii glycoproteins on various neuronal celt types that do not normally express these proteins at detectable levels. wong et al. (1984) demonstrated a dramatic increase in the expression of the mhc class i glycoprotein in astrocytes, neurons, oligodendrocytes and microglia following the delivery of ifn-y to the cns of 2-dold mice. direct injection of ifn-y into the cns has also been demonstrated to increase the expression of mhc class i on rat endothelial and ependymal cells and class ii on microglia, ependymal, and perivascular cells (sethna and lampson, 1991) . continuous iv infusion of ifn-~f into lewis rats for a period of 3 d induced mhc class ii expression on microglia, ependymal and endothelial cells of large blood vessels (steiniger and van der meide, 1988) . these findings support the earlier work of momburg et at. (1986) who reported that the iv infusion of ifn-y into mice resulted in class ii expression in many organs, including the brain, where "round cells in the vicinity of meningeal blood vessel," presumably microglia, were class ii immunoreactive. ifn-y has also been suggested to play a role in the breakdown of the blood-brain barrier and the recruitment of inflammatory cells into the cns. seth_ha and lampson (1991) reported that a single injection of ifn-y into the rat basal ganglia induced the infiltration of cd4+ t-cells into the perivascular space and monocytes/ macrophages, and presumptive natural killer cells into the brain parenchyma. these authors suggested that such cellular recruitment may be facilitated by ifn-y-induced changes in the endothelial cells, resulting in an alteration in the permeability of the blood-brain barrier. moreover, simmons and willenborg (1990) , using direct injection of ifn-~. into the spinal cord of rats, reported an inflammatory response similar in pattern to that observed in early eae with the accumulation of inflammatory cells in the meninges and around spinal cord veins also suggestive of a compromised blood-brain barrier. tjuvajev et ai. (1995) reported an ifn-y-induced breakdown of the blood-brain barrier in an in vivo brain tumor model, and huynh and dorovini-zis (1993) demonstrated that brain endothelial cells undergo dramatic morphological, functional, and permeability changes in response to ifn-y, suggesting that this cytokine alters bloodbrain barrier function. steffen et al. (1994) demonstrated increased expression of vascular cell adhesion molecules (vcam) and intercellular adhesion molecule-1 (icam-1) on the brain endothelium of sjl mice experiencing eae, and antibodies to these molecules inhibited binding of peripheral lymphocytes to inflamed brains. furthermore, mccarron et al. (1993) showed that ifn-y significantly increased the adhesion of mgp-specific encephatitogenic t-ceils to mouse cerebrovascular endothelial cells, and that this effect correlated with the induction of icam-1 expression by the endothelial cells. the studies discussed above consistently indicate a role for ifn-y in the inducement of expression of mhc and cell adhesion molecules, and the recruitment of inflammatory cells; however, the infusion of ifn-y into the cns has also been implicated in a variety of other processes. for example, yong et al. (1991) reported that the direct administration of ifn-~/ into the adult mouse brain following corticectomy resulted in an increase in trauma-initiated gliosis. brosnan et al. (1989) , using the visual pathway of the rabbit, demonstrated that the injection of ifn-y significantly reduced neural conduction and induced subtle axonal pathology. in addition, the simultaneous injection of ifn-7 and a myelin/oligodendrocyte glycoprotein antibody significantly delayed cervical somatosensory evoked potentials, and caused massive demyelination in the spinal cord (vass et al., 1992) . vass et al. (1992) also reported an ifn-y-induced increase in mhc expression, which accompanied spinal cord demyelination. in complement with various delivery systems previously discussed, we have used a transgenic approach to examine the effects of ifn-y on the developing nervous system, (corbin et al., 1996) . transgenic mice were generated in which the expression of ifnq was volume 14, i997 placed under the transcriptional control of the mbp gene (mbp/ifn-y transgenic animals). transgenic mice generated with tl-ds construct have a shaking/shivering phenotype that is similar to that observed in naturally occurring mouse models of hypomyelination (e.g., sb_iverer, jimpy, quaking), and these transgenic animals have dramatically less cns myelin than control animals. reactive gliosis and increased macrophage/microglia f4/80 immunostaining were also observed. additionally, mhc class i and class ii mrna levels were increased in the cns of mbp/ifn-y transgenic mice, and the increase in mhc class i mrna expression was detected in both white and gray matter regions. surprisingly, cerebelfar granule cell migration was also disrupted in these animals. these results strongly support the hypothesis that ifn-~, is a key effector molecule in irnmtme-mediated demyelinating disorders, and suggest that the presence of this cytokine may also disrupt the cytoarchitecture of the developing nervous system. the mechanism by which ifn-7 exerts its action within the cns remains unresolved. one idea concerning the potential cellular site of action of ifn-y in cns demyelinating disorders is that ifn-1 activates macrophages/ microglial cells, which in turn mediate cytotoxic effects on oligodendr~ytes (fig. 1) . tnf-(z, which has previously been shown to be toxic to oligodendrocytes in vitro, is expressed by ifn-y-activated macrophages/microglia (selmaj et al., 1991) . more recent in vitro experiments suggest that the microglial cell toxicity to otigodendrocytes is mediated through cellsurface expression of tnf-cz and the production of nitric oxide (merrill et al., 1993) . nitric oxide is very labile, such that to elicit their cytotoxic effects, the activated microglia need to be in intimate contact with the target oligodendrocytes. oligodendrocytes undergo necrotic cell death following exposure to nitric oxide (mitrovic et al., 1995) . in support of the hypoth~ esis that nitric oxide is important in the pathogenesis of immune-mediated demyelinating diseases, it has been shown that nitric oxide synthase mrna levels are increased in mice experiencing eae (koprowski et al., 1993) , and nadph-diaphorase histochemical staining, a marker for nitric oxide production, as well as nitric oxide synthase rnrna levels were found to be elevated in the brains of ms patients (b6 et al., 1994) . ifn-y may also have a direct deleterious effect on oligodendrocytes (fig. i) , which have been shown to express ifn-7 receptors (torres et al., 1995) . we have examined the effects of ifn-7 on oligodendrocytes using the moch-1 cell line, which was derived from an oligodendroglioma that developed in a transgenic line of mice and expresses a variety of features of oligodendrocytes (hayes et al., 1992; li et al, 1995) . when moch-1 cells are cultured in medium containing low concentrations of volume 14, 1997 serum (1% fetal bovine serum [fbs]) they extend multiple, thin, branched processes and have phase-bright cell bodies, which are typi ~ cal features of cultured primary oligodendrocytes (reviewed in popko et al., 1994) . furthermore, these cells are immunoreactive against antibodies to galactocerebroside, a glycolipid marker of mature oligodendrocytes, and abundantly express myelin protein genes (hayes et al., 1992) . mochq cells cultured in 1% fbs or chemically defined medium do not express detectable levels of the astrocyte marker glial fibrillary acidic protein (gfap). when moch-1 cells are cultured in 1% fbs medium containing ifn-7. however, they transform to flat, astrocyte-like cells with enlarged cell bodies and appear more adhesive ~ in addition to these morphological changes, ifn-1 stimulates the expression of abundant levels of the astrocyte marker gfap, as well as slightly elevated levels of mbp and plp mrna. ifn-7 also induces an enormous increase of mhc class i expression, as well as a comparatively modest increase of mhc class ii mrna expression in mochq cells. the morphological and molecular changes mc)ch-1 cells undergo in response to ifn-7 suggest that ifn-7 may induce a direct effect on oligodendrocytes, or a subpopulafion of these cells, in vivo . recent studies further support the hypothesis that ifn-y has a direct, deleterious effect on oligodendrocytes. using a morphological assay, oligodendrocytes in vitro were shown to undergo apoptotic cell death at very high frequency following exposure to relatively low concentrations of ifn-7 (vartanian et al., 1995) . the addition of rnicroglia to the culture did not significantly increase the frequency of otigodendrocyte death, further supporting a direct effect of the cytokine. interestingly, vartanian et ai. (1995) also detected apoptotic oligodendrocytes, along with ifn-7, in the advancing margin of active ms plaques, but not in the chronic portion of the plaque or in adjacent unaffected white matter. agresti et al. (1996) have also observed deleterious effects of ifn-y on cultured oligodendrocytes. in the absence of cell death, they observed a decrease in the ability of oligodendrocyte progenitors to divide and differentiate, as well as changes in myelin protein gene mrnas and a general metabolic depression. these effects were shown to be fully reversible following cytokine withdrawal (agresti et al., 1996) . the harmful effects of ifn-y on myelination might be mediated through the induction of mhc expression in oligodendrocytes, which normally do not express these molecules at an appreciable level. in support of this possibility, transgenic mice in which mhc class i expression has been targeted to oligodendrocytes are severely hypomyelinated (turnley et al., 1991b; yoshioka et al, 1991; turnley and morahan, 1995) . this hypomyelh~ation occurs in the absence of immune celt infiltration, suggesting that mhc class i expression in oligodendrocytes is sufficient to disrupt myelination. recently; power et al. (1996) examined these transgenic animals in more detail and demonstrated that cell-surface expression of mhc class i in oligodendrocytes of these mice was minimal moreover, immunocytochemistry for f~2-microglobulin, which is required for cellsurface expression of mhc class i, revealed that expression of ~2-microglobulin was undetectable in oligodendrocytes and limited to microglia in lhe cns. power et al. (1996) suggest that the accumulation of mhc class i protein m the cytoplasm of the oligodendrocytes, owing to the absence of f~2-microglobulin, may interfere with normal myelin protein trafticking, leading to the observed hypomyelination in these mice. in vitro, oligodendrocytes at all stages of differentiation can be induced by ifn-7 to express cell-surface mhc class i (suzumura et al., 1986; turnley et al., 1991a; massa et al., 1993) . nevertheless, it is unclear what the consequences of oligodendroglial expression of mhc class i are. as mentioned previously, cd8+ t-cells are present in the cns of ms patients (martin et al., 1992; utz and mcfarland, 1994) . it is, however, unknown whether oligodendrocytes in vivo can present antigen to these cells. in contrast, many investigators have searched for, but failed to demonstrate, ifn-7mediated induction of mhc class ii on oligodendrocytes in culture (wong et al., 1984; suzumura et al., 1986; turnley et al., 1991a; satoh et al., 1991b) , suggesting that oligodendrocytes are refractory to mhc class ii induction. calder et al. (1988) demonstrated that oligodendrocyte precursors (o-2a progenitor cells) can be induced to express mhc class ii by ifn-~/, but that maturation into oligodendrocytes leads to loss of inducibility. nevertheless, bergsteinsdottir et al. (1992) were able to induce mhc class ii expression in a subset of mature oligodendrocytes in culture with ifn-7 in the presence of the synthetic glucocorticoid dexamethasone. although gtucocorticoids are normal cns constituents (birmingham et al., 1984; kumar et al., 1989) , the in vivo significance of these in vitro observations is unclear. another activity of ifn-t that might be relevant to the pathogenesis of immune-mediated demyelinating disorders concerns the ability of this cytokine to stimulate the expression of mat-shock (stress) proteins. these are ubiquitously expressed proteins that are believed to play a role as chaperones in normal protein synthesis and degradation (reviewed in jindal, 1996) . the expression of these proteins increases following cell stress, and this response is thought to be protective. heat-shock proteins are highly conse~rved across divergent species and are very irnmunogenic. it has been demonstrated that there is increased heat shock protein expression in ms and eae lesions, and importantly, that there is an immune response to [he stress proteins in these disorders (selmaj et al., 1992; prabhaker et al., 1994; gao et al, 1995; van noort et al., 1995) . it has also been demonstrafed that 1fn-'f is capable of potentiating the induction of stress protein synthesis (morange et al., 1986; ferm et al., 1992) , such that this effect might facilitate the induction of the immune response to the heat-shock proteins in immune-mediated disorders. the characterlzation of the heat-shock response in these disorders should provide further insight into the role these proteins play in the pathogenesis of disease. a potential direct effect of ifn-? on oligodendrocytes in immune-mediated demyelinating disorders is consistent with recent studies that examined biopsy specimens from patients with acute ms (rodriguez and scheithauer, 1994) . they observed degeneration of inner myelin loops with preservation of outer loops and axons in early plaques, as well as areas in which well-myelinated and demyelinated axons were in close proximity. these observations suggest that the early pathological lesions that occur in ms are compatible with a primary disturbance in the myelinating function of oligodendrocytes, not a nonspecific inflammatory reaction that affects the myelin sheath, as might occur if microglia were activated subsequently to phagocytose the myelin sheath. astrocytes, which have been shown to express the receptor for ifn-7 (rubio and de felipe, 1991) , respond to the presence of this cytokine in a variety of ways. as mentioned previously, mhc class i expression on astrocytes increases substantially in the presence of ifn-7 (wong et al., 1984; mauerhoff et al., 1988; massa et al., 1993) . this expression appears functional in that astrocytes are susceptible to lysis by cytotoxic t-cells in an antigen-specific manner (skias et al., 1987) . ifnq,-induced mhc class ii expression by astrocytes has also been demonstrated in vitro and in vivo (fontana et al, 1984; fierz et al., 1985; pulver et al., 1987) , although fine in vivo levels are low relative to that detected on microglial cells (reviewed in benveniste, 1992) . the biological significance of the antigen-presenting potential of astrocytes remains to be resolved in vivo. there is also evidence that suggests that ifn-7 plays a role in the reactive astrogliotic response, particularly in immune-mediated disorders. reactive astrogliosis is the response in which astrocytes express increased levels of gfap, undergo hypertrophy, and proliferate (eng, 1988) . there are dramatically elevated levels of geap expression in the cns of the volume i4, 1997 mbp/ifn-7 transgenic animals, although it is unclear whether this represents a direct response of astrocytes to i~-7 or an indirect response elicited by the myelin abnormalities that occur in these mice (corbin et al., 1996) . moreover, as mentioned, yong et al. (1991) showed that if1n<7, as well as a number of other cytokines (balasingam et al., 1994) , increased trauma-induced gliosis in mice. these investigators also demonstrate that ifn-? promotes the proliferation of adult human astrocytes in vitro (yong et al., 1991) . nevertheless, in a follow-up study, yong et al. (1992) were unable to reproduce these results using mouse astrocytes, suggesting that there are species differences in the astrocytic response to ifn-7. ifn-7 has also been shown to increase the expression of intercellular adhesion molecules on astrocytes. human fetal (frohman et al., 1989) and adult (satoh et al., 1991a) astrocytes, as well as mouse astrocytes (satoh et al., 1991b) , have been shown to express icam-1 in the presence of ifn-7. such expression might facilitate the interaction of these cells with lymphocytes. ifn-~,-stimulated human and rat astrocytes have also been shown to express vcam-1, possibly suggesting a role in lymphocytic infiltration across the blood-brain barrier into the cns (rosenman et al., i995) . although ifn-7 readily induces the expression of mhc molecules in astrocytes, oligodendrocytes, and resident cns microglia, !fn-7-induced expression of mhc molecules in neurons is under tighter control. early evidence demonstrating the ability of brain cells, including neurons, to express mhc molecules includes experiments in which ifn-y was either added to mixed brain cultures or i~ected directly into the cns (wong et al., 1984 (wong et al., , 1985 . as detected by immunohistochemical analysis, the addition of ifn-7 to brain cells in culture results in 9% of neurons expressing mhc class i, whereas direct injection into the cns results in approx 15% of neurons expressing mhc class i. moreover, the response of the olb21 neuronal cell line to ifn-? has been investigated (joly and oldstone, 1992) . the olb21 cell line was developed by retroviral-mediated oncogene delivery to cells from the olfactory bulb (ryder et al., 1990) . in these cells, ifn-7 stimulates the expression of mhc class i both at the mrna level and on the cell surface. furthermore, in the presence of ifn-7, the peptide transporters ham 1 and ham 2, which are involved in the processing of class i antigens, are induced in these cells, whereas an increase in the expression of ~2-microglobulin also occurs (joly and oldstone, 1992) . in other studies, cell lines derived from two medulloblastomas, which are immunoreactive against neurofilament antibodies, but unreactive against gfap and s-100 antibodies, have also been shown to empress mhc class i and class ii antigens following exposure to ifn-y (tamura et al., 1989) . although these studies further demonstrated that cells of neuronal origin are intrinsically capable of expressing mhc molecules and suggest that this expression may serve a functional role, the physiological conditions under which functional neuronal mhc expression can occur have not yet been fully elucidated. toward this goal, neumann et al. (1995) have combined whole-cell patch-clamp recording techniques with single-cell rt-pcr analysis to demonstrate that in the presence of ifn-7, both mhc class i and 132-microglobulin are significantly more inducible on electrically silent neurons compared to neurons spontaneously firing action potentials. this result suggests that functional mhc class i expression may occur only in neurons that are no longer biologically active. this would then allow these cells to be recognized and killed by cytotoxic t-lymphocytes, thus permitting active virus to be cleared from inactive neurons. this result also suggests that active neurons may not possess the potential to express functional mhc, thus allowing neurons to survive at the cost of continued viral infection (joly et al., 1991) . future investigation in this area will likely yield additional insight into our understanding of the functional interactions between neurons and cells of the immune system. although the majority of the studies addressing the effect of ifn-~, on neurons have been directed at examining the expression of mhc glycoproteins, several studies have reported other ifn-~,-induced neuronal consequences. as previously discussed, brosnan et al. (1989) reported a significant reduction in neural conduction and modest axonal pathology in the visual system of the rabbit following injection of ifn-,f. recently, mcmillian et al. (1995) reported that the treatment of rat primary basal forebrain mixed cultures with ifn-7 decreased the number of choline acetyl transferase (chat) immunopositive neurons, whereas the number of chat immunonegative neurons was unaffected. this selective neuronal killing was shown to be mediated through ifn-~,-induced microglia activation. moreover, birdsall (1991) demonstrated that following exposure to ifn-~[, two human neuroblastoma cell lines, but not cultured human cortical neurons, expressed icam. ifn-y also enhanced tnf-el-induced binding of these cells to neutrophils. finally, collins (1995) demonstrated that susceptibility to viral infection of a human cerebral cortical neuronal cell line was dramatically increased following treatment with human ifn-~, and that this increased susceptibility was owing to membrane expression of hla class i molecules. ifn--f has also been shown to affect neuronal differentiation in vitro. chang et al. (1990) demonstrated that ifn-~, retarded the death of cultured rat sympathetic neurons following nerve growth factor (ngf) deprivation. moreover, ifn-y has been shown to potentiate ngfstimulated differentiation of pc12 cells (improta et al., 1988) . barish et al. (1991) also demonstrated that ifn-y increased the differentiation of cultured cortical and hippocampal neurons. nevertheless, it is unlikely that the developing nervous system is exposed to appreciable levels of ifn-7 under normal conditions, such that the in vivo relevance of these observations is unclear. in contrast, there is in vivo evidence that suggests that ifn-~, might have a detrimental effect on the developing nervous system. as mentioned earlier, transgenic mice in which ifn-y was targeted to the cns demonstrate a disruption in cerebellar granule cell migration (corbin et al., 1996) . during development, cerebellar granule cells migrate from the external granule cell layer (egl), through the molecular layer, to the internal granule cell layer (igl) along processes extended by radial gliai cells (chuong, 1990) . in transgenic mice expressing ifn-? within the cns, many granule cells are still observed in the egl and in the molecular layer. this observation is reminiscent of other mouse mutants (e.g., weaver, staggerer) in which the interaction between granule cells and radial glia is disrupted (chuong, 1990) . perhaps the ectopic expression of ifn-~, in the cerebellum of transgenic mice has a deleterious effect on lhe interaction between granule cells and radial glia, thus disrupting the migration of these cells from the egl to the igl. further investigation is warranted to characterize the potentially deleterious developmental effects of ifn-g on the cns in more detail. recently, meda et al. (1995) suggested a role for ifn-y in alzheimer's disease. the senile plaques that develop in alzheimer's patients are characterized by the extracellular deposits of [3-amyloid protein. meda et al. (1995) demonstrated that ifn-y acted synergistically with fj-amyloid in the activation of microglia, which produce tnf-o~ and nitric oxide. in coculture experiments, it was shown that microglia activated by ifnq, and ~-amyloid caused neuronal injury. clearly, these are exciting findings that lead to the speculation that ifn-~/might also be involved in other neurodegenerative disorders. ifn-? is a multifunctional cytokine involved in regulating a variety of immune responses. this cytokine also has a multitude of effects in the cns (table 2) . although normally excluded from the cns, this cytokine is present during many disorders in which the cns is affected, including the most common human demyelinating disease, ms. the presence of table 2 effects of ifn-? on the cns disruption of myelination exacerbation of reactive gliosis disruption of the blood-brain barrier recruitment of inflammatory cells from the periphery induction of mhc expression stimulation of macrophage/microglial cells disruption of cerebellar granule cell migration this cytokine in the cns leads to an abundance of pathological effects, including decreased myelination, increased gliosis, disruption of blood-brain barrier func~on, increased mhc expression, stimulation of macrophage/microglial activation, and disruption of cerebellar granule cell migration. taken as a whole, these observations demonstrate that ifn-? can either directly or indirectly mediate many of the cns pathological effects that are observed in disorders in which immune cell traffic in the cns increases. understanding the mechanism(s) by which ifn-~,r exerts its actions on cns function will undoubtedly prove useful toward generating rational therapeutic approaches for the treatment of cns disorders in which this cytokine plays a harmful role. we thank numerous colleagues for many helpful discussions that guided the formulation of the ideas presented here. we also thank kathy toews for assistance in preparing the manuscript. the ifn-? work in brian popko's laboratory has been supported by the national institutes of health (nih) research grant roi ns34939, and the national multiple sclerosis society research grant rg 2089. j. d. is supported by a training grant hd07201 from the nih. b. p. is a recipient of an nih research career development award (ns 01637) from ninds. reversible inhibitory effects of interferon-y and tumouar necrosis factor-o: on oligodendroglial lineage cell proliferation and differentiation in vitro molecular cloning and expression of the human interferon-? receptor experimental allergic encephalomyelitis: a usefid model for multiple sclerosis ligand-induced assembly and activation of the gamma interferon receptor in intact cells reactive astrogliosis in the neonatal mouse brain and its modulation by cytokines, f neurosci gamma-interferon promotes differentia* tion of cultured cortical and hippocampal neurons increased production of interferon gamma and tumor necrosis factor precedes 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kinase jak2 of a mutant cell line defective in the interferon-gamma signal transduction pathway interferon-like virus-inhibitor induced in human leukocytes by phytohernagglutinin cytokines and murine autoimmune encephalomyelitis: inhibition or enhancement of disease with antibodies to select cytokines, or by delivery of exogenous cytokines using a recombinant vaccinia virus system inducible expression of h-2 and ia antigens on brain cells interferon-gamma induces the expression of h-2 and la antigens on brain ceils r (1991) y-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo differential proliferafive response of human and mouse astrocytes to gala-interferon ~1) try_ ~nsgewic mouse model for central nervo~is system demyelknation role of interferon-y in immune cell regulation the t lymphocyte in experimental allergic encephalomyelitis key: cord-268835-catuja6c authors: libbey, jane e.; mccoy, lori l.; fujinami, robert s. title: molecular mimicry in multiple sclerosis date: 2007-12-31 journal: international review of neurobiology doi: 10.1016/s0074-7742(07)79006-2 sha: doc_id: 268835 cord_uid: catuja6c one of the most common demyelinating central nervous system (cns) diseases in humans is multiple sclerosis (ms). the disease can be very debilitating with vision loss, motor and sensory disturbances, and cognitive impairment. the clinical course may present as a relapsing‐remitting disease course, a progressive disease course, or a combination thereof. the etiology of ms is unknown. though many viruses have been shown to be associated with ms, no one virus has ever been demonstrated to be the cause of ms. in addition, ms is thought to have an autoimmune component. molecular mimicry is one hypothesis put forth which could reconcile the diverse pathology and etiology of ms. molecular mimicry occurs when peptides from pathogens share sequence or structural similarities with self‐antigens. infection with various pathogens, each with its individual molecular mimic to a cns antigen, may explain the inability of investigators to link one specific virus to ms. molecular mimicry may be mediated through human leukocyte antigen class i‐ and class ii‐restricted t cells and antibodies, which may explain the diversity in phenotype. aspects of molecular mimicry will be discussed in relation to each of these immune system components. examples of various molecular mimics will be discussed with a particular focus on the cns and ms. molecular mimicry alone may not be able to induce disease; priming of the immune system by infection with a pathogen that carries a molecular mimic to self may have to be followed by a later nonspecific immunologic challenge in order for disease to be initiated. recent research into this priming and triggering of disease will be discussed in relation to an animal model for ms. multiple sclerosis (ms) is the most common demyelinating disease in humans. ms has prevalence rates between 50 and 100 per 100,000 caucasians; other ethnic groups have somewhat lower prevalence rates and women are more azicted than men by a 2:1 ratio (kurtzke, 1997) . the inflammatory demyelinating lesions characteristic of ms are limited to the central nervous system (cns) (dejong, 1970) . in most instances, ms patients have oligoclonal immunoglobulin (ig)g bands in the cerebral spinal fluid (csf), and a mild mononuclear pleocytosis may also be present. clinical features of the disease include vision loss, motor and sensory disturbances, and cognitive impairment. the clinical course of ms can include relapses and remissions and may be progressive in nature. ms has been proposed to be mediated by autoreactive cns-specific cd4 þ t cells (markovic-plese and mcfarland, 2001; noseworthy et al., 2000) . however, significant numbers of cd8 þ t cells are found in ms lesions (hayashi et al., 1988; sobel, 1989) and are likely to be involved in pathogenesis. there have been many studies which attribute the neuroinflammation and neurodegeneration in ms to cd8 þ t cells, cd4 þ t cells, or antibody recognition of self. in this chapter, we summarize some salient points on each of these components of the immune system as they relate to molecular mimicry and disease. however, first we will provide background information and define molecular mimicry in relation to other key concepts. molecular mimicry was first hypothesized to be a potential mechanism for the initiation of autoimmune disease in the early 1980s. it is said to occur when peptides from viral or bacterial (zabriskie, 1986) proteins share sequence or structural similarities with self-peptides. this similarity could stimulate autoreactive immune responses causing the production of self-reactive antibodies. fujinami, oldstone, and colleagues (fujinami and oldstone, 1985; fujinami et al., 1983) initially showed that a cross-reactive epitope between the hepatitis b virus polymerase and the encephalitogenic epitope of myelin basic protein (mbp) for the rabbit could induce an experimental autoimmune encephalomyelitis (eae)-like disease when used as an immunogen. for eae to occur, this cross-reaction had to occur at the level of induction/activation of autoreactive cd4 þ t cells. this was the first demonstration of autoimmune disease induced by a viral peptide (fujinami and oldstone, 1985) . in the past, peptide similarities had been identified by computer searches for similar amino acid sequences; however, based on more recent studies, molecular mimicry has also been shown to occur with incomplete sequence matching provided the major histocompatibility complex (mhc) and t-cell receptor (tcr) contact motifs are preserved (lang et al., 2002) . additionally, peptide elution studies have found that mhc molecules can potentially bind hundreds of diverent peptides (hunt et al., 1992) . the work by lang et al. (2002) suggested that molecular mimicry occurs rather frequently having shown that diverent peptides bound to class ii molecules can lead to cross-reactivity by the same tcr provided the complexes have similar charge distribution and overall shape. the flexibility of tcr recognition plays a major role in forming the t-cell repertoire through thymic selection, and is highly important in protecting the host against potential pathogen-derived antigens that are more wide ranging than the limited number of memory t cells (casrouge et al., 2000) . tcr degeneracy raises the potential for cross-reactivity between pathogen-derived and self-antigens. the various forms of molecular mimicry are illustrated in fig. 1 . there are many molecular mimics that have been identified in ms studies, both viral and bacterial, as shown in table i , which supports the hypothesis that molecular mimicry frequently occurs. the welsh and selin laboratories have investigated molecular mimicry, or the sharing of immunologic cross-reactive epitopes, between viruses, as opposed to between a pathogen and its host, using such viruses as lymphocytic choriomeningitis virus (lcmv), pichinde virus (pv), vaccinia virus (vv), and murine cytomegalovirus (mcmv) chen et al., 2001; welsh et al., 2000) . they coined the term ''heterologous immunity'' to describe the protection provided by one fig. 1. molecular mimicry with and without sequence homology. the two peptides shown in (a) share sequence homology as well as t-cell receptor (tcr) contact sites (marked as an ''x''). the peptide sequences in (b) have the same overall shape and tcr contact residues, but do not share sequence homology. in (c), the sequences share tcr contact sites only; they do not share sequence homology and the overall shape is slightly diverent. molecular mimicry may still occur due to the core region of tcr recognition; however, this recognition may be lower aynity. infection against a diverent viral infection at a later time (chen et al., 2001) . this protection to the second infection was mediated by cd8 þ t cells and the production of interferon (ifn). they also found that for cd8 þ t cells, cross-reactivity impacts t-cell kinetics and the hierarchy of t cells responding to epitopes encoded by the infecting virus, which changes t-cell responsiveness (chen et al., 2001; selin et al., 1998) . foreign proteins generally have several epitopes capable of being presented by mhc molecules; however, the cellular immune response is usually focused toward a narrow subset of these epitopes. this narrow response is known as immunodominance. when dominant epitopes have been deleted from a pathogenic organism, other epitopes emerge as dominant ( yewdell and bennink, 1999) . this phenomenon of immunodominance can be avected by prior infection with an organism encoding an epitope that generates t cells that cross-react with a second pathogen . the skewing of t-cell immunodominance may be beneficial or harmful to the host. additional examples of cross-reactivity resulting in altered immune response (sospedra et al., 2005) myelin basic protein acinetobacter calcoaceticus 4-carboxymuconolactone decarboxylase a chlamydia pneumoniae (lenz et al., 2001) epstein-barr virus (lang et al., 2002) davies et al. (1996) . include: primary infection with influenza virus followed by epstein-barr virus (ebv ), human papillomavirus and human coronavirus, and that which occurs between diverent strains of dengue virus (kim et al., 2005) . for example, dengue virus serotypes encode subdominant epitopes that share sequence homology between them. when a host with prior immunity to one of these serotypes is infected with another viral serotype, the weakly shared epitope dominates the immune response resulting in better recognition of the first serotype. this is due to memory t cells specific for the shared epitope proliferating and expanding, giving the subdominant epitope a distinct advantage (mongkolsapaya et al., 2003) . unfortunately, not all cases of heterologous immunity are as predictable. for instance, previous infection with lcmv has been shown to protect mice against vv infection resulting in reduced viral titers and a change in the t-cell-dependent pathology (chen et al., 2001; selin et al., 1998) . in contrast, prior vv infection has little evect on lcmv immunity. it has been reported that vv infection of lcmv-immune mice results in expansion of mhc class i-restricted cd8 þ t cells with diverent epitopes dominating the response (chen et al., 2001; kim et al., 2002 kim et al., , 2005 selin et al., 1998) . following vv infection, lcmv-immune mice had a reduction in the frequency of lcmv-specific cd8 þ t memory cells to some epitopes while others remained constant or increased (kim et al., 2005) . heterologous immunity and molecular mimicry may be able to explain the low concordance rates for ms in monozygotic twins. with a 30% concordance rate for this group, it is clear that ms has a genetic component but that environmental factors also play a role in disease pathogenesis (ebers et al., 1986; mumford et al., 1994; sadovnick et al., 1993) . perhaps a series of infections that generate immune reactivity to otherwise subdominant epitopes mimicking self-proteins are required in a genetically predisposed individual in order for disease to occur. this response or t-cell reactivity would be diverent for identical twins since their tcrs are rearranged randomly. an alternate hypothesis currently being explored states that environmental factors in fetal or neonatal stages of life cause genetically susceptible individuals to be primed for autoimmune disease. the déjà vu theory takes this step further stating that there is an initial (fetal or neonatal) viral infection that persists and primes the individual for autoimmune disease provided a subsequent infection shares t-cell epitopes with the persisting virus (merkler et al., 2006) . in support of this, a study by rothwell et al. (1996) of patients with type-1 diabetes found that disease prevalence was higher in patients born in the spring and early summer than during the winter months. however, this hypothesis is still controversial as studies from slovenia (ursic-bratina et al., 2001) and israel (laron et al., 2005) had similar results with higher prevalence rates for type-1 diabetes in individuals born in the spring and early summer, but an analysis of data from germany showed opposite results with fewer type-1 diabetes cases born between april and september (neu et al., 2000) . thus far, mhc class ii-restricted cd4 þ t-cell responses to self have been more extensively investigated in ms patients and animal models than the mhc class i-restricted cd8 þ t-cell response. however, clinical and experimental research indicates that cd8 þ t-cell reactivity could be involved in the pathology of ms (wekerle and lassmann, 2005) . cytotoxic t lymphocytes (ctls) could destroy oligodendrocytes leading to demyelination. in vitro experiments have shown that oligodendrocytes can be induced by ifn-to express mhc class i antigen but not class ii antigen (grenier et al., 1989) . in both ms and theiler's murine encephalomyelitis virus (tmev) infection, apoptosis of oligodendrocytes has been observed (dowling et al., 1997; fujinami, 1996, 1999; tsunoda et al., 1997) . tmev belongs to the family picornaviridae. infection with tmev causes an extensive demyelinating disease in the cns of persistently infected mice fujinami, 1996, 1999) . we monitored cytotoxic t-cell responses in sjl/j mice, which are susceptible to tmev-induced demyelinating disease. we found that spleen cells and t-cell clones from tmev-infected mice were highly cytotoxic to uninfected syngeneic cells, but not to allogeneic cells following stimulation with tmev-infected antigen-presenting cells. these autoreactive cells were found to be cd3 þ cd8 þ by flow cytometry and antibodyblocking cytotoxicity assays ( tsunoda et al., 2005) . double chamber 51 cr release assays showed that direct cell-to-cell contact was required for lysis. it was also found that cell killing was mediated by the fas-fasl pathway (tsunoda et al., 2002) . intracerebral adoptive transfer of activated tmev-induced autoreactive cells and t-cell clones into naive mice resulted in degenerating lesions in the spinal cord, suggesting that these mhc class i-restricted cd8 þ t cells could play an evector role in cns pathology in this model (tsunoda et al., 2005) . we went on to show that these ctls could be eyciently induced by a recombinant vv encoding the tmev viral capsid proteins (tsunoda et al., 2006) . we hypothesized that the autoreactive ctls recognize tmev capsid proteins that share molecular mimicry with cns antigen. we are in the process of narrowing down the tmev capsid epitope and characterizing the mouse cns antigen that is the target of these ctls. previous studies on memory cd8 þ t cells suggested that these cells were a resting, nondividing population lying in wait until reexposure to the antigen that resulted in their initial diverentiation ( jamieson and ahmed, 1989) . other studies suggest that memory cd8 þ t cells continuously undergo a low level of homeostatic division for a long period of time following antigen clearance (kos and mullbacher, 1993; tough and sprent, 1994; zimmerman et al., 1996) . this homeostatic division can be augmented by type i ifn, interleukin (il)-15, and ifn inducers such as poly (i:c) as well as viral infection (tough et al., 1996; zhang et al., 1998) . one study used mice whose immune system had been reconstituted with labeled splenocytes from lcmv-immune mice and examined the fate of these cells following poly (i:c) treatment or infection with lcmv, pv, and vv (kim et al., 2002) . previous reports from this group indicated that lcmvimmune mice were partially resistant to pv and vv and that lcmv-immune splenocytes could provide resistance to both pv and vv following adoptive transfer into naive animals; however, this resistance was lost if cd8 þ t cells were depleted (selin et al., 1998) . in the kim et al. (2002) study, it was found that the heterologous viruses (pv and vv) had the capacity to induce several cycles of proliferative expansion of memory cd8 þ t cells which altered the antigen hierarchy of t cells specific to earlier pathogens. lcmv infection resulted in a greater than 300-fold increase in the number of cd8 þ t cells with a majority of cells being lcmv specific. in contrast, poly (i:c) treatment only resulted in limited cell division without a net increase in cell number or changes in the hierarchy. this phenomenon is likely due to poly (i:c) induction of apoptosis, which ovsets cell division (kim et al., 2002) . therefore, it was concluded that memory cell division with a net increase in number is characteristic of antigenspecific stimulation while division without an increase in cell number is a characteristic of cytokine-induced bystander stimulation. interestingly, it was also found that ctls generated against either lcmv or pv were not capable of lysing cells infected with the heterologous virus, which indicated little cross-reactivity between the viruses . however, ctls from pv-infected lcmvimmune mice were able to lyse cells infected with either virus (selin et al., 1994) . it was found that pv encodes a subdominant epitope that shares six of eight amino acids with an lcmv epitope . the fate of memory cells was assessed following pv infection of mice reconstituted with lcmv-immune cells (kim et al., 2005) . results indicate heterologous infection caused an increase in the number of t cells specific for the subdominant pv epitope. in contrast, there were considerable diverences between individual lcmv-immune mice in terms of what epitope-specific t cells were stimulated by vv infection. even though these responses were unpredictable, patterns of epitope recognition did emerge, with three epitopes more likely to be dominant. these diverences in epitope-specific response are thought to be due to private specificities of the tcr repertoire generated in each mouse following random rearrangement of the variable (v), diversity (d), and joining ( j) tcr gene segments (kim et al., 2005) . studies examining the composition, maintenance, and alteration of the t-cell memory pool have important implications for ms, and may help to explain the low concordance rates seen in identical twins. a novel transgenic mouse model was created by oldstone and colleagues (evans et al., 1996) to examine whether molecular mimicry to a protein expressed in the cns could lead to autoimmune disease. in this model, mice were generated that express nucleoprotein (np) or glycoprotein from lcmv under the control of the mbp promoter as self in oligodendrocytes (evans et al., 1996) . these mice were infected with the armstrong strain of lcmv and monitored for clinical signs of disease. it was found that lcmv infected tissues in the periphery but not the cns, and that a vigorous cd8 þ ctl response cleared virus from all tissues by 7-14 days postinfection. at 3-weeks postinfection, the transgenic mice had 150-300 cd8 þ t cells per sagittal section of brain while nontransgenic control mice had less than 30 lymphocytes per section. perivascular cuyng was seen in the transgenic mice and lymphocytes were seen in the parenchyma, brain stem, and spinal cord. interestingly, brains of the transgenic mice infected with lcmv were found to contain these elevated levels of cd8 þ t-cell infiltrates 1 year after infection. at 3-months postinfection, a clustering of cd4 þ and cd8 þ t cells was found predominately in the white matter of the cns including the corpus callosum, internal capsule, fimbria hippocampus, brain stem, and spinal cord. clinical signs, including ruzed fur, weight loss, and balance diyculties, were also evident in 75% of the transgenic mice at 3-months postinfection. this study also investigated the evect of a second infection with lcmv or vv encoding the np transgene on the transgenic mice 6 weeks after the initial lcmv infection. stimulation of the memory response by these viruses resulted in enhanced pathology including an increase in the ratio of cd8 þ to cd4 þ t lymphocytes and a greater likelihood that these infiltrates would be localized to the white matter as shown by immunohistochemical staining (evans et al., 1996) . this enhanced disease pathology, characterized by demyelination and motor dysfunction, following secondary viral infection is similar to human cns autoimmune diseases, and these results suggest that infection by a virus that shares immune determinants with a protein expressed in oligodendrocytes can induce a chronic inflammatory disease of the cns. to further validate the role viruses may play in the induction of autoimmune disease, a molecular mimicry model utilizing an h-2d b -restricted immunodominant epitope of lcmv, np 396-404 was used to identify structurally homologous murine self-proteins (hudrisier et al., 2001) . the mhc class i-restricted cd8 þ t-cell response against the lcmv np protein has been shown to be directed against this immunodominant np 396-404 epitope. typically in an lcmv-infected mouse that has cleared the virus, there is activation and proliferation of cd8 þ t cells which stay at a high level throughout the animal's life despite the fact that there is no detectable virus or viral antigens. these ctls represent a potential source of autoreactivity, especially if their cytolytic activity remains functional and intact. six nonameric sequences from endogenous proteins which shared structural and functional homology with lcmv np 396-404 were identified through a database search. five of these peptides were shown to have high h-2d b -binding aynity and shared the main tcr contact site with lcmv np 396-404 . three of these five peptides also shared an auxiliary tcr contact residue and one of these, tumor necrosis factor (tnf) receptor i 302-310 , also shared a third residue with the lcmv peptide resulting in a marked functional similarity between the self and viral epitopes. despite the presence of tcr contact residues, none of the epitopes identified were able to stimulate antiviral ctls in cytotoxicity assays. however, these peptides did behave as antagonists of lysis by lcmv-specific ctls which indicated that they were capable of interacting with the tcr, though the aynity was relatively low. low aynity recognition of self-mhc promoted thymocyte survival, and cells cultured with peptides were maintained for more than 2 months, suggesting these epitopes were in fact low-aynity molecular mimics. importantly, these self-peptides were shown to allow lcmv-specific and potentially autoreactive cd8 þ t cells to maintain their cytolytic function in the absence of viral antigens over a period of months (hudrisier et al., 2001) . in the 1930s rivers and colleagues (rivers and schwentker, 1935; rivers et al., 1933) published reports on induction of encephalomyelitis using cns homogenates; and throughout the 1950s and 1960s research continued on myelin proteins in eae and ms. in the 1940s it was shown that injection of myelin proteins with adjuvant into naive animals could cause relapsing-remitting, acute, or chronic encephalomyelitis (freund et al., 1947; kabat et al., 1946 kabat et al., , 1947 kopelov and kopelov, 1947; morgan, 1946 morgan, , 1947 morrison, 1947) . early studies also determined that eae could be transferred by adoptive transfer of cd4 þ t cells (pettinelli and mcfarlin, 1981; zamvil and steinman, 1990) . mhc class ii was found to have a genetic link with a variety of autoimmune diseases including ms ( jersild et al., 1973a,b) . mhc class ii molecules present selfpeptides to cd4 þ t cells; therefore, cd4 þ t cells are thought to play a major role in the initiation and/or progression of many autoimmune diseases. several animal models of ms and human studies using blood and csf from ms patients implicate cd4 þ t cells, mimicry, and various microbes such as chlamydia pneumoniae, influenza virus, torque teno virus (ttv ), ebv, and human herpesvirus 6 (hhv-6) (croxford et al., 2005; lang et al., 2002; markovic-plese et al., 2005; sospedra et al., 2005; sriram et al., 1999; tejada-simon et al., 2003) . c. pneumoniae has been associated with ms (sriram et al., 1999) . in one study, csf from 17 patients with relapsing-remitting ms, 20 patients with progressive ms, and 27 patients with other neurological diseases was tested for c. pneumoniae (sriram et al., 1999) . bacterial dna was detected in 97% of these ms patients compared to only 18% of patients with other neurological diseases. they were also able to isolate c. pneumoniae from the csf of ms patients in addition to identifying anti-c. pneumoniae antibodies. this work inspired lenz et al. (2001) to identify a protein from c. pneumoniae, cnp0483, with similarity to mbp. cnp0483, an mbp 68-86 homologue peptide, was used to sensitize lewis rats. animals began showing signs of disease approximately 12-days postsensitization. pathologically, perivascular cuyng and mononuclear cell infiltrates were found in the spinal cords of cpn0483-immunized rats. additionally, splenocytes from cpn0483injected mice that had been cultured with peptide caused disease when they were transferred into naive animals. interestingly, the cpn0483 peptide shares only seven amino acids with mbp 68-86 . this small degree of similarity happens to constitute a structural motif that permits interaction of the peptide with mhc class ii gene products (lenz et al., 2001) . influenza virus is an example of a virus infection where there is flexibility in tcr recognition and the degree of sequence and structural similarity necessary for cross-reaction. one group derived a cd4 þ t-cell clone from an ms patient's peripheral blood mononuclear cells (pbmcs) during an acute respiratory infection with influenza-a (markovic-plese et al., 2005) . they determined that this t-cell clone, gp5f11, reacted with the immunodominant influenza hemagglutinin (flu-ha) epitope 306-318. gp5f11 was found to be a high avidity pathogenspecific t-cell clone that had demonstrated cross-reactivity against 14 flu-ha variants, 11 viral, 15 human, and 3 myelin-derived peptides. of the 11 viral mimics, it was found that equine influenza-a ha peptide had greater than 70 times higher stimulatory potential than the human influenza peptide, suggesting that there is a high potential for cross-recognition even for a t-cell clone with a stringent (high avidity) tcr response. of 12 potentially stimulatory myelinderived peptides, 2 myelin oligodendrocyte glycoprotein (mog) peptides and one 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase (cnpase)-derived peptide were found to be extremely potent stimulators of the t-cell clone. interestingly, the cnpase-derived peptide had no sequence similarity to the flu-ha peptide (markovic-plese et al., 2005) . this broad range of cross-reactivity of a single tcr would assure protection against much more than the original infecting influenza virus with the potential side evect of autoimmune responses against self-antigens having structural or sequence similarity to pathogenic peptides. another series of experiments found that one cd4 þ t-cell clone (mn19) isolated from the csf of an ms patient during an exacerbation was stimulated by arginine-enriched protein domains from common viruses as well as the nonpathogenic ttv (sospedra et al., 2005) . the t-cell clones used in this experiment were found to be expanded during exacerbations of ms and reduced during periods of remission. biometric analysis was used to predict stimulatory peptides for the t-cell clones and then peptides were selected from human infectious agents. many peptides were identified using this method; however, following normalization only two related dna viruses were selected. these viruses were ttv and ttv-like mini virus, both of which are recognized as ubiquitous, nonpathogenic viruses in the human population. there were several stimulatory peptides identified from ttv, and surprisingly, the majority were mapped to a 74-amino acid sequence in the n-terminal region of open reading frame 1. this n-terminal region is enriched in positively charged amino acids, mainly arginine. other stimulatory peptides were also identified for the t-cell clone from other human viruses, including adenovirus and papillomavirus. the adenovirus peptide was from the pvii protein which is enriched in arginine and performs a histone-like function. the papillomavirus peptides were found in the minor capsid protein l2, which is enriched in basic amino acids. human stimulatory peptides from the human cns proteins adrenergic receptor -1b, -1a, and -2c, arginine-rich protein, and dopamine d2 receptor were also identified for the mn19 t-cell clone. interestingly, arginine-rich domains are frequent in viruses as well as eukaryotes and prokaryotes (sospedra et al., 2005) . this recognition of an arginine-enriched conserved domain may be involved in inducing and perpetuating autoimmune responses against arginine-rich self-antigens. ebv has long been associated with ms and viral reactivation is linked to disease activity (bray et al., 1983; larsen et al., 1985) . ebv is a large dna virus that causes mononucleosis in humans and has been shown to persist in b cells. infection with ebv late in life has been correlated with an increased risk of developing ms. lang et al. (2002) used a cd4 þ t-cell clone isolated from an ms patient with a relapsing-remitting disease course to examine the tcr contact surfaces of a cross-reactive tcr that recognizes both the mbp 85-99 peptide and an ebv dna polymerase peptide (ebv 627-641 ). they found that mbp 85-99 was recognized in the context of drb1*1501 and ebv 627-641 was recognized in the context of drb5*0101 based on relative binding aynities (lang et al., 2002) . drb1*1501, drb5*0101, as well as dqb1*0602 are mhc class ii alleles of the dr2 haplotype that have been shown to be risk factors for ms. transgenic mouse studies determined that spleen cells from tcr-drb1*1501 double-transgenic mice only responded in the form of proliferation and cytokine production to the mbp 85-99 peptide, while spleen cells from tcr-drb5*0101 double-transgenic mice only responded to the ebv 627-641 peptide. spleen cells from triple-transgenic mice with tcr-drb1*1501-drb5*0101 responded to both the mbp 85-99 and ebv 627-641 peptides. these transgenic mice recognized the mbp and ebv peptides in the context of two diverent mhc class ii molecules. next, the crystal structures of both of these mhc-peptide complexes were determined and compared. it was found that the tcr contact surfaces of the drb5*0101-ebv 627-641 and drb1*1501-mbp 85-99 complexes were structurally very similar. the nature of mhc class ii binding to peptide, with the peptide chain held in a highly conserved extended conformation, is thought to allow for structural mimicry in tcr recognition (lang et al., 2002) . clinical studies extending the in vitro work of lang et al. (2002) on ebv have found that cd4 þ t cells present in the csf of an ms patient cross-react with ebv 627-641 and the immunodominant peptide mbp 85-99 (holmøy et al., 2004) . the presence of ebv 627-641 -specific cd4 þ t cells in the csf proves that ebv-specific t cells can gain access to the intrathecal compartment and suggests that they could target mbp in the cns (holmøy et al., 2004) . another virus that encodes proteins with peptide mimics to human brain proteins is hhv-6, the agent responsible for infantile exanthem. the virus has a tropism for cd4 þ t cells and can reactivate from its latent state following immunosuppression (braun et al., 1997; salahuddin et al., 1986) . challoner et al. (1995) found that oligodendrocytes from ms patients expressed hhv-6 virion proteins. a protein found in both the a and b variants of hhv-6, known as u24, has amino acid sequence similarity with mbp. two 13-mer peptides corresponding to residues 1-13 of the hhv-6 u24 protein and mbp 93-105 were synthesized and used to determine whether t cells would cross-react with the two peptides or were specific for only one of the sequences (tejada-simon et al., 2003) . t-cell cultures from pbmcs of ms patients were incubated with mbp 93-105 or hhv-6 u24 1-13 for 1 week, tested for specificity, and characterized as mbp or hhv-6 reactive or cross-reactive. the frequency of t cells reactive to both hhv-6 and mbp peptides was determined to be significantly higher in ms patients than in the control group (tejada-simon et al., 2003) . the cytokine profiles of the peptide-specific and crossreactive t-cell lines generated from ms patients were found to display a th1 phenotype, predominately producing ifn-and tnf-, but not il-4 and il-10, regardless of peptide specificity. these cells were shown to be cd4 þ by flow cytometry. the presence of oligoclonal antibodies in the csf of patients with ms is a consistent immunologic marker of the disease (link, 1978; link and kostulas, 1983) . two or more oligoclonal bands consisting of igg are routinely seen by isoelectric focusing followed by immunoblotting of csf in up to 95% of ms patients (link and huang, 2006) . each band represents antibody, though identification of the antigen that these antibodies bind has been diycult in part because of the low amount of antibodies contained in the csf. some of the antigens to which the oligoclonal igg antibodies have been found to be specific include mbp, hhv-6, measles, rubella, varicella-zoster virus, and herpes simplex virus 1 (hsv-1) (cruz et al., 1987; derfuss et al., 2005; reiber et al., 1998) . one method that has been used as an alternative source of antigens to study the specificity of antibodies is phage-displayed random peptide libraries (rpl) (cortese et al., 1996; dunn, 1996; smith, 1991) . the peptides in these libraries are referred to as mimotopes because it is not required for them to have sequence similarity, but rather they mimic binding properties or conformation of natural epitopes. there have been reports of mimotopes from rpl selection that were recognized by antibodies from the csf of ms patients (cortese et al., 1996 (cortese et al., , 1998 dybwad et al., 1997) . interestingly, some of these antibodies were detected in sera from normal individuals as well as ms patients, suggesting that the mimotopes mimic common antigens (cortese et al., 2001) . one mimotope family, ms17, was chosen because of its reactivity with csf antibodies and high frequency of reactivity with the sera of ms patients. two csf samples from ms patients that recognize ms17 were tested for reactivity in an enzyme-linked immunosorbent assay (elisa) to neurotropic viruses, including ebv, measles, mumps, rubella, hsv-1, and cytomegalovirus (cortese et al., 2001 ). both csf samples tested positive against measles and hsv-1; however, only the reactivity to hsv-1 was competitively inhibited by ms17 phage. in addition, whole cell extract from an hsv-1-infected cell line preincubated with csf from the ms patients completely abolished recognition of the phage (cortese et al., 2001) . a consensus sequence profile was then derived from multiple alignments of all hsv-1 isolates, and this was used to search for homology between eight members of the ms17 family and hsv-1 protein sequences. the highest score from this profile came from the n-terminal region of the hsv-1 envelope glycoprotein b (gb). the ms17 mimotope corresponds to the sequence of hsv-1 gb between amino acid positions 474-482. antibodies against ms17 were raised in rabbits, and extensive characterization of these antibodies demonstrated that the ms17 phage is both an antigenic and immunogenic mimic of the hsv-1 gb epitope. using the anti-ms17 antibodies in place of the csf samples, the antibodies were found to specifically interact with an as yet uncharacterized protein present in protein extract from the brain of an ms patient (cortese et al., 2001) . these studies represent an important step in identification of the natural antigens that antibodies from ms patients recognize as well as providing an example of molecular mimicry between a viral and cns epitope that is mediated through antibody. the rpl technique has the potential to aid in explaining the pathogenesis of autoimmune diseases. monoclonal antibodies generated against various virus-specific proteins have been shown to cross-react with host cell components, thus serving as additional examples of molecular mimicry at the level of antibodies. studies have shown that monoclonal antibodies generated against viral proteins from measles, hsv-1, and vv react with host cell components (dales et al., 1983; fujinami et al., 1983) ; common determinants existed between viruses and cellular-or tissue-specific elements (srinivasappa et al., 1986) . in another report, a monoclonal antibody, h8, to the tmev daniels (da) strain reacted with both tmev viral protein-1 and lipid-like moieties including galactocerebroside, a major component of myelin (fujinami et al., 1988) . in mouse brain cultures, cells that bound the tmev monoclonal antibody, h8, were also labeled with antibody to mbp (yamada et al., 1990) . this double staining indicated that the h8 antibody recognized epitopes on oligodendrocytes. when the antibody was injected into mice with eae, h8 caused an increase in the area of demyelination within spinal cords. furthermore, a competition elisa for galactocerebroside and tmev found that sera from mice contained antibody with the same specificity as h8. these results indicate that the immune response which generates antibodies specific for tmev that cross-react with myelin and oligodendrocytes could contribute to demyelination through an antibody-mediated process ( yamada et al., 1990) . there are several other examples of molecular mimics at the level of antibodies in autoimmune diseases other than ms. they include guillain-barre syndrome in which antibody cross-reactivity between peripheral nerve and ganglioside has been shown (ogino et al., 1995; yu et al., 2006) , and rheumatic heart disease in which antistreptococcal antibodies cross-react with n-acetyl--d-glucosamine, myosin, and other self-epitopes (guilherme et al., 2006; mertens et al., 2000) . in addition, autoantibody development is seen in systemic lupus erythematosus (poole et al., 2006) . ebv has been associated with lupus through serological and dna studies and antibodies specific for epstein-barr nuclear antigen-1 (ebna-1) cross-react with lupus-associated autoantigens (poole et al., 2006) . alternately, infection with human t-lymphotropic virus type-1 (htlv-1) can cause htlv-1associated myelopathy/tropical spastic paraparesis (ham/tsp), an immunemediated disease of the cns (lee et al., 2006) . molecular mimicry has been found in antibodies that cross-react with heterogeneous nuclear ribonucleoprotein a1 (hnrnp a1), a protein found in cns neurons, and the htlv-1 tax protein. two core epitopes within the c-terminal region of hnrnp a1, both functionally important regions, were found to react with antibodies purified from ham/ tsp patients. additionally, monoclonal antibodies raised against htlv-1 tax protein also reacted with the two core epitopes from hnrnp a1 (lee et al., 2006) . potentially, infection with viruses that have molecular mimicry with cns antigens can prime autoreactive immune cells specific for the cns in hosts. our laboratory has explored whether a virus having molecular mimicry with self-cns antigens could induce an autoimmune disease in mice which had previously been inducible only by injection of cns antigen in complete freund's adjuvant (cfa) (theil et al., 2001) . three recombinant vvs were constructed: encoding myelin proteolipid protein (vv plp ), encoding myelin-associated glycoprotein (vv mag ), and encoding glial fibrillary acidic protein (vv gfap ). mice infected with vv plp , vv mag , or vv gfap showed no clinical or histological signs of cns disease. this suggests that molecular mimicry alone cannot result in high enough numbers or activation of cns-specific autoimmune cells for induction of cns disease. thus, 5 weeks after the first infection, when vv was cleared, we challenged mice nonspecifically with cfa to activate cns-specific autoimmune cells (bystander activation). clinically, some mice showed paralysis in the tail, similar to eae, following the cfa challenge. at 1-month post-cfa challenge, mice were sacrificed and cns tissues were examined for pathological changes. all mice (15/15) were found to have inflammatory lesions with cd3 þ t cells in the cns (theil et al., 2001) . as a negative control, mice were infected with vv sc11 , a recombinant vv that encodes -galactosidase, which has no known molecular mimicry with cns antigens; following cfa challenge, no inflammatory changes were seen in the cns (theil et al., 2001) . these data indicate that infections with viruses encoding molecular mimics can substantially prime animals for autoimmune disease and at a later time a nonspecific immunologic challenge could initiate the disease. as reviewed by trinchieri (1995) , mcmv infection in mice causes a significant burst of il-12, which promotes the development of th1 cells. therefore, we have tested whether vv plp -sensitized mice developed clinical disease following a second, unrelated viral challenge with mcmv. preliminary findings suggest this to be the case. five out of nine mice primed with vv plp and challenged with mcmv were found to have meningitis and perivascular cuyng in the cns. in contrast, mice primed with vv plp and challenged with the wild-type strain of vv were found to have no obvious lesions. in another experiment, mice primed with vv plp and challenged with mcmv showed marked weight loss and had righting reflex disturbances, compared with mice injected with phosphate-buvered saline (pbs) or vv sc11 followed by mcmv challenge (ikuo tsunoda, jane e. libbey, and robert s.fujinami, unpublished data). thus far, the definitive experiments to determine whether disease in this model is caused by mhc class i or class ii cells have not been completed. however, these experiments are important in that they provide a working model mirroring what may be occurring in human patients with ms. molecular mimicry has been found in many autoimmune diseases on a variety of levels. there is significant evidence that autoreactive cd4 þ and cd8 þ t cells as well as autoantibodies can contribute to disease progression in various animal models of ms and other autoimmune diseases. however, it has yet to be shown that a single molecular mimic is responsible for initiation of disease. none of the autoimmune diseases described 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roles in guillain-barre syndrome and related diseases rheumatic fever: a model for the pathological consequences of microbial-host mimicry the t lymphocyte in experimental allergic encephalomyelitis potent and selective stimulation of memory-phenotype cd8 þ t cells in vivo by il-15 visualization, characterization, and turnover of cd8 þ memory t cells in virus-infected hosts we wish to thank ikuo tsunoda, md, ph.d., for many helpful discussions and ms. kathleen borick for her excellent preparation of the chapter. this work was supported by nih grant ai0581501. key: cord-276587-ynionj5r authors: hwang, mihyun; bergmann, cornelia c. title: alpha/beta interferon (ifn-α/β) signaling in astrocytes mediates protection against viral encephalomyelitis and regulates ifn-γ-dependent responses date: 2018-04-27 journal: j virol doi: 10.1128/jvi.01901-17 sha: doc_id: 276587 cord_uid: ynionj5r the contribution of distinct central nervous system (cns) resident cells to protective alpha/beta interferon (ifn-α/β) function following viral infections is poorly understood. based on numerous immune regulatory functions of astrocytes, we evaluated the contribution of astrocyte ifn-α/β signaling toward protection against the nonlethal gliaand neuronotropic mouse hepatitis virus (mhv) strain a59. analysis of gene expression associated with ifn-α/β function, e.g., pattern recognition receptors (prrs) and interferon-stimulated genes (isgs), revealed lower basal mrna levels in brain-derived astrocytes than in microglia. although astrocytes poorly induced ifnβ mrna following infection, they upregulated various mrnas in the ifn-α/β pathway to a higher extent than microglia, supporting effective ifn-α/β responsiveness. ablation of the ifn-α/β receptor (ifnar) in astrocytes using mgfapcre ifnar(fl/fl) mice resulted in severe encephalomyelitis and mortality, coincident with uncontrolled virus replication. further, virus spread was not restricted to astrocytes but also affected microglia and neurons, despite increased and sustained ifnα/β and isg mrna levels within the cns. ifn-γ, a crucial mediator for mhv control, was not impaired in infected mgfapcre ifnar(fl/fl) mice despite reduced t cell cns infiltration. unexpectedly however, poor induction of ifn-γ-dependent major histocompatibility complex (mhc) class ii expression on microglia supported that defective ifn-γ signaling contributes to uncontrolled virus replication. a link between sustained elevated ifn-α/β and impaired responsiveness to ifn-γ supports the novel concept that temporally limited early ifn-α/β responses are critical for effective antiviral ifn-γ function. overall, our results imply that ifn-α/β signaling in astrocytes is not only critical in limiting early cns viral spread but also promotes protective antiviral ifn-γ function. importance an antiviral state established by ifn-α/β contains initial viral spread as adaptive immunity develops. while it is apparent that the cns lacks professional ifn-α/β producers and that resident cells have distinct abilities to elicit innate ifn-α/β responses, protective interactions between inducer and responder cells require further investigation. infection with a gliaand neuronotropic coronavirus demonstrates that astrocytes mount a delayed but more robust response to infection than microglia, despite their lower basal mrna levels of ifn-α/β-inducing components. lethal, uncontrolled viral dissemination following ablation of astrocyte ifn-α/β signaling revealed the importance of ifn-α/β responses in a single cell type for protection. sustained global ifn-α/β expression associated with uncontrolled virus did not suffice to protect neurons and further impaired responsiveness to protective ifn-γ. the results support astrocytes as critical contributors to innate immunity and the concept that limited ifn-α/β responses are critical for effective subsequent antiviral ifn-γ function. v iral infections of the central nervous system (cns) are rare but can lead to rapid mortality or long-term neurological disabilities even if acute encephalitis is resolved (1) . early essential host defense mechanisms involve induction of alpha/beta interferon (ifn-␣/␤) and signaling through the ifn-␣/␤ receptor (ifnar) to upregulate ifn-stimulated genes (isgs). isg expression is associated with numerous biological activities, including antiviral and immunomodulatory pathways, e.g., interference with translation, apoptosis, and enhanced major histocompatibility complex (mhc) class i antigen (ag) presentation (2) (3) (4) . while the ifn-␣/␤ response is critical in stemming cns viral replication and spread, it is rapidly downregulated and insufficient to eliminate virus in the absence of subsequent adaptive immune responses (5) (6) (7) . moreover, the efficiency of the innate response in limiting viral spread sets the stage for the effectiveness of subsequent adaptive immunity (2) (3) (4) 8) . detection of viruses and induction of ifn-␣/␤ are initiated by pattern recognition receptors (prrs), which differ in subcellular localization and are specialized to recognize distinct molecular entities in virus structural components, genome, and/or replication intermediates (2, 8, 9) . prrs which recognize rna viruses include endosome-associated membrane-bound toll-like receptors (tlrs), mainly tlr3, and the cytoplasmic retinoic acid inducible gene 1 (rig-i)-like receptors (rlrs) and melanoma differentiationassociated antigen 5 (mda5). specialized prr adapter proteins transmit signals to activate ifn response factor 3 (irf3), irf5, and irf7, which translocate to the nucleus to induce ifn-␤ and a subset of ifn-␣ genes (9) . secretion of ifn-␣/␤ and signaling through ifnar subsequently activate isgs and the "antiviral state." prr activation also activates nuclear factor b (nf-b)-regulated genes, thereby inducing proinflammatory cytokines and chemokines to facilitate recruitment of innate and adaptive effector cells (2, 9) . virtually all nucleated cells are able to induce and respond to ifn-␣/␤, including cns resident cells (5, 6) . the cns does not harbor plasmacytoid dendritic cells, which are potent peripheral ifn-␣/␤ inducers (3, 10) , and therefore has to rely on intrinsic induction of ifn-␣/␤. cns resident cells have been shown to differ widely in the repertoire as well as magnitude of basal and inducible transcripts encoding prrs and factors associated with the ifn-␣/␤ pathway (5) (6) (7) 11) . as ifn-␣/␤ signaling can rapidly induce genes involved in virus sensing and their signaling components, cell types which are not effective initial ifn-␣/␤ inducers may nevertheless contribute to the overall innate antiviral activity by efficient ifn-␣/␤ signaling and amplification of the ifn-␣/␤ response (5, 11, 12) . the interdependence of cns cells in achieving an ifn-␣/␤-dependent antiviral state thus requires further investigation. microglia and astrocytes are the cns resident cells most prominently involved in early innate responses to injury, autoimmune attack, or infection (6, 7, 11, 12) . in infections these early responses are essential to attract peripheral immune cells and limit viral dissemination (2, 3, 8) . importantly, not only productively infected but also abortively infected astrocytes mount innate responses that contribute to protection (3, 13, 14) . cns infections by several neuronotropic rna viruses, including la crosse virus, rabies virus, vesicular stomatitis virus (vsv), and theiler's murine encephalomyelitis virus (tmev), suggested that astrocytes, not neurons, are the main source of ifn-␤ expression and contributors to virus control through both tlr and rlr activation pathways (14) (15) (16) . both microglia and astrocytes express various prrs, and their sentinel function is supported by their rapid response to microbial infection (5, 7, 10, 14, 17, 18) . in addition to participating in direct antiviral functions, astrocytes regulate cns homeostasis, support neuronal function, and participate in formation and maintenance of the blood-brain barrier (bbb) (19, 20) . both astrocytes and ifn-␣/␤ actively participate in bbb function to restrict cns entry of molecules, cells, or pathogens (19, 21) . ifn-␣/␤ signaling specifically to astrocytes promotes bbb integrity in the cerebellum and protects from west nile virus infection (22) . thus, both astrocyte ifn-␣/␤ induction and signaling regulate the outcome of infection within the cns at different functional levels. the essential role of ifnar signaling in limiting viral dissemination within the cns is supported by neurotropic virus infections of ifnar-deficient (ifnar ϫ/ϫ ) mice (3, 5, 6, 23). however, caveats for ifnar ϫ/ϫ mice are the reduced basal levels of factors involved in the ifn-␣/␤ pathway as well as loss of innate myeloid cell dynamic activity in response to infection (11, 24, 25) . further, while much information on cns innate responsiveness is derived from primary glial and neuronal cultures, the in vivo studies of ifn-␤ induction highlight how unsuspected players, such as abortively infected astrocytes, contribute to pathogen control (14) . based on the prominent role of astrocytes in participating in innate responses, this study set out to assess how ifnar ablation in astrocytes affects pathogenesis in a gliaand neuronotropic coronavirus infection. the murine coronavirus mouse hepatitis virus (mhv) strain a59 infects microglia, astrocyte, neurons, and oligodendroglia following intracranial (i.c.) administration (26, 27) . although mhvs are at best poor inducers of ifn-␣/␤ (28) (29) (30) , they do induce ifn-␤ in microglia/macrophages (18) . importantly, even the low levels of ifn-␣/␤ are essential to prevent viral dissemination and mortality (31, 32) . the studies here reveal distinct patterns of basal and inducible levels of mrnas encoding components of the ifn-␣/␤ pathway in astrocytes and microglia isolated from naive and infected adult mouse brains. despite expressing lower baseline mrna levels, astrocytes upregulated ifn-␣/␤ pathway gene expression to a greater extent than microglia, supporting effective ifn-␣/␤ responses. ablation of ifn〈r in astrocytes using mgfapcre ifnar fl/fl mice resulted in severe encephalomyelitis and mortality by 7 days postinfection (p.i.). this contrasted with mild clinical symptoms and no fatalities in infected control ifnar fl/fl mice. uncontrolled viral spread throughout the cns parenchyma of mgfapcre ifnar fl/fl mice not only was associated with increased astrocyte infection but also affected neurons and microglia, despite overall elevated and sustained levels of mrnas for ifn-␤ and ifn-␣ genes and isgs. ifn-␥, a crucial mediator of mhv control in the cns, was not impaired, despite reduced t cell cns infiltration. unexpectedly however, defective ifn-␥ signaling was implicated by impaired induction of ifn-␥-dependent mhc class ii expression on microglia. overall our results imply that ifn-␣/␤ signaling in astrocytes not only is critical in limiting cns viral spread but also promotes lymphocyte-derived protective antiviral ifn-␥ function. mhv strain a59 induces type i ifn in the cns coincident with viral replication. to evaluate the kinetics of mhv a59 replication relative to ifn␣/␤ and ifn␥ mrna levels in the cns, brains from uninfected and infected wild-type (wt) c57bl/6 mice were harvested out to day 21 p.i. virus replication was monitored by expression of viral rna encoding the n protein (a59 n), which is present on genomic and all subgenomic rnas (33) . viral n mrna was most abundant between days 3 and 5 p.i. and declined by day 7 p.i. (fig. 1 ), but it remained detectable out to day 21 p.i. ifn␤, ifn␣4, and ifn␣5 mrnas, the most abundant ifn␣ mrnas expressed following mhv a59 infection (34) , also reached maximal levels between 3 and 5 days p.i. and dropped significantly by day 7 p.i. (fig. 1 ). while ifn␤ mrna was still elevated at day 10 p.i., ifn␣4 and ifn␣5 mrnas had declined to basal levels by day 7 p.i. ifn␥ mrna was already detectable at day 3 p.i. but did not peak until 5 to 7 days p.i. expression levels declined by day 10 p.i. but remained elevated above background out to day 21 p.i. (fig. 1) . ifn␣/␤ mrna levels thus peaked together with viral mrna, whereas the ifn␥ mrna peak was delayed and coincided with t cell infiltration (31) . astrocytes exhibit distinct induction of and responsiveness to ifn-␣/␤ compared to microglia. although mhv a59 replicates in glia and neurons, it induces ifn-␣/␤ only in microglia, not astrocytes, using primary cell cultures (29) . to assess the relative induction of and responsiveness to ifn-␣/␤ in astrocytes and microglia in vivo, we used infected gfap-gfp mice to isolate cd45-negative, green fluorescent protein (gfp)-positive (cd45 ϫ gfp ϩ ) astrocytes and cd45 int cd11b ϩ microglia by fluorescenceactivated cell sorting (facs) ( fig. 2a ). measurement of viral n relative to glyceraldehyde-3-phosphate dehydrogenase (gapdh) mrnas in either glia population revealed viral replication in both microglia and astrocytes at days 3 and 5 p.i. and a decline by day 7 p.i. (fig. 2b ). while increased viral n mrna in microglia relative to astrocytes at days 3 and 5 p.i. did not reach statistical significance, overall viral n mrna levels mirrored those in total brain. the same populations of purified cells were used to measure transcript levels of the ifn␣/␤ genes, genes regulating ifn-␣/␤, signaling and selected antiviral isgs (fig. 2c) . in naive mice, constitutive expression of these genes was lower or undetectable in astrocytes compared to microglia. following infection, ifn␤ mrna was upregulated by day 3 p.i. in microglia but was not upregulated until day 5 p.i. and was less robust in astrocytes. in contrast, ifn␣4 and ifn␣5 mrnas were not significantly upregulated in microglia but were increased prominently in astrocytes by day 5 p.i. consistent with the decline in viral rna, ifn␣/␤ mrnas dropped to baseline levels by day 7 p.i. (fig. 2c ). transcripts for components required for ifn-␣/␤ signaling showed no significant differences between astrocytes and microglia throughout days 3 to 7 p.i. (fig. 2c) . while infection mediated a decline in ifnar1 mrna relative to basal levels in microglia by day 5 p.i., it did not alter expression levels in astrocytes. stat1 mrna levels varied between cell preparations and showed no significant changes throughout infection in either cell type. irf9 transcripts were not affected by virus infection in microglia but increased modestly in astrocytes. in contrast, individual isgs were regulated distinctly not only between the glia populations but also within each glia type over time (fig. 2c) . mhv cns infection has been shown to strongly induce ifn-induced protein with tetratricopeptide repeats (ifit1 and ifit2) and 2=,5=-oligoadenylate synthetase 2 (oas2) transcripts (11, 31) . although ifit1 mrna was increased in both populations by day 3 p.i., the relative induction was significantly higher in astrocytes than in microglia at all time points. in contrast, ifit2 mrna was induced more prominently in microglia at day 3 p.i., reached similar levels in both cell types at day 5 p.i., and dropped thereafter in both populations. lastly, oas2 mrna showed peak upregulation in microglia by day 3 p.i. and modest induction in astrocytes (fig. 2c) . under the assumption that viral mrna levels reflect similar replication in both glial populations, these data support microglia as superior initiators of ifn-␤ production relative to astrocytes following mhv a59 infection in vivo; these findings are consistent with results from primary neonatal cell cultures (18, 29) . moreover, delayed ifn␤ upregulation in astrocytes suggested that their sensitivity to viral replication is enhanced once viral sensors are elevated via ifn-␣/␤ signaling. this notion was supported by delayed, yet more extensive, upregulation of the ifn␣ as well as the ifit1 and ifit2 genes compared to that in microglia. isg upregulation further appeared to be independent of direct changes in ifnar, stat1, or irf9 transcript levels. many prr-encoding genes are themselves isgs and are upregulated during cns infections (3, 11, 35, 36) . mda5, rig-i, and tlr3 have all been established as prrs contributing to ifn-␣/␤ induction in mhv-infected microglia/macrophages and oligodendrocytes (18, 27, 37, 38) . basal mda5, rig-i, and tlr3 transcript levels were all significantly lower in facs-purified astrocytes than in microglia (fig. 3) . nevertheless, astrocytes rapidly upregulated mda5, rig-i, and tlr3 mrnas to levels observed in microglia as early as day 3 p.i. (fig. 3) , when total ifn␣/␤ mrna peaked in the brain (fig. 1 ). relative to basal levels, the fold increase in astrocytes exceeded that in microglia. all three prr transcripts declined by day 7 p.i., reflecting the decline in ifn-␣/␤. with the exception of mrna encoding ikk, a kinase involved in both induction and ifnar signaling, constitutive levels of transcripts for prr signal transduction factors represented by irf3 and irf7 were also lower in astrocytes (fig. 3) . expression of the cellular kinase ikk mrna was gradually increased in both glial populations over time up to day 7 p.i., with increases more pronounced in microglia (fig. 3) . irf3 mrna expression was decreased in microglia following infection and remained unchanged in astrocytes, contrasting with upregulation of irf7 mrna in both cell types between days 3 to 5 p.i. and downregulation thereafter (fig. 3) . these data are consistent with constitutive irf3 expression but inducible expression of both ikk and irf7 following infection or ifn-␣/␤ treatment of non-cns cells (39) . overall, the data suggest that initial production of ifn-␤ in microglia results in upregulation of select isgs in astrocytes, thereby amplifying ifn-␣/␤. however, it remains unclear whether astrocytes induce ifn-␤ directly via virus-derived prr activators or via mediators released from necrotic or apoptotic cells in the inflamed cns environment. regardless, the results demonstrate that astrocytes are sensitive responders to ifn-␣/␤ and active participants in the ifn-␣/␤-induced amplification loop. ifnar signaling in astrocytes is essential to control mhv a59 infection. given the prominent response of astrocytes to ifn-␣/␤ following mhv a59 infection in vivo, we determined the protective role of astrocyte-mediated ifnar signaling against infection using mgfapcre ifnar fl/fl mice. disease onset was similar in mgfapcre ifnar fl/fl and ifnar fl/fl mice starting at day 4 p.i. however, in contrast to mild clinical symptoms in ifnar fl/fl mice, mgfapcre ifnar fl/fl mice developed severe encephalitis and succumbed to infection by day 7 p.i. (fig. 4a ). the 4-to 5-day-delayed mortality compared to that of ifnar ϫ/ϫ mice infected with a similar virus dose (31) supported astrocytes as critical, but not the only, contributors to early ifnar-dependent protection. to evaluate whether disease severity and mortality in mgfapcre ifnar fl/fl mice were correlated with impaired viral control, infectious virus was measured in brain supernatants. the virus loads were comparable in both groups at day 3 p.i. but reached ϳ5-log 10 -higher levels in mgfapcre ifnar fl/fl mice than in ifnar fl/fl mice by day 5 p.i. (fig. 4b ). the anatomical localization and distribution of viral n protein were also evaluated by histology. compared to small isolated residual foci of viral ag-positive cells in brains of ifnar fl/fl mice at day 6 p.i., mgfapcre ifnar fl/fl mice exhibited extensive dissemination with increased intensity of viral ag staining throughout the brain (fig. 4c ). high-magnification images indicated that virus was not limited to cells consistent with glial morphology but also was in cells with typical neuronal morphology in mgfapcre ifnar fl/fl mice (fig. 4c, insets) . as mhv a59 is hepatotropic following peripheral infection and can spread to the liver following i.c. infection of immunocomby rt-pcr. data represent the means ϯ sem from two experiments, each comprising 5 or 6 pooled brains per time point, and were analyzed by the unpaired two-tailed student t test and two-way anova. *, significance between microglia and astrocytes; #, significance compared to each respective naive population: * and #, p ͻ 0.05; ** and ##, p ͻ 0.01; *** and ###, p ͻ 0.001. promised mice (26), we also surveyed liver for viral n mrna. the levels of viral n relative to gapdh transcripts were elevated in livers of mgfapcre ifnar fl/fl compared to ifnar fl/fl mice at day 6 p.i. (values of 82 ϯ 48 versus 5 ϯ 3) (data not shown). however, overall viral n mrna levels were significantly higher in the cns (9,013 ϯ 998 versus 230 ϯ 55, respectively) (see fig. 8 ). there was also no evidence of necrotic foci by gross visual examination (data not shown). mortality was thus attributed to overwhelming virus spread within the cns. mhv a59 infects multiple cns cell types, including microglia, astrocytes, and neurons (28, 31) . to evaluate to what extent the absence of ifn-␣/␤ signaling in astrocytes predisposes astrocytes over other cell types to enhanced infection, brains from infected mgfapcre ifnar fl/fl and ifnar fl/fl mice were analyzed for viral n protein in combination with antibodies (abs) specific for neurons (neun), astrocytes (glial fibrillary acidic protein [gfap]), and microglia/macrophages (iba1). characteristic staining and colocalization patterns at day 4 p.i. in wt mice are depicted in fig. 5a . microglia and neurons showed prominent perinuclear cytoplasmic n protein localization, with some extension into processes in microglia. in contrast, viral ag was only occasionally detected in the cytoplasm of astrocytes and was localized mainly proximal to, but not overlapping with, astrocytic processes. in this context it is critical to note that the anti-gfap ab used to identify astrocytes stains main stem branches but not the cytoplasm or fine processes of astrocytes and poorly stains gray matter astrocytes (20, 40) , suggesting that viral protein may localize to astrocytic processes poorly detected by anti-gfap ab. total n protein staining revealed an ϳ3-fold-higher positive area in mgfapcre ifnar fl/fl than in ifnar fi/fi mice as determined by pixels per observed field at day 4 p.i.; the difference increased to ϳ40-fold by day 6 p.i., when virus already declined in ifnar fi/fi mice (fig. 5b) . keeping the limitations of gfap staining in mind, colocalization of viral ag with gfap was overall increased at day 6 p.i. furthermore, when the relative proportion of the dual-positive area within the total viral ag-positive pixel area was assessed, percentages were similar in both mouse strains at day 4 p.i. but were 2-fold higher in mgfapcre ifnar fl/fl mice than in controls at day 6 p.i. (fig. 5c ). viral ag-positive microglia were also overall increased in mgfapcre ifnar fl/fl mice at day 6 p.i. however, the relative proportion of the dually viral ag-and iba1-positive area per total viral ag-positive area was comparable between groups at both days 4 and 6 p.i. (fig. 5d) . to evaluate the relative infection of astrocytes versus microglia, we also measured the proportions of viral ag-positive area relative to gfap-or iba1-positive areas. while infection of microglia increased from ϳ20% to ϳ30%, it increased from ͻ5% to 15% in astrocytes in mgfapcre ifnar fl/fl mice between days 4 and 6 p.i. (fig. 6) . although infected neurons were increased in mgfapcre ifnar fl/fl compared to ifnar fl/fl mice throughout the brain by day 4 p.i., they declined significantly in both groups by day 6 p.i. remaining elevated neuronal infection in mgfapcre ifnar fl/fl mice at day 6 p.i. was still particularly evident in lower and distal parts of the brain (pons and medulla) (fig. 5e) . analysis of apoptosis to explain the decline in virus-infected neurons indicated and microglia in brain cortex of both mouse groups at day 6 p.i. bar graphs show the proportion of n ϩ gfap ϩ or n ϩ iba1 ϩ per total n ϩ area, respectively. insets show infected astrocytes at higher magnification and arrows indicate infected microglia. (e) representative staining of viral n protein in neurons in brain cortex and brain stem at days 4 and 6 p.i. the bar graph shows average numbers of virus-infected neurons per brain section from 2 or 3 whole brain sections per mouse (n ϭ 3 mice). arrows indicate virus-infected neurons. scale bar, 50 m. (c to e) left and right panels indicate ifnar fl/fl and mgfapcre ifnar fl/fl mice, respectively. the data in panels b and c represent the means ϯ sem from 6 or 7 fields in 2 or 3 slides from 3 mice per group and were analyzed by the unpaired two-tailed student t test and two-way anova. *, significance between ifnar fl/fl and mgfapcre ifnar fl/fl mice; #, significance between days 4 and 6 p.i. in the same group: *, p ͻ 0.05; ** and ##, p ͻ 0.01; ***, p ͻ 0.001. overall sparse but more abundant neuronal apoptosis in mgfapcre ifnar fl/fl mice (fig. 7a ). occasional apoptotic cd3 ϩ lymphocytes were also observed with elevated frequency in mgfapcre ifnar fl/fl mice (fig. 7b ). while these results likely underestimate the extent of viral infection in astrocytes, they support that the absence of astrocyte ifnar signaling leads to increased viral dissemination most prominently within astrocytes but also to microglia and neurons. abrogated ifnar signaling in astrocytes does not impair overall expression of ifn-␣/␤, isgs, chemokines, and cytokines. to assess whether uncontrolled virus spread was attributable to overall impaired innate immune responses, we determined transcripts for ifn-␣/␤ and isgs in relation to viral n mrna levels in brains of both mouse groups (fig. 8 ). viral n mrna levels were similar at day 3 p.i. but increased significantly in mgfapcre ifnar fl/fl mice compared to ifnar fl/fl controls by day 6 p.i. (fig. 8) , consistent with increased infectious virus by day 5 p.i. (fig. 4b) . elevated viral n mrna correlated with increased ifn␤, ifn␣4, and ifn␣5 transcripts at day 6 p.i. (fig. 8) . to assess how elevated ifn-␣/␤ affected downstream ifnar signaling, we further compared mrna levels of the isgs (ifit1, ifit2, isg15, mx1, pkr, and oas2). while these transcripts were all similar in both mouse groups at day 3 p.i., ifit1, ifit2, and mx1 mrnas were increased 2-to 3-fold in mgfapcre ifnar fl/fl mice by day 6 p.i. isg15, pkr, and oas2 mrnas were not affected or reduced relative to those in ifnar fl/fl controls (fig. 8) . ifnar abrogation on astrocytes thus did not impair global ifn␣/␤ or select isg expression in the infected cns. however, innate immunity by ifnar-competent cells was clearly insufficient to stem viral dissemination throughout the cns in mgfapcre ifnar fl/fl mice. astrocytes are also potent inducers of chemokines and cytokines (13, 41) , and a subset of chemokines, e.g., the neutrophil chemoattractant cxcl1, can be negatively regulated by ifn-␣/␤ (42). we therefore evaluated how the absence of ifnar on astrocytes modulates expression of select nf-b-dependent chemokines and the proinflammatory cytokine interleukin 6 (il-6) (43, 44) . transcripts for cxcl1 and ccl2, potent chemoattractants for neutrophils and monocytes, respectively, were similar at day 3 p.i. but were significantly upregulated at day 6 p.i. (fig. 9) , consistent with increased viral replication and ifn-␣/␤-mediated downregulation of cxcl1 by astrocytes (42, 45) . increased il6 mrna expression at day 6 p.i. (fig. 9 ) further indicated that ifnar abrogation on astrocytes does not impact proinflammatory cytokine induction. in contrast, ccl5 mrna, which is prominently expressed by t cells during mhv infection (46) , was significantly reduced compared to that in ifnar fl/fl mice at day 6 p.i. dysregulation of these proinflammatory factors suggested that myeloid cells may be recruited in favor of lymphocytes in mgfapcre ifnar fl/fl mice, thereby contributing to morbidity and uncontrolled viral spread. infected mgfapcre ifnar fl/fl mice exhibit increased cns neutrophil accumulation and reduced t cell infiltration. flow cytometry revealed that altered chemokine expression patterns indeed affected the composition of inflammatory cells within the cns. consistent with elevated cxcl1 mrna, neutrophils in mgfapcre ifnar fl/fl brains were increased by ϳ8-fold at day 6 p.i. relative to those in ifnar fl/fl mice, although numbers in both groups were already reduced compared to those at day 3 p.i. (fig. 10a) . differences in macrophages were not statistically significant (fig. 10b) . as both cd4 and cd8 t cells are essential to reduce infectious mhv, with ifn-␥ playing a prominent antiviral role (33, 47) , we further assessed if impaired t cell recruitment and ifn-␥ production within the cns is associated with lack of virus control. indeed, both cd4 and cd8 t cells were reduced in the cns of mgfapcre ifnar fl/fl mice at day 6 p.i. (fig. 10c) . however, similar ifn-␥ levels (fig. 10d) , despite reduced t cell numbers, suggested that increased viral replication leads to increased ag presentation, thereby triggering more or prolonged ifn-␥ production by t cells in mgfapcre ifnar fl/fl mice (48, 49) . impaired access of t cells to the cns parenchyma was ruled out as an explanation of loss of viral control, as we found no differences in anatomical distribution of cd3 ϩ t cells within perivascular and parenchymal sites between the two groups (data not shown). uncontrolled viral replication in mgfapcre ifnar fl/fl mice despite similar ifn-␥ production thus implied that viral replication overwhelms the t cell response or that ifn-␥ signaling is impaired. determination of ifn-␥-specific responses in vivo is complicated by the fact that many factors upregulated by ifn-␥ are also induced by ifn-␣/␤ (50). nevertheless, a prominent indicator of ifn-␥ signaling in microglia/macrophages is induction of the transcription factor class ii transactivator (ciita), a master regulator of mhc class ii expression (51, 52) . interestingly, ciita transcripts were barely induced in brains of mgfapcre ifnar fl/fl mice, in contrast to robust upregulation in control mice, at day 6 p.i. (fig. 10e) . analysis of mhc class ii expression on microglia by flow cytometry confirmed that the very modest ciita mrna induction impaired ifn-␥-dependent mhc class ii upregulation. only ϳ16% of microglia expressed mhc class ii in mgfapcre ifnar fl/fl mice, compared to ϳ76% in ifnar fl/fl mice, and the mean fluorescence intensity, reflecting expression levels, per cell was also lower (fig. 10f) . other ifn-␥-dependent factors include the chemokines cxcl9 and cxcl10 (53, 54) . while cxcl9 expression is inducible mainly by ifn-␥, cxcl10 is inducible by ifn-␣/␤, ifn-␥, and tumor necrosis factor (tnf) (42, 55, 56) . furthermore, cxcl9 is produced by macrophages/microglia and endothelial cells, but not astrocytes, whereas cxcl10 is induced prominently in astrocytes during inflammation (53, 57) . assessment of cxcl9 and cxcl10 mrnas showed similarly low levels in both groups at day 3 p.i. (fig. 10g) . however, unlike ifnar fl/fl mice, mgfapcre ifnar fl/fl mice failed to upregulate cxcl9 mrna by day 6 p.i. in contrast, cxcl10 mrna levels were elevated by ϳ2.5-fold in mgfapcre ifnar fl/fl mice by day 6 p.i. but did not increase in ifnar fl/fl mice. given that mgfapcre ifnar fl/fl mice do not express ifnar on astrocytes, cxcl10 mrna upregulation is likely driven by ifn-␥ and/or tnf. overall, these results implied that microglia, but not astrocytes, are significantly compromised in ifn-␥ responsiveness in mgfapcre ifnar fl/fl mice. preexposure to ifn-␣/␤ modifies ifn-␥ responsiveness in myeloid cells. one explanation for the differential responsiveness to ifn-␥ in microglia versus astrocytes in mgfapcre ifnar fl/fl mice is that prolonged ifnar signaling via elevated and sustained ifn-␣/␤ expression may skew subsequent ifn-␥ responsiveness. this is supported by an antagonistic effect of ifn-␣/␤ on ifn-␥ responses in macrophages following ifn treatment or listeria infection (58) (59) (60) . we therefore used the viral rna mimic poly(i·c), a synthetic double-stranded rna (dsrna) analog recognized by tlr3 as well as rig-i/mda5, to induce ifn␣/␤ mrna in bone marrow-derived macrophages (bmdm) and monitor ifn-␥ responsiveness. previous in vivo data revealed that poly(i·c) administration into the cns of mice induced ifn␣/␤ mrna by 4 h, which was sustained out to 12 h (11). bmdm were thus pretreated with poly(i·c) for 4 or 12 h and subsequently treated with ifn-␥ for 18 h. ifn-␥-only treatment was included as a positive control. poly(i·c) alone did not induce ciita mrna at 4 h and induced it only sparsely by 12 h. however, while ifn-␥ treatment alone effectively induced ciita mrna, it failed to induce ciita mrna in cells pretreated with poly(i·c) for either 4 or 12 h (fig. 11) . similarly, poly(i·c) alone only sparsely induced cxcl9 mrna at 4 h compared to induction by ifn-␥ treatment alone, and short poly(i·c) pretreatment suppressed cxcl9 mrna induction by ifn-␥. assessment of an inhibitory effect of prolonged poly(i·c) exposure was preempted by induction of cxcl9 mrna levels similar to those with ifn-␥. finally, treatment with poly(i·c) alone was superior to ifn-␥ alone in upregulating cxcl10 mrna, with saturation already achieved at 4 h. these results are consistent with poly(i·c)-mediated upregulation of cxcl10 via ifn-␣/␤ and tnf and preempted use of cxcl10 as a target gene to assess suppressive functions on ifn-␥ signaling in this experimental setting. overall, these data indicate that preexposure of macrophages/ microglia to a virus-induced inflammatory milieu including ifn-␣/␤ has the potential to negatively regulate subsequent ifn-␥ signaling. however, the magnitude of ifn-␣/␤mediated suppression of ifn-␥-dependent gene induction depends on other regulatory elements in the respective target gene promoter. these results reveal that the duration and/or level of cell exposure to ifn-␣/␤ can substantially diminish responsiveness to temporally lagging ifn-␥, thereby reducing ifn-␥ protective functions. the ability to induce and respond to ifn-␣/␤ varies between cell types as well as between types of virus, but the contribution of different cns cell types to mount ifn-␣/␤-mediated protection in vivo are less well explored (6) . our studies comparing the capacity of adult astrocytes relative to microglia to respond to mhv a59 infection revealed that astrocytes expressed lower basal levels of prrs and signaling components to induce ifn-␣/␤ and isgs than microglia, consistent with published gene array data (61) . nevertheless, despite a delay in response to infection, astrocytes exhibited strong upregulation of prrs, ifn␣s, and select isgs, supporting that astrocytes are well positioned to respond to paracrine ifn-␣/␤ to amplify their innate antiviral response. the critical role of ifnar signaling in astrocytes to block viral spread was confirmed by rapid virus dissemination throughout the cns upon abrogation of ifnar signaling in astro-cytes. thus, although basal expression levels of ifn-␣/␤ signaling components have been shown to determine the responsiveness of cells to insults (11, 62) , our data reveal that cells expressing low basal levels of ifn-␣/␤ pathway components can nevertheless trigger a protective innate immune response through paracrine ifn-␣/␤ signaling. delayed mortality of infected mgfapcre ifnar fl/fl mice compared to ifnar ϫ/ϫ mice implied that other cell types, including microglia, can produce and respond to ifn-␣/␤. this is clearly demonstrated by elevated and sustained ifn␣/␤ as well as select isg expression despite ifnar deletion on astrocytes. however, vast dissemination of virus throughout the cns clearly indicated that ifnar signaling specifically in astrocytes plays a dominant role in host protection, which could not be compensated by ifnarcompetent glia and neurons. while microglia appeared to exhibit some autocrine action as suggested by less overall infection relative to that in astrocytes, neurons in the brain stem appeared to be most susceptible and vulnerable to infection in the absence of astrocyte ifnar signaling. although we cannot exclude that inherently reduced activation of innate viral components in neurons may contribute to their susceptibility, our data indicate that overwhelming infection of ifnar-deficient astrocytes increases adjacent neuronal infection. west nile virus infection of neuronal cultures demonstrates that neurons have the capacity to mount innate viral responses via tlr and irf activations (63, 64) . moreover, neurons have also been demonstrated to induce ifn-␤ following tmev and la crosse virus infection in vivo (65) . nevertheless, these responses were very sparse and focal. in contrast, subsequent studies of la crosse virus infection in ifn-␤ reporter mice showed that apparently noninfected astrocytes and microglia in close proximity to infected neurons were prominent ifn-␤-producing cells, while ifn-␤ expression by infected neurons was rare (16) . more recent data demonstrate that ifn-␤-producing astrocytes appeared to be transiently or abortively infected during various infections associated with neuronal tropism (14) . the spatial extent of innate protection induced in an infected environment may thus depend on the prominent infected cell type. as both microglia and astrocytes have far-reaching processes, release of ifn-␣/␤ from these cells may provide more extensive protection than release from neuronal cell bodies. two independent studies with vsv indeed indicate that locally induced ifn-␤ in the olfactory bulb can trigger isgs in distal parts of the brain, although one indicates astrocytes and the other neurons as the prominent ifn-␤ inducers (15, 66) . regardless, the vast increase of virus in mgfapcre ifnar fl/fl mice supports the notion that the inability of astrocytes to clear virus, rather than loss of direct isg effects on other cns cells, is responsible for loss of viral control. in addition to uncontrolled virus, elevated neutrophil accumulation may contribute to tissue damage and rapid morbidity of mgfapcre ifnar fl/fl mice. increased neutrophil cns infiltration correlated with increased mrna expression of the neutrophil chemoattractant cxcl1 (41) and its downregulation by ifn-␣/␤ and ifn-␥ (42, 45, 67, 68) . neutrophils are early innate responders, which contribute to bbb breakdown and tissue damage via secretion of matrix metalloproteinases (mmps) and a wide range of other degrading enzymes released upon degranulation (69, 70) . during autoimmune-mediated cns inflammation, neutrophils have also been shown to release cytokines which mature antigen-presenting cells and subsequently reactivate t cells (71) (72) (73) . elevated cxcl1 mrna as well as increased levels of mrnas encoding il-6 and ccl2, two other proinflammatory factors prominently induced in astrocytes (74) , further suggested that the nf-b pathway initiated by ppr activation was intact in infected mgfapcre ifnar fl/fl mice. finally, our results show that extensive virus spread in the absence of protective astrocyte ifnar signaling cannot be controlled by t cells. although previous studies of cxcl9 and cxcl10 monoclonal antibody (mab) blockade to reduce cxcr3-dependent t cell recruitment showed a reduction in t cell migration and loss of viral control in a related mhv infection (75, 76) , subsequent studies in cxcl9-and cxcl10-deficient mice indicated that cxcl10 is more critical for effective adaptive immunity (57) . impaired t cell accumulation despite unimpaired cxcl10 mrna in mgfapcre ifnar fl/fl mice was thus unanticipated. while an overwhelming virus load may lead to aginduced t cell exhaustion and death (77) , dysregulation of t cells requires future investigation. regardless of reduced t cell numbers, their initial activity as monitored by ifn-␥ secretion was not impaired. ifn-␥ signaling is essential for mhv control within the cns by both exerting direct antiviral effects and enhancing mhc class i and ii upregulation (78) . however, reduced induction of ifn-␥-dependent ciita and cxcl9 mrnas showed that microglia were significantly impaired in ifn-␥ responsiveness. in contrast, increased cxcl10 mrna expression in the absence of ifnar on astrocytes implied that ifn-␥ signaling in astrocytes remained intact. the notion that elevated and sustained ifn-␣/␤ signaling acts as a negative regulator of ifn-␥ signaling was supported by earlier reports showing antagonistic effects of ifn-␣/␤ on ifn-␥-induced activation of macrophages (58-60). one possible mechanism may involve downregulation of the ifn-␥ receptor (ifngr), as shown during listeria infection (58) . our in vitro studies further supported selected downregulation of ifn-␥ responsiveness by ifn-␣/␤ via pretreatment of bmdm with the dsrna mimic poly(i·c). ifn-␣/␤-dependent sensitization of microglia/macrophages to subsequent ifn-␥ responses may provide a mechanism to limit excessive immune responses leading to tissue damage. in this context it is of interest to note that both ifn-␣/␤ and ifn-␥ responses are tightly controlled by negative regulators, including phosphatases (79) . cross talk between ifn-␣/␤ and ifn-␥ responses may thus provide additional levels to protect the host from exacerbated proinflammatory responses. overall, our data demonstrate a vital protective role of astrocyte-mediated ifn-␣/␤ signaling in host protection from neurotropic coronavirus-induced encephalomyelitis. further, the link between sustained elevated ifn-␣/␤ and impaired responsiveness to ifn-␥ supports the novel concept that temporally limited early ifn-␣/␤ responses are critical for effective antiviral ifn-␥ function. mes/j (gfap-gfp) mice were originally purchased from the jackson laboratory (bar harbor, me) and backcrossed to c57bl/6 mice for at least 10 generations. b6.cg-tg (gfap-cre) 77.6mvs/j (stock number 012887; mgfapcre) mice were also purchased from the jackson laboratory (bar harbor, me). ifnar fl/fl mice engineered to contain loxp sites flanking exon 10 of the ifnar gene were originally produced on the 129/ola background in u. kalinke's laboratory (paul-ehrlich-institut, langen, germany) as described previously (80) and were backcrossed onto the c57bl/6 background in r. schreiber's laboratory (washington university school of medicine, st. louis, mo) as described previously (81) . ifnar fl/fl mice were crossed with mgfapcre ϩ/ϫ mice to generate mgfapcre ϩ/ϫ ifnar fl/fl (mgfapcre ifnar fl/fl ) mice, which were crossed with ifnar fl/fl mice for experimentation. offspring were genotyped for both the presence of the cre recombinase gene and exon 10 deletion from the ifnar gene by pcr using the following primers: cre, 5=-gtccaatttactgaccgtac acc-3= (forward) and 5=-gttattcggatcatcagctacacc-3= (reverse); ifnar flox, 5=-tgctttgaggagc gtctgga-3= (forward) and 5=-catgcactaccacaccaggcttc-3= (reverse). cre-negative ifnar fl/fl littermates were used as wt controls (ifnar fl/fl ). all mice were housed under specific-pathogen-free conditions at an accredited facility at the cleveland clinic lerner research institute. mice of both sexes were infected intracranially (i.c.) at 6 to 7 weeks of age with 2,000 pfu of the hepato-and neurotropic mhv a59 strain expressing enhanced green fluorescent protein (egfp), kindly provided by volker thiel (kantonal hospital, st. gallen, switzerland) (82) . mhv a59 was propagated on delayed brain tumor (dbt) astrocytoma monolayers, and virus titers were determined as described previously (83) . virus in the cnss of individual mice was measured by plaque assay on dbt cells using clarified supernatants from brain homogenates prepared as described previously (84) . clinical disease severity was graded daily using the following scale: 0, no disease symptoms; 1, ruffled fur; 2, hunched back or inability to turn upright; 3, severe hunching/wasting or hind limb paralysis; 4, moribund condition or death (31) . all animal procedures were approved by the institutional animal care and use committee of the cleveland clinic (phs assurance number a3047-01) and were conducted in compliance with the guide for the care and use of laboratory animals from the national research council. brains from mice perfused with cold phosphate-buffered saline (pbs) were homogenized in dulbecco's pbs (dpbs) (ph 7.4) using tenbroeck tissue homogenizers as described previously (84) . homogenates were centrifuged at 450 ϫ g for 10 min at 4°c. cells were resuspended in rpmi containing 25 mm hepes (ph 7.2), adjusted to 30% percoll (pharmacia, uppsala, sweden), underlaid with 1 ml 70% percoll, and centrifuged at 850 ϫ g for 30 min at 4°c. cells were collected from the 30%/70% interface, washed with rpmi, counted, and suspended in fluorescence-activated cell sorting (facs) buffer (0.1% bovine serum albumin in dpbs). fc␥ receptors were blocked with 1% mouse serum and rat anti-mouse cd16/32 mab (clone 2.4g2: bd biosciences, san diego, ca) for 20 min on ice prior to staining with fluorescein isothiocyanate (fitc)-, phycoerythrin (pe)-, peridinin chlorophyll protein (percp)-, or allophycocyanin (apc)-conjugated mabs specific for cd45 (clone 30-f11), cd8 (clone 53-6.7), cd4 (clone gk1.5), ly6g (clone 1a8), cd11b (clone m1/70), and mhc class ii (clone m5/114.15.2) (all from bd bioscience, mountain view, ca) and f4/80 (serotec, raleigh, nc) in facs buffer. samples were analyzed on a bd accuri c6 plus instrument (bd biosciences). forward-and side-scatter signals obtained in linear mode were used to establish a gate containing live cells while excluding dead cells and tissue debris. data were analyzed using flowjo 9 software (tress star inc., ashland, or). microglia and astrocyte isolation for gene expression analysis. brains from 5 or 6 naive or mhv a59-infected gfap-gfp mice at days 3, 5, and 7 p.i. were homogenized using a neural tissue dissociation kit (papain, miltenyi auburn, ca) following the manufacturer's protocol. brain homogenates were prepared for 30/70% percoll gradients to isolate cells as described above. cns cells were blocked with mouse serum and anti-cd16/32 mab as described above prior to staining with cd45 and cd11b in facs buffer. microglia and astrocytes were sorted based on their cd45 int cd11b ϩ and cd45 neg gfp ϩ phenotypes, respectively, using a facs aria iii (bd biosciences) and facs diva software (bd biosciences). sorted cells were resuspended in trizol (invitrogen, carlsbad, ca) and stored at ϫ80°c. gene expression analysis. rna from brains or sorted cells was extracted using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer's instructions. following treatment with dnase i using a dna-free kit (ambion, austin, tx), cdna was synthesized using moloney murine leukemia virus reverse transcriptase (invitrogen) in buffer containing 10 mm deoxynucleoside triphosphate mix, 250 ng random hexamer primers, and oligo(dt) (1:1 ratio) (invitrogen). rna expression was assessed using either sybr green master mix (applied biosystems, foster city, ca) or taqman fast master mix (applied biosystems, foster city, ca) as described previously (31) . the following primers were used with sybr green master mix: for the gapdh gene, 5=-catggcct tccgtgttccta-3= (forward) and 5=-atgcctgcttcaccaccttct-3= (reverse); for cxcl9, 5=-tgcac gatgctcctgca-3= (forward) and 5=-aggtctttgagggatttgtagtgg-3= (reverse); for cxcl10, 5=-ga cggtccgctgcaactg-3= (forward) and 5=-gcttccctatggccctcatt-3= (reverse); for the viral nucleocapsid (n) gene, 5=-gccaaataatcgcgctagaa-3= (forward) and 5=-ccgagcttagccaaaacaa g-3= (reverse); for il6, 5=-acacatgttctctgggaaatcgt-3= (forward) and 5=-aagtgcatcatcgttgt tcataca-3= (reverse); for oas2, 5=-aaaaatgtctgcttcttgaattctga-3= (forward) and 5=-tgtgcct ttggcagtggat-3= (reverse); for ifnar1, 5=-cccaaggcaagagctatgtc-3= (forward) and 5=-tctgaa cggcttccagaact-3= (reverse); for ikk, 5=-ccagaagattcagtgttgtttgg-3= (forward) and 5=-tca ttgtagctgagccctg-3= (reverse); for mda5, 5=-gacaccagaattcaagggac-3= (forward) and 5=-gc cacacttgcagataatctc-3= (reverse); and for rig-i, 5=-gtcagcacaaaccacaacc-3= (forward) and 5=-gtctcaaccactcgaatgtc-3= (reverse). taqman fast master mix and taqman primers/probes gene expression in total tissue mrna or glial populations was normalized to the respective gapdh mrna expression in each mrna preparation and converted to a linearized value using the formula: 2e(c t gapdh ϫ c t gene ) ϫ 1,000. immunohistochemistry and immunofluorescence. brains from pbs-perfused mice were fixed with 10% neutral zinc-buffered formalin and embedded in paraffin for viral n protein analysis. the distribution of viral antigen (ag) was determined using mab j3.3 specific, for the carboxyl terminus of the viral n protein, as the primary mab, biotinylated horse anti-mouse igg as the secondary ab, and streptavidinconjugated horseradish peroxidase and 3,3=-diaminobenzidine substrate (vectastain-abc kit; vector laboratories virus-infected cells were identified using mab j3.3 (31) in combination with cell type-specific markers for microglia/macrophages using rabbit anti-iba1 astrocytes using rabbit anti-glial fibrillary acidic protein (anti-gfap all images were analyzed using fiji version 1.0. quantification of viral n protein staining areas and the relative proportion of colocalization with gfap or iba1 reactivity were calculated per field of interest using image-pro plus version 7.0. infected neurons were enumerated by counting entire brain sections. for immunohistological analysis, representative data are presented from 6 or 7 separate fields per mouse with 3 or 4 mice per group per time point. ifn-␥ elisa. ifn-␥ in brain supernatants was measured by enzyme bone marrow was collected from femurs and tibiae of 8-to 10-week-old mice, passed through a cell strainer, and centrifuged at 400 ϫ g for 5 min at 4°c. cells were suspended in bmdm medium (dulbecco modified eagle medium [dmem], 10% fcs, 20% l929 conditioned medium as a source of colony-stimulating factor 1, 0.1% gentamicin, 1% sodium pyruvate) and seeded at a density of 5 ϫ 10 6 cells in 10 ml bmdm growth medium into 10-mm 2 tissue culture dishes. an additional 5 ml bmdm medium was added 3 days later viral diseases of the central nervous system regulation of type i interferon responses antiviral type i and type iii interferon responses in the central nervous system regulation of antiviral t cell responses by type i interferons type i interferon response in the central nervous system brain heterogeneity leads to differential innate immune responses and modulates pathogenesis of viral infections immune surveillance of the cns following infection and injury pathogen recognition and inflammatory signaling in innate immune defenses pathogen recognition and innate immunity innate antiviral signalling in the central nervous system oligodendroglia are limited in type i interferon induction and responsiveness in vivo basal 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microglia in vitro factors supporting intrathecal humoral responses following viral encephalomyelitis cxcr3 ligands: redundant, collaborative and antagonistic functions tnf activates an irf1-dependent autocrine loop leading to sustained expression of chemokines and stat1-dependent type i interferon-response genes astrocytederived cxcl10 drives accumulation of antibody-secreting cells in the central nervous system during viral encephalomyelitis induction of ifn-alphabeta enables listeria monocytogenes to suppress macrophage activation by ifn-gamma agonist and antagonist effects of interferon alpha and beta on activation of human macrophages. two classes of interferon gamma receptors and blockade of the high-affinity sites by interferon alpha or beta contrasting effect of alpha/beta-and gamma-interferons on expression of macrophage ia antigens an rna-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex constitutive type i interferon modulates homeostatic balance through tonic signaling measure and countermeasure: type i ifn (ifn-alpha/beta) antiviral response against west nile virus tlr8: an innate immune receptor in brain, neurons and axons neurons produce type i interferon during viral encephalitis long-distance interferon signaling within the brain blocks virus spread cxcr2-mediated tumor-associated neutrophil recruitment is regulated by ifnbeta ifn-gamma protects from lethal il-17 mediated viral encephalomyelitis independent of neutrophils neutrophils promote mononuclear cell infiltration during viral-induced encephalitis how neutrophils kill microbes neutrophils amplify autoimmune central nervous system infiltrates by maturing local apcs the contribution of neutrophils to cns autoimmunity cytokine-regulated neutrophil recruitment is required for brain but not spinal cord inflammation during experimental autoimmune encephalomyelitis increased astrocyte expression of il-6 or ccl2 in transgenic mice alters levels of hippocampal and cerebellar proteins expression of mig (monokine induced by interferon-gamma) is important in t lymphocyte recruitment and host defense following viral infection of the central nervous system the t cell chemoattractant ifn-inducible protein 10 is essential in host defense against viral-induced neurologic disease high antigen levels are the cause of t cell exhaustion during chronic viral infection perforin-mediated effector function within the central nervous system requires ifn-gamma-mediated mhc up-regulation shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated jak/stat pathway type i interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia type i interferon is selectively required by dendritic cells for immune rejection of tumors mouse hepatitis virus liver pathology is dependent on adp-ribose-1љ-phosphatase, a viral function conserved in the alpha-like supergroup pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies distinct cd4 t-cell effects on primary versus recall cd8 t-cell responses during viral encephalomyelitis we thank michael diamond (washington university school of medicine, st. louis, mo) for kindly providing ifnar1 fl/fl mice on the c57bl/6 background. we also sincerely thank jennifer powers for exceptional facs purification and the cleveland clinic lerner research institute imaging core and john peterson for assistance with confocal microscopy. key: cord-022395-rk31pwoa authors: schuller-levis, georgia; kozlowski, piotr b.; kascsak, richard j. title: central nervous system: viral infection and immune-mediated inflammation date: 2012-12-02 journal: xenobiotics and inflammation doi: 10.1016/b978-0-12-628930-5.50019-9 sha: doc_id: 22395 cord_uid: rk31pwoa nan locally disabled for long periods of time (reese and karnovsky, 1967; hirano etal, 1970) . a peculiarity of the bbb is that it excludes macromolecules from entering the cns but allows megastructures such as entire hematogenous inflammatory cells to cross the barrier without opening the tight junctions (hirano et al, 1970; azarelli et al, 1984; lossinsky et al, 1989; raine et al, 1990; powell, 1991) . nonetheless, the entry of hematogenous inflammatory cells into the cns appears to be strictly controlled by the ecs. an initial event in the inflammatory reaction in the cns is the egress of hematogenous cells into the extravascular milieu of the cns parenchyma ( figure 1 ). only the previously sensitized inflammatory cells, i.e., lympho cytes, can invade the cns (raine et al, 1990; powell, 1991) . the prerequisite for transendothelial passage is the attachment of sensitized cells to the luminal surface of endothelial cells. a growing body of evidence suggests that lymphocytes and monocytes (but not neutrophils) attach to parajunctional endothelial cell receptors and subsequently insert pseudopodial pro jections into tubular, channel-like openings in the endothelial cells (lossin sky et al, 1989; powell et al, 1991) . these channels serve as eventual conduits for the transendothelial cell passage into the extravascular space. numerous studies have shown a great variety of specific membrane recep tors on endothelial cells that are critical for the binding of hematogenous cells. these highly specific receptors include several distinct classes of adhesion molecules. these receptors include the immunoglobulin super family (icam-1 and -2, lfa-3, vcam-1, endocam-1, and ncam-1); the integrins (lfa-1 or c d l l a / 1 8 , mac-1, lpam-1, 0 1 , β2, β3 families); the lec-cam family for lectins ; cadherins (ρ, ν, e-cadherins), and the hermes group of antigens (cd-44) (harlan, 1985; dustin and springer, 1988; lassmann et al, 1991 , staunton et al, 1990 yednock, 1985) . the expression of these receptors, also called addressins, on the endothelial surface is under the control of several cytokines, some of which are present only in local inflammatory response and absent under normal conditions. neutrophils, which also require addressins for adherence, are able to enter the cns by opening and passing through a tight junction between adjacent endothelial cells (broadwell, 1989) . second, there are structural cells of the cns that, in addition to their "main" roles, may, under certain conditions, play a role in antigen presenta tion and/or processing. these cells include astrocytes, endothelial cells, and microglial cells (fontana et al, 1984) . under normal conditions, only a small portion of meningeal, perivascular dendritic cells and resident mi croglial cells may express low levels of class ii major histocompatibility complex (mhc) antigens. the expression of these antigens on various cell types seems to be related to the severity of the inflammatory response. in mild inflammation, class ii mhc antigen expression is upregulated mostly on microglia cells; only in severe inflammation can some expression of these antigens be seen on astrocytes. gamma interferon (ifn-γ) is known to stimulate in vitro the expression of class ii mhc antigens on endothelial cells, microglial cells, and astrocytes (wong et al., 1984; hirsch et al., 1983; fierz et al., 1985) . the nonhematogenous cells that are native to the cns have a very limited role in cns inflammation. neurons, oligodendroglia cells, and ependymal cells, if not affected directly by an infectious agent, appear to be inert bystanders, even in the midst of severe inflammation. astrocytes perform a variety of functions within the cns. astrocytic processes are a vital part of the bbb, and the cells themselves maintain the homeostasis of the neuropil (figure 2 ). astrocytic cells are the main cells involved in scar formation following cns injury. they are now considered an accessory cell to the immune system of the cns (hertz etal., 1990; yong et al., 1991; rodriguez et al., 1987) . they express mhc antigens, may act as antigen-presenting cells (apcs), and secrete cytokines. in inflammation, astrocytic cells (especially type 1) may be stimulated by laf-and icam-1, by cytokines from τ cells, macrophages, microglial cells, and/or other astrocytes. il-2 stimulates proliferation of astroglia in experimental brain injury. in vitro, il-1, tumor necrosis factor (tnf), and ifn-γ stimulate proliferation of astrocytes. ifn primes astrocytic cells to the effect of il-2 by secretion of tnf, il-1, il-3, il-6, and lymphotoxin. the endothelial cells are not only an anatomical and functional barrier between the blood and the brain; they are also pivotal cells in inflammation. ecs not only regulate the influx of cells and solutes, but they may also act as apcs, and the luminal surface of ecs may be the site of antigen presenta tion and recognition. class i mhc molecules necessary for antigen recogni tion can be found on all cells constituting the bbb, whereas class ii mhc can be induced by ifn-γ stimulation. class ii mhc antigens expressed on ecs increase with the increased severity of inflammation, suggesting that the mhc expression is progressively turned on and may, under certain conditions, lead to amplification of local inflammation. ecs activation in chronic (and not necessarily severe) inflammatory processes may not be a secondary consequence of a local process but may, in fact, initiate and perpetuate the inflammation (hertz et al., 1990; gimborne and beviaqua, 1988; montovani and dejana, 1989; cotran, 1987) . it also appears that the endothelial cell may be a pivotal cell in autoim mune inflammatory reaction (with local intrathecal antibody formation) without the involvement of extraneuronal antigens or infectious agents. ecs may present antigens that are specific for the cns and attract sensitized τ cells. τ cells may attach to cns antigens and perpetuate inflammation by secreting lymphokines (ifn, il-2) and attract even more lymphocytes. this critical mass of lymphocytes and lymphokines may open the bbb, which allows τ cells to enter the cns parenchyma to recruit β cells, which then locally produce antibodies against the cns antigens presented by endothelial cells. microglia cells are considered immune-competent cells that are native to the cns but whose origin is still unclear (graeber and streit, 1990) . they are found throughout the cns and constitute from 5 to 20% of all cns cells (figure 3 ). microglia cells share common surface antigens with mono cytes and macrophages but not with neuroectodermal cells. the functions of microglial cells include phagocytosis, presentation of antigens, produc tion of il-1, and stripping of synaptic buttons (deafferentation) from disthe antigen is also present on brain resident microglial cells. note the cell surface staining and presence of the dichotomically branching processes. cells are evenly spaced, and do not form a connection with each other. anti-ln-1 monoclonal antibody, avidin-biotin technique, 70-/ltm-thick vibratome section; nuclei were counterstained with hematoxylin, x 470. tressed neurons. there is a low level of expression of class ii mhc antigens on microglial cells under normal conditions, and this expression can be dramatically increased with infusion of ifn-γ (graeber and streit, 1990; tribolet de et al, 1984; hayes et al, 1987; hayes et al, 1988; delisle et al, 1986; hickey and kimura, 1988; mcgeer et al, 1987; mcgeer et al, 1988) . microglia cells also express the cd4 molecule, which is considered an entry point for the human immunodeficiency virus (hiv) (watkins et al, 1990; bourdial et al, 1991; levy, 1988; michaels et al, 1988 ). yet another unique feature of the cns is the presence of cns-specific antigens such as myelin basic protein (mbp), glial fibrillary acidic protein (gfap), or neuron-specific enolase (nse). such proteins can be the focus of autoimmune responses within the cns. this autoimmune response can be a major contributing factor to pathology and clinical disease (see later discussions on viral-induced autoimmune response and experimental aller gic encephalomyelitis). a strong body of evidence now suggests that some diseases of the cns have an immunologic pathogenesis (paterson, 1977 (paterson, , 1978 (paterson, , 1979 (paterson, , 1982 . ex amples include experimental allergic encephalomyelitis (eae), acute dis seminated encephalomyelitis (adem), and multiple sclerosis (ms). acute and chronic relapsing eae can be induced in laboratory animals by an injection of cns tissue, cns myelin, myelin basic protein, or more recently, t-cell lines specific for nervous system antigens. adem occurs in humans and animals after an antecedent virus infection (see later discussion on viral induced autoimmunity) or vaccination with a living virus or a vaccine containing nervous tissue. ms is an acute or chronic progressive remitting disorder of humans characterized by inflammatory cell infiltration and demyelination. eae is a useful autoimmune disease model for the study of mechanisms of cellular immune reactions in the cns and has been considered an experi mental model of ms since the earliest descriptions by rivers et al (1935) . in recent years, reproducible small animal models of chronic eae have become available (stone and lerner, 1965; wisniewski and keith, 1977; massanari, 1980; lubin et al, 1981; brown et al, 1982) . these small animal models cover the full spectrum of ms pathology (wisniewski et al, 1982; lassmann and wisniewski, 1979) and allow the study of the pathogenesis of chronic inflammatory demyelinating plaque formation. progress on the pathogenesis and treatment of eae has been advanced by the selection and maintenance of permanent autoantigen mbp-specific t-cell lines. τ cells can now be isolated from animals with eae that will mediate eae in the recipient animal. this isolation is possible as a result of progress in the characterization of immune cells subsets, using monoclonal antibodies, and the definition of their growth requirements (i.e., il-2) (ben-nun et al, 1981) . it is not clear how the sensitized τ cells present in the circulation of eae animals, cross the bbb and recognize an antigen or antigens that are located in the major dense lines of the myelin sheath. one possible explanation of this phenomenon is that the low rate of physiological ex change of lymphocytes that appears to exist between the cns and the circulation allows the entry of the sensitized τ cells (lassmann et al, 1991; wekerle et al, 1986; wekerle et al, 1991) . interaction between inflammatory cells and the endothelial cells lining the bbb is an important initial event during inflammation of the cns. the expression of la and of the endothelial leukocyte adhesion molecule (elam-1) on endothelial cells in the cns are some of the earliest changes detected to date in brains of animals with eae (rose et al, 1991) . although the etiology of ms is still unknown, the similarity of ms to remitting-relapsing eae suggests that ms, like eae, might be the conse quence of either a direct or indirect autosensitization to myelin antigens (i.e., mbp and proteolipid apoprotein) (rose et al, 1991) . as a result of the application of advances in molecular biology, such as the polymerase chain reaction, and developments in cellular immunology, such as the ability to grow t-cell clones, great progress has been made in the pathogenesis and treatment of ms (steinman, 1991) . recent reports that most encephalitogenic (bp-specific) t-cell clones derived from mice or rats use common variable region genes of the rearranged t-cell receptor (tcr) have intensified the search for analogous associations in humans (burns et al, 1989; urban et al, 1988) . steinman (1991) has recently reviewed the strong parallels between t-cell receptor (tcr) usage in the pathogenesis of eae and tcr usage in mbp, and specific τ cells in ms peripheral blood and τ cells in demyelinated plaques in ms brains. the development of strategies for selective immunotherapy in eae was based on these similarit ies. this therapy uses monoclonal antibodies on either class ii molecules of the mhc or tcr-variable regions or peptides that compete with hla class ii molecules or vaccination against tcr-v regions. for example, vaccination of mice with appropriate tcr peptides is highly effective at inducing both cellular and humoral response to tcr and at inhibiting eae (howell et al, 1989; vanderbark et al, 1989) . monoclonal antibodies that bind only the complex of bp and i-as inhibited eae in h-2s mice when injected within -2 to + 1 0 days of disease induction (aharoni et al, 1991) . sriram and carroll (sriram and carroll, 1991) have shown that in vivo treatment with i-a antibodies at the earliest sign of eae results in decreased homing of radiolabeled cells to the brain. these data suggest that class ii antigens play an important role in cellular migration across the bbb. other potential therapies for eae include the use of cytokines (see cyto kine section) and suppressor τ cells. although mechanisms involved in eae remission are unclear, suppressor τ cells have been postulated to play a role in the prevention of autoimmunity (ofosu-appiah and mokhtarian, 1991) . an adaptively transferred suppressor t-cell line, obtained from spleens of mice that recovered from eae, was able to downgrade eae in mice subsequently challenged with mbp-activated τ cells. immunologically important cytokines are produced chiefly by lympho cytes and macrophages. inflammation in the nervous system is likely to be the same as in other organ systems, with a few modifications. because of the bbb and the macromolecular nature of cytokines, some of these important inflammatory molecules are likely to be cns resident derived. recent studies have also indicated astrocytes, microglia, and endothelial cells as possible additional sources of cns-derived cytokines (fontana et al, 1982; frei et al, 1985; giulian et al, 1985; frei et al, 1989; sawada et al, 1989) . immunocytochemical techniques have demonstrated the presence of a variety of cytokines (see earlier discussion) (i.e., mif, ifn-γ, tnfa, il-1,2,3) during chronic relapsing eae . il-1, il-2, tnfa, and il-6 have also been shown in the serum and/or csf of eae animals and/or ms patients (merril et al, 1989; gallo et al, 1988; gijbels et al, 1990) . a. tgf-/31 and il-1 transforming growth factor β (tgf-β) has pleotrophic effects. evidence to date includes the possible role of down-regulation of the ifn-γ induction of class ii mhc expression on the inductive phase of the immune response, as well as inhibition of cytotoxic t-cell development and antagonism of tnf at the effector end of the immune response (racke et al, 1991; wahl et al, 1988; rook et al, 1986; ranges et al, 1987; czarniecki et al, 1988; schluesener, 1990; shalaby and ammann, 1988) . when administered dur ing eae induction, tgf-/31 slightly delays the onset of disease (kuruvilla et al, 1991) . however, when given during remission, tgf-/31 prevents the occurrence of relapses in relapsing eae, suggesting an anti-inflammatory effect of tgf-/31. when tgf-j81 was preincubated in vitro, activation and proliferation of myelin basic protein-specific lymph node cells in vitro were decreased and severity of the clinical course was reduced (racke et al, 1991) . recent data indicate that il-1 may promote cns inflammation, and that blocking il-1 activity may prove beneficial in the treatment of cns inflammatory disease. for example, eae in the lewis rat has been shown to be exacerbated by il-ια. in addition, the disease was significantly delayed and attenuated by il-1 receptor antagonist (il-lra) (jacobs et al, 1991) . il-1 receptor antagonists have been used to ameliorate other inflamma tory diseases such as rheumatoid arthritis and septic shock, and colitis in a rabbit model (cominelli et al, 1990; ohlsson et al, 1990; wakabayashi et al, 1991; arend and dayer, 1990; higgins and postlethwaite, 1991) . several structural variants of il-lra have been described (arend, 1991) that might be of interest in exploring the therapeutic potential of il-lra. administration of il-lra into the lateral ventricle of rabbit brains has been reported to block il-l-induced non-rapid-eye movement sleep as well as il-l-induced fever (opp and krueger, 1991) . the similarities of macrophages and microglia are striking (merz et al, 1987) . for example, il-1, il-6, and tnfa are known to be produced by monocytes and macrophages. in the cns, microglia have been implicated as a major contributor to the production of il-1, il-6, and tnf (woodroofe et al., 1991) . using a continuous microdialysis probe, woodroofe et al. (1991) showed a 15-fold increase in il-1 over a 24 to 48 hr period and a slight increase in il-6 at day 1, as a result of mechanical trauma to the brain. another potential cellular source of il-1 is the astrocyte. however, the authors have immunocytochemical evidence that in this model the astrocyte response appears much later-at day 7. an increase in peripheral benzodi azepine receptors after local injection of il-1 and tnfa has been demon strated on glial cells, but not on neurons (bourdial et al, 1991) , raising the possibility of a sequential mechanism involving the activation of microglia, the release of il-1 and tnfa, and the promotion by these cytokines of the astroglial reaction. administration of human serum amyloid a to mice inhibited fever in duced by ril-1/3 or rtnfa in vivo, whereas the addition of human serum amyloid a to murine hypothalamic slices inhibited il-1/3-or tnfa-induced prostaglandin e2 production. these data suggest a possible feedback rela tionship between serum amyloid a and cytokines (shainkin-kestenbaum et al, 1991) . injection of il-1 results in an increased release of corticotropin-releasing factor (ohgo et al, 1992; besedovsky et al, 1986; sapolsky et al, 1987; uehara et al, 1987) . in addition, il-ια injected into the third ventricle of castrated rats inhibited the pulsatile release of luteinizing hormone (rettori et al, 1991) . in a recent report, palazzolo et al (1990) suggest that the central and neuroendocrine effects of il-1 are most likely produced through changes in neurotransmitter metabolism in the brain. in another report, mohankumai et al. (1991) provide evidence that il-1 stimulates the release of catecholamines (e.g., dopamine and its metabolite, dihydroxyphenylacetic acid (dopac) from discrete hypothalamic nuclei of conscious, freely mov ing rats. the precise role that il-1 may play in neuroimmunomodulation is still not clear. the important known role of the hypothalamicpituitary-adrenocortical (ηρα) axis in regulating inflammation mandates further studies of the role of il-1 as a neuroimmunomodulator. another potential interaction of the nervous system and the immune system is the involvement of cytokines in neurogenic inflammation (kim ball, 1991) . in the kimball model, substance p, a neuropeptide, is at the center, interacting with fibroblasts, mast cells, β cells, and macrophages. in addition to substance p, other neuropeptides, neurotransmitters, and other vasoactive amines act in concert with cytokines to affect immunologic mechanisms. b. il-2, il-6, tnfa, and ifn-γ the roles of established monokines (il-6 and tnfa) and t-cell products (ifn-γ and il-2) on cns pathology and physiology are stimulating areas of ongoing research. tnf appears to be a major cytokine involved in cellular injury. axon and myelin abnormalities have been demonstrated in vitro by tnf (selmaj and raine, 1988) . after il-2 perfusion in rats, tnf was noted to coincide with myelin damage. interestingly, il-2 perfusion has been shown to compromise the bbb (ellison and merchant, 1991) . several struc tural variants of il-lra have been described (arend, 1991) that might be of interest in exploring the therapeutic potential of il-lra. several phosphodi esterase inhibitors (e.g., theophylline, pentoxifylline, and 3-isobutyl-lmethylxanthine) have been shown to suppress tnfa synthesis (endres et al., 1991) . high tnf levels have been reported to be associated with several manifestations of malaria (kremsner et al, 1991; shaffer et al, 1991) , and decreasing tnf, levels with recovery. in a murine model of cerebral malaria, pentoxifylline, which reduces tnf, given for 10 days after infection, pro tected the mice from development of cerebral malaria (kremsner et al, 1991) . one of the pleotrophic effects of il-6 is a neurotrophic effect that has been described in viral diseases (frei, 1989) and improved survival of mes encephalic catachoaminergic and septal cholinergic neurons (hama et al., 1991) . mice infected with lethal lymphocytic choriomeningitis (lcm) virus as well as lcm carrier mice have been shown to be correlated with high levels of secretion of il-6 (maskophidis et al., 1991) . of interest is the possible role of il-6 in the formation of alzheimer amyloid plaque (baurer and strauss, 1991) . bauer et al. have shown a potent human proteinase inhibitor, a-2 macroglobulin (a2m), after stimulation with il-6 (ganter et al, 1991) . subsequently, they examined whether a 2 m and il-6 could be detected in alzheimer disease (ad) brain. they report that ad cortical senile plaques display strong a2m and il-6 immunoreactivity, with no such immunoreactivity found in age-matched control brains. the data indicate that neuronal cells are the site of a2m synthesis in ad brains. among the best known pleotrophic effects of ifn-γ is its ability to induce the expression of class ii mhc molecules on several cell types. class ii mhc antigens are known to be essential in antigen presentation. studies have shown that ifn-γ can induce expression of class ii mhc molecules on astroglia (sedgwick et al., 1991) . however, sedgewick et al. (1991) were unable to show that astroglial cells act as stimulators of c d 4 + τ cells and therefore postulated another cell type as the major antigen-presenting cell in cns inflammation. infection with mouse hepatitis virus (mhv), has been shown to block expression of mhc molecules on murine cerebral endothelial cells (see later discussion) (joseph et al., 1991) . joseph et al. postulate possible release of cytokines by endothelial cells as a mechanism for blocking ifn-induced class ii antigens. several additional cytokines have been reported that may play a role in cns pathology and physiology. growth factors and, to some extent, il-1/3 and tnfa appear to increase nerve growth factor synthesis and secretion by astrocytes (yoshida and gage, 1992) . cultured astrocytes have been reported to express macrophage colony-stimulating factor activity at a high level, which may be important in the replication of microglial precursors (alliot et al, 1991) . growth differentiation factor-1, a recently described member of the trans forming growth factor β superfamily, has been reported to be restricted almost exclusively to the cns (lee, 1991) . this extracellular signaling factor is postulated to play a general role in nervous system maintenance and function. of interest is one of two platelet-activating factor (paf) antago nists, which has been shown to also inhibit il-l/3-induced acth secretion (rougeot et al, 1991) . this suggests that il-1 ηρα secretion may be medi ated, at least in part, by the production of paf. virus infection of the cns can result in a wide range of pathogenic outcomes: a rapid, severely acute form; a chronic, progressive form; a reversible, nonproductive, latent type; or various intermediate stages. dis ease is a direct result of interaction between host and infecting virus. host parameters include age, genetic makeup, and immune competency, whereas viral factors include class and strain of agent, target specificity, and genomic variation. the phenotypic expression of disease is a complex multifactorial interaction among these parameters. the first step in the disease process involves entry of the virus into the cns. various host protective barriers prevent direct viral entry into the cns and force indirect routes of infection. agents that cause viremia can enter by replication in the endothelial cells that line the blood vessels. viruses known to enter by this route include the toga viruses, enteroviruses, and certain retroviruses. viruses can also invade from the bloodstream by entering the stroma and the choroid plexus through the fenestrated capillary and endothelium and either infecting or being transported across the cho roid plexus into the csf. recently, it has been demonstrated that fibroblastlike cells isolated from the human choroid plexus can be infected with hiv (harouse et al, 1989) . once in the csf, the virus can infect ependymal cells lining the ventricles and invade the underlying cns tissue. the virus can also enter through "trojan horse" mechanisms, being transported within circulating leukocytes such as lymphocytes or macrophages/mono cytes, which can contain such viruses as measles, mumps, canine distem per, lentiviruses, or togaviruses. viruses can also infect peripheral neurons at such sites as the neuromuscular junction and can be axonally transported toward the cns. rabies and herpes simplex may utilize such a route (john son, 1984) . herpes simplex may also gain entrance to the cns by means of the sensory neurons of the olfactory system. once within the cns, viruses encounter a differentiated cell population with complex, functionally integrated cell-to-cell interactions. the highly specialized cytoplasmic membranes of this cell population allow for great variation in virual receptor sites and in the abilities of cells to support viral replication. viral tropism to cells within the cns involves both cell and viral receptor molecules . polioviruses display a particular affinity for anterior horn motor neurons, whereas the rabies virus normally prefers neurons of the limbic system (johnson, 1980) . infections with orthomyxoviruses or paramyxoviruses usually involve the selective infection of ependymal cells (wolinsky et al, 1976) . the polyomavirus, which is the causative agent of progressive multifocal leukoencephalopathy (see later discussion), produces a lytic infection of oligodendrocytes and a nonpermissive infection of astrocytes (richardson, 1961) . the la antigen receptors present on a variety of cell types have been implicated as viral receptors (inada and mims, 1990) . cellular cd4 protein interacts with hiv gp 120 and is the receptor for this virus on lymphocytes and monocytes (wigdahl and kunsch, 1990) . several studies suggest the presence of the cd4 receptor on cells within the cns (dewhurst et al, 1987) . the specific affinity of rabies virus to acetylcholine receptors serves to facilitate uptake and transfer of virus to the cns and to determine neuronal specificity (lentz and burrage, 1982) . as stated earlier, one potential outcome of cns viral infection is rapid acute disease, usually including encephalitis and/or meningoencephalitis. this can be accompanied by perivascular inflammation involving polymor phonuclear cells followed later by macrophages, lymphocytes, and microg lia. during infection by certain viruses, i.e., poliovirus, the inflammatory response consists of a meningeal reaction, perivascular cuffing, and paren chymal infiltration. polymorphonuclear leukocytes are seen early, with a later shift to predominantly mononuclear cells. microglial cell infiltration and neuronophagia are also seen (johnson, 1982) . pathogenesis is normally a two-step process consisting of viral-mediated cell destruction as well as immune-mediated, viral-induced cellular destruc tion. viral antigens are normally good immunogens, and host immune surveillance directs a t-cell-dependent immune response (burns, 1975) . such a response is directed primarily against infected cells that express viral or altered cell surface proteins. antibodies can function as intermediaries in the destruction of infected cells by means of complement-mediated cytotox icity or antibody-dependent cellular cytotoxicity or by serving as opsinizing factors. cytotoxic τ cells can destroy such cells directly or release lympho kines to recruit, activate, and/or sensitize other lymphoreticular elements. cytolytic τ lymphocytes (ctls) are an important effector in antiviral immu nity (zinkernagel and doherty, 1986) . such cells can recognize foreign viral antigens on cell surfaces only in the context of self mhc structures. until recently, the hla class i molecules were thought to be the primary, if not the only, hla recognition structure for ctls. studies of measles and epstein-barr virus (ebv) infection suggest that hla class ii molecules can also serve as recognition sites, further expanding the potential action of ctls. viral antigens recognized by ctls have also been expanded beyond the traditional cell surface molecules (braciale and braciale, 1986) . prelimi nary data indicate that recognition of internal viral polypeptides and per haps even nonstructural gene products may be a common feature of antivi ral ctls. coronavirus mhv-4 can selectively block gamma-interferoninduced class ii antigen expression on cerebral endothelial cells (joseph etal, 1991) . studies by other investigators have shown class i modulation on mouse glial cells by other related coronaviruses (suzumura etal., 1986) . in such a manner, viruses may be able to evade immune-mediated events that occur at the level of the bbb. the virus is able to avoid host immune surveillance and to enter the cns. such events probably also prevent late immune-mediated demyelinating disease, which is absent in mhv-4 virus infection (see later discussion). immunodeficiency states, such as aids or iatrogenic immunosuppres sion in transplant recipients, may sometimes modify the inflammatory response of the cns to pathogens. in aids, especially at the terminal stage of disease, the picture of cns inflammation, i.e., in toxoplasma gondii encephalitis or in progressive multifocal leukoencephalopathy caused by jc virus, can differ from that seen in nonimmuno-suppressed individuals. the inflammation seems muted, and the destructive component of the process (fulminant necrosis) may dominate. this may serve as another example that the inflammatory response within the cns is partly depen dent on the peripheral immune system. as discussed earlier, immune surveillance in response to viral infections can lead to virus-induced immune response to normal host components. during acute infections, this response can lead to increased tissue damage. during chronic or persistent infections, such responses can perpetuate or be the primary cause of the disease process. involvement can take the form of humoral response (autoantibodies) as well as cell-mediated autoimmune (cmai) response (ter meulen, 1989) . such autoimmunity may involve a phenomenon known as molecular mimicry (srinvasappa et al, 1986) , an immune process in which molecules coded for by dissimilar genes share similar structures. in such a manner, antiviral antibodies may bind to host antigens as well as to antigens of the virus. many examples of such mimicry have now been identified: measles virus phosphoprotein and keratin ; vaccinia virus hemagglutin and vimentin (dales et al., 1983) ; fusion protein of measles and heat shock protein (shesheberadarin and norby, 1984) ; hiv gpl20 and neuroleukin (mizarachi, 1989) . in an analysis of monoclonal antibodies to 11 different viruses, 4% of such anti bodies cross-reacted with host cell determinants (srinvasappa et al., 1986) . generation of cytotoxic cross-reactive effector lymphocytes or antibodies would recognize "self proteins" located at target cells. the virus need not be present for such events to take place. myelin basic protein exhibits a significant degree of homology with several viral proteins, including hepati tis β virus polymerase. inoculation of hepatitis β virus polymerase peptide into rabbits caused perivascular infiltration localized to the cns, reminis cent of eae (fujinami and oldstone, 1985) . several other mechanisms are potentially operational in virus-induced autoimmune disease. viruses may disrupt normal immune regulation by direct interaction with cells of the reticuloendothelial system (res). as discussed earlier, many viruses can replicate in these cell types, leading to destruction of lymphocyte subpopulations or stimulation of autoreactive clones. viruses may incorporate host molecules into their envelope or coat, or they may insert, modify, or expose other cellular components on the cell surface. immune pathological events may also be triggered by induction of class ii mhc antigens on the surface of infected cns cells. mhc antigens are expressed only at low levels or not at all on the majority of cns cells (hart and fabre, 1981 ). astrocytes exposed in vitro to measles or corona virus jhm begin to express mhc class ii antigens, which are further enhanced in the presence of tnf (massa et al, 1987 ). an analogous situation in vivo would certainly facilitate ctl-mediated autoimmune response. postinfectious encephalomyelitis has been associated with a wide range of virus infections including measles, mumps, rubella, vaccinia, and herpes zoster and certain respiratory viral infections (johnson and griffin, 1986) . neuropathological changes resemble those seen in eae and appear to be a direct result of an mbp-directed immune response (ter meulen, 1989) . coronavirus jhm causes acute demyelinating encephalitis by selective in fection of oligodendrocytes. lymphocytes from sick animals that are pas sively transferred to syngeneic animals produce lesions in the recipients that resemble those in individuals with allergic encephalomyelitis (ter meulen, 1989) . viral infection of oligodendrocytes appears capable of inducing an autoimmune response against the myelin proteins produced by these cells. in measles-induced encephalomyelitis, there is little evidence that virus invades the cns. deregulation of autoreactive cells may occur secondarily to viral infection of lymphoid cells. demyelinating disease caused by theiler's murine encephalomyelitis virus (tmev), a nonbudding enterovirus, may result from a virus-specific, delayed-type hypersensitivity (dth) (clatch et al., 1986) . lymphokines produced by mhc class ii-restricted, tmev-specific, dth τ cells primed by interaction with tmev-infected macrophages would recruit and activate additional macrophages, leading to nonspecific macrophage-induced damage. in contrast to the autoimmune mechanisms described above, persistent viral infections can be a consequence of the ability of the virus to avoid immune surveillance. in the face of excessive viral antigen, both virusspecific antibodies and ctls are suppressed or rendered ineffective. vi ruses may be able to down regulate appropriate recognition molecules on the surface of immune cells. expression of virus surface proteins involved in immune recognition may also be reduced. reduced expression of glyco protein has been observed during persistent infections with arenaviruses, paramyxoviruses, retroviruses, and rhabdoviruses (oldstone, 1989) . antibody-induced antigenic modulation can lead to measles virus persistent infection in the cns (fujinami and oldstone, 1984) . presumably, this mod-ulation can occur in body compartments that are devoid of the effector molecules of complement such as the cns. subacute sclerosing panenceph alitis (sspe) results from a persistent cns infection with defective measles virus (wechsler and meissner, 1982) . a defect in viral envelope genes prevents expression of viral antigen on cell surfaces. infection is propagated by cell-to-cell transmission of virus, allowing the agent to avoid immune surveillance. viruses can also replicate in the cellular constituents of the immune system and in such a way disable specific immune responsiveness. viruses thus persist within cells that are ordinarily utilized to provide clearance. viruses such as measles and hiv can infect lymphocytes and/ or macrophages, resulting in generalized immunosuppression. such immu nosuppression leaves the host susceptible to infection by a wide range of other microorganisms, leading to the opportunistic infections often associ ated with hiv and other such viruses. viruses can also avoid immune surveillance through latency, a state of reversible, nonproductive infection. viral genetic information can remain within the host in either an integrated or an episomal state. retroviruses such as hiv use reverse transcriptase to insert their dna proviral form into host-cell dna; this integration step is a prerequisite for the replication of these rna viruses (mahry, 1985) . herpes simplex virus (hsv), which resides in sensory ganglion cells, integrates its dna directly into host nuclear dna. the dna of other dna viruses such as ebv or jc agent, the polyomavirus that causes progressive multifocal leukoencephalopathy, remains latent in a nonintegrated episomal form. latent infections estab lished by these viruses may result from a lack of host factors that are critical for the expression of viral early gene products (garcia-blanco and cullen, 1991) . the subsequent activation of specific cellular transcription factors in response to extracellular stimuli can induce the expression of virus and lead to cns disease. herpesviruses such as hsv-1, hsv-2, or vzv cause initial acute peripheral infection followed by a latent infection of neurons. in this environment, sheltered from immune surveillance, the virus can remain for the life of the host. hsv-1 can be activated by a number of seemingly unrelated stimuli such as physical or emotional stress or damage to adjacent tissue. reactivation probably results from a signal transduction event that directly or indirectly induces hsv-1 gene transcription (leib et ah, 1989) . latently ebv-infected β cells can also persist for the life of the individual. if immediate early viral gene expression does not occur, ebv may establish a latent infection (miller, 1985) . both activated τ lymphocytes and nondividing macrophages are able to support the synthesis and integra tion of hiv-1 proviruses (fauci, 1988) . resting τ cells are nonpermissive and do not support replication or integration. resultant unintegrated viral intermediates may persist in a viable yet transcriptionally inert form for extended periods of time. in quiescent macrophages or in τ cells in a resting state, cellular factors critical for proviral transcription may not be active. postintegration latency in hiv-1 may result from inefficient proviral tran scription and from a suboptimal amount of rev, a viral-coded transactivation regulatory protein (pomerantz et al, 1990) . stimulation of these cells in some manner could induce cellular transcription factors increasing rev which leads to productive virus replication. the rare demyelinating disor der, progressive multifocal leukoencephalopathy (pml) (see earlier discus sion), is caused by the reactivation of latent polyomavirus ( j c v ) in the oligodendroglia cells of affected individuals (mazlo and tariska, 1980) . this disease is usually associated with an underlying disorder of the res system in which immune responsiveness is impaired. loss of immune surveillance allows reexpression of virus and cytocidal effects on oligodendrocytes, lead ing to demyelination. the final type of interaction of virus within the cns is a chronic persistent infection that does not elicit an immune response. wild mouse ecotropic immune leukemia virus (mulv) induces a progressive form of hind limb paralysis in a natural population of mice as well as in laboratory animals (gardner et al., 1973) . pathologically, the disease is characterized by the absence of host inflammatory response at the site of tissue destruction and by a picture of neuronal degeneration, spongiform gray matter lesions, and gliosis. paralysis is a direct result of viral replication, most notably in anterior horn motor neurons, but virus can also infect endothelial and glial cells. there is no virus-mediated ctl or antibody response. the cns is devoid of inflammatory infiltrates or deposits of igg or complement. in genetically susceptible laboratory strains of mice, ability to induce disease is age depen dent and involves tolerance to the virus. passive immunization with anti bodies to mulv virus can prevent paralysis. neuronal destruction and spongiosis may be a direct result of the interaction of viral gene expression in the neuron. recent evidence suggests that a posttranscriptional step in virus envelope protein synthesis is impaired and that neurological disease may be a consequence of abortive replication of virus within neurons (sharpe et al, 1990) . such abortive infections can make the presence of virus difficult to detect. analogies to other retrovirus cns infections, notably hiv and htlv-1, suggest that similar mechanisms may be operational in cns disease caused by these agents. another group of infectious agents that establish cns disease in the absence of inflammatory response are the agents of the transmissible spon giform encephalopathies (tses) or prion diseases (carp et al, 1989) . unlike the retroviral disease described above, in which immune response to virus is age dependent and the disease process may include tolerance, there is no evidence for the ability of the host to generate any immune response to the agents of tse. these agents cause creutzfeldt-jakob disease (cjd), gerstmann-straussler-scheinker syndrome (gss), and kuru in humans and are associated with scrapie, bovine spongiform encephalopathy (bse), chronic wasting disease, and transmissible mink encephalopathy (tme) in animals. the agents that cause these diseases are poorly characterized, but it is certain that they do not exhibit properties of known viruses or virus like agents. these agents replicate peripherally in the res system, notably in spleen and lymph nodes. agent crosses into the cns, leading to spon giosis, gliosis, and neuronal destruction in the absence of any inflammatory response, similar to that described above for the retroviruses. modulation of the immune system only affects the agent given peripherally. immuno logical compounds, given close to the time of peripheral infection, that stimulate the immune response shorten incubation periods, whereas com pounds that suppress the immune response extend the incubation period. treatment with dextran sulfate (farkuhar and dickinson, 1986 ) extends disease and may mediate its effect through interaction with macrophages. recent studies suggest the importance of follicular dendritic cells (kitamoto et al., 1991) in agent peripheral replication. similar dendritic cells are in volved in antigen presentation and virus replication in infections caused by other viruses. similar to evidence described for the neurotropic retroviruses, pathology within the cns may include abnormal protein processing, in this case, the production of an abnormal host-coded protein termed prp (prusiner, 1991) . both agent replication and spongiform change are associ ated with the presence of this protein within the cns. in conclusion, the inflammatory response within the cns appears to have several aspects. first, the cns itself is unique from other organs in the presence of bbb with specialized endothelial cells, the presence of cnsspecific cells (e.g., microglia or astrocytes), and the presence of cns-specific antigens. second, infectious agents such as viruses comprise a wide spec trum of agents with varied neural tissue virulence and varied specificity of agents to certain cns cell types. some of these agents may produce fulmi nant disease with severe, if not lethal, cns 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microglia in cytokine production lymphocyte homing receptor: relationship between the mel-14 antigen and cell surface lectin expression and modulation of hla-dr on cultured human adult astrocytes fibroblast growth factors stimulate nerve growth factor synthesis and secretion by astrocytes mhc restricted cytotoxic τ cells: studies on the biological role of polymorphic major transplantation antigens determining t-cell restriction specificity, function and responsiveness key: cord-270084-xs0pbcip authors: stohlman, stephen a.; sussman, mark a.; matsushima, glenn k.; shubin, richard a.; erlich, stephanie s. title: delayed-type hypersensitivity response in the central nervous system during jhm virus infection requires viral specificity for protection date: 1988-09-30 journal: journal of neuroimmunology doi: 10.1016/0165-5728(88)90007-0 sha: doc_id: 270084 cord_uid: xs0pbcip abstract the jhm strain of mouse hepatitis virus (jhmv) elicits an i-a-restricted delayed-type hypersensitivity (dth) response mediated by a thy-1+, lyt-1+, and cd4+ t cell. adoptive transfer of these polyclonal cd4+ t cells from immunized mice prevents death in lethally infected recipients without significantly reducing virus titer in the central nervous system (cns). these observations raise the possibility that the recruitment of mononuclear cells into the cns may play a critical role in survival from a lethal cns infection. transient dth response to nonviral antigens induced an accumulation of monocytes in the cns that was maximal at 48 h post-challenge and virtually resolved by 5 days post-challenge. by contrast the induction of prolonged dth responses resulted in the accumulation of a large number monocytes that persisted in the cns for at least 5 days post-challenge. neither type of dth reaction suppressed virus replication or prevented death from concomitant lethal jhmv infection. many viral infections are accompanied by delayed-type hypersensitivity (dth) responses; however, the role of the dth response in viral pathogenesis or resistance is unclear (liew, 1982) . dth is an antigen-specific response mediated by t cells which express the cd4 cell surface marker and respond to antigen presented in the context of major histocompatability complex (mhc) class ii molecules. antigen-induced activation of tdt h cells results in the release of lymphokines, increased vascular permeability and the local accumulation of antigen-nonspecific monocytes (liew, 1982; askenase and van loveren, 1983; geczy, 1984) . our laboratory has previously shown that the adoptive transfer of cd4 ÷ cell tdt h clones specific for the neurotropic jhmv strain of mouse hepatitis virus protects mice from death due to a lethal infection . protection requires only a small number (5 x 104) of these clonal t cells, is mhc class ii (i-a) restricted, is prevented by immunosuppression of the host and results in the accumulation of large numbers of perivascular mononuclear cells . these characteristics are consistent with the induction of a dth response (liew, 1982) . the data suggest that the dth-stimulated local accumulation of monocytes within the central nervous system (cns) may play a pivotal role in providing protection to the host from a lethal infection. in addition, protection was not accompanied by a reduction of virus in the cns, which suggests that the dth response may play a role in the establishment of persistent jhmv infection in the cns, leading to chronic jhmvinduced demyelination (stohlman and weiner, 1981) . in comparison with the well-defined roles of both antibody in the neutralization of infectious virus and cytotoxic t lymphocytes (ctl) in the lysis of infected cells, the role of dth-inducer t cells in providing antivirai effector function is unclear; however, the recruitment of antigen-nonspecific bone marrow-derived monocytes to the site of viral infection is a primary effect of their activation (allison, 1974; liew, 1982) . the cd4* phenotype and mhc restriction of tdt h cells are identical to t cells that provide help in the augmentation of both antiviral antibody and the ctl responses. although adoptive transfer of jhmv-specific cd4 + t cell clones prevents death from lethal jhmv infection, it does not result in a significant increase in serum antiviral antibody titer, suggesting that protection is not due to t cell-mediated help provided to antibody producing b cells . ctl responses are pivotal in protection and reduction of viral titer in several systemic infections (sethi et al., 1983; byrne and oldstone, 1984; morrison et al., 1986) . although jhmv-specific ctl have been recently described (kyuwa et al., 1988) , it is not clear if they play a significant role in protection from lethal jhmv infection. since the jhmv-specific dth response in the cns confers survival to mice undergoing an otherwise fatal infection, it is important to determine if protection is mediated via the action of the bone marrow-derived monocytes recruited into the cns and/or other antigen-nonspecific components of the response. in this paper we demonstrate that jhmv elicits an /-a-restricted, cd4* t cell-mediated dth response that provides protection from lethal infection upon adoptive transfer to naive recipients. however, the induction of dth responses by nonviral antigens resulting in the recruitment of monocytes into the cns during a lethal jhmv infection is not protective. this suggests that the antigen-nonspecific components of a dth response in the cns are not critical to survival from lethal jhmv infection. a/j, a.sw, balb/c, c57bl/6, c57bl/10, bi0.a(2r), b10.a(5r), b10.br, b10.mbr, cba, c3h/he, sjl and swr mice were purchased from the jackson laboratory (bar harbor, me) at 6 weeks of age. mice were used as recipients in the adoptive transfer experiments within 6 days of arrival. sera obtained from representative mice were tested for jhmv antibody by enzyme-linked immunosorbent assay (elisa) as previously described (fleming et al., 1986 ) and found to be seronegative. the ds small plaque variant of the neurotropic jhmv strain of mouse hepatitis virus (mhv) was used for intracerebral (i.c.) infection (stohlman et al, 1982a) . the dl large plaque variant of jhmv was used for intraperitoneal (i.p.) immunizations (stohlman et al., 1982a) . both viruses elicit comparable immune responses following i.p. injection (unpublished data). viruses were propagated in dbt cells, a continuous murine astrocytoma, as previously described (stohlman and weiner, 1981) . mice were inoculated i.c. with approximately 5 x 103 plaque-forming units (pfu) of jhmv in a volume of 0.03 ml. animals succumbing to this infection have acute encephalomyelitis with evidence of acute and chronic demyelination (stohiman and weiner, 1981; stohlman et al., 1982a; erlich et al., 1987) . mice were immunized by injecting approximately 106 pfu of jhmv i.p. in a volume of 1.0 ml. five days after primary immunization with jhmv, mice were injected in the right footpad with 25 ~1 of a lysate of jhmv-infected cells prepared as previously described (woodward et al., 1984) . the same protocol was used for immunizations with 150/~g of keyhole limpet hemocyanin (klh; calbiochem, la jolla, ca) or 150 #g of ovalbumin (ova; sigma, st. louis, mo) in 1.0 ml of phosphate-buffered saline (pbs), ph 7.4. mice immunized with sheep red blood cells (srbc; colorado serum, denver, co) received 1 x 10 9 srbc i.p. immunized mice were challenged 5 days later with 150 ~tg of klh, 250 /~g of ova or 10 7 srbc injected into the right footpad in a volume of 25 ~1. control antigens (25/~1) were injected in the left footpad. the dorso-ventral thickness of the footpad was measured 24 h after challenge. data are presented as the thickness of the right foot minus the thickness of the left foot plus or minus one standard error of the mean. statistical significance was determined by two-tailed student's t-test. for adoptive transfer experiments, the spleens were removed aseptically from mice 5 days post-immunization, teased, and the cells washed twice with dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal calf serum (fcs; gibco laboratories, grand island, ny). the cell concentration was adjusted to 5 × 107 per ml in dmem containing 10% fcs. a total of 2 x 108 cells were incubated on columns of nylon wool (fenwall, morton grove, il) for 60 min at 37 ° c. nonadherent cells were eluted and washed twice prior to intravenous (i.v.) transfer of 5 x 107 cells to recipient mice. flow cytometric analysis revealed that approx. 85% of these cells were thy-l.2 +. for the dth response, mice were challenged with antigen in the footpad on the same day and the dth response determined as described above. to determine survival, mice were challenged with 5 x 103 pfu of jhmv injected i.c. on the same day. nylon wool-purified t cells were depleted of thy-l.2 and cd4 bearing cells using monoclonal antibodies ho-13-14 and g.k. 1.5, respectively. both antibodies were obtained from the american type culture collection (rockville, md). depletions of lyt-l.2 and lyt-2.2 (cd8) bearing cells were performed with monoclonal antibodies purchased from cedarlane laboratory (westbury, ny). briefly, 107 cells per ml were incubated for 60 min on ice with each antibody, washed in dmem containing 1% bovine serum albumin and incubated at 37°c for 60 rain with prescreened rabbit serum (cedarlane laboratory) as a source of complement. cells were resuspended to the original viable cell concentration and transferred to recipient mice i.v. the isolation and characterization of the jhmv-specific t cell clones have been previously reported (woodward et al., 1984) . briefly, popliteal lymph node cells were obtained from c57bl/6 mice immunized with a lysate of jhmv-infected dbt cells emulsified in complete freund's adjuvant. the t cells were cloned by limiting dilution and maintained in rpmi 1640 medium supplemented with 10% fcs, 100 u/ml of penicillin, 50/~g/ml of streptomycin, 2 mm glutamine, 5 x 10 -5 m 2-mercaptoethanol, jhmv antigen, irradiated (2000 rad) syngeneic spleen cells as a source of antigen-presenting cells, and partially purified mouse interleukin-2 (woodward et al., 1984) . the clone used in this report is designated 4bi0 and has the cell surface phenotype thy-l.2 +, lyt-l.2 +, cd8-and cd4 + (woodward et al., 1984; stohlman et al., 1986) . one passage prior to use, the 4b10 clone was cultured in the presence of ultraviolet-inactivated jhmv antigen to prevent carry-over of infectious virus. viable cells were isolated by centrifugation on lympholyte m (cedarlane laboratories), and 1 x 10 6 cells were transferred i.c. to recipients on the same day as i.c. challenge with 5 x 103 pfu of jhm. viral titers were determined 5 days post-infection as previously described (stohlman and weiner, 1981) . briefly brains were removed aseptically from sacrificed mice, placed in ice-cold dulbecco's pbs, ph 7.4, and homogenized manually using 7 ml tenbrock tissue homogenizers. homogenates were clarified by centrifugation at 800xg for 7 rain at 4°c. virus titer in the supernatants was determined by adsorbing 0.2 ml of 10-fold serial dilutions onto monolayers of l2 cells in 24-well plates (falcon plastics, oxnard, ca). after incubation for 60 min at 37°c, the inoculum was removed, followed by the addition of 1.0 ml dmem supplemented with 2% fcs. viral titers were determined 48 h later from duplicate or triplicate assays of groups of three of four mice and the results expressed as the mean titer per gram of tissue. the pathogenesis of jhmv infection and the dth response in the cns were monitored by histological evaluation of representative coronal sections. brains were fixed by immersion in clark's fixative for 3 h, embedded in paraffin and sectioned at 7/~m. tissue sections were stained with ehrlich's hematoxylin and eosin (h&e), dehydrated and mounted in permount prior to light microscopy. it has been suggested that jhmv induces a relatively poor dth response in some inbred strains of mice, including the c57bl/6 strain (kyuwa et al., 1984) . to compare the jhmv-induced dth response in the c57bl/6 mice used in our previous studies to that in other mouse strains, groups of 6-week-old mice of various strains were immunized with jhmv and challenged 5 days later. table 1 shows that there are indeed strain-specific differences in responsiveness to jhmv using an identical immunization and challenge protocol. however, c57bl/6, which were previously reported to be low responders (kyuwa et al., 1984) , are clearly high responders. the jhmv-induced dth response is not linked to h-2, since cba (h-2 k) and a.sw (h-2 s) responded, while c3h (h-2 k) and sjl (h-2 s) mice showed a minimal or no response, as previously reported (kyuwa et al., 1984; stohlman et al., 1985) . although the basis for the discrepancy in these results is unclear, c57bl/6 mice are clearly responders in the immunization and challenge protocol described in this report. to define the characteristics of the cells mediating the dth response to jhmv and the nonviral antigen klh, spleen cells from immunized mice were adoptively transferred to naive recipients. mice were immunized by i.p. injection with either klh or jhmv. spleens were removed 5 days later and the nylon wool-purified cell population transferred i.v. to naive recipients. antigen was injected on the same day and the dth response measured 24 h later. table 2 shows that the transfer of nylon wool-purified spleen cells from mice primed with either klh or jhmv results in an antigen-specific dth response in naive recipients. complement-mediated antibody a results expressed as the difference of the right footpad minus left footpad 5-one standard error of the meanx 10 -2 mm as described in materials and methods. b differences observed between swr and c3h/he or sjl were significant by student's t-test (p < 0.025). none 28.2 + 4.3 c only 25.9 5-5.4 thy-l.2 + c 5.3 + 1.2 cd4 + c 7.2 5-3.8 none 22.6 + 4.7 c only ¢ 22.2 5-5.5 cd8 + c 22.0 5-6.2 thy-1 + c 6.8 5-2.8 lyt-i + c 9.8 + 1.7 cd4 + c 7.5 :t: 4.4 20.5 5-6.2 3.35-2.1 4.5 5-0.6 9.5+2.1 a results expressed as the difference of the fight footpad minus the left footpad + one standard error of the meanx 10 -2 mm as described in materials and methods. b six days after immunization, spleens were removed and 1 x 106 nylon wool-purified spleen cells were transferred i.v. to naive recipients. c = complement. ,l the difference observed between positive dth responders (c57bl/6 untreated) and low dth responders (anti-lyt-i treated c57bl/6) was significant (p = 0.005). depletions identify the phenotype of the dth-inducer t cells elicited in response to both antigens as thy-1 +, cd4 +. in addition, the jhmv-specific dth-inducer t cells were lyt-1 ÷ and cd8-. this is identical to the phenotype of the previously characterized jhmv-specific tdt h cell clones which mediate both the dth response and the protection from lethal jhmv infection (woodward et al., 1984; stohlman et al., 1986) . to ensure that the active cells derived from the jhmv-immunized donors had the appropriate pattern of genetic restriction, nylon wool-purified t cells from jhmv-immunized c57bl/6 (h-2 b) donors were adoptively transferred to congenic strains and dth responses were measured 24 h later. table 2 also shows that c57bl/6 and b10.a(5r) mice, both of which are i-a b, responded, while b10.a(2r), b10.br and b10.mbr mice, which are not histocompatible at i-a with the donor cells, did not respond. these data show that histocompatibility at i-a is necessary for responsiveness and thus confirm that these cells have both the surface phenotype and genetic restriction typical not only of a tdt~ cell (liew, 1982) but also of the jhmv-specific tdt h cell clones previously described (woodward et al., 1984; stohlman et al., 1986) . the adoptive transfer of the jhmv-specific t cell clone 4b10 elicits a dth response in naive mice and provides them with protection from lethal infection . jhmv titer in the cns was not reduced in the protected mice, and there was no difference between mice receiving 4b10 cells and untreated controls in jhmv titer or kinetics during the entire course of the infection . mice were either injected in the footpad with t cell clone 4b10 (1 x 105 per recipient) mixed with antigen for the dth response or injected i.c. (1 x 106 cells per recipient) and challenged i.c. with jhmv to determine survival and virus replication. the data in table 3 (experiment no. 1) confirm our initial observations. we have previously shown that injection of hen egg lysozyme-specific t cell clones plus antigen i.c. has no effect on a lethal jhmv infection . table 3 also shows that the i.v. adoptive transfer of nylon wool-purified cells from jhmv-immunized donors (5 x 10 7 cells per recipient) was similarly capable of inducing at dth response, as well as providing protection from death due to lethal infection without reducing jhmv titer in the cns. protection was accompanied by an increase in mononuclear cell infiltration and perivascular cuffing in the brain parenchyma (fig. 1a) , compared to jhmv-infected mice not receiving cells from immunized donors (fig. 1b) . these data suggest that the adoptive transfer of polyclonal dth effector t cells from jhmv-immunized mice provides protection similar to the adoptive transfer of dth-effector t cell clones. to determine if the dth reaction itself provides protection against lethal jhmv infection, dth responses to the nonviral antigens ova, klh or srbc were tested for their ability to prevent death due to a concurrent jhmv infection. survival was monitored for 21 days post-infection. all mice died between days 10 and 12 post-infection. h viral titers were determined 5 days after challenge. ¢ average response of 22 naive mice challenged with jhmv, klh, ova or srbc. d t cell clone 4b10 (1 × l0 s) was mixed with jhmv or control antigen and injected into the left or right footpads respectively. dth was measured 24 h later. c t cell clone 4b10 (1 × 106) was injected i.c. followed by i.c. injection with 5 × 103 pfu of jhmv as previously described . f nylon wool-purified splenic t cells (5 × 107) from donors immunized 5 days earlier were transferred i.v. to naive recipients. recipients were challenged immediately in the footpads with jhmv or control antigens. dth was measured 24 h later. g recipients were prepared as described in footnote f and challenged i.c. on the same day with 5 × 103 pfu of jhmv. h mice were immunized i.p. with antigen (klh, ova or srbc) 5 days prior to challenge either in the footpads to determine dth response or i.c. with antigen and jhmv to determine survival. i not tested. j mice were immunized i.p. with klh 5 days prior to challenge with kfca as described in footnote h post-immunization, mice were infected with jhmv and challenged i.c. with the appropriate nonviral antigen. following 5 additional days, half of the mice in each group were sacrificed for histological examination and, in the case of klh, to determine the virus titer in the cns. the other half of each group was monitored for survival. none of the immunized mice challenged with these nonviral antigens survived the concurrent jhmv infection, nor was there a reduction in cns viral titer following challenge with klh (table 3 ). all mice died between 10 and 12 days post-infection, suggesting that the concurrent dth responses had essentially no effect upon survival. histological examination of both infected and uninfected mice immunized with these antigens and challenged i.c. revealed a peak of mononuclear infiltration within the cns at 48 h following secondary challenge. however, the responses in uninfected mice had diminished to almost undetectable levels by the fifth day post-infection, indicating that the response to these nonviral antigens was transient. in comparison, jhmv infection results in a progressive increase in cellular infiltration in the cns that is initially evident by 4 days post-infection ( fig. 1a ; weiner, 1973) . thus, it is possible that the dth-induced presence of monocytes within the cns during the initial stages of jhmv infection was not of sufficient magnitude or duration to suppress virus replication or provide any substantial measure of protection. histological examination of mice protected from a lethal jhmv infection via the adoptive transfer of cd4 ÷ t cell clones showed a dramatic increase in the mononuclear infiltration into the brain parenchyma compared to mice receiving jhmv only. the increase was noted by 5 days post-infection (unpublished data). klh, which resulted in the most vigorous dth response as measured by footpad swelling, also induced the most vigorous cellular infiltration into the cns (data not shown). to approximate the prolonged mononuclear infiltration seen during infection, klh was emulsified in freund's complete adjuvant (kfca), and injected into klh-immunized mice. this resulted in a prolonged bilateral accumulation of mononuclear cells in the cns that was not localized to the area of injection (fig. 1c ). this vigorous response to kfca in klh-immunized mice had no apparent adverse clinical effects, and no deaths occurred. by contrast, naive mice receiving kfca exhibited little perivascular cuffing or cellular infiltration in the cns. table 3 (experiment no. 3) shows that kfca also elicited a vigorous dth response in the footpad (70.0 :t: 11.0 × 10 -2 mm) in klh-immunized mice, compared to naive mice (11.3 5:8.0 × 10 -2 mill). the phenotype of the tot n cell reactive to kfca is cd4 ÷ and thy-1 ÷ (data not shown), identical to the cell responsible for the dth response to klh (table 2) . kfca injected i.c. into klh-immunized mice co-infected with jhmv produced a prolonged cellular infiltration and perivascular accumulation of mononuclear cells consistent with a typical dth response (fig. 1d) ; however, it was unable to either confer survival or suppress virus replication (table 3, experiment no. 3) . this further indicates that a dth-induced accumulation of monocytes, even throughout the peak period of virus replication in the cns, was not protective. in addition, no change in survival time was noted in klh-immunized mice receiving kcfa i.c. with a concurrent jhmv infection compared to naive mice. infection of mice with jhmv results in an acute encephalomyelitis with both acute and chronic ongoing demyelination (lampert et al., 1973; stohlman and weiner, 1981; erlich et al., 1987) . although demyelination has been reported in immunosuppressed mice (weiner, 1971) , relatively little is known about the contribution of the immune system in prevention of lethal infection, clearance of infectious virus or establishment or maintenance of latent jhmv infection in the cns. we have sought to determine if the protection afforded jhmv-infected mice by the adoptive transfer of jhmv-specific cd4 ÷ t cells ) is due to the dth-induced accumulation of antigen-nonspecific monocytes within the cns. dth responses are initiated by the interaction of cd4 ÷ t cells with antigen in an /-a-restricted manner, resulting in both t proliferation and the release of a variety of lymphokines. this is followed by an increase in vascular permeability and the local accumulation of bone marrow-derived antigen-nonspecific monocytes which produce a variety of monokines and hydrolytic enzymes (nash and geli, 1981; liew, 1982; askanase and van loveren, 1983; geczy, 1983) . we have determined that jhmv elicits a vigorous dth response in c57bl/6 mice that is mediated by an lyt-1 ÷, cd8-, cd4 ÷, thy-1 ÷ cell and shows the appropriate genetic restriction to i-a. we have further shown that the adoptive transfer of these nylon wool-purified polyclonal dth-inducer t cells protects mice from a lethal infection without reducing the virus titer in the cns, suggesting that they are functionally similar to the cd4 ÷ tdt n cell clones . to determine if the accumulation of antigen-nonspecific monocytes in the cns provides protection to the host from a lethal jhmv infection, dth reactions were induced in the cns with non-jhmv antigens concomitant with a lethal jhmv infection. transient responses early in infection were unable to suppress virus replication or confer survival to lethally infected mice, suggesting that the presence of monocytes early in infection did not alter the outcome of a lethal infection. kfca was retained in the brain parenchyma and induced a persistent and panencephalitic dth response in klh-immunized mice. mice exhibited perivascular cuffs of mononuclear cells resembling the response in jhmv-specific cd4 ÷ t cell clone recipients . however, in spite of clear evidence of a dth response to kfca by both footpad swelling and histological examination of the cns, these mice also succumbed to a lethal jhmv infection. this indicates that the presence of increased numbers of monocytes throughout the infection was also unable to alter the course of the lethal disease. these data also suggest that the lymphokines released by the dth-inducer t cells, the monokines and hydrolytic enzymes released by the monocytes and the loss of vascular permeability do not contribute directly to survival from a lethal jhmv infection. cells of the monocyte/macrophage lineage have been implicated as the pivotal cells in natural resistance to a number of viral infections, including two other strains of mouse hepatitis virus (mogensen, 1979) . these studies have relied on either the in vitro growth of virus in macrophages, the ability of macrophages to suppress virus growth in a second susceptible cell type or the in vivo effects of anti-macrophage treatments such as carrageenan or silica particles (allison, 1974; mogensen, 1979) . although there are numerous virus infections in which macrophages are believed to play a role in survival from lethal infections, it has recently been shown that mononuclear phagocytes do not play an important role in the clearance of lymphocytic choriomeningitis virus (lehman-grube et al., 1987) . jhmv replicates equally well in vitro in macrophages from both resistant and susceptible strains of mice (stohlman et al., 1982b) . this is in contrast to both the mhv-2 and mhv-3 strains, whose in vitro replication in macrophages correlates with mouse strain-specific resistance (bang and warwick, 1970; virelizier and allison, 1976 ). in addition, macrophages from naive mice can suppress the replication of jhmv in a second susceptible cell line in vitro; however, we suggested that this suppression was not of critical importance in survival, since macrophages from both susceptible and resistant strains of mice suppressed virus replication to the same extent (stohlman et al., 1982b) . it has been suggested that dth responses may be superfluous or even detrimental to the host due to the local increase in extracellular fluids and tissue injury induced by the accumulation of monocytes (liew, 1982; askenase and van loveren, 1983; townsend, 1985) . our study suggests not only that the antigen-nonspecific components of the dth response are not detrimental, but also that they do not play a major role in either viral clearance or protection from a lethal infection. mice undergoing either transient or prolonged dth responses in the cns to nonviral antigens had the same mean survival time as animals receiving jhmv only, suggesting that these responses may in fact be superfluous to the immunological events that interact to provide protection from a lethal infection. the data in this report demonstrate that the nonspecific components of the dth response do not provide a substantial measure of protection; however, the mechanism of protection afforded by the jhmv-specific t cell clones is not clear. in addition, mice surviving infection in the absence of virus reduction via the adoptive transfer of dth-inducer t cell clones, nylon wool-purified dth-inducer t cells or neutralizing monoclonal antibodies have persistent jhmv infections with ongoing chronic demyelination (buchmeier et al., 1984; stohlman et al., 1986; unpublished observation) . neither the dth-inducer t cell clones or the nylon wool-purified t cells from mice immunized with jhmv exhibit demonstrable cytotoxic activity against virus-infected targets expressing mhc class i or class ii molecules. histological analysis of the cns of mice protected by the adoptive transfer of t cell clone 4b10 suggests that mice are protected from death due to the inhibition of viral replication in neurons (unpublished data). a similar phenomenon has been previously described following protection mediated by the adoptive transfer of anti-jhmv monoclonal antibodies (buchmeier et al., 1984) . these mechanisms may be different since protection afforded by dth-inducer t cell clones does not alter the antiviral antibody response ; however, the effect, i.e., the reduction of neuronal infection appears to be similar. with the demonstration that the antigennonspecific components of the dth response do not constitute a major protective mechanism in jhmv infection, efforts are now being directed toward understanding the role of viral-specific immunity in survival from lethal jhmv infection. on the role of mononuclear phagocytes in immunity against viruses delayed-type hypersensitivity: activation of mast cells by antigen specific t-cell factors initiates the cascade of cellular interactions mouse macrophage as host cells for the mouse hepatitis virus and the genetic basis of their susceptibility murine hepatitis virus-4 (strain jhm) induced neurological disease is modulated in vivo by monoclonal antibody biology of cloned cytotoxic t lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo experimental neuropathology of chronic demyelination induced by a jhm virus variant (ds) pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies the role of lymphokines in delayed-type hypersensitivity reactions genetic control of delayed-type hypersensitivity to mouse hepatitis virus in mice characterization of mouse hepatitis virus-reactive t cell clones mechanism of demyelination in jhm virus encephalomyelitis mechanism of recovery from acute virus infection. iv. the questionable role of mononuclear phagocytes in the clearance of lymphocytic choriomeningitis virus from spleen of mice regulation of delayed-type hypersensitivity to pathogens and alloantigens role of macrophages in natural resistance to virus infections differences in antigen presentation to mhc class i-and class ii-restricted influenza virus-specific cytolytic t lymphocyte clones protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice the delayed hypersensitivity t cell and its interactions with other t cells protection of mice from fatal herpes simplex virus type i infection by adoptive transfer of cloned virus-specific and h-2 restricted cytotoxic t lymphocytes chronic central nervous system demyelination in mice after jhm virus infection murine coronaviruses: isolation and characterization of two plaque morphology variants of the jhm neurotropic strain macrophage antiviral activity: extrinsic versus intrinsic activity the defect in delayed-type hypersensitivity of young adult sjl mice is due to a lack of functional antigen-presenting cells in vivo effects of coronavirusspecific t cell clones: dth inducer cells prevent a lethal infection but do not inhibit virus replication macrophage response to herpes simplex encephalitis in immune competent and t cell-deficient mice correlation of persistent mouse hepatitis virus (mhv-3) infection with its effect on mouse macrophage cultures pathogenesis of demyelination induced by mouse hepatitis virus (jhm virus) production and characterization of t cell clones specific for mouse hepatitis virus, strain jhm: in vivo and in vitro analysis we thank john fleming and wendy gilmore for helpful discussions, cindy segal for assistance with the histology and carol flores for preparation of the manuscript. this investigation was supported by u.s. public health service grants ns00789, ns18146, ns7149 and at22691 and grant rg1449 from the national multiple sclerosis society. key: cord-266078-h53zpjab authors: mcguckin wuertz, kathryn; treuting, piper m.; hemann, emily a.; esser-nobis, katharina; snyder, annelise g.; graham, jessica b.; daniels, brian p.; wilkins, courtney; snyder, jessica m.; voss, kathleen m.; oberst, andrew; lund, jennifer; gale, michael title: sting is required for host defense against neuropathological west nile virus infection date: 2019-08-15 journal: plos pathog doi: 10.1371/journal.ppat.1007899 sha: doc_id: 266078 cord_uid: h53zpjab west nile virus (wnv), an emerging and re-emerging rna virus, is the leading source of arboviral encephalitic morbidity and mortality in the united states. wnv infections are acutely controlled by innate immunity in peripheral tissues outside of the central nervous system (cns) but wnv can evade the actions of interferon (ifn) to facilitate cns invasion, causing encephalitis, encephalomyelitis, and death. recent studies indicate that stimulator of interferon gene (sting), canonically known for initiating a type i ifn production and innate immune response to cytosolic dna, is required for host defense against neurotropic rna viruses. we evaluated the role of sting in host defense to control wnv infection and pathology in a murine model of infection. when challenged with wnv, sting knock out (-/-) mice displayed increased morbidity and mortality compared to wild type (wt) mice. virologic analysis and assessment of sting activation revealed that sting signaling was not required for control of wnv in the spleen nor was wnv sufficient to mediate canonical sting activation in vitro. however, sting-/mice exhibited a clear trend of increased viral load and virus dissemination in the cns. we found that sting-/mice exhibited increased and prolonged neurological signs compared to wt mice. pathological examination revealed increased lesions, mononuclear cellular infiltration and neuronal death in the cns of sting-/mice, with sustained pathology after viral clearance. we found that sting was required in bone marrow derived macrophages for early control of wnv replication and innate immune activation. in vivo, sting-/mice developed an aberrant t cell response in both the spleen and brain during wnv infection that linked with increased and sustained cns pathology compared to wt mice. our findings demonstrate that sting plays a critical role in immune programming for the control of neurotropic wnv infection and cns disease. introduction through subcutaneous inoculation from the bite of an infected mosquito. a parallel form of infection using sub-cutaneous challenge of wnv in a mouse model has been shown to replicate the progression, tissue involvement, and pathology of wnv infection that occurs in humans [19] [20] [21] [22] . in the mouse model, viral replication occurs at the subcutaneous site of entry followed by infection of the draining lymph node and splenic infection [19] . these processes first trigger innate immune activation in peripheral tissues outside of the central nervous system (cns) through viral recognition by the rig-i-like receptors to induce irf3 activation and the production of types i and iii interferon (ifn) [23] [24] [25] [26] . innate (rlr) immune defenses triggered by rlr signaling and ifn actions serve to restrict the tissue tropism of wnv and are essential for protection against neuroinvasion [19, 23, 24, [27] [28] [29] [30] [31] [32] [33] [34] . type i and iii ifn are essential to inform the innate and adaptive immune interface to balance development of effective immunity, protect the blood-brain barrier, and limit immune-related pathology in the cns [19, 23, 24, [35] [36] [37] [38] [39] . in particular, type i ifn-dependent cytokine and chemokine signaling cascades are essential for functional development of the cytotoxic cd8+ t cell response, as well as its regulatory t cell (tregs; foxp3+ cd4+ t cells) counterpart [24, 36, 37, [39] [40] [41] [42] . while cd8+ t cells are required for controlling both peripheral and cns viral load, cd4+ t cells, specifically tregs, are essential for preventing symptomatic disease in the cns [40] [41] [42] [43] . the adaptor protein, stimulator of interferon genes (sting), has also been implicated in host defense against wnv [44] [45] [46] . sting was first described as an essential defense mechanism against both rna and dna viruses [47, 48] . since then, sting has been recognized for its role in responding to cytoplasmic dna and mediating subsequent innate immune activation and ifn production. however its role in the defense against rna viruses is poorly understood [47] [48] [49] [50] [51] [52] [53] [54] . intriguingly, multiple rna viruses, including dengue virus, yellow fever virus, hepatitis c virus and coronaviruses, direct viral evasion strategies to disrupt the sting signaling pathway, reflecting a likely role for sting in host defense against rna viruses [52] . sting was found to be required for host defense during infection with influenza a virus, as well as dengue virus, a closely related flavivirus to wnv [55] [56] [57] . additionally, during infection with related flavivuses including japanese encephalitis virus (jev) and zika virus, sting deficiency led to increased neuropathology in vivo and in vitro, suggesting a critical role for sting in cns defense [58, 59] . the role for sting in the cns has been implicated in multiple other neurodegenerative diseases including aicardi-goutières syndrome, sterile immune mediated cns pathology and during chronic cns diseases [14, 16, [60] [61] [62] [63] [64] [65] [66] . in this study, we investigated the hypothesis that sting plays a regulatory role in the immune response against wnv, thereby restricting viral neurotropism and neuropathology. we show that sting is essential for host defense against wnv in a mouse in vivo model of infection. clinical and pathological analyses demonstrate a novel role for sting in conferring cns defense against wnv in vivo. we found that tonic levels of type i ifn were decreased in sting-/-bone marrow derived macrophages (bmdm) and linked with increased susceptibility to wnv infection. following infection, we observed heightened immune responses in vitro and in vivo concomitant with increased viral load. sting deficiency led to the development of an aberrant adaptive immune response, with decreased activation of cd8+ cells and t regulatory cells (tregs) in the spleen, and decreased cd4+ t cell numbers resulting in an altered cd4/cd8 t cell ratio in the cns coupled with cns disease. our observations imply an essential role for sting within the interface between the innate and adaptive immune responses for effective immune programming in the control of wnv infection and cns disease. previous studies demonstrated that mice defective in sting signaling experienced increased mortality during wnv infection, yet the linkage of sting to immune response programming for defense against wnv has not been defined [46] . using genetically knocked-out tmem173 (sting-/-) mice [67] , we first performed a survival analysis to confirm the role of sting in host survival during wnv infection (fig 1a) . c57b/6j (b6, wt) and sting-/-mice were infected through subcutaneous virus challenge via foot-pad injection and monitored for 18 days post infection (dpi). mice were scored daily for morbidity, marked as loss in body weight ( fig 1b) and overall increased clinical score (fig 1c) . consistently, between 8-12 dpi, mice either met euthanasia criteria (terminal; t) or went on to survive (survivors; s) through 18 dpi (study end-point) (fig 1d) . using this model, we confirmed the occurrence of increased susceptibility to wnv infection in the complete absence of sting (figs 1a and s1a), similar to what was previously described in sting gt/gt mice [46] . we also observed significantly increased clinical severity scores in the sting-/-mice that persisted until the study-endpoint, when wt mice had returned to a base-line clinical score (fig 1b and 1c) . additionally, we monitored mice daily for the duration of the experiment until they either met euthanasia criteria or at the study end-point, day 18 post infection. results from each mouse were analyzed to determine if there were differences in clinical signs between wt and sting-/-mice. notably, sting-/-mice displayed increased neurological signs of disease, characterized by loss of balance, reduced muscle tone and reflexes predominantly in the pelvic limbs and increased paresis and paralysis, implicating more severe damage to the hind-brain and spinal cord (fig 1e and 1f ). in order to determine if there was a survivor bias in the clinical data, we retrospectively stratified the data into cohorts of mice that met euthanasia criteria (terminal; t) or ones that survived until day 18 post-infection (survivors; s), the pre-determined study end-point ( fig 1d, 1g and 1h ). by doing so, we found that significant differences in body weight loss and clinical scores between wt and sting-/-mice were only observed in the survivor cohort and not in the terminal cohort. while there is an essential role for sting in host survival during acute infection (figs 1a and s1a), these data implicate an additional prolonged requirement for sting in both prevention and recovery from neurological pathology. when we examined cns pathology, we found that in both wt and sting-/-mice, pathological scores were significantly increased in the spines of the survivor cohort, with a trend toward increased scores in the brains and spines of the terminal cohort ( fig 1d, 1i and 1j ). intriguingly, while sting-/-terminal mice displayed increased cns pathology, wt mice that met terminal criteria had unexpectedly low clinical scores, suggesting that they met euthanasia criteria for reasons independent of severe encephalitis. during necropsy, we observed that the gastrointestinal (gi) tract of terminal mice exhibited gross distension or other aberrant phenotypes including stool compaction, disintegration and in some cases severe reduction in size or collapse of the gi tract (s1b fig) . pathologic analysis confirmed that terminal mice display increased gi pathology that included microbiome overgrowth and neuronal degeneration and loss in the myenteric ganglia, particularly in sting-/-(s1c and s1d fig) . previous studies have indicated that gi manifestations during wnv infections exist in both mice and humans, and are positively correlated to increased neurotropism and mortality [15] [16] [17] 22] . this outcome may imply that wt mice are meeting euthanasia criteria following wnv infection due to severe gi disease rather than severe cns involvement as previously thought. further, these results demonstrate that sting plays a systemic role in host defense against wnv, with increased frequency of mortality and pathology occurring in the cns and gi tract in sting-/ mice. together, these results show an essential role for sting in host survival and neuropathological defense in the cns during wnv infection. to determine if sting is required for viral control in the cns, we challenged mice with wnv via footpad injection and examined tissue viral load at 4 dpi (peak of peripheral viremia) and 8 dpi (peak of detectible virus in the cns) (fig 2a) . viral titer of macrodissected brains and extracted spinal cords were examined by plaque assay individually for each mouse in the cohort (fig 2a) . as expected, virus was not detected at 4 dpi in the cns but by 8 dpi virus was clearly detected in different cns regions. virus was not consistently found in the cns of all mice nor in every tissue examined. there was however, a consistent trend toward increased numbers of infected mice with detectible virus in the cns as well as increased viral titers in the cns of sting-/-mice compared to wt. to determine if there was detectible virus in the brains of terminal vs survivor mice, tissues from retrospectively sorted mice utilized for pathological analysis (fig 1i and 1j) were immunostained for the presence of wnv antigen ( fig 2b) . wnv foci were found in the brains of wt and sting-/-terminal mice but were not apparent in wt or sting-/-survivors, suggesting that either the virus had cleared or that surviving mice did not have cns infection. neuronal death was assessed by tunel stain in both wt and sting-/ survivors. here we observed enhanced neuronal apoptotic death in the sting-/-cohort, suggesting sting may have a direct or indirect role in neuronal defense in the cns (fig 2c) . in order to determine if sting is required for neuronal defense against wnv, primary cortical neurons were isolated and cultured, followed by infection with wnv to determine viral growth kinetics under conditions of single and multi-step growth ( fig 2d) . surprisingly, no difference was detected between in wnv replication in wt and sting-/primary cortical neurons (fig 2d) . to determine if the actions of sting might be restricted to the cns for wnv protection, we performed an intracranial virus inoculation bypassing the role of the peripheral immune response and physical barriers such as the blood-brain barrier to directly infect the brain with wnv (fig 2e) . at 4 dpi, there was no difference in cns viral load found in wt vs sting-/-mice nor was viral load different between sting-/-and wt mice. taken together, our observations imply that the role of sting is not limited to mediating viral control in the cns. it is possible that sting is therefore required in the development of a protective immune response in the periphery such that in the absence of sting the immune response is aberrantly programmed, leading to cns immunopathology. given that sting deficiency was associated with enhanced mortality (see fig 1) without a significant increase in cns viral burden (fig 2) , we considered that sting deficiency could result in defective antiviral innate immune signaling and lead to loss of viral control in the periphery, thereby leading to enhanced morbidity and mortality. we first tested the role of sting in bmdms, as macrophages are a tropic cell and key modulator of peripheral viral control during wnv infection ( fig 3a) [19] . as expected, wnv levels were significantly increased by 24 and 48 hours post inoculation (hpi). unexpectedly however, sting-/-bmdm had increased innate immune and inflammatory gene expression, including enhanced level of type i ifn expression during wnv infection ( fig 3b) . we then examined the spleens of infected mice to determine if there was an overall loss of viral control manifested as increased viral load over wt. as expected, virus was detected at 4 dpi in both wt and sting-/-. surprisingly however, there was no difference in 4 dpi viral titers between wt and sting-/-, nor was there a sustained virologic response in sting-/-mice ( fig 3c) . these data indicate that peripheral loss of viral control does not occur in the absence of sting (fig 3c) . similarly, viral rna was detected equally in spleens of infected wt and sting-/-mice at 4 dpi, but the virus was largely cleared from the spleen by 8 dpi (fig 3d) . in the cns however, we observed a trend toward increased viral rna and innate immune gene expression at 8 dpi in wnv-infected sting-/-mice, similar to that observed in bmdm (fig 3a and 3d) . these data were unexpected as we initially predicted that sting deficiency would reduce innate immune activation based on the known role of sting signaling in ifn induction. these data demonstrate that innate immune activation and the inflammatory response are exacerbated in both in vitro and in vivo sting deficient models, possibly culminating in enhanced immunopathology in sting-/-mice. the canonical sting sensing pathway is dependent on upstream recognition of dna dangeror pathogen-associated molecular patterns (damp, pamp) such as dna viruses, cell-free or mitochondrial dna, by cyclic gmp-amp synthase (cgas). in mammals, cgas binding to dsdna activates its synthase activity to produce a cyclic di-nucleotide, cgamp (cyclic guanosine monophosphate-adenosine monophosphate), which binds to sting, initiating downstream activation of sting by phosphorylation, sting relocalization from diffuse cytosolic to punctate pattern, and subsequent induction of innate immune signaling and ifn production [47, 48, 53, 68, 69] . during rna virus infections however, the role for sting defense has not been well-characterized. to evaluate the activation of sting during wnv infection, we utilized a recently described telomerase reverse transcriptase human foreskin fibroblasts (hff) model to assess activation of endogenous sting by phosphorylation and relocalization from the cytoplasm to the perinuclear space during wnv infection [70] . transfection of interferon-stimulated dna (isd; calf-thymus dna) into hffs initiated re-localization of sting as previously reported by 3hpi [48, 70] . intriguingly however, sting was not relocalized in wnv infected cells (fig 4a) . it is possible that the kinetics of sting activation are different from isd activation of sting as compared to wnv infection, so we performed a time course experiment to detect sting activation by phosphorylation status [71] , assessing a range of 1-24 hpi at moi = 1 ( fig 4b) . similar to what was observed by ifa, sting phosphorylation was not observed at any time point during wnv infection, although phosphorylated stat1 and wnv protein was detected at 24 hpi, suggesting virus replication and innate immune signaling were occurring normally ( fig 4b) . to determine if activation was dependent on viral load, we infected hff with a moi = 1 and moi = 10 of wnv, but also observed no sting activation as measured by phosphorylation ( fig 4c) . these data suggest that sting is not canonically activated during wnv infection in hff cultures and reveals a potential noncanonical role for sting in host defense during infection with wnv. in order to determine if there was a systemic change in the innate immune profile in sting-/-mice, we examined the cytokine and chemokine profile in the serum of wt and sting-/-mice at the peak of peripheral viremia (4 dpi) and cns viral burden (8 dpi). we found that mock infected sting-/-mice had an increased basal production of multiple cytokines and chemokines at 4 dpi. we also observed significant increases in il33, il4, il6, il15, mcsf, gro-alpha, while at 4 dpi ip-10 (cxcl10) was decreased in sting-/-compared to wt mice (s2 fig). while these cytokines have multiple roles in immune modulation, a common role among them is in activation and recruitment of t cells. these data suggest that sting is required for regulation of immune cytokine and chemokines that program immune cell trafficking and actions during wnv, as has been shown for sting in cancer immunity and autoimmune signaling [53] . to determine if sting is required for proper programming of the t cell response during wnv infection, we examined splenic t cells from wt and sting-/-mice at 8 dpi, a time point when the adaptive immune response is established in wt mice [24] . we observed a reduction in the frequency of cd8+ t cells, along with a trend toward decreased numbers of t cells in the spleens of sting-/-mice compared to wt during wnv infection (fig 5b) . additionally, within the cd8+ t cell subset (fig 5c) , there was a significant decrease in frequency of activated (cd44+) and cxcr3+ t cells, and we observed a consistent trend of decrease in the frequency of wnv-specific cd8+ t cells in the spleens of sting-/-mice compared to wt, suggesting that sting is required for optimal anti-wnv cd8+ t cell responses. we also observed a significant increase in the frequency of cd4+ t cells in sting-/-mice (fig 5b) , with a corresponding trend toward increased absolute cell numbers. while we observed a trend toward differences in the absolute number of most cell populations examined between wt and sting-/-mice, we found that significant differences most typically occurred in cell frequencies, suggesting that the balance of t cells subsets may be skewed in the absence of sting. in particular, we found skewing within the t regulatory cell (foxp3+) populations ( fig 5e-5g) , with significant deficits in ki67+, cd44+ and cd73+ tregs, cd44 and cd73 + tregs. these data suggest that sting is required for modulating t cell responses and t cell frequencies during wnv infection that lead to a protective rather than pathogenic outcome. because of the heightened innate immune profile and aberrant programming of the t cell responses in spleens of sting-/-mice, we examined the cns-specific t cell profile across mouse lines. histological analyses revealed trends toward increases in cns immune cellularity, both in the form of perivascular and parenchymal mononuclear infiltrate, suggesting the cns pathology may be immune-mediated (fig 6a) . we then performed a cd3 ihc stain in the brains of survivors, we found increased clusters of cd3 infiltrate in the hind and midbrain regions (fig 6b) co-localized with robust lesions. in serial slices of the same tissues, we did not observe wnv staining by ihc in sting-/-survivors (fig 2) , however we did observe continued gliosis, suggesting that a potential immunopathology may occur in the brain of sting-/-mice infected with wnv. previous studies indicated that cellular infiltrate in the brain is predominantly comprised of cd3+ t cells during wnv infection [72] . therefore, we characterized t cell responses of wt and sting-/-mice in the cns on 4 dpi to examine baseline differences at 8 dpi when wnv and leukocytes are both present in the cns (fig 6j) . lymphocyte and t cell responses in both mock and wnv-infected mice were comparable at 4 dpi, indicating that there was no gross difference in the cns between wt and sting-/-mice ( fig 6c and 6d ). by 8 dpi however, we found statistically significant decreases in the frequency and numbers of cd4+ t cells in sting -/-mice ( fig 6f) . although there was no difference in the total numbers of cd8+ t cells, there was a statistically significant increase in the frequency of cd8+ t cells in the cns of sting-/-mice, likely due to overall trend of decreased numbers of lymphocytes in the brain (fig 6c-6e ). by 8 dpi, these changes resulted in a significantly decreased cd4/cd8 ratio of t cells, indicating an imbalanced t cell response to wnv in the cns of sting-/-mice ( fig 6i) . of cells that made it to the brain by 8 dpi, no differences were found in the absolute number of activated (cd44+) or wnv-specific (ns4b tetramer+) cd8 + t cells (fig 6g and 6h ), foxp3+cd25+cd4+ t cells (fig 6k) in the brain. these data suggest that sting is not essential for recruitment of wnv-specific cytotoxic t cells in the cns, however it may be required for balancing the cytotoxic vs immunosuppressive adaptive response. furthermore, it is also possible that the enhanced recruitment of cells to the cns is in response to damage caused by the virus, aberrant immune signaling, or both. this outcome would suggest that sting plays an essential role in modulating the balance between immunopathogenic and immunoprotective response in the cns during wnv infection. the increase in clinical disease and pathological damage observed in the sting-/-versus wt mice, particularly in survivors, could be due to an aberrant immune response resulting in cns damage after initial viral insult. we found that cns pathology in wt mice is largely restricted to the cortex and meninges, while sting-/-mice display increased pathology in the cerebellum and hind/mid brain regions in addition to the cortex and meninges (fig 7a and 7c) . these data correlate with the increased cd3 staining observed by ihc in sting-/-mice (fig 6b) , also noted as the same brain regions where wnv is often detected by ihc (fig 2b) . these observations suggest that sting plays a role in directing or maintaining the t cell response to specific loci within the cns or that initial viral infection led to increased sting is required for host defense against neuropathological west nile virus infection recruitment of a localized adaptive immune response that resulted in immunopathology. furthermore, pathology in the spine was more diffuse, suggesting that sting has a widespread protective role in the cns during wnv infection (fig 7b and 7d) . these observations led us to investigate if there was a localized polarization of microglia or infiltrating macrophages in cns regions toward an m1 or m2 phenotype (fig 7e) . microglia have the highest levels of sting (tmem173) expression observed in any cell within the adult mouse [73, 74] and it is possible that in the absence of sting, microglia are aberrantly polarized, enhancing immunemediated pathology. to examine this possibility, we assessed the expression of m1 (cxc1 and il6) and m2 (pparg, arg1, chil3 and retnla 1) associated genes by rt-qpcr in different regions of the cns. in wt mice, we found that cxcl1 (marking an m1 phenotype) was present in the brain stem by day 8 post infection, and retnla 1 expression (marking an m2 phenotype) occurred in both the mock and 4 dpi tissues within the brain stem and sub-cortex (containing the thalamus) regions of the brain (fig 7e) . this profile suggests that cns homeostasis includes a localized m2 phenotype that is induced to a m1 phenotype in wt mice following wnv infection. in sting-/-mice however, we found a widespread increase in the m1 response gene expression (marked by cxcl1 and il6) with the highest expression observed in the brain stem and spinal cord. simultaneously, there was also a corresponding increase in pparg and chil3 (marking the m2 phenotype), with no clear difference in arg1 expression and an overall trend toward decreased expression of retnla. these observations reveal a widespread increase in both m1 associated genes, with altered regulation of the m2 associated genes in sting-/-mice, potentially resulting in aberrant balance of the m1 and m2 polarization in the cns. to determine where in the cns sting is actually localized and if this tissue localization overlaps with the location of the cellular infiltrate noted histopathologically or with expression of innate immune genes, we utilized the allen brain institute database to search for sting (tmem173) localization in the mouse brain [75] . within the brain, sting expression is found within the olfactory bulb, thalamus/midbrain, brainstem and cerebellum, as well as low levels throughout the cortex, overlapping areas that are affected most severely by wnv infection (s3) [14, 75] . these regions of brain affected correlate with the clinical signs we observed including loss of balance, tremors, and loss of motor function (figs 1e and 7c-7e). furthermore, these areas of sting expression overlap with the brain regions where altered regulation of m1 or m2 gene expression were most readily observed, implicating a role for sting in polarization of either or both microglia and macrophages in the cns. cumulatively, these data suggest that sting has an essential role in maintaining immune response homeostasis and immune programming in initial defense against wnv infection. without sting, immunopathology occurs, leading to exacerbated cns disease and clinical sequelae. recent years have seen a marked increase in the global health threat presented by emerging and re-emerging encephalitic viruses, particularly those with increased neurotropism and neuropathology such as wnv [1, 3, 10, 76, 77] . previous studies indicated an important role for sting in host survival during wnv infection [46] , however it is unclear what role sting plays in conferring host defense against rna viruses [52, 54] . here, we demonstrate that sting is essential to prevent host morbidity and mortality during wnv infection where it plays a role in immune homeostasis and programming. however, sting is not canonically activated in vitro upon infection with wnv, revealing a novel function for sting during infection with rna viruses. furthermore, we show that sting is essential for host neuropathological defense against wnv through regulation of the innate-adaptive immune interface in vivo. we found that sting deficient mice exhibit increased mortality and morbidity including increased and sustained neurological clinical signs, particularly in mice that survive infection (fig 1) . these data were corroborated by pathological analysis, which also revealed distinct differences in cns pathology. intriguingly, there seems to be a stratification in clinical and pathological findings between the sting-/-mice that meet euthanasia criteria and those that go on to survive. survivorship bias has been previously reported in the wnv model, with these data further implicating this bias as a critical factor to consider when performing time course vs. end-point experiments [78] . unexpectedly, these studies also revealed that there was minimal cns pathology in wt mice that met euthanasia criteria. it is typically assumed that mice meeting euthanasia criteria do so because of neuroinvasion and subsequent encephalitis. our data instead indicates that both wt and sting-/-terminal mice have severe gross gi abnormalities, with corroborating abnormalities by histopathology, which may be the proximate cause of morbundity and meeting euthanasia criteria (s1). gi complications during wnv have been previously described, however further study is necessary to understand the implications of gi pathology on wnv induced morbidity and mortality [15] [16] [17] 79] . recently it has been shown that during wnv infection causes delayed gi transit, dependent on infiltrating antiviral cd8+ t cells [80] . furthermore, both in this model and in a lung model where sting exhibits a gain-of-function mutation, t cell-dependent chronic tissue damage occurs, supporting our findings that sting may play a broad and significant role in communicating between the innate and adaptive immune responses [80, 81] . together, these data demonstrate an essential neuroprotective role for sting during wnv infection, potentially through a cellular mediated mechanism instead of the canonical interferon antiviral function typically attributed to sting. wnv typically is cleared through development of an innate immune response and effective t cell immunity [19] . to prevent progression to neuroinvasion, both the innate and adaptive immune response are critical to control wnv viremia and prevent viral induced pathology [19-21, 24, 82, 83] . because the known function of sting is to initiate a type i ifn response to both pamps and damps, we anticipated that the type i ifn response would be diminished both in vivo and in vitro explaining the increased viral loads. surprisingly, we actually observed an increased inflammatory and antiviral innate immune response in sting-/-mice in the cns during wnv infection. this same increase in the cytokine-chemokine response was also observed in bmdm (fig 3) and in serum of infected mice (fig 5) . these outcomes were highly unexpected as the most commonly described role for sting is known as initiating a type i ifn response [46-48, 53, 54] . in particular, sting was shown previously to facilitate the actions of the elf4 transcription factor to promote type i ifn expression from wnv-infected cells wherein loss of sting associated with reduced ifn and isg expression (49) . while we observed significant increases in ifn and isg expression in bmdm lacking sting, it is likely that sting imparts cell type-specific actions for regulation of innate immune signaling, similar to other pathogen recognition receptors that govern innate immune signaling against wnv, likely explaining this discrepancy between studies [19] . it is also important to note that our studies employed sting-/-mice produced through classical gene targeting approach [48] while the previous study used sting gt/gt mutant mice produced from n-ethyl-n-nitrosourea mutagenesis and encoding a t596a point mutation of sting [84] , highlighting that genetic differences between mouse lines might impact findings. importantly, both mouse lines exhibit increased susceptibility to lethal wnv infection, and together reveal expanded roles for sting in immune regulation during wnv infection. our data also suggest that sting has a role in controlling wnv replication and tropism, as we found increased viral loads in bmdm, as well as a trend toward increased viral load in the cns, particularly in the hindbrain regions, but not in the spleens of infected mice lacking sting (figs 2 and 3) . the trend toward increased virus in the cns of sting-/-mice could either suggest increased susceptibility of the virus in the cns, delayed clearance of the virus after entering the cns, or possibly a combination of the two. variation observed within strains could be the result of harvesting mice at set time points instead of following them until a determination if they would survive or meet euthanasia criteria, highlighting the potential import of survivorship bias within this model. it does not appear that the requirement for sting in viral control is restricted to neurons or the cns, as no difference was observed in the viral load of sting-/-primary cortical neurons or intracranial infection (fig 2) . this outcome suggests that while there is a peripheral requirement for sting in conferring cns protection, it is not due to complete inhibition of viral control in the periphery. intriguingly, base-line expression of type i ifn and isgs were significantly reduced in sting-/-bmdm compared to wt, but not other inflammatory genes (fig 3) . it is possible that this reduction in baseline ifn allows wnv to establish an earlier and more robust infection, that is later controlled by the rig-i dependent antiviral response [23, 34] . however, we favor that sting plays a role in innate immune homeostasis, as in its absence the control of the inflammatory response is lost (figs 4 and 7) , thus leading to immune-mediated pathology. this function for sting may explain why we had a trend but not significant increase in viral load in the cns; it is possible that virus is able to establish a stronger infection in the cns earlier on but is cleared through an exacerbated innate inflammatory and antiviral response in the absence of sting. alternatively, it is possible that in the absence of sting clearance of the virus takes longer due to an ineffective immune response. following either of these events subsequent t cell recruitment is likely, but in a manner that leads to enhanced immunopathology and lack of recovery from clinical illness. in addition to its role in mounting a type i ifn response to pamps and damps, recent studies demonstrated an essential role for sting in developing antitumor t cell responses [53] . these studies suggested that dead and dying cells are phagocytosed by dendritic cells, which requires sting to present antigen and produce a type i ifn signaling cascade that informs and develops the adaptive immune response. this outcome could also implicate a requirement for sting in microglial-dependent phagocytosis of dead and dying cells, with subsequent sting-dependent polarization and release of soluble factors that effectively recruit and maintain a protective cellular response in the cns. upon examining the cns of infected mice, particularly in sting-/-with ongoing signs, we observed increases in mononuclear cellular infiltrate, implicating possible immunopathology. previous studies have shown that there is an essential requirement for both cd8+ and cd4+foxp3+ (treg) t cells to control wnv and prevent immunopathology [42, 43, 72] . cd8+ t cells in particular are essential for wnv clearance, however without an adequate treg response or appropriate balance of cd4+ and cd8+ t cells an uncontrolled cytotoxic t cell response could result in immune mediated pathology. examining the programming of the adaptive immune response in spleens (fig 5) we found that expression of ki67, cd44 and cd73 in splenic foxp3+cd4+ tregs were impaired, implicating a role for sting in the proliferation, activation and suppressive potential of tregs. upon examining the brains of mice at baseline (4 dpi) and following infection (8 dpi), we observed no differences at baseline between wt and sting-/-mice, however the total cd4+ t cells and the cd4/cd8 ratio was significantly decreased in sting-/-mice, suggesting that there is a defective recruitment or maintenance of t cells in the brain (fig 6) . these data in combination with enhanced cns pathology suggest that the cytotoxic effect of cd8+ t cells may not be controlled adequately in the absence of sting. it is also possible that increases in cellular response within the cns recruit an enhanced protective cellular response as a result of viral damage or aberrant immune signaling. consistent with this either of these options, we found that in sting-/-survivors there were large clusters of cd3+ cells (fig 6) as well as other cellular infiltrate (fig 7) in the same vicinity as we observed increased pathology and where sting is localized in the brain (fig 7) . recently, a noncanonical sting-dependent signaling pathway was described where multiple cell types initiated an innate immune response following il1b release in response to mitochondrial dna release in the cytoplasm [70] . furthermore, this sting-induced response to il-1b was essential for the control of dengue virus infection, a flavivirus related to wnv [70] and that this response is linked with protection against wnv neurovirulence in vivo [70, 83] . thus, it is intriguing to speculate that noncanonical sting activation in response to proinflammatory cytokine signaling serves to direct immune programming that protects against viral neuroinvasion and cns pathology during wnv infection. in summary, our study reveals that that sting is required for immune response programming to restrict wnv infection and neuropathogenesis. all animal experiments were approved by the university of washington institutional animal care and use committee (iacuc) guidelines as per protocol #4158-05 and follow the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. invasive infections and manipulations were performed under anesthesia and every effort was made to limit suffering. c57bl/6j (wt) and tmem173-/-(sting-/-) mice were genotyped and bred under specific pathogen-free conditions in the animal facility at the university of washington. sting-/mice were gifted by the stetson lab, who generated them as previously described [67] followed by speed congenics to bring them to a 99.4% c57bl/6j background. additional c57bl/6j (wt) mice were purchased from jackson laboratories, bar harbor, me. both male and female mice, ages 8-11 weeks were represented in both the control and infected groups. mice for primary cortical neurons (wt and sting-/-) were set up as timed breeders and embryos were harvested. mice were monitored daily and assigned a clinical score to describe overall well-being and signs of hind-limb dysfunction (paresis). clinical scores (cs) of (0) without clinical signs, or (1-6) dependent on severity of clinical signs presented. cs = 1: ruffled fur, lethargic; no paresis; cs = 2: very mild to mild paresis (in 1 or more hind limbs with minimal gait disturbance or limb-dysfunction); cs = 3: frank paresis involving at least one hind limb and/or eye conjunctivitis; cs = 4: severe paresis and/or paresis in both hind-limbs; cs = 5: true paralysis; cs = 6: moribund. additionally, mice were observed daily for the presence or absence of various specific signs. each mouse was scored as either exhibiting the clinical sign (yes = 1) or not, (no = 0). each sign was monitored through the duration of the experiment and the results were graphed as the average daily score/mouse. results of clinical signs monitored represent the entire population until they reached euthanasia criteria, at which point the remaining mice continued to be scored until day 18 post infection or study end point. clinical signs monitored daily include: lethargy (l), ruffled fur/decreased of grooming, hunched, paresis/ paralysis (any degree of severity), tremors, abdominal (ab) distension/gi distress, loss of balance, increased reflex/tone in limbs (fore and/or hind) and tail, decreased reflex/tone in limbs and tail. the clinical scoring system incorporated signs based off of predicted involvement of different anatomical regions within the cns and was created using modifications of various previously described scoring systems for experimental autoimmune encephalomyelitis [86] [87] [88] [89] . similar neuroanatomic regions were examined pathologically in an attempt to correlate clinical and neurological phenotype of disease. subcutaneously-infected mice were monitored for 18 days post infection (dpi). euthanasia criteria was determined as a clinical score � 5 for 2 or more consecutive days, or 20% loss in body weight. a clinical score of 6 (moribund) or respiratory distress resulted in immediate euthanasia. mice meeting euthanasia criteria were identified as terminal (t) and were euthanized by co 2 asphyxiation followed by cervical dislocation. mice who did not meet euthanasia criteria were monitored until end point (18 dpi) were identified as survivors (s). all remaining s mice were euthanized at the end of study (18 dpi) as described above. mice used for morbidity and mortality analysis were necropsied when meeting euthanasia (t) criteria, or study end (s). after euthanasia by co 2 , a complete necropsy was performed and tissues were collected and immersion fixed in 10% neutral buffered formalin [90] . the head was removed and skull cap lifted, leaving the brain within the skull cavity during fixation. the spine was fixed in situ in order to preserve the mesenteric ganglia. histological preparation hematoxylin and eosin (h&e) and immunohistochemical (ihc) staining was performed by the uw histology and imaging core (hic) and the vanderbilt university medical center translational pathology shared resource (tpsr). primary pathological analysis was performed on the cns (brain and spine) and gastrointestinal (gi) tract by a board-certified veterinary pathologist (pmt) (supplemental methods table 1 ). in the brain, the following changes were scored on a subjective 0-4 scale of increasing severity: perivascular inflammation, parenchymal inflammation, hemorrhage, neuronal necrosis, and meningitis. in the spinal cord the presence (1) or absence (0) of mononuclear inflammation was documented from 5 different sections of the spine (c1-c5, c6-t2, t3-l3, l4-s2, s3) for a maximum score of 5 per mouse. for the enteric nervous system (ens), the degree of mononuclear cells present in the myenteric ganglia, extent to the changes and any secondary gi lesions such as dilation or mucosal change were scores on a on a subjective 0-4 scale of increasing severity. ihc staining of wnv (vrl w1015) and cd3+ t cells (mca1477 abd serotec) were performed by the uw histology core. verowho (european collection of authenticated cell cultures; ecacc) cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with 10% fbs, 1mm sodium pyruvate, 2mm l-glutamine, antibiotic/antimycotic solution and non-essential amino acids (complete dmem; cdmem) and split using 0.25% trypsin following pbs wash. hff cells were kindly gifted from stetson lab and were grown in cdmem. cells were split using 0.05% trypsin following pbs wash. bone marrow was collected from sting-/-and wt mice and frozen in 10% dmso/90% fbs. to generate bone marrow derived macrophages (bmdm), bone marrow stocks were thawed, washed and resuspended in cdmem containing [50 μm] bme and [40ng/ml] murine mcsf (mmcsf). cells were cultured for 7 days in non-tc coated plates, then scraped, washed with pbs and seeded at 1e6 cells/well in 12-well tc coated plates with cdmem+bme+mmcsf. cells were infected or transfected the next day. wnv-tx biological isolates (2002) were utilized for in vivo work, while wnv-tx ic (infectious clone) stocks were utilized for cell culture (in vitro) studies. working stocks were propagated in vero-e6 (american type culture collection; atcc) and titered by standard plaque assay on verowho and bhk21 (american type culture collection; atcc) cells as previously described [24] . single-use aliquots from the same viral stock lot were prepared and utilized for all experiments described here. age and sex-matched 8-11 week old mice were anesthetized by isofluorane and inoculated subcutaneously (s.c.) in the right rear footpad with 100 pfu wnv-tx 2002 (wnv-tx) diluted in 40 ul pbs, administered via 1ml insulin needle. mice were monitored daily for clinical score and loss of body weight. euthanasia criteria was determined as a clinical score � 5 for 2 or more consecutive days, or 20% loss in body weight. a clinical score of 6 (moribund) or significant respiratory distress resulted in immediate euthanasia. mice were anesthetized with ketamine/xylazine, the top of the head was cleaned with etoh, and the mouse was then restrained manually on a solid surface. the site of injection was approximately halfway between the eye and ear, and just off the midline, in the medial posterior region of the top of the skull. the injection was done with a 29g needle using a hamilton syringe into the cerebral cortex. following infection, mice were monitored for revival from anesthesia and monitored daily for clinical score and loss of body weight. euthanasia criteria was determined as a clinical score � 5 for 2 or more consecutive days, or 20% loss in body weight. a clinical score of 6 (moribund) or significant respiratory distress resulted in immediate euthanasia. to determine the viral load from in vivo tissue samples, mice were terminally anesthetized using ketamine/xylazine mixture followed by cardiac perfusion with 30-40 ml pbs. kidney(s) and spleen were collected whole; brains were harvested and macrodissected into four anatomical regions, including the cerebellum, cortex, sub-cortex, and brainstem [91] ; spinal cords were collected by perfusion with pbs. tissues were harvested into 1 ml pbs on ice in percelly's tubes with ceramic beads. following harvest, tissues were homogenized (percellys 24) 5500/1x 20s/5 min and centrifuged at 4˚c/5 min/10k rpm. supernatant was collected and analyzed by plaque assay on vero-who cells (0.5% agarose overlay, 3% neutral red counter stain after five days post inoculation; plaques counted 10-15h post staining). cells were inoculated with wnv in serum-free media and the inoculum left for 1hr rocking at 37˚c. inoculum was removed, cells washed 1x and media replaced with cdmem. at the indicated time-points, supernatant was collected for virologic and cytokine analysis; cells were treated with ripa buffer for wb analysis (or) with rlt for total cellular rna isolation. primary cerebral cortical neuron cultures were generated from e15 wt and sting-/embryos as previously described [92] and maintained in serum free neurobasal-a medium (life technologies 21103-049) with b27 supplement (gibco 17504-044). neuron cultures were used for virologic experiments after 7 days in vitro. cortical neuron cultures were infected at moi 0.001 with wnv-tx [32] . multistep growth curve experiments were performed as described [93] and quantified via plaque assay using bhk21-15 cells. mice were euthanized in an isoflurane chamber followed by cardiac perfusion with 30-40 ml pbs. tissues were harvested; right kidney and spleen were collected whole; brains were harvested and macrodissected into four anatomical regions, including the cerebellum, cortex, sub-cortex, and brainstem [91] ; spinal cords were collected via pbs perfusion. tissues were harvested into 1 ml rnalater and stored at 4˚c for a minimum of 1 week to stabilize the rna. tissues were removed from rnalater solution and transferred to 1 ml trireagent in percelly's tubes with ceramic beads at rt. following harvest, tissues were homogenized in a percelly's homogenizer (5500/1x20s/5 min) followed by centrifugation (4˚c/10k rpm/5min). rna isolated with the ribopure kit from trireagent using per manufacturer's instructions. cdna was generated from 350 ng rna using iscript kits with random primers per manufacturer's instructions. cellular and viral gene analysis was assessed by sybr green rt-qpcr using an abi viia7 and analyzed as the linear fold change (2^-dct) over a housekeeping gene (gapdh) from wt mock infected sample or mouse (table 2 ). cells were harvested in rlt and total cellular rna isolated for rt-qpcr analysis using qishredders and the quiagen rneasy kit per the manufacturer instructions. cdna was generated from 100 ng total rna using the iscript kit per manufacturer instructions using their provided oligo(dt) and random primers. cellular and viral genes were analyzed by sybr green rt-qpcr using an abi viia7. primers for bmdm experiments described above. protein extracts from cells were prepared in ripa buffer. 7-15 ng protein lysate was analyzed by 4-20% gradient sds-polyacrylamide gel electrophoresis by immunoblotting, using 5% bsa blocking buffer and nitrocellulose membranes. the following antibodies were utilized: wnv ns3 (r&d baf2907), actin (c4; emd mab1501), stat1 (cst 9172p), sting (cst d2pzf), pstat1 (y701; cst 58d6), psting (cst d7c3s). 8e4 (or) 8x10^4 hff cells were seeded onto glass coverslips in a 24-well plate. the following day, cells were infected with wnv at moi = 1 or transfected with calf-thymus dna (ctdna; isd) (thermo fisher, waltham, ma, usa) at 3ug/ml final concentration using lipofectamine 3000 and following the manufacturer's protocol. 24h after wnv infection or 3h after ctdna transfection cells were fixed with 4% paraformaldehyde for 15min at room temperature (rt). cells were permeabilized with 0.1% triton x-100 for 5min at rt. after blocking the cells for 30min with 3% bsa in pbs, immunofluorescent staining was performed overnight at 4˚c with the following primary antibodies: rabbit-anti-sting (1:100, gifted by glen barber), mouse-anti-dsrna (j2, 1:800, scicons, budapest, hungary). nuclei were counterstained with 4',6-diamidino-2-phenylindole, dihydrochloride (dapi, thermo fisher). fluorophore coupled secondary antibodies (thermo fisher) were applied for 1h at rt. after washing with pbs samples were mounted onto glass slides using prolong gold (thermo fisher). images were acquired with a nikon eclipse ti confocal microscope equipped with a 60x oil immersion objective using the nikon confocal software. insets were captured with 4x enlargement of 600x images. images were merged and processed using the nikon confocal analysis software (nikon, melville, ny, usa). mice were euthanized by isoflurane and perfused with 30-40ml pbs to ensure systemic removal of blood and residual intravascular leukocytes. spleens were homogenized and single cell suspensions were treated with ack lysis buffer to clear any remaining red blood cells, washed and resuspended in facs buffer (1x pbs, 0.5% fbs). cells were plated at 1e6 cells/ well and stained for surface markers 15 minutes on ice. cells were then fixed, permeabilized (foxp3 fixation/permeabilization concentrate and diluent, ebioscience) and stained intracellularly with antibodies for 30 minutes on ice. flow cytometry was performed on a bd lsrii machine using bd facsdiva software. analysis was performed using flowjo software. the following directly conjugated antibodies were used: b515-foxp3, b710-cxcr3, g575-ki67, g610-cta-4, g666-cd127, g780-klrg1, r660-ns4b tet, r710-cd45, r780-cd44, uv395-cd8, uv730-cd3, v450-cd73, v610-cd4, v655-cd25, v510-live/dead. cells were counted by hemocytometer using trypan blue exclusion. brains were harvested into rpmi and mechanically suspended using a 70um strainer. each brain suspension was added to hypertonic percoll to create a 30% percoll solution, vortexed then centrifuged at 1250 rpm for 30 minutes at 4˚c. following centrifugation, the supernatant was aspirated and cell pellet treated with ack lysis buffer to remove any residual red blood cells. cells were then washed and filtered through a 70um nylon mesh to remove residual debris and resuspended in facs buffer. cells were counted using beads during facs analysis. cells were plated at 1e6 cells/ well and stained for surface markers 15 minutes on ice. cells were then fixed and extracellularly stained with antibodies for 30 minutes on ice. flow cytometry was performed on a bd lsrii machine using bd facsdiva software. analysis was performed using flowjo software. the following directly conjugated antibodies were used for fig 7cb: gross pathology scores of the gi tract from necropsied mice. mice were visually examined at necropsy and scored. scores were assigned to each mouse ranging from 0 (normal gi tract) to 3 (grossly distended or aberrant morphology). n = 3-9 per condition; students t-test (unpaired). p = 0.05 � . c: pathological analysis was performed on randomly selected representative mice. sections of the gi were scored including sections from: 1) the duodenum and upper jejunum; 2) jejunum; 3) ileum; 4) cecum; 5) colon; 6) stomach. graphed as the mean sum of all scores. n = 1-2 per condition. d: representative hematoxylin and eosin-stained small intestinal sections. mock tissues were unremarkable with readily detectable myenteric ganglia (black ovals) and normal scant intestinal contents (inserts). survivor mice have mild enteritis and minimal changes in the myenteric ganglia (ovals) with normal intestinal contents. in contrast, terminal mice have myenteric ganglia with neuritis, degeneration and neuronal loss. there is bacterial overgrowth and exudative material within the intestinal contents (inserts at lower right of each image). in the sting-/mice, there is readily observable vacuolation of the inner tunica muscularis (arrow), dilation of lacteals (asterisk) and intramucosal hemorrhage and lymphocytic and proliferative enteritis. figure 7) : sting localization in the mouse brain. sting localization in the brain is centralized to the hind/mid-brain, hippocampus, primary motor-cortex and olfactory bulb in the brain. square: midbrain/thalamus region. oval: hindbrain (cerebellum and brain-stem). image is from the allen institute for brain science. emerging and reemerging neurologic infections emerging viral infections of the nervous system west nile virus: review of the literature epidemiology of neuroinvasive arboviral disease in the united states division of vector-borne diseases ncfe, zoonotic infectious diseases cdc. west nile virus and other arboviral diseases-united states estimated cumulative incidence of west nile virus infection in us adults neurologic manifestations and outcome of west nile virus infection lessons from the west nile viral encephalitis outbreak in new york city, 1999: implications for bioterrorism preparedness the outbreak of west nile virus infection in the new york city area in 1999 emergence and re-emergence of viral diseases of the central nervous system neurocognitive and functional outcomes in persons recovering from west nile virus illness neuroinvasive disease and west nile virus infection west nile fever characteristics among viremic persons identified through blood donor screening immune surveillance of the cns following infection and injury gastrointestinal manifestations of acute west nile virus infection in humans systemic distribution of west nile virus infection: postmortem immunohistochemical study of six cases clinical characteristics and functional outcomes of west nile fever clinical spectrum of muscle weakness in human west nile virus infection west nile virus infection and immunity a mouse model of west nile virus infection a mouse model of chronic west nile virus disease evaluation of a mouse model for the west nile virus group for the purpose of determining viral pathotypes the essential, nonredundant roles of rig-i and mda5 in detecting and controlling west nile virus infection ips-1 is essential for the control of west nile virus infection and immunity interferon-lambda restricts west nile virus neuroinvasion by tightening the blood-brain barrier cell-specific irf-3 responses protect against west nile virus infection by interferon-dependent and -independent mechanisms immune responses to west nile virus infection in the central nervous system the innate immune playbook for restricting west nile virus infection a systems biology approach reveals that tissue tropism to west nile virus is regulated by antiviral genes and innate immune cellular processes nile virus evades activation of interferon regulatory factor 3 through rig-i-dependent and -independent pathways without antagonizing host defense signaling establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda5 signaling through ips-1 resistance to alpha/beta interferon is a determinant of west nile virus replication fitness and virulence alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival distinct rig-i and mda5 signaling by rna viruses in innate immunity irf-3, irf-5, and irf-7 coordinately regulate the type i ifn response in myeloid dendritic cells downstream of mavs signaling pattern recognition receptor mda5 modulates cd8+ t cell-dependent clearance of west nile virus from the central nervous system the rig-i-like receptor lgp2 controls cd8(+) t cell survival and fitness deficient ifn signaling by myeloid cells leads to mavs-dependent virus-induced sepsis a temporal role of type i interferon signaling in cd8+ t cell maturation during acute west nile virus infection role of cd8+ t cells in control of west nile virus infection cd4+ t-cell responses are required for clearance of west nile virus from the central nervous system cd8+ t cells mediate recovery and immunopathology in west nile virus encephalitis tregs control the development of symptomatic west nile virus infection in humans and mice pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity panviral specificity of ifn-induced genes reveals new roles for cgas in innate immunity elf4 is critical for induction of type i interferon and the host antiviral response sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity recognition of cytosolic dna by cgas and other sting-dependent sensors sting and the innate immune response to nucleic acids in the cytosol the cgas-sting defense pathway and its counteraction by viruses message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection sting: infection, inflammation and cancer cgas and sting: at the intersection of dna and rna virus-sensing networks virus-cell fusion as a trigger of innate immunity dependent on the adaptor sting influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses denv inhibits type i ifn production in infected cells by cleaving human sting sting mediates neuronal innate immune response following japanese encephalitis virus infection zika virus infection activates sting-dependent antiviral autophagy in the drosophila brain circulating nucleic acid analysis: diagnostic applications for acute pathologies activation and regulation of cellular inflammasomes: gaps in our knowledge for central nervous system injury the concentration of cell-free dna in focal epilepsy the value of serial plasma nuclear and mitochondrial dna levels in patients with acute ischemic stroke systemic challenge with the tlr3 agonist poly i:c induces amplified ifnalpha/beta and il-1beta responses in the diseased brain and exacerbates chronic neurodegeneration sting-mediated type-i interferons contribute to the neuroinflammatory process and detrimental effects following traumatic brain injury self-dna, sting-dependent signaling and the origins of autoinflammatory disease autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna interleukin-1β induces mitochondrial dna release to activate innate immune signaling via cgas-sting phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation the neuropathology of west nile virus meningoencephalitis. a report of two cases and review of the literature tmem173 (sting) cellular expression single-cell transcriptomics of 20 mouse organs creates a tabula muris allen mouse brain atlas: allen brain institute zika virus as an emerging global pathogen: neurological complications of zika virus epidemiology and neurological complications of infection by the zika virus: a new emerging neurotropic virus end-point disease investigation for virus strains of intermediate virulence as illustrated by flavivirus infections oral antibiotic treatment of mice exacerbates the disease severity of multiple flavivirus infections sting-associated lung disease in mice relies on t cells but not type i interferon rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity il-1beta signaling promotes cns-intrinsic immune control of west nile virus infection the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides dopamine: a parallel pathway for the modulation of spinal locomotor networks a neuropathological analysis of experimental autoimmune encephalomyelitis with predominant brain stem and cerebellar involvement and differences between active and passive induction b cells promote induction of experimental autoimmune encephalomyelitis by facilitating reactivation of t cells in the central nervous system the more the merrier? scoring, statistics and animal welfare in experimental autoimmune encephalomyelitis experimental autoimmune encephalomyelitis in the mouse mouse necropsy ripk3 restricts viral pathogenesis via cell death-independent neuroinflammation neuronal cxcl10 directs cd8+ t-cell recruitment and control of west nile virus encephalitis propagation, quantification, detection, and storage of west nile virus we thank daniel stetson, lauren aarreberg, sunil thomas, aimee sekine and megan de la riva (university of washington) for critical discussion, technical assistance and reagents. we thank glen barber (university of miami) for providing sting antibody for immunostaining. key: cord-003738-el0wyu74 authors: zhang, qingxiu; zhu, wen; xu, fei; dai, xuejiao; shi, ligen; cai, wei; mu, hongfeng; hitchens, t. kevin; foley, lesley m.; liu, xiangrong; yu, fang; chen, jie; shi, yejie; leak, rehana k.; gao, yanqin; chen, jun; hu, xiaoming title: the interleukin-4/pparγ signaling axis promotes oligodendrocyte differentiation and remyelination after brain injury date: 2019-06-21 journal: plos biol doi: 10.1371/journal.pbio.3000330 sha: doc_id: 3738 cord_uid: el0wyu74 the repair of white matter damage is of paramount importance for functional recovery after brain injuries. here, we report that interleukin-4 (il-4) promotes oligodendrocyte regeneration and remyelination. il-4 receptor expression was detected in a variety of glial cells after ischemic brain injury, including oligodendrocyte lineage cells. il-4 deficiency in knockout mice resulted in greater deterioration of white matter over 14 d after stroke. consistent with these findings, intranasal delivery of il-4 nanoparticles after stroke improved white matter integrity and attenuated long-term sensorimotor and cognitive deficits in wild-type mice, as revealed by histological immunostaining, electron microscopy, diffusion tensor imaging, and electrophysiology. the selective effect of il-4 on remyelination was verified in an ex vivo organotypic model of demyelination. by leveraging primary oligodendrocyte progenitor cells (opcs), microglia-depleted mice, and conditional opc-specific peroxisome proliferator-activated receptor gamma (pparγ) knockout mice, we discovered a direct salutary effect of il-4 on oligodendrocyte differentiation that was mediated by the pparγ axis. our findings reveal a new regenerative role of il-4 in the central nervous system (cns), which lies beyond its known immunoregulatory functions on microglia/macrophages or peripheral lymphocytes. therefore, intranasal il-4 delivery may represent a novel therapeutic strategy to improve white matter integrity in stroke and other brain injuries. introduction matter components. however, the potential effects of il-4 on white matter integrity after ischemic stroke remain elusive. in the current study, we discovered that il-4 is essential for white matter repair after stroke. intranasal delivery of il-4 nanoparticles after stroke robustly promoted oligodendrogenesis, enhanced white matter integrity, and improved long-term functional recovery. complementary studies in cell cultures and animals revealed that il-4-mediated oligodendrogenesis was achievable even in the absence of microglia/macrophages in vitro or after microglia/macrophage significant depletion in vivo. furthermore, il-4 directly promoted opc differentiation into mature oligodendrocytes in a peroxisome proliferator-activated receptor gamma (pparγ)-dependent manner. these collective findings suggest that activation of the il-4/ pparγ signaling cascade may represent a novel recovery-enhancing strategy to promote longterm outcomes after ischemic stroke. to explore the potential cellular targets for il-4, we used flow cytometry to assess expression of il-4rα on various types of cells in the ischemic brain. consistent with previous reports [15, 21] , il-4rα was expressed on cd45 − cd11b − gfap + astrocytes, cd45 − cd11b − neun + neurons, cd45 intermediate cd11b + microglia, cd45 high cd11b + macrophages, and other infiltrated immune cells (cd45 + cd11b − ) 3 d after 60-min mcao (fig 1a and 1b) . notably, il-4rα was also expressed in oligodendrocyte lineage cells (fig 1a-1d ). different markers, including a 2 b 5 , o4, and o1/galactocerebroside (galc), were used to identify oligodendrocytes at different differentiation stages. il-4rα was detected in cd45 − cd11b − o4 + cells (fig 1a-1d ), which were identified as preoligodendrocytes and premyelinating oligodendrocytes, and cd45 − cd11b − o1 + cells (fig 1a and 1b) , which were identified as premyelinating oligodendrocytes or mature oligodendrocytes [22] . the percentages of il-4rα + o4 + cells were higher in the ipsilateral ischemic hemisphere (fig 1c and 1d) , suggesting a potential direct effect of il-4 on white matter cells. imagestream analyses confirmed that il-4rα was expressed on both a 2 b 5 + opcs and o4 + preoligodendrocytes/premyelinating oligodendrocytes, as well as cd11b + microglia/macrophages in the ischemic hemisphere ( fig 1e and s1a-s1c fig) . in light of these results, we evaluated whether il-4 influenced white matter integrity after ischemic stroke. wt and il-4 ko mice were subjected to transient focal ischemia induced by 60-min mcao. dual staining for myelin basic protein (mbp, a major myelin protein) and smi32 (a marker of demyelinated axons) was performed to assess the lesion in white matter (fig 1f-1h ). the overall immunofluorescence intensity of mbp staining decreased in the infarct border along three white matter-rich brain regions (the external capsule [ec] , the cortex, and the striatum) (see "total" values in panel of the right of fig 1f) at 14 d after mcao, indicating a significant loss of myelin protein after stroke. additionally, the overall immunofluorescence of smi32 increased in these three regions, resulting in a significant increase in the smi32/mbp ratio in wt mice after stroke (fig 1g) . il-4-deficient mice displayed exacerbation of the loss in mbp ( fig 1f) and a further increase in the smi32/mbp ratio compared to wt mice (fig 1g) , suggesting that il-4 is important in maintaining axonal myelination or promoting axonal remyelination after stroke. stroke impacts both gray and white matter. in order to confirm the effect of il-4 in white matter, we explored the remyelination capacity of il-4 in a model of lysophosphatidylcholine/lysolecithin (lpc)-mediated demyelination using ex vivo organotypic cerebellar slice cultures. this toxicant-based experimental model of demyelination, although not attempting to fully mimic a disease with complex etiology and pathogenesis, has proven extremely useful for understanding the biology of remyelination [23] . slices collected from wt and il-4 ko mice exhibited comparable extents of demyelination, as revealed by the loss of mbp staining along neurofilamentheavy (nfh + ) axonal fibers, at 3 d after lpc (fig 2a) . partial remyelination was achieved by 7 d following lpc in wt brain slices, but il-4 deficiency significantly delayed remyelination (fig 2a and 2b) , as shown by lower mbp staining intensity ( fig 2c) and less mbp + axonal coverage ( fig 2d) . il-4 supplementation significantly enhanced remyelination in wt brain slices and rescued remyelination capacities in il-4 ko brain slices (fig 2a, 2c and 2d ). il-4 liposome nanoparticles were prepared to facilitate the penetration of il-4 into the ischemic brain. the mean particle size of il-4 protein-loaded liposome was 122.6 ± 2.4 nm with a and the ratio of smi32 to mbp immunofluorescence intensity (g) in ipsilesional ec, cortex, striatum, and the total for all three areas quantified. n = 6-8/group. data are normalized to the intensities of contralateral hemispheres. � p < 0.05, �� p < 0.01, ��� p < 0.001. one-way anova and bonferroni post hoc. (h) representative images for mbp (green) and smi32 (red) double immunostaining in the peri-infarct areas in brains from sham, wt mcao mice, and il-4 ko mcao mice 14 d after mcao. scale bar: 50 μm. data associated with this figure can be found in the supplemental data file (s1 data). cns, central nervous system; ec, external capsule; fsc-w, forward scatter width; gfap, glial fibrillary acidic protein; il-4r, interleukin-4 receptor α; ko, knockout; mbp, myelin basic protein; mcao, middle cerebral artery occlusion; mfi, mean fluorescence intensity; opc, oligodendrocyte progenitor cell; ssc-a, side scatter area; wt, wild-type. lpc (500 μg/ml) was added in wt or il-4 ko organotypic cultures for 17 h to induce demyelination. il-4 (20 ng/ml) or vehicle was added after lpc removal. culture slices were fixed 3 d and 7 d after lpc treatment. control cultures were maintained in normal culture media for 7 d. (a) mbp (green) and nfh (red) staining was performed to label myelin and axonal fibers, respectively. scale bar: 50 μm. (b) mbp and nfh double staining and imaris 3d reconstruction. arrows indicate myelination of individual axonal fibers that are magnified in the images to the right. scale bar: 15 μm. (c) myelin integrity was measured as the intensity of mbp immunostaining. (d) the myelination index was calculated as the % mbp + myelin coverage along the entire nfh + fibers. n = 5-7 slices/group. � p < 0.05, �� p < 0.01, ��� p < 0.001. one-way anova and bonferroni post hoc. data associated with this figure can be found in the supplemental data file (s1 data). il-4, interleukin-4; ko, knockout; lpc, lysophosphatidylcholine/lysolecithin; mbp, myelin basic protein; nfh, neurofilament heavy; wt, wild-type. polydispersity index of approximately 0.212 ± 0.004 and a zeta potential of 4.05 ± 1.35 mv, confirming high stability of the nanodispersion. the il-4 nanoparticles were applied through the nasal route (50 μg/kg body weight), which has been increasingly utilized for cns drug delivery because of its capacity to bypass the blood-brain barrier and avoid first-pass metabolism [24] . intranasal administration of il-4 nanoparticles effectively increased il-4 protein levels in the corpus callosum (cc) and ec, cerebral cortex, and striatum 12 h after il-4 application (fig 3a-3c ). il-4 remained elevated in cortex and striatum until 24 h after delivery (fig 3b and 3c ). c57bl/6j mice were subjected to 60-min mcao and randomly assigned to receive il-4 nanoparticle (50 μg/kg body weight) or control nanoparticle (vehicle) treatment. nanomaterials were intranasally delivered at 6 h after mcao and repeated at 1-7 d, 14 d, 21 d, and 28 d after mcao (fig 3d) . magnetic resonance imaging (mri) on these animals was performed serially at 9.4 t on days 2, 14, 21, and 28 after mcao. t 2 -weighted images showed no significant difference in the brain lesion volume of il-4-versus vehicle-treated mice (fig 3e and 3f) , indicating comparable gross tissue damage across treatment groups. histological staining of the neuronal marker neun further demonstrated comparable tissue loss 35 d after stroke in il-4-and vehicle-treated mice (fig 3e and 3g) . serial whole-brain in vivo diffusion tensor imaging (dti) scans were collected 14 d, 21 d, and 28 d after mcao. the same animals were then killed at 35 d after stroke, and the fixed brains were subjected to ex vivo dti scanning (fig 4a and 4b , s2a and s2b fig) . longitudinal observation of fractional anisotropy (fa) maps and calculation of the ratio of fa(ipsilateral)/ fa(contralateral) demonstrated progressive white matter injury (ratio < 1) with a slow recovery in the ec of vehicle-treated stroke mice at 14-35 d after stroke, which was significantly improved by day 28 following stroke in il-4-treated mice (fig 4c) . this trend was observed until day 35 in the ex vivo fa values (fig 4c) . further analysis of the 35 d dti images revealed that il-4 treatment enhanced white matter integrity, as revealed by elevated fa values in the white matter-enriched ec and internal capsule (ic) (fig 4d, 4e and 4g ). in addition, il-4 treatment significantly reduced radial diffusivity (rd, λ ? ) in the ec ( fig 4f) and ic (fig 4h) , which likely indicates improved myelin integrity [25] . no significant differences between il-4 and vehicle treatment groups were observed in axial diffusivity (ad, λ || ), a measurement for axonal integrity (s2c and s2d fig) . to further explore the biological basis of these dti results, we compared the dti results with immunohistochemical staining of mbp performed in the same animals. there were remarkable similarities between the fa maps and mbp images (fig 4d, 4i and 4j). il-4 treatment significantly increased the mbp + area in the ec (fig 4d and 4i ). there was a significant positive correlation between the mbp + areas and the fa values in the ec (fig 4j) . the quantification of mbp staining confirmed a significant increase in mbp intensity in the ischemic brains of il-4-treated mice compared to vehicle-treated mice (fig 4k) . to determine whether il-4 can improve axonal myelination after stroke, myelin thickness was measured in the ec using transmission electron microscopy (tem) (fig 5a) . frequency histograms revealed much greater reduction of axon diameters in the mcao + vehicle group compared to the mcao + il-4 and sham groups, suggesting higher axonal degeneration in stroke mice without il-4 treatment (fig 5b) . scatter plots of the g-ratio (the ratio of the inner axonal diameter to the total outer diameter of myelinated fiber) as a function of axon diameter in sham mice or stroke mice treated with vehicle or il-4 revealed significant differences in myelin thickness (average g-ratio: sham = 0.72 [n = 183 axons from 3 animals]; mcao + vehicle = 0.79 [n = 233 axons from 3 animals]; mcao + il-4 = 0.73 [n = 176 axons from 3 animals]) ( fig 5c) . significant increases in the g-ratio were observed in both low-and high-caliber axons at 35 d after mcao, indicating a reduction in myelin thickness ( fig 5d) . il-4 post-treatment significantly reduced the g-ratio of the middle-sized (diameter = 400-800 nm) and large (diameter > 800 nm) axonal fibers, suggesting that il-4 may maintain axonal myelination or promote axonal remyelination after stroke. the g-ratio in small axonal fibers (diameter < 400 nm) displayed a trend toward reduction with il-4 treatment ( fig 5d) . saltatory conduction of action potentials along myelinated axons relies on normal organization of the nodes of ranvier (nor), which are rich in sodium and potassium ion channels. double immunofluorescent analyses of nav1.6 (a nodal sodium channel protein) and contactin-associated protein (caspr, a paranodal cell adhesion protein) revealed the nor in the ec (fig 5e) . typical nor are characterized by punctate nav1.6 staining at nodes between the paranodal staining of caspr. the total numbers of normal nor decreased significantly after stroke in the ipsilateral ec compared to the contralateral ec (fig 5f) , and this was accompanied by a decrease in the caspr + paranode length (fig 5g) . il-4 treatment significantly increased the number of nor and the paranode length in the ischemic ec (fig 5f and 5g) , suggesting an improvement in nor structure. the paranodal gap between pairs of caspr staining remained unchanged between groups ( fig 5h) . as il-4 enhanced the structural integrity of white matter after mcao, we further evaluated white matter function by measuring the transmission of action potentials in the cc and ec (fig 5i) . evoked compound action potentials (caps) display an early peak representing fast-conducting myelinated axons (n1, fig 5j) [26] . the amplitude of the n1 segment was significantly reduced 35 d after mcao (fig 5k) , indicating impaired conduction through myelinated axons. il-4-treated mice exhibited less reduction in the n1 amplitude after mcao, suggesting myelin preservation or improved remyelination of axons compared to the vehicle-treated subjects. sensorimotor and cognitive functions were assessed by investigators blinded to group assignments. as shown in fig 6a and 6b , il-4-treated and vehicle-treated stroke mice showed comparable reductions in latencies to contact and remove adhesive tape from the paw at 3 d after stroke, suggesting similar initial deficits in dexterity across groups. however, at later time points starting from 7-10 d after stroke, il-4 treatment reduced the time spent touching and removing tapes in stroke mice, culminating in improved performance in the adhesive removal test (fig 6a and 6b ). long-term sensorimotor performance in the rotarod test after mcao was also improved in il-4-treated mice ( fig 6c) . furthermore, il-4-treated mice exhibited long-term improvements in cognitive function in the morris water maze test, as manifested by a reduction in the latency to find the hidden platform (improved spatial learning, fig 6d and 6e ) and increased time spent in the target quadrant when the platform was removed (improved memory, fig 6d and 6f ). there was no difference in swimming speed among different treatment groups, indicating similar gross motor skills across groups ( fig 6g) . next, we measured the potential correlation between white mater integrity and behavioral performance after stroke in vehicle or il-4-treated mice. the results revealed significant negative correlations between mbp intensity and the latency to touch ( fig 6h) and remove ( fig 6i) the adhesive tape at 35 d after stroke, confirming an association between histological observations of white matter integrity and long-term functional recovery of behavioral endpoints. successful differentiation of opcs into myelinating oligodendrocytes is important for remyelination [11] . thus, we compared the regeneration of myelin-producing oligodendrocytes in mcao mice with or without il-4 treatment. 5-bromo-2 0 -deoxyuridine (brdu) was injected at 3-6 d after mcao to label newly generated cells. mature oligodendrocytes were recognized by immunostaining with anti-adenomatous polyposis coli (apc) (or cc1) antibodies [27] . compared to vehicle-treated mice, a significantly higher ratio of brdu + apc + newly generated oligodendrocytes was observed in il-4-treated mice 35 d after mcao (fig 7a and 7b ). the total numbers of brdu + cells and the total numbers of apc + cells were also significantly increased in il-4-treated mice compared to vehicle-treated mice (fig 7c and 7d ). it has been reported that higher concentrations of the apc antibody may detect some populations of astrocytes in certain pathological conditions [27] . however, no apc signal was detected in glial fibrillary acidic protein (gfap) + astrocytes in the ischemic brain in our preclinical model of stroke (s3 fig) . il-4 is known to promote microglia m2 polarization, which in turn enhances opc differentiation and remyelination [28] . we observed in our model that il-4 treatment robustly promoted microglia/macrophage polarization toward an anti-inflammatory phenotype (cd206 + iba1 + ) at 7 and 14 d after stroke (s4 fig). microglia depletion through genetic targeting or pharmacological treatment is a widely used approach to evaluate the contribution of this population to various biological processes [29] . therefore, we chose plx5622, an inhibitor of colony-stimulating factor 1 receptor, to deplete microglia/macrophages and identify their role in il-4-afforded oligodendrogenesis ( fig 7e) [30] . consistent with previous reports [30] , plx5622 achieved approximately 90% depletion efficacy within a short period. dietary intake of plx5622 for 7 d dramatically reduced the number of microglia in the normal brain significant depletion of microglia/macrophages did not change the number of brdu + apc + cells or total brdu + cells compared to microglia/macrophage-competent stroke mice (fig 7f-7h ). il-4 treatment increased the numbers of brdu + apc + cells or total brdu + cells after stroke in mice (h) quantification of the number of brdu + cells in the peri-infarct areas. n = 7-8/group. � p < 0.05, ��� p < 0.001. one-way anova followed by bonferroni post hoc. data associated with this figure can be found in the supplemental data file (s1 data). apc, adenomatous polyposis coli; brdu, 5-bromo-2 0 -deoxyuridine; ctx, cortex; ec, external capsule; il-4, interleukin-4; mcao, middle cerebral artery occlusion; str, striatum. fed with the control diet. il-4 treatment exhibited a trend (p = 0.086) toward lower capacities to increase the number of brdu + apc + newly generated oligodendrocytes in microgliadepleted mice versus microglia-competent mice, supporting a potential role of microglia in il-4-afforded oligodendrogenesis. importantly, il-4 treatment still resulted in a significant enhancement of brdu + apc + oligodendrocyte regeneration in microglia/macrophagedepleted stroke mice (fig 7f and 7g ). considering the expression of il-4r on oligodendrocyte linage cells, these results indicate that il-4 may exert microglia/macrophage-independent effects on oligodendrocyte regeneration after demyelination. consistent with a previous study in a model of multiple sclerosis [31] , we found that repeated intranasal il-4 administration did not change t-lymphocyte subpopulations or b lymphocytes in the blood (s6a-s6c fig consistent with a direct effect of il-4 on oligodendrocytes, in vitro studies showed that il-4 at a concentration range of 5-100 ng/ml directly stimulated the differentiation of neuron-glia antigen 2 (ng2) + opcs into mbp + mature oligodendrocytes without the aid of microglia ( fig 8a and 8b ). il-4 at concentrations of 10 or 20 ng/ml induced opc differentiation that was comparable to the effects of a t3 cocktail typically used to induce opc differentiation in vitro. the effect of il-4 on opc differentiation was indeed il-4r-dependent, because specific il-4rα neutralizing antibodies (il-4rabs) blocked the il-4-induced increase in mrna expression of the mature oligodendrocyte marker mbp (fig 8c) . il-4 at a concentration range of 5-100 ng/ml had no effect on opc survival (s7a and s7b microarray analyses were performed to further elucidate the mechanisms underlying the promotion of oligodendrogenesis by il-4. opcs were treated with pbs or il-4 (20 ng/ml) for 3 d. total rna extraction from cell pellets was used for the affymetrix clariom s rat array. principal component analysis (pca) showed clustering of samples in a treatment-dependent manner ( fig 8d) . in total, 996 differentially expressed genes (degs) were identified (false discovery rate [fdr] < 0.05; and fold change > 2), out of which 495 were up-regulated and 501 were down-regulated in il-4-treated opcs relative to pbs-treated opcs (fig 8e, s1 and s2 tables). the gene ontology (go) enrichment analysis was then performed using the metascape analysis [32] (http://metascape.org). the top 20 enriched go terms in three categories (biological processes, cellular components, and molecular functions) were identified in the upregulated degs by il-4 treatment (s8a fig). a network plot further captured the relationships between the main regulated biological processes, which were related to cell adhesion and motility, cytokine production, cell responses to external stimuli, cellular component organization, and cell death (fig 8f) . the enriched go terms among the down-regulated degs in il-4-treated opcs represent biological processes such as developmental growth, small molecule biosynthetic process, vessel development, and responses to nutrients and peptides (s8b fig). these results indicated changes in cell mobilization, growth, differentiation, and defense in il-4-treated opcs. a kyoto encyclopedia of genes and genomes (kegg) pathway analysis 8g) . the ppar transcription factor assay confirmed that pparγ activity, but not pparα or pparδ activities, was up-regulated in opcs upon il-4 treatment ( fig 8h) ; these effects were abolished when il-4rα was blocked with a neutralizing antibody ( fig 8i) . furthermore, immunostaining (fig 8j and 8k ) and western blotting (fig 8l-8n, and s9 fig) demonstrated that the pparγ inhibitor gw9662 blocked il-4-induced expression of mature oligodendrocyte markers (mbp or myelin oligodendrocyte glycoprotein [mog]), suggesting that pparγ activation is essential for the opc differentiation induced by il-4. finally, opc-specific pparγ ko (pparγ-opc ko) mice were used to confirm the importance of pparγ signaling in il-4-enhanced oligodendrogenesis in an in vivo stroke model ( fig 9a) . to confirm the ko of pparγ specifically in opcs, pdgfrα + cd45 − opcs, glast + cd45 − astrocytes, cd45 + immune cells, and other cd45 − glast − pdgfrα − cns cells were sorted from the brains collected from pparγ-opc ko mice at 9 d after 4-hydroxytamoxifen injections. the dna extracts were subjected to pcr using primers flanking the loxp site and primers detecting the cre cdna. the loxp band was abolished in opcs because of the targeted deletion after cre-induced recombination, while remaining intact in astrocytes, immune cells, and other cns cells sorted from the same brain ( fig 9b) . the expression of cre was detected in the opcs of the pparγ-opc ko mice ( fig 9b) . deletion of pparγ specifically in opcs exerted no impact on il-4r expression on o4 + oligodendrocytes (s10 fig). vehicle or il-4 treatment in pparγ-opc ko mice resulted in similar neuronal tissue loss (fig 9c and 9d ) and white matter lesion (fig 9c, 9e and 9f) 35 d after stroke. the lack of pparγ expression in opcs did not significantly impair oligodendrocyte replacement after stroke, as shown by comparable numbers of brdu + apc + cells in the ischemic brain from pparγ-opc ko mice and control mice at 35 d after mcao (fig 9g and 9h) . however, the absence of pparγ in opcs reduced the capacity of il-4 to increase the number of newly generated brdu + apc + oligodendrocytes (fig 9g and 9h ). consistent with a reduced capacity to promote oligodendrogenesis, il-4 failed to improve long-term sensorimotor (fig 9i and 9j ) or cognitive (fig 9k-9m ) deficits after stroke in the absence of pparγ. il-4/pparγ signaling improves white matter repair after stroke the ischemic brain, which is attributed, at least partly, to its release from injured neurons or from infiltrated immune cells [19] . cerebral il-4 levels then return to baseline within 1 wk after stroke [16, 19] . in patients, serum levels of il-4 are known to change following an acute stroke [33] . furthermore, il-4 gene polymorphisms are related to susceptibility to ischemic stroke and subsequent functional outcomes [34, 35] . finally, a recent study reported the expression of il-4r surrounding axonal filaments in postmortem brain tissue from patients with multiple sclerosis [31] . although these clinical and experimental data are correlative, the present series of in vivo and in vitro studies firmly establish a causal link between il-4 repletion and white matter integrity in preclinical stroke models. deficiency of endogenous il-4 an improvement in white matter integrity might be achieved in two ways: preservation of preexisting white matter structures or enhancement of white matter repair. our data favor the latter as the predominant mechanism of action for il-4. first, longitudinal dti revealed similar reductions in fa values shortly after stroke in vehicle or il-4-treated mice, indicating comparable degrees of initial white matter injury during the early/acute phase. a significant improvement in white matter microstructure was only observed in il-4-treated animals in later stages of recovery (28 and 35 d after stroke). these results strongly suggest a delayed effect of il-4 on white matter repair, rather than preservation shortly after injury. second, electron microscopy (em) and histological studies revealed structural signs of remyelination in the ischemic brain, including thinner myelin segments than before stroke, and reduced paranode length with caspr/nav1.6 staining [36] . each of these structural alterations were significantly improved by il-4 treatment, indicating superior remyelination. third, il-4 treatment increased the number of new, mature oligodendrocytes (apc + brdu + ) in the ischemic brain 35 d after stroke. such improvements in oligodendrogenesis are critical for successful remyelination. finally, the lpc model of demyelination-remyelination unequivocally confirmed the white matter-specific effects of il-4 in remyelination. as expected, the il-4-mediated reconstitution of myelin structure culminated in functional improvements in neural conduction and behavioral performance, despite no changes in the degree of gray matter loss. although il-4 is important for white matter repair after brain injury, the il-4 ko mice do not display deficits in myelin coverage in organotypic brain slice cultures under physiological conditions. this observation is consistent with the view that different mechanisms control myelin sheath formation during normal development and remyelination [36] . although our t2 scanning did not show significant reductions in brain lesion size under conditions of il-4 repletion, there was a trend toward lesion volume reduction at 14-35 d after stroke. as the il-4 treatment was initiated 6 h after stroke, it remains possible that il-4 may provide some degree of early protection [16] . it is also possible that some neun + neurons remaining within the gray matter of mice without il-4 might not be physiologically functional, although they are still histologically present, perhaps because their communication via white matter tracts is significantly impeded. a previous study using mixed glial cultures demonstrated that the addition of il-4 promotes the differentiation of opcs into mbp-expressing oligodendrocytes in vitro, an effect mainly attributed to il-4-mediated anti-inflammatory effects [37] . an additional conclusion emerging from our studies is that il-4 enhances white matter repair at least partially independent of its well-established anti-inflammatory effects on microglia/macrophages. in il-4-treated stroke mice, we observed an anti-inflammatory phenotypic change in microglia/ macrophages, which is known to promote remyelination after cns injury [28] . however, dramatic depletion of microglia/macrophages in the ischemic brain did not abolish the capacity of il-4 to enhance oligodendrocyte replacement, supporting the involvement of microglia/ macrophage-independent mechanisms. in vitro studies then confirmed that il-4 can also directly target white matter components and promotes opc differentiation into mature oligodendrocytes in the absence of microglia/macrophages. previous fate mapping studies revealed that opcs are responsible for generating new oligodendrocytes during remyelination [38, 39] . however, the differentiation of opcs is frequently impaired in lesioned brains, mainly because of nonpermissive environmental cues [40] [41] [42] . we found that il-4 treatment may overcome these obstacles for opc differentiation in the ischemic brain, both directly by promoting opc differentiation and indirectly through immunomodulation. notably, endogenous oligodendrocyte differentiation has been observed until as late as 3 mo after cns injuries [39] , and this prolonged effort may provide a sufficiently wide therapeutic window that allows delayed il-4 treatment to restore white matter and encourage long-term stroke recovery. in the present study, we report a previously undefined mechanism of il-4, namely that it directly induces opc differentiation by activating pparγ signaling. pparγ is a master transcription factor known to promote opc differentiation and maturation though manifold mechanisms involving enhanced myelin lipid synthesis, reduced oxidative stress, and promotion of mitochondrial function [43, 44] . our previous work has shown that rosiglitazone, a pparγ agonist, enhanced opc proliferation and increased the number of newly generated mature oligodendrocytes after mcao [45] . in addition, pparγ may interact with histone deacetylases to modulate cell differentiation [46] . we demonstrated here that pparγ inhibition abolished the effect of il-4 on opc differentiation in vitro and that opc-specific ko of pparγ in vivo reduced il-4-enhanced oligodendrocyte replacement in the ischemic brain. these collective results indicate that il-4-induced oligodendrocyte differentiation/maturation relies at least partially on the activation of pparγ activity in opcs. precisely how il-4/il-4r engages pparγ activity is not yet known, but several studies suggest that il-4 may activate pparγ in macrophages and other cell types through signal transducer and activator of transcription 6 (stat6) signaling [37, 47] . whether this pathway is also operational in opcs lies beyond the scope of the present study but warrants further investigation. aside from the cell-specific effects of il-4 on opcs, we note that il-4r is widely distributed in many types of cns cells as well as infiltrated immune cells. several lines of evidence from our study support the capacity of il-4 to target multiple types of cells in addition to oligodendrocytes. first, we did not observe significant differences in the density of brdu + apc + cells between wt and pparγ-opc ko mice following mcao, suggesting that endogenous il-4 is able to promote white matter integrity by targeting other types of cells. second, although pparγ inhibition eliminated the effect of il-4 on opc differentiation in cultures, the absence of pparγ in opcs reduced, but did not abolish, the capacity of il-4 to increase the number of newly generated oligodendrocytes in vivo. these results suggest that pparγ expression in opcs is an important but not exclusive mechanism for il-4-induced oligodendrogenesis in preclinical stroke. indeed, we observed that il-4 treatment robustly promoted microglia/macrophage polarization toward an anti-inflammatory m2phenotype, as mentioned above. il-4-stimulated microglia have been shown to release il-4 induced 1 (il4i1), which in turn augments remyelination [48] . thus, direct and indirect effects on microglia/macrophages may contribute to the white matter repair afforded by il-4. it has also been reported that the effects of il-4 on adaptive immune responses contribute partially to recovery from neurotrauma [49] . in our hands, however, repeated intranasal il-4 administration did not change t-lymphocyte subpopulations or b lymphocytes in blood and did not alter the composition of infiltrated lymphocytes. these data dispute the involvement of adaptive immune responses in il-4-afforded neuroprotection in our stroke model. aside from its roles in immunoregulation, il-4 can also directly target other types of cns cells. for example, a recent study reported that the activation of il-4r signaling in neurons may increase axonal outgrowth and ameliorate axonal pathology in a model of multiple sclerosis [31] . in this context, it is important to note that we did not observe improvements in the dti measurement for axonal integrity (ad, λ || ). in other disease models, multiple factors including inflammation, cellular infiltration, and gliosis may contribute to ad at chronic stages of disease. therefore, the measurement of λ || appears to lack sufficient resolution to distinguish axonal damage/repair versus confounding cellular responses over the course of progressive demyelination [50] . on the other hand, our em data demonstrated an increase in axon diameter in il-4-treated mice compared to vehicle-treated controls, supporting a beneficial role of il-4 in maintaining axonal structure. these structural data were supported by our electrophysiological studies of axon conductance. preservation of axonal structure and electrical function might provide an important feedback signal for opc differentiation and guide remyelination [51, 52] , preventing wallerian degeneration of neurons after ischemia and enhancing white matter repair. in addition to these mechanisms, astrocytes are known to produce trophic factors in response to il-4 stimulation, including brain-derived neurotrophic factor (bdnf) and nerve growth factor (ngf), both of which are also critical for brain function and brain repair [53] . a series of in vitro studies assessed the effective concentrations of il-4 in various types of cns cells. for example, il-4 regulates microglia activity at concentrations of 1-20 ng/ml [19, 54] . il-4 inhibits inflammatory responses and boosts trophic factor release in astrocytes at 5-10 ng/ml [53] . il-4 protects neurons against excitotoxicity and stimulates axonal outgrowth in vitro at 50 ng/ml [31] . in the present study, il-4 directly stimulated opc differentiation within a concentration range of 5-100 ng/ml. these collective studies reveal similar effective il-4 concentrations (in nanoscales) in glial and neuronal cells of the cns. indeed, it is precisely the multitargeting capacities of low-dose il-4 that may render it an excellent therapy for cns injuries characterized by a wide array of pathological mechanisms. the ultimate implementation of preclinical research into clinical treatments requires extensive information on safety, optimal dose, route, and schedule of administration, among other variables. in the present study, il-4 stimulated opc differentiation in cultures at a concentration range of 5-100 ng/ml, and the optimal concentration was 10-20 ng/ml. although there is neither a mathematical nor empirical method to accurately convert in vitro concentrations to in vivo intranasal dosage, these in vitro data support the capacity of il-4 to directly target opcs at low concentrations. previous preclinical studies have similarly documented therapeutic effects of low doses of il-4 in ischemic stroke and other neurological diseases. for example, subcutaneous il-4 application at 2 μg/kg/d [19] or intracerebroventricular application of il-4 at 60 ng/d [16] significantly improved long-term outcomes after stroke. vogelaar and colleagues reported that intrathecal or intranasal (1 μg/d) il-4 treatment ameliorated clinical signs and reversed disease progression in a model of multiple sclerosis [31] . those studies, however, did not measure il-4 concentrations in the brain after treatment. here, we delivered il-4 intranasally at a dose of 50 μg/kg/d (about 1.25 μg/d based on 0.025 kg average body weight), which significantly elevated il-4 levels in key parts of the brain. this dose is similar to the effective intranasal dose for vogelaar's study and improved long-term functional recovery in our stroke model. repeated dosing regimens or controlled continuous release was employed for all in vivo il-4 treatment because of its rapid clearance and inactivation. the use of liposome-entrapped peptides has been reported to protect the cytokine and enhance its tissue delivery [55, 56] . consistent with those findings, we demonstrated that il-4 liposome nanoparticle delivery elevated il-4 levels in the brain for at least 24 h. further characterization of the optimal regimen of il-4 treatment for stroke would accelerate its clinical translation. despite growing awareness of the importance of white matter restoration in poststroke rehabilitation, there is no effective therapy to preserve white matter function or to improve its repair in humans. our study has uncovered a novel role of il-4 in poststroke white matter recovery-a role that lies beyond its well-known immunoregulatory functions. furthermore, we have shown that il-4 promotes oligodendrogenesis and white matter repair in a pparγdependent manner. therefore, it seems reasonable to continue to test il-4 as a therapeutic strategy for functional recovery after ischemic stroke. in particular, the intranasal route enables cns il-4 delivery, which is expected to significantly reduce the risk of peripheral side effects and expedite the eventual translation of il-4 treatment for stroke victims. all animal procedures were approved by the university of pittsburgh institutional animal care and use committee (approval number: 18032546), performed in accordance with the national institutes of health guide for the care and use of laboratory animals, and reported in accordance with the animal research: reporting in vivo experiments (arrive) guidelines. all efforts were made to minimize animal suffering and the number of animals used. all animals were housed in a temperature-and humidity-controlled facility with a 12-h light/dark cycle. food and water were available ad libitum. transient cerebral ischemia was induced by intraluminal occlusion of the left middle cerebral artery (mca) for 60 min, as described previously [57] . sham-operated animals underwent the same anesthesia and exposure of arteries without mcao. briefly, mice were anesthetized with 3% isoflurane in a 30% o 2 /68.5% n 2 o mixture until they were unresponsive in the tail-pinch test. mice were then fitted with a nose cone blowing 1.5% isoflurane in a 30% o 2 /68.5% n 2 o mixture under spontaneous breathing for anesthesia maintenance. an 8-0 monofilament with a silicon-coated tip was introduced into the common carotid artery, advanced to the origin of the mca, and left in place to limit mca blood flow for 60 min. rectal temperature was controlled at 37.0 ± 0.5˚c using a temperature-regulated heating pad during surgery. regional cerebral blood flow was measured in all stroke animals using laser doppler flowmetry. animals that did not display at least 70% reduction in regional cerebral blood flow during mcao compared to pre-ischemia levels were excluded from further experimentation. surgeries and quantification were performed by investigators blinded to animal genotypes and experimental grouping. animals were randomly assigned to sham or cerebral ischemia groups and received randomized treatments using a lottery drawing box. a grand total of 287 mice (64 sham-operated and 223 ischemic mice) were used in this study, including 26 mice that were excluded from further assessments, either because of death after ischemia or failure of ischemia induction. the mortality during mcao surgery was 7.6% in wt male mice (12/158), 14.3% in il-4 ko male mice (1/7), 11.1% in microglia/macrophage-depleted mice (3/27), and 12.5% in pparγ-opc ko mice (3/24). there was no significant difference in mortality rate across groups. lecithin (150 mg) and cholesterol (15 mg) were dissolved in 5 ml of a chloroform-methanol mixed solution (3:1, volume:volume). a lipid layer was formed after evaporating the mixed solvent under vacuum distillation. recombinant il-4 protein (1 mg, peprotech, rocky hill, nj, usa) was dissolved in 5 ml of water and then added to the lipid layer for hydration for about 30 min. the mixture was homogenized by ultrasonic probes for 15 min in an ice bath and then filtered through a 0.45-μm membrane. the free il-4 protein was removed by ultrafiltration with centrifugal filter units (100-kda cutoff). the particle size and zeta potential of il-4 protein-loaded liposomes were determined by dynamic light scattering (dls). the concentration of il-4 protein was determined by the bicinchoninic protein determination kit (thermo fisher scientific, pittsburgh, pa, usa). animals were randomly assigned to receive vehicle or il-4 (50 μg/kg body weight, intranasal) nanoparticle treatment starting 6 h after mcao and repeated at 1-7 d, 14 d, 21 d, and 28 d postsurgery. briefly, under isoflurane anesthesia, five 2-μl drops (total 10 μl) of il-4 nanoparticles were applied alternately into each nostril with a 2-min interval between drops. control animals received an equal volume of vehicle control using the same anesthesia and delivery regimen. in order to label proliferating cells, animals were intraperitoneally injected with the thymidine analogue brdu (50 mg/kg, b9285, sigma, st. louis, mo, usa) twice a day (with an interval of at least 8 h) for 4 consecutive days, beginning at 3 d after mcao. behavioral tests were performed by an individual blinded to experimental groups. sensorimotor functions were measured 3-35 d after mcao. the rotarod test. briefly, mice were forced to run on a rotating drum (iitc life science, woodland hills, ca, usa) with speeds starting at 4 rpm and accelerating to 40 rpm within 400 s. three consecutive trials were conducted for each mouse with an interval of 15 min. the time at which a mouse fell off the drum was recorded as the latency to fall. data were expressed as mean values from three trials. the adhesive removal test. the adhesive removal test was performed to assess tactile responses and sensorimotor asymmetries. two 2 × 3-mm adhesive tapes were applied to lesioned forepaws. tactile responses were measured by recording the time to initially contact the ipsilateral paws, as well as the time to remove the adhesive tape, with a maximum observation period of 120 s. morris water maze test. cognitive function was analyzed with the morris water maze test 23-28 d after mcao, as described previously [16] . in the learning test, a square platform (11 × 11 cm 2 ) was submerged 2 cm beneath the water surface in a circular pool (diameter = 109 cm) of opaque water. mice were placed into the pool from one of the four locations and allowed to locate the hidden platform for 60 s. each mouse was trained on 3 trials (with randomly assigned starting positions) per day to locate the platform for three consecutive days before mcao. at the end of each trial, the mouse was placed on the platform or allowed to stay on the platform for 30 s with prominent spatial cues displayed around the room. the time spent to reach the platform (learning phase) was recorded. three trials were performed on each day. the memory test was performed on day 28. the platform was removed, and a single 60-s probe trial was conducted. time spent in the goal quadrant where the platform was previously located was recorded. animals were euthanized and perfused with saline followed by 4% pfa (sigma-aldrich; st. louis, mo, usa) in pbs. brains were cryoprotected in 30% sucrose in pbs. coronal brain sections (25 μm) were sliced on a freezing microtome, and six equally spaced coronal brain sections encompassing the mca territory were stained with a rabbit anti-neun antibody (abn78, 1:500; millipore, burlington, ma, usa) or rabbit anti-mbp antibody (mbp ab40390, 1:500, abcam, cambridge, ma, usa). loss of neun + or mbp + tissue was analyzed by a blinded observer using nih image j software. the infarct volume with edema correction was calculated as the volume of the contralateral hemisphere minus the noninfarcted volume of the ipsilateral hemisphere. coronal brain sections were subjected to immunofluorescence staining. briefly, floating brain sections were blocked with 5% donkey serum in 0.3% triton x-100 in pbs (pbst) for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4˚c. after three washes in 0.3% pbst, sections were incubated with the appropriate secondary antibodies for 1 h at room temperature. the process was repeated once for double staining. image analyses were performed on one or two randomly selected microscopic fields in the peri-infarct areas of the ec, two in the cortex, and two in the striatum of each section; two sections covering the infarct area were assessed for each mouse brain. the recorded images were loaded onto image j (nih) and were manually quantified by 2 observers blinded to grouping. positively stained cells were electronically labeled with the software to avoid duplicated counting. the infarct area was identified as the region in which the majority of dapi-stained nuclei were shrunken. the border of the infarct was determined by the loss of neun staining and the accumulation of iba1 + microglia/macrophage or brdu + apc + oligodendrocytes on adjacent sections. we defined the peri-infarct area as the tissue that covers a radial distance of 200-300 μm from the border of the infarct. animals were euthanized and perfused with cold saline. brains were dissected, and the ipsilateral (left) and contralateral (right) hemispheres were collected. brains were dissociated into a single-cell suspension using a gentlemacs dissociator (miltenyl biotec) following the manufacturer's instructions. the suspension was passed through a 70-μm cell strainer (fisher scientific, pittsburgh, pa, usa) and resuspended in 30% percoll (ge health). single-cell suspensions were separated from myelin and debris by centrifugation (500g, 30 min, 18˚c) on a 30%-70% percoll gradient. cells at the interface were collected and washed with hank's balanced salt solution (hbss; sigma-aldrich, st. louis, mo, usa) containing 1% fetal bovine serum (sigma-aldrich, st. louis, mo, usa) and 2 mm edta (sigma-aldrich, st. louis, mo, usa) before staining. blood cells were prepared as described previously [57] . single-cell samples were first incubated with antibodies to surface antigens for 30 min on ice at 4˚c in the dark. after two washes, cells were fixed and permeabilized with an intracellular staining kit (thermo fisher ebioscience, pittsburgh, pa, usa) according to the manufacturer's protocol. caps were measured in the ec. mice were decapitated after co 2 euthanasia, and brains were removed. coronal slices (350 μm thick) were prepared −1.06 mm from bregma and transferred to an incubation chamber containing pregassed (95% o 2 /5% co 2 ) artificial cerebrospinal fluid (acsf; 126 mmol/l nacl, 2.5 mmol/l kcl, 1 mmol/l na 2 h 2 po 4 , 2.5 mmol/l cacl 2 , 26 mmol/l nahco 3 , 1.3 mmol/l mgcl 2 , 10 mol/l glucose [ph 7.4]) for 30 min at 34˚c and then for 1 h at room temperature (25˚c). brain slices were then perfused with acsf at a constant rate (3-4 ml/min) at 25˚c. for recording, a bipolar stimulating electrode (intertip distance, 100 μm) was placed across the cc at about 0.9 mm lateral to the midline. a glass extracellular recording pipette (5-8 mo tip resistance when filled with acsf) was placed in the ec, 0.25, 0.5, 0.75, and 1 mm lateral from the stimulating electrode. the recording at 0.75 mm from the stimulating electrode was reported. the cap signal was digitized (digidata 1500b plus humsilencer; molecular devices, san jose, ca, usa), amplified (×1 k), and recorded by the axoclamp 700b amplifier (molecular devices, san jose, ca, usa). the signal was then analyzed by pclamp 10 software (molecular devices, san jose, ca, usa). the input-output curves were generated by increasing the stimulating intensity by 0.25-ma intervals from baseline, up to 2 ma. the amplitude of the n1 component of the cap (representing myelinated fibers) was calculated as the variance from the first peak to the first trough. tem was used to measure myelin thickness in the cc/ec area. mice were perfused with icecold saline, followed by 4% pfa and 2.5% glutaraldehyde in 0.1 mol/l pbs buffer. the cc/ec tissue near the site of ischemia was microdissected into 1-mm 3 blocks. these specimens were immersion-fixed for 24 h in 2% glutaraldehyde. tissue was then rinsed in pbs and fixed in 1% osmium tetroxide in 0.1 m pbs for 45 min. samples were dehydrated in increasing concentrations of acetone and embedded in araldite resin. ultrathin 60-nm sections were cut on a leica uct ultramicrotome with a diamond knife (diatome, wetzlar, germany), stained with uranyl acetate and lead citrate, and viewed using a jem1400 tem (jeol, akishima, japan). two sections from each animal were analyzed. three to 5 images (600 μm 2 each) were acquired in randomly selected areas within the cc/ec from each section at a magnification of 50,000× and analyzed with image j by an investigator blinded to experimental groups. at least 50 random axons per animal were analyzed by tracing the axonal circumference and the whole fiber circumference in a blinded fashion. g-ratios were calculated as the ratio of the inner axonal diameter to the total outer diameter (axonal diameter + total myelin sheath thickness). for in vivo mri, mice were initially anesthetized with 3% isoflurane and then positioned on an animal cradle with a stereotaxic head holder. anesthesia was maintained between 1% and 1.5% isoflurane throughout the experiment. respiration and temperature were monitored continuously, and temperature was maintained by a warm animal heating system (sa instruments, stony brook, ny, usa). mri was performed on a 9.4 t/30-cm aviii hd spectrometer (bruker biospin, billerica, ma, usa) equipped with a 12-cm high-performance gradient set and using an 86-mm quadrature rf transmit volume coil and a 2-channel receive surface rf coil and paravision 6.0.1. a t 2 -weighted rare sequence was used to visualize the infarct, with the following setup: repetition time (tr)/echo time (te) = 4,000/40 ms, field of view (fov) = 20 × 20 mm, acquisition matrix = 256 × 256, 16 slices with a slice thickness of 0.5 mm, 4 averages, and a rare factor = 8. the dti data were collected using an echo planar imaging (epi)-dti imaging sequence with the same geometry as the t 2 -weighted scan, using the following setup: tr/te = 2,300/22 ms, fov = 20 × 20 mm, acquisition matrix = 128 × 128, 16 slices with a slice thickness of 0.5 mm, 8 averages, 2 segments, 5 a0 images and 30 noncolinear diffusion images, δ/δ = 10/3 ms, and a b-value = 1,000 s/mm 2 . perfusion-fixed brains were left in the skull and imaged ex vivo using a bruker av3hd 11.7 t/89-mm vertical-bore microimaging system equipped with a micro2.5 gradient set capable of 1,500 mt/m, a 20-mm quadrature rf resonator, and paravision 6.0.1 (bruker biospin, billerica, ma, usa). dti data were collected using a multislice spin-echo sequence with 5 a0 images and 30 noncolinear diffusion images with the following setup: te/tr 22/2,800 ms, 2 averages, 160 × 160 matrix, 16 × 16-mm fov, 25 slices, 0.5 mm slice thickness, bvalue = 3,000 s/mm 2 , and δ/δ = 11.0/5.0 ms. this setup has been established for the acquisition of high-resolution dti scanning in small animals [8, 58, 59] . dti data were analyzed with dsi studio software (http://dsi-studio.labsolver.org/). rois were drawn manually in a blinded manner encompassing the ec and ic in the ipsilesional and contralesional hemispheres to determine fa, ad, and rd. dec, fa, and rd maps were generated by dsi studio software. in order to verify cre-induced deletion of flanked pparγ exons, a pair of primers (forward: 5 0 -tgtaatggaagggcaaaagg; reverse: 5 0 -tggcttccagtgcataagtt) that flank the downstream loxp site were used to perform pcr with genomic dna. program for pcr was 98˚c for 1 min; (98˚c for 15 s; 60˚c for 15 s; 72˚c for 15 s) × 38, and then 72˚c for 1 min. a pair of primers that detect the cre cdna (forward: 5 0 -gcggtctggca gtaaaaactatc; reverse: 5 0 -gtgaaacagcattgctgtcactt) were used as a positive control to confirm the absence of pcr band in opc was not due to failed pcr protocol itself. microarray experiment was performed in the genomics research core in the university of pittsburgh. total rna was prepared from il-4-or pbs-treated opcs using trizol (qiagen) according to manufacturer's instruction. preparation of cdna was performed using the wt plus reagent kit (thermo fisher) according to the manufacturer's instructions. reverse transcription was performed using 100 ng total rna and dntp-t7 random primers. second-strand synthesis and in vitro transcription created amplified crna. cdna from a second reverse transcription reaction was fragmented and end-labeled with biotin for hybridization to the affymetrix clariom s rat array. following overnight (16 h) hybridization at 45˚c with rotational mixing at 60 rpm, arrays were processed on a genechip 450 fluidics station using manufacturer-specified protocols. to remove unbound sample, arrays were first washed with nonstringent wash buffer a. the genechips were then stained for 10 min in stain cocktail 1. buffer a was again used to wash off excess stain. signal amplification was achieved by 10-min incubation with stain cocktail 2 followed by a second 10-min incubation with stain cocktail 1. the chip was washed with high-stringency buffer b and filled with array holding buffer before being removed from the fluidics station and scanned using the genearray 3000 scanner. first-level image analysis was performed using affymetrix expression console. four biologic replicates were performed for each experimental condition. data resulted in 23,188 detected probes that were used for subsequent analysis using transcriptome analysis console (affymetrix) and metascape analysis [32] (http://metascape.org). the go network plot is visualized using cytoscape, in which terms with a similarity > 0.3 are connected by edges. each node represents an enriched term and is colored by its cluster id. primary opc cultures were isolated with the differential attachment method. briefly, brains of mixed-sex sprague dawley rat pups (postnatal day 1-2) were triturated after removing the meninges and blood vessels. the brain tissues were dissociated with trypsin (0.01%) at 37 c for 15 min. after washing with ice-cold dmem, the cells were passed through a 70-μm filter and plated onto poly-d-lysine-coated t-flasks filled with culture media (dmem/f12 containing 10% heat-inactivated fetal bovine serum, 2 mm l-glutamine, 1 mm sodium pyruvate, 100 μm nonessential amino acids, 50 u/ml penicillin, and 50 μg/ml streptomycin) [45] . cells were grown to confluence (12) (13) (14) ) in a humidified incubator at 37˚c and with 5% co 2 . microglia were removed by shaking the mixed glia-containing flasks for 1 h at 180 rpm. after removing microglia, flasks were subjected to shaking at 200 rpm overnight to separate opcs from the astrocyte layer. opcs were maintained for 3-5 d in a serum-free basal defined medium (bdm: dmem, 0.1% bovine serum albumin, 50 μg/ ml human apo-transferrin, 50 μg/ml insulin, 30 nm sodium selenite, 10 nm d-biotin, 10 nm hydrocortisone) containing 10 ng/ml pdgf and 10 ng/ml bfgf. for oligodendrocyte induction, opcs were stimulated with t3 and cntf (10 ng/ml). the medium was exchanged every 2 d. protein was isolated from in vitro cultures. western blots were performed using the standard sds-polyacrylamide gel electrophoresis (page) method. the primary antibodies used in this study include rabbit anti-mbp (cell signaling technology, 78896, 1:500, danvers, ma, usa), mouse mog (santa cruz, sc-166172, 1:200, santa cruz, ca, usa), and mouse anti-β-actin (a2228, 1:20,000; sigma-aldrich, st. louis, mo, usa). in brief, polyvinylidene difluoride (pvdf) membranes were blocked in 5% nonfat milk for 1 h at room temperature and then incubated at 4˚c overnight with primary antibodies. next, the membrane was incubated with secondary antibodies for 1 h at room temperature (1:10,000, li-cor biosciences, lincoln, ne, usa), and scanned with li-cor odyssey infrared imaging system 9201-550u (li-cor biotechnology, lincoln, ne, usa). imagej software was used for gel analyses. nuclear extracts were prepared from opcs using ne-pe nuclear and cytoplasmic extraction reagent kit (thermo fisher, 78833, pittsburgh, pa, usa). ppar activity was assessed using the ppar (α, δ, γ) transcription factor assay kit (abcam, ab133113, toronto, on, canada) or the pparγ transcription factor assay kit (cayman chemical, 10006855, ann arbor, mi, usa) following the manufacturers' instructions. the cerebellum and attached hindbrain were isolated from p9 c57bl/6j mouse pups, sectioned sagittally at 300 μm on a mcilwain tissue chopper, and plated onto millipore-millicel-cm mesh inserts (thermo fisher scientific, pittsburgh, pa, usa) in 6-well culture plates with media containing 50% basal medium eagle (sigma, st. louis, mo, usa, b1522), 25% heatinactivated horse serum (gibco 26050-070), 25% earle's balanced salt solution (gibco 24020-117), 6.5 mg/ml glucose (sigma g8769), 1% penicillin-streptomycin, and 1% glutamax. media were changed every 2-3 d. to induce demyelination, cultures were treated with lpc (sigma, 0.5 mg/ml) for 17 h. slices were washed in media for 10 min and treated for 3 or 7 d with pbs or il-4 (20 ng/ml, il-4 was added to fresh media every other day). slices were fixed in 4% pfa for 1 h. rabbit anti-mbp antibodies (santa cruz, santa cruz, ca, usa, sc-13914; 1:200) and mouse anti-nf-h antibodies (abcam, cambridge, ma, usa, ab8135, 1:1,000) were applied for 48 h at 4˚c. fluorescence-conjugated secondary antibodies were applied overnight at 4˚c. slices were mounted onto glass slides and coverslipped with fluoromount-g. confocal microscopy (fluoview fv1000; olympus, tokyo, japan) was used to capture images. mbp staining intensity was analyzed with imaris software (bitplane ag). morphology reconstructions were carried out with the imaris filamenttracer module. quantitative assessment of myelination was performed in a blinded manner using the imaris colocalization module. thresholds for colocalization of red (nfh + axon) and green (mbp + myelin) channels were calculated automatically and maintained across different images from the same batch of staining. a colocalization channel was created and channel statistics were calculated automatically. the myelination index was calculated as the percent of colocalized areas in the total nfh + red pixels above threshold. for each condition, at least 6 slices collected from three independent experiments were analyzed. two to three fields were imaged from each slice. sample sizes for animal studies were determined by power analyses based on pilot studies or the literature. results are presented as mean ± standard error of the mean (sem). graphpad prism software (version 8.1.0, la jolla, ca, usa) was used for statistical analyses. the student's t test was used for comparison of two groups for continuous variables with normal distributions. the mann-whitney u rank sum test was used for continuous variables with nonnormal distributions. the differences in means among multiple groups were analyzed using one-way anova. differences in means across groups with repeated measurements over time were analyzed using the repeated-measures anova. when the anova showed significant differences, pairwise comparisons between means were tested by post hoc bonferroni tests. the correlation analyses between continuous data with normal distribution were performed using pearson correlation analysis. spearman rank correlation analyses were used to test correlations between data sets with nonnormal distributions. in all analyses, p < 0.05 was considered statistically significant. clinical prediction of functional outcome after ischemic stroke: the surprising importance of periventricular white matter disease and race the axon-glia unit in white matter stroke: mechanisms of damage and recovery demyelination as a rational therapeutic target for ischemic or traumatic brain injury white matter injury in ischemic stroke oligodendrocytes and ischemic brain injury tissue plasminogen activator prevents white matter damage following stroke. the journal of experimental medicine oligodendrocyte degeneration and recovery after focal cerebral ischemia tgfalpha preserves oligodendrocyte lineage cells and improves white matter integrity after cerebral ischemia lifelong cortical myelin plasticity and age-related degeneration in the live mammalian brain focal cerebral ischemia activates neurovascular restorative dynamics in mouse brain nogo receptor blockade overcomes remyelination failure after white matter stroke and stimulates functional recovery in aged mice ascl1 lineage cells contribute to ischemiainduced neurogenesis and oligodendrogenesis the role of oligodendrocyte precursor cells expressing the gpr17 receptor in brain remodeling after stroke the age-related decrease in cns remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation regulation of learning and memory by meningeal immunity: a key role for il-4. the journal of experimental medicine interleukin-4 is essential for microglia/macrophage m2 polarization and long-term recovery after cerebral ischemia. stroke; a journal of cerebral circulation il-4 knock out mice display anxiety-like behavior. behavior genetics /pparγ signaling improves white matter repair after stroke role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus neuronal interleukin-4 as a modulator of microglial pathways and ischemic brain damage increased brain injury and worsened neurological outcome in interleukin-4 knockout mice after transient focal cerebral ischemia il-4 in the brain: a cytokine to remember temporal oligodendrocyte lineage progression: in vitro models of proliferation, differentiation and myelination remyelination in experimental models of toxin-induced demyelination quantitative analysis of drug delivery to the brain via nasal route dysmyelination revealed through mri as increased radial (but unchanged axial) diffusion of water myelinated and unmyelinated axons of the corpus callosum differ in vulnerability and functional recovery following traumatic brain injury the oligodendrocyte-specific antibody 'cc1' binds quaking 7 m2 microglia and macrophages drive oligodendrocyte differentiation during cns remyelination an updated assessment of microglia depletion: current concepts and future directions microglia are required for protection against lethal coronavirus encephalitis in mice fast direct neuronal signaling via the il-4 receptor as therapeutic target in neuroinflammation meta-and orthogonal integration of influenza "omics" data defines a role for ubr4 in virus budding reduced il-2 but elevated il-4, il-6, and ige serum levels in patients with cerebral infarction during the acute stage functional polymorphisms of interleukin 4 and interleukin 10 may predict evolution and functional outcome of an ischaemic stroke association of variable number of tandem repeat polymorphism in the il-4 gene with ischemic stroke in the chinese uyghur population regenerating cns myelin-from mechanisms to experimental medicines il-4-induced peroxisome proliferator-activated receptor gamma activation inhibits nf-kappab trans activation in central nervous system (cns) glial cells and il-4/pparγ signaling improves white matter repair after stroke ng2 glia generate new oligodendrocytes but few astrocytes in a murine experimental autoimmune encephalomyelitis model of demyelinating disease chronic oligodendrogenesis and remyelination after spinal cord injury in mice and rats myelin impairs cns remyelination by inhibiting oligodendrocyte precursor cell differentiation hyaluronan accumulates in demyelinated lesions and inhibits oligodendrocyte progenitor maturation chondroitin sulfate proteoglycans in demyelinated lesions impair remyelination peroxisome proliferator-activated receptor gamma (ppargamma): a master gatekeeper in cns injury and repair peroxisome proliferator-activated receptor-gamma agonists promote differentiation and antioxidant defenses of oligodendrocyte progenitor cells rosiglitazone promotes white matter integrity and long-term functional recovery after focal cerebral ischemia cyclin d1 inhibits peroxisome proliferator-activated receptor gamma-mediated adipogenesis through histone deacetylase recruitment stat6 transcription factor is a facilitator of the nuclear receptor ppargamma-regulated gene expression in macrophages and dendritic cells il4i1 augments cns remyelination and axonal protection by modulating t cell driven inflammation mhcii-independent cd4+ t cells protect injured cns neurons via il-4 diffusion tensor mri as a biomarker in axonal and myelin damage neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors functional il-4 receptors on mouse astrocytes: il-4 inhibits astrocyte activation and induces ngf secretion microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia cytokines in liposomes: preliminary studies with il-1, il-2, il-6, gm-csf and interferon-gamma effect of il-4 and il-12 liposomal formulations on the induction of immune response to bovine herpesvirus type-1 glycoprotein d adoptive regulatory t-cell therapy protects against cerebral ischemia selective role of na(+) /h(+) exchanger in cx3cr1(+) microglial activation, white matter demyelination, and post-stroke function recovery tissue plasminogen activator promotes white matter integrity and functional recovery in a murine model of traumatic brain injury key: cord-002021-67ao8chx authors: kim, seong bum; choi, jin young; uyangaa, erdenebileg; patil, ajit mahadev; hossain, ferdaus mohd altaf; hur, jin; park, sang-youel; lee, john-hwa; kim, koanhoi; eo, seong kug title: blockage of indoleamine 2,3-dioxygenase regulates japanese encephalitis via enhancement of type i/ii ifn innate and adaptive t-cell responses date: 2016-04-18 journal: j neuroinflammation doi: 10.1186/s12974-016-0551-5 sha: doc_id: 2021 cord_uid: 67ao8chx background: japanese encephalitis (je), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic je virus (jev). indoleamine 2,3-dioxygenase (ido) has been identified as an enzyme associated with immunoregulatory function. although the regulatory role of ido in viral replication has been postulated, the in vivo role of ido activity has not been fully addressed in neurotropic virus-caused encephalitis. methods: mice in which ido activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. neuroinflammation was evaluated by central nervous system (cns) infiltration of leukocytes and cytokine expression. ido expression, viral burden, jev-specific t-cell, and type i/ii interferon (ifn-i/ii) innate responses were also analyzed. results: elevated expression of ido activity in myeloid and neuron cells of the lymphoid and cns tissues was closely associated with clinical signs of je. furthermore, inhibition of ido activity enhanced resistance to je, reduced the viral burden in lymphoid and cns tissues, and resulted in early and increased cns infiltration by ly-6c(hi) monocytes, nk, cd4(+), and cd8(+) t-cells. je amelioration in ido-ablated mice was also associated with enhanced nk and jev-specific t-cell responses. more interestingly, ido ablation induced rapid enhancement of type i ifn (ifn-i) innate responses in cd11c(+) dendritic cells (dcs), including conventional and plasmacytoid dcs, following jev infection. this enhanced ifn-i innate response in ido-ablated cd11c(+) dcs was coupled with strong induction of prrs (rig-i, mda5), transcription factors (irf7, stat1), and antiviral isg genes (mx1, mx2, isg49, isg54, isg56). ido ablation also enhanced the ifn-i innate response in neuron cells, which may delay the spread of virus in the cns. finally, we identified that ido ablation in myeloid cells derived from hematopoietic stem cells (hscs) dominantly contributed to je amelioration and that hsc-derived leukocytes played a key role in the enhanced ifn-i innate responses in the ido-ablated environment. conclusions: inhibition of ido activity ameliorated je via enhancement of antiviral ifn-i/ii innate and adaptive t-cell responses and increased cns infiltration of peripheral leukocytes. therefore, our data provide valuable insight into the use of ido inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses. japanese encephalitis (je) is an acute zoonotic, mosquitoborne disease caused by je virus (jev), a single-stranded, positive-sense rna (~11 kb, monopartite, linear) virus belonging to the family flaviviridae and the genus flavivirus [1] . infection with neurotropic flaviviruses of the je serotype, which include je, murray valley encephalitis, st. louis encephalitis, and west nile virus (wnv), results in debilitating neurological disorders in a significant proportion of clinical cases [2, 3] . je is a leading cause of viral encephalitis manifested by extensive neuroinflammation in the central nervous system (cns) and disruption of the blood-brain barrier (bbb). in humans, the clinical presentation of jev infection ranges from mild febrile illness to severe meningoencephalitis [4] . due to rapid changes in climate and demography, vector-transmitted je poses an increasing threat to global health and welfare with nearly 70,000 cases reported annually [5] [6] [7] . the incubation period of je ranges from 5 to 15 days, and most jev infections in endemic regions manifest as mild febrile, subclinical disease which leads to protective adaptive immune responses [4] . however, approximately 25-30 % of je cases, mostly in infants, are lethal and 50 % of cases result in permanent neuropsychiatric sequelae [4] . thus, je is considered more fatal than encephalitis caused by wnv infection, which has a fatality rate of 3-5 % (1100 deaths/ 29,000 symptomatic infections) [7, 8] . currently, more than 60 % of the world's population inhabits je endemic areas, such as eastern and southern asia, and the virus is spreading to previously unaffected regions, including indonesia, pakistan, and northern australia [5, 6] . however, despite the importance of je, little is known regarding potential therapeutic strategies for regulating je progression. indoleamine 2,3-dioxygenase (ido) has been identified as an enzyme associated with powerful immunoregulatory function, likely derived from its enzymatic activity, which leads to catabolism of the essential amino acid l-tryptophan (l-trp) [9] [10] [11] [12] [13] [14] . therefore, ido-mediated depletion of l -trp and the resulting metabolites (l-kynurenine, l-kyn) induces an immunosuppressive environment through provoking tolerogenicity of antigenpresenting cells (apcs), t-cell anergy, and immune cell death [9, 10] . ido can be induced in a variety of cell types, including dendritic cells (dcs) [15] , macrophages [16] , and epithelial cells [17] . these cell types play an important role in controlling viral replication and facilitating antigenspecific adaptive immune responses [9, 10] . in various tissues, ido activity has been induced by several cytokines after viral infection, and its enzymatic activity can be blocked using the pharmacological competitive inhibitor, 1-methyl-d,l-tryptophan (1-mt) [18] . thus, inhibition of ido with the competitive inhibitor 1-mt may be a promising strategy for enhancing immune responses in various viral infection models, including human immunodeficiency virus (hiv) and influenza virus [19, 20] . also, ido ablation was shown to suppress viral replication through the upregulation of type i interferon (ifn-i) production in a retrovirus-infected murine model [21] . although the regulatory role of ido in viral replication has been reported in a few studies using an in vivo viral infection model, the in vivo role of ido activity is not fully understood in viral encephalitis caused by infection with neurotropic viruses such as jev and wnv. the molecular pathogenesis of viral encephalitis, including je, remains unclear. however, je is considered an immunopathological disease because cns invasion by jev drives the stimulation of microglia/glia and infiltrating leukocytes, leading to indirect death of neuron cells via the uncontrolled secretion of pro-inflammatory cytokines (such as il-6 and tnf-α) and soluble mediators [22, 23] . furthermore, jev infects and kills neuron cells directly in the cns. this complicated progression of je after viral infection in the host prompted us to explore the role of ido in je progression. here, we found that ido expression in myeloid cells and in neurons of the lymphoid and cns tissues was closely associated with clinical signs of je. furthermore, the inhibition of ido activity using genetic ablation or 1-mt provided enhanced resistance to je, along with a reduced viral burden and the early and increased cns infiltration of myeloid and lymphoid leukocytes. je amelioration was also associated with enhanced nk and antigen-specific t-cell responses. more interestingly, the rapid enhancement of ifn-i innate immune responses in cd11c + dcs (conventional and plasmacytoid dcs) and neuron cells likely contributed to the protective role of ido ablation in je. therefore, our data suggest that the inhibition of ido with pharmacological inhibitors could be a promising therapeutic strategy for regulating je progression. all animal experiments described in the present study were conducted at chonbuk national university according to the guidelines set by the institutional animal care and use committees (iacuc) of chonbuk national university and were pre-approved by the ethical committee for animal experiments of chonbuk national university (permission code 2013-0028). the animal research protocol used in this study followed the guidelines set up by the nationally recognized korea association for laboratory animal sciences (kalas). all experimental protocols requiring biosafety were approved by institutional biosafety committees (ibc) of chonbuk national university. c57bl/6 (h-2 b ) mice (4-5 weeks old) were purchased from samtako (o-san, korea). ido (h-2 b ) knockout (ko) mice were obtained from the jackson laboratory (bar harbor, me, usa). all mice were genotyped and bred in the animal facilities of chonbuk national university. jev beijing-1 strain was obtained from green cross research institute (suwon, korea) and propagated in the mosquito cell line, c6/36, using dmem supplemented with 2 % fbs, penicillin (100 u/ml), and streptomycin (100 u/ml) [24] . the c6/36 cultures were infected with jev beijing-1 at a multiplicity of infection (moi) of 0.1 and were incubated in a humidified co 2 incubator for 1 h at 28°c. after absorption, the inoculum was removed, and 7 ml of a maintenance medium containing 2 % fbs was added. approximately 6-7 days post-infection (dpi), cultures of host cells showing an 80-90 % cytopathic effect were harvested. the virus stocks were titrated using either a conventional plaque assay or a focus-forming assay and were stored in aliquots at −80°c until use. the mabs used for flow cytometric analyses and other experiments were obtained from ebioscience (san diego, ca, usa) or r&d systems (minneapolis, mn, usa): fluorescein isothiocyanate (fitc)-conjugated anti-cd4 (rm4-5), cd8 (53-6.7), ly-6g (1a8), b220 (ra3-6b2); phycoerythrin (pe)-conjugated anti-mouse-cd11b (m1/70), foxp3 (fjk-16s), cd154 (mr1); peridinin chlorophyll protein complex (percp)-conjugated anti-ly-6c (hk1.4); pe-cyanine dye (cy5.5)-conjugated anti-mouse ifn-γ (xmg1.2); pecyanine dye (cy7)-conjugated anti-mouse nk1.1 (pk136), il-5 (trfk4); and allophycocyanin (apc)-conjugated antimouse-cd3ε (145-2c11), cd25 (pc62.5), cd49b (dx5), ly-6g (gr-1), tnf-α (mp6-xt22), il-4 (bvd6-24g2), and il-17a (ebio17b7). the peptides of i-a b -restricted epitopes (jev ns1 132-145 [tfvvdgpetkecpd] and ns3 563-574 [wcfdgprtnail]) and h-2d b -restricted epitopes (jev ns4b 215-223 [savwnstta]) were chemically synthesized at peptron inc. (daejeon, korea). jev-specific primers for the detection of viral rna (jev10,564-10,583 forward, 5′-ccc tca gaa ccg tct cgg aa-3′ and jev10,862-10,886 reverse, 5′-cta ttc cca ggt gtc aat atg ctg t-3′) and primers specific for cytokines, ifn-i (ifnα/β), and rlrs, irfs, isgs (table 1) were synthesized at bioneer corp. (daejeon, korea) and used for pcr amplification of target genes. quantitative real-time rt-pcr for viral burden and cytokine expression viral burden and the expression of cytokines (il-6, tnf-α, and ifn-α/β), cc chemokines, and ido in inflammatory and lymphoid tissues were determined by conducting quantitative sybr green-based real-time rt-pcr (real-time qrt-pcr). mice were infected intraperitoneally (i.p.) with jev (3.0 × 10 7 plaque-forming unit (pfu)) and tissues, including brain, spinal cord, and spleen, were harvested at 2 and 4 dpi following extensive cardiac perfusion with hank's balanced salt solution (hbss). total rna was extracted from tissues using easy-blue (intron, inc., daejeon, korea) and subjected to real-time qrt-pcr using a cfx96 real-time pcr detection system (bio-rad laboratories, hercules, ca, usa). following reverse transcription of total rna with the high-capacity cdna reverse transcription kits (applied biosystems, foster, ca, usa), the reaction mixture contained 2 μl of template cdna, 10 μl of 2× sybr premix ex taq, and 200 nm primers, yielding a final volume of 20 μl. the reactions were denatured at 95°c for 30 s and then subjected to 45 cycles of 95°c for 5 s and 60°c for 20 s. after the reaction cycle was complete, the temperature was increased from 65 to 95°c at a rate of 0.2°c/15 s, and the fluorescence was measured every 5 s to construct a melting curve. a control sample that contained no template dna was run with each assay, and all determinations were performed at least in duplicates to ensure reproducibility. the authenticity of the amplified product was determined by melting curve analysis. all data were analyzed using the bio-rad cfx manager, version 2.1 analysis software (bio-rad laboratories). viral burden was expressed by the copy number of viral rna per microgram of total rna, after calculating the absolute copy number of viral rna in comparison with the standard cdna template of viral rna. the levels of l-kynurenine in the sera and brain homogenates following jev infection were measured via hplc after deproteination using a c18 reverse phase column [25] . the levels of l-kynurenine in the sera and brain homogenates were expressed as micromolars and picomoles per milligram tissue, respectively. levels of l-kynurenine were used for an in vivo index of ido enzyme activity. mice infected with jev were perfused with 30 ml of hbss at 3 or 5 dpi via cardiac puncture of the left ventricle. brains were then harvested and homogenized by gently pressing them through a 100-mesh tissue sieve, after which they were digested with 25 μg/ml collagenase type iv (worthington biochem, freehold, nj, usa), 0.1 μg/ml trypsin inhibitor nα-p-tosyl-l-lysine chloromethyl ketone, 10 μg/ml dnase i (amresco, solon, oh, usa), and 10 mm hepe in hbss for 1 h at 37°c, under shaking conditions. cells were separated using an optiprep density gradient (18/10/5 %) with centrifugation at 800×g for 30 min (axis-shield, oslo, norway), after which cells were collected from the 18 to the 10 % interface and washed twice with pbs. cells were counted and stained for cd11b, ly6g, ly6c, cd3ε, cd4, cd8α, dx5, and nk1.1 with directly conjugated antibodies (ebioscience) for 30 min at 4°c. finally, the cells were fixed with 10 % formaldehyde. data collection and analysis were performed with a facscalibur flow cytometer (becton dickson medical systems sharon, ma, usa) and flowjo (tree star, san carlos, ca, usa) software. jev-specific cd4 + and cd8 + t-cell responses were determined by intracellular cd154 [26, 27] , ifn-γ, and tnf-α staining in response to stimulation with jev epitope peptides. surviving mice infected with 3.0 × 10 7 pfu jev were sacrificed at 7 dpi, and splenocytes were prepared. the erythrocytes were depleted by treating single-cell suspensions with ammonium chloride-containing tris buffer (nh 4 cl-tris) for 5 min at 37°c. the splenocytes were cultured in 96-well culture plates (5 × 10 5 cells/well) in the presence of synthetic peptide epitopes (ns1 132-145 , ns3 563-574 , and ns4b 215-225 ) for 12 and 6 h, in order to observe cd4 + and cd8 + t-cell responses, respectively. monensin at a concentration of 2 μm was added to antigenstimulated cells 6 h before harvest. cells were washed twice with pbs and surface-stained with fitc-anti-cd4 or cd8 antibodies for 30 min at 4°c, and then washed twice with pbs containing monensin. after fixation, the cells were washed twice with permeabilization buffer (ebioscience) and stained with pe cy5.5-anti-ifn-γ or apc-anti-tnf-α in permeabilization buffer for 30 min at room temperature. intracellular cd154 was detected by the addition of cd154 mab to culture media during peptide stimulation. finally, the cells were washed twice with pbs and fixed using fixation buffer. sample analysis was performed with a facscalibur flow cytometer (becton dickson medical systems) and flowjo (tree star) software. intracellular staining for analysis of cd4 + th1, th17, and treg cells to monitor cd4 + th subsets, mice were infected i.p. with 3.0 × 10 7 pfu of jev, sacrificed at 5 dpi, and splenocytes were prepared. splenocytes were then cultured in 96-well culture plates (10 6 cells/well) with pma/ionomycin (th1 and th17) in the presence of monensin (2 μm) for 5 h at 37°c. the stimulated cells were washed twice with pbs and surface stained with fitc-anti-cd4 for 30 min at 4°c and then washed twice with pbs containing monensin. after fixation, the cells were washed twice with permeabilization buffer (ebioscience) and stained with percp-anti-ifn-γ and apc-anti-il-17α in permeabilization buffer for 30 min at room temperature. finally, the cells were washed twice with pbs and fixed using fixation buffer. to monitor treg cells, splenocytes were stained by surface staining for fitc-anti-cd4 markers for 30 min on ice and then fixed with fixation/permeabilization concentrate buffer (ebioscience) for 6 h at 4°c. after fixation, the cells were washed twice with permeabilization buffer (ebioscience) and stained with pe-anti-foxp3 in permeabilization buffer for 30 min at room temperature. the sample analysis was performed using a facscalibur flow cytometer. bone marrow-derived conventional dcs (bmdcs), plasmacytoid dcs (pdcs), and macrophages (bmdms) were prepared from bm cells from bl/6 and ido ko mice, as described earlier with some modifications [24] . briefly, for bmdcs and pdcs, bm cells (3 × 10 5 cells/ml) from femurs and tibiae were cultured in rpmi 1640 supplemented with gm-csf (2 ng/ml) plus il-4 (10 ng/ml) and flt3-l (10 ng/ ml), respectively. on day 3, another 6 ml of fresh complete medium containing gm-csf plus il-4 and flt3-l was added, and half of the medium was changed on day 6. on day 8, non-adherent and loosely adherent dcs were harvested by vigorous pipetting. cells were then characterized by flow cytometric analysis, which revealed that the culture generally consisted of >85 % cd11c + cells (25 % cd11c + cd11b + and 65 % cd11c + cd8α + ), and >90 % cd11c + pdca-1 + b220 + . bmdms were prepared by culturing bone marrow cells in dmem supplemented with 30 % l929 cell-conditioned medium (lccm) as a source of macrophage colony-stimulating factor (m-csf). on day 3, another 6 ml of fresh complete medium containing 30 % lccm was added, and half of the medium was changed on day 6. the cultured cells were harvested following an 8-day incubation and were analyzed by flow cytometry. the prepared bmdms were composed of >85 % f4/80 + cells that consisted of 99.2 % f4/80 + cd11b + and~1 % f4/80 + cd11c + cells. prepared bmdcs, pdcs, and bmdms were infected with jev at mois of 0.1, 1.0, and 10 for analysis of viral replication and 10 moi for evaluating cytokine expression. primary microglia cells were recovered from the brains of fetal bl/6 and ido ko mice (1-3 days old), as described previously [28] . primary microglia cells recovered from the brains via trypsin + edta digestion were initially seeded on poly-d-lysine/laminin-coated plates in dmem containing 10 % fbs and mechanically isolated from glia cells by a brief duration of shaking (100 rpm, 1 h). primary microglia cells were infected with jev at mois of 1.0 and 10 for analysis of viral replication and 10 moi for the evaluation of cytokine, ifn-i, rlr, and isg gene expression. primary cortical neurons were prepared from 15-day-old embryos, as described previously [24] . cortical neurons were seeded in 12-well poly-d-lysine/laminin-coated plates in dmem containing 5 % fbs and 5 % horse serum for 24 h, and then cultured for 4 days with neurobasal medium containing b27 supplement and l-glutamine (invitrogen, carlsbad, ca, usa). primary cortical neurons were infected with jev at mois of 0.001 and 0.01 for analysis of viral replication and 0.1 moi for the evaluation of cytokine expression. generation of bm chimeric mice and determination of serum ifn-β bl/6 mice (5 weeks old) and ido ko mice were irradiated with one dose of 900 rads. within 12 h, recipient mice were reconstituted with 10 7 donor bm cells derived from bl/6 and ido ko mice. the recipient mice were given sulfamethoxazole and trimethoprim in their drinking water for 10 days after irradiation. mice were infected with jev 4-6 weeks after irradiation. a commercial elisa kit (pbl biomedical laboratories, piscataway, nj, usa) was used to measure levels of secreted ifn-β protein in sera according to the manufacturer's protocol. all data were expressed as the average ± standard deviation, and statistically significant differences between groups were analyzed by unpaired two-tailed student's t tests for ex vivo experiments and immune cell analyses or anova and post hoc test for multiple comparisons of the mean. the statistical significance of viral burden was evaluated using the mann-whitney test or unpaired two-tailed student's t test. kaplan-meier survival curves were analyzed with the log-rank test. a p value of ≤0.05 was considered significant. all data were analyzed using prism software (graphpad prism4, san diego, ca, usa). to determine whether ido expression changes during je progression, several tissues obtained from jevinfected bl/6 mice were used to evaluate ido mrna expression at the early stage of infection (from 0 to 3 dpi) prior to the presentation of neurological disorders. as shown in fig. 1a , jev infection induced no apparent changes in the expression of ido in the examined tissues, including lymphoid (spleen, mesenteric ln, bone marrow), extraneural (liver), and cns tissues (brain, spinal cord) prior to the onset of neurological disorders. in general, infected mice showed clinical signs starting with generalized piloerection, paresis, and rigidity, which then progressed to severe neurological signs, such as postural imbalance, ataxia, and generalized tonic-clonic seizure, by 4 to 5 dpi. thus, we were interested in testing whether ido expression varies between mice showing clinical signs, such as paralysis, and mice displaying no clinical signs. to this end, mice were divided into two populations showing either paralysis or no paralysis 5 dpi, the point at which approximately 30-40 % of infected mice showed neurological disorders, and the expression of ido mrna was evaluated in several tissues. interestingly, enhanced induction of ido expression was observed only in lymphoid tissues (spleen and bone marrow) and the cns (brain and spinal cord) of mice showing paralysis compared to mice showing no paralysis (fig. 1b) . however, other extraneural tissues, including mesenteric ln and liver, showed no apparent increases in expression of ido in paralyzed mice. ido expression at the protein level was also confirmed by western blotting using total lysates derived from several tissues. in agreement with the enhanced induction of ido mrna expression in paralyzed mice, mice showing paralysis displayed an apparent increase in the expression of ido protein in lymphoid tissues (spleen, bone marrow) and neural tissues (brain and spinal cord) (fig. 1c) . dcs and macrophages in peripheral tissues are the primary target cells of jev, and neuron cells support jev replication after the virus gains access into the cns. also, microglia, tissue-resident macrophages in the cns are believed to regulate neuroinflammation caused by various insults. therefore, we evaluated ido expression in primary myeloid-derived dcs, pdcs, macrophages, microglia, and primary cortical neuron cells after jev infection. conventional dcs (bmdcs), pdcs, macrophages (bmdms), microglia, and neuron cells displayed different dynamic patterns of ido expression, depending on the time after jev infection (fig. 1d) . pdcs showed the most rapid expression of ido, with levels peaking at 24 h pi, whereas bmdcs displayed a delayed expression pattern with the levels peaking at 48 h pi. ido was also expressed in macrophages with gradual and moderate increases in levels up to 72 h pi, and microglia showed basal levels of ido expression up to 24 h pi and increased levels thereafter. neuron cells showed a gradual increase in ido expression with approximately fivefold higher levels at 72 h pi compared to levels in the mockinfected group. this result indicates that the primary target cells in the periphery and cns tissues can actively express ido with different intrinsic patterns in response to jev infection. collectively, these results indicate that ido expression in myeloid cells and neurons of the lymphoid and cns tissues is closely associated with the clinical signs of je. notably, ido expression in the neural tissues (brain and spinal cord) was likely to be coupled with neurological disorders such as paralysis. blockage of ido provides enhanced resistance to je because ido expression in lymphoid and neural tissues was closely associated with je progression, we were interested in investigating the role of ido during je progression. bl/6 mice infected with jev showed gradually elevated release of the tryptophan catabolite l-kynurenine by cells expressing functional ido enzyme activity in the sera and brain homogenates relative to basal levels in ido ko mice (fig. 2a) , which suggests that ido expression increases during je progression. also, we examined and compared the susceptibility of ido-deficient (ko) mice to je caused by neurotropic jev infection with that of wild-type bl/6 mice (fig. 2b) . the ablation of ido resulted in a markedly increased survival rate of 55 % in ido ko mice vs. 18 % in bl/6 mice after jev infection (3.0 × 10 7 pfu) (left curve in fig. 2b) , thereby signifying significantly enhanced resistance to je. likewise, ido ko mice showed delayed signs of neurological disorder starting 5-6 dpi compared to bl/6 mice, which displayed signs of neurological disorder sooner and in a higher proportion of animals (middle graph in fig. 2b) , and ido ablation resulted in less change in body weight (right graph in fig. 2b) . these results indicate that , and liver (liv), 1, 2, and 3 days following jev infection. b comparison of ido expression between aparalytic and paralytic hosts. ido expression was determined by real-time qrt-pcr using total rna extracted from several tissues in mice showing a neurological disorder such as paralysis (paralytic) and mice showing no paralytic symptoms (aparalytic) 5 dpi. c detection of ido protein during je progression. ido protein levels in several tissues of jev-infected mice were evaluated by western blot using a monoclonal antibody specific for ido. differences in ido protein levels between aparalytic and paralytic hosts were evaluated after mice were divided into groups showing either aparalytic (ap) or paralytic symptoms (p) 5 dpi. d ido expression in primary myeloid cells, microglia, and cortical neurons after jev infection. bone marrow-derived dcs (bmdcs), plasmacytoid dcs (pdcs), macrophages (bmdms), microglia, and primary cortical neurons were infected with jev (10 moi for bmdcs, pdcs, bmdms, and microglia; 0.01 moi for neurons) and ido expression was determined by real-time qrt-pcr at the indicated time points. the levels of ido expression were expressed as fold relative to mock-infected cells (0 h). *p < 0.05; **p < 0.01; ***p < 0.001 compared to levels in the indicated groups ido ablation ameliorates je progression. furthermore, we tested the effect of the ido-specific inhibitor 1-methyl-[d]tryptophan (1-mt) on je progression. as suggested by our other results, oral treatment with 1-mt resulted in reduced mortality, delayed neurological disorder, and less change in body weight compared to untreated bl/6 mice (fig. 2c) . therefore, this result strengthens our finding that ido ablation provides enhanced resistance to je. to better understand je progression in ido-ablated mice, we examined viral burden in peripheral lymphoid and cns tissues after jev infection. ido ko mice were observed to contain less amount of virus, with around a tenfold decrease in the spleen and brain compared to levels in bl/6 mice (fig. 2d) . ultimately, these results suggest that ido ablation ameliorates je progression by regulating the viral burden in peripheral lymphoid and cns tissues. cns infiltration by cd11b + ly-6c hi monocytes is a hallmark of cns inflammation caused by neurotropic viral infection [29] . these cells migrate into the infected brain, where they differentiate into dc, macrophage, and microglia populations [30] [31] [32] [33] . although the potential contribution of cd11b + ly-6c hi monocytes to neuroinflammation remains controversial, cns infiltration by cd11b + ly-6c hi monocytes is believed to support their protective role during lethal neuroinflammation [34] [35] [36] [37] . therefore, in order to better understand the amelioration of je in ido ko mice, we examined cns infiltration by leukocytes, including cd11b + ly-6c hi monocytes, during je progression. wild-type bl/6 and ido ko mice contained comparable levels of cd11b + ly-6c hi monocytes and cd11b + ly-6g hi granulocytes in the brain before jev infection. however, the frequency of cd11b + ly-6c hi monocytes gradually increased in the cns of ido ko mice during je progression (fig. 3a) . also, cns infiltration by cd11b + ly-6g hi granulocytes was transiently increased in ido ko mice 3 dpi, after which the ly-6g hi granulocyte frequency was comparable in both bl/6 and ido ko mice 5 dpi. similarly, the accumulated total number of cns-infiltrated ly-6c hi monocytes and ly-6g hi granulocytes was increased in ido ko mice, compared to those of bl/6 mice (fig. 3b) . furthermore, because cd4 + th1, cd8 + , and nk cells may play beneficial roles in controlling je progression [38] [39] [40] [41] , we examined cns infiltration of nk, cd4 + , and cd8 + tcells. as with cns infiltration by ly-6c hi monocytes, cns infiltration by nk, cd4 + , and cd8 + t-cells was observed to gradually increase in ido ko mice compared to levels observed in bl/6 mice (fig. 3c, d) . notably, cd8 + t-cells showed marked infiltration with threefold increased levels in the cns of ido ko mice. with respect to cns inflammation, the expression of cytokines and chemokines within the cns may be required to fully explain encephalitis, because encephalitis caused by neurotropic viruses is indirectly derived from cns degeneration caused by robust immunological responses, such as the uncontrolled secretion of cytokines, including tnf-α, and the resultant activation of microglia and astrocytes [22, 23] . therefore, we examined the expression of tnf-α and cc chemokines in the cns. our results revealed that the expression of cc chemokines was comparable in both bl/6 and ido ko mice, except that the expression levels of tnf-α and ccl2 were modestly decreased in ido ko mice (fig. 3e) . this result implies that cc chemokine expression may not play a role in increased cns infiltration by ly-6c hi monocytes, nk, cd4 + , and cd8 + t-cells. collectively, these results suggest that ido ablation allows for early and increased cns infiltration by myeloid and lymphoid leukocytes, which may mediate the early control of viral replication in the cns. antiviral innate nk cell activation is believed to play an important role in regulating je progression through the control and clearance of jev in extraneural and neural tissues [38] . therefore, in order to characterize the immunological parameters associated with control of jev replication in ido ko mice, we examined and compared nk cell responses in both bl/6 and ido ko mice. because jev was administered intraperitoneally, analysis of the spleen provides insight into how ido modulates the innate immune and inflammatory responses during the early phase of infection. analysis of splenic nk cells revealed that bl/6 mice exhibited a reduction in cd3 − nk1.1 + dx5 + nk cells following jev infection (fig. 4a) , as (see figure on previous page.) fig. 2 blockage of ido enhances resistance to je and reduces viral burden. a levels of l-kynurenine in the sera and brain of bl/6 and ido ko mice. following jev infection, levels of l-kynurenine in the sera and brain homogenates were estimated by hplc at different time points. b susceptibility of idoablated mice to je. bl/6 and ido ko mice (4 to 5 weeks old, n = 25-30) were inoculated i.p. with jev (3.0 × 10 7 pfu) and examined over 15 days for their survival. c enhanced resistance to je by ido inhibition. bl/6 mice were infected i.p. with jev and administered an ido inhibitor (1-mt, 1 and 2 mg per mouse) every day. left graph, curve showing survival rates; middle graph, proportion of mice showing neurological disorders during je progression every 6 h from 4 to 11 dpi; right graph, changes in body weight. changes in body weight were expressed as the mean percentage ± sd of body weight relative to the time of challenge. d viral burden in lymphoid and inflammatory tissues during je. viral burden in the spleen and brain of infected mice was assessed by real-time qrt-pcr at the indicated time points post-infection. the viral rna load was expressed as viral rna copy number per microgram of total rna (n = 5-7). *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups or in bl/6 control mice if wild-type mice previously showed decreased number of nk cells [24] . however, ido ko mice showed less of a reduction in the number of cd3 − nk1.1 + dx5 + nk cells. consequently, an increase in the total number of splenic nk cells was detected in ido ko mice compared to bl/6 mice. moreover, when the activation of nk cells was evaluated by the production of ifn-γ and granzyme b from nk cells, the frequency and the total number of cd3 − nk1.1 + dx5 + nk cells producing ifn-γ and granyzme b were apparently increased in ido ko mice (fig. 4b, c) . therefore, this result indicates that increased activation of nk cells plays a role in the early control of jev replication, thereby resulting in the amelioration of je progression. in addition to nk cells, adaptive immune responses specific for jev antigen are required for the regulation of je progression through peripheral control of jev replication [38] [39] [40] [41] . furthermore, ido may in some cases affect antigen-specific antibody responses [42, 43] , which contribute to the control of jev dissemination and replication in the brain. our data revealed that ido ablation induced no significant changes in serum igm and igg specific for jev antigen (fig. 5a) . because ido is known to suppress t-cellmediated adaptive immune responses in a range of clinically relevant syndromes, including autoimmune, allergic, and infectious diseases [9, 10] , we also evaluated cd4 + and cd8 + t-cell responses specific for jev antigen in surviving bl/6 and ido-ablated mice 7 dpi. ido ablation resulted in moderately increased jev-specific cd4 + t-cell responses when cd4 + t-cell responses were evaluated by intracellular cd154, ifn-γ, and tnf-α staining in response to stimulation with two epitope-peptides (ns1 132-145 and ns3 563-574 ) derived from jev (fig. 5b) . consistent with this finding, the total number of cd4 + t-cells producing ifn-γ or tnf-α in response to jev epitope stimulation was higher in idoablated mice (fig. 5c) . furthermore, somewhat interestingly, ido-ablated mice displayed markedly increased cd8 + tcell responses with three-to fivefold higher levels, compared to bl/6 mice (fig. 5d) , and consistently contained a higher total number of jev-specific cd8 + t-cells producing the frequency and number of ly-6c hi monocytes and ly-6g hi granulocytes in the cns. the frequency (a) and total number (b) of ly-6c hi monocytes and ly-6g hi granulocytes in the cns were determined by flow cytometric analyses 3 and 5 dpi using vigorous heart perfusion. values presented in the representative dot plots denote the average percentage of the indicated population after gating on cd11b + cells (n = 4-5). c, d accumulated number of nk cells, cd4 + , and cd8 + t-cells in the cns. total accumulated number of nk cells (cd3 − nk1.1 + dx5 + ), cd4 + (cd3 + cd4 + ), and cd8 + (cd3 + cd8 + ) t-cells in the cns were enumerated by flow cytometric analysis 3 and 5 dpi. e the expression of tnf-α and cc chemokines in the cns. the expression levels of tnf-α and cc chemokines were determined by real-time qrt-pcr using total rna extracted from brain tissue 2 dpi. data show the average ± sd of the indicated cell populations derived from at least five mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups ifn-γ or tnf-α in response to a jev cd8 + t-cell epitope (ns4b 215-223 ) (fig. 5e) . these results indicate that the increased cd4 + and cd8 + t-cell responses generated in ido-ablated mice could contribute to the control of je progression during the late stage of infection. cd4 + cd25 + foxp3 + treg cells may contribute to controlling lethal neuroinflammation caused by neurotropic viruses [44] . in contrast, il-17 + cd4 + th17 cells play a critical role in autoimmune and virus-caused immunopathologic diseases by facilitating pathologic consequences through neutrophil recruitment [45, 46] . the generation of cd4 + cd25 + foxp3 + tregs is reciprocally linked with il-17 + cd4 + th17 and ifn-γ + cd4 + th1 cells [47, 48] . because ido may regulate the equilibrium of cd4 + foxp3 + tregs and il-17 + cd4 + th17 cells in inflammatory diseases [49, 50] , we examined the generation of each cd4 + th subset, including cd4 + foxp3 + tregs, il-17 + cd4 + th17, and ifn-γ + cd4 + th1 cells, early during je progression. our results revealed that ido-ablated mice showed no significant alterations in the frequency or number of cd4 + cd25 + foxp3 + tregs, compared to wild-type bl/6 mice (fig. 6a, b) . in contrast, ifn-γ + cd4 + th1 cells were detected at higher levels in ido-ablated mice, and the frequency and number of il-17 + cd4 + th17 cells were not changed by ido ablation (fig. 6c, d) . therefore, this result suggests that the increase in ifn-γ + cd4 + th1 cells in ido-ablated mice contributes in part to the control of je progression during the early stage of infection. myeloid cells, including dcs and macrophages, are the primary target cells of jev infection in the peripheral tissues and function to regulate the spread of virus to distant tissues such as the cns [23] . furthermore, myeloid cells can produce ifn-i via prr recognition upon jev infection, which plays a crucial role in controlling viral replication [51, 52] . additionally, ido ablation appears to regulate viral replication via ifn-i production [21] . because viral loads at the periphery in ido-ablated mice were lower than in wild-type bl/6 mice, we evaluated the contribution of dc subsets (conventional and plasmacytoid) and macrophages to the ifn-i innate immune responses caused by jev infection in the idoablated environment. in order to assess the role of ido in inducing the ifn-i innate response of jev-infected dc subsets and macrophages, bmdcs, pdcs, and macrophages (bmdms) prepared from ido-ablated mice were infected with jev and used to evaluate viral replication and the induction of ifn-i and pro-inflammatory cytokines. interestingly, ido-ablated bmdcs and pdcs, but not bmdms, showed significantly lower jev replication (fig. 7a-c) . notably, bmdcs derived from ido-ablated mice exhibited impaired jev replication throughout the examination period compared to replication in cells from wild-type bl/6 mice. furthermore, the inhibition of jev replication in bmdcs and pdcs derived from idoablated mice was closely associated with enhanced expression of ifn-i (ifn-α/β) following jev infection (fig. 7d) . ido-ablated bmdcs and pdcs, but not bmdms, showed rapid induction of ifn-α/β in response to jev infection compared to levels measured in cells from wild-type bl/6 mice. however, it was curious that pdcs showed less apparent regulation of viral replication than bmdcs, despite pdcs inducing stronger ifn-i innate responses. presumably, this discrepancy is related to cell-intrinsic properties or other mechanisms involved in regulating viral replication. in addition, rapid and increased induction of il-6 and tnf-α mrnas was observed upon jev infection in ido-ablated bmdcs (fig. 7e) . collectively, these results imply that rapid and increased ifn-i innate responses in cd11c + dc subsets (conventional dcs and pdcs) may contribute to the early control of viral replication in the absence of ido. to further characterize ifn-i innate responses in jev-infected dc subsets derived from ido-ablated mice, we also measured the induction levels of isg genes. we specifically focused on pattern recogni). our results revealed that bmdcs and pdcs derived from ido-ablated mice showed rapid and enhanced expression of the stat1, oas1, mx1, mx2, and isg genes with slightly different patterns at 24 h pi, whereas bmdms derived from ido-ablated mice showed either no alteration or a slight decrease in the expression of prrs, ifr3 and irf7, and isg genes (fig. 8) . also, it was interesting that bmdcs and pdcs derived from ido-ablated mice displayed early enhanced induction of prrs (rig-i and mda5) and their transcription factor (irf7) at 24 h pi. this indicates that rapid and increased expression of isg genes in jev-infected bmdcs and pdcs follows the inhibition of jev replication via enhanced expression of ifn-α/β. collectively, ido ablation appears to provide rapid and increased responses of ifn-i innate immunity in myeloid-derived dcs and pdcs upon jev infection, thereby contributing to the amelioration of je through early control of viral replication. microglia cells are cns-resident macrophages that play an important role in regulating the neuroinflammation caused by sterile and non-sterile insults [53] . bystander damage caused by pro-inflammatory mediators released from microglia likely contributes to the exacerbation of je progression. furthermore, because ido-ablated microglia are considered to show stronger inflammatory responses after the virus gains access to the cns, we examined the innate immune responses of primary microglia cells derived from the brain of fetal bl/6 and ido ko mice in response to jev infection. our results revealed that primary microglia recovered from the brain of ido-ablated mice showed similar innate immune responses to jev infection as shown in macrophages derived from bm cells of ido ko mice. ido-ablated microglia were observed to allow jev replication with moderately but not significantly lower levels compared to those of bl/6 mice (fig. 9a) . consistent with this, ido-ablated microglia displayed no apparent enhancement of ifn-i innate responses when the expression of ifn-α/β and isg genes (isg49, isg54, isg56) was examined (fig. 9b, c) . also, no significant differences in the expression of rlrs and transcription factors irf3 and irf7 were observed between microglia derived from ido ko and bl/6 mice (fig. 9d, e) , and both idoablated and wild-type microglia showed comparable expression of pro-inflammatory cytokines tnf-α and il-6 in response to jev infection (fig. 9f ) . therefore, these results indicate that microglia could not provide dominant contribution to the regulation of jev (see figure on previous page.) fig. 5 ido ablation enhances cd4 + and cd8 + t-cell responses specific for jev antigen. a jev e-specific igm and igg response. sera were collected from surviving mice 7 dpi and used in elisa to detect igm and igg levels specific for the jev e protein. the data show the average ± sd of the jev e-specific igm and igg levels derived from surviving mice (n = 6-8). b, c cd4 + t-cell response specific for jev antigen. the splenocytes prepared from surviving mice (n = 4-5) were stimulated with the jev epitope peptides of cd4 + t-cells (ns1 132-145 and ns3 563-574 ) for 12 h. the frequency (b) and absolute number (c) of cd4 + t-cells specific for the jev epitope peptides were determined by intracellular cd154, ifn-γ, or tnf-α staining, combined with surface cd4 staining. d, e cd8 + t-cell response specific for jev antigen. the frequency (d) and absolute number (e) of cd8 + t-cells specific for the jev epitope peptide (ns4b 215-223 ) were determined by intracellular ifn-γ or tnf-α staining after an 8-h stimulation with peptide. values in the representative dot plots denote the average percentage of the indicated cell population, and the bar charts show the average ± sd of the values derived from at least four mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups dissemination and je progression in the cns under an ido-ablated environment. neurons are the main target cell of jev replication within the cns, and their death is a key factor in the pathogenesis and neurological sequelae of jev [23] . furthermore, neuron cells have also been shown to produce antiviral ifn-i in response to viral infection and are consequently involved in controlling viral replication in the cns [54, 55] . therefore, we examined viral replication and ifn-i innate immune responses in primary cortical neuron cells generated from wild-type bl/6 and ido-ablated mice after jev infection. consistent with the results obtained from bmdcs and pdcs, primary cortical neurons derived from ido-ablated mice showed transiently reduced jev replication (fig. 10a) . this transient reduction of jev replication in ido-ablated neurons was associated with significantly increased induction of ifn-i (ifn-α/β), relative to the levels measured in cells from wild-type bl/6 mice (fig. 10b) . also, it seemed that the expression of antiviral isg genes (isg49, isg54, isg59) in ido-ablated neurons followed ifn-i innate responses and the transient reduction in viral replication (fig. 10c) , whereas the expression levels of transcription factors (irf3, irf7), but not prrs (rig-i, mda5), were decreased by jev infection (fig. 10d, e) . therefore, these results suggest that the ablation of ido could provide enhanced ifn-i innate immune responses in neuron cells to regulate the spread of jev in the cns. the frequency and number of cd4 + foxp3 + tregs in the spleen of ido-ablated mice. the frequency (a) and absolute number (b) of cd4 + foxp3 + treg cells in the spleen of wild-type and ido ko mice were determined by flow cytometric analysis 5 dpi. the values in the representative dot plots show the average percentage of cd25 + foxp3 + cells in cd4 + t-cells; the bar charts denote the average number ± sd of cd4 + foxp3 + tregs in the spleen derived from at least four mice per group. c, d the frequency and number of ifnγ + cd4 + th1 and il-17 + cd4 + th17 cells in the spleen of ido-ablated mice. the frequency and number of ifn-γ + cd4 + th1 and il-17 + cd4 + th17 cells were determined by intracellular cytokine staining of the splenocytes prepared from wild-type and ido ko mice 5 dpi in response to pma + ionomycin stimulation. the values in the representative dot plots show the average percentage of the indicated cell populations after gating on cd4 + t-cells; the bar charts denote the average number ± sd of ifn-γ + cd4 + th1 and il-17 + cd4 + th17 cells in the spleen derived from at least four mice per group. **p < 0.01 compared with levels in the indicated groups ido ablation in hsc-derived leukocytes alleviates je via enhancement of the ifn-i innate response our results support the idea that ido ablation ameliorates je progression by provoking potent antiviral ifn-i innate responses in myeloid-derived dcs and pdcs at the periphery. therefore, we were interested in confirming whether myeloid cells derived from hematopoietic stem cells (hscs) play a dominant role in regulating je progression in ido-ablated mice via the induction of enhanced ifn-i innate responses. to this end, we used a bm chimeric model of wild-type bl/6 and ido-ablated mice. in support of our hypothesis, our results revealed that myeloid cells derived from hscs played a dominant role in conferring amelioration of je in the ido-ablated environment. specifically, wild-type bl/6 recipients of ido ko bm donor cells (ko-wt) showed enhanced resistance to je, compared to the ido ko recipients of wild-type bl/6 bm donor cells (wt-ko) and wild-type bl/6 recipients of wild-type bl/6 bm donor cells (wt-wt) (fig. 11a) . furthermore, ko-wt and ko-ko bm chimeric models showed less change in body weight after jev infection compared to the other bm chimeric models (fig. 11b) . also, potent and rapid ifn-i innate immune responses were observed in ko-wt and ko-ko bm chimeric models compared to those observed in the wt-wt bm chimeric model, as evaluated by the determination of serum ifn-β (fig. 11c) . this result indicates that myeloid cells derived from hscs of ido-ablated mice play a dominant role in the ifn-i innate response in ido ko hosts. collectively, it appears that the ablation of ido in myeloid cells derived from hscs has an important role in ameliorating je by inducing a potent and rapid ifn-i innate immune response. in this study, ido expression from myeloid and neuron cells located in extraneural and neural tissues during je progression appears to be closely associated with clinical signs such as neurological disorders. furthermore, we demonstrated that inhibition of ido activity through genetic ablation or use of an ido inhibitor (1-mt) enhanced the resistance to je progression induced by jev infection. in our mechanistic studies on the role of ido in regulating je fig. 8 the expression of rlr, irf, and isg genes in bmdcs, bmdms, and pdcs following jev infection. bmdcs, bmdms, and pdcs recovered from wt and ido ko mice were infected with jev at a moi of 10 and used to analyze the induction of irf, isg, and rlr genes at 24 and 48 h pi. the expression of each irf, isg, and rlr gene was normalized to that of β-actin after determining the mrna levels by real-time qrt-pcr and displayed as the average of at least four independent samples, according to the indicated color on a log 2 scale (see figure on previous page.) fig. 7 ifn-i innate immune responses of ido-ablated myeloid-derived cells after jev infection. primary bone marrow-derived conventional dcs (bmdcs), plasmacytoid dcs (pdcs), and macrophages (bmdms) recovered from bl/6 and ido ko mice were infected with jev at mois of 0.1, 1.0, and 10 for viral replication and 10 for cytokine expression. jev replication and the expression of cytokines and ifn-α/β were evaluated by real-time qrt-pcr using extracted total rna. a-c jev replication in bmdcs, bmdms, and pdcs. d, e ifn-α/β, il-6, and tnf-α expression in bmdcs, pdcs, and bmdms. the bar charts show the average ± sd of the values derived from bmdcs, bmdms, and pdcs assayed in quadruplicates. *p < 0.05; **p < 0.01; p < 0.001 compared with levels in the indicated groups progression, ido-ablated mice showed early and increased cns infiltration by myeloid cells (ly-6c hi monocytes and ly-6g hi granulocytes) and lymphoid cells (cd3 − nk1.1 + dx5 + nk, cd4 + , and cd8 + t-cells), which were associated with a reduced viral burden in the cns. ido ablation increased the activity of cytolytic effector cells following jev infection, due to the enhancement of ifn-γ or granzyme b-producing nk cells and jev-specific cd8 + t-cell responses in ido-ablated mice. in addition, the enhanced production of ifn-γ from cd4 + th1 cells in response to jev antigen might contribute to early viral clearance from extraneural and neural tissues of ido-ablated mice. also, somewhat intriguingly, our data revealed that potent and rapid ifn-i innate immune responses in cd11c + dcs and neuron cells following jev infection could effectively regulate the spread of jev in ido-ablated mice. this finding was supported by the results that ido ablation in myeloid cells derived from hscs played a dominant role in ameliorating je and that ido-ablated hsc-derived leukocytes were major contributors to ifn-i innate responses in the host. collectively, our data suggest that the ablation of ido activity with specific inhibitors, such as 1-mt, could be a valuable therapeutic tool for the treatment of je. ido is considered a negative regulator of the immune system because ido activity in apcs, including dcs and macrophages, is a potent mechanism for inducing immunosuppression and tolerance. therefore, it is probable that the attenuation of immunosuppressive ido activity leads to enhanced innate and adaptive immune responses for viral clearance. indeed, the inhibition of ido activity with a specific inhibitor resulted in enhanced cd4 + th1type and cd8 + t-cell responses specific to the influenza virus [20] . furthermore, the inhibition of ido activity reportedly modifies the immunodominance hierarchy by increasing the number of cd8 + t-cells specific to a subdominant epitope derived from the influenza virus. the ablation of ido also suppresses viral replication in retrovirus-infected mice through ifn-i production [21] . these findings strongly support our data that ido ablation ameliorates je progression through enhanced type i/ ii ifn innate and adaptive immune responses. although the in vivo role of ido after viral infection has been investigated in a few studies using murine infection models, the regulatory role of ido in neuroinflammation caused by neurotropic viruses, such as jev, has not been addressed to date. to the best of our knowledge, our findings provide the first evidence that ido inhibition could regulate the in vivo progression of viral encephalitis caused by neurotropic viruses such as jev and wnv. the molecular pathogenesis of je remains unclear, but je is considered an immunopathological disease since uncontrolled over-activation of microglia/glia and infiltrated leukocytes after cns invasion of jev drives neurological disorders [22, 23] . although cns infiltration by peripheral innate and adaptive immune cells appears to play a critical role in host defense against infections with neurotropic viruses such as jev, control of cns infiltration is also required because excessive or inappropriate cns infiltration can cause profound damage. therefore, the outcome of je pathogenesis appears to depend on the complicated interplay between virus-induced tissue damage and host immune response against jev antigens. cns infiltration by cd11b + ly-6c hi monocytes is a hallmark of neuroinflammation caused by neurotropic viruses. these cd11b + ly6c hi monocytes migrate into the inflamed cns, where they differentiate into dcs, macrophages, and microglia to regulate neuroinflammation [30] [31] [32] [33] [34] [35] [36] [37] . although conflicting roles have been postulated for ly-6c hi monocytes in cns inflammation, cns infiltration by cd11b + ly-6c hi monocytes is essential in the control of neuroinflammation caused by neurotropic viruses, which supports their protective role during cns inflammation [34] [35] [36] [37] . notably, the differentiation state of ly-6c hi monocytes that infiltrate into the cns appears to affect the progression of neuroinflammation caused by various insults [56] [57] [58] . supportively, ido ablation was observed to lead to enhanced cns infiltration of cd11b + ly-6c hi monocytes, which may contribute to a beneficial outcome in je. furthermore, early and increased infiltration of nk, cd4 + , and cd8 + t-cells in the cns of ido-ablated mice appears to play a role in reducing the viral burden, because infiltration of nk, cd4 + th1, and cd8 + t-cells producing ifn-γ is required for early clearance of the virus from the cns [38] [39] [40] [41] . after peripheral introduction of jev via mosquito bites, jev initially replicates in its primary target cells, including dcs and macrophages, at the periphery and subsequently gains entry into the cns through the bbb. presumably, jev-specific cd4 + th1 and cd8 + t-cell responses generated in the peripheral lymphoid tissues of ido-ablated mice would likely provide more effective control of viral replication, thereby reducing the amount of virus supplied from the periphery. in support of this notion, our data revealed that the viral burden in the extraneural and neural tissues of ido-ablated mice were lower than those in wild-type bl/6 mice. the depletion or adoptive transfer of specific cell populations of nk and cd8 + t-cells was previously reported to provide no significant contribution to host survival and viral clearance [38] . furthermore, co-transfer of immune cd4 + and cd8 + tcells, but not individual transfer of either t-cell subpopulation, significantly contributed to a favorable je outcome and promoted host survival [40] . these facts suggest the possibility that enhancement of broad immunity, including nk, cd4 + th1, and cytotoxic cd8 + t-cells, in idoablated mice accounts for the effective regulation of early viral clearance in extraneural and neural tissues, thereby providing protection against je progression without tissue injury. however, ido ablation did not result in any significant change in the humoral immune responses specific for jev antigen. the effects of ido on the humoral immune response appear to be variable, depending on the experimental model used [42, 43] . because b-cell-deficient (μmt) mice uniformly succumbed to viral encephalitis caused by flaviviruses in a previous study, one obvious conclusion is that an early and sustained humoral immune response is absolutely essential to surviving infection with jev and other related flaviviruses [59] . this notion suggests a limitation that ido inhibition may not provide the perfect protection against je progression due to the lack of substantial enhancement in the humoral response against jev antigen. thus, further studies on how to enhance the humoral immune response are needed in the context of ido inhibition. the requirement of ifn-γ in the recovery from infections with flaviviruses has been shown to be variable. ifn-γ provides an early protective immune response against a virulent north american isolate of wnv [60] and mouseadapted strains of dengue virus [61, 62] , whereas ifn-γ is dispensable in the control of infection with less virulent strains of wnv [63] or of yellow fever virus [64, 65] . additionally, ifn-γ shows only a modest protective role against murray valley encephalitis [66] . similarly, in the je model, fig. 11 ido ablation in hsc-derived leukocytes alleviates je via enhancement of the ifn-i innate response. bm cells from wt or ido ko mice were grafted to lethally irradiated wt or ido ko recipient mice, which were then infected with jev (3.0 × 10 7 pfu). a susceptibility of ido ko bm chimeric models to je. infected recipient mice (n = 12) were examined over 18 days to determine the survival rate. b changes in body weight. data are expressed as the average percentage of body weight relative to the time of challenge. c systemic ifn-β levels in an ido ko bm chimera. the amount of serum ifn-β was determined by elisa at the indicated time points. *p < 0.05; **p < 0.01 compared with levels in wt-wt bm chimera 48 h pi. #p < 0.05; ##p < 0.01 compared with levels in wt-wt bm chimera 72 h pi il-12-mediated induction of ifn-γ has been reported to result in suppressed protective immunity [67] , whereas ifn-γ was associated with a beneficial effect on the outcome of je [38] . our data favor the latter result, showing a beneficial role of ifn-γ in je progression, because ido ablation markedly increased ifn-γ-producing nk, cd4 + th1, and cd8 + t-cells. ifn-γ is believed to play diverse roles in infectious diseases, including the activation and polarization of cd4 + th cells, upregulation of fas in infected target cells, upregulation of mhc i and ii-restricted ag-presentation pathways, macrophage activation, and direct antiviral activity that overlaps with activities triggered by ifn-i [68] . also, ifn-γ produced from cd4 + th1 and cd8 + t-cells in ido-ablated mice is believed to induce the maturation of ly-6c hi monocytes, which may contribute to better je outcomes [69, 70] . however, ifn-γ-producing nk cells do not appear to significantly contribute to host survival, because nk-cell-depleted mice showed no change in viral burden or survival [38] . furthermore, flaviviruses including wnv exhibit immune escape from nk cell attack via the upregulation of mhc-i in infected cells [71] . in contrast, antigen-specific cd8 + t-cell cytolytic activity on infected target cells is thought to play a crucial role in disease recovery, given that depletion of cd8 + t-cells resulted in an increased viral burden in the cns [40] . supportively, our data revealed that ido ablation provided marked enhancement of cd8 + t-cells producing ifn-γ and tnf-α upon jev ag stimulation. in addition, ido ablation induced an increase in cd4 + th1 cells producing ifn-γ at the early stage, but il-17 + cd4 + th17 and cd4 + foxp3 + tregs were not altered by the ablation of ido. collectively, these findings suggest that ifn-γ produced from cd4 + th1 and cd8 + t-cells play an important role in regulating je progression in ido-ablated mice. the role of ido in antiviral activity has been postulated in different in vitro experiments. for example, ido activity in astrocytes is induced by the tlr3 ligand polyinosinicpolycytidylic acid, which is involved in antiviral function via the exhaustion of the essential amino acid l-trp in the environment [72] . this finding is inconsistent with our data, but it is notable that this finding was derived from an in vitro model. the intriguing result in the present study was that ido-ablated cd11c + dcs, including conventional and plasmacytoid dcs, showed potent and rapid ifn-i innate immune responses to jev infection. also, ko-ko and ko-wt bm chimeric models showed systemic ifn-β production at levels higher than in wt-wt and wt-ko bm chimeric models. this supports the notion that myeloid cells derived from hscs, including cd11c + dcs, may dominantly contribute to the rapid ifn-i innate immune response. this result is strengthened by the finding that ido ko and 1-mt-treated mice showed induction of a markedly increased ifn-i innate response in the spleen following retrovirus infection [21] . conventional and plasmacytoid dcs exhibited potent and rapid ifn-i innate responses with different dynamic patterns in the expression of prrs, transcription factors, and isg genes following jev infection. these different ifn-i innate responses in both types of cd11c + dcs are thought to be derived from the intrinsic properties of the specific cell type. although we did not investigate the detailed mechanism by which idoablated cd11c + dcs displayed a potent ifn-i response to jev infection, our data suggest the involvement of prrs (rig-i, mda5) and transcription factor irf7 because the expression levels of rig-i, mda5, and irf-7 were rapidly enhanced in ido-ablated cd11c + dcs. also, considering that only a small fraction (10-20 %) of myeloid-derived cells are infected by jev [73] , uninfected myeloid-derived cells are thought to contribute substantially to the induction of antiviral isgs through early stimulation of the stat1 transcription factor which induces isg49, isg54, and isg56 [24] . also, it is worth noting that primary cortical neuron cells derived from ido-ablated mice showed potent ifn-i innate responses resulting in delayed replication of jev. although the induction patterns of prrs and their transcription factors in neuron cells differed from those of cd11c + dcs following jev infection, the induction of isgs in ido-ablated neurons definitely followed potent ifn-i innate immune responses, such as ifn-αβ production. however, because cortical neurons are relatively insensitive to the antiviral effects of ifn-i [74] , the regulation of viral replication in primary cortical neurons by ifn-i innate responses may ultimately show some limitations. indeed, our data indicating that primary cortical neuron cells derived from ido ko mice showed transient inhibition of viral replication support this speculation. therefore, further studies are needed to define the mechanistic and functional intermediates that link ido to the regulation of ifn-i innate immune responses in different cell types. je pathogenesis in the murine model may be affected by the route of administration, dosage, and strain of the virus, and the virus propagation conditions used [23] . ido was expressed with different dynamic patterns in conventional and plasmacytoid dcs, macrophages, and neuron cells upon jev infection. thus, it is thought that je pathogenesis may also be affected by how the innate responses of specific cell types are exhibited after jev infection. rapid ifn-i/ii innate immune responses are considered more crucial in the control of je progression than adaptive tcell responses, which take time to develop. considering that ido ablation provided early and enhanced ifn-i/ii innate responses and subsequent adaptive t-cell response in the host, the inhibition of ido may be a valuable tool for 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with protective hiv-specific cd8(+) t-cell responses and disease progression the regulation of the treg/th17 balance by mesenchymal stem cells in human systemic lupus erythematosus ido upregulates regulatory t cells via tryptophan catabolite and suppresses encephalitogenic t cell responses in experimental autoimmune encephalomyelitis leukocytederived ifn-alpha/beta and epithelial ifn-lambda constitute a compartmentalized mucosal defense system that restricts enteric virus infections the yin and yang of viruses and interferons systemic inflammation and microglial activation: systematic review of animal experiments neurons produce type i interferon during viral encephalitis soluble amyloid triggers a myeloid differentiation factor 88 and interferon regulatory factor 7 dependent neuronal type-1 interferon response in vitro activation of invariant nkt cells in early phase of experimental autoimmune encephalomyelitis results in differentiation of ly6chi inflammatory monocyte to m2 macrophages and improved outcome induction of inhibitory central nervous system-derived and stimulatory blood-derived dendritic cells suggests a dual role for granulocytemacrophage colony-stimulating factor in central nervous system inflammation the blood-brain barrier induces differentiation of migrating monocytes into th17-polarizing dendritic cells b cells and antibody play critical roles in the immediate defense of disseminated infection by west nile encephalitis virus gamma interferon plays a crucial early antiviral role in protection against west nile virus infection ifngamma production depends on il-12 and il-18 combined action and mediates host resistance to dengue virus infection in a nitric oxide-dependent manner interferondependent immunity is essential for resistance to primary dengue virus infection in mice, whereas t-and b-cell-dependent immunity are less critical cd8(+) t cell-mediated immune responses in west nile virus (sarafend strain) encephalitis are independent of gamma interferon yellow fever virus encephalitis: properties of the brainassociated t-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for cd4+ lymphocytes a mouse model for studying viscerotropic disease caused by yellow fever virus infection role of type i and type ii interferon responses in recovery from infection with an encephalitic flavivirus suppression of immune response and protective immunity to a japanese encephalitis virus dna vaccine by coadministration of an il-12-expressing plasmid interferons: success in anti-viral immunotherapy ifn-gamma, as secreted during an alloresponse, induces differentiation of monocytes into tolerogenic dendritic cells, resulting in foxp3+ regulatory t cell promotion timing and intensity of exposure to interferon-gamma critically determines the function of monocyte-derived dendritic cells mhc class i up-regulation by flaviviruses: immune interaction with unknown advantage to host or pathogen astrocyte indoleamine 2,3-dioxygenase is induced by the tlr3 ligand poly(i:c): mechanism of induction and role in antiviral response impaired cross-presentation of cd8alpha + cd11c + dendritic cells by japanese encephalitis virus in a tlr2/ myd88 signal pathway-dependent manner pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons challenges in the discovery of indoleamine 2,3-dioxygenase 1 (ido1) inhibitors research progress of indoleamine 2,3-dioxygenase inhibitors this study was supported by a national research foundation of korea (nrf) grant funded by the korean government (misp) (2013r1a4a1069486). the funder had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript. cns infiltration by ly-6c hi monocytes. also, ido ablation may accelerate viral clearance from the host at a later stage via the enhancement of jev-specific ifn-γ-producing tcell responses. therefore, these results suggest that inhibition of ido activity by specific inhibitors [75, 76] may be a promising tool for therapeutic and prophylactic strategies against je. the authors declare that they have no competing interests.authors' contributions sbk and jyc conceived the study and discussed the data with ske. eu, amp, and fmah conceived the study and contributed to the experimental design. jh, syp, jhl, and kk contributed to the reagent/materials/analysis and tools and provided critical conceptual guidance. sbk and ske wrote the paper with contributions from all authors. all of the authors reviewed and approved the final version of the manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-017958-18nnwoav authors: chan, andrew; gold, ralf title: apoptotic cell death in experimental autoimmune encephalomyelitis: apoptosis of effector cells as a safe mechanism in the termination of an autoimmune inflammatory attack date: 2005 journal: experimental models of multiple sclerosis doi: 10.1007/0-387-25518-4_24 sha: doc_id: 17958 cord_uid: 18nnwoav particularly in the vulnerable cns with a low capacity for regeneration specialized mechanisms must be active for the fast and gentle elimination of dysregulated autoaggressive immune cells. in eae, local apoptosis of autoimmune t-cells has been identified as a safe means for the removal of these unwanted cells. t-cell apoptosis in situ followed by phagocytic clearance of apoptotic remnants by glia assures a minimum of detrimental bystander damage to the local parenchyma and down-regulates the local inflammatory reaction. the pharmacological augmentation of local apoptosis of inflammatory effector cells might gain therapeutic importance also in human neuroimmunological diseases such as multiple sclerosis. multicellular organisms face the task of safely disposing unwanted cells under physiological and pathological conditions. the active and tightly regulated cell death program during apoptosis is considered to play a major role in the physiological control of cell turnover while at the other end dysregulation of apoptosis has been shown to contribute to the pathogenesis of different autoimmune diseases. 508 during the last decade the role of apoptosis also in neuroimmunological diseases has been more clearly defined. in eae and experimental autoimmune neuritis (ean), especially apoptosis of the pathogenic effector cells has been thoroughly investigated. this review will briefly introduce definitions and detection methods for apoptosis. we will focus on apoptotic cell death of effector t-cells in eae, proposed mechanisms of t-cell apoptosis, functional consequences and finally its potential therapeutic implications. apoptosis is a specialized, morphologically and biochemically distinct form of eukaryotic cell death. introduced in 1972 by kerr and colleagues, the term apoptosis served to describe a set of morphological features commonly observed during cell death in various tissues and cell types which were different to those observed during necrotic cell death (1) . the stereotypic series of cellular changes comprise blebbing of the plasma membrane and condensation of chromatin at the periphery of the nucleus, followed by disintegration of the cell into multiple membrane-enclosed vesicles. in contrast to the lytic processes observed during necrosis, cell death by apoptosis is not associated with secondary inflammatory tissue responses, e.g. towards potentially harmful intracellular contents of the dying cells (2) . thus, apoptosis is considered to provide a safe and gentle cell death mechanism. in vivo, apoptosis also appears to be a fast and efficacious mechanism for the elimination of unwanted cells, with completion of the cell death program within 4-5 hrs at least in certain tissues and under defined conditions (3). as the traditional definitions of apoptosis were mainly based on morphological criteria, electron microscopy served as the gold standard in the detection of apoptosis. meanwhile, the better understanding of underlying biochemical processes during apoptosis has led to the introduction of an array of detection methods at the molecular level (4, 5) . for example, intravital internucleosomal dna-fragmentation which generates fragments of 180 bp and multiples thereof can reliably be detected using tunel or in situ nick translation techniques. the identification of a set of aspartate-directed cysteine proteases (caspases) whose activation underly many of the observed morphological changes during apoptosis has further broadened the spectrum of detection methods. thus, either the activated caspases or their proteolytic cleavage products (e.g. cytokeratin 18, poly(adp-ribose)-polymerase parp, app, actin cleavage products) can be identified. also specific alterations of the cell membrane (e.g. loss of a24. apoptotic cell death in experimental autoimmune encephalomyelitis 509 phospholipid asymmetry detected with annexin-v) or mitochondrial changes (e.g. cytochrome c release, "permeability transition") serve as the basis for commercially available detection methods for apoptosis. however, many of these detection methods have to be interpreted cautiously (4) . for example dna-fragmentation does not only occur during apoptosis, but also during necrosis. since different intracellular pathways lead to typical apoptotic features, a combination of different molecular detection methods is feasible. thus, typical morphological features of apoptosis can also occur in the absence of oligonucleosomal dna-fragmentation as well as in enucleated cells (6,7). t-cells autoreactive to cns-antigens such as mbp can also occur in healthy individuals (8, 9) . albeit more often in ms-patients, surges of increased frequencies of circulating myelin-reactive t-cells can also be observed in healthy subjects, possibly driven by cross-reactive environmental antigens (10) . these activated autoreactive t-cells are capable of entering the cns-parenchyma and thus have the potential to induce a local immune response when they encounter their specific antigen in the context of appropriate restriction molecules (11, 12) . therefore, specialized anatomic barriers such as the blood brain barrier or the absence of lymphatic drainage are not sufficient to prevent immune-mediated damage in the cns, arguing for other mechanisms in the physiological control of autoreactive inflammatory cells in situ. local apoptosis of pathogenic t-cells has been identified as a major immunological defense mechanism in the "immuneprivileged" cns, leading to an inhibition of inflammation or, once it has encroached, to rapid and non-destructive elimination of the inflammatory infiltrate (review in (13, 14) . first reports on cell death by apoptosis in inflammatory brain lesions of lewis rat eae were from pender and colleagues (15) . using morphological criteria the majority of the dying cells appeared to be lymphocytes and oligodendrocytes. apoptosis of ~13-t-cells was subsequently confirmed by the same group using pre-embedding immunolabelling techniques (16) . by combined t-cell immunohistochemistry, molecular labeling techniques and ultrastructural criteria, schmied and coworkers analyzed t-cell apoptosis in the spinal cord quantitatively during the time course of different rat eae models (17) . of all apoptotic cells, 64% were identified as t-lymphocytes, mostly expressing the ~[3-t cell receptor. while another 9% of the apoptotic cells were classified as oligodendrocytes, apoptosis of macrophages was only rarely observed. however, a considerable proportion of apoptotic cells could not be identified immunocytochemically, due to the advanced degeneration of the cells. during lewis rat cell transfer (at)-eae using mbp-specific t-cells the degree of t-cell apoptosis was minimal at early stages on day 4, but peaked at the time of recovery from disease at day 7 with apoptosis rates of up to 49% (figure 1). also in the active disease model prevalence of t-cell apoptosis in situ was highest when animals had recovered from the disease. apoptosis of inflammatory cells in situ also occurs in chronic relapsing eae models, especially during clinical relapses (15, 19, 20, 21) . in the cns of adoptively transferred chronic relapsing eae in sjl/j mice, apoptosis of cd4+ t-cells and microglia/brain macrophages could readily be observed, while oligodendrocytes and astrocytes did not exhibit tunel positivity (19) . t-cell apoptosis in situ has not only been identified in eae but also in coronavirus-mediated encephalomyelitis (22) . apoptosis of t-lymphocytes also occurs in inflammatory human brain lesions, most prominently in acute disseminated leukoencephalomyelitis (adem) (23) , an acute monophasic disease which closely mimics pathological changes of acute eae. t-cell apoptosis can also be observed in active ms lesions albeit to a lesser extent, possibly due to the chronicity of the disease (24) . the similar degree of tcell apoptosis in the acutely autoimmune inflamed rodent (eae) and human in contrast to the high t-cell apoptosis rate observed in the cns, the pns and other peripheral tissues appear to have a lower capacity to induce t-cell apoptosis locally. t-cell apoptosis could also be observed in sciatic nerves of lewis rats with active or at-ean (26) . whereas the time course was similar to eae, the extent of t-cell apoptosis was markedly different: highest levels were also found during recovery, with typical t-cell rates of approximately 10% (figure 1). however, in spite of this lower apoptosis rate, also in the pns local t-cell apoptosis can be regarded as an efficient mechanism of cell elimination. thus, given a time frame of 4-5 hrs for the completion of apoptosis, one could assume that up to 50% of an inflammatory infiltrate would be eliminated within 24 hrs. negligible degrees of t-cell apoptosis were found in t-cell mediated autoimmune diseases of muscular tissue. t-cell apoptosis was less than 0.5% in cd8-t-cell dominated experimental autoimmune myositis and biopsies of different human myopathies of presumed autoimmune etiology (27, 28) . thus, t-cell inflammation in muscle is not cleared by apoptosis in situ, which could contribute to the non-self-limiting nature of these diseases. even in hiv-associated polymyositis and -neuropathy there is virtually no relevant t-cell apoptosis in situ, in spite of the pathophysiological relevance of t-cell apoptosis in hiv-infection (29) . also t-cell infiltrates in the skin do not appear to be eliminated by local apoptosis. in lewis rat eae, there was negligible t-cell apoptosis in the dermal tissue adjacent to the sensitization site, despite a heavy t-cell infiltration (17) . furthermore, in skin biopsies of patients with dermatomyositis and lupus erythematosus, t-cell apoptosis was virtually absent (30)(chan, gold, unpublished observations). given the obvious disparity in t-cell apoptosis between the "immunoprivileged" cns and other non-immunologically protected sites, tissue-specific local mechanisms must be active in the cns, that lead to a very efficient apoptotic clearance of pathogenic t-cells. moreover, using mbp-and ovalbumin-specific t-cells, it appeared that mbp-specific t-cells were readily eliminated from the cns by apoptosis, while t-cells specific for ovalbumin, an antigen not present in the lewis rat, survived in the cns and recirculated to peripheral lymphoid organs. however, these studies could not exclude that also ovalbumin specific tcells underwent apoptosis in the cns. also, altered physicochemical properties of collapsed apoptotic cells could have precluded their quantitative recovery from spinal cord during gradient centrifugation. moreover, other mbp-reactive t-cell receptor elements besides v]38.2, proven to be encephalitogenic were not analyzed in these studies (33, 34) . using t-cells stably expressing specific genomic markers, lassmann and colleagues could demonstrate that both mbp-specific and ovalbuminspecific t-cells undergo apoptosis in the cns of eae-animals (35) . in addition, the occurence of t-cell apoptosis during eae in bone marrow chimeras with different mhc-haplotypes of the resident glial cells and the passively transferred t-cells indicates that t-cell apoptosis is not dependent on antigen-specific mechanisms (35) . thus, both encephalitogenic, antigenspecific t-cells as well as secondarily recruited bystander t-lymphocytes appear to undergo apoptosis in situ. the molecular mechanisms leading to t-cell apoptosis in the cns in situ are as yet incompletely understood (14) . in vivo, the tnf-receptor-1 (tnfr1)mediated death pathway appears to play a major role in the induction of tcell apoptosis in the cns (36) . thus, in eae of tnfri-or tnf/lymphotoxin-deficient mice, local t-cell apoptosis was decreased. tnf-c~ has been demonstrated to be a potent inducer of t-cell apoptosis (37) . following this line, administration of anti-tnf-c~ antibody decreases local t-cell apoptosis in eae of lewis rats undergoing high-dose antigentherapy with mbp, where the release of high amounts of tnf-c~ can be observed in situ (38) . t-cell apoptosis in the cns may also be mediated via the fas/fas ligand (cd95/cd95l)-pathway (review in (14, 39) ). thus, one proposed mechanism is the ligation of t-cell cd95 by cd95l expressed by host cells (41) . since t-cell apoptosis appears to be tissue specific, resident glial cells might sensitize t-cells towards cell death directly or through soluble factors. antigen presentation to mbp-specific t-cells by rat microglia results in tcell apoptosis in vitro, which can be prevented by exogenous il-2 (42) . also astrocytes, which act as non-professional antigen presenting cells (apc), render t-cells susceptible to apoptosis induced by glucocorticosteroids in vitro (43) . glucocorticosteroids markedly increased t-cell apoptosis when added to t-cell astrocyte co-cultures during late t-cell activation stages whereas there was no effect when thymus cells were used as antigen presenters. these results could argue for a scenario, where an infiltrating t-cell is primed for an apoptotic stimulus by a resident nonprofessional apc, and subsequently undergoes apoptosis under hormonal influences of the microenvironment or systemic changes. however, t-cell apoptosis during recovery from eae also occurs in the cns of adrenalectomized animals, arguing for additional mechanisms (44) . among others, further proposed mechanisms of local cns t-cell apoptosis comprise the action of reactive oxygen intermediates and nitric oxide, or cell death due to deprivation of growth factors such as il-2 (45, 46) . yet, the precise role of the different putative mechanisms as well as other to date unknown cns-specific factors in the induction of t-cell apoptosis remain to be elucidated. the high degree of apoptosis of inflammatory cells in the autoimmune inflamed human and rodent cns raises the need for an efficient and tightly controlled removal mechanism for these unwanted cells. a key event in the resolution of an inflammatory infiltrate is the nonphlogistic and thus safe phagocytic clearance of dying, yet intact leukocytes undergoing apoptosis (review in (47, 48) . apoptosis labels unwanted cells with signals that direct the recognition, uptake and subsequent degradation by tissue-specific phagocytes, thus preventing the spilling of potentially harmful contents and inhibiting secondary immune responses directed towards the dying cells. in addition to the mere clearing of cell remnants, the ingestion of apoptotic cells also actively elicits phagocyte responses that modulate immune reactions and inflammation. in lewis rat eae, phagocytosis of apoptotic lymphocytes by macrophages/microglia, oligodendrocytes and astrocytes in situ has first been described by electron microscopy (49) . using an in vitro phagocytosis model, we have further investigated the phagocytosis of apoptotic t-cells by different glial cell elements and its functional consequences (50) . lewis rat microglia efficiently phagocytose specifically apoptotic, encephalitogenic mbp-specific t-cells. this process is differentially regulated by thl-/th2type cytokines (51) . the phagocytosis of apoptotic t-cells by lewis rat microglia is more efficient than by other glial cell elements such as astrocytes. moreover, phagocytosis of apoptotic t-cells leads to a profound downregulation of microglial immune functions with a suppression of proinflammatory cytokines and microglial t-cell activation, thus silencing the microglial phagocyte in the inflammatory context (52) . figure 2 . t-cells undergoing apoptosis exhibit specific "eat me" recognition signals for phagocytes. phagocytosis by microglia is differentially regulated by cytokines ("waste disposal"). the uptake of apoptotic cells leads to a down-regulation of different microglial immune-functions ("modulation of immune responses"). astrocytes have a lower capacity to phagocytose apoptotic cells and may provide a "backup" mechanism. modified from (50) . in vitro, rat microglia/brain macrophages have also been demonstrated to phagocytose apoptotic neurons via lectin-, integrin-, and phosphatidylserinedependent mechanisms (53) . 515 current therapeutic approaches in presumably immune-mediated, demyelinating diseases of the cns such as ms aim at the rapid termination of the inflammatory process, thereby hastening clinical recovery and potentially also preventing subsequent demyelinative and axonal tissue damage (54) . glucocorticosteroids (gs) are potent anti-inflammatory drugs, whose therapeutic efficacy in neuroimmunological diseases has especially been established in immune neuropathies, ms, lupus erythematosus and cerebral vasculitis. intravenously administered high-dose gs serve as the current mainstay in the therapy of acute ms-relapses (55) . gs can mediate their pleiotropic anti-inflammatory effects via different mechanisms, e.g. modulation of cell activation, cytokine expression, secretion of inflammatory mediators, leucocyte migration and the reduction of local edema (56) . these effects are mediated via the cytosolic gs-receptor (gsr) and can be blocked by the gsr antagonist ru 468 at low gs concentrations. at higher concentrations, gs appear to induce cell death directly, possibly via nongenomic, physicochemically mediated effects on the plasma membrane (56) . in lewis rat eae, iv. methylprednisolone (mp) therapy administered at the clinical disease maximum increased t-cell apoptosis in the spinal cord in situ and decreased t-cell infiltration (57) . this effect was clearly dosedependent with a dosage of at least 10 mg/kg body weight mp required to achieve an increase of t-cell apoptosis and a marginal decrease in t-cell infiltration. higher dosages of 50 mg/kg body weight mp were superior in both respects, and were also effective in mild eae, in contrast to the lower dosage of mp. the strong apoptosis-promoting effect of gs has also been demonstrated on peripheral blood leukocytes (pbl) of ms-patients (58) . after intravenous high-dose corticosteroid treatment, apoptosis of pbls was markedly augmented in different ms-subgroups, predominantly affecting cd4-positive t cells. also in the peripheral nervous system, high-dose gs augment t-cell apoptosis (59) . in lewis rat ean, a 4-5-fold increase of t-cell apoptosis could be observed in the sciatic nerve after therapeutic administration of gs (10 mg/kg body weight prednisolone). similar results could be observed in lewis rat experimental autoimmune myositis (eam), a model for human idiopathic myosites of presumed autoimmune-inflammatory etiology (27) . here, a clear increase of apoptotic endomysial t-cells could be demonstrated, with up to 50% of these apoptotic t-cells being cd8-positive. this indicated that glucocorticosteroids also induce cd8-t-cell apoptosis, even in organs where normally t-cell apoptosis does not occur. the type 1 interferon (ifn) ifn-~, is one of the current mainstays in the immunomodulatory therapy of ms (60) . ifn-~ appears to affect particularly inflammatory aspects of ms, with a reduction of the relapse rate and positive effects on inflammatory changes observed in mri scans. in the experimental models, ifn-[3 has been shown to inhibit progression of relapsing-remitting eae (61), while early discontinuation of ifn-~ led to exacerbation of active eae (62) . while the exact mechanism of action of ifn-~ is still unknown, numerous putative mechanisms have been proposed (review in (63)). whether the therapeutic benefit of ifn-[3 is also based on the induction of apoptosis in inflammatory cells is still controversially discussed (64, 65) . in lewis rat at-eae, no major increase of t-cell apoptosis in situ could be observed after treatment with ifn-[3 (66) . however, ifn-[3 clearly increases the phagocytosis of apoptotic, encephalitogenic, mbp-specific t-cells by microglia in vitro (chan, a., seguin, r., submitted). thus, theoretically ifn-13 could help to engulf and eliminate t-cells driven into apoptosis by other therapeutically used agents, e.g. corticosteroids. antigen-specific therapy with cns or pns-autoantigens has been shown to be effective in eae and ean (67, 68) . the underlying mechanism appears to involve t-cell receptor reengagement at an appropriate stage of the cell cycle which leads to t-cell apoptosis (69) . in lewis rat cell transfer (at)-eae, high dose antigen (guinea pig mbp) therapy not only led to apoptotic cell death of invading t-cells in situ but also of resident oligodendrocytes (38) . this was accompanied by a profound modulation of the cytokine network with a rapid induction of the thl-type cytokines tnf-~ and ifn-~, as well as inducible nitic oxide synthase. neutralization of tnf-c~ in vivo led to a decrease in t-cell and oligodendrocyte apoptosis but did not change the beneficial clinical effect of high-dose antigen therapy. this argues for a central role of tnf-c~ as a mediator of local apoptosis which may have different functional effects. similar results could also be observed in antigenspecific therapy of lewis rat at-ean using p2-protein (70, 71) . apoptosis of inflammatory effector cells constitutes an effective protective mechanism in the autoimmune-inflamed cns. thus, the selective a24. apoptotic cell death in experimental autoimmune encephalomyelitis 517 induction of apoptosis in pathogenic cells is an attractive therapeutic target in neuroimmunological diseases. however, apoptosis of resident cns cells represents also a major mechanism in the pathogenesis of different neurodegenerative disorders, where selective blockade of apoptotic pathways may be benificial. therefore, the identification of the molecular, tissue-and cell-specific factors that lead to local apoptosis remain to be elucidated in order to take advantage of this phylogenetically old and important defense mechanism. apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics apoptosis and programmed cell death in immunity determination of the length of the histological stages of apoptosis in normal liver and in altered hepatic foci of rats detection of apoptosis in tissue sections programmed cell death: alive and well in the new millennium apoptotic death in epithelial cells: cleavage of dna to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation cell nucleus and dna fragmentation are not required for apoptosis fine specificity and hla-restriction of myelin basic protein-specific t cell lines from multiple sclerosis patients and healthy individuals myelin autoreactivity in multiple sclerosis: recognition of myelin basic protein 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experimental autoimmune neuritis (ean) of the lewis rat multiple sclerosis treatment lnterferon-]3 inhibits progression of relapsing-remitting experimental autoimmune encephalomyelitis discontinuation of treatment with ifn-beta leads to exacerbation of experimental autoimmune encephalomyelitis in lewis rats. rapid reversal of the antiproliferative activity of ifn-beta and excessive expansion of autoreactive t cells as disease promoting mechanisms the effects of interferon-!3 on cytokines and immune responses interferon-[31b augments activationinduced t-cell death in multiple sclerosis patients no induction of apoptosis by ifn-beta in human antigen-specific t cells interferon-beta treatment of experimental autoimmune encephalomyelitis leads to rapid nonapoptotic termination of t cell infiltration t cell deletion in high antigen dose therapy of autoimmune encephalomyelitis antigen therapy eliminates t cell inflammation by apoptosis: effective treatment of experimental autoimmune neuritis with recombinant myelin protein p2 systemic antigen in the treatment of tcell-mediated autoimmune diseases role of tnf-alpha in high-dose antigen therapy in experimental autoimmune neuritis: inhibition of tnf-alpha by neutralizing antibodies reduces t-cell apoptosis and prevents liver necrosis schwann cell apoptosis in experimental autoimmune neuritis of the lewis rat and the functional role of tumor necrosis factor-alpha the key: cord-268341-103xf3dw authors: parra, beatriz; hinton, david r.; lin, mark t.; cua, daniel j.; stohlman, stephen a. title: kinetics of cytokine mrna expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis date: 1997-07-07 journal: virology doi: 10.1006/viro.1997.8613 sha: doc_id: 268341 cord_uid: 103xf3dw abstract the potential role(s) of cytokines in the reduction of infectious virus and persistent viral infection in the central nervous system was examined by determining the kinetics of cytokine mrna expression following infection with the neurotropic jhm strain of mouse hepatitis virus. mice were infected with an antibody escape variant which produces a nonlethal encephalomyelitis and compared to a clonal virus population which produces a fulminant fatal encephalomyelitis. infection with both viruses induced the accumulation of mrnas associated with th1and th2-type cytokines, including ifn-γ, il-4, and il-10. peak mrna accumulations were coincident with the clearance of virus and there was no obvious differences between lethally and nonlethally infected mice. tnf-α mrna was induced more rapidly in lethally infected mice compared to mice undergoing a nonfatal encephalomyelitis. rapid transient increases in the mrnas encoding il-12, inos, il-1α, il-1β, and il-6 occurred following infection. nonlethal infections were associated with increased il-12, il-1β, and earlier expression of il-6, while lethal infections were associated with increased inos and il-1α mrna. these data suggest a rapid but differential response within the central nervous system cells to infection by different jhmv variants. however, neither the accumulation nor kinetics of induction provide evidence to distinguish lethal infections from nonlethal infections leading to a persistent infection. accumulation of both th1 and th2 cytokines in the central nervous system of jhmv-infected mice is consistent with the participation of both cytokines and cell immune effectors during resolution of acute viral-induced encephalomyelitis. introduction (wesselingh et al., 1994) . variations between viral infections resulting in cns the goal of the immune response during viral infection inflammation prompted an examination of the temporal is to limit replication via induction of both nonspecific and induction of cns cytokines during fatal and nonfatal cns specific antiviral effectors. acute viral infections of the ceninfections by variants of the jhm strain (jhmv) of mouse tral nervous system (cns) result in vigorous, but in some hepatitis virus (mhv). in immunocompromised hosts instances limited, host immune responses (sedgwick and jhmv replicates unchecked in the cns demonstrating dorries, 1991) . in contrast to responses in the periphery the importance of immune effectors in limiting cns virus where limiting virus replication can generally be carried replication ; houtman and out with minimal regard to tissue damage, within the cns fleming, 1996b; . effector checks and balances minimize inflammatory-mediated mechanisms implicated in protection and clearance of damage while limiting viral-induced cytopathology. al-jhmv from the cns include cell-mediated immunity and though a wide range of immune effectors are often induced, both neutralizing and nonneutralizing antibodies. jhmv predominant anti-viral mechanisms appear related to the provides an interesting paradigm of acute viral encephapathogenesis strategy of the individual agent. for example, litis not only because of its associated demyelination infection of mice with lymphocytic choriomeningitis virus (weiner, 1973; lampert et al., 1973) but also because induces a predominant cd8 / cytotoxic t lymphocyte (ctl) some immune effector mechanisms prevent death via response (lehmann et al., 1988) . by contrast, resolution of directly reducing cns virus replication while other immeasles virus-encephalitis in mice is mediated by cd4 / t mune effectors prevent death without significantly altercells and correlates with the local production of ifn-g ing virus replication hout-(finke et al., 1995) . finally, resolution of sindbis virus-inman and fleming, 1996b; . a duced encephalitis is related to induction of neutralizing common theme appears to be prevention of neuronal infection by reducing viral load or preventing neuronal infection, most likely via cytokines. tion and clearance of jhmv from the cns are not yet and rarely neurons (2.2v-1). these viruses contrast to the predominantly neuronotropic oblv-60 variant previously clear. the antiviral effects of cd8 / t cells appear to be due to direct lysis of infected cells; however, cd8 / and examined (pearce et al., 1994; . cd4 / t cells may also exert antiviral activity indirectly material and methods via cytokine secretion (biron, 1994) . neither infected neurons nor oligodendrocytes appear susceptible to major mice and viruses histocompatibility (mhc) class i-mediated killing in vivo, c57bl/6 mice were purchased from the jackson laboconsistent with the inability of jhmv-specific ctl to clear ratory (bar harbor, me) at 6 weeks and maintained in virus from infected oligodendroglia (stohlman et al., the university of southern california vivarium. all mice 1995b). furthermore, clearance of jhmv from the cns were used at 7 weeks of age. to produce a lethal infecis inhibited, but not abolished, in mice genetically defition, mice were infected by intracerebral inoculation (i.c.) cient in perforin-mediated cytolysis (lin et al., 1997) . with 100 pfu of the plaque-purified dm isolate of jhmv these data suggest the possibility that cytokines contrib(stohlman et al., 1982) in a volume of 32 ml. this virus has ute to either clearance or protection from jhmv infection. the plaque size and pathogenesis similar to the parental during jhmv infection of the cns there is an abrupt suckling mouse brain pool of jhmv originally described increase in mrna encoding interleukin-1 (a and b), ilby weiner (1973) and produces a lethal encephalomyeli-6, tumor necrosis factor (tnf)-a, and interferon (ifn)-g, tis with minimal demyelination apparent at the time of at the time of maximal decrease in virus replication and death. to produce a sublethal infection, mice were inmononuclear cell infiltration (pearce et al., 1994) . no ifnfected with 25 pfu of the 2.2v-1 monoclonal antibodyg mrna was detected in immunodeficient mice, sugderived neutralization-resistant variant of jhmv (fleming gesting this cytokine may be important during viral clearet al., 1986) . this variant replicates predominantly in oliance (pearce et al., 1994) . consistent with this concept, godendroglia producing a flaccid paralysis. although vimice treated with anti-ifn-g are more susceptible to ral antigen is cleared from survivors by 30 days postinfec-jhmv, while administration of ifn-g provides protection tion (p.i.), viral rna persists for at least 12 months (adami (smith et al., 1991) . il-6, tnf-a, and type 2 nitric oxide et al., 1995) . groups of at least 3 mice were sacrificed synthase (inos) have also been detected in the cns at various times p.i. immunosuppression was induced during acute jhmv infection (sun et al., 1995; by lethal irradiation (850r) 24 hr prior to infection. shamet al., 1995a; while il-1b, il-6, tnf-a, infected mice were injected i.c. with 32 ml of sterile endoand inos were detected in the cns of chronically intoxin-free phosphate-buffered saline (pbs). fected mice (sun et al., 1995) . the complex interactions of multiple immune effector mechanisms during jhmv virus titration infection may reflect both the relative immune privilege virus titers were determined by plaque assay using of the cns (sedgwick and dorries, 1991) as well as the monolayers of dbt cells as previously described (stohlspecific tropism of the virus for cns cell types. neuroman et al., 1982) . one-half of the brain was homogenized tropic mhv isolates differ in tropism and include viruses using tenbrock tissue homogenizers in 2.0 ml of dulbecwith predominant tropisms for astrocytes, microglia, and co's pbs, ph 7.4. the remaining half was taken for histooligodendroglia as well as neurons (fleming et al., 1986; pathology or rna extraction (see below). following cenperlman and reis, 1987; kyuwa and stohlman, 1990; trifugation at 1500 g for 7 min at 4њ, supernatants were pearce et al., 1994; stohlman et al., 1995a) . the balance assayed immediately or frozen at 070њ. data presented between limiting viral replication and preserving cns are the average titer of groups of three or more mice. function occasionally results in incomplete viral clearance and a persistent cns infection which may or may antibody titration not involve the continued presence of infectious virus jhmv-specific igm, igg1, and igg2a antibodies were quantitated by elisa as previously described (lin et al., 1996b) . persistence of infectious virus correlates with the 1997) using rabbit anti-mouse igm, igg1, or igg2a antipresence of ctl escape variants (pewe et al., 1996) . bodies (cappel, costa mesa, ca). concentrations of se-to understand the complex interrelationships between rum antibodies were expressed as the highest dilution encephalitis, protection, and viral clearance leading to a with o.d. values three times above background level. persistent infection of the cns, the expression of pro-neutralizing antibodies were tested in serum as preand anti-inflammatory cytokine mrnas in the cns of viously described (lin et al., 1997) . mice undergoing either lethal or sublethal jhmv infection were compared. the two jhmv chosen for study infect histology either primarily microglia and astrocytes, less frequently oligodendroglia and neurons (dm) or primarily oligoden-histopathologic analysis was performed as previously described (stohlman et al., 1995a) . briefly, tissues were droglia, much less frequently microglia and astrocytes, fixed for 3 hr in clark's solution (75% ethanol, 25% glacial with 32 p-atp-labeled internal oligonucleotide probes. membranes were washed (three times; 21 ssc, 0.1% acidic acid) and embedded in paraffin. sections were stained with hematoxylin and eosin or luxol fast blue. distri-sds; room temperature), exposed to storage phosphor screens (molecular dynamics, sunnyvale, ca), and bution of jhmv antigen was examined by immunoperoxidase staining (vectastain-abc kit; vector laboratory, burlin-scanned using a phosphorimaging scanner (molecular dynamics). game, ca) using the anti-jhmv mab j3.3 specific for the viral nucleocapsid protein (fleming et al., 1983) . radioactive signals of cytokine cdna were quantified and normalized to the house-keeping enzyme hypoxanthine phosphoriboxyltransferase (hprt) values to adjust cytokine mrna expression for differences in cdna as previously described (cua et al., 1995 (cua et al., , 1996 . the sample with the highest specific brains were processed individually to prevent contamination. rna was isolated from half brains by homogeni-activity was designated the 100% maximal response and values for the remainder were derived as percentage of zation at room temperature in guanidinium isothiocyanate using tenbrock tissue homogenizers as previously the highest value. data shown are mean values for 3-4 mice at each time point { 1 standard deviation. described (cua et al., 1995) . samples were sheared prior to centrifugation through 5.4 m cesium chloride at 100,000 g for 18 hr to isolate rna. the cdna were pre-results pared using avian myeloblastosis reverse transcriptase acute and subacute jhmv-induced encephalitis (promega, madison, wi) and oligo dt primers (promega) for 60 min at 42њ. expression of cytokine mrna was fatal encephalomyelitis induced by jhmv is associated with minimal demyelination (kyuwa and stohlman, determined by semiquantitative pcr analysis, following procedures previously described (cua et al., 1995 (cua et al., , 1996 (cua et al., ). 1990 houtman and fleming, 1996b) . this contrasts with infection by 2.2v-1 which produces an acute nonfatal en-pcr was performed using amplitaq dna polymerase (perkin-elmer, branchburg, nj) and specific cytokine cephalomyelitis with extensive demyelination (fleming et al., 1986; wang et al., 1990) . although both viruses primers for ifn-g, il-1a, il-1b, il-4, il-6, il-10, tnf-a (murphy et al., 1993; cua et al., 1996) , and il-12p40. the replicated rapidly to high titer in the cns (fig. 1a) , jhmvinfected mice succumbed within 8 days while 2.2v-1-sequences of the il-12p40 oligonucleotides primers and probe used are as follows: 5 primer, gac cct gcc infected mice underwent a subacute disease with little or no mortality (fig. 1b) . peak 2.2v-1 replication was at cat tga act ggc; 3 primer, caa cgt tgc atc cta gga tcg; oligoprobe, tgt ctg cgt gca agc tca day 3 while the peak of jhmv replication was delayed until day 5. 2.2v-1 clearance began at day 5 p.i. and by gga. amplification was carried out using 35 cycles of one denaturation step at 94њ (45 sec), primer annealing day 7 virus was nearly undetectable. by contrast, titers in jhmv-infected mice initially decreased at day 7 p.i. at 59њ (45 sec), extension step at 72њ (1.5 min), followed by a final extension step for 7 min. for il-4 and inos a and detectable virus was still present in the cns of moribound mice at day 8 p.i. (fig. 1a ). during lethal jhmv nested pcr was performed by using internal primers in a second round of pcr (25 cycles) under the conditions infection, virus replication within the cns is not reduced as rapidly as in mice which survive infection ( fig. 1a ) described above. the oligonucleotide primers used in the second pcr for il-4 were the corresponding se-consistent with the notion that rapid clearance correlates positively with protection. consistent with these findings, quences for the 5-primer and the probe described by cua et al. (1996) . the nucleotide sequence for the il-4 immunohistologic examination of the brains of jhmvinfected mice at day 7 showed abundant viral antigen oligonucleotide probe was ttg aag gag gtc aca gga gaa ggga (sideras et al., 1987) . the 5 and 3 outer in regions of encephalitis while only focal residual viral antigen was found in 2.2v-1-infected animals (fig. 2) . en-primer sequences for inos were gcc ttc cgc agc tgg gct gt and atg tgg tag cca cat ccc gag cephalitis was prominent in mice infected with either 2.2v-1 or jhmv and no differences in the amount or distri-cc, respectively (lyons et al., 1992) . internal 5 and 3 inos primers were agc tac tgg gtc aaa gac aag bution of mononuclear cell infiltrates were found at day 7 (fig. 2) . no serum neutralizing antibodies were de-agg ct and the 3 outer primer, respectively. the oligonucleotide probe consisted of the sequence ctc cct tected in either group by day 9 postinfection, even though the virus titer in the cns had declined over 3 log 10 (lin tcc gaa gtt tct ggc agc a. for quantification, pcr products were diluted in dena-et al., 1997; data not shown). in contrast to neutralizing antibodies, igm was first detected at day 5 post-2.2v-1 turing solution (0.4 n naoh, 25 mm edta), neutralized with tris-hci (1.0 m; ph 8.0), and transferred to 0.45 infection (data not shown) and both igg1 and igg2a were detected as early as 7 days p.i. (fig. 1c) . the igg1 and mm nytran membranes (schleicher & schuell, keene, nh) using a minifold i dot blot apparatus (schleicher & igg2a response suggest the absence of a shift toward either a th1-or th2-type response reported to be in-schuell). membranes were hybridized (60њ; overnight) volved in the response to sindbis virus-induced encepha-deficient mice, which showed no evidence of ifn-g mrna, suggests tnf-a may also contribute to inos litis (wesselingh et al., 1994) . mrna induction (colasanti et al., 1995; gazzinelli et al., 1993) . consistent with this notion, tnf-a mrna was first proinflammatory cytokines detected at day 3 in mice undergoing a lethal infection the mrna encoding ifn-g increased in both groups and at day 5 in mice sublethally infected (fig. 3c ). similar of mice through day 5 postinfection, consistent with the to the kinetics of ifn-g, tnf-a mrna increased until rapid accumulation of both nk and t cells in the cns of death of lethally infected mice. in mice undergoing a infected mice (williamson et al., 1991; williamson, 1992) sublethal infection, tnf-a mrna declined following the (fig. 3a) . no ifn-g mrna was detected in either shampeak of virus replication and approached baseline levels infected mice or in infected immunodeficient mice. durby 14 days p.i. ing the lethal jhmv infection ifn-g mrna did not in-similar to both tnf-a and inos, il-12 is secreted from crease between day 5 and day 7. however, in mice macrophages during the induction of cell-mediated imundergoing a sublethal infection the level of ifn-g mrna munity and protects from a number of viral infections continued to increase to day 7 and remained elevated, via a ifn-g-dependent mechanism (ozmen et al., 1995; suggesting the possibility that ifn-g is important followorange and biron, 1996) . no il-12 mrna was found foling infection with a jhmv variant tropic for oligodendroglowing sham infection; however, il-12 mrna increased lia. even though ifn-g mrna increased during the early rapidly and peaked at 3 days following both infections phase of infection, a sharp transient increase in inos (fig. 4a ). increased il-12 mrna also occurred in immu-mrna was detected at day 5 p.i. in mice with a lethal nodeficient mice at 3 days p.i., suggesting a direct reencephalomyelitis (fig. 3b) . only a slight increase was sponse to infection which may be related to the recently detected in mice undergoing subacute encephalomyelidescribed ifn-g-independent induction of il-12 (heinzel tis. interestingly, infection of immunodeficient mice with et al., 1996) . il-12 mrna levels decreased after day 3 jhmv induced the accumulation of inos mrna to apand nearly approached base line levels found in uninproximately 50% the level found in infected immunocomfected mice by 14 days p.i. petent mice, suggesting a direct response to viral infec-the il-1b mrna level found at day 1 p.i. declined by 3 days p.i., consistent with induction of an early transient tion. the increase in inos and tnf-a mrna in immunoincrease in il-1b mrna in sham-infected mice (fig. 4b ). subacute infection (fig. 4c ) and then declined but never returned to baseline. in lethally infected mice the peak il-1b mrna peaked at day 5 following sublethal infection and subsequently declined as virus was cleared of il-1a mrna was delayed (day 5 p.i.) and then declined as the animals succumbed to infection (fig. 4c ). il-6 from the cns. following a lethal infection, the quantity of il-1b mrna increased from day 3 p.i. until death. il-mrna peaked at day 5 postinfection in lethally infected mice and declined by day 7 as virus was cleared from 1a mrna peaked at day 3 p.i. in the mice undergoing a the cns (fig. 4d) . in contrast to the lethal infection, the of il-4 mrna expression following acute and subacute infections showed that the levels increased in parallel levels of il-6 mrna increased rapidly and peaked at day 3 p.i. following subacute infection. the level then through day 7 p.i. (fig. 5a ). in 2.2v-1-infected mice, the level of il-4 mrna continued to increase until day 9 p.i. declined rapidly by day 5 and had reached baseline levels by day 9 p.i. no il-6 mrna was detected in sham-and then declined slightly by day 14. infected mice, suggesting a rapid response to virus infection. very low levels of il-6 mrna were detected in im-discussion munodeficient mice infected with either virus. jhmv produces an acute cns infection associated with several immune effector mechanisms, including th2-related cytokines both cd4 / and cd8 / t cells houtman and fleming, 1996b) . kinetic analysis of cellular igg1 and igg2a virus-specific antibodies were detected in survivors of jhmv infection; however, there appeared cns infiltrations during jhmv infection of mice shows that nk cells accumulate prior to cd8 / t cells, which in to be little relationship between induction of antibody and control of jhmv infection within the cns. induction of both turn precede accumulation of cd4 / t cells and macrophages (williamson et al., 1991; williamson, 1992) . there isotypes suggest that th1 and th2 cytokines are induced by jhmv infection. the kinetics of il-10 mrna induction is no direct evidence for a role of nk cells in suppressing jhmv replication (houtman and fleming, 1996a) ; how-was of interest due to the association of il-10 with reduced th1 activity in vitro and with remission during exper-ever, cd8 / ctl appear to be critical immune effectors (williamson and stohlman, 1990; stohlman et al., 1995b) . imental allergic encephalomyelitis (kennedy et al., 1992) . il-10 mrna was first detected at day 3 p.i. in lethally recent analysis of jhmv pathogenesis in mice deficient in perforin suggests that in addition to cytolytic effectors infected mice, but not until day 5 postinfection in the cns of the mice undergoing a subacute encephalitis. however, other immune components also contribute to sterilizing immunity (lin et al., 1997) . similarly, the adoptive transfer at the time most lethally infected mice were about to succumb to infection (day 7), there was no difference in the of virus-specific cd4 / t cells to jhmv-infected mice demonstrates that some clones protect via reducing viral peak levels of il-10 mrna between the two groups. the kinetics of il-10 mrna accumulation differed between the replication (yamaguchi et al., 1991) , while others protect without reducing virus replication , groups; il-10 mrna accumulation in mice undergoing a sublethal infection was slower and remained at peak lev-suggesting that cytokines may play an important role in providing sterile immunity. els until day 9 p.i., prior to declining to near basal levels by day 14. no il10 mrna was detected in the cns of in general the kinetics of cytokine mrna expression correlated with the temporal presence of cns infiltrating sham-infected or infected immunodeficient mice. no il-4 mrna was detected following a single amplification dur-mononuclear cells. many cytokine transcripts, with the exceptions of il-12, il-1a, and il-6, were maximally ex-ing lethal or sublethal jhmv infections. however, after a second amplification, low abundant mrnas were de-pressed by 7 day p.i., near the peak inflammatory cell infiltration and during the elimination of virus from the tected (fig. 5a) . no il-4 mrna was detected in either sham-infected or infected immunosuppressed mice fol-cns (williamson et al., 1991; williamson, 1992) . previous data using the oblv-60 jhmv variant which has a selec-lowing two amplifications (data not shown). the kinetics tive tropism for neurons suggested a correlation between increasing time following subacute infection, consistent with the resolution of encephalitis. it is interesting that ifn-g induction, t cell accumulation, and reduction of virus replication (pearce et al., 1994) . the semiquantita-the cns of mice with active macrophage-mediated demyelination (day 14 p.i.) showed little evidence of tnf-tive kinetic analysis of ifn-g mrna in the cns of mice undergoing both lethal and sublethal jhmv infections a mrna, consistent with the inability of anti-tnf-a to prevent jhmv-mediated demyelination (stohlman et al., supports the positive correlation between ifn-g and viral clearance. however, the oblv-60 jhmv variant is 1995a). a surprising number of mrnas peaked relatively early cleared from the cns of ifn-g-deficient mice , consistent with ifn-g exhibiting poor in vitro following jhmv infection. the mrnas encoding inos, il-12, il-1a, il-1b, and il-6 peaked either prior to or anti-jhmv activity (zhang et al., 1997) and inability of rifn-g to inhibit cns virus replication (smith et al., 1991) . coincident with initiation of viral clearance. in most cases (except inos mrna) the levels were either higher or these data contrast with other viral-induced encephalopathies in which ifn-g plays a significant role (kundig et increased more rapidly in the mice undergoing subacute infections. accumulation of inos mrna was first de-al., 1993; finke et al., 1995) , including some (yu et al., 1996) , but not all (wesselingh et al., 1994) , neuronotropic tected in mice undergoing a lethal infection coincident with the initial detection of ifn-g mrna. however, the viruses. the kinetics of ifn-g mrna induction suggests that it may play a more prominent role in the pathogene-mrna levels declined as virus replication declined, suggesting a direct effect of virus on inos induction. in sis of jhmv variants with predominant tropisms for microglia, astrocytes (jhmv), or oligodendroglia (2.2v-1). contrast to lethal infections, inos mrna lagged detection of ifn-g in mice undergoing subacute infections and the isotype diversity of the anti-jhmv antibody response suggests that both th1 and th2 subsets of cd4 / increased to less than 50% the level detected in mice undergoing a lethal infection. similar to the recent data t cells are activated during infection. il-4 mrna accumulation in the cns corresponds to infiltration of th2 cells demonstrating low levels of inos in the cns of both nude mice and mice deficient in ifn-g , (cua et al., 1995) and kinetic analysis suggests that t cells expressing th2 cytokine profiles are recruited into inos mrna in immunodeficient mice was approximately 50% the levels detected in the cns of intact mice at day the cns with nearly equal kinetics in both lethally and sublethally infected mice (fig. 5a) . il-4 increases the 3 p.i. although jhmv is susceptible to inhibition by inos in vitro, inos is not associated with in vivo protection severity of encephalitis (ikemoto et al., 1995) and could potentially play a role in jhmv persistence via inhibition . il-12, predominantly produced by cells of the myelo-of viral clearance (moran et al., 1996) . in support of the recruitment of th2 cells, il-10 mrna also increased with monocytic lineage, is associated with the induction of th1 cd4 / t cells (brunda, 1994) . il-12 mrna peaked kinetics similar to those of ifn-g and il-4. whether this difference in detection of th2 cytokines is due to differ-early (day 3) in mice undergoing both lethal and sublethal jhmv infections. however, no significant differences ences in mouse strains or the selective tropism of the virus is not known. it is interesting that although il-10 is were found comparing mrna levels in immunodeficient mice to intact mice. this may suggest that jhmv infection secreted by activated microglia in vitro (lodge and sriram, 1996) , no il-10 mrna was detected in sham-induces transcription of il-12 mrna in cns cells. in addition, 2.2v-1 infects predominantly, but not exclu-infected or immunodeficient mice. this contrasts with other cytokine mrna detected in either sham-infected sively, oligodendroglia, while jhmv infects predominantly microglia and astrocytes. the relatively higher or virus-infected immunodeficient hosts (see below). tnf-a mrna is induced following jhmv infection level of il-12 mrna in 2.2v-1-infected mice suggests the possibility that oligodendroglia transcribe il-12 mrna in (pearce et al., 1994; stohlman et al., 1995a; sun et al., 1995) and tnf-a is present during both the acute and response to jhmv infection, similar to the induction of il-12 mrna following measles virus infection of oligo-persistent jhmv infections. tnf-a mrna is not translated in jhmv-infected cells (stohlman et al., 1995a ), al-dendroglia (yamabe et al., 1994 . during both the lethal and sublethal infections the il-though it may be secreted by adjacent but not infected cells. in addition, inhibition of tnf-a, which prevents 1a mrna peaks appear to coincide with replication and not clearance, suggesting that infection induces a rapid experimental autoimmune encephalitis (ruddle et al., 1990) , has no effect on either jhmv-induced encephalitis induction of il-1a mrna. these data contrast to the association of il-1a mrna and the clearance of the oblv-or demyelination (stohlman et al., 1995a) . as anticipated, based on the relative tropism of the two viruses analyzed, 60 variant of jhmv (pearce et al., 1994) , suggesting an additional difference in cytokine responses depending tnf-a mrna accumulated initially in the cns of mice infected with jhmv. however, by day 5 p.i. there was on the tropism of the virus analyzed. il-1b mrna, previously detected in the cns of jhmv-infected mice little difference in the levels of tnf-a mrna in the two groups. finally, the level of tnf-a mrna decreased with (pearce et al., 1994) , increased directly after infection at day 1 p.i. however, the level was approximately the same suggests it may play a positive role in reducing the extent of cns inflammation thereby inadvertently contributing as the level detected in sham-infected mice, suggesting it was induced by trauma. in all mice the levels subse-to persistent infection. some aspects of our data, i.e., the rapid induction of il-12 mrna in mice infected with 2.2v-quently dropped by day 3 p.i. the levels of il-1b mrna peaked at day 5 following 2.2v-1 infection and at day 7 1, suggest that infection of specific cell types may influence the induction of cytokine mrna (yamabe et al., following jhmv infection, suggesting il-1b mrna was also induced by infection. analysis of the levels in immu-1994) . this supports the notion that the cytokine mrna patterns more closely reflect diversity of the immune re-nodeficient mice were consistent with the notion that infection, and not immune infiltrates, contributed the ma-sponse to an individual agent, although differential secretion of cytokines following infection of unique cns cell jority of the il-1b mrna levels. il-6, another pleiotropic cytokine with numerous ef-types cannot be ruled out (benveniste, 1992; sun et al., 1995) . while the kinetics of ifn-g, il-4, and il-10 showed fects on immune responses (van snick, 1990) , was also detected early following both lethal and sublethal infec-little difference between the groups undergoing lethal or sublethal infections, mrnas encoding il-6 and il-1b tions. by contrast, il-6 mrna was also only detected at 6 days p.i. with the neuronotropic oblv-60 jhmv variant either appeared more rapidly (il-6) or accumulated to higher levels (il-1b) following infection with 2.2v-1 virus. (pearce et al., 1994) . kinetic analysis shows that the levels of il-6 mrna peaked at day 3 post-2.2v-1 infection by contrast the induction of inos and il-1a mrnas were increased in mice undergoing a lethal infection. these and at day 5 post-jhmv infection. interestingly, analysis of the mrna levels in the immunodeficient mice showed data suggest that an early induction of il-6, and possibly il-1b, are associated with sublethal infection or the dif-virtually no induction of il-6 mrna, suggesting that in contrast to il-1a and il-1b an intact immune response ferent tropisms exhibited by these two jhmv variants. however, during both infections the mrna levels de-was required for il-6 mrna induction. rapid induction of il-6 mrna following jhmv infection is consistent with creased as virus was cleared. similarly, there appears to be an inverse correlation between a rapid induction other models of viral-induced encephalitis in which it also precedes ifn-g (moskophidis et al., 1991) . although of inos mrna and sublethal disease, consistent with the recent demonstration that although inos is protective in both il-6 and il-10 are cofactors for ctl induction (chen and zlotnik, 1991; takai et al., 1988) , kinetic analysis is vitro, inhibition of inos activity in vivo appears to have no effect on jhmv pathogenesis . taken consistent with the notion that il-6, and not il-10, may be involved in the induction or recruitment of jhmv-specific together, kinetic analyses of the induction of cytokine mrna during the lethal and sublethal jhmv infections ctl. jhmv infection induces il-6 secretion from both brain endothelial cells and astrocytes following in vitro are consistent with the accumulation of both th1-and th2-associated cytokines and support the interaction of infection with jhmv (joseph et al., 1993) , consistent with data showing that it is produced by resident cns cells multiple cellular and soluble effector mechanisms whose balance may be critical in providing protection and steri-following infection with lymphocytic choriomeningitis virus (frei et al., 1989) . it is interesting that il-6 mrna lizing immunity. peaks first in mice infected with the 2.2 v-1 variant compared to jhmv, which infects a significantly larger num-acknowledgments ber of astrocytes. the rapid induction of il-6 and il-we thank wen-qiang for excellent technical assistance. this work 1b following infection with 2.2v-1 is consistent with the was supported by grant ns18146 from the national institutes of health. induction of these mrna in oligodendroglia infected by escherichia coli lipopolysaccharide and tumor necrosis factor alpha a paradigm for virus-induced demyelinating disease. trends microbiol. 5, 9-14. stimulation recovery from acute virus infection. role of cytotoxic t lymphocytes in the eliminatiation factor self-antigen-intion of lymphocytic choriomeningitis virus from spleens of mice duced th2 responses in experimental allergic encephalomyelitis (eae)-resistant mice exposure to t helper 2 cytokines in vivo before encounter with antigen selects for perforin-mediated cytolysis regulation of microglial activation t helper subsets via alterations in antigen-presenting cell function gamma interferon is a major mediator of antiviral defense in experiing and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv-4) rus-infected mice production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of in-weiner on the cellular source and function of interleukin 6 produced in the central nervous system in viral disease. ase chain reaction method in schistosoma mansoni infected mice an absolute and restricted requirement for il-12 in natural killer cell ifn-g production and antiviral bral toxoplasmosis is induced by in vivo neutralization of tnf-a and correlates with the down-regulated expression of inducible nitric defense the in vivo oxide synthase and other markers of macrophage activation ifng independent production of il-12 during murine endotoxemia cytokine induction during t-cell mediated clearance of mouse hepa-immunol dissociation of demyelination titis virus from neurons in vivo the astrocyte is a target cell in mice and viral clearance in congenitally immunodeficient mice infected with murine coronavirus jhm pathogenesis of mouse hepatitis virus-induced demyelination cytotoxic t cell-resistant variants are selected in a virus-induced small amounts of exogenous il-4 increase the severity of demyelinating disease grunencephalitis induced in mice by the intranasal infection of herpes simplex virus type 1 interleukin-6 induction in vitro in mouse brain endothelial cells and astrocytes allergic encephalomyelitis the immune system response by exposure to mouse hepatitis virus (mhv-4, jhm) igg1 induction factor: a single molecular entity with multiple of mice with experimental autoimmune encephalomyelitis reveals that il-10 mrna expression correlates with recovery 89-100. dependent ifn-g exerts an antiviral effect in the central nervous system but not in peripheral solid organs pathogenesis of a neurotropic of two plaque morphology variants of the jhm neurotropic strain murine coronavirus strain jhm in the central nervous system. seminar virol mechanisms of demyelination in jhm virus encephalomyelitis. electron microscopic cells prevent a lethal infection but do not inhibit virus replication tumor necrosis between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus mouse hepatitis virus-specific cytotoxic t lymphocytes protect from lethal infection without eliminating virus from the central williamson hepatitis virus strain jhm virus-specific t cells in the central nervous activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus b cell stimulatory factor-2 is involved in the differentiation of yamabe 8, gene expression in measles-infected adult human glial cells protection of mice from a lethal coronavirus infection in the central induced by murine hepatitis virus, jhm strain (mhv-4) is immunologically mediated pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) role of interferon-g in immunity to herpes simplex virus expression of gamma interferon by a coronavirus defectivealphavirus encephalitis suggests a predominant type 2 t cell response effective clearance of key: cord-001332-dp6vzgef authors: hosking, martin p.; lane, thomas e. title: elr(+) chemokine signaling in host defense and disease in a viral model of central nervous system disease date: 2014-06-17 journal: front cell neurosci doi: 10.3389/fncel.2014.00165 sha: doc_id: 1332 cord_uid: dp6vzgef intracranial infection of the neurotropic jhm strain of mouse hepatitis virus (jhmv) into the central nervous system (cns) of susceptible strains of mice results in an acute encephalomyelitis, accompanied by viral replication in glial cells and robust infiltration of virus-specific t cells that contribute to host defense through cytokine secretion and cytolytic activity. mice surviving the acute stage of disease develop an immune-mediated demyelinating disease, characterized by viral persistence in white matter tracts and a chronic neuroinflammatory response dominated by t cells and macrophages. chemokines and their corresponding chemokine receptors are dynamically expressed throughout viral infection of the cns, influencing neuroinflammation by regulating immune cell infltration and glial biology. this review is focused upon the pleiotropic chemokine receptor cxcr2 and its effects upon neutrophils and oligodendrocytes during jhmv infection and a number of other models of cns inflammation. intracranial infection of susceptible mice with the jhm strain of mouse hepatitis virus (jhmv) causes an acute encephalomyelitis followed by a chronic demyelinating disease. jhmv, after initially infecting ependymal cells lining the ventricles, rapidly disseminates to astrocytes, oligodendroglia, and microglia throughout the brain and spinal cord (wang et al., 1992) . although inflammatory virus-specific t cells are efficient in controlling viral replication through the secretion of ifn-γ and cytolytic activity, sterile immunity is not achieved. viral protein and/or rna persist within oligodendroglia and drive continual t cell and macrophage infiltration, leading to chronic neuroinflammation and demyelination. histological features associated with viral persistence include the development of an immune-mediated demyelinating disease similar to the human demyelinating disease ms; both t cells and macrophages are critical mediators of disease severity, contributing to myelin damage (cheever et al., 1949; perlman et al., 1999) . through the course of acute and chronic jhmv-induced neurologic infection, there is a coordinated expression of chemokines and chemokine receptors that regulate inflammation, contributing to both host defense and disease exacerbation. among the chemokines expressed during infection are members of the elr(+) chemokine family cxcl1 and cxcl2. cxcl1 and cxcl2 are potent chemoattractants for peripheral mononuclear cells (pmns), binding and signaling through their receptor cxcr2 (wolpe et al., 1989; moser et al., 1990; schumacher et al., 1992; marro et al., 2012; weinger et al., 2013) . moreover, pmns have been shown to enhance central nervous system (cns) inflammation by disrupting blood brain barrier (bbb) integrity in animal models of spinal cord injury (sci; tonai et al., 2001; gorio et al., 2007) , autoimmune demyelination (carlson et al., 2008) , and jhmv-induced encephalomyelitis (zhou et al., 2003) , while blocking or silencing of cxcr2 signaling mutes inflammation and tissue damage in mouse models in which pmn infiltration is critical to disease initiation (kielian et al., 2001; belperio et al., 2005; londhe et al., 2005a,b; strieter et al., 2005; gorio et al., 2007; wareing et al., 2007; carlson et al., 2008) . cxcr2 is also expressed by oligodendrocytes (omari et al., 2005) , and cxcl1 promotes the proliferation and positional migration of oligodendrocyte precursor cells (robinson et al., 1998; robinson and franic, 2001; tsai et al., 2002; filipovic and zecevic, 2008) . further, both cxcr2 and cxcl1 are expressed within active ms lesions (omari et al., 2005 (omari et al., , 2006 . how and whether cxcr2 and its cognate ligands regulate immune and glial cell function during acute and chronic disease of the cns is the focus of this review. following jhmv infection, mrna for the chemokine receptor cxcr2 and its associated ligands cxcl1 and cxcl2 are significantly upregulated within the acutely infected cns, peaking at 3 days pi ( figure 1a) . cxcl1 expression was localized to astrocytes (gfap-positive) within the parenchyma and associated with the microvasculature (figure 1b) , consistent with previous observations (lane et al., 1998; omari et al., 2006; rubio and sanz-rodriguez, 2007) . the expression of the cxcr2 ligands within the cns closely paralleled neutrophil emergency release into the circulation and infiltration into the cns; cxcr2-expressing neutrophils were detectable as early as 1 day pi and peaked at 3 days pi within both the periphery and the cns (hosking et al., 2009) . to determine whether cxcr2-signaling controlled neutrophil infiltration into the cns, jhmv-infected mice were treated with either cxcr2 antiserum or control serum (nrs). neutralization of cxcr2 almost completely abrogated neutrophil infiltration into the cns (figures 1c,d) . without infiltrating neutrophils, permeabilization of the blood-brain barrier was impaired (hosking et al., 2009 ) and subsequent inflammatory cell infiltration was significantly reduced. mice treated with cxcr2 neutralizing antiserum were incapable of controlling viral replication, and 100% of all infected mice succumbed to viral infection within 11 days and this was associated with an impaired ability to control cns viral replication (figures 1e,f) . moreover, total and virus specific cd4 + and cd8 + t cell infiltration into the cns was diminished. notably, cxcr2 neutralization did not alter the peripheral generation of virus-specific t cells, indicating that the increased mortality and diminished ability to control viral infection within the cns is likely associated with the dampened access of t cells into the cns parenchyma (hosking et al., 2009 ). collectively, these data demonstrate that during viral infection of the cns, cxcr2 and its associated chemokines function to nonredundantly attract neutrophils into the cns, where they are required to permeabilize the blood-brain barrier, thus facilitating subsequent inflammatory cell infiltration and control of viral replication. neutrophils are amongst the earliest inflammatory infiltrate into the cns following experimental autoimmune encephalitis (eae) induction, and their presence precedes axonal damage, demyelination, and clinical disease (carlson et al., 2008; soulika et al., 2009; wu et al., 2010) . neutralization of either cxcr2 (carlson et al., 2008) or cxcl1 (roy et al., 2012) potently reduces neutrophil infiltration into the cns and reduces bbb permeability, thereby significantly delaying the onset and peak of clinical symptoms. neutrophils also infiltrate into the cns during the first week following cuprizone feeding, and their early presence in the cns is absolutely necessary for the subsequent demyelination observed within the corpus callosum (liu et al., 2010a) . cxcr2 deficient mice or bone marrow chimeric mice, where myeloid cells lack cxcr2, or neutrophildepleted mice are resistant to cuprizone induced demyelination (liu et al., 2010a) . interestingly, although neutrophils are also critical for lymphocytic choriomeningitis virus (lcmv)and pilocarpine-induced bbb permeabilization and subsequent seizures (fabene et al., 2008; kim et al., 2009) , they are dispensable for seizures during theiler's murine encephalomyelitis virus (tmev; libbey et al., 2011) , underlining the fact that neutrophils are not the only cell type capable of mediating permeabilizing the bbb. to this point, resident monocytes, astrocytes, and cd8 + t cells are all capable of direct permeabilization (savarin et al., 2010 (savarin et al., , 2011 johnson et al., 2012) . nevertheless, cxcr2-directed neutrophil infiltration into the cns is a key determinate for subsequent inflammatory cell infiltration in a variety of cns models of viral infection, demyelination, and autoimmunity. how chemokine receptor signaling contributes to chronic neurologic diseases has largely been considered within the context of targeted leukocyte recruitment into the cns (liu et al., 2000 (liu et al., , 2001a glass and lane, 2003; hosking et al., 2009 ). however, numerous resident cell types of the cns also express chemokine receptors under non-inflammatory and inflammatory conditions (reviewed in bajetto et al., 2001; ubogu et al., 2006) , indicating that these cells are capable of responding to specific chemokine ligands. thus, chemokine signaling may participate in either repair and/or exacerbation of pathology following insult, injury, or infection of the cns (liu et al., 2001b; kerstetter et al., 2009; omari et al., 2009) . following jhmv infection, mrna transcripts for cxcr2 as well as its ligands cxcl1 and cxcl2 are significantly upregulated, persisting until at least 21 days pi within the spinal cord (figure 2a) . cxcl1 expression was localized to gfap+ astrocytes within the white matter (figure 2b) , suggesting that cxcr2, besides attracting neutrophils during early acute viral infection, may also alternatively function during chronic demyelination. to determine whether cxcr2 signaling was beneficial or pathogenic, mice persistently infected with jhmv were treated with anti-cxcr2 or control serum (nrs) from day 12-20 p.i. cxcr2 neutralization significantly delayed spontaneous clinical recovery ( figure 2c) . correspondingly, spinal cords from anti-cxcr2 treated mice revealed significantly greater areas of demyelination (figures 2d,e) . importantly, cxcr2 neutralization during chronic jhmv infection did not affect inflammatory cell infiltration into the cns (hosking et al., 2010) . cxcr2 neutralization was also associated with an increase of apoptotic oligodendrocytes and oligodendrocyte precursor cells within white matter tracts of the spinal cord ( figure 2f ; hosking et al., 2010) . to determine whether or not cxcr2 could directly prevent jhmv-mediated apoptosis, cultured oligodendroglia were infected with jhmv in vitro and treated with varying concentrations of cxcl1. in accordance with previous observations (liu et al., 2003 (liu et al., , 2006 zhang, 2005, 2007) , jhmv-infected oligodendrocytes readily underwent apoptosis (figure 2g) , and western blotting confirmed activated caspase 3, cleaved poly adp ribose polymerase (parp) (a caspase 3 target), and muted expression of bcl-2 ( figure 2i) . cxcl1, in a dose-dependent manner, prevented jhmv-mediated apoptosis ( figure 2g) . moreover, activated caspase 3 and cleaved parp were undetectable in cxcl1-treated cultures ( figure 2i) . notably, cxcl1 was incapable of rescuing cxcr2 deficient cultures from jhmv-mediated apoptosis (figures 2h,i) . cxcr2 also prevents ifnγ-and cxcl10-mediated apoptosis of murine or human oligodendroglia cultures (tirotta et al., 2011 (tirotta et al., , 2012 . collectively, these data suggest that cxcr2, during chronic viral infection of the cns, prevents oligodendrocyte the role for cxcr2 signaling during eae and a variety of toxin-induced demyelination models has also been studied. raine and colleagues (omari et al., 2009 ) have shown that cxcl1, when inducibly expressed by astrocytes after the onset of eae, reduces peak disease severity, reduces total demyelination, and increases the onset of remyelination. moreover, transgenic cxcl1 was associated with greater proliferation (presumably of oligodendrocyte precursors) throughout the spinal cord white matter (omari et al., 2009) . conversely, ransohoff and colleagues (liu et al., 2010b) have demonstrated, using a series of bone marrow chimeras, that parenchymal cxcr2 deficiency on radio-resistant cells promotes faster recovery from eae, cuprizone-induced demyelination, and in vitro lysotecithin-induced demyelination. notably, initial clinical severity, inflammation, and/or demyelination in all three models of demyelination and repair were similar regardless of whether parenchymal cells possessed cxcr2; accelerated recovery was associated with initial increases in oligodendrocyte precursor cells, followed by an increased density of mature myelinating oligodendrocytes (liu et al., 2010b) . similar results were observed following cxcr2 chemical anatagonism during eae and in vivo lysolecithin-induced demyelination (kerstetter et al., 2009) . the jhmv-induced model of viral-induced encephalomyelitis provides an important tool in defining molecular and cellular mechanisms that regulate neuroinflammation during both host defense and disease progression. our research on chemokines and chemokine receptors has revealed important roles for these molecules in orchestrating cns inflammation in response to jhmv infection. we and others have found unique and pleiotropic roles for elr+ chemokine signaling via cxcr2 in moderating neutrophil infiltration and protecting oligodendroglia from apoptosis in response to exposure to virus and proinflammatory cytokines. ongoing research in our laboratory continues to focus on the role of elr(+) chemokine signaling on oligodendroglia during jhmv-induced neuroinflammation. it will be important to analyze the effects of selectively ablating cxcr2 on oligodendroglia during jhmv-induced demyelination, while simultaneously manipulating the cellular sources of elr-positive chemokines in the cns that may promote neuroprotection during chronic jhmv-induced disease. chemokines and their receptors in the central nervous system. front. neuroendocrinol cxcr2/cxcr2 ligand biology during lung transplant ischemiareperfusion injury the th17-elr+ cxc chemokine pathway is essential for the development of central nervous system autoimmune disease a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin a role for leukocyte-endothelial adhesion mechanisms in epilepsy the effect of cxcl1 on human fetal oligodendrocyte progenitor cells functional expression of chemokine receptor ccr5 on cd4(+) t cells during virus-induced central nervous system disease reparixin, an inhibitor of cxcr2 function, attenuates inflammatory responses and promotes recovery of function after traumatic lesion to the spinal cord a protective role for elr+ chemokines during acute viral encephalomyelitis cxcr2 signaling protects oligodendrocytes and restricts demyelination in a mouse model of viral-induced demyelination cd8 t cell-initiated blood-brain barrier disruption is independent of neutrophil support inhibition of cxcr2 signaling promotes recovery in models of multiple sclerosis cxc chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses myelomonocytic cell recruitment causes fatal cns vascular injury during acute viral meningitis dynamic regulation of alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease interleukin-6, produced by resident cells of the central nervous system and infiltrating cells, contributes to the development of seizures following viral infection cxcr2-positive neutrophils are essential for cuprizone-induced demyelination: relevance to multiple sclerosis myelin repair is accelerated by inactivating cxcr2 on nonhematopoietic cells expression of mig (monokine induced by interferon-gamma) is important in t lymphocyte recruitment and host defense following viral infection of the central nervous system the t cell chemoattractant ifn-inducible protein 10 is essential in host defense against viral-induced neurologic disease frontiers in cellular neuroscience www.frontiersin.org neutralization of the chemokine cxcl10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis expression of cellular oncogene bcl-xl prevents coronavirus-induced cell death and converts acute infection to persistent infection in progenitor rat oligodendrocytes murine coronavirus-induced oligodendrocyte apoptosis is mediated through the activation of the fas signaling pathway induction of caspase-dependent apoptosis in cultured rat oligodendrocytes by murine coronavirus is mediated during cell entry and does not require virus replication role of the mitochondrial signaling pathway in murine coronavirus-induced oligodendrocyte apoptosis cxcr2 is critical for dsrna-induced lung injury: relevance to viral lung infection cxcr2/cxcr2 ligand biological axis impairs alveologenesis during dsrna-induced lung inflammation in mice cxcr2 signaling and host defense following coronavirus-induced encephalomyelitis neutrophilactivating properties of the melanoma growth-stimulatory activity role for cxcr2 and cxcl1 on glia in multiple sclerosis cxc chemokine receptors on human oligodendrocytes: implications for multiple sclerosis neuroprotection and remyelination after autoimmune demyelination in mice that inducibly overexpress cxcl1 coronaviruses: hepatitis, peritonitis and central nervous system disease chemokine gro1 and the spatial and temporal regulation of oligodendrocyte precursor proliferation the chemokine growth-regulated oncogene-alpha promotes spinal cord oligodendrocyte precursor proliferation cxcl1 can be regulated by il-6 and promotes granulocyte adhesion to brain capillaries during bacterial toxin exposure and encephalomyelitis induction of the cxcl1 (kc) chemokine in mouse astrocytes by infection with the murine encephalomyelitis virus of theiler monocytes regulate t cell migration through the glia limitans during acute viral encephalitis mmp9 deficiency does not decrease blood-brain barrier disruption, but increases astrocyte mmp3 expression during viral encephalomyelitis highand low-affinity binding of gro alpha and neutrophil-activating peptide 2 to interleukin 8 receptors on human neutrophils initiation and progression of axonopathy in experimental autoimmune encephalomyelitis the role of cxcr2/cxcr2 ligands in acute lung injury ifn-gamma-induced apoptosis of human embryonic stem cell derived oligodendrocyte progenitor cells is restricted by cxcr2 signaling cxcr2 signaling protects oligodendrocyte progenitor cells from ifn-gamma/cxcl10-mediated apoptosis a neutrophil elastase inhibitor (ono-5046) reduces neurologic damage after spinal cord injury in rats the chemokine receptor cxcr2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration the expression and function of chemokines involved in cns inflammation sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv-4) leads to a characteristic distribution of demyelination cxcr2 is required for neutrophil recruitment to the lung during influenza virus infection, but is not essential for viral clearance the chemokine receptor cxcr2 and coronavirus-induced neurologic disease identification and characterization of macrophage inflammatory protein 2 extensive infiltration of neutrophils in the acute phase of experimental autoimmune encephalomyelitis in c57bl/6 mice neutrophils promote mononuclear cell infiltration during viral-induced encephalitis this work was funded by national institutes of health (nih) grant r01 ns41249 to thomas e. lane and t32 hl007195-34 to martin p. hosking. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-001396-rpnuauwz authors: blanc, caroline a; rosen, hugh; lane, thomas e title: fty720 (fingolimod) modulates the severity of viral-induced encephalomyelitis and demyelination date: 2014-08-20 journal: j neuroinflammation doi: 10.1186/s12974-014-0138-y sha: doc_id: 1396 cord_uid: rpnuauwz background: fty720 (fingolimod) is the first oral drug approved by the food and drug administration for treatment of patients with the relapsing-remitting form of the human demyelinating disease multiple sclerosis. evidence suggests that the therapeutic benefit of fty720 occurs by preventing the egress of lymphocytes from lymph nodes thereby inhibiting the infiltration of disease-causing lymphocytes into the central nervous system (cns). we hypothesized that fty720 treatment would affect lymphocyte migration to the cns and influence disease severity in a mouse model of viral-induced neurologic disease. methods: mice were infected intracranially with the neurotropic jhm strain of mouse hepatitis virus. infected animals were treated with increasing doses (1, 3 and 10 mg/kg) of fty720 and morbidity and mortality recorded. infiltration of inflammatory virus-specific t cells (tetramer staining) into the cns of fty720-treated mice was determined using flow cytometry. the effects of fty720 treatment on virus-specific t cell proliferation, cytokine production and cytolytic activity were also determined. the severity of neuroinflammation and demyelination in fty720-treated mice was examined by flow cytometry and histopathologically, respectively, in the spinal cords of the mice. results: administration of fty720 to jhmv-infected mice resulted in increased clinical disease severity and mortality. these results correlated with impaired ability to control viral replication (p < 0.05) within the cns at days 7 and 14 post-infection, which was associated with diminished accumulation of virus-specific cd4+ and cd8+ t cells (p < 0.05) into the cns. reduced neuroinflammation in fty720-treated mice correlated with increased retention of t lymphocytes within draining cervical lymph nodes (p < 0.05). treatment with fty720 did not affect virus-specific t cell proliferation, expression of ifn-γ, tnf-α or cytolytic activity. fty720-treated mice exhibited a reduction in the severity of demyelination associated with dampened neuroinflammation. conclusion: these findings indicate that fty720 mutes effective anti-viral immune responses through impacting migration and accumulation of virus-specific t cells within the cns during acute viral-induced encephalomyelitis. fty720 treatment reduces the severity of neuroinflammatory-mediated demyelination by restricting the access of disease-causing lymphocytes into the cns but is not associated with viral recrudescence in this model. multiple sclerosis (ms) is a neurodegenerative inflammatory disease of the central nervous system (cns), which leads to demyelination and progressive neurological disability [1, 2] . fty720, also called gilenya/fingolimod, is an oral drug recently approved by the food and drug administration (fda) for treatment of patients with the relapsing-remitting form of ms [3] [4] [5] [6] [7] [8] . fty720 is an immunomodulatory drug that has shown to reduce both acute relapses but also new lesion formation as well as disability progression and brain volume loss in ms patients [9] . the mechanisms for how fty720 functions are not yet defined; however, the phosphorylated active form of fty720 (fty720p) is a sphingosine-1-phosphate (s1p) receptor modulator that inhibits egress of lymphocytes from lymph nodes [9] [10] [11] . it is thought that this leads to a dampening of autoreactive t cells specific for myelin antigens infiltrating into the cns. importantly, fty720, due to its lipophilic nature, penetrates the blood-brain-barrier and readily enters the cns parenchyma [9] . furthermore, fty720p is detected in situ, suggesting that it may influence the biology of resident cells of the cns [9] . fty720 has been shown to improve disease severity in experimental autoimmune encephalomyelitis (eae), an autoimmune model of neuroinflammation and demyelination commonly used as a model for ms [12] [13] [14] . indeed, therapeutic administration of fty720 in eae models is associated with reduced neuroinflammation and improved motor skills [12] [13] [14] [15] . in addition to eae, viral models of demyelination are also relevant tools for studying the pathogenesis of neuroinflammatory-mediated demyelination. for example, infection of susceptible mice with the neurotropic jhm strain of mouse hepatitis virus (jhmv) results in an acute encephalomyelitis followed by chronic demyelination. like ms, components of the immune system, such as t cells and macrophages, are important contributors to white matter destruction [16] [17] [18] . moreover, jhmv-infected mice undergoing chronic demyelination show similar clinical and histologic disease profiles compared to ms patients [19] [20] [21] . as viruses are considered to be a contributing cause of ms [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] , jhmv infection of the cns offers not only an excellent model for studying the immunopathological mechanism driving demyelination in ms patients but also can provide insight into effects of ms therapeutics within the context of viralinduced demyelination. we have evaluated the effects of fty720 on both host defense and disease progression in jhmv-infected mice. our findings reveal that fty720 treatment resulted in increased mortality associated with impaired ability to control viral replication. fty720 did not alter anti-viral effects of t cells, e.g. cytokine secretion or cytolytic activity, but affected lymphocyte egress from draining cervical lymph nodes and accumulation of virus-specific t cells within the cns. therefore, fty720 treatment mutes effective anti-viral immune responses following infection with a neurotropic virus by dampening trafficking of virus-specific t cells to the cns. conversely, administration of fty720 to jhmv-infected mice reduced the severity of demyelination by limiting infiltration of inflammatory t cells into the cns. age-matched (5 to 7 weeks) s1p1 egfp knock-in mice (c57bl/6 background) [36] and c57bl/6 mice were anesthetized with an intra-peritoneal (i.p.) injection of 150 μl of a mixture of ketamine (western medical supply, arcadia, ca, usa) and xylazine (phoenix pharmaceutical, saint joseph, mo, usa) in hank's balanced salt solution. mice were injected intra-cranially (i.c.) with 150 plaque forming units (pfu) of jhmv (strain v2.2-1) suspended in 30 μl saline [37] . clinical severity was assessed using a previously described four-point scoring scale [16] . fty720 (2-amino-2-[2-(4-octylphenyl) ethyl]-1,3-propanediol, hydrochloride) and fty720p (2-amino-2[2-(4-octylphenyl) ethyl]-1,3-propanediol, mono dihydrogen phosphate ester) were purchased from cayman chemical co (ann arbor, mi, usa). administration of fty720 or the vehicle was performed by daily i.p. injections of 100 μl starting at day 5 post-infection (p.i.). for analysis of viral titers, mice were sacrificed at defined time points. one half of each brain was removed as well as spinal cords. these were homogenized and used in a plaque assay performed using dbt mouse astrocytoma cell line [38] . experiments for all animal studies were reviewed and approved by the university of utah and the university of california, irvine institutional animal care and use committee. immunophenotyping of the cellular infiltrate present within cervical lymph nodes, brains and spinal cords of infected mice was accomplished by homogenizing isolated tissue and generating a single-cell suspension for analysis by flow cytometry as previously described [39] [40] [41] . in brief, isolated cells were fc blocked with anti-cd16/32 1:200. the following antibodies were used for immunophenotyping: apc-conjugated rat anti-mouse b220 for b cells; apc-conjugated rat anti-mouse cd4 for cd4+ t cells; pe-conjugated rat anti-mouse cd8 and apc-conjugated rat anti-mouse cd8 for cd8+ t cells; pe-conjugated rat anti-mouse interferon-gamma (ifn-γ) for intracellular cytokine staining; pe-cy7-conjugated rat anti-mouse cd45, apc-conjugated rat anti-mouse cd19 and pe-conjugated rat anti-mouse cd138 for antibody secreting cells; m133-147 tetramer-pe for virus specific cd4+ t cells and s510-518 tetramer-pe for virus-specific cd8+ t cells. cell isolates for ifn-γ intracellular staining were cultured in 200 μl rpmi-1640 supplemented with 10% fetal bovine serum, l-glutamine and penicillinstreptomycin, and stimulated ex vivo with the immunodominant cd4 epitope (m133-147) or the immunodominant cd8 (s510-518) and golgi stop for 6 h followed by intracellular staining [42, 43] . the cells were then fixed and permeabilized by using a bd cytofix/cytoperm plus kit and then stained for intracellular ifn-γ for 30 min at 4°c [44] . immunophenotyping of lymphocytes was performed following red blood cell lysis on blood samples collected with heparin-coated syringes by heart puncture from s1p1 egfp knock-in mice. cells were then fc blocked with anti-cd16/32 1:200 and stained with pe-conjugated rat anti-mouse cd3. samples were then analyzed on a bd lsr ii flow cytometer. splenocytes were isolated from mice at day 8 following i.p. infection with 2.5 × 10 5 pfu of the dm strain of mouse hepatitis virus (mhv-dm). enriched populations of cd4+ and cd8+ t cells, isolated according to the manufacturer's instructions (cd4 and cd8 isolation kits, miltenyi biotec, auburn, ca, usa), were labeled with the fluorescent dye, carboxyfluorescein diacetate succinimidyl ester (cfse) (life technologies, grand island, ny, usa), at 2.5 μm final concentration. then 1 × 10 6 total cells per well were incubated with fty720p 100 nm or vehicle and stimulated with 5 μm final peptide concentration of cd4+ t cell immunodominant epitope m133-147, cd8+ t cell immunodominant epitope s510-518, or non-specific ova control, and cultured for 72 h at 37°c, 5% co 2 in complete media. cells were then washed and the fc receptor blocked with 1 × pbs containing 1% bsa and a 1:200 dilution of rat anti-mouse cd16/32 antibody (pharmingen, san jose, ca, usa). next, cells were stained for surface antigens using apcconjugated rat anti-mouse cd4 and cd8 (pharmingen, san jose, ca, usa), according to the viral peptide stimulation condition, for 45 min at 4°c. cells were analyzed and the data assessed as described above. spleen-derived cd8+ t cells were analyzed for lytic activity at day 8 following i.p. infection of c57bl/6 mice with 2.5 × 10 5 pfu of mhv-dm. a cd8+ t cell-enriched population of cells was obtained via negative selection through use of a magnetically labeled antibody specific for the cd8 antigen followed by passage over a magnetic column (miltenyi biotec, auburn, ca, usa) [45] . the numbers of s510-518-specific cd8+ t cells were determined by tetramer staining and these cells were used as the effector population. rma-s cells, a murine lymphoma cell line that presents viral peptides to cytotoxic t lymphocytes (ctl), were cultured at a density of 10,000 per well in flat-bottomed 96-well format tissue culture plate (corning life sciences, tewksbury, ma, usa) and pulsed overnight with 5 μm of the immunodominant cd8 peptide specific for mhv spike (s) glycoprotein, spanning amino acids 510 to 518 (s510-518, bio-synthesis, lewisville, tx, usa). cd8 t-cells, exposed to either fty720p (100 nm) or vehicle alone, were then plated with rma-s cells at effector-to-target (e:t) ratios ranging from 20:1 to 2.5:1. co-cultures were incubated for 4 h at 37°c in 5% co 2 at a final volume of 200 μl per well. the amounts of lactate dehydrogenase (ldh) released from lysed cells were determined using a cytotox 96 non-radioactive cytotoxicity assay (promega, madison, wi, usa). the percentage of ctl-mediated lysis was determined as specified by the manufacturer's protocols. spleen-derived cd4+ and cd8+ t cells from mhv-dm infected mice [39] were analyzed for cytokine secretion. cd4+ and cd8+ t cells were isolated as described above using an isolation kit according to the manufacturer's instructions (miltenyi biotec, auburn, ca, usa). then 1 × 10 6 t cells per well on a round bottom 96-well plate were incubated for 48 h at 37°c in 5% co 2 in the presence of fty720p 100 nm or vehicle. supernatants were then collected and an elisa was performed for the following cytokines: ifn-γ and tumor necrosis factor alpha (tnf-α). samples were run in triplicate in accordance with the manufacturer's directions (r&d systems, minneapolis, mn, usa). clinical severity was assessed using a previously described four-point scoring scale [16] . spinal cords were isolated at defined time points and fixed overnight with 4% paraformaldehyde at 4°c. spinal cords were separated into 12 coronal sections, cryoprotected in 20% sucrose and embedded in optimum cutting temperature (o.c.t) formulation (vwr, radnor, pa, usa) [46] . next 8-μmthick coronal sections were cut and sections were stained with luxol fast blue (lfb). areas of total white matter and demyelinated white matter were determined with image j software. demyelination was scored as a percentage of total demyelination along the entire length of the spinal cord. an h&e stain was performed to determine the extent of inflammation. spinal cord sections were scored using a four-point scale to assess neuroinflammation [16] . fty720 treatment of jhmv-infected mice increases clinical disease severity and impairs control of viral replication s1p1 egfp knock-in mice c57bl/6 mice [36] were infected i.c. with jhmv (150 pfu) and subsequently treated with increasing concentrations (1, 3 or 10 mg/kg) of fty720 via i.p. injection administered daily starting at day 5 p.i. mice were scored daily until day 21 p.i. fty720 treatment resulted in increased severity of clinical disease with the greatest effects occurring at 3 mg/kg and 10 mg/kg doses (p < 0.05) compared to vehicle-treated mice ( figure 1a ). in accordance with clinical data, fty720-treated mice exhibited increased mortality in a dose-dependent manner ( figure 1b ). by day 21 p.i., <30% of mice treated with 10 mg/kg fty720 survived while mice treated with either 3 mg/kg or 1 mg/kg exhibited 40% and approximately 60% survival, respectively ( figure 1b) . based on the morbidity and mortality data, 3 mg/kg fty720 was used for subsequent in vivo studies. fty720 treatment resulted in increased viral burden within the brain and spinal cord as determined by plaque titer at days 7 and 14 p.i.(p ≤ 0.05) compared to vehicle-treated control mice ( figure 1c,d) . however, at later times p.i. the majority of mice treated with fty720 had reduced viral titers below the level of detection (approximately 100 pfu/g) within the brain and spinal cord ( figure 1c,d) . these findings indicated that the increase in mortality following administration of fty720 correlated with impaired ability to control viral replication within the cns and argues that either trafficking of virus-specific lymphocytes is impaired and/or anti-viral effector functions are negatively affected. t cell anti-viral effector function and fty720 treatment t cell responses, including proliferation, secretion of ifn-γ and ctl activity, are critical in controlling jhmv replication within the cns [47] [48] [49] [50] [51] [52] [53] . fty720p treatment (100 nm) had no appreciable effect on dampening proliferation of either cd8+ t cells specific for the immunodominant epitope for the spike (s) glycoprotein spanning amino acid residues 510-518 (s510-518) [42] or cd4+ t cells recognizing the matrix (m) glycoprotein peptide 133-147 (m133-147) [43] (figure 2a,b) . lymphocytes were isolated from spleens of jhmv-dm infected mice day 8 p.i., pulsed with either m133-147 or s510-518 peptides and treated with fty720p (100 nm) to determine if cytokine secretion was affected. fty720 treatment did not affect secretion of either ifn-γ or tnf-α compared to vehicletreated cultures ( figure 2c ). finally, fty720 treatment of cd8+ t cells did not affect lytic activity compared to controls ( figure 2d ). these findings argue that fty720 treatment does not dampen anti-viral t cell effector functions. we next determined if fty720 affected s1p1 expression on circulating lymphocytes in jhmv-infected mice. administration of fty720 reduced s1p1 on circulating cd3+ lymphocytes (p < 0.001) compared to vehicle-treated controls at day 7 p.i. (figure 3a,b) . examination of t cell infiltration into the cns of fty720-treated mice infected with virus indicated reduced frequency of cd4+ t cells (p < 0.05) at day 7 p.i. although cd4+ t cell trafficking was not affected at days 14 and 21 p.i ( figure 3c,d) . further, fty720 treatment did not affect cd8+ t cell infiltration into the cns at days 7, 14 and 21 p.i. (figure 3c ,e). infiltration of virus-specific cd4+ and cd8+ t cells, as determined by intracellular ifn-γ staining in response to treatment with immunodominant cd4+ and cd8+ viral epitopes [42, 43] , was diminished at days 7 (p < 0.01) and 14 (p < 0.05) following fty720 treatment in comparison to vehicle-treated mice ( figure 4a,b) . by day 21 p.i., infiltration of virus-specific cd4+ t cells, but not virus-specific cd8+ t cells, was also reduced (p < 0.05) in fty720-treated mice compared to control animals ( figure 4b ). infiltration of antibody secreting cells (ascs) (cd45 − cd19 low cd138+) into the cns of jhmv-infected mice was not reduced at either days 7, 14 or 21 p.i. following fty720 treatment compared to control mice ( figure 4c,d) . these findings indicate that administration of fty720 negatively impacts recruitment of t lymphocytes into the cns in response to jhmv infection. as an s1p1 functional antagonist, fty720 disrupts the s1p gradient in lymph nodes thereby trapping lymphocytes [4, 5] . to establish if this occurs during ongoing neuroinflammation in response to infection with neurotropic jhmv, draining cervical lymph nodes (dclns) were examined at defined times p.i. following treatment with fty720. administration of fty720 revealed an increase in size and weight of dclns in fty720-treated mice compared to control mice at day 7 p.i. (figure 5a,b) . immunophenotyping lymphocytes in dclns by flow cytometry revealed an increased frequency of b220+ b cells ( figure 5c ), cd4+ t cells ( figure 5d ) and cd8+ t cells ( figure 5e ) in mice treated with fty720 compared to controls, indicating increased retention of lymphocytes in lymphatic tissue in response to s1p antagonism. to investigate the potential effect of fty720 on spinal cord neuroinflammation and demyelination in jhmvinfected mice, an evaluation of the severity of white matter damage was performed at day 21 p.i. spinal cord inflammation was reduced (p < 0.05) within spinal cords at day 21 p.i. as assessed by h&e staining (figure 6a,b) . moreover, lfb staining revealed a significant (p < 0.05) reduction in demyelination in response to fty720 treatment compared to control mice ( figure 6a,b) . immunophenotyping of infiltrating lymphocytes in the spinal cord day 21 p.i. revealed a decrease in cd8+ t cell as well as cd4+ t cell percentages (p <0.05) in fty720-treated mice compared to vehicle-treated mice ( figure 6c,d) . this suggests that reduction in the severity of demyelination c a d e b figure 5 increased lymphocyte retention in dclns following fty720 treatment of jhmv-infected mice. s1p1 egfp mice were i.c. infected with jhmv (150 pfu) and treated daily with fty720 (3 mg/kg) beginning on day 5 p.i. on days 7, 14 and 21 p.i., dclns were isolated to examine size, weight and immunophenotype lymphocyte population by flow cytometry. (a) representative image depicting the increase in size of dclns obtained from fty720-treated mice compared to vehicle control-treated mice at day 7 p.i. (b) average weight of dclns increased in response to fty720 treatment compared to vehicle (p < 0.05). treatment with fty720 resulted in increased retention of b220+ b cells (c), cd4+ t cells (d) and cd8+ t cells (e) at defined times p.i. data in panels b to e represent average ± sem obtained from two or three independent experiments with a minimum of four mice/group. *p <0.05, *** p <0.001. cln, cervical lymph node. dcln, draining cervical lymph node. discussion fty720/fingolimod was the first oral treatment approved by the fda for relapsing forms of ms [10, 54, 55] . numerous clinical trials highlighted fty720 efficacy as demonstrated by benefits for relapses and magnetic resonance imaging (mri) lesions [9] . in addition, disability progression was impacted and there was a reduction in brain volume loss in ms patients in response to treatment [56, 57] . although the mechanisms by which fty720 exerts a protective effect are not defined, it is generally accepted that the main mode of action is via an immunomodulatory effect by restricting lymphocyte circulation from lymph nodes to the cns. dampened accumulation of activated lymphocytes in the cns in response to fty720 treatment most likely accounts for reduced mri lesion activity and this is supported in preclinical animal studies using eae [14, 15, 58] . whether the reduction in brain volume loss is also dependent upon reduced neuroinflammation or a direct neuroprotective effect has not been determined. recent evidence argues that fty720 exerts a neuroprotective effect as animals in which the receptor s1p1 is selectively ablated on astrocytes are resistant to the protective effects of fty720 treatment following induction of eae, although s1p1 remains expressed on circulating lymphocytes [12, 59] . however, more recent studies by cahalan et al. indicate that s1p1 antagonism reverses eae without acting on s1p1 expressed within the cns, supporting the notion that restricting lymphocyte egress from lymphatic tissue is sufficient to diminish disease severity [60] . moreover, the maintenance of egress inhibition is not required for efficacy in eae if brain levels of agonist are maintained in the steady state. full efficacy is achieved with inhibition of egress for only 30% of the 24-h dosing interval with complete recovery of circulating lymphocytes. direct effects within the cns were demonstrated for neurons, astrocytes and the blood-brainbarrier, and on the inhibition of migration of lymphocytes from perivascular cuffs into the parenchyma [61] . we chose a model of viral-induced neurologic disease to determine if fty720 treatment affects host defense and disease progression. our rationale for using the jhmv model of acute encephalomyelitis and demyelination to assess the therapeutic benefit of fty720 is based on the fact that the overwhelming majority of preclinical animal models examining the mode of action for fty720 is derived from eae, yet how this drug affects models of viral-induced cns disease are not well characterized. in addition, viral infections have long been thought to have a role in either initiating or contributing to relapse in ms patients [28] [29] [30] [31] [32] [33] [34] 62] . how treatment with fty720 influences outcomes in response to viral infection is highlighted by recent clinical studies detailing the emergence of herpes zoster and associated neurologic complications in ms patients during fty720 treatment [63, 64] . these findings suggest immunosuppression may arise in response to fty720 treatment resulting in re-emergence of persistent viruses. however, administration of fty720 to mice infected with lymphocytic choriomeningitis did not ameliorate persistence, indicating that the outcome may be dictated, in part, by the virus and sites of infection [65] . related to these observations are studies demonstrating that treatment with fty720 or other s1p1 agonists dramatically affects cytokine production and disease outcome in mice infected with influenza virus, indicating the immunomodulatory effects of such treatment [66] . with regards to viral models of demyelination, pachner and colleagues [67] demonstrated that administration of fty720 had no effect on clinical disease progression or viral load within the cns using theiler's murine encephalomyelitis virus model of demyelination. these findings are in contrast with findings using eae, in which fty720 treatment reduced clinical disease severity accompanied by limited infiltration of immune cells into the cns [13] [14] [15] 68, 69] . our findings show that fty720 treatment for jhmvinfected mice increased clinical disease severity as well as mortality. importantly, these findings correlate with impaired ability to control viral replication within the cns. the muted host defense resulting from s1p receptor antagonism was not the result of dampened anti-viral t cell effector responses, e.g. proliferation, cytokine secretion or ctl activity, but rather an inability of lymphocytes to migrate and accumulate within the cns effectively. indeed, administration of fty720 increased retention of t and b lymphocytes within the dclns, consistent with earlier reports that blocking s1p receptors disrupts lymphocyte egress from secondary lymphatic tissue [70, 71] . although viral titers were elevated within (see figure on previous page.) figure 6 fty720 treatment reduces the severity of jhmv-induced demyelination. (a) representative lfb and h&e-stained spinal cord images showing an overall reduction in the severity of inflammation and demyelination within white matter tracts (dashed lines) in jhmv-infected egfp s1p1 mice treated with fty720 (3 mg/kg) compared to vehicle control-treated mice at day 21 p.i. (b) fty720 treatment results in reduced neuroinflammation (p < 0.05) and demyelination (p < 0.05) compared to mice treated with the vehicle control. flow analysis of spinal cords demonstrates reduced entry of both cd8+ (c) and cd4+ t cells (d), while infiltration of virus-specific t cells was not affected. data in panel b represent two independent experiments with a minimum of ten mice/group. data in panels c and d are presented as average + sem and represent two independent experiments with a minimum of five mice/group. *p <0.05. the scale bar in panel a represents 200 μm. ssc, side scatter. the cns of fty720-treated mice, surviving mice were able to reduce the amount of virus below the level of detection and this lasted to day 28 p.i., arguing that viral recrudescence does not occur in this model. administration of fty720 either prophylactically or therapeutically in models of eae results in reduced lymphocyte penetration into the cns, which is associated with a reduction in the severity of demyelination [14, 15, 58] . similarly, our results show that the effects of fty720 treatment on cns inflammation in jhmvinfected mice correlates with a reduction in the severity of spinal cord demyelination. examination of the posterior funiculus and lateral white matter columns of fty720treated mice compared to controls showed an overall reduction in lesion size, demonstrating that in addition to reducing myelin damage in eae, fty720 is also effective in limiting the spread of demyelination in a viral model of ms. fty720 has also been shown to prevent axonal damage in eae [58] . fty720 in combination with other drugs in eae or cerebellar slice cultures has been shown to augment remyelination, supporting a regenerative potential [72, 73] . these findings support that fty720 may act directly upon resident cells of the cns promoting protection and repair. this is supported by elegant studies from chun and colleagues [12] that showed attenuation in eae and a loss of fty720 efficacy in conditional null mouse mutants lacking s1p1 in astrocytes. these findings highlight that fty720-mediated protection in eae occurs via a nonimmunological mechanism and suggest that targeting s1p signaling within the cns may be relevant for recovery for both eae and ms patients. whether extensive axonal sparing and/or remyelination occurs following fty720 administration to jhmv-infected mice is not known at this time and is an area of ongoing work. in this study we demonstrate that fty720 mutes effective anti-viral immune responses by preventing migration and accumulation of virus-specific t cells within the cns during acute viral-induced encephalomyelitis. fty720 treatment reduces the severity of neuroinflammatorymediated demyelination by limiting t cell egress from lymph nodes thereby reducing lymphocyte infiltration into the cns. fty720 did not alter anti-viral effects of t cells, e.g. cytokine secretion or cytolytic activity. multiple sclerosis multiple sclerosis: prospects and promise mechanisms of fingolimod's efficacy and adverse effects in multiple sclerosis fingolimod in multiple sclerosis: mechanisms of action and clinical efficacy bar-or a: clinical immunology of the sphingosine 1-phosphate receptor modulator fingolimod (fty720) in multiple sclerosis oral fingolimod (fty720) in multiple sclerosis: two-year results of a phase ii extension study fingolimod is a potential novel therapy for multiple sclerosis fingolimod, a sphingosine-1 phosphate receptor modulator, increases bdnf levels and improves symptoms of a mouse model of rett syndrome fingolimod therapy for multiple sclerosis fingolimod (fty720): discovery and development of an oral drug to treat multiple sclerosis alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists fty720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (s1p1) modulation amelioration of experimental autoimmune 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reduced neurologic disease in a viral model of multiple sclerosis functional analysis of the cc chemokine receptor 5 (ccr5) on virus-specific cd8+ t cells following coronavirus infection of the central nervous system keirstead hs: remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the mhv model of multiple sclerosis coronavirus infection of the central nervous system: host-virus stand-off inhibition of interferon-γ signaling in oligodendroglia delays coronavirus clearance without altering demyelination mhv infection of the cns: mechanisms of immune-mediated control ifn-γ is required for viral clearance from central nervous system oligodendroglia control of central nervous system viral persistence by neutralizing antibody mechanisms of central nervous system viral persistence: the critical role of antibody and b cells ctl effector function within the central nervous system requires cd4+ t cells fingolimod (fty720): a 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cerebellar slices the authors wish to acknowledge the excellent technical assistance of edna hingco and colleen worne. this work was supported, in part, by national institutes of health grant r01 ns041249 and generous support from the hausman family foundation and dawn beattie. competing interests cab and tel declare they have no competing interests. hr is a cofounder and member of the scientific advisory board for receptos, inc.authors' contributions cab designed and conducted the experiments, analyzed and interpreted the data, and wrote the manuscript. tel designed the research, analyzed and interpreted the data, and wrote the manuscript. hr assisted in data interpretation and wrote the manuscript. all authors read and approved the final manuscript. key: cord-002119-kl431ev6 authors: garcia, elisa; aguilar-cevallos, jorge; silva-garcia, raul; ibarra, antonio title: cytokine and growth factor activation in vivo and in vitro after spinal cord injury date: 2016-06-23 journal: mediators inflamm doi: 10.1155/2016/9476020 sha: doc_id: 2119 cord_uid: kl431ev6 spinal cord injury results in a life-disrupting series of deleterious interconnected mechanisms encompassed by the primary and secondary injury. these events are mediated by the upregulation of genes with roles in inflammation, transcription, and signaling proteins. in particular, cytokines and growth factors are signaling proteins that have important roles in the pathophysiology of sci. the balance between the proinflammatory and anti-inflammatory effects of these molecules plays a critical role in the progression and outcome of the lesion. the excessive inflammatory th1 and th17 phenotypes observed after sci tilt the scale towards a proinflammatory environment, which exacerbates the deleterious mechanisms present after the injury. these mechanisms include the disruption of the spinal cord blood barrier, edema and ion imbalance, in particular intracellular calcium and sodium concentrations, glutamate excitotoxicity, free radicals, and the inflammatory response contributing to the neurodegenerative process which is characterized by demyelination and apoptosis of neuronal tissue. traumatic spinal cord injury (sci) is a complex, lifedisrupting medical condition due to the detrimental effects on social, familiar, and personal life, which include in the majority of cases permanent paralysis due to the low regenerative capacity of the central nervous system (cns). sci triggers a series of interconnected mechanisms that can be divided into the primary and secondary injury. the direct and immediate physical disruption of neurons, glial cells, and blood vessels makes up the primary injury. in turn, the secondary injury consists of a cascade of autodestructive cellular and molecular mechanisms that exacerbate the primary injury and lead to an enlargement of the initial area of trauma [1] [2] [3] [4] . several mechanisms take part in this latter phase of the injury, including vascular disruption, increased blood-spinal cord barrier permeability, ionic dysregulation, edema, excessive intracellular calcium concentration, glutamate excitotoxicity, lipid peroxidation, an autoreactive inflammatory reaction, and apoptosis [5] . ultimately, the sum of these processes causes cell death, demyelination, and axonal degeneration at the epicenter of injury and the surrounding regions. these cellular and molecular changes that occur early after sci alter gene expression profiles, which is characterized by a significant upregulation of genes with roles in transcription, inflammation, and signaling proteins [6] . evidence suggests that the consequent inflammation mediated by cytokines, growth factors, and related molecules plays a role in both the damage and repair of injured neural tissue [7] [8] [9] . the critical balance between these processes plays a major participation in the progression and outcome of a neurodegenerative process [10] . cytokines encompass a large family of small signaling proteins involved in intercellular communication that are normally associated with the immune response and its 2 mediators of inflammation modulation but have pleiotropic effects in the physiology of health and disease including cellular growth, survival, and differentiation. these molecules, which can be classified as peptides, proteins, or glycoproteins, are secreted by numerous cells and can be grouped into a proinflammatory or antiinflammatory category on the basis of the final balance of their effects [10] . subsequently, growth factors are proteins synthesized by a wide variety of cells that stimulate cellular survival, chemotaxis, proliferation, and differentiation [11, 12] . the aim of this review is to expose the role of cytokines and growth factors within the pathogenesis of sci, since the study of these molecules could bring to light novel potential therapeutic targets that could reduce the degenerative processes that occur after sci. cord injury barrier. the blood-cns vascular barriers consist of complexes of adherence junction proteins and tight junctions, astrocyte endfeet, perivascular microglia, pericytes, and continuous capillary endothelial cells embedded in the basement membrane that separate and protect the cns from metabolites and neurotoxic substances present in the systemic circulation [13] [14] [15] . this infrastructure allows the blood brain barrier (bbb) and blood spinal cord barrier (bscb) to regulate the transport of molecules, the interaction between the cns and the immune system, and helps maintaining homeostasis in the brain and spinal cord. one of the earliest events ensuing traumatic sci is the disruption of the bscb by a mechanical force that destroys neural tissue and tears neuronal and endothelial cell membranes [5] . the resulting inflammatory response disturbs the microenvironment of the spinal cord, alters vascular permeability, facilitates the entry of peripheral immune cells, and exposes the adjacent noninjured tissue to potentially noxious molecules [16, 17] . these molecules include early inflammatory cytokines such as interleukin 1 (il-1 ) and tumor necrosis factor (tnf ); in addition, they might include nitric oxide (no • ), reactive oxygen species (ros), elastase, and matrix metalloproteinase-9 (mmp-9) [17] . the importance of the bscb is evidenced by the positive correlation between increased barrier disruption and improved motor locomotion 14 days after sci [18] [19] [20] . an additional consequence of such disruption is a series of regulatory changes in the transport systems for selective cytokines that may induce regenerative or destructive effects. in particular, there is an upregulation of the transport system of tnf after sci that remains saturable despite bscb disruption. the increase of tnf takes place before other cytokines in sci and is mediated by the receptor-based transport composed by tnfr1 (p55) and tnfr2 (p75) [21] . tnf has a role in inflammation, myelin destruction, apoptotic neuronal cell death, and astrocyte toxicity. nevertheless, this cytokine is also capable of stimulating neurite outgrowth, secretion of growth factors, and tissue remodeling [21] . it has been suggested that tnf has a dual role: deleterious in the acute phase, but beneficial in the chronic phase after sci [22] . similarly, leukemia inhibitory factor (lif) utilizes a transport system mediated by lifr (gp190), which is upregulated by barrier disruption, but remains saturable despite this event [21, 23] . lif is involved in the activation of microglia/macrophages and in the proinflammatory response in sci [24] . contrastingly, lif has been shown to prevent oligodendrocyte apoptosis in mice with sci after overhemisection, notably contralateral to the spinal cord lesion, through the induction of the jak/stat and akt signaling pathways as well as by potentiating the expression of the antiapoptotic molecule, ciap2. reduced oligodendrocyte apoptosis after sci with lif administration resulted in a substantial decrease in demyelination shown by the preservation of lamellated myelin surrounding viable axons and deposition of the degraded myelin basic protein. the data suggest that lif signals survival in oligodendrocytes after sci, prevents the secondary wave of demyelination, and thereby reduces inhibitory myelin deposits and enhance locomotor recovery [25] . imbalance. immediately after contusive sci, the rupture of the blood-cns barrier causes water to accumulate in the extracellular compartment and results in the production of neural tissue edema [26, 27] . this is a process that may aggravate the initial injury and result in paraplegia or even death [13] . the subsequent increment in vascular permeability and the formation of edema could also be in part mediated by the vascular endothelial growth factor (vegf) and proto-oncogene tyrosine-protein kinase (src/c-src) which exists downstream of vegf [28] . it is worth noting that administration of vegf has resulted in an increase in permeability of the bscb from the acute to chronic phase, which is interesting since it is regarded to be a component involved in angiogenesis, neurogenesis, and locomotor recovery [29] . as the secondary injury progresses, this fluid accumulation in the cns becomes characterized by ionic imbalance, which consists of an increase in the intracellular concentration of na + and ca 2+ , in conjunction with an elevated extracellular concentration of k + and mg + [30] [31] [32] . consequently, the na + and ca 2+ ions attract water molecules into the cell and cause edema. the resulting fluid accumulation then propels the compression of adjacent tissues and the development of ischemia, which leads to more autodestructive phenomena such as free-radical production, lipid peroxidation, and inflammation. it is important to note that the edema that occurs after contusive sci is directly related to the initial trauma and motor dysfunction experienced by the affected individual [27, 33] . astrocytes are the principal regulators of water transport in the cns, where they are additionally linked to the maintenance of ion homeostasis, spatial buffering of extracellular potassium, calcium signal transduction, adult neurogenesis, and neurotransmitter uptake and release [34] [35] [36] . a molecule expressed in astrocyte endfeet, astrocyte processes, and the basolateral membrane of ependymal cells is aquaporin 4 (aqp4), the predominant water channel in the cns [36] . recent studies indicate that aqp4 regulates the beforementioned astrocytic functions [36] . moreover, the absence of aqp4 has been shown to reduce proinflammatory cytokines in astrocytes such as tnf and interleukin-6 (il-6) after cns injury [37] . it is important to mention that the role of aqp4 in the resolution of edema is still under debate [37] . nevertheless, evidence demonstrates that aqp4 has an essential role in the formation and distribution of edema and that it is intrinsically involved in the development of the inflammatory process after an insult to the cns [37] . on the other hand, neurons regulate synaptic transmission and neural plasticity by the activation of membrane receptors and channels in adjacent neurons. released neurotransmitters can bind to inhibitory (gaba)ergic receptors or excitatory glutamate receptors such as amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (ampa), n-methyl-daspartate (nmda), kainate, and metabotropic receptors [38] . in the locomotor networks of the spinal cord, ca 2+ activated, apamin-sensitive k + channels (sk) control the firing of constituent neurons and regulate the locomotor rhythm. voltagegated ca 2+ channels (vgccs), such as n-type ca 2+ channels, are considered the main activators of sk channels [39] , which during early development play a role in neurite outgrowth and functional neuromuscular synapse organization [40] . nmda receptors, besides controlling evoked neurotransmitter release, also play a role in the activation of sk channels in dendrites [39, 40] . sk channels have been found to regulate hippocampal synaptic plasticity, learning, and memory, particularly sk2 channels [41] . synaptic transmission involves ca 2+ and employs calmodulin (cam) dependent kinases (camkiiv), protein kinase c, protein kinase a, ip3 kinase, ca 2+ -dependent phosphatase calcineurin b, cyclic amp phosphodiesterase, adenylyl cyclase, lca 2+ -dependent neuronal nitric oxide synthase (nos), and calpains, which are ca 2+ activated proteases [42, 43] . in the first few minutes following sci, oxidative stress, lipid peroxidation, and membranous deposition of protein aggregates take place. these processes impair ca 2+ pumps and cell membrane channels, including those present in the endoplasmic reticulum. this downregulation is evidenced by an increased concentration of cytosolic ca 2+ from extracellular pools and intracellular ca 2+ storages [44] . in normal conditions, the energy-dependent ca 2+ buffering system within axons removes the excess ca 2+ . however, when adenosine triphosphate (atp) is depleted by the excessive energy demands of demyelination, this normal ca 2+ buffering fails and the level of intracellular ca 2+ rises until it becomes toxic [44] . the result is the chaotic activation of processes such as proliferation, differentiation, apoptosis, and gene transcription in cells [45] . in addition to the before-mentioned channels, axons also have a high concentration of voltage-gated na + channels spread along the length of their bodies. thus, when axonal demyelination occurs, there is a dramatic increase in na + influx into the cell during the action potential propagation. the elimination of such an excess concentration of intracellular na + can come at a steep metabolic expense in a similar fashion to ca 2+ removal, since the na + /k + atpase maintains the na + electrochemical gradient by atp consumption [46, 47] . when atp levels fall below a certain threshold, there is a concomitant increase in the intra-axonal concentration of na + and ca 2+ . consequently, glutamate is released, and the na + /ca 2+ exchanger, which normally pumped out 1 ca 2+ in exchange for 3 na + , is reversed [46, 47] . it is also important to mention that the subsequent release of atp after the lesion increases in peritraumatic areas for 6 or more hours [48] . this excessive release of atp by the traumatized tissue after sci is followed by the activation of high affinity purinergic p2x receptors. it is important to note that the p2x7 receptors may also contribute to the excessive influx of ca 2+ since they are upregulated in response to the atp release induced by sci. this might explain why spinal cord neurons respond to atp with excessive firing, followed by irreversible increases in ca 2+ that end up in cell death [49, 50] . furthermore, p2x7rs have been associated with cells of the immune system that mediate cytotoxic cell death (because of changes in transmembrane ion fluxes, swelling, and vacuolation) and those that mediate inflammatory responses, including proinflammatory mediators such as il-1 and tnf [49, 50] . glutamate receptors are involved in the excitatory neurotransmission of the mammalian cns, where they participate in various changes in the efficacy of synaptic transmission, and induce excitotoxic damage in a variety of acute and chronic neurological disorders [51, 52] . the process of excitotoxicity refers to the excessive receptor activation by this excitatory amino acid that results in neuronal death [53] . just 15 min after sci, glutamate levels at the epicenter and surrounding regions become six times higher than physiological levels due to the overstimulation of ionotropic receptors and the massive increase of intracellular ca 2+ and na + . this glutamate influx provokes overexcitation and endotoxicity by the secondary increase of intracellular ca 2+ and the activation ca 2+ dependent signaling pathways as previously mentioned [54] [55] [56] . moreover, the augmented expressions of genes related to neurotransmitter receptors (nmda, ampa, ach, gaba, glur, and kainate) increase demyelination and oligodendrocyte destruction [57, 58] . an important mechanism for the reduction of excessive extracellular glutamate is the activity of glutamate transporters such as glial glutamate transporter 1 (glt-1) and glutamate aspartate transporter (glast), which are primarily expressed by astrocytes [59] . unfortunately, the excitotoxicity induced by the extracellular glutamate concentration is enhanced by the reduced uptake by astrocytes and the microglia release tnf , il-1 , and ros that exacerbated the neural damage [60] . tnf and il-1 have been shown to cause oligodendrocyte death when the latter are placed in coculture with both astrocytes and microglia. both cytokines inhibit glutamate transporters in astrocytes and thus expose oligodendrocytes to an excessive glutamate concentration. it is important to note that antagonists of ampa/kainate glutamate receptors such as nbqx (2,3-dioxo-6-nitro-7-sulfamoilbenzo(f)quinoxalina) and cnqx (6-cyano-7-nitroquinoxaline-2,3-dione) blocked il-1 toxicity towards oligodendrocytes [61] . tnf causes excitotoxicity through a series of interconnected, deleterious mechanisms. first, microglia release this cytokine in the inflammatory response, which induces additional release of tnf . in turn, it causes the release of glutamate that acts on metabotropic receptors of microglia and stimulates more tnf release. subsequently, astrocytes are stimulated to release glutamate, which is not effectively transported back into the soma. lastly, the rise in the excitatory/inhibitory ratio causes the excessive ca 2+ entry and excitotoxic neuronal death previously described. the consequent neuronal death caused by the excessive glutamate concentrations further stimulates microglia to remain in an active state, which includes the production and release of tnf in a vicious cycle [53] . tnf potentiates cytotoxicity by glutamate through an increased localization of glutamate receptors such as ampa and nmda while decreasing inhibitory gaba receptors on neurons [62] , which explains why nbqx blocked tnf toxicity to oligodendrocytes [61] . in the destruction of neurons, nerve fibers, glial cells, and blood vessels at the site of injury, where approximately 30% of neurofilament constitutive proteins are degraded in 1 h, and 70% are lost within 4 h after the injury [63] . proteins such as cathepsin b, y, and s, members of the cysteine lysosomal proteases and papain superfamily, have been linked to neurofilament destruction. this link results from the fact that cathepsin b can degrade myelin basic protein, cathepsin y can produce a bradykinin, and cathepsin s can degenerate extracellular molecules through inflammatory mediators. in particular, only cathepsin s is able to retain its activity after prolonged incubation at neutral ph, more than 24 h [64, 65] . the expression of this protease is restricted to cells of the mononuclear phagocytic system such as microglia and macrophages [64] . a basement membrane heparan sulfate proteoglycan (hspg), perlecan, which was found to promote mitogenesis and angiogenesis, can be degraded by cathepsin s in vitro. hspgs have roles in adhesion, protease binding sites, and growth factor regulation as is the case for basic fibroblast growth factor (bfgf) [66] . furthermore, cathepsin s degrades laminin, fibronectin, collagens, and elastin at acidic or neutral ph [65] . it is known that tnf , interferon-(ifn ), il-1 , and granulocyte macrophage colony stimulating factor (gmcsf) stimulate the release of active cathepsin s into an environment with a neutral ph [65] . subsequently, a change in lipid metabolism and the homeostasis of lipid mediators is an alternate route by which genes are thought to modulate the susceptibility of nervous tissue to trauma. interestingly, altered protein cleavage, one of the main driving forces of protein aggregation in neurodegenerative disorders, can be further enhanced by trauma occurring in the presence of specific lipid-binding proteins, important molecules in charge of the distribution of lipids and the transport of cholesterol among cells in the cns. apolipoprotein e (apoe) is one particular example of this phenomenon, since a reduction in its availability causes a reduction in the recovery after neurotrauma or an ischemic insult. apoe fragments are produced by traumainduced proteolytic cleavage, which, in turn, might disrupt the cytoskeleton by the phosphorylation of tau and the promotion of neurofibrillary tangles. at the same time, apoe4 increases the inflammatory effect of neurotrauma by a significant increase of il-6, tnf , and no • in the injured tissue [67, 68] . radicals. microvascular disruption, ionic imbalance, increased intracellular calcium levels, glutamate excitotoxicity, mitochondrial dysfunction, arachidonic acid breakdown, and the activation of inos contribute to the formation of free radicals (fr) [69] . fr are reactive molecules produced by the metabolism of the cell that possess an unpaired electron, which easily reacts with biomolecules by oxidizing them [70] . a fr is made up of sulphur (s), nitrogen (n), chloride (cl), or carbon (c). these elements associate with oxygen and form other fr such as no • . metals such as fe, mn, co, ni, and cu can also be considered fr since they have unpaired electrons [71, 72] . many of these molecules are either reactive oxygen species (ros) such as delta and sigma oxygen ( the mechanical reduction of the superoxide anion mediated by nad(p)h oxidases causes the anion to react with no and form a neurotoxic compound known as peroxynitrite [73] . at physiologic ph, peroxynitrite first reacts with proteins and phospholipids and then breaks down into other cytotoxic products such as no • , nitrogen dioxide (no 2 ), and oh − radicals. hall and braughler demonstrated the occurrence of early posttraumatic lipid peroxidation (lp) as early as 5 min after injury. lp is a mechanism that disrupts the normal structure and function of the lipid bilayers that surround the cell and membrane-bound organelles. when peroxynitrite or other fr takes an electron off a polyunsaturated lipid, it generates a lipid radical (l • ) that can further interact with molecular oxygen and yield a lipid peroxyl radical (loo • ). then, if the resulting lipid peroxyl radical loo • is not reduced by antioxidants, lp associated with sci induces early damage to the spinal microvascular endothelium (within 2-3 h). as a direct consequence of this damage, there are crater formation, platelet adherence, leucocyte presence, and the formation of microemboli, events that are concurrent with the reduced blood flow to the white matter of the spinal cord. the damage to the myelin sheath unhinged a demyelination process that is the particularity of a neurodegenerative process [74] . the cns is particularly sensitive to lp because of its high content of peroxidation-susceptible lipids (arachidonic, linoleic, and docosahexaenoic acid) and the primarily radical-mediated oxidative protein damage. considering the timeframe of the injury, the oxidative damage to dna and lipids, in addition to protein nitration, is observed within the first week after injury [75] [76] [77] [78] [79] [80] [81] . one of the degradation products of peroxynitrite, no • , alters the mitochondrial electron transport chain and induces the production of fr. these molecules have direct deleterious effect on enzymes with iron-sulfur clusters in their catalytic core, such as ubiquinone succinate [82] . after sci, the concentration of no • increases 3 to 5 times more than baseline levels and reaches its peak at 12 h. meanwhile, there is an increased production of inducible nitric oxide synthase (inos) and peroxynitrite [83] . the resulting elevated no • concentration induces cell damage and lipid peroxidation, increases vascular permeability, and causes edema [84] . hence, due to its involvement in the previous processes, no • participates in the development of the excessive glutamate and calcium concentrations that induce excitotoxicity [85] . it is known that no • is produced by different synthases. however, only inos is capable of producing a high concentration of no • for a prolonged period of time [86] . collectively, astrocytes, neutrophils, monocytes, and microglia induce the expression of inos at the presence of proinflammatory stimuli such as lipopolysaccharide (lps), ultraviolet radiation (uv), and tnf , il-6, il-1, and ifn [87] . in some studies, the expressions of inos and its protein activity were found 3 h, 4 h, 24 h, and 72 h after sci [83, 88, 89] . the inflammatory response is a characteristic phenomenon of innate immunity that does not require a previous exposition to the agent but does increase substantially with subsequent expositions as the response becomes specific and direct. cellular immunity consists of specialized cells that can recognize, endocyte, and eliminate different types of microorganisms or noxious substances. on the other hand, the humoral response is composed by soluble macromolecules that circulate in the blood and extracellular liquid that acts upon the pathogen [90, 91] . sci presents different patterns of gene expression depending on the cell type and activation phase [92] . numerous studies have suggested that the inflammatory response in sci is beneficial, because it can eliminate tissue debris and induce the release of various neurotrophic factors [17, 93, 94] . nevertheless, this inflammatory response tends to go out of control when it exacerbates autoreactive mechanisms that cause neural destruction. traumatic sci triggers inflammatory reactions in which various types of cells, cytokines, and neuroprotective/neuroregenerative molecules are involved [95] . 2.6.1. cells of the inflammatory response. immediately after the rupture of the blood-spinal cord barrier, the consequent inflammatory response involves the participation of chemical mediators, and resident (astrocytes and microglia) and peripheral (macrophages, lymphocytes) immune cells [96, 97] . additionally, oligodendrocytes, neurons, and endothelial cells participate in the cellular response after sci [98] , in which microglia and endothelial cells function as antigenpresenting cells (apc) [96] . throughout the inflammatory response, the infiltration of immune cells is the principal contributor to neural degeneration [95] . these cells are guided to the lesion site from the periphery by the effect of chemokines and cytokines that are mainly released by microglia, astrocytes, and peripheral macrophages, which make up the principal resource of these molecules in the lesion site [5, [99] [100] [101] [102] . the released cytokines include il-1 , il-1 , il-6, tnf , gm-csf, and lif [60] . the neurons of human patients expressed these molecules, 30 min after sci, and microglia, 5 h after the lesion; however, the expression decreased by the 2nd day [103] . similar results were obtained in mice and rats since the expression by local neurons was found at 1 h, and at 6 h by microglia, which decreased to baseline on day 1 after sci [104] . the expression of tnf and il-1 by microglia and astrocytes was identified 5-15 min after the lesion. the peak expression was at 1 h for tnf and 12 h for il-1 [104] . after sci, two waves of cellular infiltration have been characterized. the first wave consists of polymorphonuclear leukocytes (pmn) that predominate throughout the first hours following the lesion. they are activated by il-1, interleukin-2 (il-2), and il-6 in particular [105] and might be mainly recruited by chemokine (c-c motif) ligand 2 (ccl2), chemokine (c-x-c motif) ligand 1 (cxcl1), and chemokine (c-x-c motif) ligand 2 (cxcl2, also called macrophage inflammatory protein 2-alpha (mip2-alpha)) [106] . these cells become apparent in the walls of veins and venules adjacent to the lesion in the first 3-4 h and can be observed inside the tissue 8-24 h after the lesion. it has been found that these cells represent 90% of the infiltrating cells 12 h after the injury [107] . the inflammatory response is evidenced by the increased quantity of leukocytes in the cerebrospinal fluid, the infiltration of pmn in the lesion site, the increment in the leukotriene levels (ltb4 in particular), and the activity of myeloperoxidase. in addition, a significant increase in the expression of intercellular adhesion molecule 1 (icam-1) can be identified, which favors the infiltration of neutrophils from the first 3 to 12 h after the lesion [108] . the second wave of infiltration is characterized by the presence of monocytes and macrophages, which can be observed around 3-4 days after sci [106] . various chemokines are known to mediate macrophage infiltration such as ccl2, cxcl1, and cxcl2 [106] . this demonstrates how important the recruitment of macrophages is after an injury to the cns [109, 110] . activated microglia become evident in the first day after sci [108] ; moreover, there is a peak in the proliferation and recruitment of microglia from day 3 to day 7 [111, 112] . the overexpression of lif has been found to cause a dramatic increase in the proliferation of microglia/macrophages and astrocyte activation [24] . the pathological proliferation of macrophages and microglia might contribute to the subsequent exacerbation of the initial damage [113, 114] , even though macrophages have an important role in the clearing of denatured dendrites [115] . microglia at the injury site rapidly express the alarmin il-1 , while infiltrating neutrophils and macrophages produce il-1 which plays a role in the infiltrating mechanism of these cells. interestingly, the expression of il-1 mediates the suppression of the survival factor tox3 (tox high mobility group box family member 3) in oligodendrocytes, which in the absence of such cytokine would provide protection of this cell population, and functional recovery after sci [7] . diverse studies have reported that the recruitment of leukocytes to the injured spinal cord is a physiological response that is associated with the production of cytokines and protein kinases that are involved in the repair of the site of lesion. neutrophils, for example, are the first cells to be recruited with the objective of clearing the lesion site from possible pathogens and cellular debris through phagocytosis. however, the activation of these cells also induces the release of a significant amount of neurotoxins such as ros, rns, chemokines, and a variety of enzymes that promote tissular destruction [105, 116, 117] . the taoka report provides evidence demonstrating that after sci the maximum peak of neutrophil migration perfectly correlates with the extent of the damage and the motor alterations observed after the lesion [105] . the infiltration of monocytes and macrophages after sci has for its objective the removal of cellular debris and the stimulation of new blood vessel and parenchymal cell formation. the infiltration of these cells regulates the activation and proliferation of t lymphocytes since they play the role of apc [117] . microglia are pluripotent cells capable of developing different phenotypes proportional to the severity of the lesion, which determines the intensity of the inflammatory response, the quantity of recruited cells, and the magnitude of the immunological response. this can be explained by the interaction between microglia and t lymphocytes, since it induces an antigen specificity that regulates the immune response and the subsequent phases [118] . microglial cells are distributed throughout the cns, where they serve as a pathological sensor that is activated in response to noxious stimuli such as physical trauma or vascular obstruction [119] . activated microglia migrate to the site of injury, proliferate, and transform from the resting phenotype (ramified cells) to amoeboid phagocytic cells [120] . in fact, after sci, activated microglia can be seen at the epicenter of the lesion initially at 12 h [60] . in addition, there is an upregulation of surface receptors such as cr3 (complement receptor type 3) and mhc ii (major histocompatibility complex) whose implications are covered in several papers of our group. these activated microglia can then release a series of cytokines, chemokines, and enzymes, which are proportional to the activating stimulus as mentioned previously. the series encompass il-1 , il-6, tnf , tgf-1 (transformation growth factor-1), m-csf [121] , inos, ngf (neural growth factor), nt-3 (neurotrophin-3), and bndf (brain neuronal derived factor) [122, 123] . interestingly, monocyte derived microglia and macrophages might be able to induce regeneration by the secretion of neurotrophic factors, particularly tgf-1 [17] . activated microglia and macrophages have been implicated in the secondary pathology that accompanies traumatic or autoimmune injuries to the brain and spinal cord [124] . the associated implications usually refer to the activation of these cells towards an inflammatory m1 phenotype; however, these cells can also be activated towards an m2 macrophage phenotype that responds to il-4 and il-13. this contrasting phenotype is characterized by the production of several extracellular matrix proteins that may promote tissue remodeling, repair, neurotrophic support, and axonal regeneration [125] [126] [127] [128] . taking into account the excessive release of glutamate and the feedback of the inflammatory response after sci, it is no surprise that microglia acquire a reactive phenotype that expresses very low quantities of the mhc ii molecules and is not capable of maintaining an adequate interaction with t lymphocytes [118, 129] . the m1 phenotype is characterized by the excessive release of no • , il-1, il-6, and tnf , which leads to toxicity. in these conditions, astrocytes and postsynaptic neurons show signs of damage, evidenced by the expression of ros, which can induce apoptosis [121] . nevertheless, activated microglia remove the cellular debris after the lesion and are capable of promoting revascularization in the site of injury, which facilitates the release of trophic factors and nutrients for the survival and proliferation of infiltrating cells in the lesion site [118] . furthermore, microglia are capable of expressing glutamate transporters, which apparently help buffer the excessive concentrations of glutamate, and consequently protect cells from toxicity [129, 130] . it is important to take the before-mentioned into account since activated microglia and peripheral macrophages make up the majority of inflammatory cells present in the site of lesion, especially since these cells are morphologically different and respond to different modulatory signals [131] in an early response of the innate immunity to the lesions of the cns, which have diverse etiologies such as ischemia and neurotrauma [132, 133] . therefore, given that in the brain and the spinal cord there is a considerable heterogeneity of macrophages, the relative contribution of one population of cells in the local inflammatory reaction can dictate whether a cascade of events initiates as a regenerative or a destructive process. this all depends on the macrophage phenotype activated, particularly microglia [117] . in regard to t-lymphocyte infiltration in humans, it might be detected months after the injury, and b-lymphocytes are not usually found [134] . on the other hand, mice models present both cell types 7 days after sci, with a peak at 42 days [135] . lymphocytes are the cells that modulate the intensity of the inflammatory response. traditionally, their participation after sci has been linked with the damage to neural tissue since they are capable of producing proinflammatory cytokines such as inf and il-1 . on one hand, inf is linked directly to neuronal destruction since it induces the expression of other proinflammatory cytokines (tnf , il-6, il-12, and il-1 ), and proinflammatory molecules such as ros and inos, since it participates in the induction of transcription factors including nf (nuclear factor kappa beta) and ap-1 (activator protein-1) [118, 136, 137] . in addition, inf is known to participate in the induction of a cytolytic response by tcd8+ (cd, cluster of differentiation) since it is the principal inductor of mhci through the phosphorylation of stat1 (signal transducers and activators of transcription-1) [138] . moreover, the chemoattractant, c-x-c motif chemokine 10 (cxcl10) recruits cd4 th1 cells via the cxcr3a receptor [95] . after the induction of protective autoreactivity, which is a strategy based on the modulation of the immune response by neural derived peptides, diverse studies have reported that the presence of t lymphocytes with a th2 phenotype in the lesion site favors functional recovery [139] [140] [141] . this is due to their ability to synthesize ngf, bdnf, and diverse neurotrophins (nt3, nt4, and nt5) [142, 143] . in a classical th1 activation pattern, however, the inflammatory response after sci can be responsible for the necrosis of the lesion site and the surrounding area [144] [145] [146] . to further explore this phenomenon the cytokine expression was analyzed in comparison to sham animals and the dominant phenotype was found to be th1 and th17 predominantly according to expectations [147, 148] . this is due to its important role in the generation of free radicals, cytokines, and chemokines that exacerbate the damage to the neural tissue for weeks or even months. it is important to point out that this noxious effect occurs when the immune response is not modulated, since it is correlated with excitotoxicity, lipid peroxidation, and the development of an autoreactive response towards neural constituents [17, 93, 139, 149, 150] . contrastingly, this response can either increase the damage to the neural tissue, promote protection [150] [151] [152] [153] , or even promote restoration of the injured tissue [126, 127, 144] as a function of neuromodulation. the autoreactivity observed after sci is characterized by the specific immune response, with lymphocytes being the only cells capable of specifically recognizing the antigens, and initiating the adaptive immune response. despite the existence of mechanisms by which these autoreactive t cells are eliminated or inactivated, these are not sufficient, and consequently they can be found in practically every healthy individual. thus, autoreactivity can be part of a normal immune response that can find its origin in several infectious and inflammatory diseases. however, when this mechanism is excessive, the result is an autoimmune disease [154] [155] [156] . there are multiple diseases that are considered to be autoimmune or to have an autoimmune component. multiple sclerosis (ms) is one of such diseases. it is an inflammatory, demyelinating disease, in which an autoimmune response to mbp [157] has been reported. interestingly, after sci, an autoreactive phenomenon similar to the pathophysiology of ms can be observed. consequently, it is well-known that the lymphocyte role after sci is fundamental, because these cells are responsible for the generation of autoimmunity in individuals with genetic susceptibility [89, 158, 159] . these events can become chronic if the proinflammatory environment is not regulated. if not regulated, the response would involve the participation of other immune cells, other signaling pathways, and other patterns of gene expression. the persistent influx of immune cells from the systemic circulation as neutrophils, macrophages, lymphocytes, basophils, and eosinophils is correlated with additional elevation of proinflammatory cytokine levels and neural tissue destruction that would unavoidably make tissue recovery more difficult [108, 160, 161 ]. cytokines comprise a large family of small signaling proteins that affect nearly every biological process including embryonic development, disease, nonspecific infection response, cognitive functions, aging, cellular growth, survival, and differentiation [10, 162] . these "cytokines," which can be classified as peptides, proteins, or glycoproteins, encompass interferons, interleukins, the chemokine family, the tumor necrosis factor family, adipokines, and mesenchymal growth factors [10, 163] . these molecules are produced by one cell and go on to act on another cell in order to bring a change in the function of the target cell. the difference with hormones is that cytokines are products of most cells while not being of a particular tissue or cell. the majority of cytokines function by binding to specific cell surface receptors; this action triggers intracellular signaling and activates transcription factors such as ap-1 and nf [162] . interestingly, the diverse properties of a single cytokine can be explained by the following mechanisms: the first mechanism involves the presence of the receptor of a certain cytokine in one particular type of cell (e.g., il-33 receptor on mast cells) [164] . the second mechanism is explained by the presence of the receptor to a specific cytokine on most cells (e.g., activation of nf by il-1, or tnf induction of cox-2). the third mechanism encompasses the ability of cytokines to induce or function as coactivators (e.g., il-18 induces ifn when il-12 is present, but when it is not, il-18 induces fas ligand) [165] . despite the fact that cytokines are studied in every discipline of biology, the effects of these molecules are mostly studied in the realm of inflammation, immunology, cancer, and atherosclerosis [162] . in these areas, cytokines can be grouped into a proinflammatory or anti-inflammatory category on the basis of the resulting balance of their added effects [10] . in the cns, cytokines have homeostatic physiologic and neuromodulatory functions. surprisingly, they also have the capability of contributing to neuronal damage and destruction when their concentration exceeds a certain threshold. one of the reasons as to why they cause such damage and destruction lies in the uncontrolled inflammatory response observed after sci, which emphasizes the reason behind the augmented study of these molecules in inflammation-related research. the upregulation of these cytokines, as well as the consequent cellular infiltration they cause, plays a crucial role in the determination of the extent of the secondary tissue damage and neural degeneration observed after the injury [95, 166, 167] . therefore, taking into account that the production and release of proinflammatory cytokines and chemokines (table 1) is the first inflammatory event that develops after sci, the importance of these molecules becomes clear [166, 167] . in regard to the realm of inflammatory cytokines, there is a clear diversity in their functions. for starters, certain molecules are capable of inducing vascular permeability and cellular fluid loss, which include components of the complement cascade (c3a and c5a), which in turn cause the release of histamine, prostaglandins, and leukotrienes from resident mast cells. specific inflammatory cytokines such as tnf , il-1, and il-6 are synthetized by various cells in the cns and are known as mediators of the peripheral immune response 8 [118, 192] il-4 (i) high levels 24 h ai, concentrations remain during 7 days and decrease 3 days ai (i) neuroinflammatory regulation in various pathological conditions (ii) confers regenerative properties to macrophages (iii) controls free radical production in peripheral macrophages and microglia [166, [193] [194] [195] [196] [197] [198] [199] il-13 (i) detected 1 day ai (i) macrophage activation onto m2 phenotype [166, 199] ip-10/cxcl10 (i) expressed locally 30 [200, [205] [206] [207] mediators of inflammation 9 [200, [205] [206] [207] mcp1/ccl2 (i) detected from 1 h ai with pl at 24 h and remains low up to 24 days ai (i) macrophage and pmn infiltration mediator [106, 184, 200, 205, 206] min: minutes; ai: after injury; pl: peak levels. mediators of inflammation [168] [169] [170] . on one hand, tnf immediately recruits neutrophils to the site of the lesion by the induction of adhesion molecules such as icam-1 and vcam-1 (vascular cell adhesion molecule-1), as it stimulates the release of il-8, which is a chemotactic factor for neutrophils. furthermore, tnf alters the permeability of endothelial cells and damages the blood-spinal cord barrier. moreover, this cytokine is able to exert cytotoxic activity towards oligodendrocytes and contributes to demyelination. in addition, tnf also stimulates the proliferation and hypertrophy of astrocytes, hereby promoting the formation of the fibroglial scar, which acts as a barrier to a possible regeneration of the cns as a biological measure of last resort in response to an uncontrolled chronic inflammation [168] [169] [170] . on the other hand, studies have shown that a direct injection of il-1 into the spinal cord leads to enhanced vascular permeability and lymphocyte recruitment. subsequently, il-6 has been found to promote the activation and infiltration of macrophages and microglia [161, 171] . in fact, it is known that il-6 is a major player in chemokine infiltration, because it has the ability to interact with other cytokines and neurotrophic factors [172, 173] . interestingly, several studies have revealed that the continuous inhibition of il-6 is detrimental to functional recovery because it also participates in axonal regeneration and gliosis, in line with the role of tnf in chronic inflammation [174, 175] . thus, it is important to take into account that the mediation of the early inflammatory tissue damage may actually worsen the functional outcome [176] . this leads to a conflict, since the role of inflammation after sci appears to be contradictory when the beforementioned and following points are taken into account [177] . on one hand, proinflammatory cytokines, il-1 and il-6, are beneficial at low concentrations due to their induction of neurotrophin expression and the mediation of leukocyte activation/recruitment to the injury site by the induction of adhesion molecules in the cell surface such as icam-1, p-selectin, and e-selectin [172, 173] . on the other hand, at higher concentrations, these inflammatory cytokines activate transcription factors such as nf , ap1, and atf, factors that stimulate the expression of neurotoxic genes, including cox-2, inos, and proinflammatory proteases in different target cells [88, 178, 179] . pan found that the mrnas of cytokines such as tnf , il-1 , il-1 , and il-6 could be detected 15 min after injury. from these cytokines, il-1 and il-1 continually reached peak levels until the 6 h but were not present from the 12 to 24 h after sci. in addition, by 4 h after contusive sci, significantly increased mrna levels of il-1a and il-6 were clearly detected by qrt-pcr [180, 181] . digging further into the time frame of expression, western blot studies found that the mature form of il-1 is expressed by the 2 h. this evidence suggests that the inflammatory cytokine is released very quickly after tissue damage. the expression of these genes was identified 1 h after contusive rat sci by cdna microarrays [57] . the procedure was then repeated in spinal cord injury patients, and the same results were observed [103] . moreover, hayashi found that after sci the mrnas of cytokines such as tnf and il-1 were upregulated in as little as 1-3 h after the lesion [148, 182, 183] . on another note, tnf mrna peaked quickly 60 min after the injury and fell slightly by the 120 min. tnf mrna remained elevated by day 1 after sci, returned to a low level by day 3, and was not detected by day 5 [184] . il-6 mrna increased slowly, reached peak levels by 6-12 h, and fell by 24 h [180] . it is important to note that the levels of these mrnas were nearly undetectable in sham-injured animals. another study found that, between 12 h and 72 h after sci, the gene expression of proinflammatory cytokines such as il-1, il-3, il-6, and their receptors was strongly upregulated [6] . tnf and il-1 induce both il-1 and tnf mrnas. consequently, the downregulation of the signaling of il-1 and tnf reduces the induction of il-1 mrna [163] . this suggests that the activity of these cytokines contributes to their own mrna regulation [163, 180] . from the 3 h and up to 24 h, tnf , il-1 , il-6, and lif were found to be strongly upregulated in and around the contused area. these cytokines were produced at the same time range. it is worth noting that another wave of expression was observed for tnf and il-1 at 14 days, which correlates with an increased blood-spinal cord barrier function [104] . in particular, the overexpression of lif has been found to cause a dramatic increase in the proliferation of microglia/macrophages and astrocytic activation [24] . tnf is released significantly faster than other proinflammatory cytokines, because this is stored in a preformed state on the cell surface and in the granules of mast cells. it is not a surprise that role of this cytokine is similar to that of il-1 given the facts stated above [185] . it is important to note that tnf is the principal promoter of wallerian degeneration since it activates resident schwann cells in the peripheral nervous system and facilitates macrophage recruitment into the injury site [186] . in addition, these macrophages release proteases, fr, and cytokines [187] . similar to the facts stated above, the extracellular expression of tnf [187] in the surrounding white matter was detected 3 h posterior to contusion sci, with a peak that took place from day 1 to day 3 [166] . thus far, the time frames of expression have been described. the following information regards the receptors of such molecular products. from the two subtypes of tnf receptor that exist, each subtype has a different distribution and presence that depends on the particular cell type. for instance, tnf-r1 is expressed constitutively on most cell types, whereas the expression of tnf-r2 in astrocytes requires induction by tnf , il-1 , and ifn [188] . a large amount of evidence indicates that tnf-r1 augments neuronal death and tnf-r2 promotes neuroprotection [189] . what has been observed in the lesion concludes that the expression of tnf-r1 and tnf-r2 is increased within 15 min after traumatic sci in adult rats and reaches its peak at 4 h for tnf-r2 and 8 h for tnf-r1. the expression of both receptor subtypes then goes on to decline after day 1 and day 3, respectively [190] . it is important to note that these receptors are initially found on the epicenter of the lesion site. posteriorly, they spread radially towards distant areas during their peak expression and later become confined to the lesion area. these receptors are expressed by several cells, which include neurons, oligodendrocytes, and astrocytes [189, 190] . these cells might work individually or synergistically to mediate the biological activity of tnf , which makes an interesting research topic, given that these receptors are known to be involved in antiapoptotic activities through the tnf-r/nf signal transduction pathway [191] . on a last note, tnf participation in the expression of inos in microglial cells [137] causes an exacerbated neural destruction as a direct consequence of the induction of the nf pathway, which can then contribute to the expression of ifn . ifn within the nervous system is classically associated with the inflammatory response after injury as mentioned in the previous paragraph [213] . this molecule is believed to be normally involved as one component of the physiological response to tissue damage and trauma. cd4+ and cd8+ t cells together with natural killer (nk) cells are the major sources of ifn . nevertheless, evidence shows that this cytokine is also produced within the nervous system by neurons and glial cells in the absence of infiltrating immune cells [214] . in various animal models, ifn promotes macrophage signaling, production of proinflammatory cytokines and chemokines, recruitment of macrophages to the cns, and the activation of cns resident and infiltrating apc populations. moreover, ifn is also the most potent inductor of mhci, and it is upregulated in the cns after injury [215] . in low concentrations, ifn may participate in the homeostasis of the synaptic circuitry [216, 217] . as previously mentioned, ifn is involved in the upregulation of mhc i, which has been shown to play an important role in the synaptic plasticity process following axotomy. furthermore, ifn has been shown to regulate phosphorylation and nuclear translocation of signal transducer and activator of transcription 1 (stat1) and to influence neuronal excitability by the expression of the peripheral nerve-type sodium channel gene pn1 [192] . it is important to note that several studies found that ifn and il-17 had the highest levels of gene expression, since this indicates that the phenotype found after sci is predominantly th1 and th17 and the ifn release could be detected from 1 h to 12 weeks, depending on microenvironment [147, 148] . interleukin-17 (il-17) is primarily produced by th17 cells and has an important role in inflammation and autoimmune disease [201] . a key regulator in its production is il-6. nevertheless, tgf-and interleukin-21 (il-21) are also capable of stimulating il-17 production. similarly, interleukin-23 (il-23) is also able to promote il-17 production just as interleukin-22 (il-22) does. in one study, serum levels of il-6, il-21, and il-23 were increased in large quantities 1 h after sci, had a peak at 24 h, and had a positive correlation with increased il-17 [202] . signal transducer and activator of transcription 3 (stat3) and rar-related orphan receptor gamma (ror ) are two transcription factors capable of mediating il-17 production and th17 differentiation. as a result, a closed circuit is formed, in which il-17, the stat3 signaling pathway, and il-17 related cytokines promote neuroinflammation as they costimulate one another. il-17 expression and production was detected from 1 h to 72 h after contusion injury [202] . stat3 is a primary transcription factor of the downstream signaling of il-6 [203] . the phosphorylation of stat3 in this pathway induces a proinflammatory gene expression that correlates with il-17 quantities in spinal cord neurons and astrocytes. interestingly, through this very same pathway, anti-inflammatory th2 cells can be suppressed by il-6 inhibition of foxp3 expression in a stat3 dependant manner. it is important to recognize that stat3 was found higher in the sci rat group, whose expression peaked at 24 h [202] . it is worth noting that il-17 antagonistic therapy in rheumatoid arthritis (ra) suggests that the inhibition of the pathological role of il-17 may be a promising therapeutic approach in humans [204] . chemokines are functionally related cytokines that induce specific actions in the immune system. they are released in response to an infection, inflammation, or trauma [184] . chemokines are grouped into two families: the family (cxc), which participates in the recruitment of polymorphonuclear cells, and the family (c-c), which provides the priming signal for macrophages, lymphocytes, eosinophils, and basophils. the family includes gamma-interferon inducible protein (ip-10/cxcl10), platelet factor 4, il-1, and melanoma growth stimulatory activity (mgsa/gro/kc) [218] . in particular, the chemokine cxcl10 has been shown to inhibit angiogenesis, growth, and chemotaxis of endothelial cells via the cxcr3b receptor. consequently, the neutralization of cxcl10 promotes angiogenesis through the expression of eight genes related to angiogenesis and vasculature remodeling after sci [95] . an important member of the family is the monocyte chemoattractant protein (mcp-1/ccl2). it is detected in astrocytes and perivascular mononuclear cells in experimental allergic encephalomyelitis (eae). mcp-1 levels are related to the parallel development of clinical disease and macrophage infiltration [205, 206] . the same case applies to macrophage inflammatory protein 1 alpha (mip-1 /ccl4) and macrophage inflammatory protein 1 beta (mip-1 ) [219] . their expression has been shown predominantly in myeloid and lymphoid cells [207] , where an increased expression of mip-1, mip-2 (cxcl2/3), and mcp-1 after sci plays a role in the inflammatory process, since these molecules recruit circulating leukocytes to the injury site [220] . mcp-1 mrna was present in the normal spinal cord, was increased 1 h after sci, peaked at 24 h, and returned to a low level by day 14. mcp-1 is expressed by astrocytes that surround white matter. in addition, mip-1 mrna was present in the normal spinal cord, where it increased at 1 h after sci, peaked from 3 to 6 h, decreased by day 1, remained unchanged until day 7, and returned to a low level by day 14. mip-1 expression in astrocytes was observed from day 3 to day 6 following injury. additionally, the expression of this molecule was found at the contusion site and in rostral and caudal sections to this location. by day 5 after injury, the expression of mip-1 returned to baseline levels. moreover, ip-10 mrna presented low levels in the normal spinal cord, increased its levels at 1 h, peaked at 6 h, and remained high up to day 5 after sci. it decreased to baseline levels by day 14 [184] . another study found the chemokines, mcp-1, mip-1, mip-1 , mip-2, and ip-10, to be expressed locally at 30 min with a peak at 6 h after sci. it is worth noting then that chemokines remain present 24 d after injury-at lower levels-in contrast with the rest of the cytokines [200] . molecules of the inflammatory response. the changes in gene expression that contribute to the secondary injury are characterized by protracted neuronal loss and neurological dysfunction. therefore, the predominant downregulation of these factors might play a role in cell survival and may lead to the development of novel interventions that promote recovery [181, 221, 222] . in order to develop a viable therapy, it is essential to identify the specific molecular pathways that become altered as a function of time after sci [223] . for instance, activated macrophages and microglia after cns injury produce various neurotrophic factors and molecules that enhance regeneration [93, 224] . however, this response highly depends on the temporal sequence that proceeds the injury [108] . this consequently indicates that there is a proper and timely regulation of inflammatory reactions that can take place and be of paramount importance to the design of therapeutic strategies involving cytokines, growth factors, or neurotrophins [98, 116] . (1) cytokines. a particular cytokine involved in this beneficial aspect of the inflammatory response is il-4. this cytokine exerts an anti-inflammatory effect after cns damage [193] [194] [195] . for instance, endogenous il-4 has been shown to participate in the regulation of neuroinflammation in various pathologic conditions [196] [197] [198] . this anti-inflammatory cytokine and its receptor subunit il-4 have a role in spinal cord trauma. this is illustrated by the high level expression of il-4 24 h after contusive sci in rats, whose elevated concentration persisted for 7 days but was decreased 3 days after sci. interestingly, on day 1 after sci, an increased expression of il-13 was observed. this is noteworthy since this interleukin shares the same receptor with il-4 for signal transduction [166, 199] . moreover, the cytokine expression of the contused spinal cord was not significantly affected by il-4 attenuation for the proinflammatory cytokine levels of il-1, il-6, and tnf . in fact, the opposite effect was observed, since the event correlated with a marked increase in the extent of macrophage quantity 7 days after sci, which was preceded by an increase in the level of mcp-1 [166] . these results suggest that the expression of il-4 regulates the extent of macrophage activation in the acute phase of the injury [166] . in addition, il-4 has been shown to exert a neuroprotective effect against microglia-mediated neuronal toxicity by the regulation of fr formation [194] . on similar lines, macrophages stimulated with il-4 are reported to be less neurotoxic and to have an increased regenerative capability. this evidence makes il-4 injections a possible therapeutic application [166] . il-10 and tgf have been reported to act as neuroprotective molecules in a manner similar to il-4 [225] . for instance, it has been shown that an intrathecal infusion of tgf is able to enhance axonal growth after spinal contusion through the epidermal growth factor receptor (egfr) that is primarily upregulated by astrocytes surrounding the lesion. here, tgf stimulates proliferation, migration, and transformation to an axon phenotype supportive of growth [226] . on the other hand, a potential treatment for certain aspects of the secondary injury such as inflammation, excitotoxic damage, and neuronal apoptosis is the administration of il-10 since its anti-inflammatory effects involve the downregulation of il-1 , il-2, il-6, tnf , ifn , matrix metalloproteinase-9, nitric oxide synthase, myeloperoxidase, and ros [227] . in addition, proapoptotic factors such as cytochrome c, bax, and caspase 3 are downregulated by the effects of il-10. other effects of this cytokine include the upregulation of antiapoptotic factors such as b-cell lymphoma 2 (bcl-2). furthermore, il-10 provides trophic support to neurons by its receptor, in addition to increased tissue sparing, neuroprotection, and functional recovery. in the nervous system, il-10 receptor expression has been found in microglia, astrocytes, and oligodendrocytes acting as antagonist for the production of proinflammatory cytokines [225, 227] . in the first moments after sci, the elevated synthesis and release of proinflammatory mediators plays a role in the secondary degeneration [103] . this might be a therapeutic opportunity. for instance, an antagonist of proinflammatory cytokines such as il-1 receptor antagonist has demonstrated a neuroprotective effect after global ischemia, excitotoxicity, and traumatic brain injury in rodents [228] . (2) growth factors. after mechanical trauma, astrocytes and neurons release fibroblast growth factor (fgf) which is thought to counteract excitotoxic or ischemic damage by the activation of antiapoptotic signals in stressed neurons [229] . acidic fibroblast growth factor (afgf) is a potent mitogenic and chemotactic agent for vascular endothelial cells, dermal fibroblasts, and epidermal keratinocytes. moreover, it has a role in the regeneration process since it contributes to angiogenesis. in the normal uninjured spinal cord, afgf mrna was found to be present in low levels. after sci (table 2) , however, the factor increased in the 1 h, stayed at that level, peaked from day 5 to day 7, and remained high from day 14 to day 21 [209] . many therapeutic strategies seek to induce a higher expression of neurotrophic factors. a particular strategy that has shown significant results is the combination of peripheral nerve grafts with afgf after transection sci in rats. this strategy induced higher il-4, il-10, and il-13 levels in the graft areas of rat spinal cords. moreover, this strategy has been shown to regulate th2 cytokine production, m2 response, and neurotrophic factor production, where the latter can indirectly regulate the inflammatory response and neural destruction [211] . it is worth noting that the use of afgf with fibrin glue in combination with surgical neurolysis for nonacute sci has been proven feasible and safe in clinical trials which have shown significant improvements in asia motor and sensory scale scores and impairment scales, neurological levels, and functional independence measures, 24 months after treatment [230] . bdnf detected 6 h ai, increases up to 6 weeks, and decreases 12 weeks ai (i) m2 macrophage phenotype induction (ii) production of several extracellular matrix proteins for tissue remodeling and repair, neurotrophic support, and axonal regeneration (iii) increases growth, angiogenic, and axonal guidance factors [125, 150, 209, 210] afgf detected 1 h ai, pl 5-7 days, and remains elevated up to 14-21 days ai (i) potent mitogenic and chemotactic agent for vascular endothelial cells, dermal fibroblasts, and epidermal keratinocytes (ii) promotes th2 cytokine and neurotrophic factor production and m2 response [209, 211] bfgf detected 1 h ai, pl at day 3, remains elevated 5-7 days, and returns to low levels 14-21 days ai (i) angiogenesis [209] tgf-detected at 24 h ai (i) immunosuppressant (ii) tissue stabilization and structural preservation (iii) neural regeneration and repair [212] min: minutes; ai: after injury; pl: peak levels. after the injury, the expression of basic fibroblast growth factor (bfgf), another growth factor involved in angiogenesis, was found in astrocytes localized at the site of contusion and in the surrounding white matter. unlike afgf, bfgf mrna was not detected in the uninjured spinal cord. it was only detected 1 h after sci, in increased quantities at 6 h, and at its peak 3 days after sci. afterwards, it remained high from day 5 to day 7, only to return to a low level by days 14 to 21 [209] . before going further, it is important to note that growth factors such as tgf-may act as immunosuppressants. moving on, 24 h after sci, genes related to growth and differentiation became present. these included tgf-, nerve growth factor (vgf), platelet derived growth factor (pdgf-), galanin, and neuropeptide y. these genes have been suggested as aids in tissue stabilization, structural preservation, repair, and regeneration after sci. for instance, increased pdgf and vgf levels after sci may prevent the death of axotomized neurons and a decrease in their energy metabolism [212] . subsequently, the increased abundance of galanin and neuropeptide-y transcripts may produce an antinociceptive effect in the injured spinal cord [231] . moreover, it is known that cannabinoid receptor 1 (cb1) is colocalized with the neuropeptide cck. in this relationship, the neuropeptide acts as an endogenous opioid antagonist [232] . therefore, the downregulation of cb1 and the expression of the cck precursor might help explain why there is a relative resistance of neuropathic pain to the analgesic action of morphine in sci patients [233] . similar results have been found in several transcripts, and the previously mentioned genes have shown an increased abundance in comparison to sham animals [57, 223, 234] . (3) neurotrophins. neurotrophins constitute a family of molecules that has assumed a central role in studies dealing with recovery after sci [235] . four members of this family are involved in neuron survival and the regeneration process after sci: ngf, brain derived neurotrophic factor (bdnf), neurotrophin-3 (nt-3), and nt-4/5. neurotrophins emit signals when they bind to low and high affinity receptors in the membrane of their target cells. for instance, the low affinity p75 receptor binds all neurotrophins [208] . another signaling method used by neurotrophins is carried out by three high affinity tyrosine kinase receptors, collectively known as trk receptors. trka, trkb, and trkc compose the trk family of tyrosine-protein kinases. these three receptors mediate the biological properties of the ngf family of neurotrophins. trka is the particular receptor for ngf, while trkb serves as a receptor for both bdnf and nt-4. lastly, trkc is the primary receptor for nt-3. however, this particular neurotrophin can activate trka and trkb receptors when present in high concentrations [236] . through semiquantitative rt-pcr in a spinal cord contusion model, it was found that the expression of neurotrophin family members and their receptors was significantly diminished 6 h after the lesion. yet, in contrast to this pattern of trk receptor expression, p75ntr showed a significant upregulation after contusive sci [237] . interestingly, an increase in bndf was observed up to 6 weeks after compression sci with a decrease 12 weeks afterwards [210] . similarly, an increased expression of growth, angiogenic, and axonal guidance factors, as well as extracellular matrix molecules, can be observed in the chronic phase (days to years) following sci [150, 209] . the series of interconnected deleterious mechanisms of the secondary injury is orchestrated by the expression of specific genes, in particular those of signaling proteins such as cytokines, chemokines, and growth factors. the 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following spinal cord injury cytokine activity contributes to induction of inflammatory cytokine mrnas in spinal cord following contusion spinal cord trauma and the molecular point of no return neuroimmunology of traumatic spinal cord injury: a brief history and overview sequential mrna expression for immediate early genes, cytokines, and neurotrophins in spinal cord injury cytokine chemokine expression in contused rat spinal cord microglia and macrophage responses in cerebral ischemia tnf alpha-still a therapeutic target anti-tnf therapy in the injured spinal cord differential expression, shedding, cytokine regulation and function of tnfr1 and tnfr2 in human fetal astrocytes modulator effects of interleukin-1 and tumor necrosis factor-on ampainduced excitotoxicity in mouse organotypic hippocampal slice cultures expression of the type 1 and type 2 receptors for tumor necrosis factor after traumatic spinal cord injury in adult rats inos and nitrotyrosine expression after spinal cord injury a single pulse of nerve growth factor triggers long-term neuronal excitability through sodium channel gene induction interleukin-10, interleukin-4, and transforming growth factor-differentially regulate lipopolysaccharide-induced production of pro-inflammatory cytokines and nitric oxide in cocultures of rat astroglial and microglial cells interleukin-4, interleukin-10, and interleukin-1-receptor antagonist but not transforming growth factorinduce ramification and reduce adhesion molecule expression of rat microglial cells interleukin-13 and -4 induce death of activated microglia evidence of an anti-inflammatory role for vasogen's immune modulation therapy neuroprotective role of microglia expressing interleukin-4 cnsderived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells interleukin-4, interleukin-13, signal transducer and activator of transcription factor 6, and allergic asthma characterization of the early neuroinflammation after spinal cord injury in mice il-17 and th17 cells the role of il-17 promotes spinal cord neuroinflammation via activation of the transcription factor stat3 after spinal cord injury in the rat stat3 and suppressor of cytokine signaling 3: potential targets in lung inflammatory responses ly2439821, a humanized anti-interleukin-17 monoclonal antibody, in the treatment of patients with rheumatoid arthritis: a phase i randomized, double-blind, placebo-controlled, proofof-concept study csf-1 gene expression and protein levels in the central nervous system and peripheral immune system of rats with experimental autoimmune encephalomyelitis (eae) do chemokines mediate inflammatory cell invasion of the central nervous system parenchyma? expression and characterization of tca3: a murine inflammatory protein p75ntr: a receptor after all gene profiling in spinal cord injury shows role of cell cycle neuronal death neuronal loss and expression of neurotrophic factors in a model of rat chronic compressive spinal cord injury acid fibroblast growth factor and peripheral nerve grafts regulate th2 cytokine expression, macrophage activation, polyamine synthesis, and neurotrophin expression in transected rat spinal cords effect of transforming growth factor 1 on spinal motor neurons after axotomy peripheral nerve injury induces endoneurial expression of ifn-, il-10 and tnf-mrna microglia as a source and target of cytokines expression of mhc class i and 2-microglobulin in rat spinal motoneurons: regulatory influences by ifn-gamma and axotomy exposure to interferonduring synaptogenesis increases inhibitory activity after a latent period in cultured rat hippocampal neurons interferon--induced changes in synaptic activity and ampa receptor clustering in hippocampal cultures two burgeoning families of platelet factor 4-related proteins: mediators of the inflammatory response chemokine expression in murine experimental allergic encephalomyelitis selective chemokine mrna accumulation in the rat spinal cord after contusion injury gene expression profiling of experimental traumatic spinal cord injury as a function of distance from impact site and injury severity markedly reduced axonal potassium channel expression in human sporadic amyotrophic lateral sclerosis: an immunohistochemical study genechip5 analysis after acute spinal cord injury in rat activated human t cells, b cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? il-10 promotes neuronal survival following spinal cord injury transforming growth factor transforms astrocytes to a growth-supportive phenotype after spinal cord injury the therapeutic role of interleukin-10 after spinal cord injury the role of inflammation in cns injury and disease neuronal vulnerability in transgenic mice expressing an inducible dominant-negative fgf receptor acidic fibroblast growth factor for repair of human spinal cord injury: a clinical trial behavioral characterization of neuropeptide y knockout mice expression of the cannabinoid receptor cb1 in distinct neuronal subpopulations in the adult mouse forebrain cholecystokinin/opioid interactions anti-inflammatory and pro-inflammatory roles of tgf-, il-10, and il-22 in immunity and autoimmunity the changing scene of neurotrophic factors structural and functional properties of the trk family of neurotrophin receptors gene expression alterations of neurotrophins, their receptors and prohormone convertases in a rat model of spinal cord contusion the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord-021452-9rukc80y authors: bergman, robert l. title: miscellaneous spinal cord diseases date: 2009-05-15 journal: consultations in feline internal medicine doi: 10.1016/b0-72-160423-4/50054-8 sha: doc_id: 21452 cord_uid: 9rukc80y nan spinal cord diseases in cats vary in the severity and in progression of neurological dysfunction. diagnosis of spinal cord disease in cats can be a challenge. clinical signs often are vague and insidious. advanced imaging techniques have improved diagnostic capabilities and recognition of new disorders. infectious inflammatory disease is the most common categorical differential diagnosis in cats with spinal cord dysfunction. other common disease categories include neoplasms, trauma, and degenerative disorders. 1 veterinarians must think beyond the more common differential diagnoses to consider unusual diseases and different diagnostic approaches. this chapter emphasizes newly recognized spinal cord diseases and provides a review of the current literature. signs of neurological dysfunction dictate the neuroanatomical localization of a lesion within the spinal cord. spinal reflexes and paraspinal hyperesthesia assist with lesion localization. localization is specified to the spinal cord regions c1-c5, c6-t2, t3-l3, and l4-s2, based on upper motor neuron or lower motor neuron signs of limb weakness. cats with spinal cord compressive disease or meningomyelitis often exhibit paraspinal hyperesthesia. pathology of the spinal cord tissue itself usually does not have hyperesthesia as a clinical sign. orthopedic, polyneuropathic, myopathic, and neuromuscular junction disorders can mimic signs of spinal cord dysfunction. careful interpretation of the neurological examination differentiates among these disorders (see chapter 49) . signalment and history aid in formulation of a list of probable differential diagnoses. young cats are more likely to be diagnosed with feline infectious peritonitis (fip), lymphosarcoma, or a congenital anomaly. middle-age and older cats may be diagnosed with nonlymphoid neoplasia or intervertebral disc disease. history is important for determining temporal onset (acute, insidious, or episodic) and progression (rapid, gradual, or static). trauma, vascular insults, and some inflammatory and neoplastic diseases present acute in onset. cats with spinal cord dysfunction require thorough physical examination and routine laboratory diagnostic testing. other disorders that cause paresis can mimic spinal cord disease; for example, neuropathy, myopathy, junctionopathy, polyarthropathy, and cardiovascular disease. routine laboratory testing consists of a complete blood count (cbc), serum chemistry profile to include creatine phosphokinase (ck) enzyme activity, and urinalysis. evaluation of ck activity aids in identification of a myopathy, which can mimic spinal cord dysfunction. patient infection with feline immunodeficiency virus (fiv) or feline leukemia virus (felv) must be identified. additional serology for infectious disease is dependent on suspicion of other diseases. survey radiography of the spine is recommended for cats with spinal cord dysfunction. sedation or general anesthesia often is necessary to allow for proper patient positioning and relaxation of the spine. some findings are nonspecific, but discospondylitis, vertebral tumors, or spinal trauma usually have more obvious radiographic abnormalities. orthogonal or multiple views are recommended strongly, because a single view may not always provide complete information about the extent of the lesion (figure 51-1). myelography consists of injection of a nonionic contrast medium (0.3 to 0.45 ml/kg of iohexol [240 mg/ml] or iopamidol [200 mg/ml]) into the subarachnoid space of the low lumbar spine (l6-l7 or l5-l6) or the cerebellomedullary cistern. myelography is an imaging technique used commonly to identify the location and extent of spinal cord compression. 2 additional information may be used when combining the myelographic findings with computed tomography (ct). magnetic resonance imaging also is a more sensitive technique for evaluation of the spinal cord tissue. cerebrospinal fluid (csf) analysis is useful for detection of evidence of spinal cord disease, particularly when an inflammatory disorder is suspected. however, in most cases, a definitive diagnosis is not provided by csf analysis alone, even in cats with overt cns inflammatory disease. 3 exceptions include finding the inciting organism in the csf (e.g., cryptococcus neoformans) or identifying neoplastic cells (e.g., lymphosarcoma). 3 collection of fluid from the lumbar region may be preferable, because csf flows in a caudal direction. 4 additional diagnostic procedures include electrophysiology, csf protein electrophoresis, serology, and exploratory surgery. infectious inflammatory diseases account for 31 per cent of all feline spinal cord diseases. 1 common infectious inflammatory spinal cord diseases include fip, cryptococcosis, felv infection, and toxoplasmosis. approximately 15 per cent of cases of meningomyelitis in cats are bacterial or suspected to be bacterial in origin. 1 bacterial infections may occur secondary to hematogenous spread or, more likely, as a result of direct extension of a local wound (e.g., cat bite abscess). 5 pasteurella spp. and staphylococcus spp. are common pathogens. discospondylitis often is caused by a bacterial infection and involves the intervertebral disc and associated vertebral endplates. discospondylitis has been reported infrequently in cats and, if not treated appropriately, may progress to severe neurological dysfunction. 6, 7 polioencephalomyelitis, an inflammatory disease of unknown cause, is associated with 8 per cent of cases of feline spinal cord disease 1 and may present with clinical signs of paraparesis. 5 although not well described in the literature, eosinophilic/histiocytic meningomyelitis accounted for 6 per cent of inflammatory spinal cord diseases in cats. 1 feline immunodeficiency virus. fiv has been reported to cause a degenerative myelopathy that can be detected histologically with changes including myelin sheath splitting and intramyelinic vacuoles. clinical signs of spinal cord dysfunction are not evident in experimentally infected cats or in cats with naturally occurring fiv infection. 8 presenting signs and pathogenesis. fip accounts for more than half of the infectious inflammatory causes of myelitis in cats, and 16 per cent of all spinal cord diseases reported in cats. 1 clinical signs of fip result from the immune response of susceptible cats when infected by a mutant form of the feline enteric coronavirus (fecv), which reproduces within macrophages. 9, 10 the dry, or noneffusive, form of the disease is associated most commonly with cns signs as opposed to the "wet" or effusive form, which involves the visceral organs and causes abdominal effusion. pyogranulomatous inflammatory lesions involve the meninges, choroid plexus, and ventricular system. the immune response associated with fip causes a vasculitis and an ependymitis that subsequently may obstruct flow of csf. 11 signs of systemic illness occur in approximately 79 per cent of cats with fip. 1 typical signs include weight loss, anorexia, intermittent fever, and ocular changes (anterior uveitis or chorioretinitis). about one third of cats with fip have presenting clinical signs of neurological dysfunction. 12 young, purebred, sexually intact male cats are at a higher risk for developing fip. 13 a genetic susceptibility of about 50 per cent exists for development of fip. 10 all cats in one study of patients with neurological signs of fip were from multiple-cat households. 9 although younger cats are most susceptible to fip infection, cats of any age can develop the disease. in a case series of cats with spinal cord-related signs, more than 75 per cent were younger than 2 years of age. 1 most cats with fip have intracranial signs but often manifest signs of spinal cord dysfunction: pelvic limb ataxia, generalized ataxia, and paraspinal hyperesthesia. in a small case series of cats with confirmed fip, four of 10 had paresis or paralysis as the presenting clinical sign. 12 paresis was evident in two of seven of these cats with diffuse fip. 12 in another study, 28 of 29 cats with fip had histological lesions that predominated in the cervical spinal cord and brain. 1 diagnosis. antemortem diagnosis of fip is difficult. diagnosis is suspected based on assimilation of history, signalment, hematology, and other supportive diagnostic tests that include serology, csf analysis, findings on imaging, and tissue biopsies. a typical history includes acquisition of the cat from a cattery or shelter, and a fever that waxes and wanes and does not improve with antibiotic therapy. 10 common hematological and biochemistry abnormalities include neutrophilia or lymphopenia, low albumin with increased globulins, or a high serum fibrinogen. 10 serological testing and polymerase chain reaction studies to assess for viral load can be beneficial. 10 serology only confirms exposure to feline coronavirus. some cats with fip may have high antibody titers, but this is not absolute. a recent study of histopathologically confirmed cases of fip found that serological testing provided further support for tissue biopsy procedures. 14 high antibody titers (1:1600) provide a 94 per cent probability of active fip infection. 14 a titer that was positive but below 1:1600 suggested only a 44 per cent probability that cats had fip. 14 the titers in 10 per cent of cats with fip were negative, which suggests a compromised immune system. 14 definitive diagnosis of fip is made by histopathology of abdominal organs obtained by tissue biopsy. immunofluorescent assay/immunohistochemistry techniques can detect presence of coronavirus antigens within macrophages. 14 diagnostic evaluation of the cns aids in an indirect diagnosis of fip. results of csf analysis often reveal a marked increase in protein concentration and a neutrophilic pleocytosis. 11, 15 comparisons of antibodies in serum and csf may provide additional information, but false negatives and false positives are possible. presence of antibodies in csf must be interpreted in light of blood-brain barrier breakdown. adjunctive comparison of other infectious disease antibody titers in serum and csf can assist with determination of intrathecal production of antibodies. 10 common abnormalities on mri and ct include presence of hydrocephalus and periventricular contrast enhancement. overall the most consistent diagnostic findings in cats with the cns form of fip include a positive coronavirus igg titer in csf, a high serum total protein concentration, and abnormalities in brain imaging. 9 treatment. no treatment has been proven effective for fip, and the long-term prognosis is poor. 10 overall mortality rate is 95 per cent. 10 supportive therapies consist of antiinflammatory doses of prednisone (1 mg/kg/day po) and immunomodulation with cyclophosphamide or interferon. a recent report found use of recombinant feline interferon combined with corticosteroids more effective in cats with the effusive form than with the noneffusive form (n = 1) of fip. 16 other therapeutic recommendations include a diet with high nutritional value and stress reduction. 10 presenting signs and pathogenesis. cryptococcus neoformans is a saprophytic fungal organism that can cause systemic or focal disease. transmission occurs through inhalation of the organism that lives in the soil or bird excrements. cns signs are reflective of meningitis or focal granuloma formation within the brain parenchyma. fungal masses within the extradural space cause secondary compression ( figure 51 -2). 17 cryptococcal infection can cause focal spinal cord disease in some cats. the mean age for cats infected with cryptococcus is 6 years; however, the age range can vary. 18, 19 approximately 58 per cent of cats diagnosed with cryptococcus spp. were considered primarily outdoor cats. 18 systemic signs are variable and commonly include depression/lethargy, fever, poor body condition, or anorexia. 18 in one case series, approximately 50 per cent of the cats with cryptococcosis had cns signs, 42 per cent had ocular signs, and 32 per cent had respiratory signs. 18 cutaneous lesions also can occur. another case series reported that only 9 per cent of cats showed signs of neurological dysfunction. in this series, nasal signs were more common. 19 clinical signs of spinal cord dysfunction, including paraspinal hyperesthesia and paresis, have been reported in at least one case series. 18 c. neoformans accounted for 9 per cent of infectious causes of spinal cord disease in cats. 1 diagnosis. csf analysis is one of the most useful diagnostic tests in cats with cns cryptococcosis. neutrophilic and eosinophilic pleocytosis often are present. in some cases, the organism is identified. diagnosis also is based on detection of capsular antigen using a latex agglutination test in serum and csf. cats with focal granulomas in the cns may have negative antigen titers. 17 latex agglutination tests can have falsenegative results (or interference), which makes definitive diagnosis difficult. 18 in these cases, cultures, cytology, or histopathology of other tissues, such as skin, may be necessary. also important is documentation of the felv and fiv status of cats with cryptococcosis, because concurrent infection may be common. 18 additionally, cats with other concurrent viral infections tend to have a higher incidence of treatment failure. 20 treatment. treatment of cns cryptococcosis consists of long-term administration of systemic antifungal agents. itraconazole and fluconazole are considered the drugs of choice. fluconazole (5 to 15 mg/kg po q12h) is recommended, because it crosses the blood-brain barrier readily and has high lipid solubility. duration of treatment ranges from 6 to 10 months; however, a longer duration may be required to prevent relapse. 21, 22 antiinflammatory doses of corticosteroids help to decrease the inflammation and edema that can worsen neurological signs during treatment. therapeutic monitoring is based on clinical response and serial serum antigen titers. antigen titers often remain positive for a considerable period of time after clinical signs have resolved. 19 cats that have a reduction in antigen titer during the course of treatment have a better prognosis. 20 surgical removal of a fungal granuloma may be considered in conjunction with antifungal therapy. 17 presenting signs and pathogenesis. toxoplasma gondii is an infrequent cause of spinal cord disease in cats; it was reported as the cause for only 3 per cent of all infectious diseases resulting in spinal cord dysfunction. 1 t. gondii is a protozoal coccidian parasite for which cats are the definitive host. transmission occurs congenitally through the placenta from an infected queen or more commonly by ingesting the organism. healthy cats may be positive on serology but rarely develop clinical disease. predisposing factors for clinical disease include immunosuppression as a result of fiv and/or felv infection, administration of corticosteroids or chemotherapy, and diabetes mellitus. toxoplasmosis causes a nonsuppurative meningoencephalomyelitis. the organism also may infect muscle and peripheral nerves. 5 systemic signs include anorexia, weight loss, fever, and pneumonia. diagnosis. definitive diagnosis of toxoplasmosis is difficult. a suspected diagnosis is based on clinical signs, the exclusion of other cns diseases, serology, and response to treatment. 11 the amount of csf pleocytosis is variable, and the cellular differential count usually consists of mononuclear cells. albuminocytologic dissociation may be the only abnormality. t. gondii-specific igg and igm can be assayed in serum and csf. paired titer evaluations may detect an increase in serum igg; however, the disease course still may be static. 11 an igm titer greater than 1:256 may indicate an active or recent infection. antibodies in csf are compared with serum antibody titers for accurate interpretation of blood contamination and intrathecal antibody production. a definitive diagnosis is made by detecting the organism in a tissue biopsy. clindamycin (12.5 mg/kg po q12h for 4 weeks) is recommended for treatment of cns toxoplasmosis. 23 an alternative drug therapy is trimethoprim-sulfonamide (15 mg/kg po q12h). 23 one author reported a fair to good outcome in three cats treated for toxoplasma-induced myelitis. 5 clinical signs can be residual and response to therapy may be slow. 11 clinical presentation and pathogenesis. feline leukemia virus, an oncogenic retrovirus, can cause spinal cord dysfunction. felv can cause myelopathy by indirect and direct pathogenic mechanisms. felv can predispose to the spinal form of lymphoma indirectly or cause a degenerative myelopathy directly. felv-associated myelopathy reflects primary pathology within the spinal cord. 24 light microscopic examination revealed swollen axons and myelin sheaths in the brain stem and spinal cord of affected cats. immunohistochemical staining revealed felv antigens in neural tissue. a previously reported case of degenerative myelopathy in a felv-positive cat may have been felv-associated myelopathy. 25 this disease is associated with chronic infection with felv. cns signs develop on average 3 years after the first positive felv test. 24 mean age of affected cats is 9 years. 24 signs of felv-associated myelopathy include progressive ataxia and hyperesthesia, and paralysis develops within 1 year after onset of paraparesis. 24 urinary incontinence occurs in a small percentage of cats. diagnosis and treatment. a suspected antemortem diagnosis is based on ruling out other diseases. positive felv tests should heighten suspicion for this disease. csf analysis usually is not helpful. 24 advance imaging studies have not been evaluated in cats with felv-associated myelopathy. myelography is normal. no treatment options have been described. neoplasia is a common cause of spinal cord dysfunction in cats. with regard to relative incidence in one case series, neoplasia affected 28 per cent of cats diagnosed with spinal cord dys-function. 1 lymphosarcoma made up 38 per cent of neoplasiarelated spinal cord cases; however, this disease is becoming less common with the reduction in incidence of felv infection. 1, 26, 27 lymphosarcoma presenting clinical signs and pathogenesis. spinal lymphoma historically has been the most common cause of spinal cord neoplasms in cats. cns lymphoma accounted for 12.1 per cent of all cases of lymphoma and, of these cases, 88 per cent had spinal cord involvement. 28 the disease is especially common in young felv-infected cats, with a mean age reported between 3.6 and 4 years. 28, 29 cats younger than 3 years of age make up approximately 70 per cent of the cases. clinical signs associated with spinal lymphoma may be associated with a focal myelopathy that can occur in any region of the spinal cord. paresis has been reported in approximately 80 per cent of cats with spinal lymphoma. 28, 29 evidence of spinal hyperesthesia may be focal or multifocal with more extensive distribution. 29 the disease course can be rapidly progressive, with some cats showing signs for a week or less. 28, 29 neurological signs are related to the location of the lymphoma. although lymphosarcoma generally is a multicentric disease, more than 85 per cent of cats with cns involvement lack systemic signs or hematological changes. 29 renal lymphoma is likely to metastasize to the cns. diagnosis. evidence for systemic disease on physical examination includes enlargement of lymph nodes and abdominal organs. a cbc may show anemia, leukopenia, and thrombocytopenia. circulating lymphoblasts may be present on a differential white blood cell count. a positive correlation between serological testing and spinal lymphoma has been reported. 28, 29 the safest and most reliable method of obtaining a diagnosis of cns lymphoma is confirmation of the presence of lymphoma in other visceral organs. bone marrow aspiration may be diagnostic for neoplasia in up to 81 per cent of cases with this disease. 29 csf analysis is not always diagnostic for lymphosarcoma because of its extradural location. one case series reported that 6 of 17 cats had neoplastic lymphocytes in the csf. 28 myelography can determine lesion extent and detect presence of extradural, intradural-extramedullary, or intramedullary involvement. an extradural lesion is the most common myelographic finding. fluoroscopic aspiration and cytology may allow definitive diagnosis of the spinal lesion. 29 mri may detect intramedullary lesions. treatment. positive felv status in cats has been shown to be a negative prognostic indicator for spinal lymphoma. 30 the prognosis for cats with paresis or paraplegia is considered poor. treatment options for spinal lymphoma consist of chemotherapy, surgical resection, and focal irradiation. 31 no superior treatment strategy for chemotherapy has been documented. currently, multidrug protocols are advocated. 27, 29 a laminectomy procedure facilitates diagnosis and decompression until other therapies can take effect. clinical presentation and pathogenesis. nonlymphoid tumors involving the spinal cord are less common in cats. tumors may be categorized based on expected locations: intramedullary, extramedullary/intradural, and extradural. intramedullary tumors are considered uncommon and make up 10 per cent of all reported spinal cord neoplasms in cats. 1 documented tumors include astrocytoma and ependymoma. 32, 33 intradural/extramedullary tumors make up 12 per cent of spinal neoplasia cases and include meningiomas, meningeal sarcomas, and malignant nerve sheath tumors. 1 feline meningiomas usually occur rostral to the foramen magnum; only 4 per cent of meningiomas found in cats affected the spinal cord. 34 levy, mauldin, kapatkin, et al reported five cases of spinal meningiomas in cats: one in the cervical region, three in the thoracic spine, and one in the lumbar spine. 36 another case report described a meningioma affecting the spinal cord at the c6-c7 spinal cord segment. 35 extradural spinal cord compression can result from spinal canal masses or tumors of the surrounding bone and vertebrae. these make up about 40 per cent of spinal cord-associated neoplasms in cats, with vertebral and bone tumors accounting specifically for 29 per cent of all spinal tumors. 1 reported tumor types include chondrosarcoma, lipoma, osteosarcoma, and multiple myeloma. 36 nonlymphoid spinal neoplasia typically occurs in older cats, with a median age of 12 years in one case series. 36 these tumors are not associated with felv or fiv infection. clinical signs of myelopathy are dependent on tumor location. focal pain and paresis are most typical. intramedullary neoplasms usually do not cause spinal hyperesthesia until later in the disease course. the clinical course of spinal neoplasms also varies. chronic progressive spinal dysfunction may be expected; however, peracute signs (e.g., pathological vertebral fracture) may present (figure 51-3) . diagnosis. the process of diagnosis of nonlymphoid tumors begins with survey spinal radiographs. evidence of bony lesions can be evident in osteosarcoma and multiple myeloma. myelography determines extent and location of spinal involvement. advanced imaging (ct and mri) may assist further with determination of lesion extent. findings on csf analysis often are nonspecific. fluoroscopic aspiration or surgical biopsy may yield a definitive diagnosis. treatment. specific treatment regimens are based on histopathological diagnosis of the tumor. treatment often consists of palliative corticosteroids (i.e., prednisone 0.5 to 1 mg/kg/day po) to control edema, and pain management. surgical removal/debulking of various tumor types has been described and may improve survival times. 36 a reasonable survival time can be expected for cats with meningiomas after surgical resection. 36 osteosarcomas may be associated with long survival times and appear to be less aggressive than the canine form of this disease. 36 a treatment regimen reported for multiple myeloma in a maine coon cat 6 years of age consisted of a combination of chemotherapy and irradiation. 37 clinical presentation and pathophysiology. spinal trauma is an important cause of spinal cord dysfunction in cats. cats have been the subject of multiple laboratory studies of spinal cord injury. [38] [39] [40] [41] [42] [43] [44] information learned from this research must be interpreted from the standpoint that the mechanism of spinal cord trauma is controlled in the laboratory environment. naturally occurring spinal trauma in cats is not a well-described phenomenon because cats often do not survive the inciting incident. spinal injury occurs more frequently in younger cats. cats (n = 69) in a retrospective study of spinal injury were 9 years of age or younger; 39 per cent were younger than 1 year of age, and 29 per cent were between 1 and 3 years of age. 45 the mean age of another study of 30 cats was 3.6 years (range 2 months to 12 years). 46 common causes of spinal trauma are height-related and vehicular injuries. in a large report of high-rise syndrome in cats, 10 per cent sustained spinal fractures or luxations. 47 other sources of trauma include dog attacks and gunshot wounds. 45, 46 compression-type fractures occur in more than two thirds of the cases with spinal fractures. 46 twenty per cent of cats with spinal injuries also have acute disc extrusions secondary to the trauma. 46 spinal cord contusion injury without evidence of fractures also may occur after a fall. 46 other injuries that occur in conjunction with spinal trauma include pneumothorax, pulmonary contusions, abdominal organ trauma, and head trauma. although all segments of the spine are susceptible to trauma, the cervical and sacral/caudal regions are much less likely to sustain injury. the thoracic and lumbar regions make up 51 per cent and 32 per cent of spinal column injuries, respectively. 45 traditionally, the most likely location for spinal column injury is at a site characterized by a transition from rigid stability to less stability, such as t13-l1 or l7-s1. this was challenged recently in a larger study consisting of 69 cats, in which 51 per cent of the spinal trauma cases occurred between t8 and t12. 45 the segments between l2-l3 and l4-l5 also were significant sites in 43 per cent of the cases. the t11 through t12 vertebrae have been reported to be affected in 45 per cent of spinal injuries in cats. 46 a case series reported by voss showed that 50 per cent of spinal fractures were located between the l3 and l6 vertebrae. 48 diagnosis. in cases of suspected spinal injury, the neurological examination is performed with caution to minimize movement of the cat with suspected spinal instability. evaluation of deep pain perception is most important with regard to determining prognosis. spinal radiography using orthogonal views should localize the lesion. radiography documents spinal alignment during that time without knowledge of the amount of displacement at the time of the injury. the entire spine should be radiographed, because multiple spinal fractures are common. advanced imaging of the spine has been recommended because plain film radiography and myelography may underestimate the degree of fractures or luxations present. 48 myelography or mri defines the extent of spinal cord compression more accurately. 46 treatment. goals of therapy are to prevent further mechanical damage to the spinal cord and reduce secondary injury processes. treatment recommendations often are adapted from laboratory studies that involve species other than cats. drawing firm conclusions for optimal treatment of spinal trauma in cats is difficult. management of a cat with spinal trauma should focus first on systemic stabilization. management consists of following the abcs of trauma. airway is assessed for patency and adequate ventilation. appropriate fluid therapy helps to maintain cardiovascular function. aggressive fluid therapy is important to maintain spinal cord perfusion. 49 hypotension is one factor shown to worsen outcome in human beings with spinal cord injury. isotonic crystalloids (lactated ringer's solution or 0.9 per cent sodium chloride) at shock doses, initially (60 ml/kg/hr iv in cats) are given to effect until heart rate, capillary refill time, and pulse quality improve. hetastarch (6 per cent) is a large molecular weight colloid that consists of a branched polysaccharide, amylopectin. its molecular properties provide a long intravascular half-life. the dose is 10 to 20 ml/kg given to effect up to 40 ml/kg/hr. hetastarch is administered intravenously to cats to effect up to 10 to 15 ml/kg; the dose is increased in 5 ml/kg increments every 5 to 10 minutes to avoid nausea and vomiting. hypertonic saline (7 per cent) also may be used to expand blood volume quickly. the dose (4 to 5 ml/kg) is administered as an intravenous bolus over 3 to 5 minutes. the disadvantage associated with the use of hypertonic saline is that it remains in the vascular space for only 15 to 60 minutes. blood products also are used to expand volume and provide increased oxygen delivery. whole blood is administered intravenously at a dose of 4 to 10 ml/kg/hr, over 4 to 6 hours in stable patients and faster in unstable patients. the goal is to restore the hematocrit to 25 to 30 per cent and albumin to more than 2 g/dl. use of high-dose methylprednisolone sodium succinate (mpss) is becoming more controversial but is still considered standard of care in human medicine. experimental spinal cord injury studies in cats have shown that increased lactate levels in spinal cord immediately after injury most likely were attributed to decreased spinal cord perfusion. 41 high doses of mpss 30 minutes after induced trauma attenuated the secondary injury process dramatically. the intent of high-dose mpss is to provide adequate tissue concentrations of steroid at the site of injury. the original dose for this regimen was 30 mg/kg iv initially, then a dose of 15 mg/kg given 2 and 6 hours later, followed immediately by a 2.5 mg/kg/hr infusion, which is continued for 42 hours. the total dose of mpss administered is 165 mg/kg. 43 recent prospective clinical studies that have used modifications of this protocol have come under intense scrutiny for various reasons, including statistical manipulation, lack of proven efficacy, and increased rates of complications in human beings and dogs. [50] [51] [52] [53] [54] administration of mpss is time dependent and has shown efficacy if administered within 30 minutes of the injury. 55 use of high-dose mpss is not recommended for administration if the time has been longer than 8 hours after sustaining the injury. supportive medical management alone is useful if spinal instability is not detected or when there is financial constraint. 56, 57 cats may not tolerate body splints. 56 strict cage confinement is relied upon for 4 to 6 weeks after the injury to initiate the healing process. 58 surgical management. surgical management of spinal trauma is recommended in cases of instability or severe spinal cord compression. 58 the timing of surgery relative to the injury is somewhat controversial; however, adequate medical stabilization before surgery is essential. early decompression has been supported in the laboratory setting in cats, but the optimal time to perform surgery still is unknown. 59 immediate surgical decompression of the affected site is a controversial subject in human spinal trauma, and some studies have not shown a benefit to early surgery. 60, 61 the technique of surgical stabilization depends somewhat on the fracture type. decompression alone may be sufficient in some cases in which instability is not present. 45 decompression was needed in cats that sustained displacement of the intervertebral disc or endplate into the spinal canal. 45 a dorsal laminectomy, preserving the articular processes, suffices for adequate decompression. common techniques of internal spinal fixation/stabilization include the use of pins with polymethylmethacrylate and spinal stapling. spinal stapling involves the use of rigid intramedullary pins that are secured to the spine after reduction of the fracture/luxation. intramedullary pins are secured to the lamina at the base of the spinous process (figure 51-4) . 62 spinal stapling is considered technically easier to perform than other described forms of internal stabilization. limited information is available for the long-term outcome. problems associated with this type of surgery include fragility of the spinous processes and migration or breakdown of implants. 45 a recent case series involving 16 cats with thoracolumbar trauma described using a figure-8 tension band technique as a modification of spinal stapling. 48 complications from this technique were not observed. 48 successful use of pins and polymethylmethacrylate to stabilize a lumbar fracture in a cat has been described. 63 this form of treatment provides significant stability, particularly for rotational forces. 64 optimal placement of pins within the vertebral body may be difficult because of the small size of typical feline vertebral bodies. complications of this procedure include pin migration, pin breakage, pneumothorax, and additional trauma to soft tissue structures. outcome for cats with spinal trauma is guarded. survival rate in one study was only 60 per cent. 46 cats that did not survive were euthanized or died within 4 days of the injury. cats with spinal fractures and absence of deep pain perception almost always have a hopeless prognosis. the return of motor function does not equate necessarily with return of voluntary urination. 48 spinal walking: from laboratory to clinics. prognosis for return of voluntary motor function in cases of absent deep pain perception generally is considered grave. however, the cat has been the subject of extensive experimental work studying the return of ambulation after spinalization, or complete transection of the spinal cord. "spinal walking" is a clinical term used for return of ambulation in an animal with no deep pain perception in the pelvic limbs. in the laboratory setting, this phenomenon is known as spinal locomotion. pelvic limbs are under no voluntary control, and the thoracic limbs move asynchronously with the pelvic limbs when on a treadmill. spinal locomotion may be evident within a few days of the injury. 66 the spinal cord generates this pattern of limb movement, which allows for the placement each foot, weight-bearing, and alterations of speed with change in treadmill velocity. 65 the animal also is capable of stepping over objects placed in its way. 66 cats have been trained to develop spinal locomotion after complete experimental spinal cord transection at t13. the underlying mechanism may be the result of a spinal locomotor generator. 66 spinal locomotion is dependent on the development and preparation of a spinal locomotor pattern generator, stimulation of cutaneous receptors, alterations of intraspinal neurochemistry, and input from the midlumbar spinal cord. 66 plasticity occurs within the spinal cord as a result of training. lesion location within the spinal cord also can affect the ability to walk; for example, a lesion at the l3-l4 spinal segment is not conducive to development of spinal locomotion. 65 spinal walking in a cat with a complete spinal cord injury is much less likely to occur without training. 67 animals with complete spinal cord transections and no training can begin to take steps within weeks of the injury. 65 cats with naturally occurring spinal trauma had a low success rate in development of spinal locomotion after injury. reasons for the low success were attributed to less controlled spinal injury and inadequate physical therapy/training. training for 30 minutes daily 5 days a week provides an 87 per cent success rate of weight-bearing in the pelvic limbs. 68 without appropriate rehabilitation the rate drops to 33 per cent. 69 variability exists among cats as to when walking movements begin to occur. 66 repeated training of a cat by placing the thoracic limbs on a nonmoving platform and the pelvic limbs on a treadmill resulted in better walking and weight-bearing ability in the pelvic limbs. 65 this process involves the use of a treadmill, tail support, and various forms of stimulation. 70 cutaneous stimulation is important for afferent sensory input. younger animals tend to have a better recovery rate for walking. 66 training activities resulted in almost all cats regaining the ability to walk. early intensive training allowed for better walking. 66 spinal locomotion is maintained only for a finite period after discontinuation of training activities and begins to show decline after 12 weeks. 71 long-term management of a deep pain-negative cat. much has been learned in cats after experimental spinal cord injury regarding optimal medical management of deep pain-negative cats. 70 bladder expression is required at a minimum of twice daily. some cats urinate without expression, but the bladder is not emptied completely. stimulation of the perineum initiates a mass reflex and partial emptying of the bladder. 70 researchers report that treadmill training also stimulates urination and defecation in cats with complete spinal cord injuries. 70 inadequate emptying of the bladder predisposes to chronic urinary tract infections 70 (see chapter 48) . suggestions for care to prevent this problem include adequate bladder expression and water intake. 70 chronic bladder infections weaken the muscular wall, further complicating manual emptying of the bladder. 70 fecal elimination usually can occur without assistance and is aided by perineal stimulation. 70 diarrhea and constipation still can occur as complications. clinical presentation and pathogenesis. intervertebral disc disease (ivdd) is recognized commonly in cats with approximately 27 published cases. 72 several case series have been published in recent years. [73] [74] [75] the incidence of ivdd as a significant clinical problem compared to other diseases that affect cats has been reported to be 0.12 per cent. 74 earlier clinical reports of disc disease in cats were postmortem studies that described cervical and, to a lesser extent, lumbar disc disease in older cats. [76] [77] [78] [79] [80] these discs were mostly hansen type ii, with bulging of the annulus fibrosus into the spinal canal, and were described as incidental findings. characteristics of a degenerated intervertebral disc suggest some degree of chondroid degeneration of the discs. 72 more recent literature describes hansen type i, with extrusion of nucleus pulposus into the spinal canal, and recognizes this type to be the most common form of disc-related spinal cord compression in cats. 74 ivdd also can occur spontaneously in cats having no history of trauma. 74 ivdd occurs more frequently in middle to older aged cats. mean age for all reported cases is 7 years. 72 the age range varies somewhat in different reports, between 3 and 9 years, 73 and 4 and 17 years (mean age of 9.8). 74 no gender or breed predilections exist for ivdd in cats. clinical signs of disc disease vary on lesion location and in severity and can consist of back pain and paresis/plegia. lesion involvement in the thoracolumbar region of the spinal cord is common. 72 cervical disc disease is uncommon in cats, with two reported cases confirmed by necropsy, and one presumed case diagnosed with mri. [81] [82] [83] disc spaces between the t11 and l2 vertebrae are affected in 50 per cent of cats with clinical signs of ivdd. 72 the l4-l5 disc interspace also is a common site in 26 per cent of the reported cats with ivdd. 72 ivdd at l7-s1 disc was described in a cat with lower motor neuron signs, flaccid tail, and urinary and fecal incontinence. 84 diagnosis. survey spinal radiographs may reveal typical evidence of disc disease: narrowed disc spaces and evidence of mineralized material in the intervertebral foramen. 74 collection of csf is performed to eliminate other potential inflammatory diseases. findings on csf analysis in cats with ivdd are not specific and may show a mild neutrophilic pleocytosis and increased protein concentration. myelography is used to localize the site of the disc extrusion/herniation more precisely. computed tomography can detect hyperdense material within the spinal canal at the affected disc space. 74 findings on mri suggestive of ivdd include evidence of dehydration of the disc with loss of signal intensity on t2-weighted sequence. 83 treatment. conservative medical management has been used successfully in cats with ivdd; however, in severe spinal cord compression, this form of treatment should not replace surgery. based on a limited number of case reports, medical management alone may result in a poor outcome. 72 conservative management still may be a better option in cases with a small amount of extruded disc material in the canal. 83 medical management usually consists of pain control with use of a combination of narcotics and corticosteroids. corticosteroid therapy (prednisone 0.5 to 1 mg/kg/day po) is used short-term in combination with strict cage confinement for 4 to 6 weeks. physiotherapy also may aid the long-term outcome of neurological function. surgical decompression for removal of extruded disc material may be accomplished with use of either a hemilaminectomy or dorsal laminectomy procedure. 73, 74 surgery offers a higher rate of success and more rapid and complete neurological recovery when compared with conservative treatment. 75, 85 many cats still have residual neurological deficits that include paresis and urinary and/or fecal incontinence. 73, 74 clinical presentation and pathogenesis. fibrocartilaginous embolism (fce), or embolic myelopathy, has been described in many species including cats (table 51-1) . [86] [87] [88] this is a rare disease in cats, with about 7 per cent of spinal cord diseases attributed to vascular causes. 1 in this disease, a small portion of fibrocartilaginous tissue, which is presumed to be intervertebral disc material, occludes the vascular supply to the spinal cord, resulting in an acute onset of asymmetrical spinal cord dysfunction. typically, fce is nonprogressive and not painful. lesions in the cervical and lumbar spinal cord regions have been reported. the mean age from the limited case reports available is 10.2 years, with a range between 8 and 12 years of age. diagnosis. diagnosis of fce is based on elimination of other causes of myelopathy. csf analysis may reveal a neutrophilic pleocytosis and an increased protein concentration. 87, 88 similar abnormalities also have been reported with intervertebral disc disease and may simply indicate necrosis. 73, 83 case reports have lacked definitive imaging results, except in one case in which myelography showed evidence of intramedullary swelling. 88 mr images can be expected to show an increased signal intensity relative to surrounding tissues of the spinal cord parenchyma on a t2-weighted sequence. treatment. treatment strategies have been extrapolated from treatment options recommended in other species with fce. this consists of high doses of mpss (if the drug can be administered within 8 hours of the onset of clinical signs), adequate fluid therapy, bladder management, and supportive care. once the cat is stabilized, physical therapy may aid in recovery. prognosis in these cases is difficult to predict because the literature in this area shows some bias as definitive diagnosis requires necropsy. prognosis is presumed guarded to fair in cats that have deep pain perception intact. syringomyelia is an abnormal fluid-filled cavity within the parenchyma of the spinal cord. hydromyelia often occurs with syringomyelia and is defined as dilation of the central canal. pathophysiology of syringohydromyelia is associated with alterations in flow of csf often secondary to a congenital anomaly, infectious disease process, or trauma. syringohydromyelia has been reported in cats but is not well described. 89 clinical signs include paraspinal hyperesthesia and paresis. the syrinx can be detected using mri. treatment usually is directed toward the underlying cause. an antiinflammatory dose of prednisone (0.5 to 1 mg/kg/day) may reduce edema and inflammatory response. clinical presentation and pathogenesis. diverticulum within the subarachnoid space results in accumulation of csf and compression of the spinal cord, which causes neurological dysfunction. these diverticula are not true cysts but rather leptomeningeal cavitations that are filled with csf. 93 spinal arachnoidal cysts have been reported to cause paresis in cats. [90] [91] [92] [93] another case report documented an intradural epithelial-lined cyst found at the vertebral body of c7 in a 2 1 /2year-old female burmese cat. 93 location of these cyst formations is variable in cats and can occur in the cervical, thoracic, and lumbar spine. the cause of arachnoidal cysts is unknown, but may be related to factors that include previous trauma, inflammation, and developmental or congenital malformations. 92 an arachnoidal cyst in a 7-year-old spayed female domestic shorthair cat with paraparesis was associated with a lordotic malalignment of the caudal thoracic spine. 92 affected cats usually are young to middle-age with a range between 2 and 7 years of age. clinical signs usually are chronic and progressive and reflect the location of the cyst. duration of clinical signs is chronic and progressive in onset. a cat in one report showed signs for only a few weeks. 90 diagnosis. diagnosis of spinal arachnoidal cysts is made using myelography, ct-myelography, or mri. the diverticulum is identified with myelography as a teardrop shape within the subarachnoid space ( figure 51-5) . 92 magnetic resonance imaging can document a spinal arachnoidal cyst on a t2weighted sequence as an area of hyperintensity. 93 treatment. reports of treatment protocols for spinal arachnoidal cysts in cats have been limited. surgical fenestration has been reported. 90,91,93 a hemilaminectomy or dorsal laminectomy is used to expose the cystic structure for dural fenestration and possible excision of the cyst. outcome in these cases has been excellent with complete recovery. residual neurological deficits may occur. histopathology of the cyst wall is recommended to rule out other lesions. palliative medical management consists of antiinflammatory doses of corticosteroids. prevalence of diseases of the spinal cord of cats intervertebral disc disease and myelography inflammatory cerebrospinal fluid analysis in cats: clinical diagnosis and outcome. australian college of veterinary scientists science week analysis of cerebrospinal fluid from the cerebellomedullary and lumbar cisterns of dogs with focal neurologic disease: 145 cases (1985-1987) spinal cord diseases in cats bacterial discospondylitis in a cat lumbar diskospondylitis and meningomyelitis caused by escherichia coli in a cat neuropathology in cats experimentally infected with feline immunodeficiency virus: a morphological, immunocytochemical and morphometric study diagnostic features of clinical neurologic feline infectious peritonitis recommendations from workshops of the second international feline coronavirus/feline infectious peritonitis symposium spinal cord disorders an axial ct image after a myelogram of the lumbar spine from a 7-year-old male castrated domestic shorthair cat with a history of progressive upper motor neuron paraparesis. the cat also had a spinal malformation inflammation and changes in cytokine levels in neurological feline infectious peritonitis epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals comparison of different tests to diagnose feline infectious peritonitis back to the cat": feline spinal cord disease use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis a cryptococcal granuloma in the brain of a cat causing focal signs feline cryptococcosis: a retrospective evaluation clinical and serologic evaluation of cats with cryptococcosis cryptococcal infection in cats: factors influencing treatment outcome, and results of sequential serum antigen titers in 35 cats cryptococcosis in cats: clinical and mycological assessment of 29 cases and evaluation of treatment using orally administered fluconazole infectious diseases of the dog and cat toxoplasmosis and neosporosis feline leukemia virusassociated myelopathy in cats degenerative myelopathy in a cat feline lymphoma and leukemia therapeutic choices for the medical management of feline lymphoma. waltham feline medicine symposium feline spinal lymphosarcoma: a retrospective evaluation of 23 cats spinal lymphoma in cats: 21 cases feline lymphoma (145 cases): proliferation indices, cluster differentiation 3 immunoreactivity, and their association with prognosis in 90 cats principles of treatment for feline lymphoma spinal cord astrocytoma in a cat a glioma in the spinal cord of a cat meningiomas in animals use of magnetic resonance imaging for diagnosis of a spinal tumor in a cat nonlymphoid vertebral canal tumors in cats: 11 cases (1987-1995) multiple myeloma in cats: variable presentation with different immunoglobulin isotypes in two cats effects of a single large dose of methylprednisolone sodium succinate on experimental posttraumatic spinal cord ischemia. dose-response and time-action analysis the neuroprotective pharmacology of methylprednisolone correlation of methylprednisolone levels in cat spinal cord with its effects on (na + k + )-atpase, lipid peroxidation, and alpha motor neuron function lactate and pyruvate metabolism in injured cat spinal cord before and after a single large intravenous dose of methylprednisolone effects of multi-dose methylprednisolone sodium succinate administration on injured cat spinal cord neurofilament degradation and energy metabolism evaluation of an intensive methylprednisolone sodium succinate dosing regimen in experimental spinal cord injury pretreatment with alpha tocopherol enhances neurologic recovery after experimental spinal cord compression injury management of spinal trauma in 69 cats survival rates and outcomes in cats with thoracic and lumbar spinal cord injuries due to external trauma high-rise syndrome in cats: 207 cases tension band stabilization of fractures and luxations of the thoracolumbar vertebrae in dogs and cats: 38 cases combined medical and surgical treatment after acute spinal cord injury: results of a prospective pilot study to assess the merits of aggressive medical resuscitation and blood pressure management methylprednisolone for acute spinal cord injury: an inappropriate standard of care is the role of steroids in acute spinal cord injury now resolved? high dose methylprednisolone in the management of acute spinal cord injury -a systematic review from a clinical perspective gastric hemorrhage in dogs given high doses of methylprednisolone sodium succinate complications of methylprednisolone sodium succinate therapy in dachshunds with surgically treated intervertebral disc disease evaluation of time-dependent spread of tissue damage in experimental spinal cord injury by killedend evoked potential: effect of high-dose methylprednisolone nonsurgical management of thoracic and lumbar spinal fractures and fractures/luxations in the dog and cat: a review of 17 cases management of vertebral column fractures in dogs and cats: 211 cases (1977-1985) spinal fracture or luxation reversible spinal cord trauma in cats. additive effects of direct pressure and ischemia an evidence-based review of decompressive surgery in acute spinal cord injury: rationale, indications, and timing based on experimental and clinical studies current use and timing of spinal surgery for management of acute spinal cord injury in north america: results of a retrospective multicenter study principles of vertebral fracture management use of pins and methylmethacrylate in stabilization of spinal fractures and luxations the rotational stabilizing effect of spinal fixation techniques in an unstable vertebral model recovery of locomotion in the cat following spinal cord lesions determinants of locomotor recovery after spinal injury in the cat locomotor capacity attributable to step training versus spontaneous recovery after spinalization in adult cats effects of training on the recovery of full weight bearing stepping in the adult spinal cat return of weight supported locomotion in adult spinal cats chronic spinal cord-injured cats: surgical procedures and management retention of hindlimb stepping ability in adult spinal cats after the cessation of step training feline intervertebral disc disease: a review of the literature intervertebral disc extrusion in six cats intervertebral disk disease in 10 cats spontaneous lumbar intervertebral disc protrusion in cats: literature review and case presentations disc protrusions in the cat: distribution of dorsal protrusions along the vertebral column disc protrusions in the cat: age incidence of dorsal protrusions disc protrusions in the cat: ventral protrusions and radial splits degeneration of the intervertebral disc in the cat protrusion of the intervertebral disc in the cat intervertebral disc syndrome in the cat intervertebral disc protrusion in a cat acute intervertebral disc extrusion in a cat: clinical and mri findings lumbosacral disc disease in a cat radiographic diagnosis: intervertebral disc extrusion in a cat fibrocartilaginous embolic myelopathy in a cat tetraparesis in a cat with fibrocartilaginous emboli fibro-cartilaginous embolism in a cat syringomyelia and hydromyelia in dogs and cats subarachnoid cyst in a cat correlative imaging findings in seven dogs and one cat with spinal arachnoid cysts spinal subarachnoid cyst in a cat intradural epithelial cyst in a cat key: cord-007603-27m9wz0i authors: rall, glenn f.; mucke, lennart; nerenberg, michael; oldstone, michael b.a. title: a transgenic mouse model to assess the interaction of cytotoxic t lymphocytes with virally infected, class i mhc-expressing astrocytes date: 2002-11-11 journal: j neuroimmunol doi: 10.1016/0165-5728(94)90163-5 sha: doc_id: 7603 cord_uid: 27m9wz0i astrocytes provide crucial support for neurons and their impairment by viruses or their interactions with anti-viral or autoimmune responses could contribute to neurological disease. we have developed a transgenic mouse model to assess lymphocyte-astrocyte interactions. the major histocompatibility complex (mhc) class i molecule, d(b), was expressed in astrocytes under the transcriptional control of regulatory sequences from the glial fibrillary acidic protein (gfap) gene. baseline cerebral mhc class i mrna levels from transgenic mice were elevated over those of non-transgenic controls, and a prominent increase in cerebral mhc class i expression occurred following focal, injury-induced astroglial activation within transgenic brains but not in non-transgenic controls. facs analysis of explant astrocyte cultures from established transgenic lines demonstrated astroglial expression of the gfap-d(b) fusion gene at the protein level. functional antigen-presenting capacity was conferred by the d(b) transgene, as virus-infected primary astrocytes obtained from transgenic balb/c mice (k(d)i(d)d(d)l(d)) expressing the d(b) molecule were lysed by d(b)-restricted anti-viral ctl. the central nervous system (cns) has been considered an immune privileged site compared with other organs by virtue of the blood-brain barrier that restricts access of macromolecules and non-activated lymphoid cells from the periphery into the cns parenchyma and because resident cns cells such as neurons, oligodendrocytes and astrocytes express negligible to undetectable levels of class i major histocompatibility (mhc) molecules (reviewed in lampson, 1987) . mhc class i molecules function to present viral protein fragments (peptides) on the surface of cells so they can be recognized as foreign and be destroyed by mhc-matched, anti-viral ctl (zinkernagel and doherty, 1974; townsend et al., 1985) . however, lack of mhc expression may not be absolute as astrocytes and oligodendrocytes can be focally induced to express mhc class i in response to certain cns infections (massa et al., 1986; suzumura et al., 1986; olsson et al., 1987; liu et al., 1989) and during inflammatory demyelinating disease (traugott and lebon, 1988) . further, activated t lymphocytes can traverse the intact blood-brain barrier and enter the brain parenchyma (wekerle et al., 1986 (wekerle et al., , 1987 hickey et al., 1991; oldstone and southern, 1992) . together, these data suggest that activated antiviral or autoimmune t cells can come in close proximity to resident cns cells, and their interactions and/or release of cytokines may result in focal induction of mhc molecules on these cns cells. to better understand the potential role of class i mhc expression and antigen presentation by resident cns ceils, we have expressed a murine class i mhc molecule (d b) in transgenic mice under the control of a variety of cns cell-specific promoters. here we report the establishment of transgenic mouse lines in which a constitutive and inducible class i mhc molecule is expressed in cns astrocytes. astrocytes provide crucial support for neurons and oligodendrocytes through such diverse functions as the production of neurotrophic factors and the elimination of neurotoxins (reviewed in eddleston and mucke, 1993) . consequently, their impairment or destruction by either antiviral or auto-immune responses would disturb cns homeostasis. notably, a variety of dna and rna viruses can infect astrocytes in vivo (epstein et al., 1984; stowring et al., 1985; gyorkey et al., 1987; mirra and del rio, 1989; carbone et al., 1991; itoyama et al., 1991; rinaman et al., 1993; epstein et al., 1994) . the gfap-d b mice should provide a useful tool to study immune interactions with virally infected astrocytes in vivo. from dr. r.b. wallace, city of hope research institute, duarte, ca); /72-microglobulin cdna from clone p /72-m2 (daniel et al., 1983) , obtained from dr. p. kourilsky, institute pasteur, paris, france; a cdna fragment of mouse beta-actin bp 480-740 (tokunaga et al., 1986) amplified from murine genomic dna by pcr; and sv40 bp 1705 -1910 (lebowitz and weissman, 1979 which identifies sv40 sequences at the 3' end of the gfap vector and serves as a marker for the transgene. male and female c57b1/6 and balb/cbyj mice of various ages (newborns to 1 year of age) were used. animals were maintained in conditions consistent with aaalac regulations throughout the course of the investigation. focal mechanical brain lesions were placed by penetration of one or both hemispheres with a sterile 27 gauge needle as described (mucke et al., 1991) . for this procedure, mice were anesthetized with methoxyflurane. standard protocols (ausubel et al., 1987; sambrook et al., 1989) were used for the construction of the gfap-d b fusion gene. all junctions created by the subcloning were sequenced prior to microinjection. fertilized oocytes were obtained from (c57b1/6 × c57b1/6) or (balb/c x balb/c) females. purification of the transgene, preparation of mice and microinjection and reimplantation of fertilized oocytes were carried out as described (mucke et al., 1991) . the entire sequence of the gfap gene was included in the construct to avoid deletion of potentially important intragenic regulatory elements (sarkar and cowan, 1991) . transgenic mice were identified by slot blot or southern blot hybridization of genomic dna extracted from tail tissue. poly(a) + rna was extracted from brains and analyzed by northern blot and sequential hybridization with different probes (mucke et al., 1991) . the following dnas were 32p-labeled with the random hexanucleotide primer method (sambrook et al., 1989) and used as probes: a partial d b cdna from clone ph 203 (reyes et al., 1982) which cross-hybridizes with both h-2 b and h-2 d mhc class i transcripts (obtained tissues were removed from transgenic and nontransgenic littermates and snap frozen in liquid nitrogen. total rna was later extracted using the gtc-acid phenol method (chomczynski and sacchi, 1987) . 500 ng of total rna was reverse transcribed (m-mlv reverse transcriptase, gibco-brl/life technologies, gaithersburg, md) at 37°c for 15 min using the random hexamer extension method (perkin-elmer cetus, emoryville, ca). the resulting cdna was then subjected to 40 cycles of pcr (60°c annealing; 72°c extension; 95°c denaturation) in the presence of taq polymerase and oligonucleotide primers designed to amplify either a product specific for the gfap-d b transgene (primers a and b) or an internal standard, gapdh (primers c and d). the sequences of the primers are as follows: a, 5' aggtt ggagc ggaga cgcat 3'; b, 5' gcgct ctggt tgtag tagcc 3'; c, 5' tggta tcgtg gaagg actca tgac 3'; d, 5' agtcc agtga gcttc ccgtf cagc 3'. primary astrocytes were isolated by a procedure modified from mccarthy and de vellis (1980) . briefly, neonatal brains were mechanically dissociated in dmem containing 15% fetal bovine serum. following 3 days of incubation in poly-l-lysine coated tissue culture flasks, cells were shaken at 37°c at 100 rotations per min for 24 h to remove non-adherent cells. when fixed with 2% paraformaldehyde in pbs and labeled with antibodies against gfap (dako, carpinteria, ca) as described (mucke et al., 1991) , at least 95% of the adherent cells stained positive for this astroglial marker. all astrocytes were used within 15 days of culture and were passaged no more than three times. for facs analysis, uninfected primary astrocytes were trypsinized, washed and incubated with medium alone or with a 1:50 dilution of primary antibodies: b22.249 r1 (anti-d b) or 30-5-7s (anti-l ~) (accurate chemical & scientific corporation, westbury, ny). binding of primary antibodies was revealed with a fitc-conjugated secondary antibody (dilution 1:200). all reactions were incubated on ice for 30 min. the cells were washed extensively and analyzed on a becton dickinson facs 4. dead cells were excluded by addition of 1 ng/ml propidium iodide to the samples prior to fluorimetry. chromium release assays were carried out as described elsewhere (oldstone, 1990) . in brief, primary astrocytes were infected with the armstrong ca 1371 clone 53b of lcmv (lcmv arm) (dutko and oldstone, 1983 ) at a multiplicity of infection (moi) of 3. this moi ensured that all cells became infected as determined by infectious center assays (not shown). 48 h later, the astrocytes were labeled with 51cr and exposed to lcmv primed h-2 matched or h-2 mismatched splenocytes at different effector-to-target cell (e:t) ratios. ctl specific for lcmv were raised by injecting 3-4-month-old c57b1/6 and balb/c mice intraperitoneally with 2 × 105 plaque-forming units of lcmv arm. splenic lymphocytes obtained from these mice 7-8 days later were used in chromium release assays. the specific 51cr release was calculated according to the following formula: experimental release -spontaneous release × 100 maximum release -spontaneous release each sample was done in triplicate and the variance within triplicates was 10% or less. different groups of astrocytes compared in ctl assays were established and processed simultaneously. to establish transgenic mice with astroglial expression of an mhc class i molecule, a minigene encoding the mhc class i antigen d b was placed under the regulatory influence of a modified murine glial fibrillary acidic protein (gfap) gene (fig. 1) . earlier work documented that the gfap gene effectively targets the expression of different proteins to astrocytes in vivo, including lacz (mucke et al., 1991) , human alpha-l-antichymotrypsin , the hiv1 coat protein, gpl20 (toggas et al., 1994) , and the cytokine il-6 (campbell et al., 1993) . for construction of the gfap-d b transgene, the 5' and 3' ends of a partial cdna encoding the greater portion of the d b molecule (reyes et al., 1982) were ligated with fragments of genomic d b sequence derived from the mo/d b clone (allen et al., 1986 ) (obtained from dr. r. flavell, yale university, new haven, ct). the resulting d b minigene consists of all exons as well as the first two introns of the d b gene. a polyadenylation signal is provided by 3' untranslated genomic d b sequence. the d b minigene was fused with the modified gfap gene (mucke et al., 1991) after addition of noti(5') and saii(3') linkers and the structure of the resulting construct was characterized by restriction analysis and sequencing of promoterminigene junctions. the gfap-d b fusion gene was then microinjected into fertilized oocytes from c57b1/6 (h-2 b) and balb/c (h-2 d) mice as described (mucke et al., 1991) . founders, separate lines of transgenic mice were established and screened for expression of the transgene. four of the five lines were derived from c57b1/6 × c57bi/6 (h-2 b) zygotes (lines nos. 162, 165, 184, 185) while one line (no. 208) was derived from a balb/c × balb/c (h-2 d) zygote. to determine tissue expression of the gfap-d b transgene, reverse transcriptase/polymerase chain reaction (rt-pcr) was done on multiple tissues from transgenic and nontransgenic littermates including brain, spleen, pancreas, kidney, thymus, liver and testes. to determine rna expression in the peripheral cns, sciatic nerve was also analyzed. two sets of primers were used. one set, primers a and b, shown in fig. 2c , was specific for gfap-d b sequences and distinguished between spliced and unspliced products due to the presence of a 190-bp intron between the two primers. the other set of primers, primers c and d, served as a pcr control, and amplified gapdh, a protein expressed in all tissues. brain rna from transgenic mice but not from nontransgenic littermates gave rise to a pcr product indicative of spliced rna ( fig. 2a) . a faint band was also detected in the thymi of these mice. no other organ, including sciatic nerve, gave rise to the transgene-derived pcr fragment. when the brain was dissected into four major regions (the cerebral hemispheres, the olfactory bulb, the cerebellum and the midbrain/brainstem), all portions of the brain resulted in the correctly spliced product (fig. 2b) . thus, expression of the transgene is primarily restricted to the brain, and is expressed throughout the brain. by northern analysis, offspring from four (lines nos. lesioned: + + the gfap-d b expression at the protein level was too low to be detected in situ by immunostaining. however, expression of the correctly folded d b molecule on the surface of astrocytes could be demonstrated by facs analysis (fig. 4) using b22.249 r1, a monoclonal antibody that detects the correctly folded d b molecule (allen et al., 1986 ). an increase in mean fluorescence (from 12.26 to 23.24) was found in primary transgenic astrocytes relative to non-transgenic astrocytes (fig. 4a, arrows) . as a control, no significant difference in mean fluorescence was identified between transgenic and non-transgenic astrocytes when an l d specific monoclonal antibody was used (fig. 4b) . . two mice received focal brain lesions, as described in materials and methods, 2 days prior to sacrifice. two unlesioned mice served to establish baseline levels of the different transcripts. a probe for beta-actin was used to assess amounts of rna per lane. the brains of gfap-d b transgenic mice compared with non-transgenic controls (fig. 3) . in normal non-transgenic mice, there was no detectable increase in the cerebral level of mhc class i mrna 48 h following focal mechanical brain injury (fig. 3) . in gfap-d b transgenic mice, however, there was a marked increase in mhc class i mrna levels in response to such injuries. this injury-induced upregulation of cerebral mhc class i expression was also found in transgenic lines nos. 162, 165 and 184 (2-6 mice analyzed per line) (data not shown). the astroglial expression of a functional, transgenederived d b molecule was demonstrated by infection of explant cultures of astrocytes from transgenic mice with lcmv for 48 h followed by incubation of astrocytes with lcmv-specific, db-restricted ctl. table 1 shows that astrocytes obtained from h-2 d balb/c mice that expressed the h-2 b d b minigene were specifically lysed by h-2 b ctl while astrocytes obtained from h-2 d mice not expressing the d b molecule were not (36% vs. 10% s~cr release at e : t ratios of 50 : 1). because the exposure to serum-containing media induces astrocytes to express endogenous mhc molecules (keane et al., 1992) , both transgenic and nontransgenic lcmv-infected astrocytes were effectively lysed by h-2-matched ctl bearing the same h-2 haplotype as the target cell (40% vs. 46% 5]cr release at e : t ratios of 50 : 1). similar results were obtained in three other independent assays using three different batches of astrocytes. in this report, mhc class i minigene to astrocytes using sequences derived from the gfap gene. transgenic mice expressing d b mhc molecules on astrocytes displayed similar survival rates as age and sex matched nontransgenic littermates over a 1-year period of observation (data not shown). further, transgenic mice expressing the mhc molecule in astrocytes showed no signs of cns impairment and histological analysis of their brains was indistinguishable from that of non-transgenic controls, suggesting that class i mhc expression alone is not sufficient to induce spontaneous neuropathology. the gfap-d b transgene is expressed in a functional manner as indicated by ctl assay (table 1) . lcmv-infected astrocytes from gfap-d b transgenic mice that are normally kdidd d were specifically lysed by anti-viral, h-2 b restricted ctl while astrocytes from normal balb/c mice were not. because the transgene-derived o b molecule is the only h-2 b mhc class i antigen that could be expressed on astrocytes of transgenic balb/c (h-2 d) mice, lysis of these astrocytes by h-2b-restricted ctl demonstrates that lcmv peptide processing and interaction with the o b minigene product is appropriate for recognition by h-2 b anti-lcmv ctl. transgenic mice which express a class i mhc gene product (k b) driven by the gfap promoter have been established previously (schonrich et al., 1991) . our model incorporates the entire gfap gene, which may include intragenic regulatory elements required for astrocyte-specific expression. further, our choice of the d b gene was chosen based on the well-characterized anti-lcmv response to target cells expressing the d b molecule. the inability to detect the o b molecule in vivo with antibody likely reflects the insensitivity of the immunohistochemical assay since d b was detected with antibody when facs analysis was used in ex vivo astrocyte preparations (fig. 4) . notably, ctl recognition and lysis despite undetectable levels of mhc class i and other immunoregulatory molecules have been noted (skias et al., 1987; oldstone et al., 1991) . a strength of this model is the inducibility of the gfap promoter, exemplified here by the increase of transgenic class i mhc levels following mechanical trauma (fig. 3) . a variety of neural injuries have also been shown to up-modulate gfap expression (smith et al., 1983; de la monte et al., 1987; manuel±dis et al., 1987; delacourte, 1990) . therefore, insults which result in an upregulation of the gfap gene should correspond with focal induction of class i mhc expression on astrocytes. experiments using allografts into the rat cns and under the kidney capsule (mason et al., 1986) have shown that rats mounted a brisk anti-graft response to the kidney graft while the cns graft rejection occurred more slowly. this suggests that the microenvironment of the cns, not the antigen-presenting capacity of the cells themselves, restricts immune recognition. further, massa has shown that brain-enriched gangliosides can suppress transcription of class i and ii mhc on astrocytes (massa, 1993) . these results indicate that some cells of the cns may be under immunosuppressive pressures that inhibit mhc expression resulting in a lack of recognition by the immune surveillance system and avoidance of immune-mediated damage. the ability to express inducible mhc molecules and cytokines in vivo in specific cns cells by using cns cell-specific promoters not sensitive to immunosuppressive factors should allow a determination of the immunopathogenic effects of aberrant mhc expression during diverse viral infections and autoimmune diseases of the cns. beta 2-microglobulin is not required for cell surface expression of the murine class i histocompatibility antigen h-2d b or of a truncated h-2d b current protocols in molecular biology neurologic disease induced in transgenic mice by cerebral over-expression of interleukin-6 borna disease virus replicates in astrocytes, schwann cells and ependymal cells in persistently infected rats: location of viral genomic and messenger rnas by in situ hybridization single step method of dna isolation by acid guanidinium-thiocyanate acid-phenol-chloroform extraction structure and expression of the mouse beta 2-microglobulin gene isolated from somatic and non-expressing teratocarcinoma cells subacute encephalomyelitis of aids and its relation to htlv-iii infection general and dramatic glial reaction in alzheimer brains genomic and biological variation among commonly used lymphocytic choriomeningitis virus strains molecular profile of reactive astrocytes -implications for their role in neurologic disease htlv-iii/lav-like retrovirus particles in the brains of patients with aids encephalopathy t-lymphocyte entry into the central nervous system early loss of astrocytes in herpes simplex virus-induced central nervous system demyelination resistance and susceptibility of neural cells to lysis by cytotoxic lymphocytes and by cytolytic granules molecular bases of the immune response to neural antigens organization and transcription of the simian virus 40 genome flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro astrocyte gene expression in creutzfeldt-jakob disease the fate of auogeneic and xenogeneic neuronal tissue transplanted into the third ventricle of rodents specific suppression of major histocompatibility complex class i and class ii genes in astrocytes by brain-enriched gangliosides viral particles induce ia antigen expression on astrocytes preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue the fine structure of acquired immunodeficiency syndrome encephalopathy the expression of major histocompatibility complex (mhc) class i antigens in the brain differs markedly in acute and persistent infections with lymphocytic choriomeningitis virus (lcmv) rapid activation of astrocyte-specific expression of gfap-lacz transgene by focal injury transgenic models to study the pathogenic role of mutated and non-mutated forms of human amyloid proteins in the development of alzheimer's disease (ad) animal virus pathogenesis. a practical approach trafficking of activated cytotoxic t lymphocytes into the central nervous system: use of a transgenic model virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response induction of class i and class ii transplantation antigens in rat brain during fatal and nonfatal measles virus infection the complete amino acid sequence of the murine transplantation antigen h-2d b as deduced by molecular cloning spatiotemporal responses of astrocytes, ramified microglia, and brain macrophages to central neuronal infection with pseudorabies virus overexpression of nef as a marker for restricted hiv-i infection of astrocytes in post mortem pediatric central nervous tissues molecular cloning: a laboratory manual intragenic sequences affect the expression of the gene encoding glial fibrillary acidic protein down-regulation of t cell receptors on self-reactive t cells as a novel mechanism for extrathymic tolerance induction susceptibility of astrocytes to class i mhc antigen-specific cytotoxicity immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis detection of visna virus antigens and rna in glial cells in foci of demyelination coronavirus infection induces h-2 antigen expression on oligodendrocytes and astrocytes neuropathologic alterations induced in brains of transgenic mice by expression of the hiv-i envelope protein gpl20 nucleotide sequence of a full-length cdna for mouse cytoskeletal beta-actin mrna cytotoxic t cells recognize fragments of influenza nucleoprotein multiple sclerosis: involvement of interferons in lesion pathogenesis cellular immune reactivity within the cns immune reactivity in the nervous system: modulation of t-lymphocyte activation by glial cells restriction of in vitro t cell mediated cytotoxicity in lymphocytic choriomeningitis virus within a syngeneic or semi-allogenic system we thank jenny price, william johnson and edward rockenstein for excellent technical support, and gay schilling for help with the preparation of the manuscript. key: cord-022163-7klzsrpu authors: broder, christopher c.; wong, kum thong title: henipaviruses date: 2016-09-09 journal: neurotropic viral infections doi: 10.1007/978-3-319-33133-1_3 sha: doc_id: 22163 cord_uid: 7klzsrpu the first henipaviruses, hendra virus (hev), and nipah virus (niv) were pathogenic zoonoses that emerged in the mid to late 1990s causing serious disease outbreaks in livestock and humans. hev was recognized in australia 1994 in horses exhibiting respiratory disease along with a human case fatality, and then niv was identified during a large outbreak of human cases of encephalitis with high mortality in malaysia and singapore in 1998–1999 along with respiratory disease in pigs which served as amplifying hosts. the recently identified third henipavirus isolate, cedar virus (cedpv), is not pathogenic in animals susceptible to hev and niv disease. molecular detection of additional henipavirus species has been reported but no additional isolates of virus have been reported. central pathological features of both hev and niv infection in humans and several susceptible animal species is a severe systemic and often fatal neurologic and/or respiratory disease. in people, both viruses can also manifest relapsed encephalitis following recovery from an acute infection, particularly niv. the recognized natural reservoir hosts of hev, niv, and cedpv are pteropid bats, which do not show clinical illness when infected. with spillovers of hev continuing to occur in australia and niv in bangladesh and india, these henipaviruses continue to be important transboundary biological threats. niv in particular possesses several features that highlight a pandemic potential, such as its ability to infect humans directly from natural reservoirs or indirectly from other susceptible animals along with a capacity of limited human-to-human transmission. several henipavirus animal challenge models have been developed which has aided in understanding hev and niv pathogenesis as well as how they invade the central nervous system, and successful active and passive immunization strategies against hev and niv have been reported which target the viral envelope glycoproteins. the genus henipavirus in the family paramyxoviridae is presently represented by three known virus isolate species hendra virus (hev), nipah virus (niv) and cedpv (cedpv) and are enveloped, single-stranded negative-sense rna viruses (wang et al. 2013b ; marsh et al. 2012 ) . hev and niv are bat-borne disease-causing zoonoses while cedpv also resides in the same bat species as does hev in nature. studies have shown that cedpv is not pathogenic in animals susceptible to hev and niv disease, nor is it known to be zoonotic. to date, bats appear to be predominant natural reservoir hosts for henipaviruses (clayton et al. 2013 ) and recently, by nucleic acid based detection surveys, there has been a signifi cant species expansion of the henipavirus ranks including at least two full genome sequences , and also a report of one henipavirus from a rodent, but to date hev, niv, and cedpv are the only virus isolates that have been reported (wu et al. 2014 ; drexler et al. 2012 ) . central pathological features of both hev and niv infection in humans and several susceptible animal species is a severe systemic and often fatal neurologic and/or respiratory disease (abdullah and tan 2014 ; wong and ong 2011 ; playford et al. 2010 ) . of additional concern in people, both viruses, but particularly niv, can also manifest as relapsing encephalitis following recovery from an acute infection resulting from a recrudescence of virus replication in the central nervous system (cns) (wong and tan 2012 ; wong et al. 2009 ). spillovers of hev have continued to occur in australia since its identifi cation, as does niv in bangladesh and india, since its recognition in malaysia, which continue to make these henipaviruses an important transboundary biological threat . niv in particular possesses several features that highlight a pandemic potential, such as its ability to infect humans directly from natural reservoirs or indirectly from other susceptible animals along with a capacity of limited human-to-human transmission (luby 2013 ) . several henipavirus animal challenge models have been developed which has aided in understanding how hev and niv invade the central nervous system de wit et al. 2014 ) , and successful active and passive immunization strategies against henipaviruses have been reported which target the viral envelope glycoproteins broder 2012 ; broder et al. 2012 ). a new paramyxovirus was isolated and identifi ed in 1994 in an outbreak of fatal cases of respiratory disease in horses and humans in the brisbane suburb of hendra, australia, and was shown to be distantly related to measles virus and other morbilliviruses (murray et al. 1995a ) . thirteen horses and their trainer succumbed to the infection by this previously unknown virus, along with the non-fatal infection of seven other horses and a stable hand. in an unrelated and only retrospectively identifi ed spillover of this same virus near mackay in central queensland, ~1000 km north of brisbane, a farmer experienced a brief aseptic meningitic illness after caring for and assisting at the necropsies of two horses that were only later shown to have died from this virus infection (hooper et al. 1996 ; rogers et al. 1996 ) . thirteen months later this individual suffered severe fatal encephalitis resulting from that initial virus infection characterized by uncontrolled focal and generalized epileptic activity (o'sullivan et al. 1997 ) . the virus was provisionally termed equine morbillivirus but was later re-named hev where the initial recognized outbreak had occurred. to date, hev has since reemerged in eastern australia on 55 occasions with more than 97 horse deaths, 2 hev antibody positive euthanized dogs, and 4 of 7 human case fatalities anonymous 2012 anonymous , 2013a anonymous , b , 2014a . although hev infection was detected in two dogs in recent years, the extent of hev transmission from bats to dogs in australia is unknown, and all recognized hev spillovers and all cases of confi rmed human infections, the horse has served as an intermediate host between the virus-shedding bat reservoir and humans. the epidemiological features and potential mechanisms at play of hev emergence and continued spillovers have been examined (plowright et al. 2011 ) and reviewed elsewhere (field et al. 2007 . niv emerged just a few years later following the initial recognition of hev. a large outbreak of encephalitis among pig farmers in peninsular malaysia began in 1998 and continued into the next year (chua et al. 1999 ). this outbreak was initially attributed to japanese encephalitis virus because it occurred among people in close contact with pigs. however, several features distinguished this outbreak from japanese encephalitis such as patients were primarily adults not children, along with household clustering of cases being noted, and many of those affl icted had previously been vaccinated against japanese encephalitis (chua et al. 1999 ) . a syncytia-forming virus in vero e6 cell culture was obtained from the cerebrospinal fl uid (csf) of two patients which cross-reacted with antibodies against hev and several patients had igm antibodies in their csf that were reactive against hev (chua et al. 1999 ) . later molecular genetic studies confi rmed the close relationship of this new paramyxovirus, termed niv, to hev (chua et al. 2000a ) . there were at least 265 cases of human infection with 105 fatalities in malaysia along with an additional 11 cases and 1 fatality among abattoir workers in singapore (chua et al. 2000a ; paton et al. 1999 ) . the chronology of the events and the epidemiological features of this outbreak, including potential causes and the factors that exacerbated this outbreak, as well as the pathological observations made in both animals and humans have been critically reviewed and recently examined elsewhere (wong and tan 2012 ; wong and ong 2011 ; chua 2003 ; pulliam et al. 2012 ). niv has not reappeared in malaysia, however nearly annual outbreaks of niv infection have now been recognized since 2001, occurring primarily in bangladesh but also india. the most recent cases of human infections occurred in early 2015 with two fatalities (anonymous 2015 ) . the spillovers of niv in bangladesh and india have had lower numbers of human infections; however the fatality rates have been notably higher from 75 to 100 %. in addition, direct transmission of niv from bats to humans from the consumption of contaminated date palm sap along with signifi cant human-tohuman transmission has now been documented homaira et al. 2010a , b ; luby et al. 2009b ) . the epidemiological details of the spillovers of both hev and niv into people since their emergence and recognition have recently been reviewed and summarized in detail luby and broder 2014 ) . there have been ~613 human cases of niv infection with 315 fatalities (reviewed in luby et al. 2009b ; broder 2012 ; anonymous 2014c anonymous , 2015 . both hev and niv are highly pathogenic in a number of mammalian species and possess several characteristics that distinguish them from all other known paramyxoviruses and are classifi ed as biosafety level-4 (bsl-4) agents. finally, although not associated with a zoonotic event, the third recognized henipavirus species as a virus isolate was recently identifi ed . urine sample collecting for pcr and virus isolation experiments were being carried out as part of fi eld studies on hev genetic diversity and infection dynamics in fl yingfox populations in queensland, australia. from these studies a syncytia-inducing virus was identifi ed in pteropus bat kidney cell culture isolated from samples collected in september 2009 from a fl ying-fox colony in cedar grove, south east queensland . molecular analysis indicated that this virus was a new paramyxovirus most closely related to hev and niv and the virus was named cedpv after the location of the bat colony sampled. animal challenge studies with cedpv in guinea pigs and ferrets which are susceptible to infection and disease with hev and niv, revealed that while cedpv replication occurred and induced neutralizing antibodies, no clinical disease was apparent ). soon after the discovery and isolation of hev, a state-wide serologic survey of 2411 horses reported no evidence of infection and only horses involved in the initial brisbane outbreak were positive (ward et al. 1996 ) . this was followed by a large serological survey conducted across eastern queensland, australia in an effort to identify the potential natural host(s) of the virus, and 5264 sera samples across 46 species, mostly wildlife, were screened and no evidence of hev neutralizing antibody was found (young et al. 1996 ) . however, the additional screening of potential animal reservoirs that overlapped the two initial but distant hev spillover events led to the testing of the four fruit bat species (fl ying foxes) native to mainland australia, and here serological evidence was found in all four species of pteropus fruit bats (young et al. 1996 ) . hev was later isolated from the gray-headed fl ying fox ( pteropus poliocephalus ) and the black fl ying fox ( p. alecto ) (halpin et al. 2000 ) . following the fi rst appearance of niv in peninsular malaysia, a serological surveillance study on samples from 324 bats across 14 species revealed the presence of niv neutralizing antibodies in island fl ying-foxes ( p. hypomelanus ) and malayan fl ying foxes ( p. vampyrus ) . a follow-up study focusing on virus isolation by collecting pooled urine samples from island fl ying foxes, as well as partially eaten fruit, reported the isolation of niv . niv has since been isolated from the urine of p. lylei in cambodia (reynes et al. 2005 ) . serological assays as a means of detection of the presence of niv and/or hev in nature, from wildlife, domestic animals and human populations, is more readily achievable as compared to either virus isolation or nucleic acid detection (mcnabb et al. 2014 ) . a number of serological surveys have been carried out over the past several years to screen for the presence of henipaviruses in bats, domestic livestock and people. the preponderance of data indicates that the pteropus bat species appear to be the major natural reservoir hosts for henipaviruses yadav et al. 2012 ; wacharapluesadee et al. 2010 ; epstein et al. 2008 ; iehle et al. 2007 ) and all bat isolates of hev, niv and also cedpv have been derived from pteropus bats (halpin et al. 2000 ; chua et al. 2002 ; reynes et al. 2005 ; rahman et al. 2010 ; marsh et al. 2012 ) (see also chap. 26). further, as natural hosts, a lack of any observed overt disease in wild bats is also in agreement with a lack of elicited clinical signs in experimentally infected pteropid bats (middleton et al. 2007 ; williamson et al. 1998 williamson et al. , 2000 halpin et al. 2011 ) . pteropus bat species are distributed as far west as madagascar, through the indian subcontinent to southeastern asia and australia, and eastwards through oceania (clayton et al. 2013 ; breed et al. 2013 ; field et al. 2001 ) . however, there is evidence of henipaviruses in wide variety of other bat species in both megachiroptera and microchiroptera suborders (hayman et al. 2008 ; peel et al. 2012 peel et al. , 2013 hasebe et al. 2012 ; wacharapluesadee et al. 2005 ; li et al. 2008 ; drexler et al. 2009 drexler et al. , 2012 . most recently, a novel henipa-like virus, mojiang paramyxovirus (mojv), was identifi ed in rats ( rattus fl avipectus ) in china by nucleic acid analysis, with a genome length of 18,404 nt; however no virus isolate was obtained (wu et al. 2014 ) . also, serological and/or nucleic acid evidence of henipa-viruses in domestic livestock and in human populations have been reported providing evidence of sporadic henipavirus spillover events and also suggesting the existence of less pathogenic-related henipavirus. these fi ndings included henipavirus presence in domestic pigs in ghana, west africa; cattle, goats, and pigs in bangladesh; horse and humans in the philippines, and human populations in cameroon, africa (ching et al. 2015 ; pernet et al. 2014 ; chowdhury et al. 2014 ; hayman et al. 2011 ) . only the incident in the philippines was associated with a disease outbreak with evidence of horse-to-human and human-to-human transmission with niv as the likely cause (ching et al. 2015 ) . genomic sequence analysis revealed that hev isolates obtained from horses and a fatal human case in 1994 were essentially identical and both were highly similar to genomic sequences later obtained from p. poliocephalus and p. alecto 2 years after the initial outbreak (halpin et al. 2000 ; murray et al. 1995b ) . also, sequence analysis of fi ve hev isolates obtained from horses in australia; murwillumbah, in new south wales (2006) , and peachester (2007), clifton beach (2007), redlands (2008), and proserpine (2008) all in queensland, revealed identical genome lengths of 18,234 nt and sequence variation across the full genomes was <1 % (marsh et al. 2010 ) . similarly, in the initial malaysian outbreak of niv, both pig and human isolates were genetically similar to those obtained some years later from island fl yingfoxes ( p. hypomelanus ) (abubakar et al. 2004 ; chan et al. 2001 ; chua et al. 2002 ; harcourt et al. 2000 ) . however, a greater diversity among niv isolates is seen when comparisons are made between the malaysian isolates to the more recent niv isolates from other areas of southeast asia. the fi rst niv isolate from outside of malaysia came from bangladesh . characterization of the genome of niv-bangladesh revealed a length of 18,252 nt, 6 nt longer than the prototype niv-malaysian isolate, with a genome homology between them of 91.8 % . also, in that study, four niv-bangladesh isolates were examined showing a 99.1 % nt homology with interstrain nucleotide heterogeneity suggesting multiple spillovers of niv-bangladesh into people from varying bat sources. a third lineage of niv was isolated from lyle's fl ying fox ( p. lylei ) in cambodia and nucleocapsid (n) gene sequence analysis revealed this isolate to be more closely related to niv-malaysia than to niv-bangladesh (reynes et al. 2005 ; wacharapluesadee et al. 2010 ) whereas an analysis of nucleic acid sequences of niv derived from human sources from an outbreak in siliguri, india in 2001 revealed an isolate similar to niv-bangladesh (chadha et al. 2006 ) and a full niv genome amplifi ed from patient lung tissue from an outbreak in 2007 in west bengal, india showed 99.2 % nt with the niv-bangladesh isolate from 2004 (arankalle et al. 2011 ). more recently, partial genome sequence analysis of niv derived from an indian fl ying fox ( p. giganteus ) obtained from myanaguri, west bengal, india, revealed an n gene with 100.0 % homology with niv sequences from those prior outbreaks in india and with niv-bangladesh sequences, and a 96.0 % identity with niv isolates from cambodia and malaysia (yadav et al. 2012 ). in addition to the demonstration of at least three distinct virus isolate lineages of niv; malaysia, bangladesh and cambodia (wang et al. 2013b ), other nucleic acid based studies have signifi cantly expanded the genus henipavirus (drexler et al. 2012 ) . nineteen newly identifi ed virus species classifi ed into the genus henipavirus have been identifi ed, along with one full genome sequence , 18,530 nt, (gh-m74a) from a bat spleen ( eidolon helvum ) from ghana confi rmed classifi cation in the genus henipavirus (drexler et al. 2012 ) . cedpv is the third recognized species of henipavirus as a virus isolate . cedpv was isolated from pooled urine samples from a colony of predominantly p. alecto also with some p. poliocephalus . the cedpv genome is 18,162 nt and its organization was shown to be similar to that of hev and niv. also, some antigenic cross-reactivity of the cedpv n protein was noted with that of niv and hev; and cedpv was shown to utilized ephrin-b2 as entry receptor (discussed in the next section). henipavirus particles are enveloped and pleomorphic, with a size ranging from 40 to 1900 nm and can vary from spherical to fi lamentous forms when imaged by electron microscopy (hyatt et al. 2001 ; goldsmith et al. 2003 ; murray et al. 1995b ) . the viral envelope carries surface projections composed of the viral transmembraneanchored fusion (f) and attachment (g) glycoproteins (fig. 1 ). henipavirus genomes are unsegmented, single-stranded, negative-sense rna (wang et al. 2013b ). at the time of their discovery, the genomes of niv and hev were the largest amongst all members of the paramyxoviridae family, a factor considered in their classifi cation into their own genus, henipavirus (wang et al. 2000 ) . this increase in genome length is primarily attributable to additional nucleotides in 3′ untranslated regions of each transcription unit except the large/polymerase (l) gene (wang et al. 2000 harcourt et al. 2000 ) . as with all characterized members of the subfamily paramyxovirinae , the hev, niv and cedpv genomes and are divisible by six, conforming to the "rule of six" which relates to the way each n protein molecule interacts with every six nucleotides (lamb and parks 2013 ; wang et al. 2013b ). the rna genome in association with the n protein is also referred to as the ribonucleoprotein core that has a characteristic herringbone appearance by electron microscopy (wang et al. 2013b ) and is contained within a lipid bilayer (envelope) that is derived from the infected host cell during virus assembly and budding (fig. 1 ) . the relative gene order is conserved as compared to other paramyxoviruses, with the n gene being fi rst, followed by the p (phosphoprotein), m (matrix), f, g and l genes in a 3′ to 5′ order (fig. 1 ). gene transcription occurs in a gradient manner because of a failure of the rna polymerase to reinitiate transcription at downstream genes and those genes located towards the 3′ end are transcribed more abundantly than genes towards the 5′ (lamb and parks 2013 ) . the n, p, and l proteins form a complex that is responsible for replication of viral rna; polymerase activity resides within the l protein (lamb and parks 2013 ) . in addition to the full-length unedited p gene product, the henipavirus p gene (the largest among the paramyxoviruses) also encodes the v and w proteins which are produced through a transcriptional editing mechanism involving addition of nontemplated g nucleotides, as well as the c protein, which is encoded by an alternative start site within the p gene (lamb and parks 2013 ) . products of the p gene can antagonize both double-stranded (ds) rna signaling and interferon (ifn) signaling (reviewed in shaw 2009 ; basler 2012 ) . the v protein functions in anti-ifn induction or dsrna signaling, similar to that of other paramyxoviruses, by targeting the helicase encoded by the melanoma differentiationassociated gene 5 (mda5) . whereas the niv w protein could also inhibit dsrna signaling but does so by nuclear translocation, targeting interferon regulatory factor 3 (irf-3) and effectively blocking both dsrna signaling via mda5 and through the cell surface expressed toll-like receptor 3 (tlr-3) signaling pathway. henipaviruses also target the paracrine signal transduction pathway that is initiated by the binding of type i ifn to the two cell surface interferon alpha and beta receptors, ifnar1 and ifnar2 which assemble into a functional receptor complex leading to the activation of signal transducers and activators of transcription (stat) factors where they later direct the expression of genes possessing an interferon stimulated response element (isre) within the nuclease (reviewed in de weerd et al. 2007 ). the henipavirus v, w and p proteins block the type i ifn signaling pathway with the niv v and p proteins forming high-molecular weight complexes in the cytoplasm with stat1, and the niv w protein targeting stat1 within the nuclease (reviewed in detail (shaw 2009 ; basler 2012 ) ). in contrast, major difference between niv and hev with cedpv was noted in that the p gene lacks both rna editing and also the coding capacity for the v protein which may be a factor that limited its observed in vitro pathogenesis . the diverse ways that niv and hev can antagonize the host interferon responses are believed to be important factors that infl uence their pathogenic potential. the henipavirus m protein, which underlies the viral membrane ( fig. 1 ) , plays a key role in organization of viral proteins during the process of virion assembly and budding from the host cell, and the niv m protein possesses the ability to bud from expressing cells independent of any other viral proteins forming virus-like particles (ciancanelli and basler 2006 ; patch et al. 2007 ). sequence motifs with the m protein have been identifi ed that may act as traffi cking signals to facilitate the budding process (patch et al. 2008 ; ciancanelli and basler 2006 ; harrison et al. 2010 ). finally, the g and f envelope glycoproteins are located on the surface of the virion, appearing as spikes projecting from the envelope membrane of the viral particle ( fig. 1 ) and are essential for the binding and entry steps of the virus into permissive host cells (reviewed in bossart et al. 2013 ; steffen et al. 2012 ). the henipavirus g glycoprotein is a homo-tetramer and responsible for attachment of the virion to entry receptors on the host cell and the f glycoprotein is a homotrimer responsible for facilitating the fusion of the viral membrane with that of the host cell (reviewed in steffen et al. 2012 ) . additional details of the henipavirus envelope glycoproteins will be discussed below with regard to cellular tropism and as the targets of antiviral strategies. the exceptionally broad species tropism of henipaviruses, as represented by niv and hev, distinguishes them from all other known paramyxoviruses (wang et al. 2013b ). in addition to their principle natural hosts, pteropid bats, niv is known to have naturally infected pigs, horses, cats, dogs and humans, and experimental infections with disease in guinea pigs, cats, hamsters, ferrets, squirrel monkeys and african green monkeys have been demonstrated. in addition, niv can also productively infect chicken embryos with severe pathology (tanimura et al. 2006 ) . hev in nature appears less transmissible and naturally acquired infections have been observed only in bats, horses, dogs and humans; however, experimentally, hev can infect and cause disease in guinea pigs, cats, hamsters, ferrets, mice and african green monkeys (reviewed in geisbert et al. 2012 ) taken together, henipavirus infections seven orders (six mammalian and one avian). the henipavirus membrane anchored envelope glycoproteins (g and f) are the mediators of virus attachment and host cell infection and a major determinant of cellular tropism. the g glycoprotein is the henipavirus attachment glycoprotein and has neither hemagglutinating nor neuraminidase activities; activities associated with many other paramyxovirus attachment glycoproteins known as hemagglutinin-neuraminidase (hn) or the hemagglutinin (h) protein (wang et al. 2013b ; lamb and parks 2013 ) . the niv and hev g glycoprotein engage host cell membrane proteins as entry receptors and bind to ephrin-b2 and ephrin-b3 (negrete et al. 2005 (negrete et al. , 2006 bonaparte et al. 2005 ; bishop et al. 2007 ). the ephrin-b2 and -b3 molecules are members of a large family of cell surface expressed glycoprotein ligands that bind to eph receptors, the largest subgroup of receptor tyrosine kinases (drescher 2002 ; poliakov et al. 2004 ). the eph receptors and their ephrin ligands comprise an important group of bidirectional signaling molecules in a variety of cell-cell interactions including those of vascular endothelial cells and are modulators of cell remodeling events within the nervous, skeletal and vascular systems (pasquale 2010 ; lackmann and boyd 2008 ) . ephrin-b2 expression is prominent in arteries, arterioles and capillaries in multiple organs and tissues (gale et al. 2001 ) while ephrin-b3 is found predominantly in the nervous system and the vasculature (reviewed in poliakov et al. 2004 ; pasquale 2008 ) . the ephrin-b2 and -b3 molecules are highly sequence conserved across susceptible hosts including human, horse, pig, cat, dog, mouse and bat with amino acid identities of 95-96 % for ephrin-b2 and 95-98 % for ephrin-b3 . the identifi cation of ephrin-b2 as a major receptor for niv and hev has aided in the understanding and clarifi cation of both their broad species and tissue tropisms, as well as the resultant pathogenic processes that are seen in humans and animal hosts (reviewed in hooper et al. 2001 ; wong and ong 2011 ) . similar to most paramyxoviruses, the henipaviruses have two membraneanchored glycoproteins that are required for virus entry. the henipavirus attachment glycoprotein (g) is a type ii membrane protein with the amino (n)-terminus oriented towards the cytoplasm and the carboxy (c)-terminus extracellular . the g glycoprotein is comprised of a stem (or stalk) and a globular head domain which binds ephrin receptors. the native conformation of g is a tetramer, which is comprised of a dimer of dimers . the crystal structures of both niv and hev g globular head domains have been determined both alone and in complex with the ephrin-b2 and -b3 receptors, revealing the exact g-receptor interactions and identical receptor binding sites; with four binding pockets in g for the residues in the ephrin-b2 and -b3 g-h loop that are highly conserved (bowden et al. 2008a (bowden et al. , b , 2010 xu et al. 2008 xu et al. , 2012 . the second protein is the fusion (f) glycoprotein that facilitates the fusion of the viral and host cell membranes. f is a type i membrane glycoprotein with an extracellular n-terminus and is a class i viral fusion protein sharing several conserved features with other viral fusion glycoproteins . f is initially expressed as a precursor (f 0 ) which forms an oligomeric trimer that is cleaved into two disulfi de bond-linked subunits (f 1 and f 2 ) by the endosomal protease cathepsin l (pager and dutch 2005 ) . unique to the henipaviruses, the processing of f 0 into its biologically active form is a multi-step process requiring recycling of f 0 from the cell surface into an endosomal compartment, mediated by an endocytosis motif present in the cytoplasmic tail of f (meulendyke et al. 2005 ; vogt et al. 2005 ) . after cleavage, the homotrimer of disulfi de bond-linked f 1 and f 2 subunits is traffi cked back to the cell surface. the f glycoprotein contains two α-helical heptad repeat domains that are involved in the formation of a trimer-of-hairpins structure which facilitates membrane merger and peptides corresponding to either heptad repeat domains can inhibit the fusion activity of f when present during the fusion process (reviewed in bossart et al. 2013 ) . the henipavirus g and f glycoproteins work cooperatively to mediate membrane fusion and particle entry into the host cell. following virus attachment to a receptor-bearing host cell, the fusion-promoting activity of the g glycoprotein is initiated by engaging ephrin receptors and the g glycoprotein then facilitates the triggering of conformational changes in f, transitioning f conformation from a prefusion to post-fusion form driving the membrane fusion process between the virion and plasma membranes, resulting in delivery of the viral nucleocapsid into the cytoplasm (reviewed in aguilar and iorio 2012 ; lee and ataman 2011 ). in a related process, virus-infected cells expressing attachment and fusion glycoproteins on their surface can fuse with receptor-bearing cells leading to the formation of multinucleated giant cells (syncytia)-a hallmark of many paramyxovirus infections including the henipaviruses (wang et al. 2013b ) . the incubation period of human niv and hev infections ranges from a few days to about 3 weeks mahalingam et al. 2012 ) . to date, there have been only seven known cases of human hev infection, so much less is known about its clinical manifestations compared to niv infection. following an infl uenza-like illness (fever, myalgia, headaches, lethargy, vertigo, cough, pharyngitis, and cervical lympadenopathy), the majority developed severe disease and died; only two patients survived (mahalingam et al. 2012 ; selvey et al. 1995 ; playford et al. 2010 ). thus the mortality was about 60 %. three patients had an acute encephalitic syndrome characterized by drowsiness, confusion, ataxia, ptosis, dysarthria and seizures and died soon after. one patient had an acute pulmonary syndrome described as a pneumonitis with chest radiograph fi ndings of diffuse alveolar shadowing (selvey et al. 1995 ) . although clinical acute encephalitis was never suspected, apart from pulmonary pathology, this patient's brain at autopsy also showed features of acute encephalitis ). interestingly, abnormal chest radiographs were also described in two other clinical encephalitis cases. in one patient following relatively mild aseptic meningitis associated with headache, drowsiness, vomiting and neck stiffness, clinical features of probable meningoencephalitis , he presented 13 months later with full blown fatal encephalitis (o'sullivan et al. 1997 ) . in retrospect, this was the fi rst case of relapsing henipavirus encephalitis. the brain magnetic resonance (mr) scans available in three acute encephalitis patients showed multifocal hyperintensive lesions in the cerebrum and brainstem, and leptomeningeal enhancement . in the case of relapsing encephalitis, extensive, predominantly cortical hyperintense lesions were observed (mahalingam et al. 2012 ). based on a large cohort of 94 patients with niv infection from a single institution, the main features of acute infection was fever, headache, dizziness, and vomiting . a majority of patients had reduced consciousness levels and signs of brainstem dysfunction. other distinctive clinical signs included segmental myoclonus, arefl exia, hypotonia, hypertension, and tachycardia. the cerebrospinal fl uid obtained from lumbar puncture showed elevated leukocyte counts and protein levels. electroencephalogram abnormalities consisting of diffuse slow waves (continuous or intermittent) with or without focal sharp waves were observed, and in general correlated with disease severity. brain mr scans of acute niv infection were characterized by disseminated, multiple hyperintense lesions mainly in subcortical and deep white matter of the cerebrum with no associated edema or mass effect or correlation with severity of neurological signs. chest radiographs were reported to be abnormal in some patients paton et al. 1999 ) . the risk factors for severe disease and poor prognosis included abnormal doll's eye refl ex, tachycardia, and the presence of virus in the cerebrospinal fl uid (chua et al. 2000b ) , and diabetes mellitus (chong et al. 2001b ) . a small number, probably <10 %, of patients with acute niv infection developed a late-onset encephalitis (in symptomatic patients with no previous encephalitis or patients with asymptomatic seroconversion) or a relapsing encephalitis (in patients with previous encephalitis) a few weeks later. although potentially fatal, the mortality at about 18 % is considerably lower that acute encephalitis . the clinical features of late-onset encephalitis and relapsing encephalitis are similar to acute encephalitis. however, some features like fever, coma, brainstem signs, segmental myoclonus and meningism were less commonly observed, while seizures and focal cortical signs were more frequent. cerebrospinal fl uid pleocytosis was common but no virus could be isolated. the brain mr scans showed confl uent geographical abnormalities, especially in the cortical gray matter that is strikingly different from acute niv encephalitis . although most niv-infected human patients presented with acute encephalitis, some 25 % of patients also presented with respiratory signs, some cases also presented as a non-encephalitic or asymptomatic infection with seroconversion (chua 2003 ) . niv infection could also take a chronic and quiescent course with neurological disease occurring later (>10 weeks) following a non-encephalitic or asymptomatic infection. a recrudescence of neurological disease, also termed relapsing encephalitis, was also observed in some patients who had previously recovered from an acute encephalitic infection. here, there is a recrudescence of virus replication in the cns. most reported cases of relapsed encephalitis presented from a few months to approximately 2 years following the initial acute infection, however two cases of relapsed encephalitis were observed in 2003 4 years later (wong et al. 2001 ; chong and tan 2003 ; tan and wong 2003 ) and the longest reported case of niv encephalitic recrudescence is 11 years (abdullah et al. 2012 ) . this recrudescence of henipavirus encephalitis was fi rst noted in the second fatal human case of hev infection which presented with similar fi ndings (o'sullivan et al. 1997 ; wong et al. 2009 ). interestingly, evidence of recrudescence of niv infection in pteropus bats has also been reported (sohayati et al. 2011 ) as well as hev infection modeling in fl ying-fox populations (wang et al. 2013a ). there is no evidence of hev shedding in people who have recovered from infection (taylor et al. 2012 ) . persistent neurological defi cits have been observed in >15 % of niv infection survivors ). in addition, recent studies have also assessed the long-term neurologic and functional outcomes of >20 individuals surviving symptomatic niv infection in bangladesh (sejvar et al. 2007 ). in bangladesh, the outcomes among 22 of 45 serologically confi rmed cases of niv infection revealed neurological sequelae in survivors, and patients who initially had encephalitis could continue to exhibit neurological dysfunction for several years (sejvar et al. 2007 ). both persistent and delayed-onset neurological sequelae were noted, including a higher proportion of persistent behavioral disturbances including violent outbursts and increased irritability among pediatric patients (sejvar et al. 2007 ). viral persistence and/or recrudescence within the cns are suspected to be at play in these individuals . the mechanisms that allow niv and hev to escape immunological clearance for such an extended period and later result in disease are unknown, and this characteristic of niv and hev has important implications for therapeutics development. hev spillovers in australia have occurred annually since 2006 and to date there have been seven human cases of which four have been fatal (playford et al. 2010 ) . all human cases of hev infection was the result of exposure and transmission of the virus from infected horses to humans. the fi rst human case presented as an acute severe respiratory disease but no clinical evidence of acute encephalitis. at autopsy, the lungs showed macroscopic evidence of congestion, hemorrhage and edema (selvey et al. 1995 ) associated with focal necrotizing alveolitis and evidence of syncytia and multinucleated giant cell formation, and viral inclusions. focal vasculitis was also noted in some pulmonary vessels. viral antigens were localized by immunostaining to alveolar type ii pneumocytes , intra-alveolar macrophages and blood vessels ). although clinical encephalitis was apparently absent, the brain pathology clearly showed acute encephalitis characterized by mild meningitis, parenchymal and perivascular infl ammation . more importantly, there was evidence of neuronal viral inclusions, vasculitis and necrotic/vacuolar plaques. viral antigens/rna were demonstrated in blood vessels, neurons (fig. 2d ) , and ependyma. mild infl ammation could also be found in the lymph node and kidney where viral antigens were detected in glomeruli and renal tubules. panels ( a , b , d , f ) from wong and ong ( 2011 ) , panels ( c , e ) from wong et al. ( 2002 ) a second fatality occurred in an individual who fi rst experienced an aseptic meningitic illness associated with drowsiness caused by hev infection acquired after assisting at the necropsies of two horses that were only later shown to have died from hev infection. approximately 13 months later this individual suffered a recurrence of severe encephalitis characterized by uncontrolled focal and generalized epileptic activity. infl ammatory lesions were only found in the cns, not in other organs obtained at ). extensive lesions were found mainly in the meninges and cerebral cortex, but focal lesions were also found in the cerebellum, pons and spinal cord. there was intense infi ltration of the parenchyma and perivascular areas by macrophages, lymphocytes, and plasma cells together with severe neuronal loss, reactive glial, and vascular proliferation. although viral inclusions were not prominent, viral antigens/rna were detected in neurons, glial, and/or infl ammatory cells. interestingly, there was no evidence of vasculitis or endothelial syncytia in the cns, as well as absence of these and other features of infl ammation in all the non-cns organs examined. in the fi rst niv outbreak in malaysia and singapore, autopsies were conducted on >30 individuals which has afforded a better understanding of the pathology of niv in comparison to that of hev infection. these autopsies were mostly in individuals, including pig farm workers and farmers, who in one way or another had contact with sick pigs. the macroscopic features were generally non-specifi c. perhaps the most distinctive microscopic feature is the disseminated vasculitis found in most organs examined, particularly in the cns and lungs. the fully developed, typical vasculitic lesion comprised focal segmental infl ammation of the vascular wall, endothelial ulceration and thrombosis (fig. 2a ) . the rare endothelial multinucleated syncytia may occasionally be found in early vasculitis (fig. 2b ) . viral antigens and nucleocapsids can be demonstrated in blood vessels. extravascular necrotic lesions and infl ammation in many organs can also be seen. in the cns parenchyma, distinct necrotic plaques (fig. 2e ) arising from vasculitis-induced vascular obstruction, ischemia and infarction and/or neuronal infection were commonly found. neurons in or around necrotic plaques and other infl amed neuronal areas often showed the widespread presence of viral antigens (fig. 2c ) . glial cells were much more rarely involved. viral inclusions in neurons in the cns and other cells in non-cns tissues were also observed. apart from vasculitis, infl ammation, necrosis, and the rare multinucleated giant cells or syncytia involving extravascular tissue in the lung, spleen, lymph node, and kidney (fig. 2f ) , were reported hooper et al. 2001 ; wong 2010 ) . the combination of disseminated, vasculitis-induced thrombosis, vascular occlusion, and microinfarction, together with direct infection of parenchymal cells suggest a unique dual pathogenetic mechanism for tissue injury in acute niv infection. this appears to hold true for acute hev infection as well. certainly in the cns, extensive virus-associated vasculopathy, with or without neuroglial infection, as a signifi cant cause of tissue injury is probably unique. the pathological features in the few autopsy cases of niv relapsing or late-onset encephalitis and the single case of hev relapsing encephalitis were similar and confi ned mainly to the cns (wong and tan 2012 ; tan et al. 2002 ) . there was extensive and severe meningoencephalitis with parenchymal and perivascular infl ammation, severe neuronal loss and reactive gliosis. viral inclusions , antigens/ rna could be detected but vasculitis were absent (wong 2010 ) . indeed, vasculitis or other vasculopathies which were readily found in the acute infection, were absent in the cns and extra-cns organs. in addition to hev and niv infection of bats (middleton and weingartl 2012 ) , detailed reviews of the disease manifestations observed in natural and experimental infections of animals with hev and niv have recently been reported (dhondt and horvat 2013 ; geisbert et al. 2012 ; wong and ong 2011 ) . as mentioned previously, natural hev infections have almost exclusively been observed in horses, and only recently have two dogs been reported hev antibody positive. whereas in addition to pigs, naturally acquired niv infection was noted in dogs, cats and horses in the initial malaysian outbreak . serological studies of natural niv infection revealed that dogs in areas associated with farms in the malaysian outbreak were susceptible to infection ). however, diseased dogs were not prevalent with only two animals examined (one dead and one sick) wong and ong 2011 ) . in bangladesh, a few cases of human niv infection were associated with sick animal contact including cows (hsu et al. 2004 ) , pigs, and goats , and recently serological evidence of henipavirus infection in cattle, goats and pigs in bangladesh has been reported (chowdhury et al. 2014 ). the development of animal models of henipavirus infection and pathogenesis has been critical for understanding henipavirus pathogenesis and also needed for the evaluation of potential vaccines and therapeutics. several well-established animal models of hev and niv infection and pathogenesis have been developed and include the guinea pig (williamson et al. 2000 ; 2001 #3773; middleton et al. 2007 ), hamster (guillaume et al. 2009 ; wong et al. 2003 ) , cat middleton et al. 2002 ; williamson et al. 1998 ) , pig (li et al. 2010 ; weingartl et al. 2005 ; middleton et al. 2002 ) , ferret (pallister et al. 2011 ; bossart et al. 2009 ), african green monkey (agm) geisbert et al. 2010 ) , squirrel monkey (marianneau et al. 2010 ) and horse (marsh et al. 2011 ). among these models, the pathogenic processes of henipavirus infection in the hamster, ferret and agm best represent the pathogenesis observed in humans; whereas the most appropriate models for livestock are the pig and horse. the syrian golden hamster and niv challenge was the fi rst successful small animal model of henipavirus infection and pathogenesis . niv infection in the hamster produced severe lesions in the brain, with animals succumbing to infection 5-9 days after intraperitoneal infection, 24 h following the development of tremors and limb paralysis. hamsters inoculated intranasally survived ~5 days longer post-challenge, displaying progressive neurological signs and breathing difficulties. vascular pathology was widespread, involving the brain and lung, with endothelial cell infection. the vascular and parenchyma lesions were consistent with cns-mediated clinical signs . another study showed that higher doses of niv resulted in an acute respiratory distress syndrome (ards) while lower doses would yield the development of neurological signs and more widespread infection throughout the endothelium . hev infection of hamsters also produces both respiratory and brain pathology, with endothelial infection and vasculitis, and direct parenchymal cell infection in the cns (guillaume et al. 2009 ). similar to niv infection in hamsters, higher doses of hev resulted in ards and lower doses produced a more neuropathogenic syndrome ). niv infection of ferrets produces both a severe respiratory and neurological disease along with systemic vasculitis following oral-nasal challenge by 6-10 days postinfection (bossart et al. 2009 ; pallister et al. 2009 ). clinical signs in infected ferrets included severe depression, serous nasal discharge, cough and shortness of breath, and tremor and hind limb paresis. pathological fi ndings included vascular fi brinoid necrosis in multiple organs, necrotizing alveolitis, and syncytia of endothelium and alveolar epithelium. severe focal necrotizing alveolitis vasculitis and focal necrosis in a wide range of tissues was observed along with signifi cant levels of viral antigen in blood vessel walls. niv antigen was present within the brain along with infected neurons, and virus isolation from the brain and other organs was reported. hev challenged ferrets, also by the oral-nasal route, rapidly progressed with severe disease 6-9 days following infection with essentially identical fi ndings as seen in nivchallenged ferrets (pallister et al. 2011 ) . the henipavirus disease processes in the ferret accurately refl ects those reported in niv-infected humans and the ferret model has been used in the evaluation of vaccines and therapeutics against henipavirus infections. the fi rst successful nonhuman primate models for both niv and hev infection were developed using the african green monkey (agm) rockx et al. 2010 ) . both niv and hev will produce a uniformly lethal disease process following low dose virus challenge by intratracheal inoculation within 7-10 days post-infection. hev and niv spread rapidly to numerous organ systems within the fi rst 3-4 days following challenge. monkeys begin to develop a progressive and severe respiratory disease ~7 days post-infection rockx et al. 2010 ) . the lungs become enlarged and with high levels of virus replication, congestion, hemorrhage, and polymerized fi brin. widespread vasculitis with endothelial and smooth muscle cell syncytia with viral antigen, along with viral genome was detected in most organs and tissues along with associated pathology. monkeys infected with either niv or hev also exhibit neurological disease signs with the presence of meningeal hemorrhaging and edema, and vascular and parenchymal lesions in the brain including infection of neurons with in the brainstem particularly involved (fig. 3 ) rockx et al. 2010 ). rockx et al. ( 2010 ) the squirrel monkey was also found to be susceptible to experimental niv infection via intravenous and intranasal routes demonstrating fi ndings similar to agm and human infection (marianneau et al. 2010 ) . vasculopathy and parenchymal cell infection were found in the cns, lungs and other organs. niv infection of pigs revealed the respiratory system as a major site of virus replication and pathology, with viral antigen and syncytia formation present in the respiratory epithelium (tracheal, bronchial, bronchiolar, and alveolar) and small blood and lymphatic vessels (middleton et al. 2002 ; hooper et al. 2001 ; wong and ong 2011 ) . virus was also observed in the kidneys and in endothelial and smooth muscle cells of small blood vessels (middleton et al. 2002 ) . cns involvement was less common, with meningitis or meningoencephalitis observed as opposed to encephalitis (middleton et al. 2002 ) . niv infection of piglets generally resulted in a mild clinical disease with fever and respiratory signs and virus replication noted in the respiratory system, lymphoid tissues and the cns (weingartl et al. 2005 ) . recoverable virus was recorded in the respiratory, lymphatic and nervous systems, and virus shedding in nasal , pharyngeal, and ocular fl uids was reported. hev infection of pigs also presents as a primarily respiratory disease in both landrace piglets and older gottingen minipigs, with possible cns involvement observed in minipigs, and similar patterns of virus shedding (li et al. 2010 ) . overall, hev appeared to cause a more severe respiratory syndrome in pigs in comparison to niv. although hev and niv disease in pigs is often less severe in comparison to other animal models, the virus does replicate and disseminate to a variety of organs along with signifi cant levels of virus shedding. natural hev infection in horses is often associated with severe disease and experimental infections are essentially uniformly fatal (marsh et al. 2011 ) . naturally infected horses appear to have an incubation period of ~8-11 days and animals initially present as anorexic and depressed with general uneasiness and ataxia, with the development of fever and sweating. respiration becomes rapid, shallow and labored with pulmonary edema and congestion, along with nasal discharge 1-3 days following the onset of clinical signs. in severe cases the airways of horses are often fi lled with a blood-tinged frothy exudate. there was hemorrhage, thrombosis of capillaries, necrosis, and syncytial cells in the endothelium of pulmonary vessels noted. viral antigen was also observed within endothelial cells across a wide variety of organs, with recoverable virus from a number of internal organs as well as from saliva and urine. neurologic clinical signs can also present (rogers et al. 1996 ) . however, in experimentally infected horses, only meningitis (with vasculitis) was noted in all animals (marsh et al. 2011 ) and viral antigen was detected in the meninges of each case. one horse in this study also presented with vasculitis of blood vessels in the brain parenchyma, and hev antigen was also identifi ed within the cerebral blood vessels of this animal (fig. 4 ) (deborah middleton, personal communication) . also, an experimental control horse in middleton et al. ( 2014 ) also had vasculitis with hev antigen in blood vessels within the brain. however, to date hev antigen has not been reported to be present in the neurons of infected horses, but this may be a sampling artefact and/or an observation exacerbated by the fact that the horses are being euthanized and the hev infection is not reaching its full pathogenic expression under experimental conditions. however, the meningitis and infl ammation of cerebral blood vessels in the experimentally infected horses may be suffi cient explanation for the clinical signs of neurological disease in naturally acquired cases of hev infection (deborah middleton, personal communication) . experimental infection of horses with niv has not been performed but the brain and spinal cord of one naturally infected horse was examined and immunohistochemical staining of viral antigen observed revealing non-suppurative meningitis . an array of viruses across many families are known to exhibit neurotropism and there are two central routes of cns invasion; hematogenous spread or via infection of nerve cells (swanson and mcgavern 2015 ; koyuncu et al. 2013 ) . many viruses that cause viremia following the establishment of an initial infection have an opportunity to breach the blood-brain-barrier (bbb); a highly selectively permeable barrier that separates the cns from the peripheral blood circulation (ransohoff et al. 2003 ) . once in the blood, a number of viruses including some herpesviruses, paramyxoviruses, retroviruses, picornaviruses, fi loviruses, and fl aviviruses can directly infect vascular endothelial cells (koyuncu et al. 2013 ) which could allow passage of virus into the cns and/or promote infl ammation and breakdown of the bbb which may also facilitate virus access to the cns (obermeier et al. 2013 ) . alternatively, some viruses can infect myeloid and lymphoid cells and these infected cells can naturally traverse the bbb delivering virus into the cns by the "trojan horse" mechanism (mcgavern and kang 2011 ). a number of neurovirulent paramyxoviruses, particularly the morbilliviruses like measles virus and canine distemper virus, but also mumps virus and newcastle disease virus, can productively infect lymphocytes (joseph et al. 1975 ; krakowka et al. 1975 ; fleischer and kreth 1982 ; hao and lam 1987 ) (see also chap. 2). these infected lymphocytes serve as a cell-associated viremia which can then lead to the delivery of virus into the cns by transmigration through bbb (lossinsky and shivers 2004 ) . cns invasion by niv and hev is a key feature of their pathogenic features in humans and as discussed earlier several animal models have also demonstrated niv and hev cns disease. the widespread and disseminated endothelial infection and vasculitis in henipavirus encephalitis strongly suggest that bbb disruption is an important, if not the most important route, for viral entry into the cns. plaque-like, groups of infected neurons were frequently observed near to infected/vasculitic vessels suggesting centrifugal viral spread from focal bbb damage. however, although niv was shown not to infect human lymphocytes and only low levels of monocyte infection have been reported, human lymphocytes could bind niv and facilitate its transfer and infection to other susceptible cells (mathieu et al. 2011 ) . the traffi cking of such cell-associated infectious niv within a host disseminates the virus and also could potentially deliver niv into cns by leukocyte transmigration . in pigs, however, niv infection of cd6+ cd8+ t lymphocyte has been observed, along with monocytes and nk cells (stachowiak and weingartl 2012 ) . cd6 is a costimulatory molecule involved in lymphocyte activation and differentiation (gimferrer et al. 2004 ) which engages activated leukocyte cell adhesion molecule (alcam/cd166) which is known to promote leukocyte migration across the bbb (cayrol et al. 2008 ) . in this instance, it was suggested that nivinfected cd6+ t cells would elaborate a strong interaction alcam expressed on microvascular endothelial cells which could determine the observed tropism of niv for small blood vessels and also facilitate cns invasion by leukocyte migration. similar studies have not been reported with hev. alternatively, some neurotropic viruses can invade the cns via infection of peripheral nerves (swanson and mcgavern 2015 ) . for example, some neurotropic viruses begin the infection process in one cell type or tissue such as the oropharyngeal and intestinal mucosa in case of poliovirus (see also chap. 1) or in myocytes at the bite site in the case of rabies virus (see also chap. 4) and both later use peripheral motor neurons and retrograde transport to infect the cns (koyuncu et al. 2013 ). in the case of some herpesviruses, initial infection of sensory neurons is followed by retrograde transport and establishment of latency in the peripheral nervous system, and fortunately anterograde transport of herpesviruses to the cns is rare (koyuncu et al. 2013 ) (see also chap. 18). olfactory receptor neurons provide a unique opportunity for neurotropic pathogens to invade the cns because of the direct exposure of dendrites to the environment within the olfactory epithelium, and a few members of several virus families, including fl aviviruses, togaviruses, and bunyaviruses are known to invade the cns via an initial infection of olfactory receptor neurons within the olfactory epithelium and once infected virus can gain access to the cns by transported anterograde transport (mori et al. 2005 ; koyuncu et al. 2013 ) . certain paramyxoviruses have also been shown capable of neuroinvasion via anterograde transport following infection of olfactory neurons (rudd et al. 2006 ; ramirez-herrera et al. 1997 ) . niv infection in pigs is often asymptomatic as discussed above. when disease was noted in naturally infected pigs, neurological disease manifested as trembling, twitches, muscle spasms, and uncoordinated gait (mohd nor et al. 2000 ) . experimental niv infection challenge of landrace female piglets by the ocular and oronasal routes revealed that virus replication occurs in the oropharnyx and then spreads sequentially to the upper respiratory tract and submandibular lymph nodes, followed by replication in the lower respiratory tract, and additional lymphoid tissues, and niv was detected in the nervous system of both sick and apparently healthy animals; including cranial nerves, trigeminal ganglion, brain, and cerebrospinal fl uid. niv invaded the cns via cranial nerves, most importantly via the olfactory nerve, as early as 3 dpi, as well as by crossing the bbb (weingartl et al. 2005 ) . one report of hev infection of landrace and gottingen minipig breeds by oronasal or nasal inoculations produced clinical signs that were primarily respiratory with suggestive neurological involvement seen only in the gottingen minipig. an aged mouse model of intranasal challenge with hev revealed that animals could consistently develop encephalitic disease, and an anterograde route of neuroinvasion of the cns via olfactory nerves was proposed (dups et al. 2012 ) , however in a follow-up study using the same model with niv-bangladesh and niv-malaysia, animals did not exhibit cns disease (dups et al. 2014 ) . as was discussed earlier, in the hamster model for both niv and hev challenge, lower doses of virus allowed for a more neuropathogenic disease state. in an elegant spatial-temporal model of niv infection in the hamster by intranasal inoculation (10 5 tcid 50 ), individual niv-infected neurons were observed extending from the olfactory bulb by 4 dpi, demonstrating direct evidence for virus transport in the cns via olfactory neurons (munster et al. 2012 ) (fig. 5 ) . at 6 dpi, meningoencephalitis was observed, characterized by multifocal men-ingeal and perivascular lymphocytic infi ltration, and in the olfactory bulb neurons and axons of the olfactory nerve layer, glomerular layer and external plexiform layer of the olfactory bulb were positive by niv antigen staining. niv dissemination from the olfactory bulb to the olfactory tubercle region was noted by 6 dpi. from olfactory tubercle region, which is highly innervated to other brain regions including the hypothalamus, thalamus, amygdala, hippocampus and brain stem, spread of niv within the cns is readily possible. similarly, in oronasal challenge models of both niv and hev in the ferret (pallister et al. 2011 ; bossart et al. 2009 ), henipavirus genome and viral antigen were consistently detected in the olfactory lobe of brains along with many animals demonstrating neurological disease such as tremors and hind limb weakness or paralysis. finally, in the agm nonhuman model of niv and hev infection described earlier, consistent neurological disease was observed even though an intratracheal route of challenge is performed, with those animals surviving longer, or those challenged with lower doses of virus, showing more severe neurological disease with signs such as tremors, paralysis and convulsions geisbert et al. 2010 ) (geisbert and broder unpublished) . however, in human niv autopsy studies, involvement of the olfactory bulb has not been demonstrated so far . presently, there are no approved therapeutics for treating hev or niv infection in people, but there have been a few approaches tested in animal models (reviewed in broder 2012 ) . ribavirin is often a fi rst line treatment course for suspected viral infections of unknown etiology, having antiviral activity against many rna and some dna viruses (sidwell et al. 1972 ) and is an accepted treatment against several viruses including respiratory syncytial virus and arenaviral hemorrhagic fevers (reviewed in snell 2001 ) . during the initial niv outbreak in malaysia, some patients were treated with ribavirin and there was some evidence that this therapy may have been clinically benefi cial (chong et al. 2001a ; snell 2004 ) . of the recorded human hev cases , three individuals were treated with ribavirin, and of these, two succumbed to disease and one survived (playford et al. 2010 ) . chloroquine, an antimalarial drug, was shown to block the critical proteolytic processing needed for the maturation and function of the hev f glycoprotein discussed earlier (pager et al. 2004 ) and could block infection in cell culture (porotto et al. 2009 ). however, chloroquine and ribavirin treatment of a hev-infected individual had no clinical benefi t (reviewed in broder et al. 2013 ) . animal studies have also revealed no therapeutic benefi t of either chloroquine or ribavirin. two studies in hamsters and one study in monkeys showed that ribavirin treatment only delayed death after virus infection (freiberg et al. 2010 ; georges-courbot et al. 2006 ; rockx et al. 2010 ) , with hev challenge monkeys treated with ribavirin having marked increases of neurological symptoms. chloroquine treatment was also unable to prevent niv disease in ferrets (pallister et al. 2009 ). also, various forms of poly(i:c) are strong inducers of ifn-α and -β production, have been explored as antiviral therapies for over 40 years. polyic 12 u is very specifi c in triggering the toll-like receptor (tlr)3 pathway (reviewed in nicodemus and berek 2010 ). polyic 12 u was shown capable of blocking niv replication, and continuous administration of polyic 12 u for 10 days beginning at the time of challenge was shown to prevent lethal niv disease in fi ve of six hamsters (georges-courbot et al. 2006 ) , suggesting that use of tlr3 agonists such as polyic 12 u, perhaps in combination with other antiviral strategies, should be explored. but for hev and niv, the development of new therapeutics and vaccines has primarily focused on targeting the attachment and infection stages mediated by the viral f and g glycoproteins. as discussed earlier, peptides, typically 30-40 residues in length that are homologous to either of the heptad repeat domains of several paramyxovirus f glycoproteins, including the henipaviruses, can potently inhibit membrane fusion by blocking the formation of the trimer-of-hairpins structure (reviewed in bossart et al. 2013 ) . the fi rst henipavirus-specifi c peptide fusion inhibitor was a 36 amino acid heptad repeat-2 sequence (niv-fc2) (bossart et al. 2001 ) analogous to the approved hiv-1 specifi c therapeutic peptide enfuvirtide (fuzeon™) . other studies showed that a heptad repeat-2 peptide from human parainfl uenza virus type-3 (hpiv3) f blocked hev mediated fusion (porotto et al. 2006 ) and a sequence-optimized and cholesterol-tagged hpiv3-based heptad repeat-2 peptide appeared effective in the niv hamster (porotto et al. 2010 ). this cholesterol-tagged antiviral peptide could also penetrate the cns and exhibit some effective therapeutic activity against niv. additional in vivo effi cacy testing of peptide fusion inhibitors as henipavirus therapeutics merits further investigation. almost without exception all virus-neutralizing antibodies to enveloped viruses are directed against the viral envelope glycoproteins on the surface of the virion particle. initial passive immunization studies were conducted in the hamster nivchallenge model and showed that antibody immunotherapy against henipavirus infection by targeting the viral envelope glycoproteins was possible. protective passive immunotherapy using either niv g and f-specifi c polyclonal antiserums, or mouse monoclonal antibodies (mabs) specifi c for the henipavirus g or f glycoproteins has been shown (guillaume et al. 2004 (guillaume et al. , 2006 (guillaume et al. , 2009 . these studies demonstrated a major role of viral glycoprotein specifi c antibody in protection from henipavirus-mediated disease (reviewed in broder et al. 2012 ). using recombinant antibody technology, henipavirus-neutralizing human mabs reactive to the g glycoprotein were previously isolated (zhu et al. 2006 ) . one mab, m102, possessed strong cross-reactive neutralizing activity against hev and niv and was affi nity maturated (m102.4) and converted to an igg1 format and produced in a cho-k1 cell line ). the m102.4 mab epitope maps to the receptor binding site of g and engages g in a similar fashion as the ephrin receptors ). the m102.4 mab can neutralize niv-malaysia, hev-1994, hev-redlands and niv-bangladesh isolates (bossart et al. 2009 ). in a post-exposure niv-challenge experiment in the ferret model, a single dose of mab m102.4 administered by intravenous infusion 10 h after lethal challenge could prevent lethal infection (bossart et al. 2009 ). the therapeutic effi cacy of mab m102.4 has also been examined in monkeys against both niv and hev challenge with a study design refl ecting a potential real life scenario that would require a post-exposure treatment geisbert et al. 2014 ) . in one study, animals were challenged intratracheally with hev and later infused twice with m102.4 (~15 mg/kg) beginning at 10, 24, or 72 h post-infection followed by a second infusion ~48 h later. all subjects became infected following challenge, and all animals that received m102.4 survived whereas all control subjects succumbed to severe systemic disease by day 8. animals in a 72 h treatment group did exhibit neurological signs but all recovered by day 16, but there was no evidence of hev-specifi c pathology in any of the m102.4-treated animals, and no infectious hev could be recovered from any tissues from any m102.4-treated subjects. a follow-up study evaluated the effi cacy of m102.4 against niv disease in the agm model at several time points after virus exposure by intratracheal challenge, including at the onset of clinical illness ). here, subjects were infused twice with m102.4 (15 mg/kg) beginning at either 1, 3, or 5 days after virus challenge and again 2 days later. all subjects became infected after challenge and all subjects that received m102.4 therapy survived infection, whereas the untreated control subjects succumbed to disease between days 8 and 10 after infection. animals in the day 5 treatment group exhibited clinical signs of disease, but all recovered by day 16. together, these studies revealed that mab m102.4 could prevent widespread henipavirus dissemination in challenged subjects, and were the fi rst successful post-exposure in vivo therapies against hev and niv in nonhuman primates. a variety of active immunization strategies for henipavirus have been examined using recombinant virus platforms, protein subunit, virus-like particles and dna vaccines. several of these strategies have only been examined in terms of their ability to generate a henipavirus-specifi c neutralizing response (kong et al. 2012 ; kurup et al. 2015 ; wang et al. 2006 ; walpita et al. 2011 ) , whereas other studies examined immune response and effi cacy in animal challenge models. the fi rst report used the hamster model and the attenuated vaccinia virus strain nyvac, using recombinant viruses encoding either the niv f or g, both individually and in combination to immunize animals, and the study revealed that complete protection from nivmediated disease was achievable and that an immune response to the viral envelope glycoproteins can be important in protection (guillaume et al. 2004 ) . another poxvirus-based vaccine was examined as a potential livestock vaccine using recombinant canarypox virus in pigs (weingartl et al. 2006 ) . here, the niv f and g glycoprotein genes were used to generate recombinant canarypox viruses (alvac) vaccine vectors and used to immunize pigs. alvac vectors expressing f and g were tested alone and in combination, and piglets were challenged intranasally with niv. here, protection from niv-mediated disease was seen in all vaccinated pigs by either alvac vector alone or in combination and that vaccinated animals shed only low levels of nucleic acid detectable virus with no isolatable virus (weingartl et al. 2006 ) . more recently, several viral vector-based henipavirus vaccines have also been examined in animal challenge studies; these have included immunizations using the vesicular stomatitis virus based platform (vsv) expressing either the niv g or f glycoprotein in the hamster model (debuysscher et al. 2014 ; lo et al. 2014 ) and also vsv-based vaccines using niv f or g in the ferret model (mire et al. 2013 ) . all these studies demonstrated that a single dose of vaccine could induced strong neutralizing antibody responses and could afford protection from niv challenge, highlighting their potential usefulness as either a livestock vaccine or one suitable in an emergency use or outbreak scenario . vaccination and challenge experiments have also been examined using an adeno-associated virus platform with niv g showing protection against challenge in the hamster model and low level crossprotection (three of six animals) against a hev challenge (ploquin et al. 2013 ) , and also a recombinant measles virus vector with niv g which showed two of two agms were protected from niv challenge (yoneda et al. 2013 ) . a protein subunit vaccine strategy for henipaviruses has been extensively examined because of the inherent safety of such an approach. soluble, secreted, oligomeric forms of the g glycoprotein (sg) from both niv and hev were developed . the hev-sg glycoprotein is a secreted version of the molecule with a genetically deleted transmembrane and cytoplasmic tail that is produced in mammalian cell culture systems and is properly n-linked glycosylated (colgrave et al. 2011 ) . hev-sg retains many native characteristics including oligomerization and ability to bind ephrin receptors (bonaparte et al. 2005 ) , and it elicits potent cross-reactive neutralizing (hev and niv) antibody responses in a variety of animals including mice, rabbits, cats, ferrets, monkeys and horses. studies using the hev-sg subunit immunogen in the cat model demonstrated that it could elicit a completely protective immune response against a lethal subcutaneous niv challenge showing that a single vaccine (hev-sg) could be effective against both hev and niv. further studies in the cat model demonstrated that pre-challenge virus-neutralizing antibody titers as low as 1:32 were completely protective from a high-dose oronasal challenge of niv (50,000tcid 50 ) ). hev-sg immunization studies in the ferret model using either 100, 20 or 4 μg doses of hev-sg formulated in cpg and allhydrogel tm could all afford complete protection from a 5000 tcid 50 dose of hev (100 times the minimal lethal dose) with no disease or evidence of virus or viral genome in any tissues or body fl uids in the 100 and 20 μg vaccine groups; and only a low level of viral genome detected in the nasal washes from one of four animals in the 4 μg vaccine group. no infectious virus could be recovered from any vaccinated ferrets. the hev-sg subunit vaccine has also been evaluated in nonhuman primates (agms). in one study, doses of 10, 50, or 100 μg of hev-sg were mixed with allhydrogel ™ and cpg and vaccine was given to three subjects in each dosing group twice, 3 weeks apart, and subjects were challenged by intratracheal administration with a tenfold lethal dose of niv (1 × 10 5 tcid 50 ) 21 days later. complete protection was observed in all vaccinated subjects. some subjects had pre-challenge niv neutralizing titers as low as 1:28. no evidence of clinical disease, virus replication, or pathology was observed. a second study examined hev-sg vaccination and protection from hev challenge in agms, and also evaluated the hev-sg subunit (100 μg doses) in allhydrogel ™ and cpg as well as formulated with only allhydrogel ™ . subjects were vaccinated twice, 3 weeks apart, and were challenged intratracheally with a tenfold lethal dose of hev (∼5 × 10 5 plaqueforming units) 21 days after the boost vaccination. none of the eight vaccinated animals showed any evidence of clinical illness, virus replication, or pathology. the study also clearly demonstrated that hev-sg-allhydrogel ™ alone is capable of providing complete protection from a hev challenge providing crucial data for supporting preclinical development as a henipavirus vaccine for use in people. the simplicity and inherent safety of the hev-sg subunit vaccine approach together with the numerous successful vaccination and challenge studies that have been carried out in multiple animal models, the hev-sg subunit vaccine was chosen for the development of an equine vaccine to prevent infection in horses and also reduce the risk of hev transmission to people. hev-sg was licensed by zoetis, inc. (formerly pfi zer animal health) and developed as an equine vaccine for use in australia. horse hev-sg vaccination and hev challenge studies were conducted in australia the bsl-4 facilities of the australian animal health laboratories (aahl) in geelong, australia . here, hev-sg was formulated in a proprietary adjuvant (zoetis, inc.) and in two initial effi cacy studies in horses, either a 50 or 100 μg dose of the same sourced hev-sg which was used in all the animal challenge studies described earlier. two additional studies used 100 μg hev-sg produced from clarifi ed cho cell culture supernatant ( zoetis, inc. ) that was then gamma irradiated. immunizations were two 1-ml doses administered intramuscularly 3 weeks apart. horses in the effi cacy studies were exposed oronasally to 2 × 10 6 tcid 50 of hev. seven horses were challenged 28 days, and three horses were challenged 194 days, after the second vaccination. all vaccinated horses remained clinically healthy after challenge showing protection with hev neutralizing titers as low as 1:16 or 1:32 pre-challenge. at study completion, there was no gross or histologic evidence of hev infection in vaccinated horses; all tissues examined were negative for viral antigen by immunohistochemistry; and viral genome was not recovered from any tissue, including nasal turbinates, pharynx, and guttural pouch. in nine of ten vaccinated horses, viral rna was not detected in daily nasal, oral, or rectal swab specimens or from blood, urine, or feces samples collected before euthanasia, and no recoverable virus was present. only in one of three horses challenged at 6 months after vaccination, low viral gene copy numbers were detected in nasal swab samples collected on post-challenge days 2, 4 and 7, a fi nding consistent with self-limiting local replication, but no recoverable virus was present ). the horse vaccine against hev (equivac ® hev) is the fi rst commercially deployed vaccine developed against a bsl-4 agent and is the only licensed treatment for henipavirus infection. to date, more than 430,000 doses of equivac ® hev vaccine have been administered to horses (zoetis, inc.). hev and niv are the fi rst and only examples of zoonotic paramyxoviruses that can infect and cause lethal disease across a broad range of mammalian species including humans and there are currently no approved treatment modalities for people. because of the potential environmental accessibility of hev and niv and their highly pathogenic characteristics, the development of effective countermeasures against these biothreats has been a major research focus over the past decade. much of this research has focused on the virus binding and entry processes, including the processing, maturation and function of the envelope glycoproteins and the attachment to host cellular receptors and the membrane fusion process. these efforts have led to the development and testing of potential vaccine candidates and antiviral therapeutics. in 2010, the m102.4 mab producing cell line was provided to the queensland government, queensland health, australia to produce the m102.4 mab for emergency use on a compassionate basis in future cases of high-risk human hev exposure. queensland health authorities have completed in may, 2016, the fi rst phase 1 clinical safety trial of m102.4 in human subjects (queensland 2013 ). to date, 11 individuals exposed to either hev in australia (10 people) or niv in the united states (1 person) have been given high-dose m102.4 therapy under emergency use protocols, and all have remained well with no associated adverse events. in addition, the vaccine against hev (equivac ® hev) is vaccine for horses that is also expected to provide a substantial health benefi t to humans, and has fi t well within the spirit of a "one health" approach for the human and animal interface and also in respect to environmental health. studies on niv and hev have also provided important model systems to examine how pathogenic viruses 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workers in singapore henipavirus neutralising antibodies in an isolated island population of african fruit bats continent-wide panmixia of an african fruit bat facilitates transmission of potentially zoonotic viruses evidence for henipavirus spillover into human populations in africa human hendra virus encephalitis associated with equine outbreak protection against henipavirus infection by use of recombinant adenoassociated virus-vector vaccines urban habituation, ecological connectivity and epidemic dampening: the emergence of hendra virus from fl ying foxes (pteropus spp.) diverse roles of eph receptors and ephrins in the regulation of cell migration and tissue assembly inhibition of hendra virus fusion simulating henipavirus multicycle replication in a screening assay leads to identifi cation of a promising candidate for therapy inhibition of nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry agricultural intensifi cation, priming for persistence and the emergence of nipah virus: a lethal bat-borne zoonosis world-fi rst hendra treatment one step closer date palm sap linked to nipah virus outbreak in bangladesh characterization of nipah virus from naturally infected pteropus vampyrus bats experimental infection of swine and cat central nervous systems by the pig paramyxovirus of the blue eye disease three or more routes for leukocyte migration into the central nervous system nipah virus in lyle's fl ying foxes a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection investigation of a second focus of equine morbillivirus infection in coastal queensland canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion mr imaging features of nipah encephalitis long-term neurological and functional outcome in nipah virus infection infection of humans and horses by a newly described morbillivirus nipah virus in the fruit bat pteropus vampyrus in sumatera, indonesia henipaviruses employ a multifaceted approach to evade the antiviral interferon response broad-spectrum antiviral activity of virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide ribavirin-current status of a broad spectrum antiviral agent ribavirin therapy for nipah virus infection evidence for nipah virus recrudescence and serological patterns of captive pteropus vampyrus nipah virus infects specifi c subsets of porcine peripheral blood mononuclear cells henipavirus mediated membrane fusion, virus entry and targeted therapeutics viral diseases of the central nervous system relapsed and late-onset nipah encephalitis nipah encephalitis outbreak in malaysia distribution of viral antigens and development of lesions in chicken embryos inoculated with nipah virus no evidence of prolonged hendra virus shedding by 2 patients endocytosis of the nipah virus glycoproteins a longitudinal study of the prevalence of nipah virus in pteropus lylei bats in thailand: evidence for seasonal preference in disease transmission vaccine potential of nipah virus-like particles recrudescent infection supports hendra virus persistence in australian flying-fox populations fields virology molecular biology of hendra and nipah viruses the exceptionally large genome of hendra virus: support for creation of a new genus within the family paramyxoviridae effi cacy of dna immunization with f and g protein genes of nipah virus negative fi ndings from serological studies of equine morbillivirus in the queensland horse population invasion of the central nervous system in a porcine host by nipah virus recombinant nipah virus vaccines protect pigs against challenge transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats experimental hendra virus infection in pregnant guinea-pigs and fruit bats (pteropus poliocephalus) a guinea-pig model of hendra virus encephalitis emerging epidemic viral encephalitides with a special focus on henipaviruses a golden hamster model for human acute nipah virus infection pathology of acute henipavirus infection in humans and animals human hendra virus infection causes acute and relapsing encephalitis nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis clinical and pathological manifestations of human henipavirus infection late presentation of nipah virus encephalitis and kinetics of the humoral immune response novel henipalike virus, mojiang paramyxovirus, in rats new insights into the hendra virus attachment and entry process from structures of the virus g glycoprotein and its complex with ephrin-b2 host cell recognition by the henipaviruses: crystal structures of the nipah g attachment glycoprotein and its complex with ephrin-b3 crystal structure of the hendra virus attachment g glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody detection of nipah virus rna in fruit bat (pteropus giganteus) from india nipah virus infection in bats (order chiroptera) in peninsular malaysia recombinant measles virus vaccine expressing the nipah virus glycoprotein protects against lethal nipah virus challenge serologic evidence for the presence in pteropus bats of a paramyxovirus related to equine morbillivirus exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody potent neutralization of hendra and nipah viruses by human monoclonal antibodies acknowledgments c.c.b. is supported nih grant ai054715-06. portions of fig. 1 were illustrated by andrew hickey. brain stem immunohistochemistry-stained images in fig. 3 were provided by thomas geisbert. brain parenchyma immunohistochemistry-stained images in fig. 4 were provided by debora middleton. key: cord-283850-kt8n6pg2 authors: steardo, luca; steardo, luca; verkhratsky, alexei title: psychiatric face of covid-19 date: 2020-07-30 journal: transl psychiatry doi: 10.1038/s41398-020-00949-5 sha: doc_id: 283850 cord_uid: kt8n6pg2 the coronavirus disease 2019 (covid-19) represents a severe multiorgan pathology which, besides cardio-respiratory manifestations, affects the function of the central nervous system (cns). the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), similarly to other coronaviruses demonstrate neurotropism; the viral infection of the brain stem may complicate the course of the disease through damaging central cardio-respiratory control. the systemic inflammation as well as neuroinflammatory changes are associated with massive increase of the brain pro-inflammatory molecules, neuroglial reactivity, altered neurochemical landscape and pathological remodelling of neuronal networks. these organic changes, emerging in concert with environmental stress caused by experiences of intensive therapy wards, pandemic fears and social restrictions, promote neuropsychiatric pathologies including major depressive disorder, bipolar disorder (bd), various psychoses, obsessive-compulsive disorder and post-traumatic stress disorder. the neuropsychiatric sequelae of covid-19 represent serious clinical challenge that has to be considered for future complex therapies. human civilisation has always co-existed with parasitic forms of life represented by bacteria and viruses that invariably took the toll of life. when social, biological and economic factors aligned, the infections became widespread reaching the level of pandemic, which caused massive death and misery; pandemics shaken the foundations of society and turned the course of history and mindset of humanity. the typhoid fever devastated athens in 490 bc thus giving the military society of sparta upper hand in peloponnesian war, the plague of justinian doomed the reincarnation of roman empire, while the black death, caused by yersinia pestis that killed a third of population of europe, instigated tectonic changes in economic relations that ultimately disposed of serfdom and feudalism and laid foundations of renaissance. the last global epidemic of spanish flu responsible for 20-50 millions deaths has coincided with first world war, internecine conflicts and birth of bolshevism, which all together brought the greatest confusion to mankind. movements of great masses of soldiers from the us brought the h1n1 influenza a virus to europe; disruption of the health services, poor hygiene associated with movements of people, devastations of war and malnutrition all sparkled the superinfection with unusually high death toll 1 . all major pandemic, being associated with severe environmental stress, affected human way of thinking and human psychological health. systematic studies aimed at identifying pathogenetic mechanisms responsible for the onset of psychiatric diseases following viral epidemics begun in 19 century. the eminent english doctor, henry holland in 1839 proclaimed that the flu was responsible "of featured impairments of mental functions almost in the same ratio of the body ……. and that the behavioural alterations were not comparable to those secondary to other fevers" 1 . eighty years later karl menniger confirmed the association between viral infection and psychiatric morbidity: "one hundred cases of mental disease associated with influenza in the recent pandemic have been studied at the boston psychopathic hospital. the variety of mental disturbance manifested is wide… they are readily classifiable into four groups: delirium, dementia praecox, other psychoses, and unclassified. of these, dementia praecox is the largest group numerically" 2 . over the years the accumulated clinical evidence has strengthened our knowledge of psychiatric features of cerebral disease. in the past few decades the interest in the putative aetiologic role of viruses has gradually enhanced to enclose not only the organic mental disorders induced by acute viral encephalitis and the slow viral infections of the central nervous system (cns) but also to encompass the so-called functional psychiatric diseases such psychosis, depression and bipolar disorder (bd). it has became universally acknowledged that combination of systemic infection, viral neurotropism and environmental stress facilitates or even induces development of psychiatric pathologies that exacerbate the course of pandemic and present a significant therapeutic challenge. the coronavirus disease 2019 (covid-19) pandemic revives a long-forgotten challenge for humanity that lived in (illusionary) mass infection free environment. grappling with the uncertainties of a newly emerged disease, against which neither vaccine nor effective treatment protocol exists, the mankind will likely subsist in a new reality for months if not years before implementation of a global remedy. how the virus interacts with our body and which are the pathophysiological scenarios for acute phase of the disease and long-lasting outcomes are the critical questions to be addressed to identify medical strategies. the covid-19 results from the infection with a novel coronavirus that was first identified in china following an initial outbreak in 2019 2 . this coronavirus, named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) belongs to group 2b of β-coronavirus family 3 . the sars-cov-2 is recognised as the seventh component of the coronavirus family and has been included in the orthocoronavirinae subfamily 4 . coronaviruses are singlestranded rna viruses generally related to respiratory illness; they also (albeit less frequently) may instigate gastrointestinal and neurological disorders in a wide variety of mammals and birds. the coronaviruses have high rates of mutation and recombination as well as a propensity of cross-species transmission 5 . the sars-cov-2 enters the cell following binding to plasmalemmal ace2 enzyme with subsequent endocytic internalisation 6, 7 . the primary targets for the virus are represented by epithelial cells of the lungs and gastrointestinal tract. endocytosis of the ace2-virus complex also leads to a depletion of plasmalemmal pool of ace2 with consequent reduction in conversion of angiotensin ii to angiotensin 1-7; the latter peptide possesses marked anti-inflammatory properties 8, 9 and the reduction of arg 1-7 significantly contributes to lung failure and the massive occurrence of pulmonary fibrosis described in patients with covid-19 10 . whether sars-cov-2 could penetrate cells through alternative routes remains unclear, although in contrast to other coronaviruses, sars-cov-2 does not bind to plasmalemmal receptors such as aminopeptidase n and dipeptidyl peptidase 11 . the clinical presentation of covid-19 is dominated by respiratory signs with less frequent occurrence of gastrointestinal symptoms. the virus invasion is not limited to these two organs, particularly considering that significant expression of ace2 is detected in other tissues, including heart, kidney, endothelium and cns 12 . viral infection of the brain 13 may have multiple neurological and psychiatric consequences, contributing to both the acute phase of disease and its potential sequelae fig.1 . the neurotropism has been well documented for several β-coronaviruses including sars-cov-1, mers-cov and the hev 67 n virus of porcine hemagglutinating encephalomyelitis [14] [15] [16] [17] [18] [19] . arguably, the primary way for sars-cov-2 is associated with ace2 expressed in neurones and in neuroglia [20] [21] [22] the ace2 expressing neural cells are found in the circumventricular organs, such as the subfornical organ, the paraventricular nucleus, the solitary tract and in the rostral ventrolateral medulla 21 . all these regions have little protection of the blood brain barrier (bbb) and all are involved in cardiovascular and respiratory regulation. the lack of bbb makes these cns sites vulnerable in many pathologies, such as various types of systemic inflammation including sepsis-associated encephalopathy, neuroinfection with bacteria, viruses or parasites, stress and autoimmune encephalitis 23, 24 . microglial cells localised in cvo seems to be in a state of chronic activation in the attempt to limit the entry of circulating neurotoxic molecules or invasive agents into the parenchyma and to preserve cerebral homoeostasis 25 . the sars-cov-2, similarly to other respiratory viruses, could gain access to cns through several routes, for example by migrating through axons of the olfactory nerve 26 . the intranasal infection of sars-cov-1 or mers-cov 27 was shown to result in rapid spread of viral particles into the brain possibly through the olfactory bulb by a retrograde axonal transport; viruses replicating in the nasal cavity may utilise the direct link with the olfactory bulb to colonise the cns. in this paradigm the virus is transported through the axons of olfactory bulb neurones with subsequent infection of the specific type of neuroglia the sustentacular cells of the olfactory bulb 28 . when the virus was administered intranasally in extremely low [2] doses, only the cns was disseminated 5 , strengthening the concept of an intrinsic neurotropism of coronaviruses. in rodents ablation of the olfactory bulb prevented viral spread following nasal infection 29 . further support for the role of nasal-olfactory route comes from clinical observations according to which anosmia develops early in sars-cov-2 infected subjects 30 . the sars-cov-2 rna was present for 20 or more days in oropharyngeal and nasopharyngeal secretion samples of 30% of covid-19 survivors, suggesting that sars-cov-2 can linger for a long time at both upper and lower respiratory tract 31 . the virus can also enter the brain through infecting endothelial cells lining brain vasculature; an electronmicroscopic analysis of the frontal lobe identified sars-cov-2 viral particles in the endothelium with some indications for virus transit to the neuropil 32 . the sars-cov-2 can enter the cns using perivascular spaces of the glymphatic system 33 . furthermore, viruses can invade the brain through other nerves, such as the trigeminal nerve, which projects nociceptive terminals to nasal cavities 34 . similarly, sensory fibres of the vagus nerve, that innervate the respiratory tract, can present another invasion route 35 . further evidence of the sars-cov-2 neuroinfection, oedema and neuronal degeneration were reported in postmortem brain samples, while in a case of encephalitis genome sequencing confirmed viral presence in the cerebrospinal fluid 36 . post-mortem analysis of nervous tissue from tissue of a 54 years-old man who died from severe respiratory failure associated with covid-19 identified sars-cov-2 viral particles in the olfactory nerve, in the gyrus rectus and in the brainstem with signs of profound damage to all elements of the tissue including glial cells, neurones, their axons and myelin 37 . it seems therefore that sars-cov-2, similarly to sars-cov-1 and mers-cov infects the brainstem, in which the respiratory neuronal circuits are located, and, by analogy, a similar infection could occur and contribute to the respiratory failure, observed in sars-cov-2 pneumonia. breathing depends on a central pattern generator located in the dorsolateral pons, in the nucleus of the solitary tract, and in ventrolateral medulla; this patter generator is responsible for respiratory rhythms and control of motor neurones innervating respiratory muscles 38, 39 . in a sub-population of covid-19 patients' respiratory failure is manifested by a decreased breathing rate with hypoxia and hypercapnia. many of these patients remain in a coma for days, despite the suspension of sedative treatment and the absence of apparent metabolic alteration, indicating viral encephalitis, which often is resolved without major sequelae 40 . this is not always the case, however, and when the extent of respiratory failure is overwhelming, patients die before the virus-induced brain damage can become evident 41 . given the viral load in the brain stem, the subsequent reduction of ace2 expression associated with the neuronal death, could lead to an alteration in baroreceptors function associated with inflammation. systemic inflammation compromises the blood-brain barrier (bbb) and floods the brain with pro-inflammatory factors. the virus may also cross the bbb at the level of the circumventricular organs or through retrograde axonal transport via olfactory bulb and infect the brain, thus instigating reactive gliosis, which leads to an increased production and secretion of cytokines and other pro-inflammatory factors. the combination of systemic inflammation, hypoxia resulting from respiratory failure and neuroinflammation may trigger or exacerbate psychiatric diseases. an increase in the sympathetic tone and a severe, life threatening rise in blood pressure 42, 43 . the encephalitis, reported as a complication of coronavirus infection, invariably affects not only the brain stem but also thalamus and white matter 26, 44, 45 . these aspects have to be taken in account by clinicians dealing with covid-19 patients displaying severe cardiovascular and respiratory failure. recognizing that respiratory symptoms may, at least in part, originate from the encephalitic damage to the brain stem, may help to design more effective treatments. the damage to the brain stem as well as to other brain structures can also result from systemic inflammation, often referred to as systemic inflammatory response syndrome or "cytokine storm" 46, 47 . at the same time the brain is a target for infectious toxic encephalopathy, associated with systemic toxaemia or hypoxia that accompany acute infectious diseases. toxic encephalopathies have massive neurological and psychiatric presentations and even cerebral oedema, which however develops without accumulation of inflammatory markers in cerebrospinal fluid 48 . in addition, systemic infection and high levels of circulating cytokines often damage microcirculation, inducing oedema and thrombosis; the tromboembolia being reported in up to 30% of patients 49 . the cytokines, furthermore, activate autonomic nerves and hypothalamic-pituitary-adrenal axis which affect blood pressure. all these factors together stipulate ischaemic damage to the brain and are associated with occurrence of strokes which further increase mortality in covid-19 patients 50 . despite the existence of bbb, the brain and the spinal cord communicate with the peripheral immune system, and hence every systemic inflammation affects the cns 51 . in the context of covid-19 the damage to bbb mediated by a massive increase in circulating pro-inflammatory factors is highly likely 52 . compromised bbb allows an inflammatory storm to engulf cns leading to functional damage. once in the brain, peripheral inflammatory molecules as well as inflammatory cells instigate neuroinflammation thus perturbing homoeostasis, altering neural networks and inducing neuronal death 53, 54 . in the initial phases of systemic inflammation, antiviral immunity can effectively blunt viral dissemination, since reactivity of neuroglia and influx of surveying t cells can remove infectious elements, preventing spread without any further tissue damage 55 . in severe covid-19, substantial release of chemokines and interleukins associated with systemic inflammation and the marked lymphopenia allow higher and a more prolonged persistence of a viral load; consequently deficient clearance of the virus together with the reactive gliosis can perpetuate neuroinflammation [56] [57] [58] . even in mild cases, sars-cov-2 pneumonia causes hypoxia, which on its own can trigger or exacerbate inflammatory response of the cns. cerebral hypoxia activates key inflammatory transcription factors, including nf-κb and hypoxia inducible factor which stimulate overproduction of pro-inflammatory messengers 59 , trigger glial reactivity 60,61 , induce mitochondrial oxidative damage, and activate promoter region of numerous mirnas, crucial for regulating gene expression during inflammation 62 . an excessive glial reactivity due to persistent exposure to pro-inflammatory cytokines also contributes to synapse loss and neuronal death 63, 64 . the impact of sars-cov-2 infection on the brain is associated with excessive physical and psychological stress that stimulates the hypothalamic-pituitary-adrenal axis thus further exacerbating neuroinflammatory status 65 . the duration and frequency of exposure to stressors impacts neuroinflammation. in this sense while a response to short and moderate stressors could be beneficial, repeated or extended exposure to strong stressors exacerbates inflammation 66 . exposure to long-lasting stress enhances inflammatory response through the release of several pro-inflammatory factors, which trigger down-stream signalling pathways, including nf-κbdependent transcription. contribution of glucocorticoids, associated with stress response, to sustaining and promoting neuroinflammation is complex, going beyond effects deriving from the activation of the signals downstream of their receptors 67 . microarray experiments demonstrated that glucocorticoids drive expression of specific gene profiles, whereas concurrent co-activation of glucocorticoid receptors and nf-κb-dependent transcription induces a peculiar pattern of gene expression different from the one resulting from separate activation of each signalling pathway 68 . neuroinflammation is a significant aetiological factor for a large number of neuropsychiatric and neuro-cognitive diseases, including neurodegenerative disorders [69] [70] [71] , depression 72 , psychosis 73 , autism 74 , drug abuse 75 , sleep disorders 76 and epilepsy 77 . the neuropsychiatric burden of this pandemic is currently unknown, but is likely to be considerable. based on the results from investigations of recent epidemics by corona respiratory viruses, sars-cov-1 and mers-cov, it is possible to assume that a significant percentage of subjects recovering from pneumonia do not fully regain their previous emotional state and cognitive abilities. indeed, a study of neuropsychiatric consequences of sars-cov-1 performed at 30-50 months after the infection demonstrated an occurrence of 40% of posttraumatic stress disorder (ptsd), 36.4% of depression, 15.6% of obsessive convulsive disorder, and an equal incidence for anxiety disorders 78 . furthermore, a metaanalysis among sars-cov-1 patients of mixed conditions showed neurocognitive deficits up to 18 months post-discharge 79 , including mild cognitive impairment 80 . given this evidence the burden of long-term post-sars-cov-2 delirium and dementia may be notable, especially for elderly subjects who are more vulnerable to postinfectious neurocognitive sequelae. the average age of the subjects with severe covid-19 is around 63 years, whereas patients under the age of fifty represent only 26% of all clinical cases. the ageing itself is the major risk factor for cognitive pathologies and neurodegeneration; severe systemic diseases as well as stress are known to provoke or accelerate cognitive decline in elderly. in the aged brain the neurogenesis is dwindling, synaptic plasticity deteriorates, metabolism is reduced and overall brain vulnerability to exogenous insults is increased. ageing of the human brain is also associated with degeneration and atrophy of microglia and astrocytes which diminishes homoeostatic and neuroprotective support and again increases brain susceptibility to pathology [81] [82] [83] [84] . infection with sars-cov-2 (even in moderate clinical cases) thus promotes cognitive disorders with emergence of delirium, acute psychosis, exacerbation of mild cognitive impairment or with accelerating of dementia associated with various neurodegenerative conditions, including alzheimer's disease (ad) 85, 86 . conceptually, neuroinflammation contributes to the pathological development of neurodegeneration and often is considered as a common, even unifying feature of neurodegeneration 87 , while brain infection and ischaemic insults by themselves can trigger neurodegenerative process and instigate dementia 88 . it is a truth universally acknowledged that systemic inflammatory challenge accelerates cognitive impairment, which implicates that the infection itself, as well as aberrations of the innate immune system, is responsible for the cognitive deficits 89 . epidemiological observations as well as neuropathological analysis support the notion of a direct correlation between systemic infections, neuroinflammation and cognitive disorders, such as delirium and ad 90, 91 . in this context, cohort studies identified pneumonia as the pre-eminent pathology responsible for hastening and boosting cognitive decline 92 . at the same time vaccinations against bacteria or viruses reduce the risk of the progressive evolution of dementia 93 . close correlation between pneumonia and delirium in the elderly is a long-standing observation, and delirium, which represents the most common event of acute brain dysfunction, is a frequent complication of cov-19 clinical progression, perhaps due to the neurovirulence, severe peripheral inflammation, profound stress; even "social distancing" and loneliness which elderly experience during pandemic contribute to psychotic episodes 94 . systemic and tissue immune response contribute to the pathophysiology of numerous neuropsychiatric diseases through modifying neurochemical environment, synaptic transmission and plasticity, synthesis and secretion of neurotrophic factors, neurogenesis, and brain connectome. in this context, the major depression disorder (mdd) is one of the most frequent neuropsychiatric disorders linked to inflammatory injury to the brain. a large body of evidence has associated depression symptoms to pro-inflammatory factors 95 and neuroglial failure 96 . this link specifically applies to subtypes of depression occurring in the elderly. ageing substantially affects the levels and the activity of pro-inflammatory cytokines in the cns. systemic infection can itself trigger major depression in elderly patients, because of agedependent decrease of immune homoeostasis 97 . in particular increased serum levels of interleukin-1β directly correlate with emergence of late life mdd 98 . similarly, a correlation has been observed between inflammatory factors and some specific symptoms, for example, high levels of tnf-α and il-2 associate with apathy and motor inhibition, whereas il-6 associates with anhedonia and suicidality 99 . the levels of cytokines decrease when patients recover normal mood levels; conversely cytokines remain elevated in patients resistant to treatment 100, 101 . severe cases of covid-19 are almost invariably accompanied with excessive host immune response, mainly characterised by a massive increase in plasma levels of il-6, which directly correlates with an unfavourable outcome of the disease 102 . at the same time abnormally high concentrations of il-6 were detected in the cerebral spinal fluid of suicide attempters 103 , of subjects suffering from either depression or schizophrenia 104 , of old depressed patients 105, 106 and of mothers with postpartum depression 107 . a large body of evidence demonstrated that changes in il-6 levels, both in plasma and in the brain, are implicated in the emergence of depression, although other factors, environmental or genetic in nature, provide an important contribution 108 . in the cns il-6 acts as a pro-inflammatory mediator, which promotes synthesis and secretion of additional inflammatory factors and acute phase proteins by astrocytes and microglia 109 . thus il-6, together with tnf-α and il-1β, can be considered as one of the primary regulators of the immune response in the brain, while astrocytes and microglia are the major responders to il-6, as well as prominent producers of il-6 stimulated by damage and pathogenassociated molecular patterns (including viruses and their components), neurotransmitters and pro-inflammatory messengers 63, [110] [111] [112] [113] . physiological plasma levels of il-6 in adults range between 1-10 pg/ml whereas in a systemic inflammation is raises to several ng/ml; 114 and even higher concentrations were reported for covid 19 115 . incidentally, high levels of il-6 have been detected in the plasma, in the cerebrospinal fluid (csf) and in the postmortem prefrontal cortex of subjects with suicidal ideation, with non-fatal suicide attempts or committed suicides 116 . at the same time no direct correlation was found between plasma and csf concentrations of il-6 in subjects with suicide attempts, nor such correlation was detected for scores of depression severity 117 . the il-6 levels in circulation also correlated with suicide endophenotypic behaviours, such as personality trait disorders, aggressivity and impulsivity 118 . this is in agreement with numerous findings proving the role of cytokines in regulating emotions and behaviours through interacting with specific brain areas and different neuronal pathways 119 . covid-19 pandemic resulted in significant changes in lifestyle and interpersonal relationships condemning many to prolonged loneliness. these conditions of psychosocial stress can also have a detrimental effect on the most fragile subjects affecting their ability to modulate emotions 120 . decreased control over impulsivity and feelings of fear in combination with inflammatory challenges to the brain might increase the risk of suicide. abnormal balance between the pro-inflammatory (il-6 and tnf-α) and anti-inflammatory cytokines in the cns and in the plasma have been repeatedly observed in patients with bd supporting the notion that neuroimmune response may be a prominent factor contributing to aetiopathogenesis of this illness 121, 122 . in acute phases of bd either during manic or depressive episode, an activation of inflammatory cascades were reported, which was considered by many, but not by all, a characteristic feature of the acute illness, rather than a persistent trait of the disease 123 . several cytokines, such as il-1β, tnf-α, il-6, interferon-γ were found to increase in circulation in acute phases of bd, with a parallel reduction in the anti-inflammatory factors il-10 and transforming growth factor β-1, especially in the manic phase 123, 124 . analysis of the presence of pro-inflammatory molecules in the csf in bd patients revealed contradictory results. somewhat high csf levels of il-8, monocyte chemoattractant protein 1(mcp-1 / ccl-2), and neurofilament light chain were detected in bd subjects, although these biomarkers did not correlate with the outcome of the disease 125 . a meta-analysis of csf cytokines content in bd subjects revealed increased levels of il-1β, the il-8 showed statistically insignificant rise and no changes for il-6 were detected 126 . the disparity between the levels of interleukins and chemokines in blood serum and in the csf is another controversial issue. it is of course tempting to assume that this discrepancy is lost in covid-19 since the overload of interleukins and chemokines, compromised bbb and activation of cns resident and invading immune cells exacerbates the neuroinflammation and promotes a bidirectional flow of inflammatory messengers through a permeable barrier. such a scenario, however, remains highly hypothetic and much more investigations and analysis are needed to reveal possible association of viral infection in general and covid-19 in particular with bd. a wide spectrum of immune system dysregulations as well as infections (together of course with genetic vulnerability, abnormalities in neurotransmission, stress and exposure to environmental factors such as childhood maltreatment) are recognised as potential pathogenetic factors of reactive psychosis 127 . enhanced inflammation in psychosis has been confirmed by meta-analyses showing increased concentrations of cytokines and their receptors in chronic schizophrenia, as well as in drug-naïve patients in their first episode of psychosis (fep) 128 . a recent study aimed at investigating pro-inflammatory cytokine profile in fep patients showed an up-regulation of il-6, tnf-α and il-1β, which was not found in healthy siblings, suggesting familiar vulnerability is not involved in generating the inflammatory-related psychotic reactions 129 . in an attempt to conceptualise the risk of the emergence of psychosis in subjects infected with sars-cov-2, it should be emphasised that high levels of il-6, correlate with reduced hippocampal size in schizophrenic subjects accounting, at least partially, for their cognitive deficits 130 . moreover, elevated levels of il-6 were detected in csf of schizophrenic subjects 104 . even more intriguing is the observation that high levels of il-6 in adolescents correlate positively with the occurrence of psychosis later in life 131 . a growing body of literature reported the occurrence of obsessions and compulsions in patients who had recently recovered from viral encephalitis 132 . already in 1930s more than a third of cases of obsessive and compulsive disorders (ocd) were recognised an organic in pathogenesis, and linked with von economo's encephalitis 133 . subsequently, neuropsychiatric literature was dotted with numerous case reports ranging from those of obsessive syndromes with post-encephalitic parkinsonism 134 , to those of post-encephalitic subjects in which diabetes insipidus coexisted with ocd 135 , to the six ocd patients with anamnesis of viral encephalitis 136 . beside these early examples, more recently, high levels of borna virus immune complexes and viral components (proteins, rna) were detected in the blood and in peripheral mononuclear cells of ocd patients, reinforcing the notion of a significant link between viral infection and ocd in predisposed subjects 137 . in this scenario, since functional neuroimaging demonstrated ocd implies alterations in the striato-thalamo-cortical circuits, it was of interest that activity of these circuits may be affected in viral infection, possibly through interferences with glutamate transmission 138 . beyond any doubt immune dysfunction plays a causative role in childhood-onset ocd where the sudden onset of obsessive compulsive signs and tics occurs in the aftermath of a streptococcal infection, with subsequent production of auto-antibodies against neuronal antigens of the basal ganglia 139 , further supporting the notion that an alteration of the immune system may be implicated in the pathobiology of these disorders. numerous investigations have shown a correlation between circulating proinflammatory cytokines levels and ocd 140, 141 . increase in blood concentrations of il-1β, il-6 and tnf-α in ocd patients was detected when compared to normal controls paired by gender, age, and educational level 142 . these findings are in agreement with results of studies investigating drug-naïve, comorbidity-free ocd subjects 143 . the observation that pro-inflammatory cytokines are increased in a study that eliminated any confounding factors, such as anxious or depressive comorbidity or the effects of psychotropic drugs, represents a more convincing support for the idea that immunological abnormalities contribute to the origin of ocd 141 . systemic inflammation which is the prominent feature of covid-19 may therefore trigger ocd in surviving subjects. extensive literature reports epilepsy and behavioural abnormalities as closely linked pathologies 144 . indeed, psychiatric diseases are more frequent in epileptic subjects than in general population irrespective of the time of seizures onset, which could occur either before or after the appearance of psychiatric disorders, suggesting a mutual relationship and potentially shared aetiology 145 . this intriguing coexistence of psychiatric features in epileptic patients does not represent a coincidence or an ordinary comorbidity but more likely it reflects interconnected pathobiological processes 146 . neuroinflammation may hint to the underlying mechanism shared by epilepsy and psychiatric disorders, albeit with distinct involvement of neuronal substrates 77 . this makes any rigid separation between epilepsy and some psychiatric disorders less stringent, and hence we included epilepsy in the discussion for the remarkable behavioural alterations and for the role of neuroinflammation in its pathogenesis. the link between epilepsy and neuroinflammation is universally recognised 77 . persistent neuroinflammatory cascade due to cytokine load and bbb damage is associated with glial reactivity, synaptic changes, and the generation of hyper-excitable networks with lower seizure threshold which all promote epileptic activity 147 . epidemiological findings have indicated neuroinfection and systemic infections as one of major cause of acquired epilepsy 148, 149 . viral encephalitis, for example, increases the risk of subsequent seizures 149 . increased concentrations of il-1 were detected in plasma and csf of different epileptic phenotypes, suggesting this cytokine seizureinducing properties 150 . changes in gabaergictransmission and reduction of astrocytic glutamate uptake may account for il-1 dependent increase in susceptibility to epilepsy 151, 152 . similarly, raised levels of il-6 were reported in both plasma and csf in patients suffering from a wide range of epileptic presentations, while this increased concentrations correlated with the severity of seizures 153 . capability of il-6 to promote epileptogenesis is further corroborated by the evidence that il-6 overexpression induces abnormal ictogenesis in mice hippocampus 154 . associations between epilepsy and covid19 have not yet been reported 155 , however, american epilepsy society already suggested that covid-19 could increase the risk of sudden unexpected death in epilepsy (sudep). there are some reports which indicate that infections, bacterial or viral may increase the risk of sudep. at present, there are no data on the association between covid −19 and sudep 156 . generally, albeit incorrectly, it is assumed that once the trauma is over and the subject is no longer under the pressure of stress, the path for steady recovery begins, since the time heals all wounds. unfortunately, this is not always the case because in susceptible subjects the active stress instigates brain processes whereby traumatic memories suddenly re-emerge and disturb the mental health. the persistence of these conditions generates the ptsd 157 . the ptsd is no longer classified among anxiety disorders; it is considered a trauma or stress-related disorder 158 . the pathogenetic link between inflammation and ptsd is well documented 159 . because of marked impact of stressors on the immune system, it is not surprising that ptsd is associated with the immune state 160 . increased concentrations of pro-inflammatory factors were observed both within systemic circulation and in the brain in the context of ptsd 161 . activation of neuroglia induced by heavy or persistent stressors can stimulate aberrant secretion of pro-inflammatory signals which could potentially facilitate the appearance of ptsd. data from meta-analyses confirm a remarkable increase in proinflammatory molecules in subjects with ptsd, including il-6, tnf-α, and il-1β [162] [163] [164] . the levels of il-10, an anti-inflammatory interleukin, have been also increased, probably in an attempt to offset the inflammatory processes triggered by stress, further highlighting a close link between inflammation, stress and ptsd 165 . the occurrence of ptsd was usually associated with occurrence of low-grade inflammation 166 . beside changes in cytokines, ptsd is also connected with enhanced nf-κb expression, this transcription factor being implicated in the inflammation process and its elevated expression correlated directly with ptsd severity 167 . moreover, ptds is usually comorbid with depression as well as with anxiety, with drug addiction and with high frequency of suicide, since all these conditions share common inflammatory mechanisms into their pathogenetic processes 168 . however, it remains to be elucidated whether in all cases the relationship is mutual and what factors, along with the inflammatory ones, play a causative role in determining the comorbidities observed. the ptsd can be a likely outcome for covid-19 sufferers. this stems not only from severity of systemic inflammation and viral invasion into the brain, but also from the gravity of stress caused by an unexpected pandemic which, for the high mortality, has a shocking value. significant number of psychotic episodes in the aftermath of spanish flu pandemic has highlighted the possibility of increasing incidence of schizophrenic disorders in subjects infected with the sars-cov-2 169 . high levels of coronavirus immunoreactivity in subjects with recent onset of psychotic episodes as well as the serious neuropsychiatric complications including auditory and visual hallucinations as well as severe delusions have been reported in covid-19 patients 120, 170, 171 . although neurodevelopmental origins of schizophrenia are generally accepted, other aetiological factors such as direct effect of a viral neuroinfection or indirect effect of immune aberrations occurring in adult subjects cannot be excluded 172 . schizophrenia is also considered as a neurodegenerative illness in adulthood, with neuronal shrinkage and loss, oligodendrocyte damage, alterations in synaptic connectivity, all likely associated with cognitive impairments [162] [163] [164] [165] [166] [167] [168] [169] [170] [171] [172] [173] . although there are no evidence directly linking covid-19 with the risk of schizophrenia, frequent occurrence of psychotic episodes highlights the need for further, more detailed investigations. the sars-cov-2 pandemic poses a long-lasting challenge, which not only affects cardio-respiratory system but links systemic infection to neuropsychiatric diseases. investigations of previous viral respiratory epidemics have demonstrated the onset of a wide range of psychiatric disorders over the course and in the aftermath of the infection. the pandemic of spanish flu in 1918-1920 instigated speculation of the causative role of viral infection in the pathogenetic mechanism of behavioural disorders in bipolar and schizophrenic subjects. karl menninger was one of the first to declare that he was persuaded that dementia praecox (as schizophrenia was called in those days) is, in majority of instances, a somatopsychosis, "the psychic manifestations of an encephalitis" 174 . in the same period, jacob kasanin and j. w. petersen suggested that "a thorough review of some of the early histories of atypical cases of schizophrenia or affective disorders may reveal a previous encephalitis" 175 . at present, there are few preliminary studies considering neuropsychiatric complications of covid-19, however, on the basis of the results of the previous epidemics of various respiratory viruses it is possible to assume an increased incidence of mental pathologies as an unwanted sequelae. not only sars-cov-2 can penetrate the brain and cause direct damage to neuronal networks, the experience of potentially lethal and untreatable covid-19 is the cause of a severe distress, which may induce long term behavioural changes or aggravate a pre-existing mental illness. here we outlined possible neuropsychiatric complications that could arise in subjects infected with sars-cov-2. patients with covid-19 could present with a wide range of neuropsychiatric symptoms, which result from systemic inflammation, cns effects of cytokines, infection of neural cells by sars-cov-2, neuroinflammation, glial dysfunction or aberrant epigenetic modifications 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10.1016/j.jcpa.2004.01.010 sha: doc_id: 270780 cord_uid: 3g381rzr abstract archive central nervous tissue from 286 cats with neurological disorders was reviewed for histological evidence of feline spongiform encephalopathy (fse), which may have occurred before it was first recognized in 1990. the following six categories of disease were identified: congenital; degenerative; inflammatory; neoplastic; fse; lesion-free. the largest category (inflammatory) contained 92 cats, of which 47 were considered to be consistent with infection by feline infectious peritonitis (fip) virus. six cats showed evidence of more than one disease process; thus, one cat with fip also had toxocara infection of the lateral ventricles and five cats with fse also showed perivascular cuffing suggestive of concurrent viral infection. in only two cases did the diagnosis on review differ significantly from the original interpretation. there was no evidence of fse before the original case was recognized in april 1990. the causes of nervous disorders in cats are well documented in standard texts (hopkins, 1992; jubb and huxtable, 1993; summers et al., 1995) , but to our knowledge there are no reports of surveys of nervous diseases in the cat. feline neurological disease accounts for approximately 10% of total cat referrals (i.e., approximately 60 cases per annum) to the feline centre, university of bristol veterinary school (gruffydd-jones, unpublished data) and a specific clinical diagnosis is made in only 30-40% of cases. in 1990, feline spongiform encephalopathy (fse) was identified as a cause of nervous disease in cats (wyatt et al., 1990 wyatt, 1991) . it was subsequently shown that fse had the characteristics of a transmissible spongiform encephalopathy (tse) (pearson et al., 1992) and that in mouse transmission studies its behaviour was similar to that of bovine spongiform encephalopathy (bse) (fraser et al., 1994) . at that time the archive of post-mortem diagnoses of feline neurological cases was briefly reviewed , but detailed histopathological examination was not undertaken. more recently, serological and molecular evidence of exposure of cats in the uk to borna disease virus (bdv) has been reported (reeves et al., 1998) . the aims of this study were (1) to extend the preliminary retrospective survey of the bristol university archive of cats with neurological disease, (2) to review the histology of cases of nervous disorders, (3) to categorize the nature of the pathological changes, and (4) to search for histological evidence that fse may have occurred in the uk before it was first recognized in 1990. archived feline specimens were obtained from the division of veterinary pathology, infection and immunity. the specimens were collected during the period 1975 to december 1998. in all, 301 cases of feline neurological disease in cats were identified, consisting of 234 cases referred from the university of bristol veterinary school, and 67 submitted mainly as formalin-fixed tissues from outside practitioners. however, in 11 cases no central nervous system (cns) material had been collected, and in a further four the original tissues, blocks or slides were unavailable. as a result, the cases from which histological sections were available for review numbered 286. the material examined, which varied between cases, is summarized in table 1 . in 1990 the sampling procedure was standardized to include brain sections from the anterior cerebrum, mid-cerebrum (thalamic level), posterior cerebrum (including the midbrain), cerebellum and medulla at the level of the obex. the breeds represented in the 286 cases were: domestic shorthair (172), siamese (27), domestic longhair (22), burmese (19), birman (13), persian (8), rex (4), miscellaneous (19) and unknown (2). paraffin wax sections (8 mm) of formalin-fixed nervous tissues were stained with haematoxylin and eosin (he) and examined by light microscopy. in two cases, brain tissue cut from the original blocks was dewaxed, processed in alcohol, and placed in 0.1 m sodium cacodylate buffer before being processed by routine methods for electron microscopy (em). the various diagnoses made were compared with those in the original reports. the histological lesions in the 286 cases were classified under the following six headings: congenital; degenerative (including feline dysautonomia syndrome [fds] ); inflammatory or lesions of infectious diseases (including feline infectious peritonitis [fip] , toxoplasmosis and cryptococcosis); neoplasia (including lymphoma); fse; no detectable lesions. the results of the 286 case reviews generally accorded with the original reports, with only occasional discrepancies. such discrepancies are mentioned in the account below. this group consisted of 12 cats, all under 2 years of age. the mean age was 18.7 weeks with a median age of 12 weeks. the most common diagnoses in this group were cerebellar hypoplasia (four cats) and lysosomal storage diseases (three cats) (fig. 1) . storage disorders were identified by the presence of many swollen abnormal neuronal cell bodies (summers et al., 1995) . in one kitten, electron microscopy and histochemical stains supported the initial diagnosis, and biochemical analysis of tissue (dr bryan winchester, personal communication) confirmed the storage disorder to be a gm 1 gangliosidosis (barker et al., 1986) . in another case the diagnosis was confirmed by thin layer chromatography but no detailed information was available. a dermoid cyst present at the level of the third thoracic vertebra of a 16-month-old cat resulted in ataxia and euthanasia . the remaining diagnoses included one case each of hypomyelinogenesis, neuraxonal dystrophy, syringomyelia and cerebellar abiotrophy. degenerative lesions other than those of fds. these accounted for 42 cats mainly mature animals but ranging in age from 5 weeks to 19 years (mean 4.5 years). the changes present included wallerian degeneration of brain or spinal cord (or both), white matter spongy degeneration, and myelin oedema. three cats with hepatic encephalopathy due to portosystemic shunts were included in this group because, although the shunt may have developed as a congenital lesion, the cns lesion was degenerative rather than of a primary congenital type. feline dysautonomia. this affected 27 cats, mainly in the younger age range (median age 12 months; mean age 18 months). the majority of cases (23) were diagnosed between 1982 and 1986, with only four cases diagnosed subsequently. diagnostic features included loss and degeneration of neuronal cell bodies within sympathetic ganglia, with a variable infiltrate of mononuclear cells (sharp et al., 1984) . brain tissue was examined in 10 of these cases, one of which showed mild vacuolation of the white matter in the obex region, but no other lesions affecting brain or spinal cord of the remaining cats with typical ganglionic lesions were identified. the 92 cats affected had a mean age of 5.7 years. they included 47 fip cases (mean age 18 months; median age 10 months). burmese cats with fip ðn ¼ 8þ were over-represented in relation to the proportion of animals of this breed in the overall study (19 of 268 cats). typical inflammatory lesions included perivascular cuffing and meningeal infiltration by mononuclear cells, gliosis, and variable neuronal degeneration suggestive of a possible underlying viral aetiology. in cats in which the inflammation tended to be pyogranulomatous, located around the lateral ventricles or in the meninges, or affecting the choroid plexus, with or without evidence of vasculitis or hydrocephalus, a diagnosis of fip was made. in one of these cats there was concurrent infection by toxocara larvae within dilated lateral ventricles (fig. 2) . of the 45 remaining cases (i.e., non-fip cats) eight had protozoal tissue cysts (presumed but not confirmed to represent toxoplasmosis; fig. 3 ) and one had cryptococcosis (confirmed by culture; fig. 4 ). protozoal infection and cryptococcal meningitis tended to affect middle-aged or older cats; however, the youngest animal with protozoal infection was only 2 months old. a single large protozoal cyst, unassociated with any inflammatory response, was identified in the hippocampus of a persian cat with fse (see below) and was considered to be an incidental finding. bacterial infection was identified in three cats, two of which had a meningoencephalitis associated with bacterial colonies; in the third case the infiltrate was suggestive of a bacterial meningitis and bacteria were identified by electron microscopy (fig. 5) . in the remaining 33 cats a diagnosis of meningitis or encephalitis, or both, was made. a specific diagnosis was not possible in many cases, but in one cat immunohistochemical examination had there may be some difficulty in distinguishing fip infection from other forms of viral infection, and eight cats were identified in which the original diagnosis had been of a meningoencephalitis but which, on review, were considered to have features consistent with a diagnosis of fip. one cat had originally been found (correctly) to have hydrocephalus, but the periventricular and choroid plexus pyogranulomatous inflammation also present had not been reported. on review, therefore, this cat was considered most likely to have been infected by fip virus. conversely, another cat, originally thought to have fip, was considered on review to have lesions of a less specific nature, due to some other, possibly viral, agent. thirty-eight cases of neoplasia were seen, of which 18 were diagnosed as lymphoma. cats with lymphoma tended to be younger (mean age 3.75 years) than those with other forms of neoplasia (mean age 6.9 years). the distribution of lymphoma within the cns is shown in table 2 . lymphoma was located within the spinal canal in 13 cats; four of which also had neoplastic infiltrates in the brain. the brain alone was affected in the remaining five cats. twelve of the 18 cats with lymphoma also showed evidence of lymphoma affecting other sites, e.g., kidney, liver, lymph node and vertebrae. in the remaining 20 cases (neoplasia other than lymphoma), meningioma was the most common tumour (12 cats; six with single cranial meningioma, two with multiple cranial meningioma, and four with spinal meningioma). there were, in addition, four cases of glioma (one of which had previously been diagnosed as an inflammatory lesion), three cases of sarcoma and one of metastatic carcinoma secondary to a bronchoalveolar carcinoma. fse was diagnosed in 24 cats, with an average age of 6 years. fse lesions were absent in the brains of 136 cats which died before recognition of the first case of fse by wyatt et al. (1990) ; after this first case, however, 23 of 149 cats examined post mortem were found to have fse. the diagnosis was confirmed by demonstrating typical lesions in the brain and spinal cord, consisting of: widespread neuropil vacuolation, particularly of the grey matter; neuronal vacuolation, with one or multiple empty vacuoles, throughout the cns but particularly in the thalamus, midbrain, obex and spinal cord; astrocytic hyperplasia and hypertrophy . in five cats, there was also evidence of mild perivascular cuffing (fig. 7) in the cerebrum or medulla, or both, and in one of these five animals focal accumulation of lymphoid cells in the meninges of the brain and spinal cord. one of these five cats also had a large protozoal cyst within the hippocampus, without any inflammatory reaction (see above). the dual presence in these five animals of fse lesions and lesions indicative of possible viral infection suggests the presence of more than one disease process. fifty-one cats (17.8% of the 286 examined) showed no lesions either at the initial examination or at the review. the sections examined were of brain alone (33 cats), brain and spinal cord (11 cats), spinal cord alone (two cats), and ganglia (two cats). detailed histological investigation of the archive indicated that in the majority of cases there was agreement between the original histological descriptions and diagnoses, and those following re-examination of the slides. however, occasional discrepancies were found. in one cat with hydrocephalus and an inflammatory infiltrate typical of fip (krum et al., 1975) , the original description did not include reference to the inflammatory infiltrate. a second cat was originally reported to have an inflammatory and degenerative lesion of the midbrain and medulla, but on review the lesion was considered to be associated with a pleomorphic tumour (possibly a glioma of the astrocytoma type). the cells were variable in appearance and it would seem were originally interpreted as purely inflammatory. other cases in which the original and final diagnoses differed were more difficult to interpret. these cats included five in the group with degenerative changes; in these animals the lesion distribution or appearance suggested viral infections such as fiv (poli et al., 1997) , although gliosis did not appear to be a feature. in addition, there may have been overlap between the inflammatory lesions associated with fip virus and with other viral agents. other techniques (e.g., immunohistochemistry) might have helped to classify these cases further. in six cats there was evidence of more than one lesion pattern or disorder. five of these cats had characteristic lesions of fse as well as evidence of perivascular cuffing suggestive of concurrent viral infection. one of these, which had a high coronavirus antibody titre suggestive of infection by fip, was reported by reeves et al. (1998) to have given a positive polymerase chain reaction (pcr) result for borna disease virus (bdv) in one of three tests on brain tissue, but this was considered an equivocal finding. the sixth cat had inflammatory lesions consistent with fip, including dilation of the lateral ventricle. in addition the lateral ventricles contained nematode (toxocara) larvae. this was probably merely an incidental finding, but it may nonetheless have contributed to the ventricular dilation. the largest combined group of cats included those with evidence of inflammatory lesions within the cns. approximately 50% (47) had lesions consistent with a diagnosis of fip (krum et al., 1975; august, 1984) . of the remainder, the majority had a non-suppurative encephalitis or meningoencephalitis suggestive of viral infection (summers et al., 1995) . bacterial infection, toxoplasmosis and cryptococcosis also produce inflammation and degeneration within the cns and may be associated with infection by other viruses, e.g., fiv or feline leukaemia virus (felv) (gerds-grogan and dayrell-hart, 1997) . in cats with cns inflammatory lesions suggestive of viral infection, immunohistochemical and molecular techniques would probably be of diagnostic value. the largest single category consisted of 51 cats for which no cns abnormality could be detected. it is possible that an underlying physiological or metabolic defect resulted in neurological signs without producing recognizable histological changes (summers et al., 1995) , or that localized lesions occurred but were not included in the histological sections. congenital lesions, not surprisingly, were seen mainly in young cats, cerebellar hypoplasia and lysosomal storage disorders being the most common findings. gm 1 gangliosidosis was confirmed in two cats, one of which showed biochemical features similar to those of gm 1 gangliosidosis type 1 (barker et al., 1986) . feline dysautonomia, also seen mainly in young cats, was first recognized in 1981 (key and gaskell, 1982) . significant numbers of cats were affected in the early 1980 s, but cases are now diagnosed infrequently. the cause is not known but a toxic or infectious aetiology is most likely (summers et al., 1995) . the cns is rarely affected and the diagnosis is made from examination of ganglia. this was reflected in the present study. degenerative conditions of the cns encompass a diverse group of disorders, some of which may be due to infectious agents. for example, white matter vacuolation may be due to fiv infection (poli et al., 1997) . metabolic causes include hepatic encephalopathy, organophosphorus intoxication and ischaemia. a limited range of cns lesions may occur in response to a wide range of factors; this fact often complicates diagnosis (summers et al., 1995) . meningioma was the most frequently diagnosed primary tumour of the cns (12 cats out of 22). these neoplasms, which occurred in the cranium or spinal canal, or both, included two cases of multiple cranial meningioma. the distribution was similar to that found in other studies (gavin et al., 1995) . lymphoma, a common cns neoplasm, tends to occur in the spinal canal more often than the cranium (wheeler, 1989) . of the 18 lymphomas identified in this study: nine were confined to the spinal canal (seven being confined to the spinal canal or meninges, and two to the cord); four were confined to the brain; one was confined to the brain meninges; and four were present in both the brain and spinal canal. the present findings are therefore in accordance with those of wheeler (1989) . neoplastic cells may be present subdurally, causing compression of the cord and secondary degeneration, or may infiltrate the parenchyma causing direct damage. eight cats in this study had meningeal or extradural lymphomatous infiltrates with secondary cord compression. lymphoma affecting the cns is often part of a generalized disease (wheeler, 1989) . this detailed study confirmed that in cats with clinical signs of neurological disease the most frequent diagnostic category was "inflammation" (32%). in 17.8% of cases, however, histological changes were absent in the cns sections examined. in six cats there was evidence of more than one disease process. furthermore, there was no histological evidence of any cat with fse before recognition of the first case in 1990 (wyatt et al., 1990 . therefore, the original detection of fse was not due merely to increased awareness of tses: it was due to the sudden appearance of a disease that had not previously existed. in five of the cats found to have fse since the original cases reported by wyatt (1991) and wyatt et al. (1991 wyatt et al. ( , 1993 , there was also perivascular cuffing suggestive of concurrent viral infection. evidence of possible dual infection has been observed in cases of bse in cattle (s. ryder, personal observation). borna disease virus is associated with a non-suppurative meningoencephalitis in horses and other species (rott and becht, 1995) and is considered by some to be the cause of "staggering disease" in cats (lundgren et al., 1995) . no specific clinical signs or neurological lesions of borna disease have yet been demonstrated in cats in the united kingdom, but this requires further investigation. feline infectious peritonitis. an immune-mediated coronaviral vasculitis gm 1 gangliosidosis (type 1) in a cat transmission of feline spongiform encephalopathy to mice central nervous system tumors feline cryptococcosis: a retrospective evaluation encephalitis associated with giant cells in a cat with naturally occurring feline immunodeficiency virus infection demonstrated by in situ hybridisation dermoid cyst of the spinal cord associated with ataxia in a cat feline neurology. part 1. intracranial disorders the nervous system puzzling syndrome in cats associated with pupillary dilatation hydrocephalus associated with the non-effusive form of feline infectious peritonitis staggering disease in cats: isolation and characterisation of the feline borna disease virus feline spongiform encephalopathy: fibril and prp studies feline spongiform encephalopathy: a review neuropathology in cats experimentally infected with feline immunodeficiency virus: a morphological, immunocytochemical and morphometric study natural borna disease virus infection in cats in the united kingdom natural and experimental borna disease in animals feline dysautonomia (the key-gaskell syndrome): a clinical and pathological study of forty cases veterinary neuropathology, mosby-year book spinal tumours in cats naturally occurring spongiform encephalopathy in cats in the uk. australian veterinary practitioner feline spongiform encephalopathy spongiform encephalopathy in a cat naturally occurring scrapie like spongiform encephalopathy in five domestic cats we thank all the colleagues (clinicians and pathologists) who contributed to the cases used in this retrospective study. we are grateful to dr bryan winchester of the institute of child health for confirming the diagnosis of gm 1 gangliosidosis. key: cord-008530-yni0poh9 authors: asensio, valerie c.; campbell, lain l. title: chemokines and viral diseases of the central nervous system date: 2004-01-07 journal: adv virus res doi: 10.1016/s0065-3527(01)56006-6 sha: doc_id: 8530 cord_uid: yni0poh9 this chapter discusses chemokines and their receptors in the evolution of viral infectious diseases of the central nervous system (cns). infection of the human cns with many different viruses or infection of the rodent cns induces vigorous host-inflammatory responses with recruitment of large numbers of leukocytes, particularly t lymphocytes and macrophages. chemokines coordinate trafficking of peripheral blood leukocytes by stimulating their chemotaxis, adhesion, extravasation, and other effector functions. in view of these properties, research efforts have turned increasingly to the possible involvement of chemokines in regulating both peripheral tissue and cns leukocyte migration during viral infection. the biological effects of chemokines are mediated via their interaction with receptors belonging to the family of seven transmembrane (7tm)-spanning, g-protein coupled receptors (gpcrs). in the normal mammalian cns, the number of leukocytes present in the brain is scant. however, these cells are attracted to, and accumulate in, a variety of pathologic states, many involving viral infection. although leukocyte migration into local tissue compartments, such as the cns, is a multifactorial process, it has become clear that chemokines are pivotal components of this process, providing a necessary chemotactic signal for leukocyte recruitment. the central nervous system (cns) has been viewed as a relatively immune privileged organ. in physiologic states, the cns contains few, if any, leukocytes, and an absence of professional antigen-presenting cells (apc); it is barren for the expression of key immune accessory molecules such as major histocompatibility molecules (mhcs), and is fortified by an effective blood-brain barrier. nevertheless, the past decade has witnessed a revision of this concept, as evidenced by the fact that activated t lymphocytes, regardless of their antigen specificity, migrate effectively through the blood-brain barrier (bbb), but also by the fact that the cns contains numerous resident cells, such as microglia, cerebral endothelial cells, and astrocytes, that can, under the appropriate conditions, acquire the expression of immune accessory molecules and may function as apc (hickey, 1999; owens et al., 1994) . significantly, in numerous pathological conditions, immune cells are readily recruited to and accumulate in the cns. infection of the human cns with many different viruses (e.g., human t cell leukemia virus type i [htlv-i]; measles virus, human immunodeficiency virus [hiv] type 1; and herpes simplex virus [hsv] type 2), or of the rodent cns (e.g., theiler's murine encephalomyelitis virus [tmev] , mouse hepatitis virus [mhv] , lymphocytic choriomeningitis virus [lcmv] and vesicular stomatitis virus) induces vigorous host inflammatory responses with recruitment of large numbers of leukocytes, particularly t lymphocytes and macrophages. this host response can be a two-edged sword that, on the one hand, is needed to control and extinguish the invading virus but, on the other, can produce immune pathology and tissue injury resulting in mental and physical debilitation, and often death, of the host organism. therefore, increasing our knowledge of the mechanisms that control the trafficking of immune cells into the cns, and knowledge of the subsequent interactions between these cells that contribute to cns damage, is an important objective. our understanding of the factors that govern leukocyte trafficking has been advanced considerably with the discovery of a large superfamily of mostly small proteins (termed chemokines) that function as leukocyte chemoattractants in immunoinflammatory states (for reviews, see bacon and oppenheim, 1998; rollins, 1997; taub and oppenheim, 1994) . chemokines coordinate trafficking of peripheral blood leukocytes by stimulating their chemotaxis, adhesion, extravasation, and other effector functions. in view of these properties, research efforts have turned increasingly to a focus on the possible involvement of chemokines in regulating both peripheral tissue and cns leukocyte migration during viral infection. however, it is clear that the scope of chemokine involvement in the pathogenesis of host/viral interactions extends well beyond leukocyte migration. with studies on hiv-1 in the vanguard, new roles for chemokines and their receptors in viral infection became apparent with the demonstration that certain classes of chemokine receptors functioned as essential coreceptors for hiv-1 binding and entry into the cell. yet more recent work has indicated that the genomes of some large viruses, belonging to the herpesvirus and poxvirus families, contain homologs of the mammalian chemokines and their receptors. these homologs may function to modulate local inflammatory responses and favor viral survival and spread. from the host perspective, accumulating evidence indicates that chemokines are plurifunctional molecules that may have a significant impact on the cns, regulating cellular communication in the developing and the normal adult cns (asensio and campbell, 1999) . here, we review the subject of chemokines and their receptors in the evolution of viral infectious diseases of the cns. as a prelude to the chemokines. atac, activation-induced chemokine-related molecule exclusively expressed in cd8 ÷ t lymphocytes; bca-1/blc, b cell-attracting chemokine/b-lymphocyte chemoattractant; gro, growth-regulated oncogene; il-8, interleukin-8; ip-10, inflammatory protein 10 kd; i-tac, interferon-inducible t cell alpha-chemoattractant; lix, lps-induced cxc chemokine; mcp, monocyte chemotactic protein; mig, monokine induced by interferon-gamma; mip, macrophage inflammatory protein; mrp-1, miprelated protein 1; pbp, platelet basic protein; pf4, platelet factor 4; rantes, regulated on activation-normal t cell expressed and secreted; sdf-1, stromal cell-derived factor-1; slc, secondary lymphoid tissue chemokine; chr., chromosome. main subject of this chapter, our discussion initially will provide the reader with a general background to the rapidly expanding area of chemokines and their receptors and the expression of these molecules in the cns. the chemokines are currently divided into 4 families ( fig. 1) : the cxc or ~ family; the cc or ~ family; the c or 7 family; and the cx3c or 5 family (zlotnik et al., 1999) . in general, within each chemokine subfamily, the individual members show considerable homology in their 130 val]~rie c. asensio and iain l. campbell amino acid sequence and often possess overlapping chemoattractant specificity. chemokines share a common three-dimensional structure including an nh2-terminal loop and three antiparallel ~ sheets that are followed by a c-terminal a helix. the amino acid backbone of chemokines belonging to the cc, cxc, and cx3c families contains four conserved cysteines, while the c family chemokine, lymphotactin, contains only two cysteines, which correspond to the first and third cysteines in the other groups. chemokines can be produced by a variety of immune cells such as t lymphocytes, macrophages, and natural killer (nk) cells, but also by organ-specific cell types such as resident glial cells in the cns (see the discussion below). depending on the chemokine and its cellular source, the expression of chemokines may be constitutive or inducible, and a new classification of chemokines, defined according to their expression pattern, has been proposed (mantovani, 1999a; sallusto et al., 1999a) . the regulation of chemokine expression can be mediated by soluble factors such as inflammatory cytokines, e.g., interleukin (il)-i, tumor necrosis factor (tnf)-a, interferon (ifn)-7, as well as by microbial products, e.g., bacterial lipopolysaccharide (lps) or hiv coat protein gp120 (baggiolini, 1998; baggiolini et al., 1997; premack and schall, 1996; strieter et al., 1996) . in contrast, counterregulatory cytokines can downregulate the production of chemokines. for example, il-10 and transforming growth factor (tgf)-~ have been shown to inhibit lps-induced microglial cell rantes mrna expression and protein release (hu et al., 1999) . the glucocorticoid dexamethasone is also a particularly potent inhibitor for cc and cxc chemokine production (tobler et al., 1992; villiger et al., 1992) . the biological effects of chemokines are mediated via their interaction with receptors belonging to the family of seven transmembrane (7tm)-spanning, g-protein-coupled receptors (gpcrs). all chemokine receptors contain two conserved cysteines, one in the nh2-terminal domain and the other in the third extracellular loop; these are assumed to form disulfide bonds critical for the formation of the ligand binding pocket. in accordance with the chemokine classification, the receptors are divided into four major groups, cxcr, ccr, xcr, and cx3cr (table i) . individual chemokine receptors may often be promiscuous, meaning that they are capable of binding several different chemokines, and conversely, individual chemokines can often bind to several different receptors. interaction between a chemokine and its receptor leads to generation of a coordinated series of signaling events such as mobilization and influx of ca 2+ and also activation of various signal transduction pathways (premack and schall, 1996) . lymphotactin a chemokines in boldface type are cross-subfamily ligand-binding receptors. bca-1/blc, b cell-attracting chemokine/b-lymphocyte chemoattractant; elc, epstein-barr virus-induced receptor ligand chemokine; ena-78, epithelial cell-derived neutrophil-activating factor-78; gcp-2, granulocyte chemoattractant protein-2; gro, growth-regulated oncogene; il-8, interleukin-8; ip-10, inflammatory protein 10 kd; i-tac, interferoninducible t cell alpha-chemoattractant; larc, liver-and activation-related chemokine; lix, lps-induced cxc chemokine; mcp, monocyte chemotactic protein; mig, monokine induced by interferon-gamma; mip, macrophage inflammatory protein; mrp-1, miprelated protein 1; pbp, platelet basic protein; pf4, platelet factor 4; rantes, regulated on activation-normal t cell expressed and secreted; sdf-1, stromal cell-derived factor-l; slc, secondary lymphoid tissue chemokine; teck, thymus-expressed chemokine. protein 10 kda); and mig (monokine induced by interferon-y), (see fig. 1 ). the presence or absence of an elr motif (glutamic acid-leucinearginine sequence) preceding the first cysteine further subdivides the cxc chemokines into elr or non-elr groups. examples of some common elr chemokines include il-8, gro-a, and mip-2, while non-elr chemokines include ip-10, mig and sdf-1. this grouping not only reflects a structural compartmentalization, but also a functional one in that elr cxc chemokines are predominantly chemoattractant for polymorphonuclear leukocytes and bind to the cxcr1 and cxcr2 receptors, while non-elr chemokines are chemoattractant for t and b cells and bind to the cxcr3 or cxcr4 receptors. in addition, members of the cxc chemokine family can either promote or inhibit anglogenesis, depending on whether they are elr or non-elr cxc chemokines, respectively. the elr-chemokines are generally produced during inflammatory responses provoked by bacterial infections. on the other hand, production of the non-elr chemokines, ip-10/crg-2 and mig, is commonly associated with viral infections where they likely function to recruit t cells and macrophages that predominate at sites of infection (amichay et al., 1996; asensio and campbell, 1997; asensio et al., 1999a; carr et al., 1998; charles et al., 1999; fisher et al., 1995; lahrtz et al., 1997; nazar et al., 1997; vanguri and farber, 1994) . this scenario is borne out by the recent demonstration of pronounced antiviral activity of mig and ip-10 when it is expressed ectopically by recombinant vaccinia viruses (mahalingam et al., 1999) . the antiviral actions of mig and ip-10 in this setting are mediated indirectly and require nk cells and ifn-a, ifn-~, and ifn-y. sdf-1 was originally isolated from bone marrow stromal cells as a pre-b cell growth-stimulating factor. in contrast to ip-10 and mig, sdf-1 is produced constitutively in many organs (e.g., heart, lung, and cns) and binds to the cxcr4 receptor. animals lacking sdf-1 or its receptor invariably die at or soon after birth and have major abnormalities affecting the immune system (an impairment in b lymphocyte development) as well as various organs including the heart and cns nagasawa et al., 1996; tachibana et al., 1998; zou et al., 1998) . these studies revealed that sdf-1 is a crucial mediator of cellular migration and patterning during organogenesis. to date, 5 cxc chemokine receptors have been described and their ligands identified (see table i ) (baggiolini et al., 1997; mackay, 1997; moser et al., 1998) . most of the early characterization of the cxc chemokine receptors was done with the il-8 receptors cxcr1 (also known as il-8ra) and cxcr2 (also known as il-8rb). cxcr2 is expressed on a variety of cells including t cells, monocytes, melanoma cells, synovial cells, neutrophils, and myeloid precursor cells. expression of cxcr1 is restricted largely to myeloid cells and neutrophils. while cxcr2 binds all the elr-chemokines, cxcr1 binds only il-8. mice lacking cxcr2 show normal development of neutrophils but have decreased numbers of myeloid progenitors (broxmeyer et al., 1996; cacalano et al., 1994) . cxcr2-deficient mice do not exhibit any other overt pathologic abnormalities. cxcr3 is the receptor for several of the non-elr cxc chemokines, ip-10, mig and i-tac. surprisingly, some cc chemokines such as eotaxin, mcp-4 (weng et al., 1998) , and 6ckine (also called exodus 2 or slc) also bind to cxcr3. through this interaction, eotaxin does not activate the cxcr3 receptor but effectively blocks ip-10 binding and prevents receptor activation. thus, these cc chemokines may serve as natural cxcr3 antagonists highlighting complexity in the regulation of chemokine actions. human cxcr3 has been shown to be produced specifically by il-2-activated cd4 ÷ t cells, b cells, and nk cells. cd8 ÷ t cell and nk cell clones have also been shown to express the cxcr3 receptor . this suggests non-elr chemokines such as ip-10 may exert a selective influence on thl and cytotoxic lymphocyte and nk cell recruitment, which may be important in the development of innate and adaptive immune responses to viral infections. cxcr4 and cxcr5 bind the other non-elr chemokines sdf-1 and bca, respectively. cxcr4, also known as lestr or fusin, serves as a major coreceptor for lymphotropic strains of hiv-1 (deng et al., 1996; feng et al., 1996) and is expressed in a variety of tissues including brain, heart, liver, and colon and in nonhematopoietic cells (lavi et al., 1997; ohtani et al., 1998) . cxcr4 mrna expression is also found in murine lymphocytes, macrophages, neutrophils, and glial cells . as indicated above, disruption of the cxcr4 gene causes defects in vascular development, hematopoiesis, cardiogenesis, and neurogenesis of the cerebellum. cxcr5, originally described as blr-1 (burkitt's lymphoma receptor-l), is highly related to the il-8 receptors (dobner et al., 1992) . cxcr5 mrna is expressed by b cell lymphomas and by granule and purkinje cells of the cerebellum. therefore, in addition to regulation of recirculating mature b lymphocytes (forster et al., 1996; forster et al., 1994; kaiser et al., 1993) , this receptor may be involved in some cns functions. however, mice lacking cxcr5 show significant phenotypic changes in lymphoid organs but not in the brain, suggesting that this receptor, unlike cxcr4, does not play an obligatory role in cns development (forster et al., 1996) . the cc or ~-chemokines contain the largest number of members and are so named because the nh2-terminal cysteines are adjacent to each other. individual cc chemokines may exert their actions on multiple leukocyte subtypes, including monocytes, basophils, eosinophils, t cells, dendritic cells, and natural killer cells. well-known members of this family include mcp-1, mip-la, c10, eotaxin, and rantes. a more comprehensive list of the members of this family can be found in fig. 1 . one of the most studied chemokines of this family is mcp-1, which attracts t cells and monocytes but not neutrophils (carr et al., 1994; matsushima et al., 1989) . mice with targeted disruption of the mcp-1 gene have defects in monocyte/macrophage recruitment in a variety of inflammatory and immunological models. conversely, transgenic mice with expression of mcp-1 targeted to pancreatic islets (grewal et al., 1997) or oligodendrocytes in the brain (fuentes et al., 1995) show an accumulation of monocytes at these sites. interestingly, these accumulating monocytes showed little evidence of activation, indicating that mcp-1 is an effective monocyte chemoattractant but lacks the ability to further activate these cells. another well-studied cc chemokine is mip-la, which induces migration of monocytes, t lymphocytes, and eosinophils (baggiolini et al., 1994) . mice with a targeted disruption of mip-la show no obvious hematopoietic abnormalities; however, they are resistant to coxsackievirus-induced myocarditis and clear influenza at a delayed rate (cook et al., 1995) . to date, 9 cc receptors (ccr1-ccr9) have been identified (table i) . ccr1 was originally designated as an mip-la/rantes receptor (gao et al., 1993; gao and murphy, 1995; neote et al., 1993) and was later shown to also bind mcp-2 and mcp-3 (benbaruch et al., 1995) . the ccr1 receptor is mainly found on circulating mononuclear cells. mice lacking ccr1 (receptor for mip-la, mip-5, mcp-2, mcp-3, and rantes) have a phenotype similar to that of mip-la deficient mice with a defect in inflammatory responses to microbial challenge such as coxsackie b and influenza viruses (gao et al., 1997) . in addition, these animals have impaired trafficking of subsets of myeloid progenitor cells. the primary receptor for mcp-1, ccr2, is expressed on monocytes, myeloid precursor cells, activated t lymphocytes, and b lymphocytes but not on neutrophils (bonecchi et al., 1999; frade et al., 1997; myers et al., 1995) . mice with targeted disruption of the ccr2 receptor have defects in monocyte/macrophage recruitment in a variety of inflammatory and immunological models (boring et al., 1998; kurihara et al., 1997; kuziel et al., 1997) . ccr3, which is the eotaxin receptor, is prominently expressed by eosinophils and is involved in the recruitment of these cells during allergic reactions. ccr4 is the main receptor for rantes and mip-la, while ccr5, expressed on monocytes/macrophages, t cells, and granulocyte precursors (alkhatib et al., 1996; deng et al., 1996) , binds mip-la, mip-1~, and rantes. the receptors ccr6 and ccr7, originally identified as orphan receptors, bind mip-3a, and mip-3~ and 6ckine respectively yoshida et al., 1997) . ccr8 binds 1-309 (goya et al., 1998) , tarc, and mip-i~ and is expressed in the thymus (zingoni et al., 1998) . ccr9, also designated as gpr-9-6, was shown to specifically bind teck (zaballos et al., 1999) . compartmentalization of cc chemokine receptor expression is emerging as an important regulatory component during the adaptive immune response. ccr5, the receptor for rantes, mip-i~, and mip-1~ is expressed by memory and activated t cells but not by naive t cells (bleul et al., 1997) . moreover, ccr5 is preferentially expressed by human thl cells, in contrast to ccr4 and ccr3, which are found predominantly on th2 cells (bonecchi et al., 1998; qin et al., 1998) . concomitant with their differential chemokine receptor expression, thl and th2 cells selectively migrate in response to the corresponding chemokine ligand. more recently, sallusto et al. showed that memory t cells can be distinguished with respect to their effector function in peripheral organs by their expression of the chemokine receptor ccr7 (sallusto et al., 1999b) . ccr7 ÷ memory t cells retain lymph node homing receptors and lack immediate effector function, whereas ccr7memory t cells express receptors for migration to inflamed tissues and possess effector function. ccr8, like ccr4 (bonecchi et al., 1998) , is preferentially expressed by activated th2 cells. ccr8 may be important in lymphocyte development as well as in th2-immune responses. polarized th2 cells have been shown to differentially express the chemokine receptors, ccr8 (zingoni et al., 1998) , ccr4 (bonecchi et al., 1998; sallusto et al., 1999a) and ccr3 (sallusto et al., 1997) . in the murine system, a recent study showed that thl and th2 cells expressed comparable levels of ccr1, ccr2, and ccr4 mrna. similar to human t helper cells, murine thl cells preferentially expressed ccr7 and ccr5, whereas th2 cells expressed more ccr3 (randolph et al., 1999) . in conclusion, depending on their antigenic stimulation and/or polarization toward thl versus th2 subsets, chemokine receptors are differentially expressed, which, in addition to their characteristic cytokine profiles, may thus further distinguish these cells (mantovani, 1999b ). lymphotactin, the lone member of the c-chemokine family, is produced by activated mouse t cells (kelner et al., 1994) . lymphotactin is unique because (1) it has only two cysteines, the second and the fourth, of the four cysteines conserved in the cxc, cc, and cx3c chemokines; and (2) the c-terminal sequence is much longer than those of other chemokines. lymphotactin, and the subsequently reported single motif cysteine (smc) chemokine-1 and the activation-induced, t-cell-derived, and chemokine-related molecule (atac), are all identical (muller et al., 1995; yoshida et al., 1995) . lymphotactin expression on activation is extremely rapid, which suggests that it has a role in the early phases of an inflammatory response. when lymphotactin is injected into peritoneum, it causes an influx of t lymphocytes and nk cells by 24h (hedrick et al., 1997) . yoshida et al. (1998) identified an orphan receptor, gpr5 (heiber et al., 1995) , as being a high-affinity functional receptor for lymphotactin this receptor was renamed xcr1. the single member of the cx3c or 8 family, named fractalkine, is also unique with a novel arrangement of three amino acids separating the first two conserved cysteines. furthermore, fractalkine exists in both soluble and membrane-bound forms (bazan et al., 1997; pan et al., 1997) . this chemokine is found on activated endothelial cells and is tethered to the membrane by a mucinlike stalk that suggests a novel role for this chemokine in juxtacrine signaling. the interaction of endothelial-cell-expressed fractalkine with its receptor, cx3cr1, on leukocytes mediates the initial capture, firm adhesion, and activation of circulating leukocytes independently of integrin or adhesion molecule involvement (fong et al., 1998) . thus, fractalkine not only induces the migration of leukocytes but also facilitates their adhesion directly to the endothelium at sites of inflammation. fractalkine has been shown to foster the migration of monocytes, t cells, and nk cells (hedrick et al., 1997; kelner et al., 1994) . the receptor for fractalkine was originally cloned from a rat brainstem cdna library and named rbsll (harrison et al., 1994) . subsequently, the human receptor was identified as v28 (raport et al., 1995) , or cmkblr1, which is highly expressed in the brain and in neutrophils, monocytes/macrophages, and t lymphocytes (combadiere et al., 1995) . imai et al. (1997) identified this molecule as being the fractalkine receptor (cx3cr1) that is expressed mainly by nk cells and monocytes and to a lesser extent in cd8 ÷ t cells. many of the large dna viruses are infamous for their ability to undermine host immunity. two genres of viruses, the herpesviruses and poxviruses, were recently shown to contain open reading frames (orfs) that encode homologs of mammalian chemokines and chemokine receptors (for detailed reviews, see ahuja et al., 1994; dairaghi et al., 1998; lalani et al., 2000; murphy, 1994; smith et al., 1997) . kaposi's sarcoma-associated herpes virus (kshv) encodes three cc-like chemokines, vmip-i, vmip-ii, and vmip-iii. the vmip-i and vmip-ii show 60% sequence identity to each other and 43% and 52% identity to mip-la, while vmip-iii is more distantly related to its viral and mammalian counterparts (stine et al., 1999) . of the three viral chemokines, the function of vmip-ii is the best characterized. receptor binding studies suggest vmip-ii is a ligand for ccr3 (boshoff et al., 1997) . in addition to being a potent inhibitor of hiv infection mediated principally through this receptor (boshoffet al., 1997), vmip-ii is able to mobilize calcium and stimulate the chemotaxis of eosinophils where ccr3 is predominantly expressed (boshoffet al., 1997) . in contrast to this agonist action on ccr3, vmip-ii also binds with a high degree of affinity to a number of other cc (ccr1, 2, 5) and cxc chemokine receptors (cxcr4), where it acts as an antagonist (kledal et al., 1997) , as well as to cx3cr1 . human monocyte chemotaxis induced by rantes, mip-i(~, and mip-i[~ is inhibited by vmip-ii, suggesting that this viral mediator may be a broad-spectrum chemokine antagonist. recently, vmip-i was shown to selectively bind to ccr8 (a cc chemokine receptor associated with th2 lymphocytes), acting as an agonist and stimulating ca 2+ mobilization on human t cells . interestingly, vmip-ii also binds to ccr8; however, this is associated with antagonistic actions . kaposi's sarcoma is an angioproliferative disorder, so it is interesting that in addition to deviation of the host chemokine response, vmip-i and vmip-ii both induce angiogenesis in a chick chorioallantoic assay (boshoff et al., 1997) . thus these viral chemokines might contribute directly to the pathogenesis of the proliferative angiopathy in kaposi's sarcoma. in addition to these chemokine homologs, herpesviruses also contain open reading frames (orfs) that share significant sequence conservation with the chemokine receptors. kshv and herpesvirus saimiri (hvs) contain orf 74 in hsv, whose sequence is most similar to the human il-8 receptors cxcr1 and cxcr2 (ahuja and murphy, 1993) . like cxcr1 and cxcr2, orf 74 binds il-8 as well as the other elr cxc chemokines, gro-(z and pf-4 (ahuja and murphy, 1993) . however unlike cxcr1 and cxcr2, orf 74 can signal in a constitutive agonist-independent manner and stimulate the proliferation of kidney fibroblasts (arvanitakis et al., 1997) . in addition to the stimulation of proliferation of nontransformed cells, orf 74 signaling may also lead to cellular transformation and tumorigenicity (bais et al., 1998) . thus, it would appear that orf 74 may contribute to the development of the transformed cell phenotype found with kshv-infected cells in kaposi's sarcoma lesions. cytomegalovirus (cmv) is a common opportunistic pathogen of immunocompromised hosts in whom the virus exhibits tropism for leukocytes. human and murine cmv possess genes that encode a variety of chemokine homologs (macdonald et al., 1997) . the murine cmv chemokine 1 and 2 (mck-1 and mck-2) peptides induce calcium signaling and adherence in peritoneal macrophages (saederup et al., 1999) . the human cmv gene ul146 encodes a protein designated as vcxc-1 and provides the first example of a virus-encoded cxc chemokine (penfold et al., 1999) . the vcxc-1 glycoprotein shows high-affinity binding to the cxcr1 and cxcr2 chemokine receptors, inducing calcium mobilization and chemotaxis of neutrophils. thus, the common property of the cmv chemokine homologs is to function as chemokine agonists. this in turn may promote leukocyte migration to sites of infection and may be responsible for more efficient dissemination of cmv in the host. in support of this notion, mck-1/mck-2 mutant cmv displays markedly reduced peak levels of monocyte-associated viremia in experimentally infected mice (saederup et al., 1999) . a gene encoding a viral homolog of ccr1 was found within the genome of the human cmv. this gene, orf us28, of human cytomegalovirus (hcmv) was shown to bind mip-la,-~, mcp-1, rantes , and mcp-3 (bodaghi et al., 1998) with higher affinity than their cognate receptors. thus, it is attractive to speculate that hcmv-infected cells expressing us28 may be used for sequestration of chemokines from the extracellular milieu. us28 can also serve as a coreceptor for hiv infection of cd4 t cells and may facilitate an interplay between hiv and hcmv (pleskoffet al., 1997) . the poxvirus molluscum contagiosum virus (mcv) infects humans, causing papules of the skin associated with virtually no immune response to the virus in the skin lesion. the genome of mcv contains an orf that encodes a protein (mc148r) that is a cc chemokine homolog (senkevich et al., 1996) . mc148r has homology to mip-la/[3 but does not induce chemotaxis and antagonizes mip-la-induced chemotaxis (krathwohl et al., 1997) . furthermore, mc148r inhibits the leukocyte response to several cc and cxc chemokines (damon et al., 1998) , indicating that it can act as a broad-spectrum chemokine antagonist. therefore, a primary function of this virus-encoded chemokine homolog might be to provide an immune evasion strategy that acts to limit the recruitment of effector leukocytes to mcvinfected epidermal cells. in conclusion, unquestionably these large dna viruses encode functional homologs of chemokines and chemokine receptors. although the precise role of the viral chemokine and chemokine receptor homologs (discussed above) in infectious processes remains to be determined, they clearly could contribute to the strategies employed by these viruses to subvert host immunity. the examples discussed here indicate that these strategies are diverse and, depending on the virus, range from wholesale suppression of leukocyte trafficking--providing immune evasion--to promoting selective leukocyte chemotaxis fostering the more efficient infection and dissemination of virus via its preferred target host cell. a concerted research effort has been mounted recently to ascertain the role of chemokines in immunoinflammatory disorders of the cns, such as multiple sclerosis (ms). as a result, it has become clear that most cells resident in the cns, including neurons, astrocytes, and microglia, can synthesize and secrete various classes of chemokines. importantly, these same cells are endowed with a variety of different chemokine receptors and therefore have the potential to respond to chemokines in their local environment. as recently speculated by us (asensio and campbell, 1999) and by others (mennicken et al., 1999) , it is likely the cns has its own chemokine ligand/receptor network, the function of which could extend well beyond the regulation of leukocyte trafficking in antiviral and other neuroimmune responses (see fig. 3 ). at present, the genes for only three chemokines, mcp-1, sdf-1, and fractalkine, are known to be expressed constitutively in the cns. evidence suggests mcp-1 is expressed in the cortex and hippocampus of the rat brain during development as well as in the adult animal (pousset, 1994) . more recently, mcp-1 expression at the protein level was shown in the human cerebellum, medulla oblongata, and pons (meng et al., 1999) . however, the function of constitutively expressed mcp-1 in the brain is unknown. the sdf-1 gene is found under physiological conditions in many organs including murine (tashiro et al., 1993) and human (shirozu et al., 1995) brain. sdf-1 has two isoforms, sdf-la and sdf-i~, that are produced by alternative splicing from a single gene (tashiro et al., 1993) . sdf-la rna is present at high levels in cultured rat astrocytes, at low levels in neurons, and is not detectable in microglia, while sdf-1~ rnais found at low levels in all these cell types . tanabe et al. (1997b) showed that sdf-la was able to induce migration of microglial cells but not astrocytes, even though the sdf-1 receptor, cxcr4, was expressed on both cell types. furthermore, human sdfla was able to induce calcium flux in cultured astrocytes (bajetto et al., 1999) . it was hypothesized that astrocyte-secreted sdf-1 could facilitate interneural cell communication . this is certainly the case during cns development, since, as noted above, knocking out cxcr4 results in a major defect in granule cell migration and cerebellar development zou et al., 1998) . the cx3c chemokine fractalkine is also found constitutively in the brain (bazan et al., 1997; pan et al., 1997) . fractalkine mrna is expressed at high levels by neurons in the olfactory bulb, cerebral cortex hippocampus, caudate putamen, and nucleus accumbens nishiyori et al., 1998; schwaeble et al., 1998) . in vitro studies have confirmed that fractalkine is constitutively expressed by neurons and, further, can be induced in astrocytes by tnf-a and il-i~ (maciejewski-lenoir et al., 1999) . in a model of peripheral nerve injury, harrison et al. have shown that fractalkine and its receptor were highly upregulated in the brain, with motor neurons being the primary source of the chemokine. fractalkine treatment of primary cultured rat microglial cells induces vigorous increases in intracellular ca 2+ levels and chemotaxis, which are inhibited by an antibody against the fractalkine (cx3cr1) receptor . although the precise function of fractalkine in the cns awaits clarification, the localization of the fractalkine receptor on microglia and the expression of its ligand by neurons clearly establish a spatial framework that could facilitate juxtacrine communication and migration between these cells (see fig. 3 ) nishiyori et al., 1998) . a majority of chemokines are not detectable in the cns under physiological conditions, but are present during diverse pathologic states including, as discussed below, viral diseases. it is beyond the scope of this chapter to detail all of the findings in these different pathologic states and the reader is encouraged to consult many excellent recent reviews dealing with this subject (asensio and campbell, 1999; glabinski and ransohoff, 1999; karpus and ransohoff, 1998; mennicken et al., 1999; ransohoff, 1997; ransohoff and tani, 1998) . it is clear from most, if not all, cases examined that glial cells represent an important source for the localized production of specific chemokines. for example, in ms, expression of ip-10 (balashov et al., 1999; sorensen et al., 1999) and mcp-1 (simpson et al., 1998; van der voorn et al., 1999) is prominent in astrocytes while mip-i(~ expression is strongly associated with macrophage/microglia (balashov et al., 1999) . in support of these clinicopathologic findings, studies in vitro demonstrate that expression of various cc and cxc chemokines can be induced in astrocytes and microglia by proinflammatory cytokines and lps. although differential expression of mcp-1 by astrocytes, and of mip-i(z by microglia, was reported for murine glial cells (hayashi et al., 1995) , this was not the case with human microglia, with expression of both these chemokines being induced in these cells by lps (mcmanus et al., 1998) . similarly, expression of ip-10 by murine astrocytes and microglia can be induced by ifn-~/or lps (luo et al., 1998; majumder et al., 1998; ren et al., 1998; vanguri, 1995) . in contrast to the glial cells, few inducible chemokines have been reported to be expressed by neuronal cells either in vitro or in vivo. how does cns chemokine expression contribute to the pathogenesis of neuroinflammatory disease states? clearly, one key role involves recruitment of leukocytes to the brain. differences in the chemokine gene expression patterns can be found in different neuroinflammatory diseases and correlate with a bias toward the involvement of particular leukocytes. in experimental and clinical bacterial meningoencephalitis, where cns lesions consist predominantly of neutrophils and monocytes, there is dominant cerebral production of the neutrophil and monocyte attractant chemokines il-8, mip-2, and groa, and mcp-1, mip-i~, and mip-i~, respectively (lahrtz et al., 1998; spanaus et al., 1997; sprenger et al., 1996) . in contrast, as discussed below, in viral meningoencephalitis, mostly lymphocytes and monocytes are found in the cns, in parallel with the cerebral expression of the chemokine genes encoding ip-10, mcp-1, and rantes, which are effective lymphocyte and monocyte chemoattractants (asensio and campbell, 1997; lahrtz et al., 1997) . transgenic studies show that individual chemokines can have a specific chemoattractant "signature." thus, cns expression of either the groa genes (tani et al., 1996) or the mcp-1 genes (fuentes et al., 1995) under the control of the myelin basic protein (mbp) promoter induces robust leukocyte infiltration of the cns that is composed predominantly of either neutrophils or monocytes, respectively. studies with intracerebral microinjection also demonstrate that the acute presence of chemokines in the cns leads to robust and cell-specific leukocyte recruitment. for example, mip-2 is more potent than il-8, ip-10, or mcp-1 in stimulating polymorphonuval]~rie c. asensio and iain l. campbell clear leukocyte recruitment to the brain (bell et al., 1996) , while the cc chemokine c10, when injected into the lateral ventricle, is able to induce a recruitment of macrophages, but not t cells (asensio et al., 1999b) . finally, neutralization of the chemokine mip-la, which is expressed in eae, is associated with a reduction in disease severity and in cns inflammation (karpus et al., 1995) . in all, these studies indicate that the qualitative makeup of chemokine production in the cns during disease can dictate the recruitment of selected leukocytes to the cns, and that targeting these molecules may provide an effective therapeutic approach to suppress cns inflammation. the cc and cxc receptors are expressed on a number of different cell types and their expression is often regulated by exogenous and endogenous stimuli. numerous in vitro and in vivo studies show that different neural cells express a variety of chemokine receptors under normal conditions and could therefore facilitate a direct interaction of these cells with chemokines. neurons have prominent expression of many chemokine receptors including cxcr2, cxcr4, ccr1, ccr4, ccr5, ccr9/10, cx3cr1, and duffy antigen-related chemokine (darc) (horuk et al., 1997; klein et al., 1999; lavi et al., 1998; meucci et al., 1998; sanders et al., 1998; vallat et al., 1998) . immunohistochemical staining of the human brain revealed cxcr1 is not detectable, whereas cxcr2 is expressed at high levels by subsets of projection neurons in different regions of the brain including the hippocampus, dentate nucleus, pontine nuclei, locus coeruleus, and paraventricular nucleus, and also in the spinal cord (horuk et al., 1996; horuk et al., 1997) . darc is expressed by subsets of endothelial cells and purkinje cells in the cerebellum, suggesting that this enigmatic receptor that lacks signaling function may have multiple roles in normal and pathological physiology, perhaps as a chemokine clearance receptor (horuk et al., 1996; horuk et al., 1997) . functional ccr3, ccr5, and cxcr4 receptors, with different patterns of distribution, were demonstrated on cultured fetal human and macaque neurons (klein et al., 1999) . expression of cxcr4 has also been detected by immunohistochemical staining on subpopulations of neurons in the normal adult brain (lavi et al., 1997; sanders et al., 1998; vallat et al., 1998) , while in the rat, levels of the cxcr4 receptor rna are higher on specific neurons, which include cerebellar purkinje cells, hippocampal hilar neurons, and cerebral cortical neurons (wong et al., 1996) . a clear theme emerging from all these studies is that neurons express a variety of chemokine receptors and that this expression in the cns exhibits marked regional and cellular diversity, suggesting that chemokines could have differential effects on different neurons. other than neurons, glial cells also express a variety of chemokine receptors. the fractalkine receptor cx3cr1 is found at high levels in rodent and human brain and shows strong localization to microglia (combadiere et al., 1998; harrison et al., 1998; imai et al., 1997) . the fact that fractalkine is expressed by neurons (as noted above), and its receptor by microglia, suggests a possible important role for this chemokine and its receptor in communication between neurons and microglia. a similar scenario may follow with sdf-1, whose receptor, cxcr4, in addition to expression by neurons, is found on astrocytes (heesen et al., 1996) and on microglial cells (tanabe et al., 1997b) . finally, the cc chemokine receptors ccr1, ccr5, and ccr3 have been shown to be expressed by astrocytes and/or microglial cells both in vitro and in vivo (boddeke et al., 1999; he et al., 1997; lavi et al., 1998; tanabe et al., 1997a; vallat et al., 1998; westmoreland et al., 1998; xia et al., 1998) . the expression of many of these chemokine receptors in the cns is not static and can be dynamically regulated by various stimuli. for example, in a model of excitotoxic brain injury induced by intracerebral injection of nmda in the rat, marked upregulation of ccr5 expression was observed by microglia and neurons (galasso et al., 1998) . in brain from alzheimer's cases, plaque-associated reactive microglia also show increased ccr5 expression . expression of the fractalkine receptor cx3cr1 is increased on perineural microglia following facial nerve axotomy in the rat . boddeke et al., have shown, by rt-pcr (reverse transcription-polymerase chain rear) analysis, that ccr1, ccr2, and ccr5 expression in cultured rat microglial cells is significantly upregulated after lps treatment (boddeke et al., 1999; spleiss et al., 1998) . the pathophysiological significance of altered cns chemokine receptor expression in disease states remains to be determined. however, it might be speculated that this provides yet another level of regulation in the already complex cellular communication processes mediated by the chemokines. cns infection, with a number of different classes of viruses, can provoke vigorous inflammatory responses with subsequent recruitment of large numbers of leukocytes (for a detailed discussion of this topic, see chapter 6 in this volume). in view of their functional properties, increasing interest has focused on the possible involvement of chemokines in regulating cns leukocyte migration during viral infection. however, as discussed below, studies of hiv-1 have revealed that chemokine involvement in the pathogenesis of host-virus interactions extends well beyond leukocyte migration. the lentivirus hiv-1 and its simian counterpart, simian immunodeficiency virus (siv), both infect the cns and are responsible for causing neurologic disease. in the case of hiv, a vast amount of research effort focused on this virus led to the discovery of new chemokine receptors that proved to be critical for understanding the process of hiv-1 entry into cells (bleul et al., 1996; feng et al., 1996; oberlin et al., 1996) . the interactions between this virus and the host cns have also been studied extensively, and more recently the focus has turned to the chemokines and their receptors. as discussed below, the findings point to a significant role for the chemokines and the chemokine receptors in hiv-1 neuropathogenesis. although cd4 was identified initially as a primary receptor for hiv-1 binding and entry, it was clear this could not account for many of the characteristics of hiv-1 infectivity, such as the existence of different strains of hiv-1 with selective tropism for either t lymphocytes (ttropic) or monocytes (m-tropic). a clue to the identity of other possible cofactors for hiv-1 entry into cells came with the discovery that the chemokines mip-la, mip-i~, and rantes produced by cd8 ÷ t cells could suppress hiv-1 infection of cultured cells (cocchi et al., 1995) . a seminal advance then came when it was shown that in addition to cd4, cxcr4 was required for t-tropic hiv-1 strains to infect host cells (bleul et al., 1996; feng et al., 1996; oberlin et al., 1996) . the identification of ccr5 as a receptor for mip-la, mip-i~, and rantes soon led to the demonstration that ccr5 served as a prominent coreceptor for cell entry by m-tropic strains ofhiv-1 (alkhatib et al., 1996; choe et al., 1996; deng et al., 1996; doranz et al., 1996; dragic et al., 1996) . the factors that govern hiv-1 entry into cells are clearly very complex, and while cxcr4 and ccr5 are the major coreceptors for ttropic and m-tropic hiv-1 strains, a large number of other chemokine receptors including ccr2b, ccr3, and ccr8, the us28 chemokine receptor homolog encoded by cytomegalovirus, as well as the orphan chemokine receptors gprl, gpr15/bob, and strl33/bonzo, can function as coreceptors for hiv-1 infection (deng et al., 1997; heiber et al., 1996; liao et al., 1997; littman, 1998; marchese et al., 1994; pleskoffet al., 1997) . the importance of the chemokine receptors for hiv-1 infection in vivo is illustrated in the case of individuals that are homozygous for a 32 base pair mutation in the ccr5 gene and who have marked resistance toward hiv infection (biti et al., 1997; o'brien et al., 1997; theodorou et al., 1997) . in addition, it has been reported that individuals with mutations in the cxcr4 and ccr2 genes also exhibit a retarded progression of hiv-l-associated disease . a significant number of patients with hiv-1 infection suffer from a variety of neurological problems known collectively as hiv-l-associated cognitive and motor disorder, or "neuroaids" (see chapter 18 in this volume). in its severest form, neuroaids can be responsible for subcortical dementia, memory impairment, and motor disorder. although the neuropathology of neuroaids is often variable, typical features include formation of multinucleated giant cells, diffuse gliosis, myelin pallor, and increased blood-brain barrier permeability. neurodegeneration, with disrupted synaptodendritic organization and loss of neurons, is observed in many but not all cases (everall et al., 1993) . the cause of neuroaids and the mechanisms that lead to brain injury are not well understood, although a combination of both host-and hiv-derived toxic factors is likely to be involved (gendelman et al., 1997) . it is clear, however, that productive hiv-1 infection of the cns is largely restricted to macrophage/microglia and infrequently to neurons or astrocytes. infection of the cns is thought to result from the entry of hiv-l-infected lymphocytes or macrophages--the subsequent presence of hiv-1 proteins in microglia and multinucleated giant cells suggests spread of the virus from the infiltrating infected leukocytes. as discussed above, resident cells of the cns are well endowed with a variety of different chemokine receptors. the levels and type of coreceptot expression are likely to be pivotal determinants of hiv-1 tropism in the brain. the coreceptors for m-tropic variants of hiv-1, ccr5, ccr3, and cxcr4 are expressed by microglia (he et al., 1997; lavi et al., 1997; vallat et al., 1998) . studies with primary cultures of human microglia have shown that ccr5 and ccr3 can serve as coreceptors with cd4 for hiv-1 infection, while cxcr4 is relatively ineffective in this capacity. microglia have very low surface-expressed cd4 (buttini et al., 1998) and this may thus preclude the efficient use of cxcr4 as a coreceptor. hiv-1 infection of microglial cultures can be inhibited by the cognate ligands mip-i~ (ccr5) and eotaxin (ccr3), further verifying the key role of the microglial-expressed cc-chemokine receptors for hiv-1 entry into cells. interestingly, the use of both ccr3 and ccr5 for hiv-1 infection of microglia contrasts with blood-derived macrophages in which ccr5, but not ccr3, functions as a coreceptor (alkhatib et al., 1996; deng et al., 1996; wu et al., 1997) . this suggests that m-tropic hiv-1 variants may arise in the brain that have distinct coreceptor requirements. in support of this, hiv-1 in the brain is typically m-tropic (korber et al., 1994; power et al., 1994) . moreover, he and colleagues (1997) demonstrated that yu2 and jrl env proteins that are cloned directly from hiv-l-infected brain use ccr5 or ccr3, together with cd4, for virus entry into transfected cells. a further determinant of hiv-1 entry and infection in the cns may be the chemokines themselves. increased levels of mip-la, mip-i~, rantes, mcp-1, ip-10, and sdf-1 have been reported in the brain or the csf of individuals with neuroaids (conant et al., 1998; kelder et al., 1998; kolb et al., 1999; letendre et al., 1999; sanders et al., 1998; schmidtmayerova et al., 1996; zheng et al., 1999b) . hiv-1 may directly regulate the expression of these chemokines. thus, infection of cultured microglia and/or astrocytes markedly increases the expression of mip-la and mip-i~ (schmidtmayerova et al., 1996) , while expression of sdf-1 rna decreases in hiv-l-infected microglia but increases in similarly infected astrocytes (zheng et al., 1999b) . in recent unpublished studies, we have observed induction of ip-10 gene expression in astrocytes in the brain of transgenic mice with astrocyte-targeted expression of hivgpl20. these transgenic mice exhibit similar neurodegenerative and other pathologic changes (e.g., astrocytosis) to neu-roaids (toggas et al., 1994) --our findings suggest that a further action of hivgpl20 is the induction of ip-10. the cns expression of ip-10 and other chemokines, such as mcp-1, correlates with viral load and increases with the progression of brain injury, indicating a possible role for these chemokines in the pathogenesis of neuroaids (kelder et al., 1998; kolb et al., 1999) . other than their potential contribution to brain injury (discussed in more detail below), the increased presence of these chemokines in the cns during hiv-1 infection may promote, via their chemoattractant activity, the further recruitment and accumulation of t lymphocytes and macrophages in the brain. in support of this idea, hiv-tat, protein-induced mcp-1 expression by astrocytes can stimulate the transmigration of monocytes across an in vitro model of the human bbb (weiss et al., 1999) . finally, since mip-la and rantes can effectively inhibit ccr5 coreceptor-mediated cell entry by hiv-1 (cocchi et al., 1996; dragic et al., 1996) , expression of these and other chemokines in the cns in neu-roaids might limit the spread of hiv-1 by antagonizing infection of target microglia. the wide distribution of chemokine receptors in the normal and hiv-infected human brain, as well as the increased expression of their cognate ligands, not only provides an avenue for hiv entry into the brain via the microglia, but might also contribute to the neuronal injury and loss that ensue. as indicated above, current evidence supports the idea that neuronal injury and death in neuroaids are consequences of indirect mechanisms involving a variety of host-derived molecules for example, cytokines and excitotoxic amino acids or hivencoded factors such as gpl20; it is probable that the host and viral products work in combination. loss of neurons, astrocytes, and microglia through apoptosis appears to be prominent in neuroaids (adle-biassette et al., 1995; gelbard et al., 1995; shiet al., 1996) and can be induced by hiv-1 infection of these cells in vitro (shi et al., 1996) . analysis of a panel of diverse hiv-1 primary isolates or strains revealed that, compared with blood-derived or t-tropic hiv-1 isolates, brain-derived m-tropic hiv-1 isolates are ineffective at inducing neuronal and astrocyte apoptosis (ohagen et al., 1999; zheng et al., 1999b) . thus, the primary determinant of neurodegeneration may not be the m-tropic hiv forms constituting the major cns reservoir of hiv-1, but rather, it resides with blood-derived viruses that are prominent during later stages of disease. furthermore, the env v3 region is the major determinant of neuronal apoptosis and also determines chemokine coreceptor usage for infection (ohagen et al., 1999) , implicating these viral coreceptors in the effector pathways that mediate neuronal damage during neuroaids. as mentioned above, the blood-derived, t-tropic hiv-1 isolates rely primarily on cxcr4 as a coreceptor, whereas the brain-derived, mtropic isolates use ccr5 and ccr3. in view of the congruity between the induction of neuronal apoptosis and chemokine coreceptor usage, and the fact that neurons express cxcr4 (as noted above), many recent studies have focused on the possible role of cxcr4 in mediating brain injury in neuroaids. hesselgesser and colleagues showed that signaling by cxcr4, following the binding of t-tropic hivgpl20, activates apoptotic cell death in human hnt neuronal cells (hesselgesser et al., 1998) . this does not appear to reflect an unusual property of hnt cells, which have a transformed phenotype, as induction of apoptotic cell death is also induced in rat hippocampal neurons exposed to hivgpl20 (meucci et al., 1998) . in these latter experiments, pretreatment of the neurons with the cognate ligand for cxcr4, sdf-1, partially protected against gpl20-induced neuronal apoptosis, suggesting that the effects of the viral product are mediated, in part, by cxcr4 binding. more recently, purified virions from t-tropic, but not m-tropic, hiv isolates were shown to induce cd4 independent neuronal signaling and apopto-sis that could be blocked by antibodies to cxcr4 (zheng et al., 1999a) . in these studies, the purified t-tropic hivgpl20 protein proved to be far less effective in affecting neuronal signaling and cell death than the whole virions. since the effects of the virions could be blocked by antibodies to gpl20 and gp41, the findings indicate that the envelope proteins in their native state are more potent mediators than their recombinant or purified derivatives. in all, the implication from these studies, which utilized either human neuronal cell lines or highly purified rodent or human primary neuronal cultures, is that direct binding of hiv gpl20 to the cxcr4 found on neurons activates apoptotic cell death in these cells. contrary to this notion, in mixed brain-cell cultures derived from embryonic rat cerebrocortex, gpl20-induced neuronal apoptosis was completely blocked by the tripeptide tkp, which blocks macrophages microglial activation (kaul and lipton, 1999) . this blockade by tkp was specific for gpl20 as sdf-l-induced neuronal apoptosis (as noted below) was unaffected by the peptide. interestingly, gpl20induced neuronal apoptosis in this model system was also blocked by the cc chemokines rantes and mip-i~, which bind to ccr5, suggesting there may be regulatory cross talk between the ccr5 and cxcr4 receptors. however, the conclusion from this study is that gpl20-induced neuronal apoptosis depends predominantly on an indirect pathway via activation of chemokine receptors on macrophages microglia. many possible explanations exist as to why there are differences between these studies with respect to the observed effects of gpl20's being conveyed by direct or indirect pathways. these include differences in the species and types of neuronal or brain-cell cultures used and the types, form, and levels ofgpl20 added to the cultures. while the question of whether the induction of neuronal apoptosis by gpl20 is consequent to interaction directly with the cxcr4 receptor on neurons or occurs indirectly, following activation of the macrophage/microglia, is unresolved, there is general agreement that hivgpl20 binding to cxcr4 can regulate a number of cellular signaling pathways (kaul and lipton, 1999; meucci et al., 1998; zheng et al., 1999a; zheng et al., 1999b) . these include inhibition of cyclic amp; activation of phosphoinositol (pi) hydrolysis, which is partially prevented by antibody to cxcr4 (zheng et al., 1999a; zheng et al., 1999b) ; activation of p38 mitogen-activated kinase (p38 mapk) (kaul and lipton, 1999) ; and increased ca 2+ mobilization (meucci et al., 1998) . hiv gpl20 neuronal apoptosis can be inhibited by inhibitors of p38 mapk (kaul and lipton, 1999) and by calcium/calmodulin-dependent protein kinase ii, protein kinase a, and protein kinase c (zheng et al., 1999a) , linking neuronal apoptosis, in part, to these signal transduction pathways. the preceding discussion indicates that hiv gpl20 neurotoxicity likely involves, in part, the chemokine receptor cxcr4, raising the possibility that hiv-1 subverts a key physiological cell death pathway. as we noted above, sdf-1/cxcr4 signaling is crucial for normal cerebellar neuronal migration and patterning during cns development. sdf-1 is expressed constitutively in the adult cns predominantly by astrocytes and to a lesser degree by neurons , raising the likelihood that this chemokine may regulate physiological processes in the adult brain. in support of this, sdf-la regulates a number of signal transduction pathways in neurons, causing decreased cyclic amp, increased pi hydrolysis, activation of p38mapk, and increased ca 2+ mobilization (kaul and lipton, 1999; meucci et al., 1998; zheng et al., 1999a) . functionally, exposure of hippocampal neurons to sdf-1 results in increased synaptic transmission, which is blocked by antibodies to cxcr4 (zheng et al., 1999a) . the similar effects of sdf-1 and hiv gpl20 in regulating cxcr4 signaling raise the question as to whether sdf-1 might also induce neuronal apoptosis. here, the findings have been conflicting. four studies revealed that sdf-1 promoted increased neuronal apoptosis (hesselgesser et al., 1998; kaul and lipton, 1999; zheng et al., 1999b) and activation of a key cell death gene, caspase 3 (zheng et al., 1999a) . in contrast to these findings, meucci and colleagues reported that, following treatment with sdf-1, there was marked protection of neurons from culture-and hiv gpl20-induced apoptosis (meucci et al., 1998) . the reason for these discrepancies is not known but again likely reflect differences in cell preparations and culture conditions. similar to hiv, siv infection of macaques can lead to the development of an aids-associated encephalitis that is virtually indistinguishable from neuroaids (raghavan et al., 1999; sasseville and lackner, 1997) . both t-tropic and m-tropic siv variants have been isolated and, like neuroaids, macrophage/microglia constitute the primary infected target cell population in the brain of siv-infected monkeys. the host immune system plays a major role in the infection of the cns with siv, where perivascular macrophages are continuously replaced via recruitment from the circulating monocyte pool through the blood-brain barrier (lane et al., 1996) . thus, infected t cells and monocytes likely enter the cns parenchyma during the asymptomatic phase of infection. in addition to leukocyte and endothelial adhesion molecules, chemokines could be critical components involved in siv infection of the brain. using immunohistochemical staining to investigate chemokine expression in the encephalitic brain of siv-infected macaques, sasseville and coworkers reported upregulation in the expression of a number of chemokines, including mip-la, mip-i~, rantes, mcp-3, and ip-10, by vascular endothelium and/or perivascular mononuclear cells . in contrast to neuro aids, mcp-1 was not elevated in the siv-encephalitic monkey brain. with the exception of mcp-1, expression of these proinflammatory chemokines in the siv-infected brain is similar to that found in the human brain in neuroaids (as noted above). however, like neu-roaids, the in vivo function of these chemokines in siv-encephalitis remains unknown; it is reasonable speculation however, to suggest that leukocyte recruitment to the siv-infected cns may be directed by these chemokines. in a follow-up study by this group, examining chemokine receptor expression in siv-aids encephalitis, the chemokine receptors ccr3, ccr5, cxcr3, and cxcr4 were found to be expressed by inflammatory cells within perivascular lesions (westmoreland et al., 1998) . in addition, expression of ccr3, ccr5, and cxcr4 was detected on subpopulations of large hippocampal and neocortical pyramidal neurons and on glial cells in both the normal and the siv-encephalitic brain. again, these findings show an overlap with those reported for the normal and the hiv-infected brain in humans (as noted above). m-tropic strains of siv utilize ccr5 as a coreceptor with cd4 for infection of macrophages deng et al., 1997) . expression of ccr5 is enriched on microglial cells in the macaque brain and may therefore provide the molecular framework for infection by siv in the monkey cns. overall, the preceding discussion highlights the overlapping expression patterns and potentially similar roles for the chemokines and their receptors in the pathogenesis of siv-encephalitis and neu-roaids. this idea is further underscored by a recent study in which chemokine receptor expression and signaling were examined in cultured neurons derived from either macaque or human brains (klein et al., 1999) . thus, both the qualitative pattern of expression and the functional behavior of the chemokine receptors, ccr3, ccr5, and cxcr4, in the two species are remarkably alike. in humans, multiple sclerosis is the most common and the bestknown cns demyelinating disorder. ms is thought to be an autoim-mune t-cell-mediated disease that is targeted at the myelin sheath. the etiology of ms is unknown (hailer, 1999) , although at one time or another, different viruses have been implicated in its pathogenesis, including epstein-barr virus (ebv), measles virus, and recently, hhv-6. the human t lymphotropic virus type i (htlv-1) infection of the cns can cause a progressive inflammatory demyelinating disease and shares many similarities with ms. in view of a possible viral role in ms, research has focused on several experimental viral models associated with the development of neuroimmune responses and primary demyelination that show many clinicopathologic similarities with ms. among the best-known and most widely studied of these experimental models are theiler's murine encephalomyelitis virus (tmev)-and murine hepatitis virus (mhv)-induced demyelinating disease. htlv-1 is the etiologic agent for adult t cell leukemia (atl) and for ham/tsp, which shows similar features to primary progressive ms (calabresi et al., 1999) . ham-tsp is a chronic progressive neurological disorder characterized by a perivascular demyelination and axonal degeneration, along with the presence of inflammatory infiltrating cells such as macrophages and cd8 ÷ t cells in the damaged areas (moore et al., 1989; umehara et al., 1993; wu et al., 1993) . these infiltrating cells produce several inflammatory mediators including the chemokine mcp-1 (umehara et al., 1993) . in addition, htlv-1infected t cell lines produce a number of chemokines including sdf-1, rantes, mip-la, and mip-i~ (arai et al., 1998; baba et al., 1996; mendez et al., 1997) . ham/tsp patients have a high frequency of cd8 ÷ ctl in their peripheral blood and cerebrospinal fluid (csf). biddison and coworkers have shown that htlv-i-specific cd8 ÷ ctl clones secrete mip-la, mip-i~, and il-16 (biddison et al., 1997) . thus, these htlv-1 ctls may be an important source of chemokines in ham/tsp, where they might contribute to the pathogenesis of this disorder. consistent with this, elevated mip-la protein was found in the csf in two of three patients with ham/tsp (miyagishi et al., 1995) . a further clinical study showed that mcp-1 was detectable on perivascular inflammatory cells and the vascular endothelium in active-chronic lesions of spinal cords of ham/tsp patients (umehara et al., 1996) . mhv belongs to the coronaviruses, a ubiquitous group of positivestranded rna viral pathogens of man and animals associated with a variety of respiratory, gastrointestinal, and neurological disorders. val]~rie c. asensio and iain l. campbell cns infection of susceptible murine hosts with neuro-adapted strains of mhv leads to a robust initial recruitment of leukocytes to the brain that follows an early peak of viral replication. this acute encephalomyelitis phase, with its resultant tissue injury, often leads to death of the host. however, in surviving animals, further viral replication is controlled by the host response and a chronic mononuclear cell inflammation, which occurs in the brain and spinal cord, is linked to a progressive demyelinating disease (buchmeier and lane, 1999; lane and buchmeier, 1997; weiner, 1973) . the mechanisms of demyelination in this model are not clear, although there is a requirement for a competent immune response. in a study of the kinetics and histological localization of chemokine gene expression in the brain and spinal cord of mhv-infected mice, lane and colleagues showed that a number of a-and ~-chemokines were expressed during acute or chronic stages of mhv-infection (lane et al., 1998) . expression of the transcripts for ip-10, mip-2, mcp-1, mcp-3, mip-1~, and rantes overlapped with the occurrence of acute viral encephalomyelitis, being present by day 3 postinfection and peaking at day 7 postinfection. during the chronic demyelinating phase of the disease, both ip-10 and rantes transcripts remained elevated. ip-10 rna colocalized with viral rna and was present in astrocytes and microglia associated with demyelinating lesions (lane et al., 1998) . with the exception of rantes, a similar pattern of chemokine expression was observed with cultured astrocytes infected with active, but not uv-inactive, mhv, indicating that viral replication in these cells can directly stimulate chemokine gene expression. therefore, in mhv infection of the brain, early viral-induced chemokine gene expression by resident cns cells such as astrocytes might promote the recruitment of t lymphocytes and macrophages and contribute to the maintenance of the chronic inflammatory response leading to demyelination. further studies in the mhv model using cd4 ko and cd8 ko mice show an important role for cd4 ÷ t-cells in the pathogenesis of inflammatory disease and demyelination (lane et al., 2000) . thus, mhvinfected cd4 ko mice have a marked reduction in the number of activated macrophage/microglia within their brains and spinal cords and significantly less demyelination. concomitant with the reduction in cns inflammatory disease in the mhv-infected cd4 ko mice, lower levels of rantes, but not other chemokine transcripts and protein were found indicating that cd4 ÷ t-cells represent one major source of rantes in the cns during mhv encephalomyelitis. administration of rantes neutralizing antisera to mhv-infected mice is associated with a significant reduction in macrophage infiltration and demyelination compared to control mice (lane et al., 2000) . these data clearly indicate that cd4 + t-cells have a pivotal role in accelerating cns inflammation and demyelination within infected mice possibly by regulating rantes expression. tmev-induced demyelinating disease is characterized by cns mononuclear cell infiltration resulting in a chronic cd4 + t-cell-mediated demyelinating disease (for more discussion, see chapter 7 in this volume). a study of the expression of cc and cxc chemokines in the cns throughout the disease course showed that expression of c10, ip-10, mcp-1, mip-la, mip-i~, and rantes mrna transcripts overlapped with the development of disease (hoffman et al., 1999) . expression of mip-i~ and mip-i~ protein was also detectable, being present in the spinal cord before the onset of disease and persisting throughout disease progression. these findings are somewhat reminiscent of those reported for mhv encephalomyelitis (discussed above) and reiterate the theme that chemokine expression in these viralinduced immune-mediated demyelinating diseases is a complex process that is clearly associated with disease progression. identifying the key chemokine protagonists in tmev-induced demyelinating disease awaits clarification. meningoencephalitis represents one of the most devastating consequences of viral infection of the cns, in which a concerted immunoinflammatory response by the host produces severe injury to the brain, leading to debilitating neurological disease and often death. in the viral meningoencephalitides, the specific mechanisms underlying the localization, extravasation, and activation of immune cells in the cns and the subsequent interactions between these cells are not well understood. studies from our own laboratory examined chemokine gene expression in lymphocytic choriomeningitis (lcm) that was induced by intracranial inoculation of mice with lcmv (asensio and campbell, 1997; asensio et al., 1999a) . following inoculation, lcmv infects and rapidly replicates in the meninges, the choroid plexus, and the ependymal membranes lining the ventricles. in this very well characterized model, immunocompetent adult mice infected with lcmv develop an acute monophasic disease characterized by the presence of infiltrating mononuclear cells in these same regions of the brain, and this leads to convulsive seizures and death by 6-8 days later (buchmeier et al., 1980; doherty et al., 1990) . infiltrating cells are predominantly t lymphocytes as well as macrophages. mhc class-i-restricted anti-lcmv cd8 ÷ cytotoxic lymphocytes (ctls), in addition to clearance of the virus, are also the primary effectors of lcm (buchmeier et al., 1980; doherty et al., 1990) . immune-compromised mice that are unable, or fail, to mount anti-lcmv cd8 ÷ ctl responses do not develop immune pathology in the brain and survive. we hypothesized that chemokines may be important regulatory signals for the cerebral recruitment and extravasation ofleukocytes in lcm (asensio and campbell, 1997; asensio et al., 1999a) . in examining this, we observed that the pattern of chemokine gene expression in lcm is dynamic and complex, with often overlapping expression of a number of different subclasses of chemokine genes. thus, by day 3 postinfection the expression of a number of chemokine genes is evident, including c10, mcp-3, mip-i~, mcp-1, crg-2/ip-10, and rantes. by day 6 postinfection the expression of all these chemokine genes increases markedly and the expression of the lymphotactin gene is also evident in the brain. a qualitatively similar but markedly decreased level of chemokine gene expression is observed in the brain of lcmv-infected athymic mice. a similar pattern of cerebral chemokine gene expression is also found in lcmv-infected ifn-y ko mice, although the levels of expression in the absence of ifn-y are about 50% of those in lcmv-infected wild-type controls. in both euthymic and athymic mice, expression of ip-10 was predominant at both early and late time points after infection and preceded detectable increases in proinflammatory and interferon cytokine gene expression and cns leukocyte recruitment in euthymic mice. in all, fig 2. the cxc chemokine ip-10 is expressed at high levels in the brain, following infection by many viruses. in this example mice were infected intracranially with lcmv, and the viral nucleoprotein (np) and ip-10 rna expression in the brain were analyzed by in situ hybridization. high expression of ip-10 rna was apparent by day 3 (a) postinfection and increased further by day 6 (b) postinfection. regional expression of ip-10 rna, on the whole, overlapped with sites of viral infection, as indicated by the expression of the lcmv-np gene. however, parenchymal expression of the ip-10 gene was also found in the absence of local lcmv-np expression. by using dual-label analysis to identify the cellular localization of ip-10 rna expression, these parenchymal cells were identified as gfap-positive astrocytes (c; arrows). from asensio, v. c., kincaid, c., and campbell, i. l. chemokines and the inflammatory response to viral infection in the 156 vall~rie c. asensio and iain l. campbell these observations, together with the finding that crg-2/ip-10, a prominently expressed chemokine gene in many different cns viral infections, is expressed by resident cns cells including astrocytes (see fig. 2 ), suggest that activation ofchemokine gene expression may be a direct, early, and localized host response to lcmv infection. these findings are consistent with the proposed involvement of chemokines as key signaling molecules for the subsequent migration of leukocytes to the cns, following lcmv infection. however, formal demonstration of the precise chemotactic, and possibly of other functions of chemokines in lcm, presently awaits verification. herpes simplex virus (hsv) is a common etiologic agent of acute focal encephalitis. pathologically, in hsv encephalitis, leukocytes infiltrate the subarachnoid space as a hallmark of meningitis and encephalitis, while csf analysis typically reveals a predominantly lymphocytic pleocytosis. to evaluate the possible contribution of chemokines to hsv encephalitis, rosler and coworkers examined the spectrum, quantity, and time course of csf chemokines in three patients with proven hsv type 1 encephalitis (hse-1) (rosler et al., 1998) . high chemokine levels were present in the csf of all hse-1 patients. peak levels occurred at the time of admission, with mcp-1 being greater than either mip-la or rantes. il-8 levels were elevated at 4 to 8 hours after admission. by contrast, plasma chemokine levels were considerably lower than csf levels, pointing to the localized nature of the cns chemokine response in these hse-1 patients. a comparison of mcp-1 levels with clinical status revealed a high reciprocal correlation. this single clinical study implicates chemokines, particularly mcp-1, in the pathogenesis of hse-1 and suggests these mediators may be useful indicators to determine the stage and severity of rise-1. experimental studies of hsv-1 in mice, following ocular infection, highlight the rapid and sustained upregulation of a number of chemokine genes including gro-a, mip-i~, mip-2, mcp-1, ip-10, and rantes (carr et al., 1998; thomas et al., 1998) . hsv stromal keratitis, associated with productive infection in the eye, results in significant accumulation of pmn, which may be driven by the presence in particular of the cxc chemokines gro-a and mip-2 (thomas et al., 1998) . a clinical survey of chemokine expression in csf in patients with viral meningitis, due to infection with paramyxoviruses or enteroviruses, implicated ip-10 and mcp-1 as important contributory factors in the accumulation of activated t cells and monocytes in the cns (lahrtz et al., 1997) . importantly, this study formally demonstrated that the ip-10 and mcp-1 levels in csf correlated with leukocyte chemotactic activity. mcp-1 was identified as an important chemoattractant for pbmcs while the combination of mcp-1 and ip-10 was required for migration of activated t cells. this study clearly establishes a direct link between the cns expression of the chemokines ip-10 and mcp-1 and cns leukocytosis in viral meningitis. in experimental studies, the paramyxovirus newcastle disease virus (ndv) induces ip-10 gene expression in infected astrocytes and microglia detectable by 3 hours postinfection (vanguri and farber, 1994) . uv-inactivated ndv proved to be as effective as live virus in inducing ip-10 gene expression and was not blocked by the protein synthesis inhibitor cycloheximide, indicating that the ip-10 gene functions as an immediate early response gene, following ndv infection. similar to ndv, measles virus (mv) is a paramyxovirus that induces ip-10 gene expression in human glioblastoma cells (nazar et al., 1997) . the promoter requirements for the induction of ip-10 gene expression are similar for ifn-~ and mv; however, these two stimuli use different combinations of dna binding factors, with stat 1 or nf~b being more important in the direct induction of ip-10 by ifn-~ or mv, respectively. while it is presently unknown whether these paramyxoviruses induce the expression of other chemokine genes, their potent ability to directly induce ip-10 expression by glial cells may constitute a significant cns source of production of this chemokine during viral meningitis. mouse adenovirus-type 1 (mav-1) is a double-stranded dna virus that causes a fatal hemorrhagic encephalopathy in c57b1/6 mice within 4-6 days (guida et al., 1995) , along with infection of cerebrovascular endothelial cells. chemokine gene expression was investigated and prominent induction of ip-10 was observed in the spleen and cns of susceptible mice, whereas mcp-1 and mip-2, respectively, were found in the spleen and brain of resistant mice (charles et al., 1999) . vascular endothelium and cns glia were identified as sites of ip-10 mrna expression in susceptible animals. in the normal mammalian cns, the number of leukocytes present in the brain is scant. however, these cells are attracted to, and accu-158 vali~rie c. asensio and iain l. campbell mulate in, a variety of pathologic states, many involving viral infection. although leukocyte migration into local tissue compartments such as the cns is a multifactorial process, it has become clear that chemokines are pivotal components of this process, providing a necessary chemotactic signal for leukocyte recruitment. generation of this signal in cns viral infection may involve localized production of proinflammatory chemokines by cells intrinsic to the brain, including the astrocytes and microglia. the activated glia have the potential to produce a spectrum of cc and cxc chemokines that may vary, depending on the nature of the invading pathogen. this in turn will determine the qualitative makeup of the leukocytes recruited to the brain. it is important to note, however, that how a chemokine that is secreted by a parenchymally located glial cell is "sensed" by circulating peripheral leukocytes is unclear at this time. roles beyond leukocyte chemoattraction are implied for the chemokines and their receptors. studies of hiv highlight the dynamic relationship between chemokine receptor usage and tropism for different isolates of hiv. on the other hand, the fact that chemokine receptor ligands are effective inhibitors of hiv entry into target cells suggests their local production may serve as a host response to limit further spread of the infectious agent. some chemokines such as ip-10 are documented as having direct antiviral functions. whether this is important in cns defense against viral infection where ip-10 is highly expressed, and whether any other chemokines have an antiviral function, remain open questions. a further consequence of the localized production of chemokines in the brain is likely the direct modulation of cns cell function. various classes of chemokine receptors are expressed by cns cells including neurons and the glia. in many respects the cns possesses its own chemokine network permitting autocrine and paracrine regulation of cellular activity. chemokines are implicated in many physiological functions including development, cell growth, angiogenesis, and cellular migration. perturbation of the cns chemokine network, resulting from viral infection, could therefore either be beneficial by promoting reparative processes or detrimental by contributing to tissue injury and loss. in conclusion, it is now apparent that, like their proinflammatory cytokine counterparts, the chemokines are important plurifunctional mediators in the host response to viral infection of the cns (see fig. 3 and color section). as such, a greater understanding of the neurocellular and viral targets and functions of the chemokines holds the promise of revealing new molecular focal points for therapeutic inter[for color reproduction, see color section.] chemokines are potentially multifunctional effectors in cns viral infections. in this schema, initial infection of the cns with a virus results in the localized production of chemokines by reactive astrocytes and microglia. some chemokines such as sdf-1 and fractalkine are produced constitutively in the cns and their production may also be altered by viral infection. increased chemokine production may then promote the further recruitment and extravasation of antiviral immune effector cells from the periphery. chemokine receptors are widely expressed by neuronal and glial cells and additional functions of the chemokines are likely. these may include direct antiviral actions (for example, by ip-10), and intercellular communication (for example, neuronally derived fractalkine can stimulate microglia). in addition, chemokines may exert detrimental actions; for example, sdf-1 can impair neuronal activity and stimulate apoptosis of these cells. m, macrophage; tc, t lymphocyte; n, neutrophil. vention that could more effectively control cns viral infections and prevent tissue injury. acknowledgments val6rie asensio is a postdoctoral fellow of the national multiple sclerosis society. the authors' studies were supported by nih grants mh 50426; mh 47680 and ns 36979 to ilc. this chapter is publication no. 13255-np, from the scripps research institute. neuronal apoptosis in hiv infection in adults chemokine receptors and molecular mimicry molecular piracy of mammalian interleukin-8 receptor type b by herpesvirus saimiri cc ckr5: a rantes, mip-la, mip-i~ receptor as a fusion cofactor for macrophage-tropic hiv-1 genes for chemokines mumig and crg-2 induced in protozoan and viral infections in response to ifn-7 with patterns of tissue expression that suggest nonredundant roles in vivo human t-cell leukemia virus type 1 tax protein induces the 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the chemokine receptor ccr8 is preferentially expressed in th2 but not thl cells recent advances in chemokines and chemokine receptors function of the chemokine receptor cxcr4 in haematopoiesis and in cerebellar development key: cord-004911-fbge8tkc authors: imrich, h.; harzer, k. title: on the role of peripheral macrophages during active experimental allergic encephalomyelitis (eae) date: 2001 journal: j neural transm (vienna) doi: 10.1007/s007020170060 sha: doc_id: 4911 cord_uid: fbge8tkc experimental allergic encephalitis (eae) is an experimental autoimmune inflammatory condition of the central nervous system (cns) that serves as a disease model for multiple sclerosis (ms). the primary effector mechanisms of the immune system leading to tissue destruction during eae remain still controversial. t-cells, microglia, and macrophages infiltrating the brain parenchyma are suggested to be involved. to clarify the role of these cells during disease lewis rats were immunised with different immunisation protocols: immunisation with myelin basic protein (mbp) in complete freunds adjuvant (cfa) containing high dose of mycobacterial components induced severe disease, whereas immunisation with low dose of mycobacterial components induced only mild disease. severely and mildly diseased animals were analysed with respect to infiltration of t-cells, macrophages and upregulation of mhc class ii molecules on microglia in the brain. all immunised rats showed high t-cell infiltration accompanied by microglia activation. the degree of disease and the infiltration of macrophages varied with dose of adjuvant. lowering the dose of adjuvant prevented the development of disease but also the influx of peripheral macrophages into the brain without affecting the peripheral t-cell response to the autoantigen. thus, appearance of (autoreactive) t-cells in the brain and microglia activation were probably not sufficient for development of disease. it can be concluded that peripheral macrophages play an essential or even key role in the pathogenesis of active eae. experimental allergic encephalomyelitis (eae) is an inflammatory disease of the central nervous system (cns) inducible in susceptible strains of rats by active immunisation with myelin basic protein (mbp) in adjuvant. target of the inflammatory response is the myelin membrane that ensheats axons, as well as the oligodendrocytes that produce myelin. histologically, eae is characterised by perivascular infiltrates that principally contain cd4 ϩ t cells and macrophages, and by upregulation of major histocompatibility complex (mhc) in the brain (owens et al., 1994; zamvil and steinman, 1990) . eae requires the development of a cell-mediated immune response within the cns. t-helper cells specific for encephalitogenic epitopes of mbp most likely initiate the inflammatory response. passing the blood brain barrier (bbb) they accumulate in the perivascular lesions in the cns together with blood born macrophages (levine et al., 1975; owens et al., 1994; zamvil and steinman, 1990) . although the pathology of eae is characterised by these infiltrating cells, the primary immune effector mechanisms responsible for the destruction of myelin membranes remain controversial. destruction might be mediated by mhc class ii-restricted cd4 ϩ t-cells via the local secretion of cytokines (sun and wekerle, 1986) or by the activation of microglia (owens et al., 1994) , or by infiltrating macrophages (huitinga et al., 1990) . for encephalitogenic t-cells it was shown that they can lyse syngeneic astrocytes in an antigen specific manner (sun and wekerle, 1986) . however, the most favoured hypothesis is that activation of microglia via infiltrating t-cells may lead to tissue damage (owens et al., 1994) . microglia is the most abundant cell population upregulating mhc class ii molecules in the brain during inflammation (hayes et al., 1987; matsumoto et al., 1986) . gamma interferon (ifng) release by infiltrating t-cells may be responsible for this "activation" of microglia. in vivo infusion or intrathecal injection of ifng leads to rapid expression of class ii molecules on microglia (steiniger and van der meide, 1988; vass and lassmann, 1990; wong et al., 1984) . in addition it was shown that microglial cells are able to drive an immune response in vitro (frei et al., 1987; panek and benveniste, 1995; suzumura et al., 1987) . in numerous cns diseases such as multiple sclerosis (ms), aids dementia complex, alzheimer's disease, and parkinson's disease it is believed that microglia plays a role in pathogenesis (dickson et al., 1993; hofman et al., 1986; mcgeer et al., 1993; münch et al., 1998) . moreover, it was shown in vitro that microglial cells are able to release a battery of toxic substances such as reactive oxygen intermediates (colton and gilbert, 1987) , proteinases (banati et al., 1993) and reactive nitrogen intermediates (boje and arora, 1992; zielasek et al., 1992) . such properties are well known for peripheral macrophages. therefore, the question arises what the role of infiltrating macrophages during cns inflammation may be. hypothesising a correlation between the pathogenic cell population and disease, different immunisation protocols were compared with regard to induction of disease and the appearance of macrophages, t-cells, and activation of microglia in the cns. these cells were characterised using a previously reported method to isolate infiltrating leukocytes from the inflamed brain imrich et al., 1994; sedgwick et al., 1991) which allows to analyse infiltrating cells by flow cytometry. the advantage of this method is the possibility to monitor the whole situation in the cns instead of only small sections analysed in histological stainings. kinetics of infiltrating macrophages and t-cells in the cns as well as kinetics of microglia activation were investigated after induction of severe and mild disease in lewis rats. the results clearly indicate that in the eae rat model strong t-cell influx into the brain parenchyma accompanied by microglia activation not necessarily leads to neurological symptoms. only in diseased animals strong macrophage infiltrates can be detected. therefore, although the resident microglia cell population shares many properties with macrophages, there may be a clear functional difference between these two accessory cell types of the immune system. specific pathogen-free lewis rats (rt1 l ) were purchased from the charles river gmbh, sulzfeld, germany. to stain cells for flow-cytometric analysis the following monoclonal antibodies (mabs) were used: ox1 anti rat leukocyte common antigen cd45 (sunderland et al., 1979) , ox6 anti rat monomorphic mhc class ii, rt1 (i-a) (mcmaster and , r73 specific for the α/ chains of the rat tcr (hünig et al., 1989) , ox8 anti rat cd8 complex (brideau et al., 1980) and w3/25 anti rat cd4 complex (williams et al., 1977) . the mab mrc ox-21 specific for the human c3b inactivator served as a control antibody (hsiung, 1982) . all mabs were obtained from serotec. phycoerythrin (pe)labeled secondary goat anti-mouse igg (gamigg) was derived from dianova (hamburg f.r.g.). active eae was induced by immunisation of rats with myelin basic protein (mbp) in complete freunds adjuvant as described previously (imrich et al., 1995) . mbp was isolated from guinea pig brains according to watanabe et al. (1983) . freunds adjuvant and mycobacterium tuberculosis were purchased from difco (detroit, mi). the control antigen keyhole limpet haemocyanin (klh) was from sigma. the antigen was emulsified in adjuvant to a concentration of 1 mg/ml. in addition mycobacterium tuberculosis was added: low dose ϭ 1 mg/ml adjuvant and high dose 5 mg/ml adjuvant. each animal received 2 ϫ 50 µl of antigen in adjuvant. clinical signs of eae were graded daily on an arbitrary scale of 1 to 6 as follows: (0) no overt disease; (1) limp tail; (2) paresis of 1 or 2 legs; (3) and (4), unilateral and bilateral hind leg paralysis, respectively; (5) bilateral hind leg paralysis and incontinence; (6) moribund (imrich et al., 1995) . inflammatory lymphocytes were isolated from the cns according to a procedure published earlier imrich et al., 1994) . briefly, animals were thoroughly perfused with pbs (200 ml) before removing the cns (ϭ brain and spinal cord). lymphoid cells were released from the cns tissue by mechanical disruption and enzymatic digestion of the tissue followed by percoll step gradient centrifugation. leukocytes were collected from interfaces of the appropriate density and sedimented in hank's buffer (170 ϫ g, 10 min, 4°c). popliteal lymph nodes were minced through a steel sieve in hank's buffer. connective tissue fragments were allowed to settle for 10 min at 4°c and lymphocytes were sedimented from the supernatant by low speed centrifugation (170 ϫ g, 10 min, 4°c). isolated lymphocytes were cultured in 96 well microtiter plates (costar) at a density of 5 ϫ 10 5 cells per well in 200 µl rpmi 1640 medium supplemented with 3% rat serum at 37°c (5% co 2 ). cells were restimulated with antigen at a final concentration of 20 µg/ml mbp or mycobacterial antigens, respectively. klh was used as a control antigen. proliferation was assessed by [ 3 h]thymidine incorporation (1 µci/well) after 48 h of culture. incorporated radioactivity was determined by harvesting the lymphocytes on glass fiber filters, lysing the cells by hypotonic wash and subsequent count of radioactive decays in a -counter (canberra packard). the stimulation index represents the ratio of incorporated radioactivities after stimulation with antigen and control antigen. lymphocytes were characterised by two color immunofluorescence and four parameter flow-cytometry (forward/side scatter, red/green fluorescence) as described earlier (imrich et al., 1995 (imrich et al., , 1994 . briefly, lymphocytes were indirectly stained using a primary mouse mab and a phycoerythrin (pe)-labeled secondary goat anti mouse igg (gαmigg) followed by another primary mouse mab labeled with fitc. to avoid capture of fitclabeled primary mab by pe-labeled secondary gαmigg normal mouse serum was used in excess to block potentially free binding capacity on gαmigg. flow-cytomeric analysis was performed using a facscan (becton-dickinson, heidelberg, germany) linked to a hewlett packard 6000 computer with the consort 30 software installed. infiltrating leukocytes were distinguished from macrophages by criteria of fsc/ssc. two gates were selected. the lymphocyte gate with the following cordinates fsc/ssc: 55/3, 54/18, 78/33, 109/33, 112/22, 106/7 and the macrophage gate with cordinates fsc/ssc: 81/4, 81/42, 84/243, 246/243, 239/32, 237/29. after labelling cells with anti tcr, no tcr positive cells were found in the macrophage gate. to determine the total number of cell subpopulations isolated from the brain, first the total number of cells collected from the gradient was counted. since the distribution of events out of 10.000 within the different gates were given by facs analysis, the total number of cells isolated from the gradient with specific fsc/ssc characteristics could be calculated. the real cell number of infiltrating leukocyte subpopulation was calculated by multiplying the percentage of positive events by the total number of cells within the specific gate. to induce active eae lewis rats were immunised with myelin basic protein (mbp) in complete freunds adjuvant (cfa) containing 5 mg/ml of mycobacterium tuberculosis (myc.t). the development of neurological symptoms was monitored by scoring of tale and limp paraplegia (imrich et al., 1995) . in fig. 1 the course of the disease is shown. clinical signs of the disease started at day 9 and reached a peak at day 14 post immunisation (14 dpi). thereafter, animals started to recover and at 18 dpi all had completely recovered from the disease. to induce this severe disease it was necessary to immunise rats with mbp in cfa containing high dose of myc.t. normally for immunisation an antigen is emulsified in cfa containing 1 mg/ml myc.t. this amount of myc.t is sufficient to induce a strong immune response (deeb et al., 1992; johnston et al., 1991; smith et al., 1992) . this concentration of myc.t in combination with the mbp batch used for these experiments was insufficient to induce severe neurological symptoms in eae. after immunisation of rats with mbp in cfa containing 1 mg/ml myc.t most of the animals did not develop disease. only 1 out of 6 animals showed mild clinical symptoms (clinical score ϭ 1) at 14 dpi ( fig. 2 however, after increasing the dose of mycobacterial antigens in cfa (from 1 to 5 mg/ml) in combination with a constant concentration of the autoantigen, seven out of seven animals developed neurological symptoms at 14 dpi ( fig. 2 lower panel) . therefore, myc.t antigens in cfa may modulate the immune response in an dose dependent manner. it seemed that a higher dose of myc.t in cfa may induce a stronger t-cell response to the autoantigen leading to severe disease in immunised animals. to test this assumption proliferation assays were performed with lymphocytes derived from draining lymph nodes of rats immunised with mbp in cfa containing high dose of myc.t and compared with specific proliferation of lymphocytes derived from animals immunised with mbp in cfa with low the average symptomatology is given by the arithmetic mean value (n ϭ 7). clinical score: (0) no overt disease; (1) limp tail; (2) paresis of 1 or 2 legs; (3) and (4) unilateral and bilateral hind leg paralysis, respectively dose myc.t. in fig. 3 one out of four proliferation assays with lymphocytes derived at 12 dpi is shown. from all experiments similar results were obtained. no difference of mbp specific t-cell proliferation was observed fig. 3 (upper panel) . in both cases, the incorporation of 3 h-thymidine in response to mbp was 3 fold higher compared to the control antigen klh. in comparison with the response against the autoantigen, the t-cell response against myc.t was much higher. however, as shown in fig. 3 (lower panel) the myc.t specific proliferation did again not differ between the animal groups. in both cases, the stimulation index was around 6 indicating that there was no decrease in the t-cell response against myc.t after decreasing the amount of myc.t antigens in cfa. grouping of animals according to severity of symptoms at maximum of disease after immunisation of rats with cfa containing low dose of mycobacterium tuberculosis (upper panel), and after immunisation of rats with cfa containing high dose of mycobacterium tuberculosis (lower panel) respectively. clinical score: (0) no overt disease; (1) limp tail; (2) paresis of 1 or 2 legs; (3) and (4), unilateral and bilateral hind leg paralysis, respectively; (5) bilateral hind leg paralysis and incontinence; (6) moribund therefore, neither the mbp specific t-cell response alone, nor the additional response against mycobacterial antigens in the periphery necessarily lead to an onset of the disease. thus, it seemed likely that the difference in the immune reaction between these two animal groups resulting in a different course of the disease may be localised in the affected tissue. since no differences in the t-cell response against the autoantigen between diseased and symptom-free rats could be detected in the periphery during eae, it had to be assumed that different numbers of cells and cell populations contributing to the inflammatory process would infiltrate the brain tissue. to monitor such differences between diseased animals and animals without symptoms, leukocytes were isolated from the cns and analysed by flow cytometry with regard to infiltrating t-cells (tcrϩ), macrophages (cd45 high ) and activation of resident microglia (cd45 low , mhc iiϩ). to characterise t-cells, leukocytes derived from the cns were labelled with anti tcr (r73) and anti cd4 (w3725) or anti cd8 (ox8). t-cells could be additionally distinguished from infiltrating macrophages by criteria of forward/side scatter. infiltrating macrophages resemble a different cell population clearly separated from lymphocytes if analysed by criteria of forward/ side scatter. additionally they were marked with anti lca (ox1) and anti mhc ii (ox6). microglial cells could not be separated from lymphocytes nor from infiltrating macrophages by criteria of forward side scatter. the lymphocyte gate as well as the macrophage gate were contaminated by microglia. nevertheless, microglia could be well distinguished from infiltrating macrophages, since it resembled the lca low cell population. activated t-cells enter the brain more frequently than naive t-cells (hickey et al., 1991) . therefore, as a control for the situation in the unaffected brain animals were immunised with keyhole lymphet haemocyanin (klh) in cfa to induce a strong immune reaction in the periphery. in these rats only 2.3% of isolated cells from the cns were t-cells (fig. 4a) . to distinguish between t-cells and infiltrating macrophages in fig. 4a cells were gated on lymphocytes by criteria of forward and side scatter. the biggest cell population found within this gate could be characterised as microglial cells, since they belong to the lca low cell population (data not shown). like the lymphocyte gate, the macrophage gate was also contaminated by microglial cells as shown in fig. 4b . but even these bigger cells do not express mhc class ii molecules. only 0.29% of microglial cells within the macrophage gate were stained with the antibody ox6 (fig. 4b) . in contrast animals immunised with mbp emulsified in cfa containing low dose of myc.t, showed a strong t-cell influx in the brain at 14 dpi. 30% of isolated cells from the brain belonged to the t-cell population in the lymphocyte gate (fig. 4c) . t-cell influx was accompanied by microglia activation, as 57% of isolated microglia expressed mhc class ii molecules in the macrophage gate (fig. 4d) . but even in the lymphocyte gate, a big proportion of microglial cells seemed to be activated in the affected tissue as indicated by upregulation of cd4 molecules on tcr negative cells (fig. 4a compared with fig. 4c) . from diseased animals immunised with mbp in cfa containing high dose of myc.t, leukocytes were isolated from the cns of rats at different stages of the disease: at the onset (12 dpi), the maximum (14 dpi) and after recovery from the disease (20 dpi). leukocytes were analysed by flow cytometry (fig. 5 ). progression to disease was characterised by t-cell influx in the brain of diseased animals. at 12 dpi 35% of isolated cells were t-cells, 27% cd4 ϩ and 8% cd8 ϩ t cells in the lymphocyte gate (fig. 5a) . t-cell influx reached a maximum (48%) just at the maximum of disease at 14 dpi (fig. 5c) and declined slowly during convalescence to 30% at 20 dpi. at any point of time analysed so far cd8 ϩ t-cells were a minority in the brain. only 8% of isolated : a,b) , compared to rats after immunisation with mbp in cfa with low dose of myc.t antigens (14 dpi) (lower panel: c,d). leukocytes extracted from the entire cns by percoll density centrifugation were pooled (from 3 animals). to differentiate between microglia and infiltrating macrophages, cells were labelled with the antibody ox1 (leukocyte common antigen ϭ lca). the lca high-positive cell population resembles infiltrating macrophages, and lca low-positive cells are resident microglia. in addition, cells were labelled with the anitbody ox6 (mhc class ii). analysed cells were gated for macrophages by criteria of forward and side scatter in fig. 4b ,d. to characterise t-cells, leukocytes were labelled with the antibody r73 (tcr α ) and the antibody w3/25 (cd4). analysed cells were gated for lymphocytes by criteria of forward and side scatter in fig. 4a ,c. similar results were seen in at least four experiments fig. 5 . facs analysis of leukocytes isolated from the brain after induction of severe disease by immunising animals with mbp in cfa containing high dose of myc.t. animals used for facs analysis at 12 dpi already showed neurological signs (clinical score 2), at 14 dpi animals suffered from severe disease (clinical score 4) and at 20 dpi only rats convalescent from severe disease (clinical score 4) were used. leukocytes extracted from the entire cns by percoll density centrifugation were pooled (from 3 animals each studied day). to differentiate between microglia and infiltrating macrophages, cells were labelled with the antibody ox1 (leukocyte common antigen ϭ lca). the lca highpositive cell population resembles infiltrating macrophages and lca low-positive cells are resident microglia. in addition cells were labelled with the anitbody ox6 (mhc class ii). analysed cells were gated for macrophages by criteria of forward and side scatter in fig. 5b ,d,f. to characterise t-cells, leukocytes were labelled with the antibody r73 (tcr α ) and the antibody ox8 (cd8). analysed cells were gated for lymphocytes by criteria of forward and side scatter in fig. 5a ,c,e. similar results have been observed in at least four experiments leukocytes expressed cd8 and tcr at 12 and 14 dpi and only 5% after recovery at 20 dpi (fig. 5a,c,e) . the leukocyte influx varies early past immunisation and later past immunisation or between animals with high or low clinical score. therefore, to allow comparison between the different animal groups, the real cell number of each cell population within the brain was calculated as described in material and methods. t-cell influx in the cns accompanied by microglia activation was higher in severe diseased animals at 14 dpi compared to undiseased animals immunised with low dose of cfa as indicated in fig. 4 and 5. but it is remarkable that the amount of t-cells in the brain of animals without symptoms was comparable to animals with relatively mild symptomatology (clinical score ϭ 2) at 14 dpi, however, only the latter developed disease as shown in table 1 . this was even true for activated microglia within the macrophage gate. in table 1 the total number of infiltrating t-cells, macrophages and the amount of activated microglia within the macrophage gate was calculated. two animal groups were compared: the one after immunisation with high dose of myc.t in the adjuvant developing disease, and the other after immunisation with low dose of myc.t without neurological symptoms. as shown in table 1 , the only difference between these two groups is the lack of peripheral macrophages in brain tissue of healthy animals. during the course of eae the majority of infiltrating leukocytes were cd4 tcells (fig. 5 ). an other cell population infiltrating the cns during disease were peripheral macrophages. as shown in fig. 5b in diseased animals immunised with high dose of myc.t at 12 dpi, 25% of leukocytes isolated from the brain falling in the macrophage gate, belonged to infiltrating macrophages. this cell population increased to 32% at the maximum of disease (fig. 5d) . in contrast to t-cells, peripheral macrophages were strongly diminished in the cns after recovery from disease. only 7% of isolated cells within the macrophage gate at 20 dpi were infiltrating macrophages (fig. 5f ). macrophages were neither detected in the brain of rats immunised with mbp in cfa containing low dose of myc.t nor in control rats immunised with klh as shown in fig. 4b,c and table 1 . peripheral macrophages were also absent from the brain of rats immunised with klh in cfa containing high dose of myc.t (data not shown). these results showed that infiltrating macrophages in brain tissue were involved in tissue destruction during active eae. as in undiseased animals no infiltrating macrophages were found in brain tissue, it is likely that peripheral macrophages may be responsible for severe tissue destruction resulting in disease. given the real number of infiltrating cells can be calculated after characterisation by facs analysis (imrich et al., 1994) , the amount of brain infiltrating t-cells as well as the amount of infiltrating macrophages were determined in animals with different disease severity. leukocytes isolated from cns of three animal groups (each n ϭ 4) with different degrees of neurological symptoms at the maximum of the disease were analysed with regard to infiltrating t-cells vs. infiltrating macrophages. in fig. 6 the t-cell/macrophage ratio is shown as calculated for all three animal groups to demonstrate the distribution of these two cell types in the affected cns tissue. in all three animal groups t-cells clearly dominated in the brain as shown in fig. 6 . while the t-cell predominance was only moderate in severely diseased animals (1.7ϫ higher than for macrophages), it was more pronounced in animals with low clinical score (4.5ϫ higher than for macrophages) and even up to 30 fold higher in animals without clinical symptoms. the aim of this study was to clarify the contribution of infiltrating t-cells and macrophages to tissue destruction during active eae. different immunisation protocols were used to induce disease with different clinical score in order to identify the infiltrating cell populations dominating in the cns of severely diseased animals. the phenomenon that induction of active eae depends on the immunisation protocol is well known. after immunisation with mbp in incomplete freunds adjuvants (ifa) lewis rats fail to develop disease (swierbors and swanborg, 1977) . in this model, however, t-cells and peripheral macrophages fail to infiltrate the brain parenchyma. after immunisation of rats with mbp in cfa containing high dose of myc.t animals develop severe disease with high influx of t-cells and peripheral macrophages in brain tissue. however, in our hands even in this table 1 . comparison of brain infiltrating macrophages mø, brain infiltrating t-cells and the amount of activated microglia within the macrophage gate. the exact cell number of these different cell populations extracted from the brain of each animal was calculated (see material and methods). two animal groups were compared: four diseased animals at 14 dpi (clinical score ϭ 2) after induction of eae with high dose of myc.t. in the adjuvant and four rats without symptoms at 14 dpi with low dose of myc.t. in the adjuvant rat dose of myc t. clinical score mø t-cells activated microglia 1 high 2 4.6 ϫ 10 5 4 ϫ 10 6 9 ϫ 10 5 2 high 2 3.4 ϫ 10 5 1.7 ϫ 10 6 8.8 ϫ 10 5 3 high 2 4 ϫ 10 5 0.8 ϫ 10 6 8 ϫ 10 5 4 high 2 5 ϫ 10 5 4.5 ϫ 10 6 10 ϫ 10 5 5 low 0 0 3.8 ϫ 10 6 12 ϫ 10 5 6 low 0 0 2.6 ϫ 10 6 7 ϫ 10 5 7 low 0 0 2 ϫ 10 6 8.6 ϫ 10 5 8 low 0 0 3.5 ϫ 10 6 9 ϫ 10 5 animal group, some of the rats did not develop disease. despite the fact that these animals showed high t-cell influx in the cns, peripheral macrophages failed to infiltrate the brain. this observation led to the animal model described in this paper. after immunisation of rats with cfa containing low dose of myc.t, and constant concentrations of the autoantigen mbp animals failed to develop severe symptoms, nevertheless, they showed strong t-cell influx in brain parenchyma. the simplest explanation of this phenomenon was the induction of a weaker t-cell response to the autoantigen. however, the situation seemed to be more complex. t-cell responses against mbp were similar in proliferation assays with lymphocytes isolated from animals immunised with mbp in cfa containing low dose of myc.t to responses in severely diseased animals immunised with mbp in cfa containing high dose of myc.t. another explanation was the induction of a strong t-cell response specific for mycobacterial antigens imported to the brain. a similiar mechanism was reported for an other animal model: in the experimental autoimmune uveoretinitis (eau) it was shown that recruitment of antigen-nonspecific t-cells plays a pivotal role in the pathogenesis (caspi et al., 1993) , therefore, it had to be assumed that also in eae high numbers of t-cells specific for mycobacterial antigens and infiltrating the brain may contribute to tissue destruction. however, after immunisation of rats with higher concentrations of myc.t the t-cell response against this antigen was similar to that of animals immunised with cfa containing low dose of myc.t. therefore, neither the mbp specific t-cell response nor a strong t-cell response against mycobacterial antigens necessarily led to an onset of the disease. fig. 6 . t-cell/macrophage ratio in leukocytes isolated from the brain of animals after induction of active eae (14 dpi) with different clinical scores. cells were extracted from the cns by percoll density centrifugation, characterised by flow cytometry and the real number of infiltrating t-cells and macrophages were calculated by multiplying the percentage of positive cells by the total mumber of leukocytes calculated for the specific gate. the ratio between t-cells and macrophages was plotted versus clinical score. vertical bars indicate the sd of the mean value derived from the t-cell/macrophage ratio of four animals with the same clinical score eae is mediated by t-cells of the th1 subtype (owens et al., 1994) . therefore, th2 cells may be generated in the current model. but even this possibility has to be excluded. a strong mbp-specific t-cell response of the th2 subtype can be induced in rats after targeting the autoantigen to bcells. these cells show a different migration behaviour and do not infiltrate the brain parenchyma (saoudi et al., 1995) . thus, th2 cells fail to pass the blood brain barrier and do not induce mhcii expression on microglia. cfa is known to induce a strong th1 response (audibert and lise, 1993) and, indeed, after immunisation of rats with mbp in cfa (low dose of myc.t), a strong t-cell influx in the brain parenchyma accompanied by microglia activation as indicated by upregulation of mhc ii on these cells, was observed. strong t-cell infiltrates of the cns, but in relatively mild disease were described in an other animal model, the experimental autoimmune panencephalitis. after immunising with another brain-specific autoantigen, the s100 , strong t-cell infiltrates were observed in the cns. despite this intense inflammatory response, the animals did not develop severe clinical disease. it is of interest that quantitative morphological studies revealed that macrophages are underrepresented in the inflammatory infiltrates caused by the adoptive transfer of s100 -specific t-cells as compared to macrophages in the disease induced with mbp-specific t-cells (kojima et al., 1994) . the failure to develop disease in the eae model after reducing the concentration of mycobacterial antigens in cfa, as well as in the described s100 model, can be explained by the absence of infiltrating macrophages but also by the failure of lytic power of infiltrating t-cells. experiments in the eae model described by huitinga et al. (1990) showed that after depletion of peripheral macrophages by toxic liposomes the onset of the disease is prevented. these data clearly support the assumption that macrophages are the principal cause of tissue destruction in the cns in eae. the liposomes used in that study could pass the blood brain barrier (huitinga et al., 1990) . therefore, it can not be excluded that after treating animals with toxic liposomes to deplete peripheral macrophages, activated microglia may be affected, too, by loosing its lytic capacity. the data presented here demonstrate that activation of microglia was not necessary to induce tissue destruction in the cns. it seems that infiltrating t-cells recruit peripheral macrophages after they encounter their antigen. indeed, in all animals with severe disease examined in the present study, strong macrophage infiltrates were detected in brain parenchyma. in contrast no infiltrating macrophages were found in brain parenchyma of rats without symptoms after immunisation. thus, the number of infiltrating macrophages in the brain parenchyma correlated well with the disease. in addition, t-cells persisted longer in brain parenchyma than macrophages. during the convalescence period and even after recovery from disease strong t-cell influx accompanied by microglia activation was detected in the brain. in contrast peripheral macrophages disappeared soon from the brain in the period of recovery. these results clearly indicate that infiltrating macrophages are responsible for tissue destruction in the brain during active eae and that microglia, although sharing many properties with peripheral macrophage, is functionally distinct from infiltrating macrophages. this finding is strongly supported by another study (berger et al., 1997) . the authors compared different animal models with respect to cell infiltrates in the cns and disease. however, there was no correlation between t-cell infiltration and severity of symptoms. the most striking neuropathologic correlate was the high absolute number of infiltrating macrophages. therefore, infiltrating macrophages are concluded to play a pivotal role in the pathogenesis of eae. it can not be excluded that t-cells contribute directly to tissue destruction, but it seems more reasonable that infiltrating t-cells may recruit macrophages which eventually cause the tissue damage. t-cells and microglia may initiate the immune reaction in the cns, but other cell types including macrophages seem to be required for development of a full blown inflammation followed by tissue destruction. adjuvants: current status, clinical perspectives and future prospects detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin b and l experimental autoimmune encephalomyelitis: the antigen specificity of t lymphocytes determines the topography of lesions in the central and peripheral nervous system microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death two subsets of rat t lymphocytes defined with monoclonal antibodies recruitment of antigen-nonspecific cells plays a pivotal role in the pathogenesis of a t cell-mediated organ-specific autoimmune disease, experimental autoimmune uveoretinitis production of superoxide anions by a cns macrophage, the microglia comparison of freund's and ribi adjuvants for inducing antibodies to the synthetic antigen (tg)-al in rabbits microglia and cytokines in neurological disease, with special reference to aids and alzheimer's disease population dynamics of lymphocyte subsets in the central nervous system of rats with different susceptibility to coronavirus-induced demyelinating encephalitis antigen presentation and tumor cytotoxicity by interferon-gamma-treated microglial cells microglia are the major cell type expressing mhc class ii in human white matter t-lymphocyte entry into the central nervous system immunoregulatory molecules and il 2 receptors identified in multiple sclerosis brain purification of human c3b inactivator by monoclonal-antibody affinity chromatography suppression of experimental allergic encephalomyelitis in lewis rats after elimination of macrophages a monoclonal antibody to a constant determinant of the rat t cell antigen receptor that induces t cell activation cervical lymphoid tissue but not the central nervous system supports proliferation of virus-specific t lymphocytes during coronavirus-induced encephalitis in rats prevention and treatment of lewis rat experimental allergic encephalomyelitis with a monoclonal antibody to the t cell receptor v beta 8.2 segment an evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits experimental autoimmune panencephalitis and uveoretinitis transferred to the lewis rat by t lymphocytes specific for the s100 beta molecule, a calcium binding protein of astroglia do neurological signs occur in experimental allergic encephalomyelitis in the absence of inflammatory lesions of the central nervous system? immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to ia-positive cells with dendritic morphology microglia in degenerative neurological disease indentification of ia glycoprotein in rat thymus and purification from rat spleen alzheimer's disease-synergistic effects of glucose deficit, oxidative stress and advanced glycation endproducts inflammatory cytokines in the brain: does the cns shape immune responses? class ii mhc gene expression in microglia. regulation by the cytokines ifn-gamma, tnf-alpha, and tgf-beta prevention of experimental allergic encephalomyelitis in rats by targeting autoantigen to b cells: evidence that the protective mechanism depends on changes in the cytokine response and migratory properties of the autoantigen-specific t cells isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system the selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits rat ependyma and microglia cells express class ii mhc antigens after intravenous infusion of recombinant gamma interferon ia-restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes purification with monoclonal antibody of a predominant leukocyte common antigen and glycoprotein from rat thymocytes mhc antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of ia antigen expression by gamma-interferon immunoregulation of experimental allergic encephalomyelitis: conditions for induction of suppressor cells and analysis of mechanism intrathecal application of interferon gamma. progressive appearance of mhc antigens within the rat nervous system adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis analysis of cell surfaces by xenogeneic myeloma-hybrid antibodies: differentiation antigens of rat lymphocytes inducible expression of h-2 and ia antigens on brain cells the t lymphocyte in experimental allergic encephalomyelitis production of nitrite by neonatal rat microglial cells/brain macrophages authors' address: h. imrich, institut für virologie und immunbiologie we thank dr. m. czub, dr. t. herrmann and dr. r. gold for critical reading of this manuscript. key: cord-007170-svsfu7fj authors: richt, j. a.; vandewoude, s.; zink, m. c.; clements, j. e.; herzog, s.; stitz, l.; rott, r.; narayan, o. title: infection with borna disease virus: molecular and immunobiological characterization of the agent date: 1992-06-17 journal: clin infect dis doi: 10.1093/clinids/14.6.1240 sha: doc_id: 7170 cord_uid: svsfu7fj borna disease virus (bdv), which seems to be distinct from all other known viruses, exhibits a unique mechanism of pathogenesis. this review highlights several aspects of the biology of infection with this virus and summarizes the preliminary characterization of the agent. studies on bdv may help to illuminate several important areas of neurobiology, including the mechanisms regulating the replication of a new type of rna virus in the nuclei of neural cells, the neuroinvasiveness and neurotropism of such viruses, their t cell-mediated immunopathology, tolerance in newborn animals to persistent viral infection of the central nervous system, and behavioral diseases and eating disorders induced by such agents. mainly in the gray matter of the brain, and pathognomonic intranuclear joest-degen inclusion bodies in neurons and astrocytes of the frontal cortex [8, 9] . mechanisms of transmission of the virus in horses and sheep are not well understood, but experimental data suggest that intranasal infection is the common route [10] . recent seroepidemiological and virological surveys of borna disease in horses have shown that bdv-specific antibodies are present in many horses without clinical signs of the disease. since most of these animals remain clinically healthy for at least 1 year, a long incubation period may be characteristic of the pathogenesis of borna disease [11] . these data suggest further that bdv is more widely disseminated than was previously thought. in fact, bdv-specific antibodies have been found in clinically healthy horses in the united states (authors' unpublished results). bdv has been only partially characterized. although the agent is known to have physical and replicative properties typical of conventional viruses [1, 12, 13] , its nucleic acid has only recently been identified as rna [14] [15] [16] [17] . bdv is highly neurovirulent in most laboratory animals and in cell cultures. it has a tropism for cells derived from the neural crest: neurons, astrocytes, and schwann cells. tissue culture cells of nonneuronal origin exhibit only low-level susceptibility to infection, but this resistance can be overcome by cocultivation with bdv-infected primary brain cultures [18] . the virus replicates noncytopathically in infected cells and is tightly cell associated. infection with bdv is persistent; cultures produce approximately one infectious unit per cell [13] . treatment of infected cells with interferon a/,3 does not influence the establishment or maintenance of persistent infection [19] . persistently infected mdck (madin-darby canine kidney) cells are widely used in indirect immunofluorescence assays for the detection of bdv-specific antibodies in serum and csf of affected animals and humans [18, 20] . the viral antigens in persistently infected cells accumulate in the nuclei (sparing the nucleoli) and in the perinuclear area of the cytoplasm and appear as punctate grains in immunofluorescence reactions. bdv particles have never been visualized in infectious material, but replication of bdv is associated with synthesis of at least three novel polypeptides with respective molecular masses of 14, 24, and 38-39 kd [21] [22] [23] [24] . antibodies to these virus-associated antigens are produced in infected animals and can be recognized readily by radioimmunoprecipitation and western blot analysis [21] [22] [23] [24] . sera from animals previously infected with bdv or immunized with bdv-specific proteins do not neutralize the infectivity of the virus [25] [26] [27] . polyclonal antibodies to all three bdv proteins are found in the serum of infected animals. monoclonal antibodies (mabs) to the 38/39-kd and 24-kd antigens have been obtained after immunization of mice with bdv-specific antigens [22, 24] or after infection of rats [22] . most of these antibodies react in western blot analyses exclusively with the protein used for immunization in lysates of infected cell cultures and infected rat brain. evidence for an antigenic relation between these two proteins has been found by protease digestion and tryptic fingerprint analysis [24] . several identical peptides were revealed after digestion of the 38/39-kd and 24-kd proteins. furthermore, the cross-reactivity of mabs to the purified 38/39-kd and 24-kd proteins indicates that these proteins share antigenic determinants [16, 24] . neither protein is glycosylated, but the 24-kd antigen was found to be phosphorylated at serine residues [24] . bdv has not yet been classified taxonomically because infectious particles have never been visualized, and only recently has this agent been identified as an rna virus [14] [15] [16] [17] 28] . since novel proteins were identified in bdv-infected cells and tissues, it was assumed that bdv-specific mrnas must also be present in infected material. thus the strategy used to isolate bdv-specific molecular clones was to construct subtractive cdna libraries from mrnas isolated from uninfected and bdv-infected cell cultures and brain [15, 16] . no specific nucleic acid probe for bdv was available; the cdna cloning was therefore done in an expression vector, and clones were screened with mabs [16] specific for the bdv-specific 38/39-kd protein [22] or with a radiolabeled subtraction probe enriched for bdv-specific sequences [15] . the bdv-specific clones isolated in our laboratory [16, 17, 28] and in that of another group [14, 15] recognized four bdv-specific rnas of 10.5, 3.6, 2.1, and 0.85 kilobases (kb), respectively, in bdv-infected rat brain; all of these rnas were enriched by polyadenylate selection [16] . lipkin et al. [15] reported that the largest rna species is 8.5 kb in size and is not polyadenylated. in vitro transcription and translation of the bdv-specific cdna clone isolated in our laboratory (clone b8) produced proteins of 24 and 14 kd that were recognized by both polyclonal and monoclonal antibodies to bdv [16, 17, 28] (figure 1). no significant similarities to any known viral or cellular sequences were detected with both the nucleotide and the amino acid sequences of clone b8 [16, 17] . in addition, southern blot hybridization with bdv-specific cdna clones isolated by both laboratories [15, 16] showed that these clones were not of cellular origin and did not hybridize to any dna species in infected material. these data and the identification of four species of rna in infected material clearly demonstrate that bdv is an rna virus [14] [15] [16] [17] 28] . furthermore, since the bdv-specific rnas in infected material are sensitive to digestion with pancreatic rnase, bdv seems to be a single-stranded rna virus [14] . oligonucleotides with negative polarity from different regions of the b8 clone all hybridized to the same four positivestrand rnas identified for the entire b8 clone [16, 17, 28] (figure 2, lane b). this result indicates that all of the nucleotide sequences in the b8 clone are present in all of the larger rnas and thus suggests that the organization of the rnas is a nested set of overlapping rnas, as in coronaviruses [29] [30] [31] . in addition, positive-polarity oligonucleotides from different regions of clone b8 hybridized to three bdv-specific rnas of 10.0, 3.5, and 1.7 kb [17, 28] (figure 2, lane d). these rnas probably represent negative-strand complements of the positive rna species. recently, negativestrand rnas have been identified in coronavirus-infected cells; these rnas are thought to be important in replication of the subgenomic rnas [30, 31] . thus, both the organization of the rnas and the presence of positive-and negativestrand rnas indicate a striking similarity of bdv to the coronavirus family, although it has not yet been resolved whether bdv is a negative-or a positive-strand rna virus. the resolution of this issue will require the isolation of bdv and the identification of the polarity of its genomic rna. bdv is infectious for a wide range of animal species, from chickens to nonhuman primates and possibly humans [1, 3, 20, 32] . the clinical manifestations and outcome of experimental infection vary among animals. rabbits develop an reproduced with permission from [28] . acute, fatal paralytic disease similar to that seen in horses within 4-6 weeks [4, 5] ; tree shrews (tupaia glis) develop an unusual, nonfatal behavioral disease characterized by aberrant social interactions [33] . several strains of rat can be infected with bdv [25] [26] [27] 34] , but only some of these strains develop disease [35] . with the exact manifestation depending on the viral variant, lewis rats can develop a biphasic neurological illness characterized by an initial hyperacute aggressive phase that is followed by a passive somnolent stage, an obesity syndrome with fertility disturbances, or paralysis followed by death [5, 25, 26, 36] . rhesus monkeys develop severe paralytic disease with retinopathy [5, 37, 38] , whereas mice and hamsters show no clinical signs despite replication of bdv in the cns [39, 40] . histologic studies of brains from animals with borna disease show typical meningoencephalomyelitis, with the most severe lesions in the frontal region of the cerebral cortex. no pathological abnormalities have been observed in infected mice and hamsters. tree shrews have been used extensively in studies of social interactions because of their well-defined behavioral pat-terns. sprankel et al. found that these animals, classified at the phylogenetic root of the primates, developed a long-lasting, persistent, productive infection in the cns and unique behavioral abnormalities after is inoculation with bdv [33] . characteristic perivascular infiltration of mononuclear cells and intranuclear joest-degen inclusion bodies in the cns were observed. housing conditions influenced the reaction of these animals to infection. the behavior of all paired animals changed dramatically. these animals showed a need for increased body contact with their partners during the first few weeks after infection. further, infected animals tended to accept their partners much more quickly in grooming social interactions than did uninfected shrews. normally passive females became as aggressive as their male partners and took poor care of their offspring. in contrast, only 25% of the infected animals maintained in solitary cages showed clinical and behavioral changes. these animals exhibited hyperactivity, with drastic shortening of resting time, and developed eating disorders (bulimia) about 4 weeks after infection. the hyperactive phase was followed by a hypoactive phase characterized by retirement into sleeping boxes and decreased interest in self-grooming. only some of these animals devel-retinopathy in humans. thus, in studies of these changes, rhesus monkeys were infected ic with bdv [5, 37, 38] . the animals developed a persistent infection in the cns and produced antibodies to bdv-specific antigens in serum, csf, and aqueous humor. pathological alterations in the cns and retina consisted of infiltrations of mononuclear cells similar to those in naturally infected animals; however, no destruction of the neuronal layers of the retina was observed [37, 38] . clinical signs started with apathy and anorexia 4-8 weeks after infection and progressed to severe neurological manifestations dominated by paralysis of the hind limbs. one animal splenectomized 11 months before infection showed signs of apathy but developed no paralysis and had fewer cellular infiltrates in the cns and the retina than did other animals [37] . these findings suggested that the pathogenesis of borna disease in primates is based on an immunopathological mechanism-an idea confirmed in studies with lewis rats and rabbits. neurotropism of bdv figure 2 . northern blot analysis of rna from rat brain. lanes a and c contain 10µg of total rna from uninfected rat brain. lanes b and d contain 10 tig of total rna from bdv-infected rat brain. lanes a and b were hybridized with a negative-sense 32p-labeled oligonucleotide (247), lanes c and d with a positive-sense 32p-labeled oligonucleotide (305). the blot was stripped and rehybridized with a chicken li-actin probe to ensure that equal amounts of rna were loaded in each lane. reproduced with permission from [28] . oped more severe neurological signs, such as ataxia and partial paralysis of the limbs [33] . the pathological changes in the brain and retina of bdvinfected animals resemble certain types of encephalitis and most studies on the pathogenesis of bdv infection have involved experimentally inoculated lewis rats. as has already been mentioned, the clinical manifestations of the infection in these animals depend on the passage history of the virus used for inoculation. thus a variety of bdv variants have been used, including strains that cause paralysis and death (similar to the disease in horses and sheep), obesity with fertility disturbances, behavioral changes, or inapparent infections. in all cases, ic inoculation of bdv into adult rats resulted in productive viral replication in the nervous system, where viral antigen was confined to astrocytes, schwann cells, and central and ganglionic neurons and their axonal-dendritic processes [10, 41, 42] . infectivity was first detected in brain homogenates 7 days after inoculation with 105-106 id50/ml by day 15 [26] . this infectivity titer in the brain was maintained throughout an observation period of 7 months (figure 3, top). similar data were obtained for black hooded rats, which were found to be resistant to the disease [35] . lower levels of infectivity (10 2-104 id50/ml) were usually found in spinal cord, adrenal glands, and ganglia of the autonomic nervous system. infectivity in the eyes was transient, lasting -4 weeks after virus appeared in the brain. loss of infectivity coincided later with degeneration and loss of retinal neurons. in immunocompetent adult rats no infectivity was found in extraneural tissue at any stage [26, 43] . neither infectious virus nor viral antigens were detectable in lung, spleen, kidney, muscle, peritoneal macrophages, or blood. the same pattern of tropism was seen in cyclophosphamide-treated rats [26, 43] (figure 3, bottom) and in athymic adult rats after ic inoculation of bdv [44] . the lack of susceptibility of cultured macrophages and spleen fibroblasts week-old rats infected with bdv and treated with cyclophosphamide (150 mg/kg) 1 day later. the persistently high levels of infectivity in the brain and eyes and the lack of infectivity in nonneural tissues are shown. rats did not respond immunologically to the virus and did not become ill. reproduced with permission from [26] . to infection with bdv demonstrated the strong tropism of the virus for cells of neural origin [26, 27] . in rats inoculated ic or intranasally at birth, bdv replicated preferentially in the cns and the peripheral nervous system but also spread to nonneural tissues. whereas infectivity in nervous tissue was maintained at a level of 10 5 -106 id50/ml, titers in the nonneural tissues such as heart, lung, liver, spleen, kidney, pancreas, and bladder were approximately two orders of magnitude lower [43] . infectious virus was shed in saliva, lacrimal fluid, and urine [10] . infectivity assays and immunocytochemical assays on sequential tissue samples obtained after inoculation from infected newborn rats showed that the agent first began to replicate in the cns and then disseminated along nerve pathways to nonneural tissues. presumably, infection in the latter tissues was initiated at the point of innervation, with subsequent spread from cell to cell. no viremia was detected in these animals. the similar pattern of dissemination noted in infected adult rats treated for prolonged periods with cyclosporin a [23, 45] may suggest that nonneural cells are innately susceptible to infection but that yet-unknown factors in the host prevent access of the virus to those cells. delineation of the scheme for the dissemination of bdv via neural pathways from and to the cns was based on the initial observation by krey et al. [46] that in rabbits spread of the virus from the brain to the retina could be prevented by ablation of the optic disk. the incubation period of the disease in rats (the period between inoculation of virus at a peripheral site and onset of illness) varied significantly with the route of inoculation [10, 27] . the onset of boma disease could be recognized only after the virus invaded the brain. disease appeared 17 days after ic inoculation, 20-24 days after intranasal inoculation, and 47 days after footpad inoculation [27] . after intranasal infection (probably the natural route), bdv entered the neural receptors in the olfactory epithelium and migrated into the brain (figure 4) [10] . similar results were obtained with inoculation into the hind footpad of adult rats (figure 5). infection and disease were prevented by sectioning of the sciatic nerve before or within 1 day after inoculation of bdv [27] . in contrast, neither iv inoculation of bdv weekly for 3 weeks nor oral inoculation of the virus resulted in infection [27] . thus bdv infection in the host resulted only after obligatory replication in nervous tissues, with subsequent dissemination to nonneural tissues via neural pathways in immunologically impaired animals. once introduced into the cns of the rat, bdv usually caused a persistent infection with continuous productive replication in the brain and spinal cord. all immunocompetent infected animals developed antibodies to bdv-specific antigens, and these antibodies coexisted with infectious virus in the cns. hyperimmune sera or csf from bdv-infected animals did not protect against infection in neutralization experiments in vivo or in vitro [26, 27] . infection of newborn rats. although infection of newborn rats resulted in persistent viral replication in the cns as well as in visceral organs, these animals developed no inflammatory response or signs of borna disease. however, "luxury" functions of the cns seem to be affected, possibly as a result of morphological changes confined to the loss of neurons from the dentate gyms of the hippocampus and of some neurons from the nuclear and photoreceptive layers of the retina [25, 26, 43] . the animals produced normal litters, and no virus was found in brain homogenates of their progeny [26] . (however, when housed in the same cage as their infected newborns, mothers can become infected [10] .) infected newborn animals developed bdv-specific antibodies late after infection [26, 43] . infection of immunocompetent rats. infected immunocompetent rats developed severe disseminated meningoencephalitis in which the most intense inflammatory reaction was centered in the gray matter of the cerebral cortex. the onset of clinical disease coincided with the appearance of meningoencephalomyelitis rather than being a direct effect of viral replication in the brain [26, 27] . the most prominent changes were dense accumulations of mononuclear cells in the perivascular spaces and throughout the neuropilcorresponding to the areas with greatest viral replication [25] [26] [27] 42] . immunocytochemical investigation of the composition of the inflammatory cell population during the course of infection revealed that macrophages and lymphocytes of the cd4+ phenotype were dominant at all stages [42, 47] . cd8 + t cells were less frequent. b lymphocytes and plasma cells became prominent during later stages of the disease, when marked parenchymal deposition of immunoglobulin developed [42] . the severity of the inflammatory reaction reached its max-imum between day 30 and day 40, during the hyperacute phase of the disease. edema and necrosis of cellular elements in the neuropil accompanied the inflammatory changes. this phase was characterized by alertness, loss of fear, and frenzied hyperactivity. infection with an "aggressive" variant led to paralysis and death during this phase. the cell loss was most extensive in the cerebrum between the frontal and temporal cortices, and this effect finally led to hydrocephalus ex vacuo in surviving animals [25, 26] . hydrocephalus progressed slowly, and, despite continuous productive replication of bdv in the brain, the inflammatory response began to decline after day 50, with only minimal inflammatory lesions detectable in the brain by day 200. there was no further loss of brain substance and no further extension of hydrocephalus after the inflammatory cells disappeared. beyond day 75, by which the inflammatory response had become mild, the animals became much calmer, spending most of the time in an inactive state and asleep. no pathological changes were found in the ependymal lining of the ventricles or the choroid plexus throughout the study [25, 26] . despite the dynamic changes in the intensity of inflammation, high levels of viral replication in the cns persisted in these animals ( figure 3, top) . pathological changes in the eyes were similar to those in the brain and developed subsequent to encephalitis. there was a degeneration of rods and cones and a gradual focal disappearance of neurons from the inner and outer nuclear layers [25, 26] . by day 100 most of the animals had become blind since the infection resulted in the complete destruction of the inner and outer nuclear layers of the retina. no inflammatory cells were found in the organs at late stages. viral variants differed in their ability to cause various clinical manifestations in the rat. this observation raises the intriguing possibility that bdv exhibits marked genetic variability and that different strains may cause different pathological manifestations of neurological disease. the obesity syndrome developed gradually in immunocompetent infected rats, starting -5 months after infection ( [48] and s. herzog and r. rott, unpublished results). the obese rats exhibited characteristic behavioral and metabolic alterations, with significant increases in the intake of food and water and the development of high levels of triglycerides, insulin, and glucose in the blood [48] . histologic examination revealed prominent hydrocephalus and meningoencephalitis. in long-term survivors, titers of infectious virus in the cns declined drastically and antibody levels decreased ( [48] and s. herzog and r. rott, unpublished results). prevention of disease in rats. in 1981 gierend and ludwig [49] reported that treatment of bdv-infected rabbits with glucocorticoids and/or cyclophosphamide resulted in a reduction in antibody production, the development of only low-intensity encephalitis, and a delayed onset of clinical disease. similar studies showed that a single ip injection of cyclophosphamide (150 mg/kg), given i day before or after ic inoculation of bdv into 4-week-old rats, prevented the onset of borna disease. these rats did not develop antibodies to bdv, encephalitis, or clinical disease [25, 26] . the level of production of virus in the cns and the neurotropism of the agent were similar in treated and untreated infected animals ( figure 3) . thus a persistent, productive type of viral replication developed in the brain and eyes of cyclophosphamide-treated rats (figure 3, bottom) without the complications of pathological effects or clinical disease. as in infected newborn rats, neurons in the dentate gyms of the hippocampus degenerated and were replaced by glial cells. since other infected neuronal groups did not degenerate, loss of these cells may have been due to down-regulation of trophic factors. in contrast, no necrosis among other cell types in the brain occurred in these animals. similarly, infected newborn, athymic, and cyclosporin a-treated adult rats failed to develop encephalitis or disease [23, 26, 36, [43] [44] [45] . these data suggested strongly that borna disease was caused by the cellular immune response to bdv in immunocompetent animals. reconstitution of bdv-specific inflammation: immunosuppressed animals. the immune basis of borna disease was confirmed in adoptive transfer experiments in which spleen or cervical lymph node cells from infected rats were transferred to infected, 4-week-old, cyclophosphamide-immunosuppressed virus carriers. after adoptive transfer these tolerant virus carriers developed bdv-specific antibodies, encephalitis, and behavioral disease (table 1). the donor note. four-week-old lewis rats were inoculated ic with bdv; some rats were treated with cyclophosphamide (150 mg/kg) 24 hours later. animals rendered tolerant by this treatment were injected iv with various cell suspensions, as indicated, and were maintained for clinical and pathological examination. nmi t cells are described in the text; ppd t cells = purified protein derivative-specific t cells. figure 6 . acetone-fixed astrocytes persistently infected with bdv were stained with tetrarhodamine isothiocyanate-labeled rat antibodies to bdv and fluorescein isothiocyanate-labeled rabbit antibodies to glial fibrillary acidic protein (gfap). arrows point to punctate accumulation of bdv antigens in the nuclei, and arrowheads show diffuse staining pattern of gfap in the cytoplasm. cells had no effect when injected into immunocompetent animals [25] . further, transfer of hyperimmune bdv-specific sera into virus carriers did not cause clinical disease [25] . with the goal of identifying the cell type responsible for inducing disease in virus carriers, a permanent t cell line, nm 1, was developed from the popliteal lymph node cells of lewis rats immunized in their hind feet with affinity-purified, bdv-specific 38/39-kd protein [47, 50] . the nm 1 cells responded specifically to the 38/39-kd bdv antigen in lymphocyte proliferation assays and did not recognize unrelated antigens. cytofluorographic studies with mabs to leukocyte differentiation antigens showed a staining pattern characteristic for cd4 + t cells. inhibition studies with mabs against selective restriction elements of the major histocompatibility complex (mhc) revealed that these cd4 + effector t cells were mhc class ii restricted [47, 50] . the latter cells were functionally characterized in studies with astrocytic cultures used as antigen-presenting cells or targets and pulsed with bdv-specific 38/39-kd protein or infected with bdv. astrocytes are known to become infected during bdv-induced encephalopathy [41, 42] , and primary astrocyte cultures are susceptible to bdv infection when inoculated with infectious rat brain homogenate (figure 6) . the persistent noncytopathic infection of astrocytes with bdv did not result in significant spontaneous expression of mhc class ii antigens, although such expression has been reported for other neurotropic viruses [51, 52] ; however, the expression of this antigen was easily induced by incubation with recombinant interferon-7 (ifn-y). in lymphocyte proliferation experiments, uninfected, ifn-7treated astrocytes were able to induce specific proliferation of nm 1 cells when inoculated exogenously with the 38/39-kd protein [50] . when persistently bdv-infected astrocytes were used as antigen-presenting cells after induction with ifn-y, only slight specific proliferation of the nm 1 cells was found. however, exogenous addition of the bdv-specific 38/39-kd protein to these cultures resulted in antigen-specific proliferation [50] . furthermore, when analyzed in a conventional cytotoxicity assay, bdv-infected astrocytes were found to be susceptible to lysis by nm 1 t cells (j. a. richt and l. stitz, unpublished results). the bdv-specific nm i cells induced acute meningoencephalitis mainly in the gray matter of the brain and produced hyperacute paralytic disease when injected iv into immunosuppressed syngeneic virus carriers [47, 50] (table i ) . the clinical signs became manifest as early as 5 days after transfer, with development of severe apathy and somnolence; most of these animals had to be killed within 24 hours after the onset of disease. inflammatory exudates contained a large number of cd4 + t cells, fewer cd8 + lymphocytes and b cells, and some neutrophils [42, 47, 50] . the animals did not go through the hyperactive phase that typically occurs during regular infection or reconstitution with spleen cells. their disease was hyperacute and rapidly fatal. the fact that disease could be induced after transfer of mhc class ii-restricted, bdv-specific, cytolytic cd4 + t lymphocytes suggested that the lesions in borna disease may be a delayed-type hypersensitivity reaction. the mechanism for the different clinical disease that resulted from reconstitution with nm 1 cells-as opposed to spleen and lymph node cells-is not understood. the nm 1 cells probably caused elaboration of unusually large amounts of acute-phase proteins and cytokines, which in turn could have caused an imbalance in neurotransmitter functions in the brain [53] . the difference in clinical disease caused by the spleen and lymph node cell suspensions may also have been due to the combination of nonselected immune cells used for transfer. carriers. the pathogenesis of borna disease has several features in common with lymphochoriomeningitis (lcm) virus infection in mice. in both cases the virus causes acute cns disease that can be prevented by immunosuppression, and this tolerance can be overcome by reconstitution with specifically sensitized cells. the similarity in the pathogenesis of the two infections applies in newborn animals. newborn mice inoculated with lcm virus become persistently infected, but their growth is severely retarded and they later die from immune complex disease [54] . newborn rats inoculated with bdv also become persistently infected, as has already been described. however, unlike their murine lcm-infected counterparts, persistent bdv-infected rats do not become ill [43] . in contrast to adoptive transfer into cyclophosphamidetreated virus carriers [25] , injection of spleen cell suspensions from diseased syngeneic rats into newborn bdv carriers did not result in inflammation or in clinical boma disease [55] . for evaluation of the role of immunologic tolerance in neonatal virus carriers, these animals were surgically joined to syngeneic normal rats via the peritoneal cavity in a manner that allowed the free flow of humoral and cellular immune elements [55] ; this scheme followed the classical experiment of billingham et al. [56] . the normal-counterpart rats were then inoculated with bdv and developed typical borna disease. examination of the tissues from the persistently infected chimera showed that these animals developed mild lesions in the central and peripheral nervous systems. adoptive transfer of the cd4+ virus-specific t cell line nm 1 into neonatal (6-week-old) infected rats also resulted in mild transient encephalitis [55] ; this development suggested that tolerance to the virus in newborn infected animals can be overcome by transfer of bdv-specific immune cells, although reconstitution was not as efficient as in cyclophosphamide-treated virus carriers. regulatory immune mechanisms present in neonatal infected animals, but not in adult rats rendered tolerant by cyclophosphamide, may be responsible for this unique mechanism of tolerance and resistance to the induction of disease. the behavioral changes in animals infected with bdv are somewhat reminiscent of some types of affective disorders in human beings. indeed, the serum and csf of some patients with psychiatric illnesses (including recurrent unipolar depression, bipolar affective disorders, and residual-type schizophrenia or personality disorders) contain bdv-specific antibodies [32, 57] . antibodies were present in 4%-7% of sera obtained from -5,000 psychiatric patients from germany, the united states, and japan. the percentage of patients seropositive was highest in a region of southern germany where borna disease has been known to be endemic among horses and sheep. approximately 1% of 1,000 randomly collected sera from otherwise hospitalized patients from this area in which borna disease is endemic contained antibodies to bdv [20] . examination of 56 acutely seropositive patients by magnetic resonance imaging revealed that 46% had cerebral lesions in one or both hemispheres, whereas no one in a seronegative control group had such lesions [58] . serological examination by bode et al. of patients infected with human immunodeficiency virus has shown an incidence of bdv antibodies of -8% [59] . the same investigators have reported a high incidence of bdv-specific antibodies among patients with chronic inflammatory neurological disorders such as multiple sclerosis [60] . the bdv specificity of the human antibodies was recently reinforced by the finding that antibodies from three of seven seropositive patients recognized the 24-kd protein expressed by a bdv-specific cdna clone [16, 28] (figure 1). these data suggest either that a bdv-like infection is prevalent in the human population or that some patients with inflammatory neurological diseases have cross-reacting antibodies that recognize antigens expressing epitopes homologous with those of bdv proteins. these observations were substantiated further by attempts to isolate bdv from the csf of three seropositive patients [20] . csf from these patients was either applied to fetal rabbit brain cells or inoculated is into rabbits, which are highly susceptible to borna disease. in cell cultures a small number of immunoreactive cell foci were found with bdv-specific antibodies 10-12 days after inoculation. however, the cells lost their antigen during subcultivation. the inoculated rabbits developed no signs of borna disease; neither lesions in the cns nor bdv-specific antigens were demonstrable in brain sections by immunohistologic techniques. nevertheless, these animals developed bdv-specific antibodies in their sera. that the brain homogenate from one rabbit was infectious for fetal rabbit brain cells was demonstrated by positive cell foci in immunofluorescence assays [20] . however, again, the antigen disappeared during attempts to propagate the agent by subcultivation of the cells. these findings can be interpreted as typical of abortive infection. although these data strongly support the hypothesis that a bdv-like agent causes infection in humans, a defined mental disorder has not been correlated with the presence of bdv-specific antibodies. the development of such a disorder may be related to various factors, such as the genetic properties of the virus; the route of infection; and the age, immune status, and genetic makeup of the infected individual. evidence that these factors play a role in the outcome of borna disease has been found in naturally infected horses and sheep as well as in experimentally infected animals. die bornasche krankheit der pferde und schafe virologisch gesicherter ausbruch der bornaschen krankheit in einer schafherde der schweiz experimentelle untersuchungen fiber die seuchenhafte gehirn-und rackenmarksentztindung der pferde (bornasche krankheit) the cerebrospinal fluid of rabbits infected with borna disease virus handbuch der viruserkrankungen untersuchungen fiber das klinische verhalten der seuchenhaften gehirnrtickenmarksentziindung (bornaschen krankheit) des pferdes nebst angaben fiber diesbeztigliche therapeutische versuche borna disease in a sheep untersuchungen fiber die pathologische histologie, pathogenese und postmortale diagnose der seuchenhaften gehirn-rtickenmarksentziindung (bornasche krankheit) des pferdes die ausbreitung der enzephalitischen reaktion bei der bornaschen krankheit der pferde und deren beziehungen zur encephalitis epidemica, zur heine-medinschen krankheit und zur lyssa des menschen. eine vergleichend-pathologische studie axonal transport of borna disease virus along olfactory pathways in spontaneously and experimentally infected rats seroepidemiologische untersuchungen zur bornaschen krankheit (ansteckende gehirn-riickenmarkentziindung) der pferde borna disease: a persistent virus infection of the central nervous system preliminary studies on the biology of borna disease virus molecular characterization of the borna disease agent isolation and characterisation of borna disease agent cdna clones a borna virus cdna encoding a protein recognized by antibodies in humans with behavioral diseases analysis of borna disease virus-specific rnas in infected cells and tissues replication of borna disease virus in cell cultures influence of interferon on persistent infection caused by borna disease virus in vitro borna disease, a possible hazard for man? isolation and characterization of a 14500 molecular weight protein from brains and tissue cultures persistently infected with borna disease virus purification and properties of an intranuclear virus-specific antigen from tissues infected with borna disease virus atypical dissemination of the highly neurotropic borna disease virus during persistent infection in cyclosporin a-treated, immunosuppressed rats antigenetic relationship and further characterization of two major borna disease virus proteins behavioral disease in rats caused by immunopathological responses to persistent borna virus in the brain pathogenesis of borna disease in rats: immune-mediated viral ophthalmoencephalopathy causing blindness and behavioral abnormalities pathogenesis of borna disease in rats: evidence that intraaxonal spread is the major route for virus dissemination and the determinant for disease incubation molecular and immunopathological studies of borna disease virus infection in rats coronaviruses and their replication coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in rna synthesis minus-strand copies of replicating coronavirus mrnas contain antileaders detection of serum antibodies to borna disease virus in patients with psychiatric disorders behavior abnormalities in tree shrews (tupaia glis, diard, 1920) induced by boma disease virus persistent, tolerant or subacute infection in borna disease virus-infected rats studies on the genetic control of resistance of black hooded rats to borna disease untersuchungen fiber die experimentelle bornavirus-infektion bei der ratte borna disease in rhesus monkeys as a model for uveo-cerebral symptoms virus-induced pigment epithelitis in rhesus monkeys. clinical and histological findings persistent borna virus infection in adult hamsters adaptation of borna disease virus to the mouse astrocytes and schwann cells are virus-host cells in the nervous system of rats with borna disease determination of immune cells and expression of major histocompatibility complex class ii antigen in encephalitic lesions of experimental borna disease replication of borna disease virus in rats: age-dependent differences in tissue distribution effect of borna disease virus infection on athymic rats inhibition of immunemediated meningoencephalitis in persistently borna disease virusinfected rats by cyclosporine a spread of infectious virus along the optic nerve into the retina in borna disease virus-infected rabbits borna disease virus-induced meningoencephalomyelitis caused by a virus-specific cd4+ t cell-mediated immune reaction escape from lethal disease in rats after borna disease virus infection: survival with obesity syndrome influence of immunosuppressive treatment on borna disease in rabbits borna disease, a progressive meningoencephalomyelitis as a model for cd4 + t cell-mediated immunopathology in the brain viral particles induce la antigen expression on astrocytes tumor necrosis factor amplifies measles virus-mediated la induction on astrocytes narayan 0, oldstone mba. neurotransmitter abnormalities in borna disease southern p. virus and immune responses: lymphocytic choriomeningitis virus as a prototype model of viral pathogenesis induction of borna disease and inflammation in rats inoculated with borna disease virus as neonates quantitative studies on tissue transplantation immunity. iii. actively acquired tolerance borna disease virus. a possible etiological factor in human affective disorders? kernspintomographische befunde bei psychiatrischen patienten mit and ohne semm-antikfirper gegen das borna disease virus-specific antibodies in patients with hiv infection and with mental disorders increased incidence of borna disease virus infection in chronically diseased patients key: cord-262281-56tbrl8a authors: hawkes, c. h.; del tredici, k.; braak, h. title: parkinson's disease: a dual‐hit hypothesis date: 2007-10-24 journal: neuropathol appl neurobiol doi: 10.1111/j.1365-2990.2007.00874.x sha: doc_id: 262281 cord_uid: 56tbrl8a accumulating evidence suggests that sporadic parkinson's disease has a long prodromal period during which several non‐motor features develop, in particular, impairment of olfaction, vagal dysfunction and sleep disorder. early sites of lewy pathology are the olfactory bulb and enteric plexus of the stomach. we propose that a neurotropic pathogen, probably viral, enters the brain via two routes: (i) nasal, with anterograde progression into the temporal lobe; and (ii) gastric, secondary to swallowing of nasal secretions in saliva. these secretions might contain a neurotropic pathogen that, after penetration of the epithelial lining, could enter axons of the meissner's plexus and, via transsynaptic transmission, reach the preganglionic parasympathetic motor neurones of the vagus nerve. this would allow retrograde transport into the medulla and, from here, into the pons and midbrain until the substantia nigra is reached and typical aspects of disease commence. evidence for this theory from the perspective of olfactory and autonomic dysfunction is reviewed, and the possible routes of pathogenic invasion are considered. it is concluded that the most parsimonious explanation for the initial events of sporadic parkinson's disease is pathogenic access to the brain through the stomach and nose – hence the term ‘dual‐hit’. sporadic parkinson's disease (pd) is the most frequent degenerative disorder of the human nervous system after alzheimer's disease. it is not known to occur spontaneously in other vertebrates and does not directly affect other organs apart from the nervous system. motor dysfunction (hypokinesia, postural imbalance, cogwheel rigidity, resting tremor) indicates the presence of disease, but also can appear in the guise of 'parkinsonism' in other disorders that are associated with a significant reduction of dopamine in the central nervous system (cns). parkinsonism may develop as a sequel to intoxication, trauma, vascular disease and infections. there are genetically based familial forms and degenerative or sporadic forms, the last of which include the tauopathy progressive supranuclear palsy (psp) and synucleinopathies such as multiple system atrophy (msa) and lewy body disease. lewy body disease has been further subdivided into pure autonomic failure, sporadic pd and dementia with lewy bodies (dlb) [1] [2] [3] [4] [5] . this review will refer mainly to the sporadic form of pd. the pathological process that underlies sporadic pd is linked to the development of a-synuclein-containing inclusion bodies in the form of lewy bodies (lbs) in perikarya and lewy neurites (lns) in neuronal processes [6] [7] [8] . of the diverse neuronal types within the human nervous system, only a few develop pathological inclusions, and this selective involvement is reflected in the regional pattern of the pathology. vulnerable cells are distributed throughout the peripheral, enteric and central portions of the nervous system (ens/cns) [9] [10] [11] [12] . all of the susceptible cells are projection neurones that generate a long and thin axon, which is unmyelinated, or poorly myelinated [13] . despite their greater prevalence with advancing age, pd-associated inclusion bodies do not occur consistently in all aged non-symptomatic cases and, as such, they are pathological rather than protective or neutral age changes. the presence of these pathognomonic inclusions is a prerequisite for the post mortem diagnosis of sporadic pd [14, 15] . these disease-related inclusions occur in symptomatic pd patients as well as in individuals who did not manifest any of the characteristic motor symptoms in life. thus, the pathological process has a pre(motor)-symptomatic and a symptomatic phase [16] [17] [18] . the term 'presymptomatic phase' implies that even when only a few lns/lbs are detectable in non-symptomatic cases, such 'incidental' inclusion bodies represent incipient pd or the harbinger of the symptomatic disease phase [14, 19] . it has been postulated that sporadic pd might be a primary disorder of olfaction, given that smell loss is an early event in the course of this disorder [20] . this theory was derived from sources based on psychology, physiology, anatomy and pathology. additional studies not only have corroborated the initial involvement of anterior olfactory structures, but also have pointed to an early involvement of the enteric nerve cell plexuses as well as of the dorsal motor nucleus of the vagus and the intermediate reticular zone in the lower brainstem [21] [22] [23] [24] [25] . to be plausible, any theory attempting to explain sporadic pd must incorporate these extra-nigral sites, which consistently become affected in the course of the disorder [9] . furthermore, any speculation regarding the cause and beginnings of pd must take into account the involvement of olfactory and autonomic systems that generally develop prior to the onset of the classical somatomotor symptoms [26] [27] [28] . this article will review the evidence for such involvement and summarize several hypotheses developed on the basis of these findings. the first case-control study that demonstrated smell abnormality in pd was conducted by ansari and johnson [29] in 22 clinically diagnosed pd patients. a subsequent larger study used detection threshold tests to amyl acetate in 78 subjects and 40 controls [30] . thresholds were reduced, but no correlation was found with age, gender or treatment with levodopa. unlike the first study, there was no association with disease duration. the next sizeable investigations using the university of pennsylvania smell identification test (upsit) showed that age-matched olfactory dysfunction did not relate to odour type, was independent of disease duration, and did not correspond with motor function, tremor or cognition [31, 32] . the authors also demonstrated that the deficit was of the same magnitude in both nostrils, and not influenced by anti-parkinsonian medication. a comparable survey was undertaken using upsit in 155 cognitively normal, depression-free pd patients, and 156 age-matched controls [33] . upsit scores for pd patients were dramatically lower than those for controls. there was no correlation between disease duration and upsit score (r = 0.07). impairment of smell sense has also been documented in pd patients using sniffin sticks [34, 35] . although the psychophysical evidence provides overwhelming support for olfactory involvement in pd, it does not completely eliminate the possibility of confounding factors from cognitive dysfunction, nor can it be determined whether the smell defect is initially peripheral or central [36] [37] [38] [39] . a further measure of smell sense is the olfactory eventrelated response (oerp) pioneered by kobal and plattig [40] , which has the advantage of minimizing the potential effect of cognitive dysfunction. an initial examination compared oerp recording of 73 pd patients with that of 47 controls of similar age and gender [33] . in 36 patients (49%), responses were either absent or unsatisfactory for technical reasons. analysis of the 37 with a measurable trace showed that, for hydrogen sulphide (h2s), a highly significant latency difference existed between diagnostic groups. similar results were obtained in 31 patients with clinically labelled pd tested by oerp to vanillin and h2s [41] . prolonged latencies were seen in these individuals whether they were taking anti-parkinson medication or not. the above-cited evidence from psychophysical and neurophysiological sources gives virtually unassailable support to the presence of olfactory dysfunction in established pd, a feature that occurs more frequently [33] than tremor (80% vs. 70%). if the previous observations about early olfactory involvement are correct, this should be reflected by tests in individuals at risk for future disease or by prospective studies of those with prior olfactory impairment [42] . montgomery and colleagues [43, 44] implemented a test battery comprising motor function, olfaction (upsit) and mood for pd first-degree relatives. there were significant differences in sons and daughters, particularly where the affected parent was the father.this work has been criticized [45] because there may have been self-selection in allegedly unaffected relatives -who may have had undisclosed motor complaints resulting in the unusually high 2-year positive prediction rate (40 out of 59 subjects). ponsen et al. [45] evaluated prospectively 78 asymptomatic firstdegree relatives of non-familial pd patients by olfactory tests and dopamine transporter scan (datscan). forty were hyposmic at baseline and, when reviewed 2 years later, four had abnormal datscans and showed clinical signs of pd. in the remaining 36 hyposmics, who displayed no sign of pd, the rate of decline of dopamine transporter binding was higher than in normosmic relatives. others [46] tested 30 patients with unexplained and isolated smell impairment to determine whether any might be in the premotor phase of pd. apart from detailed olfactory testing, subjects were evaluated by datscan and transcranial sonography of the substantia nigra. eleven displayed increased (that is, abnormal) echogenicity on transcranial sonography characteristic of pd. ten subjects volunteered for datscan and, of them, five had abnormal scans and an additional two were borderline, suggesting that they might be in a presymptomatic phase of pd. two of the five scanpositive patients have now developed clinically confirmed pd (hummel, pers. comm., 2006) . the first long-term, community-based prospective study has been published, in connection with these relations [47] . the authors used the cross-cultural brief smell identification test (bsit) [48] in 263 healthy japanese-american men aged 71-95 years who participated in the honolulu-asia ageing study. after 7-year follow-up, 19 men developed pd at an average latency of 2.7 years from baseline assessment. adjustment for multiple confounders gave relative odds for pd in the lowest tertile of bsit score of 4.3 (95% ci 1.1-16.1; p = 0.02), thus indicating a moderate predictive power of olfactory testing. in the same cohort, those who later died underwent brain autopsy to detect the presence of brainstem lbs [49] . of 163 autopsied men without clinical pd or dementia, 17 were found to have incidental lbs. those who scored in the lowest tertile of the bsit were significantly more likely to have lbs at autopsy. potentially conflicting findings were obtained in a study of 70 male twin pairs discordant for pd [50] , 62 of whom agreed to undergo olfactory tests. at baseline, the authors confirmed impaired upsit scores in the affected twins, but not in their brothers who were rated normal. after a mean of 7.3 years, 28 brothers were still alive and, of them, 19 were retested using the 12 odour bsit, whereby 2 had developed pd. neither had impaired upsit at baseline, but the average decline in upsit percentile score in both was greater than the remaining 17, who had not developed the disease. it is suggested that smell testing may not be a reliable predictor. the dropout rate was unusually high and smell assessment was by different methods, that is, initially by upsit (40-item) then by bsit (12-item). on the first occasion, the test was unsupervised; moreover, a 7.3-year interval may have been too short. despite such evidence, theories regarding olfactory dysfunction as a characteristic symptom of pd have had difficulty gaining acceptance, because 10-20% with a clinical diagnosis of pd have normal smell identification. in some pd patients with normal upsit scores, the oerp was absent or delayed [33] , which implies that the olfactory deficit may be more frequent than indicated by upsit. pathology-based studies indicate that the diagnostic error rate for pd is 10% or higher, and that olfactory identification is sometimes abnormal in other diseases showing parkinsonism [51] . mistaken diagnosis of pd occurs most often in msa, psp, vascular parkinsonism and, occasionally in individuals with essential tremor. all these disorders may be characterized by either normal or slight impairment of olfaction. it is not yet known whether persons with normal upsit score are the very patients who have received an incorrect diagnosis, but prospective studies would resolve this issue. an understanding of the applied anatomy of the vagus is necessary in view of its early and severe pathological involvement. three major components of the vagus can be distinguished in the medulla oblongata: (i) the ambiguus nucleus, a motor nucleus that innervates muscles of the palate, pharynx, larynx and heart; (ii) the dorsal motor nucleus, which harbours the preganglionic visceromotor neurones that control the postganglionic parasympathetic nerve cells in internal organs of the chest and abdomen; and (iii) the complex of nuclei accompanying the solitary tract. the parvocellular nuclei of the latter complex receive taste information and an abundance of visceral sensations. the dorsal motor nucleus is damaged severely and early on in pd, the solitary tract nuclei exhibit little change until later stages, and the nucleus ambiguus remains intact [21] (figure 1 , right). the gastrointestinal tract is supplied by two major nerves: the vagus, which is for excitatory parasympathetic control, and sympathetic nerves, which are inhibitory. there is abundant evidence of pathological involvement in both systems, as detailed below, and clinically features of this pathology become increasingly obvious [52] [53] [54] [55] . dysphagia is a common feature sometimes demonstrable in asymptomatic individuals [56] . in one survey of 17 autopsy-confirmed cases of pd, swallowing difficulties appeared after an average period of 10 years from onset of initial extrapyramidal symptoms [57] . a variety of mechanisms account for swallowing difficulty, with evidence of dysfunction at either oropharyngeal or oesophageal level. although dysphagia may relate to mechanical problems (e.g. zenker's diverticulum) or dry mouth, the majority can be explained on the basis of vagal dysfunction. reduction of appetite and weight loss are common features that typically affect more advanced cases, and this may also relate to impairment of smell and taste [58] , or to poor diet, increased energy expenditure or depression [59] . although many patients drool saliva, the consensus view is that the volume of saliva produced is either unchanged or decreased, although it may be increased by levodopa preparations [60] . gastric dysfunction is likewise a recognized feature of established pd [61, 62] . these investigators reported frequent bloating or nausea suggesting delayed gastric emptying ,and this phenomenon has indeed been confirmed by measurement of gastric emptying time in patients whether they are receiving levodopa or not [63] . while little is known about small intestinal motility in pd, there are several studies relating to colonic disorder. [154] . (a-b, right hand side) to show the topographical distribution pattern of a-synuclein pathology in the human medulla in stages 1 and 2 of sporadic parkinson's disease, adapted and reproduced in part with permission from del tredici et al. [21] . amb, ambiguus nucleus; dmo, dorsal motor nucleus of the vagus (marked in green); gig, gigantocellular reticular nucleus, iop, inferior olivary nucleus, principal subnucleus; irz, intermediate reticular zone; rob, nucleus raphes obscurus; sol, solitary tract; xii, motor nucleus of the hypoglossal nerve. if ens involvement and disease of both the parasympathetic and sympathetic systems is an early event in pd, there should be relevant symptoms in the premotor phase. there is presently only one investigation in support of this, namely the prospective study of bowel habit in elderly patients who took part in the honolulu heart program [64] . this comprised 6790 men without extrapyramidal disease at enrolment, followed up for 24 years, of whom 96 developed pd. the adjusted risk of pd in those with less than one bowel movement per day, compared with those with one or more per day, was increased almost threefold (or 2.7; 95% ci 1.3, 5.5), implying that constipation is a harbinger of pd [27] . prevalence estimates of constipation in pd have been inconsistent because of variable definitions of constipation, but a conservative estimate would be at least 20% [65] . colonic transit time is raised from control values of around 20 h to 44-130 h [66,67] , probably worsening as the disease advances. in established pd, several investigators have found reduced cardiac uptake of the noradrenaline analogue, metaiodobenzylguanidine (mibg) [68] [69] [70] [71] [72] . this procedure evaluates postganglionic noradrenergic cardiac sympathetic function. heart rate variability may be abnormal in pd and is thought to reflect involvement of both sympathetic and parasympathetic pre-and postganglionic neurones [73] . incidental lbs have been found in the cervical sympathetic ganglia and cardiac plexus [74] . in another study, it was clearly shown that tyrosine hydroxylase-immunoreactive nerve fibres in the heart had almost entirely disappeared in patients with pd (and dlb), whereas they were well preserved in all those with psp and pure alzheimer's disease [75] . only one patient with pd displayed abnormalities in the sympathetic ganglia, which indicates that cardiac sympathetic denervation precedes neuronal loss there. autonomic failure presaging a parkinsonian syndrome has been shown in two patients [76] , but, to date, there have been no long-term prospective epidemiological studies of cardiac function. one investigation showed a significant correlation between mibg uptake and olfactory disorder in 26 patients with pd (but not msa), thereby confirming the close association of these two modalities [77] . a variety of sleep disorders are recognized in established pd [78] , of which the most studied are excessive daytime sleepiness and rapid eye movement sleep behaviour disorder [79] . the pathophysiology is not understood fully but most likely reflects cellular changes found in the parkinson neuropathological stages 1 and 2, involving the reticular formation, coeruleus/subcoeruleus complex, pedunculopontine nucleus and hypothalamus [80] . in the prospective honolulu-asia ageing study [81] , 43 of 3078 men aged 71-93 years developed pd over a 7-to 10-year period, and those reporting excessive daytime sleepiness were approximately three times more likely to develop pd (or 3.3; 95% ci 1.4, 7.0). according to one source [82] , 11/29 subjects with unexplained rem sleep behaviour disorder (rbd) subsequently developed parkinsonism, complementing the finding of decreased striatal transporter uptake in rbd by others [83] . in other studies of olfaction and sleep [84] [85] [86] , many rbd patients had significantly impaired smell function, once more implying that olfaction and rbd are early features. a difficulty with most of these studies, however, is the lack of post mortem confirmation. the investigation by iranzo and co-workers [87] is of particular interest in this regard. apart from corroborating the suspected status of rbd as a precursor of pd, they document the time interval between onset of rbd and clinically manifest sporadic pd. in seven patients, the latent period was on average 12 years (range 3-17 years). given that rbd is a sign of pd stage 2 pathology, this estimate provides a lower time limit for the presymptomatic phase and suggests that the earliest evidence of pd pathology (stage 1) would be 15-20 years before the onset of typical clinical manifestations. braak and colleagues [22] performed detailed pathoanatomical analyses of 41 cases of pd by a-synuclein immunostaining. a similar approach was taken in 69 subjects who had no pd-associated somatomotor symptoms in life but displayed lns and/or lbs (incidental cases). a third group consisted of 58 age-and gender-matched cases without lns or lbs and no history of neurological or psychiatric illness. the results of the cross-sectional study led the authors to propose that the pd-related pathological process in the cns progresses, apparently without remission, through presymptomatic and symptomatic disease phases [22] . a subsequent study identified lns and lbs in enteric nerve cell plexuses in both presymptomatic and clinically diagnosed pd cases [88] (figure 2 ).this led to the dual-hit hypothesis 603 suggestion that the pathological process begins at two sites simultaneously, that is, in the olfactory bulb/anterior olfactory nucleus and within enteric nerve cell plexuses. following damage of these predilection sites, the preganglionic parasympathetic projection neurones of the dorsal motor nucleus of the vagus (figure 3 ) and, shortly thereafter, the post-and preganglionic sympathetic projection neurones in the coeliac ganglion and intermediolateral nucleus of the spinal cord may become drawn into the disease process [89] . next to show the lesions are superordinate supraspinal centres such as the coeruleus/subcoeruleus complex, magnocellular portions of the reticular formation, and posterior raphe nuclei ( figures 4b and 1, right) . additional cns regions might then follow successively: central subnucleus of the amygdala [90] [91] [92] [93] , pars compacta of the substantia nigra, and magnocellular nuclei of the basal forebrain [23] . in other words, all of the vulnerable sites do not become involved at the same time but, rather, in a predictable topographic sequence. within the brain, the disease process displays a stereotypical caudal-rostral advance from the lower brainstem through basal portions of the mid-and forebrain, finally reaching the cerebral cortex ( figure 4a) . a distinctive lesional pattern, including neuronal loss, emerges. with one exception [94] , subsequent studies have confirmed, for the most part, the results of this pathoanatomical study [95] [96] [97] [98] , and interrater reliability of the pathological samples was high [99] . pd-associated a-synuclein-containing inclusion bodies as yet have not been found in the olfactory epithelium of autopsied pd patients [100] . nasal biopsy specimens from seven patients with symptomatic pd [101] were compared with four anosmic controls using antibodies against olfactory marker protein (omp), neurotubulin, protein gene product 9.5 (pgp 9.5) and mrna for omp. irregular areas of olfactory epithelium were positive for pgp 9.5 and neurotubulin but mostly negative for omp, although mrna for omp was found in the olfactory cleft and respiratory mucosa. in this small series, there was no clear difference between pd and anosmic controls; however, sections were not examined for presence of lns/lbs, and it could be argued that those with anosmia may have been in the presymptomatic phase of pd. daniel and hawkes [102] examined olfactory bulbs and tracts in eight controls as well as eight patients with a clinical and pathological diagnosis of pd taken from the united kingdom parkinson's disease brain bank. all pd cases contained lbs, which were most numerous in the anterior olfactory nucleus but also were found in mitral cells, the first projection neurones to receive input from the bipolar neurones in the olfactory epithelium. it was subsequently shown that loss of neurones in the anterior olfactory nucleus correlated with disease duration [103] . one report [104] suggested that expression of tyrosine hydroxylase in the olfactory bulb is increased 100-fold, and that the consequent excess of dopamine might explain the hyposmia that develops in pd. braak and colleagues [22] confirmed the presence of pd-related lesions in mitral cells and tufted neurones of the olfactory bulb and in projection neurones of the anterior olfactory nucleus, which is dispersed throughout the olfactory tract. a tightly woven network of lns rapidly develops within the anterior olfactory nucleus. from there, the pathology tends to spread slowly into more remote olfactory sites (olfactory tubercle, piriform and periamygdalear cortex, entorhinal cortex of the ambient gyrus) [105] without advancing into non-olfactory cortical areas [8, 22, 24] . it is known that ens lesions occur in symptomatic cases of pd [106] [107] [108] [109] [110] . as in the brain, only a few of the many neuronal types within the ens [111] [112] [113] are prone to develop pd-associated lesions (figure 2a,b) . once again, the vulnerable cells apparently are projection neurones with a long and unmyelinated axon [13] . the inhibitory nitrergic vasoactive intestinal polypeptide neurones have been shown to develop lns and lbs [109, 110] . axons containing a-synuclein from the submucous plexus have been seen to protrude through the muscle layer of the mucosa, extending widely and ramifying within the mucosal lamina propria [88] (figures 2c,d and 3) . axons of affected ens cells contain thread-like or spindle-shaped lns of varying size. in the mucosal lamina propria, the unmyelinated axons are only micrometers away from the body's innermost environment, their only protection being a single layer of epithelial cells (figures 2d and 3) . such involvement of the enteric nerve cell plexuses may represent a particularly early event -if not the earliest vagal-associated event -because lns were observed both in clinically diagnosed cases and in non-symptomatic individuals with pd-related brain lesions limited to the lower brainstem [88] . in summary, the studies indicate that early involvement of the ens with widespread and thinly distributed lesions throughout the walls of the upper gastrointestinal tract is accompanied by mild cns lesions. it remains to be seen whether ens lesions also occur in the absence of lesions in the brain. the very first brainstem lesions appear in the dorsal motor nucleus of the vagus [21] . the preganglionic parasympathetic projection neurones there generate long and thin unmyelinated axons [114, 115] . pathological a-synuclein aggregates are detectable in both proximal intramedullary and peripheral portions of these axons. other nuclei in the dorsal vagal area, including the nucleus gelatinosus, area postrema, and the nuclei surrounding the solitary tract, are minimally affected or uninvolved. the catecholaminergic melanoneurones in this area and those in the intermediate reticular zone [116] remain intact, at least initially [21] . these neurones do not project to the periphery but to higher levels of the brain [117] . the multipolar motorneurones of the ambiguus nucleus have thickly myelinated axons and remain free of lns/lbs. pd-associated pathological changes in sympathetic pathways (intermediolateral column of the spinal cord, coeliac ganglion and other peripheral sympathetic ganglia) have been studied and, to some extent, characterized already [74, 118] . recently, such lesions also have been shown in presymptomatic patients dying of non-neurological causes, thereby confirming the early involvement of the autonomic system [89, 119, 120] . all cases examined also displayed pd-related involvement of the lower brainstem, which, in turn suggests that the spinal cord abnormalities within the sympathetic nervous system may occur after the parasympathetic preganglionic projection neurones of the vagus have become affected. it is unlikely that the lesions in the olfactory system and in other predilection sites within the ens and cns develop independently of each other. a more plausible explanation is that a common pathologic insult, a hitherto unknown neurotropic factor or pathogenic substance, induces the disease and triggers the sequential involvement of vulnerable regions. anatomical analysis reveals a continuous chain of long-axoned and sparsely myelinated projection neurones that interconnect not only the olfactory epithelium but also the ens with the brain (figure 3 ) and, within the brain, all of the vulnerable regions [23] . after uptake, such a neurotropic pathogen might utilize these pathways and progress within the nervous system by way of axonal transport and trans-synaptic transmission [121] [122] [123] [124] neuroactive substances, including neurotropic viruses, unconventional pathogens with prion-like properties, or slow neurotoxins, usually are taken up at synapses, where they are frequently controlled by receptor-mediated endocytosis. the substances are transported to the cell body via the axon [125] [126] [127] [128] . neurotropic viruses that pass from the surroundings into axons of susceptible nerve cells can be prevented from doing so by the existence of a myelin sheath, which functions as a physical barrier against virus penetration [129] . thus, the absence of a myelin sheath around axons of the first neurones in the potential chain of vulnerable projection neurones may facilitate entrance and damage by viruses or other pathogens [125, 127, [130] [131] [132] . in addition, most of the neuronal types located within the cns are protected against uptake of substances from the extracellular milieu beyond the cns by the blood-brain barrier. only axons of nerve cells that project from the olfactory epithelium into the cns and those that project from the cns into the periphery, such as the preganglionic parasympathetic and sympathetic fibres, lack such a protective barrier. accordingly, an intravenous injection of horseradish peroxidase (hrp) results in retrograde labelling of the dorsal motor vagal nucleus [133] . despite enormous gaps in the present state of knowledge, the main line of reasoning that favours a 'dual-hit' pd-related process (with olfactory and enteric means of access) is that both are in close and constant contact to the (potentially hostile) outer environment. from the enteric plexuses, the prospective pathogen may gain access retrogradely to the cns via parasympathetic pathways (vagus; figure 3 ) and, thereafter, through post-and preganglionic sympathetic fibres. once in the cns, the disease process could ascend from spinal cord and lower brainstem through vulnerable portions of the basal mid-and forebrain until it reaches the cerebral cortex ( figure 4a ). from the olfactory epithelium, on the other hand, the pathogen would follow an anterograde route, to reach medial amygdala, olfactory tubercle, as well as piriform and periamygdalear cortex. the possibility that pd might be of viral aetiology was formally addressed by elizan and casals [134] . the inability to transfer pd to primates and the lack of viral antibodies in the then newly described guam pd-dementia individuals, who were usually anosmic [135] , argued against a viral aetiology for pd, although the theory was not entirely discounted. influenza virus has been associated with pd, as discussed in detail by takahashi and yamada [136] . despite many negative studies that searched for direct evidence of influenza a in pd [137] [138] [139] [140] [141] , nearly all pointed out that influenza a behaves as a persistent virus possibly capable of initiating autoimmunity. on the basis of human and experimental models, these authors proposed that the virus shows a predilection for the substantia nigra, cerebellum and hippocampus, and may be responsible for the formation of lbs. parkinsonism is seen on rare occasion during or after infection with herpes simplex encephalitis [142] , and a link between chronic herpes simplex type 1 encephalitis, pd and tic doloureux has been suggested [143] . there is no evidence of antibodies to herpes simplex virus (hsv) type 1 and 2 when compared with measles or cytomegalovirus antibodies in the serum and spinal fluid of pd subjects [144, 145] . similar findings were documented by elizan and casals [134] . one unconfirmed report suggests an association between pd and coronavirus infection on the basis of enzyme-linked immunosorbent assay in cerebrospinal fluid in 20 patients [146] . there is recent evidence that diseases of intermediate type hypersensitivity (asthma, allergic rhinitis, seasonal rhinitis) may be associated with pd [147] . retrospectively, the authors reviewed medical records in 196 cases of pd and 196 healthy controls and found a significant, approxi-dual-hit hypothesis 607 mately twofold increase of prior intermediate-type sensitivity disorder in general, but particularly for allergic rhinitis (or 2.9; 95% ci 1.3, 6.4; p < 0.01). there was a trend towards protection against pd in those who had used anti-inflammatory drugs. it is suggested that patients with pd might initiate an inflammatory response that could be directed towards the cns. there is some evidence of impaired smell sense in those with allergic rhinitis [148] , raising the possibility that allergic rhinitis might facilitate entry of a pathogen from the nose into the olfactory bulb and tract. altered protein handling may be a factor in the pd-associated pathogenic process. many viruses, e.g. epstein-barr virus, encode proteins that exploit the ubiquitin-proteasome system to regulate virus latency and allow the persistence of infected cells in immunocompetent hosts [149, 150] . there is longstanding evidence that neurotropic virus can enter the brain via the nasal route. it was shown that hsv type 1 placed intranasally in 6-week-old mice was detectable in the trigeminal root entry zone and olfactory bulbs 4 days later [151] . in some mice, virus which had entered the olfactory bulb, spread via axons as far as the temporal lobe, hippocampus and cingulate cortex. another study in the rat showed that hrp applied intranasally can be transported to the bulb, anterior olfactory nucleus, as well as to cholinergic neurones of the diagonal band, serotonergic raphe neurones, and noradrenergic cells of the locus coeruleus [152] . it is not widely appreciated that direct connections exist between primary olfactory areas and the substantia nigra. for example, application of hrp into the olfactory tubercle results in anterograde labelling of the ventral tegmental area, the pars reticulata of the substantia nigra, and anterior olfactory nucleus. as anticipated, there was retrograde labelling in the olfactory bulb and anterior olfactory nucleus [153] . blessing and colleagues [154] studied connections of the vagus using hsv type 1 in the rat (figure 1, left) . live hsv-1 was injected into the cervical vagus, and its distribution was examined using polyclonal antiserum. on day 2, virus was detected in glial cells of the area postrema, in nuclei of the solitary tract, the vagal dorsal motor nucleus, spinal trigeminal nucleus, and the great raphe nucleus, whereas the hypoglossal nucleus was spared, as in pd. on day 3, there was rostral spread to involve the locus coeruleus, parvocellular reticular nucleus and periaqueductal grey, but not the substantia nigra. hsv-1-positive neurones were seen in an oblique area corresponding to the human intermediate reticular zone [114] . this route of infection is shown in figure 1 (left) and should be compared with the similar distribution of pd-associated brainstem pathology (figure 1 , right). there are clear differences between this animal model and the pathology in humans. for instance, in contrast to the situation in pd, the trigeminal nerve in the rodents is involved and the substantia nigra is spared. nevertheless, the rostral migration of hsv-1 has remarkable similarity to the suggested progression of the a-synuclein pathology in pd ( figure 1 ) and reaffirms speculation that a neurotropic agent may utilize the vagus as a means of entering the medulla [22, 23] . the experiments of blessing and co-workers [154] are of additional interest in that they show that the first site of attack is in glial cells, which raises the possibility that residual virus in these cells may be responsible for the slow progression of pd. transvagal spread in animal models has been reported for other neurotropic enteroviruses, reovirus, pseudorabies virus and haemagglutinating encephalomyelitis virus [132] . the same group was unable to introduce cns infection in mice with influenza a virus through non-autonomic routes, such as the anterior chamber of the eye, the brachial plexus, knee joint, sciatic nerve and hindlimb footpad. conversely, pseudorabies virus spreads though both somatic and autonomic nerves. a strain of hong kong influenza virus inoculated intranasally in mice reached the cns through afferent fibres of the olfactory, vagal, trigeminal and sympathetic nerves following replication in the respiratory mucosa [155] . intranasally inoculated avian influenza virus in mice gave rise to lesions in lung and brainstem [156] . the main areas involved were the nuclei of the solitary tract and the trigeminal ganglia. although haematogenous cns spread is well documented in many viral strains, the findings of these experiments indicate that influenza viruses have preferential routes of access to the cns along the vagus and olfactory nerves following replication in the lungs, but the pattern of brainstem involvement is variable; that is, the trigeminal nerve is sometimes involved, at other times spared. these observations have some parallels with the pattern of proposed cns invasion in pd. although no virus mirrors exactly the pathological profile seen in pd, it is possible that a particular viral strain might display a predilection for the olfactory and vagal routes of entry. we propose that an unknown neurotropic pathogen initiating the pathological process underlying sporadic pd adopts a two-pronged attack on the nervous system: anterogradely, via olfactory pathways; and retrogradely, via enteric plexuses and preganglionic vagal fibres. if the pathogen is viral, then brain entry via the nasal route or uptake through the gastrointestinal tract has ample precedent, as described above. a nasal infection would have direct access to the olfactory nerve; infected saliva or mucous could be swallowed and reach the upper digestive tract to infect axons of meissner's plexus and, after transneuronal passage, ascend retrogradely in preganglionic parasympathetic fibres of the vagus nerve to the lower brainstem. direct access to the medulla via the viscerosensory fibres of the vagus in the pharynx or via the trigeminal nerve, while anatomically appealing, is not compatible with virtual sparing of the solitary tract and trigeminal nuclei during the entire course of the disorder. our observations relate to the commonest, sporadic variety of pd. as mentioned earlier, not all neuropathologists are in accord with the frankfurt pd classification [94, 95] , the main objection being (in a few cases) the absence of medullary or pontine lewy pathology where the substantia nigra was affected. another study used the frankfurt staging procedure to group an autopsy cohort into: preclinical (stages 1 and 2); early (stages 3 and 4, 35% with clinical pd); and late (stages 5 and 6, 86% with clinical pd) cases [157] . preclinical compared with early or latestage cases should progressively be more elderly at the time of sampling, but this feature was not observed. despite this, the proposed sequential progression of lewy pathology, and the 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visceral afferent nerve critical appraisal of the braak staging of brain. pathology related to sporadic parkinson's disease the authors wish to thank ms inge szász-jacobi for expert assistance with the graphics. this work was made possible in part by funding from the deutsche forschungsgemeinschaft (dfg) and hilde-ulrichs foundation (florstadt-staden, germany). key: cord-008523-avkgldnp authors: perlman, stanley; wu, gregory f. title: selection of and evasion from cytotoxic t cell responses in the central nervous system date: 2004-01-07 journal: adv virus res doi: 10.1016/s0065-3527(01)56029-7 sha: doc_id: 8523 cord_uid: avkgldnp cytotoxic cd8 t lymphocytes (ctls) are critical for the clearance of noncytopathic viruses from infected cells. this chapter discusses one mechanism used by viruses to persist—namely, the selection of a variant virus in which changes in the sequence of a ctl epitope abrogate recognition. the unique features of cytotoxic cd8 t cell function in the central nervous system (cns) are discussed. the role of ctl escape mutants in the viral evasion of the immune system and subsequent disease progression in non-cns infections are summarized. the immune response in the cns is similar to the response in extraneural tissue, but several aspects of the activation of the immune response, cellular trafficking, and antigen presentation are unique to the cns. although the cns has classically been considered a site of immune privilege, surveillance of the normal cns by circulating, activated lymphocytes occurs, with a limited number of lymphocytes being present in the normal cns at any given time. in mice infected with mouse hepatitis virus and in some humans persistently infected with human immunodeficiency virus type1, hepatitis b virus or hepatitis c virus, ctl escape mutants play an important role in virus amplification and disease progression. the immune response in the cns is similar to the response in extraneural tissue, but several aspects of activation of the immune response, cellular trafficking, and antigen presentation are unique to the cns (reviewed in lassmann, 1997; lassmann et al., 1991; matyszak, 1998; williams and hickey, 1995) ). the function of cytotoxic cd8 t cells in the inflamed cns and the major histocompatibility complex (mhc) class i antigen expression in this tissue are most pertinent for the topic of this review and will be preferentially discussed in this section. although the cns has classically been considered a site of immune privilege, surveillance of the normal cns by circulating, activated lymphocytes occurs, with a limited number of lymphocytes being present in the normal cns at any given time (hickey, hsu, and kimura, 1991) . however, considerable data suggest that the immune response in the cns is initiated not in the parenchyma but, rather, in draining lymph nodes located in the deep cervical tissue (cserr and knopf, 1992) . dendritic cells, critical for antigen presentation, are virtually absent from the normal cns, suggesting that virus antigen must spread to the draining lymph node tissue before activation can occur (hart and fabre, 1981; matyszak, 1998; matyszak and perry, 1996) . in support of this contention, influenza virus inoculated directly into the cns parenchyma does not induce an immune response until virus spreads to the cerebrospinal fluid (csf) and, soon thereafter, to the draining lymph nodes (stevenson et al., 1997a; stevenson et al., 1997b) . once the immune response has been initiated, breakdown of the blood-brain barrier, presence of plasma proteins, and recruitment of leukocytes characterize the cns inflammatory response. notably, cells with the phenotype of dendritic cells are detected as part of the cellular infiltrate in inflammatory processes and are believed to contribute to sustaining the immune response during chronic inflammation (matyszak, 1998; matyszak and perry, 1996) . within the cns, cd8 and cd4 t cell effector functions are virtually the same as in peripheral sites of inflammation. cd8 t cells are responsible for the majority of cytotoxic activity and exhibit three effector mechanisms: perforin/granzyme-mediated killing; fas/fas ligand-mediated killing; and cytokine secretion (liu, young, and young, 1996; smyth and trapani, 1998) . in one well-documented example, perforin is critical for the cd8 t cell response to lcmv, both peripherally and in the cns (kagi et al., 1994) . fas/fasl interactions have not been documented to be critical for cd8 t cell effector function in the cns, but fasl expression by infiltrating cd4 t cells has been impli-cated in oligodendrocyte death in inflammatory settings such as experimental allergic encephalomyelitis (eae) and multiple sclerosis (ms) (d'souza et al., 1996; sabelko et al., 1997; waldner et al., 1997) . secretion of interferon-~/(ifn-~/) and of other cytokines are of critical importance in orchestrating multiple components of the cellular and humoral immune responses. furthermore, in some cases, cytokines are capable of directly inhibiting viral replication. for example, ifn-• appears to be critical for virus clearance from infected oligodendrocytes in mice infected with mouse hepatitis virus (parra et al., 1999) . in another example, genetic disruption of ifn-~ or its receptor inhibits virus clearance in strains of mice normally resistant to infection with theiler's murine encephalomyelitis virus (tmev) (fiette et al., 1995) . ctls also release chemokines through granule exocytosis, thereby sustaining the immune response by contributing to the influx of other inflammatory cells (reviewed in baggiolini, dewald, and moser, 1997; luster, 1998) . the influx of both antigen-specific and nonspecific cells may contribute to virus clearance but also has the potential for increasing damage to nearby uninfected cells (bystander damage). this mechanism has been postulated to be significant in the pathogenesis of several cns diseases with inflammatory components, including demyelination in animals with virus-induced or autoimmune demyelination (houtman and fleming, 1996b; lassmann, 1997) . one major difference between the cns and extraneural tissue is found in mhc class i antigen expression. in extraneural tissue, mhc class i antigen is expressed constitutively by most cells. however, in the uninflamed cns, constitutive expression of mhc class i and ii antigen is infrequent and is confined to a subset of endothelial cells (mhc class i) and macrophages/microglia (mhc class ii) (reviewed in lampson, 1995; shrikant and benveniste, 1996) . expression of these molecules by astrocytes, oligodendrocytes, or neurons is not believed to occur in the normal cns. however, this conclusion may need to be modified since, in a recent study, the coordinated expression of mhc class i antigen (heavy chain and ~2-microglobulin) and of a t cell receptor component (cd3~) by neurons during development was postulated to be important for synapse formation (corriveau, huh, and shatz, 1998) . mhc class i and class ii expression are upregulated in the cns in multiple pathological settings with infectious or noninfectious etiologies (also reviewed in lampson, 1995; shrikant and benveniste, 1996) . as discussed above, mhc class i expression by antigen-presenting cells resident in the cns is not involved in initiation of the ctl response but is likely to be critical for its perpetuation. expression of mhc class i molecules by infected cells is also required for recognition and lysis by activated antigen-specific cd8 t cells. the most convincing data show that mhc class i and class ii antigens are upregulated on microglia/macrophages in inflammatory diseases of the cns, including ms and tmev-and mhv-induced demyelinating encephalomyelitis. t cell costimulatory molecules such as b7-1 and b7-2 are also upregulated on these cells and, in some cases, activated microglia/macrophages isolated from the cns can directly stimulate antigen-specific t cells proliferation (bo et al., 1994; matyszak, 1998; pope et al., 1998; xue et al., 1999) . mhc class i and class ii expression by astrocytes and oligodendrocytes has been demonstrated in vitro, although, in some cases, only after exposure to proinflammatory cytokines, such as interferon-7 (lampson, 1995; shrikant and benveniste, 1996) . whether astrocytes and oligodendrocytes upregulate mhc class i and class ii expression in the inflamed cns remains quite controversial, with expression of these molecules being reported in some studies (lampson, 1995; shrikant and benveniste, 1996) . in most recent studies, mhc antigen expression by astrocytes and oligodendrocytes in inflamed tissue was not detected (e.g., pope et al., 1998; xue et al., 1999) , but these negative conclusions must be tempered because a biologically significant level of expression might not be detectable with presently available assays. mhc class i antigen upregulation on neurons has not been reported in pathological conditions, although a recent set of elegant studies demonstrated the expression of these molecules following inhibition of electrical activity (neumann et al., 1995; neumann et al., 1997) . if verified in the infected cns, these results would suggest that only neurons sufficiently damaged by infection with a virus or another infectious agent would be recognized by antigen-specific ctls. furthermore, this mechanism would minimize destruction of infected, but still functionally active, neurons. although there is no agreement on the specifics, these studies clearly demonstrate that mhc class i and ii antigens are expressed on cns-derived cells in vitro and in vivo during inflammatory processes. as a consequence, they provide a framework for interpreting experiments that describe the selection of ctl escape mutants in the cns of virus-infected mice. occurring outside the cns the first experimental evidence for virus escape from ctl recognition by mutation of a cd8 t cell epitope was provided by pircher et al., (1990) . in mice that were transgenic for a t cell receptor (tcr) specific for lymphocytic choriomeningitis virus (lcmv), infection with lcmv resulted in the rapid appearance of virus mutated in the target ctl epitope. infection of tcr transgenic mice, but not of immunocompe-tent b6 mice, with mutant lcmv resulted in a delay in the kinetics of virus clearance. similar results were obtained when wild-type mice were infected with lcmv variants in which some or all of the appropriate ctl epitopes were mutated (lewicki et al., 1995; moskophidis and zinkernagel, 1995) . notably, lcmv escape mutants are not generated in immunocompetent mice after infection with wild-type virus. in these mice, ctls recognizing multiple epitopes are detected, suggesting that an immune response directed against several epitopes precluded the selection of ctl escape mutants, at least in settings in which virus clearance was relatively efficient. a subsequent study suggested that ctl escape mutants may emerge and become the predominant virus in human populations in which a single hla allele is overrepresented, and in which an immunodominant epitope is restricted by that allele. the strain of epstein-barr virus (ebv) circulating in humans residing in the coastal regions of papua new guinea contains a mutation in an immunodominant cd8 t cell epitope that diminished binding to the hla-all molecule (de campos-lima et al., 1994) . hla-all is present at a high frequency in this population, suggesting that the selection of virus encoding this mutation was immune-driven. however, this conclusion may not be warranted since the same mutation is found in virus circulating in the highland populations of papua new guinea, even though the hla-all allele is not overrepresented in that group (burrows et al., 1996) . the studies of lcmv-infected mice suggested that efficient virus clearance precluded the selection of ctl escape mutants. conversely, less efficient virus clearance would be predicted to facilitate the emergence of these mutants. therefore, the potential contribution of ctl mutants to pathogenesis was examined next in several persistent infections, including human and nonhuman primates chronically infected with the human immunodeficiency virus (hiv-1), hepatitis b virus (hbv), and hepatitis c virus. the emergence of ctl escape mutants during the course of persistence was documented in each of these infections (franco et al., 1995; mcmichael and phillips, 1997; weiner et al., 1995) . the most convincing data suggesting a role for these variants in disease progression comes from studies in which the emergence of ctl escape mutants are documented early during the infection. selection at early times postinfection is consistent with a role in delaying the elimination of virus from the infected host. in one study, borrow et al. (1997) described an hiv-infected patient in which the initial ctl response was directed at a single hla b44-restricted epitope. early in the course of the infection, a mutation in a presumptive anchor residue was detected, leading to a loss of recognition by the patient's ctls. shortly thereafter, the ctl response spread to addi-stanley perlmanandgregoryewu tional, presumably less immunodominant, epitopes. these responses were able to control the virus for a short period, but were, ultimately, inadequate because the patient exhibited a rapid rate of disease progression with a fatal outcome. in another study, mutations resulting in ctl escape were identified in the nefgene in a patient during primary hiv infection. these mutations abrogated recognition by nef-specific ctls harvested from the patient, suggesting that their selection was also immune-driven and that it contributed to virus persistence (price et al., 1997) . in still another study, ctl escape mutants were identified in hivinfected patients several years after the primary infection, coincident with increased virus replication and decreased cd4 t cell counts . it is uncertain, in these cases, whether ctl escape caused disease progression or whether it was a consequence of increasingly ineffectual control of virus replication by other components of the immune system, such as antibodies and cd4 t cells. ctl escape mutants that emerge only after immune surveillance has diminished may still contribute to disease progression in hiv-infected patients, even if they are not the primary cause for the loss of control by the immune system. ctl escape mutants were also selected in an hiv-infected individual treated with ctls directed against a single epitope (koenig et al., 1995) , thereby showing that, as in lcmvinfected tcr transgenic mice, a strong, monospecific ctl response facilitated their appearance. ctl escape variants are occasionally detected in patients chronically infected with hbv, usually in the context of a narrowly focused, strong immune response (bertoletti et al., 1994a; bertoletti et al., 1994b) . a more common finding in patients infected chronically with hbv is the lack of a significant ctl response (rehermann et al., 1995) , suggesting that variation in hbv-specific t cell epitopes is important in only a minority of patients. similarly, ctl escape mutants were detected in a chimpanzee chronically infected with hepatitis c virus (weiner et al., 1995) , but persistence appears to be associated, most commonly, with a weak ctl response (rehermann et al., 1996) . mutations in an epitope or in its flanking sequences can result in escape from surveillance by cd8 t cells, by affecting any one of several steps along the pathway used to generate peptides for presentation by mhc class i molecules. antigen processing involves proteolytic cleav-age by proteosomes in the cytoplasm and transport of the resulting peptides to the endoplasmic reticulum for binding to the mhc class i molecule. mutations in sequences flanking a ctl epitope may affect proteolytic cleavage or transport into the endoplasmic reticulum. only a few examples of mutations affecting either of these processing steps have been described (eisenlohr, yewdell, and bennink, 1992; ossendorp et al., 1996) . in one example, comparison of the sequence of an immunodominant ctl epitope encoded by two related murine leukemia virus families (akv/mcf and friend/moloney/rauscher [fmr] ) showed that a single amino acid change present in the fmr strains abrogated cd8 t cell recognition (ossendorp et al., 1996) . the mutation did not affect binding of peptide to the mhc class i molecule or immunogenicity of a peptide corresponding to the variant epitope. rather, the loss of recognition occurred only after endogenous processing of antigen and resulted from alterations in proteosomal processing, which were shown using purified 20s proteosomes in vitro. these conclusions assume that processing in vitro mimics what occurs in the cell and that other differences in the amino acid sequence between the two viruses did not contribute to the observed differences. mutations in sequences flanking a ctl epitope are difficult to detect, and therefore may be more important in ctl escape than is appreciated at present. more commonly, mutations in ctl epitopes that affect binding to the mhc molecule, or affect recognition by the tcr, have been identified. some of the mutations described above, in hiv-1 infected individuals, diminish binding to the mhc class i molecule or result in increased rates of dissociation from the mhc molecule (reviewed in mcmichael and phillips, 1997) . mutations that impair binding to the mhc molecule are the simplest to interpret and, depending on the diminution of binding, will completely abrogate recognition by epitopespecific cd8 t cells. mutations that affected binding to tcrs were first reported in studies describing selection of lcmv ctl escape mutants in mice transgenic for an lcmv-specific tcr (pircher et al., 1990) . peptides mutated in residues that directly contact the tcr have also been described in virus isolated from hiv-infected patients . these mutations may result in partial or complete loss of recognition by autologous ctls. during the course of these studies on viral variation, mutations were identified in viral rna and dna isolated from patients persistently infected with hiv-1 or hbv that not only diminished recognition by epitope-specific cd8 t cells, but also inhibited recognition of wild-type epitope if both epitopes were present in the infected cell (bertoletti et al., 1994b; klenerman et al., 1994) . the mechanism of this phenomenon (tcr antagonism) is not completely understood, but is believed to involve delivery of an altered signal to the cd8 t cell by the mutated peptide/mhc class i complex, resulting in nonresponsiveness or diminished responsiveness to the target agonist peptide (sette et al., 1996) . although tcr antagonism has only been demonstrated by using ctl clones, it has also been documented to occur when variant and wild-type epitopes are presented by different cells, a scenario that presumably mimics the situation in the infected host (meier et al., 1995) . another mechanism similar to antagonism is interference with ctl priming (plebanski et al., 1999) . in this case, a variant epitope does not inhibit ctl effector function but, rather, the generation of activated antigen-specific ctls from naive precursor cd8 t cells. this mechanism, identified in humans and mice infected with malaria, has not yet been described in any viral disease. one of the best examples of a pathological setting in which ctl escape mutants contribute to virus persistence and the development of disease is that of mice infected with the neurotropic coronavirus, mouse hepatitis virus--strain jhm (mhv-jhm). selection of escape mutants was not anticipated because immune selective pressure in the cns, a site that is relatively protected from immune surveillance, might be predicted to be less than in the periphery. neurotropic coronaviruses, including mhv-jhm, the a59 strain of mhv (mhv-a59) and mhv-3, cause acute and chronic infections of the cns in susceptible mice and rats (reviewed in houtman and fleming, 1996b; lane and buchmeier, 1997; stohlman, bergmann, and perlman, 1998) . mhv-jhm is highly neurovirulent and infection of susceptible mice results in widespread infection of neurons with a rapidly fatal outcome. however, more generally interesting are those model systems in which mhv-jhm and mhv-a59 are induced to cause either acute or chronic demyelination. general strategies for modifying the acute infection have been recently reviewed (perlman, 1998; stohlman, bergmann, and perlman, 1998) , and two general approaches will be briefly described here. in the first, mice are infected with attenuated virus. attenuated virus was cloned from pools of virus harvested from suckling mouse brain (stohlman et al., 1982) . alternatively, viral mutants were selected by chemical mutagenesis or by treatment with neutralizing antibody directed against the surface (s) glycoprotein (dalziel et al., 1986; fleming et al., 1986) . in each case, the variant virus causes acute encephalitis in only a small percentage of mice but is able to persist and cause demyelination in most survivors. in mice infected with these variants, infectious virus is, in general, cleared by 2-3 weeks after inoculation. clinical symptoms are most evident within the same time frame. mice that survive the infection slowly recover, although evidence of ongoing demyelination can be detected for several months after the acute infection has resolved. in the second approach, mice are infected with wild-type mhv-jhm and protected from the acute encephalitis by administration of anti-mhv antibody or mhv-specific cd4 or cd8 t cells (buchmeier et al., 1984; stohlman et al., 1986; yamaguchi et al., 1991) . this intervention also results in decreased infection of neurons, but does not prevent infection of cells in the white matter or demyelination. virus clearance is enhanced by treatment with protective antibodies or t cells in some studies (buchmeier et al., 1984; yamaguchi et al., 1991) , but not all . in a variation of the latter approach, suckling c57bl/6 (b6) mice are protected from acute encephalitis with nursing by dams previously immunized to mhv-jhm . the suckling mice are protected from acute encephalitis and remain asymptomatic for 3-8 weeks. at that time, a variable percentage (30-90%) develop histological evidence of demyelination and clinical signs of hind-limb paralysis. immunization of the dams may be accomplished either by active immunization with infectious virus , or by passive infusion of neutralizing antibody (pewe, xue, and perlman, 1997) , and with similar outcomes. the temporal course of this disease is very different from that observed in other models of mhv-jhm-induced demyelination, because mice are asymptomatic for several weeks before developing disease, and because infectious virus is not cleared. in a series of reports, pewe et al showed that escape from the cytotoxic cd8 t cell response was a major factor in the disease progression in these mice (pewe et al., 1996; pewe, xue, and perlman, 1997; pewe, xue, and perlman, 1998) . ctl escape mutations were detected in all mice that developed clinical disease several weeks postinfection in this model. in b6 mice, two cd8 t cell epitopes, encompassing residues 510-518 (s-510-518) (h-2db-restricted) and 598-605 (s-598-605 (h-2kb-restricted), of the s glycoprotein are recognized (bergmann et al., 1996; castro and perlman, 1995) . these two epitopes are located within a region of the protein that is prone to both mutation and deletion (termed the "hypervariable region") (banner, keck, and lai, 1990; 228 stanley perlman and gregory f. wu parker, gallagher, and buchmeier, 1989) . the location of the epitopes within a region that can tolerate mutation, without loss of function, most likely contriljutes to the selection of ctl escape mutants. mutations have only been detected in epitope s-510-518, the immunodominant epitope of the two , and not in epitope s-598-605. mutations are not detected in regions of the genome flanking epitope s-510-518 or in most mhv-specific cd4 t cell epitopes. the one possible exception is that a mutation is detected in a cd4 epitope encompassing residues 328-347 of the s glycoprotein (s-328-347) in several mice, although wild-type epitope is also detected in the same animals. the significance of this mutation is not known, because it does not appear to decrease recognition by cd4 t cells specific for the epitope . mutations have been detected in amino acids at positions 2-7 of epitope s-510-518, with only a single variant generally being detected in any individual infected mouse (pewe, xue, and perlman, 1997) . a summary of mutations detected thus far is shown in fig. 1 . based on the crystallographic structure of the h-2d b molecule and on mutagenesis studies (hudrisier et al., 1996; young et al., 1994) , some of these mutations are predicted to affect binding to the mhc class i molecule, whereas others inhibit binding to the tcr of the cd8 t lymphocyte. mutations in epitope s-510-518 are selected within 10-12 days postinfection, a time at which virus is not cleared in other mhv infections (houtman and fleming, 1996a; lin et al., 1999) . detection of ctl escape mutants at a time before virus clearance is expected to be complete is consistent with a role for these mutants in virus persistence. notably, epitope s-510-518-specific ctl activity can be detected in the cns as early as 5 days postinfection, prior to the detection of ctl escape mutants (unpublished observations). mutations are also not detected in infected mice with severe combined immunodeficiency (scid mice), suggesting that their selection is cd8 t cell-driven (pewe, xue, and perlman, 1997) . in this model, mice that do not develop hind-limb paralysis by 60-80 days postinfection remain asymptomatic. mutations are only occasionally detected in asymptomatic mice at 60-80 days postinfection suggesting that escape from cd8 t cell surveillance correlates well with virus amplification in b6 mice but is not sufficient for the development of clinical disease. as discussed above, the role of ctl escape mutants in viral pathogenesis is controversial. these mutants are not expected to be relevant to pathogenesis if the ctl response is directed at several ctl epitopes, as is believed to occur in many human infections. it has also been postulated that, even if a single epitope is immunodominant, a t cell summary of mutations detected in epitope s-510-518 in virus isolated from mice with hind-limb paralysis. a panel of peptides resulting from single nucleotide changes in the sequence of the wild-type epitope were tested in cytotoxicity assays. only those changes resulting in a 20-fold decrement in activity are included in the figure. the amino acids listed above the wild-type sequence have been detected in virus isolates, whereas those listed below the wild-type sequence (underlined) have not yet been detected. the l to p change in position 3 results in only a 4-fold decrease in recognition but has been detected in a minority of cdna clones in single mice with hind-limb paralysis (marked with parentheses). *mhc (m) or tcr (t) contact-based on published reports (hudrisier et al., 1996; hudrisier et al., 1995; young et al., 1994) . response that is comprised of many cd8 t cell clonotypes would preclude ctl escape, because a single mutation would affect recognition by only a subset of the epitope-specific t cells (ishikawa et al., 1998) . to determine the biological significance of the mutations that were detected in mhv-infected mice, several additional experiments were performed. first, a panel of variant epitopes were analyzed in cytotoxicity assays, using either lymphocytes derived from the cns of mice with acute encephalitis directly ex vivo or bulk populations of splenocytes cultured in vitro for 2 weeks with peptide s-510-518 pewe et al., 1996) . this panel comprised all the peptides resulting from single nucleotide changes in epitope positions 2-7 and 9. these assays showed that nearly all of the mutations that were 230 stanley perlman and gregory f. wu selected in vivo resulted in a complete or substantial loss of recognition by either population of lymphocytes. several additional mutations also resulted in a nearly complete loss of recognition by these lymphocytes, but have not yet been detected in vivo (fig. 1 ) strikingly, some mutations in position 9, a secondary anchor for binding to the mhc molecule, resulted in loss of recognition, but were never selected in infected mice. whether this apparent selectivity reflects a sampling error, or is in fact biologically driven (i.e., results in detrimental changes in rna or protein structure), remains to be determined. the biological significance of these mutations was also addressed by infecting maternal antibody-protected suckling c57bl/6 mice with virus mutated in epitope s-510-518. mice infected with this virus do not recognize epitope s-510-518 but still respond to epitope s-598-605. variant virus-infected mice have significantly greater mortality and morbidity when compared to mice infected with wild-type virus (pewe, xue, and perlman, 1998) . these results suggest that the cd8 t cell response to epitope s-510-518 is an important component of the host response to mhv-jhm, and support the idea that escape from this response is beneficial to the virus and results in increased virus replication and disease progression. these results suggest that the ctl response to epitope s-510-518 is monospecific, because single mutations in residues important for binding to either mhc antigen or the tcr result in substantial loss of activity in cytotoxicity assays. the monospecificity of the response is consistent with a monoclonal or an oligoclonal response to the epitope. to directly assess the diversity of the t cell response, tcr v~ element usage and the complexity of the complementarity determining region 3 (cdr3) were determined. the tcr is a heterodimer consisting of an a and a ~ chain. the great diversity in the t cell response results from the large number of different v, d, and j elements in the germ line, coupled with imprecise joining at the v-d and d-j junctions ([3 chains) or v-j junction (a chain). the cdr3 encompasses the junctional region of the a and 13 chains and makes direct contact with the mhc/peptide complex. analysis of a monoclonal or oligoclonal t cell response should reveal usage of only one or a small number of different v~ and va elements and minimal heterogeneity of the cdr3. sequence analyses of the cdr3 were facilitated by the use of soluble mhc/peptide tetramers specific for epitope s-510-518 (tetramer s-510). soluble mhc class i/tetramers were first described by altman et al., and have been shown, in several subsequent studies, to detect antigen-specific t cells with high sensitivity and specificity (e.g., altman et al., 1996; flynn et al., 1998; murali-krishna et al., 1998) . epitope s-510-518-specific cd8 t cells were isolated directly from the cns of mice with acute encephalitis by sorting with a fluorescentactivated cell sorter (facs) after staining with anti-cd8 antibody and tetramer s-510. the tetramer s-510-positive t cells were analyzed initially for v~ usage. the results showed usage of a large number of different v[~ elements by cd8 t cells responding to the epitope, with preferential usage of some v~ elements occurring, specifically v~8 and v[~13 . analysis of individual cdr3 sequences from a subpopulation of tetramer s-510-positive cells, those expressing the v~13 element, revealed substantial heterogeneity. the cdr3 sequences were shown to fit, approximately, a logarithmic distribution (fisher, corbet, and williams, 1943; taylor, kempton, and woiwod, 1976) . from this distribution, it was possible to calculate that approximately 300-500 epitope s-510-518-specific cd8 t cell clonotypes were present in the cns of a mouse with acute encephalitis. similar measurements using lymphocytes harvested from the cns of mice with chronic demyelination revealed that approximately 100-900 different clonotypes were present in these animals. previous studies suggested that either 3,000 (butz and bevan, 1998) or 600 (bousso et al., 1998) precursor cd8 t cells for any single epitope were present in an individual mouse. our calculation of the number of t cells recognizing epitope s-510-518 agrees more closely with the lower estimate. notably, none of the wild-type epitope s-510-518 rna, or little of it, is present in the cns of mice with chronic demyelination, but cd8 t cells specific for the epitope are still detectable. whether this represents stimulation by residual wild-type antigen (which may be cleared more slowly than viral rna (knopfet al., 1998; zhang et al., 1992) , or by variant sequence remains to be determined. furthermore, many of the [~ chain cdr3 sequences were detected in more than one mouse. in the case of mice with chronic demyelination, very few cdr3 sequences were unique to a single animal. thus, unlike other experimental systems (bousso et al., 1998) , the repertoire of cd8 t cell tcrs recognizing epitope s-510-518 is not unique to each naive animal but is, in large part, common among all c57bl/6 mice. this lack of variability may facilitate the emergence of ctl escape mutants in a large fraction of infected mice. these results suggest that ctl escape mutants are critical in virus persistence and in the development of clinical disease in mice infected with mhv-jhm. these results also show that ctl escape mutants are selected in the presence of a polyclonal, but monospecific, cd8 t cell response. as already mentioned, the importance of ctl escape 232 stanley perlman and gregory f. wu mutants in other infections is much less certain, suggesting that the following features, present in the mhv-jhm-infected cns, must predispose to the selection of these mutants: 1. ctl escape mutants are observed in mhv-infected rodents only when b6 mice are inoculated at the suckling stage with virulent wildtype virus and protected from acute disease by nursing by immunized dams. inoculation with virulent mhv-jhm and use of b6 mice are essential for disease to develop. chronic demyelination does not develop if suckling mice are inoculated with the less neurovirulent a59 strain of mhv (unpublished observations). in most other models of mhv persistence, older mice are inoculated with attenuated strains. persistence is manifested by the presence of viral rna and ongoing demyelination in the absence of infectious virus (adami et al., 1995; houtman and fleming, 1996a; rowe et al., 1997) . ctl escape mutants have not been detected in these models, possibly because clearance of infectious virus is so rapid . a normal immune system is also required, however, because even when clearance is delayed in mice infected with attenuated virus, as occurs in immunodeficient mice, ctl escape mutants are not commonly selected . 2. infection with wild-type virus at the suckling stage (10-14 days old) is also crucial in this model. three-week-old b6 mice inoculated with wild-type virus and protected from acute encephalitis by passive administration of small amounts of neutralizing antibody are protected from acute encephalitis, but they do not develop hind-limb paralysis at later times (sun and perlman, 1995) . demyelination can be detected in the spinal cords of these mice at 2 months postinfection. these mice have not formally been assessed for the presence of ctl escape mutations at late times after infection, but they clearly do not develop a clinical disease consistent with the selection of ctl escape mutants. these results suggest that inoculation of mice with virulent virus at the suckling stage is essential for the selection of ctl escape mutants or, as a minimum, for their clinical manifestation. this requirement for inoculation of suckling mice may reflect the immaturity of the immune system. a consequence of infecting mice at 10 days of life may be suboptimal clearance of virus. consistent with this possibility, after inoculation at the suckling stage, low levels of infectious virus can be detected at 40 days postinfection in balb/c mice (unpublished observations) . balb/c mice never develop evidence of hind-limb paralysis or other clinical disease at any time and develop completely normally (castro et al., 1994; . 3. another consequence of inoculation of mice with an immature immune system may be an aberrantly polarized immune response. a striking feature of the immune response in most c57bl/6 mice infected with mhv at the suckling stage is the lack of an appreciable antibody response, when they are assayed by elisa or in neutralizing assays (jacobsen and perlman, 1990; . the cd4 and cd8 t cell response in maternal antibody-protected mice that develop chronic demyelination is proinflammatory and readily detected (castro et al., 1994; . this strongly polarized, cell-mediated immune response in the presence of a defective humoral response may also predispose to the selection of ctl escape mutants. in support of this, balb/c mice inoculated at the suckling stage in the maternal antibody model mount a significant antibody response and do not develop hind-limb paralysis (castro et al., 1994; . 4. mhv infection of congenic b10.a(18r) (kbddl d) mice results in the development of hind-limb paralysis, albeit at a lower frequency than in either b6 or c57bl/10 mice (castro et al., 1994) . two ctl epitopes, s-598-605 and an la-restricted epitope encompassing residues 318-326 of the nucleocapsid (n) protein (n-318-326) , are recognized in these mice. the immunodominant epitope recognized in b6 mice, epitope s-510-518, is not recognized in b10.a(18r) mice since these mice do not encode the h-2d b allele. virus isolated from b10.a(18r) mice with hind-limb paralysis was evaluated for the presence of mutations in epitopes s-598-605 and n-318-326, and none were detected. the development of hind-limb paralysis in b 10.a(18r) mice, in the absence of mutations in either ctl epitope, suggests that a factor expressed on the c57bl/6 background (decreased production of antibody?) may contribute to the increased amount of virus replication observed in these mice. the outgrowth of ctl escape mutants in b6 mice, in conjunction with this other putative factor, results in a much higher rate of clinical disease than is observed in b10.a(18r) mice in which ctl escape mutants are not selected. 5. a ctl response directed at a single immunodominant epitope enhances the likelihood that ctl escape mutants will be selected. in b6 mice, two mhv-specific epitopes are recognized. cytotoxicity assays using cns-derived lymphocytes from mice with acute encephalitis, directly ex vivo (castro and perlman, 1995) , and limiting dilution assays using splenocytes harvested from mice intraperitoneally immunized with mhv , suggested, in both cases, that the two epitopes were recognized by similar numbers of cd8 t cells. more recent measurements, using mhc class i peptide/tetramers to detect antigen-specific cells or methods to detect interferon-~ production after stimulation with peptide, confirmed 234 stanley perlman and gregory f. wu these results (unpublished observations). however, much more peptide s-598-605 is required to sensitize target cells for lysis. thus, the ctl response in these mice may be functionally directed at a single epitope. conversely, a ctl response to a single immunodominant epitope is not sufficient for ctl escape mutants to be selected. the ctl response in balb/c mice is directed at a single epitope (n-318-326), yet these mice do not develop hind-limb paralysis in the maternal antibody-protection model (castro et al., 1994) , and ctl escape mutants are not selected (unpublished observations). the ability of the virus to tolerate mutations in the part of the protein that contains the target cd8 t cell epitope is also important. epitope s-510-518 is in a region of the s protein prone to deletion and mutation, while the balb/c epitope is located in a highly conserved region of the n protein. as mentioned above, mutations in this epitope are not detected in b10.a(18r) mice, even though the background of this strain may be favorable for the selection of ctl escape mutants. 6. mhv-jhm is neurotropic and the infection caused by this agent is confined, for the most part, to the cns. as discussed above, the processes of initiation of an inflammatory response, trafficking to the site of inflammation, and antigen presentation within the cns differ in several aspects from similar processes occurring at extraneural sites. in particular, mhc class i expression is minimal in the normal cns, and while clearly upregulated within the infected cns, determining the cellular sites of expression remains an area of active research. therefore, while it is speculative at present, it is formally possible that infection within the cns results in a modified immune response that increases the likelihood that ctl escape mutants will be selected during the course of an infection. what can be concluded from these studies about the role of ctl escape mutants in the pathogenesis of viral infections in the cns? the following conclusions may also be applicable for ctl escape mutants arising in extraneural tissue. first, ctl escape mutants are not commonly selected in most infections. one explanation for this is that the selection of ctl escape mutants is uncommon if the cd8 t cell response is directed against several epitopes. however, a cd8 t cell response directed against one epitope or a few, has been observed in several human and experimental infections (cole, hogg, and woodland, 1994; flynn et al., 1998; lehner et al., 1995; moss et al., 1991; murali-krishna et al., 1998; wallace et al., 1999) --suggesting that an immunodominant response is not unusual. nevertheless, ctl escape mutants are not detected in most experimental settings even when only one epitope is immunodominant, or a few are. second, the recognition of an epitope by a diverse population of cd8 t cell clonotypes does not prevent emergence of these variants. what does appear to be true, however, is that the diversity of the response matters less than whether the different cd8 t cell clonotypes recognize the same or different parts of the mhc/peptide complex. a response narrowly focused on one part of the complex will facilitate the selection of ctl escape mutants. the ctl response to epitope s-510-518 is very sensitive to changes in residues 4 and 6 pewe et al., 1996) , two residues important for binding to the tcr (young et al., 1994) . strongly focused recognition of central residues of an immunodominant h-2kb-restricted epitope, which is recognized in sendai virus-infected b6 mice, has also been reported (cole, hogg, and woodland, 1995) . third, in mice infected with mhv and in some humans persistently infected with hiv-1, hbv, or hepatitis c virus, ctl escape mutants play an important role in virus amplification and disease progression. in most cases, however, it is likely that there would not have been sufficient time for selection of ctl escape mutants if virus clearance were rapid and complete. thus, selection of ctl escape mutants in the absence of any other factor is probably not sufficient to prevent rapid virus clearance from an infected host. in mhv-infected b6 mice, their selection appears to be facilitated by a suboptimal humoral immune response. this deficient antibody response may be a consequence of infection of suckling mice in the presence of maternally derived antibody. maternal immunization has been shown to impair immune responses in other infections (wang et al., 1998) , although it has never been shown to affect selectively humoral, and not cellular, immunity. fourth, an interesting aspect of this model system is the number of questions that it raises about the dynamics of mhv growth and cellular tropism in the cns. unlike the situation in mice chronically infected with tmev, in which macrophages are the main reservoir for virus (lipton, twaddle, and jelachich, 1995) , no single type of cns cell has been identified as the predominant target in mice persistently infected with mhv. the selection of ctl escape mutants in the cns of mhv-infected mice demonstrates that mhv infection of a cell expressing mhc class i antigen is a critical part of the pathogenic process. astrocytes, macrophages/microglia, and oligodendrocytes are all infected in these animals xue et al., 1999) . of all of these cells, only macrophages/microglia express detectable levels of mhc class i antigen in mhv-infected mice with chronic demyelination (xue et al., 1999) . one interpretation of these results is that macrophages/microglia are in fact the primary site of productive virus infection, with replication in oligodendrocytes or astrocytes being of secondary importance or abortive. infection of the latter two cells might be crucial for the development of clinical disease, but might be less important for propagation of the virus. alternatively, astrocytes or oligodendrocytes might express mhc class i antigen, albeit at levels below the detection limits of assays presently available. escape from cd8 t cell surveillance might occur in these cells, thereby resulting, simultaneously, in increased virus replication and disease progression. infection with mhv affects mhc class i antigen expression by astrocytes in vitro and in newborn mice but this has not been demonstrated in vivo in older mice (correale et al., 1995; gilmore, correale, and weiner, 1994; suzumura et al., 1988; suzumura et al., 1986) . conversely, proof that astrocytes or oligodendrocytes were the most important sites of mhv-jhm replication and, therefore, the sites where selection of ctl escape mutants occurred, would provide evidence that these cells express mhc class i antigen. evolution of mouse hepatitis virus (mhv) during chronic infection: quasispecies nature of the persisting mhv rna phenotypic analysis of antigen-specific t lymphocytes human chemokines: an update a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus variability of persisting mhv rna sequences constituting immune and replication-relevant domains the jhm strain of mouse hepatitis virus induces a spike protein-specific db-restricted ctl response cytotoxic t lymphocyte response to a wild type hepatitis b virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope natural variants of cytotoxic epitopes are t-cell receptor antagonists for antiviral 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variants of the jhm neurotropic strain in vivo effects of coronavirus-specific t cell clones: dth inducer cells prevent a lethal infection but do not inhibit virus replication spread of a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes induction of glial cell mhc antigen expression in neurotropic coronavirus infections coronavirus infection induces h-2 antigen expression on oligodendrocytes and astrocytes diversity statistics and the logseries model fas-and fasl-deficient mice are resistant to induction of autoimmune encephalomyelitis the cytotoxic t-cell response to herpes simplex virus type 1 infection of c57bl/6 mice is almost entirely directed against a single immunodominant determinant effect of passive immunization or maternally transferred immunity on the antibody response to a genetic vaccine to rabies virus persistent hepatitis c virus infection in a chimpanzee is associated with emergence of a cytotoxic t lymphocyte escape variant traffic of hematogenous cells through the central nervous system antigen specificity of cd4 t cell response in the central nervous system of mice infected with mouse hepatitis virus depletion of blood-borne macrophages does not reduce demyelination in mice infected with a neurotropic coronavirus protection of mice from a lethal coronavirus infection in the central nervous system by adoptive transfer of virus-specific t cell clones the three-dimensional structure of h-2d at 2.4 a resolution: implications for antigen-determinant selection directional and compartmentalised drainage of interstitial fluid and cerebrospinal fluid from the rat brain immunology taught by viruses this work was supported in part by grants from the national institutes of health (nih) (ns 36592 and ai43497) and from the national multiple sclerosis society. g.f.w. was supported in part by a national research service award from the nih. key: cord-302796-zi3p2k1b authors: cardona, astrid e.; li, meizhang; liu, liping; savarin, carine; ransohoff, richard m. title: chemokines in and out of the central nervous system: much more than chemotaxis and inflammation date: 2008-05-08 journal: journal of leukocyte biology doi: 10.1189/jlb.1107763 sha: doc_id: 302796 cord_uid: zi3p2k1b actions of chemokines and the interaction with specific receptors go beyond their original, defined role of recruiting leukocytes to inflamed tissues. chemokine receptor expression in peripheral elements and resident cells of the central nervous system (cns) represents a relevant communication system during neuroinflammatory conditions. the following examples are described in this review: chemokine receptors play important homeostatic properties by regulating levels of specific ligands in blood and tissues during healthy and pathological conditions; chemokines and their receptors are clearly involved in leukocyte extravasation and recruitment to the cns, and current studies are directed toward understanding the interaction between chemokine receptors and matrix metalloproteinases in the process of blood brain barrier breakdown. we also propose novel functions of chemokine receptors during demyelination/remyelination, and developmental processes. the central nervous system (cns) is characterized by a relatively immunosuppressive environment as a result of the lack of lymphatic drainage, resident dendritic cells (dc), and mhc expression [1] . moreover, the blood brain barrier (bbb) restricts inflammatory leukocyte trafficking into the cns [2, 3] . however, the cns has the potential of developing efficient innate, adaptive, and regulatory immune responses. during neuroinflammatory disorders, such as multiple sclerosis (ms), the maintenance of this specialized environment is disturbed, and the loss of the bbb integrity is a critical event in disease pathogenesis. bbb disruption allows leukocytes to traverse the vessel wall, accumulate within perivascular spaces, and then progress across the glia limitans into the parenchyma to initiate a destructive inflammatory response [4] . these events are strictly regulated by adhesion molecules, chemokines, and matrix metalloproteinases (mmps) [5] [6] [7] . the chemokine system is comprised of ϳ50 molecules and 20 receptors in humans, with orthologs in other mammalian species [8, 9] . the chemokine ligand superfamily is divided into subgroups, of which the largest are the cc chemokines (28 members) and the cxc chemokines (16 members) . chemokine subgroup members, encoded in multigene arrays, are functionally related and signal to corresponding families of chemokine receptors, which are g-protein-coupled receptors (gpcrs) and act specifically through pertussis toxin-sensitive g ␣i components. gpcrs are drug targets [10] , and the biotech/pharmaceutical industry has mounted substantial efforts to modulate chemokine receptor activity, heightening the medical importance of chemokine-mediated regulation of homeostatic and inflammatory processes. first identified by their ability to mediate leukocyte chemoattraction in vitro, chemokines are now recognized to govern a wide array of functions during inflammation and immunity. over the past decade, chemokine receptors have been localized to various cell types other than blood leukocytes [11, 12] . of particular interest in the cns, specific chemokine receptors have been detected on microglia, astrocytes, oligodendrocytes, neurons, and brain microvasculature. as an important corollary statement, the use of immunohistochemistry needs always to be controlled by appropriate studies in receptor-deficient knockout mice where available, given the notorious lack of specificity with these reagents. further, in situ hybridization also needs to be used to confirm findings. it is important to define the roles of chemokines and their receptors in healthy mice and in models of neural pathology. we will describe experimental approaches we use to study chemokine biology in vivo during normal and neuroinflammatory conditions and report our observations regarding the roles of chemokine receptors in ligand homeostasis; demyelination/ remyelination; their connection to mmps and bbb breakdown; and their participation during developmental processes (fig. 1) . chemokine-binding molecules (a.k.a., nonsignaling or "decoy" receptors) and chemokine sequestration three chemokine receptor-like molecules-d6, the duffy antigen receptor for chemokines (darc), and chemocentryxchemokine receptor (ccx-ckr; also known as ccrl1)have the capacity to bind chemokines without evoking the prototypical cellular responses [13, 14] such as chemotaxis or activation. these atypical receptors sequester chemokines and regulate their bioavailability and therefore, are critical regulators of inflammation [15] . the duffy blood group antigen was described over 50 years ago and later recognized as the entry receptor on erythrocytes for some malarial parasites [16] . the duffy antigen was named darc after it was shown that it has the unusual capability to bind a broad spectrum of chemokines of cc and cxc subfamilies [17, 18] . darc is expressed by endothelial cells [19, 20] and is accordingly involved in transferring chemokines across the endothelium, and as a result of the abundance red blood cells (rbcs), darc is seen as a receptor involved in clearance of chemokines in the blood. d6 is an extremely promiscuous receptor [21] , binding to at least 12 chemokines of the cc subfamily, all of which are inflammatory chemokines [13, 21] . mice lacking d6 exhibited exaggerated, cutaneous, inflammatory responses and an increased susceptibility to the development of skin cancer [22] . unexpectedly, d6-deficient mice were also resistant to induction of experimental autoimmune encephalomyelitis (eae) as a result of impaired encephalitogenic responses [23] . analyses of skin tissues at the immunization site in d6 ϫ/ϫ mice showed cd11cϩ aggregates, suggesting that dc might be trapped at the site of antigen injection, therefore limiting antigen presentation [23] . contrasting d6-binding properties, ccx-ckr binds the homeostatic chemokines ccl19, ccl21, ccl25, and human cxcl13 [24, 25] . ccx-ckr is predicted to modulate homoeostatic lymphocyte and dc trafficking, key migratory events in acquired immune responses that are directed by ccx-ckr-binding chemokines, which reveal functional and biochemical diversity within the chemokine receptor family and provide insights into novel mechanisms of chemokine regulation. for the past 5 years, we have been studying the role of the fractalkine receptor cx3cr1 during cns inflammation [26] . fractalkine is a unique cns chemokine present on neuronal membranes and capable of being released as a soluble protein by constitutive or stress-activated a disintegrin and metalloproteinase-family protease activity [27] [28] [29] . fractalkine exerts its functions by binding to cx3cr1 on microglial cells. although they are mainly produced in the cns, fractalkine and cx3cr1 also have a distinctive peripheral pattern of expression. fractalkine is found at low levels in endothelial and some epithelial cells of selected tissues such as kidney, lung, prostate, and heart but not spleen or liver [30] . cns endothelial cells do not express fractalkine. circulating monocytes and nk cells express cx3cr1 [31] . in our studies, we determined the levels of soluble fractalkine in the normal, healthy mouse brain, and we found that the levels of soluble fractalkine in wild-type and cx3cr1 -/mice were dramatically different. naïve, cx3cr1-deficient mice exhibited over 50 times more soluble fractalkine in the brain and in the serum compared with normal, wild-type mice [32] . these observations led us to hypothesize that signaling chemokine receptors are required for ligand homeostasis. we continued our studies by determining the levels of ligands in various chemokine receptor-defifig. 1 . understanding the biology of chemokine receptors. the focus of our research is schematically presented, and the four different areas integrate some complex observations that suggest key regulatory functions of chemokine receptors. our observations suggest that signaling chemokine receptors play a role in clearance of specific ligands with important implications for receptors that bind to more than one ligand, and where blockade approaches are proposed. cxcr2 is explored in the context of demyelinating models, and preliminary data show that the absence of cxcr2 in the cns is associated with enhanced tissue repair. also, cxcr2 is presented as an important chemoattractant for neutrophils during jhm strain of mouse hepatitis virus (jhmv) infection, and we are currently studying the role of neutrophil-derived mmp9 in bbb integrity. developmental studies are used to examine the role of cxcr7 during neurogenesis. cient strains (fig. 1) . we analyzed mouse lines lacking chemokine receptors, including members from the cc and cxc receptor family, such as ccr2, cxcr2, and cxcr3, in addition to cx3cr1/ccr2 double-deficient mice. these chemokine receptor-deficient lines allowed us to determine the levels of constitutive ligands such as cx3cl1 and inflammatory ligands such as ccl2, ccl3, cxcl1, and cxcl10 in circulation (serum) and tissues during healthy conditions. our results indicated that deficiency of chemokine receptors is associated with increased levels of the corresponding ligands [32] . ccr2 -/mice exhibited approximately three times more circulating ccl2 when compared with wild-type. cxcr2 -/mice showed levels of circulating cxcl1 and cxcl2 that were more than 30-fold increased when compared with wildtype mice. circulating levels of cxcl10 in naïve, wild-type and cxcr3 -/mice were undetectable. however, after inducing eae, we found that cxcr3 -/mice exhibited significantly higher cxcl10 levels. examination of chemokine levels in cns tissues, where cx3cl1 and cxcl1 are expressed, also revealed that these ligands were significantly higher in cx3cr1 ϫ/ϫ and cxcr2 ϫ/ϫ mice, respectively, when compared with wild-type mice [32] . expression analysis showed that cx3cl1 mrna levels were comparable in cx3cr1 ϫ/ϫ and wild-type mice, indicating that increased cx3cl1 was not caused by accumulation of mrna. reconstitution of cx3cr1and cxcr2-deficient mice with wild-type bone marrow substantially restored chemokine homeostasis, supporting the notion that chemokines are cleared from the periphery and in some instances, from cns tissue by cells expressing specific receptors [32] . the differences observed in ligand concentration in the various chemokine receptor-deficient mice analyzed are not clear but might be a result of the degree of promiscuity of the receptor or the relative expression levels of ligand and alternate receptors. it is well documented that chemokine functions can be suppressed by chemokine receptor desensitization and internalization upon ligand binding [33, 34] . therefore, we examined whether ligands present at high concentrations could alter the expression of an alternate receptor when the cardinal receptor is absent. we performed a ccl3-binding assay by flow cytometry in pbmc and resident peritoneal macrophages from ccr2 -/mice to analyze the functional availability of ccr1. we found the proportion of ccl3-bound cells was reduced significantly in ccr2 ϫ/ϫ mice when compared with wild-type mice [32] . therefore, excess ccr2 ligands, such as ccl7, which bind ccr1, can down-regulate this receptor. involvement of chemokine receptor in homeostasis of specific ligands came from studies that show rapid use of ccl2 by wild-type macrophages and increased amounts of ccl2 in ccr2 ϫ/ϫ mice in response to alloantigen [35] . moreover, our group reported that ccl2 is consumed by ccr2ϩ migrating pbmc in a human bbb model [36] . these observations support an important biological role of signaling chemokine receptors as scavengers of specific ligands (fig. 1 ). mmps, a large family of proteolytic enzymes, are involved in the degradation of protein components of the extracellular matrix, such as collagen, elastin, fibronectin, and laminin [37, 38] . these highly regulated enzymes are implicated in a variety of physiological (angiogenesis, bone remodeling, etc.) and pathological processes, in particular, in the pathogenesis of cns inflammatory disorders [37] . increased expression of several mmps has been found in the serum, cerebrospinal fluid, and cns of ms patients [39] . various mmps are also increased in the cns during experimental ms mouse models [40, 41] . in addition, intracerebral injection of mmps to healthy mice induces bbb breakdown and leukocyte recruitment [42] , whereas mmp inhibition blocks the onset or the development of eae and even reverses the disease [43, 44] . among the mmps, mmp9 is of particular interest, as it is increased in the serum of ms patients, especially during clinical relapse, and correlates with disease activity on gadolinium-enhanced brain magnetic resonance imaging [45, 46] . furthermore, young, mmp9-deficient mice are resistant to eae [47] . these findings implicate mmp9 in bbb disruption. in collaboration with drs. cornelia bergmann and steve stohlman, we are evaluating the role of mmp9 in bbb disruption using the murine model of cns demyelination induced by neurotropic coronavirus (jhmv) infection [48] . jhmv infection along with theiler's murine encephalomyelitis virus (tmev) represent excellent viral models to study mechanisms involved in demyelinating diseases, such as ms. cns infection with the neurotropic strain of mouse hepatitis virus (mhv) induces an acute inflammatory response, followed by a chronic cns infection with ongoing demyelination. in contrast to tmev infection, infectious mhv remains undetectable during the chronic phase of jhmv infection, although viral antigens and rna are present. furthermore, chemokine and mmp expression during jhmv infection [48, 49] is well characterized and provides the basis to study individual factors contributing to parenchymal cns infiltration by leukocytes. it is known that mmp9 protein levels are increased during jhmv infection [50] , and this correlates with neutrophil recruitment. however, mmp9 mrna is not altered, unlike mmp3 and mmp12 mrna, which appear up-regulated [49] . neutrophils store mmp9 within their granules and are abundant, inflammatory cells entering the cns early during infection, and their infiltration correlates with a rapid decline in bbb integrity. to study the role of neutrophils and mmp9 in bbb disruption during jhmv infection, neutrophils were depleted using anti-gr1 antibody (rb6-8c5) [51] . neutrophil depletion resulted in reduction of leukocytes in the brain, lessened bbb disruption, and absence of mmp9 activity, suggesting a role of this enzyme in bbb breakdown. however, a direct implication of mmp9 in mediating the loss of bbb integrity was not demonstrated. to address this question, mmp9-deficient mice were infected with jhmv, and preliminary data revealed decreased leukocyte infiltration in infected mmp9 ϫ/ϫ mice compared with wild-type controls (c. savarin, cornelia bergmann, stephan stohlman, unpublished results). analyses of bbb permeability need to be performed to confirm the direct role of mmp9 in bbb disruption. we are interested in characterizing the cell type(s) responsible for mmp9 release during jhmv infection. the anti-gr1 antibody rb6-8c5, classically used for neutrophil depletion, recognizes ly-6g and ly-6c, two distinct surface antigens expressed by neutrophils (ly6g high ) and immature bone marrow-derived macrophages (ly6c high ) [52] , suggesting that anti-gr1 antibody treatment depletes neutrophils and recruited monocytes. importantly, macrophages constitute a second population present in the cns at the peak of neutrophil infiltration following jhmv infection [48] and represent a potential source of mmp9 [39, 40] . to assess the specific contribution of mmp9 release by neutrophils and/or macrophages in bbb disruption, jhmv pathogenesis will be studied in cxcr2-and ccl2-deficient mice. using cxcr2 ϫ/ϫ mice, a chemokine receptor that binds cxcl1 and cxcl2 [53] , the two major neutrophil chemoattractants in jhmv-induced cns inflammation, we will be able to confirm neutrophil involvement in bbb disruption during jhmv infection. previous studies [54] showed that jhmv infection in ccr2-deficient mice was associated with increased mortality and absence of t cell infiltration, which are the primary effectors in viral clearance of infected cns resident cells [55, 56] . the ccr2 ligand, ccl2, has also been implicated in chemoattraction of t cells to the inflamed cns [57] . however, our preliminary results showed that migration of cd4 and cd8 t cells into the cns of ccl2-deficient mice was similar to control mice, suggesting that ccl2 is not implicated in cns t cell recruitment during jhmv infection. by contrast, a significant reduction of f4/80 ϩ macrophage infiltration was observed, indicating an important contribution of ccl2 in macrophage migration within the cns following jhmv infection. these observations confirmed the relevance of using ccl2-deficient mice to study the implication of macrophages in mmp9 release and bbb disruption. cxcr2 is expressed by peripheral monocytes and neutrophils and in the cns, is present on oligodendrocyte progenitor cells (opcs) in the brain and spinal cord [58] . one of the most studied ligands for cxcr2, cxcl1 is expressed by spinal astrocytes and acts as a potent promoter of opc proliferation. cxcr2 mediates monocyte arrest under flow conditions [59] and in the cns, governs migratory arrest of oligodendrocyte precursors and proliferative responses during development [58, 60] . therefore, in addition to the properties of chemokines in migration described originally, cxcr2/cxcl1 interactions control the positioning of opcs in the developing spinal cord by arresting their migration. global lack of cxcr2 is associated with relative resistance to demyelination we are interested in defining the function of cxcr2 during myelin repair. for this research, we use cxcr2 ϫ/ϫ mice, initially generated in 1994 on a balb/c background, to study myelopoiesis [61] . we crossed cxcr2 ϫ/ϫ mice for eight generations to the swxj (h2q/s) and c57bl/6 background to determine the role of cxcr2 during demyelination/remyelination processes in vivo using the mouse models of eae, cuprizone intoxication, and focal demyelination by lysolecithin (lpc) injection (l. liu, abdelmadjid belkadi, taofung hu, k. choi, lindsey a. darnall, robert h. miller, r. m. ransohoff, unpublished data). we found that cxcr2 ϫ/ϫ mice were relatively resistant to induction of eae. disease onset, kinetics, mortality, and peak day of disease severity in the rare cxcr2 ϫ/ϫ mice that developed signs of eae were equivalent to that seen in cxcr2 ϩ/ϩ littermates. interestingly, recovery from the initial attack of eae was faster and more robust in cxcr2 ϫ/ϫ mice, and disease scores in the resolution phase were significantly lower than in cxcr2 ϩ/ϩ mice. six weeks after cuprizone feeding, a significant difference in the tissue area affected by demyelination was observed in wild-type mice (60% demyelinated tissue) versus cxcr2 ϫ/ϫ mice (10% demyelinated tissue). similar observations were obtained 7 days after lesion induction by lpc injection, and wild-type mice exhibited a 30% demyelinated area compared with 10% in cxcr2 ϫ/ϫ mice. we proposed the hypothesis that cxcr2 mediates pathogenic effects during eae at two separate levels. first, cxcr2 acts as an "arrest receptor" for myeloid cells promoting accumulation of inflammatory effectors in the cns, as reported for atherosclerosis models. second, the cxcr2 ligand cxcl1 expressed by reactive astrocytes at lesion edges [62] [63] [64] may arrest opcs and block the remyelination process by precluding opc entry into the lesions, thereby suppressing lesion repair. we proposed that eae lesions might resolve more efficiently in mice lacking cxcr2 in the opcs. we addressed this issue by constructing radiation bone marrow chimerae. following sublethal irradiation, bone marrow from heterozygous mice was used for reconstitution, giving rise to chimeric cxcr2 ϩ/ϫ 3 cxcr2 ϩ/ϩ and cxcr2 ϩ/ϫ 3 cxcr2 ϫ/ϫ mice. both of these chimeric mice express cxcr2 on hematopoietic cells, but cxcr2 ϩ/ϫ 3 cxcr2 ϫ/ϫ mice selectively lack cxcr2 on opcs (and other radio-resistant cells). we found strong evidence that absence of cxcr2 in the cns was associated with improved lesion repair in eae. cxcr2 ϩ/ϫ 3 cxcr2 ϩ/ϩ and cxcr2 ϩ/ϫ 3 cxcr2 ϫ/ϫ mice had comparable incidence of eae, indicating that loss of function of cxcr2 on leukocytes caused reduced incidence of eae in cxcr2 ϫ/ϫ mice. the day of onset, peak disease (reflecting kinetics of neurological impairment), and severity of eae were all equivalent in both groups of mice. however, as seen previously in cxcr2 ϫ/ϫ mice, the timing and extent of recovery were accelerated in mice lacking cxcr2 in the cns. this result suggested that the absence of cxcr2 in the cns led to improved repair of eae lesions. this notion was supported by the observation that lesion size in cxcr2 ϩ/ϫ 3 cxcr2 ϩ/ϩ and cxcr2 ϩ/ϫ 3 cxcr2 ϫ/ϫ mice was comparable at the peak of eae, correlating to the equivalent disease scores at this stage. however, during the recovery phase (10 days after onset of neurological signs), we found significantly smaller lesions in cxcr2 ϩ/ϫ 3 cxcr2 ϫ/ϫ mice. furthermore, after cuprizone intoxication, we found equal demyelination in cxcr2 ϩ/ϫ 3 cxcr2 ϫ/ϫ and cxcr2 ϩ/ϫ 3 cxcr2 ϩ/ϩ mice at 4, 5, and 6 weeks after cuprizone treatment. however, cxcr2 ϩ/ϫ 3 cxcr2 ϫ/ϫ mice exhibited enhanced remyelination 7 days after removing cuprizone compared with cxcr2 ϩ/ϫ 3 cxcr2 ϩ/ϩ mice. a similar result was found after lpc injection (l. liu, a. belkadi, t. hu, k. choi, l. darnall, r. h. miller, r. m. ransohoff, unpublished data). therefore, blocking cxcr2 might confer neuroprotection and anti-inflammatory effects. signaling pathways by cxcl12 binding to cxcr4 and cxcr7 cxcl12 and cxcr4 represent a chemokine receptor pair that plays multiple functions during developmental and pathological processes. cxcr4 was the first known receptor for cxcl12. previous studies [65, 66] demonstrated abnormal migration of cerebellar neurons in cxcr4-or cxcl12-deficient mice, suggesting a role of the cxcl12-cxcr4 axis in cns development. binding of cxcl12 to cxcr4 causes the activation of a series of downstream signaling pathways that regulates cell proliferation, migration, and survival in the cns [67] . recently, an orphan receptor, previously termed rdc1, was shown to be the second receptor for cxcl12, according to the gene structure, chromosomal location, and its high affinity to cxcl12 [68, 69] . this new chemokine receptor is now termed cxcr7. interestingly, cxcl11, another cxc chemokine formerly known as ifn-inducible t cell ␣ chemoattractant, can also bind cxcr7 with high affinity. some of our efforts are concentrated in understanding the role of cxcr7 in the cns in relationship to the various cxc ligands identified so far. although cxcr7 is expressed in various cell types of peripheral tissues as well as the cns [69] , its role in peripheral tissues is poorly understood. binding of cxcl12 to cxcr7 provides cultured, receptor-bearing cells with a growth and survival advantage and increased adhesive properties [69, 70] . however, signaling pathways triggered by binding of cxcl12 to cxcr7 remain controversial with a number of mutually contradictory published results [68 -73] . in cancer biology, cxcr7 may be envisioned as an interesting target, as its expression at the mrna level is increased in malignant gliomas compared with the normal brain tissues. by in situ hybridization, it was confirmed that cxcr7 expression is confined to vessels of gliomas but not normal cns tissue. furthermore, it was demonstrated that cxcr7 is also expressed on tumor-associated vasculature in breast and lung tumors. enhanced expression of cxcr7 in tumor cells not only promotes tumor growth but also enhances the progression of metastases. it is plausible that cxcr7 works by sequestering cxcl11 or cxcl12 [74] , making cxcr7 and cxcr4 potential targets for intervention in tumor biology. of relevance, specific small molecular compounds for cxcr7 inhibited the growth of human tumor cells in mouse tumor models [69] . therefore, blockade approaches to inhibit cxcr7, and its effects on metastatic spreading are a major area of research. consistent with previous observations [69] , our mrna expression studies have demonstrated that cxcr7 is expressed during the early development of cns [71] . however, we did not observe significant differences in the distribution of major cns cell populations between cxcr7 ϫ/ϫ and wild-type mice. hippocampus, dentate gyrus, and cerebellum of cxcr7 ϫ/ϫ mice appeared normal. in the developing spinal cord and dorsal root ganglia at embryonic day 15.5 (e15.5), normal neurogenesis occurs, and expression of cxcr4 is maintained in the cxcr7 ϫ/ϫ neural tube (e9.0 -e12.5). these observations indicate that cxcr7 is not directly involved in neurogenesis. it is important to note that there are many developmental defects associated with cxcr4 deficiency; such defects include, among others, distorted laminar structure and abnormal migration of cells from the proliferative, external granule layer in the developing cerebellum, with establishment of proliferating aggregates and absence of foliation [65] . in zebrafish, an important role for cxcr7 has been revealed recently in the development of the lateral line [75, 76] , a mechanosensory system comprised of sensory organ neuromasts. all neuromasts originate from a migrating primordium, which moves from head to tail during the development of a lateral line. cxcr7 is expressed in the posterior-lateral line, and a complementary expression pattern between cxcr7 and cxcr4 was described. in particular, expression of cxcr7 is strong in the posterior part but absent in the leading half of primordium, and expression of cxcr4 is mainly detected in the leading part of primordium [75, 76] . morpholino knockdown of cxcr7 leads to the deficient migration of primordium that eventually affects the distribution of neuromasts. other studies also support this observation by showing that antisense knockdown of cxcr7 specifically affects the migration of trailing cells, resulting in tissue stretching [76] . further studies are needed to delineate in detail the downstream mechanisms of cxcr7 during mammalian development. understanding the mechanisms that govern the expression and regulation of chemokines and their receptors is crucial to define the consequences of chemokine receptor blockade for therapeutical purposes. chemokine receptor-deficient murine models are instrumental in defining the roles of chemokines during in vivo inflammatory conditions. our results implicate signaling chemokine receptors in consumption and clearance of specific ligands. for some receptors, the promiscuity of ligand binding can alter their expression if alternate ligands are present in high levels. the observation of increased ligand levels in chemokine receptor-deficient mice carries relevance for therapeutic application of chemokine biology [77] . in particular, chemokine elevation may represent a valuable biomarker for the efficacy of blocking molecules, and more importantly, this phenomenon needs to be taken into account where unexpected (although not necessarily detrimental) consequences might arise. our studies show that global deficiency of cxcr2 is associated with relative resistance to demyelination. however, using three different in vivo models, we found that reconstitution of cxcr2 on hematopoietic cells "rescued" demyelination. blockade of cxcr2 might promote remyelination in ms lesions, as well as reducing inflammation. therefore, blocking cxcr2 might confer neuroprotection and anti-inflammatory effects. among therapeutic strategies under investigation for treatment of ms, several groups focus on preventing leukocyte infiltration within the cns. mmp9, by its implication in bbb disruption, constitutes a potential and promising target for ms treatment (fig. 1) . moreover, treatment of eae with mmp inhibitors showed interesting findings, but some of these inhibitors were then abandoned because of side-effects as a result of the lack of inhibitor selectivity [78 -80] . indeed, mmps can also display beneficial roles, particularly in tissue repair [81] . regarding these data, it is important to know the exact role and source of mmps during ms pathogenesis, before using mmp inhibitor treatment. our data, by demonstrating a direct effect of mmp9 in bbb disruption and identifying which cell type is implicated in mmp9 release, would bring additional information essential for potential ms therapeutic strategies. cxcr7 and cxcr4 are also targets for inhibition for clinical intervention, particularly in cancer biology. our efforts are concentrated in defining the specific actions of chemokine receptors in the brain and their linkage with peripheral elements during neuroinflammatory conditions with the hope that successful clinical, therapeutic approaches might be developed. what is immune privilege (not)? the ins and outs of t-lymphocyte trafficking to the cns: anatomical sites and molecular mechanisms three or more routes for leukocyte migration into the central nervous system what is the blood-brain barrier (not)? molecular mechanisms involved in t cell migration across the blood-brain barrier the expression and function of chemokines involved in cns inflammation chemokines and matrix metalloproteinase-9 in leukocyte recruitment to the central nervous system chemokines: immunology's high impact factors the many roles of chemokines and chemokine receptors in inflammation chemokines and their receptors: drug targets in immunity and inflammation chemokine receptors in neuroinflammation chemokines and chemokine receptors: multipurpose players in neuroinflammation cloning and characterization of a novel promiscuous human ␤-chemokine receptor d6 purification and biochemical characterization of the d6 chemokine receptor chemokine sequestration by atypical chemokine receptors erythrocyte receptors for (plasmodium knowlesi) malaria: duffy blood group determinants identification of a promiscuous inflammatory peptide receptor on the surface of red blood cells regulation of chemotactic networks by enhanced expression of duffy antigen in the lungs during suppurative pneumonia expression of the duffy antigen/receptor for chemokines (darc) by the inflamed synovial endothelium cloning and characterization of a novel murine ␤ chemokine receptor, d6. comparison to three other related macrophage inflammatory protein-1␣ receptors, ccr-1, ccr-3, and ccr-5 the atypical chemokine receptor d6 suppresses the development of chemically induced skin tumors cutting edge: the silent chemokine receptor d6 is required for generating t cell responses that mediate experimental autoimmune encephalomyelitis characterization of mouse ccx-ckr, a receptor for the lymphocyte-attracting chemokines teck/mccl25, slc/mccl21 and mip-3␤/mccl19: comparison to human ccx-ckr cutting edge: identification of a novel chemokine receptor that binds dendritic cell-and t cell-active chemokines including elc, slc, and teck control of microglial neurotoxicity by the fractalkine receptor tumor necrosis factor-␣-converting enzyme (adam17) mediates the cleavage and shedding of fractalkine (cx3cl1) tumor necrosis factor-␣-converting enzyme mediates the inducible cleavage of fractalkine fractalkine is expressed by smooth muscle cells in response to ifn-␥ and tnf-␣ and is modulated by metalloproteinase activity the transmembrane form of the cx3cl1 chemokine fractalkine is expressed predominantly by epithelial cells in vivo analysis of fractalkine receptor cx(3)cr1 function by targeted deletion and green fluorescent protein reporter gene insertion scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues pathways for internalization and recycling of the chemokine receptor ccr5 chemokine receptor internalization and intracellular trafficking ccr2 regulates the level of mcp-1/ccl2 in vitro and at inflammatory sites and controls t cell activation in response to alloantigen modulating ccr2 and ccl2 at the blood-brain barrier: relevance for multiple sclerosis pathogenesis matrix metalloproteinases and diseases of the cns the role of matrix metalloproteinases in autoimmune damage to the central and peripheral nervous system differential matrix metalloproteinase expression in cases of multiple sclerosis and stroke elevation of matrix metalloproteinases (mmps) in multiple sclerosis and impact of immunomodulators differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states cxc chemokines generate age-related increases in neutrophil-mediated brain inflammation and blood-brain barrier breakdown suppression of experimental allergic encephalomyelitis in the lewis rat by the matrix metalloproteinase inhibitor ro31-9790 effective treatment of models of multiple sclerosis by matrix metalloproteinase inhibitors serum gelatinase b, timp-1 and timp-2 levels in multiple sclerosis. a longitudinal clinical and mri study serum mmp-9 and timp-1 levels are related to mri activity in relapsing multiple sclerosis resistance of young gelatinase b-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions coronavirus infection of the central nervous system: host-virus stand-off expression of matrix 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acts as a t-lymphocyte chemoattractant the chemokine receptor cxcr2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration gro family chemokines are specialized for monocyte arrest from flow the chemokine growth-regulated oncogene-␣ promotes spinal cord oligodendrocyte precursor proliferation neutrophil and b cell expansion in mice that lack the murine il-8 receptor homolog murine experimental autoimmune encephalomyelitis: a model of immune-mediated inflammation and multiple sclerosis role for cxcr2 and cxcl1 on glia in multiple sclerosis multiple sclerosis. oligodendrocyte survival and proliferation in an active established lesion function of the chemokine receptor cxcr4 in hematopoiesis and in cerebellar development impaired b-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in cxcr4 or sdf-1 deficient mice multiple roles of the chemokine cxcl12 in the central nervous system: a migration from immunology to neurobiology the chemokine sdf-1/cxcl12 binds to and signals through the orphan receptor rdc1 in t lymphocytes a novel chemokine receptor for sdf-1 and i-tac involved in cell survival, cell adhesion, and tumor development essential but differential role for cxcr4 and cxcr7 in the therapeutic homing of human renal progenitor cells disrupted cardiac development but normal hematopoiesis in mice deficient in the second cxcl12/sdf-1 receptor the role of cxcr7/rdc1 as a chemokine receptor for cxcl12/sdf-1 in prostate cancer proteolytic processing of cxcl11 by cd13/aminopeptidase n impairs cxcr3 and cxcr7 binding and signaling and reduces lymphocyte and endothelial cell migration control of chemokine-guided cell migration by ligand sequestration the chemokine sdf1a coordinates tissue migration through the spatially restricted activation of cxcr7 and cxcr4b control of cell migration in the development of the posterior lateral line: antagonistic interactions between the chemokine receptors cxcr4 and cxcr7/rdc1 pharmaceutical targeting of chemokine receptors reversal of experimental autoimmune encephalomyelitis with a hydroxamate inhibitor of matrix metalloproteases matrix metalloproteinases in multiple sclerosis: is it time for a treatment trial functional roles and therapeutic targeting of gelatinase b and chemokines in multiple sclerosis metalloproteinases: mediators of pathology and regeneration in the cns key: cord-002209-xs6qigg4 authors: kıray, hülya; lindsay, susan l.; hosseinzadeh, sara; barnett, susan c. title: the multifaceted role of astrocytes in regulating myelination date: 2016-09-17 journal: exp neurol doi: 10.1016/j.expneurol.2016.03.009 sha: doc_id: 2209 cord_uid: xs6qigg4 astrocytes are the major glial cell of the central nervous system (cns), providing both metabolic and physical support to other neural cells. after injury, astrocytes become reactive and express a continuum of phenotypes which may be supportive or inhibitory to cns repair. this review will focus on the ability of astrocytes to influence myelination in the context of specific secreted factors, cytokines and other neural cell targets within the cns. in particular, we focus on how astrocytes provide energy and cholesterol to neurons, influence synaptogenesis, affect oligodendrocyte biology and instigate cross-talk between the many cellular components of the cns. astrocytes were long considered secondary to neurons in central nervous system (cns) function, and erroneously dismissed as "brain glue" (glia is the greek term for glue). research over the past two decades, however, has shown astrocytic roles extending to a range of brain functions far beyond basic physical and metabolic neuronal support (sofroniew and vinters, 2010) . astrocytic regulation of myelination was first hypothesised by műller in 1904, who claimed that the demyelinating disease, multiple sclerosis (ms), was rooted in astrocytic dysfunction (müller, 1904; williams et al., 2007) . evidence has since continued to grow supporting the premise that astrocytes could be important in regulating myelination (sofroniew and vinters, 2010; williams et al., 2007; barnett and linington, 2013; moore et al., 2011) . glial fibrillary acidic protein (gfap) has been used extensively in the study of astrocytes. increased gfap expression has been associated with astrocyte reactivity in cns lesions and is a pathological hallmark of disease and/or injury. fig. 1 illustrates astrocytes immunolabelled with gfap and nestin, another marker thought to label reactive astrocytes (kamphuis et al., 2012) . in experimental allergic encephalomyelitis (eae), a widely used animal model of ms, where demyelination is induced by myelin antigens, administered together with adjuvant that contains bacterial components (traugott and lebon, 1988; tsukada et al., 1991; villarroya et al., 1996) , gfap expression was seen on more numerous and much larger astrocytic processes in chronic lesions compared to normal appearing white matter (webster et al., 1985; eng et al., 1971) . thus, the degree of gfap immunoreactivity appears to reflect the level of reactive astrogliosis. this was reviewed in detail by sofroniew and vinters (2010) , who described a continuum of phenotypic changes, that range from mild to severe, the latter resulting in glial scar formation (sofroniew and vinters, 2010; nash et al., 2011a) . attempts have also been made to define the astrocyte phenotype in more detail along this continuum (liberto et al., 2004) . it has been suggested that mild astrogliosis is associated with astrocyte "activation" and severe astrogliosis is associated with "reactivity", with the former promoting recovery of cns function after injury and the latter walling off the injured area and preventing repair (liberto et al., 2004) . although activated astrocytes have been associated with less detrimental effects on the cns and reactive astrocytes as more damaging, it is clear that these properties are not all or nothing and reactive astrocytes can fig. 1 . expression of astrocyte reactivity markers. rat neurosphere-derived astrocytes cultured on pll-coated glass coverslips express the reactivity markers gfap (green) and nestin (red). astrocytic effects on re/myelination can be classified into 4 main groups. they contribute to re/myelination by: 1) providing an energy source (lactate) and cholesterol for neurons. glucose taken up by endothelial cells lining the blood brain barrier is later transferred to astrocytes which transform it to glycogen, which can then be used to produce lactate. 2) playing a role in synaptic signal transmission by regulating the fluid, ph/ion (e.g. potassium, k + ), glio/neurotransmitter homeostasis and contributing to synapse modulation through secreted molecules, such as thrombospondins (thbss) . 3) affecting the survival, proliferation and maturation of oligodendrocytes by secreting growth factors, some of which are regulated by iron homeostasis provided by astrocytes. chemokines may also influence oligodendrocyte membrane ensheathment of axons. 4) altering reactivity status through their release of chemokines/cytokines, which in turn affects the cross-talk between all neural cells including microglia. also be beneficial to cns repair. interestingly, in studies using gfap null mice, it was seen that animals had abnormal myelination, nonmyelinated axons in the optic nerve with an age related reduction in myelin thickness (liedtke et al., 1996) . non-conservative mutations in the gfap gene have also been linked to the white matter brain disorder, alexander disease (brenner et al., 2001; li et al., 2002) . therefore, the evidence for direct or indirect astrocytic roles on re/myelination has been established both by in vitro and in vivo studies. considering the limitations in the current treatments for demyelinating cns diseases and injuries, it is crucial to identify other approaches to regulate myelination in search of novel strategies for repair. astrocytes have been shown to promote myelination through their supportive roles on neuron survival and maintenance of neuronal activity, and their direct action on proliferation, differentiation and migration of oligodendrocytes (fig. 2) . this review will focus on the interaction of astrocytes with neural cells to synergistically promote myelination. it is apparent that astrocytes can affect myelination under a range of normal and pathological conditions, but it is important to understand how this is regulated. many molecules can trigger or even regulate astrogliosis, including large polypeptide growth factors and cytokines (john et al., 2003; moore et al., 2011) , mediators of innate immunity such as lipopolysaccharide (lps) and other toll-like receptor ligands (farina et al., 2007) , neurotransmitters (bekar et al., 2008) , purines, reactive oxygen species, and molecules related to hypoxia and glucose deprivation (swanson et al., 2004) . for the purpose of this review we will focus on cytokines and chemokines. although these compounds are primarily considered in the context of chemotaxis in immune cells, here we will highlight their roles on astrocyte activation and reactivity. these molecules can be produced in an autocrine or paracrine fashion by various cell types in the cns including neurons, oligodendrocyte lineage cells, microglia, pericytes and endothelial cells. not only do these factors influence astrocyte phenotype but they also can affect a range of neural and immune cell types. 2.2. astrocyte activation (mild astrogliosis): a pro-reparative phenotype? astrocytes can be activated directly or indirectly. for example, in response to injury, microglia become activated and release the cytokine interleukin 1β (il-1β, herx et al., 2000) , which is an early injury signal (auron, 1998) . the delay of astrocyte activation in mice lacking il-1β, as well as in mice lacking il-1 type 1 receptor suggests that microglial activation is necessary for astrocyte activation (herx et al., 2000) . it has also been suggested that ciliary neurotrophic factor (cntf; a member of the il-6 family of cytokines)treated astrocytes in vitro had a phenotype that was more supportive of cns repair and thus are, by definition, activated (albrecht et al., 2002; . under cntf treatment, astrocytes upregulate expression of classical reactivity markers such as gfap, vimentin, and clusterin, show cellular and nuclear hypertrophy, and increase their proliferation rate (winter et al., 1995; levison et al., 1996; 1998; hudgins and levison, 1998) . there is a growing body of evidence for the promotion of neuronal survival by cytokine-activated astrocytes, potentially through secretion of various neurotrophic growth factors in the vicinity of neurons including nerve fig. 3 . simplified schematic of the effects of cytokine-activated astrocytes on re/myelination. astrocytes can be influenced by various cytokines to change their reactivity status to one that falls within the continuum of phenotypes, namely more activated or reactive; both of which will secrete factors that can modulate myelination in a positive or negative way. astrocytes with more severe reactivity present increased gfap expression, proliferation rate, and cellular hypertrophy with a more stellate morphology. the milder "activated" astrocytes can secrete a range of factors including; neurotrophic factors, growth factors, and cytokines that will stimulate re/myelination by promoting neuronal survival, neurite outgrowth, neurogenesis, and/ or oligodendrocyte precursor cell (opc) survival, proliferation, and/or maturation. conversely astrocytes that tend to have a more severe "reactive" phenotype, possibly induced by proinflammatory cytokines/cns tissue damage, may secrete cytokines and chemokines that lead to myelin and oligodendrocyte damage in vitro, suppress remyelination, delay disease recovery in experimental autoimmune encephalomyelitis (eae), and suppress myelination in myelinating embryonic rat mixed spinal cord cultures. however, these reactive scar forming astrocytes can also protect cns tissue by preventing immune cells from invading and exerting a pro-inflammatory response and have been shown to even ameliorate eae. growth factor (ngf), brain-derived neurotrophic factor (bdnf), activity dependent neurotrophic factor (adnf), hepatocyte growth factor (hgf), leukaemia inhibitory factor (lif), fibroblast growth factor-2 (fgf-2) and cntf (schwartz and nishiyama, 1994; rudge et al., 1995; uchida et al., 1998; dreyfus et al., 1999; messersmith et al., 2000; albrecht et al., 2002; fig. 3 ). moreover, cultured spinal cord astrocytes, treated with cntf, support the survival of a significantly greater number of ventral spinal motor neurons and promote neurite outgrowth better than unstimulated astrocytes (albrecht et al., 2002) . other researchers have shown that cytokine-activated astrocytes can promote neurogenesis, possibly by stimulating the differentiation of neural stem cells (nscs) residing in the subventricular zone and the dentate gyrus in adult animals (liberto et al., 2004) . because these multipotent nscs can migrate beyond their sites of origin and can later differentiate into oligodendrocytes, neurons and microglia, they have the potential to enhance recovery from cns injury and disease. importantly, activated astrocytes have also shown positive effects on myelination. our own investigations have shown that cntf activated astrocytes can promote the percentage of myelinated fibres in cns rat cultures (nash et al., 2011b) . further evidence of this has been shown in mice infected with the a-59 strain of the mouse hepatitis virus (mhv-a59), an animal model for ms (jordan et al., 1989 , messersmith et al., 2000 . these animals have been shown to secrete increased levels of cntf during the remyelination phase and cntf mrna is induced in the remyelinating regions in cells exhibiting astrocytic features (albrecht et al., 2003) . it is suggested that the increase in il-1β levels at early stages of cns pathology stimulates the induction of cntf mrna and protein in astrocytes (stöckli et al., 1991; guthrie et al., 1997; dallner et al., 2002; liberto et al., 2004) , a phenomenon which appears to be important for remyelination (herx et al., 2000) . this could be due to fgf-2 signalling as cntf treatment elevates astrocytic levels of fgf-2 mrna significantly, whereas, il-1 β shows no effect (albrecht et al., 2003) . since fgf-2 can enhance opc proliferation (albrecht et al., 2003) , it may produce more opcs for subsequent myelination (redwine et al., 1997; messersmith et al., 2000) . moreover, if the gp130 receptor, the ubiquitous signal transducer for cntf and all il-6 family members, is genetically removed from astrocytes, astrocyte survival was poor, there was a reduction in the development of astrogliosis, and larger areas of demyelination formed with a greater proinflammatory t cell response (haroon et al., 2011) . therefore, cntf seems to be an important cytokine involved in astrocyte reactivity and myelination. interestingly, il-1β can also stimulate the astrocytic production of another il-6 family cytokine, lif, (aloisi et al., 1994) , which has been shown to promote survival and differentiation of oligodendrocytes (khan and de vellis, 1994; mayer et al., 1994; bugga et al., 1998) . lif also decreases disease severity when exogenously administered in both chronic and relapsing-remitting eae mice (aloisi et al., 1994; butzkueven et al., 2002; ishibashi et al., 2006) . positive effects of lif on the survival and maturation of oligodendrocytes also provides evidence for the positive roles of lif on myelination (khan and de vellis, 1994; mayer et al., 1994; bugga et al., 1998) . however, other pro-inflammatory cytokines such as tumour necrosis factoralpha (tnf-α) and interferon-gamma (ifn-γ) have also been shown to potentiate reactive astrogliosis (yong et al., 1991 john et al., 2003 as discussed below. in contrast to their positive effects on myelination, astrocytes can also have a more detrimental effect on cns repair via the secretion of chemokines/cytokines when in a more severe, reactive state (fig. 3) . one such cytokine is tnf-α, which has been shown to induce myelin and oligodendrocyte damage in vitro (selmaj and raine, 1988) . tnf-α mrna expression in ms plaques positively correlates with the extent of demyelination and has been shown to be present in microglia/macrophages and to a smaller percentage of astrocytes (bitsch et al., 2000) . on the other hand, studies have shown tnf-α expression is mainly associated with gfap positive fibrous astrocytes in chronic active ms lesions at the lesion edge (hofman et al., 1989) as well as foamy macrophages and endothelial cells (selmaj et al., 1991) . however, the fact that astrocytes appear as the major or minor tnf-α expressing cell types in ms lesions might be because astrocytes internalize the protein in a receptor-mediated manner (aránguez et al., 1995; kuhlmann et al., 2006) rather than producing it themselves as suggested by hofman et al. (1989) and bitsch et al. (2000) . moreover, it is possible that astrocytes require a longer period of time to become reactive upon injury and only produce tnf-α first at the lesion edge of acute ms plaques and later both at the lesion edge and in the lesion centre of chronic active plaques (selmaj et al., 1991) . in situ hybridisation for tnf-α mrna has been detected in gfap-positive astrocytes in mice suffering from pneumococcal meningitis (izadpanah et al., 2014) which also suggests that astrocytes can indeed produce tnf-α in cns pathologies. since tnf-α effects the maturation of oligodendrocytes (cammer and zhang, 1999) , remyelination failure in the cns lesions could be because tnf-α prevents the in situ differentiation of oligodendrocytes. interestingly, direct cell contact between pre-oligodendrocytes (preols) and astrocytes has been shown to be a prerequisite for tnf to induce apoptosis in preols of rodent mixed glial cultures (kim et al., 2011) . nevertheless, it is possible that tnf-α increases production of pdgf in demyelinated spinal cord lesions of mhv-a59-injected mice (redwine and armstrong, 1998; frost et al., 2003) as suggested by the increase of pdgf-β transcription and pdgf-αβ protein levels in embryonic human astrocytes upon tnf-α treatment (silberstein et al., 1996) . therefore, astrocytes might have a positive role on remyelination both by producing tnf-α and by secreting pdgf upon stimulation with tnf-α. pdgf could in turn support the survival and enhance the proliferation of opcs in demyelinating lesions (woodruff et al., 2004; vana et al., 2007) . consequently, it is yet difficult to conclude whether reactive astrocytes associated with increased tnf-α levels in cns lesions are predominantly stimulatory or inhibitory to remyelination. ifn-γ, another cytokine found in ms plaques, has been reported to not only activate astrocytes, but is also expressed by reactive astrocytes and by immune cells that astrocytes have stimulated (pulver et al., 1987; traugott and lebon, 1988; miljkovic et al., 2007; hashioka et al., 2009; ionescu et al., 2011) . similar to tnf-α, ifn-γ has been shown to suppress remyelination and to delay disease recovery in transgenic eae mice, where ifn-γ expression by astrocytes was stimulated temporally in the recovery stage (lin et al., 2006) . astrocyte-directed expression of ifn-γ in transgenic mice has also resulted in regional hypomyelination and selective disruption of brain histogenesis, which led to ataxia and shorter life span (laferla et al., 2000) . furthermore, knocking down ifn-γ receptor expression in astrocytes three days before immunization suppressed eae and demyelination by inhibiting inflammatory cell infiltration (ding et al., 2015) . these animals presented lower mean clinical scores even when the receptor silencing was initiated after disease onset or at disease peak (ding et al., 2015) . despite the abovementioned evidence suggesting inhibitory roles for reactive astrocytes on myelination, hindinger et al. (2012) have proposed that ifn-γ signalling in astrocytes is indispensable for the alleviation of eae since levels of demyelination and axonal loss are increased during acute eae in mice with an astrocytic expression of a dominant negative allele for ifn-γ receptor. nevertheless, their approach blocked ifn-γ signalling in astrocytes without decreasing the expression of ifn-γ receptor, which would lower the levels of ifn-γ available for immunoregulatory cells; whereas, ding et al. (2015) have knocked down the expression of the receptor itself. therefore, blocking or lowering ifn-γ signalling in astrocytes with a carefully planned strategy might provide new disease-modifying treatments that will limit demyelination. reactive astrocytes also secrete c-x-c motif chemokine 10 (cxcl10, ransohoff et al., 1993) , particularly around active ms lesions (omari et al., 2005; carter et al., 2007) . cxcl10 mrna expression increases significantly during peak disease and decreases during the recovery phases in animal models of ms (godiska et al., 1995; glabinski et al., 1997; fife et al., 2001) . a direct effect of cxcl10 on the inhibition of myelination was shown in dissociated rat spinal cord cells plated on astrocytes treated with cxcl10 and its neutralizing antibody. in these experiments cxcl10 identified by microarray analysis, was upregulated in an astrocyte phenotype that was inhibitory to cns myelination in vitro. specifically, cxcl10 appeared to inhibit oligodendrocyte process extension (nash et al., 2011b) . consequently, cytokines stand out as an important family of molecules to activate astrocytes and to initiate different forms of astrocyte reactivity that could be either beneficial or inhibitory for the cns milieu in terms of re/myelination. it should be noted that the secretion of such pro-inflammatory cytokines that can contribute to the lack of remyelination within ms plaques is not restricted to reactive astrocytes since other glial and inflammatory cell types will also secrete them. moreover, the up regulation of such cytokines by reactive astrocytes can also be protective for cns injury. for example, the astrocytic scar can restrict leukocyte migration from within areas of damaged tissue into the otherwise healthy non damaged cns tissue in close proximity to the scar protecting it from immune mediated damage (faulkner et al., 2004; okada et al., 2006; herrmann et al., 2008; voskuhl et al., 2009 ). a vital supportive role played by astrocytes following injury is the provision of an energy source, which is important if axons are to be myelinated. this energy is metabolized from glucose which enters the brain via the endothelial cells lining the blood brain barrier (bbb), which are in close contact with astrocytes. unlike endothelial cells, astrocytes biochemically transform glucose into glycogen, the principal source of stored energy in all cell types (pellegri et al., 1996; pfeiffer-guglielmi et al., 2003) . in addition, it has been suggested that astrocytes under low glucose concentrations can degrade stored glycogen into lactate which in turn increases extracellular lactate levels to provide energy for nearby axons when deprivation occurs after injury (tekkök et al., 2005) . the lactate derived from astroglial glycogen via glycolysis is transferred directly to the axon at the node of ranvier (hirrlinger and nave, 2014) . the importance of lactate during demyelination is only just emerging. whether astrocytes maintain the energy levels of only axons as previously thought, or if this extends to oligodendrocytes as well, is an interesting concept. oligodendrocytes are known to consume lactate at higher levels than other cns cells, therefore making them an important user of any lactate production. furthermore, promotion of myelination via oligodendrocytes has been shown when endogenous lactate is applied (rinholm and bergersen, 2012) therefore at least some astrocytic lactate production may be targeted to myelinating oligodendrocytes (sánchez-abarca et al., 2001; rinholm et al., 2011) . however, energy regulation in the cns is more complex as recent evidence has shown that oligodendrocytes in turn can transfer glycolysis products such as lactate to axons via monocarboxylic acid transporters (mct1, mct2), which reside in internodal myelin and the axonal compartment (fünfschilling et al., 2012) . cholesterol is essential to every cell in the body as it is an important component of cellular membranes. in the cns it is vital for normal brain development, is a precursor to many signalling molecules such as steroid hormones, and importantly, is a major structural component of myelin sheaths (siegel et al., 1999) . the bbb prevents the transport of either hepatic or dietary cholesterol, meaning that cholesterol must be derived by de novo synthesis within the cns (orth and bellosta, 2012) . astrocytes are proposed to be one of the primary cellular sources of cholesterol (pfrieger and ungerer, 2011) and mediate its secretion by their expression of several apolipoproteins, molecules that bind cholesterol (boyles et al., 1985; lin et al., 1986; xu et al., 2006; kurumada et al., 2007) . there is sufficient evidence to suggest that there is horizontal transfer of cholesterol (boyles et al., 1985; lin et al., 1986; xu et al., 2006; kurumada et al., 2007) with both astrocytes and oligodendrocytes producing cholesterol to maintain myelin sheath formation and neurons. this transfer between cells is critically relevant to neurodegenerative diseases, since the availability of cholesterol is thought to be an essential rate limiting factor to myelin production (may et al., 2004; liu et al., 2010) . therefore, in addition to their roles in providing energy to neurons, astrocytes also emerge as important cholesterol-suppliers in the cns, which is vital for myelination. a further function of astrocytes is the removal of excitotoxic molecules from the extracellular space, thus supporting neuronal survival. they can actively remove excitotoxic glutamate and convert it to glutamine by increasing their levels of glutamate transporters and glutamine synthetase (faden et al., 1989; eng et al., 1997; krum et al., 2002) , thereby preventing neuronal cell death during brain pathology. astrocytes can also release gliotransmitters such as glutamate, purine, gaba and d-serine into the synaptic cleft upon excitation by changes in neuronal synaptic activity and can thereby regulate neuronal excitability (parpura et al., 1994; bezzi et al., 1998; mothet et al., 2000; coco et al., 2003; halassa et al., 2007) . neurotransmitter released from the neuronal synapse can reach adjacent astrocytes, stimulating increases in intracellular ca 2+ concentrations, which then leads to the secretion of gliotransmitters (porter and mccarthy, 1997) . these regulatory molecules then can feedback to presynaptic nerve terminals to modulate synaptic neurotransmission (araque et al., 1998) . these observations have even given rise to the currently accepted 'tripartite synapse' hypothesis, where astrocytes form an active, integral regulatory component of the synapse (araque et al., 1999; halassa et al., 2007) . recently, electron microscopy has also shown microglia interacting with neuronal synapses (tremblay et al., 2010) and playing a role in synapse maturation, synaptic remodelling, and synaptic activity (ji et al., 2013) . evidence has shown that microglia secrete immune factors that play an important role in synaptic connections and illustrates the complexity of cross-talk between neural cells and the immune system. these researchers have suggested a change in name from tripartite synapse to the quad-partite synapse (schafer et al., 2013; wu et al., 2015) . in addition to playing a role in synaptic signal transmission, astrocytes can also modulate synapses. it has been demonstrated that astrocytes secrete molecules such as thrombospondins that might be required for the formation, function and pruning of developing synapses (ullian et al., 2001; christopherson et al., 2005) . both presynaptic and postsynaptic activity in purified rat retinal ganglion cultures have been enhanced in the presence of astrocytes; and immunohistochemistry of rat brain cryosections from various developmental stages have shown that glial generation precedes the appearance of synapses (ullian et al., 2001) . astrocytes might also be functional in synaptic remodelling and pruning in healthy or diseased adult cns (barres, 2008) . because synaptic signal transmission can trigger astrocytes to secrete the cytokine lif, which in turn increases the number of myelinated axons in dorsal root ganglion cultures co-cultured with oligodendrocytes (ishibashi et al., 2006) , the support and maintenance of healthy signal transmission appears important for the regulation of myelination. furthermore, astrocytes may contribute to synaptic transmission by supporting maintenance of the synaptic interstitial fluid by regulating ion, ph and transmitter homeostasis. astrocyte processes contain transporters for potassium uptake and aquaporin 4 water channels (nielsen et al., 1997; rash et al., 1998; amiry-moghaddam et al., 2003; solenov et al., 2004) , which maintain the transmitter homeostasis of the synaptic interstitial fluid (steinhäuser et al., 1994; gundersen et al., 1996; mennerick et al., 1996; bergles and jahr, 1997; fujita et al., 1999) . connexin channels and connexin proteins are important candidates which play a role in the regulation of neuronal activity and survival. for example, astroglial connexins decrease neuronal excitability by removing extracellular potassium and glutamate; while also providing metabolic supply to neurons (wallraff et al., 2006; rouach et al., 2008; froger et al. 2010; pannasch et al., 2011) . on the other hand, the elevation of connexin expression at lesion sites in cns pathologies might also be associated with other, possibly protective, roles (nagy et al., 1996; koulakoff et al., 2008; kuchibhotla et al., 2009; mei et al., 2010; karpuk et al., 2011) . studies using connexin knockout animals allude to the importance of connexins in promoting communication between astrocytes and between astrocytes and oligodendrocytes on myelin integrity. myelin damage has been observed in cx47, cx43/cx30 and cx30/cx47 single/double knockout mice (menichella et al., 2003; odermatt et al., 2003; lutz et al., 2009; tress et al., 2012) . interestingly, the level of oligodendrocyte gap junction connexins cx47 and cx32 were reduced both within and around lesions during early stages of inflammatory demyelination in eae mice, (traugott and lebon, 1988; tsukada et al., 1991; villarroya et al., 1996; meeuwsen et al., 2003) . these mice also presented decreased expression of cx43, the major astrocytic partner of cx47, when spinal cord sections were analysed immunohistochemically (markoullis et al., 2012) . cx43 expression was increased at late eae stages, where remyelination was observed, leading to the suggestion that astrocytic protein cx43 might play an important role in recovery from neuroinflammation (markoullis et al., 2012) . in the 1980s advancements were made on the ability to grow purified cultures of glial cells, with raff and colleagues in particular developing techniques to purify opcs from the optic nerve, a tissue devoid of neuronal cell bodies (raff et al., 1983) . with the development of these culture techniques it was shown that astrocytes play important roles in opc differentiation (noble and murray, 1984; noble et al., 1988; raff et al., 1988; noble et al., 1989) and in the rate of oligodendrocyte axonal ensheathment (watkins et al., 2008) . further in vitro studies, where conditioned medium collected from primary astrocyte monolayers was incubated with other neural cells, showed enhanced neuronal survival, proliferation of opcs, and protection of oligodendrocytes from stress (noble and murray, 1984; yoshida et al., 1995; yamamuro et al., 2003; zhu et al., 2006; arai and lo, 2010) . it is likely that astrocytes support opc survival and proliferation by providing soluble factors such as platelet derived growth factor (pdgf) and fgf-2 (bögler et al., 1990; ferrara et al., 1988; pringle et al., 1989) . astrocytes are important providers of secreted growth factors, for both neuronal and glial proliferation and survival. for example, cntf, although shown to be important in the activation of astrocytes, is constitutively expressed by white matter astrocytes, and is a key player in opc survival and maturation in vitro and in vivo as discussed earlier (stöckli et al., 1991; dallner et al., 2002; stankoff et al., 2002; cao et al., 2010) . cntf has also been reported to enhance the migration of subventricular zone-derived progenitors (vernerey et al., 2013) , protect oligodendrocytes from apoptosis, and decrease myelin destruction in demyelinating pathological conditions (linker et al., 2002) . in studies when cntf was injected subcutaneously at the remyelination phase of cuprizoneinduced demyelination, an increase was seen in myelin oligodendrocyte glycoprotein (mog) expression in the cerebral cortex (salehi et al., 2013) . moreover, intraperitoneal injections of cntf and intravenously transplanted mesenchymal stem cells that overexpress cntf resulted in a reduced loss of neurons and disease severity, and increased neuronal functional recovery in eae mice (kuhlmann et al., 2006; lu et al., 2009) . however, in these experiments it is difficult to see if the effect is on the activation status of the astrocytes as discussed above, or the direct effect on the opc. astrocytes have also been shown to exhibit a crucial role in opc remyelination via their iron exporter ferroportin (fpn) in mice, where focal demyelination was induced by the injection of lysophosphatidylcholine (lpc) into their spinal cords (schulz et al., 2012) . in these astrocyte-specific fpn ko mice, fewer remyelinating axons and a reduction in opc proliferation were observed following lpc-induced demyelination compared to control animals. this could either be due to direct effects on opcs through limited iron supply or indirect effects via iron-deficient microglia, which expressed significantly lower levels of tnf-α and il-1β when stimulated by lps compared to control microglia (schulz et al., 2012) . because the expression of fgf-2 and insulin-like growth factor-1 (igf-1) was significantly upregulated by il-1β and tnf-α, respectively, and the expression of transforming growth factor beta (tgf-β) was stimulated by il-1β in purified astrocyte cultures, it has been suggested that iron-deprivation in the milieu would lower the expression of il-1β and tnf-α in microglia and thus lead to reduced growth factor expression in astrocytes, which would in turn render opc proliferation and possibly differentiation (schulz et al., 2012) . as discussed throughout this review astrocytes can play important roles not just in myelination during development, but also in remyelination in adult tissue after cns injury. their reparative roles might be related to their level of reactivity, so it is important to identify markers that can define these different astrocyte phenotypes although at present these are not easy to define. one approach is to use microarrays. as described, nash et al. (2011b) identified cxcl10 as inhibitory to myelination, but others using a lower-scale cdna array that contained probes for cytokines, chemokines, growth factors and their receptors have identified other pro-inflammatory cytokines including tnf-α, il-1β, or ifn-γ (meeuwsen et al., 2003) . another large array was carried out on gfp-astrocytes purified at various time points after the onset of two models of disease, namely ischemic stroke (middle cerebral artery occlusion) and neuroinflammation induced by lps injection (zamanian et al., 2012) . the resulting data suggested that astrocytes could present different mrna expression profiles depending on the insult despite the presence of reactive gliosis in both types of cns damage. however, although there was upregulation of a core set of genes (lipocalin 2 and serpina3n), it was clear that changes in the astrocyte after injury are highly heterogeneous, and that changes in astrocyte activity may depend on the injury type. hopefully, more specific markers of "good" or "bad" astrocytes for cns repair will be identified and allow more specific identification of how these astrocytes influence repair. there is abundant evidence to suggest that astrocytes contribute to re/myelination mainly by: 1) providing the right conditions for neurons to myelinate by i) supplying neurons with energy and cholesterol, ii) removing excitotoxic molecules from the extracellular environment, and iii) regulating the fluid, ion, ph, and neuro/gliotransmitter homeostasis. 2) playing a role in the survival, proliferation, maturation and function of oligodendrocytes and the migration of opcs into the lesioned areas in the cns. 3) influencing microglia. the manner by which astrocytes affect myelination can often be seen to correlate with its level of reactivity. astrocyte reactivity can be induced by the milieu of cytokines present after injury, which can be beneficial or inhibitory in re/myelination depending on the context and severity of the 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developmental changes, and cellular localization of cntf-mrna and protein in the rat brain transfer of glycogen-derived lactate from astrocytes to axons via specific monocarboxylate transporters supports mouse optic nerve activity interferon-gamma and ia antigen are present on astrocytes in active chronic multiple sclerosis lesions microglial interactions with synapses are modulated by visual experience panglial gap junctional communication is essential for maintenance of myelin in the cns tumor necrosis factor and interleukin-1 in the csf and sera of patients with multiple sclerosis histological investigation of spinal cord lesions in the spinal hyperostotic mouse (twy/twy): morphological changes in anterior horn cells and immunoreactivity to neurotropic factors control of synapse number by glia plateletderived growth factor promotes repair of chronically demyelinated white matter ciliary neurotrophic factor controls progenitor migration during remyelination in the adult rodent brain baumann n. myelin-induced experimental allergic encephalomyelitis in lewis rats: tumor necrosis factor alpha levels in serum and cerebrospinal fluid immunohistochemical expression in glial cells and macrophages of optic nerve and spinal cord reactive astrocytes form scar-like perivascular barriers to leukocytes during adoptive immune inflammation of the cns the impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating cns coculture system immunocytochemical study of myelinassociated glycoprotein (mag), basic protein (bp), and glial fibrillary acidic protein (gfap) in chronic relapsing experimental allergic encephalomyelitis (eae) astrocytes-friends or foes in multiple sclerosis? a role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury platelet-derived growth factor regulates oligodendrocyte progenitor numbers in adult cns and their response following cns demyelination microglia: dynamic mediators of synapse development and plasticity profile and regulation of apolipoprotein e (apoe) expression in the cns in mice with targeting of green fluorescent protein gene to the apoe locus possible involvement of astrocytes in neuroprotection by the cognitive enhancer t-588 gamma-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo neurotrophic effects of conditioned media of astrocytes isolated from different brain regions on hippocampal and cortical neurons genomic analysis of reactive astrogliosis astrocyte-conditioned medium protecting hippocampal neurons in primary cultures against corticosterone-induced damages via pi3-k/akt signal pathway this work was funded by the wellcome trust (hk, wt083434), medical research council (sl, mr/j004731/1) and medical research scotland (sh, phd-718-2013). key: cord-280987-uhxk5b1b authors: turtle, l.; solomon, t. title: encephalitis, viral date: 2014-05-01 journal: encyclopedia of the neurological sciences doi: 10.1016/b978-0-12-385157-4.00375-4 sha: doc_id: 280987 cord_uid: uhxk5b1b encephalitis is inflammation and swelling of the brain, often caused by an acute viral infection, or a paraor postinfectious phenomenon known as acute disseminated encephalomyelitis (adem). the most common sporadic viral cause is herpes simplex virus, but arthropodborne viruses, such as japanese encephalitis virus, are a major cause in some settings, and rabies in others. herpes simplex encephalitis is treated with acyclovir; rabies and japanese encephalitis are preventable with vaccines. with the reduction in measles through vaccination, the incidence of adem, which often follows measles, has also reduced. encephalitis refers to the inflammation of the brain. the term includes both viral infections of the brain with predominantly gray matter disease and acute disseminated encephalomyelitis (adem), an immune-mediated demyelinating disease. in addition, other immune-mediated forms of encephalitis associated with antibodies to voltage-gated potassium channels, or nmethyl-d-aspartate (nmda) receptors, are being recognized with increasing frequency; being not viral associated, they are not discussed further here. clinically, acute viral encephalitis and adem usually manifest with fever, severe headache, neck stiffness, alterations of consciousness, focal neurological signs, and often seizures, especially in children. the pathology of acute viral encephalitis is characterized by mononuclear inflammatory cells in a perivascular distribution in the gray matter and meninges and neural cell destruction with neuronophagia. the pathology of adem or postinfectious encephalomyelitis is characterized by perivenular mononuclear cell inflammation and demyelination predominantly in the white matter of the brain and the spinal cord. both forms of encephalitis are linked primarily with viral infections, but both are also associated with some bacterial infections. rarely, acute encephalitis may be due to fungal and parasitic infection, and adem may be associated with vaccines. the two forms of encephalitis occur because of different spectra of viruses and involve different mechanisms of pathogenesis. following some general comments on viral pathogenesis, the two forms of encephalitis are discussed separately. viruses can be released by infected humans or animals in saliva, respiratory droplets, breast milk, feces, urine, semen, vaginal secretions, blood, or tissue. viruses have no mobility but must penetrate strong barriers to enter the body of a susceptible human host. the skin is covered by a layer of keratinized cells that will not sustain virus replication, but arthropods and animal bites, needles, and tissue transplantation can breach this barrier directly ( table 1) . the mucous membranes of the respiratory, gastrointestinal, and genitourinary tract are protected by secretory immunoglobulins (ig), and all are monitored by macrophages that phagocytize viruses and other particles. in addition, the respiratory tract has a mucous coating and cilia that beat the film containing inhaled particles outward and away from the epithelial cells of the lower respiratory tract, and the gastrointestinal and genitourinary tracts have extremes of acidity. a few nonenveloped, acid-resistant viruses (adenoviruses, enteroviruses, parvoviruses, and reoviruses) can survive passage through the acidity and solvents of the gastrointestinal tract. once permissive host cells are infected in the subcutaneous tissue, the mucous membranes, or the hematopoietic system (particularly macrophages), viruses replicate usually locally before there is invasion of the central nervous system (cns). spread into the cns is via nerves or the blood ( table 2) . for many years, the nerves, both peripheral and olfactory, were regarded as the sole portals into the cns, and early experimental studies documented the neural transmission of rabies virus, herpes simplex virus, and polioviruses. nerve endings have receptors for some viruses, and the viruses are transported by the retrograde axon transport system to the cell bodies, where replication is possible. this transport occurs in the peripheral nervous system along motor and sensory fibers and also within the cns, exemplified by the movement of polioviruses between spinal cord motor neurons and corresponding cortical neurons. the olfactory pathway is a unique venue for neural spread because the olfactory rods extend out from the olfactory mucosa and the central processes of the olfactory neurons synapse within the cns. thus, a single neuron can provide transport from the ambient environment to the cns. despite this direct connection, few aerosol or respiratory infections have been found to invade through the olfactory route. although early studies concluded that the blood-brain barrier was impenetrable by viruses, most viruses do invade the cns from the blood. some viruses infect cerebral endothelial cells or choroid plexus epithelial cells and 'replicate before spreading' into the cns. others cross uninfected vascular endothelial cells into the brain in endocytotic vesicles or within infected mononuclear cells (e.g., human immunodeficiency virus (hiv)). invasion from blood depends on the magnitude and duration of the viremia. viruses in the blood are promptly removed by the reticuloendothelial system and, similar to other particles, speed of clearance is size dependent. small viruses ( e.g., flavivirus such as the west nile virus) are removed more slowly, and many larger viruses avoid clearance by growth in leukocytes or adhesion to red cells. within the cns, specific cell populations may be vulnerable to specific viruses. for example, rabies virus infects only neurons, and early in infection neurons that modify behavior are selectively affected. this explains the unique clinical syndrome of vigilance and agitation. similarly, polioviruses selectively infect and destroy motor neurons causing the syndrome of flaccid paralysis. arboviruses infect primarily neurons, especially in the basal ganglia, and herpes simplex virus infects neurons, glia, and endothelial cells. jc virus selectively lyses oligodendrocytes causing demyelinating disease. enteroviruses and mumps virus infect primarily meningeal and ependymal cells; therefore, they usually cause benign meningitis and only rarely are associated with encephalitis. neuroinvasiveness, neurotropism, and neurovirulence are features of viruses that need to be differentiated. neuroinvasiveness is the ability to penetrate the cns. traditionally, neurotropism referred to the ability to infect neural cells other than neurons and is distinct from neuronotropism, which indicates the ability to infect neurons; however, in more recent literature, the term neurotropism is applied to both types of infection. neurovirulence relates to the ability to cause disease. thus, mumps virus that can often be recovered from spinal fluid during uncomplicated parotitis is highly neuroinvasive, is neurotropic in ependymal cells but not neuronotropic, and has a low level of neurovirulence. in contrast, most arbovirus infections are asymptomatic or cause only fever, malaise, and minor systemic symptoms. on rare occasions when arboviruses infect the brain, they usually cause encephalitis with a significant death rate; thus, these viruses are not highly neuroinvasive but are highly neurotropic (and neuronotropic) and neurovirulent. it must be noted that neuroinvasiveness and neurovirulence are also dependent on a myriad of host factors, such as age, immune responses, and genetic constitution. the incidence of acute encephalitis is estimated between 3.5 and 7.4 per 100 000 persons per year (which is approximately one-third the incidence of viral meningitis). encephalitis has been associated with approximately 100 different viruses that cause disease of variable severity ( table 3) . encephalitis is more common in children than in adults and varies by gender, season, and geographical location. for example, cns complications of mumps virus infections are three times more common in males than females. seasonally, enterovirus infections are more common in summer and early fall, lymphocytic choriomeningitis virus infections are more common in winter, and arbovirus infections are more common during the feeding season of the transmitting arthropod. there is marked geographical variation in the incidence and causes of viral encephalitis. for example, rabies causes more than 25 000 deaths per year in india but only 1-5 deaths in the us. each year a variety of arboviruses cause 200-2000 cases of encephalitis in the us, tickborne encephalitis virus causes a few thousand cases in europe, whereas japanese encephalitis virus causes approximately 68 000 cases in asia. clues to the causative agent may be found in a history of travel, family illnesses, recreational activities, sexual history, animal exposures, and past immunization. general examination may show a rash in enterovirus, herpesvirus 6 and 7, and west nile virus infections; herpangina in coxsackievirus infections; adenopathy in hiv, epstein-barr virus, and cytomegalovirus infections; parotitis in mumps or lymphocytic choriomeningitis virus infections; or pneumonitis in adenovirus, lymphocytic choriomeningitis, or nipah virus infections. the classic clinical symptoms and signs of encephalitis are fever, headache, nuchal rigidity, depression of consciousness, focal neurological signs, and often seizures. occasionally, fever is not present at the time of presentation, stupor may obscure a complaint of headache, or nuchal rigidity may be absent; thus, no finding is absolute. spinal fluid examination is critical for diagnosis. typically, a mononuclear cell pleocytosis, modest elevation of protein, normal glucose concentration, and often increased pressure are found. early in disease, polymorphonuclear cells may predominate or fluid may be acellular, but within 24 h a mononuclear cell response usually evolves. in some infections, mild depressions of glucose concentration are present. the presence of red cells may suggest herpetic encephalitis, but the finding is neither consistent nor specific. rabies and herpes simplex virus encephalitis usually present with characteristic syndromes. rabies may present as either an ascending paralysis simulating the guillian-barré syndrome or an altered mental status with periods of confusion with bizarre behavior and autonomic signs progressing to intense agitation with aerophobia and hydrophobia. acute flaccid paralysis can also be a feature of flavivirus encephalitides, such as west nile encephalitis, japanese encephalitis, and tickborne encephalitis. herpes simplex virus encephalitis usually localizes to one or both temporal lobes, which may manifest initially as subtle anomia, followed by bizarre behavior, hallucinations, aphasia, and quadratic visual field defects. imaging studies typically show lowdensity lesions in one or both temporal lobes with enhancement and mass effect. only approximately a half of patients with encephalitis with temporal lobe signs prove to have herpes simplex virus encephalitis. arboviral encephalitis, however, often causes thalamic lesions on imaging. more than 90% of patients with herpetic encephalitis have temporal lobe changes, but herpes simplex virus causes only 10% of cases of encephalitis (even fewer in some geographical regions) and other agents may occasionally mimic these symptoms and signs. diagnosis of herpes simplex virus encephalitis is important because it is the most common cause of fatal nonepidemic encephalitis worldwide, and there is specific treatment that can reduce the fatality rate approximately from 70% to 20%. the second diagnostic imperative is to rule out nonviral disease that can masquerade as viral encephalitis and that requires specific therapies (table 4) . establishing a definitive diagnosis can also have significant public health implications depending on the agent identified. diagnosis is dependent on identifying virus in spinal fluid or brain tissue or on serological tests that often require a convalescent serum too late to aid in acute management. in herpes simplex and other herpesvirus infections, polymerase chain reactions can identify specific viral dna in spinal fluid. in many arbovirus infections, igm-capture enzyme-linked immunoassays on spinal fluid can quickly identify specific viral infections. epidemiology historically, adem accounted for one-third of cases of acute encephalitis. vaccine practices have reduced or eliminated the major causes ( table 5) . cessation of vaccinia infections to prevent smallpox and institution of immunizations for measles, mumps, rubella, and varicella viruses have eliminated the majority of cases of adem. indeed, in nations with successful immunization policies, the majority of cases are now related to nonspecific upper respiratory infections. a history is often obtained that an exanthem or a nonspecific respiratory or gastrointestinal illness preceded the encephalitis symptoms by 5 days to 3 weeks. for example, postmeasles encephalomyelitis typically begins 4-8 days after the rash when the child has defervesced and is returning to normal activities. headache, fever, and depressed consciousness develop abruptly, often with focal neurological signs and seizures. postvaricella encephalitis is commonly limited to acute cerebellar ataxia. the spinal fluid may show a mild pleocytosis and protein elevation, but in one-third of patients these analyses remain normal. assays for myelin basic protein are often positive, consistent with the demyelinating nature of the disease. magnetic resonance imaging is the more definitive test, often showing multifocal white matter lesions with enhancement suggesting inflammatory lesions of similar age. in adem, virus isolation or serological studies are of little aid in diagnosis. a similar clinical-pathological syndrome follows immunization with rabies and japanese encephalitis vaccines prepared in neural tissues and, in the case of rabies vaccines, has been shown to be associated with an immune response to myelin basic protein. similar lymphoproliferative responses to myelin basic protein have been shown in postmeasles encephalomyelitis and in postvaricella cerebellar ataxia, indicating an analogous autoimmune mechanism. indeed, in adem after measles, the virus cannot be found in the brain by virus isolation, immunocytochemical staining, or in situ hybridization; in this disease, encephalitis occurs without evidence of neuroinvasion. specific drug treatments are available only for herpesvirus infections. the incidence of hiv-associated encephalitis has been reduced by antiviral therapy, but whether this represents only a postponement of cns disease is yet to be determined; recent data suggest that many people with hiv will progress to some form of neurocognitive deterioration. rabies is unique in that postexposure treatment with vaccine and immune globulin can reduce the hazard of disease, but once clinical symptoms have developed, no treatment is effective. vaccines can prevent measles, mumps, rubella, chickenpox, japanese encephalitis, and tickborne virus infections. an additional bonus of measles immunization has been elimination vast reduction in cases of subacute sclerosing panencephalitis. arbovirus encephalitis. central nervous system, overview. encephalitis/leukoencephalitis, acute hemorrhagic. encephalomyelitis, acute disseminated. herpesviruses, human. measles virus, neurological complications of. meningitis, viral acute encephalitis viral infections of the nervous system encephalitis and infectious encephalopathies viral encephalitis: a clinician's guide key: cord-010016-fs8pjy1z authors: webb, h. e.; fazakerley, j. k. title: can viral envelope glycolipids produce auto‐immunity, with reference to the cns and multiple sclerosis? date: 2008-05-12 journal: neuropathol appl neurobiol doi: 10.1111/j.1365-2990.1984.tb00335.x sha: doc_id: 10016 cord_uid: fs8pjy1z many viruses, with lipid envelopes derived from the host cell membranes, have been implicated in the aetiology of multiple sclerosis (ms), and epidemiological studies support an infectious agent. alternatively the disease is thought by other workers to be auto‐immune in nature, and recently much attention has been focused on immunological sensitivity to glycolipids in ms patients. in this paper it is proposed that cns demyelination could arise in susceptible individuals (hla type) from an immune response to glycolipids, triggered by the carrier effect of one or more enveloped neurotropic viruses. viruses have been thought to be involved in such diseases as multiple sclerosis (ms) for many years. the proof of this still escapes the medical profession. many different viruses including measles (adams et al., 1970; salmi et al., 1973; miyamoto et al., 1976 nakajima, 1973), herpes (catalano, 1972) , coronavirus (burks et al., 1979) , and canine distemper virus (cook, dowling & russell, 1979) have been implicated by the presence of anti-viral antibodies. several viruses have been isolated including herpes simplex (gudnadottir et al., 1964) and parainfluenza type 1 (ter meulen et al., 1972) . paramyxo-like virus inclusions have been seen (prineas, 1972; pathak & and canine distemper virus is implicated epidemiologically. the number of viruses implicated in ms has, if anything, confused the issue of a viral aetiology. the possible role of viruses in ms is well reviewed by ter meulen & stephenson (1983) . rogers et al. (1967) isolated several viruses from kuru infected chimpanzee brains. from such observations it has become quite clear over the years that viruses enter the brain easily, can remain there throughout life, and in many cases produce no disturbance. in any virus infection with a viraemia, virus from the cerebral capillaries can be transported easily across the basement membrane by pinocytosis and enter the brain parenchyma (pathak & webb, 1974) . neurotropic viruses enter the brain before there is any inflammation. the fact that these viruses cross the blood-brain barrier, whereas smaller particles such as ferritin do not, is of interest. pathak & webb (1974) suggested that this was because the virus in the blood represented virus which had replicated in the host, and so would have host membrane in its envelope and be considered as 'self'. the cells of the immune response, lymphocytes, plasma cells, macrophages, and occasional polymorphonuclear cells, enter later by diapedesis across the basement membrane and an inflammatory response develops. many of the neurotropic viruses are 'budding' viruses. they incorporate lipids from the membranes of the host cell into their envelope. on return to the peripheral circulation, such virions could present the cns glycolipids in their envelopes in an actively antigenic form to the immune system, and so trigger an immune response, resulting in cns auto-immunity. this mechanism would encompass both those of the virus and the auto-immune (experimental allergic encephalomyelitis, eae) models of cns demyelination, reconciling the two schools of thought in relation to such diseases as ms. to date most physico-chemical analyses of viral antigenicity have concentrated on viral proteins. some workers have also looked for proteins or glycoproteins of the host cell which may have become incorporated into the viral envelope, but these have proved to be negligible. viruses represent virally coded proteins in their envelope and do not make use of host coded membrane protein. glycolipids may be highly antigenic when incorporated into the envelope of budding viruses and by contrast these are host membrane glycolipids. many of the viruses, which at one time or another have been thought to have been involved in ms pathogenesis, are capable of multiplying in the cells of the cns, mature by budding through the cell membranes and take host cell glycolipids into their envelope. in the paramyxoviridae which includes measles, mumps, canine distemper, parainfluenza types 1 to 4 and sendai virus, release of mature virions takes place by budding and 2040% of the dry weight of the virion is lipid (nakajima & obara, 1967) . klenk & chopin (1969 , 1970 cultured para-influenza virus type sv5 in four different host cells with different lipid compositions, and determined that the lipid composition of the viral envelope closely resembled that of each of the host cell membranes from which it was derived. that the lipids of the viral envelope resemble those of the host cell from which the virus is derived has been demonstrated for many viruses including, mumps virus (soule, marinetti & morgan, 1959 ), influenza virus (kates et al., 1961 , blough & merlie, 1970 , sinbis virus (hirschberg & robbins, 1974; quigley, rifiin & reich, 1971 ), semliki forest virus (renkonen et al., 1971; laine et al., 1972) and venezuelan equine encephalomyelitis virus (heydrick, comer & wachter, 1971) . this has been shown by many workers. harboe, schoyen & bye-hansen (1966) showed that fowl plague and influenza virus grown in entodermal cells of the chick chorio-allantoic membrane could be inhibited by antibody prepared in rabbits against normal uninfected allantoic membrane. feinsod, spielman & swaner (1975) showed that sindbis virus which had replicated in aedes aegypti mosquitoes was neutralized by immune serum made against whole body extracts of uninfected a . aegypti. this serum did not neutralize sindbis virus grown in vero cells. steck, tschannen & schaefer (1981) showed that the 'neurotropic' strain of vaccinia virus when given to mice would produce an immune reaction, in which antibodies binding to normal uninfected myelin and oligodendrocytes were detectable. however, if the 'dermatotropic' strain of virus was used, no antibodies against cns tissue were produced. reilly & schloss (1971) demonstrated that friend leukaemia virus buds from erythrocyte membranes taking red blood cell membrane components into its envelope. cox & keast (1973) showed that such a virus could trigger a reaction leading to the haemolysis of normal uninfected erythrocytes. almeida & waterson (1969) using a corona virus which had grown in chicken fibroblasts, showed that immune sera made against this virus in chickens labelled only the viral protein spikes. if, however, the immune serum was made in rabbits, against the 'chicken fibroblast' derived virus, the resulting antiserum labelled both the viral protein spikes and the intermediate envelope lipids, and thus demonstrated the chicken host cell origin of the viral envelope. in addition the 'chick fibroblast' derived virus could be neutralized by immune serum made in rabbits against uninfected chick fibroblasts. rook & webb (1970) observed that lymphocytes of mice which were reactive against tick borne encephalitis virus (langat tp21) destroyed both langat tp21 infected, and to a lesser but significant extent, uninfected cultured mouse glial cells suggesting that, in the process of immunizing the donor mouse with langat tp21, cell mediated immunity to some component of normal brain membranes had been produced. semliki forest virus (sfv) is an alphavirus of the togaviridae, and is an enveloped virus that matures by budding from the cell membranes. the virus derives its envelope lipids from these membranes (renkonen et al., 1971; luukkonen, kaariainen & renkonen, 1976) . mice infected intra-peritoneally develop demyelination which is maximal between days 14 and 21 postinfection, after the immune response has cleared detectable virus from both blood and brain. the demyelination is focal and can occur throughout the cns (kelly et al., 1982) including the optic nerves and the spinal cord (illavia, webb & pathak, 1982; pathak, illavia & webb, 1983) . the demyelination is dependent upon t-lymphocytes probably cytotoxic cells (jagelman et al., 1978; fazakerley, amor & webb 1983; pathak et al., 1983) and probably results from an immune reaction against viral antigens on the surface of oligodendrocytes or myelin. oligodendrocytes do not appear to be destroyed during an avirulent sfv (a7) infection although an immune attack could change the cellular activity from myelin maintainance to that of cell repair, and by default allow degeneration of the myelin. good remyelination occurs by day 35 postinfection, although the myelin does not return completely to normal. zlotnik, grant & batter-hatton (1972) have shown chronic active gliosis with spongiform change in mouse brain 2 years after avirulent sf virus infection. a possible explanation is that brain-derived sfv returns to the peripheral circulation and initiates a n immune reaction against cns glycolipids which contributes to this long term pathological change. in support of this hypothesis, brain derived sfv has been shown to react significantly in an elisa against immune serum raised to galactocerebroside, gangliosides and particularly to glucocerebroside (webb et al., 1981) . in addition, the antiglucocerebroside serum coupled to either ferritin or protein-a gold, successfully labels sfv budding from brain cell cultures (n. evans, personal communication). the host cell plasma membranes are not labelled by this antiserum, which is appropriate since sfv grown in vitro buds extensively from the internal cell membranes (erlandson et al., 1967) and glucocerebroside is a marker of internal but not external membranes. although the alphaviruses, sindbis and semliki forest virus, are serologically unrelated to the flaviviruses, langat virus (tp21) and west nile virus, they both multiply well in cns cells (illavia & webb, 1968; precious, webb & bowen, 1976; herzberg, 1976) . since all are budding viruses they will have a similar host derived viral envelope provided the virus replicates in the same cell type. in our laboratory we have infected mice with the non-lethal encephalitogenic alphaviruses, sindbis or sfv a7(74), and then challenged these animals intracerebrally at weekly intervals for 7 weeks with the normally 100% lethal flavivirus, langat virus (tp21), or with west nile virus. up to and probably after 35 days following infection by either alphavirus there was a significant protection to flavivirus. seven days after langat or west nile virus challenge of the alphavirus infected mice, brain virus titres were significantly lower than in mice given flavivirus alone. it was felt that protection initially might be due to interferon release but none was measureable more than 5 days after the first alphavirus infection (oaten, bowen & webb, 1976; oaten, webb & jagelman, 1980) . at times after the second week it was considered that protection might be due to the first virus, the alphavirus, multiplying in the brain and taking brain cell membrane components into its envelope, which could be antigenic and stimulate humoral and cell mediated immunity. the second infecting virus, the flavivirus, by replicating i n similar cells may also have incorporated the same cell membrane components into its envelope, and thereby be partially neutralized by the previously induced immune response. cell membrane glycolipids are most likely to be involved in such crossreactivity. in tissue culture, anticerebroside antibodies have been shown to produce demyelination of myelinated axons (dubois-dalcq, niedieck & buyse, 1970; fry et al., 1974) . raine et al. (1981) tested the ability of antisera against whole white-matter myelin basic protein and galactocerebroside to demyelinate myelinated cultures of mouse spinal cord. the effects of the anti-whole white matter antibody and the antigalactocerebroside antibody were identical; both produced demyelination, whilst the anti-myelin basic' protein antibody had no effect. lumsden (1972) and leibowitz & gregson (1979) suggested that antibody to glycolipids might be present in patients with cns disease. nagai et al. (1976) reported that lesions of the cns and peripheral nervous system could be produced by immunizing rabbits and guinea-pigs with ganglioside gm, and gd,,. saida et al. (1979) produced an experimental allergic neuritis by immunization with galactocerebrosides. more recently konat et al. (1982) produced an experimental 'ms-like' disease in rabbits by immunizing them with bovine brain gangliosides. an immune response to glycolipids can thus result in demyelination. arnon et al. (1980) found that antibodies to glycolipids were present in 40% of ms patients' sera as tested by liposome lysis. antibodies to gm, and gm, gangliosides were present. offner, konat & sela (1981) showed that multisialogangliosides, particularly gt1 and g,,,, were powerful stimulators of active e-rosetting lymphocytes from ms patients. sela, konat & offner (1982) demonstrated the presence of elevated ganglioside levels in serum and peripheral blood lymphocytes from ms patients in remission compared with controls. ilyas & davison (1983) using an e-rosette assay showed hypersensitivity to gangliosides in ms patients. some response to myelin basic protein was also obtained, but this also occurred in patients with other cns disturbances. however, the reactivity to gangliosides appeared to be particularly specific to the ms patients. the ganglioside-stimulated e-rosettes could be inhibited by cyclosporin a (davison & ilyas, 1982), which blocks receptors for hla-dr antigens on tcells (palacios & moller, 1981) and prevents interleukin production. the tlymphocytes of ms patients thus appear to be sensitive to glycolipids. more attention needs to be paid to the antigenicity of cns glycolipids and in particular to the antigenicity of viral envelope glycolipids. it is an intriguing possibility that cns demyelination in diseases such as ms, arises as a result of an auto-immune reaction against specific glycolipids, induced by the carrier effect of a budding neurotropic virus. the presence of antibodies and reactive t-lymphocytes to glycolipids in ms patients, alternatively might only reflect the release of these components during cns damage. however, it is unlikely that glycolipids released in this way would produce an immune response as in most cases release of tissue components directly into the circulation does not provoke the production of auto-antibodies (roitt, 1980; allison, 1971) . for example, destruction of thyroid tissue by doses of therapeutic radio-iodine, does not initiate thyroid auto-immunity, nor does damage to the liver in alchoholic cirrhosis result in the production of mitochondria1 antibodies, as seen in auto-immune primary biliary cirrhosis. to initiate autoimmunity it is a prerequisite that the antigen is presented correctly to the immune system. thus, to initiate autoimmune thyroiditis in rabbits the thyroid antigens were inoculated in freund's adjuvant (rose & witebsky, 1968) . similarly to produce eae in rabbits with glycolipids the antigen was emulsified in freund's adjuvant (konat et al., 1982) . this probably provides the required immunological carrier effect. a carrier effect is essential when considering glycolipids, since these behave immunologically as haptens, (marcus & schwarting, 1976; rapport & graf, 1969) . theoretically, numerous neurotropic budding viruses could provide a carrier effect for cns glycolipid haptens, thus leading to an antiglycolipid immune response and demyelination in susceptible individuals (hla type). such an hypothesis for the pathogenesis of ms would encompass a host of eligible budding cns viruses (simplified in figure 1 ) and the many viruses implicated by epidemiology, serology, isolation or microscopy could all be involved. this may be reflected in the finding that antibodies isolated from different plaques within the same ms figure 1 . the possible role of recurrent infections of the cns in ms in genetically susceptible individuals (hla type). a neurotropic enveloped virus, for example, measles (l), enters the brain and replicates in the cells of the cns including, e.g. the oligodendrocytes. the envelope of the budding virus is derived from the lipids of the host cell membranes. glycolipids in the envelope of virions returning to the blood may be antigenic, in association with the viral proteins which may act as carrier determinants. glycolipid sensitized lymphocytes then enter the brain by diapedesis and attack either the myelin directly or the myelin supporting cells. this results in demyelination and clinical relapse. after some time suppressor t-cells are generated and control the reaction resulting in remission. at a later date a second, e.g. a coronavirus (2) or a third influenza virus (3), or a previous virus infection which has become latent and now re-activated, enters, replicates in the brain, and returns to the circulation, presenting the same brain specific glycolipid(s) in its envelope. the immune response is restimulated resulting in a second, third, fourth or fifth relapse. remission intervenes as the tsuppressor cells control the response after each restimulation by virus. i n this way any number of enveloped neurotropic viruses could be involved in initiating and restimulating a n autoimmune response to the same brain cell membrane specific glycolipid(s). semliki forest virus is included in the figure because it produces immune mediated demyelination in experimental infection of mice. the figure represents a simplified concept of the foregoing hypothesis. the argument could be applied to other organisms, e.g. mycoplasma pneumoniae, whose membrane constituents react with antibodies made against cerebrosides and indeed have been shown to react with antibodies produced i n the csf of multiple sclerosis patients. 0 oligodendrocytic lipid membrane; 0 measles; 0 corona; 0 influenza; 0 arbovirus sfv. n: nucleus of oligodendrocyte; t: t-lymphocyte; b: b-lymphocyte; a: axon. brain m a y be t w o different viruses (nordal, vandvik & norrby, 1978) . relapse and remission, as seen in ms, could be a function of new v i r u s infection (or re-activation of latent virus), and the activity of t-suppressor lymphocytes, directed against the virus induced antiglycolipid response. it is of relevance that a functional abnormality of virus induced t-suppressor lymphocytes has already been demonstrated in ms patients (neighbour & bloom, 1979 ). nile virus following pretreatment with sindbis, semliki forest and chikungunya virus multi-sialo brain gangliosides are powerful stimulators of active erosetting lymphocytes from multiple sclerosis patients cyclosporin-a blocks receptors for hla-dr antigens on t-cells possible mechanisms for the transport of semliki forest virus into and within mouse brain paramyxovirus-like inclusions in brain of patient with severe multiple sclerosis the identification and role of cells involved in c.n.s. demyelination in mice after semliki forest virus infection: a n ultrastructural study. immunology of nervous system effect of two defined strains of semliki forest virus on cultures of suckling mouse brain cells paramyxo-like particles associated with acute demyelination in chronic relapsing multiple sclerosis phospholipid composition of rous sarcoma virus, host cell membranes and other enveloped rna viruses immunochemical reaction of lipids demyelination in vitro the erythrocyte as virus carrier in friend and rauscher virus leukemias the lipid class composition of semliki forest virus and of plasma membranes of the host cells latent viruses in chimpanzees with experimental kuru key: cord-018034-gx5c9mk8 authors: nan title: cell and tissue reactions date: 2006 journal: forensic neuropathology and associated neurology doi: 10.1007/3-540-28995-x_4 sha: doc_id: 18034 cord_uid: gx5c9mk8 similar types of tissue reaction result as a final common pathway from a wide array of different internal brain pathophysiological states and external insults. since these cellular and tissue reactions are largely independent of the specific type of insults, they are, therefore, non-specific. the tissue reactions are to be differentiated according to their specific pathogenetic mechanisms, though these mechanisms as well as the phenomena are overlapping as demonstrated in fig. 4.1; brain ischemia as a type of metabolic disturbance, edema, intracranial pressure, necrosis, herniation and inflammation are influencing themselves and are dependent on each other. some will be mentioned again in later chapters as viewed from different forensic aspects; therefore, a certain redundancy is unavoidable. immediately following, we offer a survey of the individual types of reaction and their fundamental pathophysiological principles and morphology. similar types of tissue reaction result as a final common pathway from a wide array of different internal brain pathophysiological states and external insults. since these cellular and tissue reactions are largely independent of the specific type of insults, they are, therefore, non-specific. the tissue reactions are to be differentiated according to their specific pathogenetic mechanisms, though these mechanisms as well as the phenomena are overlapping as demonstrated in fig. 4 .1; brain ischemia as a type of metabolic disturbance, edema, intracranial pressure, necrosis, herniation and inflammation are influencing themselves and are dependent on each other. some will be mentioned again in later chapters as viewed from different forensic aspects; therefore, a certain redundancy is unavoidable. immediately following, we offer a survey of the individual types of reaction and their fundamental pathophysiological principles and morphology. if brain volume increases, both blood and csf are displaced until the intracranial pressure (icp) increases. the consequent pression of the brain against the inelastic dura mater (fig. 4 .2) and the skull can lead to a lethal series of complications in clinical neurology. the following remarks are based largely on ironside and pickard (2002) . brain volume depends on the following factors: 1. water content (cerebral hydration). the brain has a normal water content of about 75%. a disturbance of the blood−brain barrier (bbb) can lead to an increase in the fluid content, with a consequent increase in brain volume. 2. intracranial blood volume (hirai et al. 1986 ). this can be driven upward by a number of factors: arterial hypertension (marshall et al. 1969 ); enhanced cerebral blood flow secondary to elevated cerebral perfusion pressure (artru et al. 1976 ); a decline in the cerebrovascular resistance of arterioles, capillaries, and postcapillary vessels (langfitt et al. 1965) due to hypercapnia, hypoxemia associated with severe elevation of arterial pressure (marmarou et al. 1997) , or due to obstruction of the venous outflow of the brain. elevated cerebral blood volume, also known as "brain swelling," is a congestive process. 3. cerebrospinal fluid (csf) pressure. the central nervous system (cns) of the average adult contains a csf volume of approximately 120−140 ml. among the causes of a rise in csf pressure is acute obstructive high pressure hydrocephalus (see pp. 54 f). a number of additional factors may also influence brain volume. the congested brain expands particularly rapidly under high arterial pressure (leech and miller 1974) . nawashiro et al. (1995) used experimental closed brain injury in rats to demonstrate a rapid and widespread increase in regional cerebral blood flow and impaired cerebral autoregulation. in humans a variety of factors act in concert after the incidence of severe brain injury. cerebral computed tomography (cct) and magnetic resonance imaging (mri) studies have shown that brain edema is the major fluid component of brain swelling (marmarou et al. 1997 (marmarou et al. , 2000 . a reactive hyperemia is an additional factor and may be the mechanism underlying mechanical/ischemic brain injury (seida et al. 1989 ). moreover, regional cerebral ischemia additionally is attributed to a compromised, leaky microvasculature rather than to vasospasm of larger vessels (schröder et al. 1998 ). the conclusion that brain swelling is due primarily to edema and not congestion of blood appears to be valid also for children (see chap. 20, pp. 415 f) . cerebral blood flow in children with severe head injuries is not substantially increased over that in uninjured children (zwienenberg and muizelaar 1999) . this has also been demonstrated following experimental generation of brain trauma in newborn and juvenile pigs (armstead and kurth 1994) . the experimental findings of biagas and coworkers (1996) , in contrast, demonstrated a delayed rise in cerebral blood flow following experimental contusion in young and adult, but not in elderly, rats. normal adult icp is less than 2 kpa (1 kpa=7.5 mm hg=7.5 torr), mild elevations in pressure range from 2 kpa to 3 kpa, moderate elevations attain 4 kpa, while major intracranial hypertension exceeds 5 kpa (miller and ironside 1997) . normal csf pressure in adults is 0−1.3 kpa, with an upper limit of 2 kpa. while a short-term rise in icp pressure of up to 10 kpa may be tolerated so long as it does not cause distortion of the brain (johnston and paterson 1974) , a mbi-induced rise of more than 8 kpa with distortion of the brain can result in herniation and brain death syndrome. the classic symptoms associated with elevated icp are vomiting, headache, papilledema, and coma. schematic demonstration of overlapping pathogenetic mechanisms that are associated with different types of tissue reactions by a causal link: metabolic disturbance, i.e., edema, increasing cerebral blood volume, perfusion pressure and herniation, i.e., brain swelling and cortical necrosis distortion or pressure on the floor of the fourth ventricle are most likely responsible for the vomiting, while stretching and distortion of the dura mater and major intracranial blood vessels, all sensitive to pain, probably account for the headache. the papilledema is not a direct result of the rise in water content, but the consequence of an accumulation of axoplasm in the optic papilla secondary to the blockade of axoplasmic flow from ganglion cells along the optic nerve. papilledema is a common symptom of chronic intracranial hypertension. it is not, however, a common feature of mbi (selhorst et al. 1985) , and its absence does not necessarily mean that icp is normal. a further increase of icp leads to a loss of consciousness and coma. elevated icp affects other organ systems as well. it often induces arterial hypertension and systolic blood pressure may climb to 40 kpa or more. the arterial hypertension is caused by an increase in sympathetic activity. cases of raised icp with myocardial involvement often exhibit pathological alterations consisting of subendocardial hemorrhage and widespread focal myocardial necrosis as well as electrocardiographic changes such as t-wave inversion and elevation of the st−segment, which point to myocardial ischemia (connor 1968) . respiratory disturbances associated with elevated icp often precede apnea. central neurogenic hyperventilation is observed in connection with the midbrain lesion. in patients with raised icp, neurogenic pulmonary edema can complicate the clinical course. the mucosa of the digestive and urogenital tracts can become hemorrhagic, eroded, and ulcerated, gastric erosions being particularly common in comatose patients with elevated icp. a fundamental distinction must be made between global and focal cerebral edema. the former follows acute systemic hypoxic events, e.g., transitory cardiac arrest or chronic hypoxia in respiratory diseases. global cerebral edema may also be associated with metabolic diseases, intoxications, and inflammation. focal cerebral edema results from focal tissue destruction or alteration of brain tissue that has undergone membrane failure in cells and vessels due to infarction or traumatic hemorrhage or tumor. the tissue surrounding the central lesion has passed only the upper threshold of electrical silence and thus retains the capacity to recover if perfusion is restored in time (harding and copp 1997) . this zone resembles the penumbra surrounding the moon in full eclipse (astrup et al. 1981) . because these tissue changes are partly reversible, they are of considerable therapeutic interest (see pp. 63 f). information on the incidence of intracranial hypertension has been gained mainly from survivors of mbi. miller and his associates (1977) reported that icp levels exceeded 2.7 kpa for 5 min or longer in 44% of 160 patients in one series and in 53% of another series of 215 patients (miller et al. 1981) . raised icp was found in more than 70% of cases in a more detailed prospective study of elevated icp in victims with severe head injury by marmarou and colleagues (1991) . jones and colleagues (1994) found elevated icp in more than 80% of 74 brain injury patients (54 severe, 17 moderate and 3 minor) undergoing artificial ventilation with icp monitoring. these findings indicate that intracranial hypertension is a common event, especially in comatose patients. certain features detected by cct are consistently associated with elevated icp. loss of the images of the third ventricle and perimesencephalic csf cisterns, and dilatation of the lateral ventricle contralateral to a mass lesion, as well as the absence of these features are no guarantee that icp will remain normal (teasdale et al. 1984; o'sullivan et al. 1994) . elevated icp is also common in patients who are comatose from causes other than brain injury (chan-dler and kindt 1976) . among the possible causes are liver failure (hepatic coma), intracranial hemorrhage (subarachnoid and intracerebral), post-hypoxemic states (cardiorespiratory arrest, near drowning), infection (meningitis, abscess, and encephalitis), as well as various other types of inflammation and intoxication. adams and graham (1976) published neuropathological criteria for determining at autopsy whether icp in victims of brain injury was elevated during life. the same team (graham et al. 1987 ) compared the nature of the brain damage in patients with and without elevated icp after suffering a non-missile brain injury who had survived long enough to receive treatment in a neurosurgical unit. pressure necrosis of the parahippocampal gyrus, an indicator of high supratentorial icp and tentorial herniation, was present in twothirds of the 434 patients in their most recent study. it was closely associated with skull fracture, brain swelling, diffuse axonal injury, hypoxic brain damage, and extensive supratentorial hematoma. the brain stem was damaged in 68% of victims with pressure necrosis, the anterior lobe of pituitary was necrotic in 45%, and there was hemorrhagic infarction in the distribution of the posterior cerebral artery in 36%. klatzo (1967) distinguishes two types of edema: vasogenic edema and cytotoxic edema (table 4 .1). this distinction continues to be of both theoretical and practical value (cf. kimelberg 1995a, b; mendelow et al. 2000) . blood vessels in the cns are uniquely restricted in their permeability. many substances are exchanged between the blood and brain parenchyma at slower rates and the concentrations in cns at steady state are lower than in other organs (lee 1982) . dyes as well as proteins, drugs, and microorganisms introduced into the csf enter the brain freely, while those introduced into the blood stream do not. this limited permeability is attributed to the bbb, a specialized feature of the cns that restricts the entry of viruses and bacteria, emigration of immune cells, and diffusion in the cns of drugs and soluble molecules from the systemic compartment. intravenously applied evans blue will bind to serum albumin and permeate the bbb only under pathological conditions. this phenomenon is demonstrated after experimentally induced ischemia of one hemisphere by means of macroscopic observation ( fig. 4.3a) and by means of fluorescence microscopy ( fig. 4 .3b). the demonstration of albumin in human brain using immunohistochemistry will only succeed during the very early postmortem interval ( fig. 4 .3d) − before a general diffusion of blood serum occurs within the brain parenchyma − as a morphological marker of the diffuse postmortem bbb disturbance. under experimental conditions an accumulation of plasma proteins in purkinje cells gencic 1980a, ikegaya et al. 2004) takes place. anatomically the bbb consists of a capillary endothelium containing intercellular tight junctions and specialized enzymes, such as transpeptidases, dehydrogenases, decarboxylases, and monoamine oxidases (reese and karnowsky 1967; brightman and reese 1969; lee 1982) . the intercellular junctions are most conspicuous near the luminal surface where the cell membranes fuse. a basement membrane in contact with astrocytic foot processes surrounds the endothelial cells. pericytes are enclosed by an envelope of the perivascular basement membrane, which splits to enclose the pericyte. brain capillaries are almost totally invested by astrocytic processes. astrocytes exert inductive actions during development and are thereby largely responsible for the special attributes exhibited by endothelial cells, such as the presence of tight junctions between the cells (abbott et al. 1992) . astrocytes and microglia both contribute to the formation of the bbb (prat et al. 2001) . there is an inverse hemodynamic relation between icp and cerebral blood flow (cbf): the higher the icp, the lower the cbf. if cerebral circulation and circulatory autoregulation are normal, a drop in icp will induce only a slight increase in cbf until a threshold level of about 8 kpa is attained. cbf is regulated by mechanisms such as compensatory dilatation of small arteries and arterioles. patients suffering from acute mbi, intracranial hemorrhage, or hypoxic brain injury need a mean arterial blood pressure above 8 kpa to maintain perfusion. the brain damage in such circumstances is associated with a rise in cerebrovascular resistance due to the vessels' spastic reactivity. baseline icp levels may even need to be higher in order to drive sufficient blood through the brain tissue (chan et al. 1992) . should cbf drop below 10 (ml/min)/100 g, potassium levels in the intercellular spaces rise, while intracellular sodium and calcium increase. cellular edema causes the cells to swell and a calcium influx triggers a series of autodestructive processes. the bbb can be compromised by the following three mechanisms (see miller and ironside 1997) : 1. enhanced vesicular transport and creation of transendothelial channels by perturbation of endothelial plasmalemma, increased pinocytic activity, the activity of free oxygen radicals, or an increase in superoxides. subarachnoid application of hemoglobin and hemoglobin degradation are known to cause brain edema (huang et al. 2002) . 2. disconnection of the interendothelial tight junctions, e.g., by substances of very high osmolarity. 3. structural or biochemical modification of the endothelial membrane that intensifies its permeability. it has also been known for a long time that the ability of certain substances to pass through the bbb depends on their specific properties: ▬ their nature regarding the capacity of the bbb for active transport (broman and steinwall 1967) ▬ their affinity for carrier molecules (lajtha 1968 ). ▬ their molecular radius (thompson 1970) . ▬ their lipid solubility (oldendorf 1977) . as shown in detail later, the permeability of the bbb also depends on age. a good example of this is bilirubin encephalopathy, which is caused by bilirubin crossing the bbb of certain nuclei during the perinatal period − a feat it is incapable of in later life − and inflicting damage on nerve cells and, to a lesser vasogenic perifocal edema in a case of a traffic accident associated with liver failure and elevated bilirubin level. a massive intracerebral hemorrhages and green-colored edema; b focal hemorrhage; perifocal as well as contralateral greencolored edema degree, on astrocytes (for further information see pp. 452 ff). thus for bilirubin at least the bbb appears to be less efficient at birth than in children or adults (haymaker et al. 1961) . in mbi with intracerebral hemorrhages and associated elevation of the bilirubin level the perifocal edema can be marked by a green color demonstrating extravascular bilirubin as demonstrated in fig. 4 .4. in senile and mentally disturbed patients, the bbb has been found to have a lower rate of transport, a reduced uptake of glucose and other nutrients, plus a diminished outflow of metabolic wastes (quadbeck 1967 (quadbeck , 1968 . the movement of water from the vascular compartment to intracellular or interstitial spaces is not regulated biochemically by the bbb since hydrostatic and more powerful osmotic gradients enable free water to diffuse passively across capillary membranes. the passage of ions and molecules of various sizes is controlled by lipid-soluble substances in the endothelial wall and by ionic channels and active pumps. the white matter of the brain is 68% water, the gray matter 80% (adachi and feigin 1966) . a rise in brain water content entails an increase in brain volume, i.e., brain edema. as a result of energy failure (deprivation of oxygen and glucose), which disables the sodium-potassium membrane atpase pump system, water accumulation within the cells follows an osmotically obliged response to an increase in intracellular sodium and loss of potassium ( fig. 4.3c) . the influx of osmotically drawn water causes swelling of the cell. the energy failure is accompanied by an influx of sodium and chloride and an efflux of hydrogen ions, potassium, and bicarbonate. there is a parallel disturbance of the voltage-and ligand-gated mechanisms that regulate the entry of calcium into the cell that initiates a calcium-mediated destructive sequence. cytotoxic edema is the result of the action of various cytotoxic agents, e.g., of cyanide or triethyl tin (also see chaps. 16, 17). brain cells can also swell without a concomitant increase in brain volume if fluid shifts from an extracellular to an intracellular space. although this does not produce an immediate rise in icp, cellular edema ultimately draws water from the vasculature into the brain, increasing brain volume and precipitating a rise in icp. ischemic edema is a cytotoxic edema whose clinical effects depend on how much and for how long cerebral blood flow is reduced (bell et al. 1985) . klatzo (1967) showed that the initial cytotoxic edema following permanent occlusion of a major blood vessel causes irreversible ischemic cell damage, resulting in a secondary vasogenic edema when endothelial cells are damaged. even temporary ischemia with subsequent reperfusion will induce reactive hyperemia and secondary endothelial damage that produces a secondary vasogenic edema (greenwood 1991) . as a whole, the brain is resistant to pure hypoxia (diminished oxygen supply) (pp. 276 ff, see also miyamoto and auer 2000) , which causes no or only partial breakdown of the bbb and which may be reversible after recovery (bakay and lee 1968; auer 2000) . anoxia (complete lack of oxygen), by contrast, results in a rapid rise in bbb permeability that becomes irreversible after just a few minutes (pp. 280 ff). if anoxia acts in combination with complete ischemia secondary to ligation of both common carotid and subclavian arteries, the bbb can retain its impermeability for as long as 3 h (broman 1949; blank and kirshner 1977) . incomplete ischemia, however, will rapidly and completely break down the bbb (bakay and lee 1965) . kimelberg and colleagues (1997) could demonstrate the potential toxic mechanisms of this type of edema on neurons. they describe a primary cytotoxic effect on astrocytes that induces astrocytic swelling. this swelling in turn leads to the release of excitatory amino acids such as glutamate, whose levels increase in extracellular spaces following the incidence of mbi (kanthan and shuaib 1995) . a rise in extracellular glutamate levels causes cell death due to an influx of ca 2+ via the neurons' activated ionotropic glutamate receptors (choi and rothman 1990) . other authors have offered a somewhat different explanation for the irreversible injury: it may be induced by simultaneously generated free radicals or extravasated plasma components that stimulate the activation of nitric oxide synthase (nos) in reactive cells. nitric oxide thus generated may contribute to diffuse degeneration of the white matter (gotoh et al. 1998 ). the accumulation of plasma proteins within neurons and microglia in combination with cytochrome-c release by astrocytes can lead to dna fragmentation and cell death (matz et al. 2001 ). klatzo's (1967) classification of brain edema has proved to be a useful aid in distinguishing between various pathogenetic mechanisms and their sequelae. in experimental and clinical practice however it must be assumed that brain swelling is caused by a combination and/or a temporal overlapping of a number of processes, as described by kimelberg et al. (1997) . non-invasive diffusion-weighted (dw) mri is able of calculating changes in the apparent diffusion coefficient (adc) of water protons in the brain (garcia et al. 1995; chu et al. 2001) . a decline in adc has been attributed mainly to a reduction of the extracellular space and a rise in intracellular volume, although other contributing factors are possible (pierpaoli et al. 1996) . in this manner cellular (cytotoxic) edema can be differentiated from extracellular (vasogenic) edema and a correlation made with the severity of injury and consequent deficit. because dw-mri (see also mendelow et al. 2000) enables edema types to be determined intra vitam under clinical conditions, current classifications of edema types could be revised in light of new findings. however, we should remember that all edema ultimately arises from the blood. it is the size of the leak in the brain vasculature that gives rise to the artificial distinction between cytotoxic and vasogenic edema, cytotoxic edema being mainly water and vasogenic edema including proteins also derived from the blood. brain swelling caused by edema, congestion or a rise in csf pressure can obliterate the subarachnoid space, flatten the gyri, reduce ventricular size (squier 1993 , fig. 4 .2), and cause herniation (see pp. 51 ff). at autopsy the white matter seems softer in consistency and paler than normal. the normal, sharp demarcation between gray and white matter is lost, often with thinning of the cortex overlying the zone of white matter edema. the arcuate fibers are spared. in rare cases of vasogenic edema associated with liver insufficiency combined with elevated bilirubin levels in serum the spread of edema may be characterized by a greenish-yellow color ( fig. 4.4) . under normal conditions bilirubin is not able to permeate the bbb, with one exception: the newborn (see pp. 452 ff). cytotoxic edema, which predominates in gray matter, is characterized by astrocytic swelling and the enlargement of perineuronal and perivascular spaces indicative of the swelling of astrocytic foot processes around neurons, capillaries, and arterioles ( fig. 4 .5). the hallmarks of vasogenic edema include swelling of pericapillary astrocytic processes and of oligodendrocyte cytoplasm, plus the spread of exudate in the extracellular space of white matter ( fig. 4.6 ). macroscopically vasogenic edema induces a slight green discoloration of the white matter. histologically, edema, vasogenic edema in particular, features extensive cytoplasmic vacuolation in the white matter with status spongiosus where a clear space surrounds small vessels and nuclei ( fig. 4.6) . ultrastructurally, few visible changes are evident in the cerebral capillaries. the brain parenchyma, in contrast, exhibits swelling of glial processes or dendrites, splits in the myelin laminae and, less often, enlargement of the extracellular space. vacuolation may be especially prominent in myelinated fiber bundles and constitute the earliest and most consistent elementary edema-induced change. following immersion fixation, however, these phenomena can often be difficult to distinguish from (postmortem) artifacts. these phenomena may be associated in the beginning with a leukocyte emigration ( fig. 4 .7a) and in the last stage with astrocytic hyperplasia and hypertrophy ( fig. 4 .7c, d). astrocytes and macrophages also ingest extravasated plasma proteins. myelin sheaths undergo increasing fragmentation and macrophages phagocytose lipid breakdown (figs. 3.8f, 9.15b, 9.16) . oligodendrocytes are much less likely to partake in the alterations of edematous brain tissue. most cases of brain edema exhibit a combination of cytotoxic and vasogenic edema. inhibition of ion pumping or secondary retrograde reaction can cause swelling of neurons. the usual reaction is neuronal shrinkage, commonly combined with swelling of neighboring glial cells, especially of astrocytic processes. irreversible changes in the myelin sheath are unequivocally manifested by the apposition of mac-rophage reaction in the form of compound granular cells. involvement of the white matter by edema of this severe degree coincides with a so-called edematous necrosis (jacob 1947) . the final phase of terminal edema can be marked by cystic alteration or glial scaring. brain swelling is one of a wide variety of neurological conditions, among them tumors, hemorrhages, and ischemia/hypoxia, that can induce an increase in icp. a rise in icp leads not only to compression of the brain, but to diminished csf volume, shifting, and herniation, as well as to secondary complications such as ischemia and hemorrhage. if not treated, icp can rapidly progress to death due to brain stem compression secondary to cerebellar or uncal herniation (meyer 1920) . focal expanding mass lesions must be distinguished from diffuse space-occupying processes. ▬ diffuse brain lesions such as inflammation, bilateral intracranial hemorrhage, total brain ischemia (cardiac arrest) or intoxication are macroscopically characterized ( fig. 4 .2) by a tension of the dura, gyral flattening, and by narrow ventricles that are symmetrically compressed. no lateral shift of midline structures is seen, but rather central herniation of the diencephalon (centrencephalic herniation) and by cerebellar tonsillar herniation and compression of the medulla oblongata. bilateral herniation may occur and various types of herniation result from caudal displacement of the brain parenchyma (for types of herniation, see below). ▬ focal intracranial processes such as abscess, tumor, infarction or subdural hemorrhage ( fig. 4 .8a, b) are also capable of inducing a life-threatening homolateral rise in icp. because they allow time for intrinsic compensatory mechanisms to operate, particularly reduced csf volume, slowly expanding focal lesions are less likely to cause an early increase in icp and brain shift. however, the distortion and herniation of the brain in such cases can be considerable. rapidly expanding focal lesions, by contrast, are more likely to produce an immediately elevated icp. brain death often supervenes in such cases before much distortion or herniation can occur. distortion of the brain results from compressive forces exerted by adjacent structures, which leads to overall expansion of the hemispheres. the dura mater may become so tense as to compress the terminal branches of the cerebral arteries, with consequent ischemic or hemorrhagic necrosis of cortical structures (lindenberg 1955) or impairment of perfusion (janzer and friede 1979) accompanied by perisulcal infarcts. continued expansion of the mass may provoke contralateral displacement of the midline structures (see chap. 7 and fig. 4 .8a). if the contralateral foramen of monro is obliterated, the contralateral lateral ventricle may become enlarged, triggering a further rise in icp. a lesion that expands in the frontal lobe may displace the free margin of the anterior part of the falx cerebri ( fig. 4 .8a, d). if a lesion expands in the temporal lobe, a disproportionately pronounced shift of the third ventricle will occur ( fig. 4 .8a), with upward displacement of the sylvian fissure and neighboring branches of the middle cerebral artery. the ultimate result of the space-occupying process is development of lateral and then downward herniation, visible at several loci: 1. falx cerebri (cingulate, or subfalcine herniation). 2. tentorium cerebelli (lateral, or uncal herniation). 3. thalamus/hypothalamus (central, or diencephalic herniation) which may result in downward displacement and hemorrhage in the midbrain and pontine tegmentum. 4. foramen magnum (tonsillar herniation). a bilateral expanding supratentorial mass can cause herniation-induced notches as well as hemorrhages of the uncal area. this in turn exerts downward pressure on the medial part of the parahippocampal gyrus toward and through the tentorial incisura (figs. 4.8b, 4.9). the clinical and morphological sed a hemorrhage in the upper frontal lobe may lead to a notch, a hemorrhage or a softening of the corpus callosum; e a rare tentorial displacement caused by an infratentorial space-occupying process is demonstrated by notches on the upper cerebellar surface (arrows) quelae of uncal herniation depend on the magnitude of the supratentorial pressure and on anatomical variations in the size of the tentorial notch, position of the brain stem within the notch, position of the oculomotor or third nerve, and the inter-oculomotor nerve angle. they also depend on the structure and course of the posterior cerebral artery, known to play a role in herniation syndromes (adler and milhorat 2002) . herniation of the parahippocampal gyrus ( fig. 4 .9) creates narrowing of the midbrain along its transverse axis and compression of the aqueduct. this pushes − in the case of a unilateral expanding mass − the contralateral cerebral peduncle against the opposite free tentorial edge ( fig. 4 .8b), pinning the ipsilateral oculomotor nerve between the petroclinoid ligament or free edge of the tentorium and the posterior cerebral artery. the lesion of the ipsilateral oculomotor nerve is associated clinically with ptosis and dilatation of the ipsilateral pupil, which becomes unresponsive to light. the elevated icp produces a wedge of hemorrhagic necrosis along the parahippocampal gyrus groove (so-called pressure necrosis, to be differentiated from "herniation contusion" − see figs. 4.8b, 4.9b) . pressure necrosis can result from an icp exceeding 5.4 kpa (see adams and graham 1976) . it is analogous to necrosis due to brain retractor pressure in neurosurgery. clinically, uncal herniation is accompanied by an abrupt worsening of the patient's neurological status, with loss of consciousness and onset of decerebrate rigidity, both due to midbrain impairment caused by pressure coming from above. compression of arteries can also cause secondary necrosis: if the anterior choroidal artery is occluded, the result can be infarction of the medial part of the globus pallidus; posterior cerebral artery occlusion can cause hemorrhagic infarction of the thalamus, of the medial and inferior surfaces of the cortex of the occipital lobe (figs. 4.10, 9.24) , and of the temporal lobe including the hippocampus. herniation of the ipsilateral cingulate gyrus under the free edge of the falx results from the unilateral growth of a mass in the frontal or parietal lobe and causes selective displacement of the pericallosal arteries away from the midline (fig. 4.8a, d) . should this compromise circulation through the pericallosal arteries, the parietal parasagittal cortex can become infarcted, which is expressed clinically as weakness or sensory loss in one or both legs. a frontal or parietal mass lesion can induce downward axial displacement of the diencephalon (fig. 4 .8a) and rostral brain stem (figs. 4.8c, 4.11a) . the consequent symmetrical herniation of both parahippocampal gyri (fig. 4.9a , b) may be manifested clinically by bilateral ptosis and failure of upward gaze. the final clinical result is loss of consciousness, decerebrate rigidity, and bilateral dilatation of the pupils with loss of the pupillary light reflex. the blood pressure rises due to increased sympathetic activity. hemorrhage or necrosis of the midbrain and/ or pons are the possible sequelae of supratentorial space-occupying processes located adjacent to the midline (figs. 4.8a, 4.11a, 9.23 ). these lesions are caused by caudal displacement and anterior-posterior elongation of the rostral brain stem and by sideto-side compression by the tentorial hernia, coupled with relative immobility of the basilar artery. progressive displacement stretches and narrows the central perforating branches of the basilar artery which supply the rostral brain stem, causing spasm, infarction or hemorrhage. an early complication of expanding masses in the posterior cranial fossa is displacement of the cerebellar tonsils through the foramen magnum (figs. 4.2d, 4.11b, c) . this may also be caused, however, by lesions occupying the supratentorial space. morphologically, the tips of the tonsils display hemorrhagic necrosis and grooving of the ventral surface of the medulla where it impinges on the anterior border of the foramen magnum. clinically these changes give rise to apnea, which can occur while the victim is still conscious. among the other common neurological deficits are decerebrate rigidity and impairment of brain stem reflexes. upward tentorial displacement (fig. 4 .8e) is produced by enlargement of an infratentorial mass in the posterior cranial fossa. both the fourth ventricle and aqueduct become compressed and displaced contralaterally. there is upward herniation of the superior surface of the cerebellum, which is distinguished clinically by the abrupt manifestation of bilateral extensor rigidity and loss of pupillary light response. a number of clinical complications associated with brain swelling, brain edema, and bbb can arise during diagnostic and therapeutic interventions in the cns carried out by physicians or nursing personnel. since the sequelae are foreseeable − and in most cases avoidable − these complications will be dealt with briefly in the following. ▬ in patients with elevated icp, a lumbar puncture of the csf can give rise to herniation. papilledema, though not always associated with icp, must be excluded before every csf puncture. should other clinical signs of high icp be present, a ct examination must be carried out prior to puncture. ▬ pharmacotherapy must not be performed without knowing whether the agent can permeate the bbb and affect the cns. this is especially true of substances such as antibiotics or cytostatics that are intended to reach and act upon the cns. other substances are not intended to reach the cns because they are toxic there; thus, the contraindication for intrathecal application of vincristine (see p. 359). ▬ if cns edema already exists, the bbb may be (pathologically) permeable to substances not intended to reach the cns, some of which can then have a toxic effect. a mbi-induced perifocal edema, for example that arises in the context of polytrauma-induced shock, can produce greenish discoloration of the perifocal edema as a consequence of an accompanying hepatic insufficiency, which can have an additional neurotoxicologic effect on the neurons. ▬ the status of the bbb may well be age dependent, its postnatal status differing from that of adults. blood group incompatibility between mother and child during pregnancy or after birth can cause bilirubin encephalopathy (see pp. 452 ff). the bbb appears to be less permeable in the elderly. ▬ a cytotoxic effect can be mediated by alcohol in mbis with consequent loss of neurons. alcohol lowers cerebral perfusion pressure (cbf) and depresses ventilation. it diminishes respiratory drive in response to elevated paco 2 levels. ethanol-induced respiratory depression and hypotension can increase the morbidity and mortality associated with brain injury. the theoretical considerations of kimelberg et al. (1997) appear to contradict these empirical findings, arguing rather that alcohol inhibits both the excitotoxin receptor function of neurons (simson et al. 1991) and the influx of ca 2+ via nmda receptor ion channels. hydrocephalus is characterized by abnormal accumulation of fluid within csf spaces, i.e., within the cerebral ventricles and subarachnoid space. by this time, there is atrophy of the brain parenchyma and additional ventricular enlargement. csf is formed by the choroid plexus at a rate that remains unchanged within a wide range of icp values: 15−25 ml/h will avert a long-lasting imbalance between its formation and absorption. elevated csf pressure is associated only with acute or obstructive hydrocephalus (see below). the subarachnoid space and ventricular system are connected via the foramina of luschka and magendie in the basal cisterns. csf is absorbed by the arachnoid villi, which do not fully develop until adolescence or young adulthood (grassman and potts 1974) . in fetuses and infants, csf is absorbed mainly through nerve roots and periventricular and arachnoid veins. external hydrocephalus ex vacuo involves diffuse loss of gray matter that gives rise to external atrophy, with dilatation of the subarachnoid space ( fig. 31.6a , b). diffuse loss of white matter can cause expansion of the ventricular system, the so-called internal hydrocephalus ex vacuo (fig. 31.6c ). the causes of the gray and white tissue damage may vary, but csf kinetics and anatomic pathways are important considerations (see also pp. 482 f). a distinction is made between normal pressure hydrocephalus of still unknown etiology (adams et al. 1965; hurley et al. 1999) , low pressure hydrocephalus, and high pressure hydrocephalus. the latter is caused by accumulation of fluid secondary to elevated csf production (hypersecretory hydrocephalus) or insufficient resorption (malabsorptive hydrocephalus or communicating hydrocephalus). disten-sion of the ventricles results from pressure-induced fluid build up in the cerebral ventricles (reversible) or from pressure-induced (irreversible) atrophy as a result of parenchymal loss. fluid may also enter the periventricular tissue (trans-ependymal resorption of csf seen on neuroimaging). normal pressure hydrocephalus can feature repeated brief episodes of elevated icp, possibly in the form of an intermittent pressure or occult hydrocephalus. normal pressure hydrocephalus can also result from a hydrocephalus ex vacuo, which is associated with primary ventricular system enlargement and white matter destruction. it is often found in victims of severe brain injury, in alcoholics, and vascular disease patients with multi-infarct dementia or other types of progressive degenerative brain disorders, especially age-dependent dementia (miller and ironside 1997) . hydrocephalus ex vacuo can also be caused by long-lasting generalized brain edema with high icp. the causes of obstructive hydrocephalus are mbi, subarachnoid hemorrhage, meningitis and arachnoid fibrosis, arnold-chiari malformation, aqueductal stenosis (for complete list see table 4 .2, for review see garton and piatt 2004) . the latter can be either congenital due to atresia or acquired due to inflammation, compression or reactive gliosis, hemorrhages or tumor. the responsible congenital abnormalities consist in most cases of replacement of the aqueduct lumen by numerous random, narrow channels or ependymal nests. external hydrocephalus, whatever the causes are, exhibits dilation of the subarachnoid space, with no increase in collagenous fibers or cellular elements, but an increase in csf. the causative encephaloclastic disease may be diagnosed on the basis of other phenomena, such as liver cirrhosis in alcoholics, or loss of neurons in the presence of plaques and tangles in senile dementia or alzheimer's disease. typical macroscopic features of internal hydrocephalus include an enlarged ventricular system ( fig. 4.12a, b) (weller and shulman 1972) , interstitial edema, disruption of the ependymal cells lining the ventricle, and axonal and myelin destruction in the periventricular white matter (del bigio 2004) . secondary changes in neurons reflect compensation to the stress or ultimately the disconnection. proliferating astrocytes and/or gliosis (fig. 4.12c) replace in part the interrupted ependymal cell line. these glial nodules appear granular or like small tumors (fig. 4.12c ) upon macroscopic inspection of the inner surface of the ventricles (fig. 4.12b) . there is also a partial reestablishment of flattened ependymal cells, and a decline in the number of axons with parallel proliferation of glial fibers in the periventricular white matter. in chronic hydrocephalus with high pressure hydrocephalus, a flattening of the gyral crests is seen (fig. 4.13a ); in addition a small reactive glial zone around the ventricular system (fig. 4.13b ) may develop which can be separated from the intact white matter at autopsy (fig. 4.13c) . in adults, the clinical symptoms of hydrocephalus are non-specific; in infants and children they may be specific (see chap. 21 − pp. 482 f) and depend on the causes and the time course of the hydrocephalus. the salient symptoms comprise psychopathological alterations such as dementia, memory disturbances, and loss of orientation, culminating in the most severe cases in loss of consciousness. the first step in diagnosing hydrocephalus involves the use of imaging techniques, mri and cct, to establish its presence. the second step seeks to determine the underlying cause, and again employs as its methods of choice mri and cct, in combination with clinico-chemical (fishman 1980 ) and cytological analysis of the csf itself (oehmichen 1976a ). "necrosis" (for review see lindenberg 1982 ) is commonly used to designate the death of tissue components, including that of cells and their processes in a defined area of blood and oxygen supply. after a severe episode of ischemia, mbi or epilepsy, it is typical to find necrotic cell death within the injury core. in addition, a substantial number of neurons in regions surrounding the injury core have been observed to die via the programmed cell death pathways due to secondary effects derived from the various types of insults (liou et al. 2003) . "apoptosis" (for review see vermes et al. 1998 ) is applied to the selective (programmed) death of one or more individual cells. apoptosis is the more active and physiological form of cell death. in necrotic cell death, a stimulus such as ischemia, hemorrhage, mechanical or chemical damage alters cell homeostasis thus causing cell death, whereas in apoptosis an internal death stimulus triggers the innate cellular suicide program, the latter (not the stimulus itself) mediating the cellular demise (beal 1995) . in the following, the various pathogeneses and mechanisms of these types of cell death will be described, along with their different morphologies and underlying molecular factors. the most common cause of necrosis is ischemia. other causes include mechanical injury (contusion fig. 4 .12a−c. internal hydrocephalus. a expanding ventricular system associated with an atrophy of white matter; b the ventricular surface is commonly marked by a granular surface structure, which (c) microscopically is characterized by multiple glial nodules which replace lost segments of the ependymal layer (h&e, magnification ×200) necrosis), toxic agents (formic acid in methyl alcohol), heat (thermocoagulation), freezing (cryosurgery), infections (poliovirus), and overexposure to ultrasound. each case, however, involves the action of additional factors independent of the type of primary traumatic event. chief among these factors are free radicals and nitric oxide (no). reaction products of no and o 2 , including potent oxidizing molecules such as peroxynitrite and nitrogen dioxide, can be more toxic than no itself. the type of brain necrosis depends in large part on the duration of local circulatory arrest: 1. transient ischemia only destroys neurons and oligodendrocytes, inducing incomplete necrosis or selective neuronal necrosis (scholz 1953 ). 2. prolonged ischemia, termed "infarction," gives rise to complete necrosis of all tissue components. morphologically cell necrosis, especially neuronal necrosis, features irreversible changes of the cytoplasm (condensation, hydropic swelling, intense eosinophilia, loss of structure, homogenization) ( fig. 4.14) and of the nucleus (pyknosis, karyolysis, karyorrhexis) (majno and joris 1995) . time course. the data vary on how soon after the onset of ischemia the first microscopic neuronal changes become evident. some authors report an interval of 30 min (jacob and pyrkosch 1951) , others 14/15 h (müller 1930) . the data may differ in part due to prolongation of the necrotic process for as long after death as brain temperature remains favorable (lindenberg 1982). prolonged ischemia-induced tissue necrosis is termed "infarction" or liquefactive necrosis. the infarcted area displays macroscopically evident pallor on h&e, nissl, and myelin preparations within 3−5 h as an indication of acidosis. a narrow halo of even greater pallor surrounds the necrotic area ( fig. 4.20a) . around the necrotic area, vessels are distended and release fluid into the infarcted tissue and surrounding tissue by way of a vascular network (perifocal edema). neurons become thorny and severely shrunken within 12−36 h, with darkly staining incrustation of their pericellular structures. a survival time of 12 h leads to homogenization of the cytoplasm and nuclear and cytoplasmic pallor. between 36 h and 48 h, the neurons disappear except for the nuclei (see fig. 4.14c) . within 1−2 h the necrotic tissue is characterized by an emigration of neutrophil leukocytes (fig. 4.15a, b) . within 18 h the necrotic area exhibits proliferation and extensive activation of microglial cells (fig. 4 .15c−f). along the infarct margin, macrophage numbers increase. hypertrophic astrocytes appear along the border zone within the brain parenchyma after 4−6 days ( fig. 4.16) . the infarct liquefies at its center and macrophages phagocytose the debris. the final stage of cortical liquefactive necrosis is termed "laminar necrosis" (fig. 4 .17a−c) associated with intense gliosis during the final phase (fig. 4.17d) . the final stage of ischemic involvement of the basal ganglia and the thalamic nuclei is a cystic necrosis (fig. 4.18) . ischemic damage of the hippocampal area is characterized by a segmental loss of neurons in the hippocampal cortex ( fig. 4.19a, b) associated with compensatory (early) microglial activation (fig. 4.19c ) and secondary gliosis (fig. 4.19d) . another type of brain tissue necrosis is the rare phenomenon "persistent coagulative necrosis" first described by spielmeyer (1922) (fig. 4.20) , which affects cell tissue elements. within gray matter the outlines of dead neurons are recognizable, their cytoplasm is intensely eosinophilic and usually contains numerous, often large, vacuoles, and the nucleus stains poorly with hematoxylin. this picture, which is recognizable within the first 4−6 h, is followed by decreasing stainability of nucleus and cytoplasm until a barely recognizable ghost cell is all that re-mains. acute and enduring deprivation of blood supply causes necrosis of cells and tissue components, the lesion remaining in a state of "coagulation" for a prolonged period. the cells appear only as shadows and the necrotic tissue persists within the brain as a foreign body and sometimes becomes encapsulated in mesenchymal tissue. the pathogenesis of this rare type of necrosis is still unknown (escolá and hager 1963; cervos-navarro and ferszt 1977) . apoptosis can be differentiated from necrosis based on differences in their pathogenesis, cell reactions, and morphologic features. apoptosis is the programmed death of a cell as regulated by specific death genes (for review see clarke 1998 ). it initiates a delayed secondary death of neurons in response to environmental changes, deficient metabolic and trophic supply, and changed gene transcription. during apoptosis, the integrity of mitochondria is compromised and various pro-apoptotic proteins are released into the cytoplasm. this results in activation of caspases, proteases that orchestrate the death of the cell (waterhouse 2003). apoptosis requires active protein synthesis (mcintosh et al. 1998; raghupathi et al. 2000) . a single cell can undergo a switch between the two types of cell death based on several pathways (mcconkey 1998; fiskum et al. 1999 ; see also leist et al. 1990 ) (for further informations, see p. 620). the characteristic morphology of apoptosis exhibits cleavage of the internucleosomal chromatin that can be identified in situ using the terminal deoxynucleotidyl transferase-mediated dutp nick end-labeling (tunel) method (gavrieli et al. 1992) . apoptosis causes pyknosis of the nucleus and condensation and shrinkage of the cell body. as it progresses, budding and karyorrhexis occur, and ultimately a breakup into clusters of apoptotic bodies (majno and joris 1995) . the time course of apoptosis following a traumatic event is as follows: about 4 h after a traumatic event, apoptosis begins, and remains demonstrable for about 3 days (yakovlev and faden 1997) . three major factors are known to participate in the apoptotic cascade of "delayed" neuronal death (for review kermer et al. 1999 ; see also huppertz et al. 1999 ): 1. immediate early gene transcription factors (cjun, jun-b, jun-d, c-fos, ap-1, atf, nf-κb) 2. proteases (calpains, caspases) 3. glutamate-mediated toxicity (free radicals, protein-kinases, ca 2+ homeostasis, second messenger systems) the death-inducing activity of the bax, bad, bid, bcl-x s family members is thought by raghupathi et al. (2000) to be in dynamic equilibrium with their survival-promoting cognates bcl-2, bcl-x l . shifts in the protective intracellular bcl-2-family-protein levels can tilt the balance toward cell death by activating the death-inducing cysteine proteases, caspases (thornberry and lazebnik 1998) . the death of single cells releases insufficient quantities of chemoattractants to allow effective concentrations of molecular species to reach the vascular endothelium. for this reason a genuine cell reaction does not occur. neighboring cells that are not professional phagocytes cannibalize the cell debris in a process specific to apoptosis (majno and joris 1995) . in inflammatory diseases, an essential factor in the resolution of the inflammatory attack is the clearance of apoptotic leukocytes by tissue-specific phagocytes (platt et al. 1998 ). this process has been termed the "safe, phagocytic clearance of dying, yet intact leukocytes undergoing apoptosis" involving rapid recognition, uptake, and degradation. microglial cells were recently shown to be capable of protecting neurons, cerebellar granule neurons in particular, from apoptosis (polazzi et al. 2001 ): molecules are released by apoptotic neurons that enable the anti-apoptotic activity of microglia. in vitro, normal microglia release molecules capable of rescuing neurons from apoptotic death. microglia, astrocytes, and oligodendroglia may participate in apoptotic or necrotic processes. the reaction of neurons highly sensitive to injuries such as ischemia, hypoglycemia, infection, and mechanical trauma are described above and classified systematically in table 4 .3. a review by rosenblum (1997) includes a comparison of various hypotheses regarding the underlying causes of "delayed" neuronal death, among them excitotoxicity, calcium, and apoptosis. rubin (1997) and abe (1999) review the phenomenon of neuronal apoptosis as it appears in various neurological diseases. astrup et al. (1981) first defined the ischemic penumbra as brain tissue perfused at a level within the thresholds of functional impairment and morphologic integrity, which has the capacity to recover if perfusion is improved. because tolerance of tissue to ischemic damage is dependent on residual flow and duration of flow disturbance (heiss and rosner 1983) , the ischemic penumbra is a dynamic process; it exists for a short period even in the center of ischemia, from which the conversion into irreversible necrosis propagates over time to the neighboring tissue. focal ischemia results in necrosis at the infarct core and activation of complex signal pathways for cell death and cell survival in the penumbra. increased expression of caspase-1, -3, -8, and -9, and of cleaved caspase-8 has been observed in the penumbra (for details, see ferrer and planas 2003) . but for a limited time interval, penumbral tissue has the potential for recovery and, therefore, is the target for interventional therapy in acute ischemic stroke (time window, heiss 2000). relatively little is known about the effects of injury of the dendritic processes of neurons. axotomy of a motor neuron induces loss of some presynaptic terminals and the retraction of processes (blinzinger and kreutzberg 1968; summer and watson 1971) . abnormalities in dendritic spines have been reportfig. 4 .21a−f. dendritic and axonal injury. a dendritic processes and neuronal perikarya are reactive with a map2 antibody which (b) lose their reactivity within a short time after an ischemic period as well as in cases of (c) mbi-induced hemorrhage; d axonal injury is demonstrable by axonal bulbs or balls using h&e stain as well as (e, f) by their reactivity to β-app antibody (magnification a ×50; b ×500, c ×100; d−f ×1,000) ed during the perinatal period in cases of developmental retardation (purpura 1975 (purpura , 1976 . li et al. (1997) showed that expression of microtubule-associated protein 2 (map2) in perikaryons and dendrites is a sensitive marker of dendritic lesions in spinal cord trauma (fig. 3.2b ; see also chap. 10 − pp. 226 f). as early as 4 h after moderate or severe compression of the spinal cord, loss of map2 immunoreactivity in dendrites and nerve cell bodies became evident in the injured segment. this phenomenon continued for the duration of the 9-day experimental period. how much map2 immunoreactivity is lost depends on how hard the cord is impacted (li et al. 1995) . the loss of immunoreactivity may result from an (impact-induced) influx of calcium, activating calcium-dependent proteolytic enzymes capable of degrading map2 (inuzuka et al. 1990 ). the same phenomena may be observed as a result of hypoxic lesions in the hippocampal area ( fig. 4.21a, b) . axonal injury is characterized by an interruption of axonal fibers (fig. 3.2b ) as demonstrable − for example − as a result of a gunshot: the axons will be fragmented (figs. 8.13b, 9.19) . moreover, axonal injury induces an anterograde (wallerian) and retrograde degeneration of the injured axons. the terms "anterograde" and "retrograde" refer to the directions of conduction of the nerve impulse along the axon, i.e., the degeneration following focal damage proceeds in a centrifugal or centripetal direction (for review see brodal 1982 ). an interrupted anterograde flow of proteins along the axon can cause the phenomenon of axonal injury. this phenomenon was once demonstrated by h&e stain and by silver staining techniques within 16−24 h after a traumatic event (strich 1956; adams et al. 1982) . but since the injured axon is selectively characterized by expression of β-amyloid precursor protein (β-app) (fig. 4 .21d−f; cf. gentleman et al. 1993; sherriff et al. 1994) , it is now routinely confirmed in 105−180 min (blumbergs et al. 1995; oehmichen et al. 1999) , in adult brains as well as in infant brains (reichard et al. 2003) . β-app expression is seen even if the axonal injury is moderate or the axotomy delayed or incomplete (povlishock 1992) . although axonal injury was long thought to be a morphological correlate of mbi, it is now known to be a non-specific phenomenon also associated with acute intoxication (nies et al. 2002) or hypoxia/ischemia (oehmichen et al. 1999) . in a recent study graham et al. (2004) evaluated the pattern of β-app immunoreactivity. the authors identified three different types: 1. a diffuse − multifocal type − in mbi, co poisoning and hypoglycemia; 2. a type corresponding to the outlines of an infarct or hematoma with evidence of raised intracranical pressure; 3. a mixture of (1) and (2) which was seen in serve mbi. it is still unclear which events trigger axonal degeneration within the cns and pns. kapoor et al. (2003) suggest a link between nitric oxide (no) and subsequent molecular events that previously have been indicated as contributors to reversible and irreversible axonal injury. waxman (2003) demonstrates the axonal death cascade induced by hypoxia, ischemia, mechanical trauma, inflammation or no. the molecular events involving sodium channels and the na + /ca 2+ exchanger lead to an increase in intracellular calcium, which can provoke axonal degeneration. axonal injury of myelinated axons is always accompanied by a demyelinating process, i.e., a loss of myelin, which will be eliminated by mononuclear phagocytes (see: fig. 9.15b) . the sequelae will be a glial scar lacking myelin as demonstrable by luxol fast blue stain (fig. 4.22a) or by immunohistochemistry using an antibody against myelin basic protein ( fig. 4.22b, c) . interrupted axons or injured neurons exhibit disintegration of the distal stump. breakdown of the distal axonal stump is known as "wallerian degeneration" and begins several days after a traumatic event. its salient morphological features are lysis of axons and myelin, schwann cell proliferation, and phagocytosis by invading macrophages. axonal and myelin degeneration follow a time course described in detail by brodal (1982) (see also p. 242). axotomy induces a characteristic centripetally directed morphological change of the neuron termed retrograde axonal degeneration. this retrograde alteration is characterized by rounding of the neuronal contours, swelling of the neuronal cytoplasm, and a decline in the number of nissl bodies at the center of the cytoplasm (central chromatolysis − see fig. 3 .1c). the nucleus becomes slightly deformed and is displaced to the periphery of the perikaryon. these changes correspond to the changed or increased neuronal gene expression after axotomy (graeber et al. 2002) . axotomy is followed in hours by rapid upregulation of the immediate early genes, such as cfos, c-jun, jun-b (haas et al. 1993) , in association with the upregulation of heat shock proteins (kalmar et al. 2002) . the one constant change associated with acute retrograde changes may be chromatolysis. these changes are sometimes accompanied by local proliferation of satellite glial cells whose time course has also been described by brodal (1982) (see also pp. 242 and 304 f). it is accepted as common knowledge that plasticityassociated molecular and structural events occur in the injured brain. these are at least partly responsible for functional recovery. increases in dendritic arborization, spine density, and synaptogenesis in both peri-injury and intact cortical areas are the potential morphological strategies that enable the brain to reorganize its neuronal circuits (keyvani and schallert 2002) . on the other hand we have to consider that the scarring process is an ineffective regenerative process which is associated with cell proliferation. the cell proliferation will be − especially within the first 48 h after the traumatic event − non cell-specific, as immunostaining with markers for immature and mature astrocytes, activated microglia, neural precursors, and mature neurons will be negative (chirumamilla et al. 2002) . nevertheless, it is also accepted that both retrograde degeneration and the axonal injury-induced bulbs and swelling of the proximal axonal stump are markers of a regeneration process. although the cns has little innate capacity for repair, this capacity does exist for the peripheral nervous system. it is not known why axons in the adult cns are not capable of better regeneration; it is known that they do in fact regenerate, as was recently reconfirmed by von euler et al. (2002) . the observations of schwab and caroni (1988; schwab 1993 ) are of major importance. they show that proteins released by oligodendrocytes inhibit axonal elongation. this inhibitory protein has since been identified and cloned (chen et al. 2000; grand-pré et al. 2000; prinjha et al. 2000) . these authors also identified the protein (nogo) of a previously unknown gene that encodes the inhibitory myelin protein in rats and humans. the myelin-derived axon outgrowth inhibitor nogo protein binds to an axonal nogo-66 receptor and at least accounts for the lack of cns repair (li and strittmatter 2003; mcgee and strittmatter 2003) . it remains unclear, however, what effect other factors may have, whether negative (e.g., the release by neurons of large quantities of glutamate following the incidence of spinal cord injury), or positive [e.g., release of tissue growth factors (ramer et al. 2000) and cytokines, or induction of the macrophage scavenger function (see also schwab 2000) ]. it is now thought that neurons are renewed throughout life from endogenous stem cells and added to the dentate gyrus. adult neurogenesis could be demonstrated in the subgranular and subventricular zones of the hippocampus (kempermann et al. 1998; cameron and mckay 1999) and the olfactory bulb (craig et al. 1999 ). these are the only zones where differentiation of neural stem cells into neurons is known to take place in the intact adult cns (reilly 2002) . because this process does not occur in the adult spinal cord, svendsen (2002) postulates that the growth of neurites can only be stimulated by newly regenerated astrocytes. additional findings suggest that adult neurogenesis is actively regulated in large part by astrocytes (reilly 2002; song et al. 2002) . gage (1998) and colleagues (kempermann et al. 1998) were able to show that exposure to an environmental challenge evokes much greater neurogenesis in old than in young animals. more recent data suggest that the old brain reacts quickly with a neurogenic response to functional challenges, a type of cellular plasticity associated with the continued sensory stimulation and activity of the aging brain (kempermann et al. 2002; mckhan 2002) . the cns was once described as an "immunologically privileged site" (medawar 1948) . this hypothesis was based mainly on the existence of the bbb (pachter et al. 2003) , which restricts diffusion of soluble molecules and the migration of immune cells out of the systemic circulation into the cns (oehmichen 1983) . pericytes, endothelial cells, microglia and astrocytes, by contributing to bbb function (see above), participate in cns immune regulation (prat et al. 2001) . meanwhile a number of basic processes are known (bauer et al. 2001 ) that explain how the cns responds to inflammatory attack by regulating local antigen presentation, by the different cell types, and by its special cytokine environment. it also eliminates inflammation via emigrating macrophages (oehmichen et al. 1979 − for review oehmichen 1982 cserr and knopf 1997) and destruction of apoptotic t-cells. in vitro and in vivo studies have elucidated the mechanisms underlying immune-mediated (via cytokines, macrophage/microglial toxins, and antibodies) tissue damage, featuring many different potential pathways. the normal cns has limited expression of major histocompatibility complex (mhc) class i antigens, primarily on the endothelium and glial cells, and no expression of mhc-ii and the various immunoactive adhesion molecules. fontana et al. (1982) were the first to point out that cytokines such as interferonγ (ifn-γ) are able to stimulate astrocytes to express mhc class ii to secrete cytokines (il-1, il-3, tnf-α), and to present antigens such as myelin basic protein (mbp) to specific t-cell lines (fontana et al. 1982 (fontana et al. , 1984 massa et al. 1987) . frei et al. (1987) focused on microglia and found that they too could respond to ifn-γ by upregulating mhc-ii. if ifn-γ or ifn-γ and tnf-α are introduced directly into the cns, there is independent, progressive upregulation of both mhc classes and of adhesion molecules, such as the intercellular adhesion molecule-1 (icam-1), which are expressed first by perivascular macrophages (hickey and kimura 1988) , subsequently by microglia and macrophages throughout the cns, and finally by astrocytes (massa et al. 1986 ). experimental induction of autoimmune encephalitis (eae) revealed that perivascular macrophages are the chief presenters of cns antigens to circulating t-cells (hickey and kimura 1988) . the role of the endothelium in antigen presentation within the cns is ambiguous. astrocytes too are thought (waksman 1997) to play an ambiguous role. when presenting antigen to specific t-cells in vitro, they are lysed. it is not known whether t-cells are induced to proliferate or to release inflammatory cytokines, or possibly both, or whether they shut down for lack of suitable co-stimulatory signals (so-called clonal anergy). the duality of the inflammatory response is crucial to host repair and defense. it may also however cause loss or impairment of function, i.e., although otherwise beneficial, inflammation may impair neuronal function (perry et al. 1999 ). instead of the bbb being a limiting factor of the cns immune response as once believed, today it is thought that the brain itself is an immune system organ (fabry et al. 1994; chao et al. 1997 ). this conclusion is based on the numerous cytological and immunological findings of the past two decades showing that the targets to be protected are neurons, axons, and myelin. in the absence of protection these can become necrotic or succumb to apoptosis, be phagocytosed and disappear. ultimately all signs of degenerative alteration can be demonstrated. there is no direct correlation however between leukocyte emigration and parenchymal cell death in vivo (schmid-schönbein et al. 1999) . glial cells outnumber neurons in the cortex, where there are eight glial cells for every neuron. among glial cells in the cortex, astrocytes comprise 80%, microglia 15%, and oligodendroglia 5% (chao et al. 1997 ). these cells possess many immunological features marking them as important immunoregulatory cells. hallmarks of cns inflammation in particular are activated microglia and astrocytes. inflammation undoubtedly serves primarily as a host defense mechanism in peripheral tissue, facilitating essential repair processes by altering local blood flow in the injured tissue, with accumulation of fluid and specialized cells. the brain possesses cellular host defense mechanisms since activated t-cells are capable of crossing the bbb (hickey et al. 1991) . cellular mediators of cns inflammation in the brain have been shown to differ with regard to type and number from those of the periphery (see below). these differences are mainly due to the brain's tight regulatory environment and the balance between inflammation-induced tissue damage and tissue repair (parsons and hunter 1999) . the specificity of the immune response appears to be controlled largely by cns antigen presentation (sedgwick and hickey 1997) , though the precise nature of the control remains unresolved. cns antigen presentation according to hart and fabry (1995) occurs outside and inside the cns, with the bbb playing a major role in the regulation of cns immune function. parsons and hunter (1999) showed that the early events leading to t-cell activation by antigenpresenting cells result from mhc binding. co-stimulatory molecules then bind to the antigen, presenting cell−t-cell complexes for further development of the cascade. mhc is expressed not only on astrocytes and microglia, but under certain conditions also on neurons and oligodendrocytes (sedgwick and hickey 1997) . mhc ii antigens are also expressed under normal conditions in a population of macrophages inhibiting the perivascular space, subarachnoid space, and choroid plexus. this expression may indicate that these cells perform a modulatory function at the blood/csf interface (matyszak et al. 1992) . under highly specific circumstances, physiological response mechanisms resembling those at the periphery also appear to take place in the brain. under pathological conditions, hematogenous cells that are absent or extremely rare under normal circumstances appear to accumulate in the brain, i.e., platelets (fig. 4.23a) , neutrophilic leukocytes ( fig. 4.23b,c) , lymphocytes (fig. 4.23d) , and macrophages. the number and type of inflammatory cells in the cns vary widely depending on the attracting stimulus or on their inherent ability to attack a cns antigen. the intravascular cells, especially the leukocytes, interact with vessel walls as determined by integrins (hynes 1992) , selectins (bevilaqua 1993) , and immunoglobulins of the supergene family (springer 1990). among the heterogeneous supergene family are mhc molecules, t-cell receptors, and icam-1. during emigration, i.e., extravasation, leukocytes initially come into loose contact with the walls of vessels of the microcirculation via selectin molecules or lectins, producing a rolling motion along the vessel wall (mcever 1994) . they are then expressed on the endothelium. the selectin molecules are e-selectin (elam-1), l-selectin (lam-1, leccam-1), and p-selectin (cd62). p-selectin plays a role in recruitment of neutrophils to the brain parenchyma (bernardes-silva et al. 2001) , while e-selectin is thought to participate in both neutrophil and cd4 + t-cell adhesion (harlan and liu 1992) . endothelial selectins can be rapidly upregulated following wounding, p-selectin within minutes (<2 h − zoppo 1997) , and e-selectin within a few hours (granger and kubes 1994; mcever 1994) . among the stimuli for expression of endothelial cell adhesion receptors are tnf-α and il-1. adhesion of leukocytes to endothelial cells is mediated by adhesion molecules such as mac-1, the intracellular adhesion molecules (icams), lymphocyte function-associated antigen-1 (lfa-1), and vascular cell adhesion molecule-1 (vcam-1), all of which, as already mentioned, are upregulated in endothelial cells. in the cns, icam-1 and vcam-1 ( baron et al. 1993 ) are constitutively expressed on perivascular cells and some astrocytes. in areas of cns inflammation they are readily upregulated on endothelial cells and astrocytes (sobel et al. 1990) , which stimulates recruitment of neutrophils to the site of injury. such neutrophil emigration can be experimentally induced by extravasal injection of cytokines such as il-1β (bernardes-silva et al. 2001 ) or tnf-α (schmid-schönbein et al. 1999 . neutrophils are the first circulating leukocytes to reach the site of injury. increase in vascular permeability is caused by the release of free radicals and lysosomal enzymes, giving rise to edema (weiss 1989) . despite mechanisms evolved to restrict entry of neutrophils into the brain parenchyma, neutrophil recruitment is clearly a feature of acute brain injury, such as that caused by stroke or mechanical trauma. numerous studies employing a transient or permanent model of focal ischemia in rats and mice have demonstrated that cerebral tissue injury is lessened by neutrophil depletion (jean et al. 1998) . a correlation between the development of cerebral edema and neutrophil recruitment has also been shown in models of mbi (schoettle et al. 1990 ). this deleterious effect of neutrophil recruitment contrasts with their beneficial function and phagocytic ability as scavenger cells in cases of bacterial inflammation and of particular importance in cases of sterile inflammation. the aforementioned recruitment of t-lymphocytes to the site of injury clearly depends on an accumulation of adhesion molecules, especially of vcam-1, in the endothelial wall. when activated, t-cells express lfa-1 and can bind icam-1 on endothelium (van kooyk et al. 1993) , thus facilitating entry of t-cells into the cns (baron et al. 1993) . few data have been published on the migratory requirements of b-cells. it is thought, however, that b-cells in their fully mature form as plasma cells have no or only limited migratory potential. immunization of rats with a foreign, non-pathogenic antigen behind the bbb was found to result in the presence of b-cells and plasma cells specific for that antigen in the cns (knopf et al. 1994) . this finding appears to indicate that, after entering, b-cells remain in the cns at least in part because they have found their specific antigen . after entering the cns, the function of t-and bcells is to recognize their antigen. among the potential antigen-presenting cells of the cns are endothelial cells and astrocytes. under inflammatory conditions, microglial and perivascular cells (members of the mononuclear phagocyte family residing in the cns) constitute the chief antigen-presenting cells. as already mentioned (pp. 27 f), a distinction must be made between activated and resident microglia, and leptomeningeal, perivascular, and choroidal macrophages. all of these cells represent different functional stages of blood monocytes (oehmichen and huber 1976; oehmichen 1976a oehmichen , b, 1978 oehmichen , 1983 cf. also perry and gordon 1997) . microglia residing in the white matter do not express the mhc i antigen, and only a few express mhc class ii (hart and fabre 1981) . leptomeningeal, perivascular, and choroidal macrophages, in contrast, do express mhc class ii. the resident microglia also feature a downregulation of other antigens, the leukocyte common antigen (lca), and ed-1 or cd4. monocytes emigrate under pathological conditions into the brain parenchyma, where their morphology and antigenic characterization both change. they now participate in the immunological process as macrophages and express mhc class ii antigens. they also scavenge cell debris and myelin fragments left over from damaged tissue. astrocytes are among the first local cells to respond to cns injury. the main responses are reactive gliosis and swelling of reactive (hypertrophic) astrocytes upregulating gfap. reactive astrocytes express acute phase reactive protein (koo et al. 1991) , mhc class ia (frank et al. 1986 ) and mhc class ii antigens (fierz et al. 1985) , il-1 (griffin et al. 1989) , plus multiple other factors (for review see norenberg 1997) . astrocytes are known to act in conjunction with cells of the immune system and to be involved in immune/ inflammatory processes. they are immunocompetent cells capable of augmenting, amplifying, and sometimes even of regulating an immune response. they produce many immune mediators and can in return be affected by them. by helping to eliminate infectious or foreign agents, astrocytes may contribute to a beneficial response. given their direct contact with leukocytes in the blood, endothelial cells constitute the ideal site for antigen recognition in the cns (sedgwick and hickey 1997) . the scarcity of t-cells in the cns may be an indication that endothelial cells are the sole site capable of adequate t-cell antigen−mhc interaction. the finding that activated t-cells can enter the cns through an intact bbb (hickey et al. 1991) , however, is a clear sign that antigen recognition at the endothelial cell surface need not occur. endothelial cells are thus regarded as major players in the inflammatory and immune response (sternberger et al. 1989 ) and simultaneously guarantee the bbb. they also enable alterations in the form of receptor-mediated events (dietrich 1999) , i.e., an inflammatory response (for details, especially on the expression of adhesion receptors, see above). endothelial cells proliferate at the site of brain wounds (fig. 4.24a) . therefore, the number of capillaries increases and − in a final phase − decreases near hemorrhages or infarcts (fig. 4 .24b, c) in association with an increase in collagen fibers, especially collagen type iv (fig. 4.24c ). this process leads to a network of collagen fibers and glial fibers, which is the last stage in the formation of a brain scar. among the inflammatory mediators are cytokines and their subgroups, chemokines, in the sense of adhesion molecules. other mediators of inflammation include effector molecules such as no, nos, reactive oxygen species (ros), and free radicals. cytokines mediate the initiation, propagation, regulation, and suppression of immune and inflammatory responses (benveniste 1999) . they are proteins with low molecular weight and are synthesized during effector phase immunity. they are secreted by cells and are also expressed on their surfaces. many different cell types are capable of producing the individual cytokines, which for their part can have a variety of effects on different cell types. usually acting locally, cytokines begin to act on target cells by binding to specific cell-surface receptors, which generally have a high affinity for their ligands. it takes only minute amounts of a cytokine to evoke a biological response. in multiple sclerosis or experimental allergic encephalitis, ifn-γ and il-2 are known to be products of activated t-cells. tnf-α and il-1 derive from activated astrocytes and macrophages. astrocytes can be activated by ifn-γ and/or tnf-α, produce il-1 and il-3, tnf-α, transforming growth factor β (tgf-β), granulocyte-macrophage colony-stimulating factor (gm-csf), and other types of molecules such as prostaglandin e 2 (pge 2 ) (for review see waksman 1997) . in human brains, tnf-α, il-1, and il-6 in particular can be induced by mbi or cerebral ischemia. brain ischemia triggers rapid production of tnf-α mrna, which peaks 6−12 h after ischemia and subsides 1−2 days later. it remains above baseline, however, for up to 5 days (barone 1999) . neuronal cells in and around the ischemic tissue acutely express tnf-α in a so-called penumbra, but it also turns up several days later in macrophages in the infarcted tissue. tnf-α triggers adhesion molecule expression on activated glial cells and the endothelium, in this manner regulating gliosis, tissue remolding, and scar formation. il-1 levels rise before and during glial activation and neuronal damage (rothwell et al. 1999 ). chemokines are chemoattractant cytokines. during inflammation they mediate leukocyte entry into the cns. among their known functions is the interaction of leukocytes with the endothelial surface, a multistep and sequential process mediated by selectin molecules by which the leukocyte rolls on the endothelium. the end result is firm adhesion. the entire process is mediated by interaction of icam-1 and vcam-1 expressed by endothelial cells and their leukocyte-associated ligands. moderate levels of icam-1 and very low levels of vcam-1, two molecules responsible for the adhesive properties of granulocytes and of t-cells, are expressed by brain endothelial cells. chemokines constitute a subgroup of small cytokines (8−10 kda) that attract certain inflammatory cell populations, among them lymphocytes, neutrophils and monocytes, to the target tissue (meeusen et al. 1996; bonecchi et al. 1998 ). the number of known chemokines and chemokine receptors continues to expand rapidly (for review see prat et al. 2001) . three classes of chemokines are known, as defined by the arrangement in the mature protein of conserved cysteine (c) residues, cxc or α-chemokines, cc or βchemokines, and of cc or γ-chemokines. astrocytes, endothelial cells (zach et al. 1997; weiss et al. 1998 ), perivascular cells, and macrophages (simpson et al. 1998 ) produce and release chemokines. ros and no are generated in astrocytes and activated macrophages (hartung et al. 1988 ). ros, no, and other free radicals are effector molecules that contribute to the inflammatory cascade and tissue damage. what role complement plays in cns damage is not clear (morgan 1999) . the enzyme no synthase (nos) synthesizes no. inducible nos (inos) is an isoform of nos that is induced transcriptionally by immunological stimuli. inos, which synthesizes large quantities of no, participates in inflammation-induced cytotoxicity. in the brain, inos message, proteins, and enzymatic activity are induced de novo by cerebral ischemia (iadecola 1999) . inos is expressed by neutrophils in permanent ischemia and in vascular cells in transient ischemia. high levels of no are synthesized by human astrocytes upon stimulation with ifn-γ, tnf-α, il-1 and potentiate il-1. dalkara et al. (1999) showed that no plays a detrimental role in experimentally induced cerebral infarction in neuronal nos knockout mice. the nos knockout infarcts 24 h after permanent vessel occlusion were 38% smaller than those of wild type. these finding seem to indicate that expression of inos is a factor contributing to ischemic brain damage. an apoptotic pathway mediates no-induced neuronal cell death and an nmda receptor antagonist blocks no-mediated neurotoxicity. neuronal cell death was shown by chao et al. (1997) to be initiated by the release of il-1 by the microglial cell, this in turn inducing the generation of astrocytes. neurons are destroyed by no via nmda receptor-linked apoptosis. cerebral trauma, ischemia, and reperfusion are known to generate hydrogen peroxide and superoxide radicals, which then produce ros and hydroxyl radicals (chan 1999) . under normal conditions and following reperfusion injury, mitochondrial respiration creates ros. microglia and astrocytes that have been activated by cytokines produce vast quantities of neurotoxic free radicals. an intramitochondrial antioxidant enzyme, manganese superoxide dismutase (mnsod), scavenges superoxide radicals and thus constitutes the first line of antioxidant defense. as already pointed out, inflammatory responses are based on an inflammatory cascade, whose details have become increasingly clear in recent years. three basic types of inflammatory response are known: sterile inflammation, cell-mediated inflammation, and antibody-mediated inflammation. these types of response are mutually exclusive, but characterized by occasional overlapping. in cases of mechanical violence, spontaneous intracerebral hemorrhage or stroke, sterile inflammation features an initial phase of infiltrating neutrophils and a second phase of infiltrating mononuclear phagocytes (bone marrow-derived monocytes, i.e., activated microglia and macrophages). macrophages and neutrophils produce cytotoxic cytokines such as tnf-α, proteolytic enzymes (anthony et al. 1997) , reactive oxygen intermediates (cross et al. 1998) , cell death-inducing surface molecules such as fasligands (d'souza et al. 1996) , or even excitotoxins (lipton 1998) . the macrophages in particular take up the scavenger function and eliminate tissue and cellular debris. they also release mediators that promote the scarring process, i.e., induce fibroblasts to produce collagenous fibers and stimulate astrocytes to proliferation and produce fibrils, aiding healing by the production of a fibrillary glial-collagenous scar. multiple sclerosis (ms) is the classic model of t-cellmediated inflammation whose inflammatory infil-trates are chiefly comprised of t-lymphocytes, fewer b-lymphocytes, as well as activated microglial cells and macrophages (brück et al. 1995; gay et al. 1997) . ms features local expression and/or upregulation of markers of t-lymphocyte and macrophage activation (brück et al. 1995) , of class i and class ii mhc antigens (traugott 1987) , of chemokines and adhesion molecules in addition to their receptors (lassmann 1998) , and of co-stimulatory molecules (windhagen et al. 1995) . diseases with an autoimmune background such as ms or virus-induced inflammatory diseases exhibit a uniform cellular and mediator profile. unlike ms, lesions associated with viral inflammation of the brain display considerable differences in topography and in their patterns of structural damage. they also vary with regard to the nature of the immune response and its associated cellular tropism. in lesions of virus encephalitis cd8 + -lymphocytes abound; in ms lesions both active and inactive cd8 + -cells usually outnumber cd4 + -cells (gay et al. 1997) . virus-infected cells generally evoke a cell-mediated immune response, although humoral mechanisms play a role as well (for review see esiri and kennedy 1997) . phagocytosis of infected cells by macrophages can also be promoted by antibodies. antibody-dependent cell-mediated cytotoxicity (adcc) is a process in which lymphocytes bearing fc receptors for igg lyse virus-infected cells bearing relatively small amounts of surface-bound antibody. the immunological specificity of the reaction derives from the antibody not the lymphocytes, which are not specific and have been designated killer cells. an important cell-mediated specific mechanism for killing infected target cells is provided by virus-specific cytotoxic t-cells. the cytotoxic effect is seen even if an antibody is lacking and is restricted by mhc class i antigens. viral antigens on the surface of infected host t-cells are recognized by virus-induced cytotoxic t-cells in association with class i mhc antigens. the infected target t-cells are then killed by these t-cells only if they share the same mhc antigens, i.e., the t-cell killing is restricted by mhc (zinkernagel and doherty 1974) . viral infection induces secretion of cytokines within the cns, either by lymphomononuclear cell infiltrates or infected brain cells. cytokines play an important role in the induction of mhc molecules. they stimulate humoral and cell-mediated immune responses by acting on immune system cells and neighboring brain cells, evoking the expression of surface recognition molecules such as mhc antigens and antiviral proteins such as mx (campbell 1991) . viral infections can provoke or amplify mhc class ii expression on the surface of astrocytes and microglial cells, which is important in light of the significance of these antigen-presenting cells in the cns. this process can occur as a direct effect of the infection even in the absence of ifn-γ, as shown for measles virus-infected astrocytes and the murine coronavirus j. howard mueller virus (massa et al. 1987) . in viral and bacterial inflammation, both the cell-mediated immune response and the humoral response are highly important, the latter also usually dominating. blood-borne dissemination of virus from the primary infection site to other organs is restricted by circulating antibodies, igg or igm. antibodies in tissue spaces can stop the spread of infection from one cell to another by neutralizing extracellular viruses. however, viruses able to fuse cell membranes, such as measles or herpes viruses, can elude this mechanism without ever being exposed to antibody. viruses can be inactivated by antibodies in a variety of ways. antibodies can assist phagocytosis by coating the surface of the virion, or they may thwart attachment of the virus to specific receptors on vulnerable cells, or in the case of enveloped viruses they may promote viral lysis via attachment and activation of complement. in the absence of antibody, direct viral lysis can also be produced by complement alone. edema can produce an increase in tissue pressure that disturbs the microcirculation of portions of the cns that are anatomically prevented from swelling by bone or tight meningeal constraints. it is thought that this mechanism contributes to the formation of necrotic lesions in transverse myelitis. the inflammatory process often has a vasculitic component (gray 1997 ) that causes occlusion of small veins and venules and is commonly associated with massive tissue damage. ischemia can contribute to the development of structural damage and functional deficits in inflammatory cns lesions. involvement of arteries and veins is rare in bacterial inflammation but the exudate is frequently accompanied by strands of fibrin. the edematous cortex exhibits large artificial perineuronal spaces and a spongiform neuropil. the cytoplasm of neurons often reveals ischemic cell necrosis and is acidophilic. if the course is subacute, fibrinoid necrosis and thrombosis may appear in a few blood vessels in the exudate, resulting in small foci of cortical necrosis. in the field of neuropathology both gross and microscopic changes can be misinterpreted. only a few aspects will be discussed here, each involving routine immersion fixation with 10% buffered formalin, dehydration and embedding in paraffin (see above). a detailed overview of the problems associated with postmortem changes, artifacts, and misinterpretation has been provided by lindenberg (1982) . autolysis, a process involving self-digestion of tissue, can cause slides of the adult brain to be discolored or poorly staining. the morphology of the changes associated with autolysis are identical with those of respirator brain (brain death). they arise if fixation is done too late, i.e., if the interval between intracranial circulatory arrest and autopsy or brain fixation is too long. depending on the ambient conditions, the brain will liquefy after a certain postmortem interval without fixation. the brain emits a foul odor if it has undergone microorganism-induced putrefaction, and the central parts of brain slides display a faint pink coloration. variably large bullous cavities ( fig. 4.25a, b) give an appearance of swiss cheese to large sections of the brain. such cavities are created by the activation of gas-producing microorganisms due to poor quality formalin. macroscopic necrosis of the cerebellar granular layer was interpreted by ikuta et al. (1963) as a postmortem phenomenon, whereas lindenberg (1982) thought that the necrotic process precedes or accompanies the onset of sublethal hypoxemia, i.e., shortly before death. neurons in the substantia nigra and locus coeruleus of the brains of infants and children up to 5 years of age normally possess no melanin pigment. this finding is neither an artifact nor pathological. the spinal cord is especially sensitive to postmortem mechanical injury in an unfixed state. in this manner the so-called toothpaste-artifact can occur (hughes 1978) . if the tissue in a segment of the spinal cord is artificially constricted, proximal portions of the cord are squeezed upward, distal portions downward, thus appearing in histological sections at the wrong level. any tissue processing produces artifacts that have to be interpreted: fixation procedures as well as staining techniques. the artifacts are dependent on the time periods between death and fixation, the duration of the fixation process, the temperatures, etc. for example the freezing process of (brain) tissue leads to crystalline vacuoles within the tissue sections ( fig. 4.26a, b) . additionally, the forensic neuropathologist fundamentally needs to be able to reliably distinguish between vital and postmortem changes (cf. oehmichen 1995) . this requires among other things the testing and use of novel histochemical and immunohistochemical staining techniques that can help to establish the length of the postmortem interval. so-called dark neurons (fig. 4.26c) can appear among otherwise normal neurons. dark neurons are irregularly contoured, shrunken neurons that are created by excessive pressure on unfixed postmortem tissue (scharrer 1938; cammermeyer 1961 cammermeyer , 1975 . apical dendrites also have a dark coloration, sometimes in combination with a corkscrew-like appearance. there is a danger of confusing dark neu-rons with ischemic cell necrosis. in our own investigations (oehmichen and gencic 1980a) we could observe that most, but not all dark neurons have a potent albumin uptake, an indication that they represent lesions of neuronal metabolism (and/or membrane) . animal experiments demonstrate that the following three types of altered neurons appear at different postmortem intervals in rats (oehmichen and gencic 1980b) : 1. shrunken, hyperchromatic neurons, whose number declines as the postmortem interval proceeds. 2. swollen and autolytic neurons, with a pale perikaryon and nucleoplasm, and an absence of nissl bodies. the nucleus can no longer be differentiated from the vacuolized and autolysing cytoplasm. the cells themselves have lost their contours and appear swollen and spherical. the swelling in particular represents the most fundamental postmortem change (whose differential diagnosis is retrograde degeneration). 3. pericellular spaces surrounding neurons may be caused by postmortem autolytic processes that mimic edema, especially in the gray matter. children two years old and younger almost invariably exhibit periventricular and perivenous accumulation of cytoplasm-poor, lymphocyte-like mononuclear cells which suggest an encephalitis (so-called pseudoencephalitis). those cell aggregations consist of neuroblasts as an indication of development, not of an inflammatory process. this age group also regularly shows a superficial granule layer of the cerebellar cortex composed of germinal cells (matrix cells). these usually disappear some time between the second and fourth years of life. in cases of sudden death with brief agony or if tissues have been poorly fixed (hirano 1981) , extensive acute swelling of oligodendroglia is common. in addition, a generalized swelling and clasmatodendrosis of astrocytes is often seen. these changes are rather slight compared to the neuronal changes. they must also be regarded as non-specific and do not constitute markers of edema. lindenberg (1982) points out that the state of the brain before circulatory arrest also plays a 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(cd86), and interleukin 12 cytokine in multiple sclerosis lesions traumatic brain injury regulates expression of ced-related genes modulating neuronal apoptosis expression of a chemotactic cytokine (mcp-1) in cerebral capillary endothelial cells in vitro immunological surveillance against altered self components by sensitized t-lymphocytes in lymphocytic choriomeningitis selectins, icams, and integrins in cns injury severe pediatric head injury: the role of hyperemia revisited key: cord-017954-vobslprh authors: croxford, j. ludovic; miyake, sachiko title: animal models for the study of neuroimmunological disease date: 2016-03-26 journal: neuroimmunological diseases doi: 10.1007/978-4-431-55594-0_3 sha: doc_id: 17954 cord_uid: vobslprh the development and use of numerous animal models of human autoimmune diseases have provided important advances in our understanding of pathogenic mechanisms of disease and provided robust and reliable models to test novel therapeutic strategies. however, few preclinical studies of therapeutic treatments have demonstrated efficacy in the clinic, possibly because of the biological differences between humans and other animals. although animal models of human disease are imperfect, it is important to understand the differences between the human disease and its animal models and to design experimental studies using animal models appropriately for the questions being asked. this review provides an overview of the currently used animal models of three human neuroimmunological diseases, multiple sclerosis, guillain-barré syndrome, and myasthenia gravis, as well as the advantages and disadvantages of each model and how they correlate or differ from their human counterpart. following decades of research, scientists have developed a large number of drugs and therapeutic agents that can be used to reduce the symptoms and severity of a number of neuroimmunological diseases such as multiple sclerosis (ms), neuritis, and myasthenia gravis. however, none of these treatment methodologies is curative, and many have a limited life span, such as interferon (ifn)-β that induces neutralizing antibodies in some ms patients [1] , or cause serious side effects (progressive multifocal leucoencephalopathy) such as monoclonal anti-very late antigen (vla)-4 antibodies (natalizumab, tysabri®) [2] and thus cause patients to drop out of clinical trials or stop taking the treatment. in addition, although many studies have dissected the intricate pathways involved in the aetiology, development, and pathogenesis of diseases such as ms, we still do not have a definitive understanding of how these diseases manifest and, indeed in the case of ms, whether it is a single disease or rather a spectrum of disorders with similar characteristics. therefore, researchers have used animal models to aid our understanding of disease pathways, immune cell functions, and pathology. an example of the importance of animal models was the demonstration that cd4 t cells specific for a myelin epitope injected into naïve animals were sufficient to induce a central nervous system (cns) demyelinating disease with cns lesions similar to those observed in ms [3, 4] . in addition, these models are a useful preclinical aid to developing novel therapeutic agents. many different animal models of neuroimmunological disorders have been developed over the last few decades with some success. however, it is important to understand the advantages and limitations of each model to ensure that the correct model is being used for the purpose of the study. in this review, we provide an overview of the currently available neuroimmunological disease animal models, describing their advantages and disadvantages as well as their relationship and correlation to the human disease they are attempting to model. although no one animal model is completely identical with human disease, they are nevertheless very useful and important tools with which to increase our understanding of neuroimmunological disease when used correctly. ms is a human, inflammatory, demyelinating disease of the cns, with characteristic demyelinating lesions containing immune cell infiltrate and activated cns-resident cells located in the white matter of the brain and spinal cord; therefore, studies to investigate its pathology are difficult and often rely on autopsy tissues. in these cases, patients might have had the disease for decades and therefore the tissues are representative of disease at the end stage. furthermore, epidemiological studies have suggested that the initiating factors for ms might be infections that occur during childhood [5] . therefore, animal models of ms are critical for the study of susceptibility, disease initiation, and pathology during the early stages of disease. experimental autoimmune encephalomyelitis (eae) is an autoimmune t cell-mediated disease of the cns mediated primarily by cd4þ t cells and a commonly used animal model for the study of ms. disease is characterized by perivascular lesions containing inflammatory infiltrates and nerve conduction block that causes reversible hind-limb paralysis. in addition, late-stage eae animals also develop axonal demyelination and loss, a pathological hallmark of ms, which leads to severe permanent disability. characterization of the immune cell infiltrate (t cells, b cells, activated macrophages, and microglia) and the pathology of perivascular demyelinating lesions have shown similarities with human ms lesions [6, 7] although in eae the spinal cord is the target organ, whereas in ms the brain is more often targeted, especially the white matter. however, eae is a useful model to study the inflammatory stage of ms and shares some characteristics of ms such as optic neuritis, increased susceptibility of females, perivascular lesions, axonal demyelination, and partial remyelination, as well as eventual axonal loss and flaccid-limb paralysis. there are two commonly used models of eae. active eae is induced by subcutaneously immunizing genetically susceptible animals, usually rodents, with myelin antigen in freund's complete adjuvant-containing mineral oil and mycobacterium tuberculosis strain h37ra. in some eae low-responder mouse strains, such as c57bl/6, additional injections of pertussis toxin are required. during the induction phase, myelin-specific t cells are activated by antigen-presenting cells (apc) presenting myelin peptide fragments in the draining lymph nodes to produce t helper 1 (th1) type cytokines, such as interferon (ifn)-γ and tumour necrosis factor (tnf)-α, which allows them to escape the lymph nodes and traffic to the cns. the effector phase of disease involves the extravasation of activated myelinspecific t cells through the blood-brain barrier and into perivascular spaces in the spinal cord. here, the encephalitogenic t cells encounter cns-resident cells mediating further stimulation of pro-inflammatory cytokines and chemokines that mediate a secondary influx of other peripheral inflammatory cells including b cells and mononuclear phagocytes. a study in eae and a viral model of ms demonstrated that apcs in the cns restimulated myelin-specific t cells to initiate epitope spreading to other myelin antigens and perpetuate disease [8] . the consequence of this pro-inflammatory cytokine milieu is the demyelination of cns axons, thought to be mediated in part by numerous mechanisms including phagocytosis by activated mononuclear cells, destructive effects of anti-myelin antibodies, the production of free radicals, and the direct cytotoxic effects of pro-inflammatory cytokines secreted by activated cd4þ t cells and monocytes. initially guinea pigs and rats were the animals of choice for eae studies. however, with the advent of transgenic and gene knockout technology, mice have become the more commonly used animal for eae studies. numerous mouse models of eae have been developed, and these are differentiated by the strain of mouse used and the immunodominant myelin peptide for that particular strain. commonly used eae-susceptible mouse strains include sjl, b10.pl, c57bl/6, c3h, swr, and biozzi abh. depending upon the immunizing myelin antigen and mouse strain used, different forms of eae can be induced. the most commonly used models are sjl mice immunized with proteolipid protein (plp) 139-151 that induces a relapsing-remitting form of disease, the most common form of ms, and c57bl/6 mice immunized with myelin oligodendrocyte protein (mog) 35-55 that initiates a chronic-progressive type of eae. other strains, such as biozzi abh mice, can be immunized with a suspension of whole biozzi abh spinal cord to develop a very reproducible relapsing-remitting disease [9] . the clinical symptoms of eae usually develop at 7-15 days post-immunization and include weight loss, loss of tail tone and gait, and eventually paralysis in either or both hind limbs. weight loss precedes the onset of disease symptoms and thus can be used as a marker for disease onset. daily observation of eae symptoms and body weight is noninvasive and is therefore a great advantage to researchers, allowing the disease process to be followed, especially when studying the effects of drug treatments. of note, some mouse strains are resistant to eae (a/j, c3h/hej, akr, nzw, and dba/2). in addition to the importance of environmental factors, it is thought that multiple predisposing genetic elements might be involved in susceptibility to ms (reviewed by ebers 1994) [10] . therefore, the backcrossing of eae-resistant mice with eae-susceptible strains has been useful for genetic susceptibility studies. the second model of eae is passive and involves the in vitro restimulation of encephalitogenic t cells with the myelin peptide used to immunize the original t-cell donor animals [11] . the successful culture of these cells usually requires the addition of th1 cytokines such as il-12 [12] . once cells have been sufficiently activated, they are intravenously administered to naïve animals, where they traffic to the cns and induce disease. the cns pathology and disease course are similar to that for active eae. the ability of mbp-specific t cells to induce eae in rats and mice was some of the earliest evidence to suggest that ms has an autoimmune aetiology [3, 4] . another evidence for an autoimmune inflammatory pathogenesis includes the presence of mhc class ii-restricted cd4þ t cells that recognize myelin antigens such as myelin basic protein (mbp), plp, and mog in ms patients as well as healthy individuals and that mhc class ii genes are associated with ms susceptibility [13] [14] [15] . because t cells are already activated when administered into naïve animals, passive eae is a suitable model for investigating the effector phase of disease. it has some advantages over passive eae as an immunization step is not required; thus, there is no antigen reserve that continually activates naïve t cells, which likely boosts the immune system nonspecifically. furthermore, the direct injection of effector t cells into naïve mice allows the definitive starting point of disease induction to be known, which might be useful for treatment studies, and finally encephalitogenic t cells can be directly tracked in vivo to study methods of extravasation into the cns and for isolation of antigen-specific t cells. early studies demonstrated that activated myelin-specific th1 cd4þ t cells secreting ifn-γ, tnf-α, and il-2 were encephalitogenic and sufficient to induce eae when transferred into naïve mice of a susceptible genetic background [16] [17] [18] . however, the knockout of genes encoding ifn-γ or tnf-α [19, 20] exacerbated eae and therefore the role of th1-induced eae was not straightforward. il-17 production by cns-infiltrating t cells is important for blood-brain barrier dysfunction and lesion formation in ms patients [21, 22] , and the genetic inhibition of il-17a in mice was sufficient to partly ameliorate eae in mice [23] . this indicated that th17 cells, an alternative effector t-cell subset to th1, might be the critical effector cells in eae. indeed, th17 cells cultured in the presence of il-23 and other cytokines become highly pathogenic and can induce eae when transferred into naïve mice [24] . th17 cells show an enhanced efficiency at inducing eae compared with th1 cells [24] and therefore might require less cell manipulation in vitro for adoptive transfer studies. although there is a predominance of eae papers investigating the role of myelinspecific cd4þ t cells in eae, ms lesions have been reported to contain greater numbers of cd8þ t cells compared with cd4þ t cells [6, 25, 26] . however, the function of cd8 t cells in ms lesions is unclear, and therefore, the study of cd8þ t cells in eae is important. a number of eae models induced by mhc class i-specific cd8þ t cells have been reported. mbp79-87-specific cd8þ t-cell clones isolated from mbp-immunized c3h wild-type mice induced eae with numerous neurological deficits (ataxia, spastic reflexes, spinning) as well as hindlimb paralysis when intravenously injected into c3h wild-type mice [27] . this form of eae was very severe and all mice were moribund by day 14. interestingly this model showed different clinical symptoms to the cd4þ t cell-mediated type of eae, but importantly had some similarities to ms, in that perivascular lesions were predominant in the brain, compared with cd4þ eae where perivascular lesions are located in the spinal cord. another study reported that mog35-55-specific cd8þ t cell lines could also induce a severe, chronic form of eae when adoptively transferred to naïve c57bl/ 6 mice [28] . in contrast to the mbp-induced cd8 t-cell model of eae, lesions were present in both the spinal cord and brain. differences in genetic background, availability of myelin antigens in the cns, or induction procedure might explain the differences observed between the two models. in contrast to these early cd8 studies, more recent investigations have indicated inhibitory roles for cd8 t cells in eae: neuroantigen-specific autoregulatory cd8þ t cells inhibited autoimmune demyelination by modulating dendritic cell functions [29] and il-15-dependent cd8þ cd122þ t cells ameliorated eae by reducing il-17 production by cd4þ t cells [30] . humanized mouse models have also been developed to study the effect of ms-related myelin epitopes presented to cd8 t cells by human hla molecules and showed that mog181-189 presented by hla-a*201 to mog-specific cd8 t cells exacerbated cd4 t cell-induced eae [31] . overall, these models of cd8 t cell-induced eae are important for determining the function of cd8 t cells during cns autoimmune disease, i.e. pathological versus inhibitory/regulatory effects, and have an advantage over cd4 t cellinduced eae in that the target organ is predominantly the brain, similar to that in ms. a number of spontaneous mouse models of eae have been developed by the transgenic expression of t-cell receptors (tcr) specific for myelin antigens (plp, mog, or mog) in t cells that overcome the negative selection of autoreactive t cells in the thymus (reviewed by croxford 2011) [32] . therefore, these models represent a more "natural" type of disease and are useful for examining the role of autoimmune t-cell activation by environmental stimuli. however, depending on the type of study involved, for example, drug efficacy testing, the use of spontaneous eae models is not recommended because of the wide variance in disease onset and severity. the advantages of eae are its robust and reproducible disease course that is useful for studying the early stages of disease initiation; the activation of immune cells, especially antigen-specific t cells; as well as mechanisms in the effector phase such as the role of resident cns cells, regulatory t cells, and other regulatory mechanisms. in addition, pathological studies at all stages of disease can provide important information regarding sites and mechanisms of demyelination, remyelination, nerve conduction block, and axonal loss. furthermore, depending upon the models used, the different mechanisms involved in relapsing vs chronic forms of disease can be studied. the reader is encouraged to read review articles describing the specific induction protocols for the numerous eae models that are available [11, 12] . the most significant disadvantage of eae is the location of pathology in spinal cord, unlike ms, where many lesions occur in the brain. furthermore, although eae is clearly an autoimmune disease, ms does not have all the hallmarks of an autoimmune disease, and evidence for a specific immunodominant myelin antigen is lacking. although cd4þ t cells are critical for disease induction in most eae models, their role in ms is less clear, where many other types of immune cells such as cd8þ t cells, b cells, and monocytes probably also have important roles. for example, b cells have little or no role in many eae models, at least at the early time points usually studied; however, their importance in ms is suggested by the beneficial effects of anti-cd20 therapy in some ms patients [33] . eae is a very useful tool to test potential new immunomodulatory therapies. although four approved ms treatments were studied in eae (natalizumab, mitoxantrone, glatiramer acetate, and fingolimod), the large majority of new treatments fail when they enter ms clinical trials. an explanation for this might be that treatment studies using the eae model do not mimic the patient disease course in the clinic. for example, the administration of drugs before the initiation of eae disease is only useful for indicating an effect on the activation of t cells and has no real clinical significance for ms treatment, where patients have often experienced symptoms for some time before treatments are started. in addition, treatments administered before the onset of symptoms that prevent paralysis are often said to prevent demyelination. however, if these treatments are immunomodulatory, then it is difficult to differentiate between their anti-demyelination and immunosuppressive effects. therefore, potential treatments should be administered after the onset of eae symptoms for clinically meaningful results. in summary, although eae is an imperfect model of ms, its correct usage by investigators is critical when studying novel therapeutic compounds. a number of etiological studies have indicated the potential role of viruses in the susceptibility, onset, and exacerbation of ms. therefore, in addition to immunization models that are useful to investigate the early immunological pathways involved in pathogenesis, a number of virus-induced demyelinating models have been developed that allow the study of the potential viral aetiology of ms. using these models a number of hypotheses of the mechanism of disease onset have been developed including molecular mimicry, epitope spreading, direct bystander activation, and release of cryptic epitopes. theiler's murine encephalomyelitis (tmev) is a single-strand rna virus that belongs to the cardiovirus group of the picornaviridae and is a natural mouse pathogen. tmev-induced demyelinating disease (tmev-idd) develops in susceptible strains of mice (sjl/j) upon intracranial injection of the bean 8386 strain of tmev, which persistently infect microglial cell populations in the cns [34, 35] . the persistent infection of tmev is thought to cause demyelination mediated by macrophage bystander destruction activated by tmev-specific cd4þ t-cell responses that target the persistent viral infection, leading to the release of myelin antigens that can activate autoreactive plp156-171-specific t cells that have escaped negative selection [36] . epitope spreading to other myelin epitopes propagates the disease, which shows similar inflammatory and demyelinating pathological hallmarks as those seen in ms patients [37, 38] . the onset of a chronic progressive demyelinating disease with no recovery or remission periods occurs around day 30-35 post-infection and continues for over 100 days. tmev-idd is characterized by the development of a spastic hind-limb paralysis and perivascular and parenchymal lesions in the spinal cord with demyelination of white matter tracts containing mononuclear cell infiltrates. although epitope spreading is thought to be involved in tmev-idd, molecular mimicry, the mistaken recognition of a pathogenic epitope for a "self" epitope due to shared amino acid sequences, is another potential mechanism for the induction of autoimmunity. early studies demonstrated that the immunization of viral peptides could stimulate myelin peptide-specific t-cell responses in vivo [39, 40] . however, the need for epitope processing and the role of a "live" viral infection to stimulate the host innate immune system are not addressed by short-length peptide immunization. therefore, recombinant tmev strains engineered to incorporate 30-mer myelin or bacterial/viral myelin mimic epitopes were used to study the potential viral induction of autoimmunity by molecular mimicry [41, 42] . infection of sjl mice with tmev engineered to express a plp mimic peptide derived from haemophilus influenzae, a natural mouse pathogen, with 6/13 homologous amino acids to plp139-151, including primary tcr and mhc class ii contact residues, induced a mild but rapid onset cns disease [42] . interestingly, the immunization of mice with the haemophilus influenzae peptide in complete freund's adjuvant failed to induce overt disease, indicating the importance of pathogen-delivered innate immune signals for the induction of disease by molecular mimicry. of note, viral peptide sequences in tmev do not share any sequence homology with plp, the immunodominant myelin peptide in tmev-idd in sjl mice, therefore indicating the role of the engineered haemophilus influenzae peptide sequence in disease induction. these studies were expanded to include tmev expressing 30-mer from murine hepatitis virus (mhv), which only shares 3/13 amino acids with plp139-151, and demonstrated the importance of a proline residue at the secondary mhc class ii contact point [43] . semliki forest virus (sfv) strain a774 is a neurotropic, single-stranded rna alphavirus of the togaviridae family that induces a demyelinating disease upon intraperitoneal injection in sjl/j mice. disease is characterized by virus-induced demyelination of the cns. despite the clearance of the virus by the immune system, maximal demyelinating lesions with the expression of ifn-γ and tnf-α are observed at 14 days post-infection up to 1 month in balb/c mice, whereas both demyelination and pro-inflammatory cytokine expression can be detected in sjl/j mice up to 1 year [44] . sfv infection induces an mbp-specific t-cell response [45] and demyelination is mediated by cd8 t cells [46, 47] . mhv, a group ii positive-strand rna coronavirus, is a natural pathogen of mice that upon intracranial injection causes an acute encephalomyelitis that develops into a chronic cns immune-mediated demyelinating disease with some clinical and pathological similarities with ms [48] . mhv can induce either acute or chronic forms of disease. the acute form is characterized by the production of pro-inflammatory cytokines and chemokines that eventually reduce mhv viral load in the cns. however, persistent infection of spinal cord white matter tracts propagates and promotes antiviral responses that cause demyelination leading to symptoms of limp tails and partial to complete hind-limb paralysis, similar to that in eae. in contrast to eae and other viral models of cns demyelinating disease, myelin-specific t cells and epitope spreading are not thought to be required for demyelination; rather it is the persistent effect of antiviral immune responses including macrophages and cd4þ and cd8þ t cells that cause chronic demyelination (reviewed by lane 2010) [49] . in summary, tmev-idd is a demyelinating model that is associated with persistent infection of the cns, whereas sfv infection of sjl mice might represent a model of ms where immune-mediated demyelination is triggered by a virus infection of the cns that is cleared efficiently by the host immune system. although some studies have indicated ms might have a viral aetiology, the mechanisms involved are unknown but might involve an infection in early life that primes autoreactive t cells by molecular mimicry or a persistent infection of the cns that causes demyelination via epitope spreading and/or molecular mimicry [50] . thus, both mouse models allow the study of how viruses might induce cns demeylinating autoimmune disease. in contrast, mhv provides a different scope for study, the underlying mechanisms that mediate host defence against an acute viral infection that later becomes chronic and which is associated with cns demyelination and neurological symptoms in the absence of myelin-specific t-cell responses. guillain-barré syndrome (gbs) is a rapid autoimmune-mediated acute disease of the peripheral nervous system, which is often caused by infection with campylobacter jejuni. clinical disease characteristics usually occur rapidly following onset, usually within 4 weeks, and include neuromuscular paralysis, progressive limb weakness, and deficits of the sensory and autonomic systems as well as cranial nerve involvement. the generation of antibodies to cell-surface gangliosides highly expressed on peripheral nerves by cross-reactivity to campylobacter jejuni outer membrane components (lipo-oligosaccharides) mediates disease [51] and is thought to be caused by postinfectious molecular mimicry (reviewed by shahrizaila 2011) [52] . two forms of gbs have been characterized: (i) a primary demyelinating form (acute inflammatory demyelinating polyradiculoneuropathy [aidp]) and (ii) those with axonal involvement (acute motor axonal neuropathy [aman], acute motorsensory axonal neuropathy [amsan]), with either the presence or absence of antiganglioside antibodies [53, 54] . the pathogenic mechanisms of the aidp form of gbs include the demyelination of the peripheral nerve myelin sheath by inflammation including upregulated adhesion molecules, pro-inflammatory cytokines, and the infiltration of cd4þ t cells and plasma cells secreting anti-ganglioside antibodies and macrophages [53] [54] [55] [56] , whereas the axonal forms of gbs (aman, amsan) usually do not include lymphocyte or monocyte involvement, rather wallerian degeneration as well as complement and anti-ganglioside antibodies that directly mediate myelin destruction [57] . disease is often acute and the recovery of motor function is common in approximately 60 % of gbs patients. however, in other cases, residual sensory deficits remain, and in extreme cases, severe alterations to the autonomic nervous system can cause death by respiratory failure, embolism, or cardiac arrest. current treatment of gbs is generally nonspecific and typically includes the administration of intravenous immunoglobulins or plasmapheresis, which reduce the duration to recovery. however, neither of these treatments is curative, and therefore, the use of animal models of gbs to identify pathogenic mechanisms and to test novel treatments is important. experimental autoimmune neuritis (ean) is a commonly used, robust, and highly predictable animal model of gbs. it was originally developed in rabbits [58] , but has since been induced in a wide variety of animals including rats, mice, rabbits, guinea pigs, and monkeys. disease is induced by the active immunization of susceptible animals with purified whole peripheral nerve myelin or specific myelin protein components (myelin protein 2 [p2], myelin protein zero [p0]) in complete freund's adjuvant [58] [59] [60] . ean in rats and mice is a monophasic acute demyelinating inflammatory disease of the peripheral nervous system [58] with many similarities to gbs including clinical, immunological, and morphological characteristics. in rats, onset of disease is observed at 12-13 post-immunization with a peak of disease severity at day 16. clinical disease is characterized by tail and limb weakness, and histopathological analyses have shown the presence of nerve oedema and demyelinated peripheral nerves accompanied by the infiltration of inflammatory cells, features which are present in gbs. symptoms are likely to be caused by a combination of nerve conduction block and the effects of demyelination. during the effector phase of ean disease, typical pro-inflammatory components including chemokines (mip-1α and mip-1β, mcp-1, rantes, and ip-10) (reviewed by fujioka 1999) [61, 62] , cytokines (reviewed by zhu 1998) [63] , and adhesion molecules (vcam-1) [64] have been shown to be upregulated. th1-type cytokines might mediate the demyelination of peripheral nerves as evidence shows that the addition of ifn-γ can enhance ean, whereas blockade of ifn-γ with neutralizing antibodies can ameliorate ean symptoms and disease course [65] . ean mouse models with severe clinical symptoms and pathological features similar to rat ean have also been reported. an early study reported the induction of ean sjl/j mice, which showed subclinical damage to peripheral nerve myelin but without clinical symptoms, in contrast to ean lewis rats, that developed typical hind-limb weakness and histopathology of the peripheral nervous system [66] . a subsequent study demonstrated that the addition of pertussis toxin to sjl/j mice immunized with bovine peripheral nerve myelin in complete freund's adjuvant enhanced the mild disease seen in the absence of pertussis toxin [67] . when immunized mice with pertussis toxin were treated with recombinant mouse il-12, the disease course duration was prolonged and recovery was delayed. histological analysis demonstrated severe demyelination of the caudae equinae and sciatic nerves during the recovery stage as well as mononuclear cell infiltration. of note, although c57bl/6 mice were initially thought to be resistant to ean induction, immunization of male c57bl/6 mice with a synthetic p0 180-199 peptide induced the clinical and pathological characteristics of acute monophasic ean [68] . as previously shown, the addition of intravenously administrated pertussis toxin increased the incidence of ean and enhanced inflammation and demyelination of peripheral nerves. similar to that seen for eae, in addition to immunization models of ean, adoptive transfer models of ean have been reported. p2-and p0-specific cd4þ t cell lines have been shown to transfer histopathologically similar ean to naïve syngeneic lewis rat recipients when injected intravenously [69] [70] [71] . however, the onset of disease was earlier (day 7) in a p2-adoptive transfer ean model compared with active immunization ean models (day 12-13). although ean is a robust, reproducible model of neuritis, sharing many pathological and clinical features with gbs, one critical disadvantage is that campylobacter jejuni infection, which is thought to be involved in a high percentage of human cases of gbs, does not induce disease in rats. furthermore, immunization of rats with various gangliosides, the main immunological target of the immune system in campylobacter jejuni-induced gbs, also does not induce disease [72] . the addition of gangliosides to immunization protocols failed to have an enhancing effect of ean severity or disease onset. although other species of animals have shown some promise in terms of developing conduction block (rabbits immunized with gm1 demonstrated sciatic nerve conduction block [73] , 33/100 chickens administered campylobacter jejuni isolated from a chinese patient showed sciatic nerve wallerian degeneration with minor demyelination) [74] , these models have not been studied extensively. therefore, the existing ean model is useful for studies related to the effector phase immune-mediated mechanisms of peripheral nerve demyelination; it is less useful for studies to determine the pathogenic mechanisms involved following campylobacter jejuni infection. myasthenia gravis (mg) is a rare neuromuscular disease that causes excessive fatigue and generalized muscle weakness that can fluctuate and is characterized by clinical symptoms including ptosis and diplopia. muscle weakness often worsens upon use but usually improves with rest. as the disease course of mg progresses, bulbar and respiratory muscle weakness worsens, which can become life-threatening, often requiring the use of mechanical ventilation by intubation. mg is thought to be a t cell-dependent antibody-mediated autoimmune disease because approximately 80 % of mg patients have autoantibodies against acetylcholine receptors (achr) [75, 76] , possibly as a consequence of the loss of "self"tolerance in the thymus [77] . achr antibodies bind to achr expressed at neuromuscular junctions (nmj) and block neuromuscular transmission. interestingly, the first experimental evidence to suggest achr antibodies might be pathogenic was demonstrated using an experimental rabbit model where immunization with purified acetylcholine receptor in complete freund's adjuvant induced the production of antibodies to acetylcholine receptor, which mediated neuromuscular blockade that caused flaccid paralysis and mg-like symptoms [78] . despite the early identification of achr antibodies as potential mediators of disease, the mechanisms involved that precede the production of achr antibodies are less clear, although involvement of the thymus has been indicated. the symptomatic treatment of mg using acetylcholinesterase inhibitors and/or immunosuppressive drugs can improve muscle function although these treatments are limited by either a reduction in efficacy over time or severe side effects. furthermore, plasma exchange and surgery in patients that develop thymoma are invasive procedures. currently, no treatments directly target the autoimmune component of disease, and therefore, there is a still a need for the further elucidation of mg pathogenesis and the development of novel drugs, which highlights the importance of using animal models of mg. experimental autoimmune myasthenia gravis (eamg) was originally induced in rabbits by immunization of highly purified achr isolated from the electric organ of electrophorus electricus emulsified in complete freund's adjuvant [78] . this induced the production of antibodies that specifically recognize achr and bind to these receptors at nmjs, subsequently blocking neurotransmission, causing muscle fatigue and weakness, which mimic the symptoms observed in human mg. since the first description of eamg, it has been induced in a wide variety of animals including rabbits [78, 79] rats [80] , mice [81, 82] , guinea pigs [80] , goats [83] , monkeys [84] , and frogs [85] . currently, rat and mouse models are the animal models of choice, as gene knockout and transgenic technology has allowed a greater in-depth investigation into the specific molecules involved in disease pathogenesis. susceptible rat strains include lewis, fischer, and wistar-munich rats, and nonresponder strains include wistar furth and copenhagen rats. it is important to note that some mouse strains have different susceptibilities to eamg; h-2 b, s haplotype strains (c57bl/6 and sjl/j) are high responders, whereas h-2 k, p haplotypes are nonresponders; therefore, it is important to determine the strain used before initiating studies [81, 86] . interestingly, rats appear to be more susceptible to eamg than mice, as usually only one immunization with achr in complete freund's adjuvant is required to induce autoantibodies to achr. in contrast, susceptible mouse strains usually require two or three immunizations with achr in complete freund's adjuvant. furthermore, disease severity in mouse eamg is reduced compared with the rat model, and therefore, this is an important consideration if the efficacy of novel therapeutic agents is to be tested. of note, susceptibility to both mg and eamg is linked to the hla/mhc region [87] . thus, eamg has many clinical and pathological similarities to human mg, especially the presence of autoantibodies that recognize and bind to achr. following immunization, t cells mediate the production of achr-specific antibodies in murine eamg as well as human mg [88] [89] [90] , and treatment with anti-cd4 or anti-ia antibodies can block the induction or induce remission of eamg [89, 91] . further evidence for a role of t cells in eamg pathogenesis was shown by the use of lymphocyte immunosuppressive agents and oral tolerance to achr that inhibited disease onset [92] [93] [94] . similar to that observed in eae, the role of pro-inflammatory cytokines including il-1, il-12, ifn-γ, and tnf-α is also important in the onset of eamg, with functions related to t-cell development, proliferation, and differentiation. studies in eamg demonstrated that treatment of eamg rats or mice with anti-tnf-α treatments reduced eamg development [95] and significantly improved established disease [96] . confirmation of a role for tnf in mg was demonstrated by a trial investigating the use of a tnf inhibitor (etanercept) in mg patients that reduced muscle weakness [97] . once the autoreactive t cells become activated, they stimulate b cells to produce and secrete anti-achr antibodies that induce the observed clinical symptoms. eamg studies have indicated that following the binding of achr antibodies to nmjs, complement activation including c3 and c9 deposition and membrane attack complex might mediate the destruction of nmj plasma membranes [98] [99] [100] . confirmation of a role for complement in eamg was shown in studies reporting that the blockade of the complement system by complement inhibitors protected rats against the induction of eamg [101] [102] [103] . importantly, the role of complement was indicated in human mg [99, 100, 104] , indicating the validity of eamg. clinical signs of active eamg (tremor, hunched posture, muscle weakness, and fatigue) usually occur 3-10 days after the second immunization, and mice are observed for signs of muscle weakness by the paw-grip test at least once a week. in addition, a number of other tests have been developed to assess the extent of disease including the quantitative measurement of muscle weakness by electromyography, the evaluation of eamg induction by the quantification of muscle achr loss, and serum anti-achr antibody levels by radioimmunoassay or elisa. therefore, active eamg is a useful model for myasthenia gravis and the extent of disease can be measured using fairly noninvasive techniques. another method for the induction of eamg is the passive transfer model, where autoantibodies specific for achr from donor achr-immunized animals are injected daily into naïve animals, and this was first shown in a rat model [83, 105] . another method for the passive induction of eamg is the injection of achr-specific antibodies isolated from the serum of mg patients [106] . interestingly, the transfer of eamg by autoreactive lymphocytes is less robust than that by autoantibodies [107] and indicates that autoreactive antibodies are probably the major mechanism involved in the onset of nmj destruction. in summary, active eamg is useful for investigating the induction phase of disease (t-and b-cell activation and autoantibody production) including loss of selftolerance, mechanisms of antigen-specific immune response induction, and their modulation by therapeutic agents (to induce tolerance or immunosuppression), whereas passive eamg is useful for the study of the effector phase of disease including igg deposition in nmjs, complement molecules, and investigating treatments using regulatory proteins to prevent the degradation of nmjs. clinical disease in the passive model of eamg is similar to that observed in active eamg and therefore serves as a useful model of mg when full activation of the immune system is not required, as is seen following active immunization protocols. although most mg patients develop autoreactive antibodies to achr, approximately 20 % of mg patients are achr antibody negative; however, 30-40 % of these mg patients have antibodies that recognize muscle-specific kinase (musk) [108] . musk is a tyrosine kinase receptor that is involved in the development of postsynaptic membranes in the nmj. however, whether musk antibodies are involved in the pathogenicity of mg is unclear and it is unknown whether they contribute to muscle weakness. therefore, the use of animal models is useful to help dissect the potential pathogenic function of musk antibodies. a recent study demonstrated that immunization of rabbits or mice with a musk ectodomain induced muscle weakness as measured by electromyographic analysis and flaccid paralysis, which was similar to that in human mg [109, 110] . however, the appearance of disease-related symptoms is longer than that for achr-active and achr-passive models of eamg, and the passive transfer of musk antibodies is less effective than the equivalent achr-passive model. therefore, musk antibody-related mg might represent a subtype of mg or reflect differences in environmental or genetic susceptibility factors. eamg is a very useful animal model for the study of the pathways involved in disease pathogenesis as well as investigating novel therapeutic strategies. importantly, eamg shares similar symptoms with mg, especially muscle weakness and fatigue. furthermore, they share such immunopathological features as achrspecific antibodies in the serum, muscle achr loss, presence of complement factors such as c3 and c9 at nmjs, and a supportive role for t and b cells as regards autoantibody production. one of the major differences between eamg and mg is the involvement of the thymus (loss of self-tolerance) in human mg, which is absent in eamg, where tolerance must be "broken" by immunization of the autoantigen in complete freund's adjuvant. although many therapeutic strategies have been demonstrated to be beneficial in eamg, few have translated to the clinic for human mg. this might be due to the human form of disease having a more complex aetiology than eamg. however, eamg still has an important role to play in preclinical studies, whether to investigate pathogenic mechanisms or potential therapeutic strategies. despite the numerous beneficial advances of researchers in each of these human neuroimmune diseases, the underlying mechanisms of many of these disease processes are still unclear and this has hindered the search for curative therapies. although the use of human tissues and samples is critical for these types of studies, often they are invasive and provide an indication of a single time point within a disease process that might have been ongoing for years or decades. therefore, it is vital to use animal models to fill in the "gaps" that cannot be analysed by research using human tissues. however, whilst animal models of disease can provide useful information, it is important to note that no single model is a perfect model of its human counterpart and each has their advantages and disadvantages. therefore, it is important to use the correct model for the study involved, i.e. immune cell activation, mechanisms of demyelination, immune cell trafficking into the target organ, induction vs effector phases of disease or routine drug testing. in addition, it is important to remember that each mouse strain used is genetically representative of a single human individual; therefore, multiple strains should ideally be used when developing novel treatments or investigating pathogenic mechanisms, to reduce the possibility of strain irregularities confounding the results. incidence and significance of neutralizing antibodies to interferon beta-1a in multiple sclerosis. multiple sclerosis collaborative research group (mscrg) 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development of experimental autoimmune myasthenia gravis anti-tnf-alpha antibodies suppress the development of experimental autoimmune myasthenia gravis treatment of experimental autoimmune myasthenia gravis with recombinant human tumor necrosis factor receptor fc protein etanercept treatment in corticosteroid dependent myasthenia gravis ultrastructural localization of immune complexes (igg and c3) at the end-plate in experimental autoimmune myasthenia gravis ultrastructural localization of the terminal and lytic ninth complement component (c9) at the motor end-plate in myasthenia gravis myasthenia gravis: quantitative immunocytochemical analysis of inflammatory cells and detection of complement membrane attack complex at the end-plate in 30 patients inhibition of acute passive transfer experimental autoimmune myasthenia gravis with fab antibody to complement c6 novel complement inhibitor limits severity of experimentally myasthenia gravis anti-c5 antibody treatment ameliorates weakness in experimentally acquired myasthenia gravis immune complexes (igg and c3) at the motor end-plate in myasthenia gravis: ultrastructural and light microscopic localization and electrophysiologic correlations passively transferred experimental autoimmune myasthenia gravis myasthenia gravis: passive transfer from man to mouse experimental autoimmune myasthenia: cellular and humoral immune responses auto-antibodies to the receptor tyrosine kinase musk in patients with myasthenia gravis without acetylcholine receptor antibodies induction of myasthenia by immunization against muscle-specific kinase myasthenia gravis experimentally induced with muscle-specific kinase key: cord-015684-q10sx1dm authors: cacabelos, ramón title: pharmacogenomic biomarkers in neuropsychiatry: the path to personalized medicine in mental disorders date: 2009 journal: the handbook of neuropsychiatric biomarkers, endophenotypes and genes doi: 10.1007/978-90-481-2298-1_1 sha: doc_id: 15684 cord_uid: q10sx1dm neuropsychiatric disorders and dementia represent a major cause of disability and high cost in developed societies. most disorders of the central nervous system (cns) share some common features, such as a genomic background in which hundreds of genes might be involved, genome-environment interactions, complex pathogenic pathways, poor therapeutic outcomes, and chronic disability. recent advances in genomic medicine can contribute to accelerate our understanding on the pathogenesis of cns disorders, improve diagnostic accuracy with the introduction of novel biomarkers, and personalize therapeutics with the incorporation of pharmacogenetic and pharmacogenomic procedures to drug development and clinical practice. the pharmacological treatment of cns disorders, in general, accounts for 10–20% of direct costs, and less than 30–40% of the patients are moderate responders to conventional drugs, some of which may cause important adverse drugs reactions (adrs). pharmacogenetic and pharmacogenomic factors may account for 60–90% of drug variability in drug disposition and pharmacodynamics. approximately 60–80% of cns drugs are metabolized via enzymes of the cyp gene superfamily; 18% of neuroleptics are major substrates of cyp1a2 enzymes, 40% of cyp2d6, and 23% of cyp3a4; 24% of antidepressants are major substrates of cyp1a2 enzymes, 5% of cyp2b6, 38% of cyp2c19, 85% of cyp2d6, and 38% of cyp3a4; 7% of benzodiazepines are major substrates of cyp2c19 enzymes, 20% of cyp2d6, and 95% of cyp3a4. about 10–20% of caucasians are carriers of defective cyp2d6 polymorphic variants that alter the metabolism of many psychotropic agents. other 100 genes participate in the efficacy and safety of psychotropic drugs. the incorporation of pharmacogenetic/ pharmacogenomic protocols to cns research and clinical practice can foster therapeutics optimization by helping to develop cost-effective pharmaceuticals and improving drug efficacy and safety. to achieve this goal several measures have to be taken, including: (a) educate physicians and the public on the use of genetic/ genomic screening in the daily clinical practice; (b) standardize genetic testing for major categories of drugs; (c) validate pharmacogenetic and pharmacogenomic procedures according to drug category and pathology; (d) regulate ethical, social, and economic issues; and (e) incorporate pharmacogenetic and pharmacogenomic procedures to both drugs in development and drugs in the market to optimize therapeutics. central nervous system (cns) disorders are the third problem of health in developed countries, representing 10-15% of deaths, after cardiovascular disorders (25-30%) and cancer (20-25%) . approximately, 127 million europeans suffer brain disorders. the total annual cost of brain disorders in europe is about €386 billion, with €135 billion of direct medical expenditures (€78 billion, inpatients; €45 billion, outpatients; €13 billion, pharmacological treatment), €179 billion of indirect costs (lost workdays, productivity loss, permanent disability), and €72 billion of direct non-medical costs. mental disorders represent €240 billion (62% of the total cost, excluding dementia), followed by neurological diseases (€84 billion, 22%). 1 senile dementia is becoming a major problem of health in developed countries, and the primary cause of disability in the elderly. alzheimer's disease (ad) is the most frequent form of dementia (50-70%), followed by vascular dementia (30-40%) , and mixed dementia (15-20%) . these prevalent forms of agerelated neurodegeneration affect more than 25 million people at present, and probably more than 75 million people will be at risk in the next 20-25 years worldwide. the prevalence of dementia increases exponentially from approximately 1% at 60-65 years of age to more than 30-35% in people older than 80 years. it is very likely that in those patients older than 75-80 years of age most cases of dementia are mixed in nature (degenerative + vascular), whereas pure ad cases are very rare after 80 years of age. the average annual cost per person with dementia ranges from €10,000 to 40,000, depending upon disease stage and country, with a lifetime cost per patient of more than €150,000. in some countries, approximately 80% of the global costs of dementia (direct + indirect costs) are assumed by the patients and/or their families. about 10-20% of the costs in dementia are attributed to pharmacological treatment, including anti-dementia drugs, psychotropics (antidepressants, neuroleptics, anxiolytics), and other drugs currently prescribed in the elderly (antiparkinsonians, anticonvulsants, vasoactive compounds, antiinfl ammatory drugs, etc). in addition, during the past 20 years more than 300 drugs have been partially or totally developed for ad, with the subsequent costs for the pharmaceutical industry, and only 5 drugs with moderate-to-poor effi cacy and questionable cost-effectiveness have been approved in developed countries. [2] [3] [4] the lack of accurate diagnostic markers for early prediction and an effective therapy of cns disorders are the two most important problems to effi ciently diagnose and halt disease progression. the pharmacological treatment of cns disorders, in general, accounts for 10-20% of direct costs, and less than 30-40% of the patients are moderate responders to conventional drugs, some of which may cause important adverse drugs reactions (adrs). in the case of dementia, less than 20% of the patients can benefi t from current drugs (donepezil, rivastigmine, galantamine, memantine), with doubtful cost-effectiveness. the pathogenic mechanisms of most cns disorders (e.g., psychosis, depression, anxiety, alzheimer's disease, parkinson's disease, huntington's disease, multiple sclerosis, etc) are poorly understood. this circumstance makes it diffi cult the implantation of a molecular intervention to neutralize causative factors. in fact, more than 80% of the 25,000 genes integrating the human genome are expressed in the cns at different periods of the life span, and only a few neurotransmitters (e.g., noradrenaline, dopamine, acetylcholine, gaba, histamine, and less than ten neuropeptides) are the actual targets of conventional psychopharmacology. common features in cns disorders include the following: (a) polygenic/ complex disorders in which genomic and environmental factors are involved; (b) deterioration of higher activities of the cns; (c) multifactorial dysfunctions in several brain circuits; and (d) accumulation of toxic proteins in the nervous tissue in cases of neurodegeneration. for instance, the neuropathological hallmark of alzheimer's disease (ad) (amyloid deposition in senile plaques, neurofi brillary tangle formation, and neuronal loss) is but the phenotypic expression of a pathogenic process in which more than 200 genes and their products are potentially involved. drug metabolism, and the mechanisms underlying drug effi cacy and safety, are also genetically regulated complex traits in which hundreds of genes cooperatively participate. structural and functional genomics studies demonstrate that genomic factors, probably induced by environmental factors, cerebrovascular dysfunction, and epigenetic phenomena, might be responsible for pathogenic events leading to premature neuronal dysfunction and/or death. pharmacogenetic and pharmacogenomic factors may account for 60-90% of drug variability in drug disposition and pharmacodynamics. about 10-20% of caucasians are carriers of defective cyp2d6 polymorphic variants that alter the metabolism of many psychotropic agents. the incorporation of pharmacogenetic/pharmacogenomic protocols to cns research and clinical practice can foster therapeutics optimization by helping to develop cost-effective pharmaceuticals and improving drug effi cacy and safety. [5] [6] [7] extensive molecular genetics studies carried out in the past 2 decades have demonstrated that most cns disorders are multifactorial, polygenic/complex disorders in which hundreds of genes distributed across the human genome might be involved (tables 40.1-40. 3). 8, 9 for example, 255 genes have been associated with dementia (table 40 .1), 205 with schizophrenia (table 40. 2), 106 with depression (table 40. 3), 107 with anxiety, 103 with stroke, 385 with different types of ataxia, 155 with epilepsiy, 83 with meningioma, 105 with glioblastoma, 27 with astrocytoma, 73 with parkinson's disease, and more than 30 genes with cerebrovascular disorders. 8, 10 many of these genetic associations could not be replicated in different settings and different populations due to many complex (methodological, technological) factors. 8, 11, 12 furthermore, the same genomic defect can give rise to apparent diverse phenotypes, and different genomic defects can converge in an apparently common phenotype, this increasing the complexity of genomic studies (e.g., patient recruitment, pure controls, concomitant pathology, epigenetic factors, environmental factors). several candidate genes for schizophrenia may also be associated with bipolar disorder, including g72, disc1, nrg1, rgs4, ncam1, dao, grm3, grm4, grin2b, mlc1, syngr1, and slc12a6. genes associated with bipolar disorder include trpm2 (21q22.3), gpr50 (xq28), citron (12q24), chp1.5 (18p11.2), gchi (14q22-24), mlc1 (22q13), gabra5 (15q11-q13), bcr (22q11), cux2, flj32356 (12q23-q24), and napg (18p11). 9 another paradigmatic example of heterogeneity and complexity is dementia, one of the most heterogeneous disorders of the cns. the genetic defects identifi ed in ad during the past 25 years can be classifi ed into three main categories: (a) mendelian or mutational defects in genes directly linked to ad, including (i) 32 mutations in the amyloid beta (aβ)(abp) precursor protein (app) gene (21q21); (ii) 165 mutations in the presenilin 1 (ps1) gene (14q24.3); and (iii) 12 mutations in the presenilin 2 (ps2) gene (1q31-q42) 8, 10, 13 (table 40 .1). (b) multiple polymorphic variants of risk characterized in more than 200 different genes distributed across the human genome can increase neuronal vulnerability to premature death 8 (table 40 .1). among these genes of susceptibility, the apolipoprotein e (apoe) gene (19q13.2) is the most prevalent as a risk factor for ad, especially in those subjects harbouring the apoe-4 allele, whereas carriers of the apoe-2 allele might be protected against dementia. 8 apoe-related pathogenic mechanisms are also associated with brain aging and with the neuropathological hallmarks of ad. 8 (c) diverse mutations located in mitochondrial dna (mtdna) through heteroplasmic transmission can infl uence aging and oxidative stress conditions, conferring phenotypic heterogeneity. 8, 14, 15 it is also likely that defective functions of genes associated with longevity may infl uence premature neuronal survival, since neurons are potential pacemakers defi ning life span in mammals. 8 all these genetic factors may interact in still unknown genetic networks leading to a cascade of pathogenic events characterized by abnormal protein processing and misfolding with subsequent accumulation of abnormal proteins (conformational changes), ubiquitin-proteasome system dysfunction, excitotoxic reactions, oxidative and nitrosative stress, mitochondrial injury, synaptic failure, altered metal homeostasis, dysfunction of axonal and dendritic transport, and chaperone misoperation 8,16-20 ( fig. 40.1 ). these pathogenic events may exert an additive effect, converging in fi nal pathways leading to premature neuronal death. some of these mechanisms are common to several neurodegenerative disorders which differ depending upon the gene(s) affected and the involvement of specifi c genetic networks, together with cerebrovascular factors, epigenetic factors (dna methylation) and environmental conditions (nutrition, toxicity, social factors, etc). 8, [16] [17] [18] [19] [20] [21] [22] the higher the number of genes involved in ad pathogenesis, the earlier the onset of the disease, the faster its clinical course, and the poorer its therapeutic outcome. 8, [16] [17] [18] [19] [20] high throughput microarray gene expression profi ling is an effective approach for the identifi cation of candidate genes and associated molecular pathways implicated in a wide variety of biological processes or disease states. the cellular complexity of the cns (with 10 3 different cell types) and synapses (with each of the 10 11 neurons in the brain having around 10 3 -10 4 synapses with a complex multiprotein structure integrated by 10 3 different proteins) requires a very powerful technology for gene expression profi ling, which is still in the very early stages and is not devoid of technical obstacles and limitations. 23 transcripts of 16,896 genes have been measured in different cns regions. each region possess its own unique transcriptome fi ngerprint that is independent of age, gender and energy intake. less than 10% of genes are affected by age, diet or gender, with most of these changes occurring between middle and old age. gender and energy restriction have robust infl uences on the hippocampal transcriptome of middle-aged animals. prominent functional groups of age-and energy-sensitive genes are those encoding proteins involved in dna damage responses, mitochondrial and proteasome functions, cell fate determination and synaptic vesicle traffi cking. the systematic transcriptome dataset provides a window into mechanisms of neuropathogenesis and cns vulnerability. 24 with the advent of modern genomic technologies, new loci have been associated with different neuropsychiatric disorders, and novel pathogenic mechanisms have been postulated. cryptic chromosome imbalances are increasingly acknowledged as a cause for mental retardation and learning disability. with subtelomeric screening, nine chromosomal anomalies and submicroscopic deletions of 1pter, 2qter, 4pter, 5qter and 9qter have been identifi ed in patients with mental retardation. 25 increased dna fragmentation was observed in non-gabaergic neurons in bipolar disorder, suggesting that non-gabaergic cell may be selectively vulnerable to oxidative stress and apoptosis in patients with bipolar disorder. 26 [17] [18] [19] [20] with laser microdissection, rna amplifi cation, and array hybridization, expression of more than 1,000 genes was detected in ca1 and ca3 hippocampal neurons under normoxic conditions. the comparison of each region under normoxic and ischemic conditions revealed more than 5,000 ischemia-regulated genes for each individual cell type. 27 microarray technology has helped to elucidate gene expression profi les and potential pathogenic mechanisms in many other cns disorders including schizophrenia and bipolar disorder, [28] [29] [30] speech and language disorders, 31 parkinson's disease, 32, 33 huntington's disease, 34 prion disease, 35 drug addiction, 36,37 alcoholism, 38 brain trauma, 39 epilepsy, [40] [41] [42] cockayne syndrome, 43 rett syndrome, 44 friedreich ataxia, 45 neuronal ceroid lipofuscinosis, 46 multiple sclerosis, 47 amyotrophic lateral esclerosis, 48 acute pneumococcal meningitis, 49 and the role of lipids in brain injury, psychiatric disorders, and neurodegenerative diseases. [50] [51] [52] interactions between genomic factors and environmental factors have been proposed as important contributors for brain neuropathology. in schizophrenia, neurodevelopmental disturbances, neurotoxins and perinatal infections, myelin-and olygodendrocytes abnormalities and synaptic dysfunctions have been suggested as pathophysiological factors. individual genotoxicants can induce distinct gene expression signatures. exposure of the brain to environmental agents during critical periods of neuronal development can alter neuronal viability and differentiation, global gene expression, stress and immune response, and signal transduction. 53 the binomial genome-neurotoxicants effect can be documented in cases of drug abuse or alcohol dependence. functional gene expression differences between inbred alcohol-preferring and nonpreferring rats suggest the presence of powerful genomic infl uences on alcohol dependence. 54 alcohol dependence and associated cognitive impairment may result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. it has been suggested that cycles of alcohol intoxication/withdrawal, which may initially activate nuclear factor-kappa b (nf-κb), when repeated over years downregulate p65 (rela) mrna expression and nf-κb and p50 homodimer dna-binding. downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently results in depression of κb regulated genes. alterations in expression of p50 homodimer/nf-κb regulated genes may contribute to neuroplastic adaptation underlying alcoholism. 55 gene expression profi ling of the nucleus accumbens of cocaine abusers suggests a dysregulation of myelin. 56 humans who abused cocaine, cannabis and/or phencyclidine share a decrease in transcription of calmodulin-related genes and increased transcription related to lipid/cholesterol and golgi/er function. 57 another important issue in the pathogenesis and therapeutics of cns disorders is the role of micror-nas (mirnas). mirnas are small (22 nucleotide), endogenous noncoding rna molecules that posttranscriptionally regulate expression of protein-coding genes. computational predictions estimate that the vertebrate genomes may contain up to 1,000 mirna genes. mirnas are generated from long primary transcripts that are processed in multiple steps to cytoplasmic 22 nucleotide mature mirnas. the mature mirna is incorporated into the mirna-induced silencing complex (mirisc), which guides it to target sequences located in 3′ utrs where by incomplete base-pairing induce mrna destabilization or translational repression of the target genes. an inventory of mirna expression profi les from 13 regions of the mouse cns has been reported. 58 this inventory of cns mirna profi les provides an important step toward further elucidation of mirna function and mirna-related gene regulatory networks in the mammalian cns. 58 the introduction of novel procedures into an integral genomic medicine protocol for cns disorders is an imperative requirement in drug development and in the clinical practice to improve diagnostic accuracy and to optimize therapeutics. this kind of protocol should integrate the following components: (i) clinical history, (ii) laboratory tests, (iii) neuropsychological assessment, (iv) cardiovascular evaluation, (v) conventional x-ray technology, (vi) structural neuroimaging, (vii) functional neuroimaging, (viii) computerized brain electrophysiology, (ix) cerebrovascular evaluation, (x) structural genomics, (xi) functional genomics, (xii) pharmacogenetics, (xiii) pharmacogenomics, (ix) nutrigenetics, (x) nutrigenomics, (xi) bioinformatics for data management, and (xii) artifi cial intelligence procedures for diagnostic assignments and probabilistic therapeutic options (table 40 .4). 2, 8, [16] [17] [18] [19] [20] [21] [22] 59, 60 all these procedures, under personalized strategies adapted to the complexity of each case, are essential to depict a clinical profi le based on specifi c biomarkers correlating with individual genomic profi les. functional genomics studies have demonstrated the infl uence of many genes on cns pathogenesis and phenotype expression (tables 40.1-40. 3). taking ad as an example, it has been demonstrated that mutations in the app, ps1, ps2, and mapt genes give rise to wellcharacterized differential neuropathological and clinical phenotypes of dementia. 8 the analysis of genotypephenotype correlations has also revealed that the presence of the apoe-4 allele in ad, in conjunction with other genes, infl uences disease onset, brain atrophy, cerebrovascular perfusion, blood pressure, β-amyloid deposition, apoe secretion, lipid metabolism, brain bioelectrical activity, cognition, apoptosis, and treatment outcome. 8, [16] [17] [18] [19] [20] [21] [22] 61 the characterization of phenotypic profi les according to age, cognitive performance (mmse and adas-cog score), serum apoe levels, serum lipid levels including cholesterol (cho), hdl-cho, ldl-cho, vldl-cho, and triglyceride (tg) levels, as well as serum nitric oxide (no), β-amyloid, and histamine levels, reveals sex-related differences in 25% of the biological parameters and almost no differences (0.24%) when patients are classifi ed as apoe-4(−) and apoe-4(+) carriers, probably indicating that genderrelated factors may infl uence these parametric variables more powerfully than the presence or absence of the apoe-4 allele; in contrast, when patients are classifi ed according to their apoe genotype, dramatic differences emerge among apoe genotypes (>45%), with a clear biological disadvantage in apoe-4/4 carriers who exhibit (i) earlier age of onset, (ii) low apoe levels, (iii) high cho and ldl-cho levels, and (iv) low no, β-amyloid, and histamine levels in blood. 8, [16] [17] [18] [19] [20] [21] [22] 61 these phenotypic differences are less pronounced when ad patients are classifi ed according to their ps1 (15.6%) or ace genotypes (23.52%), refl ecting a weak impact of ps1-and ace-related genotypes on the phenotypic expression of biological markers in ad. ps1related genotypes appear to infl uence age of onset, blood histamine levels and cerebrovascular hemodynamics, as refl ected by signifi cant changes in systolic (sv), diastolic (dv), and mean velocities (mv) in the left middle cerebral arteries (mca). 19 ace-related phenotypes seem to be more infl uential than ps1 genotypes in defi ning biological phenotypes, such as age of onset, cognitive performance, hdl-cho levels, ace and no levels, and brain blood fl ow mv in mca. however, when apoe and ps1 genotypes are integrated in bigenic clusters and the resulting bigenic genotypes are differentiated according to their corresponding phenotypes, an almost logarithmic increased expression of differential phenotypes is observed (61.46% variation), indicating the existence of a synergistic effect of the bigenic (apoe + ps1) cluster on the expression of biological markers, apparently unrelated to app/ps1 mutations, since none of the patients included in the sample were carriers of either app or ps1 mutations. 19, 61, 62 these examples illustrate the potential additive effects of ad-related genes on the phenotypic expression of biological markers. furthermore, the analysis of genotype-phenotype correlations with a monogenic or bigenic approach documents a modest genotype-related variation in serum amyloid-β (abp) levels, suggesting that peripheral levels of abp are of relative value as predictors of disease-stage or as markers of disease progression and/ or treatment-related disease-modifying effects. 19, 61, 62 the peripheral levels of abp in serum exhibit an apoe-dependent pattern according to which both apoe-4(+) and apoe-2(+) carriers tend to show higher abp levels than apoe-4(−) or apoe-3 carriers 19,61-63 ( fig. 40 est concentration of serum histamine is systematically present in apoe-2(+) and apoe-4(+) carriers, and the highest levels of histamine are seen in apoe-3(+) carriers ( fig. 40.3 ). central and peripheral histaminergic mechanisms may regulate cerebrovascular function in ad, which is signifi cantly altered in apoe-4/4 carriers. 19, [61] [62] [63] [64] [65] [66] these observations can lead to the conclusion that the simple quantifi cation of biochemical markers in fl uids or tissues of ad patients with the aim of identifying pathogenic mechanisms and/or monitoring therapeutic effects, when they are not accompanied by differential genotyping for sample homogenization, are of very poor value. differential patterns of apoe-, ps1-, ps2-, and trigenic (apoe + ps1 + ps2) cluster-related lymphocyte apoptosis have been detected in ad. fas receptor expression is signifi cantly increased in ad, especially in apoe-4 carriers where lymphocyte apoptosis is more relevant. 19, 67 it has been demonstrated that brain activity slowing correlates with progressive gds staging in dementia 8, 16, [18] [19] [20] (fig. 40.4 ). in the general population subjects harbouring the apoe-4/4 genotype exhibit a premature slowing in brain mapping activity represented by increased slow delta and theta activities as compared with other apoe genotypes. in patients with ad, slow activity predominates in apoe-4 carriers with similar gds stage 8,16,18-20 ( fig. 40.4) . ad patients harbouring the apoe-4/4 genotype also exhibit a dramatically different brain optical topography map refl ecting a genotype-specifi c differential pattern of neocortical oxygenation as well as a poorer activation of cortical neurons in response to somatosensory stimuli ( fig. 40 .5). all these examples of genotype-phenotype correlations, as a gross approach to functional genomics, illustrate the importance of genotype-related differences in ad and their impact on phenotype expression. 8, [16] [17] [18] [19] [20] [21] [22] 62, 63 similar protocols are applied to schizophrenia, depression, anxiety and other neuropsychiatric disorders. most biological parameters, potentially modifi able by monogenic genotypes and/or polygenic cluster profi les, can be used in clinical trials for monitoring effi cacy outcomes. these parametric variables also show a genotypedependent profi le in different types of dementia (e.g., ad vs. vascular dementia). for instance, striking differences have been found between ad and vascular dementia in structural and functional genomics studies. 8, [16] [17] [18] [19] [20] [21] [22] 62, 63 our understanding of the pathophysiology of cns disorders has advanced dramatically in the last 30 years, especially in terms of their molecular pathogenesis and genetics. drug treatment of cns disorders has also made remarkable strides, with the introduction of many new drugs for the treatment of schizophrenia, depression, anxiety, epilepsy, parkinson's disease, and alzheimer's disease, among many other quantitatively and qualita-tively important neuropsychiatric disorders. improvement in terms of clinical outcome, however, has fallen short of expectations, with up to one third of the patients continuing to experience clinical relapse or unacceptable medication-related side effects in spite of efforts to identify optimal treatment regimes with one or more drugs. 68 potential reasons to explain this historical setback might be that: (a) the molecular pathology of most cns disorders is still poorly understood; (b) drug targets are inappropriate, not fi tting into the real etiology of the disease; (c) most treatments are symptomatic, but not anti-pathogenic; (d) the genetic component of most cns disorders is poorly defi ned; and (e) the understanding of genome-drug interactions is very limited. with the advent of recent knowledge on the human genome 69,70 and the identifi cation and characterization of many genes associated with cns disorders, 8, 19 as well as novel data regarding cyp family genes and other genes whose enzymatic products are responsible for drug metabolism in the liver (e.g., nats, abcbs/ mdrs, tpmt), it has been convincingly postulated that the incorporation of pharmacogenetic and pharmacogenomic procedures ( fig. 40 .6) in drug development might bring about substantial benefi ts in terms of therapeutics optimization in cns disorders and in many other complex disorders, assuming that genetic factors are determinant for both neuronal dysregulation (and/or neuronal death) 8,16-22 and drug metabolism. [71] [72] [73] fig. 40.6 effi cacy and safety issues associated with pharmacogenetics and pharmacogenomics (adapted from r. cacabelos 19, 20 ) however, this fi eld is still in its infancy; and the incorporation of pharmacogenomic strategies to drug development and pharmacological screening in cns disorders is not an easy task. the natural course of technical events to achieve effi cient goals in pharmacogenetics and pharmacogenomics include the following steps: (a) genetic testing of mutant genes and/or polymorphic variants of risk; (b) genomic screening, and understanding of transcriptomic, proteomic, and metabolomic networks; (c) functional genomics studies and genotype-phenotype correlation analysis; and (d) pharmacogenetics and pharmacogenomics developments, addressing drug safety and effi cacy, respectively. 8, [16] [17] [18] [19] [20] [21] [22] [74] [75] [76] [77] with pharmacogenetics we can understand how genomic factors associated with genes encoding enzymes responsible for drug metabolism regulate pharmacokinetics and pharmacodynamics (mostly safety issues). [78] [79] [80] with pharmacogenomics we can differentiate the specifi c disease-modifying effects of drugs (effi cacy issues) acting on pathogenic mechanisms directly linked to genes whose mutations determine the disease phenotype. [16] [17] [18] [19] [20] [21] [22] [74] [75] [76] [77] the capacity of drugs to reverse the effects of the activation of pathogenic cascades (phenotype expression) regulated by networking genes basically deals with effi cacy issues. at present, the terms pharmacogenetics and pharmacogenomics are often used interchangeably to refer to studies of the contribution of inheritance to variation in the drug response phenotype 73 ; however, from historical and didactic reasons (until a more suitable and universal defi nition can be established) it would be preferable to maintain the term of pharmacogenetics for the discipline dealing with genetic factors associated with drug metabolism and safety issues, whereas pharmacogenomics would refer to the reciprocal infl uence of drugs and genomic factors on pathogenetic cascades and disease-associated gene expression (effi cacy issues). [18] [19] [20] [21] [22] [74] [75] [76] [77] the application of these procedures to cns disorders is a very diffi cult task, since most neuropsychiatric diseases are complex disorders in which hundreds of genes might be involved 8, [16] [17] [18] [19] [20] [21] [22] [74] [75] [76] [77] (tables 40.1-40.3 ). in addition, it is very unlikely that a single drug be able to reverse the multifactorial mechanisms associated with neuronal dysfunction in most cns processes with a complex phenotype affecting mood, personality, behaviour, cognition, and functioning. this heterogeneous clinical picture usually requires the utilization of different drugs administered simultaneously. this is particularly important in the elderly population. in fact, the average number of drugs taken by patients with dementia ranges from six to more than ten per day depending upon their physical and mental conditions. nursing home residents receive, on average, seven to eight medications each month, and more than 30% of residents have monthly drug regimes of nine or more medications, including (in descending order) analgesics, antipyretics, gastrointestinal agents, electrolytic and caloric preparations, central nervous system (cns) agents, anti-infective agents, and cardiovascular agents. 81 in population-based studies more than 35% of patients older than 85 years are moderate or chronic antidepressant users. 82 polypharmacy, drug-drug interactions, adverse reactions, and non-compliance are substantial therapeutic problems in the pharmacological management of elderly patients, 83 adding further complications and costs to the patients and their caregivers. in 2000-2001, 23.0-36.5% of elderly individuals received at least 1 of 33 potentially inappropriate medications in ten health maintenance organizations (hmos) of the usa. 84 although drug effect is a complex phenotype that depends on many factors, it is estimated that genetics accounts for 20-95% of variability in drug disposition and pharmacodynamics. 79 under these circumstances, therapeutics optimization is a major goal in neuropsychiatric disorders and in the elderly population, and novel pharmacogenetic and pharmacogenomic procedures may help in this endeavour. [16] [17] [18] [19] [20] [21] [22] [74] [75] [76] [77] the pharmacogenomic outcome depends upon many different determinant factors including (i) genomic profi le (family history, ethnic background, disease-related genotype, pharmacogenetic genotype, pharmacogenomic genotype, nutrigenetic genotype, nutrigenomic genotype), (ii) disease phenotype (age at onset, disease severity, clinical symptoms), (iii) concomitant pathology, (iv) genotype-phenotype correlations, (v) nutritional conditions, (vi) age and gender, (vii) pharmacological profi le of the drugs, (viii) drug-drug interactions, (ix) gene expression profi le, (x) transcriptomic cascade, (xi) proteomic profi le, and (xii) metabolomic networking ( fig. 40.7) . the dissection and further integration of all these factors is of paramount importance for the assessment of the pharmacogenomic outcome in terms of safety and effi cacy (figs. 40.8 and 40.9). more than 80% of psychotropic drugs (table 40 .5) are metabolized by enzymes known to be genetically variable, including: (a) esterases: butyrylcholinesterase, paraoxonase/arylesterase; (b) transferases: n-acetyltransferase, sulfotransferase, thiol methyltransferase, thiopurine methyltransferase, catechol-o-methyltransferase, glutathione-s-transferases, udp-glucuronosyltransferases, glucosyltransferase, histamine methyltransferase; (c) reductases: nadph: quinine oxidoreductase, glucose-6-phosphate dehydrogenase; (d) oxidases: alcohol dehydrogenase, aldehydehydrogenase, monoamine oxidase b, catalase, superoxide dismutase, trimethylamine n-oxidase, dihydropyrimidine dehydrogenase; and (e) cytochrome p450 enzymes, such as cyp1a1, cyp2a6, cyp2c8, cyp2c9, cyp2c19, cyp2d6, cyp2e1, cyp3a5 (table 40 .6) and many others. 19, 20 polymorphic variants in these genes can induce alterations in drug metabolism modifying the effi cacy and safety of the prescribed drugs. 85 drug metabolism includes phase i reactions (i.e., oxidation, reduction, hydrolysis) and phase ii conjugation reactions (i.e., acetylation, glucuronidation, sulfation, methylation). 80 the principal enzymes with polymorphic variants involved in phase i reactions are the following: cyp3a4/5/7, cyp2e1, cyp2d6, cyp2c19, cyp2c9, cyp2c8, cyp2b6, cyp2a6, cyp1b1, cyp1a1/2, epoxide hydrolase, esterases, nqo1 (nadph-quinone oxidoreductase), dpd (dihydropyrimidine dehydrogenase), adh (alcohol dehydrogenase), and aldh (aldehyde dehydrogenase). major enzymes involved in phase ii reactions include the following: ugts (uridine 5′-triphosphate glucuronosyl transferases), tpmt (thiopurine methyltransferase), comt (catechol-o-methyltransferase), hmt (histamine methyl-transferase), sts (sulfotransferases), gst-a (glutathion s-transferase a), gst-p, gst-t, gst-m, nat2 (n-acetyl transferase), nat1, and others. 86 polymorphisms in genes associated with phase ii metabolism enzymes, such as gstm1, gstt1, nat2 and tpmt are well understood, and information is also emerging on other gst polymorphisms and on polymorphisms in the udp-glucuronosyltransferases and sulfotransferases. the typical paradigm for the pharmacogenetics of phase i drug metabolism is represented by the cytochrome p-450 enzymes, a superfamily of microsomal nonsteroidal anti-infl ammatory drug drug-metabolizing enzymes. p450 enzymes comprise a superfamily of heme-thiolate proteins widely distributed in bacteria, fungi, plants and animals. the p450 enzymes are encoded in genes of the cyp superfamily (table 40 .6) and act as terminal oxidases in multicomponent electron transfer chains which are called p450containing monooxigenase systems. some of the enzymatic products of the cyp gene superfamily can share substrates, inhibitors and inducers whereas others are quite specifi c for their substrates and interacting drugs. [18] [19] [20] [71] [72] [73] [78] [79] [80] there are more than 200 p450 genes identifi ed in different species. saito et al 87 provided a catalogue of 680 variants among eight cyp450 genes, nine esterase genes, and two other genes in the japanese population. the microsomal, membrane-associated, p450 isoforms cyp3a4, cyp2d6, cyp2c9, cyp2c19, cyp2e1, and cyp1a2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. about 60-80% of the psychotropic agents currently used for the treatment of neuropsychiatric disorders are metabolized via enzymes of the cyp family, especially cyp1a2, cyp2b6, cyp2c8/9, cyp2c19, cyp2d6 and cyp3a4 (table 40 .5). cyp3a4 metabolizes more drug molecules than all other isoforms together. most of these polymorphisms exhibit geographic and ethnic differences. [88] [89] [90] [91] [92] [93] [94] these differences infl uence drug metabolism in different ethnic groups in which drug dosage should be adjusted according to their enzymatic capacity, differentiating normal or extensive metabolizers (ems), poor metabolizers (pms) and ultrarapid metabolizers (ums). most drugs act as substrates, inhibitors or inducers of cyp enzymes. enzyme induction enables some xenobiotics to accelerate their own biotransformation (auto-induction) or the biotransformation and elimination of other drugs. a number of p450 enzymes in human liver are inducible. induction of the majority of p450 enzymes occurs by increase in the rate of gene transcription and involves ligand-activated transcription factors, aryl hydrocarbon receptor, constitutive androstane receptor (car), and pregnane x receptor (pxr). 93, 95 in general, binding of the appropriate ligand to the receptor initiates the induction process that cascades through a dimerization of the receptors, their translocation to the nucleus and binding to specifi c regions in the promoters of cyps. 95 cyps are also expressed in the cns, and a complete characterization of constitutive and induced cyps in brain is essential for understanding the role of these enzymes in neurobiological functions and in age-related and xenobiotic-induced neurotoxicity. 96 assuming that the human genome contains about 20,000-30,000 genes, at the present time only 0.31% of commercial drugs have been assigned to corresponding genes whose gene products might be involved in pharmacokinetic and pharmacodynamic activities of a given drug; and only 4% of the human genes have been assigned to a particular drug metabolic pathway. supposing a theoretical number of 100,000 chemicals in current use worldwide, and assuming that practically all human genes can interact with drugs taken by human beings, each gene in the human genome should be involved in the metabolism and/or biopharmacological effect of 30-40 drugs; however, assuming that most xenobiotic substances in contact with our organism can infl uence genomic function, it might be possible that for 1,000,000 xenobiotics in daily contact with humans, an average of 350-500 xenobiotics have to be assigned to each one of the genes potentially involved in drug metabolism and/or xenobiotics processing. to fulfi l this task a single gene has to possess the capacity of metabolizing many different xenobiotic substances and at the same time many different genes have to cooperate in orchestrated networks to metabolize a particular drug or xenobiotic under sequential biotransformation steps (figs. 40.7 and 40.8). numerous chemicals increase the metabolic capability of organisms by their ability to activate genes encoding various xenochemical-metabolizing enzymes, such as cyps, transferases and transporters. many natural and artifi cial substances induce the hepatic cyp subfamilies in humans, and these inductions might lead to clinically important drug-drug interactions. some of the key cellular receptors that mediate such inductions have been recently identifi ed, including nuclear receptors, such as the constitutive androstane receptor (car, nr1i3), the retinoid x receptor (rxr, nr2b1), the pregnane x receptor (pxr, nr1i3), and the vitamin d receptor (vdr, nr1i1) and steroid receptors such as the glucocorticoid receptor (gr, nr3c1). 97 there is a wide promiscuity of these receptors in the induction of cyps in response to xenobiotics. indeed, this adaptive system acts as an effective network where receptors share partners, ligands, dna response elements and target genes, infl uencing their mutual relative expression. 97,98 the most important enzymes of the p450 cytochrome family in drug metabolism by decreasing order are cyp3a4, cyp2d6, cyp2c9, cyp2c19, and cyp2a6. [85] [86] [87] 94, 99, 100 the predominant allelic variants in the cyp2a6 gene are cyp2a6 * 2 (leu160his) and cyp2a6del. the cyp2a6 * 2 mutation inactivates the enzyme and is present in 1-3% of caucasians. the cyp2a6del mutation results in no enzyme activity and is present in 1% of caucasians and 15% of asians. [18] [19] [20] 86 the most frequent mutations in the cyp2c9 gene are cyp2c9 * 2 (arg144cys), with reduced affi nity for p450 in 8-13% of caucasians, and cyp2c9 * 3 (ile359leu), with alterations in the specifi city for the substrate in 6-9% of caucasians and 2-3% of asians. [18] [19] [20] 86 the most prevalent polymorphic variants in the cyp2c19 gene are cyp2c19 * 2, with an aberrant splicing site resulting in enzyme inactivation in 13% of caucasians, 23-32% of asians, 13% of africans, and 14-15% of ethiopians and saoudians, and cyp2c19 * 3, a premature stop codon resulting in an inactive enzyme present in 6-10% of asians, and almost absent in caucasians. [18] [19] [20] 86, 101 the most important mutations in the cyp2d6 gene are the following: cyp2d6 * 2xn, cyp2d6 * 4, cyp2d6 * 5, cyp2d6 * 10 and cyp2d6 * 17. [18] [19] [20] 96, 102 the cyp2d6 * 2xn mutation gives rise to a gene duplication or multiplication resulting in an increased enzyme activity which appears in 1-5% of the caucasian population, 0-2% of asians, 2% of africans, and 10-16% of ethiopians. the defective splicing caused by the cyp2d6 * 4 mutation inactivates the enzyme and is present in 12-21% of caucasians. the deletion in cyp2d6 * 5 abolishes enzyme activity and shows a frequency of 2-7% in caucasians, 1% in asians, 2% in africans, and 1-3% in ethiopians. the polymorphism cyp2d6 * 10 causes pro34ser and ser486thr mutations with unstable enzyme activity in 1-2% of caucasians, 6% of asians, 4% of africans, and 1-3% of ethiopians. the cyp2d6 * 17 variant causes thr107ile and arg296cys substitutions which produce a reduced affi nity for substrates in 51% of asians, 6% of africans, and 3-9% of ethiopians, and is practically absent in caucasians. [18] [19] [20] 86, 96, 102 the cyp2d6 enzyme, encoded by a gene that maps on 22q13.1-13.2, catalyses the oxidative metabolism of more than 100 clinically important and commonly prescribed drugs such as cholinesterase inhibitors, antidepressants, neuroleptics, opioids, some β-blockers, class i antiarrhythmics, analgesics and many other drug categories, acting as substrates, inhibitors or inducers with which most psychotropics may potentially interact (table 40 .5), this leading to the outcome of adrs. [18] [19] [20] 86, 96, 103 the cyp2d6 locus is highly polymorphic, with more than 100 different cyp2d6 alleles identifi ed in the general population showing defi cient (poor metabolizers, pm), normal (extensive metabolizers, em) or increased enzymatic activity (ultra-rapid metabolizers, um). 100,104 most individuals (>80%) are ems; however, remarkable interethnic differences exist in the frequency of the pm and um phenotypes among different societies all over the world. [18] [19] [20] 89, [91] [92] [93] [94] 102 on the average, approximately 6.28% of the world population belongs to the pm category. europeans (7.86%), polynesians (7.27%), and africans (6.73%) exhibit the highest rate of pms, whereas orientals (0.94%) show the lowest rate. the frequency of pms among middle eastern populations, asians, and americans is in the range of 2-3%. [16] [17] [18] [19] [20] 94 cyp2d6 gene duplications are relatively infrequent among northern europeans, but in east africa the frequency of alleles with duplication of cyp2d6 is as high as 29%. 73 the most frequent cyp2d6 alleles in the european population are the following: cyp2d6 * 1 (wild-type) (normal), cyp2d6 * 2 (2850c > t)(normal), cyp2d6 * 3 (2549a > del)(inactive), cyp2d6 * 4 (1846g > a)(inactive), cyp2d6 * 5 (gene deletion)(inactive), cyp2d6 * 6 (1707t > del)(inactive), cyp2d6 * 7 (2935a > c)(inac-tive), cyp2d6 * 8 (1758g > t)(inactive), cyp2d6 * 9 (2613-2615 delaga)(partially active), cyp2d6 * 10 (100c > t)(partially active), cyp2d6 * 11 (883g > c) (inactive), cyp2d6 * 12 (124g > a)(inactive), cyp2d6 * 17 (1023c > t)(partially active), and cyp2d6 gene duplications (with increased or decreased enzymatic activity depending upon the alleles involved). [16] [17] [18] [19] [20] [104] [105] [106] in the spanish population, where the mixture of ancestral cultures has occurred for centuries, the distribution of the cyp2d6 genotypes differentiates 4 major categories of cyp2d6-related metabolizer types: (i) extensive metabolizers (em)( * 1/ * 1, * 1/ * 10); (ii) intermediate metabolizers (im)( * 1/ * 3, * 1/ * 4, * 1/ * 5, * 1/ * 6, * 1/ * 7, * 10/ * 10, * 4/ * 10, * 6/ * 10, * 7/ * 10); (iii) poor metabolizers (pm)( * 4/ * 4, * 5/ * 5); and (iv) ultra-rapid metabolizers (um)( * 1xn/ * 1, * 1xn/ * 4, dupl). in this sample we have found 51.61% ems, 32.26% ims, 9.03% pms, and 7.10% ums. 20, [74] [75] [76] [77] the distribution of all major genotypes is the following: * 1/ * 1, 47.10%; * 1/ * 10, 4.52%; * 1/ * 3, 1.95%; * 1/ * 4, 17.42%; * 1/ * 5, 3.87%; * 1/ * 6, 2.58%; * 1/ * 7, 0.65%; * 10/ * 10, 1.30%; * 4/ * 10, 3.23%; * 6/ * 10, 0.65%; * 7/ * 10, 0.65%; * 4/ * 4, 8.37%; * 5/ * 5, 0.65%; * 1xn/ * 1, 4.52%; * 1xn/ * 4, 1.95%; and dupl, 0.65%. 20, [74] [75] [76] [77] in some instances, there is association of cyp2d6 variants of risk with genes potentially involved in the pathogenesis of specifi c cns disorders. when comparing ad cases with controls, we observed that ems are more prevalent in ad ( * 1/ * 1, 49.42%; * 1/ * 10, 8.04%)(total ad-ems: 57.47%) than in controls ( * 1/ * 1, 44.12%; * 1/ * 10, 0%)(total c-ems: 44.12%). in contrast, ims are more frequent in controls (41.18%) than in ad (25.29%), especially the * 1/ * 4 (c: 23.53%; ad: 12.64%) and * 4/ * 10 genotypes (c: 5.88%; ad: 1.15%). the frequency of pms was similar in ad (9.20%) and controls (8.82%), and ums were more frequent among ad cases (8.04%) than in controls (5.88%). 20, 74, 75, 77 we have also investigated the association of cyp2d6 genotypes with ad-related genes, such as app, mapt, apoe, ps1, ps2, a2m, ace, agt, fos, and prnp variants. 20, 74, 75, 77 no app or mapt mutations have been found in ad cases. homozygous apoe-2/2 (12.56%) and apoe-4/4 (12.50%) accumulate in ums, and apoe-4/4 cases were also more frequent in pms (6.66%) than in ems (3.95%) or ims (0%). ps1-1/1 genotypes were more frequent in ems (45%), whereas ps-1/2 genotypes were over-represented in ims (63.16%) and ums (60%). the presence of the ps1-2/2 genotype was especially high in pms (38.46%) and ums (20%). a mutation in the ps2 gene exon 5 (ps2e5+) was markedly present in ums (66.67%). about 100% of ums were a2m-v100i-a/a, and the a2m-v100i-g/g genotype was absent in pms and ums. the a2m-i/i genotype was absent in ums, and 100% of ums were a2m-i/d and ace-d/d. homozygous mutations in the fos gene (b/b) were only present in ums, as well. agt-t235t cases were absent in pms, and the agt-m174m genotype appeared in 100% of pms. likewise, the prnp-m129m variant was present in 100% of pms and ums. 20, 74, 75, 77 these association studies clearly show that in pms and ums there is an accumulation of ad-related polymorphic variants of risk which might be responsible for the defective therapeutic responses currently seen in these ad clusters. 20, [74] [75] [76] [77] it appears that different cyp2d6 variants, expressing ems, ims, pms, and ums, infl uence to some extent several biochemical parameters, liver function, and vascular hemodynamic parameters which might affect drug effi cacy and safety. blood glucose levels are found elevated in ems ( * 1/ * 1 vs. * 4/ * 10, p < 0.05) and in some ims ( * 4/ * 10 vs. * 1xn/ * 4, p < 0.05), whereas other ims ( * 1/ * 5 vs. * 4/ * 4, p < 0.05) tend to show lower levels of glucose compared with pms ( * 4/ * 4) or ums ( * 1xn/ * 4) (table 40 .7). the highest levels of total-cholesterol are detected in the ems with the cyp2d6 * 1/ * 10 genotype (vs. * 1/ * 1, * 1/ * 4 and * 1xn/ * 1, p < 0.05). the same pattern has been observed with regard to ldlcholesterol levels, which are signifi cantly higher in the em-* 1/ * 10. in general, both total cholesterol levels and ldl-cholesterol levels are higher in ems (with a signifi cant difference between * 1/ * 1 and * 1/ * 10), intermediate levels are seen in ims, and much lower levels in pms and ums; and the opposite occurs with hdlcholesterol levels, which on average appear much lower in ems than in ims, pms, and ums, with the highest levels detected in * 1/ * 3 and * 1xn/ * 4 (table 40 .8). the levels of triglycerides are very variable among different cyp2d6 polymorphisms, with the highest levels present in ims ( * 4/ * 10 vs. * 4/ * 5 and * 1xn/ * 1, p < 0.02). these data clearly indicate that lipid metabolism can be infl uenced by cyp2d6 variants or that specifi c phenotypes determined by multiple lipid-related genomic clusters are necessary to confer the character of ems and ims. other possibility might be that some lipid metabolism genotypes interact with cyp2d6-related enzyme products leading to defi ne the pheno-genotype of pms and ums. no signifi cant changes in blood pressure values have been found among cyp2d6 genotypes; however, important differences became apparent in brain cerebrovascular hemodynamics (table 40 .9). in general terms, the best (2) 0.88 ± 0.07 (6) 0.56 ± 0.02 ( ldl-cholesterol levels, than in ims ( * 4/ * 10, p < 0.05); and diastolic velocities (dv) also tend to be much lower in * 1/ * 10 and especially in pms ( * 4/ * 4) and ums ( * 1xn/ * 4), whereas the best dv is measured in * 1/ * 5 ims. more striking are the results of both the pulsatility index (pi = (sv-dv)/mv) and resistance index (ri = (sv-dv)/sv), which are worse in ims and pms than in ems and ums (table 40 .9). these data taken together seem to indicate that cyp2d6-related ad pms exhibit a poorer cerebrovascular function which might affect drug penetration in the brain with the consequent therapeutic implications. [16] [17] [18] [19] [20] [74] [75] [76] [77] some conventional anti-dementia drugs (tacrine, donepezil, galantamine) are metabolized via cyp-related enzymes, especially cyp2d6, cyp3a4, and cyp1a2, and polymorphic variants of the cyp2d6 gene can affect the liver metabolism, safety and effi cacy of some cholinesterase inhibitors. 107, 108 in order to elucidate whether or not cyp2d6-related variants may infl uence transaminase activity, we have studied the association of got, gpt, and ggt activity with the most prevalent cyp2d6 genotypes in ad (table 40 .10). globally, ums and pms tend to show the highest got activity and ims the lowest. signifi cant differences appear among different im-related genotypes. the * 10/ * 10 genotype exhibited the lowest got activity with marked differences as compared to ums (p < 0.05 vs. * 1xn/ * 1; p < 0.05 vs. * 1xn/ * 4). gpt activity was signifi cantly higher in pms ( * 4/ * 4) than in ems ( * 1/ * 10, p < 0.05) or ims ( * 1/ * 4, * 1/ * 5, p < 0.05). the lowest gpt activity was found in ems and ims. striking differences have been found in ggt activity between pms ( * 4/ * 4), which showed the highest levels, and ems ( * 1/ * 1, p < 0.05; * 1/ * 10, p < 0.05), ims ( * 1/ * 5, p < 0.05), or ums ( * 1xn/ * 1, p < 0.01) ) ( table 40 .10). interesting enough, the * 10/ * 10 genotype, with the lowest values of got and gpt, exhibited the second highest levels of ggt after * 4/ * 4, probably indicating that cyp2d6-related enzymes differentially regulate drug metabolism and transaminase activity in the liver. these results are also clear in demonstrating the direct effect of cyp2d6 variants on transaminase activity 20,77,109 (table 40 .10). (2) 16.28 ± 7.40 (11) 18.14 ± 6.79 (17) intermediate metabolizers * 1/ * 3 22.33 ± 1.52 (3, 4) 24.66 ± 10. 59 22.00 ± 8.71 * 1/ * 4 21.76 ± 3.57 (5, 6) 21.88 ± 8.40 32.23 ± 25.53 * 1/ * 5 18.33 ± 2.33 (7, 8) 16.16 ± 5.60 (12, 13) 18.50 ± 6.47 ( no clinical trials have been performed to date to elucidate the infl uence of cyp2d6 variants on the therapeutic outcome in ad in response to cholinesterase inhibitors or other anti-dementia drugs. to overcome this lack of pharmacogenetic information, we have performed the fi rst prospective study in ad patients who received a combination therapy with (a) an endogenous nucleotide and choline donor, cdp-choline (500 mg/day), (b) a nootropic substance, piracetam (1,600 mg/day), (c) a vasoactive compound, 1,6 dimethyl 8β-(5bromonicotinoyl-oxymethyl)-10α-methoxyergoline (nicergoline) ( (fig. 40.10 ). among ems, ad patients harbouring the * 1/ * 10 genotype responded better than patients with the * 1/ * 1 genotype. the best responders among ims were the * 1/ * 3, * 1/ * 6 and * 1/ * 5 genotypes, whereas the * 1/ * 4, * 10/ * 10, and * 4/ * 10 genotypes were poor responders. among pms and ums, the poorest responders were carriers of the * 4/ * 4 and * 1xn/ * 1 genotypes, respectively. 20, 77, 109 from all these data we can conclude the following: (i) the most frequent cyp2d6 variants in the spanish population are the * 1/ * 1 (47.10%), * 1/ * 4 (17.42%), * 4/ * 4 (8.37%), * 1/ * 10 (4.52%) and * 1xn/ * 1 (4.52%), accounting for more than 80% of the population; (ii) the frequency of ems, ims, pms, and ums is about 51.61%, 32.26%, 9.03%, and 7.10%, respectively; (iii) ems are more prevalent in ad (57.47%) than in controls (44.12%); ims are more frequent in controls (41.18%) fig. 40 .10 cyp2d6-related therapeutic response to a multifactorial treatment in alzheimer's disease over a 1-year period (adapted from r. cacabelos 77, 109 ).patients received a combina-tion therapy for 1 year, and cognitive function (mmse score) was assessed at baseline (b) and after 1, 3, 6, 9, and 12 months of treatment. than in ad (25.29%), especially the * 1/ * 4 (c: 23.53%; ad: 12.64%) and * 4/ * 10 genotypes (c: 5.88%; ad: 1.15%); the frequency of pms is similar in ad (9.20%) and controls (8.82%); and ums are more frequent among ad cases (8.04%) than in controls (5.88%); (iv) there is an accumulation of ad-related genes of risk in pms and ums; (v) pms and ums tend to show higher transaminase activities than ems and ims; (vi) ems and ims are the best responders, and pms and ums are the worst responders to a combination therapy with cholinesterase inhibitors, neuroprotectants, and vasoactive substances; and (vii) the pharmacogenetic response in ad appears to be dependent upon the networking activity of genes involved in drug metabolism and genes involved in ad pathogenesis. [16] [17] [18] [19] [20] [74] [75] [76] [77] 109, 110 taking into consideration the available data, it might be inferred that at least 15% of the ad population may exhibit an abnormal metabolism of cholinesterase inhibitors and/or other drugs which undergo oxidation via cyp2d6-related enzymes. approximately 50% of this population cluster would show an ultrarapid metabolism, requiring higher doses of cholinesterase inhibitors to reach a therapeutic threshold, whereas the other 50% of the cluster would exhibit a poor metabolism, displaying potential adverse events at low doses. if we take into account that approximately 60-70% of therapeutic outcomes depend upon pharmacogenomic criteria (e.g., pathogenic mechanisms associated with ad-related genes), it can be postulated that pharmacogenetic and pharmacogenomic factors are responsible for 75-85% of the therapeutic response (effi cacy) in ad patients treated with conventional drugs. [16] [17] [18] [19] [20] [74] [75] [76] [77] 109, 110 of particular interest are the potential interactions of cholinesterase inhibitors with other drugs of current use in patients with ad, such as antidepressants, neuroleptics, antiarrhythmics, analgesics, and antiemetics which are metabolized by the cytochrome p450 cyp2d6 enzyme. 111 although most studies predict the safety of donepezil 112 and galantamine, 107 as the two principal cholinesterase inhibitors metabolized by cyp2d6-related enzymes, 113, 114 no pharmacogenetic studies have been performed so far on an individual basis to personalize the treatment, and most studies reporting safety issues are the result of pooling together pharmacological and clinical information obtained with routine procedures. 103, [115] [116] [117] in certain cases, genetic polymorphism in the expression of cyp2d6 is not expected to affect the pharmacodynamics of some cholinesterase inhibitors because major meta-bolic pathways are glucuronidation, o-demethylation, n-demethylation, n-oxidation, and epimerization. however, excretion rates are substantially different in ems and pms. for instance, in ems, urinary metabolites resulting from o-demethylation of galantamine represent 33.2% of the dose compared with 5.2% in pms, which show correspondingly higher urinary excretion of unchanged galantamine and its n-oxide. 118 therefore, still there are many unanswered questions regarding the metabolism of cholinesterase inhibitors and their interaction with other drugs (potentially leading to adrs) which require pharmacogenetic elucidation. it is also worth to mention that dose titration (a common practice in ad patients treated with cholinesterase inhibitors; e.g., tacrine, donepezil) is an unwise strategy, since approximately 30-60% of drug failure or lack of therapeutic effi cacy (and/or adr manifestation) is not a matter of drug dosage but a problem of poor metabolizing capacity in pms. additionally, inappropriate drug use is one of the risk factors for adverse drug reactions (adrs) in the elderly. the prevalence of use of potentially inappropriate medications in patients older than 65 years of age admitted to a general medical or geriatric ward ranges from 16% to 20%, 119 and these numbers may double in ambulatory patients. overall, the most prevalent inappropriate drugs currently prescribed to the elderly are amiodarone, long-acting benzodiazepines and anticholinergic antispasmodics; however, the list of drugs with potential risk also include antidepressant, antihistaminics, nsaids, amphetamines, laxatives, clonidine, indomethacin, and several neuroleptics, 119 most of which are processed via cyp2d6 and cyp3a5 enzymes. 120 therefore, pre-treatment cyp screening might be of great help to rationalize and optimize therapeutics in the elderly, by avoiding medications of risk in pms and ums. there are substantial differences between individuals in the effects of psychotropic drugs in the treatment of neuropsychiatric disorders. pharmacogenetic studies of psychotropic drug response have focused on determining the relationship between variation in specifi c candidate genes and the positive and adverse effects of drug treatment. 121 more than 200 different genes are potentially involved in the metabolism of psychotropic drugs infl uencing pharmacokinetics and pharmacodynamics. of all genes affecting drug metabolism, effi cacy and safety, the cyp gene family is the most relevant since more than 60% of cns drugs are metabolized by cytochrome p450 enzymes. [122] [123] [124] approximately, 18% of neuroleptics are major substrates of cyp1a2 enzymes, 40% of cyp2d6, and 23% of cyp3a4; 24% of antidepressants are major substrates of cyp1a2 enzymes, 5% of cyp2b6, 38% of cyp2c19, 85% of cyp2d6, and 38% of cyp3a4; 7% of benzodiazepines are major substrates of cyp2c19 enzymes, 20% of cyp2d6, and 95% of cyp3a4 (table 40 .5). approximately, 80% of patients with resistant depression, 60% of patients non-responsive to neuroleptics, and 50-70% of patients with paradoxical responses to benzodiazepines are carriers of mutant variants of the cyp2d6, cyp2c9 and cyp3a4 genes, falling within the categories of poor or ultra-rapid metabolizers. other genes infl uencing psychotropic drug activity include the following: abcb1 ( [128] [129] [130] [131] [132] [133] [134] [135] [136] [137] [138] (table 40.5) . historically, the vast majority of pharmacogenetic studies of cns disorders have been addressed to evaluate the impact of cytochrome p450 enzymes on drug metabolism. [125] [126] [127] furthermore, conventional targets for psychotropic drugs were the neurotransmitters dopamine, serotonin, noradrenaline, gaba, ion channels, acetylcholine and their respective biosynthetic and catalyzing enzymes, receptors and transporters 121 ; however, in the past few years many different genes have been associated with both pathogenesis and pharmacogenomics of neuropsychiatric disorders. some of these genes and their products constitute potential targets for future treatments. new developments in genomics, including whole genome genotyping approaches and comprehensive information on genomic variation across populations, coupled with large-scale clinical trials in which dna collection is routine, now provide the impetus for a next generation of pharmacogenetic studies and identifi cation of novel candidate drugs. [139] [140] [141] cyclic nucleotide phosphodiesterases (pdes) are a family of enzymes that degrade camp and cgmp. intracellular cyclic nucleotide levels increase in response to neurotransmitters and are down-regulated through hydrolysis catalyzed by pdes, which are therefore candidate therapeutic targets. camp is a second messenger involved in learning, memory, and mood, and cgmp modulates brain processes that are controlled by the nitric oxide (no)/cgmp pathway. the analysis of snps in 21 genes of this superfamily revealed that polymorphisms in pde9a and pde11a are associated with major depressive disorder. in addition, remission on antidepressants was associated with polymorphisms in pde1a and pde11a. according to these results, it has been postulated that pde11a (haplotype gaacc) has a role in the pathogenesis of major depression. 142 another example is the purinergic receptor gene p2rx(7), located in a major linkage hotspot for schizophrenia and bipolar disorder (12q21-33), which has been associated with bipolar disorder, but nine functionally characterized variants of p2rx(7) did not show association with schizophrenia. 143 the possible role of a tag snp (the 1359g/a polymorphism) of the gene encoding the cannabinoid receptor type 1 (cnr1) has been investigated in schizophrenics treated with atypical antipsychotics. no difference in 1359g/a polymorphism was observed between patients and control subjects, and no relation-ships were noted between this polymorphism and any clinical parameter considered as potential intermediate factor; however, the g allele was signifi cantly higher among non-responders vs. responsive patients, suggesting that the g allele of the cnr1 gene could be a pharmacogenetic rather than a vulnerability factor for schizophrenics. 144 synaptic dysfunction is a potential pathogenic factor in schizophrenia. cholesterol is an essential component of myelin and has proved important for synapse formation and lipid raft function. it has been demonstrated that the antipsychotic drugs clozapine and haloperidol stimulate lipogenic gene expression in glioma cells in culture through activation of the sterol regulatory element-binding protein (srebp) transcription factors. recently, the action of chlorpromazine, haloperidol, clozapine, olanzapine, risperidone and ziprasidone on srebp and srebp-controlled gene expression (acetyl-coa acetyltransferase 2, acetoacetyl-coa thiolase, acat2; 3-hydroxy-3-methylglutaryl-coa reductase, hmgcr; 3-hydroxy-3-methylglutaryl-coa synthase 1, hmgcs1; fdps; sterol-c5-desaturase like, sc5dl; 7-dehydrocholesterol reductase, dhcr7; low density lipoprotein receptor, ldlr; fatty acid synthase; farsenyl diphosphate synthase, fasn; stearoyl-coa desaturase, delta-9-desaturase, scd1) has been investigated in different cns human cell lines, demonstrating that antipsychotic-induced activation of lipogenesis is most prominent in glial cells and that this mechanism could be relevant for the therapeutic efficacy of some antipsychotic drugs. 145 rgs2 (regulator of g-protein signaling 2) modulates dopamine receptor signal transduction. functional variants of this gene (rgs2-rs 4606 c/g) may infl uence susceptibility to extrapyramidal symptoms induced by antipsychotic drugs. this snp is located in the 3′-regulatory region of the gene, and is known to infl uence rgs2 mrna levels and protein expression. 146 furthermore, rgs4 (regulator of g protein signaling 4) genotypes predict both the severity at baseline symptoms and relative responsiveness to antipsychotic medication. 147 tardive dyskinesia is characterized by involuntary movements predominantly in the orofacial region and develops in approximately 20% of patients during long-term treatment with typical antipsychotics. polymorphic variants of cyp1a2, cyp2d6, and drd3 genes have been associated with tardive dyskinesia in schizophrenics. 148, 149 in contrast, the haplotype t-4b-glu of the endothelial nitric oxide synthase (nos3) gene (-786t > c in the promoter region, 27-bp variable number of tandem repeats in intron 4, glu298asp in exon 7) might represent a protective haplotype against tardive dyskinesia after long-term antipsychotic treatment. 150 the t102c variant in the serotonin 2a receptor (htr2a) and the ser9gly variant in the dopamine d3 receptor (drd3) were associated with a risperidone response to exacerbated schizophrenia. the patients with t/t in the htr2a gene show less clinical improvement than do those with t/c or c/c. the c allele is more frequent in responders. when combinations of both polymorphisms are considered, patients who have t/t in the htr2a gene and encode ser/ser or ser/gly from drd3 gene have a higher propensity to non-responsiveness compared to other subjects, suggesting that the htr2a t102c variant could be a potential indicator of clinical improvement after risperidone treatment. 151 there is a signifi cant relationship between a promoter region polymorphism in the serotonin transporter gene and antidepressant response, as well as for associations between candidate neurotransmitter receptor genes and second generation antipsychotic drug response. 121 polymorphic variants of several serotonin receptor subtypes seem to be involved in the efficacy and symptomatic response of schizophrenic patients to atypical antipsychotics. for instance, the −1019 c/g polymorphism of the htr1a receptor gene is associated with negative symptom response to risperidone in schizophrenics. 152 interaction between comt and notch4 genotypes may also predict the treatment response to typical neuroleptics in patients with schizophrenia. 153 the effi cacy of iloperidone in patients with schizophrenia has been associated with the homozygous condition for the rs1800169 g/g genotype of the ciliary neurotrophic factor (cntf) gene. 154 dopamine receptor interacting proteins (drips) are pivotally involved in regulating dopamine receptor signal transduction. two snps in the dopamine receptor interacting protein gene, nef3, which encodes the drip, neurofi lament-medium (nf-m), were associated with early response (rs1457266, rs1379357). a 5 snp haplotype spanning nef3 was over-represented in early responders. since nef3 is primarily associated with dopamine d1 receptor function, it is likely that both genes cooperate in eliciting genotype-specifi c antipsychotic response. 155 the improvement in the positive and negative syndrome scale (panss) positive subscore was found signifi cantly greater in patients homozygous for the a1287 allele of the slc6a2 (solute carrier family 6 (noradrenaline transporter), member 2) gene, and smaller in patients homozygous for the c-182 allele of the slc6a2 gene, suggesting that these polymorphisms of the noradrenaline transporter gene are specifi cally involved in the variation of positive symptoms in schizophrenia. 156 weight gain is a problem commonly found in patients treated with neuroleptics, tricyclic antidepressants, and some antiepileptics (e.g., valproic acid). the adipocyte-derived hormone, leptin, has been associated with body weight and energy homeostasis, and abnormal regulation of leptin could play a role in weight gain induced by antipsychotics. the leptin gene promoter variant g2548a was associated with clozapine-induced weight in chinese patients with chronic schizophrenia. 157 likewise, studies in caucasians suggest that genetic vulnerability in the leptin gene (−1548g/a) and leptin receptor (q223r) may predispose some individuals to excessive weight gain from increased exposure to olanzapine. 158, 159 the development of selective type 5 metabotropic glutamate receptor (mglu5) antagonists, such as 2-methyl-6-(phenylethynyl)-pyridine (mpep) and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (mtep), has demonstrated the potential involvement of these receptors in several cns disorders including depression, anxiety, epilepsy, parkinson's disease, drug addiction, and alcoholism. treatment with mpep and mtep can induce gene expression related to atp synthesis, hydrolase activity, and signaling pathways associated with mitogen-activated protein kinase (mapk) in the frontal cortex, this constituting another potential therapeutic target in some neuropsychiatric disorders. 160 a new marker (rs1954787) in the grik4 gene, which codes for the kainic acid-type glutamate receptor ka1, has been associated with response to the antidepressant citalopram, suggesting that the glutamate system plays a role in modulating response to selective serotonin reuptake inhibitors (ssris). 161 glycogen synthase kinase-3β (gsk3b) activity is increased in the brain of patients with major depressive disorders. inhibition of gsk3b is thought to be a key feature in the therapeutic mechanism of antidepressants. four polymorphisms of the gsk3b gene [rs334555 (−50 t > c); rs13321783 (ivs7 + 9227 a > g); rs2319398 (ivs + 11660 g > t); rs6808874 (ivs + 4251 t > a)] have been genotyped in chinese patients with major depression. gsk3b tagt carriers showed poorer response to antidepressants. 162 lithium has been used for over 40 years as an effective prophylactic agent in bipolar disorder. response to lithium treatment seems to be, at least in part, genetically determined. it has been suggested that lithium exerts an effect on signal transduction pathways, such as the cyclic adenosine monophosphate (camp) pathway. association studies in patients with bipolar disorders revealed that creb1-1h snp (g/a change at 2q32.3-q34) and creb1-7h (t/c change) may be associated with bipolar disorder and lithium response. 163 dna oligonucleotide microarrays have been used to evaluate gene expression in the substantia nigra of patients with parkinson's disease (pd). sporadic pd is characterized by progressive death of dopaminergic neurons within the substantia nigra, where cell death is not uniform. the lateral tier of the substantia nigra (snl) degenerates earlier and more severely than the more medial nigral component (snm). genes expressed more highly in the pd snl included the cell death gene, p53 effector related to pmp22, the tnfr gene, tnfr superfamily, member 21, and the mitochondrial complex i gene, nadh dehydrogenase (ubiquinone) 1-beta subcomplex, 3, 12 kda (ndufβ3). genes that were more highly expressed in pd snm included the dopamine cell signaling gene, cyclic adenosine monophosphate-regulated phosphoprotein, 21 kda, the activated macrophage gene, stabilin 1, and two glutathione peroxidase (gpx) genes, gpx1 and gpx3. this gene expression profi le reveals that there is increased expression of genes encoding pro-infl ammatory cytokines and subunits of the mitochondrial electron transport chain in glial cells, and that there is a decreased expression of several glutathione-related genes in the gnl, suggesting a molecular basis for pathoclisis. 164 these fi ndings may contribute to open new therapeutic avenues in pd, where glial cells might represent potential targets to halt disease progression. pharmacological inhibition of cyclic-dependent kinase 5 (cdk5) protects neurons under distinct stressful conditions. in ad and amyotrophic lateral sclerosis deregulation of cdk5 causes hyperphosphorylation of tau and neurofi lament proteins, respectively, leading to neuronal cell death. by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionisation-time of fl ight (maldi-tof)-mass spectrometry, several phosphoproteins that are modulated by cdk5 inhibitors have been identifi ed. these phosphoproteins include syndapin i which is involved in vesicle recycling, and dynein light intermediate chain 2 which represents a regulatory subunit of the dynein protein complex, confi rming the role of cdk5 in synaptic signaling and axonal transport. other phosphoproteins detected are cofi lin and collapsing response mediator protein, involved in neuronal survival and/or neurite outgrowth. selective cdk5 inhibitors can also block mitochondrial translocation of pro-apoptotic cofi lin. phosphoproteome and transcriptome analysis of neurons indicate that cdk5 inhibitors promote both neuronal survival and neurite outgrowth. 165 these compounds might represent novel therapeutic alternatives in neurodegenerative disorders. despite the promising results obtained with structural and functional genomic procedures to identify associations with disease pathogenesis and potential drug targets in cns disorders, it must be kept in mind that allelic mrna expression is affected by genetic and epigenetic events, both with the potential to modulate neurotransmitter tone in the cns. 166 epigenetics is the study of how the environment can affect the genome of the individual during its development as well as the development of its descendants, all without changing the dna sequence, but inducing modifi cations in gene expression through dna methylation-demethylation or through modifi cation of histones by processes of methylation, deacetylation, and phosphorylation. 167 cumulative experiences throughout life history interact with genetic predispositions to shape the individual's behaviour. 167 epigenetic phenomena can not be neglected in the pathogenesis and pharmacogenomics of cns disorders. studies in cancer research have demonstrated the antineoplastic effects of the dna methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid, of current use in epilepsy. 168 novel effects of some pleiotropic drugs with activity on the cns have to be explored to understand in full their mechanisms of action and adjust their dosages for new indications. both hyper-and hypo-dna methylation changes of the regulatory regions play critical roles in defi ning the altered functionality of genes (mb-comt, maoa, dat1, th, drd1, drd2, reln, bdnf) in major psychiatric disorders, such as schizophrenia and bipolar disorder. 169 this complexity requires a multifactorial approach to overcome the hurdles that cns drug development faces at the present time. 170 polymorphic variants in the apoe gene (19q13.2) are associated with risk (apoe-4 allele) or protection (apoe-2 allele) for ad. 8, [18] [19] [20] for many years, alterations in apoe and defects in the apoe gene have been associated with dysfunctions in lipid metabolism, cardiovascular disease, and atherosclerosis. during the past 25 years an enormous amount of studies clearly documented the role of apoe-4 as a risk factor for ad, an the accumulation of the apoe-4 allele has been reported as a risk factor for other forms of dementia and cns disorders. 8, [18] [19] [20] apoe-4 may infl uence ad pathology interacting with app metabolism and abp accumulation, enhancing hyperphosphorylation of tau protein and nft formation, reducing choline acetyltransferase activity, increasing oxidative processes, modifying infl ammation-related neuroimmunotrophic activity and glial activation, altering lipid metabolism, lipid transport and membrane biosynthesis in sprouting and synaptic remodelling, and inducing neuronal apoptosis. 8, [18] [19] [20] different apoe genotypes confer specifi c phenotypic profi les to ad patients. some of these profi les may add risk or benefi t when the patients are treated with conventional drugs, and in many instances the clinical phenotype demands the administration of additional drugs which increase the complexity of therapeutic protocols. from studies designed to defi ne apoe-related ad phenotypes, 8, [16] [17] [18] [19] [20] [21] [22] 62, 63, [74] [75] [76] [77] 109, 110 several confi rmed conclusions can be drawn: (i) the ageat-onset is 5-10 years earlier in approximately 80% of ad cases harbouring the apoe-4/4 genotype; (ii) the serum levels of apoe are the lowest in apoe-4/4, intermediate in apoe-3/3 and apoe-3/4, and highest in apoe-2/3 and apoe-2/4; (iii) serum cholesterol levels are higher in apoe-4/4 than in the other genotypes; (iv) hdl-cholesterol levels tend to be lower in apoe-3 homozygotes than in apoe-4 allele carriers; (v) ldl-cholesterol levels are systematically higher in apoe-4/4 than in any other genotype; (vi) triglyceride levels are signifi cantly lower in apoe-4/4; (vii) nitric oxide levels are slightly lower in apoe-4/4; (viii) serum abp levels do not differ between apoe-4/4 and the other most frequent genotypes (apoe-3/3, apoe-3/4); (ix) blood histamine levels are dramatically reduced in apoe-4/4 as compared with the other genotypes; (x) brain atrophy is markedly increased in apoe-4/4 > apoe-3/4 > apoe-3/3; (xi) brain mapping activity shows a signifi cant increase in slow wave activity in apoe-4/4 from early stages of the disease (fig. 40.4) ; (xii) brain hemodynamics, as refl ected by reduced brain blood fl ow velocity and increase pulsatility and resistance indices, is signifi cantly worst in apoe-4/4 (and in apoe-4 carriers, in general, as compared with apoe-3 carriers); (xiii) lymphocyte apoptosis is markedly enhanced in apoe-4 carriers; (xiv) cognitive deterioration is faster in apoe-4/4 patients than in carriers of any other apoe genotype; (xv) occasionally, in approximately 3-8% of the ad cases, the presence of some dementia-related metabolic dysfunctions (e.g., iron, folic acid, vitamin b12 defi ciencies) accumulate in apoe-4 carriers more than in apoe-3 carriers; (xvi) some behavioral disturbances (bizarre behaviors, psychotic symptoms), alterations in circadian rhythm patterns (e.g., sleep disorders), and mood disorders (anxiety, depression) are slightly more frequent in apoe-4 carriers; (xvii) aortic and systemic atherosclerosis is also more frequent in apoe-4 carriers; (xviii) liver metabolism and transaminase activity also differ in apoe-4/4 with respect to other genotypes; (xix) blood pressure (hypertension) and other cardiovascular risk factors also accumulate in apoe-4; and (xx) apoe-4/4 are the poorest responders to conventional drugs ( fig. 40.11 ). these 20 major phenotypic features clearly illustrate the biological disadvantage of apoe-4 homozygotes and the potential consequences that these patients may experience when they receive pharmacological treatment. 2, 4, 8, [16] [17] [18] [19] [20] [21] [22] 62, 63, [74] [75] [76] [77] 109, 110 fig. 40.11 apoe-related cognitive performance in patients with alzheimer's disease treated with a combination therapy for 1 year (adapted from r. cacabelos 77, 109 ). patients received a combination therapy for 1 year, and cognitive function (mmse score) was assessed at baseline (b) and after 1, 3, 6, 9, and 12 months of treatment. several studies indicate that the presence of the apoe-4 allele differentially affects the quality and size of drug responsiveness in ad patients treated with cholinergic enhancers, neuroprotective compounds or combination therapies; however, controversial results are frequently found due to methodological problems, study design, and patients recruitment in clinical trials. from these studies we can conclude the following: (i) multifactorial treatments combining neuroprotectants, endogenous nucleotides, nootropic agents, vasoactive substances, cholinesterase inhibitors, and nmda antagonists associated with metabolic supplementation on an individual basis adapted to the phenotype of the patient may be useful to improve cognition and slow-down disease progression in ad. (ii) in our personal experience the best results have been obtained combining (a) cdp-choline with piracetam and metabolic supplementation, (b) cdp-choline with piracetam and anapsos, (c) cdp-choline with piracetam and cholinesterase inhibitors (donepezil, rivastigmine), (d) cdp-choline with memantine, and (e) cdpcholine, piracetam and nicergoline. (iii) some of these combination therapies have proven to be effective, improving cognition during the fi rst 9 months of treatment, and not showing apparent side-effects. (iv) the therapeutic response in ad seems to be genotypespecifi c under different pharmacogenomic conditions. (v) in monogenic-related studies, patients with the apoe-2/3 and apoe-3/4 genotypes are the best responders, and apoe-4/4 carriers are the worst responders ( fig. 40.11 ). (vi) ps1-and ps2-related genotypes do not appear to infl uence the therapeutic response in ad as independent genomic entities; however, app, ps1, and ps2 mutations may drastically modify the therapeutic response to conventional drugs. (vii) in trigenic-related studies the best responders are those patients carrying the 331222-, 341122-, 341222-, and 441112-genomic clusters. (viii) a genetic defect in the exon 5 of the ps2 gene seems to exert a negative effect on cognition conferring ps2+ carriers in trigenic clusters the condition of poor responders to combination therapy. (ix) the worst responders in all genomic clusters are patients with the 441122+ genotype. (x) the apoe-4/4 genotype seems to accelerate neurodegeneration anticipating the onset of the disease by 5-10 years; and, in general, apoe-4/4 carriers show a faster disease progression and a poorer therapeutic response to all available treatments than any other polymorphic variant. (xi) pharmacogenomic studies using trigenic, tetragenic or polygenic clusters as a harmonization procedure to reduce genomic heterogeneity are very useful to widen the therapeutic scope of limited pharmacological resources. [4] [5] [6] [16] [17] [18] [19] [20] [21] [22] 62, 63, [74] [75] [76] [77] 109, 110 apoe infl uences liver function and cyp2d6-related enzymes probably via regulation of hepatic lipid metabolism. 20, 42, [74] [75] [76] [77] it has been observed that apoe may infl uence liver function and drug metabolism by modifying hepatic steatosis and transaminase activity. there is a clear correlation between apoe-related tg levels and got, gpt, and ggt activities in ad. 20, [74] [75] [76] [77] 171 both plasma tg levels and transaminase activity are signifi cantly lower in ad patients harbouring the apoe-4/4 genotype, probably indicating (a) that low tg levels protect against liver steatosis, and (b) that the presence of the apoe-4 allele infl uences tg levels, liver steatosis, and transaminase activity. consequently, it is very likely that apoe infl uences drug metabolism in the liver through different mechanisms, including interactions with enzymes such as transaminases and/ or cytochrome p450-related enzymes encoded in genes of the cyp superfamily. 20, [74] [75] [76] [77] 109, 171 when apoe and cyp2d6 genotypes are integrated in bigenic clusters and the apoe + cyp2d6-related therapeutic response to a combination therapy is analyzed in ad patients after 1 year of treatment, it becomes clear that the presence of the apoe-4/4 genotype is able to convert pure cyp2d6 * 1/ * 1 ems into full pms (fig. 40.12) , indicating the existence of a powerful infl uence of the apoe-4 homozygous genotype on the drug metabolizing capacity of pure cyp2d6-ems. 20, 74, 75, 109 behavioral disturbances and mood disorders are intrinsic components of dementia associated with memory disorders. 60, [172] [173] [174] the appearance of anxiety, depression, psychotic symptoms, verbal and physical aggressiveness, agitation, wandering and sleep disorders complicate the clinical picture of dementia and add important problems to the therapeutics of ad and the daily management of patients as well. under these conditions, psychotropic drugs (antidepressants, anxyolitics, hypnotics, and neuroleptics) are required, and most of these substances contribute to deteriorate cognition and psychomotor functions. apoe-related polymorphic variants have been associated with mood disorders 175, 176 and panic disorder. 177 gender, age, dementia severity, apoe-4, and general medical health appear to infl uence the occurrence of individual neuropsychiatric symptoms in dementia, and medical comorbidity increases the risk of agitation, irritability, disinhibition, and aberrant motor behavior. 178 a positive association between apoe-4 and neuropsychiatric symptoms 179 and depressive symptoms in ad has been reported, 180 especially in women. 181 in other studies, no association of apoe-4 with behavioral dyscontrol (euphoria, disinhibition, aberrant motor behavior, and sleep and appetite disturbances), psychosis (delusions and hallucinations), mood (depression, anxiety, and apathy), and agitation (aggression and irritability) could be found. 182 some authors did not fi nd association of apoe-4 with major depression in ad 183, 184 or in patients with major depression in a community of older adults, 185 but an apparent protective effect of apoe-2 on depressive symptoms was detected. 186 others, in contrast, found that apoe-4 was associated with an earlier age-of-onset, but not cognitive functioning, in late-life depression. 187 apoe−/− mice without human apoe or with apoe-4, but not apoe-3, show increased measures of anxiety. 188 differences in anxiety-related behavior have been observed between apoe-defi cient c57bl/6 and wild type c57bl/6 mice, suggesting that apoe variants may affect emotional state. 189 histamine h3 autoreceptor antagonists increase anxiety measures in wildtype, but not apoe−/−, mice, and apoe defi cient mice show higher sensitivity to the anxiety-reducing effects of the h1 receptor antagonist mepyramine than wildtype mice, suggesting a role of h3-autoreceptormediated signaling in anxiety-like symptoms in this ad-related animal model. 190 in humans, apoe-4 carriers with deep white matter hyperintensities in mri show association with depressive symptoms and vascular depression. 191 . reduced caudate nucleus volumes and genetic determinants of homocysteine metabolism accumulate in patients with psychomotor slowing and cognitive defi cits, 192 and older depressed subjects have persisting cognitive impairments associated with hippocampal volume reduction. 193, 194 depressive symptoms are also associated with stroke and atherogenic lipid profi le. 195 some multifactorial treatments addressing neuroprotection have shown to be effective in reducing anxiety progressively from the fi rst month to the 12 month of treatment. 109 the anxiety rate was declining from a baseline hrs-a score of 10.90 ± 5.69 to 9.07 ± 4.03 (p < 0.0000000001) at 1 month, 9.01 ± 4.38 (p < 0.000006) at 3 months, 8.90 ± 4.47 (p < 0.005) at 6 months, 7.98 ± 3.72 (p < 0.00002) at 9 months, and 8.56 ± 4.72 (p < 0.01) at 12 months of treatment (r = −0.82, a coef.: 10.57, b coef.: −0.43). 109 similar striking results were found in depression, suggesting that improvement in mood conditions can contribute to stabilize cognitive function or that neuroprotection (with the consequent stabilization or improvement in mental performance) can enhance emotional equilibrium. 20, 74, 75, 109 at baseline, all apoe variants showed similar anxiety and depression rates, except the apoe-4/4 carriers who differed from the rest in a signifi cantly lower rates of anxiety and depression (figs. 40.13 and 40.14). remarkable changes in anxiety were found among different apoe genotypes (fig. 40.13) . practically, all apoe variants responded with a signifi cant diminution of anxiogenic symptoms, except patients with the apoe-4/4 genotype who only showed a slight improvement. the best responders were apoe-2/4 > apoe-2/3 > apoe-3/3 > apoe-3/4 carriers (fig. 40.13) . the modest anxiolytic effect seen in apoe-4/4 patients might be due to the very low anxiety rate observed at baseline. concerning depression, all apoe genotypes improved their depressive symptoms with treatment except those with the apoe-4/4 genotype which worsen along the treatment period, especially after 9 months (fig. 40 .14). the best responders were patients with apoe-2/4 > apoe-2/3 > apoe-3/3 > apoe-3/4, and the worst responders were patients harbouring the apoe-4/4 genotype 20,74,75,109 (fig. 40.14) . the optimization of cns therapeutics requires the establishment of new postulates regarding (a) the costs of medicines, (b) the assessment of protocols for multifactorial treatment in chronic disorders, (c) the implementation of novel therapeutics addressing causative factors, and (d) the setting-up of pharmacogenetic/ pharmacogenomic strategies for drug development. 20, [74] [75] [76] [77] 109 the cost of medicines is a very important issue in many countries because of (i) the growing of the aging population (>5% disability), (ii) neuropsychiatric and demented patients (>5% of the population) belong to an unproductive sector with low income, and (iii) the high cost of health care systems and new health technologies in developed countries. despite the effort of the pharmaceutical industry to demonstrate the benefi ts and cost-effectiveness of available drugs, the general impression in the medical community and in some governments is that some psychotropics and most anti-dementia drugs present in the market are not costeffective. 20, [74] [75] [76] [77] 109 conventional drugs for neuropsychi-atric disorders are relatively simple compounds with unreasonable prices. some new products are not superior to conventional antidepressants, neuroleptics, and anxiolytics. there is an urgent need to assess the costs of new trials with pharmacogenetics and pharmacogenomics strategies, and to implement pharmacogenetic procedures to predict drug-related adverse events. 20, 74, 75, 109 cost-effectiveness analysis has been the most commonly applied framework for evaluating pharmacogenetics. pharmacogenetic testing is potentially relevant to large populations that incur in high costs. for instance, the most commonly drugs metabolized by cyp2d6 account for 189 million prescriptions and us$12.8 billion annually in expenditures in the us, which represent 5-10% of total utilization and expenditures for outpatient prescription drugs. 196 pharmacogenomics offer great potential to improve patients' health in a cost-effective manner; however, pharmacogenetics/pharmacogenomics will not be applied to all drugs available in the market, and careful evaluations should be done on a case-by-case basis prior to investing resources in r&d of pharmacogenomic-based therapeutics and making reimbursement decisions. 197 in performing pharmacogenomic studies in cns disorders, it is necessary to rethink the therapeutic expectations of novel drugs, redesign the protocols for drug clinical trials, and incorporate biological markers as assessable parameters of effi cacy and prevention. in addition to the characterization of genomic profi les, phenotypic profi ling of responders and non-responders to conventional drugs is also important (and currently neglected). brain imaging techniques, computerized electrophysiology, and optical topography in combination with genotyping of polygenic clusters can help in the differentiation of responders and non-responders. the early identifi cation of predictive risks requires genomic screening and molecular diagnosis, and individualized preventive programs will only be achieved when pharmacogenomic/pharmacogenetic protocols are incorporated to the clinical armamentarium with powerful bioinformatics support. 18-20,74,75.109 an important issue in ad therapeutics is that antidementia drugs should be effective in covering the clinical spectrum of dementia symptoms represented by memory defi cits, behavioural changes, and functional decline. it is diffi cult (or impossible) that a single drug be able to fulfi l this criteria. a potential solution to this problem is the implementation of cost-effective, multifactorial (combination) treatments integrating several drugs, taking into consideration that traditional neuroleptics and novel antipsychotics (and many other psychotropics) deteriorate both cognitive and psychomotor functions in the elderly and may also increase the risk of stroke. 198 few studies with combination treatments have been reported and most of them are poorly designed. we have also to realize that the vast majority of dementia cases in people older than 75-80% are of a mixed type, in which the cerebrovascular component associated with neurodegeneration can not be therapeutically neglected. in most cases of dementia, the multifactorial (combination) therapy appears to be the most effective strategy. [18] [19] [20] [74] [75] [76] [77] 109 the combination of several drugs (neuroprotectants, vasoactive substances, acheis, metabolic supplementation) increases the direct costs (e.g., medication) by 5-10%, but in turn, annual global costs are reduced by approximately 18-20% and the average survival rate increases about 30% (from 8 to 12 years post-diagnosis). there are major concerns regarding the validity of clinical trials in patients with severe dementia. despite the questionable experience with memantine, 199 simi-lar strategies have been used to demonstrate the utility of donepezil in severe ad. 200 this kind of studies bears some important pitfalls, including (a) short duration (<1 year), (b) institutionalized patients, (c) patients receiving many different types of drugs, (d) non-evaluated drug-drug interactions, (e) side-effects (e.g., hallucinations, gastrointestinal disorders) that may require the administration of additional medication, (f) lack of biological parameters demonstrating actual benefi ts, and (e) no cost-effectiveness assessment, among many other possibilities of technical criticism. [18] [19] [20] 109, 201 some of these methodological (and costly) problems might be overcome with the introduction of pharmacogenetic/ pharmacogenomic strategies to identify good responders who might obtain some benefi t by taking expensive medications. major impact factors associated with drug effi cacy and safety include the following: (i) the mechanisms of action of drugs, (ii) drug-specifi c adverse reactions, (iii) drug-drug interactions, (iv) nutritional factors, (v) vascular factors, (vi) social factors, and (vii) genomic factors (nutrigenetics, nutrigenomics, pharmacogenetics, pharmacogenomics). among genomic factors, nutrigenetics/nutrigenomics and pharmacogenetics/pharmacogenomics account for more than 80% of effi cacy-safety outcomes in current therapeutics. [18] [19] [20] 74, 75, 77, 109 some authors consider that priority areas for pharmacogenetic research are to predict serious adverse reactions (adrs) and to establish variation in effi cacy. 202 both requirements are necessary in cns disorders to cope with effi cacy and safety issues associated with either current cns drugs and new drugs. 121, 138 since drug response is a complex trait, genome-wide approaches (oligonucleotide microarrays, proteomic profi ling) may provide new insights into drug metabolism and drug response. genome-wide family-based association studies, using single snps or haplotypes, can identify associations with genome-wide signifi cance. 203, 204 to achieve a mature discipline of pharmacogenetics and pharmacogenomics in cns disorders and dementia it would be convenient to accelerate the following processes: (a) educate physicians and the public on the use of genetic/genomic screening in the daily clinical practice; (b) standardize genetic testing for major categories of drugs; (c) validate pharmacogenetic and pharmacogenomic procedures according to drug category and pathology; (d) regulate ethical, social, and economic issues; and (e) incorporate pharmacogenetic and pharmacogenomic procedures to both drugs in development and drugs in the market to optimize therapeutics. 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cord-104265-kcygxo7h authors: brabb, thea; von dassow, peter; ordonez, nadia; schnabel, bryan; duke, blythe; goverman, joan title: in situ tolerance within the central nervous system as a mechanism for preventing autoimmunity date: 2000-09-18 journal: j exp med doi: nan sha: doc_id: 104265 cord_uid: kcygxo7h multiple sclerosis (ms) is believed to be an autoimmune disease in which autoreactive t cells infiltrate the central nervous system (cns). animal models of ms have shown that cns-specific t cells are present in the peripheral t cell repertoire of healthy mice and cause autoimmune disease only when they are activated by immunization. t cell entry into the cns is thought to require some form of peripheral activation because the blood–brain barrier prohibits trafficking of this tissue by naive cells. we report here that naive t cells can traffic to the cns without prior activation. comparable numbers of t cells are found in the cns of both healthy recombinase activating gene (rag)(−/)− t cell receptor (tcr) transgenic mice and nontransgenic mice even when the transgenic tcr is specific for a cns antigen. transgenic t cells isolated from the cns that are specific for non-cns antigens are phenotypically naive and proliferate robustly to antigenic stimulation in vitro. strikingly, transgenic t cells isolated from the cns that are specific for myelin basic protein (mbp) are also primarily phenotypically naive but are unresponsive to antigenic stimulation in vitro. mononuclear cells from the cns of mbp tcr transgenic but not nontransgenic mice can suppress the response of peripheral mbp-specific t cells in vitro. these results indicate that naive mbp-specific t cells can traffic to the cns but do not trigger autoimmunity because they undergo tolerance induction in situ. activation of self-reactive t cells is one of the first steps in the development of autoimmune disease. the pool of autoreactive t cells available for activation is limited to a large extent by mechanisms of tolerance induction that eliminate self-reactive t cells (1) . animal models of autoimmune disease, however, demonstrate that some self-reactive t cells remain in the periphery. these t cells are described as "ignorant" because they do not cause autoimmune disease when encountering self-antigen in vivo unless the self-antigen is presented in an immunostimulatory context (2, 3) . experimental allergic encephalomyelitis (eae), 1 an animal model of multiple sclerosis (ms), illustrates this phenomenon. eae is induced by immunization with central nervous system (cns) antigens such as myelin basic protein (mbp) in cfa (4) . recognition of mbp in this context activates naive t cells circulating in the periphery, which then enter the cns and initiate destruction of myelin (5) . in the absence of experimental intervention, the mbp-specific t cells are not activated and do not induce eae. cns immune privilege is believed to be one of the main components in maintaining ignorance in mbp-specific t cells. immune privilege is maintained within the cns by the blood-brain barrier that limits cns trafficking by leukocytes (6) (7) (8) . previous studies have suggested that only activated t cells are able to cross the blood-brain barrier and perform immune surveillance of the cns (7, 9, 10) . our studies use tcr transgenic mice specific for mbp1-11 (11, 12) , the predominant epitope targeted in eae in b10.pl mice (13) . mbp1-11-specific t cells in the periphery of tcr transgenic mice are primarily naive and proliferate readily to mbp in vitro (11, 14) . eae can be induced in these mice by administration of pertussis toxin or 872 in situ tolerance within the cns can occur spontaneously in an environment with increased microbial exposure (14) . in the studies reported here, we analyzed the ability of the mbp1-11-specific t cells to traffic to the cns in the absence of any exogenous stimulation. surprisingly, comparable numbers of t cells were isolated from the cns of both mbp-specific tcr transgenic mice and wild-type mice. in contrast to wild-type mice, however, most of the mbp-specific t cells found in the cns of young mice exhibited a naive phenotype rather than an activated or memory phenotype. similar observations were made in tcr transgenic models specific for nonself antigens. tcr transgenic models differ significantly from wild-type mice in that tcr transgenic mice have few activated/memory peripheral t cells. these results indicate that, in the absence of activated t cells in the periphery, naive t cells can traffic to the cns. despite the trafficking of naive mbp-specific t cells through the cns, the majority of mbp tcr transgenic mice do not develop autoimmunity. we show that the lack of spontaneous eae is due in part to suppression of the responses of naive mbp-specific t cells within the target organ. furthermore, cns cells from mbp tcr transgenic mice are able to suppress the response of nontolerant peripheral mbp-specific t cells in vitro. these data demonstrate that cns autoimmune disease is regulated in part by the induction of tolerance to cns antigens in situ. mice. mbp tcr1 transgenic mice (b10.pl-h2 u -h2-t18 a [73ns]/sn-tgn [tcr ␣ ]/bljg and b10.pl-h2 u -h2-t18 a [73ns]/sn-tgn [tcr ␤ ]/c14jg [11] ) and mbp tcr2 transgenic mice (12) were backcrossed onto b10.pl (73 ns)/sn for at least 11 generations. b10.pl (73 ns)/sn, c57bl/6j, and lymphocytic choriomeningitis virus (lcmv) tcr transgenic mice (b6, d2-tgn (tcr lcmv) 327sdz [15] ) were obtained from the jackson laboratory. ovalbumin (ova) tcr transgenic mice (c57bl/6-tgn [tcr ova] 1100mjb [16] ) and e ␣ 52-68:i-a b (tea) tcr transgenic mice (17) were provided by dr. michael bevan (university of washington, seattle, wa) and dr. alexander rudensky (university of washington, seattle, wa), respectively. do11.10 tcr transgenic mice were provided by dr. anne o'garra (dnax research institute, palo alto, ca). mice were maintained in a specific pathogen-free facility. the animal care committee of the university of washington approved all procedures used in this report. antibodies. the following monoclonal antibodies were used for flow cytometry: pe-conjugated anti-tcr ␤ (clone h57-597), hamster anti-igg (clone ha4/8), rat anti-igg (clone r35-95), anti-cd3 (clone 17a2), anti-v ␤ 8.1,8.2 (clone mr5-2), and anti-v ␣ 2 (clone b20.1); fitc-conjugated anti-cd44 (clone im7), anti-igg (clone r35-95), anti-cd3 (clone 500-a2), anti-cd4 (clone gk1.5), anti-cd8 (clone 53-6.7), anti-tcr ␤ (clone h57.597), and anti-cd45rb (clone 16a); biotin-conjugated anti-tcr ␤ (clone h57.597), anti-cd49d (clone r1-2), anti-igg (clone r35-95), anti-v ␤ 8 (clone mr 5-2), anti-cd45r (clone ra3-6b2), anti-cd8 (clone 53-6.7), anti-v ␣ 2 (clone b20.1), anti-cd4 (clone rm 4-5), anti-cd45rb (clone 16a), and anti-cd62l (clone mel-14); and purified anti-cd16/cd32 (fc ␥ iii/ii receptor, clone 2.4 g2). all monoclonal antibodies were purchased from bd pharmingen except anti-cd3, which was purchased from caltag. biotin-conjugated primary monoclonal antibodies were detected by streptavidin-conjugated tricolor from caltag. flow cytometry. single-cell suspensions (10 6 ) of splenocytes, ln cells, or cns cells (10 5 ) were resuspended in 10 l of whole mouse serum and anti-cd16/cd32 (0.05 g/ml) for 15 min at room temperature (rt). samples were then incubated for 30 min at rt in the dark with primary antibodies diluted in 50 l of facs ® staining buffer (fsb [0.05% sodium azide and 5% fcs; atlanta biologicals] in pbs). cells were washed, resuspended in 50 l of secondary antibody diluted in fsb, and incubated for 20 min at rt. cells were washed and resuspended in either 300 l of fsb or fixed in fsb with 0.37% formaldehyde. cells (at least 3 ϫ 10 5 per sample) were analyzed with a facscan™ flow cytometer (becton dickinson). fluorescence is plotted on a log scale. lymphocyte isolation. mice were anesthetized with a mixture of xylazine (7 mg/kg), ketamine (100 mg/kg), and pentobarbital (50 mg/kg) diluted in saline and administered by intraperitoneal injection. deeply anesthetized mice were perfused with 50 ml of heparinized pbs via the left ventricle of the heart. the spinal cords were removed via insufflation and the brain was dissected. both tissues were minced, dissociated with a 5-ml syringe plunger, and strained through wire mesh (small parts). after centrifugation, cells were resuspended in 37% percoll (amersham pharmacia biotech), centrifuged at 2,118 g for 15 min, washed, and counted. to prepare cells for proliferation experiments, pooled cns samples were washed twice with 37% percoll, resuspended in 30% percoll, and layered over 70% percoll. cells harvested from the gradient interface were washed and resuspended in complete media (dmem; gibco brl) supplemented with essential amino acids (irving scientific), 9.5% fcs, 1.9 mm l -glutamine, 0.95 mm sodium pyruvate, 43 m ␤ -mercaptoethanol, 95 u/ml penicillin g, 95 g/ml streptomycin sulfate, and 0.1 mm nonessential amino acids. the amount of contamination of the cns mononuclear cell preparation with peripheral cells was assessed using proflavin staining. proflavin is a nuclear dye that crosses the endothelium of most blood vessels easily but does not cross the blood-brain barrier (18, 19) . in two separate staining experiments in which proflavin hydrochloride (20 g/ml) was administered intravenously, ͼ 80% of the cns mononuclear cells did not stain positively for proflavin (data not shown). splenocytes were purified over a lympholyte m gradient (cedarlane) and rbcs were lysed before staining. lns (cervical, inguinal, brachial, and axillary) were teased with tweezers and filtered through wire mesh (small parts) to create a single-cell suspension. in some experiments, cervical and inguinal lns were harvested separately. t cell stimulation assay. single-cell suspensions of ln or cns mononuclear cells were plated at 1.5 ϫ 10 4 v ␣ 2 ϩ t cells/ well (mbp tcr1, tea tcr) or 2 ϫ 10 4 tcr ϩ t cells/well (mbp tcr2, d011.10, and nontransgenic) in 96-well plates in complete media. apcs (10 6 /well) consisted of splenocytes from the appropriate wild-type mouse depleted of tcr ϩ cells by panning with anti-thy 1.2 (clone 30-h12; bd pharmingen)-coated plates (20) , or by magnetic bead depletion using biotinylated anti-tcr and streptavidin-coated dynabeads (dynal). mbp tcr1 and mbp tcr2 t cells were stimulated in the presence or absence of 30 m mbp ac1-11 peptide (research genetics) and t cell-depleted apcs. tea t cells were stimulated in the presence or absence of 5 g/ml e ␣ 52-68 peptide (a gift from dr. alexander rudensky) and t cell-depleted apcs. d011.10 t cells were stimulated in the presence or absence of 1 m ova 323-339 peptide (a gift from dr. craig beeson, university of washington, seattle, wa) and t cell-depleted apcs. 5-bromo-2 јdeoxyuridine (brdu; 25 g/ml; sigma-aldrich) was added after 48 h of culture and cells were harvested 12-16 h later. proliferation was assessed by staining the cultured cells with pe-labeled anti-v ␣ 2 (for mbp tcr1 and tea transgenic mice) or anti-tcr monoclonal antibody (for mbp tcr2 and do11.10 tcr transgenic mice), cell permeabilization, and staining with fitc-labeled anti-brdu monoclonal antibody (clone 3d4; bd pharmingen) as described previously (21, 22) . for assessment of anergy in cns t cells, stimulations were performed as described above except in the presence or absence of 50 u il-2/ml (23) . statistics. the student's t test was used for a comparison of the means between sample groups. error bars represent one sd. animals. the high incidence of spontaneous eae in recombinase activating gene (rag) ϫ / ϫ mbp tcr transgenic mice suggests that t cells may be activated by encountering mbp epitopes in vivo. activation of mbp-specific t cells within the cns is only possible if some naive t cells gain access to this tissue. to investigate this possibility, we isolated lymphocytes from the brain and spinal cord of clinically healthy, 4-7-wk-old, well perfused, mbp tcr1 transgenic mice on the rag ϩ / ϩ background. lymphocytes from individual animals were analyzed to avoid mixing cells from any mice that had preclinical spontaneous eae with cells from healthy mice. surprisingly, the number of t cells per gram of cns tissue isolated from young, healthy mbp tcr1 transgenic mice was comparable to the number isolated from nontransgenic control mice ( fig. 1 ). this result was unexpected because entry into the cns is believed to be limited to activated t cells (7, 9, 10) , and tcr transgenic mice typically have few activated t cells in the periphery (14, 24) . a second mbp tcr transgenic model (mbp tcr2) also demonstrated normal numbers of t cells within the cns compared with age-matched nontransgenic mice ( fig. 1 ). of eight mbp tcr2 transgenic mice analyzed, only one mouse exhibited an abnormally high number of t cells (1.5 ϫ 10 6 t cells/g, 100-fold higher than the average t cell count). data from this mouse were not included in fig. 1 because of the possibility that this animal was developing spontaneous disease. the number of t cells in the cns of rag ϫ / ϫ mbp tcr transgenic mice was also analyzed to determine if the expression of endogenous tcrs influenced the ability of transgenic t cells to enter the cns. the number of t cells isolated from the cns of rag ϫ / ϫ mbp tcr transgenic mice were comparable to the number isolated from nontransgenic mice and rag ϩ / ϩ tcr transgenic mice (fig. 1) . t cells in the cns of young tcr transgenic mice are predominantly naive. previous studies in nontransgenic mice indicated that only activated t cells cross the blood-brain barrier (7, 9, 10) . to investigate whether the t cells found in the cns of tcr transgenic mice also display an activated phenotype, t cell populations in 4-7-wk-old nontransgenic (controls) and rag ϫ / ϫ mbp tcr transgenic mice were analyzed using antibodies specific for cell-surface activation/memory markers. in nontransgenic mice, the majority of cns t cells expressed high levels of cd44 as well as low levels of cd45rb (fig. 2 ) and high levels of cd49d (data not shown), consistent with an activated/ memory phenotype. peripheral t cells from nontransgenic mice expressed significantly lower levels of these activation/memory markers compared with cns t cells ( p ϭ 0.002). these observations are consistent with earlier studies showing that the cns is preferentially trafficked by activated t cells. our analysis also revealed that the ratio of cd8 ϩ /cd4 ϩ t cells is more than twofold higher in the cns than in the spleen in nontransgenic mice (data not shown). the basis for this cd8 ϩ enrichment in the cns is not clear; however, it may reflect the fact that a higher percentage of cd44 high t cells are found in the cd8 ϩ peripheral t cell subset compared with the cd4 ϩ peripheral t cell subset (28 ϯ 3.5% versus 19 ϯ 3.9%, respectively, n ϭ 4) in wild-type mice. the phenotype of t cells in both the periphery and cns of rag ϫ / ϫ mbp tcr transgenic mice was strikingly different from the phenotype seen in nontransgenic mice. the highly restricted tcr repertoire in rag ϫ / ϫ tcr transgenic mice limits the ability of these t cells to be activated by environmental antigens. therefore, the percentage of cd44 high splenocytes in both rag ϫ / ϫ mbp tcr1 and rag ϫ / ϫ mbp tcr2 transgenic mice was 10-fold lower than the percentage in nontransgenic mice (fig. 2) . unexpectedly, the majority of t cells isolated from the cns of rag ϫ / ϫ mbp tcr1 and mbp tcr2 transgenic mice also exhibited a naive phenotype characterized by low levels of cd44 (fig. 2 ) and cd49d (data not shown), and primarily intermediate to high levels of cd45rb (fig. 2) . the data described above suggest that reducing the number of activated t cells in the periphery does not diminish trafficking to the cns, but instead allows more naive t cells to gain entry. this hypothesis predicts that the cns of other rag ϫ/ϫ tcr transgenic mice should also contain similar numbers of t cells as the cns of wild-type mice, and that the cns t cells in the tcr transgenic mice should display a naive phenotype. to test these predictions, cns t cells were isolated from two other rag ϫ/ϫ tcr transgenic models that were not specific for cns antigens. in rag ϫ/ϫ lcmv and rag ϫ/ϫ ova tcr transgenic mice, the number of t cells isolated per gram of cns tissue was again comparable to the number of t cells isolated from nontransgenic control mice (data not shown). t cells isolated from the cns of both of these transgenic models also express low levels of cd44 and cd49d, and intermediate to high levels of cd45rb ( fig. 2 and data not shown). in each rag ϫ/ϫ tcr transgenic model except rag ϫ/ϫ mbp tcr1 transgenic mice, the percentage of naive t cells (cd45rb high , cd44 low ) found in the cns is ͼ80% compared with ͻ30% in nontransgenic mice (fig. 2) . rag ϫ/ϫ mbp tcr1 transgenic mice are unique in that they appear to contain a higher percentage of cd45rb low t cells in the cns than the other transgenic models, and consequently a lower percentage of naive t cells. interestingly, mbp tcr1 transgenic mice on a rag ϩ/ϩ background also differ significantly from mbp tcr2 transgenic mice in their incidence of spontaneous eae. in a conventional animal facility, the incidence of spontaneous eae is 45% in mbp tcr1 mice but only 11% in mbp tcr2 transgenic mice (25) . in our analyses of t cell phenotype, we also examined the expression of cd62l, an ln homing receptor typically expressed on naive t cells (26, 27) . in the periphery of both nontransgenic and tcr transgenic mice, cd62l was predominantly expressed on naive t cells (defined by expression of cd44, cd49d, or cd45rb) as expected. surprisingly, in nontransgenic and tcr transgenic models, cd62l expression was observed on cns t cells that exhibited activation/memory markers as well as on naive t cells, and did not correlate with either phenotype. cns lymphocytes were enriched for cd62l low cells compared with splenocytes in nontransgenic animals (cns, 47 ϯ 7%; spleen, 10 ϯ 7%; n ϭ 3), rag ϫ/ϫ mbp tcr2 transgenic mice (cns, 46 ϯ 11%; spleen, 7 ϯ 2%; n ϭ 3), rag ϫ/ϫ ova transgenic mice (cns, 64 ϯ 10%; spleen, 10 ϯ 1%; n ϭ 4), and rag ϫ/ϫ lcmv transgenic mice (cns, 52 ϯ 17%; spleen, 9 ϯ 4%; n ϭ 3). this general enrichment of cd62l low cells in the cns and lack of correlation with expression of activation markers suggest that cd62l is not strictly a naive t cell marker, and its downregulation in some cases may occur independently of t cell activation. mbp-specific t cells convert to memory cells with age. the observation that young rag ϫ/ϫ mbp tcr1 transgenic mice have a similar percentage of cd45rb low t cells in the cns and spleen as nontransgenic mice suggested that interactions with the mbp-specific tcr on these cells promotes the accumulation of t cells with a memory phenotype. antigenic stimulation could be provided by mbp itself, by a cross-reactive self-antigen, or environmental antigen. in wild-type mice, the percentage of memory t cells increases with age in the peripheral lymphoid compartments, presumably due to an increased number of encounters with environmental antigens (28) . we hypothesized that a similar increase in the number of memory t cells should be observed in mbp tcr1 transgenic mice if t cells in these animals are continuously interacting with antigen. analyzing t cell populations in the spleen and cns of mbp tcr1 transgenic mice and nontransgenic mice as a function of age tested this prediction. rag ϩ/ϩ instead of rag ϫ/ϫ mbp tcr1 transgenic mice were analyzed because older rag ϫ/ϫ mbp tcr transgenic mice exhibit a 100% incidence of spontaneous eae (12) . in contrast, rag ϩ/ϩ mbp tcr1 transgenic mice exhibit eae primarily between 5 to 10 wk of age, but very few cases of eae are observed in mice ͼ69 d of age (14) . older mbp tcr1 transgenic mice (ͼ69 d) exhibited a significant increase in the percentage of activated/memory cells (cd45rb low , cd44 high ) in both the cns and the periphery compared with young mbp tcr1 transgenic mice (p ϭ 0.0006; fig. 3 and table i ). furthermore, the age-dependent decrease in naive t cells was significantly greater in mbp tcr1 transgenic mice than in nontransgenic mice (p ϭ 0.0003). a similar age-dependent decrease in naive t cells is seen in mbp tcr2 transgenic mice (data not shown). several observations support the idea that the increase in memory t cells in rag ϩ/ϩ mbp tcr transgenic mice results from interactions via the mbp-specific tcr rather than interactions with environmental antigens via endogenously rearranged tcrs. first, the absolute number of cns t cells per gram of tissue in older mbp tcr transgenic mice is significantly greater than the number found in older nontransgenic mice (1.4 ϫ 10 4 cns t cells in nontransgenic mice, n ϭ 11; 6.0 ϫ 10 4 cns t cells in mbp tcr1 mice, n ϭ 10, p ϭ 0.0007; 7.1 ϫ 10 4 cns t cells in mbp tcr2 mice, n ϭ 3, p ϭ 0.007). this observation suggests that expression of the mbp tcr results in more antigenic stimulation than the diverse repertoire of tcrs expressed in nontransgenic mice. second, the absolute number of both total mbp-specific (v ␣ 2 ϩ ) t cells and cd45rb low v ␣ 2 ϩ t cells increases with age within the cns in mbp tcr1 transgenic mice, suggesting that the increase in memory t cells does not simply reflect an in-crease in t cells with endogenously rearranged tcr chains (data not shown, p ϭ 0.00004). these data are in contrast to results from mhc class ii-restricted tcr transgenic mice specific for a nonself antigen, pigeon cytochrome c (24) . in this model, there was an increase in peripheral memory t cells as the animals aged; however, this increase was completely in the transgene-negative t cells rather than the transgene-positive t cells. our data demonstrate that as mbp tcr transgenic mice age, an increasing number of t cells expressing mbp-specific tcrs are activated and converted to a memory phenotype in the absence of spontaneous eae. tolerance is induced in mbp-specific naive t cells that enter the cns. the observation that activated/memory mbp-specific t cells are found in higher numbers in the in situ tolerance within the cns cns of healthy, older transgenic mice than in younger mice was inconsistent with the fact that the incidence of spontaneous eae is significantly higher in younger rather than older mice. therefore, we investigated the possibility that, in contrast to activated t cells, naive mbp-specific transgenic t cells entering the cns undergo tolerance in situ and become unresponsive to mbp. cns t cells isolated from 14-17 healthy mbp tcr1 transgenic mice were pooled and cultured in vitro with mbp ac1-11. t cell-depleted splenocytes from wild-type mice were added as apcs. v ␣ 2 ϩ t cells isolated from the cns of mbp tcr1 transgenic mice did not proliferate in response to antigen (fig. 4, a and b , p ϭ 0.004). in contrast, v ␣ 2 ϩ t cells from lns of the same animals plated at the same density as the cns t cells proliferated robustly in response to mbp ac1-11. cns t cells isolated from mbp tcr2 transgenic mice also did not proliferate in response to antigen, whereas ln t cells proliferated strongly (fig. 4 b) . to determine whether the induction of nonresponsiveness in t cells within the cns was unique to t cells specific for mbp, two different mhc class ii-restricted tcr transgenic mouse models specific for non-cns antigens were analyzed using the same experimental protocol. both cns and ln t cells harvested from tea tcr transgenic mice specific for the mhc class ii e␣ peptide and do11.10 tcr transgenic mice specific for ovalbumin proliferated equally when stimulated with e␣ peptide and ova peptide, respectively, in vitro (fig. 4 b) . thus, nonresponsiveness of t cells from the cns was observed only when the t cells were specific for a cns antigen. previous studies of tolerance to tissue-specific antigens have implicated draining lns as the site for induction of t cell tolerance. therefore, we investigated the possibility that nonresponsiveness may be induced in the mbp-specific t cells by presentation of mbp epitopes in the cervical lns that are believed to drain the cns (29, 30) . in the experiments in fig. 4 b, t cells from multiple peripheral lns draining different tissues were pooled together such that nonresponsiveness in the cervical lns might not have been detected. fig. 4 c compares the proliferative responses to mbp ac1-11 of v ␣ 2 ϩ t cells isolated from the cervical and inguinal lns of mbp tcr1 transgenic mice. t cells from both types of lns proliferated strongly to mbp peptide, supporting the idea that induction of tolerance in mbpspecific t cells occurs within the cns itself. to investigate the mechanism of tolerance induction in the cns, we first determined if the mbp-specific t cells isolated from the cns were anergic. cns t cells from both mbp tcr1 and mbp tcr2 transgenic mice were harvested and stimulated with mbp ac1-11 and irradiated apcs in the presence and absence of il-2. cns t cells isolated from both transgenic models remained nonresponsive to antigen even in the presence of il-2, whereas ln t cells from the same mice proliferated robustly (data not shown). therefore, the lack of proliferation observed with mbp-specific t cells from the cns was not due to an ability to produce il-2. in all experiments described so far, no more than 21 mbp tcr transgenic mice were pooled per experiment. in one additional experiment, 36 mbp tcr1 tcr transgenic mice were pooled in order to expand the number of wells exposed to il-2. in this experiment, both cns and ln t cells proliferated in the presence and absence of il-2 (data not shown). no proliferation was detected in any of the other experiments using mbp tcr transgenic cns t cells (n ϭ 5). therefore, we suspect that the unusually large number of mice pooled in this experiment included a mouse with subclinical eae. the incidence of spontaneous eae in the colony from which these mice were obtained is 15%. t cells in the cns of mice with subclinical eae will already be activated and would be expected to proliferate in vitro. we next investigated whether the mononuclear cells isolated from the cns of mbp tcr transgenic mice could actively suppress the responses of t cells isolated from the periphery of the same mice that have not undergone tolerance induction. in control experiments, ln t cells (2 ϫ 10 4 ) from mbp tcr1 or tcr2 transgenic mice were cultured with and without mbp peptide and either irradiated apcs alone or with irradiated apcs and cns cells isolated from 15 to 17 nontransgenic mice. the transgenic ln t cells proliferated strongly to mbp ac1-11 in both the presence and absence of cns mononuclear cells isolated from nontransgenic mice (fig. 5) . these results confirm our observations made with the tea and do11.10 tcr transgenic mice that cns mononuclear cells do not generally suppress t cell proliferation. interestingly, cns cells isolated from mbp tcr transgenic mice had a very different effect in these experiments. proliferation of mbpspecific ln t cells was significantly inhibited upon incubation with cns cells isolated from mbp tcr transgenic mice compared with incubation with peptide and irradiated apcs alone (fig. 5, p ϭ 0.004) . these data are the results of four independent experiments using cns cells from mbp tcr transgenic mice. thus, mononuclear cells present only in mbp tcr transgenic mice and not nontransgenic mice are capable of mediating bystander suppression of nontolerant mbp-specific peripheral t cells. our studies show that multiple mechanisms are responsible for maintaining the immunologically privileged status of the cns. previous work indicated that immunological privilege was the result of the specialized structure of the blood-brain barrier that limited t cell trafficking to activated t cells (7, 9, 10) . our data support the notion that t cells found in the cns of wild-type mice are predominantly activated/memory t cells. however, recent experiments in sheep demonstrated that naive lymphocytes infused directly into the blood could circulate through the cerebral spinal fluid (csf [31] ). our experiments suggest a basis for reconciling these apparently conflicting findings. we show that the same number of t cells are found in the cns of nontransgenic, tcr transgenic, and even rag ϫ/ϫ tcr transgenic mice that have very few activated t cells in the periphery. thus, a similar number of t cells traffic through the cns of healthy animals regardless of the number of activated t cells present in the periphery. the cns of nontransgenic mice is enriched in activated/memory t cells, suggesting that activated t cells have a competitive advantage over naive t cells in crossing the blood-brain barrier. however, as the number of activated t cells in the periphery decreases, more naive t cells are able to enter the cns. this is most clearly illustrated in rag ϫ/ϫ tcr transgenic mice in which there are very few activated t cells in the periphery and the vast majority of t cells in the cns exhibit a naive phenotype. the increased ability of activated/memory t cells to cross the blood-brain barrier may explain our finding in nontransgenic mice that more cd8 ϩ t cells are found within the cns than cd4 ϩ t cells. in the periphery of nontransgenic mice, the activated/memory t cell population contains a greater percentage of cd8 ϩ than cd4 ϩ t cells. the cd44 high t cell population in the periphery has been shown to have a higher percentage of cd8 ϩ t cells relative to cd4 ϩ t cells in other models as well (32) . alternatively, experiments in a different system suggest that cd8 ϩ t cells may preferentially traffic through the cns (33) . in these studies, dendritic cells and moth-cytochrome c peptide were injected into the csf of mhc class iirestricted moth-cytochrome c peptide tcr transgenic mice. preferential recruitment of mhc class i-restricted cells to the cns was observed even though mhc class ii molecules presented the stimulating antigen. these findings and our results are both consistent with enhanced re-circulation or accumulation of cd8 ϩ t cells through the cns. an important implication of our results is that circulation of autoreactive naive t cells through the cns represents a potential trigger of autoimmune disease. although the degree of trafficking through the cns by naive t cells is greater in rag ϫ/ϫ tcr transgenic mice than in nontransgenic mice, some trafficking of the cns by naive t cells occurs continuously in nontransgenic animals. we demonstrate that initiation of autoimmunity by these naive t cells in the cns is specifically prevented by the induction of tolerance in situ. naive mbp-specific t cells isolated from the cns are unresponsive to antigen while mbp-specific t cells from the periphery of the same animals are fully functional in response to antigen stimulation. induction of tolerance is unique to mbp-specific t cells present in the cns, as cns t cells isolated from tcr transgenic mice specific for non-cns antigens are not tolerized and proliferate robustly in response to antigen in vitro. previous studies of t cell tolerance to tissue-specific antigens indicated that tolerance occurred in lns draining the relevant tissue. for example, t cells specific for antigens expressed in the pancreas undergo deletion within the lns draining the pancreas (34) (35) (36) (37) (38) . in contrast, we demonstrate that induction of tolerance in mbp-specific t cells occurs within the target organ itself rather than within a lymphoid tissue. first, we confirmed that our cns t cell isolation procedures from mbp tcr transgenic mice produced t cells that were present behind the blood-brain barrier in vivo without significant contamination of peripheral t cells (see materials and methods). second, we examined t cells isolated from cervical lns for evidence of tolerance because these lns are believed to drain the cns (29, 30) . in situ tolerance within the cns we found that t cells from these lns proliferated as robustly as t cells from other lns and significantly more than t cells from the cns. thus, tolerance does not appear to occur in the draining lns but rather occurs in the cns itself. it is not yet clear whether in situ tolerance induction is unique to the cns and constitutes an aspect of immune privilege of this organ, or whether tissue-specific tolerance occurs in other organs as well. exposure of naive t cells to antigens within parenchymal tissues has been suggested as a basis for some forms of neonatal tolerance. t cell trafficking through extra-lymphoid tissues is thought to occur more extensively in neonates than in adult mice. however, in at least one example the tolerance induced in neonates by increased trafficking of peripheral tissues was systemic tolerance and did not result in the presence of tolerant t cells exclusively in the relevant tissue (39) . in contrast to these findings, we demonstrate that the tolerance to mbp within the cns is tissue-specific rather than systemic. our investigation of the mechanism of tolerance induction in naive mbp-specific t cells entering the cns showed that tolerance does not occur through downregulation of tcr or cd4 because the expression of these receptors on cns t cells is comparable to that observed on peripheral t cells (data not shown). inhibition of proliferation of cns mbp-specific t cells is not mediated by inhibitory factors found in the csf or brain during inflammation that appear to modulate t cell activity (40, 41) . these factors operate within the microenvironment of the inflamed cns and would not be present in our in vitro cultures. in addition, naive cd4 ϩ t cells specific for non-cns antigens proliferate equally well when they are isolated from either the cns or lns, and it is only t cells from mbp tcr transgenic mice that are nonresponsive. our data suggest instead that the interaction of naive mbpspecific t cells with antigen in the cns results in an irreversible state of nonresponsiveness that is maintained even in the absence of soluble factors found in cns microenvironments during inflammation. the nonresponsiveness induced in mbp-specific t cells in situ was not reversed by addition of exogenous il-2 in vitro; however, in vivo induction of t cell anergy is not necessarily overcome by addition of exogenous il-2 (42) (43) (44) (45) (46) . influenza hemagglutinin-specific tcr transgenic t cells that have been anergized in vivo have been shown to contain high levels of il-4 and il-10 mrna, suggesting that the anergic t cells may function as regulatory t cells (46) . this phenomenon could explain our finding that mononuclear cells isolated from the cns of mbp tcr transgenic mice are capable of suppressing the proliferation of nontolerant peripheral mbp-specific t cells in vitro. investigation is underway to determine which cell types mediate the suppression and whether this bystander suppression is mediated by soluble factors and/or is cell contact dependent. despite tolerance induction of naive mbp-specific cns t cells, spontaneous eae still occurs in a percentage of the mbp tcr transgenic mice, primarily within an age window of 5-12 wk (14) . interestingly, our studies show that at the age when the incidence of spontaneous eae in mbp tcr1 transgenic mice declines, the number of mbp-specific t cells in the cns of these mice doubles and their phenotype converts to that of an activated/memory t cell. this increase in activated/memory t cells is due in part to expression of the mbp-specific tcr because the number of memory t cells is greater in the cns of older tcr transgenic mice than in the cns of age-matched nontransgenic mice, and much of this increase in mbp tcr transgenic mice can be accounted for by an increase in mbpspecific (v ␣ 2 ϩ ) memory cells. thus, although t cells in older mbp tcr transgenic mice appear to have encountered antigen more frequently than t cells in older nontransgenic mice, this encounter is not correlated with initiation of disease. spontaneous eae may be prevented in older mbp tcr transgenic mice by the development of nontransgenic cd4 ϩ regulatory t cells that have been shown to be critical in prevention of spontaneous eae (12, 47, 48) . these regulatory t cells must not function by decreasing exposure of mbp-specific t cells to antigen since memory mbp-specific t cells accumulate in older mbp tcr transgenic mice, but rather the regulatory t cells must change the outcome of this interaction. in summary, our experiments suggest a model in which naive mbp-specific t cells that escape thymic or peripheral tolerance traffic at a low frequency through the cns in normal individuals. these t cells are prevented from initiating cns autoimmune disease by in situ tolerance when they encounter antigen within the cns. the spontaneous autoimmune disease that occurs in some mice may be caused by a failure of tolerance at several levels. peripheral events could activate mbp-specific t cells either by immunization, as is the common mechanism for the induction of eae, or via molecular mimicry mediated by an infectious agent (49) (50) (51) (52) (53) . once activated, the mbp-specific t cells are no longer susceptible to tolerance in situ and can initiate autoimmune disease. alternatively, autoimmunity could develop because tolerance occurring within the cns is circumvented. potential mechanisms for overcoming tolerance in the cns include an infection that may increase costimulation and mhc expression in situ, genetic defects in the tolerance mechanisms, or peripheral infections that may influence antigen presentation within the cns through the release of soluble factors. these experiments reveal a new form of immunoregulation of cns-specific t cells that must be considered in the study of the pathogenesis of cns autoimmunity. tolerating the nervous system: a delicate balance self-ignorance in the peripheral t-cell pool tolerance and ways to break it experimental autoimmune (allergic) encephalomyelitis: induction, pathogenesis, and suppression grune and stratton immunology of multiple sclerosis and experimental allergic encephalomyelitis immunologically privileged sites traffic of hematogenous cells through the central nervous system lymphocyte targeting of the central nervous system: a review of afferent and efferent cns-immune pathways cellular immune reactivity within the cns t-lymphocyte entry into the central nervous system transgenic mice that express a myelin basic protein-specific t cell receptor develop spontaneous autoimmunity high incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein t cell receptor transgenic mice t cell epitope of the autoantigen myelin basic protein that induces encephalomyelitis triggers of autoimmune disease in a murine t-cell receptor transgenic model for multiple sclerosis tolerance induction in double specific t-cell receptor transgenic mice varies with antigen t cell receptor antagonist peptides induce positive selection deficient positive selection of cd4 t cells in mice displaying altered repertoires of mhc class ii-bound self-peptides hematic and fluid barriers in the optic nerve hematic and fluid barriers of the retina and vitreous body in vitro assays for mouse lymphocyte function turnover of naive-and memory-phenotype t cells a functionally compromised intermediate in extrathymic cd8 ϩ t cell deletion differential tolerance is induced in t cells recognizing distinct epitopes of myelin basic protein antigen-independent changes in naive cd4 t cells with aging tolerance and autoimmunity in tcr transgenic mice specific for myelin basic protein a cell-surface molecule involved in organ-specific homing of lymphocytes entry of naive cd4 t cells into peripheral lymph nodes requires l-selectin differences in the expression profiles of cd45rb, pgp-1, and 3g11 membrane antigens and in the patterns of lymphokine secretion by splenic cd4 ϩ t cells from young and aged mice lymphocyte targeting of the central nervous system: a review of afferent and efferent cns-immune pathways cervical lymphatics, the blood-brain barrier and the immunoreactivity of the brain: a new view cerebral spinal fluid lymphocytes are part of the normal recirculating lymphocyte pool pgp-1hi t lymphocytes accumulate with age in mice and respond poorly to concanavalin a disproportionate recruitment of cd8 ϩ t cells into the central nervous system by professional antigen-presenting cells peripheral tolerance of cd4 t cells following local activation in adolescent mice constitutive class i-restricted exogenous presentation of self antigens in vivo class i-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive cd8 ϩ t cells the peripheral deletion of autoreactive cd8 ϩ t cells induced by cross-presentation of self-antigens involves signaling through cd95 (fas, apo-1) ontogeny of t cell tolerance to peripherally expressed antigens control of neonatal tolerance to tissue antigens by peripheral t cell trafficking the susceptibility of mice to immunemediated neurologic disease correlates with the degree to which their lymphocytes resist the effects of brain-derived gangliosides normal cerebrospinal fluid suppresses the in vitro development of cytotoxic t cells: role of the brain microenvironment in cns immune regulation in vivo induction of anergy in peripheral v beta 8ϩ t cells by staphylococcal enterotoxin b reduced cd3-mediated protein tyrosine phosphorylation in anergic cd4 ϩ and cd8 ϩ t cells subset-specific analysis of calcium fluxes in murine aids modifications of cd8 ϩ t cell function during in vivo memory or tolerance induction interleukin 10 secretion and impaired effector function of major histocompatibility complex class ii-restricted t cells anergized in vivo regulatory cd4 ϩ t cells expressing endogenous t cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis cd4 ϩ t cells prevent spontaneous experimental autoimmune encephalomyelitis in anti-myelin basic protein t cell receptor transgenic mice amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity sequence homology between certain viral proteins and proteins related to encephalomyelitis and neuritis molecular mimicry in t cell-mediated autoimmunity: viral peptides activate human t cell clones specific for myelin basic protein myelin basic protein and human coronavirus 229e cross-reactive t cells in multiple sclerosis molecular mimicry and immune-mediated diseases the authors thank eric huseby, antoine perchellet, and audrey seamons for critical reading of the manuscript, and jessica weber for assistance in animal husbandry. key: cord-018042-qt7055fw authors: müller, marcus; campbell, iain l. title: chemokine actions in the cns: insights from transgenic mice date: 2008 journal: central nervous system diseases and inflammation doi: 10.1007/978-0-387-73894-9_10 sha: doc_id: 18042 cord_uid: qt7055fw historically the central nervous system (cns) has been viewed as a relatively immune sheltered tissue. under physiological conditions the cns is devoid of leukocytes, including professional antigen presenting cells (apc), is deficient in key immune accessory molecules such as major histocompatibility molecules (mhc) and is protected by an effective blood brain barrier. significantly, however, in numerous pathological states including infectious diseases and autoimmune disorders (e.g. multiple sclerosis) immune cells are effectively recruited to and infiltrate in the cns. this immune response can be a two-edged sword required on the one hand to control infection and facilitate tissue repair and regeneration but on the other causing tissue injury that can result in life threatening complications. therefore, understanding the mechanisms that control the trafficking of leukocytes to the cns and the subsequent interactions between these cells that contribute to tissue injury has significant implications. chemokines following such insults can be mediated by a number of factors including microbial products (e.g., lps) and host defense molecules such as cytokines (e.g., il-1, ifn-γ and tnf) . cells intrinsic to the nervous system, including neurons, the macroglia and microglia, all have the ability to produce different chemokines (reviewed in asensio and campbell (1999) and ubogu et al. (2006) ). moreover, the surfaces of these same cells are decorated with a variety of different chemokine receptors. thus, the cns seemingly has its own chemokine ligand/receptor network, which is consistent with the growing awareness that the function of chemokines could extend well beyond the regulation of leukocyte trafficking in the brain (asensio and campbell, 1999; mennicken et al., 1999) . so it can be seen that the cns biology of the chemokines is likely to extend well beyond the singular function of leukocyte chemotaxis. a number of important questions are therefore raised concerning the functions of the chemokines in the cns. some key questions include: (1) do the presence of chemokines in different disordered states reflect their primary involvement or is an epiphenomenon? (2) is there a cause and effect relationship between the production of a particular chemokine (or chemokines) and the development of specific molecular and cellular neuropathological alterations? (3) what are the mechanisms that underlie chemokinemediated actions in the cns? (4) could manipulation of the chemokines or their receptors be used as a rational means for therapeutic intervention? to begin to address these questions experimental approaches are required that are non-invasive and organism-based. of particular significance in this regard has been the application of genetic engineering technology that has permitted the germline transmission of foreign genes (transgenes) in the mouse. this is accomplished via a fusion gene construct (transgene) in which the gene of interest is placed under the transcriptional control of a cell-specific promoter. the transgene is then introduced (most commonly by microinjection) into the pronucleus of fertilized eggs, which are then implanted in the oviduct of pseudopregnant recipient mice. the transgenic approach has a number of advantages over other approaches such as intracerebral infusion or in vitro cell culture. transgenic modeling allows the prolonged, reproducible delivery of a specific pure protein to specific cells in the intact cns. the actions of the transgene product are exerted and can be assessed in a milieu where the anatomic and physiologic interactions of the cns are preserved. moreover, individual lines of mice in which the transgene is stably integrated into the genome can be developed and provide an unrestricted source of animals identical for the introduced genetic and molecular alteration, hence permitting systematic multi-level analysis of pathological, electrophysiological, neuroendocrinological and behavioral manifestations. for more detailed reviews of these techniques see (galli-taliadoros et al., 1995; campbell and gold, 1996; campbell et al., 1998) . the application of genetically-manipulated animal models to study the cns biology of the chemokines and their receptors has emerged as an important experimental approach. in this chapter, we will discuss findings from studies based on the use of cns-chemokine transgenic models. in particular, we will focus on the contribution that these models have made to our understanding of the basic functions and mechanisms of actions of chemokines in the cns. as mentioned in the preceding, a great many chemokines belonging in particular to the cxc and cc families are known to be induced in the brain during a variety of different pathologic states (asensio and campbell, 1999; ransohoff, 2002; rebenko-moll et al., 2006; ubogu et al., 2006) . the consequences of chemokine expression in the unmanipulated cns and in some cases in stimulus-evoked disease models have been examined in transgenic mice with cns-targeted expression of specific chemokines. a summary of the salient features of transgenic models in which cns-specific promoter constructs were used to drive expression of a specific chemokine gene is given in table 1 and will be outlined in more detail in the following commentary. the chemokine cxcl1 (also known as gro-α/kc/n51) contains an n-terminal elr-amino acid motif and belongs to the cxc-chemokine family. cxcl1 binds to the cxcr2 receptor found predominantly on the surface of neutrophils. not surprisingly, cxcl1 is a potent neutrophil chemoattractant. that this chemokine has a wider function in the cns is supported by numerous studies. cells intrinsic to the cns including neurons (horuk et al., 1997) , astrocytes (danik et al., 2003; flynn et al., 2003) , microglia (filipovic et al., 2003; flynn et al., 2003) , oligodendrocyte precursor cells (nguyen and stangel, 2001) and mature oligodendrocytes (omari et al., 2006) have been reported to have the cxcr2 receptor. a role for cxcr2/ cxcl1 interaction is documented in rodents in the development and maintenance of the oligodendrocyte lineage, myelination, and white matter in the neonatal and adult cns (padovani-claudio et al., 2006) . the cns function of this chemokine was examined in transgenic mice by placing the cxcl1 gene under the transcriptional control of the myelin basic protein (mbp) promoter . in the resulting transgenic mice (termed mbp-kc) transgene-encoded mrna levels were coincident with the developmentallyregulated pattern of endogenous mbp, peaking at 2-3 week of age, before declining to low levels characteristically seen in adult brain. during the period of maximal transgene expression, the brain of mbp-kc mice was heavily infiltrated with neutrophils that accumulated in perivascular, meningeal and parenchymal sites. at this time, despite loss of blood brain barrier (bbb) integrity and focal gliosis, there was no evidence of brain tissue destruction nor did affected animals display abnormal neurological signs. these results underscore the ability of cxcl1 to promote neutrophil migration to and extravasation in the cns. however, the absence of any tissue destruction suggests that this chemokine does not activate the neutrophils. curiously, in one high transgene expressing line, from > 40 days of age, mice exhibited a syndrome of progressive neurological dysfunction consisting of slowing (boztug et al., 2002) of the righting reflex, clumsiness, increase of rigidity of the hindlimbs and tail and profound truncal instability. the major neuropathological findings were florid microglial activation and bbb disruption without dysmyelination. despite the severity of this neurological disorder (which resulted in premature death), there was no histological evidence of damage to neurons, myelin or axons. although the basis for the neurological dysfunction developing in the mbp-kc mice is unknown, tani et al. (1996) speculated that this might be due to the glial and bbb perturbations. it is also possible as the authors suggested that chronic exposure of neurons to the chemokine may directly compromise their function. in support of such a mechanism, cxcl1 can enhance both stimulus-evoked and spontaneous postsynaptic currents , and increase neurotransmitter release and reduce long-term depression , in purkinje neurons. on the other hand, despite accumulating evidence that cxcl1 influences oligodendrocyte precursor cell and differentiated oligodendrocyte function both in vitro and in vivo, surprisingly, mbp-kc mice were reported to exhibit normal cns myelination both in the acute and chronic disorders . cxcl10 (also known as ip-10) belongs to a sub-family of chemokines that include cxcl9 (mig) and cxcl11 (itac) within the cxc family. unlike cxcl1, the chemokines in this sub-family lack the n-terminal elr-amino acid motif. expression of the cxcl9, cxcl10 and cxcl11 genes is induced by the cytokine ifn-γ and all three chemokines bind to a common receptor, cxcr3. high levels of cxcr3 can be found principally on activated cd4 + th1 and cd8 + t-cells and nk-cells (taub et al., 1993 (taub et al., , 1996a hopkins and rothwell, 1995; liao et al., 1995; loetscher et al., 1996; cole et al., 1998) . in keeping with this cellular receptor distribution, cxc9, cxcl10 and cxcl11 all promote the trafficking of these immune cells in vitro and are strongly implicated in the generation of type i immune responses associated with anti-viral host defense, transplant rejection and autoimmunity (liu et al., 2005) . evidence from rodent studies indicate that a functional cxcr3 receptor exists on microglia (rappert et al., 2004) and neurons (nelson and gruol, 2004) . descriptive studies confirm the presence of high levels of cxcl10 rna and protein, particularly in astrocytes and neurons, in a variety of experimental and human neurological diseases including viral infection and autoimmune diseases (klein, 2004; liu et al., 2005) . a transgenic mouse model was developed in which the gfap promoter was used to accomplish astrocyte-targeted production of cxcl10 (see fig. 10 .1) (boztug et al., 2002) . astrocyte-localized production of the gfap-transgene encoded cxcl10 rna was confirmed while production of cxcl10 at the protein level was demonstrated by immunoblotting. the levels of these transgene-encoded products were shown to be similar to levels of the endogenous cxcl10 induced following viral infection of the mouse brain confirming the pathophysiological relevance of this model. these gfap-cxcl10 transgenic mice bred normally and appeared physically unimpaired. histological evaluation of the brain revealed a predominantly subarachnoidal and meningeal accumulation of mixed leukocytes with only minor parenchymal infiltration (see fig. 10 .2). the accumulation of leukocytes in the brain of these transgenic mice was markedly amplified by systemic immune challenge after immunization with complete freund's adjuvant and pertussis toxin. immunophenotypic characterization of these infiltrates revealed surprisingly, that the majority of infiltrating leukocytes generation of transgenic mice with cxcl10 gene production targeted to astrocytes. a cdna encoding murine cxcl10 was inserted downstream of the murine gfap promoter and upstream of a human growth hormone (hgh) polyadenylation signal sequence. this transgene construct was microinjected into the germ line of mice to generate a stable transgenic line. the presence of cxcl10 gene expression in astrocytes (lower panel) was confirmed by in situ hybridization for cxcl10 rna combined with immunohistochemistry for gfap protein. note that some cxcl10 rna positive astrocytes (black arrows) extend processes (blue arrows) to a blood vessel (asterisk) that is close by (see color plates). were neutrophils and macrophages with a minority being represented by cd + tcells. these observations suggest that constitutive astrocyte production of cxcl10 in the cns does not provide an effective signal for the recruitment of t-cells from the periphery. this conclusion is in keeping with the findings from more recent reports that found that mice deficient for cxcl10 exhibit similar clinical and pathological outcomes as wild type controls for experimental autoimmune encephalomyelitis (eae-a cd4 + t-cell-mediated disease) (klein et al., 2004) as well as murine hepatitis virus encephalitis (stiles et al., 2006) . the finding that neutrophils were preferentially recruited to the cns of the gfap-cxcl10 transgenic mice was unexpected since these cells were not known to have cxcr3. this point was confirmed by the studies of boztug et al. (2002) who used fluorescence activated cell sorting analysis to show that the neutrophil population present in the cns of the gfap-cxcl10 transgenic mice were cxcr3 negative. moreover, the results of a survey of the cerebral expression of a large number of other chemokines including elr-cxc chemokines known to have neutrophil chemoattractant properties proved unremarkable. therefore, the basis for the preferential accumulation of neutrophils in the brain of the transgenic mice is unknown. in all, these findings argue for a cxcr3-independent chemoattractant mechanism mediated by cxcl10 for the recruitment of neutrophils to the brain. it is interesting to note that an alternative receptor for cxcl10 has been reported on endothelial and epithelial cells (soejima and rollins, 2001) . however, the identity of this novel cxcl10 binding moiety and its functional signature is currently unknown. of further note in these gfap-cxcl10 transgenic mice was the lack of any gliosis or degenerative features that would be expected if there existed a destructive immunoinflammatory response in the cns. so while constitutive, astrocyte-targeted production of cxcl10 can promote the recruitment of leukocytes to the cns, this chemokine lacks the ability to further influence these cells, in particular, to drive a functional immune response. moreover, although there are reports that a functional cxcr3 exists on neurons (nelson and gruol, 2004) and microglia (rappert et al., 2004) in the rodent cns, there would seem to be little effect of cxcl10 on these cells in this transgenic model. a possible explanation for this lack of effect of cxcl10 is that there is desensitization to the chemokine through cxcr3 downregulation. in support of this idea, hippocampal slices from wild type but not gfap-cxcl10 transgenic mice in response to acute exposure to recombinant cxcl10 protein were shown to exhibit altered synaptic plasticity that was cxcr3dependent (vlkolinsky et al., 2004) . interestingly, the role of cxcl10 as an effective t-cell chemoattractant may depend on the tissue in which the chemokine is produced. in contrast to the cns, transgenic production of cxcl10 targeted to the beta cells of the islets of langerhans induced spontaneous infiltration of large numbers of cd4 + and cd8 + t-cells around the islets (rhode et al., 2005) . moreover, islet cxcl10 production markedly accelerated a stimulus-evoked autoimmune process by enhancing the migration of antigen-specific t-cells to the islets resulting in the development type i diabetes. the chemokine ccl2 (also known as mcp-1 and je) was first identified as a monocyte attracting chemokine (rollins, 1991; rollins et al., 1991) but also has been shown to attract a wide variety of other leukocytes, including, activated t-cells, dendritic cells, mast cells and basophils (taub et al., 1996b; gunn et al., 1997; siveke and hamann, 1998) . although ccl2 binds to a number of cc receptors the primary receptor for this chemokine is ccr2. cells within the cns including astrocytes and microglia have been shown to be positive for ccr2 (banisadr et al., 2002) , while the ligand is produced by astrocytes (ransohoff et al., 1993; glabinski et al., 1996) . ccl2 is implicated in the pathogenesis of a wide variety of experimental and human neurodegenerative and immune-mediated disorders (cartier et al., 2005) . the first transgenic model developed to achieve ccl2 production in the cns of mice employed the mbp-promoter to drive expression of the ccl2 gene in oligodendrocytes (fuentes et al., 1995) . temporal expression of transgene-encoded ccl2 rna followed that of endogenous mbp peaking between the second and third post-natal week before declining. in addition, ccl2 protein was demonstrated to be present in the brain of the transgenic but not wild type mice during peak transgene gene expression. in concordance with the temporal profile of transgene-encoded ccl2 production, discrete monocytic cell infiltrates were observed throughout the brain predominantly in perivascular sites in the meninges, choroid plexus, and brain parenchymal white matter. the intensity of these infiltrates could be increased as well as further parenchymal infiltration stimulated, following systemic lps administration to the mbp-mcp1 transgenic mice. importantly, there was no evidence of either spontaneous or stimulus-evoked cns injury, particularly in white matter, in these transgenic mice. the observations in this model confirmed the potent monocyte chemoattractant properties of ccl2 and further indicate that while ccl2 can provide a signal for the recruitment of these cells to the cns, the chemokine does not directly stimulate these cells to engage in an active inflammatory process. however, a more recent study using these transgenic mice revealed that a systemic injection of pertussis toxin can induce a reversible encephalopathy with clinical signs, including tremor, inactivity, limb clasping and weight loss (toft-hansen et al., 2006) . encephalopathic signs were correlated with a more diffuse distribution of monocytes, which were less restricted to the perivascular space suggesting a role for the inflammatory process in the clinical symptoms. however the pathological basis for the physical impairments in these mice was not determined and it therefore remains unclear whether tissue injury was also involved. increased levels of metalloproteinases were found in pertussis toxin treated mbp-ccl2 transgenic mice, suggesting a critical need for metalloproteinases for the parenchymal infiltration of the monocytes. weight loss and parenchymal infiltration, but not perivascular accumulation, were markedly resolved by the broad-spectrum metalloproteinase inhibitor bb-94/batimastat. the findings suggest that a critical event in inducing disease in this model is metalloproteinase-dependent leukocyte migration across the astroglial basement membrane of the blood-brain barrier, which is induced by pertussis toxin. as noted above, astrocytes but not oligodendrocytes have been found to be a major source of ccl2 in various cns pathologies. the production of ccl2 under the control of the mbp-promoter follows a similar temporal pattern as the endogenous gene and produces a spike of ccl2 activity. the delivery of maximal transgene-derived ccl2 in this model occurs during a developmentally sensitive period before subsiding making this model less relevant for studying the effects of ccl2 on the adult brain. in order to address these issues, huang and co-workers generated transgenic mice with astrocyte-targeted expression of the ccl2 gene driven by the human gfap promoter (huang et al., 2002 . these gfap-ccl2 transgenic mice produced ccl2 in the cns at levels similar to those produced during eae, yet, these animals exhibited little spontaneous inflammation. this contrasts with the mbp-ccl2 transgenic mouse model in which widespread and marked perivascular accumulation of monocytes was seen. the explanation for this difference between these models is not clear but it may reflect the chronic and more generalized production of high levels of ccl2 in the case of the gfap-ccl2 transgenic mice. however, systemic immunization with complete freund's adjuvant and injection of pertussis toxin induces a transient encephalopathy with elevated levels of ifn-γ and il-2 in the gfap-ccl2 transgenic mice. interestingly, the encephalopathy was less severe in ccl2 transgenic mice on a t-cell deficient background, indicating the involvement of t-cells and not only monocytes in the observed encephalopathy (huang et al., 2002) . moreover, this phenotype was shown to be dependent on ccr2 indicating that under these conditions ccr2 is the principal receptor for ccl2 in the murine cns. although a number of studies have suggested that ccr2 activation in the periphery downregulates th1-t-cell development and can augment th2-t-cell development (daly and rollins, 2003) , the findings in the gfap-ccl2 transgenic mouse indicate that in the cns, ccl2 may recruit th1 t-cells or direct t-cell polarization toward type 1 cytokine production. this concept is also supported by the finding that mice deficient for the ccl2 receptor, ccr2, are resistant to the development of the cd4 + t-cell-mediated autoimmune disease, eae (fife et al., 2000) . however, in findings that are difficult to reconcile with these conclusions, it has been reported that gfap-ccl2 transgenic mice developed significantly milder eae disease than littermate controls (elhofy et al., 2005) . antigen-specific t-cells recovered from the gfap-ccl2 transgenic mice showed decrease proliferative response to autoantigen and secreted less ifn-γ and had lower levels of il-12 receptor rna. these studies suggest that rather than augmenting th1 cells, that chronic ccl2 production in the cns impairs the function of these cells. although it has been possible to produce stimulus-evoked encephalopathy in mbp-and gfap-ccl2 transgenic mice by systemic innate immune challenge these disorders are largely transient and therefore do not model the increased levels of cns ccl2 that have been reported to occur in chronic neurological diseases such as human immunodeficiency virus type 1-associated dementia, amyotrophic lateral sclerosis, and multiple sclerosis. however, huang et al. (2005) report from a more recent study that gfap-ccl2 transgenic mice develop a spontaneous, delayed encephalopathy from > 7 months of age. these mice presented with loss of weight, a hunched posture, slow response to tactile stimuli and a flaccid tail. some animals even developed a hind-limb paralysis at > 12 months of age. the brain exhibited perivascular infiltrates made up of activated macrophages, microglia with morphological activation, and impaired bbb. surprisingly, no evidence was found for demyelination or of reduced neurons, axons, or synapses of neural components. microglia in the gfap-ccl2 transgenic mice, despite the appearance of activation and increased cd45 immunoreactivity, were found to be defective in their response to environmental stimuli in vitro. the authors propose that chronic elevation of ccl2 in the cns leads to desensitization of ccr2 and microglial dysfunction. further support for the notion that chronic ccl2 leads to microglial dysfunction comes from a study by yamamoto et al. who generated bigenic gfap-ccl2/tg 2576 mice that, in addition to chronic ccl2, also overproduce a mutant amyloid precursor protein (app) in the cns (yamamoto et al., 2005) . these workers found a significant increase in amyloid deposition in ccl2/app bigenic mice compared with app singly transgenic mice. despite being accompanied by an increase in lesion-associated macrophages/microglia, evidence is provided suggesting that these cells show reduced phagocytic function leading to accelerated amyloid deposition. these findings underline the importance of considering the participation of chemokines and chemokine driven monocyte accumulation and function in the pathogenesis of degenerative cns diseases. the chemokines ccl19 and ccl21 have been implicated in the migration of lymphocytes and are produced in secondary lymphoid organs such as the lymph nodes. both these chemokines bind to ccr7 and ccr11. mice lacking ccr7 have impaired migration of lymphocytes into secondary lymphoid organs (forster et al., 1999) . ccl21 but not ccl19 has also been reported to bind to cxcr3 (rappert et al., 2002) . ccl19 and ccl21 are localized in venules surrounded by ccr7 positive inflammatory cells in the brain and spinal cord of mice with eae and functional studies suggest that ccl19 and ccl21 are involved in t lymphocyte migration into the central nervous system during eae (alt et al., 2002) . however, recent studies in ccr7 deficient mice suggest that ccl19 and ccl21 function may be dispensable in eae (pahuja et al., 2006) . this interpretation is complicated in the case of ccl21 since this chemokine could still play a role in eae in ccr7 deficient mice via cxcr3 on encephalitogenic t-cells. moreover, ccl21 was reported to be produced by neurons in the murine brain following ischemic insult, highlighting the existence of an intrinsic source of this chemokine in the cns that could interact with microglia via cxcr3 . to study the biological role of the chemokine ligands ccl19 and ccl21, transgenic mice were developed that expressed either ccl19 or ccl21 in oligodendrocytes of the cns using an mbp-promoter construct (chen et al., 2002a) . as expected, transgene-encoded ccl19 and ccl20 mrna was developmentally regulated and peaked at 2-3 weeks of age. expression of ccl19 or ccl21 protein in the cns of the transgenic mice was confirmed by immunohistochemical staining. ccl19 producing transgenic mice did not show any clinical or histological phenotype, which led to the conclusion that ccl19, at least from diffuse cerebral expression, is not able to attract leukocytes into the brain or cause a cns pathology. in contrast, transgenic mice with mbp-promoter driven production of ccl21 developed a rapid onset encephalopathy with tremor, ataxia and weight loss. most of the ccl21 producing animals died within 4 weeks. studies in vitro documented a chemotactic effect of ccl21 for thymocytes, naïve t cells, dendritic cells and b cells and not for macrophages and neutrophils (hedrick and zlotnik, 1997; nagira et al., 1997; kellermann et al., 1999) . however, cerebral infiltrates in ccl21 producing transgenic mice consisted almost exclusively of neutrophils and eosinophils. these infiltrates were associated with gliosis, microglial activation and demyelination. infiltrating lymphocytes were not observed-even in areas in which the bbb was disrupted. the lack of lymphocyte infiltration is a notable finding because transgenic expression of ccl21 in the pancreas induced lymphocyte influx and led to the formation of lymph node like structures (fan et al., 2000; chen et al., 2002b) . it would thus appear that tissue-specific factors that might include for example, specific adhesion molecules and other cofactors, are required for ccl21 to induce lymphocyte recruitment. as pointed out by the authors the phenotypic differences observed between mbp-ccl21 and mbp-ccl19 transgenic mice is very interesting given that these chemokines are functionally related and bind to the same cell surface receptors, ccr7 and ccr11. however, and as noted above ccl21 but not ccl19 also binds to cxcr3, which in the case of murine microglia produces functional alterations in these cells (rappert et al., 2002) . interestingly, the same workers have reported that neurons in the ischemic brain produce ccl21 (biber et al., 2001) . these observations have led to the suggestion that ccl21 and cxcr3 may mediate neuron-glia interactions during disease conditions. it is speculated that ccl21 derived from transgenic oligodendrocytes in the brain of the mbp-ccl21 mice may have interacted with cxcr3 on microglia inducing an inflammatory response that led to the influx of inflammatory cells into the cns. while further studies, such as crossing the mbp-ccl21 transgenic mice with cxcr3 deficient animals, would help to clarify this mechanism, as noted in the preceding discussion, transgenic mice with astrocyte-targeted production of the primary cxcr3 ligand cxcl10 do not share the severe neurological phenotype of the mbp-ccl2 mice. an important constraint with the transgenic approach that needs to be considered relates to the spatial and temporal control of transgene expression. the production of chemokines in pathologic states such as in eae is regulated in a temporal fashion that precedes and then overlaps with the severity of the disease process. moreover, the spatial pattern of expression of chemokines such as cxcl10 is focally restricted to the immunoinflammatory lesions. the production of chemokines achieved in pathologic states therefore would favor the establishment of highly localized chemoattractant gradients that then guide the trafficking of leukocytes. in contrast, in the transgenic model the chemokine is produced more chronically and more diffusely throughout the cns. in addition to the potential for receptor down-regulation and desensitization of the ligand response, it is likely that any chemoattractant gradient in this milieu would be much weaker in strength and more widely distributed. technically achieving more acute and focal expression of the chemokine to better replicate pathologic states in the adult brain using transgenic modeling is clearly desirable but difficult to achieve at this time. although constructs have been engineered that permit the temporal control of transgene expression in the cns, more precise spatial targeting that would allow for highly localized production of the chemokine in neural cells such as astrocytes is not yet possible. despite the drawbacks noted in the preceding discussion, it is clear that development of molecular genetic methods that allow for the targeted manipulation of gene expression in an intact organism has been important in contributing to our understanding of the actions of chemokines in the cns in physiologic and pathologic states (see table 1 ). promoter-driven expression of different biologically relevant chemokines in the cns of transgenic mice highlight the ability of some but not all of these molecules to stimulate the recruitment but commonly not the functional activation of specific subsets of leukocytes to the cns. with certain chemokines such as cxcl10 and ccl21, their transgenic production in the cns led to the accumulation of unexpected leukocyte phenotypes that could not have been predicted based on either in vitro chemotaxis assays or the known cellular distribution of the receptor. this to some extent reflects the complexity of the actions and interactions between chemokines and their receptors many of which we know to be promiscuous for the ligands they bind. moreover, differences in the immunophenotype of leukocytes recruited to the cns versus peripheral tissues following organspecific transgene driven chemokine production indicates that tissue-specific influences can clearly determine how a chemokine will behave in that milieu. there are still considerable gaps in our understanding of chemokine biology and neurobiology. however, ongoing and future studies employing approaches such as transgenic modeling no doubt will begin to fill these gaps. functional expression of the lymphoid chemokines ccl19 (elc) and ccl 21 (slc) at the blood-brain barrier suggests their involvement in g-protein-dependent lymphocyte recruitment into the central nervous system during experimental autoimmune encephalomyelitis chemokines in the cns: plurifunctional mediators in diverse states distribution, cellular localization and functional role of ccr2 chemokine receptors in adult rat brain ischemia-induced neuronal expression of the microglia attracting chemokine secondary lymphoid-tissue chemokine (slc) functional expression of cxcr3 in cultured mouse and human astrocytes and microglia leukocyte infiltration, but not neurodegeneration, in the cns of transgenic mice with astrocyte production of the cxc chemokine ligand 10 transgenic modeling of neuropsychiatric disorders transgenic models to study the actions of cytokines in the central nervous system chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases the many roles of chemokines and chemokine receptors in inflammation central nervous system inflammation and neurological disease in transgenic mice expressing the cc chemokine ccl21 in oligodendrocytes ectopic expression of the murine chemokines ccl21a and ccl21b induces the formation of lymph node-like structures in pancreas, but not skin, of transgenic mice interferon-inducible t cell alpha chemoattractant (i-tac): a novel non-elr cxc chemokine with potent activity on activated t cells through selective high affinity binding to cxcr3 monocyte chemoattractant protein-1 (ccl2) in inflammatory disease and adaptive immunity: therapeutic opportunities and controversies widely expressed transcripts for chemokine receptor cxcr1 in identified glutamatergic, gamma-aminobutyric acidergic, and cholinergic neurons and astrocytes of the rat brain: a single-cell reverse transcription-multiplex polymerase chain reaction study transgenic expression of ccl2 in the central nervous system prevents experimental autoimmune encephalomyelitis molecular mechanisms involved in t cell migration across the blood-brain barrier cutting edge: ectopic expression of the chemokine tca4/slc is sufficient to trigger lymphoid neogenesis cc chemokine receptor 2 is critical for induction of experimental autoimmune encephalomyelitis gro-alpha and cxcr2 in the human fetal brain and multiple sclerosis lesions regulation of chemokine receptor expression in human microglia and astrocytes ccr7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1 gene knock-out technology: a methodological overview for the interested novice cxc chemokines interleukin-8 (il-8) and growth-related gene product alpha (groalpha) modulate purkinje neuron activity in mouse cerebellum chemokine monocyte chemoattractant protein-1 is expressed by astrocytes after mechanical injury to the brain monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation identification and characterization of a novel beta chemokine containing six conserved cysteines cytokines and the nervous system. i: expression and recognition expression of chemokine receptors by subsets of neurons in the central nervous system pertussis toxin-induced reversible encephalopathy dependent on monocyte chemoattractant protein-1 overexpression in mice chronic expression of monocyte chemoattractant protein-1 in the central nervous system causes delayed encephalopathy and impaired microglial function in mice the cc chemokine receptor-7 ligands 6ckine and macrophage inflammatory protein-3 beta are potent chemoattractants for in vitro-and in vivo-derived dendritic cells regulation of neuroinflammation: the role of cxcl10 in lymphocyte infiltration during autoimmune encephalomyelitis ifn-inducible protein 10/cxc chemokine ligand 10-independent induction of experimental autoimmune encephalomyelitis human mig chemokine: biochemical and functional characterization chemokine receptor cxcr3: an unexpected enigma chemokine receptor specific for ip10 and mig: structure, function, and expression in activated t-lymphocytes chemokines and chemokine receptors in the cns: a possible role in neuroinflammation and patterning molecular cloning of a novel human cc chemokine secondary lymphoid-tissue chemokine that is a potent chemoattractant for lymphocytes and mapped to chromosome 9p13 the chemokine cxcl10 modulates excitatory activity and intracellular calcium signaling in cultured hippocampal neurons expression of the chemokine receptors cxcr1 and cxcr2 in rat oligodendroglial cells role for cxcr2 and cxcl1 on glia in multiple sclerosis alterations in the oligodendrocyte lineage, myelin, and white matter in adult mice lacking the chemokine receptor cxcr2 experimental autoimmune encephalomyelitis develops in cc chemokine receptor 7-deficient mice with altered t-cell responses modulation of the neurotransmitter release in rat cerebellar neurons by gro beta the chemokine system in neuroinflammation: an update astrocyte expression of mrna encoding cytokines ip-10 and je/mcp-1 in experimental autoimmune encephalomyelitis secondary lymphoid tissue chemokine (ccl21) activates cxcr3 to trigger a cl-current and chemotaxis in murine microglia cxcr3-dependent microglial recruitment is essential for dendrite loss after brain lesion chemokines, mononuclear cells and the nervous system: heaven (or hell) is in the details islet-specific expression of cxcl10 causes spontaneous islet infiltration and accelerates diabetes development je/mcp-1: an early-response gene encodes a monocyte-specific cytokine recombinant human mcp-1/je induces chemotaxis, calcium flux, and the respiratory burst in human monocytes chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells t helper 1 and t helper 2 cells respond differentially to chemokines a functional ifn-gamma-inducible protein-10/cxcl10-specific receptor expressed by epithelial and endothelial cells that is neither cxcr3 nor glycosaminoglycan t cell antiviral effector function is not dependent on cxcl10 following murine coronavirus infection neutrophil infiltration, glial reaction, and neurological disease in transgenic mice expressing the chemokine n51/kc in oligodendrocytes the effects of human recombinant mip-1 alpha, mip-1 beta, and rantes on the chemotaxis and adhesion of t cell subsets human interferon-inducible protein-10 induces mononuclear cell infiltration in mice and promotes the migration of human t lymphocytes into the peripheral tissues and human peripheral blood lymphocytes-scid mice beta chemokines costimulate lymphocyte cytolysis, proliferation, and lymphokine production metalloproteinases control brain inflammation induced by pertussis toxin in mice overexpressing the chemokine ccl2 in the central nervous system the expression and function of chemokines involved in cns inflammation acute exposure to cxc chemokine ligand 10, but not its chronic astroglial production, alters synaptic plasticity in mouse hippocampal slices overexpression of monocyte chemotactic protein-1/ccl2 in beta-amyloid precursor protein transgenic mice show accelerated diffuse beta-amyloid deposition the authors studies referred to in this article were funded by usphs grants mh 62231 and ns044905. key: cord-016954-l3b6n7ej authors: young, colin r.; welsh, c. jane title: animal models of multiple sclerosis date: 2008 journal: sourcebook of models for biomedical research doi: 10.1007/978-1-59745-285-4_69 sha: doc_id: 16954 cord_uid: l3b6n7ej to determine whether an immunological or pharmaceutical product has potential for therapy in treating multiple sclerosis (ms), detailed animal models are required. to date many animal models for human ms have been described in mice, rats, rabbits, guinea pigs, marmosets, and rhesus monkeys. the most comprehensive studies have involved murine experimental allergic (or autoimmune) encephalomyelitis (eae), semliki forest virus (sfv), mouse hepatitis virus (mhv), and theiler’s murine encephalomyelitis virus (tmev). here, we describe in detail multispecies animal models of human ms, namely eae, sfv, mhv, and tmev, in addition to chemically induced demyelination. the validity and applicability of each of these models are critically evaluated. multiple sclerosis (ms) affects about 350,000 people in the united states and is a major cause of nervous system disability in adults between the ages of 15 and 45 years. the symptoms are diverse, ranging from tremor, nystagmus, paralysis, and disturbances in speech and vision. extensive demyelination is seen in the neuronal lesions. the clinical heterogeneity of ms, as well as the finding of different pathological patterns, suggests that ms may be a spectrum of diseases that may represent different pathological processes. 1 this has led to the development of many different animal models, including rodents and nonhuman primates, that reflect the pathological processes and could allow for the development of therapeutic approaches. at the present time, the exact etiological mechanism in humans is not clear; however, several animal models are available providing insight into disease processes. the relative inaccessibility and sensitivity of the central nervous system (cns) in humans preclude studies on disease pathogenesis, and so much of our understanding of infections and immune responses has been derived from experimental animal models. the experimental systems include theiler's virus, mouse hepatitis virus, and semliki forest virus infections of laboratory rodents. additional information has been obtained from studies of experimental infections of other animals that result in demyelination, notably maedi-visna virus in sheep and canine distemper virus in dogs. in humans and animals, most natural cases of demyelinating disease are rare complications of viral infections. one possible reason for the low incidence of demyelination following viral infections could be the low efficiency of neuroinvasion. however, a correlation between cns infection and clinical disease is difficult to determine. the role of genetics and environmental factors in ms is complex. factors such as geographical location, ethnic background, and clustering in temperate climates all contribute to susceptibility. individuals with a north european heritage are statistically more susceptible to ms than those from a more tropical environment and it is more common in women. 2 epidemiological data indicate that ms is not a single-gene disorder and that additionally environmental factors contribute to the disease. 3 data from genetic studies indicate that although mhc genes clearly contribute to disease susceptibility and/or resistance, it is probable that a combination of environmental factors may additionally contribute to disease development in genetically predisposed individuals. to understand the initiating factors and progression of ms, researchers have turned to experimental model systems. since this disease cannot be recreated in a tissue culture system, much effort has been directed to the use of laboratory animals. those animal models should mirror the clinical and pathological fi ndings observed in human ms. ideally, the animal model should be in a species that is easy to handle, inexpensive, can be kept in large numbers, and is easily bred in laboratory conditions. the most frequently used animals are laboratory rodents, including mice, rats, guinea pigs, and hamsters. one of the most useful aspects of laboratory rodents as animal models of disease is the vast array of inbred strains of the species available, most notably in experimental mice. additionally, very valuable information has been obtained from studies using larger animals including sheep, dogs, cats, and nonhuman primates. models of ms fall into two main groups: viral and nonviral. viral models are immensely relevant since epidemiological studies suggest an environmental factor, and almost all naturally occurring cns demyelinating diseases of humans and animals of known etiology are caused by a virus. these include in humans, subacute sclerosing panencephalitis (sspe)-caused by measles or rubella viruses, progressive multifocal leukoencephalopathy (pml)-caused by jc virus, and human t lymphotrophic virus-1 (htlv-1)-associated myelopathy (ham)-caused by htlv-1; in animals, these include visna virus in sheep and canine distemper in dogs. however, no one virus has consistently been associated with human ms, although it is likely that more than one virus could trigger the disease. of the nonviral models of ms, experimental allergic encephalomyelitis (eae) is the most widely studied. eae is characterized by inflammatory infiltrates in the cns that can be associated with demyelinating lesions. in eae, the disease is initiated by the extraneural injection of cns material, or purified myelin components, emulsified in an adjuvant, the most commonly employed one being complete freund's adjuvant containing mycobacterium tuberculosis h37ra. however, no naturally occurring autoimmune correlate of this experimental disease is known, although it is extensively researched as a model of ms, with the reasoning that ms may be such a disease. the most widely studied models of ms are the experimental infections of rodents resulting in an inflammatory demyelinating disease in the cns, such as theiler's virus, mouse hepatitis virus, and semliki forest virus. 4 each of these infections gives rise to lesions of mononuclear cell inflammatory demyelination throughout the brain and spinal cord but not in the peripheral nervous system. as such, this histopathology correlates with human ms, although it does not preclude the fact that the viruses could gain access to the cns via the peripheral nervous system. these viral models demonstrate how a virus can easily reproduce cns disease, which is comparatively rare in humans, and how this can be influenced by many factors including both genetic and immunological. experimental studies in induced animal models have the advantage over studies in spontaneous models in that the onset and progression of the disease can be controlled. although it has been proposed that some autoimmune diseases may have a viral etiology, virus-induced autoimmunity is a controversial subject. epidemiological studies of ms provide strong evidence for the involvement of a viral etiology in the onset of disease. theiler's virus-induced demyelination, a model for human ms, bears several similarities to the human disease: an immune-mediated demyelination, involvement of cd4 + helper t cells and cd8 + cytotoxic t cells, delayed type hypersensitivity responses to viral antigens and autoantigens, and pathology. indeed this mouse model may provide a scenario that closely resembles chronic progressive ms. theiler's murine encephalomyelitis virus (tmev) is a picornavirus that causes an asymptomatic gastrointestinal infection, followed by occasional paralysis. there are two main strains of tmev, the virulent strains and persistent theiler's original (to) strains. the virulent gdvii strains of theiler's virus are highly neurovirulent and when injected intracranially, cause death by encephalitis within 48 h. gdvii strains also cause differing forms of paralysis depending on the route of inoculation (see table 69 -1). from these studies it appears that the gdvii virus may gain access to the cns by retrograde axonal transport rather than by a hematogeneous route. 5 infection of susceptible strains of mice with the persistent to strains bean, da, ww, or yale results in a primary demyelinating disease that closely resembles human ms. 6 infection of resistant strains of mice with bean does not result in demyelinating disease, since these mice are able to clear virus from the cns. susceptible mice fail to clear virus from the cns, possibly resulting from poor natural killer (nk) cell and cytotoxic t lymphocyte (ctl) responses. persistent viral infection of the cns is required for demyelination. following the intracranial injection of susceptible mice with bean, virus replicates both in the brain and spinal cord. 7 one month postinfection, viral titers decrease, and high levels of neutralizing antibodies are detected (figure 69-1) . at this point in the disease, neurons may become infected with virus and mice may develop a nonprogressive flaccid paralysis of the forelimbs and/or hindlimbs. 8 this is sometimes referred to as a polio-like disease, but this is confusing since flaccid paralysis in mice infected with poliovirus is progressive and normally results in death. in the late phase of the disease, astrocytes, oligodendrocytes, and macrophage/microglial cells become infected with virus. also in the demyelinating disease there is both b and t cell autoimmunity, directed against myelin and its antigenic components. genetics of persistent infection and demye-linating disease all inbred mouse strains inoculated intracerebrally with tmev show early encephalomyelitis, but not all strains remain persistently infected. resistant strains normally 9 this trait is under multigenic control, with h-2 mhc class i genes being the most prominent. additionally, several non-h-2 quantitative trait loci (qtl) have been identified within the same h-2 haplotypes that control persistence. there is generally a good correlation in inbred strains between susceptibility to three phenotypes (viral load, pathology, and symptoms), suggesting that variations in both demyelination and clinical disease may result from how each mouse strain can control the viral load during the persistent infection. 10 using b10 congeneic and recombinant strains of mice, susceptibility to disease has been mapped to the h-2d region. 11 furthermore, resistant haplotypes are dominant and the same locus controls viral load during persistence and demyelination. currently, 11 non-h-2 susceptibility loci have been identified as having an effect on susceptibility to theiler's virus-induced disease (tvid) (see table 69 -2). the mechanism(s) of tvid may be different for different mouse strains, but most of the information has come from studies of sjl/j mice infected with the da or bean strain of virus. the virus infects oligodendrocytes, and the resulting demyelinating disease could be due, in part, to the virus killing oligodendrocytes directly or by the virus-specific cd8 + ctls present in the lesions. 7 a series of experiments has demonstrated that demyelination correlates with the presence of a cd4 + t cell-mediated response against viral epitopes. these cells secrete cytokines such as interferon (ifn)-γ that activate both microglial cells and invading monocytes, which subsequently secrete factors such as tumor necrosis factor (tnf)-α and thus can cause "bystander" demyelination. activated macrophages ingest and degrade damaged myelin. autoantibodies 12,13 and myelin-specific cd4 + t cells have been shown in sjl/j mice several months after intracranial inoculation. 14, 15 epitope spreading in these mice commences with recognition of a proteolipid protein (plp) epitope, and then progresses to additional plp epitopes and then to myeloid basic protein (mbp) epitopes. 14 a direct demonstration that disease can be maintained on a purely autoimmune footing, after infection has been eradicated, has not been shown. immunity and theiler's virus the first response to viral infection is the production of type i interferons, which are critical for viral clearance. ifn-α/β receptor knockout mice injected with tmev die of encephalomyelitis within 10 days of infection. nk cells are activated early in infection with certain viruses. in tmev infection susceptible sjl mice have a 50% lower nk cell activity in comparison to the highly resistant c57bl/6 mice. this low nk activity in sjl mice is in part due to a defect in the thymus impairing the responsiveness of nk cells to stimulation by ifn-β. the pivotal role of nk cells in early tmev clearance is demonstrated by the finding that resistant mice depleted of nk cells by monoclonal antibodies to nk 1.1 develop severe signs of gray matter disease. 16 in the early disease, both cd4 + and cd8 + t cells have been shown to be important in viral clearance. in early disease cd4 + t cells are required for b cells to produce antibodies for viral clearance. 12 these cd4 + t cells secrete ifn-γ, which in vitro inhibits tmev replication and has a protective role in vivo. cd8 + t cells are also important in viral clearance, as demonstrated by the finding that cd8 + t cell-depleted mice fail to clear virus and develop a more severe demyelinating disease. 17 cd8 + t cells also provide protection against tvid when adoptively transferred to a tvid-susceptible strain, balb/c.anncr. thus, cd8 + t cells are implicated in viral clearance and resistance to demyelination. higher ctl activity has been demonstrated in tvid-resistant c57bl/6 mice as compared to resistant sjl/j mice. 18 these ctls may play an important role in viral disease since they may recognize viral determinants and/or they may inhibit delayed type hypersensitivity responses. the relative roles of th1/th2 cells in tvid are very complex, and a simple picture of a th1 or th2 polarization during infection may not be apparent. a pathogenic role for th1 cells during late demyelinating disease is demonstrated by the finding that both tvid correlates with delayed type hypersensitivity responses to tmev and that the depletion of cd4 + t cells during late disease results in the amelioration of clinical signs. high levels of the proinfl ammatory th1 cytokines ifn-γ and tnf-α in late disease correlate well with maximal disease activity. evidence demonstrating the protective role of th2 in tvid has been shown in experiments in which skewing the immune response toward th2 immunity in tmev infection diminishes the later demyelinating disease. 19 however, other studies have shown that the th1/th2 balance did not explain the difference in susceptibility to tvid. th1 cytokines are generally pathogenic during late demyelinating disease, whereas th2 cytokines are protective. 20 and is characterized by abnormally thin myelin sheaths in relation to axon diameter. to date, however, there are few reliable data on the frequency of remyelination in ms patients. stimulation of remyelination is a potential treatment for ms. the tmev model of ms can be used to study remyelination using remyelinationpromoting antibodies. in this remyelination model, sjl/j mice, aged 4-8 weeks, are injected with a 10 µl volume containing 200,000 pfu of daniel strain intracerebrally. all animals develop mild encephalitis, which resolves within 14 days after the injection. the infected mice then develop the chronic demyelinating disease that gradually progresses over several months. to study remyelination, mice that had been infected with tmev for 6 months receive a single intraperitoneal injection of 0.5 mg (∼0.025 g/kg body weight) of a recombinant remyelinating antibody (rhigm22) in phosphate buffered saline (pbs). 21 in one study, 82.8% of lesions in animals treated with rhigm22 showed retraction of varying degrees, presumably the effect of remyelination in these lesions. the direct binding of rhigm22 to demyelinated lesions is consistent with the hypothesis that these antibodies work directly in the lesions, probably by binding to the cns glia to induce remyelination. thus, this murine tmev model can also be used as a model with which to examine different modes of remyelination. mouse hepatitis virus (mhv) is a member of the coronaviridae, a group of large positive sense enveloped rna viruses. depending on the strain of virus used, mhv causes a variety of diseases including enteritis, hepatitis, and demyelinating encephalomyelitis. 22 infection of mice with the neutrotropic jhm strain of mhv causes encephalitis, followed by chronic demyelination. virus is not cleared from the cns, resulting in a persistent infection. after intracerebral or intranasal infection with mhv, virus enters the brain and causes encephalitis. 23 intranasal infection with mhv-jhm or -a59 leads to viral spread through the olfactory bulb and along the olfactory tracts, as well as along the trigeminal nerve to the mesencephalic nucleus. 23 up to 4 days postinfection (pi) early viral spread is via specific neural pathways and neural connections. viral titers peak at about day 5 pi in the brain and later in the spinal cord and virus is cleared by days 8-20 pi. 24 however, viral antigen is still detectable up to day 30 pi. additionally, viral rna is detectable in the brain as late at 10-12 months postinfection, although the amount of rna decreases with time. liver infection can occur after any route of infection (in, ic, ig, or ip), with viral titers peaking at day 5 pi and hepatitis developing during the first 1-2 weeks. 25 cns demyelination develops as active mhv infection resolves. the lesions observed are histologically very similar to those observed in ms patients. these mhv lesions are characterized by primary demyelination accompanied by naked axons, 23 and are found scattered throughout the spinal cord. 26 the peripheral nervous system is not affected. chronic lesions are associated with lipid-laden macrophages, scattered lymphocytes, and perivascular cuffing. these chronic lesions can persist as late as day 90 pi, and demyelinating axons can be seen as late as 16 months postinfection. chronic diseases in mhv-infected mice are associated with ataxia, hindlimb paresis, and paralysis, followed by a recovery. this animal recovery is mediated by cns remyelination, beginning anywhere from 14 to 70 days pi. c57bl/6 mice (h-2 b ) are susceptible to mhv infection. in this murine model adult mice (of weight 20-22 g) are anesthetized by inhalant anesthesia and receive an intracerebral injection of approximately 500 pfu of a neurotropic mhv strain in a volume of approximately 20 µl of pbs. this intracerebral injection of mhv results in a biphasic disease: an acute encephalomyelitis with myelin loss, followed 10-12 days later by an immunemediated demyelinating encephalomyelitis with progressive destruction of the cns. 27 there is an 80-90% survival rate of mice injected with this mhv, with animals usually succumbing during the first 2 weeks of acute infection. animals surviving this acute stage show a 95% chance of survival. 28 control animals injected intracerebrally with sterile pbs show no clinical signs or histological defects. electron micrographs of demyelinating lesions show that macrophage processes slip between layers in the myelin sheath, implying that macrophages could indeed be mediating demyelination. 29 the appearance of macrophages within the cns also correlates with the development of lesions. additionally, they do not appear in large numbers in the absence of lymphocytes, so it is possible that myelin damage is caused by a nonmacrophagedependent mechanism and that macrophages may only clear up the damaged myelin. in contrast to other mouse models of demyelination, there does not appear to be a clear role for any single lymphocytic or monocytic subset mediating the demyelination. rather, it appears that a balance of immune components may be necessary for viral clearance and that various pathways, both immune and nonimmune, may cause the ensuing demyelinating events. recently, progress has been made in further identifying the immune cells required for demyelination. experimental infection of severe combined immunodeficiency (scid) mice, lacking t cells, results in fulminate encephalitis without demyelination. 30 adoptive transfer of splenocytes from syngeneic immunocompetent mice into infected scid mice results in demyelination within 7-9 days posttransfer. additional experiments indicated that either cd4 + or cd8 + t cell subsets are capable of initiating this process. however, mice that receive splenocytes depleted of cd4 + t cells survive longer and develop more demyelination than mice receiving splenocytes depleted of cd8 + t cells. thus, experimental scid mice demonstrate that the roles of each t cell subset in demyelinating diseases are not equal. 31 ifn-γ is a critical mediator of homeostasis and infl ammation in ms and many of its rodent models. bone marrow chimera mice have been used to address the role of ifn-γ in bystander demyelination mediated by cd8 + t cells. these chimeras as rodent models for jhm have addressed a hypothesis that ifn-γ produced by cd8 + t cells, and not from other sources, was the critical component in mediating bystander demyelination. this chimeric approach did not compromise ifn-γ production by cells such as nk cells and dendritic cells, thus preserving the innate immune response to the virus. 32 the results demonstrated that ifn-γ produced by these innate cells was unable to initiate the demyelinating disease, even in the context of activated cd8 + t cells lacking only the ability of produce ifn-γ. these findings highlight the role that cd8 + t cells have in demyelination in jmh-infected mice. 33 it has been demonstrated that ifn-γ is critical in other animal models of demyelination and in ms. semliki forest virus (sfv) is an alphavirus of the togaviridae. the virus has been isolated from mosquitoes, but the natural host is unknown. sfv is a single-stranded positive strand rna virus that has been cloned and sequenced. the most commonly studied strains used in adult mice are the virulent l10 strain and the avirulent a7(74) strain. both of these strains are avirulent in neonatal and suckling mice by all routes of infection. experimental infection of mice with sfv is widely used as a model to study the mechanism of virus-induced cns disease. sfv has the advantage of being neuroinvasive as well as neurotropic, thus allowing studies of viral entry into the cns and the integrity of the bloodbrain barrier (bbb). following intraperitoneal injection with 5000 pfu sfv in 0.1 ml pbs containing 0.75% bovine serum albumin, 34 all strains replicate in muscles and other tissues, resulting in a plasma viremia. virus then crosses the cerebral vascular endothelial cells, resulting in infection of neurons and oligodendrocytes. 35 in neonatal or adult mice, infection with virulent strains results in widespread infection that is lethal within a few days. in contrast, infection of mice with the a7(74) strain results in a cns infection, and infectious virus is cleared from the brain by day 10. infiltrating mononuclear cells are observed 3 days pi and peak at about day 7. focal lesions of demyelination throughout the cns are observed 10 days pi and peak between 14 and 21 days pi. 36 sfv-induced demyelinating diseases have been widely studied following intraperitoneal injection of adult mice with the a7(74) strain of the virus. following intraperitoneal injection, virus is detected in the brain by 24 h. viral titers then rise, but rapidly decline following initiation of the immune response. interestingly, although infectious virus can be detected only up to day 8 pi, realtime polymerase chain reaction (rt-pcr) studies detect viral rna up to day 90 pi. 37 thus, it is possible that there is persistence of viral antigen(s). disturbance of the bbb occurs between 4 and 10 days pi, which corresponds to the increase in inflammatory cell infi ltration and reduction in viral titer and which may be related to the influx of cells or cytokine-mediated effects. the presence of macrophages, activated microglia, and the proinfl ammatory cytokines tnf-α, ifn-γ, interleukin (il)-1α, il-2, il-6, and granulocyte-macrophage colony-stimulating factor (gm-csf) during sfv-induced demyelination, in addition to enhancing the infl ammatory response, may also play a role in controlling viral infection since il-6, ifn-γ, and tnf have direct antiviral activity. 38 additionally, ifn-γ and tnf production peripherally coincides with sfv-induced encephalitis in sjl and b6 mice. interestingly, these same cytokines predominate in ms lesions. 39 an intense inflammatory response characterized by perivascular cuffi ng is apparent histologically from 3 days. demyelination, as demonstrated using luxol fast blue staining of sections, is apparent by 14 days. however, small focal lesions of demyelination can be observed using electron microscopy by day 10. a striking feature of sfv infection appears in the optic nerve, where there are demyelinating lesions and changes in visually evoked responses and axonal transport. 40 this optic neuritis also occurs in human ms. it appears that sfv-induced demyelination in this mouse model is accompanied by neurophysiologically demonstrable visual deficits very similar to those found in ms patients. thus, this may provide a very useful animal model for research into ms. the advantages of this model are that genetic and environmental factors can be readily controlled, while the low cost and fast reproductive rate make experimental design considerably easier. no demyelination is observed following sfv infection of scid mice or athymic mice. in the absence of specific immune responses, scid mice infected with sfv a7(74) have a persistent viremia, a persistent and restricted cns infection, and no lesions of demyelination. comparison of the infection to that in nu/nu and balb/c mice and studies on the transfer of immune sera show that immunoglobulin m (igm) antibodies clear the viremia but not the brain virus and that infections of brain virus can be reduced by igg antibodies. these igg antibodies can abolish infectivity titers in the brain but cannot remove all viral rna. 41 adoptive cell transfer studies and administration of anti-cd8 antibodies demonstrate that demyelination following sfv infection is dependent on cd8 + t cells. 4 this is consistent with the finding that the cns inflammatory infiltrate is dominated by cd8 + t cells. this fi nding is analogous to that in ms and is in contrast to that in eae, where cd4 + cells predominate. 42 in the eae autoimmune model of ms, studies suggest that a th1 cytokine profile predominates. another point of difference between the eae model and the sfv model is shown in the th1/th2 profiles. following infection with sfv, th1 and th2 cytokines were detected in the cns and both were present throughout the time course studied, indicating that there was no bias of th response in the cns, nor were changes apparent with time. 43 the experimental disease eae has been investigated in many strains of animals including mice, rats, guinea pigs, rabbits, marmosets, and rhesus monkeys. eae is an autoimmune infl ammatory disease of the cns and is characterized by perivascular and subpial inflammatory infiltrates and demyelinating lesions. the disease is usually initiated by injection of autoantigens emulsifi ed in an adjuvant. the progression and pathology of lesions observed depend on the type of antigen used in the injection, the method of injection, and the strain of animal used. because of its very nature, eae as a model of ms does not address certain pertinent questions relating to ms, such as age-related onset of disease or epidemiology. a major difference between eae and viral models of ms is that in eae the inflammatory response is directed to autoantigens. a feature of the eae model is that the course of the disease can be relapsing and remitting. studies of eae have been used to identify antigenic determinants on components of myelin. using bioinformatic technology these determinants have been used to search available databases of viral and bacterial proteins. results indicate numerous viral and bacterial protein segments with probable sequence similarity to myelin basic protein determinants. 44 experimental allergic encephalomyelitis in rabbits eae has been induced in rabbits by footpad inoculation with rabbit spinal cord homogenate, resulting in hindlimb paresis or paralysis. 45 rabbits with 5-day paraplegia showed increased spinal cord incorporation of radioactive drugs administered in the epidural space. thus, this demyelinating disease process may expose the spinal cord to larger amounts of sub-stances administered neuraxially. it is therefore possible that this rabbit model could be used to investigate the incorporation of radioactive therapeutic drugs in the epidural space. experimental allergic encephalomyelitis in guinea pigs guinea pigs have also been investigated to determine whether they may serve as useful eae models of ms. the interest in guinea pigs stems from the fact that group 1 cd1 glycoprotein homologues, which in humans present foreign and self lipid and glycolipid antigens to t cells, are not found in mice and rats but are present in guinea pigs. in this guinea pig model, animals have been sensitized for eae, and cd1 and mhc class ii expression has been measured in the cns. in normal guinea pigs low level mhc class ii occurred on meningeal macrophages and microglial cells, whereas immunoreactivity for cd1 was absent. in the eae cns, however, the majority of infiltrating cells were mhc ii + and microglia showed increased expression, whereas cd1 immunoreactivity was detected on astrocytes, b cells, and macrophages. minimal cd1 and mhc ii coexpression was detected on inflammatory cells or glia. thus, in this guinea pig eae model group 1 cd1 molecules are upregulated in the cns on subsets of cells distinct from the majority of mhc iibearing cells. 46 this expression of cd1 proteins in such eae lesions broadens the potential repertoire of antigens recognized at these sites and highlights the value of this guinea pig model of human ms. experimental allergic encephalomyelitis in rats rats were injected with spinal cord homogenate or the encephalitogen; myelin basic protein induced eae in genetically susceptible dark agouti (da) rats but not in albino oxford (ao) rats. here 8-to 12-week-old rats were immunized in either or both hind footpads with 0.1 ml antigenic emulsion containing 100 µg rat spinal cord tissue in complete freund's adjuvant (cfa). 47 rats are monitored from day 5 after inoculation and the severity of disease was assessed by grading tail, hindlimb, and forelimb weakness, each on a sale of 0 (no disease), 1 (loss of tail tonicity), 2 (hindlimb weakness), 3 (hindlimb paralysis), to 4 (moribund or dead). clinical disease in susceptible strains of rats is apparent in all animals, and the onset of disease occurs at day 11 postinjection. at the peak of the clinical manifestation of eae there is a marked increase in the level of infiltration of cells accompanied by a lack of activation in susceptible da rats, whereas it remains elevated in resistant ao rats. at the peak of clinical disease da rat spinal cords contain high levels of cd4 + t cells. da rats also contained 10 times as many live cd4 + t cells as ao rats. astrocytosis, as an indication of cns reaction to the presence of infl ammatory cells, was clearly observed in both rat species. microglial activation persists in resistant ao rats, whereas activation is downregulated in da rats. in this model it is speculated that at the peak of disease, infiltrating monocytes and macrophages are the main antigen-presenting and effector cells. rat eae may also be induced by the injection of xenogeneic myelin. for example, 8-to 12-week-old lewis rats injected in both hind footpads with an emulsion containing 100 µg of guinea pig myelin basic protein and cfa develop acute eae. also chronic relapsing eae (cr-eae) may also be induced in this rat model using a regimen of intraperitoneal injections of 4 mg/kg of cyclosporin a. 48 pathology studies indicate that in acute and cr-eae, mcp-1 and its receptor ccr2 are significantly upregulated throughout the course of cr-eae and that a large number of macrophages infiltrated the cr-eae lesion. this suggests that macrophages recruited by mcp-1 and ccr2-expressing cns cells are responsible for the development and relapse of eae. thus, in this rat model, in addition to t cells, macrophages are another target for immunotherapy studies for neurological autoimmune diseases. a more recent development of a rat eae model involves using human mbp as antigen. here eae was induced by the immunization of female wistar rats with human mbp. it was found that most of the rats developed tail tone loss and hindlimb paralysis together with demyelination, infiltrative lymphocyte foci, and "neurophagia" in the cortex of cerebra and in the white matter of the spinal cord. 49 this study further demonstrated that this rat model of eae induced by human mbp resembles many features of human ms and may promise to be a better animal model for the study of ms. 49 the use of cfa is not a prerequisite for the development of rat eae. for example, eae can be induced in 10-to 16-week-old da rats by a single hind footpad injection of an encephalitogenic emulsion consisting of rat or guinea pig spinal cord homogenate (sch) in pbs. 50 the reason for not wanting to use cfa is that in itself it induces a strong inflammatory response and exerts numerous immunomodulatory properties. additionally, cfa induces a strong anti-purified protein derivative (ppd) response and may induce adjuvant arthritis, another autoimmune disease. the susceptibility of da rats to eae induction with sch depends upon the origin of the cns tissue, the homologous tissue being the more efficient encephalitogen. da rats that recovered from eae that had been induced with homologous sch without adjuvant and then immunized with the encephalitogenic emulsion containing cfa developed clinical signs of the disease. neurological signs in rechallenged rats were milder, but first signs appeared earlier. the earlier onset of eae observed in da rats after rechallenge has been attributed to the reactivation of memory cells. taken together, these experiments demonstrate that eae can be effi ciently and reproducibly induced in da rats without the use of cfa. this experimental model for understanding the basic mechanisms involved in autoimmunity within the cns, without the limitations and inherent problems imposed by the application of adjuvants, may represent one of the most reliable rodent models of ms. the rat as an experimental model could be used to evaluate new immunotherapies of eae. these include antigen-induced mucosal tolerance, treatment with cytokines, and dendritic cellbased immunotherapy. the ideal treatment of diseases with an autoimmune background such as ms should specifically eliminate autoreactive t cells without affecting the integrity of the immune system. one way to achieve this would be to induce immunological tolerance to autoantigens by the oral or nasal administration of autoantigen. several studies have shown that nasal administration of soluble antigens results in peripheral tolerance by immune deviation or the induction of other regulatory mechanisms. in the rat model this tolerance has been investigated using synthetic peptides of mbp, mbp68-86, 87-99, and 110-128. nasal administration of the encephalitogenic mbp68-86 or 87-99 suppresses eae. mbp68-86 and 87-99 given together had synergistic effects in suppressing eae and reversed ongoing eae. a problem, however, of antigen-specific therapy by the nasal route is that one antigen, or peptide, may be effective in inducing tolerance in one strain of animal but not in another. one way of treating ongoing eae may be the use of an altered peptide ligand with high tolerogenic efficacy when administered nasally. 51 cytokines have been widely used in disease prevention and treatment. cytokine immunotherapy in ms could employ one or two basic strategies: first, to administer immune response downregulatory cytokines, or second, to administer inhibitors of proinfl ammatory cytokines. the nasal route of administering these cytokines has been studied in the rat eae model. nasal administration of low doses of antiinflammatory or regulatory cytokines such as il-4, il-10, or tumor growth factor (tgf)-β1 inhibits development of rat eae when given before or on the day of immunization, but by differing mechanisms. nasally administered il-10 reduced both peripheral immune responses and microglia activation in the cns, whereas nasal administration of il-4 or tgf-β1 triggered the activation of dendritic cells (dcs). however, nasal administration of cytokines alone fails to treat ongoing lewis rat eae. interestingly, nasal administration of mbp68-86 + il-4 or mbp68-86 + il-10 suppresses ongoing eae in lewis rats. the suppression of eae by mbp68-86 + il-10 is associated with the induction of a broad lymphocyte hyporesponsiveness. although this combined administration of autoantigen plus cytokine may be effective in treating rat eae, the applicability of this to human ms is severely limited by the lack of knowledge of the pathologically relevant autoantigen(s) in ms. dcs not only activate lymphocytes, but also induce t cell tolerance to antigens. 52 use of tolerogenic dcs is thus a possible immunotherapeutic strategy for treatment of eae, and indeed this has been studied in some detail. however, mbp68-86-pulsed dcs only prevented the development of eae and failed to treat ongoing eae in lewis rats. 53 in an attempt to treat ongoing eae, splenic dcs have been isolated from healthy lewis rats and modifi ed in vitro with cytokines ifn-β, il-2, il-10, or tgf-β1. upon subcutaneous injection into lewis rats on day 5 pi with mbp68-86 + fca, ifn-β or tgf-β1-modifi ed dcs promoted immune protection from eae. the common marmoset callithrix jacchus is an outbred species characterized by a naturally occurring bone marrow chimerism. the marmoset is a primate phylogenetically close to humans, and has been studied as an animal model for ms. 54 eae can be induced in the common marmoset by the injection of human brain white matter, dispersed in demineralized water to a concentration of 30 mg/ml and emulsified with cfa containing 0.5 mg/ml of mycobacterium butyricum h37a. monkeys are injected intracutaneously with 600 µl of emulsion into the dorsal skin at several locations. clinical disease in this model is scored daily on a scale from 0 to 5: 0 = no clinical signs; 0.5 = apathy, loss of appetite, and an altered walking pattern without ataxia; 1 = lethargy and/or anorexia; 2 = ataxia; 2.5 = paraparesis or monoparesis and/or sensory loss and/or brainstem syndrome; 3 = paraplegia or hemiplegia; 4 = quadriplegia; and 5 = spontaneous death attributable to eae. 55 here the onset of disease, as measured by clinical scores, is variable among animals between 7 and 13 weeks postinoculation. additionally, the maximal clinical scores are variable among animals and range between 2 and 4. on histopathological examination, large plaques of demyelination are observed in the white matter of cerebral hemispheres, mainly localized around the wall of lateral ventricles, in the hemispheric white matter, corpus callosum, optic nerves, and optic tracts. the demyelinated areas show a moderate or severe degree of inflammation characterized by perivascular cuffs of mononuclear cells. in the spinal cord, widespread demyelination is also observed. areas of demyelination involve the ventral, lateral, and dorsal columns of the spinal cord, especially in the outer part of the spinal tracts. thus, pathology in the marmoset model is characterized by infl ammation, demyelination, and astrogliosis. interestingly, this model demonstrates the presence of axonal damage in demyelinating plaques. indeed, axonal damage and loss are well-known events in ms. in ms, axonal damage appears to be an early event, related to an acute inflammation. in the marmoset eae, axonal damage also occurs in areas of acute and early inflammation and demyelination. this eae in c. jacchus is of special interest because of the resemblance of this model to the human disease, and the similarity between the immune systems of marmosets and humans. the type of clinical signs of eae in marmosets depends largely on the antigens used for disease induction. sensitization of marmosets to human myelin induces a relapsing-remitting, secondary-progressive disease course. 56 lesions in this model represent all stages present in chronic ms. marmosets inoculated with mbp develop only mild inflammatory disease unless bordetella pertussis is used with the encephalitogen. cns demyelination critically depends on the presence of antibodies to myelin oligodendrocyte glycoprotein (mog), a minor cns component. marmosets sensitized to a chimeric protein of mbp and proteolipid protein (of myelin) develop clinical eae only after the autoimmune reaction has spread to mog. marmosets immunized with recombinant human mog 1-125 do not develop relapsing-remitting disease but only chronic-progressive disease. 57 during the asymptomatic phase of this primary progressive-like disease, which can last from 2 to 20 weeks, brain lesions are detectable using magnetic resonance imaging (mri), but are not expressed clinically. the induction of eae with mbp or white matter tissue homogenate (wmh) has been well established in rhesus monkeys (macaca mulatta). the rhesus monkey was the first animal species in which eae was deliberately induced. 58 that autoimmunity to brain antigens could induce paralytic disease was confirmed by studies in rhesus monkeys given repeated inoculations of brain homogenates. 58 mog-induced eae has also been produced in this nonhuman primate species 59 that is a highly relevant model for the human disease. the close similarity of the human and rhesus monkey immune system is illustrated by the high degree of similarity between the polymorphic mhc and t cell receptor genes between these two primates. to produce this mog-induced eae, monkeys are injected, under anesthesia, with a total of 1 ml of 1:1 emulsion composed of 320 µg mog in pbs and cfa at 10 sites into the dorsal skin. overt clinical signs are scored daily according to the following criteria: (0) no clinical signs; (0.5) loss of appetite, apathy, and altered walking; (1) lethargy, anorexia, substantial reduction of the general condition, and loss of tail tonus; (2) ataxia, tail biting, sensory loss, and/or blindness; (2.5) incomplete paralysis of one (hemiparesis) or two sides (paraparesis); (3) complete paralysis of one (hemiplegia) or two sides (paraplegia); (4) complete paralysis (quadriplegia); and (5) death. the onset of clinical disease varies between animals, and occurs at days 15-23 after encephalitogenic challenge. all monkeys, however, develop clinical disease and all achieved a score of 4 on clinical severity. the current available panel of nonhuman primate eae models may reflect the spectrum of inflammatory demyelinating diseases in the human population. these eae models can therefore be used to investigate pathogenic mechanisms and to develop more effective therapies. the most widely studied animal model of eae is that of the mouse. in common with other animal models of eae, disease induction varies depending on both the sex of the animals, the mouse strain used, as well as the origin of the spinal cord encephalitogen. in this model mice, aged 6-8 weeks, are immunized subcutaneously in four sites over the back with 200-400 µg of guinea pig mbp emulsified in equal volumes of cfa containing 200-400 µg heat-killed mycobacterium tuberculosis. 60 mice also receive 200 µg of pertussis toxin in 0.2 ml pbs intraperitoneally at the time of immunization and 48 h later. mice are then scored daily for clinical signs of eae for at least 35 days as follows: 0, no clinical signs; +1, limp tail or waddling gait with tail tonicity; +2, ataxia or waddling gait with tail limpness; +3, partial hindlimb paralysis; +4, total hindlimb paralysis; and 5, moribund/death. for each strain of mice there is variation in day of onset of disease, varying from day 7 to day 22 postinfection; incidence of disease, varying from 30% to 100% of animals; incidence of mortality, varying from 0% to 40% of animals; and mean clinical scores, varying from 0 to 1.6. many mouse strains have been employed in the study of eae, and while the sjl strain has been most frequently used to model gender differences in both disease onset and severity, the sjl model has some limitations due to its diminished cd4 + t cell repertoire. certain susceptible strains of mice, such as fvb mice, show a relapsing-remitting course of disease that bears some resemblance to ms. fvb mice therefore may serve as a mouse strain into which various transgenes may be introduced for the purpose of studying their influence on eae and for exploring new therapeutic approaches. 61 since eae is a well-studied disease in mice, mimicking many clinical and pathological features of ms, including cns infl ammation and demyelination, it is of significance that it can also be used as an appropriate model to study ms-related pain. it has been clearly demonstrated in sjl that in both "active" and "passive" eae, there is an initial increase in tail withdrawal latency (hypoalgesia) that peaked several days prior to the peak in motor defi cits during the acute disease phase. during the chronic disease phase, tail withdrawal latencies decreased and were significantly faster than control latencies for up to 38 days postimmunization. thus, it is possible to use both murine active and passive eae as models for ms-related pain. 62 while specific immunotherapeutic strategies are effective in experimental model systems, translation to the human disease has genetically been poorly tolerated or has proved to be ineffective. this conflict may in part be due to the model systems used as well as the poor correlation of in vitro findings compared to those observed in vivo. in biozzi abh mice, which express the novel mhc class ii a, eae occurs following immunization with myelin proteins and peptide epitopes of these proteins; however, only plp peptide 56-70, mog peptide 8-21, or spinal cord homogenate reproducibly induces chronic relapsing eae (creae) with infl ammation and demyelination. 63 creae provides a wellcharacterized reproducible system to develop therapeutic strate-gies during established relapsing autoimmune neurological disease and is pertinent to ms. in creae in abh mice, relapse and progression of disease are associated with emergence and broadening of the immune repertoire due to release of myelin antigens following myelin damage. 64 thus, this creae model in biozzi abh mice is very well suited as a model with which to examine the effect of therapeutic strategies in a dynamic system. disease susceptibility in human ms is associated with three mhc class ii alleles in the hla-dr2 haplotype, drb1*1501, drb5*0101, and dqb1*0602. 65 an autoimmune pathogenesis has been hypothesized in which one or more of these mhc class ii molecules presents cns-derived self-antigens to autoaggressive cd4 + t cells, which infiltrate the cns initiating an infl ammatory response. however, the target autoantigens in ms are unknown. immunization of mice with myelin or other brainassociated proteins induces eae, a disease resembling ms both clinically and pathologically. the proteins, mbp, plp, and mog, components of the myelin sheath, are candidate antigens. 66 indeed, t cells that are reactive to these antigens have been demonstrated in ms patients. 67 mice expressing the human hla-dr2 (drb1*1501) molecule are capable of presenting peptides from all these three ms candidate autoantigens. it is possible in ms that while t cells responding to one of these antigens may initiate the disease, epitope spreading and the recruitment of t cells with additional specificities, as the disease progresses, could lead to infl ammatory responses to several proteins resulting in an escalation of the autoimmune response. 68 transgenic mouse models of multiple sclerosis are now well established. the following are two examples of such transgenic models. first, ms is associated with hla class ii molecules hla-dr2, -dr3, and -dr4. in humans it is difficult to analyze the individual roles of hla molecules in disease pathogenesis due to the heterogeneity of mhc genes, linkage disequilibrium, the influence of non-mhc genes, and the contribution of environmental factors. however, the specific roles of each of these class ii molecules can be addressed using transgenic models expressing these hla genes. this model could prove useful in deciphering the role of hla molecules and autoantigens in ms. 69 second, while eae has been a valuable model for the immunopathogen-esis of ms, it has sometimes been difficult to reconcile the fi ndings and therapies in the rodent models and the cellular and molecular interactions that can be studied in human disease. humanized transgenic mice offer a means of achieving this, through the expression of disease-implicated hla class ii molecules, coexpressed with a cognate hla-class ii-restricted, myelin-specifi c t cell receptor derived from a human t cell clone implicated in disease. such transgenic mice could provide an excellent model for studying epitope spreading in a humanized immunogenetic environment and for testing of immunotherapies. 70 the majority of the current therapies being planned for phase ii and iii trials in ms were first examined in eae. thus a particularly pertinent question is whether eae is a suitable relevant research tool for ms? some researchers believe that while eae is a useful model of acute human cns demyelination, its contribution to the understanding of ms is limited. 71 eae is an acute monophasic illness, as compared to ms, which is a chronic relapsing disease, and may be more suited as a model of acute disseminated encephalomyelitis (adem). drawbacks of the eae model include the following: (1) the nature of the inflammatory response in eae as compared to ms; (2) th-1-mediated disease in eae as compared to ms; (3) differences in the pathology between eae and ms; and (4) pitfalls in extending immunotherapies from eae to ms (see table 69 -3). consequently it may be concluded that the clinical picture of eae presented depends not only on the animal species used, but also on the route of administration of the encephalitogen and the nature of the encephalitogen, mbp, plp, or mog. it is thus possible that these eae models are somewhat imprecise methods to study the pathogenesis of ms or to develop therapeutic strategies. the nonhuman primate eae models are of primary importance for the safety and efficacy of testing new therapeutics for ms that may not work sufficiently well in species distant from humans, such as rodents. questions concerning the immunogenicity of biological therapeutics have also been addressed in nonhuman primates. many biological therapeutics, such as anti-cd4 antibodies 72 and altered peptide ligands, 73 have been investigated in rodents. although some of these therapeutics have been effective in treating eae in rodents, they have proven to be partially effective, or in some cases detrimental, in ms patients. 74 this ultimately raises the question of whether rodent models are the appropriate animal models for testing new therapeutic strategies for use in human ms. there are several examples, in humans and animals, of demyelinating diseases not associated with viral infections, such as demyelination associated with vitamin deficiency or toxins. many different animal models of eae have been studied using various mri techniques. 75 the clinical features of such models depend greatly upon the route of inoculation of the encephalitogen as well as the species and strain of animal used. inoculation routes such as subcutaneous, footpad, or intraperitoneal are not helpful in determining the onset or location of the lesion in the brain or spinal cord. thus, to create demyelinating lesions of precisely known locations and time courses, stereotaxic techniques are used to inoculate animals with chemicals that induce demyelinating lesions in the brain. several chemicals, such as ethidium bromide, cuprizone, and lysophosphatidylcholine (lpc), when injected directly into nerves or into the cns, produce lesions of demyelination. for demyelination studies with lpc, male wistar rats are anesthetized with sodium pentathal and fixed in a rat head restraining stereotaxic surgical table, head shaved, a burr hole created, and 0.2 µl of a 1% lpc solution in isotonic saline injected using an injector cannula. then lpc is infused at the rate of 0.05 µl/min for the next 4-5 min. the cannula is then removed and the burr hole closed using bone wax. rats are then observed daily and histological studies are carried out from day 3 to day 15 after lpc injection to cover the entire process of disease evolution. 76 using this lpc-induced demyelination it is possible to observe the complete pathological process of demyelination and remyelination in this animal model of ms. demyelination can be observed with the maximum value occurring on day 10. after day 10, remyelination starts with a reduction in edema. this model could be particularly useful for studying remyelination. one prominent feature of all chemically induced lesions is that the demyelinating lesions, and subsequent remyelination, can be studied without the interference of immune mechanisms. this has a tremendous advantage over virally induced models of ms: since no virus was inoculated none can remain to affect the remyelination. the use of animal models to investigate the pathogenesis of neuroinflammatory 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depletion on the pathogenesis of theiler's murine encephalomyelitis virus infection in cba mice characterization of b lymphocytes present in the demyelination lesions induced by theiler's virus investigation of the role of autoimmune responses to myelin in the pathogenesis of theiler's virus-induced demyelinating disease persistent infection with theiler's virus leads to cns autoimmunity via epitope spreading role of natural killer cells as immune effectors in encephalitis and demyelination induced by theiler's virus the role of cd8+ t cells in the acute and chronic phases of theiler's virus-induced disease in mice quantitative, not qualitative, differences in cd8+ t cell responses to theiler's murine encephalomyelitis virus between resistant c57bl/6 and susceptible sjl/j mice inhibition of theiler's virus mediated demyelination by peripheral immune tolerance induction in vivo evaluation of remyelination in rat brain by magnetization transfer imaging a human antibody that promotes remyelination enters the cns and decreases lesion load as detected by t2-weighted spinal cord mri in a virus-induced murine model of ms persistent viral infections weiss sr the organ tropism of mouse hepatitis virus a59 in mice is dependent on dose and route of inoculation cd4+ and cd8+ t cells are not major effectors of mouse hepatitis virus a59-induced demyelinating disease cellular reservoirs for coronavirus infection of the brain in beta2-microglobulin knockout mice autoimmune and virus induced demyelinating diseases. a review dynamic regulation of alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the mhv model of multiple sclerosis oligodendrocytes and their myelin-plasma membrane connections in jhm mouse hepatitis virus encephalomyelitis macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination, following murine infection with a neurotropic coronavirus cd4 and cd8 t cells have redundant but not identical roles in virus-induced demyelination cd8+ t cr+ and cd8+ tcr-cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers bystander cd8 t-cellmediated demyelination is interferon-γ-dependent in a coronavirus model of multiple sclerosis a role for α4-integrin in the pathology following semliki forest virus infection replication of the a7(74) strain of semliki forest virus is restricted in neurons demyelination induced in mice by avirulent semliki forest virus. ii. an ultrastructural study of focal demyelination in the brain long-term effects of semliki forest virus infection in the mouse central nervous system a case for cytokines as effector molecules in the resolution of virus infection the adhesion molecule and cytokine profi le of multiple sclerosis lesions physiological deficits in the visual system of mice infected with semliki forest virus and their correlation with those seen in patients with demyelinating disease role of immune responses in protection and pathogenesis during semliki forest virus encephalitis isolation and characterization of cells infiltrating the spinal cord during the course of chronic relapsing experimental allergic encephalomyelitis in the biozzi ab/h mice characterization of the cellular and cytokine response in the central nervous system following semliki forest virus infection probable epitopes: relationships between myelin basic protein antigenic determinants and viral and bacterial proteins acute experimental allergic encephalomyelitis increases lumbar spinal cord incorporation of epidurally administered [(3)h]-d-mannitol and [(14)c]-carboxyl-inulin in rabbits upregulation of group 1 cd1 antigen presenting models in guinea pigs with experimental autoimmune encephalomyelitis: an immunohistochemical study neurological response after induction of experimental allergic encephalomyelitis in susceptible and resistant rat strains upregulation of monocyte chemotactic protein-1 and cc chemokine receptor 2 in the central nervous system is closely associated with relapse of autoimmune encephalomyelitis in lewis rats evaluation of a rat model of experimental autoimmune encephalomyelitis with human mbp as antigen induction of experimental autoimmune encephalomyelitis in dark agouti rats without adjuvant inhibition of experimental autoimmune encephalomyelitis by inhalation but not oral administration of the encephalitogenic peptide: influence of mhc binding affi nity dendritic cells and the control of immunity rat models as tools to develop new immunotherapies eae in the common marmoset callithrix jacchus demyelination and axonal damage in a non-human primate model of multiple sclerosis histopathological characterization of magnetic resonance imaging-detectable brain white matter lesions in a primate model of multiple sclerosis: a correlative study in the experimental autoimmune encephalomyelitis model in common marmosets (callithrix jacchus) experimental allergic encephalomyelitis in the new world monkey, callithrix jacchus observations on the attempts to produce acute disseminated allergic encephalomyelitis in primates rhesus monkeys are highly susceptible to experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein: characterization of immunodominant t-and b-cell epitopes sex differences in experimental autoimmune encephalomyelitis in multiple murine strains eae susceptibility in fvb mice hyperalgesia in an animal model of multiple sclerosis encephalitogenic and tolerogenic potential of altered peptide ligands of mog and plp in biozzi abh mice native myelin oligodendrocyte glycoprotein promotes severe chronic neurological disease and demyelination in biozzi abh mice hla class ii associated genetic susceptibility in multiple sclerosis: a critical reevaluation progress in determining the causes and treatment of multiple sclerosis t cell reactivity to multiple myelin antigens in multiple sclerosis patients and healthy controls humanized animal models for autoimmune diseases identifi cation of t cell epitopes on human proteolipid protein and induction of experimental autoimmune encephalomyelitis in hla class ii-transgenic mice disease-related epitope spread in a humanized t cell receptor transgenic model of multiple sclerosis experimental allergic encephalomyelitis: a misleading model of multiple sclerosis the use of monoclonal antibodies for treatment of autoimmune disease an altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis the ups and downs of multiple sclerosis therapeutics gadolinium enhancement in acute and chronic progressive eae in the guinea pig sequential diffusion-weighted magnetic resonance image study of lysophosphatidyl choline-induced experimental demyelinating lesion: an animal model of multiple sclerosis the authors would like to thank dana parks for expert secretarial assistance with this manuscript. key: cord-264794-bgygebgx authors: lundgren, a.-l. title: feline non-suppurative meningoencephalomyelitis. a clinical and pathological study date: 1992-11-30 journal: journal of comparative pathology doi: 10.1016/0021-9975(92)90015-m sha: doc_id: 264794 cord_uid: bgygebgx abstract a spontaneous neurological disease in cats characterized by behavioural and motor disturbances was investigated by clinical, morphological and immunological methods. neuropathological examination showed a marked inflammatory reaction in the cerebral leptomeninges and the grey matter of the brain. in the white matter, the reaction was moderate. the changes consisted of perivascular cuffing by mononuclear cells and neuronal damage. the brain stem (thalamus, mesencephalon, caudal colliculus) was most severely affected. the spinal cord and its leptomeninges were involved to a lesser degree. the histopathological picture as well as the laboratory findings suggests a viral cause of the disease. the morphology of the disease and serological as well as immunohistochemical results indicate that this disorder is different from previously known feline viral encephalitides. feline non-suppurative meningoencephalomyelitis comprises a group of diseases which apparently are related. the syndrome seems to be geographically widespread since cases have been reported from australia (borland and mcdonald, 1965) , the united states (vandevelde and braund, 1979) and switzerland (hoff and vandevelde, 1981) . suspected cases have also been recorded in morocco (martin and hintermann, 1952) and sri lanka (mcgaughey, 1953) . a similar clinical condition affects lions, tigers and other large cats (flir, 1973; melchior, 1973; gutter, wells and baskin, 1983; truyen, stockhofe-zurwieden, kaaden and pohlenz, 1990 ). in 1974, kronevi, nordstrgm, moreno and nilsson reported the occurrence in sweden of a feline neurological disorder with the histological features of a non-suppurative meningoencephalomyelitis. thirty cats were affected, seven of which were examined post .mortem. these showed, throughout the brain and spinal cord, mononuclear perivascular cuffing, gliosis and meningitis. the lesions were most pronounced in the brain stem. the authors thought the disease was a specific entity, but did not express an opinion as to its aetiology. serological screening for viral agents was not performed by kronevi et al. (1974) and efforts to isolate a virus yielded negative results. since the report by kronevi et al. (1974) , the disease has become recognized in certain parts of sweden and is referred to as "staggering disease". in 1992, strijm reviewed the clinical findings in 33 cases of staggering disease recorded from 1988 till 1990. the author performed serological screening for feline leukaemia virus (felv), feline immunodeficiency virus (fiv) , feline coronavirus (fcov), toxoplasma gondii and borrelia burgdorfeeri in some of the diseased cats. the results were negative with the exception of one cat, which was positive for antibodies to fiv. there have been doubts as to whether staggering disease is a specific entity. it has been argued that the syndrome may include several aetiologically unrelated conditions affecting the central nervous system of cats, e.g. toxoplasmosis (hirth and nielsen, 1969) and the cerebral form of feline infectious peritonitis (slauson and finn, 1972; kornegay, 1978) . in order to establish whether staggering disease is a specific entity, it was decided to perform a systematic and thorough neuropathological examination of a larger number of cats than were included in the original report by kronevi et al. (1974) . furthermore, it was decided to discuss the possible aetiology of the disease based on modern knowledge of feline infectious agents. the present investigation details the pathological and clinical features of staggering disease and includes a search for aetiological agents with the use of immunohistochemistry and serology. from january 1990 till march 1992 a total of 316 cats was referred to the department of pathology, faculty of veterinary medicine, uppsala, for post mortem examination. sixty-three of the cats were diagnosed as having staggering disease. from this group, 25 cases that were clinically well documented were chosen for the present investigation. clinical examination and treatment of most of the cats took place at the university clinics, faculty of veterinary medicine, uppsala. the author performed clinical examination of six cats as well as interviews with their owners. fifteen cats were examined by several small animal veterinarians at the university clinics. four cats were clinically examined at private veterinary clinics and afterwards referred to the department of pathology for necropsy. one cat died of the disease. the others were killed by pentobarbital overdose. all cats with staggering disease were investigated post mortem by the author. tissue specimens were fixed in buffered 10 per cent formalin for light microscopy. the brain and spinal cord were removed from all cats for examination. coronal sections of the brain included the frontal, parietal, temporal and occipital lobes, basal ganglia, hippocampus, thalamus, mesencephalon, caudal colliculus, cerebellum and obex. from the spinal cord, sections were taken from cervical, thoracic and lumbar segments. the number of cases in which the different parts of the brain and spinal cord were examined are shown in table 3 . in 12 cases, portions of the proximal sciatic nerve were taken for histological examination. portions of the eyes and sartorius muscles were taken from eight cats. the cranial mesenteric ganglion was examined in four cases. samples were removed from the liver in 25, the kidney, heart and spleen in 24, the lung, mandibular gland and small intestine (jejunum) in 22, the pancreas in 20 and the retropharyngeal lymph nodes in five cats. paraffin wax sections were cut 4 l.trn thick and stained with haematoxylin and eosin (he). sections of the basal ganglia, hippocampus, thalamus, mesencephalon, caudal colliculus, obex and the spinal cord were stained with luxol fast blue, nissl, giemsa, phosphotungstic acid haematoxylin, gomori's reticulin and lendrum's stain for inclusion bodies in selected cases. to estimate the inflammatory changes, the presence of perivascmar cuffs and inflammatory nodules consisting of lymphoid cells and macrophages (as described by leestma, 1991) was recorded in each examined section of the central nervous system (table 3) . perivascular cuffs were counted in at least 5 low power fields (lpf) with a diameter of 5 mm and rated + = 1 to j/lpf, + + = 4 to g/lpf, + + + = > g/lpf. the thickness of the perivascular cuffs was rated from 1 to 4 where 1 cone cell thick, 2 = 2 to 3 cells, 3 = 4 to 6 cells, 4= more than 6 cells. the size of the inflammatory nodules was rated from 1 to 3 where 1 =a few cells, 2=moderate cluster of cells, 3 = extensive cellular infiltration. the presence of inflammation in the leptomeninges was graded from 1 to 3 with regard to the degree of cellular infiltration where 1 = slight, 2 = moderate, 3=severe. in five cats, material from the parietal lobe, basal ganglia, thalamus, mesencephalon, caudal colliculus, cerebellum and pons were fixed in buffered 10 per cent formalin for 48 h and then examined for the presence of pseudorabies virus antigen, canine distemper virus (cdv) antigen and toxoplasma gondii antigen, by the peroxidase-antiperoxidase (pap) immunohistochemical method (sternberger, 1979) with polyclonal primary rabbit antibodies kindly su rijksuniversiteit, ghent, belgium (pseudorabies), lied by p. de groot c. rmell swedish national bacteriological laboratory (cdv) institute ( toxoplasma gondii). and a. uggla, swedish national veterinary blood chemistry and csf analysis complete blood analysis was performed in 11 cats. the tests included haemoglobin concentration, total white blood cell count (wbc) and differential cell count, packed cell volume (pcv) and alanine aminotransferase and urea concentrations. in three other cats, only haemoglobin concentration and wbc were analysed. cerebrospinal fluid (csf) of seven cats was obtained by cisternal puncture and examined for colour, turbidity, protein content, total red (rbc) and white blood cell (wbc) counts and differential cell count. serology serum from 11 cats was tested for the presence of felv antigen and antibodies to fiv using an enzyme-linked immunosorbent assay (elisa) kit (cite-combo-test, idexx corp., portland, me, u.s.a.). s erum was tested for antibodies to fcov in nine cats with an elisa kit supplied by svanova biotech, uppsala, sweden. in seven cats, serum was tested for antibodies to borrelia burgdorferi with an indirect immunofluorescent antibody (ifa) test (burgess, 1986 ) with a human borrelia burgdorferi isolate as antigen. in five cats, serum was tested for antibodies to tick-borne encephalitis virus (tbev) by the haemagglutination inhibition (hi) method with l-day-old chicken erythrocytes starting with serum diluted 1 in 10 (kunz, hofmann and dippe, 197 1) . some of the cats with staggering disease came from urban, but mostly (68 per cent) from rural surroundings of uppland and malardalen, sweden. cases of the disease occurred throughout the year, but most often from december to may. the cats were aged 1 to 12 years, the mean age being 4.8 years. fifteen cats were male (60 per cent), twelve of them neutered. twenty-one cats (84 per cent) were short or long haired domestic cats, two were siamese, one abyssinian and one a norwegian forest cat. twenty cats (80 per cent) were allowed to roam freely outdoors. one cat came from a home where two other cats previously had been affected by staggering disease. four cats came from households where there were other cats unaffected by the disease. one cat had experienced several upper respiratory tract infections during a period of 9 months before showing signs of staggering disease. three to 6 months before showing the first signs of staggering disease, eight cats (32 per cent) were infested with ticks. the vaccination status of most cats was unknown; six had been vaccinated against feline panleukopenia. the most striking clinical manifestations (table 1) were a stiff staggering gait, inability to jump up and down normally and into-ordination of the hindlegs followed by weakness and paresis. some cats lost their ability to retract their claws. in addition to these motor disturbances, many cats showed mental changes. cats previously shy became social and affectionate, mewing more than usual, while cats that were customarily cheerful and affectionate became introverted and shy. aggressive behaviour was rare. depression, loss of appetite and dehydration were seen in many cases. less frequent signs included pruritus, hypersensitivity to sound and light, increased salivation, impaired vision, staring gaze, hyperaesthesia, constipation, tremor, circling and seizures. the rectal temperature was measured in 19 cats. nine of these (47.4 per cent) were febrile ( > 39*5"c). in the final stages of the disease the hindlegs were paralysed. all cats remained conscious to the end. most cats were treated with antimicrobial drugs such as tetracycline, ampicillin and chloramphenicol. intravenous fluids and b-vitamins were administered in many cases. some cats were given corticosteroids. despite treatment, the condition of the cats deteriorated and most cats had to be killed after 1 to 4 weeks illness. two cats were severely ill from the onset of the disease and had to be put down in less than a week. a few cats recovered incompletely. the only cat who died naturally of the disease did so after 4 weeks. a moderate leukopenia (2.6 to 4.1 x 10" cells per 1) was found in five of 14 cats examined. in two of the cats with leukopenia, the differential cell count was normal. the other three cats with leukopenia showed mild neutropenia and mild lymphocytosis; the neutrophil-lymphocyte ratio being 32:58, 32:59 and 33:66, respectively (normal range of the neutrophil count: 35 to 75 per cent; normal range of the lymphocyte count: 20 to 55 per cent). one cat in the final stage of the disease exhibited a wbc count of 15.4 x 10' cells per 1 and a neutrophil-lymphocyte ratio of 93: 1. haemoglobin concentration (normal value 80 to 150 g per 1) and pcv (normal value 24 to 45 per cent) were normal except for slightly raised values in dehydrated animals. alanine aminotransferase (normal value < 1.2 pkat per 1) was moderately raised in four cats (n= 12). urea concentration (n= 11) was normal (4.5 to 13-o mmol per 1) in 10 cats and slightly raised in one cat (14-o mm01 per 1). cerebrospinal fluid (table 2 ) was transparent and colourless in all cases, showed a moderate elevation in protein content (normal value < 25 mg per dl) in six cats and an increased wbc count (normal value < 5 cells per pl) in four cats. all sera examined for antibodies against fiv and presence of felv antigen were negative. six of the cats tested for antibodies against fcov were negative (titre < 1 in 10). in two cats the fcov antibody titre was 1 in 10 and 1 in 320, respectively. all sera examined for antibodies against borrelia burgdorferi and tbev were negative. pathology central nervous system. one cat that had been ill for 10 months exhibited a slight thickening and opacity of the cerebral leptomeninges. in the other cases, the brain and spinal cord did not show any abnormal changes on naked eye inspection. histopathological examination revealed throughout the central nervous system a non-suppurative inflammation characterized by perivascular mononuclear cuffing, presence of inflammatory nodules and neuronal degeneration in all cats. perivascular cuffs occurred within virchow-robin spaces and rbc= red blood cells, * after correction for blood contamination (fenner, 1989 around small vessels (fig. 1) . the cell population of the cuffs consisted of lymphoid cells, histiocytes, occasional plasma cells and macrophages laden with lipid-like material. the cuffs were one to six cells thick and sometimes seemed to compress the lumen of the vessels. the endothelial cells were swollen, but vascular thrombosis and vasculitis were not seen. neurones adjacent to cuffed vessels sometimes appeared shrunken and uniformly acidophilic. perivascular cuffing was less prevalent and less severe in the white matter than in the grey matter. inflammatory nodules consisting of aggregates of lymphoid ceils and macrophages, as described by leestma (1991) , were frequently seen in the grey matter. neuronal degeneration and neuronophagia (fig. 2 ) occurred in all parts of the cns except for the cerebellum. inclusion bodies were not identified. some large neurones, such as the hypoglossal nerve cells, contained small, cytoplasmic, eosinophilic granules. these were not further characterized. meningitis was present over all parts of the brain, but was most prevalent over the cerebral cortex and cerebellum (fig. 3) . spinal cord meningitis was slight and sometimes absent. the meningeal inflammatory cells were of the same type as in the cuffs. in the cat that had been ill for 10 months there was a moderate fibrosis of the cerebral leptomeninges. on the basis of classification and scoring of lesions, a localization pattern emerged (table 3 ; fig. 4 ). the most severe inflammatory changes were seen in the grey matter of the brain stem (thalamus, mesencephalon, caudal colliculus), basal ganglia and hippocampus. moderate inflammation was observed in the cerebral cortex. parenchymal lesions in the cerebellum were slight or absent, whereas cerebellar meningitis was severe. in the medulla oblongata, lesions were not as extensive as in the more proximal sections of the brain stem. at all levels of the spinal cord, inflammatory changes were moderate and mostly confined to grey matter. the lesions occurred in the ventral and dorsal horns with no apparent predilection for either place. degeneration of myelin was sometimes observed in the spinal ventrolateral tracts. the spinal nerve roots did not show any abnormalities. immunohistochemical examination by the pap method for the antigens of pseudorabies virus, cdv and toxoplasma gondii yielded negative results. positive control sections were stained and examined in all cases. other tissues. five cats were in poor physical condition. the urinary bladder of two cats was severely distended with urine. marked constipation of the rectum was evident in four cats. one cat had multiple small cysts in the cortex of the kidneys. other gross lesions were not observed. the liver of three cats showed varying degrees of fatty change. the cortex of the kidney of 12 cats exhibited small interstitial collections of lymphocytes, histiocytes and plasma cells. the retropharyngeal lymph nodes showed follicular hyperplasia and abundant accumulation of lymphocytes in cortex and paracortex in five cats. in eight cats, the lymph follicles of the spleen were reduced in number. the germinal centres, although large in size, were markedly lacking cells and in some cases showed degenerative changes of the central area (karyorrhexis, karyolysis and formation of an amorphous eosinophilic material). depletion of lymphoid cells was evident in the marginal zones of the follicles and in the periarteriolar lymphatic sheath (t cell area). small perivascular lymphocytic accumulations were seen in the cranial mesenteric ganglion of two cats (n = 4). the eyes and skeletal muscle showed no pathological changes. there were no abnormalities in the sciatic nerve of any of the cats examined. neuropathological examination of the cats of the present study showed a marked inflammatory reaction in the cerebral leptomeninges as well as in the grey matter of the brain and spinal cord. in the white matter, inflammatory changes were moderate. the reaction was characterized by perivascular mononuclear cuffing, neuronal damage and presence of inflammatory nodules consisting of lymphoid cells and macrophages. these changes are typical of viral infection (leestma, 1991) . the findings of degeneration and depletion of lymphoid cells in the follicles of the spleen also seem to point to an infectious agent. the laboratory findings, including leukopenia and elevations in protein content and wbc count of the csf, are not specific for viral infection, but at least suggest that the disease in the cats of this study is infectious in its nature. the abundance of neurological signs in cats with staggering disease is a reflection of a widespread failure of the nervous system, typically seen in disseminated inflammations. the most notable viral agents causing progressive multifocal involvement of the cns in cats are feline infectious peritonitis virus (fipv), feline leukaemia virus (felv) and feline immunodeficiency virus (fiv). any of these viruses as well as some other agents known to cause cns disorders in cats could be the cause of staggering disease. the central nervous system form of feline infectious peritonitis (fip) is manifested clinically in personality changes, posterior paresis, nystagmus and seizures (pedersen, 1976; kornegay, 1978) . lesions in the brain and spinal cord consist of pyogranulomatous meningitis, encephalitis and ependymitis (slauson and finn, 1972; legendre and whitenack, 1975; kornegay, 1978) . inflammation of choroid plexuses and the mesencephalic aqueduct results in blockage of normal csf flow and secondary hydrocephalus (krum, johnson and wilson, 1975; bar-lough and summers, 1984) . in the present investigations, cns lesions showed a different pattern of localization compared with fip. there was no predilection for choroid plexuses and ependyma. vasculitis, a common finding in fip, was not present. although cases of pure neurological fip exist, most cats also have involvement of the eyes and/or other organs (kornegay, 1978) . in the present investigation, all major lesions were confined to the cns. the results of serological investigation for coronavirus antibodies do not support the view that fipv or other coronaviruses are plausible aetiological agents in feline non-suppurative meningoencephalomyelitis. the finding of a coronavirus antibody titre of 1 in 320 in one cat is not significant since titres of this size are also seen in many cats that have had previous infections with feline enteric coronavirus (fecv), a virus closely related to fipv (pedersen, 1991a) . feline leukaemia virus (felv) has been reported as a cause of encephalitis in cats (sottiaux and pialat, 1989) . however, felv is more commonly associated with epidural lymphosarcoma, which is usually manifested as acute posterior paresis or paralysis (pedersen, 1991 b) . neither the serological results nor the clinical and histopathological findings in the cats with staggering disease indicate a felv infection. feline immunodeficiency virus (fiv) has emerged as an important cause of neurological disease in cats (dow, poss and hoover, 1990; sparger, 1991) , often in association with clinical syndromes typical of an immunodeficient state (chronic stomatitis, enteritis, dermatitis, etc). neurological signs include psychotic behaviour, dementia, seizures and ataxia. brain lesions are confined to the thalamus and midbrain, sparing the cerebral cortex and cerebellum (dow et al., 1990) . non-suppurative encephalitis with perivascular mononuclear cuffing and glial nodules is commonly observed. antibodies against fiv were not found in the cats of the present study. seroconversion in fiv infection may be delayed for months or even years (sparger, 1991) . also, cats in the terminal stages of fiv infection will sometimes show undetectable concentrations of antibody. however, the absence of clinical signs of immunodeficiency in cats with staggering disease argues against fiv as a causative agent. the nervous system in cats is susceptible to feline panleukopenia virus infection during the prenatal and early postnatal period, resulting in cerebellar hypoplasia (greene and scott, 1990) . both clinical and histopathological data (csiza, de lahunta, scott and gillespie, 1972) are different from the cats of this report. in addition, six of the cats with staggering disease had been vaccinated against feline panleukopenia. sweden is a rabies-free country and staggering disease has neither the clinical signs nor histological features typical of rabies. aujeszky's disease (pseudorabies), which is fatal in carnivores (dow and mcferran, 1963) , is excluded because of differences in the clinical picture, absence of intranuclear acidophilic inclusion bodies in neurones and astroglia and negative immunohistochemical staining for virus antigen. canine distemper virus (cdv) has been reported as a cause of encephalomyelitis in tigers (blythe, schmitz, roelke and skinner, 1983) . domestic cats are susceptible to experimental infection with cdv, but natural disease has not been reported in cats (appel, sheffy, percy and gaskin, 1974) . immunohistochemical staining for cdv antigen yielded negative results in cats with staggering disease. encephalitis has been induced experimentally in cats with the viruses of newcastle disease (luttrell and bang, 1958) ) b orna disease (ihlenburg, 1966) , near eastern equine encephalomyelitis (daubney and mahlau, 1957) and human poliomyelitis (salvioli, gotti and sternini, 1952) . it is not known whether these viruses naturally infect domestic cats, but there are no reported cases in the literature. keane, parent and little (1987) inoculated one cat intracerebrally and six cats intravenously with powassan virus of the flavivirus serogroup. none of the cats developed neurological signs although histological lesions of a nonsuppurative encephalomyelitis were observed in two cats. tick borne encephalitis virus (tbev) of the flavivirus group is transmitted by the tick ixodes ricinus in sweden. most of the cats of the present study were allowed to roam freely outdoors in regions harbouring ixodes ricinus and there is a possibility that this tick could be the vector of the aetiological agent causing staggering disease. however, cats with staggering disease have tested negative for antibodies to tbev, which indicates that this virus and probably other related viruses of the flavivirus serogroup can be excluded as the primary cause of the disease. toxoplasma gondii, the most important non-viral infectious agent causing cns disease in cats, is ruled out because of negative immunohistochemical staining for 1. gondii antigen, absence of tissue cysts and absence of extraneural lesions. borrelia burgdorferi, a cause of arthritis and possibly meningitis in dogs (greene, 1990) has not been reported as a cause of neurological disease in cats. all cats tested for antibodies to borrelia burgdorfeeri in the present study were negative. in conclusion, the neuropathological features as well as the serological and immunohistochemical results in the cats with staggering disease are not compatible with any of the known spontaneous feline viral diseases affecting the cns. neither are they compatible with toxoplasmosis. the feline meningoencephalomyelitis of the present investigation is doubtless identical with that described by kronevi ef al. (1974) and shows many similarities to the cases of unknown aetiology reported in switzerland by hoff and vandevelde ( 198 1) . six cases of polioencephalomyelitis in cats were reported in the united states (vandevelde and braund, 1979) . clinical signs included ataxia, tremors and seizures. although the pathological lesions in the cns were qualitatively similar to the lesions in the cats of the present study, the localization pattern was different. in the american cats, the most severe lesions were observed in the spinal cord and consisted of a marked degeneration and loss of neurones as well as white matter degeneration. in a german safari park, an outbreak of fatal encephalomyelitis in lions and tigers occurred in the early 1970s (flir, 1973; melchior, 1973) . the clinical as well as the histopathological picture in these large cats closely resembled that of our cats. many lions became more affectionate and infantile and assumed a strange staring gaze. this increased affection, a phenomenon often observed in staggering disease, is not reported as a conspicuous feature of any of the known feline infectious cns disorders; neither is it mentioned in the other reports of feline meningoencephalomyelitis of unknown aetiology cited above. the histological features in the lions and tigers were those of non-suppurative polioencephalomyelitis, with the most severe lesions in the brainstem. efforts to isolate virus from the cns failed. in recent years, a further outbreak of the disease was observed in the same park (truyen et al., 1990) . attempts to isolate a virus from the cns again failed, but feline herpes virus type 1 (fhv-1) was isolated from the tonsil of one lion. this was interpreted as an infection superimposed on the encephalomyelitis as a result of reduced resistance and was not regarded as the primary cause of the disease. fhv-1 is well known as the cause of feline rhinotracheitis. what is against fhv-i as the aetiological agent in staggering disease is the fact that fhv-i is regarded mainly as a breeding cattery problem, the virus being transmitted through intimate contact between cats (pedersen, 1991c) . in contrast, staggering disease is almost unheard of-in breeding catteries and does not appear to be as contagious as feline rhinotracheitis. kittens are more severely affected by fhv-1 than adult cats, while staggering disease has not been observed in cats younger than 6 months. the role of fhv-1 in feline cns disorders has not yet been clarified, however, and the possibility that fhv-1 or related herpesviruses could be involved in the cause of staggering disease remains open. in conclusion, some previously recorded cases of idiopathic, feline, non-suppurative meningoencephalomyelitis presented clinical and pathological similarities to our cases suggesting a common or related aetiology. viral infection is the most likely cause of staggering disease. the possibility of an arthropod-borne mode of transmission has been suggested. given the consideration that most cats were freely roaming outdoors, rodents could be alternative sources of infection since they are the hosts of some neurotropic viruses, e.g. theiler's virus and lymphocytic choriomeningitis virus. another possible mode of transmission may be through bites and scratches exchanged between cats in fights over territory. inoculation by bite wounds has been shown to be the major mode of transmission of feline immunodeficiency virus (fiv) and the fact that male cats are more often affected by staggering disease than females gives some support to the theory that staggering disease could be transmitted by bites in a similar way. further serological and virological investigations in order to identify the cause of the presently described feline non-suppurative meningoencephalomyelitis are required. this work is now in progress. canine distemper virus in domesticated cats and pigs encephalitis due to feline infectious peritonitis virus in a twelve-week-old kitten chronic encephalomyelitis caused by canine distemper virus in a bengal tiger feline encephalomyelitis experimental inoculation of dogs with borrelia burgdorferi. zentralblatt f$r bakteriologie spontaneous feline ataxia near-eastern equine encephalomyelitis aujeszky's disease in the dog and cat feline immunodeficiency virus: a neurotropic lentivirus the neurologic evaluation of patients encephalomyelitis bei grosskatzen. deutsche tier&tliche wochenschrifi feline panleukopenia. in infectious diseases of the dog and cat lyme borreliosis. in infectious diseases of the dog and cat neurologic disease in three cats at the audubon park zoo pathology of feline toxoplasmosis non-suppurative encephalomyelitis in cats suggestive of a viral origin experimentelle prufung der empfanglichkeit der katze fur das virus der bornaschen krankheit california serogroup and powassan virus infection of cats feline infectious peritonitis: the central nervous system form feline ataxia due to nonsuppurative meningoencephalomyelitis of unknown aetiology hydrocephalus associated with the noneffusive form of feline infectious peritonitis viral infections of the nervous system feline infectious peritonitis with spinal cord involvement in two cats newcastle disease encephalomyelitis in cats infectious myelitis of cats: preliminary communication sur i'existence, au maroc, d'une maladie contagieuse du chat, non encore d&rite: la myilite infectieuse meningo-enzephalitis beim lowen und tiger feline infectious peritonitis: something old, something new feline infectious peritonitis virus infection feline leukemia virus infection feline herpesvirus type-l infection effetti polimorfi nel gatto da inoculazione di materiale poliomielitico. rivista dell'lstituto sieroterapico italiano meningoencephalitis and panophthalmitis in feline infectious peritonitis meningo-encephalite associte a une infection felv chez un chat. pratique medicale et chirurgicale de i'animal de compagnie feline immunodeficiency virus immunocytochemistry, 2nd edit vingelsjuka hos katt a case report: encephalitis in lions. pathological and virological findings polioencephalomyelitis in cats the author is grateful to the staffs of the departments of virology, pathology and bacteriology of the swedish national veterinary institute for carrying out the serological and immunohistochemical investigations. thanks are due to dr per bierke for assistance with evaluation of the lymphatic tissues and to drs egenvall, stavenborn and tistedt of the department of medicine and surgery, faculty of veterinary medicine, for performing clinical examination of cats with staggering disease. this work was supported by grants from the albert hjarre foundation, agria insurance company and the swedish fund for research without animal experiments. key: cord-272981-8gahvdt0 authors: wege, helmut; watanabe, rihito; ter meulen, volker title: relapsing subacute demyelinating encephalomyelitis in rats during the course of coronavirus jhm infection date: 1984-08-31 journal: journal of neuroimmunology doi: 10.1016/0165-5728(84)90022-5 sha: doc_id: 272981 cord_uid: 8gahvdt0 abstract temperature-sensitive mutants of the murine coronavirus jhm induced a subacute demyelinating encephalomyelitis (sde) in young rats. neurological symptoms were associated with marked lesions of primary demyelination in the white matter of the central nervous system (cns), and developing after an incubation time of several weeks to months. many rats survived this infection and recovered completely from this cns disease. among 43 survivors of sde, 9 rats developed a relapse 27–153 days after onset of the first attack. neuropathological examination of these animals revealed areas of fresh demyelination together with old remyelinated lesions. viral antigens were detectable in the neighbourhood of fresh lesions and in some cases infectious virus was re-isolated from rats revealing low antibody titers to jhm virus. these results demonstrate that mutants of jhm virus can induce a relapsing demyelinating disease process, associated with a persistent infection, which possesses some similarities to chronic experimental allergic encephalomyelitis. pathogenesis are unknown. the most prominent example is multiple sclerosis (ms) which is thought to be associated with an immune pathological process p~sibly acting in concert with a virus infection (waksman 1981) . it has been shown that an autoimmune reaction against cns tissue can lead to 6hronic relapsing demyelination as seen in chronic experimental allergic encephalomyelitis (eae) whereas the role of a virus infection in the induction of such a condition has not so far been directly demonstrated (mcfarlin et al. 1974; paine et al. 1978; lassmann and wisniewski 1979; traugott et al. 1982) . it is there.fore desirable to develop animal models of relapsing demyelination associated with a virus infection in order to analyse the mechanisms by which virus-induced neuropathological changes may occur. viruses can induce demyelination which is interpreted as either the consequence of cell death in the course of viral replication, or the result of dysfunction in persistendy infected oligodendrogiia cell*, lmmunopathologieal reactions could also play a role in virus-induced demyelination as studies with theiler's virus infection in mice suggest. relapsing demyelination associated with a clinical diseac, e as seen in chronic eae has been postulated in virai infections of the cns, especially in man, but not studied experimentally. in the following we describe a model in which certain mutants of muri1~e coronavirus jhm cause clinical relapsis of a subacute demyelinating encephalomyelitis (sde) in rats. after virus infection in rats inflammatory disseminating cns lesions of marked demyelination develop accompanied by clinical signs of a subacute disease after varying incubation times. a considerable percentage of diseased animals survive and recover from this infection and some come down with a second attack of sde after a period of weeks to months. such animals show both, fresh demyelinating lesions with infiltrations, and remyelina~.ed areas in the brain and spinal cord. infectious jhm viru:, can be reisolated from brain tissue and virus antigens demonstrated in brain cells. these findings demonstrate that a clinically recognizable relapsing cn5 disease can develop in association with exacerbations of demyelination in the. course of a persistent viral infection of brain tissue. outbred specific pathogen-free rats (chbb/thom) were purchased from "rhomae (biberach, f.r.g.'. lewis rats were obtained from the zentralinstitut ftir versuchstiere (hannover, f.r.g.). 10-15 day old rats received 4 x 103 pfu of jhm virus in 30 t~l tissue culture medium into the left brain hemisphere using a dispmser syringe. the temperature-sen:;ifive (ts) mutants ts6, ts42 and is43, which were selected from the routine coronavirus jhm after growth in present, of fluoroura~.il have been previously described (wege et ai. 1983) . the virusct; were propagated on monolayers of sac(-) cells at 340c with eagle's minimal essential medium (1*.~em) and 5% fetal calf serum. samples of brain and spinal cord were homogenized immediately a:fter dl~,sec',ion and adsorbed for 1 h at 34°c on monolayers of sac(.) cells in 24 wel~ cluster plates (wege et al. 1983 ). the number of wells in which syncytia developed was scored after 3 days incubation at 34°c. the supernatar~ts from positive wells were harvested, pooled ~md titrated at 34°c as well as 39.5°c to measure the temperature sensitivity. cell~ from reisolation attempts wltich yielded only very few or sl~dl syncytia were trypsinised, mixed with normal sac(-) cells and passaged further. to measure neutralizing antibodies, the s(:ra were inactivated for 30 rain at 56°c. mixed in serial dilutions with 100 pfu of wild type jhm virus (f'mal volume 200 ~1) and incubated :~or 1 h at 4°c. neutralization was assayed using sac(-) cells in 24-well microplates. the cultures were incubated for 20 h at 37°c without agar overtay. microplaques were counted after staining with may-grtinwald and giemsa, the serum dilution resulting in 50~ plaque reduction (ndso/0.1 ml) was thus calculated. total 51l~4-virus ~_,'.:ibodies were titrated using a solid phase ei~yme immunoassay (elisa), performed according to standard procedures ~fvoller et al. 1976). antigen was prepared from infected or control sac(-) cells, wl'fich were disrupted by dounce homogenisation in rsb (tris-hcl 20 mm, ph 7.4; sodium chloride 100 ram, magnesium chloride 5 mm) and 0.2~ nonidet p40. the homogenate wes centrifuged 5 min at 10000 × g and the antigens were pelleted by t~ltracentrifugation at 100000 × g for 90 min. antigens and indicator immunoglobulins (horseradish peroxidase-lab¢lled anti-rat globulin, dako) were c~dibrated by bh3ck titrations for reproducible sensitivity. the positive anti-jhm virus serum was prelmred by immunisation of rats with density gradient purified virus (wege et ~d. 1979 ) and h~ a neutralisation titre 1 : 1250. the antigen block titrations were c~dibrated with a serum dilution of 1:409 to give a netto absorbance value (we mean of virus antigen-coated wells minus mean of control antigen-coated wells) of 1.0. flat bottom microplates (dynatech) were coated with antigen and control antigen in 0.05 m sodium carbonate buffet, ph 9.6 overnight at 4°c (100/~l/well), and unbound proteins removed by washing with tris buffer (0.05 m, ph 7.4) containing 0.15 m sodium chloride and 0.1~ tween 20. serum dilutions were made in the same buffe~ containing 5~ newborn calf serum and 0.002~ phenol red. after incubation for i h at 37°c the plates were washed again, then incubated with peroxidase-laladled rabbit anti-rat globulin and after a final wash orthophenylene diamine (0.5 ngg/ml) in citric acid (35 mm, dinatrium hydrogen-phosphate 66.6 mm, bydrogene peroxide 0.01~, ph 5) was added as substrat¢. the reaction was stopped after i h at reran temperature by addition of 50 btl sulfuric acid (3 m) and the colour measured with a multichamiel spectrophotometer (titertek, flow labs.) at 492 rim. the endpolnt of the titration (elisa fitre) was defined as that serum dilution which yielded a nett,~ absorbance value more than 3-fold higher than the negative control sera at a dilution of 1 : 10. the positive and negative serum pool which was used for the calibration of the assay by block titrations was included in each experiment. tissue was fixed ia buffered 10~ formalin and embedded in paraffin for histological examinati,~n. sections were stained with hematoxylin-eosin and luxol fast blue. for upon embedding, animals were perfused wi~h 2% g)utaraldehyde and 2% paraformaldehyde, 0.5% sucrose in 0.1 m phosphate buffer solution. the tissue was postfixed by osmi~jm, dehydrated, embedded in upon and 1/zm sections were stain~ with toluidine flue for light microscopy. ultrathin sections were stained with uranylacetate and lead citrate for electron microscopy. appropriate tissue blocks were fixed in buffered 2~ paraformaidehyd¢ for 4 h to overnight, washed by graded sucrose, finally by 10~ glycerol and 20~ sucrose in 0.1 m phosphate buffer at the ~.emperature of 4°c, embedded in oct, frozen by liquid nitrogen and kept at --70°c until 10-#m sections were prcparecl by cryostat. the pap method (sternberger et al 1979) was applied by using rabbit anti-jhm .serum (having a neutralization titre of 1 : 1860) at a dilution of 1 : 1000. the rabbit had been immunised with density gradient purified jhm virus (~vege et al. 1979 ) and the serum was absorbed with acetone-extracted rat brain powder. for control staining, normal preimmune rabbit serum and an anti-measles virus rabbit serum was used. as a second antibody anti-rabbit igg (dako z147) and pap complex (dako z113) were inc!uded. counter staining was achieved with methyl green. intracerebral inoculation of 10-15 days-old thomae or lewis rats with temper~. ture-sensitive (ts) mutants was followed by the developn~cnt of sde after an incubation period of several weeks (wege et al. 1983 ). this disease started quite often with a mild to moderate hindleg weakness with ataxic gait which causes the rat considerable difficulties in righting itself up when flipped onto its back. at v~ariable times up to several days these signs were followed by severe hindleg weakness leading to hindleg paraly:~is. at such a stage no voluntary movement of the hindlimbs could be observed although some reflex stiffening occurred. approximately 60-70% of the disea.,:ed animals died within a few days after onset of disease, whereas the surviving animals recovered completel:/, or with a stabilized disability. the most pron~inent histologic~ll finding consisted of demyelinating plaques located in the white matter ( fig. 1) primarily of the oplic nerve, midbrain, ports, cerebellum and spinal cord. within the demyelinated plaque, axons and neurons were well preserved. in ~,ddition, cell infiltrations were observed consisting; of iymphocytes, plasma cells and macropkages. infectious virus could be isolate~ by conventional methods, and viral antigens were easily detectable in glia cells in the neighbourhood of demyehnatiag plaques (nagashima et al. ; sorensen et al. 1980 sorensen et al. , 1982 wege et al. 1983 ). fi 8,1, large demyelinating plaque located in the white matter of the spinal c~rd in a lewis rat with sde. hematoxylin-eosin and luxol fast blue staining, x 28. of particular interest is the observation that some of the ar.imals whic~ survived and recovered from sde revealed later a second attack of this disease. of 276 rats infected with different ts-mutants c f jhm virus 57% developed clin~ical signs of sde within an incubation pe~od of 14-.158 days. 36% of the diseased an~-'lals survived this infection. among the 56 survivors of sde, 13 rats continuously exhibited clinical signs of hindleg paresis or paralysis. the remaining ,*3 animals were apparently healthy. 1~ ut 9 of these animals developed z~ second attack of sde 2-;8 weeks after recovery from the first attack. as documented in table 1 these animals had developed their first attack of sde after an incubation period of 14-33 da2/s. clinical symptoms of this i ~ ~ ' • first attack lasted from ~ to 25 days until they recovered. in all cases recc,ved' was complete and no disability w~.s noted. these animals came down with a second attack 41-173 days p.i., however, wi~h an averetge incubation time between onset of the first attack of disease and its exacerbation, of 61 days. the clinical picture of the secor, d attack was similar to the first~ except the course seemed to be more severe. since these animals were needed for experimental analysis it is not known whe~.her they would have recovered again. the histopathological investigation of relapsing sde in these rats revealed the following changes as documented in ]rig. 2. all rats studied showed fresh demyelinating lesions with infiltrations of mononuclear cells primarily ic~:ated in spinal cord and brain stem. in the same animals, old lesions were also 10und in pons, thalamus, cerebellum or spinal cord. these old lesions in the whit,; raatter revealed extended remyelination of the cns and/or pns type. in some cases proliferation of astroglia was observed. a very pronounced fresh, demyelinated lesion in ;lt: spinal cord at the same level as a remyelinated area is shown in fig. 2a . thi,; rat developed a relapse of sde 138 days p.i. (no. 8 in table 1 ). a higher magnification of a thin section from the demyelinated plaque clearly reveals naked ~txons in the presence of macrophages (fig. 2c ). an extended area of remyelination by schwann cells (pns type) was noted at the same level of this spinal cord. higher mal~;nification and electron microscopy clearly demonstrated the association of schwann calls with axons and the basal membrane (figs. 2b,e,f,g). even in the remyelinated areas infiltrating macrophages were detectable. furthermore, remyelinated areas of the cns type were found at different lejels of this spinal cord (fig. ld) . in addition, remyelinated areas of the pns type and macrophage infiltrations with myelin debris were encountered in the pons. attempts to re-isolate jhm virus from these ammals yielded different results (table 2 ). despite the fact that all animals tested revealed viral antigens ~n gila cells fig. 2 . relapse of sde 138 days p.;. following inoculation of ts43. the lewis. rat was infected at an age of 10 days. a: large demyelinated plaque in the anterior and lateral area of white matter (;~'row heads). "[he anterior white matter is unusually swollen by edema which leads to deformation of the grey matter. however, in this area neurons are well preserved. in the posterior column (surrounded by arrows) pns type remyelination was observed. hematoxylin-eosin and luxol fast blue staining of paraffin-embed~k,d section. the rat was perfused with glutaraldehyde-paraformaldehyde solution. × 64. b: same level as a, higher magnification from remyelinated area. numerous nu,cle:i of schwaxm oils are detectable indicating a pns type remyelination. note infiltration by macroph~ges (arrow heads) even in this area. x420. c: sa nc level of spinal cord with demyelinating plaque as a, l-p.m section after embedding in epon. naked axons (arrow heads) and infiltration by macropha-es (arrows) with myelin debris indi*:ate fr~,h demyelinafion. stained with toluidine blue. x 625. d: thoracic spinal cord, upper level. remyelination of the cns type is indica,led by thinly myelinated ~xoas (arrow heads), l-pm section after embedding in epoe., stained with toluidi~e blue. ×550. e: 1-p m section after embedding in epon from same level with remyelination of pn s type as a am~. b. nu, lei of schwann cells associated with axons. macrophage infiltration (arrow heads), x 324. • md g: electron microscopy of remyelinated area (,.ame level as a and b). schwann cells in asso.lation with axons arc surrounded by a basal memarane. × 4400 and × 26 0(.~:. in the neighbourhood of demyelinating plaques (fig. 3) , onty fro:~ 2 animals (animal nos. 6 and 7) was infectious virus obtained after homogenization of spinal cord and brain specimens. only non-infectious virus was recovel,ed from the other animals. sac(-) cells which were used for those isolation attempts, develotw.d cy~p~thic effects consisting of giant ~ell formation, a typical cpe of corom,v;rus irffection, in which v~tal antigens were present. no infectious virus was detectable in these cultm'es, however, either in the supernatant or in a cell-associated form. a non-~elding persistent infection was apparently established in these tissue cu!t*~,re cells. with regard to these findings, the immune response of these animals against'. jhm virus is of interest. all animals tested except ?, revealed high antibody titers against jhm virus detectable hy elisa and neutralitjng assay. only serum samples from rat nos. 6 and 7 showed a low reaction in the elisa and no detectal,01,: neutralizing antibodies. yet, i~ fectious virus was isolated only from these 2 animals which lacked neutralizing antibodies. this suggests that the failure to recover infectious virus from the other rats might ,be related to the pr,sence of neutralizing, antibodies which inactivate free infectious virus. diseussio. intracerebral inoculation of 10-15-days-old thomae or lewis rats with certain neurotropic ts.m~ttants of murine coronavirus jhm leads to the d,velopment of a suba~te demyel~rating enc, phalomyelitis which may or may not b, f~ltm./ufimals surviving this infection can recover completely and reveal no clinical signs of disability, but under conditions which are presently not understood a ~concl attack of this cns disease can develop wlfich is very similar to the tirst or~e. these observetions demon.,;trate that jhm-persistent infection in tat. cn:~ tissue can remit in demye.linating diseases, with remission and relaf;e of cli mica] symptoms, in connection with exacerbations of neuropaihological changes. evidence for chronic, recunent demyelination by coronavirus jhm has been obtaine~l hi infections of mice i but no significant clinical changes were observed m this animal (hemdon et a~. 1975; haspel et al. 1978; knobler et al 1981 knobler et al , 1982 stohlman.and weiner 1981) .~ a biphasic dkease course with clinical symptoms and recurrent demyelination has been described in theiler's virus infection of mice (lipton and dal canto 1976; dal canto and lipton 1979; dal canto 1982) . in thj,s infection, young adult n~e develop a flaccid paralysis from which the majority of animals recover. survi,dn 8 mice subsequently come down after weeks with a spastic, paralyric gait ditovder. in the early dim the grey matter is predominantly involved, resembling human poliomyelitis, whereas in ',he later disease primary demyelination it found relawd to perivascular mononuclear cell infiltrations. in this virus infection the early aqd late disease are neuropathologically quite differew,, whereas in jhm viru~induced recurrent demyelinafion the first and second diseas,~ are cliuically and netx-opathologically very similar. some evklence has been pro'~ided that theiler virus induces an immune-lmthologtcal reaction which could contribute to demyelinafion. immunmuppreudon decreases or prevents de~2ielination, bm the exact pathogenetic raechanitm is unknown. the induction of a progressive demyelinating, or relapsing demyelinating, disease process, in jhm virus-infected rats has ~me parallels to chronic eae, an animal model based on sensitation ~8ainst cns tissue extracts or myelin basic protein (mcfarlin et al. 1974; raine et al. 1978; lassmanll and wisniewski 1979; traugott et al. 1982 . al. 1981; vandevelde et al. 1982) . these data suggest that different virus infections of the cns can be associated with an autoimmune response against brain tissue. on the other hand, it is co~tceivable that the virus infection of cns ~lls also contributes co the relapsing disease course. it has been shown in visna infection in sheep that the antivirai immune response is not able to control fl~ cn$ infection, in the presence of antibodies to visna virus, mutants continuously develop which cannot be neutralized by the ~ntibodies directed against the parenud1 virus (n~rayan e~ al. 1978). the emergence, of each new mutant permits the infection of n~v ar~ts of the brain lead!',~g to inflammatory reactions and cns damage. in coronavir~s infection of rats, jhm virus can be re-isolated after several months of persistence, so far, the antigenicity and biological properties of such re-isolated virus have not been compared to those of the virus used for inoculation. in relation to other i~,,rsistent infections of the cns such as subacute selerosing panencephalitis, it is rem~kablc tha~ ir fectious jhm virus can be isolated: this suggests that antigenic change~ may occur during persistency. the occurrence of chronic viral infection, the relapsing clinical course witil the presence of old and fresh dernyetinating plaques and the evidence of a cell.media£ed immune reaction to myelin basic protein during the fi~'st attack of sde (wal~nabe et al. 1983) , suggest that this dis~as~ process is a result of a very complex virus.host interaction, in which host factors play an important role. it is conceivable tha~ the autoimmune reaction against elyelin basic protein may also perpetuate the d~sease, even when the virus is no longer demonstrab!, in cns tissue. the features ol thi~ coronavirus jhm-asscciated cns disease in rats reveal tome dmilarities to multiple sclerosis in man and ~t is hoped fllat the analysis of the events which lead to thi~ animal disease will be of relevance in our understanding of other relapsin,~, de~ myelinating processes. uncoupl~ relationship between demyelination and primary infection of myelinating cells in theiler's virus encephalomyelitis recurrent demyelination in chronic central nervous system il~rection produced by theiler's routine encephalomyelitis virus temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination mo,se hepatitis vir,.u~-htduced re,mint demyelination selective localization of wild type and mutant mouse hepati~s ~rus ohm) strain antigens in ciqs tissue by fluorescence, light and e~ectron microscopy virus persistence and r'~curring demyelination produced by a temperature-sensitive mutant of mhv-4 chronic relapsing experimental allergic encephalomyelitis --clinicopathological comparison with multiple sclerosis theiler's virus induced demyelination --prevention by immunomppression corona virus induced subacute demyelinat* ing encephalomyelitis in rats --a morphological analysis demyelinating encephalomyehtis induced by a long.term corona virus infection in rats virus mutation during slow infection --temporal development and characterization of mutants of visna virus recovered from sheep chronic relapsing experimental encephalomyeliti!s --cns plaque development in umuppressed and ~uppressed animals in vivo and in vitro models of demyelination in vivo and in vitro models of demyelinating disease endogenous factors influel~ing derrtyelinating di3ease caused by mouse hepatitis virus in rats and mice induction of antimyelin and antioligoden&~ocyte antibodies by vaecinia virus chronic central nervous system demyelination in mice after jhm virus infection, ~euroiosy chronic relapsing experimental autoimmune e~halomyelitis --treetmant with combinations of myelin components promotes clinical and struclural recovery immunological and pathological fmdin~ in demyelimttinl~ ancephnhtis associated with canine distemper virus infection microplate enzyme immunoatsays for the immuncdiasnosis of virus infections current trends in multiple sclerolds research, lmjnunolosy today adoptive trar~fer of eae-like iedo~ by bm~p s~jnmlar~ed lymphocytes from ran witl corcnavirus-induced demyelinafing encephalomyelitis struc/ural polypeptid~ of the routine cc~ronavirus jhm neurovinllenc¢ of routine coronavirus jhm wmperatur~.-sensitive mutants in rats we thank margarete sturm, hanna wege and renate abt for excellent technical assistance and helga kriesinger for typing the manuscript. key: cord-000725-rafwlw0t authors: hindinger, claudia; bergmann, cornelia c.; hinton, david r.; phares, timothy w.; parra, gabriel i.; hussain, shabbir; savarin, carine; atkinson, roscoe d.; stohlman, stephen a. title: ifn-γ signaling to astrocytes protects from autoimmune mediated neurological disability date: 2012-07-27 journal: plos one doi: 10.1371/journal.pone.0042088 sha: doc_id: 725 cord_uid: rafwlw0t demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (ms). cells resident within the central nervous system (cns) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. astrocytes, the most abundant cns cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. experimental autoimmune encephalomyelitis (eae) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (ifn-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. inhibition of ifn-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (bbb) integrity or the composition of the acute cns inflammatory response. nevertheless, increased demyelination at peak acute disease in the absence of ifn-γ signaling to astrocytes correlated with sustained clinical symptoms. following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of ifn-γ signaling to astrocytes in neuroprotection. diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. the cns infiltrating leukocyte composition was not altered; however, decreased il-10 and il-27 correlated with sustained disease. these data indicate that astrocytes play a critical role in limiting cns autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of ifn-γ receptors. cns resident cells are targets of autoimmune mediated damage but also active participants in disease development, progression and control [1, 2] . however, their contributions to neuroprotection and regulation by inflammatory mediators are not well defined. cns insults, including autoimmune disease, initiate rapid astrocyte activation characterized by cellular hypertrophy and increased intermediate filament glial fibrillary acidic protein (gfap) expression [3] [4] [5] . astrocytes form a physical barrier surrounding areas of inflammation initially limiting bystander tissue damage [6, 7] . however, this barrier subsequently impedes axonal regeneration contributing to sustained disability [3, 5, 8] . innate and adaptive pro-inflammatory astrocyte functions include production of pro-inflammatory cytokines, reactive oxygen species, chemokines, and matrix metalloproteinases [3] [4] [5] . by contrast, secretion of anti-inflammatory cytokines and scavengers of reactive oxygen species, as well as inhibition of both microglial activation and tumor necrosis factor (tnf) secretion, all support an astrocyte mediated anti-inflammatory function [3] [4] [5] . therefore, astrocyte activation constitutes a ubiquitous, yet heteroge-neous response associated with both promoting and inhibiting cns repair [3] [4] [5] . ms and eae are both associated with t cells secreting ifn-c (th1 cells) and il-17 (th17 cells) which play complex, not fully understood roles in disease initiation and progression [2, 9] . in vivo and in vitro evidence indicates that suppression of encephalitogenic t cell proliferation within the cns during eae and activation of anti-inflammatory programs are in part mediated via astrocytes [4, 5] . similar to astrocytes, ifn-c mediates both proand anti-inflammatory functions during autoimmune disease [10] . early ifn-c induced effects are pro-inflammatory; ifn-c facilitates inflammatory cell access, shapes their composition, increases major histocompatibility complex (mhc) expression, contributes to macrophage and microglia activation, and initiates oligodendrocyte death [11] . similarly, ifn-c mediated protection during eae is also multifaceted [12] [13] [14] [15] . it acts as a negative regulator of neutrophil accumulation, th17 cell activation, il-1r signaling, protease secretion, and chemokine activity. it also inhibits proinflammatory cytokine secretion via induction of suppressor of cytokine secretion (socs) proteins, facilitates t cell apoptosis and protects oligodendrocytes via inducing endoplasmic reticulum (er) stress responses [10, 11, 16, 17] . this highlights the critical role of a single mediator in both promoting disease but also limiting inflammatory mediated damage required to initiate repair cascades. based on the gatekeeper functions of astrocytes and the diverse biological effects of ifn-c, we set out to determine how ifn-c signaling specifically to astrocytes influences cns autoimmune disease. the results demonstrate that ifn-c signaling to astrocytes had no profound effects on initial disease progression, but played an essential protective role during the transition from acute to chronic disease. clinical remission induced by ifn-c signaling to astrocytes coincided with reduced demyelination, axonal degeneration, and astrocyte activation. the ifn-c receptor is expressed ubiquitously; however, these data reveal astrocytes as the primary target and mediator of the well established antiinflammatory activity of ifn-c within the cns. to understand the role of ifn-c signaling to astrocytes during the pathogenesis of cns autoimmune disease, eae was induced in transgenic mice expressing a signaling deficient dominant negative ifn-c receptor 1 specifically on astrocytes (gfapcr1d mice) [18] . peripheral activation of self reactive t cells in gfapcr1d mice was similar to wt mice (data not shown). this is consistent with both the cns restricted transgene expression in the gfapcr1d mice as well as the similar t cell activation following peripheral immunization with a non-self antigen [18] . following immunization the incidence of disease, initiation of clinical symptoms, and initial symptom progression were unaltered by the inability of astrocytes to respond to ifn-c ( fig. 1a ; table 1 ). in addition, neither immunized group exhibited clinical symptoms of atypical eae associated with the absence of ifn-c [19] . however, clinical symptoms in gfapcr1d mice began to diverge from wt mice at , day 12 post immunization (p.i.) prior to the peak of clinical disease. in both groups clinical disease peaked at , day 14 p.i., but severity was increased from a score of 3.1 in wt mice to 4.0 in the gfapcr1d group ( fig. 1a ; table 1 ). gfapcr1d and wt mice were compared at the peak of acute disease to determine if ifn-c signaling altered astrocyte activation or cns inflammation. astrocyte hypertrophy and gfap expression ( fig. 1b) were similar in both groups, indicating no overt effects of ifn-c on initial astrocyte reactivity. furthermore, neither the extent of inflammation nor the anatomic distribution of inflammatory cells was altered (fig. 1b) . flow cytometry confirmed no difference in the overall extent of cd45 hi inflammatory cells recruited into the cns ( fig. 2a) . furthermore, percentages of cd4 + t cells ( fig. 2a) , cd8 + t cells and macrophages within the infiltrates were also similar (fig. 3) . in contrast to the association between increased eae severity and neutrophil accumulation in the global absence of ifn-c [14, 15] , only a small percentage of neutrophils were identified in the cns of both groups by flow cytometry (fig. 3) ; their low presence was confirmed by the inability to identify cells with the characteristic morphology of neutrophils in the brain by histopathology (fig. 1b) . the absence of increased neutrophils in the cns of gfapcr1d mice during acute disease suggested that clinical disease was aggravated by a mechanism distinct from global ifn-c deficiency. in addition to the similar frequency of cd4 + t cells in the brains of the two groups ( fig. 2a) , myelin oligodendrocyte glycoprotein (mog) reactive cd4 + t cells secreting ifn-c were also similar ( fig. 2b ). by contrast, mog specific cd4 + t cells secreting il-17 ( fig. 2b ) and foxp3 + regulatory cd4 + t cells (tregs) were decreased in gfapcr1d mice compared to wt mice (fig. 2c) . reduced th17 and tregs may be attributed to increased ifn-c [10, 16] . alternatively, as ifn-c is protective in eae [12] [13] [14] [15] , increased disease severity may reflect reduced bioavailable ifn-c due to sequestration of ifn-c binding to the dominant negative receptor. however, measurement of ifn-c in cell free supernatants derived from dissociated brains at the peak of acute disease demonstrated protein levels of 2.160.2 ng/brain in wt mice versus 3.660.5 ng/brain in gfapcr1d mice (n = 3; p,0.05). as overall frequencies of mog reactive cd4 + t cells secreting ifn-c were similar, increased ifn-c in the brains of gfapcr1d mice suggested enhanced secretion at the cellular level. although a contribution of nk or cd8 t cells could not be excluded, these potential sources of ifn-c were unlikely due to their low frequencies (nk ,5% and cd8 + t cells , 5%, see fig. 3 ) and their equivalent frequencies in the wt and gfapcr1d mice. furthermore, reduced th17 cell and treg frequencies were consistent with suppression of these populations due to elevated ifn-c [10, 16, 20] . inflammation in spinal cords from the gfapcr1d mice was also similar to wt controls at the peak of clinical symptoms (fig. 4) . although numbers and distribution of cd4 + t cells were also similar (4.661.3/mm 2 in gfapcr1d mice vs. 3.860.1/ mm 2 in wt mice; fig. s1 ), spinal cords of gfapcr1d mice exhibited a ,2-fold increase in demyelination (fig. 4) . areas of demyelination encompassed 7.361.0% of spinal cord white matter in gfapcr1d mice versus 3.860.9% in wt mice (p#0.01). furthermore, the increase in demyelination was associated with a prominent loss of axons in gfapcr1d mice compared to controls (fig. 4) . despite elevated demyelination and axonal loss in the absence of ifn-c signaling to astrocytes, spinal cords showed no evidence of differential astrocyte activation by either immunohistochemistry (fig. 4 ), or differences in gfap mrna expression during the peak of acute disease (fig. 5 ). although ccl5, il-1 and tnf mrna expression were increased in the spinal cords of the gfapcr1d mice, no differences in ifn-c, inos, cxcl10 or il-6 mrna were consistent with similar inflammation (fig. 5) . these data suggest that the initial disease pathogenesis, reflected by an increased demyelination in spinal cords, but not brain, during ascending paralysis is dampened by ifn-c signaling to astrocytes. subsequent to peak disease severity the clinical symptoms in wt mice began a modest decline by day 16 p.i. (fig. 1a ). by contrast, gfapcr1d mice not only exhibited increased severity of clinical symptoms following day 14 p.i., but the continued disease escalation was associated with increased mortality ( fig. 1a ; table 1 ). sustained morbidity and significant mortality in gfapcr1d mice after day 30 p.i. implied a critical role for ifn-c induced astrocyte signaling in neuroprotection and limiting disability. a similar absence of clinical remission was found in a preliminary experiment comparing eae sjl mice carrying the gfapcr1d gene (data not shown). these data suggest that astrocyte responses to ifn-c are protective, irrespective of genetic background. escalating disease in gfapcr1d mice coincided with focal areas of intense inflammation in spinal cords, which contrasted with the more diffuse inflammation in wt mice (fig. 6 ). demyelination was also increased with myelin loss encompassing 5.761.8% of spinal cord area in gfapcr1d mice versus 2.361.4% in wt mice (p#0.05) at day 35 p.i. (fig. 6 ). the demyelinated areas further exhibited enhanced axonal damage in gfapcr1d mice (fig. 6) , supporting a correlation between enhanced tissue damage, sustained morbidity and increased mortality ( fig. 1a ; table 1 ). astrocyte activation associated with areas of myelin loss is a prominent finding in the cns of both patients with ms and rodents with eae. although demyelination was increased in the cns of gfapcr1d mice, the extent of astrocyte activation associated with spinal cord white matter lesions was similar in both groups ( fig. 6 ; ,60 gfap + cells/mm 2 ). by contrast, the frequency of activated astrocytes in spinal cord grey matter areas that were not associated with demyelinated lesions, was increased in gfapcr1d mice with 101.5623.0 gfap + activated astrocytes/mm 2 versus 25.5615.5 in wt mice (p,0.001) (fig. 7) . a similar increase in astrocyte activation within grey matter distal to white matter lesions was also detected in gfapcr1d sjl mice during chronic eae (data not shown). flow cytometric analysis during chronic disease revealed an ,4fold increase in cd45 hi inflammatory cells confirming increased inflammation in the absence of ifn-c signaling to astrocytes (fig. 8a ). similar to the acute disease, relative percentages of neutrophils, macrophages, cd4 + and cd8 + t cells were all similar (fig. 3) . furthermore, no differences in expression of activation markers on cns derived cd4 + t cells, including cd69, cd122, cd127, fas, fasl, icos and cd152 were evident between the groups (data not shown). the percentages of mog reactive th1 cells were also not altered in the cns of gfapcr1d relative to wt mice (fig. 8b ) and the decreased percentage of potentially destructive th17 cells identified during acute disease (fig 2) , was also sustained during chronic disease (fig. 8b ). equivalent expression of il-7, il-23 and tgf-b mrna (fig. 8c ) suggested that the decrease in th17 cells was dependent upon an increase in ifn-c and not related to a defect in activation or maintenance signals [20] . increased inflammation and sustained mhc class ii expression on microglia (fig. 8a ) further suggested that ifn-c signaling to astrocytes down regulates inflammation universally without altering the composition of the cns infiltrates. this concept is supported by sustained composition of cns infiltrates during inflammation induced astrocyte apoptosis [21] . the inability to restrain ongoing inflammation during eae in gfapcr1d mice was evident at multiple levels. astrocyte activation was sustained consistent with increased expression of gfap mrna. ccl2, ccl5 and cxcl10 mrna levels were increased (fig. 8c ). the expression of mrna encoding potentially destructive immune mediators including il-1, il-6, inos, and tnf were all increased (fig. 8c ). ifn-c was ,2-fold higher in the cns of gfapcr1d mice (fig. 9) . lastly, the level of the antiinflammatory cytokine il-10 was reduced ( fig. 9 ), suggesting limited availability of il-10 may be a key in prolonging astrocyte activation and inflammation [22] . a possible link between reduced il-10 and the inability of ifn-c signaling to astrocytes is provided by the capacity of ifn-c to induce il-27 in astrocytes [23] , thereby promoting il-10 production. indeed il-27 in the cns of the gfapcr1d mice was significantly reduced compared to wt mice during chronic disease (fig. 9 ). by contrast, il-12 ( fig. 9) , an indirect suppressor of cns inflammation by promoting ifn-c production [24] , was similar in the cns of both gfapcr1d and wt mice. astrocytes derived from gfapcr1d and wt mice were stimulated with ifn-c to confirm ifn-c dependent il-27 secretion by astrocytes [25] . while ifn-c induced il-27 secretion by astrocytes from wt mice, il-27 secretion was significantly reduced in cultures derived from gfapcr1d mice (fig. 10) . these data support the possibility that ifn-c mediated il-27 secretion by astrocytes regulates eae effector t cell function, inflammation and tissue destruction via induction of il-10; however, this does not exclude the possibility that the inability of astrocytes to respond to ifn-c could directly or indirectly dysregulate a variety of other immunomodulatory functions [4, 5, 17] . in the eae model of ms, ifn-c functions as a proinflammatory cytokine in the early stages of disease [10, 11, 26 ], yet it also assumes a prominent anti-inflammatory role during the transition to remission [10, [12] [13] [14] [15] . protection has been attributed to a variety of potentially interrelated mechanisms including limiting neutrophil accumulation, th17 cell activation, il-1r signaling, matrix metalloproteinase secretion, pro-inflammatory cytokine secretion and chemokine activity; in addition ifn-c facilitates t cell apoptosis and protects oligodendrocytes from death via an er stress response [10, 11, 17] . the activities of astrocytes during autoimmunity also range from pro-to antiinflammatory [3] [4] [5] . however, to what extent a direct action of ifn-c on astrocytes contributes to inhibitory mechanism is unclear. the data herein demonstrate that among the multiple early functions of astrocytes imposed by innate responses are largely pro-inflammatory during eae [3, 6, 7, 26] . for example, astrocytes contribute to the loss of bbb integrity via secretion of reactive oxygen species, chemokines and pro-inflammatory cytokines [3] [4] [5] . this pro-inflammatory milieu is associated with initial axonal damage prior to accumulation of self reactive t cells [27] , which further promotes immune mediated damage. blocking the pro-inflammatory transcription factor nf-kb in astrocytes improves recovery during chronic eae [28, 29] . supporting an initial pro-inflammatory role of astrocytes dependent on innate, not ifn-c responsiveness, inhibition of ifn-c signaling to astrocytes did not influence eae onset or incidence, initial disease progression, astrocyte activation, or bbb integrity as indicated by similar entry of inflammatory cells into the brain. by contrast, an inflammation dampening effect of ifn-c became evident during the onset of disease remission. astrocytes limit ongoing inflammation and pathogenic processes at several levels. they not only facilitate repair by inhibiting inflammatory cell entry into the cns parenchyma [6, 7, 30] , but also down regulate t cell effector function and proliferation [4, 5, 21] . for example, eae in gfap deficient mice results in more severe and widespread inflammation [31] . in addition, t cell-astrocyte interactions as well as astrocyte secretion of an unidentified soluble product, distinct from nitric oxide, prostaglandins, or tryptophan metabolism, suppress t cell proliferation [4, 5] . t cell proliferation was also not inhibited via defective il-2 release, despite the suggestion that t cell-astrocyte interactions facilitate secretion of anti-inflammatory cytokines [4, 5] . lastly, astrocytes may limit inflammation by triggering apoptosis in t cells [32] . nevertheless, the role of ifn-c signaling in these potentially anti-inflammatory, protective mechanisms is not clear. elevated ifn-c in the cns of gfapcr1d mice during both acute and chronic disease excluded limited ifn-c due to sequestration by the transgenic receptor as a mediator of increased clinical severity and mortality. indeed, enhanced ifn-c production may reflect an attempt to compensate for the loss of ifn-c dependent astrocyte mediated control of inflammation [33] . sustained expression of pro-inflammatory cytokines, particularly il-6 and tnf, thus represents a primary mechanism underlying ongoing tissue destruction in gfapcr1d mice. il-6 is known to mediate neurological dysfunction [34] and astrocytes are the predominant source of il-6 during cns autoimmune disease. furthermore, its secretion is down regulated by ifn-c induced socs activity [17] . on the other hand, sustained tnf implicated dysregulated activation of microglia or macrophages via increased ifn-c or il-6, as tnf is predominantly secreted by activated cns macrophages and microglia during eae [35, 36] . however, the down regulation of microglia activation, including tnf secretion, following interaction with activated astrocytes [37] questions this notion. while both il-6 and tnf may thus contribute to sustained pathological changes, the source of tnf remains unclear. similarly, astrocytes are a potential source of the ifn-c induced chemokine cxcl10 during eae, one of the chemokines controlling t cell recruitment [38] . however, the increased expression of cxcl10 coupled with the inability of the astrocytes in the gfapcr1d mice to respond to ifn-c suggests altered microglia activation and secretion of cxcl10 [38] or activation of cxcl10 transcription via an independent signaling pathway mediated by tnf or type 1 interferons [39, 40] . importantly, pathology further correlated with decreases in both tregs and il-10, but not with increased antigen specific th17 cells, although astrocytes are critical targets of il-17 [41] . although it is possible that the increased ifn-c in the cns influenced peripheral activation of antigen specific th17 cells, previous data demonstrated no evidence for expression of the transgene in peripheral organs, including lymphoid organs [18] . il-10, secreted by a variety of cells types including cd4 + t cells, cd8 + t cells and tregs [42] , inhibits both acute and chronic eae [43, 44] . the decrease in this anti-inflammatory cytokine suggests that the induction of il-27 secretion is a critical aspect of astrocyte responses to ifn-c, thus promoting il-10 secretion by t cells [23, 25] . however, our data is unable to distinguish the contribution of paracrine versus autocrine ifn-c induced signals to astrocytes on il-10 production, as il-10 also regulates astrocyte activation [22] , and can itself be secreted by astrocytes to exert autologous functions [45] . in addition to mediating an imbalance of protective versus detrimental cytokines, our results demonstrate that ifn-c signaling to astrocytes limits the extent of astrocyte activation during chronic eae, specifically in grey matter. activated astrocytes, especially within and adjacent to areas of demyelination are a prominent feature of white matter plaques associated with both chronic ms and eae [3] [4] [5] . astrocytes protect neurons and oligodendroglia, but also form glial scars which inhibit regeneration after neuronal injury [3, 5, 8, 20] . the absence of reactive astrocytes increases axonal regeneration after injury [46] , consistent with the concept that limiting astrogliosis is critical for axonal regeneration after neuronal injury. while the significance of sustained astrocyte activation in grey matter tracks is unclear in our model, it is consistent with increased axonal damage, and suggests possible damage to axons outside the lesions, which may not be manifested by histological analysis. limiting inflammation following either infection or during an autoimmune attack is a prerequisite for the initiation of repair. this is critically important within the cns which has limited regenerative capacity. in summary, our data identify astrocytes as prominent targets underlying ifn-c mediated suppression of chronic cns inflammation [19] . the data further provide a link between sustained inflammatory responses, enhanced demyelination and axonal degeneration associated with loss of neurological function during eae and chronic progressive ms. although the inability of astrocytes to respond to ifn-c did not alter disease in the brain, engagement of the ifn-c receptor on astrocytes in spinal cord limits demyelination and functions in a neuroprotective capacity. current therapies for ms are primarily focused on antiinflammatory and immunomodulatory approaches and have been partially successful in treating acute episodes. the identification of astrocytes as critical responders and mediators of ifn-c signaling in limiting cns autoimmune disease may provide insights into new approaches to limit long term progression to disability. brains and spinal cords from perfused mice were homogenized separately as previously described [47, 48] . homogenates were centrifuged at 4506g for 7 min at 4uc. supernatants were stored at -80uc for cytokine determination (see below). cells were resuspended in 30% percoll (amersham biosciences, piscataway, nj) and isolated by centrifugation (8006g for 30 min at 4uc) onto 70% percoll cushions. non-specific binding was inhibited by incubation with anti-cd16/cd32 (2.4g2; bd biosciences, san diego, ca) and a 10% mixture of normal goat, human, mouse and rat serum for 10 min on ice. fitc, pe, percp, and apc conjugates with monoclonal antibodies (mab) used to identify and quantify microglia and inflammatory cells were: cd4 (gk1.5), cd8a (53-6.7), cd45 (30-f11), mhc class ii (2g9), ly6g (1a8) (bd biosciences), and f4/80 (serotec, raleigh, nc). microglia and inflammatory cells were distinguished based on differential cd45 expression. cd4 + t cells were identified as cd45 hi cd4 + , cd8 + t cells as cd45 hi cd8 + , macrophages as cd45 hi f4/80 + and neutrophils as cd45 hi ly6g + mhc class ii -. mog specific induction of cytokines was determined by stimulation of 1610 6 cns cells with 20 mg/ml peptide for 6 h at 37uc with golgistop (bd biosciences) added for the last 4 h. intracellular cytokines were detected with fitc-anti-ifn-c (clone xmg1.2; bd biosciences) and pe-anti-il-17 (clone tc11-18h10; bd biosciences). intracellular foxp3 was detected by staining for cell surface markers, followed by permeabilization with fixation/ permeabilization reagent (ebioscience, san diego, ca) and incubation with pe-labeled anti-foxp3 (fjk-16s; ebioscience). cells were analyzed on a facscalibur flow cytometer (bd biosciences) using cellquest pro software (bd biosciences). data was analyzed using flowjo (7.6.1) software (tree star inc., ashland, or). cytokines were determined by elisa using antibody pairs and recombinant cytokine standards from bd bioscience. il-27 was measured using quantikine mouse il-27p28 immunoassays (r&d systems inc., minneapolis, mn). following anesthesia, mice were perfused with pbs (ph 7.2). brains and spinal cords were fixed with clark's solution (75% ethanol and 25% glacial acetic acid), and embedded in paraffin. spinal cords were divided into 6 sections prior to embedding, corresponding to cervical, thoracic and lumbar levels. cross sections (6 mm), were stained with either hematoxylin and eosin (h&e) or luxol fast blue (lfb). immunoperoxidase staining was used to identify activated astrocytes with anti-gfap (abcam, cambridge, ma) and axonal integrity with smi-31 and smi-32 mab (sternberger monoclonals inc., lutherville, md) followed by visualization using vectastain abc kit (vector laboratories, burlingame, ca) and 3,39-diaminobenzidine (sigma-aldrich). sections from at least 3 separate experiments containing at least 3 mice per group were reviewed in a blinded manner. numbers of gfap + cells in spinal cord were determined in 15 non-overlapping 406 fields (0.2 mm 2 ) in white matter and gray matter. stained spinal cord sections of all 6 levels on individual glass slides were scanned (406) and digitally imaged at high resolution with an aperio scanscope (vista, ca). aperio software was used to quantify areas of demyelination within the white matter tracks of each of the 6 sections per individual mouse. for analysis of cd4 + t cells spinal cords were embedded in tissue-tek o.c.t. (andwin scientific, tryon, nc), flash frozen in liquid nitrogen and stored at 280uc. blocks were warmed to 220uc prior to cutting 10 mm sections by cryostat. following fixation with acetone for 2 min at 4uc, non-specific antibody binding was blocked with cyto q background buster (innovex biosciences, richmond, ca) for 15 min. sections were stained with anti-cd4 (l3t4) antibody (vector laboratories) diluted in cyto q immuno diluent (innovex biosciences) followed by biotinylated rabbit anti-rat, peroxidase abc reagent and visualized with novared substrate (all from vector laboratories). sections were counter stained with hematoxylin to visualize the nuclei. spinal cord sections were scanned (406) and digitally imaged at high resolution with an aperio scanscope. mixed glial cultures (,70% astrocytes) were established from neonatal gfapcr1d and wt mice as previously described [47] . il-27 secretion was determined 48 h after rifn-c (100 ng/ml) stimulation. frozen tissues were homogenized in trizol (invitrogen, carlsbad, ca) and cdna prepared as described [47, 48] . quantitative real-time pcr was performed using 4 ml of cdna and sybr green master mix (applied biosystems, foster city, ca) in duplicate on a 7500 fast real-time pcr system (applied biosystems). expression levels were normalized to ubiquitin or gapdh using the following formula: statistical significance was determined by two-tailed student's t test. a value of p,0.05 was considered statistically significant. figure s1 cd4 + t cell recruitment into the spinal cord during acute eae. cd4 + t cells were visualized in 10 mm frozen sections of spinal cords from wt and gfapcr1d tg mice at day 18 p.i. immunoperoxidase stain (novared chromogen with hematoxylin counterstain). scale bars = 50 microns. (tif) multiple sclerosis: a complicated picture of autoimmunity the immunology of multiple sclerosis astrocytes: biology and pathology astrocytes in the tempest of multiple sclerosis astrocytes-friends or foes in multiple sclerosis leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice reactive astrocytes form scar-like perivascular barriers to leukocytes during adaptive immune inflammation of the cns regeneration beyond the glial scar autoimmune t cell responses in the central nervous system how interferon-gamma keeps autoimmune diseases in check the integrated stress response prevents demyelination by protecting oligodendrocytes against immune-mediated damage intrathecal delivery of ifn-gamma protects c57bl/6 mice from chronicprogressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes interferon-gamma confers resistance to 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early times after repeated local cytokine treatments astrocyterestricted ablation of interleukin-17-induced act1-mediated signaling ameliorates autoimmune encephalomyelitis regulation and functions of the il-10 family of cytokines in inflammation and disease il-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of il-10-and il-4-deficient and transgenic mice central nervous system expression of il-10 inhibits autoimmune encephalomyelitis multiple sclerosis: cytokine receptors on oligodendrocytes predict innate regulation axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination distinct regulation of mhc molecule expression on astrocytes and microglia during viral encephalomyelitis we thank bruce trapp for helpful discussions, wenqiang wei, eric barron and ernesto barron for assistance with the histopathology and jeffrey tarcy, kate stenson and maria ramirez for assistance with mouse husbandry and immunological assays. key: cord-277679-sc9hugxr authors: khateb, mohamed; bosak, noam; muqary, maryam title: coronaviruses and central nervous system manifestations date: 2020-06-23 journal: front neurol doi: 10.3389/fneur.2020.00715 sha: doc_id: 277679 cord_uid: sc9hugxr sars-cov-2 is a highly pathogenic coronavirus that has caused an ongoing worldwide pandemic. emerging in wuhan, china in december 2019, the virus has spread rapidly around the world. corona virus disease 2019 (covid-19), which is caused by sars-cov-2, has resulted in significant morbidity and mortality. the most prominent symptoms of sars-cov-2 infection are respiratory. however, accumulating evidence highlights involvement of the central nervous system (cns). this includes headache, anosmia, meningoencephalitis, acute ischemic stroke, and several presumably post/para-infectious syndromes and altered mental status not explained by respiratory etiologies. interestingly, previous studies in animal models emphasized the neurotropism of coronaviruses; thus, these cns manifestations of covid-19 are not surprising. this minireview scans the literature regarding the involvement of the cns in coronavirus infections in general, and in regard to the recent sars-cov-2, specifically. coronaviruses constitute a group of single-stranded rna viruses that cause respiratory, hepatic, enteric, and neurological illness. the severity of symptoms is highly variable, and the range of animal species affected is broad, including humans (1) (2) (3) . in general, coronaviruses are classified into four major genera: alphacoronavirus, betacoronavirus (βcov), gammacoronavirus, and deltacoronavirus (3, 4) . over the last two decades, two novel βcov strains caused severe human illness (5, 6) : the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). both strains were associated with high morbidity and mortality rates, and the lack of successful treatments. in december 2019, the novel severe acute respiratory syndrome coronavirus (sars-cov-2) emerged in wuhan, china. corona virus disease 2019 (covid-19) has since become a worldwide pandemic, with high rates of mortality (7) (8) (9) . the sequence, pathogenesis and cellular entry mechanisms of the sars-cov-2 are similar to those of the sars-cov (10) (11) (12) . the major clinical manifestations of sars-cov-2 infection are respiratory due to pulmonary complications (13) (14) (15) (16) (17) . symptoms can be mild, including fever, headache, cough, dyspnea and myalgia; or severe, such as acute respiratory distress syndrome (ards), which sometimes develops about 1 week into the illness and may result in death (7-9, 13-17). central nervous system (cns) complications of covid-19 infection have not been systematically investigated or analyzed. anosmia several cross sectional studies (35, 36) the neurotropism potential of coronaviruses was demonstrated in previous studies (10, 18) . for example, sars-cov is believed to enter the brain primarily via the olfactory bulb, resulting in rapid infection with transneuronal spread and minimal cellular infiltration (19) . this may cause neural dysfunction especially at the cardiorespiratory centers in the medulla, as was demonstrated in mice transgenic for human ace2 (20) . considering the above, we screened the available literature regarding possible neurological manifestations and complications of coronaviruses in general, and of the recent sars-cov-2 specifically. table 1 summarizes human manifestations of sars-cov-2 and other coronaviruses in the cns. below we describe the evidence of cns disorders that have been linked with coronaviruses. multiple sclerosis (ms) is a chronic inflammatory demyelinating disease of the cns. the immune reactions involved in ms are attributed to both genetic and environmental factors (37, 38) . a possible link between coronaviruses and demyelinating disorders, including ms, has been reported (39) (40) (41) . coronavirus strains were suggested to initiate immunopathogeneic events leading to cns demyelination. some strains were even proposed as an experimental model for ms to further study the mechanisms of virus-induced demyelination (42) (43) (44) . in animals, the murine coronavirus jhm was demonstrated to cause demyelination (45) . the ability of coronaviruses to produce demyelination in experimentally infected mice has led to studies that investigate the possibility of human coronaviruses in ms brain tissues. interestingly, nucleic acids of the human coronavirus 229e strain were detected in the cns tissue of four of eleven ms patients, indicating neurotropism of the 229e strain (46) . moreover, coronavirus-like particles under electron microscopy were found in autopsies from two ms patients (47) . murray et al. (48) demonstrated that direct intracerebral administration of coronaviruses might cause demyelinating disease in primates. using in situ hybridization, they detected coronavirus rna sequences in the brain and in demyelinating plaques in 12 of 22 ms patients (49) . in contrast, other studies that used polymerase-chain-reaction (pcr) with specific primers for the two human coronaviruses 229e and oc43 did not show any difference between the ms and the control groups in coronaviral rna detection in brain tissues (41, 50) . moreover, antigenic assessment by comparing levels of coronavirus antibodies failed to support the claim of coronaviruses as an etiology for ms. this is because no significant differences were found between samples of ms patients and control subjects (51) . multiple mechanisms were proposed to explain demyelination by coronaviruses in animal models. jhm virus-induced demyelination was largely correlated with cytopathogenic properties of the virus for oligodendrocytes (52) . the molecular basis of this process was linked to the e2 sub-region (45) . additional mechanisms subsequently emerged. one of them involves molecular mimicry between coronaviruses and myelin as a basis for the autoimmune reaction leading to crossreactivity of t-cells (53, 54) . furthermore, rna recombination demonstrated that the s gene of the coronavirus mouse hepatitis virus (mhv) is related to certain molecular aspects of demyelination. this indicates the potential role of viral envelope s glycoproteins in autoimmune-induced demyelination (43) . mutations in the spike glycoprotein of human coronavirus oc43 (hcov-oc43) modulated the disease from chronic encephalitis to flaccid paralysis and demyelination (55) . other studies emphasized the importance of t cells, especially cd8, in the process of demyelination in mice infected with coronaviruses (56, 57) . accordingly, gamma-interferon was shown to lead to demyelination mediated by cd8 cells. nevertheless, kim et al., showed that the specific antibodies against the coronavirus jhm in animal models were sufficient to induce demyelination in the absence of t cells. they also showed that the destruction of myelin in these cases resulted from the complement system, as well as from fc receptor-dependent mechanisms (58, 59) . additional mechanisms related to chemokines like cxcl10 and chemokine receptor ccr5 were also described (60, 61) . coronaviruses have been associated with other demyelinating pathologies like acute or subacute disseminated encephalomyelitis (adem) in humans (31, 62) . adem is a rare acute inflammatory demyelinating disease that may follow viral infections. intracerebral inoculation of the human coronavirus hcov-oc43 into balb/c mice caused acute encephalitis with cellular death by necrosis and apoptosis (63) . several months of follow-up of viral rna led to the conclusion that viral persistence could be associated with increased neuronal degeneration, resulting in neuropathology and motor deficits. in line with these experimental findings, in a child who presented with adem, cerebrospinal fluid (csf) tested positive for hcov-oc43, using pcr (31) . several patients infected with mers-cov were reported to demonstrate a severe neurological syndrome; one of them met the criteria for adem (30) . another report described a patient who was diagnosed with the presumably post/para-infectious syndrome of bickerstaff 's brainstem encephalitis (34) . recently, a link between sars-cov-2 infection and adem was described in one woman (29) . acute monosymptomatic optic neuritis (amon) is sometimes an initial symptom of ms; the implication of coronaviruses in the etiology of ms is described above. however, a study that compared the presence of coronavirus rna in the csf of individuals with amon and controls revealed no significant differences (64) . encephalitis is a serious and potentially fatal neurologic illness characterized by cns inflammation. the etiologies of the majority of those affected are viral, yet identifying the specific causal organism can be difficult. morfopolu et al., described an 11-month-old boy with severe combined immunodeficiency who had symptoms of acute encephalitis. the conventional diagnostic pcr assay was negative. eventually, using three independent methods, the hcov-oc43 in the child's brain tissue was identified (22) . clinical presentation suggestive of encephalitis was also reported in a 59-year-old woman infected with sars-cov (23) . the patient developed status epilepticus, which followed a few hours of disorientation during hospitalization in the intensive care unit (icu) due to respiratory symptoms. ct scan of the brain did not detect any abnormality. csf did not contain any cells but was positive for sars-cov using real-time pcr analysis. one of the early symptoms of covid-19 infection is headache. the first documented patient with meningoencephalitis as a result of sars-cov-2 infection had neck stiffness and transient generalized seizures (21) . sars-cov-2 rna was not detected in the nasopharyngeal swab but was detected in his csf. parkinson's disease is a common neurodegenerative disease that primarily affects the basal ganglia system. disruption of motor functions is one of its main clinical manifestations. csf seropositivity for several strains of coronaviruses has been reported in parkinson's disease (65) . however, this correlation was not confirmed or replicated over nearly three decades. acute flaccid paralysis (afp) is a life-threatening syndrome characterized by dramatic weakness in muscles, often respiratory and bulbar muscles, as a result of damage to lower motor neurons. when secondary to peripheral nerve involvement, the syndrome is usually a manifestation of gullian-barre syndrome. the latter was reported to be related to mers-cov (34) , and also to sars-cov-2 (66), but is out of the scope of the current review of cns involvements. in other cases of afp, the paralysis is due to damage at the level of the anterior horn of the spinal cord, and termed acute flaccid myelitis. in such cases, other etiologies, especially various infections, were described (67) . a similar clinical picture can be secondary to acute myelopathy, most commonly the result of inflammatory diseases of the spinal cord, either as part of adem presentation or isolated acute transverse myelitis. turgay et al., described a 3-year-old girl who was admitted with lower respiratory tract infection and afp, in the presence of normal nerve conduction studies and electroneuromyography (ncs-emg) and csf content. magnetic resonance imaging (mri) was unavailable in the managing center. co-infection with human coronaviruses 229e and oc43 was detected by realtime pcr analysis of nasal swab samples (33) . interestingly, the girl showed clinical improvement after treatment with ivig. similarly, a 66-year-old man who presented with afp and urinary and bowel incontinence, which developed about 1 week after fever and subsequent respiratory symptoms, was eventually diagnosed with covid-19 (32) . no csf, ncs-emg, or spinal mri were obtained due to pandemic-related reasons, but the authors emphasized that the sensory clinical features implied spinal involvement. following treatment that combined antibiotic agents, anti-viral drugs, dexamethasone and ivig, the patient exhibited clinical improvement within a rather short follow-up period. loss of appetite has been reported in many patients with covid-19; and anosmia (loss of the sense of smell) is recognized as a common and early symptom (10, 18, 35) . as previously mentioned, sars-cov-2 has neuro-invasive propensity, which has been demonstrated as a common feature of coronaviruses. this supports the possibility that the virus infects the olfactory bulb in the early stages, and progresses from there to various brain regions in later stages. the exact route by which sars-cov-2 enters the cns is still poorly understood. coronaviruses have been reported to first invade peripheral nerve terminals, eventually reaching the cns via a trans-synaptic transfer (18, 68) . viral antigens have been detected in brainstem nuclei such as the nucleus of the solitary tract and nucleus ambiguus (8, 10, 69) . this indicates possible dysfunction of the cardiorespiratory center in the brainstem as a complication of coronaviruses infection. disruption of the function of cns respiratory centers in the brainstem is believed to exacerbate respiratory failure and impair the state of consciousness of individuals infected by coronaviruses. ane is a rare complication of viral infections like influenza, which may be caused by intracranial cytokine storms that break down the blood-brain-barrier (70) . evidence from the covid-19 pandemic suggests that for a subgroup of patients with severe presentation, this condition might be complicated with cytokine storm syndrome (71) . radiological diagnosis of ane was reported in a woman with covid-19 who presented with altered mental status (24) . accumulating evidence implies a possible link between infection with the novel sars-cov-2 and acute ischemic stroke (ais). in general, infectious diseases of the respiratory system are a possible and independent risk factor for plaque rupture that results in ais (72, 73) . the cytokine storm caused by coronavirus infection increases the risk of thrombosis in general, and of ais specifically (9, 71, 74) . moreover, changes in platelet count and d-dimer were reported in cov-sars2 infection (9) . an italian study showed an increase in the ratio of arterial as well as venous thromboembolic events in patients with covid-19 (75) . of 388 patients in that study, ais presented in nine (2.5%). interestingly, the reason for hospitalization in six of the nine patients was the neurological presentation of ais. an observational study of 184 patients hospitalized in the icu with covid-19 demonstrated that five of the surviving 78 had ais (25) . similarly, a case series was published of five sars-cov-2 patients presenting with acute ais secondary to large vessel occlusion (lvo) (26) . all five patients were younger than 50 years old, two of them were without risk factors for stroke. in another case series of covid-19 patients with ais during their hospitalization (27) , all six had risk factors for stroke, and were detected with lvo. interestingly, three of the six patients demonstrated cerebral ischemia in vascular multiple territories. notably, five of these patients had been positive to lupus anti-coagulant. this supports previous studies that suggested that infection with sars-cov-2 triggered production of antiphospholipid antibodies (76) . in a case series of four patients with covid-19 who presented with ais, two of them had lvo of the middle cerebral artery (28) . taken together, the evidence suggests that infection with coronaviruses might increase the risk of ais. additional neurological signs, reported in patients with severe sars-cov-2 with ards include myalgia (29, 77) , diffuse corticospinal tract signs with enhanced tendon reflexes and ankle clonus, and dysexecutive syndrome (78) . interestingly, in the study that described the latter manifestations, imaging findings included perfusion abnormalities in all the patients, leptomeningial enhancement in most of them, and cerebral ischemia in about 23% (78) . however, these results should be interpreted cautiously, as they may represent, at least in part, different effects of the critical illness state, regardless of the sars-cov-2 etiology. in general, the evidence available on the direct relations between respiratory neurotropic viruses and psychiatric disorders is relatively sparse. specifically, a positive association was described between infection with coronaviruses (nl63 strain), as reflected by seropositivity, and mood disorders (79) . an additional serological study demonstrated a rise in the level of antibodies against the same strain of coronaviruses in individuals with recent onset of psychosis (80) . early reports from the ongoing covid-19 pandemic suggest psychiatric morbidities that have some resemblance to those reported in the early phase of the sars outbreak. patients are reported to experience fear, loneliness, anger and general distress, which have been primarily attributed to the need for quarantine; and infection symptoms such as fever and cough (81) . moreover, adverse effects of treatments such as insomnia from corticosteroids, and psychotic disorder from chloroquine, might exacerbate patients' mental status (82, 83) . additional reasons for psychiatric morbidities include experiencing adverse effects of treatment during hospitalization, uncertainty regarding prognosis and undergoing icu care. therefore, infection with of sars-cov-2 is considered a traumatic experience; this enhances anxiety and substantial mental distress. in the immediate aftermath of the sars epidemic, various psychiatric morbidities were reported. these included depression, adjustment reactions, anxiety, agitation, psychotic symptoms, delirium, and even higher suicidality (82, 84, 85) . in the early recovery phase, up to 35% of sars survivors showed signs of anxiety, depression or both (84, 86) . importantly, post-traumatic stress reactions were identified in sars survivors in the early, 2-4 week period, following discharge. furthermore, about 45% of the respondents in that outbreak were diagnosed with at least one psychiatric disorder during the study period. psychiatric disorders such as major depression, post-traumatic stress disorder (ptsd) and adjustment disorder were described during the 6 months after patients' discharge (84, 85) . cumulative incidence of any psychiatric disorder during the 3 years following the sars outbreak was reported to reach 59% in sars survivors (86) . for example, the cumulative incidence rates for ptsd and depressive disorders were about 48 and 44%, respectively (86) . similarly, the prevalence of psychiatric morbidity at 30 months after sars infection was reported to reach up to one-third of the total survivors who presented with signs of various psychiatric diagnoses. at that timepoint, about 26 and 16% had ptsd and depressive disorders, respectively (86) . the above highlights the urgency of addressing mental health care needs related to the current pandemic of covid-19, particularly for those with comorbid mental disorders. lessons learned during the sars outbreak should be applied to designing strategies in the novel covid-19 outbreak (87). accumulating evidence from the current sars-cov-2 pandemic, together with literature on other coronaviruses, suggest that infection with coronaviruses may be related to cns manifestations or complications, including anosmia, acute ischemic strokes, viral meningoenchephalitis, acute necrotizing encephalopathy, acute flaccid paralysis, and other presumably post/para-infectious syndromes. in addition, patients with covid-19 may be particularly prone to mental disorders including ptsd, major depression and adjustment disorder. therefore, we believe these patients should be carefully monitored for clinical signs suspecting cns involvement such as headache, seizures, sensory or motor deficits, and altered mental state. early diagnosis of cns manifestations in individuals with covid-19 may be crucial for their overall prognosis. a multidisciplinary approach should be emphasized by health authorities, which would provide urgent mental health support for the affected, and also for health care workers. mk initiated the idea, screened the available literature about neurological manifestations in coronavirus infections, created and designed the manuscript. mm created the section on psychiatric sequels of coronavirus infections and contributed to the final revisions. nb contributed to the overall manuscript design. all authors contributed to the article and approved the submitted version. coronavirusesdrug discovery and 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financial relationships that could be construed as a potential conflict of interest.copyright © 2020 khateb, bosak and muqary. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-278684-txlvla0j authors: gonzalez–dunia, daniel; sauder, christian; de la torre, juan carlos title: borna disease virus and the brain date: 1998-01-30 journal: brain res bull doi: 10.1016/s0361-9230(97)00276-1 sha: doc_id: 278684 cord_uid: txlvla0j viruses with the ability to establish persistent infection in the central nervous system (cns) can induce progressive neurologic disorders associated with diverse pathological manifestations. clinical, epidemiological, and virological evidence supports the hypothesis that viruses contribute to human mental diseases whose etiology remains elusive. therefore, the investigation of the mechanisms whereby viruses persist in the cns and disturb normal brain function represents an area of research relevant to clinical and basic neurosciences. borna disease virus (bdv) causes cns disease in several vertebrate species characterized by behavioral abnormalities. based on its unique features, bdv represents the prototype of a new virus family. bdv provides an important model for the investigation of the mechanisms and consequences of viral persistence in the cns. the bdv paradigm is amenable to study virus–cell interactions in the cns that can lead to neurodevelopmental abnormalities, immune-mediated damage, as well as alterations in cell differentiated functions that affect brain homeostasis. moreover, seroepidemiological data and recent molecular studies indicate that bdv is associated with certain neuropsychiatric diseases. the potential role of bdv and of other yet to be uncovered bdv-related viruses in human mental health provides additional impetus for the investigation of this novel neurotropic infectious agent. to establish a persistent infection in its host, a virus has to meet two essential requirements. first, it must avoid clearance by the host immune surveillance. second, it must adopt a noncytolytic strategy of multiplication. to achieve these goals, viruses have developed a plethora of strategies [1, 52] . the central nervous system (cns) has intrinsic properties that favor the establishment of viral persistence [116] , namely: (a) the existence of the blood-brain barrier and the paucity of immune elements in the brain, that provide the cns with an immunologically isolated environment; (b) viral replication is often restricted in cells of neuroectodermal origin, thus facilitating persistence; (c) viral spread over long distances within the cns is facilitated by the intricate networks of cell processes and cell interactions. evidence provided by epidemiological and clinical data, together with virological studies, have led to the hypothesis that chronic viral infections of the cns contribute to human mental disorders of unknown etiology. it is therefore of interest to identify relevant infectious agents, as well as to understand the mechanisms by which viruses persist in the cns and interfere with brain functions. borna disease virus causes cns disease in a broad range of vertebrate animal species that is manifested by behavioral abnormalities and diverse pathology. bdv provides an important paradigm for the investigation of the mechanisms and consequences of viral persistence in the cns. studies on this viral system will contribute to the elucidation of immune-mediated pathological events involved in virally induced neurological disease, as well as the mechanisms whereby viruses induce neurobehavioral disturbances in the absence of the hallmarks of cytolysis and inflammation. moreover, serological data and recent molecular epidemiological studies indicate that bdv can infect humans, and is possibly associated with certain neuropsychiatric disorders. based on its unique biological and genetic characteristics, bdv represents the prototype of a new family of viruses. consequently, investigations on bdv may provide new insights into virus-cell interactions in the cns. the prospect of finding other bdv-like viruses, some of them possibly clinically relevant, provides further impetus for the study of this novel neurotropic infectious agent. the purpose of this review is to discuss our present knowledge of bdv persistence in the cns. we will first briefly describe the biology of bdv, with special focus on the recent developments regarding its molecular biology. we will then examine the interaction between bdv and the cns. finally, we will discuss the evidence supporting a possible role of bdv in certain human mental disorders. of borna, in saxony, where many horses from a cavalry regiment died during an epidemic in 1895. early studies showed that bd could be transmitted by using brain homogenates from diseased horses, demonstrating the infectious nature of the disease [101, 125, 155, 188] . sensitivity to detergents, ultraviolet light, and organic solvents, together with filtration studies suggested that bd was caused by an enveloped virus, the borna disease virus. besides horses and sheep, infections with bdv have been documented in cattle, cats, donkeys, rabbits, and ostriches [23, 35, 128, 130, 172] . sporadic cases of bd have also been reported in several other animal species [172] . moreover, experimental infections have revealed the remarkable wide host range of bdv, which includes birds, rabbits, and rodents, as well as nonhuman primates [5, 77, 90, 105, 124, 126, 150, 172, 177, 203, 205] . bdv infection leads, after a variable incubation period, to diverse pathological manifestations that depend on the species, immune status and age of the host, route of infection, and the particular strain of virus used for infection. the adaptation of bdv to small laboratory animals, such as the rabbit and the rat, has facilitated more detailed studies on bdv pathogenesis and the neurobehavioral alterations accompanying the infection. filtration experiments estimated that bdv particles had a size of 80 to 125 nm [155] . recent electron microscopy studies have shown that preparations of cell-free infectious bdv contain enveloped particles of roughly spherical morphology, approximately 100 nm in diameter [74, 230] . intracytoplasmic virus-like particles with similar characteristics have also been observed in thin sections of bdv-infected cells [42] . all bdv isolates are characterized by being highly neurotropic and by having a noncytolytic strategy of replication [87] . cells and tissues of nonneural origin exhibit only a low susceptibility to infection. the paucity of cell-free virus associated with bdv infection has long hampered its molecular characterization. the establishment of persistently infected cell lines and the use of molecular genetic approaches has enabled the recent progress in the molecular characterization of this infectious agent. these studies have demonstrated that bdv is a nonsegmented, negativestranded (nns) rna virus [43, 50, 123, 216] . the cloning and complete sequencing of two bdv isolates has uncovered the genomic organization of bdv [31, 44, 48, 183] . the genome is about 8.9 kb long, with complementary 3ј and 5ј untranslated regions at its termini (fig. 1) . the antigenomic polarity contains information for at least five open reading frames (orf). similarly to other members of the mononegavirales, the genome can be divided in three main gene blocks: block 1 codes for the nucleoprotein and polymerase cofactors, represented by the bvp40 (orf i) and bvp24 (orf ii) proteins of bdv; block 2 codes for the matrix and virus envelope proteins, whose likely counterparts in bdv are the bvp16 (orf iii) and bvp56 (orf iv) proteins, respectively; and block 3 codes for the viral polymerase, identified as orf v in the bdv genome. the molecular biology of bdv has been the subject of recent reviews [30, 48, 183] and is not the focus of this review. we will only briefly describe here the main features that distinguish the replication and gene expression regulation of the bdv genome. the bdv nucleoprotein (np) bvp40 is present at high levels in infected cells and tissues. this protein is likely encoded in two isoforms of 38 and 40 kda [81, 94, 165] . this may be related to the presence of two in-frame initiation codons in the bvp40 gene sequence. the bvp24 protein is acidic, with a high ser/thr content and is phosphorylated at serine residues [94, 211, 212] . these features are consistent with the phosphoprotein (p) transcriptional activator found in other nns rna viruses. the transcription unit encoding bvp24 can also direct the synthesis of a polypeptide of 10 kda (bvp10). recent data from our laboratory indicate that bvp10 is present in infected cells. the orf encoding bvp10 starts 46 nucleotides upstream from bvp24 and overlaps, in a different frame, with the 213 first nucleotides of orf ii (fig. 1) . the function of bvp10 is presently unknown. a similar situation has been described for the p gene of sendai virus [46] and vesicular stomatitis virus [202] . bdv orf iii (bvp16) likely represents the bdv matrix (m) protein. in contrast to other nns rna viruses, bdv m protein is glycosylated and data suggest that it might be present at the surface of the virion envelope [111] . orf iv is predicted to encode for a polypeptide of 56 kda (bvp56). sequence features suggest that this protein is a viral surface glycoprotein (gp). recent reports have provided experimental evidence that bvp56 is involved in virus entry [74, 185] . bvp56 is present as two forms in bdv-infected cells [74] . one form of approximately 84 kda (gp-84) corresponds to the full-length product encoded by orf iv and accumulates in the endoplasmic reticulum. the molecular weight of this polypeptide, higher than 56 kda, is due to glycosylation. a shorter product of 43 kda (gp-43) corresponds to the c-terminus of gp-84 and is generated via cleavage by the cellular protease furin [73] . moreover, gp-43 is present at the surface of infected cells. both gp-84 and gp-43 are associated with infectious virions. these features indicate a novel maturation pathway for a nns rna virus surface gp and, hence, for the assembly of bdv particles [74] . orf v is capable of encoding a polypeptide with a predicted molecular mass of 180 kda, whose deduced amino acid sequence displays strong homology with the nns rna viral polymerases (l protein family) [44] . this homology is particularly high in the case of the conserved putative catalytic domain. bdv has the property, unique among known animal nns rna viruses, of a nuclear site for the replication and transcription of its genome [43] . consistent with this finding, bdv ribonucleoproteins (rnp) are found in the nucleus of persistently infected cells [43] . as with other nns rna viruses, bdv rnp are infectious upon transfection of susceptible cells [43] . bdv exhibits a complex transcriptional pattern in infected cells. subgenomic messenger rnas (mrnas) encoding bvp40 and bvp24 are monocistronic. in contrast, mrnas encoding the m, gp, and l proteins are polycistronic. in the other nns rna viruses, these polypeptides are usually encoded by monocistronic mrnas. in addition, bdv does not exhibit the configuration of transcription termination signal, intergenic region, and transcription initiation signal that is characteristically present at the gene boundaries of nns rna viruses [213] . bdv transcription units and transcriptive signals frequently overlap (fig. 1) . moreover, bdv utilizes the host splicing machinery to generate some of its mrnas [45, 186] . genomic stability is another striking characteristic of bdv. the two bdv complete genomic sequences yet determined have more than 95% homology at the nucleotide level [31, 44] . this is remarkable for rna virus isolates with different origin and passage history in animals and cultured cells. these sequences are also very similar to those obtained from field cases of bd in different animal species [21, 184] , as well as to sequences derived from human cases of bdv infection [29, 49, 108, 180] . this extraordinary sequence conservation, uncommon for an animal nns rna virus, may indicate that there is a prevalent and stable species of bdv in nature, with the ability to infect several animal species, including humans. the reasons for the great stability of the bdv genome are unknown. interestingly, sequence analysis reveals that the l proteins of bdv and sonchus yellow net virus, a plant nucleorhabdovirus that also replicates in the nucleus, are the most distantly related to the l proteins from animal rhabdoviruses, raising intriguing questions about the evolutionary origins of bdv [44] . gonzalez-dunia, sauder and de la torre bdv is unique not only for its extraordinary wide host range, combined with a remarkable genomic stability, but also in various aspects of its biology. expression of the bdv compact genome is regulated by an overlap of transcription units and transcriptive signals, an overlap of orf, a readthrough of transcription termination signals and rna splicing. the concurrent use of such a diversity of strategies for the regulation of gene expression is unique among known nns rna viruses. taken altogether, this has led to the proposal that bdv represents the prototype of a new taxon of nns rna viruses [48, 183] . the portal of entry for bdv infection has not formally been identified. however, field observations have led to the hypothesis that a primary route of infection might be through the nose and the olfactory neuroepithelium [172] . animals may become infected by contact with saliva, excretions, nasal secretions, or by exposure to contaminated food or water. contact experiments have suggested that healthy carrier animals might represent a source of infection, because the disease can be transmitted between horses and sheep living in the same stable [172] . oral infection has been achieved in several animal species, suggesting that the virus could also be transmitted by the oral/gastric route [83, 130] . finally, although formal evidence is lacking, colostrum and milk could play a role in the infection of the newborn [172] . experimental infection of the rat has allowed the dissection of the dissemination pathway of bdv [36, 140] . after initial replication in the neurons located at the site of entry, bdv likely migrates intraaxonally in an anterograde or retrograde direction towards the cns. the pathway of dissemination of bdv following intranasal inoculation of the rat has been thoroughly analyzed [140, 190] . the virus initially replicates in the neuroreceptor cells of the olfactory epithelium. subsequently, the virus gains access to the cells of the main olfactory bulb, including the tufted and mitral cells, as well as the periglomular cells, the granule cells, and the short axon neurons of the internal granular layer of the main olfactory bulb. following invasion of these sites, the virus will reach the efferent targets of the main olfactory bulb, like the anterior olfactory nucleus, the prepiriform cortex, the olfactory tubercle, the olfactory amygdala, and the entorhinal cortex. further spread is then possible into numerous diencephalic and telencephalic areas, through polysynaptic neuronal connections. likewise, experimental infection into the foot pad of a rat results in a retrograde transport of bdv, through the sciatic nerve, and then from neuron to neuron into the cns [36] . moreover, evidence of oral infection suggests that the nerve endings innervating intestinal regions are suitable for uptake of the virus and its transport to the cns [130] . alternatively, oral uptake of bdv may also result in the infection of the gut epithelium. the virus could then gain access to the surrounding lymphoid tissue, infect target blood cells, and be transported by the bloodstream into the cns. the location of the initial site of entry will condition the incubation period preceding the onset of overt bd, reflecting the time required for viral transport into the brain. a similar mode of dissemination has been described for rabies virus, another nns rna virus [76] . both rabies and bdv spread along neuronal chains. the manner in which these chains are utilized is determined by the natural connections of neurons. such a mode of spread is compatible with a synaptic transfer of these viruses. moreover, the lack of detection of mature virus particles at any stage during bdv propagation has led to the hypothesis that viral spread ensues in the form of bare rnp [43, 76] . this hypothesis has been further strengthened by the demonstration that rnp are infectious [43] . within the cns, bdv will exhibit a preferential tropism for the limbic system, including the hippocampus [37, 78, 140] . this area will eventually harbor the highest viral load regardless of the initial mode of infection, even if viral spread is not restricted to the limbic system and will diffuse throughout the cns [78] . hence, bdv is also found in the hypothalamus, thalamus, and cerebral cortex. viral antigen accumulates in the nucleus, the perikaryon, and processes of the infected neurons [77] . the nucleolus, however, remains free of antigen. in the nuclei of infected neurons, aggregates of virus-specific material are likely to form the joest-degen inclusion bodies that characterize bdv-infected cells [100] . the topography of the infection in the hippocampus is particularly interesting. the stratified distribution of viral markers coincides with that of some excitatory amino acids receptors, in particular the glutamate receptors [78] . also, specific neurotransmitter pathways are affected by bdv infection [122, 198, 200] . this could indicate that these receptors are used by the virus or by viral rnp for their transneuronal transfer. besides neurons, glial cells are also permissive for bdv. virus antigen and rna appear rather early in astrocytes, followed by oligodendrocytes, ependymal cells, as well as schwann cells in the peripheral nervous system [37] . it is probable that glial cells become secondarily infected after bdv release by the neurons. in agreement with this hypothesis, purkinje cells are the only cells in the cerebellum containing bdv antigen early after infection. later on, glial cells also become infected [8] . this is also reminiscent of findings described in other biphasic cns diseases caused by rna viruses, such as theiler's virus infection of the mouse. in this case, the acute phase is characterized by an active viral replication in neurons. subsequently, the virus accesses glial cells in the spinal cord, which is required for the establishment of persistence [97, 99] . in the late stages of infection, bdv diffuses centrifugally, probably by using an anterograde axonal transport, and viral markers have been detected in peripheral nerves of all tissues and organs. consequently, many tissues and organs become positive for viral markers [78, 190, 231] . these include lachrymal, salivary or sebaceous glands and, as a rule, any ectodermal/epithelial tissue that can be reached by bdv through central or peripheral innervation. however, it is likely that extra neural tissue will become eventually infected only if the virus is delivered through the peripheral axons for a long time. the dissemination of bdv within the host could effectively ensue in the form of rnp. however, horizontal transmission of bdv would likely require shedding of mature, enveloped particles with the ability to initiate infection in a new host. despite numerous attempts, such particles have never been detected at any stage during bdv infection [76] . therefore, the extremely low levels of infectious virus shed by infected animals may explain the poor rate of contagion of bdv. neuropathogenesis of the natural bdv infection. the neuropathogenesis of naturally occurring bd has been best characterized in the horse, where more than 80% mortality is observed. there is also increasing evidence that horses can be subclinically infected with bdv [7, 84, 117, 146] . borna disease is defined as a nonpurulent polioencephalomyelitis [77, 101, 188] . the inflammatory cells form massive perivascular cuffs, as well as more diffuse tissue infiltrates. inflammatory infiltrates are present predominantly in the gray matter, but can also invade the underlying white matter. reactive astrocytosis usually follows the acute phase of the disease. interestingly, despite this strong inflammatory reaction, neuronal degeneration is limited. horses may develop dimness of vision and partial or complete blindness, as a result of the degeneration of the optic nerve and neurons in the retina, likely caused by the lymphocytic infiltration [182, 233] . however, bdv may also replicate in the retina without exerting any overt neuronal damage [84] . the brain regions most affected by the inflammatory response are the olfactory bulb, the hippocampus, and the caudate nucleus [102] . inflammation is moderate in the spinal cord and absent in the cerebellum [101] . the colocalization of cell infiltrates with sites of high viral expression suggests that the inflammatory reaction is due to the presence of viral antigen. the neuropathological features of bd in sheep are very similar to those described in horses [15, 16, 101] . symptoms observed during natural bdv infection are summarized in table 1 . neuropathogenesis of the experimental bdv infection. a broad spectrum of animals can be experimentally infected with bdv ( table 1 ). the rat is the most commonly used model for the study of bdv pathogenesis. the age, immune status, and genetics of the rat, as well as viral factors can influence the course of infection [85, 86, 88, 125, 150, 151] . adult rats infected intracerebrally or intranasally with bdv usually develop an immune-mediated biphasic behavioral disease. rats display clinical signs and a histopathological picture very similar to that described for the naturally infected horse [36, 38, 39, 90, 150, 151] . the onset of clinical signs, including movement abnormalities, aggressive and hyperactive behavior, coincides with the appearance of an inflammatory reaction in the brain that reaches its maximum of severity between days 30 and 40 after infection. parts of the limbic system, the cerebral cortex, and the olfactory bulb exhibit the strongest inflammatory reaction, which is limited to the gray matter. some level of inflammation is also observed in the basal ganglia, the diencephalon, and to a lesser degree, in the midbrain and medulla. this extensive inflammatory reaction leads to neuronal destruction that in some cases may cause a hydrocephalus. as with the infection of horses, the cerebellum rarely shows signs of inflammation. high virus replication is observed in the eyes of bdv-infected rats, which is accompanied by retinitis and leads to blindness due to the neuronal loss in the retina. the initial aggressive hyperactive behavior is followed by apathy, somnolence, and depression. eventually, some animals may also develop obesity [150] . this chronic phase is characterized by a steady decline in the inflammatory reaction, despite continuous high viral load in the cns. to our knowledge, this has 650 gonzalez-dunia, sauder and de la torre not been observed in other viral infections. the mechanisms underlying this delayed immune-tolerance are unknown. it is possible that sustained expression of high levels of viral antigens may result in the exhaustion of high affinity virus-specific t-cells [143, 217] . in contrast to the situation depicted above, bdv neonatal infection of the rat causes a life-lasting persistent infection that is characterized by the lack of a cellular immune response to bdv and the absence of clinical signs of bd [38] . this infection has been designated as a ''persistent, tolerant infection of the newborn'' (pti-nb) rat [38] . nevertheless, the humoral response to bdv is not significantly impaired in pti-nb rats [38, 150] . brains of pti-nb rats harbor a viral load comparable to that found in rats with acute bd [36, 88, 150, 207] , illustrating the noncytolytic replication of bdv. although pti-nb rats do not show clinical signs, they exhibit distinct deficiencies in emotional and cognitive functions [9, 57] , as well as physiological and neurodevelopmental abnormalities [8, 9] . hence, the pti-nb rat provides a valuable model to investigate the consequences of bdv infection in the cns without immune-mediated damage (see sections 3.3 and 3.4). bd as an immune-mediated neurological disease. immunological mechanisms frequently contribute to the pathophysiology of virus-induced cns diseases [1] . experimental bd provides a valuable model to study t-cell-mediated immunopathology in the cns [125, 204] . the cell-mediated immune response to bdv plays an essential role in the development of bd. this has been shown by studies on immunocompromised animals. bdv infection of rats treated with cyclophosphamide, cyclosporin a, or of athymic rats does not lead to inflammation and development of disease [88, 151, 209] . however, adoptive transfer of spleen cells from bd rats into bdvinfected immunosuppressed recipients results in inflammatory changes in the brain and concomitant bd [151] . in contrast, transfer of immune serum does not lead to disease, demonstrating that antibodies are not involved in the immunopathological process induced by bdv [90] . studies on pti-nb rats provide further evidence for the role of the cell-mediated immune response in the development of bd. bdv replicates in the thymus of pti-nb rats [176] . as with other viruses, early expression of viral antigens in the thymus can promote clonal deletion of t-cells that would normally respond to the infectious agent [96] . thus, the lack of bdv-specific precursor t-cells in the spleen and lymph nodes likely explains the absence of an inflammatory reaction in the cns of pti-nb rats. pathogenic role of cd4 ϩ and cd8 ϩ t-cells in bd. both cd4 ϩ and cd8 ϩ t-cells are present in the cns cell infiltrates and contribute to the immune-mediated pathology associated with bd [19, 204] . the role of cd4 ϩ t-cells in bd was supported by the isolation of a bdv-specific cd4 ϩ t-cell line with the ability to induce disease upon adoptive transfer into bdv-infected immunosuppressed recipients [167, 169] . adoptive transfer of these cd4 ϩ t-cells into immunocompetent uninfected rats did not cause encephalitis or disease, demonstrating that this bdv-specific tcell line was not encephalitogenic per se. recent evidence indicates that a direct cytotoxic activity of cd4 ϩ t-cells plays a rather limited, if any, role in bd [197] . although bdv-specific cd4 ϩ t-cells can exhibit major histocompatibility complex (mhc) class ii restricted cytotoxic activity in vitro [167, 168] , there is not, however, convincing evidence of such an activity in vivo [162, 197] . it should be noted that cd4 ϩ t-cells may acquire cytotoxic activity during in vitro cultivation [65] . in addition, there is no evidence of mhc class ii expression in neurons, the main cell type destroyed by the inflammatory response [38, 167, 206] . furthermore, cd4 ϩ t-cells are present predominantly in perivascular cuffs, whereas few reach the brain parenchyma, where the immune-mediated damage occurs [20, 197] . besides, adoptive transfer of noncytolytic virus-specific cd4 ϩ cells into bdv-infected immunosuppressed recipients can lead to the development of bd only if cd8 ϩ t-cells enter the brain [163] . development of disease in these recipients can be prevented by treatment with monoclonal antibodies that specifically block cd8 ϩ t-cell activity [163, 208] . conversely, treatment of bdv-infected rats with anti-cd4 ϩ antibodies does not effectively prevent inflammation and tissue damage in the cns [162] . taken together, these findings suggest that cd4 ϩ t-cells contribute to the pathogenesis of bd predominantly as t-helper cells [20, 163, 197] . nevertheless, a potential lytic role of virus-specific cd4 ϩ t-cells cannot be completely excluded [197] . in particular, astrocytes may express mhc class ii antigens [53, 162] , thus being potential targets for cd4 ϩ t-cells. cd4 ϩ t-cells have been shown to play a role in measles-and coronavirus-induced encephalomyelitis [121, 132] . however, these cases are considered as examples of virus-induced autoimmunity, illustrated by the ability of these cells to trigger an inflammatory reaction in the cns of uninfected animals. on the other hand, there is ample evidence that cd8 ϩ t-cells act as effector cells in the immune-mediated pathological reaction observed in bd. elimination, or functional blocking, of cd8 ϩ t-cells prevents both bdv-induced neurological symptoms and histopathological changes in the brain [20, 162, 197, 207, 208] . in addition, lymphocytes isolated from rat brains during acute bd exhibit mhc class i-restricted cytotoxic t-lymphocyte (ctl) activity in vitro [162] . adoptive transfer of these lymphocytes into immunosuppressed bdv-infected recipients results in the extravasation of cd8 ϩ t-cells in the brain parenchyma, leading to immunopathological and clinical signs similar to experimental bd [197] . therefore, neuronal damage seen in bd appears to be mediated by the cytotoxic activity of cd8 ϩ t-cells present in the brain parenchyma of bdv-infected rats. cd8 ϩ ctl can destroy cells presenting virus-derived peptides in the context of mhc class i presentation [232] . evidence suggests that mhc class i antigens are expressed at the surface of astrocytes and neurons in the cns of bdv-infected rats, concomitantly with the peak of cd8 ϩ t-cell infiltrate in the cns and the onset of neuronal degeneration [20, 37, 206] . under normal conditions, cells of the cns express very low, if any, mhc antigens, but their expression can be upregulated during pathological situations [55, 58] . it has been proposed that virally-infected neurons avoid destruction by ctl because of the lack of mhc class i expression [103, 157] . however, synaptically silent neurons may express mhc class i molecules [154] . bdv is one of the rare cases in which it has been shown that a viral infection of the cns can induce neuronal expression of mhc class i in vivo [20, 55, 69, 70, 162] . the molecular mechanisms responsible for bdv-induced mhc class i expression by neurons are presently unknown. it is possible that bdv infection of neurons directly causes upregulation of mhc class i expression. alternatively, induction of neuronal mhc class i expression may be due to the action of cytokines secreted by inflammatory cells or virally-infected glial cells [54, 55, 113] . certain cytokines, including interferon (ifn) ␥ or ␤ and tumor necrosis factor ␣ (tnf␣) can upregulate mhc class i expression on neurons in vitro and to some extent in vivo [58, 224] . evidence suggests that bdv-infected astrocytes could secrete ifn ␣/␤, which, in turn, could induce mhc class i expression in neurons [162] . neurons in the cns are terminally differentiated cells without the capacity of proliferation. consequently, destruction of virallyinfected neurons by the immune response can cause an irreversible loss of cells whose functions are required for normal brain performance. the existence of the blood-brain barrier significantly restricts the degree of immunological surveillance and the vigor of the immune response in the cns. however, activated virus-specific ctl can access the brain parenchyma. bdv-mediated induction of mhc class i expression on neurons can then be detrimental for the host by allowing destruction by ctl. furthermore, this bdv-specific ctl activity is unable to control the infection and viral load is not significantly decreased after regression of the immune response. thus, the immune-mediated damage is likely to 652 gonzalez-dunia, sauder and de la torre have more devastating consequences than those directly due to the mere bdv persistence in neurons. role of cytokines in bdv pathogenesis. cytokines and free radicals have recently gained attention as other mediators of immunopathology. there is increasing evidence that certain stimuli, including viral and bacterial infections, can upregulate cytokine expression in the cns, resulting in tissue damage and neurological disease [34, 136, 171, 225] . along with activated lymphocytes and macrophages present in immune infiltrates, resident cns cells like neurons, astrocytes, and microglia are potential producers of cytokines [171] . proinflammatory cytokines including il-1␣, tnf␣, and il-6 can cause neuronal damage [118, 171] . levels of il-6, tnf␣, and il-1␣ mrnas are significantly increased in bd rat brains and their expression correlates with the degree of inflammation and severity of neurological signs [190] . moreover, production of ifn ␥ by infiltrating cd8 ϩ t-cells could induce mhc class ii expression on astrocytes and microglia. this, in turn, would enhance cd4 ϩ t-cell activity and contribute to the immunopathology of acute bd. it has also been proposed that the decline of inflammation during the chronic phase of bd might be mediated by cytokines such as ifn ␥ [190] . free radicals such as nitric oxide, generated by the inducible nitric oxide synthase (inos), can also cause direct neuronal damage [112, 153] . increased cns expression of inos mrna is observed in rats with bd. levels of inos mrna correlate with the severity of neurological signs and degree of inflammatory lesions [2, 229] . the finding of up to 30-fold increased amounts of nitric oxide in bdv-infected rat brains suggests a possible contribution of nitric oxide to bd pathogenesis [92] . however, it is also possible that the inos mrna expression in the cns merely reflects a nonspecific activation of microglia, thus being more of a secondary than a primary pathogenic importance. bdv is considered as a noncytolytic virus in vitro, because infected cultured cells do not exhibit impaired growth or survival [87] . analysis of the situation in animals with bd is complicated by the massive inflammatory response accompanying the infection. thus, it is difficult to distinguish cell and tissue damage directly caused by bdv replication from the damage due to the antiviral immune response. the pti-nb rat model (see above) has provided a system to study virus-induced structural and functional cns alterations without inflammation [38] . studies on pti-nb rats have suggested that bdv can induce a selective damage on specific neuronal populations [9] . however, this restricted neuronal degeneration is not considered as a ''classical'' cytopathic effect, but is rather due to an interference of bdv infection with the postnatal development of cns areas that experience extensive postnatal maturation. one of the most noticeable morphological features of the pti-nb rat brain is the considerable cerebellar hypoplasia induced by bdv perinatal infection. also, the dentate gyrus in the hippocampus undergoes progressive degeneration. these traits are accompanied by a prominent and widespread astrocytosis [204] , defined as an increase in the number and size of cells expressing the glial fibrillary acidic protein (gfap), an astrocyte-specific marker [60] (see also the increased levels of gfap mrna expression in pti-nb rats shown in fig. 2 ). other perinatal virus infections, including lymphocytic choriomeningitis virus (lcmv), rat parvovirus and reovirus type iii, also induce damage to the cerebellum [139, 158, 166] . the cerebellum developmental arrest has been attributed in these cases to either an immune-mediated, or a virus-induced lysis of the dividing imma-ture cerebellar granule cells. the characteristics of bdv-induced cerebellar hypoplasia have been detailed in a recent report [8] . the arrest of cerebellar development occurs between 7 and 14 days postinfection. this coincides with a progressive atrophy of the granule cell layers. in contrast to the other viral systems mentioned above, the granule cells are not infected by bdv. purkinje cells are the predominant, if not the only, cell type infected in the cerebellum at the early stages of its development [8] . therefore, it appears that bdv-induced cerebellar damage is caused by different mechanisms than those proposed with other perinatal virus infections. evidence indicates that purkinje cells play a main role in supporting the multiplication, maturation, and migration of the granular cells [195] . this role could be altered by bdv infection. the fig. 2. tissue factor (tf) messenger rna levels are markedly increased in the cns of bdv persistently infected (pti-nb) rats. total rna was isolated from brains of pti-nb rats (lanes 1 to 3) and sham infected control rats (lanes 4 and 5), and analyzed by northern blot hybridization for the expression of tf and glial fibrillary acidic protein (gfap). viral rna was detected in bdv-infected rats using a bvp40 cdna probe. levels of tf mrna were normalized to those of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (gapdh). see also [75] . massive astrocytosis observed in pti-nb rats might also be involved in the cessation of granule cell migration, especially given the key role of astrocytes in providing the physical track upon which the granule cells migrate [82] , as well as neurotrophic support to surrounding cells [60] . the hippocampus consistently harbors the highest viral load in pti-nb rat brains [37, 78, 140] . despite considerable replication of bdv in this area, there is little effect on the lifespan of the infected neurons. one notable exception is the dentate gyrus (dg), whose neurons are progressively destroyed [38] . the dg is formed primarily during the postnatal period. although birth of cells in the dg begins embryonically, the vast majority (ͼ85%) of dg granule neurons are generated after birth [79] . hence, neuronal multiplication and migration will occur in this area well into adulthood. this represents a unique characteristic for neurons of the mammalian adult brain, that are usually considered as a whole as postmitotically differentiated cells [79] . dentate gyrus degeneration has also been observed following persistent neonatal infection with lcmv [160] . it has been shown that degeneration of the dentate gyrus and abnormalities in synaptic function occur after lcmv clearance from the granule cells. electrophysiological measurements demonstrated that these cells exhibit a persistent hyperexcitability. this was due to a decreased inhibition, usually mediated by a subpopulation of gaba interneurons that were affected by the infection [160] . the mechanisms underlying bdv-induced dg degeneration are presently unknown. given the noncytolytic effect of bdv on other neuronal populations, one could hypothesize that bdvmediated degeneration of dg granule neurons is likely due to either (a) the unique characteristics of these mitotically active neurons, or (b) the disruption of pathway(s) essential for dg maturation [214] . these questions warrant further investigations, especially considering the importance of the dg and hippocampus in learning and memory. in this respect, bdv represents an important model to investigate the pathogenesis of cns disease linked to abnormal cns development and to better understand the regulation of cns plasticity. the effector mechanisms involved in bdv-induced neuronal damage are still poorly understood. however, bdv infection is not likely to cause direct neuronal destruction but triggers instead a series of secondary events leading to cell damage within the cns. most of the cns alterations induced by bdv infection of immunocompetent adult rats can be attributed to the virus-induced cellular immune response. however, bdv-mediated impairment of brain activity can also occur without the hallmarks of inflammation and widespread cytolysis, owing to the virus' ability to cause changes in neuronal organization and to interfere with brain cell functions required to maintain cns homeostasis. the pti-nb rat offers a unique system to study the mechanisms underlying these virus-cns cell interactions. astrocyte functions are essential to neurons [60] . it is, therefore important to investigate the consequences on brain function associated with the prominent astrocytosis that characterizes pti-nb rat brains. we have already mentioned the key role of astrocytes in providing a substrate for neuronal migration during brain development. the astrocytic network participates in brain information processing in cooperation with neuronal networks [47] . astrocytes also play roles in the elimination of neurotoxins and in the production of various cytokines, which can act as neuronal differentiation factors [159] . cytokines can contribute to the regulation of neuronal neurotransmitter gene expression and, hence, can dramatically alter synaptic function [171, 225] . these changes can, in turn, contribute to complex animal behavior as well as to plasticity processes accompanying learning and memory. expression of high levels of proinflammatory cytokines [190] and of complement cascade components [56] have been documented in the rat cns during acute bd (see above). these molecules were likely to have been produced by inflammatory cells invading the brain parenchyma. therefore, it was difficult to assess the intrinsic response of resident cns cells. to separate these two events, morimoto et al. used dexamethasone treatment of rats after bdv infection [141] . this synthetic glucocorticoid effectively prevented inflammation in the cns of bdv-infected rats. however, several proinflammatory cytokines such as tnf␣ and il-6 were still induced in the brains of dexamethasone-treated rats, suggesting that these might have been produced by cns resident cells. however, dexamethasone treatment of rats per se may have a profound action on the cytokine expression pattern of cns cells [181] . studies on pti-nb rats may provide valuable information regarding the contribution of cns resident cells to disturbances in cytokine gene expression caused by bdv. perinatal bdv infection of the rat provides a system to dissect the elements involved in bdv-induced alterations of cns homeostasis without inflammation. so far, very few studies have addressed such questions in this model. recently, a bdv-induced upregulation of tissue factor (tf) was demonstrated in the cns of pti-nb rats [75] . tf, a transmembrane receptor, is the primary initiator of the coagulation protease cascade that results in the generation of the protease thrombin [62] . besides, tf appears to have pleiotropic roles and has been implicated in signal transduction, angiogenesis and brain function [33, 61, 175, 228] . in the cns, tf is primarily, if not solely, produced by astrocytes [59] . tf expression is markedly increased during persistent infection with bdv, both in vivo and in vitro [75] (fig. 2) . this upregulation is due to bdv infection of astrocytes, and is mediated by an increased transcription of the tf gene and a stabilization of tf mrna. the significance of this finding is still a matter of speculation. however, because this phenomenon occurs without hemorrhage, inflammation, or disruption of the blood-brain barrier, it raises interesting questions about the consequences of tf upregulation and suggests that tf may fulfill functions other than hemostasis in the cns. there is increasing evidence that proteins of the coagulation and fibrinolysis systems may function in the cns independent from blood clotting, such as regulating normal brain development and defending the brain against damage caused by stroke, trauma, and other injuries [131] . any imbalance in the protease activities may contribute to neuronal damage. tf is the primary initiator of the thrombin activation system, a protease that has been implicated in several neurodegenerative diseases, such as alzheimer's disease [131, 135] . interestingly, thrombin is predominantly expressed by dopaminergic neurons of the mesencephalon [221] . therefore, this may suggest a link between tf upregulation and the dopaminergic abnormalities observed in bd rats (see section 3.5). these findings suggest that upregulation of tf expression due to bdv infection may play an important role in the cns response to this viral assault and perhaps also in the bdvmediated disturbances of cns function. whether cns expression of other genes, including cytokines, is specifically altered by bdv infection remains to be established. behavioral abnormalities caused by bdv infection (table 1) were first described in naturally infected horses and sheep [125] . the acute phase of disease is characterized by movement disorders, accompanied by excitability and hyperactivity. the later stages of the disease are frequently manifested by somnolence, gonzalez-dunia, sauder and de la torre depression, apathy, and anorexia. mortality rates are as high as 80 -100% in horses and 50% in sheep. animals that survive remain chronically infected and recurrent episodes with exacerbation of behavioral alterations may occur [83] . both host and viral factors influence significantly the behavioral abnormalities associated with bdv infection. for example, hamsters and black-hooded rats do not develop apparent behavioral or neurological symptoms, despite the presence of virus in the cns [5, 85] . strain specific differences in the susceptibility to disease have also been observed in mice. thus, although bdv induces encephalitis to a similar degree in mrl/ϩ and mrl/lpr mice, only mrl/ϩ mice develop behavioral abnormalities [177] . this dissociation between inflammation and symptoms indicating the onset of the disease may reflect differences in the composition and immunological properties of the cellular infiltrate [177] . the host genetic background likely contributes to these differences. alternatively, host factors could also modulate the vulnerability of brain cells to the inflammatory reaction [78] . bdv-infected tree shrews illustrate important aspects of the consequences that bdv persistent infection may have in the behavior of highly developed animals [203] . tree shrews (tupaia glis) are phylogenetically classified at the root of the primates and exhibit a complex behavioral repertoire [201] . tree shrews inoculated with bdv develop a persistent infection that is frequently associated with behavioral disorders. interestingly, housing conditions have a strong influence on the behavioral manifestations after infection. only a small number of animals kept in isolation show behavioral abnormalities, whereas all paired animals display profound sociosexual behavioral disturbances. these include a tendency to accept partners more quickly, a need for increased body contact, as well as a reversal in the role of the sex partners and an impaired breeding behavior of the females. some animals kept in isolation also show neurological symptoms (table 1 ). in these cases, an initial hyperactive phase, that coincides with the peak of the neurological symptoms, is followed by a phase of decline. all these animals recover, but relapse of clinical symptoms can occur in some cases. it is unknown why clinical symptoms are observed only in some animals, especially because all have comparable viral load in the brain and similar titers of serum antibodies to bdv [203] . the behavioral alterations exhibited by bdv-infected tree shrews are controlled by the limbic system and can be summarized as a disinhibition toward the environment [203] . interestingly, evidence indicates that virus-specific lesions in neurons of the limbic system represent a hallmark of bdv infection. behavioral disturbances associated with bdv infection have been thoroughly studied in the rat model. intracerebral inoculation of the adult immunocompetent rat causes a persistent infection that is associated with the previously described immune-mediated biphasic neurobehavioral disease (see section 3.2.2). bd rats develop a progressive movement and behavior disorder, accompanied by hyperactivity. several studies have focused on the pharmacology and neurochemistry of this hyperactive syndrome [198, 199, 200] . based on similarities with the behavioral disturbances caused in rats by neurotoxic or electrolytic lesions in the frontal cortex or its catecholamine afferents [215] , it has been proposed that bdv-induced disturbances might be due to alterations of the dopamine system. dopaminergic activity is enhanced in the prefrontal cortex of bd rats, revealed by high levels of 3,4-dihydrophenylacetic acid, the primary dopamine metabolite. yet, dopamine receptor density is not affected in the prefrontal cortex [199] . analysis of dopamine receptor density in rostral striatal areas, such as the nucleus accumbens, revealed a loss of dopamine reuptake sites correlated with decreased numbers of d 2 and d 3 receptors binding sites [200] . the nucleus accumbens is a primary site of integration between limbic and motor information and has been involved in the control of motor, cognitive, emotional, and reward processes [119] . thus, abnormalities in the mesocorticolimbic dopaminergic network may constitute the neural substrate of hyperactivity in bd [200] . furthermore, similarities between these alterations and those observed in manic depressive or schizophrenic patients [161, 187, 223] may be an important pathophysiologic feature linking the bd rat model to human psychiatric diseases. besides dopamine, alterations in other neurotransmitter systems have also been described in bd rats and include changes in mrna levels of cholecystokinin, glutamic acid decarboxylase, and somatostatin [122] . the behavioral abnormalities observed in bd rats are likely to be a consequence of the inflammatory response. in particular, the high levels of cytokines produced by the immune infiltrates during the acute phase of disease could cause behavioral changes, including hyperactivity and aggressiveness [34, 171] . the correlation of cytokine levels in bd rat brains with the degree of inflammatory reaction and severity of neurological symptoms supports this hypothesis. it should be reminded that the hippocampus frequently harbors a high viral load [37, 78, 140] , thus becoming a primary target for the virus-specific inflammatory reaction. this brain structure plays essential roles in governing emotion, learning and memory [10] . therefore, cytokine-mediated disturbances of hippocampal functions can cause severe behavioral abnormalities [17, 89] . the later phase of bd is characterized by the decline of the inflammatory reaction and by symptoms like apathy and somnolence. this likely reflects the neuronal damage caused by the immune response. in contrast to bdv-infected immunocompetent adult rats, pti-nb rats develop a chronic infection with no overt signs of classic bd. however, pti-nb rats exhibit distinct cognitive, behavioral and physiological abnormalities (table 1) [9, 57] . pti-nb rats show impaired cognitive functions in different learning paradigms, including spatial discrimination and discriminated avoidance tasks [9, 57] . results from open field and neophobia tests revealed emotional disturbances in pti-nb rats, manifested as a reduced resting behavior and decreased anxiety. moreover, these animals displayed hyperactivity when placed in a novel environment [9] . reduced body weight and length were also observed in pti-nb rats, compared to age-and sex-matched uninfected controls. impaired growth did not seem to be related to disturbances in growth hormone physiology, because levels of growth hormone, insulin growth factor type 1, and glucose were unchanged in pti-nb rats [9] . these differences in growth appeared only after weaning, suggesting that the feeding behavior might have been affected by perinatal bdv infection. in addition, pti-nb rats exhibited an altered preference for a saline solution when it was paired with either water, saccharin, or quinine solutions [9] . the mechanisms underlying this altered salt taste preference are unknown. disturbances in the postnatal maturation of the cerebellum and hippocampus induced by bdv, which have been reviewed above, probably contribute to the neurobehavioral abnormalities observed in these animals. clinical, epidemiological, and virological data indicate that viruses can persist in the cns and induce progressive neurological disorders, which are associated with diverse pathological manifestations. these include alterations in behavior and cognition [138, 142, 227] . these findings have led to the hypothesis that viruses can contribute to neuropsychiatric disorders whose etiology remains unknown [227] . the wide host range of bdv, to-gether with the observation that bdv-infected animals exhibit behavioral disturbances resembling some human mental disorders, prompted studies aimed at determining an association of bdv with these disorders. seroepidemiological studies. seroepidemiological studies conducted in different laboratories over the last 10 years have consistently shown an increased bdv seroprevalence in neuropsychiatric patients (table 2) . however, considerable variations in prevalence rates have been described, ranging from 1.6 to 30% in psychiatric patients and 0 to 3.5% in controls (table 2) . moreover, follow-up studies on psychiatric patients revealed a high bdv seroprevalence (21.1%), reaching up to 37% among patients with major depression [25] . increased bdv seroprevalence has also been reported in immunosuppressed hiv-infected patients from europe and thailand [6, 26] , individuals with parasitic infections in africa [26] , as well as patients with neurological disorders [13] . these serological findings should be cautiously evaluated because of the following reasons: (a) in many cases, only limited information was provided about the composition of the subject group analyzed. thus, factors such as the geographic area, the heterogeneity of diagnoses, and clinical status of patients may have significantly influenced these results. for example, a slightly higher percentage of seropositives is found in patients' sera derived from areas in southern germany, where bdv is endemic in animals [12, 14, 173] (table 2) ; (b) differences in the experimental procedures used to detect antibodies to bdv in human sera may also have affected the results. in particular, the immunofluorescence assay used in many of these studies [25] [26] [27] is considered to be unreliable [110, 218] . the use of immunofluorescence assays for bdv serology poses difficulties because of the very low bdv antibody titers in human sera [22] . furthermore, sera from neuropsychiatric patients often contain autoantibodies that react with nuclear structures [67, 120, 194] . thus, the nuclear localization of bdv antigens in infected cells makes necessary the use of appropriate controls to discriminate between bdv-specific staining and nuclear staining due to autoantibodies. increased specificity and sensitivity are now provided by western blot assays that use either bdv-infected cell extracts [66, 218] , or recombinantly expressed bdv antigens [108, 180] . interestingly, results from studies conducted using this improved serologic test have still shown a significantly higher seroprevalence in neuropsychiatric patients (6.5-30%) compared to nonpsychiatric control groups (0 -1.4%) [66, 108, 180, 218] . detection of bdv antigens and rna in human peripheral blood mononuclear cells. the cloning and molecular characterization of bdv [31, 44] has allowed the development of specific and sensitive procedures for the detection of bdv rna in biological samples. using reverse transcriptase-pcr (rt-pcr) procedures, bdv rna can be detected in the peripheral blood mononuclear cells (pbmc) of infected rats [176, 193] . this finding led to the use of a similar approach to examine the prevalence of bdv in humans. thus, bdv rna was first detected in the pbmc from four out of six hospitalized psychiatric patients (inpatients) [29] . this initial study has been followed by more comprehensive molecular epidemiological investigations. bdv rna prevalence of 37% (22 of 60) [108] , or 10.9% (6 of 55) [95] , has been documented in pbmc of japanese neuropsychiatric inpatients. studies on japanese healthy blood donors revealed a viral prevalence of 4.6% (8 of 172) [108] and 0% (0 of 36) [95] . studies on german neuropsychiatric patients revealed a bdv rna prevalence in pbmc of 50% (13 of 26), whereas 0% (0 of 23) of normal controls were positive for viral rna [180] . these studies have also shown that patients harboring detectable levels of viral rna in their pbmc are frequently bdv seronegative; conversely, bdv-seropositive individuals have frequently nondetectable levels of viral rna [29, 108, 180] . recently, a high bdv seroprevalence and viral rna in pbmc have been detected in blood samples from japanese patients diagnosed with chronic fatigue syndrome (cfs) [109, 148] . bdv antibodies were detected in 33.7% (30 of 89) cfs patients, but only in three cases of 100 healthy controls. bdv rna was detected in 16% (10 of 62) cfs cases and 4.6% (8 of 172) control cases. patients with cfs frequently exhibit neuropsychiatric symptoms, including severe depression. the etiology of cfs is unknown, but evidence suggests the involvement of viral infections [93] . thus, these preliminary and intriguing results deserve further investigation. the number and nature of the infected cells in pbmc are still controversial. in persistently infected rats, the prevalence of bdvinfected cells in pbmc has been estimated at 1 per 5 ϫ 10 6 cells [176] . the need of highly sensitive rt-pcr procedures to detect bdv rna in pbmc from humans or bdv-infected animals indicates an extremely low viral load in pbmc. however, some investigators have reported that 10 to 17% of the monocytes of bdv-positive patients express bdv antigens [28, 29] . this would suggest that about 1% of the pbmc are infected with bdv. in this case, even assuming a very low viral load per cell, detection of bdv rna should not require the use of nested rt-pcr. the reasons for this apparent discrepancy remain to be determined. data from the rat model suggest that bdv is present in a very small fraction of circulating stromal cell precursors, rather than in the monocytes [176] . pbmc could have become infected during passage through the brain, or they may represent primary targets with the ability to spread the infection into the cns, a situation that has been proposed for other neurotropic viruses [64, 149] . intravenous injection of rats with cell-free bdv is not followed by the establishment of a persistent infection [36] . this argues against cells within the pbmc being primary targets for bdv. sequence analysis revealed a high degree of conservation of both inter-and intrapatient bdv sequences, as well as a close genetic relationship between human and animal-derived bdv sequences [29, 49, 180] . this finding is consistent with the remarkable genetic stability of bdv in animals, which has been dealt with in a previous section. however, a higher sequence variability has been reported in human bdv sequences derived from japanese patients' pbmc [106] . this might reflect strain differences due to geographical location. nevertheless, this higher variability may have been overestimated by the experimental procedures used to determine these sequences (see discussion in [180] ). the high genetic stability of bdv is an uncommon finding for an rna virus. because the mutation frequencies of rna viruses exceed by more than a million fold those of their eukaryotic hosts, extremely rapid virus evolution is anticipated and frequently observed [91] . however, rna viruses can also display long-term stasis both in nature and in laboratory conditions, as a result of selection for fit master sequences in rather constant environments [91, 219] . the high sensitivity of nested rt-pcr allows the detection of very low levels of target sequences. this method is also prone to artifacts due to inadvertent contaminations with laboratory sources of bdv. moreover, the high degree of bdv sequence conservation proscribes the use of sequence analysis to pinpoint cases of contamination. these concerns can be addressed by following well-established guidelines to avoid contamination problems during rt-pcr assays [137, 180] . nevertheless, the low level of viral rna in pbmc, close to the sensitivity threshold of nested rt-pcr, can hamper the reproducibility of results between laborato-656 gonzalez-dunia, sauder and de la torre ries [179] . it is worth noting that although the detection of bdv rna in pbmc provides a valuable diagnostic tool, it does not necessarily reflect the viral load in the cns. for example, pti-nb rats harbor extremely low levels of bdv rna in their pbmc, whereas high levels of viral antigen and rna are expressed in the cns [176, 193] . isolation of human bdv. recently, bdv has been isolated from pbmc of three german psychiatric inpatients with affective disorders [24] . the isolation was achieved by cocultivation of pbmc with a human oligodendroglia cell line, followed by serial passages of these cells. rescue of human bdv required the use of pbmc samples with strong viral antigen expression and at least 11 passages of the initial cocultivation [24] . these requirements to achieve virus isolation may explain why initial attempts to isolate bdv from humans had been unsuccessful [173] . sequence analysis of the three human bdv isolates revealed a very close relationship with known bdv animal strains [49] . this high sequence conservation, together with the number of passages necessary to isolate the virus, has raised concerns about possible contamination with laboratory strains and stresses the need of further studies to confirm these findings. detection of bdv antigens and rna in the human brain. given the hypothesis that bdv contributes to human mental disorders, it should be expected that bdv also infects the human cns. some investigators have used rt-pcr to search for bdv sequences in autopsy brain material from cases of schizophrenia and affective disorders [189, 192] . these initial studies failed to detect bdvpositive samples. however, a recent study has documented the detection of bdv antigen and rna in human autopsy brain samples [51] (fig. 3) . the initial selection of the cases analyzed in this study was based on histopathological features resembling those observed in chronically bdv-infected animals. in particular, damage to the hippocampus and astrocytosis were used as screening criteria. among 600 autopsy cases examined, five were found with sclerosis of the hippocampus and astrocytosis as the main histopathological findings. these patients presented with a clinical history of mental disorders manifested as severe depression and memory loss. in four of these five patients, immunohistochemistry and in situ hybridization revealed the presence of viral antigen and rna in neurons and astrocytes within the hippocampal formation. in contrast, bdv markers were not found in control cases [51] . detection of viral rna required the use of nested rt-pcr, suggesting that failure to detect bdv rna in previous studies, using single round rt-pcr, might have been related to lack of sensitivity. these findings demonstrate that bdv can persist in the human cns, providing the basis for more comprehensive studies aimed at determining whether bdv persistence in the cns is associated with specific human mental disorders. bdv rna has also been detected recently in clinical samples of brain tumors [147] . viral rna was detected in 5 of 16 specimens with grade 4 glioblastomas, but in none of 21 lower grade astrocytoma specimens. patients with malignant brain tumors, especially glioblastoma multiforme, are frequently severely immunosuppressed [170] . these findings provide additional support to a possible relationship between immunosuppression and levels of bdv expression. further studies are required to clarify this issue. a recent report [177b] has described the detection of bdv rna in postmortem brain samples from individuals with mental disorders. this study reported the detection of bdv p gene transcripts in 9 out of 17 patients with schizophrenia and 2 out of 5 patients with bipolar disorder. the data discussed above provide strong evidence that bdv can infect humans and persist in the cns. the establishment of an association between bdv and certain neuropsychiatric disorders awaits confirmation from more comprehensive molecular epidemiological studies. however, it should be emphasized that finding such an association does not prove the contribution of bdv to the pathophysiology of these mental disorders. in addition, bdv has been associated with both schizophrenia and affective disorders, raising the question of how the same infectious agent could be involved in two different mental disorders. schizophrenia and affective disorders are chronic and complex cns diseases, manifested by multiple signs and symptoms that tend to recur, often in a cyclical fashion [3, 164] . epidemiological data indicate that environmental factors, such as stress, contribute to these disorders [3, 41, 71, 72, 164, 178] . there is compelling evidence that viral infection of the brain represents an important risk factor [227] . moreover, findings in bdv-infected animals suggest that the biology of bdv is compatible with its potential role in these mental disorders [198] . bdv-infected animals display a diverse range of symptoms, including neurobehavioral alterations with cyclic episodes. the remarkable heterogeneity in disease phenotype associated with bdv infection depends on both host and viral factors, as well as other exogenous factors. thus, stress can contribute to recurrent disease episodes in bdv chronically infected animals [83, 134] . neurostructural abnormalities have been associated with schizophrenia and affective disorders. these affect several brain regions but are predominantly localized to medial temporal lobe structures. however, functional alterations suggest that the disease process may also affect other brain areas, involve integrative sensory function and motor coordination [227] . abnormalities within the hippocampal formation are frequently observed in these disorders [3, 18, 191] . studies using high resolution volumetric magnetic resonance imaging have shown that affective disorders are associated with structural changes in the hippocampus [191] . it has been suggested that the long-term overexpression of glucocorticoids, frequently seen in patients with major depression, could cause degeneration of hippocampal neurons, which express high levels of glucocorticoids receptors [178] . in the case of schizophrenia, hippocampal alterations are thought to reflect an altered process of cell migration during brain development, possibly triggered by an early viral infection [18] . bdv has a predominant tropism for limbic system structures [37, 78, 140] and perinatal infection causes hippocampal damage. interestingly, magnetic resonance imaging studies have suggested that cerebral abnormalities occur more frequently in bdv-seropositive psychiatric patients [11, 218] . moreover, pti-nb rats display cerebellar hypoplasia. the cerebellum is linked with limbic structures through multisynwere analyzed for the expression of bvp40 antigen and glial fibrillary acidic protein (gfap). both hs and ad cases exhibited a prominent astrocytosis as determined by gfap expression. bvp40 antigen was detected in the hs case but not in the ad case. see also [51] . scale bar: 40 m. gonzalez-dunia, sauder and de la torre aptic circuits, which have been implicated in mood regulation [196] . there is also evidence of a decreased size of the cerebellum in patients with affective disorders [63, 152, 220] . altered neurotransmitter functions affecting mainly catecholamines and especially dopaminergic systems have been implicated in the pathophysiology of human mental diseases [80, 164, 226] . this altered neurotransmission activity is thought to affect the regulation of limbic system functions, which, in turn, can lead to the various endocrine disturbances observed in these disorders [71, 72] . bdv infection causes distinct cns dopamine disturbances [199, 200] . bdv can also affect other neurotransmitter systems [122] , or the expression of neurotrophic factors required to maintain brain homeostasis. it is worth noting that bdv infection induces a prominent and chronic astrocytosis that was also observed in the autopsy brain samples from human cases identified as positive for bdv antigen and rna [51] . bdvinduced disturbances in astrocyte functions may contribute to neuronal dysfunction by affecting complex interactions within neural networks (see section 3.4). major human mental disorders, including schizophrenia and affective disorders, likely involve a concert of genes with varying penetrance among individuals that interact with exogenous factors to generate a variety of clinical phenotypes [164] . bdv can represent an exogenous factor contributing to these disorders. viral variants, even single amino acid substitutions, can exhibit remarkable differences in their expression pattern in cells and tissues of the infected host, that are associated with diverse disease phenotypes [98, 210] . similarly, genetic differences among individuals play a critical role in the outcome of a viral infection [32] . thus, it is conceivable that depending on the individual's genetic vulnerability and the properties of specific viral variants, bdv infection could contribute to distinct types of mental disorders. the source of bdv and routes for human infection are also germane to the role of bdv in neuropsychiatric disorders. evidence suggests that the nose is a main site of virus entry in animals [172] . sporadic evidence has suggested the possibility of transmission from bdv-infected animals to humans [14, 222] . the close genetic relationship between bdv sequences derived from humans and animal strains provides support for this hypothesis. in this regard, it is of interest that domestic cats are susceptible to bdv, representing potential carriers of bdv [129, 145] . the detection of bdv rna in a significant percentage (4.2 to 5%) of blood donors' pbmc raises the concern of a possible transmission by blood transfusion [107] . as with other neurotropic viruses, a small number of bdv-infected cells present in a blood sample could reach the brain and establish a persistent infection within the cns. depending on the genetic susceptibility of the host and on viral factors, this persistent infection may have diverse clinical consequences, manifested after a variable period of incubation. one should keep in mind that stress, a common finding in psychiatric patients, can induce immunosuppression by altering glucocorticoids levels [144] . this, in turn, could enhance susceptibility to infectious agents, including bdv. the molecular analysis of human biological samples to detect bdv poses considerable technical difficulties. comprehensive molecular epidemiological studies aimed at determining the prevalence of bdv in neuropsychiatric patients from different geographic areas will help to evaluate the significance of bdv as a potential human pathogen. these studies will require the use of standardized methods for the detection of viral antibodies and rna sequences in human biological samples, as well as uniform criteria for the clinical diagnoses. persistence of viruses effect of neurotropic virus infection on neuronal and inducible nitric oxide synthase activity in rat brain mood disorders: introduction and overview borna disease virus: a possible etiologic factor for human affective disorders? arch persistent borna virus infection in adult hamsters unusually high seroprevalence of borna disease virus in clade e human immunodeficiency virus type 1-infected patients with sexually transmitted diseases in thailand varied prevalence of borna disease virus infection in arabic, thoroughbred and their cross-bred horses in iran developmental injury to the cerebellum following perinatal borna disease virus infection early and persistent abnormalities in rats with neonatally acquired borna disease virus infection the hippocampus: functional and structural correlations expanded nuclear magnetic resonance studies in borna disease virus seropositive psychiatric patients and control probands findings with nuclear magnetic resonance tomography in psychiatric patients with and without serum antibodies to the virus of borna disease possible significance of borna disease for humans borna disease virus: possible causal agent in psychiatric and neurological disorders in two families die enzootische enzephalitis des schafes die enzootische encephalitis des schafes. vergleichende experimentelle untersuchungen über die seuchenhafte gehirnrückenmarksentzündung der pferde und schafe reduced long-term potentiation in the dentate gyrus of transgenic mice with cerebral overexpression of interleukin-6 schizophrenia: brain structure and function presence of cd4ϩ and cd8ϩ t cells and expression of mhc class i and mhc class ii antigen in horses with borna disease virus-induced encephalitis immune-mediated brain atrophy. cd8ϩ t cells contribute to tissue destruction during borna disease sequence analyses of the p24 gene of borna disease virus in naturally infected horse, donkey and sheep human infections with borna disease virus and potential pathogenic implications borna virus infections in cattle associated with fatal neurological disease first isolates of infectious human borna disease virus from patients with mood disorders borna disease virus infection and affective disorders in man human infections with borna disease virus: seroprevalence in patients with chronic diseases and healthy individuals borna disease virus-specific antibodies in patients with hiv infection and with mental disorders a novel marker for borna disease infection borna disease virus genome transcribed and expressed in psychiatric patients molecular biology of borna disease virus genomic organization of borna disease virus host susceptibility to viral disease tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation cytokines in viral diseases borna disease in naturally infected cattle pathogenesis of borna disease in rats: evidence that intra-axonal spread is the major route for virus dissemination and the determinant for disease incubation borna disease virus replicates in astrocytes, schwann cells and ependymal cells in persistently infected rats: location of viral genomic and messenger rnas by in situ hybridization borna disease: association with a maturation defect in the cellular immune response astrocytes and schwann cells are virus-host cells in the nervous system of rats with borna disease the encephalitic reaction in borna disease virus infected rhesus monkeys the neuroendocrinology of depression and chronic stress virus-like particles in mdck cells persistently infected with borna disease virus borna disease virus (bdv), a nonsegmented rna virus, replicates in the nuclei of infected cells where infectious bdv ribonucleoproteins are present sequence and genome organization of borna disease virus rna splicing contributes to the generation of mature mrnas of borna disease virus, a non-segmented negative strand rna virus the sendai virus p gene expresses both an essential protein and an inhibitor of rna synthesis by shuffling modules via mrna editing neuronal activity triggers calcium waves in hippocampal astrocyte networks molecular biology of borna disease virus: prototype of a new group of animal viruses sequence characterization of human borna disease virus molecular characterization of the borna disease agent detection of borna disease virus antigen and rna in human autopsy brain samples from neuropsychiatric patients the anatomy of viral persistence: mechanisms of persistence and associated disease determination of immune cells and expression of major histocompatibility complex class ii antigen in encephalitic lesions of experimental borna disease direct evidence that interferon-beta mediates enhanced hla-class i expression in measles virus-infected cells differential up-regulation of hla class i molecules on neuronal and glial cell lines by virus infection correlates with differential induction of ifn-beta expression of c1q, a subcomponent of the rat complement system, is dramatically enhanced in brains of rats with either borna disease or experimental allergic encephalomyelitis learning deficiencies in borna disease virus-infected but clinically healthy rats regulation of mhc class i and beta 2-microglobulin gene expression in human neuronal cells. factor binding to conserved cis-acting regulatory sequences correlates with expression of the genes astrocytes are the primary source of tissue factor in the murine central nervous system. a role for astrocytes in cerebral hemostasis molecular profile of reactive astrocytes-implications for their role in neurologic disease the structural biology of expression and function of tissue factor the molecular biology of initiation of coagulation by tissue factor reduction of cerebellar volume in major depression: a controlled mri study infection of monocytes during measles acquisition of specific cytotoxic activity by human t4ϩ t lymphocytes in culture detection of borna disease virus-reactive antibodies from patients with affective disorders by western immunoblot technique autoimmune phenomena in schizophrenic patients in the beginning the horse is sad''-a historical abstract of borna disease gamma interferon expression and major histocompatibility complex induction during measles and vesicular stomatitis virus infections of the brain dhib-jalbut, s. major histocompatibility complex class i expression on neurons in subacute sclerosing panencephalitis and experimental subacute measles encephalitis clinical and biochemical manifestations of depression. relation to the neurobiology of stress (1). new engl clinical and biochemical manifestations of depression. relation to the neurobiology of stress (2). new engl mechanism of bdv entry into cells characterization of borna disease virus p56 protein, a surface glycoprotein involved in virus entry expression of tissue factor is increased in astrocytes within the central nervous system during persistent infection with borna disease virus rabies and borna disease. a comparative pathogenetic study of two neurovirulent agents borna disease of horses. an immunohistological and virological study of naturally infected animals borna disease-neuropathology and pathogenesis neuronal birth and death mood disorders: biochemical aspects purification and properties of an intranuclear virus-specific antigen from tissue infected with borna disease virus mechanism of glial-guided neuronal migration in vitro and in vivo die bornasche krankheit der pferde und schafe ein beitrag zur epizootiologie der bornaschen krankheit beim pferd studies on the genetic control of resistance of black hooded rats to borna disease replication of borna disease virus in rats: age-dependent differences in tissue distribution replication of borna disease virus in cell cultures effect of borna disease virus infection on athymic rats progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin 6 in the brain persistent, tolerant or subacute infection in borna disease virus-infected rats genetic diversity of rna viruses local nitric oxide production in viral and autoimmune diseases of the central nervous system viruses, neurosis and fatigue borna disease virus p24 and p38/40 synthesized in a baculovirus expression system: virus protein interactions in insect and mammalian cells borna disease virus and the consumption of raw horse meat abrogation of tolerance to a chronic viral infection chimeric theiler's virus with altered tropism for the central nervous system a single amino acid change determines persistence of a chimeric theiler's virus role of vp2 amino acid 141 in tropism of theiler's virus within the central nervous system ü ber eigentümliche kerneinschlüsse der ganglienzellen bei der enzootischen gehirn-rückenmarksentzündung der pferde untersuchungen über die pathologische histologie, pathogenese und postmortale diagnose der seuchenhaften gehirn-rückenmarksentzündung (bornasche krankheit) des pferdes weitere untersuchungen über die seuchenhafte gehirn-rückenmarksentzündung (bornasche krankheit) des pferdes mit besonderer berücksichtigung des infektionsweges und der kerneinschlüsse viral persistence in neurons explained by lack of major histocompatibility class i expression detection of antibodies against borna disease virus in sera and cerebrospinal fluid of horses in the usa adaptation of borna disease virus to the mouse sequence variability of borna disease virus open reading frame ii found in human peripheral blood mononuclear cells prevalence of borna disease virus rna in peripheral blood mononuclear cells from blood donors demonstration of human borna disease virus rna in human peripheral blood mononuclear cells possible correlation between borna disease virus infection and japanese patients with chronic fatigue syndrome no evidence of borna disease virus-specific antibodies in multiple sclerosis patients in germany characterization of a borna disease virus glycoprotein, gp18 in vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases augmentation of major histocompatibility complex class i and icam-1 expression on glial cells following measles virus infection: evidence for the role of type-1 interferon multifocal retinopathy in borna disease virus infected rabbits virus-induced pigment epithelitis in rhesus monkeys. clinical and histological finds persistence of rna viruses in the central nervous system seroepidemiologische untersuchungen zur bornaschen krankheit (ansteckende gehirn-rückenmarkentzündung) der pferde cytotoxic mechanism of tumor necrosis factor-alpha mesocorticolimbic dopaminergic network: functional and regulatory roles immunoglobulins, autoantibodies and other serum fractions in psychiatric disorders induction of autoimmune reactions to myelin basic protein in measles virus encephalitis in lewis rats neurotransmitter abnormalities in borna disease isolation and characterization of borna disease agent cdna clones borna disease (bd), a slow virus infection-biological properties of the virus borna disease: a persistent virus infection of the central nervous system the cerebrospinal fluid of rabbits infected with borna disease virus natural borna disease in domestic animals other than horses and sheep clinically diseased cats with nonsuppurative meningoencephalomyelitis have borna disease virusspecific antibodies staggering disease in cats: isolation and characterization of the feline borna disease virus a borna-like disease of ostriches in israel a new link in the brain's defenses inducibility of ia antigen on astrocytes by murine coronavirus jhm is rat strain dependent der nachweis von latent infizierten pferden, schafen und rindern und deren bedeutung als virusreservoir bei der bornaschen krankheit weitere untersuchungen zur bornaschen krankheit der pferde und schafe tissue factor antigens in senile plaques of alzheimer's disease cytokines in inflammatory brain lesions: helpful and harmful control of contamination associated with pcr and other amplification reactions. molecular bio-products viruses and behavioral changes: a review of clinical and experimental findings cerebellar hypoplasia in neonatal rats caused by lymphocytic choriomeningitis virus axonal transport of borna disease virus along olfactory pathways in spontaneously and experimentally infected rats intrinsic responses to borna disease virus infection of the central nervous system research on the viral hypothesis of mental disorders virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector t cells glucocorticoids and immune functions demonstration of borna disease virus rna in peripheral blood mononuclear cells derived from domestic cats in japan demonstration of borna disease virus rna in peripheral blood mononuclear cells from healthy horses in japan expression of borna disease virus messages in clinical samples from patients with brain malignant tumors demonstration of borna disease virus rna in peripheral blood mononuclear cells derived from japanese patients with chronic fatigue syndrome detection of measles virus genome directly from clinical samples by reverse transcriptase-polymerase chain reaction and genetic variability behavioral disease in rats caused by immunopathological responses to persistent borna virus in the brain pathogenesis of borna disease in rats: immune-mediated viral ophthal-662 gonzalez-dunia, sauder and de la torre moencephalopathy causing blindness and behavioral abnormalities cortical atrophy in schizophrenia and mania: a comparative ct study nitric oxide as a secretory product of mammalian cells induction of mhc class i genes in neurons borna disease and enzootic encephalomyelitis of sheep and cattle description of feline nonsuppurative meningoencephalomyelitis (''staggering disease'') and studies of its etiology mechanism and consequence of viral persistence in cells of the immune system and neurons the pathogenesis of parvovirus-induced cerebellar hypoplasia in the syrian hamster mesocricetus auratus. fluorescent antibody, foliation, cytoarchitectonic, golgi and electron microscopic studies neuronal differentiation factors/cytokines and synaptic plasticity persistent dentate granule cell hyperexcitability after neonatal infection with lymphocytic choriomeningitis virus in vivo d2 dopamine receptor density in psychotic and nonpsychotic patients with bipolar disorder lysis of major histocompatibility complex class i-bearing cells in borna disease virus-induced degenerative encephalopathy immunopathogenic role of t-cell subsets in borna disease virus-induced progressive encephalitis genetic tools for unraveling the nature of a complex disorder genomic organization of the structural proteins of borna disease virus revealed by a cdna clone encoding the 38-kda protein reovirus type iii encephalitis-a virologic and ultrastructural study borna disease virus-induced meningoencephalomyelitis caused by a virus-specific cd4ϩ t cell-mediated immune reaction borna disease virus-infected astrocytes function in vitro as antigen-presenting and target cells for virus-specific cd4-bearing lymphocytes borna disease, a progressive meningoencephalomyelitis as a model for cd4ϩ t cellmediated immunopathology in the brain modulation of t-cell function by gliomas cytokines and the nervous system ii: actions and mechanisms of action natural and experimental borna disease in animals borna disease, a possible hazard for man? detection of serum antibodies to borna disease virus in patients with psychiatric disorders binding of human factor viia to tissue factor induces cytosolic ca 2ϩ signals in j82 cells, transfected cos-1 cells, madin-darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor hematologic consequences of borna disease virus infection of rat bone marrow and thymus stromal cells borna disease virus in mice: host-specific differences in disease expression lipkin, w. i.; and the bornavirus study group why stress is bad for your brain detection of borna disease virus (bdv) antibodies and bdv rna in psychiatric patients: evidence for high sequence conservation of human blood derived bdv rna characterization of mechanisms involved in transrepression of nf-kappa b by activated glucocorticoid receptors untersuchungen über das klinische verhalten der seuchenhaften gehirn-rückenmarksentzündung (bornaschen krankheit) des pferdes nebst angaben über diesbezügliche therapeutische versuche the remarkable coding strategy of borna disease virus: a new member of the nonsegmented negative strand rna viruses sequence conservation in field and experimental isolates of borna disease virus biochemical and functional analysis of borna disease virus g protein rna splicing in borna disease virus, a nonsegmented, negative-strand rna virus human brain d1 and d2 dopamine receptors in schizophrenia, alzheimer's, parkinson's, and huntington's diseases die ausbreitung der encephalitischen reaktion bei der bornaschen krankheit der pferde und deren beziehungen zu der encephalitis epidemica, der heine-medinschen krankheit und der lyssa des menschen. eine vergleichend-pathologische studie a search for borna virus-specific genome in human nerve tissue by reverse transcriptase polymerase chain reaction (rt-pcr) kinetics of virus spread and changes in levels of several cytokine mrnas in the brain after intranasal infection of rats with borna disease virus hippocampal atrophy in recurrent major depression polymerase chain reaction (pcr) search for viral nucleic acid sequences in schizophrenia borna disease virus in peripheral blood mononuclear and bone marrow cells of neonatally and chronically infected rats autoantibodies to dna in multicase families with schizophrenia local control of granule cell generation by cerebellar purkinje cells the anatomy of mood disorders-review of structural neuroimaging studies induction of degenerative brain lesions after adoptive transfer of brain lymphocytes from borna disease virus-infected rats: presence of cd8ϩ t cells and perforin mrna behavioral disturbances and pharmacology of borna disease prefrontal cortex dysfunction in borna disease virus (bdv)-infected rats a neural substrate of hyperactivity in borna disease: changes in brain dopamine receptors behavior of tree shrews a small highly basic protein is encoded in overlapping frame within the p gene of vesicular stomatitis virus behavior alterations in tree shrews (tupaia glis, diard 1820) induced by borna disease virus immunopathogenesis of borna disease borna disease in rhesus monkeys as a models for uveo-cerebral symptoms transforming growth factor-beta modulates t cell-mediated encephalitis caused by borna disease virus. pathogenic importance of cd8ϩ cells and suppression of antibody formation atypical dissemination of the highly neurotropic borna disease virus during persistent infection in cyclosporine a-treated, immunosuppressed rats preventive effects of early anti-cd4 or anti-cd8 treatment on borna disease in rats inhibition of immune-mediated meningoencephalitis in persistently borna disease virus-infected rats by cyclosporine a a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with the ability to cause growth hormone deficiency syndrome antigenic relationship and further characterization of two major borna disease virus-specific proteins niemann, h. the 24k protein of borna disease virus evolution of negativestranded rna viruses effects of adrenalectomy on spatial memory performance and dentate gyrus morphology paradoxical reinforcing properties of apomorphine: effects of nucleus accumbens and area postrema lesions a borna virus cdna encoding a protein recognized by antibodies in humans with behavioral diseases tolerance by exhaustion borna disease virus and schizophrenia molecular evolution of eastern equine encephalomyelitis virus in north america computed tomography in schizophreniform disorder and other acute psychiatric disorders cellular localization of thrombin receptor mrna in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mrna borna disease virus antibodies among workers exposed to infected ostriches positron emission tomography reveals elevated d2 dopamine receptors in drug-naive schizophrenics inducible expression of h-2 and ia antigens on brain cells cytokine production in the central nervous system neurochemical, viral and immunological studies viruses, schizophrenia and bipolar disorders tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice severity of neurological signs and degree of inflammatory lesions in the brains of rats with borna disease correlate with the induction of nitric oxide synthase borna disease virus: immunoelectron microscopic characterization of cell-free virus and further information about the genome detection of borna disease virus rna in naturally infected animals by a nested polymerase chain reaction mhc-restricted cytotoxic t cells: studies on the biological role of polymorphic major transplantation antigens determining t-cell restriction-specificity, function, and responsiveness handbuch der viruskrankheiten ii we wish to especially acknowledge beatrice cubitt for critical discussions and for her long standing expert assistance. this work was supported by nih grant ns 32355-02. dgd is supported by the institut pasteur and cs is a fellow of the german stipendienprogramm infektionsforschung, dkfz, heidelberg. this is publication 10776-np from the scripps research institute. all authors contributed equally to this review. key: cord-299051-5r2s8z1a authors: tsuhako, maria heloisa; augusto, ohara; linares, edlaine; chadi, gerson; giorgio, selma; pereira, carlos a. title: tempol ameliorates murine viral encephalomyelitis by preserving the blood–brain barrier, reducing viral load, and lessening inflammation date: 2010-03-01 journal: free radic biol med doi: 10.1016/j.freeradbiomed.2009.12.013 sha: doc_id: 299051 cord_uid: 5r2s8z1a multiple sclerosis (ms) is a progressive inflammatory and/or demyelinating disease of the human central nervous system (cns). most of the knowledge about the pathogenesis of ms has been derived from murine models, such as experimental autoimmune encephalomyelitis and viral encephalomyelitis. here, we infected female c57bl/6 mice with a neurotropic strain of the mouse hepatitis virus (mhv-59a) to evaluate whether treatment with the multifunctional antioxidant tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) affects the ensuing encephalomyelitis. in untreated animals, neurological symptoms developed quickly: 90% of infected mice died 10 days after virus inoculation and the few survivors presented neurological deficits. treatment with tempol (24 mg/kg, ip, two doses on the first day and daily doses for 7 days plus 2 mm tempol in the drinking water ad libitum) profoundly altered the disease outcome: neurological symptoms were attenuated, mouse survival increased up to 70%, and half of the survivors behaved as normal mice. not surprisingly, tempol substantially preserved the integrity of the cns, including the blood–brain barrier. furthermore, treatment with tempol decreased cns viral titers, macrophage and t lymphocyte infiltration, and levels of markers of inflammation, such as expression of inducible nitric oxide synthase, transcription of tumor necrosis factor-α and interferon-γ, and protein nitration. the results indicate that tempol ameliorates murine viral encephalomyelitis by altering the redox status of the infectious environment that contributes to an attenuated cns inflammatory response. overall, our study supports the development of therapeutic strategies based on nitroxides to manage neuroinflammatory diseases, including ms. multiple sclerosis (ms) is a progressive inflammatory and/or demyelinating disease of the human central nervous system. with an incidence of 1 per 1000 individuals, ms is the most prominent chronic neurological disease of young adults in the moderate climate areas of the northern and southern hemispheres [1] . while there is a clear genetic influence [2, 3] , complex diseases such as ms are likely to involve inherited arrays of genetic polymorphisms, each exerting a slight effect on disease establishment and progression [4] . environmental factors are also recognized as contributors to ms. although the view that infectious agents encountered early in life may prime or trigger the disease process that manifests later in life is widely supported, incontrovertible evidence for a direct viral or bacterial trigger of ms has yet to be provided [1, [5] [6] [7] . because of the relative inaccessibility and sensitivity of the human central nervous system (cns), most of the knowledge about the pathogenesis of ms has been derived from experimental animal models, such as murine experimental autoimmune encephalomyelitis (eae) and murine viral encephalomyelitis [1, [6] [7] [8] . although the triggers of ms remain under scrutiny, it is widely accepted that an immune-mediated inflammatory response induced by lymphocytes, macrophages, and microglia in the cns participates in the pathogenesis of the disease [1, [6] [7] [8] . the resulting inflammatory environment favors high rates of superoxide anion and nitric oxide formation through nadph oxidase and inducible nitric oxide synthase (inos), respectively [9] [10] [11] . these primary radicals lead to species that are more reactive toward biomolecules, such as peroxynitrite, nitrogen dioxide, carbonate radical, and myeloperoxidase compound i. they, in turn, may propagate the inflammatory cascade and/or trigger degenerative cascades leading to nitrooxidative damage of the neural tissue [9] [10] [11] . accordingly, the presence of 3-nitrotyrosine residues in proteins, a marker of nitrooxidative damage, has been consistently demonstrated in acute and chronic active ms lesions of animal models and human patients (see, for instance [8, [12] [13] [14] ). a particularly effective compound for deactivating nitrogen dioxide, carbonate radical, and the enzyme myeloperoxidase is the cyclic nitroxide radical tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) [15] [16] [17] [18] [19] [20] [21] [22] [23] . under physiological conditions, the nitroxide tempol (tpno • ) acts as a multifunctional antioxidant because it reacts with diverse biological oxidants and reductants while being recycled through the oxammonium cation (tpno + ) and hydroxylamine derivative (tpnoh), respectively. after several catalytic cycles, tempol is eventually consumed by recombination reactions with specific radicals, such as tyrosyl and thiyl radicals and/or metabolized [15, 19, 21, 23] . these nearly catalytic antioxidant properties are likely to contribute to the efficiency of tempol in reducing tissue injury in animal models of inflammation (reviewed in refs. [15, 24] ). to the best of our knowledge, there are no peer-reviewed studies reporting the effects of tempol on animal models of ms, although a patent has been recently published (wo 2009/023707 a1). in this case, the described examples were obtained with the murine eae model. here we show a striking effect of tempol in ameliorating murine viral encephalomyelitis induced by a neurotropic strain of the mouse hepatitis virus (mhv-59a) [6, 7, 25, 26] . by monitoring disease scores, mouse survival, cns integrity, viral load, and markers of inflammation, we also provide mechanistic clues to the ameliorating action of tempol. all reagents were purchased from sigma, merck, or fisher and were analytical grade or better. all solutions were prepared with distilled water purified with a barnstead milli-q system. strain a59 of coronavirus mhv (mhv-59a) was obtained from the laboratoire de virologie (strasburg, france). the virus was propagated and plaque assayed on l929 cell cultures in modified eagle's medium containing 10% fetal bovine serum at 37°c, as previously described [27] . infection of mice and tempol treatment spf female c57bl/6 mice (5-6 weeks of age) were obtained from our animal facilities, and the experiments performed followed the approved animal guidelines (committee of animal ethics, instituto de química, universidade de são paulo). the animals were intracranially inoculated (ic) into the left cerebral hemisphere with 20 μl of a solution containing the mhv-59a virus (500 pfu) diluted in phosphate-buffered saline (pbs) containing 0.75% bovine serum albumin (bsa) or with pbs plus bsa (control animals) [25, 26, 28] . the injection was located in the deep cortical layer of the parietal cortex close to the corpus callosum as verified by pilot experiments in thaw-mounted brain sections of animals injected with evan's blue. tempol (24 mg/kg) was administered intraperitoneally (ip) in two doses on the first day (5 min before and 2 h after mhv-a59 inoculation) and in daily doses for 7 days between 9:00 and 10:00 am; these animals also received tempol (2 mm) in the drinking water ad libitum [15, 29] . untreated animals were those that were inoculated with the virus and received pbs ip instead of tempol. sham animals were those that received pbs plus bsa intracranially instead of the virus and were not treated with tempol. mice were examined for clinical symptoms and scored as previously described [26] . in summary, normal mice scored 0; mildly sick animals (showing piloerection, hunched posture, and lethargy) scored 1; moderately sick mice (showing hind-or fore-limb weakness/paresis, lethargy) scored 2; severely sick animals (hindor fore-limb paralysis, lethargy, depletion, ataxic gait) scored 3; moribund mice scored 4; and dead animals scored 5. mice were euthanized with a lethal dose of thiopental (ip) for the collection of tissue samples and to interrupt the experiment. at day 7 after infection, mice were euthanized with a lethal dose of thiopental (ip) and perfused intracardially with 10 ml of 10% buffered formalin. the brains were removed, formalin-fixed, paraffin-embedded, and stained with hematoxylin and eosin, to evaluate infiltration, and using weil for demyelination assessment [30] . mouse brain and spinal cord tissues were collected, weighed, and homogenized in dmem supplemented with 10% fetal bovine serum and 50 μg/ml gentamycin. the homogenates (10-100 mg/ml) were serially diluted and plaque assayed on l929 cells [27] . analysis of tempol and its hydroxylamine derivative by electron paramagnetic resonance (epr) epr spectra were recorded at room temperature on a bruker er 200 d-src upgraded to an emx instrument. mice were sacrificed 10, 15, and 25 min after tempol administration (24 mg/kg; ip). brain tissues were collected, homogenized in pbs (1.5 ml/g tissue), and subjected to epr analysis before (to determine tempol levels) and after addition of 1 mm ferricyanide (to determine tempol plus hydroxylamine levels) [29] . the tempol concentration in the brain was estimated by double integration of the epr spectrum and comparison with that of a standard tempol solution scanned under the same conditions. to determine the levels of tempol in the brain of animals receiving 2 mm tempol in the drinking water, the animals were sacrificed between 9:00 and 10:00 am and the brain tissues were collected, homogenized, and analyzed by epr as described above [29] . sodium fluorescein was used as a tracer as previously described with some modifications [31] . briefly, mice received 100 μl of 10% sodium fluorescein in pbs ip and, 10 min later, were anesthetized, bled, and transcardially perfused with a minimum of 30 ml of pbs/ heparin (1000 units/l) and pbs. blood, brain, and spinal cords were collected for further analysis. the brains were washed with pbs and photographed. the spinal cords were weighed and homogenized in pbs (0.5 ml/spinal cord) and centrifuged (14,000 g, 2 min). a sample of the clarified supernatant was used to determine total protein concentration with a bio-rad kit. in parallel, 0.3 ml of the clarified supernatant was mixed with 1 ml of 15% trichloroacetic acid and centrifuged (10,000 g, 10 min). after the addition of 0.125 ml of 5 m naoh to 0.5 ml of the supernatant, the fluorescence was determined by using a fluorolog fluorimeter (spex) at 437 nm excitation and 508 nm emission. sodium fluorescein standards (50-1000 ng/ml) were used to determine fluorescein contents in the samples. blood was centrifuged and the levels of fluorescein in the sera were determined as above. fluorescein uptake from the circulation into the spinal cord tissue is expressed as (μg fluorescence spinal cord tissue/ mg protein)/(μg fluorescence sera/μl blood) to normalize values for blood levels of the marker [31] . at 7 days after infection, mice were euthanized with a lethal dose of thiopental (ip) and perfused intracardially via the left ventricle with 20 ml of pbs followed by 20 ml of 10% buffered formalin. brains and spinal cords were removed and kept in 10% buffered formalin (16-18 h) . tissue sections were paraffin-embedded and coronal sections (3 μm) were obtained and affixed onto slides. the slides were sequentially deparaffinized, rehydrated, and treated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. then, the antigens were retrieved by incubating the slides at 100°c with citrate buffer, ph 6.0, for 1 min in a pressure cooker (in the case of anti-nitrotyrosine and mac-2) or with tris-edta buffer, ph 8.0, for 1 min in a vegetable steamer (for anti-inos). finally, the slides were incubated at 4°c overnight with primary antibodies diluted in pbs/0.1% bsa. the employed antibodies and dilutions were mouse monoclonal to nitrotyrosine (abcam, uk; 1:400), rabbit polyclonal to inos (abcam; 1:1500), and monoclonal anti-mouse mac-2 (cedarlane, canada; 1:50,000). after extensive washing with 0.5% tbs-tween, the sections were incubated at 37°c in a humidified chamber with novolink max polymer (novocastra, uk) according to the instructions of the manufacturer for anti-nitrotyrosine and anti-inos antibodies. in the case of mac-2 antibody, the sections were incubated with avidin biotinylated peroxidase complex (abc), using the abc vectastain elite kit (vector laboratories, burlingame, ca, usa; 1:200) according to the instructions of the manufacturer. the avidin-biotin complex was visualized with 3,3′diaminobenzidine (sigma, st. louis, mo, usa) and hydrogen peroxide. counterstaining was performed with harris's hematoxylin (merck, darmstadt, germany). to assess nonspecific staining, the samples were similarly treated, but in the absence of the primary antibodies. the mac-2 (marker of macrophage cells), inos, and 3-nitrotyrosine immunoreactivity was measured in regions of the forebrain by semiquantitative bilateral image analysis (two sections/mouse; three animals) implemented on a kontron-zeiss ks400 image analyzer (germany) as previously described [32] . briefly, a television camera from the microscope acquired the image. the fields were selected within the brain regions of cortical areas, amygdaloid nuclei, hippocampus, and corpus callosum, bilaterally. the size of the sampled fields was 8.96 × 10 4 μm 2 (40×) for cortical areas, amygdaloid nuclei, and hippocampus and 1.21 × 10 4 μm 2 (63×) for corpus callosum. after shading correction, the following discrimination procedure was performed. the mean value of the gray matter (mgv ± standard error) in areas of the brain devoid of specific labeling was taken as the background. gray values darker than the background were considered to correspond to specific labeling and discriminated. the specific mgv was taken as the difference between the specific and the background values. the glass value was left constant at 200 mgv. the morphometric (area) and the microdensitometric measurements (specific mgv) indicate the amount of immunopositive profiles in the sampled fields. only the area of immunolabeling was presented. at 7 days after infection, mice were euthanized with a lethal dose of thiopental (ip) and the spinal cord tissues were collected and immediately frozen in liquid nitrogen. the frozen tissues were pulverized with a pestle and mortar containing liquid nitrogen. then, they were homogenized at 4°c with lysis buffer (0.18 ml/spinal cord) containing 50 mm tris (ph 7.4), 0.5% triton x-100, 5 mm edta, protease inhibitors (10 μg/ml trypsin inhibitor, 1 mm sodium orthovanadate, 10 μg/ml benzamidine, and 5 μg/ml antipain hydrochloride), and protease inhibition cocktail for tissues (1/2500 v/v; sigma). the homogenate was centrifuged at 4°c and 1000 g for 10 min and the supernatant was stored at −80°c until analysis of total protein and nitrated protein (protein 3-nitrotyrosine). total protein in the samples was determined by the bradford method with a bio-rad kit. samples of spinal cord homogenates (0.5 μg) were transferred onto a nitrocellulose membrane by vacuum for slot blots. assessment of blotted protein was performed by ponceau s staining as previously described [33] . the destaining was done with distilled water, and the membrane was blocked with 5% nonfat dried milk (2 h) before exposure to primary antibody solution for 1 h (mouse monoclonal antinitrotyrosine, 1/1000; abcam). primary antibody was detected by 1 h incubation with secondary antibody (peroxidase-conjugated rabbit polyclonal to mouse igg, 1/5000; abcam). slot blots were revealed with chemiluminescence reagents (amersham), and relative quantification of 3-nitrotyrosine was performed by densitometry (imagequant version 5.2; molecular dynamics) [15, 29] . mice were euthanized at 1 and 7 days after infection (for cd4 and cd8 analysis) and at 7 days after infection (for tnf-α and ifn-γ analysis) with a lethal dose of thiopental (ip), and the spleens and/or spinal cords were immediately removed. total rna was extracted using trizol reagent (invitrogen), following the instructions of the manufacturer. rna concentration was determined by uv spectroscopy at 260 nm. total rna was reverse transcribed into cdna with the super script first-strand synthesis system for rt-pcr (invitrogen), following the instructions of the manufacturer. amplification of cdna was performed according to the instructions of the manufacturer (pcr master mix; promega) in a thermal cycler (mj research, ptc 200). the protocols for ifn-γ, tnf-α, cd4, cd8, and β-actin transcript detection were as follows: in the case of ifn-γ, predenaturation at 94°c for 4.5 min and cycles of denaturation at 94°c for 20 s, annealing at 58°c for 20 s, and extension at 72°c for 30 s (repeated for 35 cycles) and a final end extension of 1 min at 72°c; for tnf-α, pre-denaturation at 95°c for 4 min and cycles of denaturation at 95°c for 20 s, annealing at 56°c for 20 s, and extension at 72°c for 30 s (repeated for 34 cycles) and a final end extension of 1 min at 72°c; for cd4 and cd8, pre-denaturation at 95°c for 15 min and cycles of denaturation at 94°c for 30 s, annealing at 60°c for 36 s, and extension at 72°c for 60 s (repeated for 40 cycles); and for β-actin, pre-denaturation at 95°c for 3 min and cycles of denaturation at 94°c for 45 s, annealing at 60°c for 45 s, and extension at 75°c for 45 s (repeated for 33 cycles) and a final end extension of 1 min at 72°c. the following primers were used for pcr amplification: ifn-γ primer (up, 5′-gctctgagacaatgaacgct-3′, and down, 5′-aaagagataatctggctctgc-3′, 227 bp) [34] ; tnf-α primer (up, 5′-tctcatcagttctatggccc-3′, and down, 5′-gggag-tagacaaggtacaac-3′, 212 bp) [35] ; cd4 primer (up, 5′-tccttcccactcaactttgc-3′, and down, 5′-aagcgagacctgggg-tatct-3′, 200 bp); cd8 primer (up, 5′-gctcagtcatcagcaactcg-3′, and down, 5′-atcacaggcgaagtccaatc-3′, 200 bp) [36] ; and β-actin primer (up, 5′-tggaatcctgtggcatccatgaaac-3′, and down, 5′-taaaacgcagctcagtaacagtccg-3′, 348 bp) [37] . pcr products were analyzed on 1.5% agarose gel and stained with ethidium bromide (0.5 μg/ml). relative quantification of the transcripts was performed by densitometry (imagequant version 5.2; molecular dynamics). data are expressed as means ± standard error. the differences between experimental groups were examined using the unpaired t test. the outcome of viral infections depends on a variety of factors, such as host age, sex, and genetics and virus strain, mutability, dose, and route of administration [6, 25, 26] . thus, we started by examining the characteristics of the mhv-59a infection in our experimental settings. spf female c57bl/6 mice (n = 12) received pbs (ip) as a surrogate for the ulterior tempol treatment and intracranial inoculation of mhv-59a (500 pfu) and were maintained under observation. one day after infection, 50% of the animals showed mild neurological symptoms, such as piloerection, hunched posture, and lethargy (score 1) (fig. 1a) . between 2 and 4 days after inoculation, some mice improved, but at least 70% showed moderate to severe neurological symptoms, such as hind-or fore-limb weakness/paresis (score 2) or hind-or fore-limb paralysis and ataxic gait (score 3). between 6 and 8 days after infection, 30% of the mice died. ten days after inoculation, 90% of the mice had died, and the survivors still presented neurological symptoms that persisted up to 60 days (fig. 1a) . histopathological analysis of brain tissues collected 7 days after inoculation showed massive inflammatory cellular infiltration throughout forebrain gray and white matter regions (fig. 1b) . inflammatory foci that were accumulating increased amounts of infiltrating cells were evident in gray matter regions, such as neocortex, hippocampus, amygdala, and diencephalon, and also in white matter structures, such as the lateral regions of the corpus callosum, internal capsule, anterior commissure, and fornix. moreover, perivenous cuffing was particularly seen in the white matter clusters, mainly associated with perivenular disruption of the myelin staining by weil (fig. 1b) . thus, we confirmed that mhv-a59 infection of spf female c57bl/6 mice models certain of the aspects of the cns inflammation associated with ms [5] [6] [7] , being useful to test the effects of tempol treatment. functional and analytical studies have demonstrated that tempol penetrates the blood-brain barrier (reviewed in [38] ). this was confirmed by epr analysis of brain homogenates of sham and mhv-59a-infected mice sacrificed 10, 15, or 25 min after tempol administration (24 mg/kg; ip; fig. 2 ). as is usual in biological fluids and tissues, most of the tempol was present in its reduced form, the corresponding hydroxylamine. indeed, the characteristic epr signal of tempol became noticeable upon addition of ferricyanide to the homogenates [29] . total tempol (nitroxide plus hydroxylamine) levels decayed quickly in vivo, attaining about 0.3 μm in brain 25 min after administration. maximum levels (about 15 μm) were measured 10 min after administration in sham mice. the lower levels of total tempol detected in infected mice (about 3 μm after 10 min) are consistent with the course of infection producing radicals that are able to react with tempol to generate redox inactive products [29] . despite the fast metabolism of tempol in mice (fig. 2) , treatment of mhv-59a-infected mice with this compound profoundly altered the outcome of the disease. for example, the neurological symptoms and cns infiltration and demyelination were greatly reduced (fig. 3) , and mouse survival was significantly increased (fig. 4) . indeed, histopathological analysis of the forebrain regions of tempol-treated mice revealed a dramatic reduction in inflammatory cell infiltration throughout the forebrain gray and white matter regions. in addition, perivenous accumulation of inflammatory cells and disruption of the weil's myelin stain were substantially reduced (compare figs. 1b and 3b). whereas 90% of untreated mice died at 10 days after infection, 70% of the mice treated with tempol survived (fig. 4) and about half of them displayed the behavior of normal mice (fig. 3a) . these striking effects were obtained with tempol administration (24 mg/kg; ip) in two doses on the first day (5 min before and 2 h after mhv-a59 inoculation) and in daily doses for 7 days; the animals also received tempol (2 mm) in the drinking water ad libitum [29] . no adverse effects of this treatment were observed in uninfected mice (fig. 4) . previously, we have shown that tempol administered in the drinking water was distributed to tissues but we did not examine cns tissues [29] . now, we determined the levels of total tempol (tempol plus hydroxylamine) in the brain, spinal cord, and plasma of mice receiving 2 mm tempol in the drinking water. these levels were similar and around 0.3 μm (data not shown). to gain insight into the mechanisms by which tempol ameliorates viral encephalomyelitis, we first examined whether the nitroxide fig. 1. (b) representative hematoxylin/eosin-and weil-stained (bottom right) sections from brains of mhv-a59-infected mice treated with tempol 7 days postinfection. the regions were labeled as co (cortical area), amyg (amygdaloid nucleus), hip (hippocampus), and cc (corpus callosum). in contrast to the brains of untreated mice (fig. 1b) , massive inflammatory cellular infiltration and loci of myelin disruption are largely absent. affected viral load in the cns (table 1 ). one day after inoculation, virus titers were undetectable by plaque assay in the cns of both untreated and tempol-treated mice. by the time the first animals died, between 6 and 7 days after inoculation, virus titers were high in both the brain and the spinal cord of untreated mice but were significantly reduced in mice treated with tempol (table 1) . next, we examined blood-brain barrier (bbb) integrity because its loss has been associated with several neurological diseases, including ms [39] . to this end, the uptake of the fluid-phase marker sodium fluorescein was assessed quantitatively and qualitatively in the spinal cords and brains of mice, respectively. the results show that 7 days after mhv-59a inoculation there was a marked increase in the permeability of the bbb of untreated mice (fig. 5) . in contrast, tempoltreated animals presented a remarkably preserved bbb. in addition to the loss of blood-brain barrier integrity, cns inflammation is a crucial event in the pathogenesis of ms. thus, we complemented the histopathological analysis of cns tissues of mhv59a-infected mice (figs. 1b and 3b) with immunohistochemical and rt-pcr analysis of markers of inflammation. immunohistochemical analysis of the brain (fig. 6 ) and spinal cord (data not shown) tissues showed that viral infection resulted in macrophage infiltration, inos expression, and protein nitration, all of which were greatly attenuated by tempol treatment. in some brain sections of infected mice, activated glial cells strongly staining for nitrated proteins were noticeable (fig. 6, bottom left) . semiquantitative image analysis of the immunostaining of the brain tissues revealed that treatment with tempol reduced macrophage infiltration, inos expression, and 3nitrotyrosine levels to about 4, 26, and 16% of those of untreated mice, respectively (fig. 7a) . quantification of 3-nitrotyrosine levels in the spinal cords of the animals by immune slot blot confirmed that tempol inhibited its levels to about 12% of those of untreated mice (fig. 7b) . treatment with tempol also greatly reduced the levels of tnf-α and ifn-γ mrnas in the spinal cords of the animals as assessed by rt-pcr (fig. 7c) . these cytokines can be produced by t cells, which are involved in the immune and inflammatory response during viral encephalomyelitis [reviewed in 6, 7] . thus, we also quantified the levels of cd4 and cd8 mrna in the spinal cords and spleens of the animals. treatment with tempol decreased the infiltration of cd4 + and cd8 + t lymphocytes into the spinal cords, although only the differences in cd8 mrna levels were statistically significant (figs. 8a and 8c). tempol did not alter the levels of cd4 and cd8 transcripts in the spleen (fig. 8a) , indicating a preserved immune status of the treated animals. naive mice presented lower levels of cd4 and cd8 transcripts (fig. 8b ) than infected animals (fig. 8a) , as can be inferred from the relative intensity of the corresponding cd4, cd8, and β-actin bands. these results demonstrate that tempol strongly inhibited the cns inflammation of mhv-59a-infected mice (figs.1, 3, 6, and 7) without compromising their immune system (fig. 8 ). the results reported here show that treatment with tempol is quite effective in ameliorating mhv-59a virus-induced murine encephalomyelitis. in our experimental setting, the neurological symptoms and cns alterations developed quickly, and 90% of the mice died 10 days after virus inoculation (figs. 1a and 4) . treatment with tempol attenuated these neurological symptoms and increased survival up to 70% (figs. 3a and 4) . notably, half of the survivors behaved as normal mice, at least when kept at a low dose of tempol in the drinking water (2 mm). not surprisingly, treatment with tempol substantially preserved the integrity of the cns (compare figs. 1b and 3b) , including the bbb (fig. 5) . the effects of tempol in preserving the neurological behavior and the cns integrity of infected mice were accompanied by a pronounced reduction in the infiltration of macrophages and cd8 + t table 1 virus titers in the cns of mhv-59a-infected female c57bl/6 mice treated or not with tempol 1 and 7 days postinfection (dpi) a virus titer (log 10 ) (pfu/mg tissue) lymphocytes into the cns (figs. 6a, 7a, and 8 ). systemic t cells did not decrease, excluding a major immunosuppressive effect of tempol ( fig. 8 ) [40] . treatment with tempol also reduced the tissue levels of markers of inflammation, such as expression of inos, tnf-α and ifn-γ transcription, and protein nitration (figs. 6 and 7). in these animals, the inhibition of protein nitration (about 86%) was higher than that of inos expression (about 74%; figs. 7a and b), indicating that tempol is acting as both an antioxidant and a regulator of inos expression [29] . the antioxidant action of tempol during the mhv-59a virusinduced encephalomyelitis was also revealed by the faster decay rate of tempol plus hydroxylamine in the brains of infected mice compared with sham mice (fig. 2) . this indicates that the infectious process generates a considerable amount of radicals, some of which are able to react with tempol to produce redox-inactive products [29] . consequently, tempol altered the redox status of the infectious environment (fig. 2) . on the other hand, it is being increasingly recognized that the redox status of extra-and intracellular microenvironments influences the outcome of infectious/inflammatory processes [41] [42] [43] . thus, it is likely that tempol attenuated the cns inflammatory response and ameliorated viral encephalomyelitis (figs. 1 and 3-8) by altering the redox status of the infectious environment (fig. 2) . in this regard, our study supports the argument that the antioxidant and anti-inflammatory actions of tempol are likely to be interrelated [29, 38, 44] . the effects of tempol on redox signaling cascades involved in inflammatory processes, however, remain poorly investigated at the molecular level [38] . at the phenomenological level, tempol has been previously shown to display anti-inflammatory properties in mouse models of infectious/inflammatory diseases. for instance, tempol administration decreased the levels of tnf-α mrna and inhibited nf-κb activation in mice subjected to carrageenan-induced pleuritis [44] and inhibited inos expression in murine leishmaniasis [29] . in the case of the mhv-59a-infected mice, treatment with tempol reduced the levels of inos protein (figs. 6 and 7) and viral load into the cns (table 1 ). this reduction in viral load contrasts with our previous observation that in reducing inos expression and protein nitration in murine leishmaniasis, tempol increases parasite load in cutaneous lesions [29] . the opposite effects of nitric oxide-derived oxidants in different infectious diseases are likely to result from the diverse modes of microbial replication and invasion of host tissues [45, 46] . in contrast with most bacteria and parasites, virus cannot be confined to limited areas by the nonspecific host defense mediated by phagocytes and their mediators. in consequence, nitric oxide-derived oxidants are more likely to cause nonspecific nitro-oxidative damage in virusinfected tissue than to eliminate the virus [47] [48] [49] . accordingly, it has been reported that infection of inos knockout (−/−) and inos (+/+) mice with mhv resulted in similar kinetics of virus clearance, but in reduced neuronal apoptosis and mortality [50] . we did not follow the kinetics of virus clearance in our experimental setting, but we did show that 7 days after infection, tempol reduced viral load, inos expression, and cns damage. thus, it is feasible that by decreasing nitro-oxidative stress, tempol attenuates the mutation rate of the virus, precluding evolution of mutants able to escape the antiviral immune responses of the host [45, 46] . additionally, tempol [41, 42] . taken together, our results indicate that tempol ameliorates murine viral encephalomyelitis by altering the redox status of the infectious environment that contributes to an attenuated cns inflammatory response (figs. 1 and 3-8) . thus, our study argues for the interrelatedness of the antioxidant and anti-inflammatory actions of tempol [29, 38, 44] . this cross talk, however, remains to be examined at the molecular level. with regard to therapeutic implications, it is important to note that the natural antioxidant uric acid has been shown to be quite effective in ameliorating eae by inhibiting cns inflammation, bbb dysfunction, and tissue damage [8, 12, 51, 52] . this discovery motivated the initiation of clinical trials with inosine, a metabolic precursor of uric acid. oral administration of inosine increases uric acid levels in both serum and cerebrospinal fluid, whereas an oral dose of uric acid is mostly degraded by uricase in the gastrointestinal tract. the ameliorating effects of uric acid in eae have been attributed to its ability to scavenge nitric oxidederived oxidants [8, 12, 51, 52] . in comparison, tempol is more effective than uric acid in scavenging nitric oxide-derived oxidants [9] , displays low toxicity in animal models [15, 16] , permeates the blood-brain barrier (fig. 2) , and inhibits the enzyme myeloperoxidase ( [20] ; see augusto et al., [53] , which has been recently associated with ms [54, 55] . in this context, the reported ameliorating effects of tempol on viral murine encephalomyelitis provide support for the development of therapeutic strategies 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mouse hepatitis virus infection of the central nervous system uric acid, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis uric acid, a peroxynitrite scavenger, inhibits cns inflammation, blood-cns barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis inhibition of the chlorinating activity of myeloperoxidase by nitroxides immunohistochemical and genetic evidence of myeloperoxidase involvement in multiple sclerosis myeloperoxidase-targeted imaging of active inflammatory lesions in murine experimental autoimmune encephalomyelitis key: cord-285493-eg2ltip6 authors: schwab, s.; herden, c.; seeliger, f.; papaioannou, n.; psalla, d.; polizopulou, z.; baumgärtner, w. title: non-suppurative meningoencephalitis of unknown origin in cats and dogs: an immunohistochemical study date: 2007-02-01 journal: j comp pathol doi: 10.1016/j.jcpa.2006.11.006 sha: doc_id: 285493 cord_uid: eg2ltip6 non-suppurative meningoencephalitis of unknown cause is a frequent finding in dogs and cats. fifty-three dogs and 33 cats with non-suppurative meningoencephalitis of unknown aetiology were examined immunohistochemically for 18 different infectious agents, including viruses, bacteria and prion protein(sc). in 14 (26%) of the dogs and 13 (39%) of the cats a causative agent was identified in the central nervous system (cns), two dogs and one cat giving positive results for two infectious agents simultaneously. the study revealed infections with known causative agents (porcine herpes virus 1, feline infectious peritonitis virus, escherichia coli) and a new disease pattern of parvovirus infection in the cns of dogs and cats. infection of the cns with feline leukaemia virus was found in a cat. five dogs and four cats gave positive results for west nile virus (wnv) antigen. in one dog, canine parainfluenza virus antigen was detected in the brain. four dogs and four cats gave positive results for encephalomyocarditis virus (emcv). the significance of the detection of wnv and emcv antigen requires further study. the aetiology remained undetermined in 39 dogs (74%) and 20 cats (61%). although it is possible that non-infectious causes play a more important role than previously thought, infections with hitherto unrecognized agents cannot be ruled out. non-suppurative meningoencephalitis in cats and dogs is usually considered to be caused by viral infections (summers et al., 1995) , and the histopathological ¢ndings consist mainly of perivascular and parenchymal in¢ltration with lymphocytes, macrophages and plasma cells, usually associated with meningitis and occasionally with in£ammation of the plexus chorioideus and the ependyma (braund, 1980; luttgen, 1988) . viruses, due to cell tropism and route of infection, often induce a typical pattern of in£ammation in the brain and spinal cord (braund, 2001) . in newborn animals, infections of the central nervous system (cns) with bacteria, including various strains of escherichia coli, are a common sequel to septicaemia, whereas in adult dogs and cats bacteria may follow a neurogenic, otogenic or rhinogenic route to enter the cns (meric,1988; summers et al.,1995) . whilst most bacterial cns infections induce a purulent in-£ammation, some bacteria, particularly listeria spp., as well as protozoa, fungi and algae, cause a non-suppurative, mainly granulomatous in£ammation of the cns (rand et al., 1994; summers et al., 1995; tipold, 1995; quesnel et al., 1997) . in the brains of beagles with granulomatous leptomeningitis, maeda et al. www.elsevier.com/locate/jcpa (1993) demonstrated e. coli and considered it to be the causative agent. if parasites enter the cns, they often induce a granulomatous in£ammation (summers et al., 1995) . in dogs, the most common parasite to cause infection of the cns is toxoplasma gondii, followed by neospora caninum and, albeit rarely, cuterebra spp. in cats, cuterebra spp. are the most common parasites to be found in the cns, followed by t. gondii in rare cases (braund, 2001) . there would also seem to be non-infectious causes of non-suppurative meningoencephalitis. thus, autoimmune and hereditary mechanisms are suspected to be the underlying cause of cns disorders such as granulomatous meningoencephalitis (gme) in dogs (harris et al., 1988; kipar et al., 1998) , pug dog encephalitis and non-suppurative meningoencephalitis in greyhounds (callanan et al., 2002) . so far no aetiological agent has been detected for feline ''staggering disease'' or so-called feline polioencephalomyelitis (vandevelde and braund, 1979; nowotny and weissenbo º ck, 1995; braund, 2001) , although some suspect staggering disease to be due to borna disease virus (bdv) infection (lundgren et al.,1995) . paraneoplastic disorders of the cns, well known in man, have not been reported in cats and dogs (braund et al.,1987) . recent studies on a limited range of infectious agents failed to identify the cause of non-suppurative meningoencephalitis in 47% and 23% of dogs and cats, respectively (rand et al., 1994; tipold, 1995; quesnel et al., 1997; k.melzer, personal communication) . to provide further information, the present study sought to determine the cause of 86 cases of non-suppurative meningoencephalitis in dogs and cats by the immunohistochemical examination of formalin-¢xed, para⁄n wax-embedded tissue. in addition to examination for known viral and bacterial pathogens and prion protein sc ,west nile virus (wnv), a recently emerging cause of disease, was included in the present study (komar, 2000; lichtensteiger et al., 2003; austgen et al., 2004) , together with encephalomyocarditis virus (emcv), an agent so far demonstrated only in the cns of pigs and rodents (nowotny, 1996; sue et al., 2003) . fifty-three canine and 33 feline cases of non-suppurative meningoencephalitis of unknown origin, collected during the period 1998^2003 by the department of pathology of the university of veterinary medicine hannover, germany, were selected. the case selection was made in the light of a preliminary investigation which revealed that 274 of 1209 (23%) dogs and 146 of 741 (20%) cats examined in a 5-year-period (1998^2002) displayed changes in the cns. non-suppurative meningoencephalitis accounted for 22% and 49% of the cns changes in dogs and cats, respectively. the cause of 49 canine and 32 feline cases remained undetermined after examination by routine diagnostic procedures. these 81 animals, together with four dogs and one cat with undetermined non-suppurative meningoencephalitis encountered in 2003, were included in this study. due to the retrospective nature of the present study, clinical data were restricted to a short statement from the records of the department of pathology in most cases (46 dogs and 30 cats). in seven dogs and three cats, no clinical history was available. archived cns sections of each case, stained with haematoxylin and eosin (he), were re-examined for in-£ammatory changes. from each animal, one section of the cerebral cortex, the hippocampus (missing in three dogs and four cats), the midbrain (missing in ¢ve dogs and ¢ve cats), the cerebellum (missing in two dogs and two cats), the brain stem (missing in 17 dogs and 13 cats) and the spinal cord (available in only seven dogs and two cats) were examined. in one canine section, a spinal cord ganglion was present. the distribution of the lesions (e.g., polio-, leuco-, and panencephalitis) was categorized according to summers et al. (1995) . the type of in£ammation was de¢ned as follows: lymphohistiocytic, if in¢ltrates consisted mainly of lymphocytes and macrophages; granulomatous, if macrophages predominated; pyogranulomatous, if neutrophils and macrophages were the main cell types; and mixed, if macrophages, lymphocytes, plasma cells and neutrophils appeared in equal quantities. the degree of histological changes were determined semiquantitatively. perivascular in¢ltrates were described as: mild (1^15 perivascular cells per â100 ¢eld, or 1^2 cell layers per vessel, or both); moderate (16^30 perivascular cells per â100 ¢eld or 2^3 cell layers per vessel, or both); and severe (430 perivascular cells per â 100 ¢eld or x3 cell layers per vessel, or both). meningeal in¢ltrates were described as mild (one or no more than a few mild in¢ltrates), moderate (one moderate in¢ltrate or several mild in¢ltrates), or severe (several moderate in¢ltrates or con£uent in¢ltrates). if suggested by histological results, slides were additionally stained with periodic acid^schi¡ (16 dogs, six cats), ziehl^neelsen (11dogs, one cat), grocott (10 dogs, four cats), luxol-fast blue (nine dogs, one cat), cresylecht-violet (two dogs, one cat), alcian-blue (one cat) or congo-red (one cat), as described by romeis (1994) . for the detection of 18 infectious agents (table 1) , the avidin^biotin-peroxidase complex (abc) method was performed, as previously described (hsu et al., 1981; wu º nschmann et al.,1997) . due to their known cross-reactivity for the detection of canine and feline herpesvirus, parvovirus and coronavirus antigen, the same antibodies were used for both species. feline tissue was not examined for canine adenovirus 1and canine tissue was not examined for feline leukaemia virus (felv). ihc for the detection of felvantigen was additionally performed on lymphoid tissue; however, such tissue was available from only 20 of the 33 cats. in one dog that showed immunoreactivity for porcine herpesvirus 1, tissue from the gastrointestinal tract was also examined, and in one dog and two cats showing immunoreactivity for emcv, myocardial tissue was examined. brie£y, specimens were dewaxed in graded alcohols and endogenous peroxidase was quenched with h 2 o 2 0.5% in ethanol.the pre-treatments applied to sections (table 2 ) depended on the primary antibody and were performed as previously described (see below). the various pre-treatments were as follows: pronase e (merck, darmstadt, germany; bahn, 1988) ; trypsin (fluka, neu ulm, germany; nietfeld et al., 1989) ; kovacevic et al., 1997) ; pepsin (dako, hamburg, germany; bahn, 1988) , proteinase k and formic acid (merck, darmstadt, germany; hardt et al., 2000) ; and microwave pre-treatment (kahveci et al., 2003) . goat serum, diluted 1 in 5 in phosphate-bu¡ered saline (pbs), served as a blocking serum. primary antibodies were, unless stated otherwise, diluted in pbs containing bovine serum albumin (bsa) 1%, and incubated overnight (12^16 h) at 4 1c ( table 2) . biotinylated goat-anti-mouse (gam-b), goatanti-rabbit (gar-b) and goat-anti-£uorescein-isothiocyanate (ga-fitc-b; directed against the fitcligated anti-feline infectious peritonitis [fip] virus antibody) antibodies served as secondary antibodies (vector laboratories, burlingame, ca, usa) and were all diluted 1 in 200 in pbs. finally, sections were incubated with abc (vector) for 30 min at room temperature. between the incubation steps, sections were thoroughly rinsed with pbs. positive antigenâ ntibody reactions were ''visualized'' by incubation with 3,3 0 -diaminobenzidine-tetrahydrochloride (dab) with h 2 o 2 0.5% for 10 min followed by mild counterstaining with haematoxylin. sections were mounted with roti-histokit (carl roth kg, karlsruhe, germany) under coverslips. in control sections the primary and secondary antibodies were replaced by pbs, control serum or isotype-speci¢c antibodies directed against irrelevant antigens. toy breeds constituted 27% (14) ). animals aged less than 1 year accounted for 38% of a¡ected dogs,37% were1^5 years old, and 21% were aged45 years. in 4% of dogs the age was unknown. the domestic shorthair breed accounted for 52% of affected cats. in 11 cats the breed was unknown. cats less than 1 year of age accounted for 27%, 26% were aged 1^5 years, and 26% were45 years old. in 21%, the age was unknown. the sexes were represented equally in both species. neurological signs, including ataxia, seizures and central blindness, were reported in 35 dogs (68%). nine dogs (17%) showed other symptoms such as vomiting and diarrhoea but lacked neurological signs. one dog died spontaneously and one under anaesthesia during routine surgery, no clinical signs having been reported before death. in seven dogs no clinical data were available. neurological signs were reported in 17 cats (52%). thirteen (39%) showed gastrointestinal or respiratory signs but not neurological signs. in three cats, one of which died spontaneously, no clinical data were available. lymphohistiocytic, perivascular in¢ltrates were found in 40 dogs (75%) and 23 cats (70%). predominantly granulomatous in¢ltrates were found in 10 dogs and two cats. mixed in£ammatory in¢ltrates appeared more often in cats (six animals) than in dogs (two animals). two cats and one dog exhibited pyogranulomatous in£ammation. in 39 dogs (74%), encephalitis was the most prominent ¢nding. in 13 dogs (25%), in¢ltrates were restricted to the meninx and a ganglioneuritis without encephalitis or meningitis was found in one animal. in 26 cats (79%), encephalitis was observed. in six cats (18%), in¢ltrates were restricted to the meninx and a chorioiditis without encephalitis was found in one animal. six dogs showed, in addition to the in£ammatory changes in the cns, mild-to-moderate vacuolation of the white matter of the cerebrum (three animals) or cerebellum (one animal) or both (two animals). luxol-fast-blue staining revealed demyelination in two of these dogs. only one of these six dogs showed an in-£ammatory in¢ltrate within the areas of vacuolation. two cats showed in addition to the in£ammatory changes in the cns, mild vacuolation of the white matter of the cerebellum (one animal; severe) or both cerebrum and cerebellum (one animal; mild). luxol-fastblue staining did not reveal demyelination. of the nine dogs without neurological signs, one showed mild, focal meningitis and two showed mild, multifocal meningitis. moderate in£ammation, consisting of multifocal or focal in¢ltrations in various brain regions (midbrain, cerebellum, meninges, choroid plexus) was observed in six dogs. in£ammatory changes were lymphohistiocytic (six cases), mixed (two cases) or granulomatous (one case). the 13 cats without neurological signs showed mild, multifocal in£ammation of the meninx (two cases), meninx and cerebellum (two), meninx and cerebrum (one), meninx, cerebrum and brainstem (one), meninx 8000 (pbs contained 20% nss but no bsa), adsorbed to rat brain powder microwave gam-b anti-prion protein sc 400 (pbs contained 10% goat serum but no bsa); no further blocking with goat serum necessary formic acid, proteinase k, microwave anti-e. coli 1600 none gar-b nss, normal swine serum; tuf, target unmasking £uid; gam-b, biotinylated goat-anti-mouse antibody; gar-b, biotinylated goat-anti-rabbit antibody; ga-fitc-b, biotinylated goat-anti-fitc antibody (see materials and methods). ã against agents listed intable 1. y in phosphate-bu¡ered saline (pbs) containing bovine serum albumin (bsa) 1%. and brainstem (one) or choroid plexus (one). also seen were mild-to-moderate in£ammation of the brainstem (one) and of the meninx and cerebral cortex in addition (one). one cat showed moderate, focal meningitis, one a moderate multifocal meningoencephalitis and one a severe, generalized choroiditis. the type of in£ammation was lymphohistiocytic in six cases, mixed in three, granulomatous in two, and pyogranulomatous in two. the gastrointestinal tissue examined in one dog, and the myocardial tissue examined in one dog and two cats, showed no signi¢cant histological changes. in 14 dogs (26%) and 13 cats (39%), viral or bacterial antigens were detected by ihc (table 3) , two dogs and one cat giving positive results for two infectious agents simultaneously. control sections containing the appropriate antigens gave positive signals, in contrast to negative controls. special histochemical stains invariably gave negative results. in12 of the14 positive dogs and nine of the13 positive cats, reactions were found only in cns regions in¢ltrated with in£ammatory cells. the remaining two dogs and four cats showed immunoreactivity only in cns regions without in£ammatory in¢ltration. of the 14 positive dogs,12 (94%), had shown neurological signs, almost always said to be severe. it was not possible, however, to relate the histological or ihc results to the clinical signs. one dog positive for emcv antigen did not show neurological signs, and for one dog positive for wnv antigen no clinical data were available. of the 13 positive cats, eight had shown neurological signs (severe in three cases). in the remaining ¢ve cats, no statement regarding the severity of signs was recorded. four cats with a positive immunoreaction showed no neurological signs. for one positive cat, no clinical data were available. in a 2-year-old male canadian shepherd dog with diarrhoea and progressive convulsions, porcine herpesvirus-1 (phv-1) antigen was demonstrated in neuronal perikarya of the gyrus parahippocampalis. no phv-1 antigen was detected in the hippocampus, cerebellum, brain stem, spinal cord, remaining part of the cerebrum, or the gastrointestinal tract. histologically, there was mild, lymphohistiocytic polioencephalitis in the gyrus parahippocampalis and occasional neuronal necrosis in the ca1 and ca2 regions of the hippocampus. parvovirus antigen was demonstrated in the cns of ¢ve greek hunting dog puppies from two litters originating from the same breeder, and in a 2-week-old cat. clinically, the dogs had shown generalized tremor and jumping movements in the hind limbs, and the neurological signs in the cat were characterized by severe ataxia, opisthotonus and convulsions. neither in the dogs nor in the cat were cerebellar hypoplasia or necrotic purkinje cells observed. histopathologically, all affected dogs showed mild to moderate lymphohistiocytic meningitis or leucoencephalitis (or both) as well as mild to moderate, and in one case severe, vacuolation in the white matter of cerebrum and cerebellum. parvovirus antigen was demonstrated in periventricular cells resembling spongioblasts, in macrophages, microglia and astrocytes, and in cells of the outer granular layer of the cerebellum (fig. 1) . the histopathological changes in the cat consisted of mild, lymphohistiocytic in£ammation of the meninges and cerebellum, without vacuolation. parvovirus antigen was detected in the cytoplasm and nucleus of numerous small and large neurons, mainly in the granular layer and outer granular layer of the cerebellum, in the grey matter of the spinal cord, and multifocally in most other brain regions (fig. 2) . in addition, parvovirus antigen was observed in macrophages, microglia, astrocytes and ependymal cells. fip virus antigen was detected in three cats aged 6,18 and 36 months.two a¡ected cats had shown increasing apathy and recurrent fever attacks (one animal) or diarrhoea and dyspnoea (one). no clinical data were available for the third cat. viral antigen was demonstrated in the cytoplasm of macrophages within the in-£amed brain regions in all three cases. histologically, lesions consisted of pyogranulomatous and mixed in¢ltrates in the plexus chorioideus and the meninges. the third cat showed moderate lymphohistiocytic leucoencephalitis in the caudal regions of the cerebrum. weak immunolabelling for feline leukaemia virus antigen was seen in microglia and astrocytes in the hypothalamus of a 9.5-year-old european shorthair cat with progressive apathy and increased salivation (fig. 3) . histopathological changes consisted of mild multifocal lymphohistiocytic meningitis and severe satellitosis in the cerebral cortex. no lymphoid tissues or other organs were available for further investigations. five dogs (8.9%) and four cats (11.8%) showed immunoreactivity for west nile virus (wnv) antigen. in one dog, canine parain£uenza virus (cpiv) antigen was also detected in the same brain region (see also below). one of these dogs, and one cat also gave positive reactions with emcv-speci¢c antibodies (see also below). in four of the ¢ve dogs, severe neurological signs, including ataxia, convulsions and central blindness, had been recorded. no clinical data were avail-able for the ¢fth dog. all four cats had shown neurological signs, reported in one case to consist of severe circling, hypermetria and blindness. in four of the ¢ve dogs and two of the four cats a moderate to severe meningoencephalitis a¡ecting the grey and white matter was observed; this varied from granulomatous, pyogranulomatous, lymphohistiocytic to ¢brinopurulent, occasionally associated with neuronal necrosis or malacia. in the remaining dog, in£ammation was restricted to the grey matter of the cerebrum and cerebellum, being associated with severe malacia in the hippocampus. one cat showed mild focal, lymphocytic polioencephalitis and moderate focal, lymphohistiocytic meningitis, as well as severe vacuolation of the cerebral white matter and severe generalized neuronal necrosis. in another cat, severe, focal, ¢brinopurulent meningitis and severe, acute neuronal necrosis in the hippocampus were observed. wnv immunolabelling was seen in the cytoplasm of various types of neurons, macrophages, astrocytes and microglia (fig. 4) . strong immunolabelling of neutrophils was observed in two cats with pyogranulomatous in£ammation (fig. 5) . these immunohistochemical reactions were in general more intense with the anti-nsp-1wnvantibody than with the anti-mep-e wnvantibody. in some cases, immunoreactivity was observed with only one of these two wnv monoclonal antibodies. one dog showed immunoreactivity in pyramidal cells of the ca1 and ca2 sector of the hippocampus, and another in the frontal cortex, respectively. a positive reaction in numerous macrophages and microglial cells in the midbrain, pons and frontal cerebrum was detected only with the anti-nsp-1 antibody. one dog showed wnv immunolabelling in numerous macrophages and microglial cells, and pyramidal cells in the cerebrum, hippocampus and midbrain, but only with the anti-nsp-1 antibody. another dog, in contrast, showed immunoreactivity in a few neurons and macrophages in an in£amed part of the midbrain, but only with the anti-mep-e antibody. one cat showed immunolabelling with both wnv antibodies in numerous neutrophils and occasional macrophages in the plexus chorioideus and laterally to the third ventricle. in this cat, small neurons, macrophages and microglial cells in the same brain region reacted only with the anti-nsp-1 antibody. in another cat, astrocytes and perivascular macrophages in the lobus parietalis reacted with both antibodies, whereas the anti-nsp-1 antibody additionally labelled pyramidal cells in the same brain region. in one cat, astrocytes and oligodendrocytes of the medulla oblongata displayed more abundant positive signals with the anti-nsp-1 antibody than with anti-mep-e. additionally, in this animal, the anti-nsp-1 antibody labelled large neurons in the medulla oblongata which failed to react with the anti-mep-e antibody. canine parain£uenza virus (cpiv) antigen was detected in the brain of a 9-year-old male collie that was also positive forwest nile virus antigen (see alsownv, above). this dog had exhibited changed behaviour and was generally weak. histopathologically, severe periventricular granulomatous meningoencephalitis was found. viral antigen was demonstrated in the cytoplasm of occasional macrophages in the hypothalamus (fig. 6) . ihc revealed encephalomyocarditis virus (emcv) antigen in the brains of four dogs (7.1%) and four cats (11.8%). in one dog and one cat, wnv-antigen was detected in addition (see also wnv, above). the dogs were aged 0.3^8 years and the cats 0.2^10 years. two domestic shorthairs, one persian and one cat of unknown breed as well as a landseer, a west highland white terrier, a bordeaux dogge and an entlebucher sennenhund were a¡ected. a granulomatous immune response was found in three of the four dogs. the remaining dog displayed a lymphohistiocytic chorioiditis and meningitis. in¢ltration with macrophages, neutrophils, lymphocytes and plasma cells or pyogranulomatous in£ammation was present in two of the four cats, and lymphohistiocytic in£ammation in the remaining two cats. in three of these four dogs, severe neurological signs had been recorded; the fourth dog had shown dyspnoea and anaemia but no neurological signs. in three of the cats, neurological signs were recorded without reference to their severity. the fourth cat showed respiratory signs only. the eight animals showed di¡erent patterns of immunoreactivity. dab precipitates were observed in the cytoplasm of perivascular and parenchymal macrophages, in the perikarya and processes of various neurons distant non-suppurative meningoencephalitis from or within the in£ammatory lesions (fig.7) , and in periventricular and submeningeal astrocytic foot processes (fig. 8) . in one dog and two cats, vessel-associated cells, probably pericytes or endothelial cells, showed immunolabelling (fig. 9) . seven of the eight animals showed immunolabelling with the emcv-speci-¢c monoclonal antibody (mab) 4f3; mab 3e5 gave results in selected cases only. one cat which was negative with the 4f3 antibody gave emcv-speci¢c reactions with mab 3e5. in one dog the results with mab 4f3 were con¢rmed with the mab 3e5. the mab 4e4, which was applied only in one cat, con¢rmed the reactions observed with the 4f3 antibody in numerous cells of the medulla oblongata and in perivascular macrophages and occasional neurons of the mediocaudal thalamus. the histologically intact myocardial tissue available from one dog and two cats was not immunolabelled with the mab 4f3. e. coli antigen was demonstrated in the brain of a 5-month old male cat. histopathologically, mild to moderate, multifocal, perivascular in¢ltration with lymphocytes, plasma cells, macrophages and neutrophilic granulocytes was observed in the cerebellum and meninges. moderate numbers of bacterial emboli in various vessels were visible in he-stained sections. e. coli antigen was demonstrated immunohistochemically in the cytoplasm of numerous perivascular, meningeal and periventricular macrophages, and in intravascular bacteria. cases remaining unclear included 39 (74%) dogs and 20 (61%) cats, no infectious agent being identi¢ed as the underlying cause of the observed non-suppurative meningoencephalitis. in the present study, cns tissue of 53 dogs and 33 cats with non-suppurative meningoencephalitis of unknown cause was investigated for the presence of 18 different infectious agents. former studies either investigated at most six agents or were based on cases selected only on the presence of neurological signs (tipold, 1995; quesnel et al., 1997; bradshaw et al., 2004; k. melzer, personal communication) . as in other studies (sorjonen, 1992; tipold, 1995) , no speci¢c canine breed predisposition was observed. cases were seen more often in toy breeds than in other breeds, but this may merely have re£ected the normal distribution of dog breeds in germany. so far, no breed predisposition for non-suppurative meningoencephalitis has been described in respect of the cat (sorjonen, 1992) . in the present study, domestic shorthair cats accounted for more than half of the feline cases, probably due to the normal feline breed distribution in germany. the cns of the majority of dogs (40/53) and cats (23/ 33) examined showed lymphohistiocytic perivascular in¢ltrates. this is generally suggestive of a viral aetiology (summers et al., 1995) but has also been reported following hypoxic or toxic damage, which also sometimes produces demyelinating lesions (braund, 1980; lee et al., 2002) . granulomatous cns in¢ltrates, observed in 10 dogs and two cats, are often considered to be associated with fungal, parasitic or bacterial infections. fungal infections occur mainly in warmer climates (gerds-grogan and dayrell-hart, 1997 ) and, like parasitic infections, were not detected in the present study. in the 10 dogs referred to above, granulomatous meningoencephalitis (gme) was suggested by the histopathological ¢ndings and distribution of lesions. in six of these cases, toy breeds were a¡ected (twowest highland white terriers, two chihuahuas, a shih tzu and a rehpinscher). interestingly, three of the a¡ected dogs belonged to large breeds (landseer, bordeaux dogge and collie), in which gme is only rarely reported (braund, 1985) . gme has been thought to be associated with viral infections including canine distemper, but no viral agent has been demonstrated in the cns so far (thomas and eger,1989; kipar et al.,1998) . e. coli antigen, as found in the brains of beagles with granulomatous leptomeningitis (maeda et al., 1993) or in association with human cases of granulomatous encephalitis (rickert et al., 2000) , was not observed in the present cases with suspected gme. the cat with abundant e. coli antigen in the brain in the present study showed perivascular in-¢ltrations, indicating septicaemia. pyogranulomatous and mixed in£ammatory in¢ltration in the cns occurred less often in dogs than in cats. in cats, this type of change is reminiscent of fip (rand et al.,1994) . in the present study, fip virus antigen was also detected in a cat showing lymphohistiocytic leucoencephalitis, a rare consequence of fip (summers et al.,1995; braund, 2001) . in dogs, pyogranulomatous or mixed in£ammatory changes in the cns are occasionally described, but no infectious agents have so far been detected (meric,1988) . it cannot be ruled out that aetiologically undetermined feline cases were due to feline immunode¢ciency virus (fiv), which leads to lymphocytic encephalitis in 30% of naturally infected cats (gunn-moore et al., 1996) . only ¢ve of the 33 cats had been examined serologically for fiv, a positive result having being obtained in one animal. in a canadian shepherd dog, porcine herpesvirus 1 (phv 1) antigen was detected in neurons of the gyrus parahippocampalis. due to a retrograde neural route of infection, phv 1 antigen usually occurs in the brain stem or spinal cord ganglions (quiroga et al., 1998; honavar and meldrum, 2002) . the dog in this study showed diarrhoea for 3 weeks, reminiscent of the rare alimentary form of aujeszky disease (pensaert and maes, 1987) ; however, no phv 1 antigen was detected in the gastrointestinal tract. only later did the animal develop neurological signs, and an unusual portal of entry of the virus to the brain remains a possibility. neither the ¢ve dogs nor the cat with parvovirus infection of the brain showed typical cns manifestations, such as cerebellar hypoplasia or death of purkinje cells (sharp et al., 1999) , or encephalomalacia (johnson and castro, 1984; agungpriyono et al., 1999) . the ¢ndings indicated, however, a potential association between parvovirus and spongiform lesions in the cns. in chronic feline leukaemia virus (felv) infection, lymphocytic in¢ltration of peripheral nerves of the spinal cord has been reported occasionally (pedersen, 1987) . in the brain, however, felv infection has been associated only with degenerative cns disease (carmichael et al., 2002) . in one cat, felv infection of microglia and astrocytes was detected. histologically, this animal showed mild, multifocal, lymphohistiocytic meningitis. since felv regularly persists for years in lymphocytes and macrophages (rohn and overbaugh,1999) , a persistent infection of glial cells seemed possible but unfortunately no lymphoid tissue, including bone marrow (in which felv-antigen can typically be demonstrated), was available. surprisingly, ¢ve (9.4%) dogs and four (12%) cats showed immunolabelling for wnv. histopathologically, these animals showed mild to moderate meningoencephalitis in the grey and white matter, mostly with granulomatous or pyogranulomatous in£ammation. in contrast, previous reports of wnv infection in birds and in mammals indicated that the grey matter was mainly a¡ected and that the histopathology was characterized by lymphoplasmahistiocytic in£ammation with small numbers of neutrophils (steele et al., 2000; cantile et al., 2001; buchweitz et al., 2003; kelly et al., 2003; lichtensteiger et al., 2003) . polyclonal wnv-speci¢c antibodies used for immunolabelling often cross-react with other £aviviruses (steele et al., 2000; kelly et al., 2003; chvala et al., 2004) . the monoclonal antibodies speci¢c for the major envelope protein e (anti-mep) and non-structural protein 1 (anti-nsp-1) used in this study (tables 1 and 2 ) crossreact with kunjin virus, also a member of the flaviviridae (scherret et al., 2001) . presumably, the anti-nsp-1 antibody is more speci¢c than the anti-mep antibody (hall, 2000) . this might explain its stronger immunoreactivity in the present study and might support the identi¢cation of a wnv infection. the a¡ected animals did not react positively for tick-borne encephalitis (tbe) virus antigen with a polyclonal speci¢c antibody that may cross-react with other £aviviruses (h. weissenbo º ck, personal communication). however, it is still possible that the positive wnv reaction was due to infection with other cross-reactive agents or represented molecular mimicry of host-derived antigens (oldstone, 1998) . the latter possibility receives some support from studies indicating that dogs and cats can be infected with wnv but usually develop only a low viraemia, without clinical signs or histopathological changes in the brain (blackburn et al.,1989; lichtensteiger et al., 2003; austgen et al., 2004) . furthermore, no evidence of wnv infections in domestic animals has so far been detected in germany. therefore, the signi¢cance of the present ¢ndings requires further studies. natural infections of dogs with canine parain£uenza virus (cpiv) are typically associated with hydrocephalus and necrotizing periventricular encephalitis, but neither viral antigen nor rna has been detected so far in spontaneous cases (baumgìrtner et al., 1982b; vieler et al.,1994; cantile et al.,1997) . after experimental infection with cpiv, viral antigen was detected in ependymal cells and neurons and in macrophages, in ferrets and dogs respectively (baumgìrtner et al., 1982a (baumgìrtner et al., , b, 1989a w. baumgìrtner, unpublished) . cpiv infection of brain macrophages was also observed in the present study in a collie with granulomatous encephalitis characteristic of gme. interestingly, this dog also showed immunolabelling of wnv antigen. it remains unclear, therefore, whether the detection of cpivantigen represented a true infection, or whether immunoreactivity was due to a cross-reaction of the antibody with degraded cellular antigens, or to molecular mimicry (oldstone,1998) . a further surprising ¢nding was the detection of emcvantigen in the cns of four dogs and four cats. emcv-speci¢c antibodies have been detected in primates, mice, rats, horses and elephants in various parts of the world, the rat being the natural host (nowotny et al.,1993; nowotny,1996; psalla et al., 2006) . in austria, a low seroprevalence has been reported in cats (nowotny,1996) , but not dogs. in germany, emcv-associated non-suppurative meningoencephalitis is reported occasionally in man, but the virus has been isolated from the cns in only a few cases (nowotny et al., 1993; larue et al., 2003) . the histopathological changes reported here varied (neuronal necrosis, mild lymphohistiocytic meningitis, severe generalized granulomatous or pyogranulomatous meningoencephalitis). in pigs with natural emcv infections, lymphohistiocytic in¢ltrates in the cns have been described (gainer et al., 1968) . experimentally infected mice and pigs also develop lymphohistiocytic, perivascular in¢ltrates in the cns, poliomeningoencephalomyelitis being the predominant feature in mice (topham et al., 1991; larue et al., 2003) . in the present study, emcv immunolabelling was observed in perikarya and processes of neurons in some animals, indicating neurotropism similar to that observed in experimental emcv infection. the emcv-positive cells, associated with cerebral vessels, may have been endothelial cells or pericytes. neither cell type has been reported to be susceptible to emcv, but endothelial and mesenchymal cells in organs other than the cns are regularly infected with emcv, as shown by papaioannou et al. (2003) in pigs. the detection of emcv antigen in macrophages in the cns of dogs and cats may support their role in viral replication and distribution, as proposed by papaioannou et al. (2003) . in natural emcv infection of pigs, interstitial myocarditis and myocardial degeneration represent the main features (papaioannou et al., 2003) . neither change was observed in any of the present cases, but myocardial tissue was available from only one dog and two cats. despite the broad spectrum of antibodies used, the aetiology of the cns lesions could be determined in only 26% and 39% of the cases in dogs and cats, respectively. due to the retrospective character of the study, a standardized clinical and neurological investigation and serum and csf laboratory procedures were not possible. in the remaining cases, infection cannot be ruled out. other agents that might have caused the observed histopathological changes in dogs include ehrlichia spp. (glaus and jaggy, 1992; maretzki et al., 1994) , leptospira spp. (rentko et al., 1992) and borrelia spp. (mandel et al.,1993; chang et al., 2001) , and in cats bartonella henselae (breitschwerdt et al., 1999) cannot be excluded. however, the high percentage of cases in which no infectious agent was identi¢ed suggests that non-infectious causes are more common than anticipated. various factors, ranging from autoimmune processes to intoxication, have already been suggested as causes of non-suppurative changes in the cns (kipar et al.,1998; , lee et al., 2002 . the immunohistochemical reactions for wnv, cpivand emcv, which were unexpected, were possibly due to molecular mimicry, a phenomenon reported for various paramyxoviruses and forjapanese encephalitis virus (jpev; shesberadaran and norrby, 1984) , which belongs to the flaviviridae. in most of the cases showing these unexpected reactions the histopathological changes were severe and often resembled gme. autoimmune mechanisms have already been discussed as a possible cause of canine gme and pug dog encephalitis (kipar et al., 1998; . other factors that may have played a role in the ¢ndings described include the following: epitope spreading vanderlugt et al., 1998) ; hereditary factors, as already suspected in pug dog encephalitis and non-suppurative meningoencephalitis in greyhounds (hinrichs et al., 1996; callanan et al., 2002) ; intoxication (lee et al., 2002) ; increased permeability of the blood-brain barrier in systemic illness, due to prostaglandins and interleukin (il)-1, il-6 or tumour necrosis factor (tnf)-a, leading to in¢ltration of the cns by in£ammatory cells (reiss et al., 2000) . in addition, it should be mentioned that, unlike paraneoplastic encephalitis, which has been described in man but not in dogs or cats (scaravilli et al., 1999) , paraneoplastic polyneuropathy has been described in dogs (braund, 1990; wagner, 2002) . as tumours in the cns or other organs were present in only one of the cases examined, paraneoplastic encephalitis seemed unlikely to have played any role in the ¢ndings. in conclusion, in a high proportion of dogs and cats with non-suppurative meningoencephalitis, immunohistochemical examination with antibodies against 18 di¡erent infectious agents failed to reveal a cause. the signi¢cance of positive immunoreactions obtained with wnv and emcv antibodies requires further investigation. in addition, primary or virus-triggered secondary immune-mediated mechanisms should be considered in many in£ammatory cns disorders of dogs and cats. subacute massive necrotizing myocarditis by canine parvovirus type 2 infection with di¡use leukoencephalomalacia in a puppy experimental infection of dogs and cats with west nile virus. emerging infectious diseases the proteolytic pretreatment of formalin-¢xed tissue in immunohistochemical diagnosis. acta histochemica, supplements canine parain£uenza virus-induced encephalitis in ferrets acute encephalitis and hydrocephalus in dogs caused by canine parain£uenza virus naturally occurring canine distemper virus encephalitis: distribution and expression of viral polypeptides in nervous tissues chlamydia pneumoniae in koronarem plaquegewebe: vermehrter nachweis bei akutem koronarsyndrom susceptibility of dogs to west nile virus: a survey and pathogenicity trial the functional signi¢cance of epitope spreading and its regulation by co-stimulatory molecules a retrospective study of 238 cases of neurological disorders of the cat granulomatous meningoencephalomyelitis remote e¡ects of cancer on the nervous system in£ammatory diseases of the central nervous system peripheral neuropathy associated with malignant neoplasms in dogs fading kitten syndrome and neonatal isoerythrolysis reverse transcriptase-polymerase chain reaction, and immunohistochemical detection of west nile virus in a clinically a¡ected dog a novel non-suppurative meningoencephalitis in young greyhounds in ireland hydrocephalus with periventricular encephalitis in the dog pathologic and immunhistochemical ¢ndings in naturally occurring west nile infection in horses feline leukaemia virus-associated myelopathy in cat experimental induction of chronic borreliosis in adult dogs exposed to borrelia burgdorferi-infected ticks and treated with dexamethasone pathology and viral distribution in fatal usutu virus infections in birds from the intranasal infection of ferrets (mustela putorius furo) with canine parain£uenza virus localization of neutralizing regions of the envelope gene of feline leukaemia virus by using anti-synthetic peptide antibodies zur diagnostik der aujeszkyschen krankheitçeine vergleichende untersuchung immunhistologischer und virologisch-kultureller nachweismethoden high mortalitiy in a florida swine herd infected with the encephalomyocarditis virus. an accompanying epizootiological survey feline cryptococcosis: a retrospective evaluation ehrlichiose beim hund: literaturu º bersicht und fallbeschreibung encephalitis associated with giant cells in a cat with naturally occurring feline immunode¢-ciency virus infection demonstrated by in situ -hybridization the emergence of west nile virus: the australian connection a comparative study of immunohistochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies simultaneous central nervous system reticulosis in two related afghan hounds. compendium on continuing education for the practicingveterinarian comparison of di¡erent methods of diagnosing borna disease in horses post mortem ein fall von nekrotisierender meningoencephalitis beim mops (pug dog encephalitisçpde) (in german epilepsy use of avidinbiotin-peroxidase complex, abc, in immunoperoxidase techniques isolation of canine parvovirus from a dog brain with severe necrotizing vasculitis and encephalomalacia a comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin ¢xed the histopathology of rectosigmoid biopsies from adults with bloody diarrhea due to verotoxin-producing the neuropathology of west nile virus meningoencephalitis immunohistochemical characterization of in£ammatory cells in brains of dogs with granulomatous 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practicing veterinarian granulocytic ehrlichiosis and meningitis in a dog canine meningitisça changing emphasis rabies-like inclusions in dogs serological studies of domestic cats for potential human pathogenic virus infections from wild rodents encephalomyocarditis-virusinfektion beim german.). wiener tierìrztliche monatsschrift description of feline meningoencephalomyelitis (''staggering disease'') and studies of its aetiology molecular mimicry and immunemediated diseases pathogenesis of encephalomyocarditis virus (emcv) infection in piglets during the viraemia phase: a histopathological, immunohistochemical and virological study feline leukaemia virus pseudorabies virus (aujeszky 0 s disease) pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats diagnostic evaluation of cats with seizure disorders: 30 cases diagnosis of aujeszky 0 s disease virus infections in dogs by use of immunohistochemistry and in-situ-hybridization clinical, cerebrospinal £uid and histological data from twenty-seven cats with primary in£ammatory disease of the central nervous system innate immune responses in viral encephalitis canine leptospirosis: a retrospective study of 17 cases pathogenic feline retroviruses: feline leukaemia virus and feline immunode¢-ciency virus mikroskopische technik, 17th edit the neuropathology of paraneoplastic syndromes hydranencephaly and cerebellar hypoplasia in two kittens attributed to intrauterine parvovirus infection three monoclonal antibodies against measles virus f protein cross-react with cellular stress proteins myelitis and meningitis pathology of fatal west nile virus infections in native and exotic birds during the susceptibility of primary cell culture neurones from rats of di¡erent ages to encephalomyocarditis, emc, virus infection granulomatous meningoencephalomyelitis in 21 dogs diagnosis of in£ammatory and infectious diseases of the central nervous system in dogs: a retrospective study indirect role of tcells in development of polioencephalitis and encephalomyelitis induced by encephalomyocarditis virus the functional signi¢cance of epitope spreading and its regulation by co-stimulatory molecules polioencephalomyelitis in cats isolation of parain£uenzavirus type 2 from the prostatic £uid of a dog immunohistochemical detection of encephalomyocarditis virus (emcv) both in formalin and ethanol-¢xed, para⁄n-embedded tissues of pigs identi¢cation of cd4+ and cd8+ t cell subsets and b cells in the brain of dogs with spontaneous acute, subacute, and chronic-demyelinating distemper encephalitis glycoprotein speci¢c immune response in canine herpesvirus infection for providng antibodies, the authors thank: prof. haas, institute of virology, university of veterinary medicine hannover, germany, prof. richt, national animal disease center, ames, iowa, usa; dr eskens, veterinìr-untersuchungsamt mittelhessen; prof. holzmann, institute of virology, university of vienna; prof. groschup, bundesforschungsanstalt fu º rviruskrankheiten dertiere, insel riems; dr randall, department of biochemistry and microbiology, university of st andrews, uk; and dr o º rvell, huddinge hospital, huddinge, sweden. thanks are also due to mrs petra gru º nig and mrs danuta waschke for excellent technical support. key: cord-017144-k7fqneup authors: oleszak, emilia l.; kuzmak, jacek; varadhachary, arun; katsetos, christos d. title: nitric oxide in tmev date: 2005 journal: experimental models of multiple sclerosis doi: 10.1007/0-387-25518-4_35 sha: doc_id: 17144 cord_uid: k7fqneup we and others have previously investigated the role of inducible nitric oxide synthase (inos) on early acute and late chronic demyelinating disease induced by theiler’s murine encephalomyelitis virus (tmev). infection of susceptible sjl mice with this virus serves as an excellent model of virus-induced demyelinating disease, such as multiple sclerosis (ms). inos transcripts and protein were detected in brains and spinal cords of tmev-infected sjl mice during early acute disease, which resembles polioencephalomyelitis. similar level of expression of inos has been found in resistant b6 mice, which develop only early acute disease. weak inos staining was detected in reactive astrocytes and in leptomeningeal infiltrates in tmev-infected sjl mice at 42 days post infection (p.i.), corresponding to early phase of chronic demyelinating disease, but not at 66 and 180 days p.i. corresponding to advanced and terminal stages of the disease, respectively. results from other laboratories demonstrated that, blocking of no by treatment of tmev-infected sjl mice with amino guanidine (ag), a specific inhibitor of no resulted in delay of late chronic demyelinating disease. however this protective effect of no inhibitor depended on the temporal phase of the disease, type of cells expressing inos and the time of administration of ag. the results from our laboratory suggests that no expressed during early acute disease is beneficial to the host through induction of apoptosis of infiltrating t cells and resolution of encephalitis, but its role in myelin/oligodendrocytes damage during late chronic demyelinating disease is not clear and it may depend on availability of superoxide and formation of peroxynitrite. . introduction theiler's virus (tmev) induces a biphasic disease in susceptible strains of mice (such as sjl). early acute disease (polioencephalomyelitis) is characterized by replication of the virus in gray matter (i-4) . this phase of disease is associated with neuronophagia and inflammatory infiltrates in the cerebral gray matter and anterior horn cells of spinal cord (4, 5) . within two to three weeks the virus is partially cleared and approximately 35 days p.i. susceptible mice develop late chronic demyelinating disease. the virus persists at very low level in microglia/macrophages, astrocytes and oligodendrocytes (1, (5) (6) (7) (8) (9) . heavy inflammatory infiltrates comprised of cd4 + and cd8 + t cells, macrophages, and few b cells are present, exclusively in the spinal cord (7, 9, 10) . demyelinating lesions are associated with perivascular and parenchymal inflammatory infiltrates. tmev-induced late chronic demyelinating disease is an excellent animal model for human ms(l, 2, 7, 9, 11) . resistant strains of mice (such as c57bl/6) develop only early acute disease, clear the virus completely and do not develop demyelinating disease. ms is the most common inflammatory demyelinating disease in humans. it has been suggested that t cells specific for a myelin component such as mbp (myelin basic protein, plp (proteolipid) and/or mog (myelin oligodendrocytes protein), and activated microglia/macrophages participate in myelin damage (12) (13) (14) (15) . epidemiological evidence suggests that ms could be triggered by virus (es) acquired before puberty. the mechanism(s) of the pathogenesis of inflammatory demyelinating diseases is not well understood. although oligodendrocytes/myelin may be damaged by a direct attack of cytotoxic t cells, other cells, including cd4 + t cells, activated macrophages, and microglia may contribute to myelin destruction by the production of cytokines, such as il-1, ifn-7, and tnf-o¢,.and reactive oxygen and reactive nitrogen species (14, 16) . nitric oxide (no) is a short lived, highly reactive molecule with free radical properties. it is synthesized by nitric oxide synthase (nos) by converting of l-arginine to l-citrulline, reviewed in (17, 18) . there are three isoforms of nos; nostype i and nos-type iii are calcium dependent and are constitutively expressed. this results in the production of physiological levels of no which acts as second messenger in the no signaling pathway in the neuronal and cardiovascular system. the nos-type ii (inos, inducible nos) is calcium independent and is produced in large amounts during innate and adaptive immune responses (19) (20) (21) (22) (23) (24) . no produced during an innate immune response clearly contributes to the killing of intracellular microorganisms and parasites (25) (26) (27) . less is known about a role of no in the generation and regulation of an adaptive immune response, including inflammation. it has been suggested that no may exhibit cytotoxic, regulatory, and immunosuppressive properties, including induction of apoptosis and autoimmune disease, reviewed in (28) (29) (30) . such a wide array of biological actions of no suggests that inos may have different functions in different cells. the immunoregulatory action of no is likely mediated through its effect on cytokines and other inflammatory molecules by regulation of the transcription factor, nf-activating protein-1 (ap-1) by targeting the th 1/th2 balance (29, (31) (32) (33) . on the other hand, no produced by microglia/macrophages could be a potent neurotoxin and mediates tnf-~ neurotoxicity toward oligodendrocytes (16, (34) (35) (36) . the toxic effect of no is likely attributed to the nitrosylation of target iron-sulfur proteins, including enzymes involved in dna replication and repair and in blocking mitochondrial respiration (37, 38) . the uncoupling of the electron transfer chain in mitochondria will result in production of oxygen free radicals. reaction of no with a superoxide will lead to formation of extremely toxic peroxynitrite (onoo) (39) (40) (41) . peroxidations of membranes and swollen oligodendrocyte cell bodies have been described in the cns of patients with ms (42) . nitrosylation of tyrosine in proteins has been demonstrated in a number of neurodegenerative and inflammatory conditions of the cns (43) (44) (45) . in the inflammatory conditions of cns, such as viral encephalitis and eae (experimental allergic encephalomyelitis), inos is expressed in macrophages, microglia and/or astrocytes, and possibly neurons but not oligodendrocytes (41, 43, (45) (46) (47) (48) (49) (50) (51) . the role of inos has been examined in a number of encephalitis's induced by viruses, such as corona, flavi, rhabdo, borna, rabies, herpes, japanese encephalitis, venezuelan encephalitis and several others, revealing that no may contribute to the pathogenesis of the disease(52-60). alternatively, no may have a protective role in a viral infection. we have examined the expression of inos transcripts and protein in the cns of tmev-infected mice during early acute and late chronic demyelinating disease (61) . both susceptible (sjl) and resistant (b6) strain of mice were infected i.c. with the da strain of tmev and infected mice were sacrificed at 0, 3, 6, 10 days p.i. (sjl and b6), corresponding to early acute disease and at 39, 42, 66-67 and t80 days p.i. corresponding to late chronic demyelinating disease (sjl only). mock-infected mice were injected i.c. with medium alone and they were sacrificed at 6 and 39 days p.i. inos transcripts were determined by rt-pcr, followed by southern blotting, as described (61); the presence of inos protein was determined by immunohistochemical staining using affinity-purified polyclonal rabbit anti-inos antibody (61) . these antibodies are inos specific and do not react with nos type i and nos type ii. although both strains of tmev-infected mice develop polioencephalomyelitis (gray matter inflammatory disease), confirmed by histological analysis (fig.l) , none of the animals showed overt neurological signs. we did not detected any transcripts for inos in brain and spinal cord of mock-infected animals examined at day 6 and 39 p.i. and very little inos transcripts were present at day 3 p.i. in contrast, high levels of expression of inos transcripts were detected in the cns of tmev-infected sjl and b6 mice at 6 and 10 days p.i. in general, higher levels of inos expression were detected in brains of sjl and b6 mice than in the spinal cords. immunohistochemical staining of cns tissue enabled the identification of cells which expressed inos during early acute disease. at 6 and 10 days p.i. inos was expressed in strains of tmev-infected mice in areas of intense inflammation in reactive astrocytes (figs. 1a & b) , monocytes/macrophages and hypertrophic endothelial cells (figs. 1c, 1d) . few perivascular monocytes-like cells in leptomeninges were also inos positive, while lymphocytes were negative (61) . normal endothelial cells and smooth muscles cells were consistently negative for inos. to confirm that reactive astrocytes were the major cell type expressing inos, we stained adjacent sections with antibodies to gfap (glial fibrillary acidic protein) or with antibodies to inos. cells which were positive for inos were also positive for gfap. the profile of inos staining in the spinal cord was similar to that described for encephalitis. we have not detected any inos-like immunoreactivity in normal interfascicular fibrous astrocytes, protoplasmic astrocytes, bergmann's glia or oligodendrocytes. rod-shaped microglia-like cells were also consistently negative, inos protein was essentially undetectable in the brain and spinal cord of naive mice. we have examined expression of inos in late chronic demyelinating disease(61), inos transcripts were detected in spinal cords of tmevinfected sjl mice at 39 days p.i., which corresponds to the beginning of inflammatory demyelinating disease (61) . less inos transcripts were detected in brains of these mice. immunohistochemical staining confirmed expression of inos protein in fibrillary astrocytes in areas of inflammation (fig.le) and in leptomeningeal infiltrate (albeit weak) at 42 days p.i. (fig.if) . inos protein was not detected in the cns of tmev-infected b6 mice at 42 and 67 days p.i. and, as expected, there were no overt pathological changes in the brains and spinal cords of these mice. in contrast, extensive inflammatory disease, demyelination and gliosis were demonstrated in tmev-infected sjl mice at day 39, 67 and 180 p.i. however, we did not detect any inos transcripts nor inos protein at day 67 p.i. (advanced phase of demyelinating disease) or at 180 days p.i. (terminal phase of disease) associated with hind leg paralysis and incontinence). foamy-myelin-laden macrophages were always negative (fig. ig ). we have demonstrated that during early acute disease induced in tmevinfected sjl and b6 mice inos is expressed in the brain and spinal cord of infected animals, predominantly in hypertrophic astrocytes, monocytes/macrophages and endothelial cells in the area of inflammation (61) . during late chronic demyelinating disease, inos has been detected in fibrous astrocytes and meningeal infiltrates of spinal cord but only during the early stages (39 days p.i.) of late chronic demyelinating disease (sjl mice) and not at 66days p.i. or at 180 days p.i. (terminal stage of the disease). we have also demonstrated that during early acute disease there is high level of apoptosis of t cells undergoing activation induced cell death, while there is paucity of apoptosis of t cells infiltrating the cns of tmev-infected sjl mice during late chronic demyelinating disease (unpublished observations). we suggest that no produced by reactive astrocytes and macrophages/microglia during early acute disease triggers apoptosis of infiltrating t cells. conversely, lack of expression of inos in the cns during late chronic demyelinating disease (days 66 and 180 p.i.) may relate to the very low level of apoptosis of these t cells. induction of apoptosis of mbp-specific t cells by no producing astrocytes (functioning as antigen presenting cells) has been described (62) . molina-holgado(63) recently reported that in brain astrocytes infected in vitro with tmev there is high expression of inos and that il-4 and il-10 down regulate expression of inos by modulation of nf-rd3 activity. we have demonstrated high level of expression of proinflammatory cytokines in the cns of tmev-infected mice during early acute and late chronic demyelinating disease (64) . however, il-4 transcripts have been detected primarily during late chronic demyelinating disease. it is therefore possible to suggest that il-4 down regulates expression of no in astrocytes in sjl mice during late chronic demyelinating disease, which may result in inhibition of apoptosis. according to this postulate, expression of inos during early acute disease would be beneficial to the host, because no could be responsible for eliminating infiltrating t cells through down regulation of bcl-2 and induction of aicd, leading to restoration of homeostasis in the cns. thus, no may function as an anti-inflammatory or immunosuppressive molecule during early acute disease. immunosuppressive effect of macrophagederived no on t cells has also been reported (25, 28, 29) . however, lack of inos expression (down regulated perhaps by il-4/nf-v,b) during late chronic demyelinating disease in tmev-infected mice may be detrimental to the host by blocking apoptosis of infiltrating t cells. on the other hand, down regulation of no production in the cns of tmev-infected mice may represent an effort of the host to curb formation of peroxinitrite, an extremely toxic molecule, which may lead to peroxidation of membranes and formation of nitrotyrosine. whether nitrotyrosine is present in the cns of tmev-infected mice is not known. no is a weak oxidant and acts rather like an antioxidant and only in reaction with superoxide it forms peroxynitrite, very toxic molecule. we and others demonstrated the expression of inos and nitrotyrosine in reactive astrocytes and monocytes/macrophages in multiple sclerosis lesions (43, 45) . in general the expression of inos and nitrotyrosine seems to depend on the extent of lesion activity (acute vs. chronic). the unexpected protective or destructive role of no in induction and progression of eae is also well known, reviewed in (65) . the effect of specific inos inhibitor, amino guanidine (ag) on mice infected with da strain of tmev has been examined (66) . treatment of tmev-infected mice starting at day 28 p.i. till day 49 p.i. significantly decreased inflammation, demyelination and axonal necrosis. this treatment was associated with decreased apoptosis of cns infiltrating cells, presumably monocytes or lymphocytes. early treatment (starting at day 7 p.i.) was not effective (66) . when tmev-infected mice were treated starting at day 14 p.i., only demyelination and axonal necrosis were decreased while the level of inflammation was not changed (66) . these results suggest that inflammation and demyelination and axonal damage may be differentially down regulated by treatment of tmev-infected mice with no inhibitors at different times of infection. the outcome of treatment may depend on the level of activation of macrophages/microglia and astrocytes, which produce no but also on availability of superoxide. the role of inos in tmev induced demyelinating disease has also been examined using bean strain of the virus (instead of da strain routinely employed in our laboratory). the bean strain of tmev replicates with different kinetics in the cns of sjl mice (67) . for example, mice infected with bean show clinical signs of disease such as extensor spasm and hind leg of paralysis at 50 days p.i., whereas, mice infected with da strain of tmev do not show these signs earlier than 180 days p.i. treatment of sjl mice infected with bean strain of tmev with ag during the effector phase of the disease (initiating treatment at 15 days p.i.) resulted in significant delay of inflammatory demyelinating disease (67) . interestingly, inos was detected at low levels in monocytes/macrophages (moma-positive) in leptomeninges and perivascular spaces, but not in astrocytes, in tmev infected mice between 0 and 15 days p.i. (67) . the maximum expression of inos was observed at day 60 p.i. in contrast, inos expression was high in the cns of sjl mice infected with da strain of tmev between 6 and 10 days p.i., but was not detectable at 66 and 180 days p.i. treatment of tmev (bean)-infected mice with ag in the induction phase (starting at day 1 p.i.) did not alter the course of the disease (67) . the authors suggested that protective effect of ag on inflammatory demyelinating disease observed in bean infected mice when treatment had begun 15 days p.i. was associated with suppression of activation and proliferation of inflammatory macrophages and lymphocytes (67) . the results of these experiments suggest that the effect of no on inflammatory demyelinating disease induced by these two strains of tmev may depend on the kinetics of disease, time of appearance of inflammatory infiltrates, type of cells which express inos and time of administration of the inhibitor. cd. lmmunobiology of tmev infection. immunology of theiler's murine encephalomyelitis virus infection animal model: theiler's virus infection in mice theiler(s virus replication in brain macrophages cultured in vitro characterization of the to strains of theiler's mouse encephalomyelitis viuses theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination characterization of the inflammatory response in the central nervous system of mice susceptible or resistant to demyelination by theiler(s virus two models for multiple sclerosis: experimental allergic encephalomyelitis and theiler's murine encephalomyelitis virus theiler's virus persists in glial cells during demyelinating disease theiler's murine encepha-lomyelitis: a model of demyelination and persistence of virus spontaneous encephalomyelitis of mice: a new virus disease immune mechanisms of theiler's virus-induced demyelination immunology of multiple sclerosis and experimental allergic encephalomyelitis mechanisms of immune injury in multiple sclerosis immunology of multiple sclerosis greenfield's neuropathology) cytokine cytotoxicity against oligodendrocytes nitric oxide synthase structure and mechanism regulation of biosynthesis of nitric oxide endogenous nitric oxide synthesis: biological functions and pathophysiology nitric oxide synthases in mammals macrophage oxidation of l-arginine to nitrite and nitrate: nitric oxide is an intermediate inflammation, immunoregulation, and inducible nitric oxide synthase the role of nitric oxide in innate immunity persisting viruses and chronic inflammation: understanding their relation to autoimmunity, lmmunol rev the microbicidal activity of interferon-gamma-treated macrophages against trypanosoma cruzi involves an larginine-dependent, nitrogen oxide-mediated mechanism inhibitable by interleukin-10 and transforming growth factor-beta effects of nitric oxide synthase inhibitors on murine infection with mycobacterium tuberculosis different susceptibility of three clinically isolated strains of cryptococcus neoformans to the fungicidal effects of reactive nitrogen and oxygen intermediates: possible relationships with virulence control of the autoimmune response by type 2 nitric oxide synthase nitric oxide; a pathogenic factor in autoimmunity nitric oxide in autoimmune disease: cytotoxic or regulatory mediator? berrih-aknin s. in vivo and in vitro apoptosis of human thymocytes are associated with nitrotyrosine formation cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappab-binding sites nitric oxide synthases: roles, tolls, and controls nitric oxide mediates glutamate neurotoxicity in primary cultures microglial cell cytotoxicity of oligodentrocytes is mediated through nitric oxide nitric oxide as a potential pathological mechanism in demyelination: its differential effects on primary glial cells in vitro dna deaminating ability and genotoxicity of nitric oxide and its progenitors nitric oxide. a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells pathological implications of nitric oxide, superoxide and peroxynitrite formation a redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitrosocompounds production of nitrite by neonatal rat microglial cells/brain macrophages lipid peroxidation products and antioxidant proteins in plasma and cerebrospinal fluid from multiple sclerosis patients expression of inducible nitric oxide synthase and nitrotyrosine in multiple sclerosis lesions role of nitric oxide in inflammation-mediated neurodegeneration inducible nitric oxide synthase and nitrotyrosine are found in monocytes/macrophages and/or astrocytes in acute, but not in chronic, multiple sclerosis activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis induction of nitric oxide synthase in demyelinating regions of multiple sclerosis brains cloning and expression of inducible nitric oxide synthase from rat astrocytes ifn-? and il-1b induce nitric oxide formation from primary mouse astrocytes expression of inducible nitric oxide synthase by neurones following exposure to endotoxin and cytokine appearance of inducible nitric oxide synthase in the rat central nervous system after rabies virus infection and during experimental allergic encephalomyelitis but not after peripheral administration of endotoxin does nitric oxide play a critical role in viral infections? coronavirus-induced demyelination occurs in the absence of inducible nitric oxide synthase disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus inducible nitricoxide synthase plays a minimal role in lymphocytic choriomeningitis virus-induced, t cell-mediated protective immunity and immunopathology inflammation is a component of neurodegeneration in response to venezuelan equine encephalitis virus infection in mice induction of nitric oxide synthase during japanese encephalitis virus infection: evidence of protective role nitric oxide and viral infection: no antiviral activity against a flavivirus in vitro, and evidence for contribution to pathogenesis in experimental infection in vivo in vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases role of nitric oxide in hiv-1 infection: friend or foe? inducible nitric oxide synthase in theiler's murine encephalomyelitis virus infection an alternative pathway of nitric oxide production by rat astrocytes requires specific antigen and t cell contact interleukin-4 and interleukin-10 modulate nuclear factor kappab activity and nitric oxide synthase-2 expression in theiler's virus-infected brain astrocytes differential expression of tgf-beta, il-2, and other cytokines in the cns of theiler's routine encephalomyelitis virus-infected susceptible and resistant strains of mice our shifting understanding of the role of nitric oxide in autoimmune encephalomyelitis: a review nitric oxide synthase inhibitor, aminoguanidine, reduces inflammation and demyelination produced by theiler's virus infection expression and potential role of inducible nitric oxide synthase in the central nervous system of theiler's murine encephalomyelitis virus-induced demyelinating disease the support of the national multiple sclerosis society (grant rg2942) and the n. eleanor dana charitable trust is greatly acknowledged. we thank mr. brad e. hoffman for assistance in editing of the chapter. key: cord-028963-u4iupl1s authors: lane, michael; yadav, vijayshree title: multiple sclerosis date: 2020-07-10 journal: textbook of natural medicine doi: 10.1016/b978-0-323-43044-9.00199-0 sha: doc_id: 28963 cord_uid: u4iupl1s nan multiple sclerosis (ms) is a disabling disease of the cns that commonly affects young and middle-aged adults. 1 physicians have recognized ms since the mid-19th century, and although there were initially no management options, there has been significant therapeutic advancement since the 1990s with multiple effective disease-modifying therapies (dmts) now available to treat ms. in ms, the dysregulated immune system attacks the protective sheath (myelin) that covers nerve fibers in multiple locations through the cns, referred to as areas of demyelination. ms pathology primarily consists of these multifocal areas of demyelination (plaques) in the brain, spinal cord, and optic nerves. in addition to the areas of demyelination, significant axonal damage affecting the cns can also occur in severe forms of ms. frequently, the nerves are permanently damaged or deteriorate concurrently with damage to myelin. inflammatory cells composed predominantly of macrophages and lymphocytes are present when there is active demyelination within ms plaques, indicating that ms is an inflammatory disease. mri provides a means of visualizing ms lesions within the brain and spinal cord (fig. 199.1) . ms signs and symptoms depend on the parts of the cns affected, and patients have varying levels of permanent disability depending on the degree of inflammation and resultant damage. clinically, ms can cause a variety of neurological problems, depending primarily on the location and severity of ms plaques (table 199.1) . many ms symptoms, such as fatigue, cognitive impairment, and heat sensitivity, however, are not easily localized anatomically and are not well understood. in about 85% of cases, ms starts with a relapsing-remitting course. 2 patients experience relapses or attacks of ms during which they develop a new neurological problem or worsening of preexisting symptoms. relapses develop over a few days or weeks, followed by a period of improvement and stability, and typically last for more than 48 hours. in between relapses, patients are in remission and clinically stable, although they may have residual permanent neurological signs and symptoms from previous relapses. relapses that cause symptoms represent only the "tip of the iceberg" of disease activity at this stage of the illness. serial mri studies in patients with ms have disclosed that new, asymptomatic ms lesions appear within the brain 5 to 10 times more commonly than symptomatic lesions, causing permanent damage that contributes to the overall ms disease burden. 3 according to the natural history data, about 50% of patients with relapsing-remitting ms (rrms) enter a progressive phase of the disease 10 to 15 years after onset. this progressive phase, called secondary progressive ms (spms), is characterized by a steady worsening of signs and symptoms. patients with spms may or may not continue to have relapses. about 15% of ms patients have progressive worsening from the onset of their illness, a form of ms referred to as primary progressive ms (ppms). although ms is rarely fatal, it is often disabling. the natural history of ms suggested relatively quick progression of the disease from onset to having ambulation difficulty and walking with a cane occurring over a median of approximately 15 years. more recent studies report a significantly longer time to reaching this disability milestone, with a median time from onset to cane of 30 years. 4, 5 early studies of ppms also reported short median time from disease onset to cane of less than 10 years. however, more recent studies show the median time of progression to be closer to 15 years. the widespread availability and use of immune-modulating medications for relapsing ms likely have played a role in the improved long-term ms prognosis. 6 up to 15% of patients with ms never develop any overt permanent disability. most patients, however, develop varying degrees of permanent neurological disability. although it can be difficult to predict which patients will progress and which patients will have benign ms, several prognostic factors for unfavorable clinical outcomes have been identified. some of these factors include older age at onset; initial symptoms involving motor function; higher initial clinical activity, including high relapse rate; and increased disease progression in the first 5 years. smoking tobacco and low serum vitamin d levels have also been determined to be predictors of poorer long-term outcome. it is estimated that ms affects approximately 2.5 million people worldwide and 400,000 people in the united states. 6 ms affects about 1 out of 1000 persons in the united states, canada, and northern europe. ms typically begins between the ages of 20 and 40 but may occur at any age. women are more commonly affected than men, with about 60% of cases being female. a strong racial influence on the risk of developing ms exists: it is most common among individuals of white and caucasian backgrounds, particularly those of northern european descent. 7, 8 typical ms is rare among asians and black africans but is relatively common among black americans. the racial predilection of ms is one piece of evidence indicating the strong influence of genes on the risk of developing ms. in addition to racial influences, there is also an interesting geographical distribution of the disease. areas with the highest prevalence are located in higher latitudes, in both the northern and southern hemispheres. 9, 10 these high-risk areas include the northern united states, canada, great britain, scandinavia, northern europe, new zealand, and tasmania. 11, 12 pathogenesis experts agree that ms results from an acquired immune dysregulation and aberrant immune activation, leading to inflammatory processes driven by macrophages and b and t lymphocytes in the cns that result in demyelination and axonal damage. activated t cells produce proinflammatory cytokines, which additionally contribute to tissue damage. increased levels of proinflammatory cytokines, such as tumor necrosis factor alpha (tnf-α), interferon (ifn)-γ, and interleukin (il)-2, have been found in the peripheral blood, csf, and brain lesions of patients with ms. [13] [14] [15] [16] [17] [18] numerous reports document the association between these cytokines and disease activity, thus implicating them as mediators of the immunopathogenesis of ms. 13, [15] [16] [17] anti-inflammatory cytokines il-4, il-5, il-10, and il-13 and transforming growth factor beta-1 (tgf-β1) 19 have down-regulatory effects on the immune system and are thought to be beneficial in ms. 20 besides the role of macrophages, lymphocytes, and cytokines, other biomarkers, such as enzymes, can play an important role in ms pathogenesis. matrix metallopeptidase-9 (mmp-9) is a group of enzymes responsible for the migration of immune cells to sites of inflammation as well as remodeling extracellular matrix, basement membrane, and other tissues in the body by breaking down collagen components in these tissues. mmp-9s are thought to play a significant role in the transmigration of inflammatory cells into the cns by aiding in the disruption of the bloodbrain barrier. 21, 22 several studies have shown higher serum levels of mmp-9 in patients with ms. 23 ms pathology is defined as having three different categories of acute white-matter demyelination (the figure discussed in this section is under reference 1 and could not be included due to copyright concerns.). 26 the most common types (patterns i and ii) show a background of mononuclear phagocytes with perivascular and parenchymal t-cell infiltration. pattern ii is also characterized by immunoglobulin and complement deposition of antimyelin antibodies that can damage myelin either by initiating complement-mediated demyelination or assisting the phagocytosis of myelin by macrophages. pattern iii is seen in approximately 25% of biopsied active lesions and consists of oligodendrocyte apoptosis, and these lesions are similar to viral, toxic, and ischemic processes suggesting a "dying-back" phenomenon. this model of the acute ms lesion appears consistent with the most prevalent theory about ms, namely, that it is an immune-mediated disease. less understood is what happens chronically after the acute phase: whether there is persistent myelin degeneration (smoldering), inflammation resolves without inflammation (chronic inactive), or if lesions are invested with a thin myelin sheath (remyelinated). genes substantial evidence indicates that genetic and racial background influence the risk of developing ms. 27, 28 caucasian and african american populations appear to be at a much higher risk of developing ms than asians. having a first-order relative (parent or sibling) with ms increases the risk of developing the disease by five-to twentyfold. perhaps the most compelling evidence of the genetic influence on the risk of developing ms comes from studies of twins where at least one twin has ms. 29 among nonidentical twins, the chances of the second twin having ms are 1% to 2%, which is similar to the risk of nontwin sibling pairs. among identical twins, the chance of the second twin having ms is only 25%, indicating the strong influence of environmental factors besides the genetic background. although the cause of ms remains unknown, more than 100 genes are thought to affect the risk of developing ms. 30 the human leukocyte antigen (hla) genes are thought to have the strongest association with ms risk, with the most evidence for hla-drb1. 31 considerable interest lies in identifying the target(s) of the pernicious inflammatory response in ms. 29 viruses and microbial agents have been postulated to be associated with an increased risk of developing ms. for decades there has been interest in the possibility that one or more viruses or other microbial infections might cause ms. this interest stems from the inflammatory nature of ms and the apparent influence of the environment on the risk of developing ms, both suggesting the possibility of an infectious etiology. in addition, a spontaneous inflammatory demyelinating disease, called japanese macaque encephalomyelitis (jme), has been observed in a colony of japanese macaques at the oregon national primate research center. this disease appears remarkably similar to ms, with similar mri white-matter lesions, clinical findings, and pathological findings. the jme white-matter lesions were cultured and revealed a previously undescribed herpesvirus. 32 although various infectious agents have been reported to be associated with ms-including measles, epstein-barr virus, distemper virus, coronavirus, retrovirus, herpes simplex, human herpesvirus-6, and chlamydia pneumoniae-there is no convincing evidence at present of a linkage between any infectious agent and ms. 33 the lack of evidence does not exclude the possibility that an infectious agent causes ms, but currently, there is no widely accepted evidence associating ms with any specific virus or other microbe. geographical and seasonal influences. the association with latitude and ms prevalence has shown mixed results and is confounded by factors that include migration, lifestyle habits, and biological and social influences. in general, there is an increased prevalence of ms in northern latitudes (high risk) and a decreased prevalence in southern latitudes (low risk). people who move from a low-risk to a high-risk area before age 15 acquire a higher risk of developing ms, whereas those who make the same move after adolescence retain a lower risk. 12, 34 these observations suggest that an environmental exposure in the first two decades of life can influence the risk of developing ms. they also suggest that early sunlight exposure, which is correlated to serum vitamin d levels, may influence the risk for developing ms. 35 although not consistent to all geographical areas, there are several european epidemiological studies showing an association between season of birth and the risk of developing ms. [36] [37] [38] these studies indicate that there appears to be a lower risk for ms for births occurring after summer and a higher risk for ms for births occurring after winter. the authors reporting these findings suggest that maternal levels of vitamin d during the third trimester of pregnancy may influence the risk for ms, with a lower risk when maternal vitamin d levels are high (summer months) and a higher risk when maternal vitamin d levels are low (winter months). taken together, there is some evidence to suggest that sunlight exposure and vitamin d levels at a young age may have an influential role in the risk of developing ms. ms. an association was first suspected based on the observation that inland farming communities in norway had a higher incidence of ms than areas near the coastline. it was discovered that the diets of the farmers were much higher in animal and dairy products than the diets of the coastal dwellers, whose diet was enriched by cold-water fish. 39 additional studies have since correlated consumption of animal fat, animal protein, and meat from nonmarine mammals with the risk for ms. 40 individual dietary components may also affect ms pathogenesis. one example is the discovery of molecular mimicry between myelin and some dietary proteins, such as butyrophilin protein in cow's milk, inducing antibodies targeted against myelin oligodendrocyte glycoprotein (mog). high salt intake was recently postulated as a risk factor for ms development and to have an association with increased relapses in people with ms. however, this association has not been validated. 41 an epidemiological review evaluating the relationship between diet and ms from 1952 through 1995 suggested that the risk of ms is significantly correlated with the following parameters: consumption of animal fat, animal protein, and meat from nonmarine mammals. 40 however, a large prospective cohort study using data from the nurses' health study and nurses' health study ii found no evidence linking the risk of ms with the intake of saturated fats. the authors did note, however, that intake of linolenic acid, an omega-3 fatty acid (o3fa), but not fish oils or docosahexaenoic acid, was associated with a trend toward a lower risk for ms. 42 using data from the same cohorts, this same group also found no relationship between intake of fruits and vegetables and the risk of ms. 43 a case-control study in canada assessing the relationship between nutritional factors and the risk of ms in 197 incident cases of ms and 202 frequency-matched controls found a positive association between animal fat intake and the risk of ms. 44 recent data highlight that vascular disease risk factors such as obesity, hypertension, hyperlipidemia, heart disease, and diabetes can deleteriously affect ms progression. the presence of obesity as a child and in early adulthood has been associated with a greater risk of developing ms. 41 because most of the vascular disease risk factors, including obesity, are influenced by dietary habits, these studies suggest that there likely is an influence of diet on the risk of developing ms. gut microbiome. the gut microbiome is an ecosystem of commensal, symbiotic, and pathogenic microorganisms that outnumber their host's genes by more than 100 times. 45 the gut microbiome and its relationship with health and disease have been subject to extensive research, and the gut microbiome has been shown to be involved in maintaining human metabolism, nutrition, physiology, immune function, and mental health. the gut-brain axis is a bidirectional neurohumoral communication system that integrates neural, hormonal, and immunological signaling between the host gut and brain activities. 46 commensal, probiotic, and pathogenic bacteria in the gastrointestinal tract can activate neural pathways and cns signaling systems and can influence the development of anxiety and depression. 47 the bidirectional relationship is reflected in the observation that stress can influence the integrity of the gut epithelium and can alter peristalsis, secretions, and mucin production, promoting changes in microbial composition. 45 the concept of a gut-brain axis suggests that modulation of the gut microbiota may be a feasible approach to the management of cns disorders with significantly less toxicity than many of the pharmaceuticals used currently. the gut microbiome has been implicated in the induction of autoimmune conditions in human diseases and experimental animal models, including inflammatory bowel disease, ankylosing spondylitis, uveitis, rheumatoid arthritis, type 1 diabetes, and experimental autoimmune encephalomyelitis (eae). [48] [49] [50] current methodologies investigating gut microbial diversity, such as next-generation, 16s rrna gene-sequencing technologies, suggest a "dysbiotic" gut microbiota in both adult and pediatric subjects with ms. [51] [52] [53] an alteration in gut microbiota has also been apparent in subjects with ms on dmts such as glatiramer acetate and interferons. 53 additionally, alterations in gut microbiota appear to be associated with relapse risk in pediatric and adult ms subjects. 51, 53 the current, but limited, knowledge of gut microbiome changes in ms are intriguing, and it remains unclear if alterations in the gut microbiome precede or are a consequence of ms pathogenesis. university of california-san francisco (ucsf) researchers suggested a causal relationship of gut microbiota and ms in a study where transplanting microbiota from ms subjects was found to exacerbate symptoms in mice with eae. 54, 55 the same group evaluated 34 sets of twins discordant for ms, revealing that transplanting gut microbiota from the ms-affected twins into transgenic mice induced cns-specific autoimmunity at a higher incidence than microbiota taken from the healthy twins. 56 although human and mouse microbiota are not directly comparable, the biological pathways represented by ms-associated bacterial taxa largely overlap. many factors, both nonmodifiable and modifiable, can influence the composition of an individual's gut microbiome. these include genotype, age, sex, disease modifying therapies (dmts) for ms such as interferon and glatiramer acetate, antibiotics, and diet. an animal model of the gut microbiome suggests that diet can rapidly influence the gut microbiome, with changes occurring within a single day of diet change from a low-fat, plant-polysaccharide-rich diet to a high-fat, high-sugar diet. 57, 58 analysis of the fecal microbiome of a high-fiber diet compared with a western diet revealed underrepresentation of enterobacteriaceae (shigella and escherichia) with an abundance of bacteria from the genus prevotella and xylanibacter, known to contain a set of bacterial genes for cellulose and xylan hydrolysis, useful in digestion in a predominantly plant-based diet. 58, 59 the enterobacteriaceae family members associated with a western diet have been shown to exacerbate small intestinal inflammation, whereas the bacteria associated with high-fiber diets, including prevotella and xylanibacter, are associated with a protective role against gut inflammation. toxins and toxicants. all environmental pollutants have demonstrated prooxidant activity, with the most toxic compounds causing the most oxidative damage. these toxicants also typically deplete the level of reduced glutathione in the brain tissue and inhibit the function of the antioxidant enzymes. both reactive oxygen and reactive nitrogen species (ros and rns) can directly oxidize and damage dna, protein, and lipids in the brain, leading to neurodegeneration. neurons are directly affected by this oxidative stress, as are the glial cells. oxidative stress activates the glial cells, leading to an increased production and release of the proinflammatory chemicals interleukin-1β (il-1β), interleukin-6 (il-6), and interleukin-10 (il-10); gamma-interferon (ifn-γ); and tnf-α. the resulting neuroinflammation is a key component in the pathobiology of multiple sclerosis. 60 activated glial cells and mast cells both appear to be responsible for the release of the proinflammatory chemical soup that fuels neuroinflammation. glial cell activation has been implicated in the pathogenesis of epilepsy, alzheimer's and parkinson's diseases, ms, motor neuron diseases (amyotrophic lateral sclerosis [als]), stroke, and mood disorders. 61 neuroinflammation is triggered by traumatic brain injury, endotoxicity (circulating levels of lipopolysaccharides), elevated blood sugar (glycation end products), stress, and a host of environmental toxicants, including air pollutants, heavy metals, and organophosphate pesticides. 62 in a case-control study of children with ms having a father who worked in a gardening-related occupation (odds ratio [or] = 2.18, 95% confidence interval [ci]: 1.14-4.16) or any household use of pesticide-related products (or = 1.73, 95% ci; 1.06-2.81) were both associated with an increased risk of developing pediatric ms. 63 an italian case-control study indicated that occupational solvent exposures could be related to the risk of ms, as both shoe/leather workers and mechanical manufacturing industry workers were found to have a twofold increase in the odds of developing ms. 64 although there is general agreement that ms is an immune-mediated disease, why people develop ms remains uncertain. epidemiological studies suggest a complex relationship between genetic and environmental factors that can influence both the risk of acquiring ms and disease progression in ms. ms remains a clinical diagnosis. 1 no single test (e.g., blood test, mri examination, csf study) is adequate to diagnose ms. the diagnosis relies on a knowledgeable physician taking a detailed history, performing a neurological examination, conducting various tests, and then making a diagnosis on the basis of all the data. among the various practitioners, trained and experienced neurologists usually accurately diagnose ms. even among neurologists, the rate of misdiagnosis of ms can be high because there are many mimics that appear similar to ms. 65 the diagnosis of ms rests on the objective demonstration of two or more areas of demyelination in the cns that have occurred more than one time, and a diagnosis cannot be based only on a patient's symptoms. means of "objective" demonstration of areas of demyelination include the neurological examination; mri of the brain and spinal cord; csf examination; and electrophysiological tests (called evoked potentials) that assess visual, auditory, and somatosensory pathways. evaluation of the csf in a patient with suspected ms includes the presence of immunoglobulin g (igg) production within the cns, as seen by qualitative (oligoclonal bands) and quantitative (total igg, igg index, and igg synthesis rate) igg changes. excluding other disorders that can masquerade as ms is critical; these include unusual causes of stroke; nutritional deficiencies; genetic leukodystrophies or leukoencephalopathies; cancer; spinal cord compression from tumors, herniated disks, or spinal canal stenosis; vascular malformations of the spinal cord; infectious etiologies; and various other inflammatory disorders of the cns (see table 199 .2 for a list). although ms was formerly difficult to diagnose, mri scanning has significantly improved the ability to diagnose ms in its early stages because it allows imaging of areas of demyelination in the brain and spinal cord and also helps exclude the presence of other diseases that might explain the patient's symptoms. the criteria used to diagnose ms are summarized in the revised 2017 mcdonald criteria table (table 199. 3). the conventional approach to treating ms includes the use of medications to control disease activity and some symptoms and rehabilitation interventions to alleviate symptoms resulting from damage to the cns. medications that help decrease ms disease activity are referred to as disease-modifying agents, or dmts. (see table 199 .4 for a complete list.) most of the dmts are approved by the u.s. food and drug administration (fda) for relapsing forms of ms. these dmts can broadly be broken down into route of delivery, including injectable medications, oral medications, and infusions. compared with a placebo, these medications decrease the relapse rate by 30% to 67%, decrease new lesion formation in the brain as detected by mri, and decrease the risk of developing a permanent neurological disability. the injectable medications form the platform therapies and include human recombinant interferon-β (avonex, betaseron, rebif, and plegridy) and glatiramer acetate (copaxone, glatopa). interferons (ifns) are cytokines that mediate antiviral, antiproliferative, and immunomodulatory processes. there are three major forms of ifns: alpha (α), beta (β), and gamma (γ), which have some overlaps in function yet are distinct, with ifn-β showing benefit in the management of ms. although the precise mechanism by which ifn-β works in ms is not certain, the immunomodulatory effects proposed to occur include inhibition of proinflammatory t-cell activation and proliferation, destruction of autoreactive t cells, cytokine modulation, and prevention of migration across the blood-brain barrier. 19 ,66 ifn-β therapy has been found to decrease annualized relapse rates by 27% to 36% and reduce disability progression by 30% to 38% in various clinical trials. glatiramer acetate is a random polymer of four amino acids that stimulates protective t cells and is the safest option of the dmts. in multiple randomized trials, glatiramer was shown to reduce the relapse rate by 28% and reduce disability accumulation (risk ratio of 0.6) compared with placebo. 67 glatiramer acetate and ifn-β therapies are frequently used as first-line therapy for ms due to an excellent safety profile. another injectable, daclizumab (zynbryta), has been taken off the market due to hepatitis risk. the oral medications include three medications, fingolimod (gilenya), teriflunomide (aubagio), and dimethyl fumarate (tecfidera). the mechanism of action of fingolimod involves the reduction of cns inflammation and axonal damage by retaining lymphocytes in the lymph nodes so that fewer are able to enter the cns. 68, 69 fingolimod has been shown to reduce relapse rates by 50%, decrease the rate of disease progression, and decrease disease activity (mri lesions). 68, 69 teriflunomide prevents the division of active immune cells, including t and b lymphocytes. 19, 70 teriflunomide has been shown to reduce relapse rates by 36% compared with placebo and to decrease the rate of brain atrophy as assessed by mri. teriflunomide remains in the bloodstream for up to 2 years after discontinuation and is associated with fetal complications in pregnancy when taken by either males or females. 19 dimethyl fumarate is based on a german psoriasis treatment that may enhance th2 cellular response through the activation of intracellular nuclear pathways. 71 dimethyl fumarate affects transcription pathways that change the balance of t helper cell profiles, causing immunosuppression. dimethyl fumarate decreases the absolute relapse risk by 56% compared with placebo, decreases the number of new lesions on mri, and decreases the rate of confirmed disability progression. 71 the fda approved-infusions include natalizumab (tysabri), alemtuzumab (lemtrada), and ocrelizumab (ocrevus). natalizumab is a monoclonal antibody against α-4 integrin that prevents inflammatory cells from entering the cns and has been shown to decrease the annualized relapse rate by 68% and reduce disease activity (new or enlarging mri lesions) by 83% over 2 years compared with placebo. 72, 73 the main risk with natalizumab is the possibility of developing progressive multifocal leukoencephalopathy (pml), which is associated with exposure to john cunningham virus (jcv). natalizumab was taken off the market shortly after its release due to the development of pml before reintroduction with recommendations to monitor jcv antibody, which correlates with risk of pml development. 19 alemtuzumab is a monoclonal antibody to cd-52 that is present on most immune cells in the body and was approved by the fda in 2014 for relapsing ms. alemtuzumab is given as two annual infusions and has been shown to reduce relapses by 55% compared with the use of ifn-β-1a. 74 ocrelizumab is a monoclonal antibody directed at b-lymphocytes and was approved by the fda in 2017 for both primary progressive ms and relapsing forms of ms. ocrelizumab has been shown to reduce annual relapses by 50% compared with ifn-β-1a, with a 95% reduction in new active mri lesions compared with ifn-β-1a. 74 ocrelizumab, in addition to being approved for the management of relapsing ms, has been shown to be effective in decreasing the accumulation of disability in patients with primary progressive ms and is the only fda-approved drug for this indication. ocrelizumab reduced confirmed disability progression by 24% compared with a placebo in those with primary progressive ms. 74 although conventional dmts are able to reduce disease activity in relapsing forms of ms, they have limitations, which include only a modest effect on prolonging time to disability, rising costs (average cost is more than $60,000 per year), and side effects (e.g., injection-site reactions and neutralizing antibodies for ifn-β; bradycardia and arrhythmia for fingolimod; birth defects with teriflunomide; flushing and diarrhea with dimethyl fumarate). corticosteroids, such as methylprednisolone, given in high doses can decrease the duration of relapses of ms but do not affect the degree of eventual recovery after relapses. 75 a number of different medications are useful for treating various symptoms of ms, such as fatigue, bladder dysfunction, and spasticity, but these medications do not reverse the damage that has already occurred or decrease disease activity. given these considerations, an effort to identify natural therapies that have benefit for people with ms is warranted. from a natural medicine standpoint, there are four major approaches to treating ms: including all four provides the most comprehensive natural medicine treatment plan, and it is recommended that these should be used in combination with appropriate conventional therapies. diet is one way to influence general health, which is important to maintain in people with a chronic disease such as ms. there is no panacea diet for ms. however, various health factors, particularly vascular disease risk factors such as hypertension, hyperlipidemia, salt intake, diabetes, and obesity, can contribute to ms disability progression. 41 these vascular risk factors are readily influenced by diet and can be modulated with intervention. swank diet. the swank diet is one of the oldest and most well known dietary interventions used by people with ms. dr. roy swank reported that a diet low in saturated fats maintained over a long period of time tends to slow disease progression, reduce the number of attacks, and decrease mortality. 76, 77 swank began treating patients with his low-fat diet in 1948. the approach to using a low-fat diet supplemented with cod liver oil is based on epidemiological studies that found a decreased incidence of ms in populations that had a low consumption of animal fats with a high consumption of cold-water fish. based on current knowledge of the pathogenesis of ms, the rationale of using the swank diet or other diets low in saturated fats in patients with ms relates to the general health benefits of such a diet and the anti-inflammatory and perhaps neuron membrane-stabilizing effects of a diet enriched with o3fas. although the consumption of red meat is significantly restricted on the swank diet, fish appears to be particularly indicated because of its excellent protein content and, perhaps more importantly, its high content of o3fas. cold-water fish such as mackerel, salmon, and herring are rich in o3fas, which include eicosapentaenoic acid (epa) and docosahexaenoic acid (dha). as reviewed in the "nutritional supplements" section, o3fas have anti-inflammatory effects that may be of benefit in ms. in addition, because optimal neuronal functioning depends on cell membrane fluidity, which in turn depends on lipid composition, optimal essential fatty acid (efa) levels may be important in exerting neuroprotective effects by maintaining healthy neuronal functioning. 77, 78 decreasing animal fats and increasing o3fas in the diet thus may improve neuronal function by modulating neuronal lipid composition. since dr. swank's observational studies, two pilot studies evaluating diet in ms have been conducted. an open-label study evaluated the effects of a diet low in saturated fats combined with fish-oil supplementation and vitamin b complex and vitamin c in newly diagnosed rrms. 79 beside dietary modifications, subjects were advised to reduce their intake of sugar, coffee, tea, and alcohol and to stop smoking. diet was monitored over 2 years by a 4-day dietary record at the end of each year, and plasma fatty acid levels were monitored at baseline, year 1, and year 2. a significant increase occurred in the intake of fish, and a reduction in food items containing saturated fats occurred after 2 years. a significant increase occurred in plasma levels of o3fas, and a significant decrease occurred in plasma omega-6 fatty acids (o6fas). over the 2 years of the study, patients had significant reductions in both relapse rates and disability. this study, however, had significant limitations because there was no comparison group. although caution is warranted in interpreting the results of this small open-label study, its outcome does concur with dr. swank's long-term studies on diet and fish-oil supplementation for people with ms. a partially blinded, randomized controlled study evaluated the effect of low-fat dietary intervention with o3fa supplementation in 31 subjects with rrms. 80 the intervention lasted for 12 months, and the primary outcome was quality of life, evaluated using the short form health survey questionnaire (sf-36). subjects were randomized into one of two groups: the low-fat diet/fish-oil group and the diet/oliveoil group. the low-fat diet/fish-oil group followed a diet that did not exceed 15% of saturated fats (percentage of total daily calorie intake) plus fish-oil capsules (daily doses of epa 1.98 g and dha 1.32 g). the diet/olive-oil group followed the american heart association's step i diet, 30% saturated fats (percentage of total daily calorie intake) plus olive-oil capsules (6 capsules of 1 g of olive oil per day). the subjects were followed for an average of 11 ± 2.9 months, and the low-fat diet/ fish-oil group maintained better quality-of-life scores for physical well-being (although not statistically significant) than the group using olive-oil supplementation. the scores on the mental health component were similar in the two intervention groups. at the 6-month time point, the olive-oil group reported an improvement in fatigue compared with the fish-oil group (p = 0.035), which continued for 12 months. for both intervention groups, relapse rates were reduced compared with the year before entering the study. this study suggests that a diet low in saturated fats with fish-oil supplementation might promote better physical and mental health for people with ms. because all subjects improved after 12 months, the study also suggests that diet modification in addition to supplementation with a "good oil" (fish oil or olive oil) may be beneficial in people with ms. the epidemiological studies suggesting a link between a high-fat diet and an increased risk of ms coupled with the results from the three low-fat-diet studies reviewed show a consistency in identifying a lowfat diet as a therapy that may benefit people with ms. a person with ms following a low-fat diet will gain general health benefits. plant-based diet that is based mainly on complex carbohydrates as the main source of energy. this diet is based on starch, with 10% of calories derived from fat, 14% from protein, and 76% from carbohydrates. animal-derived products, dairy products, and oils are restricted from the mcdougall diet. paleolithic diet. the paleolithic (paleo) diet is based on the idea that humans are better equipped to handle the diet consumed by their paleolithic ancestors. the paleo diet is a component of the wahls protocol for ms management developed by dr. terry wahls. the diet consists of nondomesticated lean meats and plant-based foods except fruits, nuts, roots, and legumes. the ratio of saturated to polyunsaturated fatty acids is 1.4 to 2.0:1. the diet consists of three cups of green leafy vegetables, three cups of sulfur-rich vegetables, and three cups of intensely colored vegetables daily. in addition, two tablespoons of o3fas and 4 oz or more each of animal protein and plant protein is to be consumed daily. no more than two servings per week of gluten-free grains/starchy foods are recommended, and gluten, dairy, and eggs are prohibited. small open-label studies have been conducted in subjects with secondary progressive ms with a multimodal intervention consisting of diet, massage, acupuncture, meditation, and implementation of the paleo diet. these limited studies showed the diet was well tolerated, and although adherence to the diet was not consistent, participants had improved fatigue. the fatigue changes were not clinically significant, and the results were further diluted by the fact that additional interventions were employed rather than just the diet. the paleo diet also has the added risk of nutritional deficiencies, including folic acid, vitamin b 1 , and vitamin b 6 , from the decreased intake of fortified cereals, as well as calcium and vitamin d deficiency from the lack of dairy intake. 41 diet can lead to inflammation via oxidative stress based on the type and amount of food that is consumed. increases in caloric intake and glycemic load, and a high intake of saturated fat, trans fat, or o6fas can lead to postprandial inflammation. inflammation is decreased by the consumption of polyphenols, o3fas, caloric restriction, and exercise. caloric restriction has shown beneficial effects on disease activity in mouse models of ms. a pilot study evaluating the effect of restricting the diet to 1700 to 1800 kcal was performed in 33 subjects with rrms and 10 subjects with ppms. although there were no differences in neurological examination results in the follow-up period of 6 months, there was a 59% reduction of activated mmp-9 in subjects with ppms and 51% reduction in subjects with rrms. 81 in another study, 60 subjects with rrms were randomized to one of three parallel groups: one group that continued a usual diet, one group with a usual diet enhanced by an initial 7-day fasting episode, and one group with ketogenic diet over the course of 6 months. subjects were asked to complete the multiple sclerosis quality of life-54 questionnaire (ms-54). the subjects who were on the ketogenic diet and those on the initial fasting diet showed an improvement in the ms-54. 82 essential fatty acids. o3fas are found in both plant and animal forms, with alpha-linolenic acid (ala) being found in plant foods and epa and dha being found in marine foods. o6fas include linoleic acid, which is found in vegetable oils, nuts, and seeds, and arachidonic acid, which is found in meat and eggs. the body can synthesize most of the fats needed from the diet. there are two essential fatty acids (efas) that cannot be synthesized in the body and must be obtained from food: ala (an o3fa) and linoleic acid (an o6fa). omega-9 fatty acids (o9fas) are monounsaturated fats, are made in the body, and are considered nonessential. o3fas in rrms. one open-label study in rrms patients (n = 10) showed a significant decrease in mmp-9 levels secreted from unstimulated immune cells after supplementing with fish-oil concentrate at 8 g/day (containing 2.9 g epa and 1.9 g dha) for 3 months. all subjects showed a decrease in mmp-9 levels whether or not they were on ms diseasemodifying medication. 83 o3fas have been shown to have immunomodulatory effects. in vitro, animal, and ex vivo human studies have reported a decrease in mrna and protein levels of a number of cytokines, including tnf-α, ifn-γ, il-1, il-2, and vcam-1. one published study documented the effects of supplementation with fish oils enriched with the o3fas epa and dha on cytokine secretion in ms. 84 in this study, levels of il-1-β, tnf-α, il-2, ifn-γ, prostaglandin e2 (pge2), and ltb4 secreted from unstimulated and stimulated immune cells in people with ms and healthy controls were evaluated. twenty subjects with ms and 15 age-matched healthy controls were supplemented with 6 g/day of fish oil containing 3 g epa and 1.8 g dha for 6 months. all subjects with ms had a stable course of ms for at least 3 months before enrollment, had not modified their diet as a consequence of developing ms, and were not on any dmts. outcome measures compared baseline levels of the cytokines mentioned to levels after 3 and 6 months of supplementation in both subjects with ms and healthy controls. after 3 and 6 months of fish-oil supplementation, there was a significant decrease in the levels of soluble il-1β (p = 0.03), tnf-α (p = 0.02), il-2 (p = 0.002), and ifnγ (p = 0.01) in the unstimulated peripheral blood mononuclear cells (pbmcs) of both groups. a significant difference was observed after 3 and 6 months of supplementation in the levels of soluble il-1β (p = 0.01), tnf-α (p = 0.02), il-2 (p = 0.003), and ifn-γ (p = 0.005) from stimulated immune cells of both groups. cytokine levels returned to baseline values after a 3-month washout period. one study has examined the use of o3fa supplementation in ms. 85 this was a large (n = 312) double-blind, placebo-controlled trial in which patients with ms were randomized to receive either 20 capsules of fish oil per day or olive oil containing 72% oleic acid for 2 years. the total daily dose of epa was 1.71 g, and the total daily dose of dha was 1.41 g. this study reported a trend in improvement in the o3fatreated subjects compared with controls; however, these results did not achieve statistical significance (p = 0.07). there are some criticisms of the study design, including that both groups in the study were advised to follow a diet low in animal fats and high in polyunsaturated fatty acids. importantly, both groups developed changes in serum fatty acid content over the 2 years of the study. the lack of a comparison of fish-oil supplementation with a placebo in patients who did not have other dietary modifications may have affected the ability of the study to detect a statistically significant therapeutic benefit of o3fa supplementation. there is some experimental basis for considering supplementation with o6fas. two studies in an animal model of ms reported that supplementation with linoleic acid, which is rich in o6fas, decreased the severity of disease 86 and reduced inflammation in the cns. 87 o6fa (linoleic acid) supplementation for the treatment of ms has been investigated in at least three double-blind clinical trials. [88] [89] [90] o6fa spread (11-23 g/day linoleic acid) was provided in comparison with oleic acid (control). disability progression at 24 months was provided from two trials encompassing 144 patients and did not show a difference. two additional studies evaluated relapse risk in 132 subjects with rrms and showed a small decrease in relapse rate at 24 months (weighted mean difference [wmd] of 0.79, ci 0.63-1.00, p = 0.05). linoleic acid (2.9-3.4 g/day) did not show a significant decrease in the rate of progression in 65 subjects with progressive ms. 19 evening primrose oil, which is rich in the o6fa gamma-linoleic acid, is commonly used by patients with ms. gamma-linolenic acid might be more effective than linoleic acid because of its easier incorporation into brain lipids and its possibly greater effect on immune function. 91 however, evening primrose oil contains low levels of gamma-linolenic acid, and the product is relatively expensive. large and prohibitively expensive amounts of evening primrose oil would have to be used to obtain adequate supplementation. in addition, a single pilot trial of evening primrose oil in ms failed to demonstrate any benefit. 88 despite its common use by patients with ms, supplementation with evening primrose oil is not recommended. 92 the data to support o3fa or o6fa supplementation are lacking, and although there may be no harm in the use of efas, there is no major effect on disease progression in ms. epidemiological studies have found that low vitamin d intake and low serum levels of vitamin d may increase the risk of ms. 93, 94 a retrospective study of serum levels of vitamin d in people with ms (n = 199) found 84% of them to be deficient. 95 studies of vitamin d in animal models of ms have shown that vitamin d has the ability to decrease immune cell-mediated inflammation and prevent disease. 96, 97 ms studies in animal models suggest that vitamin d may have a beneficial role in ms by affecting the ability of inflammatory cells to enter the cns. 98 numerous human studies have evaluated both vitamin d supplementation and associations between serum vitamin d levels and biomarkers of ms disease progression. one open-label study (n = 16 subjects with ms) using oral calcitriol at a target dose of 2.5 mcg/dl found the intervention safe and tolerable up to a year of supplementation. 99 another study examined the seasonal variation in the serum levels of vitamin d in people with ms (n = 103) and healthy controls (n = 110) and found these levels to be significantly higher in the summer compared with the winter in both cohorts. 99 this study also observed that higher circulating levels of vitamin d in women were correlated with lower ms-related disability. a pilot study evaluating 29 subjects with rrms found a positive correlation between serum vitamin d levels and levels of the anti-inflammatory cytokine il-4. 100 in another 1-year prospective study, vitamin d supplementation and increases in serum vitamin d concentrations resulted in a significant decrease in annual relapses in ms subjects. this was observed in subjects who had vitamin d levels below 50 nmol/l. 19 thus emerging evidence from both animal studies and human studies suggests that vitamin d may have potential beneficial effects in ms. given that 30% to 50% of the general population may be deficient in vitamin d, 101 experts believe that serum levels of vitamin d should be evaluated to assess and treat vitamin d deficiency in ms. experts recommend vitamin d supplementation to target a serum level of (40-60 ng/ml or 75-150 nmol/l). 19 lipoic acid. oral lipoic acid (la) is an over-the-counter supplement that has been investigated as an antioxidant, anti-inflammatory, and neuroprotective agent in ms. la and its reduced form, dihydrolipoic acid (dhla), are potent antioxidants with multiple modes of action. la/ dhla can regenerate other antioxidants, such as glutathione, vitamin c, and vitamin e; serve as an ros scavenger; repair oxidative damage; and chelate metallic ions involved in oxidative injury. dhla acts by restoring reduced levels of other antioxidants, such as glutathione, and by repairing oxidative damage. [102] [103] [104] la is absorbed from the diet and synthesized de novo; it readily converts intracellularly to dhla. both la and dhla are present in both extracellular and intracellular environments. 105 in an animal model of ms, la has been shown to suppress the development of disease by preventing inflammatory t cells from entering the cns. [106] [107] [108] the immunomodulatory effects of la include inhibition of t-cell production of mmp-9, inhibition of the expression of the adhesion molecules icam-1 and vcam-1, and stimulation of camp by the prostaglandin receptors ep2 and ep4. 106, 107, 109 one small double-blind, placebo-controlled study evaluated the optimal dosing of oral la in rrms. 110 in this study, 37 subjects were randomized to one of four groups: (1) placebo, (2) la 600 mg twice a day, (3) la 1200 mg once a day, and (4) la 1200 mg twice a day. the study found that la given at 600 mg twice a day was barely measurable in serum, whereas la given at the dose of 1200 mg once daily showed significantly higher serum levels. the study also found an association between higher la serum levels and lower mmp-9 levels and higher la serum levels with lower soluble icam-1 levels. the investigators concluded that oral la between 600 and 1200 mg can be measured in serum and that higher serum levels of la are associated with an increased immunomodulatory activity that may benefit people with ms. a pilot double-blind, placebo-controlled study of daily oral 1200 mg la in 51 patients with spms revealed a decreased annualized rate of brain volume over the course of 2 years in the la-treated subjects. 111 there was also a trend of improved 25-foot walk time in the group that received la. larger studies are under way to further investigate the association of la and whole-brain atrophy in progressive ms. biotin. biotin, also known as vitamin h or vitamin b 7 , is a watersoluble b vitamin that is taken orally and is known to have multiple functions in energy metabolism and fatty acid synthesis. it is known to participate in myelin synthesis by activating the enzyme acetyl-coa carboxylase. high-dose biotin (300-600 mg daily) has been studied in both primary and secondary progressive ms. an initial pilot study of 23 patients with progressive ms was performed, and 91.3% of the patients had an improvement in clinical measurements. 112 patients with ms with a progressive decline in disability score over the previous 2 years were randomized to either placebo or a formulation termed md10003, a highly concentrated oral form of biotin. 113 md10003 was given at 100 mg three times daily over a 12-month period, and 12.6% of treated patients had an improvement in disability score by 1 point on the expanded disability status scale (edss), versus none of the placebo group. although these studies have revealed promising results, there have been negative results associated with biotin. a study completed in italy including 41 patients with progressive ms treated with high-dose biotin resulted in a significant increase in relapse rate. 114 many of these patients had ppms and had never experienced an ms relapse before. high-dose biotin has also been shown to interfere with various laboratory tests, resulting in a number of adverse events, including at least one death. 115 some of the laboratory values affected include measures of cardiac injury and thyroid function. 116 cognitive impairment affects up to 40% to 50% of people with ms, 117, 118 and there are currently no effective symptomatic therapies for cognitive dysfunction in ms. ginkgo biloba has been evaluated for cognitive impairment in alzheimer's disease, with mixed findings. a recent meta-analysis of these studies reports that g. biloba is safe, but the benefits for cognitive impairment and dementia are not predictable. 119 there is one randomized, placebo-controlled pilot study evaluating the effects of a standardized g. biloba extract on cognitive performance in 43 subjects with ms. 120 subjects were randomized to receive 120 mg of g. biloba twice a day or placebo for 12 weeks. the outcomes of the study included several neuropsychological tests, including the stroop test, which is a measure of attention and executive function. subjects receiving g. biloba showed significantly improved performance on the stroop test as well as improvement in subjective reports of cognitive deficits compared with the placebo group. this pilot study also showed that g. biloba extract was safe and well tolerated. although the studies evaluating g. biloba in people with dementia have shown mixed results, this supplement has been shown to be safe and well tolerated in many clinical trials. the pilot study in ms also demonstrated that g. biloba standardized extract given at 120 mg twice a day for 12 weeks was safe in people with ms. given its safety profile and limited evidence on improving attention and executive function in ms, g. biloba standardized extract may benefit ms patients with cognitive impairment. cannabinoids are a group of compounds found in the plant cannabis, also known as marijuana. the major psychoactive constituent in cannabis is delta-9-tetrahydrocannabinol (thc). thc binds to cannabinoid receptors (cb) in the cns and acts as a partial agonist to both cb1 and cb2 receptors. cannabidiol (cbd) is a nonpsychoactive constituent in cannabis and the major constituent in the plant. it is thought to decrease the clearance of thc by affecting liver metabolism. it binds to both cb1 and cb2 receptors in the cns, with a higher affinity to the cb2 receptor. cannabinoids can be delivered orally (e.g., cannabis extract, synthetic thc), through mucosa (e.g., cannabis extract oral spray, nabiximols [sativex]), and smoked. in a review of nine controlled studies evaluating a combination of thc and cbd (marinol) for spasticity in ms, it was found that thc-cbd was well tolerated and improved patient self-reports of spasticity, although objective measures for spasticity such as the ashworth score did not show significant improvement compared with a placebo. 121 the authors report that side effects were mild in both the treatment and placebo groups. the authors concluded that there was a significant improvement in patient-reported spasticity with the combination of thc-cbd and that thc-cbd was well tolerated in ms. unlike the dietary supplements discussed, marinol is a controlled substance and requires a prescription in the united states. in the past, ms patients were often advised not to exercise because increased body temperature and nerve fiber fatigue resulting from exercise were thought to induce transient symptomatic worsening and provide no long-term benefit. however, research has since shown that regular exercise is beneficial for people with ms. [122] [123] [124] [125] [126] [127] [128] four studies and two meta-analyses reported that compared with an ms nonexercise group, the ms exercise group demonstrated improvement in the subjects' reports of fatigue, quality of life, well-being, and walking ability. 123, 124, 127, 129 a systemic review of 26 studies on the effects of exercise, physical activity, and physical fitness on cognitive-performance outcomes in people with ms suggested beneficial effects of physical fitness, physical activity, and regular exercise on cognitive performance in people with ms. 130 in addition to direct effects on ms, regular exercise provides benefits in multiple facets of health, including cardiovascular risk; metabolic functioning, including blood sugar maintenance; mental health and mood; bone mineralization; and reduced risk for falls. 132 ms represents only one aspect of a person's health, and balancing other factors by maintaining a healthy lifestyle is crucial. in summary, evidence indicates that regular exercise is beneficial in ms and should be part of any natural medicine approach to treating ms. patients with ms often report that stress worsens their ms symptoms and consequently triggers an exacerbation. in a review of the scientific literature reporting associations between psychological stress and worsening of ms symptoms, an expert panel concluded that there was a possible relationship between antecedent stress and either ms onset or exacerbations. 133 a prospective longitudinal study of patients with ms designed to examine the relationship between stressful life events, psychological stress, and disease activity as measured by mri found that increased conflicts and disruptions in routine were followed by an increased risk of developing new brain lesions 8 weeks later. 134 perceived stress in patients with ms has been associated with ms exacerbations in a number of studies. 135 given the emerging evidence that antecedent stressors may contribute to the development of new lesions in ms and that perceived stress is associated with ms exacerbations, therapies that reduce stress are highly recommended in ms. there are very few scientific studies evaluating mind-body interventions such as yoga, meditation, and prayer in ms. 136 mind-body techniques using meditation, yoga, and slowed breathing to reduce stress in cancer patients have shown that these therapies are effective in decreasing stress, improving quality of life, and improving sleep. 137 a randomized controlled study investigated the effect of stress management therapy (smt) in mri outcomes in people with ms. 130 experienced therapists administered 16 individual 50-minute smt sessions over a 24-week period followed by a 24-week period of observation. smt first consisted of six sessions focused on relaxation, teaching problem-solving skills, cognitive restructuring, and enhancement of social support. participants were then able to tailor their treatment using option modules, including management of cognitive problems, communication, assertiveness, fatigue management, anxiety reduction, management of sexual dysfunction, and management of insomnia. mris were obtained at 8-week intervals during the 24-week active-treatment period. although the study was underpowered and enrolled fewer patients than originally planned, significantly more patients who underwent smt (76.8%) were free of lesions versus the controls (54.7%). there are two reported pilot studies evaluating the use of tai chi in people with ms. 125, 126 to evaluate the effects of training in the principles of "mindfulness of movement" from tai chi/qi gong in ms, 16 patients with secondary progressive ms were divided into 8 matched pairs. each pair was randomized into a mindfulness group or a usual-care group (i.e., standard medical care for ms). although there was no difference between groups in measures of balance, there was a significant improvement in the mindfulness group in self-reported measures of ms-related symptoms. 125 in a nonrandomized uncontrolled pilot study, 19 people with ms underwent tai chi training twice weekly for 8 weeks. outcomes measures compared pretraining with posttraining scores. posttraining outcomes demonstrated an improvement in walking speed, hamstring flexibility, and subjects' reports of well-being and quality of life. 126 there has been one randomized controlled study evaluating yoga in ms. 138 sixty-nine subjects were randomized to one of three groups: (1) wait-list control (n = 22), (2) exercise (n = 26), and (3) yoga (n = 21). those in the yoga group showed significant improvements in quality of life and physical measures compared with those randomized to the exercise or wait-list control group. there has been one randomized study evaluating a mindfulness-based intervention (mbi) in ms. 139 one hundred and fifty subjects were randomized to an mbi (n = 76) or usual care (n = 74), with an intervention period of 8 weeks and a 6-month postintervention follow-up. subjects randomized to mbi underwent training that included a 2½ hour session, once a week, for 8 weeks and one 7-hour session on saturdays (jon kabat-zinn's mindfulness-based stress reduction program). 140 subjects receiving usual care were offered mbi training after completing outcome assessments at the time equivalent of the mbi intervention and at 6 months postintervention. compared with the usual-care group, subjects randomized to mbi showed significant improvements in quality of life, fatigue, anxiety, and depression. mind-body interventions show promise of benefit in ms and offer a nonpharmacological therapy that can be effective in reducing stress, improving fatigue, and improving quality of life in ms. we believe that a natural medicine approach to ms management should be personalized for each individual, including dietary modification, nutritional supplementation, incorporation of exercise, and stress reduction techniques, complementary to the conventional medical approach including the use of dmt. although the data supporting the benefits of each of these individual complementary therapies in ms management are limited, natural medicine approaches when used in conjunction with the approved ms dmts have the potential to improve the general health of patients and may provide specific benefit in helping control the disease and improve symptoms. although there is no one diet for ms that has a proven efficacy, a few diets, such as the swank diet (low saturated fat, with 15 g/day or less of saturated fat intake; unsaturated fat intake of a minimum of 20 g/day and maximum of 50 g/day; other details as described earlier), mcdougall diet (very low-fat, strictly plant-based diet primarily based on starch, with ∼10% of calories to be derived from fat, 14% from protein, and 76% from carbohydrates), and modified paleo diet (nondomesticated, lean meats and plant-based foods except fruits, nuts, roots, and legumes; three cups each of green leafy, sulfur-rich, and intensely colored vegetables daily, two tablespoons of o3fas, 4 oz or more each of animal and plant protein to be consumed daily) have some limited evidence of benefit with fatigue, quality of life, and possibly mortality (long-term swank diet studies) in ms. the common theme of all of these diets is that patients with ms should limit processed foods and consume fresh, high-quality, whole foods. the key difference between the mcdougall diet and the modified paleo diet is the consumption of animal food, which is an area of unresolved controversy. various oral supplements have been studied in ms, including fish oils, vitamin c, vitamin d, biotin, lipoic acid, and g. biloba. except for the stronger data showing possible beneficial effects of vitamin d supplementation, other supplements have not yet shown convincing benefit in ms management. biotin supplementation remains currently under investigation, and because of its potential to cause interference with certain blood-based laboratory tests, careful use in clinical practice is warranted. an antioxidant, lipoic acid has convincing potential to decrease the rate of brain-volume loss in those with spms, and hence its supplementation may be considered. our recommendation for vitamin d 3 and lipoic acid supplementation is as follows: • vitamin d 3 : 2000 to 8000 units/day with the goal of achieving blood levels at an ideal range of 40 to 60 ng/ml 25-hydroxy d3 (supplementing with vitamin d requires attention to vitamins a and k2). • consider lipoic acid 1200 mg daily. the type and amount of exercise should be tailored to the patient. mild to moderate exercise for at least 30 minutes three times a week is recommended for most people with ms. types of exercise recommended for ms can include walking, stretching, bicycling, low-impact aerobics, stationary cycling, swimming or water aerobics, yoga, and tai chi. strategies to prevent overheating can include the use of air-conditioning and a cooling vest that can 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sclerosis: a pilot study lipoic acid in secondary progressive ms: a randomized controlled pilot trial high doses of biotin in chronic progressive multiple sclerosis: a pilot study md1003 (high-dose biotin) for the treatment of progressive multiple sclerosis: a randomised, double-blind, placebo-controlled study high-dose biotin increases inflammation and relapse in ppms. paper presented at european committee for treatment and research in multiple sclerosis the fda warns that biotin may interfere with lab tests: fda safety communication high-dose biotin treatment for secondary progressive multiple sclerosis may interfere with thyroid assays cognitive dysfunction in multiple sclerosis. i. frequency, patterns, and prediction cognitive dysfunction in early-onset multiple sclerosis: a reappraisal after 10 years ginkgo biloba for cognitive impairment and dementia ginkgo biloba for the improvement of cognitive performance in multiple sclerosis: a randomized, placebo-controlled trial whole plant cannabis extracts in the treatment of spasticity in multiple sclerosis: a systematic review recommendations for physical activity in patients with multiple sclerosis exercise and multiple sclerosis: physiological, psychological, and quality of life issues effects of a short-term exercise training program on aerobic fitness, fatigue, health perception and activity level of subjects with multiple sclerosis mindfulness of movement as a coping strategy in multiple sclerosis. a pilot study improving quality of life for people with chronic conditions: the example of t'ai chi and multiple sclerosis extended outpatient rehabilitation: its influence on symptom frequency, fatigue, and functional status for persons with progressive multiple sclerosis effects of an aquatic fitness program on the muscular strength and endurance of patients with multiple sclerosis effect of exercise training on walking mobility in multiple sclerosis: a meta-analysis effect of exercise training on quality of life in multiple sclerosis: a meta-analysis systematic, evidence-based review of exercise, physical activity, and physical fitness effects on cognition in persons with multiple sclerosis health benefits of exercise. cold spring harbor the relationship of ms to physical trauma and psychological stress: report of the therapeutics and technology assessment subcommittee of the american academy of neurology psychological stress and the subsequent appearance of new brain mri lesions in ms stress and multiple sclerosis mind-body interventions: applications in neurology mindfulness-based stress reduction as supportive therapy in cancer care: systematic review randomized controlled trial of yoga and exercise in multiple sclerosis multiple sclerosis pharmacogenetics: personalized approach towards tailored therapeutics full catastrophe living: using the wisdom of body and mind to face stress, pain, and illness key: cord-010187-ymhcfyxx authors: gromeier, matthias; lu, hui-hua; wimmer, eckard title: mouse neuropathogenic poliovirus strains cause damage in the central nervous system distinct from poliomyelitis date: 2005-03-25 journal: microb pathog doi: 10.1016/s0882-4010(05)80002-6 sha: doc_id: 10187 cord_uid: ymhcfyxx poliomyelitis as a consequence of poliovirus infection is observed only in primates. despitea host range restricted to primates, experimental infection of rodents with certain genetically well defined poliovirus strains produces neurological disease. the outcome of infection of mice with mouse-adapted poliovirus strains has been described previously mainly in terms of paralysis and death, and it was generally assumed that these strains produce the same disease syndromes in normal mice and in mice transgenic for the human poliovirus receptor (hpvr-tg mice). we report a comparison of the clinical course and the histopathological features of neurological disease resulting from intracerebral virus inoculation in normal micewith those of murine poliomyelitis in hpvr-tg mice. the consistent pattern of clinical deficits in poliomyelitic transgenic mice contrasted with highly variable neurologic disease that developed in mice infected with different mouse-adapted polioviruses. histopathological analysis showed a diffuse encephalomyelitis induced by specific poliovirus serotype 2 isolates in normal mice, that affected neuronal cell populations without discrimination, whereas in hpvr-tg animals, damage was restricted to spinal motor neurons. mouse neurovirulent strains of poliovirus type 2 differed from mouse neurovirulent poliovirus type 1 derivatives in their ability to induce cns lesions. our findings indicate that the characteristic clinical appearance and highly specific histopathological features of poliomyelitis are mediated by the hpvr. our data lead us to conclude that the tissue tropism of mouse-adapted poliovirus strains in normal mice is fundamentally different from that of poliovirus in hpvr-tg mice and primates, and that this is indicative of an as yet unknown mechanism of adsorption and uptake of the virus into cells of the murine cns. poliomyelitis is a rare neurological complication of primate infection with poliovirus (pv), a member of the picornavirus family, genus enterovirus. ingestion of virulent particles results in intestinal uptake, presumably through m-cells, 1 initial viral replication in subjacent lymphatic tissue, spread to deeper cervical and mesenteric *author to whom correspondence should be addressed. 0882-4010/95/040253+15 $08.00/0 o 1995 academic press limited lymph nodes (lymphatic phase), and ultimately entry of the virus into the bloodstream (viremic phase). 2 the latter is a prerequisite for passage of the blood brain barrier 3 and subsequent lytic infection of motor neurons. only a small proportion of pv infections lead to a distinctive neurological syndrome that ranges in severity from transient flaccid monoparesis to progressive flaccid paraplegia with respirato.ry impairment and sometimes bulbar involvement. 4 the hallmark of poliomyelitis histopathology is selective damage to anterior horn motor neurons along the entire spinal cord. spinal neurons outside the motor neuron system are characteristically spared, 5 in spite of their close anatomical relationship to the target of polioviral attack. the predominant molecular determinant of pv tropism is the hpvry the nucleotide sequence of hpvr identified it to be a member of the immunoglobulin superfamily, whose four mrna isoforms are the result of alternative splicing events, and give rise to different receptor molecules. 6'8 hpvrc~ and hpvr& are integral membrane proteins with divergent cytoplasmatic domains, whereas hpvr/~ and hpvr7 are secreted molecules lacking the putative transmembrane domain. 6 the hpvr is a highly glycosylated protein with an apparent molecular weight of 80kda2 the animal model for poliomyelitis in hpvr-tg mice showed pv-induced damage of comparable anatomical distribution as in primates, 1°'11 an observation confirming views of the hpvr as the critical determinant conferring pv susceptibility. unexpectedly, hpvr mrna and hpvr-related proteins were shown to be present in a wide variety of tissue homogenates not known to be sites of pv replication, ~'1° an observation suggesting additional limiting factors of pv susceptibility, mrnas specifying the simian 12 and murine ~3 homologues to hpvr have been isolated, but only the monkey-specific pvr can promote pv infection. ~2 attempts to use rodents as possible models of pv-induced disease resulted in the isolation of mouse-neurovirulent (ran) strains. a 1937 pv serotype 2 field isolate from lansing, michigan, [pv2(l)], which caused a syndrome described as 'polioencephalitis' upon intracerebral injection in the cotton rat, tm provided the first system of pv encephalitogenesis in the wild type (wt) mouse. ~s histopathological analyses of murine infection with the rodent-passaged pv2(l) isolate described a pattern of damage in accordance with concepts of primate poliomyelitis. ~6 a structural element conferring mouse neurovirulence to pv2(l) was determined to map in the capsid region. ~7 sufficient in causing this effect was a segment within the bc-ioop of vp1 since mouse avirulent pv type 1 (mahoney) [pvi(m)], after transposition of the lansing bc-ioop, caused neurological disease in mice. 18' 19 surprisingly, point mutations in distant regions of vp1 and vp2 could be shown to exert a mn phenotype. 2°.2~ the histopathological features of neurological disease caused by these genetically well defined mn pv strains have not been reported thus far. in addition to the selection of wt pv strains expressing a mn phenotype in mice, attenuated pv strains with a mouse host range phenotype but reduced neurovirulence have also been described [pv type 2, strain w222]. the attenuated (att) phenotype of pv2(w2) is reminiscent of the sabin strains of pv that express an att phenotype for primates. 23 the genetic determinants of viral neurotropism have been studied in several viral systems. most frequently, these determinants mapped to the envelope gene; examples are the alphaviruses sindbis virus 24 we have infected swiss-webster-, and icr-mice with a variety of mn virus isolates of different serotypical origin. type 2 strains pv2(l), pv2(mef-1), a recent pv2 isolate from india (ind), and pv2(w2) caused a fatal diffuse encephalomyelitis in these mice with considerable differences in intensity between individual viral strains. pvl(lsa), a mouse-adapted derivative of pvl(m), 37 caused a characteristic non-progressive panmyelitic syndrome. we have also studied the histopathology of murine infection with pvi(m), carrying a single mutation in position 54 of capsid protein vp1 that we constructed according to a published report. 2~ infections with each of these strains did not follow the stereotypic course of predictable progression seen in pv-induced poliomyelitis in hpvr-tg icr-mice. syndromes with distinct and consistent features followed intracerebral infection with each group of strains. these syndromes could be separately characterized and distinguished from hpvr-mediated poliomyelitis, on both clinical and histopathological grounds. poliovirus isolates and experimental animals. pv transgenic icr-mice expressing the hpvr ~° were a generous gift from a. nomoto (the university of tokyo, japan). swiss-webster-and normal icr-mice were obtained from taconic (germantown, ny, u.s.a). ultraviolet (uv) irradiation conditions. uv irradiation was performed in a uv-stratalinker (stratagene, lajolla, ca, u.s.a) a suspension of pv was irradiated at a distance of ca. 10 cm with an intensity of ca. 3.5 j/m z. virus was exposed to the irradiation source in ice-cooled plastic dishes at a solution depth of ca. 1ram. the loss of infectivity was confirmed in a plaque assay. poliovirus intracerebral infections. virus preparations were used to infect tg or normal mice with the desired input titer by intracerebral inoculation. mice were anesthetized, and a 25 gauge hypodermic needle was used to inject maximally 30 pl of virus suspension in dulbecco's minimal essential medium (dmem). the point of injection was the middle on the median between the ear pinnacle and the eye. infected mice were regularly observed for symptoms. none of the treated animals showed any external signs of damage or neurological disturbances in 24 h following injection. tissue processing, sectioning, and staining. affected animals in the final stage of their disease were sacrificed according to approved protocols, and their bodies were immediately perfused with 15 ml of phosphate buffered saline (pbs), followed by 15 ml of 4% neutral buffered paraformaldehyde. the brain and spinal cord were removed and fixed for 2 h at room temperature in the fixative used for perfusion. tissue specimens were rinsed for 30 rain in icecold pbs, then placed in 70% ethanol over night. dehydration was achieved through a scheme of gradually increasing ethanol concentration, followed by clearing in toluene for 2 h and infiltration with paraffin at 57.5°c for 2 h. neural tissues were embedded and cut on a rotary microtome at a thickness of 10/~m. tissue sections were placed on microscopic slides treated transgenic mice expressing the pv receptor tm were infected intracerebrally with pvi(m) with an amount of virus ranging from 10 4 to 5x 10 6 plaque forming units (pfu). all infected mice developed a characteristic neurological syndrome displaying stereotypic clinical features irrespective of input titer of virus and differing only with regard to onset of symptoms (table 1) . once visible signs of functional impairment were apparent, the progression of disease followed a predictable course. two to five days after intracerebral injection, initial signs were invariably a flaccid paralysis of the lower extremities and tail (fig. 1a) . the condition was rapidly progressive leading to complete immobilization and respiratory difficulty within 48 h. respiratory distress caused visible signs of bobbing of the head, strenuous respiratory effort, insufficient thoracic excursions, and nasal flaring. animals in the final stage of the disease were killed and their cns tissues processed according to standard procedures. histopathological analysis of the cns of pvl(m)-infected hpvr-tg mice revealed the spinal pathology of poliomyelitis described in earlier reports. 1°' 11 selective loss of anterior horn motor neurons along the entire spinal cord was invariably present (fig. 2b) . foci of virally-induced damage in the higher cervical cord extended into the brain stem and were accompanied by minor signs of inflammation in a predominantly perivascular distribution. characteristically, apart from a clearly defined lesion in the pyramidal cell layer of the hippocampal formation, lesions above the brain stem were absent in all cases analyzed. the appearance of initial lesions in the lumbar spine, distant from the injection site without evidence of damage to cortical motor neurons or descending tracts, indicated that virus had been disseminated via the hematogenous or cerebrospinal fluid route. twenty eight day old swiss-webster-and icr-mice were injected intracerebrally with pvi(m), pv2(l), pv2(mef-1), pv2(ind), or pv2(w2), with amounts of virus ranging from 104 to 5x 106. three groups of four animals each with different genetic backgrounds, swiss-webster and icr, were injected with the same viral strain. it is important to note that we were unableto detect clinical or histological evidence for differences in susceptibility towards pv infection between both outbred mouse strains used. therefore, in the following text, the term 'normal mouse' will refer to swiss-webster mice. all mn pv strains assayed caused poliomyelitis when injected into hpvr-tg mice (clinical and histopathological details are described later). none of the normal mice injected with pvi(m) showed clinical signs of neurological damage, whereas inoculation of type 2 pv strains produced signs of cns infection ( table 2) . mice infected with pv2(mef-1) and pv2(ind) were most severely affected. initial symptoms appeared 2-5 days post infection (p.i.) in all animals but they were inconsistent regarding the sites of manifestation and quality of functional impairment. motor symptoms consisted predominantly of spastic weakness involving the lower or upper extremities in a random fashion ( table 2) . paraplegic animals had a characteristic posture marked by kyphoscoliotic deformity (fig. 1b) . the progression of motor symptoms did not follow any apparent topographical scheme. pareses frequently were accompanied by gait ataxia or motor incoordination. clinical signs of respiratory involvement as described above usually did not appear during the course of the disease ( table 2 ). the clinical course proceeded to a terminal stage within 4 days after onset of symptoms. preterminal animals were severely emaciated once functional impairment supervened, and refused intake of fluid and food offered within their reach. compared to pv2(mef-1) and pv2(ind) a proportionally lower number of pv2(l)infected animals died (table 1) . their clinical syndrome was less variable, but also diverged from the regular pattern of disease localization and progression seen in poliomyelitic hpvr-tg mice. motor symptoms similar to those in pv2(mef-1) infected littermates dominated the clinical picture. progression of motor involvement did not follow any recognizable pattern and it only rarely included respiratory function ( table 2 ). eventually immobilization led to quick deterioration of the starving animals. pv2(w2) was previously reported to be of attenuated neuropathogenicity with respect to pv2(l) in mice. 44 accordingly, the proportion of infected animals that developed disease, and the severity and pace of progression of symptoms, were less than in animals infected with other pv type 2 isolates (table 1 ). this observation confirms the attenuated nature of the strain." the spectrum of functional deficits observed was identical to pv2(l)-induced disease. likewise, there was considerable variability in the sites involved in production of symptoms, course of progression, the clinical discrepancies between wt pv-induced poliomyelitis in hpvr-tg mice, and mouse-adapted pv2 infections in normal mice, were confirmed by histopathological differences. the severity and variability of clinical signs secondary to infection with the pv2 strains in normal mice was consistent with the histopathological findings. extent, morphology and localization of viral lesions in swiss-webster-and icr-mice were comparable. the range of vitally-induced damage encompassed a variety of lesions indiscriminately affecting structures within the cerebral hemispheres and the entire spinal cord. characteristically, spinal pathology was patchy and irregular, and it did not follow the consistent pattern of damage to the entire cord seen in pvinfected hpvr-tg mice. unexpectedly, anterior horn motor neurons remained frequently unaffected in normal mice infected with mn pv2, although severe pathological changes within the spinal cord were apparent (fig. 2c,d) . in contrast, routine poliomyelitis in hpvr-tg mice invariably led to complete and exclusive elimination of the motor neuron population in the spinal cord with only minor infiltrative changes ( fig. 2b) . unlike the limitation of vitally-induced lesions in pv infections of hpvr-tg mice to the spinal cord, pv2-induced disease involved the entire cns. microglial nodules accompanied by mixed lymphocytic and neutrophilic infiltrates were scattered throughout the brain and were found within the insular, piriform and temporal cortex (fig. 2f) , and the basal ganglia and thalamus (fig. 2h) . these structures were never affected in infected hpvr-tg mice (fig. 2e,g) . perivascular cuffing and dense infiltration of perivascular and periventricular parenchyma were distributed ubiquitously in the cns. the cerebral white matter was a frequent site of viral lesions. lymphocytic infiltrates within the cerebral or cerebellar peduncles, the internal capsule, or the long descending tracts and posterior columns within the upper cervical cord, occasionally caused rarefaction necrosis with secondary demyelination (fig. 21) . immunohistochemical staining for viral proteins with a polyclonal anti-pv2 antiserum revealed positive signals in those structures frequently involved by virallyfig. 3 . signs of widespread lesions and diffuse encephalomyelitis were most commonly associated with the isolates pv2(mef-1) and pv2(ind), whereas in pv2(l) cases a myelitic pattern of disease predominated over encephalitis. parallel to the clinical findings, overall cns damage induced by pv2(w2) was moderate in comparison with the other serotype 2 isolates. neurological damage focused on the spinal cord with rare cerebral involvement. spinal pathology qualitatively resembled the pv2 panmyelitis described above but the extent and number of lesions were reduced (data not shown). to analyze the clinical course of murine infection with pv2 in mice expressing the hpvr, tg mice were inoculated intracerebrally with pv2(mef-1) or pv2(l). the resulting clinical picture featured signs typically seen in murine poliomyelitis. the onset of disease was marked by a flaccid paraparesis, followed by ascending progressive symptoms as in poliomyelitis. all affected animals developed a preterminal dyspneic stage before they were killed. associated histopathology showed elements characteristic of hpvr-mediated poliomyelitis, as well as pv2-induced encephalomyelitis. anterior horn motor neurons had changes typical for poliomyelitis. in addition, there was almost always severe hemispheric involvement, never associated with hpvr-tg murine poliomyelitis resulting from infection with pvi(m). lesions equivalent to those seen in pv2-induced encephalomyelitis were distributed in the same widespread indiscriminate manner throughout the cns. gray matter structures were affected as well as cerebral white matter (data not shown). two groups of ten 28-day-old swiss-webster-and icr-mice were each infected intracerebrally with pvi(ls-a) at a range of 105 to 5 x 106pfu. as in previous assays, there was no notable difference in clinical signs and histopathological features in mice with different genetic background. all infected animals developed a specific neurological syndrome with stereotypical onset, clinical course, functional deficits, and pattern of progression. four days p.i., mice showed the first signs of an insidious spastic paraparesis (fig. 1c) . no ascending motor deficits occurred, respiratory function remained unaffected, and there were no fatalities. intracerebral inoculation of pvl(ls-a) was required to produce symptoms, since intravenous injection of virus with concomitant intracerebral needle puncture produced no neurological impairment. intracerebral inoculation of equal particle numbers of pvi(ls-a) virus, inactivated by uv-irradiation, also caused no neurological signs of cns damage. this finding indicated viral replication to be a prerequisite for pvi(ls-a) neuropathogenicity. histopathology of the neurological syndrome caused by this variant centered exclusively on the spinal cord. extensive infiltrates, originating from spinal blood vessels, invaded both the gray and white matter. pathologic changes involved the entire spinal cord, but did increase in extent of damage caudally (fig. 4a,b) . there was no apparent predilection for any cell subtype. motor neuron damage was seen within areas of destructive necrotic tissue change secondary to the inflammation. at no time during the infection could damage to motor neurons within the thoracic or cervical cord be distinguished (fig. 4b) , despite prominent inflammation affecting these regions. spinal sections taken from animals which underwent partial reversal of neurological defects, 4 weeks p.i., revealed complete regression of infiltrative changes and preservation of motor neuron populations (fig. 4c ). a mutant pvi(m) virus, which had previously been reported to exert neuropathogenic potential in normal mice 21 was tested in a similar manner as the above mentioned strains. pvl(vp1-54) carried a point mutation at position 54 (p1054s) within capsid protein vp1. intracerebral inoculation of each four swiss-webster-and icr-mice with at least i x 109pfu resulted in visible signs of neurological damage in 50% of affected animals in both groups. with the genetic variant constructed in this laboratory, this large inoculum was required to induce clinical signs. the nature of the neurological syndrome was partly obfuscated by the vigorous host response to the application of excess viral antigen, and the resulting clinical deterioration. sick mice showed prominent systemic signs of disease such as ruffled fur, reduced activity, and emaciation. symptoms of minor motor impairment were present in half of the diseased mice, but the limb pareses were difficult to assess in quality due to the general weakened state of the animal. mild limb weakness did not progress with any apparent disease pattern. severely weakened, emaciated animals succumbed to the effects of infection, without the clear causal relationship of neurological disease and fatal outcome seen in poliomyelitis, hpvr-tg mice injected with pv1(vp1-54) developed a neurological condition indistinguishable from pvi(m) related poliomyelitis (data not shown). similar to clinical findings, the histopathologic features induced by pvl(vp1-54) were less defined than in the other described infections. there was no supraspinal involvement, and spinal cord sections at all levels displayed widespread parenchymal invasion and reactive migroglial proliferation. no targeted attack against specific cell populations was apparent (fig. 4d) . histopathologic evidence for a primary thoracic and cervical cord affection was not present. with the rare exception of certain serotype 2 pv strains, e.g. pv2(mef-1) and pv2(l), naturally occurring pv strains can only infect primates, and even pv2(mef-1) and pv2(l) are mouse neurovirulent only when artificially inoculated by intracerebral injection. the narrow host range of pv results from its stringent dependence on the express exactly the same cell tropism in the murine cns as they do in the primate cns unlikely and, hence, that mn pvs would cause the same specific disease syndrome (poliomyelitis) in normal mice as they do in primates. to shed some light on these complex problems we have compared the disease syndromes caused by a variety of different pv strains in hpvr-tg and normal mice. we have come to the conclusion that mn pv strains do not produce poliomyelitis in normal mice. the occurrence of poliomyelitis in hpvr-tg mice corroborates the function of the hpvr as a major determining factor in the pathogenesis of the peculiar histopathological and clinical features of this disease. 1°'11 poliomyelitis in tg mice followed a stereotypic course: initial flaccid pareses of the lower limbs, followed by cranial progression, involving respiratory function, and fatal outcome. in contrast, the clinical symptoms resulting from mn pv infections in normal swiss-webster-and icr-mice could not be explained by classical terms of the syndrome poliomyelitis. infection of normal mice with mn pv strains caused a diffuse encephalomyelitis with highly variable neurological symptoms appearing in random order, at topographically unrelated sites. mouse neuropathogenicity was shared by several serotype 2 field isolates, the mouse adapted attenuated strain pv2(w2), the pvi(m) derivative pvi(ls-a), and pvi(m) with a mutation in residue 54 of capsid protein vpi. interestingly, the type 2 pv field isolates pv2(mef-1) and pv2(ind), have been reported to be mn without a lengthy process of adaptation, whereas other type 2 [pv2(l)] and type 1 strains [pvi(ls-a)] were passaged in the rodent cns before they acquired the mn phenotype. mn type 1 or type 3 pv field isolates have not been described. the molecular basis underlying this peculiar property of type 2 pv strains is not understood. earlier reports stressed the similarity between primate poliomyelitis and encephalomyelitis caused by pv2(l) in normal mice. 16 comparative studies at that time were impeded by the lack of a murine model of hpvr-mediated poliomyelitis. viral antigen was shown to be expressed in spinal anterior horn motor neurons, ~8 but in situ hybridization revealed the presence of viral rna in cells in posterior portions of the spinal cord as well as the white matter. "s previous analyses of pv neurovirulence using mn strains were based on the assumption of a similar pathogenesis of mn pv infections in wt mice and in primates. one parameter to assess the potential of viral neurovirulence is the ldso value, and in earlier studies, animals showing evidence of neurological functional impairment were generally killed without further histopathological analysis (see for example, ref. 17) . our results suggest that the expression of the mn phenotype of pv strains in mice should not be compared to the expression of pv neurovirulence in primates, the normal host. indeed, it appears that the expression of a mn phenotype in wt mice can result from very different genetic changes in pv strains, and that each genetic change can produce different syndromes. this is apparent not only from various type 2 mn strains, but also from the type 1 mn strain pvi(ls-a) [a derivative of pvi(m) whose genotype has been recently elucidated38]. histopathological changes within the cns should therefore be monitored when murine infections with mn pv strains are studied. a major determinant for the mn phenotype of pv2(l) is the bc-ioop of vp1 ~7 a surface protrusion of about 10 amino acids located at the apex of the poliovirion 46 that functions as a neutralization antigenic site. 47 exchange of the vp1 bc-ioop of the mouse-inert pvi(m) with the bc-ioop of pv2(l) produces the mn phenotype, ~8' 19 an observation that prompted speculation that the vp1 bc-ioop is involved in the recognition of a putative mouse receptor. no evidence for this hypothesis has been obtained as yet. strain pvi(ls-a), a mn derivative of pvi(m), accumulated 54 point mutations during passage in non-human tissue, of which 10 mutations led to amino acid exchanges in the capsid region28 these capsid mutations, however, were insufficient to produce the mn phenotype. instead, the mutations in capsid protein vp1 plus, surprisingly, five mutations in the coding region of the non-structural protein 2a "r°, a proteinase, produced mouse neurovirulence. the syndrome pv1 (ls-a) ir~duced in normal mice, however is distinct from the encephalomyelitis caused by type 2 pv strains. a pathological entity different from those observed with all other mn poliovirus strains, wes seen with pv1(vp1-54), a virus that we constructed according to a previous report. 21 pv1(vp1-54) is a mn derivative of pvi(m) that carries only a single amino acid exchange in capsid protein vp1 (p1054s). the mutation, located at an interpentameric interface inside the capsid, was suggested to destabilize the virion in the mouse cns, but the neurological syndrome caused by infection with pv1(vp1-54) was not histopathologically characterized. 21 infection of mice with pv1(vp1-54) led to neurological damage only after intracerebral inoculation of excess viral antigen. the poorly defined histopathological features of cns involvement lacked characteristics of specificity observed in hpvr-mediated poliomyelitis. the genotypic differences between mn pv strains and the clinical syndromes they cause, which differ from hpvr-mediated poliomyelitis, challenge the hypothesis of common determinants of a mn phenotype. rather, it appears that a multitude of non-related genetic elements affect this phenotypic marker in various ways. as indicated before, the nature of the receptor(s) used by the mn pv strains in the mouse cns remains to be determined. available data suggest that the mouse homologue to the hpvr does not serve as surrogate for the mn pv strains. 13 it is more likely that the mn pv strains enter neuronal tissue via cell-surface protein(s) unrelated to hpvr. if so, it is not surprising that the syndromes produced by these viral strains are distinct from poliomyelitis. in recent years picornaviruses other than poliovirus have been implicated as causative agents of neurological disease. these include enterovirus (ev) 70, 48 the etiologic agent of acute hemorrhagic conjunctivitis, or ev 71. 49 however, it has also been reported that the encephalomyelitis caused by the newly emerging human pathogen ev 71 did not always resemble poliomyelitis. s° the evolution of enterovirus strains with new neuropathogenic properties distinct from previously non-neuropathogenic ancestors might be due to adaptation to new receptor entities. the mouse-neurotropic pv variants with altered cell tropism constitute a precedent for the emergence of non-poliomyelitic picornaviral cns disease. we thank a. nomoto for the generous gift of the hpvr-tg mouse strain used in this study and b. jubelt and o. kew for kindly providing pv type 2 strains. we are grateful to p. coyle for critical review of this manuscript. we thank n. peress for helpful discussion and d. colflesh for help with microscopic imaging. this work was supported in part by nci grant ca28146, and nih grants ai15122 and ai32100. m.g. is a recipient of a grant from the stipendienprogramm infektionsforschung, heidelberg, germany. poliovirus 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hybrids simultaneously expressing antigenic determinants from all three serotypes poliovirus infection of cyclophosphamide-treated mice results in persistence and late paralysis: i. clinical, pathologic, and immunologic studies molecular pathogenesis of type 2 poliovirus in mice the three-dimensional structure of poliovirus at 2.9 ,a, resolution antigenic structure of picornaviruses radiculomyelitis following acute haemorrhagic conjunctivitis an apparently new enterovirus isolated from patients with disease of the central nervous system enterovirus type 71 infections: a varied clinical pattern sometimes mimicking paralytic poliomyelitis key: cord-295041-5vpawtef authors: jakhmola, shweta; indari, omkar; chatterjee, sayantani; jha, hem chandra title: sars-cov-2, an underestimated pathogen of the nervous system date: 2020-09-28 journal: sn compr clin med doi: 10.1007/s42399-020-00522-7 sha: doc_id: 295041 cord_uid: 5vpawtef numerous clinical studies have reported neurological symptoms in covid-19 patients since the spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), apart from the atypical signs of pneumonia. angiotensin-converting enzyme-2 (ace-2), a potential receptor for sars-cov-2 entry, is expressed on various brain cells and cerebral parts, i.e., subfornical organ, paraventricular nucleus, nucleus of the tractus solitarius, and rostral ventrolateral medulla, as well as in non-cardiovascular areas such as the motor cortex and raphe. the resident cns cells like astrocytes and microglia also express ace-2, thus highlighting the vulnerability of the nervous system to sars-cov-2 infection. additionally, transmembrane serine protease 2 (tmprss2) and furin facilitate virus entry into the host. besides, the probable routes of virus entry into the nervous system include the hematogenic pathway, through the vagus, the olfactory nerve, or the enteric nervous system. however, the trajectory of sars-cov-2 to the brain needs investigation. furthermore, a th17-mediated cytokine storm is seen in covid-19 cases with higher levels of il-1β/2/7/8/9/10/17, gm-csf, ifn-γ, tnf-α, cxcl-10, mcp1, and mip1α/β. some cytokines can cross the blood-brain barrier and activate the brain’s immune cells to produce neural cytokines, leading to neuronal dysfunctions. nonetheless, most of the neurological conditions developed due to viral infections may not have effective and registered treatments. although, some antivirals may inhibit the virus-mediated pathogenesis and prove to be suitable in covid-19 treatment. therefore, clinicians’ and researchers’ collective expertise may unravel the potential of sars-cov-2 infection to prevent short-term and long-term cns damage. the initial cases of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection appeared in december 2019 in hubei province, china [1] . since then, it has become a global threat. besides systemic and respiratory ailments, 36 .4% of coronavirus disease of 2019 (covid19) patients developed neurological symptoms [2] . additionally, taste, smell, and visual impairments are reported in several cases of covid-19 [2] . sars-cov-2, a human cov (hcov) belongs to β-coronaviruses, and various clinical and pre-clinical studies have reported potential neurovirulent properties of these viruses [3] . furthermore, the presence of sars-cov-2 in cerebrospinal fluid (csf) of covid-19 patients is confirmed through genome sequencing [4] ; however, experimental evidence is needed to validate virusmediated neurological damage. moreover, acute necrotizing hemorrhagic encephalopathy (ane) was observed in brain computed tomography and magnetic resonance imaging of a covid-19 patient [5] . this rare complication is often associated with intracranial cytokine storms and points towards the indirect mode of sars-cov-2 influence on the brain [5] . also, a detailed study of brain tissue distribution of angiotensin-converting enzyme-2 (ace-2), a potential receptor for sars-cov-2 entry [6] , may shed light on potential sars-cov-2-induced neurological alterations. elaborate ace-2 expression studies state that the receptor is preferentially expressed in the endothelium, vascular smooth muscle cells, and on the surface of a variety of the central nervous system (cns) and peripheral nervous system (pns) cells [7] [8] [9] . additional plausible entry routes to the brain may include the hematogenic pathway, transmission through the vagus, the olfactory nerve, or the enteric neuron ( fig. 1a) [10] . in brief, here we recapitulate varied aspects of covid-19-related neurological manifestations. this article is part of the topical collection on clinical outcomes of virus-mediated brain dysfunction: more prevalent than acknowledged? the association of viruses with neural disorders is widely popular, although the relativity is still disputed. neurodegenerative diseases, affecting approximately 37 million people worldwide, include degenerative ailments of the nervous system-the brain, spinal cord, and nerves [11] . numerous genomic and proteomic studies unravel the similarities between virusmediated and classical neurodegeneration or neuropathies [12, 13] . viruses introduce alterations in the functioning of neurons directly or indirectly. the neurotropic viruses afflict neurons through cell lysis, necrosis, or apoptosis [14] . indirectly, the viruses damage the neurons by manipulating or attacking the host immune responses. in the cns, the virus can activate both the adaptive and innate immune responses [15] . common pathways involved in the activation of the immune responses include the tlr mostly 3, 7, and 8 mediated damage, the release of free radicals, and inflammation [15] . although, not always does the cns immune response lead to detrimental outcomes as they usually assist in repair and regeneration [15] . multiple studies mention the corroboration of infectious respiratory organisms as causative agents of various neurological diseases [16] . respiratory syncytial virus (rsv) is known to infect the lower respiratory tract, cause infections in the immunocompromised patients, and target the cns [16] . often the virus is detected in the csf samples of the patients exhibiting symptoms like seizures and convulsions, along with signs of ataxia, hormonal dysfunction, and encephalopathies [16] . also, in vivo studies demonstrate the movement of the virus intranasally to the cns [10] . another respiratory virus that affects the infants and has neurovirulent abilities is the human metapneumovirus (hmpv) [17] . the virus is substantially detected in encephalopathic patients' csf samples, although studies demonstrating the virus' neuroinvasive properties are to be conducted [18] . furthermore, hendra virus (hev) and nipah virus (niv) affect humans and cause lung damage, pneumonia, along with hemorrhagic and necrotizing alveolitis [19] . typical signs of neurological disturbance, including convulsions, seizures with motor deficits, and febrile encephalitic syndrome, are observed due to infection caused by these zoonotic viruses [19] . animal studies show the olfactory nerve to be the main route to the cns [20] . also, the flu-causing influenza viruses account for numerous seasonal epidemics with a severe lethality rate, approximately a million cases per year [21] . additionally, the viruses also affect the brain and are linked to encephalitis, febrile seizure, acute necrotizing encephalopathy, and syndromes like the reye syndrome and guillain-barré syndrome [22] . according to some animal studies, the influenza virus can alter the brain homeostasis by traveling to the brain through the vagus nerve or the olfactory route [23, 24] . intriguing, its association with parkinson's disease (pd) and multiple sclerosis (ms) is also mentioned [25] . many encephalitis lethargica and postencephalitic parkinsonism cases followed by the 1918 "spanish" flu pandemic, caused by influenza a (h1n1), make the involvement of the flu virus evident [26] . viruses like the enteroviruses polioviruses (pv), coxsackieviruses (cv), echoviruses, and human rhinoviruses (hrv) are known to invade the cns [27] . studies describe hrv-induced meningitis and cerebellitis [27] . ev-a71 (hand-foot-mouth disease (hfmd)) and d68 outbreaks are associated with neurological complexities like myelitis (afm), meningitis, and encephalitis [27] . the hcovs can aggravate various neuropathologies. hcovs are related to the neuroinvasive animal covs like porcine hemagglutinating encephalomyelitis virus, feline cov, and the mouse hepatitis virus, which is used to generate ms models [28, 29] . furthermore, a study conducted to demonstrate the relation between the hcovs (229e and oc43) with ms and other neurological disorders involves identifying viral rna in human brain autopsies [30] . importantly, cov-oc43 and cov-229e are found in the csf of pd patients [31] . however, detailed studies are needed to differentiate the mere presence and virus-associated disease alterations. in addition, association of sars-cov is not just limited to the virus in the bloodstream may infect the peripheral immune cells. these infected leukocytes may traverse the blood-brain barrier (bbb) composed of specialized tight junctions, endothelial cells, pericytes, and astrocytes. in addition, the virus may also cross the bbb which could be severed due to the action of the cytokines or may enter the cerebrospinal fluid (csf) by direct interaction with the brain microvascular endothelium cells. both the mechanisms result in alterations in the brain homeostasis and aggravate cytokine production within the cns (ii) several viruses like hsv and influenza viruses are known to infect the olfactory epithelial membrane. sars-cov-2 may also infect and damage olfactory sensory neurons (osns) in the epithelium lining. the damage may be direct or due to the production of cytokines produced by the accessory cells in the olfactory system. the virus may anterogradely reach the olfactory bulb through the cribriform plate. finally, the virus may potentially gain entry into the cns through the mitral cells along the olfactory tract. (iii) alpha herpesviruses (e.g. hsv-1, prv) and polio virus (pv) along with rabies viruses (rv) may migrate to the cns through the peripheral nerves. (i) viruses may infect the mucosal epithelium following infection of the axonal termini of the peripheral nerves. the virus may spread to the spinal cord through retrograde axonal transport. (ii) viruses infect the smooth muscle cells and spread through the neuromuscular junctions (nmj) from muscles into the sensory/motor neurons of pns ganglia. (iv) the gastrointestinal epithelium expresses ace-2 receptors. therefore, the cells may be easily infected by the virus. the virus may directly invade the enteric nervous system or indirectly it may prime the immune cells which may result in delayed neurological impairment. b sars-cov-2-mediated cytokine storm. after attachment and entry into the epithelial cells through ace-2 receptor, the virus may activate the pro-inflammatory pathway through tlr or nf-κb signaling followed by the formation of inflammasome. various pro-inflammatory cytokines and chemokines released due to this autonomous intrinsic defense mechanism include ccl-2, ccl-4, cxcl-10, and il-6. these proteins attract various immune cells in the circulation like the monocytes, macrophages, t cells, and neutrophils at the site of infection. additionally, the situation is worsened by production of tnf-β, il-6, il-4, il-12, and il-23 by the t lymphocytes, which further accumulate the immune cells establishing a pro-inflammatory feed-back loop. these cytokines may damage the bbb and activate astrocytes and microglia, the cns resident immune cells. in response, the activated microglia and astrocytes produce il-1β, il-6, tnf-α, and il-8. elevated levels of these inflammatory cytokines can impart neurotoxic effects leading to neuronal dysfunction and various cns disease-associated pathologies lungs; instead, it is known to infect many organs, including the cns [7, [32] [33] [34] . the real-time quantitative pcr assay targeting the polymerase (orf1ab) and nucleocapsid region of the sars-cov confirmed the presence of sars-cov in csf and serum of the infected patients [35, 36] . a report suggests the association of status epilepticus with sars [35] . hospitalized children with the acute encephalitis-like syndrome were positive for anti-sars-cov igm [37] . sars-cov is associated with demyelinating pathology and found in the brain parenchyma of ms patients [5, 28] . neurological symptoms are also associated with mers-cov [38] . these examples impress on the connection of hcovs with neurological dysfunctions. therefore, the association of neurological complications with sars-cov-2 is not surprising. according to a case report of sars-cov-2 infection, virus rna was determined in the patient's csf; however, the nasopharyngeal swabs tested negative [4] . currently, evidence to state the neuropathogenesis of the sars-cov-2 in covid-19 remains scarce. nevertheless, reports suggest that sars-cov-2 can cause meningitis and encephalitis [4] . variable neurological symptoms are displayed by the covid-19 patients like pns symptoms, including hypogeusia, hyposmia, hypoplasia, and neuralgia vertigo, and cns dysfunctions like cephalgia, impaired consciousness, seizures, ataxia, and acute cerebrovascular disease, with headache and dizziness being the most common [39, 40] . neurological manifestations are common in many covid-19 patients like anosmia, an early covid-19 symptom [2, [41] [42] [43] . though seizures are seldom reported in covid-19 patients, and usually indicate an ischemic stroke, meningitis, or cerebral hypoxia, its association with comorbidities like hypocalcemia or drugs remains elusive [44] . the neurological alterations caused by the virus may result from direct cns/pns attack or indirect influence on various organs that later affect the nervous system. for example, hypertension, common covid-19 comorbidity, results in blood-brain barrier (bbb) impairment and may enhance the risk of covid-19-related cerebral complexities [45, 46] . a hypothesis relates neuronal damage to the respiratory stress from deteriorated lung conditions [47] . the oxygen deprivation may result in multiple organ failure and may affect the brain [47] . besides, patients considered during the earlier studies of the sars-cov pandemic displayed axonal motor sensory neuropathy and myopathy [48] . however, it remains unclear if the illness was virus-mediated or an outcome of high drug doses [48] . nevertheless, the effect of the sars-cov-2 on pns is noteworthy as guillain-barre, miller-fisher syndrome, and polyneuritis cranialis are reported in covid-19 fig. 1 (continued) [49] [50] [51] [52] [53] [54] . development of rhabdomyolysis, neuralgia, and myalgia in sars-cov-2-infected patients further support the virus' ability to affect pns [52] [53] [54] . a study reported elevated creatinine kinase and muscle pain in 10.7% of patients with severe covid-19 [2] . furthermore, some covid-19 patients with neurological symptoms might have a prior history of neurological complications or maybe treated for viral infections. hence, it is necessary to treat such cases using drugs with properties of high bioavailability in the brain. we have summarized information like the mode of action and brain or csf/plasma ratio of a few antivirals, which have shown promising outcomes in covid-19 treatment (table 1) [70, 71] . the use of efficient bbb penetrating drugs may be preferred during this pandemic to minimize the onset of neurological consequences of sars-cov-2 infection. indirectly the viruses may damage the neurons by manipulating or attacking the host immunity [15] . in the cns, sars-cov-2 can activate both the adaptive and innate immunity [15] . t helper cell 17 (th17)-mediated cytokine storm, evident in virus infections, is seen in covid-19 with neurological manifestations (fig. 1b) [72, 73] . clinical studies report systemic inflammation involving enhanced cytokines, particularly il-1β, il-6, il-10, granulocyte colony-stimulating factor, granulocyte-monocyte colony-stimulating factor, c-x-c motif chemokine ligand 10 (cxcl10), mcp-1, macrophage inflammatory proteins 1-α, and tumor necrosis factor α in covid-19. additionally cd4+ and cd8+ t cell lymphopenia and decreased secretion of ifn-γ in severe cases of covid-19 are reported (fig. 1a) [28, 37, 72] . intriguingly, a study suggests that an ms patient undergoing ocrelizumab (an immunosuppressive drug) therapy diagnosed positive for covid-19 does not display serious complications [74] . the increased levels of cytokines may escalate vascular and bbb permeability and inflammation [74, 75] . this information supports the hypothesis that increased bbb permeability allows virus entry into the cns, leading to covid-19-related neurological complexities. some cytokines released in the circulation can cross the bbb and activate the resident brain immune cells like microglia and astrocytes to produce neural cytokines, further worsening the condition (fig. 1b) [76] . astrocytes regulate a wide variety of functions, which may aggravate neuroinflammation. microglia mature into macrophages and may engulf the neighboring neurons on activation [77, 78] . furthermore, microglia are the primary source of pro-inflammatory cytokines, nitric oxide, prostaglandin e2, and reactive oxygen and nitrogen species [77] . microglia express ace-2, along with ace and at1 [79] . these receptors play a significant role in microglia activation and balance the pro-inflammatory or antiinflammatory effects [80] . more specifically, sars-cov-2 infection can hamper the ace-2-mediated signaling, creating a glitch in the at1 receptor-mediated path, thereby inducing a pro-inflammatory response [80] . in vivo studies suggest induction of pro-inflammatory cytokines in microglia and the mouse brain and spinal cord [81] . the situation becomes dreadful when the pro-inflammatory substances produced by astrocytes and microglia fenestrate the bbb [77, 78] . besides, sars-cov infects the myeloid cells and manipulates the innate immune system to ease its propagation to other tissues (fig. 1a) [82] . these persistently infected leukocytes act as reservoirs of the neuroinvasive hcov and can be held responsible for long-term neurological sequelae [83] . therefore, [68] , pd [69] *csf/serum ratio. hand hiv-associated neurocognitive disorders, pd parkinson's disease, rsv respiratory syncytial virus. the brain to plasma ratio or csf to plasma ratio has been denoted for each drug assuming that brain penetration is similar between rodents, non-human primates, and human patients the possibility of such cases of persistent sars-cov-2 infection may appear in the future. notably, peripheral inflammatory reactions observed in covid-19 may result in symptoms of neurological disorders [84] . cytokine storms may influence the cns and enhance the severity of covid-19 patients to develop ane, meningitis, and hemorrhage [5, 85] . therefore, it is necessary to identify the mechanism behind sars-cov-2induced cytokine storms and the course of release of the cytokines during the infection. the contribution of the proinflammatory cytokines alone and the direct tissue damage caused by the virus needs to be addressed. the indirect influence of systemic inflammation on the cns by targeting the proinflammatory mediators will be worth investigating. it is found that ace-2 is expressed in various brain regions, like the subfornical organ, the nucleus of the tractus solitarius, and rostral ventrolateral medulla, as well as in non-cardiovascular areas such as the motor cortex and raphe [86] . according to a spatial distribution analysis, ace-2 is expressed in substantia nigra and brain ventricles [87] [88] [89] . the protein's cell type distribution revealed both excitatory and inhibitory neurons, pericytes and endothelial cells, and glial cells like astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex express ace-2, unlike the cells in the region of the prefrontal cortex [9] . additionally, the hippocampus has few ace-2 expressing cells [9] . studies report that angiotensin ii downregulates the expression of ace-2 in neonatal rat cerebellar or medullary astrocytes [90] . therefore, the predominant expression of ace-2 in the brain hints towards the virus's potential to infect the cns. furthermore, brain endothelial and smooth muscle cells of the blood vessels express ace-2 [7] . the virus may enter into the cns through the hematogenic pathway, subsequently crossing the bbb [91] . a post-mortem study of the frontal lobe of a covid-19 patient reports virus presence in neurons and capillary endothelial cells [92] . infection of endothelial cells may allow the virus to pass from the respiratory tract to the blood. the virus in the peripheral system can move into the cerebral circulation, where the blood's sluggish movement may facilitate the viral s protein interaction with the ace-2 expressed on the endothelial lining of the brain (fig. 1a) [93] . another speculated entry route for sars-cov-2 may be through the enteric nervous system upon infection of enterocytes [94, 95] . enterocytes express high magnitudes of ace-2 [7] . once inside the brain, the virus can infect the neural cells, astrocytes, and microglia. these cells express ace-2, thus initiating the viral budding cycle followed by neuronal damage and inflammation (fig. 1a) [96] . moreover, multiple transcriptome studies show and validate ace-2 expression levels in various non-neuronal cells of olfactory mucosa [97] . studies support the viral susceptibility of the mucosal cells, sustentacular cells, bowman's cells, and olfactory stem cells [98, 99] . loss of smell in covid-19 is marked by potential deterioration of olfactory stem cells and other accessory cells [98] . also, a high-throughput single-cell expression study mentioned no ace-2 expression in olfactory covering glia, microvillar cells, and immature or mature olfactory sensory neurons [100] . it is speculated that sars-cov-2 on binding may stimulate olfactory receptor neurons (orns) to exert an exaggerated immune response. earlier studies with sars-cov have established infection of the brain through orns [101] . studies describing the transneuronal/transsynaptic movement of the sars-cov already exist. rabies viruses can take over the vesicular axonal transport machinery to disseminate in the brain (fig. 1a) [102] . human herpesvirus-6 (hhv-6) propagates in olfactory endothelial (oe) cells before invading the brain [93] . these studies enable to predict and support the movement of sars-cov-2 through the vesicular axonal pathway in an anterograde fashion through the olfactory nerve and facilitate brain infection [102] (fig. 1a) . also, the virus may directly reach the csf around the olfactory nerve fibers from oe cells [82] . a probable trajectory of sars-cov-2 to the brain may be via high-ace-2-expressing non-neuronal oe cells to low-ace-2-expressing mature orns along the olfactory axons. this mechanism highlights the ace-2 independent process of virus spread. lastly, the expression of transmembrane serine protease 2 (tmprss2) in human olfactory mucosa may further worsen the case of sars-cov-2 infection [97] . a study demonstrates that respiratory epithelial cells express tmprss2 without ace-2 [103] . the mosaic distribution of tmprss2 in mature orns is reported [104] . therefore, the virus can preferentially gain entry into the pns through one of the two epithelial cell types in the nose, either the goblet cells or the ciliated cells. tmprss2, in collaboration with furin, accelerates sars-cov-2 entry [105] . furin, a host serine endoprotease, is particularly of neurological relevance. in general, furin can activate neuronal growth factors and influence cns homeostasis [106] . however, upon attachment of sars-cov-2 with ace-2, the enzyme generates an active s protein through irreversible cleavage of the precursor protein [105] . the protein s1/s2 subunits separate, which subsequently facilitate virus entry into the host [105] . thus, exploring the possible avenues of sars-cov-2 entry and impact on cns is the need of the hour. various clinical reports have made the association of sars-cov-2 with neurological dysfunction prominent. covid-19-associated neurological severity is primarily associated with cytokine storms. the earlier identified sars-cov is already known to suppress the host antiviral response and activate the pro-inflammatory pathways. briefly, it would be crucial to analyze the ifn-antagonizing and inflammasome-activating properties of sars-cov-2. furthermore, the interaction of sars-cov-2 and ace-2-expressing neuronal/glial cells may facilitate virus entry into the nervous system through different routes. thus, the nervous system's involvement in covid-19 may be more than the current situation apprehends, therefore referring to the virus as an underestimated pathogen. medical expert clinicians and researchers' collaboration may address the enhanced incidents of neural dysfunctions in infected individuals. after identifying initial neurological damages, careful monitoring of covid-19 patients in the long term is also necessary. acknowledgments we thank the ministry of human resource and development and department of biotechnology, govt. of india for fellowship to shweta jakhmola and omkar indari, respectively, in the 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human lung cells furin at the cutting edge: from protein traffic to embryogenesis and disease publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-253797-a9lmfaho authors: eddleston, m.; mucke, l. title: molecular profile of reactive astrocytes—implications for their role in neurologic disease date: 1993-05-31 journal: neuroscience doi: 10.1016/0306-4522(93)90380-x sha: doc_id: 253797 cord_uid: a9lmfaho abstract the central nervous system responds to diverse neurologic injuries with a vigorous activation of astrocytes. while this phenomenon is found in many different species, its function is obscure. understanding the molecular profile characteristic of reactive astrocytes should help define their function. the purpose of this review is to provide a summary of molecules whose levels of expression differentiate activated from resting astrocytes and to use the molecular profile of reactive astrocytes as the basis for speculations on the functions of these cells. at present, reactive astrocytosis is defined primarily as an increase in the number and size of cells expressing glial fibrillary acidic protein. in vivo, this increase in glial fibrillary acidic protein-positive cells reflects predominantly phenotypic changes of resident astroglia rather than migration or proliferation of such cells. upon activation, astrocytes upmodulate the expression of a large number of molecules. from this molecular profile it becomes apparent that reactive astrocytes may benefit the injured nervous system by participating in diverse biological processes. for example, upregulation of proteases and protease inhibitors could help remodel the extracellular matrix, regulate the concentration of different proteins in the neuropil and clear up debris from degenerating cells. cytokines are key mediators of immunity and inflammation and could play a critical role in the regulation of the blood-central nervous system interface. neurotrophic factors, transporter molecules and enzymes involved in the metabolism of excitotoxic amino acids or in the antioxidant pathway may help protect neurons and other brain cells by controlling neurotoxin levels and contributing to homeostasis within the central nervous system. therefore, an impairment of astroglial performance has the potential to exacerbate neuronal dysfunction. based on the synopsis of studies presented, a number of issues become apparent that deserve a more extensive analysis. among them are the relative contribution of microglia and astrocytes to early wound repair, the characterization of astroglial subpopulations, the specificity of the astroglial response in different diseases as well as the analysis of reactive astrocytes with techniques that can resolve fast physiologic processes. differences between reactive astrocytes in vivo and primary astrocytes in culture are discussed and underline the need for the development and exploitation of models that will allow the analysis of reactive astrocytes in the intact organism. the molecular prothe characteristic of reactive astrocytes should help define their function. the purpose of this review is to provide a summary of molecules whose levels of expression differentiate activated from resting astrocytes and to use the molecular profile of reactive astrocytes as the basis for speculations on the functions of these cells. at present, reactive astrocytosis is defined primarily as an increase in the number and size of cells expressing glial sbriliary acidic protein. jn r&q this incmase in gliai tibrillary acidic prolix-~tive cells reflects ~~orninan~y pbenotypic changes of resident astrnglia rather than migration or proliferation of such cells, upon activation, astrocytes upmodulate the expression of a large number of malecules. from this molecular profile it becomes apparent that reactive astrocytes may benefit the injured nervous system by participating in diverse biological processes. for example, upregulation of proteases and protease i~i~to~ could help remodei the extrac&ular matrix, regulate tire concentration of different proteins in the neuropil and ciear up debris from degenerating &is. cytokines are key mediators of immunity and inflammation and could play a critical role in the regulation of the blood+entral nervous system interface. neurotrophic factors, transporter molecules and enzymes involved in the metabolism of excitotoxic amino acids or in the antioxidant pathway may help protect neurons and other brain cells by controlling neurotoxin levels and contributing to homeostssis within the central nervous system. therefore, an impairment of astroglial performance has the potentia1 fo exacerbate neuronai dysfunction. based on the synopsis of studies presented, a number of issues become apparent that deserve a more extensive analysis. among them are the relative contribution of microglia and astrocytes to early wound repair, the characterization of astroglial subpopulations, the specificity of the astroglial response in different diseases as well as the analysis of reactive aatrocytes tith techniques that can resolve fast physiologic processes. dieerences between reactive astrocytes in e&o and primary astrocytes in culture are discussed and underline the need for the development and exploitation of models that will allow the analysis of reactive astrocytes in the intact organism. 1, i~~du~i~n physiologic and pathologic processes." one of the most remarkable ~ha~~~~s~~ of astrocytes is their astrocytes make up a substantial proportion af vigorous response to diverse neurologic insults, a the cns and participate in a variety of important feature that is well conserved across a variety of different species. the astroglial response (see section 2) *to whom correspondence should be addressed. occurs rapidly and can brz de&&d within one hour abbr&atiorts: see over. of a focal mechanical trauma.201 prominent reactive 1.5 astrocytosis is seen in aids demerttk" a variq of other viral infections,'"' prion-associated spongiform en~phaiopat~ies~ rii in~ammatory dcmyel~na~~ng disease,"'.'53 acute traumatic brain injury,'" and such neurodegenerative diseases as alzheimcr's disease." '.hh the prominence of astroglial reactions in various diseases, the rapidity of the astroglial response and the evolutionary conservation of reactive astrocytosis indicate that reactive astrocytes fulfill important functions for the cns. yet, the exact rofe reactive abbrsviahzs: act, antichymotrypsin: ad, alzheimer's disease; adc, alds dementia complex; als, amyotrophic lateral sclerosis; app, amyloid /? protein precursor; @a4 (aaf-421, amyloid 13 protein (amino acids i-42); bdnf, brain-derived neuro~ophi~ factor; ca" i, calcium ionophore; ca ii, carbonic anhydrase ii; cad, carbamyl phosphate synthetase ii/aspartate transcarbamylase/dihydroorotase: cd, cluster designation; cjd, creutzfeld-jakob disease; cmv, cytomegalovirus; cona sup, supematant from concanavalin a-stimulated macrophages; &pm. central pontine myelinolysis: cpt-camp. g"(~-ebiorop~enyi thief adenosine 3'-5-cyclic monopbosphae; protein kinase c; pma, phorbol-iz-mvristate-1 z-ace&e; pn, proteese nexin; ptbbs, peripheral type benzodiazepine binding site; sgp, sulphated glycoprotein: sspe, subacute sclerosing pane?~~p~a~itis~ sub p, substance p; tgf, transforming growth factor; timp, tissue inhibitor of metailoprotease; 'us, tpa induced sequences; tmev, theiler's murine enccphalomyelitis virus; tnf, tumour necrosis factor; tpa, 12-o-tetradecannyl-phorbol-13-acetate; t-pa, tissue-type plasminogen activator; u-pa, urokinase-type plasminopn activator; trauma, focal mechanical or electrolytic destruction of cns tissue; vw', vasoactive intestinal peptide: vla, fi 1 integrin famity. astrocytes piay in the injured c'ns has so l'al-rzn~~~ncd efusive. assuming that the hiotugical funcrions of iwctive astrocytes are reffected in the proteins the!, express, this review aims to further our undcrstanding of these cells by providing a synopsis ot' rcccnt studies examining the molecular profile of activated astrocytes. the cns responds to neural injuries with an increase in the number and size of cells expressing glial fibrillary acidic protein (gfap), a phenomenon generally referred to as reactjve astrocytosis. gfap is an intermediate filament cytoskefetal protein expressed primarily by astrogliaz9 and represents the prototypic marker of astroglial activation.z8s60 however, despite its prominent upmodulation in response to diverse injuries, the precise function of the gfap molecule remains unciear. suppression of gfap expression in gliaf ceil lines with antisense mrnas suggests that gfap may be necessary for tbe formation of stable glial processes in response to neuronai signals."" it will be interesting to assess the functional role of gfap in vivo by ablating gfap in experimental animals with the help of homologous recombination, expression of anti-sense mrnas or ribozymes. it sboutd be noted that it has not yet been established if an increased level of gfap expression and/or turnover is, in fact, a reliable indicator of astroglial activity in general. for example, in normal rodent brains, astrocytes of the glial limitans and the hippocampal formation show higher levels of gfap mrna and gfap immunostainin~ than astrocytes of other brain regions.".'si.i5".1h7.?"1 this raises the question whether these heterogenous levels of gfap expression reflect particular functional demands placed upon specific astroglial subpopulations and whether they correlate with a general increase in the functional activity!ltl~tabolism of the strong& gfaf-positive cells. it should also be noted that using increased gfap expression as the basis for the definition of astroglial activation will exclude any subpopulation of astrocytes that responds to neural injury without gfap expression. pending further experimental evaluation of these issues we have considered the induction of gfap expression to be the main indicator of astroglial activation. 'the origin of the increased number of gfapexpressing cells that appear in response to ncurologic insults has been the subject of intense discussion over the last decade, specific&y, the debate has focused on the question of whether reactive astrocytosis represents primarily the prol~ferat~on/migration of gfap-positive cells or the phenotypic change of local astrocytes. studies using double-labeling with gfap antibodies and bromodeoxyuridiae or tritiated thymidine to identify dividing astrocytes have shown that, ar least in acute lesions, mitotic division (pro-for the majority of gfap-positive cells that appear in response to the injury (for review, see ref. 211) . furthermore, we have been unable to find convincing in vivo evidence that mature gfap-positive astrocytes of adult brains are able to migrate effectively. hence, it is likely that the appearance of gfappositive astrocytes in regions of acute neural injury represents primarily a change in the phenotype of resident astroglia. it can, however, not be excluded that astroglial proliferation contributes more significantly to chronic astrocytosis. in many instances, the phenotypic changes seen in reactive astrocytes may reflect a substantial increase in astroglial metabolism and protein synthesis, consistent with a "healthy" cellular hypertrophy in response to increased physiologic demands. in other situations, however, astroglial swelling may result from pathologic processes that afflict the astrocyte itself (for review, see ref. 210). the current literature on reactive astrocytosis is extensive. we have attempted a comprehensive review of this subject using rigid selection criteria to produce a practical synthesis that will be easily amenable to consultation. to construct a list of molecules expressed by activated astrocytes we have included information drawn from two types of studies. the first are studies carried out in vivo where the expression of a particular molecule or its mrna was co-localized to reactive astrocytes by immunochemical staining or in situ hybridization. for inclusion into the table clear evidence for astroglial expression was required, for example co-labelling with gfap or demonstration of electron-microscopic features typical of astrocytes. this should ensure that the molecules in question were indeed found in astrocytes rather than in other injury-responsive cns cells, in particular microglia. the combination of immunostaining with in situ hybridization can also help differentiate between accumulation in astrocytes of molecules actually synthesized by these cells and those produced elsewhere and subsequently taken up by the astrocyte. many interesting leads on the induction of potential astroglial molecules, particularly enzymes, have come from studies on bulk brain extracts. however, because these studies usually do not provide direct proof that astrocytes form the main cellular source of the identified molecules in the patholo~~lly altered cns, they have not been included in this review. the second class of information comes from in vitro studies. since the isolation of enriched astrocyte cultures by mccarthy and de vellis192 and subsequent refinements, a great deal of experimental work on astrocytes has been carried out in vitro. experiments indicating the upregulation of a molecule by a certain factor in vitro may offer clues as to what happens during reactive astrocytosis in the cns, especially if this factor is known to occur in pathological conditions. however, while tissue culture studies often provide important leads they can also sometimes be misleading. therefore, because so much of our current knowledge on astrocytes is based on in vitro studies we would like to address a few caveats that should be kept in mind when considering the molecular profile of cultured astrocytes. a major consideration is the imperfect purity of primary astrocyte cultures because current techniques for purifying astrocytes usually produce cultures of 90-99% purity. contamination with microglia is particularly problematic because these cells also respond to neural injuries and secrete a number of biologically active molecules such as cytokines. at present, the most definitive assay for determining the cell source of most molecules is the combination of immunostaining with in situ hybridisation but this has been carried out only rarely (for an example, see ref. 287). for inclusion into table 1 , we have favored studies that have addressed the issue of culture purity. the adult cns is characterized by the close interaction of many different cell types both through actual cell contact and secretion of factors. thus a further problem is that cells in nearly pure primary culture have been released from these interactions. this point is illustrated by the fact that astrocytes in tissue culture have different morphologies depending on whether they are cultured alone or with other neural cells. cultured alone, they bear few processes, however when co-cultured with neurons they develop multiple processes. 'lo the physiolo~c behavior of astrocytes is also dependent on the presence of other neural cells. cocultivation of astrocytes with neurons induces calcium channel activity in astrocytes which is undetectable in pure astrocyte cultures or in astrocytes co-cultured with oligodendrocytess6 many protocols for the establishment of primary astrocyte cultures include an early exposure of the cells to relatively high concentrations of serum. this represents a major difference from the situation in vivo where astrocytes are shielded from blood-derived factors by the blood-brain barrier. in essence there are numerous variables in culture conditions that could dramatically influence the molecular profile of astrocytes in vitro and alter the astroglial responsiveness to further stimulation. cloned lines of immortalized glial cells such as the rat glioma cell line c6 can circumvent the problem of culture impurity and have yielded an enormous amount of interesting data. however, they differ from astrocytes in viva in many respects, even more so than primary astrocytes. as an example, astrocytes of the adult cns have only a limited proliferative potential'72~2'1 and this is reflected to some extent in primary culture. in contrast, immortalized cell lines often proliferate vigorously having been released from many controlling influences, including in some cases contact inhibition. therefore, findings obtained with immortalized glial cell lines have not been included in table 1 io,11 io,11 10 10 9 9 9 9 10,ll io,11 10 9 9 9 9 10,ll io,11 10 io,11 10,ll 10 10 9 9 9 9 10 10 10 10 9 the assignment of molecules to a specific functional category was introduced to facilitate consultation of the table. note, however, that this assignment is somewhat arbitrary as a number of molecules can exist in different forms or fulfill functions in different categories. for example, there is evidence that components of the amyloid fi protein precursor (app) could function as a protease inhibitor2'4s273 or as a serine proteasg2' while the structure of the whole precursor molecule resembles that of a cell-surface receptor '38. in addition, it seems likely that other functions will be identified for many of the above molecules, some of which may be more relevant to the cns than those they are currently assigned. because gfap is a well established marker for reactive astrocytes and colocalization with gfap was required for inclusion into the table this molecule has not been listed. separation of inducer molecules/conditions by commas indicates that each inducer was effective when tested in isolation, whereas a plus sign indicates that synergistic effects were observed when both inducers were combined. references in square brackets [] contradict the previously quoted reference. for definitions, see abbreviations list. the transition of astrocytes from the resting to the activated state is associated with the expression of new molecules not normally detectable in quiescent astroglia as well as with the upmodulation of factors that are found in resting astrocytes at lower levels. table 1 lists a number of molecules whose expression in astrocytes increases upon astroglial stimulation and, hence, may provide a molecular profile of reactive astrocytosis. from this table, it appears that reactive astrocytes are equipped with a large armamentarium of molecules that allows them to participate in many important biologic functions. in the subsequent sections we will speculate how the expression of specific groups of molecules could relate to the function of reactive astrocytes. as outlined above astrocytes undergo dramatic changes upon activation which are likely to have functional consequences. it remains, however, controversial if the induced changes are generally beneficial or detrimental in nature (reviewed in ref. 231). on one hand, it is conceivable that the increase in cytoskeletal proteins within reactive astrocytes may assist wound repair by stabilizing the tissue surrounding neural injuries. the glial scar formed by reactive astrocytes may also help to wall off areas of tissue necrosis, excluding non-neural cells from the cns parenchyma and appears to fill in the space that results from neuronal 10~s.~~ on the other hand, it has been suggested that the glial scar may form a barrier that could hinder regenerative processes such as neurite outgrowth.2~~232 central neurons do not regenerate effectively after injury. the studies of aguayo and colleagues indicate that this is due not to an intrinsic inability of these neurons to regenerate but to the environment present within the cns.* electron-microscopic analysis of regenerating axons revealed that arrest of axonal growth in the cns occurs in the immediate vicinity of reactive astrocytes. 175 this observation together with the finding that reactive astrocytes in vivo express molecules which inhibit neurite extension in vitro'93 suggests that astrocytes can actively inhibit regeneration. while it is difficult to prove that dense gliotic scars do not mechanically block axonal growth, in vitro evidence suggests that astrocytes themselves are not necessarily inhibitory to regeneration (reviewed in refs 111, 182) . furthermore., reactive astrocytes do not prevent pc12 cells from extending neurites over glial scars in optic nerve explants.'j3 most conclusively, the in vivo experiments of gage and kawaja showed that in the presence of ngf (produced by transplanted fibroblasts), reactive astrocytes could, in fact, provide a substrate for the growth of sympathetic neurites.'" these findings demonstrate that, at least in certain experimental situations, astrocytes do not inhibit but may even promote regeneration. a role for reactive astrocytes in regeneration and tissue repair is also supported by their molecular profile (see table 1 ) which suggests both a production of, and interaction with, the extracellular matrix. in vivo astrocytes express extracellular matrix molecules such as laminin, chondroitin-6-sulphate proteoglycan and glial hyaluronate adhesion protein, a hyaluronate binding protein. in vitro, they are also able to secrete glycosaminoglycans.*j35 reactive astrocytes may interact with extracellular matrix and other cns cells via adhesion molecules such as embryonic neural cell adhesion molecule and cytotactin/tenascin. transforming growth factor (tgf)-/l i has been shown to be increased in reactive astrocytes after cns stab wounds."' logan and colleagues proposed that astroglial secretion of tgf-fl 1 may attract fibroblasts into the lesion site, regulate their deposition of extracellular matrix proteins and synthesis of degradative enzymes, and play a role in controlling angiogenesis in the scar. hence, astrocytes may be important in controlling the deposition of scar tissue after injury and its vascularization."' the production of proteases and protease inhibitors might allow astrocytes to further remodel the extracellular matrix at sites of neural injury and to clear up the debris of degenerating cells. while the activity of these molecules would thus assist in wound repair it is also conceivable that astroglial proteases or protease inhibitors have detrimental effects in certain pathologic conditions. the production of calcium activated proteases by reactive astrocytes has, for example, been implicated in the degeneration of neurons after ischemia, and in the production of the amyloid /3 protein,'" a protein that accumulates abnormally in the brains of patients with alzheimer's disease. destruction or degeneration of white matter tracts in the cns leads to the release of large quantities of myelin lipids. apolipoprotein e (apoe) is a major constituent of both low-and high-density lipoproteins and plays an important role in lipid transport and metabolism. within the cns apoe is constitutively produced by astrocytes,"~202~'5' and the astroglial expression of apoe has been found to be upmodulated during reactive astrocytosis."' astrocyte-derived apoe may help deliver lipids to other cns cells for membrane biosynthesis and facilitate the removal of cholesterol into the periphery. consistent with the latter possibility is the increase in plasma apoe levels observed during the active phase of experimental allergic encephalomyelitis (eae).'"' a demyelinating disease of the cns. one of the major functions proposed for reactive astrocytes is the initiation of immune responses within the cns (e.g., see ref. 112 ). when treated with factors such as interferon-y, astrocytes in vitro are induced to express molecules involved in immune responses, for example major histocompatibility complex (mhc) antigens and adhesion molecules such as intercellular adhesion molecule 1. cultured astrocytes are able to present antigens to mhc class i and to mhc class ii restricted t lymphocytes80~8'~'74,26' and to produce many different cytokines. in addition, a number of in vivo immunohistochemical studies have reported the expression of mhc molecules on small numbers of reactive astrocytes in different pathologic conditions (see table i ). taken together, these findings support speculations that (i) antigen presentation by mhc expressing astrocytes and astroglial production of cytokines might play a crucial role in cns-immune interactions; and that (ii) interactions are mediated primarily by microglia rather than by astrocytes.".'*' '23~'4'~'ch~'5x~'rr these studies indicate that astrocytes probably do not function as the main antigen presenting cells in the cns and argue against a major role for astrocytcs in the initiation of immune-mediated neurologic diseases. however, as outlined below, astrocytes may still have important regulatory effects on inflammatory and immune responses directed at the cns. the interaction of the cns with blood-borne factors and cells is of paramount importance in the pathogenesis of a number of neurologic diseases. this interaction is controlled, in part. by the blood-brain barrier which is formed by the unique properties of the cns endothelial cells. astrocytes are in intimate contact with these cells by their endfeet processes**' and several lines of evidence suggest that they may participate in the control of the blood-cns interface. astrocytes could influence the entry of hematogenous cells into the cns as well as their intraparenchymal activity through the secretion of cytokines. as indicated in table 1 , astrocytes appear to produce a large number of cytokines and inflammatory mediators in vitro. unfortunately. in gw confirmation of these findings is lacking in most cases and the possibility of microglial contamination of astrocyte cultures has not always been addressed rigorously. however, the few in vivo studies that are available support the postulate that astroglial cytokine production may be involved in the pathogenesis of viral and immune mediated neurologic diseases. for example, wahl and colleagues*" have shown that reactive astrocytes in hiv-1 infected brains express tgfp and speculate that this cytokine enhances the recruitment of hiv-l -infected monocytic cells. hence, the astroglial tgf,0 production could both contribute to the inflammatory changes seen in hiv-i associated encephalomyelitis and also increase the spread of cell-borne virus in(to) the cns. it should be noted in this context, however, that many cytokines appear to fulfill a multitude of functions (for review see ref. 26.5 ) and that their effects in the intact adult cns are only now beginning to be defined." it is, therefore, perhaps not too surprising that the effects of cytokines in specific neurologic diseases have been difficult to predict.'0.'9.'74.'4h proteases and protease inhibitors could be used by astrocytes to regulate the concentration of a variety of proteins in the parenchyma, including cytokines and proteases derived from the blood or from other brain cells. such a role has recently been suggested for protease nexin 1,5'*'27 a protease inhibitor found to be increased in reactive astrocytes.'*' in vitro data indicate that protease-protease inhibitor complexes can induce the synthesis of acute phase proteins in response to injury'q~'s2 and stimulate the directed migration of neutrophils. is because reactive astrocytes express both cathepsin g-like protease and alphalantichymotrypsin-like protease inhibitor activities (abraham et al., unpublished observations) such complexes may form around reactive astrocytes where they would directly or indirectly increase the release of cytokines and acute phase proteins from astrocytes, endothelial cells, microglia or blood derived in head trauma and intracerebral hemorrhage the blood-cns interface is acutely disrupted. this disruption causes red blood cells to extravasate, lyse and release iron-containing compounds into the cns. consequences of such lesions include focal encephalomalacia, hemosiderin deposition and occasionally the development of recurrent seizures. studies in experimental animals suggest that some of the clinical sequelae of brain trauma are related to the induction of free radicals by the iron moieties within extravasated blood, and the subsequent peroxidation of lipids. 282 the expression of transfer&, which mobilizes and transports iron, and its receptor in reactive astrocytes 55395*2'5 suggests that these cells may help diminish excess iron loads around sites of tissue injury. the blood-brain barrier shields the cns from toxic metals present within the blood. however, in a number of locations the blood-brain barrier is leaky. *' surrounding these sites one finds a class of gfap-positive cells termed gomori astrocytes (reviewed in ref. 245) which may have an important role in controlling metal toxicity. these cells increase in number after irradiation256 and accumulate silver, mercury and lead after systemic administration of these compounds. 245 gomori astrocytes express metallothionein,289 a protein which can bind to heavy metals such as cadmium and mercury, detoxifying them in the process. the protein is inducible by heavy metals in various tissues and there is some evidence that this occurs in astrocytes after cadmium administration.*08 tissue factor or tissue thromboplastin is a transmembrane glycoprotein that functions as the initiator of the coagulation protease cascade. in the brain tissue factor is expressed predominantly in astrocytes.'*" in view of the apposition of astroglial endfeet with cns endothelial cells (see above), tissue factor could help astrocytes form a "hemostatic envelope" around the vascular system of the cns. the upregulation of tissue factor expression by reactive astrocytes in nonhemorrhagic conditions such as scrapie suggests that tissue factor may fulfill additional functions within the cns. while it has long been realized that astrocytes secrete factors that promote the growth and prolong the survival of neurons in explant culture,16 so far only a limited number of astroglial molecules that exert trophic effects on neurons have been identified. however, it seems likely that this small group represents the tip of the iceberg. as outlined below some astroglial neurotrophic factors may act directly on neurons whereas others could benefit neurons indirectly through the support of other cns cells. both nerve growth factor (ngf) and basic fibroblast growth factor (bfgf) act as survival and neurite extension factors for some types of cultured neurons.'99~278~286 astrocytes, in contrast to microglia, are ablear to secret ngf in vitro.*" after trauma, ngf levels are increased in both the optic nerve"' and the hippocampus,'60~28' and in a separate study, the cellular source of ngf was shown to be astrocytes. i4 astrocytes also produce bfgf in vitro in response to various factors, and in alzheimer's disease and lesioned brain bfgf has been localized to reactive astrocytes (see table 1 ). recent evidence from tissue culture studies suggests that growth factors such as ngf and bfgf are able to protect central neurons against hypoglycemic/excitotoxic insults by stabilizing neuronal calcium hemostasis.48'90'9' reactive astrocytes produce insulin-like growth factor-1 (igf-1) after ischemia.94j63 because igf-1 has neurotrophic effects,46.94 this astroglial response may help diminish neuronal loss. igf-1 also stimulates oligodendrocyte development and myelination in vitro.m work with mice transgenic for igf-1 supports a similar role for the molecule in vivo.47a consistent with the postulated role of igf-1 in myelination, both igf-1 and its receptor decrease to minimal levels in the adult brain.'3'8,33 reactive astrocytes have recently been shown to express igf-1 '49 concomitant with the expression of the igf-i receptor by immature oligodendrocytes around the lesion.'49 this raises the possibility that reactive astrocytes play an important role in the remyelination of the adult cns. however, reactive astrocytes expressing tumor necrosis factor 01 (tnfcr) have been identified in multiple sclerosis lesions.'28~248 while there is no direct evidence for a role of astrocyte-derived tnfa in demyelination in vivo, this cytokine has been shown to be toxic to oligodendrocytes in culture. 234,249 consequently, it remains undecided at this point if the role of astrocytes in inflammatory demyelinating diseases is beneficial or detrimental. high concentrations of excitatory neurotransmitters are extremely toxic to neurons (reviewed in ref. 52). evidence is increasing that the neuronal death or impairment that follows acute neurologic insults (e.g. hypoxia/ischemia, mechanical trauma, prolonged seizures) may, in the large part, be mediated by an increase in the extracellular concentration of excitatory amino acids such as glutamate. a role for glutamate toxicity has also been proposed in more chronic neurologic diseases such as alzheimer's disease,'47,'xy aids dementia (reviewed in ref. 173) , sulfite oxidase deficiency, guam amyotrophic lateral sclerosis and huntington's disease (reviewed in ref. 52). in the presence of high glutamate levels, removal of astrocytes from mixed cultures quickly leads to neuronal cell death.2"."8.260 in vitro studies suggest that amino acid transmitters may be removed from the extracellular space by astrocytic uptake mechanisms (reviewed in refs 79, 113, 132) . astrocytes also contain glutamine synthetase which converts glutamate to glutamine and helps detoxify ammonia in the cns. this enzyme has been shown to be upmodulated in reactive astrocytes in pathologic conditions.4'.2"" hence, it is possible that astrocytes participate in the removal of neurotoxins by both enhanced uptake and metabolic turnover. the recent cloning of the transporters for gaba and the amines, noradrenalin, serotonin and dopamine (for review see refs 254, 272) should supply molecular tools that will help in understanding the role of reactive astrocytes in regulating other neurotransmitters. in a number of recent studies, heyes and his colleagues have provided evidence that the nmda receptor agonist quinolinic acid is involved in the pathogenesis of the neurologic dysfunction that can be associated with hiv-i infection and other inflammatory diseases of the nervous system.'"."' i20 because the quinolinic acid metabolizing enzymes, 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase, have been localized to astrocytes in vivo, '50~'5' it is conceivable that the expression of these enzymes increases in astrocytes responding to inflammatory lesions. while an upmodulation of these enzymes in reactive astrocytes has apparently not yet been documented in the literature such an astroglial response could serve important protective functions in a variety of neurologic diseases. free radicals form another group of chemicals that could be extremely toxic to the nervous system'02,'0' and the ability to eliminate or control these entities may be critical after neurologic insults such as cerebral hemorrhage. 282 while this issue does not appear to have been directly studied in reactive astrocytes, there is evidence that astrocytes may play a role in the antioxidant defense system. the biopigments biliverdin and bilirubin are potent antioxidants.258 they are synthesized by a pathway which is rate-limited by the heme oxygenase isozymes ho-1 and ho-2. ho-i is expressed by astrocytes in culture72 and induced in glial cells after heat shock to the rat brain.74 apolipoprotein d, proposed also to be involved in the production of antioxidants, appears to be expressed by astrocytes in the normal cns" and increases in the peripheral nervous system after injury.'" antioxidant enzymes such as superoxide dismutase and catalase have been proposed to be induced in reactive astrocytes in alzheimer's disease."" if the above studies are confirmed by double-labeling of reactive astrocytes it would be interesting to know if the antioxidant enzymes are induced solely to protect the astrocytes themselves or whether they are also secreted to influence the environment of other neural cells. in this review we have constructed a molecular profile of reactive astrocytes and drawn conclusions from this profile on the functions reactive astrocytes may fulfill in neurologic diseases. as a result we have hypothesized that activated astroglia may benefit the damaged nervous system by participating in several important biologic processes such as the regulation of neurotransmitter levels, the repair of the extracellular matrix, control of the blood-cns interface, transport processes, and trophic support of other cns cells. the detectability of specific molecules depends not only on their absolute levels but also on the sensitivity of the assays used, i.e. the inability to detect certain markers does not necessarily exclude their presence. consequently, it cannot be excluded that "resting" astrocytes also fulfill some of the functions assigned to reactive astrocytes but at a lower level. we would like to emphasize that our extrapolation of the functions of reactive astrocytes from the molecules they express is speculative and based on current knowledge. it seems likely that other functions will be identified for many of these molecules, some of which may be more relevant to the cns than the ones they are currently assigned. we also expect that the ongoing discovery of cns-specific genes (see ref. 198 for review) and the development of novel molecular probes/assays will significantly expand the molecular profile of reactive astrocytes. in the majority of cns diseases clinical signs and symptoms are related most directly to an impairment of neuronal functions. while little evidence exists that the activity of reactive astrocytes is directly detrimental to the nervous system, it is conceivable that an impairment of astroglial performance could exacerbate neuronal dysfunction. this pathogenetic scenario may, for example, exist in hepatic encephalopathy (see ref. 210 for review), scrapie in which prions appear to accumulate first in astrocytes" or in aids dementia where viral or macrophage-derived products could interfere with astroglial functions such as neurotrophic support and/or elimination of excitotoxins,36.37,49.173,228 an inspection of table 1 reveals that reactive astrocytes express a number of molecules that are typically produced by hematogenous cells. this observation could reflect the evolutionary response of the cns to two different types of selective pressures. neurophysiologic processes that occur within seconds. there appears to be a need for the cns to restrict for example, the response of neurons to electrical the access of hematogenous cells as evidenced by the stimulation was shown to be accompanied by rapid blood-brain barrier and the delayed invasion of ca*+ oscillations within astrocytes in hippocampal neutrophils and monocytes after injections of lps slice preparations. 62 astrocytes themselves are also into the brain parenchyma when compared with capable of responding to neurotransmitters (reviewed peripheral sites.' on the other hand, early stages of in refs 19, 26) . because of their close association with wound repair within the cns may depend on the nodes of ranvier,32*'% perinodal astrocytes may be fast action of those factors which are released into in a particularly suitable position to influence neuroperipheral wounds by hematogenous cells. recent physiologic processes. it is possible that rapid evidence suggest that astrocytes are able to respond responses of astrocytes are of greater functional to neural injury with great rapidity.'"*'31s"' therefore, importance in neurologic diseases than the molecular astrocytes may fulfill some of the functions that are changes that occur over hours or days. yet, this type carried out by invading hematogenous cells during of response cannot be detected with conventional wound repair in peripheral sites. we would like to histopathologic methods. the application of novel emphasize at this point that the response of the cns neurophysiologic and cell biologic techniques should to neurologic injury involves many cell types in allow a high chronologic and spatial resolution of addition to astrocytes and that the assignment of astroglial responses and is expected to substantially certain functions to astrocytes by no means excludes further our understanding of astroglial functions the participation of other cells. an assessment of the in health and disease. we suspect that this type of relative contributions of microglia and astrocytes to analysis will reveal "reactive astrocytosis" to be a early wound repair within the cns should be a much more dynamic process than is currently particularly fruitful subject for future studies. conceptualized. recent data indicate that subpopulations of astrocytes can be distinguished both at the molecular'7*'64,'83~'97 and functiona16',**' levels. in leukocyte research the development of molecular markers has revealed a great functional diversity among cells that appear morphologically very similar. it seems likely that ,future molecular studies will also reveal a functional heterogeneity of reactive astrocytes that far surpasses their morphologic differences. it will be particularly interesting to find out whether there are subpopulations of astrocytes that respond to some neurologic disease processes but not to others. in a similar vein, it needs to be determined whether diverse neurologic diseases provoke the expression of the same set of astroglial molecules or whether the astroglial response is specific, with different molecules being expressed by astrocytes responding to different neurologic insults. we would like to end this commentary by pointing out the imbalance between in vitro and in vivo studies in astroghal research. judged by the number of in vitro vs in vivo studies (see table 1 ), much greater efforts appear to have been placed on the extensive analysis of astrocytes in culture than on the in vivo confirmation of existing in vitro findings. however, reactive astrocytes in the adult brain and primary astrocytes in ccl1 culture differ in many respects and results obtained in vitro and in vivo often do not overlap (see table 1 and, for an example, ref. 170) . it is, therefore, to be hoped that future research will complement the vigorous efforts made in cell culture systems with the development and exploitation of models that allow the analysis of reactive astrocytes in the intact organism. it should also be pointed out that the response of astrocytes to neurologic insults has so far been documented primarily by immunohistochemical staining and in situ hybridization. this methodologic approach provides a static image of the molecular profile of reactive astrocytes and does not allow the resolution of fast physiologic changes. recent evidence suggests that astrocytes participate in central nervous system myelination in transgenic mice ngf and bfgf protect rat hippocampal and human cortical neurons against h~o~y~~c damage by stabilising calcium homeostasis human immunodeficiency virus can productively infect cultured human glial cells &expression of glial fibrillary acidic protein and vimentin in the central and peripheral nervous systems of the twitcher mutant. gfii i 11990) protease nexin-i localization in the human brain suggests a protective role against extravasated serine proteases glutamate neurotoxicity and diseases of the nervous system tumor necrosis factor-alpha production by astrocytes. induction by lipopoly~~ha~de, ifn-gamma, and il-lbeta characterization and differential distribution of the three major human protein kinase c isozymes (pkc alpha, pkc beta, and pkc gamma) of the central nervous system in normal and alzheimer's disease brains a histochemical study of iron, transferrin, and ferritin in alxheimer's diseased brains neuronal modulation of calcium channel activity in cultured rat astrocytes regulation and selective expression of ly-6a/e, a lymphocyte activation molecule, in the central nervous system glial cell-specific mechanisms of tgf-81 induction by il-1 in cerebral cortex transforming growth factor-beta 1 (tgf-beta i) expression and regulation in rat cortical astrocytes heterogeneity of the glial fibrillary acidic protein in gliosed human brain filament proteins in rat optic nerves undergoing wallerian degeneration: localization of vimentin, the fibroblastic 100-a filament protein, in normal and reactive astrocytes neuronal activity triggers calcium waves in hippocampal astrocyte networks macrophages can modify the non~~issive nature of the adult mammalian central nervous system castration enhances expression of glial fibrillary acidic protein and sulfated glycoprotein-2 in the intact and lesion-altered hippocampus of the adult male rat subacute encephalomyelitis of aids and its relation to htlv-iii infection general and dramatic glial reaction in alzheimer brains glial heterogeneity may define the three-dimensional shape of mouse mesencephalic dopaminergic neurons astrocytes and microglia in human brain share an epitope recognized by a b-lymphocyte-specific monoclonal antibody (ln-1) scrapie-associated priori protein accumulates in astrocytes during scrapie infection neuropathological changes in scrapie and alzheimer's disease are associated with increased expression of apoli~protein e and cathepsin d in astrocytes regulation of heat shock protein synthesis in ral astrocytes heme oxygenase is a heat shock protem and pest protein in rat astroglial cells astrocytes are the primary source of tissue factor in the murine central nervous system-a role for astrocytes in cerebral hemostasis selective autoregulation of endothelins in primary astrocyte cultures: endothelin receptor-mediated potentiation of endothelin-1 secretion normal and heat-induced patterns of expression of heme oxygenase-i (hsp32) in rat brain: hyperthermia causes rapid induction of mrna and protein astrocyte lineage. ii. mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin-and gfp-containing intermediate filaments. &vi astrocytes as antigen-presenting cells. i. induction of ia antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation the role of astrocytes in the interaction between the immune and nervous system characterizatjon of glutamate uptake systems in astrocyte primary cultures from rat brain astrocytes as antigen-presenting cells. part ii: unlike h-zk-dependent cytotoxic t cells, h-2ia-restricted t cells are only stimulated in the presence of interferon gamma astrocytes present myelin basic protein to encephalitogenic t-cell lines production of prostaglandin e and an interleukin-i like factor by cultured astrocytes and c6 glioma cells expression of class ii major histocompatibiiity antigens on reactive astrocytes and endothelial cells within the gliosis surrounding metastases and abscesses on the cellular source and function of interleukin-6 produced in the central nervous system in viral diseases the induction of inter~llular adhesion molecule 1 @cam-l) expression on human fetal astrocytes by interferon-gamma, tumor necrosis factor alpha, lymphotoxin, and interleukin-1: relevance to intracerebral antigen presentation astrocytes and intracerebral immune responses interleukin-1 beta and tumor necrosis factor-alpha synergistically stimulate nerve growth factor (ngf) release from cultured rat astrocytes subacute spongiform encephalopathies: transmissible cerebral amyloidoses caused by unconventional agents lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microghal cells expression of microtubule-associated protein 2 by reactive astrocytes laminin and heparan sulphate proteoglycan in the lesioned adult mammalian central nervous system and their possible relationship to axonal sprouting localization of the cd44 glycoprotein to fibrous astrocytes in normal white matter and to reactive astrocytes in active lesions in multiple sclerosis ameboid microglia as effecters of inflammation in the central nervous system a role for igf-i in the rescue of cns neurons following hypoxic-ischemic injury changes in ghal cell markers in recent and old demyelinated lesions in central pontine myelinolysis 11984) dibutvrvl cvclic amp causes intermediate filament accumulation and actin reorganisation in astrocytes basic fgf in adult rat brain: cellular distribution and response to entorhinal lesion and ~rnb~a-fornix transection brain interleukin 1 and s-10 immunoreactivity are elevated in down syndrome and alzheimer disease laminin-like antigen in rat cns neurons: distribution and changes upon brain injury and nerve growth factor treatment reactive oxygen species and the central nervous system (1992) neuroactive kynurenines in lyme borreliosis substance p and astrocytes: stimulation of the cyclooxygenase pathway of arachidonic acid metabolism phorbol diester tpa elicits prostaglandin e release from cultured rat astrocytes leukotriene production by cultured astroglial cells primary rat astroglial cultures can generate leukotriene b4 recombinant interleukin-i beta stimulates eicosanoid production in rat primary culture astrocytes neuronal regulation of astroglial morphology and proliferation in vitro astrocytes: auxiliary cells for immune responses in the central nervous system? role of astrocytes in compartmentation of amino acids and energy metabolism brain macrophages synthesize interleukin-1 and interleukin-i mrnas in vitro quinolinic acid in cerebrospinal fluid and serum in hiv-i infection: relationship to clinical and neurological status sustained increases in cerebrospinal fluid quinohnic acid concentrations in rhesus macaques (macaca muluttu) naturally infected with simian retrovirus type-d increased cerebrospinal fluid quinolinic acid, kynurenic acid, and l-kynurenine in acute septicemia cerebrospinal fluid and serum neopterin and biopterin in d-retrovirus-infected rhesus macaques (mucnca muiutta): relationship to clinical and viral status increased ration of quinolinic acid to kynurenic acid in cerebrospinal fluid of d retrovirus-infected rhesus macaques: relationship to clinical and viral status cerebrospinal fluid quinolinic acid concentrations are increased in acquired immune deficiency syndrome graft-vs.-host disease elicits expression of class i and class ii histocompatibility antigens and the presence of scattered t lymphocytes in rat central nervous system perivascular microglial cells of the cns are bone marrow derived and present antigen in vivo expression of ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the lewis rat infection of human t-lymphotropic virw type i to astrocytes in vitro with induction of the class ii major histocompatibility complex expression of ia antigen by cultured astrocytes treated with gamma-interferon ) c-fos proto-oncogene expression in astrocytes associated with differentiation or proliferation but not depolarization the nroloneed oresence of elia-derived nexin, an endogenous protease inhibitor, in the hippocampus after ischemia-induced delayed neur&al death tumor necrosis factor identified in multiple sclerosis brain immunoregulatory molecules and il-2 receptors identified in multiple sclerosis brain gfap mrna levels following stab wounds in rat brain biochemical and immunocvtochemical changes in alial fibrillarv acidic protein after stab wounds primary cultures of murine astrocytes produce c3 and factor b, two components of the alternative pathway of complement activation immunocytochemical localization of gd3 ganglioside to astrocytes in murine cerebellar mutants temporal expression of mouse glial fibrillary acidic protein mrna studied by a rapid in situ hybridization procedure production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus laminin is induced in astrocytes of adult brain by injury transforming growth factor-beta 1 in the rat brain: increase after injury and inhibition of astrocyte proliferation transforming growth factor-beta 1 stimulates expression of nerve growth factor in the rat reactive gliosis models of neuronal injury in aids: another role for the nmda receptor? flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro astrocytes block axonal regeneration in mammals by activating the physiological stop pathway a time course for the focal elevation of synthesis of basic fibroblast growth factor and one of its high-affinity receptors (flg) following a localized cortical brain injury enhanced expression of transforming growth factor 81 in the rat brain after a localised cerebral injury ngf gene expression in actively growing brain glia novel astrocytic protein in multiple sclerosis plaques production of hemopoietic colony-stimulating factors by astrocytes permissive and non-permissive reactive astrocytes: immunofluorescence study with antibodies to the glial hyaluronate-binding protein astroglial cell contributions to neuronal survival and neuritic growth synthesis and release of neuroactive substances by glial cells viral particles induce ia antigen expression on astrocytes tumor necrosis factor amplifies measles virus-mediated ia induction on astrocytes analysis of ia induction on lewis rat astrocytes in vitro by virus particles and bacterial adjuvants hyperinducibility of ia antigen on astrocytes correlates with strain-specific susceptibility to experimental autoimmune encephalomyelitis immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to ia-positive cells with dendritic morphology beta-amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity fibroblast growth factor and glutamate: opposing actions in the generation and degeneration of hippocampal neuroarchitecture glia protect hippocampal neurons against excitatory amino acid-induced degeneration: involvement of fibroblast growth factor. ht preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue reduction of neurite outgrowth in a model of glial scarring following cns injury is correlated with the expression of inhibitory molecules on reactive astrocytes the regulation of proenkephalin expression in a district population of glial cells induction of interleukin-1 and tumor necrosis factor alpha in brain cultures by human immunodeficiency virus type 1 a novel type of glial cell associated with nodes of ranvier in rat optic nerve fibrous and protoplasmic astrocytes are biochemically and developmentally distinct spinal cord reconstruction immunohistochemical determination of protein kinase c expression and proliferative activity in human brain tumors production of cytotoxic factor for oligodendrocytes by stimulated astrocytes immune response gene products (ia antigens) on glial and endothelial cells in virus-induced demyelination enhanced release of plasminogen activator inhibitor(s) but not of plasminogen activators by cultured rat glial cells treated with interleukin-1 accumulation of extracellular glutamate and neuronal death in astrocyte-poor cortical cultures exposed to glutamine glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture ia expression in chronic relapsing experimental allergic encephalomyelitis induced by long-term cultured t cell lines in mice cytokine-induced expression of intercellular adhesion molecule-l (icam-1) in cultured human oligodendrocytes and astrocytes expression and induction of intercellular adhesion molecules (icams) and major histocompatibility complex (mhc) antigens on cultured murine oligodendrocytes and astrocytes heterogeneous induction of 72-kda heat shock protein (hsp72) in cultured mouse oligodendrocytes and astrocytes production of tumor necrosis factor-aloha bv microglia and astrocytes in culture glial fibrillary acidic protein and vimentin in the experimental glial reaction of the rat brain gomori-positive astrocytes: biological properties and implications for neurologic and neuroendocrine disorders rat astrocytes express interferon-gamma immunoreactivity in normal optic nerve and after nerve transection immunohistochemical study of glial reaction and serum-protein extravasation in relation to neuronal damage in rat hippocampus after &hernia identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions tumor necrosis factor mediates myelin and oligodendrocyte damage in oitro alterations in plasma lipoproteins and apolipoproteins in experimental allergic encephalomyelitis expression of beta-amyloid precursor protein in reactive astrocytes following neuronal damage immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis neuroscience. vehicles of inactivation regulation of nerve growth factor (ngf) synthesis in the rat central nervous system: comparison between the effects of interleukin-1 and various growth factors in astrocyte cultures and in uiuo periventricular gomori-positive glia in brains of x-irradiated rats rat ependyma and microglia cells express class ii mhc antigens after intervenous infusion of recombinant gamma interferon bilirubin is an antioxidant of possible physiological importance macrophages in the peripheral nervous system and astroglia in the central nervous system of rat commonly express apolipoprotein e during development but differ in their response to injury glial uptake of excitatory amino acids influences neuronal survival in cultures of mouse hippocampus ia-restricted encephalitogenic t lymphocytes mediating eae lyse autoantigenpresenting astrocytes coronavirus infection induces h-2 antigen expression on oligodendrocytes and astrocytes increase in basic fibroblast growth factor immunoreactivity and its mrna level in rat brain following transient forebrain ischemia astrocytes produce interferon that enhances the expression of h-2 antigens on a subpopulation of brain cells the cytokine handbook acidic fibroblast growth factor-like immunoreactivity in brain of alzheimer patients regulation ofplasminogen activators and type-i pfasminogen activator inhibitor by cyclic amp and phorbol ester in rat astrocytes multiple sclerosis: involvement of interferons in lesion pathogenesis 19%) interferon-gamma and ia antigen are present on astrocytes in active chrontc multiple sclerosis lesions on the presence of &positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation neurotrans~itter t&porters (plus): a promising new gene family nexin-ii. a potent aatichymotrypsin, sbbws identity to'amyloid beta-protein precursor lntrathecal application of interferon gamma. progressive appearance of mhc antigens within the rat nervous system induction and regulation of class ii major bjsfo~ompatibilit~ complex mrna expression in astrocytes by interferon-~rna and tumor necrosis factor-alpha simultaneous expression of glial fibrillary acidic (gfa) protein and neuron-specific enolase (nse) by the same reactive or neoplastic astrocytes macrophage-and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome fibrobiast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension surmression bv antisense mrna demonstrates a reouirement for the glial fibrillary acidic protein in the formation of'stable astrocyti processes in response to neurons changes induced in astrocyte cathensin d bv cvtokines and leuueutin. f. ~e~r~~~rn. fil increased p-nerve growth factor messenger rna and protein levels in neonatal rat hippocampus following specific cholinergic lesions iron-induced lipid peroxidation and brain injury responses inducible expressian of h-2 and ia antigens on brain cells common immunologic determinant between human irnrn~n~e~~ienc~ virus type 1 gp4i and astrocytes immunohi~och~i~al detection of laminin and vimentin in the thafamic vb nucleus after ablation of somatosensory cortex in the rat 19n2) the biology and mechanism of action of nerve growth factor fibrobfast growth factors stimulate nerve growth factor synthesis and secretion by astrocytes cooperative regulation of nerve growth factor synthesis and secretion in frbrobfasts and astrocytes by fibroblast growth factor and other cytokines glial immunoreactivity for metallothionein in the rat brain regulation of brain-derived neurotrophic factor and nerve growth factor mrna in primary cultures of hippocampal neurons and astrocytes the reaction of astrocytes to acute virus infections of the central nervous system 195. 198. 206. negro a., tavella a.. facci l., callcgaro l. and skaper s. d. (1992) path. 135, 827-833. 220. peitsch m. c. and boguski m. s. (1990) is apolipoprotein da mammalian bilin-binding protein? new biol. 2, 197-206. 221. perraud f., besnard f., pettmann b.. sensenbrenner m. and labourdette g. (1988) effect of acidic and basic tibroblast growth factors (afgf and bfgf) on the proliferation and the glutamine synthetase expression of rat astroblasts in culture. gjiu 1, 124 131. 222. peters a., palay s. l. and webster h. def. (1991) academic press, orlando. 231. reier p. j., eng l. f. and jakeman l. key: cord-021772-5v4gor2v authors: levine, gwendolyn j.; cook, jennifer r. title: cerebrospinal fluid and central nervous system cytology date: 2019-05-31 journal: cowell and tyler's diagnostic cytology and hematology of the dog and cat doi: 10.1016/b978-0-323-53314-0.00014-6 sha: doc_id: 21772 cord_uid: 5v4gor2v nan analysis of csf is an important adjunctive diagnostic tool in the workup of patients with cns disease and must be interpreted within the context of the patient's history, clinical signs, clinicopathological data, imaging studies, and other ancillary diagnostics. rarely is csf solely used to provide an etiological diagnosis (exceptions include cytological visualization of infectious agents or overtly neoplastic cells), but analysis may significantly narrow the field of pathophysiological differentials, guiding further diagnostic and therapeutic options. csf analysis is most sensitive in detecting inflammatory disease. 3 positive findings in csf tend to be more diagnostically helpful compared with negative findings but are often nonspecific because many different diseases may cause a common csf pathology (e.g., neutrophilic pleocytosis). 4 occasionally, the magnitude of change within the csf may be as instructive as the character of the change (e.g., a marked increase in protein concentration raising diagnostic concern for feline infectious peritonitis, marked neutrophilic pleocytosis raising diagnostic concern for steroid-responsive meningitis-arteritis in a young dog in pain). 4 more frequently, however, specific disease etiologies will present with csf changes of variable character and magnitude. csf that falls within laboratory reference intervals should never be used to rule out a differential diagnosis because negative findings may represent early or mild disease, disease suppressed or masked by therapeutic intervention, or a disease process that does not present within the particular area of the extracellular space being sampled. csf analysis may or may not correlate with imaging studies; a retrospective study of 92 cats receiving magnetic resonance imaging (mri) for spinal signs showed that abnormal csf was not a predictor for abnormal mri. 5 in another study, approximately 25% of dogs with intracranial signs and inflammatory csf had normal brain mri results. 6 the conventionally accepted theory of csf secretion and transport is based on the concept of active transport of ions within the ventricular ependymal cells and choroid plexi, subsequent passive flow of fluid, and circulation and drainage of csf into dural venous sinuses. these ideas have recently come under scrutiny as potentially simplistic and inconsistent with the past 100 years of experimental evidence. 7 analysis of past experiments, coupled with new data, supports a "global production" hypothesis-that instead of exclusive formation within the ventricles, csf is continually created and reabsorbed diffusely by cerebral capillaries that have slight variances in hydrostatic and osmotic pressure. canine studies have documented csf production within the ventricular system and the sas. 2 a study of 158 dogs with focal, noninflammatory disease showed that in cases of spinal lesions, csf was more likely to be abnormal if collected from the lumbar cistern, that is, caudal to the lesion. 12 this observation may be explained by presupposing cranial to caudal flow of csf, but the traditionally held theory of csf flow has recently been contested. 7 in canines, csf collected from the cerebromedullary cistern generally has lower microprotein concentrations compared with samples collected from the lumbar cistern. 13 blood contamination may be more pronounced in lumbar collection, as the desired subarachnoid space is more difficult to enter and yields a smaller volume of fluid that tends to flow more slowly. 9, 10 moreover, hemodilution may contribute to increased measured protein concentration. 13 rare instances of csf contamination with hematopoietic precursors have only been reported from lumbar sites. 14 a low, but potentially catastrophic, risk for puncturing the cervical spinal cord or caudal brainstem exists during cerebromedullary collection. because the spinal cord length is variable, spinal cord puncture is a possibility during lumbar collection, but it is associated with less severe adverse effects compared with injury following cisternal puncture. in a case series of four accidental cisternal parenchymal punctures (documented by using mri), three of the four patients suffered neurological decompensation and subsequently had to be euthanized. 15 the following equipment should be assembled: anesthesia and monitoring equipment, clippers, aseptic preparation materials for the skin, sterile gloves, and a spinal needle with stylet. for cerebromedullary cistern collection in dogs weighing less than 25 kg and for cats, a 22-gauge, 1.5-inch spinal needle is usually adequate, and a 22-gauge, 2.5-inch spinal needle is recommended for dogs weighing greater than 25 kg. for lumbar puncture, a 22-gauge spinal needle up to 6 inches long may be required for obese or extremely large patients. if available, fluoroscopic equipment may aid in the acquisition of cisternal or lumbar csf. at the authors' institution, fluoroscopy is often used before cisternal csf acquisition in toy-breed dogs to exclude the possibility of subclinical atlantoaxial subluxation. the anesthetized patient is placed in lateral recumbency (it is generally easier for a right-handed clinician to have the patient in right-lateral recumbency, and vice versa), with the neck and back flush to the edge of a sturdy table. for collection from the cerebromedullary cistern, the neck is flexed such that the dorsum of the muzzle is 90 degrees to the long axis of the body (if needed, stabilizing the endotracheal tube to prevent kinking and deflating the cuff to prevent tracheal trauma), and the snout is propped up slightly, if necessary, to keep it parallel with the table and not angulated from the sagittal plane. 10 a wide area (3-5 cm) around the atlanto-occipital joint (beyond atlas wings and axis spinous process and to the external occipital protuberance) is shaved and aseptically prepared, and landmarks are palpated with a gloved, nondominant hand. 9 the needle is inserted at the intersection of two imaginary perpendicular lines that run (1) along the dorsal midline (dividing the patient sagittally) from the occipital protuberance to the cranial spinous process of the axis (c2) and (2) across the craniolateral aspects of the wings of the atlas (c1) (dividing the patient craniocaudally). for lumbar collection, the pelvic limbs are brought forward into full flexion, and the needle is inserted cranial and parallel to the dorsal spinous process of l6 for dogs and l7 for cats, advancing the needle until the ventral aspect of the vertebral canal is encountered; the needle is then retracted slightly and csf is collected from the ventral sas. 10, 16 the pelvic limbs may be kicked or may twitch slightly during collection because of irritation of the cauda equina or spinal cord parenchyma. for either location, once landmarks are palpated, the needle is held stably with the dominant hand and very slowly advanced, stylet in place. the heel of the dominant hand may be supported against the table. for cisternal collection, it is important to advance the needle toward the point of the nose without angulation. the stylet is removed with the nondominant hand every 2 to 3 mm to check for fluid within the needle hub, waiting a few seconds. it is common to feel a decrease in resistance to forward needle movement once the thecal space is entered. if bone is hit or frank hemorrhage is observed from the needle, it should be withdrawn slowly and collection reattempted. 9 if clear or slightly blood-tinged fluid is observed, advancement of the needle is stopped, and open tubes are placed directly under the needle hub to collect freely falling drops. csf is collected passively and should not be aspirated. there are no significant objective data regarding the maximal amount of csf that may be collected in dogs. several authors claim that it is safe to collect 0.2 milliliters (ml) of csf per kilogram of body weight (1 ml/5 kg); in other species much higher volumes of csf per body weight are acquired standardly. 17 in general, 0.5 to 1 ml of csf is adequate for routine diagnostic tests, including cell counts, protein concentration, and cytological analysis. larger volumes are necessary for additional diagnostics (cultures, titers, polymerase chain reaction [pcr], flow cytometry, protein electrophoresis, etc.). two sets of tubes should be readied and ideally handled by an assistant. an ethylenediaminetetraacetic acid (edta)-treated (purple-top) tube is used for cell counts, flow cytometry, and pcr testing for organisms, and plain (red-top) tubes are used for protein concentration, culture, or immunologic assays. 10 some sources indicate that plain tubes are recommended, as edta could increase protein concentration. if csf analysis will occur rapidly (within 1 hour), collection into a plain tube is adequate, whereas preservation of cells may be improved with collection into edta if analysis will be delayed. if low volume is present, priority is given to the edta tube. if csf appears red, then iatrogenic hemorrhage (puncture of a dural vessel) or actual cns hemorrhage has occurred. in this instance, the first few drops are allowed to collect into the first set of tubes, and the second set of tubes are reserved for the latter portion of the sample, as iatrogenic hemorrhage tends to clear over time. if the hemorrhage does clear, a decision may be made about discarding the first set of tubes or keeping them for ancillary testing not affected by the hemorrhage. after collection, the needle is withdrawn without the stylet, and the csf within the needle is allowed to drip into one of the tubes or is placed in an additional plain tube and saved for culture. as with other clinicopathological and cytological samples, evaluation of a fresh specimen is preferred to minimize cellular degradation, to which csf is particularly vulnerable because of its relatively low protein concentration. sample degradation will affect cell differential count to a greater extent than the total nucleated cell count or the protein concentration. 18 a study of 30 canine csf samples with pleocytosis concluded that delay of analysis up to 8 hours was unlikely to alter interpretation, especially in samples with protein concentrations above 50 milligrams per deciliter (mg/dl). 18 preservative should be added to low protein samples unless analysis is to be completed within 60 minutes (see next section), and a dilutional effect must then be factored into cell counts. 18 samples to be shipped to a reference laboratory overnight should be kept at refrigeration temperature and shipped with ice packs for analysis within 48 hours. 9, 16 the reference laboratory should be prenotified to ensure prompt analysis. if analysis is likely to be delayed by more than 1 hour and the csf sample has a protein concentration less than 50 mg/dl, one of the following may be added as a protein source to maintain cellular integrity: (1) hetastarch (add 1:1 volume), (2) fetal calf serum (3.7 g/dl protein; add 20% by volume), or (3) autologous plasma or serum (fresh or frozen; 11% by volume ≡ one drop from 25-gauge needle (approximately 0.03 ml) mixed into 0.25 ml csf). 10, 19, 20 the sample should be labeled with the protein source and amount added to the sample. one study demonstrated better preservation of mononuclear cells in canine samples when fetal calf serum was used instead of hetastarch. 18 all samples should be refrigerated at 4°c to minimize cellular degradation. a hemocytometer may be employed in practice to count nucleated cells and erythrocytes. both sides of the cover-slipped hemocytometer are loaded with unstained csf, which is then placed in a humidified container for 10 to 15 minutes to allow cells to settle on the glass. because the fluid is unstained, the microscope condenser is lowered to improve contrast. erythrocytes and nucleated cells are differentiated by size, refraction, granularity, and smoothness of plasma membrane. 21 some laboratories stain csf samples with new methylene blue (nmb), as leukocytes will take up stain, whereas erythrocytes remain unstained, making differentiation of leukocytes (specifically small lymphocytes) and erythrocytes easier (fig. 14.1) . 22 a small volume of csf is drawn into a capillary tube coated with nmb or a tube that has a small volume of nmb followed by an air pocket. 22 the tube containing nmb and csf is gently rocked back and forth, allowing the cells to take up some stain without diluting the csf with a volume of nmb. 22 the hemocytometer is then loaded, and each population is counted and totals are calculated, as follows: neubauer chamber: (1) both areas of large nine squares are counted, and the average of the number of leukocytes and erythrocytes is found; (2) the average is multiplied by 9 to get the cells per microliter (cells/μl). 10 the advia 120 (siemens medical solution, fernwald, germany) hematology instrument has been validated for analyzing canine csf samples and shows excellent correlation with manual methods used in dogs with increased total cell counts (pleocytosis), but the instrument may overestimate the cell count in samples without pleocytoses and has not been validated for the identification of eosinophils. 23 the automated differential count is also more accurate at higher cell numbers and thus should be compared with a traditional manual differential. the advia 2120 hematology analyzer displayed satisfactory agreement with the standard hemocytometer method. 24 validation experiments using 67 canine samples showed a sensitivity of 100% and specificity of 89% for accurately identifying samples with pleocytosis when manual counting was considered the gold standard (>5 cells/μl). 24 the instrument tended to be less accurate at lower (within reference interval) nucleated cell counts. 24 erythrocytes may be a source of interference, as a red blood cell (rbc) count of 250 cells/μl was shown to elevate the nucleated cell count. 24 with regard to differential cell count, the instrument performed better in the presence of pleocytosis, whereas monocytes were overcounted at lower nucleated cell counts. 24 automated cell counts thus should not replace a manual differential but may be used as another level of quality control. automated instruments cannot recognize altered cell types, such as atypical neoplastic cells. measurement of csf specific gravity is not considered to be helpful because of low sensitivity for detecting abnormalities. 12 csf microprotein may be semiquantitatively measured by using urine dipsticks that detect albumin. this assay has a lower detection limit of 100 mg/ dl; therefore, it has low sensitivity for mild to moderate csf protein concentration elevations (30 mg/dl to 100 mg/dl). false-positive or false-negative reactions may occur if the dipstick reads at trace or 1+, but this method is useful if other techniques are not available. 11 reference laboratories apply a similar but more sensitive methodology to measurement of csf microprotein as that of serum protein, using the trichloroacetic acid method, the ponceau s red dye-binding method, or the coomassie brilliant blue method. 22 csf globulin production is typically screened for with the pandy reaction. in this test, a few drops of csf are added to 1 ml of 10% carbolic acid solution, and the resulting turbidity is graded 0 to 4+. any pandy score above zero is considered elevated. globulin concentration below 50 mg/dl will be undetectable with either test. 10, 21 protein electrophoresis and immunoelectrophoresis may be performed on csf and serum for maximum fractionation. 25 the utility of protein electrophoresis or immunoelectrophoresis of csf lies in discriminating altered blood-brain barrier (bbb) permeability from increased localized production of immunoglobulin, which may be suggestive of (but not specific for) a disease entity for which an electrophoretic pattern has been established. cytological analysis is a critical component of csf evaluation because the differential count (percentages) of cells may be abnormal, even if the total nucleated count is within reference interval. cytology also enables examination for neoplastic cells, infectious agents, and evidence of prior hemorrhage. it may also serve as a quality control point, allowing for correlation between observed cellularity and the total count generated by a hemocytometer or an automated analyzer. because of its low cellularity, csf must be concentrated before cytological smear preparation. use of an in-house sedimentation chamber (sörnäs procedure) may be very useful and preserves cell-free fluid for ancillary testing. 10 this technique will recover approximately 60% of total cells, which is sufficient for analysis. 16 a syringe barrel (with the tip and needle aseptically removed with a scalpel blade) is turned upside down and the smooth, top side is placed in warm petroleum jelly and then onto a clean slide. once a seal has formed, fresh csf (at least 0.5 ml) is placed in the syringe and allowed to sit for 30 minutes. 16, 21 then, the supernatant is aspirated carefully with a pipette so as not to disturb the bottom layer contacting the slide. the syringe barrel is removed, and any excess csf is carefully absorbed with a small piece of filter paper or paper towel. the slide is completely and rapidly air-dried without heat (inadequate drying results in cellular distortion), excess petroleum jelly removed with a scalpel blade, and the slide is stained with routine romanowsky stains (e.g., diff-quik). if csf is sent to a reference laboratory, a cytological slide will likely be prepared using cytocentrifugation (500-1000 revolutions per minute [rpm] for 5-10 minutes, either onto a slide coated with albumin or with the addition of 0.05 ml of 30% albumin for improved cell capture) for maximal concentration of nucleated cells onto one slide. 16 cytocentrifuged cytology may show excellent cellular detail, but the preparation may enlarge cells slightly and create an artifactual foamy or vacuolated appearance. 16 slides are air-dried and stained with conventional romanowsky stains. multiple cytospin preparations may be made to yield 200 intact nucleated cells for classification. as it is rare for etiologic agents to localize only within the cns, all cases of suspected infection may be aided diagnostically by fine-needle aspiration (fna) cytology, biopsy with histopathology, culture of nonneural lesions, or all of these. 21 bacterial culture and sensitivity testing of csf is recommended for most cases of neutrophilic pleocytosis, given the appropriate clinical index of suspicion for a septic lesion. even when organisms are visualized on csf cytology, speciation and susceptibility testing may help guide prognostic and treatment decisions. alternatively, bacterial or fungal culture may be negative regardless of cytological observation of organisms. 10, 20 it must be remembered that bacterial cns infection is highly uncommon in dogs and cats compared with other domestic animal species. 26 advanced techniques for neurological disease diagnosis are expanding rapidly. enzyme-linked immunosorbent assay (elisa)-based assays for antibody detection and pcr-based assays for nucleic acid detection of several medically important microbes have been developed for use on csf and may be instructive in the diagnosis of viral, rickettsial, protozoal, or fungal diseases. 20 a large canine study that included a subset of 16 dogs with neoplastic or inflammatory disease showed that csf titer provided diagnosis in 25% of cases. 3 antibody assays should be interpreted cautiously because the presence of antibody may indicate prior exposure or vaccination rather than active infection. moreover, compromise to the bbb in states of inflammation may translate to the presence of antibodies within the csf without local production. occasionally cross-reactive antibodies may be present that do not represent presence of the disease agent under assessment. similarly, specimens for pcr should be submitted to a laboratory with strict quality control to minimize false-negative and false-positive results. poor collection technique may result in false-positive results, especially for bacterial species that are ubiquitous in the environment. 27 as with other aspects of csf analysis, a negative pcr result does not definitively rule out the presence of a pathogen because of the sampling limitation of a small portion of the extracellular space. 20 csf contains glucose, electrolytes, neurotransmitters, and enzymes, but these substances are not measured routinely, although this measurement represents a rapidly expanding area of research in the effort to give clinicians better tools for diagnosing patients and determining prognoses. csf enzymes originate from the bloodstream, the cns, or cells within csf. 10 one study of 34 cats with noninflammatory cns disease showed that measurement of csf activities of lactate dehydrogenase (ldh), aspartate aminotransferase (ast), and creatine kinase (ck) were not diagnostically sensitive but may be useful in detection of acute injury. 28 multiple studies have correlated elevations in csf ck activity with poor prognosis in dogs with neurological disease or spinal cord injury. 29, 30 immunoassays for vascular endothelial growth factor (vegf) and s-100 calcium-binding protein have shown elevations of both molecules in the csf of experimentally induced hypothyroid dogs, suggesting endothelial and glial contribution to increased bbb permeability in this population. 31 myelin basic protein (mbp) has been found to be elevated in lumbar csf in dogs with degenerative myelopathy, supporting the conclusion that it is a demyelinating lesion. 32 mbp concentration is elevated in the csf of dogs affected by intervertebral disk herniation (ivdh) and has been found to be an independent predictor of poor prognosis. 33 beta-2-microglobulin, a major histocompatibility complex i (mhc-i)-associated molecule, has been assayed by using elisa and found to be elevated in the csf of dogs with ivdh and inflammatory disease and also positively correlated with normal total nucleated cell count (tncc). 34 the amino acids tryptophan and glutamine have been found to be elevated in the csf of dogs with portosystemic shunts because of abnormal ammonia metabolism. 35 one study found increased oxytocin in the csf of dogs with spinal cord compression, where it is believed to have an analgesic effect. 36 gamma-aminobutyric acid (gaba) and glutamate neurotransmitter concentrations have been measured in dogs with epilepsy. 37 normal csf is clear and colorless, with few cellular elements and a protein concentration approximately 200 to 300 times less than that of plasma or serum. red or yellowish coloration indicates prior lesional hemorrhage or iatrogenic hemorrhage during collection. in the latter case, a pellet of rbcs will be present after centrifugation. true xanthochromia (yellowish color of hemoglobin breakdown products) that does not clear on centrifugation, cytological evidence of erythrophagia, or both indicate prior hemorrhage into the subarachnoid space. 20 increased bilirubin leakage into the sas or high concentrations of csf protein (>100-150 mg/dl) may cause xanthrochromia. 21 increased turbidity of the sample may be caused by increased number of cells present (>400 rbcs/μl or >200 nucleated cells/μl) but is usually not affected by mild changes. 10, 11 cell counts tncc is fewer than 5 cells/μl in the dog and fewer than 8 cells/μl in the cat, and elevation above this range is termed pleocytosis. 10 grading of pleocytosis is somewhat subjective: in one reference, "mild" was defined as 6 to 50 cells/μl; "moderate" as 51 to 1000 cells/μl; and "marked" as more than 1000 cells/μl. 4 depending on laboratory-specific reference intervals, normal protein concentration is usually less than 25 to 30 mg/dl for cisternal csf and less than 45 mg/dl for lumbar csf. 10, 20 approximately 80% to 95% of csf protein is albumin, and 5% to 12% of csf total protein comprises gammaglobulins. 2 eighty percent of csf protein is transferred from plasma, with the remainder produced within the cns. the latter population includes molecules also produced by other organs and proteins unique to the csf that may potentially be used as markers of cns tissue damage. experimental evidence and earlier literature support a gradient of increasing protein concentration from cranial to caudal within the subarachnoid space, which has been attributed to slower flow and greater blood-csf permeability caudally. 12 normal csf is acellular or contains small numbers of small lymphocytes (figs. 14.2 and 14.3) and large mononuclear cells (macrophages, ependymal lining cells, meningothelial lining cells, choroid plexus cells) (figs. 14.4 and 14.5). large mononuclear cells may be vacuolated and contain phagocytized material ( fig. 14.6) . a low frequency of nondegenerate neutrophils (<25%), which are usually indicative of blood contamination during collection, may be present. 38 a study of 359 samples of canine csf found a 7.5% incidence of meningeal, choroid plexus, ependymal, endothelial cells, or all of these. 39 no correlation existed between the presence of these cells and the presence of pleocytosis, elevated protein concentration, or the primary disease etiology. 39 thus it is postulated that the presence of these cells is an artifact of collection and should not be overinterpreted. the authors recommended the term "surface epithelial cells" for the combined grouping (which cannot be distinguished cytologically), although not all of these cells (meningeal, endothelial) are of epithelial origin. 39 occasionally, anucleate superficial squamous epithelial cells may be seen; these may be caused by contamination from the skin (fig. 14.7 ). occasionally, small amounts of granular, foamy extracellular material are present and are consistent with myelin or myelin-like material, which will stain positively with luxol fast blue stain. this material may consist of myelin fragments, which are generated from demyelination, or may consist of myelin figures (a nonspecific term for layered phospholipids exfoliated from damaged cells). 40 the two cannot be distinguished with light microscopy. the significance of this material remains unclear because it may be observed in samples from patients with no discernible cause. a study of 98 canine cerebromedullary and lumbar csf samples showed 20% incidence of myelin-like material, with a higher percentage in samples from the lumbar cistern or from small dogs (<10 kg). 41 the presence of the material was not correlated with case outcome. 41 similarly, in a study of 61 cavalier king charles spaniels with chiari-like malformations, myelinlike material was observed in 57% of lumbar csf collections and 12% of cerebromedullary collections. 42 thus myelin-like material may be a procedural artifact or may be consistent with a demyelinating (e.g., canine distemper virus, degenerative myelopathy) or potentially necrotizing disorder (e.g., ivdh, other spinal trauma, or a necrotic neoplasm). 40, 41 normal csf should not contain erythrocytes, but hemodilution is a common occurrence. varying reports on the effect of blood contamination on tncc, leukocyte differential, and protein concentration have been published. [43] [44] [45] [46] deciding whether increased tncc or protein concentration is the result of hemodilution alone or a significant change concurrent with hemodilution necessarily remains, to an extent, a subjective assessment and must be critically evaluated in light of the magnitude of csf findings along with the other pertinent facts of the case. correction formulas for csf parameters in the face of hemodilution (e.g., adding 1 nucleated cell/μl per 100 or 500 rbcs/μl) are unreliable. 45, 46 in a recent study of 106 canine csf samples without pleocytosis (tncc <5/μl) but containing at least 500 rbcs/μl, the mean percentage of neutrophils (45.2% versus 5.7%), percentage of samples with eosinophils present (36.8% versus 6.8%), and mean protein concentration (40 mg/dl versus 26 mg/dl) were found to be significantly increased in the samples with blood contamination when compared with controls. 47 significant rbc contamination warrants repeat sampling, if possible. marked hemorrhage or evidence of prior hemorrhage (erythrophagocytosis, xanthochromia, hemosiderin-laden macrophages) may be useful in the diagnosis of cns trauma, which may be accompanied by neutrophilic to mixed cell pleocytosis and mild increase in protein concentration. 4 elevated protein concentration in csf (>30 mg/dl) may occur with or without pleocytosis, and in the absence of pleocytosis is termed albuminocytological dissociation (acd). high protein concentration may be the result of leakage of plasma or cellular proteins across the bbb, localized production of immunoglobulin, localized tissue damage or necrosis, decreased clearance of protein into the venous sinuses, obstruction of csf circulation, or all of the above. as such, it is a nonspecific change that indicates cns damage or hyperproteinemic disease and is consistent with disease of any etiology (e.g., trauma, metabolic, infectious, inflammatory, degenerative, or neoplastic). caution should be exercised when diagnosing acd if the sample is hemodiluted (>500 rbcs/μl). 47 as is true for pleocytosis, inflammation of the meninges and superficial regions of parenchyma will result in greater csf protein elevations than for lesions that are more remote from the sas. occasionally, an abnormal leukocyte differential (shifted from mononuclear predominance to neutrophil predominance) without pleocytosis occurs. this may only be detected if cytological analysis (after sedimentation or cytocentrifugation of csf) is performed. increased percentages of neutrophils may occur in early or mild inflammatory disease, noninflammatory cns disease, disease that is remote from the sas or sampling site, or in cases of hemodilution. an increased proportion of neutrophils is present when neutrophils comprise greater than 25% of all nucleated cells, and increased percentage of eosinophils occurs when eosinophils comprise greater than 1% of the differential. 10 when present (with or without increased tncc), neutrophils should be evaluated for toxic change, degenerative change, and intracellular organisms or other inclusions ( fig. 14.8 ). increased percentage of neutrophils without pleocytosis has been associated with healthy dogs, blood contamination, degenerative disk disease, neoplasia, cerebrovascular accident, fracture, cns aspergillosis, and fibrocartilaginous embolism (fce). 10, 42, 48 a study of 61 cavalier king charles spaniels with chiari-like malformation documented that those with syringomyelia were more likely to have an increased percentage of neutrophils, but it was not reported whether this subpopulation also had a concurrent pleocytosis. 42 in another study, cats with cns neoplasia had increased percentage of neutrophils or lymphocytes without a pleocytosis. 28 although not a classic pattern, infectious or inflammatory disease should not be ruled out if increased neutrophils are visualized without pleocytosis. increased percentage of eosinophils has been reported in parasitic and protozoal diseases, such as neospora caninum infection. 38 one cat with eosinophilic meningoencephalitis (eme) of unknown etiology had an increased percentage of eosinophils and lymphocytes without pleocytosis. 49 the specific diseases mentioned in the next section on various categories of pleocytosis are a survey of the current literature and meant to be a helpful starting point in the generation of particular differential diagnoses. thus disease entities are listed in the section under which they are most commonly present, but it is important to note that for all disease entities, variability in the nature and the magnitude of pleocytosis may emerge in a particular patient at a particular point in time. wherever possible, other categories of pleocytosis that have been reported for a disease have been mentioned. generally, pleocytoses are defined by the cell type that comprises 70% or more of the nucleated cell population. if all cell types are 50% or less, the pleocytosis is classified as a mixed cell pleocytosis. and if, for example, lymphocytes are greater than 50% but less than 70%, some pathologists will classify the pleocytosis as mixed cell, lymphocyte predominant. a pleocytosis will be classified as eosinophilic if eosinophils compose at least 10% to 20% of the nucleated cell population. 11 bacterial meningoencephalomyelitis. bacterial infections of the cns are unusual and represent a small portion of neutrophilic pleocytoses. typically, this pleocytosis is severe (could be over 1000 cells/μl), neutrophilic, and accompanied by significantly elevated protein concentration, but the cell population may change to mononuclear during the course of treatment. 10, 20, 50 rare instances of brain abscessation secondary to sepsis (which may be a sequela of iatrogenic immunosuppression) may result in marked neutrophilic pleocytosis, markedly elevated protein concentration, visualization of bacterial organisms (see fig. 14.8) , and abnormal mri findings. 51 staphylococcus intermedius was cultured from the csf of a dog presenting with a retrobulbar abscess and neurological signs. 52 the csf showed a moderate neutrophilic pleocytosis (75 cells/μl) and borderline elevation in protein concentration (30 mg/dl). 52 local extension of severe otitis interna resulting in meningoencephalitis and ventriculitis in a dog has been reported. 53 this patient exhibited a severe neutrophilic pleocytosis (3672 cells/μl) and protein elevation (>400 mg/dl). 53 pasteurella multocida meningoencephalomyelitis in a kitten was characterized by marked neutrophilic pleocytosis (981 cells/μl) with mild protein elevation (31 mg/dl) and rare extracellular and intracellular bacterial rods. 54 bacterial culture and susceptibility testing are recommended but may yield false-negative results if organisms are not circulating in the extracellular space or if prior antibiotic therapy had been given. serology and csf-pcr (using organism-specific or universal bacterial [ub] pcr) are recommended. 27, 54 cryptococcosis in dogs. cryptococcus spp. are a large genus of systemic dimorphic fungi with a predilection for cns tissue, which is infected hematogenously or via direct penetration of the cribriform plate. only two species at this time are medically important: (1) cryptococcus neoformans (var. neoformans and var. grubii) and (2) cryptococcus gattii. in a recent study of 31 dogs with cryptococcosis, 68% had cns infection, with neurological signs being the most common reason for presentation. 55 dogs and cats with cryptococcosis typically have pleocytoses and elevated protein concentrations, but pleocytoses may be variably neutrophilic, eosinophilic, mononuclear, or mixed. in a recent study of 15 dogs with cns cryptococcosis, organisms were found in 11 of 15 csf samples (figs. 14.9 and 14.10). 56 all affected dogs had pleocytoses that were mixed to mononuclear, whereas cats tended to have neutrophilic pleocytoses. 56 of the samples, 11 of 12 also had increased protein concentrations (mean 494 mg/dl), which were significantly higher than in cats in the same study (mean 45 mg/ dl). 56 capsular antigen latex agglutination testing on serum or csf is highly sensitive and specific and is recommended if cryptococcosis is suspected but organisms are not visualized cytologically. 57 this test may yield negative results if disease is present but localized (i.e., within the respiratory tract), so appropriate clinical signs should guide testing. culture of csf may also be helpful and may distinguish c. neoformans from c. gattii with the use of selective media. the finding of inflammatory foci on mri may be supportive of the presence of fungal disease; cryptococcosis may result in mass lesions, meningitis, or pseudocyst formation. cryptococcosis is the most common systemic fungal disease of cats and is believed to infect the cns less frequently than in the dog. a recent study found that 42% of 62 cats with cryptococcosis had cns infection, but respiratory signs were still a more common reason for presentation. 55 mild to marked neutrophilic or mononuclear pleocytosis may occur, with variable and occasionally normal protein concentrations. 4 a study of cats with cns cryptococcosis showed organisms in 9 of 11 of the csf samples, and a majority of cases (9 of 10) had neutrophilic pleocytosis and increased protein concentration (8 of 10). 56 eosinophilic pleocytosis may also occur. capsular antigen latex agglutination testing on serum or csf is recommended for confirmation of cryptococcus spp. infection, with rare false-negative reactions if disease is highly localized. fungus that has been visualized in canine csf and may be extracellular or within leukocytes. 58 ehrlichiosis. neutrophilic pleocytosis has been reported in cases of granulocytic ehrlichia spp. in dogs (fig. 14.11 ). 61 neurological signs are uncommon in this disease, and affected dogs may display features ranging from ataxia to seizures. (fip) has been traditionally linked to marked csf changes, but the current literature paints a somewhat more varied picture. one study of natural fip infection showed neutrophilic pleocytosis (as defined by >50% neutrophils) in the majority (7 of 11) of cases, with fewer cases of mononuclear (3 of 11; as defined by >80% mononuclear cells) and mixed cell (1 of 11) pleocytosis, all of variable severity. 4 most cases (7 of 9) also had differing degrees of elevated protein concentrations. 4 diagnosis was confirmed by histopathology or suggested by elevated feline coronavirus antibody titers and reduced albumin-to-globulin ratios in both serum and body cavity effusions. 4 a slightly older study of 16 csf samples (natural and experimental infections) showed pleocytosis in 2 of 16 cases (neutrophilic and lymphocytic) and elevated protein concentration in 4 of 16 cases. 62 in a larger study of 67 cats with fip or non-fip disease, incidence of pleocytosis was highest in the neurological fip group, but 20% of these patients did not have a pleocytosis. 63 additionally, protein concentrations were variably elevated and not statistically different in fip compared with non-fip neurological disease. 63 another study of 12 cats with cns fip showed 8 of 12 with unspecified pleocytosis and 3 of 12 with elevated protein concentration. 64 in cats with cns disease, sensitivity of feline coronavirus (fecov) immunoglobulin g (igg) in csf for the diagnosis of fip was 60%, and specificity was 93%, with a positive predictive value of 75% and a negative predictive value of 87% (fip prevalence in this population was 25.6%). 63 definitive diagnosis of this disease remains challenging, with virus identification (pcr or immunohistochemistry) accompanied by pyogranulomatous inflammation in tissues being the gold standard. hypergammaglobulinemia, elevated serum α 1 -acid glycoprotein (agp), mri abnormalities (typically involving the ventricular lining and meninges), and positive feline coronavirus igg titer or pcr from serum, tissue, or csf are supportive but not specifically diagnostic, and negative findings do not rule out disease. 20, 63, 65 toxoplasmosis in cats. cats are the definitive hosts for toxoplasma gondii and may be subclinically infected; thus, diagnostics should only be performed on patients with appropriate clinical signs. cats typically present with mild neutrophilic or mononuclear pleocytosis and normal to mildly elevated protein concentration, but marked protein elevation may occur. 4 mild lymphocytic pleocytosis is also reported. 65 diagnosis may be confirmed by direct visualization of organisms in csf, aspirates of other inflammatory foci, histopathology of affected tissues, or fecal examination. serology must be interpreted cautiously because igg may remain elevated for up to 6 years after exposure. therefore, paired serum igm-igg titers, indicating acute exposure, or documentation of rising serum igg titers are more useful, but the latter is difficult to document in the advanced state of disease. 65, 66 spinal epidural empyema in dogs. epidural empyema is an uncommon disease in dogs, resulting from pyogenic infection in the epidural space. one study showed 4 of 5 dogs with neutrophilic pleocytosis of variable magnitude (11-342 cells/μl). 67 no organisms were visualized on any of the samples. 67 except for one case with a lumbar csf protein concentration of 726 mg/dl, protein elevations were modest. 67 three csf samples were cultured with no growth, and two dogs for which follow-up csf was obtained showed resolution of pleocytosis. 67 these results are not surprising, as the dura likely provides a barrier to prevent infection extending from the epidural space to the subarachnoid space. reported in a young cat with a marked neutrophilic pleocytosis with intracellular and extracellular merozoites observed on csf cytology. 68 diagnosis was confirmed with decreasing paired serologic titers, and speciation to the level of sarcocystis dasypi or sarcocystis neurona was conducted with pcr from blood. 68 a case of systemic acanthamoeba spp. infection in a young boxer, diagnosed post mortem, had antemortem csf with marked neutrophilic pleocytosis (4956 cells/μl), marked increase in protein concentration (259 mg/ dl), and subnormal csf iga concentration (33 mg/dl; reference interval 35-270 mg/dl). 69 postmortem pcr for the organism was positive on extraneural tissue but not on csf or spinal cord. 69 the patient had been deliberately immunosuppressed on the basis of a preponderance of evidence of steroid-responsive meningitis arteritis at initial presentation and thus may have been infected either before or opportunistically after treatment. 69 another case report of canine cerebellar balamuthia mandrillaris infection (diagnosed post mortem with immunohistochemistry) displayed a marked neutrophilic pleocytosis (234 cells/μl), but other cases with lymphocytic pleocytosis have been reported. 70 because of tissue encystment, it is suggested that extraneural tissue be used for immunohistochemistry or pcr for antemortem confirmation of amoebic infection; pcr of csf may be diagnostic but is not widely available. 69, 70 two dogs with aberrant spinal migration of spirocirca lupi nematodes had moderate to marked neutrophilic to mixed or eosinophilic pleocytoses (800 cells/μl with 91% neutrophils; 180 cells/μl with 60% neutrophils, 30% eosinophils). 71 steroid-responsive meningitis arteritis. steroid-responsive meningitis arteritis (srma) is presumptively an immune-mediated disease of mainly young, medium-and large-breed dogs: beagles, boxers, bernese mountain dogs, weimaraners, and nova scotia duck tolling retrievers are overrepresented. 20 csf analysis is important in diagnosis and typically features a moderate to marked neutrophilic pleocytosis (a left shift may be present) and markedly elevated protein concentration. chronically, pleocytosis may change to a more mononuclear or mixed population (fig. 14.12 ) and may become mild or even fall into reference intervals. 72 a study of 20 affected dogs showed neutrophilic pleocytosis in 12 of 20 cases and mononuclear pleocytosis in 8 of 20 cases. 72 concurrent elevations of serum and csf iga titers (elevated igg and igm fractions may be present), serum concentration of cross-reactive protein (crp), or serum α 2 -macroglobulin is diagnostically supportive but not specific. 20, 50 increases in iga have been linked to a t-helper 2 (th2)-dominated immune response driven by elevated interleukin-4 (il-4) and decreased il-2 and interferon-gamma (ifn-γ). 73 serum amyloid a (saa), serum agp, and serum haptoglobin may also be elevated. 74 another study of 36 dogs with srma reported statistically significant elevations of csf and serum crp, but not serum α 2 -macroglobulin, in dogs with srma compared with other neurological diseases. 75 in a study of 20 dogs, serum crp was positively correlated with csf tncc. 72 additionally, serum haptoglobin and serum and csf iga remained increased throughout successful treatment, indicating that these parameters are more useful for diagnosis than for monitoring therapy. 72 serum and csf concentrations of crp and saa have been documented to fall significantly during treatment, and repeat measurement of serum crp or saa may be used to guide therapy and predict relapse, which is less invasive and more sensitive than repeat csf sampling. 72, 74, 75 rare cases have been documented in cats with marked mononuclear or mixed pleocytosis and mild to moderate protein concentration elevations. 4 intervertebral disk herniation. csf from patients with ivdh may be extremely variable; data indicate that csf findings correlate with location of sampling, disk herniation location, chronicity of the lesion, and severity of spinal cord injury. bearing this in mind, it is no surprise that some reports in the literature state that neutrophilic, lymphocytic, mixed, and mononuclear pleocytoses are most common in dogs with ivdh. 12, 30, 76 a study of 423 cases of ivdh showed 51% with pleocytosis, of which 31% were neutrophilic, 41% were lymphocytic, 20% were mixed, and 7.4% were mononuclear. 76 of all cases, 71% had elevated protein concentrations. 76 interestingly, a larger number of cases of lymphocytic pleocytosis were observed in the samples analyzed more than 7 days after onset of clinical signs. 76 the magnitude of pleocytosis, in general, was also shown to decrease with increasing time between clinical onset and sampling, and this observation has been corroborated by other studies. 12, 76 prior treatment with corticosteroids was observed to reduce the number of observed lymphocytes in csf. 76 the authors also found a higher incidence of pleocytosis in thoracolumbar disease (61%) compared with cervical disease (23%), but this may have been caused by exclusive sampling of lumbar csf closer to the lesion. 76 ivdh is rare in cats and has been reported to feature mild mixed cell pleocytosis and elevated protein concentration. 4 patients typically present with nonpainful, progressive, asymmetrical neurological signs. as only histopathology is confirmat ory, it is a multimodal diagnosis of exclusion. a study of 32 dogs with presumptive fce, based on history, clinical signs, imaging, and outcome, showed 53% with normal csf, 25% with acd, and 19% with mild to moderate pleocytosis (7-84 cells/μl; median 12/μl). 77 pleocytoses were neutrophilic or mixed. 77 one study of 36 confirmed cases in dogs showed that 64% had normal csf and the remainder displayed mild changes. 78 another study looking at five dogs suggested that pleocytosis may be marked, up to 529 cells/μl. 3 fce is much less common in cats. in general, the disease process and clinical signs are similar to those in dogs, with the exception that the disease presents in cats in middle or older age, usually with cervical spinal cord signs. a case series of five cats showed csf ranging from normal to marked neutrophilic pleocytosis with moderately elevated protein concentration and variable correlation to clinical outcome. 79 the case with the most severe csf changes had extensive myelomalacia at necropsy. 79 it was suggested in this study that csf is more likely to be abnormal if collected closer to the lesion and that mri is helpful for localization and in supporting the diagnosis. 77, 79 thiamine deficiency in cats. thiamine deficiency is a rare nutritional disorder of patients fed noncommercial, misformulated commercial, or irradiated diets. two case reports showed increased percentage of neutrophils or mild neutrophilic pleocytosis, presumptively from cerebrocortical necrosis. 4 diagnosis is based on history, response to treatment, mri features compatible with the disease (cortical and brainstem hyperintensities), or histopathology. 80 spaniels with chiari-like malformation showed that 40% of dogs with concurrent syringomyelia and cisternal csf sampling had mild (up to 15 cells/μl) pleocytoses and increased percentages of neutrophils compared with the subpopulation without syringomyelia, but it was not specifically documented whether pleocytoses were, in fact, neutrophilic or mixed with an increased percentage of neutrophils. 42 a positive correlation was also seen to exist between tncc and syrinx size. 42 neoplasia. it is important to perform csf in neurology patients with suspected neoplasia, as definitive diagnosis may be achieved if neoplastic cells are directly observed via cytology. inflammatory pleocytoses or elevated protein concentrations are common in patients with cancer, tend to be mild to moderate in magnitude, and may represent paraneoplastic inflammation, compromise of the bbb, lesional necrosis, or all of these. 28 normal csf is also a common finding in cases of neoplasia. moreover, in the absence of overtly neoplastic cells, no defined patterns connect specific tumors with specific types of inflammatory pleocytoses. neutrophilic pleocytosis of unspecified magnitude was found in the csf of 2 of 11 cats with spinal lymphoma and in 3 of 7 cats with nonlymphoma spinal neoplasia (astrocytoma or osteosarcoma). 81 additionally, the remaining four cats with nonlymphoma spinal tumors (meningioma, peripheral nerve sheath tumor, plasma cell tumor) had either normal csf or acd of unspecified magnitude. 81 metastatic tumors to the cns should also be considered in a patient with neurological signs. eosinophilic meningoencephalitis of dogs. eme is an idiopathic diagnosis of exclusion that is typically steroid responsive and is postulated to be triggered by an underlying hypersensitivity, allergy, or self-limiting infection. the disease may be overrepresented in rottweilers and golden retrievers. 82 a study of 23 dogs with eosinophilic pleocytosis (defined by >20% eosinophils) showed 16 cases of idiopathic eme, 4 cases of infectious disease (c. neoformans, n. caninum, baylisascaris procyonis), and 3 cases of ivdh. 83 the magnitude of pleocytosis or the percentage of eosinophils could not be used to distinguish infectious versus eme cases, although ivdh cases tended to have milder pleocytoses (<84 cells/μl). 83 in about half the eme cases, mri showed abnormal findings. 83 peripheral eosinophilia may or may not be present. highly suggestive of protozoal (toxoplasmosis, neosporosis), fungal (cryptococcosis), parasitic (including cuterebra spp., dirofilariasis), and algal (protothecosis) infections and also rarely in cases of canine distemper and rabies viruses. 10, 84 eosinophils have also been found in cases of granulomatous meningoencephalomyelitis (gme). 85 eosinophilic pleocytosis has been documented in bacterial encephalitis as well. 21 gondii infection tend to have neurological or neuromuscular signs. case reports are sporadic; documentation of mild acd (58 mg/dl) and also a report of mild lymphocytic or eosinophilic pleocytosis (35 cells/μl) with an elevated protein concentration of 77 mg/dl exist in the literature. 86 it is important to rule out other potential causes of the neurological signs because immunocompetent dogs tend to clear subclinical infections, and therefore paired serum igm-igg titers or sequential serum igg titers are preferable to a single serum igg titer. to the author's knowledge, no data on the life span of canine igg antibodies exist. reports in the literature are conflicting with regard to the cross-reactivity of t. gondii antibodies to other agents, such as n. caninum. 20,86 pcr testing for toxoplasma in serum, tissue, or csf is diagnostic. 86, 87 rabies. pleocytoses may be lymphocytic and of varying severity. ancillary antemortem diagnostics include viral pcr on saliva or csf and the saliva antigen latex agglutination test. in a study of 15 dogs under quarantine for suspected natural infection (subsequently confirmed positive), 13 of 15 were saliva-pcr positive, and 4 of 15 (27%) were csf-pcr positive. 88 all animals with positive results on csf were also positive on saliva, and interestingly 100% correlation was seen between positive csf-pcr and the dull clinical presentation (all aggressive clinical presentations were csf-pcr negative). 88 negative testing should never exclude diagnosis because viral load is highest within salivary glands and brain parenchyma. 88 frequently made by exclusion when coupled with appropriate clinical signs. csf and mri findings are variable and may be normal in the acute stage of disease before inflammation has peaked. 89 in a study of 32 dogs with noninflammatory distemper, half (15 of 32) had normal csf. 85 a study of eight dogs with natural infection (confirmed by cns tissue-pcr and histopathology) showed lymphocytic pleocytosis in all samples and normal protein concentrations. 90 another case (confirmed by tissue-pcr and csf-pcr) in a 7-month-old dog displayed marked (554 cells/μl) lymphocytic pleocytosis and a normal protein concentration. 89 because this is a demyelinating disease, myelin-like material, which is amorphous, granular, pink, foamy, and stains positively with luxol fast blue, may be present. 40 pcr testing of csf, serum, urine, epithelial or tonsillar tissue is available, and immunohistochemistry on biopsy specimens of nasal mucosa, haired skin, or footpad is 88% to 96% sensitive for detection of viral antigen. 91 is present, it is likely to be lymphocytic. pleocytoses are typically mild to moderate, but severe lymphocytic pleocytoses have been reported. 89 main differential diagnoses include other viral diseases, gme, or chronic bacterial infection. extranigral signs related to the gastrointestinal or the respiratory system, if present, may be helpful in distinguishing this disease from gme. 89 in a study comparing four dogs with chronic cdv, six dogs with acute cdv, and controls, dogs with chronic cdv had markedly elevated csf igg concentration. 92 the igg region was polyclonal, including a population of neutralizing antibodies for cdv. 92 fungus acquired through inhalation, and most cases in the united states are observed in the southwestern region of the country. signs tend to involve respiratory or skeletal systems, and cns involvement is rare. one dog had a mild to moderate lymphocytic pleocytosis. 57 complement fixation (detecting igg) or tube precipitation (detecting igm), or agar-gel immunodiffusion serological testing is recommended for confirmation. necrotizing meningoencephalitis. meningoencephalitis has been subcategorized as necrotizing meningoencephalitis (nme) and necrotizing leukoencephalitis (nle) on the basis of histopathological appearance. both nle and nme are believed to have an immune-mediated basis, and recent data support that in pugs with nme, canine leukocyte antigen gene aberrations exist. 93 meningoencephalitis is rapidly progressive and affects a variety of generally young to middle-aged toy-breed dogs, including the pug, shih tzu, papillon, maltese, chihuahua, yorkshire terrier, french bulldog, pekingese, west highland white terrier, boston terrier, japanese spitz, and miniature pinscher breeds. 20, 94 a study of csf from 14 pugs with nme showed 12 of 14 with pleocytoses of varying severity (mean 120 cells/μl). 95 of these dogs, 66% had a lymphocytic pleocytosis, 17% had a mononuclear pleocytosis, and 17% had a mixed cell pleocytosis (fig. 14. 13) 95 ; 11 of 14 dogs had elevated protein concentrations (mean 88.4 mg/dl). 95 another study of three dogs showed one with acd and two with moderate to marked (40-220 cells/μl) neutrophilic to lymphocytic pleocytosis. 94 mri findings may help support a diagnosis, but only histopathological examination of lesions provides definitive proof. other. four cats with ischemic encephalopathy had mild (<10 cells/μl) lymphocytic pleocytosis. 28 another study of feline ischemic encephalopathy reported one cat with normal csf and another with mononuclear to mixed pleocytosis (26 cells/μl). 96 a report of two cats with cerebrovascular disease (infarction or stroke) showed one with mononuclear pleocytosis and the other with acd. 97 cerebrovascular disease was correlated in several other cases (without csf data) to hepatic lipidosis or fip. 97 other. a case report of a pug with a mild mononuclear pleocytosis (8 cells/μl), mild elevation in protein concentration (89 mg/dl), evidence of hemorrhage, and direct visualization of angiostrongylus vasorum helminth larvae is found in the literature. 102 eosinophilia was not observed within the csf or peripheral blood. 102 the organism is endemic in europe and canada among foxes and canids, mainly causing respiratory signs or coagulopathy; neurological signs are typically caused by hemorrhage. 102 two dogs with paraparesis and pyogranulomatous lumbar masses (one intradural, one extradural) had lumbar csf with mild mixed cell pleocytosis (lymphocytes and nondegnerate neutrophils) or lymphocytic pleocytosis. 103 these patients were serologically pcr positive for bartonella vinsonii subsp. berkhoffi and presented with a nodular dermatosis. 103 a dog with neurological signs and hepatozoon canis infection showed marked lymphocytic pleocytosis (243 cell/μl) with mildly elevated protein concentration (37 mg/dl). 104 organisms were not visualized in csf but were found on cytology of peripheral blood, lymph node, bone marrow, and bony lesions. serology and positive pcr from a bone marrow sample were diagnostic. 104 intrathecal contrast administration. contrast media or pharmacological agents, such as epidural anesthetics, may introduce preanalytical error into csf samples, artificially raising tncc and protein concentrations. 105 in a study of 17 healthy dogs given either iopamidol or metrizamide for emg, same-day csf sampling showed that 8 of 17 developed a mild to moderate mononuclear to mixed mononuclear or neutrophilic pleocytosis (6 of 8 were from iopamidol). 106 in the same study, 3 of 17 (all metrizamide) developed mild protein elevation, but mean protein concentration for both groups stayed within the reference interval. 106 in dogs given metrizamide, 7 of 8 had an increased pandy score after emg, which was considered a false-positive result because of the contrast agent. 106 these data, plus histopathology from the same population, showed that the contrast agents caused low-grade leptomeningeal inflammation with no statistical difference between the two agents studied. 106 another similar study over 30 days showed that post-emg csf changes reversed after approximately 5 days. 107 granulomatous meningoencephalitis. gme is a progressive immune-mediated disease that is overrepresented in females, toybreed dogs, and terriers. 20 it is a diagnosis of exclusion and has clinical presentations and mri findings that may be similar to various infectious and neoplastic diseases. csf may be unaffected or may display a mononuclear to mixed pleocytosis and protein concentration elevations, both of varying severity (figs. 14.14 and 14.15). in a study of 188 csf samples from dogs with inflammatory neurological diseases, marked pleocytosis (>1000 cells/μl) was found in cases of srma, bacterial encephalitis, or gme. 85 pleocytosis may also be lymphocytic or neutrophilic. 10 csf protein electrophoresis may be helpful, as several cases have been shown with increased β-globulin and gammaglobulin fractions. 108 most of the diseases described previously in this chapter may manifest as mixed cell pleocytoses, depending on the time interval between disease onset and csf sampling, disease severity, and previous treatment administered. a mixed cell pleocytosis would be expected to occur during transition between different phases of the inflammatory response, where certain cells may predominate at specific times after injury. is typically rare and may involve chorioretinitis or focal cerebral granuloma in the cat. 109 a study of two dogs with systemic blastomycosis and neurologic signs showed mild mixed cell pleocytosis (8 cells/μl, mononuclear predominant; and 15 cells/μl, lymphocytic predominant). 110 using csf cytology or culture to diagnose the organism may be unrewarding. agar-gel immunodiffusion serologic testing has high sensitivity and specificity for canine antibodies and is recommended if appropriate clinical signs (respiratory signs or lymphadenopathy) are present. 57 agar-gel immunodiffusion testing is less sensitive (25%-33%) in the cat, as indicated by a limited number of reports. 57 urine antigen enzyme immunoassay (eia) has good sensitivity for dogs and has been used successfully on at least one cat. 109 eia may also be performed on csf. cytology of nasal, pulmonary, or dermal lesions is more likely to yield direct visualization of organisms. lymphoma. lymphocytic pleocytosis of inflammatory origin may be difficult to distinguish from lymphoma exfoliating into the csf (fig. 14.16 ). the size of lymphocytes and morphological atypia may be helpful, although these may be challenging to differentiate from artifactual morphological changes secondary to cytospin preparation. cats with neoplasia may have lymphocytic pleocytoses (suggestive of lymphoma), mild to moderate mononuclear to mixed cell pleocytoses (suggestive of nonlymphoma tumors), or normal csf. one study examined six cases of feline cns or multifocal lymphoma, which displayed pleocytoses of variable magnitude, absent to mildly elevated protein concentrations, and neoplastic cells visualized in 5 of 6 of the csf samples. 4 in this study, eight cats with cns signs that were ultimately diagnosed with nonlymphoma tumors (e.g., meningioma, carcinoma, nerve sheath tumor) had mild csf protein elevations and either normal tncc (1 of 8) or mild to moderate mononuclear or mixed cell pleocytosis (7 of 8). 4 another study of 11 cats with spinal lymphoma showed neoplastic cells visualized in one case and hemodilution, acd, or neutrophilic pleocytosis in the remainder of cases. 81 a case report of feline multiple myeloma involving lumbar vertebrae and associated soft tissues exhibited cisternal csf with an elevated protein concentration of 290 mg/dl and mild pleocytosis (8 cells/μl) consisting of a majority of neoplastic plasma cells. 111 diagnosis was further confirmed by abnormal urine protein electrophoresis and bone marrow aspiration. 111 histiocytic malignancies. malignant histiocytosis or histiocytic sarcoma tumor cells in canine csf have been documented in two recent case reports; csf cytology displayed marked mononuclear pleocytoses (>500 cells/μl) and mild to moderately elevated protein concentrations (<135 mg/dl). 112, 113 tumor cells phenotypically resembled macrophages, displayed multiple criteria of malignancy, and reacted positively to cd1c on immunocytochemistry, compatible with interstitial dendritic cell origin. 112, 113 necropsy was confirmatory and found no evidence of neoplasia outside of the cns. 112, 113 a case report of a gliomatosis cerebri (gc) neoplasm in a middle-aged poodle showed csf with a mild lymphocytic pleocytosis (20 cells/μl) and protein concentration elevation. 114 on histopathology, lymphocytelike perivascular cuffing and meningitis were noted. other case studies of canine gc have reported normal csf or mild acd. 115 in a study of 56 dogs with intracranial meningioma, in which csf analysis was performed, 29% had normal csf, 45% had acd, and 27% had pleocytosis (2 of 3 of these neutrophilic pleocytosis; 1 of 3 unspecified), with the overall incidence of neutrophilic pleocytosis at 18%. 116 in this study, a positive correlation existed between elevated tncc and anatomical localization of the lesion to the caudal (versus middle or rostral) portion of the cranial fossa, and no association between pleocytosis and necrosis within the lesion was found. 116 these findings contradict prior reports of a high percentage of abnormal csf findings in meningioma, and the authors reported that concurrent glucocorticoid therapy in some of the patients may have negatively biased the data. 11, 116 a study of 26 dogs with spinal meningioma showed no cases with exfoliating tumor cells, 62% with mild pleocytosis up to 47 cells/μl (mean 11 cells/μl), and normal or variably elevated protein concentrations up to 836 mg/dl (mean 212 mg/dl). 117 both cisternal and lumbar csf samples were evaluated in this study and not found to be significantly different. 117 interestingly, tumors of the lumbar region displayed higher mean tncc and protein concentrations compared with tumors of the cervical area (24 versus 4 cells/μl and 158 versus 98 mg/dl, respectively), which the authors postulated may be reflective of a higher number of lumbar csf samples with proximity to the lesion. 117 other neoplasms. a case report of canine csf with 240 cells/μl was characterized by atypical neoplastic round cells that were confirmed on immunocytochemistry and immunohistochemistry to be from a metastatic mammary carcinoma. 118 inflammatory cells were of low numbers and were of a mixed population. 118 a study of csf from 25 dogs with choroid plexus tumors showed direct observation of tumor cells in 47% of the cases of carcinoma. 119 mild to moderate mixed-cell pleocytosis was present in all cases of papilloma and in half of the carcinomas; when pleocytosis was present, no difference in magnitude existed between benign and malignant tumors. 119 all cases had elevated protein concentrations, with median concentration for carcinoma being significantly higher (108 mg/dl) than median concentration for papilloma (34 mg/dl). 119 a cutoff protein concentration of 80 mg/dl yielded a sensitivity of 67% and a specificity of 100% for detection of choroid plexus carcinomas. 119 another case report of canine choroid plexus carcinoma had a mononuclear pleocytosis of 165 cells/μl, mildly elevated protein concentration of 30 mg/dl, and numerous tumor cells visualized. 120 a rise in the availability of stereotactic brain biopsy has facilitated increased cytological assessments of cns lesions. this technique offers several advantages, although significant equipment investment and time to perfect techniques is required. stereotactic biopsy often offers application accuracy for targeting lesions that approximate 3 mm or less in all directions. in one study, diagnostic accuracy of stereotactic biopsy specimens submitted for histopathology (i.e., agreement with specimens obtained via open approaches) exceeded 90%. 121 in experienced hands, stereotactic biopsy is believed to be a relatively low-morbidity procedure. a b 20um 20um cytological interpretation of brain biopsy specimens acquired via stereotaxy or open approaches may be challenging and does require a tumor that exfoliates well, a surgeon willing to provide multiple samples, and a cytologist with expertise in this area. 122 a study of 42 canine and feline cases of biopsy-or necropsy-confirmed cns lesions showed squash-prep smear cytology to have 76% sensitivity in accurately determining diagnosis, with an additional 14% of cases having partial correlation between cytology and histopathology. for the remaining 10% of cases, cytological interpretation did not correlate with final diagnosis. 123 cytological interpretation of cns lesions may be very difficult, and biopsy with histopathological examination is recommended to confirm all diagnoses. it is important for cytological samples to be prepared in the same manner each time to avoid introducing additional cytological variations that the pathologist has to read through. some authors recommend wet-fixation of tissues followed by staining with hematoxylin and eosin (h&e). 122 at the authors' institution, cns cytological samples are air-dried and stained with diff-quik or a modified wright stain. the reader is referred elsewhere for a complete discussion of normal cns cytology. 124 clinical imaging findings, and signalment, should be considered carefully and may help the pathologist to formulate a list of potential differential diagnoses. it must be kept in mind that primary tumors may metastasize to the cns, and these should be included in the differential diagnoses, where appropriate. meningiomas are composed of neoplastic cells arising from the meningothelial cells of the leptomeninges of the cns. 125 these tumors are the most common primary cns tumors of dogs and cats. 126 histologically, these neoplasms are classified into at least nine subtypes based on appearance, and some tumors may be characterized by more than one pattern (fig. 14.17 ). 125 cytologically, smears are often characterized by spindle-shaped cells draped around vessels and arranged in large whorling structures (fig. 14.18 ). some cells may contain nuclei that display intranuclear cytoplasmic pseudoinclusions, but this is not a feature reliably seen on a majority of tumors (fig. 14.19 ). 127 as a whole, this group represents the second most common primary cns neoplasm seen in dogs and cats. 126 glial tumors are more a b common than meningiomas in brachycephalic breeds. 126 glial tumors arise from the supporting cells of the cns. astrocytomas are found most frequently in the cerebral hemispheres, although they have been reported to occur in various locations throughout the cns. 125 astrocytomas arise from transformed astrocytes and are characterized cytologically by high cellularity, a high degree of nuclear pleomorphism, and fibrillar cytoplasmic processes. 125 tumor cells will stain positively for glial fibrillary acid protein (gfap). 125 oligodendrogliomas are derived from transformed oligodendrocytes and are found within the gray or white matter of the cns, with the highest incidence in the cerebral hemispheres. 125 cytological preparations are characterized by large numbers of blood vessels surrounded by neoplastic cells (fig. 14.20) . 125 neoplastic oligodendrocytes have small amounts of eosinophilic cytoplasm surrounding uniformly round nuclei. 125 ependymomas are derived from the ependymal lining cells found on the surface of the ventricular system of the brain and central canal of the spinal cord. 125 these tumors are rare and are found most often in the lateral ventricles. 125 cytologically, smears are characterized by neoplastic cells palisading around branching vascular structures. 125 cells are cuboidal to columnar in shape with high nuclear-to-cytoplasmic (n:c) ratios and eccentrically placed nuclei. 125 choroid plexus tumors arise from the modified ependymal lining cells that contribute to the production of csf. they are more common in dogs than in cats. 125 papillomas and carcinomas have a very similar cytological appearance and may only be reliably differentiated on the basis of histopathological examination. 123 cytological preparations contain polygonal cells arranged in rafts, columns, or papillary projections around capillary structures (fig. 14.21 ). 125 medulloblastoma arises within the cerebellum and is a type of primitive neuroectodermal tumor derived from a germinal neuroepithelial cell. 125 cytologically, preparations are highly cellular, composed of individual round cells that are large in size and have moderate to high n:c ratios. the appearance of these cells is reminiscent of large lymphocytes or histiocytes (fig. 14.22 ). nephroblastoma is a unique tumor arising in the spinal cord of young dogs (under age 4 years), usually between the t10 and l2 spinal cord segments. 125 the cytological appearance of this tumor has been described in a recent report and is characterized by three populations of cells: (1) high n:c ratio blastemal cells, veterinary neuroanatomy and clinical neurology the function, composition and analysis of cerebrospinal fluid in companion animals: part i-function and composition cerebrospinal fluid analysis and magnetic resonance imaging in the diagnosis of neurologic disease in dogs: a retrospective study inflammatory cerebrospinal fluid analysis in cats: clinical diagnosis and outcome clinical and magnetic resonance imaging findings in 92 cats with clinical signs of spinal cord disease magnetic resonance imaging findings in 25 dogs with inflammatory cerebrospinal fluid the formation of cerebrospinal fluid: nearly a hundred years of interpretations and misinterpretations cerebrospinal fluid collection, examination, and interpretation in dogs and cats cerebellomedullary cerebrospinal fluid collection in the dog the function, composition and analysis of cerebrospinal fluid in companion animals: part ii-analysis cerebrospinal fluid analysis analysis of cerebrospinal fluid from the cerebellomedullary and lumbar cisterns of dogs with focal neurologic disease: 145 cases (1985-1987) comparison of total white blood cell count and total protein content of lumbar and cisternal cerebrospinal fluid of healthy dogs bone marrow contamination of canine cerebrospinal fluid iatrogenic brainstem injury during cerebellomedullary cistern puncture collecting, processing, and preparing cerebrospinal fluid in dogs and cats canine neurology: diagnosis and treatment. saunders effects of time, initial composition, and stabilizing agents on the results of canine cerebrospinal fluid analysis analysis of cerebrospinal fluid from dogs and cats after 24 and 48 hours of storage conventional and molecular diagnostic testing for the acute neurologic patient cerebrospinal fluid analysis in the dog: methodology and interpretation cerebrospinal fluid analysis evaluation of the advia 120 for analysis of canine cerebrospinal fluid automated flow cytometric cell count and differentiation of canine cerebrospinal fluid cells using the advia 2120 high resolution protein electrophoresis of 100 paired canine cerebrospinal fluid and serum bacterial meningoencephalomyelitis in dogs: a retrospective study of 23 cases (1990-1999) a case of canine streptococcal meningoencephalitis diagnosed using universal bacterial polymerase chain reaction assay clinical, cerebrospinal fluid, and histological data from thirty-four cats with primary noninflammatory disease of the central nervous system critical evaluation of creatine phosphokinase in cerebrospinal fluid of dogs with neurologic disease associations between cerebrospinal fluid biomarkers and long-term neurologic outcome in dogs with acute intervertebral disk herniation blood-brain-barrier disruption in chronic canine hypothyroidism measurement of myelin basic protein in the cerebrospinal fluid of dogs with degenerative myelopathy cerebrospinal fluid myelin basic protein as a prognostic biomarker in dogs with thoracolumber intervertebral disk herniation beta-2-microglobulin levels in the cerebrospinal fluid of normal dogs and dogs with neurological disease cerebrospinal fluid glutamine, tryptophan, and tryptophan metabolite concentrations in dogs with portosystemic shunts oxytocin content of the cerebrospinal fluid of dogs and its relationship to pain induced by spinal cord compression cerebrospinal fluid gammaaminobutyric acid and glutamate values in dogs with epilepsy cerebrospinal fluid analysis significance of surface epithelial cells in canine cerebrospinal fluid and relationship to central nervous system disease cerebrospinal fluid from a 6-yearold dog with severe neck pain prevalence and significance of extracellular myelin-like material in canine cerebrospinal fluid evaluation of cerebrospinal fluid in cavalier king charles spaniel dogs diagnosed with chiarilike malformation with or without concurrent syringomyelia effects of iatrogenic blood contamination on results of cerebrospinal fluid analysis in clinically normal dogs and dogs with neurologic disease reference intervals for feline cerebrospinal fluid: cell counts and cytologic features differences in total protein concentration, nucleated cell count, and red blood cell count among sequential samples of cerebrospinal fluid from horses effects of blood contamination on cerebrospinal fluid analysis cytologic interpretation of canine cerebrospinal fluid samples with low total nucleated cell concentration, with and without blood contamination clinical features and magnetic resonance imaging findings in 7 dogs with central nervous system aspergillosis clinical, cerebrospinal fluid, and histological data from twenty-seven cats with primary inflammatory disease of the central nervous system inflammatory diseases of the spine in small animals brain abscess and bacterial endocarditis in a kerry blue terrier with a history of immune-mediated thrombocytopenia central nervous system infection with staphylococcus intermedius secondary to retrobulbar abscessation in a dog cerebral ventriculitis associated with otogenic meningoencephalitis in a dog meningoencephalomyelitis caused by pasteurella multocida in a cat clinical features and epidemiology of cryptococcosis in cats and dogs in california: 93 cases (1988-2010) clinical signs, imaging features, neuropathology, and outcome in cats and dogs with central nervous system cryptococcosis from california fungal infections of the central nervous system in the dog and cat disseminated histoplasmosis in cats: 12 cases (1981-1986) treatment of thoracolumbar spinal cord compression associated with histoplasma capsulatum infection in a cat clinicopathologic and diagnostic imaging characteristics of systemic aspergillosis in 30 dogs ehrlichiosis in a dog with seizures and nonregenerative anemia diagnostic features of clinical neurologic feline infectious peritonitis use of anti-coronavirus antibody testing of cerebrospinal fluid for diagnosis of feline infectious peritonitis involving the central nervous system in cats use of albumin quotient and igg index to differentiate blood-vs brain-derived proteins in the cerebrospinal fluid of cats with feline infectious peritonitis the cat with neurological manifestations of systemic disease. key conditions impacting on the cns feline toxoplasmosis: interpretation of diagnostic test results spinal epidural empyema in seven dogs sarcocystis sp. encephalomyelitis in a cat multisystemic infection with an acanthamoeba sp. in a dog another case of canine amoebic meningoencephalitis-the challenges of reaching a rapid diagnosis spinal intramedullary aberrant spirocerca lupi migration in 3 dogs steroid responsive meningitisarteritis: a prospective study of potential disease markers, prednisolone treatment, and long-term outcome in 20 dogs pathogenetic factors for excessive iga production: th2-dominated immune response in canine steroidresponsive meningitis-arteritis the role of acute phase proteins in diagnosis and management of steroid-responsive meningitis arteritis in dogs concentrations of acutephase proteins in dogs with steroid responsive meningitis-arteritis lumbar cerebrospinal fluid in dogs with type i intervertebral disc herniation magnetic resonance imaging findings and clinical associations in 52 dogs with suspected ischemic myelopathy fibrocartilaginous embolism in dogs fibrocartilaginous embolic myelopathy in five cats reversible encephalopathy secondary to thiamine deficiency in 3 cats ingesting commercial diets tumors affecting the spinal cord of cats: 85 cases (1980-2005) clinical and clinicopathological features of non-suppurative meningoencephalitis in young greyhounds in ireland cerebrospinal fluid eosinophilia in dogs what is your diagnosis? cerebrospinal fluid from a dog. eosinophilic pleocytosis due to protothecosis diagnosis of inflammatory and infectious diseases of the central nervous system in dogs: a retrospective study emergency presentations of 4 dogs with suspected neurologic toxoplasmosis use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs realtime pcr analysis of dog cerebrospinal fluid and saliva samples for ante-mortem diagnosis of rabies cerebrospinal fluid from a 7-month-old dog with seizure-like episodes clinicopathological findings in dogs with distemper encephalomyelitis presented without characteristic signs of the disease immunohistochemical detection of canine distemper virus in haired skin, nasal mucosa, and footpad epithelium: a method for antemortem diagnosis of infection production of immunoglobulin g and increased antiviral antibody in cerebrospinal fluid of dogs with delayed-onset canine distemper viral encephalitis necrotizing meningoencephalitis of pug dogs associates with dog leukocyte antigen class ii and resembles acute variant forms of multiple sclerosis necrotizing meningoencephalitis in five chihuahua dogs epidemiology of necrotizing meningoencephalitis in pug dogs cerebrospinal cuterebriasis in cats and its association with feline ischemic encephalopathy feline cerebrovascular disease: clinical and histopathologic findings in 16 cats clinical characterization of a familial degenerative myelopathy in pembroke welsh corgi dogs detection of neospora caninum tachyzoites in canine cerebrospinal fluid detection of neospora caninum tachyzoites in cerebrospinal fluid of a dog following prednisone and cyclosporine therapy necrotizing cerebellitis and cerebellar atrophy caused by neospora caninum infection: magnetic resonance imaging and clinicopathologic findings in seven dogs angiostrongylus vasorum causing meningitis and detection of parasite larvae in the cerebrospinal fluid of a pug dog bartonella-associated meningoradiculoneuritis and dermatitis or panniculitis in 3 dogs hepatozoonosis in a dog with skeletal involvement and meningoencephalomyelitis cerebrospinal fluid response following metrizamide myelography in normal dogs: effects of routine myelography and postmyelographic removal of contrast medium cerebrospinal fluid changes after iopamidol and metrizamide myelography in clinically normal dogs transient leakage across the blood-cerebrospinal fluid barrier after intrathecal metrizamide administration to dogs cerebrospinal fluid protein electrophoresis: a clinical evaluation of a previously reported diagnostic technique cerebral blastomyces dermatitidis infection in a cat clinical and magnetic resonance imaging features of central nervous system blastomycosis in 4 dogs multiple myeloma with central nervous system involvement in a cat antemortem diagnosis of localized central nervous system histiocytic sarcoma in 2 dogs cerebrospinal fluid from a 10-yearold dog with a single seizure episode oligodendroglial gliomatosis cerebri in a poodle gliomatosis cerebri in six dogs characteristics of cisternal cerebrospinal fluid associated with intracranial meningiomas in dogs: 56 cases (1985-2004) canine intraspinal meningiomas: imaging features, histopathologic classification, and long-term outcome in 34 dogs neoplastic pleocytosis in a dog with metastatic mammary carcinoma and meningeal carcinomatosis choroid plexus tumors in 56 dogs (1985-2007) choroid plexus carcinoma cells in the cerebrospinal fluid of a staffordshire bull terrier ct-guided brain biopsy using a modified pelorus mark iii stereotactic system: experience with 50 dogs primary canine and feline nervous system tumors: intraoperative diagnosis using the smear technique squash-prep cytology in the diagnosis of canine and feline nervous system lesions: a study of 42 cases canine and feline cytology-e-book: a color atlas and interpretation guide tumors in domestic animals what is your diagnosis? intracranial mass in a dog a true "triphasic" pattern: thoracolumbar spinal tumor in a young dog key: cord-312064-hza70mur authors: borrow, p; welsh, c j; tonks, p; dean, d; blakemore, w f; nash, a a title: investigation of the role of delayed-type-hypersensitivity responses to myelin in the pathogenesis of theiler's virus-induced demyelinating disease. date: 1998-04-17 journal: immunology doi: nan sha: doc_id: 312064 cord_uid: hza70mur the contribution of autoimmune responses to the pathogenesis of theiler's virus-induced demyelinating disease was investigated. delayed-type hypersensitivity responses to myelin were examined in both symptomatic and asymptomatic mice at different times post-infection, in order to determine whether autoreactivity correlates with the development of demyelination. the results indicate that although autoimmune responses probably do not play a major role in the initiation of demyelination at early times post-infection, autoreactivity to myelin antigens dose eventually develop in symptomatic animals, perhaps through the mechanism of epitope spreading. autoimmunity to myelin components is therefore an additional factor that may contribute to lesion progression in chronically diseased animals. theiler's murine encephalomyelitis virus (tmev) is a picornavirus which induces a biphasic disease of the central nervous system (cns) following intracranial inoculation of susceptible strains of mice.' an acute, polio-like disease with grey matter pathology occurs during the first month post-infection, and is followed in surviving animals by a chronic demyelinating disease which represents a valuable model for multiple sclerosis (ms) in man (reviewed in ref. 2) . primary demyelinating lesions associated with inflammatory cell infiltrates are found in the spinal cord, and are accompanied by clinical s~gns of spastic paralysis. the pathogenesis of theiler's virus-induced demyelinating disease (tvid), is still not completely understood: by analogy with other virally induced demyelinating diseases of the cns, and hypotheses that have been proposed for the aetiology of ms, direct viral damage and/or immunopathology may be involved. tmev typically infects oligodendrocytes in vitro3 and can produce demyelination directly in vivo, e.g. in nude mice where virus replication proceeds unchecked in the absence of t-cell responses.4'5 genetically susceptible immunocompetent strains of mice fail to clear virus from the cns and infected oligodendrocytes, astrocytes and microglia/macrophages can be detected throughout the chronic disease;6'7 however, virus replication appears to be restricted,8'9 and there is evidence to suggest that much of the demyelination in these animals is immunopathologically mediated. morphological studies indicate a close correlation between inflammatory cell infiltrates and areas of demyelination,10 and the lesions have a distribution and appearance similar to those observed in experimental autoimmune encephalomyelitis (eae),7 where t cell-mediated damage is known to be of pathogenic importance. immunosuppression has been shown to reduce incidence/severity of the chronic disease phase, and the fact that anti-ia""2 and anti-cd4 antibodies13 also reduce demyelination, suggests that class ii-restricted cd4' t cells are involved in disease pathogenesis. however, immunogenetic experiments indicate that at least one of the genes important in determining disease susceptibility/resistance maps to major histocompatibility complex (mhc) class 1,14-16 suggesting that a class i-restricted (cd8+ t cell-mediated) response also plays a role in tmev infection. in vivo t cell depletion experiments, work with p2-microglobulin knock-out mice (which lack functional cd8+ t cells) and adoptive transfer experiments have implicated cd8+ t cells in viral clearance from the cns.17-2' cd8+ t cells may also modulate the demyelinating phase of the disease,'7'19'21 perhaps by the production of cytokines, e.g. interferon-'y (ifn-y), for which both protective and pathogenic roles have been suggested in tmev infection.2325 immunopathological responses within the cns may be directed against virus-encoded determinants and/or autoepitopes. initial evidence for the involvement of virus-specific responses in disease pathogenesis came from the correlation 478 0 1998 blackwell science ltd observed by clatch et al.,26 between the ability of different inbred mouse strains to mount an antiviral delayed-type hypersensitivity (dth) response after intracranial infection with tmev and their susceptibility to demyelinating disease. more recently, it has been shown that priming of dth responses to tmev and adoptive transfer of cd4+ virusspecific t cells into tmev-infected recipient mice both increase the incidence and accelerate the onset of clinical disease27 and it has been demonstrated that induction of peripheral tolerance to viral antigens will inhibit the development of demyelinating disease.28 virus-specific cd4' t cells thus clearly play an important role in the pathogenesis of tvid. however, it is also possible that as in other animal models of demyelinating disease such as eae or that produced in mice/rats infected with the jhm strain of coronavirus mouse hepatitis virus (mhv),29 autoimmune responses directed at myelin components may also be induced and make some contribution to the demyelinating disease. the work described here addresses the question of whether or not this is the case in tvid. autoimmune t-cell responses are examined over time postinfection in two strains of mice of differing disease susceptibility, in order to reveal whether autoimmune reactivity correlates with disease development, and hence what role autoimmunity may have in disease induction and/or progression. two inbred strains of mice were used in this study: sjl mice, which are highly susceptible to tvid, and cba mice, which are of intermediate susceptibility. the latter mouse strain was used in addition to the former because its lower disease incidence provided the opportunity to compare clinically healthy with diseased animals even at late times post-infection. female cba mice (department of pathology, cambridge university, cambridge, uk) and sjl mice (obtained from olac, bicester, uk) were infected when 4-5 weeks old. the bean 8386 strain of tmev was a gift from dr h. l. lipton (northwestern university, evanston, il). virus was grown in bhk-21 cells, and the culture supernatant containing infectious virus was aliquoted and stored at -70°before use. the viral titre was determined by plaque assay on bhk-21 cells.30 infection ofmice and assessment ofclinical signs of demyelination mice were anaesthetized with ether and injected intracranially (i.c.) into the right cerebral hemisphere with 104 plaque forming units (pfu) of bean in a 20-j1 volume; control animals were injected i.c. with 20 jl of phosphate-buffered saline (pbs). after infection (day 0) all animals were examined twice weekly for the development of clinical signs indicative of demyelinating disease. clinical signs were scored on a scale from 0 to 6, where 0 indicates a healthy animal and 1-6 represent gradually increasing severity of signs as follows: 1, ruffled fur and/or hunched posture; 2, ruffled fur and hunched posture plus unsteady gait; 3, very unsteady gait, weak grasp response when placed on wire grid, and sometimes slight hind limb monoparesis; 4, severe hind limb weakness and/or paralysis, weight loss (especially cba mice); 5, paraparesis of hind limbs (forelimbs sometimes involved), severe weight loss, frequently diarrhoea; 6, moribund/dead. mice were anesthetized with ether and perfused via the left ventricle with 4% glutaraldehyde in phosphate buffer ph 7-2. the fixed spinal cords were removed and cut into four or five blocks which were post-fixed with 1% osmium tetroxide, dehydrated with graded ethanol and embedded in taab resin (taab laboratory equipment, aldermaston, uk). full face coronal sections were cut at 1 jim from one end of each block and stained with alkaline toluidine blue. histological assessments were made without prior knowledge of experimental protocol or disease classification. myelin-specific dth testing dth testing was performed on groups of 5-10 tmev-infected mice and sham-injected age-matched controls at different times post-infection. mice to be dth tested were selected at random from the available animals at that time-point with a clinical score > 3 (sjl mice) or either > or < 3 as appropriate (cba mice). each animal was only dth tested once during the course of the experiment. ear pinna thickness was measured with a mitutoyu engineer's micrometer and then the left ear was injected intradermally in the dorsal surface with 10 jg mouse myelin in 20 pbs, and the right ear was simultaneously injected with pbs only. twenty-four hours after challenge the increase in thickness of each ear over prechallenge measurements was determined and the results were expressed as the myelin-specific increase (i.e. increase in myelin-injected ear minus increase in pbs-injected ear) in units cm x l0-1. ear swelling reactions showed typical dth kinetics, i.e. the maximal swelling occurred at hr. an analysis of variance was used to determine the statistical significance of dth test results. development of demyelinating disease in tmev-infected sjl mice sjl mice are highly susceptible to the development of chronic demyelinating disease following infection with tmev. this is illustrated by the results of the experiment shown in fig. 1 , where a group of 50 sjl mice were infected i.c. with tmev and their clinical signs were monitored over time. the bean 8386 strain of tmev used here was tissue culture-adapted, and produced only mild (if any) signs of tmev-induced early acute grey matter disease, but over the first 6 months postinoculation, almost all ( days post-infection from 0 (healthy) to 6 (moribund) (see the materials and methods for details). histological analysis of the cns of sjl mice with different clinical scores revealed that inflammatory demyelinating lesions (fig. 2a) were consistently found in the brain and spinal cord of animals with a clinical score of three or more (i.e. exhibiting moderate to severe signs of demyelinating disease). analysis of myelin-specific dth responses in sjl mice undergoing tmev-induced demyelinating disease to determine whether autoimmune t-cell responses may be contributing to the pathogenesis of the chronic demyelinating disease exhibited by bean-infected sjl mice, at both early times (4 and 8 weeks) and late times (36 and 48 weeks) postinfection, groups of 7-10 animals exhibiting moderate to severe clinical signs of disease were tested for dth reactivity to myelin. the use of whole myelin rather than individual myelin components for dth testing allowed simultaneous detection of responses against a mixture of potential oligodendrocyte autoantigens. the results obtained are shown in fig. 3a . mice in the early stages of the disease (tested 4 and 8 weeks post-infection) did not exhibit significant myelin-specific dth reactivity; but by contrast chronically diseased animals (tested at 36 and 48 weeks post-infection) did show dth reactivity to myelin. analysis of variance of the myelin-specific increases in ear thickness revealed that the dth response mounted by diseased animals 36-48 weeks post-infection was statistically significant (p<0 05). this response did not reflect the spontaneous development of myelin-autoreactivity in sjl mice with age, since the myelin-specific response made by agematched uninfected control mice at each time point did not differ appreciably from that mounted by uninfected sjl mice at time 0. cba mice are less susceptible to the development of demyelinating disease following infection with tmev a group of 50 cba mice infected with the bean strain of tmev were also scored for the development of clinical signs of disease over time post-infection as described above for sjl mice (fig. 1) . whereas almost all of the tmev-infected sjl mice had developed clinical signs of disease by 4-5 months post-infection ( fig. 1) only 68% of similarly-infected cba mice were found to have done so even by 1 year post-infection (fig. 1) . in the cba mouse strain, therefore, both apparently healthy and symptomatic animals could easily be identified even at late times post-infection. histological analysis of the cns of cba mice exhibiting clinical signs of different severity revealed that as with sjl mice, demyelinating lesions could consistently be found in the brain and spinal cord of animals with a clinical score of three or more (i.e. with moderate to severe symptoms of disease) (fig. 2b) . animals with lower clinical scores failed to show pronounced histological abnormalities. the lesions in cba mice (fig. 2b) were less extensive than those in sjl mice (fig. 2a) . sjl mice had more pronounced cellular infiltrates with inflammatory cells in areas where no demyelination was evident. axonal degeneration was also a feature of the demyelinating lesions in sjl mice. by contrast in the cns of cba mice, the inflammatory infiltrates were much more closely localized to the areas of demyelination and there was no evidence of axonal degeneration. at different time-points following infection with tmev, groups of five cba mice which were either exhibiting moderate-severe clinical signs of disease or that appeared healthy/ mildly symptomatic were tested for dth reactivity to myelin (fig. 3b) . the results obtained in the groups of cba mice with a clinical score > 3 paralleled those obtained in the sjl mouse strain (where all animals tested had clinical scores in this range): i.e. significant myelin-specific dth responses were not observed in mice tested at early times post-infection (which had only recently developed clinical scores indicative of demyelinating disease), but highly significant (p<0.001) responses were olserved in chronically-diseased animals (nine mice tested 36-4l weeks following infection). by contrast, cba mice which remained relatively asymptomatic over the entire course of the infection did not exhibit myelin-specific dth reactivity even when tested at very late times (five mice tested at 36-48 weeks) post-infection. this demonstrates that myelin-specific dth responses are developed only in animals undergoing demyelinating disease. 1998 blackwell science ltd, immunology, 93, 478-484 we have examined t-cell responses to myelin in tmevinfected mice of different strains at various times post-infection to determine whether correlations exist between the development of autoreactivity and demyelination that might indicate a causative link. minimal dth responses following peripheral challenge with myelin were observed in the first few months post-infection. however, when animals were tested at very late times post-infection, it was found that symptomatic but not asymptomatic animals of both strains did then possess significant myelin-specific dth reactivity. we have also obtained similar results in cba and sjl mice infected with the da strain of theiler's virus (data not shown). it thus appears that autoimmune responses to myelin are not of importance in the initiation of early demyelinating disease in tmev-infected mice. however, they are induced at very late times postinfection, although only in animals in which there has already been damage to myelin within the cns. it is possible that dth to myelin could contribute to lesion progression during chronic disease. early experiments to examine whether autoimmune responses are induced in tmev-infected mice involved unsuccessful attempts adoptively to transfer demyelinating disease from tmev-infected animals to naive syngeneic recipients with spleen cell preparations33 (our unpublished observations). in this paper, we directly tested cell-mediated autoimmune responses in tmev-infected mice. in agreement with results of other groups who also tested dth responses34'35 and spleen cell proliferative responses33'36 to myelin and myelin components over the first 2-3 months following infection of mice with tmev, we were unable to detect autoimmune t-cell responses in the periphery at early times post-infection. it is always possible that myelin-specific responses develop within the cns of tmev-infected mice at these times which are not detected peripherally. musette et al., observed that there was a selective expansion of t cells with receptors composed of particular vijp combinations in the spinal cord but not the spleen of tmev-infected sjl/j mice, suggesting that a local antigen-driven response was occurring, although they did not address the specificity of this response. however, even if autoimmune responses are initiated in the cns over the first few months post-infection, they do not appear to make a significant contribution to the demyelinating disease process at this time, as the induction of tolerance to spinal cord homogenate did not influence the course of demyelinating disease over the first 60 days post-infection. 36 autoreactive t-cell responses have not previously been examined in the later stages of the chronic demyelinating disease induced by tmev in mice. however, some evidence that autoimmunity may develop in tmev-infected mice at late times post-infection was reported by cash et al.,7 who studied the specificity of immunoglobulin secreted by b cells isolated from the cns of sjl/j mice infected 3-7 months previously with tmev-da, and found antibodies which reacted with two non-viral white matter components which were present only in infected animals. in addition, serum autoantibodies to myelin have been detected in bean-infected cba mice.13 in the present study, we tested autoreactive t-cell responses and found that dth responses to myelin were readily detected in the periphery of diseased but not asymptomyelin-specific dth reactivity of sjl mice at different times following infection with tmev. groups of 7-10 sjl mice and age-matched sham-infected controls were tested just prior to infection with tmev, and at the indicated times following infection, for dth reactivity to myelin as described in the materials and methods. all infected animals used in this experiment had a clinical score of .3 (i.e. exhibited moderate-severe clinical signs of disease indicative of cns demyelinating disease). data for the mice tested prior to infection and all of the sham-infected age-matched control mice at subsequent time-points is recorded as the 0 time-point; data at subsequent timepoints represents the results obtained with infected mice at the indicated time post-inoculation. the results shown are the mean myelin-specific increase in ear thickness of each group of animals; the bars indicate one standard error above and below the mean. *indicates a dth response to myelin shown to be significantly greater than the control response (p<0'05). (b) myelin-specific dth reactivity of cba mice at different times following infection with tmev. at the indicated times post-infection, groups of five cba mice which were asymptomatic or exhibiting mild disease signs (open bars); or groups of five mice with a clinical score of 3 i.e. exhibited moderate-severe clinical signs of disease indicative of cns demyelinating disease (black matic animals at late times post-infection. the fact that only mice undergoing demyelinating disease developed autoreactive dth responses suggests that the autoimmune responses did not represent cross-reactivity with viral antigens, but rather were related to the demyelinating disease process. autoimmune responses probably arise in tmev-infected mice undergoing demyelinating disease via determinant spreading. 36 this phenomenon, which involves a change in t-cell specificity over time so that a broader array of epitopes start to be recognized, has been demonstrated in (sjl x blo.pl)fj mice suffering from eae.39 by analogy, in theiler's virusinfected mice, the initial immune reaction within the cns is mainly anti-viral, but as the myelin damage increases, later in the disease process, autoimmune responses may arise by intermolecular epitope spreading. this may account for the fact that the myelin-specific t-cell responses are only detected at later time-points. two pathological events which take place in the cns of tmev-infected mice that probably play key roles in the process of epitope spreading to autoantigens are damage to cns myelin and the induction of mhc expression. some oligodendrocyte destruction may be a direct result of the viral infection4'5 but immune-mediated attack may be of greater importance. histologically, there is evidence of myelin sheath degradation and uptake by macrophages in lesions in the cns of tmev-infected mice. myelin antigens may be released to the periphery and autoimmune t cells primed there which then traffic back into the cns and are retained following recognition of their specific antigen; or autoreactive t cells might potentially be induced within the cns itself. in either case, mhc regulation within the cns is critical to allow recognition of autoantigens to occur there. up-regulation of mhc class i, and also mhc class ii antigens (in disease-susceptible mouse strains), has been demonstrated within the cns of tmev-infected mice.21'40 some viruses are able to achieve mhc induction by direct infection of cns cells, e.g. the coronavirus mhv-jhm, which up-regulates mhc class ii expression on infected astrocytes.41 mhv-jhm induces a demyelinating cns disease in lewis rats. in this model diseased rats show t-cell reactivity to myelin basic protein29 and the induction of mhc class ii expression on glial cells and the presentation of degraded myelin is postulated to be a key factor in the autoimmune disease. whilst the bean strain of tmev is unable directly to up-regulate mhc class ii expression on astrocytes in a similar way,42 cytokines produced by t cells infiltrating the cns to mediate control of virus replication at early times post-infection, e.g. ifn-'y, will induce mhc expression on cns cells. correlations have been observed between the ability of ifn-y to induce mhc class ii expression on astrocytes42 and -cerebrovascular endothelial cells43 from different mouse strains and their susceptibility to tmev-induced demyelinating disease. in these mouse strains, chronic mhc up-regulation on cns cells in the context of autoantigen release will also set the stage for development of autoimmune responses. bars) (nine symptomatic mice were tested at 36-48 weeks postinfection) were tested for dth reactivity to myelin. *indicates a dth response to myelin shown to be significantly greater than the control response (p<0.001). details as for (a). the contribution that the autoimmune t-cell responses we have observed in mice undergoing tmev-induced demyelinating disease makes to lesion spread and disease progression at late times post-infection remains unclear. there did not seem to be any correlation between clinical score and the strength of the dth response to myelin autoantigens in individual sjl mice tested at 36-48 weeks (not shown), but as clinical scores from 3 to 5 did not correlate precisely with increasing extent of demyelination within the cns, such a correlation would perhaps not have been expected. this question thus remains unresolved. in summary, we report that in a cns demyelinating disease triggered by a known viral infection, autoreactive t-cell responses ultimately develop in chronically diseased animals as a secondary phenomenon, the pathogenic significance of which has not been defined. this finding has important implications for hypotheses which have been proposed as to the aetiology of ms in man. ms is known to involve t cellmediated damage to the cns; but the process by which this is triggered and the specificity of t cells involved are currently unclear. t cell reactivity to myelin basic protein has been detected in the peripheral blood and/or cerebrospinal fluid of some ms patients,44-47 although this is not always observed,48 and such responses have been reported in some control subjects with other diseases that affect the cns.49 it has been suggested that these autoreactive t cells are central to disease production in ms. however, it is possible that as in tmev-induced demyelination, they in fact represent only an epiphenomenon, being induced in some individuals only after damage to myelin has been produced by other mechanisms, which have yet to be identified. theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination immunology of theiler's murine encephalomyelitis virus infection theiler's virus in brain cell cultures: lysis of neurons and oligodendrocytes and persistence in astrocytes and macrophages da strains of theiler's murine encephalomyelitis virus induces demyelination in nude mice mechanism of theiler's virus-induced demyelination in nude mice identification of theiler's virus infected cells in the central nervous system of the mouse during demyelinating disease observations on demyelinating lesions induced by theiler's virus in cba mice theiler's virus rna and protein synthesis in the central nervous system of demyelinating mice minus-strained rna synthesis in the spinal cords of mice persistently infected with theiler's virus primary demyelination in theiler's virus infection: an ultrastructural study partial suppression of theiler's virus-induced demyelination in vivo by administration of monoclonal antibodies to immuneresponse gene products (ia antigens) monoclonal anti-i-a antibody reverses chronic paralysis and demyelination in theiler's virus-infected mice: critical importance of timing of treatment the effect of l3t4 t cell depletion on the pathogenesis of theiler's murine encephalomyelitis virus infection in cba mice theiler's murine encephalomyelitis virus-induced demyelinating disease in mice is influenced by the h-2d region: correlation with tmev-specific delayed-type hypersensitivity the interaction of two groups of murine genes determines the persistence of theiler's virus in the central nervous system fvb mice trangenic for the h-2db gene become resistant to persistent infection by theiler's virus the role of cd8 + t cells in the acute and chronic phases of theiler's virus-induced disease in mice theiler's virus infection of 02-microglobulin deficient mice class i-deficient resistant mice intracerebrally inoculated with theiler's virus show an increased t cell response to viral antigens and susceptibility to demyelination abrogation of resistance to theiler's virus-induced demyelination in h-2b mice deficient in beta-2-microglobulin differential expression of h-2k and h-2d in the central nervous system of mice infected with theiler's virus adoptively transfered cd8 + t lymphocytes provide protection against tmev-induced demyelinating disease in balb/c mice alteration in the level of interferongamma results in acceleration of theiler's virus-induced demyelinating disease theiler's virus infection of 129sv mice that lack the interferon alpha/beta or interferon gamma receptors gamma interferon is critical for resistance to theiler's virus-induced demyelination class iirestricted t cell responses in theiler's murine encephalomyelitis virus (tmev)-induced demyelinating disease. ii. survey of host immune responses and central nervous system virus titers in inbred mouse strains class ii-restricted t cell responses in theiler's murine encephalomyelitis virus-induced demyelinating disease. vi potentiation of demyelination with and characterization of an immunopathologic cd4 + t cell line specific for an immunodominant vp2 epitope inhibition of theiler's virus-mediated demyelination by peripheral immune tolerance induction adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelination encephalomyelitis preparation and characterization of encephalomyocarditis (emc) virus endogenous cyclic amp-stimulated phosphorylation of a wolfgram protein component in rabbit central nervous system myelin protein measurement with the folin phenol reagent serum and cells from theiler's virus-infected mice fail to injure myelinating cultures or to produce in vivo transfer of disease class ii-restricted t cell responses in theiler's murine encephalomyelitis virus (tmev)-induced demyelinating disease. part 1. cross-specificity among tmev substrains and related picornaviruses, but not myelin proteins t lymphocyte repertoire in theiler's virus encephalomyelitis-the nonspecific infiltration of the central nervous system of infected sjl/j mice is associated with a selective local t cell expansion class iirestricted t cell responses in theiler's murine encephalomyelitis virus (tmev)-induced demyelinating disease. iii. failure of neuroantigen-specific immune tolerance to affect the clinical course of demyelination characterization of b lymphocytes present in the demyelinating lesions induced by theiler's virus determinant spreading and the dynamics of autoimmune t-cell repertoire evolution of the t-cell repertoire during the course of experimental immune-mediated demyelinating diseases immune response gene products (ta antigens) on glial and endothelial cells in virus-induced demyelination viral particles induce la expression on astrocytes susceptibility to theiler's virusinduced demyelinating disease correlates with astrocyte class ii induction and antigen presentation correlation between susceptibility to demyelination and interferon gamma induction of major histocompatibility complex class ii antigens on murine cerebrovascular endothelial cells in vitro cell-mediated immunity of cerebrospinal fluid lymphocytes to myelin basic protein in primary demyelinating diseases expansion of antigen-specific t cells from cerebrospinal fluid of patients with multiple sclerosis anti-myelin basic protein autoreactive t lymphocytes in healthy subjects and multiple sclerosis patients human t cell response to myelin basic protein in multiple sclerosis patients and healthy subjects myelin basic protein and proteolipid protein reactivity of brain and cerebrospinal fluid-derived t cell clones in multiple sclerosis and postinfectious encephalomyelitis cell-mediated immunity to myelin associated glycoprotein, proteolipid protein and myelin basic protein in multiple sclerosis key: cord-315656-asvf4roo authors: wu, junjiao; tang, yu title: revisiting the immune balance theory: a neurological insight into the epidemic of covid-19 and its alike date: 2020-10-15 journal: front neurol doi: 10.3389/fneur.2020.566680 sha: doc_id: 315656 cord_uid: asvf4roo as the pandemic of covid-19 is raging around the world, the mysteriousness of severe acute respiratory syndrome-coronavirus 2 (sars-cov-2) coronavirus is being revealed by the concerted endeavors of scientists. although fever and pneumonia are typical symptoms, covid-19 patients exhibit multiple neurological complications. in this interim review, we will summarize the neurological manifestations and their potential causes in covid-19. similar to the other two fatal respiratory coronaviruses, sars-cov and middle east respiratory syndrome coronavirus (mers-cov), sars-cov-2 also shows to be neuroinvasive that may spread from the periphery to brain, probably by the retrograde axonal transport. the invaded viruses may directly disrupt the complex neural circuits, and raise a chronic activation of immune responses. in another hand, multiple organ failure in severe covid-19 is caused by the systemic acute immune responses, and unsurprisingly caused the brain inflammation and led to encephalitis. however, in the central nervous system (cns), the activation of resident immune cells including microglia and astrocytes may lead to chronic immune imbalance, which underlies the potential long-term effects in synaptic changes and neuropsychiatric impairments. the neuroinvasive biology also provides a possible link with the braak staging of neurodegenerative diseases such as parkinson's disease (pd). although with considerable advances, the neurotropic potential and chronic neurological effects caused by sars-cov-2 infections merit further investigations. the ongoing spread of covid-19 disease, is the first pandemic ever caused by coronaviruses in the human history, as announced by the world health organization (who) in march 2020. the ferocious virus, isolated as a new strain of zoonotic coronavirus named as severe acute respiratory syndrome-coronavirus 2 (sars-cov-2), has rapidly spread with over 23.2 million confirmed cases and 0.8 million deaths globally as of aug 23 2020 (johns hopkins university). the pandemic has exhausted the entire worlds' personal protective equipment and medical ventilators, and is also strenuously hurting the global economy and raising considerable social issues. as we are in the midst of this ongoing pandemic, it has gathered the concerted efforts of clinicians, public health experts, virologists, immunologists, and other scientists to understand the virus's biology and blocking agents. so far, a myriad of urgent endeavors has been maneuvered, aiming to reveal the multiple aspects of this wily virus, ranging from the genomic structures, sensing receptors to the development of specific medicines and vaccines. structurally, sars-cov-2 is a single-stranded rna virus, whose genome contains 29,891 nucleotides in size and 12 putative functional open reading frames (orfs) (1) . of those translated proteins, the spike proteins located on the virus surface mediate the virus entry into host cells (2, 3) . mechanistically, the spike of sars-cov-2 senses the angiotensin converting enzyme 2 (ace2) receptor (2) (3) (4) (5) , the same as sars-cov, which normally helps regulate blood pressure and anti-atherosclerosis (6) . this binding, in concert with host proteases, principally tmprss2, facilitates the virus getting through the cell membrane by endocytosis (4), followed by hijacking the host cell's translation machinery and producing massive virus copies and further invading new cells. as ace2 is typically enriched in type ii alveoli cells, the lung tissue becomes the major organ affected by the virus (7) . the typical symptoms of covid-19, unsurprisingly, are fever, cough, and pneumonia, which probably lead to acute respiratory distress syndrome (ards) and acute lung injury, as described in around 20% of covid-19 patients (8) . along with sars-cov broke out in 2003 and middle east respiratory syndrome coronavirus (mers-cov) since 2012, sars-cov-2 is the third coronavirus that can cause severe respiratory diseases. genomic analysis shows that sars-cov-2 is in the same β-coronavirus clade as sars-cov and mers-cov, and shares a highly homologous sequence with sars-cov (9). scientists thus have put great efforts in clarifying how it resembles and differs from sars-cov and mers-cov at multiple levels that may shed light on the covid-19 therapeutics and drug repurposing. specifically, the similarity goes to the systemic organ injury and cytokine storm in severe situations. although the symptoms in lungs are manifested at an early stage, they can be extended to multiple organs including the blood vessels, heart, gut, kidneys, testicles, and brain, which are well-known to express ace2 and are potential targets of covid-19 (10) . unlike the outbreak of sars and mers, the emerging single-cell rna-sequencing (scrna-seq) during recent years is rapidly advancing our ability to comprehensively map the cell types with ace2/tmprss2 expression (11) (12) (13) (14) (15) . it is shown that, besides pneumocytes, ace2 receptors are present in various cell types including the nasal epithelial cells, oral mucosa epithelial cells, cholangiocytes, intestine enterocytes and, importantly, immune cells such as b cells, natural killer t cells, monocytes, and macrophages (11, 13, (16) (17) (18) (19) . notably, ace2 is also expressing in the brain, in which eight cell types were identified including excitatory neurons, inhibitory neurons, oligodendrocyte progenitors, oligodendrocytes, microglia, astrocytes, pericytes, and endothelial cells (13) . however, other studies showed contradictory results that glial cells may not express ace2, but instead might express non-canonical docking receptors such as basigin (bsg) or neuropilin-1 (nrp1) to facilitate viral cell entry and replication (20, 21) . nevertheless, the present of ace2 receptors in multiple organs underlies the systemic impairment by sars-cov-2 infection. coronavirus infection has been associated with neurological manifestations such as stroke, seizure, convulsions, mental confusion, and encephalitis (22, 23) . during the outbreak in 2003, sars-cov could induce neurological diseases such as polyneuropathy, encephalitis, and aortic ischemic stroke (24) . the virus itself has been detected in the cerebrospinal fluid (csf) samples, and even the brain of deceased patients (25, 26) . in 2012, nearly 20% of patients with mers-cov infection developed neurological symptoms, including ischemic stroke, paralysis, unconsciousness, guillain-barre syndrome, and other infectious neuropathy (27) . it is thus not surprising to see neurological manifestations in covid-19 patients as well (28) (29) (30) . in general, covid-19 neurological manifestations could be classified into two categories: central nervous system (cns) symptoms and peripheral nervous system (pns) symptoms. cns symptoms included headache, dizziness, acute cerebrovascular disease, ataxia, disturbance of consciousness, and epilepsy. however, pns symptoms are less severe and manifested as neuralgia, hypoplasia, hyposmia, and hypogeusia. notably, respiratory illness in covid-19 patients may also result from the direct role of sars-cov-2 in respiratory control nuclei in the brain (31) . interestingly, still many patients who present with severe neurological complications have hardly any respiratory symptoms, suggesting a rather heterogenous neurological responds among individuals, and that neurological manifestations did not appear concomitantly with respiratory symptoms. in a retrospective series of 214 covid-19 patients at a hospital located in the epicenter of wuhan, china, neurologic symptoms were recorded in 78 patients (36.4%) included headache and disturbed consciousness, and 6 patients had strokes (32) . half of the patients in strasbourg, france by severe sars-cov-2 infection was associated with encephalopathy, prominent agitation and confusion, and some of them had single acute ischemic strokes after brain imaging (33) . in japan, a covid-19 patient was brought in by the ambulance due to a convulsion accompanied by unconsciousness, which was diagnosed with aseptic meningitis/encephalitis (34) . notably for this case, the specific sars-cov-2 rna was detected from the csf sample. similarly, a medical team at a hospital in beijing, china confirmed the presence of sars-cov-2 in the csf of a 56-year-old patient with covid-19 by genome sequencing, thereby clinically verifying viral encephalitis (35) . notably, unlike encephalopathy, the acute stroke is most likely caused by endothelial injury due to a pro-inflammatory hypercoagulable state post sars-cov-2 infection (36, 37) . hence in china, the neurological symptoms have been added into the diagnosis and figure 1 | proposed neuroinvasion routes and immune responses in covid-19. upon infections by sars-cov-2 coronaviruses, covid-19 patients exhibit multiple neurological complications, which might be due to the effects through the direct pathway and the indirect pathway. (i) the neuroinvasive properties of sars-cov-2 underlies the retrograde axonal transport in the direct pathway. specifically, sars-cov-2 viruses may go upward through the olfactory nerve across the cribiform plate and to the brain, or alternatively, start from the gastrointestinal system to invade the enteric nervous system and finally the brain. several other invasion routes for sars-cov-2 may include blood-borne diffusion through the blood-brain barrier, blood-cerebrospinal fluid barrier and meningeal cerebrospinal fluid barrier. those invaded viruses may directly disrupt the complex neural circuits, and raise a chronic activation of immune responses. (ii) multiple organ failure in severe covid-19 is caused by the systemic acute immune responses, the cytokine storm, and unsurprisingly caused the brain inflammation and led to encephalitis. however, the potential long-term effects in synaptic changes and neuropsychiatric impairments in key brain regions should not be neglected. this is probably caused by the activation of cns immune cells that renders chronic immune imbalance. treatment protocol for 2019 novel coronavirus pneumonia (the 7th trial edition), released by national health commission & state administration on march 3, 2020, which reminds us of taking nucleic or genomic tests with csf samples and carefully handling with neurological complications to reduce the fatality of critical care patients. those neurological manifestations observed in covid-19 patients are reminiscent of neuroinvasive potential of sars-cov-2 virus, like the other zoonotic coronaviruses sars-cov and mers-cov (31) . an increasing number of patients with covid-19 reported a sudden loss of smell (anosmia) or taste (dysgeusia) (38, 39) that may serve as initial manifestations and warning signs for possible subsequent cns involvement. given that ace2 is highly expressed in nasal epithelial cells (11) , people speculate that nose might be the first stop during the invasion of viruses, which then go upward through the olfactory nerve across the cribiform plate, and to the brain (29) (figure 1 ). one recent study showed that, based on bulk and single-cell rna sequencing, ace2 expressed in supporting and stem cells in the human/mouse olfactory epithelium, as well as vascular pericytes in the mouse olfactory bulb, however, was not detected in olfactory sensory or bulb neurons (40) . furthermore, autopsy studies of covid-19 patients found that olfactory epithelium showed prominent leukocytic infiltrates in the lamina propria and focal atrophy of the mucosa, and olfactory nerve fibers in the lamina propria were lack of myelin, suggestive of axonal damage (41) . however, the clear evidence is still lacking to confirm whether the olfactory neuropathy is due to direct viral infection or mediated by perturbing supporting non-neural cells. the occurrence of diarrhea, as another covid-19 symptom, suggests that the gastrointestinal system is a possible alternative pathway to invade the enteric nervous system and finally the brain (42) (figure 1) . several invasion routes for coronaviruses have been postulated (28, 43) , including retrograde axonal transport, blood-borne diffusion through the blood-brain barrier (bbb), blood-cerebrospinal fluid barrier, and meningeal cerebrospinal fluid barrier (44) . once in the brain, those viruses may directly destroy the complex organization of neural circuits through neuronal damage, and raise a chronic activation of the inflammatory responses. apart from the direct infection of the brain, sars-cov-2 may cause neurological disorders indirectly by triggering an over-activated immune responses, characterized as cytokine storm. cytokines are chemical signaling molecules that summon immune cells and mediate a balanced immune response, however, in the cytokine storm, levels of certain cytokines soared far beyond the required levels so that the recruited immune cells began to attack healthy tissues and caused catastrophic organ failures. vital research suggests that for many patients who died from covid-19, the fatal blow may be their own immune system rather than the virus itself. the initiation of cytokine storm is a common complication caused by fatal respiratory coronaviruses, like sars-cov and mers-cov, and is the major cause of morbidity (45) . studies have shown that increased numbers of pro-inflammatory cytokines (such as il-1β, il-6, il-12, ifn-γ, ip10, and mcp1) in the serum of severe sars patients are associated with lung inflammation and extensive lung injury (46) . similarly in 2012, it was reported that mers-cov infection can induce substantially elevated concentrations of pro-inflammatory cytokines such as il-6, ifn-γ, tnf-α, il-15, and il-17 (47, 48) . ongoing studies have also been revealing the features of cytokine storms in covid-19 patients. for most severe patients with covid-19, the levels of pro-inflammatory cytokines soared in the serum, similar to that in sars and mers, including il-6, il-1β, il-2, il-8, il-17, g-csf, gm-csf, ip10, mcp1, mip1α, and tnf-α (8, (49) (50) (51) (52) (53) . in addition, patients admitted to the intensive care unit (icu) had higher g-csf, ip10, mcp1, mip1a, and tnf-α concentrations than patients not admitted to the icu, suggesting that cytokine storm is associated with disease severity (8) . high levels of pro-inflammatory cytokines could cause shock and tissue damage, leading to respiratory failure, or multiple organ failure. they mediate extensive lung pathology, resulting in massive infiltration of neutrophils and macrophages, diffuse alveolar injury and the formation of clear membranes and diffuse thickening of the alveolar wall (54) . thus, it is urgently needed therapeutics based on suppressing cytokine storms. in the clinical practice, anti-inflammatory agents have been frequently used for the treatment of patients with severe illness by virus infections. for instance, corticosteroids were ever used in treating patients with sars, which have actually saved many lives and families. however, long-term use of this powerful broad immunosuppressant can cause various complications such as increased cholesterol, brittle bones, cataracts, as well as depression that may greatly reduce the quality of life. more unfortunately, the latest evidence from sars and mers patients shows that receiving corticosteroids has no effect on mortality, but delays viral clearance (8, 55, 56) . therefore, according to the who's interim guidelines, corticosteroids should not be routinely given systemically. it is noteworthy that, il-6, one of the cytokines elevated in response to sars-cov-2 was the most reported in multiple clinical groups. for instance, in a series of 99 covid-19 cases from hospitals in wuhan and shanghai, china, half of the patients show elevated il-6 levels (57). another group investigated the immune responses and cytokine release from patients in chongqing, china. they found that il-6 was higher in 76.19% of severe patients, whereas that was seen in only 30.39% of mild patients (58) . it echoes that the elevated serum il-6 correlates with pneumonia, ards, and adverse clinical outcomes (59) (60) (61) . elevated serum c-reactive protein, which is regulated by il-6, also serves as a biomarker of severe coronavirus infection (62) . based on this fact, drugs such as tocilizumab, satralizumab, and sarilumab, as il-6 receptor (il-6r)-targeted monoclonal antibodies (mabs), might prove beneficial for the treatment of covid-19 (63) . indeed, controlled clinical trials are underway around the world to test the treatment of il-6 and il-6r antagonists for covid-19 patients with severe respiratory complications. preliminary results from the study of 21 severe covid-19 patients receiving tocilizumab in anhui province, china are encouraging (64) . all patients have recovered from fever within the first day of tocilizumab treatment (64) . other clinical trials are also underway in different countries. although the efficacy of tocilizumab in covid-19 patients with ards requires further evaluation in a larger randomized controlled trial, this encouraging clinical trial suggests that neutralizing mabs against other pro-inflammatory cytokines such as il-1 and il-17 might also be useful (54) . for urgently treating the soaring number of severe patients, the chinese diagnosis and treatment protocol for 2019 novel coronavirus pneumonia (the 7th trial edition) has updated a guideline for taking immunotherapy: for patients with extensive lung lesions and severe cases who also show an increased level of il-6 in laboratory testing, tocilizumab can be used for treatment. although with exciting benefits, the inhibition of il-6 pathway works mostly for severe cases, the long-term treatment strategy against the sars-cov-2 infection requires the rapid development of effective anti-viral drugs and, more importantly, vaccines. the systemic cytokine storm caused the multiple organ failure, and unsurprisingly triggered the hyperinflammatory responses frontiers in neurology | www.frontiersin.org inside the cns that further exacerbated the neurological pathology. the spreading of infected leukocytes might across the compromised bbb, caused by increased pro-inflammatory cytokines, from the periphery to the brain. previously, for most cases of sars, autopsy detections of affected brain tissue samples have shown signs of extensive edema, microglial hyperplasia, neuron necrosis, nerve demyelination, as well as massive infiltration of monocytes and lymphocytes (65) . based on recent autopsy results, brain hyperemia and edema, partial neuron degeneration, as well as inflammatory cell infiltration in perivascular regions were also detected in covid-19 patients from china (66) . the persistence of coronavirus infection and its ability to infect macrophages, microglia, and astrocytes in the cns are particularly critical in the pathogenesis of encephalitis (30) . notably, a neurotropic virus could directly activate glial cells and induce a pro-inflammatory phenotype (67) . studies have confirmed that primary glial cells cultured in vitro released a large number of pro-inflammatory factors, such as il-6, il-12, il-15, and tnf-α, upon coronavirus infection (22) . glial cells, as resident immune cells of the cns, normally take a role in maintaining the homeostasis, responding promptly to cns injuries such as trauma, ischemia, and infection, and also providing support and protection for neurons. particularly, microglia are initially activated to clear the invaded pathogens by secreting pro-inflammatory mediators, followed by promoting tissue reconstitution and inflammation resolution. microglia have been demonstrated to protect against lethal coronavirus encephalitis in mice (68) . during the early days after infection, microglia were required to limit mouse hepatitis virus (mhv) replication and subsequent morbidity and lethality. the chemical depletion of microglia led to increased viral replication in the brain and caused ineffective t cell responses, reminiscent of the critical role of microglia in the early innate responses to virus infections (68) . however, in addition to protective effects, microglia may also mediate hippocampal presynaptic membrane damage through complement system, resulting in long-term memory impairment and cognitive decline in patients with encephalitis, caused by coronavirus or human immunodeficiency virus (hiv) infection (69) . thus, beyond the acute cytokine storm, the activation of immune cells in the brain might cause chronic inflammation and brain damages in covid-19 patients. taken together, in the short-term, sars-cov-2 infections may cause the cns inflammation and lead to encephalitis. potential long-term effects, such as changes in mood and cognitive behavior, and continuous changes in the expression of genes that regulate synaptic activity in key brain regions should not be neglected. moreover, this speculation has drawing increasing attentions of clinicians and neuroscientists (21, (70) (71) (72) (73) . hence, prognostic research on potential and longitudinal potential covid-19-related neuropsychiatric diseases is crucial in disease surveillance and evidence-based treatment strategies (74) . the multiple organ failure in covid-19 is associated with the acute immune imbalance, whereas the chronic immune imbalance in the cns, either by invaded virus or by infiltrated immune mediators, might be happening (figure 1 ). an emerging hypothesis states that the inflammation caused by viral infection may trigger and propagate chronic neuronal dysfunction, which is an event before the clinical onset of multiple neurodegenerative diseases such as parkinson's disease (pd) and alzheimer's disease (ad) (75) . notably, the chronic immune imbalance is the shared hallmark for neuropsychiatric and neurodegenerative diseases, due to the uncontrolled skewing of glial phenotypes into detrimental states (76) . experimental vaccination of mice with h5n1 influenza virus can mimic many aspects of pd-like initiation and pathogenesis (77, 78) . the continued inflammation that follows in the viral trajectory leads to dysfunction or degeneration of dopaminergic neurons in the midbrain, just as seen in pd patients (77, 78) . therefore, it would be interesting to probe the relationship between the immune responses upon coronavirus infections and neurodegeneration/ neuropsychiatric impairments. the similar set of sustained elevated pro-inflammatory cytokines or chemokines, typically ils, cxcls, and tnf, that trigger the cytokine storm of covid-19 are also frequently detected in the csf and autopsy brain samples (79) (80) (81) , which is critical in the development and progression of numerous neurodegenerative disorders. since the role of neuroinflammation and specific inflammatory mediators have been recently extensively reviewed in respective diseases including pd, ad, amyotrophic lateral sclerosis (als), huntington's disease (hd), and multiple sclerosis (ms) (82-86), we will not discuss in much details but give some examples. for ad, pro-inflammatory factors are responsible for the increased amyloid precursor protein (app) production and amyloid-β (aβ) load, as well as tau hyperphosphorylation, the hallmarks of ad. specifically, tnf-α can increase aβ burden by promoting β-secretase production and increased γ-secretase activity (87) . elevated il-6 levels have been shown to activate cdk5, a kinase that phosphorylate tau proteins (88) . such extensive neuroinflammation thus would cause neuronal death that leads to cognitive impairment and dementia. alpha-synuclein (α-synuclein), a major component of lewy bodies in the pathogenesis of pd, plays an important role in mediating innate and adaptive immunity (89) . particularly, the mutant forms of α-synuclein in pd could induce microglial activation, releasing various pro-inflammatory cytokines (il-6, il-1β, and tnf-α etc.) and cxcl12, by recognizing toll-like receptors (tlrs) (90, 91) . similarly, for als, the aggregated proteins as mutated superoxide dismutase (msod1), caused motoneuron injury and triggered microgliosis in spinal cord cultures by releasing pro-inflammatory cytokines and free radicals (92) . overall, the aggregated proteins among multiple neurodegenerative diseases including α-synuclein, aβ, and msod1, can initiate a pro-inflammatory responses that lead to a sustained imbalance of neuroinflammation and neuronal loss due to the persistent protein aggregations (76) . in addition, the nerve demyelination was also observed in both sars-cov and sars-cov-2 infected patients, resembling the pathology of ms, which is also tightly associated with neuroinflammation (85) . a similar pattern of elevated proinflammatory factors (il-6, il-8, tnf etc.) was recorded in the csf samples of ms patients with severe gray matter damage (93) . interestingly, other neuropsychiatric diseases such as schizophrenia, bipolar disorders, depression, and among others, are also tightly linked with the neuroinflammatory responses (94) . for instance, the levels of pro-inflammatory mediators including il-6, il-8, il-1β, and tnf-α in the csf or peripheral blood are obviously higher in schizophrenia patients (95, 96) . microglia that release pro-inflammatory factors such as tnf-α can promote the release of glutamate to induce oligodendrocyte dysfunction, resulting in abnormal neural networks in the brain of schizophrenic patients (97) . notably, the altered mental status and neuropsychiatric presentations were recorded in covid-19 patients and other coronavirus infected diseases (98, 99) . above all, the neuroinflammation imbalance toward proinflammatory states shows to be a shared hallmark of various neurological diseases, hence, the cns infiltrated immune mediators in covid-19 patients would probably take part in the chronic pathogenesis process and bring about certain irreversible neuronal impairments. in another hand, given that sars-cov-2 viruses have invaded the cns and can be detected in the csf, their direct effects in the chronic modifications of neural circuits worth further investigations. also, it is intriguing to address whether the infection increases the risk of developing chronic neurodegenerative diseases. the braak hypothesis regarding the etiology of sporadic pd proposes that neurotropic viruses entering the nasal cavity and gastrointestinal tract may trigger lewy pathology, which then spreads to the cns through transneuronal transport, resulting in neurodegeneration in critical brain nuclei (100) . recent studies have confirmed the nasal-brain and gut-brain deliveries in the pathogenesis of pd (101, 102) . interestingly, the symptoms of anosmia and diarrhea of covid-19 patients indicate the nasal and digestive system as the routes of viral infection, which may echo the braak staging evidence that the prodromal or preclinical stage of pd is characterized by olfactory and gastrointestinal symptoms (103) . even though covid-19 respiratory tract infections and cardiovascular events are the main causes of death, the clinical awareness of neurological impairments can reduce the mortality of infected patients. to reduce the risk of those neurological complications, further investigations are needed to determine specific risk factors or protective determinants of neurological events. although recovery from the acute phase of infection can of course be relieved from a public health perspective and help stop the spread of infection, the long-term neurological effects of the disease must also be considered. so far, mounting studies have reported various neurological manifestations, however, the neurotropic potential and chronic neurological effects of the sars-cov-2 virus remains to be fully addressed. currently, the urgent need for treating covid-19 severe patients is still suppressing cytokine storms and balancing the immune system, particularly also in the cns. unfortunately, no specific medicine against covid-19 has been developed till now. apart from using mabs such as tocilizumab, satralizumab, and sarilumab, a recent study reported that dexamethasone, a corticosteroid used widely for its anti-inflammatory and immunosuppressant effects, showed to reduce the mortality by 1/3 among patients receiving invasive mechanical ventilation and by 1/5 among patients receiving oxygen by other means, but had no effects for patients without receiving respiratory support (104) . however, this drug, as mentioned earlier as other corticosteroid drugs, was also under critical concerns of side effects. different drugs work depending on the severity of disease and the timepoint for delivery. adding the need of treating neurological complications, the therapeutic strategy becomes more complicated. it is possible that the anti-neuroinflammatory drugs that used for treating neurodegenerative diseases might be repurposed, due to their capability of crossing the bbb. however, the candidate drugs and doses would be really dependent on each individual, since neurological complications were heterogenous among populations, and importantly, their safety for normal people infected with sars-cov-2 will also await clinical trials to be proven. additionally, another method to alleviate the fierce immune responses is employing the anti-inflammatory and antiapoptotic effects of mesenchymal stem cells (mscs), which can repair lung epithelial cell damage and facilitate alveolar fluid clearance (54) . so far, they are still in clinical trials and are waiting for evaluation. in the other way, 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creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-253201-r6vsa0pw authors: nazari, s.; azari jafari, a.; mirmoeeni, s.; sadeghian, s.; heidari, m. e.; asarzadegan, f.; puormand, s. m.; alikhani, k.; ebadi, h.; fathi, d.; dalvand, s. title: central nervous system manifestations in covid-19 patients: a systematic review and meta-analysis date: 2020-07-22 journal: nan doi: 10.1101/2020.07.21.20158691 sha: doc_id: 253201 cord_uid: r6vsa0pw background: at the end of december 2019, a novel respiratory infection, initially reported in china, known as covid-19 initially reported in china, and later known as covid-19, led to a global pandemic. despite many studies reporting respiratory infections as the primary manifestations of this illness, an increasing number of investigations have focused on the central nervous system (cns) manifestations in covid-19. in this study, we aimed to evaluate the cns presentations in covid-19 patients in an attempt to identify the common cns features and provide a better overview to tackle this new pandemic. methods: in this systematic review and meta-analysis, we searched pubmed, web of science, ovid, embase, scopus, and google scholar. included studies were publications that reported the cns features between january 1st, 2020, to april 20th, 2020. the data of selected studies were screened and extracted independently by four reviewers. extracted data analyzed by using stata statistical software. the study protocol registered with prospero (crd42020184456). results: of 2353 retrieved studies, we selected 64 studies with 11282 patients after screening. most of the studies were conducted in china (58 studies). the most common cns symptom of covid-19 were headache (8.69%, 95%ci: 6.76%-10.82%), dizziness (5.94%, 95%ci: 3.66%-8.22%), and impaired consciousness (1.9%, 95%ci: 1%-2.79%). conclusions: the growing number of studies have reported covid-19, cns presentations as remarkable manifestations that happen. hence, understanding the cns characteristics of covid-19 can help us for better diagnosis and ultimately prevention of worse outcomes. ). at the end of december 2019, a novel respiratory syndrome, currently known as covid-19, was reported in wuhan city, hubei province, china, and the first sign of this 2019 novel coronavirus infection (2019-ncov, covid-19) was pneumonia [1] [2] [3] [4] [5] [6] [7] . this new infection rapidly spread worldwide, and an increasing number of infected cases and deaths have been reported globally [8, 9] . hence, the covid-19 outbreak was officially considered as a public health emergency of international concern (pheic) by the world health organization (who) emergency committee [10, 11] . severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a zoonotic pathogen and can transmit from infected animals (such as bats and snakes) to humans eventually leading to epidemics and pandemics through human-to-human transmission [11, 12] . most cases of covid-19 have shown respiratory symptoms ranging from cough to dyspnea and respiratory failure as well as the typical signs and symptoms of infection such as fever and fatigue [7, [13] [14] [15] . however, a growing number of covid-19 patients are presenting with different combinations of the central nervous system (cns) manifestations [16] [17] [18] . several case reports have indicated the presence of various cns complications, including encephalitis, stroke, meningitis, and encephalopathy in covid-19 patients [19] [20] [21] [22] . furthermore, a large observational study carried out by mao et al. show the prevalence of the cns presentations such as dizziness, headache, impaired consciousness, acute cerebrovascular disease, ataxia, and seizure [16] . therefore, awareness of the different aspects of the short and long-term effects of this virus on the central nervous system could decently guide scientists. in this systematic review and meta-analysis, we assessed the cns manifestations in covid-19 cases. ("wuhan coronavirus" or "wuhan seafood market pneumonia virus" or "covid19 virus" or "covid-19 virus" or "coronavirus disease 2019 virus" or "sars-cov-2" or "sars2" or "2019-ncov" or "2019 novel coronavirus" or "2019-ncov infection" or "2019 novel coronavirus disease" or "2019-ncov disease" or "coronavirus disease-19" or "coronavirus disease 2019" or "2019 novel coronavirus infection" or "covid19" or "covid-19" or "severe acute respiratory syndrome coronavirus 2" or "coronavirus*") and ("manifestation, neurologic" or "neurological manifestations" or "neurologic manifestation" or "neurological manifestation" or "neurologic symptom" or "cns" or "brain" or "neuro*" or "headache" or "dizziness" or "ataxia" or "epilepsy" or "seizure" or "migraine*" or "csf" or "cerebrospinal fluids" or "fluid, cerebrospinal" or "fluids, cerebrospinal" or "cerebro spinal fluid" or "cerebro spinal fluids" or "fluid, cerebro spinal" or "fluids, cerebro spinal" or "spinal fluid, cerebro" or "spinal fluids, cerebro" or "stroke" or "vertigo" or "consciousness" or "impaired consciousness" or "coma" or "cerebrovascular disease" or "acute cerebrovascular disease" or "encephalitis") alone or in combination with or and and operators. the desired data was recorded using an excel spreadsheet form that included the title, first author, year and month of publication, type of study, country, total sample size, the sample size of male and female, study design, demographic characteristics, exposure history, clinical manifestation, cns symptoms, and any reported comorbidity. we assessed the quality of included studies (a.aj. s.m, s.s, s.s, and m.h), based on the nih quality assessment tool for observational cohort and case series studies [24] . this instrument assessed the quality of included studies based on the research question, study population, the participation rate of eligible persons, inclusion and exclusion criteria, sample size justification, analyses, reasonable timeframe, exposure, outcome measures, outcome assessors and, loss to follow-up. data from included studies were extracted for the number of events and total patients to perform a meta-analysis. cochrane's q test and the i 2 index were used to assess heterogeneity among selected studies. heterogeneity was categorized as low (below 25%), moderate (25%-75%), and all analysis were performed using stata statistical software, version 13 (statacorp). as illustrated in (figure 1), a total of 2353 studies were retrieved after a systematic search in the aforementioned databases. (table 2) , the proportion of . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.21.20158691 doi: medrxiv preprint patients with travel history to wuhan, wuhan related exposure, and living in wuhan was 30.43%, 3.83%, and 4.74%, respectively. in addition, the proportion of patients with travel history to other infected areas and contact with patients was 1.17% and 13.33%, respectively. mortality was assessed in 25 studies with a pooled incidence rate of 10.47%. the incidence rate of positive females and males were 46.4% (95% ci: 43%-49.8%) and 49.5% (95% ci: 45.7%-53.3%), respectively. 36.1% (95% ci: 27.9%-44.8%) of infected patients were in the severe, critical, or intensive care unit condition. in addition, the incidence rate of mortality and survival were 10.47% (95% ci: 5%-17.3%) and 81.43% (95% ci: 65.7%-93.2%), respectively. based on the results shown in (table 3 and the highest incidence rate among cns symptoms of covid-19 patients was for headache (8.69% with 95% ci: 6.76%-10.82%), followed by dizziness (5.94%, 95%ci: 3.66%-8.22%), and impaired consciousness (1.9% with 95% ci: 1%-2.79%). table 4 ) shows comorbidities that were reported in 60 studies including 6959 patients. the highest incidence rate in comorbidities were hypertension with 23.54% (95% ci: 19.14%-27.94%), diabetes mellitus (11.68% with 95% ci, 0.98%-13.57%), cardiovascular disease (11.66% with 95% ci: 0.89%-14.35%), and cerebrovascular diseases (3.47% with 95% ci: 2.29%-4.85%). (table 4) reported in a growing number of studies [87] . in addition to the common symptoms in covid-19, several cns symptoms such as headache and impaired consciousness have been observed in infected patients [16] . while most investigated the respiratory symptoms of covid-19, mao et al. specifically examined the prevalence of neurological manifestations ranging from cns to peripheral nervous system (pns) and neuromuscular symptoms in an observational study on covid-19 patients [16] . they demonstrated cns presentations ranging from dizziness and headache to impaired consciousness, acute cerebrovascular disease, ataxia, and seizure [16] . based on the possible neuroinvasive potential of covid-19, in this systematic review and meta-analysis, we analyzed those evidence indicating the involvement of cns. we assessed 11687 covid-19 adult patients from six countries. we reported that covid-19 patients commonly showed cns symptoms, including headache, dizziness, and impaired consciousness. headache (8.69%) were the most common cns symptoms, followed by dizziness (5.94%) and impaired consciousness (1.9%). there are two main routes of cns entry of covid-19 (hematogenous and peripheral nerves route) leading to cns infection and inducing various symptoms such as meningitis and encephalitis. in the hematogenous route, the virus infecting respiratory tracts can reach the cns through the bloodstream via overcoming a strict obstacle known as the blood-brain barrier (bbb) [18, [88] [89] [90] [91] [92] [93] . they also may enter the cns through circumventricular organs, those cns organs lacking the bbb [94] . the second route, a peripheral nerve, can provide the virus with a retrograde route in to access the cns via an axonal transport machinery [18, [88] [89] [90] [91] [92] [93] . in accordance with this finding, some previous studies on other types of coronaviruses indicating that coronaviruses can reach the brain via cranial nerves (e.g., olfactory, trigeminal nerve terminals in the nasal cavity) [88, [95] [96] [97] . such a neuroinvasive propensity is supported by reporting of patients exhibiting smell impairment, as a hallmark of covid-19 infection due to the involvement of the olfactory nerve [16, 87] . the covid-19 infection mechanism requires the virus attaching to its receptor called angiotensin-converting enzyme 2 (ace2) expressed in various tissues ranging from endothelial cells of the cardiovascular system, airway epithelia, kidney cells, small intestine, and lung parenchyma [98] [99] [100] . there exists a wealth of evidence that supports the expression and distribution of the ace2 in the cns [100] [101] [102] [103] [104] [105] [106] . hence, ace2 may be a potential target of covid-19 upon the entrance into the cns, triggering its effects on cns tissue [92] . the . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . presence of the virus in the central nervous system is also supported by some evidence reporting covid-19 in the csf of the infected cases [19, 21] . in our meta-analysis, the mortality rate of covid-19 cases with at least one cns symptom was 10.47%, which is much higher than the mortality rate of the general infected population [107] . such a mortality rate can indicate the importance of careful monitoring of cns manifestations in covid-19 patients. moreover, in mao's study, the prevalence of cns manifestations were higher in severe cases of the illness associated with higher mortality [16] . although, this can be due to the effect of covid-19 on the brain stem and suppression of the cardiorespiratory control centers causing respiratory failure and death [108] , other possible reasons such as cytokine storm and hypoxia have been suggested to explain the high mortality rate in cases with cns manifestations. cytokine storm as an immune system response during covid-19 infection could enhance the permeability of the blood brain-barrier (bbb) [109, 110] . infection of airway tissues by covid-19 in severe infection leads to impaired gas exchange, subsequently causing cns hypoxia resulting in neural dysfunction [111] . this hypoxic condition associated with severe infection disrupts the bbb through elevation of some factors like nitric oxide (no) and inflammatory cytokines [112] . more precisely, all of these factors mentioned above may contribute to making the bbb more permeable to the virus. therefore, in the severe condition of infection, covid-19 easily enters the cns via disrupted bbb and puts the brain at risk leading to the manifestation of cns features [113] [114] [115] [116] . furthermore, recent studies have shown that covid-19 can accelerate the formation of the blood clot in the blood vessels, increasing the risk of cerebrovascular diseases in covid-19 patients [117, 118] . hence, because the brain is nourished by a network of blood vessels, this could be indicative of the importance of cerebral vasculature investigations on the cns symptoms in the covid-19 infection. in a nutshell, attention to the cns aspects of covid-19 infection has outstanding benefits for clinician's understanding of a very serious complication of this infection. at this point in time, researchers have mainly focused on finding medicinal treatments for respiratory symptoms of covid-19. however, it is necessary to investigate the various cns manifestations of covid-19 since they are associated with increased severity and mortality [16] . not only respiratory system dysfunction, but also impairment of respiratory control centers in the cns (brain stem) can induce acute respiratory failure [108, 119] . therefore, considering all effective factors, it can provide clinicians to choose the best way in an attempt to manage this pandemic more efficiently. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.21.20158691 doi: medrxiv preprint there are several limitations in our systematic review and meta-analysis. since in this ongoing pandemic, most of the investigations have conducted on typical signs and symptoms of covid-19. thus the number of studies on the atypical complications of covid-19, such as cns presentations are partially low. moreover, there exist many covid-19 preprint papers that have not yet undergone peer review. additionally, five studies included in our meta-analysis reported headache and/or dizziness as one symptom in covid-19 cases. because we were not sure that headache and/or dizziness is resulted from headache or is a consequence of the dizziness, it would be challenging to categorize headache and/or dizziness in the subgroup of dizziness or headache. hence, in our meta-analysis, it was not reported as a cns manifestation and are implied as a separate symptom (table 3). covid-19 is a global problem that currently affects millions of people. this highly pathogenic virus can affect various parts of the human body. although the respiratory tract has been mainly targeted by covid-19, the central nervous system can be affected significantly. in addition, patients with more severe illness showed more cns symptoms, which may bring on worsen clinical conditions. this study achieved an important estimation for the incidence of neurological abbreviation . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.21.20158691 doi: medrxiv preprint cns: central nervous system; covid-19: coronavirus disease 2019; sars-cov-2: severe acute respiratory syndrome coronavirus 2; pheic: public health emergency of international concern; who: world health organization; prisma: preferred reporting items for systematic reviews and meta-analyses; pns: peripheral nervous system; bbb: blood brain barrier; ace2: angiotensin-converting enzyme 2; ci: confidence interval . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.21.20158691 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.21.20158691 doi: medrxiv preprint figure 1 . the process of surveying, screening, and selecting the articles for this systematic review and meta-analysis based on prisma guideline . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . created with biorender.com) . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 22, 2020. . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july 22, 2020. . https://doi.org/10.1101/2020.07.21.20158691 doi: medrxiv preprint . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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barrier in hypoxic-ischemic conditions nervous system involvement after infection with covid-19 and other coronaviruses specific eeg encephalopathy pattern in sars-cov-2 patients brain mri findings in patients in the intensive care unit with covid-19 infection cerebrovascular and neurological dysfunction under the threat of covid-19: is there a comorbid role for smoking and vaping? altered covid-19 receptor ace2 expression in a higher risk group for cerebrovascular disease and ischemic stroke covid-19-related stroke central neurogenic respiratory failure: a challenging diagnosis retrospective china, wuhan 41 11 / 30 good hsih case series india key: cord-294812-nnlzwaf1 authors: desforges, marc; le coupanec, alain; brison, élodie; meessen-pinard, mathieu; talbot, pierre j. title: neuroinvasive and neurotropic human respiratory coronaviruses: potential neurovirulent agents in humans date: 2014-03-12 journal: infectious diseases and nanomedicine i doi: 10.1007/978-81-322-1777-0_6 sha: doc_id: 294812 cord_uid: nnlzwaf1 in humans, viral infections of the respiratory tract are a leading cause of morbidity and mortality worldwide. several recognized respiratory viral agents have a neuroinvasive capacity since they can spread from the respiratory tract to the central nervous system (cns). once there, infection of cns cells (neurotropism) could lead to human health problems, such as encephalitis and long-term neurological diseases. among the various respiratory viruses, coronaviruses are important pathogens of humans and animals. human coronaviruses (hcov) usually infect the upper respiratory tract, where they are mainly associated with common colds. however, in more vulnerable populations, such as newborns, infants, the elderly, and immune-compromised individuals, they can also affect the lower respiratory tract, leading to pneumonia, exacerbations of asthma, respiratory distress syndrome, or even severe acute respiratory syndrome (sars). the respiratory involvement of hcov has been clearly established since the 1960s. in addition, for almost three decades now, the scientific literature has also demonstrated that hcov are neuroinvasive and neurotropic and could induce an overactivation of the immune system, in part by participating in the activation of autoreactive immune cells that could be associated with autoimmunity in susceptible individuals. furthermore, it was shown that in the murine cns, neurons are the main target of infection, which causes these essential cells to undergo degeneration and eventually die by some form of programmed cell death after virus infection. moreover, it appears that the viral surface glycoprotein (s) represents an important factor in the neurodegenerative process. given all these properties, it has been suggested that these recognized human respiratory pathogens could be associated with the triggering or the exacerbation of neurological diseases for which the etiology remains unknown or poorly understood. viral infections of the respiratory tract represent a major problem for human and animal health around the world. these respiratory infections induce the most common illnesses [1] and are a leading cause of morbidity and mortality in humans worldwide, especially children, the elderly, and immune-compromised individuals [2] [3] [4] . on a statistical basis, children represent a highly susceptible group, as they may experience multiple infections each year until they reach the age of 10 [5] . respiratory viral infections also represent an economic threat for agriculture, especially in cattle, swine, and poultry production. the idea that viruses can cause respiratory tract infections has been demonstrated since the early 1930s [2] . nevertheless, with the help of modern diagnostic tools, a significant number of new respiratory viruses have been discovered since the beginning of the twenty-first century and it is estimated that there are about 200 antigenically distinct viruses able to cause infection of the respiratory tract, especially in infants and children [6] . in fact, it is now believed that viruses cause 95 % of respiratory diseases in children and infants, and about 30-40 % in the elderly [2] . although the airway epithelial cells in the respiratory tract represent a first line of defense against pathogens, they can be targeted by several different respiratory viruses that can infect them as a way to penetrate the human host. several infections are self-limited and the infection remains local as the virus is cleared by the immune system in the respiratory tract with minimal clinical consequences. however, in some circumstances, viruses can avoid the immune response and cause more severe respiratory diseases [1] or even spread to other tissues, including the central nervous system (cns), where they could induce other types of pathologies [7] . neuroinvasive and neurotropic viruses: associated neuropathologies induce or participate in the induction of neurological diseases (neurovirulence) [8] . in humans, a long list of viruses possess these ''neuroproperties'' and infection often leads to acute encephalitis, which can be fatal depending on virus tropism [9] . rabies virus [10] , herpes simplex virus (hsv) [11] , and arthropod-borne flaviviruses [12] can induce encephalitis in humans. chronic human neurological diseases may also be linked to viral infection. in acquired immunodeficiency syndrome (aids) dementia and related disorders, human immunodeficiency virus (hiv) induces neurodegeneration [13] , which can result in motor dysfunctions and possibly cognitive impairments [14] . progressive multifocal leukoencephalopathy (pml) is a human demyelinating disease [15] where prolonged immunosuppression leads to reactivation of latent polyoma jc virus (jcv) [16] . subacute sclerosing panencephalitis (sspe), a progressive fatal neurological disease, is caused by cns persistence of measles virus [17] . human t-cell lymphotrophic virus (htlv-1) causes progressive tropical spastic paraperesis/htlv-1-associated myelopathy (ptsp/ ham) in 1-2 % of infected individuals [18] and hsv-1 and human herpes virus 6 (hhv-6) were proposed to cause or exacerbate alzheimer's disease (ad) [19] . as mentioned above, respiratory viral agents also have the capacity to invade the cns where they will infect resident cells and potentially be neurovirulent in inducing a neuropathology. several of these recognized respiratory pathogens can gain access to the cns, where they can eventually cause health problems in humans. respiratory syncytial virus (rsv), the most common pathogen to cause lower respiratory tract infection in infants worldwide [20] , is one such neuroinvasive respiratory agent that has been detected in the cerebrospinal fluid (csf) of patients [21, 22] and that was associated with convulsions [23] , febrile seizures, and encephalitis [24] . furthermore, it was recently shown that rsv can spread from the airways to the cns in mice after intranasal inoculation, and that it induces behavioral and cognitive impairments [25] . measles virus (mv) from the paramyxoviridae family is another common virus that causes a disease of the respiratory airways associated with fever, cough, and congestion. however, mv infection also induces other symptoms including a characteristic rash and koplik's spots [26] in the oral mucosa. one of two most important sequelae associated with mv infection is immunosuppression, which facilitates infection by opportunistic bacteria or parasites that can lead to pneumonia or diarrhea. a second type of rare but significant sequelae is long-term cns disease [26] . postinfectious encephalomyelitis (pie) or acute disseminated encephalomyelitis (adem) occurs in 1 of 1,000 measles cases in children and adolescents. measles inclusion body encephalitis (mibe) is a second cns complication that can arise after a mv infection in immune-compromised patients. finally, subacute sclerosing panencephalitis (sspe) is a third form of cns disease associated with mv infection. it is a slow progressive neurological disease that appears 6-10 years after infection in about 4-11 cases per 100,000 cases of measles (for review see [27] ). hendra virus (hev) and nipah virus (niv) are two members of the genus henipavirus (hnv), also from the paramyxoviridae family. they represent important emerging viruses that were discovered in the 1990s [28] . fruit bats are the natural reservoir and both viruses can be transmitted to humans from intermediate reservoirs such as pigs and horses [29] . the hnv causes acute and severe respiratory disease in humans, including necrotizing alveolitis with hemorrhage, pulmonary edema, and pneumonia [28] . neurological signs of pathology include confusion, motor deficits, seizures, febrile encephalitic syndrome, and reduced level of consciousness. moreover, neuropsychiatric sequelae have been reported but it is not known whether postinfectious encephalomyelitis occurs following infection [29] . the use of animal models showed that the main route of entry into the cns is the olfactory nerve [30] . influenza virus comes in three types: a, b, and c. types a and b cause the flu syndrome, characterized by chills, fever, headache, sore throat, and muscle pains [31] , and are responsible for seasonal epidemics that affect 3-5 million humans, of which 250,000-500,000 cases are lethal each year [32] . although influenza type c virus is less frequent in humans, it is also distributed around the world where it mainly infects infants and young children; most of the time infection results in mild upper respiratory tract illnesses. however, complications associated with lower respiratory tract infection do occur in children under 2 years of age [33, 34] . human influenza a virus is of particular interest because of its capacity to recombine and rearrange with its avian and porcine counterparts to generate new emerging viruses that are introduced into the human population and that may lead to pandemic outbreaks associated with significant morbidity and mortality, as it was observed at least four times during the twentieth century and already once in the twenty-first century [32, 35] . influenza virus type a is highly contagious and, even though most infections are localized to the upper respiratory tract, some more severe cases may result in pneumonia [36] and even complications involving the cns [37] . several studies have shown that influenza a can be associated with encephalitis, reye's syndrome, febrile seizure, acute necrotizing encephalopathy, and possibly acute disseminated encephalomyelitis (adem) in humans [31, [38] [39] [40] [41] . making use of murine models, it has also been shown that influenza a virus could reach the cns through the olfactory nerve route and alter hippocampal morphology or expression of synaptic regulatory genes while impairing cognition and emotional behavior [42, 43] . influenza a virus was also described as a factor which may increase the risk of parkinson's disease (pd) [37] . among the different respiratory viruses, coronaviruses are important pathogens of humans and animals, causing a range of symptoms, including in the cns. coronaviruses, a family of enveloped positive-stranded rna viruses with a characteristic crown-shaped appearance, are widespread in nature and can infect several different species [44] , in which they cause mainly respiratory and enteric pathologies, with neurotropic and neuroinvasive properties in various hosts including humans, cats, pigs, rodents, and fowl [45] [46] [47] [48] . they are taxonomically grouped in the family coronaviridae, within the order nidovirales, and they are classified within four different genera, namely alpha, beta gamma, and deltacoronaviruses [49, 50] . they form a group of enveloped viruses that have the largest genome among rna viruses. this non-segmented 30 kb positive single-stranded polyadenylated rna possesses 4 or 5 genes encoding structural proteins (s, e, m, n; he for the genus betacoronaviruses) and several genes encoding non-structural proteins. the spike protein (s) is a type-1 glycosylated transmembrane protein responsible for the recognition of the cellular receptor used by the virus to infect a susceptible cell. the envelope (e) protein is a small structural protein anchored in the viral envelope, and which has a role in the assembly of the virion; it appears to be responsible for the adequate curving of the viral envelope. the membrane (m) protein possesses three transmembrane domains and interacts with all the other structural viral proteins and therefore helps to shape and maintain the virion structure. the nucleocapsid (n) protein associates with the viral genome and plays an essential role in encapsidating it into a helical nucleocapsid within the viral particle. the hemagglutinin-esterase (he) is only present in most species of the betacoronavirus genus. like the s protein, it is a transmembrane protein which forms homodimers and which interacts with different types of sialic acid, associated with an apparent role in hemagglutination. it also possesses an acetylesterase function, which may be important early during infection or during the release of viral particles from the infected cells at the end of the replication cycle of the betacoronaviruses [48] . as mentioned above, coronaviruses are widespread in nature and can infect several different animal species, in which very often they are both respiratory and enteric pathogens. although several animal coronaviruses induce severe enteric diseases, they also often reach the respiratory tract, where they can be associated with mild to severe diseases. some examples of coronaviruses that can infect livestock or poultry, in which they have a respiratory tropism, are transmissible gastrointestinal virus (tgev) and its associated s protein deletion mutant: porcine respiratory coronavirus (prcov) and porcine hemagglutinating encephalitis virus (phev), which infect swine; bovine coronavirus (bcov), which infects cattle; and infectious bronchitis virus (ibv), which infects chicken [51] . several of these animal coronaviruses cause severe economic burden to poultry, swine, and cattle industries worldwide. other animal coronaviruses that have a respiratory tropism are feline coronavirus (fcov); canine respiratory coronavirus (crcov); and bat cov, which infects the bat species miniopterus [51] . among the respiratory animal coronaviruses, fcov and phev have been associated with neurological diseases. neurological symptoms may occur in cats infected with a highly virulent fcov variant, designated fipv [52, 53] . neurological disease appears partially immune-mediated and may result in uncontrolled secretion of cytokines [54] that leads to diverse pathological manifestations including meningitis [55] and even spinal cord involvement [56] . furthermore, there is often a small amount of infectious fcov present in brain tissue [52] . the phev was isolated from the brains of suckling pigs suffering from encephalomyelitis several years ago in canada [57] and the disease could be reproduced experimentally in piglets following intranasal inoculation [58] . after oronasal infection, it was shown that the virus first infects epithelial cells of the respiratory tract and small intestine and, using retrograde neuronal spreading via peripheral nerves, was able to enter the cns [59] . more recently, the neuroinvasiveness and neurotropism of the virus were again demonstrated in a murine model, where phev induced a poor inflammatory reaction in the cns and infected cells showed no cytopathological changes [60] . the last, but not the least, example of another animal coronavirus, i.e., neuroinvasive, neurotropic, and neurovirulent, is mouse hepatitis virus (mhv). this virus is a subspecies of the species murine coronavirus (mucov) [49] , which represents a collection of viral strains with different tropism, including respiratory for the mhv-1 strain, and neurotropic for the mhv-jhm and mhv-a59 strains. in susceptible mice, these two latter strains of mhv induce a demyelinating disease that resemble human multiple sclerosis (ms) [61] . coronaviruses are all molecularly related in structure and mode of replication [62] . therefore, the close structural and biological relatedness of hcov to the neurotropic animal coronaviruses has led to speculation about possible involvement of hcov in neurological diseases. till now, no clear specific association has ever been made between coronaviruses and any known human neuropathology. however, hcov-229e and hcov-oc43 [63] [64] [65] [66] , as well as sars-cov [67, 68] , were shown to be neuroinvasive and neurotropic. human coronaviruses (hcov) usually infect the upper respiratory tract, where they are mainly associated with common colds. however, in more vulnerable populations such as newborns, infants, the elderly, and immune-compromised individuals, they can also reach the lower respiratory tract, where they could instead be associated with pneumonia, exacerbations of asthma, respiratory distress syndrome or even severe acute respiratory syndrome (sars) [44, 69] . ever since their discovery in the late 1960s, coronaviruses able to infect humans were neglected by the international medical community. however, when a variant emerged from animals in southeast asia to cause the first pandemic of the twentyfirst century: the severe acute respiratory syndrome or sars, these apparently innocuous viruses suddenly became ''more interesting.'' indeed, the 2002-2003 sars pandemic was caused by a coronavirus variant that appears to have emerged from a bat reservoir to infect palm civets, sold live in open markets, which served as intermediate reservoirs before crossing into humans. moreover in the fall of 2012, 10 years after the sars episode, the world health organization (who) warned the international medical community, that a sars-like disease affected individuals that traveled from the arabian peninsula to the united kingdom, which may indicate a possible resurgence of sars. however, using molecular sequencing, it was rapidly shown that this new respiratory coronavirus was genetically different from sars-cov, underlining the importance of a molecular approach in making a viral diagnostic. it is now recognized that the new epidemic is caused by a new coronavirus from the genus betacoronavirus that was first named hcov-emc (for human coronavirus-erasmus medical center), human betacoronavirus 2c, and ncov or ncov (for novel coronavirus), and that is now known under the official name mers-cov: the middle-east respiratory syndrome coronavirus [50] . as of august 30, 2013, who indicated that the mers-cov has spread to eight different countries, where 108 laboratory-confirmed cases of individuals have been identified as infected by the mers-cov, with 50 deaths [70] . although coronaviruses that infect humans are mainly recognized respiratory pathogens, as they usually first target respiratory and mucosal surfaces, infectious particles, antigens or rna, were detected in other tissues than the respiratory tract, including the cns. the detection of hcov rna in human brain samples clearly demonstrates that these respiratory pathogens are naturally neuroinvasive in humans and suggests that they establish a persistent infection in human cns [63] . furthermore, we have shown that these viruses are able to establish a persistent infection in human cells representative of the cns [64, 65] and that hcov-oc43 rna could be detected for at least a year in the cns of infected mice that survived the virus-induced acute encephalitis [71] . therefore, an apparently innocuous human respiratory pathogen may persist in the human cns as a component of what is proposed to be a «viral flora» of the brain, like hsv in a large proportion of the population. it would therefore be possible that such a persistent infection may become a factor or cofactor of neuropathogenesis in genetically or otherwise predisposed individuals. human coronaviruses were first isolated in the mid-1960s from patients with upper respiratory tract disease [72] . until the end of the twentieth century, only two serological groups, represented by strains oc43 and 229e, were known to infect humans and they were recognized as respiratory pathogens responsible for up to 30 % of common colds [72] . over the last 10 years, sars has generated renewed interest in coronaviruses that led to the discovery of new coronaviruses that can infect humans: sars-cov [73, 74] , hcov-nl63 [75] , hcov-hku1 [76] and mers-cov [77] . among these six coronaviruses, at least hcov-229e and hcov-oc43, as well as sars-cov, possess neuroinvasive properties as viral rna [63] or infectious virus [67, 68] can be detected in human brains. to our knowledge, there exist no reports on the detection of hcov-hku1, hcov-nl63, and mers-cov in the human cns. on the other hand, neurological symptoms have been described in association with both hcov-hku1 and hcov-nl63 [78] and a recent report, which evaluated mers-cov cell tropism, suggest that, among several cell lines representative of different tissues and organs, this virus seems to be able to infect the neuron-committed human cell line nt2 [79] . viruses may enter the cns through two distinct routes: hematogenous dissemination or neuronal retrograde dissemination. hematogenous spread involves the presence of a given virus in the bloodstream and retrograde viral spread toward the cns occurs when a given virus infects neurons in the periphery and uses the transport machinery within those cells to gain access to the cns [80] . in order to be neuroinvasive, viruses such as hcov-229e, hcov-oc43, and sars-cov may use both entry routes from the periphery. the hematogenous route involves the presence of a given virus in the blood, where it can either remain free for a period of time before it can infect the endothelial cells of the blood-brain-barrier (bbb), or infect leukocytes that will become some sort of viral reservoir for dissemination to other sites. both situations occur during hiv infection of the cns. indeed, hiv-infected leukocytes that migrate through the bbb (called the trojan horse [81] ), is one of the route of spread, and direct infection of endothelial cells of the bbb is also possible even though the viral replication is at a low level [82] . infection of human monocytes/macrophages by hcov-229e and hcov-oc43 was reported [83, 84] and infection by hcov-229e of murine dentritic cells expressing the human aminopeptidase n [85] suggests that human coronaviruses may use these cells to disseminate to other tissues, including the cns, where they could be associated with other type of pathologies. sars-cov was also shown to be able to infect human monocytes/macrophages [68, 86] . moreover, monocytederived dendritic cells are also susceptible to infection by sars-cov [87] . human primary monocytes are activated following infection by hcov-229e [83] . since they eventually become macrophages as they invade tissues, this activation suggests that hcov-229e-infected monocytes would serve to facilitate their passage toward other tissues including the cns, especially in immunecompromised individuals, as this was observed for murine cytomegalovirus (mcmv) [88] . the fact that hcov-229e could only infect partially immunecompromised transgenic mice [89] suggests that hcov-229e could take advantage of an immune-suppressed environment and disseminate to the cns within susceptible individuals. the establishment of a persistent infection in a human leukocytic cell line [83] is also consistent with the possibility that monocytes/ macrophages serve as a reservoir and vector for this neuroinvasive hcov [63] . the sars-cov also infects monocytes/macrophages [68, 86] and dendritic cells, in which it modulates innate immunity [87] . these cells could also serve as a reservoir for the virus to reach and maintain itself in the cns. our results indicate that hcov could also infect human endothelial cells of the bbb in culture (unpublished data) and it has been speculated that sars-cov could do the same after viremia [90] . therefore, the neuroinvasive hcov could use the hematogenous route to penetrate into the cns. the second form of any viral spread toward cns is through neuronal dissemination, where a given virus infects neurons in periphery and uses the machinery of active transport within those cells in order to gain access to the cns [80] . after an intranasal infection, both hcov-oc43 [91] and sars-cov [92] were shown to infect the lungs in mice and to be neuroinvasive as hcov-oc43 [93, 94] and sars-cov [95] were detected in the cns of susceptible mice. therefore, these two coronaviruses may use both the hematogenous and transneuronal route toward the cns. furthermore, as shown in fig. 1 , once in the brain, hcov-oc43 can disseminate from the olfactory bulb to the cortex and we have previously shown that it could also reach the medulla, while the cerebellum remained almost uninfected. the hippocampus represents another specific structure infected by hcov-oc43 in the brain (fig. 1) and once in this region of the brain, the virus appears to spread by a transneuronal route before it eventually reaches the spinal cord [48] . neuroinvasive viruses can damage the cns as a result of misdirected host immune responses (virus-induced neuroimmunopathology) and/or viral replication, which directly induces damage to cns cells (virus-induced neuropathology). in acute encephalitis, viral replication occurs in the brain tissue itself, possibly causing destructive lesions of the gray matter [47] . as mentioned above (section ''neuroinvasive and neurotropic viruses: associated neuropathologies''), chronic human neurological diseases may also be linked to viral infection. however, in several cases of these chronic diseases, it is difficult to ascertain a role for any given virus, in part due to the difficulty of establishing the time at which these viruses become involved. also, the four koch's postulates for disease induction dictate whether a particular infectious agent causes a specific disease [96] . however, several viral infections, especially slow viral infections related to diseases that are rare manifestations of an infection, represent situations where koch's postulate should be modified to better adapt to the situation [97, 98] . a series of new criteria, adapted from sir austin bradford hill's criteria for causation [98] , has been elaborated by giovannoni and collaborators [99] and should replace koch's postulates when one wants to evaluate the relevance of any given virus in relation to ms etiology [99] or any other long-term human neurological diseases potentially related to a viral infection as well, including infection by human coronaviruses. the presence of hcov-229e and hcov-oc43 was detected in various neurological diseases in humans, including pd and ms [63] and adem [100] . multiple sclerosis truly represents a human neurological disease where an infectious agent or agents may play a triggering role, with viruses the most likely culprit in genetically predisposed individuals [101] . there is a presumption that several neurotropic viruses could be involved in ms pathogenesis but that they may do so through similar direct and/or indirect mechanisms [102] [103] [104] [105] . however, research has not yet led to a direct link to any specific virus or other microbes with ms. association of coronaviruses with ms was suggested in numerous reports that are reviewed elsewhere [48] . one of these reports demonstrated a significant association of colds with ms exacerbations and a significant association of hcov-229e infection in ms patients [106] and another report on the association of viral infections and ms [107] commented that seasonal hcov infection patterns do fit the observed occurrence of ms exacerbations. furthermore, the case of these human coronaviruses in the cns may represent a new example where the traditional koch's postulates should be replaced by the previously cited adapted hill's criteria [99] . more than a decade ago, we experimentally confirmed that hcov-oc43 and hcov-229e were naturally neuroinvasive in humans. although viruses were also detected in some control brains, there was a significantly higher prevalence of hcov-oc43 in brains of ms patients [63] . moreover, these data, in association with the observation that autoreactive t-cells recognized both viral and myelin antigens in ms patients but not in controls [108, 109] during infection by hcov-oc43 and hcov-229e, suggest that the immune response may participate in the induction or exacerbation of neuropathologies such as ms in genetically or otherwise susceptible individuals (data summarized in table 1 ). furthermore, even though the use of the immunosuppressive drug cyclosporin a in hcov-oc43-infected mice resulted in a faster onset of encephalitis, suggesting a role for t-cells in viral clearance and survival with no related immunopathology [71] , it was shown that in recombination activation gene (rag) knock-out mice, hcov-oc43-induced encephalitis could be partially mediated by the t-cell response to infection [93] . the participation of different types of t-cells has been shown to play a significant role in the demyelinating neurological disease induced by the adapted from arbour et al. [63] and boucher et al. [108] numbers in the upper portion indicate the number of individuals positive for viral rna and numbers in parenthesis indicate the total number of individuals tested numbers in the lower portion indicate the number of t-cell clones obtained. monospecific describes clones that react against a single antigen and cross reactive describes clones that react both with hcov and myelin antigens murine cov, in particular for strain mhv-jhm [110] , which represents the murine counterpart of hcov-oc43. more recently, making use of another mouse model, we showed that hcov-oc43 induced immune cell infiltration and cytokine production in mouse cns and that this response was significantly higher after infection by variants which harbor point mutations in the viral surface glycoprotein (s) [111] . importantly, these s point mutations were acquired after persistent infections of human neural cell lines [112] . moreover, we also showed that infection by the s mutants is linked to glutamate excitotoxicity [113] . therefore, as this increase in cytokine production may induce damages to the neurons [114] that can be associated with problems in glutamate homeostasis, which in the end may create glutamate excitotoxicity [115] , it can contribute to neuronal degeneration associated with hind-limb paralysis, as illustrated in fig. 2 , and possible demyelination [111, 113] . the outcome of the observed degeneration of neurons may eventually lead to the death of these essential cells. as previously mentioned, infection of neurons by itself may also participate in the process of cell death by directly generating a cytotoxic insult related to viral replication and/or to the induction of different cell death pathways. when present in the murine cns, hcov-oc43 infects neurons in several different regions of the brain [48] (fig. 1 ) before reaching the spinal cord. infection of these essential cells induces their degeneration [111, 113] , as observed by aberrant state of neurofilament phosphorylation, a situation that often leads to cell death and that could be directly induced by viral replication. furthermore, using two model cell lines of differentiated human neurons, we demonstrated that programmed cell death (pcd) was induced after hcov-oc43 infection [116, 117] and that the inhibition of viral replication was also in direct correlation with increased cell survival, suggesting that infection and production of new infectious viruses directly participate in the process of degeneration and eventual death of neurons. our results indicate that the underlying mechanisms appear to involve different cellular factors and pathways, including caspase-independent apoptosis, parthanatos, and necroptosis, three forms of programmed cell death (pcd), which are reviewed elsewhere by the nomenclature committee on cell death (nccd) [118] . these cell death pathways can act separately but may also interact in response to a stimulus (including a viral infection), as they share some of the cellular factors involved in the overall process that leads to cell death and that often converges toward mitochondria [118] . figure 3 is a tentative representation of the various pathways and cellular factors involved during hcov-oc43-induced pcd of infected neurons. it is based on our data [111, 113, 116, 117] and on the scientific literature that describes some molecular pathways (parthanatos, necroptosis and apoptosis) and cellular factors, including calcium overload, endoplasmic reticulum (er) stress, excitotoxicity, poly(adp-ribose) polymerase (parp), calpain and oxidative stress related to the formation of reactive oxygen species (ros) involved in mitochondrial dysfunction, and eventual neurodegeneration and neuronal cell death [118] [119] [120] . virus-cell interaction is always important in the regulation of cell response to infection. for hcov-oc43, we clearly showed that the viral s glycoprotein is an important factor of neurovirulence and neurodegeneration of infected cells [111, 113, 116] . this was similarly shown for coronavirus strains mhv-a59 and mhv-jhm, which represent subspecies of the mucov species, the murine counterpart of hcov-oc43 as they are both members of the betacoronavirus genus. indeed, several reports and reviews have, over the years, described that s protein of this neuroinvasive and neurotropic murine coronavirus is a major factor associated with neurovirulence during encephalitis and the eventual demyelinating disease in susceptible mice [121, 122] . our more recent data also demonstrated that the he protein is an important factor for the production of infectious hcov-oc43, suggesting an attenuation of the eventual spread of viruses deficient in fully active he protein into the cns [123] . therefore, as the infection of neuronal cells apparently directly participate in the induction of neuronal death, by abrogating the production of infectious virus, the he protein of hcov-oc43 could play a role in neurovirulence of hcov-oc43, like it does for mhv [124] . (1) hallmarks of apoptosis, including relocalization of the activated pro-apoptotic protein bax (bcl-2 associated protein x) from the cytosol to the mitochondrial membrane, cytochrome c release from mitochondria toward the cytosol, dna fragmentation, and activation of caspases -3 and -9, are observed during infection of human neurons. however, using a pan-caspase inhibitor (z-vad-fmk), cell death is not abrogated after infection, suggesting a caspase-independent type of apoptosis. (2) relocalization of the mitochondrial protein aif (apoptosis-inducing factor) toward the nucleus (taif) is observed after infection and participates in dna fragmentation in conjunction with cypa (cyclophilin a) and histone h2ax. the aif is known to be activated during caspase-independent apoptosis. however, aif is also involved in parthanatos, another form of pcd potentially associated with neurodegeneration. as they are synthesized by the poly(adp-ribose) polymerase (parp) during a neuronal stress, including during hcov-oc43 infection, polymers of adpribose (par) may relocalize toward mitochondria and participate in the activation and relocalization of aif toward the cytosol before it reaches the nucleus. cyclophilin d (cypd) inhibition decreases aif release from mitochondria and abrogates cell death induced by infection. (3) aif release from mitochondria may be induced through its truncation (taif) by activated calpain, which is usually activated by a rise in the mitochondrial calcium concentration. (4) this increase in calcium concentration may be linked with either an important entry from the extracellular milieu (for instance during excitotoxicity) or with a release of calcium from the endoplasmic reticulum (er) following induction of er stress. both situations are probably taking place after infection of neurons by hcov-oc43. the increase in calcium concentration in mitochondria may also induce production of reactive oxygen species (ros) that can be harmful for mitochondria and hence neurons. (5) the presence during infection of an inhibitor (nec-1) of the receptor interacting protein kinase-1 (rip-1), significantly increases cell survival and partially abrogates viral replication, suggesting that necroptosis, a third form of pcd which involves rip-1 and rip-3 downstream of the tumor necrosing factor (tnf) receptor family (in the form of the death-inducing signaling complex (disc)), may play a role in hcov-oc43-induced neuronal death. solid arrows indicate experimental data and dashed arrows represent possible pathways based on the current literature (see text for details) as mentioned above, sars-cov is also neuroinvasive and neurotropic in humans [67, 68] and it could therefore be associated with the development of a neurological disease. furthermore, the involvement of sars-cov in cns infections was underscored by the findings that made use of transgenic mouse models expressing the human angiotensin-converting enzyme-2 (the cellular receptor used by sars-cov to infect susceptible cells). indeed, using these mice, it was shown that sars-cov could invade the cns after an intranasal infection primarily through the olfactory bulb [95] or even after an intraperitoneal infection [125] , with concomitant neuronal loss [95, 125] ; a phenomenon that can eventually lead to neurological problems. the presence of coronaviruses in the human central nervous system is now a recognized fact as they appear to be part of a viral flora of the brain, with potential neuropathological consequences in genetically or otherwise susceptible individuals, with or without additional environmental insults. knowledge of mechanisms and consequences of virus interactions with the nervous system is essential to better understand potentially pathological consequences and design intervention strategies that are appropriate to encephalitis or exacerbations of other types of neurological diseases for which a given virus is involved. in that regard, hill's criteria adapted by giovannoni 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induce acute inflammation and chronic demyelination in the spinal cord and optic nerves mediated by axonal spread following intracranial inoculation in mice, with pathologic features similar to the human demyelinating disease multiple sclerosis. spinal cord demyelination is also induced following intranasal inoculation with neurotropic mhv strains, however much higher viral doses are required as compared to intracranial inoculation. recently, it was shown that intranasal administration of low concentrations of proteins leads to significant, rapid accumulation of protein in the optic nerve and in the eye, with only low levels reaching spinal cord and other brain regions. thus, we examined whether intranasal inoculation with mhv at doses equivalent to those given intracranially could induce optic neuritis—inflammation, demyelination and loss of retinal ganglion cells (rgcs) in the optic nerve with or without inducing spinal cord demyelination. four week old male c57bl/6j mice were inoculated intracranially with the recombinant demyelinating strain rsa59, or intranasally with rsa59 or the non-demyelinating strain rsmhv2 as control. one month post-inoculation, mice inoculated intracranially with rsa59 had significant myelin loss in both spinal cord and optic nerves, with significant loss of rgcs as well, consistent with prior studies. as expected, intranasal inoculation with rsa59 failed to induce demyelination in spinal cord; however, it also did not induce optic nerve demyelination. no acute inflammation was found, and no viral antigen was detected, in the optic nerve or retina 1 day after inoculation. results confirm the neurotropic effects of rsa59 following intracranial inoculation, and suggest that direct infection with axonal transport of virus from brain to spinal cord and optic nerve is required to induce demyelinating disease. these studies suggest that mhv does not selectively concentrate in optic nerve and retina to sufficient levels to induce demyelination following intranasal inoculation. intracranial inoculation should continue to be considered a preferred method for studies of mhv-induced optic neuritis and central nervous system (cns) demyelinating disease. neurotropic strains of mouse hepatitis virus (mhv) induce acute inflammation and chronic demyelination in the spinal cord and optic nerves mediated by axonal spread following intracranial inoculation in mice, with pathologic features similar to the human demyelinating disease multiple sclerosis. spinal cord demyelination is also induced following intranasal inoculation with neurotropic mhv strains, however much higher viral doses are required as compared to intracranial inoculation. recently, it was shown that intranasal administration of low concentrations of proteins leads to significant, rapid accumulation of protein in the optic nerve and in the eye, with only low levels reaching spinal cord and other brain regions. thus, we examined whether intranasal inoculation with mhv at doses equivalent to those given intracranially could induce optic neuritis-inflammation, demyelination and loss of retinal ganglion cells (rgcs) in the optic nerve with or without inducing spinal cord demyelination. four week old male c57bl/6j mice were inoculated intracranially with the recombinant demyelinating strain rsa59, or intranasally with rsa59 or the non-demyelinating strain rsmhv2 as control. one month post-inoculation, mice inoculated intracranially with rsa59 had significant myelin loss in both spinal cord and optic nerves, with significant loss of rgcs as well, consistent with prior studies. as expected, intranasal inoculation with rsa59 failed to induce demyelination in spinal cord; however, it also did not induce optic nerve demyelination. no acute inflammation was found, and no viral antigen was detected, in the optic nerve or retina 1 day after inoculation. results confirm the neurotropic effects of rsa59 following intracranial inoculation, and suggest that direct infection with axonal transport of virus from brain to spinal cord and optic nerve is required to induce demyelinating disease. these studies suggest that mhv does not selectively concentrate in optic nerve and retina to sufficient levels to induce demyelination following intranasal inoculation. intracranial inoculation should continue to be considered a preferred method for studies of mhv-induced optic neuritis and central nervous system (cns) demyelinating disease. neurotropic strains of mhv have been extensively used to induce neuroinflammation mediated acute and chronic demyelinating disease of cns. depending upon route of inoculation and strain of mhv, different regions of the cns are affected. inoculation with experimental neurotropic strains jhm and mhv-a59 induces a biphasic disease with acute meningoencephalitis (in first 10-14 days post inoculation) followed by subacute and chronic inflammatory demyelinating disease (stohlman and weiner, 1981; lavi et al., 1984; das sarma et al., 2000) . both jhm and mhv-a59 strains of mhv cause some subacute and chronic inflammatory demyelination in the brain, but a much larger disease burden in the spinal cord. translocation of virus from initial site of inoculation in brain to spinal cord occurs by trafficking of virus particle in neural and glial cells sun and perlman, 1995) . intranasal as well as intracranial inoculation of jhm has been shown to induce similar symptoms in balb/c mice (robbins et al., 1991) . similarly, intracranial inoculation has been well used as a method for mhv-a59 to cause the biphasic disease (das sarma et al., 2000 , 2002 , while a higher dose of mhv-a59 is required to reach the same level of inflammation in ceacam-/-mice when inoculated through the nasal route (blau et al., 2001; hemmila et al., 2004) . with intracranial inoculation, the inflammation is not limited to brain and spinal cord. mhv-a59 and recombinant strain rsa59 cause inflammation in optic nerve with subsequent demyelination of optic nerve and rgc loss (shindler et al., 2008 (shindler et al., , 2011 . studies of isogenic recombinant strains rsa59 and rsmhv2 of demyelinating strain (mhv-a59) and non-demyelinating strain (mhv2), respectively, containing enhanced green fluorescent protein (egfp) have elaborated the mechanisms of demyelination and axonal loss and have helped in tracking and tracing of virus in vitro as well as in vivo (das sarma et al., 2009) . rsa59 can cause demyelination, but rsmhv2 cannot, which makes it a suitable control to determine the cellular and molecular basis of demyelination in mice. following intranasal inoculation of mice, mhv accesses the cns through the olfactory nerve and spreads from the olfactory system (jacobsen and perlman, 1990; perlman et al., 1990) into structures of the limbic system and their brainstem connections. this has led investigators to suggest that interneuronal transport is one mechanism of viral spread during acute encephalitis (barthold, 1988; lavi et al., 1988; , and studies showing spread of virus sequentially from cerebral hemispheres to brainstem to spinal cord provide further support for this interneuronal transport mechanism. similar axonal transport of virus from brain to spinal cord (das sarma et al., 2009) , as well as from brain to optic nerve (shindler et al., 2008 (shindler et al., , 2011 , has been reported following intracranial inoculation with mhv-a59 or rsa59 and may serve as one mechanism for virus to avoid immune surveillance; however, axonal spread to optic nerve has not been well examined following intranasal inoculation. different intra-and extracellular pathways may help facilitate viral transport across olfactory or respiratory epithelial barriers. endocytosis into olfactory sensory neurons followed by intraneuronal transport to the olfactory bulb, or transcellular transport to the lamina propria via sustentacular cells, have been suggested as potential intracellular pathways (kristensson and olsson, 1971; broadwell and balin, 1985; thorne et al., 1995; doty, 2008; kristensson, 2011) . delivery of large molecular weight biological therapies (e.g., stem cells, gene therapy vectors, and large proteins) to the cns via intranasal administration has been explored as a potential method to treat multiple cns diseases/disorders including parkinson's and alzheimer's diseases, multiple sclerosis, seizures, strokes, and psychiatric disorders (costantino et al., 2007; neuwelt et al., 2008; dhuria et al., 2010) . spread of smaller peptides through rodent brain following intranasal administration occurs rapidly, with diffuse brain distribution and greatest levels found in olfactory bulbs and trigeminal nerves, just 1 h after treatment. igf-1 (mw = 7.65 kda) is one of the most studied proteins using intranasal delivery to the cns (thorne et al., 2004) . even entry of some high molecular weight proteins such as vegf (mw = 38.2 kda) to the cns has been shown following intranasal administration (yang et al., 2009) . recently, it has been shown that proteins in a complex biologic therapy, st266, administered via the intranasal route in rats reached the cns within 30 min, and st266 proteins were detected in the vitreous and the optic nerve at markedly higher concentrations than in the brain (khan et al., 2017) , suggesting a rapid, direct nose-to-optic nerve delivery method for proteins. whether viruses can follow similar pathways to preferentially spread to optic nerve at low inoculation titers has not been reported, but if such pathways are present, the rapid spread of virus could provide an additional mechanism for immune evasion and therefore promote viral infection at lower inoculation titers. we hypothesized that rsa59 can be used to induce optic neuritis when inoculated intranasally at lower doses than required to induce brain and spinal cord disease due to enhanced viral spread to optic nerve. mice were inoculated with rsa59 and rsmhv2 as control, both intranasally as well as intracranially at equivalent concentrations to compare if both routes of administration result in the same optic nerve pathology. four-week-old virus-free c57bl/6j mice were purchased from the jackson laboratory (barharbor, me, usa). all animal procedures and care were conducted in accordance with ethical guidelines approved by the institutional animal care and use committee at the university of pennsylvania. rsa59 and rsmhv2, the isogenic recombinant strain of mhv-a59 and mhv2, respectively, were used as previously described (das sarma et al., 2009) . rsa59 and rsmhv2 are each engineered to express enhanced green fluorescence protein (egfp), thus allowing viral antigen detection by fluorescence without immunohistochemical staining (das sarma et al., 2002) . mice were monitored daily for signs of mhv induced neurologic up to 26 days (chronic stage) post-infection. 50% ld50 doses of strains rsa59 (70,000 pfu), and rsmhv2 (1000 pfu) were used to inoculate 4-week-old, mhv-free, c57bl/6j mice (jackson laboratory). desired pfu of viruses were prepared in a total volume of 20 µl in pbs and were pipeted as intranasal drops noninvasively every 2 min to alternating nares until all 20 µl were delivered, with simultaneous occlusion of the opposite naris. drops were placed at the opening of the nares, allowing them to be snorted into the nasal cavity. the mice for day 26 studies were also inoculated intracranially as a positive control for disease pathogenesis with rsa59, as in prior studies (das sarma et al., 2009) . control mice mock-infected with pbs were inoculated in parallel. animals were euthanized (3 mice per group) at day 1 post-inoculation (p.i.) and day 26 p.i. at 1 and 26 days p.i., tissues, including whole eyes, optic nerves, brains, spinal cords, and livers, were isolated from both mock-and virus-infected mice. for paraffin sectioning, eyes and optic nerves were fixed for 15 min after dissecton in 4% paraformaldehyde (pfa) while brains, spinal cords, and livers were fixed in 4% pfa overnight. five micrometer sections were cut for routine cns pathology staining following fixation and tissue processing. sections were stained with luxol fast blue (lfb) to detect myelin loss in spinal cord and optic nerve as in prior studies (shindler et al., 2008) . demyelination was scored based on detection of focal white matter areas lacking lfb staining using a relative three-point scale. areas of demyelination were quantified using a 0-3 point scale, where 0-no demyelination; 1-rare foci of demyelination; 2a few foci of demyelination; and 3-large (confluent) areas of demyelination. all slides were coded and read in a blinded manner. serial sections from eye, optic nerve, and brain were stained by the avidin-biotin-immunoperoxidase technique (vector laboratories) using 3, 3-diaminobenzidine as substrate, and a 1:100 dilution of anti-iba1 (wako, richmond, va, usa), and 1:40 dilution of antiviral nucleocapsid antiserum (mouse monoclonal anti-n; nucleocapsid protein of mhv-jhm, monoclonal clone 1-16-1, kindly provided by julian lebowitz, texas a&m, college station, tx) as primary antibodies. control slides from mock-infected mice were incubated in parallel. rgc immunolabeling and counting was performed as in prior studies (khan et al., 2017) . briefly, eyes removed at the time of sacrifice were fixed with 4% pfa. retinas were isolated and whole-mounted on glass slides, washed several times in pbs, permeabilized at −70 • c in 0.5% triton x 100 solution, then thawed and washed again in 0.5% triton x 100. retinas were incubated overnight with goat anti-brn3a (rgc marker) antibody (santa cruz biotechnology) diluted 1:100 in blocking buffer (pbs containing 2% bovine serum albumin and 2% triton x 100). after washing in pbs, retinas were incubated for 1 h with alexa fluor-488 anti-goat secondary antibody diluted 1:500. retinas were then washed and mounted with vectashield mounting medium for fluorescence. photographs of rgcs were taken in 12 standardized fields at 1/6, 3/6, and 5/6 of the retinal radius from the center of the retina in four quadrants at 40x magnification. rgcs were counted in each field by a blinded investigator using image-pro plus 5.0 (media cybernetics, silver spring, md) software. four week old c57bl6/j mice were inoculated with 50% ld50 doses of rsa59 or rsmhv2 by intranasal administration or by intracranial injection, or mock transfected by intranasal administration of solution without virus. pathology was assessed from lfb stained cross-sections of spinal cord isolated from mice at day 26 (peak of demyelination) p.i. rsa59, when injected intracranially, induced significant myelin loss within formed demyelinating plaques, [average demyelination score 1.33 ± 0.2357; (mean ± se); n = 3 mice (9 sections/group); p < 0.0001 vs. control] as in prior studies (figures 1j-l) . as expected, mice infected intracranially with rsmhv2 did not show any significant myelin loss (data not shown). interestingly, no demyelination plaques were observed in any level of spinal cord sections of rsa59 infected mice when given via the intranasal route (figures 1d-f) . similarly, as expected, no myelin loss was observed in intranasally mockinfected (figures 1a-c) or rsmhv2-infected mouse spinal cord (figures 1 g-i) . intracranial, but not intranasal, inoculation with rsa59 induces optic neuritis mice inoculated intracranially with rsa59 have been found to exhibit retrograde axonal transport of virus from the lateral geniculate nuclei along the optic nerve into the retina, and can cause optic nerve inflammation and demyelination (shindler et al., 2011) , whereas rsmhv2 does not. to investigate whether intranasal rsa59 administration can induce optic nerve demyelination similar to intracranial inoculation, 5 µm thick serial optic nerve sections from the same mice shown in figure 1 were stained with lfb. rsa59, when infected intracranially, induced significant myelin loss with notable demyelinating plaques in optic nerves, (figure 2 ) as previously observed (shindler et al., 2008) . similar to spinal cord, little or no demyelinating plaques were observed in optic nerve sections of rsa59 infected mice when injected intranasally, which was comparable to rsmhv2 and mock infected mice. demyelinating optic neuritis induced by intracranial inoculation with rsa59 has been shown previously to lead to neuronal damage with loss of rgcs (khan et al., 2014) . to examine whether intranasal infection with demyelinating strains of mhv figure 1 | comparative demyelination study between intracranially inoculated and intranasally infected mouse spinal cords. serial thoracic (left column), cervical and lumbar (center and right columns) cross sections of spinal cord from post-inoculation day 26 intranasally mock-infected (a-c), rsa59-infected (d-f), and rsmhv2-infected (g-i) mice, and intracranially rsa59-inoculated mice (j-l) were stained with lfb (scale bar = 50 µm). marked area indicate typical demyelinating plaques found in spinal cord white matter. average demyelination score is 1.33 ± 0.2357; (mean ± se) (m); n = 9/group; data comparisons were done by one-way anova and tukey's multiple comparison post-hoc testing with graphpad prism 6.0 software. ****p < 0.0001. results in neuronal loss, retinas were isolated from the same mice shown in figure 1 , and rgcs were labeled and counted in a blinded fashion. intracranially rsa59-infected mice had significantly fewer surviving rgcs compared to mock-infected mice, whereas mice infected intranasally with either rsa59 or rsmhv2 did not show rgc loss (figure 3) . proteins rapidly accumulate at high concentrations in optic nerve and in the eye following intranasal administration (khan et al., 2017) . thus, the potential for mhv viruses to similarly spread figure 2 | comparative optic nerve demyelination study between intracranially-and intranasally-infected mice. representative optic nerve sections from chronic stage (day 26 p.i.) mock-infected (n = 6) (a), rsa59 intrancranially-infected (n = 6) (b), rsa59 intranasally-infected (n = 5) (c), and rsmhv2 intranasally-infected (n = 5) (d) mice stained with lfb show demyelination only in rsa59 intracranially-infected mice (scale bar = 100 µm). the relative level of demyelination scored by a blinded investigator showed significant demyelination in optic nerves of mice inoculated intracranially with rsa59, but not in mock-infected (control) mice nor in mice infected intranasally with either rsa59 or rsmhv2 (*p < 0.05 vs. all other groups) (e). data comparisons were done by one-way anova and tukey's multiple comparison post-hoc testing with graphpad prism 6.0 software. to optic nerve and retina was assessed 1 day following intranasal inoculation. four-week-old, mhv-free, c57bl/6j mice were inoculated intranasally with 50% of the ld50 dose of rsa59 or rsmhv2, and mice were euthanized 1 day later. retinas and optic nerves were isolated, sectioned, and immunostained with anti-viral nucleocapsid antisera to detect viral spread. no significant staining was observed in any of the retinas or optic nerves from mock-infected (n = 6), rsa59-infected (n = 6), or rsmhv2-infected (n = 6) mice (figure 4) . as shown in prior studies (shindler et al., 2011) , viral antigen does not reach the retina within 1 day following intracranial inoculation with rsa59 (data not shown). viral antigen is found in the retina 6 days after intrancranial inoculation (figure 4) , while no antigen is detectable in retina following intranasal inoculation at day 1 (figure 4 ) or any later time points (data not shown). to further confirm that intranasal rsa59 administration fails to induce optic neuritis, acutely, optic nerve sections were immunostained for the microglial/macrophage marker iba1. previously, it has been observed that intracranial inoculation with rsa59 induces acute optic nerve inflammation containing almost entirely activated microglia/macrophages (shindler et al., 2011) 3-6 days post-inoculation. to study whether intranasal figure 3 | rsa59 infection induces rgc loss. representative photos illustrate the decreased rgc numbers in eyes of mice inoculated intracranially with rsa59 (b) compared to mock-infected control mice (a). mice inoculated intranasally with rsa59 (c) or rsmhv2 did not show rgc loss (d) (scale bar = 50 µm). the total number of labeled rgcs present in 12 standardized retinal fields was counted. the average number of surviving rgcs/eye (n = 6/group) shows intracranial rsa59 induced a significant decrease in rgc numbers compared to control mice (**p < 0.01). neither rsa59 nor rsmhv2 induced rgc loss compared to control mice when administered intranasally. rgc numbers in rsmhv2-infected mice were significantly higher than in mice intracranially inoculated with rsa59 (*p < 0.05) (e). data comparisons were done by one-way anova and tukey's multiple comparison post-hoc testing with graphpad prism 6.0 software. inoculation rapidly induces similar optic nerve inflammation, optic nerve sections were stained with anti-iba1 antibody. sections from mock-infected mice were used to demonstrate resting levels of microglial staining. iba-1 staining did not reveal any increased numbers of microglia/macrophages in optic nerve sections following intranasal viral inoculation as compared to mock-infected (figure 4) . to confirm that lack of viral antigen detection in the optic nerve represents a failure of the virus to spread to optic nerve and retina, and not a failure to detect viral antigen, viral antigen was also examined in olfactory bulb sections from the same mice by autofluoresence of egfp (figures 4 j,k) as well as immunostaining with anti-viral nucleocapsid antisera (figures 4 l,m) . the current studies compared effects of rsa59 infection via two different routes of inoculation on the development of demyelinating disease in the optic nerve and spinal cord. results suggest intracranial inoculation is the best method to induce neuroinflammation. in the current study, intracranial infection of rsa59 lead to chronic stage inflammation and demyelination in both the optic nerve and spinal cord. this result is consistent with earlier studies where intracranial inoculation of rsa59 or its parental strain mhv-a59 led to demyelination and axonal loss in spinal cord ) and optic nerve (shindler et al., 2008 (shindler et al., , 2011 . induction of optic neuritis by intracranial inoculation of rsa59 is dependent on retrograde transport of viral antigen along rgc axons that occurs over several days and results in late stage demyelination (shindler et al., 2011) as seen again in the current study. together, spinal cord and optic nerve pathology observed after intracranial inoculation of rsa59 in the current study confirm that the viral titer used retains its previously identified ability to induce cns demyelinating disease. while intrancranial inoculation was shown previously to induce optic neuritis (shindler et al., 2008 (shindler et al., , 2011 , effects of intranasal inoculation with rsa59 on optic nerve pathology were not reported. in the current study, intranasal infection by rsa59 at the same dose used for intracranial inoculation was not able to induce any neuropathogenesis, either in optic nerve or spinal cord. earlier studies with several other strains showed that intranasal inoculation of mhv can cause cns disease, but much higher concentrations of virus were used than in intracranial inoculation studies. mhv-jhm enters the central nervous system (cns) via the olfactory after intranasal inoculation . the intranasal inoculation of jhm strain can lead to encephalitis and demyelination. the oblv strain of mhv can infect the main olfactory bulb (schwob et al., 2001) . intranasal inoculation of mhv-a59 can lead to hepatitis with measurable viral load in brain as well (hemmila et al., 2004) . these studies did not report optic nerve pathogenesis following intranasal inoculation although it is not known if that was examined. there are several possible reasons that we did not see retinal infection or optic nerve inflammation and demyelination lesions after intranasal inoculation. most likely, the dose of virus may have been too low to cause the pathogenesis. this finding was not unexpected in the spinal cord, where previous studies showed higher doses were necessary. however, based on the high levels of protein accumulation in optic nerve and eye following intranasal inoculation (khan et al., 2017) , it was anticipated that rsa59 would also preferentially accumulate in the eye, but results suggest that higher doses are likely required. alternatively, the intranasal inoculation may be less effective for inducing optic neuritis and retinal lesions than the intracerebral route because of the longer distance required for the virus to travel to the eye if it travels via axonal transport and neuronneuron spread similar to what has been observed following intracranial inoculation (das sarma et al., 2009; shindler et al., 2011) . the precise mechanisms mediating spread of a virus or a drug from the nose to various cns regions are not fully elucidated. at least three steps are necessary following intranasal administration (1) crossing the epithelial barrier in the nasal cavity, (2) transport from nasal mucosa to site of brain entry, likely across the cribiform plate, (3) transport from the site of brain entry to other anatomical regions. alternatively, they may be absorbed into the systemic circulation and gain secondary access to the cns through the blood brain barrier. the speed at which proteins were reported to reach the eye and optic nerve, 30 min after intranasal administration (khan et al., 2017) , suggests hematogenous spread is very unlikely, and that intraaxonal transport is even more unlikely. it was hypothesized that perhaps some pathway of local diffusion or lymphatic pathway may allow rapid protein diffusion over the relatively short distance from absorption through the cribiform plate to optic nerve (khan et al., 2017) . for rsa59, prior intracranial studies demonstrate that the virus can use axonal transport machinery to spread intraneuronally (das sarma et al., 2009) , thus it is likely that a similar mechanism would be used after intranasal administration and entry into the olfactory nerve. the path from there to optic nerve is not direct, and thus may require much higher titers of virus to occur at a pathologic level, or may not be possible at all. these hypothesized mechanisms may explain why we were not able to see viral staining day 1 post infection whereas the drug st266 and other small molecules can be found in optic nerve as early as 30 min post intranasal inoculation (dhuria et al., 2010; khan et al., 2017) . intranasal administration provides a potential non-invasive method for delivering material to the cns. interestingly, complex mixtures containing physiologic concentrations of multiple proteins show that protein can rapidly accumulate in the eye and optic nerve following intranasal delivery, suggesting a direct nose to eye diffusion pathway that remains to be fully elucidated (khan et al., 2017) . the current results show that rsa59 does not follow a similar pattern of accumulation in the optic nerve, suggesting that viral particle may be too large or complex to follow the same pathway, or may actively enter neurons locally and restrict their movement to intraneuronal axonal transport. nonetheless, the ability of neurotropic mhv viruses to infect different cells, translocate throughout the cns, and induce inflammatory demyelination, continues to provide a reproducible model to study optic nerve and spinal cord demyelinating disease following intracranial inoculation. thus, intracranial inoculation should continue to be considered a preferred method for studies of mhv-induced optic neuritis and cns demyelinating disease. ms, rk and kd performed the wet lab experiments and wrote their contributions. ks and jd designed the studies and wrote the paper. delhi and dupre fellowship, multiple sclerosis society international federation (msif), uk for a fellowship. this work was supported by the department of biotechnology bt/pr20922/med/122/37/2016 to jd. ks thanks research to prevent blindness, nih grant ey015014, and the f. m. kirby foundation. the authors thank iiser kolkata and university of pennsylvania, philadelphia, for support. the funders had no role in the study design, data collection, and interpretation, or the decision to publish the work. two neurotropic viruses, herpes simplex virus type 1 and mouse hepatitis virus, spread along different neural pathways from the main olfactory bulb the olfactory nerve and not the trigeminal nerve is the major site of cns entry for mouse hepatitis virus, strain jhm olfactory neural pathway in mouse hepatitis virus nasoencephalitis targeted disruption of the ceacam1 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localization of virus and antibody response in mice infected persistently with mhv-jhm intranasal delivery of a novel amnion cell secretome prevents neuronal damage and preserves function in a mouse multiple sclerosis model sirt1 activating compounds reduce oxidative stress mediated neuronal loss in viral induced cns demyelinating disease microbes' roadmap to neurons uptake of exogenous proteins in mouse olfactory cells limbic encephalitis after inhalation of a murine coronavirus mhv-a59 pathogenesis in mice strategies to advance translational research into brain barriers effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain spread of mhv-jhm from nasal cavity to white matter of spinal cord. transneuronal movement and involvement of astrocytes ocular tropisms of murine coronavirus (strain jhm) after inoculation by various routes intranasal inoculation with the olfactory bulb line variant of mouse hepatitis virus causes extensive destruction of the olfactory bulb and accelerated turnover of neurons in the olfactory epithelium of mice macrophage-mediated optic neuritis induced by retrograde axonal transport of spike gene recombinant mouse hepatitis virus experimental optic neuritis induced by a demyelinating strain of mouse hepatitis virus chronic central nervous system demyelination in mice after jhm virus infection spread of a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes quantitative analysis of the olfactory pathway for drug delivery to the brain delivery of insulin-like growth factor-i to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration direct transport of vegf from the nasal cavity to brain the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer sm and handling editor declared their shared affiliation at time of review.copyright © 2018 singh, khan, dine, das sarma and shindler. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-299949-kmn53e2z authors: schultz, kimberly l.w.; griffin, diane e. title: immune responses to viruses in the cns date: 2016-05-09 journal: encyclopedia of immunobiology doi: 10.1016/b978-0-12-374279-7.14022-6 sha: doc_id: 299949 cord_uid: kmn53e2z for recovery from infection, the immune response in the central nervous system (cns) must eliminate or control virus replication without destroying nonrenewable, essential cells. thus, upon intracellular virus detection, the infected cell must initiate clearance pathways without triggering neuronal cell death. as a result, the inflammatory response must be tightly regulated and unique mechanisms contribute to the immune response in the cns. early restriction of virus replication is accomplished by the innate immune response upon activation of pattern recognition receptors in resident cells. infiltrating immune cells enter from the periphery to clear virus. antibodies and interferon-γ are primary contributors to noncytolytic clearance of virus in the cns. lymphocytes are retained in the cns after the acute phase of infection presumably to block reactivation of virus replication. for recovery after virus infection of the central nervous system (cns), the essential, nonrenewable nature of neurons requires a fine-tuned immune response that controls virus replication without damaging neuronal function. damage can result directly from virus replication or from the host immune response to infection. functional impairment or loss of neurons following infection can be fatal or leave survivors with neurological sequelae including cognitive deficits, seizures, or paralysis (hart et al., 2014; griffiths et al., 2013; silverman et al., 2013; ooi et al., 2008; sauder et al., 2001; finley et al., 1955) . thus, the immune responses required for successful clearance and control of virus infections in the cns are often distinct from those required for clearance from other organs and are characterized by noncytolytic, virus-specific processes. this strategy preserves cns function and minimizes the likelihood of autoimmunity. many viruses can infect the cns, including dna viruses, plus-and minus-strand rna viruses, and retroviruses, leading to varying outcomes from disease. dna viruses, such as herpesviruses (reviewed in koyuncu et al., 2013) , often establish a latent infection as opposed to rna viruses that generally lack a nuclear phase for their replication cycle and cause acute disease. in this article, we will focus on rna virus infections in the cns (table 1) . much of our knowledge about the immune response to neurotropic viruses comes from studying well-characterized mouse models of infection. studies have investigated the course of disease and immune response both in immunocompetent mice and animals deficient in specific components of the immune response. these studies have provided detailed knowledge of the role of each arm of the immune response in control of virus replication and spread, virus clearance, and in immunopathology. in all infections, outcome of infection is dependent on the age and genetic background of the mouse and the strain of the virus used. for simplicity, we will focus on the most commonly studied strains of each virus family and infection of mature mice. detailed studies of immune responses to neurotropic viruses have included neuronal infections with rabies virus, flaviviruses, and alphaviruses, as well as infection of multiple cell types with natural mouse pathogens such as theiler's murine encephalomyelitis virus (tmev), mouse hepatitis virus (mhv), and lymphocytic choriomeningitis virus (lcmv). the immunological processes required for virus clearance from the cns are cell type and virus specific. experimental approaches to define these clearance mechanisms are dependent on the transient depletion of specific immune cell populations and on the use of mice that have selective deficiencies in various components of the immune system. because of the interdependent relationships of components of the immune system in the development of an immune response, deficiencies of one type of cell or molecule may affect several facets of the immune response, making it difficult to identify specific effectors that are crucial for in vivo clearance. infection is rarely initiated in the cns because viruses must invade the cns from initial sites of infection in the periphery with induction of the immune response in peripheral lymphoid tissues. entry of viruses, immunoglobulins, and immune cells from the blood is restricted by the blood-brain barrier (bbb), a selectively permeable barrier with tight junctions between cerebrovascular endothelial cells that are supported by astrocytes ( figure 1 ). the bbb separates the parenchyma of the cns from the circulating blood and serves as a physical blockade to bloodborne infections of the cns. however, the endothelial barrier is more permeable at certain sites in the cns (e.g., choroid plexus) and inflammation increases permeability to allow immune cell infiltration along with opportunities for virus entry. historically, routes of cns infection have been deduced from data obtained by histological staining at early times after infection or disruption of a potential route of infection. entry routes are not mutually exclusive, as multiple routes have been described for some viruses. recently, new techniques such as intravital microscopy and clarity preparation of infected brains have been developed that may lead to new insights on the mechanisms of cns entry (yang et al., 2014; chung et al., 2013; mcgavern and kang, 2011) . in general, virus entry is either from the periphery by neuronal axonal transport or from the bloodstream across the vascular endothelium. sensory and motor neurons extend their processes into the periphery and provide a point of entry for some neurotropic viruses replicating in peripheral tissue. expression of viral receptors on neuromuscular junctions facilitates entry of poliovirus, adenovirus, and rabies virus into the cns (salinas et al., 2010) . olfactory neurons that project into the respiratory mucosal epithelium can provide a direct route to the brain for alphaviruses (phillips et al., 2013; powers and logue, 2007; charles et al., 1995) , flaviviruses (yamada et al., 2009; monath et al., 1983) , coronaviruses (barnett and perlman, 1993) , paramyxoviruses (munster et al., 2012) , bunyaviruses (bennett et al., 2008) , and occasionally influenza virus (van riel et al., 2014) . hematogenous entry occurs when a virus directly infects bbb endothelial cells or infects leukocytes that cross the bbb providing entry by a 'trojan horse' mechanism (neal, 2014; wilson, 2013; rhoades et al., 2011; kim, 2003; haase, 1986) . the cns is relatively protected from immunologic activity. in addition to the physical protection by the bbb, the brain parenchyma has no lymphatic vessels or professional antigenpresenting cells, low expression of major histocompatibility complex (mhc) molecules, and active maintenance of an immunologically quiescent state. however, the exclusion of immune cells and the role of active immune signaling in the cns has been redefined recently (schwartz et al., 2013; muldoon et al., 2013; elmer and mcallister, 2012; hernangómez et al., 2012) . resident cells in the nervous system, including neurons, play an active role in the immune response (schultz et al., 2014; o'donnell et al., 2012; chakraborty et al., 2010; daffis et al., 2008a; castorena et al., 2008; daffis et al., 2007; commonly used in mouse models of viral encephalitis. jackson et al., 2006) . additionally, memory t cell and b cell are found in the cns long after infectious virus has been eliminated (phares et al., 2013; metcalf et al., 2013; wakim et al., 2010; wilson et al., 2010) . resident cells monitor the cns for infection and initiate and control inflammation when infection occurs. microglial cells, the resident macrophages of the cns, express the cd200 receptor (cd200r), trem2, cd172a, and cd45 and are kept in a quiescent state through interactions with electrically active, healthy neurons expressing cd200, hsp60, cd47, and cd22 and through the production of neurotrophins (chavarría and cárdenas, 2013; ransohoff and cardona, 2010; hoek et al., 2000) . local production of the antiinflammatory cytokines transforming growth factor (tgf)-b and il-10 by astrocytes, pericytes, and meningeal cells further inhibits cellular activation (schwartz et al., 2013; fabry et al., 1995; johnson et al., 1992) . activated t cells cross the bbb into the cns for immunological surveillance upon interactions with p-selectin on endothelial cells, but leave or die if antigen is not encountered (irani and griffin, 1996; wekerle et al., 1991 wekerle et al., , 1986 . the innate immune response initiated by resident cells in the cns upon virus infection is the first line of defense ( figure 2(a) ). detection of infection occurs through activation of cellular pattern recognition receptors that include the toll-like receptors (tlrs), rig-i-like receptors (rlrs), and nod-like receptors (nlrs). microglia, as the professional immune cells of the cns, express all of the known pattern recognition receptors. additionally, neurons, astrocytes, and to a lesser extent oligodendrocytes, express selected pattern recognition receptors and thus contribute to innate immune signaling (kigerl et al., 2014) . engagement of tlrs and rlrs activates the transcription factors irf-3, irf-7, and nfkb that control expression of type-i interferon (ifn)-a and ifn-b. for many cns infections, ifn production is critical for early control of infection. mice deficient in type-i ifn signaling, ifnar1 à/à , have increased virus replication and mortality upon infection by a variety of viruses including sindbis virus (sinv), west nile virus (wnv), lcmv, and vesicular stomatitis virus (samuel and diamond, 2005; byrnes et al., 2000; ryman et al., 2000; müller et al., 1994) . moreover, pretreatment with ifn is protective (frolov et al., 2012; lucas et al., 2003; grieder and vogel, 1999; després et al., 1995a) . ifn signaling must be tightly regulated as excess ifn, particularly ifn-a, can be neurotoxic (nallar and kalvakolanu, 2014; reyes-vázquez et al., 2012) . in contrast, ifn-b coordinates the immune response and is generally neuroprotective (mclaurin et al., 1995) . autocrine and paracrine binding to the ubiquitously expressed ifna/b receptor initiates jak/stat signaling and directs ifn-stimulated gene (isg) expression. isgs restrict virus replication in infected cells and establish an antiviral state in neighboring cells to limit virus spread. although ifn signaling stimulates the expression of hundreds of isgs, antiviral effects oligodendrocyte: glial cell that produces the myelin sheath around neuronal axons and promotes conduction neuron: electrically active cell that transmits signals from the periphery and within the cns astrocyte: glial cell that secretes neuroprotective factors and is associated with maintenance of the bbb microglial cell: bone marrow-derived macrophage-lineage glial cell that is the resident 'macrophage' of the cns; constantly surveys the cns and becomes immunologically active upon pathogen detection blood brain barrier (bbb): endothelial cells connected by tight junctions that form a highly selective barrier between the circulating blood and the cns parenchyma of these proteins are both virus specific and tissue specific (cho et al., 2013a; diamond and gale, 2012; schoggins et al., 2011; zhao et al., 2011) . for instance, the isg ifit2 restricts wnv in some regions of the brain, but did not affect replication in the cerebral cortex, spinal cord, or periphery (cho et al., 2013b) . engagement of nlrs by infecting viruses can initiate inflammasome formation in the cns. the inflammasome activates caspase-1 to cleave precursors of the proinflammatory cytokines il-1b and il-18. secretion of mature il-1b and il-18 helps to orchestrate the inflammatory response to infection. the magnitude and timing of the inflammatory response must be controlled to limit damage to bystander cells. inflammasome-mediated signaling has varying affects during cns infections (prow and irani, 2008; sergerie et al., 2007; liang et al., 1999) . inflammasome activation during wnv infection is protective, as mice deficient in inflammasome components have increased virus replication in the brain and decreased survival (kumar et al., 2013; ramos et al., 2012) . in contrast, inflammasome activation in microglia and astrocytes contributes to increased immunopathology and possibly bystander neuronal death during japanese encephalitis virus infection (kaushik et al., 2012; das et al., 2008) . neurons are active contributors to the innate immune response during virus infection as has been demonstrated in cultures of primary and immortalized neurons (schultz et al., 2014; farmer et al., 2013; cho et al., 2013a; peltier et al., 2013; castorena et al., 2008; delhaye et al., 2006; préhaud et al., 2005) . in response to alphavirus, flavivirus, and bunyavirus infections, mature neurons rapidly activate irf-3 and irf-7 to induce expression of type-i ifn, limit virus replication, and preserve neuronal function (schultz et al., 2014; farmer et al., 2013; peltier et al., 2010; daffis et al., 2008b; castorena et al., 2008; daffis et al., 2007) . additionally, il-1b synergizes with ifn-b to control wnv replication in neurons (ramos et al., 2012) . the combination and importance of each innate immune signaling pathway in response to infection is likely cell type and virus specific. in addition to factors that control virus replication, infected cells produce factors that activate astrocytes and microglia, upregulate expression of mhc molecules on microglial cells, increase expression of adhesion molecules including intercellular adhesion molecule 1 and vascular adhesion molecule 1(vcam1) on capillary endothelial cells to direct leukocyte infiltration to the site of infection, and modulate the inflammatory response. cytokines and chemokines important for these processes are induced in a virus-specific manner but often include ifn-g, il-1, il-6, il-10, il-12, tumor necrosis factor (tnf), ccl1, ccl2, ccl5, cxcl9, and cxcl10 (kulcsar et al., 2014; tun et al., 2014; lee et al., 2013; hayasaka et al., 2013; metcalf et al., 2013; ramos et al., 2012; stubblefield park et al., 2011; shrestha et al., 2006; klein et al., 2005; burdeinick-kerr and griffin, 2005; bergmann et al., 2004; chang et al., 2000; liang et al., 1999) . these factors facilitate recruitment of circulating leukocytes across the bbb and into the cns. in addition to controlling virus replication in cns cells, innate immune signaling initiates the virus-specific adaptive immune response. infiltration of mononuclear inflammatory cells into the cns typically begins 3-4 days after infection (figure 2(b) and 2(c)). t cell and b cell trafficking into the cns is promoted by neuronal expression of the chemokine cxcl10 that binds to cxcr3 on activated t cell and b cell (phares et al., 2013; zhang et al., 2008; klein et al., 2005) . additionally, proper trafficking of t cells to appropriate brain regions is promoted by signaling through ccr5 and cxcr4 (mccandless et al., 2008) . cells first accumulate in the perivascular areas and then infiltrate the parenchyma in the regions of virus infection. essentially, all components of the cellular immune response are detected in the infiltrate: natural killer (nk) cells, antigen-specific cd4 þ and cd8 þ t cells, b cells, and monocytes/macrophages (peña et al., 2014; zhao et al., 2013; lee et al., 2013; chang et al., 2000; rowell and griffin, 1999; parra et al., 1997; pearce et al., 1994; wesselingh et al., 1994) . the uninfected cns does not have professional antigen-presenting cells capable of activating naïve t cells, but dendritic cells are detected in the cns during inflammation after either entering from the circulation or developing from a subpopulation of activated microglia. presentation of viral peptide antigen in association with the appropriate mhc molecules, predominantly expressed on glial cells, retains activated t cells in the cns (kimura and griffin, 2000; irani and griffin, 1996; suzumura et al., 1988) . the continued presence of viral protein antigens promotes long-term retention of virus-specific b cells (phares et al., 2013; metcalf et al., 2013) . virus is cleared from the cns in a multistep process that must first stop cell-to-cell spread and eliminate cell-free infectious virus (figure 2(b) ). this phase of viral clearance can be assessed by measurement of infectious virus but, as neutralizing antibody is produced, virus clearance is best assessed by quantitative measurement of viral nucleic acid. initially, local production of type-i ifn reduces cell-to-cell spread through paracrine antiviral signaling. infectious virus is neutralized by antibody produced by b cells that enter the cns and interact with viral glycoproteins on the infected cell surface. additionally, ifn-g, interacting with ifn-g receptors expressed on the surfaces of infected cells, inhibits virus production (phares et al., 2013; metcalf et al., 2013; stewart et al., 2011; hooper et al., 2009; tschen et al., 2006; binder and griffin, 2001; ubol et al., 1995; levine et al., 1991) . for full recovery, virus-infected cells or viral genomes need to be cleared from the cns. in peripheral tissues, virus-infected cells are usually eliminated by virus-induced or immunemediated cytolysis. clearance of virus-infected cells in the cns becomes a more complicated process due to the nonrenewable and essential nature of neurons and the important role of glial cells in maintaining neuronal function. if the immune system destroys the infected cell, then the outcome of infection will be the same as if the virus caused cell death. however, if infected cells are allowed to survive, there must be a clearance mechanism that inhibits synthesis of viral nucleic acid and proteins and eliminates viral genomes. if clearance is not complete, mechanisms are needed to avoid progressive or relapsing disease. these processes must be tightly regulated to prevent immune-mediated damage to both infected and uninfected cells during the response to cns infection. for instance, the cd8 þ t cell response can be detrimental during wnv infection (szretter et al., 2012; wang et al., 2003) . during fatal encephalomyelitis due to infection with a neurovirulent strain of the alphavirus sinv, infiltration of th1 and th1/th17 cd4 þ t cells is associated with a rapidly fatal paralytic disease. this response is modulated by il-10 produced by intrinsic cells of the cns and by infiltrating regulatory t cells (kulcsar et al., 2014) . il-10 also plays a protective role during coronavirus and flavivirus infections of the cns (tun et al., 2014; hayasaka et al., 2013; trandem et al., 2011) . generally, clearance of rna viruses from neurons occurs through noncytolytic antibody and cytokine-mediated mechanisms to preserve neuronal function. this process has been studied both in virus-infected mice and in cultured neurons. in mice, the clearance of sinv is a two-phase process (metcalf and griffin, 2011) . infectious virus is rapidly cleared during the first week after infection and then viral rna is cleared slowly over the next 30-60 days followed by persistence of a low level of rna. cd8 þ t cells followed by cd4 þ t cells and b cells enter the cns during the first phase when infectious virus is cleared. in the second phase, t cell and b cell are retained in the cns during viral rna clearance with overall larger numbers of cd4 þ t cells and b cells than cd8 þ t cells. numbers of immune cells in the cns gradually decrease with decreasing rna, but the resident populations are steadily enriched in those that are virus specific. within 2 months after sinv infection, most antibody-secreting cells (ascs) produce sinvspecific igg (metcalf and griffin, 2011; tyor et al., 1992) . antibody that mediates virus clearance from neurons is often directed against viral structural proteins on the infected cell surface and has been most completely analyzed for cells infected with sinv (hooper et al., 2009; levine et al., 1991) . in addition to neutralizing free virus, antibody to the sinv e2 glycoprotein can bind to the surface of infected cells and may direct intracellular signaling to control virus replication (després et al., 1995b; levine and griffin, 1992) . the antiviral effect of this antibody does not require complement or phagocytic cells, but is dependent on bivalent antibody, implying that cross-linking of viral proteins at the cell surface results in intracellular inhibition of virus production (ubol et al., 1995; levine et al., 1991) . antibody acts by unknown mechanisms to suppress virus replication and restore host protein synthesis, membrane potential, and type-i ifn responsiveness (després et al., 1995a,b) . cd8 þ t cells can exert antiviral effector functions either through a noncytotoxic, cytokine-mediated, or a cytotoxic pathway. the most effective noncytotoxic cytokine identified is ifn-g and the cytotoxic effector pathways involve perforin and granzymes or cd95 (fas)-cd95l interaction. cd8 þ t cells can be activated by interactions with mhc class i complexes on neurons (chevalier et al., 2011) or through cross-priming interactions with mhc class i molecules on surrounding glial cells. t cells recruited into the cns during sinv infection facilitate, but are not necessary, for rna clearance (rowell and griffin, 2002; kimura and griffin, 2000) . ifn-g alone can clear sinv from motor neurons, but not from cortical or hippocampal neurons which require antibody (burdeinick-kerr and griffin, 2005; binder and griffin, 2001) . ifn-g production by t cells is also important for clearance of mhv, borna disease virus, and measles virus infections from the cns (o'donnell et al., 2012; stubblefield park et al., 2011; richter et al., 2009; templeton and perlman, 2008; pearce et al., 1994) . clearance of wnv is dependent on cd8 þ t cells and monocytes, not antibody (sitati et al., 2007; shrestha et al., 2006; klein et al., 2005) . monocyte-derived cells likely signal to t cells, leading to optimal activation necessary for virus clearance (durrant et al., 2013) . t cells clear virus through ifn-g production or through cytotoxic methods, such as perforin (shrestha et al., 2006) . cytotoxic t cells are targeted to infected neurons upon upregulation of prodeath molecules (fas or trail ligand) and increased mhc class i expression (shrestha et al., 2012; chevalier et al., 2011; shrestha and diamond, 2007) . neurons infected with virulent strains of rabies virus upregulate fasl and b7-h1 to inhibit t cell function and prevent virus clearance and the virus-induced inflammatory response (lafon et al., 2008; baloul et al., 2004) . additional upregulation of the nonclassical mhc class i molecule hla-g on infected neurons may promote tolerance . the best-studied examples of glial cell infections in mice are the picornavirus tmev and coronavirus mhv. failure to clear the acute infection by susceptible strains of mice leads to persistent production of infectious virus and immune-mediated demyelinating disease. thus, these infections have become models for the human demyelinating disease multiple sclerosis. tmev has an early encephalitic phase, which mainly involves infection of neurons, followed by persistent infection of glial cells. in resistant strains of mice, virus clearance is dependent on a rapid cd8 þ t cell response (lindsley and rodriguez, 1989) . in susceptible strains, infectious virus is cleared from the neurons, but not from microglia, astrocytes, or oligodendrocytes. establishment of a persistent infection involves failure of all stages of the immune response, beginning with innate immune signaling through tlr3 and tlr2, proinflammatory molecule expression (il-6), and regulation of infiltrating t cells (jin et al., 2010; so and kim, 2009 ). il-6 inhibits cytotoxic t cell function and apoptotic death by preferential induction of il-17-producing th17 cells (hou et al., 2009) . additionally, il-17 and il-6 synergistically promote expression of prosurvival bcl family members that facilitate survival of virus-infected cells (hou et al., 2014) . mhv infects a wide range of cell types including macrophages, microglia, astrocytes, and oligodendrocytes. the early adaptive response to mhv infection is characterized by expression of the chemokines cxcl9, cxcl10, ccl2, ccl3, and ccl5 and their receptors ccr1, ccr5, and cxcr3 by microglia and astrocytes (lane et al., 1998) . cxcl10 expression is important for recruitment of t cells (phares et al., 2013) . proinflammatory cytokine expression (i.e., decreases as the percentage of virus-specific cd8 þ t cells and expression of t cell support molecules (i.e., cxcl10, ccl5, and ifn-g) increases (lane et al., 1998; parra et al., 1997) . ifn-g plays a key role in dampening mhv replication and orchestrating t cell infiltration, along with maximal expression of mhc molecules on microglia and macrophages (whitman et al., 2009; bergmann et al., 2004 bergmann et al., , 2003 parra et al., 1997) . infiltrating cd4 þ t cells accumulate around blood vessels and provide supporting factors for infiltrating cd8 þ t cells that invade the parenchyma at the site of infection (phares et al., 2011; stohlman et al., 1998) . granzyme b-positive cd8 þ t cells target mhc class i-positive, infected cells for cytolysis, but effector function may be specific for the targeted cell type lin et al., 1997) . ifn-g is particularly important for clearance from oligodendrocytes, whereas perforin and cd8 þ t cell-mediated cytolysis is important for the clearance of virus from astrocytes and microglia (gonzález et al., 2005; bergmann et al., 2003; parra et al., 1999; lin et al., 1997; stohlman et al., 1995) . oligodendrocyte killing by cd8 þ t cells results in demyelination during the acute phase of infection (templeton and perlman, 2008) . cytolytic function declines concomitant with loss of viral antigen lin et al., 1999) . viral rna persists within the cns for over 12 months, regardless of the presence of nonanergic, virus-specific cd4 þ and cd8 þ t cells retained in the cns (phares et al., 2011 (phares et al., , 2010 ramakrishna et al., 2004; bergmann et al., 1999) . additionally, persistent oligodendrocyte infection and the consequent immune response are associated with chronic demyelination. long-term control of virus infection (figure 2(c) ), regardless of infected cell type, is characterized by virus-specific antibody, a lack of infectious virus, and low levels of viral rna that are detected by sensitive methods such as qpcr (metcalf and griffin, 2011; stewart et al., 2011; appler et al., 2010; fragkoudis et al., 2008; tschen et al., 2002; tyor et al., 1992) . although control of the acute phase of mhv infection by the adaptive response is independent of antibody, long-term production of antibody is necessary to prevent reactivation of infection lin et al., 1999) . as the bbb does not allow antibody to efficiently enter the cns from the periphery, ascs must either be continuously recruited to the cns or maintained in the brain parenchyma to produce antibody locally (metcalf et al., 2013; stewart et al., 2011; hooper et al., 2009; diamond, 2003; diamond et al., 2003; ramakrishna et al., 2002; tyor et al., 1992; levine and griffin, 1992; parsons, 1989) . antibody controls persistent infection in the cns through a multifaceted defense strategy that preserves neuronal function following infection. the need for continued antibody production in the cns is highlighted in studies where passive transfer of antibody blocked recrudescence only during the time of treatment levine and griffin, 1992; levine et al., 1991) . molecular cues in the brain microenvironment orchestrate asc recruitment, retention, and maturation. ascs remain in the cns to block reactivation of virus replication. the mechanisms for recruitment and retention have been characterized during mhv and sinv infection (metcalf and griffin, 2011; marques et al., 2011; lin et al., 1999; tyor and griffin, 1993) . the chemokine receptor cxcr3 and its ligand cxcl10 are critical for recruitment of ascs to the cns during mhv infection (phares et al., 2013; gil-cruz and perez-shibayama, 2012; marques et al., 2011) . cxcl10 is also elevated during sinv, tmev, and rabies virus infections, although its role in asc recruitment has not been defined (rainey-barger et al., 2011; kuang et al., 2009; phares et al., 2006; hoffman et al., 1999) . infiltrating b cells early in infection are naïve/early activated but progress to a more differentiated, isotype-switched phenotype. consequently, asc that first enter the cns produce igm, likely contributing to neutralization of free virus. through the course of infection, igg predominates with iga also present and declining levels of igm (phares et al., 2014; metcalf and griffin, 2011; tschen et al., 2002; tyor and griffin, 1993) . long-term expression of b cell activating factor (baff) and a proliferating-inducing ligand (april) in the brain likely support asc survival (metcalf et al., 2013; metcalf and griffin, 2011; phares et al., 2011; tschen 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mohammadi, soheil; moosaie, fatemeh; aarabi, mohammad hadi title: understanding the immunologic characteristics of neurologic manifestations of sars-cov-2 and potential immunological mechanisms date: 2020-09-01 journal: mol neurobiol doi: 10.1007/s12035-020-02094-y sha: doc_id: 262786 cord_uid: otxpc46a similar to its predecessors, coronavirus disease 2019 (covid-19) exhibits neurotrophic properties, which lead to progression of neurologic sequelae. besides direct viral invasion to the central nervous system (cns), indirect cns involvement through viral-mediated immune response is plausible. aberrant immune pathways such as extreme release of cytokines (cytokine storm), autoimmunity mediated by cross-reactivity between cns components and viral particles, and microglial activation propagate cns damage in these patients. here, we review the currently available evidence to discuss the plausible immunologic pathways that may contribute to the development of covid-19 neurological complications, namely alzheimer’s disease, parkinson’s disease, stroke, multiple sclerosis, guillain-barre syndrome, seizure, and brainstem involvement. in december 2019, the outbreak of novel coronavirus disease 2019 (covid-19) emerged in wuhan, china. to date, the outbreak has turned into a pandemic, infecting millions of people globally [1] . although the virus, also known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), mainly manifests as an acute respiratory infection [2] , recent evidence suggests that 36% of affected patients exhibit neurological sequelae [3] . neurologic symptoms are more common among severe cases of the disease, regarding that 84% of the intensive care unit (icu)-admitted sars-cov-2 patients exhibited at least one neurologic symptom [4] . in line with these, brain magnetic resonance imaging (mri) investigations in sars-cov-2 patients show multifocal hyperintense white matter lesions and cortical signal abnormalities (particularly in the medial temporal lobe) on fluid-attenuated inversion recovery (flair), along with intracerebral hemorrhagic and microhemorrhagic lesions, a n d l e p t o m e n i n g e a l e n h a n c e m e n t [ 5 , 6 ] . electroencephalogram (eeg) studies demonstrate high amplitude monomorphic delta waves, indicating the central nervous system (cns) involvement in sars-cov-2 patients [7] . also, functional magnetic resonance imaging (fmri) is recently proposed as an independent predictor of neurologic outcomes [8] . sars-cov-2 patients present with elevated plasma levels of neurofilament light chain protein (nfl) and glial fibrillary acidic protein (gfap), which are known as biochemical indicators of neuronal injury and glial activation, respectively [9] . also, postmortem brain autopsies demonstrate virus invasion to different brain regions, including the hypothalamus and olfactory bulb, accompanied by neural death and demyelination [10, 11] . several pathogenic mechanisms have been proposed for the neurological deficit in sars-cov-2: indirect cns involvement through systemic inflammation, direct invasion of the virus into the cns, multi-organ failure, hypoxia, sepsis, etc. [12, 13] . in this article, we aim to revisit the possible pathomechanisms that may contribute to development of neurologic complications in patients afflicted with this virus with a substantial focus on immunological pathways. angiotensin-converting enzyme 2 (ace-2) [19] . further investigations in rodents show that ace-2 is expressed in brain regions like cortex and raphe and brainstem, predominantly in neurons [19] . also, spatial distribution analysis reveals that ace-2 is highly expressed by neurons and glial cells in different human brain regions, including middle temporal gyrus, cingulate gyrus, substantia nigra, choroid plexus, and, to a lesser extent, hippocampus [20] . a c e -2 i s a t y p e i t r a n s m e m b r a n e metallocarboxypeptidase that acts as a receptor for sars-cov as well as sars-cov-2 [21] . binding of viral spike (s) protein to ace-2 receptors accompanied by proteolytic cleavage of viral spike protein mediated by transmembrane serine protease 2 (tmprss2) facilitates cell entry ( fig. 1 ) [21] . systemic inflammatory response syndrome (sirs) is defined as excessive host immune response against noxious stimuli (e.g., viral infection), through which the primary protective role of cytokine release turns into a detrimental response against host tissues, leading to impaired integrity of capillary walls and end-organ dysfunction [22] . sirs accounts for the neurologic sequelae found in sars-cov patients [23] . sars-cov infects macrophages, monocytes, and dendritic cells and upregulates the expression of proinflammatory cytokines (e.g., tumor necrosis factor-α (tnf-α), interleukin-6 (il-6)), and inflammatory chemokines fig. 1 three phases of cns infection by sars-cov-2. first, the virus directly invades the brain through vascular or retrograde axonal transport within the cribriform plate, and thus the viral load increases in the csf. second, the virus infects, replicates in, and kills neural cells through ace-2 receptors and tmprss2, a facilitator of viral entry. finally, in the third phase, the immune-mediated response against the virus can indirectly involve the brain, although the viral replication declines in this last phase. cns central nervous system, ace-2 angiotensin-converting enzyme 2, tmprss2 transmembrane serine protease 2. adopted from https://www.dpz.eu/de/infothek/wissen/coronaviren.html, by markus hoffmann. adopted with permission (c-c motif chemokine ligand 2 (ccl2), c-c motif chemokine ligand 3 (ccl3), c-c motif chemokine ligand 5 (ccl5), c-x-c motif chemokine 10 (cxcl10)) [24] . complicated cases of sars-cov exhibit higher levels of proinflammatory cytokines like il-6 and interferon-γ (ifn-γ), making them more susceptible to neurologic complications [25] . studies on postmortem cases indicate that lymphocytes and monocytes infiltrate in brain vessel walls, exacerbating the neuronal degeneration and demyelination process [17] . last but not least, indirect immunofluorescence staining and cell-based enzyme-linked immunosorbent assay (elisa) study in the serum of sars-cov patients reveal the presence of igg and igm autoantibodies as well as complement cytotoxicity against human epithelial and endothelial cells [26] . this finding implies a role for autoimmunity in postinfectious complications of sars-cov. middle east respiratory syndrome (mers), another respiratory infection from the cov family, was first reported in saudi arabia in 2012. nearly 20% of mers-cov patients develop neurologic manifestations during the disease course [17] . however, the neurologic sequelae do not always occur in the infection process and might manifest after a delay of 2 or 3 weeks [17] . stroke, encephalopathy, seizure, gbs, and brainstem involvement could accompany mers infection [16, 17] . brain mri in neurologically affected mers-cov patients reveal hyperintense lesions in white matter and subcortical frontal, parietal, and temporal lobes, along with basal ganglia and corpus callosum [27] . the mechanisms of neurological involvement in mers-cov are almost similar to sars-cov, with a surge in circulating cytokine levels and a role for autoreactive autoantibodies [28, 29] . sars-cov-2 invasion throughout the cns can be divided into three phases: first, the virus directly invades the brain through vascular or retrograde axonal transport within the cribriform plate, and thus, the viral load increases in the csf [4] . second, the virus infects, replicates in, and kills neural cells through ace-2 receptors [30] . third, the immunemediated response against the virus can indirectly involve the brain, although the viral replication declines in this final phase [31] (fig. 1) . recent evidence shows that the third phase (also known as sirs) accounts for the majority of cns disturbances mediated by sars-cov-2 [31] . sars-cov-2 invades astrocytes, macrophages, and microglia in cns and induces a proinflammatory state characterized by increased levels of inflammatory mediators including interleukin-1β (il-1 β), interleukin-2 (il-2), il-2 receptor (il-2r), interleukin-4 (il-4), il-6, interleukin-10 (il-10), interleukin-18 (il-18), ifn-γ, tnf-α, granulocyte colony-stimulating factor (gcsf), monocyte chemoattractant protein 1 (mcp-1), macrophage inflammatory protein 1-α (mip-1α), cxcl10, ccl2, and c-reaction protein (crp) [25] . also, sars-cov-2 induces in vitro expression of inflammatory cytokines such as il-6, interleukin-12 (il-12), interleukin-15 (il-15), and tnf-α in glial cells [17] . severe cases of the disease compared with non-severe cases tend to increase in circulatory levels of il-2, il-2r, il-6, il-10, ifn-γ, tnf-α, ccl2, procalcitonin (pct), crp, erythrocyte sedimentation rate (esr), and white blood cell (wbc) and neutrophil counts and decrease in total lymphocyte, cd4+ t lymphocyte, and cd8+ t lymphocyte counts, while b cell count remains in the normal range [24, 32, 33] . also, antibodies (namely igg, igm, and iga) against viral particles rise in the disease course [34] . seroconversion of igm and igg antibodies begins 4 days after the symptom onset [35] . although the median time for seropositivity against the receptor-binding domain of the virus in the serum is equal for different isotypes, igg is estimated to remain positive in the serum for 75 days, that is the longest period comparing with iga and igm, lasting for 51 and 47 days, respectively [34] . of note, the social isolation and the resultant stress exacerbate the aforementioned cytokine release in affected patients [25] . cytokine storm is a term used for the extreme release of interferons, tumor necrosis factors, chemokines, interleukins, and other mediators in a hyperactive and injurious manner against host tissues [36] . cytokine storm is attributed to sars-cov-2 as well as sars-cov infection and accompany with a spectrum of sirs manifestations, including hypotension, tachycardia, tachypnea, and fever, leading to adverse clinical outcomes [36] . cytokine storm involves the cns, regarding the fact that most cytokines are either produced in the brain tissue or able to cross the blood-brain barrier in the condition that released cytokines (namely, il-1β, il-6, and tnf-α) disrupt the integrity of blood-brain barrier [37] . so, brain autopsy studies of postmortem sars-cov-2 patients demonstrate the infiltration of monocytes, macrophages, and t-lymphocytes into the vessel walls ( fig. 2) [15] . also, interferon-α2 (ifn-α2) and ifn-γ upregulate the expression of ace-2 receptors and, thus, facilitate the entry of the virus into host cells in-vitro [38] . cytokine storm results in neural death, activation of microglia, disruption of synaptic plasticity, and impairment in neurotransmitter metabolism. cytokine storm is also a predictor of hippocampal atrophy [12] . cytokine storm stimulates the secretion of glucocorticoids through manipulation of the hypothalamic-pituitary-adrenocortical axis; nevertheless, the resultant increase in glucocorticoids is not able to suppress the inflammation due to impaired negative feedback mechanism [25] . last but not least, the cytokine storm might lead to delayed immune dysregulation after the disease course, manifested by persistent inflammation or immunosuppression, which might cause delayed complications [25] . cns features of sars-cov, mers-cov, and sars-cov-2 are summarized in table 1 . regarding the widespread influence of sars-cov-2mediated immune response on cns, we discuss the inflammatory correlates of neurologic sequelae of sars-cov-2 in the following sections. brain areas like the hippocampus and midbrain are vulnerable to direct sars-cov-2 invasion and induction of alzheimer's disease (ad) and parkinson's disease (pd), respectively [18] . the aberrant immune response characterized by a surge in cytokine levels (e.g., il-6) derived by sars-cov-2 accelerates the process of neurodegeneration that may contribute to the development of neurodegenerative diseases [42] . the mentioned aberrant immune response may be a consequence of infection of intestinal mucosa and modulation of the gut microbiome by sars-cov-2, which can trigger neuroinflammation and neurodegeneration [24] . interestingly, modulation of the gut microbiome plays a role in the pathophysiology of ad and pd [43, 44] . seemingly, other coronaviruses like human coronavirus oc43 (hcv-oc43) may trigger delayed neural degeneration in animal models [45] . alzheimer's disease ad, the most prevalent neurodegenerative disorder, is characterized by the accumulation of intraneuronal aggregates of hyperphosphorylated tau and extracellular beta-amyloid plaques [37, 46, 47] . considering the role of cholesterolbinding protein (apolipoprotein e (apoe)), a potential link between ad and sars-cov-2 infection is implicated. serum cholesterols bind to apoe receptors and induce ace-2 receptor transport to the cell surface [48] . super-resolution imaging study demonstrates that high cholesterol levels confer a 2-fold increase in sars-cov-2 entry sites and thus facilitate the viral entry [48] . apoe and ace-2 are highly expressed in alveolar type ii cells, and the apoe e4 allele modulates the pro-/anti-inflammatory state in macrophages [49] . on the other hand, apoe, acting in conjunction with apoc1 and apoj, transports cholesterol to maintain the myelin and neural membranes, dendritic reorganization, and synaptic turnover [50] . apoe functions as a beta-amyloid chaperone through the transport of beta-amyloids to the lysosomes [50] . apoe e4 allele associates with increased formation and deposition of beta-amyloid, and apoe e4-positive mice exhibit increased accumulation of tau aggregates [50] . in line with these, homozygote apoe e4e4 not only acts as a risk factor for severe sars-cov-2 infection but also confers a 14-fold increase in susceptibility to ad [49] . a growing body of evidence implicates that neuroinflammation may be an origin of ad. persistent systemic inflammation can activate glial cells like microglia and astrocytes. activated microglia secrete pro-inflammatory cytokines (e.g., il-1β, il-6, il-12, tnf-α) [37, 51] . we hypothesize that not only the persistent systemic inflammation caused by sars-cov-2 may act as a trigger for microglial activation but also large amounts of pro-inflammatory cytokines secreted in response to this viral infection may aggravate neurodegeneration leading to ad. elevated levels of tnf-α associate with a 4-fold increase in cognitive dysfunction [37] . increased il-1β and il-6 mitigates microglial phagocytosis of β-amyloid plaques and thus impairs synaptic plasticity and memory [19, 25] . ifn induces synaptic degeneration as a mediator of post-viral inflammatory response. it is worth knowing that βamyloids can encircle viral particles and enhance the aforementioned ifn-mediated response [52] . on the other hand, nod-, lrr-, and pyrin domain-containing protein 3 (nlrp3) inflammasomes play a role in microgliamediated il-1β release in ad. sars-cov-2 open reading frame 3a (orf3a) protein stimulates nlrp3 inflammasomes, activation of which accelerates the neurodegeneration process in ad [12] . pd, the second most common neurodegenerative disorder, is characterized by the degeneration of dopaminergic neurons of substantia nigra pars compacta, along with microscopic findings of intracellular α-synuclein aggregates [53] [54] [55] [56] . autoantibodies against α-synuclein, gangliosides, pigment neuromelanin ma1/ma2, leucine-rich glioma inactivated 1 (lgi1), and glutamic acid decarboxylase (gad65) are found in the serum of pd patients, which implicate a role for autoimmunity in the pathogenesis of pd [53] . knowing that sars-cov-2 may remain latent in neurons, we hypothesize that autoimmunity in sars-cov-2 infection due to crossreactivity mediated by antiviral antibodies may lead to delayed progression of pd [28] . this hypothesis is supported by the fact that anti-cov antibodies are found in the csf of pd patients [28] . we also postulate that aberrant cytokine release into the csf of sars-cov-2 patients may increase the risk of progression into pd by aggravating the neurodegeneration process, considering the fact that pd patients exhibit elevated levels of pro-inflammatory cytokines (including il-1β, il-6, tnf-α, and ifn-γ) in csf. cytokine storm activates microglia, which are implicated in the pathophysiology of pd [57] . moreover, the extreme release of cytokines into the csf mediated by sars-cov-2 dysregulates the balance between production, release, and reuptake of neurotransmitters including dopamine, which possibly contributes to the development of pd [25] . moreover, the common finding of anosmia (total loss of sense of smell) in pd and sars-cov-2 reinforces their association. anosmia is a common finding in neurodegenerative diseases [58] , and over 90% of pd patients develop anosmia or hyposmia (partial loss of sense of smell). anosmia may precede motor symptoms of pd and thus can be used as a marker of underlying neurodegeneration [59] . although mri studies of pd patients show that volume and sulcus depth of olfactory bulb decrease, biopsy samples of olfactory epithelium are normal in these patents [60] . these findings suggest that the observed anosmia in these patients is due to a disturbance in central and not peripheral processing of smell [60] . knowing that autopsy studies of postmortem pd patients suffering from anosmia show abundant deposition of lewy bodies in the olfactory bulb, anosmia is attributed to the distribution of lewy bodies from the medulla in early stages of pd [61] . however, in later stages, cognitive deficits and cholinergic denervation may underlie the development of anosmia in these patients [60] . on the other hand, a recent review reports a nearly 23% prevalence for anosmia in sars-cov-2 patients [62] . several explanations are suggested to shed light on the underlying cause of anosmia in sars-cov-2 patients. at first, it was hypothesized that the local inflammation and the resulting coryza and sinusitis account for anosmia in these patients. however, recent imaging studies demonstrate no signs of sinusitis in sars-cov-2 patients. moreover, the presence of anosmia in the absence of other symptoms suggests that coryza cannot be the reason for olfactory dysfunction in sars-cov-2 [15] . indeed, milder inflammation and inflammatory infiltrations in sars-cov-2 infection comparing with sars suggest a different route of infection through which the virus transmits via infected macrophages to the supporting cells of the olfactory epithelium and olfactory bulb, leading to impairment of olfactory receptors [63] . several findings support this latter hypothesis: first, olfactory epithelial cells (particularly sustentacular cells) and olfactory bulb cells express ace-2 and tmprss2 [64, 65] . second, postmortem studies show that the virus can invade the brain through the olfactory epithelium and olfactory bulb [66] . third, positron emission tomography-computed tomography (pet-ct) study in sars-cov-2 patients with anosmia demonstrate declined orbitofrontal cortex activity, suggesting neural dysfunction [67]. forth, the genome sequence of the virus was found in the olfactory mucosa and olfactory bulb, indicating axonal transport [66] . the latter hypothesis, also known as post-viral olfactory dysfunction, is reported in a case of sars-cov as well and is more prevalent among patients with a history of the neurologic disease [64] . maintenance of viral load in the olfactory epithelium in post-viral olfactory dysfunction can disrupt the regenerative capacity of neurons [64] . both ischemic and hemorrhagic strokes are being increasingly reported in sars-cov-2 patients and are the most common findings in neuroimaging studies (table 2) [40, 75] . the prevalence of stroke is estimated to be between 0.2 and 1% of all infected patients [76] , often proposed as the most common neurologic manifestation in sars-cov-2 patients [77] . high incidence of stroke in sars-cov-2 patients under 50 years old without cardiovascular risk factors accompanied by worse outcomes in sars-cov-2-mediated stroke comparing with other strokes indicates that this viral infection precipitates the pathways leading to stroke [76] . postmortem autopsy studies of sars-cov-2-mediated stroke demonstrate hyperemic and edematous brain areas [76] . also, micro-and macrothrombosis are found in other organs such as lung and kidney [76] . these findings indicate that coagulopathy may underlie the occurrence of stroke in these patients. severe inflammatory response mediated by sars-cov-2 can upregulate pro-coagulative factors [17] . recent investigations suggest that blood d-dimer, crp, fibrinogen, and wbc count increases in sars-cov-2 patients, which propagate the hypercoagulable state. moreover, a group of sars-cov-2 patients present positive lupus anticoagulant, anti-β2-glycoprotein-1, and anticardiolipin (marker of antiphospholipid syndrome) antibodies, which may precipitate stroke [15] . another key contributor to stroke is the vascular wall injury, which induces the release of tissue factor [17] . damage to vascular walls may occur as a consequence of direct viral invasion and replication in the vascular epithelium, also known as endotheliitis [66] . endotheliitis due to sars-cov-2 is reported in other organs such as lung, heart, kidney, and bowel but not yet investigated in cns [15] . also, the aberrant immune response mediated by sars-cov-2 may provide the driving force for vascular wall damage [23] . this process takes place as a component of secondary hemophagocytic lymphohistiocytosis, characterized by inflammatory-mediated tissue damage through hyperactivation of macrophages and lymphocytes, leading to microangiopathy [15] . several other mechanisms may increase the risk of stroke in sars-cov-2 infection. the cytokine storm mediated by sars-cov-2 precipitates microthrombosis [41] . in fact, elevated expression of pro-inflammatory cytokines (namely il-1β, il-6, and tnf-α) increases the risk of ischemic and hemorrhagic strokes [78] . also, anti-cytokine therapies against these mediators protect from recurrence of stroke in experimental models [78] . moreover, systemic inflammation can ignite atrial fibrillation and rupture the carotid plaques, leading to ischemic stroke [15] . also, sars-cov-2 downregulates the expression of ace-2 receptors, which degrade angiotensin ii. so, the increase in angiotensin ii and the resulting activation of the renin-angiotensin-aldosterone system (raas) system promotes sympathetic tone, oxidative stress, and inflammation and triggers fibrotic events [79] . multiple sclerosis (ms) is a chronic disabling disorder characterized by demyelination of white matter and, to a lesser extent, gray matter of cns [51] . recently, the first casereport of ms after sars-cov-2 infection was presented in the literature [80] . a sars-cov-2 patient without a history of in flair images along with abnormalities in frontal, [5] neurological symptoms developed visual acuity and field defects and hyperreflexia. orbital and brain mri investigations demonstrated enhancement of left optic nerve accompanied by demyelinating lesions in supratentorial periventricular areas of occipital and temporal lobes (table 2) . interestingly, csf pcr analysis of sars-cov-2 was negative, while csf oligoclonal igg and igm were positive, conferring a postinfectious immunological response as an etiology for this phenomenon [80] . an autoimmune inflammation mediated by activated microglia and cytotoxic t-cells against oligodendrocytes underlies the myelin degeneration in ms [51] . elevated levels of pro-inflammatory cytokines (e.g., tnf-α, il-6, ifn-γ, il-1β) are found in the csf of ms patients suffering from either white matter or gray matter deficits [24] . tnf-α is isolated from active areas of neurodegeneration in postmortem ms brains [57] . overexpression of tnf-α stimulates apoptosis of oligodendrocytes and infiltration of immune cells into the csf, while intrathecal injection of tnf-α monoclonal antibodies prevents from the development of ms in animal models of experimental autoimmune encephalomyelitis (eae) [57] . il-6 maintains the balance between cytotoxic and regulatory t-cells. overproduction of il-6 inhibits the differentiation of t-cells into regulatory t-cells and thus promotes the inflammatory state and demyelination in mice [57] . in line with this, il-6 deficient mice are resistant to ms, and administration of il-6 receptor (il-6r) antibodies inhibits the myelin degeneration in eae mice [57] . moreover, the aberrant release of il-6 disrupts the blood-brain barrier integrity and facilitates the infiltration of cytotoxic t-cells [57] . last but not least, il-1β receptor (il-1βr) antibodies antagonize the demyelination process in rat models of eae, which suggests a role for il-1β in the pathogenesis of ms [81] . after all, knowing that the aforementioned cytokines surge as a result of sars-cov-2 mediated inflammation, we postulate that persistent inflammation in sars-cov-2 may propagate myelin destruction and lead to delayed progression of ms. moreover, it is hypothesized that an autoimmune reaction mediated by the cross-reaction between viral particles and myelin basic protein may provide the driving force for neural demyelination. this hypothesis is supported by the fact that genome of other coronaviruses like cov-oc43 and cov-229e, as well as their antibodies, are isolated from cns of ms patients, and coronavirus-like particles are found in perivascular cuffs of human ms brain [82] . in fact, the virus might lie dormant in astrocytes and oligodendrocytes and trigger the autoimmunity mediated by molecular mimicry. interestingly, intracranial inoculation of murine coronavirus (mouse hepatic virus (mhv)), induces optic neuritis and focal encephalitis leading to chronic demyelination in mice [83] . in this case, both innate and adaptive immune responses comprising of activation of microglia, b cells, cd4 t cells, and [74] cd8 t cells along with the direct viral toxicity are proposed to mediate myelin stripping [68, 70] . gbs i s a pos t-in fectiou s autoimmune caus e of polyneuropathy in predisposed individuals that leads to flaccid paralysis of lower extremities, along with autonomic and sensory deficits [69] . autoimmunity is the milestone of gbs pathophysiology. neurotrophic macrophages present bacterial or viral particles to lymphocytes in peripheral nervous system (pns) [69] . the resultant immune response (i.e., complement and macrophage activation, cytokine release, and free radical production) accompanied with the molecular mimicry between pathogen epitopes and host gangliosides such as n-acetylgalactosaminyl gd1a (galnac-gd1a), ganglioside d1a (gd1a), ganglioside (gm1), and ganglioside m1b (gm1b) and myelin proteins result in acute neural damage [69] . gbs is reported in other post-viral infections, including influenza, enteroviruses, zika, sars-cov, mers-cov, and sars-cov-2 [13] . several cases of gbs due to sars-cov-2 infection are presented in the literature with the varying onset of gbs symptoms from a day before to 3 weeks after presentation of sars-cov-2 symptoms [71, 84] . enhancement of cervical and cranial roots (e.g., optic, oculomotor, and facial nerves) in mri studies along with presence of axonal and demyelinating patterns in nerve conduction studies are suggestive of gbs in sars-cov-2 patients (table 2 ) [71, [84] [85] [86] . several findings indicate that the occurrence of gbs in sars-cov-2 is mediated by an autoimmune response mediated by molecular mimicry and not the direct viral invasion. binding of sars-cov-2 spike protein to cell surface occurs not only through ace-2 receptors but also through sialic acid-containing glycoproteins and gangliosides. considering the fact that anti-ganglioside antibodies are found in the serum of sars-cov-2-triggered gbs patients, a cross-reactivity between sars-cov-2-binding gangliosides and peripheral nerve glycolipids are implicated [87] . also, recent experiments are not able to detect the viral genome in csf of a high proportion of post-sars-cov-2 gbs patients [23, 39, 72] . moreover, the administration of intravenous immunoglobulin (ivig) resolves symptoms and improves the outcomes in post-sars-cov-2 gbs patients [59] . other plausible theories may underlie the development of gbs in sars-cov-2. a shift in immune response toward pro-inflammatory cytokines (e.g., il-1β, il-6, tnf-α, and ifn-γ) occurs in acute stages of gbs and mediate neural degeneration [69] . similarly, elevated levels of il-1β and il-6 are found in the csf of gbs patients [69] . so, the release of pro-inflammatory cytokines in sars-cov-2 may propagate the neurodegeneration leading to gbs. also, the sars-cov-2-mediated cytokine storm may result in disruption of blood-brain barrier integrity and infiltration of lymphocytes leading to gbs [73] . recent retrospective, case series, and case report studies indicate that sars-cov-2, as well as sars-cov and mers-cov patients, is prone to encephalopathy and seizure [88] . brain mri studies of these patients demonstrate multifocal involvement of cortex and white matter (including periventricular areas), along with multiple hyperintense areas in t2-weighted and flair images in temporal, frontal, and occipital lobes and hippocampus (table 2 ) [74, 89] . consequences of sars-cov-2 infection, including organ failure, hypoxia, metabolic disturbance, and direct viral invasion into the cns, are proposed to explain the observations of encephalopathy and seizure in these patients [88] . however, most findings are in favor of immune-mediated encephalopathy and seizure. except for two case reports, all the remaining studies are not able to detect the viral genome in the csf of these patients [41] . seemingly, most patients exhibit a dramatic response to plasmapheresis and steroid therapy, which indicates a role for autoimmune-mediated injury [23] . as mentioned before, the aberrant release of pro-inflammatory cytokines like il-1β, il-6, and tnf-α disrupts blood-brain barrier permeability, and the resulting infiltration of wbcs and activation of microglia and astrocytes damages neurons and glial cells [24, 41] . also, it is speculated that the viralmediated production of autoantibodies against glial cells might be responsible for neural injury [74] . transgenic mice infected by sars-cov and mers-cov exhibit brainstem involvement due to trans-synaptic projection of the virus throughout the olfactory epithelium, olfactory bulb, thalamus, and finally the brainstem [16] . the virus might target the primary respiratory oscillator (known as pre-bötzinger complex) in the brainstem and result in respiratory failure in these animal models [16] . seemingly, acute respiratory failure due to brainstem dysfunction is reported in sars-cov-2 patients [90] . also, a recent case study reports post-infectious brainstem involvement presenting with involuntary movements, myoclonus, and hyperekplexia after gradual respiratory symptom relief in a case of sars-cov-2 [91] . postmortem autopsy studies elucidate areas of hyperemia and edema and neurodegeneration in the brainstem [90] . mri findings in a case of brainstem involvement demonstrate high signal lesions in bilateral pons in t2-flair images ( table 2 ) [92] . these findings are in favor of brainstem involvement due to direct viral invasion. however, the absence of viral genome in csf along with the presence of inflammatory mediators (namely tnf-α and interleukin-8 (il-8)) and pleocytosis in csf in a group of patients with brainstem dysfunction suggests that the aberrant immune response might underlie the brainstem dysfunction in sars-cov-2 [90] . sars-cov-2 pandemic, beginning in december 2019, has now become a global concern. although the acute respiratory manifestations of the disease are greatly considered, reports of neurologic sequelae are dramatically increasing. a recent cohort study of sars-cov-2 patients reported a 4.3% prevalence for neurologic symptoms, other than anosmia and headache [93] . also, para-clinic investigations support the idea of post-sars-cov-2 cns involvement ( table 2) . although the cns complications might be a consequence of direct viral invasion into the cns, recent findings are in favor of immune-mediated cns involvement [31] . sars-cov-2 genome sequences are frequently undetectable in the csf [93] . also, recent studies suggest that blood-brain barrier integrity is disrupted, and lymphocytic pleocytosis and wbc infiltration into the cns occur in these patients [93] . interestingly, the presence of auto-antibodies against gangliosides in sars-cov-2 patients suggests that an autoimmune phenomenon mediated by the virus might underlie the neurologic symptoms [93] . as a result, we propose that the aberrant immune response mediated by sars-cov-2 is responsible for the majority of acute or chronic neurological manifestations. in fact, sars-cov-2 infection increases the risk of alzheimer's disease, parkinson's disease, stroke, multiple sclerosis, guillain-barre syndrome, seizure, encephalopathy, and brainstem involvement through different immune pathways including fig. 3 the pathomechanisms of immune-mediated cns involvement in sars-cov-2 infection. sars-cov-2 induces neurodegeneration through cytokine storm, autoimmunity, microglial and inflammasome activation, and gut microbiome modulation. these pathways lead to development of alzheimer's disease in susceptible individuals and worsen the severity of parkinson's disease in affected patients. inflammatorymediated vascular wall injury, atrial fibrillation, carotid plaque rupture, and upregulation of pro-coagulative factors may increase the risk of stroke in these patients. cytokine storm and autoimmunity induced by cross-reactivity between viral particles and cns components may contribute to the development of autoimmune disorders of cns, like guillain-barre syndrome. also, the aforementioned mechanisms exacerbate symptoms in multiple sclerosis patients. cytokine storm induces microglial activation and wbc infiltration into the cns and, thus, precipitates seizure and encephalopathy. cns central nervous system, wbc white blood cell. adopted from https://www.dpz.eu/de/infothek/wissen/ coronaviren.html, by markus hoffmann. adopted with permission cytokine storm, autoimmunity, microglial and inflammasome activation, gut microbiome modulation, wbc infiltration into the cns, inflammatory-mediated vascular wall injury, and upregulation of pro-coagulative factors (fig. 3) . precise investigation and follow-up of sars-cov-2 patients is warranted in this issue, as the delayed presentation of these complications may lead to underdiagnosis. immunomodulatory drugs such as cytokine-blocking therapies (e.g., il-6r monoclonal antibodies, tnf-α inhibitors, il-1β antagonists), antibodies against viral spike proteins and viral binding receptors (ace-2), anti-inflammatory drugs like nonsteroidal antiinflammatory drug (nsaid), and prophylactic antibodies are under investigation in the prevention and treatment of sars-cov-2 [94, 95] . however, the efficiency of these drugs to decline the burden of short-term and long-term neurological manifestations of sars-cov-2 is yet to be investigated. a systematic review of covid-19 epidemiology based on current evidence di napoli r (2020) features, evaluation and treatment coronavirus (covid-19) neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan, china neurologic features in severe sars-cov-2 infection brain mri findings in patients in the intensive care unit with covid-19 infection brain mri findings in 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associated with sars-cov-2 evolution and resolution of brain involvement associated with sars-cov2 infection: a close clinical-paraclinical follow up study of a case immune-mediated neurological syndromes in sars-cov-2-infected patients covid-19: the inflammation link and the role of nutrition in potential mitigation therapeutic strategies in the development of anti-viral drugs and vaccines against sars-cov-2 infection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no conflict of interest. key: cord-288111-0ufc54kw authors: ter meulen, volker title: autoimmune reactions against myelin basic protein induced by corona and measles viruses date: 2006-12-17 journal: ann n y acad sci doi: 10.1111/j.1749-6632.1988.tb27062.x sha: doc_id: 288111 cord_uid: 0ufc54kw nan when rats are infected with jhm virus at the age of 21 to 35 days, they develop either acute encephalomyelitis ( ae) or subacute demyelinating encephalomyelitis ' the experiments described in this manuscript were supported by deutsche forschungsgemeinschaft. the acute disease has an average incubation period of 6-12 days and follows a rapidly progressing clinical course characterized by paralysis and early death. examination of the neurologic tissues of these animals reveals changes limited to gray matter of both the brain and spinal cord, consisting of necrosis of the neurons and consequent infiltration by granulocytes, lymphocytes, and macrophages. viral antigen is evident in neurons and glia, and the culprit virus is readily isolated from the brain and spinal cord tissues. in contrast, sde has an incubation period of 2 weeks to 8 months, followed by a slow onset of disease characterized by paralysis of the hind legs and ataxic gait, eventually culminating in tetraplegia. the most prominent neuropathologic changes include primary demyelination in the white matter of the optic nerves, midbrain, pons, cerebellum, and spinal cord. within the demyelinated plaques are well-preserved axons and neurons. there is also perivascular cuffing by lymphocytes and other mononuclear cells, as well as infiltration by macrophages. here, too, the viral antigen can be recognized in the tissues, but it is primarily found in the glia and in the proximity of the plaques. the virus can also be isolated by conventional techniques, regardless of the length of the incubation period. many of these animals eventually recover and show evidence of remyelination. some animals that survive sde and recover later experience a second attack of the neurologic disease! between 60 and 120 days following the first episode of disease, the animals get sick again, with identical symptoms that are now more severe. histopathologic abnormalities consist of new demyelinative lesions infiltrated by mononuclear cells located primarily in the brain stem and the spinal cord. ?aoreover, there are old lesions where they were previously noted, revealing extensive remyelination indicative of a repair mechanism that had developed after the initial disease. in contrast to the first attack of sde, following the second attack, isolation of virus is a rarity, even as viral antigen is readily detected in glia and the region surrounding the plaque. because jhm infection in the lewis rat resembled some characteristics of experimental allergic encephalomyelitis (eae), we decided to compare the infection with this virus to that of another inbred rat, bn, which is resistant to eae. weanling bn rats can be infected with the jhm virus, but the majority of these animals remain clinically well despite histologic changes of subacute demyelinating encephalomyelitis. virus can be isolated from their brains, but only up to 10 days after infection, even as fresh cns lesions continue to develop and viral antigen persists beyond that period. in contrast to sde in lewis rats, in bn rats the main neuropathologic lesions consist of small nodular demyelinated plaques predominantly located in the periventricular white matter, an area in which lewis rats only rarely develop changes. these nodular lesions, which differ from the demyelinated plaque seen in infected lewis rats, contain microglia, phagocytes, and astrocytes with swollen cytoplasm. in addition, the inflammatory response is expressed mainly by plasma c e k 5 obviously, therefore, an important pathogenic mechanism in response to the jhm infection depends on the genetic characteristics of the animal. infection of weanling lewis rats with neurotropic measles virus cam/rbh results in either acute measles encephalitis or subacute measles encephalitis (same)! the acute encephalitis is characterized by a short incubation period and focal infiltration by mononuclear cells of gray matter of both cerebral hemispheres and basal ganglia. there are usually few or no degenerative changes. same develops in about 20% of the infected animals, after an incubation period of 3 weeks to 3 months. the earliest sign of the disease is an arrest of weight gain or even weight loss. this is followed by generalized hyperexcitability, unsteadiness, abnormal posture, paresis of the limbs, and occasional seizures. this phase of the disease lasts for several days up to 3 weeks and has a case fatality rate of 50%. histopathologic changes consist of prominent lymphomonocytic perivascular cuffing. there are no demyelinated plaques. 'the survivors recover completely and remain well for at least 8 months. infection of weanling bn rats results in acute encephalitis in a small percentage only; the majority develop clinically silent encephalomyelitis (cse). the histopathologic lesions in animals with cse consist of inflammatory lesions, widespread proliferation of the glial cells, and multicystic parenchymal degeneration. infectious virus is easily isolated from the cerebral hemisphere of both types of rats with acute encephalitis. however, in contrast, neither in same in the lewis rats nor in cse in the bn rats could infectious virus be isolated by conventional techniques or by co-cultivation, despite the detection of viral antigen. this failure of isolation could be attributed to restriction of gene expression of the measles virus occurring at the level of virus envelope proteins. as a result, infectious virus particles cannot be assembled, because the matrix protein required for the budding process is not synthesized. at no time during the course of the disease has virus been recovered from their lungs, liver, kidneys, spleen, or thymus. host response, both humoral and cell-mediated immunity (cmi), plays a major role in controlling virus infections. specific antibodies neutralize extracellular virus and protect against reinfection; in some circumstances they also eliminate infected host cells by antibody-dependent cytotoxicity. cmi reactions, however, are the predominant mechanism that destroys infected cells. individuals with an inherited defect in the cmi develop severe complications of those infectious diseases that tend to be benign in normal individuals. in cns infections, intrathecal synthesis of virus-specific antibodies is an important defense mechanism and is well documented in human patients.'-'' it is the b lymphocytes that are responsible for the intrathecal synthesis of the antibodies, which are of an oligoclonal nature as determined by agarose electrophoresis or isoelectric focusing. it is possible to determine viral specificity of the igg clones in the csf specimens by immunoprint fixation i' or immunoblot te~hnique.'~ this determination is important in diagnosis. in rats infected with either j h m or measles virus that develop sde or same, the same diagnostic techniques have been applied by us'5-" with the following findings. in the lewis rats there was only an occasional intrathecal virus-specific response despite the presence of oligoclonal igg. it is therefore probable that these immunoglobulins of restricted heterogeneity are directed against nonviral antigens, which are likely to be antigens of the central nervous tissues. conversely, in the bn rats the oligoclonal intrathecal antibodies were specific against the virus used in the original infection. it is likely, therefore, that in the bn rat these antibodies tend to play a protective role, whereas in the lewis rat no such protection is afforded. an analysis of the cmi response in sde and same revealed a reaction against the jhm, or measles, virus and in the diseased lewis rats a reaction also against myelin basic protein (mbp).'"'' lymphocytes, whether collected from the spleen, thymus, or peripheral blood, all had a proliferative response in the presence of mbp, akin to that in eae. moreover, when lymphocytes from rats with sde or same were infused intravenously into normal rats, the recipients developed symptoms of eae within 5 days, consisting of hypersensitivity to touch and a slightly ataxic gait. histologic examination of the cns tissues of these animals revealed perivascular cuffing with mononuclear cells in the white matter of the spinal cord, where the lesions were in the dorsal columns-the pons, the cerebellum, and the thalamus. infected bn rats did not develop such an autoimmune reaction when tested either by in vitro analysis or by the adoptive transfer of lymphocytes.' we can therefore conclude that the cellular autoimmune reaction is of pathogenic significance in the infected lewis rat. our knowledge of the specific immune responses in the cns to viral infections is quite incomplete. the cns is an immunologic island, considered a privileged site. in order for the lymphocytes to find an antigen in the cns, they must invade its domain and, once there, they must secure a mechanism for identification of the antigens. recent evidence has been brought forth that astrocytes can act as cells presenting antigens.2" on the basis of the information, we endeavored to investigate the effects of the two viruses on astrocytes. both viruses can induce class i1 molecules on cultured astrocytes,2'.22 a property independent of viral replication in the astrocytes because viruses inactivated by ultraviolet irradiation are also effective. apparently this capacity to induce the ia antigen depends on direct interaction of the viruses with the cell membranes of the astrocytes. this interpretation has been derived from the observation that monoclonal antibodies directed against the e2 glycoprotein of the jhm virus or against the hemagglutinin of the measles virus prevent the induction of class i1 antigens. it appears, therefore, that either the viruses bind to specific receptors on the cell surface or the cells phagocytize the viruses and the expression of the ia antigen follows. this mechanism is independent of gamma-interferon. it may be similar to the one described for bacterial endotoxin.23 the phenomenon of ia induction by these two neurotropic viruses has important implications on the mechanisms of pathogenesis. until recently it was assumed that gamma-interferon released by activated t lymphocytes was indispensable to the induction of ia on certain antigen-presenting including astrocytes. because the brain lacks lymphatic drainage and the so-called blood-brain barrier restricts traffic of lymphocytes and macromolecules, gamma-interferon would not be readily available for the induction of ia, especially in the early phase of the infection. what apparently happens is that the viruses themselves induce ia expression on the astrocytes, enabling these cells to present the viral antigens to the lymphocytes and allowing the host to mount an effective immune response to control the infection. although the means by which viruses induce t-lymphocyte responses against host antigens are still unclear, there are a number of speculations to account for this phenomenon. 1. the fact that viruses require living cells for replication has major consequences for the host. during replication, the viruses can incorporate into their envelopes host antigens and they insert, modify, or expose internal cellular antigens on the cell surface. these heretofore "hidden" antigens, now exposed, could appear foreign to the host's immune system. 2. the viruses can interact with the immune regulatory systems by destroying subpopulations of lymphocytes or by stimulating generation of lymphocyte clones that are autoreactive. many viruses are lymphotropic, as exemplified by measles and epstein-barr virus. measles virus replicates in t or b lymphocytes, which leads to lymphocyte destruction and subsequently to an immunosuppressive effect clinically documented by abolition of skin reactions in delayed hypersensitivity. epstein-barr virus infects and transforms human b lymphocytes. the transformed, or immortalized, cells could under certain circumstances secrete antibodies reacting with cellular constituents. 3. molecular mimicry could also be operating. an immune response might be raised against certain viral antigens that cross-react with some native host antigens. a variety of viral sequences, including those of measles virus, were recently compared by computer analysis with myelin basic protein ( mbp).26 the comparison showed amino acid homology between viral proteins and mbp. this observation led to an interesting experiment in which a rabbit was immunized with a synthetic peptide from such a sequence of the hepatitis b virus polymerase?' peripheral blood lymphocytes from the immunized rabbits proliferated in the presence of either mbp or hepatitis b polymerase, and their cns tissue revealed similar neuropathologic changes similar to those in eae. in this context it is of interest to note that in postinfectious encephalomyelitis in man associated with rubella, varicella, or measles, an mbp-specific lymphoproliferative response has been observed?' 4. an autoimmune response could be triggered by the development of an antiidiotypic antibody. in a reovirus model these antibodies directed to type i11 hemagglutinin react with receptors for reovirus on the surfaces of the lymphocytes and nerve cells, and it is possible that this could trigger an autoimmune reaction.29 5 . the last possible mechanism for the development of an immunopathologic reaction in the course of a cns viral infection is the induction by the virus of class i1 antigens on astrocytes and a consequent delayed type hypersensitivity reaction (dth) in genetically susceptible animals. in order for the helper t lymphocytes to recognize viral antigens, class i1 antigens probably have to be present on the astrocytes. however, in extremely high constitutive levels of class i1 expression on astrocytes, an inappropriate or excessive presentation of self-antigens and viral antigens may develop, similar to that in autoimmune processes directed against the thyroid gland.'" this mechanism could well play a role in the jhm-and measles-virus-induced cns disease in lewis rats, because as recently shown by us," these hyperexpressions of ia molecules on astrocytes after contact with gamma-interferon or viral particles are genetically regulated. jhm and measles virus infections in rats are models of a persistent viral infection of the cns with and without demyelination, associated with a cell-mediated immune reaction to mbp. availability of inbred susceptible and resistant rat strains, which react differentially to viral and host antigens, makes possible a variety of experimental permutations aimed at defining the causal mechanisms of cns diseases. these mechanisms can be studied from the points of view of molecular biology and immunology, because both viruses used are well characterized and there are appropriate immunologic markers for the lymphocytes and the brain cells of these rats. this approach can be expected to bring us towards an understanding of the pathogenesis of persistent cns infections that are associated with demyelination and mediated by immunologic reactions. our hope is that information gained from these animal models will have a bearing on studies of related human diseases, particularly parainfectious encephalitis and multiple sclerosis. corona virus induced subacute demyelinating encephalomyelitis in rats: a morphological analysis demyelinating encephalomyelitis induced by a long-term corona virus infection in rats relapsing subacute demyelinating encephalomyelitis in rats in the course of 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encephalitis in ,lewis rats astrocytes present myelin basic protein to encephalitogenic t-cell lines viral particles induce l a antigen expression on astrocytes tumor necrosis factor amplifies measles virus-mediated ia induction on astrocytes b cell activation. 111. â�¬3 cell plasma membrane depolarization and hyper-ia antigen expression induced by receptor immunoglobulin cross-linking are coupled regulation of murine macrophage ta antigen expression by a lymphokine with immune interferon activity regulation of murine macrophage ia expression by an immune interferon-like lymphokine: inhibitory effect of endotoxin sequence homology between certain viral proteins and proteins related to encephalomyelitis and neuritis amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity measles encephalomyelitis: clinical and immunologic studies identification of a hemagglutinin-specific idiotype associated with reovirus recognition shared by lymphoid and neural cells epithelial cells expressing aberrant mhc class 11 determinants can present antigen to cloncd human t cells hyperinducibility of ia antigen on astrocytes correlates with strain-specific susceptibility to experimental autoimmune encephalomyelitis acknowledgment i thank dr. michael katz for helpful discussions and for editing the manuscript. the secretarial assistance of helga kriesinger is gratefully acknowledged. key: cord-309109-c5hajb6k authors: matthews, a. e.; weiss, s. r.; paterson, y. title: murine hepatitis virus—a model for virus-induced cns demyelination date: 2002 journal: j neurovirol doi: 10.1080/13550280290049534 sha: doc_id: 309109 cord_uid: c5hajb6k most murine hepatitis virus (mhv) strains, as their name suggests, infect the liver. however, several murine strains are tropic for the central nervous system (cns) and cause encephalitis with subsequent cns demyelination. the cns demyelination shares pathological similarities with human cns demyelinating diseases such as multiple sclerosis (ms). these viruses are, therefore, used to study the role of the immune system in viral clearance from the cns, in cns demyelination, and in remyelination. nevertheless, it is still unclear exactly how mhv induces demyelination and to what extent the immune system plays a role in this pathology. here we review this field in the context of the immune response to mhv in the liver and the cns focusing on studies that have been published in the past 5 years. in weanling mice compared to adult mice (weiner, 1973) . most studies are performed on 4-to 6-weekold mice. both mhv-a59 and jhm infect, to some extent, oligodendrocytes, astrocytes (lavi et al, 1987; stohlman et al, 1995a) , neurons (parra et al, 1997; lavi et al, 1999) , hepatocytes (weiner, 1973; navas et al, 2001) , macrophages, including kuppfer cells (even et al, 1995; stohlman et al, 1995a) , thymic epithelial cells (knobler and oldstone, 1987; godfraind et al, 1995) , and the endothelial cells which line blood vessels (joseph et al, 1995; lavi et al, 1999) . mhv-a59 has also been shown to infect b cells (coutelier et al, 1994) , and mhv-jhm has been detected in ependymal cells (stohlman et al, 1995a) . except for glial cells and neurons, these cell populations closely match those demonstrated to express the known mhv receptor bgp1a (biliary glycoprotein 1a), a member of the carcinoembryonic antigen (cea) family, which in turn is a member of the immunoglobulin superfamily (williams et al, 1991) . the bgp1a receptor has been identi ed on hepatocytes, macrophages, epithelial cells (such as those in the intestine), endothelial cells, b cells, and a small proportion of thymic epithelial cells (williams et al, 1991) . a major determinant of tropism is the spike (s) protein (phillips et al, 1999; navas et al, 2001) , suggesting that variations in mhv receptor (such as posttranslational modi cations or expression of different isoforms) may affect recognition by different infectious mhv can be found in astrocytes, oligodendrocytes, and neurons after acute infection . late after infection, virus is detectable only as low levels of antigen or rna (lavi et al, 1984b) . in animals with viral persistence due to the absence of ifn-°, antigen is detected mainly in oligodendrocytes (parra et al, 1999) . in mice infected in the presence of protective maternal antibody, antigen persists in astrocytes as well as other unidenti ed cells (perlman and ries, 1987) . these studies demonstrate that virus can persist in glial cells. after intracerebral (i.c.) or intranasal (i.n.) infection with mhv-a59, virus enters the brain and causes encephalitis . intranasal infection with mhv-jhm or -a59 leads to viral invasion through the olfactory bulb and along olfactory tracts, as well as a slower approach along the trigeminal nerve to the mesencephalic nucleus perlman et al, 1989) . early spread of i.) appears to be along speci c neural pathways in the cns (perlman et al, 1989 ) and, therefore, through neural connections. later widespread infection can be blocked by passive transfer of antibody, suggesting spread through the blood, cerebrospinal uid, and/or interstitial uid (perlman et al, 1989) . liver infection can occur after any route of infection (i.n., i.c., i.g., or i.p.) with mhv-a59 . mhv-a59 liver titers peak 5 dpi after i.c. infection (lavi et al, 1984b) and hepatitis develops during the rst 1 to 2 weeks . mhv-jhm replicates transiently in the lungs, blood, and cervical lymph nodes after i.n. infection, and occasionally is found in the thymus and bone marrow (barthold and smith, 1992) . as would be expected in an infection that comes under immunological control, viral titers peak around 5 d.p.i. in the brain (parra et al, 1997) , slightly later in the spinal cord (matthews et al, 2001) , and virus is cleared by 8-20 d.p.i. (sutherland et al, 1997) . the kinetics of viral antigen expression follow a pattern similar to that observed of viral titers during acute infection (wang et al, 1992) , but viral antigen is still detectable up to 30 d.p.i. (lavi et al, 1984a) . viral rna is detectable in the brain as late as 10-12 months postinfection, although the amount of rna decreases with time (lavi et al, 1984a) . however, immunosuppression of cell-mediated immunity by drugs, radiation, or t-cell depletion at late time points after infection does not result in viral recrudescence from this rna pool (stohlman and weiner, 1981) . the blood-brain barrier is a major factor of the cns that limits immune function by preventing the easy entry of serum antibodies and infectious organisms in the absence of cns in ammation (cserr et al, 1992) . although it has been thought that the blood-brain barrier allows the entry of only activated t cells, a recent study demonstrated that naive t cells can enter the cns if there are few activated t cells in circulation (brabb et al, 2000) . it has been suggested that immunosuppressive cytokines such as transforming growth factor-beta, alpha-melanocyte stimulating hormone, and interleukin-10 (il-10) may limit or alter immune function within the cns (gordon et al, 1998; lipton and gonzales-scarano , 1978) . work with t cells responding to sindbis virus infection of the cns demonstrated that t cells lose their ability to proliferate and down-regulate il-2 production after entering the brain, supporting the hypothesis that the environment of the cns alters immune function (irani et al, 1997) . in addition, the brain fosters viral antigen or nucleic acid persistence, which is not observed in most peripheral organs and seems to be attributable at least in some instances to inhibited immunemediated clearance (kristensson and norrby, 1986) . the poor regenerative potential of neurons and the sensitivity of the cns to small lesions may have contributed to the development of a cns environment which downregulates the more destructive aspects of the immune response. the cells of the immune system successfully enter the cns after mhv infection. mononuclear cells of bone marrow origin peak 7 d.p.i. they then decline, even in animals that do not control the infection (williamson et al, 1991) . in the absence of t cells in nude mice, infectious mhv-jhm is not cleared with normal kinetics (houtman and fleming, 1996) , demonstrating that t cells are important for clearance of mhv. cd8 c t cell response to mhv cd8 c t cells are critical for the early control of mhv infection in the cns and for prevention of mortality (gombold et al, 1995; stohlman et al, 1995b; sutherland et al, 1997) . virus-speci c cd8 c t cells peak 7 days after mhv infection in the brain (williamson et al, 1991) . the ability to detect mhv speci c cd8 c t cells in the draining cervical lymph nodes (cln) or the spleen varies parra et al, 1999) . this variability may be due to differences in the speci c injection sites. virus delivered to the parenchyma results in a delayed peripheral immune response compared to virus delivered to the ventricles and cerebrospinal uid (stevenson et al, 1997) . cd8 c t cells recognize peptides presented on cell surface mhc class i molecules. although neurons do not express mhc class i and therefore are not anticipated targets of cd8 c t cells, mhc class i is present on endothelial cells and microglia (horwitz et al, 1999) . in addition, class i expression can be induced in astrocytes and oligodendrocytes in culture if exposed to factors secreted by cells infected with mhv-a59 (suzumura et al, 1986) . after mhv-a59 infection of cd8 c t-cell-de cient mice, viral antigen clearance was delayed in microglia, lymphocytes, endothelial cells, and meningeal cells but not in neurons or glia . in another study, transfer of cd8 c t cells suppressed mhv-jhm antigen expression in astrocytes and microglia but not oligodendrocytes (stohlman et al, 1995a) . cd8 c t cells can function through the lytic molecule perforin, through apoptosis induced by fas ligand (fasl), or by secreting cytokines such as ifngamma and tnf-alpha (kajino et al, 1998) . all of these mechanisms may be productively used in the immune response to mhv. in the cns of mice decient for perforin, viral clearance is delayed, thereby implicating a role for perforin in viral control . ef cient viral clearance in fas-de cient mice demonstrated that fasl-mediated lysis is not required for viral clearance. nonetheless, mice lacking both fasl-mediated apoptosis and perforin have delayed clearance compared to mice just lacking perforin, suggesting that fas-fasl interactions can contribute to viral control although they may not be very important (parra et al, 2000) . both cd4 c and cd8 c t cells persist in the cns for up to 34 d.p.i., with cd8 c t cells being more prominent in the parenchyma both early and late after infection (stohlman et al, 1998a) . the number of cd8 c t cells decreases with time and decreasing viral rna marten et al, 2000) . at 45 d.p.i., about 48% of these cells are still virus-speci c, similar to the percentage of virus-speci c t cells observed during acute infection. many of these chronically present cells secrete ifn-gamma, but cytolytic activity is gradually lost . although activated , mhv-speci c, cd8 c t cells transferred to infected mice can control viral replication, they are not suf cient to clear infectious virus from oligodendrocytes (stohlman et al, 1998b) . the presence of cd4 c t cells is necessary for transferred cd8 c t cell protection. indeed, these cells appear to be important for cd8 c t-cell lytic activity and survival in the cns, perhaps through the secretion of il-2 (stohlman et al, 1998b) . virally activated cd4 c t cells protect against lethal viral infections after transfer, but as with transferred cd8 c t cells, they are not suf cient to clear the virus (stohlman et al, 1986) . depletion of either cd8 c or cd4 c t cells results in the inability of mice to control acute viral titers in the brain (williamson et al, 1990; sutherland et al, 1997) . thus, both cd4 c and cd8 c t cells are important for control of acute viral infection in the cns. unlike cd8 c t cells, which accumulate in the parenchyma, cd4 c t cells tend to accumulate perivascularly and in subarachnoid spaces (stohlman et al, 1998a) , locations rich in mhc class ii positive macrophages and microglia (hickey and kimura, 1988; braun et al, 1993) . interestingly, cd4 c t cell numbers peak in the cns at 9 d.p.i., a little later than cd8 c t cells (williamson et al, 1991) , and persist in the cns after viral clearance marten et al, 2000) . the effector function of many lymphocytes involves the production and secretion of cytokines. mrna from a wide number of in ammatory cytokines can be detected in the brain 3-7 days following i.c. infection with mhv-jhm. these include interferon (ifn) alpha and beta (wang et al, 1998) , interleukin (il)-1 alpha, il-1 beta, il-6, il-2, and tnf-alpha (parra et al, 1997) . immuno uorescence of mhv-jhm infected spinal cords demonstrated the presence of tnf-alpha protein as well as il-1 beta and il-6 in cells with the morphology of macrophages early after infection and in uninfected astrocytes late after infection . both th1 (ifn-gamma) and th2 (il-4, il-5, and il-10) cytokine mrnas increase after infection with various strains of mhv, peaking 7 to 9 days after mhv-jhm infection (stohlman et al, 1995a; parra et al, 1997) . in several studies mhv infection resulted in a predominantly igg2a antibody isotype pro le (coutelier et al, 1987; fleming et al, 1989) supporting the idea of a predominantly th1 response, although in another study igg2a did not predominate over igg1 (parra et al, 1997) . these data, combined with the presence of both th1 and th2 cytokine mrnas, suggest that neither t-helper cell type necessarily predominates, although a th1 pro le is slightly more common. mhv-jhm infection of mice de cient for ifn-gamma revealed that ifn-gamma is not required for optimal antibody production or for ctl responses. ifn-gamma is, however, necessary for clearance of virus from the oligodendrocytes (parra et al, 1999) . clearance of an mhv strain that primarily infects neurons is delayed in the absence of ifn-gamma, suggesting that it may affect viral control in other cell types as well without being absolutely required for clearance (lane et al, 1997) . in mice decient for il-10, mhv-jhm causes a transient increase in viral titers 7 d.p.i. and greater mortality than in wild-type mice despite normal kinetics of viral clearance, which is thought to be due to an increase in in ammatory cytokines (lin et al, 1998) . the role of b cells in the control of mhv has been studied in rats and mice. in lewis rats, the transfer of b cells into irradiated recipients decreased mhv-jhm titers in the cns (schwender et al, 1999) . igm, the rst detectable antibody isotype, rst appears 6 d.p.i. with mhv-jhm i.c. in mice (stohlman et al, 1986; williamson et al, 1991) . neutralizing antibodies are detected in the serum by 9 d.p.i. with mhv-jhm given i.c., but were not suf cient to control viral titers within 11 d.p.i. in the absence of cd8 c t cells . similarly, after mhv-a59 infection, serum antibody is detectable 7 d.p.i. and reaches maximal levels 14-56 d.p.i. (lavi et al, 1984b) . by electron microscopy, plasma cells can be observed in the cns as late as 90 d.p.i. with mhv-jhm (stohlman and weiner, 1981) . antibody mostly of isotype igg2a is detected after infection (lardans et al, 1996) , although sometimes igg2a and igg1 eventually reach equivalent levels (parra et al, 1997) . transfer of virusspeci c antibodies increases survival, presumably by decreasing peak viral levels, delaying neuronal infection, and/or decreasing neuronal infection (fleming et al, 1989; yokomori et al, 1992) . the contribution of humoral immunity to viral clearance and persistent infection in the cns has been investigated using mice de cient in secreted antibodies or b cells bergmann et al, 2001; matthews et al, 2001) . mice homozygous for disruption of the ig mu gene (mumt mice) lack b cells and develop acute disease in the cns with similar kinetics and severity to wild-type c57bl/6 mice after mhv-jhm infection or mhv-a59 infection (matthews et al, 2001) . viral clearance during acute infection is similar in both groups. however, although virus is quickly cleared from the cns of wildtype mice, it reemerges and persists in the cns of mumt mice. b-cell-de cient mice have been shown to have impaired t cell immunity against a number of viral infections including mhv-jhm (bergmann et al, 2001) , possibly due to their absence as antigenpresenting cells in these mice. to clarify how b cells mediate viral clearance in the cns, we compared the role of b cells as antigen-presenting cells, using mice with b cells that are unable to secrete antibody, with that of secreted antibody, using mumt mice reconstituted with mhv-a59 immune immunoglobulin (matthews et al, 2001) . mumt mice that received a59-speci c antibody had decreased virus in the cns, whereas mice with b cells decient in antibody secretion did not clear virus from the cns. these data suggest a major role for immune antibody in controlling virus replication in the cns. nonlymphocytic immune response to mhv natural killer (nk) cells (asialogm1 c cells) peak in the cns at 7 days after i.c. inoculation with mhv-jhm. the 7 d.p.i. cns mononuclear cells are actively cytotoxic on the classic nk target cell line yac-1 when assayed ex vivo (williamson et al, 1991) . in the absence of t cells, a transient decrease in viral titers 5 to 7 d.p.i. has been attributed to the nk cell response (williamson et al, 1990) . after immunosuppression by cyclophosphamide, in mice infected with a high dose of mhv-jhm, death occurs more quickly (3 d.p.i.) than in untreated mice (6-7 d.p.i.) (weiner, 1973) . this observation suggests that the innate immune response that expands in response to infection, such as the nk cells, can be important in the early control of viral infection. macrophages are susceptible to infection by mhv (wijburg et al, 1996) , although only a subset is infected initially. it has been proposed that cd4 c t cells are necessary as a source of rantes to attract monocyte/macrophage in ltration of the cns . the role of macrophages in the immune response is dif cult to pinpoint. depletion of blood-borne macrophages had little effect on cns demyelination (xue et al, 1999) . on the other hand, depletion of macrophages before i.v. infection with a highly lethal isolate of mhv-a59 resulted in earlier mortality (3-5 d.p.i.) associated with increased viral titers in the spleen and liver (wijburg et al, 1997) . macrophage depletion before i.n. infection with mhv-jhm also resulted in rapid death (xue et al, 1999) . the early time point of mortality suggests that macrophages help control viral titers before the t and b cell populations are fully activated and effective. the immune response that controls mhv infection in the liver is similar in some ways to that important in the cns. pre-existing antibody can protect from lethal liver infections and decrease liver lesions, so antibody is capable of controlling liver viral titers (buchmeier et al, 1984) . however in contrast to the importance of antibody in controlling mhv-a59 replication in the cns, antibody is not required for viral clearance from the liver. in mumt mice, which lack mature b lymphocytes, and in igm-tg mice with b cells that do not secrete antibody, infectious virus was cleared from the livers with similar kinetics to wild-type mice after i.c. or i.h. infection (matthews et al, 2001) . cell-mediated immunity is required for the control of infection in the liver. depletion of cd4 c or cd8 c t cells has been shown to result in increased mhv-jhm titers in the liver and prolonged accumulation of infectious virus to 5 d.p.i. when both t cell populations were depleted, viral titers were not controlled within 7 d.p.i., although the mice did subsequently recover (kyuwa et al, 1996) . these data suggest that t cells play a role in controlling viral titers, even before 5 d.p.i. beta2-microglobulinde cient mice, which lack cd8 c t cells, have delayed clearance of virus from the liver . ifn-gamma-de cient mice experience greater mortality than do wild-type mice after i.p. infection with mhv-jhm or mhv-a59 (kyuwa et al, 1998; schijns et al, 1996) . virus persists up to 48 d.p.i. in the liver and large lesions develop in these mice, although there is no chronic increase in liver enzyme activity indicative of loss of liver function. therefore, ifn-gamma is critical for viral control in the liver. in the absence of ifn-gamma, cd8 c t cell depletion further increased the severity of disease (kyuwa et al, 1998) . therefore, cd8 c t cell functions other than ifn-gamma production are also important for viral control. cns demyelination develops as active mhv-a59 or mhv-jhm infection resolves. these lesions are histologically very similar to those observed in ms patients. cns lesions are predominantly created by primary demyelination as evidenced by the intact, naked axons present in these lesions, although axonal damage is also detected . the peripheral nervous system is not affected (lampert et al, 1973) . lesions are scattered randomly throughout the spinal cord (lampert, 1978) . chronic lesions are associated with lipid-laden macrophages (presumably full of myelin), scattered lymphocytes, and naked axons. viral particles are not detected. astrocytic reactions and perivascular cuf ng are also sometimes found in lesions (weiner, 1973; stohlman and weiner, 1981) . chronic lesions can persist as late as 90 d.p.i. (stohlman and weiner, 1981) , and scattered demyelinated axons can be detected even 16 months after infection (herndon et al, 1975) . early demyelination after high-dose, lethal mhv-jhm infection is associated with polymorphonuclear cells, mononuclear cells, and extracellula r myelin debris. degeneratin g axons are sometimes detectable (lampert et al, 1973) . nevertheless, even in necrotic lesions formed during lethal infections, some axonal preservation is observed (weiner, 1973) . chronic disease in mhv-infected animals is characterized by a single major episode of demyelination associated with the development of ataxia, hind limb paresis, and paralysis (lavi et al, 1984b) , following which animals usually recover. recovery is mediated by cns remyelination, sometimes accompanied by peripheral nervous system remyelination occurring in the cns (takahashi et al, 1987) . remyelination has been reported to begin anywhere from 14 to 70 d.p.i. (kristensson and norrby, 1986; takahashi et al, 1987) . as early as 2 to 3 weeks postinfection, there is a dramatic increase in mbp mrna at the edges of lesions suggestive of early remyelination before histological detection (kristensson and norrby, 1986) . at 28 d.p.i., replicating oligodendrocytes were detectable near lesions (herndon et al, 1977) . it is thought that remyelination involves oligodendrocyte precursor o-2a cell proliferation (godfraind et al, 1989) . how does cns demyelination occur? this is perhaps the major question posed with the mhv model of ms. neither acute encephalitis nor rna persistence is suf cient to cause demyelination, as demonstrated with two mutants of mhv-a59 (leparc-goffart et al, 1998; das sarma et al, 2000) . infectious virus does not have to be successfully cleared for demyelination to occur (houtman and fleming, 1996) . transfer of cd8 c t cells during acute infection results in no subsequent demyelination, although whether this treatment changes viral rna persistence is unclear (stohlman et al, 1995a) . early mhv-jhm-induced demyelination is rare in immunode cient transgenic scid and rag1 knockout mice or irradiated mice, demonstrating a strong if not absolute requirement for lymphocytes (houtman and fleming, 1996; wu and perlman, 1999) . electron micrographs of demyelinating lesions demonstrate that macrophage processes slip between layers in the myelin sheath, suggesting that macrophages could be the direct mediators of demyelination (powell and lampert, 1975) . macrophages localize to demyelinating lesions, and their appearance correlates with the development of lesions. they do not appear in great numbers in the absence of lymphocytes, consistent with the observation that lymphocytes are important for demyelination (wu and perlman, 1999; lane et al, 2000) . despite the visual evidence that macrophages invade myelin sheaths, it is possible that macrophages could be localizing to areas of demyelination to clean up the damaged myelin caused by a nonmacrophage dependent mechanism. depletion of blood-borne macrophages does not affect the severity of demyelination, although similar techniques do affect demyelination in eae and theiler's virus-induced demyelination (huitinga et al, 1995; rossi et al, 1997; xue et al, 1999) . however, macrophages that are assumed to be derived from microglia or perivascular macrophages are present in the cns of these depleted mice and may still play a role beyond that of scavenging for disintegrating myelin (xue et al, 1999) . t cells could mediate demyelination through direct lysis of oligodendrocytes or through in ammation mediated by cd4 c t cell-dependent in ammation as demonstrated in experimental autoimmune encephalomyelitis . certainly t cells persist in the cns after virus is cleared and during active demyelination (marten et al, 2000) . there is a signi cant amount of data demonstrating that neither cd4 c nor cd8 c t cells are speci cally required for demyelination. demyelination occurs during acute infection of t cellde cient nude mice. beta-2-microglobulin-de cient mice cannot express mhc class i and therefore do not develop mature cd8 c t cells. i-a b -de cient mice lack mhc class ii and therefore do not develop mature cd4 c t cells. both of these strains of t cell-de cient mice demonstrate early mhv-jhm induced demyelination (houtman and fleming, 1996) . infection with mhv-a59 of beta-2 microglobulin-de cient mice and mice ef ciently depleted of cd8+ t cells also results in demyelination, although less frequently than in wild-type mice (gombold et al, 1995; sutherland et al, 1997; lavi et al, 1999) . partial depletion of cd4 c and cd8 c t cells does not inhibit chronic demyelination either, although in this study t cells were not depleted until 7 d.p.i. (sutherland et al, 1997) . a few studies suggested that the absence of cd4 c or cd8 c t cells resulted in decient demyelination. cd4 c or cd8 c t cell-de cient mice infected with mhv-jhm were less likely to develop acute demyelination, but the mice that did develop demyelination had similar severity to that observed in wild-type mice (houtman and fleming, 1996) . cd4 c t cell-de cient mice infected with mhv-jhm developed acute and chronic demyelination, but it was signi cantly less severe than that seen in cd8 de cient or wild-type mice . when splenocytes were transferred to immunodecient rag1 knockout mice, either cd4 c or cd8 c t cells were suf cient to generate signi cant levels of acute demyelination, although cd4 c t cells were less ef cient at inducing demyelination than cd8 c cells (wu et al, 2000) . these data suggest that each of these t-cell subpopulations has the capability to mediate cns demyelination after mhv infection and may induce slightly different levels or pathways of demyelination. on the other hand we have observed (matthews et al, 2002) moderately sized, typical demyelinating lesions as well as atypical small round necrotic lesions in mhv-a59 infected rag1 knockout mice, suggesting that, although t cells may be required for robust demyelination, other mechanisms must also be at work. knockout mice have been used to demonstrate that the mechanism by which demyelination takes place requires neither perforin nor fas-fasl interactions parra et al, 2000) . furthermore, removing tnf-alpha, il-10, or ifn-gamma through depletion or the use of genetically de cient mice did not decrease demyelination (stohlman et al, 1995b; lin et al, 1998; parra et al, 1999) . chemokines, however, may play a role in demyelination. gene expression for a number of chemokines has been found in the cns of mice undergoing chronic demyelination (lane et al, 1998) . of these the c-c chemokine rantes has been shown to possibly play a role in mhv-induced demyelination . systemic depletion of rantes with rantes-speci c antisera resulted in a signi cant reduction in macrophage in ltration and demyelination in c57bl/6 mice infected with mhv-4. rantes is a pro-in ammatory chemokine produced by t cells, platelets, and endothelia that acts as a chemoattractant for a variety of lymphocytic and myeloid cell types including monocytes and granulocytes. lane and colleagues believe that the reduced demyelination observed in mhv-infected cd4 ¡=¡ mice may be due to the absence of cd4 c t cell-derived rantes . thus, rantes produced by cd4 c t cells may attract macrophages into the cns during viral infection. activated macrophages are a potent source of toxic nitric oxide intermediates produced by inducible nitric oxide synthases (inos). inhibition of one of these, nos-2, has been shown to reduce mhv-induced demyelination (lane and buchmeier, 1999) , providing further evidence for a role for pro-in ammatory innate immune mechanisms in cns demyelination in this mouse model. among the cxc chemokines expressed in the cns during mhv-induced demyelinating disease is ifninducible protein 10 (ip-10), suggesting a possible role for this chemokine in demyelination (lane et al, 1998) . however, ip-10 has also been shown to be crucial for control of mhv viral replicatio n and recovery from acute encephalitis in the c57bl/6 mouse . administration of anti-ip-10 antibody led to a signi cant reduction in t cells in ltrating the cns of mhv-infected mice, decreased levels of ifn-°in the cns and increased mortality of infected mice . thus, although chemokinemediated in ammatory responses in the cns may contribute to demyelination, as with all immune response elements examined, they also play a vital role in viral clearance. b cells and the antibodies they secrete could potentially also mediate demyelination. however, no correlation has been found in mhv-infected mice between antibody titers and severity of demyelination (koolen et al, 1987) . in some animal models of cns demyelination, antibodies against self-antigens in the brain induce or exacerbate demyelination (brehm et al, 1999; genain et al, 1999) but there is no evidence for the induction of antibodies that recognize host proteins after mhv infection of the cns. we have examined the role of b cells in demyelination using b cell-de cient mumt mice (matthews et al, 2002) . in infected b cell-de cient mice, robust demyelination not only occurred but was also statistically more severe than in wild-type animals 30 and 60 d.p.i. this increase in demyelination could be associated with the persistence of infectious virus in the absence of b cells. in mice lacking antibody fc receptors or complement pathway activity, thereby lacking the ability to utilize antibody effector functions, virus did not persist yet demyelination was similar to that observed in wild-type mice. antibody has also been suggested to facilitate remyelination. however, in mumt mice, remyelination was still detected. therefore, we nd no evidence that b cells are important for cns demyelination, nor are they required for remyelination. another hypothesis that has been proposed is that mhv invades the oligodendrocytes or cells that provide critical functions to the oligodendrocyte and damages them directly, thereby causing oligodendrocyte death or retraction of the myelin sheath. early work on mhv-jhm using electron microscopy and immuno ourescence demonstrated that the virus infects oligodendrocytes early and possibly late after infection, and thereby supported this hypothesis (lampert et al, 1973; powell and lampert, 1975; wu and perlman, 1999) . however, the degenerating oligodendrocytes detected during early paralysis rarely actually contained viral particles (powell and lampert, 1975) . viral particles were also detected in astrocytes 7 d.p.i., and virus could potentially damage oligodendrocytes by disrupting the function of these cells . interestingly, mice immunosuppressed using cyclophosphamide with resultant loss of antibody production and decreased perivascular in ammation still displayed small demyelinating lesions (weiner, 1973) . likewise, lipid-laden macrophages and occasional small lesions were detected in rag1 knockout mice (wu and perlman, 1999) , and scid mice can develop demyelination, if rarely (houtman and fleming, 1996) . direct virally mediated demyelination is unlikely to be the sole cause of demyelination, however, because virus in the presence of t cells more reliably induces demyelinating lesions (wang et al, 1992) . it is clear that demyelination after mhv infection is a complex issue. it is possible that both direct viral destruction and immune mediated destruction contribute to the development of demyelinating lesions. past research has focused on dissecting the immune response to mhv in order to determine the role of single components in the demyelinating phase of the disease. however, in contrast to other mouse models of demyelination, there does not appear to be a clear role for any one particular lymphocytic or monocytic subset that mediates the demyelination. rather, it appears that a balance of immune components may be required for viral clearance and that various immune and nonimmune pathways may mediate the subsequent demyelinating events. it is this balance that determines the level of encephalitis in acute infection and sets up the conditions that lead to demyelination. one model that would account for the involvement of a variety of immune elements in demyelination would associate cns pathology with free radical production by in ammatory cells of the myeloid lineage. these may be attracted into the cns by chemokines secreted by viral speci c cd4 c t cells. in this model cytokines secreted by cd8 c t cells would assist in the state of activation of macrophages and astrocytes and therefore in uence the level of cns disease. such a model for cns pathology has been implicated for eae (koprowski et al, 1993; lin et al, 1993) and borna disease virus-induced cns pathology (hooper et al, 2001) . future studies are required to determine if this mechanism also prevails in mhv-induced demyelination. in this review we have described studies in which modulating the immune response has resulted in perturbation of the balance between replication/spread of the virus and clearance with consequential changes in acute disease and demyelination. knowledge of the contribution of different components of the disease would be useful in manipulating this balance so that robust viral clearance does not result in chronic cns demyelination. viremic dissemination of mouse hepatitis virus-jhm following intranasal inoculation of mice inverted immunodominance and impaired cytolytic function of cd8 c t cells during viral persistence in the central nervous system impaired t cell immunity in b cell-de cient mice following viral central nervous system infection in situ tolerance within the central nervous system as a mechanism for preventing autoimmunity cellular component s of the immune barrier in the spinal meninges and dorsal root ganglia of the normal rat: immunohistochemical (mhc class ii) and electronmicroscopic observations epitope speci city of demyelinating monoclonal autoantibodies directed against the human myelin oligodendrocyte glycoprotein (mog) murine hepatitis virus-4 (strain jhm)-induced neurologic disease is modulated in vivo by monoclonal antibody b lymphocyte and macrophage expression of carcinoembryonic antigen-related adhesion molecules that serve as receptors for murine coronavirus igg2a restriction of murine antibodies elicited by viral infections afferent and efferent arms of the humoral immune response to csf-administered albumins in a rat model with normal blood-brain barrier permeability demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus mouse hepatitis virus infection of mice causes long-term depletion of lactate dehydrogenase-elevatin g virus-permissive macrophages and t lymphocyte alterations monoclonal antibodies to the matrix (e1) glycoprotein of mouse hepatitis virus protect mice from encephalitis identication of autoantibodies associated with myelin damage in multiple sclerosis in vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice thymus involution induced by mouse hepatitis virus a59 in balb/c mice mouse hepatitis virus a59-induced demyelination can occur in the absence of cd8 c t cells normal cerebrospinal uid suppresses the in vitro development of cytotoxic t cells: role of the brain microenvironment in cns immune regulation mouse hepatitis virus-induced recurrent demyelination. a preliminary report regeneration of oligodendroglia during recovery from demyelinating disease perivascular microglial cells of the cns are bone marrow-derived and present antigen in vivo coronaviridae: the viruses and their replication the central nervous system inammatory response to neurotropic virus infection is peroxynitrite dependent detailed in vivo analysis of interferon-gamma induced major histocompatibility complex expression in the central nervous system: astrocytes fail to express major histocompatibility complex class i and ii molecules dissociation of demyelination and viral clearance in congenitally immunode cient mice infected with murine coronavirus jhm macrophages in t cell line-mediated, demyelinating, and chronic relapsing experimental autoimmune encephalomyelitis in lewis rats regulation of brainderived t cells during acute central nervous system in ammation organ speci c endothelial cell heterogeneity in uences differential replication and cytopathogenicity of mhv-3 and mhv-4. implications in viral tropism fas-and perforinindependent mechanisms of cytotoxic t lymphocyte virus persistence and recurring demyelination produced by a temperature-sensitive mutant of mhv-4 infection and involution of mouse thymus by mhv-4 induction of demyelination by a temperature-sensitive mutant of the coronavirus mhv-a59 is associated with restriction of viral replication in the brain in vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases nucleocapsid or spike protein-speci c cd4 c t lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of cd8 c t cells persistence of rna viruses in the central nervous system role of cd4 c and cd8 c t cells in mouse hepatitis virus infection in mice autoimmune and virus induced demyelinating diseases. a review mechanism of demyelination in jhm virus encephalomyelitis. electron microscopic studies dynamic regulation of ®-and -chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease inhibition of nitric oxide synthase-2 reduces the severity of mouse hepatitis virusinduced demyelination: implications for nos2/no regulation of chemokine expression and in ammation a central role for cd4 c t cells and rantes in virus-induced central nervous system in ammation and demyelination disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus polyclonal b lymphocyte activation induced by mouse hepatitis virus a59 infection cellular reservoirs for coronavirus infection of the brain in beta2-microglobulin knockout mice detection of mhv-a59 rna by in situ hybridization the organ tropism of mouse hepatitis virus a59 in mice is dependent on dose and route of inoculation experimental demyelination produced by the a59 strain of mouse hepatitis virus determinants of coronavirus mhv pathogenesis are localized to 3 0 portions of the genome as determined by ribonucleic acid-ribonucleic acid recombination coronavirus mouse hepatitis virus (mhv)-a59 causes a persistent, productive infection in primary glial cell cultures targeted recombination within the spike gene of murine coronavirus mouse hepatitis virus-a59: q159 is a determinant of hepatotropism antibody prevents virus reactivation within the central nervous system the role of il-10 in mouse hepatitis virus-induced demyelinating encephalomyelitis mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis nitric oxide localized in the spinal cords of mice with experimental allergic encephalomyelitis: an electroparamagneti c study central nervous system immunity in mice infected with theiler's virus local neutralizing antibody response cutting edge: the t cell chemoattractant ifn-inducible protein 10 is essential in host defense against viral-induced neurologic disease contributions of cd8 c t cells and viral spread to demyelinating disease neither b cells nor t cells are required for cns demyelination in mice persistently infected with mhv-a59 antibody is required for clearance of infectious murine hepatitis virus a59 from the cns but not the liver coronavirus infects and causes demyelination in primate central nervous system murine coronavirus spike protein determines the ability of the virus to replicate in the liver and cause hepatitis molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain a59 kinetics of cytokine mrna expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis ifn-gamma is required for viral clearance from central nervous system oligodendroglia contributions of fas-fas ligand interactions to the pathogenesis of mouse hepatitis virus in the central nervous system spread of a neurotropic murine coronavirus into the cns via the trigeminal and olfactory nerves the astrocyte is a target cell in mice persistently infected with mouse hepatitis virus pathogenesis of chimeric mhv-4/mhv-a59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence oligodendrocytes and their myelin-plasma membrane connections in jhm mouse hepatitis virus encephalomyelitis role of macrophages during theiler's virus infection exacerbated viral hepatitis in ifn-gamma receptorde cient mice is not suppressed by il-12 modulation of acute coronavirus-induced encephalomyelitis in gamma-irradiated rats by transfer of naṏve lymphocyte subsets before infection virus dissemination through the brain parenchyma without immunologic control apoptosis of jhmv-speci c ctl in the cns in the absence of cd4 c t cells ctl effector function within the central nervous system requires cd4 c t cells mouse hepatitis virus-speci c cytotoxic t lymphocytes protect from lethal infection without eliminating virus from the central nervous system tumor necrosis factor expression during mouse hepatitis virusinduced demyelinating encephalomyelitis in vivo effects of coronavirus-speci c t cell clones: dth inducer cells prevent a lethal infection but do not inhibit virus replication chronic central nervous system demyelination in mice after jhm virus infection activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus spread of a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes cd4 c and cd8 c t cells are not major effectors of mouse hepatitis virus a59-induced demyelinating disease coronavirus infection induces h-2 antigen expression on oligodendrocytes and astrocytes post in ammatory remyelination in the spinal cord of mice infected with mouse hepatitis virus, jhm strain sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv-4) leads to a characteristic distribution of demyelination coronavirus mhv-a59 causes upregulation of interferon-beta rna in primary glial cell cultures pathogenesis of demyelination induced by a mouse hepatitis role of spleen macrophages in innate and acquired immune responses against mouse hepatitis virus strain a59 role of virus-speci c cd4 c cytotoxic t cells in recovery from mouse hepatitis virus infection receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins effective clearance of mouse hepatitis virus from the central nervous system requires both cd4 c and cd8 c t cells characterization of brain-in ltrating mononuclear cells during infection with mouse hepatitis virus strain jhm cd4 and cd8 t cells have redundant but not identical roles in virus-induced demyelination macrophage in ltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus depletion of blood-borne macrophages does not reduce demyelination in mice infected with a neurotropic coronavirus hemagglutinin-esterase-speci c monoclonal antibodies alter the neuropathogenicity of mouse hepatitis virus key: cord-346339-y7z1sa8y authors: baumgärtner, wolfgang; löscher, wolfgang title: re-emergence of neuroinfectiology date: 2016-01-11 journal: acta neuropathol doi: 10.1007/s00401-016-1535-3 sha: doc_id: 346339 cord_uid: y7z1sa8y nan infections of the central nervous system (cns) represented an area of major concern in the pre-antibiotic and pre-vaccination era. following the wide-spead introduction of antibiotic therapy and the implementation of national vaccination programs, many infectious diseases appeared to be successfully contained, and the health threat posed by infectious pathogens seemed to belong to the past. however, in recent years the number of reported cases of infectious agents causing cns infections in the form of emerging and re-emerging diseases has been increasing [9] . among the most devastating infectious diseases of the cns that plague today's world are cerebral malaria, rabies, toxoplasmosis, bacterial meningitis, arbovirus encephalitis and human immunodeficiency virus-associated neurological diseases [4, 5, 9, [20] [21] [22] . viral and bacterial cns diseases represent an important but relatively neglected area of medicine in developing countries [8] . similarly, parasiteinflicted diseases of the cns represent a major threat to public health indeveloping countries; this threat is also present in the western world but the disease burden is less [4, 5] . overall, the burden of infectious cns diseases is reinforced by the fact that survivors may suffer from life-long etiology, discover the underlying mechanism and design prevention and intervention strategies. the cause of an inflammatory cns disease frequently remains undetermined. approximately 30 % of patients with suspected cns infection lack an etiological diagnosis and one-third of these patients ultimately die as a result of the illness [19] . however, present-day researchers have acquired a new diagnostic tool, namely, metagenome sequencing, and this tool is increasingly being applied to the identification of pathogens [18] . intensified search with this tool and with new technologies have resulted in the identification of various pathogen vectors, including bats, domestic pets and wildlife animals, for different agents, such as the hendra and borna viruses, japanese encephalitis infection and middle east respiratory syndrome (mers) coronavirus [7, 8, 10, 21] . this issue of acta neuropathologica includes a cluster of three review articles on neuroinfectiology with special emphasis on viral and bacterial diseases as well as the role of these diseases in epilepsy [6, 13, 23] . these reviews reflect the plethora and diversity of causes, consequences and reaction patterns in neuroinfectiology. the review by ludlow et al. [13] depicts emerging and re-emerging neurotropic and non-neurotropic viruses that cause cns diseases and sheds some light on the mechanisms underlying both the direct and indirect as well as the immediate and delayed consequences of these diseases. viral diseases of the cns, many arising from zoonotic pathogens, can be caused by a variety of viruses, including the bunyavirus, nipah virus, hendra virus and rabies, polio, tick-borne encephalitis, herpes and measles viruses [7, 9, 21, 22] . viral cns infections are commonly caused by mosquito-borne viruses, such as the west nile, chikungunya and japanese encephalitis viruses, which have in recent times expanded their geographic range. in addition, some viruses, including the bat henipaviruses nipah virus and hendra virus, the borna virus as well as the japanese encephalitis virus, have crossed the human species barrier (spill-over infection) [10, 21, 22] . various bacteria, including streptococcus pneumoniae, neisseria meningitidis, haemophilus influenzae, enterohemorrhagic escherichia coli (ehec) and listeria monocytogenes, are among the most common bacterial causes of cns diseases in humans. others, such as streptococcus suis, are zoonotic pathogens with great regional differences as formidably described by the review of doran et al. [6] . in africa's so-called "meningitis belt", which stretches from senegal to ethiopia, outbreaks of meningitis due to meningococcal disease caused by neisseria meningitidis occur regularly, killing thousands and infecting tens of thousands each year [20] . complications and long-term sequelae after the initial bacterial infection include epileptic seizures, hydrocephalus, infarction, herniations and persistent defects after healing. noteworthy, septic patients may develop septic encephalopathy, a potentially irreversible acute cerebral dysfunction which is clinically characterized a slowing of mental processes, impaired attention, memory dysfunction, delirium and/or coma [24] . epilepsy with recurrent unprovoked (spontaneous) seizures may be a serious consequence of cns infections [23] . the current terminology for the concept(s) and underlying cause(s) of epilepsy refers to three categories, i.e. genetic, structural/metabolic and unknown. this categorization replaces the previously used terms of idiopathic, symptomatic and cryptogenic. epilepsy resulting from various processes, including traumatic brain injury, neoplasms, ischemic or hemorrhagic stroke and cns infection, belong to the structural/metabolic category. congenital and developmental issues and genetic conditions are mostly associated with the development of epilepsy in younger patients, whereas infection, head trauma and tumors leading to epilepsy may occur at any age. in survivors of cns infections, the risk of unprovoked seizures is approximately 7 % in developed countries, but much higher in resourcepoor countries. in their review, vezzani et al. [23] comprehensively describe the infectious diseases and sterile (non-infectious) inflammatory responses, as well as the associated underlying complex pathogenetic mechanisms, which result in the development of epilepsy. these authors also describe those factors which play a critical role during epileptogenesis and which should be considered in prevention strategies. the causes of degenerative, inflammatory and behavioral disorders, including alzheimer disease, parkinson disease, multiple sclerosis, guillain-barré syndrome, encephalitis lethargica, congenital malformations, schizophrenia and bipolar disorder, are largely unknown. however, recent studies have been (re)-focusing on the role of neuroinflammation and infections as driving forces. in addition, new concepts in which infections early in life are considered to be predisposing factors with clinical manifestation(s) decades later are now being studied [11, [13] [14] [15] . factors contributing to the emergence of new pathogens include changes in human demographics and behavior, intensification of international travel (tourism), commerce (global trade), increased economic development and land use, increasing importation of infected animals and exotic pet trade and the altered migration of vectors (such as birds and arthropods) and their adaption in new environments, in part facilitated by global warming [9, 17] . faced with complex patterns of global changes, the researcher analyzing the interconnections among humans, companion animals, livestock and wildlife requires integrated approaches. these complex interactions and their conceptual interpretation depend on a multidisciplinary and cross-sectoral approach involving experts in both human and veterinary medicine as well as those in ecology, as envisioned by the one health-one medicine concept [12, 26] . in addition to opportunistic infections, activation of silent-primarily non-pathogenetic-agents may represent a future threat as potent drugs are available to treat autoimmune disease, organ transplants and cancer. in addition, more patients with congenital immunodeficiency live longer and may be exposed to these pathogens. similarly, the gut microflora has been shown experimentally to play an important role as a trigger of cns inflammation [3] . furthermore, t cells become licensed in the lung to enter the cns [16] . these different modes of pathogen-host or immune system interaction may represent future avenues by which to study the pathogenesis of inflammatory and degenerative lesions in the nervous system, as outlined in the recent review by bauer et al. [2] which focuses on progressive multifocal leukoencephalopathy and immune reconstitution inflammatory syndrome. neuroinfectiology represents an emerging multidisciplinary field which centers on the complex interactions between cns and pathogen-associated cellular and molecular processes, inflammation, immune responses, degeneration, stem cell homeostasis as well as tissue repair and regeneration. in order to combat this challenge extensive cross-fertilization between scientists from various fields is needed. new pathogens associated with cns involvement have emerged in recent years. these represent a major threat to public health, are of great economic relevance and represent a medical challenge due to the lack of appropriate diagnostic and treatment strategies [9] . global trading, tourism and ecological and demographic changes will contribute to outbreaks of diseases due to these emergent agents. globalization of human travel and industrial exchange facilitates the spread of infections worldwide within a short time period. in addition, developments of immunomodulatory drugs to treat immune-mediated disease might cause opportunistic infections or activation of latent agents. new molecular detection systems will improve our ability to rapidly diagnose and recognize emerging and re-emerging pathogens and the host genetic factors involved in disease susceptibility, but the development of new strategies for diagnosis, prevention and treatment of neurological disorders will only be efficiently addressed by an interdisciplinary approach bridging the fields of neuroscience and infection medicine. moreover, little is known about the role of pathogen-related predisposing factors, mechanisms causing acute disease in individuals and factors resulting in long-term cns disturbances. overall, a more conceptual understanding of neuroinfectiology is pivotal for the development of successful prevention and treatment strategies. future studies in neuroinfectiology will address questions relating to the mechanisms of direct and indirect as well as acute, delayed and long-term damage, the role of misdirected immune responses in lesion initiation and the progression as well as prevention of cns infection by developing appropriate intervention strategies and potential beneficial approaches for tissue regeneration. infectious agents associated with schizophrenia: a meta-analysis progressive multifocal leukoencephalopathy and immune reconstitution inflammatory syndrome (iris) commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelination kristensson k (2009) infectious diseases of the nervous system and their impact in developing countries parasitic diseases of the central nervous system host-pathogen interactions in bacterial meningitis emergence and re-emergence of viral diseases of the central nervous system an orthopoxvirus-based vaccine reduces excretion after mers-cov infection in dromedary camels neuroinfectiology. subspecialty with a future a variegated squirrel bornavirus associated with fatal human encephalitis gobal research priorities for infections that affect the nervous system teaching "one medicine, one health neurotropic virus infections as the cause of immediate and delayed neuropathology emerging roles of pathogens in alzheimer disease neuropsychiatric sequelae of nipah virus encephalitis t cells become licensed in the lung to enter the central nervous system the ecology of emerging neurotropic viruses assembly of viral genomes from metagenomes burden of neuroinfectious diseases on the neurology service in a tertiary care center tackling meningitis in africa emerging viral infections of the central nervous system: part 1 emerging viral infections of the central nervous system: part 2 infections, inflammation and epilepsy progress in clinical neurosciences: sepsis-associated encephalopathy: evolving concepts guillain-barré syndrome from "one medicine" to "one health" and systemic approaches to health and well-being acknowledgments the research underlying this editorial was in part supported by niedersachsen-research network on neuroinfectiology (n-rennt) of the ministry of science and culture of lower saxony, germany. key: cord-020770-wpub7krf authors: benmamar-badel, anouk; owens, trevor; wlodarczyk, agnieszka title: protective microglial subset in development, aging, and disease: lessons from transcriptomic studies date: 2020-04-03 journal: front immunol doi: 10.3389/fimmu.2020.00430 sha: doc_id: 20770 cord_uid: wpub7krf microglial heterogeneity has been the topic of much discussion in the scientific community. elucidation of their plasticity and adaptability to disease states triggered early efforts to characterize microglial subsets. over time, their phenotypes, and later on their homeostatic signature, were revealed, through the use of increasingly advanced transcriptomic techniques. recently, an increasing number of these “microglial signatures” have been reported in various homeostatic and disease contexts. remarkably, many of these states show similar overlapping microglial gene expression patterns, both in homeostasis and in disease or injury. in this review, we integrate information from these studies, and we propose a unique subset, for which we introduce a core signature, based on our own research and reports from the literature. we describe that this subset is found in development and in normal aging as well as in diverse diseases. we discuss the functions of this subset as well as how it is induced. the term "microglia" was brought to the scientific community's attention a century ago with its first use by pio del rio-hortega (1), who strived to distinguish them from oligodendrocytes. his early work also highlighted their phagocytic ability, as well as their potential to undergo morphological changes. this early description led the community to consider microglial cells as a homogeneous population, even though the first description of a microglial subset ("satellite microglia") appeared as early as 1919 (1) . microglia originate from yolk-sac progenitors that start migrating toward the fetus around midpregnancy. these progenitors reach the embryonic brain around embryonic day (e) 9.5-e10.5 (2, 3) until the formation of the blood-brain barrier around e13.5-e14.5 in the mouse, and between the 4th gestational week to the 24th gestational week in the human (4, 5) . as such, they are among the first cells to colonize the developing brain, and they participate in central nervous system (cns) development. for instance, they contribute to refine brain wiring through enhancing both synapse formation (6, 7) and elimination (8, 9) , they modulate axonal growth (10, 11) , they secrete factors promoting neuronal progenitors survival (12) helping with neuronal positioning (11, 13) , and they participate in the clearance of live and apoptotic cells during development (14) . microglia also take on physiological functions in the adult cns, as they constantly sense their immediate environment, in a so-called "never-resting state" (15, 16) . our knowledge of microglial physiology and process motility relies heavily on studies in anesthetized animals. understanding of microglial functions in the steady state is challenged by a recent study showing that microglial process motility and morphology are affected by the wakefulness state of mice (17) . aside from this surveillance immune function, they are also fundamental for regulation of social behavior, learning, and memory, as these functions are impaired upon their depletion and restored after repopulation (18) . microglial roles in injury and disease contexts have been investigated extensively, with new advances contributing to deepen our understanding of microglia and their effect on other glial cells [reviewed in greenhalgh et al. (19) ]. these physiological functions advanced our view of microglia, from being initially thought of as exclusively sentinel cells reacting in the context of injury. this dated view on microglia led to the superposition of macrophage m1/m2 phenotypes onto them (20), which was an early attempt to grasp the extent of microglial diversity. this classification is however mostly obsolete nowadays, as it was proved to be simplistic and disconnected from in vivo reality (21) . indeed, the variety of functions microglia take on in space, time, and health states along with reports of sex differences in microglial function have led the community to infer a greater microglial heterogeneity than initially thought. with the progress of technology, investigating such diversity has become possible, notably through the development of high-throughput techniques such as mass cytometry and with the recent advances in transcriptomic studies with single-cell rna-sequencing (rnaseq). these technologies allowed the identification of microglial signatures linked to their "activation state." in 2014, butovsky et al. described a "homeostatic" microglial signature, comparing microglia with monocytic populations and other cns cells (22) . this signature includes genes such as p2ry12, fclrs, tmem119, hexb, mertk, cx3cr1, csf1r, etc. that have been used in numerous studies thereafter to identify microglial cells. this was a fundamental step in distinguishing resident microglia from other tissue-resident macrophages and infiltrates in disease context. this "homeostatic" signature was more recently revised and extended to developmental stages in addition to adulthood by matcovitch-natan et al. (23) . in this study, single-cell rna-seq helped associate the microglial signature identified at each different age to the potential functions these cells take on during life. they pinpointed three different temporal stages of development, each linked to a particular signature: early microglia associated with proliferation and differentiation, pre-microglia related to neuronal development, and adult microglia. it has recently been suggested that microglial heterogeneity peaks early during development and then reaches a minimum in the homeostatic adult brain, only to regain diversity in old age (24) . in addition, some microglial subtypes have been based on surface markers and sometimes function [discussed in stratoulias et al. (25) ]. this has been mostly achieved through systematic transcriptional investigation of microglia in different contexts. however, because every study is done with different techniques (microarrays, bulk rna-seq, single-cell rna-seq, etc.), on different kinds of samples (whole brain, sorted microglia based on different gating strategies, microdissected microglia, sorted nuclei, etc.), and in different animal models, there is a risk for confusion of data. we believe that there is a need for an overview-by looking at the big picture, common patterns can be identified between studies that might otherwise have been overlooked. in this review, we summarize and interpret transcriptomic studies on microglia from development, homeostasis, and disease states to bring to light a subpopulation common to all these different states. we discuss the factors inducing this subpopulation and its functional importance in all of the studied conditions. finally, we provide a core signature for this subset and propose to systematize and unify the naming of this microglial subpopulation to clarify the literature and avoid redundancy in future studies. we propose to use a name already used in numerous studies and that accounts for these cells' expression signature: cd11c+ microglia. for long, microglia have been considered simply as macrophages, due to the belief that all macrophages emerged from the bone marrow. consensus that a subset of microglia expressed cd11c was therefore at first difficult to achieve. cd11c was widely accepted as a marker for dendritic cells (dcs), to the extent that some studies have used it as the sole identifier for dcs. added to this was the constant difficulty of discriminating cnsresident parenchymal microglia from blood-derived myeloid cells, with which they share many markers [reviewed in amici et al. (26) ]. until recently, it was indeed not possible to reliably discriminate microglia, especially activated microglia, from blood-derived monocytic myeloid cells, using morphology or routine myeloid markers. panels of differentially expressed genes that can be used to distinguish microglia including tmem119 (27) and the homeostatic marker p2ry12 (22) were however recently identified and validated in both homeostatic and disease conditions (28) . to our knowledge, the first observation of microglia expressing cd11c was made in human multiple sclerosis (ms) tissue by immunohistochemical analysis (29) . one, however, cannot be completely certain of the exclusive microglial nature of the cells identified in this study based on the markers used and our current knowledge of myeloid cell marker expression patterns. the first report to explicitly identify cd11c+ cells in the cns as microglia came from butovsky et al. in 2006 (30) . they identified populations of cd11c+ cells in a mouse model for alzheimer's disease (ad) as microglia, based on their location and co-expression of isolectin b4 and cd11b, although these cells showed a dendritic morphology. the major point of interest in that study was the observation that all mhc-ii+ microglia that engulfed amyloid β in the brain of glatiramer acetate (ga)vaccinated transgenic (tg)-ad mice co-expressed cd11c. also, relevant to our subsequent studies, these cells could be stained with an antibody specific for insulin-like growth factor 1 (igf1). a "gold standard" for microglial identification remains their relatively low level of expression of cd45 in flow cytometry analyses (31) . in the course of study of glial responses in the dentate gyrus to axonal transection in the entorhinal cortex (the perforant path lesion model), we noted a subpopulation of cd45 low cd11b+ cd11c+ cells in flow-cytometry profiles of cells isolated from lesion-reactive hippocampus. their functional significance and whether they derived intraparenchymally or by immigration from bone marrow were not determined (babcock and owens, unpublished). exactly similar cells were then observed in cuprizone-demyelinated corpus callosum (32, 33) . these were described to express slightly higher levels of cd45 than their cd11c− counterparts, while remaining within the cd45 low gate (33, 34) . in addition, they did not express ccr2 characteristic for infiltrating leukocytes and expressed high levels of cx3cr1 supporting their microglial status (33) . further analysis showed that cd11c+ microglia were also induced in experimental autoimmune encephalomyelitis (eae) (33) (34) (35) ) and a mouse model for neuromyelitis optica (nmo) (33) , as well as during postnatal development (24, (35) (36) (37) . in older studies, ambiguity in assigning cd45 levels resulted in cd11b+ cd11c+ populations in cns of mice with eae or infected with toxoplasma gondii being identified as dcs (38) , although, with hindsight, consideration of bimodal cd45 profiles allows that at least some of them may have been microglia. the fact that cd11c+ microglia express slightly higher cd45 levels than resting microglia may have contributed to uncertainty, and claims that dcs derived from microglia (38, 39) may need re-evaluation. relative cd45 levels as detected by flow cytometry are not as useful for histological discrimination. depending on the antibodies and staining protocols used, microglia may even not be detected as cd45+ cells, or else cannot be distinguished from other cd45 hi cells. similarly, cd11c promoter-driven fluorescent reporter transgenic mice cannot discriminate between the many cell types that can express or upregulate cd11c without co-staining for lineage-specific markers. identification of cd11c+ microglia in such mice relies on interpretation of sometimes fortuitous observations that include consideration of a cell's morphology and location. using an eyfp-cd11c transgenic strain, bulloch et al. identified a small fraction of cd11c+ microglia that were immunoreactive for mac-1, iba1, cd45, and f4/80 (40) . the parenchymal juxtavascular iba1+ cd11b+ gfp-cd11c+ cells described by prodinger et al. in a cd11c-gfp reporter mouse likely included microglia, although in a non-diseased mouse, they would only account for around 2% of them (41) . flow-cytometric analysis confirmed cd45 low gfp-cd11c+ cells in the cns of these mice (42) . the fact that they were mhc ii-negative likely reflects that they derived from non-diseased tissue, unlike the eae-derived cells that we described (34) . typical microglia markers and their functions are listed in table 1 . even before microglia were formally identified, the presence of fat-laden cells had been reported and suggested to be a part of (49) the normal developing cns (50) (51) (52) , and to participate in either cell death processes (53) or myelin formation (54) (55) (56) . early after the initial description of microglial cells, neuroanatomists began to track and map microglia in the cns. del rio-hortega was the first to describe "fountains of microglia" in the developing brain, having amoeboid morphology and being preferentially located in the white matter (57) . already in 1925, penfield reported that what he describes as "neuroglia of mesodermal origin" "were variously considered to be normal and having to do with myelination or to indicate an abnormal inflammatory process" (58) . in the mid-to late 1970s, with del rio-hortega's "fountains of microglia" in mind, these cells were investigated again using light and electron microscopy. most studies describe round, amoeboid, highly vacuolated cells with fat-containing granules, which are found in developing white matter, particularly along unmyelinated axonal tracts in the corpus callosum of rabbits (59) , rats (60), mice (61) , birds (62) , fish (63) , and humans (64) , as opposed to more highly ramified cells present in the gray matter. in all these studies, amoeboid or ovoid-shaped microglia invade the white matter before disappearing when increasing numbers of ramified microglia colonize the gray matter (peaking around postanatal day (p) 5 and disappearing around p10 to p15 in rodents). multiple studies support this finding and extrapolate their potential function, stating either that they have enhanced phagocytic abilities for the elimination of apoptotic material coming from normal developmental cell death or that they participate in myelination (59, 60, (65) (66) (67) (68) . this involvement in myelination was reinforced by a study by pont-lezica et al. showing that microglial alteration early in development leads to impaired corpus callosum fasciculation (11) . their phagocytic abilities along with their morphology provoked debates regarding their origin (68) , their fate (66), and even their microglia status with some studies modifying the nomenclature by referring to them as "brain macrophages" rather than "amoeboid microglia" (67, 68) . with the new notion of microglial phenotypes emerging, these early amoeboid microglia were hypothesized to have higher "activation" levels before becoming "deactivated" in a controlled manner, as this was believed to be temporarily helpful to scavenge debris coming from developmental cellular death. to corroborate this hypothesis, hristova et al. attempted the first phenotypic analysis of these cells, and reported expression of high levels of integrins alpha x (itgax, cd11c), alpha 4 (itga4), alpha 5 (itga5), and beta 2 (itgb2) in microglia from periventricular white matter in comparison to cortical microglia at p7 by staining quantification in iba1+ cells (37) . in addition, in situ hybridization clearly showed transient igf1 and colonystimulating factor 1 (csf1) mrna expression within microglial cells in the corpus callosum and periventricular white matter until approximately two postnatal weeks (37) . in this study, expression of igf1 and csf1 by microglia were hypothesized to play a protective role, preventing axonal damage for instance, which has since then been confirmed in a study by ueno et al. (12) . this finding was reinforced by our own study showing that microglial cells expressing high levels of itgax and igf1 are present in the white matter (cerebellum and corpus callosum) of developing mouse brains particularly between p3 and p5 where they make up almost 20% of all microglia and decrease in numbers already at p7 before being almost completely undetectable by p28 (35) . presence of igf1-expressing microglia in these locations in p5 brains was further confirmed by in situ hybridization (69) . we performed rna-seq on these cells between p3 and p5 after facs-sorting based on cd45 dim cd11b+ cd11c+ gating comparing them to their cd11c− counterparts. we identified a robust neurodevelopmental gene signature for developmental cd11c+ microglia, including factors involved in astrocyte and neuronal differentiation, tissue remodeling, and myelinogenesis accompanied by downregulation of immune function-related genes. of note, itgax, itga4, csf1, and igf1, which were highlighted in the hristova study, were also part of this signature. importantly, we demonstrated that igf1 expression by cd11c+ microglia during development is crucial for primary myelination. indeed, selective deletion of igf1 specifically from cd11c+ cells led to myelination defects in p21 brains (35) . interestingly, all neonatal microglia expressed neuroectodermal genes including nestin. a concomitant study by hagemeyer et al. similarly identified amoeboid microglia in the developing white matter of the corpus callosum and cerebellum particularly between p1 and p8 before being almost undetectable by p14 (70) . interestingly, they used a mac-3 staining to identify these cells, reminiscent of a study by valentino and jones who reported mac-3 expression in "fountain microglia" in a footnote (68) . they identified a signature akin to the one we found (38 genes in common out of 61 upregulated genes including itgax, csf1, and igf1) by comparing "fountain microglia" from corpus callosum at p7 with cortical microglia at the same age by whole-genome microarray (70) . of note, the study underscores that many of the most upregulated genes were related to a primed or activated microglial phenotype and they confirmed cd11c expression in the "fountain of microglia" cells with a reporter mouse. in addition, by depleting all microglia during the critical period of the first postnatal week, they showed that the number of oligodendrocyte progenitor cells was reduced and a long-lasting effect on myelination was induced into adulthood (70) , in line with our own results. two recent studies used single-cell rna-seq to elucidate microglial heterogeneity during development (24, 36) . the barres lab study used deep single-cell rna-seq on microglial cells sorted based on cd11b+ gating and cd45 levels from six different brain regions at e14.5, p7, and p60 (24). they found a cluster of cells they named "proliferative region-associated microglia" (pam), mainly found at p7 in the white matter, that have an amoeboid morphology and phagocytose newly formed oligodendrocytes (24) . in addition, they reported enhanced expression of igf1 and itgax in this cluster compared to any other at p7 or other time points. these cells were observed as early as e17.5 in the embryonic brain, their numbers peaking around p7 and were almost absent from p14 brains (24) . all these features fit with cd11c+ microglia from our study and the historical "fountain of microglia" cells. the stevens lab used high-throughput rna-seq on microglial cells from the whole brain sorted based on a cd45 dim cd11b hi cx3cr1 hi gating at e14.5, p4-5, p30, p100, and p540 and in injury contexts, prioritizing high numbers of cells over depth of sequencing (36) . they identified a cluster of cells exclusive for the p4-5 time point, which have an amoeboid morphology, express phagocytosis-related genes, and are restricted to the corpus callosum and cerebellum, associating closely with axonal tracts, which they named "axon tractassociated microglia" (atm) (36) . again, the features of this subset resembled closely the features of cd11c+ microglia and "fountain of microglia" cells described above. interestingly, their study showed no evidence for a sex bias, the number of cells associated to this cluster being similar for neonatal female and male pups (36) . in addition, anderson et al. (71) described gene signatures of retinal microglia in p7 mice, 60% of which were found to express cd11c. the microglial signature in the p7 retina fit the signature associated to developmental cd11c+ microglia as itgax, lpl, clec7a, and igf1 were enriched in sorted cd11c hi vs. cd11c low cells at p7, whereas p2ry12 and tmem119 were downregulated (71) . we therefore hypothesize that cd11c+ microglia, fountains of microglia, pams, and atms, although described in different studies by different methods under different names, actually represent the same population of cells. comparison of the transcriptomic signature found in each of these studies leads to a core signature of 11 genes found in all four studies (gpnmb, itgax, spp1, fam20c, fabp5, hpse, igf1, folr2, csf1, and anxa5) and 28 additional genes found in at least three of these studies (atp6v0d2, slpi, cd28, crip1, lgals1, anxa2, vat1, ifitm2, gm1673, plaur, s100a1, colec12, clec7a, atf3, atp1a3, ephx1, nceh1, lpl, pld3, plin2, aplp2, ccl3, bnip3, ccl9, gpx3, slc16a3, lag3, and lilrb4) (figure 1) . interestingly, csf1, one of the genes of the core signature, has been identified as one of the prominent genes characteristic of the pre-microglia homeostatic signature (23) . these 39 genes constitute the "developmental signature" of the microglial population described in this section. of note, homeostatic microglia markers, such as tmem119, p2ry12, sall1, tgfbr1, fcrls, and cx3cr1, have been shown to be expressed by this subset, although in most reports at slightly lower levels than in adult microglia or other neonatal microglia (24, 35, 36, 70) . later in this review, we will refer to this population as "developmental cd11c+ microglia". features of this population include peak numbers between p3 and p7, amoeboid morphology, phagocytic abilities, and location in white matter (figure 1 ). in addition, studies mentioned in this section clearly reveal a critical functional role of developmental cd11c+ microglia in the myelination process. their presence in high numbers in the white matter makes them strategically placed in both space and time to take on that role. the aforementioned data support their involvement in phagocytosis of newly formed oligodendrocytes, probably linked to the proper establishment of primary myelination (24, 35, 36, 70) . two of the studies show the long-term importance of these cells on oligodendrocytes and myelination later in life (35, 70) . although the number of common genes in the developmental signature might appear low, we would argue that this is probably due to discrepancies in the transcriptomic techniques used (microarray, bulk rna-seq, high-throughput single-cell rna-seq, deep single-cell rna-seq), as well as the isolation techniques used (facs-sorting based on various gatings, presence or absence of perfusion, whole brain dissection, or region microdissection) (see table 2 ) [discussed in (76) ]. however, similarities in the localization, colonization kinetics, morphology, and functional role leave little room for doubt regarding the uniqueness of the population described. recent studies have described the homeostatic adult brain as the state with lowest microglial heterogeneity (24) . in addition, most high-throughput studies investigating adult microglia in steady state generally report very homogeneous populations in the homeostatic clusters, whether by mass cytometry (77) or single-cell rna-seq (36) , characterized by robust expression of classical microglial homeostatic markers. however, in a cd11c-eyfp reporter mouse, yfp-expressing cells have been found throughout the brain and retina in adulthood. although initially thought to be dcs (40), they have since then been shown to exhibit a phenotype resembling microglia (41, 78) . interestingly, a particular abundance of these cells is found in ventral areas of the brain, white matter tracts, and areas of adult neurogenesis (78) . this is in line with a report that clec7a+ microglia are found in neurogenic niches in the adult mouse (24) , showing that in the homeostatic adult brain, microglia with a phenotype similar to developmental cd11c+ microglia could remain in low number in selected areas. consistent with this, a subset of microglia (also positive for tmem119 and p2ry12) expressing higher levels of cd11c was found in the human subventricular zone and thalamus (79) . in reporter mice, expression of cd11c has been shown to not always follow the expression of the yfp reporter and should therefore be taken cautiously (78) . the existence of cd11c-expressing microglia has however been confirmed in the adult homeostatic brain (around 2% of total microglia) (33-35, 42, 80, 81) . similarly, a small population of cells from the choroid plexus of adult mice was shown to be transcriptionally distinct from other choroid plexus cells and border-associated brain macrophages. this population named "kolmer's epiplexus cells" closely resembles microglial cells and was associated with enriched expression of spp1, apoe, and igf1 (82). although itgax was not among the significantly upregulated genes in this study, cd11c+ cells expressing low levels of cd45 have previously been described in the choroid plexus of adult mice (78) . change in microglial gene expression and phenotype in steadystate aging has been studied extensively. although reports agree on the changes in morphology and general phenotype of microglia toward dystrophic microglia (deramification, cytorrhexis, and fragmentation) in aging [reviewed in (83) ], genomic studies have given discrepant results, with some arguing for shift toward neuroprotection (84) and others highlighting a "primed phenotype" with higher immune activation (85) . that said, having a second look at datasets from various studies brings to light common highly expressed genes in aged microglia compared to young microglia: spp1, clec7a, igf1, lpl, axl, apoe, lgals3, itgax, cst7, etc. are indeed found across several studies (84) (85) (86) , although not all and not always in the same range of upregulation (87) . in a later study, holtman et al. related the "primed" microglial signature they found from two aging models (one physiological aging model and one accelerated aging model) to the study by hickman et al. and found a high correlation between the datasets (88) . high-throughput single-cell methods are a good way to decipher complex populations with mixed subsets. a masscytometry study revealed that a specific subset of microglia emerges during aging that overexpresses surface cd11c and cd14, clec7a, and cd68 as compared to other microglia figure 1 | cd11c+ microglia signature in developmental stages. during development, cd11c+ microglia have an amoeboid morphology and localize close to white matter tracts, essentially in the corpus callous and cerebellum. they are present early during embryonic development and their numbers peak between p3 and p7. comparison of genes upregulated in four studies (24, 35, 36, 70 ) reveals a common signature for developmental cd11c+ microglia of 39 genes upregulated in at least three of the studies (bold dark outline). genes shared with the disease signature in figure 2 are in bold. the venn diagram was generated using the online tool venny (72). at the same age, although they downregulate cx3cr1 and mertk (77, 89) . cd11c expression of microglia in the white matter and caudal areas of the cns of aged mice was also shown using immunohistochemistry (90) . this study also reports expression of clec7a in white matter tracts of aged animals and reports numerous changes in white matter microglia associated with aging. similarly, single-cell rna-seq revealed that several populations of microglia that were present in younger age at very low numbers become increasingly prevalent with aging. one of these populations (referred to as oa2) is characterized by genes from the developmental signature and genes classically associated with neurodegeneration (spp1, lpl, lgals3, lilrb4, (98) , possibly indicating the presence of cd11c+-microglia-like cells in the repopulating clusters they described. this is reinforced by our study in which cd11c+ microglia could be found in repopulating microglia clusters after genetic microglial depletion (35) . however, in contrast to zhan et al. our analysis did not show neonatal-like, neurodevelopmental gene signature in repopulated microglia (35) . the low extent of overlap between these studies and our newly defined neonatal cd11c+ microglia could be explained by heterogeneity of repopulating microglia, diluting the signal from cd11c+ microglia in bulk rna-seq studies. microglia activation is a common feature in many neurological disorders including inflammatory, demyelinating, and degenerative diseases, as well as glioma and injury. although microglia activation may have deleterious consequences, it has also been shown in many instances to exert protective and regenerative effects. it is now becoming clear that there is an emergence of cd11c+ microglia population in pathological conditions. in this section, we will discuss the importance and the role of this cell subset in several neurological diseases. for decades, it has been known that microglia localize around aβ plaques, and engulf aβ in ad, showing their importance in the disease. in recent years, interest in these cells has increased, largely due to a wave of transcriptomic and genome-wide association (gwas) studies. in addition, a majority of ad risk genes are related to microglia, including triggering receptor induced on myeloid cells 2 (trem2) [reviewed in mcquade and blurton-jones (105)]. despite the enormous amount of data generated, no consensus has yet been reached on whether microglia are protective or detrimental in neurodegeneration. some of the attempts to resolve this issue involved comparing transcriptomes of microglia sorted from healthy, aged, and diseased brains. the study by holtman et al. cited above identified a microglial signature found not only in aging models but also in disease models including the app/ps1 ad model and the sod1 model for amyotrophic lateral sclerosis (als) (88) . the common genes included itgax, clec7a, axl, lgals3, and apoe, indicating the presence of a cd11c-expressing microglial population in these models. the gene module described in this study mostly contained genes related to phagocytosis and cell proliferation, with tissue protective elements (88) . with a similar strategy, other studies demonstrated that microglia from aging brains and from amyloidosis (app/ps1) and tauopathy (aav-tau p301l) shared a common gene signature including cst7, itgax, gpnmb, clec7a, lpl, lgals3, apoe, and spp1 (86). similar results were also obtained by krasemann et al. in the app/ps1 model. such shared microglial characteristics led to the term "microglial neurodegenerative phenotype (mgnd) signature" (75) . this is also in line with the presence of cd11c-expressing microglia in these models, with a phenotype similar to the one found in physiological aging. the presence of cd11c+ microglia around aβ plaques has been shown in several studies (30, 73, 74, 106, 107) . a recent study by kamphuis et al. extensively investigated the localization, proliferation status, and transcriptome of cd11c+ vs. cd11c− microglia in app/ps1 mice (73) . importantly, this study also highlighted a steady increase in cd11c transcripts in brains of app/ps1 and 3xtg-ad mice with aging as plaques appear, as well as in hippocampal samples from ad patients, although it declines in the later stages of the disease (73) . the transcriptomic signature of cd11c+ microglia, when compared to their cd11c− counterparts, showed increased expression of gpnmb, fabp5, spp1, igf1, itgax, gm1673, cst7, cox6a2, apoe, ch25h, clec7a, lilrb4, csf1, axl, lpl, sulf2, egr2, anxa5, cd68, timp2, and ctsb among others. many of these genes are common with the developmental signature of cd11c+ microglia described above or with the signatures found in whole brain "primed" microglial signatures (73) . these findings further support that the "primed" microglia phenotype described in many studies recapitulates the cd11c+ microglia signature diluted among cd11c− counterparts. the robustness of the signature is hardly surprising, considering that cd11c+ microglia make up for 23% of all iba1+ cells in the aged app/ps1 brain (73) . of note, strong upregulation of some cd11c+ microglia signature genes, including itgax, clec7a, and cst7, was even detectable in whole tissue samples from cortex and hippocampus in ad models (73, 108) . high-throughput single-cell studies also contributed to our understanding of microglial populations in ad rodent models. the same study that identified cd11c and cd14 surface expression by mass cytometry on a microglia population emerging in aging also identified a similar population in app/ps1 brains (77) . single-cell rna-seq studies identified three microglial signatures in neurodegeneration models: the disease-associated microglia (dam) signature (74), the late response microglia signature (109) , and the activated response microglia (arm) signature (80) that emerge in the 5xfad, ck-p25, and app nl−g−f models for ad, respectively. all three studies described cell clusters showing nearly identical microglia populations, similar to the cd11c+ microglia signature observed in the kamphuis study. importantly, all of the dam cells were cd11c+ (74) with highly overlapping gene signatures uncovered by bulk sequencing of sorted cd11c+ microglia (73) . microglia with characteristics from the arm cluster are present in low numbers (ca. 2%) even in wild-type mice at young age, increasing as part of normal aging to reach up to about 12% of all microglia (80), consistent with observations discussed above of cd11c+ microglia in the steady state in adult and aging mice. arm microglia are however most evident in app nl−g−f mice where they outnumber all other microglial clusters reaching 52% of all microglia at 21 months of age (80) . this is in line with increases in cd11c+ microglia reported in other studies. importantly, the signature observed in cd11c+/dam/mgnd/arm microglia is enriched for known ad risk genes (80) . of note, this transcriptomic signature is similar to that induced by retinal degeneration (110) . cd11c+ microglia have been demonstrated to be beneficial for and to correlate with increased aβ uptake and induction of igf1-mediated neurogenesis in an animal model of ad (30) . in addition, abundance of igf1-expressing microglia around aβ plaques was recently confirmed by in situ hybridization in an ad model (69) . functional analyses led to discrepant results suggesting either protective, immunosuppressive function as well as enhanced capacity for uptake and lysosomal degradation of aβ (73) , or pathogenicity via possible contribution to local arginine deprivation and subsequent neurodegeneration (111) . butovsky's group also proposed a detrimental role for these cells due to ameliorated aβ deposition in 4-month-old trem2-deficient mice that lack cd11c+ microglia (75) . however, the role of trem2 is not clear, since other data show either protective or detrimental roles for this protein depending on the age of the animals (75, (112) (113) (114) . nonetheless, all these studies demonstrate lack of microglial proliferation and clustering around plaques in trem2-deficient animals, thus allowing for more dispersed aβ localization in ad models (75, (112) (113) (114) (115) (116) . this can be detrimental due to aβ spreading that is not limited by microglia clusters, ultimately leading to severe axonal dystrophy (114) . moreover, it has been demonstrated that in trem2-deficient animals older than 8 months, the aβ burden is enhanced as compared to 4-month-old animals, suggesting that trem2 signaling is necessary for limiting advanced stage pathology (117) . thus, cd11c+ microglia may actually be beneficial and protective in later stages of the disease as proposed by keren-shaul et al. (74) . human data further support this hypothesis since loss-of-function mutations in trem2 have been identified as a strong risk factor for the development of ad and other neurodegenerative diseases [reviewed in mcquade and blurton-jones and ulland and colonna (105, 118) ]. collectively, cd11c+ microglia (also referred to as primed microglia, late response microglia, dam, arm, or mgnd) are a well-defined population of cells that show adaptation predominantly for phagocytic clearance of apoptotic/necrotic neurons and limiting aβ spreading. given that ad risk genes are enriched in this population (80) , mutations in such genes may have an impact on the ability of cd11c+ microglia to cope with aβ plaque burden, either promoting or limiting ad pathology. als is a disease affecting motor neurons leading to their degeneration. microglial contribution to the disease has been established since a robust microglial activation has been found in both patient and transgenic mouse tissue (119, 120) . in addition, many risk factors for the disease have been shown to be expressed by microglia in the cns, reinforcing the idea of an involvement of these cells in the disease (121) . microglial activation in the disease arises from accumulation of misfolded protein, and, similarly to observations made in other disease contexts, microglia have been reported to play a beneficial role in the pre-symptomatic phase of the disease before shifting to detrimental roles in the advanced disease state (122) . however, microglial depletion in the context of als has not been found to increase survival (123) , leading to the idea that both functions might be concomitant, constantly counteracting each other. interestingly, a study from 2013 analyzed the transcriptome of microglia sorted from mice carrying an als-associated mutation and found a particular signature for these cells at the end stage of the disease compared to microglia from healthy brains (124) . once again, among the top regulated genes were genes related to huntington's disease, ad, and parkinson's disease (mapt, psen2, apoe, etc.). the signature found in this study includes both factors reported to be beneficial in the context of als (igf1, grn, trem2, tyrobp, etc.), and factors known to be detrimental (mmp12, optn, cybb, etc.), as well as some like spp1, gpnmb, and itgax recurrently found in neurodegenerative diseases. microglia were also found to upregulate surface cd11c. microglia from sod1 mice were also found to fit the abovementioned mgnd signature, in addition to expressing clec7a levels increasingly during disease progression (75) . neuron degeneration and nerve injury have been linked to microglia in various models for traumatic brain injury (tbi) (125) , spinal cord injury (sci) (126) , nerve injury (93) , and ischemic stroke (127) . much like in inflammation models, microglial contribution in all of these models is still rather unclear and they may play a double role considering their association with both beneficial and detrimental effects. studying microglia in context of inflammation can get quite complicated due to massive infiltration of peripheral immune cells, notably monocytes and macrophages, occurring subsequently to tbi (128) , sci (129) , and stroke (127, 130, 131) . in a study comparing the transcriptomics of microglia and macrophages after ischemia in rats, it was reported that microglia played a detrimental role and macrophages played a beneficial role with regard to recovery, based on their expression of classical inflammation markers (132) . investigation of the genes enriched in microglia three days after middle cerebral artery occlusion compared to sham controls, however, revealed spp1, gpnmb, lgals3, fabp5, and axl among others, fitting with the potential presence of cd11c+ microglia-like cells in this context, diluted among other microglia. consistent with this, ccl2 mrna was found to be increased in microglia and macrophages at this time point (132) , an aspect that has been associated with the emergence of cd11c+ microglia (81) . another study, conducted in a model of phototrombic stroke on whole tissue, actually showed upregulation of gpnmb, itgax, and clec7a in a cluster associated with early response (133) , which the authors related to the dam phenotype (74) . in a study of facial nucleus axotomy, the authors also related the observed microglial phenotype (134) to the dam phenotype, as well as to a phenotype found in the ck-p25 model (109): 72 genes were regulated in common between all three studies representing almost 75% of all genes upregulated in the facial nucleus axotomy model. interestingly, in an sci transcriptomic study, a profile of microglia reminiscent of the cd11c+ phenotype was identified (with upregulation of gpnmb, spp1, lpl, apoe, igf1, lgals3, and itgax among others) and persisted in a full transection model, whereas it contracted concomitantly to recovery in a hemisection model (135) , indicative of the transitory nature of this subset. conversely, in tbi, the microglial signature was further from the cd11c+ microglia signature, although itgax was among the upregulated genes 14 and 60 days post-injury, possibly indicating once again a dilution of the signature in all microglia (136) . in addition, considering the difficulty associated with gating out macrophages from microglia in a context of extensive infiltration, macrophage contamination of the sorted samples cannot be excluded in these studies, potentially complicating interpretation of the observed transcriptomes. ms is an inflammatory, demyelinating disease of the cns that can be modeled by eae or toxin-induced demyelinating models. recent advancement in our understanding of the disease points toward important roles for microglia in the pathomechanism. although the evidence supporting their implication in initiation and facilitation of the disease is strong (95) , there is a growing body of evidence for their protective functions including involvement in remyelination (137) . we have identified cd11c+ microglia during eae accounting for around 10% of total microglia in whole cns (33, 34) . of note, this subset is even more abundant in the spinal cord at the peak of the diseases reaching up to 60% of total microglia (wlodarczyk, unpublished) . the emergence of the cd11c+ microglia is a dynamic process starting at the onset, reaching a maximum at the peak and contracting in the chronic phase of eae (77, 138) . these cells are localized in the demyelinated spinal cord lesions (33) . cd11c+ microglia from eae again showed upregulation of similar genes as in neurodegenerative models including itgax, gpnmb, spp1, etc. (35) . a similar signature was confirmed by krasemann et al. (75) . in addition, deep analysis of genes that were upregulated in cd11c+ microglia population pointed to their involvement in immune responses (35) . a key aspect of neuroinflammation in eae is the recruitment and reactivation of encephalitogenic t cells to express their effector functions. many cell types are implicated in this process, including blood-derived dcs and monocytes/macrophages but also parenchymal microglia (139) . in eae, cd11c+ microglia express mhci, mhcii, and costimulatory molecules cd80/cd86 (34, 140) , which is in line with recent highthroughput mass-cytometry reports (77, 138) . we have provided evidence that cd11c+ microglia are able to induce similar proliferative response of encephalitogenic cd4+ t cells as blood-derived professional antigen-presenting cells (32, 34) . interestingly, in contrast to cd11c+ blood-derived cells and cd11c− microglia, cd11c+ microglia completely lacked mrna expression for il-23 (34) that is known to induce gm-csf-producing cd4+ t cells, critical for eae pathology (141) . this indicates that although cd11c+ microglia alone might contribute to t cell expansion, they are unlikely to induce pathogenic t cell responses. importantly, a subsequent study showed that they were a major source of message for myelinogenic igf1, suggesting that they might exert protective roles in eae (33) . this is supported by our recent study showing that stimulation of csf1r with its ligands during symptomatic eae significantly reduced demyelination and ameliorated disease progression most likely through induction of cd11c+ microglia (81) . moreover, decreasing cd11c+ microglia by blocking of trem2 signaling (as discussed below) led to increased severity of eae and exacerbated demyelinating lesions in the spinal cord (142) , further supporting protective roles of cd11c+ microglia. microglia are known to contribute to remyelination by creating an environment supporting opc recruitment and differentiation by phagocytosing myelin debris, secreting growth factors and modulating extracellular matrix [reviewed in lloyd and miron (137) ]. circumstantial evidence for remyelinating properties of cd11c+ microglia includes our first demonstration of the expansion of these cells in cuprizone-demyelinated corpus callosum (32) . a microarray study by olah et al. identified a pro-remyelinating microglial signature that includes several genes reminiscent of the cd11c+ microglia characteristics described above (itgax igf1, clec7a, apoe, spp1) (143) . moreover, cd11c immunoreactive microglia were present in remyelinating corpus callosum (32) . a similar microglial signature was later confirmed in both demyelination and remyelination phases (144) . conversely, microglia expressing the cd11c+ microglia signature including apoe, axl, igf1, lyz2, itgax, and gpnmb were identified by single-cell transcriptomics in both deand remyelinated lesions (145) . recently, cuprizone-mediated demyelination was shown to be alleviated in mice lacking microglial sirpα that have increased numbers of cd11c+ microglia, pointing to their protective role (89) . in line with the induction of cd11c+ microglia (81) , stimulation of csf1r ameliorated cuprizone-induced demyelination (146) . another line of evidence comes from the influence of trem2 deficiency, which leads to absence of cd11c+ microglia in adult mice (74, 75) , on remyelination after cuprizone demyelination. the data indicate that trem2 deficiency had no impact on the initial demyelination, but affected subsequent remyelination when the cuprizone treatment was prolonged, most likely by impairing myelin removal as well as myelin regeneration, which further supports a protective role for cd11c+ microglia in this paradigm (144, 147) . additionally, it was reported that microglial necroptosis in circumstances of lysophosphatidylcholine demyelination leads to repopulation by pro-regenerative cd11c+ microglia, as blocking of this mechanism prevented remyelination (148) . of note, demyelination induced by mouse hepatitis virus also led to enrichment of cd11c+ microglial gene signature in the spinal cord (149) . taken together, association of cd11c+ microglia to white matter (89) as well as their role in primary myelination strongly support their importance in induction and facilitation of remyelination. this opens the possibility for induction of innate repair programs in diseased cns via promotion of the emergence of cd11c+ microglia. very early studies identified microglial cells close to gliomas to resemble the amoeboid form described during development and to take on phagocytic functions (58) . more recent studies have shown that parenchymal microglia are attracted to the tumor in glioma-affected brains, representing up to 30% of the tumor mass (150) . microglia associated to the tumor have been termed glioma-associated microglia/macrophages (gam). these cells initially exhibit beneficial anti-tumor abilities but have been found to be hijacked by the tumor to exert tumor-promoting functions [reviewed in li and graeber (151) ]. a study from 2015 identified a signature for gams, and emphasized their high expression of spp1 and gpnmb (152) . they compared this signature to classical macrophage activation markers (m1/m2) and concluded a lack of overlap between the gam signature and these classical phenotypes. of note, the signature also includes genes such as itgax, fabp5, and clec7a among others recurrently found in disease signatures (152) . considering the similarities observed in gene expression from the different studies aforementioned, we compared the transcriptomic signatures obtained in studies comparing specifically microglia sorted based on a typical marker for this specific subset of microglia or from single-cell rna-seq (three of the ad studies and one eae study, figure 2 ). we found a core disease signature for microglia consisting of 89 genes shared between all four studies (figure 2) . itgax being once again a part of this signature and with clarity in mind, we will refer to this signature as the "cd11c+ microglia disease signature" henceforth. once again, the microglial nature of this subset is supported by expression, although slightly lower than in homeostatic microglia, of tmem119, cx3cr1, p2ry12, sall1, and tgfbr1 among other homeostatic genes (35, (73) (74) (75) . over the years, advancements in technology have allowed the scientific community to investigate cells and cell populations in increasingly detailed ways, particularly at the molecular level. this investigation has been done using a multiplicity of different conditions and models, leading to increasing amounts of data generated. although invaluable, this work has also led to redundancy in the microglial profiles that were identified (154) . our investigation led us to define two particularly strong signatures for cd11c+ microglia in development (figure 1 ) and in disease (figure 2) . interestingly, li et al. (24) as well as anderson et al. (71) related the developmental microglia signature observed in their studies to the dam microglial signature. these similarities prompted us to compare the signatures we identified from the literature. comparison of the developmental signature and the disease signature resulted in defining of a "core" signature common to cd11c+ microglia across all contexts, which consists of 22 genes: ank, anxa5, aplp2, atp1a3, clec7a, colec12, csf1, ephx1, fabp5, fam20c, gm1673, gpnmb, hpse, igf1, itgax, lilrb4, lpl, nceh1, plaur, pld3, plin2, and spp1 (figure 3 and supplementary table 1) . interestingly, the protein network linked to these genes had significantly more links than what can be expected, indicating at least a partial biological connection between these genes (figure 3) . further investigation of the physiological function of the proteins related to the genes present in the core signature revealed their involvement in lipid metabolism, cell migration and proliferation, and, to a lesser extent, immune function (supplementary table 1) . as expected, all of these proteins had been associated with various brain diseases (supplementary table 1 ). of note, many of these proteins assume similar function or have been found to interact directly or indirectly with each other (supplementary table 1 ). further investigation of these genes and proteins in link with one another would most likely unveil interesting mechanisms underlying cd11c+ microglia function. although described previously as different microglial subsets, we argue that the robust core signature we have identified can be found for this subset across all these different stages. we suggest that the differences in this subset observed between conditions reflect methodological discrepancies ( table 2) or microenvironment-linked context-specific changes and the subset's own phenotypic plasticity in coping with these variations, rather than fundamental differences in cell lineage. the dynamics of cd11c+ microglia seem tightly spatiotemporally regulated. they first emerge during the first postnatal week, peaking at p5 and gradually decreasing as animals age, being barely detectable in the healthy adult cns (33-35, 42, 80, 81) to increase again in aging or disease (33, 73, 81, 85, 89) . importantly, none of the studies that have investigated induction of inflammation by means of lipopolysaccharide, poly(i:c), or other immune challenges could recapitulate the robust cd11c+ signature found in steady state and disease and injury contexts (86, 88, 90, 124, 155, 156) . below, we present factors that participate in controlling the induction of this population (figure 4) . one candidate that has been extensively studied with regard to cd11c+ microglia is the trem2 pathway. trem2-deficient animals were shown to downregulate the cd11c+ microglia signature in cuprizone-induced demyelination (144) and in an ad model (113) . in addition, in the study from the amit lab, trem2 deficiency in an ad mouse model led to an arrest of microglia in an intermediate state between the homeostatic state and the cd11c+ microglia stage. barely any microglia in these mice exhibited the cd11c+ microglial signature (74) . this suggests that cd11c+ microglia induction is a two-step process, where the first step, to leave the homeostatic state, is trem2-independent and the second step, to reach the complete cd11c+ microglia phenotype, is trem2-dependent. these observations were confirmed by krasemann et al. in another trem2-deficient ad model (75) . similarly, apoedeficient mice exhibit lower numbers of cd11c+ microglia in ad, als, and ms mouse models (75, 80) . this is suggestive of a positive feedback loop, as this population itself strongly upregulates apoe (75) . surprisingly, the barres lab showed that induction of cd11c+ microglia during postnatal development in contrast to adulthood is trem2-apoe-independent (24) . a similar trem2 independence of cd11c+ microglia induction was shown in the developing retina (71). figure 2 | cd11c+ microglia signature in disease states. in diseased cns, cd11c+ microglia adopt an amoeboid, reactive morphology. in ad, they are found surrounding aβ plaques. similarly, in ms and als models and in injury, they are found around and in the lesions. in glioma, they are found mixed with tumor cells. cd11c+ microglia numbers in diseased cns vary considerably, ranging from 10 to 50% of all microglia. comparison of genes upregulated in four studies (35, (73) (74) (75) reveals a common signature for cd11c+ disease microglia of 89 genes upregulated in at least three of the studies (bold dark outline). genes shared with the developmental signature in figure 1 are in bold. raw data for the krasemann study were obtained using the gene expression omnibus database and the differential gene expression analysis was performed using the debrowser package in r (153) . the venn diagram was generated using the online tool venny (72). krasemann et al. highlighted phagocytosis of apoptotic neurons and monocytes as a trigger for the induction of the cd11c+ microglia phenotype (75) . of note, induction of this phenotype was not observed upon microglia exposure to escherichia coli, zymosan particles (75), or microparticles (marczynska et al., unpublished), suggesting that induction of cd11c+ microglia is a tightly controlled reaction to local cell damage or apoptosis, rather than to phagocytosis itself. interestingly, microglial necroptosis in demyelination models leads to brain repopulation with cd11c+ microglia from nestin+ resident microglia (148) . similarly, nestin+ microglia colonizing the brain after microglia ablation expressed surface cd11c (98) . the gene expression in repopulating microglia highly overlapped with the cd11c+ microglia signature. we showed that genetic or toxin-induced ablation of neonatal cd11c+ cells led to their instant repopulation (35) . whether the observed concomitant decrease of cd11c− microglia (35) reflects induction of cd11c+ phenotype in cd11c− cells by phagocytosis of dying microglia has not been determined. interestingly, a dramatic decrease in cd11c+ microglia was observed in the postnatal retina of mice deficient in bax, a pro-apoptotic gene that is essential for developmental death of neurons (71) . this emphasizes that apoptotic cells are a strong and common inducer of cd11c+ microglia regardless of age and condition. this is also in line with several studies where developmental cell death has been linked figure 3 | core cd11c+ microglia signature. considering similarities between the transcriptomic signatures and functions in the cd11c+ microglia subset in development and in disease, we compared both signatures to obtain a core of genes upregulated in this subset across all conditions. we observe overlap of 20% of the genes between both signatures, corresponding to 22 shared genes. upon interrogating the string database (szklarczyk d, gable al, lyon d, junge a, wyder s, huerta-cepas j, simonovic m, doncheva nt, morris jh, bork p, jensen lj, von mering c. string v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. nucleic acids res. 2019 nov; 47:d607-613.), we observed that the network formed by the proteins corresponding to the genes in the core signature had significantly more interactions than expected from a similar set of random proteins, indicating that these proteins related to the genes in the core signature are at least partially biologically connected. the thickness of the edges linking the different genes is proportional to the strength of the evidence linking the two proteins. the venn diagram was generated using the online tool venny (72) . to microglial entry in the developing cns (61) . in addition, retinal cd11c+ microglia were resistant to depletion induced by either csf1r deficiency or blocking, contrary to their cd11c− counterparts. in line with this, our own data showed that despite using several depletion regimens, cd11c+ microglia could not be depleted from postnatal brain as they were immediately repopulated (35) . we have shown that both populations of adult microglia (cd11c+ and cd11c−) express equal levels of csf1r (33) . importantly, stimulation of this receptor by its ligands, interleukin (il)-34 and csf1, induced a significant increase in cd11c+ microglia numbers, with faster kinetics for il-34 (81) . moreover, such stimulation induced ccl2 in the brain, and we showed that overexpression of ccl2 leads to a dramatic expansion of cd11c+ microglia in a ccr2independent manner (81). butovsky et al., on the other hand, showed that another cytokine, il-4, can induce cd11c+ expression on aβ pretreated microglia (30, 157) . moreover, they demonstrated that ga vaccination leads to an increase of cd11c+ microglia surrounding aβ plaques and suggested that this was induced by t-cell-derived il-4 (30). recently, the emergence of cd11c+ microglia in the adult brain has been shown to be homeostatically controlled by sirpα/cd47 interaction. genetic ablation of sirpα in microglia or global lack of cd47 equally resulted in increased numbers of cd11c+ microglia, suggesting that microglial sirpα suppresses cd11c expression in the same cells (89) . here, we have demonstrated that the subpopulation of microglia described in many recent studies (and named pam, atm, fountain of microglia, dam, arm, mgnd, and late response microglia) indeed reflects the characteristics of cd11c+ microglia, originally identified over a decade ago. thus, we believe that a unification of the nomenclature by referring to the microglial subset expressing the described signature, from development to old age, as cd11c+ microglia is a necessary step to progress our understanding of microglia biology. this subset emerges in development before contracting during adulthood but is triggered to re-emerge in aging as well as in the context of disease or tissue injury (figure 4) . the summary of the data that mentioned microglia showing the aforementioned signature strongly points to the importance figure 4 | cd11c+ microglia as a subset of microglia present through life and across conditions. our investigation leads us to believe that cd11c+ microglia represent a subset of microglia characterized by a robust signature of 22 genes expressed by this subset at any age and in various disease states. emergence of this subset is induced by various factors including signaling through the trem2-apoe pathway, cell death, il-4 signaling, and cytokine signaling through csf1r inducing ccl2, and is inhibited by cd47/sirpα signaling. in physiological conditions, cd11c+ microglia account for around 15% of all microglia, before contracting to 2% in adulthood and being re-induced by aging at levels similar to development. in disease states, their numbers oscillate between 10 and 50%. we argue that despite the numerous names given to this subset across conditions, it is unique and should be referred to as "cd11c+ microglia". of cd11c+ microglia in primary myelination during cns development as well as their protective, remyelinative, and regenerative capacities in cns pathology. this opens new perspectives for therapeutic targeting of microglia in neurological conditions. aw and ab-b designed the manuscript. ab-b analyzed the transcriptomic data and prepared the figures. ab-b, aw, and to wrote and approved the manuscript. tercer elemento" de los centros nerviosos. i. la microglía en estado normal. ii. intervención de la microglía en los procesos patológicos (células en bastoncito y cuerpos gránulo-adiposos). iii. naturaleza probable de la microglía fate mapping analysis reveals that adult microglia derive from primitive macrophages microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain entry and distribution of microglial cells in human embryonic and fetal cerebral cortex microglial dynamics during human brain 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host transcriptome from demyelinating spinal cord of murine coronavirus-infected mice the brain tumor microenvironment the molecular profile of microglia under the influence of glioma glioma-associated microglia/macrophages display an expression profile different from m1 and m2 polarization and highly express gpnmb and spp1 debrowser: interactive differential expression analysis and visualization tool for count data the kaleidoscope of microglial phenotypes microglia transcriptome changes in a model of depressive behavior after immune challenge single-cell transcriptomics reveals distinct inflammation-induced microglia signatures microglia can be induced by ifn-γ or il-4 to express neural or dendritic-like markers the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.00430/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 benmamar-badel, owens and wlodarczyk. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-345254-glm2dxhh authors: hwang, mihyun; phares, timothy w; hinton, david r; stohlman, stephen a; bergmann, cornelia c; min, booki title: distinct cd4 t-cell effects on primary versus recall cd8 t-cell responses during viral encephalomyelitis date: 2015-02-13 journal: immunology doi: 10.1111/imm.12378 sha: doc_id: 345254 cord_uid: glm2dxhh cd4 t-cell help is not a universal requirement for effective primary cd8 t cells but is essential to generate memory cd8 t cells capable of recall responses. this study examined how cd4 t cells affect primary and secondary anti-viral cd8 t-cell responses within the central nervous system (cns) during encephalomyelitis induced by sublethal gliatropic coronavirus. cd4 t-cell depletion before infection did not impair peripheral expansion, interferon-γ production, cns recruitment or initial cns effector capacity of virus-specific cd8 t cells ex vivo. nevertheless, impaired virus control in the absence of cd4 t cells was associated with gradually diminished cns cd8 t-cell interferon-γ production. furthermore, within the cd8 t-cell population short-lived effector cells were increased and memory precursor effector cells were significantly decreased, consistent with higher t-cell turnover. transfer of memory cd8 t cells to reduce viral load in cd4-depleted mice reverted the recipient cns cd8 t-cell phenotype to that in wild-type control mice. however, memory cd8 t cells primed without cd4 t cells and transferred into infected cd4-sufficient recipients expanded less efficiently and were not sustained in the cns, contrasting with their helped counterparts. these data suggest that cd4 t cells are dispensable for initial expansion, cns recruitment and differentiation of primary resident memory cd8 t cells as long as the duration of antigen exposure is limited. by contrast, cd4 t cells are essential to prolong primary cd8 t-cell function in the cns and imprint memory cd8 t cells for recall responses. a major role of cd4 t cells during infections is to support priming, programming and/or effector function of cd8 t cells. help can be provided by cytokine production or regulation of co-stimulatory factors, such as cd40/cd40 ligand via antigen-presenting cells. 1, 2 nevertheless, the requirement for cd4 t-cell help in primary cd8 t-cell responses is not universal and depends on the in vivo milieu during initial t-cell activation. primary cd8 t-cell responses against infectious agents are mostly cd4 t-cell independent, whereas responses to non-inflammatory stimulation or non-replicating vaccines are dependent on cd4 t-cell help. [3] [4] [5] [6] irrespective of the requirement for cd4 t-cell help for primary cd8 t-cell responses, it is accepted that cd4 t-cell help is necessary for the generation of memory cd8 t cells capable of efficient recall responses. 5, 7, 8 cd4 t cells also play a key role in optimal cd8 t-cell expansion in the draining lymph node (ln), subsequent mobilization of activated cd8 t cells into inflamed tissues, as well as their maintenance and survival at effector sites. 1, [9] [10] [11] [12] while imprinting of cd4 t cells on cd8 t-cell function and survival has been extensively studied in peripheral and balb/c (h-2 d ) mice, which resolves into a persistent infection associated with chronic demyelination. 13 initial activation of adaptive immunity occurs in the draining cervical ln (cln). 14 activated cd4 and cd8 t cells subsequently cross the blood-brain barrier and enter the cns, where they are re-stimulated to secrete interferon-c (ifn-c), express granzyme b, and lyse virus-infected target cells. 9, 13 cd8 t cells are the major effectors controlling viral load via both ifn-c and perforin-mediated mechanisms. [15] [16] [17] nevertheless, sustained viral rna indicates persistence at low levels. 18 the role of cd4 t cells is complex because they not only promote cd8 t-cell function and survival within the cns 9,10 and directly contribute to viral control, but also enhance pathology. [19] [20] [21] [22] [23] a recent study to assess whether cd4 t cells influence cd8 t cells at the activation or effector stage during jhmv infection revealed that cd4 t cells not only enhance cd8 t-cell expansion in the cln during priming, but also exert helper function within the cns by locally promoting cd8 t-cell effector function and survival. 9 cd8 t cells were incapable of controlling virus in the cns without cd4 t cells, even when primed in the presence of cd4 t cells. 9 the latter results were obtained in h-2 b mice, in which the dominant cd8 t-cell response is directed to an epitope in a hypervariable region of the viral spike (s) protein restricted to h-2d b . 24 in the present report, we set out to assess the extent of cd4 t-cell imprinting not only on primary cd8 t-cell responses, but also on memory formation and recall cd8 t-cell responses in the cns. balb/c mice were chosen for these studies because they mount a prominent h-2l d restricted cd8 t-cell response to an epitope in the highly conserved nucleocapsid (n) protein, which is expressed at much higher levels than the s protein, 25, 26 potentially leading to distinct t-cell activation requirements. an accelerated cd8 t-cell response to the n relative to s epitope is indicated by earlier detection of n-specific relative to s-specific responses in cln of infected balb/c 14 and c57bl/6 9 mice, respectively, as well as an early preponderance of n-specific over s-specific cd8 t cells in the cns of jhmv-infected (balb/c 9 c57bl/6) f 1 mice. 26 moreover, adoptive transfers indicate that virusspecific cd8 t cells induced in the context of h-2 d have more potent antiviral activity than virus-specific cd8 t cells induced in the context of h-2 b . 15, 27 surprisingly, herein we show that peripheral expansion of virus-specific cd8 t cells was not impaired in the absence of cd4 t cells in balb/c mice, as distinct from c57bl/6 mice. furthermore, cd4 t-cell help during priming was dispensable for cns accumulation and initial function of primary virus-specific cd8 effector t cells. however, uncontrolled cns virus replication in the absence of cd4 t cells ultimately resulted in loss of ifnc production, higher cd8 t-cell turnover, and inability to acquire an effector memory phenotype. nevertheless, the unhelped cd8 t-cell phenotype was rescued when virus replication was controlled by transfer of memory cd8 t cells, indicating that the unhelped cd8 t-cell phenotype during primary responses is regulated by antigen load, rather than lack of cd4 t-cell imprinting. by contrast, unhelped memory cd8 t cells mounted poor recall responses when transferred into cd4 t-cell-sufficient mice and could not be sustained in the cns, despite efficient virus control. mice, virus and cd4 t-cell depletion balb/c (h-2 d ) mice were obtained from the national cancer institute (frederick, md). all mice were used at 6-7 weeks of age and infected intracranially in the right hemisphere with 2000 plaque-forming units of the glia tropic monoclonal antibody (mab) -derived 2.2v-1 variant of mouse hepatitis virus strain jhm (jhmv). 28 virus titres were determined as previously described. 28 briefly, clarified supernatants from individual brain homogenates were used to measure virus by plaque assay on a murine astrocytoma cell line, designated dbt. plaques were counted after 48 hr incubation at 37°. clinical disease was scored daily as described elsewhere: 28 0, healthy; 1, ruffled fur and hunched back; 2, hind limb paralysis/ inability to turn to upright position; 3, complete hind limb paralysis and wasting; 4, moribund/dead. cd4 t cells were depleted by intraperitoneal injection with 250 lg purified anti-mouse cd4 mab (gk1.5; bioxcell, west lebanon, nh) at days à2 and 0 of infection. cd4 t cells remained below 0á3% in mab-treated mice for at least 21 days. controls received the same amount of isotype control anti-b-gal mab (gl113). memory cd8 t cells were generated by intraperitoneal injection of thy-1.1 balb/c mice with 2 9 10 6 plaqueforming units of jhmv. donor mice were treated with anti-mouse cd4 or control mab at day à2 and 0 relative to intraperitoneal immunization for comparative analysis of 'unhelped' versus 'helped' cd8 t cells. after 3-4 weeks splenic donor cd8 t cells were isolated by negative selection using fitc-labelled anti-cd4 (clone rm4-5), cd19 (clone 1d3), mhc class ii (clone m5/114.15.2), fcc iii/ii receptor (clone 2.4g2) and nk1.1 (clone pk136), in combination with anti-fitc-coated magnetic beads (miltenyi biotec, inc., auburn, ca) and transferred into either naive balb/c mice at day 1 before intracranial infection (1á2 9 10 6 to 1á5 9 10 6 /mouse) or at 5 days after intracranial infection (0á25 9 10 6 /mouse) as indicated. all procedures were carried out under animal protocols approved by the institutional animal care and use committees of cleveland clinic foundation. for lymphocyte isolation from the cns, brains and spinal cords were removed from mice perfused with cold pbs (ph 7á2). tissues were homogenized in rpmi-1640 containing collagenase (1 mg/ml; roche, indianapolis, in) and dnase i (1 mg/ml, roche) using gentlemacstm tubes, a gentlemacs dissociator (miltenyi biotec), and cell strainers (bd falcon, durham, nc). homogenates were centrifuged at 450 g for 10 min at 4°. the cells were resuspended in cold pbs, adjusted to 30% percoll (ge healthcare, uppsala, sweden), underlayed with 70% percoll and centrifuged at 850 g for 30 min at 4°as described previously. 9 mononuclear cells were collected from the 30%/70% interface, washed with rpmi-1640 and counted before analysis. peripheral lymphocytes were isolated from cln. rna from brains of naive and jhmv-infected mice was extracted using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer's instructions and cdna was subsequently generated by superscript iii rtase (invitrogen) with oligo-dt(12-18) primers (invitrogen). taqman primers/probes specific for gapdh (mm03302249_g1), ifn-c (mm01168134_m1) and cxcl9 (mm00434946_m1) were purchased from applied biosystems (foster city, ca) and rna levels were determined using steponeplus tm real-time pcr systems (applied biosystems) and stepone tm software v2.1 (applied biosystems). gene expression was normalized to gapdh expression and converted to a linearized value using the formula: [2e(ct gapdh à ct gene )] 9 1000. single-cell suspensions were blocked with rat anti-mouse cd16/32 mab (clone 2.4g2: bd biosciences, san diego, ca) for 20 min on ice before staining. for four-or fivecolour flow cytometry, cells were stained with fitc-, phycoerythrin (pe) -, pe-cy5-, allophycocyanin-and pe-cy7-conjugated mab specific for cd45 (clone 30-f11), cd8 (clone 53-6.7), klrg1 (clone 2f1), cd127 (clone a7r34) and mhc ii (clone m5/114.15.2) (all from ebioscience, san diego ca) in pbs containing 0á1% bsa. for intracellular staining for ki67 (clone b56; bd biosciences), cells were stained with surface molecules before permeabilization with fix (0á4% pfa)/perm buffer (pbs containing 0á1% bsa and 0á1% saponin). virus-specific cd8 t cells were detected with pe-cy7-conjugated anti-cd8 and pe-conjugated l d /pn class i tetramer 26 at 0á1 lg/ 0á5 9 10 6 to 1á0 9 10 6 cells as directed by the supplier (nih tetramer core facility, atlanta, ga). for virus-specific ifn-c production by cd8 t cells, cells were cultured with or without 1 lm pn peptide for 5-6 hr with 1 ll of golgi stop (bd bioscience)/ml. after stimulation, cells were stained for cd8 surface expression, fixed and permeabilized to detect intracellular ifn-c (clone 4s.b3; ebiosience). samples were analysed on a facs lsrii (bd biosciences). forward and side scatter signals obtained in linear mode were used to establish a gate containing live cells, while excluding dead cells and tissue debris. data were analysed using flowjo (9.3.3) software (tree star inc., ashland, or). for cytotoxicity assays target cells were prepared by pulsing ammonium-chloride-potassium lysing buffer (invitrogen) -treated splenocytes with the immunodominant l d -restricted pn peptide (5 lm) for 2 hr at 37°. peptidepulsed and untreated targets were then labeled in 50 nm cfse and 2á5 nm cfse, respectively. equal numbers of peptide pulsed (cfse high ) and control (cfse low ) targets were mixed together. total target cells were plated into a v-bottom 96-well plate at 10 3 per/well, and virus-specific cd8 t cells were added at effector : target ratios of 1 : 1, 2 : 1 or 4 : 1. target cells incubated alone were used as negative controls. the plate was centrifuged (300 g for 1 min) to optimize cell contact and incubated for 5 hr at 37°. the proportion of cfse high and cfse low cells was determined by flow analysis. specific lysis was calculated with the formula: [1 à (ratio of targets only/ratio of targets + t cells)] 9 100. brains and spinal cords from pbs-perfused mice were fixed with 10% neutral zinc-buffered formalin and embedded in paraffin. sections were stained with either haematoxylin & eosin or luxol fast blue (lfb) to determine inflammation and myelin integrity, respectively, as described previously. 9 distribution of viral antigen was determined by immunoperoxidase staining (vectastain-abc kit; vector laboratory, burlingame, ca) using the anti-jhmv mab j3.3 specific for the c-terminus of the viral n protein as the primary antibody, and horse antimouse as the secondary antibody (vector laboratory) as described elsewhere. 9, 16, 17 results are expressed as the mean ae sem for each group of mice. results are expressed as the mean ae sem for each group of mice. statistics were determined using unpaired two-tailed student's t-test and verified using two-way analysis of variance (anova) with bonferroni post-test. any variance between statistical evaluations resulting in a change from significant to non-significant or vice versa are indicated in respective figure legends (figs 1,6,8 ). for the data sets indicated in legends, graphs were plotted using a graphpad prism 5.0 software (graphpad software, inc., la jolla, ca). to assess whether cd4 t-cell help for cd8 t cells during jhmv cns infection is dependent on the nature of the viral antigen and genetic background, balb/c mice were treated with depleting anti-cd4 or irrelevant anti-b-gal mab before infection. distinct from h-2 b mice, 9 the absence of cd4 t cells did not affect expansion of virusspecific cd8 t cells in the cln (fig. 1a) . interferon-c production by virus-specific cd8 t cells was also not affected ( fig. 1b) , confirming a redundant role of cd4 t cells in priming effector cd8 t cells in h-2 d mice. these results prompted us to further examine the efficacy of primary anti-viral cd8 t cells within the cns, when primed in the absence (unhelped) or presence (helped) of cd4 t cells. disease onset and severity were initially similar in cd4depleted and control infected balb/c mice (fig. 2a) . however, whereas controls recovered after day 10 postinfection (p.i.) with a survival rate > 90%, cd4-depleted mice exhibited progressively increased clinical symptoms, with mortality beginning at day 25 p.i. and culminating at 100% before day 40 p.i. (fig. 2a,b) . the sustained symptoms leading to mortality were associated with uncontrolled virus replication (fig. 2c ). whereas infectious virus in the brain progressively declined to undetectable levels by day 21 p.i. in control mice, cd4depleted mice failed to control infectious virus after day 7 p.i. (fig. 2c) . these data demonstrated a critical role of cd4 t cells in controlling cns virus replication at least to day 14 p.i., when viral clearance in both the h-2 b and h-2 d genetic backgrounds is independent of humoral immunity. 29, 30 consistent with the inability to clear infectious virus from the brain, immunohistochemistry revealed an increased number of virus-infected cells in cd4-depleted mice at day 21 p.i. (fig. 3a) . interestingly, in the brain many of the virus-infected cells exhibited the morphology of neurons (fig. 3a, inset) . by contrast, only very rare virus-infected cells were detectable in the brains of con-trol mice at day 21 p.i. virus-infected cells were also more abundant in spinal cords of cd4-depleted mice compared with controls (fig. 3b) . in cd4-depleted mice virus-infected cells were predominantly glia in appearance (fig. 3b , left inset) with occasional infected neurons (fig. 3b, right inset) . in control mice the spinal cords showed only rare virus-infected cells with the appearance of macrophages (fig. 3b, inset) . in contrast to the increased number of virus-infected cells and disease severity, overall inflammation in the spinal cord and the extent of demyelination were comparable between the groups (fig. 3c) . as t cells are the primary mediators of jhmv control in the cns during the first 14 days p.i. 29, 30 the inability of cd4-depleted mice to reduce viral load suggested impaired cd8 t-cell recruitment or function. in balb/c mice infected with a lethal jhmv strain, activated cd8 t cells gained access to the cns independent of cd4 t cells; however, their numbers in the cns were reduced by~50%. 10 following sublethal jhmv infection of balb/ c mice, cd4 t cells were more prevalent than cd8 t cells in the cns at day 5 p.i. and accumulated to peak numbers by day 7 p.i. (~2á5 9 10 5 ), similar to cd8 t cells (~5 9 10 5 ). 29 to distinguish whether cd4 t cells affected cd8 t-cell recruitment and/or effector function following sublethal infection, cns accumulation of virusspecific cd8 t cells was monitored. cd4 t-cell depletion did not alter the proportion of virus-specific t cells within the cd8 t-cell population infiltrating the brain or spinal cord (fig. 4a , data not shown). while the frequencies of virus-specific cd8 t cells were equally low at day 5 p.i., peak frequencies were reached by day 7 p.i. in both groups. moreover, the absolute numbers of virus-specific cd8 t cells were also similar at all times p.i. (fig. 4b) , indicating that cd4 t cells were not required for virusspecific cd8 t-cell recruitment or retention. in the spinal cord virus-specific cd8 t cells were also virtually identical throughout infection, irrespective of the presence or absence of cd4 t cells during priming (fig. 4b) . the failure to clear infectious virus in the absence of cd4 t cells (fig. 2c ) therefore suggested either that the effector functions of cd8 t cells within the target tissue are impaired, or that cd4 t cells contribute directly to virus control. the primary anti-viral mediators reducing infectious jhmv within the cns are t-cell-derived ifn-c and perforin. [15] [16] [17] the ifn-c controls virus directly in oligodendrocytes and is also critical to up-regulate mhc class i on oligodendrocytes as well as mhc class ii on microglia and macrophages. 17, 31, 32 perforin-mediated cytolysis specifically controls virus in microglia/macrophages, but not oligodendrocytes. 15 we therefore examined potential defects in effector functions of brain-derived unhelped virus-specific cd8 t cells by measurement of ex vivo cytolytic activity and ifn-c expression. cytolytic capacity of both unhelped and helped cd8 t cells showed no differences between the groups at day 7 p.i. (data not shown). similarly, cd4 t-cell depletion did not impair the frequency of ifn-c-producing cells in brain-derived cd8 t cells at day 7 p.i. (fig. 5a) . at day 14 p.i. the frequency of ifn-c-producing unhelped cd8 t cells remained similar to day 7 p.i., but was reduced relative to helped cd8 t cells. however, by day 21 p.i. the frequency of ifn-c-producing virus-specific cd8 t cells in the unhelped group declined to < 30%, whereas it was sustained at~45% in controls. in addition to reduced frequencies, the extent of ifn-c production as assessed by mean fluorescence intensity (mfi) was also lower in the unhelped groups at days 14 and 21 p.i. (fig. 5a) . in spinal cords the frequencies of ifn-c-producing cd8 t cells were highest at day 7 p.i. and declined in both groups out to day 21 p.i., with no statistically significant differences between both groups (data not shown). although a trend towards reduced ifn-c secretion on a per cell basis was indicated by reduced mfi in the unhelped cd8 t-cell population, differences were not significant (data not shown). these results suggested that cd4 t cells support sustained ifn-c expression by virusspecific cd8 t cells under conditions of prolonged antigen exposure. no defects in initial generation and cns recruitment of ifn-c-producing virus-specific cd8 t cells suggested that the failure to control infectious virus was attributed to inefficient triggering of t-cell effector function in vivo. interferon-c mrna expression in the brain was indeed reduced in the cns of cd4-depleted mice compared with controls (fig. 5b) . to assess functional ifn-c protein in vivo we further monitored ifn-c dependent up-regulation of cxcl9 mrna and mhc class ii on microglia. decreased levels of cxcl9 mrna supported reduced ifn-c activity (fig. 5b) . moreover, mhc class ii expression was barely detectable on microglia at day 5 p.i. in cd4-depleted mice, but was already up-regulated on 70% of microglia in controls (fig. 5c) . although > 98% of microglia in both groups expressed mhc class ii by day 7 p.i., overall expression levels assessed by mfi were lower in cd4-depleted mice. cd8 t cells therefore produced sufficient ifn-c in vivo to up-regulate mhc class ii on microglia by day 7 p.i. in the absence of cd4 t cells. however, ifn-c levels were insufficient to achieve optimal mhc class ii induction, cxcl9 mrna up-regulation, or unhelped cd8 t cells do not acquire a memory precursor effector phenotype these data indicated that maintenance of ifn-c-producing cd8 t cells in the cns appears optimal when cd4 t cells are present during priming and/or the effector phase. upon activation, cd8 t cells undergo a complex differentiation programme determined by the nature of inflammatory signals. 33, 34 however, two effector cell subsets whose differential expression of the interleukin-7 receptor a chain (cd127) and killer cell lectin-like receptor g1 (klrg1) is associated with fate determination and development of memory cells are common to many infections or immunizations. up-regulation of klrg1 on effector t cells directly coincides with the magnitude of t-bet expression and serves as a marker of terminally differentiated effector cells. 33, 34 by contrast, cd127 is down-regulated upon activation, and activated cells with sustained cd127 expression survive the cd8 contraction phase to form the memory pool. 35 hence, during differentiation cd8 t cells expressing a cd127 à klrg1 + phenotype are generally considered to be short-lived effector cells (slec), whereas a cd127 + klrg1 à phenotype is indicative of long-lived memory precursor effector cells (mpec). 34 we therefore examined whether the absence of cd4 t cells at priming alters the differentiation phenotype of cns infiltrated cd8 t cells based on cd127 and klrg1 expression (fig. 6a) . although virus-specific cd8 t cells were low at day 5 p.i. (fig. 4) , the majority had downregulated cd127 (85% versus 70%, in cd4-depleted versus control mice, respectively; data not shown). however, at this early time, only 15% of virus-specific cd8 t cells had a cd127 à klrg1 + slec phenotype in cd4depleted mice, while~30% of helped cd8 t cells displayed this terminal effector phenotype (fig. 6b) . although this profile suggested a more activated virusspecific cd8 t-cell population in cd4-sufficient mice, these early differences resolved by day 7 p.i., when the relative populations of slec were similar at~17%, irrespective of cd4 t cells (fig. 6b) . by days 14 and 21 p.i. the proportion of klrg1 + cells and slec within virusspecific cd8 t cells had dropped significantly in both groups, although slec continued to be higher in cd4depleted mice (fig 6b) . cd127 + klrg1 à mpec were similarly low in both groups at day 5 p.i. and gradually increased throughout infection. however, whereas~70% of helped virus-specific cd8 t cells exhibited an mpec phenotype by day 21 p.i., this population only reached 30% in cd4-depleted mice (fig. 6b) . a minor fraction ranging from 5% to 20% of virus-specific cd8 t cells expressed both cd127 and klrg1 throughout infection in both groups. whereas this population increased with time p.i. in cd4-depleted mice, it decreased in controls that had cleared virus (fig. 6b) . cd127 expression on klrg1 + cells has been observed under conditions of repetitive antigen re-stimulation and may mark long-lived effector memory t cells. 36, 37 overall, the proportion of klrg1-expressing jhmv-specific cd8 t cells was relatively low compared with differentiation of slec during infection with viruses replicating in non-cns tissues, e.g. following lymphocytic choriomeningitis virus infection or following malaria parasite immunization. 34, 38, 39 weak klrg1 expression may be attributed to the unique cns environment and/or the redundant role of interleukin-12, a strong inducer of klrg1, during jhmv infection. 40 overall, these results are consistent with the notion that early differences in virus-specific cd8 population arise from the absence of cd4 t cells during priming, whereas the significantly reduced efficacy to acquire an mpec phenotype, coincident with retention of a larger proportion of slec, is driven by ongoing viral antigen stimulation. the impaired ability of unhelped virus-specific cd8 t cells to acquire an mpec phenotype, yet the absence of a preferential decline of unhelped relative to helped cd8 t cells, is consistent with the notion that ongoing exposure to viral antigen drives continual renewal. 34, 41 we therefore compared the homeostatic turnover of virus-specific cd8 t cells in the cns of cd4-depleted and control mice during acute and persistent infection. consistent with similar numbers of virus-specific cd8 t cells within the cns, their proliferation was comparable at~85% at day 5 p.i. (fig. 6c) . the proportions of proliferating virus-specific cd8 t cells within the cns gradually declined in both groups starting at day 7 p.i. however, 50% of unhelped cd8 t cells still showed evidence of proliferation at days 14 and 21 p.i., whereas proliferation by helped cd8 t cells had declined to~25% (fig. 6c) . these data suggest that elevated virus load sustains proliferation, yet also promotes activation-induced cell death. indeed, while the frequency of apoptotic virus-specific cd8 t cells was similarly high in both groups at day 5 p.i., it remained significantly higher in cd4-depleted mice at day 14 p.i. (fig. 6c) . therefore, in the absence of cd4 t cells continued proliferation by virus-specific cd8 t cells was balanced by activation-induced cell death, resulting in comparable total numbers of virus-specific cd8 t cells within the cns. similar phenotypic subsets, as well as activity of helped and unhelped virus-specific cd8 t cells in the cns at day 7 p.i. suggested that cd4 t cells do not imprint peripheral cd8 t-cell activation or initial cns effector function. to eliminate the variable effect of viral load in altering cd8 t cells, cd4-depleted infected mice received jhmv-specific memory cd8 t cells to reduce viral load. 15 memory cd8 t cells were derived from thy-1.1 mice immunized with jhmv 3-4 weeks post immunization and transferred into jhmv-infected cd4-depleted or control thy-1.2 recipients at day 5 p.i. this strategy allowed activation of endogenous cd8 t cells and monitoring of both donor and recipient t cells based on the thy-1 congenic marker. both cd4-depleted and control recipients developed mild signs of paralysis, and completely recovered from clinical symptoms by day 16 p.i. without mortality (fig. 7a) . donor memory cd8 t cells prevented mortality consistent with the inability to recover infectious virus at day 16 p.i. flow cytometric analysis at day 16 p.i. confirmed essentially equivalent cns recruitment of total as well as virus-specific thy-1.1 donor cd8 t cells in both cd4-depleted and control recipients (fig. 7b,c) . the vast majority of donor cd8 t cells recruited to the cns were virus specific (60-70%), despite constituting only 2-3% of the donor population before transfer (data not shown). virus-specific recipient thy-1.2 cd8 t cells were slightly higher in the brain of cd4-depleted mice (fig 7d) , whereas those in spinal cord were similar (fig. 7d , data not shown) confirming that endogenous cd8 t-cell populations were not skewed by the transfer (fig. 7d) . importantly, > 60% of virus-specific endogenous cd8 t cells in the cns of cd4-depleted mice expressed a cd127 + klrg1 à mpec phenotype at day 16 p.i. (fig 7e) . the slec proportion was reduced to < 2%, showing a similar phenotype distribution as in control mice (fig. 7e) . the relative mpec and slec populations were also similar in spinal cords in both recipient groups (data not shown). the capacity of endogenous unhelped cd8 t cells to produce ifn-c was also restored to that of helped cd8 t cells (fig. 7f) . these findings suggest that the altered phenotype of primary cns cd8 t cells in cd4-depleted mice is mainly driven by sustained viral load, rather than lack of cd4 t-cell help or imprinting. in peripheral infections, cd4 t-cell imprinting on cd8 t cells is most evident during memory recall responses. 7, 8 we therefore determined if cd4 t-cell imprinting is critical for cd8 t-cell recall responses and survival within the cns. to generate unhelped or helped donor memory cd8 t cells, thy-1.1 mice were either treated with anti-cd4 or control mab before jhmv immunization. unhelped or helped memory thy-1.1 cd8 t cells were transferred into naive thy-1.2 recipients 1 day before infection. mice with no donor t cells served as controls. recipients of helped memory cd8 t cells displayed enhanced virus control at day 5 p.i., but all groups controlled infectious virus by day 14 p.i. (fig. 8a) . however, in contrast to their helped memory counterparts or primary cd8 t cells (figs 1 and 4) , unhelped virus-specific memory cd8 t cells were significantly impaired in expansion in the draining cln, as well as early accumulation in the cns, (fig. 8b) . the relative proportion and total numbers of thy-1.1 + cd8 t cells was significantly lower in the unhelped, relative to helped, donor populations in both cln as well the cns at days 5 and 7 p.i. (fig 8b,c) . the difference was most apparent at day 5 p.i. in the cns and was largely resolved by day 14 p.i. moreover, the number of virusspecific donor cd8 t cells capable of ifn-c production was reduced in both cln and cns of recipients of unhelped versus helped memory cd8 t cells at days 5 and 7 p.i. (fig. 8d) . despite an apparent equilibration towards similar numbers of unhelped versus helped donor cd8 t cells in the cns by day 14 p.i., survival of the unhelped memory cd8 t cells was profoundly reduced by day 35 p.i. during the chronic phase. although helped donor thy-1.1 + cd8 t cells comprised 25% of the total cd8 t-cell population, the proportion of unhelped cd8 t cells had declined > 98% (fig. 8e) . this vast reduction was also reflected by the total numbers of unhelped versus helped thy-1.1 + donor cells within the cns (fig. 8e) . unhelped memory cells also failed to survive in the draining cln (data not shown). nonetheless, despite their low number, the relative proportion of ifn-c-producing cells was comparable between the groups (fig. 8f) . these results suggest that cd4 t-cell help at priming plays an essential role in generating long-lasting memory t cells following recall responses within the cns. numerous studies on the role of cd4 t cells in regulation of cd8 t-cell immunity reveal that help for functional primary cd8 t-cell responses is dependent on the pathogen and possibly the target tissue. for example, cd4 t-cell help is dispensable for activation and differentiation of naive cd8 t cells during lymphocytic choriomeningitis virus 8 during herpes simplex virus infection. 43 irrespective of the diverse effects of cd4 t-cell help on primary cd8 tcell responses, memory cd8 t cells generated in the absence of cd4 t cells are typically defective in recall responses. 5, 7, 11 our studies demonstrate that cd4 t cells differentially influence cd8 t-cell immunity during primary and recall responses mounted to jhmv infection in h-2 d mice and that the necessity of cd4 t-cell help during priming is distinct from infected h-2 b mice. 9 cd8 t cells primed in the absence of cd4 t cells in h-2 d mice had no defects in expansion or ifn-c expression in the periphery similar to vaccinia virus infection of h-2 b mice. 5 accumulation and subsequent retention of virus-specific cd8 t cells within the cns also remained unaffected by the absence of cd4 t cells in h-2 d mice. moreover, cytolytic activity was similar and ifn-c-producing virus-specific cd8 effector cells were equally abundant in the cns regardless of the presence or absence of cd4 t cells during priming and acute infection. cns cd8 t cells were nevertheless incapable of reducing infectious virus when primed without cd4 t cells, potentially because of delayed and suboptimal mhc up-regulation in the absence of ifn-cproducing cd4 t cells. a role for cd4 help in maintaining effector function during prolonged antigen exposure was indicated by the decline in both the percentage and i. density plot shows representative staining of donor thy-1.1 + cd8 t cells in the brain. bar graphs represent absolute numbers of helped or unhelped donor thy-1.1 + cd8 t cells. (f) cns-derived cells were incubated with pn peptide to determine ifn-c-producing thy-1.1 + cd8 t cells. data represent the mean ae sem. statistical significance was determined using an unpaired student's t-test. *p < 0á05; **p < 0á01; ***p < 0á001. evaluation by analysis of variance showed slightly increased statistical significance for values in (b) for sc and cln at day 7 and in (d) for cln at day 5. by contrast, values in (b) for brain at day 14 and cln at day 7, in (c) for brain and sc at day 5 and cln at day 14, as well as (d) for brain at day 5 and 14, and sc and cln at day 7 did not reach statistically significant differences. the failure to clear infectious virus from the cns in cd4-depleted mice also resulted in higher proliferation and apoptosis of virus-specific cd8 t cells within the cns, consistent with the inability to acquire an mpec phenotype. nevertheless, equilibration of viral load in cd4-depleted mice and controls by transfer of memory cd8 t cells revealed that the unhelped cd8 t-cell phenotype was prominently driven by viral load rather than the absence of cd4 t-cell imprinting. the mechanisms underlying the differences in cd4 tcell imprinting during the priming phase in h-2 b versus h-2 d mice are unclear, but highlight critical epitope-specific and genetic influences. the inability to detect jhmv replication in cln suggests a minimal influence of virusdriven pro-inflammatory responses, especially as type i ifn induction by jhmv is very low. 44, 45 differences may rather reside in the magnitude and kinetics of antigen processing or peptide presentation as l d restricted n protein-specific responses appear to arise earlier compared with d b -restricted s protein-specific responses. 26 moreover, the breadth of cd4 t-cell responses appear to differ, as numerous h-2 b epitopes have been defined, while they appear more limited in h-2 d mice. 46, 47 irrespectively, uncontrolled viral replication despite intact accumulation of virus-specific cd8 t cells was the consequence of cd4 t-cell depletion in both h-2 b and h-2 d mice; 9,10 while an early defect in cns cd8 t-cell responses in infected h-2 b mice suggested a local beneficial effect of cd4 t cells on cd8 t cells, 9 a similar but more subtle effect in h-2 d mice cannot be excluded. however, we have been unable to distinguish a direct contribution of cd4 t cells to viral control, e.g. via early ifn-c secretion, from an indirect contribution to cd8 t-cell function involving antigenpresenting cells or soluble mediators. these findings imply that primary cd8 t-cell anti-viral immunity during encephalitis may vary significantly in an outbred population based on provision of cd4 t-cell help at multiple stages and anatomical locations. nevertheless, irrespective of potentially distinct mechanisms, cd4 t cells appear essential for effective local anti-viral cd8 t-cell function in the cns. a number of studies have also revealed that cd4 t cells facilitate the generation of functional memory cd8 t cells during recall responses. 7, 8, 42 similar to peripheral infections, the role for cd4 t cells in cd8 t-cell recall responses was essential following jhmv cns infection. compared with their helped counterparts, jhmv-specific unhelped memory cd8 cells expanded less efficiently in the cln, were delayed in cns accumulation, and exhibited reduced survival. a potential mechanism underlying cd4 help is tumour necrosis factor-related apoptosisinducing ligand (trail). 48 since helped cd8 t cells are capable of down-regulating trail expression, they are less vulnerable to trail-mediated apoptosis. 49 however, we previously reported that purified cd8 t cells from the cns of cd4-depleted h-2 b mice had only slightly increased transcript levels of trail mrna compared with controls. 9 whether trail expression influences the limited expansion and survival of unhelped memory cd8 t cells in h-2 d mice remains to be examined. distinct from many peripheral infections, both cd4 and cd8 t-cell effector functions are associated with demyelination and clinical disease during jhmv encephalomyelitis. infection of oligodendrocytes, the primary targets of jhmv infection, is a prerequisite for demyelination but is insufficient to induce myelin stripping without t cells. 13, 15 in this context it is of interest to note that uncontrolled virus replication during persistence did not exacerbate demyelination in cd4-depleted mice. this contrasts with increased demyelination associated with similarly uncontrolled viral replication in mice deficient in humoral immunity 29 and supports the notion that cd4 t cells promote pathology. 19, 23 finally, it is interesting to note that uncontrolled viral replication in cd4-depleted h-2 d mice resulted in viral spread to cells with neuronal morphology. while neuronal infection is sparse in immune competent mice, it is clearly evident under conditions of uncontrolled virus replication in infected ifn-a/b receptor-deficient, b-cell deficient, as well as scid mice, 15, 30, 45 potentially contributing to the lethal phenotype. in summary, these data demonstrate that the roles for cd4 t cells in generating fully functional effector/memory cd8 t-cell responses following cns virus infection could be manifold depending on the stages of activation and differentiation, effector site of cd8 t-cell function, as well as genetic background. identifying the cellular mechanism by which cd8 t-cell immunity is regulated by cd4 t cells will provide a key insight into understanding cd4-cd8 cooperation for the development of effective primary as well as memory responses within the cns. revealing the role of cd4 + t cells in viral immunity t-cell help for cytotoxic t lymphocytes is mediated by cd40-cd40l interactions a novel helper role for cd4 t cells cd4 + t cells are required to sustain cd8 + cytotoxic t-cell responses during chronic viral infection requirement for cd4 t cell help in generating functional cd8 t cell memory longitudinal requirement for cd4 + t cell help for adenovirus vector-elicited cd8 + t cell responses defective cd8 t cell memory following acute infection without cd4 t cell help cd4 + t cells are required for secondary expansion and memory in cd8 + t lymphocytes cd4 t cells promote cd8 t cell immunity at the priming and effector site during viral encephalitis ctl effector function within the central nervous system requires cd4 + t cells cd4 + t cells are required for the maintenance, not programming, of memory cd8 + t cells after acute infection cd8 + t lymphocyte mobilization to virusinfected tissue requires cd4 + t-cell help coronavirus infection of the central nervous system: host-virus stand-off kinetics of virus-specific cd8 + tcell expansion and trafficking following central nervous system infection perforin and c interferon-mediated control of coronavirus central nervous system infection by cd8 t cells in the absence of cd4 t cells mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis ifn-c is required for viral clearance from central nervous system oligodendroglia evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection cd4 and cd8 t cells have redundant but not identical roles in virus-induced demyelination effective clearance of mouse hepatitis virus from the central nervous system requires both cd4 + and cd8 + t cells cd4 t cells contribute to virus control and pathology following central nervous system infection with neurotropic mouse hepatitis virus a central role for cd4 + t cells and rantes in virus-induced central nervous system inflammation and demyelination memory cd4 + tcell-mediated protection from lethal coronavirus encephalomyelitis the jhm strain of mouse hepatitis virus induces a spike protein-specific db-restricted cytotoxic t cell response characterization of the ld-restricted cytotoxic t-lymphocyte epitope in the mouse hepatitis virus nucleocapsid protein inverted immunodominance and impaired cytolytic function of cd8 + t cells during viral persistence in the central nervous system bystander cd8 t cell-mediated demyelination after viral infection of the central nervous system pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies mechanisms of central nervous system viral persistence: the critical role of antibody and b cells antibody prevents virus reactivation within the central nervous system inhibition of interferon-c signaling in oligodendroglia delays coronavirus clearance without altering demyelination perforinmediated effector function within the central nervous system requires ifn-c-mediated mhc up-regulation heterogeneity and cell-fate decisions in effector and memory cd8 + t cell differentiation during viral infection inflammation directs memory precursor and short-lived effector cd8 + t cell fates via the graded expression of t-bet transcription factor selective expression of the interleukin 7 receptor identifies effector cd8 t cells that give rise to long-lived memory cells stimulation history dictates memory cd8 t cell phenotype: implications for prime-boost vaccination cd27 stimulation promotes the frequency of il-7 receptor-expressing memory precursors and prevents il-12-mediated loss of cd8 + t cell memory in the absence of cd4 + t cell help pathogen-induced inflammatory environment controls effector and memory cd8 + t cell differentiation short-lived effector cd8 t cells induced by genetically attenuated malaria parasite vaccination express cd11c interleukin-12 (il-12), but not il-23, deficiency ameliorates viral encephalitis without affecting viral control antigen-independent memory cd8 t cells do not develop during chronic viral infection cd4 t cells are required for cd8 t cell survival during both primary and memory recall responses cd4 + t cells are required for the priming of cd8 + t cells following infection with herpes simplex virus type 1 oligodendroglia are limited in type i interferon induction and responsiveness in vivo type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd8 t cells antigen specificity of cd4 t cell response in the central nervous system of mice infected with mouse hepatitis virus immunogenicity of jhm virus proteins: characterization of a cd4 + t cell epitope on nucleocapsid protein which induces different t-helper cell subsets enhancement of proliferation and downregulation of trail expression on cd8 + t cells by il-21 cd4 + t-cell help controls cd8 + t-cell memory via trail-mediated activation-induced cell death we sincerely thank wenqiang wei, eric barron, ernesto barron and jennifer powers for exceptional technical assistance. mh designed and performed the experiments, analysed the results, drafted the figures and manuscript. twp and drh participated in experiments, analysed the results and drafted the figures. sas, ccb and bm designed the study, interpreted the data and edited the manuscript. this work was supported by the national institutes of health grant po1 ns064932 and cancer center support grant p30 ca014089. there are no financial conflicts of interest to declare. key: cord-290566-tmsocyfc authors: chitnis, tanuja title: the role of cd4 t cells in the pathogenesis of multiple sclerosis date: 2007-05-25 journal: int rev neurobiol doi: 10.1016/s0074-7742(07)79003-7 sha: doc_id: 290566 cord_uid: tmsocyfc t lymphocytes play a central role in the pathogenesis of multiple sclerosis (ms) (zhang et al., 1992). both cd4+ and cd8+ t cells have been demonstrated in ms lesions, with cd4+ t cells predominating in acute lesions and cd8+ t cells being observed more frequently in chronic lesions (raine, 1994). additionally, t cells are found in all four of the described histopathologic subtypes of ms (lucchinetti et al., 2000). activated myelin‐reactive cd4+ t cells are present in the blood and cerebrospinal fluid (csf) of ms patients; in contrast, only nonactivated myelin‐reactive t cells are present in the blood of controls (zhang et al., 1994). the success of several t‐cell‐targeted therapies in ms reinforces the importance of the role of the t cell in ms pathogenesis. here, we outline basic concepts in cd4+ t‐cell immunology and summarize the current understanding of the role of cd4+ t cells in the pathogenesis of ms. t lymphocytes play a central role in the pathogenesis of multiple sclerosis (ms) (zhan g et al. , 1992) . both cd4 þ and cd8 þ t cells have been demon strated in ms lesions, with cd4 þ t cells predom inating in acu te lesions and cd 8þ t cells being obser ved more frequen tly in chronic lesions (raine, 1994) . additiona lly, t cells are found in all four of the described histopathologic subtypes of ms (lucchinetti et al., 2000) . activated myelin-reactive cd4þ t cells are present in the blood and cerebrospinal fluid (csf) of ms patients; in contrast, only nonactivated myelinreactive t cells are prese nt in the blood of controls (zhan g et al. , 1994) . the succes s of several t-cell-targeted therapies in ms reinforces the importance of the role by the cd3 antigen. the cytoplasmic tail of the cd3 proteins contains one copy of a sequence motif important for signaling functions, called the immunoreceptor tyrosine-based activation motif (itam). phosphorylation of the itam initiates intracellular signaling events. the interaction of mhc-peptide complex with t cells, while necessary, is insuycient for t-cell activation. additional classes of molecules are involved in t-cell antigen recognition, activation, intracellular signaling, adhesion, and traycking of t cells to their target organs. two signals are required for t lymphocyte activation. according to this ''two-signal'' model (bretscher and cohn, 1970) , ''signal 1'' consists of the interaction of the tcr with antigen, presented by the mhc on the surface of apcs. ''signal 2'' consists of the engagement of costimulatory receptors on the tcell by ligands present on the surface of apcs (alegre et al., 2001; bretscher, 1999) . after contact with specific antigen-mhc complex and adequate costimulatory signals, t cells begin to proliferate, diverentiate, and deliver a series of signals, enabling evector functions to other cells such as b cells and nk cells. t cells can thereby orchestrate the immune response. costimulatory molecules may deliver either a stimulatory (positive) or an inhibitory (negative) signal for t-cell activation (brunet et al., 1987) . examples of molecules delivering a positive costimulatory signal for t-cell activation include the b7-cd28 and cd40-cd154 pathways. examples of molecular pathways delivering a negative signal for t-cell activation include b7-ctla4 and pd1-pd ligand. the delicate balance between positive and negative regulatory signals can determine the outcome of a specific immune response. importantly, in the absence of adequate costimulatory signals, t cells can die or become anergic in vitro, and thus, fail to initiate an evective immune response in vivo. therefore, manipulation of costimulatory signals represents an important mechanism to inhibit immune-activation. on exposure to an antigen, antigen-specific t cells proliferate and diverentiate into evector t cells (sprent and surh, 2002) . the vast majority of evector t cells undergo apoptosis as the immune response progresses, and the few lymphocytes that survive become long-lived memory t cells (dutton et al., 1998) . memory t cells are specific to the antigen encountered during the primary immune response and react rapidly and vigorously on reencounter with the same antigen. functionally, in terms of activation requirements, memory t cells can be activated by lower concentrations of anti-cd3 (byrne et al., 1988) , require less costimulation by anti-cd28 (kuiper et al., 1994) , and readily secrete more evector cytokines (bird et al., 1998; ehlers and smith, 1991; lee et al., 1990) than naive t-cell counterparts, indicating a state of hyperresponsiveness. molecules primarily involved in cell migration into tissues include chemokines, integrins, selectins, and matrix metalloproteinases (mmps). chemokines constitute a large family of chemoattractant peptides that regulate the vast spectrum of leukocyte migration events through interactions with chemokine receptors. the integrin family includes vascular cell adhesion molecule-1 (vcam-1), intercellular adhesion molecule-1 (icam-1), and leukocyte function antigen 3 (lfa-3), cd45, and cd2. the integrin family also mediates t-cell adhesion, facilitates interaction with the apcs, as well mediates adhesion to nonhematopoietic cells such as endothelial cells and guides cell trayc. l-selectins facilitate the rolling of leucocytes along the surface of endothelial cells and function as a homing receptor to target peripheral lymphoid organs. the mmps are a family of proteinases, secreted by inflammatory cells, which digest specific components of the extracellular matrix, thereby facilitating lymphocyte entry through basement membranes, including the blood-brain barrier (bbb). the th cells play a critical role in the orchestration of the immune response, in part, through the production of cytokines that provide secondary signals to other cells in the immune cascade. two major types of th cell responses have been described. th1 cells produce il-2, tnf-, and interferon (ifn)-, while th2 cells produce il-4, il-5, il-10, and il-13. a th3 cell that primarily secretes tgf-has been described in the context of oral tolerance to myelin antigens (fukaura et al., 1996; hafler et al., 1997) and in other immune-mediated settings (minguela et al., 1999) . these subsets of t cells interregulate each other's development, with th1 cytokines suppressing th2 diverentiation and vice versa (fig. 1) . a subset of t cells that predominantly produces il-17 has been described (yao et al., 1995) . these cells are believed to represent a distinct subset from ifn--producing th1 cells, evidenced by the dependence of th il-17 cells on il-16 and tgf-for diverentiation (bettelli et al., 2006; mangan et al., 2006; veldhoen et al., 2006) and il-23 for expansion (aggarwal et al., 2003; langrish et al., 2005) , as opposed to th1 cells which are dependent on il-12 and il-2, respectively, for diverentiation and expansion. both th1 and th2 cytokines have been shown to suppress the development of th17 cells (harrington et al., 2005; park et al., 2005) . an intriguing relationship between the generation of pathogenic th17 and regulatory cd4þcd25þfoxp3þ cells has recently been demonstrated. tgfis critical for the diverentiation and generation of cd4þcd25þfoxp3þ regulatory t cells; however, if additionally exposed to il-6 during culture, these cells diverentiate into th17 cells (bettelli et al., 2006) . tanuja chitnis traditionally, th cell subsets have been distinguished by their patterns of cytokine production, however identification of distinguishing surface molecule markers has been a major advance in the field. t-cell-, ig-, and mucin-domaincontaining molecules (tim) represent an important family of molecules which encode cell-surface receptors involved in the regulation of th1-and th2-cellmediated immunity. tim-3 is specifically expressed on th1 cells and negatively regulates th1 responses through interaction with the tim-3 ligand, galactin-9, also expressed on cd4þ t cells (monney et al., 2002; sabatos et al., 2003; zhu et al., 2005) . tim-2 is expressed on th2 cells and appears to negatively regulate th2 cell proliferation, although this has not been fully established. tim-1 is expressed on th2 cells > th1 cells and interacts with tim-4 on apcs to induce t-cell proliferation (meyers et al., 2005) . intracellular signaling mechanisms provide the link between the binding of the cytokine with its receptor and the evect of the cytokine on cellular function. the janus kinase and signal transducer and activator of transcription (jak/stat) family of transducer/transcription-activating factors plays a critical role in the signaling of many cytokine receptors. cytokine binding to the specific receptor activates the jak molecule associated with the receptor, causing phosphorylation of tyrosine residues, and binding of the jak molecules to its receptor. this facilitates binding of stat proteins to the phosphorylated receptor, which subsequently dissociates from the receptor, dimerizes, and activates transcription of genes containing specific cis-regulatory stat-binding sequences. diverent cytokine receptors are associated with diverent jak/stat proteins. the il-12 receptor is associated with jak-2 and stat3 and stat4 (jacobson et al., 1995) . the il-4 receptor is associated with jak-1-3 and stat6 (kaplan et al., 1996; takeda et al., 1996) (fig. 1 ). mice deficient in stat6 display a reduction in th2 cytokine production, decreased il-4-induced b-cell proliferation and reduced ige (kaplan et al., 1996; takeda et al., 1996) . in contrast, stat4 plays a pivotal role in th1 immune responses. stat4 is activated after il-12 interacts with the il-12 receptor, inducing transcription of ifn(jacobson et al., 1995) . mice deficient in stat4 lack il-12-induced ifn-production and th1 diverentiation (kaplan et al., 1996; thierfelder et al., 1996) , and display a predominantly th2 phenotype (kaplan et al., 1996) . t-bet is a th1-specific t box transcription factor that directly controls the expression of the hallmark th1 cytokine, ifn-, and il-12r2 expression, thus facilitating th1 cell diverentiation (szabo et al., 2000) . the transcription factor c-maf enhances il-4 production and represents an important step in the induction of th2 cells (ho et al., 1996) . gata-3 was found to be an stat6-independent inducer of th2 diverentiation, and is located upstream of c-maf, thus representing a master switch in th2 development and commitment (ouyang et al., 2000) . th17 diverentiation is independent of stat4 and stat6 signaling (park et al., 2005) ; however, it has been demonstrated that stat3 signaling is activated by both il-6 and il-23, and binds to il-17 gene promoters (chen et al., 2006) . socs-3 is a major regulator of il-23-mediated stat3 phosphorylation and subsequent th17 generation (chen et al., 2006) . several populations of regulatory or suppressor t cells have been described in humans. these include cd4þcd25þfoxp3 regulatory t cells (baecher-allan et al., 2001; dieckmann et al., 2001; levings et al., 2001; stephens et al., 2001; yagi et al., 2004) , cd8þcd28à t cells (koide and engleman, 1990) , il-10producing th2 cells (bacchetta et al., 1994) , and tgf--producing th3 cells (kitani et al., 2000; roncarolo and levings, 2000) . regulatory t cells suppress t-cell proliferation through a variety of mechanisms, including the production of immunosuppressive cytokines, or through t-t-cell interactions. several studies have demonstrated that these cells play an important role in the control of the immune 48 tanuja chitnis response in multiple sclerosis (ms) and that the function of regulatory t cells may be enhanced by immunomodulatory therapies (crucian et al., 1995; fukaura et al., 1996; hafler et al., 1997; karaszewski et al., 1991; viglietta et al., 2004) . cd4þ t cells are believed to play a central role in the pathogenesis of ms. in this section, we summarize the prevailing theory of the pathogenesis of ms, and evidence for the role of cd4þ t cells from both human ms and animal models of disease. much of the current understanding of the potential mechanistics and role of cd4þ t cells in ms comes from the animal models simulating features of ms. experimental autoimmune encephalomyelitis (eae) is an inflammatory central nervous system (cns) demyelinating disease, and may be induced in several animal types via immunization with myelin proteins or peptides. disease is primarily mediated by myelin-reactive th1 cells, which precipitate an inflammatory, demyelinating response within the cns (chitnis et al., 2001b) . transfer of myelin basic protein (mbp)-specific t-cell clones restricted to class ii (ia) antigens of the mhc into naive recipient animals causes a similar inflammatory demyelinating disease (zamvil et al., 1985) . eae reproduces many of the clinical and immunologic aspects of ms, and has been widely used to study the mechanisms of cd4þ t-cell priming and response to myelin components (bettelli et al., 1998; chitnis et al., 2001a) as well as to test potential therapies for ms (aharoni et al., 1999; yednock et al., 1992) . theiler's murine encephalomyelitis virus-induced demyelinating disease (tmev-idd) model is a virally mediated model of cns inflammatory demyelination, with some resemblance to ms and is induced by direct cns infection of the neurotropic tmev picornavirus, initially resulting in an immune-mediated reaction primarily involving tmev-specific cd4 and cd8 t cells (clatch et al., 1986; rodriguez et al., 1996) . however, during the chronic stages of disease, t-cell reactivity to host myelin peptides has been observed, indicating epitope spreading has occurred, causing secondary t-cell responses to myelin breakdown products, and resulting in a disseminated autoimmune response . a summary of t-cell immunology related to animal models of ms is beyond the scope of this chapter, and can be found elsewhere in this volume or in alternate sources (chitnis and khoury, 2003a,b) ; however, selected topics in ms that are illuminated by animal studies are discussed. the prevailing theory of the etiology of ms is that of ''molecular mimicry'' whereby cd4þ t cells activated by a foreign antigen cross-react with myelin antigens. activated myelin-reactive cd4þ t cells are present in the blood and cerebrospinal fluid (csf) of ms patients; in contrast, only nonactivated myelinreactive t cells are present in the blood of controls (zhang et al., 1994) . sequences in mbp has been shown to resemble several viral sequences, and in some cases, crossreactive t-cell responses have been demonstrated. although no pathogen has definitely been proven to be the cause of ms, it is conceivable that certain pathogens serve as molecular mimics to cns components or play a role in the activation of myelin-specific cd4þ t cells. examples of cross-reactive t cells with mbp antigens include human herpesvirus 6 (hhv-6) (tejada-simon et al., 2003) , staphylococcal enterotoxin antigens , coronavirus (talbot et al., 1996) , influenza virus hemagglutinin (markovic-plese et al., 2005) , and epstein-barr virus (ebv) (lang et al., 2002) . proteolipid protein (plp) shares common sequences with haemophilus influenzae (olson et al., 2001) , while semliki forest virus (sfv) peptides mimic epitopes of myelin oligodendrocyte glycoprotein (mog) (mokhtarian et al., 1999) . these activated t cells are then thought to migrate to the cns, where they undergo reactivation in response to nascent myelin antigens. the reactivation of t cells heralds an inflammatory response within the cns, resulting in more tissue damage and release of secondary antigens. subsequent t-cell reactivity to secondary antigens is termed ''epitope spreading.'' evidence of epitope spreading has been demonstrated in animal models of ms (mcmahon et al., 2005; vanderlugt et al., 1998) , and may play an important role in the pathogenesis of the human disease. although this is the most widely accepted paradigm of ms pathogenesis, and is supported by evidence from studies discussed in this chapter, there still remain many unanswered questions, which include the identity of the initiating foreign cross-reactive antigen(s), the identity of the initiating self-antigen directed t-cell response, and the nature of epitope spreading within the cns. moreover, the paucity of cd4þ t cells in certain pathological subtypes of ms questions whether alternate mechanisms may predominate in subsets of this heterogeneous disease. because of the focus on myelin proteins and, in particular, mbp as a potential autoantigen in ms (allegretta et al., 1990; chou et al., 1992; zhang et al., 1994) , considerable interest has developed in the role of t-cell responses to mbp. moreover, ms disease-associated mhc class ii allele, drb1*1501 has been shown to be evective in presenting mbp peptide to t-cell clones isolated from ms patients (wucherpfennig et al., 1995 (wucherpfennig et al., , 1997 . activated myelin-reactive cd4þ t cells 50 are present in the blood and csf of ms patients; in contrast, only nonactivated myelin-reactive t cells are present in the blood of controls (zhang et al., 1994) . furthermore, mbp-reactive t cells isolated from the csf of ms patients display increased expression of the il-2 receptor (zhang et al., 1994) , consistent with a previously activated or memory phenotype. in ms patients, but not in healthy controls, these cells can be activated in the absence of cd28-b7 costimulation, thus implying that they have been previously activated in vivo (markovic-plese et al., 2001; scholz et al., 1998) . mbp-reactive t cells from ms patients were found to be less responsive to ctla-4 blockade compared to those from healthy controls (oliveira et al., 2003) , signifying that in ms patients these t cells are not subject to the normal regulatory mechanisms. although the contribution of mbp-reactive t cells to the pathogenesis of ms is currently unknown, their diverential phenotype and costimulatory requirements indicate a memory and potentially dysregulated cell population. t-cell reactivity to other myelin proteins and peptides in ms has been explored. studies examining plp t-cell responses demonstrated t-cell proliferation to certain epitopes (pelfrey et al., 1994) , with some diverential reactivity when compared to controls (markovic-plese et al., 1995; zhang et al., 1994) . t-cell responses to recombinant mog appeared to be similar in ms patients and healthy controls (diaz-villoslada et al., 1999) , although other studies demonstrated increased reactivity (kerlero de rosbo et al., 1993) or altered t-cell properties (van der aa et al., 2003a) in ms patients. other studies have examined t-cell responses to myelin oligodendrocyte basic protein (mobp) (holz et al., 2000) or 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase (cnpase) (muraro et al., 2002) , with some reactivity demonstrated in t-cell lines isolated from select ms patients. t-cell reactivity to other cns antigens has not been fully explored due to the technical diyculties with performing and interpreting the results of such assays. many studies currently employ strategies to expand t cells using nonspecific methods or mixtures of myelin peptides. studies in patients with postinfectious encephalomyelitis or acute disseminated encephalomyelitis (adem) have consistently found robust t-cell reactivity to myelin peptides in both the blood and csf (hafler et al., 1987; hemachudha et al., 1988; pohl-koppe et al., 1998) , and suggest an intriguing relationship in the pathophysiology of adem and ms. studies examining tcr repertoire in ms patients have demonstrated a bias for use of chain variable region (v) 5.2 and 5.3 (kotzin et al., 1991; lozeron et al., 1998; oksenberg et al., 1993) , and this has led to the exploration of tcr v 5.2/5.3-targeted therapies. tcr vaccines employing tcr v 5.2 peptides are the role of cd4 t cells in the pathogenesis thought to exert their evects by enhancing the function of regulatory t-cell populations recognizing tcr determinants (vandenbark, 2005; vandenbark et al., 1996) , and are currently undergoing pilot studies in ms. phase ii trials using antibodies specifically targeting the v 5.2/5.3 sequence of the tcr have shown some success and no significant adverse evects olsson et al., 2002) . however, other groups (gran et al., 1998; lozeron et al., 1998; musette et al., 1996) have found predominant usage of other v chains, indicative of potential limitations with tcr v 5.2-targeted therapies. studies in twins demonstrated similar selection of tcr v chains in concordant twins in response to mbp, when compared to discordant twins or controls, suggesting a genetic basis for the evolution of self-antigen t-cell responses (utz et al., 1993) . ms patients treated with autologous hematopoietic stem cell transplantation demonstrated an increase in naive compared to memory cd4þ t cells, with increased tcr diversity indicative of broader clonal phenotypes 2 years following therapy (muraro et al., 2005) . a separate study ascertained similar findings and found that mbp-reactive t cells demonstrated broader epitope recognition following reconstitution (sun et al., 2004) . pathologically, ms lesions are characterized by perivascular infiltrates of cd4þ and cd8þ t cells and macrophages (prineas and wright, 1978; traugott et al., 1983b) . cd4þ and cd8þ t cells and macrophages are also found toward the periphery of the lesion and in the normal appearing white matter (traugott et al., 1983a) . cd4þ t cells were shown to predominate in acute lesions, while cd8þ t cells were observed more frequently in chronic lesions (raine, 1994) . similar densities of cd3þ t cells have been demonstrated in all four of the histopathologic subtypes of ms, although type iii and iv lesions are additionally characterized by prominent oligodendrocyte degeneration (lucchinetti et al., 1996) . attempts to isolate t-cell clones from the brains of ms patients failed to show either mbp or plp reactivity (hafler et al., 1987) . tcr analysis from ms lesions demonstrated a broad tcr v and v repertoires in active lesions, while fewer tcr v genes were detected in chronic plaques and control samples (wucherpfennig et al., 1992) . other studies demonstrated restricted tcr specificities, with rearranged v 5.2 genes found in the brains of all patients who were hla drb1*1501, dqa1*0102, dqb1*0602, and dpb1*0401 positive, suggesting that mhc class ii genotype may play a role in vdj rearrangements in ms lesions (oksenberg et al., 1993) . t cells in parenchymal ms lesions lacked ccr7, indicating a diverentiation of central-memory t cells into evector memory cells presumably on restimulation by antigen within the cns (kivisakk et al., 2004) . in summary, multiple studies have demonstrated the presence of cd4þ t cells in ms lesions, arguing for a central role in ms pathogenesis. the lack of 52 tanuja chitnis consensus regarding t-cell specificities suggest a heterogeneity in t-cell responses at the time of analysis, which may be a result of epitope spreading in chronic disease. costimulatory pathways deliver a positive or negative signal for t-cell activation, and thus represent an important control step in the immune response. the cd28/ctla-4-b7-1/2 family of costimulatory molecules represents an important step in the activation of cd4þ t cells. lesions in the cns of patients with ms were found to be exclusively associated with the expression of b7-1 in perivenular lymphocytes, while b7-2 was expressed on macrophages in both ms and in other neurological diseases (windhagen et al., 1995) . peripheral blood mononuclear cells (pbmcs), isolated from ms patients, showed increased expression of b7-1 on both cd4þ and cd8þ cell patients with rapidly progressive disease, compared to those with stable disease, or normal controls (mena and rohowsky-kochan, 1999) . in a separate study, b7-1 expression localized to b cells was found to be increased during ms relapses, and treatment with ifn-1b reduced the number of b7-1-expressing b cells but increased the number of b7-2 monocytes (genc et al., 1997) . in total, these observations suggest that the b7-cd28-ctla4 pathway is activated in ms, and that b7-1, in particular, may play an important role in regulating disease activity. genetic polymorphisms of costimulatory molecules may contribute to disease susceptibility. three ctla-4 gene polymorphisms were found in ms patients, but not in healthy controls (ligers et al., 1999) , however no association was found with disease course or severity (masterman et al., 2002) . in an olmstead county study, two polymorphisms were associated with the presence of ms (kantarci et al., 2003) . the canadian collaborative study found no association of ctla-4 polymorphisms with the disease course of ms (dyment et al., 2002) . the exon 1 a/g polymorphism was associated with the presence of oligoclonal bands in the csf (fukazawa et al., 1999) . thus, dysregulation of ctla-4 signaling may contribute to susceptibility to ms. a phase i safety study of ctla4ig (repligen-rg2077) as well as a multicenter study of ctla4ig (bms-188667) for ms are ongoing. interaction of cd40 on apcs with cd154 on t cells induces apc production of il-12, a major factor in th1 cell diverentiation (kelsall et al., 1996) . expression of both cd40 and cd154 were increased in lesions from postmortem ms brains compared with controls, with cd40 found predominantly on macrophages and microglia, while cd154 colocalized with the cd4 t-cell marker (gerritse et al., 1996) . expression of cd154 was found to be higher in peripheral blood monocytes isolated from spms compared with rrms or the role of cd4 t cells in the pathogenesis healthy controls (filion et al., 2003; jensen et al., 2001) , and was reduced by ifn-treatment (teleshova et al., 2000) . pbmcs from spms patients produced more il-12 and ifn-when restimulated in vitro, compared with healthy controls (karni et al., 2002) . in summary, these studies indicate that the cd40-cd154 pathway is important for the regulation of th1 cytokine production in ms. clinical trials with an anti-cd154 antibody (biogen) in autoimmune disease such as itp and lupus were terminated because of the occurrence of thromboembolic events. another formulation of the antibody (idec pharmaceuticals) is under investigation. a phase i clinical trial in ms patients was recently performed with good safety data, and therapeutic evects are currently under investigation. programmed death-1 (pd-1) is expressed on t cells and is a negative regulator of t-cell activation. pd-1 polymorphism was shown to be a genetic modifier of the progression of ms, and this may relate to pd-1-mediated inhibition of t-cell activation (kroner et al., 2005) . the tim family of molecules are important cell-surface markers as well as costimulatory regulators of th1/th2 responses. in ms patients, csf t-cell clones demonstrated reduced levels of tim-3 and t-bet and secreted higher amounts of ifn-than did those from control subjects, indicating that tim-3 may represent an important regulator of th1 responses in ms (koguchi et al., 2006) . in summary, there is significant evidence that costimulatory molecules represent an important step in the control of t-cell activation in ms and are viable therapeutic targets. in the context of ms, th1 cytokines are thought to mediate disease, while th2 cytokines are believed to play a protective role. however, as our understanding of the disease evolves, it is clear that this paradigm is not absolute. moreover, evidence of a distinct lineage of th17-producing t cells has led to reevaluation of the role of th1 cytokines in ms. th1 cytokines are predominantly found in the brains of ms patients, while a paucity of th2 cytokines, in particular tgf-, and il-10 was observed (cannel la an d rain e, 1995 b; hofma n et al. , 1986; woodroof e and cuzner, 1993). studies using semiquantitative rt-pcr and immunocytochemistry found increased expression of b7-1 and il12p40 in acute ms plaques, compared with samples isolated from inflammatory infarcts (windhagen et al., 1995) . il-6, ifn-, and tnf-were expressed by cells located in the perivascular cuvs, suggesting that in acute ms lesions, inflammatory cells are the most important source of these cytokines (woodroofe and cuzner, 1993) . expression of tnf-was localized to macrop hages, microglia , and astrocyt es (cannel la and rain e, 1995 b; 54 tanuja chitnis hofman et al., 1989; selmaj et al., 1991) in chronic-active lesions. a separate study found that il-2 was expressed predominantly in association with perivascular inflammatory cells examining acute ms lesions (hofman et al., 1986) . contrary to the th1/th2 paradigm of ms, high levels of il-4 were expressed in both acute and chronic-active ms lesions with no obvious correlation to the resolution of the lesion (cannella and raine, 199 5b) . studies using rn a microarra ys in ms brains at autopsy found increased transcripts of genes encoding for il-6, il-17, and ifn(lock et al., 2002) , indicating a potential role for both th1 and th17 cells in proinflammatory responses in ms. several studies have demonstrated enhanced production of the hallmark th1 cytokine, ifn-, from pbmcs restimulated ex vivo in ms patients compared to controls comabella et al., 1998) . clinical attacks correlated with increased ifn-production in vitro (beck et al., 1988) . in a similar study, ifnwas found to blunt increased production of ifn-during relapse (becher et al., 1999) . tcr-mediated ifn-and il-10 secretions are increased in relapsingremitting (rr) and secondary progressive (sp) patients, but not in primary progressive disease, suggesting a dysregulation of this signaling pathway in certain ms subtypes (balashov et al., 2000) . interestingly, spms patients also exhibit seasonal variations of ifn-production with increased expression in autumn and winter months compared with spring and summer months, which was not observed in normal controls . a progressive course of ms was found to be significantly more frequent in carriers of the ifn-receptor-2 allele arg64 (schrijver et al., 2004) . administration of ifn-to ms patients precipitated clinical attacks, confirming the role of ifn-as a proinflammatory cytokine in ms (panitch et al., 1987a,b) . studies of the prototypic th2 cytokine il-4 in ms are limited, il-4 was expressed in high levels in both acute-and chronic-active ms lesions (cannella and raine, 1995a) . high frequencies of t-cell clones reactive to mbp-and plpexpressing il-4 were found in ms patients compared with untreated patients (chou et al., 1992) . increased expression of il-4 secretion by cd3-stimulated pbmcs was demonstrated in spms patients treated with cyclophosphamide/ methylprednisolone compared with untreated patients . thus, the role of il-4 in the pathogenesis of ms is unclear, however may be associated with responses to therapy. studies examining th17 cell activity in ms found that dendritic cells from ms patients secrete elevated amounts of il-23 and express increased levels of il-23p19 mrna, and are associated with increased t-cell production of il-17 (vaknin-dembinsky et al., 2006) . a japanese study examining cytokine expression found that csf levels of il-17, il-8, and il-5 were significantly higher in opticospinal-ms patients than in conventional rrms patients, and may be associated with pronounced neutrophilic infiltrates typically found in the opticospinal disease variant (ishizu et al., 2005) . alterations in the function of several populations of regulatory t cells have been demonstrated in ms. induction of regulatory antigen-specific th3 cells through oral tolerance with myelin proteins has been described (fukaura et al., 1996; hafler et al., 1997) . deficiency in the cd8þcd28à subset of suppressor cells has been demonstrated in ms patients (crucian et al., 1995) . moreover, increases in -adrenergic receptor density on cd8þcd28à cells were found in ms compared with controls (karaszewski et al., 1991) . much attention has focused on the cd4þcd25þ subset of regulatory t cells. induction of cd4þcd25þ t cells is controlled by the transcription regulator foxp3 which also serves as a cell-specific marker (hori et al., 2003) . defects in the evector function of cd4þcd25þ regulatory t cells have been demonstrated in ms patients (viglietta et al., 2004) . thus, defects in regulatory t-cell populations may facilitate the development and/or progression of autoimmunity. t-cell migration into the cns in ms is believed to follow the sequence of capture, rolling, activation, adhesion strengthening, and finally transmigration through the bbb. the specifics of these events may vary depending on the region of the cns, as well as the activation state of t cells and microvasculature. two processes appear to be important in t-cell migration into the cns: (1) migration of t cells from the blood into the csf with interactions with dendritic elements on the luminal surface of the choroid plexus resulting in immunosurveillance and (2) t-cell migration through inflamed endothelial bbb and interactions with perivascular apcs within the virchow-robbins space (engelhardt and ransohov, 2005) . evidence exists for both of these processes in ms, however the relative contribution of each is unclear, and may depend on the stage and subtype of disease. adhesion molecule icam-1 is expressed on the inflamed bbb in ms, while lfa-1 is expressed on infiltrating t cells, suggesting an important role for this pathway in t-cell migration in ms (bo et al., 1996) . inhibition of interactions between integrin molecule 41 present on the surface of t cells with vcam-1 present on endothelial cells of the bbb was shown to suppress the development of eae (yednock et al., 1992) . this led to the development of an 41-integrin antibody, natalizumab, which has been evective in reducing ms relapses (miller et al., 2003) . however, clinical trials with a natalizumab led to several cases of progressive multifocal leukoencephalopathy, resulting in the reevaluation of the role of such drugs in ms therapeutics (kleinschmidt-demasters and tyler, 2005; langer-gould et al., 2005; van assche et al., 2005) . cxcr3 is postulated to be the major chemokine involved in the traycking of t cells from the blood to the csf for immunosurveillance in both normals and ms patients (kivisakk et al., 2002) . studies of chemokine expression on peripheral blood t cells in ms patients found a positive correlation between cxcr3 expression on blood t cells (eikelenboom et al., 2002) and csf t cells (sindern et al., 2002) on mri measures of disease activity in ms patients. t-cell migration is an important step in the pathogenesis of ms, as confirmed by the success of 41-integrin antibody therapy (natalizumab). however, evective blockade of cns immunosurveillance has produced profound adverse evects, as evidenced by the development of pml in three treated patients. the risk of infection and emergence of tumors due to blockade of immunosurveillance must be balanced with therapeutic evect when utilizing such powerful therapeutics. much attention has focused on the presence of axonal damage in ms plaques as a substrate for chronic progressive disease (trapp et al., 1998 (trapp et al., , 1999 . although mediators of axonal damage include cytokines, complement, antibody, and nitric oxide, t cells may play a significant role both in neurodegeneration and in neuroprotection. the presence of cd8þ t cells, but not cd4þ t cells, in the ms lesion correlates with axonal damage (bitsch et al., 2000) . in the tmev model of ms, demyelination but no axonal damage was found in the cns of mhc class i-deficient mice after infection with theiler's virus (rivera-quinones et al., 1998) , suggesting that axonal damage is class i mediated. these mice also displayed a deficiency of cd8-positive cells in their cns lesions. similarly, in vitro, t cell-mediated neurotoxicity was dependent on ifn--induced expression of mhc class i (medana et al., 2001) . collectively, these studies suggest that class i expression in the cns, particularly on neurons, may enhance a cytotoxic cd8þ t-cell response. in vitro coculture of human fetal neurons with okt3-activated cd4þ or cd8þ t cells has been found to produce apoptosis of neurons (giuliani et al., 2003) . this process required physical contact of the cells, as demonstrated by transwell experiments, and was not dependent on mhc i. protection could be conferred by blocking cd40 on both t cells and neurons, and fasl on neurons. attention has focused on the regulatory role that neurons may play on t cells. in a lewis rat model of eae, motoneurons were demonstrated to engulf t lymphocytes through a process consistent with emperipolesis (smith et al., 2000) . neuronal production of tgf-has been shown to play a significant role in the induction of cd4þcd25þfoxp3þ regulatory t cells in a murine eae model (liu et al., 2006) . these studies have important implications for understanding the t cell-neuronal interactions in ms. although mbp-reactive t cells have been implicated in the pathogenesis of ms (zhang et al., 1992 (zhang et al., , 1994 , an intriguing study has shown that passive transfer of mbp-reactive t cells resulted in protection of the retinal ganglion cell body after optic nerve crush injury in vivo (moalem et al., 1999) . interestingly, transfer of t cells specific for other antigens was not protective. furthermore, expression of mrna for several nerve growth factors, including brain-derived neurotrophic factor (bdnf), was upregulated after antigen-activation of these t cells, while the injured nerve expressed mrna for nerve growth factor receptors (moalem et al., 2000) . two major types of th cell responses have been described, based on cytokine secretion. th1 cells produce the cytokines ifn-and tnf-, while th2 cells produce il-4, il-5, and il-10. eae has been associated with a th1 response, while th2 cytokines are generally protective. in support of the above findings, a separate study demonstrated that the presence of myelin-reactive th2 cells conferred neuroprotection in organotypic hippocampal slice cultures (wolf et al., 2002) . these findings have several implications for ms. first, myelinreactive t cells may be neuroprotective under certain conditions including trauma, suggesting that the ms brain may be more susceptible to inflammationinduced damage. second, the local production of neurotrophic factors by inflammatory cells may be neuroprotective. however, in contrast to these findings, other studies have shown that mbp-tcr transgenic mice sustained more cns damage and inflammation following traumatic spinal cord injury, compared with wildtype controls (jones et al., 2002) . t-cell infiltrates were localized to areas of demyelination and axonal loss, suggesting that autoreactive t cells can trigger autoimmune demyelination in the setting of trauma under certain circumstances. therefore, the therapeutic potential of neuroprotective t lymphocytes should be entertained only with caution. glatiramer acetate (ga) is an established treatment for rrms. its mechanism of action relies, in part, on the migration of th2 cells into the cns (aharoni et al., 2000 (aharoni et al., , 2002 , where they presumably downregulate local inflammatory responses. interestingly, these t cells have been shown to produce bdnf in the eae model (aharoni et al., 2003) , and ga-reactive t cells harvested from ms patients treated with ga produce bdnf on restimulation in vitro (ziemssen et al., 2002) . however, the role of bdnf in neuroprotection in ms or eae has not been established. thus, the role of this and other neurotrophic factors requires further exploration. the success of several t cell-targeted therapies in ms reinforces the importance of the role of the t cell in ms pathogenesis. of the six approved therapies for ms, the evects of ga and natalizumab can be directly related to modulation 58 tanuja chitnis of t-cell function. modulate some t-cell functions including t-cell migration and th1 cytokine production (yong, 2002) , while mitoxantrone is a cytotoxic agent that nonspecifically abrogates t-and b-cell proliferation. an altered peptide ligand (apl) may be defined as ''any peptide that serves as a receptor ligand in which substitutions of a single or multiple amino acids lead to changes in the functional outcome of receptor signaling'' (bielekova and martin, 2001) . apls have most commonly been used as tcr ligands to alter t-cell responses to presumed immunogenic or target antigens presumably resulting in immune suppression or immune deviation as well as induction of a regulatory t-cell population reactive to the apl itself, which then serves to downregulate the inflammatory disease process through bystander suppression. glatiramer acetate (ga; copolymer-1; copaxone ) is an fda approved therapy for the treatment of rrms. ga is an apl that was originally developed to mimic mbp. it is composed of a random sequence of the amino acids glutamic acid, lysine, alanine, and tyrosine present in a specific molar ratio (0.14:0.34:0.43:0.09). copaxone is administered by daily subcutaneous injection, and in a phase iii clinical trial was found to reduce relapse frequency by 29%, as well as decrease the incidence of new gadolinium enhancing lesions on mri (ge et al., 2000; johnson et al., , 2001 . despite its crude resemblance to mbp, evidence from several studies showing ga stimulates several nonmyelin antigen t-cell lines suggests that ga acts as a ''universal'' or degenerate t-cell antigen (duda et al., 2000) . ga has been shown to inhibit responses to mbp-specific t-cell lines in vitro (racke et al., 1992) , and in vivo treatment with ga induces a hyporesponsiveness to this antigen (schmied et al., 2003) . interestingly, ga-reactive t-cell lines isolated from both treated patients as well as untreated controls were found to cross-react with a variety of peptides, suggesting degenerate antigenicity. in addition, th2 cytokine deviation was noted in ga-reactive t-cell lines (duda et al., 2000) . in the eae model, th2-producing ga-reactive t cells were shown to accumulate in the cns and attenuate disease (aharoni et al., 2000) . thus, the principal mechanism of action of copaxone may be the induction of th2 responses, which exert bystander suppression of inflammation within the cns. apls targeting mbp have been widely studied in ms because of the interest in this potential autoantigen. an apl to mbp 87-99 peptide was shown to be evective in ameliorating disease in the eae model (karin et al., 1994 ). an initial phase i clinical trial tested four doses of an apl to mbp 83-99 administered subcutaneously for 4 weeks demonstrated no safety concerns (bielekova and martin, 2001) . two phase ii trials using mbp 83-99 apls were initiated: a small nih-based trial tested the highest dose of apl cgp77116 (50 mg) administered weekly for 9 months. three of eight patients developed atypical ms exacerbations, characterized by a high gadolinium-enhancing lesion load, tumefactivetype lesion, or a flaccid paralysis with inflammatory involvement of the peripheral the role of cd4 t cells in the pathogenesis nervous system . 2/3 of exacerbations, correlated with enhanced reactivity to mbp . a second larger multicenter study testing three doses of apl nbi-5788 (5, 20, or 50 mg) versus placebo in 144 total patients was terminated because of the occurrence of apl-induced systemic hypersensitivity reactions in 9% of enrolled patients (kappos et al., 2000) . in both studies, enhanced ex vivo t-cell responses to apl were observed following treatment. in patients who developed hypersensitivity reactions, enhanced th2 responses to the apl could be demonstrated (kappos et al., 2000) . further phase ii studies investigating the evects of low-dose apl (nbi-5788) are currently undergoing safety evaluation. t-cell vaccination strategies attempt to eliminate pathogenic t cells through the enhancement of regulatory immune responses to autoreactive t cells. this approach requires the isolation of autoreactive t-cell clones from the individual patient's blood or csf, and subcutaneous reinjection in the form of an immunizing vaccine. pilot trials of t-cell vaccination with autologous mbp-specific t cells from peripheral blood, in 28 rrms and 26 spms patients, demonstrated a modest reduction in posttreatment relapse rate (medaer et al., 1995; zhang et al., 2002) . in this study, the frequency of gadolinium-enhancing lesions was largely unchanged posttreatment. a second pilot trial using autologous mbp and mogreactive t-cell vaccines in 20 rrms nonresponders demonstrated a significant reduction in relapse rate (p ¼ 0.026) as well as gadolinium enhancing and t2 lesion load (achiron et al., 2004) . in both studies, no serious adverse events were noted. in a small study utilizing myelin-reactive cd4þ t cells derived from autologous csf, no adverse evects were observed in any of the five treated patients (van der aa et al., 2003b) . further larger phase ii trials using t-cell vaccination are planned. tcr vaccination strategies target tcr sequences believed to be critical in the immunopathogenesis of ms. in ms, v 5.2/5.3þ has been identified as a dominant tcr variable region sequence involved in mbp t-cell reactivity (kotzin et al., 1991; lozeron et al., 1998; oksenberg et al., 1993) . tcr vaccines are thought to exert their evects by enhancing the function of regulatory t-cell populations recognizing tcr determinants (vandenbark, 2005; vandenbark et al., 1996) . tcr peptides derived from the v 5.2 region of the tcr have been used as a vaccine in ms patients. in a double-blind pilot study, 23 patients were treated with weekly to monthly injections of the peptide. all patients carried the hladrb1*1501 allele. enhanced t-cell responses to the immunizing peptide correlated with clinical improvement (bourdette et al., 1994; vandenbark et al., 1996) . t-cell responses to mbp trended downward in responders. no major adverse events were observed in treated patients. atm-027 is an antibody specifically targeting the v 5.2/5.3 sequence of tcr. results from a multicenter phase ii study in 47 ms patients treated with a run-in regimen of atm-027 monthly for 6 months, showed no significant reduction in new gadolinium mri 60 tanuja chitnis lesions posttreatment despite a significant reduction in v 5.2/5.3þ t cells olsson et al., 2002) . ms relapses occurred in three treated patients, however no other adverse events directly related to drug were observed. these negative results suggest that there is considerable variability in v profiling in individual ms patients. an alternative explanation is that by the time the disease presents clinically, epitope spreading has occurred, negating the use of a single v-depleting agent. cd4 is a cell-surface marker of th cells. in a randomized phase ii doubleblind trial, an anti-cd4 antibody (cm-t412) was administered intravenously to 35 rrms and spms patients (van oosten et al., 1997) . administration of the antibody resulted in a rapid and sustained reduction in circulating cd4þ t cells. infusion-related side evects including nausea, fever, and tachycardia were limited to 24-h postinfusion. after 9 months, treated patients demonstrated an approximately 40% reduction in relapse rate compared to placebo controls, however there was no significant change in the number of gadolinium-enhancing lesions on mri. although anti-cd4 therapy was evective in reducing relapse rate, lack of eycacy on primary mri measures has led to questions regarding the evectiveness in ms. the jak/stat family of transducer/transcription-activating factors plays a critical role in the signaling of many cytokine receptors. 3-hydroxy-3-methylglutaryl coenzyme a (hmg-coa) reductase inhibitors such as atorvastatin has been shown to exert its protective evects in eae through the induction of stat6 phosphorylation and secretion of th2 cytokines, with concomitant inhibition of stat4 phosphorylation and secretion of th1 cytokines (youssef et al., 2002) . a pilot clinical trial of simvastatin in ms showed positive results and a larger trial of atorvastatin is under way. inhibition of molecular pathways involved in t-cell migration has been evective in reducing ms relapses (miller et al., 2003) , however clinical trials with an 4-integrin antibody led to several cases of progressive multifocal leukoencephalopathy, resulting in the reevaluation of the role of such drugs in ms therapeutics (kleinschmidt-demasters and tyler, 2005; langer-gould et al., 2005; van assche et al., 2005) . in conclusion, we have summarized the evidence for the central role of cd4þ t cells in the pathogenesis of ms, which represents the work of many teams of investigators over many years. although much progress has been made in the field, which has led to important therapeutic advances, several questions remain unanswered, including the nature of the initiating t-cell responses and the role of cd4 t cells in the pathogenesis the mechanisms of propagation of the disease within the cns. the field of t-cell biology remains an integral part of the ms question, and further advances will undoubtedly lead to improved treatment strategies for patients. t cell vaccination in multiple sclerosis relapsing-remitting nonresponders patients interleukin-23 promotes a distinct cd4 t cell activation state characterized by the production of interleukin-17 copolymer 1 acts against the immunodominant epitope 82-100 of myelin basic protein by t cell receptor antagonism in addition to major histocompatibility complex blocking specific th2 cells accumulate in the central nervous system of mice protected against experimental autoimmune encephalomyelitis by copolymer 1 oral treatment of mice with copolymer 1 (glatiramer acetate) results in the accumulation of specific th2 cells in the central nervous system glatiramer acetate-specific t cells in the brain express t helper 2/3 cytokines and brain-derived neurotrophic factor in situ t-cell regulation by cd28 and ctla-4 t cells responsive to myelin basic protein in patients with multiple sclerosis high levels of interleukin 10 production in vivo are associated with tolerance in scid patients transplanted with hla mismatched hematopoietic stem cells cd4þ cd25 high regulatory cells in human peripheral blood increased interleukin 12 production in progressive multiple sclerosis: induction by activated cd4þ t cells via cd40 ligand seasonal variation of interferon-gamma production in progressive multiple sclerosis defective regulation of ifn and il-12 by endogenous il-10 in progressive ms interferongamma secretion by peripheral blood t-cell subsets in multiple sclerosis: correlation with disease phase and interferon-beta therapy increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger ov exacerbations? il-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of il-10-and il-4-deficient and transgenic mice reciprocal developmental pathways for the generation of pathogenic evector th17 and regulatory t cells antigen-specific immunomodulation via altered peptide ligands encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase ii clinical trial with an altered peptide ligand helper t cell diverentiation is controlled by the cell cycle acute axonal injury in multiple sclerosis. correlation with demyelination and inflammation distribution of immunoglobulin superfamily members icam-1, -2, -3, and the beta 2 integrin lfa-1 in multiple sclerosis lesions immunity to tcr peptides in multiple sclerosis. i. successful immunization of patients with synthetic v 5.2 and v 6.1 cdr2 peptides a theory of self-nonself discrimination a two-step, two-signal model for the primary activation of precursor helper t cells a new member of the immunoglobulin superfamily-ctla-4 diverential activation requirements for virgin and memory t cells the adhesion molecule and cytokine profile of multiple sclerosis lesions tim-2 regulates t helper type 2 responses and autoimmunity selective regulatory function of socs3 in the formation of il-17-secreting t cells cytokine shifts and tolerance in experimental autoimmune encephalomyelitis role of costimulatory pathways in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis cd28-independent induction of experimental autoimmune encephalomyelitis evect of targeted disruption of stat4 and stat6 on the induction of experimental autoimmune encephalomyelitis frequency of t cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis characterization of theiler's murine encephalomyelitis virus (tmev)-specific delayed-type hypersensitivity responses in tmevinduced demyelinating disease: correlation with clinical signs elevated interleukin-12 in progressive multiple sclerosis correlates with disease activity and is normalized by pulse cyclophosphamide therapy alterations in levels of cd28à/cd8þ suppressor cell precursor and cd45roþ/cd4þ memory t lymphocytes in the peripheral blood of multiple sclerosis patients autoreactivity to myelin antigens: myelin/oligodendrocyte glycoprotein is a prevalent autoantigen ex vivo isolation and characterization of cd4(þ)cd25(þ) t cells with regulatory properties from human blood glatiramer acetate (copaxone) induces degenerate, th2-polarized immune responses in patients with multiple sclerosis t cell memory no evidence to support ctla-4 as a susceptibility gene in ms families: the canadian collaborative study diverentiation of t cell lymphokine gene expression: the in vitro acquisition of t cell memory chemokine receptor expression on t cells is related to new lesion development in multiple sclerosis the ins and outs of t-lymphocyte traycking to the cns: anatomical sites and molecular mechanisms b7-2) and cd40l expression in relapsing and progressive multiple sclerosis induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-beta1-secreting th3 t cells by oral administration of myelin in multiple sclerosis patients ctla-4 gene polymorphism may modulate disease in japanese multiple sclerosis patients glatiramer acetate (copaxone) treatment in relapsing-remitting ms: quantitative mr assessment increased cd80(þ) b cells in active multiple sclerosis and reversal by interferon beta-1b therapy cd40-cd40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis vulnerability of human neurons to t cell-mediated cytotoxicity detection of skewed t-cell receptor v-beta gene usage in the peripheral blood of patients with multiple sclerosis myelin basic protein and proteolipid protein reactivity of brain-and cerebrospinal fluid-derived t cell clones in multiple sclerosis and postinfectious encephalomyelitis oral administration of myelin induces antigen-specific tgf-beta 1 secreting t cells in patients with multiple sclerosis interleukin 17-producing cd4þ evector t cells develop via a lineage distinct from the t helper type 1 and 2 lineages immunologic studies of patients with chronic encephalitis induced by post-exposure semple rabies vaccine the proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4 immunoregulatory molecules and il 2 receptors identified in multiple sclerosis brain tumor necrosis factor identified in multiple sclerosis brain myelin-associated oligodendrocytic basic protein: identification of an encephalitogenic epitope and association with multiple sclerosis control of regulatory t cell development by the transcription factor foxp3 intrathecal activation of the il-17/il-8 axis in opticospinal multiple sclerosis interleukin 12 signaling in t helper type 1 (th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (stat)3 and stat4 increased t cell expression of cd154 (cd40-ligand) in multiple sclerosis copolymer 1 reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase iii multicenter, double-blind placebo-controlled trial. the copolymer 1 multiple sclerosis study group copolymer 1 the role of cd4 t cells in the pathogenesis 65 reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase iii multicenter, double-blind, placebo-controlled trial pathological cns autoimmune disease triggered by traumatic spinal cord injury: implications for autoimmune vaccine therapy ctla4 is associated with susceptibility to multiple sclerosis impaired il-12 responses and enhanced development of th2 cells in stat4-deficient mice induction of a non-encephalitogenic type 2 t helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase ii trial. the altered peptide ligand in relapsing ms study group increased lymphocyte beta-adrenergic receptor density in progressive multiple sclerosis is specific for the cd8þ, cd28à suppressor cell reversal of experimental autoimmune encephalomyelitis by a soluble peptide variant of a myelin basic protein epitope: t cell receptor antagonism and reduction of interferon gamma and tumor necrosis factor alpha production il-18 is linked to raised ifn-gamma in multiple sclerosis and is induced by activated cd4þ t cells via cd40-cd40 ligand interactions interleukin-12 production by dendritic cells. the role of cd40-cd40l interactions in th1 t-cell responses reactivity to myelin antigens in multiple sclerosis. peripheral blood lymphocytes respond predominantly to myelin oligodendrocyte glycoprotein antibodymediated suppression of v5.2/5.3(þ) t cells in multiple sclerosis: results from an mrimonitored phase ii clinical trial activated self-mhc-reactive t cells have the cytokine phenotype of th3/t regulatory cell 1 t cells t-cells in the cerebrospinal fluid express a similar repertoire of inflammatory chemokine receptors in the absence or presence of cns inflammation: implications for cns traycking expression of ccr7 in multiple sclerosis: implications for cns immunity progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta-1a for multiple sclerosis dysregulated t cell expression of tim3 in multiple sclerosis diverences in surface phenotype and mechanism of action between alloantigen-specific cd8þ cytotoxic and suppressor t cell clones preferential t-cell receptor beta-chain variable gene use in myelin basic protein-reactive t-cell clones from patients with multiple sclerosis a pd-1 polymorphism is associated with disease progression in multiple sclerosis diverences in responsiveness to cd3 stimulation between naive and memory cd4þ t cells cannot be overcome by cd28 costimulation a functional and structural basis for tcr cross-reactivity in multiple sclerosis progressive multifocal leukoencephalopathy in a patient treated with natalizumab il-23 drives a pathogenic t cell population that induces autoimmune inflammation functional and ontogenetic analysis of murine cd45rhi and cd45rlo cd4þ t cells human cd25þ cd4þ t regulatory cells suppress naive and memory t cell proliferation and can be expanded in vitro without loss of function the ctla-4 gene is associated with multiple sclerosis neuron-mediated generation of regulatory t cells from encephalitogenic t cells suppresses eae gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis t cell receptor v 5 and v 17 clonal diversity in cerebrospinal fluid and peripheral blood lymphocytes of multiple sclerosis patients heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination distinct patterns of multiple sclerosis pathology indicates heterogeneity on pathogenesis transforming growth factor-beta induces development of the t(h)17 lineage t cell recognition of immunodominant and cryptic proteolipid protein epitopes in humans cd4þ cd28-costimulation-independent t cells in multiple sclerosis high level of cross-reactivity in influenza virus hemagglutinin-specific cd4þ t-cell response: implications for the initiation of autoimmune response in multiple sclerosis ctla4 dimorphisms and the multiple sclerosis phenotype epitope spreading initiates in the cns in two mouse models of multiple sclerosis depletion of myelin-basicprotein autoreactive t cells by t-cell vaccination: pilot trial in multiple sclerosis transection of major histocompatibility complex class i-induced neurites by cytotoxic t lymphocytes expression of costimulatory molecules on peripheral blood mononuclear cells in multiple sclerosis tim-4 is the ligand for tim-1, and the tim-1-tim-4 interaction regulates t cell proliferation a controlled trial of natalizumab for relapsing multiple sclerosis persistent infection with theiler's virus leads to cns autoimmunity via epitope spreading implication of th1, th2, and th3 cytokines in liver graft acceptance autoimmune t cells protect neurons from secondary degeneration after central nervous system axotomy production of neurotrophins by activated t cells: implications for neuroprotective autoimmunity molecular mimicry between a viral peptide and a myelin oligodendrocyte glycoprotein peptide induces autoimmune demyelinating disease in mice th1-specific cell surface protein tim-3 regulates macrophage activation and severity of an autoimmune disease t cell response to 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase (cnpase) in multiple sclerosis patients thymic output generates a new and diverse tcr repertoire after autologous stem cell transplantation in multiple sclerosis patients expansion of a recurrent v 5.3þ t-cell population in newly diagnosed and untreated hla-dr2 multiple sclerosis patients selection for t-cell receptor v beta-d beta-j beta gene rearrangements with specificity for a myelin basic protein peptide in brain lesions of multiple sclerosis ctla-4 dysregulation in the activation of myelin basic protein reactive t cells may distinguish patients with multiple sclerosis from healthy controls a virusinduced molecular mimicry model of multiple sclerosis depletion of v 5.2/5.3 t cells with a humanized antibody in patients with multiple sclerosis stat6-independent gata-3 autoactivation directs il-4-independent th2 development and commitment exacerbations of multiple sclerosis in patients treated with gamma interferon treatment of multiple sclerosis with gamma interferon: exacerbations associated with activation of the immune system a distinct lineage of cd4 t cells regulates tissue inflammation by producing interleukin 17 identification of a second t cell epitope of human proteolipid protein (residues 89-106) recognized by proliferative and cytolytic cd4þ t cells from multiple sclerosis patients myelin basic protein reactive th2 t cells are found in acute disseminated encephalomyelitis macrophages, lymphocytes, and plasma cells in the perivascular compartment in chronic multiple sclerosis copolymer-1-induced inhibition of antigen-specific t cell activation: interference with antigen presentation the dale e. mcfarlin memorial lecture: the immunology of the multiple sclerosis lesion absence of neurological deficits following extensive demyelination in a class i-deficient murine model of multiple sclerosis the balance between persistent virus infection and immune cells determines demyelination the role of diverent subsets of t regulatory cells in controlling autoimmunity interaction of tim-3 and tim-3 the role of cd4 t cells in the pathogenesis 69 ligand regulates t helper type 1 responses and induction of peripheral tolerance in vitro evidence that subcutaneous administration of glatiramer acetate induces hyporesponsive t cells in patients with multiple sclerosis expansion of autoreactive t cells in multiple sclerosis is independent of exogenous b7 costimulation polymorphisms in the genes encoding interferongamma and interferon-gamma receptors in multiple sclerosis identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions expression of chemokine receptor cxcr3 on cerebrospinal fluid t-cells is related to active mri lesion appearance in patients with relapsing-remitting multiple sclerosis immune deviation following pulse cyclophosphamide/methylprednisolone treatment of multiple sclerosis: increased interleukin-4 production and associated eosinophilia autoimmune encephalomyelitis ameliorated by ampa antagonists t cell memory human cd4þ cd25þ thymocytes and peripheral t cells have immune suppressive activity in vitro characteristics of t-cell receptor repertoire and myelinreactive t cells reconstituted from autologous haematopoietic stem-cell grafts in multiple sclerosis a novel transcription factor, t-bet, directs th1 lineage commitment impaired il-13-mediated functions of macrophages in stat6-deficient mice myelin basic protein and human coronavirus 229e cross-reactive t cells in multiple sclerosis cross-reactivity with myelin basic 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interferon-beta and glatiramer aetate in ms the hmg-coa reductase inhibitor, atorvastatin, promotes a th2 bias and reverses paralysis in central nervous system autoimmune disease t-cell clones specific for myelin basic protein induce chronic relapsing paralysis and demyelination autoreactive t cells in multiple sclerosis increased frequency of interleukin 2-responsive t cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis activation and clonal expansion of human myelin basic protein-reactive t cells by bacterial superantigens t cell vaccination in multiple sclerosis: results of a preliminary study the tim-3 ligand galectin-9 negatively regulates t helper type 1 immunity glatiramer acetate-specific t-helper 1-and 2-type cell lines produce bdnf: implications for multiple sclerosis therapy. brain-derived neurotrophic factor key: cord-320617-ucm7wx8b authors: b’krong, nguyen thi thuy chinh; minh, ngo ngoc quang; qui, phan tu; chau, tran thi hong; nghia, ho dang trung; do, lien anh ha; nhung, nguyen ngoc; van vinh chau, nguyen; thwaites, guy; van tan, le; van doorn, h. rogier; thanh, tran tan title: enterovirus serotypes in patients with central nervous system and respiratory infections in viet nam 1997–2010 date: 2018-04-12 journal: virol j doi: 10.1186/s12985-018-0980-0 sha: doc_id: 320617 cord_uid: ucm7wx8b background: enteroviruses are the most common causative agents of human illness. enteroviruses have been associated with regional and global epidemics, recently, including with severe disease (enterovirus a71 and d68), and are of interest as emerging viruses. here, we typed enterovirus a-d (ev) from central nervous system (cns) and respiratory infections in viet nam. methods: data and specimens from prospective observational clinical studies conducted between 1997 and 2010 were used. species and serotypes were determined using type-specific rt-pcr and viral protein 1 or 4 (vp1, vp4) sequencing. results: samples from patients with cns infection (51 children – 10 csf and 41 respiratory/rectal swabs) and 28 adults (28 csf) and respiratory infection (124 children – 124 respiratory swabs) were analysed. twenty-six different serotypes of the four enterovirus species (a-d) were identified, including ev-a71 and ev-d68. enterovirus b was associated with viral meningitis in children and adults. hand, foot and mouth disease associated enteroviruses a (ev-a71 and coxsackievirus [cv] a10) were detected in children with encephalitis. diverse serotypes of all four enterovirus species were found in respiratory samples, including 2 polio-vaccine viruses, but also 8 cv-a24 and 8 ev-d68. with the exception of ev-d68, the relevance of these viruses in respiratory infection remains unknown. conclusion: we describe the diverse spectrum of enteroviruses from patients with cns and respiratory infections in viet nam between 1997 and 2010. these data confirm the global circulation of enterovirus genera and their associations and are important for clinical diagnostics, patient management, and outbreak response. electronic supplementary material: the online version of this article (10.1186/s12985-018-0980-0) contains supplementary material, which is available to authorized users. enteroviruses are non-enveloped single stranded rna viruses of the genus enterovirus within the family of picornaviridae. seven species of enterovirus are associated with human disease: enterovirus a-d and rhinovirus a-c. while rhinoviruses commonly cause mild respiratory illness, enteroviruses a-d (evs) are a significant cause of morbidity and mortality worldwide. prior to being reclassified as ev a-d, evs were originally classified as polioviruses (pv) 1-3, coxsackieviruses (cv) a1-24 and b1-6, echoviruses (e) 1-33 and numbered enteroviruses (68-121) [1] . in addition to the surveillance system for poliomyelitis, some countries have established comprehensive surveillance programs for circulating non-polio evs [2, 3] . in recent years, surveillance activities have been enhanced in response to the emergence of hand, foot, and mouth disease (hfmd) causing enteroviruses in the asia-pacific region [4] and the global spread of ev-d68 causing respiratory infections [5, 6] . ev infections are often asymptomatic, but may also result in a diverse spectrum of clinical illness, varying from mild febrile illnesses to severe disease of the cutaneous, gastrointestinal, respiratory, cardiovascular, and central nervous system (cns) [7, 8] . generally, ev a is associated with herpangina and hand, foot and mouth disease (hfmd), ev b with herpangina and sporadic and epidemic viral meningitis or encephalitis, ev c with poliomyelitis and ev d with respiratory infections [2, [9] [10] [11] . in viet nam, since 2005, various serotypes of ev a, most commonly enterovirus a71 (ev-a71), coxsackievirus a16 (cv-a16), cv-a10, and cv-a6 have been associated with outbreaks of hfmd [12, 13] and evs have also been frequently detected in aetiological studies of cns and respiratory infections [14] [15] [16] [17] [18] . in the majority of aetiological studies only generic rt-pcr is used for detection of enteroviruses [14-16, 18, 19] . information about specific enterovirus serotypes circulation and their associated clinical phenotypes therefore remains sparse. here we report the clinical associations and serotyping results of evs that were previously detected in our studies of cns and respiratory infections in southern and central viet nam between 1997 and 2010. data and samples from 5 prospective observational clinical studies on cns (n = 3) and respiratory (n = 2) infections, conducted in viet nam between 1997 and 2010 were used [14-16, 18, 19] . patients were enrolled according to studyspecific case definitions for cns infection, acute respiratory infection and lower respiratory tract infection. the three cns studies were conducted to determine the etiology of cns infections in children in children's hospital 1 in ho chi minh city [hcmc] (2004), and in adults in the hospital for tropical diseases (1997 diseases ( -2008 and in a network of 12 provincial hospitals in the southern and central part of viet nam (2007) (2008) (2009) (2010) . in these three studies, csf was obtained from all participants, while throat and rectal swabs were only taken from children. the two respiratory infections studies were conducted to study lower respiratory tract infection in hospitalized children under 2 years of age in two main paediatric hospitals in hcmc (2009-2010) and the antibiotic use in out-patients with acute respiratory infections in children's hospital 1 in hcmc (2009-2010). respiratory swabs were taken from all enrolled children. patients positive for generic ev rt-pcrs in csf or swabs from these studies and with diagnostic specimens available were included in this study. the geographical distribution of included patients is shown in additional file 1: figure s1 . viral rna was extracted from clinical samples using either the magna pure 96 platform (roche applied science, darmstadt, germany) or the qiaamp viral rna mini kit (qiagen gmbh, hilden, germany) following the manufacturer's instructions. ev-a71 specific one-step real-time rt-pcr ev-a71 detection and typing of samples from patients with cns infection was performed using real-time rt-pcr as described previously [12] . in brief, 2 μl of viral rna were subjected to one-step real-time rt-pcr reaction using the superscript iii one-step qrt-pcr system with platinum taq dna polymerase (invitrogen, carlsbad, ca, usa). the cycling conditions included one cycle of 60°c for 3 min, followed by 15 min at 53°c and 2 min at 95°c, and 45 cycles of 15 s at 95°c, 1 min at 53°c (including data acquisition) and 15 s at 72°c. nested rt-pcr for viral protein 1 (vp1) and complete viral protein 4 and partial viral protein 2 (vp4/vp2) was carried out using previously described assays [20] [21] [22] . vp1 rt-pcr of samples from patients with cns infection was primarily performed using the assay described by leitch et al., [22] and alternatively, the vp1 assay described by oberste et al., [20] . the vp4/vp2 assay described by mirand et al., [21] was applied to all samples from patients with respiratory infection and to samples from patients with cns infection with negative vp1 rt-pcrs. there was no major modification of the original assays except the use of superscript iii reverse transcriptase with platinum taq (invitrogen) in the first round rt-pcr and hotstar taq dna polymerase (qiagen) in the second round pcr and the adaptation of thermal cycling conditions as per supplier's instructions. in the three assays, 5 μl of viral rna were used in the first round one-step rt-pcr and then 0.5 μl of the first round pcr product was transferred to the second round pcr reaction. pcr products of the 2nd round pcr were analysed in agarose gel, purified with cold ethanol and were then subjected to dna sequencing using the big dye terminator v3.1 cycle sequencing kit (applied biosystems inc., foster city, ca, usa). nucleotide sequences of the vp1 and vp4/vp2 were assembled using vector nti ® express software v7.1 (thermo fisher scientific, waltham, usa). all the sequences have been submitted to genbank (mh021887-mh021955). multiple sequence alignments were performed using bioedit software v7.0.9 (ibis therapeutics, ca, usa) and with the inclusion of reference prototype sequences obtained from genbank. neighbour-joining trees were constructed in mega software v7.0.26 (www. megasoftware.net) using the maximum composite likelihood nucleotide substitution model with 1000 bootstrap replicates. serotype assignments for vp1 and vp4/vp2 sequences were performed using an automated phylogenetic-based enterovirus typing tool developed by kroneman et al., [23] available at http://www.rivm.nl/mpf/enterovirus/ typingtool/. because the automated typing tool can determine vp4/vp2 sequences at species level only [23] , further serotype determination for vp4/vp2 sequences was based on highest nucleotide identity score (hnis) and highest amino acid sequence similarity (haass) with reference prototypes [24, 25] . in case of discrepancy, final assignment was based on haass and further confirmed using blast [21, 26] . statistical analyses were performed using spss v23.0 (spss, inc., chicago, il, usa). categorical variables were compared using chi-square or fisher's exact tests and continuous variables were compared using mann-whitney u test. two-sided p values ≤0.05 were considered significant. samples from a total of 203 patients were included in this study, including from 79 patients with cns infection and 124 with respiratory infection. when analyzing for the monthly distribution of cases, there was no clear peak among cns cases, whereas two peaks of respiratory cases were found in april and november (fig. 1 ). among patients with cns infection (n = 79), 51 were children (median age 2 years, interquartile range [irq]: 1-5) and 28 were adults (20 years, irq: 17-29.5). all adults had ev detected in csf and 93% (26/28) had meningitis as discharge diagnosis. of the 51 children, 10 had ev detected in csf, and the remaining (41/51) had ev detected in respiratory and/or rectal swabs. encephalitis was the most common discharge diagnosis (30/41). fifteen deaths were noted, and all were children with ev detected in swabs only (table 1) . among patients with respiratory infections, the median age was 12.7 months (iqr: 5.7-24.3). inpatients (n = 65) were younger than outpatients (n = 59), due to study enrolment criteria ( table 2) . bronchiolitis (as assessed at the discretion of treating physicians) was the most common clinical diagnosis (57% in outpatients and 61% in inpatients). thirty-five percent of inpatients had a clinical diagnosis of pneumonia ( table 2) . in patients with cns infection, co-infection was more common and was found in 27% (14/51) children versus 11% (3/28) adults (table 1) . most co-infected children (13/14) had ev detected in swabs only. of the 15 documented fatal cases, 7 were serologically confirmed to have co-infection with dengue virus (denv; n = 3) and japanese encephalitis virus (jev; n = 4) (additional file 4: table s1 ). among the 3 co-infected adults, 2 had cytomegalovirus (cmv) dna detected in csf and 1 had positive csf culture with mycobacterium tuberculosis (table 1) . in both patient groups of respiratory infection, there were high rates of co-infection with other respiratory viruses. co-infection with rhinovirus (rhv; 69%) and respiratory syncytial virus (rsv; 27%) was more common than other viruses, including adenovirus (adv; 14%), parainfluenza viruses (piv; 9%), human metapneumovirus (hmpv; 9%), human bocavirus (bov; 7%), human coronavirus (hcov; 4%), influenza virus (flu; 2%), and parechovirus (pev; 1%). the rate of coinfection with rhv and rsv was significantly higher in the inpatient group as compared to the outpatient group (p < 0.001) ( table 2) . additional file 2: figure s2 ) and vp4/vp2 (n = 16; additional file 3: figure s3 ) sequencing. ev b was the only species detected in adults and the most common species in both swabs and csf from children (table 3) . ev-a71 was the most commonly detected single serotype, in 9 swabs and 1 csf of 10 children. three of these children had a clinical diagnosis of hfmd while the other 7 children had a clinical diagnosis of encephalitis (additional file 4: table s2 ). there was no specific serotype associated with fatal outcome (additional file 4: table s1 ). among patients with respiratory disease 13 different serotypes of four enterovirus species were identified by vp4/ vp2 sequence analysis including a (n = 2, 2 serotypes), b other discharge diagnoses included sepsis (n = 5), dengue haemorrhagic fever (n = 1), colitis (n = 1), diarrhea (n = 1) metabolic disease (n = 1), and hepatitis (n = 1) c statistical comparison between children and adults with ev detection in csf (n = 11, 7 serotypes), c (n = 10, 3 serotypes), and d (n = 8, 1 serotype). cv-a24 (n = 8) and ev-d68 (n = 8) were the two most common serotypes detected (table 3) and these viruses had a close genetic relationship in phylogenetic tree analyses (additional file 3: figure s3 ). there is limited information on circulating enterovirus serotypes and associated clinical phenotypes from the asia pacific region, including viet nam. such knowledge is essential for laboratory diagnostics, patient management and future outbreak response. here, the diversity of enterovirus serotypes belonging ev a-d was assessed in samples from patients with cns and respiratory infections. some of these serotypes, including ev-a71 and ev-d68 have recently been recognized as emerging viral pathogens with pandemic potential. ev-a71 is the most important pathogen of hfmd epidemics in the asia-pacific region with over 2 million cases reported annually [27, 28] . here, ev-a71 was detected in 10/51 (9 swabs and 1 csf) children with cns infections, three of those had a clinical diagnosis of hfmd of which two died [13] . in 9/10 cases, ev-a71 was the only pathogen detected in diagnostic samples. these data are in accordance with previous reports showing that ev-a71 has the potential to cause brain stem encephalitis and death in children under 5 years of age [27] , but is rarely detected in csf [29] . ev-d68 has recently been recognized as an important cause of respiratory illness [30] . ev-d68 emergence was first reported from two children's hospitals in the usa in august 2014 [31] and, shortly after, from countries across the americas, europe, and asia [30] . here, ev-d68 accounted for 8/31 successfully typed enteroviruses from patients with respiratory illness recruited between 2009 and 2010. it remains unknown why ev-d68 has been circulating in viet nam for some time but has not been associated with outbreaks as observed in the usa and other countries in southeast asia. we have recently reported an in depth phylogenetic analysis of these ev-d68 viruses showing that their introduction into viet nam may have already occurred in 2008 [32] . cv-a24 has been associated with herpangina, acute flaccid paralysis and epidemics of acute haemorrhagic conjunctivitis [33] . in the present study, it was one of the most common enteroviruses: 8/31 typed viruses from patients with respiratory illness. however, the circulation of this particular enterovirus serotype has not been reported from viet nam. ev b was found in csf of both children and adults and 3/4 children and 11/13 adults had a clinical diagnosis of meningitis (additional file 4: table s4 ), in accordance with the known clinical spectrum of ev b [9, 10] . ev a was found in 2 csf of children and both had a clinical syndrome of encephalitis (additional file 4: table s4 ). it is likely these children had hfmd associated rhombencephalitis, in line with reports from the region starting in [27] . remarkably, none of the 8 children who died had either ev or any other pathogen detected in csf suggesting other etiology and/or emphasizing the diagnostic challenges of encephalitis [14, 34] . in addition to ev-a71, other common serotypes in cns infections cases included e-4 and e-30 (both ev b). these two viruses have frequently been involved in outbreaks of aseptic meningitis worldwide [35, 36] . while e-30 has been confirmed to be the cause of a meningoencephalitis outbreak in northern viet nam previously [37] , e-4 has yet been associated with any local epidemic of viral meningitis among children and young adults. pv-1 and 2 (ev c) were detected in swab specimens of 3 children: one with cns and two with respiratory illness. as children were under 2 and sequences had 99-100% of vp4/vp2 identity to sabin vaccine strains [38] these were considered to be derived from oral polio vaccine. data from this study showed that among patients with cns infection, children had a higher rate of co-infection than adults. here, this was due to the fact that both swabs and csf were used in the diagnostics for children while only csf was used for adults. in addition, children may be more vulnerable to viral infection than adults, particularly to the locally endemic viruses jev (n = 10) and denv (n = 4) [14] . as jev and denv infections are commonly associated with severe disease and mortality, the contribution of ev to the disease phenotype of these cases is uncertain. similarly, in patients with respiratory infection, the clinical significance of ev infection should be interpreted with caution because of the high rate of co-infection with more common respiratory viruses [39] . all the serotyping assays used in the current study, including the ev-a71 specific rt-pcr, vp1 and vp4/ vp2 sequencing have previously shown their efficient performance in clinical samples. the application of these assays in samples of patients with cns infection would help to improve serotyping yield. however, the overall serotyping success rate for patients with cns infection in the current study was low (58%) as compared to previous studies [21, 22] . this may be due to low viral load or nucleic acid degradation after long-term storage. the low serotyping success rate (25%) of respiratory samples can be explained by the sole use of vp4/vp2 sequencing assay. however, sample degradation and false positivity for ev using generic pcr among rv co-infected patients (68.5%) could not be excluded. other drawbacks of the current study included the small sample size, the sporadic and expanded geographical distribution of cases that hampered our further analyses for serotypeassociated seasonality and geographics [7] . despite these limitations, however, our study revealed diverse enterovirus serotypes have been circulating in viet nam in association with respiratory and cns infections. our study illustrates the circulation of diverse enterovirus serotypes belonging to four species (a-d), and their association with respiratory and cns infections in viet nam. these data are important for patient management, laboratory diagnostics and future outbreak response. additional file 1: figure s1 . the geographic distribution of cns and respiratory cases included in the current study (tiff 892 kb) additional file 2: figure s2 . phylogenetic tree analysis of partial vp1 enteroviral sequences. a middle-point rooted tree of partial vp1 sequences (330 bp) showing the genetic relationship among species and serotypes of the current study (filled triangles) and with reference prototypes. table s1 . demographic, clinical and diagnostic information of the 15 death cases. table s2 . demographic, clinical and laboratory information of 10 cases with ev-a71 infection. table s3 . vp4/vp2 nucleotide and amino acid similarity scores. epidemiology of meningitis and encephalitis in the united states from 2011-2014 the impact of increased molecular diagnostics hand, foot, and mouth disease in china, 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cerebrospinal fluid direct identification of human enterovirus serotypes in cerebrospinal fluid by amplification and sequencing of the vp1 region an automated genotyping tool for enteroviruses and noroviruses a comparison of the vp1, vp2, and vp4 regions for molecular typing of human enteroviruses molecular identification of 13 new enterovirus types, ev79-88, ev97, and ev100-101, members of the species human enterovirus b basic local alignment search tool virology, epidemiology, pathogenesis, and control of enterovirus 71 epidemiological and clinical characteristics of children who died from hand, foot and mouth disease in vietnam enterovirus 71 infection in children with hand, foot, and mouth disease in shanghai, china: epidemiology, clinical feature and diagnosis global emergence of enterovirus d68: a systematic review severe respiratory illness associated with enterovirus d68 -missouri and illinois enterovirus d68 in viet nam two outbreaks of acute hemorrhagic conjunctivitis in africa due to genotype iii coxsackievirus a24 variant variations in cerebrospinal fluid viral loads among enterovirus genotypes in patients hospitalized with laboratory-confirmed meningitis due to enterovirus predominance of enterovirus b and echovirus 30 as cause of viral meningitis in a uk population a new variant of echovirus 4 associated with a large outbreak of aseptic meningitis an approach for differentiating echovirus 30 and japanese encephalitis virus infections in acute meningitis/encephalitis: a retrospective study of 103 cases in vietnam vaccinederived polioviruses and the endgame strategy for global polio eradication detection of respiratory viruses in gargle specimens of healthy children we are indebted to ms. le kim thanh for providing supports in sample storage. the research leading to these results has received funding from the wellcome trust of great britain [101104/z/13/z and 204904/z/16/z]. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. le van tan is a wellcome-trust intermediate fellow in public health and tropical medicine. all data generated or analysed during this study are included in this published article. hrvd, lvt and ttt: conceived and designed the study. nttcb, nnn, lvt and ttt: did laboratory testing and analysed the test results. nnqm, ptq, tthc, hdtn, lahd, nvvc and gt: provided research materials, enrolled patients and took samples. nttcb, lvt, hrvd and ttt: drafted and reviewed earlier versions of the manuscript. all authors read and approved the final manuscript. the original studies were reviewed and approved by the local institutional review boards of all enrolling hospitals and the oxford tropical research ethics committee (oxtrec), university of oxford, oxford, united kingdom. written informed consent was obtained from parent or legal guardian of each participant. subsequent approvals for use of original samples for serotyping of enterovirus was obtained from the institutional review boards from the enrolling hospitals when not part of the original protocols. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-304084-ervaxqph authors: chang, luan-yin; hsiung, chao a; lu, chun-yi; lin, tzou-yien; huang, fu-yuan; lai, yu-han; chiang, yu-ping; chiang, bor-luen; lee, chin-yun; huang, li-min title: status of cellular rather than humoral immunity is correlated with clinical outcome of enterovirus 71 date: 2006 journal: pediatr res doi: 10.1203/01.pdr.0000238247.86041.19 sha: doc_id: 304084 cord_uid: ervaxqph we valuated specific cellular and humoral immune response of cases of enterovirus 71 (ev71) infection and correlated immune response with clinical outcome. after obtaining informed consent, we enrolled 30 ev71 cases including 7 cases with brainstem encephalitis plus pulmonary edema, 12 cases of cns (cns) involvement and 11 uncomplicated cases. we measured antibodies specific to ev71, lymphocyte proliferation response and ev71-stimulated cellular response of th1/th2 cytokines and chemokines. the 7 ev71 cases involving brainstem encephalitis plus pulmonary edema had a significantly lower phytohemagglutinin stimulation index than other cases (p = 0.04). after ev71 stimulation of peripheral mononuclear cells, there was a significant increase in cellular th1 cytokine (γ-interferon) and proinflammatroy cytokines. however, cases with pulmonary edema had significantly lower cellular γ-interferon (p = 0.04), lower cellular il-1β (p = 0.04), lower cellular il-6 (p = 0.04), lower cellular tumor necrosis factor-α response (p = 0.04), and lower cellular macrophage inflammatory protein-1α (p = 0.04) response compared with other cases. their titers of ev71 neutralizing antibodies demonstrated no difference among cases. these results suggest lower ev71-specific cellular response may be associated with immunopathogenesis of ev71-related pulmonary edema. o utbreaks of enterovirus 71 (ev71) encephalitis have been reported from the united states, europe, australia, japan, brazil and malaysia (1-10), since it was originally characterized from a 1969 california outbreak (1) . before 1998, there were three large outbreaks with dozens of fatal cases occurring in bulgaria, hungary, and malaysia (l975, l978, and 1997, respectively) (3, 5, 10) . the largest and most severe ev71 epidemic to date occurred in taiwan in 1998 (11) (12) (13) (14) (15) (16) . a total of 129,106 cases of hand-foot-and-mouth disease and herpangina (hfmd/ha) were reported (15) . severe neurologic complications and/or pulmonary edema were reported in 405 cases, and 78 children died (15) . most ev71 fatalities were cases of fulminant pulmonary edema (15) . however, ev71 infection causes very diverse symptoms, ranging from none (about 71%) to fatality (about 0.05%) (17, 18) . it remains unknown why different hosts of the same ev71 infection have such a range of clinical outcomes (17, 18) . perhaps this range is related to virulence or load of the virus, or particular host factors. to date no relationship has been found between ev71 genotypes and clinical outcome (19, 20) , and ev71 virulence factors have not been clarified. it is possible that host factors, especially host immune response, may be of ultimate importance to clinical outcome. to clarify severe ev71 infection pathogenesis, we investigated factors of cellular versus humoral immune response and correlated this with clinical outcome. committee approved this study and informed parental consent was obtained, 30 ev71 cases of different severity were enrolled. ev71 infection was confirmed by positive ev71 isolation and/or positive ev71 specific igm at the onset of their disease. the cns involvement was indicated in four types of cases. those with aseptic meningitis had headache and irritability along with cerebrospinal fluid (csf) pleocytosis (ͼ5 leukocytes/l) and without an altered level of consciousness. the second type of cases involved encephalitis had altered level of consciousness plus cerebrospinal fluid (csf) pleocytosis. poliomyelitislike syndrome was defined as acute limb weakness and decreased reflex and muscle strength. finally, cases with encephalomyelitis had the occurrence of both encephalitis and poliomyelitis-like syndrome. laboratory studies. ev71-specific humoral immunity: for ev71-specific humoral immunity, ev71 neutralization antibody and igm were measured. ev71 igm was measured at the onset of disease and ev71 neutralization antibody was evaluated at the same time with the measurement of cellular immunity. the laboratory method for measuring ev71 neutralizing antibody followed the standard protocol of neutralization test in microtiter plates (21) . serum samples were inactivated at 56°c for 30 min and then serially diluted from 2-to 1,024-fold. we mixed and incubated (37°c, 2 h) 50 l of each diluted serum with 50 l containing one hundred 50% tissue culture infective dose (tcid 50 ) of ev71 strain tw/2272/98 (genbank accession number af119795) in microtiter plates (19) two replicate wells were used per serum dilution. then microtiter plates were seeded with 100 l of rhabdomyosarcoma cells (8 ϫ 10 4 cells per ml) and incubated (37°c 5% co 2 atmosphere, 2-7 d). each test was run with cell control, serum control and virus back titration with 100 -0.1 tcid 50 . cytopathic effect was observed under an inverted microscope after incubation, and serotiter was determined when cytopathic effect was observed in 1 tcid 50 of the virus back titration. microtiter plates were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet. seropositivity was defined as serotiter ն 8. for ev71 igm detection, ev71 isolate tw/2086/98 was amplified and purified as an antigen for use in -capture elisa, whose sensitivity and specificity was 91.5% and 93.1%, respectively (22) . cellular immunity: proliferation response, cellular th1/th2 cytokine and chemokine response. to isolate peripheral blood mononuclear cells (pbmcs), blood samples were heparinized and subjected to ficoll-hypaque (pharmacia diagnostics ab, uppsala, sweden) gradient centrifugation. cells at the interface were removed carefully and washed twice with pbs. the isolated pbmcs were cultured in 96-well round-bottom microplates (3 ϫ 10 5 cells per well in 0.2 ml of culture medium) in rpmi 1640 medium supplemented with 1 mm glutamine, 1 mm sodium pyruvate, 50 m 2-me, 100u/ml penicillin, 0.1 mg/ml streptomycin, and 2% human ab serum (biocell laboratories inc., rancho domingnez, ca). in addition, pbmcs were stimulated with phytohemagglutinin (pha) (8 g/ml) or different concentrations of ev71 whole virus antigen (1, 5, and 10 g/ml) tw/2272/98 (genbank accession number af119795) or in the absence of antigen (served as the control). ev71 whole virus antigen was purified by 15-45% sucrose gradient centrifugation (25,000 rpm for 4 h at 4°c) and the concentration of virus was measured by use of bio-rad protein assay reagents. after 3 d of incubation, supernatant was collected and assayed for production of th1/th2 (t helper 1/t helper 2) cytokines (␥-interferon, il-1␤, il-2, il-4, il-5, il-6, il-10, il-13 and tnf-␣) and chemokines (eotaxin, il-8, ip-10, mcp-1, mcp-2, mcp-3, mcp-4, mip-1␣ and rantes), which were measured with protein array kits (fast quant microspot assay, shleicher & schuell, dassel, germany). all the assays were triplicate. the detectable levels for il (interleukin)-1␤, il-2, il-4, il-5, il-6, il-10, il-13, ␥-interferon and tnf-␣ (tumor necrosis factor-␣) were 3-3,000, 3-3,000, 4 -3,000, 10 -3,000, 3-3,000, 30 -12,000, 100 -12,000, 30 -12,000, and 4 -3,000 pg/ ml, respectively. the detectable level for ip-10 (interferon ␥-inducible protein-10) was 12.2-12,500 pg/ml and that for eotaxin, il-8, mcp (monocyte chemoattractant protein)-1, mcp-2, mcp-3, mcp-4, mip (macrophage inflammatory protein)-1␣ and rantes (regulated on activation, normal t expressed and secreted) was 2.4 -2,500 pg/ml respectively. proliferation response was measured at the end of the culture period. after 6 d of culture, 1 mci of 3 h-thymidine (amersham, buckinghamshire, england) was added to each well for 18 h, and the incorporated radioactivity was measured by ␤-counter (parkard instrument co., inc., meriden, ct). data are taken from triplicate wells as mean number of counts per minute (cpm). stimulation index was calculated by the formula of the cpm incorporated in response to antigen divided by the cpm incorporated in the absence of antigen. in each time of lymphocyte proliferation response, pbmcs of one healthy individual were stimulated with phytohemagglutinin (pha) (8 g/ml) and used as the experimental control, and their pha stimulation index was ranged from 8 -45. statistics. data were analyzed with sas statistical package (version 9.1, sas institute, cary, nc). because there was limited case number and values were not distributed normally, we used kruskal-wallis test and mann-whitney u-test for continuous variables. 2 test was used for categorical data. wilcoxon signed rank test was used to compare cellular cytokine and chemokine response with or without ev71 antigen stimulation. to prevent the confounding effects of age and sex on the immune response, multiple logistic regression analysis was used to calculate the adjusted p values for cellular and humoral immunity after concomitant adjustment of age and sex. p ͻ 0.05 was considered statistically significant. immune work-up was completed with 30 ev71 cases. this included 7 cases with brainstem encephalitis plus pulmonary edema, 12 cases of cns (cns) involvement including 5 of septic meningitis, 5 of encephalitis, 1 of poliomyelitis-like syndrome and 1 of encephalomyelitis, and 11 uncomplicated cases of hand-footand-mouth disease or herpangina. in 22 cases, we isolated the virus itself, and detected positive igm of ev71; the remaining 8 cases were negative for the virus but positive for ev71 igm at the onset of disease. their median (range) age at the onset of disease was 0.55 (0. 16 -1.6) year for the 7 cases with brainstem encephalitis plus pulmonary edema, 2.4 (1.3-4.5) years for 12 cases of cns involvement and 1.8 (0.37-5.0) years for 11 uncomplicated cases; the age at the onset of disease was significantly younger for the group with brainstem encephalitis plus pulmonary edema than the other two groups (p ϭ 0.007 with kruskal-wallis test). the male-to-female ratio was 3:4 for the 7 cases with brainstem encephalitis plus pulmonary edema, 7:5 for 12 cases with cns involvement and 6:5 for the 11 uncomplicated cases (p ϭ 0.80 with 2 test). the median (range) interval between their disease onset and enrollment in this study was not significantly different among the three groups: 1.9 (1.1-2.9) years for the 7 cases with brainstem encephalitis plus pulmonary edema, 2.5 (0.7-5.2) years for 12 cases with cns involvement, and 2.6 (0.7-2.7) for 11 uncomplicated cases (p ϭ 0.17 with kruskal-wallis test). the median (range) age at this immune study was 3.1 (1.7-3.8) years for the 7 cases with brainstem encephalitis plus pulmonary edema, 5.8 (3.5-7.3) years for 12 cases with cns involvement, and 4.5 (2.0 -8.1) years for 11 uncomplicated cases (p ϭ 0.005 with kruskal-wallis test). among the seven cases of brainstem encephalitis plus pulmonary edema, one recovered completely, one had sequelae of right upper-limb paralysis plus scoliosis, and five had polio-like sequelae and hypoventilation with ventilator support. all remaining 11 cases of cns involvement as well as the 12 uncomplicated cases recovered without neurologic sequelae. ev71-specific neutralizing antibody titers. ev71-specific humoral immunity is shown in fig. 1 , and neutralizing antibody titers did not differ significantly among all the three groups of different severity (p ϭ 0.567 with kruskal-wallis test). age ͻ3 y and gender did not affect the neutralizing antibody titers significantly, either (p ϭ 0.68 and p ϭ 0.83 with mann-whitney u-test, respectively). cell-mediated immunity. pha-stimulation index is shown in fig. 2 . ev71 cases with pulmonary edema had significantly lower pha-stimulated lymphocyte proliferation (median only one (14%) of the pulmonary edema cases had a pha-stimulated index over 19 (p ϭ 0.04 with 2 test), whereas 13 (57%) of non-pulmonary edema cases did. age ͻ3 y and gender did affect the phastimulation index significantly (p ϭ 0.89 and p ϭ 0.48 with 2 test, respectively). after adjusted with age and gender, cases of pulmonary edema was still significantly associated with lower rate of pha-stimulated index over 19 (p ϭ 0.02 with multiple logistic regression analysis). ev71 stimulation index was dose-related. higher doses of ev71 antigen stimulation induce higher lymphocyte proliferation response. although ev71-specific lymphocyte proliferation was higher in uncomplicated cases, no statistical difference in lymphocyte proliferation was found between pulmonary edema cases and other cases (p ϭ 0.48, p ϭ 0.33, p ϭ 0.59 for ev71 concentration 1, 5, or 10 g/ml, respectively). the highest ev71 stimulation index levels are shown in fig. 3 . the median ev71-stimulated index of all 30 cases was 3.59. four (57%) of the 7 pulmonary edema cases had an ev71-stimulated index over 3.59, whereas 11 (48%) of 23 non-pulmonary edema cases did (p ϭ 0.95 with 2 test). overall only 60% (18/30) had peak ev71 siͼ ϭ 2, and included 4 (57%) of pulmonary edema cases, 6 (50%) of cns cases, and 8 (73%) of uncomplicated cases (p ϭ 0.53 with 2 test). age ͻ3 y and gender did affect the ev71-stimulation index significantly (p ϭ 0.11 and p ϭ 0.47 with 2 test, respectively), either. cellular cytokine and chemokine response after ev71 stimulation. difference in the cytokine and chemokine response of peripheral mononuclear cells with and without ev71 antigen stimulation is shown in table 1 . in comparison with condition of absence of ev71 antigen, ev71 antigen stimulation induced a significant increase in cellular th1 cytokine (␥-interferon) but a significant decrease in th2 cytokine (il-5). there were a ␥-interferon median increase of 42 pg/ml and an il-5 median decrease of 1.45 pg/ml. as well, there was an increase of the pro-inflammatory cytokines il-6 (median increase, 500 pg/ml), il-1␤ (median increase, 36 pg/ml), and tnf-␣ (median increase, 86 pg/ml). overall, ev71 stimulation had a positive effect on cellular th1 cytokine and pro-inflammatory cytokine response, but no effect or a mildly negative effect on th2 cytokines. pha stimulation index among ev71 cases with different severity (ev71 cases with pulmonary edema, ev71 cases with cns involvement, and uncomplicated ev71 cases). ev71 cases with pulmonary edema had a significantly lower pha stimulation index (p ϭ 0.04, measured to compare the percentages of a response over the median level of increase of all the ev71 cases by using likelihood ratio 2 test). peak enterovirus 71 (ev71) stimulation index among ev71 cases with different severity (ev71 cases with pulmonary edema, ev71 cases with cns involvement, and uncomplicated ev71 cases). all peak ev71 stimulation index were statistically identical among the three groups (p ϭ 0.31 with kruskal-wallis test) the numbers in parenthesis are the ranges of the difference for peripheral mononuclear cellular cytokine and chemokine response with and without ev71 antigen stimulation. * the "-" denotes decreased response after ev71 stimulation. p-values were measured to compare the difference of cellular cytokine/chemokine response with or without ev71 antigen stimulation by using wilcoxon signed rank test. ns, not significant; tnf-␣, tumor necrosis factor-alpha; ip, interferon ␥-inducible protein; mcp, monocyte chemoattractant protein; mip, macrophage inflammatory protein; rantes, regulated on activation, normal t expressed and secreted. after ev71 stimulation, certain cellular chemokine responses significantly increased, including ip-10 (median increase, 87 pg/ml), mcp-2 (median increase, 1,051 pg/ml), mcp-3 (median increase, 897 pg/ml), mcp-4 (median increase, 7.25 pg/ml), mip-1␣ (median increase, 1,635 pg/ml), and rantes (median increase, 325 pg/ml). after observing the significant increase in cellular response of ␥-interferon, il-1␤, il-6, tnf-␣, ip-10, mcp-2, mcp-3, mcp-4, mip-1␣ and rantes, and the significant decrease in il-5, we compared the cellular response of these cytokines/ chemokines with severity of cases. cases with pulmonary edema were found to have significantly lower cellular ␥-interferon ( fig. 3; p ϭ 0.04) , lower cellular il-1␤, lower cellular il-6, lower cellular tnf-␣ response and lower cellular mip-1a response in comparison with other ev71 cases. in cases with pulmonary edema the percentage of increase over the median level of cellular ␥-interferon, il-1␤, il-6, tnf-␣ and mip-1␣ was lowest ( table 2 ). uncomplicated ev71 cases had highest percentages of increase over the median level of cellular ␥-interferon, il-1␤, il-6, and tnf-␣ ( table 2 ). age and gender did not affect their cellular cytokine response significantly. after age and gender adjustment, the percentage of increased cellular cytokines was still significantly different among the three groups with different clinical severity (p ϭ 0.02 for ␥-interferon, il-1␤, il-6, and tnf-␣) (fig. 4) . in contrast, 67% (8/12) cases with cns involvement and 64% (7/11) uncomplicated cases had decrease over the median level of cellular il-5 response after ev71 stimulation whereas none of the pulmonary edema cases had decrease of cellular il-5 response (p ϭ 0.01). in general, ev71 antigen enhanced cellular th1 cytokine, pro-inflammatory cytokine and chemokine response, and reduced cellular th2 cytokine (il-5) response in most cases of cns involvement and uncomplicated cases. however, ev71 antigen had little effect on cellular cytokine and chemokine response in most cases with brainstem encephalitis plus pulmonary edema. this study suggests that cellular immunity rather than humoral immunity may be associated with the clinical outcome of ev71 infections. age and gender may influence the immunity, however we demonstrate cellular immunity (pha stimulation index) and cellular cytokine (␥-interferon, il-1␤, il-6, and tnf-␣) response were significantly weaker in ev71 cases with pulmonary edema than the other ev71 cases after age and gender adjustment. on the contrary, the humoral immunity (ev71-specific neutralizing antibody) did not differ significantly among cases with different clinical severity. this study was an exploratory study with the small number of the cases and was not investigated prospectively, so the results * the "-" denotes decreased response after ev71 stimulation. p-value was measured to compare the percentages of cases with a response above the median level by using likelihood ratio 2 test among the three groups. tnf-␣, tumor necrosis factor-alpha; ip, interferon ␥-inducible protein; mcp, monocyte chemoattractant protein; mip, macrophage inflammatory protein; rantes, regulated on activation, normal t expressed and secreted. . cellular interferon-␥ response after ev71 stimulation at the concentration of 10 g/ml) among ev71 cases with different severity (ev71 cases with pulmonary edema, ev71 cases with cns involvement, and uncomplicated ev71 cases). ev71 cases with pulmonary edema had significantly lower interferon-␥ response than the other ev71 cases (p ϭ 0.04, measured to compare the percentages of a response over the median level of increase of all the ev71 cases by using likelihood ratio 2 test). immunopathogenesis of ev71 infection must be validated by further prospective immunologic studies in ev71 cases or host immune genetic studies. in the most severe ev71 cases with pulmonary edema there is lower cellular th1 cytokine coupled with a lower lymphocyte proliferation response. higher incidence of most severe ev71 diseases were found in children under 3 y of age (15, 16) , and this might be explained by their weaker cellular immunity. we hypothesize that lower cellular immunity may delay viral killing or clearance, thus resulting in viral dissemination, sustained systemic inflammatory response and subsequent pulmonary edema. host genetic factors may also play an important role and will require further investigation. yang kd et al. reported that patients with ev 71 meningoencephalitis had a higher frequency of g/g genotype with a polymorphism of the cytotoxic t lymphocyte antigen-4 at position 49 of exon 1 than did control subjects without meningoencephalitis. in these cases there was no difference in specific ev 71 neutralizing antibody titers in the 2 groups (23). in our study, humoral immunity did not affect the clinical outcome of ev71 infection, either. results from the two studies suggest that younger children with genetics involving decreased cellular rather than humoral response may be linked to severe ev 71 infection. in this study control for differences in the ages of patients was tried in each of three presentations, but truly valid conclusions regarding differences in cell-mediated immune response in the brainstem encephalitis plus pulmonary edema group can only be established using age matched controls in the cns involvement and uncomplicated disease groups. another query is whether maternal ev71 could have been important in later cellular immune response antibody making the lower cellular ev71 immune response of the youngest subjects, as maternal antibody might be after administration of live viral vaccines. however, we had a seroepidemiology study, done in 1999 (18) , which showed that 94% (177/189) of 3-to 6-month-old children and 92% (265/287) of 7-to 12month-old children did not have ev71 neutralizing antibody. the 8% of 7-to 12-month-old children might get their antibody through natural ev71 infection rather than maternal antibody. the results suggested that even the youngest group of ev71 cases seldom had maternal ev71 antibody, so their lower cellular immunity might be related to other causes rather than maternal antibody. our previous studies reported that patients with pulmonary edema had dramatically high blood values of il-1␤, il-6, tumor necrosis factor-␣, white blood cell counts, and glucose levels (24, 25) . these findings suggest that a combination of cns and systemic inflammatory response may trigger ev71related cardiopulmonary collapse (24) . another study of patients with pulmonary edema showed significant immunomodulator elevation as well as lower circulating cd4 ϩ t-cells, cd8 ϩ t-cells, and natural killer cells (26) . they also suggested that the combination of an extensive systemic and cns inflammatory response and lymphocyte depletion appears to be responsible for the pathogenesis of ev71associated pulmonary edema (26) . suggested treatments have varied. since those cases with pulmonary edema had lower cellular immunity, a rational treatment may involve regimens to enhance cellular immunity. in the case of poliomyelitis, ifn-␥ treatment of age-dependent poliomyelitis-susceptible mice protected them from paralytic disease (27) . recent studies have also shown that interferon-␥ synergizes with ifn-␣/␤ to inhibit the replication of both rna and dna viruses. scagnolari et al. investigated the effects of ifns on the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov), finding that although sars-cov is only moderately sensitive to ifn-␤ and weakly sensitive to ifn-␣ and ifn-␥, in combination they have a strong synergistic effect on virus replication (28) . these two studies imply treatments for severe ev71 infection, but further animal or clinical trial is mandatory to prove the efficacy of immunomodulators. in addition, use of an antiviral agent to decrease replication may also be beneficial (29) . animal studies are ongoing on this topic. in the liu et al. study, an early administration of recombinant mouse interferon-␣ protected the mice against ev71 infection and in vitro analysis of virus-induced death showed that human type i interferons exerted a direct protective effect on ev71, so interferons may play an important role in controlling ev71 infection and replication (30) . currently there is no vaccine for ev71. since the major cytokine response of mononuclear cells after ev71 challenge is th1 cytokines, induction of th1 cellular response may be critical to vaccine development for prevention of severe ev71 disease. therefore, an ideal vaccine should trigger adequate immunity in both cellular and humoral systems. in conclusion, we found that cellular immunity rather than humoral immunity may be related to the clinical outcome of ev71 infections. ev71 cases with decreased cellular immunity plus lower cellular ␥-interferon and other cytokine/ chemokine response may be prone to disseminated ev71 infection and subsequent pulmonary edema. an apparently new enterovirus isolated from patients with disease of the central nervous system new enterovirus type associated with epidemic of aseptic meningitis and/or hand, foot, and mouth disease epidemiological, clinical and pathomorphological characteristics of epidemic poliomyelitis-like disease caused by enterovirus 71 outbreaks of hand, foot, and mouth disease by enterovirus 71: high incidence of complication disorders of central nervous system virological diagnosis of enterovirus type 71 infections: experiences gained during an epidemic of acute cns diseases in hungary in 1978 enterovirus type 71 infections: a varied clinical pattern sometimes mimicking paralytic poliomyelitis enterovirus 71 infections and neurologic disease: united states, 1977-1991 role of enterovirus 71 in acute flaccid paralysis after the eradication of poliovirus in brazil deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: clinical and pathological characteristics of the disease fulminant neurogenic pulmonary edema with hand, foot and mouth disease deaths among children during an outbreak of hand, foot and mouth disease-taiwan sentinel surveillance of enterovirus 71 clinical features and risk factors of pulmonary edema after enterovirus 71-related hand, foot, and mouth disease an epidemic of enterovirus 71 infection in taiwan neurological complications in children with enterovirus 71 infection transmission and clinical features of enterovirus 71 infections in household contacts in taiwan risk factors of enterovirus 71 infection and associated hand-foot-mouth-disease/herpangina in children genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in taiwan enterovirus 71 from fatal and nonfatal cases of hand, foot and mouth disease epidemics in malaysia, japan and taiwan in 1997-1998 enterovirus responses of igm for enterovirus 71 infection altered cellular but not humoral reactions in children with complicated enterovirus 71 infections in taiwan pro-inflammatory cytokine reactions in enterovirus 71 infections of the central nervous system different pro-inflammatory reactions in fatal and non-fatal enterovirus 71 infections: implications for early recognition and therapy pathogenesis of enterovirus 71 brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema inhibition of virus-induced age-dependent poliomyelitis by interferon-gamma increased sensitivity of sars-coronavirus to a combination of human type i and type ii interferons design, synthesis, and structure-activity relationships of pyrazolo[3,4-d]pyrimidines: a novel class of potent enterovirus inhibitors type i interferons protect mice against enterovirus 71 infection key: cord-349285-zmp7sw5q authors: koh, kyung‐nam; im, ho joon; chung, nak‐gyun; cho, bin; kang, hyoung jin; shin, hee young; lyu, chuhl joo; yoo, keon hee; koo, hong hoe; kim, hee‐jin; baek, hee jo; kook, hoon; yoon, hoi soo; lim, young tak; kim, heung sik; ryu, kyung ha; seo, jong jin title: clinical features, genetics, and outcome of pediatric patients with hemophagocytic lymphohistiocytosis in korea: report of a nationwide survey from korea histiocytosis working party date: 2014-07-03 journal: eur j haematol doi: 10.1111/ejh.12399 sha: doc_id: 349285 cord_uid: zmp7sw5q background: we analyzed a nationwide registry of pediatric patients with hemophagocytic lymphohistiocytosis (hlh) in korea to assess the clinical and genetic features and treatment outcomes in pediatric hlh. methods: the korea histiocytosis working party retrospectively analyzed data on 251 pediatric patients diagnosed with hlh between 1996 and 2011. results: in the study cohort, 25 cases were categorized with familial hlh, 64 with presumed secondary hlh, and 162 with unspecified hlh. of 217 evaluable patients, 91 (42%) had concomitant epstein–barr virus infection. of 238 evaluable patients, central nervous system (cns) involvement, which was more frequent in the familial group, was evident in 81 cases (34%). genetic tests revealed a predominant unc13d mutation with a high incidence of two recurrent splicing mutations (c.118‐308c>t and c.754‐1g>c). the 5‐yr overall survival rate was 68% (38% in the familial group and 81% in the presumed secondary group). the 5‐yr overall survival rate among 32 patients who underwent allogeneic hematopoietic stem cell transplantation was 64%. in multivariate analysis, a younger age at diagnosis, severe transaminasemia, and a coagulation abnormality were independent prognostic factors for survival. responses during initial treatments were also significant indicators of outcome. conclusion: our study showed the unique predominance of a unc13d mutation and vulnerability to epstein–barr virus infection in korean children with hlh and emphasizes the prognostic significance of age, liver dysfunction, and treatment responses in this disease. a multicenter prospective trial that builds on the present results is warranted to identify subgroups of patients with a poor prognosis and identify optimal treatments. hemophagocytic lymphohistiocytosis (hlh) is a potentially fatal disease caused by dysregulated immune responses and overwhelming inflammation (1) . hlh can be categorized into two distinct forms: primary or familial hlh (fhl) and secondary hlh. flh can be further subcategorized into five subtypes, fhl1 to fhl5, according to causative genes. mutations in prf1, unc13d, stx11, and stxbp2 have been linked to fhl2, fhl3, fhl4, and fhl5, respectively (1) (2) (3) . secondary hlh is triggered by infections, malignancy, or rheumatic disease, without a known genetic predisposition. interestingly, ethnic differences in genetic susceptibility to fhl and vulnerability to epstein-barr virus-associated hlh have been reported (1, 4) . the establishment of diagnostic criteria and the use of immunochemotherapy with or without appropriate allogeneic hematopoietic stem cell transplantation (hsct) have significantly improved survival outcomes in patients with hlh (5) (6) (7) (8) . early recognition and the prompt initiation of treatment are mandatory for an improved prognosis. however, reliable outcome predictors for hlh and risk-defined treatment strategies remain to be adequately established. although our understanding of hlh has increased significantly, a large-scale, nationwide study of this heterogeneous disease is still lacking. our present study was conducted to analyze a korean national registry of pediatric hlh and thereby investigate the epidemiologic features and ethnic characteristics of korean pediatric patients with hlh, as well as the general clinical features and prognostic factors of hlh. the korea histiocytosis working party retrospectively collected data on children and adolescents with hlh from member hospitals. a total of 251 patients diagnosed with hlh between 1996 and 2011 were registered from 22 korean institutions. the case report form included information on demographic characteristics, clinical, laboratory, and radiological findings at presentation, genetic mutation analysis, type of treatment and responses to treatment, allogeneic hematopoietic stem cell transplantation, and survival outcomes. patients with malignancy-associated hlh were not registered in this study. classification hlh was diagnosed based on the diagnostic criteria proposed by the histiocyte society in 1991 and updated in 2004 (6, 9) . since 2006, the korea histiocytosis working party has recommended that patients who present with suspicious clinical and laboratory features for hlh should undergo genetic testing for this disorder at a central laboratory, irrespective of their age. in our present study, patients who were found to have a genetic abnormality or early-onset disease (≤2 yr) with family history without genetic evaluation were designated as having familial hlh. patients whose genetic testing for unc13d, prf1, stx11, and stxbp2 revealed no genetic abnormality and who had no family history of hlh were designated as having presumed secondary hlh. patients who could not be defined as having familial or secondary hlh due to unavailability of genetic test and data were designated as having unspecified hlh. cns involvement was defined as either of the presence of neurological symptoms or pleocytosis and/or proteinosis in cerebrospinal fluid (csf), or the demonstration of abnormalities on magnetic resonance imaging (mri) such as high signal intensity lesions, hemorrhage, atrophy, and leptomeningeal enhancement. a t-test was used to compare differences between parametric variables. a chi-square test or fisher's exact test was used to assess differences between groups. the kaplan-meier method was used to estimate survival probabilities, and a log-rank test was used to test the prognostic significance of various risk factors. multivariate cox analysis was performed to assess associations between clinical variables and prognosis. p < 0.05 were considered statistically significant. all statistical analyses were performed using spss version 21.0 (statistical package for the social sciences; ibm, armonk, ny, usa). demographic data and clinical features of 251 patients with hlh at their initial presentation are shown in table 1 . twenty-five patients (10%) were categorized with familial hlh based on family history and/or genetic mutation, 64 (25%) with presumed secondary hlh, and 162 (65%) with unspecified hlh. patients with clinical symptoms or signs suggesting ch ediak-higashi syndrome, griscelli syndrome, or hermansky-pudlak syndrome were not reported. in our total hlh cohort, 123 patients were male and 128 were female (male : female ratio = 0.96) with a median age at diagnosis of 3.2 yr (range, 0-18.7 yr) and 43 patients younger than 12 months (17%). the proportion of infants younger than 12 months was significantly higher in the familial hlh group than in the presumed secondary hlh group (p < 0.001). the median age at diagnosis of the familial group was significantly younger than that of the presumed secondary hlh group (0.4 yr vs. 3.5 yr; p < 0.001). the proportion of patients with familial hlh tended to decrease as the age of the patients increased with 10% of the patients over 5 yr of age (3/29) having genetic mutations consistent with fhl. notably, 59% of the infant patients (16/27) were shown to have a genetic background associated with hlh. in particular, young infants younger than 6 months of age were highly likely to have a genetic predisposition to hlh (14/20, 70%) ( fig. 1) . at initial presentation, nearly all of our patient subjects had a fever, and most had hepatosplenomegaly. splenomegaly was more prevalent in the familial hlh group than in the presumed secondary hlh group (p = 0.018). of 233 evaluable patients, 41 (17.6%) had neurological symptoms. the proportion of patients with neurological symptoms was not different between the familial and presumed secondary hlh groups. the characteristics of laboratory and histopathological findings are shown in table 1 . most patients had bicytopenia with predominant anemia and thrombocytopenia, with significantly higher incidence of anemia (p = 0.016), thrombocytopenia (p = 0.01), and bicytopenia (p = 0.044) in the familial hlh group than in the presumed secondary hlh group. elevated lactate dehydrogenase (ldh) levels (>500 iu/l) were more common (p = 0.043) and transaminasemia marginally more common (p = 0.087) in the presumed secondary hlh group. the differences of other laboratory findings were statistically not significant between the two groups. notably, many patients had liver dysfunction, with elevated levels of aspartate transaminase (ast) and/or alanine transaminase (alt) (>200 iu/l), hyperbilirubinemia (>3.0 mg/dl), and hypoalbuminemia (<2.5 g/dl), and more than a quarter of the patients had significant coagulation abnormalities (aptt >60 s). also, most patients had elevated levels of serum ldh. the cns involvement in our hlh cohort is summarized in table 1 . of 238 evaluable patients, 81 (34%) had cns involvement. csf and mri abnormalities were observed in more than 30% of the evaluable patients. patients with familial hlh had more frequent cns involvement with more frequent mri abnormalities (p = 0.007) and csf proteinosis (p = 0.007) than those with presumed secondary hlh, whereas the proportion of patients with clinical neurological symptoms was not different between the two groups. the rate of concomitant ebv infection was significantly lower in patients with cns involvement than those without (30% vs. 47%; p = 0.021). of 217 evaluable patients for ebv, 91 (42%) had concomitant ebv infection. ebv infection tended to be more common in the presumed secondary hlh group than in the familial hlh group, but without statistical significance (42% vs. 20%; p = 0.116), and was significantly associated with concomitant hyperbilirubinemia (46% vs. 29%; p = 0.014). genetic testing for unc13d, prf1, and stx11 was carried out for 85 patients and testing for stxpb2 for 23 patients, with 19 (22%) showing genetic mutations. the most common genetic mutation was in unc13d (unc13d mutations in 15 and prf1 mutations in 4). no patient had mutation in stx11 and stxbp2. among 15 patients with unc13d mutations, 13 had biallelic mutations and 2 had monoallelic mutations. of 28 mutations in unc13d, 24 were splicing mutations, 3 frameshift mutations, and 1 nonsense mutation. notably, 2 recurrent splicing mutations-c.118-308c>t (ivs1-308c>t) (7/15) and c.754-1g>c (ivs9-1g>c) (8/15)-predominated, with either mutation found in 80% of the patients with unc13d mutations (12/15). detailed data on mutations were separately reported (10). all two patients, whose detailed data on prf1 mutations were available, had same frameshift mutation of c.1090_1091delct (p.leu364glufs*93) (1 with biallelic and 1 with monoallelic mutation). the majority of our study patients (167 of 222 evaluable patients) were treated with hlh-94-or hla-2004-based immunochemotherapy (5, 6) , whereas 9% of these cases (21/ 222) were treated with other treatments such as methylprednisolone, intravenous immunoglobulin, or antithymocyte globulin; 15% of the patients (34/224) had no treatment due to spontaneous resolution or rapid progression of the disease ( table 2) . data for analysis of treatment response were available in 175 among 188 patients with hlh who received treatment. eighty patients (46%) achieved complete resolution, and 43 (25%) showed improvement. in contrast, 27 (15%) of these patients had reactivated or persistent disease with minimal or no improvement after 8 wk of initial treatment, and 25 (14%) patients died of refractory disease ( table 2 ). of the total cohort of 251 enrolled hlh patients, 45 (18%) died within 8 wk of diagnosis, and 32 (13%) beyond 8 wk, leading to a 5-yr overall survival (os) rate of 68% ( fig. 2a) . the 5-yr os rate was significantly poorer in the familial hlh group than in the presumed secondary hlh group and unspecified hlh group (38% vs. 81% vs. 68%; p = 0.001; fig. 2b ). the results of univariate analysis on the putative prognostic factors for os according to the type of disease are shown in figure 1 distribution of familial and presumed secondary hlh by age for 89 classifiable patients. table 3 . overall, a younger age, cns involvement, severe transaminasemia (ast or alt >800 iu/l), severe cholestasis (total bilirubin >6 mg/dl), and coagulation abnormalities were significant indicators of a poor prognosis. in the familial hlh group, only a coagulation abnormality was a marginally significant prognostic factor, and cns involvement and age were not found to be significantly associated with survival outcome. in the presumed secondary hlh group, a younger age, severe transaminasemia, a coagulation abnormality, and cns involvement were found to be predictors of poor survival outcomes. overall, disease status and treatment response represented by the ferritin level after 8 wk of treatment were found to be significant indicators of outcome. disease status after 8 wk was an indicator of poor outcome in both the familial and presumed secondary hlh groups, whereas the ferritin levels after 8 wk were significant indicators in the familial group, not in the presumed secondary group. overall, multivariate cox regression analysis revealed that a younger age at diagnosis (hazard ratio [hr] = 2.40; p = 0.002; 95% confidence interval [ci], 1.37-4.22), severe transaminasemia (hr = 1.82; p = 0.028; 95% ci, 1.07-3.11), and a coagulation abnormality (hr = 2.52; p < 0.001; 95% ci, 1.51-4.20) are independent prognostic factors for survival. thirty-two of the patients with hlh in our study cohort underwent allogeneic hematopoietic stem cell transplantation (hsct). the reasons for using hsct were familial hlh in 10 cases, active disease after the initial treatment in 11, and reactivated disease in 11. whereas nine patients received transplants from matched related donors, 22 patients received transplants from unrelated donors. graft sources included the bone marrow in 15 patients, granulocyte colony-stimulating factor-mobilized peripheral blood in 9, and cord blood in 6. busulfan-(n = 26) or total-body irradiation-based (n = 1) myeloablative conditioning (mac) regimens were used in 27 and fludarabine-based reduced-intensity conditioning (ric) regimens in 5. twenty-four patients had non-active disease at the time of hsct, while 8 had active disease. three patients developed graft failure. all three patients, who developed graft failure, had received cord blood graft after mac in 2 and ric in 1. no patient received donor lymphocyte infusion or a stem cell boost. the 5-yr os rate after allogeneic hsct was 64%. the type of donor, conditioning regimen, and the status prior to hsct did not influence the outcome, while cord blood graft tended to be associated with a poorer outcome, but without statistical significance (p = 0.115) (fig. 3) . seven patients died of transplant-related causes (graft failure in three, infection in two, heart failure in one, hemorrhage in one, and graft-versushost disease in one), and two patients died of refractory or reactivated disease. our current study represents the first nationwide assessment of hlh in korea and describes the epidemiological and clinical characteristics of korean patients with this disorder. figure 2 the 5-yr overall survival rate of 251 patients with hlh in the overall group (a) and according to the type of disease (b). our results emphasize the prognostic significance of cns involvement and liver dysfunction (including severe cholestasis, severe transaminasemia, and coagulation abnormalities) and the predictive role of the treatment response, which was represented by the decline in the ferritin level. moreover, our present findings confirmed the predominance of unc13d mutations in korean patients with fhl and the high incidence of ebv-associated hlh in korea. fhl is usually diagnosed during infancy or early childhood. our current analyses revealed a median age at diagnosis of fhl of 0.4 yr, with patients younger than 1 yr old highly likely to have a genetic cause associated with hlh. thus, genetic evaluations should be mandatory for all patients who present with hlh during infancy or early childhood. however, our study findings also showed that older children might have the genetic mutation consistent with fhl. indeed, 10% of patients in our hlh cohort older than 5 yr of age were shown to have a genetic background. previous studies have reported that adolescents and even older adults may have a genetic predisposition to hlh (11) (12) (13) (14) . thus, all cases of suspicious symptoms, regardless of age, should be evaluated for familial hlh, even when triggering factors are found. clinical and laboratory features at presentation in our cohort are comparable to previous reports (15) (16) (17) (18) . notably, 97% of patients were found to have hemophagocytosis, which is higher than previous studies (17, 18) . this is likely to be due to limited availability of nk-cell activity test and measurements of scd25, which are elements of diagnostic criteria for hlh. patients, who were found to have hemophagocytosis, thus meeting the diagnostic criteria for hlh without those two tests, were more likely to be included in the present study. therefore, high percentage of hemophagocytosis in our study does not emphasize the presence of hemophagocytosis as a necessary requisite for diagnosis of hlh. cns involvement has been reported to be a critical factor for predicting the outcomes of patients with hlh (19) (20) (21) . our current data indicate that cns involvement is more common in the familial group, thus suggesting that genetic susceptibility plays an important role in the infiltration of activated lymphocytes and macrophages into the cns. cns involvement was found to be a significant prognostic factor in our presumed secondary hlh group but was not associated with a poor outcome in the familial group. thus, prognostic significance of cns involvement according to the type of hlh should be evaluated in a larger study. in our present study cohort, 42% of patients with hlh had an ebv infection. this finding is a further example of the high incidence of secondary hlh associated with ebv infection in an asian population (4, 22) . this unique vulnerability to ebv infection in asian patients with hlh suggests that different genetic backgrounds can contribute to the development of the disease, even in cases of secondary hlh. in addition, hlh patients with documented ebv infection had a lower rate of cns involvement and a higher rate of cholestasis, which suggests the tropism of organ involvement in ebv-associated hlh. a future study should include further genetic testing for sh2d1a, xiap/birc4, and itk, which have been implicated in ebv-associated lymphoproliferative disorders, to differentiate hlh from these disorders presenting as hemophagocytic syndrome (23) . our present study findings confirmed that unc13d is the gene most commonly mutated in korean patients with fhl, which was also suggested by a previous pilot study (24) . ethnic differences have been reported in relation to the genetic predisposition to each subtype of fhl (1, 25, 26) . interestingly, the most commonly mutated gene for fhl in the neighboring countries of korea, japan and china is prf1 (26, 27) . two recurrent splicing mutations-c.118-308c>t and c.754-1g>c-were found in nearly three-quarters of the patients in our current study cohort, which suggest the presence of founder effects of these mutations in the development of hlh in korea (10) . in contrast, c.1596 + 1g>c, the most common mutation in japanese fhl3 patients, and c.2346_2349delggag and c.753 + 1g>t, the most common mutations in european fhl3 patients, were not found in our series, which suggest a geographic or ethnic specificity of the mutations causing each fhl subtype (28, 29) . our current analyses showed a 5-yr os rate of 68%, which is comparable with previous studies (4, 5) . patients with familial hlh in our series showed a significantly poorer outcome. most deaths occurred within the first 2 months of the disease in our presumed secondary hlh group, whereas many patients with fhl succumbed to the refractory or reactivated disease after the early period of the disease. hsct was able to rescue a substantial portion of the patients with fhl or refractory disease. notably, the use of a cord blood graft was associated with a poor outcome. thus, the optimal conditioning regimen according to a graft source should be evaluated in a future study. our present study incorporates the inevitable limitations of being long-term and retrospective in design, such as missing data, inconsistent evaluations, and the use of heterogeneous treatments. limitations of this retrospective study include the incompleteness of testing for stxbp2 mutation and the absence of testing for mutations in sh2d1a, xiap/birc4, and itk, which was why we adopted the term 'presumed' secondary hlh for the patients without mutations in unc13d, prf1, and stx11. this leaves the possibility of the presence of cryptic genetic mutation in the patients designated as having presumed secondary hlh, thus requiring cautious interpretation of our results. another limitation of the present study is that a larger portion of patients, who were enrolled during early period of the study, has not been tested for genetic mutation and was thus designated as having unspecified hlh. there are no reliable clinical criteria to distinguish familial and secondary hlh. therefore, we could not further define the patients with hlh without genetic evaluation or family history, even though they were reported to have possible triggering causes. albeit these limitations, our observations of discrete clinical features between familial and presumed secondary groups and prognostic factors evaluated in our overall cohort could provide clinical implications for a future research agenda. in conclusion, our current analyses have revealed a unique genetic susceptibility to fhl3 and vulnerability to ebv infection in korean children with hlh. age, liver dysfunction, and treatment response were found to be significant indicators of a poor prognosis. serial monitoring of changes in the ferritin levels during the initial treatment of hlh may enable the disease status to be followed and allow for the prediction of treatment outcomes, thus helping to identify the subgroup of patients who need more intensive treatment. a multicenter prospective trial is warranted to identify the subgroups of patients with hlh with poor prognoses and find optimal treatment for these cases. in addition, the optimal intensity and duration of treatment for patients with presumed secondary hlh need to be investigated. hemophagocytic lymphohistiocytosis: advances in pathophysiology, diagnosis, and treatment advances in understanding the pathogenesis of hlh diagnostic evaluation of patients with suspected haemophagocytic lymphohistiocytosis nationwide survey of hemophagocytic lymphohistiocytosis in japan treatment of hemophagocytic lymphohistiocytosis with hlh-94 immunochemotherapy and bone marrow transplantation hlh-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis reduced-intensity conditioning significantly improves survival of patients with hemophagocytic lymphohistiocytosis undergoing allogeneic hematopoietic cell transplantation fludarabine-based reduced-intensity conditioning regimen for hematopoietic stem cell transplantation in primary hemophagocytic lymphohistiocytosis diagnostic guidelines for hemophagocytic lymphohistiocytosis founder effects in two predominant intronic mutations of unc13d, c.118-308c>t and c.754-1g>c underlie the unusual predominance of type 3 familial hemophagocytic lymphohistiocytosis (fhl3) in korea late onset of familial hemophagocytic lympho-histiocytosis genotype-phenotype study of familial haemophagocytic lymphohistiocytosis type 3 familial hemophagocytic lymphohistiocytosis type 3 diagnosed at school age: a case report hemophagocytic syndrome in elderly patients with underlying autoimmune diseases hemophagocytic lymphohistiocytosis: an update on diagnosis and pathogenesis hemophagocytic lymphohistiocytosis in infancy and childhood hemophagocytic lymphohistiocytosis in texas: observations on ethnicity and race experience with hemophagocytic lymphohistiocytosis/macrophage activation syndrome at a single institution frequency and spectrum of central nervous system involvement in 193 children with haemophagocytic lymphohistiocytosis central nervous system (cns) involvement is a critical prognostic factor for hemophagocytic lymphohistiocytosis hematopoietic stem cell transplantation in hemophagocytic lymphohistiocytosis: a single-center report of 48 patients infection-and malignancy-associated hemophagocytic syndromes. secondary hemophagocytic lymphohistiocytosis x-linked lymphoproliferative syndromes and related autosomal recessive disorders unc13d is the predominant causative gene with recurrent splicing mutations in korean patients with familial hemophagocytic lymphohistiocytosis mutation spectrum in children with primary hemophagocytic lymphohistiocytosis: molecular and functional analyses of prf1, unc13d, stx11, and rab27a characteristic perforin gene mutations of haemophagocytic lymphohistiocytosis patients in japan screening the prf1, unc13d, stx11, sh2d1a, xiap, and itk gene mutations in chinese children with epstein-barr virus-associated hemophagocytic lymphohistiocytosis genotype-phenotype study of familial haemophagocytic lymphohistiocytosis due to perforin mutations correlation between phenotypic heterogeneity and gene mutational characteristics in familial hemophagocytic lymphohistiocytosis (fhl) this study was supported by a grant (2013e6301400) from korea centers for disease control and prevention. none to declare. key: cord-017499-51yy7y9n authors: freye, enno; levy, joseph victor title: mechanism of action of opioids and clinical effects date: 2008 journal: opioids in medicine doi: 10.1007/978-1-4020-5947-6_2 sha: doc_id: 17499 cord_uid: 51yy7y9n nan the opium poppy probably reached china about the seventh century a.d. through the efforts of arab traders who advocated its use for medicinal purposes. in chinese literature, however, there are earlier references to its use. the noted chinese surgeon hua to of the three kingdoms (220-264 a.d.) used opium preparations and cannabis indica for his patients to swallow before undergoing major surgery. the opium poppy, papaver somniferum, is an annual plant, i.e., the plant matures one time, and does not regenerate itself. new seed must be planted each season. from a small seed, it grows, flowers, and bears fruit (a pod) only once. the entire growth cycle for most varieties of this plant takes about 120 days. the tiny seeds (like the seeds on a poppy seed roll) germinate quickly in warm air and sufficient soil moisture. in less than 6 weeks, the young plant emerges from the soil, grows a set of four leaves, and resembles a small cabbage in appearance. the lobed, dentate (jagged-edged) leaves are bluish-green with a dull gray or blue tint. the major legal opium production areas in the world today are in governmentregulated opium farms in india, turkey, and tasmania (australia). the major illegal growing areas are in southwest asia (afghanistan, pakistan, and iran) and in the highlands of mainland southeast asia (burma, laos, vietnam, and thailand)popularly known as the golden triangle ( figure ii-3) . opium poppy is also grown in colombia, mexico, and lebanon. opium poppies containing small amounts of opium alkaloids were, at one time, widely grown as an ornamental plant and for seeds in the united states. the opium poppy control act of 1942 declared the possession of this plant illegal. from the cut capsule latex is exuded, which is collected and further processed in order to gain the different ingredients ( figure ii-2) . within the secreted latex collectors will find the major constituents of opium poppy, which are as follows: 1. morphine (10%-17%), the most important alkaloid, which was discovered by the pharmacist sertürner in a small town of einbeck, located in lower saxonia in germany in 1803. he decided to name the extract from the opium poppy morphine ( figure 3. thebaine (0.5%-2%) a precursor of many of semi-synthetic opioid agonists (i.e. etorphine, oxymorphone) and antagonists (naloxone, naltrexone, diprenorphine, cyprenorphine), mixed agonist/antagonists (nalbuphine) as well as the partial agonist buprenorphine ( figure c ). 4. benzylisoquinolines are a group of agents, which do not interact with the opioid receptor. the most important one is papaverine (0.5%-1%) a phosphodiestrase inhibitor, which relaxes the smooth muscle, and noscapine (2%-9%), which is used as a cough suppressant ( figure d) . raw or cooked opium contains more than 35 different alkaloids, including morphine, codeine, and thebaine ( figure ii-4) . in mainland southeast asia, the morphine alkaloid alone accounts for approximately 10% of the total weight of opium. heroin manufacturers must first extract the morphine from the opium, before converting the morphine to heroin. the extraction is a simple process, requiring only a few chemicals and a supply of water. morphine sometimes is extracted from opium in small clandestine laboratories, which are typically set up near the opium poppy fields. since the morphine base is about one-tenth the weight and volume of raw opium, it is desirable to reduce the opium to morphine before transporting the product from the field to a heroin laboratory. the following is a step-by-step description of morphine extraction in a typical mainland southeast asian laboratory. an empty 55-gallon oil drum is placed on bricks about a foot above the ground and a fire is built under the drum. thirty gallons of water are added to the drum and brought to a boil. ten to fifteen kilograms of raw opium are added to the boiling water. with stirring, the raw opium eventually dissolves in the boiling water, while soil, leaves, twigs, and other non-soluble materials float in the solution. most of these materials are scooped out of the clear, dark brown liquid opium solution. slaked lime (calcium hydroxide) or, more often, a readily available chemical fertilizer with a high content of lime, is added to the solution. lime will convert the water-insoluble morphine alkaloid into water-soluble calcium morphenate. (other opium alkaloids do not react with lime to form water-soluble calcium salts, as does morphine.) codeine is an opium alkaloid that is slightly water-soluble and some codeine will be carried over with the calcium morphenate in the liquid. otherwise, for the most part, the other alkaloids will become a part of the sludge. as the solution cools, the morphine solution is scooped from the drum and poured through a filter. cloth rice sacks are often used as filters and can then be squeezed in a press to remove most of the solution from the wet sacks. liquid saponated cresol (lysol) is commonly added to the solution to facilitate filtering. the morphine-rich solution is then poured into large cooking pots and reheated but, this time, not boiled. ammonium chloride (a powder) is added to the heated calcium morphenate solution to adjust the alkalinity to a ph of 8 to 9, and the solution is then allowed to cool. within 1 or 2 h, morphine base precipitates (crashes) out of the solution and settles to the bottom of the cooking pot. the solution is then poured off through cloth filters. any solid morphine base chunks in the solution will remain on the cloth. the morphine base is removed from both the cooking pot and from the filter cloths, wrapped and squeezed in cloth, and then dried in the sun. when dry, the crude morphine base is a coffee-colored coarse powder. this form of morphine is commonly known by the chinese term pi-tzu in mainland southeast asia. if morphine base is to be stored or transported to another location, it may be pressed into blocks. crude morphine base is generally 50%-70% morphine, and is an intermediate product in the heroin process. addicts do generally not use this morphine base. this crude morphine base may be further purified (and changed to morphine hydrochloride) by dissolution in hot water and hydrochloric acid, then adding activated charcoal, reheating, and filtering. the solution is filtered several times before being allowed to cool. as the solution cools, morphine hydrochloride precipitates out of the solution and settles to the bottom. the precipitate is trapped (or captured) by filtration. if the morphine hydrochloride is to be stored or transported to another location, it may be pressed into bricks. morphine hydrochloride (often tainted with codeine hydrochloride) is usually pressed into brick-sized blocks in a press and wrapped in paper or cloth. the most common block size is 2 in. by 4 in. by 5 in., and weighs about 3 lb (1.3 kg). it takes a full day to extract morphine from opium. as described in the preceding paragraphs, the chemicals used to isolate morphine from opium (known as extraction) include calcium hydroxide (slaked lime) and ammonium chloride. the precursor chemical normally used in the conversion of morphine to heroin (known as acetylation) is acetic anhydride. chemical reagents used in the conversion process include sodium carbonate and activated charcoal. chemical solvents needed are chloroform, ethyl alcohol (ethanol), and ethyl ether. other chemicals may be substituted for these preferred chemicals, but most or all of these preferred chemicals are readily available from smugglers and suppliers. laboratory equipment includes large chinese cooking woks, measuring cups, funnels, filter paper, litmus paper, and enamel (or stainless steel) pots. only the most sophisticated heroin laboratories use glass flasks, propane gas ovens, vacuum pumps, autoclaves, electric blenders, venting hoods, centrifuges, reflux condensers, electric drying ovens, and elaborate exhaust systems. it is common to find portable, gasoline-powered generators at clandestine heroin conversion laboratories. generators are used to power various electrical devices. heroin synthesis from morphine (either morphine base or morphine hydrochloride) is a two-step process that requires between 4 and 6 h to complete . heroin base is the intermediate product. typically, morphine hydrochloride bricks are pulverized and the dried powder is then placed in an enamel pot. acetic anhydride is added, which then reacts with the morphine to form heroin acetate. (this acetylation process will work either with morphine hydrochloride or morphine base.) the pot lid is tied or clamped on, using a damp towel for a gasket. the pot is carefully heated for about 2 h, below boiling, at a constant temperature of 85 c (185 f). it is never allowed to boil or to become so hot as to vent fumes into the room. tilting and rotation agitate the mixture until all of the morphine has dissolved. when cooking is completed, the pot is cooled and opened. during this step, morphine and the anhydride become chemically bonded, creating an impure form of diacetylmorphine (heroin). water is added to the thick, soupy mixture and the mixture is stirred as the heroin dissolves in the solution. sodium carbonate (a crystalline powder) is dissolved in hot water and then added slowly to the heroin solution until effervescence stops. this precipitates heroin base, which is then filtered and dried by heating in a steam bath. for each kilogram of morphine, 685 g-937 g of crude heroin base is formed, depending on the quality of morphine. the tan-colored heroin base (about 70% pure heroin) may be dried, packed, and transported to a heroin-refining laboratory, or it may be purified further before conversion to heroin hydrochloride (a water-soluble salt form of heroin) at the same site. mainland southeast asian heroin base is an intermediate product that can be further converted to either smoking heroin (heroin no. 3) or injectable heroin (heroin no. 4). to make heroin no. 3, the crude base is mixed with hydrochloric acid, resulting in heroin hydrochloride (hcl). adulterants, including caffeine, are added after this conversion. for each kilogram of crude heroin base, about 1 kg of caffeine is used. various flavorings such as quinine hydrochloride or strychnine hydrochloride are sometimes added to heroin no. 3. next, the wet paste mix is stirred to dryness over a steam bath. the resulting dry heroin no. 3 will be in the form of coarse lumps. the lumps are crushed and passed through a mesh sieve, and the grains (pieces) are then packaged for sale. the entire process takes about 8 h and requires only minimal skill. while extra attention to stirring is required to assure dryness, one person can prepare 1 kg of heroin no. 3 during this time. the reaction of morphine with acetic anhydride produces heroin acetate. to the heroin acetate mixture in the pot, water is added and mixed by stirring. a small amount of chloroform is added. the mixture is stirred and then allowed to stand for 20 min. doing so dissolves highly colored impurities and a red, greasy liquid is formed at the bottom of the container. the water layer is carefully poured off and saved in a clean pot, leaving the red grease in the pot. in a clean pot, activated charcoal is stirred into the aqueous solution and is filtered to remove solid impurities. the decolorizing effects of the charcoal, combined with the chloroform treatment, will leave a light yellow solution. the use of charcoal is repeated one or more times, until the solution is colorless. sodium carbonate (a crystalline powder) is dissolved in hot water and then added slowly to the heroin solution until effervescence stops. this precipitates the heroin base, which is then filtered and dried by heating on a steam bath. the heroin base is heated until dried. the powder should be very white at this stage. if not white, the base is redissolved in diluted acid, treated repeatedly with activated charcoal, re-precipitated, and dried. the ultimate purity and color of the resulting heroin hcl will depend largely on the quality of the heroin base. the heroin base is then dissolved in ethyl ether. conversion to the hydrochloride salt is achieved by adding hydrochloric acid in ethanol to the heroin mixture. the heroin then precipitates. the process of extracting morphine from opium involves dissolving opium in boiling water, adding lime (calcium oxide), or slaked lime (calcium hydroxide), or limestone (calcium carbonate) to precipitate non-morphine alkaloids, and then pouring off the morphine in solution. ammonium chloride is then added to the solution to precipitate morphine from the solution. the chemicals used to process opium to morphine have a number of legitimate purposes and are widely available on the open market. an empty oil drum, some cooking pots, and filter cloths or filter paper are needed. in the united states, opium preparations became widely available in the nineteenth century and morphine was used extensively as a painkiller for wounded soldiers during the civil war. the inevitable result was opium addiction, contemporarily called the army disease or soldier's disease. these opium and morphine abuse problems prompted a scientific search for potent, but nonaddictive, painkillers. in the 1870s, chemists developed an opium-based and supposedly nonaddictive substitute for morphine. the bayer pharmaceutical company of germany was the first to produce the new drug in large quantities under the brand name heroin. this product was obtained by acetylation of morphine. soon thereafter studies showed heroin to have narcotic and addictive properties far exceeding those of morphine. although heroin has been used in the united kingdom in the treatment of the terminally ill, its medical value is a subject of intense controversy. among the commonly known classes of opioids/opiates being used in practice are morphine, codeine, heroin, and the antagonist naloxone ( figure ii-6 ). morphine by itself is still made from opium and although there is a major first-pass effect (i.e. degradation by liver enzymes), oral administration is still possible, but requires substantial dosage increase. codeine, which is also taken orally, has a strong ability to inhibit coughing, but it induces less analgesia. among the phenylpiperidines a number of synthetic compounds have entered the market. the most known is meperidine/pethidine (demerol®), which is very similar to morphine, but is more efficacious when given orally for the control of pain. another derivative is loperamide (imodium®), an agent being used as a common antidiarrheal, which does not enter the brain, as it is incapable of crossing the blood-brain barrier. hence it is not abused and therefore is sold as a doc (drug over the counter). contrary, fentanyl (sublimaze®) is an opioid, which is at least 200 times as potent as morphine. this agent is used with nitrous oxide or droperidol (a neuroleptic) in intravenous anesthesia (neuroleptanesthesia), but it is also a used in a transdermal patch for the control of chronic pain. another known opioid is methadone, which has a good oral efficacy, a much longer half-life than morphine (8-12 h) , and in regard to its effect much like morphine. it is used for treatment of heroin addiction and for the control of chronic pain. a methadone congener, which is being used solely in the methadone substitution programs is laam ( -levoacetylmethadol), only needs to be taken once every 72 h. the opioid propoxyphene (darvon®) has figure ii-6. molecular structure of different opioid ligands with agonistic or antagonistic properties the lowest analgesic potency (0.02 times morphine). it is almost always given together with aspirin for the control of mild to moderate pain. it is very popular clinically due to misplaced concerns about the abuse potential of codeine. it is interesting to note that all commonly used opioids have a similar structure in regard to their terminal morphine ring and the distance between the ring and the n-substitution. such common traits suggests that opioids must have a common structure in order to interact with a specific receptor site ( figure ii-7) . thus, central analgesics mediate their action by means of an interaction with specific opioid receptor sites located within specific areas of the central nervous system, which are engaged in the transmission of nociceptive afferences and the identification of pain. there, opioids act as agonists at highly definite receptor sites, and there is general agreement on the existence of at least three types of opioid receptor sites (table ii-1). 1. the morphine mu receptor ( ) at which the prototype morphine binds, 2. the kappa receptor ( ) at which the prototype agonist is ketocyclazocine, and 3. the delta receptor ( ) at which the prototype endogenous opioid ligand enkephalin binds. opioid receptors are distributed widely in brain and found in spinal cord and peripheral sensory and autonomic nerves. there are the three well-characterized members of the opioid receptor family, designated by the greek symbols , and . the more recently discovered orl1 receptor is placed with this family due to its high degree of structural homology. these receptors were renamed op1, op2, op3 and op4, respectively, by an international union of pharmacology (iuphar) nomenclature committee in 1996 [1] . this nomenclature has proved unpopular. the nomenclature (x-opioid peptide receptor) has been proposed giving , mu or mop; , delta or dop; , kappa or kop and orl1 or nop receptors. in order to keep matters straightforward the original nomenclature is used in the following chapters. the products of endogenous opioid peptide genes activate opioid receptors physiologically: proenkephalin (giving methionine-and leucine-enkephalin; metenk and leu-enk, respectively; figure ii-8), prodynorphin (dynorphins a and b and -neo-endorphin) pro-opiomelanocortin ( -endorphin) and pronociceptin (nociceptin, also known as orphanin fq). met-enk and leu-enk have highest affinity for -receptors, less affinity for , and very low affinity for -receptors; the dynorphins have preferential affinity for -receptors, but bind to the and types with high affinity; -endorphin binds with high affinity to and receptors, but has little affinity for receptors. all the peptides are full agonists at their cognate receptors. endomorphin-1 and -2, derived from an unknown precursor, are endogenous peptides with high selectivity for -receptors. these peptides are unusual in that they are partial agonists. none of the proenkephalin, prodynorphin or pro-opiomelanocortin peptide products or the endomorphins displays affinity for the orl1 receptor. similarly, the orl1 receptor agonist nociceptin has no appreciable affinity for , or receptors. the four receptor types have been cloned and shown to be 7-transmembrane receptors activating g proteins of the pertussis-toxin insensitive g i/o family, but including g z. evidence for subtypes of , and opioid receptors exists, but the molecular basis for the observed functional and pharmacological differences is unclear. putative 1 and 2 receptors are differentiated by several agonist and antagonist ligands. however, there is only one receptor gene, the protein product of which has properties of the putative 2 receptor. the distinction between the proposed 2 and 2 receptors is based largely on the apparent preferential blockade of the 1 type by the antagonist, naloxonazine [2] . there is only one cloned receptor gene, corresponding to the putative 1 receptor, but several forms of the -receptor mrna arising from alternative splicing have been reported. the receptors these encode differ at the end of the c-terminal tail and show subtle differences in the binding profile of opioid ligands; a role for the variants is not known. the cloned -receptor, with high affinity for u69593 is the 1 subtype. the proposed 2 and 3 subtypes are poorly defined in both molecular and pharmacological terms (table ii-2) . a recent explanation for subtypes has evolved with the identification of opioid receptor heterodimers or hetero-oligomers that appear to have properties different from the monomeric receptors. an interesting addition to ligands that bind to the 1 receptor is the hallucinogen salvinorin-a. this is a highly efficacious and potent agonist, but is most unusual in that it has no nitrogen atom. endogenous opioid systems have a functional role in modulating pain perception; opioid agonists are therefore potent analgesics. opioid receptors are also present in hypothalamus (figure ii-9), where they influence temperature regulation and control of hormonal secretion. in the forebrain, endogenous opioids are involved in behavioral reinforcement and appear to play a role in anxiety and in the expression of emotions. in addition, opioids influence gastrointestinal and autonomic nervous system function. originally, a fifth binding site, the sigma receptor, was included in this group. however, actions mediated through this receptor are not reversed by naloxone so it is not a true opioid receptor. the -receptors have been further sub-classified into two distinct subtypes (1 and 2), as have the -receptors. kappa receptors have been divided into 1, 2, and 3 sub-types. recently, several of these receptors have been cloned successfully. in animal models, some laboratories have cloned up to 10 -receptor subtypes [4] . however, the functional significance of these "spliced variants" remains unclear at present. originally suggested by martin and coworkers [5] , all three opioid receptor types mediate different opiate effects as they figure ii-9. difference in topographic density of the three opioid receptor sites within the central nervous system. adapted from [3] normally serve endogenous opiates (the endorphins, dynorphins, and enkephalins; table ii-1): 1. activation of the mu receptors ( ) results in analgesia, euphoria, respiratory depression, nausea, gi slowdown, and miosis. receptors of this type are mostly located in periaquaductal gray (pag), spinal trigeminal nucleus, caudate and geniculate nuclei, thalamus, and spinal cord. 2. binding at the kappa receptors ( ) induces modest analgesia, dysphoria, feelings of depersonalization and disorientation, miosis, and mild respiratory depression. these receptors are mainly found in basal ganglia, nucleus accumbens, ventral tegmentum, cortex, hypothalamus, periaqueductal grey, the spinal cord, and in the periphery. 3. occupation of the delta receptors ( ) results in anxiolysis and central pain relief, although its overall significance is not all that well understood. they are mainly found in the nucleus accumbens and the limbic system (table ii-2) . molecular biology techniques have enabled the primary amino acid sequence of the human -, -, and -opioid receptors to be determined. the pharmacological and functional properties of the cloned receptors, the development of "knockout" animals, which are deficient in a receptor or part of a receptor, and the manipulation and substitution of various amino acids in critical domains of the various opioid receptors have provided new insights in opioid action. in this regard, the three opioid receptor genes, encoding mu (mor), delta (dor), and kappa (kor) have been cloned. the binding affinities of a range of opioids to the -, -, and -opioid receptors and also to the cloned orphinan receptor have been examined in animals. the animal data indicate that while the commonly prescribed opioids (agonists and antagonists) bind preferentially to the -receptor, they also interact with all three receptor types. morphine and normorphine (a minor metabolite of morphine) show the greatest relative preference for the -receptor. methadone (which also has some nmda-receptor blocking activity) shows significant binding to -receptors, while buprenorphine, and to a lesser extent naloxone, avidly binds to all three receptor types. there is evidence (albeit inconsistent) that the d-enantiomer of methadone blocks the nmda receptor [6] . the binding affinity of buprenorphine to the receptor is much greater than that of naloxone, which explains why the latter only partially reverses buprenorphine overdose. animal data also indicate that codeine and diamorphine have very poor binding to opioid receptors, which reinforces the possibility that both are prodrugs where the pharmacologically active species are morphine [7] and 6-monoacetyl morphine, respectively [8] . oxycodone may also act through an active metabolite, though there are some data, which suggest that this is not the case [9] . pethidine is considered to be a potent -receptor agonist, but it does bind weakly to all three opioid receptors (table ii-1) . ketobemidone has a lower affinity for the -receptor than does morphine, but it shows greater discrimination for this receptor compared to -receptors. the binding of both of these opioids to the -receptor is similar [10] . this difference in opioid action is also mirrored in the difference in affinity of various narcotic ligands interacting with the three relevant opioid receptor sites (table ii-3) . it should be noted that some of those ligands, either pure antagonists, mixed agonist/antagonists or partial agonists, are characterized by displacement potency at a specific receptor site. from the above binding and displacement values it can be seen, that opioid practically bind to all three receptor sites, however with different affinity. the preference in binding to one receptor site manifests itself in the visible clinical effect, which may either be of agonistic or of antagonistic nature. the binding of morphine, methadone, buprenorphine, and naloxone to the cloned human -receptor shows excellent congruence with the animal data [16] . fentanyl shows a similar binding affinity, while codeine demonstrates greater binding affinity to the cloned human receptor (table ii-3; figure ii -10). thus, for these commonly administered opioids, there is no great variability in their affinity for the humanreceptor. the clinical relevance of these data is that different opioids act in different ways. from anecdotal clinical experience there is considerable interindividual adapted from [11, 12, 13, 14, 15] variability in response to each opioid and this reinforces the need to assess an individual's response to opioid analgesia carefully. it also is premature to extrapolate from laboratory data, which in many instances have not yet been replicated, to the clinic. however, data increasingly inform the clinical use of these drugs and will be particularly relevant to new approaches to their use such as "opioid switching". figure ii-10 shows the putative analgesic effect mediated by the main -opioid receptor depicting that higher affinity also correlates closely with analgesic potency. but aside from -receptor interaction, analgesia can also be mediated through a -receptor and a -receptor site. the classification of different opioid receptor types is based on the original description by martin and coworkers from 1976 [5] . the effects presumed to be mediated at -receptors have been defined as a result of both human and animal studies, while the effects mediated at -receptors derive predominantly from animal models. receptors mediate analgesia that persists in animals made tolerant to -agonists. the -agonists produce less respiratory depression and miosis than -agonists. it is assumed that opioid receptors mediate opioid receptors are found in several areas of the brain, particularly in the periaqueductal grey matter, and throughout the spinal cord ( figure ii-9 ). supraspinal systems have been described for -, -, and -receptors, whereas -and -receptors modulate pain at the spinal level [3, 17, 18] . the different distribution of the various opioid subsites suggests different mechanisms of action in the mediation of analgesia. thus, -selective opioids like morphine, fentanyl and sufentanil, due to the high density of binding sites, mediate their main action within the brain stem and the midbrain. due to their close vicinity to respiratory and cardiovascular regulating centers in the brain stem, selective -opioids accordingly induce a marked depression of respiration and blood pressure. on the other hand, due to the main distribution of the -receptors within the cortex (lamina v, vi) [19] it is conceivable that these ligands induce a lesser respiratory and cardiovascular depressive effect. as a consequence and contrary to -ligands, -ligands induce a marked sedative appearance. in addition, there is a lesser addiction liability with -ligands, which is easily derived from the fact that the relevant areas in the limbic system show a low concentration of -binding sites. also, the lesser analgesic potency of -ligands is enlightened by the fact that most of the -selective receptors can be found in the deep lamina vi of the cortex. since their dendrites retrograde descend to the thalamus, all ascending nociceptive input is modified, resulting in a depression of nociceptive afferences and a reduction in arousal. certain dendrites of petrosal cells of the cortex also descend down to the brain stem, whereby the activating, ascending reticular system (ars) is affected resulting in a reduction of vigilance [20] . in summary, due to the dissimilarity of distribution of the three opioid receptor subtypes with the spinal cord and the supraspinal areas of the cns, a functional differentiation can be expected. this effect is reflected in difference of binding affinities with the brain where 22% of all receptor sites are referred to the -, 36% to the -and 42% to the -opioid receptor [20, 21] . the present understanding of the effect profiles of opioid receptors, however, remains incomplete, as new advances make it clear that their disposition and structure are extremely complex. opioids inhibit pain signals by different mode of actions: • inhibition of ca ++ -influx into the buttons of the presynaptic cell (e.g. the one releasing substance p; figure ii -11) . this is because ca ++ -influx is necessary for neurotransmitter release, whereby opioids reduce or prevent substance p from being released. • acting as an inhibitory neurotransmitter, since the opioid hyperpolarizes the postsynaptic cell by enhancing k + -flow out of the neuron, which makes it more difficult for all incoming nociceptive afferences to stimulate the next neuron, and thus more difficult to send painful information. • moderation of central perception of painful information in the limbic system so as to make it less aversive when it is perceived. several facts have led to the assumption that opioids interact with specific binding sites in the cns. a slight molecular substitution at the side chain of the morphine molecule structure results in considerable changes of potency (table ii-5) . whereas pethidine (meperidine, usp), a piperidine derivative, may be considered a weak analgesic, fentanyl, a piperidine derivative, is about 100-300 times more potent than morphine. the opioid antagonists levallorphan and naloxone are noted for a low and a analgesic effect, respectively. furthermore, only the levorotator (levo-) isomers of opioids, which appear in their natural form (i.e. compounds which, when in solution, rotate plane-polarised light to the left) are pharmacologically active (e.g. levorphanol). their dextrorotatory (dextro-) isomers, which can be synthesized in the laboratory (e.g. dextrophane), shows a negligible pharmacological effect. both substances are structurally the mirror image of each other ( figure ii-12) . in this context it is important to note that only the levo-stereoisomer of the racemic mixture is the pharmacologically active ingredient. this observation supports the part ii figure ii-11. mechanism of action of opioids at the central nervous system. by binding at the same receptor site as the endogenous opioids (i.e. enkephalins, endorphins), the release of excitatory neurotransmitters such as acetylcholine and glutamate is decreased thereby reducing the receiving cells excitatory input. the degree of opiate receptor binding is proportionally to the net release of excitatory transmitters and the reduction of depolarization produced by the arriving nociceptive nerve impulse. this enkephalin inhibitory system normally modulates the activity of the ascending pain pathways within the spinal cord and the brain. opioid agents act by binding to unoccupied enkephalin receptors, thereby potentiating the analgesic effects of the system notion that stereroselectivity of an opioid analgesic is a prerequisite in order to bind to the opiate receptor site, thus inducing analgesia. based on their interactions with the various receptor subtypes, opioid compounds can be divided into agonist, agonist/antagonist, and antagonist classes (table ii-4) . by definition an agonist is a drug that has affinity for and binds to cell receptors to induce changes in the cell that stimulate physiological activity. the agonist opioid drugs have no clinically relevant ceiling effect to analgesia. as the dose is raised, analgesic effects increase in a log linear function, until either analgesia is achieved or dose-limiting adverse effects supervene. efficacy is defined by the generally opioids exist in optical isomers, which are a mirror image in the molecular form. only the levorotatory (levo)-isomer, which in solution rotates plane-polarized light to the left, produces the characteristic analgesic effect of an agent. the dextrorotatory isomer is totally inactive. this sterospecificity of opiate action supports the concept of selective receptor binding to a site, which is able to distinguish in "handedness or goodness of fit" of an opioid molecule maximal response induced by administration of the active agent. in practice, this is determined by the degree of analgesia produced following dose escalation through a range limited by the development of adverse effects. potency, in contrast, reflects the dose-response relationship. potency is influenced by pharmacokinetic factors (i.e. how much of the drug enters the body systemic circulation and then reaches the receptors) and by affinity to drug receptors. the concepts of efficacy and potency are illustrated in the following figure, which shows the dose-response curves for two drugs with a full agonistic and a partial agonistic action. if the logarithm of dose is plotted against response an agonist will produce an s-shaped or sigmoid curve. the efficacy of the two drugs, defined by maximum response is the same. the full agonist produces the same response as a partial agonist but at a lower dose, and therefore is described as more potent ( figure ii-13 ). an antagonist by definition is an agent that has no intrinsic pharmacological action but can interfere with the action of an agonist. competitive antagonists bind to the same receptor and compete for receptor sites, whereas non-competitive antagonists block the effects of the agonist in some other way. contrary the mixed agonist/antagonists analgesics can, in turn, be subdivided into the mixed agonist/antagonists and the partial agonists, a distinction also based on specific patterns of drug-receptor interaction. both the partial agonist and the agonist/antagonist drugs have a ceiling effect for analgesia, and although they produce analgesia in the opioid-naive patient, in theory they can precipitate withdrawal in patients who are physically dependent on morphine-like drugs. for these reasons, they have been considered generally to have a limited role in the management of patients with cancer pain. the mixed agonist/antagonist drugs produce agonist effects at one receptor and antagonist effect at another. pentazocine is the prototype agonist/antagonist: it has agonist effects at the -receptors and weak to medium antagonistic action at the figure ii-13. typical dose-response curves of a full agonist, a partial agonist and an antagonist on opioid-related effects -receptor thus in addition to analgesia, pentazocine may produce -mediated psychotomimetic effects not seen with full or partial agonists. when a mixed agonist/antagonist is administered together with an agonist, the antagonist effect at the -receptor can generate an acute withdrawal syndrome. a partial agonist has low intrinsic activity (efficacy) so that its dose-response curve exhibits a ceiling effect at less than the maximum effect produced by a full agonist ( figure ii-11) . buprenorphine is the main example of a partial agonist opioid. increasing the dose of such a drug above its ceiling does not result in any further increase in response. this phenomenon is illustrated in the figure in which buprenorphine is the partial agonist. the full agonist is more potent than the partial agonist (in the lower part of the curve it will produce the same response at a lower dose), but is less effective than both coadministered ligands because of its ceiling effect. when a partial agonist is administered together with an agonist, displacement of the agonist can cause a net reduction in pharmacological action, which may be sufficient to generate an acute withdrawal syndrome. while this is a theoretical possibility with morphine and buprenorphine, no such interaction has been reported clinically. similarly, it has been suggested that the effects of morphine may be blocked in a patient switched from buprenorphine, because of the prolonged action of buprenorphine and the assumption that it will "antagonize" the effect of morphine. this has been one of the reasons, why buprenorphine has not been in cancer pain management. however, the recent development of a transdermal formulation of buprenorphine may encourage its use in chronic cancer pain (and chronic noncancer pain). an analgesic ceiling with buprenorphine is only reached at doses of 8-16 mg or more in 24 h [22, 23] . when used in usual recommended doses (e.g., two patches of 70 g/h of transdermal buprenorphine, equivalent to 3-4 mg per 24 h) buprenorphine can be considered a full -agonist since at these doses its effect will lie on the linear part of the dose-response curve [24] . relative potency is the ratio of the doses of two analgesics required to produce the same analgesic effect. by convention the relative potency of each of the commonly used opioids is based upon a comparison with 10 mg of parenteral morphine. data from single-and repeated-dose studies in patients with acute or chronic pain have been used to develop an equianalgesic dose table (table ii-5) that provides guidelines for dose selection when the drug or route of administration is changed. the information contained in the equianalgesic dose table does not represent standard doses, nor is it intended as an absolute guideline for dose selection. many variables may influence the appropriate dose for an individual patient, including intensity of pain, prior opioid exposure in terms of drug, duration, and dose (and the degree of cross-tolerance that this confers), age, route of administration, level of consciousness, metabolic abnormalities (see below), and genetic polymorphism in the expression of relevant enzymes or receptors. in addition, a substitution at the side chain, for example the substitution of a methyl-group by an allyl-group or the substitution by a cyclopropyl-group results in the new opioid antagonist naloxone, diprenorphine and naltrexone respectably, or mixed agonists/antagonists (nalorphine, levallorphane), which have the capability of antagonizing the effect of the parent compound ( figure ii-14) . similarly, when the n-methyl group of the highly potent opioid oxymorphone or the pure agonist etorphine (1000 times of morphine) is replaced by a cyclopropylmethyl group, the highly potent antagonists naltrexone and diprenorphine are derived. these compounds are 2.5 times as potent as naloxone and while the former is used as an oral preparation in the rehabilitation of the earlier opiate addict, the latter is used in veterinary medicine for the reversal of immobilization of wild animals. in addition, diprenorphine is also the original substance of buprenorphine where additional three methyl groups are incorporated in the molecule ( figure ii-15 ). such minor changes in the molecular structure and their resultant major pharmacological effect suggest, that similar to hormones and catecholamines, opioids bind with specific receptor sites which results in the characteristic effects such as analgesia. various research groups corroborated this hypothesis almost simultaneously. pert and snyder [17] , terenius [25] and kosterlitz [26] were the first research group to identify selective binding sites in the cns using radioactive labeled opioids. these so-called opiate binding sites were found mainly in neuronal structures and nervous pathways involved in the transmission of nociceptive signals such as the first relay station of pain transmission, the substantia gelatinosa of the spinal column. in the posterior horn the impulse is passed over to the second neuron while, simultaneously, descending nerve fibers from the reticular system (the cortico-and reticulo-spinal tract) induce either a facilitation or an attenuation of pain transmission, which results in a modulation of pain impulses at the spinal level ( figure ii-11 ). the course of pain transmission is to the contralateral side of the spinal cord where impulses have already undergone a distinct separation. it is the paleospinothalamic pathway, consisting of nonmyelinated c-fibers, which mediate the excruciating, dull pain component, which is difficult to localize as it ends in the nonspecific nuclei of the medial thalamus [27] . en route, the paleospinothalamic tract sends off afferent fibers to the midbrain area, such as the periaqueductal grey matter and the reticular formation [28] . the pathway ends in intralaminar nuclei of the thalamus and the nucleus limitans, a patch of pigmented nerve cells border the mesencephalon ( figure ii-16) . from there, subcortical pain pathways link with the pallidum, the alleged psychomotoric center that sends fibers to all areas of the brain hemisphere. the neospinothalamic pathway, in contrast, consists of myelinated a 2 -fibres, which transfer impulses to the nucleus ventrocaudalis-parvocellularis (n.v-c parvocellularis). from there pain sensations are projected to the postcentral gyrus, which enables the patient to localize the source of pain ( figure ii-16) . both, the central grey matter and the pallidum are characterized by a dense accumulation of opiate binding sites [29, 28] . it is worth noting that nervous pathways transmitting the dull, chronic and less pinpointed pain components are more affected by opioids, while , is an important relay station in the mediation of nociceptive afferents to higher pain modulating and discriminating centers of the cns, which is necessary for the nonspecific feeling of pain and is closely coupled with emotions the neospinothalamic pathway conveys the sharp and well localized pain components which accompany any injury and are always the first to be perceived. the indefinable, dull, emotional component is perceived later, giving pain its negative character. this separation in pain pathways is of special importance. impulses from the fast pathway usually antagonize the mediation of slow afferent impulses on all levels in the cns: substantia gelatinosa and reticular formation, as well as the specific and the nonspecific projecting nuclei of the thalamus [30] . opioid binding sites, as they are visualized with receptor-binding techniques, strikingly map the paleospinothalamic pain pathway ( figure ii-17) . furthermore, there is a high density of opioid binding sites in various other parts of the brain [3, 17, 18, 31]: 1. the corpus striatum, being part of the limbic and the extrapyramidal motor system, is responsible for triggering opioid-induced muscular rigidity. it is not only the regulatory center for locomotion but it is also the center for the regulation of attention and perception. 4. the nucleus solitary tract in the brain stern, which is the origin of the noradrenergic dorsal pathway bundle, which is in command of vigilance and the cough reflex. 5. the nuleus amygdala, being part of the limbic system, is in charge of the mediation of euphoria (or "kick") when opioids are used for other purposes than pain. 6. the locus coeruleus being the origin of the neurosympathetic system in the brain stem, regulates peripheral vasodilatation. 7. lastly, a dense accumulation of opioid binding sites is found in the substantia gelatinosa at the dorsal horn of the spinal cord. -current thinking is that effective opioid analgesics work through stimulating mu ( ) receptors, which also produce euphoria. -euphoria is mediated by the actions of opiates at a cluster of brain areas that include the nucleus accumbens and ventral segmental area. dopamine influx seems to cause subjective pleasure, or euphoria. -opioids may have a "disinhibiting" (inhibition of inhibitory neurons) effect that allows greater dopamine influx. because the main property of opioids is the blockade of nociceptive transmission in the mesencephalon (i.e. the nucleus limitans and the limbic system) the following effects can be observed: 1. no pain (analgesia), since any sensation is not identified as painful. 2. a lack of the negative emotional component of pain. on the contrary, euphoria may result and pain is no longer experienced as an emotional distress, even though pain impulses are transmitted via the ventrocaudal-parvocellular nucleus to the postcentral cortex. 3. during the opioid-induced pain-free state, the site of pain, however, still can be localized. as a consequence pain has lost its negative character and is no longer experienced or perceived as uncomfortable and distressing. in contrast to the analgesics that have a peripheral site of action (e.g. acetylsalicylic acid; asa), opioids act at the relay station of nociceptive-propagating pathways at the synapse of nerve conduction. within the nerve, pain impulses are transmitted as a change in electric conduction. and in order to guarantee maintenance of the nociceptive impulse, the excitatory impulse releases a neurotransmitter at the terminal nerve. due to its chemical configuration, the transmitter fits exactly into a binding site at the opposite nerve ending resulting in an increase of excitability and a change in the electrical nerve conduction. opioids have the property of binding to specific receptor sites at pre-and post terminal nerve endings resulting in an inhibition of a release of the excitatory neurotransmitter. the continuity of the impulse is interrupted, the nociceptive signal is no longer transmitted and thus can no longer be perceived as such ( figure ii-11 ). due to the difference in stereoconfiguration, opioids differ in their affinity (i.e. goodness of fit) at these binding sites ( figure ii-18 ). this explains why different opioids are characterized by a large variety in potency. in addition, opioids also differ in their intrinsic activity (i.e. the degree of conformational change of the receptor site) resulting in different intracellular effects. taken together affinity and intrinsic activity results in the efficacy of a drug within the system ( figure ii-19) . thus, binding properties are reflected in varying analgesic potencies. contrary, the intensity of binding with the receptor site (i.e. the intensity with which the opioid adheres to the binding site) is reflected in the duration of effects (table ii-6 and figure ii-20) [32, 14, 33] . for instance opioid analgesics such as sufentanil or lofentanil have an exceptional goodness of fit to the opioid receptor site, which results in high potency. on the other hand, the low dissociation coefficient from the receptor of buprenorphine or lofentanil is characterized by a long duration of action, while the high association coefficient demonstrates increase of affinity to the binding site. contrary to agonists, antagonists are able to displace an opioid from its receptor binding site and take up his position. displacement is only possible because the antagonist has a greater affinity to the binding site. therefore, affinity of an opioid antagonist is expressed in its antagonistic potency. naloxone or naltrexone have a very high affinity to the receptor and easily displace an opioid whereas levallorphan is five times weaker ( figure ii-21) . in order to induce a similar antagonistic effect, a higher dose of levallorphan is necessary. in order to induce increasing effects with opioids, the goodness of fit not only is a prerequisite. of additional importance is the conformational change the receptor undergoes after binding, which is expressed in the "the intrinsic activity". an opioid must, therefore, not only fit to the receptor; it must also induce a chain reaction in the transmembrane receptor domain resulting in a net effect ( figure ii-19) . the reaction after opioid binding seems to depend on the side chain of the molecule. thus it appears that one portion of the opioid molecule provides the binding to the receptor whereas another portion is responsible for the induction of a conformational change (i.e. intrinsic activity), which will either be of agonistic or antagonistic nature. in a sensitive and specific opiate-receptor assay, the guinea pig ileum with its dense accumulation of receptor binding sites, it was possible to demonstrate receptor affinity and pharmacological efficacy ( figure ii-22) . this assumption is underlined by the effects induced by "pure" opioid antagonists such as naloxone or naltrexone, which also have a good fit with the receptor site, however when given on their own do not induce an analgesic effect. for instance, if naloxone is given by itself, the compound does not induce effects similar to its parent compound oxymorphone ( figure ii-14) . also, in contrast to a potent opioid like fentanyl, the antagonist naloxone has a lower dissociation coefficient resulting in a shorter duration of action, which may result in a reoccurrence of an opioid-like effects such as respiratory depression. however, due to its high source: adapted from [33, 34, 35] part ii figure ii-20. difference in affinity and intrinsic activity of various opioids. note, that codeine has a similar intrinsic activity as sufentanil. however, due to the higher affinity of the latter the net analgesic potency is much larger association coefficient (i.e. affinity), it induces a rapid displacement of the agonist and a reversal of all opioid effects. on the other hand mixed agonist/antagonists, such as pentazocine, nalorphine, levallorphan, nalbuphine and butorphanol, demonstrate characteristics, which enable them to displace a pure agonist at the receptor site (antagonistic effect), but at the same time when administered by themselves, they induce opioid related effects such as analgesia and respiratory depression (agonistic effects; table ii-7) . such dual activity is only possible by means of their intrinsic activity at two distinct and different receptor sites: one the antagonistic activity at the -and its agonistic action art the -receptor site. and lastly, partial agonists like meptazinol and buprenorphine induce their analgesic potency via the -opioid receptor. although having a high affinity, their analgesic ceiling effect at the higher dose range is due to a lesser intrinsic activity, resulting in a lesser net analgesic appearance than pure agonists. such difference in the characteristic traits of opioids can be summarized as follows: 1. the affinity to the receptor (displacement properties or extrinsic activity) 2. the intensity of binding to the receptor (duration of effect) 3. the ability to change the conformation of the receptor (intrinsic activity) 4. the competitive potency (antagonism) 5. the degree of metabolism (duration of effect) note the relatively high antagonistic potency of buprenorphine, however, is due to its high affinity to the receptor site resulting in the displacement of a ligand at the preoccupied receptor site. similarly like a hormone or other extracellular "first messengers" that bind to its receptor on a cell surface, a signal is transmitted or "transduced" to the cells interior, thus setting a series of events that produce a biological response. such "events" include both chemical reactions and physical reactions like a conformational change in the protein molecules. the biological responses include cell differentiation, altered metabolism and cell growth and division. there are three signaling pathways that share many of the same intracellular events. each pathway is characterized by its receptor and by the cascade of intracellular events that lead to a biological response. each receptor has an extracellular, transmembrane, and intracellular component and the binding of a ligand to the receptor represents the "primary message". the term "secondary messenger" is used for those mediators that diffuse from one part of the intracellular space to its spatially removed target. among these secondary messengers are adenosine-3,-5-cyclic phosphate (c-amp). many integral membrane glycoprotein membranes share a seven transmembrane alpha-helix motif ( figure ii-23) . the ß-adrenergic receptor, whose natural ligands are epinephrine and norepinephrine, is an example of such receptors. similarly in the opiate receptor, binding of a ligand presumably initiates a conformational change in the membrane protein that is transmitted to the cell interior. this physical reaction can then facilitate other physical or chemical reactions, which are conveyed to ion channels, resulting in a change of transmembrane ion flow. the transduction of the signals from external messengers, including opiate ligands involves intracellular heterotrimeric g-proteins, which are bound to the inner cell (plasma) membrane, a secondary messenger system, involving cyclic amp, and a target response. as the name implies, these proteins are trimers, consisting of an , , and subunit. they are bound to the inner membrane and the subunit can bind the guanine nucleotides, gtp and gdp. g-proteins are involved in vision, smell, cognition, hormone secretion and muscle contraction in humans, and in mating in yeast. there are more than 100 receptors (not including odor receptors) that utilize g-proteins, and there are at least 20 members of the g-protein family, with each member having its characteristic , , and subunits. while the subunit is different for each g-protein, the ß/ pair can be the same. however, all of the g-proteins share a similar structure. in regard to the opioid receptor, specifically the g-proteins transmit the signal from the intracellular part of the receptor to the effector. adenylyl cyclase (ac), which is an inner membrane-bound enzyme, regulates the production of the secondary messenger, adenylyl cyclase. other effectors that are g-proteindependent include additional enzymes, like cyclic gmp phosphodiesterase, and transmembrane ion channels ( figure ii in its resting conformation, the g-protein consists of a complex of the three subunit chains and a gdp molecule bound to the alpha subunit. the alpha subunit is in close proximity to the intracellular part of the transmembrane receptor and, when a ligand binds to the receptor, the change in its conformation causes it to bind to the g-protein at the alpha subunit. this results in an exchange of bound gdp for gtp, which is more abundant in the cell than gdp. gtp causes a conformational change in the alpha subunit, thus "activating" it so that the alpha subunit dissociates from the -pair. the alpha subunit diffuses along the membrane until it binds to an effector, thereby activating it. the alpha subunit is also a gtpase, so the signal transduction is regulated at this level by hydrolysis of gtp to gdp and inorganic phosphate. such hydrolysis can occur spontaneously or upon interaction with a gtpase activating protein, "gap". the gdp-alpha subunit complex then binds to the ß/ complex to reform the original trimeric protein. since the stimulation of the external receptor can activate a number of g-proteins, signal amplification can occur. while this is a desired response in many instances, control at this level is needed to modulate it. g-proteins, then, are nano-switches when they turn on the effector by binding of the alpha subunit and turning it off when the gtp is hydrolyzed. the duration of production of secondary messenger, like cyclic amp, is determined by the rate of hydrolysis. in this sense, the g-protein acts as a nano-timer. although there is controversy over the role of the ß/ subunits in modulation of signals, it is likely that there are both inhibitory and stimulatory effects. if different receptors act on the same g-protein, or if different g-proteins act on the same effector, the potential exists for a "graded" response to an extracellular signal. if the same receptor acts on many g-proteins, or if one g-protein acts on many effectors, then there may be many simultaneous responses to the primary messenger. following binding the g-proteins activates the membrane-bound effector, adenylyl cyclase (ac). this enzyme catalyzes the synthesis of cyclic amp resulting in the formation of atp, camp and pyrophosphate. because this molecule is freely diffusing through the cytoplasm, it is a "secondary messenger" (figure ii-25). the reverse reaction, the formation of atp from camp and pyrophosphate, is catalyzed by a specific phosphodiesterase. camp is involved in a number of physiologic processes. for the breakdown of glycogen, stimulation of the ß-adrenergic receptor involves activation of adenylyl cyclase and synthesis of cyclic amp. the activity of camp-dependent protein kinase (capk) requires camp in order to phosphorylate ser and thr residues on other cellular proteins. glycogen phosphorylase is activated by capk, making glucose-6-phosphate available for glycolysis. adenylyl cyclase activity is regulated at a number of levels, including modulation of gtpase activity of ga, phosphodiesterase activity, and protein phosphatases. inhibitory g proteins, gi, are analogous to the stimulatory g proteins, gs, except for the exchange of gtp by gdp by the -subunit and the subsequent inhibitory action of gia on adenylyl cyclase. rather, it has been determined that ligand induced dimerization is the mechanism through which the receptor ptks are activated. this dimerization brings the tyrosine kinase catalytic domain on each receptor into close enough arrangement so that each kinase can phosphorylate tyr residues in the other's tyrosine kinase domain. such activated catalytic domains can then phosphorylate tyrosines outside of the catalytic domains, which can then modify other intracytoplasmic proteins, either by phosphorylation or by other means. all these changes are reversed with an overexpression of activation when an opioid is antagonized by a specific antagonist such as naloxone with activation of the excitatory nmda-(n-methyl-d-aspartate) receptor, resulting in a rebound with an increase in transmission of stimuli ( figure ii-27) . the next step in the signaling pathway involves activation of an inner membranebound monomeric g protein known as ras, which initiates a series of kinase reactions that ultimately carry the signal to the transcriptional apparatus of the nucleus. ras, being a g protein, is activated when its bound gdp in the resting state is replaced by gtp. it, too, has gtpase activity, but the half-life is too slow to allow for effective regulation of a signal. another gtpase activating protein, gap, increases the rate of gtp hydrolysis by ras. a "kinase cascade" ensues, involving raf (a ser/thr kinase), map kinase (also known as mek, which is both a tyr kinase and ser/thr kinase, and a family of proteins known as mapks or erks. opioids induce a variety of clinical relevant effects, which can be subdivided in being advantageous and/or even detrimental. one of the major consequences following opioid administration is that of analgesia, or antinociception. and while nsaids induce their antiniociceptive effect via cyclooxygenase (cox) inhibition, local anesthetics selectively block ion-channels, thus inhibiting the transmission of nociceptive efferent to the higher pain modulating centers in the cns. contrary, opioids bind to those areas, which not only are involved in transduction but also in the modulation and identification of painful afferences. although the majority of opioids are able to induce a maximal analgesic effect, the dosages necessary to induce such a result differ significantly. for instance, an opioid like sufentanil part ii needs a much lower dosage than the less potent opioid morphine. this is due to the higher affinity and intrinsic activity of sufentanil, suggesting that only a lesser portion of receptors needs to be occupied in order to induce the desired effect. however, a high analgesic potency necessarily does not reflect a better efficacy. this is because in certain painful conditions, some opioids are more efficacious than others. on the other hand, not all painful conditions, as the patient expresses them, can be treated successfully with an opioid. therefore, before starting an opioid therapy it is mandatory to evaluate the kind of painful condition the patient has, use the specific opioids as indicated, and avoid those painful states where opioids are contraindicated or result in a lesser therapeutic effect. however, there is the general position: in intense to severe, excruciating pain, opioids are the sole agents, which are able to induce sufficient analgesia -pain from muscular dysfunction. in patients who present pain of myofacial nature, opioids are contraindicated since they will not result in an alleviation of nociception. due to muscle spasm or an increase in tension physical therapy presents the first defense line in the therapeutic approach. this is accompanied by the administration of a benzodiazepine, which induces a muscle relaxant effect and/or the injection of a corticoid together with a local anesthetic (0.5% bupivacaine) in so-called trigger points ( figure ii-28) . trigger points are typical points which are sore and from which the pain radiates to referred areas. such points can be felt as knots or bumps under the palpating finger, which can be moved over the underlying musculature. following the in injection of the local anesthetic the circulus vituosus of increased muscle tension and myofacial pain is interrupted. local ischemia is alleviated and local accumulation of pain mediating substances is flushed out. a typical example is tension-type headache, which is the moist common type of headache. originating from increased stress, it is accompanied by emotional factors and fear. thus the painful condition can be considered of psychosomatic nature. -pain of neurogenic or deafferentiation origin, also termed as complex regional pain syndrome (crps), this type of pain is mostly seen after injury of peripheral nerves leading to spontaneous and paroxysmal discharges. such pain typically is seen as post-herpetic pain, central pain after stroke, diabetic peripheral neuropathy, phantom limb pain, traumatic nerve avulsion, trigeminal neuralgia, lumbosacral plexopathy, all being circumscribed as neuropathic pain. it originates proximal of the peripheral nociceptor ( figure ii-29) , and characteristically is due to a dysfunction or lesion of the peripheral nerve fibers and/or centrally located nervous structures. typically this type of pain is accompanied by a sensory deficit: it is of a burning, shooting, stabbing, piercing, tearing or electricshock-like, paroxysmal and vice-like nature. this pain is of paresthetic, hypoor hyperesthetic quality often is refractory to any opioid therapy. the causes for such painful conditions may be quite different: compared to a normal situation (top), there is spontaneous ectopic firing from increased sodium-channel activity after peripheral nerve injury (middle), which eventually results in central sensitization with stimulus independent pain (bottom). modified from [36] ectopic spontaneous discharges originating from a lesioned or a cut nerve, this results in an upregulation of sodium-channels being the source of spontaneous discharge. continuous nociceptive barrage later results in central sensitisation and "wind-up" (figure ii-29 ). partial denervation with spontaneous discharge of nerve activity is followed by an induced release of a nerve growth factor (ngf). such release induces sprouting of fibers into adjacent afferent somatic nerve fibers resulting in an enlargement of the receptive field and an increased conduction of nociceptive impulses to higher pain centers ( figure ii-30 other topical formulations with capscaicin or emla cream present additional therapeutic options for treatment of neuropathic pain. in addition, transcutaneous electrical stimulation (tens) or even spinal cord stimulation (scs) may present an alternative and effective strategy, resulting in the attenuation of pain. in the latter technique, analgesia is induced by the electrically induced release of endogenous opioids (enkephalins, endorphins, dynorphin) in the spinal cord and within the hypothalamus activating the descending serotoninergic and noradrengergic pathways. -opioids in visceral painful condition. another type of pain, which cannot be treated sufficiently with an opioid, is visceral pain. such a painful condition may arise from the intestine (e.g. the irritable bowel syndrome or ibs) or pain emerging from other internal organs such as the gall bladder, the urinary tract or pain following an appendectomy, cholecystectomy or hysterectomy ( figure ii-33 ). due to the participation of smooth muscles in such a condition a peripheral analgesic with a muscle relaxant effect can be of advantage. because the sympathetic nervous system to a major part is involved in such a condition, therapeutic implications incorporate a ganglionic blocker, surgical sympathectomy, or intravenous conduction anesthesia with guanethidine. -psychosomatic painful conditions, if treated with an opioid, in the long run are bound to end in opioid resistance. such a painful condition is mainly seen in the depressive patient or it may even be a premonitoring sign of schizophrenia. however, pain can also be part of a conversion-neurotic syndrome [37, 38] , where aside from pain fear, phobia, and obsessive-compulsive symptoms are the dominant elements. painful conditions being sensitive to opioids all agonizing painful states, which are due to • posttraumatic • postoperative • ischemic, or of • tumor origin, can be treated sufficiently with an opioid. the rational for the therapy with a central analgesic is related to the fact, that the nociceptive impulses are transmitted via specific pain afferents reaching supraspinal areas. by blockade of specific receptor sites within the brain, opioids induce a reduction and/or result in a total blockade of nociceptive impulses. opioids therefore present the main line of defense in all medium to severe painful conditions. when using opioids one has to realize that this group of ligands, besides their beneficial analgesic effect at the same time also induces a detrimental respiratory depression. this is a mayor drawback when using opioids in acute pain and is directly proportional to their analgesic potency. for instance a potent opioid such as fentanyl already is able to induce respiratory depression in the lower dose range. however, a less potent opioid like codeine or tramadol, even when given in dosages higher than their therapeutic margin, will not induce a clinically relevant respiratory depressive effect ( figure ii-34) . because opioids given for alleviation of chronic pain are given orally and in a controlled release formulation, there is no acute rise in opioid plasma level, which otherwise would induce respiratory impairment. in addition, chronic pain patients cannot be considered as being opioid naïve. their respiratory center already has developed some degree of habituation, being less sensitive to the opioid agent. those opioid ligands, which inherit a lesser respiratory depressive effect, however, are characterized by a comparable reduced analgesic potency. also, a pure -type ligand such as morphine, fentanyl or sufentanil is characterized by a doserelated decrease in respiration until total apnea becomes apparent. contrary, the potent partial agonist buprenorphine with increasing doses demonstrates a ceiling effect, which is seen at a dose of 2 g/kg ( figure ii-35) . typically, when administering high dosages of potent -type ligands such as fentanyl or alfentanil, a time related sequence of effects on respiration can be observed. the progression of respiratory depression is a characteristic trait, which develops within seconds to minutes: 1. a reduction in respiratory rate (bradypnea) with a partial compensation of tidal volume. 2. a respiration, which is only kicked off by external stimuli, such as noise or pain. changes in tidal volume (liter/min) in subjects being exposed to increasing dosages of the pure -type ligand fentanyl and the partial agonist buprenorphine. note, the ceiling effect in respiratory depression at higher doses of buprenorphine. adapted from [22] 3. a short period where respiration is forgotten, originally termed in europe as "oublie respiratoire", as it was observed in the early times of neuroleptanesthesia when using fentanyl together with a neuroleptic agent for the induction and maintenance of anesthesia. at this stage, however, the patient can be ordered to take deep breaths. 4. the total apnea, where in spite of any external stimuli or the command to breathe the patient spontaneously will not be able to take a deep breath. he needs immediate respiratory assistance. this centrally-induced opioid-related respiratory depression is due to a blockade of the respiratory regulating centers within the brain stem (pons and medulla), resulting in a lesser sensitivity to an increase in arterial pco 2 and/or a reduction of arterial po 2 [39, 40] . in addition the activating reticular system (ars), which descends down into the brain stem, acts as a regulatory pacemaker for the inspiratory center, by which respiratory depressive effect of opioids are affected. this is reflected in the clinics when in addition to an opioid a benzodiazepine is added, which by depressing vigilance, results in an immediate cessation of respiration [41] . any opioid-induced respiratory depression instantaneously and effectively can be reversed by the administration of a specific opioid antagonist such as naloxone. because of the higher affinity of the antagonist, naloxone displaces the agonist from the receptor site (competitive antagonism), and after binding respiratory depression is reversed and normal ventilation is instigated ( figure ii-36) . clinically, an opioid-related respiratory depression is reversed by titrating the dose of naloxone necessary to • initiate a sufficient spontaneous respiration, however • avoiding an acute abstinence syndrome with tachycardia and hypertension, and at the same time • remaining a sufficient level of analgesia during reversal one should consider the half-life of naloxone, which is between 20 and 30 min [42, 43] . therefore "remorphinisation" with a reoccurrence of respiratory depression may appear if the half-life of the agonist is longer than the antagonist, or if high concentrations of the agonist are still circulating in the blood plasma [44] . following successful reversal it therefore is mandatory to administer an additional dose of naloxone intramuscularly, which acts like a depot, or hook the patient up to a continuous intravenous drip of a naloxone solution, sufficient for long-term receptor occupation by the antagonist. all these procedures, however, do not replace the need for a continuous surveillance, which is necessary in order to detect any possible gradual development of respiratory impairment. respiratory depression can also be reversed by a mixed agonist/antagonist such as nalbuphine. although being less potent than naloxone, it however is one of the mixed ligands having a sufficient antagonistic potency (table ii-8) . at the same time the ligand has a 3-fold longer duration of action than [47, 48] , while the lesser antagonistic potency results in a more gradual and not in abrupt displacement, resulting in lesser sympathetic overdrive [49] ( figure ii-37) . another pure antagonist, nalmefene shows the longest duration of antagonism with up to 8 h of action [51] . because of its high antagonistic potency (2.5-fold of naloxone) an acute abstinence syndrome can be induced if the necessary dose is not titrated to patients need [52] . it had been proposed that different receptor sites in the cns mediate opioid-related analgesia and respiratory impairment [53] . such difference has also been demonstrated for fentanyl-analogues in the animal [54] , suggesting a clinical relevance for reversal of respiratory impairment, however at the same time maintaining antinociception. such connotation was further corroborated by experimental data where the selective antagonist naloxonazine was able to reverse opioid induced analgesia, but not respiratory impairment. this led to the assumption that -opioid subreceptors are involved in the mediation of opioid-induced respiratory depression (i.e. 1 and 2 ) [55, 56] . clinical data seem to underline this assumption, as low doses of sufentanil demonstrated a lesser respiratory depressive effect and a higher analgesic potency when compared to fentanyl. such difference in action reportedly is due to a disparity in receptor affinity to -subsites, with a higher affinity to the 1 -and a lesser affinity to the 2 -receptor [57] . other experiments, however, suggest that the co-binding of -selective ligands to the -receptor results in respiratory impairment. subanalgesic doses of the -selective ligand d-ala 2 -d-leu-enkephalin, when co-administered with morphine, induced a potentiation of analgesia, while another -ligand d-ala 2 -met-enkephalinamid produced a reduction in analgesia [58] . such -related differentiation in efficacy was also seen with the potent ligand sufentanil. there respiratory impairment was reversed while at the same time maintaining antinociception using the highly selective -antagonists naltrindol and naltribene respectively [59] ( figure ii-38) . the implication of / -receptor interaction is further supported by receptor binding studies, where sufentanil demonstrates higher -selectivity than fentanyl (table ii-9) . such putative interaction between -and -receptors is further corroborated when co-administering intrathecally a -and a -selective ligand resulted in a potentiation of effects [61] . from such data it can be concluded that a coupling mechanism between the -and -opioid receptor not only seems to result in an increase in analgesia, but at the same time also seems to cause respiratory depression. such coupling mechanisms may result in a modulation to potentiation of effects whereby it is still uncertain whether both sites independently operate from each other or whether the -receptor only accentuates the effects induced by -binding. besides a direct action of opioids on the sensitivity of the respiratory center to changes of arterial po 2 and pco 2 , also centrally-induced sedative effects very likely influence respiration. such sedative effects can be derived with the aid of the electroencephalogram where clinically different potent opioids qualitatively induce a different in the eeg pattern. since such eeg changes are dose-related, one is able to derive a dose-relationship. at the same time such eeg-changes reflect the bioavailability of centrally active agents acting on nervous structures of the cns, depicting the effect-concentration site [62, 63] . thus, following intravenous administration of an agent, it is not the plasma concentration, which is responsible for a centrally induced effect. more importantly, it is the actual concentration of the opioid at the receptor site, which is affected significantly by issues such as distribution of an agent, its lipophilicity, or the present brain perfusion. therefore vigilance changes can be considered as important aspects in an opioidrelated respiratory impairment being derived in two relevant experiments: 1. wakefulness by itself already is a fact resulting in sufficient respiration. this could be demonstrated nicely in volunteers where hyperventilation and the resultant hypocapnia resulted in a rhythmic respiratory pattern. if however, the same volunteers were asleep or in anesthesia, hypocapnia was followed by apnea [64] . 2. in the animal laryngeal stimulation during anesthesia resulted in apnae, without, however, initiating a cough reflex. being awake, a cough reflex without apnae was induced following laryngeal stimulation [65] . 3. there is a close exponential correlation of the physiologic regulatory mechanism affecting respiration. this had been demonstrated after sufentanil application in the canine, whereby increasing dosages of a selective antagonist not only dose-related reversal of sufentanil-induced hypercarbia and hypoxia with the two selective -antagonists naltrindol (nti) and naltribene (ntb) respectively, in the canine. due to the higher lipophilicity of naltribene being able to pass through the blood-brain barrier, there is a superior reversal effect. in spite of increasing doses of the antagonist there is a blockade of response to the electrically induced evoked potential, which is only reversed by the highly specific -antagonist cyprodime. adapted from [60] reversed the depressed respiratory drive but at the same time induced an increase of power in the high frequency beta band (13-30 hz) of the eeg ( figure ii-39) , reflecting increase in vigilance [66] . 4. clinically such sedative related respiratory depression can also be derived in patients, when cumulative dosages of an opioid reach a point where the respiratory center "forgets" to respond adequately by initiating deep breaths (oublie respiratoire). this is seen in classical neuroleptanalgsia where the patient's vigilance can be increased to a point by external stimuli (e.g. pain, auditory stimuli) resulting in the initiation of an inspiratory effort [67] . from all these data it can be derived that the simultaneous binding of opioid within the activating reticular system (ars) in the brain stem, vigilance is depressed, which secondarily affects the response of the respiratory center following hypercapnia. at such instances the overall mesencephalic reticular control mechanism is no longer able to adequately respond to a stimulus and only with an increase in vigilance there is an accelerated reactivity, being able to sufficiently respond to an increase in arterial pco2. since the reticular mechanism is coupled with reticulo-cortical afferences, such changes can be derived from cortical changes in the eeg. such a "forgotten" reaction to sufficiently respond to a given stimulus [65] is also seen in the clinical environment when a benzodiazepine is given on-top of an opioid resulting in a further deterioration of respiratory drive. this is because a benzodiazepine depresses the reaction of the ars, and the concomittant reduction in vigilance results in a lessened reaction to external stimuli, producing a clinically relevant suppression of respiration. in general prolongation of respiratory depression after opioid administration has to be expected when the following agents are coadministered: 1. all agents that inhibit the biotransformation of opioids such as contraceptives, anti-tumor agents, anti-arrhythmics, antidepressants, systemically administered antimycotics, neuroleptic drugs, and volatile anesthetics [68, 69, 70, 71, 72] . by inhibition of conjugation of glucoronide and oxidative dealkylation, the necessary metabolic pathways for degradation and termination of activity of most agents, a prolongation of action has to be expected. 2. all agents, which are able to displace the opioid from protein binding within the plasma, resulting in a higher portion of the pharmacologically active agent. preparations such as cumarine derivatives, and phenylbutazone, which when coadminstered are prone to result in a prolongation of effects [73, 74, 75, 76] . 3. in addition, hypoproteinemia and acidosis of the blood, both of which result in lesser protein binding, cause a higher concentration of non-bound opioid in the blood plasma. such increase in plasma concentration now is able to bind to the receptor site with an increase of efficacy and a longer duration of action [77] . following opioid-based anesthesia, several factors cause an overhang of opioid action, which may even result in a "re-morphinisation" and the re-occurrence of respiratory impairment: 1. the excessive intramuscular premedication with an opioid, which may act like a depot. 2. the premedication with a long-acting benzodiazepine, which is able to induce a reduction in vigilance lasting into the postoperative period. 3. the uncritical intraoperative use of high concentrations of a volatile anesthetic, which results in a lesser biodegradation of the opioid. 4. the intraoperative administration of fractional doses of an opioid, which results in an accumulation. due to the fact that a portion of each dose of an intravenously administered opioid is also taken up by peripheral sites (e.g. fatty tissue, musculature, skin, internal organs) there is an accumulation of the agent, which act like a depot. from there the drug later diffuses into the blood-stream, resulting in a prolongation of effects ( figure ii-40 ). 5. an insufficient loading dose of the opioid, which may result in the necessity of re-administration of small amounts of the drug intraoperatively with consequent peripheral accumulation. 6. long-term intravenous administration of an opioid by drip, resulting in the increase of the agent in the peripheral compartment with later recirculation into the blood stream. 7. the combination of opioids with different half-lifes, which may result in an unforeseen potentiation of effects. 8. uncritical administration of bicarbonate resulting in alkalosis of the blood, which induces a faster release of the opioid from the peripheral compartment. 9. a non-corrected hypovolemia, which coincides with lesser protein binding of the agent and a higher portion of the free active compound. 10. uncritical use of a selective antagonist such as naloxone, not considering that its half-life is shorter than the agonist, resulting in a later reoccurrence of respiratory impairment. the sedative effect of opioids goes in hand with their capability to induce sleep (lat. hypnos). such an effect is mostly seen with the mixed agonist/antagonists, while morphine takes a medium position ( figure ii-41) . the hypno-sedative effect of opioids is useful in premedication and during postoperative analgesia, where a sedated status of the patient is advantageous. in contrast to a potent sedative nature of mixed agonist/antagonists, the pure -type ligand fentanyl is characterized by a very low sedative potency. when in the beginning of use of neuroleptic analgesia for anesthesia, fentanyl was used together with the neuroleptic agent droperidol, often patients reported of intraoperative "awareness". although being an obligatory part of anesthesia, sleep, was not sufficiently maintained throughout the whole procedure. therefore in order to guarantee a sufficient level of sleep in patients receiving a fentanyl-based anesthetic technique, an additional hypnotic (propofol), a benzodiazepine (midazolam), a neuroleptic agent (i.e. dehydrobenzperidol), or a volatile anesthetic (sevoflurane, desflurane, or enflurane) has to be given on-top the opioid. nowadays the problem of awareness again has gained much attention [78] , since the technique of total intravenous anesthesia (tiva) with remifentanil and [84] propofol, completely omitting nitrous oxide (n 2 o), often results in an insufficient level of sleep with awareness. typically pure -ligands such as bremacozine and tifluadom (table ii-3) , which in comparison to morphine have a 2-fold analgesic potency [79, 80] , do not induce a respiratory depressive effect [81] . their lack in respiratory impairment is due to the selective binding in deep layers of the cortex [19, 82] , where in comparison to the brain-stem, a more than 50% higher concentration of -binding sites is found [3, 83] . their predominant sedative effect is due to centripetal fibers descending down from deep layers of the cortex to the thalamus, thus decreasing the nociceptive input [20] . although having the advantage of an increased sedative effect combined with the lack in respiratory impairment, clinically, the use of -ligands had to be abandoned. this is because of their intense dysphoric side effects, which lasts for several hours. in addition, their analgesic potency, in comparison to pure -ligands is much lower. therefore such agents cannot be regarded as suitable for intraoperative use, where an intense nociceptive barrage can only be blocked by a potent -opioid. only the mixed agonist/antagonists (e.g. nalbuphine, butorphanol), which exert their analgesic action through binding at the -site, currently are in clinical use mainly for postoperative analgesia [85, 86] . this is because cumulative dosages, contrary to a typical -ligand like morphine, result in a ceiling effect for respiratory depression ( figure ii-42 ). in addition, because of their wide margin of safety, high dosages part ii figure ii-42. ceiling effect of respiratory depression following cumulative doses of the mixed agonist/antagonist nalbuphine. in contrast to morphine, at a certain dose there is no further increase in the degree of respiratory impairment. adapted from [89] have been advocated in balanced anesthesia where the opioid resulted in an up to 70% reduction in mac (minimal alveolar concentration) of the volatile agent [87, 88] . opioids in general induce a dose-related hyposedative component, which is mirrored in the electroencephalogram by an increase of activity in the slow -with concomitant decrease of power in the fast -domain. however, when giving a large bolus dose of fentanyl (7-10 g/kg body weight) alfentanil (50 g/kg body weight) morphine (3-10 mg/kg body weight) or sufentanil (2-3 g/kg body weight) an immediate dominance of delta-waves in the eeg becomes evident, being accompanied by sleep. for instance, such effects clinically are seen when high-dose opioid anesthesia is used in cardiac patients for the induction of anesthesia. such a sleepinducing effect is due to a short-term blockade of all afferences being switched in the activating reticular system (ars) of the mesencephalon. aside from a blockade within the nucleus limitans a deep level of analgesia is initiated [90] . such a "narcotic component", with dominance of delta-activity in the eeg, and contrary to equi-analgesic doses of fentanyl, it is more apparent after sufentanil [91] , which makes this agent more suitable for the induction of cardiac patients ( figure ii-43) . following induction with a potent -ligand such as fentanyl or sufentanil the initial "narcotic component" later transforms into a "pure analgesic component". this is because the opioid is redistributed, which results in a lesser concentration within the cns and a lesser binding in areas within the ars. at this stage there is a note, a pronounced delta activation after injection and a lesser arousal reaction induced by laryngoscopy and intubation, which in the sufentanil group reflected in a lesser decline of power in the slow delta-domain of the eeg. adapted from [92] dominance in the -band (7-13 hz) of the eeg, which is stable, not being affected by any nociceptive stimuli [93, 94] . clinically, such an effect has been described for the precursor of fentanyl, the opioid phenoperidine [95] and for fentanyl [96] . after a period of 10-15 min the deep narcotic component changes into a sedative state, which is stable and cannot be reversed to desynchronization by any nociceptive stimulus. during such "analgesic state" the patient again is able to respond to verbal commands, while at the same time having a deep analgesic level ( figure ii-44) . without the addition of nitrous oxide, such patients are awake, however, nociceptive afferents are not able to modulate the ars, the endotracheal tube is tolerated while at the same time nociception is only sensed as a touch. such phenomena are due to afferents ascending along the spinothalamic tract, which directly ascend to the postcentral cortical area by which the impulse can be localized. collaterals, which ascend through the nucleus limitans within the limbic system and convey nociception, are sufficiently blocked by the opioid and the patient does not perceive pain ( figure ii-45) . the opioid receptor system bordering the fourth cerebral ventricle and the underlying activating reticular system is the relevant anatomical structure in mediating sedation. selective perfusion of increasing concentrations of the opioid fentanyl in the awake canine induced a dose-related enlargement of slow-wave high amplitude delta-activity within the eeg, characterized by a sleep-like behavior ( figure ii-46) . this effect was reversed by the levo-isomer of naloxone inducing an arousal reaction. it, however, was not reversible by the dextro-isomer of the antagonist [98] . the physiological significance of opioid receptors in the control of vigilance is also reflected in the high density of opioid binding sites in the mesencephalon [99] . physiologically this is mirrored by an arousal reaction following intense acoustic or a nociceptive stimulus, both of which induce a reversal from the low frequency delta-to high frequency beta-activity in the eeg ( figure ii-47) . in summary, it is concluded that opioids primarily affect the limbic system, the specific site for inducing the negative component of nociception. lastly such assumption is underlined by the result from mc kenzie and coworkers in the animal where the opioids morphine and pethidine were not able to sufficiently block any pain related nervous transmission from the mesencephalon to the higher cortical areas [100] . in contrast, both ligands were able to block nociceptive transmission from the mesencephalon to hippocampal areas of the limbic system, the part of the cns, which is responsible for the identification of pain, causing the negative, grief, stinging and an intense emotional feeling associated with pain ( figure ii-48) . such differences in pain modulation were corroborated in patients undergoing stereotactic, painful stimulation within specific areas of the cns [101] . nociceptive afferents of the spinothalamic tract end in the nucleus ventrocaudalis parvo-cellularis part ii figure ii-46. selective perfusion of increasing doses of fentanyl through the fourth cerebral ventricle of the awake canine. note, the direct sedative (delta-synchronisation in the eeg) effect via the underlying ars. this effect is mediated through opioid receptors located on the floor of the ventricle, since it was reversible with naloxone (desynchronisation with beta activation in the eeg). adapted from [98] thalami, from where they further ascend to different cortical areas. since these nuclei reflect a specific somatotopic differentiation, electrical stimulation within this area induced painful sensations in different parts of the body. decoding of painful afferents was only possible when the stimulating electrode was placed within the nucleus limitans, where collaterals of the spinothalamic tract switch to the limbic system. there stimulation induced a less well-localized, however, intense unspecific displeasure [102] . following the administration of high dosages of opioids with different potency, epileptogenic activities in the eeg with tonic-clonic seizures can be induced in the animal. pethidine (meperidine), morphine, alfentanil, fentanyl and sufentanil when administered in doses above 20, 180, 5, 4 and 4 mg/kg body weight respectively, induced epileptogenic discharges [103] . because such massive dosages are never used in anesthesia or for analgesia in acute or chronic pain, epileptogenic effects, although being cited in the literature after fentanyl [104, 105] and sufentanil [106] are of insignificant nature. this is because the clinical picture resembles tonicclonic seizures, however, in the eeg no such discharges could be derived [107] . therefore, those high doses of opioids, which induced epileptogenic activity in the rat [108] or the canine [103] are far off from therapeutic range. thus, in general, an epileptogenic activity of opioids can be canceled out. one exception is the use of high dosages of pethidine (meperidine), where the metabolic product norpethidine is a potent epileptogenic compound, which especially in the newborn is able to induce epileptogenic activity [109] . the cause for the few observations of a pseudoepileptogenic activity of opioids when being administered within the therapeutic range very likely is due to a desinhibition of the cortical motor center within the cns, as this phenomenon was observed during the induction of anesthesia or following a decline in plasma concentration. such assumption is underlined by part ii figure ii-48 . two main components of painful afferents: localization of nociception (cortical area) and the initiation of the negative component (i.e. the limbic system within the mesencephalon). opioids mainly modify the latter area "epileptogenic activity" during the induction of anesthesia with the pure hypnotic etomidate, where desinhibition of the motor cortex activity was the cause of cortical discharges [110] . each cough involves a complex reflex arc beginning with the stimulation of sensory nerves that function as cough receptors. there is evidence, primarily clinical, that the sensory limb of the reflex exists in and outside of the lower respiratory tract. although myelinated, rapidly adapting pulmonary stretch receptors (rars), also known as irritant receptors, are the most likely type of sensory nerve that stimulates the cough center in the brain, afferent c-fibers and slowly adapting pulmonary stretch receptors (sars) also may modulate cough. rars, c-fibers, and sars have been identified in the distal esophageal mucosa; however, studies have not been performed to determine whether they can participate in the cough reflex. although gastroesophageal reflux disease can potentially stimulate the afferent limb of the cough reflex by irritating the upper respiratory tract without aspiration and by irritating the lower respiratory tract by micro-or macroaspiration, there is evidence that strongly suggests that reflux commonly provokes cough by stimulating an esophageal-bronchial reflex. each involuntary cough involves a complex reflex arch beginning with the stimulation of sensory nerves in the airway epithelium that function as "cough receptors." efferent impulses from these receptors are conducted by means of the vagus nerve to the "cough center" in the brain stem. because cough can be voluntarily suppressed, controlled, or initiated, there also can be afferent input from the cerebral cortex. the function of this "cough center" is to receive these impulses and produce a cough by activating efferent nervous pathways to the diaphragm and laryngeal, thoracic, and abdominal musculature. the possibility that there might be afferent input other than the vagus nerve and cerebral cortex was based on clinical observations described in case reports and a few animal studies [111] . histologic studies of the respiratory tract in both animals and humans have revealed sensory nerve endings within the basal layer of the epithelium and between epithelial cells of the larynx, trachea, and bronchi [111] . these nerve endings are thought to be cough receptors. they contain neuropeptides, such as substance p and calcitonin-gene related peptide (cgrp), which mediate neurogenic inflammatory events in the airways. these sensory nerve endings have been found to be most numerous in the posterior wall of the trachea, at branching points of large airways, and less numerous in the more distal, smaller airways. none have been found beyond terminal bronchioles. it is not known for certain which type of afferent nerve mediates cough. a model that summarizes the current understanding of cough is schematically depicted in the figure. it shows the myelinated, rapidly adapting pulmonary stretch receptors (rars), also referred to as irritant receptors, as the most likely type of sensory nerve that stimulates the cough center in the brain. both mechanical and chemical stimulation of rars have been shown experimentally to cause cough. another type of sensory nerve in the airways, c-fibers, may also participate in regulating cough. they are unmyelinated, vagal afferent fibers that may be activated by the same triggers as rars. their activation releases neuropeptides locally that may secondarily stimulate cough by activating rar nerves. however; impulses transmitted by c-fibers alone probably do not stimulate cough, because experimental evidence has shown that they inhibit cough centrally in the brain. a third type of sensory nerve, the slowly adapting pulmonary stretch receptors (sars), may modulate cough. although these nerves do not directly respond to chemical and mechanical triggers, they do appear to be activated by the deep breath of a cough and may enhance cough by making the expiratory effort more forceful. in addition to mechanical and chemical stimuli, cough has been caused in animals by thermal and electrical provocation. the sites most sensitive to all stimuli are the larynx, trachea, and cannulae of the larger airways. outside of the lower respiratory tract, cough receptors have been demonstrated histologically only in the hypopharynx [111] . however, it has been inferred from clinical studies that sensory nerve endings subserving the cough reflex via the vagus nerve probably exist in the extemal auditory canals and eardrums, hypopharynx, pericardium, stomach, and esophagus, because stimulation of these sites has been reported to cause cough [111] . based on the fact that cough can be voluntarily initiated, postponed, and/or suppressed, this provides evidence that there also can be afferent input from the cerebral cortex. in addition to directly stimulating cough by carrying impulses from cough receptors to the cough center, vagal afferents may indirectly provoke cough by another mechanism. they may stimulate neurotransmitter release or mucus secretion from airway submucosal glands that, in turn, stimulate the cough reflex [111] . the existence of a discrete central cough center is controversial. what is known is that afferent pathways first relay impulses to an area in or near the nuc1eus tractus solitarius. these impulses then are integrated into a coordinated cough response in the medulla oblongata of the brain stem, probably separate from the medullary centers, which control breathing. although electrical stimulation studies of different areas in the medulla have evoked cough in animals, suggesting that the cough center is diffusely located [111] a discrete cough center still may exist, because these electrical stimulations may have activated afferent pathways of the cough reflex. the motor outputs trom the cough center are in the ventral respiratory group, with the nucleus retro-ambigualis sending impulses via motoneurons to the respiratory skeletal muscles and the nucleus ambiguus sending impulses to the larynx and bronchial tree. more specifically, the efferent impulses of the cough reflex are transmitted from the medulla to the expiratory musculature, through the phrenic nerve and other spinal motor nerves, and to the larynx through the recurrent laryngeal branches of the vagus nerves ( figure ii-49) . vagal efferents also innervate the tracheobronchial tree and mediate bronchoconstriction [111] . although stimulation of cough and bronchoconstriction can be experimentally separated using nonpermeant anions to stimulate cough without bronchoconstriction, these two phenomena normally are activated simultaneously to facilitate the most effective cough. bronchoconstriction may improve clearance of secretions by narrowing the cross-sectional area of the airways, thereby increasing the velocity of air leaving the patients lower respiratory tract during the expiratory phase of coughing [111] . experimentally, it has been shown in animals that the efferent pathways of the cough reflex are anatomically distinct and separate from the efferent pathways of normal spontaneous ventilation. blockade of the cough reflex arch by means of opioids, known as the antitussive action, refers to the fact that they suppress this protective reflex. this is of benefit during anesthesia and/or in patients being artificially ventilated in the intensive care unit (icu) because it results in the tolerance of an endotracheal tube. however, the antitussive potency differs significantly among the various opioids ( figure ii-50) . the action is not related to a specific receptor site, because a stereoselective action of opioids in regard to their antitussive effect could not be demonstrated. in addition, reversal with the selective antagonist naloxone is less selective [112] . the mode of action is a blockade of the cough center within the brainstem. three of the most commonly used suppressors of the cough reflex are hydrocodone, codeine and hydromorphone. all of them are characterized by low analgesic potency, they model for afferent limb of cough reflex in airways. myelinated. rapidly adapting pulmonary stretch receptors (rars) and unmyelinated c-fibers are sensory nerves that participate in the afferent limb of the cough reflex. mechanical and chemical stimuli activate sensory nerve enelings in the epithelial layer. rars appear to be the main type of sensory nerve stimulating cough centrally. although c-fibers may inhibit cough centrally, neuropeptides released in the periphery upon stimulation of c-fibers may indirectly stimulate cough by activating rars. slowly adapting pulmonary stretch receptors (sars) do not respond to irritant stimuli that initiate cough but may enhance cough centrally by making expiratory muscular effort more forceful demonstrate a negligible dependence liability, and they are common components in doc (drugs over the counter) cough medicine. a similar antitussive potency, however, is also seen with the more potent opioids such as diamorphine, fentanyl or sufentanil. the latter are used in an opioid-based anesthetic regimen or in icu patients who are in need of ventilatory support. when a potent opioid such as sufentanil is used, the patient is adapted much easier to the respiratory cycle of the ventilator resulting in lesser doses of additional sedative agents. morphine in this regard has a much weaker antitussive activity while pethidine (meperidine) and all mixed agonist/antagonists are characterized by a negligible antitussive action ( figure ii-50) . it can be summarized that potent opioids also inherit a marked antitussive effect, while weak opioids and especially the mixed opioid analgesics are unable to sufficiently suppress the cough reflex. during the induction of anesthesia, while injecting an intravenous bolus dose of a potent opioid such as fentanyl or sufentanil, often a cough reflex is initiated. such part ii dependence or addictive liability (how addictive a drug is likely to be) depends upon how quickly the drug enters/leaves the brain. also, it is directly proportional to the analgesic potency and it depends on the opioid receptor sites with which the ligand interacts. for instance, members of mixed agonist/antagonists (i.e. nalbuphine, pentazocine, butorphanbol) demonstrate a predominant interaction with the -opioid receptor, which is characterized by a low dependence liability ( figure ii-51 ). in addition, development of dependence also is related to the speed for instance, an opioid like buprenorphine is characterized by a low dissociation constant reflecting long binding to the receptor, a long duration of action and a slow separation from the receptor. the definition of addiction is based in two different terms: 1. physiological dependence produced by repeated drug-taking that is characterized by a withdrawal syndrome, when drug is removed (e.g. alcohol, opiates). 2. psychological dependence produced by repeated drug taking that is characterized by obsessions and compulsive drug-seeking behaviors; results in a detrimental impairment in physical, mental or social functioning. there are five classes of abused psychoactive drugs 1. opioids produce a dream-like state; effects include: analgesic (reduction in pain), hypnotic (sleep inducing), euphoria (sense of happiness or ecstasy) using morphine, heroin, or the cough suppressant codeine. 2. depressants produce feelings of relaxation/sedation and a dream-like state, anxiolytic (anxiety-reducing) and hypnotic effects; reduce central nervous system activity. members of the class are alcohol, barbiturates, and benzodiazepines. 3. stimulants increase alertness, arousal, and elevated mood; activate central nervous system (sympathomimetic = mimic the activation of the sympathetic nervous system). members of this class are cocaine, amphetamines, nicotine, caffeine. 4. psychedelics produce distortions of perception and an altered sense of reality. typical representatives are lsd, psilocybine, mescaline, mdma (ecstasy), and pcp (phencyclidine). 5. marijuana with one of its major active ingredient thc produces feelings of well-being and sense of acuity (sharpness). it also can produce feelings of relaxation. also, it is recognized, that opioids, which demonstrate a fast onset of action or due to their galenical preparation (injection, smoking) result in an immediate high plasma concentration, followed by an instantaneous receptor binding, results in a "kick" with an euphoric feeling. such preparations therefore are more prone to induce a behavior pattern of abuse. therefore addictive liability or how addictive a drug is likely to be, depends upon how quickly the drug enters/leaves the brain (table ii-10) . most importantly, the tendency of opioids to result in an abuse is very much linked to the fact of why and when the opioid is ingested. for instance, an opioid taken only for the mere pleasure will rapidly result in the development of dependency. contrarily, if an opioid is taken for the attenuation of pain, the likelihood to develop an abuse behavior is very low. aberrant drug related behavior has to be suggested when the following signs of abuse are obvious: in physical examination. a routine physical examination can elucidate common complications of heroin use or assist in diagnosing opioid dependence. chronic intravenous use can be confirmed by the presence of "track" marks, which are callouses that follow the course of a subcutaneous vein. these are caused by repeated injections into adjacent sites over an accessible vein. tracks are often found in easily accessible body areas, such as the backs of the hands, antecubital fossae, on the legs, or in the neck. signs of recent injection may be found in unusual places in patients attempting to hide their sites of injection. a thorough examination for tracks or recent injection sites should include looking between the table ii-10. factors that influence how quickly a drug will enter the brain 1. chemical structure: how fatty is the drug (i.e. its lipophilicity)? does the drug have nutrients that our brain uses? 2. how fast does the drug cross the blood brain barrier (bbb) lipophilic drugs (i.e. heroin, fentanyl) cross the bbb much faster than hydrophilic agents (i.e., morphine); they mimic nutrients our brain needs and can "slip" through transporters in the bbb. 3. route of administration: is the drug entering directly into the blood stream or is it entering first into the stomach? the routes of administration increase the likelihood that a drug enters the blood stream whereby it increases the addictive liability of the drug. intravenous injection > smoking/snorting > sublingual application > inhalation via nostrils and lungs because they have abundant blood capillaries and more blood supply under the tongue and the lung respectively. fingers and toes, under the fingernails and toenails, in the axillae, breast veins, and the dorsal vein of the penis. one complication of drug use that can be found on examination is nasal septal perforation from repeated intranasal insufflation (especially when cocaine is mixed with heroin and snorted). a heart murmur may indicate subacute bacterial endocarditis, a complication of intravenous injection without using good sterile technique. posterior cervical lymphadenopathy may suggest early viral infection, especially with hiv. hepatic enlargement may indicate acute hepatitis; a small, hard liver is consistent with chronic viral hepatitis due to hepatitis b or c virus, which are common among injection drug users who share needles. signs of opioid intoxication may include pinpoint pupils, drowsiness, slurred speech, and impaired cognition. signs of acute opioid withdrawal syndrome include watering eyes, runny nose, yawning, muscle twitching, hyperactive bowel sounds, and piloerection. on the other hand the following behaviors are more suggestive of an addiction disorder: • selling prescription drugs • prescription forgery • stealing or "borrowing" drugs from others • injecting oral formulations • obtaining prescription drugs from nonmedical sources • concurrent abuse of alcohol or illicit drugs • multiple dose escalations or other non-compliance with therapy despite warnings • multiple episodes of prescription "loss" • repeatedly seeking prescriptions from other clinicians or from emergency rooms without informing the prescriber or after warnings to desist • evidence of deterioration in the ability to function at work, in the family, or socially that appear to be related to drug use • repeated resistance to changes in therapy despite clear evidence of adverse physical or psychological effects from the drug the following behavior pattern is less suggestive of an addiction disorder. • aggressive complaining about the need for more drugs. • drug hoarding during periods of reduced symptoms. • requesting specific drugs. • openly acquiring similar drugs from other medical sources. • unsanctioned dose escalation or other noncompliance with therapy on one or two occasions. • unapproved use of the drug to treat another symptom. • reporting psychic effects not intended by the clinician. • resistance to a change in therapy associated with "tolerable" adverse effects with expressions of anxiety related to the return of severe symptoms. and while addiction is characterized by a compulsive drug-using and drug-seeking behavior that interferes with normal functioning and causes use of the drug despite increasingly damaging consequences, there are different mechanisms from dependence as addicts can experience intense cravings for drugs even years after sobriety (after body set-points, etc. should have adjusted back to normal). the nervous pathways involved in the development of dependence is the mesolimbic-dopaminergic reward system, where direct activation (i.e. cocaine, nicotine, alcohol) or inhibition of the inhibitory gabaergic neurons in the ventral tegmental area (vta) directly project to dopaminergic neurons in the nucleus accumbens (i.e. opioids such as heroin) results in an increased release of dopamine in the nucleus accumbens and stimulation of the prefrontal area resulting in euphoria. on the other hand, insufficient release of dopamine in this area results in dysphoria, which is seen in abstinence ( figure ii-52 ). in the acute phase, opioids inhibit adenylyl cyclase and camp. over time, the downstream transcription factor (creb) is activated which increases adenlyly cyclase production ( figure ii-53) . chronic opioid use leads to upregulation of camp and creb, directing to tolerance, dependence and withdrawal symptoms. the significance of intracellular changes in creb is supported by data in mice without creb, which are less likely to develop addiction. also, recent research has also implicated an opioid peptide, dynorphin, in this pathway. besides initial changes in creb, chronic administration of an opioid results in the induced increased formation of peptides syntheses fosb within the nucleus accumbens. such overexpression of fosb increases the sensitivity to cocaine and opioids resulting in an increased likelihood of relapse. in general an opioid-related dependency has to be distinguished from a dependency of the barbiturate-, alcohol-or cocaine-type. this is because they all result in different psychopathological and withdrawal symptoms. the latter is a set of physiological reactions that occur in response to removal of a drug following repeated treatment; often (although not always), the reactions are opposite those produced by the drug itself. in the beginning it is the pleasure seeking behavior that results in repetitive drug administration until finally, the drugs of abuse that produce physical dependence (e.g., opiates or alcohol), results in the avoidance of the unpleasant withdrawal syndrome can contribute to repeated drug-taking ( figure ii-54 ). the main physiological consequence of nausea and emesis is the removal of toxins, which is an important protective reflex mechanism being induced during food intoxication. it however, is also seen after radiation or chemotherapy, after the administration of an opioid-based anesthetic regimen or during long-term therapy for alleviation of chronic pain. about 20% of all patients experience nausea and/or emesis after opioid anesthesia. the cause of such reaction is a stimulation of the chemoreceptor biochemical changes induced during addiction and the development of tolerance, which is followed by withdrawal when an antagonist is administered or by lack in maintenance dosages trigger zone (ctz), which lies in close vicinity to the emetic center, bordering the fourth cerebral ventricle, above the area postrema ( figure ii-55 ). this area is richly supplied with dopaminergic, histaminergic, serotonergic (5-ht 3 ), and cholinergic receptor sites, being the origin of metabolic or drug induced vomiting [113] . contrary to the other areas within the cns, the ctz is characterized by leaking capillaries (windowed capillaries), through which opioids as well as toxins can disseminate. such anatomical difference indicates that this area does not contain the usual blood-brain barrier (bbb). being located within the dorsal part of the activating reticular formation (ars), all visual, cortical and limbic efferences, as well as efferences of nearby nuclei of the vasomotor center and the center for salivation and respiratory control are switched, resulting in a controlled succession during vomiting. once the vomiting center is stimulated by any of the efferent stimuli, a coordinated sequence of events is commenced: • stop of rhythmical contractions of the stomach, followed by an accumulation of food in the abdomen, resulting in. • retroperistaltic action. • contraction of the cardia with increase of pressure in the stomach. • due to the coordinated contractions of diaphragm, intercostal muscles and the rectus abdominis muscle, food is being expelled forcefully via the opened orifice of the stomach, the dilated esophagus and the opened glottis. because the ctz shows a dense accumulation of serotonin receptors, the serotoninantagonist ondansetron (zofran®) is able to induce an antiemetic effect [114, 115] . other agents, which are given for reversal of emesis, are metoclopramide, and/or the neuroleptic agents haloperidol, triflupromazine, or alizapride-hcl, all of which interact through direct binding with the dopaminergic d 2 -receptor. another antiemetic is diphenhydramine, which exerts its action via binding at the cholinergic and histaminergic receptors ( figure ii-56) . postoperative nausea and emesis (ponv), however, still present a problem specifically related to anesthesia. in a large survey with over 2000 patients and using multivariance analysis, the following main risk factors for ponv were identified: • female sex • young age • history of ponv/motion sickness • nonsmoking status • long duration of anesthesia each 30 min increase in duration increases ponv risk by 60%, and a baseline risk of 10% is increased to 16% after 30 min [116] . the type of operation, the addition of nitrous oxide (n 2 o), high age and/or the addition of an opioid to the anesthetic regimen, in comparison to the above risk factors, had a lesser impact on the incidence on ponv [117, 118] . commonly the key strategic antiemetic agents for reducing patients ponv are as follows (figure ii-56): the 5-ht 3 -receptor antagonists are used for both the prevention and treatment of ponv and have a low side-effect profile. they are given toward the end of surgery for greatest efficacy, and are more effective in preventing vomiting than in preventing nausea. dolasetron, granisetron, and ondansetron all have favorable side-effect profiles. no evidence has revealed differences in efficacy and safety among the 5-ht 3 -receptor antagonists used for the prophylaxis of ponv. a recent study demonstrated the equivalent efficacy and safety of granisetron and ondansetron when these agents were used in combination antiemetic therapy. in this study, low-dose granisetron (0.1 mg) plus dexamethasone 8 mg was found to be not inferior to ondansetron 4 mg plus dexamethasone 8 mg in patients undergoing abdominal hysterectomy with general anesthesia. the combinations prevented vomiting in 94% and 97% of patients, respectively, in the first 2 h after tracheal extubation, and in 83% and 87% of patients, respectively, in the 24 h after extubation. dexamethasone has been found to be effective for the management of ponv and their proposed mechanism of action is that of membrane stabilization and inhibition of inflammation. use of this agent is controversial because of its alleged association with delayed wound healing. it has a slow onset but a prolonged duration of action, and therefore it is advised that dexamethasone be administered upon induction of anesthesia. the most commonly used dose for adults is 8-10 mg i.v. smaller doses of 2.5-5 mg have also been used and found to be as effective. based on a quantitative, systematic review of the data, no adverse side effects, especially delayed wound healing, have been noted following a single antiemetic dose of dexamethasone [119] . the neuroleptic drug droperidol, a butyrephenone derivative, is widely used for ponv prophylaxis and is comparable with ondansetron as a prophylactic antiemetic. similar to haloperidol it acts as a dopamine antgonist at the ctz and the area postrema. for greatest efficacy, droperidol is administered at the end of surgery or concomitantly with morphine via patient-controlled analgesia systems. the use of low doses (0.625-1.25 mg) of droperidol has not been associated with the typical side effects of higher doses of this drug (hypotension, extrapyramidal symptoms, sedation, akathisia, dysphoria). in 2001, the food and drug administration began requiring that droperidol labeling include a "black box" warning stating that the drug may cause death or life-threatening events resulting from qtc prolongation and the possibility of life-threatening torsades de pointes. the labeling requirement was based on 10 reported cases associated with droperidol use (at doses of 1.25 mg) during its approximately 30 years on the market [120] . however, no case reports in peer-reviewed journals have linked droperidol with qtc prolongation, cardiac arrhythmias, or death at the doses used for the management of ponv. also, in a randomized, doubleblind, placebo controlled trial, droperidol was not associated with a significant increase in the qtc interval in comparison with saline solution [121] . in another recent study, droperidol did not increase the qtc interval any more than did ondansetron [122] . transdermal scopolamine (transderm scop® 1.5 mg), an antimuscarinic ganet, works by blocking the cholinergic receptor. it has an antiemetic effect when applied the evening before surgery or 4 h before the end of anesthesia preventing the patient from post-discharge nausea, vomiting and retching. the phenothiazines, promethazine, and prochlorperazine act both as d 2 -and the h 1 -receptor antagonist. they also inhibit histamine receptors and possibly cholinergic receptors in the gut. both have been shown to be effective antiemetics when administered intravenously at the end of surgery. all three drugs may cause sedation, dry mouth, and dizziness. metoclopramide is benzamide that blocks d 2 -receptors both centrally and peripherally in the gastrointestinal tract increasing gastric emptying. the antihistamines, especially diphenhydramine act on both the ctz and the vestibular pathways of the inner ear. at higher doses however, they can prolong general anesthesia and recovery times. consensus guidelines agree that patients at high or moderate risk for ponv are most likely to benefit from prophylaxis. patients at low risk for ponv are usually not candidates for prophylaxis unless their condition may be compromised by the medical sequelae or vomiting. those at moderate risk for ponv should receive antiemetic monotherapy or combination therapy. those at high risk should receive combination therapy with two or three antiemetics from different classes. drugs with different mechanisms of action can be combined for optimal efficacy. for example, the 5-ht 3 -receptor antagonists (more effective against vomiting) can be combined with droperidol (more effective against nausea). a multimodal approach that incorporates both baseline risk reduction and antiemetic therapy should be adopted for ponv prophylaxis. a recent prospective, double blind, randomized, controlled trial compared three strategies for the prevention of ponv in patients undergoing laparoscopic cholecystectomy: (1) a multimodal approach using ondansetron, droperidol, and total intravenous anesthesia (tiva) with propofol; (2) a combination of ondansetron and droperidol, with isoflurane and nitrous oxide-based anesthesia; and (3) tiva with propofol alone. the complete response rate was higher in the multimodal group (90%) than in the combination group (63%) or tiva-only group (66%), as was the degree of patient satisfaction. nausea and vomiting may persist in some patients after they leave the postanesthesia care unit (pacu). after medication and mechanical causes of ponv have been excluded, rescue therapy with antiemetics can be initiated. for patients who received no prophylaxis, low-dose therapy with 5-ht 3 -receptor antagonists may be initiated. consensus guidelines also recommend low-dose therapy with a 5-ht 3 -receptor antagonist for patients in whom dexamethasone prophylaxis has failed. for patients in whom initial 5-ht 3 -receptor antagonist prophylaxis has failed, a 5-ht 3 -receptor antagonist rescue therapy should not be given within the first 6 h after surgery. similarly, patients in whom prophylactic combination therapy with a 5-ht 3 -receptor antagonist plus dexamethasone has failed should be treated with an antiemetic from a different class. as a general guideline, patients who experience ponv within 6 h after surgery should be treated with an antiemetic other than the one used for prophylaxis. for the treatment of patients who experience ponv > 6 h after surgery, drugs from the prophylactic antiemetic regimen may be repeated, except for dexamethasone and transdermal scopolamine, which have a longer duration of action. also, propofol may be used in small doses (20 mg as needed) for the treatment of ponv in a supervised environment. the preliminary results of a recent analysis support the recommendation that a rescue antiemetic should be from a class other than that of the original antiemetic agent [123] . this analysis of a previous trial reported that in patients who failed prophylaxis with ondansetron or droperidol, promethazine was significantly more effective in controlling ponv than the original agent. dimenhydrinate was also more effective than droperidol in patients who failed prophylaxis with droperidol. in summary, the first step in the management of ponv is to identify surgical patients at high or moderate risk for ponv, then reduce baseline risk factors in these patients. combination antiemetic therapy is recommended for patients at high risk for ponv for patients at moderate risk, monotherapy or combination therapy may be considered. a multi modal approach for the prevention of ponv including the use of antimetics with different mode of action, hydration and tiva with propofol has been shown to be most effective. patients who have not received prophylaxis and experience ponv can be treated with a low dose of a 5-ht 3 -receptor antagonist. in patients who fail prophylaxis treatment with an antiemetic, another agent than the one used for prophylaxis is recommended. opioids can induce muscular rigidity, which is due to an increased tone of the striatal muscle. especially, the muscles of the thoracic cage and of the abdomen show this rigidity, a phenomenon, which is observed after the bolus injection of a potent opioid, such as the fentanyl series (i.e. fentanyl, sufentanil, alfentanil and remifentanil; figure ii-57) . increase in muscle tone is directly correlated to the -receptor interaction, because mixed agonist/antagonists and highly selective -opioid antagonists (i.e. ctap), but not -(e.g. nor-binaltorphine) nor -antagonists (e.g. naltrindole) were able to reverse such muscular rigidity [124, 125] . in addition, administration of the selective antagonist methylnaltrexone in the nucleus raphe pontis was able to reverse increased muscle tone after alfentanil in the animal [126] suggesting this nucleus is an additional important site of action of opioids to induce rigidity. clinically this rigidity is a disadvantage because it results in an insufficient ventilation of the patients and is characterized by the following features [127, 128] : • it appears shortly after intravenous injection of a potent opioid. • it can be induced especially in the elderly patient population. • it is potentiated by nitrous oxide (n 2 o). • it is more likely to develop in patients with parkinson's disease. the anatomical correlate by which opioids induce muscular rigidity is the striatum, and, being part of the basal ganglion system, it has the task to control locomotion ( figure ii-58) . within the striatum there is a dense accumulation of opioid binding sites, which interact with dopaminergic d 2 -receptors. similar as in parkinson's disease, there is a reduction in the dopamine level with an ensuing imbalance of the cholinergic transmitter system, both of which are in balance with each other and a necessary prerequisite for the control of muscle tone [129] . while in parkinson's disease, increased muscle tone is induced by decrease of dopaminergic neurons in the striatum, opioid-induced rigidity is due to an enhanced degradation of the transmitter dopamine resulting in a functional deficit of a sufficient level in the nigro-striatal pathway [130] . the exact mode of action of opioids to reduce dopamine level within the nigrostriatal system and induce muscular rigidity, very likely is induced by inhibition of tyrosine hydroxylase, the necessary enzyme for the synthesis of dopamine [131] . due to the interconnection with the inhibitory gabaminergic system, output of gaba in the pallidum declines ( figure ii-58) . this, in turn, causes an overactivity of cholinergic neurons projecting to thalamic neurons. from here the area 6a of the cortical premotor center is activated and the corticospinal tract leads efferents to the anterior horn of the spinal cord [132] . although opioids do not directly affect muscle tone, rigidity rapidly can be reversed by the injection of a competitive or non-competitive muscle relaxant [133] . although the increased efferent output at the neuromuscular junction is not reduced, muscle relaxants induce their action by inhibiting the binding of acetylcholine at the motor endplate ( figure ii-59) . because the gabaminergic system in the putamen is involved in the mediation of opioid-induced muscular rigidity, any increase in gabaminergic transmission can also ease this side effect. such a notion has been supported by results, where a benzodiazepine reduced the increased muscle tone, an effect that could be reversed by the specific benzodiazepine antagonist flumazenil [125] . in addition, significance of the neurotransmitter dopamine in basal ganglia of the cns, which are involved in the regulation of muscle tone 1 = pallidum externum; 2 = putamen; 3 = nucleus caudatus; 4 = thalamus; 5 = hypothalamus; 6 = lobus parietalis; 7 = central grey; 8 = corticospinal tract; 9 = inhibitory dopaminergic pathway; 10 = thalamo-cortical neurons; 11 = substantia nigra since neighboring 2 -receptors interact with the substantia nigra, opioid-related rigidity could be attenuated by the additional administration of the 2 -agonist dexmedetomidine [135] . the miotic action of opioids on the pupil is an easily recognizable and quantifiable effect in man. the neural pathways responsible for regulating pupil size are reasonably well defined. yet, the mechanisms behind this and related effects of opioids on the eye in humans and laboratory animals have just begun to be explored. opioid-induced miosis in the human, dog and rabbit is thought to be mediated through the central nervous system. this action is a specific opioid effect as demonstrated by its antagonism by naloxone. theories have been advanced suggesting comparison between groups p < 0.05 p < 0.01 figure ii-59. alfentanil-induced truncal rigidity in patients following induction of anesthesia. in comparison to the rapid bolus injection of the drug, slow injection over a period of 2 min resulted in a significant lesser reduction of thoracic compliance. the increase in truncal rigidity was instantly reversed by a low dose (25 mg/70 kg body weight) of the fast acting muscle relaxant succinylcholine. adapted from [134] that morphine produces its effects by direct stimulation of the edinger-westphal (preganglionic parasympathetic) nucleus [181] . an alternative view has been postulated that morphine depresses cortical centers, which normally inhibit the edinger-westphal nucleus. others have suggested that miosis is caused by stimulation of opioid receptors located on the iris sphincter, although this opinion seems to be in the minority. the exact site, or sites, of action within the cns, which are responsible for opioid-induced miosis remain obscure. it is generally accepted, however, that sympathetic innervation is not essential, the miotic effect being entirely dependent on the integrity of the parasympathetic system. for example, lee and wang [182] have shown that dogs with a sectioned oculomotor nerve fail to show miosis even with a 30 fold increase in the dose of morphine. in contrast, dogs show normal responses following sympathectomy. while local application of a muscarinic antagonist (scopolamine) that blocks the pupil sphincter completely abolishes the pupillary effects of morphine in the rabbit, application of a sympathetic neuronal blocker (guanethidine) or of an alpha-adrenergic antagonist (phentolamine) that block the pupil dilator had no effect in those experiments. thus, pharmacologic dissection of the autonomic innervation of the pupil suggests that opioid-induced miosis is mediated solely through the parasympathetic system. other cns structures may also be involved in opioid-induced miosis. lee and wang [182] have shown that removal of the cerebral hemispheres potentiates the miotic action of morphine in the dog. they interpreted this effect as being a reflection of the loss of tonic inhibition originating in the occipital lobes. the latter are known to play a regulatory role in pupillary function, particularly with respect to the near-response (accommodation, convergence and miosis) and, hence, their removal might be expected to alter the pupillary response to drugs. the same authors also observed that acute or chronic optic nerve section did not alter the miotic response to morphine in dogs. in humans, it was shown that morphine produced a dose-related miosis under conditions of low ambient light. taken together, these findings suggest that morphine may cause miosis through more than one mechanism. the main neural structures, which are thought to regulate pupillary size are found in the midbrain, mainly the pretectal area and the edinger-westphal nucleus of the oculomotor complex. because neuronal unit activity in the edinger-westphal nucleus has been shown to correlate with light-induced pupillary constriction. opioids therefore depress or abolish spontaneous and light-induced firing of pupilloconstrictor neurons in the pretectal area, while the opposite effect is observed in the edinger-westphal nucleus where a marked increase in spontaneous firing rate resulting in madriasis. it is because of this increase in activity that certain animal species (rat, cat, monkey) demonstrate an opioid-induced mydriasis. in addition, the brain stem region regulating pupil size is known to have multiple inputs, including the cortex and midbrain, and several others can be assumed to exist. depression by morphine of tonic inhibitory input trom the cortex may partially account for the miosis observed by lee and wang [182] . these findings suggest that opioids may act directly on the neurons subserving the parasympathetic light reflex. also, in contrast to other workers, morphine has no local action on the iris. for example, lee and wang [182] could not produce miosis by injecting 20% of an effective systemic dose of morphine (1 mg) directly into the anterior chamber of the eye in dogs. although opioid binding sites have been found in the retina of the rat, cow, toad and skate, opioids injected into the anterior chamber may stimulate retinal receptors in some species, causing miosis via reflex parasympathetic output. following oral ingestion, but also after the systemic administration, opioids also bind to selective receptors located within the intestinal tract. the physiological significance of peripherally located opioid binding sites within the intestine is that of regulation of the propulsive transit. the intestine with a total surface of nearly 400 m 2 is an underestimated important anatomical site as it has a high accumulation of neuronal tissue, which has been termed the enteral nervous system (ens), which acts like a second brain. since there is a close interconnection of the ens with the cns via the vagus nerve, regular impulses to and from the ens are being exchanged. anatomically the intestine is surrounded by two separate syncytial, netlike nervous structures. one is the myentericus plexus (auerbach) located between the longitudinal and the circulatory muscle fibers ( figure ii-60) . the second is the submucosal meissner plexus, located between circulatory and the submucosal muscle fibers. within the auerbach plexus of the intestinal tract, there is a balance between the cholinergic and enkephalinergic neurons: binding of systemically applied opioids to enkaphalinergic receptor sites results in an inhibition of transit followed by constipation. contrarily, cholinesterase inhibitors induce an accumulation of acetylcholine at ach-receptors with an increase in motility and an enhancement of transit. presently, however, not very much is known of the long-term effect of central analgesics on opioid-receptors within the myenteric plexus, and if opioid ligands induce only a constipating effect, whether they also depress the immune system in the intestine or result in a distress the neuroregulatory and endocrine function. while analgesia, respiratory depression, bradycardia, antitussive action and miosis all are centrally induced opioid effects, the most relevant peripheral opiod action is that of constipation [109] [136] . this is most relevant in patients with chronic pain taking an opioid for its attenuation. being one of the major side effects, it often results in the necessity to take a laxative on a routine basis. the cause for such constipation is the constriction of the pylorus resulting in a delay in emptying of the stomach [137] . however, most important, opioids induce a constriction of the small intestine resulting in a delay of the propulsive transit. because selective opioid binding sites are mainly located in the small intestine, opioids inhibit the release of local acetylcholine [136, 137, 138] , followed by a concomitant loss of coordinated propulsive movements of the gut. a constipating effect of opioids on the large intestine is of significantly lesser degree, because this part of the intestinal tract contributes to a much lesser extent to the overall constipating effect. this is because continuous propulsive movements are not seen on this area, and contrary to the small intestine, the percentage of enkephalinergic neurons is significantly lower [139, 140] . in addition, enkephalin derivatives are able to inhibit transit in the small while at the same time increasing contractions in the large intestine [141] . systemicallyselective applied opioids therefore primarily interact with enkephalinergic neurons in the antrum, the duodenum, and the small intestine, all of which results in a delay of transit [142, 143, 144, 145] . the constipating effect of an opioid can be reversed by a selective peripheral acting antagonists such as methylnaltrexone [146, 147] or alvimopane [148] . opioids, which interact primarily with the -opioid receptor induce a lesser constipating effect [148, 149] , while -selective ligands induce no effect on gastrointestinal transit [150] . comparable to a ketamine-or a volatile anesthetic based regimen, an opioid-based anesthesia results in a longer delay of gastrointestinal emptying in the postoperative period [151, 152] (figure ii-61) . this, however, is clinically of little significance, as the potential constipating effect does not last longer than 48 h after anesthesia. contrarily to many other anesthetics, opioids in general do not depress the cardiovascular system. this is also reflected in the higher therapeutic range (ld 50 /ed 50 ) being derived in the animal (table ii11) . such data can also be conveyed to the human, since a wide therapeutic margin of safety is directly correlated with a lack in cardiovascular impairment. while carfentanil, with a potency twice that of sufentanil, is solely used in veterinary medicine for the immobilization of wild animals [153] , lofentanil (20fold potency of fentanyl), due to its intensive receptor binding, is characterized by a duration of action of 24 h [154] . both fentanyl derivates are not in clinical use, because the high potency and the intense receptor binding would be difficult to handle in patients. from the table, however, it is obvious that the higher the selectivity to the receptor site, and the higher the potency, the lesser the amount of cardiovascular depression [33, 35, 103, 155, 156, 157] . following the injection of potent opioids, bradycardia is the most prominent cardiovascular effect seen in patients. this is due to a direct central stimulation of the nucleus nervi vagi and a typical effect of -ligands. thereafter, a reduction of the sympathetic drive is initiated resulting in an overexpression of parasympathetic activity. also, a direct peripheral negative inotropic activity with a potentiation of acetylcholine release at the sinus node of the heart is discussed [158] . the increase in vagal tone and the reduction of sympathetic drive on the peripheral vasculature results in a decline of mean arterial pressure. a reduction of sympathetic tone on vessel tone and a reduction of resistance is also termed as "pooling" of circulating blood volume. such a reduction of peripheral resistance in certain cases may be of benefit for the patient, as it is accompanied by a reduction in afterload of the heart [159, 160] . bradycardia, the reduced peripheral resistance (i.e. afterload of heart) as well as the pooling effect with a reduction of preload of the heart, can be of benefit for a patient with myocardial infarction. this is because those three variables are major determinants in myocardial oxygen consumption (mvo 2 ) [160, 161] . it, however, should be noted that the sympatholytic action with pooling of blood volume induced by potent opioids might demask a previously compensated hypovolemic condition in a patient resulting in significant hypotension. for instance, in patients with multiple trauma a reduced dose of the opioid should be given, either diluted or slowly injected while measuring blood pressure continuously. in general, however, especially in polytraumatized patients, opioids are of benefit, as they reduce the stress-related release of hormones and particularly of angiotensin ii, maintaining the effect of circulating catecholamines on the vasculature. opioid-related bradycardia with an accompanying hypotension can rapidly be reversed with increasing doses of the vagolytic agent atropine (0.25-05-1.0 mg/kg body weight). the incidence and the severity of such a drop in blood pressure cannot be foreseen. it is related to the autonomic basal tone of the patient and the dose of the injected potent -ligand ( figure ii-62) . depending on the product, the autonomic basal tone of, and the applied dosages to the patient, either parasympathetic (inhibitory) and/or a sympathetic (excitatory) symptoms are induced (table ii12) . such clinical effects can be diminished by atropine, an -blocker (e.g. phenoxybenzamine), a ß-blocker (e.g. propranolol), and a ganglionic blocker (e.g. hexamethonium) respectively [103] . the stimulatory effects of opioids can also be explained in the laboratory where stimulation of cyclic amp formation, phosphinoside hydrolysis, and the elevation of intracellular calcium, resulting from mobilization of calcium stores and by stimulating influx, which leads to an increased neurotransmitter release and neurotransmission [163] . thus, at the cellular level these changes may underlie the opioid stimulatory effect. in addition, such stimulation is also discussed as playing a part in the development of tolerance to opioid drugs [164] . the effect of increasing doses (mg/kg body weight) of potent opioids on the cardiovascular system of the canine, where low amounts result in parasympathetic activation, and high to massive doses induce an increase of sympathetic drive. adapted from [103] in opioid-based anesthesia, vagal-or sympathetic-induced side effects can be reduced or eliminated by the following techniques: 1. the preliminary administration of atropine (up to 1 mg/kg body weight). 2. the simultaneous administration of a volatile anesthetic (n 2 o, enflurane, desflurane, sevoflurane). 3. the simultaneous use of a neuroleptic agent (e.g. droperidol, haloperidol). 4. the simultaneous use of a benzodiazepine (e.g. diazepam, midazolam, lorazepam). 5. the simultaneous use of a hypnotic (e.g. barbiturate, etomidate, propofol). adapted from [103, 162] all these agents induce a depression of cns activity in different areas of the central nervous system, which results in equilibrium of the autonomic nervous system discharge, thus, reducing the overshoot of sympathetic and/or parasympathetic tone ( figure ii-63) . mixed agonist/antagonists, when given in dosages above the therapeutic range, induce a cardiostimulatory sympathomimetic effect, which purportedly is induced via stimulation of -receptor sites [165] . as a result, tachycardia, an increase in peripheral vascular resistance, and an increase in pulmonary artery pressure are induced (table ii13) , all of which increase myocardial oxygen consumption (mvo2). therefore agonist/antagonists should not be given above their therapeutic range in patients with mi or with a preexistent cardiovascular disease [166] . a malfunction at the atrio-ventricular node in the myocardium, followed by prolongation of the p-q interval is a phenomenon, which can be induced in patients demonstrating a preexisting abnormal conduction system in the heart. such prolongation manifests itself especially when potent opioids are being administered (fentanyl, sufentanil), whereby the opioid-induced acetylcholine release induces a stimulation of vagal activity. thus, patients already having a prolongation of p-q time or who present a sick-sinus syndrome, extreme bradycardia has to be anticipated, which could result in concomitant cardiac arrest. in order to prevent such a scenario, the opioid should not be given as a bolus, but rather as a diluted solution. in addition, the solution should be injected slowly over a long period of time neuroleptics block afferents from entering the ascending reticular formation, which increase vigilance; tranquillizers protect the hippocampus from an excitatory activation, while barbiturates, hypnotics and volatile anesthetics primarily block the cerebral cortex from arousal table ii13 . different cardiovascular effects of -ligands, mixed agonist/antagonists, and partial agonists resulting in a decrease (⇓) or an increase (⇑) blood pressure heart rate pulmonary artery pressure adapted from [166, 167, 168] of at least 2 min. if, however, extreme bradycardia is recognized on the monitor, atropine is the agent of choice (0.5-1.0 mg/kg body weight) for rapid reversal. in very extreme cases, the antiarrythmic agent metaproterenol may become necessary, as it is able to increase atrio-ventricular conduction. high doses of methadone or its derivative -levoacetylmethadol (laam) may result in life threatening torsades de points with the potential of ensuing ventricular fibrillation. predisposing factors for the development of such a situation are a prolongation of atrio-ventricular conduction time, hypopotassemia, and/or the simultaneous intake of agents, which inhibit metabolism of the opioid (e.g. tricyclic antidepressants, imidazol derivatives, antimalaria agents, or antihistaminics). a direct negative inotropic effect on the myocardium has been demonstrated in the isolated papillary muscle and in the langendorff preparation of the heart for a variety of opioids [169, 170] . such direct effects, however, are not of clinical significance, because such a depression is only evident in concentrations above the therapeutic range. in addition, compensatory cardiovascular and the autonomic regulatory mechanisms come into play when an opioid is given to a subject. following the intravenous injection of pethidine (meperidine, usp), hypotonia and syncope may result. because of the atropine-like molecular structure of this agent, tachycardia, as well as reflex bradycardia can be observed [171] . for the reason of these potential side effects pethidine should not be given to patients with myocardial infarction [109] . in addition, it is observed that in a shock-like situation, due to the release of endogenous opioids (enkephalins, endorphins), the additional administration of an exogenous opioid results in an additional occupation of opioid binding sites within the myocardium. this aspect is followed by a negative inotropic effect with an unfavorable consequence on hemodynamics [172] . some experimental work has postulated a putative direct negative inotropic effect of n 2 o in an opioid-based anesthetic regimen [173] . since this is mainly seen when n 2 o is given in concentrations above 50% with a resultant drop in fio 2 , this very both agents dose-dependently reduce adrenaline-induced ventricular extrasystoles. adapted from [180] pvc-premature ventricular countraction likely is due to an insufficient myocardial oxygen supply. in addition, high concentrations of n 2 o have a direct vasodilatory effect, resulting in a reduction of venous return to the heart and a drop in blood pressure [174] . it therefore is advocated that in patients receiving opioid anesthesia with a preexisting cardiovascular disease, the optimal concentration in fio 2 should be around 0.5. opioids also have been demonstrated to induce an anti-arrhythmic effect. this has been shown in the animal for meptazinol [175] and in experimental coronary artery occlusion, using fentanyl, sufentanil and carfentanil respectively 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neuroleptiques en cours d'intervention part ii drug interactions of clinical significance with opioid analgesics drug interaction and side effects index™ biotransformation von fentanyl. ii. akute arzneimittelinteraktion -untersuchungen bei ratte und mensch the magnitude and the duration of respiratory depression produced by fentanyl and fentanyl plus droperidol in man a quick guide to common drug interaction, in patient care plasma protein binding of phenytoin after cholecystectomy and neurosurgical operations intravenous fentanyl kinetics morphine and phenytoin binding to human plasma protein in renal and hepatic failure plasma concentrations of fentanyl in normal surgical patients and those with severe renal and hepatic disease reduction in the incidence of awareness using bis monitoring bremazocine: a potent, long-acting opiate kappa-agonist tifluadom (kc-5103) induces suppression and latency changes of somatosensory-evoked potentials which are reversed by opioid antagonists 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patienten eeg findings in neuroleptanalgesia central nervous effects of neuroleptanalgesia as induced by haloperidol and phenoperidine exzitatorische und inhibitorische phänomene am zentralnervensystem, verursacht durch fentanyl perfusion of the fourth cerebral ventricle with fentanyl induces naloxone reversible hypotension, bradycardia, baroreflex depression and sleep in unanaesthetized dogs regional distribution of opiate receptor binding in monkey and human brain the effects of morphine and pethidine on somatic evoked responses in the midbrain of the cat, and their relevance to analgesia wechselwirkungen zwischen dem system der schnellen schmerzempfindung und dem des langsamen über die zweiteilung der schmerzempfindung und des schmerzgefühl, in schmerz comparative study of cardiovascular, neurological, and metabolic side effects of eight narcotics in dogs genralized grand mal seizure after recovery from uncomplicated fentanyl-etomidate anesthesia another case of grand mal seizure after fentanyl aministration tonic-clonic activity after sufentanil seizure-like movements during fentanyl infusion with absence of seizure activity in a simultaneous eeg recording the effects of high-dose fentanyl on cerebral circulation and metabolism in rats opioid analgesics and antagonists, in the pharmacological basis of therapeutics diagnose and treatment of symptoms of the respiratory tract 3 h-codeine binding in the guinea pig lower brain stem physiology and pharmacology of vomiting treatment of postoperative nausea and vomiting after outpatient surgery with the 5-ht3 antagonist ondansetron comparison of ondansetron versus placebo to prevent postoperative nausea and vomiting in women undergoing ambulatory gynecologic surgery coinsensus guidelines for managing postoperative nausea and vomiting postoperatives erbrechen -ein score zur voraussage des a meta-analysis of nausea and vomiting following maintenance of anaesthesia with propofol or inhational agents dexamethasone for the 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droperidol on the dopamine metabolism of the rat striatum morphine katalepsy in the rat: relation to striatal dopamine metabolism tyrosine hydroxylation in the rat striatum after fentanyl and droperidol in vivo decrease of neocortical choline acetyltransferase after lesion of the globus pallidum in the rat attenuation of fentanyl-induced truncal rigidity die lungencompliance wird durch die rasche injektion von alfentanil beeinträchtigt dexemedetomidine, acting through central alpha2-adrenoceptors, prevents opiate-induced muscle rigidity in the rat gastrointestinal effects of opioids naloxone and morphine inhibit gastric emptying of solids effect of synaptic transmission blockade on morphine action in the guinea pig myenteric plexus enkephalin-like immunoreactivity in the human gastrointestinal tract autoradiographic localisation of opiate receptors in rat small intestine influence of opiates on colonic motility relative involvement of mu, kappa, and delta receptor mechanisms of opiate-mediated antinociception in mice inhibition of gastrointestinal transit by morphine in rats results primarely from direct drug action on gut opioid sites effect of morphine on gastric emptying a comparison of the effect of oral controlled release morphine and intramuscular morphine on gastric emptying methylnaltrexone prevents morphine-induced delay on oral-cecal transit time without affecting analgesia: a double-blind randomized placebo-controlled trial oral methylnaltrexone for opioid-induced constipation effects of adl 8-2698, a peripherally restricted mu opioid anntagost, on gut motility in methadone and laam-dependent patients with opioidinduced constipation. a dose-ranging study the effects of morphine and nalbuphine on intestinal transit in mice peptide opioid antagonist seperates peripheral and central opioid antitransit effects die gastro-coekale transitzeit nach fentanyl/midazolam-im vergleich zur enfluran-und ketamin/midazolam-narkose keine hemmung der intestinalen 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and analgesic efficacy of the agonist-antagonist opioids a review of its pharmacological properties and therapeutical uses, in new drug series contractile responses to morphine, piritramid and fentanyl: a comparative study of effects on the isolated myocardium myocardial opiate receptor activity is stereospecific, independent of muscarinic receptor antagonism, and may play a role in depressing myocardial function utilisation de la pentazocine comme analgesique pour le traitement des douleurs post-operatoires. etude comparative entre le pethidine, la piritramide et la pentazocine, in utilisation de la pentazocine en anesthesie et reanimation hemodynamic changes following corticosteroid and naloxone infusion in dogs subjected to hypovolemic shock without resuscitation nitrous oxide as an adjunct to narcotic anesthesia does nitrous oxide or a reduced fi02 alter the hemodanymic function during high dose sufentanil anesthesia? the antiarrhythmic effect of meptazinol effects of high doses of fentanyl on myocardial infarction and cardiogenic shock in the dog the antifibrillatory effect of fentanyl, sufentanil, and carfentanbil in the acute phase of local myocardial ischemia in the dog antifibrillatory action of the narcotic agent fentanyl protective effect of the vagotonic action of morphine sulfate on ventricular vulnerability les effets anti-arrythmiques des opiaces. comparison avec un beta-bloqueur chez le chien. cah d'anesthesiol opposite pupillary effects in the cat and the dog after microinjection of morphine, normorphine and clonioline in the edinger-westphal nucleus mechanism of morphine -induced miosis in the dog key: cord-274315-08mk8a86 authors: disano, krista d.; stohlman, stephen a.; bergmann, cornelia c. title: an optimized method for enumerating cns derived memory b cells during viral-induced inflammation date: 2017-06-15 journal: journal of neuroscience methods doi: 10.1016/j.jneumeth.2017.05.011 sha: doc_id: 274315 cord_uid: 08mk8a86 abstract background cns inflammation resulting from infection, injury, or neurodegeneration leads to accumulation of diverse b cell subsets. although antibody secreting cells (asc) within the inflamed cns have been extensively examined, memory b cell (bmem) characterization has been limited as they do not secrete antibody without stimulation. moreover, unlike human bmem, reliable surface markers for murine bmem remain elusive. new method using a viral encephalomyelitis model we developed a modified limiting dilution in vitro stimulation assay to convert cns-derived virus specific bmem into asc. comparison with existing methods stimulation methods established for lymphoid tissue cells using prolonged stimulation with viral lysate resulted in substantial asc loss and minimal bmem to asc conversion of cns-derived cells. by varying stimulation duration, tlr activators, and culture supplements, we achieved optimal conversion by culturing cells with tlr7/8 agonist r848 in the presence of feeder cells for 2days. results flow cytometry markers cd38 and cd73 characterizing murine bmem from lymphoid tissue showed more diverse expression patterns on corresponding cns-derived b cell subsets. using the optimized tlr7/8 stimulation protocol, we compared virus-specific igg bmem versus pre-existing asc within the brain and spinal cord. increasing bmem frequencies during chronic infection mirrored kinetics of asc. however, despite initially similar bmem and asc accumulation, bmem prevailed in the brain, but were lower than asc in the spinal cord during persistence. conclusion simultaneous enumeration of antigen-specific bmem and asc using the bmem assay optimized for cns-derived cells enables characterization of temporal changes during microbial or auto-antigen induced neuroinflammation. antibody (ab) secreting cells (asc) and serum ab are essential immune components that neutralize pathogens during infection (igg + bmem) or independent responses (igm + bmem) following initial antigen exposure and can persist independent of antigen within secondary lymphoid tissue (slt) for years (kurosaki et al., 2015; taylor et al., 2012) . although bmem do not spontaneously secrete ab, minimal stimulation requirements, including t cell help and/or secondary encounter of antigen can trigger rapid differentiation into antigen-specific asc or re-seed gc, thereby promoting isotype switching and somatic hypermutation (kurosaki et al., 2015; zuccarino-catania et al., 2014; pape et al., 2011; dogan et al., 2009; hebeis et al., 2004; aiba et al., 2010) . in addition to contributing to long-lived humoral immunity, bmem function as potent antigen presenting cells and as immune modulators by secreting cytokines (shimoda and koni, 2007; duddy et al., 2007; adlowitz et al., 2015; lund, 2008; lino et al., 2016) . although studies of b cells within the inflamed cns have commonly focused on asc and ab specificity, b cell populations accumulating during cns infection, injury, and neurodegeneration are diverse and include bmem (duddy et al., 2007; niino et al., 2009; krumbholz et al., 2012; michel et al., 2015; metcalf and griffin, 2011; cepok et al., 2006; phares et al., 2014; dang et al., 2015; ankeny et al., 2009) . however, the role of bmem within the inflamed cns is relatively unexplored due to limitations of reliable surface markers, particularly in murine models. human bmem are classically distinguished by expression of cd27, a protein belonging to the tumor necrosis factor receptor (tnfr) family that provides signals regulating entry into plasma cell lineage (klein et al., 1998; tangye et al., 1998) . more detailed human bmem phenotyping revealed heterogeneous populations, including cd27 − bmem, and markers have expanded to include specific patterns of cd38, cd21, cd24, cd19, b220, fcrh4 and cd25 (amu et al., 2007; küppers, 2008; sanz et al., 2008) . while this panel has aided in identifying several reliable markers of human bmem, murine bmem characterization is limited by the low frequency of bmem and minimal expression of cd27 (xiao et al., 2004; anderson et al., 2007; liu et al., 1996; ridderstad and tarlinton, 1998) . although several markers including cd73, cd38, cd80 and pd-l2 have been proposed to define at least five subsets of bmem, these markers are also expressed by several other b cell phenotypes within slt (zuccarino-catania et al., 2014; anderson et al., 2007; conter et al., 2014; tomayko et al., 2010) . moreover, our own studies of murine b cell subsets in the central nervous system (cns) have revealed unique patterns of activation markers compared to peripheral b cell counterparts, further complicating identification of cns b cell subsets based on well-defined slt markers (disano et al., 2017) . for example, cd80, a marker of cd4t cell help and a proposed marker defining subpopulations of bmem within slt, was found on multiple b cell phenotypes within the cns (disano et al., 2017) . bmem analysis in vivo has largely relied on protein immunizations in b cell receptor (bcr) transgenic mice to increase bmem frequencies, or on antigenic challenge in naïve recipients of adoptively transferred antigen-specific b cells. both in vitro and in vivo bmem to asc conversion has been shown to require proliferation (slifka and ahmed, 1996b; cao et al., 2010; pinna et al., 2009; tangye and hodgkin, 2004; bernasconi et al., 2002; kometani et al., 2013) . quantitative assessment of bmem frequency and antigen specificity thus include lengthy elisa based limiting dilution assays (lda) requiring 2-3 weeks of stimulation or shorter 3-6 day in vitro stimulation methods to convert bmem into asc, which are measured by conventional elispot (slifka and ahmed, 1996b; cao et al., 2010; pinna et al., 2009; amanna and slifka, 2006; jahnmatz et al., 2013; walsh et al., 2013; crotty et al., 2004; buisman et al., 2009) . these methods to define bmem antigen specificity and relative frequencies have focused on peripheral blood or slt using tlr agonists to stimulate in vitro bmem conversion to asc. to the best of our knowledge these approaches have not been applied to cns-derived bmem which are exposed to a vastly distinct microenvironment. prolonged isolation procedure of lymphocytes from the cns as well as their prior in vivo exposure to toxic factors may require fine-tuning methods to define bmem kinetics and specificity during cns infection, injury, and neurodegeneration. in the present study, we analyzed bmem marker expression on cns infiltrating b cells and optimized in vitro stimulation methods to enumerate virus-specific bmem in the cns using neurotropic coronavirus jmhv-induced encephalomyelitis. in this model, virus introduced into the brain spreads to spinal cords (wang et al., 1992) . although t cells clear infectious virus from both organs within 14-16 days post infection (p.i.), virus establishes persistence characterized by low levels of persisting viral rna and elevated levels of chemokines and cytokines predominantly in spinal cords (phares et al., 2014) . asc emerging within the cns after initial viral control maintain persisting viral rna at low levels and prevent viral recrudescence (lin et al., 1999; marques et al., 2011) . isotypeunswitched igg − b cells accumulating early during infection are progressively replaced by more differentiated igd − igm − isotypeswitched bmem and asc (phares et al., 2014) . asc are recruited directly to brain and spinal cord in a cxcr3/cxcl10 dependent manner (marques et al., 2011) . although the initial percentage of asc within total b cells is similar in brain and spinal cords, asc accumulate faster and to a higher percentage in spinal cord during viral persistence (phares et al., 2014) . while igg + bmem emerge in the brain (phares et al., 2014) , their relative recruitment to spinal cords, specificity and potential local conversion to asc remains unknown. distinct cd38 and cd73 expression patterns among cns infiltrating b cells relative to slt counterparts limited bmem identification by flow cytometry. furthermore, in vitro bmem stimulation protocols optimized for splenocytes failed to convert cns bmem, suggesting cns-derived bmem succumb to cell death. this was supported by reduced pre-existing asc using similar culture conditions compared to direct ex vivo elispot asc. comparison of tlr7/8 and tlr9 agonists as bmem activators, supplementation with feeders and il-2, as well as reduced culture length revealed optimal cns-derived bmem conversion is achieved by 2 day stimulation with the tlr7/8 agonist r848 and irradiated splenocyte feeders. bmem analysis during jhmv infection indicated bmem accumulated prominently during chronic infection, similar to asc, and revealed similar igg secretion levels as asc. however, ratios of asc to bmem were inverted when comparing brains and spinal cords. overall, this protocol provides an optimized assay to define bmem specificity, quantity, and isotype within inflamed cns tissue. wild type (wt) c57bl/6 mice were purchased from the national cancer institute (frederick, md). six-to seven-week old mice were infected intracranially with 1000 plaque forming units (pfu) of the gliatropic monoclonal ab derived variant of jhmv designated j.2.2v-1 (fleming et al., 1986) . cells isolated from brains and spinal cords were used for in vitro stimulation and elispot assays. for immunization and splenic b cell analysis, six to seven-week-old mice were injected intraperitoneally (ip) with 1 ml of jhmv dm (9.8 × 10 6 pfu/ml). all animal procedures were approved by the institutional animal care and use committee of the cleveland clinic and were conducted in compliance with the guide for the care and use of laboratory animals from the national research council. mice were perfused transcardially with 1 × pbs (cleveland clinic research institute cell services core, cleveland, oh) prior to decap-itation. the skin was removed to expose the skull and a midline incision was made. the skull was then removed to resect the brain. following brain dissection, the dorsal skin along the spinal column was removed to cut the spinal column at the level of the iliac crest. the spinal cord was flushed at the caudal opening of the spinal column with 1 × pbs using a 10 ml syringe and an 18 gauge needle. mononuclear cells from spleen, brain, or spinal cord were isolated from pooled organs of 3-4 mice per time point. spleens were dissociated mechanically and red blood cells lysed. for cell isolation from brains or spinal cords, tissues were minced and digested in 5 ml rpmi 1640 (cleveland clinic research institute cell services core, cleveland, oh) supplemented with 10% fetal calf serum (fcs) (hyclone, logan, utah), 100 l of collagenase type i (100 mg/ml; worthington biochemical corporation, lakewood, nj) and 20 l (200u) of dnase i (25 mg/ml) (roche, indianapolis, in) for 40 min at 37 • c. collagenase activity was terminated by addition of 0.1 m edta (ph 7.2) at 37 • c for 5 min. following centrifugation, cells were resuspended in rpmi supplemented with 2% fcs, adjusted to 30% percoll (ge healthcare life sciences, pittsburgh, pa) and underlayed with 70% percoll. after centrifugation for 30 min at 850g, mononuclear cells were recovered from the 30/70% percoll interface and washed with rpmi supplemented with 2% fcs. cells subjected to flow cytometric analysis were resuspended in fluorescent-activated cell sorter (facs) buffer (pbs with 0.5% bovine serum albumin), whereas cells for in vitro stimulation were resuspended in rpmi 1640 containing 2 mm l-glutamine, 2 mm non-essential amino acids, 1 mm sodium pyruvate, 25 g/ml gentamicin, 5 × 10 −5 m 2-mercaptoethanol and 10% fcs (rpmi complete). cells were incubated in facs buffer supplemented with 1% mixed serum containing mouse serum (thermo fisher scientific, waltham, ma), goat serum (atlanta biologicals, flowery branch, ga), and horse serum (vector laboratories, youngstown, oh) at 1:1:1 and 0.5 l rat anti-mouse fc␥iii/ii mab (2.4g2; bd bioscience, san jose, ca) per 10 6 cells for 20 min on ice prior to staining. expression of cell surface markers was determined by staining on ice for 30 min with ab specific for cd45 (30-f11; percp-cy5.5), cd19 (1d3; pe-cf594), cd73 (ad2: pe-cy7) (bd biosciences, san jose, ca), igm (eb131-15f9; pe), igd (11-26; apc), and cd38 (90; pe) (ebioscience, san diego, ca). cells were analyzed on a bd lsrii flow cytometer (bd biosciences, san jose, ca) using flowjo (version 9.7.6) software (tree star, ashland, or). doublet exclusion and live gating were applied as previously described (disano et al., 2017) . dead cells comprised less than 10% of total sc or brain cells. for all results, plots are representative of 3-4 independent experiments. splenocytes from naïve c57bl/6 mice were used as feeder layers during b cell stimulation. following red blood cell lysis and washing, resuspended splenocytes were irradiated with a dose of 3000 rad using a shepherd irradiator (jl shepherd and associates, san fernando, ca). for stimulation with viral lysate, 60 × 10 6 splenocytes in 10 ml rpmi complete were mixed with 0.5 ml jhmv lysate (2 × 10 6 pfu/ml) and incubated for 1 h (h) at 37 • c prior to irradiation. irradiated feeders with or without viral lysate were washed three times at 450 × g for 5 min and resuspended at 5 × 10 5 cells in 0.1 ml rpmi complete or rpmi complete containing either 5 g/ml cpg or 1 g/ml r848 (invivogen, san diego, ca) and supplemented or not with 10 ng/ml recombinant mouse il-2 (biolegend, san diego, ca). feeders with various stimulating agents were then plated into 96-well flat-bottom tissue culture plates (corning, tewksbury, ma). to block proliferation of stimulated cns derived effector cells, cell suspensions were also irradiated at 3000 rad, washed, and resuspended in rpmi complete prior to stimulation. effector cells from cln, spleens, brains, or spinal cords of jhmv infected wt mice were resuspended at a starting concentration of 1 × 10 (pape et al., 2011 ), 1.25 × 10 (taylor et al., 2012 , and 1.25 × 10 4 cells per 0.1 ml rpmi complete containing 5 g/ml cpg or 1 g/ml r848 with or without il-2 10 ng/ml, respectively. twofold dilutions (3 wells per dilution; 12 total wells per condition) were plated into 96-well flat-bottom tissue culture plates containing 5 × 10 5 irradiated feeder splenocytes and incubated for 10 h or 2, 3, 4, or 5 days at 37 • c and 5% co 2 . after stimulation, cells were washed three times with 0.2 ml prewarmed 37 • c rpmi complete per well and centrifuged at 190 × g for 5 min. after the last wash, cells were resuspended in 0.2 ml rpmi complete per well and transferred to elispot plates. jhmv-specific igg asc were measured by elispot assay as previously described (disano et al., 2017) . briefly, 96-well pvdf multiscreen hts ip plates (emd millipore, billerica, ma) were coated with jhmv dm (∼5 × 10 5 pfu/well) overnight at 4 • c. serial dilutions of cells plated in triplicate were incubated for 4 h at 37 • c and 5% co 2 . asc was detected by sequential incubation with biotinylated rabbit anti-mouse igg (0.5 g/ml; southern biotech, birmingham, al) overnight at 4 • c, streptavidin horseradish peroxidase (1:1000; bd biosciences, st. louis, mo) for 1 h at room temperature, and filtered 3,3 -diaminobenzidine substrate (sigma-aldrich, st. louis, mo) in 0.3% hydrogen peroxide. brown spots were visible within 2-4 min and the reaction was terminated using cold tap water. spots were counted using an immunospot elispot reader (cellular technology ltd., shaker heights, oh). minimum and maximum spot size cutoffs were set to 0.0009 mm 2 and 0.2295 mm 2 , respectively and spots were analyzed using diffuse processing and spot separation size of 3.00-5.00. following automated counting, wells were re-counted manually for exclusion of artifacts. wells containing ≥4 spots scored positive for virusspecific asc. for analysis, 3-5 wells within a linear dilution range were averaged for each stimulation condition. data were analyzed using prism (version 6.0) software (graph-pad). statistical significance between the experimental groups was assessed using a two-tailed paired t-test. in all cases, a p value of <0.05 was considered significant. data is representative of 2-3 experiments with 3-4 pooled mice per experiment. several phenotypic markers have been described to distinguish murine bmem from mature naïve or other b cell subsets in lymphoid organs, including cd80, pd-l2, cd38 and cd73 (anderson et al., 2007; tomayko et al., 2010) . however, previous comparative analysis of temporally matched cervical lymph node (cln) and cns b cell subsets for expression of the activation marker cd80 and the gc b cell marker gl7 revealed distinct patterns (disano et al., 2017) . for example, a larger fraction of several cns b cell phenotypes expressed cd80 compared to peripheral counterparts and expression was sustained, unlike transient expression in cln. this suggested that conventional phenotypic patterns characterizing b cell subsets, including bmem, in slt are insufficient to reliably mark similar subsets in the cns. we therefore asked if cd38 and cd73, common markers for murine bmem within slt, showed similar expression patterns on b cells derived from cln versus brain during viral encephalomyelitis. the transmembrane receptor cd38 is involved in apoptosis, activation, differentiation, and proliferation (vences-catalán and santos-argumedo, 2011). cd38 is highly expressed on naïve mature b cells and is downregulated during activation. its expression is lowest on gc b cells and isotype-switched asc. by contrast, isotype-switched bmem exhibit prominent cd38 expression, while expression on gc independent igm + bmem is unclear (anderson et al., 2007; ridderstad and tarlinton, 1998; vences-catalán and santos-argumedo, 2011) . distinct from cd38, expression of cd73, a surface glycoprotein regulating extracellular atp and adenosine levels, is low on naïve b cells but upregulated on antigen experienced and gc b cells (conter et al., 2014) . cd73 expression in slt delineates several subpopulations of bmem and is absent amongst plasmablasts and plasma cells (conter et al., 2014; tomayko et al., 2010) . cd45 hi cd19 + b cells from the brain at day 38 p.i. as well as four distinct differentiation subsets defined by their surface ig as naïve mature b cells (igd + igm + ), activated b cells (igd int igm + ), pre-gc/gc/isotype-unswitched (igd − igm + ) bmem and isotypeswitched bmem/asc (igd − igm − ) were thus compared to analogous cln day 14 p.i. subsets for cd38 and cd73 expression (fig. 1) . these time points were chosen to reflect abundant b cell subsets in the respective organs. fig. 1a shows representative gating strategies of the four populations with naïve b cells represented by region 1, activated b cells by region 2 igd − igm + bmem as region 3 and isotype-switched bmem/asc as region 4. analysis focused on day 14 p.i. when gc formation in cln is prominent and on day 38 p.i. when more differentiated isotype-switched b cells dominate over less differentiated b cells in the brain during jhmv persistence (phares et al., 2014; disano et al., 2017) . consistent with a minor population of b cells forming gcs and thus a predominant igd + igm + naïve b cell population in cln throughout infection ( fig. 1a ) (disano et al., 2017) , the vast majority of total cln b cells (>90%) expressed cd38 at day 14 p.i. (fig. 1b) . while cd38 expression marked all naïve b cells, cd38 progressively decreased on b cells transitioning to an isotype-switched phenotype (fig. 1b) . cd38 expressing cells were reduced to 70% in igd int igm + b cells, to 27% in igd − igm + pre-gc/gc b cells and 13% in igd − igm − isotype-switched cells ( fig. 1b ; populations 1,2,3, and 4, respectively, in panel a), which comprise a relatively small proportion of cd19 + cells within the cln (disano et al., 2017) . brain cd19 + b cells revealed overall reduced cd38 expression (65%) compared to the cln (fig. 1c ). while igd + igm + b cells also all expressed cd38 (99%), a smaller proportion of more differentiated cells downregulated cd38 compared to their cln counterparts. although expression was progressively lower relative to the naïve population in activated (igd int igm + ), pre-gc/isotype-unswitched bmem (igd − igm + ), and isotype-switched b cells (igd − igm − ), the percentage of cd38 + cells remained >50% even in the isotype-switched population (fig. 1c) . the pattern of cd73 expression was even more diverse than cd38 between b cell subsets in cln and brain ( fig. 1d and e) . in cln, the proportion of cd73 + cells in total cd19b cell was <10%, coincident with sparse expression in naïve igd + igm + b cells. expression remained low on igd int igm + b cells (∼10%; fig. 1d ), but was increased on igd − igm + cells (41%). the proportion of cd73 + cells was the highest (80%) in isotype-switched igd − igm − b cells. this profile is consistent with cd73 upregulation on antigen experienced and gc b cells (conter et al., 2014) . relative to cln, cd73 expression was 4-fold higher (41%) among cns infiltrating cd19 + b cells, reflecting higher proportions of activated and isotypeswitched b cells accumulating in the cns during persistence. however, with the exception of naïve b cells, cd73 was differently regulated on the corresponding b cell subpopulations within the brain (fig. 1e) . igd int igm + b cells segregated into a population of no or low expressors and ∼50% cd73 high expressors. the igd − igm + b cells were also split into a cd73 non-expressing and expressing phenotype, although cd73 expression levels were slightly lower than in igd int igm + b cells. isotype-switched igd − igm − b cells exhibited an even more pronounced decline in both the proportion and intensity of cd73 expression (fig. 1e) . whether the distinct patterns of cd38 and cd73 expression on cns versus cln b cells reflects preferential recruitment or local influences remains unclear. nevertheless, the data clearly indicate that activation or bmem markers characterizing b cell subsets in lymphoid tissue are not suitable to identify cns b cell subsets. culture conditions designed to quantify virus-specific bmem have largely been optimized using cells derived from lymphoid tissue and rely on various in vitro stimulation strategies to convert bmem into asc, which are then measured by elispot in a separate plate. in vitro this process requires proliferation and thus prolonged stimulation more than 24 h, the minimum time required to trigger division by bmem (slifka and ahmed, 1996b) . for instance, following lcmv infection maximal virus-specific bmem conversion into asc was initially achieved by stimulating splenocytes from lcmv infected mice at serial dilutions with irradiated lcmv infected carrier splenocytes as a source of viral antigen for 6 days p.i (slifka and ahmed, 1996b) . although in vitro stimulation often relies on nonspecific polyclonal activators as infected cells often provide insufficient antigen stimulus, specific antigen stimulation is more selective as it minimizes excessive b cell proliferation and bystander ab production to irrelevant antigens (hebeis et al., 2004) . to apply virus-specific in vitro stimulation lda to jhmv infection, we initially infected mice ip, harvested splenocytes at day 28 p.i. and stimulated serial cell dilutions in 96-well flat bottom culture plates with irradiated feeders pre-incubated with viral lysate for 4, 5, or 6 days at 37 • c. after in vitro stimulation, and washing to remove virus and antibody, cells were transferred to virus-coated elispot membrane plates and incubated at 37 • c for 4 h to detect virus-specific igg asc. asc quantified after stimulation reflect both pre-existing asc and asc derived from bmem. to quantify preexisting asc, serial splenocyte dilutions were subjected to a direct ex vivo 4 h elispot assay. decreased asc frequencies following incubation with viral lysate for 4 or 6 days relative to 5 days (data not shown), revealed 5 days stimulation was optimal for asc survival and bmem conversion following peripheral jhmv infection. however, splenocyte stimulation with feeders pre-incubated with viral lysate alone did not increase asc frequencies relative to ex vivo elispot analysis (fig. 2a) . these results suggested viral lysate provided insufficient stimulation to mediate bmem conversion. bmem proliferation and conversion to asc can be stimulated using pokeweed mitogen (pwm) or tlr agonists, including synthetic oligonucleotide cpg (tlr9 agonist) and r848 (tlr7/8 agonist), which selectively convert bmem, but not naïve b cells into asc (cao et al., 2010; pinna et al., 2009; jahnmatz et al., 2013; walsh et al., 2013; crotty et al., 2004; hawkins et al., 2013) . we therefore initially used cpg as a common polyclonal activator to specifically enhance conversion of splenic bmem and not naïve b cells from jhmv immune mice. cpg was added to either irradiated feeders alone or feeders pre-incubated with viral lysate to stimulate jhmv bmem for 4, 5, or 6 days as described above. elispot analysis revealed 5 days stimulation was superior to 4 or 6 days in achieving highest asc frequencies (data not shown). furthermore, optimal cpg mediated bmem to asc conversion was independent of viral lysate ( fig. 2a) . virus-specific igg asc frequencies were 2-fold higher (∼300) following stimulation compared to direct ex vivo elispot analysis (∼150). lastly, splenocyte cpg stimulation fig. 1 . bmem markers on cns infiltrating b cells have distinct surface expression from the periphery. brain and cln cells isolated from pooled organs of jhmv infected mice at day 14 (cln) and day 38 (brain) p.i. were analyzed for cd38 and cd73 expression. (a) representative gating strategy for cd19 + infiltrating b cells in the brain and gating of igd + and igm + subsets in the brain and cln. representative density plots depict cd38 (b-c) and cd73 (d-e) expression among total cd19 + , naïve igd + igm + , and igd − igm − isotype switched b cells within the cln (b, d) and brain (c, e). representative histograms depict cd38 or cd73 expression among naïve igd + igm + , activated igd int igm + , igd − igm + , and isotype-switched asc/bmem igd − igm − b cells within the cln and brain. data is representative of 3-4 independent experiments. , or spinal cord (c) of infected mice at day 28 p.i. were stimulated for 5 days under various conditions including: 1) cpg, 2) irradiated feeders pre-incubated with viral lysate, 3) cpg with irradiated feeders, or 4) cpg with irradiated feeders pre-incubated with viral lysate. after culture, virus-specific igg secreting asc were enumerated by elispot using virus coated plates. pre-existing asc numbers were determined by 4 h (h) direct ex vivo elispot. data represents the mean ± sem asc per 10 6 cells based on cells plated prior to cpg stimulation from 3 to 4 pooled mice. asc frequencies per animal were determined using the average frequencies of 3-5 wells showing spots within linear dilution range. data are representative of 2 independent experiments. significant differences between conditions at 4 h and 5 days indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. for 5 days in the absence of feeders decreased asc recovery compared to direct ex vivo elispot analysis suggesting feeders provide survival factors. the same in vitro stimulation method used for jhmv immune splenocytes was applied to analyze virus-specific igg bmem in brain and spinal cord following intracranial jhmv infection. moreover, surface igg + cd138 − b cells indicative of bmem are detectable at day 28 p.i. during persisting jhmv infection (phares et al., 2014) . to examine bmem specificity, single cell suspensions from brains and spinal cords at day 28 p.i. were serially diluted and stimulated under optimal conditions previously determined for splenic b cells using cpg for 5 days in the presence of irradiated feeders with or without viral lysate. cells were then transferred to elispot plates and assessed for virus-specific igg asc (fig. 2b) . we focused on igg as the vast majority (∼70%) of isotype switched virus specific asc in the cns secrete igg isotype ab, with a minor contribution of iga secreting asc (tschen et al., 2002) . pre-existing virus-specific asc were measured in a direct 4 h ex vivo elispot assay. in contrast to splenocytes, neither brain or spinal cord cells revealed bmem to asc conversion, as indicated by no increase in spots relative to the numbers obtained from direct ex vivo elispot analysis. this finding was independent of addition of viral lysate. rather than increasing, virus-specific asc after cpg stimulation were actually 3-fold lower in the brain (∼1 × 10 3 asc) and 2-fold lower in the sc (∼2 × 10 3 asc) relative to pre-existing asc (fig. 2b,c) . this suggested that cell death during strong stimulation and prolonged culture of cns cells or loss in the washing/transfer procedure leads to premature attrition of pre-existing asc. this is supported by studies with lcmv, which have suggested direct ex vivo asc assays may yield higher asc frequencies than those observed after prolonged culture, thereby complicating calculations and potentially underestimating asc converted from bmem (slifka and ahmed, 1996b) . the discrepancy in bmem conversion after stimulation in the spleen versus cns implied that while the cns environment can sustain bmem or asc survival, survival is limited when explanted. we therefore assessed how distinct in vitro stimulation conditions including the tlr agonists cpg (tlr9) and r848 (tlr7/8) affect cns derived pre-existing virus-specific igg asc. to minimize cell death due to prolonged culture, incubation was limited to 2 days, which increased asc frequencies compared to 4 or 5 days stimulation in initial studies (data not shown). pre-existing asc were measured in serial dilutions of cns-derived cells at day 28 p.i. using four conditions: a) direct ex vivo 4 h elispot, b) stimulation with r848 or cpg for 10 h, which is insufficient time for bmem to differentiate into asc (slifka and ahmed, 1996b) , c) stimulation with r848 or cpg supplemented with feeders for 2 days, but irradiation of cns cells to block cellular division (slifka and ahmed, 1996b) and lastly d) culture with splenocyte feeder cells only for 2 days to assess whether feeder-derived supplements sustain pre-existing asc. following the distinct culture conditions, serially diluted cells were transferred to elispot membranes and assessed for virusspecific igg asc 4 h later. asc obtained from in vitro stimulation controls were compared to pre-existing asc obtained by direct ex vivo elispot (fig. 3) . fig. 3 only includes data from r848 stimulation as cpg and r848 stimulation produced interchangeable results (data not shown). 10 h culture with r848 already reduced asc frequencies relative to ex vivo asc numbers to ∼30% in both brain and spinal cord-derived cells. however, 2 day stimulation conditions did not further reduce pre-existing asc numbers. all culture conditions yielded ∼1 × 10 3 pre-existing asc within the brain and ∼5 × 10 3 within the spinal cord, which represents a ∼3-fold reduction compared to direct asc assays. these consistent control results suggested multiple wash steps and transfer to elispot membranes reduce asc recovery ∼3-fold. importantly, the differing in vitro controls abrogating bmem conversion all resulted in similar virus-specific igg asc, demonstrating reproducible calculation of pre-existing asc. exposure of pre-existing asc to the same culture conditions as bmem thus underestimates asc but provides a more valid approach to assess asc derived from bmem conversion relative to pre-existing asc, than comparison to asc numbers obtained from direct ex vivo elispot. to optimize cns derived bmem conversion, we next examined the effects of tlr agonists r848 and cpg under various culture conditions using cns cells from persistently infected mice at days 28-38 p.i. r848 stimulation of human pbmcs and murine splenocytes has been recently shown to enhance bmem conversion compared to cpg stimulation, suggesting r848 may be a superior stimulating agent (jahnmatz et al., 2013) . bmem conversion was evaluated relative to pre-existing asc numbers obtained from irradiated cns cells stimulated for 2 days under the same conditions (fig. 4a ). as expected, feeder cells themselves showed no detectable asc even when cultured with tlr agonists (fig. 4a). fig. 3 . in vitro stimulation protocol reduces asc numbers. cells isolated from brain or spinal cord of infected mice at day 38 p.i. were stimulated for 10 h or 2 days with r848 in the presence of irradiated splenocyte feeders as indicated. direct ex vivo asc quantification was conducted using 4 h incubation on elispot plates. for 10 h asc controls, cns cells were cultured with splenoctye feeders with r848 stimulation. for 2 day (d) asc controls, splenocyte feeders were cultured with irradiated cns cells with r848 stimulation or cns cells without stimulation. after culture, virus-specific igg secreting asc were enumerated by elispot using virus coated plates. data represents the mean + sem asc per 10 6 cells prior to stimulation from 3 to 4 pooled mice. asc frequencies per animal were determined using the average frequencies of 3-5 wells showing spots within linear dilution range. data are representative of 1-2 independent experiments. coculture of brain-derived cells with feeders alone improved recovery of virus-specific asc, relative to no feeders, supporting feeders provide survival factors. however, feeders did not significantly enhance asc frequencies in spinal cord-derived b cells (fig. 4a) , suggesting they may be less prone to death than brain counterparts. cpg stimulation resulted in a modest increase of virus-specific asc in brain derived, but not spinal cord-derived cells compared to feeders alone. by contrast, r848 stimulation significantly increased virus-specific asc by 2-3-fold relative to pre-existing asc in both brain and spinal cord cells. irradiation of cns effector cells reduced cpg and r848 asc frequencies back to feeder only culture frefig. 4 . 2 day r848 stimulation provides optimal asc conversion for cns-derived bmem. (a) cells isolated from brain or spinal cord of infected mice at day 38 p.i. were incubated with irradiated feeders plus r848 or cpg for 2 days as indicated. (b) cells isolated from brain or spinal cord of infected mice at day 28 p.i. were stimulated with cpg or r848 with or without il-2 in the presence of irradiated splenocyte feeders for 2 or 3 days. after culture, virus-specific igg secreting asc were enumerated by elispot using virus coated plates. data represents the mean + sem asc per 10 6 cells based on cells plated prior to stimulation from 3 to 4 pooled mice. asc frequencies per animal were determined using the average frequencies of 3-5 wells showing spots within linear dilution range. data are representative of 2 independent experiments. significant differences between conditions in panel a indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. in panel b significant differences comparing conditions either at day 2 or day 3 indicated by * p ≤ 0.05, ** p ≤ 0.01, and ¶ p ≤ 0.05, respectively; significant differences comparing conditions between days 2 and 3 indicated by # p ≤ 0.05. quencies (fig. 4a) . these results confirmed that increased asc frequencies following tlr stimulation indeed resulted from bmem proliferation and conversion. importantly, as bmem proliferation is only initiated after 24 h, the 2 day stimulation period limits proliferation to 1-2 divisions thereby minimizing artificially high bmem frequencies due to numerous divisions (slifka and ahmed, 1996b; tangye and hodgkin, 2004; bernasconi et al., 2002) . supplementation of media with il-2 either using cona conditioned medium or recombinant il-2 has been shown to enhance cell survival and bmem conversion in in vitro bmem stimulation assays (slifka and ahmed, 1996b; walsh et al., 2013) . to test whether cns bmem conversion could be further increased, we expanded stimulation times to 3 days and supplemented media with il-2. however, neither increased culture duration nor addition of il-2 to cpg stimulation enhanced bmem to asc conversion in brain derived cells, independent of stimulation for 2 or 3 days (fig. 4b) . furthermore, il-2 only improved asc recovery after 3 day, but not 2 day culture in cpg stimulated spinal cord cells. while prolonged culture time modestly increased brain derived virus-specific asc following r848 stimulation, il-2 addition had no enhancing effects. prolonged stimulation also had no beneficial effects on bmem conversion in r848 stimulated spinal cord cells and il-2 only modestly increased asc after 3 days stimulation. overall, these results confirmed superior stimulation by r848 compared to cpg, independent of prolonged culture or il-2 addition. we therefore chose r848 stimulation for 2 days as optimal for bmem conversion in cns derived cells. jhmv infection induces virus-specific igg asc expansion within the draining cervical lymph nodes (cln) at day 14 p.i coincident pre-existing asc numbers are calculated from r848 stimulated irradiated cns cells incubated with splenocyte feeders. bmem numbers are calculated based on total asc numbers after r848 stimulation minus pre-existing asc numbers. data represents the mean ± sem asc per 10 6 cells based on cells plated prior to nonspecific stimulation from 3 to 4 pooled mice. mean values are calculated from 5 wells showing spots within the linear dilution range. two independent experiments revealed similar asc/bmem kinetics throughout infection. (c) spot size of brain or spinal cord-derived asc following direct ex vivo 4 h incubation (asc) and 10 h or 2 day r848 stimulation. r848 counted highlights asc counted for 2 day r848 stimulation with gc formation (disano et al., 2017; tschen et al., 2002) . virusspecific igg initially accumulates in the brain and spinal cord, but preferentially increases in the spinal cord, the site of viral persistence and low, but ongoing inflammation (phares et al., 2014) . increased virus specific igg in spinal cords by day 21 p.i. correlates with the overall increased fraction of asc compared to the brain (phares et al., 2014; phares et al., 2016) . using the assay conditions optimized above, we assessed bmem numbers relative to asc in the cns during jhmv infection at the onset of asc emergence at day 14 and during persistence at day 35 p.i. (tschen et al., 2002) . reproducibility and kinetics of virus-specific igg bmem and pre-existing asc was evaluated in the brain and spinal cord in independent experiments (fig. 5a, b) , as biological variability in immune responses to infection and efficiency in cns cell isolation can lead to differences in absolute frequencies between multiple experiments. pre-existing asc were calculated using the average of 4-5 wells in a linear dilution range from irradiated cns cells cultured with r848 and feeders. virus-specific igg bmem were calculated by subtracting the average pre-existing asc from the average number of asc obtained after r848 stimulation. virusspecific asc progressively accumulated within the cns during persistence, with elevated frequencies in spinal cord at day 35 p.i., confirming previous results (phares et al., 2014; tschen et al., 2002) . virus-specific bmem frequencies in the brain were comparable to asc at day 14 p.i. (∼500 cells), and increased between 5-8-fold by day 35 p.i. (fig. 5a, b) . furthermore, bmem surpassed asc frequencies in the brain by 1.2-2-fold by day 35 p.i. spinal cords also revealed similarly low frequencies of bmem and asc at day 14 p.i., with both increasing by day 35 p.i. although the increase relative to day 14 p.i. ranged between 3-5-fold, virus-specific asc frequencies were overall 3-fold higher than bmem. while the ratio of asc to bmem was ∼0.7 in the brain, it was inverted at 3.0 in spinal cord when averaging results from 2 separate experiments. furthermore, in contrast to preferential accumulation of asc in the spinal cord during chronic infection, bmem were similar within the brain and spinal cord at day 35 p.i. it remains unclear whether the site of enhanced viral persistence preferentially affects asc over bmem accumulation, or whether the spinal cord environment can mediate local conversion of bmem to asc. detection of virus-specific bmem in the cns led us to examine bmem derived asc igg secretion capacity compared to pre-existing asc. spot diameter and intensity reflect both ig secretion rates and affinity, with large and intense spots indicative of more differentiated, high affinity asc (sibley et al., 2012; thomson, 2005; feske et al., 2012; karulin and lehmann, 2011) . therefore, we investigated ig secretion by quantifying spot size of virus-specific igg derived from converted asc after 2 days r848 stimulation versus pre-existing asc from direct ex vivo elispot (henn et al., 2009) . as an additional control, asc spot size after 10 h r848 stimulation was compared to control for effects of in vitro stimulation on pre-existing asc spot size (fig. 5c ). quantification revealed no significant difference in asc spot size after 10 h stimulation versus direct ex vivo elispot (data not shown). more importantly, virusspecific igg spot size from brain derived asc measured directly by ex vivo elispot assays and those asc formed 2 days post r848 stimulation was also similar at a mean spot size of 30-40 m 2 (fig. 5c, d) . similarly, spot size analysis of spinal cord-derived cells revealed no significant difference between asc after r848 stimulation compared to direct ex vivo elispot. nevertheless, the mean spot size was 40-50 m 2 , suggesting enhanced igg secretion for asc and well. (d) mean ± sem asc spot size following direct ex vivo elispot (asc) and asc after 2 day r848 stimulation (r848) in the brain and spinal cord (sc). data are representative of 2-3 independent experiments. significant differences between brain and spinal cord indicated by * p ≤ 0.05, and ** p ≤ 0.01. bmem in the spinal cord relative to brain (fig. 5c, d) . the in vitro stimulation assay established for cns derived bmem is therefore a useful tool to quantify and determine bmem specificity and can be adapted to assess isotype variants and ig secretion levels during heterologous cns inflammation models. the presence of b cells with multiple differentiation phenotypes in the cns following various insults, including infection, has reinvigorated research into mechanisms supporting their accumulation, function, and specificity. the recruitment of asc into the cns and their specificity following viral encephalomyelitis is well documented in animal models (tschen et al., 2002; metcalf et al., 2013) . asc have also been detected in the csf of patients afflicted by viral encephalitis and multiple sclerosis (krumbholz et al., 2012; burke et al., 1985; jacobi et al., 2007; kapoor et al., 2004; linnoila et al., 2016; skoldenberg et al., 1981) . however, the presence of bmem and their specificity has not been studied extensively due to methodological limitations in determining specificity. as bmem require minimal stimulation to convert to asc compared to naïve b cells (kurosaki et al., 2015) , they are ideal candidates to locally differentiate and sustain ab responses. persisting antigen, t helper cells, cytokines, and tlr agonists all present in the inflamed cns have the potential to induce bmem differentiation into asc (kurosaki et al., 2015; hebeis et al., 2004; aiba et al., 2010; geffroy-luseau et al., 2011) . during viral encephalitis, antigen specific bmem may locally contribute to humoral responses and serve a protective role in controlling infectious virus. however, bmem directed against self-antigens during neurodegeneration, autoimmunity, or injury, may enhance pathogenic responses. jhmv induced acute encephalomyelitis resolving into persistence has provided an excellent model to study progression and maintenance of humoral responses within the cns. the accumulation of isotype-switched igg + cd138 − cd19 + b cells, characteristic of bmem, in both the brain and spinal cord during persistent infection led us to assess their specificity using established bmem to asc conversion protocols developed for b cells in lymphoid organs. however, several unsuccessful trials to apply these procedures to cns b cells prompted us to tailor culture conditions to enhance bmem to asc conversion as well pre-existing asc survival. to yield high numbers of viable cns cells which retain all classical b cell surface markers, the protocol uses collagenase-based digestion of cns tissue. the initial lda in vitro culture conditions mainly differ from other lda stimulation protocols by utilizing shorter r848 mediated tlr7/8 stimulation periods to circumvent b cell loss presumably due to cell death. moreover, addition of irradiated feeder cells improved survival of asc/bmem, while il-2 supplementation has no beneficial effect. although the shortened 2 day stimulation period minimized cell death, significant cell loss in the wash and transfer process was evident by a similar reduction in pre-existing asc after both 10 h and 2 day stimulation compared to direct ex vivo elispot. although ex vivo stimulation directly on elispot membranes thus appears ideal to minimize cell loss, prolonged incubation of cells on elispot membranes leads to increased background, artefacts due to cell debris, and increased spot density due to high levels of ab secretion. an obvious caveat of the in vitro stimulation on 96 well flat bottom culture plates is thus that bmem and asc frequencies may be underestimated by 3fold as indicated by our control experiments with pre-existing asc. in vitro stimulation controls inhibiting bmem conversion are thus necessary to standardize cell loss and compare the ratio of bmem to pre-existing asc. another caveat of bmem stimulation prior to elispot analysis is the use of polyclonal stimulators such as tlr agonists, which induce rapid proliferation regardless of antigen specificity, thereby potentially overestimating bmem numbers based on asc conversion. in vitro stimulation utilizing feeders presenting virus antigen would limit non-specific, rapid proliferation resulting in a more accurate assessment of virus-specific bmem. however, incubation of virus lysate with feeders was insufficient to convert bmem in our system presumably due to limiting virus dose. nevertheless, fairly short stimulation times of 2 days in addition to use of virus coated elispot plates limit proliferation and the potential for skewed virus-specific igg asc within total igg asc. although future studies aim to optimize stimulation using feeders incubated with distinct viral antigens to induce more physiologically relevant bmem conversion, a caveat in the jhmv system is that spike protein binds to ceacam1, which is highly expressed on b cells and itself a signaling molecule (williams et al., 1990; greicius et al., 2003; khairnar et al., 2015) . in summary, our optimized lda in vitro stimulation elispot assay provides a tool for simultaneous analysis of cns derived bmem and asc during neuroinflammatory diseases, including microbial infection, autoimmunity, or tissue injury. the relatively short 2.5-3 day combined in vitro stimulation lda/elispot procedure is more efficient than previously described 2-4 week stimulation elisa based bmem assays, which measure secreted ab. the assay allows for assessment of bmem antigen specificity, isotype and kinetic alterations throughout disease, and are complementary to enumerating bmem based on surface marker profiles. comparative assessment of bmem in slt and the cns will aid in understanding the relationship between peripheral bmem and cns accumulation during disease and identifying tissue specific therapeutics to either enhance or diminish bmem during neuroinflammatory diseases. expansion of activated peripheral blood memory b cells in rheumatoid arthritis, impact of b cell depletion therapy, and biomarkers of response preferential localization of igg memory b cells adjacent to contracted germinal centers quantitation of rare memory b cell populations by two independent and complementary approaches the human immunomodulatory cd25+ b cell population belongs to the memory b cell pool new markers for murine memory b cells that define mutated and unmutated subsets b cells produce pathogenic antibodies and impair recovery after spinal cord injury in mice maintenance of serological memory by polyclonal activation of human memory b cells long-term presence of memory b-cells specific for different vaccine components virus-specific antibody-producing cells in blood and cerebrospinal fluid in acute japanese encephalitis an optimized assay for the enumeration of antigen-specific memory b cells in different compartments of the human body accumulation of class switched igd-igm-memory b cells in the cerebrospinal fluid during neuroinflammation cd73 expression is dynamically regulated in the germinal center and bone marrow plasma cells are diminished in its absence tracking human antigen-specific memory b cells: a sensitive and generalized elispot system meningeal infiltration of the spinal cord by non-classically activated b cells is associated with chronic disease course in a spontaneous b cell-dependent model of cns autoimmune disease activated gl7(+) b cells are maintained within the inflamed cns in the absence of follicle formation during viral encephalomyelitis multiple layers of b cell memory with different effector functions distinct effector cytokine profiles of memory and naive human b cell subsets and implication in multiple sclerosis handbook of elispot: methods and protcocols pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies tlr9 ligand induces the generation of cd20+ plasmablasts and plasma cells from cd27+ memory b-cells ceacam1 is a potent regulator of b cell receptor complex-induced activation quantal and graded stimulation of b lymphocytes as alternative strategies for regulating adaptive immune responses activation of virus-specific memory b cells in the absence of t cell help modulation of single-cell igg secretion frequency and rates in human memory b cells by cpg dna, cd40l, il-21, and cell division quantitation of intrathecal antibodies in cerebrospinal fluid of subacute sclerosing panencephalitis, herpes simplex encephalitis and multiple sclerosis: discrimination between microorganism-driven and polyspecific immune response optimization of a human igg b-cell elispot assay for the analysis of vaccine-induced b-cell responses human memory b cells: memory b cells of a special kind persistence of west nile virus (wnv) igm antibodies in cerebrospinal fluid from patients with cns disease handbook of elispot: methods and protocols ceacam1 induces b-cell survival and is essential for protective antiviral antibody production human immunoglobulin (ig)m+igd+ peripheral blood b cells expressing the cd27 cell surface antigen carry somatically mutated variable region genes: cd27 as a general marker for somatically mutated (memory) b cells repression of the transcription factor bach2 contributes to predisposition of igg1 memory b cells toward plasma cell differentiation b cells and antibodies in multiple sclerosis pathogenesis and therapy memory b cells antibody prevents virus reactivation within the central nervous system csf herpes virus and autoantibody profiles in the evaluation of encephalitis cytokine-producing b cells: a translational view on their roles in human and mouse autoimmune diseases tracing the development of single memory-lineage b cells in a highly defined immune response cytokine-producing b lymphocytes-key regulators of immunity cxcr3-dependent plasma blast migration to the central nervous system during viral encephalomyelitis alphavirus-induced encephalomyelitis: antibody-secreting cells and viral clearance from the nervous system recruitment and retention of b cells in the central nervous system in response to alphavirus encephalomyelitis b cells in the multiple sclerosis central nervous system: trafficking and contribution to cns-compartmentalized inflammation memory and naive b-cell subsets in patients with multiple sclerosis different b cell populations mediate early and late memory during an endogenous immune response progression from igd+ igm+ to isotype-switched b cells is site specific during coronavirus-induced encephalomyelitis cxcl13 promotes isotype-switched b cell accumulation to the central nervous system during viral encephalomyelitis clonal dissection of the human memory b-cell repertoire following infection and vaccination kinetics of establishing the memory b cell population as revealed by cd38 expression phenotypic and functional heterogeneity of human memory b cells mhc-restricted b-cell antigen presentation in memory b-cell maintenance and differentiation elispot refinement using spot morphology for assessing host responses to tuberculosis herpes simplex encephalitis: a serological follow-up study. synthesis of herpes simplex virus immunoglobulin m, a, and g antibodies and development of oligoclonal immunoglobulin g in the central nervous system long-term humoral immunity against viruses: revisiting the issue of plasma cell longevity limiting dilution analysis of virus-specific memory b cells by an elispot assay humoral immunity due to long-lived plasma cells divide and conquer: the importance of cell division in regulating b-cell responses identification of functional human splenic memory b cells by expression of cd148 and cd27 a germinal center-independent pathway generates unswitched memory b cells early in the primary response handbook of elispot: methods and protocols cutting edge: hierarchy of maturity of murine memory b cell subsets recruitment kinetics and composition of antibody-secreting cells within the central nervous system following viral encephalomyelitis cd38 through the life of a murine b lymphocyte optimization and qualification of a memory b-cell elispot for the detection of vaccine-induced memory responses in hiv vaccine trials sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv-4) leads to a characteristic distribution of demyelination purification of the 110-kilodalton glycoprotein receptor for mouse hepatitis virus (mhv)-a59 from mouse liver and identification of a nonfunctional, homologous protein in mhv-resistant sjl/j mice cd27 is acquired by primed b cells at the centroblast stage and promotes germinal center formation cd80 and pd-l2 define functionally distinct memory b cell subsets that are independent of antibody isotype this work was supported by us national institutes of health grant ns086299. the funding source had no involvement in the study design, writing of the manuscript, decision to submit, or collection, analysis, and interpretation of data. we sincerely thank mi widness and dr. alice valentin-torres for preparation of splenocyte feeder cells and mi-hyun hwang for viral cns infection. key: cord-021500-sy6lnt7b authors: jean harry, g.; toews, arrel d. title: myelination, dysmyelination, and demyelination date: 2007-05-09 journal: handbook of developmental neurotoxicology doi: 10.1016/b978-012648860-9.50007-8 sha: doc_id: 21500 cord_uid: sy6lnt7b nan normal functioning of the nervous system involves the transmission, processing, and integration of information as nervous impulses. impulse transmission along axons is greatly facilitated by the presence of myelin, the compact multilamellar extension of the plasma membrane of specialized glial cells that spirals around larger axons. in the central nervous system (cns), oligodendroglial cells are responsible for the synthesis and maintenance of myelin, whereas schwann cells subserve this role in the peripheral nervous system (pns). schwann cells produce a single segment of myelin (called an internode), whereas oligodendroglial cells furnish multiple myelin segments around different axons, although only one segment for a given axon. there are periodic interruptions along the axons between adjacent myelin internodes; termed nodes of ranvier, these short intervals where axons are not enveloped by myelin are vital for normal nervous system function (see later this chapter). myelin is an electrical insulator, and the periodic interruptions at the nodes allow for rapid and efficient transmission of nervous impulses. in unmyelinated axons, impulse transmission involves a wave of membrane depolarization that moves down the axon in a continuous sequential manner. however, in myelinated axons, the myelin internodes function as high-resistance insulators, so the excitable axonal membrane, containing a high concentration of voltage-sensitive sodium channels, is exposed only at the nodes of ranvier. impulse conduction thus involves excitation at the nodes only, and the impulse jumps from node to node (saltatory conduction) (see funch and faber, 1984; waxman et al., 1989; and morell et al., 1994, for details) . saltatory conduction is much more rapid and requires much less energy for membrane repolarization than conduction in unmyelinated axons. myelin thus greatly increases the efficiency of the nervous system, facilitating conduction while conserving metabolic energy and space. it is not difficult to imagine how even minor loss of myelin or perturbations in its structure and/or function could have deleterious effects on normal nervous system function. structural aspects of the process of myelination are most easily illustrated in the pns. each myelin-forming schwann cell produces an elaborate specialized extension of its plasma membrane, which is wrapped spirally around a segment of one axon (fig. 1 ). schwann cells, the glial cells of the pns, are derived from the portion of the neural epithelium that gives rise to the neural crest (le douarin, 1982) . during development, schwann cells invade the developing nerves, where they migrate along bundles of axons, proliferate (probably in response to an axonal mitogen; webster and favilla, 1984) , and segregate the axons individually within invaginations on the surface of schwann cells. as they cease migrating, they synthesize a basal lamina (billings-gagliardi et al., 1974) , composed of laminin, merosin, type iv collagen, fibronectin, nidogen/entactin, and heparan sulfate proteoglycan (sanes and cheney, 1982; tohyama and ide, 1984; bannerman et al., 1986; leivo and engvall, 1989; sanes et al., 1990) . as the axons continue to enlarge, the larger axons become further segregated so that a single schwann cell envelops a single axon. after the plasma membrane of the schwann cell has completely enclosed the axon, the external surfaces of the plasma membrane fuse to form a structure known as the mesaxon. the mesaxon then elongates and spirals around the axon, eventually resulting in a "jelly-roll" structure consisting of double layers of the schwann cell plasma membrane. myelin internodes can be as much as 2 mm long and contain 5 mm of myelin spiral (friede and bischhausen, 1980) . myelin compaction occurs as cytoplasm is extruded and the cytoplasmic faces are condensed to produce the dark major period line visible in electron micrographs (fig. 2) . the juncture of what was originally the outer faces of the apposing plasma membranes form the lighter appearing intraperiod line. mature myelin thus has a characteristic compact multilamellar structure, but cytoplasmic inclusions continu-figure 2 electron micrograph of compact myelin from the mammalian cns. although there are minor ultrastructural differences, compact pns myelin has a similar ultrastructural appearance. note the alternating pattern of darker major dense lines and paler intraperiod lines, originally formed by fusion of apposing surfaces of the inner and outer leaflets, respectively, of the oligodendroglial plasma membrane. cytoplasm-containing internal mesaxons can be seen on two of the myelinated axons. ous with the perikarya cytoplasm of the schwann cells are also present ( figs. 1 and 2 ). in addition, the myelin internode contains several ultrastructurally and biochemically distinct membrane domains, including the outer plasma membrane of the myelin-forming cell and the compact myelin itself, as well as the cytoplasmcontaining schmidt-lanterman incisures, paranodal loops, nodal microvilli, and outer and inner mesaxons. the latter cytoplasm-containing structures provide connections with the perikaryal cytoplasm, and are vital for myelin maintenance. the process of myelination in the cns is similar, except that a single oligodendroglial cell extends a number of processes from its cell body; each process then envelopes and myelinates a single segment of a given axon (fig. 3) . much of the local membrane assembly to give mature, compact myelin occurs within the oligodendroglial cytoplasmic processes (waxman and sims, 1984) . the size of the fibers and the thickness of the sheaths are very different in the pns and the cns, but the overall surface area of myelin generated by an oligodendrocyte around multiple axons may be no larger than that formed by a schwann cell around a single internode. the term oligodendrocyte, meaning "few processes," is actually somewhat of a misnomer, as a given oligodendrocyte may myelinate anywhere from less than five axons up to dozens of axons (butt and ransom, 1989; bjartmar et al., 1994) . as in the pns, the onset of myelination is preceded by proliferation of oligodendroglia. as development continues, both the diameter and length of the axons increase, and this is associated with a corresponding increase in nodal length as well as increases in myelin thickness. thus, despite its compact highly ordered appearance, myelin continues to expand in all planes during growth and development. in general, myelination follows the order of phylogenetic development, with the peripheral nerves myelinating first, then the spinal cord, and finally the brainstem, cerebellum, and cerebrum. there is, however, considerable overlap in this progression. in addition, each fiber tract may have its own spatiotemporal pattern of myelination, so that the degree of myelination may differ in different fiber tracts at a given developmental stage. for example, myelination in the spinal cord proceeds in a rostral-caudal gradient, whereas in the optic nerve, myelination progresses with a retinal to chiasmal gradient. although not necessarily an absolute prerequisite for function, in general, fiber tracts are myelinated before they become fully functional. myelination is a major metabolic and structural event that occurs during a relatively brief but precisely defined period in the normal progression of events involved in nervous system development. in both the cns and pns, an enormous amount of myelin membrane is formed (some pns axons may have as many as 100 layers) and this membrane must be maintained at a considerable distance from the supporting glial cell body. the surface area of myelin per adult oligodendrocyte in the rat brain has been calculated to be 1-20 x 105/zm 2, several orders of magnitude greater than the perikaryal membrane surface area of about 100 /zm 2 (pfeiffer et al., 1993) . myelinating glial cells are thus maximally stressed in terms of their metabolic and synthetic capacity during this time, with each cell synthesizing myelin equivalent to up to 3 times the weight of its perikarya each day . because of these very high levels of synthetic and metabolic activity, these myelinating cells are especially vulnerable to nutritional deficits and/ or to toxic insults or injuries during this period. the tightly programmed sequence of events eventually resulting in the formation of mature compact myelin and the consequent initiation of impulse transmission is regulated by interactions between axons and glial cells at numerous stages (see waxman and black, 1995, for detailed discussion) . during the early stages of myelination, the axon is loosely ensheathed by processes arising from immature, relatively undifferentiated glial cells. loose glial ensheathment of axons in the optic nerve is seen in the rat beginning at postnatal day 6. this is followed by spiral wrapping of the axon by oligodendroglial processes that form compact myelin. in some tracts, the immature myelin sheath is initially close to the oligodendroglial cell body (remahl and hildebrand, 1990) , but with maturation the sheath is displaced radially and often is connected to the cell body by only a thin cytoplasmic bridge. a single oligodendrocyte can myelinate axons of various diameters in their vicinity and can form myelin sheaths of different thicknesses around axons of differing diameter. the development, maturation, and maintenance of the myelin sheath is dependent on both the normal functioning of the myelinating glial cells and the integrity of its relationship to the axon it ensheaths. during development, physical features of the myelin sheath, such as thickness and number of lamellae, are not preprogrammed within the myelin-forming cells, but rather depend on local regulation by the axon, with larger axons having thicker myelin sheaths (waxman and sims, 1984) . myelin internodal distance is also matched to fiber diameter (hess and young, 1952) and the internodal distance:diameter ratio is different for fibers in different tracts. there is some evidence that myelination is initiated when a developing axon reaches a "critical diameter." however, myelination occurs over a range of axonal diameters (fraher, 1972) and at various times along a single axon (waxman et al., 1972; waxman, 1985) . the signal for initiation of myelination thus appears to be specific for particular axons, or for specific domains along axons. the axonal membrane contains molecules that trigger mitogenesis in schwann cells and oligodendrocytes (salzer et al., 1980a; 1980b; devries et al., 1983; chen and devries, 1989) and regulate the rate and degree of myelin formation (black et al., 1986; waxman 1987a,b) . although some schwann cells go on to myelinate axons, others only ensheath bundles of unmyelinated axons. these nonmyelinated schwann cells express distinct molecular markers such as the low-affinity nerve growth factor receptor (ngf-r), neural cell adhesion molecules (n-cam and l1), and growth associated protein-43 (gap-43), but none of the myelin specific proteins (see following sections, as well as mirsky and jessen, 1990; curtis et al., 1992) . transplantation studies have shown that the axon determines the phenotype of the schwann cell (aguayo et al., 1976; weinberg and spencer, 1976) . thus, there are a number of potentially distinct physical interactions between schwann cells and axons as schwann cells proliferate, migrate, ensheath axons, and form myelin sheaths during development of the pns. although the axon influences and helps direct the formation of myelin, the myelin sheath also significantly defines features of the axon. the premyelinated axon is electrically excitable (foster et al., 1982; waxman et al., 1989) and the loss of excitability in the internodal axonal membrane that occurs with myelination involves an active suppression of na + channels by the overlying oligodendrocyte or myelin sheath (black et al., 1985 (black et al., , 1986 . proliferation of oligodendrocyte precursors in the optic nerve is dependent on axonal electrical activity in that the blockage of optic nerve electrical activity by transection or exposure to tetrodotoxin resulted in a dramatic loss of oligodendrocyte precursor cells (barres and raft, 1993) . potassium channels may also participate in myelin formation; the potassium channel blocker, tea + was shown to be effective in eliminating myelination in spinal cord explants while leaving axonal conduction and synapse formation intact. the ontogenic development of the myelinating schwann cell lineage has been relatively wellcharacterized, particularly in rodents. most of our knowledge derives from cell-culture studies, but in vivo developmental studies in normal as well as in transgenic and gene knock-out mice have also proved useful. understanding of the events and stages involved is of potential clinical relevance, not only with respect to various toxic neuropathies and to pns nerve regeneration, but also because schwann cell precursors may be attractive schwann cells of the pns originate from primitive neural crest cells, proliferative multipotential cells also capable of differentiating into neurons or melanocytes. the first step along the schwann cell lineage gives the schwann cell precursor, a proliferative cell that becomes associated with many axons and expresses the lowaffinity nerve growth factor receptor (ngf-r), growth-associated protein 43 (gap-43), and the neural cell adhesion molecules n-cam and l1. the subsequent "committed" schwann cell becomes associated with progressively fewer axons and expresses, in addition to the previously noted markers, s-100 protein (from this stage onward, all schwann cells express s-100). committed schwann cells develop into either nonmyelinating schwann cells, which remain associated with several axons and express galactocerebroside (galc) in addition to the previous markers, or into myelinating schwann cells. myelinating schwann cells progress through a proliferative "premyelinating" stage, characterized by transient expression of suppressed camp-inducible pou-domain transcription factor (scip), followed by a "promyelinating" galc-positive stage, becoming associated with a single axon in the process. the final differentiation into a mature myelinating schwann cell involves candidates for transplantation to facilitate remyelination and repair in injured cns. a detailed discussion of this subject is beyond the scope of this chapter, but the reader is referred to several comprehensive reviews on this subject if more details are desired (jessen and mirsky, 1991; gould et al., 1992; mirsky and jessen, 1996; zorick and lemke, 1996) . schwann cells develop from neural crest cells and most of the key developmental stages in schwann cell maturation seem to depend on axon-associated signals. schwann cell precursors give rise to immature schwann cells, which have a distinct phenotype (fig. 4a ). these cells then develop into either myelin-forming or nonmyelin-forming schwann cells, under the control of reversible processes regulated by axon-associated signals. even mature schwann cells show a high degree of plasticity. following a demyelinating insult, schwann cells dedifferentiate into more primitive precursor cells, but generally do not die. when appropriate conditions present themselves (such as the regrowth of axons that occurs following a nerve-crush injury), these cells can proliferate, reestablish contact with axons, redifferentiate to the myelinforming phenotype, and remyelinate the axons, thereby restoring normal function. it is possible that primitive neural crest cells enter the schwann cell lineage only when they first encounter axons in the developing nerves, and that a signal related to axonal contact guides them down the path to mature schwann cells. such signals may involve members of the neu-differentiation factor (ndf) family. members of the ndf growth factor family, including glial growth factor (ggf), heregulin, acetylcholine-inducing activity (aria), and neuregulin, are alternatively spliced products of a single gene, and these molecules are emerging as important regulators of schwann cell lineage development (dong et al, 1995; zorick and lemke, 1996) . alternatively, it is possible that selected neural crest cells have already entered the schwann cell lineage before they encounter axons (see mirsky and jessen, 1996, for discussion) . investigation of which transcription factors regulate schwann cell development is a current area of active study. among factors likely to play significant roles are the zinc-finger transcription factors krox-20 (topilko, 1994) , scip (see zorick and lemke, 1996) , pax3 (kioussi et al, 1995) , and possibly c-jun (stewart, 1995) . a number of signalling molecules also regulate schwann cell proliferation and myelination, including insulin-like growth factor-1 (igf-1), which promotes expression of a myelinating phenotype in cell culture, and transforming growth factor/3s (tgf-/3), which inhibit myelin formation. the latter may be involved in generating the nonmyelinating schwann cells, which ensheath smaller pns axons. the developmental lineage of the oligodendrocyte, the myelin-producing cell of the cns, is also relatively well characterized, particularly in rodent in vitro systems (fig. 4b ). oligodendrocytes originate as neuroectodermal cells of the subventricular zones and then migrate, proliferate, and further differentiate into mature, postmitotic myelin-forming oligodendroglial cells. their development is discussed only briefly here, but a more comprehensive review is available (warrington and pfeiffer, 1992) . panels of cell-and stage-specific antibodies have proved especially useful in characterizing the sequential expression of various developmental markers, and this has allowed identification of distinct phenotypic stages, each characterized by its proliferative capacity, migratory ability, and distinct morphologies. primitive precursor cells differentiate into proliferative, migratory bipolar o2a progenitor cells. these cells are bipotential, being capable of differentiating into either astrocytes or oligodendrocytes. the oligodendrocyte lineage progresses through several additional stages, including an immature oligodendrocyte expressing galactocerebroside, sulfatide, and cyclic nucleotide phosphodownregulation of ngf-r, gap-43, n-cam, and l1 expression, with upregulation of expression of galc and myelin proteins, and in vivo, the synthesis and elaboration of myelin. because of the high degree of plasticity of schwann cells, most of the developmental steps shown are reversible. (modified from mirsky and jessen [1996] and zorick and lemke [1996] ). (b) myelinating oligodendroglial cells of the cns originate from neuroectodermal cells of the subventricular zones of the developing brain. the earliest precursor cells recognized to date (pre-gd3 stage) are proliferative, unipolar cells that express the embryonic neural cell adhesion molecule (e-ncam). these cells develop into gd3 ganglioside-expressing proliferative bipolar cells, termed o-2a progenitor cells because they are capable (in culture, at least) of developing into either "type 2" astrocytes or oligodendrocytes. development continues through a postmigratory but proliferative multipolar pro-oligodendroblast (pro-ol) and a pre-galc stage, characterized by lack of expression of galc. the onset of terminal oligodendroglial cell differentiation (immature ol stage) is identified by the surface appearance of a subset of "myelin components," consisting of the lipids galc and sulfatide, as well as the enzyme cyclic nucleotide phosphodiesterase (cnp). immature ols then undergo final differentiation into mature oligodendrocytes (mature ol), characterized by regulated expression of myelin components such as mbp and plp and by the synthesis and elaboration of sheets of myelin membrane. diesterase (all myelin components), finally arriving several days later at the mature oligodendrocyte stage. mature oligodendrocytes express all of the myelinspecific proteins and are capable of myelin synthesis in vitro in the absence of axons. it is worth noting that oligodendroglial cells show some developmental plasticity, and this may be of clinical relevance with respect to cns remyelination. in addition, a small population of oligodendrocyte progenitors persist in the adult rat brain (see wolswijk and noble, 1995, for details) , and these constitute a potential source of myelin-forming cells for cns remyelination. immature, cycling oligodendroglial progenitor cells endogenous to adult white matter are capable of remyelinating cns axons following lysolecithin-induced demyelination (gensert and goldman, 1997) . also, 02-a progenitor cells from mice subjected to coronavirusinduced demyelination show increased phenotypic plasticity and enhanced mitotic potential, properties that may be linked to the efficient remyelination that occurs following the demyelinating phase of this disease (armstrong et al., 1990) . manipulation of these progenitor cells by various factors (see later this chapter) thus may also be useful in promoting remyelination in a clinical context. as is the case for schwann cells in the pns, development of oligodendrocytes is governed by a number of growth factors, including platelet-derived growth factor (pdgf), basic fibroblast growth factor (bfgf), igf-1, tgf-/3, and nerve growth factor (ngf), as well as several cytokines (see pfeiffer et al., 1993) . oligodendrocytes differ from schwann cells in that they can be induced to produce myelin in culture in the absence of axons. this raises the question of the extent to which neurons and their axons influence oligodendrocytes with respect to development and myelination in vivo. it is, however, difficult to imagine the lack of significant interaction between these two cells in the developing nervous system, and in fact, many such interactions are known. neurons produce many of the growth factors involved, and they are known to modulate steady-state levels of myelin components as well as mrna levels for these components (singh and pfeiffer, 1985; macklin et al., 1986; kidd et al., 1990; barres and raft, 1993,) . as in the pns, production of myelin by oligodendrocytes requires the coordinated synthesis of massive amounts of myelin components. the marked upregulation of myelin-specific proteins, as well as of enzymes involved in synthesis of myelin lipids (see later this chapter), reflects corresponding increases in abundance of the respective mrna transcripts, suggesting that most regulation of the program for myelination occurs at the level of transcription (see hudson, 1990 , for review). a possible candidate for the coordinate control of cns myelination is myelin transcription factor 1 (myti), a zinc-finger dna-binding protein first identified by its ability to recognize the myelin proteolipid protein (plp) gene (kim and hudson, 1992) . myti mrna transcripts are most abundant in oligodendrocyte progenitor cells, suggesting that this factor acts at a very early stage in the regulation of transcription for myelinogenesis (armstrong et al, 1995) . myelin of both the cns and pns has a distinctive composition that differs somewhat from that of most cellular membranes. it is the major component of white matter of the cns, accounting for about half the dry weight of this tissue, and is responsible for its glistening white appearance. the same is true for larger nerves of the pns, such as the sciatic nerve. myelin in situ has a water content of about 40%, and is characterized by its high lipid content (70-85% of its dry mass) and its correspondingly low protein content (15-30%). most biologic membranes have a much higher protein:lipid ratio, usually somewhere around unity. the insulating properties of myelin, vital to its physiological function, are related to this high lipid content. the high lipid content of myelin also results in a buoyant density less than that of other biologic membranes, and advantage can be taken of this to isolate myelin with a high yield and a high degree of purity (norton and poduslo, 1973a) . the lipid content of cns myelin (table 1) is characterized by high levels of galactolipids (about 32% of lipid dry weight), including galactocerebroside (gal-c) and its sulfated derivative, sulfatide, and cholesterol (about 26% of total lipid weight), with phospholipids accounting for most of the remainder (norton and cammer, 1984; dewille and horrocks, 1992; morell et al., 1994) . plasmalogens, phospholipids having a fatty aldehyde linked to the c1 of glycerol instead of a fatty acid, are especially prominent in myelin. ethanolamine plasmalogens and phosphatidylcholine (lecithin) are the major phospholipid species. gangliosides are also present in minor amounts. pns myelin has a similar composition, although there are quantitative differences (see smith, 1983) . pns myelin has less cerebroside and sulfatide and more sphingomyelin than cns myelin. these differences are minor, however, relative to the larger differences in protein composition discussed later. the protein composition of myelin is relatively simple in that a few major structural proteins account for the bulk of the total protein ( table 2 ). the plp and the myelin basic proteins (mbp) together account for about 80% of the protein content of cns myelin (nornorton and cammer, 1984; morell et al., 1994; and morell and toews, 1996b , for references and additional details. bother lipids are also present in myelin, including gangliosides, galactosyl diglycerides, and fatty acid esters of cerebroside; although not shown in the table, polyphosphoinositides may account for up to 7% of total myelin lipid phosphorus (see morell et al., 1994) . ccalculated from data in norton and cammer (1984) , using 800 and 750 as average molecular weights for phosphoglycerides and sphingomyelin, respectively. aabbreviations: epg, ethanolamine phosphoglycerides; cpg, choline phosphoglycerides; spg, serine phosphoglycerides; ipg, inositol phosphoglycerides. eprimarily ethanolamine plasmalogens. fvalue includes both spg and ipg. ton and cammer, 1984; morell et al, 1994) . in contrast, p0 protein, a protein not found in the cns, accounts for more than half the total protein of pns myelin. aonly major proteins are shown; see text for discussion of other myelin proteins, and morell et al (1994) and newman et al. (1995) for additional references and details. babbreviations: plp, proteolipid protein; mbp, myelin basic protein; cnp, cyclic nucleotide phosphodiesterase; mag, myelin-associated glycoprotein; pmp-22, peripheral myelin protein-22. c composite values representative of adult mammalian myelin. dalthough mrna for this protein has been detected, the protein itself, if present at all, is present in myelin at only low to undetectable levels. mbp, p2-protein, and pmp-22 account for most of the remainder of pns myelin proteins. plp, the major protein component of cns myelin, is present in pns myelin at only very low levels, if at all (see later). in both cns and pns myelin, there are a number of other minor but integral protein components, and the list will continue to grow as research continues. these include structural proteins and proteins involved in cell-cell interactions, as well as a large number of enzymes, receptors, and second messenger-related proteins. all of these have vital roles in maintaining the complex structure of myelin and/or in its function. characteristics of some myelin proteins, including selected aspects of their gene structure and expression, follows, but it is necessarily brief. for a more detailed discussion of individual myelin proteins, see lemke (1988) , morell et al. (1994) , campagnoni (1995) , and newman et al. (1995) . it is worth noting at this point that the composition of myelin changes during development, with the first myelin deposited having a somewhat different composition than that present in adults (norton and cammer, 1984) . in the rat brain, myelin galactolipids increase by about 50%, and phosphatidylcholine decreases by a similar amount. similar changes have been noted in human myelin as well. other minor changes in lipids and gangliosides also occur. the protein portion of myelin also changes somewhat during development; both mbp and plp increase during development, whereas the amount of higher molecular weight proteins decreases. myelin basic proteins (mbps) are highly basic proteins of related isoforms derived from alternative splicing of a single gene. the mbp gene consists of seven exons distributed over about 32 kb of chromosome 18 in the mouse (roach et al, 1985) and human (sparkes et al, 1987) and chromosome 1 in the rat (koizumi et al., 1991) ; at least six transcripts are expressed via alternative splicing of rna (table 3 ). the mbp gene is actually a "gene within a gene," being part of a much larger (approx. 105 kb in mice and 179 kb in humans) transcriptional unit, called the golli-mbp gene pribyl et al, 1993) . portions of the golli-mbp gene are expressed outside the nervous system, including the immune system, although the exact function of these gene products remains unknown. this may be of relevance to clinical disorders related to autoimmunity against mbp. expression of the various mbp protein products is also very complex; in addition to alternative splicing of a number of exons, there is also considerable transcriptional and posttranscriptional control (see campagnoni, 1995, for details) . this complexity of gene expression is augmented by posttranslational protein modifications, including loss of c-terminal arginine, n-acylation, glycosylation, phosphorylation, methylation, deamination, and substitution of some arginine residues with citrulline (toews and morell, 1987; smith, 1992; morell et al, 1994) . mbps are extrinsic membrane proteins localized to the cytoplasmic membrane surface (major dense line) of myelin of both the cns and pns. in the cns this protein accounts for approximately 30% of the total myelin protein, whereas in the pns it accounts for only 18% (greenfield et al, 1980) . myelin lipids can promote mbp self-association, suggesting it may exist as oligomers on the cytoplasmic surface of the myelin membrane (smith, 1992) . it has been suggested that stabilization and maintenance of the myelin structure may be due to specific associations between mbps and sulfatides and gangliosides (ong and yu, 1984; yu, 1989, 1992; mendz, 1992; smith, 1992) . proteolipid protein (plp) and dm20 integral membrane proteins with several transmembrane domains. plp is the most abundant protein in cns myelin (50%), and although mrna for this protein is present in the pns, the protein itself is present at only very low levels in pns myelin and its function in pns myelin is unknown kamholz et al., 1992) . a report (garbern et al, 1997 ) of a human plp null mutation phenotype characterized by a demyelinating peripheral neuropathy suggests that plp/dm20 is necessary for proper myelin formation in the pns as well as the cns. this report also demonstrates by immunoelectron microscopy the presence of plp in compact pns myelin (however, see also puckett et al., 1987) . like mbp, plp is one of the products of alternative splicing of a single gene, having a molecular weight of approximately 25kda. dm20, a second isoform that migrates as a 20kda band on sds gel electrophoresis, is identical to plp except for the deletion of amino acid residues 116-150 (macklin, 1992) . the plp-dm20 gene, located on chromosome x in the mouse, rat, and human, is approximately 17 kb in length and consists of seven exons. the alternative splicing of this gene to give plp and/or dm20 is developmentally regulated, with the dm20 splice product predominating during early myelination. some mutations of the plp/dm20 gene (e.g., jimpy mice; see later this chapter) result in developmental abnormalities prior to any myelination, suggesting that gene may be involved in other functions besides myelination. in addition, expression is not confined to myelin-producing oligodendrocytes in the cns. the role of protein products of this gene outside the nervous system remain unknown, however. missense mutations of the plp/dm20 gene give rise to a host of cns pathologies, the most devastating being pelizaeus-merzbacher disease (pmd) (see seitelberger, 1995) . in some forms of pmd, there is complete deletion of the plp/dm20 gene (raskind et al, 1991) . the described single base pair deletion in humans leads to the absence of plp/dm20 expression; this produces a disease similar to, but less severe than, classic pmd, but also involving a progressive demyelinating peripheral neuropathy (garbern et al, 1997) . a number of both positive and negative cis-acting elements, as well as some trans-acting factors, have been identified for this gene (see campagnoni, 1995, for details) . in its orientation in the myelin membrane, the extracellular domains of plp may be instrumental in stabilizing the intraperiod line of myelin . in addition, plp may play an active role as an ion channel (toews and morell, 1987; lees and bizzozero, 1992) . as noted previously, dm20 is generally a relatively minor product of the plp gene but it shows a pattern of developmental regulation distinct from plp. it is expressed earlier in development than plp and is the major plp gene product in the developing embryo (ikenaka et al., 1992; macklin, 1992; timsit et al., 1992) . its presence in "premyelinating" glial cells and in cells outside the glial cell lineage suggest a possible functional role unrelated to myelination. myelin-associated glycoprotein (mag) is the principal glycoprotein of central nervous system myelin (for review, see milner et al, 1990; quarles et al, 1992) . mag is heavily glycosylated and is specific to myelin sheaths, with an especially high concentration in the periaxonal regions of both cns and pns myelin. it is a member of the immunoglobulin gene superfamily (sutcliffe et al, 1983; lai et al., 1987; salzer et al, 1987) . examination of the extracellular, n-terminal domains suggest that mag is most closely related to the cell adhesion molecules n-cam, l1, and contactin. in the pns, mag immunostaining is seen in glial membranes of the schmidt-lantermann incisures, paranodal loops, and mesaxons (trapp, 1990) and is distinctly absent from the compact myelin sheath. it is thought that mag plays a major role in membrane-membrane interactions during myelin formation and maintenance quarles et al, 1992; morell et al., 1994) . it is presumed to be involved in the adhesion of the myelin sheath to the axonal plasmalemma and in membrane spacing (trapp, 1990) , and it has been implicated in various peripheral neuropathies (mendell et al, 1985; tatum, 1993) . mag exists as two isoforms (l-mag and s-mag) that are derived by alternative splicing from a single gene. l-mag is produced almost exclusively in the cns and is the predominant variant during early development and active myelination (campagnoni, 1988; trapp, 1990) . s-mag is the major isoform in the adult cns and in the pns at all ages. it is thought that the differences in distribution within the cns and pns may be associated with either the phosphorylation or other posttranslational modifications (e.g., sulfation of oligosaccharide moieties, acylation of the transmembrane domain) altering interactions with the cytoskeleton (trapp, 1990; quarles et al, 1992) . homotypic interaction may be operational in schmidt-lantermann incisures, paranodal loops, and mesaxon membranes in pns myelin, whereas heterotypic interactions with axolemmal constituents may mediate glia-axon adhesion trapp, 1990; quarles et al., 1992) . 2'-3'-cyclic nucleotide-'-phosphodiesterase (cnp) is localized within oligodendrocytes in the cns and within schwann cells in the pns. one of the earliest markers for cells of the oligodendroglial lineage, cnp is an enzyme that hydrolyzes 2',3'-cyclic nucleotides to form 2'-nucleotides exculsively. because no physiologically relevant substrate molecules have been found in myelin, however, this enzymatic activity may actually be vestigial and unrelated to its function in myelin. the current view of cnp is that it may be a key component of an interactive protein network within oligodendroglial cells, possibly involved in extension of processes (e.g., see braun et al, 1990) . it is isoprenylated, suggesting possible involvement in signal transduction processes (braun et al., 1991) . furthermore, the presence of potential nucleotide-binding domains on cnp (sprinkle, 1989) suggest it might exert regulatory influence on various cellular processes such as growth and differentiation by serving as a link between extracellular signals and intracellular effector molecules. its exact function within myelin and oligodendroglial cells, however, remains unknown. glycoprotein (mog) mylein/oligodendrocyte glycoprotein (mog) is localized primarily at the external surfaces of the myelin sheath and oligodendrocytes. it is developmentally regulated, appearing with the onset of myelination as one of the last myelin protein genes expressed (scolding et al., 1989) . features of the protein structure suggest it may be a member of the immunoglobulin gene superfamily (gardinier et al., 1992) . because anti-mog antibodies can cause demyelination in vivo (schluesener et al., 1987) , it has received attention for a potential role in autoimmune-mediated demyelination such as experimental autoimmune encephalomyelitis (eae) and multiple sclerosis (gunn et al., 1989; bernard and kerlero de rosbo, 1991) . oligodendrocyte-myelin glycoprotein (omgp) is one of the minor protein components of myelin that appears in the cns during the period of myelination. it is highly glycosylated and appears to be specific to oligodendrocytes and myelin membranes in the cns (mikol and stefansson, 1988) . the protein is anchored to the membrane through a glycosylphosphatidylinositol intermediate. a subpopulation of omgp molecules contain the human natural killer cell antigen-1 (hnk-1) carbohydrate (mikol et al, 1990b) . the presence of a tandem leucine repeat domain in the predicted polypeptide sequence and the apparent presence of omgp at the paranodal regions of the myelin sheath have lead to speculation that its major role is as an adhesion molecule that mediates axon-glial cell interactions (mikol et al., 1990a) . p0 protein (peripheral myelin protein zero) accounts for more than 50% of the protein in peripheral nerve myelin (ishaque et al., 1980) . this has lead to the suggestion that p0 is the pns equivalent of plp in the cns, although the properties of these two proteins are very different. p0 is a transmembrane protein with a glycosylated extracellular domain, a single membrane spanning region, and a highly basic intracellular domain (lemke, (lemke and axel, 1985) . it is thought to play an important role in the compaction of myelin through the homotypic interaction of molecules on adjacent myelin lamellae (lemke, 1988) . like mag, it is a member of the immunoglobulin superfamily. unlike many of the other myelin protein genes, expression of the p0 gene is highly restricted to schwann cells. the p0 gene contains six exons distributed over about 7 kb in both rats and mice (lemke, 1988; you et al, 1991) . based on transgenic experiments, elements regulating its expression appear to reside in first 1.1 kb of the 5'-flanking region (messing et al, 1992) . mutation of the p0 gene in humans is associated with charcot-marie-tooth disease, type 1b, an inherited peripheral neuropathy (hayasaka et al., 1993; kulkens et al., 1993) . peripheral nerve protein p2 is a basic protein distinct from mbp. interest in p2 arose from its ability to induce experimental allergic neuritis, a demyelinating disease of the pns (kadlubowski and hughes, 1980) . it has sequence homology to cellular retinol and retinoic acidbinding proteins (crabb and saari,1981; eriksson et al., 1981) and fatty acid-binding proteins (fabps) (veerkamp et al., 1991) , and has high affinity for oleic acid, retinoic acid, and retinol (uyemura et al., 1984) . the p2 protein gene belongs to an ancient family of fabps that diverged into two major subfamilies (medzihradszky et al., 1992) . p2 mrna levels parallel myelination during development as well as the levels of microsomal enzymes involved in fatty acid elongation. this suggests the p2 protein may be involved in fatty acid elongation or in the transport of very long-chain fatty acids to myelin (narayanan et al, 1988) . peripheral myelin protein-22 (pmp-22) is a glycoprotein with an apparent molecular weight of about 22 kda (kitamura et al., 1976; smith and perret, 1986 ). the rat and human genes have been cloned (spreyer et al., 1991; welcher et al, 1991; hayasaka et al., 1992) ; although these have a high homology to the growth arrestspecific mrnas for gas 3 and pasii-glycoprotein, the function of pmp-22 in pns myelin remains unknown. the pmp-22 gene maps to mouse chromosome 11 and a point mutation in this gene is apparently responsible for the autosomal dominant mutation in the trembler (tr) mutant mouse (suter et al, 1992a,b) . in humans, the gene maps to chromosome 17; this gene is duplicated in patients with charcot-marie-tooth disease, type 1a, and this alteration is presumably related to the pathology (patel et al., 1992; valentijn et al., 1992; see campagnoni, 1995, and newman et al., 1995 for additional references and discussion). the rat pmp-22 gene, the expression of which is largely confined to the pns, is developmentally regulated in schwann cells, where its expression coincides with myelination (spreyer et al, 1991; snipes et al., 1992) . mrna expression is coordinately down-regulated along with other myelin proteins during tellurium-induced primary demyelination and during degeneration induced by nerve transection or crush; message levels are consequently up-regulated along with other myelin genes during remyelination if it occurs (spreyer et al., 1991; toews et al, 1997) . although myelin initially was believed to be metabolically inert (due largely to the very slow metabolic turnover of some of its components) and to function exclusively as an electrical insulator, it is now known that the picture is considerably more complex and interesting. all protein and lipid components of myelin turn over with measurable turnover rates (see benjamins and smith, 1984; morell et al, 1994) . although some structural components of myelin are indeed relatively stable metabolically, with half-lives of several months, there is also a very rapid turnover of some myelin components. the phosphate groups modifying myelin basic protein in compact cns myelin turn over with half-lives of minutes or less (desjardins and , and the phosphate groups on myelin polyphosphoinositides also show a very rapid turnover rate. at least 40 different enzyme activities have been documented in cns myelin (newman et al, 1995) . in addition to cnp described previously, these include enzymes related to second messenger signalling, as well as associated receptors and g-proteins (larocca et al., 1990) . a number of enzymes involved in myelin lipid metabolism are also present, including those for phospholipid synthesis and catabolism. noteworthy among the latter are phospholipase c activities for polyphosphoinositides, and these may have important roles in signal transduction mechanisms in myelin (ledeen, 1992) . also of note are a cholesterol ester hydrolase, and udp-galactose : ceramide galactosyltransferase, the terminal enzyme in biosynthesis of galactocerebroside, the most "myelinspecific" lipid. various proteases, protein kinases and phosphatases, and transport-related enzymes are also present; of particular interest with respect to the transport-related enzymes are carbonic anhydrase (cammer et al, 1976) and na+,k+-atpase (zimmerman and cammer, 1982) . these enzymes may be involved in controlling k + levels at nodes of ranvier (lees and sapirstein, 1983) and/or in removal of carbonic acid from metabolically active axons. the cell surface area of myelin-forming cells is so large as to suggest the need for specialized structures and mechanisms for transporting components between the perikaryon and the remote extensions. there is indeed a great deal of protein and lipid transport and targeting within the myelinating glial cell, and cytoskele-tal elements play important roles in these processes. p0, mag, and laminin (a secreted extracellular matrix component ; cornbrooks et al, 1983) are synthesized and modified in the rough endoplasmic reticulum (rer) and golgi membranes of the perinuclear cytoplasm and then sorted into different carrier vesicles upon exit from the trans golgi network (trapp et al, 1993) . these proteins must be transported over millimeter distances prior to insertion into their proper surface membrane locations. various proteins enriched in compact myelin reach their proper destinations via different mechanisms. for example, p0 reaches compact myelin by vesicular transport, whereas it is the mrna for mbp that is translocated, with synthesis of mbp occurring close to the site of its insertion into the forming myelin (colman et al, 1982; trapp et al, 1987; griffiths et al, 1989) . as noted previously, the cytoskeleton plays a major role in transport and assembly of myelin. in myelinating schwann cells, microfilaments are enriched beneath the membranes of the schmidt-lanterman incisures, the outer and inner mesaxons, and portions of the outermost compact myelin lamellae and schwann cell plasma membrane (trapp et al., 1989; zimmerman and vogt, 1989; kordeli et al, 1990) . it is thought that interactions between mag and microfilaments play a role in membrane motility during myelination (trapp and quarles, 1982; trapp et al, 1984; martini and schachner, 1986; salzer et al, 1987) . mag colocalizes with microfilaments at membranes that move during internodal growth (trapp et al, 1989) . a role for mag in myelin wrapping and spacing is supported by studies showing precocious spiral wrapping by myelinating schwann cells transfected with additional copies of mag (owens et al, 1990) , and impaired or prevented wrapping when mag expression is reduced or eliminated in schwann cells (owens and bunge, 1991; mendell et al., 1985) . microfilaments also help to define and maintain organelle-rich channel regions and organelle-free nonchannel regions of the myelin internode. the channel regions are important to formation and maintenance of the myelin internode and to intracellular transport of myelin components. they include the external cytoplasmic channels, schmidt-lanterman incisures, para nodal loops, and periaxonal cytoplasm. microfilaments associated with the abaxonal plasma membrane and the adaxonal periaxonal membrane may have multiple functions in dealing with the external environment, including endocytosis (blok et al., 1982) , pinocytosis (phaire-washington et al, 1980) , and exocytosis ( john et al, 1983; koffer et al., 1990) , as well as stress resistance. just as they do in axons, microtubules may also subserve a role in the directional movement of organelles within glial cells. post-golgi vesicles transported in this way could be involved in the growth, turnover, or modification of compact myelin at distant sites. microtubules are the largest of the cytoskeletal filaments and provide a dynamic substrate for organelle trafficking and structural organization (for review see dustin, 1984; kirschner and mitchison, 1986; schroer and sheetz, 1991) . they are present in all major cytoplasmic compartments of the myelin internode, but are excluded from compact myelin (peters et al., 1991) . in myelinating schwann cells, microtubules are crucial to the transport of myelin proteins and organelles (trappet al, 1995) . this function is determined by the orientation and organization of microtubules, which in turn are influenced by axons (kidd et al., 1994) . during the formation of the myelin sheath, contact with a myelin-inducing axon results in a more complex microtubule organization (kidd et al., 1994) . as in axons, microtubules are inherently unstable and oscillate between phases of elongation and collapse (dustin, 1984; kirschner and mitchison, 1986 ). the extent of depolymerization and repolymerization is determined by complex assembly/ disassembly kinetics and can be influenced by modifications such as binding of maps (sloboda et al., 1976; pryer et al., 1992) . microtubule disassembly causes marked accumulation of p0, mag, and laminin in the perinuclear cytoplasm of myelinating schwann cells (trapp et al., 1995) . because of this, chemically induced neurotoxicity involving microtubules may lead to alterations not only in axons, but also in myelin and myelinating cells as well. in myelinating schwann cells, the major intermediate filament is vimentin (dahl et al., 1982; schachner et al., 1984; kobayashi and suzuki, 1990) . in most cells, intermediate filaments are considered to have a structural role in mechanically maintaining cell shape against external forces (klymkowsky et al., 1989; skalli and goldman, 1991) it has been proposed that vimentin intermediate filaments interact with microfilament-associated molecules and with microtubules in resisting stress (wang et al., 1993) . it is thought that intermediate filaments play a role in the process of myelination in the pns because myelinating schwann cells contain abundant intermediate filaments and the content of these filaments between myelinating and nonmyelinating schwann cells can vary substantially. because the structure and composition of myelin is unique, its formation involves activation of a set of unique genes (see lemke, 1988 , for details). these genes include those related to induction of myelination (e.g., glial-specific receptors for differentiation signals), those involved in controlling and directing the initial deposition of myelin (e.g., axon-glial cell-adhesion molecules), and those involved in actual production of compact myelin (e.g., structural proteins of myelin). genes for enzymes involved in synthesis of lipids enriched in myelin are also preferentially activated as well. the process of myelination is a highly regulated event that begins postnatally during the first few weeks of life in the rodent brain and within the third fetal trimester in the human spinal cord. in the cns of rodents, maximum levels of synthesis of myelin components and actual accumulation of myelin occurs at about 3 weeks of age (norton and poduslo, 1973b; norton and cammer, 1984; morell et al., 1994) , and although myelin accumulation continues for an extended time, the rate of synthesis declines considerably by about 6 weeks of age. this time course is similar to the profile of expression of myelin protein genes (see campagnoni and macklin, 1988) . in the rodent pns, myelination begins at about birth, peaks at about 2 weeks, and then decreases to a low basal level by the end of the first month (webster, 1971) . as is the case in the cns, the pattern of myelin synthesis and accumulation is closely paralleled by expression of mrna for pns myelin protein components (lemke and axel, 1985; stahl et al, 1990 ) and for enzymes involved in synthesis of major myelin lipids (hydroxymethylglutaryl-coenzyme a [hmg-coa] reductase, the rate-limiting enzyme in cholesterol biosynthesis; and ceramide galactosyltransferase, the ratelimiting enzyme in cerebroside biosynthesis) (lemke and axel, 1985) . regulation of the expression of myelin genes occurs at a number of different levels including promotor choice, transcription, mrna splicing and stability, translation, and posttranslational processing (for reviews see campagnoni, 1988; campagnoni and macklin, 1988; lemke, 1988; nave and milner, 1989; ikenaka et al, 1991; mikoshiba et al, 1991; campagnoni, 1995) . the synthesis and assembly of myelin has been examined by measuring incorporation of radioactive precursors into myelin components both in vivo and in tissue slices, by measuring the in vitro activities of enzymes involved in synthesis of myelin components, by examining levels of expression of mrna species for myelinrelated genes, and by actual isolation and analysis of myelin (for reviews see benjamins and smith, 1984; dewille and horrocks, 1992; morell et al., 1994) . after individual myelin components have been synthesized, they must be assembled to form mature compact myelin. some components, such as plp, which is synthesized on bound polyribosomes in the oligodendroglial perikaryon, show a time lag of about 45 min between their synthesis and their appearance in myelin, reflecting the time required for transport from their site of synthesis to the forming myelin. other components, such as mbp, show only a short lag, in keeping with their synthesis of free polyribosomes in oligodendroglial processes, near the actual site of myelin assembly. in keeping with this difference, studies have shown that myelinating cells spatially segregate mrna species for myelin-specific proteins (see trapp et al., 1987) . mrna for mbp is transported to near the sites of myelin assembly before the protein is synthesized gillespie et al., 1990) , whereas plp mrna is present in a perinuclear location. individual lipids also show different kinetics of entry into myelin following synthesis, and some of this may be due to synthesis in, or movement through, different intracellular pools benjamins and iwata, 1979 ; for review, see benjamins and smith, 1984) . the mature myelin internode contains several ultrastructurally and biochemically distinct membrane domains that include the outer plasma membrane of the myelin-forming cell and its attached compact myelin, as well as the schmidt-lanterman incisures, paranodal loops, nodal microvilli, the periaxonal membrane, and the membranes of the outer and inner mesaxons (see figs. 1 and 3) . some of these membrane domains are compositionally distinct, containing different structural proteins and differing in lipid composition as well. with respect to the pns, p0, mbp, and pmp-22 are enriched in compact pns myelin (trapp et al., 1981; omlin et al., 1982) , whereas plp and mbp predominate in compact cns myelin. the exact mechanisms by which individual components are targeted to their respective membrane domains and other molecular aspects of actual myelin membrane assembly are not well understood, but this continues to be an active area of investigation. it seems clear that cytoskeletal elements are closely involved in the intracellular sorting and transport of myelin components, as discussed in a previous section, and they presumably also function in the actual process of myelin assembly. in the mouse, terminal differentiation of myelinforming cells occurs mostly after birth, following establishment of the basic wiring of the nervous system. many neurologic mutations result in dysmyelination, the inability of myelin-forming glial cells to assemble qualitatively and/or quantitatively normal myelin (see quarles et al., 1994; nave, 1995) . because myelination is a postnatal event in rodents, this inability to assembe normal myelin is not immediately lethal, and the animals usually survive for at least several weeks. mutations that affect myelin are characterized behaviorally by abnormalities such as shivering, ataxia, and frequently including seizures; these signs of abnormal nervous system function begin at about the time when myelin accumulation becomes significant, probably an indication of the importance of myelin for motor control and normal brain function. in general, myelin-deficient mice possess a mutant gene for some structural myelin protein (see lemke, 1988) . the major known "myelin-deficient" mutations in mice are described as follows, as are some related transgenic and "knockout" mouse models. the shiverer mouse (shi, mouse chromosome 18) was one of the first neurologic mouse mutants examined at the molecular-genetic level (roach et al., 1983) . affected homozygotes lack any detectable mbp and fail to make normal cns myelin. the behavioral phenotype of this mouse is first observed within the second postnatal week, when a general body tremor, which becomes more pronounced with intentional movements, develops (biddle et al., 1973; chernoff, 1981) . the shivering behavior derives from a loss of spinal motor and reflex control and increases with age, often progressing to include seizures. the life span of the shiverer mouse is limited to approximately 6 months of age. histologic examination shows severe dysmyelination throughout the entire cns, but with normal-appearing pns myelin (privat et al., 1979; kirschner and ganser, 1980; rosenbluth, 1980) . at the ultrastructural level, the pattern of dysmyelination is dominated by a severe lack of myelin. the myelin-like structures that are occasionally present are loosely wrapped around the axon and the intracellular adhesion zone of the extended cell process that normally forms the "major dense line" of myelin cannot be discerned . the lack of proper myelin sheath formation may be the result of a defect in myelin compaction or a related process, because oligodendroglia appear to be normally differentiated. histologic evidence of dysmyelination is supported biochemically by a dramatic reduction of all major myelin proteins, and more specifically by the complete lack of mbp. this is the result of a large (20 kb) genomic deletion encompassing exons 3-7 of the mbp gene (or exons 7-11 of the larger golli-mbp gene) resulting in no coding capacity for any of the mbp isoforms (roach et al., 1985; molineaux et al., 1986) . the tremoring phenotype can be cured by a number of manipulations associated with restoration of mbp expression, indicating that the amount of mbp available to the oligodendrocytes is a rate-limiting step in the assembly of cns myelin. successful approaches include reintroduction of the entire wild-type mbp gene into the germ line of the shiverer mouse , increasing the transgene copy number shine et al., 1990) , and the reintroduction of a mbp minigene encoding only the smallest (14 kda) mbp isoform (kimura et al., 1989) . a shiverer-like phenotype can also be generated in normal mice by specifically down-regulating the amount of mbp mrna available for protein synthesis via transgenic expression of the mbp gene in "anti-sense" orientation under the control of its cognate mbp promotor (katsuki et al., 1988) . similarly, the formation of antisense mbp mrna is the presumed primary defect of the myelin-deficient (shi-mld) mouse mutant, an allele of the shiverer mutation on chromosome 18 (doolittle and schweikart, 1977; popko et al., 1988) . the presence of this antisense rna is thought to reduce and dysregulate the amount of normal mbp mrna functionally available, thereby resulting in a level insufficient for normal myelin formation (fremeau and popko, 1990; tosic et al., 1990) . the dysmyelination is less severe in the myelin deficient mouse when compared to the shiverer mouse. isolated white matter tracts in cns have a 3-4-fold increase in sodium channel density and it has been suggested that some myelin-associated molecule absent from the shiverer white matter tracts could cause a down-regulation of either the synthesis or accumulation of sodium channels in myelinating axons (noebels et al., 1991) . the lack of dysmyelination in the pns is thought to be due to the substitution of p0, the major integral protein of pns myelin, for the structural function of mbp (lemke and axel, 1985) . the mammalian plp gene is linked to the x chromosome and defects in this gene are associated with neurologic abnormalities in the mouse and with pelizaeus-merzbacher disease in humans. in the mouse, three mutations have been characterized: the jimpy (jp), myelin synthesis-deficient (jp msd), and rumpshaker (rsh). each derives from a point mutation that alters the structure of the encoded protein. in the jimpy mouse, the mutation is a single nucleotide change in the plp gene that inactivates the splice-acceptor site of intron 4. the last a-helical transmembrane domain is replaced by an aberrant carboxy terminus, and the resulting abnormally folded protein is degraded in the endoplasmic reticulum shortly after synthesis, failing to reach the golgi apparatus for further processing and transport (roussel et al, 1987) . the cns is nearly completely devoid of myelin, with less than 1% of the axons ensheathed; pns myelin, however, is ultrastructurally intact (sidman 1964; her-schkowitz et al, 1971) . there are only a few layers of abnormally thin myelin around cns myelin, consisting of either uncompacted membrane whorls or compacted myelin with abnormal ultrastructure (duncan et al, 1989) . the major cause of the dysmyelination in the jimpy mouse seems to be a lack of differentiated oligodendrocytes. the proliferation rate of oligodendrocyte precursor cells is increased but an abnormally high rate of apoptotic cell death eliminates most of these maturating oligodendrocytes (skoff, 1982; knapp et al, 1986; barres et al., 1992) . islands of myelinated fibers are formed by the few oligodendrocytes that escape degeneration and developmental arrest. the behavioral phenotype is evident at 2 weeks of age and consists of general body tremor and ataxia; the animals die with seizures and convulsions by about 4 weeks of age. heterozygous jimpy females, which are mosaics with respect to the x-linked plp gene, display normal behavior. in the allelic mouse mutant, jimpy msd, there are similar ultrastructural alterations in myelin as seen in the jimpy mouse, but about twice as many glial cells escape premature degeneration (billings-gagliardi et al, 1980) . the rumpshaker mutant is the result of a novel mutation of the plp gene (schneider et al, 1992) and displays a phenotype very different from the jimpy and the jimpy msd. rumpshaker mice have more myelin than other dysmyelinated mutants and the degree of dysmyelination varies among cns regions, with early myelinated regions appearing normal whereas late myelinating regions are severely hypomyelinated. the oligodendrocytes appear differentiated and most escape apoptotic cell death resulting in a normal complement of mature oligodendrocytes (griffiths et al, 1990) . the rumpshaker mutation appears to allow the oligodendrocyte to survive but somehow interferes with its ability to normally deposit plp in the myelin membrane (schneider et al, 1992) . although sparse, some myelin sheaths subsist in the rumpshaker mutants and these show selective immunostaining for dm20 (schneider et al., 1992) . these findings suggest that dm20 may serve a critical purpose in glial cell development that is distinct from any function in myelin formation and maintenance. p0-deficient mice have been generated by homologous recombination of the p0 gene in mouse embryonic stem cells with the cloned gene and subsequent generation of germline chimeric mice (giese et al, 1992) . animals lacking one functional copy of the p0 gene are phenotypically normal, but the homozygotes develop a behavioral phenotype by the third week of life. these mice show a body tremor and dragging movements of the hindlimbs. there is no evidence of paralysis or sei-zures and the mutants have a normal life span. histologically, the deficit is characterized by the inability of the schwann cell to assemble a compacted multilamellar pns myelin sheath. the high degree of variability in the pathology is thought to be due to the intervening actions of other proteins such as cell adhesion molecules (mag; n-cam), and perhaps other myelin proteins. using promoter and regulatory regions of the p0 gene in a fusion gene construct, schwann cells were destroyed when they began to express p0, after associating in a 1 : 1 ratio with axons (messing et al, 1992) . the behavioral phenotype of the schwann cell-ablated mice was similar to the phenotype displayed in the homogygous p0 mutants. a proliferation of nonmyelin-forming schwann cells was induced along with skeletal muscle atrophy. the trembler mouse (tr; mouse chromosome 11) contains a mutation of the pmp-22 gene, which results in pns dysmyelination. the pmp-22 gene encodes an integral membrane protein specific to schwann cells (22 kda peripheral myelin protein) believed to be important for normal schwann cell development (spreyer et al, 1991; welcher et al., 1991) . histologically, the majority of large caliber axons in the sciatic nerve are devoid of a myelin sheath and, if present at all, these are abnormally thin and relatively uncompacted (henry and sidman, 1988) . the total number of schwann cells is dramatically increased at the time of segregation of axons, and myelin deposition is arrested. in the absence of pns myelin, the mice display a behavioral phenotype characterized by a coarse-action tremor that begins at the end of the second postnatal week and results in moderate quadriparesis and a waddling gate. under controlled conditions, these animals can experience a normal life span. the quaking mouse (qk; mouse chromosome 17) is the result of an autosomal recessive mutation (sidman et al., 1964) . homozygous mice carrying the viable quaking allele (qkv/qkv) show the typical motor coordination signs of dysmyelination in the absence of seizures and have a normal life span. the myelin deficiency, characterized by fewer than normal myelin lamellae, is predominately in the brain and spinal cord, although a lack of normal compaction and enlarged intraperiod lines of some myelinated fibers has been noted in the pns as well (trapp, 1988) . interestingly, the distribution of mag is shifted from the innermost myelin layer facing the axon to throughout the compact myelin sheath. myelin mutants in a number of other species besides humans and mice have also been described (see duncan, 1995 , for detailed discussion). these include x-linked mutations in the dog (shaking pup, griffiths et al., 1981) , pig (type a iii hypomyelinogenesis congenita, blakemore et al., 1974) , rat (myelin-deficient; dentinger et al., 1982; jackson and duncan, 1988) , and rabbit (paralytic tremor, taraszewska, 1988) , as well as the autosomal recessive taiep mutant rat . as noted previously, myelination is a critical process in the maturation of the nervous system. it involves the synthesis of an enormous amount of specialized membrane within a relatively short period of time. much of the myelin in both the cns and the pns is formed during a relatively short "developmental window" (first few years in humans; first 30 days of age in rodents), and this period is preceded by a burst of myelinatingcell proliferation. during these time periods, a large portion of the nervous system's metabolic capacity is devoted to myelinogenesis. during these "vulnerable periods," the process of myelination is especially susceptible to perturbations such as toxic insults, nutritional deficiencies, genetic disorders of metabolism, viral infections, substances of abuse, and other environmental factors (for review, see wiggins, 1986) . insults occurring during the period of proliferation of myelinating cells may be especially disruptive, as this may lead to an irreversible deficit of myelin-forming cells and consequent permanent hypomyelination. perturbation of myelination at a later stage may result in a myelin deficit that can be reversed. depending on the timing of the insult, a myelin deficiency can result from alterations related to several different developmental events, including failure of myelin-forming cells to proliferate, reduction of axonal development resulting in fewer and/ or smaller axons to myelinate, and decreased formation of myelin at time of maximal synthesis. the morphologic term myelinopathy describes damage to white matter or myelin, and disorders of myelin can be classified by a number of different factors. such factors include the preferential effects on either the cns or on the pns or an involvement of both systems. in addition, effects on myelin can sometimes be delineated as the result of a primary effect on myelin itself or the myelinating glial cell. myelin loss due to a primary insult to myelin or the myelinating cell are termed primary demyelination. there are a number of factors relevant to selective targeting of various toxic or metabolic in-suits to myelin (for discussion, see morell et al, 1994; morell and toews, 1996a ). an intact axon is a prerequisite for maintenance of normal myelin; alterations in myelin due to an effect on the neuron or the underlying axon is termed secondary demyelination. secondary demyelination is an inevitable consequence of serious damage to neurons supporting myelinating axons or to axonal transcetion or crush (wallerian degeneration). however, the distinction between primary and secondary demyelination is often somewhat vague; the basis for this distinction usually involves morphologic evidence of the initial target site. the term hypomyelination is used to describe developmental alterations of myelination in which an insufficient amount of myelin accumulates. hypomyleination can be the result of disease processes, undernutrition, or toxic insult. the term dysmyelination, when used in its strictest sense, refers to certain inborn errors of metabolism in which a block in the breakdown of a myelin lipid causes accumulation of myelin of an abnormal composition (which eventually leads to a collapse and degeneration of myelin), but it is also in wide use as a general descriptor of situations characterized by any abnormalities in myelin. some specific myelinopathies that are preferential to developing organisms are discussed later in this chapter. additional toxicants have been demonstrated to disturb myelin in the adult animal and the morphologic descriptions and mechanistic processes involved have been previously discussed (morell, 1994; morell and toews, 1997) . these include tellurium, diphtheria toxin, 2'3'dideoxycytidine, vigabatrin, carbon monoxide, triethyltin, lead, hexachlorophene, cuprizone, and isoniazid. in the human infant, several studies have provided evidence supporting the concept of a critical period from birth to about 2 years of age, during which time the nervous system is most vulnerable to malnutrition. the production of neurons is virtually completed by about the midpoint of gestation, but glial cell production continues through the end of gestation into the second postnatal year. the vulnerability of the developing nervous system to various factors is determined by the developmental stage of the cellular activities targeted by a specific insult. the effects of an agent or condition may vary depending on the agent, the time of insult during development, and the species under study (dobbing, et al, 1971) . general factors such as undernutrition can have maximal effects on processes that are most active during what has been called the "brain growth spurt" (dobbing and sands, 1979) . depending on the developmental process ongoing at the time of exposure, alterations can be produced in either the number of neurons and extent of axonal arborizations, the number of glial cells, or the degree of myelination. myelination in both the cns and pns is sensitive to nutritional factors (see wiggins, 1986; blass, 1994) . brains of rats undernourished from birth contain a lower amount (20% deficit) of total lipids, cholesterol, and phospholipids and a 50% deficit in cerebrosides (benton et al, 1966) . following severe nutritional deprivation during lactation and post-weaning, total brain galactolipids, cholesterol, and lipid phosphorus showed a slower rate of accumulation (krigman and hogan, 1976) . lipid phosphorus and cholesterol levels recovered by adulthood, whereas galactolipids remained at a 60% decreased level. myelin recovered from undernourished rats was normal in lipid composition but siginificantly reduced in total amount (fishman et al., 1971) . a reduced proportion of basic and proteolipid protein was seen in myelin isolated from undernourished rats at postnatal days 15 and 20, but the composition was similar to normals by postnatal day 30. at all time points examined, myelin yield was 25% less than normal levels (wiggins et al., 1976) . these studies suggested that undernutrition produced a delay in myelin maturation. morphologic examination of animals undernourished from birth showed a decreased number of mature oligodendroglia and poorly stained myelin (bass et al., 1970; krigman and hogan, 1976) . the number of myelin lamellae per axon and the number of myelin lamellae for a given axon diameter were both lower (krigman and hogan, 1976) . some studies suggest that in some cases, myelination is able to catch-up and achieve normal levels once unrestricted feeding is initiated. rats deprived by an increased litter size rapidly gained body and brain weight and normal brain lipid composition within 3 weeks after weaning to an unrestricted diet (benton et al, 1966) . however, nutritional deprivation during the first 21 days of life resulted in reduced levels of total brain lipids, cerebrosides, cholesterol, and plp, and this deficit persisted through 120 days of age (bass et al., 1970) . similar persistent myelin deficits were found in brains of rats subjected to either moderate or severe food deprivation during the first 30 days of life (toews et al., 1983) . although metabolic studies showed that after 6 days of free feeding following 20 days of postnatal starvation, incorporation of labeled precursors into myelin proteins was higher than in animals starved for the entire 26 days, and it was still depressed relative to controls (wiggins et al., 1976) . severe underfeeding in rats from 1 to 14 days of age resulted in a lasting significant deficit in myelin, even with rehabilitation (wiggins and fuller, 1978) . overall, these studies point to the possibility of irreversible deficits in myelin resulting from nutritional deficiencies during development. additional studies suggest the most vulnerable period for myelin may be the time of oligodendroglial proliferation. animals deprived during this period are left with a permanent deficit of myelinforming cells, resulting in irreversible hypomyelination (wiggins and fuller, 1978) . apparently, once normal numbers of oligodendroglia have been formed, the process of myelin formation itself is somewhat more capable of nutritional rehabilitation. similar effects can be found with a specific nutritional manipulation of depleting protein in the diet. in rats subjected to a protein and calorie deficiency during gestation and lactation, glial numbers were greatly reduced, and by postnatal day 19 the majority of cells in the corpus callosum appeared to be glioblasts rather than differentiated oligodendroglia (robain and ponsot, 1978 ). an early postnatal protein deficiency resulted in reduced levels of brain myelin and an altered myelin composition in rats (nakhasi et al., 1975) . in myelin from offspring of rats maintained on a 4% protein diet during lactation, an excess of high molecular weight proteins and a deficiency of plp in heavy myelin was found at postnatal day 17, with normal protein composition seen at 53 days (figlewicz et al., 1978) . the mag persisted in its higher molecular weight form longer than normal, suggesting that protein deficiency results in a delay in development and maturation of the myelination process (druse and krett, 1979) . animals raised on a fat-deficient diet are able to synthesize all fatty acids except the essential fatty acids (linoleic and linolenic acid families). essential fatty acid deficiency induced prenatally in the mothers and postnatally in the offspring resulted in lower brain weights (white et al., 1971) , and a low level of galactolipid and plp (mckenna and campagnoni, 1979) . in the optic nerve of essential fatty acid-deficient rats, vacuolation, intramyelinic splitting, and wallerian degeneration were present (trapp and bernsohn, 1978) . several studies have demonstrated hypomyelination in the offspring of copper-deficient mothers (dipaolo et al., 1974; prohaska and wells, 1974) . in the third generation of mice maintained on a copper-deficient diet, the offspring showed approximately 60% decrease in myelin yield with the major glycoprotein shifted to a higher molecular weight (zimmerman et al., 1976) . thyroid hormones influence the temporal onset of myelination and its compositional maturation. neonatal thyroidectomy in rats results in a lasting reduction of total cerebroside in the brain and a 30% reduction in myelin yield (balazs et al., 1969) . it is thought, however, that hypothyroidism does not exert a specific effect on myelin but rather delays myelin development and matu-ration (dalal et al, 1971; walters and morell, 1981) . whereas hypothyroidism resulted in a 1-2 day delay of myelinogenesis with prolonged immature myelin formation, it eventually attained a normal composition, although the myelin deficit persisted. a classic example of differential susceptibility of the developing organism to the effects of an environmental chemical is that of inorganic lead exposure. children are more vulnerable to lead in terms of external exposure sources, internal levels of lead, and timing of exposures during development. at high exposure levels, lead induces encephalopathy in children and can be life threatening. experimental animal studies have allowed examination of various specific target sites and processes of development susceptible to lead toxicity . during development, the process of cns myelination shows an increased vulnerability to lead exposure. the amount of lead that accumulates in the brain of the developing animal during lactation can be as much as 4 times higher than brain levels in the lactating dam receiving lead in the drinking water. under these conditions, myelin was significantly reduced; however, the relationship between the axon diameter and myelin lamellae remained normal, suggesting that the hypomyelination was the result of altered axonal growth (krigman et al., 1974) . direct administration of lead via to pups intubation from 2 to 30 days of age resulted in a reduction of myelin accumulation in the forebrain and optic nerve. these effects were not due to undernutrition, as the accumulation of brain myelin was decreased significantly relative to controls undergoing a similar degree of malnourishment (toews et al., 1980) . in developing rats, there is a synergistic interaction between lead exposure and mild malnutrution induced by milk deprivation with respect to decreasing the normal developmental accumulation of myelin (harry et al., 1985) . this interaction appeared to be more prevalent in females as compared to males. the decrement in myelin induced by development exposure to inorganic lead is a long-lasting effect that persists into adulthood (toews et al., 1980 (toews et al., , 1983 . myelination is not necessarily the most sensitive target for lead as low doses sufficient to produce some microscopically discernible hemorrhagic encephalopathy in the cerebellum of young rats did not depress myelination (sundstrom and karlsson, 1987) ; this hemorrhagic encephalopathy may be related to concentration of lead in brain capillaries (toews et al., 1978) . the basic cns change induced by exposure to tet is a massive cerebral edema, restricted primarily to the white matter (magee et al., 1957; torak et al, 1970) , with the formation of intramyelinic vacuoles (jacobs et al., 1977) . the pathologic effect varies with the age of the animal (suzuki, 1971) . young rats exposed to tet develop severe spongious white matter similar to that seen in the adult, but with the absence of major clinical signs seen in the adult (suzuki, 1971; blaker et al., 1981) . it is thought that the severe paralysis seen in the adult animal is due in part to the intracranial pressure developed during severe edema, whereas the open cranial sutures in the young rat may allow for edema in the absence of increased pressure. when newborn rats are exposed to tet, brains became swollen and petechial hemorrhages are observed, particularly in the cerebellum. necrotic cells were found diffusely throughout the brain (watanabe, 1977) . when older (postnatal day 8) animals were exposed, both the hemorrhagic and necrotic changes occurred, but damage was also seen in the myelinated fibers of the brain stem and cerebellum. although the morphologic alterations in myelin dissipate with time, biochemical evidence suggests that the amount of myelin produced is decreased and that this myelin deficit persists through adulthood (blaker et al., 1981; toews et al., 1983) . chronic exposure to tet from 2 to 30 days after birth decreases myelin yield and cerebroside content (55%) and 2',3'-cyclic nucleotide 3'-phosphohydrolase activity (20%) (blaker et al, 1981) . in studies using radioactive tracer, smith (1973) demonstrated that it is the newly forming cns myelin that is preferentially susceptible to degradation by tet. interestingly, administration of tet to quaking mice did not produce intramyelinic edema (nagara et al, 1981) . when young animals are exposed to trialkyllead, the process of myelination is inhibited (konat and clausen, 1976) . unlike triethyltin, this impairment in myelinogenesis is not accompanied by edema of white matter. the impairment appears to be primarily in the deposition of myelin rather than in the program for myelination, as the protein composition of forebrain myelin isolated from triethyllead-intoxicated young rats was normal (konat and clausen, 1978) . in vitro studies suggest that the alteration involves posttranslational processing and transport of integral membrane proteins, processes particularly important for myelin proteins during development (konat and clausen, 1980; konat and offner, 1982) . hexachlorophene (2,2'-methylenebis-3,4,6-trichlorophenol) is an antimicrobial agent that has been used previously in soaps and detergents, as well as in the bathing of newborn babies to prevent bacterial infections (herter, 1959; powell et al., 1973; for review, see towfighi, 1980) . both cns and pns myelin show a severe white matter edema following exposure, and young rats are more vulnerable than adults (towfighi et al, 1974) . in young rats, edema of the myelin sheath becomes evident after postnatal day 15, probably because the myelin membrane provides a hydrophobic reservoir for accumulation of this toxic compound and thereby becomes a significant site for fluid accumulation (nieminen et al., 1973) . developmental exposure results in a decrease of the normal accumulation of myelin during development (matthieu et al., 1974) . in 22-dayold rats nursed by mothers fed hexachlorophene, there was a decrease in myelin yield, yet the myelin composition remained normal. abnormal "dissociated" myelin accounted for about 10% of the total myelin and contained the typical myelin constituents with the exception of mag, which was absent (matthieu et al., 1974) . degeneration caused by this antimetabolite involves myelin, neurons, astrocytes, and oligodendroglia. in young animals injected with 6-aminonicotinamide, the pns shows a selective swelling of schwann cell cytoplasm at the inner surface of the myelin sheath. the nerve is compressed by the swelling and results in an overgrowth of the myelin sheath (friede and bischhausen, 1978) . young ducklings fed a diet containing ihn developed a wobbling gait and head tremor after 2 weeks, progressing to ataxia and inability to stand (lampert and schochet, 1968) . examination of the cns showed spongy degeneration of the myelin-containing white matter. cuprizone (bis-cyclohexanoneoxalyhydrazone) is a copper chelator that results in cns demyelination following dietary exposure to weanling mice. the loss of myelin can reach as much as 70% in white matter regions of the cerebrum (carey and freeman, 1983) . deficits in adenosine triphosphate (atp) production secondary to reduced activity of cytochrome oxidase (a copperrequiring enzyme) may lead to alterations in energyrequiring ion transport mechanisms, but the underlying reason for targeting of this compound to cns myelin is not clear. interestingly, cuprizone inhibits carbonic anhydrase, an enzyme present in myelin, and this inhibition takes place well before any demyelination is observed (komoly et al., 1987) . in experimental studies of cuprizone neurotoxicity, mrna for mag, a protein located at the myelin-axonal interface, is downregulated during demyelination and returns to normal levels following cessation of exposure (fujita et al, 1990) . the mrna for this glycoprotein exists in two major splice variants that are both severely downregulated. on recovery, one splice variant returns to normal levels whereas the other shows an accumulation above control levels. prolonged exposure to cuprizone (9 weeks or longer in mice) results in irreversible demyelination (tansey et al., 1996) , possibly due to death of oligodendrocytes and/or oligodendrogilal precursor cells. exposure of weanling rats to a diet containing tellurium (element 52) leads to a highly synchronous demyelinating peripheral neuropathy (lampert and garrett, 1971; duckett et al., 1979; said and duckett, 1981; takahashi, 1981; bouldin et al, 1988) . when tellurium exposure is discontinued, there is rapid and synchronous remyelination. although tellurium toxicity in humans is rare, this model is of considerable interest as a system for studying the manner in which a specific metabolic insult can lead to demyelination. because there is little or no associated axonal degeneration, it has also proved useful for examining events and processes related to pns remyelination, independently of processes related to axonal regeneration. inclusion of 1-1.5% elemental tellurium in the diet of weanling rats leads to a primary segmental demyelination of about 20-25% of myelinating internodes in the sciatic nerve, but with sparing of axons (bouldin et al, 1988; harry et al, 1989) . this demyelination results in a peripheral neuropathy characterized by hindlimb paresis and paralysis. older rats are much more resistant to tellurium, and the cns is generally not affected, although some pathologic alterations can be induced with prolonged exposure periods. the nature of the underlying metabolic insult has been delineated. tellurium blocks cholesterol synthesis, specifically by inhibiting the enzyme squalene epoxidase, an obligate step in the cholesterol biosynthesis pathway (harry et al, 1989; wagner-recio et al, 1991; wagner et al, 1995) . tellurite, a water-soluble oxidized metabolite of the administered insoluble element, is the active species in vitro, effective at micromolar concentrations in a cell-free system (wagner et al., 1995) . the organotellurium compound dimethyltelluronium dichloride, (ch3)zteci2, is also effective in inhibiting squalene epoxidase in cul-tured schwann cells and in inducing demyelination when administered intraperitoneally (goodrum, 1997) . presumably, the resulting cholesterol deficit in schwann cells eventually leads to an inability to maintain preexisting myelin and to assemble new myelin; this in turn leads to the observed demyelination. although the telluriuminduced inhibition of cholesterol biosynthesis is systemic, deleterious effects are confined largely to the sciatic nerve. in the liver, which supplies cholesterol for most body tissues, the resulting intracellular cholesterol deficit results in a marked upregulation of the cholesterol biosynthetic pathway (toews et al, 1991b; wagner-recio et al, 1991; wagner et al, 1995) , presumably via well-characterized feedback mechanisms (see goldstein and brown, 1990, for review) . this allows normal levels of cholesterol synthesis in this tissue despite considerable inhibition of one of the steps in the synthesis pathway, and normal levels of lipoprotein-associated circulating cholesterol are maintained. however, unlike many other tissues, the sciatic nerve cannot use circulating cholesterol; all cholesterol required for myelin in the sciatic nerve must be synthesized locally (jurevics and morell, 1994) . this fact, coupled with the great demand for cholesterol in the rapidly myelinating pns at the time of tellurium exposure, may account for the specificity of toxicity observed. expression of mrna for myelin proteins is markedly down-regulated during the demyelinating phase of tellurium neuropathy, as is gene expression for enzymes involved in synthesis of lipids enriched in myelin (toews et al, 1990 (toews et al, , 1991a (toews et al, ,b, 1997 . the latter include hmg-coa reductase, the rate-limiting enzyme in cholesterol biosynthesis. although this enzyme is markedly upregulated in the liver (as expected from the telluriuminduced intracellular sterol deficit), it is down-regulated in the sciatic nerve in concert with other myelin-related genes (toews et al., 1991b) . failure to up-regulate the cholesterol biosynthesis pathway in the sciatic nerve is, in fact, probably the major underlying reason for the preferential susceptibility of this tissue. the co-ordinate down-regulation for myelin-related genes seen following exposure to tellurium suggests that gene expression of all proteins involved in myelin synthesis and assembly may be under the co-ordinate control of the overall program for myelination (see morell and toews, 1996a; toews et al, 1997 , for discussion). the coordinate downregulation of myelin gene expression takes place in all myelinating schwann cells and not just in those undergoing demyelination (toews et al., 1992) . thus, this downregulation is not just a secondary response to injury but rather reflects the co-ordinate control of myelin gene expression. when tellurium exposure is discontinued, there is co-ordinate up-regulation of these messages during the remyelinating period. thus, tellurium toxicity specifically leads to pns demyelination because (1) synthesis of cholesterol, a major myelin lipid, is severely inhibited; (2) unlike other tissues, peripheral nerve cannot up-regulate the synthesis of cholesterol in response to the tellurium-induced cholesterol deficit; (3) because the pns is isolated from the circulation by barriers, it cannot use circulating cholesterol derived from the diet or from synthesis in the liver; and (4) there is a particularly high demand for cholesterol in the myelinating pns at the time of tellurium exposure. the process of myelination by oligodendroglia in the cns and by schwann cells in the pns represents a complex series of metabolic and cell-biologic events involving intercellular recognition and interaction, adhesion, synthesis, sorting and assembly of specialized myelin membranes, compaction of myelin lamellae, and axonal (and possibly glial) ion channel reorganization. this entire process must be completed during an intense burst of metabolic activity at a specific predetermined interval during development, and failure to complete this program of myelination within the proper "developmental window" may have permanent deleterious effects. because myelin-forming cells are operating at near their metabolic capacity, they are especially sensitive to toxic or other types of insults during this "vulnerable period" of nervous system development, and deficiencies in myelin, either qualitative or quantitative, may result. these myelin deficiencies can result from underlying alterations of various developmental events, including failure of myelin-forming cells to proliferate in normal numbers, reduction of axonal development, and/ or decreased or altered formation of myelin at its normal time of maximal synthesis. the timing of any toxicant exposure or other insult can also differentially effect one or more of these events necessary for normal myelination. although these processes are best examined in the developing nervous system, a clearer understanding of the biochemistry, molecular biology, and cell biology of these events is also of particular relevance with regards to remyelination in injured adult nervous tissue. further delineation of the underlying nature of insults that result from toxic, genetic, nutritional, or other perturbations will also be useful in better understanding these vital processes. multipotentiality of schwann cells in cross-anastomosed and grafted unmyelinated nervesmquantitative microscopy and radioautography 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thickness morphological correlates of functional differentiation of nodes of ranvier along single fibers in the neurogenic electric organ of the knife fish sternarchus low density of sodium channels supports action potential conduction in axons of neonatal rat optic nerve the geometry of peripheral myelin sheaths during their formation and growth in rat sciatic nerves development of peripheral nerve fibers studies on the control of myelinogenesis. ii. evidence for neuronal regulation of myelin production a myelin protein is encoded by the homologue of a growth arrest-specific gene brain recovery from essential fatty acid deficiency in developing rats myelination: a critical stage in development early postnatal starvation causes lasting brain hypomyelination myelin synthesis during postnatal nutritional deprivation and subsequent rehabilitation in vitro studies of the development, maintenance and regeneration of the oligodendrocyte-type-2 astrocyte (o-2a) lineage in the adult central nervous system dna sequence, genomic organization, and chromosomal localization of the mouse peripheral myelin protein zero gene: identification of polymorphic alleles hypomyelination in copper-deficient rats atpase activities in myelin and oligodendrocytes isolated from the brains of developing rats and from bovine brain white matter membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of ranvier in electric ray and rat schwann cell differentiation key: cord-298847-szezd2vb authors: jacomy, hélène; talbot, pierre j title: vacuolating encephalitis in mice infected by human coronavirus oc43 date: 2003-10-10 journal: virology doi: 10.1016/s0042-6822(03)00323-4 sha: doc_id: 298847 cord_uid: szezd2vb involvement of viruses in human neurodegenerative diseases and the underlying pathologic mechanisms remain generally unclear. human respiratory coronaviruses (hcov) can infect neural cells, persist in human brain, and activate myelin-reactive t cells. as a means of understanding the human infection, we characterized in vivo the neurotropic and neuroinvasive properties of hcov-oc43 through the development of an experimental animal model. virus inoculation of 21-day postnatal c57bl/6 and balb/c mice led to a generalized infection of the whole cns, demonstrating hcov-oc43 neuroinvasiveness and neurovirulence. this acute infection targeted neurons, which underwent vacuolation and degeneration while infected regions presented strong microglial reactivity and inflammatory reactions. damage to the cns was not immunologically mediated and microglial reactivity was instead a consequence of direct virus-mediated neuronal injury. although this acute encephalitis appears generally similar to that induced by murine coronaviruses, an important difference rests in the prominent spongiform-like degeneration that could trigger neuropathology in surviving animals. although the etiology of most neuroautoimmune, neuroinflammatory, and/or neurodegenerative diseases remains unclear, virus infections could directly trigger neurodegeneration or initiate a cns-directed inflammatory process leading to central nervous system (cns) damage, or a combination of both. indeed, parkinson's disease, alzheimer's disease, amyotrophic lateral sclerosis (als), and multiple sclerosis (ms) could actually represent infectious diseases (calne et al., 1986; kristensson, 1992; kirk and zhou, 1996; allen et al., 1996; hayase and tobita, 1997; klein et al., 1999; boucher et al., 2001; jubelt and berger, 2001; giraud et al., 2001; sola et al., 2002) . moreover, psychiatric disorders were also investigated as a possible consequence of viral infections (waltrip et al., 1995; lewis, 2001) . the vertebrate cns was long thought to be inaccessible to cells of the immune system or to viruses. however, the presence of virus in the cns is more frequent than expected and viral detection in the cerebrospinal fluid of patients suggests the ability of viruses to cross the bloodbrain barrier (koskiniemi and vaheri, 1989; georgsson, 1994) . in fact, neuroinvasive viruses can damage the cns and produce neurological disease in sensitive hosts, due to the misdirected immune response of the host (virus-induced immunopathology) and/or viral replication in cells of the brain (virus-induced cytopathology). nevertheless, primary infections of the brain are not common and viruses are the leading cause of encephalitis, which results from either direct infection (acute encephalitis) or the immune response to an infection (postinfectious encephalitis or acute demyelinating encephalomyelitis). in acute encephalitis, viral replication occurs in the brain tissue itself, causing destructive lesions of the gray matter: this was reported after herpes simplex, rabies, or some arbovirus infections (rupprecht et al., 2002; shoji et al., 2002) . therefore, the knowledge of infectious agents involved in neurological diseases and mechanisms underlying the induction of neuropathology by these pathogens will be invaluable for preventing and developing novel clinical interventions. coronaviruses are enveloped positive-stranded rna viruses that infect multiple species of mammals, including man, causing diseases that range from encephalitis to enteritis. human coronaviruses (hcov) are recognized respiratory pathogens responsible for up to 35% of common colds (mcintosh, 1996; myint, 1994) and also involved in nocosomial infections (sizun et al., 2000) . they have occasionally been associated with other pathologies, such as pneumonia, meningitis, and enteritis (riski and hovi, 1980; resta et al., 1985) . moreover, hcov have the ability to replicate and persist in human neural cells (bonavia et al., 1997; arbour et al., 1999a,b) and to have neuroinvasive properties (burks et al., 1980; murray et al., 1992; stewart et al., 1992; arbour et al., 2000) . this has stimulated research on their possible involvement in neurological disorders. of the two known hcov serotypes, designated oc43 and 229e, hcov-oc43 is antigenically related to murine coronaviruses (mhv) . given that, under certain conditions, mhv causes experimental cns inflammatory demyelination that pathologically resembles ms (bailey et al., 1949; lampert et al., 1973; weiner, 1973; wang et al., 1990) , the related human coronavirus represents a logical target for investigation. in the present study, we report the development of a mouse model to characterize in vivo hcov-oc43-mediated neuropathogenesis. we describe the acute disease induced by hcov-oc43 infection, which resulted from neuronal infection and loss. this animal model constitutes a tool to study neuroinvasive and neurovirulence properties of a common cold virus and the mechanisms underlying the development of a diffuse vacuolating meningoencephalitis, an emerging medical problem (shoji et al., 2002; whitley and gnann, 2002) . balb/c and c57bl/6 mice were selected in view of their relative susceptibility to both respiratory and enteric strains of mhv (barthold and smith, 1987) . we tried different inoculation routes to establish the neurotropic and neuroinvasive properties of hcov-oc43 in mice. an intraperitoneal inoculation with a virus dose of 10 5 tcid 50 revealed that hcov-oc43 virus infection could be lethal until 8 days postnatal (dpn) and the same doses were nonlethal at 21 dpn. with an intraoral inoculation of 10 4 -10 5 tcid 50 of virus, we were unable to reveal the presence of virus or virus gene products in any tissue tested (brain, spinal cord, heart, lung, liver, and spleen), even by rt-pcr. mice were susceptible to intranasal (in) inhalation of the hcov-oc43 solution at 10 4 -10 5 tcid 50 . this infection was lethal in 1-week-old c57bl/6 mice. however, 21 dpn mice infected this way did not show clinical signs of pathology, but 4 of the 8 animals were found positive for viral rna by rt-pcr analyses. viral rna was mainly found in the cns but some mice also showed virus rna in the spleen (data not shown). therefore, virus could spread from the periphery to the cns after in inhalation. having shown hcov-oc43 neuroinvasive properties, we chose for the remaining experimentation to use intracerebral inoculation (ic) so as to favor a cns infection. the ic route results in a more reproducible infection, a better control of viral doses introduced into the brain. the correlation between viral infectious dose and 100% mortality in the two strains of mice after inoculation is shown in table 1 . mice became less susceptible with age and were resistant at 35 dpn for c57bl/6 and at 28 dpn for balb/c mice. we then determined the optimal experimental conditions to obtain a sublethal dose that still allowed virus replication and virus-induced cns pathology in 21-day-old mice. viral dose was administered under deep anesthesia and was determined to be 10 l of a virus solution containing 10 tcid 50 for c57bl/6 and 10 5 tcid 50 for balb/c mice aged 21 dpn. under these conditions, inoculated mice developed signs of acute disease characterized by loss of weight, apathy, ruffled fur, humped posture, and wasting (figs. 1a and b) . animals showed atrophy of skeletal muscles and occasionally exhibited paralysis of their forelimbs. during the second week postinfection, some of the animals recovered and clinical signs totally disappeared. for others, pathological signs increased and led to death. the infected animals became anorexic, inactive, and dehydrated, increasing percentages of mortality. we established survival curves for each mouse strain (fig. 1c) . eighty percent of the c57bl/6 mice died within the first 15 days postinfection and only 20% of balb/c mice died during this period, even after receiving a higher viral dose. moreover, mice inoculated with supernatants from cell cultures infected with brain tissue from affected mice developed the same disease, demonstrating that the virus was responsible for pathology. viral rna could be detected in the brain as early as 24 h postinfection, and after 2 to 3 days in the spinal cord. all c57bl/6 mice were positive for hcov-oc43 rna during the first 11 days postinfection and during the first 9 days for balb/c mice ( figs. 2a and b) . a screening of viral repnote. this dose (expressed in tcid 50 ) is in function of mouse age (days postnatal; dpn) at the time of inoculation. lication was performed by rt-pcr in a variety of tissues and results obtained indicated that infection was restricted to the cns during the first 9 days postinfection. after that, in the most affected mice, viral rna was also found in heart, spleen, and lungs, and at lower levels in liver and muscles between the 11th and 13th days postinfection in c57bl/6, suggesting a viremic spread or transneuronal transmission (fig. 3b) . the presence of hcov-oc43 rna was detectable in the brain until 11 days postinfection for 40% of balb/c mice and until 15 days postinfection for 25% of c57bl/6 mice. no viral rna could be found from tissues harvested after these times postinfection. it was also confirmed that the rt-pcr assay designed to specifically detect hcov-oc43 was indeed specific and could not have detected an enzootic mhv strain (fig. 3c) . infectious virus appeared around 3 days postinfection and could be isolated from the cns of c57bl/6 mice during the first 2 weeks postinfection ( fig. 2a) . the highest levels of infectious virions were found between 5 and 9 days postinfection (fig. 2c) . in balb/c mice, virus was detectable at 1 day postinfection and reached the highest titer around 3 days postinfection (figs. 2b and c). the highest infectious titers observed were 10 8 tcid 50 /g for brain and 10 6 tcid 50 /g for spinal cord extracts. no infectivity could be detected at and after 13 days postinfection for c57bl/6 and 9 days postinfection for balb/c mice. viral proteins were found in the brain and spinal cord of c57bl/6 mice between 5 and 11 days postinfection (fig. 3a) and were undetectable after 10 days postinfection. we detected two forms of the n protein, as already noted in 8 dpn hcov-oc43-inoculated mice (jacomy and talbot, 2001) or after mhv-4 infection (talbot et al., 1984) . blood collected at different time points after infection revealed that serum contained antibodies specific for hcov-oc43. humoral immunity started to appear at 1 week postinfection and increased during the first month postinfection, and antiviral antibodies were still present at 4 months postinfection, as shown by indirect immunofluorescence on infected hrt-18 cells (data not shown). no immunofluorescent cells were seen with serum obtain from control mice. histochemical labeling of viral distribution at different times after infection revealed that virus infection initiated by ic inoculation was quickly disseminated throughout the cns. cells positive for viral antigens were first observed at hcov-oc43-infected mice gained weight normally during the first 5 days after infection, after which they all lost weight during the acute phase of the disease. the more affected mice lost more weight more rapidly than less affected mice and died during this period. after 9 days postinfection, mice which survived gained weight to reach the weight of control animals around 21 days postinfection. (c) survival curves of mice after hcov-oc43 infection. balb/c mice received a higher dose than the c57bl/6 mice, 10,000 tcid 50 versus 10 tcid 50 . however, c57bl/6 were less resistant, with 80 versus 20% of death after infection. 3 days postinfection in the gray matter of the brains of c57bl/6 mice. at this time, microglial activation was still undetectable as assessed by mac-2 immunostaining. at 1 week postinfection, hcov-oc43 had spread to all cns regions, predominantly in the entire cerebral cortex, the striatum, the hippocampus, the hypothalamus areas, the colliculus superior, and the brain stem, including the spinal cord (figs. 4 and 5). the cerebellum was frequently spared, but purkinje cells were found positive for virus in some animals. astrogliosis revealed by gfap staining increased and activated microglial cells started to appear along the ventricles (figs. 4f, g, and h). activated microglial cells were not observed in the cns of noninfected control mice at any time during investigation, as monitored by the absence of staining for mac-2, a marker not expressed in nonreactive microglia (walther et al., 2000) . in the spinal cord at 7 days postinfection, an hcov-oc43-specific mab labeled sensory and motor neurons, and microgliosis and astrogliosis were also detected in infected regions (fig. 5) . the progression of the infection was accompanied by identical neuropathologic features in the two strains of mice: neurons exhibited severe signs of pathology, most of them showing necrosis and vacuolation. this started by the development of small and round empty vacuoles in the cytoplasm, which increased in size (figs. 4c, d, and e). these spongiform-like lesions were seen primarily within the neuronal cell bodies, the neuropil being generally unaffected (fig. 6c ). this feature was never observed in noninfected brain (fig. 6a) . ultrastructurally, numerous cells presented cytoplasm disorganization without lysis of the cellular membranes. degenerative changes included cytoplasm rarefaction, dilatation of the rough endoplasmic reticulum (rer), and disaggregation of polyribosomes leading to the appearance of free ribosomes. hematoxylin-eosin staining also revealed the presence of degenerated neurons with picknotic or small densely stained nuclei and eosinophilic cytoplasm (figs. 6c, e, h, and i). at an advanced stage of disease, loss of neurons was pronounced and was particularly evident in ca1 and ca3 hippocampal layers ( fig. 6d -i). histological examination of the brain or spinal cord revealed scattered infiltration of inflammatory cells, starting by mononuclear cell infiltrations (fig. 6b ) and perivascular cuffing. some macrophage-mediated elimination (neufig. 2 . hcov-oc43 infectious virus and rna in the cns of 21 dpn mice. (a) 100% of brains from c57bl/6 mice inoculated ic with 10 tcid 50 of hcov-oc43 were positive for viral rna between 3 and 11 days postinfection. only 25% of the surviving mice were found positive after 15 days postinfection and rna was not found thereafter. infectious virus appeared later and disappeared before elimination of viral rna. between 5 and 11 days postinfection, 100% of brains contained infectious virus. (b) detection of hcov-oc43 rna in the brain of balb/c mice inoculated ic with 10 5 tcid 50 of hcov-oc43 revealed that 100% of these mice were positive until 9 days postinfection. infectious virus was detectable in all mice only during the first 3 days postinfection and gradually fewer mice were found positive. (c) histogram representing the amount of infectious virus detected in five brains from the two strains of mice at different intervals postinfection. the limit of the detection assay was 10 0.5 tcid 50 . ronophagia) was also encountered. in the spinal cord, viral particles observed 7 days postinfection at the electron microscopic level were mostly localized in the cell cytoplasm, closely associated with the golgi apparatus or in extracellular spaces (fig. 7) . viral replication and transneuronal passage occurred in a stepwise fashion that utilized existing cellular processes. when hcov-oc43 replication and spread reached maximal levels, around 9 days postinfection, astrogliosis and microgliosis progressively increased in all infected regions of the cns until the death of the animal (figs. 4g and h). therefore, a correlation between pathological signs of disease observed in mice and morphological injury of the brain was apparent. clearance of mhv from the cns appears to involve t cells (sussman et al., 1989) and age-acquired resistance to virus could be abolished in immunosuppressed animals (zimmer and dales, 1989) . therefore, we examined the effect of immunosuppression on hcov-oc43-mediated neuropathogenesis. the immunosuppressive effects of cyclosporin a (csa) are clearly established (borel et al., 1976) . as csa causes a specific reversible inhibition of immunocompetent lymphocytes (preferentially t cells) and inhibits gene transcription for certain cytokines, in particular il-2 (kupiec-weglinski et al., 1984; elliott et al., 1984; shevach, 1985) , we investigated whether csa could modify the course of the acute hcov-oc43 infection on the development of cns lesions or on viral replication. it is known that csa injected into mice at 50 mg/kg/day induces neurotoxicity (hypocellular and disorganized organs), whereas csa at 12.5 mg/kg/day induces no abnormalities and spread to all organs (boland et al., 1984) . therefore, csa doses were selected to avoid cytotoxic effects and mortality in mice and were in accordance with immunosuppression-inducing doses described in the literature (bolton et al., 1982; pasick et al., 1992) . control mice treated with csa at a daily dose of 20 mg/kg did not show any apparent adverse effects: they gained weight normally and did not present ruffled fur or lethargy. for csa-treated and untreated mice, the kinetics of weight loss was similar after hcov-oc43 infection. nevertheless, immunosuppression by csa slightly precipitated the disease but increased mortality (fig. 8) . this was more pronounced in mice treated with csa at 20 mg/kg/day where 100% of mice died, whereas only 80% of oil-treated mice succumbed to hcov-oc43 infection. infection of mice by hcov-oc43 was dependent on a number of variables, including dose, route of inoculation, age of the host, and its genetic background. indeed, our results show striking susceptibility differences between two strains of mice: balb/c mice were more resistant than c57bl/6. moreover, resistance increased with age in the two strains of mice. this suggests that susceptibility to human coronavirus neuropathogenesis may be linked to genetic factors. our study also confirms that human coronaviruses have neuroinvasive properties in mice, which was first shown in newborn mice (barthold et al., 1990) , and that such neuroinvasion is possible even after maturation of the immune system (king et al., 1992) , which is consistent with their detection in human brain (burks et al., 1980; murray et al., 1992; stewart et al., 1992; arbour et al., 2000) . even though our study does not confirm a specific route of entry into the cns, a transneuronal route already demonstrated for mhv (lavi et al., 1988; barthold et al., 1990; perlman et al., 1990a) constitutes a likely possibility. twenty-one days postnatal mice infected by ic inoculation of hcov-oc43 developed signs of acute disease characterized by apathy, hunched posture, ruffled fur, and tremors, comparable to pathological signs described after mhv infection (kristensson et al., 1986) . following ic inoculapathologic symptoms and mortality even with very high viral doses (lavi et al., 1986) . the clinical signs of pathology after hcov-oc43 ic inoculation coincided with the peak in virus yields observed at approximately 5 to 9 days postinfection for c57bl/6 mice. this indicates that virus replication in the cns apparently played a major role in the establishment of the pathology. infected mice showed extensive inflammatory responses characterized by mononuclear perivascular cuffing, neuronophagia, and a great number of reactive glial cells in the infected regions. to investigate whether infiltration of inflammatory cells contributed to neurodegeneration or if infectious virus was directly responsible for vacuolating lesions and neuronal death, we evaluated the effect of treating animals with cyclosporin a, a powerful immunosuppressant drug. with the dose used (10 or 20 mg/kg/day), csa is known to be distributed extensively throughout the body and not to cause neurotoxicity in mice (boland et al., 1984) . immunosuppression precipitated human coronavirus-induced disease and increased the percentage of acute death (80 vs 100%). under csa treatment, neurons also presented vacuolation and degeneration. therefore, the pathology observed following hcov-oc43 infection was likely not immunologically mediated, unlike that induced by mhv-a59 and mhv-jhm (sussman et al., 1989; wang et al., 1990) , although experiments with immunodeficient mice of the same genetic background will be needed to definitely address this question with hcov-oc43. moreover, macrophage/microglial reactivity was delayed when related to infection and appeared only when the virus was present in most parts of the brain. the inflammatory response and macrophage/microglial cell recruitment seem to be strongly correlated with virus clearance, as was also demonstrated after mhv infection (sussman et al., 1989) . some strains of mhv, including a59 and jhm, are neuroinvasive in rodents, eliciting either an acute encephalitis or a chronic paralytic disease (for review, see perlman, 1998) . unlike the slow neurodegenerative disease caused by mhv (bailey et al., 1949; lampert et al., 1973; weiner, 1973; lavi et al., 1984; wang et al., 1990) , hcov-oc43 resulted in a productive and cytotoxic infection of neuronal cells in the cns, which led to neurodegeneration. group of 10 c57bl/6 mice treated with cyclosporin a at 20 mg/kg/day (oc43/20 mgcsa) and at 10 mg/kg/day (oc43/10 mgcsa) became more susceptible to hcov-oc43 infection, with 100 and 90% of death after infection versus 80% in non-csa-treated animal. infected c57bl/6 mice treated with oil alone (oc43/oil) presented similar survival curves as previously reported, with 80% of death after infection. noninfected hcov-oc43 mice treated with csa at 20 mg/kg/day (control/20 mgcsa) illustrated that csa was not toxic under these conditions. having previously demonstrated a persistent infection of hcov-oc43 in primary murine cns cell cultures and in the cns of mice inoculated at 8dpn (jacomy and talbot, 2001) , and given the observations that mhv antigens or rna were still detectable in the spinal cord several weeks after infection (woyciechowska et al., 1984; perlman et al., 1990b) , we expected to detect a persistent infection in the cns of infected 21 dpn mice. however, rt-pcr analysis revealed that viral rna could not be detected after the second week postinfection, suggesting a nonpersistent infection of hcov-oc43 virus in 21 dpn mice. histological analysis of infected 21 dpn mice showed virus spread throughout the brain and spinal cord, as we had previously described for 8 dpn mice (jacomy and talbot, 2001) . neurons remained the major cellular targets for virus, which was probably disseminated from neurons to neurons by a cell-to-cell transport, as was described after mhv infection (lavi et al., 1988; . nevertheless, even though neurons were susceptible to mhv infection, oligodendrocytes and astrocytes represented the major infected cell types (perlman and ries, 1987; . this may explain why the pathology observed in mice was different after infection with these two antigenically related coronaviruses: mhv can cause demyelination (bailey et al., 1949; lampert et al., 1973; weiner, 1973; wang et al., 1990 ) with a spongy state observed in the white matter (lavi et al., 1984) , whereas hcov-oc43 encephalitis is accompanied by vacuolating degeneration of the gray matter. the latter lesions were not detected in a previous study (pearson and mims, 1983; barthold et al., 1990) , probably because mice were younger and died within 3 to 7 days postinfection. spongiform cellular changes were occasionally reported after mhv infection. for example, vacuolation was observed in the subthalamic-nigral region after ic inoculation of mhv-a59 (fishman et al., 1985) , and foci of vacuolation were observed in the hypothalamus, cerebellar peduncles, and pons regions after in inoculation of mhv-s ( barthold and smith, 1983) . nevertheless, these degenerative changes were not commonly observed after mhv infection and were restricted to small cell populations, even after infection with 300-to 600-fold higher virus doses into c57bl/6 mice. interestingly, the appearance of clear round vacuoles and neuronal death represents a hallmark of cns degeneration observed in prion diseases (prusiner, 1998) . nevertheless, mitochondrial disease (mckelvie et al., 1991) , leigh's disease (kimura et al., 1991; agapitos et al., 1997) , pick's disease (deruaz et al., 1993) , or alzheimer's disease (duffy et al., 1988; budka et al., 1995) also display spongiform cns lesions, which are independent of the prion protein. pathways and causal start points of transmissible spongiform encephalopathies or acute encephalitis remain unknown. occasionally, viral infections of the cns were described to induce spreading spongiosis, such as in human t cell leukemia virus associated myelopathy (htlv-1) or in hiv encephalitis (rhodes, 1987; goldwater and paton, 1989) . in rodents, mutants of vsv virus (rabinowitz et al., 1976) and moloney murine leukemia virus (mo-mulv) (gardner et al., 1973; czub et al., 1994) were shown to experimentally induce spongiosis. histologically, vacuolar degeneration induced by hcov-oc43 was mainly restricted to the neuronal cell bodies, whereas that caused by prion or retrovirus first affected the neuropil. moreover, the inflammatory response was very limited in prion encephalopathy, whereas hcov-oc43 induced extensive brain inflammation. these indicate different mechanisms underlying vacuolation and neuronal death. interestingly, noninflammatory neuropathologies have been considered evidence against a viral etiology. infections by opportunistic pathogens such as respiratory or enteric virus in immuosuppressed patients (hiv, transplantation, and cancer chemotherapy) may also cause cns pathology. it has been reported that immunocompromised patients have increased incidences of malignancies induced by viral infection (penn, 1987) . therefore, severe cases of encephalitis have devastating effects on the brain and spinal cord functions. given our previous observations of hcov-oc43 persistence in human brain (stewart et al., 1992; arbour et al., 2000) , we propose that respiratory pathogens with a neurotropic and neuroinvasive potential could be associated with neurodegenerative disease in susceptible individuals. this animal model of human coronavirus neuropathogenesis may prove helpful in the characterization of coronavirus-induced neurodegeneration in surviving infected animals and of transneuronal virus spread to the cns. moreover, susceptibilities of endothelial cells and leukocytes to viral infection need to be investigated as a possible alternate route of virus entry into the cns. finally, possible explanations for our observations of striking differences in susceptibility to hcov-oc43 infection of two strains of mice remain to be investigated. even though mice are not the natural host for hcov-oc43 infections, they may provide data that, together with studies using human neural cell cultures and post-mortem brain tissue, may contribute to our understanding of the underlying mechanisms and neuropathological consequences of coronavirus infections in humans. the oc43 strain of hcov was originally obtained from the american type culture collection (atcc, rockville, md), plaque-purified, and grown on the human rectal carcinoma cell line hrt-18 as previously described . hcov-oc43 virus stocks (10 6 tcid 50 / ml) were kept at ϫ80°c. to determine the susceptibility of mice to hcov-oc43 infection, two different strains of mhv-seronegative female mice (balb/c and c57bl/6, from jackson laboratories, bar harbor, me, u.s.a.) were inoculated. inoculations were performed on mice at various days postnatal (1, 8, 15, 21, 28, 35 dpn) using 10 l of various dilutions of the initial virus stock and using different inoculation routes: intraperitoneal, intraoral, intranasal, and intracerebral, to investigate hcov-oc43 infection parameters, in particular its neuroinvasive property. the viral dose was administrated ic under deep anesthesia of ketamine-xylamine (ketamine at 200 mg/kg and xylamine at 10 mg/kg). in the present study, we chose to infect 21 dpn mice with an ic inoculation of 10 l containing 10 tcid 50 of hcov-oc43 for c57bl/6 and 10,000 tcid 50 for balb/c mice. twenty mice of each strain were used to establish survival curves. every 2 days postinfection, five animals from two groups of infected mice of each strain were sacrificed and processed for detection of viral rna, viral proteins, and infectious virus. moreover, two infected mice of each strain were perfused every 2 days for histological analysis during the first month postinfection. for each experiment, age-matched control animals, which had received a virus-free solution containing culture medium from the hrt-18 cell line, were used. to confirm that hcov-oc43 was responsible for the observed pathology, we infected hrt-18 cell cultures with brain homogenates, prepared as described below, from infected mice. cell-free supernatants harvested 4 days later were confirmed to contain infectious virus and 10 l was reinoculated ic into other animals. cyclosporin a (sigma chemical co., st. louis, mo) was rehydrated in pure ethanol as specified by the manufacturer. it was then dissolved before use in olive oil to favor free diffusion of hydrophobic cyclosporin molecules through the plasma membrane into the cytoplasm (handschumacher et al., 1984) , and heated for 2 h at 60°c. mice received a subcutaneous injection of 10 or 20 mg/kg/day. this drug was administered 1 day prior to virus inoculation and daily thereafter for 10 days. three groups of 10 female c57bl/6 mice were infected with 10 tcid 50 of hcov-oc43 and 8 control females were inoculated with hrt-18 medium. two groups of hcov-oc43-infected mice received a single daily injection of csa, either 10 or 20 mg/kg/day. four control mice and the third group of hcov-oc43-infected mice received injection of olive oil alone. the four remaining control mice were treated with csa at 20 mg/kg/day, to verify the absence of csa toxicity. brain and spinal cords were dissected, homogenized in 10% (w/v) sterile pbs, centrifuged at 4°c, 20 min at 1000 g; then supernatants were immediately frozen at ϫ80°c and stored until assayed. the extracts were processed for the presence and quantification of infectious virus by an indirect immunohistochemistry assay, as previously described (bonavia et al., 1997) . hcov-oc43-susceptible hrt-18 cells were inoculated with serial logarithmic dilutions of each tissue sample in a 96-well linbro plate (icn biomedical canada ltd., costa mesa, ca). after 4 days of incubation at 33°c in 5% (v/v) co 2 , cells were washed in pbs and fixed with 0.3% (v/v) hydrogen peroxide (h 2 o 2 ) in methanol for 30 min. after washing with pbs, they were incubated for 2 h at 37°c in 1/1000 dilution of an ascites fluid from mouse mab 1-10c.3, directed against the nucleocapsid protein of hcov-oc43 (arbour et al., 1999b) . afterwards, cells were washed in pbs and hrp goat antimouse immunoglobulins (dako, diagnostics canada inc., mississauga, on) were added and incubated for 2 h at 37°c. antibody complexes were detected by incubation in 3.3јdiaminobenzidine tetrahydrochloride solution (dab, sigma), with 0.01% (v/v) h 2 o 2 . mice were perfused by intraventricular injection of 4% (v/v) paraformaldehyde, under deep ketamine-xylazine anesthesia. brains and spinal cords were removed and tissue blocks were left in the fixative for 24 h. coronal sections from brain and segments from cervical and lumbar spinal cord were sectioned at a thickness of 40 m with a lancer vibratome. serial sections were collected in 0.05 m trisbuffered saline (tbs) and were then incubated overnight with primary antibodies, as previously described (jacomy and bosler, 1996) . for viral antigens, we used 1/1000 dilutions of ascites fluids of the 4-e11.3 hybridoma that secretes monoclonal antibodies specific for the nucleocapsid protein of the serologically related hemagglutinating encephalomyelitis virus of pigs (bonavia et al., 1997) . astrocytes were identified with a rabbit anti glial fibrillary acidic protein antibody (gfap, dako) diluted 1/500, microglia/macrophages by an ascites fluid of the rat mac-2 antibody (atcc) diluted 1/1000. then, sections were rinsed and processed with vectastain abc kit (vector laboratories, burlingame, ca). labeling was revealed with 0.03% (w/v) dab solution (sigma) and 0.01% (v/v) h 2 o 2 , which yielded a dark brown product. some sections were counterstained with the classical cresyl violet stain. to further investigate histological changes occurring in mouse brains, half hemispheres from control and infected animals were paraffin-embedded and 10-m sections were stained with hematoxylin-eosin. this was performed by the pathology department, animal resources centre, mcgill university (montréal, québec, canada). samples for electron microscopy were postfixed for 2 h with 2% (v/v) osmium tetraoxide in 0.1 m phosphate buffer at ph 7.5, dehydrated in graded ethanol series, and eponembedded as previously described (jacomy and bosler, 1996) . one-micron sections were stained with toluidine blue and examined by light microscopy. subsequent ultrathin sections were collected on collodion-coated single-slot grids, stained with lead citrate, and examined with transmission electron microscope. blood from infected or control mice were collected at 1, 2, 3, or 4 weeks and at 2, 3, and 4 months postinfection. sera were collected and kept at ϫ20°c until use for the detection of antibodies against hcov-oc43 by indirect immunofluorescent labeling of infected hrt-18 cells. briefly, hrt-18 cells cultured on 12-well slides were infected by hcov-oc43 and fixed 4 days later in cold methanol and then kept at ϫ20°c until needed. at the time of the assays, slides were incubated 1 h at room temperature with serum from control and infected mice, diluted 1/100, 1/500, and 1/1000. after several washes in pbs, slides were incubated 1 h at 37°c with alexa fluor 488 f(abј) 2 fragments of goat antimouse igg (hϩl), at a dilution of 1/15,000 (molecular probes, inc., eugene, or) and observed under a fluorescence microscope. tissues were homogenized in sub buffer, containing 8 m urea, 0.5% (w/v) sds, and 2% (v/v) ␤-mercaptoethanol and then centrifuged for 15 min at 4°c, in a microfuge at 13,000 g and supernatants were collected, as previously described (jacomy et al., 1999) . samples (5 g total protein) were fractionated on a 7.5% polyacrylamide gel (sds-page) and either visualized by coomassic blue staining or transferred to nitrocellulose for western blot analysis. nitrocellulose membranes were preincubated in 5% (w/v) skimmed milk powder in ts buffer (0.05 m tris, ph 7.4, 0.15 m nacl) and then incubated overnight at 4°c with 4e11.3 antiviral mab. after several washes with ts buffer containing 0.05% (v/v) tween 20, membranes were incubated 1 h with peroxidase-conjugated anti-mouse igg diluted 1/1000 (dako). bands were visualized using a western blot chemoluminescent kit (super signal, pierce, rockford, md). tissues were dissected every 2 days postinfection. total rna was extracted by homogenization in trizol (gibco-brl, burlington, ca). for rt-pcr, one pair of hcov-oc43 primers was designed to amplify a region containing 305 nucleotides (primers o1 and o3) of the gene coding for the n protein (arbour et al., 1999b) . the target sequences were specific to hcov-oc43 and did not amplify mhv. the suitability of rna for rt-pcr amplification was verified by an rt-pcr specific for a housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (gapdh) using a pair of gapdh primers amplifying a region containing 833 nucleotides (arbour et al., 1999b) . one pair of mhv primers was also designed to amplify a conserved region of the mhv n protein gene. primers were 5ј-cctctactgtaaaacct-gatatgg-3ј and 5ј-ctaatttagatccaaagaaga-agc-3ј, corresponding to nucleotides 677-700 and 868 -991, respectively. approximately 5 g of rna was reverse transcribed with expand moloney murine leukemia virus reverse transcriptase (gibco-brl) and the cdna products were incubated in 20 pmol of each sense and antisense primers, 2.5 mm mgcl 2 , 1ϫ pcr buffer (10 mm tris-hcl, ph 8.3; 50 mm kcl), and 0.4 mm of each deoxynucleotide triphosphate, heated at 94°c for 5 min and 60°c (hcov-oc43) or 50°c (gapdh and mhv) for 5 min. after the addition of expand high-fidelity pcr system dna polymerase (rtaq, 5000 u/l; amersham pharmacia biotech inc., baie d'urfé, qc), 30 amplification cycles of 2 min at 72°c, 1 min at 95°c, and 2 min at 60°c (hcov-oc43) or 50°c (gapdh and mhv) were performed. ten microliters of this 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a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes t-cellmediated clearance of mouse hepatitis virus strain jhm from the central nervous system western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus galectin-3 is upregulated in microglial cells in response to ischemic brain lesions, but not to facial nerve axotomy borna disease virus and schizophrenia demyelination induced by murine hepatitis virus jhm strain (mhv-4) is immunologically mediated pathogenesis of demyelination induced by mouse hepatitis virus viral encephalitis: familiar infections and emerging pathogens acute and subacute demyelination induced by mouse hepatitis virus strain a59 in c3h mice in vivo and in vitro models of demyelinating diseases xxiv. the infectious process in cyclosporin a treated winstar lewis rats inoculated with jhm virus we thank annie boucher, inrs-institut armand-frappier, for the critical review of the manuscript and francine lambert for excellent technical assistance. we also thank dr. serge dea (who tragically passed away on january 3, 2003), inrs-institut armand-frappier, for the generous gift of the 4-e11.3 antibody, and dr. yves robitaille, mcgill university, for constructive comments on neuropathology. we also thank the mcgill university animal resources centre for their help with some histology. this work was supported by grant mt-9203 from the canadian institutes of health research (institute of infection and immunity). key: cord-297448-aiorjsyh authors: atkinson, jeffrey r.; hwang, mihyun; reyes-rodriguez, angel; bergmann, cornelia c. title: dynamics of virus-specific memory b cells and plasmablasts following viral infection of the central nervous system date: 2019-01-04 journal: j virol doi: 10.1128/jvi.00875-18 sha: doc_id: 297448 cord_uid: aiorjsyh humoral responses within the central nervous system (cns) are common to many neurotropic viral infections, with antibody (ab)-secreting cells (asc) contributing to local protection. however, a role for virus-specific memory b cells (bmem) within the cns is poorly explored due to lack of robust phenotypic or functional identification in mice. this study takes advantage of the progeny of mice expressing tamoxifen-inducible cre recombinase (cre-ert2) under the aicda promoter crossed with rosa26-loxp-tdtomato reporter mice (aid(cre)-rosa26(tdtomato)) to monitor b cells having undergone activation-induced cytidine deaminase (aid)-mediated somatic hypermutation (shm) following neurotropic coronavirus infection. aid detection via tdtomato expression allowed tracking of virus-specific asc and bmem in priming and effector sites throughout infection. in draining lymph nodes, tdtomato-positive (tdtomato(+)) asc were most prevalent prior to germinal center (gc) formation, but total tdtomato(+) b cells only peaked with robust gc formation at day 14 p.i. moreover, their proportion of bmem dominated over the proportion of asc throughout infection. in the cns, tdtomato(+) cells started emerging at day 14 p.i. while they initially comprised mainly bmem, the proportions of asc and bmem became similar as tdtomato(+) b cells increased throughout viral persistence. delayed tamoxifen treatment demonstrated ongoing cns recruitment of tdtomato(+) b cells, mainly asc, primed late during gc reactions. overall, the data support the idea that virus-induced b cells exhibiting shm require peripheral gc formation to emerge in the cns. ongoing gc reactions and regional signals further regulate dynamics within the cns, with preferential maintenance of tdtomato(+) b cells in spinal cords relative to that in brains during viral persistence. importance the prevalence and role of antigen-specific bmem in the cns during viral encephalomyelitis is largely undefined. a lack of reliable markers identifying murine bmem has made it difficult to assess their contribution to local antiviral protection via antigen presentation or conversion to asc. using reporter mice infected with neurotropic coronavirus to track virus-specific bmem and asc, this report demonstrates that both subsets only emerge in the cns following peripheral gc formation and subsequently prevail. while early gc reactions supported preferential bmem accumulation in the cns, late gc reactions favored asc accumulation, although bmem outnumbered asc in draining lymph nodes throughout infection. importantly, virus-specific b cells undergoing sustained gc selection were continually recruited to the persistently infected cns. elucidating the factors governing temporal events within gcs, as well as regional cns cues during viral persistence, will aid intervention to modulate cns humoral responses in the context of infection and associated autoimmune pathologies. n eurotropic viral infections are associated with the accumulation of multiple b cell subsets within the central nervous system (cns) whose composition alters with time, indicating ongoing turnover and differentiation (1) (2) (3) . during infection with a gliatropic mouse hepatitis virus (mhv) derived from the jhm strain (jhmv2.2-1), igdpositive (igd ϩ ) (naive/transitional) b cells prevail early, but they decrease coincident with increasing proportions of cd138 ϩ antibody (ab)-secreting cells (asc) and cd138 ϫ igg ϩ isotype-switched memory b cells (bmem) as germinal centers (gcs) are formed in draining lymph nodes (1, 2) . whether the turnover in the cns involves replacement by newly recruited b cells derived from the periphery or local expansion/differentiation of b cells recruited during acute infection is largely unresolved. while asc in the blood are transiently migratory following antigen (ag)-driven differentiation, bmem recirculate for extended periods of time (4, 5) . this supports circulating bmem as a potential replenishing source of asc in the cns during persistent infections. however, their contribution to humoral immunity in the cns following infection or autoimmunity remains poorly characterized. ag-specific b cells develop into asc through two distinct pathways. in the first, ag-activated b cells rapidly differentiate into short-lived asc in extrafollicular foci (6) (7) (8) (9) . in the second pathway, b cells activated by ag and receiving cd4 t cell help at the t cell-b cell follicle border form follicular gcs, where they undergo affinity maturation and isotype switching, ultimately generating ag-specific bmem, as well as long-lived igg asc. the generation of these igg asc and bmem is significantly impaired in the absence of gcs (10) (11) (12) (13) (14) . gc reactions are marked by class-switched ig and somatic hypermutation (shm), thereby imprinting long-lived asc and bmem (15) (16) (17) (18) . these processes are both mediated by the enzyme activation-induced cytidine deaminase (aid), which introduces mutations in dna by deaminating the cytidine base to create uracil. uracil-dna glycosylase and apurinic endonuclease then act to excise uracil, and the gaps are filled in by dna repair mechanisms (12, 19, 20) . based on its shm-inducing activity, aid expression constitutes a b cell-specific marker of ag-induced affinity maturation. while the vast majority of aid-dependent affinity maturation occurs within gcs, resulting in class-switching to igg (21) , there is evidence for extrafollicular, gc-independent shm resulting in the production of ag-specific asc and abs, including those of the igm isotype (12, 22) . following gliatropic mhv cns infection, t and b cell responses are initiated in cervical lymph nodes (cln), and local cns ab production is crucial for preventing recrudescence of persisting virus following initial t cell-mediated immune control (23, 24) . using the gliatropic mhv-jhmv2.2-1 variant, we have recently demonstrated that b cells in the cns transition from a predominantly naive/early-activated igd ϩ igm ϩ population at day 7 p.i. to an igd-negative (igd ϫ ) igm ϩ and increasingly igg ϩ population, including cd138 ϩ asc, by day 21 p.i. (1, 2) . moreover, the b cell coreceptor cd19, which lowers the ag-specific activation threshold and promotes peripheral gc formation, is required for accumulation of cd138 ϩ asc in the cns (14) . these data suggested that the vast majority of plasmablasts and igd ϫ b cell subsets in the cns have undergone shm driven by viral ag-specific b cell receptor (bcr) activation and do not comprise bystander b cells recruited via ag-independent proinflammatory signals. however, identification of virus-specific asc or bmem generally depends on enzymelinked immunosorbent spot assay (elispot) techniques using virus or viral lysates as the capture ag, potentially underestimating their numbers. measurement of virusspecific bmem is especially biased by culture conditions, as b cells require nonspecific stimulation in vitro to convert into asc for subsequent quantitation by elispot (25, 26) . to better characterize the proportions of virus-specific bmem and asc accumulating in the cln and the cns following viral encephalomyelitis, we took advantage of mice expressing tamoxifen-inducible cre recombinase (cre-ert2) under the aicda promoter crossed with rosa26-loxp-tdtomato reporter mice to obtain progeny in which aidexpressing cells can be identified by fluorescence following tamoxifen administration (4, 27) . analysis of humoral responses to protein ag in aid cre -rosa26 eyfp mice confirmed that the vast majority of enhanced yellow fluorescent protein (eyfp)-expressing b cells were indeed specific for the immunizing ag (4). these dually transgenic reporter mice are thus suitable tools to phenotypically monitor the dynamics and tissue distribution of b cells having undergone virus-induced, aid-mediated shm. this study used the mhv-a59 strain, a neurotropic mhv that is less pathogenic than jhmv2.2-1, to determine the frequency, longevity, and distribution of virus-specific asc and bmem in the cln and cns of infected aid cre -rosa26 tdtomato mice using the cd19 ϩ tdtomato ϩ igd ϫ cd138 ϩ and the cd19 ϩ tdtomato ϩ igd ϫ cd138 ϫ phenotype, respectfully. tamoxifen administration at the onset of infection and throughout day 28 p.i. revealed that tdtomato ϩ b cells only accumulated in the cns following peripheral gc formation and continued well into the chronic infection phase. early gc-independent tdtomato ϩ asc in the cln did not appear to migrate to the cns. notably, an overall larger proportion of tdtomato ϩ b cells accumulated earlier and at higher frequencies in spinal cords than in brains. while bmem dominated the tdtomato ϩ population in cln throughout gc activity, they vastly exceeded asc at early but not later stages of viral persistence. the administration of tamoxifen during chronic disease, starting at day 20 p.i., revealed that ϳ50% of asc and ϳ25% of bmem were recruited from later peripheral gc reactions by 28 days p.i., accounting for nearly the entire increase in virus-specific b cells observed in the cns between days 21 and 28 p.i. overall, the results show that the vast majority of asc recruited to both the brain and spinal cord were virus specific, with limited accumulation of asc with heterologous specificity. in contrast, the fraction of virus-specific cells within the bmem population was substantially higher in spinal cords than in the brain. these data indicate that b cell subset accumulation during the persistent phase of infection is controlled by peripheral gc-driven events, as well as cns regional signals. virus-specific tdtomato ؉ asc preceded gc b cells and gc formation. following infection with gliatropic mhv-jhmv2.2-1, adaptive immune responses are initiated within cln, consistent with lymphatic cns drainage into this site (28) . moreover, virus-specific asc measured by elispot peak at ϳday 14 p.i., coincident with defined anatomical gc formation in cln (1, 29) . in contrast, the peak in total asc monitored phenotypically precedes virus-specific asc by ϳ7 days p.i. (23) . importantly, both total and virus-specific asc only start emerging in the cns at day 14 p.i. and increase thereafter. we therefore questioned whether early asc expansion is driven by agindependent innate immune signaling, as previously shown for influenza virus and west nile virus (wnv) infection (30, 31) , or whether it is ag driven but insufficient to monitor via elispot assay. aid cre -rosa26 tdtomato mice were thus treated with tamoxifen coincident with mhv-a59 inoculation to jointly track the emergence of tdtomato ϩ cells as a marker for ag-primed bmem (cd19 ϩ tdtomato ϩ igd ϫ cd138 ϫ ) and asc (cd19 ϩ tdtomato ϩ igd ϫ cd138 ϩ ) in cln. naive mice treated with tamoxifen 2 days prior to analysis were used as controls to assess baseline tdtomato ϩ b cells. the flow cytometry gating strategy depicting tdtomato ϩ cells within total b cells and b cell subsets is shown in fig. 1 . tdtomato ϩ b cells in cln of naive mice treated with tamoxifen constituted ϳ1.5% of total cd19 ϩ b cells (fig. 1 ). of these, only ϳ35% were igd ϫ , indicative of basal gc reactivity. the minor proportion of basal tdtomato ϩ cells is consistent with a small fraction of gl7 ϩ cells, driven by constitutive activation by endogenous ag (4, 32) . untreated mice showed no aid-driven tdtomato reactivity. following infection, the frequency of tdtomato ϩ b cells within total cd19 ϩ b cells increased to ϳ5% at day 14 p.i., with the vast majority being igd ϫ and only a small population expressing the asc marker cd138 (fig. 1a) . evaluation of tdtomato expression within total igd ϫ cd138 ϫ or cd138 ϩ cells indicated the vast majority were recently induced to express aid (fig. 1c ). this was confirmed by monitoring the expansion of b cells expressing gl7, an activation marker for pre-gc and gc b cells (33) . the fraction of gl7 ϩ cells was only slightly smaller than that of tdtomato ϩ cells at day 14 p.i. moreover, virtually all gl7 ϩ cells were tdtomato ϩ (fig. 1d) , consistent with gc formation (4, 32) . to confirm that the majority of tdtomato ϩ cd138 ϩ asc were indeed fig 1 gating strategy for quantification of aid-induced, tdtomato ϩ , virus-specific b cell subsets in cln via flow cytometry. naive mice were treated with tamoxifen 2 days prior to analysis and mhv-infected mice from day 0 virus specific, we directly compared the frequencies of cd138 ϩ tdtomato ϩ b cells obtained by flow cytometry to virus-specific elispot analyses in cln and brains at day 14 p.i. (fig. 1e) , when virus-specific asc peak in cln and emerge within the cns following mhv-jhmv2.2-1 infection (4, 14, 32) . in this experiment, the sum of virusspecific igg-and igm-secreting asc approximated the number of tdtomato ϩ cd138 ϩ cells at both anatomical sites, supporting their virus specificity. having confirmed expansion of tdtomato ϩ b cells with a largely igd ϫ phenotype following virus cns infection, we monitored tdtomato ϩ b cell subsets in cln throughout acute infection (day 7 p.i.) into the persistent phase (days 14 to 28 p.i.). the proportion of tdtomato ϩ cells within total b cells was already increased at day 7 p.i. compared to that in naive mice, steadily rose to ϳ7% by day 21 p.i., and remained stable thereafter (fig. 2) . the gl7 ϩ b cell fraction was not increased until day 14 p.i. and reached maximum levels of ϳ5% at day 21 p.i. (fig. 2) . moreover, the fraction of gl7 ϩ b cells expressing tdtomato increased to 80% by day 7 p.i. and plateaued at ϳ90% by day 14 p.i. (fig. 2) . these data are consistent with the formation of well-defined gc at day 14 p.i. and segregation into structurally mature gc defined by dark-and light-zone domains by day 21 p.i. following infection with the jhmv2.2-1 virus variant (1). moreover, the results confirmed aid-dependent shm and class switch recombination (csr), typical of gc b cells, and showed maintenance of gc activity out to day 28 p.i. during chronic infection. the increased proportion of gl7 ϩ cells by day 14 p.i., as well as their tdtomato expression, support the idea that induction of gl7 ϩ is specifically induced by viral ag (fig. 2 and 3 ). contrasting with the progressively increasing proportions of tdtomato ϩ and gl7 ϩ cells after day 14 p.i., cd138 ϩ asc proportions within cln b cells peaked at day 7 p.i. (fig. 2) , suggesting that they were largely gc independent. moreover, the fraction of asc rapidly declined and only slowly increased again by day 28 p.i. (fig. 2 ), similar to previous results during mhv-jhmv2.2-1 infection (14, 23) . surprisingly, however, 50% to 60% of asc at day 7 p.i. were tdtomato ϩ , compared to ϳ5% in naive mice, and these percentages were sustained throughout day 28 p.i. (fig. 2) . these results are consistent with virus-activated aid expression and shm prior to robust gc formation. however, it could not be discerned whether tdtomato ϩ asc at later times comprised a distinct or more differentiated gc-derived subset. importantly, the kinetics of igd ϫ cd138 ϫ bmem did not mirror those observed for asc. total bmem did not show increased frequencies within cd19 ϩ cln b cells in infected compared to naive mice until day 21 p.i. (fig. 2 ). bmem and asc exhibited similar fractions of tdtomato ϩ cells in naive cln (ϳ7%). however, unlike asc, bmem did not demonstrate a rapid increase in the frequency of tdtomato ϩ cells at day 7 p.i. despite this initial divergence, the tdtomato ϩ fraction in bmem increased to ϳ50% at day 14 p.i. and remained stable out to day 28 p.i. (fig. 2 ). the production of ag-primed asc and bmem thus displayed discrete kinetics in cln following mhv infection. finally, while the fraction of tdtomato ϩ cells in phenotypically naive igd ϩ cells increased by day 14 p.i. and remained elevated compared to that in naive mice, it remained ͻ4%, suggesting rapid differentiation into igd ϫ memory cells ( fig. 2) (1). figure 3 provides an overview of the temporal kinetics of absolute numbers of tdtomato ϩ cells relative to those of gl7 ϩ gc b cells, cd138 ϩ asc, and igd ϫ cd138 ϫ bmem throughout infection. total cd19 ϩ b cells were stable throughout day 14 p.i. and decreased thereafter, reflecting overall decreased cellularity of cln following intracranial (i.c.) mhv infection. although the percentages of tdtomato ϩ b cells slowly increased throughout days 7 to 21 p.i. (fig. 2) , the absolute numbers of tdtomato ϩ cells were most abundant at day 14 p.i., reflecting peak total cd19 ϩ cells (fig. 3a) . the numbers of gl7 ϩ b cells, with the vast majority being tdtomato ϩ , mirrored the kinetics of gc formation (1). total asc peaked at day 7 p.i. (ϳ2.0 ϫ 10 4 ), prior to maximal gl7 ϩ b cells (ϳ8.0 ϫ 10 4 ), reflecting peak cd138 ϩ percentages within the b cell population. by day 28 p.i., asc declined rapidly, reaching only ϳ2 ϫ 10 3 (fig. 3a) . the high proportion (ϳ65%) of asc expressing tdtomato at day 7 p.i. thus surpassed aidexpressing asc numbers during maximal gc differentiation after day 14 p.i. (fig. 3a) . these early asc are consistent with a population of largely gc-independent, igm ϩ , long-lived asc, which have been described to exhibit aid-dependent affinity maturation in other models (12) . in stark contrast to asc, bmem demonstrated a progressive increase in tdtomato ϩ cell numbers in the cln, peaking at day 14 p.i. (ϳ1.1 ϫ 10 5 ) and incrementally decreasing by day 28 p.i. (ϳ4.8 ϫ 10 4 ) (fig. 3a) . however, the frequency of tdtomato ϩ cells within bmem remained constant during chronic infection. the decrease in tdtomato ϩ bmem supports the notion of egress into circulation following peak gc formation. interestingly, the dynamics of tdtomato ϩ bmem in the cln closely resemble those of gl7 ϩ gc b cells, suggesting a relatively constant production of ag-primed bmem throughout the formation and contraction of gcs (fig. 3a) . however, as was the case with asc, it remains unclear whether the tdtomato ϩ bmem are more differentiated or exhibit increased affinity. the numbers of tdtomato ϩ igd ϩ b cells were consistently low (ͻ3.0 ϫ 10 4 ) (data not shown). the distribution of b cell subsets within cln tdtomato ϩ b cells is summarized in fig. 3b . the total numbers of cd19 ϩ tdtomato ϩ b cells increased significantly by day 14 p.i., slowly declined until day 21, and reached background levels by day 28 p.i. of note, at day 14 p.i., they comprised mainly igd ϫ cd138 ϫ bmem (ϳ72%) and a smaller proportion of cd138 ϩ asc (ϳ8%). the large proportion of bmem relative to that of asc was maintained throughout day 28 p.i. despite overall lower levels of tdtomato ϩ asc relative to the levels of bmem, asc kinetics following gc formation mirrored those of virus-specific igg asc determined by elispot following heterologous mhv-jhmv2.2-1 infection (14) . tdtomato ؉ cells localized predominantly to gcs in cln. to affirm that the majority of tdtomato ϩ b cells were indeed imprinted by gc reactions, cln sections from infected, tamoxifen-treated aid cre -rosa26 tdtomato mice were assessed for anatomical gc formation at days 7, 14, and 21 p.i., corresponding to times from emergence of gcs to mature gc formation following cns infection with the more pathogenic gliatropic mhv-jhm strain (1). at day 7 p.i., aid cre -rosa26 tdtomato mice already exhibited small foci of tdtomato ϩ b cells within follicles, indicative of early gc formation. tdtomato ϩ b cells were also evident at extrafollicular locations, consistent with gcindependent expansion of tdtomato ϩ asc (fig. 4) . by day 14 p.i., tdtomato ϩ b cells formed large foci typical of gc morphology within follicles; these structures were maintained through day 21 p.i. (fig. 4 ) and day 28 p.i. (data not shown). overall, these kinetics of gc formation are similar to those of infection with mhv-jhm2.2v-1 (1). moreover, although tdtomato ϩ asc and bmem (fig. 3b) had already emerged at day 7 p.i., the maximum gl7 ϩ tdtomato ϩ populations nevertheless coincided with mature gc formation (fig. 3) , suggesting that the vast majority of virus-specific b cells were gc derived. virus-specific asc and bmem accumulated progressively in the cns following peripheral gc formation. to assess how the kinetics of tdtomato ϩ b cells in cln fig. 1 and represent the mean values ϯ sem for individual mice from 2 separate experiments, each comprising 3 to 4 individual mice per time point. statistically significant differences from the results for naive mice (day 0 p.i.), determined by unpaired t test, are denoted as follows, with asterisks in red or black denoting significant differences in the tdtomato ϩ or total population, respectively: *, p ͻ 0.05; **, p ͻ 0.01; ***, p ͻ 0.001. sively increased throughout day 28 p.i. (fig. 5 and 6 ). however, whereas the proportion of tdtomato ϩ cells within the total cd19 ϩ population did not exceed 20% in brains, it reached nearly 80% in spinal cords at day 28 p.i. (fig. 5 and 6 ). cd138 ϩ asc started to accumulate at day 14 p.i. and increased progressively out to day 28 p.i. in both brains and spinal cords ( fig. 5 and 6) . furthermore, the percentage of tdtomato ϩ cells within asc increased more extensively within spinal cords than in brains, reaching ϳ98% compared to ϳ70%, respectively, by day 28 p.i. (fig. 5a and 6 ). igd ϫ cd138 ϫ bmem numbers in brains and spinal cords remained unaltered or even decreased compared to the numbers in naive mice during acute infection out to day 14 p.i. brains only showed a marginal increase in bmem at day 21 and more significant elevation by day 28 p.i. however, the relative proportion of tdtomato ϩ cells remained ͻ20% (fig. 5a) . in contrast, igd ϫ cd138 ϫ bmem in spinal cords were significantly elevated at day 21 p.i. and reached 6-fold-higher levels than in naive mice by day 28 p.i. distinct from the results for brains, the percentage of tdtomato ϩ cells was already higher at day 14 p.i. and increased progressively, reaching ϳ70% by day 28 p.i. (fig. 5a and 6 ). the differences in the proportions of tdtomato ϩ b cells were thus much more pronounced in bmem than in asc when comparing brain and spinal cord populations. these data suggested that viral-ag-experienced, gc-derived b cells accumulated preferentially in spinal cords, potentially outcompeting bystander or less differentiated b cells. similar to the periphery, the frequency of tdtomato ϩ cells within igd ϩ b cells remained ͻ2% and never exceeded 1 ϫ 10 2 cells in either cns tissue ( fig. 5b and 6) . comparison of total numbers of tdtomato ϩ cells and their compositions of asc and bmem in brains and spinal cords over time following infection revealed overall similar kinetics of virus-specific b cell accumulation (fig. 5b) . however, as indicated above, the spinal cord harbored vastly more tdtomato ϩ b cells at 21 and 28 days p.i. moreover, the vast majority of tdtomato ϩ b cells in the cns were igd ϫ cd138 ϫ bmem out to day 21 p.i., reflecting their dominance in cln. however, irrespective of their low proportions in cln throughout persistence, asc increased in both brains and spinal cords, resulting in proportions approximately equal to those of bmem by day 28 p.i. (fig. 5b) . virus-specific tdtomato ؉ b cells localized to the cns parenchyma. previous histological examination of b cell localization within the cns following infection with a nonlethal variant of mhv-jhm revealed cd138 ϩ cells, as well as igm ϩ and igg ϩ cells, scattered prominently within white matter or perivascular sites (23, 29) . moreover, in the brain, more differentiated igg ϩ b cells preferentially accumulated in the parenchyma with a scattered rather than clustered pattern (1), while igd ϩ cells appeared restricted to meningeal and perivascular sites. histological evaluation of spinal cords for tdtomato ϩ cells at day 21 p.i. supported prominent localization to white matter tracks , determined by unpaired t test, are denoted as follows, with asterisks in red or black denoting significant differences in the tdtomato ϩ or total population, respectively: *, p ͻ 0.05; **, p ͻ 0.01; ***, p ͻ 0.001. (fig. 7) . moreover, tdtomato ϩ cells were not associated with laminin-positive areas marking perivascular matrix and were found in select areas of the spinal cord, where they exhibited a scattered distribution; tdtomato ϩ cell clusters were very rare. virus-specific b cells were continually recruited to the infected cns. the data described above demonstrated that virus-specific tdtomato ϩ b cells progressively (4), we monitored aicda mrna levels in cln relative to the levels in brains and spinal cords throughout infection (fig. 8) . ighg mrna levels were assessed as a measure of asc igg secretion (2) . in cln, aicda mrna levels increased between days 5 and 7 p.i. and were further elevated by day 21 p.i., corresponding with gc formation. in contrast, although a statistically significant increase in aicda mrna was evident in brains and spinal cords at day 14 p.i., the levels were overall ϳ4 orders of magnitude (10 4 ) lower than in cln. ighg mrna levels increased prominently in cln by day 14 p.i., reflecting gc-matured asc. in the cns, ighg mrna levels also did not increase significantly until day 14 p.i., and the levels increased further by day 21 p.i. moreover, the levels were ϳ10-fold higher in spinal cords than in brains, consistent with higher igg secretion. these mrna kinetics were similar to those in mhv-jhmv2.2-1-infected mice (2) . while ighg mrna levels were overall lower in the cns than in cln, the ϳ100-fold-higher ratio of aicda relative to ighg mrna levels in cln versus spinal cords (ϳ1 ϫ 10 ϫ3 versus 5 ϫ 10 ϫ5 , respectively) confirmed minimal local aid activity in the cns. to support ongoing recruitment from protracted gc reactions typical of viral infections, we initiated administration of tamoxifen to infected aid cre -rosa26 tdtomato mice at day 20 p.i. this approach ensures that only those b cells primed or undergoing continuous differentiation after day 20 p.i. are marked by tdtomato expression. any accumulation of tdtomato ϩ b cells in the cns after day 20 p.i. would imply that these cells were derived from protracted peripheral gc reactions sustained during the chronic phase. cln, brain, and spinal cord cells were thus assessed for the presence of tdtomato ϩ asc and bmem cells by flow cytometry 8 days after initial tamoxifen administration (day 28 p.i.). slightly reduced frequencies of tdtomato ϩ b cells within total cd19 ϩ b cells in cln in mice receiving tamoxifen starting at day 20 p.i. compared to those starting at day 0 p.i. did not reach statistical significance (fig. 9) . furthermore, mice receiving tamoxifen treatment at day 20 p.i. revealed a frequency of ϳ30% tdtomato ϩ cells in the cd138 ϩ asc population, similar to the frequency in mice receiving tamoxifen continuously from day 0 p.i. (fig. 9) , indicating that nearly all the virus-specific asc present in the cln during chronic disease had undergone affinity maturation after day 20 p.i. in established gcs. importantly, the frequencies of tdtomato ϩ cells within the gl7 ϩ gc phenotype b cells were similar, at ϳ80% (fig. 9) . these results support continual selection and turnover of virus-specific b cells in the cln, consistent with ongoing gc reactions during chronic infection. in the brains and spinal cords of infected mice, the frequency of tdtomato ϩ cells in total cd19 ϩ b cells was significantly decreased in mice receiving tamoxifen beginning at day 20 p.i. compared to the frequency in mice receiving treatment from day 0 p.i. (fig. 9) . the frequencies of cd19 ϩ b cells within cd45 hi infiltrates were not significantly different between treatment groups in either the brains or spinal cords (data not shown). nevertheless, these mice exhibited appreciable accumulations of tdtomato ϩ b cells, reaching 10% and 20% of the frequencies observed in the control group in the brains and spinal cords, respectively (fig. 9) . interestingly, the reductions in tdtomato ϩ populations were more prominent in igd ϫ cd138 ϫ bmem than in cd138 ϩ asc in both brains and spinal cords. overall, these results are consistent with ongoing peripheral activation, differentiation, and migration of virus-specific asc and bmem to the cns during chronic infection. production of virus-specific ab within the cns is associated with protection during infection with rna viruses (24, 34) . furthermore, although both igm ϩ and igg ϩ b cells are recruited to the cns, their specificity or derivation from ongoing gc reactions remains largely unexplored. we therefore infected aid cre -rosa26 tdtomato reporter mice with mhv-a59 to monitor the expansion and distribution of b cells having undergone shm as an indicator of viral-ag-induced activation. our results demonstrate that tdtomato ϩ b cells in draining lymph nodes were already evident at day 7 p.i., prior to journal of virology peak gl7 ϩ activated and gc b cells, as well as defined gc structures evident at day 14 p.i. this early, gc-independent, tdtomato ϩ population was comprised of similar proportions of cd138 ϩ asc and igd ϫ cd138 ϫ bmem. moreover, cd138 ϩ tdtomato ϩ asc peaked at day 7 p.i., accounting for ϳ65% of total asc. while the early expansion of asc prior to gc formation confirmed results in blimp-gfp mice infected with the more pathogenic, sublethal mhv-jhmv2.2-1 variant (23), these asc were previously interpreted to result from innate bystander activation, as they were undetectable in virus-specific elispot assays (14) . however, their high frequency of tdtomato expression indicates that these asc are viral ag specific but produce ab with insufficient affinity to be detected by virus-specific elispot. this notion is supported by mhv-jhmv2.2-1 infection of cd19-deficient mice (14) , in which a significant reduction in early asc within cln at day 7 p.i. was consistent with the function of cd19 to lower the ag-driven b cell activation threshold. interestingly, somatic mutations introduced by aid may not be viral ag selected but may still be specific, as previously reported in a subset of long-lived, gc-independent, igm-secreting asc induced by vaccination or peripheral infection (12) . nevertheless, independent of shm evident at day 7 p.i., these asc do not appear to be migration competent, as tdtomato ϩ asc do not emerge in the cns until day 14 p.i. irrespective of migration capacity, these results are consistent with preferential retention of ag-induced, long-lived igm asc in the spleen following peripheral immunization or infection (12) . while small numbers of tdtomato ϩ b cells emerged in the cns during acute infection, their cns accumulation accelerated substantially out to day 28 p.i. following the formation of peripheral gcs at day 14 p.i. mature gcs in cln were sustained out to at least day 28 p.i. (data not shown), but tdtomato ϩ b cell numbers declined after day 14 p.i., consistent with the death of low-affinity clones during ongoing affinity maturation and/or egress from lymphoid tissue into circulation. the progressive increase in tdtomato ϩ b cells in the cns thus supported the idea of ongoing egress of gc-matured b cells from cln and subsequent migration to the persistently infected cns. overall, these results suggested that gcs are important for licensing migration of ag-specific b cells to sites of inflammation. the idea of imprinting of migratory capacity by gcs was supported by the increasing percentages of tdtomato ϩ cells within the asc and bmem populations, which ultimately represented a majority within both subsets. however, a lower proportion of tdtomato ϩ cells within bmem in brains compared to the proportion in spinal cords suggests that additional events dictated by the regional cns environment control the recruitment or survival of ag-specific b cells. overall, the preferential accumulation of tdtomato ϩ b cells throughout persistence demonstrated that recently activated b cells display enhanced cns migratory capacity compared to that of preexisting, non-mhv-specific asc or bmem with heterologous specificity. the relative distributions of ag-specific asc and bmem within the priming and effector sites have not been characterized during any neurotropic infection to our knowledge. in cln, asc and bmem within the tdtomato ϩ b cell population were distributed similarly prior to gc formation. moreover, the vast majority of gl7 ϩ cells were tdtomato ϩ at day 7 p.i, indicative of an activated pre-gc phenotype. similar to gc-independent, igm-secreting asc with ag-induced shm (12) , gc-independent, unswitched as well as switched bmem have also been described during the early primary response to r-phycoerythrin (r-pe) immunization (33) . however, gl7 ϩ cells were sparse at day 7 p.i., and their increase by day 14 p.i. coincided with overt gc formation. at this time, the composition of tdtomato ϩ b cells was prominently skewed toward bmem, with the proportion of asc dropping below 10% after day 14 p.i., despite a more mature gc structure. the relative abundances of virus-specific tdtomato ϩ bmem and asc in both brains and spinal cords largely reflected those in draining ln at days 14 and 21 p.i., with tdtomato ϩ bmem prevailing over asc. surprisingly, however, asc accounted for nearly 50% of tdtomato ϩ b cells by day 28 p.i. the steadily increasing proportion of asc in the cns by day 28 p.i. suggested more efficient cln egress and/or migration of asc than of bmem during ongoing gc reactions. these findings are overall consistent with a temporal switch observed in the output of gcs following model ag immunization: gcs are initially dedicated to generating bmem and only switch to asc output after peak bmem expansion (35) . however, limiting viral ag in cln during persistence may reduce asc over bmem output in cln. nevertheless, the progressive asc emergence relative to that of bmem in the cns over time supports the notion that early gc reactions are dedicated to preferential production of bmem with the capacity to migrate to the cns. asc are known to have a cxcr4-dependent migratory window, when they preferentially home to the bone marrow following peripheral immunization or infection (36) (37) (38) (39) . however, cxcr3/cxcl10 interactions are essential in mediating asc migration and access to the cns parenchyma (23) . in contrast, bmem remain prominent in secondary lymphoid organs and recirculate (40, 41) . while bmem accumulating in the cns are characterized by the expression of chemokine receptor ccr7 (2), the chemokines essential for their cns migratory behavior in vivo have not been characterized. differential chemokine receptor responsiveness during protracted gc reactions may thus further imprint preferential cns recruitment of asc during ongoing viral persistence. ongoing egress of tdtomato ϩ b cells from cln during late gc reactions was indeed supported by tamoxifen administration at day 20 p.i. analysis of b cells 8 days later revealed that ϳ20% of tdtomato ϩ b cells were derived from b cells initiating aiddriven tdtomato expression after day 20 p.i. their derivation from the periphery was supported by similar percentages of tdtomato ϩ cells within total cd19 ϩ b cells, gl7 ϩ b cells, and cd138 ϩ asc at day 28 p.i., independent of the administration of tamoxifen throughout acute infection or starting at day 20 p.i. whether these tdtomato ϩ b cells are derived from cells already expressing aid and undergoing further affinity maturation after day 20 p.i. or from newly primed b cells recruited to gcs remains to be determined. nevertheless, the less prominent reductions of tdtomato ϩ cells in asc compared to the reductions in bmem in both brains and spinal cords following late compared to early tamoxifen treatment is consistent with the notion that ongoing recruitment primarily involves asc still undergoing shm after day 20 p.i. these data are also consistent with preferential output of migratory asc by day 28 p.i. we have excluded de novo aid expression within the cns based on previous analysis of mrna transcript levels from igd ϫ cd138 ϫ bmem and cd138 ϩ asc isolated from the cns at 21 days p.i. (2) , as well as igd ϩ b cells at day 7 p.i. nevertheless, we cannot exclude the possibility that the relative increase in asc over bmem in the cns may also be driven by local conversion of bmem into asc by persistent viral ag, ongoing pattern recognition receptor mediation, or cytokine stimulation. overall, our data demonstrate for the first time that virus-specific b cells exhibiting shm only emerge in the cns following peripheral gc formation. moreover, the accumulation of asc and bmem appears to be regulated by temporal events within gcs, as well as cns regional cues during viral persistence. while early gc reactions supported preferential bmem accumulation in the cns, late gc reactions preferentially mediated asc migration to the cns. importantly, virus-specific b cells expressing aid in peripheral gcs sustained during cns viral persistence are continually recruited to the chronically infected cns. while these data do not support involvement of bystander recruitment of preexisting bmem or asc with irrelevant specificity to cns inflammation, they do predict that sustained gc reactivity may recruit de novo b cell activation to ags released from damaged tissue. such b cells may thus give rise to autoreactive abs or promote autoreactive t cells, as recently demonstrated in multiple sclerosis (42) . mice, virus infection, and tamoxifen administration. c57bl/6 mice were purchased from charles river laboratories (wilmington, ma). aicda creert2 mice and rosa26 loxp-tdtomato mice [b6.cg-gt(rosa)26sor tm14(cag-tdtomato)hze /j; ai14] were generously provided by claude-agnes reynaud and dimitrios davalos, respectively, and have been previously described (4) . progeny of the cross between aid creert2 and rosa26 loxp-tdtomato mice were termed aid cre -rosa26 tdtomato mice. mice were housed at the cleveland clinic lerner research institute under pathogen-free conditions. all animal procedures were executed in accordance with approved guidelines by the cleveland clinic lerner research institute institutional animal care and use committee. male and female mice of 6 to 8 weeks of age were infected by intracranial (i.c.) injection with 2,000 pfu of hepato-and neurotropic mhv-a59, kindly provided by volker thiel (university of bern, bern, switzerland) (43) . infected animals were evaluated daily for clinical signs utilizing the following scale: 0, healthy; 1, hunched back and ruffled fur; 2, inability to correct to upright position or partial hind limb paralysis; 3, complete hind limb paralysis and wasting; 4, moribund or deceased. to induce tdtomato expression, mice were administered 3 mg of tamoxifen (sigma-aldrich, st. louis, mo) dissolved in corn oil (20 mg/ml) via oral gavage every other day. treatment was initiated at day 0 p.i. unless otherwise stated. mononuclear cell isolation and flow cytometric analysis. cells were isolated from the cns as described previously (13, 44) . briefly, brains or spinal cords harvested from phosphate-buffered saline (pbs)-perfused mice were mechanically homogenized in dulbecco's pbs using ice-cold tenbroeck grinders. the resulting suspensions were centrifuged at 450 ϫ g for 7 min at 4°c, supernatants stored at ϫ80°c for subsequent analysis, and cells resuspended in rpmi medium. cells were adjusted to 30% percoll (pharmacia, piscataway, nj), underlaid with 1 ml 70% percoll, and collected from the 30%-70% percoll interface following centrifugation at 850 ϫ g for 30 min at 4°c. after washing, cells were resuspended in fluorescence-activated cell sorter (facs) buffer (0.5% bovine serum albumin [bsa] in phosphate-buffered saline [pbs] containing a 10% serum mixture comprised of mouse, goat, and horse serum [1:1:1] and rat anti-mouse fc␥iii/ii monoclonal ab [mab] [2.4g2; bd bioscience, san diego, ca]) for 20 min on ice. cells were then stained with mab specific for cell surface markers cd45 (30-f11), cd19 (1d3), cd138 (281-2), t cell and b cell activation antigen (gl7) (all from bd pharmingen), and igd (11-26c; affymetrix, inc., san diego, ca). cells were then washed with facs buffer, fixed with 2% paraformaldehyde, and analyzed on a bd lsr ii or facsaria iii flow cytometer. the resulting data were analyzed with flowjo software (tree star, inc., ashland, or). elispot. asc were determined by enzyme-linked immunosorbent spot assay (elispot) as described previously (1, 13, 14) . briefly, sterile white 96-well filter plates with 0.45-m-pore-size hydrophobic polyvinylidene difluoride (pvdf) membrane (merck millipore, billerica, ma) were stripped with 70% 10 6 pfu/well) supernatant for approximately 16 h at 4°c. wells were washed with washing buffer (0.05% tween 20 in 1ϫ pbs) and blocked with 5% fetal calf serum (fcs) in rpmi medium for 2 h at 37°c. blocking buffer was replaced with cell suspensions at serial dilutions in triplicate in rpmi medium. plates were incubated at 37°c for 4 h and then washed thoroughly to remove cells. after the addition of biotinylated rabbit anti-mouse igg (0.5 g/ml plates were incubated for ϳ16 h at 4°c, subsequently washed with washing buffer, and incubated with streptavidin-horseradish peroxidase for 1 h at 25°c. following washes with washing buffer and 1ϫ pbs, respectively, diaminobenzadine (dab) solution was added to visualize spots. once spots had developed sufficiently, the reaction was stopped by flushing wells with h 2 0 and the plates left to dry in the dark. the plates were scanned and spots quantified via immunospot (cellular technologies ltd., shaker heights, oh). threshold criteria for spot size were defined as between 0.0009 and 0.2257 mm 2 optimum cutting temperature) compound (sakura finetex, torrance, ca) and sectioned at 10 m (cln) or 12 m (spinal cords) using a leica cm3050 s cryostat ca) overnight at 4°c. sections were then washed with pbs and incubated with secondary ab using alexa fluor 647 goat anti-rat and alexa fluor 488 goat anti-hamster abs (life technologies, grand island, ny) for 1 h at room temperature. spinal cords were stained as described above using rabbit anti-mouse laminin polyclonal ab (cedarlane quantitative real-time pcr gene expression analysis. cln, brains, and spinal cords harvested from individual mice were snap-frozen, treated with 1 ml trizol quantitative real-time pcr was performed using applied biosystems gene expression assays with taqman universal master mix on a 7500 fast real-time pcr system (applied biosystems, foster city, ca). mrna levels of glyceraldehyde 3-phosphate dehydrogenase (gapdh), activation-induced cytidine deaminase (aicda), and immunoglobulin gamma heavy chain (ighg) were determined using taqman primers (applied biosystems) activated gl7ϩ b cells are maintained within the inflamed cns in the absence of follicle formation during viral encephalomyelitis progression from igdϩ igmϩ to isotype-switched b cells is site specific during coronavirus-induced encephalomyelitis recruitment and retention of b cells in the central nervous system in response to 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centers cxcr3-dependent plasma blast migration to the central nervous system during viral encephalomyelitis control of central nervous system viral persistence by neutralizing antibody an optimized method for enumerating cns derived memory b cells during viral-induced inflammation limiting dilution analysis of virus-specific memory b cells by an elispot assay visualizing antibody affinity maturation in germinal centers drainage of cells and soluble antigen from the cns to regional lymph nodes cns viral infection diverts homing of antibody-secreting cells from lymphoid organs to the cns type i ifn receptor signals directly stimulate local b cells early following influenza virus infection early b-cell activation after west nile virus infection requires alpha/beta interferon but not antigen receptor signaling a splenic igm memory subset with antibacterial specificities is sustained from persistent mucosal responses a germinal center-independent pathway generates unswitched memory b cells early in the primary response alphavirus-induced encephalomyelitis: antibody-secreting cells and viral clearance from the nervous system mechanisms of central nervous system viral persistence: the critical role of antibody and b cells long-term antibody production is sustained by antibody-secreting cells in the bone marrow following acute viral infection humoral immunity due to long-lived plasma cells a coordinated change in chemokine responsiveness guides plasma cell movements homing of antibody secreting cells recruitment of memory b cells to lymph nodes remote from the site of immunization requires an inflammatory stimulus memory b cells memory b cells activate brain-homing, autoreactive cd4ϩ t cells in multiple sclerosis mouse hepatitis virus liver pathology is dependent on adp-ribose-1љ-phosphatase, a viral function conserved in the alpha-like supergroup inverted immunodominance and impaired cytolytic function of cd8ϩ t cells during viral persistence in the central nervous system this work was supported by u.s. national institutes of health grant ns086299. the funding source had no involvement in the study design, writing of the manuscript, decision to submit, or collection, analysis, and interpretation of data.we sincerely thank claude-agnes reynaud for generously providing us aicda creert2 mice and jennifer powers for her flow cytometry expertise. key: cord-324619-y7gilopu authors: alam, s.b.; willows, steven; kulka, marianna; sandhu, jagdeep k. title: severe acute respiratory syndrome coronavirus‐2 may be an underappreciated pathogen of the central nervous system date: 2020-07-15 journal: eur j neurol doi: 10.1111/ene.14442 sha: doc_id: 324619 cord_uid: y7gilopu severe acute respiratory syndrome coronavirus‐2 (sars‐cov‐2) causes a highly contagious respiratory disease referred to as covid‐19. however, emerging evidence indicates that a small, but a growing number of covid‐19 patients also manifest neurological symptoms, suggesting that sars‐cov‐2 may infect the nervous system under some circumstances. sars‐cov‐2 primarily enters the body through the epithelial lining of the respiratory and gastrointestinal tracts, but under certain conditions this pleiotropic virus may also infect peripheral nerves and gain entry into the central nervous system (cns). the brain is shielded by various anatomical and physiological barriers, most notably the blood‐brain barrier (bbb) which functions to prevent harmful substances, including pathogens and pro‐inflammatory mediators, from entering the brain. the bbb is composed of highly specialized endothelial cells, pericytes, mast cells and astrocytes that form the neurovascular unit, which regulates bbb permeability and maintains the integrity of the cns. in this review, we briefly discuss potential routes of viral entry and the possible mechanisms utilized by sars‐cov‐2 to penetrate the cns, either by disrupting the bbb or infecting the peripheral nerves and using the neuronal network to initiate neuroinflammation. furthermore, we speculate on the long‐term effects of sars‐cov‐2 infection on the brain and in the progression of neurodegenerative diseases known to be associated with other human coronaviruses. although the mechanisms of sars‐cov‐2 entry into the cns and neurovirulence are currently unknown, the potential pathways described here might pave the way for future research in this area and enable the development of better therapeutic strategies. on december 27 th , 2019 the chinese center for disease control and prevention announced that it had detected a cluster of patients in wuhan, china who had developed severe pneumonia of unknown etiology, later termed covid-19 (coronavirus disease of 2019) (1) (2) (3) . these patients were described as presenting with mainly fever, with a few patients having accepted article difficulty in breathing and on january 8 th , 2020 the causative agent of covid-19 was identified as severe acute respiratory syndrome coronavirus-2 (sars-cov-2) (4). in the subsequent six months, sars-cov-2 has become a worldwide pandemic, infecting over eleven million people and killing more than 540,000 patients worldwide (https://www.jhu.edu/; https://coronavirus.jhu.edu/), while also crippling the world economy. although covid-19 was first described as a respiratory disease, new data shows that sars-cov-2 can infect almost every organ, resulting in an ever-increasing list of symptoms. in particular, neurological symptoms and disturbances in the central nervous system (cns) are common in many covid-19 patients, and may be a predictor of disease severity. in this review, we examine some of the most recent data of covid-19-associated neurological disease and the possibility that sars-cov-2 may be infecting the cns. in particular, we examine the possible mechanisms of viral entry through the blood-brain barrier (bbb) and the peripheral nerves and we discuss the possible long-term consequences of viral infection in the brain by surveying data from closely related, neurotropic viruses. since viral infection of the cns often has long-term neurological implications for patients, the possibility that these types of infections could lead to neurodegenerative diseases is discussed. even in the early months of the covid-19 pandemic, physicians observed that a significant subset of patients positive for sars-cov-2 presented with neurological complications, sometimes accompanied with respiratory distress. in february 2020, li et al. suggested that since sars-cov-2 shared significant similarities to severe acute respiratory syndrome coronavirus (sars-cov), it was entirely possible that sars-cov-2 could similarly penetrate the brain and cns of infected patients through synapses in the medullary cardiorespiratory center and thereby cause respiratory failure (5) . quickly thereafter, several studies of severely ill covid-19 patients in wuhan described neurological symptoms including autopsy observations of deceased patients which showed brain tissue edema and partial neuronal degeneration (6) . in a retrospective study of hospitalized patients with laboratory confirmed sars-cov-2 infection in wuhan, china, 36.4% of patients exhibited neurological symptoms (7), such as dizziness (16.8%) and headache (13.2%), while neurological symptoms were more common in severe versus non-severe patients (45.5% vs 30.3%). several symptoms accepted article were more specifically associated with severe disease, such as impaired consciousness (14.8% in severe vs 2.4% in non-severe), acute cerebrovascular disease (5.7% vs 0.8%), and skeletal muscle injury (19.3% vs 4.8%). other studies also found incidences of headache, dizziness and confusion in 5-9% of hospitalized patients (8) and a single study from wuhan, china reported similar rates (~5%) of acute cerebrovascular disease in severely affected covid-19 patients (9) . a retrospective analysis of deceased patients in china found a high rate of disorders of consciousness upon admission to the hospital, suggesting neurological complications were an indicator of poor prognosis (10) . in the past -six months, we have learned that the loss of taste and smell is an early sign of sars-cov-2 infection, affecting approximately 5% of chinese patients (7), 30-40% of european patients (11) (12) (13) , and is most prevalent in young women (12, 13) . this observation is especially significant since it has been suggested that human coronaviruses, such as sars-cov (in mice) and hcov-oc43 (in mice and humans) enter the brain through the olfactory bulb (14) . a recent cross-sectional study reported that the cumulative incidence of covid-19 was higher in patients with active epilepsy as compared to control subjects without epilepsy (15) . another study showed that plasma biomarkers of cns injury, namely neurofilament light chain protein (nfl), a marker for neuronal injury and glial fibrillary acidic protein (gfap), a marker for astrocytic injury were significantly elevated in patients with moderate and severe covid-19 (16). histopathological examination of brain specimens from a cohort of 18 covid-19 patients showed acute hypoxic injury in the cerebrum and cerebellum and loss of neurons in the cerebral cortex, hippocampus and cerebellar purkinje cell layer (17). a retrospective study from china found that covid-19 patients over 60 years old and with neurologic comorbidities were at a higher risk of developing neurologic impairments such as impaired consciousness and cerebrovascular accidents (18). sars-cov-2 belongs to the betacoronavirus genus in the coronaviridae family (19). with the exception of hcov-229e and hcov-nl63 (alphacoronaviruses), human coronaviruses (hcovs) belong to the betacoronavirus genus. all human coronaviruses have animal origins (20) and cross-species transmission appears to happen when low affinity binding occurs in receptors closely related between host species. two strains of sars-cov isolated from palm civets, for example, had high affinity for civet receptor angiotensin converting enzyme ii (ace2) and high infectivity in civet cells but these same two strains of sars-cov had low affinity for human ace2 and therefore low infectivity in human cells (21). similar to sars-cov, this article is protected by copyright. all rights reserved sars-cov-2 also binds to human ace2 with high affinity and is likely the principal entry route into human respiratory cells (22, 23). similar to sars-cov, sars-cov-2 also requires the transmembrane serine protease 2 (tmprss2) for spike protein priming and entry into the host cell (22), although it might vary depending on the cell type (24, 25). therefore, cells that express ace2 and tmprss2, such as the glia and neurons, would be plausible targets for sars-cov-2 infection (26) . infections are associated with headache, dizziness, axonopathic polyneuropathy, myopathy, ischemic stroke, ataxia, febrile seizures, convulsions, loss of consciousness and encephalomyelitis encephalitis (14) . in fact, some of these viruses have been found in the cns of their hosts (31) (32) (33) (34) (35) . sars-cov has been isolated and cultured from the brain of a severe acute respiratory syndrome (sars) infected patient, providing evidence that sars-cov is able to infect the human brain (32) . hcov-oc43 has been associated with fatal encephalitis in immunocompromised pediatric patients, demonstrating direct evidence for the presence of viral proteins and rna in neuronal cells in the autopsied brain tissue (33, 35) . a first case of sars-cov-2 meningitis/encephalitis has been reported recently, where rna has been detected in the cerebrospinal fluid (csf) (34) , suggesting that sars-cov-2 can invade the cns. similar to these neurotropic hcovs, sars-cov-2 infection in the lungs of some covid-19 patients may also lead to entry into the cns and this could occur via two main pathways: i) infection of peripheral nerves and retrograde axonal transport; and/or ii) hematogenous spread and infection of the cells of the blood-brain barrier. the peripheral organs are highly innervated with neurons that link the peripheral nervous system (pns) with the cns and neuroinvasive viruses have evolved several strategies to access the cns via the transneural route (19, 31, 36) . for example, viruses can manipulate the neuronal cytoskeletal machinery of the microtubules and accepted article molecular motors, such as dynein and kinesin to traffic virions via a retrograde and anterograde transport route, respectively from the pns into the cns (36) . after replication in the neuronal cell body, fully assembled viral particles are released into the synaptic cleft and infection is spread to presynaptic neurons. as will be discussed in more detail below, most rna viruses, including a number of coronaviruses have been shown to enter the cns by utilizing the transneural pathway (19, 31) . given the fact that sars-cov-2 is an rna virus and has substantial similarity with other coronaviruses (that belong to the same family of viruses), we hypothesize that it might deploy similar entry routes to access the cns (figure 1 ). viruses can also spread to the spinal cord from the trigeminal nuclei via the reticular formation and the reticulo-spinal tract (45) and this trans-synaptic transfer has been reported for avian bronchitis virus (a gammacoronavirus) (46, 47). experimental studies using transgenic mice revealed that both sars-cov and mers-cov, when inoculated intranasally, entered the brain, possibly via the olfactory nerves, and thereafter rapidly spread to some specific brain areas including thalamus and brainstem (29, 48) . therefore, if viral replication in the nose is accepted article sufficiently high, it is possible that these high viral titres could infiltrate the olfactory nerve. nasal swabs of symptomatic and asymptomatic covid-19 patients have higher viral loads than throat swabs (3, 49) , suggesting these cells might be the loci of viral replication and possible reservoirs for dissemination within the nasal cavity to the olfactory nerve. the lungs and spreads to the brain via the retrograde axonal transport in the vagus nerve (46, 50) . the vagus nerve travels through the neck and thorax to the stomach and it connects the lung to the gut and brain, also referred to as lung-gut-brain axis (51) . since the lungs represent a major reservoir of sars-cov-2 infection, at least in the early stages of covid-19 disease, it is possible that sars-cov-2 could use the vagus nerve to enter the cns and travel throughout the lung-gut-brain axis, potentially interfering with all of these systems at different time-points during infection. this may explain why some patients experience a combination of gastrointestinal, neurologic and lung symptoms throughout the course of infection (52) . the gut is a highly innervated organ with a complex gut microbiome and this complex network biochemically interacts with the host cns, also known as the gut-brain axis. in fact the gut has often been referred to as a neurologic organ since it is innervated by five different classes of neurons: intrinsic enteric neurons, vagal afferents, spinal afferents, parasympathetic efferents and sympathetic efferents (53) . if sars-cov-2 gains access to this highly complex innervated network, it is possible that it could use this network to penetrate the cns (54). this article is protected by copyright. all rights reserved neuroinflammation. the fact that many of these cytokines can promote vascular permeability and leakage resulting in bbb dysfunction, suggests that infection in the gut could be another plausible route by which sars-cov-2 could penetrate the brain. into the brain via retrograde transport (61) . phev binds to ncam expressed on the surface of medullary neurons to enter into the cns. since, sars-cov-2 belongs to the same genus as phev (betacoronavirus), it is possible that it might also deploy neuronal ncam to enter the cns. additionally, previous studies have shown that ace2 is present on skeletal muscles (62) . hence, sars-cov-2 might bind to ace2 receptors present on skeletal muscles and enter the cns via retrograde transport. in support of this argument, it has been shown that 19.3% of patients with severe covid-19 related neurological manifestations had skeletal muscle injury (7). the prevalence of myalgia between 11-50% and muscle weakness related to covid-19 has been reported in several studies (63) (64) (65) . acute myositis has also been recognized as a manifestation of covid-19 on mri scans (66) and in at least one specific case, an afebrile covid-19 patient was hospitalized and did not present any upper and lower airway symptoms, but had elevated creatine kinase and c-reactive protein levels, suggestive of muscle this article is protected by copyright. all rights reserved inflammation (66) . during the 2015 mers-cov outbreak in the republic of korea, four patients in a cohort of 23 (17.4%) experienced neuropathies and limb weakness, neurological complications that lasted months after initial infection (67) . it is possible that covid-19 patients that have experienced sars-cov-2 infection of the cns via the neuromuscular junction could experience similar complications and long-term follow-up of these patients should be prioritized. normally, viral entry from the blood into the cns is restricted by the blood-brain barrier (bbb), which forms a structural and functional barrier between the peripheral circulation and the cns. the bbb is comprised of highly specialized cerebrovascular endothelial cells, pericytes, mast cells and astrocytes that function together as a neurovascular unit to maintain homeostasis (68, 69) . the total length of brain capillaries in humans is approximately 600 km with a surface of 15-25 m 2 , providing a large area for viral invasion (70) . the neurovascular unit serves as the gate-keeper of the cns that protects the brain by regulating the cerebral blood flow and limiting the access of pathogens, this article is protected by copyright. all rights reserved interleukins (75) . the cytokine storm associated with sars-cov-2 infection results in increased secretion of pro-inflammatory cytokines and chemokines such as il-6, tnf-α, mip1-alpha, ip-10 and g-csf as well as c-reactive protein and ferritin (64) . these cytokines and chemokines can bind to specific receptors on the cerebral microvascular endothelium leading to bbb breakdown, neuroinflammation and encephalitis. the loss of bbb integrity could loosen the tjs between the endothelial cells paving the way for paracellular traversal of sars-cov-2 into the cns (figure 2a) . a recent study on japanese encephalitis virus (jev, an rna virus) suggests that paracellular mode of trafficking could be one of the potential routes of entry into the cns (76) . jev-infected mast cells release chymase, a vasoactive protease, which cleaves tj proteins, including zona occludens-1 and 2, claudin-5 and occludin, breaking down the bbb and facilitating entry of jev into the cns. some neurotrophic viruses, such as jev and wnv can enter into the cns via the bloodstream in a process known as viremia (77) . in the "trojan horse" strategy of neuroinvasion, the virus hides inside innate immune cells, hiv infects cd4-positive t-cells and utilizes chemokine ccr5 as a co-receptor to enter the cns (85, 86) . additionally, hiv also infects cd16-positive monocytes to travel across the bbb and infect brain microglia leading to chronic inflammation and eventually neuronal damage and dementia (36) . human cytomegalovirus (hcmv) (87, 88) , enteroviruses including poliovirus (89) and flaviviruses (90) have also been shown to infect different types of leukocytes and to use them as a reservoir for hematogenous dissemination toward the cns. it has been shown that ace2 receptor is expressed on hematopoietic cells, including monocytes and lymphocytes. t-cells via dipeptidyl peptidase 4 (91, 92) . it is possible that sars-cov-2 could infect monocytes and lymphocytes and traffic across the bbb and further infect neural cells. more recently cd147, also known as basigin and emmprin (extracellular matrix metalloproteinase-inducer) has been implicated as an alternative entry receptor for sars-cov-2, which is expressed on activated lymphoid, myeloid, epithelial and neuronal cells in the cns (93, 94) . long-term neurologic effects of covid-19? it is still too early to reliably predict the possible longterm health effects of sars-cov-2 infection but some general predictions are possible. since there is substantial population variability in symptoms associated with acute sars-cov-2 infection, the long-term consequences of covid-19 will also be variable, likely depending on sex and age (at the time of infection) of the patients. however, since other human coronavirus infections cause autoimmune and neurodegenerative diseases in their hosts, it is possible that accepted article these may also occur after sars-cov-2 infection. for instance, hcov-oc43 and hcov-229e antigens and rna have been detected in the csf and brain tissues of multiple sclerosis patients, where viral rna persists in the absence of infectious virus (31, 105) . another study showed that antibodies against hcov-oc43 and hcov-229e were found in the csf of parkinson's disease (pd) patients (106) . patients infected with other neurotropic viruses, including wnv, jev, hiv, h5n1 and iv develop parkinsonian symptoms, including tremor, rigidity, and bradykinesia and infection with these viruses may increase the risk of developing pd (107) . therefore, there is a possibility that the long-term presence of viral components in the brain of covid-19 patients may induce chronic innate and adaptive immune responses that eventually lead to autoimmune and neurodegenerative diseases such as alzheimer's disease (ad), parkinson's disease (pd) and multiple sclerosis in susceptible individuals. the pathological hallmarks of many chronic neurodegenerative diseases are selective, such as progressive loss of neurons, accumulation of misfolded/aggregated proteins like amyloid-beta and alpha-synuclein as well as neuroinflammation (108) . as discussed above, covid-19 patients present with olfactory and gastrointestinal issues and it is noteworthy that idiopathic olfactory dysfunction and vagal dysfunction are both early and common symptoms of preclinical pd and precede several years before the onset of classical motor dysfunction (109) . with regards to pd etiology, braak et al. had hypothesized that pd progresses through the nervous system in different stages (110) , which was later revised as the 'dual-hit' hypothesis by hawkes et al. (111) . according to the 'dual-hit' hypothesis, a neurotropic viral pathogen likely enters into the brain through the olfactory pathway or the enteric nervous system and leads to misfolding and aggregation of alpha-synuclein protein and its translocation to the midbrain by 'prion-like' propagation throughout the brain as the disease progresses. in an earlier study barnett et al. had showed that mhv entered the brain through the olfactory system and infected dopaminergic neurons in the substantia nigra and ventral tegmental area, respectively (112) . interestingly, mhv produced a widespread infection of the a10 group of neurons. it also infected neurons of the a8 and a9 groups, suggesting that mhv can damage the nigrostriatal and mesolimbic dopamine pathway. a growing body of literature has now revealed that majority of the proteins involved in neurodegenerative diseases are transported in exosomes (113) . therefore, it is possible that exosomes play an important role in the transport of misfolded alpha-synuclein across the bbb into the brain parenchyma where it acts as 'seeds' in the recipient neural cells, spreading throughout the brain by a 'prion-like' mechanism. chronic neurodegenerative diseases develop over decades with distinct preclinical, prodromal and clinical phases and the long-term impacts of sars-cov-2 on younger patients is not known but this article is protected by copyright. all rights reserved could be a serious concern. if sars-cov-2 alters the long-term impacts on the cns as described, they may manifest over years or decades. as recovered covid-19 patients age, there might be an upsurge in the number of neurodegenerative diseases. therefore, we propose that patients who have recovered from the acute phase of the sars-cov-2 infection, should be continuously monitored throughout their lives for the development of neurological symptoms associated with neurodegenerative disease. at this time there are no therapeutics or vaccines approved by the us food and drug administration (fda) to specifically cure, treat or prevent covid-19. however, the fda continues to issue emergency approvals for "off-label" drugs to treat severe covid-19 patients. these drugs include antiviral drugs (remdesivir, chloroquine and hydroxychloroquine) and immunomodulators (tocilizumab, canakinumab and anakinara), and some of them may contribute to neurological dysfunction. for example, chloroquine and hydroxychloroquine, which were commonly used earlier in the pandemic, could potentially increase the likelihood of neurological disorders, especially in the elderly (114) . the use of hydroxychloroquine has been associated with neuropsychiatric manifestations including irritability, nervousness and psychosis, possibly by its ability to cross the bbb (115) . corticosteroids, another common group of medications used to treat severe cases of covid-19, have also been associated with psychiatric symptoms, especially when administered at high doses (116) . the use of immunomodulators, such as tocilizumab (monoclonal antibody to il-6 receptor), canakinumab (monoclonal antibody to il-1β) and anakinara (il-1 receptor antagonist) are in clinical trials to dampen cytokine responses in severely ill covid-19 patients, however they have poor bbb penetration into the cns (117) . the use tocilizumab has been associated with multifocal cerebral thrombotic microangiopathy and adverse neurological effects on the cns (118). finally, mechanical ventilation was, and continues to be, the principal medical intervention used to treat severe covid-19 patients. however, some recent studies are raising the possibility that the use of mechanical ventilation can contribute to neurological injury, which can fuel further lung damage (119). the current pandemic has spread globally to every country, infected millions of people and remains a major public health concern. while governments around the world are using accepted article various measures, such as lockdowns, quarantine and contact tracing to control the spread of infection, it is worth noting that these approaches might significantly impact patients with chronic neurologic disorders in unintended ways. for example, the covid-19 quarantine in italy created uncertainty and confusion about the availability of clinical services and continuity of care among pd patients (120) . negative effects on mental health have been observed in those whose daily life was disrupted by the various public health measures (121) . many drugs used to treat people with chronic neurological diseases are being repurposed to fight covid-19 (122) (123) (124) , potentially leading to shortages and contributing to extra stress and worsening of disease symptoms. although we are rapidly learning about sars-cov-2 and its methods of infection, there is still much to learn. in this review, we have extrapolated information from other neurotropic viruses to make some predictions and it is clear that sars-cov-2 has the potential to infect the cns and cause long-term neurologic damage in covid-19 patients. the next few years will be critical if we are to determine the long-term effects of this pandemic on the neurological health of this population. we urge governments to create a framework and a national registry of patients who have been infected by sars-cov-2. patients, particularly those with neurological symptoms, must be tracked regularly and consistently at ongoing time points over their life time using advanced neuroimaging and biochemical analysis of biomarkers to map the degenerative process. to this end, the spanish neurological society has implemented a registry of neurological manifestations in patients with confirmed covid-19. román and colleagues have emphasized the need of covid-19 international neurological databanks to report all cases of new-onset, acute, delayed, and any long-latency neurological disorders associated with sars-cov-2 infection during the covid-19 pandemic (125). the 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with or without quarantine managements we thank the national research council canada for intramural funding. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-292255-zafnq8gl authors: trojano, m.; avolio, c. title: chapter 8 environmental factors and their regulation of immunity in multiple sclerosis date: 2016-12-31 journal: translational neuroimmunology in multiple sclerosis doi: 10.1016/b978-0-12-801914-6.00008-8 sha: doc_id: 292255 cord_uid: zafnq8gl abstract in multiple sclerosis (ms), environmental factors and genetic traits cooperate in the induction of the chronic activation of immune cells to produce the brain pathology. epidemiology has focused on different environmental risk factors but certainly virus infection, smoking, vitamin d levels, and sunlight exposure are the most relevant. what is certainly less clear is the way in which these external factors are able to induce and sustain the internal pathology process of the disease. epigenetics has been recently focused on trying to shed light on this aspect. as a matter of fact epigenetic changes are highly sensitive to environmental factors that therefore may influence the susceptibility to the disease by acting through epigenetic modifications. in this chapter we discuss the most relevant environmental factors and how they may affect the immune response in ms. finally, we discuss the possible role of the microbiota in inducing autoimmunity in ms. multiple sclerosis (ms) is an inflammatory/neurodegenerative disease of the central nervous system (cns) in which both genetic and environmental factors cooperate in the chronic activation of immune cells to produce oligodendrocyte and neuron damage. epidemiological studies have identified several environmental risk factors in ms, such as exposure to certain viruses and smoking or even lack of exposure to sunlight with a subsequent reduced vitamin d production. these factors are associated with the susceptibility in developing ms but they could also influence the disease course. however, no single risk factor per se appears to be responsible for the development of the disease, but a multifactorial interplay is most likely. because of this complex interplay, it is quite difficult to define the real impact of each single factor and in this respect the only way to proceed is to design large enough studies with highquality data. 1, 2 however, what is certainly even less known is the way in which these external factors are able to induce and sustain the internal pathology process of the disease. in this chapter we try to provide an overview of the most relevant environmental factors and how they may affect the immune response in ms. though the etiology of ms remains as yet unknown, 3 its pathogenesis has been quite extensively investigated and mostly clarified since it is widely accepted that activated peripheral immune cells enter the cns to produce the pathology. 4 the initial dysfunction can also occur within the cns and it can include mitochondrial dysfunction in neurons or oligodendrocytes, axonal energy insufficiency, or even damage to other neural organelles such as peroxisomes. 5 in such a case, whichever the initial injury, the leakage of cns antigens into draining lymph nodes activates t cells that address and enter the cns, inducing inflammation, demyelination, and oligodendrocyte loss as well as axonal/neuronal injury and loss. professional antigen-presenting cells (apcs) such as dendritic cells are needed to activate t cells. the apcs, either from the periphery or from the cns, migrate to lymph nodes, carrying the antigen (a short segment of the pathogen) bound to major histocompatibility complex (mhc class i for cd8 + and ii for cd4 + commitment) on their cell surface. in the lymph nodes, the antigen is presented to naïve t cells through a t-cell receptor (tcr) recognizing the antigen/mhc combination. this trimolecular complex (mhc/antigen/tcr) constitutes a first signal, but a second signal, mediated by costimulatory molecules (eg, b7 on apcs and cd28 on t cells) is needed for full activation of the t cells, their proliferation, and subsequent differentiation into effector cells. cd4 + t cells are crucial in ms as they can differentiate into proinflammatory t helper (th) 1 or 17 subsets, antiinflammatory th2 cells, or into cells with regulatory/antiinflammatory properties (tregs), depending on the microenvironment and cytokine milieu. 6 in ms patients there is a tendency to generate either th1 or th17 subsets, which in addition to being proinflammatory 7 may have neurotoxic effects, 8 whereas the regulatory/antiinflammatory th2 and tregs subsets are reported to be deficient in ms. 9 cd8 + t cells also have relevant roles in ms tissue damage. 10 b cells also importantly produce disease pathology in ms and this is supported by various evidence including the effectiveness of monoclonal antibody therapies that target the b-cell antigen such as cd20, 11, 12 ii. other patho-mechanisms the oligoclonal bands in the cerebrospinal fluid commonly reported in ms patients, and b-cell follicular-like structures found in the meninges of secondary progressive ms patients. 13 in addition to the pathogenetic role in the production of antibodies targeting cns structures, 14 b cells may play additional roles such as antigen presentation and help for t cells. 15 once activated, immune cells upregulate different adhesion molecules and adhere to endothelial cells of postcapillary venules in the cns. they then cross the endothelial cell barrier by means of the proteolytic activity of the matrix metalloproteinases (mmps), first migrating across the endothelial basement membrane and then the parenchymal basement membrane or glia limitans, and finally they enter the cns parenchyma. as a matter of fact mmps have been reported to be upregulated in ms. 16 upon entering the cns parenchyma, t cells are reactivated through repeated antigen presentation by apcs such as microglia, macrophages, b cells, and dendritic cells. activated immune cell subsets, as well as inflammation and demyelination, also induce neuronal injury and loss by producing free radicals, glutamate, and other excitotoxins, proteases, and cytokines. 8,17 it is therefore quite evident that ms has the characteristics of both an inflammatory/demyelinating and a neurodegenerative disease in terms of pathology but this is also clear in terms of clinical presentation, course, and accumulated disability in patients. even if not an inherited disorder, genetic factors are certainly implicated in the disease susceptibility and this is especially evident from studies demonstrating the increased risk of ms in relatives of patients with ms, with a higher risk the closer the individuals are related to the patients. 18, 19 several genetic loci, such as the hladrb1 on chromosome 6, have been reported to be associated with an increased risk for ms. 20 nevertheless, effort has been focused on epigenetic mechanisms that may influence the pathophysiology of ms. epigenetics is the study of mechanisms that alter the expression of genes without altering the dna sequence. dna methylation, histone modification, and microrna (mirna)-associated posttranscriptional gene silencing are the three most investigated epigenetic mechanisms. even if epigenetic changes are passed from parent to offspring through the germ line, they are highly sensitive to environmental factors that therefore may really influence the susceptibility to the disease by acting through epigenetic modifications. 21, 22 dna methylation 23 consists of the addition of a methyl group to the carbon-5 of a cytosine residue in dna through the intervention of enzymes called dna methyltransferase (dnmt). dnmt1 maintains dna methylation patterns during dna replication and localizes to the dna replication fork, where it methylates nascent dna strands at the same locations as in the template strand. 24 dnmt3a and dnmt3b intervene in the de novo methylation of unmethylated and hemimethylated sites in nuclear and mitochondrial dna, respectively. 24, 25 especially in mammals, dna methylation usually occurs at cpg sites (where a cytosine nucleotide is followed by a guanine nucleotide) that can be found with up to several hundred dinucleotide repeats, therefore called cpg islands and mostly found in gene promoter regions. the methylation or hypermethylation of cpg islands in promoter regions has been reported to block the expression of the associated gene. 26 dna methylation is the best investigated physiological epigenetic mechanism so far. 27 mainly in mammalian cells, histone proteins interact with dna to form chromatin, the packaged form of dna. histones are octamers consisting of two copies of each of the four histone proteins: h2a, h2b, h3, and h4. each histone octamer has 146 bp of the dna strand wrapped around it to shape one nucleosome, the basic unit of the chromatin. histone proteins can be modified 23 by posttranslational changes such as acetylation, methylation, phosphorylation, ubiquitination, and citrullination. since these histone modifications produce changes to the structure of chromatin they may affect the accessibility of the dna strand to transcriptional enzymes, therefore inducing either activation or repression of genes associated with the modified histone. 28 acetylation, mediated by histone acetyltransferases and deacetylases, is currently the most investigated and hence the most clarified histone modification. acetylation of histones generally results in the upregulation of transcriptional activity of the associated gene, whereas deacetylation of histones contributes to transcriptional silencing. 29 single-stranded, noncoding mirnas are widely represented in cells either from plants or animals. 30 the transcripts undergo several posttranslation changes, either in the nucleus or in the cytoplasm, to generate mature and functional mirnas. moreover, in the cytoplasm itself, mature mirnas associate with other proteins to form the rna-induced silencing complex (risc), in which the mirna imperfectly pairs with cognate mrna transcripts. the target mrna is then degraded by the risc, preventing its translation into protein. 31, 32 such mirna-mediated repression of translation 23 is utilized in many cellular processes, namely differentiation, proliferation, and apoptosis, as well as other key cellular mechanisms. 33, 34 current knowledge on the role of epigenetic mechanisms in ms mostly comes from pathological studies, either from biopsies or autopsies, focusing on active demyelinating or chronic lesions, but also from studies of patients with ms, either with a relapsing-remitting (rr), chronic primary progressive (pp), or secondary-progressive (sp) course. 35 patient brain biopsy samples show that active and inactive ms lesions have distinct mirna profiles. as a matter of fact, the mirnas mir-155, mir-34a, and mir-326 are highly upregulated in active ms lesions compared with inactive lesions and normal white matter from healthy controls. 36 the differentiation of t cells, especially th17 cells, is influenced by epigenetic mechanisms and mir-155 and mir-326 are also associated with t-cell differentiation. [37] [38] [39] [40] the expression of mir-155 is upregulated in macrophages, t cells, and b cells in response to ligand binding to toll-like receptors (tlrs) and inflammatory cytokines, suggesting that it is involved in inflammatory processes. 41 mice that are deficient in mir-155 are highly resistant to the development of the experimental autoimmune encephalomyelitis (eae), the animal model for ms, 41 and silencing of mir-155 by administering an antisense oligonucleotide before induction of eae attenuates the severity of symptoms. 42 moreover, expression of mir-326 is upregulated in mice with eae; in vivo silencing of this mirna results in attenuation of eae symptoms and reduced numbers of th17 cells. 43 others have shown that in untreated ms (ppms, spms, or rrms) and healthy controls, two other mirnas, mir-17 and mir-20a, are downregulated in all three forms of ms. 44 these two mirnas inhibit t-cell activation, and their downregulation in patients with ms, therefore, might contribute to a net increase in t-cell differentiation, including differentiation into th17 cells. especially in progressive ms, the evidence for involvement of epigenetic changes comes from a study showing an association between dna methylation and neuronal cell death and in fact the overexpression of dnmt3a, an enzyme involved in de novo dna methylation, induced apoptosis. 45 as far as histone modification is concerned, the citrullination of myelin basic protein (mbp) has an important role in the pathophysiology of ms. 46 mbp is a major component of myelin in the cns, and can be modified in several ways after translation. in biopsy samples from ms patients, normal-appearing white matter shows increased levels of citrullinated mbp as compared with levels in healthy controls and patients with alzheimer's disease. 47 citrullinated mbp is less stable than unmodified mbp, which suggests that citrullination might contribute to myelin breakdown and eventually to the development of an autoimmune response to mbp. 48 finally, brain biopsy material from progressive ms patients and controls without neurological disease show an increase in histone h3 acetylation in oligodendrocytes within chronic ms lesions, whereas oligodendrocytes within early-stage ms lesions show marked histone h3 deacetylation. 49 increased histone h3 acetylation in oligodendrocytes is associated with impaired differentiation and, therefore, with impaired remyelination. since epigenetic changes are highly sensitive to environmental influences, it is likely that the effects of environmental risk factors in ms might be mediated by changes in patients' epigenetic profiles. migration studies have contributed to provide evidence that a viral infection may trigger the development of ms. 50 it has been shown that people migrating from a high-risk country for ms to a low-risk one are at lower risk of developing ms than they would be in their country of origin. whereas those migrating from a low-risk country to a high-risk one keep the low risk of their country of origin, their children have a risk comparable to the country where they emigrate, 51 especially in those migrating before the age of 15, 52 suggesting that infection at a young age may predispose to the later development of ms. in addition to these migration studies, some classical studies on the incidence and prevalence of ms have suggested that there may have been ms epidemics in several locations, such as in the faroe islands after ii. other patho-mechanisms the second world war, 53 and the increase of incidence in the shetland islands 54 and sardinia 55 have been taken to suggest that an infectious agent may be involved in the pathogenesis of ms. different hypotheses have been proposed to explain how viral infections are associated with ms. 56 according to the bystander activation hypothesis, autoreactive t cells are activated by nonspecific inflammatory molecules occurring during infections, such as cytokines, superantigens, and tlr ligands. 4 the molecular mimicry hypothesis, instead, postulates that upon exposure to a pathogen, the pathogen/mhc conformation on an apc bears molecular similarity to that of an endogenous peptide, such as an mbp fragment presented within an mhc. 57 if appropriate costimulation occurs, it results in the expansion and differentiation not only of the pathogen-reactive t cells, a proper immune response, but also the expansion of mbp-reactive t cells, an improper response. if both pools differentiate into th1 or th17 proinflammatory subsets, these can become reactivated within the cns to promote pathology. in fact, t-cell lines isolated from ms patients demonstrate cross-reactivity between mbp and coronavirus 58 or epstein-barr virus (ebv) 59 antigens. furthermore, a significant degree of crystal structural similarity has been shown between the drb5*0101-ebv peptide complex and the drb1*1501-mbp peptide complex at the cell surface for tcr recognition. 60 further immunological evidence in the association of ebv with ms has been provided. the follicular-like structures under the meninges include b cells that are infected with ebv in many patients. 61 ms patients have antibodies that cross-react between mbp and ebv, a possible additional mechanism by which anti-ebv antibodies may disrupt myelin. 62 furthermore, ebv-reactive cd8 + t cells that are restricted by hla-b7, a common allele in ms, are dysregulated in ms 63 and the cd8 + t-cell deficiency in ms impairs the capacity to control ebv infection with the result that ebv-infected b cells accumulate in the cns where they produce pathogenic autoantibodies and provide survival signals to autoreactive t cells. 64 ebv infection is certainly associated with changes in epigenetic profiles in infected cells but so far this has been evaluated especially in tumors and, as a result, several types of tumor are associated with prior ebv infection, probably due to promoter hypermethylation (and, therefore, repression) of tumor suppressor genes. 65 there is still a lack of evidence for these aspects in ms. despite molecular similarity between several other pathogens and a number of myelin peptides and other molecules within the cns frequently occurring, there is a high probability that these pathogens can induce improper expansion of cns-reactive t cells to promote pathology within the cns and hence no single infectious agent may be uniquely associated with ms. both epidemiological and clinical studies have recognized smoking as an environmental risk factor for ms. 50 smoking increases the relative incidence rate of ms in current smokers compared to nonsmokers, with a dose-response dependent on the number of packs smoked per year. 66 smoking also has an impact on inflammatory outcomes in ms. patients with a clinically isolated syndrome have an increased risk of conversion to clinically definite ms in smokers compared to nonsmokers. 67 ms smokers have more gadolinium-enhancing lesions, a greater t2-lesion load, and more brain atrophy than nonsmokers, 68 as well as a quicker ii. other patho-mechanisms increase in t2-lesion volume and brain atrophy in an average follow-up period of time. 69 as far as the disease progression is concerned, the data are quite discordant since smoking is in some cases reported not to be associated with the risk of sp or with that of reaching expanded disability status scale (edss) 4.0 or 6.0 70 ; in others it is reported to be associated with a greater risk of sp course 69, 71 or even with an increase in edss scores during two years of follow-up. 72 in conclusion, smoking may have more influence in the early disease course than in the late disease stages of ms. how smoking increases the risk of ms is still a matter of debate and even whether or not cigarette smoke contains mutagens that can affect long-lasting immunity, but smoking has been demonstrated to induce an immunosuppressant state. 73 nevertheless, cigarette smoking induces immune functions and an interaction between smoking and genes regulating immune functions has been reported. 74 it would be relevant to figure out whether constituents of tobacco alter signaling through the aryl hydrocarbon receptor, a transcription factor affected by polycyclic aromatic hydrocarbons and polychlorinated dioxins, since the latter regulates t-cell polarization and alters the course of eae. 75 it is almost certain that smoking affects ms by upregulating mmps since immune cells and biological fluids of smokers tend to upregulate several mmps 76 and these may facilitate immune-cell entry to the cns parenchyma. when comparing mri scans from smokers and nonsmokers with ms, more contrast-enhancing lesions are evident among the smokers, suggesting more severe blood-brain barrier damage. 68 smoking so far has been reported to be associated with changes in epigenetic profiles in patients with cancer, especially inducing silencing of tumor suppressor genes, mostly through dna methylation. 77 smoking is also associated with changes in mirna expression profiles in spermatozoa, 78 and with altered histone modifications resulting from reduced levels of histone deacetylase 2 in macrophages. 79 in ms there is no evidence in this respect but no doubt these mechanisms are worth investigating in the disease. ms is more prevalent in regions of higher latitude 80 where an increase of female/male rate incidence has been also demonstrated in the 2000s. 81 this phenomenon seems to be associated with a decreased sunlight (uv) exposure and the subsequent reduced vitamin d production. 82 it has been shown that the risk of developing ms decreases with increasing serum 25-hydroxy-vitamin d levels in a prospective case-control study. 83 among various suspected environmental factors in ms, the lack of uv exposure has been found to be the most significant risk factor for ms. 50, 84 moreover, vitamin d may influence the disease course of ms since lower vitamin d levels have been demonstrated to be associated with higher levels of disability 85 and an association between higher levels of vitamin d and decreased risk of relapses has also been reported. 86 finally, some authors provide data showing that vitamin d supplementation may be an effective treatment for ms since high-dose vitamin d treatment in ms tends to decrease relapses. 87 the possible sequence of events linking sunlight exposure with ms is most likely based on the conversion, due to ultraviolet b radiation (290-320 nm), of cutaneous 7-dehydrocholesterol to previtamin d 3 , which then spontaneously gives origin to vitamin d 3 . 88 the latter then undergoes two hydroxylations, by d-25-hydroxylase (cyp2r1) in the liver and ii. other patho-mechanisms 25-hydroxyvitamin d-1α-hydroxylase (cyp27b1) in the kidney, to produce the biologically active form of vitamin d, 1,25-dihydroxyvitamin d 3 . variants of the cyp27b1 gene have been reported to be associated with increased risk of ms 89 and others have confirmed the association of ms with two vitamin d-related genes, cyp27b1 and cyp24a1, 20 while a vitamin d response element lies close to the promoter region of hla-drb1, the main risk allele for ms. 90 different mechanisms of action of vitamin d that may impact different steps of the disease immunopathogenesis have been reported. vitamin d either suppresses the maturation and activity of apcs, including dendritic cells, or increases their tolerogenic phenotype. 91 cd4 + t helper cells are also affected by vitamin d, with a reduced production of proinflammatory th1 and th17 cells 92 while that of th2 cells is increased. 93 vitamin d treatment induces treg activity 92 and reduces proinflammatory molecules produced by stimulated monocytes. 94 in eae, vitamin d has proved to be effective either given as preventive 95 or therapeutic treatment. 96 vitamin d can enter the cns to exert its immune-regulating properties while its possible neuroprotective role is more uncertain. certainly, the enzymes necessary to synthesize the bioactive 1,25-dihydroxyvitamin d 3 are present in the brain 97 and abnormal brain development has been observed in rats deficient in vitamin d during gestation. moreover, mice with gestational vitamin d deficiency have impaired learning in adulthood. 98 in vitro, vitamin d is able to reduce glutamate excitotoxicity to cortical, cerebellar, or hippocampal neurons. 99 whether such vitamin d neuroprotective experimental evidence is valid in human ms still remains to be elucidated. finally, it is quite evident that vitamin d may correct many of the immune abnormalities seen in ms, nevertheless which mechanisms are the most relevant to its therapeutic efficacy or whether such mechanisms include its actions within the cns are as yet unclear. some evidence also exists to suggest vitamin d might influence epigenetic mechanisms. 1,25-hydroxyvitamin d 3 has been reported to affect histone modification in cancer: studies in human colon cancer cells have shown that vitamin d induces the expression of jmjd3, the gene encoding lysine-specific demethylase 6b, which specifically demethylates lysine 27 of histone h3. 100, 101 as far as ms is concerned, the potential relevance of vitamin d-induced histone modification is suggested by a study showing that binding of 1,25-hydroxyvitamin d 3 to the vitamin d receptor leads to suppression of transcription of the proinflammatory cytokine il-17, via recruitment of histone deacetylase 2 to the il17a promoter region. 102 despite infection agents having long been investigated as possible triggers of autoimmunity in ms, their involvement still remains a matter of debate. studies have focused on the involvement of resident commensal microbiota in cns autoimmunity. 103 humans are colonized by a myriad of microbes, including bacteria, archaea, fungi, eukaryotes, and viruses both in mucosal surfaces and in the skin and are collectively termed microbiota. 104 such microbial organisms mostly belong to two large phyla, the bacteroidetes and the firmicutes. the microbiota may generally have beneficial functions to the host, but may influence the physiology and/or pathology of the host. 105 studies in eae have clarified that the microbial flora contributes to the cns-specific autoimmune disease. 106, 107 in fact, spontaneous eae incidence has been found to be strongly ii. other patho-mechanisms reduced in tcr transgenic mice kept in germ-free (gf) conditions and therefore not having resident microbes. 108 but, eae severity is also reduced in gf mice immunized with myelin peptide antigen in complete freund's adjuvant. 109 moreover, antibiotics have been found to affect disease severity by altering the gut flora. 110, 111 nevertheless, it remains unclear how and when these agents may become detrimental. since the microbiota has an impact on the host's immune system, 105 it is likely to shift the balance between protective and pathogenic immune responses. indeed, antibiotic-mediated protection from eae has been associated with a decreased production of the proinflammatory cytokine il-17 in the gut-associated lymphoid tissue, thus altering the function of invariant natural-killer t cells, 111 but also with an increase in the tregs. 110 cns-reactive immune cells can be activated by commensal microbiota either through molecular mimicry or through a bystander activation mechanism, as proposed for other infectious pathogens. however, so far no cns-mimicry epitope derived from gut bacteria has been identified, whereas the current data provide more evidence in favor of a bystander activation hypothesis. it is likely that the th17 cells generated in the gut are a result of bystander activation of apcs and that their secreted cytokines can drive naïve t cells toward proinflammatory phenotypes. nevertheless, it has been reported that specific commensal microbial species may induce either th-17 or tregs cells both in the intestine as well as at peripheral sites. 112, 113 so far, there is no clear evidence supporting the involvement of the gut microbiota either in the incidence or in the pathogenesis of ms; however, indirect data suggest a potential implication especially when considering dietary factors, which can rapidly alter gut microbial signatures. 114 at the time of writing the pathophysiological mechanisms that mediate the effects of environmental risk factors on susceptibility to ms or the course of this disease are still unknown. it is quite intriguing though, that the most important environmental risk factors for ms seem to be clearly associated with changes in epigenetic profiles and more research is certainly required to establish whether epigenetic mechanisms can truly mediate the effects of these risk factors. finally, the microbiota also deserves to be taken into consideration as an external factor favoring the disease, given the relevant implications it has in controlling the host's immune system. environmental risk factors for multiple sclerosis. part ii: 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onset of disease and severe cortical pathology humoral autoimmunity in multiple sclerosis b cells in multiple sclerosis: connecting the dots mmps in the central nervous system: where the good guys go bad a reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis risks of multiple sclerosis in relatives of patients in flanders age-adjusted recurrence risks for relatives of patients with multiple sclerosis genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals epigenetic transgenerational actions of environmental factors in disease etiology epigenetic changes in patients with multiple sclerosis eukaryotic cytosine methyltransferases dna methyltransferases dnmt3a and dnmt3b are essential for de novo methylation and mammalian development genomic dna methylation: the mark and its mediators genomic patterns of dna methylation: targets and function of an epigenetic mark epigenetic histone code and autoimmunity epigenetics and autoimmunity rna meets chromatin micrornas in cell proliferation, cell death, and tumorigenesis mammalian micrornas: a small world for fine-tuning gene expression micrornas in vertebrate physiology and human disease regulatory mechanisms of micrornas involvement in cancer the natural history of primary progressive multiple sclerosis microrna profiling of multiple sclerosis lesions identifies modulators of the regulatory protein cd47 t cell activation induces a noncoding rna transcript sensitive to inhibition by immunosuppressant drugs and encoded by the proto-oncogene regulation of the germinal center response by microrna-155 microrna-155 is a negative regulator of activation-induced cytidine deaminase papavasiliou fn shhh! silencing by microrna-155 microrna-155 promotes autoimmune inflammation by enhancing inflammatory t cell development silencing microrna-155 ameliorates experimental autoimmune encephalomyelitis microrna mir-326 regulates th-17 differentiation and is associated with the pathogenesis of multiple sclerosis micrornas mir-17 and mir-20a inhibit t cell activation genes and are under-expressed in ms whole blood epigenetic regulation of motor neuron cell death through dna methylation the role of citrullinated proteins suggests a novel mechanism in the pathogenesis of multiple sclerosis myelin in multiple sclerosis is developmentally immature peptidyl argininedeiminase 2 cpg island in multiple sclerosis white matter is hypomethylated changed histone acetylation patterns in normal-appearing white matter and early multiple sclerosis lesions environmental factors and their regulation of immunity in multiple sclerosis migrant studies in multiple sclerosis risk of multiple sclerosis related to age at immigration to israel multiple sclerosis: variation of incidence of onset over time in the faroe islands multiple sclerosis in the orkney and shetland islands. ii: the search for an exogenous aetiology incidence of multiple sclerosis in macomer, sardinia, 1912-1981: onset of the disease after 1950 viral triggers of multiple sclerosis molecular mimicry as an inducing trigger for cns autoimmune demyelinating disease myelin basic protein and human coronavirus 229e crossreactive t cells in multiple sclerosis cross-reactivity of autoreactive t cells with mbp and viral antigens in patients with ms a functional and structural basis for tcr cross-reactivity in multiple sclerosis epstein-barr virus latent infection and baff expression in b cells in the multiple sclerosis brain: implications for viral persistence and intrathecal b-cell activation combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross-react with myelin basic protein and ebv antigen hla-b7-restricted ebv-specific cd8 + t cells are dysregulated in multiple sclerosis cd8 + t-cell deficiency, epstein-barr virus infection, vitamin d deficiency, and steps to autoimmunity: a unifying hypothesis epigenetic dysregulation of the host cell genome in epstein-barr virusassociated neoplasia cigarette smoking and incidence of multiple sclerosis smoking is a risk factor for early conversion to clinically definite multiple sclerosis smoking is associated with increased lesion volumes and brain atrophy in multiple sclerosis smoking and disease progression in multiple sclerosis cigarette smoking and progression in multiple sclerosis cigarette smoking and the progression of multiple sclerosis smoking is associated with progressive disease course and increased progression in clinical disability in a prospective cohort of people with multiple sclerosis impact of smoking on inflammation: overview of molecular mechanisms smoking and two human leukocyte antigen genes interact to increase the risk for multiple sclerosis control of t(reg) and t(h)17 cell differentiation by the aryl hydrocarbon receptor smoking and matrixmetalloproteinases, neutrophil elastase and myeloperoxidase in chronic periodontitis cigarette smoking behaviors and time since quitting are associated with differential dna methylation across the human genome smoking induces differential mirna expression in human spermatozoa: a potential transgenerational epigenetic concern? cigarette smoking reduces histone deacetylase 2 expression, enhances cytokine expression, and inhibits glucocorticoid actions in alveolar macrophages latitude is significantly associated with the prevalence of multiple sclerosis: a meta-analysis geographical variations in sex ratio trends over time in multiple sclerosis vitamin d and multiple sclerosis serum 25-hydroxyvitamin d levels and risk of multiple sclerosis a quantitative analysis of suspected environmental causes of ms association of vitamin d metabolite levels with relapse rate and disability in multiple sclerosis higher 25-hydroxyvitamin d is associated with lower relapse risk in multiple sclerosis a phase i/ii dose-escalation trial of vitamin d3 and calcium in multiple sclerosis modulation of the immune system by uvradiation: more than just the effects of vitamin d? rare variants in the cyp27b1 gene are associated with multiple sclerosis expression of the multiple sclerosis-associated mhc class ii allele hla-drb1*1501 is regulated by vitamin d 1,25-dihydroxyvitamin d3 is an autonomous regulator of the transcriptional changes leading to a tolerogenic dendritic cell phenotype immunomodulatory effects of vitamin d in multiple sclerosis predominance of th2 polarization by vitamin d through a stat6-dependent mechanism 25-dihydroxyvitamin d3 inhibits cd40l-induced pro-inflammatory and immunomodulatory activity in human monocytes 1,25-dihydroxyvitamin d3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis 1,25-dihydroxyvitamin d3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis vitamin d in the healthy and inflamed central nervous system: access and function developmental vitamin d deficiency alters learning in c57bl/6j mice vitamin d hormone confers neuroprotection in parallel with downregulation of l-type calcium channel expression in hippocampal neurons vitamin d has wide regulatory effects on histone demethylase genes kdm6b/jmjd3 histone demethylase is induced by vitamin d and modulates its effects in colon cancer cells 1,25-dihydroxyvitamin d3 ameliorates th17 autoimmunity via transcriptional modulation of interleukin-17a microbial view of central nervous system autoimmunity the human microbiome project the immune system and the gut microbiota: friends or foes? remote control-triggering of brain autoimmune disease in the gut commensal gut flora and brain autoimmunity: a love or hate affair? acta neuropathol commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelination proinflammatory t-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis role of gut commensal microflora in the development of experimental autoimmune encephalomyelitis nkt cell-dependent amelioration of a mouse model of multiple sclerosis by altering gut flora induction of colonic regulatory t cells by indigenous clostridium species induction of intestinal th17 cells by segmented filamentous bacteria diet rapidly and reproducibly alters the human gut microbiome key: cord-349135-it5ahzj3 authors: lane, t. e.; hardison, j. l.; walsh, k. b. title: functional diversity of chemokines and chemokine receptors in response to viral infection of the central nervous system date: 2006 journal: chemokines and viral infection doi: 10.1007/978-3-540-33397-5_1 sha: doc_id: 349135 cord_uid: it5ahzj3 encounters with neurotropic viruses result in varied outcomes ranging from encephalitis, paralytic poliomyelitis or other serious consequences to relatively benign infection. one of the principal factors that control the outcome of infection is the localized tissue response and subsequent immune response directed against the invading toxic agent. it is the role of the immune system to contain and control the spread of virus infection in the central nervous system (cns), and paradoxically, this response may also be pathologic. chemokines are potent proinflammatory molecules whose expression within virally infected tissues is often associated with protection and/or pathology which correlates with migration and accumulation of immune cells. indeed, studies with a neurotropic murine coronavirus, mouse hepatitis virus (mhv), have provided important insight into the functional roles of chemokines and chemokine receptors in participating in various aspects of host defense as well as disease development within the cns. this chapter will highlight recent discoveries that have provided insight into the diverse biologic roles of chemokines and their receptors in coordinating immune responses following viral infection of the cns. coronaviruses are classified on the basis of several fundamental characteristics, including nucleic acid type, a lipid envelope, and their distinctive morphology [42, 64, 79] . all members have characteristic petal-shaped proteins extending from the virion surface. coronaviruses infect numerous vertebrate hosts including humans, chickens, pigs, and mice, causing a wide variety of disorders involving a number of different organ systems; however, there are specific tropisms for the cns, lungs, gastrointestinal tract, and liver [42, 64, 79] . receptor use among the varied coronaviruses is restricted to several well-defined proteins. human coronavirus infections result in acute enteritis as well as 15% of common colds indistinguishable from those caused by other viruses [42, 64, 79] . more recently, a human coronavirus has been indicated to be the etiologic agent for severe acute respiratory syndrome (sars). sars is a potentially lethal disease and is recognized as a health threat internationally [43] . the first murine coronavirus strain (mouse hepatitis virus, mhv), was isolated in 1949 [12] . mhv is a pathogen of wild mice, and natural infection is due to horizontal transmission, resulting in acute hepatitis with death in young animals and a variable course of persistent gastrointestinal tract infection in adults [79] . mhv is not an endemic mouse virus, but infects mouse colonies sporadically. it is very closely related to some human coronaviruses both at the genomic and protein levels. for example, human sera often contain antibody reactive to mhv. therefore, characterizing the immune response to murine coronaviruses may provide important insight to mechanisms of control and elimination which may have important implications with regards to understanding the immune response to human coronaviruses such as the sars coronavirus. coronavirus genomes are single-stranded positive-polarity rna molecules, larger than the size of any other known stable rna, ranging from 27 kb for the avian infectious bronchitis virus, to 31 kb for murine coronaviruses [50] . genomic rna is infectious, contains a cap structure at the 5 -end and poly(a) at the 3 -end. the genome is organized into seven or eight genes, each containing one or more open reading frames (orf) separated by intergenic sequences that contain the signals for the initiation of transcription of the subgenomic viral messenger (m)rna species. upon entry, the viral rna encodes an rna polymerase that transcribes the genome into a negative-stranded rna [50] . the latter serves as templates for positivesensed genomic rna and subgenomic mrnas. important viral structural proteins include the envelope glycoproteins (s) that bind to receptors on cell membranes [42, 64, 79] . analysis of monoclonal antibody neutralization escape variants demonstrated that the viral s protein controls cellular tropism in vivo and the role of the s protein in tropism has recently been confirmed using stable recombinant viruses in which all genes except the s protein gene were held constant [9, 82] . the protective immune response to mhv infection is characterized predominantly by cell-mediated immunity during acute infection. a number of unique aspects of cns viral infection have been described by analysis of the interactions between mhv and the immune response. antibody, although protective if administered prior to infection, is not present in the serum of infected mice until after the vast majority of virus has been cleared from the cns [56, 84] . following infection, neutrophils, macrophages, and nk cells are rapidly recruited into the cns, followed by t cells and b cells [104] . inflammation is accompanied by a progressive loss of blood-brain barrier (bbb) integrity that is apparent as early as 4 days post-infection. the initial influx of innate effectors is important in facilitating t cell infiltration, as well as regulating viral replication [104] . however, the ability to survive mhv infection appears to be predominantly due to an effective t cell-mediated response [103] . recent data have confirmed that cell-mediated immunity is critical during acute infection [53, 55, 74, 76, 92] ; however, the ability to prevent viral recrudescence is associated with the continued presence of plasma cells in the cns secreting neutralizing antibody [56, 84] . the major effectors of anti-viral immunity are virus-specific cd8 + t cells. cytotoxic t lymphocyte (ctl) induction following mhv infection of the cns has been shown to require cd4 + t cell help [92] . although the pre-cise mechanism or mechanisms by which cd4 + t cells assist cd8 + t cells have yet to be completely determined, recent studies have demonstrated that cd4 + t cells are important in preventing apoptosis of ctl entering the cns parenchyma [92] . in addition, the quality of the ctl response is cd4 + t cell-dependent [92] . an important concept derived from analysis of mhv infection is that although cd8 + t cells are the most prominent effectors for viral clearance during the acute infection, the mechanisms which control virus replication differ with the type of cns cell infected. cytolysis is important for the control of viral replication in microglia/macrophages and astrocytes while interferon (ifn)-γ is the critical effector responsible for control of virus replication in oligodendroglia [73] . the demonstration that cd8 + ctl suppresses viral replication by two separate effector mechanisms, which function within the cns in a cell type-specific manner, is an important new concept. viral persistence in white matter tracts results in a chronic demyelinating disease in which foci of demyelination are associated with areas of viral rna/antigen [51] . clinically, mice develop loss of tail tone and a partial to complete hind-limb paralysis. as a result of the clinical and histologic similarities between mhv-induced demyelination and the human demyelinating disease multiple sclerosis (ms), the mhv system is considered a relevant model for studying the underlying immunopathologic mechanisms contributing to immune-mediated demyelinating diseases [51] . a variety of different mechanisms have been postulated to contribute to mhv-induced demyelination. several studies suggest that mhv-induced demyelination involves immunopathologic responses against viral antigens expressed in infected tissues [30, 31, 37, 47] . although virus-specific antibody is considered important in suppressing viral recrudescence [84, 85] , it may also have a role in promoting demyelination [48] . mhv infection of immunosuppressed or immunodeficient mice results in high titers of virus within the cns and death but not robust demyelination [53, 105] . adoptive transfer of mhv-immune splenocytes results in demyelination to the infected recipients, suggesting a role for immune cells in amplifying demyelination [30, 31] . additional evidence for t cells in contributing to demyelination is provided by wu et al. [105] who demonstrated that both cd4 + and cd8 + t cells are important in mediating myelin destruction. in support of this are studies derived from our laboratory demonstrating that adoptive transfer of mhv-specific cd4 + or cd8 + t cells to mhv-infected rag1 −/− mice results in demyelination [30, 31] . however, demyelination was more severe in recipients of cd4 + t cell compared to cd8 + t cell recipients, and this supports a more important role for cd4 + t cells in amplifying demyelination in this model. indeed, we have demonstrated that mhv-infected cd4 −/− mice displayed a significant reduction in the severity of demyelination compared to cd8 −/− and immunocompetent wildtype mice, suggesting an important role for cd4 + t cells in amplifying the severity of white matter destruction [53] . while t cells are generally considered important in driving demyelination in mice persistently infected with mhv, the mechanisms by which these cells participate in disease may vary and depend upon various factors including the ability to secrete interferon (ifn)-γ [80, 81] . while conventional cd4 and cd8 αβ t cells are generally viewed as the primary t cell type important in disease, γδ t cells have also been shown to participate in demyelination in mhv-infected athymic mice [16] . in addition, we and others have found that macrophages/microglia are also important in contributing to demyelination [29, 32, 53, 59, 105] . the collective evidence points to a role for inflammatory t cells in contributing to macrophage/microglial infiltration and activation which ultimately results in myelin destruction. current evidence suggests that demyelination in mhv-infected mice is not the result of epitope spreading and induction of an immune response against neuroantigens as has recently been reported to occur during theiler's virus-induced demyelination [69] . however, adoptive transfer of t cells from mhv-infected rats to naïve recipient's results in demyelination [100] . whether a similar response occurs in mhv-infected mice and what the contributions are to demyelination is not clear at this time. chemokines represent a family of low molecular weight (7-17 kda) proinflammatory cytokines that are divided into four subfamilies based on structural and functional criteria [14, 60, 94] . the two major subfamilies are the cxc and cc chemokines. the cxc subfamily is structurally characterized by two conserved cysteine residues that are separated by an amino acid, while the cc subfamily is structurally characterized by conserved cysteine residues adjacent to one another. lymphotactin, the sole member of the c family, is chemotactic for t cells [44] . the cx 3 c chemokine, fractalkine, is unique in that it is expressed on the surface of cells as well as being secreted into the surrounding environment [5] . chemokines have been shown to selectively attract distinct leukocyte populations during periods of inflammation in various disease models. the cxc chemokines function primarily in attracting neutrophils, yet have a limited effect on t lymphocytes and monocytes [14, 60, 94] . however, there are exceptions to this rule in that cxc chemokines that lack the glutamic acid-leucine-arginine (elr) motif on the amino terminus are chemotactic for t cells. for example, the non-elr chemokine cxcl10 is a potent chemoattractant for activated t cells and nk cells and functions by binding to cxcr3 expressed on the surface of these cells [40, 83, 102, 106] . however, cxcl10 does not exert a chemotactic effect on neutrophils [19] . the cc chemokines are thought to attract t cells, monocytes, and macrophages, but not neutrophils [14, 60, 94] . the cc chemokine ligand 5 (ccl5) is able to attract both t cells and macrophages by binding to one of several cc chemokine receptors including ccr1 and ccr5 [14, 60, 94] . furthermore, there is increasing evidence that chemokines, such as ccl3, influence other immune system activities including t h 1/t h 2 development and t cell proliferation [46, 95] . chemokines function by binding to seven-transmembranespanning g protein-coupled receptors. the chemokine receptors are divided into those that preferentially bind cxc and cc chemokines. in addition, cc and cxc chemokine receptors are capable of binding more than one cc or cxc chemokine, respectively. a variety of cell types including lymphocytes and macrophages, as well as resident cells of the cns such as neurons, astrocytes, and microglia, express chemokine receptors [60, 94] . instillation of mhv into the cns of susceptible mice results in a wellorchestrated expression of chemokine genes, and the expression pattern correlates with the level of inflammation and disease [52] . early (~1-3 days) following infection, transcripts for cxcl10 and ccl3 are detected within the cns, suggesting an important role in initiation of immune responses (see following section; table 1 ). by day 6 post-infection (p.i.), virus has spread throughout the brain parenchyma, and a robust inflammatory response, characterized primarily by cd4 + and cd8 + t cells and macrophages, is established within the brain. chemokines expressed at this time include cxcl9, cxcl10, ccl2, ccl3, ccl4, ccl5, and ccl7 (mip-2) ( table 1) . analysis of chemokine receptor expression by both rnase protection assay (rpa), immunostaining, and flow cytometry reveals that ccr1, ccr2, ccr5, and cxcr3 are the prominent receptors expressed within the cns at various stages of disease ( table 2) . chemokine transcripts are detected almost exclusively in areas in which virus is present, indicating a localized response to infection and subsequent spread of the virus throughout the parenchyma. in situ hybridization indicates that astrocytes are the primary cellular source for many chemokines during the acute stage of disease [52] . infection of primary cultures of mouse astrocytes with mhv and evaluating chemokine gene expression by rpa provide additional support for astrocytes as an important cellular source of chemokines in this model [52] . moreover, viral replication appears to be a necessary prerequisite for inducing chemokine expression, as infection of astrocytes with inactivated virus results in a muted chemokine expression profile. additional analysis revealed that both infected and noninfected astrocytes are capable of secreting chemokines following instillation of virus into the brain, indicating that viral infection is not required for chemokine gene synthesis by target cells. these data indicate that a factor or factors (possibly type i interferons) derived from infected cells are capable of functioning in both an autocrine and paracrine manner and regulate chemokine gene expression in response to viral infection. other cell types that may also secrete chemokines following mhv infection include resident microglia/inflammatory macrophages as well as neurons [52, 75] . by day 12 p.i., mhv-infected mice that have survived the acute stage of disease develop an immune-mediated demyelinating disease. mice have cleared infectious virus (as determined by plaque assay) by 12 days, yet viral rna and protein can be detected within white matter tracts for months after infection. as the level of cns infiltration subsides following reduction of viral burden there is a corollary reduction in the expression of chemokine transcripts. analysis of chemokine message expression within the brains and spinal cords of mhv-infected mice during the demyelinating phase of disease (days 12 and onward) indicates that cxcl10 and ccl5 are the two prominent chemokines expressed [52] . in situ hybridization for chemokine transcripts indicated expression was limited primarily to areas of viral persistence within white matter tracts undergoing active demyelination [52] . similar to what was found during acute disease, astrocytes were determined to be the cellular source of cxcl10 at this stage of disease whereas inflammatory cells, presumably cd4 + t lymphocytes, expressed ccl5. more recent data now indicate that mhv-infected astrocytes treated with ifn-γ can also express ccl5 mrna transcripts and protein (t.e. lane, unpublished observations). chemokine receptors expressed during chronic demyelination include cxcr3 and ccr5, which are capable of binding cxcl10 and ccl5, respectively. indeed, we have recently determined that the majority (~90%) of infiltrating virus-specific cd4 + and cd8 + t cells express cxcr3 (t.e. lane, unpublished observations). the presence of dendritic cells (dcs) within the cns has been debated for quite some time. however, a series of recent studies clearly indicates that during induction of an autoimmune demyelinating disease, there exists the presence of cell types within the brain that clearly have characteristics of dcs [34, 65] . in addition, emerging evidence points to a previously unappreciated role for chemokines in activating and inducing the migration of differing populations of dcs in response to microbial infection of the cns [22, 23] . these cells may be important in initiation and/or maintenance of disease by participating in the activation of t cells. given the potential importance of this population of cells with regards to linking innate and adaptive immune responses following viral infection of the cns, we investigated whether dc-like cells were present within the cns in response to mhv infection. in brief, our findings clearly indicate that a dc-like population of cells is detectable within the cns as early as day 2 p.i. with mhv [96] . the activation/maturation of these cells as well as the ability to accumulate within the draining cervical lymph node (cln) appeared to be dictated by localized expression of ccl3 [96] . moreover, the ability of cultured dcs to secrete cytokines associated with the development of a t h 1 response such as interleukin (il)-12 was profoundly altered in the absence of ccl3 [96] . the importance of ccl3 signaling and the evolution of an effective t cell response was further confirmed by the demonstration that in the absence of ccl3 signaling, robust anti-viral effector responses, e.g., cytokine production and ctl activity, were dramatically compromised following mhv infection of ccl3 −/− mice [95, 96] . collectively, these studies highlight a previously unappreciated role for the importance of chemokine signaling and dc maturation/activation following mhv infection of the cns. moreover, these studies demonstrate that generation of effective t cell responses relies upon ccl3 signaling to successfully combat mhv infection. ccl3 is a chemoattractant for both t cells and macrophages and has been implicated in host defense following infection with a wide variety of microbial pathogens. mice deficient in ccl3 production exhibit increased susceptibility to disease following infection with paramyxovirus [17] , influenza virus [15] , and coxsackievirus, as well as other microbial pathogens [67, 72] . in all cases, alterations in an effective host response correlated with a paucity in leukocyte accumulation at sites of infection. although originally thought to participate in defense by attracting effector cells to infected tissue, recent reports also suggest that ccl3 expression is important in coordinating a t h 1 response [46] . numerous studies now indicate that dcs are capable of expressing various chemokines including ccl3 [21, 66, 77, 78] . moreover, dc precursors express the ccl3 receptors ccr1 and ccr5 and are capable of responding to ccl3 in vivo and in vitro resulting in both mobilization and maturation [24, 108] . indeed, flesch and colleagues have demonstrated an important role for ccl3 in dc-dependent priming of ctl to viral antigens [24] . using ccl3 −/− mice, we have demonstrated a role for ccl3 in regulating trafficking as well as antiviral effector functions following mhv infection of the cns [95] . specifically, our experiments revealed an important role for ccl3 signaling in tailoring t cell responses that allowed for egress out of draining cervical lymph nodes and trafficking into the cns. although generation of antigen-specific cd8 + t cells was not impaired following mhv infection of ccl3 −/− mice, a significant percentage of cd8 + t cells retained expression of lymph-node homing receptors cd62l (l-selectin) and the cc chemokine receptor 7 (ccr7) and did not display a dramatic increase in mrna transcripts for either cxcr3 or ccr5, two receptors which are important in allowing mhv-specific t cells access to the cns [95] . moreover, adoptive transfer of ccl3 −/− cd8 + t cells into mhv-infected rag1 −/− mice (which express ccl3 following mhv infection) resulted in homing back to secondary lymphoid organs, suggesting that lack of ccl3 imprinted on these cells carries an inability to remodulate surface tissue homing receptors. analysis of antiviral effector functions also revealed that ccl3 −/− cd8 + t cells displayed overall muted cytolytic activity as well as expression of ifn-γ when compared to ccl3 +/+ cd8 + t cells [95] . collectively, these studies highlight that, in addition to chemotactic function, chemokines influence specific lymphocyte responses and ultimately effector functions that are required for optimal host defense against microbial pathogens. cxcl9 and cxcl10 attract activated t lymphocytes following binding to cxcr3. analysis of cxcl9 and cxcl10 mrna expression within the cns of mhv-infected mice revealed that cxcl10 was clearly detectable by day 1 p.i. and was prominently expressed at days 7, 12, and 35 p.i. [52] . in contrast, cxcl9 transcripts were only detected at days 7 and 12 p.i. [58] . these data suggested that both cxcl9 and cxcl10 might be important in host defense by attracting antiviral t lymphocytes into the cns. in support of this is the observation that administration of neutralizing antibodies specific for either cxcl9 or cxcl10 to mhv-infected mice during the acute stage of disease results in a dramatic increase in mortality [57, 58] . additionally, this treatment also resulted in a significant decrease in numbers of cd4 + and cd8 + t lymphocyte infiltrating into the cns which correlated with decreased expression of ifn-γ and increased levels of virus [57, 58] . mhv infection of cxcl10 −/− mice supported and extended our previous work on antibodymediated neutralization of cxcl10 in that mhv-infected cxcl10 −/− mice display reduced t cell infiltration into the cns accompanied by reduced ifn-γ secretion and increased viral burden [18] . therefore, the collective evidence points to pivotal roles for both cxcl9 and cxcl10 as important sentinel molecules in promoting a protective response following mhv infection of the cns by attracting t cells into the cns that participate in elimination of virus. ccl5 is a t cell and macrophage chemoattractant that has been shown to influence leukocyte migration during periods of inflammation. upon mhv infection of the cns of mice, ccl5 transcripts and protein are readily detected within the brain [52] . initial studies in which cd4 −/− or cd8 −/− mice were infected with mhv indicated an overall reduction in ccl5 mrna transcripts within the brains of cd4 −/− mice, suggesting that cd4 + t cells were either a primary cellular source for ccl5 and/or influenced the expression of ccl5 by resident and inflammatory cells [53] . we now know that both inflammatory cd4 + t cells as well as astrocytes are capable of expressing ccl5 following instillation of mhv into the cns [32, 53] . furthermore, treatment with neutralizing anti-ccl5 antisera results in diminished t cell and macrophage accumulation within the cns, suggesting that in this model ccl5 is capable of regulating trafficking of these two populations of cells [32] . ccr5 is a member of the cc chemokine receptor family that is expressed on various hematopoietic cells including lymphocytes and macrophages [86] . chemokines that are capable of binding to ccr5 include ccl3, ccl4, and ccl5 [7, 68, 86] . recent studies have clearly indicated that ccr5 expression correlates with leukocyte trafficking to sites of inflammation as well as regulating the immune response following microbial infection. for example, mice deficient in ccr5 (ccr5 −/− ) exhibit altered t cell activity and impaired macrophage function [88, 109] . furthermore, macrophage trafficking in response to antigen is impaired in ccr5 −/− mice, indicating that ccr5 is required for migration of this population of cells [45] . given that both t cells and macrophages express ccr5 following mhv infection of the cns and these cells clearly influence outcome in response to infection, we have defined the contributions of ccr5 to both host defense and disease in response to mhv infection. using an adoptive transfer model in which virus-expanded t cells are transferred into mhv-infected rag1 −/− mice, we have been able to examine how ccr5 expression influences trafficking of t cells into the cns. transfer of ccr5 +/+ -derived cd4 + t cells to mhv-infected rag1 −/− mice resulted in cd4 + t cell entry into the cns and a reduction in viral titers within the brain [30] . these mice also displayed robust demyelination correlating with macrophage accumulation within the cns. conversely, cd4 + t cells from ccr5 −/− mice displayed an impaired ability to traffic into the cns of mhv-infected rag1 −/− recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. analysis of chemokine receptor mrna expression by m133-147-expanded ccr5 −/− -derived cd4 + t cells revealed reduced expression of ccr1, ccr2, and cxcr3, indicating that ccr5 signaling is important in increased expression of these receptors which aid in trafficking of cd4 + t cells into the cns. collectively these results demonstrate that ccr5 signaling is important to migration of cd4 + t cells to the cns following mhv infection. with regards to the role of ccr5 in cd8 + t cell trafficking, comparable numbers of virus-specific cd8 + t cells derived from immunized ccr5 +/+ or ccr5 −/− mice were present within the cns of mhv-infected rag1 −/− mice following adoptive transfer, indicating that ccr5 is not required for trafficking of these cells into the cns [30] . rag1 −/− recipients of ccr5 −/−derived cd8 + t cells exhibited a modest yet significant (p≤0.05) reduction in viral burden within the brain that correlated with increased cytolytic activity and ifn-γ expression. histologic analysis of rag1 −/− recipients of either ccr5 +/+ or ccr5 −/− -derived cd8 + t cells revealed only focal areas of demyelination with no significant differences in white matter destruction. these data indicate that ccr5 signaling on virus-specific cd8 + t cells modulates antiviral activities but is not essential for entry into the cns. finally, mhv infection of ccr5 −/− mice resulted in a dramatic reduction in macrophage (defined as cd45 high f4/80 + dual-positive cells) accumulation within the brains, and this correlated with a significant reduction in the severity of demyelination compared to ccr5 +/+ mice. collectively, these data suggest that ligand binding, e.g., ccl5 and/or ccl3, and signaling via ccr5 results in macrophage migration and infiltration into the cns. however, we have previously demonstrated that ccl3 is expressed only at low levels during acute disease and is not detectable during chronic demyelination, whereas robust expression of ccl5 is detected during both phases of disease, and this suggests that ccl5 is the primary ccr5 signaling chemokine in this model. this is supported by earlier studies that showed an important role for ccl5 in attracting macrophages into the cns following mhv infection [53] . therefore, the data presented in this study suggest that one mechanism by which ccl5 contributes to demyelination is via attracting macrophages into the cns through ccr5-mediated signaling pathways. additional evidence supporting this is provided by the observation that even in the presence of increased ccl5 expression at day 12 p.i., demyelination is reduced in ccr5 −/− mice. ccl2 is capable of regulating the pathobiology of various inflammatory diseases including ms and atherosclerosis [1, 8, 28, 33, 35, 61] . in addition to its potent chemoattractant effect on monocytes and macrophages, ccl2 also influences t h 2 polarization in response to certain antigenic challenge [36, 41, 46, 99] . the influence of ccl2 on t cell polarization may be due to the fact that ccl2 is constitutively expressed within secondary lymphoid tissue and would be capable of affecting cellular responses following exposure to antigen [36] . thus, available evidence indicates that expression of ccl2 is capable of influencing both innate as well as adaptive immune responses by regulating monocyte and t cell responses, respectively. analysis of chemokine receptor expression following mhv infection reveals that ccr2 is expressed by endogenous cells of the cns as well as by inflammatory t cells and macrophages, indicating a role for these receptors in regulating both the immune response and disease development [13, 31] . indeed, mhv-infection of ccr2 −/− mice resulted in a dramatic increase in mortality and enhanced viral recovery from the brain that correlated with reduced t cell and macrophage entry into the cns compared to viral infection of ccr2 +/+ mice [13] . mhv infection of ccl2 −/− mice does not result in a similar disease phenotype as observed in ccr2 −/− mice. this was somewhat surprising as ccr2 is currently the only known functional receptor for ccl2. specifically, ccl2 −/− mice were able to clear virus from the brain in a similar time frame as wildtype mice, and this correlated with the ability to generate antigen-specific t cells [39] . the deficiency in ccr2 −/− mice to clear virus from the brain is not the result of an inherent inability to generate an effective adaptive immune response to virus, as ccr2 −/− mice had a similar frequency of antigenpresenting cells (apc) and virus-specific t cells present within draining cln compared to either ccl2 −/− or wildtype mice. our findings from mhv infection of ccl2 −/− mice indicated that while ccl2 does influence leukocyte migration into the cns in response to viral infection, ccr2 is clearly more influential in directing t cell trafficking into the cns. in support of the role for ccl2 in promoting leukocyte migration into the cns of mhv-infected mice are recent studies by perlman and colleagues demonstrating that localized ccl2 expression within the cns promotes macrophage infiltration [47] . these data highlight the possibility that ligand(s) other than ccl2 are important in signaling through the ccr2 receptor. alternatively, it is possible that ccr2 signaling by either endothelial cells and/or astrocytes regulates the permeability of the bbb, as recently suggested by stamatovic and colleagues [91] . expression of chemokines has been associated with demyelinating plaque lesions present in ms patients [3, 4, 26, 27] . elevated levels of chemokines, notably cxcl10, were found in the cerebral spinal fluid (csf) of ms patients during periods of clinical attack [25, 89] . indeed, the concentration of cxcl10 within the csf of ms patients correlated with numbers of inflammatory cells and the severity of clinical disease [2, 89, 90] . moreover, when cxcl10 levels decreased, there was a corresponding decrease in inflammation and disease severity [89] . astrocyte expression of cxcl10 has been reported in active plaque lesions present in ms patients, and the majority of t cells infiltrating into the cns of ms patients express the cxcl10 receptor, cxcr3. collectively, these studies highlight a potentially important role for cxcl10 in the pathogenesis of demyelinating diseases such as ms by attracting cxcr3-expressing t cells into the cns and support targeting chemokines and their receptors for therapeutic intervention in the treatment of ms [10, 54, 70, 90] . studies from animal models of ms support this notion by demonstrating that blocking of cxcl10 often results in diminished disease severity accompanied by a marked reduction in neuroinflammation. for example, several recent reports indicate that treatment with anti-cxcl10 neutralizing antibodies resulted in delayed disease onset and diminished neuroinflammation in mice with the autoimmune demyelinating disease experimental autoimmune encephalomyelitis (eae) [20] . these studies support the idea that localized expression of cxcl10 within the cns amplifies disease severity by attracting cxcr3-expressing t cells into the cns. once present, these cells enhance neuroinflammation by secreting additional chemokines as well as cytokines that can activate resident glia cells. importantly, these studies also implicate cxcl10 as a potential therapeutic target and suggest that alternative cxcr3 ligands, e.g., cxcl9 and cxcl11, do not exert a prominent effect on t cell infiltration into the cns. however, the role of cxcl10 in contributing to neurologic disease in eae has been questioned by results indicating that cxcl10 may actually exert a protective effect in mice with eae [49, 71] . antibody-mediated neutralization following induction of eae in rats resulted in increased disease severity, and this was associated with smaller draining lymph nodes and increased numbers of cd4 + t cells infiltrating into the cns [71] . in addition, cxcl10 −/− mice exhibited increased clinical disease severity following immunization with myelin peptides, and this correlated with diminished lymph node sizes although t cell infiltration into the cns was not dramatically altered when compared to wildtype mice [49] . in these particular eae models in which mice are immunized peripherally with antigen, cxcl10 expression within secondary lymphoid tissue is considered important in dictating disease outcome by serving to retain lymphocytes and tailoring t cell responses. moreover, these findings highlight the different roles of cxcl10 in regulating cellular immune responses in different models of neuroinflammation and emphasize the need for a better understanding of how signaling by this chemokine regulates inflammation and disease. as indicated, we have determined that mhv infection of the cns results in an orchestrated expression of chemokine and chemokine receptor genes that are regulated, in large part, by the viral burden. similar to ms patients, cxcl10 is expressed primarily by astrocytes in areas undergoing demyelination, suggesting an important role in the pathogenesis of demyelination by attracting cxcr3-expressing t cells into the cns [52, 59] . indeed, our laboratory was the first to demonstrate that treatment of mice with established demyelination and paralysis with anti-cxcl10 neutralizing antibody resulted in a significant reduction in cd4 + -but not cd8 + -t cells present within the cns, and this correlated with improved motor skills and a reduction in the severity of demyelination [59] . moreover, the dramatic regain of movement in anti-cxcl10-treated mice corresponded with more than 80% of previously demyelinated axons undergoing remyelination, indicating that removal of cxcl10 promoted an environment capable of remyelination. in addition to reduced numbers of cd4 + t cells within the cns, there was a paucity of macrophage infiltration into the cns of anti-cxcl10treated mice that correlated with a dramatic reduction in the levels of the macrophage-chemoattractant ccl5. these data were consistent with previous studies indicating that cd4 + t cells were the major source for ccl5 in mhv-infected mice undergoing demyelination [53, 59] . the influence of cxcl10 in contributing to t cell responses was also examined. t cells isolated from secondary lymphoid tissue of mice treated with anti-cxcl10 displayed muted expression of ifn-γ in response to viral antigen when compared to t cells isolated from control mice, suggesting that cxcl10 also serves to influence t cell effector functions during chronic disease (t.e. lane, unpublished observations). we have previously determined that ccl5 mrna transcripts and protein are present within the cns of mhv-infected mice during chronic demyelination, indicating a potentially important role for this chemokine in promoting inflammation [52, 53] . in order to assess the functional role of ccl5 in participating in viral-induced immune-mediated demyelination, mhv-infected mice were treated via intraperitoneal (i.p.) injection with anti-ccl5 monoclonal antibody (mab) following onset of clinical disease and demyelination. such treatment resulted in a significant (p≤0.05) reduction in the severity of clinical disease compared to mice treated with an isotype (igg 1 )-matched antibody [32] . upon removal of anti-ccl5 treatment, clinical disease returned to mice such that there was no difference between the two experimental groups of mice. immunophenotyping the cellular infiltrate of mice treated with anti-ccl5 revealed reduced t cell and macrophage infiltration into the cns that is consistent with our earlier studies that ccl5 attracts these cells into the cns of mice with chronic demyelination. further, analysis of the severity of demyelination in experimental groups of mice indicated that anti-ccl5 treatment resulted in a significant (p<0.05) reduction in the severity of demyelination compared to control-treated mice. a picture is slowly evolving from our experiments designed to test the functional contributions of cxcl10 and ccl5 to chronic demyelination within mhv-infected mice. antibody targeting of the t cell chemoattractant cxcl10 in mhv-infected mice selectively affects cd4 + t cell accumulation within the cns accompanied by improved motor skills and a reduction in the severity of demyelination [59] . in contrast, ccl5 is capable of attracting both cd4 + and cd8 + t cells into the cns. it is also important to emphasize that our data on ccl5 and cxcl10 inhibition with regards to t cell and macrophage trafficking are corollary and it is possible that alternative scenarios exist. for example, studies by bergmann and colleagues suggest that during persistent mhv infection there is limited to no trafficking of t cells from the periphery into the cns. rather, upon entry during acute encephalomyelitis a certain percentage of cd4 + and cd8 + t cells is retained and participate in disease [62, 93] . in this instance, cxcl10 expression would not be functioning as a t cell chemoattractant but rather to influence specific biologic functions of t cells as well as potentiating the retention of t cells within the cns. in support of this, it is possible that cxcl10 serves to enhance cd4 + t cell proliferation, as several recent studies indicate that cxcl10 is important in contributing to t cell proliferation [18, 71, 101] . it is unlikely that cxcl10 contributes to t cell survival, as cxcl10 −/− mice do not display any abnormalities with regards to t cell half-life nor do we see any increase in numbers of apoptotic t cells following anti-cxcl10 treatment. in addition, narumi et al. [71] speculate that cxcl10 actually serves to retain cxcr3 + t cells within tissues and this influences disease severity. therefore, the selective reduction in cd4 + t cells within the cns of mhv-infected mice may not be the result of impaired trafficking. rather, either cd4 + t cells are not undergoing a steady-state turnover or are actually migrating out of the cns in the absence of signals specifying their retention. in addition, recent studies indicate an important role for cxcl10 in imparting effector functions to t cells. for example, salomon and colleagues demonstrated that anti-cxcl10 treatment improved joint swelling in a rodent model of arthritis and this correlated in part with an altered t h 1/t h 2 balance, suggesting that cxcl10 expression promotes and maintains a t h 1 state in t cells in this model [87] . similarly, we have shown that mhv-infection of cxcl10 −/− mice results in diminished ifn-γ expression by virus-specific t cells, supporting the idea that cxcl10 expression serves to maintain a t h 1-like state in t cells [18] (t.e. lane, unpublished observations). ccl5 signaling also modulates cytokine production by t cells following antigenic challenge. in support of this is our demonstration that inhibition of ccl5 signaling results in enhanced ifn-γ expression by virus-specific t cells, supporting the idea that ccl5 expression serves to regulate a t h 1-like state in t cells [32] . moreover, ablation of ccl5 signaling also modifies the cytolytic activity of mhv-specific cd8 + t cells [30] . this chapter highlights mechanisms by which chemokines participate in both host defense and disease progression in response to mhv infection of the cns. an overview of the potential functional role for select chemokines in linking innate and adaptive immune responses in response to viral infection of the cns is provided in fig. 1 . in brief, following mhv infection there is robust expression of chemokines by infected astrocytes including ccl3 that contribute to the maturation/activation of local dcs, which ultimately enables migration to draining cervical lymph nodes. activated dcs present antigen to t cells as well as secrete chemokines such as ccl3 and cxcl10 that enhance polarization to a t h 1 response. in turn, mhv-specific t cells express chemokine receptors including cxcr3 and ccr5 that enable them to traffic into the cns as a result of localized expression of ligands cxcl9 and cxcl10 (ligands for cxcr3) as well as ccl5 (ligand for ccr5). in addition, our contention is that expression of ccr2 by endothelial cells of the bbb is also important in increasing the permeability of this structure. with regards to chronic disease, mhv persistence within the cns results in chronic expression of cxcl10 and ccl5 which together contribute to the maintenance of a chronic inflammatory disease by attracting both t cells and macrophages (fig. 2) . local secretion of cxcl10 and ccl5 may also contribute to demyelination by enhancing specific t cell effector functions including (1) secretion of ifn-γ that activates local inflammatory macrophage and resident microglia, as well as directly damaging oligodendrocytes and (2) increasing ctl activity by cd8 + t cells. in addition, immature dc-like cells may also be susceptible to infection and secrete ccl3 (b) that functions in a paracrine and autocrine manner to bind to ccr1 expressed on immature dc-like cells. as a result of ccl3 signaling and mhv infection, the dc-like cells undergo maturation and activation (c) resulting in a remodulation of the plasma membrane characterized by decreased expression of ccr1 accompanied by increased expression of ccr7 as well as major histocompatibility complex (mhc) class i and ii. ccr7-expressing, activated dcs home to the draining cervical lymph node (d). upon entry, activated dcs express a variety of soluble factors including ccl3 and cxcl10 (e) that activate and enhance polarization of virus-specific t cells to a t h 1 phenotype (f). activated t cells exit the lymph node via the efferent lymph (g), enter the blood stream, and migrate to the cns via expression of the chemokine receptors cxcr3 and ccr5 (h) fig. 2a -f chemokines and mhv-induced demyelination. persistent mhv infection within astrocytes leads to chronic cxcl10 and ccl5 expression (a) that serves to recruit cxcr3 + and ccr5 + t cells into the cns (b). in addition, activated cd4 + t cells secrete ccl5 that enhances macrophage migration into the cns (c). we believe that cxcl10 may also influence t cell effector functions within the cns, including ctl activity (d) and ifn-γ secretion (e), leading to macrophage activation. both ifn-γ production and ctl activity may enhance tissue destruction as well as macrophage activation that amplifies myelin destruction (f) clearly, these observations indicate that chemokine signaling is an integral component involved in eliciting protective immunity in response to viral infection of the cns. conversely, our studies also indicate that chronic localized secretion of select chemokines ultimately amplifies disease severity through maintaining inflammation within the cns. importantly, studies derived from the mhv system demonstrate that antibody targeting of select chemokines offers a powerful approach towards delineating the functional contributions of these molecules in a model of immune-mediated demyelination. further, these studies highlight the relevancy of such an approach in treating human neuroinflammatory and demyelinating diseases such as ms. chemokines in pathology and medicine ccr5(+) and cxcr3(+) t cells are increased in multiple sclerosis and their ligands mip-1alpha and ip-10 are expressed in demyelinating brain lesions involvement of beta-chemokines in the development of inflammatory demyelination the levels of chemokines cxcl8, ccl2 and ccl5 in multiple sclerosis patients are linked to the activity of the disease a new class of membrane-bound chemokine with a cx3c motif natural killer cells in antiviral defense: function and regulation by innate cytokines molecular cloning and functional expression of murine je (monocyte chemoattractant protein 1) and murine macrophage inflammatory protein 1alpha receptors: evidence for two closely linked c-c chemokine receptors on chromosome 9 decreased lesion formation in −/− ccr2−/− mice reveals a role for chemokines in the initiation of atherosclerosis murine hepatitis virus-4 (strain jhm)-induced neurologic disease is modulated in vivo by monoclonal antibody chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases cd8+ t-cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin lack of ccr2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus structure-activity relationships of chemokines requirement of mip-1 alpha for an inflammatory response to viral infection virus-induced demyelination in nude mice is mediated by gamma delta t cells the chemokine macrophage-inflammatory protein-1 alpha and its receptor ccr1 control pulmonary inflammation and antiviral host defense in paramyxovirus infection ifngamma-inducible protein 10 (ip-10; cxcl10)-deficient mice reveal a role for ip-10 in effector t cell generation and trafficking mig and ip-10: cxc chemokines that target lymphocytes cxcl10 (ifn-gamma-inducible protein-10) control of encephalitogenic cd4+ t cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis rantes-induced chemokine cascade in dendritic cells phenotype and functions of brain dendritic cells emerging during chronic infection of mice with toxoplasma gondii brain dendritic cells and macrophages/microglia in central nervous system inflammation monocyte inflammatory protein-1 alpha facilitates priming of cd8(+) t cell responses to exogenous viral antigen serum and csf levels of mcp-1 and ip-10 in multiple sclerosis patients with acute and stable disease and undergoing immunomodulatory therapies rantes: a genetic risk marker for multiple sclerosis chemokine network in multiple sclerosis: role in pathogenesis and targeting for future treatments chemokines and disease reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor ccr5 following infection with a neurotropic coronavirus functional analysis of the cc chemokine receptor 5 (ccr5) on virus-specific cd8+ t cells following coronavirus infection of the central nervous system functional expression of chemokine receptor ccr5 on cd4(+) t cells during virus-induced central nervous system disease antibody targeting of the cc chemokine ligand 5 results in diminished leukocyte infiltration into the central nervous system and reduced neurologic disease in a viral model of multiple sclerosis mcp-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein b dendritic cells permit immune invasion of the cns in an animal model of multiple sclerosis monocyte chemoattractant protein-1 control of th2 polarization by the chemokine monocyte chemoattractant protein-1 high-magnitude, virus-specific cd4 tcell response in the central nervous system of coronavirus-infected mice bystander cd4 t cells do not mediate demyelination in mice infected with a neurotropic coronavirus differential roles of ccl2 and ccr2 in host defense to coronavirus infection blockade of cxcr3 receptor:ligand interactions reduces leukocyte recruitment to the lung and the severity of experimental idiopathic pneumonia syndrome monocyte chemoattractant protein-1 synthesis by murine lung fibroblasts modulates cd4+ t cell activation coronaviridae: the viruses and their replication sars-associated coronavirus the purification and characterization of a lymphokine chemotactic for lymphocytes-lymphotactin cutting edge: role of c-c chemokine receptor 5 in organ-specific and innate immunity to cryptococcus neoformans differential cc chemokine-induced enhancement of t helper cell cytokine production viral expression of ccl2 is sufficient to induce demyelination in −/− rag1−/− mice infected with a neurotropic coronavirus virus-specific antibody, in the absence of t cells, mediates demyelination in mice infected with a neurotropic coronavirus ifn-inducible protein 10/cxc chemokine ligand 10-independent induction of experimental autoimmune encephalomyelitis the molecular biology of coronaviruses murine coronavirus infection: a paradigm for virus-induced demyelinating disease dynamic regulation of alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease a central role for cd4(+) t cells and rantes in virus-induced central nervous system inflammation and demyelination cxcr3-binding chemokines: novel multifunctional therapeutic targets mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis antibody prevents virus reactivation within the central nervous system the t cell chemoattractant ifn-inducible protein 10 is essential in host defense against viral-induced neurologic disease expression of mig (monokine induced by interferon-gamma) is important in t lymphocyte recruitment and host defense following viral infection of the central nervous system neutralization of the chemokine cxcl10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis chemokines-chemotactic cytokines that mediate inflammation the role of mcp-1 (ccl2) and ccr2 in multiple sclerosis and experimental autoimmune encephalomyelitis (eae) role of viral persistence in retaining cd8(+) t cells within the central nervous system differences in systemic and central nervous system cellular immunity relevant to relapsingremitting multiple sclerosis diagnostic virology epitope spreading initiates in the cns in two mouse models of multiple sclerosis virally stimulated plasmacytoid dendritic cells produce chemokines and induce migration of t and nk cells macrophage inflammatory protein-1 alpha is a critical mediator of host defense against invasive pulmonary aspergillosis in neutropenic hosts cloning and characterization of a novel murine macrophage inflammatory protein-1 alpha receptor persistent infection with theiler's virus leads to cns autoimmunity via epitope spreading chemokines and chemokine receptors: potential therapeutic targets in multiple sclerosis neutralization of ifn-inducible protein 10/cxcl10 exacerbates experimental autoimmune encephalomyelitis the role of macrophage inflammatory protein-1 alpha/ccl3 in regulation of t cell-mediated immunity to cryptococcus neoformans infection ifn-gamma is required for viral clearance from central nervous system oligodendroglia contributions of fas-fas ligand interactions to the pathogenesis of mouse hepatitis virus in the central nervous system measles virus infection induces chemokine synthesis by neurons cytokine induction during t-cell-mediated clearance of mouse hepatitis virus from neurons in vivo cutting edge: differential chemokine production by myeloid and plasmacytoid dendritic cells differential migration behavior and chemokine production by myeloid and plasmacytoid dendritic cells coronaviruses: hepatitis, peritonitis and central nervous system disease cd4 t-cell-mediated demyelination is increased in the absence of gamma interferon in mice infected with mouse hepatitis virus cutting edge: cd8 t cell-mediated demyelination is ifngamma dependent in mice infected with a neurotropic coronavirus multiple regions of the murine coronavirus spike glycoprotein influence neurovirulence the chemokine receptors cxcr3 and ccr5 mark subsets of t cells associated with certain inflammatory reactions mechanisms of central nervous system viral persistence: the critical role of antibody and b cells control of central nervous system viral persistence by neutralizing antibody molecular cloning and functional characterization of a novel human cc chemokine receptor (ccr5) for rantes, mip-1beta, and mip-1alpha targeting the function of ifn-gamma-inducible protein 10 suppresses ongoing adjuvant arthritis defects in the generation of ifn-gamma are overcome to control infection with leishmania donovani in cc chemokine receptor (ccr) 5-, macrophage inflammatory protein-1 alpha-, or ccr2-deficient mice expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients multiple sclerosis: a study of cxcl10 and cxcr3 co-localization in the inflamed central nervous system monocyte chemoattractant protein-1 regulation of blood-brain barrier permeability ctl effector function within the central nervous system requires cd4+ t cells the art of survival during viral persistence chemokines, inflammation and the immune system cc chemokine ligand 3 (ccl3) regulates cd8(+)-t-cell effector function and migration following viral infection the cc chemokine ligand 3 regulates cd11c+cd11b+ cd8alpha-dendritic cell maturation and activation following viral infection of the central nervous system: implications for a role in t cell activation cxc chemokine ligand 10 controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells distinct roles for ip-10/cxcl10 in three animal models, theiler's virus infection, eae, and mhv infection, for multiple sclerosis: implication of differing roles for ip-10 effect of c-c chemokine receptor 2 (ccr2) knockout on type-2 (schistosomal antigen-elicited) pulmonary granuloma formation: analysis of cellular recruitment and cytokine responses adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis chemokine monokine induced by ifngamma/cxc chemokine ligand 9 stimulates t lymphocyte proliferation and effector cytokine production cxcr3 and its ligands participate in the host response to bordetella bronchiseptica infection of the mouse respiratory tract but are not required for clearance of bacteria from the lung effective clearance of mouse hepatitis virus from the central nervous system requires both cd4+ and cd8+ t cells characterization of brain-infiltrating mononuclear cells during infection with mouse hepatitis virus strain jhm cd4 and cd8 t cells have redundant but not identical roles in virus-induced demyelination antibody-mediated blockade of the cxcr3 chemokine receptor results in diminished recruitment of t helper 1 cells into sites of inflammation identification of a cd4+ t cell epitope within the m protein of a neurotropic coronavirus mobilization of dendritic cell precursors into the circulation by administration of mip-1alpha in mice impaired macrophage function and enhanced t cell-dependent immune response in mice lacking ccr5, the mouse homologue of the major hiv-1 coreceptor the authors wish to thank craig walsh for helpful discussion. key: cord-297325-fbilhauu authors: savarin, carine; bergmann, cornelia c.; gaignage, melanie; stohlman, stephen a. title: self-reactive cd4(+) t cells activated during viral-induced demyelination do not prevent clinical recovery date: 2015-11-11 journal: j neuroinflammation doi: 10.1186/s12974-015-0426-1 sha: doc_id: 297325 cord_uid: fbilhauu background: microbial infections have been implicated in initiating and enhancing severity of autoimmune diseases including the demyelinating disease multiple sclerosis (ms). nevertheless, the incidence of both acute and persisting viral infections without evidence of autoimmune sequelae suggests that this process is well controlled. the conditions promoting or stemming self-reactive (sr) t cells following viral-induced tissue damage thus need to be better defined. using a non-fatal viral mouse model of encephalomyelitis associated with demyelination and disability, yet ultimate clinical improvement, this study set out to monitor uptake and presentation of endogenous myelin antigens, as well as induction and fate of sr t cells. methods: activation and central nervous system (cns) recruitment of myelin-specific cd4 t cells was analyzed by flow cytometry during encephalomyelitis induced by a glia tropic murine coronavirus. potential antigen-presenting cells (apc) ingesting myelin were characterized by flow cytometry and their ability to activate sr t cells tested by co-culture with carboxyfluorescein succinimidyl ester (cfse)-labeled myelin-specific cd4 t cells. endogenous sr t cell kinetics was analyzed within both cervical lymph nodes and cns by enzyme-linked immunospot (elispot) following viral infection. results: the data demonstrate the presence of apc capable of activating sr t cells in both draining lymph nodes and the cns temporally correlating with overt demyelination. while both the cns-infiltrating myeloid population and microglia ingested myelin, only cns-infiltrating apc were capable of presenting endogenous myelin antigen to sr t cells ex vivo. finally, sr t cell activation from the endogenous t cell repertoire was most notable when infectious virus was controlled and paralleled myelin damage. although sr t cell accumulation peaked in the persistently infected cns during maximal demyelination, they were not preferentially retained. their gradual decline, despite ongoing demyelination, suggested minimal re-stimulation and pathogenic function in vivo consistent with the lack of autoimmune symptoms. conclusions: the results demonstrate the potential for cns tissue destruction to induce and recruit sr t cells to the injury site and support a host suppressive mechanism limiting development of autoimmunity. multiple sclerosis (ms) is an autoimmune disease of the central nervous system (cns) characterized by demyelination, axonal loss, and increasing disability [1, 2] . while the etiology of ms remains unknown, various genetic and environmental factors have been associated with ms pathogenesis [3] [4] [5] . among the environmental cues, viral infections, most prominently epstein-barr virus and human herpes virus type 6, have been linked to initiation or progression of disease [4, [6] [7] [8] [9] ; however, their causal nature remains unclear. anti-viral antibodies and viral antigens have been detected in brain and cerebrospinal fluid of ms patients [10] [11] [12] . nevertheless, active virus replication has yet to be demonstrated in the cns of ms patients. by contrast, infections have even been proposed to limit autoimmunity [13] . indeed, the decreased incidence of infectious diseases in the past decades inversely correlates with an increased incidence of autoimmune diseases, including ms [14] . for example, viral infection in an animal model of type i diabetes prevents disease development [15, 16] . similarly, mice bred under conventional conditions are less susceptible to diabetes than mice bred in a pathogen-free environment [14] , supporting the concept that microbiome burden may reduce the incidence of autoimmunity [17] . therefore, the role of viral infections in autoimmune diseases is complex and remains largely elusive. activation of self-reactive (sr) immune cells by epitope spreading, molecular mimicry, cryptic antigen, and bystander activation have all been proposed to explain how viral infections may ultimately result in autoimmunity [18, 19] . this is exemplified by the biphasic disease induced by theiler's murine encephalomyelitis virus (tmev) [20] . minimal clinical deficits appear during the acute phase of tmev infection, characterized by neuronal infection. the protective anti-viral immune response fails to completely eliminate infectious virus, resulting in chronic infection of microglia and macrophages [21] , ascending paralysis and progressive demyelination [22] . whereas initial tissue destruction occurs as a bystander effect of the anti-viral response, paralysis and demyelination are associated with autoimmunity mediated by sr t cells activated via epitope spreading during chronic infection [18, 22] . tmev infection thus provides a paradigm for autoimmunity associated with chronic infection and sustained inflammation. by contrast, demyelination induced by infections with the neurotropic mouse hepatitis virus (mhv) strain jhm (jhmv) or the related dual hepato-and neurotropic strain mhv-a59 suggests that persistent cns infections, even those associated with myelin destruction, do not necessarily result in clinically apparent cns autoimmune disease. although a vigorous cd8 + t cell-and th1-dominated immune anti-viral response eliminates infectious virus [23] , viral antigen and rna persist within the cns without evidence of active virus replication [24, 25] . demyelination requires both infection of oligodendrocytes and adaptive immunity and is initiated by t cell-mediated virus control. similar to chronic tmev infection, persistent mhv infections are associated with ongoing demyelination. however, following acute viral-induced tissue injury, sustained demyelination is balanced by myelin repair, associated with clinical improvement [23, 26, 27] . the absence of disease progression during chronic mhv infection suggests that autoimmune responses are either not initiated or suppressed. nevertheless, during acute mhv-a59 infection, sufficient myelin-derived self-antigen (ag) is presented in cervical lymph nodes (cln) to support proliferation of exogenously added sr t cells. however, little or no sr t cell proliferation occurred within the cns, suggesting that t cells activated in cln during acute infection migrate to the cns but are unable to promote a local autoimmune response. the present study set out to determine whether ag-presenting cells (apc) in the cns support sr t cell proliferation and whether endogenous sr t cells are induced during jhmv-induced demyelination. the data demonstrate that sr t cell activation is agdriven and mediated by a population of cd11b + apc present within both the cln-and the cns-infiltrating cells. despite myelin uptake and expression of major histocompatibility complex (mhc) class ii molecules, microglia do not support sr t cell activation. importantly, the kinetics of cd11b + cell-mediated sr t cell activation parallels demyelination and correlated with myelin-specific t cells derived from the endogenous host t cell repertoire. nevertheless, despite sustained demyelination and cns inflammation, the sr t cell response declined during viral persistence, suggesting that chronic jhmv infection establishes an environment which supports ongoing clinical improvement and regulates autoimmune responses. wild-type (wt) c57bl/6 mice were purchased from the national cancer institute (frederick, ms, usa). myelin oligodendrocyte glycoprotein (mog)-specific t cell receptor (tcr) transgenic (2d2) mice expressing the congenic marker cd90.1 [28] were obtained from dr. v.j. kuchroo (harvard university, boston, ma). ovalbumine (ova)-specific tcr transgenic (ot-ii) mice expressing the congenic marker ly5.1 were provided by dr. b. min (cleveland clinic, cleveland, oh). mice expressing the green fluorescent protein (gfp) under the myelin proteolipid protein (plp) promoter (plp-gfp) [29] were obtained from dr. w.b. macklin (university of colorado, denver, usa). all transgenic mice were bred and maintained at the biological resources unit of the cleveland clinic lerner research institute under sterile conditions. all procedures were performed in compliance with the cleveland clinic institutional animal care and use committee approved protocol number 2013-1131. peptides were obtained from bio-synthesis (lewisville, tx, usa) and included m 133-147 (gtvyvrpiiedyht), mog (mevgwyrspfsrvvhlyrngk), and mbp 60-80 (shhaartthygslpqksqr). mice were infected between 6 and 7 weeks of age in the left hemisphere with 1000 pfu of the glia tropic jhmv neutralizing monoclonal antibody (mab)-derived 2.2v-1 variant [30] . mice were assessed daily for clinical disease severity according to the following scale: 0, healthy; 1, hunched back and ruffled fur; 2, partial hind limb paralysis or inability to maintain the upright position; 3, complete hind limb paralysis; and 4, moribund or dead. anesthetized mice were perfused with phosphate-buffered saline (pbs). single-cell suspension was prepared from the cln by mechanical disruption through a 70-μm cell strainer in rpmi 1640 medium containing 25 mm hepes (ph 7.2) supplemented with 10 % fetal calf serum (fcs). brain and spinal cord were homogenized using a ten-broeck tissue grinder. tissue homogenates were adjusted to 30 % percoll (pharmacia, uppsala, sweden). a 1-ml 70 % percoll underlay was added prior to centrifugation at 850 g for 30 min at 4°c. cns-derived cells were recovered from the 30-70 % interface, washed, and resuspended in roswell park memorial institute (rpmi) 1640 medium 10 % fcs. splenocytes were isolated from naïve mice by mechanical disruption and red blood cells eliminated using gey's solution. ninety-six-well filtration plates (millipore, billerica, ma, usa) were coated overnight at 4°c with anti-interferon-γ (ifn-γ) capture ab (10 μg/ml; bd biosciences, san diego, ca, usa). serial dilutions of clnand cns-derived mononuclear cells pooled from a minimum of eight mice per time point were cultured in triplicate in rpmi complete (rpmi 1640 medium containing 2 mm l-glutamine, non-essential amino acid, 1 mm sodium pyruvate, 25 μg/ml gentamicin, and 5 × 10 −5 m 2-mercaptoethanol) supplement with 10 % fcs in the presence of irradiated splenocytes (2.5 × 10 5 cells/well) pre-incubated with or without 1 μm peptide for 2 h at 37°c. after 36 h at 37°c, spots were visualized by sequential incubation with biotinylated anti-ifn-γ mab (5 μg/ml; bd biosciences) overnight at 4°c, horseradish peroxidaseconjugated streptavidin (bd biosciences) for 1 h at room temperature, and 3,3′-diaminobenzidine substrate (sigma aldrich, st. louis, mo, usa). spots were counted using a ctl immunospot analyzer (cellular technology ltd., shaker heights, oh, usa). spots detected in wells with no stimulation (no peptide) were subtracted and results presented as the number of ifn-γ-secreting cells normalized to 10 6 input cells. non-specific binding was blocked with mouse serum and anti-mouse fcγiii/ii mab for 15 min on ice. cells were stained for surface markers for 30 min on ice using fluorescein isothiocyanate (fitc), phycoerythrin (pe), peridinin chlorophyll protein complex (percp), or allophycocyanin (apc)-conjugated mab (all from bd biosciences) specific for cd45 (clone ly-5), cd4 (clone gk1.5), cd8 (clone 53-6.7), cd25 (clone pc61), cd44 (clone im7), cd45.1 (clone a20), cd90.1 (clone ox.7), cd11b (clone m1/70), cd80 (clone 16-10a1), and cd86 (clone gl-1). cells were washed twice in facs buffer (pbs, 1 % bovine serum albumin (bsa)) prior analysis. for proliferation study, bromodeoxyuridine (brdu, 1 mg/mouse) (bd biosciences) was injected i.p. 24 h prior to sacrifice. mononuclear cells from cln and cns were prepared as described above, stained for surface markers followed by intracellular brdu according to the manufacturer's instructions using the fitc brdu flow kit (bd biosciences). data were analyzed using a facscalibur flow cytometer (bd biosciences) and flowjo software (tree star inc., ashland, or, usa). cd4 + t cells were enriched from naïve 2d2 (tcr transgenic mice specific for mog or ot-ii (tcr transgenic mice specific for ova) mice by negative selection using the cd4 + t cell isolation kit ii (miltenyi biotec inc., auburn, ca, usa), according to the manufacturer instructions. naïve (cd25 − cd44 lo ) cd4 + t cells were then purified by cell sorting (facsaria, bd biosciences). sub-lethally irradiated (450 rad) wt recipients received equal numbers (10 6 cells) of purified naïve ot-ii and 2d2 cd4 + t cells by intravenous injections and were challenged with virus 2 weeks after adoptive transfer. cln, brains, and spinal cords were isolated at various times post-infection (p.i.) from pbs-perfused mice. cln were mechanically disrupted through a 70-μm cell strainer as described above, whereas brains and spinal cords were finely minced with a razor blade. suspensions were digested in rpmi 1640 medium containing 10 % fcs, 0.5 % collagenase (100 mg/ml) (roche, basel, switzerland), and 1 % dnase i (1 mg/ml) (sigma aldrich) for 40 min at 37°c. collagenase was then inactivated by addition of 1 % 0.1 m edta for 5 min at 37°c. following centrifugation at 400 g for 7 min at 4°c, cln-derived cells were directly resuspended in facs buffer for staining, whereas cns-derived cells were isolated using percoll gradients prior to staining as described above. cln-derived cd11b − and cd11b + cells, cnsinfiltrating cd45 hi cd11b + , microglia (cd45 lo cd11b + ), and cd45 hi cd11b − cells were purified using a cell sorter (facsaria) and resuspended in rpmi complete 10 % fcs. naïve cd4 + t cells were purified from splenocytes of naïve 2d2 mice using the cd4 + cd62l + t cell isolation kit ii (miltenyi biotec) according to the manufacturer instructions. naïve 2d2 cd4 + t cells were then stained with carboxyfluorescein succinimidyl ester (cfse, 1.25 μm) (molecular probes, carlsbad, ca, usa) and cultured in a 96-well plate in the presence of cln-derived cd11b − or cd11b + cells, cns-derived cd11b − or cd11b + cells, or microglia (t cells/apc ratio 1:1) in rpmi complete 10 % fcs for 4 days at 37°c. rat anti-mhc class ii blocking mab (clone m5/114) (abcam, cambridge, ma) or mog 35-55 peptide (10 μm) were added at the initiation of the cultures for some experiments. t cell proliferation was assessed by measuring the percentage of cfse dilution by flow cytometry. mice were perfused with ice-cold pbs followed by 4 % paraformaldehyde (pfa). spinal cords were dissected, fixed for 1 h in 4 % pfa at 4°c, and then incubated with sucrose gradients as follows: 30 min with 15 % sucrose at room temperature, 30 min with 20 % sucrose at 4°c, and, finally, overnight with 30 % sucrose at 4°c. tissues were stored in cryoprotection solution until preparation of 30μm microtome sections. after antigen retrieval with 0.01 m sodium citrate buffer ph 6.0, sections were treated with 1 % triton x-100 for 30 min, treated with blocking solution (pbs, 1 % bsa, 10 % normal goat serum) for 30 min, and stained with rabbit anti-mouse iba1 (wako, richmond, va) and mouse anti-mouse plp (millipore) primary mabs overnight at 4°c. alexa fluor 488 goat anti-rabbit (invitrogen, carlsbad, ca) and alexa fluor 594 goat anti-mouse (invitrogen) were added for 1 h at room temperature. sections were mounted with vectashield mounting medium with 4′-6-diamidino-2-phenylindole (dapi) (vector laboratories) and analyzed using a leica tcs confocal microscope. results represent the mean ± sem and are plotted using graphpad prism software. statistics were calculated using a two-tailed unpaired student's t test, anova with bonferroni post-test, and dunn's multiple comparison test, and p values <0.05 were considered statistically significant. infection with the mhv-a59 strain suggested that acute encephalomyelitis provides a milieu capable of supporting proliferation of transferred mog-specific t cell receptor (tcr) transgenic t cells within the cln [31] . however, neither their reactivation within the cns, prolonged survival, or potential to induce autoimmunity have been explored. to determine whether sr cd4 + t cells are retained during chronic infection, mogspecific 2d2 cd4 + t cells were transferred to sublethally irradiated wt mice prior to jhmv infection. by enhancing engraftment of donor t cells, this approach increased sr t cells to numbers amenable to flow cytometric analysis, while maintaining a host anti-viral immune response. bone marrow-derived inflammatory (cd45 hi ) cells were minimal within the cns of recipients prior to infection (fig. 1a) , indicating non-specific activation and that cns recruitment was prevented by intact blood brain barrier. at day 7 p.i., maximal antiviral t cell responses [24, 25] coincided with a decreased percentage of transferred sr t cells in cln (fig. 1b, c) . grafted sr t cells were undetectable within the cns at day 7 p.i. following jhmv infection (fig. 1b, c) in contrast to their early migration into the cns during acute mhv-a59 infection [31] . nevertheless, transferred sr t cells were present in the cns of jhmv-infected mice by day 14 p.i. (fig. 1b, c) ; furthermore, similar proliferation of grafted sr t cells and host cd4 + t cells suggested identical activation (fig. 1d) . although the kinetics differed, these data are consistent with cns recruitment of sr t cells during mhv-mediated demyelination, independent of the virus strain and tropism [31] . importantly, retention of transferred sr t cells at slightly declining frequencies within the total cns cd4 population out to day 30 p.i. (fig. 1b, c) negated preferential expansion/survival during chronic viral infection. the absolute numbers of grafted sr cd4 + t cells gradually declined (fig. 1c) concomitant with contraction of the overall cd4 + t cell population, supporting a lack of ongoing self-ag-driven survival. furthermore, retention of sr t cells within the cns did not alter disease severity out to 30 days p.i. (fig. 1e) . within the cln, transferred sr t cells comprised~40 % of activated cd44 hi cells (data not shown) and their absolute numbers remained stable during ongoing chronic jhmv infection (fig. 1c) . limited proliferation of sr t cells within the cns during acute mhv-a59 infection [31] suggested the possibility of non-specific t cell recruitment. to determine the specificity of recruitment into the cns, heterologous ova-specific t cells were co-transferred with sr t cells to sub-lethally irradiated mice. prior to infection, the frequencies of both the sr-and ova-specific t cells were similar in cln (fig. 1f ) and blood (data not shown), demonstrating equivalent survival. following jhmv infection, the relative percentages of both the sr-and ova-specific t cells were reduced in the cln coincident with the expansion of virus-specific t cells. however, the decline in transferred sr t cells was less dramatic, suggesting enhanced survival cues via agspecific activation (fig. 1f ) . consistent with an absence of non-specific peripheral activation, ova-specific t cells were not detected in the inflamed cns at day 14 p.i. by contrast, transferred sr t cells constituted a discrete population (fig. 1f ) , confirming migration of peripherally activated cells and supporting agdependent cns retention of sr cd4 + t cells. cln constitute the major site of peripheral lymphoid drainage from the cns [32, 33] . this is supported by activation of jhmv-specific t cells within the cln [34] and detection of myelin antigens in ms patients and rodents with experimental autoimmune encephalomyelitis (eae) [35] [36] [37] , as well as following demyelination induced by oligodendrocyte death [38] . myelin, either in a soluble form or associated with apc, drains to cln, where it can potentially activate naïve sr t cells [36, 39] via presentation by cd11b + cells, comprising both dendritic cells (dc) and macrophages [40] . during eae, the most efficient population-presenting myelin antigen in the cns is cd11b + cd11c + [41, 42] . cd11b + cells represent a small subset (between 2.5 and 4 %) of the cln population, which only varies slightly throughout the course of jhmv infection (fig. 2a) . importantly, cd11c + cells constitute the vast majority of cd11b + cells (~70 %) and their frequencies within the cd11b + population remain constant between days 7 and 30 p.i. (fig. 2a) . we therefore tested unfractionated clnderived cd11b + cells for the presentation of self-ag following jhmv infection. cd11b + and control cd11b − cells were purified from the cln at different times p.i. and were co-cultured with cfse-labeled mog-specific cd4 + t cells as a source of sr t cells. neither cd11b + nor cd11b − cells isolated at day 7 p.i. supported sr t cell activation (fig. 2c) . however, by day 10 p.i., when myelin damage becomes apparent, both cd11b + and cd11b − cells initiated minimal t cell proliferation, which reached statistical significance relative to unstimulated conditions (no apc) for cd11b + cells (fig. 2c) . activation of sr t cells by the cd11b + population increased at days 14 and 21 p.i. concomitant with enhanced demyelination [23, 43] , while the cd11b − population retained only a minimal ability to support sr t cell proliferation (fig. 2b, c) . the ability of clnderived cd11b + cells to support sr t cell proliferation declined, but was sustained, by day 30 p.i., while the minimal ability of the cd11b − population dropped to below detection by 30 days p.i. (fig. 2c) . the presence of myelin ag within the cln by day 14 p.i. supports the possibility that endogenous sr t cells could be activated following jhmv-induced demyelination. the absence of clinically apparent autoimmunity, coincident with a decline rather than progressive increase in sr t cells in the cns during chronic jhmv infection (fig. 1c) , suggested that sr t cells fail to be continuously reactivated within the cns. in the inflamed cns, resident microglia and myeloid cells constitute heterogeneous populations of apc potentially capable of presenting self-ag [40] . to determine whether cnsinfiltrating myeloid cells, comprising macrophages and dendritic cells, and/or microglia are capable of processing and presenting self-ag within the cns following infection, both potential apc populations were purified based on differential cd45 expression [44, 45] at distinct times p.i. and tested for the ability to support sr t cell activation in the absence of exogenously added ag. infiltrating myeloid cells at day 7 p.i. (fig. 3a) were comprised of~30 % cd11c + cells, a proportion that gradually increased during the course of infection to reach~55 % at day 30 p.i. (fig. 3a) . at the peak of acute inflammation (day 7 p.i.), neither the cd45 hi cd11b + , cd45 hi cdllb − , nor microglia populations supported t cell proliferation (fig. 3c) . however, sr t cell proliferation was modestly increased by the cd11b + apc subset at day 10 p.i. (fig. 3c) and prominently by day 14 p.i. (fig. 3b, c) , similar to the cln (fig. 2) . on a per cell basis, the ability of infiltrating cd11b + cells to support proliferation remained relatively stable during chronic jhmv infection (fig. 3c) . although cd11b − cells appeared to promote minimal proliferation, their activity remained near threshold levels throughout the infection. similar to the cd11b − population, microglia were unable to support sr t cell proliferation at any time p.i. (fig. 3b, c) . the kinetics of sr cd4 + t cell activation by cns-infiltrating cd11b + cells correlate with viralinduced myelin destruction as a source of self-ag [43] . in addition, anti-mhc class ii ab blocked proliferation, supporting mhc class ii-dependent t cell activation (fig. 3d) rather than non-specific proliferation signals derived from the inflamed cns environment (fig. 1d) . mhc class ii expression was compared on microglia and infiltrating cd11b + cells by flow cytometry to examine whether the inability of microglia to support t cell proliferation correlated with differential mhc class ii expression. while no differences were noted in the percentage of class ii-expressing cells within the populations (fig. 4a) , microglia were inferior to infiltrating cd11b + cells in their level of class ii expression based on mhc class ii mean fluorescence intensity (mfi) (fig. 4a) , even at the peak of ifn-γ release at day 7 p.i. [25] . similarly, the mfi of the co-stimulatory molecule cd86 was slightly lower on microglia compared to i. c percentages of proliferating 2d2 cells co-cultured with media only (no apc), cd11b − , and cd11b + cells isolated from the cln of jhmv-infected mice at days 7, 10, 14, 21, and 30 p.i. data represent the mean ± sem with n = 3-12 pooled mice per time point from three separate experiments. #p < 0.05 and ###p < 0.001 depict significant differences between no apc and cd11b + cells, whereas § § §p < 0.001 depicts significant differences between cd11b − and cd11b + cells determined by a two-way anova with bonferroni post-test. significant differences between time points are indicated by **p < 0.01 and ***p < 0.001 using dunn's multiple comparison test infiltrating cd11b + cells, while no differences were observed for cd80 expression levels (fig. 4b) . moreover, while mhc class ii expression was sustained at similar levels on microglia, it was further upregulated between day 14 and 21 p.i. on infiltrating cd11b + cells (fig. 4a) . to assess whether myelin ag added to the cell culture could overcome the inability of microglia to activate sr t cells, cd11b + apc populations were pre-incubated with mog peptide. under these conditions, microglia induced proliferation of sr cd4 + t cells as well as infiltrating cd11b + cells (fig. 4c) . these data demonstrate that while class ii levels on microglia suffice to present the inability of microglia to support sr t cell proliferation may reflect a defect in myelin uptake or ag processing. myelin engulfment by iba1 + cells supports myelin uptake by microglia and/or infiltrating myeloid cells during jhmv-induced demyelination (fig. 5a) . however, the amoeboid morphology of microglia during inflammation prevents histological distinction between myelin in macrophages versus microglia (fig. 5a) . mice expressing gfp under the plp promoter were therefore infected to quantify gfp as a surrogate marker for myelin ingestion by infiltrating cd11b + cells versus microglia. flow cytometry revealed gfp within both infiltrating myeloid cells (cd45 hi cd11b + ) and microglia (cd45 low cd11b + ) (fig. 5b, c) . percentages of infiltrating myeloid cells and microglia containing gfp were relatively stable between days 7 and 30 p.i. (fig. 5b) , with a higher proportion of microglia containing gfp, supporting their primary role in demyelination [46] . in contrast to percentages, mfi analysis showed that the amount of gfp differed throughout the course of infection (fig. 5c) . infiltrating cd11b + cells exhibited peak uptake at 14 days p.i., which slowly decreased at later time points (fig. 5c) , but remained higher than gfp mfi in the control cd45 hi cd11b − population, as well as cd45 hi cd11b + cells isolated from naïve animals (fig. 5c) . by contrast, a gradual increase in gfp was observed within microglia throughout the course of jhmv infection independent of auto-fluorescence (fig. 5c) . these data imply that both the infiltrating myeloid and microglial populations phagocytoze myelin during viral-induced demyelination. therefore, the inability of microglia to support sr t cell activation is less likely to reside in fig. 5 myelin debris within cns cells. a plp (red) and iba1 (green) stainings on spinal cord tissue isolated from wt-infected mice at day 21 p.i. and analyzed by confocal microscopy. scale bar, 10 μm. (i) 3d reconstruction image view, yz (ii), and xz (iii) projections show plp + myelin in close contact and engulfed (arrow head) by iba1 + cells. b percentage of cd45 hi cd11b + and microglia (cd45 low cd11b + ) containing gfp between days 7 and 30 p.i. during infection of plp gfp mice. c mean gfp fluorescent intensity (mfi) analyzed by flow cytometry in cns-infiltrating cd45 hi cd11b − and cd45 hi cd11b + cells and microglia (cd45 low cd11b + ) between days 0 to 30 p.i. hashed bars represent microglia autofluorescence detected within infected wt mice between days 7 and 30 p.i. data represent the mean ± sem from two separate experiments with n = 3 per time point per experiment a defect in myelin uptake, rather than inefficient ag processing and/or presentation. myelin damage, uptake and subsequent presentation of self-ag by potential apc, and sustained cns inflammation without overt clinical evidence of autoimmune disease following jhmv infection pose a dilemma. plausible explanations may be that sr t cells are either not, or only minimally, induced or that they are not reactivated within the cns. indeed, demyelination triggered by oligodendrocyte death fails to initiate autoimmunity, despite myelin ag drainage to cln [38] . to determine if jhmv-induced demyelination results in the activation of endogenous sr t cells, cd4 + t cell responses to the h-2 b restricted encephalitogenic myelin epitopes mog and mbp were assessed by enzyme-linked immunospot (elispot) to detect lowfrequency responder cells. responses to the h-2 b -restricted immuno-dominant viral m 133 epitope [47] were used as positive controls. analysis focused on ifn-γproducing cd4 + t cells, as jhmv infection induces a vigorous th1 response with negligible il-17 or il-9 components (data not shown), which is supported by no affects on pathogenesis in the absence of il-23 [48] . virus-as well as myelin-specific t cell frequencies were lower than 9 and 5 positive cells per 10 6 , respectively, within the cln and spleen of naïve animals (fig. 6a) . within cln, the frequency of virus-specific cd4 + t cells peaked at day 7 p.i., declined by day 14 p.i., and remained relatively stable up to day 60 p.i. (fig. 6a) . within the cns, frequencies of virus-specific t cells were~10 higher than cln at day 7 p.i., continued to increase at day 14 p.i., and declined as the overall inflammatory response resolved due to virus control (fig. 6a) . sr t cells specific for both the immuno-dominant mog and myelin basic protein (mbp) epitopes within the cln were above baseline throughout acute and chronic jhmv infection, albeit at very low frequencies (fig. 6a) . maximum sr t cell frequencies were observed at day 30 p.i. (fig. 6a) with responses near detection limits in a large proportion of infected mice throughout infection (fig. 6b) . a very limited number of infected mice harbored sr t cells within the cns during acute infection at days 7 and 14 p.i. however, by day 21 p.i., the number of responder mice exhibiting sr t cells within the cns reached 100 % (fig. 6b) , coincident with peak frequencies of both mog-and mbp-specific t cells between days 21 and 30 p.i. (fig. 6a) . these data are consistent with maximal clinical disease throughout days 14-21 p.i. in all infected mice (fig. 6c) . although the sr t cell frequencies in the cns were variable among individual mice, and always reduced relative to virus-specific t cells, they were increased relative to the cln at day 21 p.i. and thereafter (fig. 6a) . importantly, cns sr t cells were sustained above baseline during resolution of clinical symptoms, with preferential stability of mog-specific t cells (fig. 6a, b) . however, compared to peak sr t cell frequencies at days 21/30 p.i., frequencies, as well as the number of mice harboring cns sr t cells, declined as clinical disease resolved (fig. 6c) . these data demonstrate that endogenous sr t cells are induced in the vast majority of jhmv-infected mice. however, they are only sustained within the cns of~60 % of mice during chronic demyelination, when lesion formation is counterbalanced by repair (fig. 6a, b) . importantly, segregation of mice harboring sr t cells within the cns after day 30 versus mice in which sr t cells were undetectable showed no differences in overall clinical recovery (fig. 6d) . these data further supported our previous observations that cns recruitment and retention of sr t cells did not alter disease severity (fig. 1e) . animal models have demonstrated that viral infections can promote the initiation as well as increase the severity of autoimmune diseases [4] . however, evidence for a viral etiology of human autoimmune diseases, including ms, remains elusive. moreover, given the extent of both acute and persisting viral infections without evidence of autoimmune sequelae, it has also been proposed that infections may even provide protection from autoimmunity. specifically, the conditions promoting or stemming activation of auto-ag-specific t cells following viralinduced tissue damage have not been extensively explored. chronic cns infection by the naturally occurring mouse pathogen tmev promotes activation of sr t cells, which mediates increasing tissue destruction accompanied by an ascending paralysis [20] . by contrast, infection with jhmv also leads to immune-mediated demyelination; however, persistent infection correlates with clinical recovery [27] . while clinical improvement throughout persistence despite ongoing demyelination supported the absence of an autoimmune component following neurotropic mhv infection, previous studies clearly demonstrate sufficient auto-ag presentation to induce sr t cell responses [31, 49, 50] . in an effort to reconcile peripheral activation of sr t cells, but no disease exacerbation, this study set out to assess kinetics of auto-ag presentation as well as the fate of sr t cells following jhmv infection. the data demonstrate the presence of apc capable of activating sr t cells in both the cln and the cns with maximal stimulating activity during the time of most overt demyelination. while both the cns-infiltrating myeloid population and microglia ingest gfp as a surrogate for myelin, only the cnsinfiltrating apc were capable of presenting endogenous , and mbp 60-80 detected in naïve animals. red bars represent the mean ± sem. significant differences between time points are indicated *p < 0.05, **p < 0.01, and ***p < 0.001. significant differences compared to baseline levels are indicated #p < 0.05, ##p < 0.01, and ###p < 0.001 using unpaired t test. b percentage of mice exhibiting ifn-γ-secreting mog and mbp-specific cd4 + t cells above maximal baseline levels in the cln and cns between days 7 and 60 p.i. c clinical symptoms following jhmv infection. data represent the mean ± sem of 32 mice. d correlation between frequencies of sr t cells at days 45 and 60 p.i. with average clinical scores, coefficient of determination r 2 = 0.1417 myelin ag to sr t cells ex vivo. finally, the results demonstrate that sr t cells are activated from the endogenous t cell repertoire when myelin destruction is evident, but infectious virus is already controlled. although sr t cells migrate into and are most prominent in the persistently infected cns during maximal demyelination, no evidence was found to suggest preferential expansion despite ongoing demyelination, consistent with minimal re-stimulation in vivo. mechanisms underlying sr t cell activation following cns infections include molecular mimicry between pathogen and self-epitopes, presentation of self-peptides from tissue damage, and dysregulation of regulatory tolerance mechanisms [51, 52] . myelin-specific t cells isolated from ms patients have been shown to cross-react with human coronavirus [53, 54] . similarly, homology was found between mhv and myelin peptides, potentially triggering autoimmune disease [55] . nevertheless, sr t cells were near detection levels within the cns during acute jhmv infection in the vast majority of mice and their frequency only increased as myelin damage increased. moreover, their numbers declined as clinical symptoms dropped due to increased repair. self-ag-specific activation of sr t cells is further supported by the lack of t cell activation with irrelevant specificity and blockade of activation by anti class ii ab, ruling out bystander events. overall, our data support the hypothesis that release of myelin debris due to jhmv-induced tissue damage results in cell-free or cell-associated myelin drainage to cln, where a population of cd11b + apc can support the activation of sr t cells. activated sr t cells access the cns at a time when the vast majority of infectious virus is already controlled, but persistence drives ongoing expression of proinflammatory molecules capable of recruiting activated cells from circulation [56] . local encounter with myelin-loaded cd45 hi cd11b + apc, but not microglia, is then poised to drive sr t cell reactivation. the apparent inability of microglia to activate sr cd4 + t cells ex vivo remains unclear as their uptake of myelin is consistent with demyelination independent of inflammatory monocyte recruitment following jhmv infection [46] . nevertheless, microglia also failed to support cd4 + t cell responses following tmev infection and induction of eae [57, 58] . in fact, several studies question the role of microglia as apc in vivo [59] despite their implication as apc based on expression of mhc class ii and costimulatory molecules in both ms patients [60] and eae [61] , as well as their ability to activate cd4 + t cells in vitro [57] . detection of myelin within iba1 + cells increased gfp mfi in microglia, as well as detection of myelin in microglia in other models [57, 58] supports self-ag uptake during jhmv infection. however, these data suggest that microglia may be poor at processing myelin ag for loading onto mhc class ii. this notion is also supported by effective priming of sr t cells using exogenous peptide, despite reduced mhc class ii expression relative to infiltrating cd11b + cells. in addition, as activated microglia can adapt to their surroundings to mediate both pro-and anti-inflammatory functions [62] , it cannot be excluded that microglia may negatively regulate sr t cell responses during chronic jhmv infection by limiting t cell proliferation or by inducing apoptosis [63] . the results also indicate a discrepancy between apc presentation capacity ex vivo, yet apparently limited sr t cell activation in vivo. although apc in cln sustain their ability to activate sr t cells throughout days 14 to 30 p.i., endogenous sr t cells are only detected at low frequencies and exhibit a brief peak at day 30 p.i. maximal accumulation of sr t cells in the cns between days 21 and 30 p.i. in all mice supports efficient egress from cln. whether frequencies in the cns are supported by ongoing recruitment or local expansion remains unclear. the capacity of cns-infiltrating cd11b + cells to activate sr t cells ex vivo in the absence of exogenous peptide indicates that apc are competent to reactivate sr t cells throughout chronic infection. however, no evidence for preferential expansion or retention of sr t within the cns relative to virus-specific cd4 + t cells suggests that the absence of sustained activation may contribute to the lack of increasing clinical symptoms during chronic infection. these results clearly differ from the tmev model, in which sr t cells aggravate pathology and clinical disease. one possible scenario is that persistent jhmv infection may establish an environment that suppresses the sr t cell response. jhmv infection induces both agspecific and foxp3 + regulatory t cells [64, 65] which are efficient at controlling autoimmunity in other settings [66, 67] . treg depletion [65] , elimination of agspecific treg [65] , and adoptive transfer of foxp3 + treg [68] during acute jhmv infection all demonstrated that, in contrast to tmev [69] , tregs have a limited effect on virus clearance. nevertheless, foxp3 + tregs play a critical role in limiting tissue destruction [68] , suggesting that they regulate the activation or effector function of sr t cells following jhmvinduced demyelination [70] . although treg control of sr t cells in vivo and their association with increased tissue destruction remains unknown, depletion of treg correlates with increased proliferation of sr t cells in the cln during acute mhv-a59 infection [31] . whether tregs act in limiting sr t cell induction during jhmv infection, which does not or only poorly replicates in cln, or whether additional factors limit sr t cell responses at the target site remains to be elucidated. this study demonstrates the activation and kinetics of sr cd4 + t cells within the endogenous t cell repertoire relative to self-ag presentation by apc following jhmv-induced demyelination. importantly, ongoing myelin loss, sustained cns-derived apc activity ex vivo, and retention of sr t cells do not lead to autoimmune disease during chronic jhmv infection. this model thus provides a unique opportunity to 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natasha towne, kate stenson, and megan mcconnell for their technical assistance and jennifer powers for the facs purification. this work was supported by the national institutes of health grant ns069690. the authors declare no financial conflicts of interest.authors' contributions cs designed and performed the experiments, collected and analyzed the data, and wrote the manuscript. ccb interpreted the data and wrote the manuscript. mg assisted with the experiments and edited the manuscript. sas designed the research, provided the materials, interpreted the data, and wrote the manuscript. all authors read and approved the final manuscript. key: cord-002757-upwe0cpj authors: sullivan, kathleen e.; bassiri, hamid; bousfiha, ahmed a.; costa-carvalho, beatriz t.; freeman, alexandra f.; hagin, david; lau, yu l.; lionakis, michail s.; moreira, ileana; pinto, jorge a.; de moraes-pinto, m. isabel; rawat, amit; reda, shereen m.; reyes, saul oswaldo lugo; seppänen, mikko; tang, mimi l. k. title: emerging infections and pertinent infections related to travel for patients with primary immunodeficiencies date: 2017-08-07 journal: j clin immunol doi: 10.1007/s10875-017-0426-2 sha: doc_id: 2757 cord_uid: upwe0cpj in today’s global economy and affordable vacation travel, it is increasingly important that visitors to another country and their physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population thwaites and day (n engl j med 376:548-560, 2017). this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population [1] . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. emerging infectious diseases are a result of a convergence of numerous factors and comprise complex interactions among multiple variables. some of those factors are human movement, land use change, encroachment and wildlife translocation, rapid transport, and climate change. several studies demonstrate that for a pathogen to persist in a population, a minimal host population size that is specific for the type of pathogen and host population. of particular relevance to emerging infections is the pattern of rapid population growth. in the tropics, before wwii, most regional ecosystems consisted of few large cities and scattered human settlements separated by large areas of cropland, pastureland, or undisturbed forest. today, the pattern is the opposite: many large cities have developed with only patches of undisturbed forest or grassland. domestic vectors have therefore expanded their population with increasing urbanization and this markedly impacts the interactions between vectors and pathogens [2] . human activities such as deforestation, use of pesticides, pollution, etc. lead to the loss of predators that naturally regulate rodent and insect populations. this contributes to emerging zoonotic diseases and explains why they occur more frequently in areas recently settled. in today's global economy and affordable vacation travel, it is increasingly important that visitors to another country and their the southern, with a reduction in the number of cold days per year, changes in rainfall (more winter precipitation and summer droughts) [4] , and together these changes increase the risk of several vector-borne diseases in new areas. climate change involves not only global warming but also changes in precipitation, wind, humidity, and the location and frequency of extreme weather events like floods, droughts, or heat waves. changes in climate produce changes in pathogens, vectors, hosts, and their living environment. increases in precipitation can increase mosquitoes for example, but heavy rainfalls may cause flooding that temporarily eliminates larval habitats and decreases mosquitoes. flooding may force rodents to look for new habitats in houses and increase the opportunities of vector-human interactions, as occurs for example in the case of epidemic leptospirosis. humidity is another very important factor of climate change in the development of vector-borne diseases. mosquitoes and ticks do not survive well in dry conditions. therefore, weather impacts infectious pathogen distribution in complex ways that are not predictable by the forecast. extreme weather events can precipitate outbreaks of infection. an increase in the frequency and intensity of natural disasters like hurricanes and tsunamis, in relation to the el niño/southern oscillation phenomenon, may result in more flooded grasslands, which favor the breeding of aedes and culex mosquitoes [5] , and impact water sanitation fostering outbreaks such as cholera. flooded areas can displace rodents leading to plague. tornados and other severe weather can stir up soil leading to infections with soil fungi leading to episodic outbreaks of invasive fungal disease such as mucormycosis such as apophysomyces trapeziformis [6] . malaria is a common disease that can vary dramatically depending on weather, and extreme weather can alter the very landscape, providing new bodies of water to support larval development. if the melting of glaciers and the polar ice caps bring coastal cities underwater, or if overpopulation and waste cause drinkable water shortages in certain regions of the world, we can expect mass migrations. these could change the patterns of infection and drive outbreaks. migrants traversing tropical forests, or feeding with meat from game or carcasses, are but two scenarios that could be envisioned for the emergence of zoonotic infectious diseases [7] . several predictive models have been developed to evaluate the impact of climate change on the emerging infectious diseases: climex, dymex, miasma models. nevertheless, it remains difficult to predict when and where pathogen behavior will deviate from its typical pattern. climate change primarily affects vector-borne diseases by increasing rates of reproduction and biting and by shortening the incubation period of the pathogen they carry. ticks have gained spread from the mediterranean basin to northern and eastern europe, as well as appearing at higher altitudes. increased survival, density, and activity have also been reported following shorter, milder winters. climate change has also resulted in more days of activity per year for mosquitoes. as temperatures rise, more parasites are viable within regions ranging from the mediterranean and tropical zones, up to the balkans, russia, scandinavia, and the uk. for some ticks and fleas, temperatures over 25°c with relative humidity of over 85% are optimal for their proliferation and activity throughout the year [4, 8, 9] . for example, dengue fever is usually limited to a tropical latitude and a low altitude, since mosquito eggs and larvae lose viability with sustained low temperatures. during unusually warm summers, however, dengue has been reported as high up as 1700 m above sea level. warmer temperatures also result in smaller adult mosquitoes, which need to bite more frequently to feed themselves and be able to lay eggs, thus increasing the rate of transmission [8] . in contrast, the incidence of malaria has followed mixed patterns of increase and decrease along recent decades, and computer models have failed to predict the spread. the explanation for this is, in part, that climate change also results in diminished survival of the vectors (warming over 34°c affects the survival of both parasites and vectors), and in part, that the effect of climate change is non-linear and complex [10] . the frequency of emerging vector-borne infections varies per changes in land use, human activity, intervention maneuvers to eradicate the vector or prevent transmission to humans, drug treatment, and vaccines. both ecologic and economic changes may bring together rodents and humans. hunting activities may change vector distribution and large-scale animal movements can impact disease distribution. impoverishment of cities and overcrowding in slums, but also reforestation, golf club development, and the urbanization of rural suburbs facilitate exposure to ticks and rodents [11] . many patients with pidd require immunoglobulin replacement. immunoglobulin products have been demonstrated to improve outcomes in hepatitis a and measles [12, 13] . at least some neutralizing antibody is present directed to rsv and group b streptococci [14, 15] . this raises three distinct issues for patients: (1) difficulty in the diagnosis of infections in travelers because locally produced immunoglobulin may have antibody titers to local infections that are not typical for other countries, (2) safety concerns about locally produced immunoglobulin, if the patient resides in the location long enough to require immunoglobulin from a local provider, (3) the decision to use locally produced immunoglobulin products to provide superior prevention of infection compared to the patient's usual product. there are limited data on each of these subjects. serologic diagnostic testing in patients on immunoglobulin therapy will be addressed below in terms of issues related to lack of specific antibody produced by the patient (potentially) after infection. the converse can also be an issue. patients arriving from countries with significant occurrences of infections unusual in their current country may have igg antibodies to those infections simply through their immunoglobulin product and not reflecting a true infectious event in the patient. this can lead to diagnostic confusion when serologic testing demonstrates the presence of antibodies due to the infusion product. patients will often ask if immunoglobulin products from other countries are held to the same rigorous standards as their home country. today, commercially produced immunoglobulin is safe and tightly regulated. all commercially produced immunoglobulin around the world has one of the time-tested viral inactivation procedures such as nanofiltration, caprylate absorption, pasteurization, solvent/detergent, vapor heating, and low ph treatment. these procedures uniformly inactivate enveloped viruses. many emerging viruses are specifically tested for their ability to withstand the purification process. much has been learned since the transmission of hepatitis c viruses through immunoglobulin products in the early 1990s [16] . nevertheless, vigilance is important. in 2009, counterfeit immunoglobulin was identified. therefore, patients should ensure that they receive only brand name products while traveling. the subject of whether a patient's interests would be best served by using a local immunoglobulin product, with antibodies to pathogens that are prevalent in the community, or have their home physician ship their usual product, for which the patient has a known tolerance is hotly debated. patients with a history of intolerance to immunoglobulin products should not switch unless necessary. however, there are compelling reasons to consider a locally produced product when patients are in a foreign country for an extended period. it is known that antibodies to west nile virus have tracked with the distribution of the virus as it has emerged in several areas [17, 18] . titers in products using donors from the usa have higher neutralizing titers to west nile virus than those using donors from europe, although there is a 400-fold difference in titers between lots from the usa [18] . similarly, protective antibodies to concerning pathogens may be optimal in locally produced immunoglobulin. it is critical to inquire where the plasma source is derived. in most countries, the utilized immunoglobulin is produced in europe or the usa. having a different name does not ensure that the plasma pool comes from a different country. most lots of immunoglobulin are produced from 3000 to 60,000 plasma donors. the infrastructure to support such an endeavor is not easy to establish in each geographic area. serologic testing is commonly used for the diagnosis of infection. this approach relies upon detection or quantitation of antibodies made by the host against specific pathogens. the presence of igm antibodies against a specific pathogen indicates recent infection, while igg antibodies against a specific pathogen indicate past infection. importantly, serologic testing can only be applied for the diagnosis of infection if the host can mount a specific antibody response to the pathogen. conversely, serology cannot be relied upon to diagnose infection in the setting of immune deficiency where there is impairment of the specific antibody response, such as in the case of primary antibody deficiencies, cellular immune deficiencies, combined immune deficiencies, and other secondary immune deficiencies affecting t and/or b cell function. in these situations, the causative pathogen must be identified by alternate means such as culture of the organism, antigen detection, or molecular approaches (nucleic acid hybridization, nucleic acid sequencing, or oligonucleotide probe arrays). molecular approaches are particularly relevant for the diagnosis of infection in patients with pidd. signal and target amplification techniques can be coupled with nucleic acid hybridization or probe assays to allow detection of pathogen dna or rna that is present in very small amounts in clinical samples. in patients with pidd, vaccines could play an important role in preventing infections with vaccine-preventable diseases. even pidd patients may generate some protective responses [19, 20] . the decision to immunize such patients or not depends on the type and severity of pidd as well as the type of vaccine to be administered (live or inactivated) ( table 1 ). in general, inactivated vaccines are safe for pidd patients while immunization with live attenuated vaccines is a known hazard to patients with serious immunodeficiencies of t cell, b cell, and phagocytic cell origin (table 1 ). in less severe pidd, the vaccine can induce adequate protection as in healthy individuals or the efficacy may be reduced [20] [21] [22] . of note, immunoglobulin replacement therapy induces passive immune protection to some vaccine-preventable infections, such as measles, mumps, rubella (mmr), and varicella. in addition, live viral vaccines have greatly reduced efficacy while on immunoglobulin replacement. therefore, vaccine administration in patients receiving regular immunoglobulin replacement treatment should be withheld until at least 3 to 11 months (depending on dose) after cessation of such treatment, if cessation and vaccination are safe. in addition, pidd patients who have received hematopoietic stem cell transplantation but have incomplete immune reconstitution or are under immunosuppression should not receive live attenuated vaccines. in general, the decision of administering live viral vaccines should be made by clinical immunology experts [23] . in developing countries where polio is still endemic and oral polio vaccine is essential for eradicating the disease, it is of utmost importance that all pidd patients and family members should not receive live oral polio (opv) because of the reported prolonged excretion of the virus for months and even years [24] . in addition, vaccine-associated paralytic polio is a real risk for some with pidd. these patients and family members should receive inactivated polio vaccine (ipv) instead of opv. similarly, the hazards of administering bacillus calmette-guerin (bcg) vaccine to pidd patients have been documented. in a series of 349 bcg, vaccinated severe combined immunodeficiency (scid) patients from 28 centers in 17 countries, 34% of scid patients developed disseminated bcg infection and had the worst outcome [25] . patients with chronic granulomatous disease vaccinated with bcg also developed local and disseminated bcg infection. recently, vaccine strains of rubella virus were found to be associated with skin granulomas in pidd patients [26] [27] [28] . siblings and household contacts of patients with suspected or diagnosed pidds should receive all the national immunization scheduled vaccines. ipv should be substituted for op in families where an antibody-deficient patient exists. yearly influenza vaccination of family members is recommended in order to reduce the risk of household-social transmission [20, 22, 29] . diseases where routine vaccination has reduced incidence can occasionally experience a resurgence in times of economic hardship with reduced attention to vaccination. diphtheria is currently seen in venezuela for this reason. war and disruption of health infrastructure are other common reasons for resurgence in vaccine-preventable diseases. in other settings, antivaccination sentiment has led to outbreaks of diphtheria, measles, and mumps. an additional consideration is that not all countries provide the same level of vaccination, and therefore, vaccine-preventable illness can still be seen regionally. these outbreaks represent a significant risk to patients with pidd. a universal consideration for patients with pidd is the concern about antibiotic resistance, which varies dramatically around the world. for certain high impact infections, the emerging antibiotic resistance patterns will be discussed below. antimicrobial resistance occurs naturally, but misuse and overuse of antimicrobials are accelerating this process. in nearly every country, antibiotics are overused and misused in people and animals leading to antibiotic resistance in every country. key resistance patterns to common bacteria include emergence of carbapenem-resistant klebsiella pneumoniae around the world with high frequency of resistance (due to different mechanisms) in the mediterranean, with greece, italy, and turkey having endemic spread of this pathogen [17] . carbapenem-resistant strains among other genera of enterobacteriaceae have also been recognized. they are particularly common in greece, but have been found widely distributed [30] . the new delhi metallo-beta-lactamasemediated resistance, which is endemic in the indian subcontinent but becoming increasingly spread worldwide, is a growing concern [30, 31] . as a common cause of urinary tract infections, colistin is the only recourse when carbapenemresistant enterobacteriaceae, and colistin resistance has recently emerged in small outbreaks throughout the world [32] . in these cases, the infection is essentially untreatable. fluoroquinolone-resistant escherichia coli, a common cause of urinary tract infections, now accounts for over half of the isolates in some asian countries [33, 34] . t he emergence of resistance to antibiotics in grampositive pathogens has become a major international problem as there are fewer, or even sometimes no effective, antimicrobial agents available for infections caused by these bacteria. methicillin-resistant staphylococcus aureus is common in many countries and in fact has spawned a nomenclature recognized by the general public: mrsa. several studies have reported resistance to the newer antimicrobial agents like linezolid, vancomycin, teicoplanin, and daptomycin [35] . thus far, these isolates appear to be uncommon and have been found in <1% of isolates in brazil, china, ireland, and italy, with nearly undetectable rates elsewhere. vancomycinresistant enterococcus (vre) is growing in frequency and can now be a cause of primary bacteremia in immunocompromised individuals [36] . a key message is that antibiotic resistance is increasing (generally) and travelers should be alerted to resistance to commonly encountered organisms, and if antibiotic prophylaxis is required, their prophylaxis is adjusted. neisseria gonorrhoeae is a specific organism for which resistance has become especially problematic. it has progressively developed resistance to virtually all antimicrobial agents since introduction of sulfonamides in mid-1930s. the current treatment guidelines recommend dual antimicrobial therapy (ceftriaxone 250-500 mg × 1 plus azithromycin 1-2 g × 1) as first-line regimen [37, 38] . although dual therapy is very effective, development of concomitant ceftriaxone and azithromycin resistance is likely to emerge [39] . the risk of untreatable n. gonorrhoeae demands better global antimicrobial surveillance system, clinical trials on combined therapy of existing drugs as well as novel agents in monotherapy, and development of a gonococcal vaccine. for pidd patients, guidance on barrier methods for the prevention of sexually transmitted diseases should be a part of any pre-travel counseling. mycobacteria tuberculosis (mtb) is an age-old pathogen with emerging resistance. drug-resistant tb, fueled by the hiv epidemic, is a global threat. in 2015, who estimated 480,000 new cases of multidrug-resistant tb (mdr-tb) and an additional 100,000 new cases of rifampin-resistant tb (rr-tb) who would also require mdr-tb treatment. treatment of mdr-tb and mycobacterium bovis disease is beyond the scope of this text and reader is referred to recent who mdr treatment guidelines [40] . regions of the world with >6% mdr tb include regions of azerbaijan, kazakhstan, russia, uzbekistan, china, georgia, and eastern europe. extensively drug-resistant tb (xdr tb) refers to mtb resistant to isoniazid, rifampin, any fluoroquinolone and at least one second-line drug. xdr tb has been reported in 105 countries. on average, 10% of patients with mdr tb have xdr tb. as is true for all types of mtb, xdr tb is contagious and small outbreaks related to person-person transmission have been reported. non-tuberculous mycobacteria (ntm) cause significant systemic infections in patients with defects of the il-12/ ifnγ/stat1 axis as well as in gata2 deficiency and can cause significant pulmonary infections in pidd patients with bronchiectasis. compared to tb, ntm is acquired from the environment and not from person-to-person transmission; therefore, acquisition of antibiotic-resistant strains is less common. however, in these individuals with pidd, ntm disease is often chronic and can be difficult to eradicate, and resistance can then easily develop during therapy. using combination of antibiotics is essential, and consultation with those familiar with treatment of treatment refractory ntm disease is recommended. aspergillus species are ubiquitous inhaled molds with worldwide distribution that cause opportunistic infections in immunocompromised patients [41] . aspergillosis also occurs in pidds associated with quantitative and/or qualitative phagocyte defects; it develops most frequently in chronic granulomatous disease (cgd) patients (prevalence,~40%), while it is seen less often in patients with gata2 deficiency, card9 deficiency, and congenital neutropenia syndromes [42, 43] . upon inhalation, aspergillus species cause invasive pulmonary disease in susceptible hosts with the exception of card9 deficiency, where aspergillosis has a predilection for extrapulmonary tissues with sparing of the lungs [44] . diagnosis is established by fungal culture and/or histopathology showing acute-angle septate hyphae and/or detection of galactomannan, an aspergillus cell wall component released during invasive infection, in serum or bronchoalveolar lavage fluid [41] . while azole-susceptible aspergillus fumigatus is still the most common infecting species in pidd patients, the emergence of azole-resistant a. fumigatus and nonfumigatus aspergillus species underscores the importance of a high index of suspicion in patients who do not respond to front-line voriconazole treatment [45] . the advent of fungal molecular diagnostics has demonstrated that patients with pidds are more prone to infections by uncommon low-virulence aspergillus species with intrinsic resistance to azole antifungal agents that do not infect patients with iatrogenic immunosuppression. these primarily include aspergillus viridinutans, aspergillus tanneri, and neosartorya udagawae, which pose major diagnostic and therapeutic challenges due to their impaired sporulation and propensity for contiguous and distant tissue spread, respectively. in addition, acquired azole resistance in a. fumigatus can be seen in patients on long-term exposure to azole drugs used as treatment and/or prophylaxis [46] . azole resistance in these strains is predominantly caused by point mutations in the lanosterol 14α-demethylase gene that encodes the cyp51a protein, the primary target of azole drugs. importantly, infection by azole-resistant a. fumigatus strains without prior exposure of patients to azole antifungals has recently emerged as an important global health concern due to the widespread use of sterol demethylase inhibitor fungicides in agriculture that results in cross-resistance with the triazole antifungals used in clinical practice [47] [48] [49] . fungicide-driven azole-resistant environmental aspergillus strains were first observed in the netherlands in 2007 and have since then been documented in other parts of europe, south and north america, the middle east, australia, africa, and asia. the prevalence of these azole-resistant aspergillus strains among clinical aspergillus strains recovered from patients in 19 european countries was reported to be 3.2%, while alarmingly in some areas >20% of recovered strains exhibited azole resistance [50] . the emergence of such aspergillus environmental strains poses serious threats to the treatment of immunosuppressed patients. mortality rates as high as 88% have been seen due to delays in diagnosis and suboptimal treatment with azole antifungals [51] . although no prospective data exist for the treatment of patients with such resistant fungi, the use of amphotericin b-and echinocandin-based regimens are preferred over azoles [52] . in the absence of azoles, the lack of alternative oral antifungal agents is particularly challenging for pidd patients such as those with cgd who require long-term suppressive antifungal treatment. candida species are commensal yeast fungi that colonize the mucosal surfaces of~60% of healthy individuals [53] . when perturbations in immunity and/or microbiota occur, candida causes opportunistic mucosal or systemic infections that depend on clearly segregated immune responses for host defense. specifically, t cells of the th17 program are critical for mucosal and phagocytes for systemic immunity [54] . indeed, a proportion of patients with cgd and complete myeloperoxidase deficiency develop systemic, but not mucosal candidiasis [42] , whereas patients with monogenic syndromes of chronic mucocutaneous candidiasis due to mutations in the il-17 signaling pathway (il17ra, il17rc, il17f, act1) or in other genes that adversely affect th17 differentiation (rorc, stat3, stat1, aire, dock8, stk4, irf8) do not develop systemic candidiasis. the only known pidd to date that results in combined mucosal and systemic candida infection susceptibility is card9 deficiency. systemic candidiasis in card9-deficient patients has a predilection for the central nervous system, associated with brain-specific impaired recruitment and effector function of neutrophils [55] [56] [57] . diagnosis of candida infections is established by culture. azole-susceptible candida albicans is still the most common infecting species in pidd patients; however, emergence of azole-resistant c. albicans is not uncommon during chronic azole use, making long-term therapy challenging due to lack of alternative oral antifungal treatment options [58] . beyond c. albicans, non-albicans candida species can rarely infect pidd patients, some of which have intrinsic resistance to azole antifungals, including candida glabrata and candida krusei [59] . most recently, candida auris has emerged as a global public health concern due to its resistance to antifungal drugs, high virulence potential, propensity for health careassociated horizontal transmission and outbreaks in health care settings, persistence in the human skin and hospital environment, inherent resilience to antiseptics, and misidentification by routine biochemical methods as other yeasts (most often candida haemulonii, but also candida famata, rhodotorula glutinis, or saccharomyces cerevisiae). c. auris was first recovered from the ear canal of a patient in japan in 2009 and has since then been reported to cause life-threatening infections and hospital outbreaks in europe, asia, africa, the middle east, and south and north america [60] [61] [62] [63] . most of the reported strains of c. auris have intrinsic resistance to fluconazole and other triazole antifungal agents, while a significant proportion of strains has elevated minimum inhibitory concentrations to amphotericin b and echinocandins, with some strains reportedly resistant to all three classes of azoles, polyenes, and echinocandins [64] . avoidance of azole antifungals is important in c. auris-infected patients, and echinocandinor amphotericin b-based regimens are preferred, guided by strain-specific in vitro susceptibility patterns. this section on vector-borne infections is a major focus of this review because the infections are often more severe in immunocompromised individuals and because there are mitigation strategies that should be considered even in the absence of defined medical treatments for infection. prevention of mosquito bites depends somewhat on the endemic species but there are generalizations. the use of a mosquito repellant such as deet, oil of lemon eucalyptus, ir3535, or picaridin is as important as using long sleeves and long pants while in an affected area. deet and picaridin are safe in pregnancy, and some data support their greater efficacy [65] . air conditioning and fans tend to keep mosquitoes away but netting at night is essential in mosquito-prone areas. light-colored clothing is less attractive to mosquitoes than dark clothing, and scented detergents and use of dryer sheets tend to attract mosquitoes, hence should be avoided. aedes species prefer to bite indoors and thrive in urban areas with small puddles of water. they bite most frequently around dawn and dusk. anopheles species have very similar behaviors. culex mosquitoes, in contrast, bite primarily at night. tick and fly bite prevention is focused on physical and chemical prevention. for ticks, physical inspection for biting ticks should also be incorporated. arthropod-borne viruses (arboviruses) are transmitted to humans through the bites of infected insects: mosquitoes, ticks, sand flies, or midges. some arboviruses can be transmitted through blood transfusion, organ transplantation, perinatal transmission, consumption of unpasteurized dairy products, or breastfeeding. there are >100 arboviruses causing human disease. most arboviral infections are asymptomatic. infectious manifestations range from mild febrile illness to severe encephalitis. arboviral infections are often categorized into two primary groups: neuroinvasive disease and non-neuroinvasive disease. tables 2 and 3 list the encephalitigenic viruses and the febrile/hemorrhagic disease causing viruses. in this section, we will highlight west nile virus, the most common of the encephalitogenic arboviruses. west nile virus is a single-stranded mosquito-borne rna virus of the family flaviviridae. the natural transmission cycle of the virus occurs in culex mosquitoes and birds. humans and horses are dead-end hosts for the virus. the most common mode of transmission to humans is through the bite of infected mosquitoes. other less common modes of transmission include blood transfusions, organ transplantations, occupational exposure in laboratories and mother-to-child transmission during pregnancy, childbirth, and breastfeeding. west nile virus has been diagnosed in >2000 people in the usa with slightly more than half having neuroinvasive disease. since 1999, >40,000 people in the usa have been infected. it is also common in africa, europe, and asia [66] . infection with west nile virus is asymptomatic in most individuals [67, 68] . the incubation period lasts for 2 to 6 days. however, it can be significantly longer in immunocompromised hosts. clinical manifestations following infection develop in 20-40% of those infected and include fever, headache, myalgia, arthralgia, vomiting, and rash. severe neuroinvasive disease leading to meningitis, encephalitis, and flaccid paralysis develops in less than 1% of infected individuals. the overall case fatality is approximately 10% which is a disproportionately high mortality in patients with encephalitis and myelitis. diagnosis of west nile virus rests on demonstration of specific antibody responses especially specific igm antibodies in the serum or csf of infected individuals by enzyme immunoassays. plaque reduction neutralization tests can differentiate cross-reactive antibodies. detection of virus in csf, blood, or tissue specimens by culture or pcr is particularly useful in immunosuppressed individuals who may have impaired serological responses. west nile virus has been reported in the context of both primary and secondary immunodeficiency. severe neurological manifestations have been reported in hiv-positive individuals, individuals receiving immunosuppressive therapy including rituximab, and individuals with pidd. infection with wnv has been reported in individuals with common variable immunodeficiency, idiopathic cd4 lymphopenia, gata2 deficiency, and a case of probable good's syndrome [69] [70] [71] . individuals with antibody defects, neutropenias, and impaired t cell responses are potentially at an increased risk of severe manifestations of wnv disease including severe neurological involvement. this section highlights the four important non-neuroinvasive arboviruses based on current geographical distribution: dengue, yellow fever, zika, and chikungunya (table 3) . approximately 100 countries/territories have reported local transmission for both chikungunya and dengue viruses [72] . dengue is due to infection with one of four dengue virus serotypes, transmitted by a mosquito (typically aedes aegypti). this febrile illness affects all ages with symptoms appearing 3-14 days after the infective bite. symptoms range from mild to high fever, with severe headache, musculoskeletal pain, and rash. severe dengue (also known as dengue hemorrhagic fever) occurs in 0.5-5% of cases and is characterized by fever, abdominal pain, persistent vomiting, bleeding, and breathing difficulty and is a potentially lethal complication [73] . paradoxically, the main risk factor for dengue hemorrhagic fever is pre-existing antibodies. early clinical diagnosis and comprehensive management by experienced clinicians increase survival. dengue is ubiquitous throughout the tropics with the highest infection rates in the americas and asia. dengue is now endemic in 100 countries, causing up to 50 million infections a year and 22,000 deaths, mainly among children. over half of the world's population inhabits areas at risk for dengue infection [74] . the presence of a. aegypti in over 125 countries potentially puts almost the whole world at risk of becoming infected with this virus. pcr is widely used as serologic methods to diagnose dengue. immunity to one serotype does not confer protection against the other three serotypes, and heterologous antibody may be a risk factor for hemorrhagic dengue [73] . the natural history of dengue has been studied in hiv patients where hiv did not appear to increase severity. there have been no reports of patients with pidd having dengue; however, dengue infection after solid transplantation has been reported [75] [76] [77] [78] with some patients having severe complications suggesting that t cell compromise in pidd could be a risk for severe disease. there are no antiviral medications utilized for dengue virus. care of patients with hemorrhagic disease requires meticulous approach to fluids and coagulation status. one dengue vaccine has been registered in several countries (cyd-tdv) for individuals from 9 to 45 (or 60) years old. it is a live attenuated recombinant tetravalent vaccine with backbone of the attenuated yellow fever 17d virus genome with the prm and e genes that encode the proteins from the four wild-type dengue viruses. the who has suggested its use in regions where seroprevalence of dengue virus of any serotype is 70% or greater, but has not recommended it to hiv-infected, immunocompromised individuals, nor pregnant or lactating women [79] . most people infected with the yellow fever virus have no illness. symptoms of yellow fever include sudden onset of fever, chills, headache, musculoskeletal pain, nausea, vomiting, fatigue, and weakness. the incubation period is typically 3-6 days, and symptoms may appear after return from travel. most people improve after the initial presentation, but 15% of cases progress to develop a more severe form of the disease, usually after a day of presumed recovery. the severe form is characterized by high fever, jaundice, bleeding, and eventually shock and failure of multiple organs [80] . yellow fever virus is an rna virus that belongs to the genus flavivirus. it is transmitted from mosquitoes after biting an infected primate. it is widely distributed in the equatorial tropics [80] . aedes species of mosquitoes are primarily responsible for transmission. large epidemics of yellow fever occur when the infection enters heavily populated areas with a high mosquito density and where most people have little or no immunity. west africa has undergone a large-scale vaccination campaign with impressive results and yellow fever is now uncommon in west africa [81] . serologic testing for yellow fever is the diagnostic standard. pcr can be performed on tissue samples. there are no published studies of yellow fever in immunocompromised people, but the elderly, very young, people with autoimmune disease, or who are post-thymectomy are at risk from the attenuated vaccine strain. thus, it seems likely that any immunodeficiency would be associated with more severe wildtype disease. currently, no specific antiviral drug for yellow fever exists. treatment of dehydration, liver and kidney failure, and fever improves outcomes. the yellow fever vaccine is highly effective; however, immunodeficient patients should not receive it. infection with zika virus is often asymptomatic. it represents a mild infection for those who have any symptoms [82] . the zika virus has been detected in urine, semen, and saliva of infected individuals, and transmission from transfusion and sexual relations has been reported. it is also detectable in breast milk, but breastfeeding-associated transmission has not been reported so far [83] [84] [85] . contact with highly infectious body fluids from patients with severe zika infection has also been suggested as a possible mode of transmission [86] . of tremendous importance is the presence of prolonged shedding of zika virus in a congenitally infected newborn [87] . the main public health risk of zika virus is microcephaly in newborns from infected mothers [88] . zika virus is capable of infecting human neural progenitor cells in vitro. infection results in disruption of cell cycle, increased cell death, and attenuated neuron growth [89] . zika is not thought to be a major risk for people with pidd (based on the experience with hiv patients, but our recognition of zika is very recent. there is no known specific treatment for zika; however, there is an important effort to develop a vaccine. chikungunya fever is an acute febrile illness caused by the alphavirus, chikungunya virus. the incubation period is usually 3-7 days after the bite of an infected aedes mosquito. there is abrupt onset of high fever, and the fevers can be biphasic [90, 91] . severe polyarthraligias develop after the onset of fever. the joint pains can affect any joint, but the pattern is usually symmetric and a true acute arthritis is not uncommon. the proportion of infected people with rash has varied across studies. when seen, the rash appears after the fever as a truncal maculopapular type of rash [92] . cervical adenopathy is another common feature of infection. death is uncommon in chikungunya, but serious complications such as myocarditis have been seen. over half of the patients report continued joint symptoms 1 year after acute illness and 12% have long-term joint symptoms [93] . chikungunya originated in central/east africa. in forests, the virus circulates in aedes mosquitoes and non-human primates. in urban centers, the virus circulates between humans and mosquitoes similar to the pattern of dengue. there have been periodic urban outbreaks in asia and africa since 1960 with an acceleration in spread since 2004 [94] . an important consideration is the periodic outbreaks with high attack rates in naïve populations. areas at risk currently are east africa, central africa, la reunion, india, and southeast asia. diagnostic testing utilizes pcr or serology. the threat to immunodeficient patients is not entirely clear. there are a few provocative cases where the immunocompromised appears to have been associated with fewer joint symptoms, but there were two patients, medically immune compromised, who had very severe disease [95, 96] . this suggests that the presentation may be atypical and the course may be severe in immunodeficient people. treatment is supportive, although chloroquine, acyclovir, ribavirin, interferon-ɑ, and steroids have limited preclinical data to support clinical trials. babesia microti (the main species in the usa) infection can be asymptomatic, but many people develop fever, chills, headache, myalgias, anorexia, nausea, or fatigue [97] . babesiosis often causes hemolytic anemia. b. microti is spread by ixodes scapularis ticks in the usa and babesia divergens (the main species in europe) is spread by ixodes ricinus. symptoms begin 1-3 weeks after a bite from an infected tick with b. divergens having a higher mortality rate and greater symptomatology compared to b. microti. the main geographic areas involved are the coastal eastern usa and cattle breeding areas throughout europe. the diagnosis is usually by inspection of a blood smear or through serology. a pcr test has just been developed. immunodeficiency, asplenia, and older age are recognized risk factors for severe disease and even death [98] [99] [100] . thus, congenital asplenia would be considered a major risk for severe disease. a combination of atovaquone and azithromycin is generally used for therapy, although clindamycin and quinine have been used with success. patients with severe illness have been treated with exchange transfusions. five different types of plasmodium (plasmodium falciparum, plasmodium vivax, plasmodium ovale, plasmodium malariae, and plasmodium knowelsi) infect humans. malaria is transmitted primarily by female anopheles mosquitoes. symptoms vary depending on the type of plasmodium involved but usually include high fever, chills, and headache. in some cases, the illness can progress to severe anemia, kidney and respiratory failure, and death. the incubation period typically ranges from 9 to 14 days for p. falciparum, 12 to 18 days for p. vivax and p. ovale, and 18 to 40 days for p. malariae. in p. vivax and p. ovale infections, relapses can occur months or even years without symptoms. p. vivax and p. ovale have dormant liver stage parasites that must be specifically eradicated through medical therapy. malaria has been a global health concern throughout history and is a leading cause of death and disease across many tropical and subtropical countries. over the last 15 years, new control measures have reduced malaria by over half [101] . the democratic republic of the congo and nigeria account for over 40% of the estimated total of malaria deaths globally. high rates of malaria are seen in india as well. nevertheless, malaria exists in most tropical regions of the americas, africa, and asia [101] . the diagnosis of malaria depends on the demonstration of parasites in the blood, usually by microscopy. the threat to immunodeficient patients is not entirely clear, but patients with hiv seem to have no additional burden of disease other than an increase in placental malaria, suggesting that t cells are not central to the defense of malaria [102, 103] . asplenia is a known risk factor for severe malaria [104] . antibodies appear to be both protective and pathologic [105, 106] . treatment and prophylaxis depend on the region of the world because the parasites and resistance are highly variable and highly dynamic. therefore, it is best to consult an infectious disease specialist familiar with the prophylaxis before travel and for treatment of acute cases. leishmaniasis is due to infection with an obligate macrophage intracellular protozoa of the genus leishmania. it causes a spectrum of disease ranging from a cutaneous ulcer to mucosal disease and the most severe form, visceral leishmaniasis (vl). the liver, spleen, and bone marrow are major sites of parasite growth and disease pathology in vl [107] . purely cutaneous leishmaniasis is most often caused by leishmania major, leishmania. tropica, leishmania aethiopica, leishmania infantum, and parasites belonging to the leishmania mexicana complex, the leishmania braziliensis complex, and the leishmania guyanensis complex. mucocutaneous disease is most often due to l. braziliensis complex, leishmania panamensis, leishmania amazonensis, and rarely by leishmania guyanensis. vl is most often caused by leishmania donovani and leishmania infantum (previously l. chagasi) [108] . cutaneous leishmaniasis can have many variations but is most often an ulcer that develops after an indolent papule. the incubation period ranges from weeks to months. the ulcer usually heals within months to years, and there can be mild adenopathy. mucocutaneous leishmaniasis follows a cutaneous ulcer and is only caused by l. braziliensis parasites. oral and respiratory mucosa are most often involved with granulomatous lesions that may be extremely destructive. vl is associated with fever, lymphadenopathy, hepatosplenomegaly, wasting, hypoalbuminemia, and pancytopenia. this picture evolves over months to years. there can be secondary immune deficiency due to the pancytopenia. the epidemiology has changed dramatically and has been impacted by climate change [109] . the sand flies that spread the parasite are affected by temperature and rainfall. in most endemic regions, leishmania has a patchy distribution due to micro-ecologic factors. poverty has been demonstrated to be a major risk factor for leishmaniasis [110] . it has been estimated that up to half a million new cases of vl occur every year, but the majority are in resource-poor countries such as bangladesh, nepal, india, sudan, ethiopia, and brazil. emergence of resistance to antimony-based drugs has also led to a major resurgence of disease. the primary reservoir for leishmania is forest rodents, but dogs are increasingly important. the growing spread of leishmania is due to a combination of factors, and now 88 countries have reported cases. immunodeficient patients are more susceptible to infection, and relapse occurs more frequently [111] . the risk of developing vl is estimated to be between 100 and 2300 times higher in hiv-infected than in non-hiv-infected individuals [112] , and these patients have higher rates of treatment failure with the illness often taking a prolonged chronic course and higher mortality rates [113] . a similar picture has been seen in patients with vl-infected post-kidney transplantation [114] . dendritic cells, t cells, and the generation of reactive oxygen species have been shown to be essential for parasite control [115] [116] [117] . pidd with impaired il-12 production have been associated with severe disease [118] . a patient with cd40l deficiency, associated with poor il-12 production, had chronic leishmania and died in spite of aggressive treatment. vl has been reported in cgd patients [119] . six cgd patients were infected by leishmania, and they developed hemophagocytic syndrome with a poor outcome for one of them [120] . the diagnosis of leishmaniasis is usually by visual inspection for parasites. immunofluorescence microscopy, direct agglutination, skin test, and pcr have been used. treatment is long-term and difficult. emerging resistance to first-line treatment is increasingly problematic. pentavalent antimonials are the mainstay of treatment in most countries, but liposomal amphotericin is widely used where resistance occurs. newer drugs with more favorable side effect profiles have been used in certain geographic settings: miltefosine, paromycin, and sitamaquine. rickettsiae are small gram-negative bacteria. they are obligate intracellular parasites, and the primary target in humans appears to be endothelial cells with subsequent thrombosis and clinical presentation of vasculitis [121] . the rickettsiaceae family, originally defined by non-specific phenotypic characteristics, was reclassified into different strains and subspecies based on gene sequencing and genetic phylogeny ( table 4 ). the clinical presentation of rickettsial disease can vary, but the classic triad of fever, rash, and headache still provides major clues for the diagnosis [122] [123] [124] . however, rash is not an obligatory sign, and the incidence of rash can range between 100% for rickettsia conorii infection,~90% for rickettsia rickettsii, 30% for rickettsia africae, and less than 10% in the case of anaplasma phagocytophilum infection. therefore, fever in patients with exposure to a potential vector should raise a concern for a rickettsial disease, especially if there is also evidence of rash, inoculation eschar, or localized lymphadenopathy. additional supporting laboratory findings can include neutropenia, thrombocytopenia, and increase in liver transaminases. the geographic distributions of rickettsioses and ehrlichioses are mostly dependent on their vector distribution [125] . as such, louse-borne and flea-borne are worldwide, reflecting the worldwide distribution of lice and fleas, with a tendency to parasite poor people in cold places and, characteristically, during wars. ticks, on the other hand, depend on their environment and most do not have a worldwide distribution. with the exception of the dog tick, vector for r. conorii in asia and north africa, r. rickettsii in the usa, rickettsia massiiae and erhlichia canis worldwide, most other tick-borne diseases are restricted to areas of the world correlating with the distribution of their vector [126] . for that reason, it should be anticipated that climate and environmental changes will affect vector distribution and its reservoir host and, hence, the geography and epidemiology of tick-borne diseases [10, 127, 128] . diagnosis presents a challenge, as it is extremely difficult to grow these organisms in culture. immunohistochemistry and pcr can be helpful. the severity of rickettsial disease varies with the causative agent and the host. r. rickettsii, rickettsia prowazekii, and orienta tsutsugamushi are considered most pathogenic. as for host factors, although severe and fatal cases have been described in healthy immunocompetent hosts [129, 130] , there is evidence to suggest that children under the age of 10 [130] and immunocompromised hosts either secondary to hematologic malignancies, immunosuppressant treatment for organ transplantation, or hiv infection are at a greater risk to develop more severe disease with higher case fatality rates [131, 132] . all rickettsiaceae are intracellular pathogens, and one could expect an increased risk for severe disease in pidds with abnormal t cell function. five to 7 days of doxycycline is the preferred treatment for non-pregnant adults and children. treatment should not be delayed while awaiting diagnostic testing [133] and can be given to children despite a minimal risk for dental staining. alternative treatments include azithromycin for mild disease [134] and chloramphenicol for pregnant women. anaplasma is an intracellular bacterium that infects wild and domestic mammals, including man. a. phagocytophilum was formerly known as human granulocytotropic ehrlighiosis but is now known as human granulocytotropic anaplasmosis [135] . e. chaffeensis infects monocytes and causes human monocytic ehrlichiosis [136] . anaplasma and ehrlichia have historically cycled within non-human enzootic hosts, and man has become infected through increasing interactions with the environment. ehrlichia and anaplasma are transmitted by ixodes species of ticks, and their ranges include the eastern usa, south central usa, and scattered regions of europe, as far north as sweden. these infections have not been seen in humans in the southern hemisphere, but there are reports of organisms being identified [137] . a less common mode of transmission is through transfusions. the symptoms of ehrlichia and anaplasma infections are similar [136] . abrupt onset of an influenza-like illness occurs about 12 days after a tick bite. ehrlichia can cause a mild rash (30% of adults and 60% of children), but rash is uncommon in anaplasma infections. highly suggestive laboratory features are leukopenia and thrombocytopenia. mortality in the general populations appears to be <5%, but icu admission is not uncommon. hemophagocytosis has been described with anaplasma [138] and ehrlichia [139] . both infections are more severe in any setting of immune compromised, including asplenia [129, 135] . the diagnosis is typically made by pcr, and doxycycline is the recommended treatment. intracellular inclusions can be seen on cbc smears (more often in anaplasma than ehrlichia). an uncommon but well described facet of these infections is that the tick vector can also transmit borrelia burgdorferi and babesia microti, and simultaneous infection with multiple organisms can occur. c. burnetii is a highly pleomorphic gram-negative coccobacillus and the causative agent of q fever. q fever is a zoonosis, and the most common reservoirs are cattle, sheep, and goats but many other animals can be infected by c. burnetii [140, 141] . when infected, these domestic animals can shed the organism in urine, feces, milk, and especially birth products. the pathogen survives within the phagolysosome of host cells, and a spore stage has been described. this spore stage explains the ability of c. burnetii to survive in unfavorable environmental conditions, and it can be an environmental risk for months to years after shedding from an infected animal. q fever is considered an occupational disease affecting people with direct contact with infected animals; however, indirect contact through exposure to contaminated animal products has also been described to cause disease outbreaks. humans are infected by inhalation of contaminated aerosols. following an average incubation period of 20 days, infected patients can present with severe headache, fever, chills, fatigue, and myalgia. other signs and symptoms depend on the organs involved. in contrast to rickettsial diseases described above, rash rarely occurs in the early stages of the disease. c. burnetii can cause a range of clinical symptoms. a self-limited febrile illness is probably the most common form of q fever. pneumonia, either atypical or severe, is also common and can be a part of acute q fever syndrome. in contrast, a variety of manifestations can be recognized in chronic q fever, including endocarditis, endovascular infection, osteomyelitis, hepatitis, interstitial pulmonary fibrosis, prolonged fever, and purpuric vasculitic rash. q fever diagnosis is based on serologic testing with indirect immunofluorescence being the best for differentiating between acute and chronic q fever (high antiphase i antigen titer). the treatment of choice for acute q fever is doxycycline, with co-trimoxazole, chloramphenicol, or rifampin being an accepted alternative. there is no agreement on the treatment for q fever endocarditis, and a combination of doxycycline with either fluoroquinolone or hydroxychloroquine is recommended. there is also controversy regarding the duration of treatment, ranging from 2 years to indefinite treatment. old evidence suggests that q fever is more common in immunocompromised patients. a french study showed higher incidence of antibodies to c. burnetii in hiv positive compared to hiv-negative patients (10.4 vs 4.1%). in addition, 5 out of 68 hospitalized patients were hiv positive (7.3%), suggesting a more frequent symptomatic disease [142] . a smaller similar study performed in central africa failed to show increased incidence of seropositivity in hivpositive patients [143] . two case reports describe severe disease in immunocompromised patients. the first was a case of fatal q fever disease in an 11-year-old male with cgd [144] . the patient was treated with broad spectrum antibiotics, but without coverage for q fever. the second case was a 53-yearold asplenic male who presented with fever, jaundice, and encephalopathy and was diagnosed with acute q fever [145] . the patient was successfully treated, but the two case reports could suggest susceptibility in cases of phagocytic disorders. the bartonellaceae are fastidious, facultative intracellular gram-negative bacilli (table 5 ). most species infect primarily non-human animals, and in most cases, human are considered incidental hosts. the documented common human pathogens include bartonella bacilliformis, bartonella henselae, and bartonella quintana, and it is believed that humans are the primary mammalian reservoir of b. quintana. infection occurs through inoculation of bartonella-infected arthropod feces into breaks in the skin. the epidemiology of bartonella infection in humans follows the distribution of the vector. as such, infection with b. bacilliformis follows the distribution of the sand fly vector (lutzomya) and is confined to the andes mountain in peru, ecuador, and colombia at heights of between 500 and 3200 m. the human body louse pediculus humanus is the vector of b. quintana, which explains the global distribution of this pathogen and worldwide outbreaks of trench fever, mostly in conditions of poor sanitation and upon exposure to body lice. trench fever was responsible for over a million infections during world war i. fever, either abrupt or indolent in onset, with a maculopapular rash, conjunctivitis, headache, myalgias (most often affecting legs), and splenomegaly was described. urban b. quintana infections occur most often among homeless and have a distinct clinical picture with fever as the most common manifestation. endocarditis occurs in many [146] . bartonella henselae is globally endemic, and domestic cats are a major reservoir. the major arthropod vector of b. henselae is the cat flea, which is responsible for cat-to-cat transmission. human infection, called cat scratch disease, is assumed to involve inoculation of bartonella-infected flea feces into the skin during a cat scratch. b. henselae causes primarily adenopathy and neurologic symptoms [147] . b. bacilliformis causes a condition with two phases: the acute phase with fever, anemia, and transient immunosuppression followed by nodular dermal eruption [148] . recently appreciated are the ongoing systemic features during the eruptive phase such as arthralgias, adenopathy, and anorexia [149] . diagnosis of bartonella-associated diseases can be achieved by direct examination of clinical material, bacteriologic culture methods, serologic and immunocytochemical studies, pcr-based assay, or combination of these methods. bartonella infection can present differently in immunocompromised hosts [150] . in addition to higher prevalence of bartonella infection in hiv patients [151] , both b. quintana and b. henselae can induce neovascular proliferation which might involve the skin, lymph nodes, and a variety of internal organs including the liver, spleen, bone, brain, lung, bowels, etc. these neovascular lesions, known as bacillary angiomatosis/peliosis (ba/bp), were initially described in hiv-infected patients with advanced disease and was later described in other immunocompromised hosts secondary to immunosuppressant treatment for solid organ transplantation or hematologic malignancy [152] [153] [154] [155] . cutaneous ba lesions can vary in presentation and can be subcutaneous, dermal nodules, single or multiple papule that can be flesh colored, red, or purple. lesions may ulcerate and bleed. they can change in number and size (millimeters to centimeters; few to hundreds) and can involve mucosal surfaces or deeper soft tissues. similar variation can be seen with visceral involvement. histologically, lesions consist of lobular proliferation of small blood vessels and neutrophilic predominant cell infiltration. the term bacilliary peliosis is used to describe bloodfilled cystic spaces mostly involving the liver, spleen, and lymph node. pathogenic bacteria can be isolated from vascular lesions. while both pathogens were associated with cutaneous lesions, only b. henselae was associated with visceral bp [156] . based on hiv literature, it is reasonable to expect an unusual presentation of bartonella infection especially in pidds involving t cell dysfunction. bartonella infection was described as a cause for hepatitis in a single cd40l deficiency patient [157] . in addition, since cases of granulomatous disease due to bartonella infection [158] [159] [160] have been described, it should be considered in the differential diagnosis of pidd with granulomatous inflammation. borrelia spp. the genus borrelia belongs to the spirochaetaceae family. it includes b. burgdorferi which causes lyme disease and species that cause relapsing fever. the latter are further divided into tick-borne species and louse-borne species. louse-borne relapsing fever (lbrf) is caused only by borrelia recurrentis and is spread by human body louse. the disease was epidemic in the early twentieth century, and it is estimated that more than 50,000 died of lbrf during world war ii. with sanitation improvement lbrf is now found only in the horn of africa and among homeless people in europe. more recently, cases of lbrf in refugees and migrants were described [161, 162] . tick-borne relapsing fever (tbrf) is caused by a group of pathogens which are maintained by and survive in softticks (orinthodoros genus). each tbrf borrelia species depends on one specific species of soft-body tick. except for australia and antarctica, tbrf species can be found in all continents. the animal reservoir includes small animals and rodents. since the spirochetes persist in the tick salivary gland, disease transmission occurs when humans intrude the tick's environment. tick bites are painless, and history of a tick bite is often missing. therefore, a history of potential exposure can be valuable. the major clinical symptom is relapsing fever. after a median incubation period of 7 days, patients present with febrile episode that can last 2-7 days, followed by afebrile period of 4-14 days. patients with tbrf can have up to 30 febrile relapses, while lbrf is usually associated with only one relapse. other symptoms include myalgia, arthralgia headaches, and vomiting, and physical findings include lymphadenopathy and splenomegaly with rash occurring only in third of the patients. a range of neurologic complications as well as systemic inflammatory response syndrome also have been described [163] . diagnosis is based on identifying the spirochetes on blood smear. sensitivity of blood smear is higher during febrile period (about 70%), and a negative blood smear does not exclude rf. in lbrf, the spirochete load can be low and specific stains can be helpful. other diagnostic methods include serologic testing, pcr, and mouse inoculation. doxycycline, tetracycline, and penicillin are the preferred treatment, with most patients treated with 7-10 days of doxycycline. jarisch-herxheimer reactions with high fever and leukopenia occur in half of the patients following the first antibiotic dose and can develop into a severe reaction with hypotension, respiratory distress, and death [163] . without treatment, tbrf carries a mortality rate of up to 10% with even higher 40% mortality rate for untreated lbrf. two cases of meningoencephalitis with borrelia miyamotoi in heavily treated immunocompromised patients have been described [164, 165] . lyme borreliosis is the most common vector-borne disease in the northern hemisphere caused by a group of at least 13 genospecies. lyme disease is a multisystem illness affecting the skin, joints, nervous system, and heart. human infection is caused mainly by three species: b. burgdorferi is the most common cause in north america but also found in europe, and borrelia afzelii and borrelia garinii which cause the disease in europe and asia. emerging infections in the mid-western usa with borrelia mayonii cause a condition similar to lyme disease. most tick species do not carry borrelia species. the vectors of all borrelia species are the ixodid tick species; this includes the deer tick, i. scapularis, in the northeast and midwest of the usa, ixodes pacificus in the west, the sheep tick, ixodes ricinus, in europe and the taiga tick, ixodes persulcatus, in asia. the ixodid tick demonstrates a complex vector ecology with preferences for different hosts in different geographical regions and at different stages of its development. more than 300 different species, including deer, rodents, birds, and reptiles, have been described. infection rates also show seasonal variation with highest rates during lyme disease follows several stages starting with localized disease at the site of inoculation, followed by dissemination stage and, later, persistent infection [166] . however, an individual patient can show highly variable disease progression with different patterns of organ involvement and disease severity. erythema migrans (em) is often seen at the site of the tick bite after 3-30 days of incubation. regional lymphadenopathy can be seen. secondary skin lesions represent hematogenous dissemination. at this stage, constitutional symptoms of general fatigue, fever and headaches, migratory musculoskeletal pain, conjunctivitis, and cardiac involvement occur. in total, 15% of untreated patients can develop frank neurologic manifestations of meningitis, encephalitis, and variable forms of neuritis with fluctuating symptoms. persistence can occur in untreated (on inadequately) patients. antibiotic refractory arthritis is well described. however, even without treatment, intermittent or persistent attacks usually resolve completely within several years. co-infection with a. phagocytophilum and b. microti can cause diagnostic confusion [167, 168] . the diagnosis of lyme disease is established based on clinical symptoms, history of potential exposure, and serologic studies. although positive culture can confirm the diagnosis, it can usually be obtained only from early em lesions. pcr testing is superior to cultures and can be performed on joint fluid samples [169] . cdc recommendations for the diagnosis of lyme disease are based on serology which might be impossible in pidd patients with abnormal humoral response. cdc guidelines require both an elisa (or comparable test) to be positive and a western blot (2 out of 3 bands (23, 39, or 41 kd) on the igm or 5 out of 10 bands on the igg (18, 23, 28, 30, 39, 41, 45, 58, 66, 93 kd) . most lyme manifestations can be treated with oral antibiotics, while patients with neurologic abnormalities and some patient with lyme arthritis require intravenous therapy [170] . doxycycline is the treatment of choice for early and disseminated disease, with amoxicillin as the second-line choice. jarisch-herxheimer-like reactions can appear in the first 24 h in about 15% of the patients. for patients with clear neurologic symptoms, 2-4 weeks of iv ceftriaxone is the most commonly used therapy. few cases of neuroborreliosis and hiv have been described with a good response to treatment. descriptions of lyme disease in pidd patients are lacking. zoonoses are infectious diseases that pass between animals and humans and span the spectrum of pathogens including viruses, bacteria, fungi, and parasites. zoonoses are very common and range from mild such as certain forms of tinea to lifethreatening infections such as rabies. some of the zoonoses that are vector-borne will be covered in other sections. risk mitigation strategies for zoonoses include patient education, proactive advice about risk scenarios, and avoidance of infected animals. several zoonoses are associated with contact with mammals such as rodents or domestic farm animals through direct contact or through contact with their feces. for instance, hantavirus infections are often associated with exposures to mouse droppings when staying in cabins in the western usa. occupational exposures can occur with buffalopox or parapoxvirus (causing orf infection) through direct contact with buffalo and goats/sheep, respectively [171] [172] [173] [174] . in general, there are very few cases of pidd with zoonotic infections acquired from mammals. however, there are a few special considerations. for instance, lymphocytic choriomeningitis virus is acquired through exposure to house mice primarily, with hamsters being a less common source of infection. both domestic and wild mice can carry the infection without exhibiting symptoms. although infection is rare, there have been severe cases in patients with t/ nk cell dysfunction, such as a case in xlp1 and cases in solid organ transplant recipients [175] . therefore, in patients with severe t/nk defects, consideration should be given to whether small rodents are appropriate household pets. tularemia is a disease of animals and humans caused by the bacterium francisella tularensis. rabbits, hares, and rodents are the main reservoirs. humans become infected through direct contact, ingestion of contaminated water, or inhalation of organisms. ticks and deer flies can also transmit the disease through bites. fever is universal, but other features depend on the mode of transmission. a patient with cgd had a complex course suggesting myeloid defects are a risk for more severe disease [176] . rabies is an almost universally fatal infection caused by contact with oral secretions from infected mammals, typically raccoons, bats, or foxes, and there is no suggestion that pidd or immune compromised modifies the prognosis. for individuals with high-risk exposures, such as those working with wildlife or traveling in endemic areas, pre-exposure prophylaxis is given with vaccination, and if an exposure occurs, rabiesspecific immunoglobulin is provided as well as vaccination. however, for those with humoral immunodeficiencies who cannot respond to the typical pre-exposure vaccination, there needs to be counsel on the additional risk without vaccination. in table 6 , several of the bacterial and viral zoonoses are summarized with their typical endemic areas, which is somewhat limited by diagnostic abilities and reporting, as well as the typical clinical scenarios, known cases in immunodeficiency and an approach to diagnosis and therapy. nipah virus causes a range of infectious phenotypes ranging from asymptomatic infection to acute respiratory distress and encephalitis. nipah virus was identified in 1999 on pig farms in malaysia, leading to identification of 257 human cases, including 105 human deaths and the culling of one million pigs [177] . the natural host is the fruit bat: pteropodidae pteropus. symptoms of infection from the malaysian outbreak were primarily encephalitic in humans, but later, outbreaks have caused respiratory illness, increasing the likelihood of human-to-human transmission. fever, headache, cough, abdominal pain, nausea, vomiting, weakness, problems with swallowing, and blurred vision were common. seizures were seen in 25% and coma in 60%. relapses of encephalitis have been described [178] . increasing infections due to nipah virus is thought to be due to an increasing overlap between bat habitats and pig sties in malaysia. all outbreaks thus far have been in india, bangladesh, or malaysia. the diagnosis of nipah virus relies on pcr of fluid samples, serology in convalescent samples, and immunofluorescence of tissue. there have been no infections of immune compromised patients reported. therapy is largely supportive, although preliminary reports of ribavirin use have been encouraging. a vaccine is under development. severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) are two zoonotic coronaviruses. the sars pandemic in 2002-2003 resulted in 8096 reported cases in 27 countries. no further sars cases were reported after the pandemic except isolated cases linked to laboratory accidents. patients usually presented with fever and respiratory symptoms, but occasionally had diarrhea and vomiting. about 20-30% of sars patients required mechanical ventilation, with a case fatality rate of about 9% [179] [180] [181] . mers was first noted in saudi arabia in 2012, and countries around the arabian peninsula are now endemic for mers-cov. patients usually present with fever, cough, chills, sore throat, myalgia, and arthralgia rapidly progressing to pneumonia with over 50% of patients requiring intensive care. about one-third of patients present with diarrhea and vomiting, and acute renal impairment is a striking feature of mers. risk factors for poor outcome include diabetes, hypertension, and renal and lung disease. cases have been exported to at least 26 countries with travel occasionally causing cluster of secondary outbreaks. one such example is the mers-cov outbreak involving 82 patients in south korea, and the median incubation period was estimated to be 7 days with a range of 2 to 17 days [182] . at the end of 2015, there were 1621 confirmed mers with a 36% mortality rate [179] [180] [181] . bats are the natural reservoirs of both sars-cov and mers-cov. sars-cov crossed the species barrier into palm civets and other animals in live animal markets in china, which then infected human, while a mers-cov ancestral virus crossed species barrier into dromedary camels. abundant circulation of mers-cov in camels results in continuous zoonotic transmission of this virus to human, while sars-cov was not found to circulate in the intermediate reservoirs, explaining sars being a one-off outbreak and mers a continuing zoonotic disease [179] . aerosolgenerating procedures such as intubation were associated with increased viral transmission of both covs resulting in nosocomial outbreaks [179] . super-spreaders are responsible for large and prolonged outbreaks [181] . the diagnosis for sars and mers include both serological tests and pcr assays that can quantify viral loads [183] . functional genetic polymorphisms leading to low serum mannose binding lectin (mbl) are associated with susceptibility to but not severity of sars in both southern and northern chinese [184] [185] [186] . mbl was shown to bind to sars-cov and inhibit the infectivity [184] , suggesting its role as first-line defense against sars-cov. although no patients with primary immunodeficiency infected with sars-cov or mers-cov were identified, likely due to the limited number of such infections, patients with t cell defect and type 1 interferon pathway defects could suffer a more severe disease course [187, 188] . virus-based and host-based treatment strategies are largely experimental with uncertain benefits. ribavirin, type 1 interferons, small molecules, and monoclonal antibodies that block covs entry have been explored [183] . passive immunotherapy and multiple candidate vaccines have been tested in various animal models. convalescent plasma immunotherapy has been considered, but clinical trials are lacking in mers [189] , while for sars a systematic review concluded convalescent serum may reduce mortality and appear safe [190] . the filoviridae family contains three known genera, the ebolaviruses, marburgviruses, and cuevavirus. ebolavirus and marburgvirus cause hemorrhagic fever syndromes in primates and humans, with high fatality rates. cuevavirus infects only bats. the ebolavirus genus contains five species, with two of the species (zaire ebolavirus and sudan ebolavirus) being responsible for the majority of cases of human disease, while marburgviruses contain two species (marburg virus and ravn virus). filoviruses are capable of replicating in a number of cell types (with the exception of neurons and lymphocytes). upon entry into the body of the host (via breaks in the skin, parenterally, or through mucosal surfaces), filoviruses employ a variety of mechanisms to evade the activity of the immune system [191] . the incubation period (interval from infection to onset of symptoms) varies from 2 to 21 days. symptoms begin abruptly, with high fever, severe headache, malaise, myalgia, diarrhea, nausea, and vomiting. a rash can occur between 2 and 7 days after onset of symptoms. hemorrhagic manifestations occur between 5 and 7 days, and fatal cases usually have some form of active bleeding. in an outbreak setting, the symptoms are unmistakable but confusion with malaria can occur early in the disease or in sporadic cases. since their original descriptions in 1967 and 1976, respectively, for marburg and ebola, there have been a number of sporadic cases and several major outbreaks. the largest marburg virus outbreak occurred in angola in 2005 (with a fatality rate of >80%), while the largest ebola epidemic happened between 2014 and 2016 in west africa (sierra leone, guinea, and liberia) and claiming over 11,000 lives (fatality rate > 40%). although not definitively proven in the case of ebola, bats are believed to be the natural animal reservoir for these viruses [192, 193] . these viruses are transmitted via contact with blood or body fluids from an infected host; notably, certain body fluids can harbor virus for weeks to months after resolution of disease. given the recent outbreak in west africa, there has been renewed interest in understanding the pathogenesis of filovirus infections and possible therapies. literature regarding how the pathogenesis of disease may be altered in patients with pidd is lacking. however, the assumption is that in the absence of an intact cellular and/or humoral immune response, the patient with a pidd may be at increased risk of mortality in the setting where mortality is already high. these viruses induce apoptosis of lymphocytes and macrophages, and there is therefore a profound secondary immune compromised [194, 195] . filoviruses can be detected in multiple body fluids via pcr. although practiced for decades, a study in guinea in 2015 failed to show a decrease in mortality among patients receiving convalescent plasma from previously infected donors [196] . a number of additional compounds (e.g., tkm-ebola, bcx4430, and gs-5734) and biologics (zmapp) have been shown to offer protection in animal models of ebola, but to date, no controlled and appropriately powered clinical trials have addressed their efficacy in humans. finally, a number of vaccines for ebola are undergoing clinical studies (including four in phase iii trials). importantly, in late 2016, the rvsv-zebov vaccine was shown to have displayed high efficacy in protecting immunized adults during the 2015 guinea ebola outbreak, and the data also suggested that the vaccine may even offer bherd immunity^to unimmunized persons in proximity to recipients of the vaccine [197, 198] . hepatitis e virus is a single-strand rna virus of the hepeviridae family. it is an important zoonotic disease in asia and africa, and fecal-oral spread is the usual route of transmission [199] . handling of pig or boar meat is a risk factor, and 2-10% of pig livers sold in grocery stores in japan and the usa are infected [200, 201] . swine represent the major reservoir, although antibodies to the virus have been found in many species [199] . the incubation period is 2 weeks to 2 months, and viremia disappears with the onset of symptoms. the mortality rate is 1-4% and can reach 20% in pregnancy [202] . acute hepatitis usually resolves but can lead to liver failure in severe cases. patients with hepatitis e posttransplant have had severe courses in some cases [203] . in immune compromised patients, the course can become chronic [204] [205] [206] . in these cases, cirrhosis develops. the diagnosis is by serology or pcr, and the treatment is supportive. prevention modalities for infections transmitted by humans are conceptually different than infection prevention for vector-borne infections. hand hygiene is extremely important, and avoidance of clearly infected people can be helpful. recognition of infections with fecal-oral transmission and the importance of water purity are critical for patients with pidd. in contrast, infections transmitted by aerosols require prevention strategies related to droplet precautions. in outbreak scenarios, if the risk to the patient is high, specific chemoprophylaxis may be considered. influenza viruses type a and b cause annual epidemic influenza, while type c causes sporadic mild influenza-like illness. patients present with sudden onset of fever, chills, and myalgia, followed by sore throat and cough. other less common features include diarrhea, acute myositis, and encephalopathy [207, 208] . co-infection with bacteria such as pneumococci results in more severe disease [209, 210] . influenza pandemics occur yearly around the world. influenza viruses infect 5 to 15% of the global population, resulting in~500,000 deaths annually [211] . the viruses circulate in asia continuously and seed the temperate zones, beginning with oceania, north america, and europe, then later seeding into south america [212] . diagnosis of influenza includes direct/ indirect immunofluorescent antibody staining for antigens in nasopharyngeal aspirates and pcr. a patient with compound heterozygous null mutations of the gene encoding irf7, a transcription factor for amplifying ifn-α/β, was reported to have life-threatening influenza during primary infection [213] . fatal influenza-associated encephalopathy in both chinese and japanese children has been reported to be associated with genetic variants of thermolabile carnitine palmitoyltransferase ii [214] . patients with scid will have prolonged viral shedding [215] . severe pandemic influenza a virus (h1n1) infection has been associated with igg2 and igg3 subclass deficiency [216, 217] . in addition, influenza infection can be more severe in pidd patients with underlying lung disease, such as bronchiectasis, and antibiotic coverage of chronic colonizing bacteria (such as pseudomonas) in this setting may be helpful. inactivated seasonal influenza vaccine should be given to pidd patients even those with humoral deficiencies as their t cell response to influenza could be normal and offer protective immunity against severe influenza [218, 219] . antiviral drugs include neuraminidase inhibitors (oseltamivir and zanamir) and adamantanes (amantadine and rimantadine), but resistance to adamantanes is widespread. measles is a single-stranded, negative-sense, enveloped (nonsegmented) rna virus of the genus morbillivirus. measles is highly communicable, transmitted by droplets, and less commonly by airborne spread. patients present with fever, cough, coryza conjunctivitis rash, and koplik spots. complications include pneumonia, acute encephalitis, and subacute sclerosing panencephalitis (sspe) [220] . diagnosis of measles includes serological tests, virus isolation, and pcr. in an outbreak, the clinical features may be sufficient for diagnosis. measles vaccine has caused severe measles in children with stat2 and ifn-α/β receptor deficiency [187, 188] , demonstrating the importance of type 1 interferon pathway in controlling measles. immune compromised of nearly any type is associated with severe disease and higher mortality [221] . t cell deficiency states are the most strongly associated with the development of giant cell pneumonia and inclusion body encephalitis, the most feared complications of measles. sspe is a slow encephalitis due to persistence of replication defective measles virus in the cns. it is most frequently seen when young infants are infected with measles and 6-10 years later, sspe becomes evident. there are case reports supporting the immune compromised as increasing the risk of sspe [222] . treatment of sspe with ribavirin has shown some improvement, but the prognosis in general with sspe is very grave. patients with cgd have defective memory b cell compartment, resulting in lower measle-specific antibody levels and antibody-secreting cell numbers, but severe disease has not been reported [223] . pidd patients may harbor the virus latently for longer than usual, leading to complications at the time of transplant [224] . specific antiviral therapy is lacking, but ribavirin has been given to severely ill and immunocompromised children. for measles post-exposure prophylaxis, intravenous immunoglobulin (ivig) is recommended for severely immunocompromised patients without evidence of measles immunity [225] . this would likely include patients with scid and hypogammaglobulinemia who are not yet on regular ivig. measles vaccine, given in a two-dose regimen, has brought down incidence enormously worldwide and the who is planning for eradication globally. enteroviruses (evs) are among the most common viruses infecting humans worldwide. evs are small non-enveloped, single-stranded rna viruses of the picornaviridae family. human evs are categorized into seven species that include hundreds of serotypes, such as polioviruses (pv), coxsackie viruses a, and b (cv-a and b), echoviruses, and human rhinoviruses (hrvs). of these species, many important serotypes are known to infect human such as pv1-3, cv-a16, cv-b3, ev-a71, ev-d68, and hrv (table 7) . non-polio enteroviruses (npevs) have a worldwide distribution. infants and young children have higher incidence of infection and a more severe course of illness than adults. the mode of transmission is mainly through fecal-oral and respiratory routes. infection occurs all around the year in tropical and subtropical regions, while in temperate climates the peak incidence of infection is during summer and fall months [226] . npevs are associated with diverse clinical manifestations ranging from mild febrile illness to severe, potentially fatal conditions. most cases are asymptomatic or have mild symptoms including fever with or without rash; symptoms of hand, foot, and mouth disease; herpangina; acute hemorrhagic conjunctivitis; upper respiratory infection; and gastroenteritis. more severe symptoms occur in infants and young children [227, 228] . acute flaccid paralysis [229] , neonatal enteroviral sepsis [230] , myocarditis/pericarditis [231, 232] , hepatitis, pancreatitis, pneumonia, and atypical hemolytic uremic syndrome [233] are severe manifestations seen in immunocompetent people. chronic infections have been seen in immunocompromised patients [234] . each virus may produce one or more of the aforementioned manifestations; however, some serotypes are often associated with particular features ( table 7) . the definitive diagnosis of npev infection relies on pcr or virus isolation from the cerebrospinal fluid, blood, stools, urine, or throat swab [229, 235] . treatment of npevs is mainly supportive since most infections are self-limited. high doses of intravenous immunoglobulin (ivig) are recommended in patients with severe symptoms. the efficacy of some new antiviral drugs (pleconaril, vapendavir, and pocapavir) is still under investigation [236] . no vaccine has been licensed yet for npevs. however, phase 3 clinical trials of inactivated monovalent ev-a71 vaccines manufactured in china showed high efficacy against ev-a71 in infants and young children [237] . patients with a variety of pidds are unusually susceptible to ev [238] . the most susceptible groups are patients with primary antibody deficiency such as xla, cvid, and hyper-igm syndrome (higms) as well as those having scid and major histocompatibility class ii deficiency [239, 240] . the most severe form of infection has been described in patients with xla due to the profound deficiency of immunoglobulins essential for viral neutralization during infection. affected patients usually present with indolent but relentlessly progressive non-necrotizing meningoencephalitis. regression of cognitive skills, flaccid quadriplegia, and deafness has been described. the reported non-neurologic presentations in xla include septicemia, dermatomyositis-like disease, hepatitis, and/or arthritis [238, 241] . the incidence of npev meningoencephalitis in large registries of xla cases is 1-4% [242] . unpublished data from the kuwait national pidd registry, which includes 271 pidd patients, showed that nine patients had documented npev infections and two died from these infections. the two deaths were seen in scid patients (personal communication with prof. waleed al-herz, md). in addition, npev meningoencephalitis and/or septicemia were reported in few cases with either primary b cell deficiency such as b cell linker (blink) protein deficiency [243] or acquired b cell deficiency following the administration of anti-cd20 (rituximab) [244, 245] . in all reports, better outcome was attributed to the early administration of high doses of ivig during npev viremia [246] . npev infection in pidd diseases remains a major threat to patients. also, the possible prolonged viral excretion and the emergence of resistant strains runs the risk of spreading infection to the surrounding community. oral polio vaccine (opv) consists of a mixture of three live attenuated poliovirus serotypes. opv induces production of neutralizing antibodies against all three serotypes, in addition to a local intestinal immune response. opv can result in vaccine-associated paralysis (vap) secondary to reversion of the vaccine strain to the neurovirulent wild-type strain. an example for such an event was demonstrated by the 2000-2001 outbreak in the dominican republic and haiti [247] , believed to be driven at least in part by undervaccination of the population, which allowed the spread of the reverted vaccine strain [248] . although rare, patients with pidd have a higher risk to develop vap. reports have shown that pidd patients with antibody deficiency can have prolonged viral replication which can persist for years and therefore theoretically increase the risk for a spontaneous reversion within the immunodeficient host [249] [250] [251] [252] . cases of vap were shown in patients with antibody deficiency and combined immunodeficiency syndromes [248, 253, 254] . therefore, opv is contraindicated in patients with pidd, and this contraindication extends to their household contacts [22] . beyond the obvious risk for the pidd patient, prolonged virus shedding also increase the risk for spreading vaccine-derived paralytic strain in the general population. bacterial infections have molded human behavior and altered societies over human history. today, largely ignored due to antibiotic susceptibility, they continue to cause misery and disease around the world. three infections are highlighted, and additional commonly encountered infections are listed in table 8 . pertussis is a respiratory infection caused by bordetella pertussis that begins after a 7-to 10-day incubation period as a minor upper respiratory infection that progresses with cough. initially intermittent, it evolves into paroxysmal coughing spells usually followed by vomiting in infants and young children. it lasts 6 to 10 weeks and can have many complications such as syncope, weight loss, rib fracture, and pneumonia. infants under 6 months are more severely affected, developing pneumonia, pulmonary hypertension, hypoxia, subdural bleeding, and seizures. death can occur, especially in young infants [255, 256] . adults typically have a prolonged cough with fewer complications [257] . it is transmitted via aerosolized droplets during close contact. people are most contagious during the catarrhal stage and the first half of the paroxysmal phase, totaling 5 to 6 weeks [258] . the introduction of whole-cell pertussis vaccine (dpt) in the 1940s in the usa reduced the incidence of the disease from 250,000 cases to around 1000 cases per year in the 1970s. a resurgence in 2012 was associated with the substitution of the whole-cell vaccine by the acellular pertussis vaccine (dtap) [258] . new strategies such as boosters with acellular pertussis for adolescents and adults with tdap and use of tdap during pregnancy seem to be effective in partially reducing the incidence of the disease [259] ; however, pertussis cases in the usa remain higher than the 1970s. the lack of persistence of antibody in the adult population means adults not only represent a reservoir for the disease but also do not provide sufficient titers to immunoglobulin products prepared from adult plasma pools. a relatively recent requirement in some countries is vaccination of adults every 10 years to maintain immunity. this should, over time, improve titers in immunoglobulin products. culture of specimens obtained by nasopharyngeal swabs is the gold standard of laboratory diagnosis due to the 100% specificity, but polymerase chain reaction (pcr) is gaining prominence due to its higher sensitivity and speed of results; serodiagnosis can be used in the late stages of the disease [259] . filamentous hemagglutinin (fha) and pertussis toxin (pt) antibodies were detected at peak measurements in pidd patients on regular ivig, although some of them had pt antibodies below the protective level as trough measurements [260] . severe pertussis cases have not been reported in pidd patients, but severe disease has been seen in malignancies [261] . antimicrobials such as azithromycin, erythromycin, and clarithromycin, if given during the catarrhal stage, may ameliorate the disease and shorten the contagious period. to avoid cases of pertussis, it is also worth emphasizing the importance of good vaccine coverage rate among the whole population, but especially among healthcare workers and family members of patients with pidd. neisseria meningitidis the onset of neisserial meningitis is associated with sore throat, headache, drowsiness, fever, irritability, and neck stiffness [262, 263] . purpuric lesions are very characteristic. this pathogen can also present with sepsis which has a 20% mortality rate as opposed to 11% mortality with a meningitic presentation. this bacterium can also cause a chronic condition referred to as chronic meningococcemia. this condition is characterized by intermittent fevers lasting 1 week to 3-4 months [264] . a non-purpuric rash is common which may evolve into purpura. arthritis, similar to that seen with gonococcus, is common. meningococcal disease primarily affects children under 5 years of age. n. meningitidis is a global pathogen [265] . there are 12 serogroups, but the majority of invasive meningococcal infections are caused by organisms from the a, b, c, x, y, or w serogroups. the annual number of invasive disease cases worldwide is estimated to be at least 1.2 million, with 135,000 deaths related to invasive meningococcal disease. serogroups b and c are responsible for most infections in europe. serogroup a has historically been the major organism in africa; mass vaccination has led to some improvement, but the emergence of group x disease is worrisome. the hajj in the middle east has seen epidemics of w-135, and vaccination is now required for hajj travelers. b and c serogroups are the most common through the americas. n. meningitidis cases occur at a rate of about 1 case per 100,000 people throughout the world [266] , but across the sahel of africa and in china, epidemics can lead to case rates of 500 per 100,000 [267] . the bacterium is a natural human commensal, with carriage rates of about 10%. diagnosis can be by clinical examination in epidemics or by gram stain and culture. complement-deficient individuals have an increased risk of neisserial disease, but not necessarily increased mortality. hiv is associated with increased disease, suggesting that t cells are also important for defense. thirdgeneration cephalosporins are typically used for treatment. penicillin, ampicillin, aztreonam, and chloramphenicol are alternatives. there is great inter-individual variability in the development of tb disease. roughly, 5% of infected individuals develop clinical disease within 2 years of infection (mostly during childhood). about 90% became latently infected without clinical disease, and the remaining 5 to 10% develop pulmonary tb later in life, either from reactivation of latent infection or reinfection. molecular epidemiology studies from high burden areas suggest more disease results from recent transmission than from reactivation of latent tb, particularly in people living with hiv [268] . acquired or inherited host factors may at least partially account for the variable disease course, resulting in increased susceptibility to mycobacteria infections [269] . pidd associated with tb and ntm infections include t cell deficiencies, gata2 deficiency, cgd, anhidrotic ectodermal dysplasia with immunodeficiency, x-linked (xl) recessive cd40 ligand deficiency, autosomal recessive (ar) stat1 deficiency, ar irf8 deficiency, and ar tyk2 deficiency. in addition, a group of disorders with a strong susceptibility to ntm, named mendelian susceptibility to mycobacterial diseases (msmd), have been recognized since the 1990s. these are rare inborn errors of ifn-γ signaling pathway that present with isolated predisposition to infections caused by weakly virulent mycobacteria such as bcg vaccine and environmental ntm, in otherwise healthy patients. the genetic defects involve impairment in the production of interferons (ar il12rβ1, ar il12p40, autosomal dominant (ad) irf8, ar isg15, xl recessive nemo) or response to interferons (ifn-γr, ad stat1, ad irf8, cgd) [270] . an acquired form exits: adults with ntm infection in thailand and taiwan were found to have high-titer anti-interferongamma antibody [271] . these individuals from southeast asia were found to have hla-drb1*1502/16:02 and dqb1*05:01/05:02. patients suspected of having pulmonary tb should have acid-fast bacilli (afb) smear microscopy and culture performed in three sputum samples. pcr for mtb can be performed [272] . the use of rapid tests facilitates early diagnosis, and the who has recently recommended their use. the only recommended rapid test for detection of tb with and without rifampicin resistance is the xpert mtb/rif assay (cepheid, sunnyvale, ca). the who recommends the xpert test for those suspected of having drug-resistant tb or in hiv; however, culture is still the mainstay and is not replaced by the xpert test. tb skin testing (mantoux testing) uses a purified protein derivative injected under the skin. its advantages are that it can be used for large-scale screening and it is cost effective. skin testing does have several disadvantages when used as a diagnostic test. reading the induration requires training and immunodeficiencies can alter the magnitude of the induration. immunosuppressed patients (hiv, organ transplant) are considered positive if the induration is ≥5 mm. some immunodeficiencies may completely ablate the response. other causes of false-negative tests are malnutrition, concurrent infection, recent live viral vaccine administration, renal failure, malignancy, medical stress, very elderly, young infants, or with a very recent infection with mtb. conversely, the results may be falsely positive if bcg has been administered. interferon gamma release assays can be used in any setting where skin testing would be done but are considered superior in settings where the patient has had bcg vaccination and, in some cases, where skin testing has been sown to have high false-negative rates. in general, tb treatment for patients with impaired immune response, including pidd, hiv infection, and immunosuppressive therapy, is based on the standard 6-month regimen consisting of a 2-month intensive phase of isoniazid, rifampin, pyrazinamide, and ethambutol, followed by 4 months of isoniazid and rifampin. decisions regarding treatment duration can be individualized, taking into account disease severity, organs involved, and response to treatment. significant pharmacological interaction can occur between rifampin-based mtb regimens and immunosuppressive drugs, such as calcineurin inhibitors or rapamycin, requiring strict monitoring of drug plasma concentrations [273] . therapy for ntm is complex with highly variable drug resistance patterns and a need for biological augmentation to effectively clear the organism. aspergillus fumigatus (see above), cryptococcus gattii, histoplasma capsulatum, coccidioides immitis (or c. posadasii), blastomyces dermatitidis, paracoccidioides brasiliensis, emmonsia pasteuriana, and penicillium (talaromyces) marneffei are environmental fungi that are endemic in certain parts of the world (table 9 ). with the exception of penicillium marneffei and emmonsia pasteuriana that only cause disease in profoundly immune compromised individuals, these fungi can cause infection in healthy individuals, ranging from mild, self-limited pulmonary disease to infection that requires antifungal therapy for eradication. on the other hand, patients with acquired defects in cell-mediated immunity such as those infected with hiv, and patients with specific monogenic disorders, particularly those involving the il-12/ ifn-γ/stat signaling pathways, scid, and gata2 depends on underlying state of immunosuppression and magnitude of environmental exposure icl idiopathic cd4 lymphocytopenia, aids acquired immune deficiency syndrome, stat1 signal transducer and activator of transcription 1, gata2 gata binding protein 2, scid severe combined immunodeficiency, cvid common variable immune deficiency, pidd primary immunodeficiency, il12rb1, interleukin-12 receptor subunit beta 1, ifngr1 gamma interferon receptor 1 deficiency, are at high risk of developing life-threatening disseminated infections by these endemic fungi following environmental exposure [42, [274] [275] [276] [277] [278] . diagnosis relies on culture of the corresponding fungus, histopathological demonstration of the fungus-specific characteristic morphologies, and/or surrogate serological and fungal antigen tests. treatment of clinical disease (as opposed to colonization) typically involves an initial induction phase with amphotericin b, followed by long-term azole maintenance therapy and secondary prophylaxis, and prognosis varies significantly depending on the fungal pathogen and underlying pidd. melioidosis is caused by b. pseudomallei, a gram-negative bacteria found in soil and water, in tropical climates of southeast asia and northern australia [279, 280] . melioidosis is an emerging, potentially fatal disease (20% mortality). b. pseudomallei can be transmitted by inhalation, ingestion, or direct contact (through open skin) with contaminated soil or water. animals (sheep, goats, swine, horses, cats, dogs, and cattle) are also susceptible to infection and cases of zoonotic transmission through direct contact of skin lesions with infected animal meat or milk have been described [280] . b. pseudomallei infections are endemic in northern australia and southeast asia. approximately 75% of reported infections occur during the rainy seasons. cases have also been reported in south pacific, africa, india, and the middle east. in temperate areas, infection is extremely rare and is predominantly imported by travellers or immigrants [281] . the incubation period of melioidosis is variable from 1 day to years, although common symptoms develop between 2 and 4 weeks after exposure. melioidosis presents most frequently in adults 40-60 years of age, but can occur in all ages, with one study reporting 5% of cases occurred in children [282] . in australia, the average annual incidence in 2001-2002 was reported as 5.8 cases per 100,000 people [279] . the incidence in indigenous australians was higher at 25.5 cases per 100,000. a case-cluster in an australian community was associated with post-cyclonic flooding. a recent review suggests that b. pseudomallei is increasingly prevalent in the americas, with a mortality rate of 39% [283] . infection in healthy individuals is uncommon, and more than 70% of cases occur in the setting of underlying conditions such as chronic renal disease, diabetes, chronic lung disease, and alcoholism. a recent review of melioidosis in travelers found that 46% of cases were acquired in thailand. symptoms usually started at 23 days (range 1-360 days) after leaving the endemic area. traveller infections were less often associated with predisposing risk factors (37.5%), diabetes mellitus being the most common (21%). melioidosis in travelers had lower mortality (17%) than infection in autochthonous cases in southeast asia [284] . pneumonia (~50-55%) is the most common presentation in adults. there is usually high fever, headache, anorexia, and myalgia. b. pseudomallei infection may also present as localized skin infection, septicemia, or disseminated infection. localized infection results in an ulcer, nodule, or skin abscess. this usually occurs from the bacterium breaching through a break in the skin. patients with renal disease and diabetes are more susceptible to sepsis. in disseminated infection, abscesses may develop in the liver, spleen, lung, and prostate. in children, primary cutaneous melioidosis is the commonest presentation (60%). bacteremia is less common in children than in adults, but brainstem encephalitis has been reported [282] . difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes [285] . diagnosis of melioidosis is primarily by isolation of the organism. identification of b. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. although various serological tests have been developed, they are generally unstandardized bin house^assays with low sensitivities and specificities. pcr assays have been applied to clinical and environmental specimens but are not widely available and sensitivity remains to be evaluated. cases of melioidosis have been reported in patients with acquired or inherited immune deficiency. melioidosis was the presenting complaint in several patients with cgd. bacteremic melioidosis was recently reported in two patients with prolonged neutropenia, who succumbed despite appropriate antibiotics [286] . it is likely that there is increased susceptibility in situations where innate or adaptive immunity is compromised. treatment is with intravenous antimicrobial therapy for 10-14 days, followed by 3-6 months of oral antimicrobial therapy. intravenous therapy with ceftazidime or meropenem is usually effective. oral therapy may continue with trimethoprim-sulfamethoxazole or doxycycline. free-living amoebas (fla) are protozoa found worldwide that do not require hosts to survive. fla do not employ vectors for transmission and are not well adapted to parasitism in humans. however, there are four genera/species that can cause human disease: naegleria (n. fowleri), acanthamoeba (multiple species), balamuthia (b. mandrillaris), and sappinia (s. pedata). all of these amoebae are capable of inducing cns disease in humans, but acanthamoeba species also cause various extra-cns infections, especially in immunocompromised hosts. the fla that are pathogenic in humans are reviewed below. naegleria are a diverse group of fla flagellate protozoans with a large number of distinct species. only one species, n. fowleri, has been shown to cause infection in humans. n. fowleri has a multi-stage life cycle with amoeboid and trophozoite-infective forms as well as a cyst form [287] . n. fowleri is found commonly in warm freshwater around the world including lakes, rivers, and hot springs. humans become can become infected when swimming or diving in contaminated water. in rare circumstances, infections have also been attributed to exposure from contaminated tap water sources when utilized for religious cleansing of the nose or irrigation of the sinuses. thus, tap water should not be used for nasal and sinus irrigation. it is not possible to become infected from drinking contaminated water or from contact with an infected person, and the amoeba is not found in salt water. after entry to the nasal cavity, the amoeba travels through the cribiform plate to the olfactory bulbs and migrates to the cerebellum, resulting in primary amoebic meningoencephalitis (pam), a rapidly fatal brain infection characterized by the destruction of brain tissue. in its initial presentation, pam can mimic bacterial meningitis, further delaying accurate diagnosis and initiation of therapies that may save the patient. overall, n. fowleri infections are rare. worldwide, most cases are reported in the usa, australia, and europe; however, in developing countries, it is suspected that a large number of cases go unreported. between 2006 and 2015, there were only 37 infections reported in the usa with 33 of the cases attributed to contaminated recreational water, 3 infections following nasal irrigation with contaminated tap water, and 1 case where a person was infected following use of a backyard slipn-slide utilizing contaminated tap water [288] . the fatality rate associated with n. fowleri infection is over 95%, and between 1962 and 2015, only 3 of the 138 infected persons in the usa have survived infection. initial symptoms of pam start 1 to 9 days after infection and can include headache, fever, nausea, or vomiting [288] . progressive symptoms can include stiff neck, confusion, lack of attention, loss of balance, seizures, and hallucinations. cardiac arrhythmias have also been observed. the infection progresses rapidly after initial onset and causes death within 1 to 12 days after exposure (mean of 9.9 days). since infection often progresses rapidly to death, there is often insufficient time to mount a robust immune response. however, both the innate (neutrophils, macrophages, and complement system) and the adaptive (both t and b cells) arms of the immune system have been shown to participate in the immune response to n. fowleri [289] . patients with pam have csf with elevated pressure that is often cloudy or hemorrhagic, with neutrophil-predominant pleiocytosis, elevated protein levels, and very low glucose. wet mounts from centrifuged csf will show motile mono-nucleated trophozoites measuring~10-25 μm in size. additionally, trophozoites can be identified with giemsa and wright stains of csf smears combined with an enflagellation test [289] . confirmation can be achieved via a variety of timeconsuming methods including an immunofluorescence assay [290] , culture of csf [291] , or pcr-based methods [292] . the optimal therapy for n. fowleri pam is still debated. the use of intravenous amphotericin b and fluconazole followed by oral administration of rifampin resulted in survival of a 10year-old child with pam [293] . another child was shown to survive following intravenous and intrathecal amphotericin b and miconazole as well as oral rifampin [294] . most recently, an adolescent girl was successfully treated with the combination of azithromycin, rifampin, fluconazole, and miltefosine [295] . prevention is critical for this highly fatal infection and warning pidd patients not to use tap water for nasal irrigation is important. since its original description in 1986, over 200 cases of b. mandrillaris infections have been described worldwidewith most cases occurring in south america and the usa. balamuthia are found in soil, and acquisition of disease has been associated with agricultural activities, dirt-biking, gardening, and swimming in contaminated water sources. b. mandrillaris is thought to enter the body of the host through breaks in the skin and or via inhalation. the organism is believed to access the cns through hematogenous spread, resulting in a chronic, insidious, but often fatal granulomatous amoebic encephalitis (gae), which has been documented in both immunocompetent and immunocompromised hosts [291, 296] . the incubation period from exposure to development of clinical symptoms is not well established and experts believe that this may occur between 2 months and 2 years. finally, an alternative mode of transmission via solid organ transplantation has also gained attention [297] [298] [299] . in many cases, gae is diagnosed post-mortem, due to delayed diagnosis or unawareness of the clinical entity. following infection by b. mandrillaris, two clinical patterns of presentation have been described. in the first pattern, patients initially develop a skin lesion that may resemble a painless plaque that may evolve into subcutaneous nodules and rarely ulcerations [300] . these patients may develop neurologic manifestations weeks to months later. histopathologic examination of these lesions typically reveals granulomatous reactions in the reticular dermis, associated with lymphocytic and plasma cell infiltrates as well as multinucleated giant cells, without distinct epidermal changes. skin lesions will harbor trophozoites, but these are scarce and often easily overlooked as they resemble histiocytes. it is believed that early diagnosis of b. mandrillaris infections in those presenting with skin lesions may prevent subsequent development of cns disease, but there have also been cases in which patients presenting with skin lesions have progressed to developing gae despite treatment. in the second pattern, patients present with cns involvement without a previously recognized skin lesion. patients presenting with gae may initially display fever, malaise, headache, nausea and vomiting, and frank lethargy. later, these symptoms evolve into visual abnormalities, cranial nerve palsies, seizures, focal paresis; as intracranial pressure builds, coma, and eventually death with tonsilar or uncal herniation ensue within 2-3 weeks [301] . upon infection with b. mandrillaris, brain endothelial cells produce the proinflammatory cytokine il-6, thereby initiating an inflammatory response [302] . moreover, the amoebic trophozoites infiltrate blood vessel walls. degradative enzymes, vessel wall infiltration, and the host inflammatory responses result in tissue necrosis and infarctions in the cerebral hemispheres, cerebellum, and the brainstem. in a mouse model of b. mandrillaris infection, cd4+ t cells were shown to be protective [303] , suggesting that patients with lowered number or dysfunction in cd4+ t cells may be more susceptible to disease by this amoeba. however, b. mandrillaris infections have been described in a variety of human hosts [304] , ranging from the young, healthy, and presumably immunocompetent to the elderly, and those with hiv, chronic corticosteroid exposure, on post-transplant immunosuppression and even patients with cvid. as such, further research is necessary to fully delineate the susceptibility of pidd patients. in patients who develop the characteristic skin lesions, recognition, testing, and treatment for b. mandrillaris may prevent subsequent gae. as such, obtaining tissue and looking for granulomas and trophozoites is quite helpful. skin biopsies can be stained via immunofluorescence or immunoperoxidase techniques to identify b. mandrillaris [305] . additionally, a pcr technique that identifies mitochondrial 16s ribosomal rna from b. mandrillaris is also available through the cdc [306] . in patients in whom the diagnosis is confirmed via skin biopsy, wide resection and medical treatment appears to prevent development of cns disease in at least a proportion of patients. in patients presenting with cns involvement, lumbar punctures reveal csf with lymphocytic pleiocytosis, low-to-normal glucose, and mildly to significantly elevated protein levels. trophozoites are not typically found in the csf, but pcr analysis may be performed. ct or mr imaging may show multiple nodules with ring enhancement; some of these nodules may also contain focal areas of hemorrhage. biopsies of brain tissues typically reveal granulomas and foamy macrophages and multinucleated giant cells surrounded by lymphocytic infiltrates. additionally, there will be areas of necrosis filled with neutrophils, multinucleated giant cells, and lymphocytes, with balamuthia trophozoites and cysts interspersed with macrophages [16] . as with the skin biopsies, immunofluorescent and immunoperoxidase stains may aid diagnosis and should be performed. unfortunately, the optimal medical management of cns disease is unknown. in the usa, a few patients have been successfully treated with a combination of fluconazole, flucytosine, pentamidine, a macrolide antibiotic (either clarithromycin or azithromycin), and one of the following agents: liposomal amphotericin b, miltefosine, sulfadiazine, or thoridazine [307] [308] [309] ; others in peru have been treated successfully with fluconazole (or itraconazole), albendazole, and miltefosine [307] . based on these case reports, most experts recommend treatment with a combination of medications (along with partial or complete resection of nodules) for a prolonged period of time to prevent further deterioration and death [307] [308] [309] [310] . acanthamoeba spp. the genus acanthamoeba contains at least 24 morphologically distinct species that live in a diverse array of habitats, including soil, salt, brackish, and fresh water. acanthamoeba spp. have also been found in humidifiers, heating and cooling unit components, jacuzzis, hot water tanks, bathrooms and drains, eye wash stations and dentistry irrigation systems, and more. acanthamoeba spp. have been isolated from reptiles, birds, and other non-human mammals, suggesting a broad distribution in the environment. acanthamoeba trophozoites feed on bacteria, but have also recently been shown to harbor a number of bacteria (including legionella and burkholderia spp., e. coli, listeria monocytogenes, vibrio cholerae, mycobacteria spp., chlamydophila, and others) and at least one virus (mimivirus) as endosymbionts. acanthamoeba infections in humans can present in a variety of ways. of primary importance are cns infections. like b. mandrillaris, acanthamoeba spp. can induce gae (described above). there is a high predilection for gae in those with hiv/aids, patients on chemotherapy, and those receiving broad spectrum antibiotics [301] . acanthamoeba are rarely found in csf, but some case reports indicate isolation of amoebae by culturing csf on bacterized agar plates. similar to gae seen with b. mandrillaris, cns histopathology may reveal edema, multiple areas of necrosis and hemorrhage, and occasional findings of angitis and blood vessel cuffing by inflammatory cells, as well as occasional trophozoites or cysts. cns disease treatment is not standardized, but several patients have been successfully treated with pentamidine, fluconazole, flucytosine, sulfadiazine, as well as miltefosine. acanthamoeba can rarely cause cutaneous infections; these lesions, like gae, are also predominantly seen in immunocompromised hosts. these lesions can start as nodules or papules on the lower extremities and develop into necrotic ulcers. histopathologic examination may reveal granulomatous dermal lesions in immunocompetent hosts, with histiocytes, as well as neutrophils and plasmacytes; trophozoites are typically visible [311, 312] . the optimal management of cutaneous disease is not known, but typically involves combinational therapy with topical (e.g., chlorhexidine, gluconate, or ketoconazole) and systemic (miltefosine, sulfadiazine, flucytosine, liposomal amphotericin b, azole antifungals, etc.) drugs. additionally, nasopharyngeal and sinus infections have been seen in people with severe compromise in immunity [313, 314] . patients typically present with purulent nasal discharge, and examination may reveal erosion of the nasal septum. nasopharyngeal disease can present concomitantly with cutaneous disease. treatment of nasopharyngeal or sinus disease is difficult and involves surgical debridement and combinations of systemic drugs. disseminated disease is also seen in immunocompromised hosts and typically involves concomitant pulmonary and cutaneous disease in the presence or absence of cns infection. keratitis readily occurs in immunocompetent hosts-with the major risk factor being contact lens wearing without proper adherence to recommended cleansing protocols. this infection less commonly presents as a result of direct inoculation with trauma. one of the most common reasons for contact lens wearers to acquire disease is due to the use of non-sterile tap water in preparing contact lens saline solutions [315] , although contaminated solutions from manufacturers have also been identified. patients will have pain and photophobia. physical exam reveals conjunctival injection and epithelial abnormalities (including pseudodendritic lesions) and stromal infiltrates [316] . the proper diagnosis can be made by staining corneal scrapings with calcofluor or wright-giemsa stains and examined by confocal microscopy, culture, or pcr analysis. prompt therapy with a combination of polyhexamethylene biguanide (or biguanide-chlorhexidine) and propamidine or hexamidine [317, 318] is indicated, but misdiagnosis and delayed therapy are common. more severe cases may also require debridement. the use of topical steroids before administration of combinational therapy may result in worse outcomes and should be avoided; however, if scleritis ensues, it may be necessary to use immunosuppressants to reduce the need for enucleation. severe and/or refractory cases may result in the need for cornea transplantation. phenotypes seen in pidd like hsv-1 and candida [337] . patients with ifnar2 deficiency seem highly susceptible to cns disease caused by mmr vaccine, an otherwise extremely rare phenomenon [187] . recently, a case of noroviral cns disease was described associated with a novel, yet unpublished pidd, suggesting that some pidds may lead to susceptibility of the cns to viruses that normally do not exhibit neurotropism (casanova jl, personal communication) . this again favors metagenomic approaches in the study of cns sequelae in pidd patients. in pidds, the cns is also more vulnerable to virally induced immunodysregulation [325] . conditions like primary hemophagocytic lymphohistiocytosis (hlh) may present as isolated cns disease or relapse only in the cns [338] [339] [340] . almost 20% all human malignancies are associated with chronic infections by hbv, hcv, hpv, ebv, hhv8/kshv, htlv-i, hiv-1, hiv-2, jcv, merkel cell polyomavirus (mcpv), helicobacter pylori, schistosomes, or liver flukes [341] . accordingly, pidd patients' malignancies are often associated with chronic infections. mcpv-associated merkel cell carcinoma has now been described in gata2 and tmc8 (ever2) deficiencies as well as other forms of pidd [330, [342] [343] [344] [345] [346] . large follow-up cohorts are needed to refute or confirm associations between novel pidds and malignancies, such as hyperactivating pik3cd and ovarian dysgerminoma or gata2 deficiency and leiomyosarcoma [329, 347] . recently, hymenolepis nana was found to have driven malignant transformation in an hiv patient. likely, other novel pidd-and pathogen-associated malignancies will be found in the future by those with an open and inquisitive mind [348] . understanding the specific infection susceptibility for each pidd allows not only a better understanding of host defense, but also allows the clinician to collaborate with the microbiology laboratory to make definitive diagnoses and provide the best therapy. reviewing all of the infections for each pidd is not within the scope of this article, but there are several infections that are unique for specific pidds and require special attention from the microbiology laboratory. three examples are provided below. g. bethesdensis is a gram-negative bacterium that was identified to cause disease in patients with cgd in 2006 [349] . g. bethesdensis is a member of the methylotroph group of bacteria, which are able to use single-carbon organic compounds as their only source of energy. they are widespread in the environment, but are rare human pathogens, and infections with g. bethesdensis have been limited thus far to patients with cgd. the organism was first detected in an adult patient with indolent and recurrent necrotizing lymphadenitis [349] . subsequently, g. bethesdensis was isolated from nine patients with cgd, primarily causing lymphadenitis, but there have been two fatalities [350] . treatment has been most effective with intravenous ceftriaxone. the microbiology laboratory should be alerted when there is concern for g. bethesdensis infection to allow for proper culture media. charcoal yeast extract (cye) agar and lowenstein jensen (lj) media are appropriate culture media. mycoplasma and ureaplasma spp. as molecular techniques are becoming more widely used to detect pathogens, the spectrum of infections that were previously only detected through serologic assays and research laboratories will increase. this is important especially for patients with pidd who have unique susceptibility to infection and may not have the ability to mount a serologic response. examples of infections in this group are those caused by mycoplasma and ureaplasma [351, 352] . these pathogens have been known to cause osteoarticular infections for those with antibody deficiency, such as xla and cvid. recently, mycoplasma orale, typically an oral commensal, has been isolated from two patients with defects in the activated pi3k delta syndrome, as chronic lymphadenitis in one and chronic splenic abscess in the other (sm holland personal communication). defects in pi3kcd and pi3kr are frequently associated with hypogammaglobulinemia and therefore would fit in the pattern of mycoplasma infections in those with humoral immunodeficiency. mycoplasma orale has also previously been reported as causing bone disease in a patient with cvid [353] . in patients with xla, helicobacter, camplyobacter, and the related flexispira bacteria that are typically isolated to the gi tract can disseminate and often lead to chronic bacteremia, ulcers, and bone infections [354] [355] [356] . xla patients have higher susceptibility than other humoral pidd and are thought to be due to the role that igm is playing in controlling the dissemination of these pathogens and potentially iga in providing mucosal immunity. these bacteria can be fastidious to grow, and therefore, when there is suspicion, identification needs collaboration with the microbiology laboratory. for instance, the blood culture media may allow growth (although with a longer incubation period), but then the organisms may need molecular techniques for identification, such as 16 s sequencing, as they will not grow on the agar plates. treatment is often difficult, requiring combination antimicrobials for prolonged periods (such as 1 year), and relapse is common. this review provides an important perspective for practicing immunologists, namely that we are a part of a global community as are our patients. this overview of emerging infections and infectious concerns for travelers serves as a foundation for practical considerations for clinicians and patients. using prevalence data, an estimation of the number of infected patients with pidd (table 10) can be developed [25, 130, [357] [358] [359] [360] . thus, the concerns addressed in this review are not theoretical but impact a considerable number of patients already. the landscape of emerging infections is by its nature highly dynamic. during the preparation of this manuscript, a mumps outbreak in the usa occurred, a new bunyavirus outbreak causing an hlh picture was reported (severe fever with thrombocytopenia 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transformation of hymenolepis nana in a human host granulibacter bethesdensis gen. nov., sp. nov., a distinctive pathogenic acetic acid bacterium in the family acetobacteraceae methylotroph infections and chronic granulomatous disease osteoarticular infectious complications in patients with primary immunodeficiencies increased susceptibility to mycoplasma infection in patients with hypogammaglobulinemia disseminated mycoplasma orale infection in a patient with common variable immunodeficiency syndrome relapsing campylobacter jejuni systemic infections in a child with x-linked agammaglobulinemia bacteremia and skin/bone infections in two patients with x-linked agammaglobulinemia caused by an unusual organism related to flexispira/helicobacter species successful approach to treatment of helicobacter bilis infection in x-linked agammaglobulinemia prevalence and morbidity of primary immunodeficiency diseases, united states epidemiological characteristics of spotted fever in israel over 26 years complications of bacille calmette-guerin (bcg) vaccination and immunotherapy and their management acknowledgements the authors would like to thank thomas krell for information regarding ivig safety and david peden for recognizing global warming as central to medical knowledge. conflict of interest the authors declared that they have no conflict of interest. pidds display wide genetic and phenotypic heterogeneity [319] . similar disease phenotypes may be caused by multiple genes, while patients' phenotypes caused by the same gene and even by the same mutations vary between individuals. importantly, after a novel pidd has been described, subsequent reports often reveal a wider variation in associated infections and cellular findings, often without clear genotype-phenotype correlations [320] [321] [322] [323] [324] . variation may be caused by mechanisms such as other contributing genes or geographical variation in infectious exposures. geographic differences seem most pronounced in intracellular and often chronic infections. while the numbers of described pidd patients increase, at first seemingly rarely pidd-associated infections turn out to be found in a significant subset of pidd patients [325] [326] [327] . for example, patients with cd40l deficiency living in endemic areas display susceptibility to bartonellosis and paracoccidioidomycosis, infections not described in european and us cohorts [157, 328, 329] .often, an infectious phenotype previously only described in secondary immunodeficiencies may reveal the possibility of an underlying primary immunodeficiency [327, 330, 331] . increasing numbers of genetic defects causing early-onset, severe, and recurrent susceptibility to commonly circulating pathogens like pneumococci, tuberculosis, herpes simplex, and influenza viruses as well as endemic protozoans like trypanosomes and fungi are being recognized, and thus, infections with unusual pathogens require a high index of suspicion for pidd [332] . in contrast, pidds may also manifest as suspected infection but sterile inflammation. for example, in inflammatory lesions like granulomas and necrotizing fasciitis where no clear pathogens are found, one needs to rule out aberrant host responses due to pidd [333] .chronic viral and fungal infections may also display novel phenotypes never or rarely seen in secondary immunodefic i e n c i e s . i n f e c t i o n s l i k e d e r m a t o p h y t o s i s a n d phaeohyphomycosis deeply infiltrating the skin and lymph nodes, occasionally extending to bones and central nervous system (cns) as well as predisposition to primary cns candidiasis and extrapulmonary aspergillus slowly revealed the full phenotypic spectrum of card9 deficiency [44, 324, 334] . chronic skin ulcers caused by hsv-1 and severe molluscum contagiosum suggest dock8 deficiency or gain-of-function mutations of stat1 [320, 323] . chronic mucocutaneous candidiasis has revealed a large group of monogenic diseases (il17ra, il17rc, il17f, stat1 (gof), rorx, act1), which may also be associated with recurrent bacterial infections or syndromic features [335] . while novel diseases by newly described viruses are being discovered, one needs awareness to suspect these in pidd patients [336] .interestingly, most novel forms of infectious disease in pidd patients have been described either in easily accessible sites like the skin or in immunologically privileged, normally sterile sites like the cns. this suggests that with the increasing use of invasive sampling and sensitive metagenomic approaches, we might find more novel infectious phenotypes. pathogens highly suggestive of certain pidds, like chronic enteroviral cns infections in xla patients are reviewed above. in hypomorphic mutations, cns seems to be especially vulnerable to chronically active and/or recurrent novel infectious bsmoldering^focal encephalitis lesions by pathogens key: cord-269861-r07osd0w authors: kim, jin hyoung; patil, ajit mahadev; choi, jin young; kim, seong bum; uyangaa, erdenebelig; hossain, ferdaus mohd altaf; park, sang-youel; lee, john hwa; eo, seong kug title: ccr5 ameliorates japanese encephalitis via dictating the equilibrium of regulatory cd4(+)foxp3(+) t and il-17(+)cd4(+) th17 cells date: 2016-07-20 journal: j neuroinflammation doi: 10.1186/s12974-016-0656-x sha: doc_id: 269861 cord_uid: r07osd0w background: ccr5 is a cc chemokine receptor involved in the migration of effector leukocytes including macrophages, nk, and t cells into inflamed tissues. also, the role of ccr5 in cd4(+)foxp3(+) regulatory t cell (treg) homing has recently begun to grab attention. japanese encephalitis (je) is defined as severe neuroinflammation of the central nervous system (cns) following infection with mosquito-borne flavivirus je virus. however, the potential contribution of ccr5 to je progression via mediating cd4(+)foxp3(+) treg homing has not been investigated. methods: infected wild-type (ccr5(+/+)) and ccr5-deficient (ccr5(−/−)) mice were examined daily for mortality and clinical signs, and neuroinflammation in the cns was evaluated by infiltration of inflammatory leukocytes and cytokine expression. in addition, viral burden, nkand jev-specific t cell responses were analyzed. adoptive transfer of ccr5(+)cd4(+)foxp3(+) tregs was used to evaluate the role of tregs in je progression. results: ccr5 ablation exacerbated je without altering viral burden in the extraneural and cns tissues, as manifested by increased cns infiltration of ly-6c(hi) monocytes and ly-6g(hi) granulocytes. compared to ccr5(+/+) mice, ccr5(−/−) mice unexpectedly showed increased responses of ifn-γ(+)nk and cd8(+) t cells in the spleen, but not cd4(+) t cells. more interestingly, ccr5-ablation resulted in a skewed response to il-17(+)cd4(+) th17 cells and correspondingly reduced cd4(+)foxp3(+) tregs in the spleen and brain, which was closely associated with exacerbated je. our results also revealed that adoptive transfer of sorted ccr5(+)cd4(+)foxp3(+) tregs into ccr5(−/−) mice could ameliorate je progression without apparently altering the viral burden and cns infiltration of il-17(+)cd4(+) th17 cells, myeloid-derived ly-6c(hi) monocytes and ly-6g(hi) granulocytes. instead, adoptive transfer of ccr5(+)cd4(+)foxp3(+) tregs into ccr5(−/−) mice resulted in increased expression of anti-inflammatory cytokines (il-10 and tgf-β) in the spleen and brain, and transferred ccr5(+) tregs were found to produce il-10. conclusions: ccr5 regulates je progression via governing timely and appropriate cns infiltration of cd4(+)foxp3(+) tregs, thereby facilitating host survival. therefore, this critical and extended role of ccr5 in je raises possible safety concerns regarding the use of ccr5 antagonists in human immunodeficiency virus (hiv)-infected individuals who inhabit regions in which both hiv and flaviviruses, such as jev and west nile virus, are endemic. the flavivirus genus, which includes mosquito-borne dengue virus, japanese encephalitis (je) virus, and west nile virus (wnv) [1] [2] [3] , is associated with significant morbidity and mortality due to fatal hemorrhagic fever and encephalitis. of the flaviviruses, japanese encephalitis virus (jev) continues to be the leading cause of viral encephalitis in asia and the western pacific. it poses an increasing threat to global health and welfare, with approximately 67,900 reported cases annually [4] . due to rapid changes in climate and demography, jev is currently spreading to previously unaffected regions such as indonesia, pakistan, and northern australia [5] . the incubation period of jev ranges from 5 to 15 days and is fatal in 25 to 30 % cases, mostly in infants, and a high proportion of patients who survive have serious neurological and psychiatric sequelae [4] , for which je is considered to be more fatal than wnv encephalitis, resulting in 3-5 % mortality (1100 death/29,000 symptomatic infections) [6] . pathologically, je is a severe neuroinflammation in the central nervous system (cns) closely associated with the disruption of the blood-brain barrier (bbb) [7] . although little is known about the pathogenesis of jev, considerable progress has been made in murine models [8, 9] . while jev infects and kills neurons directly in the cns, cns invasion of jev causes the stimulation of microglia/glia and infiltrated leukocytes, leading to indirect neuronal killing via oversecreting pro-inflammatory cytokines (such as il-6 and tnf-α) and soluble mediators that can induce neuronal death [10, 11] . this notion implies that je is an immunopathological disease caused by uncontrolled overactivation of innate and adaptive immune cells, resulting in neurological disorders in the cns. therefore, adequate cns infiltration and activation of peripheral immune cells is considered to play a critical role in protecting hosts from viral encephalitis such as je. indeed, cns infiltration and activation of peripheral leukocytes during je can cause profound damage if the reaction is excessive or inappropriate [12] . therefore, balanced cns infiltration and activation of peripheral leukocytes should be achieved to have a favorable prognosis of je without tissue injury. chemokine-mediated influx of peripheral leukocytes into the cns is believed to clear infection, but also be responsible for deleterious bystander neuronal damage associated with morbidity and, in some cases, increased mortality. for example, cxcr3-deficient mice are found to have enhanced cns viral titers and mortality following wnv infection [13] , while these mice are protected from lethal infection of lymphocytic choriomeningitis virus (lcmv) or cerebral malaria [14, 15] , suggesting that the final outcome of encephalitis will depend on the nature of the pathogen and a range of host factors. likewise, ccr5 plays a critical role in recovery from flavivirus encephalitis via appropriate cns migration of peripheral leukocytes, including nk cells and cd4 + /cd8 + t cells [16] [17] [18] . indeed, the important role of ccr5 in human host responses to wnv encephalitis was demonstrated by a retrospective cohort study involving persons homozygous for ccr5δ32 [19] , a loss-of-function mutation found in 1-2 % of caucasians [20] . compared to individuals without the mutation, persons carrying a homozygous ccr5δ32 allele have an increased risk of symptomatic wnv infection. in view of the large number of human infections caused by flaviviruses and their global distribution, there are concerns about the potential adverse outcomes of ccr5 antagonist use for incurable infectious diseases, including human immunodeficiency virus (hiv). furthermore, cd4 + foxp3 + regulatory t cells (tregs), which regulate excessive immune responses, are preferentially accumulated over effector t cells at sites of disease due to homing signals such as ccr5 [21] [22] [23] [24] . ccr5dependent homing of cd4 + foxp3 + tregs at infectious sites in parasitic pathogen infection models has been shown to promote pathogen persistence by regulating the magnitude of pro-inflammatory responses and the equilibrium between il-17 + cd4 + th17 and cd4 + foxp3 + tregs [23, 24] . recently, a putative role for cd4 + foxp3 + tregs in the pathogenesis of fatal acute inflammatory diseases caused by flaviviruses has been suggested in the context of their regulatory function [25, 26] . however, the role of cd4 + foxp3 + tregs in flavivirus encephalitis remains elusive due to a lack of direct evidence. presumably, ccr5-dependent recruitment of cd4 + foxp3 + tregs may affect the progression of viral encephalitis via their regulatory function. to address the direct regulation of je by cd4 + foxp3 + tregs in ccr5dependent homing context, we examined the role of ccr5 in je progression using ccr5-deficient (ccr5 −/− ) mice in this study. our results revealed that ccr5 −/− mice had exacerbated je, ultimately resulting in high mortality without altering cns viral burden, nk response, or t cell response compared to ccr5 +/+ mice. however, the increased susceptibility of ccr5 −/− mice to je was closely associated with decreased ratio of infiltrated cd4 + foxp3 + treg to il-17 + cd4 + th17 in the cns. this was directly confirmed by the fact that injection of sorted ccr5 + cd4 + foxp3 + tregs into ccr5 −/− mice provided ameliorated je without affecting cns infiltration of il-17 + cd4 + th17 cells or inflammatory ly-6c hi monocytes. therefore, our data suggest that ccr5 could dictate je progression by tightly regulating the balance between infiltrated cd4 + foxp3 + tregs and il-17 + cd4 + th17 cells in the cns. animals c57bl/6 (h-2 b ) mice (4-to 6-week-old female or male) were purchased from samtako (o-san, korea). ccr5 deficient (ccr5 −/− ) mice and foxp3 gfp knock-in mice (h-2 b ), which co-express egfp and regulatory t cellspecific transcription factor foxp3 under the control of an endogenous promoter, were obtained from jackson laboratories (bar harbor, me). ccr5 −/− ·foxp3 gfp mice were generated by crossing ccr5 −/− mice with foxp3 gfp knock-in mice. all mice were genotyped and bred in the animal facilities of chonbuk national university. jev beijing-1 strain was obtained from the green cross research institute (suwon, korea) and propagated in a mosquito cell line (c6/36) using dmem supplemented with 2 % fetal bovine serum (fbs), penicillin (100 u/ml), and streptomycin (100 u/ml) [27] . c6/36 cells were infected with jev beijing-1 at a multiplicity of infection (moi) of 0.1 and incubated in a humidified co 2 incubator at 28°c for 1 h. after absorption, the inoculum was removed and 7 ml of maintenance medium containing 2 % fbs was added. at approximately 6-7 days postinfection (dpi), cultures of host cells showing 80-90 % cytopathic effect (cpe) were harvested. virus stocks were titrated by conventional plaque assay or focusforming assay and stored in aliquots at −80°c until use. monoclonal antibodies used for flow cytometric analysis and other experiments were obtained from ebioscience (san diego, ca) or bd biosciences (san diego, ca), including fluorescein isothiocynate (fitc)-conjugated anti-cd3ε (154-2c11), ly6g (1a8), cd8 (53-67), phycoerythrin (pe)-conjugated anti-mouse cd11b (m1/70), foxp3 (fjk-16s), ifn-γ (xmg1.2), f4/80(bm8), granzyme b (ngzb), peridinin chorophyll protein complex (percp)-conjugated anti-mouse ly6c (hk 1.4), pecyanine dye (cy7)-anti-mouse nk1.1 (pl136), allophycocyanin (apc)-conjugated anti-mouse cd45(30-f11), il-17 (ebio17b7), tnf-α (mp6-xt22), biotinconjugated anti-mouse il-10 (jes5-16e3), and cd49b (dx5). peptides of the defined i-a b -restricted epitopes jev ns1 132-145 (tfvvdgpetkecpd), ns3 563-574 (wcfdgprtnail), and h-2d b -restricted epitope jev ns4b 215-223 (savwnstta) were chemically synthesized at peptron inc. (daejeon, korea). jev-specific primers for viral rna detection and primers specific for cytokines, chemokines, and transcription factors (table 1) were synthesized at bioneer corp. (daejeon, korea) and used for pcr amplification of target genes. quantitative real-time rt-pcr for determination of viral burden and cytokine expression viral burden and the expression of cytokines (il-1β, il-6, il-10, il-17, ifn-γ) and chemokines (ccl2, ccl3, ccl4, ccl5, cxcl1, cxcl2) in inflammatory and lymphoid tissues were determined using quantitative sybr greenbased real-time rt-pcr (real-time qrt-pcr). mice were intraperitoneally (i.p.) infected with jev (3.0 × 10 7 pfu). tissues including brain and spleen were harvested at 3, 4, 5, and 7 dpi following extensive cardiac perfusion with hank's balanced salt solution (hbss). total rnas were extracted from tissues using easyblue (intron, inc., daejeon, korea). reverse transcription of total rnas was performed using high-capacity cdna reverse transcription kits (applied biosystems, foster, ca). these complementary dnas (cdnas) were used for real-time qpcr using a cfx96 real-time pcr detection system (bio-rad laboratories, hercules, ca). the reaction mixture contained 2 μl of template cdna, 10 μl of 2× sybr primix ex taq, and 200-nm primers at a final volume of 20 μl. the reactions were denatured at 95°c for 30 s and then subjected to 45 cycles of 95°c for 5 s and 60°c for 20 s. after the reaction cycle was completed, the temperature was increased from 65 to 95°c at a rate of 0.2°c/15 s, and the fluorescence was measured every 5 s to construct a melting curve. a control sample containing no template dna was run with each assay, and all determinations were performed at least in duplicates to ensure reproducibility. the authenticity of amplified product was determined by melting curve analysis. viral rna burden in infected samples was expressed as viral rna copies per microgram of rna. the expression levels of cytokines and chemokines were normalized to βactin. all data were analyzed using bio-rad cfx manager version 2.1 analysis software (bio-rad laboratories). mice infected with jev were perfused with 30 ml of hbss at 3, 5, and 7 dpi via cardiac puncture of the left ventricle. the brains were harvested and homogenized by gently pressing them through 100-mesh tissue sieves, after which they were digested with 25 μg/ml of collagenase type iv (worthington biochem, freehold, nj), 0.1 μg/ml trypsin inhibitor nα-p-tosyl-l-lysine chloromethyl ketone, 10 μg/ml dnase i (amresco, solon, oh), and 10 mm hepe in hbss at 37°c for 1 h with shaking. cells were separated using optiprep density gradient (18/10/5 %) centrifugation at 800×g for 30 min (axis-shield, oslo, norway), after which cells collected from the 18 to 10 % interface were washed twice with pbs. cells were then counted and stained for cd11b, ly6g, ly6c, cd45, f4/80, cd3, cd4, cd8, and nk1.1 using directly conjugated antibodies (ebioscience) at 4°c for 30 min. finally, these cells were fixed with 10 % formaldehyde. data collection and analysis were performed with a facs calibur flow cytometer (becton dickson medical systems, sharon, ma) and the flowjo (tree star, san carlos, ca) software, respectively. the activity of nk cells was assessed by their capacity to produce ifn-γ following brief stimulation with pma and ionomycin (sigma-aldrich). briefly, splenocytes were prepared from ccr5 +/+ and ccr5 −/− mice at 2 dpi and stimulated with pma and ionomycin (pma at 50 ng/ml, ionomycin at 750 ng/ml) in the presence of monensin (2 μm) to induce the expression of ifn-γ for 1 h. after stimulation, cells were surface-stained with fitc-antimouse-cd3ε, pe-cy7 anti-mouse nk1.1, biotin-conjugated anti-mouse pan-nk cells (cd49b) [dx5] antibodies, and streptavidin-apc at 4°c for 30 min. cells were then washed twice with facs buffer containing monensin. after fixation, cells were permeabilized with 1× permeabilization buffer (ebioscience) and stained intracellularly with pe anti-mouse ifn-γ (xmf1.2) antibody in permeabilization buffer at room temperature for 30 min. after cells were washed with pbs twice, analysis was performed using a facs calibur flow cytometer and flowjo software. to monitor cd4 + and cd8 + t cell responses specific for jev, surviving mice were sacrificed at 7 dpi and splenocytes these splenocytes were then cultured in 96-well culture plates (5 × 10 5 cells/well) with synthetic peptide epitopes (ns1 132-145 , ns3 563-575 , or ns4b 215-225 ) in the presence of anti-cd154-pe for 12 h or for 6 h to evaluate cd4 + orcd8 + t cell responses, respectively [28, 29] . monensin (2 μm) was added to the antigenstimulated cells 6 h before harvest. cells were washed with pbs twice and surface-stained with fitc-anti-cd4 or cd8 antibodies at 4°c for 30 min, followed by washing twice with pbs containing monensin. after fixation, cells were washed twice with permeabilization buffer and stained with pe-anti-ifn-γ and apc-anti-tnf-α antibodies in permeabilization buffer at room temperature for 30 min. finally, cells were washed twice with pbs and fixed using fixation buffer. samples were analyzed using a facs calibur flow cytometer and flowjo software. intracellular staining for analysis of cd4 + th1, th17, and treg cells to monitor cd4 + th subsets, mice were infected i.p. with 3.0 × 10 7 pfu of jev and sacrificed at 3 and 5 dpi. brain leukocytes and splenocytes were prepared and cultured in 96-well plates (10 6 cells/well) with pma/ionomycin (th1 and th17) in the presence of monensin (2 μm) at 37°c for 5 h. stimulated cells were washed twice with pbs and surface-stained with fitc-anti-cd4 at 4°c for 30 min. after washing twice with pbs containing monensin and fixation, cells were washed twice with permeabilization buffer (ebioscience, sandiego, ca) and then stained with percp-anti-ifn-γ and apc-anti-il-17α in permeabilization buffer at room temperature for 30 min. after washing twice with pbs, cells were fixed with fixation buffer. to monitor treg cells, brain leukocytes and splenocytes were surface-stained with fitcanti-cd4 markers on ice for 30 min, followed by fixation with permeabilization concentrate buffer (ebioscience, ssan diego, ca) at 4°c for 6 h. after fixation, cells were washed twice with permeabilization and stained with peanti-foxp3 in permeabilization buffer at room temperature for 30 min. sample analysis was performed with a facs calibur flow cytometer. purification and trafficking analysis of ccr5 + cd4 + foxp3 + treg cells ccr5 + cd4 + fopx3 + treg cells were isolated from the spleen of foxp3 gfp knock-in mice using a facs aria sorter (becton dickson, palo alto, ca) with a final cell purity of ≥95 %. ccr5 + cd4 + fopx3 + treg cells were resuspended at density of 10 7 cells/ml in rpmi 1640 complete medium containing 10 % fbs, 1 % l-glutamine, 1 % nonessential amino acids, and 1 % penicillin/streptomycin. ccr5 + cd4 + foxp3 + treg cells (2 × 10 6 cells/mouse) were injected intravenously into jev-infected ccr5 −/− mice at 3 dpi. after injecting donor cells, brain and spleen tissues were harvested at 5 dpi. infiltrated cells were analyzed for the presence of gfp-labeled cells using a facs calibur flow cytometer. ccr5 − cd4 + fopx3 + treg cells were purified from ccr5 −/− ·foxp3 gfp mice and adoptively transferred into ccr5 −/− mice for the control group to ccr5 + cd4 + foxp3 + treg-recipients. in some experiments, il-10-producing ccr5 + cd4 + foxp3 gfp tregs were detected by intracellular il-10 staining combined with surface staining with ccr5 and cd4. all data are expressed as averages ± standard deviation. statistically significant differences between groups were analyzed using an unpaired two-tailed student's t test for leukocyte population analysis and in vitro experiments or anova and post hoc testing for multiple comparisons of the means. the significance of differences in viral burden and in vivo cytokine gene expression was evaluated by mann-whitney test or unpaired two-tailed student's t test. kaplan-meier survival curves were analyzed using the logrank test. a p value ≤0.05 was considered to indicate statistical significance. all data were analyzed using the prism software (graphpad prism 4, san diego, ca). ccr5 is essential for protection against je but dispensable for control of viral replication the chemokine receptor ccr5 is believed to play a critical role in recovery from flavivirus encephalitis via efficient leukocyte trafficking to the brain [16] [17] [18] . ccr5 is also a key mediator to recruit cd4 + foxp3 + tregs known as regulatory cd4 + t cell subset to dampen exacerbated inflammation such as viral encephalitis [21] [22] [23] [24] . although the role of cd4 + foxp4 + tregs in flavivirus encephalitis remains elusive, ccr5-dependent recruitment of cd4 + foxp3 + tregs may play certain roles in the control of encephalitis progression caused by flavivirus infection. to address this issue of ccr5 in flavivirus encephalitis, we first confirmed the role of ccr5 in je progression using ccr5-deficient (ccr5 −/− ) mice. after ccr5 +/+ and ccr5 −/− mice were infected with jev, surviving mice were monitored until 15 dpi (fig. 1a) . mice in both groups showed similar clinical signs, starting with generalized piloerection, paresis, and rigidity and followed by progression into severe neurological signs such as postural imbalance, ataxia, and generalized tonic-clonic seizure from 4 to 6 dpi. however, ccr5 ablation resulted in marked increases in mortality after showing neurological disorders, with a mortality rate of 100 % for ccr5 −/− mice vs. 54 % for ccr5 +/+ mice after jev infection (3.0 × 10 7 pfu). likewise, ccr5 −/− mice showed a rapid increase in the frequency of neurological disorder starting from 3 to 4 dpi after jev infection (3.0 × 10 7 pfu) with greater body weight loss, compared to ccr5 +/+ mice (fig. 1b, c) . however, the viral burden in the extraneural lymphoid tissue (spleen) and cns (brain and spinal cord) of ccr5 −/− mice was not increased compared to that of ccr5 +/+ mice (fig. 1d) . therefore, these results indicate that ccr5 ablation could result in an increased susceptibility to je progression irrespective of viral replication. to further characterize cns inflammation caused by jev infection in ccr5 +/+ and ccr5 −/− mice, we assessed the infiltration of cd11b + ly-6c hi monocytes and cd11b + ly-6g hi granulocytes into cns, because infiltration of these cell populations derived from the myeloid cell lineage has been used to evaluate cns inflammation [30] . our results revealed that a markedly higher frequency of infiltrated cd11b + ly-6c hi monocytes and cd11b + ly-6g hi granulocytes was retained in the brain of ccr5 −/− mice at 3, 5, and 7 dpi, compared to that in the brain of ccr5 +/+ mice (fig. 2a) . similarly, the absolute number of cd11b + ly-6c hi monocytes and cd11b + ly-6g hi granulocytes infiltrating the brain of ccr5 −/− mice was increased two-and threefold at 5 dpi, respectively (fig. 2b) . moreover, microglia cells contribute to the progression of encephalitis caused by some neurotropic viruses, such as wnv [31, 32] . thus, four-color (cd11c/cd11b/cd45/f4/80) staining was used to distinguish resting from activated microglia. based on the cns myeloid cell classification method of ford et al. [33] , equivalent percentages and similar absolute numbers of resting microglia (cd11c − cd11b hi cd45 int f4/80 + ) were detected in both ccr5 +/+ and ccr5 −/− mice. however, activated microglia/macrophages (cd11c − cd11b hi cd45 hi f4/80 + ) and other myeloidderived leukocytes (cd11c − cd11b int cd45 hi f4/80 + ) in the brain of ccr5 −/− mice were detected at higher frequencies and absolute numbers compared to the brain of ccr5 +/+ mice (fig. 2c, d) . these results indicate that ccr5 ablation could exacerbate je by enhancing the accumulation of data are average percentages ± sd of body weight relative to that at the time of challenge. d viral burden in lymphoid and inflammatory tissues during je. viral burden in lymphoid (spleen) and inflammatory tissues (brain and spinal cord) of infected mice (n = 5-6) were assessed by real-time qrt-pcr at the indicated time points. viral rna load was expressed as viral rna copy number per microgram of total rna (n = 5-7). *p < 0.05; **p < 0.01 compared to the levels in the corresponding groups inflammatory monocytes and granulocytes in the cns, along with activation of microglia. ccr5 is also considered to be involved in the recruitment of lymphoid lineagederived cells, including cd4 + , cd8 + t cells, and nk cells [34, 35] , which may play a beneficial role in the control of je progression [36] [37] [38] . to better understand cns inflammation in ccr5 −/− mice following jev infection, cd4 + and cd8 + t cells, and nk cells were enumerated in the brain. infiltration of both cd4 + and nk cells was evidenced by a transient increase in ccr5 +/+ mice at 3 dpi, after which the total number of cd4 + and nk cells in ccr5 +/+ and ccr5 −/− mice was comparable at 5 and 7 dpi. this result implies that cd4 + t and nk cells might not predominate in the control of je that has already progressed, because infected mice usually showed clinical signs at around 4-5 dpi. however, cd8 + t cells infiltrated the brain of ccr5 −/− mice at gradually increased levels up to 5 dpi compared to that in the brain of ccr5 +/+ mice (fig. 2e) , indicating that enhanced infiltration of cd8 + t cells is closely associated with je progression. in terms of cns inflammation, the expression of cytokines and chemokines within the cns is required for encephalitis, because encephalitis caused by neurotropic viruses is indirectly derived from cns degeneration, due to robust immunological responses such as uncontrolled secretion of cytokines and chemokines, which results in the activation of microglia and astrocytes [10, 11] . therefore, we examined the expression of cytokines and chemokines in the cns. we found that ccr5 ablation resulted in early and increased expression of pro-and anti-inflammatory the frequency and number of ly-6c hi monocytes and ly-6g hi granulocytes in the brain. ccr5 +/+ and ccr5 −/− mice were inoculated i.p. with jev (3.0 × 10 7 pfu), and the frequency (a) and total number (b) of ly-6c hi monocytes and ly-6g hi granulocytes in the cns were determined by flow cytometric analysis at 3, 5, and 7 dpi using vigorous heart perfusion. values in representative dot-plots denote the average percentage of the indicated population after gating on cd11b + cells. c, d resting and activated microglia/macrophage number in the cns. the number of resting (cd11c − cd11b hi cd45 int f4/80 + ) and activated (cd11c -cd11b hi cd45 hi f4/80 + ) as well as other myeloid-derived leukocytes (cd11b int cd45 hi f4/80 + ) was enumerated using flow cytometric analysis at 3 dpi. values in representative dotplots denote the average percentage of the indicated population after gating on cd11c − f4/80 + cells. e accumulated number of nk cells, cd4 + , and cd8 + t cells in the cns. total accumulated number of nk cells (cd3 − nk1.1 + dx5 + ), cd4 + (cd3 + cd4 + ), and cd8 + (cd3 + cd8 + ) t cells in the cns were enumerated using flow cytometric analysis at 3, 5, and 7 dpi. data are averages ± sd of the indicated cell populations derived from at least three independent experiments (n = 4-5). *p < 0.05; **p < 0.01; ***p < 0.001 compared with the levels of the indicated groups cytokines at 3 dpi. however, ifn-γ expression was higher in ccr5 +/+ mice at 3 dpi, compared to in ccr5 −/− mice. the early increase in cns infiltration of cd4 + and nk cells likely led to earlier and higher expression of ifn-γ. in addition, it was interesting that the expression levels of some cytokines were reversed at 5 dpi (fig. 3a) . notably, the anti-inflammatory cytokine il-10 was expressed at higher levels in ccr5 +/+ mice at 5 dpi, whereas il-17, which is produced by cd4 + th17 cells, was expressed at a lower level in ccr5 +/+ mice compared to those in ccr5 −/− mice at 5 dpi. with regard to chemokine expression, cc chemokines were expressed at higher levels in ccr5 −/− mice 4 dpi compared to those in ccr5 +/+ mice. interestingly, the expression levels of cxc chemokines, cxcl1 and cxcl2, were enhanced in ccr5 −/− mice at 3 dpi, and such expression was reversed at 5 dpi (fig. 3b) . collectively, these results indicate that the expression levels of pro-/antiinflammatory cytokines and cc/cxc chemokines in the cns of ccr5 +/+ and ccr5 −/− mice could change dynamically depending on the progression of je. because the cns expression pattern of cc and cxc chemokines in ccr5 +/+ and ccr5 −/− mice differed according to je progression, this cc/cxc chemokine expression may affect the migration of myeloid and lymphoid cells, including monocytes, granulocytes, nk, and t cells in both ccr5 +/+ and ccr5 −/− mice. therefore, to better understand the recruitment of myeloid and lymphoid cells in the cns, we kinetically examined the number of myeloid and lymphoid cells in the spleen and blood of ccr5 +/+ and ccr5 −/− mice depending on je progression. in addition, analyzing the spleen could provide insight into how ccr5 modulates innate and inflammatory responses immediately after infection. analysis of myeloid cd11b + and lymphoid cd8α + dc subsets revealed that both ccr5 +/+ and ccr5 −/− mice exhibited a similar reduction in the spleen (fig. 4a) , as wildtype mice have been previously shown to have a transiently decreased number of myeloid and lymphoid dcs due to jev infection [27] . however, ccr5 −/− mice had higher numbers of inflammatory cd11b + ly-6c hi monocytes and ly-6g hi neutrophil in the spleen up to 5 dpi, compared to ccr5 +/+ mice (fig. 4b) , indicating that ccr5 −/− mice experienced a severe inflammatory reaction in the spleen. also, the absolute number of splenic cd3 -nk1.1 + dx5 + nk cells was transiently decreased in both ccr5 +/+ and ccr5 −/− mice, but ccr5 +/+ mice had a higher number of cd3 − nk1.1 + dx5 + nk cells in the spleen at the early phase (1 and 2 dpi), compared to ccr5 −/− mice (fig. 4c) . interestingly, our results revealed that the frequency and number of nk cells producing ifn-γ were increased in ccr5 −/− mice, rather than in ccr5 +/+ mice, when the activation of nk cells was evaluated by assessing their production of ifn-γ (fig. 4d) . these data indicate that nk cells might not be involved in the amelioration of je in ccr5 +/+ mice. furthermore, our results revealed that the number of ly-6c hi monocytes and ly-6g hi neutrophils in the blood followed the infiltration trends of ly-6c hi monocytes and ly-6g hi neutrophils in the brain. ccr5-ablated mice had higher numbers of ly-6c hi monocytes and ly-6g hi neutrophils in the blood up to 5 dpi, compared to ccr5 −/− mice (fig. 4f) . also, nk cells in the blood of ccr5 −/− mice were detected at transiently lower levels at the early phase (1 and 2 dpi), after which they were comparable in ccr5 +/+ and ccr5 −/− mice (fig. 4g) . cd4 + t cell numbers were transiently higher number in the blood of ccr5 +/+ mice than in ccr5 −/− mice at 2 and 3 dpi, but cd8 + t cells accumulated to higher levels in ccr5 −/− mice, rather than ccr5 +/+ mice, at up to 5 dpi (fig. 4h) . therefore, these results suggest that ccr5 ablation is not involved in the migration of myeloid and lymphoid cells from the blood into the brain during je progression. antiviral adaptive immune responses, including those mediated by effector antigen-specific cd4 + and cd8 + t cells, are required for the regulation of je progression through the control and clearance of jev in extraneural lymphoid tissues and the cns [36] [37] [38] . although both ccr5 +/+ and ccr5 −/− mice infected with jev exhibited neurological disorders at 4-5 dpi, which is before functional adaptive immune responses were fully induced, we examined the generation of jev-specific cd4 + t cell responses in surviving ccr5 +/+ and ccr5 −/− mice at 7 dpi using intracellular cd154 staining combined with intracellular cytokine ifnγ staining, because cd154 + cd4 + t cells could enable us to enumerate viable jev-specific cd4 + t cells in response to stimulation with epitope peptides [28, 29] . as shown in fig. 5a , b, similar levels of jev-specific cd154 + cd4 + t cells were detected in ccr5 +/+ and ccr5 −/− mice. however, the frequencies of jev-specific cd4 + t cells producing ifn-γ were higher in ccr5 +/+ mice upon stimulation with the epitope peptides ns1 132-145 and ns3 563-574 , compared to those in ccr5 −/− mice. also, ifn-γ + cd4 + t cell numbers were higher in the spleen of ccr5 +/+ mice upon , and cd4 + /cd8 + t cells (h) were determined using flow cytometric analysis at the indicated time points. data are averages ± sd of values derived from at least three independent experiments (n = 3-5). *p < 0.05; **p < 0.01; ***p < 0.001 compared to the levels in the corresponding groups stimulation with jev epitope peptides at 7 dpi, compared to ccr5 −/− mice (fig. 5c, d) . presumably, this increase in ifn-γ + cd4 + t cells specific for jev ag may contribute in part to the control of je progression in ccr5 +/+ mice at a later phase. in contrast, the frequency and total number of jev-specific cd8 + t cells producing ifnγ and tnf-α in response to stimulation with the cd8 + t cell epitope ns4b 215-223 were higher in ccr5 −/− mice than in ccr5 +/+ mice (fig. 5e, f) . this result was inconsistent with the enhanced cns infiltration of cd8 + t cells. taken together, these results suggest that antiviral jev-specific cd8 + t cells may not be key players in the control of je progression in ccr5 +/+ mice at the early phase (4-5 dpi). however, ifn-γ + cd4 + t cells specific for jev ag appear to play a role in the control of je progression in ccr5 +/+ mice at the later phase (7-8 dpi). skewed il-17 + cd4 + th17 responses of ccr5-ablated mice during je progression nk and cd8 + t cells did not appear to play a dominant regulatory function in already progressed je because ccr5 +/+ mice failed to show enhanced cns infiltration of nk or cd8 + t cells at 5 dpi, compared to ccr5 −/− mice. in addition, a stronger jev-specific cd8 + t cell response was elicited in ccr5 −/− mice at 7 dpi, rather than ccr5 +/+ mice. although the ifn-γ + cd4 + th1 response specific for jev ag was stronger in ccr5 +/+ mice, adaptive jev-specific cd4 + t cell responses take some time to develop. therefore, adaptive jev-specific cd4 + t cell responses may contribute to the control of je progression only at a later stage. in our results, dynamic changes in the expression of pro-/anti-inflammatory cytokines, particularly il-10 and il-17, were observed in the cns during je progression at 3 and 5 dpi. therefore, we evaluated the dynamic response of cd4 + th subsets that produce typical pro-or anti-inflammatory cytokines: cd4 + th1 expressing ifn-γ, cd4 + th17 expressing il-17, and cd4 + foxp3 + tregs expressing il-10. first, we examined the frequency and total number of cd4 + foxp3 + tregs in the spleens of ccr5 +/+ and ccr5 −/− mice at 3 and 5 dpi, a time point at which the mice showed dynamic changes in cytokine expression. ccr5 +/+ mice exhibited a significantly higher frequency and number of cd4 + foxp3 + tregs at both 3 and 5 dpi, compared to ccr5 −/− mice (fig. 6a) . however, ccr5 −/− mice showed an increased frequency and total number of cd4 + th1 expressing ifn-γ and cd4 + th17 expressing il-17 in the spleen at both 3 and 5 dpi, compared to ccr5 +/+ mice (fig. 6b) . this result indicates that ccr5 −/− mice had a skewed response of these cd4 + th subsets at fig. 6 early skewed il-17 + cd4 + th17 response of ccr5-ablated mice during je progression. a, b frequency and number of cd4 + foxp3 + tregs, ifn-γ + cd4 + th1, and il-17 + cd4 + th17 cells in the spleen of ccr5-ablated mice. c, d frequency and number of cd4 + foxp3 + tregs, ifn-γ + cd4 + th1, and il-17 + cd4 + th17 cells in the brain of ccr5-ablated mice. the frequency and absolute number of cd4 + foxp3 + tregs (a, c), ifn-γ + cd4 + th1, and il-17 + cd4 + th17 cells (b, d) in the spleen (a, b) and brain (c, d) of ccr5 +/+ and ccr5 −/− mice were determined by flow cytometric analysis at 3 and 5 days following jev (3.0 × 10 7 pfu) infection. cd4 + foxp3 + tregs were detected with intracellular foxp3 and surface cd4 staining, and the frequency and number of ifn-γ + cd4 + th1 and il-17 + cd4 + th17 cells were determined by intracellular cytokine staining in response to pma + ionomycin stimulation of splenocytes or brain leukocytes prepared from ccr5 +/+ and ccr5 −/− mice. values in representative dot-plots are the average percentage of foxp3 + cells, ifn-γ + and il-17 + in cd4 + t cells. e expression of transcription factors by cns-infiltrated cd4 + t cells. after vigorous heart perfusion, sorted cd4 + t cells from cns-infiltrated leukocytes were briefly stimulated with pma plus ionomycin for 3 h. the expression of transcription factors of cd4 + th1, th2, th17, and tregs was determined by real-time qrt-pcr using total rna extracted from stimulated cd4 + t cells. data are averages ± sd of values derived from at least three independent experiments (n = 3-4). *p < 0.05; **p < 0.01; ***p < 0.001 compared with the levels of the indicated groups the early stage of je progression. to further define the skewed response of the cd4 + th subsets in ccr5 −/− mice, we examined cns-infiltrated cd4 + th subsets at 3 and 5 dpi during je progression. ccr5 +/+ mice showed a rapidly increased frequency and absolute number of cnsinfiltrated cd4 + foxp3 + tregs; levels were two-to threefold higher than those of ccr5 −/− mice (fig. 6c) . also, ccr5 +/+ mice exhibited a moderately increased number, but not frequency, of cd4 + th1 expressing ifn-γ in the cns at 3 and 5 dpi, whereas a markedly increased frequency and number of cd4 + th17 expressing il-17 were detected in the cns of ccr5 −/− mice; being approximately tenfold higher than those of ccr5 +/+ mice (fig. 6d) . moreover, cd4 + t cells sorted from the cns of ccr5 +/+ mice showed higher expression of the transcription factors t-bet and foxp3, which are involved in the differentiation of cd4 + th1 cells and tregs, compared to those from the cns of ccr5 −/− mice (fig. 6e ). in contrast, cd4 + t cells sorted from the cns of ccr5 −/− mice had higher expression of the cd4 + th17 transcription factor rorγt and il-17, compared to ccr5 +/+ mice. these results suggest that ccr5 ablation results in a skewed il-17 + cd4 + th17 response in both extraneural lymphoid tissue and the cns, which leads to reduced cns infiltration of cd4 + foxp3 + tregs during je progression, which is closely associated with exacerbation of je in ccr5 −/− mice. although our results suggest that the increased number of cd4 + foxp3 + tregs in extraneural lymphoid tissue and the cns of ccr5 +/+ mice is associated with mild je, we did not provide direct evidence regarding whether the enhanced response of cd4 + foxp3 + tregs plays a beneficial role in je progression. to address this issue, ccr5 + cd4 + foxp3 + tregs purified from ccr5 +/+ mice were injected i.v. into ccr5 −/− mice at 3 dpi, and the recipient mice were examined in terms of mortality and clinical signs up to 15 dpi. as shown in fig. 7a , ccr5 −/− recipients of ccr5 + cd4 + foxp3 + tregs showed a reduced mortality rate (around 50 %) comparable to that of ccr5 +/+ mice. however, ccr5 −/− mice that received ccr5 − cd4 + foxp3 + tregs purified from ccr5 −/−. foxp3 gfp mice showed high susceptibility to je, with a mortality rate of 90 %, similar to that of ccr5 −/− mice. in addition, a reduced proportion of mice showing neurological disorders was observed in ccr5 −/− recipients of ccr5 + cd4 + foxp3 + tregs, even though ccr5 −/− recipients of ccr5 + cd4 + foxp3 + tregs exhibited clinical signs starting at a similar time post-infection to those of ccr5 −/− mice and ccr5 − cd4 + foxp3 + treg recipients (fig. 7b) . also, ccr5 −/− recipients of ccr5 + cd4 + foxp3 + tregs showed no apparent reduction in body weight during je progression compared to ccr5 −/− mice receiving no tregs (fig. 7c) . these results suggest that ccr5 + cd4 + foxp3 + tregs purified from ccr5 +/+ ·foxp3 gfp mice could ameliorate je progression in ccr5 −/− mice, in contrast with ccr5 -cd4 + foxp3 tregs purified from ccr5 −/−. foxp3 gfp mice. to better understand je regulation in ccr5 −/− mice after injection with ccr5 + cd4 + foxp3 + tregs, the viral burden in extraneural lymphoid tissues and the cns was determined at 5 dpi. our results revealed that injection of ccr5 + cd4 + foxp3 + tregs resulted in no change in the viral burden in ccr5 −/− recipients. this result indicates that ccr5 + cd4 + foxp3 + tregs could regulate je progression without changing the viral burden (fig. 7d) . instead, ccr5 −/− recipients of ccr5 + cd4 + foxp3 + tregs showed enhanced expression levels of antiinflammatory cytokines, including il-10 and tgf-β, in the brain and spleen at 5 dpi, compared to ccr5 −/− mice receiving no tregs. however, il-17 expression was not changed in the brain and the spleen of ccr5 + cd4 + foxp3 + treg-injected ccr5 −/− recipients (fig. 7e, f) . collectively, these results indicate that ccr5 + cd4 + foxp3 + tregs injected into ccr5 −/− mice ameliorate je progression by enhancing the expression of anti-inflammatory cytokines. ccr5 + cd4 + foxp3 gfp tregs produce il-10 to ameliorate je without altering cns infiltration of ly-6c hi monocytes, cd4 + th1, or th17 ccr5 + cd4 + foxp3 gfp tregs injected into ccr5 −/− mice were detected in the cns and the spleen of recipients, whereas ccr5 −/− recipients receiving ccr5 -cd4 + foxp3 + tregs contained ccr5 − cd4 + foxp3 gfp tregs at a very low frequency (fig. 8a, b) . this result indicates that adoptively transferred ccr5 + cd4 + foxp3 gfp tregs were successfully infiltrated into the lymphoid and inflamed tissues, compared to ccr5 − cd4 + foxp3 gfp tregs. to further characterize cns inflammation in ccr5 −/− recipients of ccr5 + tregs, the infiltration of cd11b + ly-6c hi monocytes and cd11b + ly-6g hi granulocytes into the cns was assessed at 5 dpi. our results revealed that ccr5 + treg-injected ccr5 −/− recipients showed no significant changes in the frequency of cd11b + ly6c hi monocytes and cd11b + ly-6g hi granulocytes during je progression, compared to those of ccr5 −/− mice receiving ccr5 − tregs (fig. 8c) . similarly, injection of ccr5 + cd4 + foxp3 gfp tregs into ccr5 −/− recipients resulted in a moderate but insignificant increase in the number of cd11b + ly-6c hi monocytes and cd11b + ly-6g hi granulocytes in the cns (fig. 8d) . this result indicates that ccr5 + cd4 + foxp3 gfp tregs regulate je progression in ccr5 −/− mice without affecting cns infiltration of cd11b + ly-6c hi monocytes and cd11b + ly-6g hi granulocytes. we also enumerated each cd4 + th subset in the cns. ccr5 −/− recipients of ccr5 + cd4 + foxp3 + tregs had a higher number of cd4 + foxp3 + tregs in the cns at 5 days after infection, compared to ccr5 −/− mice that did fig. 7 ccr5 + cd4 + foxp3 + tregs ameliorate je in ccr5-ablated mice. a susceptibility of ccr5-ablated mice to je following adoptive transfer of ccr5 + cd4 + foxp3 + tregs. ccr5 + cd4 + foxp3 + and ccr5 − cd4 + foxp3 + treg cells were purified from ccr5 +/+. foxp3 gfp and ccr5 −/−. foxp3 gfp mice and adoptively transferred to ccr5 −/− mice (n = 10) at 2 days following jev (3.0 × 10 7 pfu) infection, respectively. surviving recipient mice were examined daily. ccr5 +/+ and ccr5 −/− mice that did not receive tregs were used as positive and negative controls, respectively. b ratio of mice showing neurological disorders in ccr5 + treg-injected ccr5-ablated mice. ccr5 −/− recipients that received ccr5 + or ccr5 − tregs were examined every 6 h from 4 to 11 dpi. c changes in body weight of ccr5 + treg-injected ccr5-ablated mice during je. data are averages ± sd of body weight relative to the time of challenge. d viral burden in lymphoid tissue and cns of treg-injected ccr5 −/− recipient. viral burden in the spleen, brain, and spinal cord of ccr5 −/− recipients was assessed by real-time qrt-pcr at 5 dpi. the viral rna load was expressed as viral rna copy number per microgram of total rna. e, f the expression of pro-and anti-inflammatory cytokines in lymphoid tissue and cns of treg-injected ccr5 −/− recipients. the expression of pro-and anti-inflammatory cytokines in the brain (e) and spleen (f) of treg-injected ccr5 −/− recipients was determined by real-time qrt-pcr at 5 dpi. data are averages ± sd of values derived from at least three independent experiments (n = 5-6). *p < 0.05; **p < 0.01 compared with the levels of the indicated groups fig. 8 ccr5 + cd4 + foxp3 + tregs ameliorate je via il-10 production without affecting the accumulation of myeloid-derived leukocytes, cd4 + th1, or th17. a, b detection of ccr5 + cd4 + foxp3 gfp tregs in the spleen and brain. adoptively transferred ccr5 + cd4 + foxp3 gfp and ccr5 − cd4 + foxp3 gfp tregs were detected by flow cytometry in the spleen (a) and brain (b) at 5 days following jev (3.0 × 10 7 pfu) infection. values in representative dot-plots denote the average percentages of ccr5 + cd4 + foxp3 gfp tregs in cd4 + t cells. c, d the frequency and number of ly-6c hi monocytes and ly-6g hi granulocytes in the cns of ccr5 + or ccr5 − treg-injected ccr5-ablated mice. the frequency (c) and number (d) of ly-6c hi monocytes and ly-6g hi granulocytes in the cns of ccr5 + (wt) or ccr5 − (ko) treg-injected ccr5-ablated mice were determined by flow cytometric analysis at 5 dpi using vigorous heart perfusion. values in representative dot-plots denote the average percentage of the indicated cell population after gating on cd11b + cells. e-h accumulated number of cd4 + th1, th17, and tregs in the cns of ccr5 + (wt) or ccr5 − (ko) treg-injected ccr5-ablated mice. the absolute number of cd4 + th1, th17, and tregs in the cns and spleen of treg-injected ccr5 −/− recipients was determined at 5 dpi. e cd4 + foxp3 + tregs in brain. f cd4 + th1 and th17 in the brain. g cd4 + foxp3 + tregs in the spleen. h cd4 + th1 and th17 in the spleen. i il-10 expression in adoptively transferred ccr5 + cd4 + foxp3 gfp and ccr5 − cd4 + foxp3 gfp tregs. the expression of il-10 in cns-infiltrated ccr5 + cd4 + foxp3 gfp and ccr5 − cd4 + foxp3 gfp tregs was evaluated by intracellular il-10 staining at 5 dpi. data are averages ± sd of values derived from at least three independent experiments (n = 3-4). *p < 0.05; **p < 0.01; p < 0.001 compared with the levels of the indicated groups not receive tregs (fig. 8e) . numbers of ifn-γ + cd4 + th1 and il-17 + cd4 + th17 cells in the cns of ccr5 + treginjected ccr5 −/− mice were not different compared to in the cns of ccr5 −/− mice, which did not receive tregs (fig. 8f) . ccr5 +/+ mice had an increased number of ifnγ + cd4 + th1 cells, but reduced number of il-17 + cd4 + th17 cells compared to ccr5 −/− mice, as shown previously (fig. 6d) . similarly, an increased number of cd4 + foxp3 + tregs was observed in the spleen of ccr5 + treg-injected ccr5 −/− recipients, compared to that of ccr5 −/− mice receiving ccr5 − tregs (fig. 8g) . however, the number of cd4 + foxp3 + tregs in ccr5 −/− recipients was lower than that in ccr5 +/+ mice. in addition, the injection of ccr5 + cd4 + foxp3 gfp tregs in ccr5 −/− mice did not affect the number of ifn-γ + cd4 + th1 and il-17 + cd4 + th17 cells in the spleen of ccr5 −/− mice during je progression (fig. 8h) . also, a higher proportion of ccr5 + cd4 + foxp3 gfp and ccr5 − cd4 + foxp3 gfp tregs adoptively transferred into ccr5 −/− mice were found to produce il-10, compared to cd4 + foxp3 − th cells (fig. 8i ). this indicates that il-10 production is comparable in ccr5 + and ccr5 − tregs purified from ccr5 +/+ ·foxp3 gfp and ccr5 −/− ·foxp3 gfp mice. taken together, these results suggest that ccr5 + cd4 + foxp3 + tregs injected to ccr5 −/− mice regulate je progression by producing il-10 and enhancing infiltration into lymphoid and cns tissues, compared to ccr5 − cd4 + foxp3 + tregs. in this study, we evaluated the role of ccr5 in je progression. the exacerbation of je in ccr5 −/− mice was typically associated with a skewed response to il-17 + cd4 + th17 cells and correspondingly reduced numbers of cd4 + foxp3 + tregs in the spleen and brain. we provided evidence that injection of sorted ccr5 + cd4 + foxp3 + tregs into ccr5 −/− mice ameliorated je progression without affecting cns infiltration of il-17 + cd4 + th17 cells, myeloid-derived ly-6c hi monocytes, and ly-6g hi granulocytes. instead, adoptive transfer of ccr5 + cd4 + foxp3 + tregs into ccr5 −/− mice increased the expression levels of two anti-inflammatory cytokines, il-10 and tgf-β, in the spleen and brain. our results suggest that ccr5 regulates the progression of viral encephalitis via governing a timely and an appropriate cns infiltration of cd4 + foxp3 + tregs and ultimately promoting survival of hosts suffering severe neuroinflammation. cd4 + foxp3 + tregs are believed to maintain host immune homeostasis by actively suppressing pathological and physiological immune responses after homing to inflamed tissues in response to the presence of foreign antigens [21] [22] [23] [24] . a putative and somewhat contradictory role of cd4 + foxp3 + tregs has been demonstrated in various models of pathogenic infections [23] [24] [25] [26] . a putative correlation between cd4 + foxp3 + treg levels and the outcome of infectious disease has been reported in wnv encephalitis because patients with symptomatic infection have lower cd4 + foxp3 + treg frequencies throughout the infection compared to asymptomatic patients [25] . in addition, a correlation of cd4 + foxp3 + tregs with the outcome of flavivirus infection has been reported; treg expansion but not absolute level was lower in children with severe dengue disease [26] . however, the factors involved in amelioration by cd4 + foxp3 + treg of severe flavivirus-induced disease are unclear. our results suggest a role for ccr5 in regulating je progression by mediating cd4 + foxp3 + treg homing, subsequently inducing skewed il-17 + cd4 + th17 responses in lymphoid and inflammatory tissues. furthermore, considering that asymptomatic and symptomatic populations have similar cd4 + foxp3 + treg frequencies prior to wnv infection, while asymptomatic patients exhibit greater treg expansion within the first 2 weeks of infection [25] , the proliferation and/or differentiation of cd4 + foxp3 + tregs in asymptomatic persons seems to be promoted by unknown factors (molecular or cellular components) derived from wnv infection. the expanded tregs then migrate into inflammatory tissues by means of homing receptors such as ccr5, thereby promoting host survival. in line with this notion, the expansion of cd4 + foxp3 + tregs in jev-infected ccr5 +/+ mice was around two-fold higher at 5 dpi, and the tlr4 signaling pathway is likely to be involved in their expansion in a je model [27] . also, the impact of ccr5 in cd4 + foxp3 + treg proliferation and its regulatory role in treg homing have been clearly demonstrated in a parasitic model [39] . ccr5-dependent recruitment of cd4 + foxp3 + tregs may dictate the magnitude of the cd4 + th1 and/or th17 subset responses to favor a detrimental or beneficial effect on pathogen persistence at the site of infection [23, 24, 40, 41] . our results favor a beneficial role for cd4 + foxp3 + tregs in ameliorating severe neuroinflammation caused by jev infection, depending on ccr5-mediated homing to inflammatory tissue. therefore, ccr5 is involved in the putative role of cd4 + foxp3 + tregs in severe flavivirus-induced diseases, such as encephalitis and hemorrhagic fever. the role of ccr5 in infectious diseases is variable in terms of its impact on pathogenesis and disease outcome. an essential role of ccr5 in ameliorating the outcome of infectious diseases has been documented in trypanosomiasis [42] , toxoplasmosis [43] , genital herpes [44] , influenza [45] , flaviviral west nile encephalitis, and je [16] [17] [18] , while a beneficial effect of ccr5 deficiency on the outcome of other infectious diseases has been postulated [23, 24, 39, 46, 47] . mechanistically, these variable outcomes of ccr5 deficiency in infectious diseases have been largely attributed to its regulatory effect on trafficking of leukocytes, including nk, cd4, cd8, and cd4 + foxp3 + treg cells to the site of infection as a consequence of the elevated immunopathology. therefore, this dichotomy in the role of ccr5 in regulating the outcome of infectious diseases prevents the generalization of our findings of the impact of chemokine receptors in disease prognosis. nevertheless, ccr5 is believed to play a crucial role in protection against severe neuroinflammation caused by flavivirus infections [16] [17] [18] . in this study, we also confirmed an essential role for ccr5 in regulating je progression. however, ccr5 deficiency failed to alter or increase the viral burden in extraneural tissue (spleen) and the cns. the activation of innate nk cells was increased in ccr5 −/− mice, rather than in ccr5 +/+ mice, as corroborated by enumeration of the ifn-γ-producing nk cells. in contrast, ccr5 +/+ mice showed a transiently higher number of cns-infiltrated nk cells with a loss of cd3 − nk1.1 + dx5 + nk cells in the spleen and blood of both ccr5 +/+ and ccr5 −/− mice after jev infection. although survived ccr5 +/+ mice displayed moderately increased responses of jev-specific cd4 + t cells at 7 dpi, ccr5 −/− mice showed a much higher frequency and number of jev-specific cd8 + t cells in response to stimulation with jev antigen. these split innate and adaptive immune responses of ccr5 −/− mice during je progression are contradictory to a previous report that nk cell responses and cd4 + as well as cd8 + t cell responses decreased in ccr5 −/− mice following jev infection [17] . the discrepancy might be due to differences in the genetic background and age of the host, strain and dosage of virus, and the route of challenge. indeed, because ccr5 +/+ and ccr5 −/− mice began to show clinical signs, such as neurological disorders, at 3-5 dpi, which is before functional adaptive immune responses were fully induced, the je model used in this study appeared to have more acute and rapid progression than that in a previous study, in which clinical signs were observed at 8-10 dpi [17] . this accelerated and rapid progression of je in ccr5 +/+ and ccr5 −/− mice might have resulted in induction of distinct nk and cd4/cd8 t cell responses in the host. early regulation of severe neuroinflammation in the cns through regulatory mechanisms such as cd4 + foxp3 + tregs and myeloidderived suppressor cells (mdsc) may be important for host survival in cases of acute and rapid progression of je. this notion is strengthened by the result that ccr5 +/+ and ccr5 −/− mice showed similar splenic cd4 + and cd8 + immune responses in a wnv infection model, to which mice are highly susceptible compared to humans [16] . also, the fact that cd4 + foxp3 + tregs can regulate the progression of wnv encephalitis in an infection model using depletion of cd4 + foxp3 + treg cells suggests an important role for cd4 + foxp3 + tregs in regulating the progression of fatal neuroinflammation caused by flaviviruses [25] . in this study, the regulatory role of cd4 + foxp3 + tregs in je progression was clarified by adoptive transfer of ccr5 + cd4 + foxp3 + tregs in ccr5 −/− mice. this is strongly supported by a recent report that tregs can ameliorate encephalitis by repressing effector t cell function [48] . however, the ccr5-mediated regulatory function of cd4 + foxp3 + tregs was likely to be relatively unimportant in je progression, because adoptive transfer of ccr5 + cd4 + foxp3 + tregs between 2 and 4 dpi ameliorated je progression, whereas cd4 + foxp3 + tregs that were adoptively transferred prior to jev infection rendered the recipients vulnerable to je (unpublished personal data). therefore, we used adoptive transfer of ccr5 + cd4 + foxp3 + tregs in ccr5 −/− mice at 3 dpi, the time point at which infected mice began to show clinical signs, such as generalized piloerection, paresis, and rigidity. although further study is warranted, ccr5 appears to play a non-committed role in je progression by regulating the trafficking equilibrium of effector leukocytes and regulatory cd4 + foxp3 + tregs, depending on disease progression. it is likely that our results discount the role of ccr5 in ameliorating je progression by cns infiltration of effector leukocytes such as nk cells, macrophages, cd4 + cells, and cd8 + t cells. it has long been assumed that leukocyte infiltration into the cns is critical for clearing virus and aiding recovery. ccr5 deficiency was associated with increased flavivirus burden in the cns but not in extraneural tissues, which was mechanistically mediated by inappropriate cns infiltration of leukocytes [16, 17] . however, the critical role of ccr5 in flavivirus pathogenesis appears to be unique in other neurotropic viruses, because infections of ccr5 −/− mice with several neurotropic viruses, such as lcmv [49] , retrovirus fr98 [50] , and mouse hepatitis virus (mhv) [51] , resulted in viral burdens in the cns similar to those of ccr5 +/+ mice. unlike earlier works on flavivirus encephalitis [16, 17] , the present study showed that the jev burden in the extraneural tissue and cns of ccr5 +/+ mice was similar to that in ccr5 −/− mice, with transiently and early increased cns infiltration of nk and cd4 + cells, but not cd8 + t cells, in ccr5 +/+ mice. although the mechanisms of increased cns infiltration of leukocytes in ccr5 −/− mice with an unchanged viral burden need to be defined, the je model used appears to affect the dynamics of leukocyte cns infiltration and the subsequent viral burden. cns trafficking of ly-6c hi monocytes and ly-6g hi granulocytes is mediated through a multistep process governed by cc and cxc chemokines. in support, the enhanced expression of cc chemokines including ccl2, ccl3, ccl4, and ccl5 appeared to facilitate cns infiltration of ly-6c hi monocytes in ccr5 −/− mice at the early phase, although whether these cells function to suppress or promote pathogenesis is unclear [52, 53] . one interesting result in this study was the reversal of cxc chemokine expression between 3 and 5 dpi. although cxcl1 and cxcl2 play a dominant role in the trafficking of ly-6g hi granulocytes, cc chemokines are also likely to be involved in cns infiltration of ly-6g hi granulocytes [54] . furthermore, increased cns infiltration of ly-6g hi granulocytes in ccr5 −/− mice is strengthened by the result that ccr5 ablation increases the recruitment of ly-6g hi granulocytes in herpetic encephalitis [55] . also, it is conceivable that cxcl2 is involved in the recruitment of granulocytic mdscs at a later stage [56] , thereby resulting in the amelioration of je progression. however, ccr5 appeared not to be involved in the migration of myeloid and lymphoid cells from the blood into the brain, because the accumulation of myeloid (monocytes, granulocytes) and lymphoid (nk, cd4/cd8 t cells) cells in the blood showed similar patterns to those in the brain. these data are in line with a previous report that ccr2 is not involved in monocyte migration from the blood into the brain [57] . ccr5 ablation may cause accumulation of ccr5 ligands (ccl3, ccl4, ccl5) via their compensation mechanism [58] , which could induce dysregulation of the migration of monocytes, nk cells, and t cells expressing cognate receptors (ccr1, ccr3). also, the appropriate adaptive cd4 + and cd8 + t cell responses can be achieved by orchestrated chemokine expression in secondary lymphoid tissues to promote contact between t and dendritic cells [59] . indeed, our results demonstrate unexpected adaptive jev-specific cd4 + and cd8 + t cell responses in ccr5 −/− mice; stronger responses of jev-specific cd4 + t cells were induced in ccr5 +/+ mice, whereas ccr5 −/− mice displayed a potent jev-specific cd8 + t cell response. ultimately, these speculations suggest that other chemokine receptors may be involved in the migration of myeloid and lymphoid cells as well as adaptive t cell responses in a ccr5-ablated environment, due to redundancy and compensation of chemokines and their receptors. in addition, the role of nk and cd8 + t cells in cns clearance of jev remains elusive, because the depletion or adoptive transfer of nk and cd8 + t cells does not contribute significantly to host survival or viral clearance [36] . also, although ifnγ + cd4 + th1 cells are believed to play a role in regulating je progression by reducing viral burden in the cns [38] , only co-transfer of immune cd4 + and cd8 + t cells, not individual transfer of either t cell subpopulation, significantly ameliorates je progression and promotes host survival [37] . these facts support the possibility that transiently and early increased cns infiltration of nk and ifn-γ + cd4 + th1 cells in ccr5 +/+ mice may not be sufficient for viral clearance from the cns, resulting in a similar cns viral burden in ccr5 +/+ and ccr5 −/− mice. therefore, balanced and orchestrated cns infiltration by innate nk and adaptive t cell subpopulations likely mediates viral clearance, thereby providing protection against je progression without tissue injury. il-17 is produced mainly by il-17 + cd4 + th17 cells. it plays a critical role in autoimmune and virus-caused immunopathologic diseases by facilitating neutrophil recruitment [60] [61] [62] . in contrast, il-17 appears to play a minor role in protective immunity against parasitic infection [63] but a more important role in fungal infection in a ccr5-ablated environment [39] . in the present study, cd4 + foxp3 + tregs did not directly regulate cns recruitment of il-17 + cd4 + th17 cells or their il-17 production. in addition, adoptive transfer of ccr5 + cd4 + foxp3 + tregs did not influence cns infiltration of ly-6c hi monocytes and ly-6g hi granulocytes. however, the anti-inflammatory cytokines il-10 and tgf-β produced by adoptively transferred ccr5 + cd4 + foxp3 + tregs might have played a role in regulating je progression. this notion is supported by the finding that insufficient antiinflammatory cytokine levels are associated with exacerbated je [64] . furthermore, our data are strongly supported by the finding that il-10 ablation exacerbates alphavirus encephalomyelitis by enhancing cns infiltration of il-17 + cd4 + th17 and ifnγ + cd4 + th1 cells, without affecting the amount of brain inflammation and viral replication [65] . indeed, the majority of adoptively transferred ccr5 + cd4 + foxp3 + tregs in ccr5 −/− mice produced il-10. furthermore, generation of cd4 + foxp3 + tregs and il-17 + cd4 + th17 cells is reciprocally regulated [60] [61] [62] . this developmental link between cd4 + foxp3 + tregs and il-17 + cd4 + th17 cells has led to speculation that these t cell subsets exist in equilibrium during inflammation and infection [66, 67] . however, this equilibrium was disturbed, thereby causing exacerbation of je progression in ccr5 −/− mice. therefore, our results provide insight into the utility of il-10 and cd4 + foxp3 + tregs for regulating je progression by maintaining the balance between cd4 + foxp3 + tregs and il-17 + cd4 + th17 cells at specific time points. ccr5 could regulate je progression via mediating the equilibrium between cd4 + foxp3 + tregs and il-17 + cd4 + th17 cells without causing tissue injury. this critical and extended role of ccr5 in flavivirus-induced diseases raises possible safety concerns regarding the use of ccr5 antagonists in hiv individuals who inhabit regions where both hiv and flaviviruses such as jev and wnv are endemic [68] . therefore, understanding the detailed role of ccr5 in the pathogenesis of flavivirus-caused encephalitis will be important to prevent any potential risk to hiv-positive individuals who take ccr5 antagonists with curative intent. neuroinvasive flavivirus infections emerging viral infections zoonotic encephalitides caused by arboviruses: transmission and epidemiology 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antigen presenting cells and causes liver damage via the il-23/il-17 axis two distinct populations of bovine il-17+ t-cells can be induced and wc1+il-17 +γδ t-cells are effective killers of protozoan parasites an insufficient anti-inflammatory cytokine response in mouse brain is associated with increased tissue pathology and viral load during japanese encephalitis virus infection interleukin 10 modulation of pathogenic th17 cells during fatal alphavirus encephalomyelitis reciprocal developmental pathways for the generation of pathogenic effector th17 and regulatory t cells in vivo equilibrium of proinflammatory il-17+ and regulatory il-10+ foxp3+ rorgamma t+ t cells maraviroc-a ccr5 antagonist for the treatment of hiv-1 infection we thank dr. yoon-young choi, center for university research facility (curf) at chonbuk national university, for sorting and analyzing cells by facs aria. this study was supported by a national research foundation of korea (nrf) grant funded by the korean government (misp) (2013r1a4a1069486). the funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. data supporting the conclusions of this article are presented in the manuscript.authors' contributions jhk and amp conceived the study and discussed the data with ske. jyc, sbk, eu, and fmah in part conceived the study and contributed to the experimental design. syp and jhl contributed to the reagent/materials/ analysis tools and provided the critical conceptual guidance. jhk, amp, and ske wrote the paper with contributions from all authors. all authors reviewed and approved the final version of the manuscript. the authors declare that they have no competing interests. ethics approval and consent to participate all animal experiments described in this study were conducted at chonbuk national university according to the guidelines set by the institutional animal care and use committee (iacuc) of chonbuk national university and were pre-approved by the ethical committee for animal experiments of chonbuk national university (permission code 2013-0028). the animal research protocol used in this study followed the guidelines provided by the nationally recognized korea association for laboratory animal sciences (kalas). all experimental protocols requiring biosafety were approved by the institutional biosafety committee (ibc) of chonbuk national university.received: 28 december 2015 accepted: 10 july 2016 • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-328763-hcbs20a0 authors: ifergan, igal; miller, stephen d. title: potential for targeting myeloid cells in controlling cns inflammation date: 2020-10-06 journal: front immunol doi: 10.3389/fimmu.2020.571897 sha: doc_id: 328763 cord_uid: hcbs20a0 multiple sclerosis (ms) is characterized by immune cell infiltration to the central nervous system (cns) as well as loss of myelin. characterization of the cells in lesions of ms patients revealed an important accumulation of myeloid cells such as macrophages and dendritic cells (dcs). data from the experimental autoimmune encephalomyelitis (eae) model of ms supports the importance of peripheral myeloid cells in the disease pathology. however, the majority of ms therapies focus on lymphocytes. as we will discuss in this review, multiple strategies are now in place to target myeloid cells in clinical trials. these strategies have emerged from data in both human and mouse studies. we discuss strategies targeting myeloid cell migration, growth factors and cytokines, biological functions (with a focus on mirnas), and immunological activities (with a focus on nanoparticles). myeloid cells play critical roles in the health and diseases of the central nervous system (cns). for example, myeloid cells constitute a significant proportion of the cells found within perivascular infiltrates in cns lesions of multiple sclerosis (ms) and its animal model, experimental autoimmune encephalomyelitis (eae) (1) (2) (3) . myeloid cells are also critically involved in the secondary damage in spinal cord injury (sci) and traumatic brain injury (tbi) (4) (5) (6) . these myeloid cells have the ability to attract other immune cells, release neurotoxic factors, phagocytose proteins and debris and promote the expansion, and polarization of antigen-specific t cells in the cns. in addition to their capacity to induce and sustain inflammation, myeloid cells are also critically involved in communication with glial cells and neurons, as well as in promoting and maintaining peripheral tolerance (7-9). ms is an inflammatory autoimmune disease wherein cells of the immune system initiate an attack against myelin in the cns that supports axonal conduction. the immune response in ms is thought to be mediated by autoreactive t lymphocytes that recognize myelin peptides. typically, demyelination is associated with an accumulation of t lymphocytes (lymphoid component of infiltrates) and monocytes/ macrophages/ dendritic cells (myeloid cells component of infiltrates) that arise from the migration of peripheral blood immune cells across the cns microvascular endothelium (10-12).as they infiltrate the cns, encephalitogenic t lymphocytes require the presence of these blood-derived antigen-presenting cells (apcs) to further sustain lymphocyte proliferation and cytokine polarization in the cns compartment (13-16). the role of these peripherally-derived myeloid cells in cns inflammation will be the focus of the present review. experimental autoimmune encephalomyelitis is a commonly utilized mouse model of ms that recapitulates many aspects of the human disease such as the cns inflammation, encephalitogenic t cell infiltration, and attack of oligodendrocytes resulting in demyelination. although not perfect, eae has allowed uncovering some of the molecular pathways governing the pathogenesis of ms such as elucidating the pathogenic role of t h 17 lymphocytes. in addition, eae models were critical in identifying and testing new therapeutic agents such as glatiramer acetate (ga) and natalizumab (17). although myeloid apcs play a prominent role in the pathogenesis of ms, there has been little consideration given to targeting these cells as an ms therapy. some of the current ms disease-modifying therapies may act on myeloid cells even if these cells were not the original intended targets (18). however, interfering directly with myeloid cell has proven to be efficacious in other diseases including psoriasis with multiple drugs targeting il-23 (guselkumab, risankizumab, and tildrakizumab) or il-12 and il-23 (ustekinumab) (19), crohn's disease and ulcerative colitis targeting il-12 and il-23 (ustekinumab) (20), rheumatoid arthritis targeting il-1 (anakinra) (21), systemic juvenile idiopathic arthritis targeting il-1β (canakinumab) (22), and many others. there are ongoing clinical trials in rheumatoid arthritis, stroke, atherosclerosis, and cancer using agents that target myeloid cells and their products. biber et al. have provided a recent comprehensive review of drugs in clinical trials targeting myeloid cells in cns diseases such as alzheimer's disease, brain tumors, and inflammatory pain, as well as for other cns diseases (23). as we will discuss in this review, multiple tools have been developed in the eae models of ms demonstrating significant regulation of disease progression by various approaches blocking myeloid cell activation and effector function, but to date, these approaches have not been tested for therapeutic efficacy in ms patients. the first strategy we will discuss is interference with peripheral myeloid cell migration to the cns. the blood-brain barrier (bbb), composed of tightly bound endothelial cells (ecs), regulates the entry of blood-borne molecules and immune cells into the cns. under physiological conditions, a limited number of peripheral blood immune cells gain access to the cns, a process called immune surveillance (24). during an inflammatory process, meningeal, and bbb-ecs amplify the migration of immune cells into the cns parenchyma, in a multistep process that involves selectins, chemokines and cell adhesion molecules (25). bbb-ecs express cell adhesion molecules such as intercellular adhesion molecule (icam)-1, vascular cell adhesion molecule (vcam)-1, activated leucocyte cell adhesion molecule (alcam), and melanoma cell adhesion molecule (mcam) which mediate at least in part, the adhesion process and the transmigration of leucocytes to the cns through their interaction with integrins αlβ2 [leucocyte function-associated antigen (lfa)-1], α4β1 [very late antigen (vla)-4], cd6, and mcam respectively (26-31). interfering with immune cell trafficking across the bbb by targeting adhesion molecules has proven to be beneficial in reducing clinical disease activity and pathological indices in ms (32). indeed, natalizumab, which blocks vla-4, the ligand of vcam-1, is reported to reduce migration of most leukocyte subtypes, including myeloid cells, into the brain. more recently, a new adhesion molecule expressed by bbb-ecs called nerve injury-induced protein (ninjurin)-1 was described (33). ninjurin-1 is a membrane protein known to interact in a homophilic manner through an extracellular residue-binding motif (34). on immune cells, ninjurin-1 was weakly expressed by lymphocytes, but highly expressed by peripheral myeloid apcs including monocytes, macrophages and dendritic cells (dcs), in humans and mice. interestingly, ninjurin-1 was also found to be expressed in ms lesions. ninjurin-1 neutralization specifically abrogated the adhesion and migration of human monocytes across a monolayer of bbb endothelial cells, without affecting lymphocyte recruitment. moreover, ninjurin-1 blockade during the course of eae reduced infiltration of peripheral myeloid cells and reduced clinical disease activity and histopathological indices of eae (33). another adhesion molecule involved in the migration of peripheral myeloid cells is junctional adhesion molecule (jam)-like (jaml). jams are type i transmembrane proteins differentially expressed at the junctions of ecs, epithelial cells, and on various leukocytes (35). similarly to ninjurin-1, jaml can interact in a homophilic manner (36). it was observed that jaml is expressed by bbb-ecs, and has an increase expression in ms lesions compared to normal appearing white matter (37). in addition, human monocytes and cd8 + t cells were found to express jaml, and its level was significantly increased on rrms patients when compared control subjects: 80 vs. 52% for monocytes, and 5.5 vs. 2.1% and for cd8 + t cells. these data reveals that jaml might be a more important adhesion molecule for monocytes than for cd8 + t cells. however, migratory capacity of both cell types was significantly compromised when jaml was blocked. chemotactic cytokines (chemokines) are secreted proteins that regulate the migration of leukocytes. chemokine receptor signaling plays a central role in cell migration during inflammatory responses in autoimmune and infectious diseases as well as in cancer. there are ∼50 chemokines and 20 receptors known at this time. blockade of ccr1 and ccr2 have been the two majors targets in a half dozen ms clinical trials (38). the chemokines ccl3 (macrophage inflammatory protein-1α-mip-1α) and ccl5 (regulated on activation, normal t cell expressed and secreted-rantes) bind to ccr1, while ccl2 (monocyte chemoattractant protein 1-mcp-1) binds to ccr2. both lymphoid and myeloid cells express ccr1 and ccr2, with monocytes/macrophages/dcs the cells where these chemokine receptors are most abundant (39-42). in animal models of ms, it was shown that ccr1-deficient animals developed a less severe disease (43), while ccr2-deficient mice were completely resistant to disease induction (44, 45), highlighting the importance of signaling through these chemokine receptors for disease initiation. in addition, it has been shown that ccr2 + ly-6c hi monocytes are rapidly recruited to the inflamed cns in eae and are crucial for the effector phase of disease. selective depletion of this specific monocyte subpopulation through engagement of ccr2 significantly reduced disease severity (46). ccr1 + and ccr2 + macrophages were both found in active ms lesions (47, 48). the role of chemokines and their receptors are now well-characterized in ms and other inflammatory diseases. the potential for a therapy targeting this signaling pathway is well-recognized. however, none of the chemokine-directed ms clinical trials has shown robust clinical efficacy. similar lack of clinical responses have also been reported in therapeutic trials targeting chemokines in other diseases such as rheumatoid arthritis, psoriasis, asthma, and many others (49). the issue may lie in the redundancy of chemokine/chemokine receptor action, in which case, it may be beneficial to develop strategies employing multiple antagonists simultaneously. as innate cells, myeloid cells express pattern recognition receptors (prrs). prrs include toll-like receptors (tlrs), rig-i-like receptors, nod-like receptors, and c-type lectin receptors (clrs) (50). selectins, which are part of the c-type lectins family, are known to play a crucial role in the control of leukocyte trafficking and homing to sites of inflammation (51). selectins are particularly important for the rolling of cells on endothelial cells, an important component of migration of myeloid cells into tissue sites of inflammation (52). more recently, it was uncovered that clec12a, a clr, was involved in facilitating binding and transmigration of dcs across the bbb in response to ccl2 chemotaxis (53). in eae, clec12a −/− mice displayed delayed disease onset and significantly reduced disease severity. additionally, in a chronic model of eae, anti-clec12a antibody treatment initiated at disease initiation also delayed onset and lessened disease severity. anti-clec12a antibody administration to mice undergoing relapsing-remitting eae after disease onset, resulted in less severe disease relapse (53). although the ligand of clec12a is currently unknown, it was suggested that the ligand is present on bbb endothelial cells (53). abundance of immune cells as well as cytokines, chemokines and immunoglobulins in ms plaques and their accumulation in the cerebrospinal fluid (csf) of ms patients, support the notion that ms is an inflammatory disorder. these observations lend support to the idea that immune cell products, especially cytokines, have an important role in both the induction and progression of ms. targeting cytokines has been a successful strategy used in therapy of other inflammatory diseases. for example, blockade of tumor necrosis factor (tnf) has shown positive results in rheumatoid arthritis and crohn's disease (54, 55) . as of december 2016, tnf inhibitors were the world's leading drug class, with sales of more than us $30 billion and used in more than seven million patients (56) . in ms, the first treatment approved for rrms was interferon (ifn)-β, thus showing that cytokines manipulation is potentially a good strategy. current data suggests that ms, and its animal model, eae, are driven by both t h 1 lymphocytes, producing ifn-γ, interleukin (il)-2 and tnf, and t h 17 lymphocytes, producing il-17, il-21, il-22, and granulocyte-macrophage colony-stimulating factor (gm-csf also known as csf-2). surprisingly, ifn-γ, il-12, il-17a, il-17f, il-21, and il-22 have all been shown to be dispensable for the development of eae [reviewed in (57) ; discussed here (58) ]. however, in 2011, the cns pathogenicity of t h 17 cells was reported to be primarily associated with their production of gm-csf (59, 60) . gm-csf production by t cells has been correlated with pathogenesis in several autoimmune diseases, including ms, rheumatoid arthritis, and myocarditis. it was reported that il-1β-and il-23-induced production of gm-csf by cns-infiltrating cd4 + t cells is essential for the induction of eae (59, 60) . gm-csf is a hematopoietic growth factor produced by a number of hematopoietic and non-hematopoietic cell types including activated cd4 + t cells, monocytes/macrophages, b cells, nk cells, endothelial cells and epithelial cells. gm-csf has a wide array of functions, notably the survival and activation of myeloid cells, the ability to induce differentiation of dendritic cells (dcs), the polarization of macrophages toward a pro-inflammatory m1 phenotype, enhanced antigen presentation, the induction of complement-and antibody-mediated phagocytosis, and the mobilization of monocytes and other myeloid populations from bone marrow to blood (61) (62) (63) . the gm-csf receptor (gm-csf rc) is a heterodimer comprised of a specific low-affinity α chain (cd116; gm-csf rα) and a common β chain (cd131; gm-csf rβ) that is shared by il-3 and il-5 (64) . the gm-csf rc is expressed in multipotent myeloid progenitor cells and continues to be expressed throughout myeloid development on monocytes, dcs, macrophages and neutrophils (65) (66) (67) . it is not expressed by t and b lymphocytes (67) . thus, most of the suspected direct effects of the gm-csf in diseases are focused on myeloid cells (peripheral and cns resident). findings related to the function of gm-csf signaling in eae pathology have been recently reviewed (68) . in brief, in eae, gm-csf is necessary for disease as gm-csf kos were found to be resistant to disease induction (69) . disease can be rescued by the administration of recombinant gm-csf. adoptive transfer using cytokine-deficient mice showed that wild-type, il-17a −/− , and ifnγ −/− t cells induced eae with similar kinetics. by contrast, gm-csf −/− t cells were incapable of inducing eae and invading the cns (59) . due to the variety of cells gm-csf can stimulate, it became important to determine the cell population in which signaling was necessary for disease. a bone marrow chimera study determined that peripheral myeloid cells, but not microglia, are key responders (59) . this corresponds with earlier observations that gm-csf administration stimulated cd11b + ly6c hi inflammatory monocytes into the circulation (70) . circulating ly6c hi monocytes traffic across the blood-brain barrier, upregulate pro-inflammatory molecules, and differentiate into central nervous system dcs and macrophages (70) . these data were confirmed recently using conditional gene targeting in which the β chain of the gm-csf receptor (csf2rb) was deleted in specific subpopulations throughout the myeloid lineages (71) . it was found that deletion of csf2rb in ccr2 + ly6c hi monocytes phenocopied the eae resistance seen in complete csf2rb-deficient mice. in humans, gm-csf levels in the csf are higher in patients with active ms than in patients in remission (72) . also, untreated ms patients had significantly greater numbers of cd4 + gm-csf + t cells and cd8 + gm-csf + t cells in peripheral blood compared with healthy controls and with ifn-β-treated ms patients (73) . in addition, ifn-β significantly suppressed gm-csf production by t cells in vitro. more recently, the canadian b cells in ms team uncovered a subset of memory b cells producing gm-csf (74) . in vitro, gm-csf-expressing b cells efficiently activated myeloid cells in a gm-csf-dependent manner, and in vivo, b cell depletion therapy resulted in a gm-csf-dependent decrease in pro-inflammatory myeloid responses of ms patients. in light of the critical role of gm-csf in the pathogenesis of ms and other inflammatory diseases, multiple tools have been developed targeting either the cytokine or the receptor. first tested in eae, it has been shown that blocking antibodies against gm-csf in chronic (c)-eae (69) or antibodies against gm-csf rα in c-eae and relapsing-remitting (rr)-eae (75) were able to prevent disease if given at the time of eae induction (day 0). mice treated with anti-gm-csf after disease onset completely recovered within 20 days of treatment in a model of c-eae (69) . therapeutic treatment with anti-gm-csf rα ameliorated progression of c-eae and resulted in a significant reduction of the relapse severity of rr-eae (75) . blockade of the gm-csf rα led to a reduction of activated mdcs, and reduced pro-inflammatory cytokine production by cd11b + ly6c + inflammatory monocytes. additionally, anti-gm-csf rα altered the expression of chemokine receptors, leading to the possibility that antibody treatment may impede cell migration (75) . logically, the next step is to test the therapeutic potential of gm-csf targeting in humans. a review of tools developed for clinical trials can be found here (76) . at this time, gm-csf blocking antibodies have been tested in rheumatoid arthritis and have shown promising results. as for ms, only one drug has been tested in clinical trials: mor-103 (also known as gsk3196165 or otilimab), a human antibody to gm-csf. the results of a phase ib clinical trial employing mor-103 in patients with relapsing-remitting or secondary-progressive ms have shown the drug to be safe and well-tolerated, although with modest efficacy (77) . at this moment, there are no ongoing clinical trials targeting gm-csf or gm-csf receptor in ms. another important growth factor regulating myeloid cell function is macrophage colony-stimulating factor (m-csf also known as csf-1). m-csf is ubiquitously produced in the steady state by a variety of cells, including endothelial cells, fibroblasts, osteoblasts, smooth muscle, and macrophages, and can be detected in plasma at ∼10 ng/ml (78) (79) (80) . the levels of circulating m-csf are upregulated in pregnancy (81) as well as in many different pathologies including cancer, autoimmune diseases and chronic inflammation (82) (83) (84) (85) (86) . m-csf stimulates progenitor cells from bone marrow and plays an important regulatory role in the survival, proliferation (in mice), differentiation, phagocytosis, and chemotaxis of myeloid cells, including monocytes, macrophages, dcs, and microglia (87) (88) (89) . the effects of m-csf are mediated by signaling through the type iii tyrosine kinase transmembrane receptor csf-1r (cd115), which is encoded by the c-fms proto-oncogene (90) . il-34 is also able to bind csf-1r with similar outcomes as to m-csf binding (88) . however, m-csf and il-34 present differences in their spatiotemporal expression patterns, and thus seem to play complementary roles in their biological activities on target cells (88, 91, 92) . csf-1r is expressed by myeloid cells such as monocytes, macrophages, dcs, and microglia, as well as by trophoblasts, neural progenitor cells and epithelial cells (93, 94) . there is ongoing debate about whether m-csf is a pro-inflammatory or pro-repair cytokine. m-csf seems to be essential for the survival and renewal of tissue-resident macrophages, but not for circulating myeloid cells. indeed, in the osteopetrotic csf1 op /csf1 op mouse, which harbor an inactivating mutation in the coding region of the csf-1 gene and are m-csf deficient, the functions and numbers of several tissue macrophage populations are altered while there is no difference in monocyte populations in the blood (95) . these findings were later confirmed in mice deficient for a specific enhancer for csf-1r gene, the fms-intronic regulatory element (fire) (96) . csf1r fire/ fire mice present a deficit in tissue resident macrophages in the brain (microglia), skin, kidney, peritoneal, and heart without significant differences in blood monocytes. during inflammation, the presence of monocytes in inflamed tissue is critical for proper immune responses, notably due to their capacity to traffic to draining lymph nodes and their ability to present antigens to t cells (2, (97) (98) (99) (100) (101) (102) (103) . while tissue resident macrophages also participate in inflammatory processes, their role in promoting tissue repair and regeneration is critical (104, 105) . for example, m-csf favors kidney and liver repair after acute injury (106) (107) (108) . moreover, m-csf is used to drive human and in mouse macrophage differentiation in vitro into an anti-inflammatory (m2) phenotype (109) (110) (111) . in eae, it was shown that peritoneal apcs treated with m-csf and pulsed with mog 35−55 , the disease initiating peptide, were able to suppress ongoing eae when injected at the time of disease initiation or significantly reduce the severity of the disease when injected at day 7 post-immunization (112) . these m-csf activated apcs were demonstrated to induce a treg profile from cd4 + t cells (cd25 + foxp3 + ) with increased secretion of il-10 and decreased secretion of il-17, ifn-γ, and tnf (112) . however, as mentioned earlier, elevated levels of m-csf are also observed in different pathologies. there are multiple publications linking m-csf/il-34 and csf-1r signaling in models of arthritis (113) (114) (115) (116) , diabetes (117) , systemic lupus erythematosus (85, 118) , cancer (119) (120) (121) , amyotrophic lateral sclerosis (122), parkinson's disease (123) , and alzheimer's disease (124) (125) (126) . in an effort to determine the role of m-csf/il-34 and csf-1r signaling in ms, different groups used potent cfms tyrosine kinase inhibitors, which block m-csf signaling. ki20227 (127) , imatinib (128) , gw2580 (128, 129) , sorafenid (128) , and plx5622 (130) are all tyrosine kinase inhibitors that have shown to effectively treat c-eae. gw2580 has the greatest apparent specificity for csf-1r vs. the other kinase inhibitors (131) . amelioration of eae using ki20227 was associated with the suppression of myeloid cell expansion in the spleen and reduction in mog-specific t-cell proliferation (127) . gw2580 and sorafenib suppressed tnf-α production by macrophages whereas imatinib and sorafenib both abrogated pdgf-induced proliferation of astrocytes (128) . plx5622 effect was associated with microglia and macrophage ablation from the white matter (130) . however, in the cuprizone model of cns demyelination, which allows study of the remyelination process with little involvement of the peripheral immune cells (132), injection of m-csf reduced demyelination by boosting microglia activity (133) . tamoxifen-induced conditional deletion of the csf-1r in microglia from cuprizone-fed mice caused aberrant myelin debris accumulation and reduced microglial phagocytic responses (89, 133) . these data indicate that m-csf plays an important role in ability of microglia to clear myelin debris and to support proper remyelination, and suggest m-csf functions as a critical factor in tissue repair. these divergent results exemplify the various functions of m-csf/il-34 and csf-1r signaling on cells. the possible contribution of m-csf signaling to both inflammatory and repair processes suggest that targeting m-csf in ms may be problematic. however, although there is an increase of myeloid cells in ms lesions, the expression of csf-1r is lower in ms lesions when compared to normal appearing white matter (134) . it is thus possible to hypothesize that a therapeutic treatment targeting m-csf in ms would primarily target peripheral myeloid cells rather than those in the cns. there are now multiple tools targeting m-csf signaling approved for human therapy, especially for cancer. imatinib was the first tyrosine kinase inhibitor approved for the treatment of chronic myelogenous leukemia (135) . imitanib is also now in clinical trials for the treatment of different pathologies, such as rheumatoid arthritis, type i diabetes and asthma, for which positive results of a phase 2 clinical trial were recently published (136) . sorafenib is approved for the treatment of primary kidney cancer and advanced primary liver cancer (137) . although there are side effects related to these inhibitors, an important advantage of tyrosine kinase inhibitors is the fact they can be administered orally to the patients. in september 2019, a phase 3 clinical trial for rrms was started testing the efficacy of evobrutinib, a bruton's tyrosine kinase inhibitor. although this is not a csf-1r inhibitor, it shows: (1) the desire to develop oral treatments in ms, and (2) the possibility of targeting tyrosine kinases in ms. bruton's tyrosine kinase are critical for b cell receptor signaling and is also involved in tlr signaling as well as inflammasome activation in myeloid cells (138) cytokines as mentioned earlier, blockade of myeloid specific cytokines il-1β, il-12, and il-23 have proven to be efficient therapies in multiple diseases such as crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, and systemic juvenile idiopathic arthritis. these cytokines are all involved in cd4 + t lymphocytes differentiation. while il-12 is critical for t h 1 induction (139), il-1β and il-23 are both involved in t h 17 differentiation and promote the encephalitogenic capacity of these cells by inducing gm-csf expression (60, 140, 141) . in eae, mice lacking il-1β, or the receptor, il-1r, developed a milder disease than wt animals (140, (142) (143) (144) (145) . moreover, specific ablation of il-1r on cd4 + t cells resulted in significantly reduced disease severity (146) , confirming the importance of il-1β signaling on t cells to induce a full eae. in addition, rats treated with an il-1 receptor antagonist (il-1ra), which blocks the biological activity of il-1β, developed milder signs of eae compared to control animals (147) . as il-1β secretion is the result of inflammasome activation, mice treated with a blocking agent for the inflammasome component nlrp3 exhibited decreased eae severity (148) . in ms, it was shown that il-1r expression is significantly higher in cd4 + t cells from rrms patients than from healthy controls (149) . il-1β expression was also found to be significantly increased in ms lesions when compared to tissue from other neurological diseases (150) . interestingly, multiple treatments used in ms [e.g., ifn-β, glatiramer acetate, and natalizumab] have shown to increase il-1ra expression and/or to decrease il-1β production (151, 152) ]. multiple tools have been developed to block il-1β activity: the recombinant il-1ra anakinra used for rheumatoid arthritis, the neutralizing il-1β antibody canakinumab used for systemic juvenile idiopathic arthritis as well as cryopyrin-associated periodic syndrome, and the soluble decoy il-1 receptor (rilonacept) also use for cryopyrin-associated periodic syndromes (153) . at this time, anakinra is the only il-1β-targeting drug in clinical testing for ms. this phase i/ii clinical trial just started a few months ago, and at this time, it is still in the recruitment phase (nct04025554). il-12 and il-23 are heterodimeric cytokines that share a common subunit il-12p40. the other subunit needed to form il-12 is il-12p35, while the other subunit to form il-23 is il-23p19. il-12 signals through the il-12 receptor (il-12r) composed of the il-12rβ1 and il-12rβ2 subunits, while il-23 signals through il-23r and il-12rβ1 (154) . thus, il-12rβ1 is required for biological response to both il-12 and il-23. when specific gene ablation was tested for the different receptor chains of il-12 and il-23, it was found that il-12rβ1 −/− mice were completely resistant to eae (155) . however, il-12rβ2 −/− mice developed severe eae, extensive inflammation and demyelination, and higher production of pro-inflammatory cytokines than wt animals (156) . finally, similar to il-12rβ1 −/− mice, il-23r −/− mice were completely resistant to eae induction (157) . as for the cytokines, mice deficient for the subunits il-23p19 or il-12p40 were resistant to eae. by contrast, mice in which the subunit il-12p35 was deleted were highly susceptible to eae (158) . in addition, treatment with anti-il-12p40 antibodies inhibited both murine and primate models of eae (159) (160) (161) . treatment with anti-il-23p19 antibodies reduced the clinical severity and prevented relapsing eae by inhibiting epitope spreading (162) . these results led to the conclusion that il-23 was a more critical factor than il-12 in the inflammatory response observed in eae. nevertheless, there are multiple studies linking both cytokines to ms pathology. it was demonstrated that peripheral blood monocytes from progressive ms patients produced increased amounts of il-12 compared to controls and that il-12 production correlated with disease activity (163) . another study showed an augmented level of il-12 mrna-expressing cells in the peripheral blood and the csf of ms patients when compared to controls (164) . there was also elevated levels of il-12p70 detected in plasma from ms patients compared to healthy individuals. a more recent report showed that both rrms and secondary progressive ms patients had increased levels of il-12p40 mrna compared with controls during the development of active lesions (165) . il-12p40 and il-23p19 have also been detected in human ms lesions (166, 167) . based on this and other data, there was hope that ustekinumab, an il-12p40 neutralizing antibody, would be efficacious for treatment of ms. however, disappointingly no clinical improvement in the treatment group compared to the placebo was found (168) . possible reasons for the failure of ustekinumab are the broad range of ms patients in the trial, many having very severe symptoms and long-standing disease. also, there may be weak bioavailability of the drug as ustekinumab may be inefficient in crossing the bbb (169) . at this time there are no ongoing trials targeting il-12/il-23 in ms despite the impressive results in the various animal models of the disease. micrornas (mirnas) are small non-coding rnas of 17-25 nucleotides that regulate gene expression by inducing mrna degradation or by interfering with translational machinery of mrnas (170) . it is predicted that more than 60% of protein-coding genes are regulated by mirnas (171) . they are key regulators of various biological processes including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and normal function [reviewed in (60) ]. extensive evidence demonstrates that mirnas play crucial roles in the development, differentiation, and function of different immune cells, such as b and t lymphocytes, dcs and macrophages (172) (173) (174) (175) . in the last few years, mirnas have drawn a lot of interest due to their involvement in the pathogenesis of cancer, inflammatory and autoimmune diseases [reviewed in (176) ]. in ms patients, expression studies using whole blood (177), pbmcs (178) , as well as brain sections (179) identified multiple deregulated mirnas. of these mirnas, three were consistently upregulated across multiple studies and directly affecting myeloid cell functions: mir-223, mir-155 and mir-146a. mir-223 is induced by the myeloid transcription factors pu.1 and ccaat/enhancer-binding protein-β (c/ebpβ) (180) . mir-223 expression is mainly confined to myeloid cells and is induced during the lineage differentiation of myeloid progenitor cells. it was shown to negatively regulate both the proliferation and activation of neutrophils (181) . moreover, mir-223 −/− macrophages exhibited enhanced pro-inflammatory m1, but decreased regulatory m2 responses (182) . it was later described that mir-223 is required for efficient m2-associated phenotype and function (183) . moreover, a low functional level of the mir-223 is essential for monocyte differentiation. in ms patients, mir-223 was found significantly increased in blood, pbmcs and active ms lesions compared with control subjects (177, 179) . during eae development, the expression level of mir-223 is dramatically increased in myeloid cell populations, but not in other cell types, and was maintained at comparable levels between disease onset and peak of disease (184) . surprisingly, although mir-223 expression is associated with m2 macrophages and microglia (183) , it was shown that mir-223 ko mice present a milder course of eae than wt mice (184) (185) (186) . reduced disease severity was also observed in adoptive transfer eae induced by transfer wt t lymphocytes into mir-223 ko recipient mice compared to transfer into wt recipient mice, demonstrating the importance of mir-223 on the apcs side rather than on the t cells side (184) . our group demonstrated that while m1-like macrophages were upregulated in ko mice, dcs showed a reduced inflammatory profile characterized by increased pd-l1 expression and decreased expression of il-1β, il-6, and il-23, all cytokines involved in differentiating and sustaining a t h 17 profile (184) . significantly, apcs from mir-223 ko mice have a comparable ability to drive t h 1 cells, but possess a reduced capacity to drive t h 17 cells (184) . moreover, it was shown that monocytic-myeloid-derived suppressor cells (mo-mdscs) isolated from mir-223 −/− suppressed t cell proliferation and cytokine production in vitro and regulated eae more efficiently than mo-mdscs derived from wt animals (186) . the enhanced suppressive function of mir-223 −/− mo-mdscs was associated with higher expression of arg1 and stat3, which are mir-223 target genes (186) . interestingly, stat3 controls the expression of pd-l1 on apcs (187), consistent with the previous observation of pd-l1 upregulation on dcs in mir-223 −/− animals. although these results point to mir-223 as a potential therapeutic target in ms, it is important to note that in a model of lysolecithin-induced demyelination, the absence of mir-223 was demonstrated to lead to impaired cns remyelination and myelin debris clearance (183) . the impaired capacity of m2 polarization by macrophages and microglia is likely a significant factor contributing to the decreased remyelination capacity in mir-223 ko mice. in particular, microglia adopting an m2 profile are critical for proper remyelination (188, 189) . thus, when targeting mir-223 in ms, it is important to keep in mind the different implications of such therapy. mir-155 has drawn a lot of attention for its possible role in ms as detailed in a recent review (190) . mir-155 has been shown to be upregulated in active ms lesions (179) as well as in cd14 + monocytes isolated from the blood of rr-ms patients compared to control donors (191) . while mir-223 expression is limited to myeloid cells, multiple immune cell populations express mir-155 such as b cells, t cells, macrophages and dcs (192) . mir-155 is found at low levels in both myeloid and lymphoid cells, but its expression is upregulated following cellular activation via antigen, toll-like receptor (tlr) ligands, and inflammatory cytokines. an important target of mir-155 is src homology 2 (sh2)-domain containing inositol-5 ′phosphatase 1 (ship-1) (193) . ship-1 is an enzyme that inhibits phosphoinositide 3-kinase (pi3k) activity, which governs cellular responses to multiple stimuli, cell proliferation and cell survival (194) . thus, it is believed that mir-155 dysregulation would have critical consequences. indeed, forced expression of mir-155 in hematopoietic stem cells by a retroviral vector leads to severe splenomegaly as well as increased myeloid cell populations in the bone marrow and in circulation (195) . in addition, it has been reported that in absence of mir-155, mice displayed altered immune responses to infectious agents, due to defective functions of b cells, t cells, and dcs (196) . focusing on myeloid populations, it was shown that dcs lacking mir-155 are less competent at inducing antigen-specific t cell activation (196) . more recently, it was demonstrated that overexpression of mir-155 in dcs is a critical event that is alone sufficient to break selftolerance in an animal model of diabetes, and promote a cd8mediated autoimmune response in vivo (197) . human cd14 + monocytes and macrophages overexpressing mir-155 exhibit increased production of pro-inflammatory cytokines, including il-1β, il-6, and tnf, and decreased production of the antiinflammatory cytokine il-10 (198) . mir-155-deficient mice display a delayed course and reduced severity of clinical symptoms of eae (199, 200) . decreased disease severity in mir-155 −/− mice was associated with reduced t h 1 and t h 17 responses. in addition to the direct effect on t cells, it was also shown that the decreased ability of mir-155 ko mice to mount inflammatory t cell responses was linked to dcs secreting less cytokines critical for driving t h 1 and t h 17 responses, mainly il-1β, il-6, il-12, il-23, and tnf (199) . mir-155 is induced in macrophages and dcs after exposure to a variety of inflammatory cytokines such as ifnβ, ifn-γ, and tnf-α (199, 201) . it is thus possible to speculate that following the first wave of inflammation, these myeloid apcs upregulate mir-155 leading to an accentuation of the inflammatory response. in addition, more recently, it has been demonstrated that mir-155 plays an essential role in driving the inflammatory phenotype of m1 macrophages (202) , which would also impact the severity of the disease. lastly, treatment with a mir-155 inhibitor after eae onset reduced the clinical disease severity (199) . considering the important role of mir-155 in driving inflammatory responses in general, and specifically in myeloid apcs, fine tuning the expression of this mirna in ms would most certainly prompt beneficial results in terms of slowing the inflammatory loop. it is noteworthy that mir-155 is the most consistent mirna found to be upregulated in ms being reported in eight independent studies (203) . a third mirna that has been shown to regulate myeloid cell activation is mir-146a. like mir-155, mir-146a is upregulated following cell stimulation and its induction is nf-κb dependent (204) . however, contrary to mir-223 and mir-155, mir-146a represses inflammatory responses by targeting two adapter proteins, tnf receptor-associated factor 6 (traf6) and il-1 receptor-associated kinase 1 (irak1), that are crucial for pro-inflammatory signaling (204) . mir-146a ko mice develop a spontaneous autoimmune disorder, characterized by splenomegaly, lymphadenopathy, and multiorgan inflammation (205, 206) . in addition, mir-146a ko mice display excessive production of myeloid cells and develop flank tumors in their secondary lymphoid organs. consistent with the repression of inflammation, mir-146a expression promotes m2-macrophage polarization by targeting notch-1 (207) . multiple studies have indicated that mir-146a plays pivotal roles in the pathogenesis of several autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and sjögren's syndrome (208) . in ms, mir-146a is upregulated in active lesions (179) , as well as in pbmcs of rrms patients (209, 210) . expression of mir-146a is reported to be significantly downregulated in glatiramer acetate treated rrms patients (210) . logically, upregulation of this mirna would seem to be beneficial in reducing the ongoing inflammation observed in ms patients leading to the possibility that the upregulation observed in ms patients is the result of the ongoing inflammation rather than a pathological expression. however, when studied in animal models of ms, there was no consensus on the suppressive effects of mir-146a. one study using the cuprizoneinduced demyelination model found that mir-146a-deficient mice displayed reduced inflammatory responses, demyelination, axonal loss, and numbers of infiltrating macrophages compared to wt controls (211) . however, a second study found that mir-146a-deficient mice developed more severe eae characterized by exaggerated t h 17 responses (212) , going along the possible beneficial effect of upregulation of this mirna in ms. more recently, it was shown that mir-146a mimic treatment of mice with rr-eae at day 14 improved neurological function, increased the number of newly generated oligodendrocytes, which may facilitate remyelination in the cns (213) . in addition, the treatment increased the number of regulatory m2 macrophages while reducing the number of pro-inflammatory m1 macrophages (213) . currently, targeting mirnas is a challenge since they control a myriad of immune and non-immune related functions. however, there is a strong interest in pursuing this approach, not only in ms, but also in many different diseases. identification of technology to target mirnas in a cell specific manner would appear to be the desired way to safely and effectively employ this targeting strategy. in the meantime, an abundance of researchers are also exploring the use of mirnas as biomarkers of diseases pathogenesis and therapy. the final strategy we will discuss is the use of nanoparticles to target myeloid cells for disease therapy which has been pioneered in our laboratory. in the recent past, many studies have focused on characterizing the ability of nanoparticles to modulate immune responses and ultimately to be used as potential therapeutics for immune-related diseases. here we will focus on the "carboxylated" poly(lactic-co-glycolic acid) (plga) nanoparticles, which are particles without any protein or peptide attached to the surface or encapsulated inside. phagocytic cells have the extraordinary ability to engulf dead cells, invading microbes and other particles, and this property of phagocyte cells led to the idea of using carriers such as apoptotic cells (214) (215) (216) , liposomes (217), extracellular vesicles (218), or nanoparticles (219) (220) (221) (222) (223) to deliver molecules to modify the immune response. we will restrict our discussion to the use carboxylated plga nanoparticles for the modulation of inflammatory monocytes for treatment of cns inflammation for multiple reasons. firstly, they can be easily manufactured under gmp conditions. secondly, they more specifically target inflammatory monocytes by their affinity of binding via the macrophage receptor of collagenous structure (marco) (224) as compared to liposomes and extracellular vesicles. thirdly, they directly carry out immune-modulatory effects on monocytes without the need for add-on agents such as would be required with liposomes. lastly, they have been proven to be safe and efficacious for use in celiac disease patients treated via intravenous infusion of gliadin encapsulating plga nanoparticles for induction of immune tolerance in a phase 1/2a clinical trial (225) . nanoparticles have diameters between 1 and 1,500 nm. smaller particles (<100 nm) are able to cross tissue barriers and traffic directly to the lymph nodes. larger particles (>100 nm) require uptake by phagocytic cells (226) . nanoparticles administered subcutaneously or intradermally may be taken up by tissue resident apcs or their precursor cells and are ultimately transported to the draining lymph nodes. systemic administration of nanoparticles favors accumulation in the organs such as the spleen and liver (227) . also, the shape of nanoparticles dictates efficiency of uptake by phagocyte cells. for example, phagocyte cells internalize spherical-shaped nanoparticles more easily than stretched-shaped structures (228) . and although positively charged particles are taken up more avidly, negatively charged particles have been shown to exhibit lower toxicity (229) (230) (231) (232) . nanoparticles can be made from different materials, metallic (e.g., silver, gold, and copper), magnetic (e.g., iron) (useful for imaging), ceramic, carbon-based, silica, lipid-based, or polymeric such as poly(amino acids), polysaccharides and poly(alpha-hydroxy acids). our group was one of the first to test the ability of 500 nm non-biodegradable carboxylated polystyrene (ps) particles to modulate immune responses in inflammatory settings in vivo. intravenous infusion of ps nanoparticles led to a reduction in trafficking of ly6c hi inflammatory monocyte into the cns and increased survival in a mouse model of west nile virus (wnv) encephalitis (224) . it was discovered that these inflammatory monocytes were redirected to the spleen of treated animals and resulted in a dramatic reduction of mortality in wnv-infected mice by preventing the release of a pro-inflammatory "cytokine storm" in the cns. robust antiinflammatory effects induced by infusion of ps nanoparticles were also observed in other inflammatory diseases such as peritoneal inflammation and inflammatory bowel disease. to enhance the clinical relevance of the nanoparticle targeting approach, we next investigated the potential of biodegradable carboxylated plga nanoparticles for regulation of myeloid cell-dependent inflammation. plga is one of the best characterized and most used biodegradable polymers. the hydrolysis of plga leads to metabolite monomers, lactic acid and glycolic acid. the two monomers are endogenous and easily metabolized by the body via the krebs cycle. there is minimal systemic toxicity associated with the use of plga (233, 234) . because plga is a safe material, it has been approved by the united states food and drug administration (fda) and european medicine agency (ema) in various drug delivery systems in humans. indeed, plga can be engineered to deliver, alone or in any combination with small-molecule drugs, proteins, peptides, dna, mirnas, and even clustered regularly interspaced short palindromic repeat (crispr) (227) . we have shown that administration of negatively charged 500 nm plga nanoparticles resulted in reduced inflammatory monocytes accumulation and overall robust beneficial effects in disease severity in multiple mouse models of inflammatory disease such as eae (224) , sci (235), tbi (236), myocardial infarction (237) , and herpes simplex virus 1 infection of the cornea (238) . the exact mechanisms behind immunomodulatory effects of plga therapy are still under investigation. however, in all these models, it has been shown plga particles are selectively recognized and bound by inflammatory monocytes. these monocytes undergo sequestration and eventual apoptosis in the spleen, culminating in reduced immune pathology at sites of inflammation. phenotypic changes were also observed on dcs and macrophages in the inflammatory sites, showing decreased expression of activation markers such as mhc ii and cd86. in the sci study, plga nanoparticle administration led to reduced m1 macrophage polarization. while our group has also shown that antigen (ag)-coupled or encapsulated plga nanoparticles can have important immunomodulatory effects (220, 222, 224, 239) , other strategies using plga nanoparticles have also been shown to regulate eae (240) . for example, cappellano et al. showed that simultaneous subcutaneous injection of plga nanoparticles loaded with either mog 35−55 or il-10 ameliorated the course of eae (241) . tgf-β, another immunoregulatory molecule, coupled to the surface of plga nanaopartlces containing plp 139−151 peptide were shown to improve the tolerogenic effect of ag-plga nanoparticles (242) . another example is maldonaldo et al. using plga nanoparticles loaded plp 139−151 together with rapamycin, an inhibitor of the mtor pathway, and demonstrating that a single dose of these particles injected at the peak of disease were able to protect from relapses (243) . also, pei et al. aimed to develop plga nanoparticles which function as a direct modulator of t cells, without the involvement of apcs (244) . for that purpose, tgf-β1 encapsulated nanoparticles were coupled with target antigens for cd4 and cd8 t cells (mog 40−54 /h-2d b -ig dimer and mog 35−55 /i-a b multimer), regulatory molecules (anti-fas and pd-l1-fc) and a "self-marker" cd47-fc (244) . these particles were injected in eae mice on day 8, 18, 28, and 38 after immunization with mog 35−55 , and induced a significant reduction in eae symptoms that lasted for more than 100 days. moreover, the authors observed a decrease of t h 1 and t h 17 mog 35−55 -specific cells as well as t c 1 and t c 17 mog 40−55specific cells, an increase of regulatory t cells, inhibition of t cell proliferation and augmentation of t cell apoptosis in the spleen (244) . in addition to regulating the immune response, plga nanoparticles have also been used as a transporter to help in the remyelination process. indeed, rittchen et al. encapsulated leukemia inhibitory factor (lif), which is a cytokine known to promote oligodendrocyte maturation thus favoring remyelination (245) . to specifically target oligodendrocytes, the lif-plga nanoparticles were coupled with anti-ng2 antibodies. the authors showed that intra-lesion delivery of lif-plga nanoparticles improved cns remyelination increasing the percentage of remyelinated axons and their thickness (245) . in conclusion, the mechanism(s) of action of plga nanoparticles are still incompletely understood, but studies in multiple models have shown their capacity to limit inflammatory events by targeting inflammatory monocytes. plga nanoparticles can also be used as delivery vectors, like liposomes and extracellular vesicles. however, a critical advantage of carboxylated plga nanoparticles, as compared to liposomes and extracellular vesicles, is their ability to act directly to modulate the function and trafficking of inflammatory monocytes based on their ability to engage the marco scavenger receptor. because of the safety record of plga nanoparticles, they can be easily translated into clinical use. in fact, cour pharmaceuticals successfully completed a phase iia clinical trial for celiac disease showing the safety and efficacy of systemic infusion of plga nanoparticles encapsulating gliadin for inducing gluten-specific immune tolerance in celiac disease patients undergoing oral gluten challenge. takeda pharmaceuticals has acquired the exclusive license for future development of this therapy for celiac and other gi diseases. cour pharmaceuticals is currently developing antigen encapsulating plga nanoparticle-based tolerance clinical programs for treatment of ms and peanut allergy and clinical programs using carboxylated "naked" plga nanoparticles targeting inflammatory monocytes for treatment of acute respiratory distress in covid-19 infection and treatment of tbi. the importance of peripheral myeloid cells in ms pathology is profound. there is an extensive presence of these cells and their products in ms lesions as well as in the csf of ms patients. studies in animal models of ms have clearly demonstrated the beneficial effects in targeting peripheral myeloid cells for the different forms of the disease. multiple tools have now been developed targeting these cells including blockade of their migration to the cns, their activation and cytokine production, their biological functions and their immunological activity (figure 1) . however, contrary to other inflammatory disorders, no drug is currently approved targeting specifically these cells in ms. multiple pro-inflammatory cytokines including gm-csf, il-1β, il-12, il-23, m-csf all represent potential ms therapeutic targets. treatments targeting these cytokines have been shown to be well-tolerated and safe in patients for different diseases. additionally, non-specific blockade of leukocyte entry to cns using natalizumab is beneficial in ms, however this carries the risk of severe side-effects from infections. however, specifically impeding the migration of myeloid cells would limit such adverse effects. clec12a, ccr1, ccr2, jam-l, and ninjurin-1 represent interesting options to inhibit cns migration of peripheral myeloid cells. altering the biological functions of myeloid cells via through mirna modulation is an appealing strategy for treating ms and other chronic inflammatory diseases. mir-146a, mir-155, and mir-223 are all upregulated on myeloid cells from ms patients. lastly, nanoparticles represent one of 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polymeric synthetic nanoparticles for the induction of antigenspecific immunological tolerance direct modulation of myelin-autoreactive cd4(+) and cd8(+) t cells in eae mice by a tolerogenic nanoparticle co-carrying myelin peptide-loaded major histocompatibility complexes, cd47 and multiple regulatory molecules myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with nanoparticles encapsulating leukaemia inhibitory factor (lif) co and also a member of the scientific advisory board and consultant for cour pharmaceutical development co as well as a shareholder.the remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 ifergan and miller. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-311845-wnk7itha authors: lubetzki, catherine; stankoff, bruno title: demyelination in multiple sclerosis date: 2014-02-05 journal: handb clin neurol doi: 10.1016/b978-0-444-52001-2.00004-2 sha: doc_id: 311845 cord_uid: wnk7itha this review, focused on demyelination in multiple sclerosis, is divided in two parts. the first part addresses the many and not exclusive mechanisms leading to demyelination in the central nervous system. although the hypothesis that a primary oligodendrocyte or myelin injury induces a secondary immune response in the central nervous system is still a matter of debate, most recent advances underline the influence of a primary immune response against myelin antigen(s), with a diversity of potential targets. whereas multiple sclerosis was long considered as a t cell-mediated disease, the role of b lymphocytes is now increasingly recognized, and the influence of antibodies on tissue damage actively investigated. the second part of the review describes the axonal consequences of demyelination. segmental demyelination results in conduction block or slowing of conduction through adaptative responses, notably related to modifications in the distribution of voltage gated sodium channels along the denuded axon. if demyelination persists, these changes, as well as the loss of trophic and metabolic support, will lead to irreversible axonal damage and loss. in this respect, favouring early myelin repair, during a window of time when axonal damage is still reversible, might pave the way for neuroprotection. demyelination as a consequence of inflammation multiple sclerosis (ms) is generally considered as an autoimmune disease, in which autoreactive t cells enter the central nervous system (cns) from the peripheral circulation and induce an inflammatory cascade resulting in demyelination and axonal loss. an extraordinary amount of literature has been accumulated since the initial experiment of rivers and schwentker in 1935 (see review by sriram and steiner, 2005) concerning the similarities and the dissimilarities between ms and its autoimmune model, experimental autoimmune encephalomyelitis (eae) . because this animal model could be induced by passive transfer of cd4þ antimyelin lymphocytes, these cells have long been considered as the primum movens of the demyelinating process in the cns of ms patients. the most common hypothesis, therefore, suggests that cd4þ lymphocytes of the th1 and th17 phenotype play a major role in demyelinating events. t-helper cells recognize their cognate myelin antigen in the context of major histocompatibility complex (mhc) class ii-bearing antigenpresenting cells (apcs), with the putative apcs being either dendritic cells at the blood-brain barrier or microglial cells (greter et al., 2005) . once entered into the brain, cd4þ th1 cells may proliferate and liberate myelinotoxic cytokines, such as interferon-g (ifn-g) and tumor necrosis factor-a (tnf-a). however, increasing evidence now suggests that, in ms, the contribution of such t-helper cells is less prominent than previously thought, and that macrophages, cd8þ t cells and b cells are major component of the inflammatory infiltrate into the lesions of both eae and ms (traugott et al., 1983; hauser et al., 1986) . moreover the deleterious impact of tnf-a and ifn-g on myelin in ms lesions has been contested (lenercept multiple sclerosis study group, 1999; lassmann, 2004) . by contrast, there is growing evidence suggesting that cytotoxic cd8 þ t cells may play a crucial role in the demyelination. oligodendrocyte and/or myelin antigens can be recognized by cd8þ t cells due to their potential for mhc class i expression under inflammatory or stress conditions (redwine et al., 2001; hoftberger et al., 2004) . also, cd8 þ t cells in the blood, cerebrospinal fluid (csf), and the lesions of ms patients have a more restricted expression of t-cell receptors than cd4 þ cells, consistent with a primary role in an antigen-restricted inflammatory response (babbe et al., 2000; jacobsen et al., 2002; skulina et al., 2004) . these cells are present in close proximity to the myelin membranes, suggesting a role in tissue damage (neumann et al., 2002) . corroborating this hypothesis, a severe model of eae was induced by adoptive transfer of anti-myelin basic protein (mbp) cd8þ t cells. this model has some interesting similarities with ms: it is characterized by perivascular inflammatory infiltrates and demyelination in the white matter, together with involvement of the gray matter and cortex; ischemic or cytotoxic injury is noted, with the presence of degenerative, apoptotic, and necrotic cells; it is improved by neutralizing antibodies to ifn-g but not to tnf-a (huseby et al., 2001) . however, this effect was not specific to mbp antigens, as myelin oligodendrocyte glycoprotein (mog) 35-55 cytotoxic t cells could also produce a severe disease in mice (sun et al., 2001) . taken together, these data suggest that cytotoxic cd8þ cells might represent a major player in ms myelin injury. it is now also well accepted that the t cells are not the only players in inducing ms lesions and demyelination, and b lymphocytes have emerged as critical actors in ms pathophysiology. this has been strongly suggested by the results of therapeutic trials showing a drastic effect on lesion formation and relapses using monoclonal antibodies against b-lymphocyte antigens, mainly cd20 (hauser et al., 2008; kappos et al., 2011) , and by the negative results obtained using ustekinumab, a monoclonal antibody specifically targeting t lymphocytes, both th1 and th17 (segal et al., 2008) . interestingly, most of the monoclonal antibodies that have been shown to be effective in ms (anti-vla4, anti-cd52, anti-cd20) all have an effect on b-cell populations. this pathogenic role of b cells in lesion formation and demyelination does not seem to be restricted to the synthesis of antibodies by plasma cells, as most of the therapeutic benefits were independent of antibodies levels, but most convincingly implies a regulatory role on t-cell function as b cells are also professional apcs (disanto et al., 2012) . to the extent that cell-mediated immunity is involved in demyelination in ms, competent apcs are an absolute requirement (prat and antel, 2005) . therefore innate immunity may play a key role in the demyelinating cascade by acting on the development and maintenance of inflammatory lesions. in the cns putative apcs are microglial or dendritic cells. whether these cells only trigger the inflammatory reaction or whether they could directly induce demyelination in ms remains an open question. in this context, a potentially primary role for innate autoimmunity in inflammatory demyelination has been proposed recently. thus, activation of apcs through aberrant activation of toll-like receptors could trigger and orchestrate an adaptive immune response to host antigens (beutler, 2004; prinz et al., 2006) . however, in contrast to this proinflammatory role, activation of microglial cells and macrophages could also favor myelin repair, through their capacity to remove debris (david and lacroix, 2003; kotter et al., 2005) . recent data have emphasized that these cells could also modulate oligodendrogenesis (butovsky et al., 2006) . a hallmark of ms is the persistence of intrathecal immunoglobulin production. however, the majority of these antibodies do not seem to be specific to neural targets, and there is no proven correlation between synthesis of oligoclonal immunoglobulin g (igg) and disease progression (walsh and tourtellotte, 1986) . nevertheless, vesicular disruption of myelin seen in highly active ms lesions was found to be associated with anti-mog and mbp antibodies, suggesting that demyelination might be causally related to the deposition of antigen-specific autoantibodies (genain et al., 1999) . such antibodies could produce demyelination by several effector mechanisms, such as antibody-dependent cell-mediated cytotoxicity, release of inflammatory mediators through stimulation of fc receptors on natural killer cells, macrophages, or mast cells, opsonization of myelin, or complement activation (archelos and hartung, 2000) . accordingly, macrophages engaged in demyelination have shown capping of surface igg located in the cleft between the clathrin-coated pit and the associated myelin debris, suggesting a specific antibody-mediated process (prineas and graham, 1981) . the deposition of antibodies on oligodendrocytes was described as a characteristic of common lesion subtypes (lucchinetti et al., 2000) , but the specificity of such deposition has been challenged, as several other pathologic conditions have shown a similar pattern (barnett et al., 2009) . experimentally, it is well known that demyelinating activity in the mog-induced eae is increased by the existence of specific anti-mog antibodies (linington et al., 1988) , especially when these antibodies recognize the native configuration (which is glycosylated) of mog (lalive et al., 2006) . such antibodies, as well as some mbp antibodies, have been detected in the serum of patients with clinically isolated syndromes and relapsing-remitting ms (berger et al., 2003; gaertner et al., 2004; lalive et al., 2006) . although some studies did not reproduce these results (lampasona et al., 2004; mantegazza et al., 2004) , high serum igg titers to native mog were detected in 40% of children with clinically isolated syndrome or acute disseminated encephalomyelitis, suggesting that in this subgroup mog might be a target of the humoral immune response (brilot et al., 2009) . however the influence of such antimyelin antibodies on demyelination, as well as their prognostic value, needs further investigation. interestingly, a pathogenic role of nmo antibody (aqp4-igg) in lesion development has been suggested in experimental models. this antibody-mediated damage occurs probably through complement-dependent astrocyte cytotoxicity and cytokine release, leading to oligodendrocyte death and demyelination (zhang et al., 2011) . a similar phenomenon has been recently described in ms. more than half of the patients displayed serum antibodies against a potassium channel expressed mainly by astrocytes, the kir 4.1 (srivastava et al., 2012) , reinforcing the view that demyelination could, at least in part, result from primary damage in other cell types such as astrocytes. closely linked to the inflammatory infiltration in the cns, several diffusible factors are possibly involved in the demyelination process. ifn-g, a th1-derived 90 c. lubetzki and b. stankoff cytokine, is expressed in ms lesions. beside its wellknown antiviral and proinflammatory action, overexpression of ifn-g in the cns could participate in demyelination. transgenic overexpression of ifn-g in the mouse by cns oligodendrocytes led to chronic demyelination that may be severe (corbin et al., 1996; horwitz et al., 1997; renno et al., 1998) . the mechanism underlying this ifn-g-mediated myelin injury may be due to induction of mhc expression in oligodendrocytes (turnley et al., 1991; horwitz et al., 1999) . however, in eae mice there is some contradictory data concerning the putative impact of ifn-g on disease evolution (sriram and steiner, 2005) . moreover, a clinical study in ms patients has reported worsening of the disease in patients receiving ifn-g (panitch et al., 1987) , although a link with increased demyelination has not been demonstrated. tnf-a is the most comprehensively studied cytokine in both eae and ms. most tnf-a overexpressing transgenic animals showed spontaneous pathology, characterized by progressive demyelination and macrophage infiltration (owens et al., 2001) . the cytopathic action of tnf-a is dependent on signaling through the p55 tnf receptor (tnf receptor i) (akassoglou et al., 1998) . nevertheless, by contrast to its potential cytopathic effect, experimental studies have shown that the second tnf receptor (p75 tnf receptor ii) might exert a protective effect on oligodendrocytes and myelin (arnett et al., 2001) and promote treg function (chen and oppenheim, 2011) . in ms, surprisingly, worsening of the disease has been reported in a clinical trial evaluating a neutralizing antibody directed against tnf-a (lenercept multiple sclerosis study group, 1999), illustrating the complex interaction of tnf-a with the pathogenesis of lesions. despite the fact that many other cytokines and chemokines have been shown to participate in the inflammatory reaction, most of them do not interact directly with demyelination (owens et al., 2001) . in mice overexpressing interleukin (il)-3 in astrocytes, however, demyelination associated with macrophage and microglial activation has been described (campbell, 1998) . several lines of evidence suggest that glutamate could also mediate injury to either the myelin or the oligodendrocyte in eae and in ms. glutamate is released in large quantities by activated immune cells, so that it could accumulate in lesions and trigger cell injury. indeed, altered glutamate homeostasis with a high-level glutaminase expression near dystrophic axons, together with decreased expression of glutamate dehydrogenase, glutamine synthetase, and glutamate transporter by oligodendrocytes in ms lesions, could contribute to deleterious accumulation of glutamate in ms brain (werner et al., 2001; pitt et al., 2003) . such excess of glutamate is thought to mediate oligodendrocyte cell death through kainate and ampa receptors expressed by the cell bodies (matute, 1998; mcdonald et al., 1998) . it has also been suggested that glutamate could mediate calcium accumulation, process retraction, and myelin injury through nmda receptors expressed by oligodendrocyte processes (karadottir et al., 2005; salter and fern, 2005; micu et al., 2006) . in addition to the long-favored hypothesis suggesting that auto-reactive t cells are generated in the systemic compartment and access to the cns where they persist and induce demyelination, it has been proposed that events within the cns could trigger the ms disease process. in their subtype characterization of ms demyelinating lesions, lucchinetti et al. (2000) already suggested that type iii and iv lesions could correspond to a primary oligodendroglial dystrophy with subsequent inflammation. especially in the type iii lesions, the preferential loss of myelin-associated glycoprotein (mag), together with oligodendrocyte nuclear condensation and fragmentation typical for apoptosis, suggests that the primary abnormality is intrinsic to oligodendrocytes. interestingly, most of these cases had a very short disease duration, reinforcing the view that, at least in some cases, this subtype could represent the initial pathogenic process of the disease. this hypothesis was illustrated by the observation of barnett and prineas (2004) , suggesting that the earliest event in lesion formation might be a caspaseindependent apoptosis of oligodendrocytes, which serves to recruit an initial innate (microglial) and a secondary adaptive (t-cell) immune response. in this study, oligodendrocyte cell death with features of apoptosis occurred prior to phagocytosis of myelin by macrophages, arguing against the long-held view that macrophages are the primary mediators of myelin destruction. the induction of a pathogenic immune reaction against white-matter antigens in response to a primary glial abnormality is a mechanism that has been reported in several types of leukodystrophy. for example, in x-linked adrenoleukodystrophy, the initial event in disease pathogenesis is induced by a primary mutation in a peroxisomal membrane protein (adrenoleukodystrophy protein: aldp) with accumulation of very long-chain fatty acids in white-matter tracts of the cns. nevertheless, the most severe phase of the disease is related to the subsequent occurrence of inflammation (berger et al., 2001) . moreover, cd8 þ cytotoxic t lymphocytes are often tightly attached to oligodendrocytes in ald tissues (moser, 2004) . similarly, in leber's hereditary optic neuropathy, which is due to mitochondrial mutations, the evolution of the disease can be influenced by an inflammatory reaction within the optic nerves or other white-matter areas (kovacs et al., 2005) . recent findings have suggested that, following a primary oligodendrocyte or myelin injury, local apcs could process myelin antigens and traffic from the cns to secondary lymphoid organs, where they may induce or enhance an adaptive demyelinating immune reaction. this hypothesis was supported by the identification of trafficking antigens in the meninges and in the cervical lymph nodes (fabriek et al., 2005; kooi et al., 2009 ). despite the fact that the antigen specificity of the t cells found in ms lesions is largely unknown, several candidate antigens, known to be capable of inducing eae, have been proposed as possible targets for the immune reaction in ms. such cns antigen-specific t cells may be normal components of the immune system, but may cause demyelination once they have undergone peripheral activation (for review, see bradl and hohlfeld, 2003) . however, to date, there is no proof that any of these autoantigens act as the antigenic target in ms. the most widely studied myelin antigens are mbp, proteolipid protein (plp), and mog. mbp is one of the most important myelin proteins in the cns, and several mbp peptides are encephalitogenic in different animal strains for eae. in humans, immunodominant peptides have also been identified, mostly in the middle section of the molecule (residues 83-102), but also in the n-and c-terminus (ota et al., 1990) . interestingly, the importance of the middle region was further supported by the finding that it is recognized in the context of a number of hla-dr molecules that are associated with ms (mainly hla drb1*1501) (martin et al., 1991; krogsgaard et al., 2000) . support for a pathogenic role of mbp-reactive t lymphocytes in ms also comes from the results of a phase ii clinical trial evaluating an altered peptide ligand of mbp. in this study, disease exacerbation in a small number of patients was associated with a strongly cross-reactive tcell response against mbp 83-99 (bielekova et al., 2000) . the primary physiologic role of mbp is thought to be maintaining (by its positive charge) the proper compaction of the myelin sheath by juxtaposing the faces of the cytoplasmic leaflets of the oligodendrocyte membrane. modifications of the mbp molecule by posttranslational events (e.g., methylation, deamidation, phosphorylation, deimination with conversion of arginines to citrullines) commonly occur and may modify the electric charge and, thus, reduce myelin stability (ridsdale et al., 1997; kim et al., 2003; harauz et al., 2004) . accordingly, the lowest cationic form of mbp (named c8, with extensive deimination of arginyl residues) was found in elevated levels in patients with ms, and the proportion of arginine loss caused by deimination is higher in acute severe ms compared to more chronic ms (moscarello et al., 1994 (moscarello et al., , 2002 wood et al., 1996) . supporting these observations are recent suggestions that a reduction in the net positive charge of mbp not only interferes with compact myelin assembly but also makes the immunodominant epitope of this protein more surface-exposed, hence more accessible to protease digestion or immune degradation (musse et al., 2006) . whereas mbp is by far the most investigated myelin antigen in both ms and eae, other candidate antigens have gained increasing attention. plp-specific clones have been isolated at various stages of ms (correale et al., 1995) and elevated frequencies of plp-specific t cells have been found in blood and csf (sun et al., 1991) . similarly, in addition to the high frequency and possible pathogenic role of anti-mog antibodies on myelin (see above), a higher proportion of t cells derived from ms sera was found to react with mog compared to controls (kerlero de rosbo et al., 1993) . moreover, several other potentially encephalitogenic myelin antigens, such as mag, have also been suggested (zhang et al., 1993) , as have ab-crystallin (van noort et al., 1995) , and transadolase-h (banki et al., 1994) . devic's disease is an example of our better understanding of disease pathogenesis, as aquaporin 4 has been identified as the target of the (mostly humoral) immune response (wingerchuk et al., 2007) . more recently, neuronal adhesion molecules expressed at the nodal and paranodal junction have been suggested as potential targets of the immune response. these targets were identified using ms sera and a proteomic screen on a glycoprotein fraction isolated from human myelin by affinity chromatography (mathey et al., 2007 ; see review by desmazières et al., 2012) . these data, which need to be confirmed on a larger population of ms, are in line with studies in ms tissue showing abnormally large aggregates of the glial isoform of neurofascin at axoglial junctions located at the periphery of demyelinating lesions (howell et al., 2006) and suggest that the axoglial junction might be an area of particular vulnerability of tissue damage. along the same line of results was the recent identification in ms patients of frequent kir 4.1 antibodies targeting mainly astrocytes (srivastava et al., 2012) , introducing astrocytes as potent actors in the demyelination process. the difficulties in proving that a target antigen is responsible for the immune reaction in ms are not surprising considering the heterogeneity of the disease and the dynamic nature of the autoimmune response (hohlfeld and wekerle, 2004) . for instance in demyelinating models, the immune response can spread to 92 c. lubetzki and b. stankoff different antigens (lehmann et al., 1993) . epitope spreading is characterized by a widening of the immune response from a single antigenic epitope to different epitopes, either on the same molecule (e.g., the intramolecular epitope spreading observed for plp) or on other molecules. interestingly, it has been shown in different demyelinating animal models that such epitope spreading could take place directly in the cns. thus, in eae, naive t cells can directly gain access to the inflamed cns and, once inside, dendritic cells may activate these t cells to initiate spreading (mcmahon et al., 2005) . however, regardless of how attractive the theory might be, the actual occurrence of epitope spreading in patients with ms has not been well documented. the possible involvement of viruses in the etiology of ms is still controversial despite much work in this area. a viral infection can influence demyelination by two main mechanisms. first, a viral infection may directly injure oligodendrocyte pathology, as seen in several animal models such as theiler's murine encephalomyelitis, jhm coronavirus (lampert et al., 1973; powell and lampert, 1975; lipton and canto, 1976; schlitt et al., 2003) , and in progressive multifocal leukoencephalopathy. despite the impressive number of viruses that have been suspected and investigated to date, however, none has ever been implicated as being causally related to the demyelination in ms. second, a viral infection may trigger an autoimmune reaction against myelin self antigens and, thus, cause subsequent demyelination, as described in postinfectious encephalomyelitis. indeed, several such mechanisms are possible, including molecular mimickry, bystander activation, and epitope spreading (grigoriadis and hadjigeorgiou, 2006) . the most popular of these is molecular mimickry, which originally referred to the presence of a sequence identity between microbe-derived peptides and certain self antigens of the host. for example, the self antigen plp 139-151 is identical to the peptide (hi 574-586 ) derived from haemophilus influenzae (croxford et al., 2005) . subsequently, this concept has been extended to include a structural resemblance between peptide-mhc complexes rather than strict identity. one example of this is the human mbpspecific t-cell receptor that can recognize either a peptide derived from mbp bound to hla-dr2b or a peptide derived the epstein-barr virus (ebv) bound to hla-dr2a (lang et al., 2002; hohlfeld and wekerle, 2004; oldstone, 2005) . this could illustrate one possible interaction between ebv and ms (another hypothesis mainly involves the influence of ebv on b-cell function or bystander effects), as ebv infection is quite universal among ms adult patients (owens and bennett, 2012) . bystander activation relates to the non-specific activation of autoreactive t cells due to a direct pathogenic effect of the virus on the target organ. in this model, viral-induced cns damage leads to the release of sequestered myelin antigens, activation of local inflammation, and finally, recruitment of autoreactive lymphocytes that activate and initiate an autoimmune injury to myelin (horwitz et al., 1998 (horwitz et al., , 1999 . finally, epitope spreading has been well described for virus-induced demyelination following infection by theiler's murine encephalomyelitis, with a spreading of the immune response to different epitopes within the myelin peptide plp (miller et al., 1997; mcmahon et al., 2005) . demyelination has important consequences for the axon, both from disturbed impulse conduction and from modification of axolemma and membrane components. in conjunction with electrophysiologic recording, experimental focal demyelination by different chemical agents such as diphtheria toxin, ethidium bromide, and lysolecithin has been used to analyze axonal function after demyelination. an area of demyelination has been shown to produce conduction block at the site of the lesion (mcdonald, 1963) , with preserved conduction distally. segmental demyelination triggers a series of adaptive responses by the axon, including changes in distribution of ion channels along the axolemma. these changes, which take 2-3 weeks to develop, may facilitate the restoration of conduction across the demyelinated segment (smith et al., 1981) . however, in this circumstance, conduction along demyelinated axons is no longer saltatory and fast but, rather, is continuous and slow . in other circumstances, conduction block may persist and such an outcome is favored by factors such as a large axon diameter (bostock and sears, 1978) , a long length of demyelination, the absence of any glial ensheathment (shrager and rubinstein, 1990) , and the presence of deleterious factors such as nitric oxide (redford et al., 1997) . in addition, demyelinated axons may also become more excitable and generate trains of ectopic impulses at the site of demyelination (smith and mcdonald, 1982) . this neurophysiology is probably associated with the positive paroxysmal symptoms that are so characteristic of ms, such as tonic spasms, paroxysmal dysarthria and ataxia, paresthesia and pain. demyelinated axons may also become active in response to mechanic deformations (smith and hall, 1980; smith and mcdonald, 1982) , and this results in the occurrence of paroxysmal symptoms such as lhermitte's sign. in addition, electrical activity in one axon can excite activity in an adjacent axon. such crossexcitation, termed ephaptic transmission, might result in transitory symptoms. however, although demonstrated between amyelinated and normal axons in a dystrophic mutant mouse, it has yet to be proven in cns demyelinated axons (smith, 1994) . changes in axonal structure and molecular organization in the early and active lesions, the axonal caliber changes at the site of the demyelinated internode. the axons in the demyelinated portions are generally thicker. this increased caliber has been linked to the fact that neurofilaments in these enlarged demyelinated axons become loosely packed, perhaps reflecting the increased permeability of demyelinated axolemma. in addition, axonal cytoskeletal proteins are modified. notably the degree of phosphorylation of neurofilaments subunits decreases. in chronic ms lesions, re-expression of the poly-sialated (psa) form of the neural cell adhesion molecule (ncam) has been reported (charles et al., 2002) . this adhesion molecule, which is widely expressed on neurons and glial cells during development, is downregulated in adult cns where its expression is restricted to areas of permanent plasticity. in ms, psa-ncam is re-expressed on some demyelinated axons within the plaques. by contrast, it is undetectable on either myelinated axons in the periplaque region or in the normalappearing white matter. most (possibly all) axons that re-express psa-ncam contain dephosphorylated neurofilaments, most likely indicating that they are chronically demyelinated axons. these findings strongly suggest that psa-ncam re-expression is a local phenomenon, thought possibly to be triggered by modification of local intracellular calcium pool, through voltage-dependent calcium channels expressed by the denuded axon (kornek et al., 2001) . during development psa-ncam has been shown, both in vitro and in vivo, to regulate myelination negatively, and removing it from the axonal surface is necessary to initiate the process of myelination (charles et al., 2000) . perhaps, therefore, by disrupting oligodendrocyte-axon interactions, the re-expression of this inhibitory adhesion molecule in chronically demyelinated axons contributes to the failure of myelin repair in ms (charles et al., 2002) . the molecular organization of the nodes of ranvier in myelinated fibers permits the rapid saltatory conduction of nerve impulses. the nodes of ranvier are separated from the internode by two distinct domains of the axolemma, the paranodal axoglial junction and the juxtaparanodal region, which are characterized by the presence of specific protein complexes. voltage-gated sodium channels (na v ), ankyrin g, nrcam, and 186-kda neurofascin are highly enriched at the node (bennett and lambert, 1999; jenkins and bennett, 2001) . at the paranodes, myelin loops are anchored to axons through septate-like junctions characterized by the enrichment of paranodin/caspr and glycosylphosphatidylinositol-anchored cell adhesion molecule contactin (einheber et al., 1997; menegoz et al., 1997; rios et al., 2000) . the juxtaparanodal region, just beyond the innermost paranodal junction, is enriched in shaker-type potassium (k v ) channels, in association with caspr2, a second member of the caspr family and the cell adhesion protein tag-1 (poliak et al., 1999; traka et al., 2002 traka et al., , 2003 . demyelination produces major modifications of the distribution of these nodal and perinodal constituents. the altered distribution of sodium channels is associated with modification in the subtype of these channels. paranodal and juxtaparanodal axonal proteins are also profoundly modified. a redistribution of na v channels along the naked axon has been reported in experimental models of demyelination and in ms lesions both by autoradiography and by immunohistochemistry (moll et al., 1991; noebels et al., 1991; craner et al., 2004a, b; coman et al., 2006) . it remains unknown whether this change is due to a na v channel synthesis or to a redistribution of na v channels from nearby nodes into the demyelinated (previously internodal) membrane. in addition to this diffuse redistribution, few broad na v channel aggregates can be detected on demyelinated axons in all ms lesions, which are threefold larger than na v channel aggregates in the periplaque areas or in the normal tissue (coman et al., 2006) . these "loose" aggregates are often associated with a diffuse na v channel distribution further down the same axon. they may represent remaining nodes, anchored by ankyrin g. alternatively, they may correspond to the phi nodes, which are seen prior to functional 94 c. lubetzki and b. stankoff remyelination , and which, therefore, might be involved in the process of myelin repair. in addition to the diffuse distribution of na v channels along denuded axons, recent studies using either subtype-specific antibodies or molecular probes have begun to identify the na v channel isoforms expressed along demyelinated axons, in ms and in experimental models of demyelination. these studies suggest that specific channel isoforms are associated with distinct physiologic functions such as the restoration of conduction or the degeneration of axons. nine genes encode distinct voltage-gated na channels (na v 1.1-na v 1.9), which differ in their amino acid sequences, their voltage dependences, and their kinetics. within the normal adult cns, na v 1.6 is the predominant sodium channel, and is clustered at the node of ranvier. during development, na v 1.2 channels are expressed diffusely along the axon prior to myelination, and subsequently at immature nodes. this immature pattern is followed by a switch from na v 1.2 to na v 1.6 at mature nodes of ranvier, by the time that myelination is complete. within ms active plaques, as in eae, it has been reported that both the na v 1.6 and the na v 1.2 channel isoforms are expressed along demyelinated axons (craner et al., 2004a, b; waxman et al., 2004) . expression of the na v 1.6 isoform is mostly associated with b-amyloid precursor protein (bapp), reflecting the dysfunction of axonal transport in damaged axons in the plaques. by contrast, the na v 1.2 isoform is associated with bappnegative (i.e., non-damaged) axons. the widespread distribution of na v 1.2 channels in uninjured axons is similar to the diffuse distribution of na v 1.2 channels along premyelinated axons, or on non-myelinated axons in adult cns. interestingly, these two isoforms differ in the currents they produce. the na v 1.2 channel isoform produces a less persistent na þ current than does the na v 1.6 isoform. perhaps the redistribution of na v 1.2 along naked axons might be an adaptive response supporting the conduction of action potentials in demyelinated axons. by contrast, the redistribution of na v 1.6 might contribute to axonal injury by inducing persistent sodium currents and triggering, thereby, a reversal of the na þ /ca 2þ exchange. if so, this mechanism might permit an influx of calcium that leads to axonal damage (waxman et al., 2004) . these potential mechanisms open important new therapeutic opportunities for neuroprotection using specific na v channel blockers. in addition to altered expression of na v 1.2 and na v 1.6 along demyelinated axon in ms, studies in eae have demonstrated increased expression of the na v 1.8 channel isoform within purkinje cells of the cerebellum. similar results have also been reported in ms patients who have experienced progressive cerebellar deficits (black et al., 2000) , suggesting that this chanelopathy might contribute to cerebellar dysfunction in ms (shields et al., 2012) . the functional importance of this upregulated na v 1.8 expression, however, has not yet been determined. in contrast to the heterogeneous distribution of na v channels, axonal proteins expressed normally at the paranode (caspr/paranodin) and at the juxtaparanode (k v channels and capsr2) are diffusely distributed along the demyelinated internode (wolswijk and balesar, 2003; coman et al., 2006) or enlarged (howell et al., 2006) . the data obtained in ms tissue are in agreement with experimental observations in dysmyelinating mutant animals (dupree et al., 1999; mathis et al., 2001; arroyo et al., 2002) . this suggests that axoglial junctions and glial contact are necessary for the maintenance of paranodin/caspr aggregation. studies of the juxtaparanodal k v channels and caspr 2 in dysmyelinating mutants have reported either mislocalization (expression at previously paranodal regions) or diffuse redistribution (dupree et al., 1999; boyle et al., 2001; mathis et al., 2001; poliak et al., 2001) . it has been suggested that the mislocalization of the k v channel may be an early event during the course of demyelination, which is followed by a diffuse redistribution in the setting of chronic demyelination. in conclusion, cns demyelination, which can be 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myelin-associated glycoprotein and myelin basic protein in patients with multiple sclerosis ex vivo spinal cord slice model of neuromyelitis optica reveals novel immunopathogenic mechanisms key: cord-320909-p93gxjm2 authors: natoli, s.; oliveira, v.; calabresi, p.; maia, l. f.; pisani, a. title: does sars‐cov‐2 invade the brain? translational lessons from animal models date: 2020-05-22 journal: eur j neurol doi: 10.1111/ene.14277 sha: doc_id: 320909 cord_uid: p93gxjm2 the current coronavirus disease (covid‐19) outbreak, caused by the novel severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2), has raised the possibility of potential neurotropic properties of this virus. indeed, neurological sequelae of sars‐cov‐2 infection have already been reported and highlight the relevance of considering the neurological impact of coronavirus (cov) from a translational perspective. animal models of sars and middle east respiratory syndrome, caused by structurally similar covs during the 2002 and 2012 epidemics, have provided valuable data on nervous system involvement by covs and the potential for central nervous system spread of sars‐cov‐2. one key finding that may unify these pathogens is that all require angiotensin‐converting enzyme 2 as a cell entry receptor. the cov spike glycoprotein, by which sars‐cov‐2 binds to cell membranes, binds angiotensin‐converting enzyme 2 with a higher affinity compared with sars‐cov. the expression of this receptor in neurons and endothelial cells hints that sars‐cov‐2 may have higher neuroinvasive potential compared with previous covs. however, it remains to be determined how such invasiveness might contribute to respiratory failure or cause direct neurological damage. both direct and indirect mechanisms may be of relevance. clinical heterogeneity potentially driven by differential host immune‐mediated responses will require extensive investigation. development of disease models to anticipate emerging neurological complications and to explore mechanisms of direct or immune‐mediated pathogenicity in the short and medium term is therefore of great importance. in this brief review, we describe the current knowledge from models of previous cov infections and discuss their potential relevance to covid‐19. highly pathogenic coronavirus (cov) infections are well-established sources of previous epidemics in humans, i.e. severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov). the novel cov named sars-cov-2, which shares a highly homological sequence with sars-cov, is responsible for the current covid-19 outbreak with more than 2 million patients diagnosed and over 146 000 deaths, which exceeds by far the total of sars and mers in 2002 and 2012, respectively [1] [2] [3] . despite the short duration of the current pandemic outbreak, several neurological and neuroradiological phenotypes have been reported [4, 5] , requiring urgent investigation into the mechanisms and etiology underlying the interplay between sars-cov-2 and the central nervous system (cns). a translational neuroscience approach is mandatory to explore the possible cns involvement in cov infections, accelerate scientific knowledge transfer to the clinical frontline and test new disease-oriented treatments. indeed, both clinical features of the previous cov epidemics (sars and mers) and lessons from animal models used in the study of sars and mers constitute valuable tools to understand the viral pathogenesis in the host and to characterize mechanisms of viral access and dissemination in the cns. meanwhile, several laboratories are rushing to study sars-cov-2 in a number of different animals, including primates, mice, rats, hamsters and ferrets [6] . here, we will provide a neurological perspective by analysing the main features of these models and point out relevant similarities and specificities in comparison to sars-cov-2. a comprehensive systematic search of medline, scopus, web of science and https://www.who.int/emerge ncies/diseases/novel-coronavirus-2019/situationreports/ was performed. in 2002, the outbreak of sars in guangdong province, china led to the discovery of sars-cov, a highly pathogenic cov, as the causative pathogen of the epidemic [7] . although the virus is primarily a respiratory pathogen, there are reports of neurological manifestations, such as epileptic seizures and encephalitis, that may suggest a cns involvement of the infection [8, 9] (table 1) . complementing these reports, post-mortem neuropathological studies have detected the sars-cov n protein and rna polymerase gene fragment in neurons of infected patients and pathological changes such as brain tissue edema and vasculitis of cerebral veins [10] . compelling evidence demonstrates that sars-cov attaches to the cell membrane by binding to human angiotensin-converting enzyme 2 (hace2), now also known to be the sars-cov-2 functional receptor [11] . human tissue studies have shown an abundant presence of these receptors not only in the epithelia of the lung and small intestine, but also in arterial and venous endothelial cells and arterial smooth muscle cells in all organs studied, including the brain [12] . based on a transgenic mouse expressing hace2, it was possible to show that angiotensin-converting enzyme 2 (ace2) is also expressed at neuronal level, namely in the cytoplasm of cell bodies [13] . distinctive properties in the structure of mouse ace2 (as compared with hace2 proteins) significantly reduce the virus tropism for mouse tissues. hence, in order to overcome this species-related difference, a transgenic model has been generated in which a vector carrying a hace2-coding sequence was introduced in wild-type mice under control of the human cytokeratin 18 (k18) promoter [14] . notably, when k18-hace2 transgenic mice were infected with sars-cov, the infection would start in the respiratory epithelium and rapidly spread to the alveoli. more importantly, neuroinvasive routes were later explored using the same model, by monitoring the kinetic profile of viral antigen [15] . strikingly, the authors showed that the viral spread started in the olfactory bulb and progressively invaded subcortical and cortical regions. such a trans-neuronal hypothesis could not apply for other infected regions, such as those brainstem nuclei that are not directly connected to the olfactory bulb. the authors raise the possibility that, once the virus is established in the brain, it might spread along specific neurotransmitter pathways or via non-neuronal routes (blood or virchow-robin spaces) [15] . overall, their results showed that, in this model, sars-cov primarily entered the brain via the olfactory nerve. alternatively, other authors theorize that cov can primarily use a hematogenous route to penetrate the cns using dendritic or white blood cells as reservoirs [16] . this presumption is based on pathological studies that have shown that monocytes and macrophages can be infected by sars-cov [17] and on cell-line studies that revealed that dendritic cells (regulators of immune responses) can be infected and impaired by this virus [18] (table 1) . multiple animal models have been explored in the context of sars, including non-human primates, hamsters, ferrets and mice ( table 2) . a comprehensive descriptive review of all suitable models is beyond the scope of this review and we refer the reader to a number of excellent reviews [19] [20] [21] . it can be inferred that there is no single ideal animal model for sars, although the evidence collected so far has significantly contributed to advancing the field. in particular, it is well established that models range from those in which only virus replication is observed (young balb/c, b6 mice) to those in which replication is accompanied by minimal signs and histopathology (such as non-human primates, ferrets and hamsters) [19] . curiously, old immunodeficient balb/c mice exhibit a clinical syndrome, supporting age as a risk factor for more severe clinical phenotypes [19] . transgenic k18-hace2 mice infected with sars-cov develop a severe pulmonary phenotype, starting in the respiratory epithelium with rapid alveolar dysfunction [14] . in this model there is a massive infiltration of macrophages and lymphocytes in the lungs, promoting a release of pro-inflammatory cytokines not only at pulmonary level, but also in the brain. in a relatively short time frame (within 5 days), k18-hace2 mice develop a severe phenotype, that includes a lethargic-like state, suggesting cns involvement. in follow-up studies in the same mouse strain, k18-hace2 [15] , the authors demonstrated an extensive involvement of the transgenic mouse brain. sars-cov produced a widespread infection involving vital brainstem nuclei, such as dorsal motor nucleus of the vagus, nucleus tractus solitarii and area postrema. this model also raised questions as to the cause of neuronal destruction and death in these animals [15] . as there was no pathological evidence of inflammation, the authors considered the possibility of apoptosis as the cause of neuronal death, although this was not confirmed [terminal deoxynucleotidyl transferase (tdt) dutp nick-end labeling (tunel)-positive cells were not detected]. it was proposed that a dysregulated cytokine response could be the cause of death in these animals. at day 4 postinfection, infected k18-hace2 mice had an upregulation of the proinflammatory cytokines interleukin (il)-1, tumor necrosis factor alpha and il-6. the authors also propose a possible direct involvement of the dorsal vagal complex, a vital region of the brain that plays an important role in orchestrating cardiorespiratory function. in fact, animals intracranially inoculated with low-dose virus exhibited limited viral spreading but succumbed rapidly [15] . overall, these data show the relevance of the transgenic approach in converting the mouse response to infection from mild to severe leading to cns human neurons are infectible [53] and ace2 neuronal expression has been identified in human cns [54] capable of infecting human neuronal cells in in-vitro cell lines [55] . ddp4 has a low expression in the brain [56] -neuropathology sars genome sequences detected in the brain in autopsies; also, edema and scattered red degeneration of neurons [17] samples not available for investigation [22, 23] . currently, mers-cov is still a relevant threat for populations in the middle east, with a high lethality (close to 35%) [2] . patients exhibit predominantly pulmonary clinical involvement in contrast to fewer patients presenting neurological manifestations such as coma, ataxia, focal motor deficits and peripheral nerve symptoms [24, 25] . unfortunately, there are no published data regarding human neuropathological findings (table 1) . the mers-cov ex-vivo models supported the clinical tropism for the pulmonary tract by showing that the virus can replicate in human lung cultures (in bronchial, bronchiolar and alveolar epithelial cells) [26] . this cell line susceptibility study also revealed that, although presenting a lower viral expression and no cytopathic effects, mers can infect human neuronal lines. dipeptidyl peptidase-4 (dpp4), also known as cd26, was identified as a functional receptor for mers-cov. dpp4 is generally expressed in human bronchiolar epithelial cells and bronchial lung tissue [27] . it can also be found in the intravascular portion of vascular endothelial cells and in the cerebrospinal fluid [28] . after identification of dpp4 as a functional receptor, which is expressed in the airway epithelia of rodents, it was expected that rodents would have been vulnerable to infection. this turned out to be wrong, as the human binding domain differs from that of rodents [29] . this limitation was overcome by developing mice expressing human dpp4 that exhibited high susceptibility to infection and displayed the features of human disease [30] , including a lethargic state, and showing high mortality and extrapulmonary involvement ( table 2 ). the authors detected a severe lung infection, but brain invasion was not seen until day 4 of infection, suggesting substantially different kinetics of mers-cov infection in the lung and brain [30] . a different animal model using human dpp4 transgenic mice studied the differences in viral replication in animals infected by a clinical aerosol transmission simulator compared with intranasal instillation-inoculated mice [31] . they found that the disease onset, lung lesion and viral replication progression were slower in the mers-cov aerosol-infected mice than in the mers-cov instillation-inoculated mice. furthermore, after aerosol infection, they detected high viral loads after 3-9 days in the lungs versus 7-9 days in the brain. again, although both lungs and brain are infected, the timing is different, with a later infection of the brain [31] . such different kinetics could suggest a hematogenous route of infection. indeed, neuroinvasive routes were not explored in either of these models. in addition, similarly to sars-cov, mers-cov has been shown to replicate in human dendritic cells and macrophages, which would support the hematogenous hypothesis [32] . a number of models have been developed and discussed in detailed review articles [33, 34] (table 2 ). in a non-human primate model of mers, de wit and colleagues inoculated rhesus macaques with mers-cov, which primarily affected the epithelium of the lower respiratory tract, giving rise to a mild-to-moderate interstitial pneumonia [35] . this model was able to replicate virus shedding and replication in tissues, as well as gene expression and cytokine and chemokine profiles. however, despite the mild clinical syndrome, no neurological signs and symptoms were reported. thus, the self-limiting nature of mers-cov infection, as transient patterns at various levels of the model, suggests that this model does not fully resemble the lethal infection observed in humans [35] . it is of note that, when macaques were immunocompromised by immunosuppressive agents, the mers-cov replicated to significantly higher titers and disseminated in other organs (cns not examined). surprisingly, histopathological alterations were reduced in the immunosuppressed animals [36] . together, these data suggest a prominent role of the host response in the manifestation of the disease. the macaque model allowed the testing of a number of potential drugs as novel therapeutics. remdesivir, an antiviral agent used also for covid-19, was able to prevent/treat the histological and radiological signatures of the disease [37] . in studies using transgenic mice expressing human dpp4, it was possible to induce features of human disease in the animals [30] . from the studied cells, pneumocytes, brain microglia, astrocytes and neuronal cells all presented high titers of virus. with regard to pathology, whereas infected mice presented an extensive pulmonary inflammatory infiltrate, the only findings in the brain were a mild perivascular cuffing [30] . however, in a different study using human dpp4 transgenic mice, a few days after the appearance of pulmonary lesions, pathological changes were documented in the brain, with dilatation and congestion of the cerebral vessels and few areas of cellular necrosis in the cerebral cortex, hippocampus and thalamus [31] . as in sars-cov-2, mers-cov infection was also shown to induce a profound acute inflammatory response within the lungs and brain of hcd26 tg mice, with upregulation of multiple genes related to the inflammatory response [30] . clinical and neuropathological features in human patients covid-19 is the most recent and dramatic pandemic, caused by sars-cov-2. registered lethality varies between european countries ranging from 1.5% in germany to over 10% in italy [1] . as in sars and mers, pulmonary clinical involvement is most prominent. however, more recently, neurological phenotypes involving central and peripheral nervous system have emerged and are being increasingly recognized, i.e. anosmia, ageusia, necro-hemorrhagic encephalitis and guillain-barr e syndrome [4] [5] 38] . so far there are no published human neuropathological findings (table 1) . the sars-cov-2 ultrastructure was recently characterized by high-resolution cryo-electron microscopy [39] . remarkably cov spike glycoprotein, by which the virus binds the cell membrane, binds ace2 with a higher affinity compared with sars-cov. in addition, most of the available antibodies to sars-cov targeting acebinding domain were unable to bind the sars-cov-2 spike protein, indicating that binding sites differ between sars-cov and sars-cov-2. such a finding indicates the urgent need for generating specific antibodies for sars-cov-2 binding domain, but might also explain the distinct pathogenic properties of sars-cov-2 [40] . in addition to ace2 receptor, sars-cov-2 uses the serine protease type ii transmembrane serine protease (tmprss2) for spike protein priming [41, 42] . very recently, in a preliminary report, brann et al. took advantage of bulk mouse whole olfactory mucosa (wom) rna sequence data derived from macaque, marmoset and human and found in both mouse and human datasets that olfactory sensory neurons do not express two key genes involved in cov-2 entry, i.e. ace2 and tmprss2 [43] . in contrast, olfactory epithelial support cells and stem cells express both of these genes, as do cells in the nasal respiratory epithelium. taken together, these findings suggest possible mechanisms through which cov-2 infection could lead to anosmia or other forms of olfactory dysfunction. moreover, these findings may question the olfactory bulb as an entry route for covs into the cns [43] . to our knowledge, no study has evaluated, so far, any type of pathway targeted at the cns or peripheral structures. clinical and pathological lessons from animal models several laboratories worldwide are accelerating attempts to develop a suitable animal model for covid-19. experimental infection with sars-cov-2 in these models provides basic information to address a number of fundamental questions regarding its pathogenicity, the interaction with the different hosts and, hopefully, establishing the criteria for prevention and care. in line with observations in sars models, non-human primates and wild-type mice infected with sars-cov-2 exhibit a relatively mild clinical disease, in spite of the evidence that quantitative reverse transcription polymerase chain reaction (rt-qpcr) revealed a massive infection of the respiratory tract [44, 45] (table 2) . rhesus macaques infected with bronchoalveolar lavage fluid obtained from an affected patient developed a histopathologically confirmed interstitial pneumonia, associated with a widespread presence of sars-cov-2 in the respiratory tract. clinical signs were mild and no viral rna was detectable by means of rt-qpcr in the blood of the primates during the whole course of infection (14 days) [44] . these findings demonstrate the causal relationship between sars-cov-2 and interstitial pneumonia, reminiscent of covid-19. moreover, and consistent with observations in sars models, bao et al. [45] used the hace2 transgenic mice and infected them with sars-cov-2 inducing interstitial pneumonia, with typical histopathological elements and, accordingly, viral antigens were found in airway epithelia. these lines of experimental evidence are relevant as they demonstrate the causal relationship between sars-cov-2 and pulmonary involvement, but, unlike in sars models, nervous system involvement was not documented in these experiments. however, it is unclear if brain tissue was systematically assessed and the most susceptible brain regions explored for direct or indirect viral presence. overall, the pathogenicity of sars-cov-2 is lower as compared with sars-cov in mice. indeed, as discussed above, hace2 transgenic mice infected with sars-cov exhibited widespread organ damage, whereas sars-cov-2, at least in this model, was confined to lungs, indicating a differential pathogenicity [45] . these studies reveal important commonalities between sars-cov-2 and sars-cov infection and identify a potential target for antiviral intervention. in fact, very recently, a tmprss2 inhibitor approved for clinical use (camostat mesylate) was tested and blocked sars-cov-2 entry into lung cells [42] . finally, the same authors were able to show that the sera from convalescent patients with sars cross-neutralized sars-2-spike-driven entry [42] . if the same effect occurred in pre-clinical models, then we would be closer to both a preventive and a diseaseoriented treatment. • major routes of cns infection: (i) spread via olfactory bulb and/or (ii) synapse-connected route to the medullary cardiorespiratory center from the mechanoreceptors and chemoreceptors in the lung and lower respiratory airways; and/or (iii) hematogenic via brain endothelial ace2 receptors. • major pathogenic pathways for cns involvement: (i) direct viral pathogenicity; and/or (ii) immunemediated pathogenicity targeting brain tissue; and/ or (iii) inflammatory involvement of brain blood vessels; and/or (iv) intravascular coagulation secondary to the systemic inflammatory response as a mayor cause of thrombosis, hemorrhage and stroke. • host individual susceptibility factors that underlie the variable severity of the disease in human patients. however, a relevant issue is also represented by gender. the clinical observation of a specific involvement of males might suggest a specific protective estrogenic effect. unravelling these points could clarify if cns contributes to respiratory failure in patients with covid-19 [15, 46] and may provide a rationale to preventive and therapeutic strategies for major neurological events such as stroke, encephalitis or other reported complications. established ex-vivo and animal models of cov infection may help to dissect pathogenicity, infective routes and nervous system targets of covs. if we manage to mimic the pathological hallmarks of covid-19 we may have the tools to test treatment efficacy and evaluate the efficacy of vaccines and therapeutics. among limitations, the following emerge as of primary importance. • neurological subtle clinical phenotypes are not easily reproducible in animal models. • neurological severe phenotypes are dependent on using specific transgenic approaches to enhance virulence, which limit direct translation to humans. • severity of the clinical features does not always parallel either the viral replication level or the histopathological findings, hinting at indirect disease mechanisms (such as inflammation and prothrombotic states) that have not been attained in the present models. • innate animal characteristics seem to influence viral infection kinetics leading to faster virus clearance. the ongoing outbreak of sars-cov-2 confirms that human covs are primarily respiratory pathogens and koch postulates have already been fulfilled in this regard [45] . previous reports suggest that sars-cov and mers-cov can occasionally cause clinically relevant cns infections. in fact, animal models suggest invasiveness of these viruses through the cns, either via the olfactory bulb or through blood dissemination of infected and activated monocytes passing through a permeable blood-brain barrier as a consequence of the systemic inflammatory response. with regard to the pathogenesis of immunemediated cns pathology, data derive broadly from mice infected with murine hepatitis virus strains, a beta-cov genetically related to human cov-oc43 [47] . briefly, three mechanisms of immune-mediated cns lesions can be recognized. (i) an excessive host response to the infection can occur resulting in a systemic inflammatory response syndrome that causes a multiple organ dysfunction (including cns). the main pathogenic mechanism in this case includes tissue 'dysoxia' due to intravascular coagulation and dysfunction of the microcirculation homeostasis. (ii) direct viral infection of immune cells, including macrophages, microglia and astrocytes in the cns, may activate glial cells that locally produce pro-inflammatory cytokines, including il-6, tumor necrosis factor alpha, il-1b and il-12 [48] . moreover, activated immune cells may contribute to tissue damage by producing toxic agents, recruiting and activating further immune cells and inducing apoptosis. immune-mediated events, either through t-cells or by means of other cytokine and chemokine pathways, may also eventually lead to demyelination. (iii) an autoimmune reaction is generated by an adaptive immune response directed against host epitopes or proteins either misrecognized by pathogen-directed antibodies or expressed by damaged tissues (and previously cryptic to the adaptive immune system) [49, 50] . in order to speed up clinically useful discoveries, it would be desirable to follow some indications such as: (i) to build a systematic, consecutive, prospective registry including epidemiological data in patients with covid-19 with attention to neurological manifestation to fully understand if sars-cov-2 infections can cause cns involvement and to what extent; (ii) to measure sars-cov-2 rna in the cerebrospinal fluid of symptomatic vs. asymptomatic patients; and (iii) to perform autoptic investigations of patients with covid-19 in order to find and characterize virus distribution across tissues (cerebral blood vessels, endothelia, glia and neurons) and neuropathological consequences such as antibody-based neuroinflammatory responses in gray and white matter, vasculitis, neuroglial death or apoptosis and ischaemic or hemorrhagic events. taking into account the fact that other covs are prone to infecting neurons in animal models as well as in humans [16, 50] we must keep an open mind regarding medium-to long-term sequelae and consequences of the acute infection. therefore, despite immune-mediated control of acute infection being attained, host-mediated immune regulatory mechanisms may fail to clear the virus potentially leading to 'chronic infections' and hence impact chronic neurological diseases, such as parkinson's disease and multiple sclerosis [51] as well as acute disseminated encephalomyelitis [52] . this calls for long-term patient follow-up in the clinics and also exploring the effect of sars-cov-2 in mouse models of neurodegenerative disorders to anticipate the occurrence of chronic sars-cov-2 cns infection. world health organization -coronavirus disease (covid-2019) situation reports world 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neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) neurological complications during treatment of middle east respiratory syndrome tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc tackling dipeptidyl peptidase iv in neurological disorders structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease the characteristics of hdpp4 transgenic mice subjected to aerosol mers coronavirus infection via an animal nose-only exposure device active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis a comparative review of animal models of middle east respiratory syndrome coronavirus infection livestock susceptibility to infection with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection guillain-barr e syndrome associated with sars-cov-2 infection: causality or coincidence? cryo-em structure of the 2019-ncov spike in the prefusion conformation potent binding of 2019 novel coronavirus spike protein by a sars coronavirusspecific human monoclonal antibody evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor non-neural expression of sars-cov-2 entry genes in the olfactory system suggests mechanisms underlying covid-19-associated anosmia infection with novel coronavirus (sars-cov-2) causes pneumonia in the rhesus macaques the pathogenicity of sars-cov-2 in hace2 transgenic mice the neuroinvasive potential of sars-cov2 may be at least partially responsible for the respiratory failure of covid-19 patients neurologic alterations due to respiratory virus infections coronavirus neurovirulence correlates with the ability of the virus to induce proinflammatory cytokine signals from astrocytes and microglia immunopathogenesis of coronavirus infections: implications for sars coronavirus infection of the central nervous system: host-virus standoff neuroinvasion by human respiratory coronaviruses detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis susceptibility of human and rat neural cell lines to infection by sars-coronavirus quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation unravelling the immunological roles of dipeptidyl peptidase 4 (dpp4) activity and/or structure homologue (dash) proteins we are grateful to drs magdalena mroczek and andrea mancini for sharing the literature on covid-19. this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. the authors declare no financial or other conflicts of interest that relate to the research covered in this article. data sharing is not applicable to this article as no new data were created or analyzed in this study. key: cord-351398-ftkrd1tj authors: stohlman, stephen a.; hinton, david r. title: viral induced demyelination date: 2006-04-05 journal: brain pathol doi: 10.1111/j.1750-3639.2001.tb00384.x sha: doc_id: 351398 cord_uid: ftkrd1tj viral induced demyelination, in both humans and rodent models, has provided unique insights into the cell biology of oligodendroglia, their complex cell‐cell interactions and mechanisms of myelin destruction. they illustrate mechanisms of viral persistence, including latent infections in which no infectious virus is readily evident, virus reactivation and viral‐induced tissue damage. these studies have also provided excellent paradigms to study the interactions between the immune system and the central nervous system (cns). although of interest in their own right, an understanding of the diverse mechanisms used by viruses to induce demyelination may shed light into the etiology and pathogenesis of the common demyelinating disorder multiple sclerosis (ms). this notion is supported by the persistent view that a viral infection acquired during adolescence might initiate ms after a long period of quiescence. demyelination in both humans and rodents can be initiated by infection with a diverse group of enveloped and non‐enveloped rna and dna viruses (table 1). the mechanisms that ultimately result in the loss of cns myelin appear to be equally diverse as the etiological agents capable of causing diseases which result in demyelination. although demyelination can be a secondary result of axonal loss, in many examples of viral induced demyelination, myelin loss is primary and associated with axonal sparing. this suggests that demyelination induced by viral infections can result from: 1) a direct viral infection of oligodendroglia resulting in cell death with degeneration of myelin and its subsequent removal; 2) a persistent viral infection, in the presence or absence of infectious virus, resulting in the loss of normal cellular homeostasis and subsequent oligodendroglial death; 3) a vigorous virus‐specific inflammatory response wherein the virus replicates in a cell type other than oligodendroglia, but cytokines and other immune mediators directly damage the oligodendroglia or the myelin sheath; or 4) infection initiates activation of an immune response specific for either oligodendroglia or myelin components. virus‐induced inflammation may be associated with the processing of myelin or oligodendroglial components and their presentation to the host's own t cell compartment. alternatively, antigenic epitopes derived from the viral proteins may exhibit sufficient homology to host components that the immune response to the virus activates autoreactive t cells, i.e. molecular mimicry. although it is not clear that each of these potential mechanisms participates in the pathogenesis of human demyelinating disease, analysis of the diverse demyelinating viral infections of both humans and rodents provides examples of many of these potential mechanisms. subsequent oligodendroglial death; 3) a vigorous virus-specific inflammatory response wherein the virus replicates in a cell type other than oligodendroglia, but cytokines and other immune mediators directly damage the oligodendroglia or the myelin sheath; or 4) infection initiates activation of an immune response specific for either oligodendroglia or myelin components. virus-induced inflammation may be associated with the processing of myelin or oligodendroglial components and their presentation to the host's own t cell compartment. alternatively, antigenic epitopes derived from the viral proteins may exhibit sufficient homology to host components that the immune response to the virus activates autoreactive t cells, i.e. molecular mimicry. although it is not clear that each of these potential mechanisms participates in the pathogenesis of human demyelinating disease, analysis of the diverse demyelinating viral infections of both humans and rodents provides examples of many of these potential mechanisms. in the last century it became clear that under unusual circumstances, viruses were able to cause demyelination in humans. viral induced demyelination in humans is most clearly associated with two uncommon chronic diseases, i.e., progressive multifocal leukoencephalopathy (pml) and subacute sclerosing panencephalitis (sspe). these demyelinating diseases are the result of infections by a papovavirus and measles virus, respectively. the fact that these disease states are both rare and are temporally remote from the acute infection fueled interest in the mechanisms of both virus persistence within the cns, the immune response within the cns and the mechanisms of viral induced demyelination. demyelinating lesions in humans may also occur rarely following systemic, most likely viral upper respiratory infections. these are collectively grouped under the umbrella of "post-infectious encephalomyelitis" (pie). interestingly, a number of viral etiologies, but no other infectious etiologies, are associated with pie. viruses implicated include measles, mumps, varicella, and influenza virus. this syndrome was also a frequent complication following subcutaneous small pox vaccination. it is interesting that pie can occur during acute infection, but it can also occur after the host's immune response appears to be in control of the infection. this fact, in addition to the usual inability to isolate the inciting virus directly from the cns, suggests an autoimmune or autoimmune-like component. indeed the pathological changes resemble some aspects of complications due to rabies virus vaccination and the autoimmune rodent model of multiple sclerosis, experimental allergic encephalomyelitis (eae). sspe is an exceedingly rare but invariably fatal, chronic progressive panencephalitis occurring in less that 1 case per million children with measles. sspe occurs 5-10 years after acute measles virus infection and is an example of a chronic defective cns viral infection (69, 70) . these are infections in which there is little evidence of infectious virus during disease, yet virus footprints can be detected. the original pathologic description of the disorder by dawson (19) described the characteristic inclusions but not the demyelination or white matter astrogliosis; characteristic features that were later described in detail (122) . the association of sspe with a viral etiology initially came from ultrastructural studies showing typical paramyxovirus in the inclusions, but no viral budding from the plasma membrane (83) . the intranuclear and cytoplasmic inclusions are found in the neurons and oligodendroglia of involved areas and vary considerably in size (3-10 microns). direct association with measles virus was demonstrated by immunohistochemical studies showing measles antigens in both nuclear and cytoplasmic inclusions (49); a finding supported by the presence of high measles virus antibody concentrations in serum and cerebrospinal fluid (csf) (17). as the name implies, there is a widespread chronic inflammatory infiltrate consisting predominantly of small lymphocytes, often in perivascular cuffs, and smaller numbers of plasma cells. neuropathologically there is widespread involvement of the cerebrum and especially the occipital lobe. in contrast to the murine models of demyelination described below, the cerebellum and spinal cord are often not involved or only show focal infiltrates. the white matter shows focal demyelination and widespread astrogliosis. direct evidence is lacking for cns infection by measles virus during acute disease. however, indirect evidence, including electroencephalographic abnormalities and csf pleiocytosis (38) in approximately 30% of patients with uncomplicated acute measles are consistent with virus gaining access to the cns during the acute infection. little is know about alterations in the homeostatic turnover of perivascular microglia during systemic infections; however, given that measles virus infects all populations of peripheral white blood cells, it is possible that the cns is infected via either the normal (or altered) turnover of perivascular microglia. rodent models have clearly shown that activated t cells enter the cns parenchyma (44), although in the absence of cognate antigen these activated cells rapidly exit the cns. in addition, b cells, both those secreting virusspecific and non viral-specific antibody are rapidly recruited into and retained within the rodent cns (34, 52). thus, a transient entry of measles virus-infected cells could provide an initial focus of cns infection. the cellular site(s) of measles virus persistence during the latent phase, defined as the period between the acute infection and clinical onset of sspe, is unknown. however, both immunohistochemical and ultrastructural analysis of sspe brains show clearly that only neurons and oligodendroglia are infected (10, 41, 104). large numbers of viral capsid structures are found, but there is a virtual absence of structures consistent with the budding of mature virions from the cell surface and the direct isolation of infectious virus is only possible by co-cultivation of infected brain cells with cells susceptible to measles virus infection (46, 79). one hallmark of sspe is the extremely high anti-measles virus antibody concentrations found in the csf (17). initially, analysis of the csf suggested the absence of antibodies specific for the m protein, which is important in viral assembly (35). furthermore analysis of the brains from sspe patients suggested the absence of immunoreactivity to the m protein (36, 37). these data suggested cns persistence was related to a defect in m protein. however, using more sensitive techniques anti-m protein antibody is detected in csf (22). furthermore, sequence analysis of the recovered measles virus genomes showed not only the presence of mutations throughout the genome, but that infected cns cells harbored full length genomes with mutations and truncated genomes containing deletions (12). as a result, these genomes lack components required for the assembly of infectious virus, consistent with the histological analysis. however, antibody does appear to be critical in the pathogenesis of sspe. in vitro analysis suggests that the anti-measles antibody response aids in persistence by stripping viral envelope proteins from the cell surface (28). furthermore, passive protection studied in rats infected with measles virus demonstrate that not only does antibody promote persistence (88, 89, 111) but it decreases viral replication at the transcriptional level (57) . the patchy demyelination associated with sspe may occur as a result of several mechanisms. although measles virus establishes an early infection primarily in neurons and oligodendroglia, the antibody response promotes viral persistence resulting in both the slow loss of infected cells and increasing csf antibody levels. indeed, oldstone (74) showed that circulating antibodies from sspe patients lysed brain cells cultured from a patient with sspe. at the same time antibody holds infectious virus in check, it also enhances the accumulation of mutations within the viral genome, facilitating persistence. current notions suggest a dynamic relationship in which infection is amplified within the cns during the clinically latent period by cell-to-cell spread, while the virus continues to accumulate additional mutations. it appears to be the accumulation of viral products in neurons and oligodendroglia, as well as neurofibrillary tangles that result in cell death (48) and in neurons and glial fibrillary tangles in oligodendroglia in some cases, demyelination. other studies have demonstrated the infiltration of cd4 + and cd8 + t cells as well as expression of inflammatory cytokines such as interferon-gamma (ifn-␥) and tumor necrosis factor-alpha (tnf-␣) suggesting that cell-mediated damage to infected cells may also play a role (45, 73) . it is likely that the demyelination is primarily a result of the direct death of oligodendroglia, resulting in primary demyelination. although secondary demyelination, as a result of direct neuronal loss may also occur, one quantitative study found that cortical thickness is not affected and there was no evidence for loss of neurons even though synaptic density was reduced (92) . pml is also a typically fatal demyelinating disease of the cns. however, in contrast to sspe, it predominantly affects immunocompromised individuals (47). in pml, multiple small foci and large regions of demyelination are present in the cerebrum, cerebellum and brain stem suggesting the evolution of smaller lesions into large confluent ones ( figure 1a) . cytologically, the lesions have characteristic features involving the oligodendroglia and astrocytes. the oligodendroglia are enlarged and show intranuclear, ground-glass inclusions ( figure 1b ). the astrocytes within the lesions are often large, reactive and atypical and may undergo mitosis. an inflammatory response is characteristically absent; however, activated microglia and lipid-containing macrophages may be seen. large lesions may also show necrosis. pml was first identified as a viral infection in 1965 based on ultrastructural studies showing large accumulations of 30 nm papovavirus-like particles in the distended nuclei of oligodendroglia (123) . two antigenically related but distinct papoviruses were initially isolated from the brains of patients with pml. one was similar to the simian vacuolating virus 40 (sv40) (113) . the other was less antigenically related to sv40 and was designated jc virus (75) . subsequently, all isolates have been similar to the original jc virus isolate. in contrast to sspe, which occurs following acute measles virus infection of naïve populations, serological studies demonstrate that jc is a ubiquitous human virus acquired early in life with no definable acute syndrome (76) . also in contrast to sspe, which is believed to establish residence within the cns during acute infection, jc virus is not detectable in brains of normal individuals or in immunosuppressed individuals which have no symptoms of pml (14, 40, 93) . this suggests that jc virus persists in a peripheral site. the pathogenesis of pml appears to progress in the following way. virus is acquired by the majority of humans during early life (76) and is maintained in an undetermined peripheral site, possibly the kidney (16), controlled by the host's immune response. the majority of adults are seropositive, but have no clinical evidence of infection. in those unfortunate individuals with underlying immunosuppressive diseases, jc virus cannot be held in check and replicates peripherally. a population-based study revealed that pml has a frequency of 5.1% in aids patients and 0.07% among patients with hematologic malignancies; however, similar clinical features of pml were found in each group (87) . the isolation of jc virus from the lymphocyte populations of aids patients without evidence of pml, suggests either that hiv-1 induced immunosuppression is particularly destructive to the mechanisms effective in inhibiting jc virus (103), or even the possibility of a synergistic influence of these two viruses (23). oligodendroglia which are not infected with hiv-1 may also contain soluble hiv-1 tat protein, a potent jc virus transactivator (108) . it appears that infection of b cells may provide the vehicle for hematogenous spread into the cns. hematogenous spread is suspected to occur based on the widespread distribution of lesions. however, the lesions show no relationship to blood vessels, suggesting that infected cells must migrate into the parenchyma to establish foci of virus replication. ultrastructural and immunohistochemical studies reveal that jc virus appears to preferentially infect oligodendroglia ( figure 1c) ; however, lesions also show infection of the enlarged astrocytes. astrocytes may be semi-permissive to infection because only a small proportion of infected cells display evidence of viral capsid protein. based on the frequency of mutations within the genome of virus isolated from the cns versus virus isolated from the periphery of the same patients (103) , it has been suggested that some alteration in viral replication may be required for cns tropism. in support of a selective tropism for oligodendroglia, infected cells predominate around the edges of the lesions. the small size of many foci may correlate with selective oligodendroglia tropism and viral induced cell death. although pml is considered to induce a primary demyelination, the center of advanced lesions show few intact axons and large lesions may become necrotic. murine models of viral induced demyelination highlight a number of aspects of cns infection and mechanisms of acute and chronic demyelination. two models in which the mouse is the natural viral host are discussed below. in both cases the virus was initially isolated from mice with spontaneous paralytic disease (2, 13, 105, 106). these viruses are from two different viral families, theiler's murine encephalomyelitis virus (tmev) is a non-enveloped positive stranded rna virus of the picornaviridae family while mouse hepatitis virus (mhv) is an enveloped positive stranded rna virus of the coronaviridae family (table 1 ). these infections of the cns provide examples of both the influence of the host's genetic background and regulation by the immune response. for example, the predominant mouse strain used for analysis of demyelination following tmev infection is the sjl strain (3, 60, 65, 66, 67) . this strain is also widely preferred for the study of eae because cns autoimmune disease can be induced with a variety of cns antigens including myelin basic protein (mbp) and proteolipid protein (plp). in addition, sjl undergo a relapsing and remitting form of autoimmune cns disease. sjl mice are used for both tmev pathogenesis and eae due to the relative resistance of many other mouse strains to these disparate cns diseases. by contrast, demyelination produced by the infection with mhv is mainly studied in c57/bl6 and balb/c mice, but not the sjl strain (101) . this is due to the absence of virion receptor expression in sjl mice (115) , making this strain resistant to mhv infection. these examples of differential responses due to host genetic backgrounds, which only partially map to the major histocompatibility complex (mhc), provide vivid examples of the complexity of responses that can lead on the one hand to viral persistence within the cns and on the other hand to very similar pathological changes, i.e., demyelination ( figure 2 ). in both cases these viruses persist within the cns following an acute episode, characterized globally as encephalomyelitis. tmev was a relatively common enteric pathogen of laboratory mice; however, naturally occurring cns infection was rare (105, 106) . experimental cns infection with the reduced neurovirulent bean or da strains results in a biphasic cns disease (60) . by contrast, infection with more neurovirulent tmev strains, i.e., gdvii and fa, generally results in a monophasic fatal disease (105, 106) . infection by all four strains results in an acute encephalomyelitis characterized by loss of anterior horn cells; however, only the bean and da strains induce a flacid paralysis. infection of mice with the reduced neurovirulent strains results in virus replication that reaches a peak followed by subsequent viral clearance from the cns ( figure 2 ). in mice genetically resistant to the chronic phase, infectious virus is com-pletely cleared. therefore the host response is able to achieve a sterile immunity. tmev is a member of the picornavirus family, a viral group that includes poliovirus and is generally considered to be primarily controlled by the humoral immune response. genetic analysis however, suggests that mhc class 1 molecules are important in resistance to tmev persistence suggesting that cd8 + t cells, possibly those cytotoxic t lymphocytes (ctl) that mediate the lysis of infected cns cells during the acute phase of infection, may play a critical role in preventing virus reactivation. indeed, ctl specific for tmev have been described, although their role in the individual mouse strains susceptible and/or resistant to tmev chronic demyelination is controversial (50). analysis of ctl responses in the early phase of virus replication shows that resistant mice make excellent ctl responses (21). by contrast, the ctl response in strains that progress to persistent infection and demyelination make relatively poor ctl responses. it has been suggested that cd8 + t cell responses to tmev contribute to demyelination during the late chronic phase of viral persistence (90) . these data suggest that tmev persistence is a direct consequence of a genetically influenced immune response unable to completely clear virus from the cns during the acute infection. histopathological findings following tmev infection of mice susceptible to chronic disease are consistent with a biphasic disease (20) (figure 2 ). in the first or neuronal phase, virus replicates rapidly within the cns, infecting cells of the thalamus, hypothalamus, and brain stem and anterior horn cells in the spinal cord. infection of white matter, meninges, choroid plexus or ependyma are not found. little demyelination or parenchymal inflammation are found during the first phase, even in mice susceptible to the late phase. therefore, the early phase of tmev cns infection resembles acute polio virus-induced encephalomyelitis with paralysis due to cytolytic infection of motor neurons resulting in the transient loss of hind limb function. the absence of inflammation is interesting because high titers of infectious virus remain at the end of the acute phase in mice that progress to the late phase of infection ( figure 2 ). in this second phase, inflammation and demyelination increase in the spinal cord and correlate with the persistence of infectious virus. lesions are most common in the lateral columns of the thoracic region and the largest may encompass the majority of the white matter. lesions are characterized by the presence of infected macrophages; however, occasionally neurons and astrocytes may also be infected. interestingly, only rarely is there infection of oligodendroglia, even though the disease is primarily one of an inflammatory demyelination. therefore, in the early lesions of the second chronic phase of tmev infection, myelin is destroyed although the oligodendroglia are not infected with virus. inflammatory infiltrates consist predominantly of macrophages and cd4 + t cells. the cd4 + t cells express the phenotype of highly activated cells, i.e., expression of the high affinity il-2r (85) . analysis of infection in cd8 + -depleted mice (9), and ␤-2 microglobulin deficient mice that lack mhc class i expression (25), indicate little support for a role of cd8 + t cells during this second phase. as disease progresses in this phase, recent data have implicated a perforin-dependent cd8 + t cell mechanism for the neurological deficits (70) . mhc class i deficient mice show decreased neurological deficits compared to wild type mice, possibly due to preservation of axons coincident with increased expression of sodium channel density (90) . consistent with an effect of cd8 + t cells on neuronal function late in infection, increased axonal damage has also been observed during the late phase of tmev persistent cns infection (62, 63, 107) . axonal damage has also been implicated in the pathogenesis of lesions in ms patients (7), possibly via an indirect effect of activated t cells on microglia (32). although activated microglia and macrophages are abundant during chronic tmev induced demyelination, it is not clear if axonal damage contributes to tmev pathogenesis or reflects the loss of axonal function due to the extensive loss of myelin. in addition to the presence of activated cd4 + t cells there is a preponderance of pro-inflammatory cytokines in the cns during chronic tmev infection (4). based on the paucity of cd8 + t cells within the inflamed cns, it appears that these cytokines are either derived from the activated cd4 + t cells or are secreted by the infiltrating macrophages or activated microglia, or both. four potential mechanisms have been proposed to explain demyelination in tmev-infected mice. first, virus infection of oligodendroglia, although rare, results in sufficient loss of cells to produce demyelination. although plausible, there appears to be too few oligodendroglia infected at any time to account for the extensive demyelination. however, it remains possible that tmev infection of oligodendroglia does contribute to the overall loss of myelin. second, the predominant anti-viral inflammatory th1 type cd4 + t cell response suggests that myelin, or the oligodendroglia itself, is damaged by the sustained presence of pro-inflammatory cytokines. this issue has been difficult to address, because most attempts to eliminate the host immune response have resulted in death due to overwhelming virus infection rather than alterations in demyelination. third, the continued secretion of ifn-␥ could maintain macrophage/microglial activation, resulting in a direct attack on myelin. this cns antigen-nonspecific induction of demyelination is termed "bystander" demyelination (118) . finally, it has also been proposed that cd8 + t cell mediated cytolysis of tmev-infected oligodendroglia could result in cell death and ultimately myelin loss. however, the small number of infected oligodendroglia along with little evidence for sustained cd8 + t cell activity in the cns during tmev persistence, suggests that this is not a predominant mechanism. the presence of demyelinating lesions in mice chronically infected with tmev correlates with high levels of cd4 + t cell mediating a virus-specific delayed type hypersensitivity reaction (15). consistent with the role of virus specific cd4 + t cells secreting pro-inflammatory cytokines in the demyelinating process, transfer of virus specific cd4 + t cells secreting th1 cytokines into infected mice increases the severity of disease (31). the majority of data support a virus-specific but indirect role of cell mediated immunity in the demyelination. initial attempts to correlate the chronic demyelinating phase of tmev infection with induction of autoimmune t cells were unsuccessful. demyelination could not be transferred with either t cells or sera from infected mice (3) . neuroantigen specific t cells responses could not be detected following tmev infection, including during the chronic demyelinating phase of infection (65) . importantly, tolerance to neuroantigen induced via the transfer of antigen-coupled syngeneic spleen cells also failed to affect the ability of tmev to induce a chronic demyelinating disease (66) . in contrast to these studies, recent data have demonstrated a progressive activation of neuroantigen specific t cells during tmev chronic demyelination (67) . there has been no evidence presented to suggest that these neuroantigen specific t cells are induced via the recognition of viral elements which cross react with host determinants (molecular mimicry) (29). although the relevance of these neuroantigen specific t cells to the progression of chronic demyelination is still not clear, the data clearly demonstrate activation of immunity to host antigens during inflammation induced by a persistent viral infection. tmev induced demyelination is predominantly a consequence of the viral specific cd4 + t cell-mediated chronic inflammatory response. infectious virus preferentially replicates in macrophages and microglia within the spinal cord. the susceptible host not only mounts a vigorous anti-viral cd4 + t cell response but also neu-tralizing antibody. it is the presence of infectious virus that induces the inflammatory response which on the one hand is unable to eliminate infectious virus from the cns; but on the other hand leads to a progressive increase in t cells which recognize potentially encephalitogenic host neuroantigen epitopes. these host antigen specific t cells do not appear to contribute significantly to the demyelinating process. why the antiviral immune effectors are unable to either eliminate infectious virus from the cns during the acute neuronal phase or control replication of infectious virus during the chronic phase of infection is not clear. however, analysis of tmev induced demyelination has provided critically important support for the concept that a chronic inflammatory response within the cns can result in the activation of autoreactive t cells (67) . in addition, analysis of this model has clearly shown that the fine antigen specificity recognized by these inflammationinduced autoreactive t cells changes with time. this suggests that broad-based immunotherapeutic approaches to inhibit chronic human autoimmune cns disease may hold greater promise than specifically targeted approaches. mhv also used to be a common enteric pathogen of laboratory mice and, similar to tmev, dissemination to the cns was a rare event. the most studied strain that causes demyelination, the jhm strain (jhmv) or mhv-4 serotype, was isolated from a mouse with spontaneous hind limb paralysis (2) . it produces an acute encephalomyelitis accompanied by primary demyelination in mice, rats and non-human primates with little or no evidence of hepatitis (13, 55, 71, 86, 95, 114) . pathogenesis is dependent upon viral dose, route of infection, the host's age and genetic background; however, prominent cns infections can be induced with the neurotropic mhv strains either via the intranasal route or by direct inoculation into the cns. the parental jhmv strain infects astrocytes, oligodendroglia, microglia and neurons. to increase the number of survivors, thereby allowing a more careful study of its pathogenesis, a number of jhmv variants, which have limited or no ability to infect neurons (i.e. the small plaque mutants, ds [24]; the temperature sensitive mutant, ts8 [39]; and the neutralizing monoclonal antibody resistant 2.2v-1 variant [26]), have been examined in detail. in general, both the more neuronotropic parental virus as well as the strains with limited or no tropism for neurons produce an acute encephalitis accompanied by acute primary demyelination. low dose infection of adult mice with the parental virus results in chronic demyelination; however, its virulence and rapid induction of death has limited its usefulness. in addition, virus replication in the cns does not appear to be compromised in the variant strains; however, the initial cellular sites of virus replication are altered. the viral surface (s) envelop glycoprotein, that contains not only the sites of binding by neutralizing antibody but also has the domain which interacts with the viral receptor, is believed to also regulate which cns cells are infected. this is based on both the analysis of neutralizing monoclonal antibody resistant variants (2.2v-1) and recombinant mhv in which the jhmv s protein was inserted into the genetic background of the more hepatotropic a59 strain (84) . however, recent data testing a different set of recombinant mhv suggest that a component other than the s protein might contribute to cns (figure 4) . following initial infection, oligodendroglia undergo both necrotic and apoptotic death; however, death is limited to apoptosis during chronic demyelination (5). interestingly, lesions contain predominantly macrophages and microglia but there is very little viral antigen ( figure 3b ). infectious virus is usually eliminated from the cns at approximately day 14 post infection (58, 59, 77, 78) , although ts8 can be recovered during chronic infection (51). following clearance of infectious virus, the number of viral antigen positive cells declines. therefore, jhmv appears to preferentially infect ependymal cells during the initial phase of infection. replication then proceeds down to the spinal cord and peripherally into the gray and white matter, with the virus infecting astrocytes, microglia and oligodendroglia. in contrast to tmev, only rarely has infectious jhmv been isolated from the cns following immune mediated clearance (51). the virus appears to establish a latent infection in astrocytes and microglia (102) but cannot be recovered following either immunosuppression or explantation of cns tissues (95, 101) . however, rare viral antigen positive cells can be detected for months within the white matter tracks, and viral specific rna can be detected in the cns for at least 1 year post infection (1). this persistent, but noninfectious virus, correlates with the presence of ongoing foci of demyelination and remyelination. in addition to jhmv, a number of mhv-induced demyelination studies have been carried out with the a59 strain which was initially isolated from a mouse with acute hepatitis. mhv-a59 exhibits both hepatotropism as well as neurotropism. at low doses of infectious virus, mhv-a59 causes an acute hepatitis and a meningoencephalomyelitis with small foci of demyelination. immunity clears infectious virus from the cns by 10 days. demyelination becomes predominant after virus is cleared (119) . at high doses, mhv-a59 produces an infection similar to the biphasic infection induced by tmev with some notable differences, including a more predominant hepatitis. however virus is cleared from the blood and liver by approximately one week post infection and the hepatitis begins to resolve. virus replicates to high titers in the brain and spinal cord but is partially controlled. infectious virus persists in the spinal cord and brain for 4 weeks post infection before complete clearance. this delayed clearance may be related to the absence of the immunodominant ctl epitope (11, 30). although no infectious virus is recovered, viral antigen can be detected in survivors for 4 months post infection. primary demyelination with axonal sparing is the predominant finding following infection with both doses of mhv-a59, although a number of mice injected with the high dose also exhibit non-inflammatory, non-obstructive hydrocephalus (56) . in contrast to tmev, there are few inflammatory cells associated with these demyelinating lesions, although similar to both tmev and the demyelination induced by jhmv, macrophages laden with intracellular myelin debris are present. mhv infection of the cns results in the induction of a vigorous local immune response confined primarily to the cns (6, 81, 97) which protect the host from the lethal effects of viral cns infection. however, similar to tmev infection of the cns, sterile immunity is not achieved. in the case of mhv this results in persistence of non-infectious virus within the cns. two potential mechanisms which limit t cell reactivity in the periphery were examined to determine if they contributed to limiting the response before sterile immunity could be achieved; however, neither il-10 nor fas/fasl interactions participated in limiting jhmv immunity (58, 79) . therefore, mhv infection is characterized by demyelination during the acute phase of replication associated with infectious virus. following the clearance of infectious virus from the cns, the second phase is best described as a latent infection. during this phase persistent virus is associated with continuing foci of demyelination. it must be noted that the absence of detectable infectious virus during this phase does not preclude that the foci of demyelination are initiated by limited amounts of local infectious virus, which are undetectable by conventional means. the observation that the amount of detectable viral footprints, i.e., antigen or viral rna, diminish with time, as do the new foci of demyelination, lends credence to the possibility that as new infectious virus is produced it is rapidly eliminated by the immune response. it is the inability of the immune response to produce a sterile immunity that has led to an examination of the mechanisms that control virus replication within the cns. the acute inflammatory response is characterized by the influx of all types of immune effectors into the murine cns, i.e., natural killer (nk) cells; cd4 + t cells, cd8 + t cells, b cells and macrophages (117) . neither nk cells, b cells, nor the antibody response, play a role in initial viral control. both the cd4 + and cd8 + t cell populations appear to be the critical elements (116) . the cd4 + t cells localize to the perivascular areas and play an as yet undefined role in the maintenance of cd8 + t cells (100) . the cd8 + t cells are the major effectors of anti-viral activity (99) . virus-specific, and apparently non-viral-specific cd8 + t cells (6), are rapidly recruited to the cns. in contrast to cd4 + t cells, they enter the parenchyma and are therefore close to the sites of virus replication. interestingly, analysis of jhmv pathogenesis has shown that cd8 + t cells use two separate antiviral effector mechanisms, dependant upon the cns cell type infected. perforin-mediated cytotoxicity controls infection of microglia and astrocytes (59) . by contrast, ifn-␥ is the antiviral mechanism responsible for controlling the infection of oligodendroglia (77) . therefore, immunity to jhmv infection of the cns is predominantly mediated by the antiviral cd8 + t cell response; however, their ability to control cns replication is high-ly dependent upon both the cd4 + t cell response and the individual cns cell types infected. tnf-␣, implicated in the progression of autoimmune demyelination (91, 94) , plays no role in either the accumulation of inflammatory cells within the cns nor in jhmv induced demyelination (98) . in addition to providing an example of cell type effector mechanisms, these data have raised a number of interesting questions concerning the interaction of the immune system with the cns. for example, why are oligodendroglia apparently refractory to mhc class i mediated cytotoxicity? if they express mhc class i molecules are they deficient in processing of the appropriate peptide? are they unable to express sufficient mhc class i molecules to initiate a cytolytic attack? do the intrinsic properties of the membrane prevent immune mediated cytotoxic attack? future analysis should shed light on these possibilities. jhmv infection has also provided insights into the interactions of the immune system and the cns in regulating viral persistence. analysis of the pathogenesis of jhmv infection in mice deficient in b cells, and therefore unable to mount an anti-viral antibody response, showed that virus replication is controlled during the acute infection, similar to the control in wild type mice (59) . however, following initial control of infectious virus, virus re-emerged within the cns of these b celldeficient mice. again, these data demonstrate that a functional t-cell immune response is unable to provide a sterile immunity following jhmv infection. importantly, the passive transfer of antiviral antibody following initial clearance completely prevented virus reactivation. these data suggest that although cell mediated immunity, predominantly the antiviral cd8 + t cell response, fails to provide sterile immunity, humoral immunity compensates by preventing the re-emergence of infectious virus during persistence. the mechanism of demyelination during jhmv infection has been controversial. initial studies showed that virus actively replicates in oligodendroglia. this suggested that oligodendroglial death due to infection resulted in primary demyelination (55, 86) . indeed, electron microscopic studies of jhmv infected cns have shown marginated nuclear chromatin in infected oligodendroglia (26), consistent with the suggestion that oligodendroglia undergo apoptotic death following infection (5). interestingly, apoptosis of oligodendroglia has also not been detected in the cns of mice with eae (8). it is clear however, that an immune component is critical for jhmv induced demyelination. immunosuppressed mice that succumb to overwhelming neuronal infection, have no detectable demyelination. reconstitution of these mice with immune cells results in demyelination (27), supporting a role for an immune component in jhmv induced demyelination. however, analysis of a mice deficient in a variety of immune components, especially those lacking both t cell populations, exhibit viral-induced demyelination (33). it appears that an inflammatory response, along with preventing rapid death due to overwhelming viral infection, is required to initiate macrophage/microglial removal of myelin (24, 42, 43, 120) . interestingly, it has recently been suggested that infiltrating macrophages are not required for demyelination, i.e., that activated microglia can remove sufficient myelin for demyelination to be evident (121) . in contrast to tmev infection, no evidence has been reported for the activation of immunity to cns self-antigens in mice infected with jhmv, although infection does induce the activation of t cells specific for self-antigen in the periphery (54) . however, self-reactive t cells capable of mediating an eae-like disease upon adoptive transfer into naïve recipients have been isolated from rats during jhmv induced subacute demyelinating encephalomyelitis (110) . whether jhmv infection of mice is capable of inducing autoreactive t cells, and what their potential contributions are to chronic demyelination in mice, remain open questions. cns infection by jhmv induces an acute encephalomyelitis with virus replication in astrocytes, microglia, oligodendroglia and rarely in neurons. the host controls infectious virus via a vigorous anti-viral immune response that is predominantly localized to the cns. this results in protection from death; however, immunity is unable to completely eliminate all traces of the virus. the result is in an acute infection characterized by immune infiltrates and primary demyelination. the demyelination most likely results from virus infection of oligodendroglia coupled with the massive influx of macrophages and activation of microglia which strip the myelin sheaths from axons leading to primary demyelination. this scenario is supported by the data demonstrating infection of oligodendroglia (26, 42, 55, 86) , the absence of demyelination following mhv infections of the cns in which oligodendroglia are not infected (26, 61) , and the absence of demyelination in mice which are immunosuppressed sufficiently to prevent macrophage infiltration (27). immunity also plays a critical role in the establishment and maintenance of viral persistence in what appears to be a latent state. the mechanism(s) of chronic demyelination are less well understood, but may relate to activation of virus replication resulting in the new foci of demyelination. the limited focal nature of the demyelination and the reduced frequency of new lesions with time during the "latent" phase are consistent with local events, possibly at the single oligodendroglial level, producing foci of new demyelination. this interpretation is consistent with temporal reductions in both viral antigen positive cells and viral rna (1, 95) . however, virus infection in cns cells types other than oligodendroglia have been noted during the latent phase (103) . these data have been interpreted to suggest that the health of the oligodendroglial cell may be affected adversely by infection of adjacent cns cell types. in addition to the pathological outcome of demyelination associated with viral infections of both humans and rodents, analysis of these viral infections have highlighted a number of common aspects. first, both human infections we describe which result in demyelination result from peripheral viral infections. sspe follows an acute measles virus infection in a naïve host while pml results from the inability of the immune response to control a persistent peripheral infection leading to dissemination to the cns. both rodent models described are based on viruses derived from a rare occurrence of dissemination into the cns, most probably from an enteric site of infection. although the immune status of the initial mice which exhibited viral mediated cns disease was not examined in either case, it is likely that these rare events were either: 1) the result of an underlying immunosuppression similar to pml or; 2) the natural selection of variants with increased neurotropism, similar to the events contributing to the evolution of both pml and sspe. in terms of the antiviral immune response, tmev infection appears to have some aspects in common with sspe. during both acute phases of infections there is little or no evidence of demyelination. although it is not clear what events predispose to sspe, tmev persists in the cns even though both cd4 + t cells and antibody responses are mounted during the acute phase. it is interesting that during sspe and tmev, neutralizing antibody is present during the virus-induced demyelination. in contrast to these viral diseases, jhmv produces demyelination during the acute infection and at least the infectious virus is controlled by the cellular arm of the immune response. however, it is the anti-viral antibody response which inhibits the jhmv reactivation following acute infection. these data suggest that either the specificity or affinity of the antibody may indeed be a critical factor in the progression of all three of these demyelinating diseases. indeed, nursing jhmv infected pups on dams immunized with jhmv prevents acute death. however a significant proportion of these mice undergo temporally distinct spontaneous virus reactivation characterized by both the presence of infectious jhmv in the cns and demyelination (82) . direct viral infection of oligodendroglia is clearly capable of producing demyelination (sspe, pml, jhmv). however, the contribution of infection or persistence in other cns cell types to the pathological processes associated with demyelination may play a substantial role, although the mechanisms are not clear. for example, analysis of the jhmv model has shown not only that specific subsets of immune effectors are critical in controlling virus infection of the major cns cell types and viral persistence, but that demyelination is absent without the immune response associated with encephalomyelitis. in addition, there is controversy concerning the infection of oligodendroglia by tmev; however, even the most optimistic estimates suggest that it is primarily the infection of myelomonocytic lineage cells which predominates. finally, advances in immunological techniques have provided evidence that during persistent tmev infection the inflammatory response, directed predominantly toward viral components, results in the eventual activation of cell mediated immunity specific for components of myelin. although it is not clear 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(1967) electron microscopic finding in two cases with inclusion bodies pathogenesis of chimeric mhv4/mhv-a59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence flow cytometric and functional analyses of cns-infiltrating cells in sjl/j mice with theiler's virus-induced demyelinating disease: evidence for a cd4+ t cell-mediated pathology oligodendrocytes and their myelin-plasma membrane connections in jhm mouse hepatitis virus encephalomyelitis aids-and non-aids-related pml association with distinct p53 polymorphism antibody-mediated modification of encephalitis induced by hamster neurotropic measles virus induction of subacute murine measles encephalitis by monoclonal antibody to virus haemagglutinin absence of neurological deficits following extensive demyelination in a class i-deficient murine model of multiple sclerosis an antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis morphometric study of the frontal cortex in subacute sclerosing panencephalitis high sensitivity detection of jc-virus dna in postmortem brain tissue by in situ pcr tumor necrosis factor mediated myelin and oligodendroglia damage in vitro chronic nervous system demyelination in mice after jhm virus infection in vivo effects of coronavirus-specific t cell clones: dth inducer cells prevent a lethal infection but do not inhibit virus replication characterization of mouse hepatitis virus specific cytotoxic t cells derived from the central nervous system of mice infected with the jhm strain tumor necrosis factor expression during mouse hepatitis virus induced demyelination mouse hepatitis virus -specific cytotoxic t lymphocytes protect from lethal infection without eliminating virus from oligodendroglia ctl effector function within the cns requires cd4+ t cells persistent infection by mouse hepatitis virus activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus detection of jc virus dna in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy subacute sclerosing leukoencephalitis: ultrastructure of intranuclear and intracytoplasmic inclusions spontaneous encephalomyelitis of mice, a new virus disease encephalomyelitis of mice extensive injury of descending neurons demonstrated by retrograde labeling in a virus-induced model of chronic inflammatory demyelination detection of hiv-1 tat and jc nucleocapsid protein, vp1, in aids brain with progressive multifocal leukoencephalopathy sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv-4) leads to a characteristic distribution of demyelination comparative analysis of coronavirus jhm-induced demyelinating encephalomyelitis in lewis and brown norway rats latent measles virus infection of the hamster central nervous system relapsing subacute demyelinating encephalomyelitis in rats during the course of coronavirus jhm infection isolation of a virus related to sv40 from patients with progressive multifocal leukoencephalopathy pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) purification of the 110-kilodalton glycoprotein receptor for mouse hepatitis virus (mhv)-a59 and identification of a non-functional, homologous protein mhvresistant sjl/j mice effective clearance of mouse hepatitis virus from the cns requires both cd4 + and cd8 + t cells characterization of brain infiltrating mononuclear cells during infection with mouse hepatitis virus strain jhm primary demyelination as a non-specific consequence of a cell-mediated immune reaction acute and subacute demyelination induced by mouse hepatitis virus strain a59 in c3h mice macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus depletion of blood-borne macrophages does not reduce demyelination in mice infected with a neurotropic coronavirus reflections on the etiology and pathogenesis of subacute sclerosing panencephalitis particles resembling papovaviruses in human cerebral demyelinating disease supported by nih grant ns18146. the authors wish to thank ernesto barron for excellent technical assistance. key: cord-329527-0rlotyz3 authors: bohmwald, karen; gálvez, nicolás m. s.; ríos, mariana; kalergis, alexis m. title: neurologic alterations due to respiratory virus infections date: 2018-10-26 journal: front cell neurosci doi: 10.3389/fncel.2018.00386 sha: doc_id: 329527 cord_uid: 0rlotyz3 central nervous system (cns) infections are one of the most critical problems in public health, as frequently patients exhibit neurologic sequelae. usually, cns pathologies are caused by known neurotropic viruses such as measles virus (mv), herpes virus and human immunodeficiency virus (hiv), among others. however, nowadays respiratory viruses have placed themselves as relevant agents responsible for cns pathologies. among these neuropathological viruses are the human respiratory syncytial virus (hrsv), the influenza virus (iv), the coronavirus (cov) and the human metapneumovirus (hmpv). these viral agents are leading causes of acute respiratory infections every year affecting mainly children under 5 years old and also the elderly. up to date, several reports have described the association between respiratory viral infections with neurological symptoms. the most frequent clinical manifestations described in these patients are febrile or afebrile seizures, status epilepticus, encephalopathies and encephalitis. all these viruses have been found in cerebrospinal fluid (csf), which suggests that all these pathogens, once in the lungs, can spread throughout the body and eventually reach the cns. the current knowledge about the mechanisms and routes used by these neuro-invasive viruses remains scarce. in this review article, we describe the most recent findings associated to neurologic complications, along with data about the possible invasion routes of these viruses in humans and their various effects on the cns, as studied in animal models. respiratory diseases caused by viral agents are one of the most critical problems in public health, as every year they are responsible for high rates of morbidity and mortality, mainly of young children, the elderly and immunocompromised individuals (talbot and falsey, 2010; tregoning and schwarze, 2010; englund et al., 2011) . the most common respiratory viruses that affect susceptible population are human orthopneumovirus-previously known as human respiratory syncytial virus (hrsv), influenza virus (iv), coronavirus (cov) and human metapneumovirus (hmpv; nichols et al., 2008) . the transmission of these viruses is mainly by contact with fomites or suspension droplets (kutter et al., 2018) . all these viruses have in common the ability to produce bronchiolitis and pneumonia, being responsible for large numbers of hospitalizations every winter season (nichols et al., 2008; talbot and falsey, 2010; tregoning and schwarze, 2010) . besides respiratory tract infections, these viruses have been associated with neurological clinical manifestations in patients with a severe occurrence of the respiratory disease (antonucci and fanos, 2005; akins et al., 2010; antonucci et al., 2010; desforges et al., 2014a; fok et al., 2015; algahtani et al., 2016) . commonly, the invasion of the central nervous system (cns) and the subsequent pathology have been more studied in infection caused by japanese encephalitis virus (jev), varicella-zoster virus (vzv), measles virus (mv) and human immunodeficiency virus (hiv), among others (koyuncu et al., 2013) . nowadays, the interest in increasing the knowledge about the characteristics and mechanisms involved in their neurological manifestations has risen. neurological abnormalities found in patients with severe respiratory illness have a spread spectrum of clinical signs, being the most reported seizures (niizuma et al., 2014; li et al., 2016) , status epilepticus (sweetman et al., 2005; vehapoglu et al., 2015) , encephalopathies (antonucci and fanos, 2005; mizuguchi et al., 2007; niizuma et al., 2014; meijer et al., 2016) and encephalitis (ng et al., 2001; niizuma et al., 2014; fok et al., 2015 ; table 1 ). the effects of each respiratory virus mentioned above in the cns infection will be discussed in detail later. for the proper functioning of the cns, it is essential to maintain homeostasis. both, the blood-brain and the blood-csf barriers play an important role in protecting the brain of free passage of unwanted molecules, pathogens and cells (mcgavern and kang, 2011) . the blood-brain barrier (bbb) is the first line of defense that prevents the entry of pathogens into the brain, and it is composed by cerebral microvascular endothelium, astrocytes, pericytes and extracellular matrix (mcgavern and kang, 2011; swanson and mcgavern, 2015a) . importantly, the brain microvascular endothelium cells (bmecs) are a cell type found in significant proportions in the bbb; in between these cells are tight junctions (tj), which controls the barrier permeability (koyuncu et al., 2013; miner and diamond, 2016) . several routes of cns invasion can be used by viral pathogens, among these are included the hematogenous route-which is the infection of the endothelium or the ''trojan horse'' mechanism-and the peripheral nerves or olfactory sensory neurons (mcgavern and kang, 2011; swanson and mcgavern, 2015b; dahm et al., 2016) . after primary infection, most neurotropic viruses can enter the bloodstream to reach cns, a process which is called viremia (gonzalez-scarano and tyler, 1987) . once inside the bloodstream, viruses can pass through the bbb by a transendothelial mechanism, which is transcytosis across bmecs and pericytes by endocytic vesicles (suen et al., 2014) . another transcellular entry method is the infection of endothelial cells, allowing the direct pass across bbb (koyuncu et al., 2013; suen et al., 2014) . besides, disruption low levels of tnf-α in csf. elevated il-6 and bdnf in csf correlates with brain damage. cappel et al. (1975) ; hirayama et al. (1999) ; ng et al. (2001) ; zlateva and van ranst (2004) ; otake et al. (2007) ; kawashima et al. (2009 kawashima et al. ( , 2012 and morichi et al. (2017) of bbb permeability by destabilization of tjs allow viral entry into the brain in a paracellular transmigration way (li et al., 2015; swanson and mcgavern, 2015b) . this event is a consequence of a systemic infection that releases inflammatory mediators-such as cytokines and chemokines-besides the matrix metalloproteinase (mmp; roe et al., 2012) . finally, the ''trojan horse'' mechanism consists in the infection of bloodstream leucocytes-mainly monocytes/macrophages-which can transmigrate via paracellular route, across the permeable bbb into the cns (suen et al., 2014) . the correct working of the organism requires continuous communication between cns and peripheral tissues. in this process, neurons play an essential role, since these cells innervate the peripheral organs, that can be used by viruses as a gate to enter the cns (swanson and mcgavern, 2015b) . neurons are polarized; this characteristic allows them to receive, process and transmit signals to other cells (koyuncu et al., 2013; swanson and mcgavern, 2015a) . some viruses can infect and migrate through the nerve ending which can be sensory or motor (swanson and mcgavern, 2015a) . for this purpose, viruses use the motor proteins dynein and kinesins-which are responsible for the retrograde and anterograde neuronal transport (swanson and mcgavern, 2015b ). an alternative route for neuroinvasion is the transport through olfactory neurons (swanson and mcgavern, 2015b) . this pathway is an excellent mechanism to access cns for viruses that enter the body intranasally (koyuncu et al., 2013) . olfactory nerve has the particularity to be in communication with the nasal epithelium and also with the olfactory bulb, the gateway to the cns (koyuncu et al., 2013; swanson and mcgavern, 2015a) . this route is commonly used by respiratory viruses that infect the cns but is not the only one, as it will be discussed later in this review. the hrsv is an enveloped, negative-sense singled stranded rna virus, which belongs to the mononegavirales order and has recently been assigned to the pneumoviridae family and the orthopneumovirus genus (afonso et al., 2016; king et al., 2018) . accordingly, this virus has also been recently renamed human orthopneumovirus, but for the purpose of these publication we will refer to it as hrsv. the main and most studied pathologies caused by hrsv are bronchiolitis and pneumonia (antonucci et al., 2010) . however, in the past years, extrapulmonary manifestations have been associated with this virus (eisenhut, 2006) . notably, there is evidence that relates hrsv infection with pathologies such as myocarditis (esposito et al., 2010) , hyponatremia (hanna et al., 2003) , hepatitis (kirin et al., 2013) and encephalopathy (ng et al., 2001) . in wallace and zealley (1970) , in a study performed in children with a febrile status, hrsv was detected, and its infection was related to neurological damage. later, cappel et al. (1975) detected viral antibodies in cerebrospinal fluids (csf) of patients that have suffered symptoms of cns infection such as seizures, convulsions and neck stiffness (figure 1) . one of the most significant findings from this report was that hrsv infection was associated with neurological abnormalities such as encephalitis (cappel et al., 1975) . a few years later, a case report of three preterm infants which were hospitalized by hrsv-induced bronchiolitis, also presented neurological abnormalities (morton et al., 1981) . despite these finding, a few years passed until, in hirayama et al. (1999) reported the case of a 3-year-old child that was hrsv-positive with clinical signs of ataxia. in the csf, a high number of leucocytes was found; however, they could not detect hrsv by polymerase chain reaction (pcr). the authors concluded that this child manifested a meningoencephalitis with cerebellitis associated with hrsv-infection (hirayama et al., 1999) . , ng et al. (2001 , performed a retrospective study where clinical data of 487 patients with bronchiolitis by hrsv infection were evaluated. the results of this analysis showed that 1.8% of the children exhibited visible clinical signs of encephalopathy, particularly seizures. another retrospective investigation that evaluated 226 patients detected that 121 were hrsv-positive and 115 hsv-negative. in the hrsv-positive cohort, about a 6.6% presented seizures; however, this number was similar for the one reported in the hrsv-negative cohort (kho et al., 2004) . in addition to this, it was found that 19.8% of the patients exhibited apnea, but no differences were found when compared with the hrsv-negative cohort (kho et al., 2004) . importantly, these data support the idea that it is relevant to analyze other symptoms associated with hrsv bronchiolitis carefully. the first detection of hrsv rna in csf was from a 4-month-old boy hospitalized by pneumonia and febrile convulsion (zlateva and van ranst, 2004 ; figure 1 ). in this study, the authors were able to identify that the hrsv strain found belonged to the serogroup b (zlateva and van ranst, 2004) . to achieve a better understanding of the effects of hrsv-infection in the cns, the csf of an 11-month-old boy that exhibited neurological abnormalities were analyzed, to evaluate the contribution of cytokines in this phenomenon (otake et al., 2007) . the results showed an increase of il-6 in the csf but not in serum, which suggests a local effect, implicating that cns cells-such as astrocytes and microglia-can be the source of these cytokines (otake et al., 2007; figure 1 ). moreover, an increase of il-6 was also found in three cases of infants younger than 2-years-old in which it was possible to detect hrsv rna-serogroup a-in csf ; figure 1 ). the authors suggest that their result support the idea of a direct invasion of the cns by hrsv . interestingly, the same group found in another cohort of children infected with hrsv the presence of viral rna, which correlated with low levels of tnf-α in csf (kawashima et al., 2012) . also, most of the patients showed an increase in the production of several chemokines such an il-8, ccl2 and ccl4 which may play an essential role in this disease (kawashima et al., 2012 ; figure 1 ). the encephalopathies caused by hrsv are classified in four groups: (1) metabolic error type; (2) cytokine storm type; (3) excitotoxicity type; and (4) hypoxic encephalopathies (morichi et al., 2011) . the encephalopathy caused by metabolic error is an abnormality of the brain function that can be reversible and involves an alteration of metabolites. remarkably, it was found in one of nine patients in this study (morichi et al., 2011) . in the second type of encephalitis, a high increase of several cytokines at systemic levels-which also affect other organs-can be detected. these were reported in only one of the patients (morichi et al., 2011) . in five of the total of patients, excitotoxic encephalopathy was found, which is characterized by febrile convulsion status epilepticus (mizuguchi et al., 2007; morichi et al., 2011) . two patients manifested encephalopathies associated with hypoxia, which is a condition that does not include any sign of the others classifications (morichi et al., 2011) . importantly, hrsv rna was found in csf in five of the nine patients analyzed, and the levels of il-6 were increased only in the patients who exhibited excitotoxic or cytokine storm encephalopathy type (morichi et al., 2011 ; figure 1) . moreover, in all the patients the levels of nitric oxide (no) were significantly increased independently of the encephalopathy type (morichi et al., 2011) . these results are consistent with the previous report of these authors where they described the finding of hrsv rna in five of eight patients and also elevated no levels when compared to influenzaassociated encephalopathies (morichi et al., 2009 ). despite the low frequency of neurological complications associated with hrsv-infection, the cases reported exhibit similar profiles, which considers elevated levels of il-6 in csf (miyamoto et al., 2013) . related to this, morichi et al. (2017) examined molecular markers in csf as a prognostic indicator of encephalopathy severity -which includes no, brain-derived neurotrophic factor (bdnf) and il-6 ( figure 1) . the analysis of these molecular markers was categorized by the encephalopathy type described earlier, in addition to the non-evaluated encephalopathy type (morichi et al., 2017) . although the authors analyzed a low number of cases, they found that in two patients with cytokine storm encephalopathy, il-6 and bdnf were significantly elevated, when compared to the control group (morichi et al., 2017) . moreover, in four patients with non-evaluated encephalopathy, the levels of no were significantly higher than in the control group (morichi et al., 2017) . the pediatric cerebral performance category scale (pcpc) score was used in order to correlate these results to the neurologic prognosis. only il-6 and bdnf correlated with pcpc scores, indicating that in more damaged patients, the secretion of these molecules is elevated (morichi et al., 2017) . these tools are nowadays an import advantage in the understanding of the neuropathies associated with hrsv-infection and help to prevent that severe cases lead to death, as was described recently (xu et al., 2018) . nowadays, the mechanisms involved in neurological complications due to hrsv-infection remains unknown. decades ago, researchers adapted the hrsv long to the brain of newborn mice to study the pathogenesis of this virus in a mice model (cavallaro and maassab, 1966; cavallaro et al., 1967) . these authors inoculated the virus intracranially several times and reported that animals exhibited clinical signs of lethargy, ataxia and tremors between the 3rd and 5th-day post inoculation (cavallaro et al., 1967) . interestingly, the authors also observed that in a few cases, mice manifested convulsions spontaneously and also died after one or 3 days (cavallaro et al., 1967) . in addition to this, histological analyses showed an association between the extensive necrosis and the liquefaction in the brain with the clinical signs of encephalitis in the mice (cavallaro et al., 1967) . by intracranial inoculation, the authors described that hrsv was not found in others organs and that mice did not exhibit a pulmonary disease (cavallaro et al., 1967) . during years, there was no report about the relationship between hrsv-infections and cns pathologies. a few years ago, li et al. (2006) described, in a study that sought to assess the persistence of infection, the ability of the virus to infect sensory neurons that innervate the lung. these authors hypothesized that hrsv infects not only pulmonary neurons but also that the g-hrsv glycoprotein can interact with the chemokine receptor for cx3cl1 (cx3cr1) expressed in these cells (li et al., 2006) . according to this, they also studied the ability of hrsv to infect primary cortical neuronal cultures and observed, by immunofluorescence, co-localization of n-hrsv protein with neuronal markers. remarkably, this was not observed when the cx3cr1 was blockade. these results suggest that hrsv can infect neurons in vitro at a low percentage (5%) and that it can also infect sensory neurons of the lungs, as reported in culture (li et al., 2006) . this work highlights the fact that hrsv can invade the cns and infects resident cells which may explain how this virus can cause neurological abnormalities in patients. to give more insights about this phenomenon, espinoza et al. (2013) evaluated the neuro-invasive ability of hrsv in a mice model performing an intranasal inoculation that differs in the methodology used in the 60s. importantly, the authors observed that viral genome and proteins could be detected in the brain of the infected mice, mainly in cortex, hippocampus and ventromedial hypothalamic nucleus (vmh) at 3 days post-infection (espinoza et al., 2013) . later they evaluated a possible route of entry for hrsv into the brain-the trojan horse mechanism-by using a blocking antibody for cd49d, which is expressed by leukocytes and is required for transendothelial transmigration of this cells (espinoza et al., 2013 ; figure 1 ). the results obtained showed a decrease of viral load in the brain of hrsv-infected mice previously treated with anti-cd49d, suggesting that this can be the entry route used by hrsv (espinoza et al., 2013) . interestingly, as hrsv proteins were found in the hippocampus-a central zone where the cognitive and behavioral process takes place-alterations in the normal function were evaluated. both hrsv-infected mice and rats were used for the evaluation of behavior and spatial learning, respectively (espinoza et al., 2013) . marble burying test was used to evaluate the mechanical digging behavior in rodents, and the data showed that a month after hrsv-infection, these mice exhibited an impairment in this behavior (espinoza et al., 2013) . moreover, hrsv-infected rats were submitted to the morris water maze test-which evaluates spatial learning-a month after the infection. the data shows that hrsv-infected rats exhibited a delay in their learning capacities, when compared to the control group (espinoza et al., 2013) . considering these results, it is suggested that hrsv-infection causes behavioral and cognitive sequelae, that have not been described yet in patients. recently, an in vitro study using neuronal n2a cells as hrsv-infection model-which are a neuroblastoma cell line that can differentiate into cells that possess neuronal characteristic-was performed (yuan et al., 2018) . the data presented by the authors indicated that this virus infects these cells and that viral titers increased up until 96 h post-infection, suggesting that hrsv replicates in this cell line (yuan et al., 2018 ; figure 1) . additionally, they evaluated if toll-like receptor 4 (tlr4) and nucleolin (c23) are able to recognize the f-hrsv protein in n2a cells, as was reported in the literature. using confocal microscopy, they found that this interaction also occurs in the hrsv-infected cells (yuan et al., 2018) . according to this, they also observed that hrsv-infection increases the protein levels of tlr4 and c23 in n2a cells (yuan et al., 2018) . to evaluate the contribution of infected neurons, in encephalopathies associated with hrsv-infection, the secretion of pro-inflammatory cytokines in the supernatant of n2a-hrsv infected cells was assessed by elisa. the data obtained showed an increase of il-6 and tnf-α in n2a hrsv-infected cells when compared to the control cells (yuan et al., 2018) . despite this new knowledge, there is no in vivo evidence that shows whether hrsv infects neurons or other resident cells. more research in this field is required to achieve a better understanding of the mechanism involved in the cns disease induced by hrsvinfection. iv is the etiological viral agent most relevant in respiratory tract infections. the influenza a (iav), b (ibv), c (icv) and d (idv) viruses belong to the orthomyxoviridae family and are the only members of their respective genus, within the unassigned order (bouvier and palese, 2008; resa-infante et al., 2010; su et al., 2017; king et al., 2018) . these viruses are enveloped, negative-sense, segmented-stranded rna and the subtypes of iav are determined by two structural proteins, hemagglutinin (ha) and neuraminidase (na; louten, 2016; su et al., 2017) . there are 18 different ha subtypes (h1-h18) described and at least 11 subtypes of na (n1-n11; louten, 2016) . based on this, any combination of ha and na proteins could be found, being relevant in human diseases the h1, h2 and h3 which are transmitted between individuals (louten, 2016) . in addition to this, when iav from animals infects naïve human individuals, antigenic shift-a process in which the re-assortment of genes segments from two subtypes of virus-takes place, sometimes leading to iv epidemics, such as the recent h5n1 epidemic (jang et al., 2009; louten, 2016; su et al., 2017) . the circulating iav that are more risk to human health are h1n1, h1n2, h2n2 and h3n2, along with ibv (louten, 2016; skowronski et al., 2018) . associations to respiratory pathologies in iv infections have been described, with neurological complications in both children and adults (goenka et al., 2014; popescu et al., 2017; paksu et al., 2018) . accordingly, clinical signs that have been observed in patients includes encephalitis (newland et al., 2003) , myelitis (salonen et al., 1997; zlateva and van ranst, 2004; xia et al., 2014; ruisanchez-nieva et al., 2017) , meningitis (liang et al., 2018) , seizures (chiu et al., 2001) and guillain-barre syndrome (sivadon-tardy et al., 2009) . one of the first reports related to the pandemic h1n1 infection in 1918. therein, the authors describe that the main neurological symptoms were detected in the nerve centers and-time after the infection-manifestations such as depression and neuritis appeared (turner, 1919) . considering this background, years later a study of h2n2 (asian influenza) showed that neurological complications incidence increased, when compared to h1n1 pandemics and remarkably iav was isolated post-mortem from the brain of a patient (kapila et al., 1958) . a posterior retrospective study revealed neuromuscular manifestations in 19% of the patients, with a wide range of clinical signs spectrum (paisley et al., 1978) . moreover, iv was detected in the csf of one patient from this study (paisley et al., 1978) . years ago, the detection of iv in csf from patients with neurological manifestations was rare. nowadays, there is more and more evidence of the ability of iv to develop neurological damage. in 2009 a new h1n1 pandemic was the causative agent of high mortality rates and exhibited increased reports of neurological complications. according to this, a retrospective study of the clinical files of 55 patients infected with h1n1 detected a 50% of visible neurological symptoms (asadi-pooya et al., 2011) . in this cohort, the most frequent neurological sign reported was headache −35% of the patients-and a few were diagnosed with severe neurological complication −9% of the patients (asadi-pooya et al., 2011) . another study performed in malaysia-that collected clinical data from pediatric hospitals during the 2009 pandemic-reported that 8.3% of children under 5 years old presented neurological manifestation, among which the 66.9% of them manifested febrile seizures (muhammad ismail et al., 2015) . importantly, 13.6% and 3.9% of children exhibited influenza-associated encephalitis and acute necrotizing encephalopathy (ane), respectively (muhammad ismail et al., 2015) . however, there was no detection of iav genetic material in the four csf samples available, whereas brainimaging showed that a few patients exhibited alterations such as cerebral edema and ane, besides the three cases where the neurological sequelae were permanent (muhammad ismail et al., 2015) . according to this information, landau et al. (2011) described that in a cohort of 74 hospitalized children, 19% of them presented neurologic complications mainly seizures. only one patient from this study was diagnosed with transverse myelitis and presented permanent sequelae (landau et al., 2011) . in addition to this, a fatal case attributed to the h1n1 pandemic infection was reported, and the clinical finding showed that the cause of death was an intracerebral thrombosis and hemorrhage with presence of the virus in the brain, but not in lungs or csf (simon et al., 2013 ; figure 2) . the knowledge not only came from epidemic iav, as these neurological signs also have been described for seasonal iav. the h3n2 and h1n1 seasonal iav have also been associated with neurological manifestations. according to this, a study described in 21 patients of a wide range of age with neurological alterations showed that the primary clinical sign was encephalitis and about 50% of the patients have sequelae (steininger et al., 2003) . moreover, although in this study the detection methodology for iv genetic material detection in csf was improved, only one sample was positive (steininger et al., 2003; figure 2 ). in another approximation to understand the etiologic agent causing myelopathy post-influenza-like syndrome, csf obtained from a patient with this disease was inoculated in several cell lines, previously reported to be permissive for the growth figure 2 | influenza virus (iv) spreads from the lungs to the cns through the vagus nerve promoting an inflammatory state. upon infection of iv, the virus reaches the lungs and, from there, it can spread into the cns by transneural route, through the vagus nerve. once set on the brain, it induces the secretion of several pro-inflammatory cytokines such as il-1, il-6, il-8 and g-csf. viral rna has been detected in csf of infected patients, and also microglial apoptosis has been described. of this virus (paiva et al., 2013) . importantly, seasonal iav h3n2 was detected in mdck cells, identified as the cause of the neurological symptoms of the patient (paiva et al., 2013) . besides the main symptoms reported for seasonal h1n1, h3n2 and ibv, altered state of consciousness are consistently detected with seizures, in patients infected with pandemics iav subtypes (newland et al., 2003; popescu et al., 2017; paksu et al., 2018) . both in this and in others studies, it has been difficult to correlate clinical signs such as csf pleocytosis with neurological manifestation; mainly due to the low number of patients that exhibit pleocytosis (paksu et al., 2018) . however, the presence of neuroradiological diagnosis suggests that the phenotype observed in patients may be a cause of direct viral invasion into the cns (paiva et al., 2013; paksu et al., 2018) . besides the neurological signs described above, a study in japan reveals that patients with neurological manifestations also had mild impairment of consciousness, typically delirium or hallucinations and abnormal behavior among others, which belongs to the neuropsychiatric disorders (manjunatha et al., 2011; mizuguchi, 2013) . accordingly, there is still necessary to perform more studies about the incidence of these neuropsychiatric manifestations, besides the direct evidence of this association with iv infection. highly pathogenic strains of iav have been used as a model to test the mechanisms involved in the cns abnormalities caused by iv-infection in humans. using an h5n3 iav that initially originated from a water bird and was adapted into chickens to increase its virulence, shinya et al. (1998) inoculated mice intranasally and evaluate its neurovirulence. the histological data collected exhibited that iav caused non-suppurative encephalitis. remarkably, they could also recover iav from the brain until day 7 post-infection (shinya et al., 1998) . years later, this group performed experiments using the same virus to elucidate the route used for entry into the cns. mice were infected intranasally or intravenously, and only the first group exhibited bronchitis and viral detection in the mucosal epithelium of the trachea to the bronchiole (shinya et al., 2000) . later, when they evaluated the brain histology, they observed that in addition to the non-suppurative encephalitis, there was an infiltration of macrophages and lymphocytes (shinya et al., 2000) . finally, the most critical finding was that viral antigens were detected in the vagal and trigeminal ganglia at day 3 post-infection (figure 2) . this event was preceded by the early infection of the nasal cavity, trachea and lungs (shinya et al., 2000) . additionally, the hypothesis of the transneural invasion was corroborated in a study performed by matsuda et al. (2004) , which showed that iav reaches cns mainly via the vagus nerve (figure 2) . moreover, through an in vitro assay-utilizing neuron cultures in a compartmentalized system-authors suggested that the mechanism of the neurotropic h5n3 to reach cns is a retrograde axonal transport (matsuda et al., 2005) . another research using the h5n1 iav (hong kong/483/97) also found that both rna and viral antigens were detected, first in the vagal and trigeminal ganglia, then later in the brainstem (park et al., 2002) . the next step in the research was to evaluate in more detail the effects of iv in the brain of challenged mice. in this context, jang et al. (2009 jang et al. ( , 2012 observed that mice challenged intranasally with h5n1(vietnam/1203/04), viral detection in cns was positive at 3 days post-infection and that the virus can infect neurons and microglia but not astrocytes (figure 2) . in addition to this, they report that h5n1 infection promotes microglial apoptosis, inducing an inflammatory state, which lasts up to 90 days post-infection, similarly to the idiopathic parkinson's disease in human (jang et al., 2009) . moreover, the authors show a loss in dopaminergic neurons in about a 17%, that began as a local immune response that could contribute to cns disease as is described in humans (jang et al., 2009) . later research demonstrated that the recovery of the neuronal lasted until 90 days post-infection and that, mainly in the substantia nigra pars compacta (snpc), the profile of cytokines was altered due to the h5n1 infection (jang et al., 2012) . interestingly, il-13 showed an early induction in the acute phase of the viral infection and then decreased rapidly, to eventually increase after 60 days post-infection (jang et al., 2012) . moreover, gm-csf, another cytokine, was not detected in the acute infection, but increased at the same time as il-13 increased, whereas cytokines such an il-1, il-6 and g-csf, among others, were only detected until 21 days post-infection (jang et al., 2012 ; figure 2 ). all the data suggest that the local immune, response mediated mainly by microglia, promotes neurons death and protein aggregation, inducing the development of neurodegenerative diseases (jang et al., 2012) . this phenomenon was also observed for h1n1 (ca/09), which promotes the microglial activation mainly in the snpc and the hippocampal dentate gyrus, however, this virus is not neurotropic (sadasivan et al., 2015) . additionally, this virus is not able to induce the disruption of bbb which is consistent with the absence of immune cells infiltration into the cns (sadasivan et al., 2015) . the iv infection has also been related to neuropsychiatric disorders. yu et al. (2014) used the neonatal model to evaluate whether iv infection might cause alterations in normal brain functions. unlike other studies described above, in this one, iv was administered intraperitoneal, as the primary focus of the research was to evaluate the systemic spread of the mouseadapted h1n1 (nws/33; yu et al., 2014) . the results showed viral detection in the hippocampus, cerebellum and cerebral cortex among other zones. in addition to this, in infected brain zones, neurons and astrocytes underwent apoptosis, which is consistent with neuroinflammation accompanied by gliosis (yu et al., 2014) . moreover, viral rna was detected in csf from adult mice, but this does not discard the possibility that in neonates, this also occurs and that iav furthermore invades the cns by crossing the blood-csf barrier (yu et al., 2014) . according to these findings, specifically the detection of the viral rna in the hippocampus, hosseini et al. (2018) recently evaluated three different mouse-adapted iavs: two non-neurotropic virus h1n1 (pr8) and h3n2 (mahk68); one neurotropic virus h7n7 (rsc35m). as they expected, no viral particles were found in the brain of h1n1-infected mice, although a few amounts of viral particles were found in h3n2-infected mice and viral detection was evident in several brain zones in h7n7-infected mice (hosseini et al., 2018) . therefore, no signs of pathological changes were detected in the brain of h1n1-and h3n2-infected mice, but in the h7n7-infected mice, there was a moderate immune cell infiltration and zones with gliosis (hosseini et al., 2018) . interestingly, when they evaluated the effects of iav infections in the behavior, no virus has affected the mice anxiety or locomotor activity whereas at 30 and 120 days postinfection, however, h3n2 and h7n7-infected mice showed an impairment of spatial learning and memory (hosseini et al., 2018) . this work proves that, unlike what is reported by jurgens et al. (2012) -which perform cognitive test and neuron morphology experiments during the acute phase of the infection-the h1n1 infection does not lead to long-term impairment in spatial memory nor affects the neuron morphology (hosseini et al., 2018) . the h3n2 subtype is not able to replicate in the cns; however, it is capable of increasing the levels of tnf-α in the hippocampus and also increase the number of microglia (hosseini et al., 2018 ; figure 2 ). on the other hand, h7n7 not only increases the level of ifn-γ and tnf-α in the cns but also alters the long-term potentiation (ltp) and disrupts the permeability of the bbb, promoting a stronger inflammatory immune response than h3n2 (hosseini et al., 2018) . based on all these data, the most important conclusion is that it is necessary to know the immune response promoted by ivs, as it has been proven that long-term alteration can be caused without cns viral replication. importantly, all the knowledge that we have today about iv infection allow us to be more prepared to diagnose and treat more efficiently patients with this infection. cov is a group of viruses that belong to the coronaviridae family and the nidovirales order. accordingly, there are four genera of cov within the coronavirinae subfamily: alphacov (acov), betacov (bcov), deltacov (dcov) and gammacov (gcov; king et al., 2012 king et al., , 2018 . their name proceeds from their characteristic crown-shape and is responsible for a wide range of respiratory and enteric diseases in several hosts, such as rodents, cats, pigs and humans (desforges et al., 2014b) . there are several human cov (hcov) described as pathogenic in humans, among which are included hcov-oc43, hcov-229e, middle east respiratory syndrome cov (mers-cov) and severe acute respiratory syndrome cov (sars-cov), all of them with their respective different genotypes (gaunt et al., 2010; cabeça et al., 2013; matoba et al., 2015) . remarkably, neurotropic and neuro-invasive capabilities have been described in several of their hosts, including humans among them, leading to symptoms such as multiple sclerosis (ms) and encephalomyelitis (lau et al., 2004; yeh et al., 2004; zlateva and van ranst, 2004; talbot and falsey, 2010) . however, the capacity of cov to infect cns in humans is not well characterized, with their detection in these samples performed mainly by detection of viral rna, exhibiting persistent infection (arbour et al., 2000; desforges et al., 2014a) . covs are enveloped viruses with a positive non-segmented single-stranded rna genome of about 30 kb of length, one of the largest among the rna viruses. they codify for four structural proteins-five in the case of some bcov-and several non-structural proteins comprised mainly on two orfs (orf1a and orf1b) that will eventually be cleaved into 15 or 16 proteins (desforges et al., 2014b) . it has been described that non-structural proteins are the leading cause of host immune system modulation and they also play a role in the replication of the genetic material of the virus (gorbalenya et al., 2006) . as described in mice, viral entry is mediated through the interaction of viral spike (s) protein and cellular cecam-1 receptor, along with other co-receptors (williams et al., 1990; bergmann et al., 2006) . from there, the virus can replicate its rna and translate it into proteins. among the cells that are permissive to mhv infection are macrophages, microglia and astrocytes (bergmann et al., 2006; jacomy et al., 2006) . remarkably, the year 2006 st-jean et al. (2006 described the recovery of an infectious hcov-oc43 with neurovirulent capacities from a full-length cdna clone inserted in a bac, with the same phenotype as a wt virus, generating an interesting methodological approach for the study of this virus. despite hcov capacities to infect cns, it has been recently characterized, its presence in human cns-related samples date back as early as 1980, where the first detection of this virus was performed in autopsy of patients with ms (burks et al., 1980) . following that, a few reports confirming the presence of this virus in samples from patients with ms was confirmed through several methods (murray et al., 1992; stewart et al., 1992) . the year 2000, through research in an autopsy samples from patients with various neurological diseases (being ms most prevalent among them). showed that a 67% were positive for hcov (with hcov-229e being twice as common as hcov-oc43; arbour et al., 2000) . moreover, the prevalence of oc43 in ms samples was statistically higher than in control patients, is the first report to provide a significant indication of the neurotropic capacity of these respiratory pathogens (arbour et al., 2000) . the first case of sars-cov infection with neurological manifestations was reported the year 2003 in a 59-year-old woman (hung et al., 2003) . she was first admitted with swinging fever, chills, productive coughing and diarrhea, which eventually lead to oxygen requirements, vomit, seizures and episodes of four-limb twitching. the respiratory failure continued until she was sedated, and ventilation was required (hung et al., 2003) . sars-cov infection was confirmed in both tracheal aspirates and csf samples, followed by ribavirin treatment, with no improvement in seizures persistence. with additional treatments, seizures were no longer detected, and she was discharged 3 weeks after admission (hung et al., 2003) . the following year, another case of sars-cov infection with detection of genetic material in csf samples was reported in a 32-year-old woman (lau et al., 2004 ; figure 3) . detection was also positive for stool specimens and peritoneal fluids. figure 3 | human coronavirus (hcov) enters the cns through the olfactory bulb, causing inflammation and demyelination. upon nasal infection, hcov can reach the cns through the olfactory bulb, as ablation of this part of the brain restricts its neurotropic capacities in mice. once the infection is set, the virus can reach the whole brain and csf in less than 7 days. accordingly, it has been described that this virus can induce demyelination. likewise, primary glial cultures have been described to secrete il-6, il-12p40, il-15, tnf-α, cxcl9 and cxcl10 upon viral infection. the patient was admitted in week 26 of pregnancy and at 7 days post-admission, mechanical ventilation was required. at day 8, she presents a sign of acute renal failure, and pregnancy termination was decided. through cesarean, a baby girl was born without further complications (lau et al., 2004) . at day 22 the patient was still sedated and on mechanical ventilation, with convulsions and loss of consciousness. starting at day 27 she was extubated and from there followed an uneventful recovery. she exhibited no further convulsions and no evident sequelae (lau et al., 2004) . organ dissemination of sars-cov in autopsy samples from patients that died of this disease was determined. the report indicates the presence of sars-cov-n protein and viral rna in the stomach, small intestine, kidney, sweat glands, parathyroid, pituitary gland, liver and cerebrum, further confirming the capacity of this virus to induce a systemic infection (ding et al., 2004) . a case report of hcov-oc43 detection in nasopharyngeal and csf samples from a child patient was performed the year 2004 (yeh et al., 2004) . the child exhibited acute disseminated encephalomyelitis, a low-prevalence cns disease that induces demyelination, being this the first case related with hcov. no other infectious agents were detected in any of the samples (yeh et al., 2004) . following this, a brief characterization of the cytokine profile in the cns, induced by sars-covinfection, was published. therein, the authors indicated that both chemokine induced by ifn-γ (cxcl9, a cxc chemokines family member) and ifn-γ-inducible protein 10 (cxcl10) were highly induced in brain samples from a deceased patient (xu et al., 2005 ; figure 3) . however, there are reports about high levels of cxcl10 in sars-cov infected patients, with no neurological manifestations. therefore the authors suggest that cxcl9 could be closely related to cns infection (xu et al., 2005) . remarkably, they also showed there that peripheral blood lymphocytes and eosinophils counts in cns-hcov-infected patients were lower when compared with respiratory-hcov-infected patients, while the opposite trend was observed for neutrophils (li et al., 2016) . these differences in the recruitment of immune cells could be related to the immune response elicited by the virus, either it is respiratory-restricted or exhibits neurotropism capabilities (li et al., 2016) . hcov capacity to reach cns after the nasal infection has been described previously in mice, particularly for hcov-oc43 (st-jean et al., 2004) . st-jean et al. (2004) reported that upon infection, viral antigens are detected in the olfactory bulb 3 days later, with no presence of virus in perivascular blood cells or any other part of the brain. after 7 days, the virus is detected throughout the whole brain tissue, indicating that it can rapidly propagate once set in cns. this replication leads to a rapid death by acute encephalitis of infected mice. remarkably, ablation of the olfactory bulb prevented the spread of mouse hepatitis virus (mhv), upon nasal infection (perlman et al., 1990) . therefore, hcov exhibits an intrinsic capability to infect neural cells and spread from cns to the periphery via a transneural route, as has also been seen for mhv (perlman et al., 1990; barthold et al., 1990 ; figure 3) . mice studies are mainly performed with mhv, a virus that belongs to the bcov genus and is genetically related to hcov-oc43; likewise, the disease at cns as elucidated by both viruses are similar, as they both induce demyelination (jacomy and talbot, 2003; bergmann et al., 2006 ; figure 3 ). jacomy and talbot (2003) were among the first to describe a mouse model to characterize the cns disease in their publication the year 2003. therein, they exhibit that balb/c and c57bl/6 mice could be infected through nasal instillation with mhv, although they chose to use intracerebral inoculation to favor cns infection (jacomy and talbot, 2003) . they also determined that viral rna could be detected in brain, heart, spleen, lungs, liver and muscles (jacomy and talbot, 2003) . likewise, the year 2004 glass et al. (2004) described a systemic non-lethal model of infection for sars-cov in c57bl/6 mice that eventually reached the brain. finally, in jacomy et al. (2006) described that hcov-oc43 could infect glial and neuronal cells of both rat and mice (figure 3) . therein, they also showed that surviving animals exhibited decreased motor functions. recently, wheeler et al. (2018) described that microglia is essential for the regulation of mhv infection, as depletion of this cell type led to faster viral replication, enhancing its capacity to avoid adaptive immunity. according to this, glial primary cultures of mhv-a59-infected cells showed an increase in the secretion of il-12 p40, tnf-α, il-15 and il-6 compared with a non-neurotropic mhv, suggesting that the infection with a neurotropic virus activates glial cells and induces a pro-inflammatory state (li et al., 2004 ; figure 3) . as described so far, covs are respiratory viruses that exhibit neurotropic capacities that not only allows them to achieve latency and avoid the immune response of the host, but also have neurological implications that can complicate the disease associated to its infection. although their mechanisms and routes to reach the cns have not been elicited yet, the detection of either viral proteins or genetic material in this issue has been confirmed thoroughly, branching the researcher's goals into acquiring new insights regarding this topic. so far, epidemiological reports have allowed to achieve this, but further work in animal models is required to fully comprehend the mechanisms that cov uses to reach cns and to achieve more suitable treatments to resolve this viral infection without an exacerbated disease. the hmpv is a new virus first reported the year 2001 in netherlands (van den hoogen et al., 2001) , and since its discovery, several epidemiological reports have placed it among the most prevalent respiratory viruses worldwide, although its disease burden has not been thoroughly characterized (hamelin et al., 2006; edwards et al., 2013) . it is responsible for respiratory illness mainly in newborns, infants and immunocompromised patients, although it can also infect healthy adults, with mild symptoms (edwards et al., 2013) . its genome is negative sense and about 13 kb length, with nine structural proteins codified on it (van den hoogen et al., 2002; schildgen et al., 2011) . hmpv belongs to the mononegavirales order, pneumoviridae family and the metapneumovirus genus (amarasinghe et al., 2018; king et al., 2018) . since it is closely related to hrsv, both in its classification and its genome, their diagnosis has been usually mistaken (edwards et al., 2013) . currently, there are no effective treatments against this virus, neither vaccines nor specifics treatments, mainly due to the lack of thorough knowledge associated with its immune response and disease pathogenesis (zlateva and van ranst, 2004; schildgen et al., 2011) . in humans, there is a handful of reports associated with encephalitis and hmpv-infections. the first approach to this topic was a report described the year 2003 in china (peiris et al., 2003) . therein, the authors describe several cases of children with acute respiratory diseases, particularly 587 patients, of which 32 were positive for hmpv rna detection (peiris et al., 2003) . although the scope of this report was not to associate hmpv with any cns abnormality, they do report five cases of children with a febrile seizure, reaching levels comparable to the ones seen in influenza, the respiratory viruses most prone to induce seizures, as they state (peiris et al., 2003) . following this, the first case in which the presence of hmpv rna in brain samples of a patient with encephalitis was reported, was the year 2005 in germany (schildgen et al., 2005) . this report described the case of a 14-month-old boy that was received in a primary care hospital with a high fever and unresponsiveness to stimulus, either verbal or tactile. previous to its internalization, during the same day he reported seizures and no spontaneous eye movement (schildgen et al., 2005) . after 10 days of hospitalization without further improvement, the child was considered to be dead and was extubated. autopsies revealed the presence of genetic material of hmpv in both brain and lungs, and no other virus, such as hrsv or hsv, were detected (schildgen et al., 2005) . concomitantly the same year, a case of encephalitis where hmpv rna was detected in nasal mucus and tracheal aspirates was reported (kaida et al., 2006) . from this point on, several cases were described where hmpv-infection was related to encephalitis, and in some cases, the detection of genetic material in different cns samples was detected. the year 2007, the death of a 6-month-old girl was reported 9 days after her admission to a healthcare unit (hata et al., 2007) . she exhibited generalized convulsions and was diagnosed with acute encephalopathy the day she was admitted, and 24 h later she fell into a coma. although there was no detection of genetic material in csf, the presence of hmpv-f protein was confirmed by rt-pcr in throat swab and urine (hata et al., 2007) . two years later, arnold et al. (2009) described in an epidemiological report the presence of nine cases of hmpv-infection related to different spectrums of cns abnormalities, distributed in two different study groups. in the first group, composed of 1474 patients, 76 samples were hmpv positive, and of those, four patients were reported with seizures. therein, they also expose data regarding hrsv, showing that 145 of the samples were positive for this virus and only one of the patients was reported with seizures. through statistical analysis, they can confirm that the frequency of hmpv patients with seizures is statistically significant, unlike the one has seen in hrsv. the second group consisted solely of patients hospitalized with high fever or any cns abnormality, ranging from the age of 6-month-old to 18 years old. in that group, they reported five patients with hmpv, where seizures were diagnosed in three (arnold et al., 2009) . they also described another patient with hmpv-infection and detection of genetic material of enterovirus in csf. remarkably, the presence of hmpv genetic material was not detected in the csf of any of the available samples. despite this, they indicate that a normal csf profile does not necessarily exclude a neurotropic mechanism, as seen for rabies encephalitis, in which only half of the csf samples are positive for csf pleocytosis (arnold et al., 2009) . likewise, the case reported above by schildgen et al. (2005) identified hmpv rna postmortem, despite normal csf cell count and a negative detection of the virus by pcr in spinal fluid (arnold et al., 2009 ). on the same line, the year 2012 the first report associated with the hmpv genetic material in csf was published (sánchez fernández et al., 2012 ). sánchez fernández et al. (2012 confirmed this detection through pcr-in a 10-year-old girl with signs of acute encephalitis-and then characterized the evolution of the disease and also included neuroimaging features therein. detection of other etiological agents, such as herpes virus and adenovirus, were negative. magnetic resonance imaging (mri) showed signs of acute encephalitis and demyelinating process mainly in the temporal and occipital lobes, but these lesions eventually spread to frontal and parietal lobes (sánchez fernández et al., 2012) . eventually, thanks to the treatment received, clinical improvement was noted and-35 days post hospitalization-she was discharged home, unlike many other cases, where these symptoms resulted in the death of the child (sánchez fernández et al., 2012) . however, some of the sequelae registered include inappropriate social abilities and infantile behavior, with severe attention-but no motor or memory-deficits (sánchez fernández et al., 2012) . more cases in which status epilepticus was reported-a single long-lasting seizure or several seizures in a specific time rangewere associated with hmpv. a 3.5-year-old girl with hmpv respiratory disease was reported the year 2013 (niizuma et al., 2014) . the year 2014, two cases were reported, a 15-month old and a 18-month-old girl, that reported hmpv infection and eventually developed respiratory failure (webster et al., 2014) . these three cases were discharged without apparent sequelae. the year 2015, the first reported case of a child with refractory status epilepticus-a non-responsive condition with a worse prognosis than commonly responsive status epilepticus-was described (vehapoglu et al., 2015) . the 4-month-old boy was received and eventually transported to pediatric icu. csf pcr analysis was negative for hsv, and nasal scraps were only positive for hmpv. as stated above, treatment with antiepileptic drugs resulted in unresponsiveness. eventually, seizures passed, and 25 days post-admission she was discharged. no evident sequelae were detected, though antiepileptic drugs were maintained for the following 6 months (vehapoglu et al., 2015) . most recently, cases of adults with acute encephalitis and hmpv detection have been described. the first report was published the year 2015 in australia, in a 47-year-old man (fok et al., 2015) . the man was found unconscious after 2 days of respiratory symptoms and immediately hospitalized. pcr testing of csf elicited no presence of classical cns pathogens (hsv, varicella zoster virus, enterovirus) and eventually nasopharyngeal aspirates were positive for hmpv and no other respiratory viruses, although at this point, not enough csf was left for further testing. no evident sequelae were detected during the following months (fok et al., 2015) . remarkably, lesions found in the mri analysis were similar to the ones described in the previously reported cases (schildgen et al., 2005; sánchez fernández et al., 2012) . then the year 2017, two reports-one associating hmpv respiratory infection, the other one indicating the presence of hmpv genetic material in csf-were published. early during that year, jeannet et al. (2017) described the case of a 61-year-old swiss man with influenza-like symptoms, which eventually suffered from a headache and seizures. mri was inconclusive, and pcr analysis was negative for csf samples, although nasopharyngeal swabs were positive for hmpv and no other respiratory virus (jeannet et al., 2017) . then, later that year, the case of a 32-year-old man was reported, although the case dated from 3 years before its publication. the man was admitted with unspecific low backache and fever, mri was normal, and no unusual symptoms were detected (tan and wee, 2017) . eventually, the patient de-saturated and respiratory failure was diagnosed, with posterior mechanical ventilation. bronchoalveolar lavages and csf pcrs were both positive for hmpv (tan and wee, 2017) . the patient was treated for 1 week with ribavirin (unspecific antiviral) and eventually respiratory symptoms subdued, although he exhibited reduced cognition and intermittent agitation as sequelae. he began rehabilitation, but there was not a significant improvement throughout the following 6 months (tan and wee, 2017) . remarkably, no studies have been performed describing the ability of hmpv to cause cns damage in mice. however, it has been described that hmpv can persist in the lung of mice after acute infection (alvarez et al., 2004; liu et al., 2009 ). this characteristic could be aiding the virus to eventually reach the cns, as the latency state is achieved through the infection of neuronal processes that innervate the lungs, as the genetic material of the virus can be detected in them (liu et al., 2009 ). moreover, as described by the authors, latent virus can be reactivated upon treatment with immunosuppressive drugs such as steroids. hmpv remains a viral agent that needs to be thoroughly characterized, as although most of its respiratory-related pathogenesis has been taken to spotlight, its capacity to achieve latency in cns cells is yet to be elicited. although there are extensive case reports that indicate neurological manifestations associated to hmpv-infection in humans, further studies are required in mice models to characterize this disease. considering that this virus was first described only 17 years ago, there is plenty of work to be done in order to fully comprehend the impact that this virus may be playing in cns-related pathologies. respiratory viruses are the leading cause of bronchiolitis and pneumonia throughout the world, affecting varying ranges of ages, but being more aggressive in children, elderly and immunocompromised individuals. most prominent respiratory viruses are hrsv, iv, cov and hmpv. remarkably, extrapulmonary symptoms have been described in these viruses, highlighting their capacity to cause neurological complications. febrile seizures, loss of consciousness, convulsion, ataxia, status epilepticus, encephalitis, myelitis, neuritis and ms are among the several extra-pulmonary symptoms that have been described. case reports of children, elderly and even adults exhibiting these symptoms have been described throughout the years for all of these viruses, bringing to spotlight the urgency to describe their neuroinvasive capacity. moreover, the detection of genetic material and even viral proteins in cns samples, such as csf or brain, is a recurrent fact described in several case reports. several ways to achieve cns have been described, including transneural and hematogenous pathways. however, the specific mechanisms responsible for dissemination of each of these viruses with neurotropic capacities into the cns, have not been thoroughly characterized yet. for instance, hrsv has been reported to reach cns via the hematogenous pathway, although other routes cannot be discarded entirely. iv can reach the brain via the transneural route, mainly by retrograde axonal transport, reaching first vagal and trigeminal nerves. likewise, cov has been described to reach the brain via olfactory bulb, spreading from this point onward into the cns and the periphery. since hmpv is an emerging virus, no studies have been performed regarding its capacity to reach cns, so further data for this virus is required. so far, several mice models have been established for every one of these viruses, allowing the acquisition of new data and the development of new insights of their neurotropic capacities and their neurological manifestations. however, further researches are still required, as many aspects of these cns pathologies remain unknown. these studies will add new approach edges to a topic that urgently needs to be characterized, as many children may currently be exhibiting these symptoms and are not being treated properly, as the respective viral infection may not be diagnosed. all authors listed have made substantial, direct and intellectual contribution to the work and approved it for publication. this work was supported by fondecyt grants n • 3180570 and n • 1150862 and the millennium institute on 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(1997) we want to thank trinidad celis for her help in the design of the figures. key: cord-324530-tac1unnp authors: andré, nicole m; cossic, brieuc; davies, emma; miller, andrew d; whittaker, gary r title: distinct mutation in the feline coronavirus spike protein cleavage activation site in a cat with feline infectious peritonitis-associated meningoencephalomyelitis date: 2019-06-26 journal: jfms open rep doi: 10.1177/2055116919856103 sha: doc_id: 324530 cord_uid: tac1unnp case summary: this report describes a cat with chronic, progressive, non-painful, non-lateralizing multifocal neurologic clinical signs associated with feline infectious peritonitis (fip). the cat initially presented as underweight, despite a good appetite, and a complete blood count showed non-regenerative anemia. three months later the cat was returned having developed ataxia and paraparesis, which then progressed over 2 months to tetraparesis, tail plegia, urinary and fecal incontinence, and titubation. histologic examination of the tissues with subsequent immunohistochemistry confirmed fip-associated meningoencephalomyelitis following necropsy. molecular analysis of the coronavirus spike protein within the tissues identified a specific, functionally relevant amino acid change (r793m), which was only identified in tissues associated with the central nervous system (ie, brain and spinal cord). relevance and novel information: this case report describes an early presentation of a cat with primarily neurologic fip, with molecular characterization of the virus within various tissues. feline infectious peritonitis (fip) is caused by feline coronavirus (fcov) and is widely considered to be one of the most significant infectious diseases to affect the feline population. [1] [2] [3] it is the most common infectious disease of the central nervous system (cns) of cats. 4 fcovs have been reported to exist as two distinct serotypes: type i (more common) and type ii viruses, 5 each with distinct biological properties. 5, 6 both fcov serotypes have distinct 'biotypes'. these are typically classified as either feline enteric coronavirus (fecv) or feline infectious peritonitis virus (fipv), with the biotypes differing based on the severity of infection in cats. [7] [8] [9] infection with fcov is common, especially in highdensity housing situations such as animal shelters and breeding facilities. 10 the fecv biotype transmits readily and causes only a mild infection, with transmission occurring via fecal-oral and possibly other routes. 7, 11 if the viral infection worsens and becomes systemic (typically infecting macrophages), then the virus is classified as the fipv biotype. 8 such viruses are believed to contain an 'internal mutation' that accounts for the altered tropism, although the nature of this mutation is not well understood. 12 clinical signs associated with the fipv biotype can be quite variable and non-specific, and can include fever, lethargy, anorexia, pica, vomiting and diarrhea. 13 these clinical signs can be present in either the 'wet', 'dry' or 'mixed' presentations. 14 the wet form of fip is characterized by an effusion in the abdominal and/or thoracic or pericardial cavities, and the 'dry' form by the presence of pyogranulomatous lesions. the 'mixed' form may present with an array of clinical signs. most commonly, neurologic clinical signs are associated with the 'dry' form but can occur with all presentations and may be the sole clinical sign observed. 12, 13 clinical features of neurologic fip can include, but are not limited to, ataxia, head tremors, seizures and/or paresis. 12, 13, 15 ocular lesions may be present with or without lesions in the cns. fip-associated pathologic changes to the cns include meningitis, encephalitis, ependymitis and choroid plexitis, often with concurrent vasculitis. 12,13 fip presenting with predominantly neurologic clinical signs provides a diagnostic challenge and definitive ante-mortem diagnosis is difficult. mri has been identified as a sensitive method of diagnosis in conjunction with clinical signs and cerebrospinal fluid analysis results such as elevated protein levels and neutrophilic pleocytosis. 16 however, such findings are still not specific to fip and may be financially prohibitive. fip may also be considered a diagnosis of exclusion, following evaluation of clinical signs, history and physical examination findings and biochemical values. 12, 13 this case report describes a cat with neurologic fip that progressed over several months. the observations and findings obtained in this case provide support that fip can present predominantly in the cns. when molecular techniques are applied to the virus, a propensity for certain mutations can be associated with specific clinical presentations or pathological changes. an intact female 8-week-old domestic shorthair cat was taken into a foster/rescue home and cohabited a house with approximately nine other cats. the facility had a periodic history of fip cases, including two deaths in the previous 4 months. the cat was co-housed in a large open sunroom containing seven litterboxes, which were cleaned once daily. the diet consisted of commercially available dry and canned food, which was separately offered in individual dishes. the cat was not rabies vaccinated, but had obtained two feline viral rhinotracheitis, calicivirus and panleukopenia vaccinations. at 14 weeks of age, the cat was presented to a general practitioner for evaluation of poor weight gain, soft stool and upper respiratory tract infection. the cat was underconditioned and weighed 2.5 lb (1.1 kg), with a body condition score (bcs) of 2/5, despite being active, alert and having a good appetite. conjunctivitis and a yellow mucopurulent discharge from the nares were noted, and the cat had a fever of 102.6°f (39.2°c). a fecal flotation was performed owing to the soft but formed stool, and no ova or parasites were detected. a complete blood count (cbc) and chemistry profile were performed (tables 1 and 2 ). the chemistry profile showed marked elevations in alkaline phosphatase, alanine transferase and phosphorus levels. a decrease in creatinine and albumin was also noted (table 1) , along with mild anemia and monocytosis. amoxicillin clavulanic acid (clavamox drops; zoetis) 62.5 mg/ml was dispensed and administered at 15.6 mg (12 mg/kg) po q12h for 10 days. blood parameters were re-evaluated at 20 weeks of age using a less defined panel and values were within the normal range (table 1) . at approximately 6 months of age, the cat returned to the general practitioner for evaluation of pelvic limb gait abnormalities that had progressed over the previous 2 weeks. examination revealed symmetric pulses in both hindlimbs and the presence of a pain response; however, less of a response was noted on the right side. paresis was observed in the right hindlimb. when the forelimbs were lifted, the cat was able to walk minimally on the hindlimbs. the cat had severe non-ambulatory paraparesis, with more severe deficits on the right side. no information about spinal reflexes was available. a cbc and chemistry panel were performed (tables 1 and 2 ). the chemistry panel revealed hypoalbuminemia, a decrease in the albumin:globulin (a:g) ratio, low creatinine values and hyperphosphatemia. the cbc revealed a slight anemia, monocytosis and thrombocytopenia. platelet clumping was noted upon microscopic evaluation. meloxicam (metacam oral suspension) was dispensed and a single 0.2 mg dose was administered orally. at 8 months of age, the cat was returned to the general practitioner due to progression of the paraparesis. the client noted further deterioration of the pelvic limb paresis, and now identified 'stiffness' in the thoracic limbs. there was no information about the pelvic limb reflexes; however, the cat had started to have occasional urinary and fecal incontinence. appetite seemed normal; however, the cat remained thin. physical examination revealed a temperature of 101.5°f (38.6°c), heart rate of 170 beats per min and respiratory rate of 30 breaths per min. bcs was 3/9; however, the weight was not noted. abdominal palpation revealed a large, easily expressible urinary bladder. neurologic examination findings revealed normal mentation with no involuntary movements such as tremors. the cat was still very ataxic and ambulatory but now tetraparetic, which was much worse in the pelvic limbs. there was no information about cranial nerve abnormalities, and the eyes and retinas were within normal limits. from a video provided by the owner (see supplementary material), tail paresis was identified. the lesion was considered to affect the cns and was localized as multifocal. a fecal flotation and direct smear were evaluated, with no ova or parasites seen. cryptococcus and toxoplasma antibody titers were performed and were negative. the tetra-ataxia and paresis significantly worsened over the next few months. additionally, the cat now had titubation, tail plegia (see video in the supplementary material) and consistent urinary and fecal incontinence. owing to the grave prognosis, the client elected humane euthanasia, at which time the cat was 10 months (40 weeks) of age. a necropsy was performed at the animal health diagnostic center, cornell university college of veterinary medicine, and this revealed no significant gross abnormalities outside of mild mesenteric lymphadenomegaly. representative sections of all organs, including the entire brain and spinal cord, were fixed in 10% neutral buffered formalin from which sections were cut, stained with hematoxylin and eosin, and analyzed via light microscopy. immunohistochemistry for fcov was carried out using monoclonal antibody fipv3-70 (1:1000), ap-anti-mouse igg and bond polymer refine red detection (leica microsystems). histologic examination revealed lesions typical of fcov infection within the cns. in the spinal cord, the leptomeninges were diffusely expanded by moderate numbers of predominantly plasma cells, admixed with fewer lymphocytes and macrophages, and surrounded by a moderate amount of edema. the underlying white matter was multifocally vacuolated with numerous dilated myelin sheaths, digestion chambers and rare spheroids (figure 1a) . at the level of the lateral aperture, the choroid plexus was expanded by large numbers of plasma cells, lymphocytes and macrophages (figure 1b) . the ependyma lining the ventricular system was effaced by a similar inflammatory population, admixed with fibrin, edema and was also forming thick perivascular cuffs often disrupting the sub-ependymal parenchyma (figure 1c) . immunohistochemistry revealed strong intracytoplasmic immunoreactivity within macrophages (figure 1d ). no fipassociated lesions were present in other organs. non-fcov comorbid histologic findings were chronic enteritis with mid-mucosal fibrosis and mesenteric lymphoid hyperplasia. molecular analysis of the viral spike protein was performed at several time points during the study. fecal samples were collected at 5 months of age (feces #1) and at 8 months of age (feces #2). following euthanasia (at 10 months of age) tissue samples were collected, along with a fecal sample (feces #3). a central 156 base pair region of the spike protein gene, including the critical s1/s2 activation site of the virus, was pcr amplified and sequenced as described in licitra et al, 17 with the following modifications: 25 μl reverse transcription pcrs were performed with qscript xlt 1-step rt pcr kit (quantbio). pcr conditions were 20 mins at 50°c, 3 mins at 95°c and 40 cycles of 10 s at 95°c, 20 s at 55°c, 40 s at 72°c, then 10 mins at 72°c. pcr products were purified using diffinity rapidtips (diffinity genomics). pcr and sequencing showed the presence of a type i fcov, based on a sequence alignment with reference genomes. the sequence information obtained from this cat is shown in figure 2 . the viral sequences from the cns (brain and spinal cord) contained specific amino acid changes compared with other samples (feces, small intestine, mesenteric lymph node and kidney). the most notable change was an arginine to methionine (r-m) substitution at the critical p1 activation position, 17 corresponding to residue 793. other changes that correlated with viruses present in the cns were present in two other positions: 770 alanine to valine (a-v); and 786 threonine to alanine (t-a). here we report clinicopathologic findings and molecular analysis of a cat with progressive neurologic clinical signs associated with fip. the cat initially presented to the referring veterinarian with respiratory signs and fever, and with abnormal liver enzyme function and anemia. at this time fip was not suspected. these initial signs resolved but were replaced by progressing neurologic signs, which led ultimately to euthanasia and submission to the study for evaluation of fcov involvement. upon euthanasia, fcov was found in various tissues in the cat, including the cns. however, histologic examination revealed fcov-associated pathology only within the cns, where there was meningoencephalomyelitis, ependymitis, choroid plexitis and vasculitis. histo logical lesions were compatible with a recent report describing meningoencephalitis in four cats with fip. 18 molecular analysis of the viral spike protein within the tissues identified a specific, functionally relevant amino acid change (r793m), which was only identified in tissues associated with the cns (ie, brain and spinal cord). the r793m mutation in the spike protein s1/s2 cleavage-activation site is a major chemical change from a basic to a hydrophobic residue, and is consistent with an elimination of furin-mediated proteolytic processing of the s protein, as seen by licitra et al, 17 and a proposed change in the activation properties and entry pathway of the virus. it is interesting to note that the r793m mutation was not present in other tissues tested in this cat at the time of necropsy but was found in our previous study (cat id #08-153990), 17 where samples were of neural origin. while biological confirmation is not available, we consider that the other changes found in the viral spike protein from central nervous tissue of this cat (a770v and t786a) are not related to changes in the activation properties and entry pathway of the virus, as they are not in defined functional regions of the spike protein and are not markedly different in their chemistry. interestingly, all samples tested from this cat contained a distinct leucine residue at position 791 (the p3 position of the furin cleavage site, typically serine or alanine as defined in licitra et al). 17 the relevance of this is currently unclear. overall, our results provide evidence that mutation of the viral spike protein is linked to fip outcome, specifically in the s1/s2 cleavage-activation site (residues 789-794). mutations leading to fip have also been linked to changes in other areas of the spike gene (position 1058) 19 and in the 3c gene. 20, 21 to compare our findings for the s1/s2 region to other proposed fip-linked mutations, we performed additional sequencing, which is summarized in table 3 . all fecal samples, as well as a kidney sample, contained methionine (m) at spike position 1058, indicating that an 'enteric' form of fcov 22 was present in the cat throughout our study. in contrast, samples from the brain and spinal cord contained leucine (l) at position 1058, indicating an fip virus. an intact 3c gene was found in feces, with the 3c gene in neural and other tissues truncated and/or deleted depending on the sample tested. this case report describes a young cat with neurologic fip in which detailed clinical and molecular characterization of the associated fcov infection was performed. while the etiology of fip remains complex and likely involves multiple mutations in the viral genome, our results indicate that a specific mutation of the viral spike protein can be associated with infection of the cns, which may explain the tropism to the cns as opposed to other organ systems. an update on feline infectious peritonitis: diagnostics and therapeutics an update on feline infectious peritonitis: virology and immunopathogenesis cns disease in the cat: current knowledge of infectious causes prevalence of diseases of the spinal cord of cats fenner's veterinary virology improving virus taxonomy by recontextualizing sequence-based classification with biologically relevant data: the case of the alphacoronavirus 1 species infectious diseases of the dog and cat a review of coronavirus infection in the central nervous system of cats and mice immunocytochemical demonstration of feline infectious peritonitis virus within cerebrospinal fluid macrophages practical overview of common infectious disease agents hagan and brunner's microbiology and infectious diseases of domestic animals diagnosis and clinical signs of feline infectious peritonitis in the central nervous system a retrospective study of the neuropathology and diagnosis of naturally occurring feline infectious peritonitis feline infectious peritonitis with neurologic involvement: clinical and pathological findings in 24 cats feline infectious peritonitis with spinal cord involvement in two cats clinicopathologic features and magnetic resonance imaging findings in 24 cats with histopathologically confirmed neurologic feline infectious peritonitis mutation in spike protein cleavage site and pathogenesis of feline coronavirus immunohistochemical studies on meningoencephalitis in feline infectious peritonitis (fip) spike protein fusion peptide and feline coronavirus virulence significance of coronavirus mutants in feces and diseased tissues of cats suffering from feline infectious peritonitis feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis acknowledgements we thank wendy wingate for help with sample collection, all members of the whittaker lab for helpful comments and support, and dr john loftus for clinical consultation and critical reading of the manuscript. video of cat at 8 months and 10 months of age. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. center.ethical approval this study involved the use of clientowned animal(s) only, and followed internationally recognized high standards ('best practice') of individual veterinary clinical patient care. ethical approval from a committee was not therefore needed.informed consent informed consent (either verbal or written) was obtained from the owner or legal guardian of all animal(s) described in this study for the procedure(s) undertaken. for any animals or humans individually identifiable within this publication, informed consent for their use in the publication (verbal or written) was obtained from the people involved.orcid id nicole m andré https://orcid.org/0000-0002-3703-5026 key: cord-317651-lsca8vt2 authors: yong, v. wee; power, christopher; forsyth, peter; edwards, dylan r. title: metalloproteinases in biology and pathology of the nervous system date: 2001 journal: nat rev neurosci doi: 10.1038/35081571 sha: doc_id: 317651 cord_uid: lsca8vt2 matrix metalloproteinases (mmps) have been implicated in several diseases of the nervous system. here we review the evidence that supports this idea and discuss the possible mechanisms of mmp action. we then consider some of the beneficial functions of mmps during neural development and speculate on their roles in repair after brain injury. we also introduce a family of proteins known as adams (a disintegrin and metalloproteinase), as some of the properties previously ascribed to mmps are possibly the result of adam activity. mmps are part of a larger family of structurally related zinc-dependent metalloproteinases called metzincins. other subfamilies of the metzincins are adams, bacterial serralysins and the astacins. metzincins use three histidine (h) residues to bind the zinc ion at their active site. these residues occur in the conserved sequence motif hexxhxxgxxhz, where z is a family-specific residue -serine in most mmps, aspartate in adams, proline in serralysins and glutamate in astacins. in addition, there is a distinct β-turn at the active site, which is delineated by a methionine residue ('met-turn') and seems to be essential for activity. there is about 20% similarity between metzincin subfamilies 2 , but identity at the catalytic domain is much higher. on the basis of substrate preference and proteindomain considerations, mmp family members (table 1) have been categorized into subgroups that include gelatinases, stromelysins, collagenases, membrane-type (mt)-mmps and 'other mmps'. however, there is much overlap in substrate specificity between subgroups. structurally, mmps are divided into three v. wee yong* ‡ , christopher power ‡ , peter forsyth* and dylan r. edwards § matrix metalloproteinases (mmps) have been implicated in several diseases of the nervous system. here we review the evidence that supports this idea and discuss the possible mechanisms of mmp action. we then consider some of the beneficial functions of mmps during neural development and speculate on their roles in repair after brain injury. we also introduce a family of proteins known as adams (a disintegrin and metalloproteinase), as some of the properties previously ascribed to mmps are possibly the result of adam activity. refs 15, 56, 100 , is by no means exhaustive and emphasizes those from the extracellular matrix. ‡ the activation of mmp12 involves the removal of the propeptide region to produce an intermediate active form (45 kda) , followed by atypical carboxy-terminal processing to produce the fully active enzyme (22 kda importance in the cns: adam10 (kuzbanian) and adam17, also known as tace (tumour-necrosisfactor-α-converting enzyme). the activity of metalloproteinases is tightly regulated, as these molecules are potent proteolytic enzymes that are capable of widespread destruction. their first regulatory step is at the level of transcription, as most mmps are not constitutively expressed but are transcribed after cell activation. transcription of many mmps is promoted by inflammatory cytokines, growth factors, chemokines, oncogenes and cell-cell or cell-matrix interactions 12 . post-translational modifications provide a second level of mmp regulation. many mmps are expressed as inactive zymogens in which the cysteine residue at the propeptide region binds the zinc ion present at the catalytic site. activating factors include the plasminogenplasmin cascade, as well as other mmps (table 1) that disrupt the interaction between cysteine and zinc (the so-called 'cysteine switch' mechanism) and then remove the propeptide region for full activation 13 . non-proteolytic compounds such as sulphydryl-reactive agents (4aminophenylmercuric acetate) and denaturants (urea) can also activate zymogens. a subset of mmps contains a cleavage site for furin-like prohormone convertases between the propeptide and catalytic domains; this subset includes the mt-mmps, which are activated during secretion and appear on the cell surface in the active form. a third means to control mmp activity is by the interaction of active mmps with tissue inhibitors of metalloproteinases (timps; see refs 14, 15 for comprehensive reviews). four timps are now known and they cause inactivation by binding to the catalytic site of mmps. interestingly, timps are also required for the activation of some mmps. in this regard, a complex formed by timp2 and the carboxyl terminus of pro-mmp2 has been found to bind mt1-mmp (mmp14) on the cell surface. an adjacent mt1-mmp molecule then removes the propeptide region of mmp2 (ref. 16 ). the pro-mmp-timp-mt-mmp trimolecular complex highlights another feature of mmp regulation: the active proteinase is found focally in the pericellular region, rather than being diffusely distributed. in addition, other means also exist to localize mmp activity to the pericellular region. for example, activated mmp2 can bind the αvβ3 integrin, whereas active mmp9 can interact with the hyaluronan receptor cd44 on the cell membrane 17 . in the case of adams, less is known about the regulation of their activity. several adams are kept in the inactive state through the interaction of a cysteine residue at the propeptide domain with zinc in the metalloproteinase module. so, as with mmps, these adams might be activated by the cysteine switch mechanism that disrupts the cysteine-zinc interaction to expose the catalytic site. adams might also be regulated at the transcriptional level. for example, interleukin-1 (il-1) can induce transcription of some adam members. little is known of the physiological that lack metalloproteinase activity, this domain might fill another purpose, such as allowing protein-protein interactions. the expression of six adams (adam9, -10, -12, -15, 17 and -19) is widespread. by contrast, some adams that are predicted to have metalloproteinase activity are expressed in a testis-specific fashion, and adam8 and -28 are predominantly haematopoietic. recently, new members of the adam family have been identified 7 . they have thrombospondin motifs and are therefore known as adamtss. all adamtss are secreted proteins. at present, at least 30 adams and 10 adamtss have been described. the literature on the role of adams in the cns is still sparse. for example, an early description of adam10 in the cns implicated this protein in oligodendrocyte and myelin biology 8 , although the molecule involved was not recognized as an adam until later. but despite the limited evidence, adams might be important proteins in the nervous system. at least 17 adams are found in the cns (ref. 9) and, in fact, some of these are expressed predominantly in the brain (for example, adam22 and -23). furthermore, many pharmacological studies on the role of mmps have failed to discriminate between mmp or adam functions. in particular, many of the inhibitors based on the functional group hydroxamate, which chelate the zinc ion from the active site of mmps, also have activity against adams (refs 10, 11) . last, the thrombospondin motif of adamtss confers degradative activity on several proteoglycans that are enriched in the cns. here, we will centre our discussion on two adams, as several lines of evidence indicate their 504 | july 2001 | volume 2 www.nature.com/reviews/neuro r e v i e w s inhibitors of adams. the crystal structure of the protease domain of human adam17 (tace) shows that, although the active-site cleft is similar to what is found in mmps, its secondary structure differs. these features might account for the finding that, whereas timp3 inhibits tace, timp1 does not. also, adam10 is inhibited by timp1 and -3, and by hydroxamates, but not by timp2 and -4 (ref. 18 ). so, timp3 might be a more general inhibitor of several adam family members, including the adamtss. it is not known if more specific, endogenous non-timp adam inhibitors exist. in the adult cns, most mmps are expressed at low or undetectable levels, although there are some exceptions. for example, rnase protection assays have revealed a high constitutive expression of mmp11 and -14 in the adult mouse brain 19 . similarly, the more sensitive polymerase chain reaction (pcr) technique has revealed the expression of mmp2, -3, -7, -9 and -13 in the normal rat spinal cord 20 . but, overall, mmps are largely absent from the normal cns and their upregulation has been reported in several neurological disorders and after injury. by contrast, in the case of adams, over 17 family members are normally expressed in the adult cns (ref. 9 ). for example, adam17 has been localized by immunohistochemistry to astrocytes and endothelial cells in adult human brain 21 , whereas adam8 has been detected in neurons and oligodendrocytes in the uninjured adult rat cns (ref. 22 ). however, the literature on the expression of adams in cns pathology is limited. we will divide our discussion of the role of mmps and adams in pathology into three parts: their role in neuroinflammation and ms, their involvement in malignant gliomas, and their participation in other neurological conditions such as stroke, viral infections and alzheimer's disease. is an immune disorder characterized by demyelination and axonal loss. the presence of proteinases in the cerebrospinal fluid (csf) of patients with ms has been known for over 20 years; some of these proteinases were recently identified as mmps. specifically, mmp9, which is absent in the csf of normal individuals, is upregulated in ms and in other inflammatory neurological diseases 23 . mmp profiles in serum and in leukocytes are also altered in ms. so, when compared to healthy controls, ms patients show increased mmp9 messenger rna in leukocytes and elevated mmp9 levels in serum. similarly, the mmp9:timp1 ratio in the serum of people with ms is higher than normal 24 . the serum mmp9 content in ms patients is higher during relapse than in an off-period 25 . by using gadolinium-enhanced magnetic resonance imaging (mri) techniques, it was found that ms patients with high mmp9 and low timp1 levels tended to worsen 26 . several groups have shown increased post-mortem expression of various mmp members (mmp2, -3, -7 and -9) in the brains of patients with ms (refs 27, 28) . cellular sources of mmps in the diseased brain include infiltrating leukocytes (lymphocytes and macrophages) and intrinsic cns cells (perivascular and parenchymal microglia, astrocytes and even neurons). although it has been less thoroughly investigated, most studies indicate that timp1 and -2 might not be altered in ms compared with controls 29 . no data are available for timp3 and -4. mmps are also dysregulated in experimental autoimmune encephalomyelitis (eae), an animal model of ms. increased expression of mmp7, -9 and -12 is observed in the rat cns immediately preceding, and in parallel with, the development of symptoms. by contrast, transcripts encoding mmp2, -3, -11 and -13 were unchanged 20 . surprisingly, the elevation of mmp7 is not noted in mouse eae, in which a prominent increase in mmp3, -9 and -12 occurs 30 . so, overall, a common elevation of mmp9 and -12 is found in ms, and in mouse and rat eae models. the correlation between elevated mmps and disease activity is probably causal, as inhibitors of metalloproteinase activity (for example, gm6001, ro-9790 and bb1101) alleviate or prevent eae (refs 1,31). also, 3-4-week-old mice that lack mmp9 are less impaired after the induction of eae when compared with wildtype animals 32 . in humans, interferon-β, a drug used against ms, attenuates mmp9 production by t cells in vitro, in agreement with the decreased capacity of t cells to traverse ecm barriers 33, 34 . in addition, ms patients treated with interferon-β show a decrease in the content of serum mmp9 and a lowering of the number of mmp9-expressing leukocytes 35 . together, these observations indicate that the antagonism of mmp function might contribute to the efficacy of interferon-β in ms. studies of ms and eae have revealed several mechanisms that help to account for the involvement of mmps in cns pathology (fig. 2) . first, there is good evidence that t cells and other leukocytes use mmps to penetrate ecm barriers, including the basement membrane that surrounds cerebral capillaries. in this regard, this process disrupts the basement membrane that surrounds the vasculature and results in blood-brain barrier (bbb) impairment. in the central nervous system, high mmp content and its indiscriminate localization result in perpetuation of an inflammatory response, which contributes to demyelination and neuronal or oligodendrocyte death. ecm, extracellular matrix. technique for the quantitation of rnas in solution. it is based on the use of a complementary radiolabelled probe that is hybridized to the target rna, after which the mixture is treated with rnase to degrade all remaining single-stranded rna. the hybridized target will be protected from digestion and can be later visualized on a polyacrylamide gel. magnetic resonance imaging imaging technique in which gadolinium is introduced as a contrast agent, allowing for short data-acquisition times, large anatomical coverage and improved image quality. cytotoxicity. given the presence of fas and fasl in the cns, mmps could affect cell survival or death by similar mechanisms. in summary, the excessive production of mmps in the cns can be neurotoxic through several mechanisms (fig. 2) . the literature on the upregulation of adams in the cns of ms patients is still in its infancy. cells of the immune system produce various adams (ref. 48 ), and it is likely that several adams will be elevated in the cns during neuroinflammation and ms. also, with the exception of adam17 described above, much remains to be explored about the consequences of the aberrant expression of adams in the cns. an extensive literature links mmps to tumour invasiveness and metastasis, as remodelling of the ecm is thought to be necessary for a tumour cell to advance. more recent studies have extended the roles of mmps to other features of tumour progression, notably proliferation, angiogenesis and survival 49 . essentially, all mmp members have been linked to cancers of various origins. so, it is not surprising that there are many reports of the increased expression of mmps in brain tumours in situ and in vitro. specifically, the upregulation of mmp2, -9 and all the mt-mmp members has been noted in highgrade specimens of glioblastoma multiforme (gbm) as compared to lower-grade cases or to non-transformed control brains [50] [51] [52] . the finding that ag3340 -an inhibitor of metalloproteinase activity -inhibits glioma growth, invasion and angiogenesis in animals 53 highlights the importance of mmps in the progression of brain tumours. mmp2, mt1-and mt2-mmp have been localized to glioma cells in addition to cns elements by in situ hybridization. by contrast, mmp9 is predominantly expressed in blood vessels at proliferative margins 51 . a simplistic interpretation of these results is that mmp2 and mt-mmps might regulate glioma invasiveness, whereas mmp9 might be crucial for angiogenesis. in this regard, the capacity of various glioma lines to migrate across a reconstituted basement membrane in vitro is correlated with the levels of mmp2 expression 54 . furthermore, the ability of the c6 gliosarcoma line to migrate along myelin is related to mt1-mmp activity 55 . as mentioned earlier, mt1-mmp facilitates the activation of mmp2, and an excessive mt1-mmp activity was recently noted in gbms (ref. 52) . similarly, factors that stimulate glioma motility, such as hepatocyte growth factor/scatter factor, increase mmp2 or mt1-mmp levels. conversely, inhibitors of mmp activity, including bb94 and bb2516, reduce glioma invasiveness in tissue culture. several mmps can be activated by the serine protease plasmin 56 . the plasmin cascade starts with binding of urokinase plaminogen activator (upa) to its receptor molecule upar. this results in upa activation and the subsequent conversion of plasminogen to plasmin by upa. it is noteworthy that the upa and upar are upregulated in gbms (ref. 57 ), endowing the tumour with the machinery to activate its own mmps the capacity of macrophages from mmp12-null mice to penetrate basement membranes is markedly diminished in vitro and in vivo 36 . also, when t cells are applied onto a monolayer of endothelial cells that overlie a barrier of collagen matrix, the addition of gm6001, an metalloproteinase inhibitor, did not prevent the t cells from traversing the endothelial barrier. however, the entry of t cells into the collagen matrix is abolished 37 . one result of disrupting the basement membrane is the breakdown of the blood-brain barrier (bbb). this has been shown experimentally by the intracerebral injection of mmps and the subsequent leakage of microvessels 31, 38 . in addition, there is a good correlation between gadolinium-enhanced mri activity in humans, an index of bbb dysfunction, and the serum content of mmp9 (refs 25, 26) . mmps have other undesirable consequences in the cns parenchyma (fig. 2) . when injected into the cns, mmps can disrupt myelin and cause demyelination 39 . furthermore, some of the fragments of the mmpmediated digestion of myelin basic protein (mbp) can induce eae when injected into rodents 31, 40 . also, human mmp9 cleaves human mbp into peptide fragments, one of which is the immunodominant epitope in humans 40 . another mechanism by which mmps might promote an inflammatory response is by the conversion of precursor, inactive molecules into their activated forms. tumour-necrosis factor (tnf)-α -a proinflammatory cytokine with several actions that include oligodendrocyte toxicity -is first made as a 26-kda membrane-associated protein that requires proteolytic conversion to the fully active 17-kda protein. although tace is known to be the protease that mediates the physiological maturation of tnf-α (ref. 41 ), it is clear that mmp7 and mt4-mmp can also efficiently mediate this conversion 42 . other molecules, such as transforming growth factor (tgf)-α, il-6, tnf receptors, l-selectins and fas ligand (fasl), are also synthesized as precursors that require processing by mmps or adams for maturation. so, in pathological conditions in which local levels of mmps are high, these molecules might significantly modulate inflammation in the cns. another pathogenic effect of mmps in the cns is neurotoxicity. vos et al. 43 reported that mmp1 is toxic to spinal cord neurons in vitro, and we found a similar neurotoxic effect of mmp2 (ref. 44 ). an alternative mechanism for mmp-mediated death might involve the phenomenon known as 'anoikis' (greek for homelessness); cells that are attached to an ecm substrate survive through integrin signalling and death ensues when the cell is detached 45 . in principle, mmps can indirectly cause cell death by degrading ecm and therefore interfering with cell attachment and integrin signalling. indeed, neuronal death in the hippocampus after kainate-induced seizures in animals has been attributed to proteases that degrade laminin 46 . finally, it has been noted that mmp7, but not mmp2, -3 or -9, acts on cell-associated fasl to convert it into soluble fasl, which induces apoptosis of epithelial cells 47 . conversely, mmp7-mediated cleavage of fasl protects sarcoma and colon carcinoma cells from chemotherapy-induced 506 | july 2001 | volume 2 www.nature.com/reviews/neuro ligand for the fas antigen, a transmembrane protein that mediates apoptosis and might be involved in negative selection of autoreactive t cells in the thymus. glioblastoma multiforme aggressive glioma, commonly of astrocytic origin, that infiltrates adjacent normal brain tissue. it accounts for about 10% of childhood brain tumours. from demented patients, were expressed in u937 monocytoid cells and human macrophages, elevated mmp2 and -7 expression was obtained compared to tat sequences from non-demented patients. these elevated mmps resulted in neuronal death 44 . collectively, the results indicate that hiv infection induces mmp expression in macrophages and that the latter are the possible sources of mmps and neurotoxicity in the cns of people with hiv-associated dementia. other viruses, including epstein-barr virus, htlv-1 and coronavirus 66 have also been reported to increase the expression of mmps in susceptible cell types. of interest, these viruses have been associated with ms pathology, raising the possibility that viral-induced demyelination might involve mmp intermediaries. mmps are also associated with alzheimer's disease, although the history has been full of false leads. alzheimer's disease is characterized by plaques that are formed mostly by the deposition of amyloid-β (aβ)a peptide derived from cleavage of the amyloid precursor protein (app), an integral membrane molecule. three proteases -α-, βand γ-secretase -are involved in app cleavage at different sites to generate aβ peptides of various lengths 67 . the predominant cleavage is mediated by α-secretase, and this mode of cleavage is thought to be non-amyloidogenic. by contrast, the combination of β-secretase and γ-secretase activities releases the amyloidogenic aβ peptide. the aspartyl protease bace (β-site app cleavage enzyme) is the best candidate to be the β-secretase 68 , whereas the γ-secretase activity depends on proteins known as presenilins. it was initially suggested that mmp2 was the α-secretase 69 , but this idea was quickly disputed. moreover, it was also suggested that mmp2 had β-secretase-like activity 70 . furthermore, the presence of immunoreactive timp in plaques of alzheimer's disease led to the speculation that an mmp was excessively produced or activated in the plaques, as timps have high affinity for mmps and would therefore localize to sites of protease activity 71 . it was subsequently suggested that the α-secretase activity was mediated by a non-mmp metalloproteinase, largely on the basis of studies using pharmacological inhibitors 72 . indeed, there is now good evidence for adam10 (ref. 73) and adam17 (ref. 74) as α-secretases. if this proves to be the case, these adams could have a protective action against alzheimer's disease, as they would funnel app towards the non-amyloidogenic pathway. it is worthwhile investigating whether the function of adams is deficient or altered in alzheimer's disease. recently, hippocampal neurons that are immunoreactive for adam1 and -2 were detected in alzheimer's disease patients but not in age-matched controls 75 . in addition, levels of these proteins were also elevated 75 . mmps are also implicated in other diseases of the nervous system, including inflammatory myopathies 76 and peripheral nerve axotomy. the involvement of mmps in degenerative diseases of the cns, including amyotrophic lateral sclerosis, is an area of increasing interest. it is likely that the list of neurological disorders associated with aberrant mmp or adam expression will grow over the years. effectively. in support of this idea, downregulation of upar in a glioma cell line led to decreased capacity to invade brain aggregates 58 . the expression of mmps by glioma cells highlights a difference between cns and systemic cancers. in the latter (for example, breast cancers), mmps are often expressed by the stroma, rather than by the tumour itself. so, several mechanisms seem to exist for bestowing glioma cells with the capacity for autonomous, sustained growth. timps are also altered in gliomas. a decrease in timp2 content in gbm compared to lower-grade tumours has been noted in some 52 , but not all 50 , studies. this decrease would potentially remove a counterbalance for mmp activity. although earlier studies noted a decrease of timp1 with increasing grades of glioma, later studies noted that it was actually upregulated in gbm (refs 50, 52) . it is worth noting in this context that timps have several properties that can contribute to the pathophysiology of cancer cells. indeed, timps have mitogenic action, and regulate survival and apoptosis independently of their inhibitory functions on mmps (ref. 14) . finally, elevated expression of various mmps has been shown for other cns tumour classes, including childhood astrocytomas, neuroblastomas and meningiomas. overall, an excess of mmp activity seems to characterize many brain tumours. the development of metalloproteinase inhibitors to treat gliomas seems warranted and some are already being tested in clinical trials. a role for adams in glioma biology remains to be defined and the related literature is limited at present 59 . a role for metalloproteinases in stroke is indicated by the finding that mmp2 and -9 are rapidly upregulated after focal cerebral ischaemia in rats 60, 61 . in humans, elevation of brain mmp9 is detected post mortem within days of infarction and, interestingly, this protein remained elevated in patients that died months after the event 62 . in another report, mmp9 was strongly expressed by neutrophils in tissues from patients up to one week after an infarct, whereas the expression of mmp2 and -7 was less marked. from one week to five years, neutrophils were absent in the lesions and the large number of macrophages present were immunoreactive for mmp2 and -7 (ref. 28 ). the elevated mmp expression might contribute to the tissue destruction in stroke. as noted earlier, mmps have the capacity to kill neurons (fig. 2) . indeed, the intravenous treatment of rats with a neutralizing mmp9 antibody one hour before vessel occlusion reduced infarct size by 28% (ref. 61 ). in addition, the size of infarcts after ischaemia in mmp9-null mice was less than that observed in wild-type controls 63 . viral infections of the cns have been increasingly associated with the production of mmps. elevated expression of mmp9 has been detected in the csf of hiv-infected patients 64 . mmp2, -7 and -9 are increased in the csf of patients with hiv-associated dementia compared to that of non-demented aids patients 65 . when brain-derived sequences of the hiv-transactivating protein tat (tyrosine aminotransferase), obtained nature reviews | neuroscience volume 2 | july 2001 | 5 0 7 dendroglial processes 84 . other metalloproteinases could also regulate myelinogenesis, but this remains to be tested. the rabbit corpus callosum expresses mmp1 and -3 before and during myelination 85 , whereas adam10 is expressed in oligodendrocytes before and during myelinogenesis 8 . in parallel with myelinogenesis, metalloproteinases also participate in axon elongation (fig. 3) . early studies noted the presence of proteolytic activity at neuronal growth cones during attachment and reattachment events 86 . some of the activity is probably contributed by metalloproteinases, as interference with mmp activity inhibited growth-cone motility 87 . inducers of neuronal differentiation and axonal outgrowth, such as nerve growth factor, laminin or retinoic acid, enhanced the expression of mmp2, -3 and -9 by dorsal root ganglion (drg) neurons, pc12 and neuroblastoma cells 88, 89 . furthermore, growth cones of pc12 cells that stably expressed mmp3 had a reduced capacity to penetrate a reconstituted basement membrane. in vivo, drosophila melanogaster flies that carry mutations in the protein kuzbanian (the mammalian homologue is adam10) show axon stalling during development 90 . in a study in which neuritic outgrowth of drg neurons that grow on top of normal adult nerves was evaluated, the slow neurite elongation was further reduced by treatment with metalloproteinase inhibitors 91 . by contrast, pretreating the nerves with recombinant mmp2 accelerated neurite growth 91 . further studies led to the conclusion that drg neurons expressed mmp2 that degraded and inactivated the neurite-inhibiting activity of chon-droitin sulphate proteoglycans present on nerves, leading to the exposure of permissive laminin for neurite outgrowth 91 . indeed, cleavage of a specific peptide bond in an ecm molecule leads to profound functional changes in other systems. for example, the cleavage of the ala586-leu587 bond in the α2 chain of laminin-5 by mmp2 induced migration of breast epithelial cells by exposing a cryptic pro-migratory site on laminin-5 (ref. 92 ). the biologically active sites in matrix molecules that become exposed after structural or conformational alterations have been called 'matricryptic sites' , and the name matricryptins has been used to describe the resulting ecm fragments that have biological activity. last, in concordance with the activity of mmp2 on proteoglycans described above, metalloproteinases might be used in the cns to destroy other inhibitory proteins. c6 glioma cells and fibroblasts transfected with mt1-mmp could digest ni250 (ref. 55 ), a nogo protein identified as one of the most potent inhibitors of axonal elongation. in this way, some mmps might act by neutralizing inhibitory proteins for axonal outgrowth. although these data implicate metalloproteinases in the creation of penetrable paths for axonal elongation (fig. 3) , metalloproteinases can also regulate guidance cues for growth cones. ephrins are guidance molecules that bind to receptor tyrosine kinases of the eph family. when the growth cone of a neuron that expresses eph receptors encounters ephrin ligands on the surface of another cell, this facilitates the adherence of the cells to each other and bidirectional signalling to occur. the although our discussion has so far revolved around the detrimental roles of metalloproteinases, it must be stressed that some of the functions of mmps in the cns might be beneficial. for example, some mmps and timps are expressed in the cns during development 77, 78 , pointing to their possible importance in brain maturation. furthermore, mmps are rapidly upregulated after nearly all types of injury to the cns, including trauma 19, 79 , indicating their possible relevance in tissue repair. in this section, we focus on the lesser-known beneficial aspects of metalloproteinases in the cns. in concordance with the classic role of mmps in modulating the motility of cells across tissue matrices, metalloproteinases might regulate the migration of precursor cells to their destinations during neural development. neural stem cells express mmp2 and all four timps (ref. 80 ), and the migration of an oligodendrocyte progenitor requires mmp activity in vitro. the protein notch affects cell-fate decisions in neurogenesis, and both adam10 (ref. 81, 82) and adam17 (ref. 83) have been shown to activate the notch signalling cascade. another role for mmps in cns development might lie in myelinogenesis, the process whereby oligodendrocytes extend several processes from their soma that reach and enwrap axons to form myelin. the initial expansion of oligodendroglial processes is immense and could require remodelling of the brain matrix by mmps. this hypothesis has been tested and oligodendrocytes were found to express mmp9 during the period of myelinogenesis. furthermore, the inhibition of mmp activity in vitro prevented the extension of oligo-508 | july 2001 | volume 2 www.nature.com/reviews/neuro r e v i e w s of mmps in the adult cns might involve their potential to affect signal transduction by, for example, linking g-protein-coupled-receptor signalling with the egf pathway. in this context, the engagement of g-proteincoupled receptors has been shown to activate a membrane-associated metalloproteinase that converts proheparin-binding egf to its mature form, triggering egf signalling 96 . regulation of synaptic plasticity could also be another function of metalloproteinases in cns injury 77 . of interest, mmp1, -2, -3 and -9 were upregulated after spinal-cord transection in the axolotl during the period of regeneration and subsided when recovery was near completion 97 . mmps could also participate in terminating inflammation in the cns, in contrast to the perpetuation of inflammation that we discussed earlier (fig. 2) . the prolonged incubation of il-1β with mmp3, and to a lesser extent mmp2 and -9, resulted in il-1β degradation 98 . furthermore, mmp2 binds to the chemokine mcp3 (macrophage chemoattractant protein 3) and cleaves its first four amino-terminal amino acids. the cleaved mcp3 binds to several chemokine receptors, acting as an antagonist 99 . so, mmps expressed in the cns might serve to abolish a chemotactic gradient for leukocyte entry. interestingly, mmp9 has been reported to truncate the amino terminus of il-8, leading to a 30-fold potentiation of il-8 activity 100 . conversely, mmp9 degraded chemokines, such as gro-α, leaving rantes (also known as scya5) and mcp2 intact 100 . in summary, metalloproteinases in the cns have clear beneficial functions during development and might also have positive effects after injury. these effects contrast with the better-described detrimental roles of metalloproteinases in the cns . the challenges associated with defining the roles of mmps in the cns are several-fold. first, it is obvious that metalloproteinases can be both friends and foes in the cns. we will therefore have to understand the balance between these states and the context that swings the pendulum between beneficial and detrimental roles. second, a given metalloproteinase might have different properties at a given condition, dependent on factors such as spatial localization and cellular source. for example, mmp9 expressed by a macrophage in the vicinity of myelin might produce demyelination, whereas the same mmp elaborated by oligodendrocytes at the tip of their processes might promote remyelination. in this context, an inhibitor of mmp9 would simultaneously curb both detrimental and reparative processes. so, the challenge is to identify all the functions of a given metalloproteinase in a particular situation and to evaluate if the net result of its inhibition is worthwhile. a third challenge is related to establishing the identity of the metalloproteinase involved in a given function. many of the pharmacological inhibitors that are available at present lack specificity towards members of a subfamily and will often antagonize the activity of members of different metalloproteinase families 10, 11 . so, when functions are ascribed to particular metalloproteinases, growth cone then overcomes these adhesive forces and breaks away from the ephrin surface. hattori et al. 93 showed that the adhesive ephrin-eph interaction is broken in vitro by adam10, which becomes activated after engagement of the eph receptor. another guidance molecule is netrin 1, which binds a receptor known as dcc (deleted in colorectal carcinoma). when axon outgrowth from embryonic dorsal spinal explants was evaluated in vitro, the facilitatory activity of netrin 1 was potentiated by ic3 and gm6001, hydroxamate metalloproteinase inhibitors 94 . it was found that dcc was shed from the cell surface by the activity of an unidentified mmp; preventing the ectodomain shedding of dcc with a metalloproteinase inhibitor resulted in responsiveness to netrin 1 (ref. 94 ). the recent report of a phenotype-based gene-trap screen to identify genes that control wiring patterns in the mouse cns further implicates metalloproteinases in axonal guidance. adam23 was one of the genes identified in this screening; its inactivation in vivo led to neurological defects, tremor and ataxia 95 . so, a beneficial role for metalloproteinases in neurogenesis and myelinogenesis during development seems to be supported by several lines of evidence. by contrast, the favourable functions (if any) of the mmps that are upregulated in cns disorders in adulthood are less clear. we have highlighted the detrimental effects of metalloproteinases in the injured adult cns (fig. 2) but some beneficial functions are also possible after injury, although they remain speculative at this point. what could they be? first, it is possible that metalloproteinases enable the migration of precursor cells to injured sites to replenish lost cells. a role in facilitating axonal regrowth, remyelination and angiogenesis is also possible on the basis of the functions of these proteases in development, as described earlier. another possible role nature reviews | neuroscience volume 2 | july 2001 | 5 0 9 bition to treat specific cns diseases? given the data that aberrant expression of mmps is correlated with conditions such as ms, we suggest that the use of metalloproteinase inhibitors is warranted. if factors such as inflammation, which cause the disease to worsen, can be curbed, then the side effects of impairing reparative activities might be a reasonable trade-off. however, much more specific inhibitors and a clearer view of which metalloproteinase member to antagonize would help towards rational therapeutics. in summary, much remains to be learned about the biology and pathology of metalloproteinases in the cns. we welcome the explosion of this field, as the rewards of an increased understanding would seem to be immense. it has to be clear, not only which metalloproteinase is involved, but also whether one is dealing with the right subfamily of metzincin metalloproteinases. clearly, the next generation of pharmacological inhibitors must be able to discriminate between subfamilies and between members of each subfamily. mice with null mutations for specific mmps and adams are already available and can help to discriminate functions of specific metalloproteinases. however, these valuable resources often show compensatory upregulation of activity of other mmps as a result of the gene deletion and the analysis of these animals must therefore be interpreted with caution. the identification of their physiological substrates is intimately related to the task of defining the functions of metalloproteinase s in the cns. although it is clear that various mmp members can act on certain ecm components in vitro (table 1) , much remains to be discovered about their natural substrates in vivo. in the case of adams, their cns substrates are largely un known and research needs to focus on this area. similarly, the ways in which metalloproteinases are transcribed and interact with other molecules, such as chemokines, remain to be elucidated. can any consensus be adopted at this point as to whether metalloproteinases should be targeted for inhimatrix metalloproteinases and diseases of the central nervous system the metzincins -topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases membrane-type 6 matrix metalloproteinase (mt6-mmp, mmp-25) is the second glycosyl-phosphatidyl inositol (gpi)-anchored mmp metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding a metalloprotease-disintegrin, mdc9/meltrinγ/adam9 and pkc δ are involved in tpa-induced ectodomain shedding of membrane-anchored heparinbinding egf-like growth factor metalloproteasedisintegrin adam 12 binds to the sh3 domain of src and activates src tyrosine kinase in c2c12 cells molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene degradation of myelin basic protein by a membrane-associated metalloprotease: neural distribution of the enzyme metalloprotease-disintegrin (adam) genes are widely and differentially expressed in the adult cns key paper that describes the existence of several adams in the cns, although the functions of many of these members remain unknown metalloprotease-disintegrin mdc9: intracellular maturation and catalytic activity tace and other adam proteases as targets for drug discovery the potential use of mmp inhibitors to treat cns diseases this details the mechanisms by which several mmp members are activated matrix metalloproteinase inhibitors in cancer therapy matrix metalloproteinases and timps relating matrix metalloproteinase structure to function: why the 'hemopexin' domain? localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for cd44-mediated tumor invasion the in vitro activity of adam-10 is inhibited by timp-1 and timp-3 interleukin-1 is a key regulator of matrix metalloproteinase-9 expression in human neurons in culture and following mouse brain trauma in vivo matrix metalloproteinase expression during experimental autoimmune encephalomyelitis and effects of a combined matrix metalloproteinase and tumor necrosis factor-α inhibitor astrocyte and endothelial cell expression of adam 17 (tace) in adult human cns tumor necrosis factor α induces a metalloprotease-disintegrin, adam8 (cd156): implications for neuron-glia interactions during neurodegeneration gelatinase in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological disorders expression of matrix metalloproteinase-9 and its inhibitors in mononuclear blood cells of patients with multiple sclerosis serum gelatinase b, timp-1 and timp-2 levels in multiple sclerosis. a longitudinal clinical and mri study serum mmp-9 and timp-1 levels are related to mri activity in relapsing multiple sclerosis the expression of tissue-type plasminogen activator, matrix metalloproteases and endogenous inhibitors in the central nervous system in multiple sclerosis: comparison of stages in lesion formation differential matrix metalloproteinase expression in cases of multiple sclerosis and stroke matrix metalloproteinases and tissue inhibitors of metalloproteinases in cerebrospinal fluid differ in multiple sclerosis and devic's neuromyelitis optica differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states matrix metalloproteinases, tumor necrosis factor and multiple sclerosis: an overview resistance of young gelatinase b-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions interferon-β decreases the migration of t lymphocytes in vitro: effects on matrix metalloproteinase-9 interferon β-1b inhibits gelatinase secretion and in vivo migration of human t cells: a possible mechanism for treatment efficacy in multiple sclerosis multiple sclerosis: pro-and antiinflammatory cytokines and metalloproteinases are affected differentially by treatment with ifn-β metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice the interrelationship of α4 integrin and matrix metalloproteinase-2 in the pathogenesis of experimental autoimmune encephalomyelitis timp-2 reduces proteolytic opening of blood-brain barrier by type iv collagenase delayed-type hypersensitivity lesions in the central nervous system are prevented by inhibitors of matrix metalloproteinases cytokine-regulated proteases in autoimmune diseases an excellent review that describes several key concepts on the generation of encephalogenic fragments after the incubation of myelin proteins with mmps. the fact that one of these fragments is the immunodominant epitope of myelin basic protein supports the concept of mmps as foes in ms a metalloproteinase disintegrin that releases tumor-necrosis factor-α from cells membrane type 4 matrix metalloproteinase (mmp17) has tumor necrosis factor-α convertase activity but does not activate pro-mmp2 cytotoxicity by matrix metalloprotease-1 in organotypic spinal cord and dissociated neuronal cultures hiv-1 tat neurotoxicity is prevented by matrix metalloproteinase inhibitors neuronal death in the hippocampus is promoted by plasmin-catalysed degradation of laminin the metalloproteinase matrilysin proteolytically generates active soluble fas ligand and potentiates epithelial cell apoptosis adam family proteins in the immune system matrix metalloproteinases: multifunctional contributors to tumor progression expression of matrix metalloproteinases and their tissue inhibitors in human brain tumors gelatinase-a (mmp-2), gelatinase-b (mmp-9) and membrane type matrix metalloproteinase-1 (mt1-mmp) are involved in different aspects of the pathophysiology of malignant gliomas roles of membrane type 1 matrix metalloproteinase and tissue inhibitor of metalloproteinases 2 in invasion and dissemination of human malignant glioma marked inhibition of tumor growth in a malignant glioma tumor model by a novel synthetic matrix metalloproteinase inhibitor ag3340 glioma invasion in vitro: regulation by matrix metalloprotease-2 and protein kinase c membranetype 1 matrix metalloprotease (mt1-mmp) enables invasive migration of glioma cells in central nervous system white matter cancer drug discovery and development: matrix metalloproteinase inhibitors in cancer therapy expression and localization of urokinase-type plasminogen activator in human astrocytomas in vivo inhibition of in vivo tumorigenicity and invasiveness of a human glioblastoma cell line transfected with antisense upar vectors the specific expression of three novel splice variant forms of human metalloprotease-like disintegrin-like cysteine-rich protein 2 gene in brain tissues and gliomas proteolytic cascade enzymes increase in focal cerebral ischemia in rat matrix metalloproteinase expression increases after cerebral focal ischemia in rats. inhibition of matrix metalloproteinase-9 reduces infarct size increased gelatinase a (mmp-2) and gelatinase b (mmp-9) activities in human brain after focal ischemia role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with bb-94 presence of matrix metalloproteinase-9 activity in the cerebrospinal fluid of human immunodeficiency virus-infected patients cerebrospinal fluid levels of mmp-2, 7, and 9 are elevated in association with human immunodeficiency virus dementia activation of glial cells by human coronavirus oc43 infection the cell biology of β-amyloid precursor protein and presenilin in alzheimer's disease β-secretase cleavage of alzheimer's amyloid precursor protein by the transmembrane aspartic protease bace a metalloproteinase inhibitor domain in alzheimer amyloid protein gelatinase a possesses a β-secretase-like activity in cleaving the precursor amyloid protein precursor of alzheimer's disease localization of tissue inhibitor of matrix metalloproteinases in alzheimer's disease and normal brain degradation of amyloid β-protein by a metalloproteinase secreted by microglia and other neural and non-neural cells constitutive and regulated α-secretase cleavage of alzheimer's amyloid precursor protein by a disintegrin metalloprotease evidence that tumor necrosis factor α converting enzyme is involved in regulated α-secretase cleavage of the alzheimer amyloid protein precursor altered cell-matrix associated adam proteins in alzheimer disease expression of specific matrix metalloproteinases in inflammatory myopathies spatiotemporal expression patterns of metalloproteinases and their inhibitors in the postnatal developing rat cerebellum differential spatial distribution and temporal regulation of tissue inhibitor of metalloproteinase mrna expression during rat central nervous system development glucocorticoid receptor-mediated suppression of activator protein-1 activation and matrix metalloproteinase expression after spinal cord injury expression and modulation of matrix metalloproteinase-2 and tissue inhibitors of metalloproteinases in human embryonic cns stem cells kuzbanian controls proteolytic processing of notch and mediates lateral inhibition during drosophila and vertebrate neurogenesis processing of the notch ligand delta by the metalloprotease kuzbanian a novel proteolytic cleavage involved in notch signaling: the role of the disintegrin-metalloprotease tace gelatinase b/matrix metalloproteinase-9 is required for oligodendroglial process extension in vivo and in vitro localization of proteinase expression in the developing rabbit brain release of plasminogen activator and a calcium-dependent metalloproteinase from cultured sympathetic and sensory neurons inhibition of retinal growth cone activity by specific metalloproteinase inhibitors in vitro ngf induction of the gene encoding the protease transin accompanies neuronal differentiation in pc12 cells induction of matrix metalloproteinase mmp-9 (92-kda gelatinase) by retinoic acid in human neuroblastoma sknbe cells: relevance to neuronal differentiation the cell surface metalloproteinase/disintegrin kuzbanian is required for axonal extension in drosophila this very elegantly conducted project shows the expression of mmp2 by neurons and the mmp-2-mediated degradation of inhibitory ecm molecules in peripheral nerves induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5 regulated cleavage of a contact-mediated axon repellent function of an axonal chemoattractant modulated by metalloproteinase activity defining brain wiring patterns and mechanisms through gene trapping in mice egf receptor transactivation by g-proteincoupled receptors requires metalloproteinase cleavage of prohb-egf matrix metalloproteinase production in regenerating axolotl spinal cord degradation of interleukin-1β by matrix metalloproteinases inflammation dampened by gelatinase a cleavage of monocyte chemoattractant protein-3 the interaction of mmp2 with a chemokine -mcp3 -converted the mcp3 into an antagonist at several chemokine receptors, thereby abolishing a directional cue for the migration of leukocytes neutrophil gelatinase b potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades ctap-iii, pf-4 and gro-α and leaves rantes and mcp-2 intact multiple sclerosis alzheimer v. wee yong is a central nervous system (cns) glial biologist and neuroimmunologist whose research has been influenced by two diseases of the cns: multiple sclerosis and malignant gliomas. several lines of his investigations have converged on metalloproteinases as mediators of tissue injury or as facilitators of repair. yong favours the concept that the precise spatial and temporal expression of specific metalloproteinase members in particular cell types determines whether these proteases serve as friends or foes in the cns.christopher power is a clinical scientist, neurologist and neurovirologist, who has been investigating the consequences of viral infections of the central nervous system (cns). his work has implicated matrix metalloproteinases (mmps) as the mediators of toxicity in the cns after infections by several viruses, including hiv. the focus of his laboratory is directed towards understanding the mechanisms by which lentiviruses (hiv and fiv) cause neuronal death through the actions of mmps.peter forsyth is a clinical scientist and neuro-oncologist, who has investigated the aberrant expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases (timps) in facilitating the growth and invasiveness of malignant glioma cells. he has used timps and other metalloproteinase inhibitors in pre-clinical testing in animal models of gliomas. forsyth has also participated in clinical trials of metalloproteinase inhibitors in gliomas.dylan edwards is interested in the roles of matrix metalloproteinases and adams (a disintegrin and metalloproteinase) in normal and pathological tissue remodelling. he focuses on metalloproteinases in angiogenesis and cancer biology, as well as the gene regulation of metalloproteinases and tissue inhibitors of metalloproteinases. • matrix metalloproteinases (mmps) and adams (a disintegrin and metalloproteinase) are part of a larger family of structurally related zinc-dependent metalloproteinases called metzincins. structurally, mmps are divided in three domains: an amino-terminal propeptide region, an amino-terminal catalytic domain, and a carboxy-terminal domain that is involved in substrate binding. adams have a prodomain, a metalloprotease region, a disintegrin domain for adhesion, a cysteine-rich region, epidermal-growth-factor repeats, a transmembrane module and a cytoplasmic tail. • the activity of mmps is tightly regulated in several ways: at the level of transcription, by post-translational modifications such as proteolysis, and through the action of endogenous tissue inhibitors of metalloproteinases. the regulation of adams is less well understood, although there is some evidence that the same three levels of regulation might control adam activity. • mmps and adams have been implicated in neuroinflammation and multiple sclerosis (ms), in the pathogenesis of malignant gliomas, and in other neurological conditions such as stroke, viral infections and alzheimer's disease. in the case of adams, their role in these pathological states has begun to be explored, but the available literature is still in its infancy. • although the detrimental roles of metalloproteinases are well documented, some of their functions in the central nervous system (cns) might be beneficial. for example, some metalloproteinases are expressed in the cns during development, pointing to a possible role in brain maturation. similarly, metalloproteinases have been implicated in myelinogenesis and axon growth. furthermore, metalloproteinases are upregulated after injury to the cns, indicating a possible relevance to tissue repair. • several challenges remain in the study of metalloproteinases and their role in brain function. it will be necessary to understand the balance between the beneficial and detrimental roles of mmps to determine whether they can be used as targets for therapeutic intervention. it will also be important to identify the physiological substrates of the different metalloproteinases, and to develop selective antagonists against the various members of the metalloproteinase families; the lack of such tools constitutes one of the main limitations to the growth of the field at present. key: cord-337365-hugenn14 authors: chen, zhuangzhuang; zhong, di; li, guozhong title: the role of microglia in viral encephalitis: a review date: 2019-04-09 journal: j neuroinflammation doi: 10.1186/s12974-019-1443-2 sha: doc_id: 337365 cord_uid: hugenn14 viral encephalitis is still very prominent around the world, and traditional antiviral therapies still have shortcomings. some patients cannot get effective relief or suffer from serious sequelae. at present, people are studying the role of the innate immune system in viral encephalitis. microglia, as resident cells of the central nervous system (cns), can respond quickly to various cns injuries including trauma, ischemia, and infection and maintain the homeostasis of cns, but this response is not always good; sometimes, it will exacerbate damage. studies have shown that microglia also act as a double-edged sword during viral encephalitis. on the one hand, microglia can sense atp signals through the purinergic receptor p2y12 and are recruited around infected neurons to exert phagocytic activity. microglia can exert a direct antiviral effect by producing type 1 interferon (ifn-1) to induce ifn-stimulated gene (isg) expression of themselves or indirect antiviral effects by ifn-1 acting on other cells to activate corresponding signaling pathways. in addition, microglia can also exert an antiviral effect by inducing autophagy or secreting cytokines. on the other hand, microglia mediate presynaptic membrane damage in the hippocampus through complement, resulting in long-term memory impairment and cognitive dysfunction in patients with encephalitis. microglia mediate fetal congenital malformations caused by zika virus (zikv) infection. the gene expression profile of microglia in hiv encephalitis changes, and they tend to be a pro-inflammatory type. microglia inhibited neuronal autophagy and aggravated the damage of cns in hiv encephalitis; e3 ubiquitin ligase pellino (pelia) expressed by microglia promotes the replication of virus in neurons. the interaction between amyloid-β peptide (aβ) produced by neurons and activated microglia during viral infection is uncertain. although neurons can mediate antiviral effects by activating receptor-interacting protein kinases 3 (ripk3) in a death-independent pathway, the ripk3 pathway of microglia is unknown. different brain regions have different susceptibility to viruses, and the gene expression of microglia in different brain regions is specific. the relationship between the two needs to be further confirmed. how to properly regulate the function of microglia and make it exert more anti-inflammatory effects is our next research direction. encephalitis is a common and serious disease, and its clinical manifestations vary from person to person. its main characteristics include altered mental status and various combinations of acute fever, seizures, neurologic deficits, cerebrospinal fluid (csf) pleocytosis, and abnormalities in electroencephalographic (eeg) [1] . although there are many reasons accounting for this disease, the most commonly identified causes are neurotropic viruses. every year in the usa, there are about seven patients per 100,000 population diagnosed as encephalitis. among all the cases, approximately half of them have unknown reasons. of the cases with a known cause, viruses represent 20-50%. herpes simplex virus (hsv) takes up 50 to 75% of identified viral cases, with varicella-zoster virus (vzv), enteroviruses, and arboviruses accounting for the majority of the remainders [2] . it is estimated that the median hospitalization charge for a patient with viral encephalitis is $89,600 for west nile virus encephalitis and $58,000 for hsv encephalitis. each year in the usa, approximately 6000 patients are hospitalized for acute viral encephalitis. the total annual cost is up to $350 million to $540 million, without counting the cost of care after discharge, costs for family caregivers, and lost earning [3] . microglia are cns-resident mononuclear phagocytic cells characterized by a unique ramified shape and distinctive gene expression [4] . most microglia are derived from a yolk sac progenitor and seed the brain early in development [5] . as resident cells, microglia are assumed to help orchestrate the immune response to pathogen infection of the brain. while often touted as immune sentinels, little is known about how or if microglia engage in this function [6] . so far, a large body of evidence stemming from both in vitro and in vivo studies have indicated that microglia are capable of responding to viral pamps [7, 8] , but the multifaceted function of microglia involved in viral encephalitis needs further understanding. recently, how microglia are recruited around infected neurons and the interaction between microglia and other cells has become a hot topic. in this review, we will talk about the function of microglia in viral encephalitis. microglia are recruited around the infected neurons by sensing extracellular atp signals microglia can sense the nucleotides released by infected neurons and are rapidly recruited around infected neurons to exert phagocytic activities in the brain (see fig. 1 ). neurons release more atp after viral infection, while the levels of atp, adp, amp, and adenosine in cell lysates decrease. noxious stimuli in neurons trigger a sustained increase in extracellular atp, which causes microglia to become activated and recruited in minutes to hours [9] . atp is a potent agonist of the p2y g protein-coupled receptor [10] . studies have shown that p2x7 or p2y12 are both abundant in microglia, and p2y12 is a microglia-specific marker in the brain [11] . extracellular atp is a strong chemotactic signal for microglia. by stimulating the p2y receptor of microglia, fig. 1 illustration of the anti-inflammatory role of microglia in viral encephalitis. the picture shows that microglia can sense atp signals through the purinergic receptor p2y12 and are recruited around infected neurons to exert phagocytic activity. microglia can exert a direct antiviral effect by producing type 1 interferon (ifn-1) to induce ifn-stimulated gene (isg) expression of themselves. ifn produced by microglia exects indirect antiviral effects by acting on other cells to activate corresponding signaling pathways it mediates the rapid response of microglia to the injury site [12] . the number of p2y12 receptors on the surface of microglia increased over twofold in response to viral infections. some experiments have built a p2x7-or p2y12-deficient mice model (p2x7 −/− or p2y12 −/− ) in vivo, the lack of p2x7 has no effect on the recruitment of microglia, whereas an absence of p2y12 resulted in> 50% reduction in the numbers of microglia recruited to infected neurons. this indicates that microglia are recruited around the infected neurons via p2y12 signaling. the cd86+ phagolysosomes of p2y12 −/− microglia decreased significantly compared with normal mice which indicates that p2y12 is essential for microglia to exert phagocytic activity [9] . microglia are key contributors to the recruitment of monocytes into the brain during viral infection. in microglia-depleted mice, the phenomenon of monocytes entering the brain is almost completely gone. cd45+ blood-borne leukocytes were seen in the brain of p2y12 −/− mice after infection, and mononuclear cell infiltration was not impaired, which indicates that microglia recruit monocytes through a p2y12 independent mechanism [9] . analysis of temporal lobe specimens in patients with herpes simplex type 1 (hsv-1) encephalitis shows that p2y12-positive microglia processes extend to hsv-1 positive cells, and there are about 1-3 activated microglia around each infected neuron. microglia appear around the cell bodies and dendrites of neurons before the mature virus particles appear in infected neuronal cells. at this stage, the neuronal membrane is intact and there is a normal chromatin structure in the nucleus. cell trajectory analysis has shown that the speed of microglia movement in mice with viral encephalitis is reduced, which is related to their tendency to stay around the infected cells [9] . typical neurological symptoms appear in mice infected with virus when microglia are completely absent, but not p2y12 deficiency [9] . consistent with this, mice with microglia depletion have higher viral loads and succumb to infection. the rapidly deteriorating neurological symptoms in microglia-depleted mice may be associated with significantly increased neuronal infection and potential microglial protective mediators such as interleukin-10 deficiency [13] . microglia play a direct antiviral effect by producing ifn-1 after recognition of virus by pattern recognition receptors (prrs) mouse hepatitis virus (mhv) is a neurotropic coronavirus. on the fourth day of viral infection, microarray analysis of infected mouse microglia showed that the ifn-1 pathway was the most upregulated [14] . ifn-1 protects the host because treatment with exogenous ifn-α or ifn-β limits viral replication, whereas the infection of mice with ifn-1 signaling deficiency can convert nonfatal coronavirus to a lethal type [15] . in the mouse model of vesicular stomatitis virus (vsv) encephalitis, it was found that infected microglia produced ifn-1, and both infected and uninfected microglia upregulated the expression of ifn regulatory factors 7 (irf7) and activated innate immunity which limits the transsynaptic spread of vsv [16] . viral infection can induce the expression of ifn-stimulated genes (isgs), thereby interfering with viral replication and promoting viral clearance [16] (see fig. 1 ). microglia express a wide range of prrs, which are important for the defense of hsv-1. virus induces expression of ifn-1 by the recognition of nucleotides and (rna-/dna-sensing) prrs [7, 8] (see table 1 for a summary). the combination of ifn-1 and the heterodimeric receptor ifnar produces a cellular response that initiates a signaling cascade that promotes heterodimers stat 1/2 nuclear translocation and transcriptional activation of isgs [17] . the rapid expression of hundreds of isgs is critical for controlling viral infections because these proteins block the entry, translation, transcription, assembly, and efflux of the virus [18] . cytosolic dsdnas can induce a strong innate immune response. gmp-amp synthase (cgas) is a cytosolic dna receptor [19] . the binding of cgas to cytosolic dna produces a second messenger, cyclic-gmp-amp (cgamp), which activates downstream sting and ultimately activates transcription factor irf3, which promotes ifn-β production [8, 20] . different types of prrs can be involved in the identification of viral infections, including membrane-associated tlrs, cytosolic rna-sensing rig-like receptors (rlrs), and dna sensors. prrs activates ifn regulatory factors 3 (irf3) through unique adaptor molecules such as tir domain-containing adaptor inducing ifn-β (trif), mitochondrial antiviral signaling (mavs), and stimulator of type i ifn genes (sting) [21] . mutations in the irf3 gene in herpes simplex encephalitis (hse) patients impaired interferon production and the tlr3-trif pathway is the most severely damaged [22] . the cgas-sting and tlr3-trif pathways are the major intrinsic sensing pathways to control hsv1 infection in the cns. microglia can induce ifn responses by recognizing viral pathogen-associated molecular patterns (pamps) through prrs. in a typical ifn-1 response pattern, prrs first induce the production of ifn-β after recognition of pamps, and then ifn-β binds to ifn-α receptor (ifnαr) to initiate irf7 gene expression in an autocrine or paracrine manner which enables a complete ifn-1 response in viral spread or secondary infections [23] . in order to simulate the natural pathway of hsv-1 entering the central nervous system by retrograde transport, the previous experiment used an ocular infection model. it was confirmed that microglia are the main source of hsv-induced ifn-1, which is induced in a cgas-sting-dependent manner. infected cells including neurons and microglia detected hsv-1 by the cytoplasmic dna receptor, the adaptor protein sting. the brain of mice lacking cgas and sting detected higher viral load, and the virus spread widely to the midbrain, hypothalamus, and preoptic area. however, studies have shown that the recruitment of microglia to the infection site is not related to sting [24] . in a model of mhv infection, depletion of microglia using colony-stimulating factor 1 receptor (csf1r) inhibitors revealed that depletion of microglia at 0-6 days of infection would result in increased mortality and a later depletion had no influences on survival. it indicated that microglia play an important role in controlling viral replication and reducing mortality in the early stage of infection [14] . upon intranasal vesicular stomatitis virus (vsv) infection, studies have found activated microglia and monocyte aggregation in the olfactory bulb. studies have also shown that microglia accumulating around the olfactory bulb form a natural immune barrier plays an important role in limiting the spread of vsv in the cns and preventing lethal encephalitis. the formation of the intrinsic microglia barrier is regulated by ifnar signaling of neurons and astrocytes and is not regulated by microglia themselves [25] . it has been previously reported that the use of brain-penetrant csf-1r inhibitor blz945 in mice can remove microglia [26] . il-34, as a csf-1r ligand, is essential for the maintenance of microglia. blz945 plays a role in specifically depleting microglia by inhibiting the interaction of csf-1r with il-34 [27] . after depletion of microglia, vsv-infected mice have higher viral load in the brain, cerebellum, and brainstem and have higher mortality, indicating that the formation of microglia barrier in the olfactory bulb is indispensable to limit the spread and spread of vsv [25] . sall1 is specifically expressed in microglia and is a negative regulator of microglia activation. sall1 is downregulated in microglia when infected by pathogens, thereby promoting microglia activation [28] . microglia confer sting-dependent antiviral activity to neurons and initiate the production of ifn-1 in astrocytes via the toll-like receptor 3 (tlr3) pathway [24] . type i ifnar signaling in astrocytes acts to stabilize the blood-brain barrier (bbb) and protect the brain from viral infections and immunopathology (see fig. 1 ). in a mouse model of west nile virus (wnv) infection, it was found that the permeability of the bbb increased in mice lacking ifnar, especially in the hindbrain, which leads to an increase in viral entry, indicating that ifnar signaling has important regulatory effects on bbb permeability in this brain region. the study found that human and mouse cerebellar astrocytes have higher expression of prrs and isgs in both steady state and ifn-induced state compared with cerebral cortical astrocytes. cerebellar astrocytes can reach maximum isg expression more quickly after receiving ifn stimulation [29] . in vitro studies have shown that ifnar pathways in brain microvascular endothelial cells (bmecs) and astrocytes can both promote the integrity of tight junction. studies have shown that ifn-1 stabilizes the integrity of bbb and promotes the formation of tight junctions by rearranging endothelial junction proteins via rho-rac signaling pathways, promoting bbb tightening and blocking the expression of il-1β and tnf-α [30] . it is well known that vascular cell adhesion molecule 1 (vcam-1) is a neuroinflammatory regulator on vascular endothelial cells [31] . ifn-1 regulates immune transport by regulating the expression of vcam-1 (cd 106) on neurovasculature [32] . ifn-β treatment of either human or mouse primary astrocyte cultures from the cerebellum, but not from the cerebral cortex, significantly downregulates vcam-1 expression [29] . drug blocking of vcam-1 binding to its ligand leukocyte adhesion molecule 4 (vla-4) has been shown to reduce pathogenic neuroinflammation and improve survival after wnv infection [33] . in ifnar-deficient mice, cerebellar vasek et al. [52] complement c3 and c3 cleavage products c3r cell surface damaging of presynaptic ends and resulting in visual-spatial processing disorders and impaired memory function infiltrating lymphocytes and macrophages were significantly reduced after treatment with vla-4 blocker [29] . loss of astrocyte ifnar signaling pathway can cause a variety of consequences, including increased expression of inflammatory cytokines and chemokines, which disrupt the blood-brain barrier during neurotropic viral infection [30] . in different brain regions, transcription, immunophenotypic, and bioenergetic properties of microglia are heterogeneous. cerebellar and hippocampal microglia maintain a stronger immune alert status than microglia in the striatum and cortex. the sensitivity of different brain regions to aging is different, and the cerebellum is the most obvious. this suggests that different brain regions have different responses to aging-related neurodegenerative lesions [34] . microglia also affect the adaptive immune response. depletion of microglia changes the response of cd4+ t cells to viral infection in the brain, the total number and percentage of cd4+ t cells decrease, and the frequency and number of tregs also decrease, indicating that microglia are crucial for fully activating virus-specific t cells. deletion of microglia in the early stages of whv infection exacerbates infection, which may be associated with loss of microglial antigen presentation function, as microglia participates in antigen presentation by upregulating tap1 and itgax. depletion of microglia can also result in the loss of major mhc ii-expressing cell type, which can also result in decreased expression of mhc ii in incoming monocytes/macrophages. because mhc ii is required for re-stimulation and activation of cd4+ t cells, a decrease in mhc ii reduces the response of virus-specific cd4+ t cells [14] . in addition to producing ifn-1, microglia can also produce other cytokines. the vitro cell culture demonstrates that cytomegalovirus (cmv)-infected astrocytes recruit microglia to the infected foci by releasing monocyte chemoattractant protein 1 (mcp-1). microglia produces tumor necrosis factor α (tnfα) and interleukin 1β (il-1β), interleukin 6 (il-6) exerting antiviral effect and inhibiting the replication of cmv in astrocytes [35] . cmv-infected microglia produce t lymphocyte chemotactic factor cxcl 10, recruiting activated t lymphocytes into the infected area, and t cells produce ifn-γ to inhibit virus replication and dissemination [36] . even with limited viral replication, cytopathic effects were evident in the hsv-infected microglia. microglia responded to nonpermissive viral infection by producing considerable amounts of tnf-α, il-1β, and ip-10. tnf-α inhibits hsv replication in astrocytes, and ip-10 possesses direct antiviral activity in neurons [37] . inflammation-induced, sting-dependent autophagy limits zika virus (zikv) infection in drosophila brains. the activation of nf-kb dependent on zikv induces expression of drosophila stimulator of interferon genes (dsting) in the brain. dsting protects against zikv infection by inducing autophagy in the brain. loss of autophagy leads to increased viral load in the brain and increases infection in drosophila [38] . multiple microbial infections activate nfkb-dependent inflammatory signaling cascades, which induce transcription of downstream antimicrobial effectors, including antimicrobial peptides (amps), thereby reducing or eliminating infection [39] . endogenous, bacterial-derived circulating dinucleotides and increased expression of sting mrna can activate sting [40] . from fruit flies to mammals, activation of antimicrobial autophagy protects cells against a variety of pathogens, including bacteria, viruses, and parasites [41, 42] . studies have shown that drosophila infected with zikv induces activation of nf-kb signaling in the imd pathway, thereby activating downstream dsting. dsting can limit intracellular pathogens by inducing autophagy [43] . autophagy protects neurons against a variety of stressors, including viral infections. autophagy clears infection by capturing pathogens, including viruses, in the cell without causing cell death, which is beneficial for mature neurons [42, 44] . previous studies used heat shock-inducible gal4 (hs-gal4) to silence autophagy core genes atg5 and atg7 in vivo [45] and found that the virus titer of zikv in drosophila is significantly increased. relish is a transcription factor of nf-kb in the imd pathway. using a validated rnai line which specifically removes relish from neurons or glial cells in vivo, known as a neuron-specific gal4 driver (elav-gal4) or a glia-specific gal4 driver (repo-gal4), respectively [46] , increased viral infection. in mammals, autophagy limits the replication or pathogenesis of herpes simplex virus type 1 (hsv-1) [47] . intracellular receptors, such as retinoic acid-inducible gene-i (rig-i) and melanoma differentiation-associated protein 5 (mda-5), collectively referred to as rig-i-like receptors (rlrs), can also detect cytoplasmic viruses nucleic acid [48] . using the advantages of drosophila, the study found that the prr-autophagy axis plays an important role in host defense [49] . fifty percent of patients with neurotropic wnv infection show chronic cognitive sequelae [50] . patients who survive after infection with wnv usually present with visual-spatial processing disorders and impaired memory function [50, 51] . studies have shown that complement c3 and c3ar mediate the loss of presynaptic ends in mouse hippocampus (see fig. 2 ). mice demonstrate spatial learning in the recovery period of neurological invasive diseases in west nile. both neurons and microglia express c3ar, which recognizes c3 cleavage products. c3 and its cleavage products attract microglia to gather around the neurons to exert phagocytosis activity and to clear the presynaptic ends. in the situation of a neurotropic virus infection, clearance of the presynaptic end may prevent trans-synaptic spread of the virus and stop abnormal signals from infected neurons [52] . infected and activated microglia in the cns persist for more than 12 months after receiving antiviral therapy in patients with hsv encephalitis [53] . this may lead to chronic damage to the cns. microglia can produce tnfα [35, 36] . local increase of tnfα in the hippocampal dentate gyrus activates astrocyte tnf receptor type 1 (tnfr1), which in turn triggers an astrocyte-neuron signaling cascade that results in persistent functional modification of hippocampal excitatory synapses which can lead to cognitive disturbance [54] . glutamatergic gliotransmission provides a stimulatory input to excitatory synapses in the hippocampal dentate gyrus. tnfα critically controls the process which may influence synaptic transmission and plasticity [55] . this suggests that microglia may also affect synaptic function through cytokines. the unprecedented re-emergence of the zika virus (zikv) virus has shocked the world, and reports on microcephaly in brazil are also increasing [56] (see fig. 2 ). in brazil, the incidence of fetal microcephaly in zikv-infected pregnant women is approximately 12.8% [57] . surprisingly, there have been increased reports of cases of simultaneous diagnosis of guillain-barré syndrome in adults with french polynesia zikv infection [58] . recent evidence suggests a causal relationship between prenatal zikv infection and severe brain abnormalities including microcephaly [59] . zikv was detected in the brain of a microcephalic fetus [60] . this means that there is a strong connection between zikv and these neurological diseases. the study found that zikv mainly infects fetal microglia and induces high levels of pro-inflammatory mediators including il-6, tnf-α, il-1β, and mcp-1. these inflammatory factors may be harmful to the fetus [61] . microglia's perivascular localization enables them to survey the influx of blood-borne components into the cns [62] . thus, microglia are highly susceptible to zikv infection. the analysis of vitro culture and microcephalic fig. 2 illustration of the pro-inflammatory role of microglia in viral encephalitis. the picture shows some harmful effects of microglia in viral encephalitis, such as mediating presynaptic membrane damage, causing fetal congenital malformations, and exacerbating the damage of cns in hiv encephalitis. microglia play an uncertain role in virus-induced aβ. the role of the ripk3 signaling pathway in microglia in viral encephalitis remains obscure fetal brain histology showed that zikv leads to activation of microglia [60] . this, in turn, causes localized neuroinflammation and is accompanied by viral disseminating to the brain parenchyma, which may lead to the development of neuronal death, especially in the cerebral cortex, causing neurological changes and microcephaly. these findings reveal that microglia play an important role in the pathogenesis of congenital zikv infection [61] . microglia are the main target of hiv-1 infection in the brain [63] . there are viral particles in the infected microglia, suggesting that they may be a potential virus reservoir [64] . the main features of hiv-1 encephalitis are the formation of multinucleated giant cells, microglia nodules, and macrophage infiltration into the central nervous system [65] . the formation of multinucleated giant cells reflects the infection of hiv-1 microglia in vitro. active infection of microglia is ultimately associated with astrogliosis, myelin pallor, and neuronal loss [65] . although there are ample evidences confirming that activated microglia have neuropathic effects in hiv-1 encephalitis, there are data suggesting that microglia may have some neuroprotective effects in the early stages of hiv disease [66] . molecular and histopathology confirm the reduction of autophagy in the brain of patients with hiv-associated dementia. hiv-1-infected microglia release neurotoxins, cytokines, viral proteins, and glutamate; activate nmda receptors on neurons; cause calcium influx, inhibit neuronal autophagy; enhance apoptotic pathways; and ultimately lead to neuronal death [67] . enhancing autophagy of neurons in patients with hiv encephalitis may become a target for future treatment. microarray analyses were performed using brain tissue samples from hiv encephalitis (hive) patients, hiv+ patients without encephalitis (hiv/noe), and hiv− controls. it was found that a large number of microglia genes have undergone significant changes during hiv infection, including immune activation and function, kinases, phosphatases, and pro-/anti-apoptotic and neurotrophic factors, indicating that the microglia function is impaired and has a pro-inflammatory tendency [68] . human cytomegalovirus (hcmv) can cause congenital encephalitis or cause encephalitis in aids patients [69] . in patients with advanced aids, hcmv-induced central nervous system infection leads to two different neuropathological patterns: microglial nodular encephalitis (mgne) and ventriculoencephalitis (ve). microglial nodular encephalitis consists of diffuse microglial cells, aggregated astrocytes, and giant cells. the formation of microglial nodules is considered to be an important cause of hcmv-related dementia in aids patients [70] . recently, a study hypothesized that, in rasmussen encephalitis (re), microglial nodules with an upregulation of tlrs provide an environment for the initiation of the later dominating t cell cytotoxicity. however, in the early stages of viral encephalitis, the interaction between microglial nodules and pathogenic t cells remains unclear [71] . in the brain, peli1 is mainly expressed in microglia. in a mouse model of lethal west nile virus (wnv) infection, peli1 promotes the production of pro-inflammatory cytokines and chemokines in microglia and promotes the entry of t cells and macrophages into the cns. peli1 plays a crucial role in wnv entry and replication in human and murine neurons and microglia (see fig. 2 ). peli1 promotes adhesion, replication, and synthesis of mature virions. autopsy of patients who died of wnv encephalitis found that peli1 was highly expressed on wnv-infected neurons and adjacent inflammatory cells. peli1-deficient microglia and neurons cause inflammation and reduce host susceptibility to lethal encephalitis [72] . microglia are the main producers of central nervous system inflammatory cytokines il-6 and tnf-α and chemokines ccl2 and cxcl10 after wnv infection [73] . the peli1-deficient mice have better resistance to wnv, reducing levels of cytokines and chemokines in the body, including il-6, tnf-α, il-1β, and il-10 and ccl2 and ccl5 [72] . peli1 expressed in microglia can promote the degradation of tnf receptor-associated factor 3 (traf3) by regulating tlr/myd88 signaling and can activate microglia during the induction of experimental autoimmune encephalomyelitis (eae) [74] . it is well known that microglia respond to viral infection by activating p38mapk [75] . in wnv-infected peli1 −/− neurons, phosphorylation levels of p38mapk and p65 decreased, indicating that peli1 plays a positive regulatory role in nf-κb and p38mapk activation [72] . peli1 facilitates wnv replication in microglia and neurons, especially in the latter, which are the major cells infected during in vivo challenge. it also positively mediates nf-κb and/or p38mapk activation in these cells and boosts a robust production of inflammatory cytokines and chemokines, which attracts more infiltration of inflammatory cells from the periphery and ultimately contributes to lethal wnv encephalitis. thus, peli1 synergistically promotes virus dissemination and inflammation in the cns [72] . under physiological conditions, neurons can produce trace amounts of amyloid-β peptide (aβ) to maintain synaptic plasticity and memory and play an antimicrobial role. the clearance of aβ is achieved by phagocytosis of microglia. hsv reactivation would trigger increased aβ production, activation of microglia, and release of pro-inflammatory cytokines (pathological state) that would sustain microglia activation and initiate a vicious circle of inflammatory responses. recurrence of this series of events during life would result in amplification and irreversible damage to hippocampus [76] . studies have shown that aβ oligomers can bind glycoproteins on the surface of herpesvirus and accelerate aβ deposition showing protective viral entrapment activity in 5xfad mouse and 3d human neural cell culture infection models against neurotropic hsv1. this indicates that aβ fibrils and deposits may be important in protecting the brain from common viral infections [77] . carbohydrate-binding promotes aβ fibrillization and leads to protofibril generation on microbial surfaces. bound protofibrils first inhibit host cell adhesion by pathogens. then, propagating aβ fibrils mediate agglutination and sequestration of microbes within fibrillar β-amyloid. when the aβ oligomer-mediated protective antimicrobial pathway is over-activated, progressive aβ deposition can cause neuroinflammation, neuropathology, and extensive neuronal death, leading to alzheimer's disease (ad), a model called antimicrobial protection hypothesis [77] . recently, a 16-year study involving more than 33,000 patients found that hsv 1 infection increased the risk of dementia by 2.56 times. and long-term use of anti-herpes drugs seems to reduce the risk of dementia in patients with hsv-1 infection. in general, patients with shorter (< 30 days) or longer (≧ 30 days) durations of anti-herpetic medications were associated with a decreased risk of dementia, and the treatment duration of ≧ 30 days was associated with a lower risk of dementia than those of a duration of < 30 days [78] . microglia have been involved in ad for more than 100 years. nissl and alzheimer first described amoebalike glial cells in various neurological states in 1904 and 1910. this amoeba-like glial cell was later confirmed to be a microglia. microglia were activated in amyloid plaque-enriched areas in alzheimer's disease brains [79] . some findings reveal that microglia serve as important physiological functions in learning and memory by promoting learning-related synapse formation through brain-derived neurotrophic factor (bdnf) signaling [80] . in the ad mouse model, a disease-associated microglia (dam) subtype was found. dam selectively aggregates around aβ and exerts phagocytosis. immunohistochemical staining of mice and human brain slices shows dam with intracellular/phagocytic aβ particles [81] . the accumulation of microglia around the aβ plaque is critical to establishing a physical barrier that limits the spread of plaque and protects surrounding neurons from the toxic effects of the plaque [82] . interestingly, in a mouse model lacking triggering receptor expressed on myeloid cells 2 (trem2), microglia could not accumulate around aβ and the survival and proliferation of microglia around the plaque is impaired. at the same time, the migration of microglia to the lesion is impaired [83, 82] . the microglia lacking trem2 has metabolic disorders, which are characterized by defects in glycolysis, atp levels, and anabolism [83] . at the same time, the signaling pathway of trem 2 is essential for phagocytosis of microglia, and the deletion of trem 2 leads to aggravated pathology [84] . more malnourished neurons were observed in the brain sections in patients carrying ad-associated trem2 variants [85] . in contrast, it has been reported that when microglia respond to aβ plaques, it activates inflammatory bodies, leading to the escalation of inflammation and the release of apoptosis-associated speck-like protein containing a card (asc) specks from inflammasomes, which in turn may initiate the seeding of new plaques [86] . moreover, excessive activation of microglia in the early stages of ad may cause excessive synaptic pruning, leading to cognitive impairment [87] . in summary, hsv-1 infection accelerates the deposition of aβ in the brain and activates microglia. and the role of aβ during viral encephalitis remains obscure and requires additional study. necroptosis is a programmed cell death coordinated by receptor-interacting protein kinases 1 (ripk1) and 3 (ripk3). mixed lineage kinase domain-like protein (mlkl) is an executioner protein that participates in the formation of an apoptotic complex that promotes cell death through cell membrane disruption and cell rupture [88] . studies have shown that the ripk3 signaling pathway can control a variety of viral infections, including hsv-1. murine but not human rip1/ rip3 directly senses hsv-1 icp6 to initiate necroptosis [89] . in the wnv virus infection model, it was found that ripk 3 inhibits viral pathogenesis by cell death-independent neuroinflammation, mice lacking ripk 3, or ripk 1 kinase activity, but not those lacking mlkl or mlkl, and caspase-8 increased susceptibility to lethal wnv infection. during wnv infection of neurons, ripk3 is activated in neurons which promotes the expression of chemokines ccl2, cxcl10, etc. and recruits infiltration of immune cells including t cells, mononuclear macrophages, and antigen presenting cells which exert antiviral effects. this process does not affect the number of microglia in cns [90] . what role does the ripk3 signaling pathway in microglia play in viral encephalitis? further exploration is needed. most of the experiments on microglia and viral encephalitis remain in the experimental stage, and many mechanisms need to be further explored before being applied to the clinic. although microglia can recognize atp released by infected neurons through p2y12 and are recruited around neurons, whether there are more other potent chemokines, further exploration is needed. although ifn-1 plays an antiviral role in most encephalitis, the optimal ifn-1 concentration and optimal application time are still undetermined. microglia can induce autophagy to exert antiviral effects, but there is insufficient evidence. microglia produce inflammatory and chemokines and induce adaptive immune responses. microglia resident in different brain regions have specific gene expression profiles, and different brain regions have different susceptibility to viruses. the relationship between the two remains undetermined, and it is expected that the innate immune response to the virus will be enhanced by changing the gene expression profile of microglia in the future. in wnv infection, microglia mediate presynaptic terminal loss in the hippocampus though recognizing c3 by c3r, c3, or c3r-specific antibodies may block this process. microglia inhibit autophagy in patients with hive, aggravating cns injury. how to inhibit or delete the function of microglia or how to enhance the autophagy of neurons will become the direction of future research. there is a strong relationship between fetal cerebellar malformation and activation of microglia in zikv infection, and it is necessary to develop microglia inhibitors that are safe for pregnant women and fetuses. pelia of microglia promotes viral replication in neurons, and moderate blockade of pelia may become a target for future treatment. in viral encephalitis, aβ produced by neurons increases the risk of dementia in patients. studies show that prolonging antiviral time will reduce the risk of dementia. the interaction between aβ and activated microglia in patients with viral encephalitis is unknown. it is unknown whether moderately enhancing the phagocytosis of microglia can reduce the risk of dementia. it needs further research. traditionally, activation of ripk3 will result in the formation of an apoptotic complex that mediates cell death. recent studies have shown that in viral encephalitis, activation of neuronal ripk3 exerts an antiviral effect in a death-independent pathway, and the ripk3 pathway of microglia requires further investigation. microglia account for 10% of the total number of cells in the adult central nervous system [91] . in a healthy cns, microglia are not "resting" but in a highly active "surveillance and rapid response" state [92] . microglia processes are dynamic, continuously scanning the environment and sampling it [93] . they are able to detect changes in ph, purines, cytokines, chemokines, amino acids, and inorganic compounds [94] . microglia are derived from the primitive macrophages of the yolk sac, which migrate to the developing central nervous system. they have the ability to self-renew, thus maintaining their number throughout life without any input from the bone marrow-derived precursor cells [95, 96] . microglia can respond to neurons, which in turn affects neuronal activity. microglia are involved in the programmed death of neurons and the apoptosis and clearance of new neurons during development. the activity of microglia promotes the pruning, elimination, and maturation of synapses [92] . microglia serve important physiological functions in learning and memory by promoting learning-related synapse formation through bdnf signaling [80] . the phagocytic activity of microglia is essential for the clearance of senescent cells and debris [97] , slowing the toxic effects of amyloid-β [98] . microglia are not only important cells that maintain the homeostasis of cns, but also respond to injury, infection, and neurodegeneration through proliferation and altered transcription and morphology [99, 92] . microglia are often touted as the first responder to cns infection and respond quickly to injury [93] . however, depending on the stimulus received, this activation profile is also different and may result in harmful or beneficial effects [100, 6] . hsv-1 can be treated, but can wnv or enteroviruses? the high morbidity and high morbidity rate of viral encephalitis have caused widespread concern. although traditional antiviral therapy 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in alzheimer's disease complement and microglia mediate early synapse loss in alzheimer mouse models mixed lineage kinase domain-like protein mlkl causes necrotic membrane disruption upon phosphorylation by rip3 rip1/rip3 binding to hsv-1 icp6 initiates necroptosis to restrict virus propagation in mice oberst a. ripk3 restricts viral pathogenesis via cell death independent neuroinflammation microglia: scapegoat, saboteur, or something else? sublime microglia: expanding roles for the guardians of the cns resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo heterogeneity of cns myeloid cells and their roles in neurodegeneration regulation of microglia development and homeostasis development and homeostasis of "resident" myeloid cells: the case of the microglia clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2 gsecretase component presenilin is important for microglia b-amyloid clearance myeloid cells in the central nervous system differential roles of microglia and monocytes in the inflamed central nervous system not applicable. not applicable. authors confirm that all relevant data are included in the article. authors' contributions zc carried out the literature review, participated in the sequence alignment, and drafted the manuscript. dz helped to draft the manuscript. gl conceived, designed, and coordinated the study and contributed to and finalized the draft. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable as no patients/participants involved in this review. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-353298-vr5hnzp8 authors: nan title: in vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination date: 1990-09-01 journal: j cell biol doi: nan sha: doc_id: 353298 cord_uid: vr5hnzp8 a demyelinating disease induced in c57b1/6n mice by intracranial injection of a coronavirus (murine hepatitis virus strain a59) is followed by functional recovery and efficient cns myelin repair. to study the biological properties of the cells involved in this repair process, glial cells were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. using three-color immunofluorescence combined with [3h]thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. we identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocyte (o-2a) progenitor cells with the o4 antibody. cultures from demyelinated tissue differed in several ways from those of age-matched controls: first, the total number of o-2a lineage cells was strikingly increased; second, the o-2a population consisted of a higher proportion of o4-positive astrocytes and cells of mixed oligodendrocyte-astrocyte phenotype; and third, all the cell types within the o-2a lineage showed enhanced proliferation. this proliferation was not further enhanced by adding pdgf, basic fibroblast growth factor (bfgf), or insulin-like growth factor i (igf-i) to the defined medium. however, bfgf and igf-i seemed to influence the fate of o-2a lineage cells in cultures of demyelinated tissue. basic fgf decreased the percentage of cells expressing galactocerebroside. in contrast, igf-i increased the relative proportion of oligodendrocytes. thus, o-2a lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. these properties may be linked to the efficient remyelination achieved in this demyelinating disease. c57b1/6n mice by intracranial injection of a coronavirus (murine hepatitis virus strain a59) is followed by functional recovery and efficient cns myelin repair. to study the biological properties of the cells involved in this repair process, glial ceils were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. using three-color immunofluorescence combined with [3h]thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. we identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocyte (o-2a) progenitor cells with the 04 antibody. cultures from demyelinated tissue differed in several ways from those of agematched controls: first, the total number of o-2a lin-cage cells was strikingly increased; second, the o-2a population consisted of a higher proportion of 04positive astrocytes and cells of mixed oligodendrocyteastrocyte phenotype; and third, all the cell types within the o-2a lineage showed enhanced proliferation. this proliferation was not further enhanced by adding pdgf, basic fibroblast growth factor (bfgf), or insulin-like growth factor i (igf-i) to the defined medium. however, bfgf and igf-i seemed to influence the fate of o-2a lineage cells in cultures of demyelinated tissue. basic fgf decreased the percentage of cells expressing galactocerebroside. in contrast, igf-i increased the relative proportion of oligodendrocytes. thus, o-2a lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. these properties may be linked to the efficient remyelination achieved in this demyelinating disease. i ~ demyelinating diseases, damage to myelin sheaths disrupts conduction of electrical impulses along axonal processes of neurons. typically, damaged myelin is not efficiently repaired in the human cns (perier and gregoire, 1965; prineas et al., 1984) . thus patients with cns demyelinating diseases, such as multiple sclerosis, frequently experience prolonged neurological dysfunction. in contrast, efficient remyelination and functional recovery is achieved in rodents in certain experimental models of cns demyelination (reviewed in ludwin, 1981) . one such model is produced by infecting c57b1/6n mice with a coronavirus (murine hepatitis virus strain a59; mhv-a59), which leads to the development of multiple cns demyelinating lesions (lavi et al., 1984; jordan et al., 1989) . this coronavirus replicates in glial cells during an early phase of the disease and is subsequently cleared from the cns (jordan et al., 1989) . infected mice exhibit clinical signs of cns demyelin-ation within the first week post infection (wpi)? by 3-5 wpi, focal areas of demyelination are present throughout the cns, with prominent lesions in the spinal cord. in the following weeks, remyelination is paralleled by functional recovery. using this model, we can analyze the processes involved in regeneration of the cells that form cns myelin. myelination in the developing cns requires the coordinated interaction of several types of glial cells (reviewed in raine, 1984; wood and bunge, 1984; raft, 1989) . in vitro studies of optic nerve from newborn rat have characterized a bipotential progenitbr cell that can give rise to oligodendrocytes or to type 2 astrocytes, 'and is therefore called an oligodendrocyte-type 2 astrocyte (o-2a) progenitor cell. type 2 astrocytes express antigens that are also present on the surface of o-2a progenitor cells. the localization and function of type 2 astrocytes in vivo is not clear at the present time. another type of astrocyte, called the type 1 astrocyte; appears before type 2 astrocytes and arises from a glial precursor distinct from the o-2a progenitor (raft et al., 1984) . type 1 astrocytes secrete growth factors that affect the proliferation, migration, and/or differentiation of o-2a progenitors (reviewed in raft, 1989; dubois-dalcq and armstrong, 1990) . upon maturation, oligodendrocytes elaborate extensive processes which wrap around cns axons to form myelin (reviewed in paine, 1984) . perinodal astrocytes, possibly type 2 astrocytes, extend processes to nodes of ranvier, which are electrically excitable regions of axons between adjacent myelin sheaths (ffrench-constant and raft, 1986b; ffrench-constant et al., 1986) . both oligodendrocytes and perinodal astrocytes seem to play a role in efficient saltatory conduction of nerve impulses; myelin acts as an electrical insulator while perinodal astrocytic processes may serve various functions associated with impulse propagation at nodes (discussed in ritchie, 1984; black and waxman, 1988) . in normal adult cns oligodendrocytes proliferate minimally (mccarthy and leblond, 1988) . attempts to induce in vitro mitosis of oligodendrocytes with exogenous growth factors have not been successful (yong et al., 1988) . however, oligodendrocytes isolated from adult cns do proliferate when cocultured with neurons (wood and bunge, 1986 ). in addition, o-2a progenitor cells isolated from adult rat optic nerve are capable of proliferating in vitro in the absence of neurons and maintain the ability to differentiate (ffrench-constant and raft, 1986a; wolswijk and noble, 1989) . these studies provide evidence that oligodendrocytes and/or o-2a progenitor cells may contribute to the regeneration of o-2a lineage cells in the adult cns. after widespread cns demyelination, surviving oligodendrocytes at the periphery of lesions do not seem to be able to elaborate myelinating processes and/or relocate to achieve efficient remyelination. proliferation of oligodendrocytes, or oligodendroctte precursor cells, during demyelination appears to be necessary to facilitate remyelination. electron microscopic autoradiographic analyses of demyelinating tissue have reported mitosis of oligodendrocytes at several stages of maturation (herndon et al., 1977; ludwin, 1979; aranella and herndon, 1984; ludwin and bakker, 1988) . a recent in vivo analysis combining immunolabeling with autoradiography has shown that o-2a progenitor cells and astrocytes that express 04 antigens proliferate early in the course of demyelination induced by coronavirus infection and that some cells generated during demyelination later became oligodendrocytes (godfraind et al., 1989) . in the present study, we have developed an in vitro system to further characterize the cells involved in the remyelination process. we have used threecolor immunofluorescence combined with autoradiography to identify specific glial cell types. in such a system we can analyze the proliferative capacity, phenotypic plasticity, and differentiation potential of the various types of glial cells which may play a key role in cns remyelination. female c57b1/6n mice, without prior exposure to mhv-a59, were obtained from the frederick cancer research facility (frederick, md). at 28 d of age each mouse was injected intracranially with 1,000 plaqueforming units of coronavirus (mi-iv-a59), which had been propagated in the 17 ci 1 line of spontaneously transformed balb/c 3t3 cells (sturman et al., 1980) . this age and inoculation dosage op "ttmized the proportion of mice developing demyelinating lesions ('069 %) relative to the incidence of acute mortality (,o11%). at 1, 3, 4, 5, or 8 wpi, mice were anesthetized with methoxyflurane and then decapitated. for each glial cell isolation, spinal cords remov&t from three infected mice exhibiting neurological dysfunction (woyciechowska et al., 1984) were combined and spinal cord tissue from three age-matched control mice was prepared simultaneously. spinal cords were minced and then dissociated according to a procedure modified from miller et al. (1985) . the tissue was incubated at 37°c with enzymes (twice for 20 rain in 0.125% trypsin with 0.02% collagenase in mem-hepes followed by one 15-min incubation in 0.05 % trypsin with 0.53 m edta), bathed in a solution that inhibited trypsin and digested free dna (0.25% soybean trypsin inhibitor, 0.002% dnase i, 0.166% bsa, and 5% fbs in dme) and passaged through pipettes (5 ml, 10 times; 1 ml, 10 times; pasteur pipette 10 times). floating cells were transferred to a 50-ml tube which was then filled with isolation medium (1 mm hepes, 50 u/ml penicillin, 50 #g/ml strep~ tomycin, and 25/zg/ml gentamycin in hbss without calcium or magnesium, ph 7.4) and spun for 10 min at 1,500 rpm in a centrifuge (glc-2b; sorvall die., newton, ct). the supernatant was removed and pelleted cells were resuspended in 10 ml of isolation medium. glial cells were separated from myelin and red blood cells using a 30% percoll gradient (hirayama et al., 1983; kim et al., 1985) . the gradient was centrifuged in an ultracentrifuge (beckman instruments, palo alto, ca) equipped with a ja20 fixed-angle rotor spun at 14,000 rpm for 45 min at 4°c. the glial cell layer, the region below the myelin cap and above the red blood cells, was transferred to a 50-ml tube that was then filled with isolation medium and spun for 10 minutes at 1,500 rpm in a centrifuge (glc-2b; sorvall div.). the supernatant was removed and the pelleted cells were resuspended (800 pl/spinal cord) in 10% fbs in dme with 584 mg/liter l-glutamine, 4.5 g/liter v-glucose, 25 #g/ml gentamycin, and 1 mm sodium pyruvate. in all cases, 200 ~tl of cell suspension was plated per 35-mm plastic dish (coated with extracellular matrix; accurate chemical & scientific corp., westbury, ny). after incubating for 1 h at 37°c to enhance attachment, the cultures were fed with 2 ml of 10% fbs-dme solution. at 1 and 3 d in vitro (die) cultures were fed with 0.5 % fbs in defined medium (dme with 584 mg/liter l-glutamine, 4.5 g/liter v-glucose, 5/~g/ml insulin, 50 /~g/ml transferrin, 30 nm selenium, 30 nm triiodothyronine, 25 #g/ml gentamycin, and 1 mm sodium pyruvate; modified from eccleston and silberberg, 1984) . the cultures were grown in this low serum defined medium to enhance the growth of oligodendrocytes relative to fibroblasts. after l, 3, or 6 die, cultures were fixed with 2% paraformaldehyde in mem-hepes for 15 min. in some experiments a terminal pulse of [methyl-3h]thymi~me (67 ci/ mmol; new england nuclear, boston, ma) was administered. for in vivo labeling, mice were injected intraperitoneally with [3i-l]thymidine (10 #ci/g body weight) 2 h before death (see fig. 1 a) . for in vitro labeling, [3h]thymidine (0.05 #ci/ml culture media) was added to cultures 20 h before fixation (see fig. 1 b) . the growth factors tested were bovine bfgf (10 ng/mi; r & d systems, minneapolis, mn), human pdgf (10 ng/mi; a and b chain heterodimer from r & d systems), and human insulin-like growth factor i (igf-i; 100 ng/mi; amgen biologicals, thousand oaks, ca). each growth factor was added individually to a culture dish at 1 div with 0.5% fbs in defined medium (see above). cultures were subsequently given a 20-h terminal pulse of [3i-i]thymidine and fixed at 3 die (see fig. 1 immunocytochemistry o-2a lineage cells were identified based upon three-color immnnofluorescence that enabled simultaneous visualization of reactivity with antigalactocerebroside (gc), anti-glial fibrlllary acidic protein (gfap), and the 04 antibody. anti-gc is a mouse monoclonal igg3, kindly provided by b. ranscht (la jolia cancer research foundation, la jolla, ca), which recognizes gc and an earlier antigen emerging on the cell surface shortly after the appearance of 04 immunostaining (ranscht et al., 1982; bansal et al., 1989) . the rabbit polyclonal anti-gfap, kindly provided by r. pruss (merrill dow pharmaceuticals, cincinnati, oh), immunostains gfap but does not react with other intermediate filament proteins . 04 is a mouse monoclonal igm, kindly provided by 1. sommer (southern general hospital, glasgow, scotland), (sommer and schachner, 1981) which recognizes sulfatide, seminolipid, and an unidentified antigen (bansal et al., 1989) . 04 and anti-gc were supernatants from hybridoma cultures containing 10% fbs in dme. the supernntants were mixed together and diluted 1:4 and 1:2, respectively, in a mem-hepes buffer solution (0.1% gelatin, 1% bsa, and 0.05% sodium azide in mem-hepos) and applied to the fixed cells for 1-2 h. 04 was visualized with rhodamine conjugated goat antimouse igm (25 t~g/mi; jackson immunoresearch laboratories, west grove, pa). anti-gc was visualized with fluorescein conjugated goat anti-mouse igg3 (16.6 tzg/ml; fischer biotech, orangeburg, ny). these secondary antibodies were also mixed together in mem-hepes buffer solution and applied for 1-2 h. after washing, the cultures were fixed with 5 % glacial acetic acid in ethanol for 10 min at -20"c to expose internal cellular antigens. this was followed by three washes in 10% fbs mem-hepes and three washes in 0.5m tris buffer. polyclonal rabbit anti-gfap was diluted 1:2,000 in a tris buffer solution (0.1% gelatin, 1% bsa, and 0.05% sodium azide in 0.5m tris buffer) for binding overnight at 4"c. anti-gfap was visualized with biotinylated donkey anti-rabbit igg (10/~g/ml in the tris buffer solution, 1-2 h; amersham chemical co., arlington heights, il) followed by streptavidin conjugated 7-amino-4-methyl-coumarin-3-acetic acid (25/zg/ ml in the tris buffer solution, 1-2 h; molecular probes, inc., eugene, or; khallhn etal., 1986). atter a final 5-rain fixation in 4% paraformaldeh)de the cultures were either coverslipped, with a solution of 20 % 0.02 m tris buffer (ph 8.2) and 80% glycerol, or processed for autoradiography (see below). immunostained cells were examined by epifluorescence with a zeiss photoscopo i11 equipped with three filter sets to simultaneously view rhodamine, fluorescein, and coumarin fluorescence. the absence of cross-reaction in the three-color immunofluorescence protocol was tested by omitting, in turn, each one of the three primary antibodies while all other steps remained unchanged. omission of each primary completely abolished binding of the corresponding secondary but did not affect signals in the other two channels. in some cultures, the presence of mixed oligodendrocyte-astrocyte phenotype cells was confirmed with a second set of prirnary antibodies. these cultures were immunostained with 04, as described above, in combination with a rat mab to gfap (lee et al., 1984) visualized with fluorescein and a rabbit polyclonal antiserum to gc, which was produced in our laboratory (according to the procedure described by benjamins et al., 1987) , and visualized with coumarin. a segment of each spinal cord was excised with a sterile razor blade before glial cell isolation. segments were fixed by overnight immersion in 1% glutaraldehyde and 2% paraformaldehyde in 0.1 m phosphate buffer. the tissue was then washed for 1 d in buffer, postfixed with 2 % osmium tetmxide for 2 h, dehydrated in graded alcohols, cleared with propylene oxide, and embedded in epon resin. transverse sections (1 ~m) were stained with toluidine blue. cultured cells, labeled either in vivo or in vitro with [3h]thymidine, were immunostained and post-fixed as described above. the specimens were then dehydrated in graded alcohols (50%, 75%, 95%; 30 seconds in each), air dried, coated with kodak ntb2 nuclear track emulsion (diluted 1:2), exposed at 4°c for 5 d, developed at 16°c in kodak d19, and fixed with kodak fixer. after several washes, cultures were coverslipped as described above. the glial cell population was characterized using three-color immunofluorescence which enabled simultaneous visualization of three antigens, each labeled by one of three different fluorochromes (rhodamine, fluorescein, or coumarin). oligodendrocytes were identified by expression of cell surface antigens recognized by anti-gc (raft et al., 1978) , cells containing intracellular filaments immunostained with anti-gfap were classified as astrocytes (biguami et al., 1972; rafter al., 1979) . type 2 astrocytes were distinguished from type 1 astrocytes by the expression of cell surface antigens recognized by the 04 antibody (trotter and schachner, 1989) . o-2a progenitors derived from adult cns were identified as cells binding 04 in the absence of gc or gfap immunolabeling (wolswijk and noble, 1989) . thus, any particular cell could be identified as an oligodendrocyte (04+ gc+ gfap-), a type 2 astrocyt¢ (04+ gfap+ gc-), an o-2a progenitor (04+ g c -gfap-), a mixed oligodendrocyte-astrocyte phenotype cell (04+ gc+ gfap+), or a type 1 astrocyte (gfap+ 0 4 -gc-). (in this paper, the term "type 2 astrocyte" will be used to denote only the 04+ gfap+ g c -antigenic phenotype and is not intended to indicate a specific localization or function in vivo.) glial cells were isolated and cultured in parallel from spinal cords of infected and age-matched control mice at several intervals after viral inoculation. these cultures contained various types of glial cells, of which o-2a lineage cells (oligodendrocytes, o-2a progenitors, type 2 astrocytes, and mixed phenotype cells) represented a small proportion of the total. the number of o-2a lineage cells was counted after 1 d in culture (fig. 2) . this number was similar for control and experimental tissue at 1 wpi (fig. 2) when demyelination was not yet detected (see fig. 3, a and b) . as demyelination and vacuolation progressed (3-5 wpi, see fig. 3 c) , o-2a lineage cells were much more abundant in cultures of the demyelinated tissue (fig. 2) . with the onset of remyelination (6-8 wpi, see fig. 3 d) , the number of o-2a lineage cells derived from lesioned tissue declined (fig. 2) . it is possible that cns inflammation and vacuolation may have facilitated dissociation of the demyelinated tissue and thereby improved the yield of growing c¢11s. yet the cultures from control versus demyelinated cns differed not only in cell the other cell types in our cultures were type 1 astrocytes, fibroblasts, and microglia. flat calls expressing gfap but not 04 antigens were identified as type 1 astrocytes. type 1 astrocytes seemed to be more prevalent in cultures of lesioned tissue. however, at 1 div such cells were usually found in dense clusters that prohibited accurate quantitation of the initial type 1,astrocyte population (fig. 4, a and c) . by 3 and 6div, type 1 astrocytes grew out from these clusters and proliferated, as assayed by [3h]thymidine incorporation (fig. 4, b and d) . flat cells presumed to be fibroblasts, due to the absence of 04, gc, or gfap immunostaining, were present and p~liferated in all cultures examined (not shown). microglia and/or macrophages were identified by their ameboid appearance, the presence of phagocytic debris within the cells, and the absence of 04 immunostaining (giulian, 1987; rieske et al., 1989) . microglial ceils are the resident macrophages of the cns and markers for distinguishing mi-croglia from blood-born macrophages, which can infiltrate the cns, are not presently available. for simplicity, cells with the phenotype of microglia and/or macrophages will be referred to as microglia throughout this paper. these cells were rare in cultures of control tissue. in contrast, the number of microglia cultured from diseased tissue increased dramatically during the period of demyelination (3-5 wpi). we identified schwann cells with an antibody to the receptor for nerve growth factor (chandler et al., 1984 ) that labeled schwann cells cultured from rat sciatic nerve, which have been shown to express this receptor (yasuda et al., 1987; distefano and johnson, 1988) . these cells were not detected in cultures of control or infected spinal cords (1 and 4 wpi). similarly, schwarm cells were rarely observed in the remyelinating lesions (fig. 3) . we then examined the relative abundances of the different 5 and 7) . the relative proportion of type 2 astrocytes (04+ gfap+ g c -) increased between 1 and 3 div, even though the cultures were maintained in defined medium with only 0.5 % fbs (figs. 5 and 7). interestingly, in cultures of neonatal rat optic nerve, 10% fbs is required to induce development of a substantial number of type 2 astrocyteg (raft et al., 1983) . in cultures of demyel~ated spinal cord tissue (5 wpi), oligodendrocytes were the predominant cell type after 1 div (fig. 5) . however, a larger percentage of the o-2a population was composed of type 2 astroeytes in these cultures as compared with cultures of control tissue. by 3 div, type 2 astrocytes became even more prevalent than oligodendrocytes. at each time point, o-2a progenitor cells represented ,,020% of the o-2a lineage population. a remarkable feature in cultures from demyelinated tissue was the presence of mixed oligodendrocyte-astrocyte phenotype cells, which were rarely seen in control cultures (fig. 5) . these cells expressed gc on their surface and contained intracellular filaments immunostained with gfap (fig. 8) . such mixed phenotype cells (04+ gc+ gfap+) were found consistently using two different antibodies to gc in combination with two different antibodies to gfap (see materials and methods). certain growth factors have been shown to have an effect on the fate of o-2a lineage cells: igf-i promotes oligodendrocyte development (mcmorris et al., 1986) , whereas bfgf inhibits myelin gene expression (mckirmon and dubois-dalcq, 1990 ). therefore, we examined whether treatment with such growth factors from 1-3 div would influence the antigenic phenotypes expressed by o-2a lineage cells isolated during the course of demyelination and remyelination (fig. 9 ). in cultures of demyelinating/remyelinating tissue exogenous igf-i increased the proportion of oligodendrocytes relative to type 2 astrocytes, whereas bfgf decreased the relative number of oligodendrocytes. pdgf did not markedly alter the ratio of type 2 astrocytes to oligodendrocytes, in agreement with the observation that pdgf allows timely differentiation of oligodendroeytes and type 2 astroeytes in cultures of developing cns tissue raft et al., 1988) . in a previous in vivo study of this demyelinating disease, o-2a lineage cells which had incorporated [3i-l]thymidine during a 2-h terminal pulse were detected in 1-1zm frozen sections (godfraind et al., 1989) . to further explore the mitotic potential of o-2a lineage cells from adult cns, we prepared cultures from spinal cords of normal and infected mice after in vivo labeling with [3h]thymidine during a 2-h terminal pulse (table i) . cells undergoing active dna synthesis were identified after 1 div by [3i-i]thymidine autoradiography combined with triple-label immunocytochemistry, as outlined in fig. 1 a. the proportion of o-2a lineage cells (oligodendrocytes, o-2a progenitors, and type 2 astrocytes) which were thymidine-labeled was extremely low in control cultures prepared from normal mice at 5 wk of age (0.52%) and at 8 wk of age (0 %). in contrast, the proportion of o-2a lineage cells (oligodendrocytes, o-2a progenitors, type 2 astrocytes, and mixed phenotype cells) that were ph]thymidine-labeled in cultures derived from infected tissue was markedly higher both in the early phase of the disease (4.96% at 1 wpi; 5 wk of age) and at the time of widespread demyelination (5.38% at 4 wpi; 8 wk of age). we next examined the proliferative capacity of the cultured cells during an in vitro pulse of [3h]thymidine (table i ). in this case proliferation in vitro was measured by adding [sh] thymidine to the culture medium at 2 div, fixing the cells 20 h later, and then identifying [3h]thymidine-labeled cells by autoradiography combined with three-fluorochrome immunocytochemistry, as outlined in fig. 1 b. the percentage of o-2a lineage cells that were labeled in vitro with ph]thymidine was approximately ninefold higher in cultures derived from diseased mice at 1 wpi than in cultures from age-matched control mice, and was increased more than threefold at 4 wpi (table i) . oligodendrocytes (fig. 10) , type 2 astrocytes (fig. 11) , o-2a progenitors (fig. 12) , and mixed phenotype cells were labeled with [3h]thymidine after the 20-h terminal pulse. remarkably, o-2a progenitors were the only cell type for which the increased percentage of [3h]thymidine-labeled cells from demyelinated tissue was highly significant (4 wpi; demyelinated = 38.3 %, control = figure 8 . a cell with a mixed oligodendrocyteastrocyte phenotype isolated from demyelinated tissue (4 wpi) which was grown in culture for 3 d, fixed, and processed for three-color immunofluorescence. (a) the cell surface membrane is immunostained with an mab against gc, visualized with fluorescein. (b) intracellular filaments are immunostained with a polyclonal antiserum recognizing gfap, visualized with coumarin. this cell was also labeled by 04, visualized with rhodamine (not shown). bar, 50 ttm. 0%; p = 0.027, as determined by paired two-tailed t test). approximately 12-14% of each of the other o-2a lineage cell types (oligodendrocytes, type 2 astrocytes, and mixed phenotype cells) were ph]thymidine-labeled in cultures derived from demyelinated tissue. although the percentage of [3h]thymidine-labeled cells within each of these phenotypes alone was not increased significantly, when combined as a single group these three phenotypes accounted for a significant proliferative response in cultures of demyelinated tissue (4 wpi; demyelinated = 13.5 %, control = 4.3 %; p = 0.029, as determined by paired two-tailed t test). thus the in vivo proliferative response of o-2a lineage cells isolated from demyelinating tissues was retained in vitro. since pdgf, igf-i, and bfgf can each induce proliferation of neonatal o-2a progenitor cells in the developing rat cns (reviewed in dubois-dalcq and armstrong, 1990), we assayed the mitogenic effect of these growth factors in our cultures of adult mouse spinal cord (see protocol outlined in fig. 1 c) . we tested each growth factor at 1, 3, 4, 5, and 8 wpi since the effect of a particular growth factor may vary during the course of the disease. in cultures of demyelinating and remyelinating tissue, none of these three growth factors caused a significant increase in the percentage of o-2a lineage cells which incorporate ph]thymidine during a 20-h terminal pulse (p = 0.691 for the difference between treatments, as determined by anova). similarly, preliminary data from control cultures indicated that treatment with pdgf, igf-i, or bfgf did not enhance the proliferation of o-2a lineage cells from normal adult mice. thus, exogenous growth factors influenced phenotype expression ( fig. 9 ) but did not enhance the proliferation of o-2a lineage cells in our cultures. in the present in vitro study, we have characterized the growth and differentiation of glial cells isolated from the cns of mice during the course of demyelination and remyfigure 9 . the relative abundance of type 2 astrocytes and oligodendrocytes in spinal cord cultures from virus-inoculated animals. the number of type 2 astrocytes and oligodendrocytes was determined as described in fig. 5 . in defined media alone, the proportion of type 2 astrocytes increases during demyelination (3-5 wpi). addition of bfgf (10 ng/nd) to the defined media between 1 and 3 div exaggerates this shift toward the type 2 astrocyte phenotype by decreasing the relative number of cells expressing gc. treatment with igf-i (100 ng/ml) induces a larger proportion of cells to express the oligodendrocyte phenotype. exogenous pdgf (10 ng/ml) did not alter the ratio of type 2 astxo~tes to oligodendrocytes. to minimize variability between experiments, the cultures were prepared in parallel for each timepoint and as one completely matched set from mice inoculated at the same time with the demyelinating virus. each value represents the ratio from cell counts in an entire 35-mm dish. a total of 1,793 cells were counted in the set of cultures. to estimate the variability that might be expected between nonmatched experiments, the ratio of type 2 astrocytes to oligodendrocytes was compared in three similar experiments of cultures grown without exogenous growth factors. the asterisks denote values which fall outside of a 95 % confidence interval of the expected variability between experiments for cultures grown without exogenous growth factors. fig. 1, a and b) . each value is the percentage (+sem) of total o-2a lineage cells (oligodendrocytes, type 2 astrocytes, o-2a progenitors, and mixed phenotype cells combined) which were [~hlthymidine-labeled (greater than 10 autoradiographic silver grains localized over each nucleus) and represents the average of 2-3 dishes. significance values were determined using the two-tailed paired t test. studies of neonatal cns tissue have shown that the growth and differentiation of o-2a lineage cells can be influenced in vitro by several polypeptide growth factors, which are synthesized in the brain (for review, see raft, 1989; dubois-dalcq and armstrong, 1990) . pdgf stimulates the proliferation of o-2a progenitor cells, which prevents premature differentiation in vitro raff et al., 1988) . figure 11 . a type 2 astroeyte cultured from demyelinated tissue (4 wpi) and processed as described in fig. 10 is identified by the presence of surface staining with 04 (a; rhodamine optics), intracellular expression of gfap (b; coumarin optics) and the absence of labeling with gc antibody (c; fluoreseein optics). the cluster of silver grains over the nucleus (d, arrow) indicates that this cell incorporated [3h]thymidine during the 20-h in vitro pulse. (e) phase contrast showing the cell processes. bar, 50 #m. fgf is also mitogenic for o-2a lineage cells (eccleston and silberberg, 1985; noble et al., 1988; besnard et al., 1989) . both fgf and epidermal growth factor inhibit expression of myelin components (sheng et al., 1989) . igf-i promotes proliferation of o-2a lineage cells while also inducing precursor cells to develop into oligodendrocytes (mcmorris and dubois-daicq, 1988) . a protein closely related to ciliary neurotrophic factor is present in developing optic nerve and induces transient expression of gfap in o-2a progenitor cells in vitro lillien et al., 1988) . whether the growth factors mentioned above have an effect on o-2a lineage cells derived from adult cns tissue is not yet clear. human oligodendrocytes isolated from normal adult brain do not proliferate in response to treatment with several growth factors, including pdgf, fgf, epidermal growth factor, and interleukin 2 (yong et al., 1988) . similarly, in the present study we found that pdgf, bfgf, or igf-i did not induce mitosis of o-2a lineage cells cultured from adult mice, as assayed by [3h]thymidine incorporation. however, a factor secreted by b104 neuroblastoma cells is mitogenic for o-2a progenitors and oligodendrocytes cultured from adult rat brain (hunter et al., 1988) . thus, normal adult o-2a lineage cells are capable of proliferating when exposed to an adequate stimulus. such a stimulus could be a factor (or factors) released in the cns during demyelination since o-2a lineage cells in the spinal cord of mhv-a59 infected mice showed increased proliferation both in vivo (godfraind et al., 1989) and in vitro (the present data). interestingly, some growth factors can influence the antigenic phenotypes of o-2a lineage cells in our cultures of demyelinating/remyelinating cns. basic fgf decreased the percentage of o-2a lineage cells expressing gc, whereas igf-i increased the proportion of oligodendrocytes relative to type 2 astrocytes. thus bfgf and igf-i might modulate phenotype expression in cns development and remyelination. proliferation of mature and immature oligodendrocytes in vivo has been described in several electron microscopic and autoradiographic studies of experimental demyelination in adult cns (herndon et al., 1977; ludwin, 1979; aranella and herndon, 1984; raine et al., 1988) . in our in vivo studies of remyelination following coronavirus-induced demyelination in mice, we have used in situ hybridization and immunolabeling techniques to analyze the expression of myelin genes and the presence of cell-type-specific antigens in oligodendrocytes and their precursors (jordan et al., 1989; godfraind et al., 1989) . we found that myelin basic protein mrna isoforms containing exon-2, which are normally abundant only during development, are reexpressed at dramatically increased levels during remyelination (jordan et al., 1990) . this recapitulation of molecular events characteristic of development suggests that newly generated oligodendrocytes are responsible for remyelination in this disease. studies using triple-label immunocytochemistry combined with autoradiography have identified o-2a progenitor cells in 1 ~m frozen sections of the spinal cord of these infected mice (godfraind et al., 1989) . o-2a progenitors and o4-positive astrocytes proliferated early in the course of the disease and some figure 12 . an o-2a adult progenitor cultured from demyelinated tissue (4 wpi) and processed as described in fig. 10 is recognized by its reactivity with the 04 antibody (a; rhodamine optics) and in the absence of staining with gc (b; fluorescein optics) or gfap (c; coumarin optics) antibodies. the cluster of silver grains over the nucleus (d, arrow) indicates that this cell incorporated [3h]thymidine during the 20-h in vitro pulse. (e) phase contrast showing the cell processes. note that numerous microglial cells are also present in this culture. bar, 50 #m. cells generated during the demyelination stage later developed into oligodendrocytes (godfraind et al., 1989) . in the present study, we demonstrate that the mitotic activity of o-2a lineage cells observed in vivo during demyelination has been maintained in vitro. this proliferation might be mediated by factors active in the culture milieu or by signals experienced in vivo which persist in vitro. the nature of the signal(s) triggering this proliferation is presently unknown. reactive astrocytes may secrete mitogenic factors while microglia can synthesize lymphokines in response to trauma (giulian, 1987; giulian et al., 1988) . since microglial cells are present in our cultures of adult spinal cord and are much more prevalent in cultures of demyelinated tissue, it is possible that o-2a lineage cells proliferate due to factors released into the culture milieu by these cells. in addition to soluble factors, denuded axons or axolemmal components, which are mitogenic for oligodendrocytes from developing cns (chen and devries, 1989), may act as mitogens for o-2a lineage cells during demyelination. denuded axons in demyelinated lesions may stimulate proliferation directly, or may "prime" o-2a lineage cells to divide in response to specific growth factors. previous studies have shown that oligodendrocytes isolated from adult spinal cord and expressing gc, but not myelin basic protein, can divide in the presence of neurons (wood and bunge, 1986) . along with o-2a progenitors, oligodendrocytes expressing gc also proliferated in our cultures from remyelinating animals. thus, proliferating oligodendrocytes could also be a major source of remyelinating cells. type 2 astrocytes and cells with a mixed oligodendrocyte-astrocyte phenotype also proliferated in response to demyelination. cells with characteristics of both o l i~ and astrocyte phenotypes have been described in several in vivo studies of demyelinating tissue (bunge et al., 1961; carrollet al., 1987; godfralnd et al., 1989; paine, 1989) . additionally, ciliary neurotrophic factor, which induces transient gfap expression in neonatal o-2a progenitors in vitro, is much more abundant in regenerating cns tissue (nieto-sampedro et al., 1983) and might possibly induce a mixed phenotype in vivo. the presence of phenotypic characteristics of both oligodendrocytes and astrocytes in the same cell indicates that some degree of plasticity is possible between these two differentiation pathways. mixed phenotype cells might be precursor cells at an intermediate bipotential stage which are responding to two differentiation signals simultaneously, or may be mature cells in a state of transdifferentiation between astrocyte and oligodendrocyte phenotypes. by either mechanism, mixed phenotype cells might provide additional ways to increase the number of oligodendrocytes available for remyelination. now that we have established and characterized an in vitro system of glial cells derived from normal and demyelinating/remyelinating adult cns, we can design future experiments to determine which of the proliferating cells provide new oligodendrocytes in remyelination and which factors enhance this pathway. since we have recently developed a similar in vitro system from adult human cns (dorn et al., 1990) , such experiments could also be performed with human oligodendrocyte lineage cells. the study of the growth and differentiation properties of the glial cell types involved in remyelination may lead to the elaboration of strategies to promote remyelination in human demyelinating diseases. mature oligodendrocytes. division following experimental demyelination in adult animals multiple and novel specificities of monoclonal antibodies o1,04 and r-mab used in the analysis of oligodendrocyte development production and characterization of high titer antibodies to 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in the rat optic nerve: in vivo evidence for two distinct astrocyte lineages injury-induced neuronotrophic activity in adult rat brain: correlation with survival of delayed implants in the wound cavity platelet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte type-2 astrocyte progenitor cell electron microscopic features of multiple sclerosis lesions continuous breakdown and regeneration of myelin in progressive multiple sclerosis plaques thy-1 antigen on astrocytes in long-term cultures of rat central nervous system glial cell diversification in the rat optic nerve galactocerebroside is a specific cell-surface antigenic marker for oligodendrocytes in culture two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides, and growth characteristics a glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium two glial cell lineages diverge prenatally in rat optic nerve platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture morphology of myelin and myelination remyelination in multiple sclerosis: immunopathologic considerations induction of oligodendrocyte proliferation and remyelination after chronic demyelination. relevance to multiple sclerosis development of oligodeodrocytes and schwann cells studied with a monoclonal antibody against galactocerebroside microglia and microglia-derived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro physiological basis of conduction in myelinated nerve epidermal growth factor inhibits the expression of myelin basic protein in oligodendrocytes monoclonal antibodies (01 to 04) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid cells positive for the 04 surface antigen isolated by cell sorting are able to differentiate into astrocytes or oligodendrocytes identification of an adult-specific glial progenitor cell the biology of the oligodendrocyte evidence that axons are mitogenic for oligodendrocytes isolated from adult animals acute and subacute demyelination induced by mouse hepatitis virus strain a59 in c3h mice cultured rat schwann cells express low aliinity receptors for nerve growth factor growth factors for human glial cells in culture we thank christine cardellechio, ray rusten, and susan wetherall for excellent technical assistance. as mentioned in the text, we are very grateful to our colleagues who donated antibodies used in this study. we also thank craig jordan and brynmor watldns for helpful comments on the manuscript.this work was supported in part by uniformed services university of health sciences grant r07403. the opinions expressed are the private views of the authors and should not be construed as official or necessarily reflecting the views of the uniformed services school of medicine or the department of defense.received for publication 15 december 1989 and in revised form 16 april 1990. key: cord-334499-fz7vrnb1 authors: templeton, steven p.; perlman, stanley title: pathogenesis of acute and chronic central nervous system infection with variants of mouse hepatitis virus, strain jhm date: 2007-06-01 journal: immunol res doi: 10.1007/s12026-007-0079-y sha: doc_id: 334499 cord_uid: fz7vrnb1 infection of mice with variants of mouse hepatitis virus, strain jhm (mhv-jhm), provide models of acute and chronic viral infection of the central nervous system (cns). through targeted recombination and reverse genetic manipulation, studies of infection with mhv-jhm variants have identified phenotypic differences and examined the effects of these differences on viral pathogenesis and anti-viral host immune responses. studies employing recombinant viruses with a modified spike (s) glycoprotein of mhv-jhm have identified the s gene as a major determinant of neurovirulence. however, the association of s gene variation and neurovirulence with host ability to generate anti-viral cd8 t cell responses is not completely clear. partially protective anti-viral immune responses may result in persistent infection and chronic demyelinating disease characterized by myelin removal from axons of the cns and associated with dense macrophage/microglial infiltration. demyelinating disease during mhv-jhm infection is immune-mediated, as mice that lack t lymphocytes fail to develop disease despite succumbing to encephalitis with high levels of infectious virus in the cns. however, the presence of t lymphocytes or anti-viral antibody can induce disease in infected immunodeficient mice. the mechanisms by which these immune effectors induce demyelination share an ability to activate and recruit macrophages and microglia, thus increasing the putative role of these cells in myelin destruction. in the absence of infection or injury, the resting state of the central nervous system (cns) is highly resistant to peripheral immune cell infiltration and activation. access to cns tissues by peripheral blood leukocytes is prohibited by tight intercellular junctions and low expression of adhesion molecules by endothelial cells of the blood brain barrier (bbb). within the cns, immune activation is further diminished through downregulation of mhc molecules by resident cns cells, and through constitutive expression of immunoregulatory molecules such as tgf-b. furthermore, secreted factors which regulate normal brain function and development exhibit immunosuppressive activity. when cns infection or injury occurs, these barriers to immune infiltration and activation are altered to allow access to circulating leukocytes and protective antibody. during cns immune responses, however, a balance between immune activation and suppression is maintained. this conserves integrity and function of non-regenerative cns tissue, while permitting immune responses against invading pathogens. tilting the balance toward immune suppression could result in persistent or chronic infection, whereas excessive immune activation could result in autoimmunity or bystander destruction and loss of function of vital cns tissue [1] [2] [3] . one pathogen that infects the cns of mice and thus challenges the local balance between immune activation and suppression is mouse hepatitis virus, strain jhm (mhv-jhm). contrary to its name, mhv-jhm is a neurotropic, not a hepatotropic, single stranded rna virus of the family coronaviridae. human coronaviruses related to mhv-jhm cause a variety of infection and disease ranging from the common cold to severe acute respiratory syndrome (sars) [1, 4] . naïve susceptible mice infected with virulent variants of mhv-jhm develop lethal acute encephalitis. however, other variants of mhv-jhm provide tools for studying both acute and chronic infection of the cns [5] . studies of cns infection with these variants have identified distinct mutations in virus structure, and have compared the effect of these structural differences on viral pathogenesis and host immune responses to infection. differences between these variants are often reflected by changes in cell tropism, neurovirulence, and in the quality of host anti-viral immune responses in the infected cns. in order to more closely compare virus structure and function with associated pathogenesis, additional mhv-jhm variants are generated through targeted recombination, with methods similar to those originally used to create mhv strain a59 recombinant viruses [6] [7] [8] [9] . initially, mutations or introduced coding sequences are generated by pcr from mhv-jhm, or from host genes encoded on plasmids, respectively. the site used for expression of non-mhv sequences is within gene 4, which is not essential for virus replication or infection [10, 11] . rna is generated from the final linearized plasmid, containing the mhv-jhm s and gene 4 sequences, by in vitro transcription. donor rna is then transfected into cells infected with a homologous recipient virus. desired recombinants are selected by host cell specificity, which is altered by donor/recipient recombination. in this manner, recombinant mhv have been generated for functional studies of mhv-jhm structural proteins, or for the production of immune effectors, and for functional studies of non-structural proteins of the human coronavirus sars-cov [12] [13] [14] . while acute infection is characterized by rapid virus spread and cns inflammation, chronic disease during mhv-jhm infection is characterized by incomplete clearance of infectious virus and concomitant development of demyelinating disease [5] . mhv-jhm infected mice with demyelinating disease serve as a relevant animal model of the human autoimmune disease multiple sclerosis (ms) [15, 16] . as in ms, demyelination induced during mhv-jhm infection is characterized by macrophage infiltration into the white matter of the cns, with subsequent destruction of the protective myelin sheath surrounding the axons of cns neuronal cells. demyelination may be induced in mice through persistent infection with attenuated mhv-jhm strains, or by partial protection from virulent mhv-jhm by mhv-specific antibody [5, 17, 18] . demyelinating disease induced during mhv-jhm infection is partly immune mediated, as mice lacking the ability to generate t cell responses fail to develop demyelination, despite high viral loads and widespread inflammation in the cns of infected mice [19, 20] . to examine this relationship between virus infection and demyelination, immunodeficient mice infected with mhv-jhm receive adoptively transferred enriched populations of t cells or other immune effectors [21, 22] . these models of mhv-jhm infection provide excellent tools for investigating both acute and chronic viral infection of the cns. however, the factors that mediate the initiation of host anti-viral immune responses, virus-induced pathology, and the regulation of these events by the specialized local environment of the cns, are only partly understood. susceptible mice infected intracerebrally with the mhv-jhm variant mhv.sd (also termed mhv-4) develop fatal acute encephalitis with a 50% mortality after inoculation with 1 plaque forming unit (pfu) [9] . in highly virulent variants of mhv-jhm, intranasal inoculation also results in uniformly fatal encephalitis in susceptible mice. the resulting pathology is generally attributed to sequence variation in the spike (s) glycoprotein. the contributions of non-s versus s genes to enhanced mhv.sd neurovirulence were determined by infection with recombinant viruses comprised of either the non-s genes of the less virulent mhv strain a59 with the s gene of neurovirulent mhv.sd, or the non-s genes of mhv-jhm with the s gene of mhv-a59 [9, [23] [24] [25] . these studies demonstrate that recombinant viruses expressing the mhv.sd s protein are more neurovirulent than mhv-a59 s glycoprotein expressing viruses. in addition to differences in mortality between mhv.sd and mhv-a59 infected mice, host immune responses also differ. mice infected with mhv.sd exhibit a prolonged innate response characterized by ifn-b production in the cns beyond 5 days post infection with decreased il-12p40 and ifn-c transcription [24] . in mhv-a59 infected mice, ifn-b transcripts decrease after 5 days post infection, with increasing ifn-c mrna coincident with increasing t cell infiltration. proinflammatory cytokines and chemokines such as il-1, il-6, mip-1a, mip-1b, and mip-2 are upregulated in the cns of mhv.sd mice in comparison to relatively low cns expression during mhv-a59 infection. adaptive immunity is suppressed during mhv.sd infection. antigen specificity of adaptive immune responses of both cd4 and cd8 t cells are determined by recognition of defined immunodominant and subdominant epitopes within mhv strains [26, 27] . the immunodominant cd8 t cell epitope recognized in mhv-jhm infected c57bl/6 mice is presented on the h-2d b class i molecule and is located in the mhv-jhm spike glycoprotein from (amino acids 510-518 (s510)). an h-2k b cd8 t cell epitope is located in the s protein of both mhv-jhm and mhv-a59 spanning amino acids 598-605 (s598). mhv-a59 infected mice mount a robust cd8 t cell response to the shared s598 epitope, while mhv.sd infected mice mount a weak response to both s598 and s510 epitopes [23, 24] . total t cell infiltration (antigen specific and non-specific) is also reduced in the cns of mhv.sd infected mice. consistent with increased inflammation in virulent mhv.sd infection, mononuclear cells expressing cd11b and fccri/iii are significantly increased in the cns of mhv.sd infected mice. these results demonstrate that mhv.sd infection of the cns results in a greater inflammatory response and a weakened cd8 t cell response when compared to mhv-a59. to evaluate the contribution of the spike (s) glycoprotein to the pathogenesis of mhv.sd infection, targeted recombination and reverse genetic techniques were used by phillips et al. to generate mhv.sd and mhv-a59 viruses that express the s glycoprotein of the other strain [9] . rempel et al. further characterized the immune responses to these viruses, using recombinant mhv-a59 viruses with the s glycoprotein of either mhv-a59 (wtr13) or mhv.sd (s4r22) [25] . infection of mice with s4r22 results in increased virulence compared to the mhv-a59 wild type recombinant wtr13. however, this increased virulence is not accompanied by a suppressed cd8 t cell response or prolonged ifn-b production, which were observed in the highly virulent mhv.sd infection. increased virulence in s4r22 infection is associated with increased cns macrophage infiltration and increased mip-1a and mip-1b transcription, while infection with either s4r22 or wtr13 results in a robust cd8 t cell response and increased cns ifn-c transcription. these results were repeated and confirmed by iacono et al., who further explored the role of background (non-s) genes by generating an mhv-jhm virus that expressed the mhv-a59 s glycoprotein. mice infected with this virus (sa59/rjhm) display an attenuated neurovirulence compared to wild type recombinant mhv-a59 (ra59). however, these mice mount a diminished antigen specific cd8 t cell response to infection similar to the suppressed response to rjhm infection. from these results, the authors concluded that although the s glycoprotein determines the neurovirulence of mhv strains, the background genes determine the extent of the cd8 t cell response. this conclusion conflicts with results obtained from infection of mice with a slightly attenuated, yet closely related mhv-jhm variant, mhv.ia [13] . similar to the neurovirulent mhv.sd, 50% of mice infected intracerebrally with 1 pfu of mhv.ia succumb to fatal encephalitis. however, in contrast to the weak response to mhv.sd infection, mhv.ia infected mice mount a robust mhv specific cd8 t cell response [28] . furthermore, attenuation in neurovirulence in mice infected with mhv.ia is attributed solely to a single amino acid change in the spike glycoprotein [13] . as with studies comparing mhv.sd and mhv-a59 virulence, examining the association of the mhv s glycoprotein with neurovirulence in mhv.ia and mhv.sd infection was performed through generation of recombinant rjhm.ia virus via targeted recombination. with this strategy, a background rjhm.ia virus expressing the spike glycoprotein of mhv.sd was generated (rjhm.sd). comparison of rjhm.ia and rjhm.sd infection revealed that the mhv.sd s expressed by rjhm.ia increases neurovirulence with accompanied increases in viral titers and lateral spread throughout the cns of infected mice. furthermore, the spike glycoprotein of rjhm.sd was shown to mediate increased receptor independent spread in infection of tissue culture cells lacking the carcinoembryonic antigen cell adhesion molecule 1 (ceacam-1, the cellular receptor for mhv-jhm entry). sequencing of these viruses indicated that the structures of the spike proteins of rjhm.ia and rjhm.sd differ by four amino acids. targeted mutation of the s protein of rjhm.ia resulting in a single change in the amino acid 310 from a serine to a glycine generated a virus (rjia.s310g) that exhibited increased neurovirulence in infected mice when compared to rjhm.ia infection. although these studies have identified structural determinants of virulence, the importance of the spike protein in the generation of contrasting immune responses to mhv.sd and mhv.ia infection is not fully understood. prolonged production of ifn-b in the cns of mhv.sd infected mice may provide one clue [24, 25] . stimulation of ifn-b production in the cns through toll like receptor 3 (tlr3) suppresses eae [29] . in certain conditions, ifn-b induces the upregulation of numerous effectors that ultimately limit t cell responses. in cultured cns cells, ifn-b production limits the capacity of antigen presenting cells to activate t cells [30] . in addition, mice deficient in ifn-b have increased antigen-specific cd8 t cell responses to peptide or dna vaccination, with decreased il-10 producing t regulatory cells (t reg ) [31] . both t reg cells and il-10 have been implicated in regulating immune responses to viral infection as well as autoimmunity [32] [33] [34] . by controlling the magnitude of anti-viral immune responses, the activity of t reg cells and il-10 effectively limit immune-mediated pathology while providing a potential opening to chronic viral infection. determining the source of ifn-b in the cns of mhv.sd infected mice is important for understanding the mechanism of mhv.sd mediated immune suppression. neurons are a major source of type i ifn in mice infected with theiler's murine encephalitis virus (tmev) [35] . furthermore, neurons inhibit t cell responses and ameliorate disease in the cns of mice with experimental autoimmune encephalomyelitis (eae), via conversion of encephalitogenic t cells into regulatory t cells [36] . determining the source of type i ifn and important associated factors in mhv.sd mediated immune suppression are the focus of current investigation. in addition to providing a model for examining structural components and determinants of virulence, targeted recombination of mhv-jhm has provided insight into the role of immunodominant epitopes in both cd4 and cd8 t cell responses to infection. persistent infection with viruses like hiv-1 or hepatitis b or c viruses may select viral mutants that evade the host cd8 t cell response [37] . these mutations are commonly selected in the immunodominant cd8 epitopes recognized by a large portion of virus-specific cytotoxic t cells, thus directly diminishing their ability to clear viral infection. these cd8 t cell epitope, or ctl escape, mutations also occur during mhv-jhm infection, allowing for viral persistence and chronic demyelinating disease [38] . mhv-jhm ctl escape mutations are selected in the immunodominant s510, but not the subdominant s598 epitope. through targeted recombination, a second high avidity cd8 t cell immunodominant epitope from lymphocytic choriomeningitis virus (lcmv gp33) [39] was added to recombinant jhm [40] . in the presence of both s510 and gp33 high avidity epitopes, demyelinating disease associated with ctl escape is prevented. in contrast to the persistent infection associated with elimination of the immunodominant mhv-jhm cd8 t cell epitope, mice infected with recombinant mhv-jhm with a single mutation in the immunodominant cd4 t cell epitope m133-147 (rj.m y135q ) exhibit milder disease with no mortality [41] . virus is ultimately cleared in rj.m y135q -infected mice and antigen specific cd8 t cell responses are equivalent to those detected in mice infected with wild type recombinant jhm. the absence of disease in rj.m y135q -infected mice is not attributed to decreases in either tnf-a or to increased th2 cytokine production in the cns. these results are striking, particularly because numerous studies have reported decreased viral clearance and increased mortality in the absence of a cd4 t cell response [19, [42] [43] [44] . future studies will be aimed at further characterizing the roles of these specific epitopes in mhv-jhm pathogenesis, the role of other epitopes such as the subdominant k b s598 epitope in ctl escape selection, and the mechanism by which the loss of the immunodominant cd4 epitope attenuates disease in infected animals. in addition to studies of acute mhv-jhm infection using the mhv.sd and mhv.ia variants, infection of mice with an attenuated variant of mhv.sd, mhv-j2.2-v1, provides a model for studying acute and chronic infection of mice with subsequent development of demyelinating disease [5, 45] . j2.2v-1 differs from mhv.sd in structure by a single amino acid in the mhv s glycoprotein. early responses to j2.2v-1 infection are similar to those in mhv-jhm-infected mice. cns inflammation allows breakdown of the bbb, permitting peripheral blood neutrophil and monocyte infiltration. concomitant with a t cell response, infectious virus is mostly cleared from the cns by 2 weeks post-infection [46] , but may remain detectable by low levels of viral rna well after viral clearance [47] . however, macrophage recruitment into areas of white matter within the cns continues in the absence of infectious virus, and mice develop plaques of demyelination in associated areas of macrophage infiltration. thus, infection of mice with mhv-j2.2v-1 provides a model for the study of persistent viral infection and chronic demyelination. ms is a debilitating human disease with a worldwide distribution, and is characterized by immune-mediated destruction of the myelin sheaths surrounding neuronal axons and, in some cases, degeneration of the axons themselves [15, 16] . ms patients can exhibit disease with several different, yet potentially overlapping, clinical and pathological profiles. due to this diversity, the etiology of ms is likely diverse as well. although not completely understood, both genetic and environmental factors play a role in disease development and progression. ms patients in remission often experience relapses after common viral infections, indicating an environmental component may be necessary to trigger disease in susceptible individuals. due to the multiple factors that promote and affect ms in humans, numerous animals models of demyelinating disease have been developed to examine particular aspects of its pathogenesis [15] . the well-established model experimental eae examines immune responses to myelin antigens in rodents. viral demyelination induced during infections with semliki forest virus (sfv), theiler's murine encephalomyelitis virus (tmev), or mhv-jhm provide models of pathogen associated demyelinating disease [15, 48] . a hallmark of ms and associated animal models of the disease is the presence of infiltrating macrophages and resident microglia in demyelinating plaques located in the white matter of the cns. both cell types are able to phagocytose myelin, and therefore are potential contributors to autoimmune tissue destruction. specific contributions of macrophages and microglia to demyelinating disease within each animal model are less clear. in models of demyelinating disease in which cns inflammation results in a breakdown of the bbb, other peripheral blood leukocytes are also present in the cns. myelin-specific t cells are present in eae lesions, while during mhv-jhm infection, t cells are predominantly specific for viral antigens. b cells play a role in demyelinating disease, as the presence of myelin specific antibody exacerbates disease in eae. professional antigen presenting cells such as dcs are also present in the cns in mice in both the eae and tmev models, and are able to prime naïve t cells in situ [49] . demyelination during mhv-jhm infection has been studied in several contexts. one particular model relies on infection of mice partially protected by nursing on mhv-jhm immune dams (suckling mouse model) [18] . in this scenario, maternal mhv-specific antibody facilitates partial viral clearance. thus, these mice survive the acute infection, but subsequently develop demyelinating disease accompanied by clinical signs of hind limb paresis. in this model, development of chronic disease is associated with mutations in the s510 cd8 t cell epitope [38] . these ctl escape mutations allow for viral persistence and chronic demyelination. emergence of ctl escape in mhv-jhm infected animals is strain dependent but mhc independent, as c57bl/6 mice allow ctl escape while balb/c or balb/b mice do not [50] . subsequent studies indicate that this difference is due to increased endogenous anti-viral antibody production, specifically due to an increase in the amount of antibody secreting, or plasma cells, in the cns of balb mice. despite the clear link between prevention of ctl escape and anti-viral antibody production in the cns of mhv-jhm infected mice, important questions remain. one important facet of ctl escape is the selection of mutations in the immunodominant d b s510, but not the subdominant k b s598 epitope. an explanation for this selection is likely found in the differences between functional avidity of the two epitopes, with the s510 epitope exhibiting higher functional avidity than the subdominant s598 epitope [26] . current studies are aimed at examining these differences by alteration of epitope avidity in recombinant mhv-jhm. a second model of demyelinating disease during mhv-jhm infection involves infection of adult mice with the attenuated mhv-j.2.2v-1 variant [45] . although generally resistant to intranasal infection, c57bl/6 mice infected intracerebrally with j2.2v-1 develop mild acute encephalitis, followed by viral clearance mediated by an anti-viral t cell response. demyelination with clinical signs of hindlimb weakness occurs during the process of virus clearance. as in other animal models of demyelinating disease, plaques of demyelination in j2.2v-1 infection are characterized by dense macrophage/microglial infiltration [15] . due to their similar function and surface marker expression, specific contributions of macrophages and microglia to demyelination during mhv-jhm infection are not well understood. chemical depletion of blood borne macrophages prior to infection does not affect disease, suggesting that microglia and/or perivascular macrophages are sufficient for demyelination to occur [51] . induction of demyelinating disease in j2.2v-1 infected mice is usually t cell or antibody mediated, as mice lacking the recombinase activating gene 1 (rag1) or severe combined immunodeficient (scid) mice do not clear virus and ultimately succumb to encephalitis with little or no demyelination [20, 52, 53] . infection of rag1-/-or scid mice, which completely lack t or b lymphocytes, provides a basis to determine the role of adaptive immune components in the development of demyelinating disease (table 1 ). j2.2v-1 infected rag1-/-mice that receive adoptively transferred splenocytes from immunocompetent mhv-jhm immunized mice regain the demyelinating phenotype [21] . depletion of both cd4 and cd8 t cells from the donor splenocyte population abrogates demyelination, while depletion of a single population does not. however, demyelination mediated by each t cell population is characterized by a distinct disease profile. infected rag1-/-mice that receive cd4 t cell-enriched splenocytes develop severe acute table 1 mediators of demyelination in mhv-j2.2v-1 infected immunodeficient mice mice mechanism reference cd4 t cells rag1-/-? [21] , [54] cd8 t cells rag1-/-cd8 ifn-c production [21] , [55] cd t cells nude, tcrb-/-ifn-c, nkg2d [61] , [62] anti-mhv ab rag1-/-fccri/iii, c activation [22] vccl2 (j2.2.ccl2) rag1-/-mac recruitment [12] encephalitis with moderate amounts of demyelination. mice that receive cd8 t cellenriched splenocytes exhibit less encephalitis and a prolonged disease course that is characterized by high levels of demyelination. furthermore, cd8, but not cd4, mediated demyelination and associated macrophage infiltration into the spinal cord white matter is significantly reduced when donor mice lack the ability to produce ifn-c [54, 55] . perforin, an essential component of ctl cytolytic activity, is not required. how ifn-c produced by cd8 t cells contributes to demyelination in mhv-jhm-infected mice is unclear. potent activation of macrophages/microglia by ifn-c likely plays a role in cell recruitment and demyelination. for example, ifn-c treatment of macrophages results in increased production of nitric oxide and increased phagocytosis. since ifn-c is required for clearance of j2.2v-1 from oligodendroglia [56] , it is possible that ifn-c produced by cd8 t cells as part of the anti-viral response results in increased activation and recruitment of myelindestroying macrophages/microglia. ifn-c also plays a role in autoimmune destruction of myelin, by enhancing cd8 t cell-mediated eae [57] . interestingly, ifn-c produced in the cns in mice with eae induces upregulation of ccl2 (mcp-1) [58] , a macrophage recruiting chemokine that also promotes infiltration into the cns during mhv-jhm infection [59] . furthermore, in a cd8 t-cell mediated model of spontaneous demyelination utilizing transgenic mice that constitutively express the costimulatory ligand cd86 on microglial cells within the cns, ifn-c receptor deficient mice exhibited no disease [60] . these studies suggest that ifn-c responsiveness by macrophages/microglia may be critical for cd8 t cell-mediated demyelination in mhv-j2.2v-1 infected mice. although transfer of cd4 or cd8 t cells induces demyelination in rag1-/-mice, j2.2 infected athymic nude mice, also lacking cd4 or cd8 t cells, develop demyelination in the absence of adoptive cell transfer [19] . however, nude mice lack only conventional ab t cells, but still retain functional cd t cells. depletion of cd t cells from infected nude mice significantly reduces demyelination [61] . demyelination in infected mice lacking the tcrb gene, which also lack conventional ab t cells, provides further proof that cd t cells can mediate demyelination [62] . furthermore, antibody depletion of ifn-c in j2.2 infected tcrb-/-mice significantly reduces demyelination, showing that ifn-c is critical for demyelination induced by cd t cells, as it is in cd8+ t cell-mediated myelin destruction. in addition to the requirement for ifn-c, recognition of nkg2d is also important for cd t cell mediated demyelination [62] . antibody blockade of nkg2d, a classic activating receptor of nk cells also expressed on a subset of mouse cd t cells, abrogates demyelination in tcrb deficient mice. therefore, cd t cells are capable of mediating demyelination during viral infection through the action of two critical effector molecules. in contrast, the role of cd t cells in eae is still controversial. depletion of cd t cells in eae results in either milder or more severe disease, depending on whether cells are depleted early or late in the inflammatory process [63] [64] [65] [66] [67] . in addition to cell-mediated demyelination, anti-mhv antibody is also capable of induction of disease. mhv-j2.2v-1 infected rag1-/-mice treated with anti-mhv antibodies develop demyelinating disease with associated white matter infiltration of macrophages/microglia [22] . although the precise role of humoral immunity in ms is not completely understood, b cells and antibody production are believed to be involved in myelin destruction [68] [69] [70] . oligoclonally expanded b cells and high levels of immunoglobulin are detected in the cerebrospinal fluid of ms patients [71] , some of which are directed against myelin components, or against common viruses such as epstein-barr [72] or varicella-zoster [73] . antibody mediated demyelination during mhv-j2.2v-1 infection requires the activating fcc receptors i and iii, since anti-mhv antibody treated infected rag1-/-mice deficient in these receptors fail to develop disease [22] . furthermore, the complement pathway may play a role in antibody-mediated demyelination, as depletion of complement by treatment of mice with cobra venom factor (cvf) results in a significant decrease in demyelination. fccr and complement may function together to activate macrophages with subsequent demyelination. adoptively transferred antibody is detectable in the cns of infected rag1-/-mice, supporting the idea of direct interaction with activating fcc receptors on macrophages. in the eae model of demyelination, mice lacking the macrophage activating fccri/iii exhibit attenuated disease, while mice lacking the inhibitory fccrii display increased disease [74] . furthermore, c3 derived complement products play a critical role in the pathogenesis of eae [75] . in addition to liver production of serum components of complement, many resident cns cells are capable of producing complement proteins. through the classical pathway, these proteins may be activated on the surface of antibody bound mhv infected cells or in mhv/antibody immune complexes, thus enhancing the recruitment and effector functions of cns macrophages/microglia. of note, rag1-/-mice also deficient in c3, the central component of the complement pathway, develop antibody-mediated demyelination (templeton and perlman, unpublished) . contrasting results between c3 deficiency and cvf depletion of complement are not surprising, since cvf exhibits considerable toxicity [76] . furthermore, recent evidence indicates that c5a may be produced independently of c3, providing a possibility that complement products downstream of c3 may play a role in demyelination in mhv-jhm infected c3-/-rag1-/-mice [77] . current studies are focused on examination of the role of complement in cns disease and in induction of immune responses to mhv-jhm infection in both immunocompetent and immunodeficient mice. inflammatory chemokines such as the macrophage chemoattractant protein ccl2 also play a role in demyelinating disease. expression of ccl2 during eae follows t cell entry into the cns [78] , and may be induced by t cell-associated increases in ifn-c [58] . ccl2 and the ccl2 receptor ccr2 are both important for recruitment of immune cells and in increased pathogenesis in eae [79] . ccl2/ccr2 also promote monocyte recruitment and viral clearance during mhv-jhm infection [59, 80] . introduction of the mouse ccl2 gene into a recombinant j2.2 (rj2.2.ccl2) virus results in an infectious virus capable of producing secreted ccl2 in infected cells in vitro [12] . rag1-/-mice infected with rj2.2.ccl2 develop demyelinating disease, whereas rag1-/-mice infected with a control virus that lacks a functional ccl2 protein (rj2.2.dccl2) do not. therefore, t cells or anti-mhv antibody are not required for demyelination in mhv-jhm-infected mice. rather, it is likely that factors induced by adaptive immune responses result in the recruitment and/or activation of macrophages/microglia into white matter areas of the cns. therefore, these cells are the final effectors in the demyelinating process and any intervention that induces their migration and activation will likely result in demyelination. further studies involve introduction of other chemoattractants into recombinant mhv-jhm, to evaluate their role in demyelinating disease, cell recruitment, generation of immune responses, and clearance of infectious virus in mhv-jhm-infected mice. studies of mhv-jhm infection of mice have provided insights into the relationship of virus phenotype to cns neurovirulence and anti-viral immune responses. the differences in infection with variants of mhv-jhm allow for examination of pathogenic mechanisms associated with both acute and chronic cns disease. recombinant virus technology has been employed to study both loss and gain of function in mhv-jhm structure, as well as the effects of host immune factors on anti-viral immune responses. using these techniques, the s gene has been identified as a primary determinant of neurovirulence in mhv-jhm infection [9, 13, 25, 79] . however, the role that virus phenotype and neurovirulence plays in the hosts' ability to generate anti-viral immune responses is less clear. some mhv-jhm variants, like mhv.ia or mhv-j2.2v-1. elicit robust anti-viral cd8 t cell responses in infected mice, while, in contrast, the highly neurovirulent mhv.sd does not [23, 24, 28] . mice which survive acute mhv-jhm infection develop demyelinating disease with associated macrophage/microglial infiltration [5] . many of the factors that mediate demyelination are capable of activating and/or recruiting macrophages (table 1) , suggesting a pathogenic role for macrophage recruitment via adaptive immune responses. future studies will aim to further understand how changes in mhv-jhm gene expression affect the pathogenesis of acute infection and demyelinating disease in the cns of infected mice. coronavirus infection of the central nervous system: host-virus stand-off immune responses to rna-virus infections of the cns basic principles of immunological surveillance of the normal central nervous system immunopathogenesis of coronavirus infections: implications for sars mouse hepatitis virus analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier reverse genetics of the largest rna viruses pathogenesis of chimeric mhv4/mhv-a59 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is ifn-c dependent in mice infected with a neurotropic coronavirus ifn-c(is required for viral clearance from central nervous system oligodendroglia a pathogenic role for myelinspecific cd8(+) t cells in a model for multiple sclerosis ifn-gamma shapes immune invasion of the central nervous system via regulation of chemokines differential roles of ccl2 and ccr2 in host defense to coronavirus infection a pathogenic role for cd8+ t cells in a spontaneous model of demyelinating disease virus-induced demyelination in nude mice is mediated by gamma delta t cells important roles for gamma interferon and nkg2d in cd t cellinduced demyelination in tcrß-deficient mice infected with a coronavirus aggravation of murine experimental allergic encephalomyelitis by administration of t-cell receptor gammadelta-specific antibody gammadelta t cells enhance the expression of experimental autoimmune encephalomyelitis by promoting antigen presentation and il-12 production gamma delta t cell regulation of 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autoimmune inflammatory damage to the central nervous system in experimental autoimmune encephalomyelitis complement and demyelinating disease: no mac needed? attenuation of experimental autoimmune demyelination in complement-deficient mice generation of c5a in the absence of c3: a new complement activation pathway central nervous system chemokine mrna accumulation follows initial leukocyte entry at the onset of acute murine experimental autoimmune encephalomyelitis targeting monocyte recruitment in cns autoimmune disease lack of ccr2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus key: cord-342204-9tgxijvn authors: nuzzo, domenico; picone, pasquale title: potential neurological effects of severe covid-19 infection date: 2020-07-03 journal: neurosci res doi: 10.1016/j.neures.2020.06.009 sha: doc_id: 342204 cord_uid: 9tgxijvn coronaviruses (covs) are large positive stranded enveloped rna viruses that generally cause enteric and respiratory diseases in humans and in animals. most human covs have recently attracted global attention to their lethal potential and great infectious capacity. a highly pathogenic cov, called covid-19 or sars‐cov2, dramatically emerged in december 2019 in wuhan, china. this new cov has caused severe pneumonia in china and rapidly spreads around the world, the covid-19 pandemic. growing evidence pieces show that viruses, such as covs, can enter the central nervous system from different pathways and inducing neurotoxicity. therefore, it is urgent to make clear whether sars-cov-2 has access to the central nervous system and can cause direct neuronal effects. moreover, a brain–lung–brain axis is been proposed from the scientific community where severe neurological dysfunction and injury are associated with lung injury, and vice versa. in this axis, virus-induced inflammation and oxidative stress could be the common mechanisms responsible for cov neurological symptoms. therefore, is important to make clear whether sars-cov-2 lung damage can cause indirect or indirect neuronal effects. covid-19 is much more than a health emergency, it has the potential to create devastating social and economic crises that will leave deep scars. nations are rushing to slow the diffusion of the virus by treating patients, limiting travel, quarantining citizens, canceling large matches, concerts, and close schools. people with covid-19 generally develop respiratory symptoms but the increasing evidence shows that some patients with a severe infection also develop neurological ailments like confusion, stroke, seizure, or loss of smell and taste. severe acute respiratory syndrome (sars) is caused by a coronavirus (cov), sars-cov-2 is a novel coronavirus identified as the cause of coronavirus disease 2019 , which is enveloped non-segmented positive-sense rna virus (subgenus sarbecovirus, orthocoronavirinae subfamily) . based on their morphology as spherical virions with a core shell and surface projections resembling a solar corona, they are termed coronaviruses. rna viruses, such as cov, infect both humans and animals (cui et al., 2019) . genetic analysis shows that sars-cov-2 has a highly homologous sequence with sars-cov (79.6%). furthermore, sars-cov-2 is 96% identical at the whole-genome level to a bat coronavirus . moreover, the entry of covid-19 into human host cells has been identified to use the same receptor as sars-cov . the recent covid-19 epidemic has been started in wuhan, china; and this highly contagious disease has spread throughout china and other part of the world (table 1) song et al., 2019; lu et al., 2020) . the symptoms of covid-19 infection usually appear after an incubation period of about five days. the adult populations, called sensitive, are at significantly increased risk of mortality (wang et al., 2019) . this effect is due to changes in aging; age-related comorbidity conditions such as heart and lung disease, diabetes, and dementia. the presence of multiple diseases in older patients may be considered as a mark of frailty, which increases the person's vulnerability to stress and impairs the multisystemic compensatory effort to restore homeostasis. the immune system of older adults presents numerous age-related changes, indicated as immune senescence (nikolich-zugich 2018). these changes concern both the innate (solana et al., 2012) and adaptive (goronzy et al., 2012; kogut et al., 2012) immune systems, as well as the immuno-response in time and space (nikolich-zugich 2018) which works effectively in young but deteriorates with age. neurotropic and neuroinvasive capabilities of coronaviruses have been described in humans. upon infection, coronavirus enters the cns, causing inflammation and demyelination (bohmwald et al., 2018) . recent studies discussed the neuroinvasive potential of sars-cov-2; in fact, some infected subjects did show neurological effects. a recent multicenter study has identified frequent (i.e. more than 80% of the cohort) gustatory and olfactory impairments in included patients. moreover, studies have shown that lung damage can be associated with brain injury and neurocognitive dysfunction and vice versa, indicating the existence of a brain-lung-brain axis (stevens et al., 2011) . in this brief review, we will discuss the evidence on the occurrence of central nervous system (cns) involvement and neurological manifestations in patients with covid-19. moreover, we have reported evidence indicating whether covid-19 might cause direct and/or indirect effect on the neuronal system or whether the eventual virus-induced neuronal damage may cause a respiratory syndrome. increasing evidence showed that coronaviruses are not always limited to the respiratory system and that they may invade the cns. in fact, detection of some rna of human-coronavirus in human brain samples clearly demonstrates that these respiratory pathogens are naturally neuroinvasive in j o u r n a l p r e -p r o o f humans and suggests that they establish a persistent infection in human cns (arbour et al., 2000) . they characterized the susceptibility of various human neural cell lines (astrocytoma cell lines u-87 mg, u-373 mg, and gl-15, as well as neuroblastoma sk-n-sh, neuroglioma h4, oligodendrocytic mo3.13, and the chme-5 immortalized fetal microglial cell lines) to an acute infection by human cov (arbour et al., 1999) . spike proteins ( therefore, the neuroinvasive capacity has been demonstrated as a common characteristic of covs, and on the base of similarity between sars-cov and sars-cov-2, it is quite likely that sars-cov-2 j o u r n a l p r e -p r o o f also possesses a like potential . although the etiology of covid-19-induced neurodegeneration remains unknown or poorly understood, it has been suggested that these human respiratory pathogens could be associated with the initiation or exacerbation of neurological diseases (desforges et al., 2014) . chen and collaborators have reported that, in wuhan city, some subjects infected with covid-19 did show neurological effects such as headache (about 8%), nausea and vomiting (1%) . moreover, in one recent paper, chinese scientists noted that on 214 covid-19 patients up to 36.4% reported neurologic symptoms such as acute cerebrovascular diseases, consciousness impairment, and skeletal muscle symptoms (mao et al., 2020) . moriguchi and collaborators report the first case of meningitis associated with covid-19. the rna of sars-cov-2 was not identified in the nasopharyngeal swab but was detected in cerebrospinal fluid by gene sequencing. a brain magnetic resonance imaging (mri) showed hyperintensity along the wall of right lateral ventricle and hyperintense signal changes in the right mesial temporal lobe and hippocampus, suggesting the possibility of covid-19 meningitis (moriguchi et al., 2020) . viruses may enter the cns by different routes involving the vasculature, the olfactory and trigeminal nerves, the cerebrospinal fluid, and the lymphatic system (desforges et al., 2020) . however, the exact route by which sars-cov enters the cns has still not been identified. the hematogenous or lymphatic route seems improbably, especially in the early stage of infection, since almost no virus particle was detected in the non-neuronal cells in the infected brain areas [ding et al., 2004; gu et al., 2005; xu et al., 2005) . different evidence shows that covs as first step invade peripheral nerve terminals, and successively their obtained access to the cns by synapse-connected route (transsynaptic transfer) (li et al., 2013) . this route has been well documented for the avian bronchitis virus (matsuda et al., 2004; chasey et al., 1976) . recently butowt and bilinska proposed that the olfactory epithelium from the nasal cavity could be a more appropriate tissue for detection of sars-cov-2 virus at the earliest stages, with respect to commonly used sputum or nasopharyngeal swabs (butowt and bilinska 2020). they point out that the different types of non-neuronal cells present in the olfactory epithelium, which express the ace2 receptor, could facilitate the binding, replication and accumulation of sars-cov-2 (butowt and bilinska 2020). this may be the underlying mechanism for the recently reported cases of smell dysfunction in patients with covid-19. the authors proposed the possibility that viral brain infection starts from the olfactory neurons (butowt and bilinska 2020). nose-to-brain route is use in biomedicine to brain drug delivery, avoiding the difficulties due to the bbb and the problems connected with systemic administration (picone et al., 2018) . the circulation of the virus in the bloodstream its interaction with the capillary endothelium j o u r n a l p r e -p r o o f and the subsequent formation of the viral particles could determine the destruction of the brain capillary endothelium allowing the access of the virus to the brain. destruction of the endothelium of the brain capillaries and hemorrhage in the brain tissue can have lethal effects long before neuronal damage occurs (mannan baig et al., 2020) . therefore, viral infection in brain may be accompanied by vascular endothelium dysfunction and brain neuroinflammation, especially in frail or older patients (toth et al., 2017) . these changes may be much worse under the hypoxic conditions of acute respiratory distress syndrome caused by covid-19. some authors suggest that the neuroinvasive potential of sars-cov-2 could play a role in the respiratory failure of covid-19 patients since the central respiratory failure progresses rapidly after the sars-cov-2 entry into the cns and damages the brainstem where the pneumotaxic center is located . in contrast, turtle says that respiratory failure caused by pneumonia is clinically distinct from that caused by brain failure and the possibility of cns entry by sars-cov-2 remains plausible, but if so, this is a very rare event (turtle et al., 2020) . however, another relevant factor linking the virus to neurological disease is that in multiple sclerosis (ms) both viruses and environmental factors are considered important for the etiopathology of disease. for this aim since 1993 viruses, have been the subject of extensive research and discussion (kurtzke 1993) . evidence for the presence of coronaviral genes in human brain tissue of ms subject has been found (murray et al., 1992; dessau et al., 2001 ). the development of a systemic inflammatory response syndrome is closely linked to severe viral infections. virus-induced oxidative stress could be mediated by an early phase of liberation of proinflammatory cytokines. in fact, the acute inflammation there is an imbalance between increased production of radicals and the availability of antioxidant molecules and may result in increased systemic oxidative stress (biswas et al., 2016) . the brain is especially vulnerable to reactive oxygen species (ros) because this tissue is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms. the death of neurons is an important point of the pathophysiological process of nervous system diseases. therefore, inflammation leads to increased levels of ros, which can induce oxidative stress at the site of inflammation (zuo et al., 2019) . on the other hand, a number of reactive oxygen/nitrogen species can initiate an intracellular signaling cascade that enhances pro-inflammatory gene expression (lorenzen et al., 2017) . thus, j o u r n a l p r e -p r o o f inflammation and oxidative stress are closely related pathophysiological events (lugrin et al., 2014) . the brain-lung-brain axis is been proposed from scientific community where severe neurological dysfunction and injury are associated to lung injury (stevens and puybasset 2011) . chronic obstructive pulmonary disease (copd) is a progressive condition characterized by airflow limitation associated with an abnormal inflammatory response. in this pathology, several systemic biomarkers of oxidative stress are available, including ros themselves (zinellu et al., 2016) . the examination of lung specimens from mild covid-19 patients showed edema, proteinaceous exudate with globules, patchy inflammatory cellular infiltration and moderate formation of hyaline membranes . in a recent review, channappanavar and perlman have examined that in the sars-cov infected animal model, marked inflammatory and immune responses may activate a "cytokine storm", and apoptosis responses and even death (channappanavar et al., 2017) . specifically, in the blood of patients infected with sars-cov-2, there is a marked increase in interleukin 1β (il-1β), interferon γ (ifn-γ), interferon-inducible protein 10 (ip-10), and monocyte chemoattractant protein 1(mcp-1), as well as il-4 and il-10 when compared to that of sars patients . collectively, the finding indicates that inflammation is a major feature in covid-19 patients. thus, excessive inflammation, depressed immune system, and an activate cytokine storm substantially contribute to the negative consequences of sars-cov-2. an excessive immune system response could induced organs failure, (cardiac, hepatic and renal systems) before than neurodegeneration (zirui tay et al., 2020) . in a cellular model, respiratory syncytial virus infection, induces ros production and consequently oxidative stress. in fact, the findings suggest an imbalance between ros production and antioxidant cellular defenses through the negative modulation of superoxide dismutase (sod) 1, 2, and 3, catalase, glutathione peroxidase (gpx), and glutathione s-transferase (gst) (hosakote et al., 2009) . taken together, these data suggest that lung inflammation could determine systemic oxidative stress. in particular, the high inflammatory capacity of sars-cov-2 could generate a high level of ros able to damage the brain. in fact, systemic oxidative stress has an important role in neurodegenerative diseases (nd) etiology (cervellati et al., 2020) . moreover, inflammatory damage to the blood-brain barrier (bbb) surface has been linked to various neurological disorders and cns infections. the neuroinflammatory signaling has been strongly linked to elevated levels of proinflammatory cytokines such as tumor necrosis factor-α (tnf-α) and interleukin-6 (il-6) (rochfort and cummins 2015) the brain is susceptible to minimal imbalances of the redox state due to its high energy and metabolic request, imbalances of the redox state favor tissue injury and j o u r n a l p r e -p r o o f neuroinflammatory mechanisms activation paving the way for neurodegeneration (martínez leo and segura campos 2019; nuzzo et al., 2014; cevenini et al., 2010) . therefore, inflammation and oxidative stress systemic, induced by sars-cov-2 lung injury, could has effect in cns causing neuronal dysfunction. another particular aspect of indirect effect of covid-19 infection is the regulation of circulating cytotoxic lymphocytes such as natural killer (nk) cells, in fact these cells are necessary for the control of general viral infection (di bona et al., 2014) . in particular, zheng and collaborators showed that the total number of nk and cd8+ t cells was decreased markedly in patients with sars-cov-2 infection. the data highlight the importance of improving the immune response of nk cells and avoiding exhaustion of cytotoxic lymphocytes at the early stage of sars-cov-2 infection . when a virus proliferates in tissue cells of lung this leads to alveolar gas exchange disorders causing hypoxia in the cns, increasing anaerobic metabolism in the mitochondria of brain cells (di carlo et al., 2012; abdennour et al., 2012) . hypoxia can cause cerebral vasodilation, swelling of brain cells, interstitial edema, cerebral blood flow obstruction, ischemia, and congestion . in this condition the brain function gradually deteriorates, drowsiness, and bulbar conjunctival edema . patients with covid-19 often suffer from severe hypoxia, hypoxia injury may cause subsequent nervous system damage [55] . in addition, for patients at particular risk of developing cerebrovascular disease, hypoxia may also induce the occurrence of acute cerebrovascular disease. although the sars-cov-2 brain infection and the related effects is not been demonstrated with certainty, this possibility remains plausible (direct brain effect). moreover, on the other side, lung injuries could have effect on cns causing neuronal dysfunction (indirect brain effect). for these considerations, we stress the need to begin the research on covid-19 actions in the cns (figure 2 ). moreover, is very important to monitor both the early and long-term neurocognitive effects of covid-19. therefore, one possibility of counteracting the covid-19 effects is the use of antiviral therapies combined with neuroprotective drugs. for example, using antiviral drugs that can cross the blood-brain barrier combined with agents that specifically target both inflammation and oxidative stress could be considered. this research received no external funding the authors declare no conflict of interest. cevenini, e., caruso, c., candore, g., capri, m., nuzzo, d., duro, g., rizzo, c., colonna-romano, g interaction brain-lungs acute and persistent infection of human neural cell lines by human coronavirus oc43 neuroinvasion by human respiratory coronaviruses does the interdependence between oxidative stress and inflammation explain the antioxidant paradox? neurologic alterations due to respiratory virus infections origin and evolution of pathogenic coronaviruses human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system neuroinvasive and neurotropic human respiratory coronaviruses: potential neurovirulent agents in humans coronaviruses in brain tissue from patients with multiple sclerosis hla and killer cell immunoglobulin-like receptors 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the chemokine mig in pathogenesis measures for diagnosing and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in wuhan functional exhaustion of antiviral lymphocytes in covid-19 patients a novel coronavirus from patients with pneumonia in china circulating biomarkers of oxidative stress in chronic obstructive pulmonary disease: a systematic review the trinity of covid-19: immunity, inflammation and intervention inflammaging and oxidative stress in human diseases: from molecular mechanisms to novel treatments key: cord-340008-2efzyki4 authors: haddadi, kaveh; asadian, leila title: coronavirus disease 2019: latest data on neuroinvasive potential date: 2020-09-17 journal: iran j med sci doi: 10.30476/ijms.2020.85980.1561 sha: doc_id: 340008 cord_uid: 2efzyki4 coronavirus disease 2019 (covid-19) is a pandemic infection. similar to other respiratory viruses, severe acute respiratory syndrome coronavirus (sars-cov-2) may enter the brain via the hematogenous or neuronal route; however, only a few reports are available on the neurological complications of covid-19. encephalopathy is a significant neurological complication of covid-19. we herein present an update on the virology, neurological pathogenesis, and neuroinvasive potential of coronaviruses and briefly discuss the latest findings on sars-cov-2 neuroinfection. the reports thus far indicate that the access of sars-cov into host cells is bolstered chiefly by a cellular receptor, angiotensin-converting enzyme 2, and that sars-cov-2 may induce some neurological manifestations via direct or indirect mechanisms. further research is required to shed sufficient light on the impact on the central nervous system and altered mental status in patients with covid-19. indeed, a better understanding of the pathways of sars-cov-2 neuroinvasion would further clarify the neurological pathogenesis and manifestations of coronaviruses and enhance the management and treatment of this group of patients. in the current epidemic era of covid-19, health care staff should strongly become aware of sars-cov-2 infection as an essential diagnosis to get away misdiagnosis and prevention of transmission. what's known • covid-19 is a pandemic whose neurological complications need clarification. similar to other respiratory viruses, sars-cov-2 may enter the central nervous system via the hematogenous or neuronal route. while facing patients with any neurological markers, medical services suppliers should consider sars-cov-2 as a differential diagnosis to avoid misdiagnosis. • there isn't any worldwide significant data about neurological manifestation and complications of covid-19 virus. we herein provide an update on the neurological pathogenesis and manifestations of coronaviruses and briefly discuss the latest findings on sars-cov-2 neuroinfection. severe acute respiratory syndrome coronavirus (sars-cov-2), a novel coronavirus, originated in china in december 2019 and rapidly progressed into an epidemic infection, such that the world health organization (who) termed this calamitous virus "coronavirus disease 2019 (covid-19)". [1] [2] [3] the majority of the early investigations focused on how sars-cov-2 attacks the respiratory system and elicits typical symptoms in most patients; nonetheless, new research disturbingly indicates that sars-cov-2 can also attack the central nervous system (cns) in a variety of ways and even cause long-term damage or death. 4 neurological complications in covid19 infection have yet to be sufficiently elucidated. patients with such complications may present with changes in their level of consciousness (e.g., acute encephalopathy). acute infections increase the risk of altered mental status, especially in elderly patients with chronic medical illnesses. [1] [2] [3] [4] [5] [6] we herein present an update on the neurological pathogenesis and manifestations of coronaviruses (covs) and briefly discuss the latest findings on sars-cov-2 neuroinfection in the limited literature. previous research shows that the central nervous system (cns) is not immune to infectioninduced changes, be they acute or latent. a wide spectrum of viruses attack the cns of humans by infecting different specific cells such as neurons and, thus, produce neural diseases. 7 in clinical practice, however, a precise evaluation of the incidence of viral infections poses a challenge. by way of example, viral presence can be detected only in up to 30 cases out of 100 000 cases in the most prevalent viruses known to induce encephalitis (i.e., herpes viruses, arboviruses, and enteroviruses). 8 some researchers have posited that the clinical picture of viral infections is often nonspecific and needs the clinician to consider various diagnoses, 7 the most significant of which are meningitis, which produces such typical symptoms as fever, neck stiffness, and photophobia; encephalitis, whose signs and symptoms can be nonexistent by may cause confusion, speech disorders, and unusual activities; focal neurological deficits such as hemiparesis and paralysis; syndromes of movement disorders; and seizure. 9 accordingly, the classification of the viral etiology of a neurological condition requires a thorough history taking and physical examination of the patient. [9] [10] [11] [12] covs are enveloped single-stranded ribonucleic acid (rna) viruses 13-15 that belong to the coronaviridae family. 16 human pathogenic covs comprise hcov-229e, hcov-oc43, hcovhku1, hcov-nl63, severe acute respiratory syndrome coronavirus (sars-cov), and the middle east respiratory syndrome coronavirus (mers-cov), [17] [18] [19] all of which are capable of creating a variety of respiratory infections such as common colds, bronchiolitis, and pneumonia. 18 numerous studies have also connected covs to cns diseases such as acute disseminated encephalomyelitis and multiple sclerosis. [20] [21] [22] history of the pathogenicity of the neuroinfection of coronaviruses covs are universal animal pathogens that can produce systemic and neurological illnesses in infected animals. covs lead to upper respiratory tract diseases 23 and are associated with gastroenteritis 24 in humans. there are, however, two reports that posit a relationship between covs and human demyelinating disease. in one of these reports, the coronavirus was separated from the brains of two patients with multiple sclerosis patients. 25 the other report was on the observation of the coronavirus via electron microscopy in the brain immunocytes of a patient with multiple sclerosis. 26 an article published in 1956 reported the intracerebral inoculation of a 10% homogenate of a mouse hepatitis virus (mhv) jhm-infected mouse brain into rhesus monkeys (macaca mulatta) induced the acute manifestations of panencephalitis. 27 immune responses mediated by t lymphocytes in the cns can be both beneficial in that they can clear inflammation mediators and harmful in that they may destroy neural tissues and activate autoimmunity and glial cells. the cns responds swiftly to coronavirus involvement by recruiting the chemokine mediators of specific t cells. the detection of antigens expressed by the major histocompatibility complex (mhc) is vital in triggering antigen-specific t cells. however, extensive t-cell stimulation and selfantigen stimulation can finally produce disorders such as demyelination. some mediators such as interferon regulatory factor 7 (ifn-7) may exert undesirable effects by employing macrophages and inducing the excessive secretion of astrocyte chemokines. [28] [29] [30] dissimilar to the cytolytic mechanisms expressed in acute infection, the immunity induced by the nonlytic humoral pathway succeeds in controlling infectious viruses through persistence. the current data appear to confirm cns infection by sars-cov. 31 some investigations have reported high serum levels of interleukin 6 (il-6), il-8, and monocyte chemoattractant protein-1 (mcp-1) in patients with sars-cov or mers-cov. 32, 33 some other studies have demonstrated considerable accumulation of il-6, il-8, and mcp-1 in the cerebrospinal fluid (csf) of patients with the cov infection of the cns. il-6 has both neurotrophic and neuroprotective properties and can increase the permeability of the blood-brain barrier. 34 a high level of il-6 causes advanced neurological disorders. 35 a study in japan on experimental mouse encephalitis verified that il-8 played a vital role in inflammatory responses such as injury to the brain. 36 mcp-1 is a chemokine that can recruit the migration of monocytes across the blood-brain barrier and invite inflammatory cells into the cns. what ensues is the entry of virus-infected cells, an increased inflammatory response, and injury to the brain, particularly encephalitis. 37, 38 virus) the existing data show that the newly disseminated coronavirus (sars-cov-2) shares similar pathogenesis with sars-cov and mers-cov in the induction of pneumonia. sars-cov-2 uses a similar receptor to enter the human host cells. [39] [40] [41] increasing evidence indicates that neurotropism is an important feature of covs. consequently, it is crucial to understand whether sars-cov-2 can increase entrance to the cns and induce neuronal damage, resulting in acute respiratory distress and death. [42] [43] [44] this neuroinvasive capability of covs has been recognized very nearly for all covs such as sars-cov, mers-cov, hcov-229e, and hcov-oc43, as well as mouse hepatitis virus and porcine hepatitis e. [45] [46] [47] neuroinvasive potential of sars-cov-2 (last update) the particular pathway by which sars-cov or mers-cov arrives in the cns has yet to be determined. nevertheless, the lymphatic or hematogenous route appears to be likely, particularly in the primary stage of the infection, when almost no particle of the virus has thus far been identified in non-neuronal cells. [31] [32] [33] growing evidence indicates that covs can reach the cns after attacking peripheral nerve elements. [44] [45] [46] in the years 2002 and 2003, some investigations reported the presence of sars-cov elements in the cns, especially in the brain neuron cells. 31, 48, 49 further studies afterward demonstrated that both sars-cov and mers-cov might intranasally, perhaps by way of the olfactory nerves, enter the brain and swiftly spread to some particular brain regions such as the brainstem and the thalamus. amongst these areas, the brainstem has been verified to be severely infected by these viruses. [50] [51] [52] the access of sars-cov into host cells is supported mostly by a cellular receptor, angiotensin-converting enzyme 2 (ace2), which is expressed in the human respiratory system, small intestine, and kidney cells. conversely, the existence of this enzyme uniquely is not adequate to make host cells vulnerable to infection. [53] [54] [55] sars-cov-2 also has comparable potential. an epidemiological study on covid-19 informed that the normal time from the first warning sign to dyspnea was five days, to hospital admission was 7 days and to the intensive care unit (icu) was eight days. consequently, the latency phase could be enough for the virus to entree and demolish the medulla oblongata. previous studies have also reported that some patients infected with sars-cov-2 showed neurological symptoms such as headache, ataxia, and convulsion. 6, 56, 57 in a recent study on 214 patients with covid-19, mao and others 6 found that almost 88% of the patients with severe infection exhibited neurological signs such as acute cerebrovascular disease and altered mental status. hence, awareness of the potential neuroinvasion can have guiding importance for the management of covid-19-induced respiratory catastrophes. therefore, similar to more respiratory viruses, sars-cov-2 can go into the brain through blood or neuronal systems. the presence of hyposmia in some patients supports the notion that the virus may enter the cns via the neuronal pathway. there has been a report of the identification of the sars-cov nucleic acid in the csf and brain tissue of patients with covid-19. 4 on march 4, 2020, jingyuan 5 described a middle age patient with pneumonia in whose csf sars-cov-2 was identified by genetic sequencing. the report also stated that despite the presence of signs and related to central nervous system damage, but brain imaging failed to expose any abnormalities and biochemical tests were normal for the csf. fortunately, however, the diagnostic test on the cerebrospinal fluid of the patient showing the existence of the sars-cov-2 coronavirus, and treatment for viral encephalitis was commenced. on march 21, 2020, filatov and others 1 described a 74-year-old man with respiratory disease, convulsion, altered mental status, and confusion who was diagnosed with covid-19. the patient, however, exhibited no csf evidence of cns infection. the authors recommended awareness concerning the manifestations of encephalopathy in patients in the acute phase of covid-19. in a review published on february 27, 2020, yan-chao and colleagues 57 of jilin university in china concluded that sars-cov-2 could contaminate nerve cells, chiefly neurons in the medulla oblongata (part of the brainstem that functions as the control center for the heart and the lungs). the injury could be related to acute respiratory failure in patients with covid-19. indeed, while the bulk of research conducted and published thus far has focused on the mechanisms whereby sars-cov-2 targets the respiratory system, more recent investigations have reported disconcerting evidence of the entrance of this new coronavirus into the cns via different ways, resulting in significant damage to this system or even death due to its infection. some investigators in china reported that more than 30% of their 214 patients with covid-19 presented with neurological signs and symptoms; they, therefore, concluded that sars-cov-2 might attack the cns through blood or retrograde neuronal routes, causing the destruction of the cns. 6 most recently, we described a female patient who referred with exclusive manifestations. our patient had altered mental status, the involvement of brain basal ganglia, most probably due to covid-19. she presented with respiratory manifestations and computed tomography scanconfirmed lung involvement typically matched by covid-19 infection. we successfully treated her with routine anti-coronavirus drugs. what all the aforementioned cases indicate is that covid-19 may have different types of cns involvement that should be considered. 5 there have also been reports of low lymphocyte counts in patients with covid-19 who had neurological manifestations by comparison with patients with covid-19 who had no such manifestations, which hints at the possibility that patients with cns involvement may have various degrees of covid-19induced immunosuppression effects, particularly in the severe phase of the disease. a study reported high d-dimer levels in severe covid-19 in comparison with non-severe covid-19, indicating that the prevalence of cerebrovascular diseases may be higher in severely ill patients. 6 muscle involvement in covid19 the latest reports on covid-19 indicate the high incidence of muscle symptoms. 2 moreover, creatine kinase and lactate dehydrogenase levels have been reported to be higher in patients with severe covid-19 than in those with nonsevere covid-19, which could be correlated with the presence of ace2 in skeletal muscles. 56 consequently, whether sars-cov-2 infects muscle cells by binding to ace2 receptors needs further research. it is also deserving of note that significantly elevated pro-inflammatory cytokines in serum can produce muscle injury. 56 stroke: in some new articles, the incidence rate of ischemic stroke in patients with covid-19 is up to 2.5%. 58 there are also reports regarding a relationship between inflammation and resultant coagulopathy by il-6 and fibrinogen due to damage to the lung alveoli. 59 the pathophysiology of the prothrombotic state subsequent to viral infections has been widely recognized. patients' susceptibility to coagulopathy and thrombotic events can be explained by prolonged stays in intensive care units and disabilities. 59, 60 guillain-barré syndrome: some reports are indicating that this syndrome could be one of the considerable neurological complications of sars-cov-2. 60 the most likely mechanism is that antibodies against some surface glycoproteins are expressed against foreign antigens that also respond to the parallel native protein structure on the neurons. nevertheless, the lack of relevant data and the complexity of establishing a causal relationship between a single pathogen and the syndrome make it extremely difficult to assess the robustness of the reported evidence. 60 cerebrospinal fluid: as yet, there is no specific csf analysis feature in covid-19 infection with neurological manifestations (especially encephalopathy), and the reported results thus far have been normal. still, diagnostic tests on the csf of patients may be essential to the diagnosis of covid-19 neuroinfection. 1, 5 new data analyses on covid-19 indicate a noticeable incidence of neurological disorders, especially in more severe infection. the neurological disorders in covid-19 may constitute a neurological tableau of a direct viral infection, neurological sequelae after the resolution of the infection, and infection in patients with chronic neurological diseases, not lease those who require immune-suppressive medications (e.g., multiple sclerosis). 6 in light of the reported findings, the neurological signs and symptoms of sars-cov-2 neuroinfection may indicate the involvement of the cns, creating such symptoms as headache, altered mental status, and epilepsy; the peripheral nervous system, creating such significant symptoms as hyposmia; and the skeletal muscles. 6 based on the findings presented here, it can be concluded that similar to other respiratory viruses, sars-cov-2 may enter the brain via the hematogenous or neuronal route. hyposmia may be deemed evidence of the neuroinvasion capability of sars-cov-2 via the olfactory route. indeed, what the ace2 theory posits is that sars-cov-2 may be responsible for some neurological manifestations. in the current epidemic era of covid-19, it is highly advisable that healthcare workers take into account sars-cov-2 infection as a differential diagnosis in patients presenting with any neurological indicators to avoid misdiagnosis and the transmission of this contagious infection. the ace2 theory indicates that sars-cov-2 may induce some neurological manifestations via direct or indirect mechanisms. finally, gaining an insight into the pathways of sars-cov-2 neuroinvasion would further clarify the neurological pathogenesis and manifestations 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current update we wish to dedicate this study to all our colleagues and volunteers that work indefatigably against covid-19, especially those in the northern iranian province of mazandaran. many thanks are also due to all patients who suffered this contagious infection. none declared. key: cord-284038-93s3ffoy authors: keyhanian, kiandokht; umeton, raffaella pizzolato; mohit, babak; davoudi, vahid; hajighasemi, fatemeh; ghasemi, mehdi title: sars-cov-2 and nervous system: from pathogenesis to clinical manifestation date: 2020-11-07 journal: j neuroimmunol doi: 10.1016/j.jneuroim.2020.577436 sha: doc_id: 284038 cord_uid: 93s3ffoy since the coronavirus disease 2019 (covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a growing body of evidence indicates that besides common covid-19 symptoms, patients may develop various neurological manifestations affecting both the central and peripheral nervous systems as well as skeletal muscles. these manifestations can occur prior, during and even after the onset of covid-19 general symptoms. in this review, we discuss the possible neuroimmunological mechanisms underlying the nervous system and skeletal muscle involvement, and viral triggered neuroimmunological conditions associated with sars-cov-2, as well as therapeutic approaches that have been considered for these specific complications worldwide. the first reports of an atypical pneumonia epidemic emerged out of wuhan, china in december 2019, and by early january 2020 the world health organization (who) started reporting on the issue (world health organization (who), 2020b). cases were associated with a novel strain of coronavirus, retrieved from lower respiratory tract samples of 4 cases on 7 january 2020, which is from the same family of viruses that are associated with severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) . subsequently the virus was named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (gorbalenya et al. , 2020) , and the disease was classified as coronavirus disease 2019 (covidamong other cranial nerves, trigeminal nerve and vagal nerve could be more plausible way of transmission. while sars-cov-2 involves lung and gastrointestinal tract very commonly, neuro-invasion through retrograde neuronal transport within vagal nerve afferents (li, bai, 2020 , toljan, 2020 has been postulated. local peripheral nerves located in the enteric nervous system, may also get infected by other cells in the gastrointestinal tract (bostancıklıoğlu, 2020 , lima et al. , 2020 , wong et al. , 2020b . experimental studies have demonstrated this retrograde route for the influenza virus (matsuda et al. , 2004) and hemagglutinating encephalomyelitis virus (hev) (andries and pensaert, 1980b) . moreover, trigeminal nerve, which usually supplies nociceptive cells in nasal cavity as well as sensory fibers in conjunctiva, might be a potential source of cns involvement. accordingly, sars-cov-2 rna has been found in patients with conjunctivitis (lima, siokas, 2020, sun and guan, 2020) . hematogenous spread, through the destruction of the blood-brain barrier (bbb), has been proposed as yet another pathway of viral invasion to the brain, as found in influenza and other coronaviruses (desforges, le coupanec, 2019 , koyuncu, hogue, 2013 , wang et al. , 2010 . this can be through the direct invasion of the cns by sars-cov-2 or infected leukocytes entering the cns (bostancıklıoğlu, 2020) . additionally, sars-cov-2 can attack angiotensin-converting (mhv) models (bleau et al. , 2015 , cabirac et al. , 1994 , cowley and weiss, 2010 . mhv studies demonstrated that coronaviruses might be capable of disrupting tight junctions of brain microvascular endothelial cells, leading to increase in permeability. mhv can cause myelitis, encephalitis and cns demyelinating disease. interestingly, mhv infected mice can be used as an experimental mouse model mimicking multiple sclerosis (mecha et al. , 2013) and causing demyelination in both brain and spinal cord. viral-like particles of sars-cov-2 were also found in post mortem brain endothelial capillary pericytes, supporting hematogenous cns infection in covid-19 patients (paniz-mondolfi et al. , 2020b) . moreover, the presence of sars-cov has been confirmed in the cerebrum of patients with sars (ding et al. , 2004) . the first and foremost important indication of viral infectivity is receptor recognition and presenting a combination of amino acids for the strongest binding of virus-host receptor. if a new virus makes a stronger bond than a prior one, it would be chosen by natural selection. rna viruses generally adapt to their hosts more rapidly, due to high mutation rates. their high adaptation capacity favors them for transmitting between animals to humans (wu et al. , 2012) . coronavirus family are all spherical or oval and have spike (s) glycoproteins throughout their envelope, which gives them the shape of a crown under electron microscopy. hence, they are named as corona (crown) viridae (schoeman and fielding, 2019, wu, xu, 2020b) . the trimeric s proteins and their receptor biding domains have a similar 3d structure and homology in both sars-cov and sars-cov-2. they both have strong affinity toward the human ace-2 receptor , ziegler et al. , 2020 . a series of mutations led to a different j o u r n a l p r e -p r o o f affinity of sars-cov receptor domains toward human ace-2 receptor. for example, there is a salt bridge between lys31 and glu35 under hydrophobic environment in human ace-2 hot spot 31 (yin et al. , 2020) . first in the civet sars-cov receptor binding domain, the 479 residues that counter reacts with hot spot 31 in ace-2 was also a lysine (yin, feng, 2020) . lysine in both sides causes steric and electrostatic interference with civet-sars-cov and ace-2 salt bridge counterpart. later as result of a point mutation (k479n) at lysine residue was substituted by asparagine, which guaranteed a stronger interaction between sars-cov and ace-2 and facilitated transmission of sars-cov to human. as such mutations happened through coronaviruses evolution, some of them were more advantageous for human-sars-cov-2 host interaction while still some are in favor of human-sars-cov. however, generally speaking, all the selected mutations in human-cov enhance their interaction with human ace-2 comparing to civet-cov (yin, feng, 2020) . ace-2 receptor is expressed in multiple tissues within human body including the cns and skeletal muscle (hamming, timens, 2004) . it is mainly detected over glial cells and neurons (baig, khaleeq, 2020 , palasca et al. , 2018 . thus, if the virus reaches out to cns or pns, neurons and glial cells would be potential targets. within the brain, ace-2 receptors are particularly present in the brainstem and medulla as part of reticular activating system function involved in regulation of cardiovascular system (xia and lazartigues, 2008) . interestingly though, expressing virus receptors, like ace for sars-cov or dipeptidyl peptidase 4 (dpp4) for mers-cov, did not seem to be the only mechanism for the host cell infection with coronavirus family. it was first postulated that the level of receptor expression in the cell is the determinant factor for its infectability but marked infection of liver by coronavirus despite very low to undetectable level of ace receptor suggested other mechanisms than ace theory j o u r n a l p r e -p r o o f (prabakaran et al. , 2004, to and lo, 2004) . another proposed pathway is through synaptic routes of nerve cells. hev, which is also a member of β-coronaviridae seems to infect cns retrogradely via peripheral sensory nerves (andries and pensaert, 1980a, hara et al. , 2009) . after infecting nerve cells hev particles bud from endoplasmic reticulum-golgi intermediate compartments. afterward they form into virion vesicles through golgi apparatus and finally are secreted into the surrounding matrix. virions would then be up taken by adjacent nerve cells (figure 1) (hara, hasebe, 2009 , steardo et al. , 2020 . viral transport through olfactory nerve seems to be a feasible channel for introducing the virus from endothelium to olfactory nerves and bulb and finally passing to brain. olfactory epithelial cells also express ace-2. this pathway also explains the anosmia caused by sars-cov-2 infection. but whether interacting with ace-2 receptor is the primary mechanism for anosmia commonly found in sars-cov infection or disruption of ciliary nasal epithelium similar to hcov-229e is key, and is yet to be determined (koyuncu, hogue, 2013 , lechien, chiesa-estomba, 2020 , troyer et al. , 2020 , wu, xu, 2020b . notably, when transgenic mice expressing human ace-2 were infected intranasally by sars-cov, the viruses were found to enter the brain by day 4 post infection primarily via the olfactory bulb resulting in a rapid, transneuronal spread to the connected areas of the brain (netland, meyerholz, 2008) . another coronavirus, hcov-oc43, has demonstrated a similar behavior (dube et al. , 2018) . in this case, the virus spreads to the piriformis cortex, brain stem, and spinal cord by day 4 post infection. interestingly, administration of zinc sulfate, that causes degeneration of the olfactory sensory neurons, almost completely stopped the virus to gain entry to the cns (dube, le coupanec, 2018) . moreover, when transgenic mice expressing human dpp4 were infected intranasally by mers-cov, brain disease was observed, with the greatest involvement noted in the thalamus and brain stem (li et al. , 2016) . the temporal course j o u r n a l p r e -p r o o f of brain tissue infection suggested retrograde virus spread from olfactory neurons. altogether, these data support the critical role of the olfactory pathway and ace-2 in the neuroinvasion process. to date, the full pathway for nerve and glial cells infections is not convincingly explained. however, in prior case reports and autopsies, sars-cov and mers-cov particles were found within neurons and glial cells as well as the cerebrospinal fluid (csf) proving that cells in the nervous system can be infected (he et al. , 2003 , lau et al. , 2004 , li, wohlford-lenane, 2016 , xu et al. , 2005 . after cell infection with coronavirus, the cell ultimate endpoint is death whether it would happen through autophagy, apoptosis, pyroptosis, elimination through innate immune cells, or other pathways (varga et al. , 2020, yang and . viral antigens were detected in respiratory brain stem centers like nucleus of the solitary tract and nucleus ambiguous. damage to these centers may be a contributor to cardiac or respiratory arrest (li, bai, 2020 , steardo, steardo jr, 2020 , xia and lazartigues, 2010 . additional effect of sars-cov-2 on cns is through systemic and local inflammatory response causing cytokines storming and immune cells reactivation (shi et al. , 2020 , steardo, steardo jr, 2020 . earlier epidemiologic studies showed that about 15% of cases might advance to severe disease and the rate is higher in people older than 65 years of age (guan et al. , 2020) . later some european countries like italy showed higher case fatalities from what china and most of other countries witnessed (onder et al. , 2020) . the determinant factors for progressing to severe stages are not completely understood; and is the most striking question. there is always a tug-of-war between viruses and host immune response. through years the host immune system either succeeds in clearing the pathogen or adapts in a way causing chronic viral infections. when a virus surpasses a species after years of co-evolution, the new host would respond to it with a more severe immune reaction that can even damage host tissues. accordingly, possible severe immune response to sars-cov-2 is expected (fung et al. , 2020 , rahman et al. , 2011 , wagstaff et al. , 2013 . there are several pathways proposed to be involved in human immune response towards sars-cov-2, and all include two general phases, innate and adaptive immune responses (figure 1). innate immune response includes activation of neutrophils, macrophages and natural killer cells and adaptive response involves cytotoxic cd8+ cells, cd4+ t helper cells and b cells (steardo, steardo jr, 2020) . what has been observed so far in severe and fatal covid-19 infection is a reduction in the absolute number of t cells as well as monocytes, eosinophils, and basophils. at the same time neutrophilic response is enhanced, leading to increased neutrophil-lymphocyte ratio. despite absolute reduction in total number of t cells, including both cd4+ and cd8+ cells, the main reduction is among memory t helpers and regulatory cells, while naïve t cells and pro-inflammatory t helper 17 cells were even boosted in number (karakike and giamarellos-bourboulis, 2019 , lagunas-rangel, 2020 , qin et al. , 2020 . because of this pro-inflammatory cell shift, immune cells hyper-react by producing excess levels of inflammatory cytokines. whether the cytokine storm is a part of the "cytokine release syndrome (crs)", or the "secondary haemophagocytic lymphohistiocytosis (shlh)" also called "macrophage activation like syndrome (mal)", the outcome is a robust increase in the highly inflammatory cytokines such as interleukin (il)-6, il-2, il-7, granulocytecolony stimulating factor (gmcsf), and tumor necrosis factor-α (tnf-α) (mehta et al. , 2020 . j o u r n a l p r e -p r o o f crs is commonly seen after car t cell therapy and sepsis following organ transplantation and is also reported after viral infections. clinical features of crs include headache, fever, encephalopathy, hypotension, coagulopathy, cytopenia and multiorgan failure , zhang, wu, 2020a . most of these features are shared with mal, which also could occur secondary to infections and hematological malignancies (karakike and giamarellos-bourboulis, 2019 ). one of the main outcomes commonly found in both crs and mal is an upsurge in il-6, which is reported to be also augmented in moderate to severe cases of sars-cov-2 infection (wan et al. , 2020) . inflammatory conditions leading to increased il-6 and tnf-α also might facilitate disruption of bbb (ichiyama et al. , 2002 , linker et al. , 2008 which in turn might be responsible for encephalitis, acute necrotizing encephalopathy and demyelination in the cns and even guillain-barré syndrome (gbs) in the pns. there are several case reports of such complications due to sars-cov-2 infection as we discuss in the next sections (alberti et al. , 2020 , mcabee et al. , 2020 , moriguchi et al. , 2020 , poyiadji et al. , 2020 , zanin et al. , 2020 . interestingly, tocilizumab (a recombinant, humanized monoclonal antibody against the il-6 receptor), which is an fda approved medication for treatment of t cell induced crs, also showed some benefit over severe cases of covid-19 infection (le, li, 2018 , xu et al. , 2020a . however, as of yet, there is not sufficient evidence to clarify the exact role of systemic inflammation versus local inflammation due to the direct viral infection or hypoxia, which is a common complication of sars-cov-2 infection. the brain and the lungs have a close inter-relation. a disease process in one would potentially cause complications of the other (abdennour et al. , 2012) . brainstem centers for respiratory and cardiovascular systems are potential targets of sars-cov-2 and neural cell death in these centers might be responsible for a central cause of respiratory/cardiovascular arrest (li, bai, 2020 , steardo, steardo jr, 2020 , xia and lazartigues, 2010 . sars-cov-2 lung infection has been reported to cause an atypical form of ards, while patients usually show relatively wellpreserved lung mechanics not matching the severity of hypoxemia. this may be due to the dysregulation of lung perfusion and hypoxic vasoconstriction, which may have a central cause as well (gattinoni et al. , 2020) . on the other hand, through a process called "infectious toxic encephalopathy" usually seen in toxic metabolic disorders or acute infections, alveolar gas exchange problem might lead to anaerobic metabolism in brain cells, and cause cns hypoxia. the hypoxemia and increased acidity within the brain causes cell swelling, interstitial edema, obstructive hydrocephalus, and increased intracranial hypertension leading to an altered mental status and even coma (abdennour, zeghal, 2012 , wu, xu, 2020b . hypoxic injury to the brain also may cause cerebrovascular accidents like stroke or seizures, and again would activate the loop of both local microglial activation and systemic inflammation mccullough, 2013, wu, xu, 2020b) . another clinical and scientific significance of sars-cov-2 infection is widespread observation of hypercoagulable state indicated by elevated d-dimer level, prolongation of prothrombin time (pt), activated partial thromboplastin time (aptt), and thrombocytopenia (violi et al. , 2020b) . coagulopathy was previously observed in infection with other coronoviridae viruses including sars and mers (giannis et al. , 2020, merad and martin, 2020) . although there are some prospective studies currently looking at incidence of thrombotic events, early studies have already confirmed increased frequency of intravascular thrombosis leading to pulmonary embolism, myocardial infarction, ischemic strokes, and even cerebral venous sinus thrombosis. j o u r n a l p r e -p r o o f a thrombotic event was sometimes reported as the first presentation of covid-19 infection (hughes et al. , 2020 , klok et al. , 2020 . in a retrospective study on 214 covid-19 patients, about 6% presented with acute cerebrovascular events, mainly ischemic strokes (cantador et al. , 2020) . a small number of stroke patients with covid-19 infection presented with cerebral hemorrhage (cantador, nunez, 2020) . recent investigation has also found that covid-19 patients have increased serum nox2 overactivation, which is an important trigger for vascular dysfunction through excess production of reactive oxygen species (violi et al. , 2020a) . interestingly, it was more up-regulated in more severe covid-19 cases and also those with thrombotic complications (violi, oliva, 2020a) . one possible underlying mechanism is the reduced expression and function of ace-2 in sars-cov-2 infected cells. ace-2 regulates the cerebral blood flow and its altered signaling can lead to subsequent hypertension and predisposition to developing hemorrhagic stroke from arterial wall rupture (sharifi-razavi et al. , 2020) . another possible mechanisms is the underlying coagulopathy induced by the infection with thrombocytopenia . since the early phases of the global pandemic, numerous studies have demonstrated clinical symptoms and signs of covid-19 which mainly include fever, cough, sore throat, dyspnea, diarrhea, nausea, vomiting, anorexia, and fatigue ( j o u r n a l p r e -p r o o f consciousness (7.5%), ageusia (5.6%), anosmia (5.1%), stroke (2.8%), nerve pain (2.3%), visual impairment (1.4%), seizure (0.5%), and ataxia (0.5%) (mao et al. , 2020 ). an important finding was the significantly higher rate of neurological manifestations (in general) and impaired consciousness, stroke and skeletal muscle injury (in particular) in patients with severe covid-19 infection than those with non-severe infection (mao, jin, 2020) . another study demonstrated that the major factor associated with neurologic complications was age over 60, which was also a strong risk factor for mortality (xiong et al. , 2020) . when patients with covid-19 infection were compared at the same level of severity, new-onset of neurologic critical events (e.g. impaired consciousness and stroke) was later found to increase the risk of death by six-fold (xiong, mu, 2020) . overall, the most common neurological symptoms described in patients are fatigue/malaise, myalgia, headache, impaired consciousness, dizziness, ageusia, and anosmia; and less common reported symptoms include visual impairment, nerve pain, occipital neuralgia, ataxia, tremor, and tic. growing number of case reports and/or series indicate that a variety of neurological conditions and post-viral triggered autoimmune complications, as we discuss below, occur in association with sars-cov-2 infection which mainly include guillain-barré syndromes (gbss) (table 2), myopathy and rhabdomyolysis (table 2) , encephalopathy, meningoencephalitis, encephalomyelitis, and myelitis (table 3) . gbss characteristically manifest with acute (< 4 weeks) ascending muscle weakness accompanied by decreased/absent deep tendon reflexes, mild-moderate sensory loss, occasionally cranial nerve involvement, and radicular or muscle pain. although gbss are commonly demyelinating (i.e. acute inflammatory demyelinating polyneuropathy [aidp]), j o u r n a l p r e -p r o o f primary axonal injury may occur, known as acute motor and sensory axonal neuropathy (amsan) or acute motor axonal neuropathy (aman). miller-fisher syndrome (mfs) is another gbs variant which is characterized by the triad of ophthalmoplegia, gait ataxia, and areflexia (rocha cabrero and morrison, 2020). gbs is considered as an autoimmune neurologic disease that can be triggered by a variety of viruses. about 70% of cases may have a viral illness 1-3 weeks prior to neurologic symptoms (wakerley and yuki, 2013) . gbs outbreak has been observed with viral epidemics including those with coronaviruses (i.e. mers-cov and sars-cov) (kim et al. , 2017, wakerley and yuki, 2013) . the first case of sars-cov-2 related gbs was reported from the major covid-19 hotspot, wuhan, china . the patient was a 61 years old woman who, one week after her trip from wuhan, developed rapidly progressive ascending limb weakness over 3 days accompanied by areflexia and later distal sensory changes. the csf study (day 4) revealed albuminocytologic dissociation and electromyography/nerve conduction study (emg/ncs) on day 5 showed a demyelinating neuropathy at early stage. laboratory results on admission were notable for lymphocytopenia and thrombocytopenia. she was treated with intravenous immunoglobulin (ivig). notably, she developed fever, cough, and pneumonia eight days after the onset of neurological symptoms. the sars-cov-2 rrt-pcr was positive in oropharyngeal swabs at that time. the patient had a full recovery after 3 weeks and the repeat test for covid-19 was negative (zhao, shen, 2020a) . overall 30 cases of gbs variants have been reported worldwide in patients with confirmed covid-19 since the pandemic (table 2), with mostly typical manifestation of rapidly progressive flaccid limbs weakness and areflexia, with and without facial muscle weakness, and distal paresthesia or numbness. cranial nerve involvements have been observed in 5 (17%) cases (bigaut et al. , 2020 , gutiérrez-ortiz et al. , 2020 , reyes-bueno et al. , 2020 j o u r n a l p r e -p r o o f al. , 2020). either unilateral or bifacial weakness was present in 12 (40%) cases. in 28 patients, covid-19 symptoms were variably present between 26 and 2 days prior to the onset of gbs symptoms, including cough (70%), fever (57%), gastrointestinal symptoms (e.g. diarrhea, nausea and vomiting, 33%), dyspnea (17%), myalgia (17%), ageusia (20%), anosmia (20%), fatigue/malaise (13%). ageusia and anosmia were also present on admission in 17% and 10% of these cases, respectively. three cases (all from spain) had mfs (gutiérrez-ortiz, méndez, 2020, reyes-bueno, garcía-trujillo, 2020). among 26 patients that underwent csf study, elevated protein levels (44 to 313 mg/dl) with normal leukocyte counts (i.e. albuminocytologic dissociation) were found in 22 (85%) patients. positive serum anti-gd1b igg antibody was reported in one patient with mfs (gutiérrez-ortiz, méndez, 2020). however, serum and csf anti-ganglioside antibodies (including anti-gm1, gq1b, and gd1b antibodies) checked in 10 and 2 cases, respectively, were negative (table 2). variable degrees of leukocytopenia (mainly lymphocytopenia; 40%) and elevated acute phase reactants (e.g. erythrocyte sedimentation rate [esr] , c-reactive protein [crp], ferritin, or fibrinogen; 40%) were also reported among these 30 cases. except for five patients (two with mfs) that did not undergo emg/ncs, 20 cases (including one with mfs) (80%) had demyelinating features (i.e. aidp), 4 (16%) had amsan, and one (4%) had aman in emg/ncs. this implies that different gbs variants could occur in association with sars-cov-2. except for 4 patients (including one mfs and one aidp case who received plasma exchange therapy), all other patients received a 5-day course of ivig (0.4 mg/kg/day, one patient received only one dose) with an additional cycle for 2 patients. overall, the outcome was favorable with partial to complete recovery in 18 (60%) cases. two patients passed away due to severe, progressive respiratory failure within 24 hours after initiation of ivig (alberti, beretta, 2020, marta-enguita et al. , 2020). an important finding in these reported cases is that covid-19 rrt-pcr was negative in all those 23 checked csf samples, indicating no active intrathecal sars-cov-2 replication or root infection. this finding combined with relatively favorable outcome post-ivig therapy and positive anti-gd1b antibody in one case may suggest an underlying autoimmune process triggered by post-sars-cov-2 viral infection in these cases. there is evidence that the sars-cov-2 s protein can bind to sialic acid-containing glycoprotein and gangliosides on cell surfaces (fantini et al. , 2020) , increasing its viral transmissibility. therefore, it is possible that crossreactivity between epitopes within the sars-cov-2 s protein-bearing gangliosides and surface peripheral nerve glycolipids may occur, serving as an underlying mechanism in sars-cov-2 triggered autoimmune gbs. accordingly, most gbs variants (aidp, aman, amsan, and mfs) have been reported in sars-cov-2 patients (table 2). checking anti-ganglioside antibodies in future cases may provide more detailed information about this hypothesis. it is also noteworthy that some of the reported gbs cases received hydroxychloroquine in addition to ivig or plasma exchange therapy. chloroquine is shown to bind to sialic acids and gm1 gangliosides preventing binding to sars-cov-2 s protein, thereby inhibiting virus entry to the cells. therefore, adjunctive therapy with chloroquine in sars-cov-2-associated gbs could be an interesting consideration in future studies. and shabarek, 2020), association of sars-cov-2 with either viral or necrotizing autoimmune myositis is still elusive. two reported cases may indirectly suggest a sars-cov-2 triggered necrotizing autoimmune myositis tong, 2020, suwanwongse and shabarek, 2020) . the first case is a man, aged 88 years, from new york, presenting with acute, painful bilateral proximal lower limb weakness and hyperckemia (13581 u/l) (suwanwongse and shabarek, 2020) who was found covid positive and started on hydroxychloroquine, and his weakness and ck levels improved one week later. the second case is a man, aged 60 years, from wuhan, with 6-day covid-positive pneumonia and fever who 7 days later, despite improvement in his clinical condition, developed painful proximal muscle weakness with hyperckemia (11842 u/l) and elevated crp, and benefited from ivig therapy (jin and tong, 2020) . a more recent study also reported six intensive care unit (icu)-admitted cases (age between 51 and 72 years old) with covid-19 who had acute flaccid quadriplegia (madia et al. , 2020) . emg/ncs showed myopathic features in all of these patients and ck levels were normal to mildly elevated (highest level of 1274 u/l), suggesting the presence of critically illness myopathy (madia, merico, 2020) . overall, these observations may necessitate pursuing more investigations such as muscle biopsy and antibody screening in some covid-19 patients with signs of skeletal muscle injury, as treatment with ivig may potentially improve functional outcomes in these patients. notably, ace-2 is shown to be expressed in skeletal muscles (cabello-verrugio et al. , 2015) ; thus, evaluation of direct sars-cov-2 infection of skeletal muscle fibers would be a highly interesting topic for future studies. human coronavirus infections have been associated with encephalopathies in the past and are known to have human neurotropic and neuroinvasive potentials, mainly through the olfactory bulb or hematogenous route, causing inflammation, and demyelination (desforges, le coupanec, 2019) . the underlying pathophysiology is still not well understood, and includes abnormal host immune responses with autoimmunity and/or direct cns damage due to viral replication and infiltration (desforges, le coupanec, 2019) . more in details, ace-2 and transmembrane protease, serine 2 (tmprss2) are documented co-receptors for sars-cov-2 entry and they are expressed in the oligodendrocytes, suggesting a direct involvement of white matter in case of encephalitis related to covid-19 infection (needham et al. , 2020 , sellner et al. , 2020 . a retrospective study from turkey (kandemirli et al. , 2020) included 235 patients in icu, 50 of which (21%) developed neurological symptoms. the authors collected mri data from 27/50 patients to show neurotropism related to covid-19. the findings included cortical flair (fluid attenuated inversion recovery) abnormalities (37%) with cortical diffusion restriction, leptomeningeal enhancement, or cortical blooming artifact in a non-specific pattern. less frequently subcortical and deep white matter flair lesions were reported. unfortunately, the findings were not correlated with the patients' symptomatology. csf data were also obtained in half of the patients with cortical flair abnormalities, showing elevated proteins, normal cell count, glucose level, igg index, oligoclonal bands and albumin as well as negative rrt-pcr for sars-cov-2 (kandemirli, dogan, 2020) . another report from uk described a patient with fever and respiratory symptoms who developed progressive unsteady gait, diplopia, limb ataxia, altered sensation in the right arm, hiccups, and dribbling when eating, found to have a rhomboencephalitis in the mri with involvement of the right inferior cerebellar peduncle (table 3) . csf showed normal protein, normal white blood cell (wbc) counts and negative bacterial j o u r n a l p r e -p r o o f culture (wong et al. , 2020a) . unfortunately, the sars-cov-2 pcr test was not administered, and there were no results for the myelin oligodendrocyte glycoprotein and aquaporin 4 antibodies sent as part of the workup. the presence of brain inflammatory changes related to covid-19 was also confirmed by neuropathology findings of foci of perivascular lymphocytes, focal leptomeningeal inflammation in brain specimens of 18 encephalopathic patients, although these findings were reported as rare and did not support an underlying diagnosis of encephalitis. immunohistochemical analyses to detect sars-cov-2 by rrt-pcr performed in the tissues were negative, and the virus was detected at low levels in only 5 patients, possibly as a result of viral direct infiltration in the brain or viral rna coming from blood (solomon et al. , 2020). increasing evidence indicates that encephalopathy is one of the several presenting symptoms or complications of covid-19. encephalitis represents an inflammatory process of the brain and surrounding tissues and its symptomatology can include altered mental status, headache, behavioral changes, psychiatric disturbances in association with focal neurological signs (e.g. paresthesia, weakness, etc.). on the other end, meningitis is an inflammatory process of the meninges and spinal cord and gives typical symptoms such as fever, headache, photophobia, phonophobia and neck stiffness. seizures could also be part of the encephalitis and meningitis presentation (asadi-pooya, 2020, sohal and mossammat, 2020). the severity of the above symptoms can vary and sometimes it is difficult to make a proper diagnosis particularly in patients with mild symptoms. to add complexity in the diagnosis and management of encephalopathies, there is the inability to distinguish the underlying process (infectious or toxicmetabolic) only based on the symptoms. indeed, many patients with severe covid-19 infection j o u r n a l p r e -p r o o f may present with altered mental status from the toxic metabolic processes due to hypoxia, electrolyte derangements, metabolic disturbances, and multiorgan failure, without necessarily presenting involvement of the cns. moreover, two cases of acute necrotizing encephalopathy (ane) in patients with covid-19 positivity from nasopharyngeal and oropharyngeal swab, but without csf pcr for sars-cov-2 data, were reported in the literature (poyiadji, shahin, 2020 , radmanesh et al. , 2020 . ane is characterized by neuroinflammation secondary to cytokine storm with multifocal symmetric lesions in the gray and white matter without direct viral damage. in addition, there is a report of possible demyelinating lesion in the white matter and globus pallidus in a 54-year-old woman admitted initially for respiratory distress due to covid-19 infection with only history of mild elevated blood pressure under treatment. her glasgow coma scale score was 14 with altered sensorium but her neurological exam was non-focal on admission, then rapidly deteriorated and required endotracheal intubation and received hydroxychloroquine in addition to azithromycin and amoxicillin/clavulanic acid. sedation was discontinued two days later but the patient remained obtunded for a long period afterwards, and this prompt neuroimaging and further investigations. brain mri eventually revealed bilateral asymmetric restricted diffusion lesions without hemorrhage or enhancement in the supratentorial periventricular white matter and globus pallidus, without involvement of the thalamus, striatum and posterior fossa. subsequent mri obtained 2 days later showed homogeneous contrast enhancement of the lesions, brain vascular images were negative. csf studies, performed twice (on admission and 9 days after), were reportedly unremarkable, including rrt-pcr for sars-cov-2. the patient was treated with steroid for suspected demyelination twelve days later after her hospitalization upon negative nasopharyngeal pcr. the patient reported residual right-side hemiplegia and there are no data about her response to steroids, which could be supportive of the j o u r n a l p r e -p r o o f diagnosis of demyelination (brun et al. , 2020) . despite that, the images were suspicious of demyelination, which has been described associated with coronavirus, both in murine animal models (wu et al. , 2000) , and in a pediatric patient with acute disseminated encephalomyelitis (adem) (yeh et al. , 2004) . however, other diagnoses could not be completely excluded in the case described above. in a retrospective observational case series from wuhan (mao, jin, 2020 ) that collected data from 214 patients with laboratory-confirmed diagnosis, 36.4% had neurological manifestations. more in detail, cns manifestation was present in 24.8% of the patients, and in particular 7.5% had encephalopathy. the authors noticed that cns manifestation were significantly more common in patients with severe infection compared to non-severe infection, with encephalopathy present in 14.8% of cases versus 2.4% (p < 0.001), respectively. the patients with severe infection were older, with higher blood pressure and with less typical symptoms such as fever or cough on admission. furthermore, those patients were more prone to develop neurological symptoms few days after the admission, with associated higher mortality rate. they also had a marked inflammatory response with higher levels of wbc counts, neutrophil counts, blood urea nitrogen, d-dimer and crp, and reduced lymphocyte and platelet counts than in those with less severe infection, pointing to a multiorgan involvement and immunosuppression as underlying pathogenic mechanism for the neurological manifestations. the study did not further investigate the etiology of the encephalopathy, either toxic-metabolic or infective. other case reports described encephalopathic patients who first presented to the hospital with new onset of seizure as manifestation of underlying meningoencephalitis in the setting of covid-19 infection (asadi-pooya, 2020 , moriguchi, harii, 2020 , sohal and mossammat, 2020 . as of now, there are no csf or serologic biomarkers available worldwide to help diagnosing cases of covid-19 with cns involvement (baig, 2020 , kandemirli, dogan, 2020 ; therefore, proper neurological evaluation of encephalopathic covid-19 patients can help early diagnosis, better tailor the treatment, and possibly improve the outcome. the workup for encephalopathic patients should not only include detailed documentation of the neurological symptoms but also electrophysiological studies (i.e. electroencephalography or eeg), csf analysis, and perhaps brain imaging (asadi-pooya and simani, 2020, liu et al. , 2020b , oxley et al. , 2020 . moreover, seizures should be suspected in case of altered mental status in patients with covid-19 since cases with clinical or subclinical seizures or status epilepticus have been reported, either as a direct consequence of the brain damage from the virus or secondary toxic-metabolic derangements (asadi-pooya, 2020). anti-epileptic drugs (aeds) should be administered in patients with seizure as initial presentation, to prevent further episodes, for a period of 6 weeks and then taper and discontinue the aed in 1-2 weeks (asadi-pooya, 2020). since these covid-19 patients are usually critically ill, intravenous formulations and aeds with less side effect on respiratory and cardiac status are recommended, such as levetiracetam and brivaracetam. moreover, since some patients may require extracorporeal membrane oxygenation which affects the pharmacokinetic of highly protein-bound aeds, phenytoin and valproic acid should be avoided (asadi-pooya, 2020). the diagnosis of covid-19 meningoencephalitis is based on clinical and laboratory studies such as csf characteristics and possibly detection of the virus in the csf. the first case of covid-19 with associated laboratory-confirmed viral encephalitis was reported in beijing in a patient j o u r n a l p r e -p r o o f with altered mental status, seizures, persistent hiccups, hyperreflexia, meningeal irritation and slow pupillary response (table 3) . notably, csf studies showed normal range wbc, glucose and protein, but an increased opening pressure and positive pcr for sars-cov-2 (oxley, mocco, 2020, sun and guan, 2020) . this case report was published in chinese and it seems to lack further clinical and laboratory data to corroborate the diagnosis. a recent paper discussed about a woman with encephalitis and no respiratory symptoms with sars-cov-2 positivity in both nasopharyngeal swab and csf . another case of meningoencephalitis from covid-19 in japan (moriguchi, harii, 2020) described a young patient who presented with headache, fatigue, fever and few day later was found unconscious with an episode of generalized tonic seizure while transported to the hospital. he had clear meningeal signs, pleocytosis in the blood, negative ct scan of the head, with sars-cov-2 detected only in the csf but not in the nasopharyngeal swab. interestingly csf showed elevated opening pressure and 12 wbc mainly mononuclear; mri showed dwi (diffusion-weighted imaging) positivity along the wall of the inferior horn of the right ventricle and flair abnormalities in the right mesial temporal lobe and hippocampus. these are the only three encephalitis cases based on our knowledge that were associated with csf viral detection. it is noteworthy that false positivity of the pcr has been reported given the risk of sample contamination from shed airborne virus with this diagnostic technique (needham, chou, 2020). on the contrary, there is also a case report of a patient who presented with fever, cough and typical multiple ground-glass opacities on ct of the lungs, who later developed focal neurological symptoms and altered mental status suggestive of meningoencephalitis (yin, feng, 2020) . notably, the throat swab was positive for covid-19 but the csf pcr was negative (yin, feng, 2020) . other csf results suggestive of viral infection were the elevated opening pressure j o u r n a l p r e -p r o o f and proteins (yin, feng, 2020) . the patient was monitored, treated with antivirals and supportive care. over time the lung infections improved, he started requiring less oxygen supplementation and concomitantly his neurological exam improved consistently. at that time two further throat swab were done and resulted negative. other case reports of patients with suspected viral meningoencephalitis with positive nasopharyngeal swab but negative csf pcr for sars-cov-2 have been reported in the literature. in the majority of the reports, csf showed increased cells, mainly lymphocytes, and elevated protein levels , dogan et al. , 2020 . more in details, a case series of 53 icu patients reported 29 subjects intubated for severe ards and no improvement of their mental status or agitated delirium after extubation with subsequent neurological workup (dogan, kaya, 2020) . neurological involvement was diagnosed in 6 of the 29 intubated patients (20.6%). these patients had increased levels of acute-phase reactants such as ferritin, crp, il-6, fibrinogen. mri findings showed white matter and cortical abnormalities and contrast enhancement compatible with meningoencephalitis in 3/6 patients. csf data revealed elevated proteins without pleocytosis in all cases with negative pcr for viruses including sars-cov-2. an underlying autoimmune etiology was suspected for both mri positive and negative patients, and they underwent treatment with plasmapheresis. improvements of the clinical status were observed in 5/6 patients, and mri findings were reversible in all 3 patients with positive mri. another case from italy (pilotto et al. , 2020) described a 60-year old man with mild respiratory symptoms that developed akinetic mutism and nuchal rigidity. mri and eeg were negative for any abnormalities. csf showed elevated protein level and lymphocytic pleocytosis, as well as increased il-6, il-8 and tnf-α but negative for sars-cov-2 and for other neurotropic viruses. covid-19 infection was established by nasopharyngeal swab. the patient was started on antibiotic and antiviral coverage j o u r n a l p r e -p r o o f initially, as well as hydroxychloroquine. an improvement was seen after high dose of steroids were initiated. patient was discharged after 5 days of iv steroids with oral prednisone taper with normal neurological examination. although the exact pathogenetic mechanism of autoimmune encephalitis in the setting of covid-19 is unclear, it may be related to cytokine storm with direct damage to the bbb and increased leukocyte migration to the brain (sohal and mossammat, 2020), as well as dysregulation of viral immunity mediated by molecular mimicry (pilotto, odolini, 2020) . a trial with immunomodulatory therapies can be crucial to diagnose autoimmune encephalitis. further case reports of patients with suspected viral or autoimmune meningoencephalitis and detailed description of their presentation and workup are also needed. the cases reported above showed that csf pcr may be not reliable for the diagnosis since sars-cov-2 dissemination in the brain can be transient and its csf titer may be extremely low (ye et al. , 2020a) . furthermore, the test is not widely available. a proper neurological examination, eeg, csf studies, and brain imaging are for now the only tools that can guide to the diagnosis of covid-19-associated meningoencephalitis, and appropriate treatment should be initiated promptly. a retrospective study of 799 patients with covid-19 reported altered mental status on hospital admission in 22% of patients who expired and 1% among those who recovered from the infection. this hints towards a possible negative prognostic factor related to encephalopathy as initial presentation. headache without associated neurological symptoms or signs was reported in 10% of the deceased patients, versus 12% of the recovered patients. metabolic derangements were more common in deceased patients than in recovered patients, and j o u r n a l p r e -p r o o f 20% of the deceased patients suffered from what was classified as hypoxic encephalopathy related to pulmonary inflammation. in this study neurological symptoms other than headache, neurological signs, and seizures were not reported or considered as possible manifestation of the disease. furthermore, no laboratory studies were carried to rule out a viral encephalitis/meningitis, underlying seizures, and non-convulsive status epilepticus. it is possible that some of the cases described to have toxic encephalopathy were indeed suffering from a viral encephalitis. one of the first case reports published at the beginning of the pandemic (filatov et al. , 2020) reported the case of a 74-year old man with chronic obstructive disease (copd), atrial fibrillation and prior cardioembolic stroke in the left posterior cerebral artery. he presented to the emergency department with fever and cough, initial negative workup for pneumonia, and initially discharged with possible copd exacerbation. he was readmitted to the emergency department after 24 hour with severe altered mental status and headache. neurology was consulted at that time and a full work up included a ct scan of the head, eeg and csf studies were conducted. the head ct scan showed the old stroke, related encephalomalacia; and csf was not suggestive for infection, although sars-cov-2 pcr was not performed. eeg showed diffuse slowing and focal sharply contoured waves in the left temporal region which raised the suspicion for subclinical seizures. he eventually tested positive for covid-19. he was treated with aeds as well as hydroxychloroquine, lopinavir, and ritonavir. unfortunately, there is no follow-up report about his response to the treatments. this case report is an example of covid-19 related toxic metabolic encephalopathy and epileptic activity from lowered seizure threshold secondary to severe underlying metabolic process (delanty et al. , 1998) . another study from france (helms et al. , 2020) showed that 84% of patients with ards and covid-19 presented with neurological signs such as agitation (69%), corticospinal tract signs (67%) and dysexecutive syndrome with inattention, disorientation, or poorly organized movements in response to command (36%). mri of the brain was performed in 13 of the 54 patients reported in the study. notably, these patients did not have any focal signs suggestive for stroke, although 23% had an underlying ischemic stroke, 62% had leptomeningeal enhancement, and all the patients who underwent perfusion imaging (11; 100%) had bilateral frontotemporal hypoperfusion. a small proportion of the patients (8 out of 54) had an eeg that showed nonspecific changes or bilateral frontal slowing; and a smaller proportion (7) acute myelitis is a known complication of viral infections, mainly attributed as an autoimmune response, although it could also be an early manifestation of other neuroimmunological disorders such as multiple sclerosis and neuromyelitis optica spectrum disorders. little is known about association between sars-cov-2 and acute myelitis. up to date, 7 cases of acute myelitis, alone (4 cases) or combined with brain involvement (3 cases), have been reported in relation to covid-19 (alketbi et al. , 2020 , munz et al. , 2020 , novi et al. , 2020 , sarma and bilello, 2020 , valiuddin et al. , 2020 , wong, craik, 2020a j o u r n a l p r e -p r o o f zanin, saraceno, 2020) . six patients (28-64 years age range, 57.1% female) variably had symptoms of covid-19 (fever, dyspnea, malaise, chills, and rhinorrhea) 25 to 2 days prior the onset of neurologic symptoms of myelitis (table 3) . sars-cov-2 rrt-pcr were checked in the csf of 5 patients and all were negative. overall, the functional outcome was favorable in 6 (85.7%) patients after treatment with either steroids or plasmapheresis. additionally, a recent case report from spain (sotoca and rodríguez-álvarez, 2020) has presented a 68 years old woman who developed a 7-day radicular neck pain, right facial numbness, left hand numbness and weakness, gait instability, and general hyperreflexia. she had fever and cough 8 days prior these symptoms. the cervical spine demonstrated a t-2 hyperintensity extending from the medulla oblongata to c7, suggestive of acute transverse myelitis. she had negative blood testing for anti-antiaquaporin-4 (aq4), -myelin oligodendrocyte glycoprotein (mog), and antiphospholipid antibodies, but had elevated protein level and pleocytosis in the csf study with no oligoclonal band, normal igg index, no anti-neuronal surface antibody, and negative sars-cov-2 pcr. however, nasopharyngeal swab for sars-cov-2 rrt-pcr was positive. she was treated with 5-day methylprednisolone (1 gr/day). few days later her symptoms worsened, and she developed bladder/bowel incontinence, bilateral hands weakness and paresthesia, and paraparesis. the repeated spinal mri showed a new area of central necrosis at the t1 level with peripheral enhancement. this case was indeed the first case of acute necrotizing myelitis in association with covid-19. she was additionally treated with plasmapheresis and 5-day methylprednisolone (1 gr/day) followed by slow tapering oral prednisone with favorable outcome after 4 weeks. the exact underlying mechanism in acute necrotizing myelitis is still elusive; however, a post-viral triggered autoimmune cytokine storm has been suggested j o u r n a l p r e -p r o o f (kansagra and gallentine, 2011) . thus, this may imply in the case of sars-cov-2, especially with observed clinical improvement after steroid and plasmapheresis therapy. there is growing evidence about psychiatric manifestations as potential complication of sars-cov-2 infection. accordingly, a worldwide exacerbation of mental health disorders during the pandemic has been reported, which includes but not limited to delirium, cognitive impairment, mood alterations, psychosis and suicide (orsini et al. , 2020) . more in details, delirium has been noticed more in 90% of covid-19 patients whose conditions require icu level of care versus 70-75% rates documented in the past (kotfis et al. , 2020) . cognitive dysfunctions have been reported to be possibly a direct consequence of covid-19 infection to the cns, and in particular to the hippocampus, which appears to be vulnerable to coronaviruses infections, with possible acceleration of hippocampal degeneration as occurs in alzheimer's disease (ritchie et al. , 2020) . cognitive impairment can be also a consequence of acute respiratory syndrome and relative hypoxia which have been associated to cerebral atrophy and ventricular enlargement (hopkins et al. , 2006) and worsening attention, executive functions and verbal memory (hopkins et al. , 2005) . anxiety, depression, post-traumatic stress disorder, insomnia and obsessive-compulsive symptomatology appear to be very common in covid-19 survivors, particularly in females, and with worsened scores on psychopathological measures in those with previous psychiatric comorbidities (mazza et al. , 2020) . a recent study reports an incidence of psychosis in infected patients between 0.9% and 4% versus a median value of 15.2 (7.7 -43.0) per 100,000 previously described (mcgrath et al. , 2004) . increased j o u r n a l p r e -p r o o f rates of suicide have been also reported, with possible contributing factors found in the social isolation/distancing, economic recession and social discrimination (thakur and jain, 2020). it is unclear whether the above psychiatric symptoms are a direct consequence of the cns viral infection (i.e. viral meningoencephalitis), cerebrovascular accidents, hypoxia, and the immunological and inflammatory responses, which may play important roles in major depressive disorder (ghasemi et al. , 2019 , wohleb et al. , 2016 and psychosis (ferrando et al. , 2020) , or whether they are related to increased psychosocial stress of this severe and potentially fatal disease and difficulties accessing to health care related to the pandemic infection (zhou and yao, 2020 ). this has been posing increased challenges in the treatment of infected patients, especially those in the icu, requiring accurate multidisciplinary approaches and early interventions to decrease overall morbidity and mortality (ojeahere et al. , 2020 ). as described in above sections, several treatment approaches have been used to treat manifestations or consequences of the sars-cov-2−related nervous system injury, such as ivig for gbs and skeletal muscle injury, iv/oral steroids and plasmapheresis for autoimmune encephalitis and acute myelitis, and aeds for seizures. with regards to medications aimed to modulate the immune response to viral infection and to induce viral clearance, antimalarial drugs (e.g. hydroxychloroquine), dexamethasone, rna-dependent rna polymerase inhibitors (e.g. remdesivir), hiv-1 protease inhibitors (lopinavir/ritonavir), and biological agents like tocilizumab, interferons and convalescent plasma have shown some beneficial effects (chibber et al. , 2020) . among these, the fda granted emergency use authorization for remdesivir as an emergency medication for severely ill hospitalized adult and pediatric patients with proved or j o u r n a l p r e -p r o o f suspected sars-cov-2 infection (lamb, 2020) . this drug, which has a broad-spectrum antiviral activity against several rna viruses, can inhibit sars-cov-2 replication, alleviate symptoms, fasten the recovery rate, and reduce mortality rate (frediansyah et al. , 2020) . more in details, the final report of the adaptive covid-19 treatment trial (actt-1), a double-blind randomized placebo-controlled trial of intravenous remdesivir in affected adults with evidence of lower respiratory tract infection, showed a median recovery time of 10 days (95% confidence interval [ci], 9 to 11) versus 15 days (95% ci, 13 to 18) in those in the placebo group. mortality estimates by day 15 were 6.7% with remdesivir versus 11.9% with placebo and those by day 29 were 11.4% with remdesivir and 15.2% with placebo (paladugu and donato, 2020) . given the lack of head to head comparison, it is unclear if remdesivir offers a superior benefit over dexamethasone, which is widely available and less expensive (mccreary and angus, 2020). the randomized evaluation of covid-19 therapy (recovery) trial has shown that dexamethasone resulted in lower 28-day mortality in covid-19 patients receiving either invasive mechanical ventilation or oxygen alone at randomization but not in those receiving no respiratory support (horby et al. , 2020) . the authors of the actt-1 trial also adjusted the data for glucocorticoid use suggesting that the benefit of dexamethasone may be additive to that of remdesivir (beigel et al. , 2020) . however, it is still unclear whether remdesivir or dexamethasone have beneficial effects on the neurological manifestation of covid-19. with regards to possible therapeutic strategies aimed to ameliorate the neuronal damage mediated by covid-19, high doses of melatonin seem promising in immunomodulation and reducing neuroinflammation, with no direct effect on viral replication or transcription. melatonin seems to act via an anti-inflammatory, anti-oxidative and immune-enhancing mechanism with ability to restore the bbb hemostasis (romero et al. , 2020). there are ongoing worldwide clinical trials for the development of a vaccine to prevent covid-19, which is not currently available and that poses some challenges due to safety, efficacy, and long-lasting effects without further risks of re-infection, particularly in the elderly population (jamwal et al. , 2020) . the covid-19 pandemic with its variety of manifestations, not only pulmonary or neurological, is an international public health emergency that requires efforts from all countries to develop effective drugs and vaccines as early as possible. evolving data indicates that patients with covid-19 may variably develop neurologic manifestations prior, during and even after the onset of common covid-19 symptoms. the commonly reported neurological symptoms and signs include dizziness, headache, myalgia, fatigue, impaired consciousness and confusion, ageusia, anosmia, neuropathic or radicular pain, occipital neuralgia, visual impairment, seizure, and ataxia. based on a growing number of case reports and series, both the cns, pns and skeletal muscles can be involved in covid-19 presenting with a variety of neuroimmunological conditions including gbss, myopathy and rhabdomyolysis, encephalopathy, meningoencephalitis, encephalomyelitis, and acute myelitis. the exact etiology of these complications remains to be fully elucidated. however, suggested mechanisms are direct sars-cov-2 infection to the nervous system, neuroinflammation, postviral triggered autoimmune response, hypercoagulability, and metabolic or hypoxic injury. in general, therapeutic strategies for covid-19 are based on three main directions: (i) targeting sars-cov-2 with antivirals, neutralizing antibodies or convalescent plasma therapy, (ii) targeting inflammatory storm using immunomodulatory medications and cytokine inhibitors, and (iii) developing vaccines to prevent the disease manifestation (for comprehensive review see j o u r n a l p r e -p r o o f journal pre-proof (vabret et al. , 2020) ). however, it is still too early to find out whether even with successful treatment of the active infection, post-viral triggered autoimmune neurological complications of covid-19 (e.g. gbs, myositis, cns demyelination, and myelitis) will be also lowered in frequency and/or severity. additional studies are clearly needed to address this issue. no conflict of interest exists in relation to the submitted manuscript. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. (27) myalgia (19), headache (10), dizziness (9), impaired consciousness (22) fever (92), cough (70), fatigue (57), anorexia (27), dyspnea (62), chest tightness (49), pharyngalgia (4), hemoptysis (4), nausea (7), vomiting (5), abdominal pain (10) leukocytosis (50) (45) myalgia (24), headache (12), dizziness (7), impaired consciousness (1) fever (90), cough (66), fatigue (45), anorexia (22), dyspnea (31), chest tightness (30), pharyngalgia (5), hemoptysis (2), nausea (10), vomiting (6), abdominal pain (12) leukocytosis (4), lymphocytopenia (5),  albumin (14) & k + (11),  ast (16), alt (19), k + (4), na + (2), ddimer (2), ldh (14), crp (14), il-1β (12), il-2r (37), il-6 (60), il-8 (8) (55) leukopenia (25) headache (6), myalgia (11.5), malaise (35) fever (98), cough (77), dyspnea (63.5), rhinorrhea (6), arthralgia (1), chest pain (2), vomiting (4), ards (67) aki (29), cardiac injury (23) (5), rhinorrhea (4), chest pain (2), diarrhoea (2), nausea/vomiting (1), ards leukocytosis (24), lymphocytopenia (35) , thrombocytosis (4), thrombocytopenia (12), anemia (51),  albumin (98) j o u r n a l p r e -p r o o f adc, apparent diffusion coefficient; aki, acute kidney injury, alt, alanine aminotransferase; ards, acute respiratory distress syndrome; ast, aspartate aminotransferase; bnp, b-type natriuretic peptide; bun, blood urea nitrogen; ck, creatine kinase; cns, central nervous system; crp, c-reactive protein; dic, disseminated intravascular coagulation; eeg, electroencephalography; esr, erythrocyte sedimentation rate; f, female; fgf, fibroblast growth factors; gcsf, granulocyte colony-stimulating factor; gmcsf, granulocyte-macrophage colonystimulating factor; icu, intensive care unit; ifn-γ, interferon-γ; il, interleukin; ip, induced protein; iqr, interquartile range; ldh, lactate dehydrogenase; mcp, monocyte chemoattractant protein; mip, macrophage inflammatory protein; pdgf, platelet derived growth factor; pro-bnp, pro-brain natriuretic peptide; pt, prothrombin time; 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(60), neutropenia (12.9), lymphocytopenia (77.6), thrombocytosis (7.1), thrombocytopenia (41.2),  albumin (78.8),  d-dimer (65.9 fever (90.5), cough (61.5), anorexia (36.2), dyspnea (29), diarrhea aidp, acute inflammatory demyelinating polyneuropathy; aki, acute kidney injury, alt, alanine aminotransferase; aman, acute motor axonal neuropathy; amsan, acute motor and sensory axonal neuropathy; ards, acute respiratory distress syndrome; ast, aspartate aminotransferase; cbc dif , complete blood counts with differential; ck, creatine kinase; cmap, compound motor action potential; crp, c-reactive protein; covid-19, coronavirus disease 2019; csf, cerebrospinal fluid; emg/ncs, electromyography/nerve conduction study; esr, erythrocyte sedimentation rate; f, female; il, interleukin; ino, internuclear ophthalmoparesis; inr, international normalised ratio; ivig, intravenous immunoglobulin; ldh, lactate dehydrogenase; lft, liver function test; m, male; nr, not reported; pt, prothrombin time; rft, renal function test; rrt-pcr, real-time reverse transcription polymerase chain reaction; sars-cov-2, severe acute respiratory syndrome coronavirus 2; siadh, syndrome of inappropriate antidiuretic hormone secretion; snap, sensory nerve action potential; wbc, white blood cell. key: cord-299967-90aknp7c authors: terry, rachael l; getts, daniel r; deffrasnes, celine; van vreden, caryn; campbell, iain l; king, nicholas jc title: inflammatory monocytes and the pathogenesis of viral encephalitis date: 2012-12-17 journal: j neuroinflammation doi: 10.1186/1742-2094-9-270 sha: doc_id: 299967 cord_uid: 90aknp7c monocytes are a heterogeneous population of bone marrow-derived cells that are recruited to sites of infection and inflammation in many models of human diseases, including those of the central nervous system (cns). ly6c(hi)/ccr2(hi) inflammatory monocytes have been identified as the circulating precursors of brain macrophages, dendritic cells and arguably microglia in experimental autoimmune encephalomyelitis; alzheimer’s disease; stroke; and more recently in cns infection caused by herpes simplex virus, murine hepatitis virus, theiler’s murine encephalomyelitis virus, japanese encephalitis virus and west nile virus. the precise differentiation pathways and functions of inflammatory monocyte-derived populations in the inflamed cns remains a contentious issue, especially in regard to the existence of monocyte-derived microglia. furthermore, the contributions of monocyte-derived subsets to viral clearance and immunopathology are not well-defined. thus, understanding the pathways through which inflammatory monocytes migrate to the brain and their functional capacity within the cns is critical to inform future therapeutic strategies. this review discusses some of the key aspects of inflammatory monocyte trafficking to the brain and addresses the role of these cells in viral encephalitis. virus infection of the brain can cause severe and lifethreatening disease. despite this, few therapies beyond intensive supportive care are available to treat patients with encephalitis [1, 2] . anti-viral drugs have been developed for some viruses that can infect the brain, such as herpes simplex virus (hsv)-1 and 2, and human immunodeficiency virus (hiv), but even with these treatments outcomes remain relatively poor [2] [3] [4] [5] . many patients succumb to disease, and survivors often suffer permanent neurological sequelae [6] [7] [8] [9] . while the development and clinical implementation of novel anti-viral drugs may improve patient outcomes, it is becoming increasingly clear that therapies targeting pathogenic elements of the host immune response may be critical for successful intervention during infection [10] [11] [12] [13] [14] . monocyte infiltration is a hallmark of central nervous system (cns) inflammation, including viral infection. these cells migrate into the infected brain, where they differentiate into dendritic cell (dc), macrophage and, arguably, microglial populations. once differentiated, these cells engage in a number of potent effector functions including antigen presentation and t cell stimulation, the production and secretion of numerous pro-inflammatory mediators as well as reactive oxygen species (ros), all of which are focused on viral containment and clearance (table 1) . however, unbalanced and poorly controlled migration and effector functions of these cells may result in immune-mediated pathology, resulting in tissue damage and destruction during some infections (table 1) . therefore, it is of high importance to understand the processes driving monocyte development, recruitment, differentiation and function, to aid in the development of novel therapeutics that inhibit immunopathological responses. monocytes are derived from hematopoietic stem cells (hsc) in the bone marrow (bm) (figure 1 ). the earliest defined precursor is the common myeloid precursor (cmp), distinguished from hsc by the expression of cd34 but not sca-1 [39] [40] [41] [42] (figure 1 ). these cells give rise to a pool of precursors called granulocyte/macrophage precursors (gmps), which express cd16/32 [39] . included within this subset is the recently defined macrophage/dc precursor (mdp), which specifically expresses high levels of the pu.1-controlled chemokine receptor cd115 (csf-1r/ m-csfr), chemokine receptor cx 3 cr 1 (fractalkine receptor), and flt-3 (cd135/flk2) [43] [44] [45] [46] [47] [48] (figure 1 ). the mdp gives rise to cd11b + , cd115 + , f4/80 + , cd11c -, ly6gmonocytes, that can be isolated from the bm and [29] inhibition of nos2 prolonged survival of wnvinfected animals [30] reviewed in [31] proteases mmp ↑ breakdown of the bbb mmp-9 −/− mice show reduced viral loads and increased survival during wnv encephalitis [32] ↑ neuronal damage/ death ↑ demyelination ↑ pro-inflammatory cytokines reviewed in [33, 34] neurotransmitters glutamate ↑ neuronal misfiring/ seizures competitive and non-competitive glutamate receptor antagonists promote survival during neurovirulent sindbis virus encephalitis [35, 36] and improved outcomes during coronavirus encephalitis [37] ↑ neuronal damage/ death ↑ production of no/ ros reviewed in [38] bbb blood brain barrier; cns central nervous system; hsv herpes simplex virus; mdp macrophage/dendritic cell precursor; mhv murine hepatitis virus; mmp matrix metalloproteinases; no nitric oxide; nos2 nitric oxide synthase-2; ros reactive oxygen species; tmev theiler's murine encephalomyelitis virus; wnv west nile virus. blood [49] [50] [51] [52] (figure 1 ). the spleen has also been identified as an important reservoir of undifferentiated monocytes that are rapidly deployed to sites of inflammation, including the ischemic heart and brain [53] [54] [55] . furthermore, a recent study has shown that cardiac infarction triggers a significant increase in numbers of mdps in the spleen, which supply monocytes throughout the duration of acute inflammation [56] . whether the spleen is a significant source of monocytes during cns infection is yet to be determined, but presents a critical area of future investigation. it is likely that both the bm and spleen are critical for supplying monocytes to the infected cns, particularly in cases of acute and severe infection, in which large numbers of these cells are rapidly deployed and recruited to the brain. monocytes are classified into two phenotypically and functionally distinct subsets the mdps give rise to two phenotypically and functionally distinct subsets of monocytes [50, 57] . ly6c hi monocytes are characterized by high expression of the chemokine receptor ccr2, adhesion molecule cd62l and low expression of the fractalkine receptor cx 3 cr 1 [48, 51, 58] . these cells have been termed 'inflammatory' because they are selectively recruited to sites of inflammation and infection in many models of disease, including atherosclerosis [59] [60] [61] [62] ; rheumatoid arthritis [63] ; experimental colitis [64] ; cardiac infarction [65] ; and cns infections including experimental autoimmune encephalomyelitis (eae) [66, 67] , amyotrophic lateral sclerosis [68] , and stroke [53] . recent studies have shown that these cells are also recruited to the virus-infected brain in animal models of hsv, hiv, murine hepatitis virus (mhv), theiler's murine encephalomyelitis virus (tmev) and a number of flaviviral encephalitides, where they give rise to macrophage, dc and, arguably, to microglial populations [11, 13, 14, 69] . conversely, ly6c lo/monocytes are smaller in size than their ly6c hi counterparts and express low levels of ccr2 and cd62l and high levels of cx 3 cr 1 [48, 51, 58] (figure 1 ). development of monocytes in the bone marrow and recruitment to the virus-infected brain. monocytes are generated from hematopoietic precursors in the bone marrow (bm). sca-1 + lin -hsc (a) give rise to cd34 + , sca-1 -cmp (b). these cells in turn give rise to a pool of precursors known as granulocyte/macrophage precursors (gmps), which express cd34 and cd16/32 (c). a fraction of these progenitors also express cd115 and cx 3 cr1 and are known as macrophage/dendritic cell precursor (mdp) (d). mdps are the direct precursors of ly6c hi inflammatory monocytes (e). mdps also give rise to circulating ly6c lo/monocytes directly, or via a ly6c hi monocyte intermediate (f). during viral encephalitis, large quantities of the chemokine ccl2 is produced by infected astrocytes, macrophages/microglia and/or neurons (g). ccl2 binds the chemokine receptor ccr2, expressed at high levels by ly6c hi inflammatory monocytes, which promotes the egress of these cells from the bm (h) into the blood, and thus recruitment from the blood into the infected central nervous system (cns) (i). here, these cells can give rise to cd45 hi ly6c hi macrophages (j) and/or cd45 int ly6c int immigrant microglia (k), although it is unclear whether ly6c int immigrant microglia are derived from a ly6c hi macrophage intermediate or directly differentiate from ly6c hi monocytes. furthermore, it is unclear whether recruited macrophages and immigrant microglia give rise to cd45 lo ly6c lo/resident microglia (l) if/when virus is cleared from the cns. in some models of viral encephalitis, ly6c hi inflammatory monocytes can also give rise to ly6c hi /cd11c + dc in the brain (m). several studies have shown that ly6c hi monocytes can give rise to circulating ly6c lo/monocytes [58, [70] [71] [72] . interest in this subset has increased substantially in the past few years [72, 73] . recent studies have described the patrolling behavior of these cells in the vasculature [73] , and have shown that in some models of disease they rapidly enter inflamed tissue and can contribute to early inflammatory responses before domination by ly6c hi monocytes [73] . in the resolution phase of some diseases, ly6c lo/monocytes are critical for wound healing and angiogenesis [50] . while apparently important in the periphery, the role of ly6c lo/monocytes during cns infection remains poorly defined, with little evidence supporting their migration into the brain during inflammation [74] . the importance of monocyte-derived cells in the pathogenesis of brain infection highlights the importance of understanding the pathway(s) through which monocytes migrate from the periphery into the brain. it is apparent that this process is regulated by cytokine/chemokine and integrin/cellular adhesion molecule interactions that facilitate emigration from the bm into the blood and entry into the cns. for example, the chemokine receptor cxcr4 and one of its ligands cxcl12 (sdf-1) directly enhance vla-4-dependent adhesion and thereby aid in retaining immature cells in the bm. deficiency in either molecule results in impaired myelopoiesis [75] [76] [77] [78] [79] [80] . in addition to cxcr4, ccr2 and its ligands, ccl2 and ccl7 (mcp-3), are a critical requirement for ly6c hi monocyte egress from the bm into the blood. ccl2/ ccr2 deficiency or blockade with antibody results in monocyte accumulation in the bm in multiple disease models, including eae, wnv and hsv encephalitides [11, 61, 67, [81] [82] [83] [84] [85] [86] [87] . monocyte recruitment into the infected brain is dependent on chemokine/chemokine receptor interactions a number of chemokines and their receptors have been implicated in the recruitment of ly6c hi monocytes from the blood and into the brain. ccr5 is expressed by ly6c hi monocytes and is important for trafficking to sites of inflammation in some models of disease. in the brain, its ligand ccl5 (rantes) expression is highly upregulated during infection/inflammation, including wnv, mhv, hsv and tick-borne encephalitis virus encephalitides [88] [89] [90] [91] [92] . another chemokine of interest that controls the trafficking of monocytes into the brain parenchyma is sdf-1/cxcl12, in conjunction with its receptor cxcr4, expressed by monocytes [93] . in animal models of cns inflammation including eae [94] , hiv [95] and wnv [96] , there is significant upregulation of cxcl12. in eae and wnv, cxcl12 has been shown to play an important role in retaining leukocytes in the perivascular space, thereby inhibiting infiltration into the parenchyma. loss of this interaction resulted in the loss of perivascular cuffs and uncontrolled infiltration of cxcr4 + leukocytes, including monocytes, into the parenchyma. [94, 96] . while it is clear that there are a multitude of soluble mediators that represent potential targets for future therapies aimed at blocking monocyte migration, the ccr2/ ccl2 axis remains the most potent pathway based on the available literature. ly6c hi /ccr2 hi monocyte recruitment into the cns in models of stroke [53] , peripheral inflammation [97] , alzheimer's disease (ad) [98, 99] and eae [67, 74, 100, 101] are all dependent on ccr2/ccl2 signaling ( figure 1 ). in the context of viral encephalitis, the ccl2/ccr2 axis is also very important. the major producers of ccl2 appear to be different depending on the infectious agent, with microglia serving as important sources during hsv infection [16, 102] , neurons in the case of wnv infection [11] and astrocytes in hiv encephalitis [103] . no matter the source of ccl2, the inhibition of ccl2 can significantly reduce the infiltration of inflammatory monocyte-derived macrophages and microglia into the infected brain [11] [12] [13] 69, 88, 102, [104] [105] [106] [107] [108] . the focus in the last decade has been heavily on the chemokines involved in monocyte trafficking, however, cellular adhesion molecules and their integrin ligands are obviously also important. in most models of viral infection, very late antigen-4 (vla-4) and leukocyte function-associated antigen-1 (lfa-1) are expressed by ly6c hi monocytes. in addition, their respective binding partner's vascular cell adhesion molecule-1 (vcam-1) and inter-cellular adhesion molecule-1 (icam-1) are usually upregulated on endothelium and other cell types in the inflamed brain [109] [110] [111] [112] [113] [114] [115] . the importance of vla-4 and vcam-1 and lfa-1 and icam-1 in the recruitment of ly6c hi monocytes to sites of inflammation is evident in experiments using gene knockout animals or specific blockade of these molecules. vla-4 and vcam-1 interactions are critical for monocyte migration to the heart in models of atherosclerosis and arterial injury [116] [117] [118] and the inflamed peritoneum [119] . vla-4 is also critical for ly6c hi monocyte infiltration of the cns in several models of inflammation, including eae and spinal cord injury [97, 109, 120] . during viral infection of the brain, we have found that recruitment of monocytes to the cns is also vla-4-dependent. vla-4 antibody neutralization significantly impairs the recruitment of ly6c hi monocytes to the infected brain, in both wnv and jev infection ( [30] , cvv et al., unpublished observations). lfa-1 and icam-1 interactions are also important for monocyte recruitment to atherosclerotic plaques [121, 122] and to the cns during eae [110] . we have shown that lfa-1 is also important for recruitment of monocytes to the wnv-infected brain, however blockade resulted in a smaller reduction in monocytes infiltration compared to vla-4 neutralization, which suggests the differential use of adhesion molecules by ly6c hi monocyte subsets which enter the wnv-infected brain [30] . in models of cns diseases, such as eae and stroke, ly6c hi monocytes have been shown to primarily differentiate into macrophage and dc populations exhibiting a m1 pro-inflammatory phenotype, which in-vitro effectively stimulates th1 and th17 responses in t cells [53, 66, 67, 74] . similarly, in models of viral encephalitis, ly6c hi monocytes have been shown to give rise to m1 pro-inflammatory cd45 hi macrophages and cd11c + dc populations, which express high levels of nitric oxide (no) and tnf during hsv, wnv, mhv, tmev and jev ( [11] [12] [13] [14] 30, 69] , cvv et al., unpublished observations). we have shown that these cd45 hi macrophages are highly effective at processing and presenting antigen and effectively stimulate t cell proliferation [30] . microglia are the resident macrophage population of the brain. similar to other tissue resident cells such as kupffer cells of the liver and langerhans cells of the epidermis, microglia originate from the yolk sac during embryogenesis, from a myeloid lineage that is independent of bm hsc and therefore distinct from that of bm-derived monocytes [123] [124] [125] . microglia can be distinguished from infiltrating monocyte-derived macrophages and dc by their low to intermediate expression of cd45 and lack of ly6c expression [11, 126] . in most infections, resident microglia play functionally distinct roles from that of monocyte-derived cells. for example, during acute wnv encephalitis, resident microglia express lower levels of proinflammatory mediators such as no, express lower levels of mhc-ii, and show a significantly reduced capacity to process antigen and stimulate t cell proliferation compared to the highly activated infiltrating macrophages [30] . in comparison, in acute tmev infection, resident microglia and infiltrating macrophages express similar levels of proinflammatory cytokines and show similar antigen processing and presentation capacity; however, in chronic stages of disease, macrophages are more efficient at stimulating t cell responses [127] . there is evidence to suggest that infiltrating monocytes have the capacity to give rise to microglial cells in some models of cns inflammation, including ad, parkinson's disease, eae, as well as in infectious models such as scrapie and bacterial meningitis [128] [129] [130] [131] [132] [133] [134] . these immigrant microglial cells appear to play distinct functional roles compared to their resident counterparts during disease. for example, immigrant microglia are more efficient at clearing amyloid plaques than resident microglia during ad [128, 135] . however, a caveat of these studies has been in the use of irradiation to generate bm chimeras to distinguish resident microglial from bmderived cells. there are currently no immunophenotypic markers that can definitively separate these two populations. as a result, the generation of chimeras can be used distinguish tissue resident and bm-derived populations. however, irradiation can disrupt the blood-brain barrier (bbb) and promote ccl2 production, resulting in the recruitment of monocytes to the cns [136] . therefore, it is difficult to conclude whether monocyte engraftment is a normal feature of disease in unperturbed animals or whether it is primarily the result of brain preconditioning by irradiation. a recent study using the parabiosis model in place of irradiated bm chimeras has shown that engraftment of monocyte-derived microglia during eae is only a transient response [137] . the parabiosis models have also been employed to show that there is no significant engraftment of monocytederived microglia in facial nerve axonomy or amyotrophic lateral sclerosis [138] . also, another recent study has compared the recruitment of monocyte-derived microglia into brain during ad, using chimeric mice generated with or without head protection during irradiation. they found that these cells do not engraft the brain of protected animals [99] . however, one major caveat of the head-protection model is the existence of bm in the skull that may be capable of reconstituting the animal. further studies are required to definitively determine whether monocyte-derived cells can give rise to microglia and if these cells truly engraft the parenchyma and remain there if/when disease is resolved. there are few studies that examine the recruitment of monocyte-derived microglia during viral infection of the cns. we have shown that in wnv encephalitis, inflammatory monocytes not only give rise to cd45 hi macrophages in the brain, but also to a cd45 int subset, which is phenotypically analogous to activated resident microglia, apart from the expression of ly6c [11, 30] . although chimeras were initially utilized to investigate this phenomenon, we further confirmed that the recruitment of these monocyte-derived cells was not the result of bbb breakdown, using methods that do not use any irradiation including bone marrow adoptive transfer studies and microparticle-based systems which track these cells with minimal perturbation of the disease system [11] . furthermore, these cells were found to contribute to the immunopathogenesis of wnv encephalitis, as ccl2 blockade significantly reduced recruitment into the cns and prolonged survival of lethally-infected animals [11] . current studies in our laboratory aim to determine whether monocyte-derived microglia truly engraft the brain parenchyma during wnv encephalitis, the functional role of these cells throughout infection, and whether these cells remain in the cns after disease is resolved. ly6c hi monocytes appear to play a paradoxical role in many disease models. for example, higher mortality rates and increased pathogen loads are seen in toxoplasma [139, 140] , listeria [83, 141] , cryptococcus [142, 143] , yersinia infections [144] , hsv-2, [145] and coronavirus [146] , as well as mhv [88] when these cells are depleted. on the other hand, ly6c hi monocytes are direct targets for pathogens such as hiv, tmev, listeria and toxoplasma [12, 69, [147] [148] [149] [150] [151] [152] . infected monocytes can be directly responsible for the dissemination of infection in a "trojan horse" fashion into the cns thereby potentiating disease and increasing potential mortality [153] [154] [155] [156] . monocytes significantly contribute to immunopathology during brain infection an arguable role of monocytes during brain infection is their potential contribution to immune-mediated pathology. in several models of cns disease, ly6c hi inflammatory monocytes cause significant damage and destruction in the brain, directly contributing to morbidity and mortality. ly6c hi monocytes contribute significantly to the pathogenesis of disease during stroke [53] . mice with ccl2 −/− and ccr2 −/− deficiency show smaller infarcts and enhanced functional outcomes relative to wild-type controls following transient cerebral ischemia [157, 158] . similarly, in models of traumatic brain injury, ccl2 −/− mice showed reductions in macrophage infiltration and lesion volume compared to wild-type mice, corresponding with improved functional recovery after injury [159] . in addition, ccr2 −/− and ccl2 −/− mice exhibit milder symptoms and, in some models, are completely resistant to the development of eae [100, 136, 160, 161] . furthermore, a recent study has shown that ly6c hi monocyte recruitment to the cns is detrimental in amyotrophic lateral sclerosis [68] . in the case of encephalitic disease, studies in our laboratory using wnv as well as others using tmev have shown that ly6c hi monocytes are recruited into the infected brain where they contribute significantly to the immunopathogenesis of disease. inhibition of inflammatory monocyte migration into the wnv or tmevinfected brain can significantly reduce morbidity and mortality [11, 12, 69, 108] . furthermore, abrogation of monocyte migration into the cns during mhv encephalitis results in the delayed onset of demyelinating disease [105] . the precise pathways through which inflammatory monovcytes contribute to pathology are still under intense investigation. however, it is clear that differentiation into effector cells such as macrophages and dc plays a substantial role. once differentiated, these cells are significant producers of no, matrix metalloproteinases (mmp) and other factors known to culminate in tissue destruction, breakdown of the bbb, as well as neuronal damage (table 1) . while in many organs such toxicity is not a major concern due to regenerative capabilities, the brain is largely comprised of many irreplaceable cellular subsets. as such not only is mortality a concern, in patients that survive serious cns inflammatory insults will often suffer long-term sequelae and neurological imbalance [6] [7] [8] [9] . although ly6c hi monocyte infiltration is a hallmark of viral encephalitis, the role of these cells in viral clearance and immunopathology is not well defined. while it is clear that these cells are critical for the control and clearance of some viruses, they are directly responsible for recruiting others into cns, or cause significant immunopathology. future studies which target monocyte development and migration to the cns in a therapeutic manner will not only provide significant insight into pathways by which monocytes are recruited to the cns, but will identify new targets for intervention during viral encephalitis. the authors declare that they have no competing interests. authors' contributions rlt drafted the manuscript. drg, cd, cvv, ilc and njck contributed to the interpretation and critical evaluation of content and revision of the manuscript. all authors read and approved the final manuscript. diagnosis and treatment of viral encephalitis viral encephalitis: familiar infections and emerging pathogens outcome of and prognostic factors for herpes simplex encephalitis in adult patients: results of a multicenter study hiv-1 and the brain: connections between 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immunodeficiency virus type 1 infection increases the in vivo capacity of peripheral monocytes to cross the blood-brain barrier into the brain and the in vivo sensitivity of the blood-brain barrier to disruption by lipopolysaccharide the ly-6chigh monocyte subpopulation transports listeria monocytogenes into the brain during systemic infection of mice ifn-gamma triggers ccr2-independent monocyte entry into the brain during systemic infection by virulent listeria monocytogenes evidence of a role for monocytes in dissemination and brain invasion by cryptococcus neoformans cd11c-and cd11b-expressing mouse leukocytes transport single toxoplasma gondii tachyzoites to the brain the role of cc chemokine receptor 2 on microglia activation and blood-borne cell recruitment after transient focal cerebral ischemia in mice effects of monocyte chemoattractant protein 1 on blood-borne cell recruitment after transient focal cerebral ischemia in mice role of ccl2 (mcp-1) in traumatic brain injury (tbi): evidence from severe tbi patients and ccl2−/− mice cc chemokine receptor 2 is critical for induction of experimental autoimmune encephalomyelitis acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the cc chemokines macrophage inflammatory protein-1alpha and monocyte chemotactic inflammatory monocytes and the pathogenesis of viral encephalitis the authors cited in this review were supported by the national health and medical research council grants 512413 and 1007757 to njck and ilc. rlt key: cord-252569-9rv1p3qh authors: zanella, m.-c.; lenggenhager, l.; schrenzel, j.; cordey, s.; kaiser, l. title: high-throughput sequencing for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis. a narrative review and clinical appraisal date: 2019-01-11 journal: clin microbiol infect doi: 10.1016/j.cmi.2018.12.022 sha: doc_id: 252569 cord_uid: 9rv1p3qh background: viral aetiologies are the most common cause of central nervous system (cns) infections. approximately one-half of cns infections remain of undetermined origin. high-throughput sequencing (hts) brought new perspectives to cns infection investigations, allowing investigation of viral aetiologies with an unbiased approach. hts use is still limited to specific clinical situations. objectives: the aim of this review was to evaluate the contribution and pitfalls of hts for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis in cns patient samples. sources: pubmed was searched from 1 january 2008 to 2 august 2018 to retrieve available studies on the topic. additional publications were included from a review of full-text sources. content: among 366 studies retrieved, 29 used hts as a diagnostic technique. hts was performed in cerebrospinal fluid and brain biopsy samples of 307 patients, including immunocompromised, immunocompetent paediatric, and adult cases. hts was performed retrospectively in 18 studies and prospectively in 11. hts led to the identification of a potential causal virus in 41 patients, with 11 viruses known and ten not expected to cause cns infections. various hts protocols were used. implications: the additional value of hts is difficult to quantify because of various biases. nevertheless, hts led to the identification of a viral cause in 13% of encephalitis, meningoencephalitis, and meningitis cases in which various assays failed to identify the cause. hts should be considered early in clinical management as a complement to routine assays. standardized strategies and systematic studies are needed for the integration of hts in clinical management. meningitis, encephalitis, and meningoencephalitis are caused by various pathogens, but viral aetiologies are the most common cause [1e4] . among these, enterovirus (ev), herpes simplex type 1 and 2 (hsv-1 and hsv-2), and varicella zoster virus (vzv) are the most frequent viruses associated with encephalitis, meningoencephalitis, and meningitis in paediatric and adult populations [1,2,4e7] . the prevalence of other viruses varies according to the geographical location and immune status of the patient. during the last two decades, the implementation of molecular assays as a complement to serological assays, immunohistochemistry, and culture have improved the diagnosis of viral central nervous system (cns) infections. nevertheless, these assays have limitations because of their targeted approach. apart from technical limitations, the diagnosis of viral cns infections is subject to several issues, such as the type of sample (cerebrospinal fluid (csf) or brain biopsy), the timeline of sample collection, and the different pathogenic mechanisms of viruses. despite technical progress, approximately one-half of encephalitis, meningoencephalitis, and meningitis cases remain of unknown origin [1, 2, 5, 7] . high-throughput sequencing (hts) has brought new perspectives to cns infection investigations. although hts has been recently integrated in encephalitis management guidelines [8] , its use is still limited to specific clinical situations or research. the contribution of hts warrants a better appraisal for further implementation in cns infection management. this narrative review aims to evaluate the contribution and pitfalls of hts for the aetiologic identification of viral encephalitis, meningoencephalitis, and encephalitis in paediatric and adult patients. a comprehensive pubmed search was conducted from 1 january 2008 to 2 august 2018 to identify human studies using the following mesh and keywords research algorithm: '((central nervous system infection or cerebrospinal fluid or central nervous system) and sequencing) and virus'. additional publications were identified from a review of full-text sources. the title and abstract of each citation were screened by two reviewers and assessed for eligibility by detailed analysis. inclusion criteria were studies including patients with encephalitis, meningoencephalitis, or meningitis of unknown origin and reporting the use of hts for the aetiologic identification of a viral origin in cns samples. exclusion criteria were reviews, animal studies, other cns diseases, and studies addressing only technical aspects. a total of 366 references were retrieved (fig. 1 twenty-nine studies were selected for qualitative analysis (19 case reports, ten case series) ( table 1) . fourteen and 13 studies concerned paediatric and adult cases, respectively, and two studies concerned both populations (table 1) . hts was performed in 307 cases (52 adults; 123 paediatric (<18 years); 132 cases with no information on age). twenty-five studies reported patient age (median age, 14 years; range, 3 months to 68 years). diagnostic criteria for encephalitis, meningoencephalitis, and meningitis were inconsistently reported. immune status was reported for 60 patients and comprised 20 immunocompromised (nine paediatric, 11 adults) and 40 immunocompetent patients (22 paediatric, eight adults). studies came from a wide range of regions: europe (ten), north america (ten), asia (six), and oceania (three) ( table 1) . when brain specimens were available, pathological examination provided proof of diagnosis of encephalitis. csf analysis results were reported in 25 patients of 20 studies, with the white blood cell count ranging from 1 to 494 cell/mm 3 in encephalitis and meningoencephalitis cases and 915 cell/mm 3 in the only meningitis case [22] ; three publications reported normal csf analysis without any description [23e25]. hts was performed on individual csf and brain specimens in 129 and 21 patients, respectively. csf samples of 162 patients were pooled for hts analysis [26e28]. hts was performed on csf and brain specimens in five patients [26,29e32] . positive results on both samples were obtained in a cache valley virus chronic meningoencephalitis case [30] and positive results on brain biopsies only were reported in two human astrovirus (hastv)-va1 and tick-borne encephalitis (tbev) cases [29, 33] . in one patient, hts analysis did not identify a viral cause, but balamuthia mandrillaris was identified in both csf and brain biopsy [32] . despite limitations because of publication bias, the overall diagnostic yield for a viral aetiology according to sample type was estimated to be higher for brain specimens (16/21 (76.2%) positive samples) than for csf samples (26/291 (8.9%) positive samples). detailed microbiological investigations performed prior to hts varied according to local practice and were not reported in seven studies [24,34e39] . in most studies, viruses identified with hts were not part of the microbiological work-up, except in three cases where hts identified a virus for which diagnostic assays were negative during routine investigation. these included a west nile virus (wnv) identified in the csf sample of a renal transplant recipient with meningoencephalitis and a negative serological assay [40] . hsv-1 was identified in an encephalitis case [35] . a vaccine strain of mumps virus was identified in a brain specimen of an immunosuppressed child with chronic encephalitis in whom (rt)-pcr for mumps on a csf sample was negative as the assay did not target vaccine strains [41] . hts was performed retrospectively in 18 studies and prospectively as part of the initial work-up in 11 case reports with an impact on the clinical management of three immunocompromised patients: a child with encephalitis associated with hastv-va1 [23] ; an adult with encephalitis associated with hastv-va1 [33] ; and an adult with chronic meningoencephalitis associated with cache valley virus [30] . turnaround times were reported in ten studies [23,31,33,36,39,40,42e45 ]. among publications in which hts was used prospectively, turnaround times ranged from 48 hours to 7 days [23, 33, 39, 40, 42, 44, 45] . hts performed on pooled or individual samples and/or subsequent confirmatory assays allowed the identification of a potential causal virus in 41 of 307 patients (13.4%), comprising 15 paediatric cases (eight immunocompromised cases), 17 adult cases (nine immunocompromised cases) and nine cases for whom age was not specified; median age was 21 years (range 3 months to 68 years). fig. 2 shows the distribution of viruses identified according to patient immune status and clinical manifestations. hts allowed the identification of viruses previously unknown or unexpected as a cause of cns infection (n ¼ 10) and thus not screened during diagnostic investigations (table 1) . these included parvovirus 4 (two) [46] , human coronavirus oc43 (one) [25] , and novel hastv-mlb2 (one) [22] identified in immunocompetent patients. a mumps virus vaccine strain (one) [41] , hastv (undetermined specie; one) [47] , hastv-va1 (four) [23, 31, 33, 43] , and hastv-mlb1 (one) [24] were identified in immunocompromised patients. a gemycircularvirus was also identified, but its causal role in encephalitis is under debate [27] . three novel viral species or strains were identified in csf samples of patients with encephalitis (table 1) : human csf-associated densovirus 1 (hucsfdv1) [37] ; cyclovirus viet-nam (cycv-vn) [28] ; and lymphocytic choriomeningitis virus (lcmv)-related arenavirus [26] . hts analysis also identified viruses known to be responsible for cns infections (n ¼ 11) and not screened or detected by routine assays (hsv-1, hsv-2, vzv, epsteinebarr virus (ebv), tbev, wnv, cache valley, saint louis encephalitis, toscana, mumps, measles, and coxsackie a9 virus) [29, 30, 34, 35, 39, 40, 42, 48, 49] (table 1) . most studies performed nucleic acid extraction protocols dedicated to rna, or rna and dna. thirteen rna and seven dna viral species were identified (table 1) . one study reported the identification of hsv-1 in a csf sample after rna extraction protocol [35] . six studies where hts analysis was not restricted to the detection of viruses resulted in the identification of bacterial (brucella melitensis and leptospira santarosai) [44, 50] , mycobacterial (mycobacterium tuberculosis) [39] , and parasitic (balamuthia mandrillaris) [32, 45] or fungal (candida tropicalis and fusarium solani and oxysporum) [38] pathogens. the use of controls was not systematically reported. nine studies reported various negative control samples, such as brain specimens without encephalitis [34] , csf samples from patients without infection [30, 32, 35, 38, 40, 45] , serum samples, and water or elution buffer [30, 44, 45, 50] . viral sequences of negative controls were not consistently described. positive controls, such as csf or serum samples positive for dna or rna viruses, were rarely reported or used [33, 35, 38, 48, 50] . to address the specificity of hts results, other techniques were performed to confirm hts results in all studies, except one [49] . (rt)-pcr assays were performed in samples from 37 patients and were positive in at least one sample in 36 patients. hts results were also confirmed with serological assays [26, 40, 41, 46] , immunohistochemistry, and in situ hybridization [25, 30, 31, 33, 39, 41, 47] . viral culture confirmed the presence of a replicative saint louis encephalitis virus in a csf sample [42] , but was unsuccessful concerning a cache valley virus [30] . hts offers the possibility of investigating viral aetiologies of cns infections by an unbiased approach when work-up according to guidelines fails to identify a causal pathogen. based on the studies retrieved, its diagnostic yield for a viral aetiology is difficult to estimate, particularly because of publication bias (high number of case reports), methodological heterogeneity, and a lack of systematic prospective studies. when focusing specifically on case series, the diagnostic yield for the identification of a viral cause was approximately 10%, but this result should be interpreted with caution in the light of the evolution of the technique from 2008 to 2017. among the studies reviewed, the hts contribution is evident not only for the identification of a potential causal virus in cns infections of unknown origin, but also in the detection of novel or divergent viruses [26, 28, 37] . similar to other techniques, the type of sample used for analysis is of particular importance. despite diverse hts protocols and publication bias, hts seemed to have a higher diagnostic yield in brain specimens than in csf samples. the diagnostic yield was particularly low in two studies where hts was performed on csf supernatant [27, 28] . hts also shows its clinical value in situations where the viral pathogenic mechanisms and specific clinical situations impairs the results of conventional assays [40] . among immunocompetent patients, hts led to the identification of viruses not previously associated with cns infections (parvovirus 4, cycv-vn, gemycircularvirus, and novel hastv-mlb2) [22, 27, 28, 46] . hts clinical impact was mainly demonstrated among immunocompromised patients, with most studies dedicated to this population. it was performed prospectively in 11 cases and led to a change in clinical management in three [23, 30, 33] . the rapid decision to perform hts, short hts turnaround times, and the efficient interpretation of results were determinant for the management of these latter patients. among immunocompromised patients, hts contributed to the detection of viruses for which no assay was performed during conventional work-up: viruses known to cause cns infections (tbev, wnv, cache valley virus, saint-louis encephalitis virus, ebv) [29, 30, 39, 40, 42] , viruses not known to be responsible for cns infections (novel hastv-va1, hastv-mlb1, human coronavirus oc43, mumps virus vaccine strain) [23e25, 31, 33, 41, 43, 47] , and novel viruses (lcmv-related arenavirus) [26] . focusing on novel hastv, hts brought new insights in our understanding of their association with cns infections [22e24, 31, 33, 43] . furthermore, all (rt)-pcr assays performed retrospectively confirmed hts results, thus highlighting the specificity of hts. in the absence of standardization, the methodological heterogeneity of studies is striking, not only concerning pre-analytic steps, but also hts per se, with the use of diverse hts platforms, single or paired protocols, as well as diverse bioinformatic pipelines and databases. the use of positive controls as quality controls was only reported in seven studies [31,33e35,38,48,50] . addressing the issue of contamination, only a few studies reported the use of negative controls [30, 32, 34, 35, 38, 40, 44, 45, 50] . viral sequences assigned to viruses not considered as the cause of cns infection were not consistently performed: 12 studies provided a description for one cns sample or more [25,27,29,30,32e35,38,40,44,50] . for most of these viral sequences, no interpretation of results was explicitly provided. among reads of viruses known to cause infections in humans, anelloviridae [51] and herpesviridae were the most described in four and seven samples, respectively; human pegivirus reads were identified in one sample. the genome of the torque teno virus, a member of the anelloviridae family, and human pegivirus have been identified in cns samples, but without any association with a cns disease so far [52e57] . other viral sequences were mostly assigned to viruses infecting plants or non-vertebrates and were considered to be reagent contaminants. the minimal description of these hts 'background' results impairs the comprehension of the composition of the cns virome. furthermore, the integration of hts results in the clinical context is of particular importance and the absence of standardization of any reporting methods precludes an objective interpretation of these results. finally, hts-negative results could be interpreted in the context of several clinical and technical aspects that could impact on the sensitivity of the method. first, from a clinical point of view, differences in diagnostic yield from a biopsy compared with csf samples could be explained by several factors: patient selection (cases of encephalitis); the type of sample (e.g. multiple pooled post-mortem brain samples); and the timeline of sampling in the context of encephalitis (biopsy positivity could possibly be less affected by time than csf). from a technical point of view, it should be considered that this narrative review includes studies from 2008 to 2018 and thus takes into account the tremendous evolution of the hts technique over this last decade. several technical issues need to be considered for the interpretation of negative results: pre-analytic steps (e.g. the use of fresh, frozen, or paraffinembedded samples for analysis, extraction protocols, fragmentation methods, library preparation, paired-end versus single-end protocols); sequencing depth; sequencing platforms (table 1) ; and the analysis of hts raw data (e.g. mapping software, viral databases, and pipeline precision). this review highlights that the use of hts in investigations concerning a viral cause of encephalitis, meningoencephalitis, and meningitis could extend not only to immunocompromised, but also to immunocompetent patients. considering the selection and publication bias of the literature reviewed here, the negative predictive value for the aetiologic identification of viral encephalitis, meningoencephalitis, and meningitis is difficult to quantify and further studies are needed. hts needs to be integrated in clinical management as a second-line technique or in parallel to first-line investigations when a standard work-up according to guidelines [8, 58] and additional investigations considering local epidemiology and specific clinical situations fail to identify a causal agent. brain biopsy should also be considered. furthermore, hts is of particular interest for the screening of a large panel of viruses, particularly to avoid a restricted screening of low-volume clinical samples, such as in paediatric patients. hts brought new perspectives to the investigations of infectious diseases. notably, its unbiased approach is of particular interest in samples that would not usually be tested in specific syndromes. its use may not only be restricted to cns samples, but also extended to other clinical samples. this is illustrated by the positive results of (rt)-pcr assays performed on blood or plasma samples collected at the time of neurological manifestations, which allowed the identification of the same virus detected by hts in cns samples (parvovirus 4, lcmv-related arenavirus, novel hastv-va1, novel hastv-mlb2) [22, 26, 31, 46] . this could be of particular interest when a cerebral biopsy cannot be performed and a disseminated infection occurs or is suspected, particularly in immunocompromised patients. an early decision to perform hts, short hts turnaround times, and an efficient interpretation of results are major issues for allowing hts to contribute to clinical management. for prospective hts use in clinical routine, this timeframe should be as short as possible for clinical decision-making. among publications in which hts was used prospectively, reported turnaround times ranged from 48 hours to 7 days [23, 33, 39, 40, 42, 44, 45] . hts use is still restricted to a limited number of diagnostic laboratories considering the cost of analysis and informatics infrastructures needed (e.g. costs of sequencing platforms, computing resources, data storage). despite the expanding use of hts in clinical microbiology, the surprisingly low number of studies retrieved for this review might be explained for several reasons, including financial limitations when considering the costs of the analysis, the need for shorter turnaround times, and the limitations cited above. addressing the question of the proof of causality, particularly in the context of pathogen discovery, lipkin proposed several criteria for pathogen causality with grading certainty according to confirmation with serological assays or culture for instance [59] . most studies confirmed hts results with (rt)-pcr assays and a few with cell culture, serological assays, and immunohistochemistry. thus, hts should be implemented in clinical routine in association with other diagnostic tests. in most studies, the approach to establish causality was not explicitly described, but was reported as the temporal association of clinical manifestations and the identification of viral sequences of a specific virus using hts on cns samples at the time of manifestations. this process was only described in few studies. similar to other molecular tests such as (rt)-pcr, the detection of viral sequences or genome in a clinical sample should be interpreted with caution in the clinical context. in a near future, the process for the establishment of causality in hts analysis should be more transparent and should comprise multidisciplinary sessions involving infectious disease specialists and bioinformatics experts, not only for results concerning viruses unexpected to cause cns infections. finally, for hts implementation in clinical routine, the question of standardization has to be addressed concerning hts protocols, data analysis algorithms, reference databases and quality controls, and further prospective studies are needed [60] . this review shows that hts contributed to the identification of potential viral aetiologies of encephalitis, meningoencephalitis, and meningitis of unknown origin in approximately 13% of cases and is of particular interest in immunocompromised patients. this unbiased or semi-unbiased approach led to the identification of novel viruses, viruses known or not expected to cause cns infections. standardized strategies are needed for the further implementation of hts in clinical management. in centres where available, the decision to perform hts should be considered early in the management of encephalitis, meningoencephalitis, and meningitis in as a second-line technique or in parallel to recommended investigations. the authors have no conflicts of interest to declare. no funding was received for this study. beyond viruses: clinical profiles and etiologies associated with encephalitis infectious encephalitis in france in 2007: a national prospective study causes of encephalitis and differences in their clinical presentations in england: a multicentre, population-based prospective study acute bacterial and viral meningitis viral infections of the central nervous system in spain: a prospective study viral meningitis etiology of aseptic meningitis and encephalitis in an adult population guidelines on the management of infectious encephalitis in adults genetic characterization of human herpesvirus type 1: full-length genome sequence of strain obtained from an encephalitis case from india analysis of an echovirus 18 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management of encephalitis: clinical practice guidelines by the infectious diseases society of america the changing face of pathogen discovery and surveillance validation of metagenomic next-generation sequencing tests for universal pathogen detection the authors would like to acknowledge rosemary sudan (geneva university hospitals, switzerland) for editorial assistance. key: cord-348746-yaf61cmx authors: foley, janet e.; leutenegger, christian title: a review of coronavirus infection in the central nervous system of cats and mice date: 2008-06-28 journal: j vet intern med doi: 10.1111/j.1939-1676.2001.tb01572.x sha: doc_id: 348746 cord_uid: yaf61cmx feline infectious peritonitis (fip) is a common cause of death in cats. management of this disease has been hampered by difficulties identifying the infection and determining the immunological status of affected cats and by high variability in the clinical, pathological, and immunological characteristics of affected cats. neurological fip, which is much more homogeneous than systemic effusive or noneffusive fip, appears to be a good model for establishing the basic features of fip immunopathogenesis. very little information is available about the immunopathogenesis of neurologic fip, and it is reasonable to use research from the well‐characterized mouse hepatitis virus (mhv) immune‐mediated encephalitis system, as a template for fip investigation, and to contrast findings from the mhv model with those of fip. it is expected that the immunopathogenic mechanisms will have important similarities. such comparative research may lead to better understanding of fip immunopathogenesis and rational prospects for management of this frustrating disease. f eline infectious peritonitis (fip) is a fatal, immune-mediated disease produced as a result of infection of macrophages by mutant feline coronavirus strains (fipvs). the severity of fip is determined by virus strain and by host-specific, partially heritable immune responses. 1 the causative agent, fip virus (fipv), is a macrophage-tropic mutant of the ubiquitous feline enteric coronavirus (fecv). [2] [3] [4] these feline coronaviruses are closely related to transmissible gastroenteritis virus (tgev) of pigs and more distantly related to mhv (a coronavirus of rodents), human respiratory coronavirus, and others. as with most rna viruses, a high rate of point or other small-scale mutations and larger scale recombination events occur. type ii fecvs are viruses that arise as a result of recombinations between type i fecv and canine coronavirus (ccv). type ii fecvs acquired a canine s gene and express a canine s gene product. 5 mutations are common in the 7b open reading frame (orf) of both type i and type ii fecvs and may be associated with reduced virulence. 4, 6 the 7b orf arose in the fecv/ccv lineage and encodes a nonstructural secretory glycoprotein of undetermined function, which is not necessary for viral replication. 7 deletions, point mutations, and frame-shift mutations leading to early truncation of the 3c orf also are common. 4, 6 because fip-defining mutations may occur in the s, 3c, and 7b genes, polymerase chain reaction (pcr) with these genes as a target cannot discriminate between benign fecv and fatal fipv. [2] [3] [4] murine hepatitis virus shares most genes with the fecvs, including m (membrane glycoprotein), e (small membrane), n (nucleocapsid), and s (spike glycoprotein), which is post-translationally modified to s1 and s2. 8 the mhv genome, however, also codes for an he protein and does not contain a 7b orf. mutations in the e and s proteins lead to attenuation of mhv. 9 a third coronavirus, hcv-229e, has been implicated in neurological disease in people. 10 fip occurs most frequently in cats younger than 3 years of age from multiple-cat homes (shelters and breeding catteries). 11, 12 one-quarter to one-third of cats with noneffusive fip have either primary neurological fip or neurological abnormalities as a part of their overall disease presentation (foley and pedersen, unpublished data). 13 the immunological and pathological characteristics of neurological fip, however, are much more stereotypic than those of systemic fip. thus, neurological fip may be useful for studying basic mechanisms of fip pathogenesis. the extent to which genetic differences among viral strains confer relative neurotropism is unknown, but some cats with severe neurological disease have mild or undetectable systemic disease. both neurological and generalized fip may present first as a nonspecific illness, with clinical signs including weight loss, weakness, fever, and lethargy. abdominal abnormalities are detected commonly on physical examination of cats with neurological fip, including mesenteric lymphadenopathy and irregular splenic and renal surfaces. 14, 15 common historical findings in cats with neurological fip include dementia, pica, seizures, inappropriate elimination, incontinence (fecal and urinary), and compulsive licking. 15, 16 neurological examination may identify ataxia, hyperesthesia, reduced consciousness, hyperreflexia, crossed-extensor reflexes, reduced conscious proprioception, caudal paresis, cerebellar-vestibular signs, or cranial nerve deficits. 14, 15, [17] [18] [19] ophthalmic lesions also are common in neurological fip, including anterior uveitis, keratic precipitates, flocculent debris in the anterior chamber, retinitis, and anisocoria. 15 murine hepatitis virus, like fipv, can produce either systemic infection or disease primarily affecting the liver. well-defined neurotropic genetic variants of mhv also occur, including a well-studied variant designated mhv-jhm, which is responsible for progressive encephalomyelitis and death in infected mice and rats. intranasal or intracranial inoculation of mhv-jhm in balb/c or c57bl/6 mice leads to rapid, fatal encephalitis. 20 the syndrome in survivors (either because the virus is attenuated or the host is resistant, vaccinated, or a pup from a vaccinated dams) consists of chronic demyelination and hindlimb paralysis 21 and has been proposed as a model for multiple sclerosis. 22 determinants of neurovirulence are found in the mhv s gene. 23, 24 a second variant of mhv, mhv-oblv60, leads to neuronal infection specifically in the anterior olfactory bulb after intranasal challenge in mice. 25 neurological disease the definitive lesion of fip is a pyogranuloma that results from immune-mediated phenomena secondary to coronaviral infection of macrophages. the most common sites are serosal, pleural, meningeal, ependymal, or uveal membranes. in the earliest stages of abdominal fip, diffuse alterations with activated mesothelial cells and a few coronavirus-infected macrophages or an exudative precipitate may be detected on serosal surfaces. 26 larger pyogranulomas become grossly visible, ranging from small lesions, often on the renal capsular surface, to severe, generalized miliary granulomatous lesions that distort renal surfaces, disseminate throughout the omentum and gastrointestinal serosa, and invade splenic and hepatic parenchyma. gross lesions of fip in the cns may be subtle, with ependymitis, thickening and opacification of meninges, and ventricular dilatation, usually in the fourth ventricle and least often in the lateral ventricles. 15 lesions occur most commonly on the ventral surface of the brain, often accompanied by secondary obstructive hydrocephalus. histopathologically, lesions in the brains of cats with fip consist of meningitis, ependymitis (ranging from mild ependymal infiltration to complete effacement of the ependymal lining by a heavy infiltrate of histiocytes and lymphocytes), periventriculitis, and choroiditis of varying severity, often superficial and oriented around the ventricles, with dense infiltrates of lymphocytes, plasma cells, neutrophils, and macrophages. 14, 15, [27] [28] [29] [30] [31] meningitis may be more severe on the ventrocaudal surfaces of the brain, especially at the base of the cerebellum and the brainstem, including the medulla oblongata. in the meninges, the inflammatory cells may have a predominantly perivascular distribution, forming cuffs around arteries (periarteritis) and infiltrating the wall of veins and venules (phlebitis), with exudation of a cell-and protein-rich edema fluid, and periventricular reactive astrocytosis. the inflammation may extend into the superficial neuropil, as well as into cranial nerve roots. 15 if lesions are deep, they usually are perivascular with scattered glial nodules. 27 hydrocephalus is seen in association with leptomeningitis, meningeal fibrosis, accumulation of cellular debris, and obstruction of the cerebrospinal fluid flow. pathologic lesions after mhv infection in the brains of rats and mice are variable, depending on viral dose and route of administration and rodent genotype, age, and immunological characteristics. 32 in severe acute jhm encephalitis, necrotizing lesions are found in the gray and white matter, with axonal changes, including disintegration of neurofilaments. 32 within the necrotic areas, perivascular neutrophilic infiltrates occur especially in the subependyma, choroid, and meninges. in acute mhv-a59 infection in cd8ϩ t-cell deficient mice, periventricular encephalitis occurs with lymphocytic infiltration into the choroid plexus, ependyma, and subependymal brain tissue. 33 lesions in mhv-oblv60 consist of local neuronal infection, some mitral cell destruction, and t-cell inflammation with astrocytosis. 25 some evidence suggests a pathological component of vasculitis, with mouse endothelial cell lines susceptible to mhv-jhm exhibiting cytopathology within several days of infection. 34 the subacute to chronic immune-modulated pathological changes that occur with manipulation of the mhv-jhm system are particularly relevant to the murine model of fip. depending on mouse strain and immunological status, mhv-jhm produces meningeal inflammation associated with t-cells and macrophages and demyelination but relatively little disease in axons. demyelination occurs within and adjacent to areas of inflammation and astrocytic proliferation. 32, 35 neutrophils and monocytes have been observed infiltrating through endothelial cell junctions and participating in the phagocytosis of myelin debris. 32 perivascular cuffing by macrophages is observed in chronic encephalitis, as in fip. the route of entry of fipv into the cns is unknown. the fipv virus probably travels hematogenously in macrophages, and 1 study reported a cat with positive immunohistochemical staining for fipv in monocytes in blood vessels of the choroid plexus. 36 once in the cns, there is little evidence that fipv enters any cells other than macrophages. foley et al 15 reported positive immunohistochemical staining (by means of a mouse monoclonal antibody against the fipv n protein) for fipv, primarily in macrophages in fip granulomas but also in some lymphocytes. virus-infected cells were numerous in some areas of intense ependymitis and choroiditis and free within the ventricular lumen, with very few positive cells in the meningeal infiltrates. no staining was observed in vascular basement membranes or in cells of the neuropil. macrophages in necrotic regions and in the center of lesions often are not infected with coronavirus. 37 neurotropic mhv strains have several routes of entry into the cns. some studies show that mhv-jhm travels up the olfactory nerve and enters the cns. 38 this is not surprising, because coronaviruses generally are epitheliotropic, and neurons share embryological origin with epithelial cells. cns infection with jhm also may occur after peripheral infection and viremia. 39, 40 receptors for neurotropic human coronavirus hcv-229e have been detected not only in human lung cells but also in human neuron, astrocyte, and oligodendrocyte cell cultures. 41 hcv-229e also infects macrophages and endothelial cells, however, suggesting hematogenous introduction into the cns, 40, 42 similar to fip. once inside the cns, the major targets for mhv are glial cells and neurons. 35 especially in neuroattenuated strains, mhv-jhm infects primarily oligodendrocytes 43 and has been reported in astrocytes. 44 mhv-4 infects mainly neurons. 20 subsequent events in chronic disease pathogenesis include recruitment of inflammatory cells and the interactions of immune cells with the virus-infected cells. macrophages, activated t-cells, and some b-cells may cross into the intact cns through the blood-brain barrier, with the potential for major cytokine upregulation. 45, 46 the immunopathogenesis of neurologic coronavirus infections after establishment of fipv in the cns, mechanisms of disease are primarily immune-mediated, involving humoral and cell-mediated immunity (cmi). coronavirus-infected macrophages can trigger massive complement activation and deposition of c3 on affected surfaces, disseminated intravascular coagulopathy, vessel necrosis, and effusion. 37, [47] [48] [49] however, immunopathogenic events in neurological fip have not been well described. both in systemic and neurological fip, antibodies (especially to the spike protein) contribute to the opsonization of viral antigen and have the capacity to mediate or enhance disease. [50] [51] [52] [53] [54] antibodies to fecv and fipv are identical. anti-fipv igg and igm-producing b-cells are present in fip lesions, at the interface of healthy tissue and granulomas, and in the serum and csf of cats with neurological fip. 26, 37 apparently, some antibody production occurs locally in the cns, in response to viral antigen in the brain. in one study, serum coronavirus titers of 16 cats with neurological fip were positive, with a median titer of 1 : 400, whereas csf titers were positive in 15 cats, with a median titer of 1 : 100. 15 two cats had high protein concentrations and increased cells, predominantly lymphocytes and neutrophils. the titer in csf was not statistically correlated with serum titer, and the ratio of serum : csf titer was not correlated with the serum : csf protein ratio. if passive leakage of protein from serum into the csf were invoked to explain the presence of antibodies, it would be expected that the total protein : anti-fipv igg ratios would be similar in both serum and csf. in contrast, csf igg titers tended to be proportionally much higher than serum titers. this finding suggests that anti-fipv igg may have been produced in the tissues of the brain in response to a locally replicating virus. although almost nothing is known about the mechanism, cell-mediated immunity has been hypothesized to be protective against fip. [50] [51] [52] [53] [54] cd4ϩ t-cells commonly are observed in fip granulomas. 37 during acute experimental multisystemic fip, apoptosis and t-cell depletion were observed in the spleen and mesenteric lymph nodes. 55 this effect was induced by heat-treated effusion fluid (presumably containing some cytokines) but not tissue culture fluid. likewise, little is known about the induction of cytokines during the course of fip. preliminary findings suggest that development of fip appears to be associated with a switch to predominantly th2 immunity, with increases in il-10 concentrations. 56 goitsuka et al 57 described increases in il-6 and il-1, but gunn-moore et al 58 reported reductions in th 2 and th 1 cytokines including il-2, il-4, il-10, and il-12, which they attributed to general immunosuppression. mildly increased amounts of il-1␣ mrna are detected inconsistently in association with lesions in cells of many organs in cats with fip. 59 roles of il-1 in this setting may include vascular endothelial activation, regulation of macrophages, il-8 and il-6, and chemotaxis. il-6 and il-1 can increase b-cell growth and differentiation and could exacerbate humoral contributions to the severity of fip. the inflammatory cytokines tnf-␣, il-1␣, and il-6 could circumvent the blood-brain barrier during systemic fip, or they could cross the blood-brain barrier after reorganization of the endothelial actin cytoskeleton. 60, 61 no information is available regarding cytokine production in neurological fip, and how cytokines in the cns compare to those in abdominal tissues of cats with generalized or effusive fip is unknown. mhv neurological disease can range from severe, acute, necrotizing, rapidly fatal encephalitis to chronic immunemediated demyelination with little encephalitis, depending on mouse strain, age, and immunocompetence. the appreciation of the principal role of the immune system in producing demyelination emerged from a series of experiments performed over several decades. profoundly immunosuppressed mice develop high concentrations of virus in the cns, but demyelination and clinical signs are minimal. immunocompetent mice have variable or even low concentrations of virus but develop marked disease. immune reconstitution in immunocompromised mice results in the development of severe demyelinating disease. if mice are pretreated with passive infusions of antibodies or t-cells or if they receive neuroattenuated mhv strains, they develop chronic, but not fatal, disease after mhv-jhm infection. 62, 63 immunocompetent c57bl/6 mice clear mhv-jhm virus from the brain but develop severe immune-mediated demyelination and paralysis. 22 in contrast, severe combined immunodeficient (scid) mice have persistent viral loads but no neurologic impairment or detectable lesions. gamma-irradiated immunocompromised mice similarly were resistant to chronic demyelination, but reconstitution of the immune system with adoptive transfer of splenocytes restored the immune-mediated lesions. 64 both cd4ϩ and cd8ϩ t-cells apparently are required to clear mhv from the cns. cd8ϩ t-cells are the predominant infiltrating leukocyte in lewis rats with mhv-jhm and paralytic disease, 65 whereas infiltration in clinically normal mhv-jhm-positive brown norway rats consists of cd4ϩ cells. ctls may kill some virally infected cells and protect mice from fatal disease, but they do not completely eliminate virus. 66 when cd8ϩ-depleted mice are reconstituted, virus load is reduced, and infection is not detected in most infected cell types, except for oligodendroglial cells. 66 mice with nucleocapsid or spike proteinspecific cd8ϩ t-cells develop chronic demyelinating disease. 21, 67 in the mhv-oblv60 model, depletion of cd8ϩ cells is associated with delayed clearance of oblv60 (but infected mice did recover). 25 ctls probably exacerbate lesions by contributing to tissue damage. mhv is relatively labile genetically (a common characteristic of rna viruses), and mutant strains with recognition sites that can evade ctl-mediated viral killing apparently increase and become the predominant viral strains in response to ctl-mediated natural selection. this feature results in disease progression in immunocompetent, but not immunosuppressed, hosts infected with these mutants. 68 several studies have suggested that apoptosis may be important in clearing virus from the cns. specific ctl recognition promotes apoptosis of mhv-infected cells. 69 in experimental allergic encephalomyelitis in rats, apoptosis appears to help control inflammation. 70 the role of cd4ϩ cells in mhv infection is also complex. nucleocapsid or spike protein-specific cd4ϩ t-cells have been shown to protect mice from coronaviral encephalomyelitis in the absence of cd8ϩ t-cells. 71 if mice with mhv-oblv60 had cd8ϩ but not cd4ϩ cells, they developed persistent infection. if they were cd8ϩ deficient, but cd4ϩ cells were normal, they had delayed clearance of the virus, suggesting a primary role for cd4ϩ cells in clearing this virus. 25 sussman et al 72 and williamson and stohlman 73 documented the requirement for cd4ϩ cells for clearance of jhm from mice. however, ␥-irradiated mice that were reconstituted with cd4ϩ cells responded to mhv-jhm challenge with earlier and more severe onset of neurological disease. 74 cd4ϩ knockout mice had less inflammation (with fewer macrophages and microglial cells) and less demyelination than did cd4ϩ competent mice. 75 the presence of mhv-jhm in astrocytes triggers a cytokine cascade that contributes to demyelination. 76 sun et al 77 documented production of tnf-␣, il-1␤, and il-6 by astrocytes in the spinal cords of mice that were chronically infected with mhv-jhm, localized to areas of virus infection and demyelination. tnf-␣ and il-6 are also produced in the brains of acutely infected mice, but the major cell producing these cytokines is the macrophage. these cytokines may help recruit t-cells and monocytes and may increase vascular permeability. 78 il-1␤ promotes leukocyte adhesion to endothelial cells, and tnf-␣ is toxic to oligodendrocytes. 63 the role of il-6 is unclear. effects attributed to il-6 include recruitment and activation of t-cells and macrophages, expansion of ctls, modulation of plasma cell differentiation, increased vascular permeability, downregulation of acute phase proteins, and contributions to immune-mediated destruction in the cns. [79] [80] [81] [82] in a study of cytokine profiles in lethally compared to sublethally affected mice with jhm, both th 1 -and th 2type cytokines, including ifn-␥, il-4, and il-10, were induced in all mice. 83 in the mice that died, tnf-␣ was induced more rapidly, and il-1␣ was increased. in mice with nonlethal infections, il-12 and il-1␤ were increased, and il-6 was expressed early. minor differences were observed in the patterns of inflammation in il-10-deficient compared to syngeneic mice, but the outcome of infection (eg, mortality and virus load) was not affected. 84 these results, combined with those of sun et al, 77 suggest that il-1␤ may allow mice to survive the early stages of the disease but may not allow for clearance of chronic infection. interferon-␥ also appears to be important for clearance of mhv infection. ifn-␥-deficient mice developed persistent jhm infection with increased clinical signs and mortality compared to mice competent to produce ifn-␥. 85 antiviral antibody and ctl responses were normal in ifn-␥-negative mice, despite the fact that ifn-␥ modulates antibody production. viral antigen occurred in oligodendrocytes and in association with cd8ϩ t-cells, suggesting that ifn-␥ is necessary for control of viral replication in oligodendrocytes. treatment of mice with anti-ifn-␥ antibody increased the mortality rate of mice, whereas immunotherapy with ifn-␥ reduced mortality and virus load. 86 mhv-oblv60 infection in immunocompetent mice induced transient mrna upregulation for cytokines il-1␣, il-1␤, il-6, tnf-␣, and ifn-␥. 25 nude mice differed in their cytokine profiles in that they lacked mrna for ifn-␥, whereas concentrations of the other cytokines remained persistently high. the authors suggested that the increased cytokines in the nude mice were produced by cns glial cells and possibly cd4ϩ and cd8ϩ subsets. ifn-␥ treatment of human neuronal cell cultures markedly increased the susceptibility of cells to infection with hcv-oc43 (although cells were susceptible to 229e without ifn-␥ treatment). 87 in this study, the hypothesized role of ifn-␥ was induction of mhc class i expression. ifn-␥ may increase superoxide dismutase and protect against oxygen radicals, kill virus directly, and increase the cytotoxicity of other effector cells. the chemokine crg-2 is expressed primarily by astrocytes during mhv infection and co-localizes with areas of viral rna and demyelination. 88 the function of crg-2 is not known, but this chemokine is also found in simian immunodeficiency viral (siv) encephalitis 89 and lymphocytic choriomeningitis. 90 with very little information available about the immunopathogenesis of neurologic fip, research performed in mice can be used as a model for fip and to contrast findings from the mhv model with those in fip. the immunopathogenic mechanisms are expected to have important similarities, but the demyelination that occurs in mhv is a more extensive pathological sequela of coronaviral infection (but not more fatal for the patient) than the focal granulomas that occur in fip. it can be hypothesized that in fip, upregulation of cytokines il-1␤, il-6, ifn-␥, and tnf-␣ may be important in mediation of destructive inflammation, but cytokines il-6 and ifn-␥ may be important in controlling viral infection, despite adverse inflammatory effects. cytokine and chemokine alterations in the brains of mice and cats with coronavirus infections may provide valuable clues to the immunopathogenesis of these diseases, as well as possible diagnostic markers and targets for immunological treatment of disease. it is tempting to consider cytokine modulation in the treatment of fip, but this application is premature without information regarding the cytokine profiles of affected cats. in addition to characterizing the concentrations of cytokines in the cns of cats with fip, it will be important to describe the timing of upregulated cytokine transcription. in mice with hmv-jhm, treatment with recombinant ifn-␥ resulted in reduced virus load in the liver but not in the brain. 86 if specific pro-inflammatory cytokines are found to be consistently high in cats with neurological fip, use of cytokine antagonists may eventually be of benefit in treatment. successful management of fip may consist of better methods of prevention as well as management of disease once it occurs. a commercial fip vaccine is available that consists of a mucosally delivered, temperature-sensitive mutant form of fipv. the vaccine virus is supposed to undergo replication only in outer oronasal cavities at low temperatures, thus triggering protective antibodies but not fip. a few controversial studies have documented reduction in fip as a result of vaccination. 91, 92 many vaccinated cats, however, fail to seroconvert with either igg or iga (foley and pedersen, unpublished data) , and independent studies failed to identify vaccine efficacy. 93, 94 other vaccines, including dna vaccines, have not progressed beyond experimental stages either because of lack of seroconversion, lack of protection, or both. in contrast with fip vaccines, vaccines against mhv have been shown to protect mice from challenge with virulent virus. a vaccine with purified spike protein 95 and synthetic s2 led to neutralizing antibodies in vivo. 96, 97 recombinant subunits against s in tobacco mosaic virus were given subcutaneously or intranasally and were protective against mhv-jhm. 98 the current standard of care for immunomodulation of fip is immunosuppressive doses of prednisone, to sustain an acceptable quality of life for as long as possible. the effects of steroids include lymphocytolysis, inhibition of arachidonic acid metabolism, reduction in cytokine rna transcription, and nitric oxide synthesis inhibition. 99 other potentially useful antiinflammatory drugs include antileukocyte antibodies and antioxidants. these treatments are only palliative. a better understanding of the fundamental 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coronavirus-induced encephalitis with a synthetic decapeptide homologous to a domain in the predicted peplomer stalk immunogenic peptide comprising a mouse hepatitis virus a59 b-cell epitope and an influenza virus t-cell epitope protects against lethal infection protective immunity against murine hepatitis virus (mhv) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an mhv epitope adjunctive therapy for bacterial meningitis: rationale for use, current status, and prospects for the future key: cord-322728-10m3xscs authors: severance, emily g.; yolken, robert h. title: chapter 29 role of immune and autoimmune dysfunction in schizophrenia date: 2016-12-31 journal: handbook of behavioral neuroscience doi: 10.1016/b978-0-12-800981-9.00029-8 sha: doc_id: 322728 cord_uid: 10m3xscs abstract in this chapter, we review data in support of the concept that immune system dysregulation is the most plausible explanation that reconciles gene by environmental interactions in schizophrenia. early investigations of this topic demonstrated aspects of aberrant activation of humoral immunity, including autoimmunity, associated with schizophrenia, whereas current research efforts have expanded this theme to include elements of innate immunity. advances in our understanding of inflammation and molecules of both the adaptive and innate immune system and their functional roles in standard brain physiology provide an important context by which schizophrenia might arise as the result of the coupling of immune and neurodevelopmental dysregulation. schizophrenia is a debilitating and complex brain disorder of unknown etiology. complicating our understanding of the causes and pathophysiology of schizophrenia is the likelihood that what we call schizophrenia is actually a heterogeneous assemblage of etiological conditions across a broad spectrum (arnedo et al., 2014) . reigning evidence supports a schizophrenia etiopathogenesis arising from and perpetuated by a multisourced genetic by environmental interaction (demjaha, maccabe, & murray, 2012; modinos et al., 2013; van os et al., 2014; tsuang, 2000) . although schizophrenia is highly heritable, the disease is polygenic, and gene studies to date have identified an enormous number of susceptibility loci (kavanagh, tansey, o'donovan, & owen, 2014 ; schizophrenia working group of the psychiatric genomics consortium, 2014). thus, the disease is thought to manifest when one or more of many possible genetic predispositions co-occurs with exposure to one or more of many possible environmental factors. relevant environmental factors can derive from a diversity of sources including exposures to infection, food-derived antigens, stress, smoking, cannabinoids, pollutants, and other toxins (allen, liu, pelkowski, et al., 2014; allen, liu, weston, et al., 2014; fine, zhang, & stevens, 2014; fineberg & ellman, 2013; fraga et al., 2011; severance, yolken, & eaton, 2014; suarez-pinilla, lopez-gil, & crespo-facorro, 2014; zhang et al., 2014) . if these exposures coincide with critical periods of fetal and neonatal brain maturation, there is the potential to aberrantly impact important brain processes including neural migration, synaptogenesis, myelination, and synaptic pruning. coinciding with these neurodevelopmental landmarks are events crucial for the instigation and maturation of innate and adaptive immunity. a possible role for immune system dysregulation in schizophrenia etiopathogenesis would reconcile both genetic and environmental hypotheses. a number of genetic loci found to associate with schizophrenia involve immune functions directly or implicate biological pathways that can influence immune function. for example, a consistently replicated locus for association with schizophrenia is the 6p22 chromosomal region that houses the major histocompatibility (mhc) locus and human leukocyte antigens (corvin & morris, 2014; purcell et al., 2009; stefansson et al., 2009) . the mhc/human leukocyte gene family functions to identify self and nonself entities and any dysfunction of these genes can render susceptibility to infectious disease, graft rejection, cancer, and autoimmunity. environmental triggers that show consistently replicated associations with schizophrenia are also those that result in immune activation. exposures to infectious pathogens, food antigens, and autoantigens have been especially well-studied risk factors for the development of schizophrenia, and special consideration is afforded to the timing, intensity, and type of immune activation elicited by these exposures (jones, mowry, pender, & greer, 2005; kirch, 1993; knuesel et al., 2014; meyer, 2013; muller, 2014; rothermundt, arolt, & bayer, 2001; torrey & peterson, 1976; . the focus of this chapter is to review some of the evidence in support of an immune and autoimmune dysfunction in the etiology, pathogenesis, and ii. neurobiology of psychotic disorders pathophysiology of schizophrenia. from a historical perspective, a recurrent immune theme predominated the early literature with a particular emphasis on schizophrenia-associated immunoglobulins and antibrain antibodies. these ideas still formulate the basis of current immune topics in schizophrenia, but over the years the scope has widened beyond the adaptive immune system to encompass also innate immunity. advances in our understanding of inflammation and mediators of both the adaptive and innate immune system and their functional roles in standard brain physiology provide an important context by which schizophrenia might arise as the result of the coupling of immune and neurodevelopmental dysregulation. the focus of this review on immune system aberrations in schizophrenia requires a review of basic knowledge of the major molecules and cells involved in the highly regulated balance of interacting innate and adaptive immune pathways. the function of the immune system is to protect the organism from disease and allow distinction between self and nonself entities, a process that is generally classified into the innate (nonspecific, always present) and adaptive (specific, triggered) immune systems. the innate immune system is composed of physical epithelial barriers, monocytes/ macrophages, dendritic cells, natural killer cells, and circulating plasma proteins. microbial invaders or compromised cells interact with recognition receptors found on monocytes/macrophages and dendritic cells. pattern recognition receptors can be cytoplasmic, membrane-bound, and secreted and include toll-like receptors, complement receptors, nucleotide-binding oligomerization domain (nod)-like receptors, pentraxins, and c-reactive protein. the adaptive immune system is composed of two immune response types: humoral (antibody) immunity and t-lymphocytemediated immunity. during activation of the adaptive immune system, binding of the invading antigen to b lymphocytes precipitates its differentiation into plasma cells that produce immunoglobulin antibodies specifically targeted to the invading antigen (alberts, 2008; rothermundt et al., 2001 ). the complement system acts in conjunction with the humoral immune system to form immune complexes with the antibody bound antigens and clear these from the body (walport, 2001a (walport, , 2001b . upon binding to monocytes/macrophages, pathogenic and other antigens also trigger the t-cell cascade, where t cells differentiate into cytotoxic t cells, t-helper cells, and natural killer cells. the lysis of cells containing the invading antigen is accompanied by the production of pro-and anti-inflammatory cytokines, signaling proteins that function in immune regulation (alberts, 2008; rothermundt et al., 2001) . dysregulation of any of these molecules, proteins, or cells at any stage of these pathways irrespective of a genetic or environmental origin can result in disorders of the immune system, which generally can take the form of inflammatory diseases, immunodeficiency, autoimmunity, or some forms of neoplasia. for complex psychiatric disorders such as schizophrenia, it is also necessary to understand how perturbations of these immune processes might impact the brain. because schizophrenia is thought to originate as a result of aberrant neurodevelopment, it is important to note that for a number of these classic immune factors, including complement, mhc, toll-like receptors, and pentraxins, additional functions in the developing brain are continuously being identified (benoit & tenner, 2011; bialas & stevens, 2013; boulanger, 2009; fourgeaud & boulanger, 2007; frodl & amico, 2014; garate et al., 2013; nagyoszi et al., 2010; pribiag & stellwagen, 2014; stephan et al., 2013; stevens et al., 2007; trotta, porro, calvello, & panaro, 2014) . it is also becoming increasingly evident that circulating endogenous peripheral immune entities may directly access the central nervous system (cns) as a result of directed regulation or compromised endothelial barriers. at the same time, it is possible that invading or resident pathogens or their products could directly exert detriment to the cns by similarly penetrating these barriers. as such, the spectrum of psychiatric dysfunctions known as schizophrenia may be the compilation of different stages of an immunoneurological intersection gone awry from both external and internal pathological molecules and pathways. early observations prepared a foundation for the studies of today where the role of immune activation is no longer questioned but understood to be the most parsimonious etiological explanation that encompasses a gene by environment landscape of schizophrenia. in this section, we will review the history of these immune associations and especially illuminate adaptive humoral immune system dysregulation because immunoglobulin abnormalities were the focus of early investigations (kirch, 1993; rothermundt et al., 2001) . although many of these early studies are inconsistent regarding the impact of any single infectious pathogen or autoimmune reaction against brain tissue, these investigations offer snapshots of how the immune process might be relevant to and influence brain function. importantly, they bring to light issues that are still relevant today and that are now studied without previous restrictions such as unrecognized disease heterogeneity, constricted study designs, and limited laboratory technologies. activation of the adaptive immune system and specifically of humoral immunity generally is manifested by changes in the levels of immunoglobulin antibodies with respect to the disease state. schizophrenia-associated changes in the levels of plasma and cerebrospinal fluid (csf) proteins were repeated findings that implicated immunoglobulins and solidified the idea that in schizophrenia, either an infectious or an autoimmune process might be occurring (amkraut, solomon, allansmith, mcclellan, & rappaport, 1973; bock & rafaelsen, 1974; burian, kubikova, & krejcova, 1964; durell & archer, 1976; fessel, 1962a fessel, , 1962b gammack & hector, 1965; hendrie, paraskevas, & varsamis, 1972; selecki, todd, westwood, & kraus, 1964; solomon, allansmith, mccellan, & amkraut, 1969; strahilevitz & davis, 1970) . of particular interest were reports that people with schizophrenia who had elevated immunoglobulin levels were also the least likely to show clinical improvement over the course of hospitalization compared with those with lower immunoglobulin levels (amkraut et al., 1973) . an infectious disease component contribution to psychotic mental disorders is often first attributed to esquirol (1845) , who suggested that the dissemination of psychoses unfolds similarly to an epidemic-like process (esquirol, 1845) . this observation was followed by other reports of psychotic epidemics in the decades following world war i and the 1918 influenza epidemic (kirch, 1993; menninger, 1919 menninger, , 1926 torrey & peterson, 1973 . the possible role of an antigen derived from a pathogenic organism such as a virus or bacteria took root in various forms and the early years of the viral hypothesis of schizophrenia is well-reviewed by peterson (1973, 1976) and kirch (1993) , with exposures to neurotropic viruses such as herpes simplex virus 1, measles, and rubella figuring prominently (kirch, 1993; torrey & peterson, 1973 . there was also an extensive literature base primarily from the 1940s to 1950s that describe a variety of antibody reactions in people with schizophrenia including the rosenow antibody-antigen skin reaction. this reaction was based on a hypothesis that several brain diseases such as epilepsy and schizophrenia were the result of alpha-hemolytic streptococci as measured by a cutaneous reaction to a streptococcal antibody or antigen that was obtained and cultured from nasopharynx samples (rosenow, 1948) . results from these studies were varied, with some showing greater immune response (cutaneous reaction) associated with schizophrenia and others showing no difference (gurassa & fleischhacker, 1958; rosenow, 1948) . we will revisit this idea of a pathogen-derived viral or bacterial source of immune activation in schizophrenia in its current form in a later section, because it is still a relevant hypothesis that is being explored with the benefit of modern tools such as high throughput sequencing. meanwhile, early literature on the topic of autoimmunity received similar effort and attention. one very early study of postmortem brain tissue identified the presence of autoantibodies to brain proteins and launched the idea that schizophrenia and other psychoses may have an autoimmune basis (lehmann-facius, 1937) . this theme continued in later decades when the role of autoantibodies to brain proteins was actively studied and disputed (boehme, cottrell, dohan, & hillegass, 1973; durell & archer, 1976; fessel, 1962a fessel, , 1962b heath, 1967; heath & krupp, 1967; heath, krupp, byers, & lijekvist, 1967a , 1967b jones et al., 2005; kirch, 1993; mellsop, whittingham, & ungar, 1973) . in some of these studies, the observation again came that levels of antibrain antibodies seemed to correlate with the intensity of psychotic symptoms and were generally higher during the early disease state and during acute attacks (glebov, 1972; gurevich, 1969; stamboliev, 1970; stoimenov, 1969) . dysregulation of the adaptive immune system and especially of humoral immunity still figures prominently in today's literature examining immune-based hypotheses for schizophrenia. speculation that medication is behind changes in immune marker levels is unavoidable; however, studies of patients who are antipsychotic naive or who have a recent onset of the disease support specific immune activation early in the course of disease, even before medication is administered (beumer et al., 2012; drexhage et al., 2010; drexhage et al., 2011; leonard, schwarz, & myint, 2012; miller, mellor, & buckley, 2012; mondelli & howes, 2014; severance et al., 2013b; steiner et al., 2012; stojanovic et al., 2014) . next we describe some current evidence available regarding schizophrenia-specific immune responses to external antigens and autoantigens. exposure to infectious disease pathogens during the pre-and postnatal period as defined by an antibody response is significantly associated with the future development of or current status of schizophrenia (arias et al., 2012; brown & derkits, 2010; buka, cannon, torrey, & yolken, 2008; fellerhoff, laumbacher, mueller, gu, & wank, 2007; mortensen et al., 2010; niebuhr et al., 2008; xiao et al., 2009; yolken et al., 2001; . we include both pre-and postnatal exposure references in this section and in a later section will review the implications on neurodevelopment of strictly maternal-occurring immune activation from a variety of sources including pathogens. pathogenic microorganisms are relevant to schizophrenia pathophysiology because they or their products can be neurotropic as well as cytotoxic or because the process of immune system activation is pathogenic in schizophrenia. certain viruses known to be neurotropic include the herpes simplex viruses, cytomegalovirus, and epstein-barr virus; these viruses are also of interest because their life cycle can contain a latent state from which they can be periodically reactivated (kirch, 1993; torrey & peterson, 1973 . to date, the strongest association of an infectious disease agent with schizophrenia is toxoplasma gondii, a neurotropic parasite, and this relationship is well-reviewed in numerous analyses and meta-analyses (arias et al., 2012; monroe, buckley, & miller, 2014; torrey, bartko, lun, & yolken, 2007; torrey, bartko, & yolken, 2012) . other pathogens that have shown significant associations with schizophrenia and psychoses also include epstein-barr virus, measles, polio, influenza, coronaviruses, human herpesvirus 2, borna disease virus, human endogenous retrovirus, and chlamydophila spp (arias et al., 2012; brown, begg, et al., 2004; dickerson, stallings, origoni, copp, et al., 2010; karlsson et al., 2001; karlsson, schroder, bachmann, bottmer, & yolken, 2004; khandaker, stochl, zammit, lewis, & jones, 2014; mednick, machon, huttunen, & bonett, 1988; perron et al., 2012; prasad, shirts, yolken, keshavan, & nimgaonkar, 2007; severance et al., 2011; suvisaari, haukka, tanskanen, hovi, & lonnqvist, 1999) . of note, exposure to the process of infection may be as or more important than the virulence or neurotropism of any single pathogen. a large study of the swedish national birth registry suggested that exposure to viral cns infections during childhood could result in the later development of schizophrenia (dalman et al., 2008) . unlike other investigations, this study did not support a link of bacterial infections with the development of subsequent psychoses. a different study, however, found that urinary tract infections (likely of bacterial origin) were found to occur with increased prevalence in schizophrenia and associated with acute relapse of psychosis (graham, carson, ezeoke, buckley, & miller, 2014; miller et al., 2013) . other conditions typically characterized by bacterial infection (sinusitis, tonsillitis, and pneumonia) were associated with the development of schizophrenia in the prenatal exposure scenario, as were genital and other reproductive infections (babulas, factor-litvak, goetz, schaefer, & brown, 2006; sorensen, mortensen, reinisch, & mednick, 2009) . it is expected that if schizophrenia in some people is the result of a specific virus or parasite, then evidence in the form of dna sequences would be found in the brain. these data, however, have thus far been elusive. the ability to efficiently search for this needle in a haystack came several years ago with the advent of highthroughput sequencing. the infancy of this field has not yet uncovered evidence for a causative pathogen, but ongoing investigations have brought about findings in unexpected places, including microbes associated with the gut microbiome. the connection between food sensitivity and propensity for schizophrenia was pioneered by f. curtis dohan, who hypothesized that wheat glutens and bovine milk caseins were broken down into bioactive exorphins that could penetrate through gut barriers, enter systemic circulation, and have access to the cns. his work was based on observations of celiac disease overlap with schizophrenia, with strong correlations of hospitalization rates for schizophrenia with wheat availability during wartime and improvement of psychotic symptoms following removal of wheat and dairy products from the diet (dohan, 1969 (dohan, , 1970 (dohan, , 1973 (dohan, , 1980 dohan, harper, clark, rodrigue, & zigas, 1984) . a recent resurgence in this field is exemplified by the numerous antibody studies that confirm an increased immune response directed at these food antigens, including a role for maternal antibodies to food antigens and the possible presence of an antigen-specific immune reaction up to 2 years before diagnosis of the disease (cascella et al., 2011; dickerson, stallings, origoni, vaughan, et al., 2010; jackson et al., 2012; karlsson et al., 2012; lachance & mckenzie, 2014; niebuhr et al., 2011; samaroo et al., 2010; severance et al., 2010; . the presence of food-derived exorphins or antibodies against them have been documented in the csf of individuals with a variety of psychoses including schizophrenia and coupled with a propensity for blood-brain and csf-brain barrier defects might implicate a neurotropic role of these peptides in the etiology or pathophysiology of the disease (axelsson, martensson, & alling, 1982; bauer & kornhuber, 1987; kirch et al., 1992; lindstrom, besev, gunne, & terenius, 1986; lindstrom et al., 1984) . autoimmune disease epidemiology and schizophrenia have been strongly linked for some time, with the first vestiges of the association coming in the form of findings suggestive of an inverse correlation between rheumatoid arthritis and schizophrenia (benros, eaton, & mortensen, 2014; eaton, hayward, & ram, 1992; . observations of a co-occurring psychosis with a number of autoimmune diseases including celiac disease, multiple sclerosis, systemic ii. neurobiology of psychotic disorders lupus erythematosus, autoimmune thyrotoxicosis, autoimmune hepatitis, and psoriasis also lent credence to the idea of an interrelated component of autoimmunity and the brain (benros et al., 2014; eaton et al., 2006) . celiac disease perhaps provides the strongest association with schizophrenia and reinforces the idea that for some, immune activation and autoimmunity have roots in the gut (baldwin, 1980; dohan, 1970 dohan, , 1973 dohan, , 1980 eaton et al., 2004) . celiac disease is a disease whereby the ingestion of wheat gluten launches an immune reaction that damages the epithelial lining of the small intestine through an autoimmune attack on tissue transglutaminase that breaks down the gluten peptide (alaedini & green, 2008; green et al., 2005; guandalini & assiri, 2014) . in the same way that the type of pathogen infection is probably not as important as the infectious process itself in causing brain pathologies such as schizophrenia, the specific type of autoimmune disease may not be the primary determinant of brain pathology. instead, the occurrence of a state of autoimmunity and its association with schizophrenia is rather likely to be a suggestion of the pathophysiology or faulty mechanism that is at work, perhaps as a disjunctive operation of an immune system pathway that has failed to function. large danish population-based studies, in fact, confirm that individuals or first-degree family members who had any history of an autoimmune disease have a 45% increased relative risk for schizophrenia (eaton et al., 2006) . the autoimmune link with schizophrenia was further solidified in an even larger investigation of this registry, and interestingly, this risk was further elevated in those with a history of an infection (benros et al., 2011) . this finding is not surprising given the fairly established literature base supporting the idea that exposure to infectious agents generates an autoimmune response (ercolini & miller, 2009) . as mentioned in a previous section, documenting and characterizing autoantibodies directed at brain proteins has been intriguing researchers for decades with generally mixed results. among the many autoantigens analyzed for an association with schizophrenia and psychosis are n-methyl-d-aspartate (nmda) receptors (deakin, lennox, & zandi, 2014; ezeoke, mellor, buckley, & miller, 2013; jones et al., 2005; masdeu et al., 2012; muller, 2014; pearlman & najjar, 2014; steiner et al., 2014; steiner et al., 2013) . this nmda receptor antibody quest was fueled by findings that antibodies to the nmda receptor were elevated in women with ovarian teratoma and psychoses-related encephalitis (dalmau et al., 2007) . other targets of autoimmune investigations include neuregulin-2, human endogenous retroviruses, cholinergic muscarinic receptors, nicotinic acetylcholine receptors, dopamine d2 receptors, mu-opioid receptors, serotonin receptors, î±-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptors, gamma-aminobutyric acid receptors, glutamic acid decarboxylase, potassium channel receptors, cardiolipin, dna, histones, and mitochondria (deakin et al., 2014; ezeoke et al., 2013; jones et al., 2005; masdeu et al., 2012) . an increased understanding of the underlying immunopathological processes and an improved characterization of reactive epitopes involved in disease pathogenesis might improve the predictive value of autoantibody assays and provide for reliable markers of disease susceptibility. a movement away from schizophrenia as a solely brain-centric disease is an active one in psychiatric research circles where an increasing awareness of the importance of the gastrointestinal (gi) tract, the body's largest immune organ, may share a bidirectional pathway with the brain. the strong association between food-based sensitivities and schizophrenia implicates the gi tract as an important site to search for immunological dysfunction. food antigen sensitivity is but one of a number of risk factors for schizophrenia that are related to gut inflammation, and this immunoglobulin g (igg) sensitivity joins other gut-related risk factors such as endothelial barrier defects, celiac disease, and exposure to t. gondii . research at this interface has shown in translational models that intestinal inflammation is a significant comorbidity of schizophrenia, and markers of this inflammation correlate with antibodies to food antigens such as gluten and casein at heightened rates in people with schizophrenia . it has been demonstrated in rodent models that the schizophrenia-associated pathogen t. gondii has many effects on the gut and during infection allows the passage of gluten peptides to translocate into circulation and provoke an antibody response (severance, kannan, et al., 2012) . in the presence of compromised epithelial and endothelial barriers, not only do foodbased peptides but also bacteria and other related harmful substances cross into the systemic circulation and generate more inflammation and propagate autoimmunity. markers of bacterial translocation are elevated in schizophrenia and also found to correlate with the antibody response to food antigens (severance et al., 2013a) . thus gut-based inflammation can be added to the growing list of studies that implicate both peripheral and cns inflammatory pathways associated with schizophrenia (dickerson et al., 2013; drexhage et al., 2010; fillman et al., 2013; fillman, sinclair, fung, webster, & shannon ii. neurobiology of psychotic disorders weickert, 2014; gibney & drexhage, 2013; leonard et al., 2012; linderholm et al., 2012; miller, buckley, seabolt, mellor, & kirkpatrick, 2011; miller et al., 2012; monji et al., 2013; muller, 2014; muller, myint, & schwarz, 2012; torrey et al., 2012; . the burgeoning field of gut brain axis analyses is the subject of investigations directed at the understanding of how gut microbes might impact neuronal connections in the cns. importantly, the gut microbiome functions to regulate the immune system. the ability of intestinal epithelial cells to actively respond to microbes is mediated by innate immune pattern recognition receptors (toll-like receptors), nod-like receptors, and helicases expressed on cell surfaces. during times of mucosal stress, gut homeostasis becomes disrupted (stockinger, hornef, & chassin, 2011) . although there are numerous reports of autism-related altered communities of the intestinal microbiome (adams, johansen, powell, quig, & rubin, 2011; finegold et al., 2010; finegold, downes, & summanen, 2012; kang et al., 2013; parracho, bingham, gibson, & mccartney, 2005; williams et al., 2011; williams, hornig, parekh, & lipkin, 2012) , studies of the microbiome in schizophrenia are scant. preliminary clinical studies report altered pharyngeal and intestinal microbiomes in individuals with schizophrenia as compared to controls (yolken & dickerson, 2014) . some insight can be gleaned from rodent studies, where manipulations of gut microbiota do in fact result in behavioral, biochemical, and molecular changes (collins, surette, & bercik, 2012; foster & mcvey neufeld, 2013; hsiao et al., 2013; stilling, dinan, & cryan, 2014) . diaz-heijtz et al. (2011) , for example, illustrated that behavioral effects accompanied changes in synaptic markers, synaptophysin and psd95, in the striatum (diaz heijtz et al., 2011) . in these rodent studies, animal phenotypes were recovered with manipulations of gnotobiotic (germ-free) animals, vagotomy, probiotics, and/or antibiotics. the ability of an extrinsically or intrinsically derived microbe, cell, protein, or other product normally found in peripheral circulation to enter to the cns renders discussion of epithelial and endothelial barriers an important topic. barrier permeability of the gut, blood-brain barrier, or blood-csf barrier (axelsson et al., 1982; bauer & kornhuber, 1987; kirch et al., 1992) can arise from a variety of environmental factors or from genetic mutations in the many biological pathways that impact this cellular architecture. barrier structures are composed of tight junctions (zonula occludens) that occur between the epithelial cells of the gi lumen of the gi tract; similar tight junction structures comprise the blood-brain barrier (deli, 2009; jong & huang, 2005) . the csf-brain and csf-blood barrier are slightly different, but these interfaces at the choroid plexus and arachnoid membrane are also relevant areas of access to the brain from the csf (laterra, keep, betz, & goldstein, 1999) . for schizophrenia, cns barrier dysfunction has been evaluated in studies of csf dynamics and is often attributed to a low-grade, systemic inflammation (bauer & kornhuber, 1987; bechter, 2013; bechter et al., 2010; kirch et al., 1992; severance, gressitt, alaedini, et al., 2015) . in conjunction with analyses of plasma and csf protein dynamics, it has been possible to detect evidence for barrier defects or restricted flow, as is particularly evident by the high prevalence of plasma-derived albumin. abnormal measures of plasma-derived albumin in the csf are noteworthy because the cns does not synthesize albumin and its elevation would require transport across the blood-brain or blood-csf barrier (tibbling, link, & ohman, 1977) . an increased albumin ratio can be indicative of either an anatomical barrier defect or a decreased csf flow rate, a dysfunction with numerous physiological causes (reiber, 1994; whedon & glassey, 2009) . the presence of pathological cns structures such as choroid plexus calcification, arachnoid cysts, and decreased brain volume all can disrupt csf flow patterns and all of these conditions have been previously associated with psychoses and schizophrenia (arango et al., 2012; kuloglu, caykoylu, yilmaz, & ekinci, 2008; laterra et al., 1999; marinescu, udristoiu, & marinescu, 2013; narr et al., 2003; reiber, 1994; rimol et al., 2012; sandyk, 1993; shiga et al., 2012; veijola et al., 2014; whedon & glassey, 2009) . although a systemic state of inflammation that might impact barrier integrities is most likely the result of immune activation from an environmental source, cellular barrier proteins and related biological pathways may also be the result of genetic associations. specific barrierrelated genes that have been significantly associated with schizophrenia include the tight junction protein claudin-5, cytoskeletal elements such as actin, haptoglobin, and nitric oxide synthetase (burghardt, grove, & ellingrod, 2014; hall, trent, thomas, o'donovan, & owen, 2014; horvath & mirnics, 2014; maes et al., 2001; sun et al., 2004; wan et al., 2007; wei & hemmings, 2005; yang et al., 2006; ye et al., 2005; zhao et al., 2014) . the etiology and pathogenesis of schizophrenia likely stem from aberrant neurodevelopment (lewis & levitt, 2002; piper et al., 2012; rapoport, giedd, & gogtay, 2012) . perinatal-occurring environmental disturbances such as maternal stress, infection, or obstetric complications may interact adversely in genetically predisposed offspring to impact neural migration, synaptogenesis, myelination, and synaptic pruning (knuesel et al., 2014) . epidemiological and preclinical studies clearly indicate that exposure to maternal immune activity is associated with pathological brain development and thus maternal immune activation has become a strong risk factor for the development of schizophrenia (bauman et al., 2014; brown & derkits, 2010; canetta et al., 2014; garbett, hsiao, kalman, patterson, & mirnics, 2012; meyer, 2013; ii. neurobiology of psychotic disorders shi, smith, et al., 2009) . specifically, maternal exposure to cytomegalovirus, herpes simplex virus type 2, influenza, rubella, t. gondii, and wheat glutens have all been documented to increase the risk of development of psychosis or schizophrenia brown, begg, et al., 2004; brown, cohen, greenwald, & susser, 2000; brown, hooton, et al., 2004; buka et al., 2008; ellman, yolken, buka, torrey, & cannon, 2009; karlsson et al., 2012; mortensen et al., 2010; pedersen, stevens, pedersen, norgaard-pedersen, & mortensen, 2011; xiao et al., 2009) . this repertoire was recently expanded to include exposure to general inflammation and innate immunity based on measures of c-reactive protein and complement c1q (canetta et al., 2014; severance, gressitt, buka, cannon, & yolken, 2014) . in this section, we will review the timelines of brain and immune development and review the evidence where these trajectories might intersect and result in brain disorders (figure 1) . neural development is a highly regulated process and since molecules and proteins of the immune system are continually being found to participate in mechanisms of normal brain development, any immune overactivation, or failure of the immune system to activate will impact brain circuitry. the immune environment during pregnancy is a complex balance aimed at preserving immune protection of both sides of the maternal-fetal interface. several good reviews are available of how this interface is skewed maternally toward inhibiting fetal immunity and regulating and maintaining a protective th2 environment over the pro-inflammatory cytotoxic th1 immune response needed to fight infectious disease (belderbos, levy, meyaard, & bont, 2013; morein, blomqvist, & hu, 2007) . maternal immunity is antibody based and functions to maintain immune tolerance in the fetus and breast-feeding neonate. as a result, all antibodies including autoantibodies are passed to the offspring during this period. furthermore, while under maternal immune protection, the antigen recognition system of the fetus is immature. once maternal-derived immune factors are depleted, the immune system of the neonate must be redirected to become competent, including a more active th1 component. maturation of the innate and adaptive immune systems is a process that occurs from the fetal stage through adulthood (belderbos et al., 2013; knuesel et al., 2014; morein et al., 2007) . molecules and proteins of the immune system are intrinsically intertwined with important brain processes during development. these processes include initial proliferation of glia and neurons, consequent migration, programmed cell death, formation of synapses, figure 1 developmental timelines of the brain and the immune system. complex disorders such as schizophrenia are thought to arise when one or more neurodevelopmental processes are interrupted because of genetic and/or environmental factors. various immune molecules, proteins, and cells such as c1q and major histocompatibility complex function in the brain during neurodevelopment, suggesting that any disruption in the immune system during pregnancy or postnatally has the ability to compound synaptic misconnections. compiled from belderbos et al. (2013) , dietert et al. (2010) , kneusel et al. (2014) , and morein et al. (2007) . ii. neurobiology of psychotic disorders myelination, and synapse pruning with the overall endpoint to establish functional neuronal circuits (knuesel et al., 2014) . here, we present the case of complement c1q as an example of an immune molecule that is highly active in the developing brain and that is also implicated in schizophrenia-associated gene and environmental studies. in the developing immune system, relevant processes include immune cell appearance, colonization, expansion, and maturation. complement c1q and mhc1 were some of the first immune molecules identified to function in synapse development and pruning in the brain (boulanger, 2009; fourgeaud & boulanger, 2007; huh et al., 2000; shatz, 2009; stevens et al., 2007) . complement pathway-related genes that have been associated with schizophrenia include the c1qb gene, complement control-related genes, and complement surface receptor gene cd46 (havik et al., 2011; zakharyan et al., 2011) . biologically, complementcontaining circulating immune complexes were elevated in individuals with schizophrenia compared to controls and a primary antigenic component of these immune complexes was often found to be casein or gluten boyajyan, khoyetsyan, tsakanova, & sim, 2008; mailian, boiadzhian, sogoian, sim, & manukian, 2005; mayilyan, weinberger, & sim, 2008; vetlugina, logvinovich, maslennikova, & vasil'eva, 1984) . finally, elevated levels of maternal c1q igg have been found to increase the odds for psychosis in offspring (severance, gressitt, buka, et al., 2014) . given that maternal igg antibodies begin transfer to the fetus at 13 weeks' gestation and approach maternal levels at time of birth (malek, sager, kuhn, nicolaides, & schneider, 1996; simister, 2003) , this study introduces the interesting possibility that autoantibodies to c1q present in the mother might interact with fetal c1q during critical periods of brain development. specifically, if the process of normal c1q-mediated synapse formation and pruning is interrupted, synaptic connections will presumably be permanently altered in the developing brain either through overpruning or through underpruning. other studies have connected the presence of maternal autoantibodies and with the development of autism spectrum disorders where maternal autoantibodies have been found to recognize brain proteins critical to the neurodevelopmental process (braunschweig et al., 2013; brimberg, sadiq, gregersen, & diamond, 2013) . this chapter provides an introduction into some of the mechanisms by which the immune system might be involved in the development of schizophrenia. if schizophrenia has an immune component, and if evidence indicates a primary rather than secondary role in disease pathogenesis, then interventions that target the immune system are warranted. toward this end, one purpose of this review was to emphasize the very diverse and multiple ways in which the immune system might impact schizophrenia. its etiology, pathogenesis, and pathophysiology may not just be a function of exposure to an infectious agent or food antigen or dysfunctional innate immunity. therefore, designing a treatment strategy to an extraordinarily heterogeneous disease is difficult. it is extremely important to be able to identify the subsets of people who have immune-related conditions and fully characterize what kind of immune anomaly is present. only in this manner can tailored treatments be evaluated. in the future, therapeutic strategies might involve monoclonal or monospecific antibodies to antagonize or inactivate antigenic or other protein targets or use of other immunosuppressive treatments. the rapid advance in the use of monoclonal antibodies for the treatment of autoimmune disorders provides hope that such therapies can also have a major impact on schizophrenia as well. dietary interventions have been successful in some instances clinically, and developmental compounds aimed to normalize gut function and 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first-episode schizophrenia are some cases of psychosis caused by microbial agents? a review of the evidence association of c1qb gene polymorphism with schizophrenia in armenian population prenatal xenobiotic exposure and intrauterine hypothalamus-pituitaryadrenal axis programming alteration transcriptome sequencing and genome-wide association analyses reveal lysosomal function and actin cytoskeleton remodeling in schizophrenia and bipolar disorder key: cord-311847-2czqs84q authors: pennisi, manuela; lanza, giuseppe; falzone, luca; fisicaro, francesco; ferri, raffaele; bella, rita title: sars-cov-2 and the nervous system: from clinical features to molecular mechanisms date: 2020-07-31 journal: int j mol sci doi: 10.3390/ijms21155475 sha: doc_id: 311847 cord_uid: 2czqs84q increasing evidence suggests that severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) can also invade the central nervous system (cns). however, findings available on its neurological manifestations and their pathogenic mechanisms have not yet been systematically addressed. a literature search on neurological complications reported in patients with covid-19 until june 2020 produced a total of 23 studies. overall, these papers report that patients may exhibit a wide range of neurological manifestations, including encephalopathy, encephalitis, seizures, cerebrovascular events, acute polyneuropathy, headache, hypogeusia, and hyposmia, as well as some non-specific symptoms. whether these features can be an indirect and unspecific consequence of the pulmonary disease or a generalized inflammatory state on the cns remains to be determined; also, they may rather reflect direct sars-cov-2-related neuronal damage. hematogenous versus transsynaptic propagation, the role of the angiotensin ii converting enzyme receptor-2, the spread across the blood-brain barrier, the impact of the hyperimmune response (the so-called “cytokine storm”), and the possibility of virus persistence within some cns resident cells are still debated. the different levels and severity of neurotropism and neurovirulence in patients with covid-19 might be explained by a combination of viral and host factors and by their interaction. coronaviruses (covs) are a group of large enveloped non-segmented positive-sense rna viruses, causing respiratory and enteric diseases in animals and humans [1] . a highly pathogenic cov, named severe acute respiratory syndrome (sars)-cov-2 (formerly known as 2019-ncov), emerged in december 2019 in the hubei region of china, and in the city of wuhan in particular. the initial cases, presenting with a dry cough, sore throat, fever, dyspnea, and bilateral lung infiltrates on chest imaging, were all linked to the wuhan's huanan seafood wholesale market, which trades fish and a variety of live animals, including bats, poultry, marmots, and snakes [2] . this novel cov has caused an outbreak viral antigen and more rapid antibody detection systems (such as the enzyme-linked immunosorbent assay) are currently being developed, although their accuracy is still limited by the relatively high rate of false-negative cases and, therefore, needs to be improved [25] . more recently, other studies are trying to propose droplet digital pcr-based methods as more effective diagnostic strategies for the identification of sars-cov-2 positive patients with low viral load [26] . to date, there is no effective treatment for patients with covid-19. adenosine analogs (e.g., remdesivir, favipiravir, ribavirin, and galidesivir) acting on the rna-dependent polymerase and blocking the viral rna synthesis are promising. chloroquine (cq) and hydroxychloroquine (hcq) can effectively inhibit sars-cov-2 in vitro, but their efficacy in vivo is under evaluation, as well as the effect of serum rich in anti-sars-cov-2 antibodies obtained from convalescent subjects [23] . very recently, a systematic review assessed the efficacy and safety of cq/hcq for treatment or prophylaxis of adult patients with covid-19 [27] . thirty-two studies were included, of which 6 randomized clinical trials (rcts) and 26 non-randomized, with a total of 29, 192 participants. overall, studies suggest that the treatment of hospitalized patients with cq/hcq may not reduce the risk of death compared to standard care. high dose regimens or combination with macrolides may be associated with harm, particularly qtc prolongation and cardiac arrhythmias [28] . post-exposure prophylaxis may not reduce the rate of infection, although the quality of the evidence is low. the authors concluded that patients should be treated with cq/hcq only if monitored and in the context of high-quality rcts [27] . therefore, rationalization of the use of these drugs is also advised [29] . other non-specific immune modulators include human immunoglobulin and corticosteroids, such as dexamethasone, a glucocorticoid that has proved to be the first life-saving drug in these patients. in particular, dexamethasone 6 mg once daily (either per os or by intravenous injection) for 10 days may result in a reduction in mortality by 1/3 in patients on ventilators and by 1/5 in those receiving oxygen [30] . other treatment options include specific monoclonal antibodies that bind the receptor-receptor domain of sars-cov-2 and antibodies that block inflammatory interleukins (il), such as tocilizumab. finally, several vaccines are under analysis and include live attenuated viruses, inactivated viruses, use of recombinant dna, and vaccines based on sars-cov-2 specific proteins and subunits [31] . until these therapeutic options are confirmed, the main measures are prevention, isolation, social distancing, frequent hand washing, and the use of personal protective equipment. some experimental and clinical studies previously performed on other covs and preclinical models seem to converge on the evidence that these viruses may have a tropism into the central nervous system (cns). seven types of covs are currently known that can infect humans and cause neurological damage [23] . in some animal and human covs (including those causing sars and mers), a neuroinvasive potential has been demonstrated [32] . although there are limitations in the epidemiological studies carried on covid-19, as well as limited case records for determining the actual incidence of these complications, some patients reported neurological symptoms, but clinical findings and pathogenic features have not yet systematically addressed. the penetration of several respiratory viruses into the cns has already been shown, a mechanism called "neuroinvasion," affecting both glia and neurons [31] . in some cases and under certain conditions, the neurotropism can cause neurovirulence, which refers to the development of neurological manifestations [33] . in the case of sars-cov-2, the existence of these phenomena is supported by previous evidence showing human cns infection from other respiratory viruses, the cns of other species infected by covs, animal and in vitro models of cns infection by human covs, and the occurrence of neurological complications in the course of other human covs infections. the aims of this review are i) to summarize the available information on the relationship between covs and the nervous system, ii) to identify the potential targets and routes of entry of sars-cov-2 into the nervous system, and iii) to describe the range of the neurological features reported to date in patients with covid-19 and the proposed pathogenic mechanisms. all the common respiratory viruses affecting humans, such as influenza, covs, and respiratory syncytial virus (rsv), can be associated with various neurological manifestations, particularly in subjects experiencing severe pulmonary symptoms [34] . for instance, the effects that rsv may cause include seizures, encephalitis, ataxia, and cerebellitis, and the virus has been detected in the cerebrospinal fluid (csf). influenza is known to cause neurological complications, such as encephalitis, myelitis, meningitis, and guillain-barré syndrome (gbs) [34] . respiratory viruses, including the cov family, affect the cns of other species, such as birds, felines, and livestock [35] . meningitis and spinal cord inflammation have been reported in cats affected by a pathogenic feline cov [36] . a 91% homology resemblance has been assessed between the human oc43 cov and the swine hemagglutinating encephalomyelitis virus (hev), which can invade the porcine brain by retrograde neuronal propagation through the peripheral nerves [37] . a subspecies of murine covs, called mouse hepatitis virus, induces a demyelinating disease resembling multiple sclerosis (ms) [35] . human covs can induce acute or persistent infections in neuronal cell lineages, neuroglia, and oligodendrocytes [38] [39] [40] . moreover, flaccid paralysis and demyelination in animal models can be caused by the human oc43 cov [41] . in particular, it has been shown that the spread of the oc43 in susceptible mice runs from the olfactory bulb to the brainstem and spinal cord, and uses the axonal transport system as the avenue for the neuron-to-neuron spread [33] . further, neuron-to-neuron propagation strategies observed in cell cultures include both passive viral particle diffusion and axonal transport [42] . sars-cov, which may enter the cns through the olfactory bulb and transneuronally spread to other brain regions, can cause neuronal death in the human ace2 receptor transgenic mouse [43] . at least four types of human covs have shown neuroinvasive capacity based on the detection of viral rna or other nucleic acids in the human brain [35] . in a 12-month-old infant with severe immunodeficiency, a case of fatal oc43 cov-related encephalitis was confirmed through rna sequencing techniques and rt-pcr in samples of brain biopsy [44] . immunohistochemical study of the brain showed a microglial reaction, t lymphocyte infiltrates, and presence of the oc43 cov nucleocapsid in neurons. in a 15-year-old adolescent with disseminated acute encephalomyelitis associated with oc43 cov infection, magnetic resonance imaging (mri) disclosed demyelination in the subcortical white matter, cerebellum, and spinal cord [45] . the oc43 cov was detected in the csf and nasopharynx secretions by using the pcr. there has also been a report of gbs associated with 229e and oc43 cov co-infection in a pediatric patient [46] . encephalitis, ischemic stroke, and polyneuropathy can result from sars-cov exposure, with viral rna detectable in the csf [47, 48] . in a necropsy study carried out on 8 victims of sars-cov, infected neurons were found in the cortex and hypothalamus, and genomic sequences of sars-cov were detected in all cases by using the rt-pcr [49] . encephalomyelitis and vasculitis may result also from mers-cov infection. a series of three patients showed that they all suffered from an altered level of consciousness, ranging from confusion to coma, along with ataxia and motor deficit [50] . at brain mri, bilateral lesions were evident in the white matter of the frontal, parietal, and temporal lobes, as well as in the basal ganglia and corpus callosum. two of these patients showed an increased protein level in the csf, while all had lymphocytopenia and severe multiple organ involvement, including kidney, liver, and the cardiovascular system [50] . during the mers-cov infection, other neurological complications were reported: brainstem encephalitis, gbs [51] , and cerebral hemorrhage in the context of thrombocytopenia and disseminated intravascular coagulation [52] . a retrospective study involving 70 mers patients reported that 8.6% had seizures, while four had gbs in a series of 23 cases. the latency of the neurological symptoms ranged from 7 and 26 days after the onset of the pulmonary disease [53] . a pubmed-based literature search was performed to find all relevant reports published until june 2020. the search queries were "covid-19 and nervous system", "brain", "neurology", "neurological", "encephalopathy", "encephalitis", "stroke", "seizures", "neuropathy". the search was also repeated by using the above-mentioned keywords and the term "sars-cov-2" instead of "covid-19". the authors selected all the articles based on the abstract and full-text examination and without a priori appraisal of inclusion/exclusion criteria. indeed, the number of studies reporting neurological complications of covid-19 is still limited and the majority include case reports/case series or retrospective samples, without a systematic specific assessment of the neurological complications. the references of the articles retrieved were also examined in search of more data. after this process, a total of 23 studies were included. figure 1 summarizes the main neurological manifestations of covid-19 and proposed mechanisms. reported: brainstem encephalitis, gbs [51] , and cerebral hemorrhage in the context of thrombocytopenia and disseminated intravascular coagulation [52] . a retrospective study involving 70 mers patients reported that 8.6% had seizures, while four had gbs in a series of 23 cases. the latency of the neurological symptoms ranged from 7 and 26 days after the onset of the pulmonary disease [53] . a pubmed-based literature search was performed to find all relevant reports published until june 2020. the search queries were "covid-19 and nervous system", "brain", "neurology", "neurological", "encephalopathy", "encephalitis", "stroke", "seizures", "neuropathy". the search was also repeated by using the above-mentioned keywords and the term "sars-cov-2" instead of "covid-19". the authors selected all the articles based on the abstract and full-text examination and without a priori appraisal of inclusion/exclusion criteria. indeed, the number of studies reporting neurological complications of covid-19 is still limited and the majority include case reports/case series or retrospective samples, without a systematic specific assessment of the neurological complications. the references of the articles retrieved were also examined in search of more data. after this process, a total of 23 studies were included. figure 1 summarizes the main neurological manifestations of covid-19 and proposed mechanisms. the available research on the neurological involvement in sars-cov-2 makes it hard to causally link a specific neurological manifestation to the viral infection. as a general rule, severe forms of covid-19 are more likely to produce neurological complications when compared to the mild forms (45.5% versus 30%). an autopsy study on deceased patients with covid-19 due to respiratory failure indicated the presence of cerebral edema and neuronal degeneration in these subjects [77] . a study in wuhan (china), reported neurological findings in 214 hospitalized patients with covid-19 [68] . another systematic study in france [60] noted neurological symptoms in 49 of 58 patients, including confusion, encephalopathy, and cortico-spinal tract signs at clinical examination, along with leptomeningeal enhancement and perfusion abnormalities on brain mri. overall, the most common neurological symptoms reported in some patients with covid-19 were headache, anosmia, ageusia, asthenia, and myalgia, followed by encephalopathy, seizures, stroke, and encephalitis [78] . elderly patients and those with previous cognitive decline, multiple comorbidities, other infections, severe medical illness, poor premorbid functional status, malnutrition, and vascular risk factors (especially hypertension) have a higher risk to show an altered level of consciousness related to covid-19 [55, 68, 79] . moreover, metabolic or endocrine derangements, including hypoor hypernatremia, hypo-or hypercalcemia, hypo-or hyperglycemia, renal and/or liver dysfunction, among others, put patients at further risk for encephalopathy. sepsis and the subsequent inflammatory and the so-called "cytokine storm" may also contribute to encephalopathy with il-6, il-8, il-10, and tumor necrosis factor α (tnfα) being implicated in confusional states [80] . finally, patients with previous neurological disorders and acute respiratory symptoms seem to be at increased risk for encephalopathy as the initial symptom of covid-19. coherently, in the study by mao et al. [68] , 15% of patients with a severe form of the disease presented an altered level of consciousness. toxic and metabolic causes, as well as the effects of drugs or hypoxia, may result in covid-19-associated encephalopathy [57] . interestingly, an electroencephalography (eeg) report on a patient with altered mental status who was unable to follow verbal orders as the presenting symptom of covid-19 showed diffuse slow waves, particularly in the left temporal region, whereas pathological findings demonstrated cerebral edema without inflammatory signs [55] . in these cases, treatment is symptomatic and includes fever control, treatment of hypoxia, and antiepileptic medications [77] . a case of covid-19-associated (confirmed by rt-pcr in a nasopharyngeal sample) acute hemorrhagic necrotizing encephalopathy has also been described [71] . brain computed tomography (ct) detected a symmetrical bilateral hypodense area in the medial thalamic nucleus, whereas mri showed contrast-enhanced hemorrhagic lesions, with multifocal and symmetrical disposition, in both thalami, insula, and the mesial region of temporal lobes [71] . although relatively rare, acute necrotizing encephalopathy can be a severe complication of some viral infections, including the influenza virus. the authors postulated that the pathogenesis might be related to the "cytokine storm" induced by covid-19 [81] . a posterior reversible encephalopathy-like syndrome, associated with transient cortical blindness, was also reported [64] . based on the available evidence, sars-cov-2 should be included in the differential diagnosis algorithm of viral encephalitis. typical symptoms are fever, headache, seizures, behavioral disorders, and altered level of consciousness. in these patients, an early diagnosis is of crucial importance to increase the survival rate, especially in those with severe pneumonia and hypoxia [23] . in a report of a 56-year-old woman from wuhan with covid-19, the brain ct remained normal but the diagnosis of encephalitis was confirmed through the isolation of sars-cov-2 in the csf using genomic sequencing techniques [75] . the case of a 24-year-old japanese man presenting with multiple generalized epileptic seizures and decreased level of consciousness led to a diagnosis of meningoencephalitis [69] . brain mri showed hyperintense areas in the right mesial region of the temporal lobe and hippocampus. while the sars-cov-2 rna was not detected in the nasopharynx, it was identified in the csf by using rt-pcr, although it was unclear if some of the patient features were present in the context of seizures due to other causes [69] . anyhow, high levels of proinflammatory cytokines in the csf can cause breakdown and increased permeability of the blood-brain barrier (bbb) which may, in turn, lead to viral invasion and clinical manifestation [71] . seizures have already been reported in cov infections, and there has been a high proportion of breakthrough seizures in patients with epilepsy who developed covid-19 [78] . nevertheless, a recent analysis of 304 patients with covid-19 [67] reported two seizure-like events only, with no confirmed cases of new-onset seizures. however, the study was limited by a lack of instrumental investigation (e.g., eeg, neuroimaging) and by its retrospective approach. a case report of a patient with no history of epilepsy who had multiple apparent tonic-clonic seizures in the context of covid-19 might be interpreted as an unmasked seizure disorder or a direct effect of covid-19 on the brain, although further confirmations are needed [63] . when compared to younger subjects without comorbidities, elderly patients with covid-19 and vascular risk factors appear to be at greater risk for developing cerebrovascular complications [23] . in a retrospective study of 221 patients [66] , 11 (5%) presented with ischemic stroke, one (0.5%) with cerebral venous thrombosis, and one (0.5%) with a cerebral hemorrhage. the risk factors for stroke in this population were: advanced age (mean age: 71.6 years), severe pulmonary disease, hypertension, diabetes, marked inflammatory or procoagulant response (e.g., increased c-reactive protein and d-dimer), and previous cerebrovascular events [66] . other researchers described five patients with stroke (80% ischemic), who had severe forms of covid-19, increased d-dimer, thrombocytopenia, and multiple organ failure [68] . notably, a study in the usa demonstrated that young patients (aged < 50 years) more likely developed large-vessel strokes in the context of covid-19, suggesting that all ages are vulnerable [70] . regarding pathomechanisms, the increased risk of cerebrovascular disease during the covid-19 infection is likely multifactorial. it has been shown that sars-cov-2 can bind to the ace2 receptor on endothelial cells, which might result in increased blood pressure. both ischemic and hemorrhagic strokes can be secondary to the increase in blood pressure, together with the presence of thrombocytopenia and coagulation disorders. the "cytokine storm" may act as another pathogenic mechanism [23] . the levels of c-reactive protein, ferritin, d-dimer, lactate dehydrogenase, and the leukocyte count have often been found to be elevated in these patients [82] . moreover, increased inflammatory markers and hypercoagulability state seem to characterize severe cases, along with a substantially enhanced risk of stroke [68] . the likelihood of ischemic or hemorrhagic stroke may be also increased by some viral-related mechanisms, including vascular endothelial cell infection and consequent vessel damage. on the other hand, it is well known that infection-associated systemic inflammation, thrombosis, or vasculitis increase the risk of stroke [83] . finally, systemic vasculitis and cns vasculitis have been demonstrated at autopsy in patients with sars-cov [84] . most patients with covid-19 complain of headaches. guan et al. [57] found that 13.6% of a series of more than 1000 patients reported headache, and in 15% of those with severe forms. the intensity of headache was generally referred to be mild, although the study did not mention whether a prior history of headache or any meningeal sign was present. in a recent case series [58] , headaches were a predominant complaint, along with fever, cough, sore throat, and breathlessness. the prevalence varies in different reports, but headache may affect up to 1/3 of patients [54, 62] . headache is a well-known clinical feature of meningitis, encephalitis, intracranial hypertension, cerebrovascular diseases, and vasculitis, whereas scarce pathophysiological data link it to covid-19. in some cases, cytokines and chemokines released by macrophages may activate nociceptive sensory neurons [85] , with a neuroinflammatory mechanism similar to that involved in pain [86] . in this scenario, screening patients for secondary causes of headache, including covid-19, is mandatory, especially for patients in whom frequency or severity of headache change, or present with systemic symptoms, or do not respond to first-line or habitual treatments. anosmia and secondarily taste disorders are commonly reported in patients with covid-19 and may appear suddenly [56] . in italy, 19.4% of patients had some form of chemosensory dysfunction [74] , whereas in a case register of twelve european hospitals, the prevalence of olfactory and gustatory dysfunction in 417 patients with covid-19 with mild-to-moderate symptoms [65] was 85.6% and 88%, respectively. notably, 12% of them declared the olfactory dysfunction as the initial symptom and 18% had no runny nose or nasal obstruction [65] . indeed, although anosmia is noted in many other respiratory infections, such as cold and influenza, in covid-19 it is typically not accompanied by nasal swelling or rhinitis [65, 74, 87] . given the reports of anosmia presenting as an early symptom of covid-19, dedicated testing may offer the potential for early detection of sars-cov-2 infection. nevertheless, the chemosensory deficit in covid19 has not yet been systematically investigated, although it is a current research "hot topic", both at the clinical-epidemiological and cellular level. initial observations and early studies suggested as possible mechanisms for anosmia in covid-19 the cleft syndrome, nasal obstruction and rhinorrhea, "cytokine storm", direct damage to olfactory receptor neurons (orns), and impairment of the brain olfactory centers. the most obvious cause would be a direct damage to orns, since other human covs (e.g., oc43) have shown to directly bind to orns. however, although anosmia is linked with human viruses causing the common cold, such as influenza and other covs (respiratory or not), the exact mechanism has not yet been clearly established. a new model of olfactory dysfunction in covid-19 has been recently drawn from the observation that sustentacular cells (suss) are the primary target of the virus and that suss infection triggers a cascade of events leading to anosmia [88] . suss express ace2 and would be infected first. impairment of sus would negatively affect orns, leading to the inhibition of the odor perception. simultaneously, rapid immune response would be induced in a subset of orns and microvillar cells, which would lead to activation of lymphocytes and macrophages and their infiltration into the olfactory epithelium, as well as secretion of proinflammatory cytokines. stem cell infection may potentially explain why a small fraction of patients with covid-19 experience long-term dysosmia [88] . of note, such a model does not imply that sars-cov-2 travels from the olfactory epithelium to the brain along the olfactory axons. indeed, no axonal transport of sars-cov-2 to the brain has been demonstrated in the hamster model during the first two weeks after infection [89] , and no viral accumulation or persistence has been reported in cerebral olfactory regions of autopsy material from patients with covid-19 [90] . on the other hand, rapid sars-cov-2 accumulation in the brain after intranasal injection was recently shown using the new humanized ace2 knock-in mouse [91] . yet, this is not synonymous with transport along olfactory axons, as other routes are also possible. if sars-cov-2 travels within the olfactory axons, this would require an ace2-independent passage of the virus from suss to orns within the olfactory epithelium. in addition, it would be relevant to examine progenitor or stem cell infection, as these olfactory epithelium cells also express significant levels of ace2. probably, also host genetic factors play a role in individual susceptibility to anosmia in covid-19 and the characterization of these factors is of particular interest [92] . regarding gustatory dysfunction in covid-19, it is known that the ability to separate flavors depends on the retronasal stimulation pathway. therefore, in patients with ageusia, retronasal olfactory dysfunction is commonly suggested, although some studies reported high ace2 expression on the oral cavity mucosa and epithelial cells of the tongue [93] . another possibility is that sars-cov-2 may have a direct effect on the taste buds or receptors [94] . gbs (i.e., acute inflammatory demyelinating polyneuropathy) can follow a gastrointestinal or respiratory infection. a molecular mimicry mechanism, in which infecting viruses likely share epitopes similar to some peripheral nerve components, is believed to occur and to stimulate autoreactive t or b lymphocytes. antibodies against the virus cross-react and bind to peripheral nerve components, thus causing neuronal dysfunction and clinical manifestations. after sars-and mers-cov infections, both gbs and acute motor axonal neuropathy have been described [32, 51] . reports of gbs in patients with covid-19 are emerging. a case series [73] reported five cases of gbs in italy, and four of them presented with lower-extremity weakness and paresthesia. patients developed symptoms five to 10 days after the onset of the viral infection. at electromyography, two patients had gbs and three acute motor axonal neuropathy [73] . another patient in iran [72] and an italian patient with the miller-fisher gbs variant [59] were also reported. in a 62-year-old patient presenting with motor weakness of the lower extremities and covid-19 symptoms, gbs associated with sars-cov-2 infection was observed a week later [76] . an increase in proteins (124 mg/dl) but not in cells was found in the csf, while at neurophysiological examination increased distal latencies and f-waves absence was detected, suggesting a severe and diffuse peripheral nerve demyelination. although the authors reported that the patient was infected by sars-cov-2 at the onset of gbs symptoms (because the patient had lymphopenia and thrombocytopenia), it cannot be excluded that covid-19 and gbs presented coincidentally [76] , and therefore further evidence is needed. some of the most frequently described non-specific symptoms are myalgia, unsteadiness, and fatigue. of note, 36.4% of 214 patients with covid-19 admitted in a wuhan hospital exhibited neurological manifestations [68] , which were categorized as "cns involvement" (24.8%), "peripheral involvement" (10.7%), and "muscular-skeletal involvement" (10.7%). among the latter, 15% of the non-severe patients reported myalgia, while 13.7% had elevated levels of creatine kinase. two cases of rhabdomyolysis (0.2%) were also described [61] . apart from a general unspecific reaction to the awareness of infection, some psychiatric illnesses might directly result from exposure to human covs. in patients with psychotic symptoms compared to non-psychiatric controls, a higher prevalence of immune reactivity for hku1 and nl63 covs was found [95] , suggesting that viral exposure may represent a comorbid risk factor in neuropsychiatric disease. however, the role that sars-cov-2 may play in the etiopathogenesis of psychiatric diseases needs to be explored. sars-cov entry into the human host cell seems to be mediated primarily by cellular receptors ace2, which are expressed in the lung, kidney, vascular endothelia, small intestine, and human airway epithelia [96] [97] [98] . conversely, mers-cov may enter human host cells primarily through the dipeptidyl peptidase-4 (dpp4) protein located in the membrane of cells of the immune system, liver, small intestine, and lower respiratory tract [99, 100] . however, ace2 or dpp4 alone are not enough to make the host cell susceptible to infections. this is particularly true when considering that sars or mers infections were also reported in the cns, where ace2 or ddp4 expression level is low under normal conditions [101] . the exact route through which sars and mers covs enter the cns is still not clear, although the glymphatic or a pure hematogenous path seems to be unlikely, particularly in the initial infection stage, during which no virus particle is detected in the brain [49, 102, 103] . however, some evidence indicates that covs might initially invade peripheral nerve terminals, and later the cns through a synapse-connected route [104] [105] [106] [107] . coherently, the sars infection seems to be able to cause significant neuronal damage without substantial inflammatory infiltration [43] . earlier studies have shown the presence of viral particles in the brain of patients with sars, located exclusively in the neurons [102, 103] . in vivo experiments using transgenic mice showed that, when sars or mers covs are given intranasally, they can enter the brain via the olfactory nerve, and quickly spread to specific brain regions, such as the brainstem and the thalamus [43, 108] . in these cases, the brain expresses ace2 receptors, that have been detected in neurons and glial cells, making them a potential target for covid-19. another important observation is that in mice infected with mers-cov with low inoculum doses, virus particles are not detected in the lung but only in the brain, which indicates that the cns infection is relevant for the high mortality of the disease [108] . however, although murine models develop cns infection, mers-cov has never been detected in the human cns, thus suggesting a different disease model [109] . viruses are present in the brain of patients with sars-covs [49] . baig et al. [24] have recently suggested a putative transcribral sars-cov-2 route to the brain and the presence of its rna in the csf would be the conclusive evidence to support the covid-19 neurovirulence. however, the pathomechanisms underlying the cns invasion seem to be more complex. a cns invasion can occur in both the initial and late phases of sars-cov-2 infection [24] . however, research is yet to determine the exact route for the entrance of the virus into the brain. a direct entry along the olfactory nerve can be considered among the potential mechanisms. in particular, the nasal olfactory epithelium is the probable site of enhanced binding of sars-cov-2. multiple non-neuronal cell types within the olfactory epithelium express two host receptors, ace2 and transmembrane protease serine 2 (tmprss2), that facilitate sars-cov-2 binding, replication, and accumulation. moreover, a subsequent brain infection beginning from the olfactory neurons might be considered, as well as the possibility that orns may initiate a rapid immune response at the early stages of the disease [110] . using a mouse model, bilinska et al. [111] determined whether cells in the olfactory epithelium expressed the receptors allowing the entry of the sars-cov-2 virus. they showed that ace2 and tmprss2 were expressed in the suss of the olfactory epithelium but not, or much less, in most olfactory receptor neurons, suggesting that suss are involved in sars-cov-2 virus entry and smell impairment. moreover, the expression of the entry proteins increased in older animals, thus possibly explaining, if verified also in humans, why older individuals are more susceptible to sars-cov-2 infection [111] . translationally, these preliminary findings suggest that damage to the olfactory epithelium may not only underlie clinical anosmia but also represent a preferential gate to the brain. namely, sars-cov-2 might spread via the transcribral route from the olfactory epithelium along the olfactory nerve to the olfactory bulb within the cns or spread retrogradely via transsynaptic transfer using an endocytosis or exocytosis mechanism and a fast axonal transport mechanism of vesicle transport moving the virus along microtubules back to neuronal cell bodies [78] . additionally, another possible transsynaptic route from the nasal respiratory epithelium to the brain via the trigeminal nerve branch has recently been hypothesized, although replication of the findings is needed [112] . the biological plausibility of the retrograde transsynaptic pathway from the peripheral nerve endings is based on the evidence that some covs appear to be capable of penetrating the cns through the cribriform plate of the ethmoid bone, even if the olfactory bulb is efficient enough to control viral invasion [113] . according to li et al. [113] , mechanoreceptors and chemoreceptors in the lung and respiratory tract can act as a possible retrograde pathway for sars-cov-2, as the nucleus of the solitary tract receives sensory information from these anatomical structures. indeed, a dysfunction of the cardiac-respiratory control centers in the medulla oblongata would aggravate the symptoms till death [113] . however, the neurogenic hypothesis of respiratory failure is not supported by other researchers [114] , as they argue that patients with covid-19 pneumonia do develop hypoxia and low co 2 levels accompanied by increased respiratory rate. while these patients can breathe spontaneously, they do it with great effort; thus, a respiratory failure resulting from a neurological origin would be characterized by a reduced respiratory rate, low oxygen levels, and high co 2 levels [114] . further virologic, histopathological, and immunohistochemical studies are necessary to demonstrate a specific neurotropism of sars-cov-2 for the brain respiratory control centers. the hypotheses behind sars-cov-2 transsynaptic propagation are further corroborated by other studies demonstrating that the virus may use a transsynaptic route for infecting the cns. one of the first pieces of evidence was provided in 1986 by gosztonyi [115] , who described the axonal transport of viral nucleic acid of some neurotropic viruses. in particular, he described that the rabies virus (rv) could be transmitted to other neurons by transsynaptic passage, without involving the complete virus replication, thus reaching various brain areas. li et al. [105] demonstrated that hev was able to propagate into cns via transsynaptic routes. namely, the peripheral inoculation of hev in both piglets and rodents results in encephalomyelitis via the primary motor cortex, where membranous-coating-mediated endo-/exocytosis events favor the hev transsynaptic transfer. in a recent review, taylor and enquist [116] deeply described the axonal route of propagation of several neuroinvasive viruses, including herpes simplex, varicella-zoster, pseudorabies, rhabdoviridae (including rv), flaviviridae, vesicular stomatitis (vsv), and theiler's murine encephalitis virus (belonging to the picornaviridae). they also described the mechanisms by which viruses were able to move in and out axons, both anterogradely or retrogradely, thanks to coupled or separate transports (mediated by vesicles or not, respectively) [116] . the anterograde and retrograde transsynaptic propagation lead researchers to adopt this viral feature for mapping the axon transports of neuronal impulses, viruses, and other factors. in this context, the transsynaptic transport of vsv has been used for tracing and mapping neuronal circuits by using specific virus-labeling techniques [117, 118] . of note, transsynaptic propagation is not a prerogative of cns viruses. for instance, the measles virus (mv) can reach the cns and cause subacute sclerosing panencephalitis, which is often fatal. the cns complications of mv infection are known to be the results of transsynaptic viral propagation thanks to the binding between mv envelope f protein and several host proteins, including hemagglutinin and neurokinin-1 [119] . taken together, these observations support the concept that some viruses, including respiratory viruses like the sars-cov-2, may propagate through the cns via a transsynaptic transport. the cell invasion of sars-cov-2 and its rapid replication seem to be supported by the ace2 receptor [120] . the damaging effects of angiotensin ii may be enhanced because of the depletion of the ace2 receptor on the cell membrane, which leads to an acute deterioration in lung function. therefore, the down-regulation of the ace2 receptor could put the hypertensive and diabetic population at higher risk for covid-19 due to the increase in angiotensin ii. a hypothesis related to this issue is that ace inhibitors, when used in patients with covid-19, can lead to an increased expression of ace2, thus probably making the cells more vulnerable to sars-cov-2 infection [120] . a study examining the risk factors for mortality in patients with covid-19 found that 40% of the deceased people presented single or multiple comorbidities, with high blood pressure being the most common (30%) [121] . the neurovirulence of sars-cov-2 could be related to the degree of expression of the ace receptor in the cns, although this receptor is expressed in endothelial cells, so it is necessary to further investigate its role in the etiopathogenesis of some neurological complications, such as stroke [23] . the viral s protein might allow the virus interaction in brain microcirculation with ace2 receptors expressed in the capillary endothelium, possibly leading to endothelial cells infection and subsequent spreading to the neurons after that the endothelial damage has occurred [24] . damage of the epithelial barrier by covs may occur, thus allowing the virus to reach the bloodstream or the lymphatic system and to spread to other tissues, including the brain. in this scenario, however, it is important to distinguish between the nasal olfactory epithelium and the nasal respiratory epithelium. while the former has been indicated as the main route for the trans-synaptic propagation of covs, the latter seems to be involved in the hematogenous propagation [78] . nevertheless, it is not well clearly understood how this could take place, although the bbb seems to be involved. two hypotheses have been proposed for the crossing of the bbb by sars-cov-2. the first mechanisms would involve the infection and transport across vascular endothelial cells, which express ace2 and, as such, are at risk for sars-cov-2 infection [78] . sars-cov-2 particles have been found in capillary endothelia and neurons of a frontal lobe specimen from an autopsy case study [122] . in particular, viral particles were packaged in intraneuronal dilated vesicles, and endocytosis or exocytosis of viral particles across endothelial cells were detected by electron microscopic imaging [78] . as soon as the virus enters vascular and neuronal cells, it might interact with ace2 on neurons, glia, and vessels, and then begin a cycle of viral budding, thus further damaging both vascular and neuronal tissue [24] . the second hypothesis is based on the so-called "trojan horse mechanism," through the infection of leukocytes that pass the bbb [35] . as lymphocytes, granulocytes, and monocytes all express ace2, the sars-cov might able to infect them [49, [123] [124] [125] , and it is likely that sars-cov-2 too may act in the same manner. moreover, the covid-19-related systemic inflammation would increase the bbb permeability, thus facilitating the invasion of the cns by the infected immune cells [126] . covid-19-related hypoxia may be responsible for indirect neuronal damage as it induces anaerobic metabolism in the cns cells, ischemia, interstitial edema, and vasodilatation in the cerebral circulation, which eventually causes stroke, syncope, and anoxic crisis [127] . the fact that covs can infect macrophages, astroglia, and microglia makes it possible for the host's immune-mediated response to playing a role. in some patients who died because of covid-19, a multiple organ failure and a hyperinflammatory syndrome (the "cytokine storm") were hypothesized as possible underlying causes [81] . in this context, a previous study in mice showed t-lymphocyte infiltration into the cns and significantly increased levels of the proinflammatory cytokine il-6 and the chemokine monocyte chemoattractant protein-1 after cov exposure [128] . finally, in genetically predisposed individuals, the persistence of covs in some cns resident cells cannot be excluded, where they would act as a cofactor of clinical exacerbations. serological techniques have identified covs in various neurological diseases, such as parkinson's disease, ms, and optic neuritis [129] [130] [131] [132] . therefore, it was proposed that a persistent cov infection might be a pathogenic factor in the development and course of some neurological diseases. for instance, infectious agents may play a triggering role in ms, with viruses being the most likely culprit in genetically predisposed individuals [133] . taken together, all the mechanisms discussed here might, at least in part, explain why and how sars-cov-2 could be moved in or within the cns despite the low expression of ace2 receptor in the brain. further studies are needed to better understand these pivotal aspects of neuroinfection. the research on the neurological manifestations of covid-19 has recently made significant progress, although the exact neuropathogenic mechanisms of sars-cov-2 are not yet completely clear. essential questions are emerging with the identification of people with covid-19 and cns involvement. although these patients may exhibit a wide range of neurological complications, it remains to be determined whether these can be an indirect and unspecific consequence of the pulmonary disease, hypoxia, or generalized inflammatory state on the cns, or if they may rather reflect a direct viral-related neuronal damage. some symptoms, such as headache and unsteadiness, are non-specific manifestations of several viral infections but, in some cases, they might accompany more severe diseases, such as meningitis, encephalitis, and stroke. a hematogenous versus a transsynaptic propagation is still debated, as well as the role of the ace2 receptor, the impact of hyperimmune response, and the viral persistence within some cns cells. the different levels and severity of human neurotropism and neurovirulence in patients with covid-19 might be explained by a combination of viral and host factors and their interaction. although researchers are yet to elucidate the real degree of neurovirulence of sars-cov-2, there is a demonstration of its presence in the csf or tissue samples at autopsy. however, in the current epidemic, some difficulties may occur in performing mri or a lumbar puncture, especially in severely affected patients and in those admitted in intensive care units. nevertheless, it remains of pivotal importance for all patients with altered consciousness or any unexplained neurological manifestation to receive an accurate neurological exam and appropriate instrumental investigations (i.e., neuroimaging, eeg, evoked potentials, csf), when necessary [134] . another warning related to this topic is that lymphopenia in immunosuppressed patients with covid-19 can be a serious risk factor; these are not only patients with cancer or with systemic autoimmune diseases but also patients with neurological disorders. indeed, taking high-dose corticosteroids or immunosuppressive/biological treatments is relevant for diseases like cerebral vasculitis, neuromyelitis optica, neurosarcoidosis, polymyositis, myasthenia gravis, or ms, and the scientific community should rapidly develop ad hoc guidelines for the management (especially in terms of reevaluation of dosages and treatment cycles) of these diseases during the covid-19 era. another relevant consideration concerns the possibility of developing anti-covid-19 drugs to cross the bbb and to selectively target the sars-cov-2 inside the brain. the role of the bbb needs to be further explored in patients with covid-19. hence, the possible neuroinvasion may be a significant mechanism to take into account for treating and preventing covid-19. in this context, the design of safe and effective brain penetrating drugs would be very helpful in preventing and possibly treating the neurological complications of covid-19, although the studies on this "cutting-edge topic" are still at their beginning [135] . the differences in the sequence of spike proteins between sars-cov and sars-cov-2 will enable scientists to identify epitopes in covid-19 patients for the development of monoclonal antibodies against this virus. basic research studies on sars-cov-2 and host interactions are the key to several unanswered questions in the prevention and control of the disease, including the challenging question of why not all patients with covid-19 show neuroinvasion and why, among those experiencing neuroinvasion, not all show neurotropism or neurovirulence [136] . moreover, the difference in terms of neurological involvement and pathomechanisms between the current pandemic and the sars and mers infections needs to be further studied. lastly, longitudinal neurological assessments of patients after their recovery will be crucial in the 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childhood multiple sclerosis: a review date: 2006-06-28 journal: ment retard dev disabil res rev doi: 10.1002/mrdd.20105 sha: doc_id: 329750 cord_uid: purunxce multiple sclerosis (ms) is an autoimmune demyelinating disorder of the central nervous system (cns) that is increasingly recognized as a disease that affects children. similar to adult‐onset ms, children present with visual and sensory complaints, as well as weakness, spasticity, and ataxia. a lumbar puncture can be helpful in diagnosing ms when csf immunoglobulins and oligoclonal bands are present. white matter demyelinating lesions on mri are required for the diagnosis; however, children typically have fewer lesions than adults. many criteria have been proposed to diagnose ms that have been applied to children, mostly above 10 years of age. the recent revisions to the mcdonald criteria allow for earlier diagnosis, such as after a clinically isolated event. however, children are more likely than adults to have monosymptomatic illnesses. none of the approved disease‐modifying therapies used in adult‐onset ms have been approved for pediatrics; however, a few studies have verified their safety and tolerability in children. although children and adults with ms have similar neurological symptoms, laboratory (cerebrospinal fluid) data, and neuroimaging findings, the clinical course, pathogenesis, and treatment of childhood onset ms require further investigation. mrdd research reviews 2006;12:147–156. © 2006 wiley‐liss, inc. m ultiple sclerosis (ms) was first described more than 170 years ago in adults. although rare, ms was recognized in children as early as 1922 [wechsler, 1922] . nevertheless, ms is still thought to be a disease of young adulthood, typically presenting between the ages of 20 and 40 years, and the diagnosis is rarely considered in children. physicians have questioned whether or not childhood ms is the same entity as seen in adults. in 1958, gall et al. published one of the earliest retrospective studies on pediatric-onset ms [gall et al., 1958] . between 1920 and 1952 , 40 children met inclusion criteria for the study. the patients demonstrated neurological signs and symptoms due to scattered lesions within the cns separated by time and space and supported by objective evidence. the study concluded that children and adults with ms have similar clinical profiles, including mode of onset, symptoms, and physical and laboratory (cerebral spinal fluid [csf]) findings. nevertheless, diagnosing ms in children is often difficult and controversial. the estimated prevalence of ms worldwide is 50 per 100,000 with 2.7-5.6% of patients presenting before the age of 15-16 years [ sindern et al., 1992; gadoth, 2003 ]. the calculated frequency of childhood-onset ms is 1.35-2.5 per 100,000 [gadoth, 2003] . ms has been diagnosed during infancy and early childhood (younger than 10 years of age) accounting for 0.2-0.7% of all cases [ruggieri et al., 1999] . there are reports of children presenting before the age of 2 years, even as early as 13 months [cole et al., 1995] . as seen in the adult population, there is a female predominance in childhood ms ranging from 2.1-3:1 [gall et al., 1958; duquette et al., 1987] . the presenting symptoms of ms in children are similar to those reported by adults. in 1987, duquette et al. reviewed 125 pediatric patients with ms who presented most commonly with either pure sensory symptoms or optic neuritis [duquette et al., 1987] . diplopia, pure motor symptoms, abnormal gait including ataxia (cerebellar or vestibular), mixed sensory and motor symptoms, and sphincter disturbances were also reported. in 1992, sindern et al. identified 31 patients with ms using poser's criteria (see diagnosis section) who presented before the age of 16 years and compared them to 72 sex-matched control patients diagnosed with ms between the ages of 20 and 40 years [sindern et al., 1992] . the most common finding at the onset of disease for both children and adults was optic neuritis, accounting for 52% and 40%, respectively. the second most common presenting symptom in children was sensory disturbance, seen in 16% of children and 15% of adults. transverse myelitis was more common in children, whereas motor symptoms were more common in adults (18%) than in children (6%). furthermore, in 71% of children, the initial presentation was rapid, resulting in admission to the hospital within a few hours to days. a longitudinal study by boiko et al. confirmed duquette's and sindern' s findings that sensory symptoms and optic neuritis were the most common initial manifestations in patients with the clinical onset of ms before the age of 16 years [boiko et al., 2002] . in 1995, poser et al. characterized the presentation of ms in adults (table 1) [poser, 1995] , and the diagnosis of ms should be considered in children presenting with similar symptoms. the clinical course of ms is divided into four subtypes: relapsing-remitting (rrms), primary progressive (ppms), secondary progressive (spms), and progressive-relapsing (prms). rrms is the most common subtype in both adults and children. there are no diagnostic tests for ms. however, a lumbar puncture is routinely performed to obtain supportive evidence of cns inflammation. in approximately 60% of patients with childhoodonset ms, the routine analysis (cell count, protein, and glucose) of csf is normal [duquette et al., 1987; dale et al., 2000] . the remainder of patients has a lymphocytic pleocytosis (typically ͻ50 cells/mm 3 ) and/or elevated protein (typically ͻ75 mg/dl) [dale et al., 2000] . intrathecal synthesis of immunoglobulin (ig), predominantly igg, is also seen in patients with ms. approximately 80% of children with ms have increased csf igg synthesis [jones, 2003] . furthermore, oligoclonal bands (ocb), markers of antibody synthesis in the cns, are present in about 85-95% of adult patients with ms [olek and dawson, 2004] . in children, ocb were present in 40 -87% of patients and may appear later during disease convalescence or relapse [sindern et al., 1992; selcen et al., 1996; dale et al., 2000; jones, 2003 ]. ocb are not specific to ms [poser, 1983; olek and dawson, 2004] . they can be found in chronic cns infections, such as subacute sclerosing panencephalitis, viral infections of the cns, autoimmune neuropathies, cervical myelopathies, and cns tumors [ cohen et al., 2000] . magnetic resonance imaging (mri) reveals asymmetric, multifocal white matter lesions on t2-weighted sequences and fluid-attenuated inversion recovery (flair) images [miller et al., 1990] . the lesions are most commonly located in the periventricular and subcortical white matter where they appear ovoid with extensions called dawson fingers [barkhof et al., 1997] . additional lesions can be seen in the cerebellum, spinal cord, basal ganglia, and thalami [dale et al., 2000] . new lesions may enhance with gadolinium administration. there are no longitudinal mri studies in childhood ms to establish whether there is progressive atrophy of the brain or the appearance of "black holes" (chronic inactive lesions). furthermore, unlike in adults, diffusion tensor imaging (dti) and magnetization transfer ratios (mtr) have not been systematically performed. finally, magnetic resonance spectroscopy (mrs) shows similar changes to those reported in adult ms patients with decreases in nacetyl aspartate (naa) reflecting neuronal loss, increases in choline reflecting remyelination, and increases in myoinositol reflecting gliosis [wolinsky and narayana, 2002] . ms remains a clinical diagnosis. in 1983, poser et al. published guidelines incorporating laboratory, neuroimaging, and neurophysiologic data into the diagnostic criteria with four proposed subtypes: clinically definite ms, laboratory-supported definite ms, clinically probable ms, and laboratory-supported probable ms (see table 2 ) . in 2001, the mcdonald criteria were introduced to facilitate and simplify the diagnosis of ms for patients between 10 and 59 years [mcdonald et al., 2001] . the authors further defined mri criteria and included both monosymptomatic disease and ppms in the clinical presentations. caution was suggested in applying these guidelines to children younger than 10 years. in fact, the sensitivity in diagnosing pediatric cases was questioned by a second panel that revised the mc-donald criteria in 2005 (see table 3 ) [polman et al., 2005] . furthermore, hahn et al. reported that many pediatric patients did not meet the mcdonald mri criteria for dissemination in space (see table 4 ) [hahn et al., 2004] . demonstrating dissemination in time (see table 4 ) is also challenging in pediatrics due to the possibility of relapses in a monophasic disease (see differential diagnosis section). nevertheless, a repeat mri performed three months after the initial study is recommended to show dissemination in time. acute disseminated encephalomyelitis (adem), multiphasic disseminated encephalomyelitis (mdem), and ms share similar clinical presentations, laboratory data, and neuroimaging abnormalities. subtle differences between the [dale et al., 2000; hynson et al., 2001; stonehouse et al., 2003 ]. in addition, hepatitis b; measles, mumps, rubella (mmr); bacille calmette-guérin (bcg); meningitis a and c; rabies; influenza; smallpox; and japanese b encephalitis vaccines, given within the six weeks prior to the onset of adem, have been suspected in triggering an autoimmune response [dale et al., 2000] . clinically, adem is more likely to present with ataxia, encephalopathy, bilateral optic neuritis, and seizures [hynson et al., 2001] . children typically have a polysymptomatic presentation with sensory, pyramidal, cerebellar, and bulbar symptoms [dale et al., 2000] . headache, fever, meningismus, and vomiting are more often associated with adem [brass et al., 2003] . unilateral optic neuritis and internuclear ophthalmoplegia are more common in ms [dale et al., 2000] . in adem and ms, the csf can be normal, although many patients have a lymphocytic pleocytosis or elevated protein. in adem, the csf white blood cell (wbc) count can be as high as 270 cells/mm 3 , with a mean around 51 cells/ mm 3 . in ms, the cell count is lower (range, 0 -130 cells/mm 3 ; mean, 18 cells/ mm 3 ) [dale et al., 2000] . the csf protein varies from 0.1 to 3.3 g/dl (mean, 0.69 g/dl) and 0.2 to 0.99 g/dl (mean, 0.38 g/dl) in adem and ms, respectively [dale et al., 2000] . ocb are seen in the csf in more than half of patients with childhood ms but can be seen in adem [dale et al., 2000; brass et al., 2003] . with considerable overlap between clinical and laboratory findings, mri is an important tool in determining the difference between adem and ms. both can affect the periventricular, sub-cortical, and deep white matter; deep gray matter; brainstem; cerebellum; and spinal cord. cortical white matter lesions are typically bilateral but asymmetric. in adem, lesions are less likely to be periventricular. also, adem more com1 yr of disease progression (retrospectively or prospectively determined) and two of the following: a) positive brain mri (9 t2 lesions or 4 or more t2 lesions with positive vep) f b) positive spinal cord mri (two focal t2 lesions) c) positive csf d note: if criteria indicated are fulfilled and there is no better explanation for the clinical presentation, the diagnosis is ms; if suspicious, but the criteria are not completely met, the diagnosis is "possible ms," if another diagnosis arises during the evaluation that better explains the entire clinical presentation, then the diagnosis is "not ms." a an attack is defined as an episode of neurological disturbance for which causative lesions are likely to be inflammatory and demyelinating in nature. there should be subjective report (backed up by objective findings) or objective observation that the event lasts for at least 24 hr. b no additional tests are required; however, if tests (mri, csf) are undertaken and are negative, extreme caution needs to be taken before making a diagnosis of ms. alternative diagnoses must be considered. there must be no better explanation for the clinical picture and some objective evidence to support a diagnosis of ms. c mri demonstration of space dissemination must fulfill the criteria derived from barkhof et al. [1997] and tintoré et al. [2000] as presented in table 4 . d positive csf determined using ocb detected using established methods (isoelectric focusing) different from any such bands in serum, or using an increased igg index. e mri demonstration of time dissemination must fulfill the criteria in table 4 . f abnormal vep of the type seen in ms. abbreviation: vep, visual-evoked potential. three of the following are required for demonstrating dissemination in space 1. at least one gadolinium-enhancing lesion or nine t2 hyperintense lesions if there is no gadolinium-enhancing lesion 2. at least one infratentorial lesion 3. at least one juxtacortical lesion 4. at least three periventricular lesions there are two ways to show dissemination in time: 1. detection of gadolinium enhancement at least three months after the onset of the initial clinical event, if not at the site corresponding to the initial event 2. detection of a new t2 lesion if it appears at any time compared with a reference scan done at least 30 days after the onset of the initial clinical event note: a spinal cord lesion can be considered equivalent to a brain infratentorial lesion, an enhancing spinal cord lesion is considered to be equivalent to an enhancing brain lesion, and individual spinal cord lesions can contribute together with individual brain lesions to reach the required number of t2 lesions. based on data from barkhof et al. [1997] and tintoré et al. [2000] . monly affects the thalami and basal ganglia, with a greater tendency for symmetry in the latter [dale et al., 2000] . in adem, a repeat mri scan performed more than two months after the onset of symptoms often shows partial or complete resolution of lesions with no new lesions. enhancement after the administration of gadolinium can be seen on the initial scan; however, no lesions enhance on the follow-up mri in adem. in ms, both new and enhancing lesions may be present when the scan is repeated, although the time to develop new lesions is unpredictable. in the absence of clinical symptoms, new findings on mri are useful in differentiating ms from adem. mdem presents a challenging dilemma in diagnosing childhood-onset ms. the clinical presentation, laboratory data, and neuroimaging features of mdem resemble adem, both of which are monophasic illnesses. however, patients with mdem have a clinical relapse after their initial illness or develop new lesions on mri, suggestive of a chronic demyelinating disease or ms. despite the presence of new lesions on mri, suggesting dissemination in time, some investigators believe that mdem and ms are separate entities. a diagnosis of mdem should be reserved for patients whose relapses are caused by the same trigger responsible for the inciting event and occur shortly after presentation or within two months of discontinuing steroids [dale et al., 2000] . ms is a neurodegenerative disease that affects young adults and children, often women. linkage and twin studies demonstrate that individuals carry a genetic susceptibility to this disease [rice, 2004] . a susceptibility locus for ms has been identified on chromosome 6, specifically the major histocompatability complex (mhc) class ii alleles human leukocyte antigens (hla) dr15 and dq6. this association is seen in all populations. in sardinians, there is an additional association with dr4, and, in turks, there is an association with dr2 and dr4. in finns, there is an association of ms with myelin basic protein (chromosome 18); however, neither this association nor an association with any other myelin genes has been noted in non-finnish populations [kenealy et al., 2003] . aside from the mhc locus, other regions of interest identified from the united kingdom study for ms susceptibility are located on chromosomes 1, 5, 6p, 7p, 14q, 17q, 19q, and xp [chataway et al., 1998] . some of the genes in these regions include tumor necrosis factor [tnf]␣, interleukin [il]-1ra, il-4, and cytotoxic t-lymphocyte-associated protein 4 (ctla-4). aside from the genetic predisposition for ms, epidemiological data indicates that an environmental factor also plays a role [compston, 2003] . for some time, an infectious agent has been suspected in triggering an autoimmune response. this theory was supported by apparent epidemics that occurred in the faroe islands and iceland following world war ii [rice, 2004] . additional support for an infectious etiology was provided by further studies that showed elevated antiviral titers (measles, rubella, mumps, varicella/zoster, ebv, influenza/parainfluenza,coronavirus, htlv-1, borna, etc) in the csf of ms patients during an acute exacerbation [ sibley et al., 1985; panitch, 1994] . presumably, the elevated titers represent nonspecific activation of b cells in the nervous system. in addition, the ms literature is replete with the isolation of viruses from the brains of patients with ms including measles, coronavirus, retroviruses, htlv-1, hhv-6, and scrapie agent. current focus on infectious agents includes ebv, hhv-6, endogenous retroviruses such as herv-w, and chlamydia pneumoniae [johnson and major, 2003] . oldstone postulated that an environmental trigger activates the immune system by "molecular mimicry" in which an infectious agent has sequence homology to a myelin protein. following the infection, tolerance is broken and an immune response ensues with the appearance of autoreactive t cells (cd4 and cd8) [oldstone, 1998] . alternatively, the pathogen activates toll receptors that then initiate the cellular immune response with the production of il-12 and il-23 [vasselon and detmers, 2002; frohman et al., 2006] . the earliest pathological change seen in an ms lesion is oligodendrocyte apoptosis with microglial activation but lacking infiltrating lymphocytes [barnett and prineas, 2004; matute and pérez-cerdá, 2005] . older lesions have perivascular infiltration by lymphocytes, plasma cells, and macrophages; loss of myelin and oligodendrocytes; axonal damage; and reactive astrocytes. chronic lesions are sharply demarcated with a hypocellular center and axonal loss, perivascular infiltration by lymphocytes, and increased number of oligodendrocytes. in chronic silent lesions, there is a loss of axons and oligodendrocytes. lucchinetti et al. have grouped the neuropathological lesions into four types, each containing t cells [lucchinetti et al., 1996 [lucchinetti et al., , 1999 [lucchinetti et al., , 2000 . type 1 is characterized by a predominance of macrophages, type ii by the deposition of immune complexes, type iii by oligodendrocyte malfunction, and type iv by oligodendrocyte death. there is insufficient data to describe the pathology of ms in children. ms is an organ-specific autoimmune disease mediated by type 1 helper t cells (t h 1) that recognize components of myelin and induce an inflammatory process by recruiting other inflammatory cells such as macrophages. in patients with ms, myelin-reactive t cells found in the blood stream produce a cytokine profile consistent with t h 1 cells. in demyelinating lesions, t h 1 cytokines, such as interferon ␥, tnf-␣, and il-2, are expressed by these leukocytes. the chemokine profile also suggests a t h 1-mediated inflammatory process. nevertheless, ms is likely to be more than a purely t h 1-mediated disease because it is likely that cd4 cells, macrophages, b cells, and a paucity of regulatory t cells also play a role [merrill, 1992; sorensen et al., 1999; frohman et al., 2006] . therapy in ms targets four different aspects of a child's illness. first, disease-modifying drugs, or immunomodulators (id), are used to alter the biological activity of the disease, thereby preventing neurological disability. second, additional medications help alleviate symptoms such as fatigue, spasticity, bladder dysfunction, and depression. third, neuroprotective agents are being studied to prevent and repair nerve injury. finally, rehabilitation is needed to overcome physical handicaps. disease modifying, symptomatic, and neuroprotective therapies will be described in this review. in evaluating effectiveness of therapies that modify the biological activity of the disease in children, a major challenge is the inability to predict the outcome of the disease and the lack of good outcome measures. the goal of any disease-altering therapy is to prevent longterm disability which evolves over many years [goodin et al., 2002] . the efficacy of the newer therapies has predominantly been studied over a short time period. moreover, the expanded disability status scale (edss) that is used as an outcome measure in adult studies has not been validated for use in children. children with ms may have cognitive dysfunction, which has not been evaluated as an outcome measure, although the ms functional composite (msfc) places some weight on mental functioning. once again, the utility of this scale has not been established in children. currently, most studies use the short-term attack rate as an outcome measure as well as mri data to assess t2 disease burden, cerebral atrophy, and the appearance of t1 black holes. although there are very few trials that have included children, in this article we review therapies that are recommended for adults and, where data is available, highlight the pediatric studies. glucocorticoids, such as intravenous (iv) methylprednisolone, are the mainstay of treatment for acute attacks or relapses in ms [goodin et al., 2002] . they suppress the immune system in many ways, such as altering cytokine profiles, inhibiting the synthesis of matrix metalloproteinases, and reducing csf antibodies to mbp and ocb [kupersmith et al., 1994] . in 1970, a multicenter trial compared adrenocorticotropic hormone (acth) (80 u/day given intramuscularly [im] for four days with a 7-day taper) against placebo in 197 patients with acute ms [rose et al., 1970] . after four weeks, the authors found that acth accelerated clinical improvement, although there was no significant difference in the outcome. in another study, acth (80 u/day for one week followed by a taper) was compared with 1 g of iv methylprednisolone for three days. in this study, there was no significant difference between the two treatment arms [thompson et al., 1989] . subsequently, a number of studies have been published using glucocorticoids for optic neuritis, most notably the optic neuritis treatment trial. this multicenter study compared iv methylprednisolone for three days followed by oral prednisone for 11 days against a 14-day course of oral prednisone and a placebo group. for both primary (visual fields and contrast sensitivity) and secondary (visual acuity and color vision) endpoints, the group that received iv methylprednisolone had an accelerated recovery of visual function compared to the placebo group. the rate of recovery for the group receiving oral prednisone was in between the iv and placebo groups. at six months, there was no difference between the treated and the placebo groups [beck, 1988] . furthermore, the group receiving oral steroids had an increased number of recurrences of optic neuritis. in addition to their use in optic neuritis, high-dose ste-roids are also known to enhance the resolution of gadolinium-positive mri lesions [barkhof et al., 1991; burnham et al., 1991] . finally, abrupt discontinuation of steroids can lead to severe clinical, radiographic, and histopathologic relapses; therefore, an oral taper is recommended. although these studies were performed in young adults with rrms and cis, iv steroids (15-30 mg/kg/day given daily for 3-5 days followed by an oral taper over 14 days) are used in children with acute attacks that impair function. interferons (ifn␤-1a and ifn␤-1b) are recombinant proteins, which inhibit the adhesion and the migration of wbc across the blood-brain barrier, thereby blocking antigen presentation and the synthesis and transport of matrix metalloproteinases [harris and halper, 2004] . in addition, they may cause a shift from a t h 1 to a t h 2 response. in adultonset ms, ifn-␤ has a beneficial effect on the clinical and radiological outcome measures. because the drug is not marketed for the pediatric population, there are no recommendations available for dosing children. for older children and adolescents, adult doses are most often used. interferon ␤-1a (inf␤-1a) is available in a weekly im injection (avonex, 30 g) or a subcutaneous (sc) injection given three times a week (rebif, 22 g or 44 g). interferon ␤-1b (inf␤-1b, betaseron, 8 million international units (miu) or 250 g) is given sc every other day. for smaller teens or children younger than 10 years, the doses are often adjusted to minimize adverse events and increase tolerability, such as starting with a half-dose of avonex or betaseron or using the lower dose for rebif. in 2006, banwell et al. retrospectively studied dosing, safety, and tolerability of ifn␤-1b in 43 children diagnosed with ms who had been treated for an average of 29.2 months [banwell et al., 2006] . treatment was initiated at full dose (8 miu or 250 g) in 15 children, all of whom were older than 10 years of age. younger children were started at 25-50% of the full dose and slowly increased; two children, both under the age of 10 years, were unable to tolerate the dose escalation. none of the children had any serious adverse events. therapy was discontinued in 25 of 43 patients after being treated for a mean duration of 111 weeks for various reasons, such as perceived lack of efficacy, cost of medication, lack of adherence, injection pain, and change in diagnosis. nevertheless, of the 38 patients with confirmed ms, the annualized relapse rate was reduced by a mean of 50%. the side effects of inf␤ in children are similar to those reported by adults. fever is the most common side effect, reported in 50% of the patients [ghezzi et al., 2005] . additional side effects include headache, myalgia, flu-like symptoms, injection site reactions, fatigue, nausea, and asthenia [waubant et al., 2001; banwell et al., 2006] . the majority of these symptoms are transient. to alleviate side effects, children may be pretreated with acetaminophen, ibuprofen, or naproxen. laboratory abnormalities, such as elevations of liver function tests, can also occur. when present, a temporary discontinuation of the medication is recommended. often, the inf␤ can be restarted without a recurrence of the elevated transaminases [banwell et al., 2006] . ga is a random polypeptide composed of four amino acids (l-glutamic acid, l-lysine, l-alanine, and l-tyrosine) resembling myelin basic protein (mbp). this drug has a number of effects on the immune system including inhibition of antigen presentation, competition and displacement of bound mbp, conversion of cd4 t cells from t h 1 to t h 2 type cells, and induction of brain-derived neurotrophic factor (bdnf) expression [teitelbaum et al., 1992; neuhaus et al., 2001; aharoni et al., 2003; azoulay et al., 2005] . it also induces antigen-specific suppressor t cells which release anti-inflammatory cytokines thereby generating tolerance to self-antigens [harris and halper, 2004] . there are no trials similar to those conducted in adults that have primarily focused on the efficacy of this drug in children with ms. there are, however, reports of using this drug in children who were given 20 mg sc daily, the standard dose for an adult. in one child treated with ga, chest pain was reported; however, no other clinical or laboratory abnormalities were identified [ghezzi et al., 2005] . although inf␤ and ga have been used in practice, the long-term tolerability, side effects, and overall efficacy in the pediatric population is not yet known. in a multicenter italian study published in 2005, ghezzi et al. focused on effectiveness and tolerability of interferons and glatiramer acetate in patients treated before the age of 16 [ghezzi et al., 2005] . sixty-five cases were reviewed. the majority was treated with avonex (38), followed by rebif (18), betaseron (16), and copaxone (9). relapses were defined as the occurrence of new symptoms lasting more than 24 hr with objective findings of cns involvement in a previously unaffected patient or the acute worsening of preexisting symptoms lasting more than 24 hr and causing an increase of at least 1 on the edss. all four of the drugs substantially reduced the relapse rate with combined data showing a decrease from 2.8 to 0.5 relapses per year and similar results for the individual medications. the change in edss was not significantly different when comparing the first and last visit in the inf␤ subgroups; however, a statistically significant difference was seen in the ga subgroup (baseline: 1.1 ϯ 0.5, posttreatment: 0.6 ϯ 0.5, p ϭ 0.007). it should be noted that the patients on ga had overall lower disease duration when compared to the other groups and edss at entry was lower than that in the avonex and rebif/betaseron groups. natalizumab is a recombinant monoclonal antibody directed against ␣4-integrin. in experimental autoimmune encephalitis (eae), the animal model for ms, the expression of t-cell surface receptors (integrins) promotes adhesion and transport of these cells through capillary endothelial cells. this antibody against ␣4-integrin blocks the adhesion of activated t lymphocytes to endothelial cells thereby preventing these cells from entering the nervous system. this is the only selective immunomodulating drug for the treatment of ms. the results from the natalizumab safety and efficacy in relapsing remitting multiple sclerosis (affirm) and safety and efficacy of natalizumab in combination with interferon ␤-1a in patients with relapsing remitting multiple sclerosis (sentinel) studies in adult patients indicate that the annualized rate of clinical relapses was reduced by 68%, the number of new and enhancing mri lesions was reduced by 83%, and a decrease occurred in progression and prolongation of the interval before neurological deterioration, demonstrating the usefulness of the drug [polman et al., 2006; rudicket al., 2006] . although natalizumab had significant short-term beneficial effects, unfortunately, three patients who received this drug developed progressive multifocal leukoencephalopathy (pml). the relative risk of developing pml in ms patients on natalizumab is 1 in 1,000 [ropper, 2006 ]. moreover, the use of this drug may have other long-term effects, such as unmasking latent viral infections as well as other diseases that are dampened by immune surveillance. in children who have a malignant course of ms, the use of this drug on a short-term basis may be warranted. campath-1h binds cd52 antigen, which is present on the surface of all b and t lymphocytes, as well as some monocytes. it is a lympholytic antibody that has been shown to prevent relapses and the formation of new mri lesions in ms; however, it does not seem to have any effect on disease progression [paolillo et al., 1999] . furthermore, when campath-1h was initially used in patients with ms, a transient worsening of symptoms occurred due to the release of cytokines and nitric oxide (no) [moreau et al., 1996] . in vitro studies demonstrated that no can cause conduction blocks that could account for the transient worsening of symptoms with treatment initiation. pretreating with steroids can avert the cytokine release. rituximab is a humanized monoclonal antibody directed against cd20 and antigens found on b lymphocytes [valentine et al., 1989 ]. b-cell proliferation, as well as an increase in the mutations of their receptors, has been shown in the csf of ms patients. the b-cell response reflects the presence of a specific antigen in the cns. thus, the b cells have become another therapeutic target in ms. rituximab, a drug that depletes b cells, is currently being investigated in the treatment of ms [reff et al., 1994; frohman et al., 2006] . mitoxanthrone is an anticancer drug that acts by intercalating into dna thereby producing dna strand breaks and interstrand crosslinking. in the immune system, it causes the elimination of lymphocytes and reduction of t h 1 cytokines. the major side effects include cardiac toxicity, presenting as a cardiomyopathy with irreversible congestive heart failure, and increased risk of developing malignant tumors. nevertheless, this drug reduced the attack rate of patients with rrms by 66%, reduced the number of gadolinium-enhancing and new lesions on the mri, and reduced the clinical rate of progression of the disease [millefiorini et al., 1997] . given the toxicity profile, this is not a first-line drug for the treatment of ms in children. cyclophosphamide is a powerful immunosuppressive agent that has been used to treat relapsing-remitting and progressive forms of ms. side effects include alopecia, nausea and vomiting, hemorrhagic cystitis, sterility, and long-term risk of malignancy. the use of iv cytoxan (400 -500 mg/day with wbc counts about 4,000 per microliter) did not show any benefit for patients with progressive ms at 1-and 2-year follow-up after the initiation of therapy [hauser et al., 1983; likosky et al., 1991] . in a canadian study using 1,000 mg of cytoxan with a 3-year follow-up of patients with progressive ms, there was no significant benefit from use of this drug [canadian cooperative ms study group, 1991] . nevertheless, in a study of 256 patients with progressive ms, younger patients derived some benefit from the use of cytoxan [weiner et al., 1993] . methotrexate acts as a folate antagonist, thereby affecting dna synthesis in immune cells. it decreases proinflammatory cytokines and enhances suppressor t-cell function. the major side effects are nausea, headache, diarrhea, liver damage, and the risk of developing non-hodgkin's lymphoma. a small, doubleblinded study of low-dose methotrexate revealed a benefit for patients with rrms but not for patients with the progressive forms of the disease [currier et al., 1993] . however, in another study of 60 patients with chronic progressive ms, low-dose methotrexate was found to be beneficial and showed a reduction in the t2 diseased burden [goodkin et al., 1995] . azathioprine is an analog of 6-mercaptopurine that inhibits purine synthesis, thereby impairing dna and rna synthesis in b cells, t cells, and macrophages. its side effects are anemia, lymphopenia, alopecia, liver dysfunction, pancreatitis, reactivation of latent infections, and the risk of developing malignancies. in a retrospective analysis of seven studies that had enrolled 793 patients, use of imuran reduced the number of relapses; however, the drug did not seem to affect the course of patients with progressive ms or their disability [yudkin et al., 1991] . cyclosporine is a potent immunosuppressive agent that selectively inhibits helper t cells. side effects include hirsutism, headaches, nausea, hypertension, edema, paresthesias, nephrotoxicity, and abdominal pain and discomfort. studies conducted in london and amsterdam showed no benefit on the relapse rate but did show some effect on slowing the progression of the disease [rudge et al., 1989] . given the side effects of this drug, its use in ms is very limited [goodin et al., 2002] . cladribine, an adenosine deaminase-resistant purine nucleoside, is a potent immunosuppressive drug that is selective for lymphocytes. side effects include nausea, diarrhea, fever, fatigue, and leukopenia. although cladribine does not have a significant effect in reducing the relapse rate, it may slow the degree of disability. in addition, it reduces the appearance of gadolinium-enhancing lesions on mri [beutler et al., 1996; rice et al., 2000] . 3-hydroxy-3-methylglutaryl coenzyme a (hmg-coa) reductase inhibitors, also called statins, have been recently studied in a variety of cns disorders, including ms. statins disrupt the activation of proinflammatory t-cells by inhibiting signals from mhc class ii molecules [neuhaus et al., 2002] . they also decrease migration of leukocytes into the cns, expression of inflammatory mediators by t-lymphocytes and in the cns [stüve et al., 2003] . statins, such as simvastatin (zocor) and atorvastatin (lipitor) have been shown to inhibit and reverse chronic and relapsing eae [stüve et al., 2003] . atorvastatin induces stat6 phosphorylation and enhances the secretion of t h 2 cytokines (il-4, -5, and -10 and transforming growth factor [tgf] ␤) while inhibiting stat4 phosphorylation and secretion of t h 1 cytokines (il-2, -12, ifn-␥, and tnf␣) [youssef et al., 2002] . in small, shortterm studies, zocor decreased the number and size of gadolinium-positive lesions on mri scans without effect on progression and disability [vollmer et al., 2004] . the immunomodulatory effects of the statins offer promise in the treatment of ms, and their usefulness is being further investigated [neuhaus et al., 2004] . vaccination therapies are currently being developed that would alter the treatment of ms. vaccinations that promote the development of tolerance have been effective in eae [robinson et al., 2003 ]. in addition, t cell and t cell receptor peptide vaccinations have been studied in humans with ms [correale et al., 2000; bourdette et al., 2005] . none of the vaccines have been studied in children. iv immune globulin (ivig) blocks fc receptors on macrophages, alters the cytokine profile, and has antiidiotypic effects. ivig is typically used as an adjunct for acute relapses; however, its recurrent use has been studied in rrms. in a multicenter, double-blind, placebo-controlled study of 148 rrms patients given ivig (0.125-0.2 g/kg) monthly for two years, a reduction in the clinical attack rate (ϫ49%) with a possible reduction in the degree of disability (not significant) was observed [fazekas et al., 1997] . in a separate study, the number of total and enhancing lesions seen on mri was decreased by more than 60% in patients treated with ivig compared with placebo [sorenson et al., 1998 ]. thus, it appears that ivig may reduce the attack rate in rrms but probably has little effect in slowing the progression of the disease. although it does not alter the long-term course in ms, plasma exchange has been used to treat acute relapses, presumably by removing harmful antibodies. several groups have investigated this particular therapeutic modality for treatment of patients with progressive ms [hauser et al., 1983] . for some patients who had not responded to iv steroids, plasma exchange performed every other day for a total of 14 days provided a greater degree of improvement when compared with a sham-treated group [weinshenker et al., 1999] . some patients receiving plasma exchange improve very rapidly, which is unlikely due to the repair of the injured tissue. instead, the rate of recovery may be due to the rapid shifts in electrolytes that result in improved axonal conduction or the possible removal of an antibody that affects transmission of electrical impulses. although fatigue is a common and debilitating symptom is adults, children rarely complain of this symptom. the mechanism for fatigue is multifactorial and includes depression, excessive effort due to muscle weakness or spasticity, re-lease of cytokines, and sleep disturbance. therapies for fatigue in ms include the use of amantadine, modafanil, and pemoline. all have been shown to have modest beneficial effect in adults. when patients have involvement of the corticospinal tracts, whether it be due to lesions in the spinal cord or higher, treatment should include physical therapy, splints to prevent contractures, and stretching exercises combined with pharmacological treatments, such as diazepam (valium), tizanidine (zanaflex), baclofen (lioreseal), and dantroline (dantrium). less well established is the use of tetrahydocannabinol. for contractures that do not respond to stretching, alternatives include serial casting, botox injections, and tenotomy. in more severe cases, a baclofen pump, or rhizotomy or myelotomy, may be considered. hemiplegia in children is disabling, particularly because of the loss of dexterity. sensory impairment further aggravates movements of the hand. such children do not use the affected hand, which results in learned nonuse of that hand. recent studies indicate that such children benefit from intensive practice and forced use; restraint of the noninvolved arm appears to improve function of the affected hand, probably due to functional reorganization of the nervous system. patients with ms have a variety of paroxysmal symptoms that last seconds to minutes and are not associated with alterations in consciousness or any electroencephalogram correlate for seizure. paroxysmal sensory symptoms and motor symptoms, such as ataxia and lhermitte's sign, respond to low doses of carbamezapine, phenytoin, and acetazolamide. heat-sensitive symptoms can respond to potassium channel blockers with the caveat that these drugs can induce seizures. this is not an uncommon symptom in some children. nonsteroidal antiinflammatory agents are recommended. if they are not sufficient, gabapentin (neurontin), carbamezapine (tegretol), or amitriptyline (elavil) can be beneficial. in ms, axonal injury occurs early in the course of the disease with eventual transection of axons. factors that have been associated with axonal injury are cytokines, no, superoxide radicals, proteases, cd8 t cells, cholesterol breakdown products, abnormal expression of sodium channels and function of the sodium-calcium exchanger, and glutamine excitotoxicity [waxman et al., 2004] . when an axon is demyelinated, there is abnormal expression of voltagegated sodium channels with increased influx of sodium in an attempt to restore conduction. to compensate for this, there is a reversal of the sodium/calcium exchanger with efflux of sodium and an influx of calcium. this could result in calcium-mediated neuronal degeneration. this hypothesis has received some support from work on eae models where sodium channel blockers, such as flecainide and phenytoin, help preserve axons [lo et al., 2003; bechtold et al., 2004] . in patients with ms, mrs has demonstrated increased glutamate concentration, providing the underpinning for considering glutamate excitotoxicity. the increased glutamate could result from a decrease in glutamate transporters in glial cells and elevation of glutaminase, a glutamate-synthesizing enzyme, in microglia [werner et al., 2001] . however, increased glutamate acting through the ␣-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (ampa) and/or nmethyl-d-aspartate (nmda) receptors, which are present on neurons and oligodendrocytes, can result in calcium-mediated cell death. riluzole, a glutamate antagonist that has been used in infants with spinal muscular atrophy, blocks nmda and sodium channels and reduces the number of t1-weighted hypointense lesions on the mri scans of patients with ms [frohman et al., 2006] . because axonal damage is a feature of ms, promoting neurite outgrowth could be beneficial. however, axonal sprouting is inhibited by activation of the nogo receptor by agonists such as nogo, oligodendrocyte-myelin glycoprotein (omgp), and myelin-associated glycoprotein (mag). thus, blocking the nogo receptor could represent a therapy that would be of value in promoting axonal sprouting [wang et al., 2002] . in acute ms plaques, there is clearcut evidence for remyelination; however, this is minimal in chronic lesions. the recruitment of oligodendrocyte precursor cells to areas of demyelination is mediated via chemokine and cytokine receptors, a pathway that appears to be intact. once attracted to areas of damage, these precursor cells recapitulate the differentiation process; however, full differentiation of these cells may be dampened by macromolecules that are negative regulators of this process, such as activation of the notch pathway due to reexpression of the ligand jagged or the nogo receptor interacting protein. in the future, both of these targets may be sites for therapeutic intervention that will aid the process of remyelination. in addition, transplantation of stem cells or oligodendroglial progenitor cells may be a consideration [john et al., 2002; mi et al., 2005; frohman et al., 2006 ]. ms is best recognized for its relapsing and remitting clinical course. in fact, in both children and adults, rrms is the most common form, followed by the secondary and primary progressive forms. however, the prognosis for pediatric ms remains controversial. the edss has been used to quantify the disability associated with ms by assigning a functional score for multiple systems (pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual, and cerebral) [kurtzke, 1983] . patients with a score of 0 have a normal neurological exam. scores between 1.0 and 3.5 are fully ambulatory, whereas 4.0 -5.5 are ambulatory for short distances without aid or rest. patients with scores greater than 6 require assistance with ambulation as well as other activities of daily living. in 2002, boiko et al. compared the time to edss of 3.0 (mild disability in at least three domains or moderate disability in one area) and 6.0 (requiring intermittent or constant unilateral assistance to walk 100 meters with or without resting) in adult-and pediatric-onset ms [boiko et al., 2002] . on average, adults had a 50% risk of reaching edss scores of 3.0 and 6.0 in 10 and 18 years, respectively, after onset whereas disability in children was much slower, taking 23 and 28 years, respectively. in addition, 53.1% of children with rrms progressed to spms after an average of 17.7 years (sd 1.17 years). the 50% risk for conversion from rrms to spms was 23 years in children, whereas it was 10 years in adults. although this data suggests a slower disease course in children, the overall morbidity is typically greater when children reach adulthood. children have higher edss scores when compared to adults with ms of the same age [ghezzi et al., 2005] . ms is under-recognized in the pediatric population and presents new challenges in diagnosis and treatment. despite significant advances in neuroimaging, ms remains a clinical diagnosis. new guidelines allow earlier diagnosis, but they have not been reliably established in children, especially those younger than 10 years of age. in 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the northeast cooperative multiple sclerosis treatment group a randomized trial of plasma exchange in acute cns inflammatory demyelinating disease multiple sclerosis: altered glutamate homeostasis in lesions correlates with oligodendrocyte and axonal damage magnetic resonance spectroscopy in multiple sclerosis: window into the diseased brain the hmg-coa reductase inhibitor, atorvastatin, promotes a th2 bias and reverses paralysis in cns autoimmune disease overview of azathioprine treatment in multiple sclerosis key: cord-353812-4oxbczqe authors: zoghi, anahita; ramezani, mahtab; roozbeh, mehrdad; darzam, ilad alavi; sahraian, mohammad ali title: a case of possible atypical demyelinating event of the central nervous system following covid-19 date: 2020-06-24 journal: mult scler relat disord doi: 10.1016/j.msard.2020.102324 sha: doc_id: 353812 cord_uid: 4oxbczqe after the novel coronavirus disease outbreak first began in wuhan, china, in december 2019, the viral epidemic has quickly spread across the world, and it is now a major public health concern. here we present a 21-year-old male with encephalomyelitis following intermittent vomiting and malaise for 4 days. he reported upper respiratory signs and symptoms 2 weeks before this presentation. two cerebrospinal fluid (csf) analyses were notable for mononuclear pleocytosis, elevated protein (more than 100 mg/dl), and hypoglycorrhachia. brain magnetic resonance imaging (mri) showed bilateral posterior internal capsule lesions extending to the ventral portion of the pons and a marbled splenium hyperintensity pattern. cervical and thoracic mri showed longitudinally extensive transverse myelitis (letm), none of which were enhanced with gadolinium. both the aqp4 and mog antibodies were negative. spiral chest computed tomography (ct) scan confirmed to covid-19 as did the high igg level against coronavirus, but the oropharyngeal swabs were negative. neurological manifestations of covid-19 have not been adequately studied. some covid-19 patients, especially those suffering from a severe disease, are highly likely to have central nervous system (cns) manifestations. our case is a post-covid-19 demyelinating event in the cns. coronavirus neuro-invasive characteristics have been identified in humans. it has been shown that severe infection with sars-cov-2 is associated with neurological manifestations such as headache, epilepsy, cerebrovascular events, and encephalitis (bohmwald et al. 2018 , asadi-pooya et al. 2020 . coronavirus probably enters the cns through the olfactory bulb, which could cause inflammation and subsequent axonal damage or demyelination (desforges and et al. 2020 ). we present a young man with covid-19 and acute encephalomyelitis with newly diagnosed possible demyelinating lesions in the cns. a previously healthy 21-year-old male with a bachelor of science was referred to the emergency room of our hospital on march 20 th , 2020. his family reported that he had a fever with chills, nonproductive cough, and a sore throat 2 weeks before admission, but no hyposmia or hypogeusia. all symptoms decreased in severity within 10 days, after which he developed significant loss of appetite, recurrent vomiting with food intolerance, and generalized malaise. following 3 days of repeated vomiting, he experienced weakness and paresthesia of the lower limbs, which continued throughout the day. the next day, family members found that he had urinary retention, increased paraparesis severity and weakness in the upper limbs; he also became drowsy. the patient had no headache, vertigo, diplopia, dysphagia, neck pain, or blurred vision. on examination, his blood pressure was 110/85 mmhg, and his pulse was 98 beats per minute. his temperature was 37.9°c, his respiratory rate 20 per minute, and his oxygen saturation 94% while he was breathing ambient air. the patient was lethargic but obeyed simple verbal commands, and there were no evidences of nuchal rigidity, kernig's or brudzinski's signs. his pupils were equally reactive to light. the muscular strength was 4+/5 in the upper limbs and 2/5 in the lower limbs. he had normal deep tendon reflexes in all four limbs and babinski's sign was absent. the position and light touch sensation were impaired in both lower limbs; additionally, he had a t8 sensory level. the abdominal cutaneous reflex was absent in all directions. a spiral chest ct scan revealed the left lung peripheral ground-glass opacities ( figure 1a ). the patient was transferred to the specialized intensive care unit designated for patients with sars-cov-2, and a nasopharyngeal swab was obtained for detection of the covid-19 genome by a real-time polymerase chain reaction. initial laboratory tests, as well as erythrocyte sedimentation rate and c-reactive protein, were normal. screening tests for human immunodeficiency virus type 1 (hiv-1) and type 2 (hiv-2) antibodies and also tests for hepatitis b virus hours after the first tap, showed 250 total nucleated cells per microliter, with 60% lymphocytes. the csf protein level was 111 mg/dl, and the glucose level was 65 mg/dl. serologic tests for covid-19 were requested, which revealed a negative result for igm, but the igg level was 1.6 (positive >1.1). at the end of the second week, the upper limb weakness improved, but the force of the lower limbs was 3+/5. the main clinical manifestation of human coronaviruses is respiratory involvement, and the leading cause of death is acute respiratory failure. however, there have been reports of extra respiratory manifestations, such as neurological findings (ashrafi et al., 2020) . recent studies suggest possible mechanisms leading to covid-19 neuroinvasive and neurotropic characteristics. the first is a direct viral injury to the cns via blood circulation or nasal epithelium (wu et al., 2020) . although there are some suggestive case reports of encephalitis (wu et al., 2020 . ye et al., 2020 ; there is no definite proof that the sars-cov-2 virus directly affects the cns. the second cause of nervous tissue damage results from the unpredictable effects of the host immune response after an acute infection. guillain-barré syndrome (gbs), as peripheral demyelination, is an example of this mechanism. some cases of covid-19-related gbs have been reported (toscano et al., 2020) , but the evidence of causality or effect is weak (toscano et al., 2020 . zhao et al., 2020 . the third mechanism is an indirect injury of the cns due to systemic disease, particularly in patients who are critically ill. the last mechanism is overactivation of the immune response, which results in cytokine release (yin et al., 2004) . according to the literature, neurotropic coronaviruses could induce a "cytokine storm" by releasing a large number of inflammatory markers (bohmwald et al., 2018) , which could activate molecular changes and also reactivate immunemediated processes (kim et al., 2017) . together, these mechanisms could induce delayed nervous system damage and neurological complications (klein et al., 2017) . studies on sars-cov-1 revealed a delayed self-reactive t-cell suppression due to viral replication, which leads to neuroinflammation, demyelination or axonal damage of the cns (savarin et al., 2017 . cheng et al., 2019 . moreover, experimental models of coronavirus-induced neurological disease have shown that sustained cns inflammation of infected animals correlates with increased demyelination (savarin et al., 2017) . recent studies have shown that the novel coronavirus appears to cross the blood-brain barrier and cause acute or delayed cns demyelination or axonal damage (desforges et al., 2020) . acute necrotizing encephalomyelitis following covid-19 is reported as a case of an acute cns injury (poyiadji et al., 2020) . moreover, a recent report revealed that cns delayed demyelinating events following covid-19 . in our case, the absence of symmetric deep-gray matter involving, hemorrhage and cavitation, and also contrast-enhancing lesions (poyiadji et al., 2020) are more suggestive of demyelination than necrotizing encephalomyelitis. according to our patient's examination, imaging, and laboratory findings, there are two differential diagnoses, including adem or neuromyelitis-optica spectrum disorder (nmosd). in the absence of histopathological evidence, we could not settle on an exact diagnosis for the patient. the presenting symptoms, including new-onset fever and drowsiness along with neurological deficit after regressing respiratory illness, suggested a post-viral adem; however, in the absence of lesions' enhancement as well as callosal involvement, adem is less likely. on the other hand, the history of sudden onset of recurrent vomiting, which could indicate the area of postrema syndrome, along with letm, and corticospinal tract and corpus callosum hyperintensities are more indicative of nmosd (wingerchuk et al., 2015) . encephalopathy, however, as a presenting symptom and hypoglycorrhachia are uncommon in nmosd. in the present study, covid-19 was not detected in csf or the nasopharynx presumably due to delayed immune-mediated cns damage that occurred after the virus was cleared. moreover, this could be explained by the low sensitivity of the system or delayed sampling . ye et al., 2020 . severe covid-19 may affect the cns and have various acute or delayed neurological complications. during the covid-19 pandemic, it is important to consider sars-cov-2 infection when seeing patients with neurological manifestations, especially those needing immune-modulator therapy, since the established recommendations are insufficient at this time. central nervous system manifestations of covid-19: a systematic review coronavirus, its neurologic manifestations, and complications neurologic alterations due to respiratory virus infections innate immune responses and viral-induced neurologic disease human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system neurological complications during treatment of middle east respiratory syndrome infectious immunity in the central nervous system and brain function sars-cov-2:"three-steps" infection model and csf diagnostic implication covid-19-associated acute hemorrhagic necrotizing encephalopathy: ct and mri features viral-induced suppression of self-reactive t cells: lessons from neurotropic coronavirus-induced demyelination guillain-barré syndrome associated with sars-cov-2 international consensus diagnostic criteria for neuromyelitis optica spectrum disorders nervous system involvement after infection with covid-19 and other coronaviruses encephalitis as a clinical manifestation of covid-19 clinical analysis of multiple organ dysfunction syndrome in patients suffering from sars sars-cov-2 can induce brain and spine demyelinating lesions guillain-barré syndrome associated with sars-cov-2 infection: causality or coincidence? this study received no funds. role of funding source: none. key: cord-334577-wb6zhovi authors: mangale, vrushali; syage, amber r.; ekiz, h. atakan; skinner, dominic d.; cheng, yuting; stone, colleen l.; brown, r. marshall; o'connell, ryan m.; green, kim n.; lane, thomas e. title: microglia influence host defense, disease, and repair following murine coronavirus infection of the central nervous system date: 2020-05-25 journal: glia doi: 10.1002/glia.23844 sha: doc_id: 334577 cord_uid: wb6zhovi the present study examines functional contributions of microglia in host defense, demyelination, and remyelination following infection of susceptible mice with a neurotropic coronavirus. treatment with plx5622, an inhibitor of colony stimulating factor 1 receptor (csf1r) that efficiently depletes microglia, prior to infection of the central nervous system (cns) with the neurotropic jhm strain of mouse hepatitis virus (jhmv) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. single cell rna sequencing (scrnaseq) of cd45+ cells isolated from the cns revealed that plx5622 treatment resulted in muted cd4+ t cell activation profile that was associated with decreased expression of transcripts encoding mhc class ii and cd86 in macrophages but not dendritic cells. evaluation of spinal cord demyelination revealed a marked increase in white matter damage in plx5622‐treated mice that corresponded with elevated expression of transcripts encoding disease‐associated proteins osteopontin (spp1), apolipoprotein e (apoe), and triggering receptor expressed on myeloid cells 2 (trem2) that were enriched within macrophages. in addition, plx5622 treatment dampened expression of cystatin f (cst7), insulin growth factor 1 (igf1), and lipoprotein lipase (lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. collectively, these findings argue that microglia tailor the cns microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair. tor 1 receptor (csf1r) that efficiently depletes microglia, prior to infection of the central nervous system (cns) with the neurotropic jhm strain of mouse hepatitis virus (jhmv) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. single cell rna sequencing (scrnaseq) of cd45+ cells isolated from the cns revealed that plx5622 treatment resulted in muted cd4+ t cell activation profile that was associated with decreased expression of transcripts encoding mhc class ii and cd86 in macrophages but not dendritic cells. evaluation of spinal cord demyelination revealed a marked increase in white matter damage in plx5622-treated mice that corresponded with elevated expression of transcripts encoding disease-associated proteins osteopontin (spp1), apolipoprotein e (apoe), and triggering receptor expressed on myeloid cells 2 (trem2) that were enriched within macrophages. in addition, plx5622 treatment dampened expression of cystatin f (cst7), insulin growth factor 1 (igf1), and lipoprotein lipase (lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. collectively, these findings argue that microglia tailor the cns microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair. • microglia augment host defense in response to cns infection by the neurotropic coronavirus jhmv. vrushali mangale, amber r. syage, and h. atakan ekiz equally contributed to this work tic chemokines including ccl5, cxcl9, and cxcl10 within the cns contribute to host defense by attracting virus-specific cd4+ and cd8+ t cells into the cns that further control viral replication through secretion of interferon-γ (ifn-γ) and cytolytic activity (bergmann et al., 2004; glass et al., 2004; glass & lane, 2003a; glass & lane, 2003b; liu et al., 2000; liu, armstrong, hamilton, & lane, 2001; marten, stohlman, & bergmann, 2001; parra et al., 1999) . antibody-secreting cells (ascs) are also capable of responding to cxcl9 and cxcl10 and aid in host defense (phares, marques, stohlman, hinton, & bergmann, 2011; . nonetheless, sterile immunity is not achieved and the majority of animals that survive the acute stage of disease develop immune-mediated demyelination in which both virus-specific t cells and macrophages amplify the severity of white matter damage associated with hind-limb paralysis (bergmann et al., 2006; hosking & lane, 2009; templeton & perlman, 2007) . defense and disease in jhmv-infected mice have been extensively studied, there is increasing interest in better understanding how resident cells of the cns contribute to these events. microglia are considered the resident immune cells of the cns and aid in a diverse array of functions including maintaining cns homeostasis as well as contributing to various disease-associated conditions (hammond, robinton, & stevens, 2018; salter & stevens, 2017; tejera & heneka, 2019; wolf, boddeke, & kettenmann, 2017) . moreover, microglia are immunologically competent and capable of rapidly responding to infection and/or damage via specific expression of surface receptors culminating in morphologic changes accompanied by secretion of proinflammatory cytokines/chemokines that function in amplifying neuroinflammation. recently, the functional role of microglia in contributing to host defense in response to cns infection with neurotropic viruses has been examined. these studies have been greatly aided by findings demonstrating that mice lacking colony stimulating factor 1 receptor (csf1r−/−) lack microglia emphasizing the importance of this signaling pathway in microglia development (ginhoux et al., 2010) . subsequent studies by green and colleagues (elmore et al., 2014) showed that blocking csf1r signaling in adult mice through administration of csf1r antagonists is also important in survival of microglia in adult mice. recent studies have employed treatment of mice with plx5622, a brain penetrant and selective antagonist of the csf1r that results in a dramatic reduction in microglia, to better understand functional roles of these cells in preclinical models of neurodegenerative disease (acharya et al., 2016; dagher et al., 2015; elmore et al., 2014; spangenberg et al., 2019) . in addition, plx5622-mediated targeting of microglia results in increased susceptibility to west nile virus (wnv) seitz, clarke, & tyler, 2018) , japanese encephalitis virus (jev) (seitz et al., 2018) , theiler's murine encephalomyelitis virus (tmev) (sanchez et al., 2019a; waltl et al., 2018) , and jhmv (wheeler, sariol, meyerholz, & perlman, 2018) arguing for a protective role for microglia against acute viral-induced encephalitis. the current study was undertaken to evaluate how microglia tailor the immunological landscape in response to jhmv infection within the brain and spinal cord at different stages of infection with regard to pathways associated with both host defense and neuropathology. we believe microglia will be critical in aiding in host defense through regulating a number of different pathways including antigen presentation and t cell activation as well as augmenting demyelination. to address this, we used a comprehensive set of analytical approaches including single cell rna sequencing (scrnaseq), flow cytometry, and histopathological techniques to assess disease outcome in jhmvinfected mice treated with plx5622 at defined times postinfection. our findings emphasize an important role for microglia in aiding in host defense in response to jhmv infection of the cns as well as influencing both the severity of spinal cord demyelination and remyelination in a model of murine coronavirus-induced neurologic disease. five-week-old c57bl/6 male mice were purchased from the jackson laboratory. mice were infected intracranially (i.c.) with 250 plaque forming units (pfu) of jhmv strain j2.2v-1 in 30 μl of sterile hanks balanced sterile solution (hbss) and animals were euthanized at days 3, 7, 12, and 21 postinfection (p.i.). clinical disease in jhmv-infected mice was evaluated using a previously described scale . to determine viral titers within brains, experimental animals were sacrificed at defined times p.i., brains isolated, homogenized and plaque assay were performed on the dbt astrocytoma cell line as described previously (hirano, murakami, fujiwara, & matsumoto, 1978 flow cytometry was performed to identify inflammatory cells entering the cns using established protocols (blanc, rosen, & lane, 2014; chen et al., 2014) . in brief, single cell suspensions were generated from tissue samples by grinding with frosted microscope slides. immune cells were enriched via a two-step percoll cushion (90 and 63%) and cells were collected at the interface of the two percoll layers. before staining with fluorescent antibodies, isolated cells were incubated with anti-cd16/32 fc block (bd biosciences, san jose, ca) at a 1:200 dilution. immunophenotyping was performed using commercially available antibodies specific for the following cell surface markers: cd4, cd8, cd11b (bd biosciences, san jose, ca), and cd45 (ebioscience, san diego, ca). the following flow cytometric gating strategies were employed for inflammatory cells isolated from the cns: macrophages (cd45 hi cd11b+) and microglia (cd45 lo cd11b+). apc-conjugated rat anti-mouse cd4 and a pe-conjugated tetramer specific for the cd4 immunodominant epitope present within the jhmv matrix (m) glycoprotein spanning amino acids 133-147 (m133-147 tetramer) to determine total and virus-specific cd4 + cells, respectively (chen et al., 2014; marro, grist, & lane, 2016a) ; apc-conjugated rat anti-mouse cd8a and a peconjugated tetramer specific for the cd8 immunodominant epitope present in the spike (s) glycoprotein spanning amino acids 510-518 (s510-518) to identify total and virus-specific cd8 + cells, respectively (chen et al., 2014; marro et al., 2016a) . data were collected using a bd lsr fortessa x-20 flow cytometer and analyzed with flowjo software (tree star inc.). immune cells were isolated as described above from brain (day 7 p.i.) and spinal cord (day 14 p.i.) and stained with dapi and apc database. expression levels and distribution of population-specific immune cell markers were then analyzed to further refine the identified clusters and expose any subpopulations that should be separated as independent clusters. once the clusters were established and identified, plots were generated using seurat, ggpubr, and fgsea r packages. mice were euthanized at defined times points according to iacucapproved guidelines and the length of spinal cord extending from thoracic vertebrate 6-10 was cryoprotected in 30% sucrose, cut into 1-mm transverse blocks and processed to preserve the craniocaudal orientation and subsequently embedded in o.c.t. (vwr, radnor, pa). eight micron (μm)-thick coronal sections were cut and sections were stained with hematoxylin/eosin (h&e) in combination with luxol fast blue (lfb) and between 4 and 8 sections/mouse analyzed. areas of total white matter and demyelinated white matter were determined with imagej software and demyelination was scored as a percentage of total demyelination from spinal cord sections analyzed (blanc et al., 2015; blanc, et al., 2014; dickey, worne, glover, lane, & o'connell, 2016; marro, grist, & lane, 2016b ). for electron microscopy (em) analysis of spinal cords, mice were sacrificed and underwent cardiac perfusion with 0.1 m cacodylate buffer containing 2% paraformaldehyde/2% glutaraldehyde. serial ultrathin sections of spinal cords embedded in epon epoxy resin were stained with uranyl acetate-lead citrate and analyzed as previously described (liu, keirstead, & lane, 2001) . images at ×1200 magnification were analyzed for g-ratio using imagej software. in adult animals there is a relationship between axon circumference and myelin sheath thickness (number of lamellae) expressed by the g-ratio (axon diameter/total fiber diameter); in remyelination this relationship changes such that myelin sheaths are abnormally thin for the axons they surround (smith, bostock, & hall, 1982 ). an abnormally thin myelin sheath, relative to axonal diameter, was used as the criterion for oligodendrocyte remyelination. absence of a myelin sheath was used as the criterion for demyelination. for most axons, two measurements were conducted with a minimum of 400 axons analyzed per experimental group. in all cases, slides were blinded and read independently by two investigators. graphpad prism was used to perform statistical analyses. data for each experiment is presented as mean ± sem. for flow cytometry analysis unpaired student's t test was used to determine significance and a p value of <.05 was considered statistically significant. wilcoxon test was used for analyzing gene expression in scrnaseq clusters and the resulting p values were corrected for multiple comparisons by holm-sidak method and a p value of <.05 was considered statistically significant. to evaluate the contribution of microglia to disease progression in jhmv-infected mice, the csf1r inhibitor plx5622 was administered as previous studies have reported this pharmacologic approach effectively depletes >90% of microglia (acharya et al., 2016; najafi et al., 2018) . mice were treated with plx5622 (1,200 mg/kg) 7 days prior to infection and continued on the drug for the duration of the experiment. treatment with plx5622 resulted in an overall increase in mortality with 25% of plx5622-treated mice surviving to day 21 p.i. whereas 75% of control-chow treated mice survived to this time ( figure 1a ). the increase in mortality in plx5622-treated mice correlated with increased viral titers within the brains and spinal cords at days 3, 7, and 12 p.i. compared with control animals; however, by day 21 p.i. viral titers were not detected (nd) in experimental groups f i g u r e 1 plx5622 treatment increases susceptibility to jhmv-induced neurologic disease. mice were fed either plx5622 or control chow for 7 days prior to i.c. infection with jhmv (250 pfu) and subsequently remained specific chow for the duration of the experiment. plx5622 treatment led to (a) increased mortality compared to control mice that was associated with an (b) impaired ability to control viral replication within the brains and spinal cords at days 3, 7, and 12 p.i. compared with control mice. representative flow cytometric data from jhmv-infected mice treated with either plx5622 or control chow and gating on microglia (cd45 lo cd11b + cells) or macrophages (cd45 hi cd11b + cells) in (c) brains at day 7 p.i., and (d) spinal cords at day 14 p.i. plx5622-treatment resulted in reduced numbers of microglia in brains and spinal cords compared with control mice. data are derived from a minimum of three independent experiments with a minimum of 3 mice/time points. data in b, c, and d are presented as average ± sem. nd, not detected; *p < .05, ****p < .0001 ( figure 1b) . we confirmed efficient microglia (cd45 lo cd11b+) depletion in plx5622-treated mice within the brain at day 7 p.i. (figure 1c) and spinal cord at day 14 p.i. (figure 1d ) using flow cytometry. plx5622 treatment did not affect numbers of macrophages (cd45 hi cd11b+) within brains and spinal cords of experimental mice (figure 1c, d) . these findings support earlier work indicating that plx5622-targeting of microglia impacts efficient immune-mediated control of viral replication following infection with neurotropic viruses sanchez et al., 2019b; seitz et al., 2018; waltl et al., 2018; wheeler et al., 2018) . our findings reveal that plx5622 treatment of mice increases susceptibility to jhmv-induced neurologic disease associated with impaired ability to control viral replication. in order to better understand the effects of plx5622 treatment on influencing the immune cell composition of the cns we used 10× genomics scrnaseq technology. experimental mice were fed either control chow or chow containing plx5622 for 7 days prior to infection and remained on chow until sacrificed at either day 7 p.i. or 14 p.i., at which point, live cd45+ cells were sorted from the brains or spinal cords, respectively. the respective tissues were found appropriate to study host defense and disease pathogenesis. the immune response to jhmv infection peaks around day 7 p.i. in the brain and ensuing spinal cord demyelination is present at day 14 p.i. we aggregated data from 4,806 cells taken from control-treated (n = 6) and 3,868 cells from plx5622-treated (n = 6) mice brain tissue at day 7 p.i. and performed unsupervised clustering analysis based on similarity of gene expression signatures using seurat single cell genomics r package (ekiz et al., 2019) (table 1) . this approach revealed 16 distinct cell clusters representative of both lymphoid and myeloid linages at day 7 p.i. (figure 2a ). to better understand the overlapping expression of marker genes and identification of cell clusters, we employed a recently described algorithm that compares the gene expression signatures of cell clusters with publicly available immgen database (ekiz et al., 2019) . as previously described (ekiz et al., 2019) , this algorithm calculates an aggregate identity score for each scrnaseq cell cluster as a measure of molecular similarity to the immgen subsets. through combinations of these two approaches, we identified three cd8+ t cell subsets [naïve, effector (eff.) , and memory (mem.)], two macrophage subsets (mac 1 and mac 2), four dendritic cell (dc) subsets (plasmacytoid, nadph [nox2], xcr1 [xcr1], ccl22 [ccl22]), and single subsets of cd4+ t cells, regulatory t cells (treg), natural killer (nk) cells, b cells, microglia, neutrophils (neuts), and monocytes at day 7 p.i. (figure 2a) . in order to verify the algorithm-assisted identification of cell clusters, we examined expression of known cellular markers in our data set; expression of these markers corresponded with the respective identities of the distinct clusters (figure 2b ,c). our initial preliminary analyses focusing on samples separately are in agreement with the results of this aggregated approach. we next analyzed differences in cd45+ cells between plx5622-treated and control mice at day 7 p.i. following jhmv infection. when data from the cellular genotypes were plotted side-byside, treatment-dependent dynamics within the tissues started to emerge (figure 3a ,b). importantly, we were able to show that control of jhmv replication within the cns is associated with infiltration of activated virus-specific cd4+ and cd8+ t cells (marten et al., 2001; pearce, hobbs, mcgraw, & buchmeier, 1994; williamson & stohlman, 1990) . at day 7 p.i., plx5622 treatment did not significantly alter cd4+ t cell infiltration yet there was an increase in cd8+ t cells (p < .05) compared with control mice as determined by flow cytometric analysis ( figure s1a ). there were no differences in virus-specific cd4+ and cd8+ t cells specific for overview of experimental conditions showing treatment, sacrifice time points, tissue collected, and total number of cd45+ cells isolated as well as reads/cell following scrnaseq analysis immunodominant epitopes present within the matrix (m) ( figure s1b) and spike (s) glycoproteins ( figure s1c ) as determined by tetramer staining (blanc, et al., 2014; marro et al., 2016b) . evaluation of defined factors associated with t cell activation at day 7 p.i. revealed reduced expression of the th1-associated transcription factor tbet (tbx21) (p < .01) and this was associated with reduced (p < .05) expression of tnf transcripts, but not ifng transcripts, in plx5622-treated mice compared to control mice ( figure 4a ). we also determined reduced expression of activation markers cd69 (cd69) and cd44 (cd44, p < .05) in cd4+ t cells from the brains of plx5622-treated mice compared to control mice at day 7 p.i. (figure 4a ). in addition, the cd4+ t cells subset from plx5622-treated mice also expressed reduced transcripts for il2ra we next performed scrnaseq on cd45+ cells enriched from the spinal cords of jhmv-infected mice treated with either plx5622 or control at day 14 p.i. using the same approach as described above using aggregated data from 2,725 cells taken from control-treated (n = 6) and 4,891 cells from plx5622-treated (n = 6) mice (table 1) mice presumably through recognition of viral antigens resulting in secretion of cytokines for example, ifn-γ that activate both resident cns cells and inflammatory macrophages/myeloid cells to secrete proinflammatory cytokines/chemokines as well as molecules damaging to oligodendrocyte function (hosking & lane, 2009; templeton & perlman, 2007) . by day 14 p.i., we detected no difference in cd4+ t cells in the spinal cords of plx5622-treated mice versus controls, though there was an increase (p < .05) in cd8+ t cells ( figure s2a) as well as virus-specific cd4+ ( figure s2b ) and cd8+ t cells cns viral infection has been identified sanchez et al., 2019a; seitz et al., 2018; waltl et al., 2018; wheeler et al., 2018) . perlman and colleagues have shown that microglia are required for optimal host defense in response to jhmv infection of the cns. targeted depletion of microglia through administration of plx5622 revealed a role in limiting mortality that was associated with impaired control of jhmv replication. the increase in susceptibility to disease did not appear to be due to altered expression of ifn-i but more likely a reflection of impaired antigen-presentation due to muted mhc class ii expression by macrophages infiltrating the cns of plx5622-treated mice and this likely resulted in dampened t cell responses . we believe the increase in expression of ifn-i response genes in plx5622-treated mice most likely reflects the overall increase in viral titers within the brains. similarly, microglia depletion led to increased mortality in mice infected with wnv associated with diminished activation of apcs and limited reactivation of virus-specific t cells that led to reduced viral clearance seitz et al., 2018) . these findings clearly implicate microglia in enhancing optimal host responses following cns viral infection, in part, by influencing antigen-presentation that affects virusspecific t cell responses. we undertook the present study to better understand how microglia contribute to host defense as well as demyelination and repair following jhmv infection of the cns using sophisticated molecular, cellular, and histologic approaches. employing plx5622 to deplete microglia, we found increased mortality associated with expression of ifn-i is critical in host defense in response to jhmv infection of the cns (athmer et al., 2018; ireland et al., 2008; vijay et al., 2017) . plx5622-mediated targeting of microglia did not affect ifn-i signaling as gsea analysis revealed ifn-α response genes were significantly enriched in both macrophages and dendritic cells within the brains of plx5622-treated mice at days 7 compared with controls ( figure 3d ,e). these findings may reflect the increase in viral titers within the cns of plx5622-treated mice at these times but also argue that microglia are not solely responsible for production of ifn-i. we detected altered t cell responses within the cns following plx5622 treatment as determined by both flow cytometry and scrnaseq. there were increased numbers of total cd8+ t cells (p < .05) as well as virus-specific cd8+ t cells within the brains of jhmv-infected mice treated with plx5622 compared to controls at day 7 p.i. we also found a trend towards increased total cd4+ t cells and virus-specific cd4+ t cells in plx5622-treated mice compared to controls although these differences were not significant. in terms of t cell activation, we detected differential responses in t cell subsets at day 7 p.i. in cd4+ t cells, there was a reduction in transcripts associated with th1-polarized activation for example, t-bet (tbx21) in plx5622-treated mice compared to controls although there were no differences in ifng transcripts in cd4+ t cells in experimental groups. plx5622-treatment also resulted in reduced expression of cd4+ t cell surface activation markers including cd44, cd69, and components of il-2 receptor in plx5622-treated mice compared with controls whereas there was an overall increased activation phenotype associated with (chabas et al., 2001; krasemann et al., 2017; ulrich & holtzman, 2016) . interestingly, expression of all three transcripts were enriched within macrophage populations suggesting a specific effect by which microglia may suppress expression and limit myelin damage. emerging studies have pointed to a protective role for microglia in limiting neuropathology and promoting repair (baaklini, rawji, duncan, ho, & plemel, 2019; lee, hamanaka, lo, & arai, 2019; lloyd & miron, 2019) . in support of this concept are recent studies from miron and colleagues (lloyd & miron, 2019) showing an important role for microglia in enhancing remyelination in a toxin-model of demyelination that is aided by microglial death and subsequent microglial repopulation; here, in this study, the absence of microglia prevents their further death and repopulation and is associated with increased white matter damage. although mechanisms by which microglia may support remyelination have not been completely defined, it is though that these cells aid in clearance of myelin debris and/or secrete growth factors/cytokines that influence maturation of oligodendrocyte progenitor cells (opcs) into mature myelin-producing oligodendrocytes. what is also clear is that microglia are heterogenous in terms of transcriptome and protein expression which is likely regulated during disease and this would influence the role of these cells in enhancing or muting disease progression and potential repair. in support of a protective role for microglia in restricting neuropathology and promoting repair is our data demonstrating that plx5622 treatment of jhmv-infected mice results in an increase in white matter damage associated with impaired remyelination (figure 7a ,b,d, and e). in addition, scrnaseq also shows reduced expression of genes encoding proteins previously associated with remyelination including cystatin f (durose et al., 2019; ma et al., 2011; shimizu et al., 2017) , insulin growth factor 1 (igf1) (hlavica et al., 2017; wlodarczyk et al., 2017; ye et al., 2002) and lipoprotein lipase (bruce et al., 2018) within macrophages isolated from the spinal cords of plx5622-treated mice (figure 7f ). these findings further support the notion that microglia may either directly or indirectly influence remyelination within the spinal cord by contributing to controlling expression of genes encoding proteins that regulate opc maturation. we are currently pursuing the functional contributions of cystatin f, igf1, and lipoprotein lipase in contributing to remyelination in jhmv-infected mice. we would caution that although demyelination was worsened and remyelination was impaired when microglia are depleted, this may the authors declare no competing financial interests. the data that support the findings of this study are available from the corresponding author upon reasonable request. kim n. green https://orcid.org/0000-0002-6049-6744 thomas e. lane https://orcid.org/0000-0003-0392-0825 elimination of microglia improves cognitive function following cranial irradiation selective packaging in murine coronavirus promotes virulence by limiting type i interferon responses central nervous system remyelination: roles of glia and innate immune cells coronavirus infection of the central nervous system: host-virus stand-off perforin and gamma interferon-mediated control of coronavirus central nervous system infection by cd8 t cells in the absence of cd4 t cells sphingosine-1-phosphate receptor antagonism enhances proliferation 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balance between cathepsin c and cystatin f controls remyelination in the brain of plp1-overexpressing mouse, a chronic demyelinating disease model saltatory conduction precedes remyelination in axons demyelinated with lysophosphatidyl choline sustained microglial depletion with csf1r inhibitor impairs parenchymal plaque development in an alzheimer's disease model microglia in neurodegenerative disorders pathogenesis of acute and chronic central nervous system infection with variants of mouse hepatitis virus, strain jhm galectin-3-mediated glial crosstalk drives oligodendrocyte differentiation and (re)myelination trem2 function in alzheimer's disease and neurodegeneration virus-induced inflammasome activation is suppressed by prostaglandin d2/dp1 signaling microglia have a protective role in viral encephalitis-induced seizure development and hippocampal damage coronavirus pathogenesis microglia are required for protection against lethal coronavirus encephalitis in mice effective clearance of mouse hepatitis virus from the central nervous system requires both cd4+ and cd8+ t cells a novel microglial subset plays a key role in myelinogenesis in developing brain microglia in physiology and disease myelination is altered in insulin-like growth factor-i null mutant mice additional supporting information may be found online in the supporting information section at the end of this article. key: cord-023143-fcno330z authors: nan title: molecular aspects of viral immunity date: 2004-02-19 journal: j cell biochem doi: 10.1002/jcb.240591009 sha: doc_id: 23143 cord_uid: fcno330z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv60). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between 5 and 11 days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd4+ and cd8+ t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd4+ and cd8+ cells were detected by immunohistochemistry at 6 days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il-2, il-3, il-4, il-5, il-6, tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il-6, tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca 92037 although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd8+ cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within 2 hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp47 was both necessary and s f i c i e n t to cause retention of class i and icp47 expression in fibroblasts caused the cells to resist lysis by cd8+ t lymphocytes. icp47 is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp47 inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp47 have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp47 associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp47 are being assessed in proteosome and tap transporter assays. gst-icp47 fusion proteins tightly bind a 8.5 kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp47 binds to a single cellular protein of =55 kda on ligand blots. these proteins are good candidates as cellular targets of icp47 and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp47 is very effective i n blocking cd8+ t lymphocyte responses in vivo, perhaps explaining the predominance of cd4+ vs. cd8+ anti-hsv ctl i n vivo. we expect that icp47 may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma2, john p. carroll2, sara r. abromson-leeman2, joseph c. laning2, martin e. dorf2, and thomas l. benjamin2. idepartment of pathology, emory university school of medicine, atlanta, ga 30322, and 2department of pathology, harvard medical school, boston, ma 02115. susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h-2 haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c3wbida mouse and the highly resistant but mhc-identical (h-2k) c57bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs7bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c3wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c3wbida x c57bwcd.i) x c57bwcdj backcross mice cosegregated with mtv-7. inheritance of mtv-7 showed perfect concordance with absence of peripheral vp6+ t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv-7 showed no evidence of recombination between pyvs and mtv-7. strongly biased usage of vp6 by (a) polyoma-specific cd8+ ctl from virus-infected c57bwcdj mice and by @) cd8+ t cells infiltrating a polyoma tumor in a virus-immune c57bwcd.i host provide further evidence that t cells bearing this mtv-7 sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv-7 sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h-2, non h-2, level of cd4+ t cells, of cd8+ t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd8+ t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd8+ t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd8+ t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd8+ t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type 5 (ad 5) and human papillomavirus type 16 (hpv16)) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad 5e1a protein to which ctl clones were directed that could eradicate ad 5e1 induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad 5e1b protein. these ad 5eib specific ctl clones, however, were as effective as the ad 5e1a specific ctl clones in the eradication of ad 5e1 induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad 5e1 induced tumor cells with the activated ras oncogen the possibility of ad 5e1b specific ctl to recognize the ad 5e1 induced tumors was eliminated whereas the ad 5e1a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv16 induced mouse tumor model an immunosubdominant ctl epitope was identified in the e7 protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv16 induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv16 induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv16, that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a *0101, a *0201, a"0301, a*1101 and a *2401). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv16 induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of 1994. class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment3.4, biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd4 + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type 1 glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 5'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph8 environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al 18083 to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about 1 pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual 5' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are 106-10'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the 2-month old infant, an rsv vaccine will need to be effective when given to 1-month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: 1) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: 2) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd8+ cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd4' and cd8+ t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd4' t cells were more pathogenic than cd8+ t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m 2 (22k) protein were predominantly cd8' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd4+ t cells, and ctl. mice primed to g protein gave rise to predominantly cd4' t cells producing th2 cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type 2 cytokines, notably il-10, appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd4 t cells and protective effects by cd8' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d 4 ' t cells and b cells (immune complexes) in maintaining cd8+ t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn 38105. the analysis of the development and memory phases of the cd4+ "helper" n h ) and cd8+ cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type 1) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose (20 mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd4+ th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd44-high, l-selectin-low, cd49d (vla-4) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd49d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd49d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus 68 which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il-2 production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b7.1 and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il-4 vs il-i2/ifny are present at the initiation of the response of either cd8 or cd4 cells they dictate that the effectors generated will be polarized either towards il-4 and il-5 secretion or il-2 and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd4 effector cells not re-exposed to ag, produce no cytokines and they die within 3-4 days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il-2 produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il-2 and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around 450 trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to 7.0, the s ikes re ain their neutral configuration (1). probably as a consequence, the viral infectivity is totally preserved after an exposure of 2 hours at p 8 6.4 an cf 37t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein (2). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position 198, which is part of antigenic site 11, is important, although not essential, for the viral virulence. similarly, the arginine 333, which belongs to antigenic site 111, is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in 3). viral strains mutated at arginine 333 have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody (4). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus (5). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il 6061 1 theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd4 antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg2a antibody; (c) activated (le., ll-2rc) t cells infiltrating the cns are exclusively of the cd4+ phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids 74-86 of the vp2 capsid protein. a thl line specific for vp274-86 exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein 0. prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie9 bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by 9 amino acids between residues 96 and 167. au of the tg(mhu2m) mice developed neurologic drsease -200 days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu2m) mice with cjd prims produced mhu2mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu2m) mice wen differenl about 10% of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane >500 days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu2m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu2m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene -8-fold was not deleterious to themiw but it did shorten scrapie incubation times from -145 d to -45 d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon 101 (corresponding to codon 102 in hurp) pmdnced spontaneous, fatal neurcdegeneration between 150 and 300 d of age in two lines of tg(mohp-pio1l) mice designated 2866 and 2247. genetic crosses of tg(moprp-p101l)2866 mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between 150 and 160 days of age. the t g~o p r p -p l o l l ) 2 8 6~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast 10 the tg2866 and 2247 mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg2862 overexpress the mutant transgene -32-fold and develop fatal neurodegeneration behveen 200 and 400 d of age. tg2862 mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu2m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md 20892-0440. cd8' lymphocytes (tcd8+) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of 8 to 10 residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors (17 residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh2-terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the 17 residue precursor than from the nh2-terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv-1 envelope protein that contain overlapping determinants each presented by different class i1 mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv-1 envelope to provide help for neutralizing antibody (ab) and cd8+ cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p18 peptide from the v3 loop of hiv-1 iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p18 segment of both iiib and mn strains of hiv-1 in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all 3 responses, neutralizing ab, ctl, and th1 helper cells. the ctl specific for the mn strain p18 peptide crossreacted with strains sc, sf2,2321, and cdc4. the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp160 that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it 10 to 100-fold more potent in binding to the class i1 mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv-1 sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a2, with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/93. cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m1) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/89 and a/hawaii/91. this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/89 virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp120 generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il-4, months after immunization were also observed . immunization of rabbits with dna encoding l1, the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type 2 (hsv-2), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv-2. dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, 1993 2593745-1749 , 2dna cell biol, 1993 the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd4 t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd8 t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd8 cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e3) codes for more than 7 polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e3 gpl9k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by 4 distinct viral polypeptides, 3 of which (the ad e3 14.7k or the complex of the 10.4k and 14.5k proteins) are coded in the e3 region. the e3 polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e3 polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e3 polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e3 14.7k or ad e3 gpl9k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e3 genes in viral constructs, several transgenic mouse lines containing the ad e3 genes have been constructed for these experiments. the e3 genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e3 transgenic animals (h-2b'd) have been transplanted allogeneically to h-2d recipients and remained viable, secreting insulin until the end of the experiment at 94 days; in contrast, control nontransgenic islets of the same genotype were rejected by 21-28 days. the e3 genes behind the native e3 promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e3 promoter of the transgene is responsive to stimulation by the ad e1a following infection with an e3 minus ad 7001 and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e3 genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a 1993 outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr03121.) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g2 encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than 200,000 cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in 1993, a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus 7.5 k promoter and the s segment under control of the 11 k promoter. the m segment, which encodes the g1 and g2 envelope proteins, was included because of our findings that: (1) immunization with vaccinia or baculovirus-expressed g1 and g2 induced a neutralizing and protective immune response in hamsters; and, (2) neutralizing antibodies to g1 or g2 could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo7 pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd8 t cells and in the induction of virus-specific ctl activity. thereafter, the cd8 t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd8 cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl-2 in the t and b cells and in c57bl/6 ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd3, thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma 01772 thfectious disease unit, massachusetts general hospital, boston, ma 021 14 â§tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied 12 or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo0 for gag and 1/8500 for envelope. cd8+ lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd4+ cells. suppression mediated by unstimulated cd8 autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd8+ cells activated by an anti-cd3-specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv-1 molecular clone (nl43) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p24 and gp160 were observed in chimpanzees infected with n u 3 and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv-1, and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to 15 years with cd4 cells > 500/mm3. methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn-1 infected autologous cd4 cells, using target cells infected with recombinant vaccinia viruses expressing hn-1 proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr3 region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for 15 years, who by qc-pcr had <i67 viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv-1 variants, and both the p17 and p24 epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr3 region of the tcr in an individual with a vigorous response to gp41 suggested that approximately 6% of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv-1 infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf-1) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il-6 and il-7 receptor genes. to investigate the role of irf-1 related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o 5 pfu of coxsackie virus 83 (cvb3). homozygous irf-1 (+) and heterozygous irf-1 (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf-1 -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf-1 regulated gene products such as inos and ifn are essential for the early defense against cvb3-induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd4 and b-cell populations and a higher proportion of macrophages and cd8 cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il2, il4 and il5, but higher levels of il6 and ifng in the elderly as compared to the young mice. the addition of mf59 to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il2, il4 and il5 levels for both young and old, while il6 and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus 8 3 (cvb3), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb3 which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb3 is able to infect normal c57bl/6 mice and immunocompromised (cd4 ko and 62m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld5os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers 3-5 days pi.. large pathological changes were detectable only in heart tissue of cd4 ko mice, and were maximal at 1-2 weeks after infection. these results suggest the possibility that cd4' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd4+ and cd8' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p56lck are resistant to coxsackievirus b3 induced heart disease. tamara martino, karen aitken, joseph penninger, jan0 nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus 83 (cvb3) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p56kk are resistant to cvb3 induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb3 replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb3induced heart disease, cytokine expression in the hearts of cvb3 infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb3 infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd4+ t cells from 62m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd4+ t cells seen in normal mice 3 days after lcmv infection, previously presumed to be due to the activity of cd8+ ctl, still occurs in 62m-/-mice. however, 621114mice show an earlier rise in percentage and absolute numbers of cd4+ t cells by 7 days after infection. cd4+ t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd8+ t cells in normal mice after lcmv infection. cd4+ ciz activity is lower in 62m-/mice and have a later peak than cd8+ ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in 621114-mice. mice can assume some of the cytotoxic functions of the cd8+ cell population from normal mice, including immune-mediated pathology. further, cd4+ t cells from 62m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd8+ t cells from normal mice. mice genetically engineered to be deficient in 62-microglobulin in conclusion, these data suggest that cd4+ t cells from am-/australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd40 and its ligand, in the presence of b cell acting cytokines, such as interleukin 4 (il-4), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd40l and il-4, did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd4ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w2 lpg, u. k. and *nationallnstirute formedica1 research, london, nw7 1aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il-3 has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il-3 on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il-3 gene was disrupted in 129/sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il-3. mice were infected intranasally with a 2 strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il-3 deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd8+ cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd4+t helper (th) cells of the -1 subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within 7 days pi. c t l s were detected by h-2 specific lysis of ldv-specific target cells. these target cells were generated by transfection of 3t3-nm cells with a retrovirus vector carrying the gene (ow 7) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected 3t3-nih cells and cultured for 5 days in the presence of il-2. the ctls had largely disappeared in mice by 30 days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about 3 days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v3000) or an isogenic single-site attenuated vee mutant (v3032) (single amino acid change at e2 glycoprotein position 209 from glu-lys) and at 1,6,24,48,72, and 96 hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il-12, ifn-y, il-2, il-4, il-6, and il-10 gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il-12, ifn-y, il-10, and il-6 were detected in the lymph node, with no changes in either il-2 or il-4; however, whereas cytokine elevations were detected by 6 hours after injection of v3000, elevations were delayed until 24 hours following infection with v3032. cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il-10, and il-12 were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il-10 and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il-12 andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il-10. to confirm the importance of the immune system in clearing rotavirus infection we infected rag 2 knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. 1990, 121:149-160) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement 4b binding protein (bc4-bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature 1988, 355:176-179) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc4bbp ( kotwal, am. biotech. lab., 1994) and has also been shown to bind to two important complement components, c3 and c4, and block the complement cascade at multiple sites in vitro (kotwal et al., science 1990, 2502327-830; mckenzie, kotwal et al., j. inf. dis. 1992 , 1661245-1250 . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas 1992, 89:628-672) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature 1993,366:748-751) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn2 mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, 1993 mol. immunol. 30:20) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h-2 haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c5deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, 1981) . various immune regulatory molecules were investigated in inbred susceptible 129/j (129) mice and resistant c57bl/6j (b6) mice which share the same mhc haplotype. when they were given 200 eid, of sendai virus intranasally, 129 mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in 86 mice. virus was eliminated from the infected lung 3 days earlier in b6 mice than in 129 mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il-2, ifn-y, il-10, and il-4 producing cells were comparable for each strains of mice, while more il-6 producing cells were present in the lung of 129 mice. however, the numbers of these cytokine producing cells peaked in 129 mice 3 days later than in b6 mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il-10, and il-6 were present in 129 mice than in 86 mice. recall cytokine production in the draining mln showed only the presence of il-2, ifn-y, il-10, and il-6, while no il-4 was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in 129 mice. therefore, the susceptibility of 129 mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th2 pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il-2, ifn-y, tnf-a or il-7 in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il-4 by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il-4 by rvv was shown to inhibit the host il-2 response required for the development of ctl's. no production was also significantly suppressed by il-4. rvvs expressing il-5 or il-6, although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i2 (il-12) doses of >i00 ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd8+ t cell expansion and ctl activation, and up to 2 log increases in viral replication [orange, wolf, and biron, j. immunol. 152:1253, 19941 . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il-12 induced tnf-cx expression and that lcmv infection synergized with il-12 for this induction. administration of antibodies neutralizing tnf reversed the il-12-induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd8+ t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il-12-treated uninfected mice and were dramatically elevated in il-12-treated virus-infected mice, including: 1) decreases in body weights; 2) elevation of circulating glucocorticoid levels: and 3) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il-12 for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a 50% decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c57bu6 mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-or pkh26-gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately 10% of the transferred bone marrow cells accumulated in spleens after 20 hrs and 30% of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn-7 mrna by in sifu hybridization. treatment with anti-agmi or anti-nk1 .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box 334, canberra, act, 2601 australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c57bu6 (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd3 antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c57bu6 mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd3 antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn-7 and il-2. in contrast spleen cells from infected c57bu6 mice produced ifn-y and l-2 to levels that were only slightly less than uninfected controls. a-6 production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd3 stimulated lymph node cells from infected mice produced elevated ifn-1 suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il-4 or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl-2 proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl-2 does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il-4 produced a fully productive vsv infection at all times analyzed, up to 3 weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells (50-1 00 fold by week 1 and 1,000 fold by week 2). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il-5 and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va 22908 we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or 8-galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed 5 or 14 days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th2 type cytokines when stimulated with rsv infected spleen cells . however, the levels of 11-5 and 1fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il-2 were higher in the bulk culture from vac-f primed animals. the il-4 and il-5 production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed 14 days after intranasal inoculation produced much lower levels of il-4 and 11-5 while the levels of il-2 and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day 3 and subside as cd8+ t cell responses are activated at day 7 post-infection. in contrast, 02m-/-mice, lacking cd8+ t cells, have dramatically elevated nk cell responses on day 7 postinfection. the 02m-/-response is evidenced by increased nk cell activity, as well as up to 5-fold increases in blast and total nki.i+cd3-cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)-2independent, whereas day 7 nk cell responses in 02m-/-mice are csa-sensitive and il-2-dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il-4 and transforming growth factor4 (tgf-0) were examined. induction of il-4 mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days 0, 3, 5, 7, 9, and 14 post-infection demonstrated delayed induction of il-4 protein as compared to ctl activation. tgf-0, evaluated in biological and elisa assays, was induced maximally at days 7 to 9 post-infection. the kinetics of tgf-0 production by cells from infected 82m-/mice was similar to that of normal mice. however, cells from 02m-/-mice produced il-4 at early but not at late times postinfection. together, these results suggest that either il-4 is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the 02m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il-1p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days 1-6 after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after 6 days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with 103 pfu of either a wild type strain of mv(chicago-1,chi-1) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over 28 days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi-1 on d4 (105.7 pfu/ third of implant), and moron d21 (103.2 pfu/ third of implant). hematoxylideosin stained sections of chi-1-infected thymuses showed marked distortion of the cortex and medulla by d4 with thymocyte poilolosis and decreased cellularity. by d14, these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd3,cd4 and cd8 were analyzed by flow cytomehy. there were significant decreases in the cd4+cd8+ cell pop-ulation by d10 with complete loss of all such cells by d28 with chi-1, and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae3) or cd15 co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d4 in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d21. only rare cells stained both with mv ha and cd2 or cd4. mv ha was not expressed over background on any cd4+ cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl-5) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd4 were found to drop dramatically. other cell surface antigens such as cd18, cd43, and cd45 were unaffected during infection with myxoma virus. further more, the downregulation of cd4 by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd4 levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p56lck associates with the cytoplasmic domain of cd4 we have also examined the association of p56lck with cd4 as well as steady state levels of p56lck during viral infection. the modulation of surface cd4 has also been described in hiv infected t cells suggesting that the loss of cell-surface cd4 may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s 1 1 o r us2-6 gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before 18 months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd8+. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p2a(paucisymptomatic) group than in the pl(asymptomatic) and the p2b-f groups (symptomatic group). the cohort of children has now been followed during 4 years, and 46 children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c 10 weeks of age) showed no deletion of their cognate vp14* t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp14+ t cells characteristic of c3h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv-1 endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i1 (pol 11) template. the promoter contains a tata box 30 bp upstream of the transcription initiation site, binding sites for several pol i1 transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol 11. in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt-2, but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i1 promoter (6 pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used (60 pglml), in the range of a typical polymerase in (pol 111) promoter (va-i). purified tax, transactivates this promoter 5-to 10-fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the 0.5 m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i1 promoter and a unique pol i11 promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i1 template containing the 21 base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i1 expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as 16 hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within 48 hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and 11 mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd54 by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within 2 h of virus infection, while an indirect mechanism, regulated by type 1 interferon (ifn), operates within 24 h of virus infection. cd54 expression increased by 4-5 fold within 2h of wnv infection on hef, and by 6-7-fold within 24h. wnv-inactivated, conditioned supematants removed from infected hef cultures after 4 h incubation did not alter cd54 expression on unqimulated hef. whereas conditioned supernatants from 24 h-infected cultures increased cd54 expression by about 1.5-2-fold after incubation for 24 h, but not after 4 h, similar to cd54 induction by 200ulml of ifn-p. increased cd54 expression on hef by wnv was also cell-cycle dependent. cd54 increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd54 by types 1 and 2 ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus 2 and 5, and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd54 expression on hef after 24 h. another alphavirus, ross river, was able to induce cd54 but only by the indirect mechanism of type 1 ifn-dependent release. poly i.c, also, increased cd54 expression to the same extent as ifn-p after 24 h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd54 expression in a manner similar to wnv. the ability of flavivhses to induce increased cd54 expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b8 patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position 3 of the pi7 gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla 68 with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position 7, not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p2microglobulin in the er results in stable cell surface expression of the trimeric mhc-1 molecule. in previous studies we showed that in hpv-16 positive cervical carcinomas there was a loss of mhc-1 protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc-1 is mediated by an hpv-16 encoded protein. human keratinocytes were transfected withvarious hpv-16 constructs including pat16, the full length genome, pat16esx the full length genome with a premature stop codon in e5, puc.et16, the e6 and e7 oncogenes only, and pkve5, expressing e5 from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for 48 hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap-1 and mhc-1 heavy chains in the cells with pat16 as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat16esx and puc.et16 lines showed comparable tap-1 and mhc-1 protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap-1 and mhc-1 mrna between the different cell lines. the data indicate that expression ofhf'v-16 e5 leads to post-transcriptional loss of mhc-1, presumably by interfering with tap. to map and characterize functional differences between e1a of ad5 and adl2, we previously constructed a series of hybrid ad5/12 e1a genes and used them with ad12 e1b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad12 e1a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd8+ as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin-10 from a variety of organisms. many of the numerous immunological properties ascribed to interleukin-10 are shared by the product of bcrfl and this has led to it being termed viral interleukin-10. in order to investigate the activity of viral interleukin-i0 (vil-10) and its interactions with the human interleukin-10 receptor we have expressed the protein in a bacterial and the eukaryotic cos-7 expression systems. the bacterially expressed vil-i0 was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt4.l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp-1. a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil-10 protein that interact with the hil-10 receptor and confer its biological activity. a number of these mutants have been expressed in the cos-7 expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil-10 that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i0 within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the 10.4k or 14.7k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the 15 ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e3l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k3l protein shows sequence similarity to the eukaryotic initiation factor 2a (eif2a) that is phosphorylated and inactivated by pkr. the k3l protein competitively binds the kinase and blocks host eif2a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin-18, tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of 125i-ifna to u937 cells and binding of 125i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for 1fnc1fp. direct binding of 1251-ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd4+ t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a20 cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase 11, wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a118083 t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa 19107. antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p2-microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids 50-57 (h2-kk), 147-155 (h2-kd) and 366-374 (h2-db). the frameshift was introduced 26 amino acids upstream of the h2-kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h2-i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h2-kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h2-dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr8 the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np 383-391 srywairtr was studied using hla b27-restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b'2705 in vitro, and for presentation to ctl clones by hla 827positive targets. even conservative amino acid substitutions of the peptide residues p 4 , 7, and 8 profoundly influenced ctl recognition, without affecting binding to hla 8'2705. these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l 4 gene segment (but not those using tcr va12) were also sensitive to p1 substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class 1. these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla 827, p i , p4 and p8 are "flag" residues with tcr accessible side chains. the e3/19k protein of human adenovirus type 2 (ad2) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e3/19k protein mediates binding to mhc molecules. apart from a stretch of approximately 20 conserved amino acids in front of the transmembrane segment, e3/19k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad2 e3/19k protein. we show that e3/19k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in 293 cells. based on the differential binding of monoclonal antibody tw1.3 and cyanogen bromide cleavage experiments, a structural model of e3/19k is proposed, in which cys 11 and cys 28 as well as cys 22 and cys 83 are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e3/19k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e3/19k molecules. mutation of the three other cysteines at position 101, 109 and 122 had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e3/19k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd8' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent -75% of the u.s. population are being tested to determine which of 5 abundant cmv proteins they recognize by a cd8' ctl response: p28, p65, p150, ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd8' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class 1 epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a2.1 developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p65 and gb together with either of two lipid-based adjuvants, commercially available d0tapm (bcehringer-mannheim) or mf5gth (chiron, emeryville, ca). our preliminary studies with hsv-2 gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa2 as a restriction element in the transgenic model. the ctl response to sendai virus in c57by6 mice is directed almost exclusively to a single h-2kb-restricted epitope derived from the virus nucleoprotein, npj24-332 (sev-9). analysis of 18 independent t cell hybridomas generated from c57by6 mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev-9 bound to kb has shown that the side chaiis of peptide residues phpi, gl484, a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi484 and a d 5 protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev-9. to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev-9 analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp4 and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep1 and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev-9. these data are consistent with a critical role for the gi94 and a d 5 in governing tcr-sev-9eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla42.1 restricted ctl from hla-a2.1 uansgenic mice was the m1 peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/8/34-specific murine ctl that recognized an hla-a2.1 restricted epitope distinct from m1. using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/8 nsl protein. because these ctl did not recognize targets infected with the a/alaska/6/77 saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a2.1 specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common 8 amino acid sequence and two distinct sets of bmding mtif residues. however, the n0name.r peptide was able to sensitize ctl for half maximal lysis at 80-2500 fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions 4 and 8 and was not recognized at any tested concentration, although it bound with higher &ity to hla-a2.1 than the peptide from a/pw8. the a/pr/8 nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c57bv6 mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd8+ immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd8' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h-2b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd8+ ctl response directed against three wellcharacterized epitopes presented by h-2db molecules: "396-404 (fqpq-ngqfi), gp33-43 (kavynfatcgi) and gp276-286 (sgven-pggycl). the h-2db motif is characterized by a sequence of 9 to 11 a.a. with two anchor residues: asn at position 5 and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the 34 lcmv peptides and 11 known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides (28/34) did not bind to db. the other 6 (including the 3 epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb2 4at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt1.aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt1.aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently2~3. rt1.a alleles are naturally linked with the tapa or the tapb allelic group4. we have set out to characterise various alleles for the rt1.a molecule, and find that, for the majority of tapaassociated rt1.a molecules, 3 acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt1.a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b8. seventeen variant peptides were examined for their binding to hla b8; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b8 positive hiv-1 infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, 16132 genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd4+ human t cell lines and clones specific for different hiv antigens (gp120, p66, p24), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct 06520 listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p60 is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p60 is processed by the host cell into the nonamer peptide p60 217-225 and is presented to cytotoxic t lymphocytes by the h-2kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p60 to show that the rate of p60 217-225 production is proportional to the amount of antigen secreted into the host cell cytosol. p60 is degraded in the host cell cytosol with a half life of 90 minutes. the appearance of p60 217-225 is coupled to the degradation of newly synthesized p60. we have determined the rate of intracellular p60 secretion and by accounting for the rate of p60 degradation we estimate that approximately 35 p60 molecules are degraded to produce one p60 217-225 epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd8+ and class i hlarestricted by the b7 molecule. all three clones recognized lllb and rf but not mn strains of hiv-1. using vaccinia vectors expressing truncated versions of the hiv-1 envelope, the clones were found to recognize an epitope within amino acids 287-364, but not including 312-328 of gp120. further mapping of the epitope with synthetic 20-mer peptides overlapping by 10, or 25-mers overlapping by 8, was unsuccessful. the sequence of the region of gp120 recognized by these clones was compared to the predicted hla-87 peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the 10 aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc 835 gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of 835 allelic variants of ebv-lcl. by 835 restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described 8'3501 peptide epitope from pp65 was not able to prime the autologous 835 ebv-lcl for killing by the pp65-specific ctl, whereas a recombinant vaccinia virus expressing whole pp65 could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp41-specific cd8' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of 835 ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b'3502 allele are recognized and killed by the pp65-specific t cell clone, and cell lines carrying 8'3501 alleles are recognized by the hiv gp41-t cell clone. we conclude that the reported cmv pp65 b"3501 restricted epitope is not correct, since the ctl in question will only recognize 6'3502 alleles in combination with the correct pp65 epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus "147-155, ha204-212 and ha210-219 was found to be similar. an 11 amino acid peptide corresponding to ha204-214 which contains the 204-212 epitope was transported at a similar efficiency as the 9 amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue 215, this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at 260.8 fmolelminute (+/-30.5). the km for this peptide was found to be 231.9nm(+/-31.8). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., 19107. previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids 147-155) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct (147-158/r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus (75 < n < 100 amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs (50-158/r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed 6r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the 6r construct was presented at a level equivalent to that seen with the wild-type 50-158/rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den-2 was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den-2 will interact with mim and lymphocytes in different manners, we used two isolates --plo46 strain (obtained from a df patient during taiwan 1981 outbreaks) and 16681 strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after 24 hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd3icdi9 mab and complement prior to adherence procedure and the purity of mim culture was checked by cd14 surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den-2 strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den2 strain. in addition, this dhf-den2 strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv-1) and lymphocytic (h9) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den2 strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den2 strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that (1) different strains of dengue virus could orchestrate quite differently with immune cells, (2) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and (3) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. (2) when viral yields were enhanced early than day5 post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv-1 using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny 10016. primary isolates of hiv-1 have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd4-based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg12 is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd4 binding site (cd4-bs) on gp120. cd4-lgg2 is a recombinant molecule in which the variable domains of both heavy and light chains of lgg2 were replaced with the first and second immunoglobulin-like domains of human cd4. both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg12 and cd4-lgg2 were added at 25 pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p24 production was measured over 14 days of culture and an end-point titer of hiv-1 in the presence and absence of added antibody was determined. both igg12 and cd4-lgg2 were found to reduce the original hiv titer from seven plasma samples with high virus titer (>250 tcid50/ml) by up to 625-fold. this is in comparison to soluble cd4 which only reduced viral infectivity by 55-fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd4-bs of hiv-1 gp120 are able to effectively neutralize primary isolates of hiv-1 and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv 16 the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of 66 overlapping synthetic peptides corresponding to the entire hpv16 capsid proteins was used to generate hyperimmune sera. several antisera against 3 different peptides were reactive with intact hpv16 capsids at titers up to 1:150.000. hiv-1 serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md 20892. previously, we showed that hiv-1 proteins conjugated to 8. abortus (ba) could generate anti-hiv-1 neutralizing antibodies in mice even after depletion of cd4* t cells. in this study a 14-mer peptide from the v3 loop of hiv-1 (mn) was synthesized 013) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v3-klh induced mainly igg1, whereas v3-ba induced all igg isotypes but lgg2a predominated. fecal extracts from mice immunized with v3-ba were shown by elsa to contain iga antibodies. sera from these mice bound gp120, expressed on the surface of infected cells. sera from mice immunized with v3-ba inhibited syncytia formed between cd4' t cells and chronically infected [hiv-i (mn)] h9 cells. inhibition of syncytia, formed by other hiv-1 lab. strains correlated with the degree of their homology with the v3 region of hiv-i (mn). to mimic the efffect of hiv-1, mice were depleted of cd4' cells using anti-l3t4 at the time of primary or secondary immunization. following primary immunization, cd4+ t cell depletion abrogated v3-klh antibody responses, whereas responses to v3-ba were retained and sera from these mice were able to inhibit gp-120mediated syncytia. in secondary responses, cd4' t cell-depletion prevented boosting to v3-klh, but v3-ba increased anti43 and syncytia-inhibiting antibodies. these results suggest that: 1. 8. abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and 2. that infection with hiv-1 with subsequent impairment of cd4' t cell function would not abrogate anti-hiv-1 antibody responses if 8. abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh61-1p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn 38 10 i, 2departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c57bl/6j(b6) mice treated with the mab mel-14 to l-selectin have been analysed following i.n. infection with sendai virus. mel-14 treatment caused a 70-90% decrease in the lymphocyte recruitment to the mediastinal (h4ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd8+ ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day 10 after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel-14 antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel-14-treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd4+ th precursor cells into lymph nodes. facs sorting for l-selectin+, 8220+, and l-selectin-, b220+ cell populations from the mln and the cln of normal b6 mice 9 days post sendai virus infection, showed that the afcs were from the l-selectin-, b220+ cell population, a population which comprised 6-10% ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv-1 infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position 582 in gp41, is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd4 binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position 281, upstream from the v3 loop, does not involve the same site and does not involve v3. substitution of thr or ile also confers resistance. replacement of the v3 loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v3 peptide of mn but is affected by regions outside v3. the other appears to be conformational and outside v3, but its functional recognition is influenced by the v3 loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids 579-613 of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus 10 to 100 fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids 603-622 of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p<0.05). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv 16 proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e6 a n d e7 genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e7 either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e7-specific and cd4+ t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e7 protein of hpv 16 was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in 4 groups of mice (1) those not challenged with e7 (2) mice not grafted but challenged with e7 protein in the ear (3) mice primed by grafting with 107 hpv e7 expressing cells and challenged with e7 protein (4) mice primed by grafting with 5 x 105 hpv 16 e7 cells on day 7, grafted again with lo7 hpv 16 e7 cells on day 14 and challenged with e7 protein in the ear. mice optimally grafted and challenged (group 3) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group 4) exhibited igg antibody levels comparable to the control group (1). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp70) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during 18 months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr1 antibodies was also investigated in 100 hepatitis c viremic individuals and 25 non-viremic patients. a high frequency of positive reaction (90%) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp120 v3 loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., 1992) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of 18 reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd3cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = ,005) and with serum rotavirus iga titers stimulated by the different reassortants (p < ,001). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, 55 individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v3 profiles were determined using synthetic proteins derived from the amino acid sequences of the v3 region of 5 laboratory strains of hiv-1 in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v3 regions of mn, sf2, ny5 and han/sc. less than 50% of individual in each group recognized the v3 peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< .001). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p24 ag inhibition assays. although most patient serum was capable of inhibiting p24 ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v3 profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at 8 days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by 15 days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at 8 days post-infection, timepoints from 30 days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about 2-fold by 15 days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd4+ lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd29 were evaluated in 49 uninfected controls (group l), 84 hiv-1 positive patients with 220% cd4+ t cells (group 2), and 47 hiv-i-infected patients with ~2 0 % cd4+ t cells (group 3). most of these subjects also had 3-color staining for cd4\cd45ro\cd45ra. the appearance of positive cd29 and cd45ro on hivinfected and uninfected cells correlated well (r=.82 p<.ool). the percentage of cells staining cd4+\cd29+.(bright plus dim) was 43.3 (95%cl 37.3-49.4) in group 1, 28.9 (27.5-30.4 ) in group 2, and 10.2(8.6-11.9) in group 3. the respective values for these groups that were cd4+\cd2gbwm was 30.6 (26.9-34.3), 20.7 (1 9.3-22.2), and 7.4(6.3-8.6). values for cd4+\cd45ro+ were 33.7 (31.8-35.5), 21.8 (20.5-23.1), and 9.9 (8.5-11.3), respectively. in single factor discriminate function tests, the %cd4+\cd29+ cells best predicted subject group (87% correct), proving to be a better discriminator than %cd4+\cd29b'h' (77% ~orrect),cd4+\cd29~"" (5 l%), cd4+\cd45ro+ (75%) and cd4+\cd45ro+\cd45ra-(63%). overall, no advantage was seen to splitting the cd4+\cd29+ cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd4+\cd45ro+ compartment into cd45ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv-1 infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl60 recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp160 were performed at month 0 and 1 and two boosts of recombinant gpl60mn/lai at month 3 and 6 in alum or incomplete freund adjuvant(1fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from 18 out of 20 donors : seven out of eighteen (39%) were found to present envelope specific cytotoxic activity at months 2, 4, 7 or 12 post immunization ; this activity was characterized as a cd3+,cd8+, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd8+ cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al 35294. mhc class i restricted cd8' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il-2 and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd8' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd4+ and cd8' t cells by their expression on the surface of distinct d45r isoforms. cd45ra is expressed on naive or virgin t cells, while cd45ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd8+ t cells. the cd8' cells were stained for cd45ro' (pe) and cd45ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a 4 hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd45ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd45ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd8+/cd45ro+ t cells. similarly, cd8+/cd45rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd8+/cd45rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd8+ t cell response in lcmv infection we have observed a profound activation and proliferation of cd8+ t cells with a 10-40 fold increase in total number peaking at day 8-9 post infection. in c57bw6 mice, most of the viral antigen is cleared by day seven, and after day 9 the total cd8+ number per spleen drops about 10-fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd8+ cell remains remarkably stable between day 7-8 of the acute infection and for many months thereafter. thus, the decline in the cd8' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd4+ cells from c57bl6 female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd4+ cells either by magnetic bead depletion of non-cd4+ cells, or by facs after staining with anti-cd4 monoclonal antibody these enriched (>90% cd4+) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + 3x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at 111600 cd4+ cells, compared to a frequency greater than 1/1ooooo in naive controls. the memory cd4+ cells were further characterized as cd45rb-low (1/472) , cd44-high (1/294), lselectin-low (1/364), and cd49d-high (vla-4-high) (v102). this is close agreement with other phenotyping studies on cd4+ memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn 38105. the immune response of influenza a and sendai-virusspecific, memory cd8' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c57bu6 mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated 5 or 6 days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd8' t cells from the ln and spleen resulting in dna fragmentation of 12-18% ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent 9 days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb41 a3 cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while 1oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb41a3 cells were treated with 500pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by 500pm of nmda was reversed by 300pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c6 on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a118083 to carol s. reiss. pediatrics, university of iowa, iowa city, ia. 52242 mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. 40.90% of suckling c57bu6 (kbdb) mice inoculated intranasally with mhv-jhm at 10 days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at 3-8 weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c57bu6 mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c57bv6 mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids 510-518 (cslwngphl, db), 598-605 (rcqifani, kb), and 1143-1151 (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c57bu6 mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of 3-4 week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of 3-4 week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after 14 days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd8+ t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases3 research units, national animal disease center, usoa, agricultural research service, ames, ia 5001 0 a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a 3.0 kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel80 gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in 4-week-old or 4-day-old pigs, lltba2 replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba2-infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba2, 4-day-old pigs were infected intranasally with lltpa2 or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about 20-fold for lltpa2. abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba2-infected pigs. while replication of lltbres in the brain progressed until death at 7 days post-infection, replication of lltpa2 in the brain ceased by 9 days post-infection and the pigs exhibited only mild clinical signs. since lltba2 is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable 07 invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type 1 (hsv-1) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv-1 in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected 293 cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb2 iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of 3-4 week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day 60 post-infection. in contrast, in a large group of cba mice studied up until 393 days post-infwtion, viral rna could be detected by both techniques in 50% of mice until as late as 268 days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca 92037 our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within 4 months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p27 production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p27 production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v1 region of the siv env gene. the majority (>95%) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v1 region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class 1 positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, 10 fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd4+ t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd4+ t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd4+ t cells with monoclonal antibody gk1.5. both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd4+ t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md 20850; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn-1 epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn-1 subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv-1 infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv-1 of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp27. because of its early expression during the hsv replication cycle, lcp27 represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein4 (nsi) and the lcp27 sequence of hsv-2. the nsi-icp27 protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing 5 pg of nsi-icp27. responder cells obtained from draining lymphnodes were re-stimulated in vitro with p815 cells lransfected with icp27 and then lesled for cytolytic activity on icp27-p815 and control p815. the induction of icp27 specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h-2d haplotype: a dd restricted epitope from the gp160 protein of hiv-1 and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc 27599 a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately 50?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd-1 mice were inoculated subcutaneously with 2 x 10' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo5 ed, of influenza h s . au 24 corn1 mice suffered severe disease and 50% died. only one of 12 ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was 246, while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, 150. in a parallel experiment, no influenza infectivity was detected in the lungs of 12 ha vector immunized mice at 4 days postchallenge. in contrast, 8/12 pbs-inoculated mice and 5/12 inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of 3.04 and 1.93 x lo6 pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h1 hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from 100 to 0.1 ug of dna was inoculated by intramuscular and intradermal routes. from 0.4 ug to 0.0004 ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a 20 week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required 250 to 2,500 times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations (4 ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the 20 weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv40 promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg5rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l 4 caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl6 e7 hpv associated cervical cancer cells express hpv16e7 protein and antibody to hpv16 e7 can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e7 protein of hpv16, and expressing e7 protein in the skin, has recently been described (1) and these mice develop spontaneous humoral immunity to e7 protein similar to patients with cervical cancer(2). to determine whether immunisation could induce immunity to e7 sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv16e7 protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv16 e7 expressing tumour cells in nrro. we then used similar immunisation with e7iquil a to induce e7 specific immunity in fvb (h-29) mice. h-2qskin @s expressing e7 were not rejected by e7 immunised h-zq mice, though immunisation induced antibody to e7, and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl6 e7 lack a tc epitope in the context of h-zq, or that expression ofe7 in the skin from the e7 transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types 16 or 18, and continued expression of the e6 and e7 gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e6 and e7 from hpv16 and 18 with the aim of inducing e6 and e7 specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e6/e7 reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv16 â�¬7 specific ctl from c57bu6 mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi 6 e7 alone, with similar recognition of the defined immunodominant h-2db restricted epitope, e7 residues 49-57. ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa 19486 mice infected with the laboratory strains of a/pr/8/34 (hln1) or the mouse adapted a/hk/68 (h3n2) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h1 and h3 strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr8 and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/92 were susceptible to subsequent infection with the a/hk/68 strain although they were resistant to weight loss and death. other strains such as a/beijing/89 or a/georgia/93 provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/68 challenge than mice previously infected with a/bei/89 and a/ga/93, and were similar to mice that had been previously infected with a/bei/92. thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h-21-ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd8+ ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day 10 after infection. there was little effect on virus clearance. as found previously with cd4-depleted h-2b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd8+ t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd8+ cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd8+ ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd8+, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd8+ responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type 1 (hsv-1) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv-1 to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day 0 and day 21 resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv-1 vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv-1. viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl47-158 and plasmodium berghei cs protein 252-260 were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam3cyj) and dissolved in a 1% dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and 10% soya oil in water. 1 wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of 3 mice intra-peritoneally or subcutaneously at 1.10 and 20 days. 7 days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard 4 hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of 1w:l the plasmodium berghei peptide encapsulated in micmpartides gave 47% iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p3ccs252-260 gave a level of lysis of 82% at an e:t ralioof1w1. these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type 3 poliovirus (shah sauketf) together with the regular type 1 and type 2 canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i1 clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp1. as previously shown for hypsin-mated type 3 poliovirus @vm alone. trypsin used to modify the type 3 component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with 10-fold concentrated type 3 component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase 1 aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type 3 poliovirus. no unexpected sideeffects were recorded phase i1 trials comprised 50 adult volunteers with at least 5 years since the last dose of poliovirus vaccine and 50 children who were due to receive the third dose of the regular immunization schedule at about 2 years. in both groups. 25 individuals received tryipv and 25 were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type 3 poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with 0.4 ug of an h1 expressing plasmid dna (pcmvri1). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at 10 days post boost protection against the lethal challenge was complete at 10 days, 1 month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv-1 t-cell epitopes: generation of hla-b*3501-restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle6 and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv-1 patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys)4 and synthetic htlv-1 peptides which seem suitable for vaccine design. htlv-1 amino acid sequences were searched for eight to 14mer patterns carrying the anchor residues of the hla-b*3501 peptide motif at positions two and eight to fourteen. 65 candidate peptides were synthesized according to the matched sequence patterns. their hla-b*3501 affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b*3501 cells. the fourth group (controls) were inoculated with h3n2 (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i1 restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h3n2 group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i1 ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp120 la1 sequence. seventeen patients participating in a phase i gp160 protocol and 13 patients participating in a phase i gp120 protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into 96 well plates containing peptides overlapping the la1 sequence of gp120, pulsed on day 7 with tritiated thymidine and harvested and counted on day 8. results: the percentage of patient's pbmcs from each trial with an lsi 2 5 to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp160 or gp120 proliferate to multiple peptides within the gp120 molecule. reactivity from the end of c1 through early c2 (lai #i 12-21 1) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v3 loop peptide (la1 #300). although the percent reactivity to the entire gp120 molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c3 (lai #319-348). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il-12 production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] 1 and 2a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna 3 proteins (3a, 3b, 3c). and ebna 3c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna 1, 2, lp or lmp 1. the ebv-specific ctl lines expressed cd3/cd8 and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class-11. these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna 3 family of proteins and lmp 2a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology,2 genetic therapy, inc., gaithersburg, maryland 20878 replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. 1 :192-200; 1994) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb274nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of 100, 50 and 10. in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: 80.3 f 1.3% vs 6.2* 0.5%. 49.6*1.6% v s 5.7*0.4%. and 22.8*3.5% vs4.8*0.5% (meanrts.e.m., n=3; p<o.ol, all comparisons) a t e:t ratios of 100, 50 and 10, respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping 20-mer peptides derived from p24. two individuals (008 and 617) had ctl against p24.14 (a(mino) a(cids) 263-282, krwiilglnknrmysftsi), two others (038 and 157) recognized p24.17 (aa293-312, frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p26.14 (8653) and p26.17 (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of 9 aa overlapping 10-mers and to test for hiv-1isiv crossreactivity. both two p24.14 epitopes (possibly restricted by hla-a2) and the p26.14 epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p24.17 epitopes is in progress. the sn p26.17 epitope was also non-crossreactive, despite only one aa difference (position 6, s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p24 in humans (johnson ji 147,1512 ,1991 , buseyne jv 67,694,1993 and to p26.17 in cynomolgus macaques (gotch jmprim 22.1 19,1993) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd-4 and cd-8 1-cells, b-cells and adherent cells, while an average of 5% of virus was associated with lps-stimulated 8-cells and adherent cells. in the second experiment the combined function of antigen presenting cells, 1-helper cells and 8-cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day 0, and decreased at days 2, 3 and 4 post-cvb3, infection relative to unlnlected animals. conclusions: these data indicate that cvb3, associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb3, modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb3,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i11 j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih 3t3 fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of 2.8 kb in hind i11 j and of 7.7 kb in hind i11 j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i11 j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the 7.7 kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv5-4 progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v 5 -4 cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv5-4 was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a 5kb transcript in northern blot analysis. a 2 kb cdna was isolated and codes f o r a novel polypeptide o f 800 amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/305/57 in in uitro assays of viral infectivity of the mouse b-lymphoma a20-1.11. we have found that although infection sensitizes the a20 cells for recognition by both class i and class i1 restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a20 cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/305/57 from a different passage history, we find that n o t only are the infected a20 cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a 10-2 the therapeutic and prophylactic antiviral efficacy of interleukin-12 (il-12) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il-12 commenced 6 hours after mice were infected w i t h hsv, and was continued daily for a total of 5 days. il-12 therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. 1 .0x106we~ells/well of both adherent and nonadherent cell types were infected with two different den-2 virus strains : (1) thiland strain isolated from the dengue hemorrhagic fever(dhf1 patient and (2) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den2 and dhf virus strain replicated much more efficiently (2.3x104pfu/ml on day 3 p.i.1 than df strain; (b) nonadherent cells were 10 times less efficient to produce viral yields than adherent cells (dhf and2 df strains peaked at 6.3~10' pfu/ml and 1.5~10 pfu/ml on day 3 p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi-2/crmn38k gene trigger an inflammatory response by 36-48 hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at 48-72 hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo 63110 murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice 2-8 months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for 14 days. by this method, 1 pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted 1:lo. scid mice were used as a second detection assay for mcmv. using 46 scid mice and different doses of virus, the lds, of mcmv in scid mice was 2-3 pfu. mcmv infections were detected when 10-25 pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of 34 spleens, 34 kidneys, and 37 salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after 10-40 days. in addition, lytic mcmv was detected in scid mice 28 days after injection with 2(10)' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the 72kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp72 mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp72 expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp72, but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp72-negative cell line showed no difference from control viruses, and the presence of hsp72 did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp70 proteins act as molecular chaperones and that hsp72 can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp70 for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw2 in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp72 does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp72 during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs7/bl6 and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days 7 and 10 postinfection, mln or spleen cells from both strains of mice produced il-2, il-6, il-10 and ifn-y, but little or no il-4 or il-5. however, following in vivo treatment with anti-ifn-y antibody, production of 1l-4 and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs7/bl6 mice showed little change in cytokine profile. an increase in il-6 and 1l-10 production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of 1l-2 and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c57/bl6 mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa 19104 rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a 505 amino acid type i membrane glycoprotein with 3 potential n-glycosylation sites at asn37, asn247, and asn319. due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble 434 amino acid form of rgp lacking a transmembrane domain (rgpt434). rgpt434 was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt434 secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt434 was secreted only when asn3l 9 was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt434. although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of 6 his residues was added to the cooh-terminus of rgpt434. this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+2-agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev-21 locus is reduced 85 to 95% compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci (1,6,10 and 11 ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a9 -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad 65. one of the major isletcell antigens, shows a high degree of homology with the 2c protein of cbv-4. studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp60 and gad65, are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p53 alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p53 expression in 31 of 52 (60%). p53 detection by ipox is usually associated with mutated p53. sscp for exons 5-9 revealed p53 alterations in 2 of 25 specimens, and loh at exon 4 was seen in only 2 of 12 evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p53. we recently reported that sv40 induces mesotheliomas in rodents, and that sv40-like sequences are present and expressed in mpm. of note, sv40 large t antigen (tag) binds and inactivates wt p53 i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p53 may result from its physicdl binding with tag. tag was detected by ipox in 32 (62%) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p53 (p2 = 0.008, fisher's exact text), and preliminary experiments suggest co-precipitation of p53 and tag. finally, we demonstrate that waf-1 is not detectable in mpm expressing wt p53 suggesting that p53 is inactive. we speculate that tag expression in mpm binds to and inactivates p53 contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in 90% of cervical carcinomas and possibly 95% of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a2 expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type 16' and hla-a2'. culture media consisted of rpmi-10% human serum supplemented in three cases with 15 u/ml il-7, in another three cases with 3 u/ml il-2 and 15 u/ml il-7, and in the final three cases with 10 u/ml il-2 and 15 u/ml il-7. the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c33a (hpv, hla-a2') transfected with either the hpv type 16 e6 or e7 genes. one patient's cells maintained with 10 uiml il-2 and 15 ulml il-7 showed hpv e7 protein specific cytotoxicity in a hla-a2 restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k-562. the ctl efficiently lysed a c33a-e7 clone but in contrast a vector only transfectant was not lysed. the lysis of c33a-e7 was blocked with a-cd3 and a-cd8 antibodies at 66% and 41% respectively. facs analysis determined that this ctl population was 92.8 % cd3'cds' and 7.3% cd3'cd4'. limited e6 recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e7 responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv-1 infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv-1 infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b8 restricted epitopes: p17-3 gag and amino acids 18-26 of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv-1 infection may be influenced by an effective host immune response against hiv-1 and/or by viral properties. to investigate the cellular immune response to hn-1 we analyzed ctl activity against different hiv-1 proteins in long-term asymptomatic hiv-1 infection as well as during rapid progression to aids. methods: 10 longterm asymptomatic individuals with cd4 counts >500 celllpl after more than 8 years of infection were selected from the amsterdam cohort study on aids versus 10 subjects who progressed to aids < 5 years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv-1 ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus 70 which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus 11 isolates from an outbreak of disease in southem india. sequence analysis within the 5' untranslated region reveals that these isolates fall into two groups that differ by -20% (equivalent diversity to that seen between between published sequences of poliovirus 1 and coxsackie a 9 virus). these two groups of viruses also differ in their cell tropism. isolates defined as group 1 by their 5'utr sequence grow equally well on ht29 cells (a human colon carcinoma cell line) and vero cells. isolates of group 2, with one exception, grow only on ht29 cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the 5' untranslated region sequence of a group 1 virus but the protein profile and cellular tropism of a group 2 virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma 021 15 polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c3-i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py4 is the mouse mammary tumor virus 7 superantigen (mtv 7 sad gene, which deletes t cells required for py tumor immunosurveillance in h-2' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i1 and pedatteck (peru). both strains are susceptible to py as 100% of infected animals develop a full profile of tumors. crosses between cs7br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech 11, yield f1 progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs7br) x c57br) and ((czech i1 x cs7br) x c57br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i1 and peru mice by pcr using primers specific for mtv 7 sag indicates that both strains are negative for proviral mtv 7 sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i1 genomic dna. these results indicate that, like the c3hibi, the pedatteck and czech i1 contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c57br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i1 mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis-2 is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis-2 suppresses primary antibody response more efficiently in infected mice. some of the fts-2 infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis-2 show a high degee of homology between this variant and the prototype f-mulv clone 57. in this study we have attempted to localize the genomic determinant of fis-2 which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis-2 and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd4) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th2 cd4 t cell subset. interestingly, when cd4 responses were observed, cd8 tcell responses were also detectable. 2 . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex-3xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp1 was strain specific, and the polymerase (3d) was the most immunogenic and cross reactive. d) a construct comprising 3d and the immunodominant vp1 epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus 1 (ehv-1; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv-1. to generate useful amounts of individual ehv-1 glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv-1 infection. au three glycoproteins induced serum (elisa) antihodies to ehv-1, and ehv-1 gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv-1, protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv-1 antigen were observed for each of the ehv-1 glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv-1. molecular pathogenesis of ural infeetiom 52-331 enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb3, infection in adolescent c3h/hesnj mice. antigen presenting cell, 1-helper cell and 8-cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo5 plaque forming units of cvb3, at day 0 and with lo7 srbc's at days 0, 2, 3 and 4 post-cvbb, infection. splenocytes were harvested 4 days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb3, was associated with 12.9% to 17.4% of cd-8 positive t-cells and w a 11 % to 26% of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a 248% and 738% under non-stimulated conditions. an average of 1 % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. 1994, in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by 24% and 15% of residues, and the 1896 nt s genome is shorter by 163 nt. we surveyed 174 reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; 27 (15.6%) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california (4), arizona (3), new mexico (l), and mexico (2). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to 15.5%. but amino acid distances were less than 2%. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd8+ ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a2+ patients, htlv i-specific cd8+ ctl restricted by hla-a2 recognize a peptide derived from the htlv i tax protein (tax 11-19 llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd8' tax 11-19 specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag 556-564 vl&sdfri) presented by hla-a2. these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c3hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd4+ t helper cells in this autoimmune response by treating mice with the cw-specific gk1.5 monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c3h mice by showing that depletion of cd4+ cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b 1o.br mice than in c3h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl57 (h2n2) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues 204-212 and 210.219. when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl57-immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha 204-219 subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha 210-219 epitope of influenza strain a i m 5 7 in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha 210-219 specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv-1 gp41 and in the n-terminus of hla class i1 beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i1 molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = 0.027 by fisher two tail analysis)in a second, 7 year-longitudinal study of 12 progre.ssors and 12 stable patients we found: (1) the production of crab was seen in 70 -80% of rapid progresson, while the true stables produce only infrequent low-titers crab. (2) in rapid pmgressors, production of crab preceded by 2-3 years the marked drop in cd4 counts. (3) crab production did not correlate with the degree of hyperglobulinemia in these patients. (4) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd4 counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn-7 receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn-1 and 11-2 production, without detectable 11-4, and with decreased 11-10 secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i1-4 producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th-2-type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord-308201-lavcsqov authors: desforges, marc; le coupanec, alain; dubeau, philippe; bourgouin, andréanne; lajoie, louise; dubé, mathieu; talbot, pierre j. title: human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? date: 2019-12-20 journal: viruses doi: 10.3390/v12010014 sha: doc_id: 308201 cord_uid: lavcsqov respiratory viruses infect the human upper respiratory tract, mostly causing mild diseases. however, in vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, causing a more severe disease (e.g., pneumonia). respiratory viruses can also exacerbate asthma and lead to various types of respiratory distress syndromes. furthermore, as they can adapt fast and cross the species barrier, some of these pathogens, like influenza a and sars-cov, have occasionally caused epidemics or pandemics, and were associated with more serious clinical diseases and even mortality. for a few decades now, data reported in the scientific literature has also demonstrated that several respiratory viruses have neuroinvasive capacities, since they can spread from the respiratory tract to the central nervous system (cns). viruses infecting human cns cells could then cause different types of encephalopathy, including encephalitis, and long-term neurological diseases. like other well-recognized neuroinvasive human viruses, respiratory viruses may damage the cns as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to cns cells (virus-induced neuropathology). the etiological agent of several neurological disorders remains unidentified. opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of these disorders whose etiology remains poorly understood. herein, we present a global portrait of some of the most prevalent or emerging human respiratory viruses that have been associated with possible pathogenic processes in cns infection, with a special emphasis on human coronaviruses. the central nervous system (cns), a marvel of intricate cellular and molecular interactions, maintains life and orchestrates homeostasis. unfortunately, the cns is not immune to alterations that lead to neurological disease, some resulting from acute, persistent or latent viral infections. several viruses have the ability to invade the cns, where they can infect resident cells, including the neurons [1] . although rare, viral infections of the cns do occur [2] . however, their incidence in clinical practice common virus, is associated with febrile illness, fever, cough and congestion [97, 98] , as well as a characteristic rash and koplik's spots [99] . in rare circumstances, significant long-term cns diseases, such as [99] post-infectious encephalomyelitis (pie) or acute disseminated encephalomyelitis (adem), occur in children and adolescents. other examples of rare but devastating neurological disorders are measles inclusion body encephalitis (mibe), mostly observed in immune-compromised patients, and subacute sclerosing panencephalitis (sspe) that appears 6-10 years after infection [100] . yet, with the exception of hiv, no specific virus has been constantly associated with specific human neurodegenerative disease. on the other hand, different human herpes viruses have been associated with alzheimer's disease (ad), multiple sclerosis (ms) and other types of long-term cns disorders [101] [102] [103] . as accurately stated by majde [104] , long-term neurodegenerative disorders may represent a "hit-and-run" type of pathology, since some symptoms are triggered by innate immunity associated with glial cell activation. different forms of long-term sequelae (cognitive deficits and behavior changes, decreased memory/learning, hearing loss, neuromuscular outcomes/muscular weakness) were also observed following arboviral infections [83, 103, [105] [106] [107] . including the few examples listed above, more than one hundred infectious agents (much of them being viruses) have been described as potentially encephalitogenic and an increasing number of positive viral identifications are now made with the help of modern molecular diagnostic methods [8, 70, [108] [109] [110] . however, even after almost two decades into the 21st century and despite tremendous advances in clinical microbiology, the precise cause of cns viral infections often remains unknown. indeed, even though very important technical improvements were made in the capacity to detect the etiological agent, identification is still not possible in at least half of the cases [110, 111] . among all the reported cases of encephalitis and other encephalopathies and even neurodegenerative processes, respiratory viruses could represent an underestimated part of etiological agents [104] . respiratory syncytial virus (rsv), a member of the orthopneumovirus genus [112] , infects approximately 70% of infants before the age of 1 and almost 100% by the age of 2 years old [113] , making it the most common pathogen to cause lower respiratory tract infection such as bronchiolitis and pneumonia in infants worldwide [32, 114] . recent evidence also indicates that severe respiratory diseases related to rsv are also frequent in immunocompromised adult patients [8, 115] and that the virus can also present neuroinvasive properties [8] . over the last five decades, a number of clinical cases have potentially associated the virus with cns pathologies. rsv has been detected in the cerebrospinal fluid (csf) of patients (mainly infants) and was associated with convulsions, febrile seizures and different types of encephalopathy, including clinical signs of ataxia and hormonal problems [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] . furthermore, rsv is now known to be able to infect sensory neurons in the lungs and to spread from the airways to the cns in mice after intranasal inoculation, and to induce long-term sequelae such as behavioral and cognitive impairments [127] . an additional highly prevalent human respiratory pathogen with neuroinvasive and neurovirulent potential is the human metapneumovirus (hmpv). discovered at the beginning of the 21st century in the netherlands [128] , it mainly causes respiratory diseases in newborns, infants and immunocompromised individuals [129] . during the last two decades, sporadic cases of febrile seizures, encephalitis and encephalopathies (associated with epileptic symptoms) have been described. viral material was detected within the cns in some clinical cases of encephalitis/encephalopathy [130] [131] [132] [133] [134] but, at present, no experimental data from any animal model exist that would help to understand the underlying mechanism associated with hmpv neuroinvasion and potential neurovirulence. hendra virus (hev) and nipah virus (niv) are both highly pathogenic zoonotic members of the henipavirus genus and represent important emerging viruses discovered in the late 1990s in australia and southern asia. they are the etiological agents of acute and severe respiratory disease in humans, including pneumonia, pulmonary edema and necrotizing alveolitis with hemorrhage [135] [136] [137] [138] . although very similar at the genomic level, both viruses infect different intermediate animal reservoirs: the horse for hev and the pig for niv as a first step before crossing the barrier species towards humans [135] . in humans, it can lead to different types of encephalitis, as several types of cns resident cells (including neurons) can be infected [139, 140] . the neurological signs can include confusion, motor deficits, seizures, febrile encephalitic syndrome and a reduced level of consciousness. even neuropsychiatric sequelae have been reported but it remains unclear whether a post-infectious encephalo-myelitis occurs following infection [141] [142] [143] . the use of animal models showed that the main route of entry into the cns is the olfactory nerve [144] and that the nipah virus may persist in different regions of the brain of grivets/green monkeys [145] , reminiscent of relapsing and late-onset encephalitis observed in humans [146] . influenza viruses are classified in four types: a, b, c and d. all are endemic viruses with types a and b being the most prevalent and causing the flu syndrome, characterized by chills, fever, headache, sore throat and muscle pain. they are responsible for seasonal epidemics that affect 3 to 5 million humans, among which 500,000 to 1 million cases are lethal each year [147, 148] . associated with all major pandemics since the beginning of the 20th century, circulating influenza a presents the greatest threat to human health. most influenza virus infections remain confined to the upper respiratory tract, although some can lead to severe cases and may result in pneumonia, acute respiratory distress syndrome (ards) [30, 149] and complications involving the cns [150] [151] [152] . several studies have shown that influenza a can be associated with encephalitis, reye's syndrome, febrile seizure, guillain-barré syndrome, acute necrotizing encephalopathy and possibly acute disseminated encephalomyelitis (adem) [153] [154] [155] [156] [157] [158] . animal models have shown that, using either the olfactory route or vagus nerve, influenza a virus may have access to the cns and alter the hippocampus and the regulation of neurotransmission, while affecting cognition and behavior as long-term sequelae [8, 69, [159] [160] [161] [162] . the influenza a virus has also been associated with the risk of developing parkinson's disease (pd) [151] and has recently been shown to exacerbate experimental autoimmune encephalomyelitis (eae), which is reminiscent of the observation that multiple sclerosis (ms) relapses have been associated with viral infections (including influenza a) of the upper respiratory tract [163] [164] [165] . another source of concern when considering human respiratory pathogens associated with potential neuroinvasion and neurovirulence is the enterovirus genus, which comprises hundreds of different serotypes, including polioviruses (pv), coxsackieviruses (cv), echoviruses, human rhinoviruses (hrv) and enteroviruses (ev). this genus constitutes one of the most common cause of respiratory infections (going from common cold to more severe illnesses) and some members (pv, ev-a71 and -d68, and to a lesser extent hrv) can invade and infect the cns, with detrimental consequences [166] [167] [168] [169] . even though extremely rare, hrv-induced meningitis and cerebellitis have been described [170] . although ev infections are mostly asymptomatic, outbreaks of ev-a71 and d68 have also been reported in different parts of the world during the last decade. ev-a71 is an etiological agent of the hand-foot-mouth disease (hfmd) and has occasionally been associated with upper respiratory tract infections. ev-d68 causes different types of upper and lower respiratory tract infections, including severe respiratory syndromes [171] . both serotypes have been associated with neurological disorders like acute flaccid paralysis (afp), myelitis (afm), meningitis and encephalitis [166, [172] [173] [174] [175] . last but not least, human coronaviruses (hcov) are another group of respiratory viruses that can naturally reach the cns in humans and could potentially be associated with neurological symptoms. these ubiquitous human pathogens are molecularly related in structure and mode of replication with neuroinvasive animal coronaviruses [176] like phev (porcine hemagglutinating encephalitis virus) [177] , fcov (feline coronavirus) [178, 179] and the mhv (mouse hepatitis virus) strains of mucov [180] , which can all reach the cns and induce different types of neuropathologies. mhv represents the best described coronavirus involved in short-and long-term neurological disorders (a model for demyelinating ms-like diseases) [181] [182] [183] . taken together, all these data bring us to consider a plausible involvement of hcov in neurological diseases. the first strains of hcov were isolated in the mid-60s from patients presenting an upper respiratory tract disease [184] [185] [186] [187] . before the severe acute respiratory syndrome (sars) appeared in 2002 and was associated with sars-cov [188] [189] [190] , only two groups of hcov, namely hcov-229e (previous group 1, now classified as alphacoronavirus) and hcov-oc43 (previous group 2, now classified as betacoronavirus) were known. several new coronaviruses have now been identified, including three that infect humans: alphacoronavirus hcov-nl63 [191] and betacoronaviruses hcov-hku1 and mers-cov [192, 193] . the hcov-229e, -oc43, -nl63 and -hku1 strains are endemic worldwide [31, 184, [194] [195] [196] [197] [198] [199] and exist in different genotypes [200] [201] [202] [203] [204] [205] [206] [207] . in immunocompetent individuals they usually infect the upper respiratory tract, where they are mainly associated with 15-30% of upper respiratory tract infections (uri): rhinitis, laryngitis/pharyngitis as well as otitis. being highly opportunistic pathogens [14] , hcov can reach the lower respiratory tract and be associated with more severe illnesses, such as bronchitis, bronchiolitis, pneumonia, exacerbations of asthma and respiratory distress syndrome [17, 31, [208] [209] [210] [211] [212] [213] [214] . the 2002-2003 sars pandemic was caused by a coronavirus that emerged from bats (first reservoir) [25] to infect palm civets (intermediary reservoir) and then humans [215] . a total of 8096 probable cases were reported and almost 10% (774 cases in more than 30 countries) of these resulted in death [216] [217] [218] . the clinical portrait was described as an initial flu-like syndrome, followed by a respiratory syndrome associated with cough and dyspnea, complicated with the "real" severe acute respiratory syndrome (sars) in about 20% of the patients [31,219]. in addition, multiple organ failure was observed in several sars-cov-infected patients [220] . in the fall of 2012, individuals travelling from the arabian peninsula to the united kingdom were affected by the middle-east respiratory syndrome (mers), a severe lower respiratory tract infection that resembled sars, leading also to gastrointestinal symptoms and renal failure among some patients [221] . molecular sequencing rapidly showed that the new epidemic was caused by a new coronavirus: the mers-cov [193, 222, 223] . mers-cov most probably originated from bats before infecting an intermediary reservoir (the dromedary camel), and also represented a zoonotic transmission to humans. phylogenetic analyses suggest that there have been multiple independent zoonotic introductions of the virus in the human population. moreover, nosocomial transmission was observed in multiple hospitals in saudi arabia [221, [224] [225] [226] [227] [228] [229] [230] . although possible, human-to-human mers-cov transmission appears inefficient as it requires extended close contact with an infected individual. consequently, most transmission have occurred among patients' families and healthcare workers (clusters of transmission). a more efficient human-to-human transmission was observed in south korea, during the 2015 outbreak of mers-cov [231, 232] . even though it has propagated to a few thousand people and possesses a high degree of virulence, mers-cov seems mostly restricted to the arabic peninsula and is not currently considered an important pandemic threat. however, virus surveillance and better characterization are warranted, in order to be prompt to respond to any change in that matter [23, [233] [234] [235] . as of october 8, 2019, the world health organization (who) reported that mers-cov had spread to at least 27 different countries, where 2468 laboratory-confirmed human cases have been identified with 851 being fatal (https://www.who.int/emergencies/mers-cov/en/). as observed for the four circulating strains of hcov [31, 194] , both sars-cov and mers-cov usually induce more [228, 236] severe illnesses, and strike stronger in vulnerable populations such as the elderly, infants, immune-compromised individuals or patients with comorbidities [31,237]. over the years, like sars-and mers-cov, the four endemic hcov have also been identified as possible etiological agents for pathologies outside the respiratory tract. indeed, myocarditis, meningitis, severe diarrhea (and other gastrointestinal problems) and multi-organ failure [220, 221, [238] [239] [240] [241] have been reported, especially in children. recent investigations on hcov as enteric pathogens demonstrated that all hcov strains can be found in stool samples of children with acute gastroenteritis; however, no evidence of association could yet be clearly demonstrated with disease etiology [242, 243] . different reports also presented a possible link between the presence of hcov within the human central nervous system (cns) and some neurological disorders among patients examined [244] [245] [246] [247] [248] [249] . like all viruses, hcov may enter the cns through the hematogenous or neuronal retrograde route. in the human airways, hcov infection may lead to the disruption of the nasal epithelium [250] and, although they bud and are released mostly on the apical side of the epithelial cells, a significant amount of viruses is also released from the basolateral side [251] . thus, although hcov infections are, most of the time, restricted to the airways, they may under poorly understood conditions pass through the epithelium barrier and reach the bloodstream or lymph and propagate towards other tissues, including the cns [33, 38, 208, 252] ; this was also suggested for other respiratory viruses that can reach the human cns, namely, rsv [8, 53] [257, 258] . moreover, persistently-infected leukocytes [252] may serve as a reservoir and vector for neuroinvasive hcov [245] . therefore, neuroinvasive hcov could use the hematogenous route to penetrate into the cns. the second form of any viral spread towards the cns is through neuronal dissemination, where a given virus infects neurons in the periphery and uses the machinery of active transport within those cells in order to gain access to the cns [35, 36] . although the olfactory bulb is highly efficient at controlling neuroinvasion, several viruses have been shown to enter cns through the olfactory route [259, 260] . after an intranasal infection, both hcov-oc43 and sars-cov were shown to infect the respiratory tract in mice and to be neuroinvasive [261] [262] [263] [264] [265] . over the years, we and others have gathered data showing that hcov-oc43 is naturally neuroinvasive in both mice and humans [244, 245, 261, 263, 266] . experimental intranasal infections of susceptible mice also indicate that, once it has invaded the cns, the virus disseminated to several regions of the brain and the brainstem before it eventually reaches the spinal cord [266] [267] [268] . furthermore, based on more recent work [269] , figure 1 illustrates the olfactory route, which is clearly the main route of neuroinvasion used by hcov-oc43, as well as the early steps of subsequent neuropropagation within the cns in susceptible mice and recapitulates the suggested equivalent pathway in humans. nevertheless, our data suggest that hcov-oc43 may also invade the cns from the external environment through other pathways involving other cranial peripheral nerves [269] , reminiscent of what was shown for other human respiratory viruses such as rsv and influenza virus [8] . therefore, on the one hand, an apparently innocuous human respiratory pathogen such as the hcov may reach the cns by different routes and induce short-term illnesses, such as encephalitis. on the other hand, it may persist in resident cells of the human cns and may become a factor or co-factor of neuropathogenesis associated with long-term neurological sequelae in genetically or otherwise predisposed individuals. because of their natural neuroinvasive potential in humans and animals, a possible association between the presence of ubiquitous human coronaviruses in the triggering or exacerbation of neurological human pathologies has often been suggested over the years. it is now accepted that hcov are not always confined to the upper respiratory tract and that they can invade the cns [220, 245, 248, 270] . as other viruses listed herein, hcov are neurotropic and potentially neurovirulent. even though no clear cause and effect link has ever been made with the onset of human neurological diseases, their neuropathogenicity is being increasingly recognized in humans, as several recent reports associated cases of encephalitis [244] , acute flaccid paralysis [271] and other neurological symptoms, including possible complications of hcov infection such as guillain-barré syndrome or adem [249, [272] [273] [274] [275] [276] [277] [278] [279] . the presence and persistence of hcov in human brains was proposed to cause long-term sequelae related to the development or aggravation of chronic neurological diseases [245] [246] [247] [248] [280] [281] [282] . given their high prevalence [31, 283] , long-term persistence and newly recognized neuropathogenesis, hcov disease burden could currently be underestimated. this suggest that better surveillance, diagnoses and deepened virus-host interactions studies are warranted in order to gather more knowledge that will make possible the development of therapeutic strategies to prevent or treat occurrences. potential short-term neuropathologies sars-cov, hcov-oc43 and -229e are naturally neuroinvasive and neurotropic in humans and therefore potentially neurovirulent [220, 244, 245, 249, 270, 271] . furthermore, animal models showed that sars-cov could invade the cns primarily through the olfactory route [265] or even after an intra-peritoneal infection [284] , and induce neuronal cell death [265, 284] . to our knowledge, no reports on the presence of the three other coronaviruses that infect humans in the cns have been published. however, neurological symptoms have been described in patients infected by all three viruses [276, 277, 285] . making use of our in vivo model of hcov neuropathogenesis, relying on the natural susceptibility of mice to hcov-oc43-the most prevalent strain among endemic hcov [210, 286] -encephalitis and transient flaccid paralysis associated with propagation towards the spinal cord and demyelination and long-term persistence in surviving mice were observed [261, [266] [267] [268] [287] [288] [289] ; thus, recapitulating the neurological afflictions reported in some patients infected by hcov [244, 249, 271, 272, [275] [276] [277] 290] . although we must interpret data obtained in rodents with all the caution dictated by the use of a non-human host, it is likely that the underlying mechanisms described will have relevance to the human situation or at least provide leads to investigate neurotropic hcov in humans. in susceptible mice, hcov-oc43 has a selective tropism for neurons in which it is able to use axonal transport as a way of neuron-to-neuron propagation [269] . these results, together with data harvested with the use of microfluidic devices (xona microfluidic), helped to elaborate a putative model of propagation adapted from tomishima and enquist [291] , in which infectious hcov-oc43 could either be assembled in the cell body or at different points along the axon using the anterograde axonal transport to propagate between neurons or from neurons to glial cells surrounding neurons in the cns (figure 2 ). furthermore, based on previous data using different mutant recombinant viruses harboring mutation in the s protein [266, 268, 269] and making use of a luciferase expressing recombinant hcov-oc43 [292] [293] [294] , we are now showing that the rate and success of virus propagation towards the spinal cord, in part through the neuron-to-neuron pathway, correlates with the exacerbation of neurovirulence ( figure 3 ). [269] and adapted from tomishima and enquist [291] . in this model, solid arrows represent fully assembled virus transport and dashed arrows represent subvirion assemblies [291] . schematic representations were assembled with the motifolio neuroscience toolkit, 2007. [292] was injected intra-nasally (i.n.) into mice. virus spread was assessed by bioluminescence imaging (bli) with the xenogen vivo vision ivis 100 imaging system (perkin-elmer) in infected anaesthetized mice placed in a light proof specimen chamber after intraperitoneal injection of d-luciferin. images were taken with a ccd camera mounted in a light-tight imaging chamber, using the acquisition software living image version 4.3.1 (caliper-lifesciences). evaluation of associated clinical scores: (levels 0 to 4: 0 is asymptomatic; 1 is mice with early hunched back; 2 is mice presenting slight social isolation, weight loss and abnormal gait; 3 is mice presenting total social isolation, ruffled fur, hunched back, weight loss and almost no movement; and 4 is mice moribund or dead (presented elsewhere; [266] ), indicate that only mice with a positive signal at both the level of the brain and spinal cord were evaluated to be at level 2 to 3. hcov-oc43 structural and accessory proteins are important for infection and some clearly represent virulence factors [38, [266] [267] [268] [269] 289, 295, 296] . using neuronal cell cultures and our murine model, we gathered data indicating that some of these proteins also play a significant role in viral dissemination [269, 296] and now aim to exploit these promising leads to fully understand the course and determinants of propagation to and through the cns and complete the neurologic portrait of short term hcov neuropathogenesis. the presence of hcov rna in the human cns establishes the natural neuroinvasive properties of these respiratory viral agents. moreover, it also suggests that they persist in human cns [245] as they do in human neural cells [297, 298] and in the cns of mice that survive acute encephalitis. these surviving mice exhibited long-term sequelae associated with decreased activity in an open field test and a reduced hippocampus with neuronal loss in the ca1 and ca3 layers [287] , reminiscent of what was observed after infection by the influenza a virus and rsv [127, 162] and to the significant loss of synapses within the ca3 region after infection by wnv [299, 300] . the precise and complete etiology of several long-term neurological pathologies still represent a conundrum. multiple sclerosis (ms) represents one such neurological disease for which an infectious agent or agents may play a triggering role, with viruses the most likely culprit in genetically predisposed individuals [301] . it has been suggested that several neurotropic viruses could be involved in ms pathogenesis but that they may do so through similar direct and/or indirect mechanisms [302] [303] [304] [305] [306] . however, although research has not yet led to a direct link to any specific virus, association of coronaviruses with ms has been suggested [307] . even though hcov-oc43 and -229e were detected in some control brains and in some brains coming from patients with different neurological diseases, there was a significantly higher prevalence of hcov-oc43 in brains of ms patients [245] . moreover, autoreactive t cells were able to recognize both viral and myelin antigens in ms patients but not in controls during infection by hcov-oc43 and hcov-229e [308, 309] . thus, the immune response may participate in the induction or exacerbation of long-term neuropathologies such as ms in genetically or otherwise susceptible individuals. furthermore, it was shown that in recombination activation gene (rag) knock-out mice, hcov-oc43-induced encephalitis could be partially mediated by the t-cell response to infection [263] . this underlines the possibility that, like its murine counterpart mhv, long term infection of the cns by hcov [245] may participate in the induction of demyelinating ms-like lesions. immune cell infiltration and cytokine production were observed in the mouse cns after infection by hcov-oc43. this immune response was significantly increased after infection by viral variants, which harbor mutations in the viral glycoprotein (s) [267] . these variants also induced glutamate excitotoxicity [268, 289] , thus increasing damage to neurons [310] and/or disturbing glutamate homeostasis [311] and thereby contributing to neuronal degeneration and hind-limb paralysis and possible demyelination [266] [267] [268] [269] . the degeneration of neurons may eventually lead to death of these essential cells by directly generating a cytotoxic insult related to viral replication and/or to the induction of different regulated cell death (rcd) pathways [312] [313] [314] . our results indicate that the underlying mechanisms appear to involve different cellular factors and pathways of rcd, described and reviewed elsewhere [38, 315] . virus-cell interactions are always important in the regulation of cell response to infection. for hcov-oc43, we clearly showed that the viral s and e proteins are important factors of neurovirulence, neuropropagation and neurodegeneration of infected cells [267] [268] [269] 296, 312] . we have also demonstrated that the he protein is important for the production of infectious hcov-oc43 and for efficient spreading between neuronal cells, suggesting an attenuation of the eventual spread into the cns of viruses made deficient in fully active he protein, potentially associated with a reduced neurovirulence [269, 295] . coronavirus accessory proteins have been extensively studied and are now considered as important viral factors of virulence implicated in pathogenesis while counteracting innate immunity [316] [317] [318] [319] . two of these accessory proteins (ns2 and ns5) produced during infection by hcov-oc43 play a significant role in virulence and pathogenesis in the mouse cns [38] . like for several other respiratory viruses, accumulating evidence now indicate that hcov are neuroinvasive in humans and we hypothesize that these recognized respiratory pathogens are potentially neurovirulent as well, as they could participate in short-and long-term neurological disorders either as a result of inadequate host immune responses and/or viral propagation in the cns, which directly induces damage to resident cells. with that in mind, one can envisage that, under the right circumstances, hcov may successfully reach and colonize the cns, an issue largely deserted and possibly underestimated by the scientific community that has impacted or will impact the life of several unknowing individuals. in acute encephalitis, viral replication occurs in the brain tissue itself, possibly causing destructive lesions of the nervous tissue with different outcomes depending on the infected regions [320] . as previously mentioned, hcov may persist in the human cns as it does in mice [245, 287] and potentially be associated with different types of long-term sequelae and chronic human neurological diseases. in their famous review on cns viral infection, published a few years ago, koyuncu et al. [35] insisted that, under the right conditions, all viruses can have access to the cns. what "under the right conditions" means certainly represents a subject of debate among virologist and physicians. nevertheless, as stated in the introduction of this review, viral factors (mutations in specific virulence genes), host factors (immunodepression, age) or a mixture of both (underlining the importance of virus-host interactions), are all good candidates to refer to if one intends to find the beginning of an explanation. a fast and accurate diagnosis would certainly improve prognosis for patients with a suspected cns infection. identification of a specific virus provides relevant information on how to treat a patient; therefore, the development of modern technologies, such as high throughput sequencing (next generation sequencing) are warranted as it represents a potentially unbiased marvelous tool for rapid and robust diagnosis of unexplained encephalitis or other types of encephalopathies or neuronal manifestations, especially in the context where more traditional techniques have failed to identify the etiological agent [21, 108, 111, 244, 321, 322] . therefore, although our attention is mainly on a few different viruses such as hsv, arboviruses and enteroviruses, it may now be the time to look at cns viral infection from another perspective. these viruses truly represent an important proportion of cns viral infection associated with encephalitis, meningitis, myelitis and long-term neurological disorders. nevertheless, accumulating evidence in the scientific literature strongly suggest that many other viral candidates could be underestimated in that matter. several human respiratory viruses are neuroinvasive and neurotropic, with potential neuropathological consequences in vulnerable populations. understanding the underpinning mechanisms of neuroinvasion and interaction of respiratory viruses (including hcov) with the nervous system is essential to evaluate potentially pathological short-and long-term consequences. however, viral infections related to diseases that are rare manifestations of an infection (like long term chronic neurological diseases), represent situations where koch's postulates [323] need to be modified. a series of new criteria, adapted from sir austin bradford hill, for causation [324, 325] was elaborated by giovannoni and collaborators concerning the plausible viral hypothesis in ms [326] . these criteria certainly represent a pertinent tool to evaluate the involvement of human respiratory viruses as a factor that could influence long-term human neurological diseases. to continue the gathering of epidemiological data is justified to evaluate the clear cause and effect link between neuroinvasive respiratory viruses and short-and long-term human neurological diseases. understanding mechanisms of virus neuroinvasion and interactions with the central nervous system is essential for different reasons. first, to 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writing of the manuscript, or in the decision to publish the results. key: cord-333186-gxs74wit authors: ashhurst, thomas myles; vreden, caryn van; niewold, paula; king, nicholas jonathan cole title: the plasticity of inflammatory monocyte responses to the inflamed central nervous system date: 2014-10-31 journal: cellular immunology doi: 10.1016/j.cellimm.2014.07.002 sha: doc_id: 333186 cord_uid: gxs74wit abstract over the last three decades it has become increasingly clear that monocytes, originally thought to have fixed, stereotypic responses to foreign stimuli, mediate exquisitely balanced protective and pathogenic roles in disease and immunity. this balance is crucial in core functional organs, such as the central nervous system (cns), where minor changes in neuronal microenvironments and the production of immune factors can result in significant disease with fatal consequences or permanent neurological sequelae. viral encephalitis and multiple sclerosis are examples of important human diseases in which the pathogenic contribution of monocytes recruited from the bone marrow plays a critical role in the clinical expression of disease, as they differentiate into macrophage or dendritic cells in the cns to carry out effector functions. while antigen-specific lymphocyte populations are central to the adaptive immune response in both cases, in viral encephalitis a prominent macrophage infiltration may mediate immunopathological damage, seizure induction, and death. however, the autoimmune response to non-replicating, non-infectious, but abundant, self antigen has a different disease progression, associated with differentiation of significant numbers of infiltrating monocytes into dendritic cells in the cns. whilst a predominant presence of macrophages or dendritic cells in the inflamed cns in viral encephalitis or multiple sclerosis is well described, the way in which the inflamed cns mobilizes monocytes in the bone marrow to migrate to the cns and the key drivers that lead to these specific differentiation pathways in vivo are not well understood. here we review the current understanding of factors facilitating inflammatory monocyte generation, migration and entry into the brain, as well as their differentiation towards macrophages or dendritic cells in viral and autoimmune disease in relation to their respective disease outcomes. monocytes, macrophages (mu), and dendritic cells (dc) are part of the 'mononuclear phagocyte system', also known as the 'reticuloendothelial system', found throughout the body. in normal tissues most mu and dc are considered to be 'tissue-resident', populating the tissue early in life, often specifically named. in the cns, the resident mu are microglia. they originate from the yolk sac [1] and are renewed in situ [2] . during inflammation, however, monocytes can migrate from the bloodstream into affected tissues, including the cns, where they differentiate into ''infiltrating'' mu or dc. whilst these resident and infiltrating cells may play prominent roles in the cns during viral or autoimmune disease, the methods by which the inflamed cns induce the mobilisation of monocytes in the bone marrow is poorly understood. moreover, the signalling events responsible for alternate monocyte or dc differentiation in the local inflammatory environment of the cns are not well described. as these signalling events dictate the nature and progression of the immune response to cns pathologies, an understanding of the mechanisms involved is crucial to identify novel targets for immune modulating therapy in these diseases. monocytes are one of the mononuclear cell types circulating in the blood that are produced in hemopoietic tissues of the bone marrow (bm) throughout life. they can be identified in human and mouse by flow cytometry, using a combination of cell surface markers. under normal conditions, the entire monocyte population is identified as cd14 + (including cd14 lo and cd14 hi subpopulations) in humans, and cd115 + cd11b + in mice and humans. in both species, two principle populations and an 'intermediate' population are identifiable. classical (also termed 'inflammatory') monocytes are cd14 hi cd16 à in humans and ly6c hi (cd43 lo ccr2 hi cx3 cr1 lo ) in mice and are the major monocyte population in the blood [3, 4] . non-classical (also termed 'patrolling') monocytes, represent a much smaller subset (approximately 10%) of blood monocytes and are cd14 lo cd16 hi in humans, and ly6c lo (cd43 hi ccr2 lo cx3 cr1 hi ) in mice [3, 4] . the intermediate group is cd14 hi cd16 hi in humans and ly6c hi cd43 hi population in mice. there are transcriptional similarities between humans and mice comparing their respective subsets, but functionally, mouse classical monocytes appear to be more related to human intermediate monocytes, based on their pro-inflammatory roles [5] . during normal hematopoiesis in mice, the mu/dc progenitor (mdp) gives rise to a pre-dc [6] and a recently-described common monocyte progenitor (cmop), distinguished from the mdp by its downregulated cd135 and upregulated ly6c, although it still lacks cd11b. the cmop differentiates into c-kit à cd115 + cd11b + ly6c + monocytes and in turn into c-kit à cd115 + cd11b + ly6c à monocytes [7] . ly6c hi monocytes are generated in the bm and during homeostasis they likely emigrate, but eventually give rise to ly6c lo (cx3cr1 hi ) monocytes [3] . under homeostatic conditions, ly6c lo cx3cr1 hi monocytes patrol the luminal side of vascular endothelium in a programmatic, albeit peripatetic manner [8] . during inflammation, however, ly6c hi monocytes emigrate from the bm along the ccr2-ccl2 axis into foci of tissue inflammation, differentiating into inflammatory mu, tipdc, or inflammatory dc, which may then migrate to the draining lymph node (dln), presumably transporting antigen acquired on the way [5, 9] . while microglia are normally self-renewing [2] , they may be supplemented and/or replenished by infiltrating ly6c hi monocytes during cns infection and/or irradiative inflammation [10, 11] and these immigrants become ly6c lo on entry into inflamed tissue [5, 9] . ly6c lo monocytes are also recruited in later stages of inflammation where they are involved in tissue repair. these cells typically differentiate into m2, i.e., anti-inflammatory, mu, supporting healing in the injured spinal cord [12] . however, the developmental connection between these 2 phenotypically similar but often temporally disparate populations is not completely clear. the mechanisms by which cns inflammation induces monocyte mobilisation in the bm are not well understood. the recruitment of bm monocytes during inflammation appears to depend on two initiating events: induction of emigration of existing bm monocytes into the circulation, and generation of new monocytes in the bm to replace the diminished population, which subsequently contribute to the emigrating monocyte population. monocyte generation relies on two processes, the 'pull' of a diminished downstream population, and/or the 'push' or direct stimulation of hematopoietic stem cells (hsc) or other progenitors. this process has been reviewed in depth elsewhere [13] , but relevant factors are considered here. ccl2 is crucial for ly6c hi inflammatory monocyte emigration from the bm [14] . recently, bm stromal cells, but not hsc, were shown to secrete ccl2 in response to low levels of circulating toll-like receptor (tlr) ligands [5] . this induced monocyte migration towards the vascular sinuses, and was dependent on myeloid differentiation primary response protein 88 (myd88) (involved in responses to tlr ligand binding in all but tlr-3 [15] ), but was independent of tnf and type-i interferon (ifn) expression. as the ccl2-expressing cells also expressed various tlr, it was suggested that these cells function to detect infection and rapidly induce monocyte emigration into the circulation. whilst this provides some insight into the mechanisms of monocyte emigration, it does not explain the 'push' signal for monocyte production. this signal is presumably provided by direct inflammatory stimulation of hsc or other progenitors, inducing increased differentiation of self-renewing hsc into downstream progenitors [13] . direct inflammatory modulation of hsc (which are ccr2 + ) by circulating tlr has been described, inducing such differentiation [16] . furthermore, soluble immune-mediators may induce bm changes; seo et al. showed that ifn-a signalling to hsc was required for the generation of ly6c hi monocytes in a model of viral pneumonia [17] and type i ifn, produced in the spleen in response to infection with listeria monocytogenes, promoted monocyte emigration from the bm [16] . interestingly, mice deficient in either myd88 or ifn-a receptor (ifnar) still had significant monocyte migration from the bm to the site of inflammation, whereas mice deficient in both did not [16] . interestingly, in these studies, ifn-c did not play an important role in monocytopoiesis. however, in a model of chronic mycobacterium avium infection, ifn-c, but not ifn-a, was found to activate hsc, resulting in differentiation into downstream myeloid and lymphoid progenitors replacing the diminished populations [18, 19] . on the other hand, in experimental autoimmune encephalomyelitis (eae), gm-csf produced by cns cells triggers monocyte mobilisation and emigration from the bm [20] . in cns-initiated mobilisation of the bm, it is unclear if there is a difference in initiating events that predisposes to the preferential differentiation of monocytes towards a mu or dc phenotype, prior to entering the cns. the fact that tlr, type-i and -ii ifn, and gm-csf each induce monocyte emigration and the production of monocytes from progenitors in different situations may explain some differences in the manner of bm mobilisation by different cns pathologies. monocyte infiltration during viral encephalitis is rapid, with lethality in animal models occurring within days of initial infiltration [10, 21] . the pathogenesis of eae, on the other hand, is chronic and/or relapsing and depends on t cell responses against myelin proteins that are initially induced by dc [22] . nevertheless, a potential connection exists between viral and auto-immune causes of cns infiltration by monocytes. in multiple sclerosis (ms), there is evidence to suggest that reactivity against myelin proteins may occur following cns viral infection, if anti-viral responses cross-react with myelin proteins [23] . the initial presence of viral rna or dna in the circulation could certainly induce monocyte emigration from the bm, via binding of intracellular rna by tlr within bm stromal cells [24] , and tlr stimulation or direct infection of hsc could induce downstream differentiation of these cells [13] . however, although tlr ligands may be present initially, recurrent episodes of ms would have few if any virus-associated tlr ligands. it is also possible that whilst type i ifn (ifn-a/b) and type ii ifn (ifn-c) are involved in both cns diseases, the innate differential production of ifn-a/b, crucial for virus control in the early response against cns viruses [25] , by a variety of cell types during infection [26] may induce monocyte mobilisation differently from that seen in ms/eae. high levels of ifn-c, which play a role in viral disease pathogenesis [27] , is associated with the later development of the adaptive immune response, and may contribute here to the continued recruitment of monocytes. in contrast to acute viral infection, in eae that progresses as a relapsing or chronic condition, ifn-c production occurs over a long period of time, similar to the role of ifn-c in hsc stimulation in chronic infection of the lung [18, 19] . as such, the differences in type-i and type-ii ifn production may differentially skew the monocyte phenotype towards a mu profile in acute infection and a dc profile in a chronic noninfectious setting. however, this has not been investigated experimentally. the separation of the cns from the peripheral circulation by the blood-brain barrier (bbb), limits the access of soluble factors and leukocytes to the cns. however, during inflammation various changes enable the recruitment of leukocytes into the brain. the process of infiltration involves detection of local chemokine gradients by susceptible migratory leukocytes in the vicinity of the affected cns parenchyma secreting the chemokines, rolling followed by firm adhesion to the local endothelium, and finally diapedesis and transmigration into the brain parenchyma [28] . chemokines produced at the site of inflammation mediate chemotaxis of leukocytes passing through the neighbouring blood vessels to that site. the chemokine receptors expressed on monocytes include: ccr2, cx3cr1, ccr1, ccr5, ccr6, ccr7, ccr8 and cxcr2 [5] . of these, ccr2, ccr1, and ccr5 appear to be among the most important for migration. the ccr2-ccl2 axis is the best-studied chemokine pathway in monocyte migration and infiltration into the cns. ccr2, which is upregulated on monocytes in a variety of cns pathologies, binds to ccl2 (or ccl7), which is produced at high levels by infected neurons in animal models of wnv encephalitis [10] and likely by glial cells in eae [29] . ccl2 neutralisation in wnv encephalitis [10] or ccl2 (or ccl7) deletion in bacterial infection [30] result in reduced monocyte recruitment. whilst this resulted in higher bacterial loads, in the latter case, reduced monocyte infiltration during wnv encephalitis led to extended, but not permanent survival in mice [10] . this highlights the severity of the immunopathology induced by infiltrating monocytes in the cns. ccl5 is highly upregulated in a variety of viral cns infections [10, 31] and binds to ccr5 (expressed on ly6c hi monocytes) and ccr1. in ms, infiltrating monocytes express both ccr1 and ccr5, with their ligands being expressed in the inflamed cns [32, 33] . ccl2 and ccl5 are both upregulated in cns infection with wnv [10, 31] . in eae, mrna and protein levels of cx3cl1 and cx3cr1 are elevated in the dorsal root ganglia and spinal cord [34] . whilst these studies were focused on neuropathic pain, they highlight the potential for recruiting cx3cr1 + non-classical monocytes to the inflamed cns during eae/ms. interestingly, in atherosclerosis, ccr2 lo monocytes did not rely on cx3cr1 to enter plaques, instead using ccr5 to some extent, while ccr2 hi monocytes used cx3cr1, ccr2, and ccr5 [35] , emphasising the ability of monocytes to adapt in different disease settings. the entry of monocytes into the cns requires their margination and initial binding to endothelium, followed by firm adhesion to enable transmigration across the bbb into the cns parenchyma. ly6c hi monocytes express a variety of cell surface molecules involved in adhesion to vascular endothelium in the cns, including l-selectin (cd62l), p-selectin glycoprotein ligand 1 (psgl1), lymphocyte function-associated antigen-1 (lfa-1), macrophage receptor-1 (mac-1), platelet endothelial cell adhesion molecule-1 (pecam-1), and very late antigen-4 (vla-4). these are reviewed in more detail elsewhere [5] . cd62l is required for entry into the inflamed peritoneum and is also critical for the migration of monocytes into the dln through high endothelial venules [36, 37] . the high expression of cd62l on ly6c hi monocytes appears to be relevant for their entry into the cns in eae [38] . this not only suggests a role for cd62l in tissue entry, but possibly that monocytes predisposed towards a dc phenotype retain cd62l expression when they migrate to the dln as efficient apc. during murine wnv encephalitis, the infiltration of pathogenic ly6c hi monocytes correlates with the upregulation of vcam-1 and icam-1 on cns vascular endothelium [39] , implicating vla-4 and lfa-1, respectively, in ly6c hi monocyte infiltration. vla-4 antibody blockade reduced monocyte infiltration by $60% and increased survival by up to 60% in infected mice. not surprisingly, as vla-4 antibody blockade is used in ms to reduce t cell infiltration, this treatment also reduced t cell infiltration into the cns in these animals, however, importantly, this was not sufficient to abrogate virus clearance. on the other hand, despite reducing monocyte infiltration by >30% in these experiments, lfa-1 blockade had no effect on survival [21] . it has been shown that patrolling of the luminal side of vascular endothelium by ly6c lo monocytes is mediated by lfa-1 [8] . this raises the possibility that interfering with lfa-1-mediated interactions might prevent ly6c lo , potentially m2 monocytes, from entering the cns, which may abrogate normal anti-inflammatory processes in viral encephalitis and enhance immunopathology mediated by infiltrating ly6c hi monocytes. however, this was not explored in these studies. this study highlights the differential function of monocyte subsets using different adhesion molecule-integrin receptor pairs, and the importance of vla-4 use by ly6c hi monocytes in cns invasion. moreover, this was the first study to demonstrate in vivo that carefully timed suppression of specific elements of the innate immune system during an acute lethal neurotropic infection could enhance survival by reducing immunopathology without interfering with the generation of immunity. inflammatory monocyte migration and subsequent differentiation into dc or mu are hallmarks of several immunopathogenic cns diseases, but the factors directing this differentiation have not been defined clearly. it has been suggested that mu and dc, as well as undifferentiated monocytes may cause immunopathology. however, the reported studies have not always distinguished unambiguously between monocytes and mu, which makes drawing firm conclusions difficult. invasion of the cns by a replicating virus results in local activation of resident microglia and astrocytes, with obvious migratory responses by these cells within the brain parenchyma, as well as the immigration of a range of leukocytes from the blood stream. this infiltrate typically contains monocytes, which differentiate into mu or activated microglial phenotypes in the brain, and these have been implicated in several diseases. thus, inflammatory mu infiltration precedes the onset and peak of disease symptoms in neurotropic coronavirus infection [40] . in lymphocytic choriomeningitis virus (lcmv), monocytes (as well as neutrophils) play a highly pathogenic role [41] . theiler's encephalomyelitis virus (tmev) infection results in major inflammatory monocyte infiltration into the cns within 48 h and ultimately induces severe monocyte-dependent cns damage, with subsequent differentiation into activated mu linked to the development of cns lesions [42] [43] [44] . several other neuroinvasive viruses, such as neurotropic mouse hepatitis virus (mhv), tick-borne encephalitis (tbe) and wnv, are associated with mu infiltration into the cns [45] [46] [47] [48] . inflammatory (ly6c hi ) monocyte differentiation into ly6c hi mu and/or activated microglia upon entry into the cns is a key feature of wnv encephalitis and plays a significant role in the pathology (and lethality) of this disease [10] . inflammatory monocytes also play a role in mediating cns damage in autoimmune diseases such as amyotrophic lateral sclerosis (als) and ms [49] . in eae, the mouse model widely used to study t cell-mediated autoimmune disease in general and ms specifically, inflammatory monocytes have a major impact. breaking peripheral tolerance to myelin proteins, leads to activation of myelin-specific t cells in secondary lymphoid organs. once these t cells arrive in the cns they become re-activated by apcs, resulting in the expression of pro-inflammatory cytokines, ifn-c, il-17, gm-csf and tnf, as well as chemokines by t cells. the circulating ly6c hi monocyte population, which expands exponentially before eae onset, represents a major proportion of the inflammatory cells in the eae cns and are dc precursors. although mu are observed in the cns of eae mice, dc are more efficient apc and activated dc co-localize with cd4 + t cells responsible for demyelination in the cns, implicating dc, rather than mu, in the amplification of responses [20, 50, 51] . while both dc and mu may be present in the diseased cns in autoimmune or viral encephalitis, mu appear to have a more prominent role in viral encephalitis, while dc are more common in autoimmune diseases. interestingly, however, dramatically reducing immigration of inflammatory monocytes into the cns at particular timepoints in either of these diseases using negatively charged microparticles, which mediate sequestration and apoptosis of these cells in the spleen, abrogates the symptomatology and in the case of wnv encephalitis, results in up to 60% survival with immunity in an otherwise lethal disease [52] . this suggests that these cells are very similar, if not the same, in the blood stream in both diseases, and that their differentiation is mediated in the cns, presumably by the prevailing milieu, despite their different fates there. alternatively, it is possible that separate mu and dc precursors may share a common receptor that mediates particle uptake, resulting in both being sequestered by the spleen with the same apoptotic outcomes. historically, monocyte-to-dc differentiation in vivo was hypothesized to be restricted to inflammatory scenarios, however, studies using fluorescent latex bead uptake to track circulating monocytes, indicated that during steady state conditions ly6c hi monocytes differentiate into cd103 + dc, whereas ly6c lo monocytes give rise to a cd11b hi dc subtype [53] . a separate study utilizing microspheres as monocyte markers, also suggests that the decision of monocytes to differentiate into mu or dc might not solely be determined by cytokines. the authors found that adding microspheres to phagocytic monocytes travelling to the lymph node (ln) induced differentiation into dcs, while monocytes staying at the site of activation became mu [54] . the differentiation of monocyte-derived dc (mddc) in the absence of inflammatory stimuli is likely a result of basal levels of signaling factors present in this environment. these studies, while useful, do not account for the confounding possibility that the microspheres themselves, once phagocytosed, may have their own influence on the subsequent differentiation of monocytes [52] . granulocyte-macrophage colony-stimulating factor (gm-csf) and macrophage-colony-stimulating factor (m-csf) are well known to influence the differentiation of monocytes in culture. gm-csf, also known as csf-2, is secreted by a broad range of cells upon stimulation with cytokines, microbial products and/or antigens, and regulates survival, differentiation, and activation of target cells such as neutrophils, monocytes, mu and dc [55, 56] . m-csf, also known as csf-1, through its receptor, cd115, influences several cell types and its effects include mediating the regulation, development, survival, proliferation and differentiation of mu [57] . culturing human peripheral blood mononuclear cells (pbmc) with m-csf alone favors the differentiation of monocytes to mu. the outcome of pbmc culture with gm-csf, on the other hand, depends on the density of gm-csf receptors (gm-csfr) on the surface of the monocytes; low gm-csfr expression is associated with mu differentiation, higher gm-csfr density with differentiation towards dc. the addition of il-4, which has a m-csf inhibiting function, overrides the effect of different levels of gm-csfr and blocks differentiation towards mu, thus favoring a dc phenotype [58, 59] . gm-csf and il-4 function by downregulating cd14 on pbmc at a transcriptional level [60, 61] . culturing human pbmc with gm-csf, ifn-c and il-4 skews the differentiation of monocytes to functional dc, which are not terminally differentiated but capable of presenting antigen to t cells [62] [63] [64] [65] . human monocyte-enriched pbmc cultured with only m-csf differentiate into immature mu but, with the addition of gm-csf and il-4, convert to immature mddc. these dc lose their phagocytic ability and become potent apc [66] . however, removal of these factors convert mddc back to a mu phenotype, indicating that the plasticity enabling differentiation into either mu or dc remains intact for some time. though culture with m-csf or gm-csf can both result in mu differentiation, mu obtained with different treatments exhibit different morphology. m-csf-stimulated mu (m-bmm) exhibit a spindle-shaped morphology, while gm-csf-derived mu (gm-bmm) appear more rounded [64, 66] . the fate of monocytes in an inflammatory milieu is markedly different from their fate under homeostatic conditions. this can be seen with adoptively-transferred ly6c hi monocytes, which give rise to regulatory mu in the non-inflamed colon, but become inflammatory dc capable of priming t cells and producing il-12, il-23, il-6 and tnf in colitic mice [67] . the fate of monocytes in inflammatory scenarios such as eae or viral encephalitis is difficult to predict due to the intricate interplay between cytokines, chemokines and receptor-ligand interactions. as would be expected, simulating an ''inflammatory'' environment by adding pro-inflammatory factors alters the differentiation outcome. in m-csf-and gm-csf-directed differentiation of bm monocytes (bmm) to mu, lps induced high levels of pro-inflammatory cytokines, il-6, tnf and il-23, from gm-bmm, whereas m-bmm produced higher levels of il-10 and ccl2. this raises the possibility that gm-csf-induced mu represent differentiation down the classical/m1 activated pathway and m-csf-induced mu represent differentiation down the alternative/ m2 regulatory pathway associated with il-10 production. these two mu populations were not terminally differentiated, as the addition or removal of m-csf or gm-csf to the relevant cultures results in an interchange between mu phenotypes [68] . inflammatory stimuli, such as lps, combined with gm-csf and il-4 result in the maturation and irreversible commitment of mddc to mature dcs [64, 69] . addition of tnf to immature dc in vitro reduces their apc capacity, possibly making them functionally mature [62] . culturing human pbmc with m-csf, il-6 and il-10 shifts monocyte differentiation from dc to mu. however, these factors do not have the capacity to convert immature dc to mu or monocytes [70] . current dogma suggests that gm-csf is crucial for the differentiation of mddc from inflammatory monocytes, and although this is true for several in vitro experiments, the in vivo scenario is markedly different. mddc differentiation in the absence of gm-csf was examined in several disease models in vivo, including infection with influenza a, streptococcus pneumonia, salmonella typhimurium, l. monocytogenes, lps stimulation and eae [71] . csf-2rb à/à and csf-2rb2 à/à mice in all these inflammatory scenarios had similar inflammatory dc numbers compared to wild type (wt) mice. these dc were identified as fully functional tipdc, indicating that gm-csf is not required for monocyte accumulation and differentiation into dc during these inflammatory conditions. the authors did however detect high levels of m-csfr and m-csf levels on dc and in inflamed tissues, respectively. removing the m-csf signal by antibody blockade of m-csf or the excision of m-csfr allele did not alter the number of ly6c hi monocytes infiltrating inflamed tissue. however, mddc numbers were significantly reduced, indicating that m-csfr signaling is likely critical for the differentiation of mddc in vivo but not for the recruitment of inflammatory monocytes [71] . the importance of m-csf in dc differentiation was also confirmed in an inflammatory skin model. uv irradiation resulted in homing of circulating gr1 hi (ly6c/g complex) monocytes to the inflamed dermis and epidermis, which differentiated into langerhans cells (lc) upon entry. m-csfr-deficient mice exhibited impaired lc differentiation and tissue mu development. although these mice had similar monocyte numbers homing to inflamed skin, they possessed significantly fewer lc, indicating that m-csfr is directly involved in monocyte-to-dc differentiation in the skin [72] . during infection, inflammatory stimuli are not only derived from pathogens (e.g. lps from bacteria) but cells from the host also contribute factors changing the microenvironment, which directly influence monocyte differentiation. the differentiation of monocytes derived from the blood occurs once they reach target tissue [72] and cross the endothelium [20] . stromal cells like fibroblasts have been shown to impact upon this differentiation. although monocytes cultured with gm-csf and il-4 yield dc [64] , the coculture of these cells with fibroblasts in steady-state conditions, skews the differentiation to mu. monocytes, which become activated when they cross the endothelium, secrete m-csf and stimulate the release of il-6 from fibroblasts. il-6 in turn increases the expression of functional m-csfr responsible for transducing the m-csf signal and thereby initiates mu differentiation [73] . however, co-culturing these human pbmc with fibroblasts, il-4, gm-csf and tnf, induces a terminally differentiated dc phenotype. this occurs because il-6 facilitates the utilization of m-csf, whereas tnf induces the internalization of m-csfr resulting in monocytes being unresponsive to autocrine il-6 and m-csf. these results suggest that the balance of the tnf/il-6 may be crucial in determining the fate of monocytes during inflammation [73] . this is further supported by the overexpression of ccl2 in the brain induced by adeno-associated virus, which results in microglial activation and elevated il-6 and gm-csf levels [74] . il-6, which is involved in monocyte-to-mu differentiation, is elevated in the cerebrospinal fluid of jev-infected patients [75] . activated murine microglia in jev and hsv infection release pro-inflammatory cytokines il-6 and il-1b [76] [77] [78] . this release of pro-inflammatory cytokines from microglia likely occurs via a rig-i-mediated pathway [79] . infiltrating mu are also a major source of il-6 in tmev infection of mice; along with the il-6 produced by microglia this might induce monocyte to mu proliferation through an autocrine loop [47, 77] . high levels of il-6 are present in the wnv-infected brain although it is not clear which cells produce it [80] . other resident cells influencing monocyte differentiation include endothelial cells in the bbb, astrocytes, microglia and oligodendrocytes, which secrete tgf-b and gm-csf in inflammatory conditions. these factors promote human pbmc (cd14 + ) differentiation to cd83 + cd209 + (dc-sign + ) dcs, which secrete il-12p70, tgf-b and il-6 [81] . another factor influencing monocyte differentiation is the presence of apoptotic cells, for example in influenza virus infection. these actively dying cells release soluble mediators, directing monocyte differentiation towards mu [82] . the activation of the caspase pathway associated with apoptosis, has also been identified as playing a role in the fate of monocyte differentiation. caspase-8 deletion arrested the m-csf-induced differentiation of bm-derived monocytes to mu [83] . caspase-8 and caspase-9 were specifically activated in human pbmc stimulated with m-csf to become mu but not by gm-csf and il-4. treatment with a broad-spectrum caspase inhibitor induced a switch from mu differentiation to death, further implicating these proteins as crucial factors in the pathway of monocyte differentiation [84] . human cytomegalovirus-stimulated monocytes relied on caspase-3 rather than the caspase-8 activation seen in m-csf-induced differentiation [85] . this raises the possibility that infected neurons undergoing apoptosis may skew the differentiation of infiltrating monocytes towards a mu phenotype. in addition to resident infected cells, the pathogen itself also influences monocyte differentiation. differentiation of monocytes into either mu or dc in vivo can be triggered indirectly by the recognition of microbial ligands by pattern recognition receptors (prr) or directly by cytokine activation. prr are a very diverse group of receptors that function by signalling from a cell membrane or cytoplasmic location, or following endocytosis of pathogens in order to destroy them. tlr are important and abundant prr on monocytes that contribute crucially to the activation of innate and adaptive immune responses. binding of microbial or viral products by tlr results in dimerization of the receptors and the subsequent triggering of intracellular signalling pathways that operate via nf-jb and map kinase pathways and result in the production and release of cytokines potentially inducing mu or dc differentiation. both tlr2/1 and il-1b receptor signalling have been implicated in monocyte differentiation through the common myd88 signalling pathway. impairing tlr-4 signalling by using tunicamycin-induced er stress to suppress nf-jb activation, markedly suppressed the ability of lps-stimulated monocytes to differentiate into mu [86] . tlr2/1-induced differentiation of monocytes to mu or dc relied on specific cytokine-receptor interactions, with il-15 and gm-csf inducing cd209 + mu and dc, respectively. il-1b activation favored the proliferation of mu over dc, while cd209 + mu proliferating from il-1b-activated culture showed enhanced phagocytosis of mycobacteria compared to tlr2/1-induced mu in culture [87, 88] . in leprosy, lesions from patients with tuberculoid leprosy (t-lep) contain both cd209 + mu and cd1b + dc, while in lepromatous leprosy (l-lep), lesions contain only cd209 + mu [88] . in this disease, where tlr2/1 becomes activated, it is the form of the developing disease that influences which effector populations differentiate from monocytes. dengue virus rna has been shown to co-localize with tlr-3 in a human monocyte cell line, which results in il-8 and ifn-a/b release. tlr-3 also plays a role in wnv, by restricting infection in neurons; its importance was confirmed in myd88 à/à mice, which show much faster viral spread in the cns than control animals. szretter et al. showed that monocyte-derived mu (and t cell) recruitment to the cns was reduced in the absence of myd88 [89] . the production of tlr-pathway inhibitors by pathogens is a widespread immune evasion method; wnv ns-1 protein for instance blocks tlr-3-induced nf-jb nuclear translocation and thereby prevents il-6 production, which plays a role in monocyte-to-mu differentiation [90] . even in eae, in the absence of pathogens, tlr have been found to influence potential mediators of monocyte differentiation. tlr can be activated by endogenous ligands, which, for example, come from dying cells and thus contribute to autoimmunity and neurodegeneration [91] . in mice immunized with mog peptide, treatment with 1,25(oh) 2 d 3 resulted in a reduction of symptoms, inflammatory cell infiltrate and tnf, ifnc and il-17 expression. these findings correlated with a reduction of eae-induced tlr expression in the spinal cord of mice after 1,25(oh) 2 d 3 treatment, especially of tlr8. testing of 1,25(oh) 2 d 3 effects in a human monocyte cell line confirmed the reduction in tlr8 expression and indicated lower mrna levels of tnf and il-1b [92] . activation of nod2, an intracellular prr, by its ligand (nodl), also stimulates monocyte responses. netea et al. showed that il-32 directly induced the differentiation of monocytes to a cell type that exhibited the morphology and functionality of a mu but possessed some dc-specific markers [93] . interestingly, nod2l activation primarily induced the differentiation of human pbmc to cd1b + dc, whereas tlr2/1 activation resulted in both mu and dc populations. the dc derived from nod2l-activated cells were superior apc to tlr2/1-induced dc. transfection with sirna resulting in the knockdown of il-32 rna, subsequently blocked the nod2l-but not tlr2/1-mediated differentiation of pbmc into cd1b + dc. in this study, increased il-32 mrna and nod2 expression in patients with t-lep correlated with higher cd1b + dc numbers present in lesions. this identifies nod2l-induced il-32 as a distinct pathway of dc differentiation in humans [94] . thus, il-1b seems to be a more potent stimulator of monocyte-to-mu differentiation, whereas tlr2/1 and especially nod2 favor differentiation to dc. interferon regulatory factors (irf), which are activated by tlr and rig-1-like receptors, are transcription factors playing a crucial role in host defense mainly by controlling the production of ifn [95, 96] . increased irf expression can be found in several cns pathologies associated with mu or dc infiltration, although the extent to which these factors may be involved in modulating the monocyte differentiation in the cns has not been examined. irf-7 is crucial for the control of wnv infection and spread to the cns [97] and along with irf-9 and -5 is upregulated in lymphocytic choriomeningitis infection [98] . irf-3 is necessary to control viral replication and il-6 production in tmev [99] and flaviviruses such as jev and dengue virus can induce irf-3, -7 and -1 in culture, respectively. although in the periphery irf-7 is mainly expressed in pdc, in the cns irf-7 can be upregulated on neurons during viral encephalitis and correlates with type i ifn production [100] . however, increased irf-7 gene expression is also present in eae cns disease progression, with the absence of irf-7 associated with increased severity of disease and mu infiltration [101] . irf-8 is well known for its role in the differentiation of non-monocyte-derived dc and maturation of myeloid dc [102] [103] [104] , as well as the normal differentiation of microglia [105] . there are several examples of irf-7 and -8 directing monocyte differentiation to mu in vitro [106, 107] . interestingly, irf-8 has been shown to negatively regulate tlr3, which is constitutively expressed upon monocyte-to-dc differentiation. irf-8 and irf-1 compete for binding, which induces tlr3 promoter activity on mddc [108] . irf-4, in turn, has been associated with monocyte-to-mddc differentiation in vitro [109, 110] . although no established link has been made between irf in the cns and monocyte differentiation, in vitro studies suggest that these factors might form a link in the sequence of events steering monocyte differentiation during viral infection or autoimmune disease. other effector cells recruited to the inflammatory milieu are stimulated to secrete factors influencing monocyte differentiation. ifn-c, which is secreted by nk cells and activated t cells, can suppress the differentiation of monocytes to dc by inducing m-csf and il-6 production from monocytes in vitro [70] . however, ifnc has also been implicated in mu induction. nk cells isolated from the blood of patients suffering from rheumatoid arthritis and psoriatic arthritis induced differentiation of monocytes into dc in a cell contact-, gm-csf-and cd154-dependent manner. these mddc differ phenotypically from dc obtained from in vitro culture of monocytes with gm-csf and il-4, but still efficiently presented antigen and activated cd4 + t cells, which were polarized toward th1 [111] . ly6c + monocytes in trypanosoma brucei brucei-infected mice gave rise to tnf-and inos-producing inflammatory tipdc (cd103 à ). il-10 treatment significantly reduced this differentiation of dc from inflammatory monocytes [112] . inflammatory monocytes expressing ly6c and producing tnf and il-12 are crucial for the defense against intestinal infection by toxoplasma gondii (t. gondii) [113, 114] . activation and function of this subset was severely impaired in t. gondii-infected cxcr3 knockout (ko) mice and disease symptoms were exacerbated. cxcr3 ko mice also show impaired recruitment of cd4 + t cells and production of ifn-c by these cells. cxcr3 was identified as important for cd4 + t cell trafficking and consequent ifn-c production in inflamed intestine. adoptively transferred ifn +/+ cd4 + t cells but not ifn à/à cd4 + t cells restored ly6c + monocyte function and activation [114] . nk cell-produced ifn-c may also regulate the differentiation of monocytes to dc, and il-12 production during intraperitoneal infection with t. gondii. these experiments identify ifn-c, produced by either nk or cd4 + t cells, as a key player in monocyte activation and differentiation to dc during inflammation [113, 114] . in the autoimmune scenario represented by eae, cxcr3 ko mice also presented with exacerbated disease; however, in contrast to the effect of cxcr3 deletion in t. gondii infection, the number of t cells trafficking to the cns was not affected. in eae and ms, infiltrating activated t cells expressing cxcr3 are attracted to cxcr3 ligands, cxcl9, cxcl10 and cxcl11 produced in and around the perivascular space and are thus restricted to this location. deletion of cxcr3 leads to uncontrolled spread of t cells throughout the cns but also reduces the recruitment of foxp3 + t cells and effector t cell interaction, and therefore results in more severe autoimmune-mediated tissue damage [115] . non-activated inflammatory monocytes and inflammatory mddc are apc capable of stimulating t cells, which in turn produce gm-csf, inducing the differentiation of inflammatory monocytes to activated inflammatory dc. activated inflammatory dc are potent apc, capable of stimulating large numbers of antigen-specific t cells, which secrete more gm-csf, tnf and ifn-c. the resulting activation of inflammatory monocytes and dc leads to increased nitric oxide (no) production. no production by inflammatory monocytes, activated by a combination of ifn-c, gm-csf and lps, has been shown to suppress cd4 + t cells cultured with mog-peptide in vitro [116] . however, it should be noted while no clearly has antiviral efficacy [80] , sustained levels in vivo may cause disease and result in substantial bystander damage during wnv encephalitis either directly [21] or indirectly, probably via ifn-c stimulation of inflammatory mu [105, 21] . another effector function of cd4 + t cells in eae was revealed by co-culture of myelin specific cd4 + t cells with monocytes from eae mice; this resulted in monocyte upregulation of mhc-ii, cd11c, cd86 and cd40, and downregulation of ly6c, indicating a shift toward dc phenotype. this was further confirmed by adding cd4 + t cells isolated directly from the spinal cord of eae mice to monocyte culture, resulting in monocytes from healthy animals upregulating dc markers and co-stimulatory molecules, becoming more granular, larger and forming dendrites [59] . nkt cells are a group of regulatory immune cells of emerging importance, which mainly recognize lipids and glycolipids presented by cd1d. their capacity to rapidly release an array of different cytokines allows them to influence the direction of the immune response. nkt cells secrete gm-csf, il-4 and ifn-c when they bind cd1d [117] , which is expressed on monocytes, inter alia. as a consequence, monocytes isolated from human blood are capable of inducing nkt cell cytokine production, which then drives monocytes to differentiate into dc [118] . a more recent study suggests that during acute neuroinflammation in eae, monocyte differentiation is skewed to m2 mu by invariant nkt cell (inkt) activation with cd1d induction and il-4 production. the switch from m1 mu to m2 mu resulted in improved disease outcome [119] . although the factors determining monocyte fate are not completely understood, studies to date strongly suggest that multiple factors at the site of differentiation, including those secreted by resident and immune effector cells, play crucial roles in this process. clear differences between mediators in an infectious setting, such as viral encephalitis, and the autoimmune response, which drives eae, likely determine the outcome of monocyte differentiation into mu or dc, respectively. these differences may also affect the mechanisms by which the cns mobilizes monocytes in the bone marrow. as monocyte differentiation can have both protective and immunopathological outcomes in cns disease, it is crucial to gain better insight into defining factors that govern differentiation to inform more tailored approaches for intervention in these diseases. fate mapping analysis reveals that adult microglia derive from primitive macrophages fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response blood monocytes consist of two principal subsets with distinct migratory properties monocyte recruitment during infection and inflammation a clonogenic bone marrow progenitor 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monocytes in vitro and experimental autoimmune encephalomyelitis in vivo interleukin-32 induces the differentiation of monocytes into macrophage-like cells nod2 triggers an interleukin-32-dependent human dendritic cell program in leprosy differential regulation of human interferon a gene expression by interferon regulatory factors 3 and 7 irf family of transcription factors as regulators of host defense interferon regulatory factor irf-7 induces the antiviral alpha interferon response and protects against lethal west nile virus infection differential regulation of interferon regulatory factor (irf)-7 and irf-9 gene expression in the central nervous system during viral infection irf3 helps control acute tmev infection through il-6 expression but contributes to acute hippocampus damage following tmev infection neurons produce type i interferon during viral encephalitis interferon regulatory factor-7 modulates experimental autoimmune encephalomyelitis in mice icsbp/irf-8 retrovirus transduction rescues dendritic cell development in vitro icsbp is essential for the development of mouse type i interferon-producing cells and for the generation and activation of cd8alpha(+) dendritic cells essential role for icsbp in the in vivo development of murine cd8alpha + dendritic cells ifn regulatory factor 8 is a key constitutive determinant of the morphological and molecular properties of microglia in the cns icsbp directs bipotential myeloid progenitor cells to differentiate into mature macrophages monocyte differentiation to macrophage requires interferon regulatory factor 7 critical role of irf-8 in negative regulation of tlr3 expression by src homology 2 domain-containing protein tyrosine phosphatase-2 activity in human myeloid dendritic cells differential expression of ifn regulatory factor 4 gene in human monocyte-derived dendritic cells and macrophages irf-4 expression in the human myeloid lineage: up-regulation during dendritic cell differentiation and inhibition by 1alpha,25-dihydroxyvitamin d3 natural killer cells trigger differentiation of monocytes into dendritic cells il-10 dampens tnf/inducible nitric oxide synthaseproducing dendritic cell-mediated pathogenicity during parasitic infection nk cell-derived interferon-gamma orchestrates cellular dynamics and the differentiation of monocytes into dendritic cells at the site of infection cxcr3-dependent cd4(+) t cells are required to activate inflammatory monocytes for defense against intestinal infection cxcr3 signaling reduces the severity of experimental autoimmune encephalomyelitis by controlling the parenchymal distribution of effector and regulatory t cells in the central nervous system plasticity of ly-6c(hi) myeloid cells in t cell regulation functionally distinct subsets of cd1d-restricted natural killer t cells revealed by cd1d tetramer staining nkt cells direct monocytes into a dc differentiation pathway activation of invariant nkt cells in early phase of experimental autoimmune encephalomyelitis results in differentiation of ly6chi inflammatory monocyte to m2 macrophages and improved outcome tma is supported by and australian postgraduate award, cvv is supported by a university of sydney international scholarship, pn is supported by an international postgraduate research scholarship. this work was supported by nh&mrc project grant 1030897 to njck. key: cord-316227-dgyxbgvg authors: geginat, jens; paroni, moira; pagani, massimiliano; galimberti, daniela; de francesco, raffaele; scarpini, elio; abrignani, sergio title: the enigmatic role of viruses in multiple sclerosis: molecular mimicry or disturbed immune surveillance? date: 2017-05-23 journal: trends immunol doi: 10.1016/j.it.2017.04.006 sha: doc_id: 316227 cord_uid: dgyxbgvg multiple sclerosis (ms) is a t cell driven autoimmune disease of the central nervous system (cns). despite its association with epstein-barr virus (ebv), how viral infections promote ms remains unclear. however, there is increasing evidence that the cns is continuously surveyed by virus-specific t cells, which protect against reactivating neurotropic viruses. here, we discuss how viral infections could lead to the breakdown of self-tolerance in genetically predisposed individuals, and how the reactivations of viruses in the cns could induce the recruitment of both autoaggressive and virus-specific t cell subsets, causing relapses and progressive disability. a disturbed immune surveillance in ms would explain several experimental findings, and has important implications for prognosis and therapy. a huge body of evidence suggests that viral infections promote ms; however, no single causal virus has been identified. multiple viruses could promote ms via bystander effects. molecular mimicry is an established pathogenic mechanism in selected autoimmune diseases. it is also well documented in ms, but its contribution to ms pathogenesis is still unclear. bystander activation upon viral infection could be involved in the generation of the autoreactive and potentially encephalitogenic t helper (th)-1/17 central memory (th1/17 cm polymorphisms associated with ms are involved in immune responses, in particular in the activation and homeostasis of t cells [6] , consistent with the concept that ms is a t cell-driven autoimmune disease. the importance of the environment in determining whether a genetically susceptible individual develops ms has been underlined by studies of monozygotic twins and of genetically susceptible individuals migrating from low-to high-risk areas. the strongest environmental risk factors are vitamin d deficiency, smoking, and viral infections [7] . interestingly, infections with helminths have been shown to have a protective effect [7, 8] . among viral infections, ebv shows the strongest association, and it was estimated that ebv-induced infectious mononucleosis increases the risk of ms to a similar degree as the strongest genetic risk factor (hla-drb1*15:01) [4, [9] [10] [11] . in addition to ebv, several other viruses have been implicated in ms [12] , in particular neurotropic viruses, including human herpes virus-6 (hhv-6) [13] , herpes zoster virus [14] and john cunningham virus (jcv) [15] , but also endogenous retroviruses [16] . based on this evidence, a possible viral etiology of ms has been proposed [9, 13, 15, 17] and continues to stimulate intense research in the field [ 6 7 1 _ t d $ d i f f ] (see outstanding questions). the risk of life-threatening jcv-induced progressive multifocal leukoencephalopathy (pml) in patients with ms undergoing therapy with natalizumab [18] , a therapeutic antibody that binds to the a4-integrin adhesion receptor and blocks lymphocyte migration to the cns, has highlighted the importance of antiviral immune surveillance of the cns. indeed, the presence of a lymphatic system in the cns has challenged the view of the cns being an immune-privileged site [19, 20] , and it is now widely accepted that the cns is surveyed and protected by antiviral t cells [21] [ 6 7 2 _ t d $ d i f f ] (box 1). given this updated view of immune responses in the cns, here we discuss different models of how viral infections could promote ms, and illustrate how a defective antiviral immune surveillance could be a driving force in its pathogenesis. although the epidemiological data clearly indicate that viral infections are a critical risk factor for ms, the underlying mechanisms are poorly understood [12] . animal models that induce experimental autoimmune encephalomyelitis (eae) in the absence of viral infections by priming pathogenic cd4 + t cells with myelin antigens are widely used to study neuroinflammation and ms [22] . self-tolerance has to be broken in these models by adjuvants such as cfa, which contain killed mycobacteria, intracellular pathogens that potently activate the innate immune system. alternative models of ms, in which demyelination is induced by neurotropic viruses, such as mouse hepatitis virus or theiler's murine encephalomyelitis virus (tmev), are less studied, but enable researchers to address how viral infections could promote ms [23] . tmev induces chronic inflammation and demyelination in the brain and, importantly, both virusspecific and myelin-reactive effector t cells are generated in this ms model [23] . thus, antiviral immune responses in the cns can result in the breakdown of self-tolerance to myelin antigens, box 1. cns immune privilege the notion that the cns is a tolerogenic, 'immune-privileged' site, where immune reactions that occur in peripheral tissues are inefficient and slow, stems from seminal studies with transplanted allogenic tissues that were not or were only slowly rejected in the brain, unless animals had been immunized previously [150] . in addition, it is well known that entry of macromolecules and immune cells into the cns from the blood is restricted by the bbb and, until recently, the cns was also believed to lack lymphatic drainage. however, the presence of a lymphatic system of the meninges in the brain and of occasionally reactivating neurotropic viruses suggest that the cns is constantly surveyed by the immune system, although in a manner that limits the type of collateral tissue damage that occurs in ms. which is normally prevented by regulatory t cells (tregs) [24] . in addition, viruses and antiviral t cells can also lead directly to damage and demyelination in the cns [23, [25] [26] [27] [28] . thus, viral ms models are highly relevant, but less studied than are those of eae, and this is one reason why the role of viruses in ms is still poorly understood. the use of myelin antigens to induce eae is consistent with demyelination in the brain of patients with ms and with the presence of autoreactive cd4 + t cells recognizing myelin-derived antigens that are restricted by the major ms risk allele, hla-drb1*15:01 [29, 30] . however, myelin-reactive cd4 + t cells are rare and were found at similar frequencies in the peripheral blood of healthy individuals and of patients with ms [30] . however, myelin-reactive t cells have more proinflammatory properties in patients with ms than in healthy donors [31, 32] , and are enriched in the csf of patients with ms shortly after an attack [32, 33] . nevertheless, myelinderived antigens are probably not the only relevant self-antigens in ms. in particular, pathogenic t cells in ms may have a degenerate t cell receptor (tcr) that cross-reacts with several structurally related self-peptides, or potentially even directly with the backbone of some mhc molecules [34] [35] [36] [37] . these autoreactive t cells have a high pathogenic potential because they could be activated by any antigen-presenting cell (apc) that expresses mhc class ii and costimulatory molecules. finally, virus-specific cd4 + and cd8 + t cells could also cause collateral damage in the cns following an antiviral immune response, in particular those that cross-react with relevant self-antigens due to a phenomenon known as 'molecular mimicry' [38, 39] . molecular mimicry is the most frequently discussed mechanism for how viruses could induce autoimmunity and ms (box 2), and excellent reviews have been published on this topic [34, 39, 40] . some tcrs recognize several similar peptides, and this cross-reactivity is relevant for autoimmunity [41] and might be exploited by pathogens, such as viruses, to avoid recognition by the adaptive immune system [42] . indeed, autoreactive t cells are either deleted in the thymus, rendered unresponsive in the periphery, or even redirected to the treg lineage that induces dominant immune suppression [43, 44] . some viral proteins contain peptide sequences that are similar to the self-proteins of their hosts [39] . in the case of ms, molecular mimicry between myelin basic protein (mbp) and the ebv latency antigen ebna-1 is well documented, since cd8 + [ 6 7 3 _ t d $ d i f f ] t cell clones isolated from patients with ms could be activated by both mbp-and ebna-1-derived peptides [38] . in addition, cd4 + t cells that cross-reacted with both ebna-1 and mbp were identified [45] , and molecular mimicry might also be exploited by hhv-6 [13] . interestingly, commensal bacteria are known to be essential for autoimmune demyelination [46] , and t cells expressing a tcr that cross-reacted with mbp and a common bacterial peptide was able to induce ms-like disease in humanized mice [37] . however, while molecular mimicry is largely accepted as the driving force in some autoimmune diseases, such as streptococcus-driven rheumatic fever, its relevance in ms is still debated [39, 40] . moreover, glossary blood-brain barrier (bbb): comprises two physical barriers of endothelial cells and the parenchymal base membrane that strongly limit the access of macromolecules and cells to the cns parenchyma. the endothelial and parenchymal basement membranes define the inner and outer limits of the perivascular space, which is filled by cerebrospinal fluid (csf), which drains macromolecules and immune cells into deep cervical lymph nodes. central memory (t cm ) and effector memory t (t em ) cells: distinguished by ccr7 expression in humans and by cd62l expression in mice; these cells home preferentially to lymphoid and nonlymphoid tissues, respectively. t em produce higher levels of proinflammatory cytokines than do t cm , but both t cm and t em contain th1, th17, and th1/17 cells. moreover, t cm express adhesion and chemokine receptors, enabling them to home to the cns. clinically isolated syndrome (cis): a first attack is classified as cis unless ms is diagnosed following the demonstration of lesion dissemination in space over time at mri or the occurrence of a second clinical attack. experimental autoimmune encephalopathy (eae): the moststudied animal model of ms. cns inflammation and disability is induced by immunization with myelin antigens and adjuvants, or by the adoptive transfer of activated, myelin-specific th cells. progressive multifocal leukoencephalopathy (pml): an often fatal demyelinating cns disease caused by uncontrolled jcv replication. it is largely limited to individuals where cd4 + t cell responses are impaired, such as patients with aids or ms, where cns immune surveillance is inhibited by natalizumab. t helper 1 (th1) and t helper 17 (th17) cells: uncommitted naïve cd4 + t cells can differentiate upon antigenic activation under the influence of different cytokines to ifn-g-producing th1-or il-17producing th17 cells. th1 cells are induced by il-12 and mediate protection against intracellular pathogens, such as viruses, while th17 cells are induced by tgf-b, ilmimicry is an evolutionary process whereby one organism acquires a similarity to another organism to obtain a survival advantage. molecular mimicry is a phenomenon whereby molecules of a pathogen, in particular peptides, are similar to peptides of its host. t cells mount an immune response when they are activated by foreign peptides, but do not normally react against self-peptides. the latter is ensured by a combination of central and peripheral tolerance mechanisms, which lead to the deletion of highly autoreactive t cells in the thymus and ensures that t cells with an intermediate autoreactivity are not aberrantly activated in the periphery. the molecular mimicry hypothesis of autoimmunity proposes that t cells cause autoimmune disease following an antipathogen immune response when they cross-react with selfpeptides from healthy, uninfected tissues. molecular mimicry is thought to be a driving force in, for example, streptococcus-driven rheumatic fever and has also been documented for the ebna-1 protein of ebv and myelin basic protein in ms. 1, il-6, and il-23, and are vital for dealing with extracellular bacteria and fungi. th1/17 cells co-produce ifn-g and il-17 and can respond to both intra-and extracellular pathogens. it was found t cell responses to antigens derived from ebv, jcv, and myelin were largely confined to t helper type 1 (th1) and th1/17 cell subsets in patients with ms [32] . therefore, cd4 + [ 6 7 4 _ t d $ d i f f ] t cell responses in ms against these viruses and myelin antigens are predominantly mediated by distinct th cell subsets rather than by virus-specific t cell clones that cross-react with self-antigens. nevertheless, th1/17 cells isolated from the csf reacted with autologous apc in the absence of exogenous antigens, and they might cross-react with peptides from other viruses or bacteria. thus, although molecular mimicry is likely to contribute to the ms risk conferred by infections, other mechanisms are also likely to be important[ 6 7 5 _ t d $ d i f f ] . in addition to cd4 + t cells, b cells and cd8 + t cells are also involved in human ms, as evidenced by their presence in the cerebrospinal fluid (csf) and in demyelinated brain lesions in patients [47] . in particular, the production of oligoclonal antibodies in the csf is highly characteristic for ms and, therefore, has been used as a supportive criterion for diagnosis [48, 49] . moreover, b cell depletion with rituximab reduces relapses in patients with rr-ms [50, 51] . this therapeutic effect could reflect the capacity of b cells to present antigens to pathogenic t cells [52] , since rituximab does not deplete plasma cells and antibody levels are poorly affected [51] . however, rituximab could also inhibit viral delivery to the brain, because b cells represent a cellular reservoir of both ebv and jcv [53] and, therefore, are a likely vehicle for these viruses to pass across the blood-brain barrier (bbb). interestingly, secondary progressive ms is characterized by tertiary meningeal lymphoid structures, which might contain infected b cells as a constant local source of ebv [54] . oligoclonal antibodies in the csf were first thought to represent non-sense igg [55] , and their physiological relevance is a matter of debate. however, several groups found that they could react with neurotropic viruses [56, 57] . conflicting results have been published on ebv-specific antibodies in the csf [9, 58] , but several groups reported that antibodies against measles, rubella, and herpes zoster viruses, known as the 'mrz reaction', are present in 80-100% of patients with ms [57] , and might predict whether patients with a clinically isolated syndrome (cis) will go on to develop ms [49] . the presence of virus-specific antibodies in the csf suggests that demyelination in ms is accompanied by antiviral immune responses. consistent with this notion, ebv-specific cd8 + [ 6 7 6 _ t d $ d i f f ] t cells are specifically expanded in the csf of patients with ms [59] , and cd8 + t cells interacting with lytically infected b cells have been identified in ms brain lesions [60, 61] . however, whether the presence of ebv in brain lesions is characteristic for ms is debated [62] . of note, several viruses persist in a latent stage, are neurotropic and, thus, might be present in the brain. moreover, herpes simplex virus (hsv) was shown to trigger the generation of autoantibodies in the brain [63] , and the neurotropic viruses hhv-6, jcv, and herpes zoster were proposed to have a role in ms [13, 15, 18] . direct detection of viral nucleic acids is limited to a minority of patients [64] , but localized viral reactivation in the parenchyma might not be necessarily detectable in the csf by standard techniques [65] . of note, many viruses, including ebv and jcv, are efficiently controlled in healthy individuals, but can cause life-threatening infections in immuno-compromised individuals [66, 67] . these findings raise the question whether patients with ms mount a somehow altered immune response against viruses, in particular in the tolerogenic environment of the cns. the association of mhc class i polymorphisms with protection is consistent with an inefficient antiviral cytotoxic immune response in patients with ms. however, so far, antiviral immune responses in patients with ms were found to be either normal or even increased, in the case of ebv [60] , and patients with ms treated with strong immune suppressants, with the notable exception of natalizumab, do not experience increased viral reactivations. nevertheless, more qualitative approaches might be required to monitor antiviral immune responses in patients with ms [54, 68] . for example, antiviral t cell responses are normally measured by ifn-g production; however, central memory t cells (t cm ), which could perform antiviral immune surveillance of the cns (see below), produce only limited amounts of ifn-g, but can be efficiently expanded with viral antigens [69] . in summary, the role of different viral infections in ms is debated, and more research is needed to understand the regulation of antiviral immune responses in patients with ms and how they might impact pathogenesis and disease progression. t cell migration from lymph nodes to the cns is required for antiviral immune surveillance and relapse a required feature of t cells to not only induce relapse, but also perform antiviral immune surveillance is their capacity to migrate from lymph nodes to the cns. access to the cns by immune cells is tightly controlled, and the brain is separated from the blood by the bbb the migration of leukocytes into different tissues is controlled by specific adhesion molecules and chemokine receptors. the a4/b1-integrin is known to be a key adhesion molecule for cns entry [70] , although th17 cells can home to the cns independently of the a4/b1-integrin [71, 72] . nevertheless, natalizumab inhibits relapses and promotes jcv reactivation and pml in patients with ms, indicating that the a4/b1-integrin has a critical role for cns homing of both pathogenic and protective, antiviral t cells. integrins have to be activated by inside-out signaling and, in human th1 cells, different chemokine receptors can induce a4/b1-driven adhesion [73] . however, the relevant chemokine receptors in cns homing and ms are debated. one candidate is ccr6, which is induced by tgf-b and proinflammatory cytokines [74] and is stably expressed on human il-17-producing th17 cells [75] [76] [77] . ccr6 was proposed to allow access of t cells to the cns via the lumbar spinal cord upon eae induction [78] or at steady state via the choroid plexus [79] , paving the way for the consecutive recruitment of additional t cells upon ensuing inflammation. inflammatory chemokine receptors implicated in cns homing and ms are cxcr3 and ccr5 [80] . these two chemokine receptors are selectively expressed on ifn-g-producing th1 and th1/17 cells, which fight viruses [81] ; the cxcr3 ligand, cxcl10/ ip-10, is induced upon viral infections in the canonical response to interferons [82] . finally, ccr7 is also implicated in t cell migration in the cns and ms [83] . this is somewhat surprising, since a key function of ccr7 on t cells is to mediate homing of naïve and ccr7-expressing t cm to lymph nodes, while homing of ccr7 à [ 6 7 7 _ t d $ d i f f ] effector memory t cells (t em ) to nonlymphoid tissues is predominantly mediated by inflammatory chemokine receptors, such as ccr5 [84] . however, relevant fractions of t cm express the a4/b1-integrin [84] , cxcr3 [69] , and ccr6 [77] , suggesting that some t cm shuttle between lymph nodes and the cns. indeed, ccr7 + t cells are highly abundant in the csf of patients with ms [85] and ccr7 ligands are expressed in the cns, including in ms lesions [86] . the expression of ccr7 and of other chemokine receptors is dynamic in antigen-activated t cells [87] and, therefore, it is uncertain whether ccr7 expression reliably identifies t cm and t em in the cns of patients with active ms [85] . nevertheless, an encephalitogenic role for t cm in ms is suggested by the therapeutic efficiency of fingolimod (fty20), which sequesters naïve and t cm in lymph nodes, but spares t em [88] . furthermore, in the csf of patients with natalizumab-treated ms, ccr7 + vla-4 + t cells were depleted, while ccr7 à vla-4 à t cells were strongly enriched [72] . since these patients had stable disease, these findings are consistent with the view that t cm -derived cells drive pathogenic cns inflammation in ms. ccr7 ligands are also crucial for protective antiviral t cell responses in the cns in mice [27] , further suggesting that the cns migrations of antiviral and pathogenic t cells rely on similar mechanisms. in summary, different migratory routes for t cells to reach the cns have been described, but the a4/b1-integrin appears to be critical for both antiviral immune surveillance and relapse, while the identities of the relevant chemokine receptors are uncertain. the eae model was instrumental for the identification of proinflammatory cytokines that can drive pathogenic cns inflammation. a seminal finding in autoimmunity was that il-23, but not the closely related cytokine il-12, has a nonredundant pathogenic role [89] [90] [91] . il-12 potently induces th1 cells, whereas il-23 induces the maturation of th17 cells, suggesting that th17 but not th1 cells are the key pathogenic cells. this concept was rapidly expanded to human organ-specific immune-mediated diseases, because single nucleotide polymorphisms (snps) in il-23r, which reduces il-23-mediated signaling [92] , were shown to have a strong protective effect in psoriasis [93] and crohn's disease [94] . however, a similar protective effect of snps in il-23r was not found for ms [95] ; instead, polymorphisms in the gene locus encoding the il-12-specific subunit p35 showed a strong association [4] [5] [6] 95] . interestingly, in viral encephalitis induced by neurotropic coronavirus in mice, il-12, but not il-23, enhanced morbidity, and this was associated with enhanced t cell ifn-g production [25] . thus, while il-23 has a nonredundant pathogenic role in eae, il-12 appears to be relevant in viral ms mouse models and possibly also in human ms [95] . in the eae model, different t cell subsets, including both th1 and th17 cells, could induce pathogenic neuroinflammation, although with different characteristics [89] . th1/17 cells that co-produce ifn-g and il-17 have high pathogenic potential and are also enriched in brain lesions of patients with ms [96] . in humans, they can be induced from not only naïve t cells with il-1b and il-23 [97, 98] , but also from conventional th17 cells with il-1b and/or il-12 [32, 76, 99] . il-17 can enhance bbb permeability [100] and has neurotoxic potential [101] ; in addition, promising results were obtained in a clinical trial with a neutralizing anti-il-17 antibody in rr-ms [102] . however, deficiency for neither il-17 [103, 104] nor ifn-g [105] completely prevents [ 6 8 1 _ t d $ d i f f ] eae induction, while gm-csf is absolutely required [106, 107] . the proposed pathogenic mechanism in eae is that dendritic cell (dc)-derived il-23 induces th17 cells to produce gm-csf, which in turn leads to the recruitment and activation of additional myeloid cells [89] . gm-csf-producing t cells are also abundant in the csf of patients with ms, but gm-csf appears to be regulated differently in humans compared with [ 6 8 2 _ t d $ d i f f ] mice [98, 108] , and is produced not only by th17 cells, but also by th1 cells [32, 108] . in summary, relevant differences exist in the regulation and pathogenicity of key proinflammatory cytokines, including il-12, il-23, and gm-csf, in eae, viral ms models, and patients with ms. an alternative mechanism that could explain a pathogenic role of viral infections in ms is bystander activation [ 6 8 3 _ t d $ d i f f ] (box 3). viruses potently induce the maturation of dcs that consequently upregulate mhc and co-stimulatory molecules [109] , thus favoring the activation of not only virus-specific, but also potentially autoreactive t cells. in addition, lytic viruses, such as jcv, can induce the death of myelin-producing oligodendrocytes [28] , inducing the release and box 3. bystander activation bystander activation is a process whereby an adaptive immune response against a specific pathogen leads to the activation not only of pathogen-specific t cells, but also of 'bystander' t cells that are not specific for the pathogen. two different mechanisms have been described: bystander t cell activation can occur in a tcr-independent fashion via homeostatic cytokines, such as il-7 or il-15. the latter allows established cd8 memory t cells and antiviral cd4 + [ 6 5 6 _ t d $ d i f f ] t cells to survive despite the high number of new effector and memory t cells that are generated to protect against a new invading pathogen. this has been well documented in the case of viral infections in mice, and tcr-independent proliferation induced by cytokines [ 6 5 7 _ t d $ d i f f ] has been documented in humans. second, bystander t cells can be activated via the tcr, because pathogens induce dc maturation and, thus, upregulate mhc and co-stimulatory molecules. this tcrdriven bystander activation is particularly important for autoreactive t cell responses, and can be further modulated by cytokines. presentation of myelin-derived self-antigens in deep cervical lymph nodes by dcs [19] ( figure 1 ). the inhibition of bystander activation of autoreactive t cells is the task of tregs [24] , but several treg subsets appear to be defective in patients with ms [110] . moreover, the ms-associated polymorphisms in genes regulating t cell activation suggest that th cells are more resistant to suppression [6] . however, naïve t cells have a high activation threshold and, therefore, it appears more likely that autoreactive memory t cells are aberrantly activated. importantly, healthy individuals harbor a population of autoreactive memory t cells that secrete il-10 in response to low-level tcr stimulation, such as self-mhc, to inhibit their own proliferation [74] . these autoreactive t cells express ccr6, possibly as a consequence of exposure to tgf-b at steady state and, thus, appear to be closely related to th17 cells [74, 77] . they are distinct from autoreactive cd25 + tregs because they do not express foxp3, and can also be distinguished from foxp3 à il-10-producing regulatory 'tr1' cells [111] [112] [113] . interestingly, some of these autoreactive ccr6 + memory t cells are specific for recall antigens, such as tetanus toxoid [74] , suggesting that they have a degenerate tcr specificity. in response to optimal tcr stimulation or to recall antigens, they behave in a similar way to conventional memory t cells, suggesting that they contribute to protective recall responses in healthy individuals [74] . intriguingly, patients with rr-ms have an expanded population of autoreactive ccr6 + t cells, which express cxcr3 and co-produce il-17 and ifn-g [32] . il-10 production is reduced in cxcr3 + ccr6 + t cells [32] , suggesting that these autoreactive th1/17 cells are more pathogenic [114] and have a reduced capacity to inhibit their own activation in response to low-level tcr stimulation [74] . notably, th1/17 cells can be induced from ccr6 + t cell precursors in response to il-1b and/or il-12 [32, 76, 99, 115] , proinflammatory cytokines that are produced by dcs in response to viruses [109, 116] . a conversion of th17 cells to ifn-g-producing th1/17 cells also occurs in inflamed tissues in mice in vivo [117] [118] [119] . in addition, the expanded th1/17 cells in patients with ms proliferated with myelin antigens, produced high levels of gm-csf and expressed the a4/b1-integrin and ccr7 [32] , indicating that these cells are potentially encephalitogenic. consistent with this hypothesis, these th1/ 17 cm cells are selectively expanded in patients with rr-ms with a high disease severity score, and might represent a cellular 'sword of damokles'. following their generation, which could be a key pathogenic event in ms, these th1/17 cm cells probably no longer require il-12, consistent with the unexpected failure of anti-il-12/23p40 antibodies to inhibit relapse in established rr-ms [120] . in summary, virus-induced dc maturation and cytokine production could lead to the generation of potentially pathogenic th1/17 cm cells from autoreactive, il-10producing cxcr3 à ccr6 + memory t cells, which are constitutively present in healthy individuals [32, 74] (figure 1 ). genome-wide association studies have identified snps in the loci of il-2ra and il-7ra as msassociated risk factors [6, 121] . these cytokine receptors not only control the homeostasis of cd4 + [ 6 8 4 _ t d $ d i f f ] regulatory and th cells, respectively [81, 122] , but are also essential for protective cd8 + t cell responses [123, 124] . notably, the risk-conferring snp of the il-7r reduces il-7r expression [6, 121] , suggesting that il-15, which controls memory t cell homeostasis together with il-7 [69, [125] [126] [127] [128] , is particularly important in patients with ms. based on t cell repopulation studies following therapeutic lymphocyte depletion with anti-cd52 antibodies, it was previously proposed that il-15-dependent t cell homeostasis might be disturbed in ms [129] . it was found that, while th1/17 cm cells were expanded in patients with ms with a high disease score, conventional th1 cells were selectively decreased, in particular th1 cm cells in patients treated with natalizumab [32] . the latter was unexpected, since natalizumab leads to the accumulation of proinflammatory t cells in the circulation [130] . notably, in healthy individuals, both th1 and th1/17 cells respond to viruses [77, 99] , including jcv [32] . conversely, in patients with rr-ms, th1/17 cm cells failed to respond to jcv, but instead proliferated spontaneously with autologous dcs [32] . consequently, the natalizumab-associated decrease in th1 cm cells might explain the risk of patients with ms developing pml following prolonged natalizumab treatment. indeed, patients with pml and ms treated with natalizumab have an impaired jcvspecific th1 response [131] , but additional studies are needed to establish whether a decrease in th1 cm cells is associated with the risk of pml. what could be the mechanism of the selective shift from th1 to th1/17 cells in the analyzed patients with ms? among cd4 + t cells, only the th1 and th1/17 subsets express high levels of t-bet [32] , which induces not only ifn-g and cxcr3 [81] , but also the il-2/15rb chain (cd122), consequently rendering t cell homeostasis sensitive to il-15 ( figure 2a ) [69, 126] . importantly, persistence of antiviral, but not of conventional, cd4 memory t cells requires il-15, and they compete with other cd122 + lymphocytes for il-15 in vivo [126] . in humans, t cm proliferate slowly in the steady state [132] , and cxcr3 + 'pre-th1' cells, which contain both th1 cm and th1/17 cm cells, express the highest levels of cd122 among cd4 + t cm , and proliferate most extensively with il-15 in the absence of tcr stimulation [32, 69, 84] . thus, th1 cm and th1/17 cm cells are expected to compete for il-15 in secondary lymphoid organs [133] , and this competition could be intensified when natalizumab limits their access to il-15rich peripheral tissues, such as bone marrow or the lungs [134] . of note, the lung is a niche for resting, myelin-reactive memory t cells in lewis rats with eae [135] . moreover, the bone marrow is a key site for memory t cell maintenance [136, 137] , and human antiviral cd4 + t cells are enriched at this site [136] . interestingly, natalizumab inhibits bone marrow homing of stem cells [138] , although it is unclear whether it also interferes with t cell homing to bone marrow or the lung [130, 139] . in lymph nodes, dcs are expected to expand preferentially autoreactive th1/17 cm cells in the absence of viral reactivation [32] ( figure 2b ). indeed, an important function of dcs is to present self-mhc to cd4 + t cells at steady state to induce naïve t cell survival [140] and allow secondary expansions of cd4 + memory t cells [141] . moreover, dcs also trans-present il-15 to lymphocytes ( figure 2b ), including cd4 + t cells [142] [143] [144] , and could induce the survival and il-15-dependent proliferation of antiviral th1 cells [126] . thus, competition for self-mhc and il-15 presented by dcs in lymph nodes is a possible mechanism whereby autoreactive, pathogenic th1/17 cm cells could expand selectively at the cost of virusspecific, protective th1 cm cells in patients with natalizumab-treated ms (figure 2 ). whatever the mechanism, such disequilibrium would render patients with ms more vulnerable not only to relapses, but also to jcv [67, 131] . in summary, cd4 + t cell homeostasis appears to be disturbed in patients with ms, but more research is needed to understand the maintenance of relapses could be triggered by viral reactivations in the cns that induces the recruitment of autoreactive bystander th1/17cells as discussed above, at least two different pathways could lead to the recruitment of pathogenic t cells to the cns, and consequently to relapses. on the one hand, autoreactive t cells could enter the cns in the absence of infections via ccr6 [78, 79] and, on the other hand, viral reactivations in the cns could induce cxcl10/ip10 and consequently attract autoreactive and/or virus-specific cxcr3 + t cells [82] . notably, since th1/17 cells co-express cxcr3 and ccr6, they could use either pathway to reach the cns. however, antiviral th1 cells that lack ccr6 expression are strongly enriched in the csf of patients with active ms [32] , suggesting that the ip10/cxcr3 axis is relevant for relapses. consistent with this notion, in the circulation of patients with ms where cns homing is blocked by natalizumab, there is a selective increase in cxcr3 + b cells [145] and cxcr3-expressing th1/17 cells [32, 130] . the requirements for cxcl10/ip10 in different animal models of ms is variable [82] , but a role for cxcl10/ip10 in ms is further suggested by the fact that it is expressed in brain lesions of patients with ms [80] , and that snps in its gene locus are associated with a worse prognosis [146] . in healthy individuals, cxcr3 + th1 and cxcr3 + ccr6 + th1/17 cells respond to viruses and express the a4/b1integrin and, thus, both could contribute to the physiological antiviral immune surveillance in the cns [147] ( figure 3a , key figure) . conversely, in patients with ms, th1 and th1/17 cells responded selectively to viral and myelin-derived antigens [32] , respectively, suggesting that, while th1 cells also mediate antiviral immune responses in patients with ms, th1/17 cells could attack uninfected tissue and promote relapses ( figure 3a ). in this scenario, relapses could be triggered by viral reactivation that lead to bystander recruitment of autoreactive th1/17 cell to the cns via ip10/cxcr3. ebv is one obvious candidate virus to induce bystander th1/17 cell recruitment, but the high frequency of jcv-specific th1 cells in the csf of patients with active ms [32] also supports a role for jcv reactivation in relapses [15] . this 'bystander recruitment model' has important implications for ms therapy, because it predicts that selective targeting of autoreactive th1/17 cells could be as efficient as natalizumab therapy, but would not induce pml if antiviral th1 cells were spared. surprisingly little is known about the signals that induce neurotropic viruses to switch from latency to a lytic stage and, therefore, stress is often suggested as a common explanation [15, 148] . in the case of jcv, tnf-a, which is produced by effector t cells upon antigenic activation, can deliver critical reactivation signals [149] . therefore, it is possible that autoreactive th1/17 cells induce de novo viral reactivation in the cns, thus fueling a vicious feed-forward loop that leads to the recruitment of new waves of pathogenic t cells and possibly also of virus-infected b cells [53] . alternatively, it is possible that autoreactive th1/17 cells home first to the cns during the remission phase of ms via ccr6, and induce then viral reactivation and the recruitment of antiviral th1 cells via ip-10 ( figure 3 however, the latter model fails to fully explain why relapses are characteristic for patients with ms, since healthy individuals also harbor autoreactive ccr6 + [ 6 8 5 _ t d $ d i f f ] t cells [74] that produce substantial amounts of gm-csf and also some il-17 [32] . in any case, the concomitant enrichment of virus-specific th1 and myelin-reactive th1/17 cells in the csf of patients with active ms further underlines the close relationship of antiviral immune responses and ms, and warrants further investigation. how infections promote autoimmune diseases is a fascinating and clinically relevant topic. in ms, the field has been dominated by the search for a single causative infectious agent and, due to strong epidemiological evidence, ebv has attracted the most attention. the intriguing molecular mimicry hypothesis has further underlined a possible role of ebv. however, accumulating evidence indicates that several neurotropic viruses, including jcv, could have a role in ms, and it is also possible that different viruses could be important in individual patients with ms. in this review, we discussed some poorly understood, intensively debated, and understudied aspects in the field. we have proposed possible mechanisms for how viral infections could generate pathogenic th1/17 cm cells from autoreactive memory cells upon bystander activation, how these th1/17 cm cells could progressively expand at the cost of protective, antiviral th1 cells in the remission phase, and how they could finally be recruited to the cns upon viral reactivation to promote relapses. notably, the molecular mimicry concept and this bystander generation/recruitment model are not mutually exclusive. however, the finding that jcv-specific and autoreactive t cells in patients with ms are largely segregated into two different subsets of th1 and th1/17 cells with different properties suggests that selective targeting of th1/17 cells could inhibit relapses without inducing pml. more research on antiviral immune responses in animal models of ms and in patients will be necessary to unravel the blood-brain barrier blood-brain barrier blood-brain barrier blood-brain barrier blood-brain barrier . antiviral t cells rapidly control the virus, and tissue repair mechanisms ensure that damage to the cns is limited. relapses could be triggered when the same immune surveillance mechanism leads to the erroneous bystander recruitment of autoreactive th1/17 cm cells to the cns in patients with multiple sclerosis (ms). in addition, infected cxcr3 + b cells could be recruited that transport viruses, such as epstein-barr virus (ebv) and john cunningham virus (jcv) to the cns. th1/17 cm cells in patients with ms, but not in healthy individuals, react with myelin-derived self-antigens and, thus, could attack healthy, 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cell survival during influenza virus infection il-15 trans-presentation regulates homeostasis of cd4( + ) t lymphocytes natalizumab treatment leads to an increase in circulating cxcr3-expressing b cells cxcl10 haplotypes and multiple sclerosis: association and correlation with clinical course t-cells in the cerebrospinal fluid express a similar repertoire of inflammatory chemokine receptors in the absence or presence of cns inflammation: implications for cns trafficking molecular basis of hsv latency and reactivation cooperative roles of nf-kappab and nfat4 in polyomavirus jc regulation at the kb control element immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye key: cord-338235-vz2d2x18 authors: pinschewer, daniel d.; schedensack, mariann; bergthaler, andreas; horvath, edit; brück, wolfgang; löhning, max; merkler, doron title: t cells can mediate viral clearance from ependyma but not from brain parenchyma in a major histocompatibility class iand perforin-independent manner date: 2010-03-30 journal: brain doi: 10.1093/brain/awq028 sha: doc_id: 338235 cord_uid: vz2d2x18 viral infection of the central nervous system can lead to disability and death. yet the majority of viral infections with central nervous system involvement resolve with only mild clinical manifestations, if any. this is generally attributed to efficient elimination of the infection from the brain coverings, i.e. the meninges, ependyma and chorioplexus, which are the primary targets of haematogeneous viral spread. how the immune system is able to purge these structures from viral infection with only minimal detrimental effects is still poorly understood. in the present work we studied how an attenuated lymphocytic choriomeningitis virus can be cleared from the central nervous system in the absence of overt disease. we show that elimination of the virus from brain ependyma, but not from brain parenchyma, could be achieved by a t cell-dependent mechanism operating independently of major histocompatibility class i antigens and perforin. considering that cytotoxic t lymphocyte-mediated cytotoxicity is a leading cause of viral immunopathology and tissue damage, our findings may explain why the most common viral intruders of the central nervous system rarely represent a serious threat to our health. viruses are the most common infectious intruders to the cns and remain a significant source of neurological morbidity and mortality worldwide. the united states centres for disease control and prevention estimate that $20 000 cases of clinically manifested viral cns infections occur in the united states each year. in addition, and vastly exceeding these numbers, many of the common viral infections 'silently' (i.e. in the absence of clinical symptoms or overt disease) involve the cns, or exhibit mild manifestations that do not require diagnostic or therapeutic intervention. hence, they are not included in the above statistics (logan and macmahon, 2008) . mumps in the pre-vaccine era is a classic example of a viral infection frequently accompanied by meningitis. cns involvement occurred in an estimated 50% of all infected individuals but mostly went unrecognized and resolved without complications or sequelae (johnstone et al., 1972; hviid et al., 2008) . similar frequencies of cns involvement were observed during acute but uncomplicated systemic infection with measles (gibbs et al., 1959; hanninen et al., 1980) . clinically, investigation of the topic has been hampered by the fact that it requires sampling of csf, an intervention difficult to justify given the mostly benign and spontaneous outcome. hence, similarly frequent mild cns involvement as in measles and mumps is also suspected for other viruses such as influenza virus (fujimoto et al., 1998) or coxsackie b virus (rubin et al., 1958) , which are nowadays more widespread. however, laboratory confirmation is not available. viral infections of the cns are classified pathologically into those restricted to the parenchyma (encephalitis) and those that predominantly affect the coverings of the cns (i.e. meninges, ependyma and chorioplexus), although some overlap of these categories is frequently observed. the majority of viral cns infections result from haematogeneous dissemination and are initially confined to the cns coverings (fields et al., 2006) . often clinically manifested as 'aseptic meningitis', this syndrome summarizes a group of disorders with a typically mild and self-limiting course of disease (adair et al., 1953; irani, 2008) . it is generally assumed that immunocompetent individuals eliminate these infections from the cns coverings before a substantial parenchymal infection can be established, and thus before substantial damage occurs. furthermore, properties intrinsic to the virus (cellular tropism, cytolytic potential) and host parameters such as age (e.g. neonatal versus adult life, thereby affecting the level of immunological competence) influence whether the virus remains restricted to the brain coverings or whether it also affects the cns parenchyma. numerous dna and rna viruses including enteroviruses, morbilliviruses, herpes viruses, lentiviruses, flaviviruses and arenaviruses are able to spread into the brain coverings in humans (lee and davies, 2007) . how they gain access to the cns has been studied extensively, and much effort has been given to elucidating virus-host interactions and immune defence in the brain parenchyma (griffin, 2003; hausmann et al., 2005; mcgavern, 2005; bergmann et al., 2006; ercolini and miller, 2006; tishon et al., 2006) . however, less is known about how infected hosts silently cleanse the cns coverings from viral infection, and thus how they prevent not only serious complications from meningitis, but also viral spread into the parenchyma. lymphocytic choriomeningitis virus (lcmv) represents the prototypic member of the arenavirus family and is widely used to study the dichotomous role of the antiviral immune response in host protection and pathogenesis ahmed and gray, 1996; oldstone, 2007) . lcmv is a natural mouse pathogen but it is also suspected to be an underestimated cause of aseptic meningitis in humans (meyer et al., 1960; jahrling and peters, 1992) . as in humans, lcmv has the capacity to infect the brain coverings of mice, and from there it gradually spreads into the parenchyma (thomsen, 2009) . intracerebral inoculation of adult mice with lcmv has been studied for decades to define the mechanisms underlying viral pathogenesis and immunopathology in the cns. after intracerebral administration most of the inoculum is drained into the systemic circulation (mims, 1960) , initiating a vigorous immune response. at the same time, the virus replicates in the leptomeninges, choroid plexus and ependymal cells (lillie, 1945; wilsnack and rowe, 1964) . lcmv is non-cytolytic in mice, and hence the ensuing cns disease is solely caused by the immune response attacking the virus infection in the cns. accordingly, t cell-depleted mice are resistant to lcmv-induced fatal choriomeningitis (cole et al., 1972) . histological examination shows massive leucocyte infiltrates accumulating in the meningeal and ventricular region of the brain 6-8 days after infection, concomitant with the onset of rapidly progressing convulsions and death. in addition to these events in the brain coverings, t cell infiltration has also been observed within the brain parenchyma, and clinical disease has mostly been attributed to the latter part of the inflammatory response (christensen et al., 2004) . cd8 + t cells have long been identified as key players indispensable for disease (cole et al., 1972; andersen et al., 1991) . yet recently the recruitment of myelomonocytic cells was also found to be an important step in pathogenesis (kim et al., 2009) , at least for the early manifestation of the disease. in contrast to the vast majority of humans with viral infection of the cns surface, intracerebral infection of mice with wild-type lcmv strains is almost invariably lethal, even at the minimal infectious dose (bonilla et al., 2002) . thus, acute lcmv meningitis in mice is only of limited use for investigating how a primary immune response can purge viruses from the brain coverings without causing overt disease. therefore, using arenavirus reverse genetics, we recently generated an attenuated lcm virus exhibiting a fundamentally different virus-host balance: recombinant lcmv expressing the surface glycoprotein of vesicular stomatitis virus (rlcmv/indg) instead of its own glycoprotein (fig. 1a) . unlike wild-type lcmv such as the armstrong strain, rlcmv/indg fails to cause disease in intracerebral-infected adult mice (pinschewer et al., 2003; bergthaler et al., 2006; merkler et al., 2006) . nevertheless, it establishes persistent infection in the cns of immunodeficient hosts owing to its non-cytolytic behaviour. as an additional important difference to wild-type lcmv, rlcmv/ indg replicates only in the cns. even in recombination-activating gene (rag)-deficient mice lacking t and b cells, interferon type i prevents viral replication in other tissues . in several other model systems, viral elimination from the cns can be affected by ongoing viral replication at additional sites, particularly when the virus overwhelms the host to cause immune exhaustion (wherry and ahmed, 2004) . thus the rlcmv/indg model is well suited to study viral cns clearance in mouse models with defects in adaptive immunity. in the present work we used this experimental model to dissect the individual contribution of key components and pathways of the adaptive immune response in purging a non-cytolytic virus infection from the cns in the absence of severe disease. our results indicate that viral elimination from ependymal cells can be achieved in a t cell-dependent manner that occurs independently of major histocompatibility complex (mhc) class i and perforin, whereas the cytolytic mechanisms of cytotoxic t cells become essential once the virus has gained access to the parenchyma, notably to glial cells. figure 1 genome organization, viral spread and blood brain barrier integrity following intracerebral infection with either lcmv armstrong or rlcmv/indg. (a) schema of the lcmv-armstrong (lmcv-arm) and rlcmv/indg genomes. both viruses consist of two single-stranded negative-strand rna segments, encoding two viral genes in ambisense orientation each. the long (l) segment encodes for the rna-dependent rna polymerase l and for the matrix protein z, while the short (s) segment carries the gp and np genes. rlcmv/indg was created by substituting the lcmv-gp gene for vesicular stomatitis virus-indg (pinschewer et al., 2003) . (b-e) c57bl/6 mice were infected intracerebrally with rlcmv/indg or lcmv-arm as indicated or were left uninfected. (b and c) viral s-segment (s seg.) rna in brain was detected at the indicated day (d) after intracerebral infection by northern blot (ethidium bromide staining of 28s rrna indicates loading control; lanes represent individual animals). (d) immunohistochemical detection of lcmv-np confirms reduced spread of rlcmv/ indg as compared to lcmv-armstrong. note that lcmv-armstrong (days 4 and 6) and also rlcmv/indg (day 6) spreads into some subependymal cells (arrows). (e) at day 6 after infection, animals were given horseradish peroxide (hrp) intravenously. one hour later, they were sacrificed for detection of horseradish peroxidase leakage into the brain parenchyma, indicative of blood brain barrier breakdown. representative pictures of horseradish peroxidase reactions are shown (n = 3-4 animals per group). scale bar for d: 100 mm; for e: 500 mm. mice c57bl/6 wild-type mice, recombination activation gene 2 deficient mice (rag à/à (chen et al., 1993a) ), b cell deficient mice (jht à/à (chen et al., 1993b) ), t cell deficient mice (tcrb à/à (mombaerts et al., 1994) ), major histocompatibility complex (mhc) class i-deficient (b2m à/à (koller et al., 1990) and k b d bà/à (perarnau et al., 1999) ), major histocompatibility complex class ii deficient mice mhcii à/à (kontgen et al., 1993) , perforin deficient mice pko (kagi et al., 1994) , tnf receptor double deficient mice (tnfr1/2 à/à (peschon et al., 1998) ), fas deficient mice (fas à/à (adachi et al., 1995) ), cd8 deficient mice (cd8 à/à (fung-leung et al., 1991) ) and interferon gamma deficient (gko (dalton et al., 1993) ) mice (on c57bl/6 background) as well as 129sv/ev wild-type mice and interferon gamma receptor deficient mice (ifngr à/à (huang et al., 1993) ) on a 129sv/ev background were bred at the institute of laboratory animal science, university of zurich and housed under specific pathogen-free conditions during all experiments. animal experiments were carried out at the university of zurich with authorization by the cantonal veterinary office and in accordance with the swiss law for animal protection, and at the university of gö ttingen with the authorization by the district government in braunschweig, in accordance with the german law for animal protection. virus stocks were prepared, infectivity was quantified and vesicular stomatitis virus neutralizing antibodies were determined as described previously (pinschewer et al., 2004) . for intracerebral inoculations, 3 â 10 3 plaque forming units of the armstrong strain of lcmv or rlcmv/indg were administered in a volume of 30 ml through the vertex of the skull using a 27 gauge needle. for intravenous infection, 2 â 10 4 plaque forming units of the armstrong strain of lcmv in a volume of 200 ml were administered into the tail vein. vesicular stomatitis virus-hyperimmune serum (pinschewer et al., 2004) was administered i.p. viral s segment ($3.4 kb) was detected by northern hybridization as described previously (pinschewer et al., 2003) . a quantitive taqman reverse transcribed (rt)-pcr protocol targeting the lcmvnucleoprotein (np) gene (to be described elsewhere) was used to quantify rlcmv/indg s segment copies in the brain of infected mice. arbitrary viral rna units were determined in a multiplex assay with a commercial kit for detection of the housekeeping gene gapdh, serving as internal reference (applied biosystems). cytotoxicity assays and enumeration of epitope-specific cd8 + t cells specific cytotoxic t cell activity of splenocytes was assayed in a 51 cr release assay and epitope-specific cd8 + t cells were enumerated using mhc class i tetramers as described (bergthaler et al., 2006) . cns tissue was prepared in hepes-glutamic acid buffer-mediated organic solvent protection effect (hope) fixative (dcs innovative) (olert et al., 2001) and embedded in paraffin as described previously (bergthaler et al., 2007) . mice were not perfused prior to tissue collection and fixation since rlcmv/indg does not replicate outside the cns, and blood contains neither free infectivity nor circulating infected cells . upon inactivation of endogenous peroxidases (phosphate buffered saline/0.3% hydrogen peroxide, 30 min) and blocking (phosphate buffered saline/10% foetal calf serum), sections were stained with primary antibodies: mouse anti-neuronal nuclei neun (chemicon international), mouse anti-glial fibrillary acidic protein (astrocytes; dako), mouse anti-nogoa [oligodendrocytes, mab11c7 (oertle et al., 2003) , kindly provided by m. e. schwab, brain research institute, zurich], rat anti-mouse cd8 (bd pharmingen), rabbit anti-ionized calcium binding adaptor-1 (microglia/macrophages; wako pure chemical industries ltd.), rabbit anti-factor viii (von willebrand factor; endothelia; abcam) and rat anti-lcmv np (vl-4, battegay et al., 1991) . bound primary antibodies were visualized either by an avidin-biotin technique with 3,3 0 -diaminobenzidine or alkaline phosphatase/anti-alkaline phosphatase as chromogens (haemalaun counterstaining of nuclei) for light microscopy or with the appropriate species-specific cy3-or cy2-conjugated secondary antibodies (all from jackson immunoresearch laboratories inc.) with 4',6-diamidino-2-phenylindole (sigma-aldrich) nuclei counterstaining for fluorescence microscopy. to assess cellular distribution of lcmv at least 130 lcmv-np + cells per staining and group (average 416 ae 123 cells) were evaluated at â400 magnification. number of lcmv-np + cells allocated to a given cellular subtypes were expressed as percent of total allocated lcmv np + cells. damage to the blood brain barrier was visualized by intravenous injection of 400 ml of 2% horseradish peroxidase (sigma) dissolved in phosphate-buffered saline (claudio et al., 1990; hawkins et al., 1990) . horseradish peroxidase leakage into the brain parenchyma was made evident in horseradish peroxidase reactions on 6 mm-thick snap-frozen sections of brain tissue. anova with bonferroni post test was used for the comparison of individual values from multiple groups. viral rna units were log-transformed for statistical analysis. differences in individual values between two groups were analysed by t-tests (unpaired, two-tailed) and virus clearance was compared in log rank tests. these analyses were performed using graphpad prism software version 4.0b. two-way anova with bonferroni's post test for a combined analysis of values from two groups in two independent experiments was performed using spss version 13.0. p-values 50.05 were considered statistically significant ( ã ) and p-values 50.01 were considered highly significant ( ãã ), whereas p-values 40.05 were considered statistically not significant. we first infected mice intracerebrally with rlcmv/indg or with its wild-type counterpart lcmv armstrong, and compared the viral burden in cns and its topographical distribution over time. northern blot analysis of lcmv armstrong-infected brain tissue detected similar amounts of viral rna on day 4 as on day 6 when the animals displayed signs of terminal disease (fig. 1b) . in addition, lcmv armstrong rna was also detectable in spleen on days 1 and 4 after infection ( supplementary fig. s1 ). by the same methods, rlcmv/indg rna became detectable no earlier than at day 6, and was eliminated by day 8 (fig. 1b and c) . in agreement with our previous findings , rlcmv/indg rna could not be detected in spleen or liver at any time point ( supplementary fig. s1 ). northern hybridization had suggested lower levels of rlcmv/indg than of armstrong in the cns (fig. 1b) , which was confirmed by histological analysis (fig. 1d ). both viruses were mostly restricted to the brain coverings (i.e. leptomeninges, ependyma and chorioplexus). however, rlcmv/indg infected far fewer cells than armstrong, both on day 4 and 6 after infection. rlcmv/indg was found in small foci and in isolated infected cells of leptomeninges and ependyma on day 4, in somewhat larger patches of infected cells of the same structures on day 6 after infection, and was cleared thereafter (days 8 and 14, fig. 1d ). in contrast, as early as on day 4 armstrong-infected cells formed a continuous layer comprising meninges, ependymal cells and choroid plexus. furthermore, a small but consistent fraction of parenchymal cells in close proximity to the ventricular ependyma (referred to as 'subependymal cells') were also infected, as previously reported (christensen et al., 2004) (arrows in fig. 1d ). we further analysed the integrity of the blood brain barrier at day 6 after infection with either lcmv armstrong or rlcmv/ indg. this time point was chosen for analysis since rlcmv/ indg rna levels and t cell infiltrates were highest ( fig. 1b and text below) and also because of terminal disease in lcmv armstrong-infected animals (fig. 1e ). extensive disruption of the blood brain barrier was found in lcmv armstrong infection, which was evident in widespread leakage of intravenously administered horseradish peroxidase into the brain parenchyma, confirming earlier observations in lcmv-infected mice (marker et al., 1984; andersen et al., 1991) . in contrast, the blood brain barrier was only very modestly affected in rlcmv/indg-infected animals. rlcmv/indg is known to persist in the brain of immunodeficient hosts, such as neonates, without causing disease (pinschewer et al., 2003; bergthaler et al., 2006; merkler et al., 2006) . hence the present data indicate that adult infection of mice with rlcmv/indg provides a useful model to study basic mechanisms of silent immune-mediated viral clearance from the brain coverings. we had previously demonstrated that the failure of rlcmv/indg to elicit fatal choriomeningitis was not due to its lack of lcmv glycoprotein (fig. 1a) as an antigenic target of the cytotoxic t cell response to lcmv armstrong (bergthaler et al., 2006) . we had also shown that intracerebral rlcmv/indg infection induces cytotoxic t cell responses of high frequency and long-lived protective capacity (bergthaler et al., 2006 ). yet lcmv armstrong-induced cytotoxic t cells reached even higher frequencies and exhibited far higher cytotoxic activity in primary ex vivo cytotoxic t cell assays (pinschewer et al., 2004) . this difference in cytotoxic t cell response rather than the viral ability to spread in the cns could thus have accounted for the differential clinical outcome after rlcmv/indg and lcmv armstrong infection. to address this possibility, we infected mice simultaneously with rlcmv/indg and/or lcmv armstrong using the intracerebral and intravenous routes in various combinations ( fig. 2a) . besides recording the clinical outcome ( fig. 2a) , we used mhc class i tetramers to monitor the magnitude of the antiviral cytotoxic t cell response (fig. 2b) , and its cytolytic activity was determined in primary ex vivo cytotoxic t cell assays (fig. 2c) . co-infection with rlcmv/ indg intracerebral and armstrong intracerebral resulted in lethal choriomeningitis analogous to armstrong intracerebral single infection. this excluded dominant negative immunomodulatory effects of rlcmv/indg on cns immunopathogenesis. furthermore we found that intracerebral administration of armstrong caused the same t cell response and disease regardless of whether or not armstrong was additionally administered intravenously. thus, we tested whether armstrong intravenous infection could drive a cytotoxic t cell response of optimal magnitude and cytolytic capacity that would trigger cns disease in animals simultaneously infected with intracerebral rlcmv/indg. although the magnitude and functionality of the peripheral antiviral cytotoxic t cell response was equivalent or even slightly higher than in armstrong single-infected mice, these animals failed to display evidence of increased morbidity, both by clinical assessment and also by testing blood brain barrier permeability analogous to the experiments displayed in fig. 1e (data not shown). hence, these results confirm that the ability of lcmv armstrong but not rlcmv/indg to cause cns disease is unrelated to differences in the cytotoxic t cell response elicited, but rather reflect differential viral load and/or distribution in cns tissues. next we analysed the contribution of the adaptive cellular and humoral immune response to silent rlcmv/indg clearance from the cns. b cell-deficient jht à/à mice exhibited unimpaired virus clearance whereas the brains of t cell-deficient tcrb à/à mice and rag à/à animals (lacking t as well as b cells) harboured considerable levels of persisting virus as assessed on day 14 of infection and thereafter ( fig. 3a and data not shown). the antiviral cd8 + t cell response to the immunodominant epitope np396 was measured in peripheral blood on day 8 using mhc class i tetramers (fig. 3b ). these responses were similarly vigorous in mice with a targeted deletion of the jh locus (jht mice) lacking b cells, and c57bl/6 control mice, but were absent in cd8 + t cell-deficient tcrb à/à and rag à/à mice, as expected, and therefore correlated with viral clearance. notably, rlcmv/indg intracerebral infection also elicited a vigorous and early virus-neutralizing antibody response ( fig. 3c and d) . its absence in jht mice did not change the clinically silent course of rlcmv/ indg infection (not shown), indicating that silent clearance of rlcmv/indg but not lcmv armstrong was unrelated to differences in the virus-neutralizing antibody responses elicited by the two viruses. it also suggested that antibodies were not essential for rlcmv/indg clearance from the cns, but a contributory role in this process was not excluded. tcrb à/à not only failed to mount cytotoxic t cell responses but in addition they displayed considerably reduced total virus-neutralizing antibody titres (igm plus igg, p50.01) and a virtual absence of the b-mercaptoethanol-resistant virus-neutralizing antibody fraction (igg, p50.01). this was presumably due to the lack of t cell help for antibody production and immunoglobulin class switch, and could also have contributed to defective viral clearance in these animals. to address this possibility, we reconstituted tcrb à/à animals with specific antibodies by intraperitoneal administration of hyperimmune serum (fig. 3e-g) . while neutralizing igg in serum was restored to virtually normal levels, viral persistence remained unaffected. in summary, these findings indicate that t cells play an essential role in the silent clearance of rlcmv/indg from the cns, a process that largely occurs independently of systemic antiviral antibodies. considering the key role of t cells in the clearance process, we dissected the contribution of mhc class i-(mhci-) and class ii-(mhcii-) restricted responses in rlcmv/indg clearance from the brain. three of four mhc class i-deficient (mhci à/à ) mice exhibited rlcmv/indg rna on day 14 after infection whereas in mice lacking mhc class ii (mhcii à/à ) virus was undetectable (fig. 4a) . the differential ability to eliminate rlcmv/indg from the cns correlated with the normal generation of virus-specific cd8 + t cells in mhcii à/à mice, whereas mhci à/à mice are devoid of a cd8 + t cell compartment (fig. 4b) . unlike in the experiment depicted in fig. 4a , a repeat experiment in mhci à/à mice resulted in invariable viral persistence. altogether, rlcmv/indg persisted in six out of seven mhci à/à mice tested, which was significantly different from the uniform clearance in c57bl/6 wild-type controls (p50.01). to corroborate these findings in an independent knockout mouse model, we assessed rlcmv/indg clearance from the cns of cd8 à/à mice, which also lack mhci-restricted t cells. virus persisted in the brains of all four cd8 à/à infected mice, further supporting a key role of mhci-restricted t cell responses in rlcmv/indg clearance from the cns ( fig. 4c and d, p50.0001 for a combined analysis of virus clearance in c57bl/ 6 versus cd8 + t cell-deficient mhci à/à and cd8 à/à mice). as a next step we analysed the individual contribution of fas, perforin, interferon-and tumour necrosis factor-for eliminating rlcmv/ indg (figs 4e-h and supplementary fig. s2 ). unimpaired virus control in mice lacking either tumour necrosis factor receptors 1 and 2 (tnfr1/2 à/à ), fas (fas à/à ), interferon-(gko) or interferon-receptor contrasted with clearly detectable viral persistence in the brain of six out of eight perforin-deficient mice tested in two independent experiments ( fig. 4e and h; data not shown; p50.01 for virus clearance in perforin-deficient mice versus c57bl/6 control mice). impaired clearance of virus from the cns of perforin-deficient mice was accompanied by normal frequencies of viral epitope-specific cd8 + t cells in peripheral blood (p = 0.69 for np396-specific cd8+ t cell frequencies in perforin-deficient mice versus c57bl/6 mice; combined analysis of figs 4f and i), as expected (badovinac et al., 2002) . this indicated that it was indeed the absence of perforin-dependent cytolytic pathways rather than a general impairment of cd8 + t cell virus clearance from ependyma to gain further insights on how rlcmv/indg persisted in the cns of perforin-deficient, tcrb à/à and rag à/à mice but not in c57bl/6 controls, we performed histological time course analyses of viral antigen and of infiltrating cd8 + t cells. four days after infection the virus was restricted to ependymal cells from where it spread into a few subependymal cells by day 6 (fig. 5a) . these early events were identical in t cell-competent perforin-deficient and wild-type animals as well as in t cell-deficient tcrb à/à and rag à/à mice. however, c57bl/6 wild-type animals eliminated the virus by day 8, whereas slowly but steadily increasing numbers of infected cells were found within the cns parenchyma of perforin-deficient, tcrb à/à and rag à/à mice on days 8, 10 frequencies of np396-specific cd8 + t cells in blood were determined on day 8 using mhc class i tetramers. tcrb à/à and rag à/à mice lack cd8 + t cells. (c and d) virus-neutralizing total ig (c) and igg (d) were determined at the indicated time points. (e-g) c57bl/6, jht and tcrb à/à mice were infected as above. an additional group of tcrb à/à mice was treated with 500 ml of vesicular stomatitis virus-immune serum on day 7. (e) viral rna in the brain was detected on day 14 by northern hybridization. (f and g) virus neutralizing total ig and igg titres in serum were determined over time. lanes in a and e and symbols in b-d represent individual mice. symbols in f-g represent the mean ae sem of three mice per group. representative results from two similar experiments are shown. n.d. = not detectable; n.s. = not significant (p40.05); ** = highly significant (p50.01). and 12. comparable virus dissemination in these three knockout strains of mice supported the notion that perforin-and mhci-dependent t cell control was primarily responsible for preventing persistence of rlcmv/indg in the brain parenchyma. the failure of perforin-deficient mice to clear rlcmv/indg was not due to a potential delay or impairment of cd8 + t cell trafficking to infected brain regions (fig. 5b) . in c57bl/6 mice, as in perforin-deficient mice, infiltrating cd8 + t cells were first detected on day 6 in similar numbers. unlike in wild-type controls, where cytotoxic t cell infiltration in the meninges and periventricular areas peaked at this time point and declined thereafter, cytotoxic t cell infiltrates in perforin-deficient mice increased even further at days 8, 10 and 12, presumably as a result of persisting viral antigen. in summary, these data show that mhci-restricted perforin-dependent mechanisms play a key role in the silent clearance of rlcmv/indg from the cns. the above analyses demonstrated similar dissemination of rlcmv/ indg in the parenchyma of perforin-deficient, tcrb à/à and rag à/à mice. however, differential infection rates were noted in the ependyma of the different mouse strains (fig. 5 , insets in day 12 images). thus we studied the cell types in which rlcmv/ indg persisted when infected mice lacked either t or b cells -specific cd8 + t cell frequencies in blood were enumerated at day 7 after infection. mhci à/à mice and cd8 à/à mice lack cd8 + t cells. symbols and lanes represent individual mice. in figure f and i, multiple comparisons were not performed since the f-test of anova failed to detect significant differences (p40.05). n.d. = not detectable; n.s. = not significant (p40.05). (rag à/à ), the entire t cell compartment (tcrb à/à ), mhci-restricted t cells (mhci à/à ) or perforin as a key component of cytolytic cd8 + t cell activity. immunohistochemical co-stains were performed 2 weeks after rlcmv/indg infection using a lcmv np-specific antibody in combination with cell type-specific markers (fig. 6 ). striking differences were noted in the ependyma on day 14, and were further supported by the kinetic analysis of virus distribution depicted in fig. 5 . unlike in the early phase of infection when the virus was mostly confined to the brain coverings (compare figs 1d and 5), mhci à/à and perforin-deficient animals displayed very few infected ependymal cells (mhci à/à 5%, perforin-deficient 2%). in contrast, ependymal cells were the most frequently infected cell type (47%) in tcrb à/à mice and rag-/-mice (48%). within the parenchyma, a similar global picture was found in all four knockout mouse strains, with the majority of infected cells identified as astrocytes that were predominantly located in periventricular areas (67% of infected cells positive for glial fibrillary acidic protein in mhci à/à mice; 60% in perforin-deficient, 31% in tcrb à/à , 29% in rag à/à mice). to a lesser degree, viral infection co-localized with the oligodendrocyte marker nogoa (mhci à/à 14%, perforin-deficient 24%, tcrb à/à 8%, 14% in rag à/à mice), with the neuronal marker figure 5 rlcmv/indg dissemination in the brain of perforin-deficient, tcrb à/à and rag à/à mice, and dense cd8 + t cell infiltrates in perforin-deficient mice. perforin-deficient (pko), tcrb à/à , rag à/à and c57bl/6 wild-type control mice were infected with rlcmv/ indg intracerebrally and were sacrificed at the indicated time points. brain tissues were processed for immunohistochemical analysis of lcmv np (a) and cd8 + t cells (b). the latter analysis was only performed for perforin-deficient and c57bl/6 mice since tcrb à/and rag à/à mice lack t cells. representative images of 2-4 animals per group and timepoint are shown. arrowheads in (a) indicate infected (lcmv np-positive) subependymal cells. insets in the day 12 timepoints of (a) show persisting infection of ependymal cells in tcrb à/à and rag à/à but not in perforin-deficient mice, whereas subependymal cells are infected in all three knockout strains. scale bars a: 100 mm; b: 50 mm. d = days post intracerebral infection. neun (mhci à/à 3%, perforin-deficient 4%, tcrb à/à 11%, 6% in rag à/à mice) and with the microglia/macrophage marker ionized calcium binding adaptor-1 (mhci à/à 11%, perforin-deficient 10%, tcrb à/à 3%, 3% in rag à/à mice). infection of endothelial cells was not detected, even when assessed in rag à/à mice ( supplementary fig. s3 ), and endothelia were therefore not included in the above analyses. taken together, these findings suggest that, unlike in the parenchyma where the cell type-specific distribution of virus is similar in all four genotypes of knockout mice tested, silent clearance from ependyma could occur in a mhci-and perforin-independent yet t cell-dependent fashion. total viral rna loads in the brain of perforin-deficient, mhci à/à , tcrb à/à and rag à/à mice are similar as determined by quantitative rt-pcr (fig. 6c) , providing evidence that within the brain parenchyma, adaptive immune pathways other than mhci-and perforin-dependent t cell clearance do not substantially influence rlcmv/indg persistence. at the same time, these rt-pcr data suggest that viral rna in the ependyma does not represent a major fraction of the total viral rna accumulating in the cns of tcrb à/à and rag à/à mice. this may suggest that ependymal cells are less permissive to viral rna amplification than the parenchyma. in contrast to viral encephalitis (i.e. infection of the brain parenchyma), viral infections generally follow a benign course if predominantly confined to the brain coverings. the underlying reasons are still poorly understood. in the current study we used a panel of gene-targeted mice to study redundant and non-redundant arms of adaptive immunity in viral clearance from cns coverings versus figure 6 the cell type-specific distribution of rlcmv/indg in the cns is different in tcrb à/à and rag à/à mice as compared to perforin-deficient and mhci à/à mice. mice of the indicated genotypes were infected with rlcmv/indg intracerebrally. fourteen days later they were sacrificed and brain tissues were processed for histological analysis of lcmv np. (a) ependyma was differentiated based on morphological criteria (arrows in top panel) combined with immunohistochemical detection of lcmv np (brown). infection of parenychmal cell types (bottom panels) was detected by combining lcmv np staining (red) with cell type-specific markers (green) in immunofluorescent co-staining as indicated. arrowheads point out colocalization of cell type-specific markers with lcmv np in immunofluorescence images. representative images from three to four animals per group are shown. scale bar: 50 mm. (b) histological images were quantified to define the proportion of each cell type within the total population of infected cells. (c) viral rna loads in the brain of the indicated mouse strains were measured by quantitative rt-pcr and are displayed in arbitrary units (see 'materials and methods' section). bars represent the mean + sd of four to seven animals per group. viral rna loads in infected c57bl/6 mice were significantly different from perforin-deficient (pko), mhci à/à , tcrb à/à and rag à/à mice (p 5 0.01), whereas the latter four were not significantly different from each other (p40.05 for all comparisons). the technical background was determined in brain tissue of uninfected mice (mean of six animals indicated as dashed line), and was not significantly different from infected c57bl/6 wild-type mice (p40.05, not shown). parenchyma. the differences discovered here offer a possible explanation for the fundamentally different clinical manifestations of viral infection in these two cns compartments. based on the present results and on earlier data, we suggest the following scenario for viral clearance in our model of 'aseptic meningitis'. blood-borne virus initially spreads to the cns coverings. both lcmv armstrong and rlcmv/indg (albeit the latter to a lesser extent) will slowly but steadily invade the brain parenchyma, spreading from cell to cell. with the onset of the adaptive immune response, ependymal viral replication is cleared in a t cell-dependent manner but independently of mhci and perforin, a non-cytolytic process causing only minimal clinical manifestations, if any. astrocytes contribute to the formation of the blood brain barrier and are close to the cns coverings, rendering them a first viral target in the parenchyma. this most likely explains the high rate of astrocyte infection in mhci à/à , pko, tcrb à/à and rag à/à mice on day 14 after infection (compare fig. 6 ), an assumption that is supported by the finding that neuronal infection becomes more prominent with time (data not shown). a reductionist model suggests that the ensuing cytotoxic t cell response eliminates the infected astrocytes by mhci-dependent, perforin-mediated cytotoxicity. unlike clearance from the cns coverings, this cytolytic process inflicts damage to the blood brain barrier (compare fig. 1e ) and, together with other factors such as the local inflammatory tissue response, will lead to breakdown of the blood brain barrier. it seems likely that, depending on the number of infected astrocytes in the blood brain barrier (i.e. virus load; high in lcmv armstrong, low in rlcmv/indg infection), the resulting damage to the blood brain barrier will vary, and thus also the extent of serum protein leakage into the parenchyma. once a certain level is reached, increased intracranial pressure will lead to the classical clinical signs of meningitis including nausea, seizures and ultimately death camenga et al., 1977; andersen et al., 1991) . in agreement with this scenario, christensen and colleagues (2004) found that delayed recruitment and accumulation of cytotoxic t cells in the brain parenchyma caused a delay in the onset of disease in lcmv-infected cxcl10-or cxcr3-deficient mice, despite normal inflammatory cell recruitment to the brain coverings (christensen et al., 2004) . the precise molecular mechanisms as to how t cells effect non-cytolytic clearance of the brain coverings remains to be addressed in future work. its occurrence independent of mhci suggests that mhcii-restricted t cell responses or non-classical t cells and their respective effector molecules may be involved. myelomonocytic cells have recently been identified as previously neglected players in lcmv-host interactions within the cns, and aside from t cells, monocyte/macrophages were also found to accumulate in the cns coverings during rlcmv/indg clearance (data not shown). their presence could be a simple consequence of inflammation but they may also contribute to clearance in a direct or indirect manner. the study of their contribution to viral clearance is complicated by considerable redundancy with other pathways, stepping in when myelomonocytic inflammatory cells are experimentally depleted (kim et al., 2009) . for similar reasons, it may be difficult to delineate the precise role of t cell effector pathways other than perforin that may contribute to clearance of rlcmv/indg from the cns parenchyma (griffin, 2003) . however, the contribution of these pathways, unlike that of perforin, may simply have remained undetected owing to a higher degree of redundancy. the model of adult intracerebral infection with rlcmv/indg offers several features that render it particularly amenable to the investigation of viral clearance in aseptic meningitis. the virus' non-cytolytic behaviour avoids complications related to viral spread in immunocompromised animals, and as reported here, its attenuation avoids the fatal immunopathological complications of wild-type lcmv infection (cole and nathanson, 1974) . on the flip-side of the coin, cytopathic effects of viral infection may also contribute to the clinical picture of 'aseptic meningitis' and are not part of the model. nonetheless, rlcmv/indg infection is ideally suited for the investigation of silent clearance, the most frequent yet often neglected outcome of viral cns infection. the present data delineate a clear dichotomy in the mechanisms underlying virus clearance from cns coverings and parenchyma. mhci-and perforin-dependence of the latter, but not the former, provides one likely explanation for the most frequently self-limiting course of viral cns infection. targeted mutation in the fas gene causes hyperplasia in peripheral lymphoid organs and liver aseptic meningitis, a disease of diverse etiology: clinical and etiologic studies on 854 cases immunological memory and protective immunity: understanding their relation breakdown of blood-brain barrier function in the murine lymphocytic choriomeningitis virus infection mediated by virus-specific cd8+ t cells programmed contraction of cd8(+) t cells after infection 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clinical and epidemiologic aspects lymphocytic choriomeningitis virus-induced central nervous system disease: a model for studying the role of chemokines in regulating the acute antiviral cd8+ t-cell response in an immuneprivileged organ cd4 t cell control primary measles virus infection of the cns: regulation is dependent on combined activity with either cd8 t cells or with b cells: cd4, cd8 or b cells alone are ineffective memory cd8 t-cell differentiation during viral infection immunofluorescent studies of the histopathogenesis of lymphocytic choriomeningitis virus infection restriction of in vitro t cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system we would like to thank c. crozier and c. bunker for critically reading the manuscript. supplementary material is available at brain online. key: cord-330553-sukrjl22 authors: stonedahl, sarah; clarke, penny; tyler, kenneth l. title: the role of microglia during west nile virus infection of the central nervous system date: 2020-08-28 journal: vaccines (basel) doi: 10.3390/vaccines8030485 sha: doc_id: 330553 cord_uid: sukrjl22 encephalitis resulting from viral infections is a major cause of hospitalization and death worldwide. west nile virus (wnv) is a substantial health concern as it is one of the leading causes of viral encephalitis in the united states today. wnv infiltrates the central nervous system (cns), where it directly infects neurons and induces neuronal cell death, in part, via activation of caspase 3-mediated apoptosis. wnv infection also induces neuroinflammation characterized by activation of innate immune cells, including microglia and astrocytes, production of inflammatory cytokines, breakdown of the blood-brain barrier, and infiltration of peripheral leukocytes. microglia are the resident immune cells of the brain and monitor the cns for signs of injury or pathogens. following infection with wnv, microglia exhibit a change in morphology consistent with activation and are associated with increased expression of proinflammatory cytokines. recent research has focused on deciphering the role of microglia during wnv encephalitis. microglia play a protective role during infections by limiting viral growth and reducing mortality in mice. however, it also appears that activated microglia are triggered by t cells to mediate synaptic elimination at late times during infection, which may contribute to long-term neurological deficits following a neuroinvasive wnv infection. this review will discuss the important role of microglia in the pathogenesis of a neuroinvasive wnv infection. knowledge of the precise role of microglia during a wnv infection may lead to a greater ability to treat and manage wnv encephalitis. west nile virus (wnv) is a neurotropic, mosquito-borne, single-stranded rna flavivirus maintained in an enzootic cycle between mosquitos, such as culex pipiens, and perching birds (order passeriformes), such as crows, jays, and finches [1, 2] . while birds are the natural reservoir for wnv, an infected mosquito may bite other mammals such as horses and humans and transmit the virus through saliva [2] . after an infected mosquito bites a human, the virus replicates within dendritic cells and macrophages of local tissue near the site of inoculation and subsequently spreads to regional lymph nodes. the resulting viremia then spreads the virus throughout the body [2] . west nile virus viremia in humans is not high enough or sustained enough to support subsequent transmission to mosquitoes, thus humans are designated "dead-end" hosts [1, 2] . west nile virus infection is now the most common cause of epidemic viral encephalitis in the united states. according to the center for disease control (cdc), since its introduction into north west nile virus-induced cns disease occurs as a result of virus crossing the blood brain barrier (bbb) following systemic infection. mechanisms of viral entry into the cns are not yet fully understood. however, theories include a cytokine-mediated increase in permeability of the bbb, hematogenous entry by infection of infiltrating immune cells, and retrograde axonal transport [7, 16, 17] . the greatest risk factor for developing wnv neuroinvasive disease is older age (>64 years), while other risk factors include hypertension and diabetes [18] . certain genetic factors also dictate increased susceptibility to neuroinvasive disease including single nucleotide polymorphisms in the oligoadenylate synthetase (oas) gene (involved in antiviral innate immunity) and genetic deficiencies in c-c chemokine receptor type 5 (ccr5), which inhibit lymphocyte trafficking into the cns [19, 20] . once within the cns, wnv directly infects neurons and several studies demonstrate that caspase 3-dependent apoptosis is an important mechanism of neuronal cell death and cns injury during wnv infection [21, 22] . the pathology of neuroinvasive wnv infection likely results from both apoptosis of neurons and secondary effects of the neuroinflammatory response, including damage to bystander cells and gliosis [22] . a critical component of the neuroinflammatory response induced by wnv infection is the activation of microglia, the innate immune cells of the cns [23] . in this review, we will discuss the role of microglia in wnv-induced cns disease as a contributor both to the protective innate immune response and to the development of long-term neurological damage. microglia are of myeloid origin and arise from the yolk sac during early development [24] [25] [26] . these cells are the resident mononuclear phagocytic cells of the cns and play multiple roles in the cns such as immune defense, maintenance of homeostasis, and synaptic pruning during early development [27] [28] [29] . importantly they continually monitor the cns for signals of injury and infection [28, 30, 31] . microglia express a spectrum of activation states in response to environmental and pathogenic cues [32, 33] . morphological changes of activated microglia include a shift from a ramified state, characterized by long extending processes, to a larger more amoeboid phenotype [34] . in addition, activated microglia display increased motility, proliferation and production of inflammatory cytokines and chemokines [29, [35] [36] [37] . in neuroinflammatory and neurodegenerative conditions, microglia are thought to contribute to pathogenicity [36, 38, 39] , however, in the case of viral encephalitis, it remains unclear whether microglia are beneficial or detrimental to disease outcomes, or possibly both. classically, macrophages and microglia have been defined as being polarized, meaning they express two major activation states: pro-inflammatory m1 and anti-inflammatory m2 [40] . while some individuals feel that this is not an accurate description of microglial activation [32] , it remains important to consider the role of microglia polarization in the pathogenesis of wnv infections. the ability to shift phenotypes allows microglia to maintain homeostasis within the cns. following infection of the cns, microglia express markers characteristic of m1 activation including increased expression of iba-1, as well as chemokines such as c-c motif cytokine 2 (ccl2), ccl3, ccl5, and ccl7 [41] . the anti-inflammatory drug minocycline reduces the expression of m1 inflammatory genes and increases the expression of m2 genes, following wnv infection of cns tissue [42] . this shift of microglia towards a more anti-inflammatory m2 state was neuroprotective [42] and suggests that m1 microglia are required for initial control of wnv infection; however, switching to an m2 phenotype may be critical in preventing excessive damage due to inflammatory processes. for the purpose of this review, we will mostly be referring to m1 microglial polarization when discussing activation of microglia. several neurotropic viruses have been shown to induce microglia activation in the cns following infection, including japanese encephalitis virus [43, 44] , dengue virus [45] , zika virus [46] , tick-borne encephalitis virus [47] , reovirus [48] , and mouse hepatitis virus [49, 50] . evidence that microglia are activated during wnv-induced cns disease in humans includes the presence of microglial nodules and increased proliferation [51, 52] . activated microglia are also seen in the cns of mice infected with wnv [53] [54] [55] and in wnv-infected ex vivo brain and spinal cord slice cultures [21, 41] . the use of ex vivo slice cultures allows for isolation of the cns immune system from the peripheral immune response. in this method, spinal cords or brains are harvested from neonatal mice and used to create 300-µm-thick slice cultures, which are incubated on culture inserts prior to infection. using this model, activated microglia were identified following wnv infection by increased expression of ionized calcium binding adaptor molecule 1 (iba1) [21, 41] , a microglia-specific marker [56] . in addition, microglia in wnv-infected ex vivo slices were increased in both size and number compared to uninfected slices and displayed an amoeboid phenotype [21, 41] . distinct microglia cell processes related to cell motility and phagocytosis of wnv-infected cells and antigenic debris were seen in wnv-infected ex vivo slice cultures (figure 1 ), including microglial cellular projections stretching over a range of distances to reach wnv-infected cells, which were often filopodial/lamellipodial in appearance ( figure 1a ), reflecting microglia with amoeboid morphology [41] . these microglia cell processes were observed contacting infected cells and initiating engulfment activity ( figure 1b -e). the formation of phagosomes was also prevalent ( figure 1f ). notably, microglia virtually never appeared to be infected themselves, despite taking in material (cells and debris) that stained positive for wnv antigens. intercellular communication via cytokine/chemokine signaling drives inflammatory responses during viral infections of the cns [40, 57] . infection of cns tissue with wnv in vivo [21, 58] and in ex vivo [41] and in vitro [23] models of wnv pathogenesis results in increased expression of proinflammatory cytokines and chemokines associated with microglial activation including c-x-c motif chemokine 10 (cxcl10), cxcl1, ccl5, ccl3, ccl2, tumor necrosis factor alpha (tnf-α), tnf-related apoptosis-inducing ligand (trail), and interleukin-6 (il-6) [40] . these cytokines are involved in inflammatory processes and activating/recruiting immune cells such as t cells [59] . inhibition of these chemokines/cytokines following treatment with minocycline resulted in decreased neuronal cell death following wnv infection of ex vivo spinal cord slice cultures [42] , suggesting they contribute to disease; however, depletion of microglia did not significantly change chemokine/cytokine expression in the brains of wnv-infected mice [60] so it is unclear what role microglia play in the production of these proteins. based on the evidence presented above, it can be concluded that microglia become activated following infection of the cns with wnv. these activated microglia can initiate an immune response that is intrinsic to the cns and does not require input from the peripheral immune system as seen in the ex vivo slice culture model. activation of the microglia is characterized by morphological changes and is associated with the increased production of inflammatory cytokines/chemokines. based on the evidence presented above, it can be concluded that microglia become activated following infection of the cns with wnv. these activated microglia can initiate an immune response that is intrinsic to the cns and does not require input from the peripheral immune system as seen in the ex vivo slice culture model. activation of the microglia is characterized by morphological changes and is associated with the increased production of inflammatory cytokines/chemokines. toll-like receptors (tlrs) are membrane spanning receptors commonly expressed by cells such as macrophages, dendritic cells, and microglia, which monitor the body for signs of infection. they play key roles in the innate immune system [61] and have the ability to recognize structurally conserved pathogen-associated molecular patterns (pamps) present on pathogens [61] [62] [63] . microglial expression of tlr3, which recognizes double-stranded viral rna, is thought to play an important role in the detection of viruses, including wnv, in the cns [64] . several studies have shown that tlr3 signaling plays a protective role in preventing lethal wnv encephalitis [54, 65, 66] . for example, mice deficient in the expression of tlr3 [63, 64] and myeloid differentiation primary response 88 (myd88), which functions downstream of tlr3 [54] , show increased susceptibility to wnv that is associated with higher viral loads in the brain and increased expression of inflammatory cytokines. while these studies present evidence for a protective effect of tlr3 following wnv infection, an earlier study showed increased survival from wnv infections in tlr3-deficient mice [65] . in this study, tlr3-deficient mice were more resistant to lethal wnv infection and showed impaired cytokine production along with enhanced viral load in the periphery. however, in the brain, viral load, inflammatory responses and neuropathology were reduced compared to wild-type, indicating tlr3 may mediate entry of wnv into the brain [67] . it has been suggested that the difference in outcome seen in tlr3-deficient mice could be explained by the distinct route of inoculation (subcutaneous versus intraperitoneal), passage history of the virus (mammalian vero cell versus insect-cell derived), and/or the virus dose [63] . more research will need to be done to achieve a full understanding of the role of tlr3 signaling in wnv encephalitis. retinoic acid inducible gene i (rig-i) and melanoma differentiation antigen 5 (mda5) are cytosolic pattern recognition receptors (prrs), which recognize viral rna products, including wnv genetic material, and are critical for initiating an innate immune defense [68, 69] . lack of either rig-i or mda5 in mice results in decreased innate immune signaling and control of viral growth. a double knockout of both receptors leads to complete loss of innate immunity gene function in wnv-infected cells and severe disease in mice [68] . microglia constitutively express both rig-i and mda5 [70] . the downstream central adaptor molecule mitochondrial antiviral-signaling protein (mavs) is also essential for control of wnv infections. a deficiency in mavs in hematopoietic cells, such as microglia and macrophages, leads to increased mortality in mice infected with wnv [71] . these findings indicate the importance of the mavs signaling pathway in the microglial response to wnv particles within the cns. as documented above, microglia recognize and become activated in response to wnv infection. however, it remains unclear if this process is essential to effective host protection against wnv or whether activated microglia contribute to pathogenesis. for example, following infection with wnv, both decreased [53] and increased [54, 64] microglial activation have been associated with increased neuronal death and susceptibility to disease. to address this disparity, the effect of wnv infection on mice lacking microglia has been investigated. mice deficient in the expression of interleukin 34 (il-34), which lack microglia but retain bone marrow-derived macrophages, have increased susceptibility to wnv, suggesting a role for microglia in antiviral defense [72] . in addition, recent studies performed in mice in which microglia were depleted using plexxicon 5622 (plx5622), an inhibitor of colony-stimulating factor 1 receptor (csf1r), demonstrated a significant increase in susceptibility to wnv-induced cns disease [60] . colony-stimulating factor 1 is critical for microglia survival [72] [73] [74] , and plx5622 has been shown to have specificity towards microglia without affecting other cell populations such as brain-specific macrophages, peripheral macrophages, or lymphocytes [49, 75, 76] . microglial depletion using plx5622 does not alter brain size, cognition, or motor function in mice [76] . to deplete microglia, mice were fed a diet containing plx5622 for 10 days resulting in greater than 80% depletion of their microglia population throughout the brain when compared to mice fed a control diet [60] . following infection of plx5622-treated mice with wnv, brain titers were significantly (10-100-fold) higher than in control mice at days 6, 9, and 10 post infection. in addition, mortality dramatically increased. of the mice infected with wnv, 100% of plx-treated microglia-depleted mice died compared to only 25% of the control fed mice [60] . these studies indicate that microglia play a critical role in controlling wnv infection and limiting wnv-induced mortality. results in wnv-infected plx5622-treated mice were remarkably similar to another recent study, which found that microglia are crucial for protection of mice from neurotropic coronavirus encephalitis and may be required for the generation of an efficient t cell response [49] . this study also showed that microglial protection was time dependent [49] . elimination of microglia using plx5622 between days 0 and 6 post-infection resulted in increased mortality in infected mice; however, microglial depletion after the first 6 days showed no effect on mortality [49] . this data indicates that microglia are essential for limiting viral growth and neuronal cell death during the initial phase of a neuroinvasive viral infection but become less important during later infection, when adaptive immune responses have developed. determination of whether microglia follow a similar time-dependent role following wnv infection in the cns and whether this is associated with changes in the t cell response remains to be determined. collectively, the studies discussed above reveal the critical role microglia play in protecting the host from wnv-induced cns disease. elimination of microglia during wnv encephalitis led to increased viral titers within the brain and increased mortality in mice. further studies are needed to determine the mechanism by which microglia protect the cns from wnv infection. potential mechanisms include phagocytosis of wnv-infected neurons, production of inflammatory cytokines/chemokines, and signaling to the adaptive immune system. matrix metalloproteinases (mmps) are a family of proteins whose major function is remodeling the extracellular complex (ec) of the bbb [77, 78] . they have also been shown to play a role in immunity by modulating the trafficking of immune cells into the cns [79] . mmp9, is expressed by microglia [78] , and has been shown to be important for wnv entry into the brain [77] . mmp9 knockout mice infected with wnv show decreased viral loads in the brain, but not the periphery, and increased survival when compared to wild-type (wt) mice [77] . resistance to lethal wnv infection coincided with an intact bbb as shown by reduced evan's blue dye leakage into the brain, indicating a role for mm9p in altering permeability of the bbb during a wnv infection [77] . an additional mechanism by which microglia may contribute to wnv entry into the brain involves intercellular adhesion molecule (icam-1), a surface glycoprotein involved in cellular extravasation into sites of inflammation. icam-1 is expressed by several cell types including microglia [80] . the expression of icam-1 on microglia may contribute to extravasation of leukocytes via the "trojan horse" method, in which the virus gains access to the cns through infected leukocytes [17, 81] . indeed, wnv infection has been shown to induce icam-1 expression in human endothelial cells and mouse brains, leading to disruption of the bbb and increased leukocyte infiltration [82] . it was also found that this process can be reversed through the use of blocking antibodies against icam-1 [82]. individuals recovering from wnv encephalitis may experience long-term neurological deficits resulting from the infection [3] . this effect of wnv infection of the cns is being extensively studied as an increased understanding of why these impairments occur will likely lead to an improvement in the long-term outcomes of recovering patients. a predominant theory is that microglia activated by memory t cells may contribute to neuronal damage following infection with wnv [83, 84] . following initiation of innate immune responses, activated microglia release cytokines that signal the peripheral immune cells to infiltrate the cns to clear the viral infection [24, 28] . cd8 + t cells mediate viral control through the secretion of effector molecules such as granzymes, perforin, and interferon-γ (ifnγ) [85] . while cd8 + t cells are necessary for proper clearance of virus from the cns [86, 87] , they also seem to contribute to neurological deficits following wnv infection [83, 84, 88] . once in the cns, cd8 + t cells will differentiate into brain resident memory t cells, which are important for protection from reinfection [85] . these t cells remain within the cns for months to years following neurotropic viral infections and studies have shown that these memory t cells promote microglia-mediated synaptic elimination following neuroinvasive wnv infections contributing to cognitive impairment. [83, 88, 89] . a recent study used a mouse model of viral encephalitis to show that t cells promote microglia-mediated loss of synapses following infection with wnv [83] . this study found an increase in the number of microglia-expressing markers indicative of activation induced by the t cell cytokine ifn-γ [83] . analysis by qpcr also revealed increased expression ifn-γ in the hippocampus following infection and throughout recovery. in addition, mice deficient in the expression of ifn-γ and wt mice were infected with wnv. mice lacking ifn-γ showed no differences in survival or weight loss when compared to wt mice [83] . however, wt mice showed special learning deficits on the barnes maze test, while ifngr1-/mice were protected from this outcome [83] . this study shows that during the process of viral clearance following wnv infection, ifn-γ produced by memory t cells can alter the function of microglia leading to elimination of synapses. the complement cascade is a component of the innate immune response, which plays a key role in defense from pathogens. during early development, microglia are involved in the process of synaptic pruning within the nervous system, which is dependent on the classical complement cascade [90] [91] [92] [93] . the mechanism by which the ifn-γ activated microglia eliminate synapses seems to be dependent on the complement system. it was found that brain regions retrieved from mice that had recovered from wnv showed increased expression of genes involved in microglial synaptic elimination by components of the complement system such as c1qa [84] . mice with either fewer microglia (due to lacking the csf1r ligand il-34) or a deficiency in the c3 complement receptor showed increased protection from microglia-mediated synapse elimination [84] . this study suggests that the complement cascade and especially microglial-c3 is responsible for synaptic elimination seen in mice following wnv infection in the brain. collectively, the studies discussed in this section provide evidence for a mechanism of neuronal damage following neuroinvasive wnv infection involving interactions in which microglia are promoted by memory t cells to eliminate synapses that have been targeted by the complement cascade. understanding the dynamics between t cells and microglia following wnv infection of the cns and how microglia may contribute to neurological sequalae could provide important insight into potential treatments for patients recovering from wnv encephalitis. microglia maintain a variety of functions within the cns, which are critical for protection from pathogens, homeostatic maintenance, and early development of the cns. these roles are summarized in table 1 . in this review, we have highlighted the important role microglia play during a neuroinvasive wnv infection. although it is evident that microglia are essential for protection from wnv, the mechanism behind how microglia limit viral growth and mortality remains unclear. the multiple and complex roles of microglia during wnv infections are pictured in figure 2 . future studies should focus on the precise mechanisms by which microglia protect from wnv and enhancing the understanding of neuroinflammatory processes that accompany neuroinvasive wnv infections. it still remains to be determined which cytokines/chemokines are produced by microglia during a wnv infection and why a decrease in the expression of inflammatory cytokines/chemokines in not observed in the cns of mice following wnv infection in the presence of plx5622. which may lead to altered outcomes when comparing human and mouse infections [96] . it is vital to the complete understanding of the role of microglia during a wnv infection to use mouse studies as a compliment to human studies and not a replacement. understanding exactly how microglia influence the progression and recovery of wnv neuroinvasive infections will allow for the development of specific treatments, which could provide the optimal outcome to patients recovering from this infection. [3] . activated microglia control viral growth and reduce mortality in mice infected with wnv [4] , likely due to a combination of microglial phagocytosis of infected neurons and the release of inflammatory cytokines, which contribute to neuroinflammation and the recruitment of lymphocytes [3, 5] . in some cases, released cytokines may contribute to wnv-induced pathogenesis. mmp9 and icam-1 expression by microglia may enhance viral entry into the cns through breakdown of the bbb and extravasation of potentially infected leukocytes [6, 7] . long term effects: following infiltration of t cells into the cns, microglia may contribute to synaptic damage triggered by ifnγ produced by memory cd8 + t cells and enhanced by the complement cascade [8, 9] . this synaptic elimination leads to neurological damage during recovery from wnv encephalitis [9] . green and red indicator arrows designate protective (green) and non-protective (red) microglial responses. [83, 84] while microglia are protective during the initial infection and vital for resolution of the infection, potentially dysregulated microglia responses driven by t cells may contribute to neurological damage and long-term impairment during the recovery stages. targeting microglial responses during both the initial and late stages of infections may offer new therapeutic options for human patients with neuroinvasive wnv infections. for example, it may be beneficial to enhance the activity of microglia during the initial infection to provide enhanced protection but then limit microglial activation during the recovery stages of infection to improve long-term outcomes. one promising treatment that may be used during the initial wnv infection to improve survival is the use of granulocyte-macrophage colony-stimulating factor (gm-csf), which promotes growth and activation of microglia, macrophages, and other myeloid cells [75, 94] . this drug is currently available (leukine) and is used to treat leucopenic patients as well as being investigated as a treatment for alzheimer's disease [95] . another study showed that the anti-inflammatory drug minocycline may be beneficial in recovery from a wnv infection of the cns [42] . most of the studies presented in this review utilized a knockout mouse model or mouse tissue model to investigate microglia. however, it is important to understand the limitations of using mouse models when studying microglia and wnv pathology. human microglia exhibit several differences to mouse microglia including increased levels of several immune genes and being longer-lived, which may lead to altered outcomes when comparing human and mouse infections [96] . it is vital to the complete understanding of the role of microglia during a wnv infection to use mouse studies as a compliment to human studies and not a replacement. understanding exactly how microglia influence the progression and recovery of wnv neuroinvasive infections will allow for the 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coronavirus infection of the central nervous system spinal cord neuropathology in human west nile virus infection virology, pathology, and clinical manifestations of west nile virus disease the immune adaptor molecule sarm modulates tumor necrosis factor alpha production and microglia activation in the brainstem and restricts west nile virus pathogenesis tlr signaling controls lethal encephalitis in wnv-infected brain west nile virus spread and differential chemokine response in the central nervous system of mice: role in pathogenic mechanisms of encephalitis microglia-specific localisation of a novel calcium binding protein, iba1 intercellular communication is key for protective ifnα/β signaling during viral central nervous system infection interleukin 17 receptor a, and glutamate signaling as well as flavivirus-specific upregulation of trna synthetases microglia in central nervous system repair after injury pharmacologic depletion of microglia increases viral load in the brain and enhances mortality in murine models of flavivirus-induced encephalitis toll-like receptors and innate immunity toll-like receptors in health and disease in the brain: mechanisms and therapeutic potential microglia recognize double-stranded rna via tlr3 toll-like receptor 3 has a protective role against west nile virus infection role of ns1 and tlr3 in pathogenesis and immunity of wnv toll-like receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis the essential, nonredundant roles of rig-i and mda5 in detecting and controlling west nile virus infection distinct rig-i and mda5 signaling by rna viruses in innate immunity characterization of retinoic acid-inducible gene-i expression in primary murine glia following exposure to vesicular stomatitis virus mavs expressed by hematopoietic cells is critical for control of west nile virus infection and pathogenesis il-34 is a tissue-restricted ligand of csf1r required for the development of langerhans cells and microglia csf1r ligands il-34 and csf1 are differentially required for microglia development and maintenance in white and gray matter brain regions csf-1 controls cerebellar microglia and is required for motor function and social interaction colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xtg-ad mice csf1 receptor signaling is necessary for microglia viability, which unmasks a cell that rapidly repopulates the microglia-depleted adult brain matrix metalloproteinase 9 facilitates west nile virus entry into the brain the role of microglia and matrix metalloproteinases involvement in neuroinflammation and gliomas matrix metalloproteinases as modulators of inflammation and innate immunity intercellular adhesion molecule-1 gene expression by glial cells. differential mechanisms of inhibition by il-10 and il-6 dual microglia effects on blood brain barrier permeability induced by systemic inflammation west nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model t cells promote microglia-mediated synaptic elimination and cognitive dysfunction during recovery from neuropathogenic flaviviruses a complement-microglial axis drives synapse loss during virus-induced memory impairment to go or stay: the development, benefit, and detriment of tissue-resident memory cd8 t cells during central nervous system viral infections update on t cells in the virally infected brain: friends and foes cd4+ t-cell responses are required for clearance of west nile virus from the central nervous system microglia in memory decline from zika virus and west nile virus infection the role of microglia in viral encephalitis: a review the complement system: an unexpected role in synaptic pruning during development and disease the classical complement cascade mediates cns synapse elimination microglia sculpt postnatal neural circuits in an activity and complement-dependent manner chapter two-complement system in neural synapse elimination in development and disease granulocyte macrophage colony stimulating factor treatment is associated with improved cognition in cancer patients gm-csf upregulated in rheumatoid arthritis reverses cognitive impairment and amyloidosis in alzheimer mice the kaleidoscope of microglial phenotypes this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest.vaccines 2020, 8, 485 key: cord-022594-fx044gcd authors: pirko, istvan; noseworthy, john h. title: demyelinating disorders of the central nervous system date: 2009-05-18 journal: textbook of clinical neurology doi: 10.1016/b978-141603618-0.10048-7 sha: doc_id: 22594 cord_uid: fx044gcd nan demyelinating disorders of the central nervous system istvan pirko and john h. noseworthy multiple sclerosis (ms) is now known to be a common malady even though it was first recognized as a distinct clinicopathological entity less than 150 years ago. 1 the lack of clear medical reports before the early 1800s is sometimes interpreted as evidence that ms is a relatively new disease. however, it is more likely that the evolution of medicine into science led to more precise observation and description of human diseases, including ms. saint lidwina of schiedam (1380-1433) developed a relapsing neurological disorder at the age of 18 and may be the first case of clinically described ms. 2 ollivier was the first to report a clinical case in the medical literature in 1824. 1 shortly thereafter, carswell illustrated a case of what is now clearly recognizable as ms in his atlas of anatomical pathology. cruveilhier published gross pathological and clinical descriptions of ms. vulpian first suggested the rubric of "sclerose en plaque" in 1866. charcot was primarily responsible for establishing ms as a unique and recognizable syndrome. 3 he also described the clinical spectrum and the histological appearance. pierre marie was the first to suggest an infectious cause of ms in 1884, a hypothesis that is still debated. toxins were also considered to be responsible in the early 1900s. a major advance toward the understanding of demyelinating diseases was the discovery of experimental allergic encephalomyelitis (eae) by rivers in 1935. 4 a variety of different demyelinating diseases have subsequently been described (table 48 -1). myelin provides insulation for axons and is necessary for saltatory conduction. it is composed of tightly wrapped lipid bilayers with specialized protein constituents. peripheral nervous system (pns) myelin is formed by the extension of schwann cells, and central nervous system (cns) myelin is produced by oligodendrocytes. the myelin coating is interrupted at regular intervals (nodes of ranvier) where the axon membrane with its concentration of voltagegated sodium channels is exposed to the extracellular environment ( fig. 48-1) . 5 the presence of myelin is essential to maintain conduction velocity; its loss or damage can lead to significantly slower conduction or conduction block. other factors affect conduction velocity including certain antibodies and chemicals like nitric oxide. in certain cases, blockade may be the initial event in the cascade of events leading to demyelination. cns and pns myelin differ in a number of important ways. schwann cells myelinate only one internodal segment from a single pns axon, whereas oligodendrocytes myelinate multiple cns axons. the proteins also differ. proteolipid protein (plp) accounts for approximately 50% of the cns myelin proteins. mutations in this highly conserved protein cause pelizaeus-merzbacher disease. protein zero is the major pns myelin protein and performs a function similar to plp in compacting the intraperiod line. myelin basic protein (mbp) makes up 30% of cns and 10% of pns myelin proteins. mbp is not an integral protein but binds to the cytoplasmic surface and is responsible for compaction at the major dense line. myelin associate glycoprotein accounts for about 1% of both peripheral and central myelin. myelin oligodendrocyte glycoprotein and cyclic nucleotide phosphodiesterase are minor constituents of cns myelin and are not found in the pns. peripheral myelin protein 22 is a minor component of pns myelin. ms is an inflammatory relapsing or progressive disorder of cns white matter and is a major cause of disability in young adults. pathologically, it is characterized by multifocal areas of demyelination, loss of oligodendrocytes, and astrogliosis but with relative preservation of axons. while demyelination is the classic hallmark of ms, axonal and neuronal injury are important aspects of the disease and are gaining more recognition. although certain clinical features are characteristic of ms, investigative studies are often needed to confirm the clinical suspicion and exclude other possibilities. recently, there have been advances in understanding the etiology, mechanisms of myelin injury, and potential for repair, and several partially effective agents are now approved for use in relapsing-remitting and secondary progressive ms. the pathogenesis and pathophysiology of ms remains incompletely understood. several mechanisms may be important to ms plaque formation: autoimmunity, infection, bystander demyelination, and heredity. although convincing proof is lacking, dietary factors and toxin exposure have been hypothesized to contribute as well. these mechanisms are not mutually exclusive, and the true pathophysiology is likely to depend on more than one of them. autoimmunity. during ontogenesis, autoreactive lymphocytes normally undergo clonal depletion, but some escape and are merely suppressed, becoming tolerant to their antigens. low levels of autoreactive t and b cells persist even in normal individuals. autoimmune disorders occur when the tolerance of these cells toward their antigen is broken. the decreased suppressor activity of circulating lymphocytes from patients with ms and other presumed autoimmune diseases may reflect loss of tolerance. 6 one potential mechanism that may break tolerance is molecular mimicry between self and foreign antigens. autoreactive t4 lymphocytes may become activated on exposure to structurally similar foreign antigens. some evidence suggests that molecular mimicry is relevant in ms. not only do several viral and bacterial peptides share structural similarities with mbp, but it has also been demonstrated that these antigens may activate mbp-specific t-cell clones derived from ms patients. 7 blood-brain barrier leakage alone may break tolerance because it gives cnsreactive lymphocytes easy access to otherwise inaccessible antigens. alternatively, a primary event such as an infection or injury may release cns antigens into the periphery, where they may activate corresponding autoreactive cells. 8 the major support for autoimmunity in the pathogenesis and pathophysiology of ms is by analogy to eae, the major animal model for ms. eae is, however, an artificial situation and there is no spontaneous autoimmune animal model of ms. while eae is the most commonly studied model of ms, several features of human ms can not be adequately captured by this model. 9, 10 over a hundred different effective treatments have been described for eae; however, almost all of them are ineffective and some are harmful in human ms. a recent editorial discusses the merits and important limitations of eae as a model for ms. 10 in eae, just like in classic human autoimmune diseases such as systemic lupus erythematosus (sle) or rheumatoid arthritis, the main target antigens are known. however, despite the discovery of several "weak" antigens in human ms, no dominant antigens have been identified to date. the only human demyelinating disease with an identified specific antigen is devic's disease (neuromyelitis optica), which appears to be a novel autoimmune chanellopathy with an antigen that is neither neuronal nor myelin related (see later discussion of devic's syndrome under neuromyelitis optica). 11 infection. the role of viral infections in the initiation and maintenance of ms has been debated for some time. several viral infections are known to cause demyelination in animals, including visna virus of goats and sheep, canine distemper virus, and theiler's murine encephalomyelitis virus. viral infections in humans can also cause demyelination (progressive multifocal leukoencephalopathy [jc papillomavirus], subacute sclerosing panencephalitis [measles virus], and human t-cell lymphotropic or leukemia virus type 1 [htlv-1]-associated myelopathy). the epidemiology of ms suggests that environmental factors may promote the disease state, possibly due to one or more viruses. a virus may be involved in the pathogenesis of ms in several ways: 1. transient or persistent infection outside the cns may activate autoreactive t cells by means of molecular mimicry or by other nonspecific means (as superantigens do). 2. transient cns infection may initiate a cascade of events that fosters autoimmunity (breach the blood-brain barrier, release cns antigens). 3. recurrent cns infections may precipitate repeated inflammation and demyelination. 4. persistent cns viral infection could either incite inflammatory reactions detrimental to oligodendrocytes or directly injure them. beyond speculation and epidemiological observations, there is insufficient evidence for a viral infection playing a causative role in ms. early serological studies are difficult to interpret because of nonspecific immune activation and resulting elevation of titers to many different viruses. many ms patients have elevated cerebrospinal fluid (csf) titers to measles and herpes simplex (hsv) viruses, but this finding appears nonspecific. virus has rarely been cultured from csf of ms patients, but a new strain of hsv (the ms strain) and a new virus (inoue-melnick virus) were first isolated from the csf of ms patients. 12, 13 newer molecular techniques to search for a viral genome in csf and brain have rejected the claim that htlv-1 is associated with ms. the finding that human herpesvirus 6 (hhv6), although present in 70% of brains from both control subjects and ms patients, is localized to the oligodendrocyte nuclei near plaques of ms patients and to oligodendrocyte cytoplasm in control subjects indicates that persistent cns viral infection is common. 14 this raises the possibility that ms may depend on an aberrant host response to this normal condition or that a defective virus that lacks the ability to evade immune detection may be to blame. more recently, measles and canine distemper virus antibodies were found elevated in blood and csf samples of ms patients, although their relationship is not clear to the disease process. in a study from denmark, patients with serological markers for late-stage epstein-barr virus (ebv) infection had a threefold increase in the likelihood of developing ms. a follow-up study from sweden failed to reach this conclusion. in general, serum samples of ms patients may contain higher titers of antibodies to the following infectious organisms: adenovirus, canine distemper virus, hsv, hhv6, and influenza, measles, mumps, parainfluenza, rubella, vaccinia, and varicella zoster virus (vzv). similarly, csf samples from ms patients may show higher titers of adenovirus; chlamydia pneumoniae; cytomegalovirus (cmv); ebv; hhv6; coronavirus; influenza viruses a and b; measles or mumps virus; mycoplasma pneumoniae; parainfluenza viruses 1, 2, and 3; respiratory syncytial virus; rubella virus; vaccinia; and vzv. there has been an interest recently in a potential link between c. pneumoniae infection and the development of ms. no direct cause-and-effect relationship has been observed between any of these infections and ms. "bystander" demyelination. immune actions may mediate myelin injury in a nonspecific manner. many soluble products of the immune response other than immunoglobulins are known or suspected to be toxic to myelin and oligodendrocytes. activated complement is capable of lysing oligodendrocytes in an antibody-independent fashion. 15 the proinflammatory cytokine tumor necrosis factor-a causes myelin disruption and oligodendrocyte apoptosis in vitro. 16 arachidonic acid metabolites may also participate in myelinolysis, and reactive oxygen species released by macrophages cause lipid peroxidation that can damage myelin. other soluble substances that are potentially toxic to myelin include nitric oxide and vasoactive amines. histological subtypes of ms lesion development. through the groundbreaking work of lucchinetti and associates in the ms lesion project, it is postulated that the formation of ms lesions follows one of four patterns. 17 patterns i and ii are related to immune-mediated damage to myelin sheaths. in pattern i, cellular mechanisms of injury seem to prevail (macrophages and t-lymphocytes) whereas in pattern ii humoral mechanisms of injury predominate (e.g., antibody and complement-mediated mechanisms). patterns iii and iv are related to oligondendrocye pathology: in pattern iii, a distal oligdendrogliopathy and apoptosis have been reported, whereas in pattern iv, primary oligodendrogliopathy and degeneration of oligodendrocytes have been described. currently these subtypes can be diagnosed only by biopsy; serum and magnetic resonance imaging (mri) markers are not yet known, although lesional t2 hypointense rims and response to plasma exchange may correlate well with pattern ii pathology. it is important to note that the patterns do not correlate with clinical subtypes of ms, with the exception of pattern iv, which has been identified only in primary progressive (ppms) patients. evidence to date suggests that the pattern of lesion formation remains the same within an individual patient; patients do not "switch" from one pattern to the other. also, the patterns do not seem to represent different chronological stages of lesion formation. gray matter involvement. it has been known since the late 19th century that ms affects both gray and white matter structures. the importance of gray matter involvement has received little attention until recently, largely due to the development of advanced mri techniques (see later) that indicate neuronal and axonal involvement even in the earliest stages of this disease. a classification system of gray matter plaques was proposed by peterson and associates 18 they described three patterns of cortical demyelination: type i lesions are contiguous with subcortical white matter lesions; type ii lesions are confined to the cortex, and are often perivascular; type iii lesions extend from the pial surface to cortical layer 3 or 4. besides cortical gray matter involvement, there is also evidence for prominent basal ganglia involvement, which can be seen in the early stages of ms, and may correlate better with motor outcome and cognitive measures than measures of white matter involvement. lucchinetti and associates demonstrated that biopsy samples from newly diagnosed demyelinating cases contain numerous infiltrating immune cells, and can be destructive. the pathological classification of cortical lesions as described by petersen can also be found in these early ms biopsy samples. approximately 20% of biopsy cases in which gray matter was also sampled had evidence of clear cortical demyelination. in 2005, an extensive histological study by kutzelnigg and associates investigated the role of cortical demyelination in all clinical subtypes of ms. 19 in this study, 52 brains of ms patients (relapsing-remitting [rr], secondary progressive [sp], and ppms) and 30 control subjects were studied using advanced quantitative morphological techniques. cortical demyelination and diffuse axonal injury in the normal appearing white matter (nawm) were reported as hallmarks of progressive forms of ms. cortical demyelination was mainly seen in the subpial layer of cortex, and was associated with significant inflammatory infiltrates in the surrounding meninges. diffuse inflammation was also found throughout the white matter of the progressive cases, associated with activation of microglia. no significant correlation was shown between focal white matter lesion load and cortical demyelination. this study defines three crucially important pathological hallmarks of ms-focal demyelinated white matter lesions, diffuse injury in the white matter, and cortical plaque formation-and concludes that white matter lesion formation predominates in active forms of ms, while cortical pathology and diffuse white matter injury characterizes the progressive forms. the authors of this landmark paper also established that these three processes are potentially independent of each other. heredity. epidemiological findings support a polygenic hereditary predisposition to ms. a number of candidate genes have been investigated, often with conflicting results. the only definitive genetic association in ms is with the serologically defined human leukocyte antigen (hla) dr15, dq6. this is one of the dr2 haplotypes, also known as dw2 in cellular terminology and drb1*1501, dqa1*0102, dqb1*0602 in molecular nomenclature. though its link to ms is well established, the risk conferred by this haplotype is small (relative risk of 3 to 4), and it is neither necessary nor sufficient for the development of ms. linkage to this locus has not been proved, indicating that it plays only a minor role in familial susceptibility. other susceptibility genes likely contribute, possibly the t-cell receptor variable b region and the igg heavy-chain variable region (especially the vh2-5 gene). but their specific roles have not been established. other genes under study have been the mbp coding gene, the ctla-4 gene on chromosome 2q33, and the interleukin-1ra associated gene, in concurrence with the hla-dr15 haplotype. mitochondrial mutations are also under investigation, and an lhon-associated mtdna mutation may be an important cofactor in developing ms in some patients. the apoe4 gene, as in alzheimer's disease, has been associated with a higher incidence of ms. on the other hand, apoe3 is considered to have neurotropic, immunomodulatory, and antioxidant properties. these findings are yet to be confirmed by larger studies. twenty percent of ms patients have at least one affected relative. only about 4% of first-degree relatives of patients develop ms, but this represents a 20-to 40-fold increase in risk compared with the general population. unaffected family members sometimes have abnormal findings on cranial mri, implying that this risk is even higher. one study of ms rates in adopted relatives of ms patients verified that the familial distribution is due to genetic factors rather than shared environment. 20 twin studies lend support to both genetic and environmental influences on ms development. genetically identical monozygotic twins are more often concordant for ms than dizygotic twins (26% and 2.4%, respectively), indicating a genetic component; 21 however, even after following monozygotic twins past age 50 or using mri data, less than 50% are concordant, suggesting a role for environmental factors. epidemiology and risk factors. ms is not a rare disease. it affects millions worldwide and approximately 400,000 in the united states alone. symptoms usually begin during young adulthood, with the peak onset at age 24. approximately 0.3% of ms cases are diagnosed before age 15. women are affected nearly twice as often as men. ms has a predilection for whites, especially those of northern european heritage. other races and ethnic populations are resistant to a variable extent. ms is virtually unknown among black africans but occurs in african-americans at half the rate of whites, possibly due to racial admixture or environmental factors. ms is rare in tropical areas, and the prevalence increases proportionally to the distance from the equator, excluding polar regions. the prevalence is less than 5 cases per 100,000 in tropical areas; in high prevalence areas it can be higher than 30 per 100,000, 22 reaching up to 100 per 100,000 in selected areas. although usually interpreted as the effect of environmental factors, the prevalence gradient is at least partially due to racial susceptibility. 23 perhaps the most incriminating evidence for the role of environmental factors in the development of ms is the changing risk with migration and the occurrence of ms clusters and epidemics. immigrant populations tend to acquire the ms risk inherent to their new place of residence. migration from high to low prevalence before the age of 15 lowers the ms risk, whereas migration after this age does not affect risk. 18 migration from low to high prevalence areas increases the risk of ms, but the effect of age is less clear. many clusters of ms have been reported. 24, 25 the occurrences of ms epidemics in iceland and the faroe islands have been proposed to be the result of exposure to a pathogen brought by british troops during their occupation in world war ii. other environmental factors associated with the development of ms include cigarette smoking (odds ratio of 1.81, ci: 1.1-2.9), animal fat intake, and deficiency of vitamin d. 26, 27 epidemiological data support the view that ms is caused or triggered by an environmental factor in persons who are genetically susceptible. the familial frequency and distribution implies that several genes contribute to susceptibility, and this is consistent with the low relative risk conferred by the genetic loci studied so far. data from clusters, migration studies, and family studies reveal that there is a latent period of some 20 years between exposure to the environmental factor and the development of clinical symptoms and that the age at exposure is around 15, the putative age at acquisition. the precise environmental events that lead to cns demyelination are uncertain. viral infection is the most plausible, but because of the nonspecific elevation of viral titers and long latent period, there is little direct evidence. minor respiratory infections precede 27% of relapses in patients with established ms. measles infection was found to have occurred at a later age in ms patients than control subjects, although the incidence of ms has not been reduced by immunization against measles. head injury and trauma have received attention as putative triggering events, but cohort studies have not verified any link. pregnancy does not alter the risk of developing ms, but it does seem to influence disease activity. the annualized relapse rate drops from approximately 0.56 to 0.12 by the third trimester, but this is offset by an increase to 1.2 in the first 3 postpartum months. most studies have found no longterm effects of pregnancy on the prognosis for progression or disability, although one did report a favorable effect. 28 a multitude of other environmental factors have been suspected to alter the risk for ms (cold climate, precipitation, amount of peat in the soil, exposure to dogs, and consumption of meat, processed meat, and dairy products), but none has been verified to be an independent risk factor. ms can cause a wide variety of clinical features. many signs and symptoms are characteristic, and a few are virtually pathognomonic for the disorder. conversely, some symptoms are atypical and some are so rare as to suggest a different diagnosis (table 48 -2). the course of the illness is also variable, but it remains a critical consideration in the diagnosis of ms. sensory symptoms are the most common presenting manifestation in ms (21% to 55%) and ultimately develop in nearly all patients. 29 loss of sensation (numbness), paresthesias (tingling), dysesthesias (burning), and hyperesthesias are common. these symptoms may occur in practically any distribution: one or more limbs, part of a limb, trunk, face, or combinations. the more distinctive sensory relapses of ms consist of the sensory cord syndrome and the sensory useless hand syndrome. a common scenario is that of numbness or tingling beginning in one foot, ascending first ipsilaterally and then contralaterally. the sensory symptoms may ascend to the trunk, producing a sensory level, or may involve the upper extremities. associated symptoms commonly include poor balance, weakness, urinary urgency, constipation, and lhermitte's sign (see later). brown-sã©quard syndrome may occur with sensory disassociation and hemiparesis. the sensory cord syndrome reflects an evolving demyelinating lesion that begins in the medial posterior column ipsilateral to the first symptoms. sensory cord syndromes are common in ms and suggest the diagnosis when they occur in young persons and remit spontaneously or in response to corticosteroids. patients with the sensory useless hand may note subjective numbness and lose discriminatory and proprioceptive function, resulting in difficulty writing, typing, buttoning clothes, and holding onto objects, especially when not looking at the hand. this problem can occur bilaterally even without lower extremity symptoms. the responsible lesion is in the lemniscal pathways either in the cervical spinal cord or in the brain stem. this syndrome usually remits over several months. the useless hand syndrome is a very specific symptom and is only rarely caused by other disorders. a large portion of ms patients have persistent sensory loss, usually consisting of diminished vibratory and position sensation in distal extremities (video 5, sensory ataxia). itching may occur in a dermatomal distribution with relapse or in paroxysms. pain is not a major manifestation of ms, but distressing lower extremity dysesthetic pain associated with spinal cord involvement, radicular pain from lesions at the root entry zone, paroxysms, and an uncomfortable sensation of pressure or tightness surrounding a leg or the trunk may be present. pyramidal tract dysfunction is common in ms and causes weakness, spasticity, loss of dexterity, and hyperreflexia (video 80, hyper-reflexia). motor deficits can occur acutely or in a chronic progression with weakness of one or more limbs and facial weakness, leg stiffness that impairs gait and balance, or extensor and flexor spasms (video 3, spastic gait). exercise or heat frequently worsens subtle deficits. muscle atrophy is usually due to disuse, but lesions of lower motor neuron fibers or of the anterior horn itself can cause a pseudoradiculopathy with segmental weakness, atrophy, and diminished reflexes. motor symptoms are presenting manifestations of ms in 32% to 41% of all cases; their prevalence is higher than 60% in long-standing ms. the initial symptom of ms is optic neuritis (on) in 14% to 23% of patients, and more than 50% experience a clinical episode of on during their lifetime. the most common manifestation is visual loss in one eye that evolves over a few days. periocular pain, especially with eye movement, usually accompanies and may precede the visual symptoms. bilateral simultaneous on is uncommon in adults, but formal visual field testing reveals unexpected defects in the clinically normal eye in a substantial number of patients. children and asian patients are more likely to have bilateral simultaneous on; it may also be seen in neuromyelitis optica (nmo) patients. examination shows an afferent pupillary defect, diminished visual acuity, subdued color perception, and often a central scotoma (video 200, afferent pupillary defect). funduscopic examination is usually normal but occasionally will reveal papillitis (more common in children) or venous sheathing. most patients begin to recover within 2 weeks, and significant visual patients with frequent and severe on events in the first 2 years were more likely to convert to nmo; they also had a higher likelihood for significant persistent vision loss. 30 cerebellar pathways are frequently involved during the course of ms, but a predominately cerebellar syndrome is uncommon at onset. the manifestations include dysmetria, dysdiadochokinesia (video 12, dysdiadochokinesis), action tremor with terminal accentuation, dysrhythmia, breakdown of complex motor movements, and loss of balance (video 14, tremor with ataxia). patients with long-standing ms may develop a "jiggling" gait and an ataxic dysarthria with imprecise articulation, scanning speech, or varying inflection, giving it an explosive character. urinary urgency, frequency, and urge incontinence (due to detrusor hyper-reflexia or detrusor-sphincter dyssynergia) result from spinal cord lesions and are frequently encountered in ms patients. the combined incidence of bowel and bladder dysfunction in ms is thought to be higher than 70%. symptoms of bladder dysfunction may be transient and occur with an exacerbation but are commonly persistent. impaired vesicular sensation causes a high capacity bladder and may lead to bladder atonia with thinning and disruption of the detrusor muscle. incontinence results in constant dribbling of urine in this irreversible condition. interruption of brain stem micturition center input sometimes leads to cocontracture of the urinary sphincter and detrusor muscles (detrusor-sphincter dyssynergia). the resulting high pressure may lead to hydronephrosis and chronic renal failure if untreated. constipation is a common problem, occurring in 39% to 53% of ms patients, especially with limited activity and spinal cord involvement. fecal incontinence is a socially devastating symptom that is often associated with perineal sensory loss in ms patients. sexual dysfunction is seldom mentioned, even though it is a frequent problem in ms. nearly two thirds of patients report diminished libido. one third of men have some degree of erectile dysfunction, and a similar percentage of women have deficient vaginal lubrication. besides direct neurological impairment, sensory loss, physical limitations, depression, and fatigue additionally contribute to sexual difficulties in ms patients. in addition, the partner's attitude and psychological factors dealing with self-image, self-esteem, and fear of rejection may also lead to impotence or loss of libido. intense vertigo associated with nausea and emesis is an occasional manifestation of ms relapse. in the absence of a clear diagnosis of ms, these symptoms are often attributed to vestibular neuronitis. patients may also develop a persistent but mild vertigo that is precipitated by movement, or this may be a residual finding after an acute relapse. internuclear ophthalmoplegia, caused by a lesion in the medial longitudinal fasciculus, is the most common cause of diplopia in ms patients (video 200, afferent pupillary defect). when symptomatic, it produces horizontal diplopia on lateral gaze that usually remits. examination discloses incomplete or slow adduction of the eye ipsilateral to the lesion and nystagmus of the contralateral eye during abduction (see chapter 9) . dissociated nystagmus may be the only finding of an old or subtle internuclear ophthalmoplegia (video 19, internuclear ophthalmoplegia). bilateral internuclear ophthalmoplegia is strongly suggestive of ms, although this rarely may occur with tumor, infarct, mitochondrial cytopathy, wernicke's encephalitis, and chiari malformation (video 229, wernicke's encephalopathy). vertical and diagonal diplopia usually results from skew deviation. nystagmus, slow saccadic movements, broken ocular pursuits, and ocular dysmetria are other eye findings produced by lesions of cerebellar and vestibular pathways (see chapters 9 and 12; video 228, saccadic dysmetria). abducens paresis occurs on occasion, but oculomotor and trochlear nerve impairment is rare. corticospinal, spinothalamic, lemniscal, vestibular, and cerebellar pathways can all be affected. cranial nerve impairment may be seen with lesions that affect brain stem nuclei or exiting and entering fibers. usually this occurs in association with other symptoms. because of the long spinal tract and nucleus, the trigeminal nerve is frequently involved (video 106, trigeminal neuralgia). facial nerve paresis does occasionally occur, but ms is an extremely rare cause of bell's palsy in patients without previous symptoms. acute unilateral hearing loss is an uncommon manifestation. dysphagia is often due to impairment of cranial nerves ix, x, and xii and generally appears late in the course of some patients. once thought uncommon, cognitive disorders are now known to be present in 40% to 70% of ms patients. 31 age, duration of ms, and physical disability do not completely predict the presence of cognitive dysfunction, but classic mri measures like the total t2-weighted lesion load does not seem to correlate well with the degree of cognitive decline. measures of cortical atrophy, venticular enlargement, and neuronal integrity seem to correlate better with the cognitive aspects of ms. the problems are often subtle and may not be detected on standard mental status evaluation. the pattern of cognitive decline is typified by decrease of episodic memory, processing speed, verbal fluency, and difficulty with abstract concepts and complex reasoning. to a lesser extent, executive functioning and visual perception, semantic memory, and attention span may also be also decreased. general intelligence is not typically affected. as expected, cortical symptoms such as aphasia, apraxia, and agnosia are unusual. homonymous hemianopia, which can be caused by cortical or subcortical lesions, is also uncommon. despite prominent cerebral white matter involvement, many of the disconnection syndromes such as alexia without agraphia, conduction aphasia, and pure word deafness have not been reported in ms patients. affective disorders are more frequent in ms patients than in the general population. these include both anxiety and depression. in long-term studies, the incidence of depression in ms patients is close to 75%. neither depression nor anxiety is related to physical or cognitive disability or mri lesion load. patients sometimes experience uncontrollable weeping or less commonly laughter incongruent with their mood. interruption of inhibitory corticobulbar fibers is responsible for these symptoms (pseudobulbar affect). fatigue is a pervasive symptom among ms patients that is not related to disability or depression. over 75% of ms patients experience fatigue during their disease course. a diurnal pattern is characteristic and follows the normal circadian pattern of body temperature fluctuations, with the worse symptoms occurring in afternoon hours (peak core body temperature) often giving way to improvement in the late evening. ms symptoms may fluctuate in a predictable fashion. transient worsening of symptoms frequently follows exercise or elevation of body temperature. one example is uhtoff's phenomenon, in which visual blurring occurs during strenuous activity or with passive exposure to heat. these episodes resolve when the body temperature cools to normal or after a period of rest. an intercurrent infection with fever can induce worsening of symptoms and may be confused with a relapse. heat sensitivity is presumably related to conduction block, as demyelinated axons are more prone to failed conduction than normal, myelinated fibers. 32 paroxysmal symptoms are characteristic of ms and are believed to be due to the lateral spread of excitation (ephaptic transmission) between denuded axons in areas of demyelination. symptoms are typically brief (seconds to 2 minutes) and recur frequently, occasionally dozens of times per day. they may be precipitated by hyperventilation, certain sensory input, or particular postures. tonic spasms (paroxysmal dystonia) most often affect the arm and leg on one side, but the face, one limb, or bilateral limbs are sometimes involved (video 20, tonic spasms). these spasms may result from lesions anywhere along the corticospinal tract. they often begin during the recovery phase after an acute relapse and remit after a few months. intense pain and ipsilateral or crossed sensory symptoms may accompany them. paroxysmal weakness occurs, but it is uncommon. a wide variety of paroxysmal sensory symptoms may occur with ms, including tingling, prickling, burning, or itching, and sharp neuralgic pain is common. trigeminal neuralgia may appear in patients with ms (video 106, trigeminal neuralgia). the occurrence of trigeminal neuralgia in a person younger than age 40 is suggestive of ms. lhermitte's sign (transient sensory symptoms usually precipitated by neck flexion) is usually described as an electrical or tingling sensation that travels down the spine or into the extremities. although quite common in ms, lhermitte's sign can also occur with a wide variety of other disorders, such as vitamin b 12 deficiency, spondylosis, chiari malformation, and tumors, and after cisplatin chemotherapy. several other paroxysmal symptoms are occasionally encountered, including paroxysmal dysarthria and ataxia, paroxysmal diplopia, and combinations of these symptoms. facial myokymia and hemifacial spasm are additional transient (lasting months) phenomena sometimes due to brain stem demyelination (video 110, facial myokymia; video 224, hemifacial spasm). trismus, kinesigenic dystonia, paroxysmal kinesigenic choreoathetosis, and segmental myoclonus have also been described in case reports of ms patients as rare and unusual examination findings. seizures occur in a larger proportion of ms cases compared to normal control subjects. a recently published review of 29 case series of ms patients with epileptic seizures yielded a prevalence of 2.3%. 33 this represents an approximately three-to sixfold increase compared to the general adult population. cortical and juxtacortical lesions may be responsible for the increased incidence of seizures in ms patients. however, such plaques are common and seizures in ms are not, which suggests that other factors may also contribute to the relationship between epilepsy and ms. focal motor seizures, possibly with secondary generalization, are the most frequent. the occurrence of seizures usually follows one of two patterns. on occasion, focal onset seizures begin early in the course of ms and later remit. the start of seizures late in the course of ms more often poses a chronic problem and may be difficult to control. the eye is the only organ outside the nervous system that is sometimes involved in ms. uveitis and retinal periphlebitis each occur in at least 10% of ms patients. in a recent study, most patients with ms-associated uveitis were white females between 20 and 50 years of age. 34 the diagnosis of ms preceded the onset of uveitis in 56%, followed it in 25%. in over 90% of the cases, the uveitis was bilateral. pars planitis was found to be the most frequent form of uveitis (over 80%), and concomitant anterior chamber inflammation was also common. usually ms-associated uveitis is benign from the standpoint of visual acuity. uveitis can involve the posterior, intermediate (pars planitis), or rarely anterior portion and resembles that seen in other inflammatory (e.g., sarcoid, reiter's syndrome, behã§et's syndrome, inflammatory bowel disease, systemic lupus erythematosus) and infectious (e.g., syphilis, tuberculosis, lyme disease) conditions. periphlebitis is seen as venous sheathing on funduscopic examination and is histologically identical to the perivascular inflammation present in brain white matter. it is interesting that inflammation commonly occurs in the retina, which has a peripheral type of myelin produced by schwann cells. there are occasional reports of peripheral nerve or nerve root demyelination in ms patients as well as central demyelination in acute inflammatory demyelinating polyradiculoneuropathy and chronic inflammatory demyelinating polyradiculoneuropathy (see chapter 49) . some of these cases may be due to the incidental occurrence of two unrelated disease processes. however, because the pns and cns share many antigens, including mbp, it is possible that an autoimmune reaction or a viral infection could involve both the cns and pns. persons with one autoimmune disorder generally have an increased risk of others. even though there are several reports of systemic and organ-specific autoimmune diseases in ms patients, population-based studies have not confirmed any increase in prevalence of these disorders among ms patients. 35 in fact, there appears to be a negative association between ms and rheumatoid arthritis. multifocal cns involvement and acute relapses, remissions, and slow progression of neurological deficits typify ms. a single episode of neurological dysfunction can be suggestive of ms if it follows the typical time course of a relapse: progression over less than 2 weeks (usually days), with or without a period of stabilization, and improvement or resolution (often over months). insidious progression of deficits localized to a single site in the cns can also be due to ms, but other causes must be excluded. the temporal course of ms can be described by one of four categories: relapsing-remitting (rr), secondary progressive (sp), primary progressive (pp), and progressive relapsing (pr). 32 many physicians use the term relapsing progressive, which encompassed patients with spms, prms, and even those with rrms who have stepwise relapse-related worsening disability. this term has recently been abandoned. other terms that relate to the course of ms but have no consensus regarding their definition are sometimes encountered. benign ms generally refers to patients who have had ms for a long time but have little or no disability. malignant ms is sometimes used to describe patients with frequent relapses and incomplete recovery but is also used in reference to patients with acute fulminant demyelinating syndromes (see later). the term clinically isolated syndrome (cis) refers to patients presenting with their first episode of region-restricted episodes of cns inflammatory demyelination. this may remain an isolated syndrome (no recurrence), it may remain a forme fruste of acute disseminated encephalomyelitis (adem), or it may be the harbinger for one of the relapsing forms of ms. the probability of recurrent demyelinating episodes (e.g., clinically definite ms) has been the subject of several important investigations, and several clinical features and test results are of predictive value. optic nerve, spinal cord, and brain stem are the most common sites of these recurrent monosymptomatic events, and the time profile follows that of ms relapses. the pathogenesis, pathophysiology, epidemiology, clinical features, associated disorders, differential diagnosis, evaluation, and management are the same as in ms. the prognosis for visual recovery after each episode of on is good, and most patients regain normal visual acuity. profound visual loss, recurrent on, and age older than 35 are associated with a higher risk for poor recovery. investigators have concluded that recurrent multifocal demyelinating episodes, fulfilling the diagnostic requirements of clinically definite ms, develop in 50% or more of patients after isolated on when follow-up is extended beyond 20 years. 36 most of this risk is incurred within the first few years, although significant risk may continue into the fourth decade after the event. children much more often develop simultaneous bilateral on and have a lower risk for subsequent ms than adults. factors that are associated with an increased risk of developing ms as a disseminated illness are the presence of venous sheathing, recurrent on, family history of ms, white race, previous vague or nonspecific neurological symptoms, and the presence of oligoclonal bands (ocbs), elevated igg index, or igg synthesis rate in csf. the severity of acute transverse myelitis is inversely related to the risk of acquiring further symptomatic demyelinating lesions. complete transverse myelitis with profound loss of motor, sensory, and sphincter function imparts a relatively low risk of 3 to 14 for the later diagnosis of ms. partial transverse myelitis with preservation of significant motor function at peak is associated with a much higher incidence of ms. although monosymptomatic brain stem demyelination is not as common as either on or acute transverse myelitis, similar conclusions have been reached. in the only study available, two thirds of these patients with cerebral white matter lesions detected on mri developed ms within 5 years, compared with none of 5 patients with normal head mri. 37, 38 a recently published 10-year follow-up of the original queen square series continues to demonstrate the value of the baseline cranial mri study in determining risk of recurrence (ms risk). in this cohort study of 81 cis patients, approximately two thirds had at least one asymptomatic lesion (54 of 81, 67%) at baseline. after 5 years of follow-up, slightly more than half with one to three asymptomatic baseline cerebral lesions had developed ms (13 of 24) compared with the majority of cases presenting with at least four baseline lesions (28 of 33, 85%). after 10 years of follow-up, the majority of patients with any asymptomatic cerebral lesions had developed definite ms (45 of 54, 83%). 39 the recently published 14-year follow-up data on this group of patients reveals that 88% of the initially mri positive patients developed ms versus 19% of the mri negative subgroup. 40 this information is helpful for treating patients in the setting of cis. differential diagnosis. only a few diseases cause neurological deficits that regress spontaneously and relapse in different areas of the cns over the course of many years. however, because of the remarkable heterogeneity of ms, many disorders may resemble ms (table 48-3) , especially in the first years of active disease. other primary idiopathic inflammatory demyelinating cns disorders may be mistaken for ms. adem usually causes monophasic cns demyelination. although it frequently involves multifocal areas of white matter simultaneously, adem cannot be reliably differentiated from the initial clinical episode of ms. fulminant brain demyelination in persons without previous symptoms of ms is more likely due to adem or other conditions (schilder's myelinoclastic diffuse sclerosis, balo's concentric sclerosis, marburg's variant of ms). neuromyelitis optica differs from ms primarily in the topography and intensity of the lesions. several systemic or organ-specific inflammatory conditions can involve the cns white matter. on, myelitis, and other syndromes sometimes occur with systemic lupus erythematosus. whether this autoimmune disease increases the risk of developing ms or causes similar syndromes by a different pathological process is unknown. sarcoidosis can affect the nervous system in several ways, including multifocal, corticosteroid-responsive white matter lesions. sjã¶gren's syndrome sometimes occurs with ms, but this may only represent a chance association. neuro-behã§et's disease has a predilection for the brain stem. occasionally, isolated demyelinating syndromes are associated with inflammatory bowel disease. a wide variety of vasculitic syndromes (e.g., primary angiitis of the cns, periarteritis nodosa, wegener's granulomatous angiitis, vasculitis associated with rheumatoid arthritis, susac's syndrome, eales'disease) may mimic ms. however, these syndromes can usually be distinguished by involvement of the cortex, seizures, early dementia, personality changes, psychosis, infarcts involving large vessel territories on mri, and lack of improvement. findings characteristic to the particular vasculitis (uveitis and vitreal hemorrhage in eales' disease, retinal and cochlear involvement in susac's syndrome, upper and lower respiratory tract involvement in wegener's granulomatosis) also aid in the correct diagnosis. a few infections must also be considered in the differential diagnosis of ms. both lyme disease and syphilis may cause multifocal white matter lesions. htlv-1 causes a chronic progressive myelopathy (htlv-1-associated myelopathy/tropical spastic paraparesis). acute or recurrent myelitis can be caused by vzv. progressive multifocal leukoencephalopathy and toxoplasma abscesses should be considered in immunocompromised patients with progressive neurological decline. bacterial endocarditis with brain abscess formation, subacute sclerosing panencephalitis, or chronic rubella encephalomyelitis may need to be considered in the appropriate circumstances. cerebrovascular disease is only rarely mistaken for ms. occasionally, an ms relapse has an abrupt onset that may mimic an infarct, especially in those not previously diagnosed with ms. the usual circumstance is that of a hemisensory or hemimotor deficit imitating a lacunar infarct. disorders with multiple cerebral infarcts (emboli, hypercoagulable states, sneddon's syndrome, cadasil, vasculitis) may produce an mri appearance and course resembling ms. vascular malformations may also produce symptoms similar to ms. additional neurological illnesses capable of producing multifocal lesions rarely mimic ms. metastatic tumors and multifocal gliomas are often cited examples, but rarely is this distinction difficult for an experienced clinician. lymphoma more commonly masquerades as ms because the lesions may involve the white matter, may be multifocal, and are corticosteroid responsive. in addition, demyelination sometimes presents as one (or a few) mass lesion(s). in this situation, biopsy may be needed for diagnosis. neoplasms can cause paraneoplastic syndromes that may be confused with ms. a high index of suspicion must be kept for older age at presentation, subacute ataxia, early dementia, and personality changes. a few metabolic disorders may resemble ms, such as vitamin b 12 deficiency, vitamin e deficiency (seen in bassen-kornzweig syndrome, hypobetalipoproteinemia, and refsum's disease), and central pontine or extrapontine myelinolysis (video 113, pontine myelinolysis). leukodystrophies are usually not difficult to distinguish from ms. krabbe's disease (galactocerebroside-b-galactosidase deficiency), metachromatic leukodystrophy (mld; arylsulfatase a deficiency), and the usual adult form of adrenoleukodystrophy (ald) and adrenomyeloneuropathy (amn) exhibit both central and peripheral dysmyelination. blood leukocyte or fibroblast culture enzyme activity levels will confirm the diagnosis of krabbe's disease and mld, and elevated levels of very long chain fatty acids occur in ald/amn. mitochondrial disorders should also be given consideration because symptoms and mri appearance may be similar to ms. a relapsing remitting disorder identical to ms is sometimes seen in patients with the mutations responsible for leber's hereditary optic neuritis (lhon). 41 this usually occurs in female patients, and there may not be a family history of visual loss. a number of rare biochemically defined illnesses and other genetic disorders may occasionally merit consideration (including cobalamin and folate dysmetabolism, adult polyglucosan body disease, hereditary spastic paraparesis, spinocerebellar degeneration, and hereditary cerebroretinal vasculopathy). 42 several additional disorders must be excluded before diagnosing primary progressive ms (ppms). spinal cord compression from spondylosis or tumor may produce chronic progressive myelopathy. chiari malformations, syringomyelia, syringobulbia, other foramen magnum lesions, spinal arteriovenous malformations, and dural fistulas may also need consideration. careful imaging readily identifies these structural abnormalities. degenerative diseases such as olivopontocerebellar atrophy may mimic ppms. mri and csf examination will help distinguish between the two. conversion reactions and somatization disorders are commonly encountered in a busy referral practice and must be accurately diagnosed to afford optimal patient management. evaluation. the diagnosis of ms is based on the demonstration of white matter lesions disseminated in time and space in the absence of another identifiable explanation. ms remains a clinical diagnosis, although mri, evoked potentials, and csf examination can help clarify less certain cases. for research purposes, various categories of ms have been defined based on the certainty of the diagnosis. 43 at least two attacks and evidence of two separate cns lesions (clinical or paraclinical) are required for the designation of clinically definite ms (cdms). two attacks and evidence of one cns lesion or one attack and evidence of two cns lesions (clinical or paraclinical) is considered clinically probable ms. cases that fulfill the criteria for clinically probable ms and have supportive csf findings are labeled as laboratory-supported definite ms. patients with a clear history of at least two attacks and supportive csf but a normal neurological examination and no paraclinical evidence of cns lesions are categorized as having laboratory-supported probable ms. suspected cases that do not fit any of these criteria may be regarded as possible ms. paraclinical evidence generally refers to abnormalities on evoked potential studies or imaging procedures. as a result of increasing availability of refined paraclinical diagnostic modalities (especially mri) and an overall better understanding of the disease process, new diagnostic criteria for ms were proposed by an international expert panel in 2001. 44 three out of four of the following findings should be present on mri: (1) one gadolinium enhancing lesion, or nine t2 hyperintense lesions; (2) at least one infratentorial lesion; (3) at least one juxtacortical lesion; and (4) at least three periventricular lesions. according to the clinical diagnostic criteria, if a patient had two or more attacks with objective evidence on examination of two or more anatomical areas involved, no additional data is required to make the definite diagnosis. however, if such diagnostic studies were done and are not supportive of a diagnosis of ms, then the diagnosis should be reconsidered. if a patient presents with a history of two or more attacks, but objective clinical evidence only suggests one lesion, the following additional data is needed to confirm the diagnosis: the disease process has to be disseminated in space as demonstrated by mri; alternatively, two or more mri-detected lesions consistent with ms plus positive csf would suffice to meet the newly defined criteria. the clinician also may elect to await a further attack implicating a different anatomical site. in case a patient had one attack, with objective clinical evidence of two or more lesions, dissemination in time as demonstrated by serial mris separated by at least 3 months or a second clinical attack would clarify the diagnosis. if a patient has a clinically isolated syndrome, or "monosymptomatic" presentation, the following criteria should be met: dissemination in space as demonstrated by mri (again separated by at least 3 months), or two or more mridetected lesions consistent with ms plus positive csf and dissemination in time on serial mri scans, or a second clinical attack. in case the patient presents with a progressive course, the presence of positive csf is required, and dissemination in space should be present, as suggested by nine or more t2-weighted brain lesions, or two or more cord lesions, or four to eight brain lesions plus one cord lesion on mri. alternatively, abnormal visual evoked potentials (veps) with four to eight brain lesions, or fewer than four brain lesions plus one cord lesion, and dissemination in time on serial mri scans, or continued progression for a year would meet the diagnostic criteria. "positive csf" according to this set of criteria is defined by either the presence of oligoclonal bands detected by established methods (preferably isoelectric focusing on agarose gel followed by immunoblotting) different from any such bands in serum, or by a raised igg index. the presence of both enhancing and nonenhancing white matter lesions on a single mr image must not be used as evidence of dissemination in time as well as space, because these can also be seen in adem. oligoclonal bands (ocbs) and an elevated igg index provide supportive csf findings. ancillary tests are frequently required to confirm the diagnosis of ms and to exclude other possibilities in uncertain cases. laboratory tests on peripheral blood can help to exclude many of the infectious and other inflammatory disorders. a chest x-ray is generally needed to assess for sarcoid or paraneoplastic disorders if these are under consideration. an ophthalmological examination may be needed to search for alternative causes of visual loss. imaging studies, csf examination, and evoked potentials are often helpful because characteristic abnormalities are frequently present. mri of the head is the most sensitive imaging study for ms ( fig. 48-3 ). focal areas of increased t2-weighted and decreased t1-weighted signal reflect the increased water content associated with demyelinated plaques. the mri appearance of ms lesions, however, is not specific and similar abnormalities may be seen in normal aging, small penetrating vessel infarcts, lyme disease, tropical spastic paraparesis/htlv-1-associated myelopathy, sarcoid, systemic lupus erythematosus, sjã¶gren's syndrome, mitochondrial cytopathies, vasculitis, and adem. the specificity for ms can be increased by consideration of lesion number, size, location, and shape. 45 this is especially important in persons older than age 50. mri characteristics, other than the ones suggested by the international criteria outlined previously, are size larger than 6 mm, oval shape (often with the long axis directed perpendicular to lateral ventricles), and locations in the periventricular area, corpus callosum, and posterior fossa. longitudinal mri studies have shown the evolution of ms lesions. 46 gadolinium enhancement, indicating blood-brain barrier disruption, sometimes precedes the development of t2-weighted lesions and typically lasts for 4 weeks in the brain (occasionally longer, especially in larger hemispheric lesions), and perhaps somewhat longer in the spinal cord. flair imaging is especially helpful for evaluating periventricular lesions that may go unnoticed on regular t2-weighted scans. the disadvantage of the technique is its relative insensitivity to posterior fossa lesions. proton density weighted images are also part of the usual sets of images used in the mr diagnostics of ms. these images can be evaluated similarly to t2-weighted images. technically, they are usually acquired together with the t2-weighted datasets, as a first echo in conventional fast spin echo sequences, where the subsequent echoes can be used for generating the t2-weighted images. new t2weighted lesions have a fuzzy border and enlarge over a few weeks. after a period of stabilization, the t2-weighted lesion regresses and becomes more sharply delineated from the surrounding white matter as edema resolves. most of the time, a residual abnormality with increased t2 weighting and decreased t1-weighted signal remains, reflecting demyelination and gliosis. the low attenuation t1 signal, or "t1 black hole," is more often seen in secondary progressive ms and is thought to represent actual tissue loss. in several well-documented cases, hypointense lesions on t2-weighted scans were described in subcortical gray matter structures in ms patients. on a molecular level these areas are thought to represent iron deposition; their significance in ms is not fully understood. the mri activity of disease, defined as either the number of new, recurrent, and enlarging lesions or the number of gadolinium-enhancing lesions, is usually higher than the clinical activity. this may be either because of the involvement of asymptomatic areas of the cns or because of a pathophysiological difference between symptomatic and nonsymptomatic lesions based on the presence or absence of axonal dysfunction. there is only poor correlation between disability and lesion load (volume of white matter abnormalities) determined by head mri. sometimes individuals have severe impairment and few mri abnormalities, and the converse may occur. this disparity is partially explained by variable spinal cord involvement, but a pathophysiological difference may account for some of the discrepancy. several mri markers of gray matter involvement correlate better than measures of white matter pathology with clinical functional outcome measures in ms. in a recent study 47 edss showed the strongest correlation with gray matter volume loss and with t1 black hole volume increase (p < 0.01); both are considered to reflect neuronal and axonal pathology. ambulatory function, assessed as the 25-feet timed walk, also correlated well with gray matter volume loss and t1 black hole volume. on normal appearing gray matter magnetization transfer ratio (mtr) histograms, normalized peak heights inversely correlated with edss in 18 rrms patients (r â¼ ã�0.65, p â¼.01). 48 in a study evaluating a number of mri parameters (including brain t1-hypointense and flair-hyperintense lesion volume, third ventricle width, brain parenchymal fraction and t2 hypointensities in the dentate nucleus), the best correlation with edss (and the only correlating parameter with 25-feet timed walk) was t2 hypointensity in the dentate nucleus. 49 in 41 ms patients, an mri study concluded that gray matter atrophy correlated with clinical status (edss, 25-feet timed walking and disease duration). 47 a study of patients with ppms and rrms showed that neocortical volume as determined by mri correlated with edss scores across all the patients, but the strength of the correlation was stronger (p < 0.05) in the ppms (r â¼ ã�0.64, p < 0.0001) than in the rrms group (r â¼ ã�0.27, p â¼ 0.04). 50 mr spectroscopy is increasingly becoming an accepted diagnostic modality, where information can be obtained about the biochemical constituents of selected voxels of interest. with this technique, a cubic volume of interest is defined based on a regular mr image set. simultaneous acquisition of multiple volume units is possible. with long echo time (te) studies, naa (n-acetylaspartate), choline, creatinine, and lactate peaks can be identified on the mr spectrum. with short te studies, myoinositol, lipids, and some neurotransmitters may be identified. the resolution of the mr spectrum (the "number of lines" in the spectrum) is proportionate to the magnetic field strength used. naa is the second most abundant amino acid constituent in the brain after glutamate. it is localized almost exclusively in neurons and axons. creatinine is used as the "constant" peak in a mr spectrum, since it is the least likely to be altered by cns-specific processes. therefore, numeric mrs data are usually presented as ratios related to creatinine. the naa/creatinine ratio is decreased in areas of axonal or neuronal loss. it correlates well with disability. it can be decreased in normal appearing white matter, also in early stages of lesion formation, thus representing a challenge to the usual dogma of axonal loss being secondary to myelin damage. the decrease of the naa/creatinine ratio may return to normal following the resolution of the acute phase. this process may be related either to reversibility of neuronal injury or to disappearance of edema in the involved areas. in general, more reduced naa peaks are seen in progressive forms of ms with more profound tissue loss. if a relatively large hemispheric lesion shows decreased naa content, similar findings may be seen in the other hemisphere in a "mirror" location. the lactate peak can be elevated in a variety of acute processes, and as such, carries relatively low specificity. the short te spectrum is used less frequently; the "mobile lipid" peak (which is thought to represent macromolecular protein fragments) is increased in areas of acute demyelination. another newer mri technique used in ms research is magnetization transfer imaging. the principle behind this imaging modality is relatively simple. in complex macromolecular systems, there is a baseline magnetization exchange in equilibrium between macromolecular protons and mobile protons. if the macromolecular protons are saturated before each excitation (and subsequent data acquisition) with a prolonged off-resonance broadband pulse, then the signal intensity of the image will be reduced owing to magnetization transfer exchange between the saturated ("bound") and free ("mobile") protons. by obtaining duplicate sets of images (with and without magnetization transfer pulse), a magnetization transfer ratio can be calculated. the ratio reflects the integrity of the macromolecular environment. it is reduced by approximately 3% to 5% in areas of edema, but it is more significantly reduced in areas of demyelination or axonal loss. if the ratio "normalizes" in a lesion, no subsequent tissue loss is usually seen on other imaging modalities. despite these advantages, the magnetization transfer imaging is technically difficult because it produces variable findings depending on the technical environment and is not universally available. it has not become an accepted and standard technique for evaluation of ms patients. it may be very useful as a marker for remyelination and tissue repair in future neuroprotective or tissue restorative trials. diffusion-weighted imaging is well known from its widespread use in the diagnosis of ischemic stroke. this technique can show early stages of ms plaque formation. the increase in apparent diffusion coefficient correlates with acute plaques, and seems to best correspond with t1-enhancing lesions; this technique may show the lesions at an even earlier stage. mri has become an important component of clinical trials in ms. because of the high sensitivity of mri for disease activity, it is reasoned that periodic mri may determine treatment efficacy more quickly than monitoring relapse rate or disability level. many studies have used mri as a secondary outcome, but clinical outcomes are still used as the primary outcome for definitive trials. additional mri techniques have also proved useful in the diagnosis of ms. mri of the spinal cord shows discrete lesions in about 80% of cdms patients. several semiautomatic methods exist to determine lesion volume, ventricle volume, or hemispheric volume. these are generally applied for research purposes only, and are not part of the usual workup or diagnostic follow-up of ms. csf evaluation remains a valuable diagnostic tool for ms. a lymphocytic pleocytosis occurs during acute exacerbations in about one third of patients, but this seldom exceeds 50 cells. eighty percent of the lymphocytes are cd3 positive. the ratio of cd4 to cd8 cells is 2:1. less than 20% of the cells are b cells. csf protein is normal in up to 60%; levels above 100 mg/dl are unusual and may suggest a different disorder. the proportion of g globulin is high owing to the synthesis of immunoglobulins within the blood-brain barrier. the majority of csf immunoglobulin is igg, although igm and iga may also be elevated. measures of intrathecal igg production have been devised that are more useful than simple g-globulin levels. the igg index and synthesis rate are elevated in 70% to 90% of cdms patients and occasionally in other disorders. agarose gel electrophoresis, or the more sensitive isoelectric focusing of csf proteins, often reveals discrete bands of immunoglobulin, each a monoclonal antibody. it is pertinent to compare serum and csf banding patterns because peripheral monoclonal gammopathies may produce csf bands. to reduce false-positive results, only unique csf ocbs should be reported. between 85% and 95% of clinically definite ms patients have ocbs; however, early in the course they are not as prevalent. once present, ocbs persist and the pattern does not vary, although new bands occasionally appear. unlike subacute sclerosing panencephalitis, in which the majority of ocbs are antibodies specific for measles virus, the antigenic specificity of ocbs in ms is unknown; they are unlikely to be pathogen specific or autoantigen directed; there is some evidence that they may be genetically determined germline antibodies. five percent to 10% of noninflammatory cns samples and 30% of inflammatory samples are also positive for ocbs. 51 more detailed recommendations about the inclusion of csf parameters to the diagnosis of ms were recently published 52 suggesting that the cell count and differential should be completed within 2 hours. the new and recommended method for the detection of ocbs includes immunoelectophorsesis on agarose gel followed by immunoblotting. the reported sensitivity of this technique is above 95%, with a specificity of 86% to 87%. in other inflammatory or infectious illnesses, ocbs are often transient features. their persistence is more suggestive of ms. the presence of myelin components, antimyelin antibodies, and kappa light chains in csf has also been used in the diagnosis of ms. however, the sensitivity and specificity of these products is less than that of ocbs. in late 2005, a new set of recommendations were published based on the first 5 years of using mcdonald's criteria in diagnosing ms. 53 the original mcdonald criteria have been incorrectly interpreted by some as mainly relying on mri for making a diagnosis of ms. in reality, the mcdonald criteria cannot even be applied without careful clinical evaluation of the patient. neurological deficits must be evident to the examiner, and must be suggestive of ms. scans that "look like" ms (and meet the criteria of barkhof and tintore) but have never been accompanied by an obvious and documented neurological examination finding do not fulfill the mcdonald criteria. there was some sympathy among the international panel members revising the mcdonald criteria to allow selected symptoms that are clearly and specifically enunciated by the patient (e.g., lhermitte's symptom, trigeminal neuralgia, numbness ascending to the waist or higher) coupled with objective paraclinical (such as imaging and csf) findings to be sufficient as an indicator of a prior or current attack needed for an ms diagnosis. however, the panel was reluctant to endorse the diagnosis of ms in the absence of any objective clinical findings, even if objective paraclinical findings are in place, at least until such a scheme is tested in prospective settings. patients with imaging and csf findings suggestive of ms but not showing any objective evidence for neurological deficits commonly seen in ms require careful clinical and radiological monitoring. until objective evidence for neurological deficits are found, ms can not be diagnosed. ms may be the correct diagnosis with less stringent imaging criteria than originally proposed; however, the panel was uncomfortable making changes that would allow mri confirmation of dissemination in space based on lower stringency imaging criteria without appropriate prospective data. most studies performed to date have been inadequately designed to address this issue. advanced imaging technologies are constantly evolving and will likely one day be shown to aid in making the diagnosis of ms. visualization of intracortical lesions, use of higher field strength magnets, and analysis of "normal appearing brain tissue," may be conrnerstones of a future mri criteria for ms. preliminary evidence suggests that "occult" damage in normalappearing white and gray matter seen with magnetization transfer, diffusion tensor imaging, or spectroscopy is an early feature of ms, whereas it likely does not occur in other demyelinating conditions such as acute disseminated encephalomyelitis and nmo. important changes have been made to the original definition of "dissemination in time" by mri. in keeping with the definition that clinical relapses must be separated by at least 1 month, it was agreed that new t2 lesions on mri should occur at least 30 days after disease onset. this means that any new t2 lesion occurring at any time point after a so-called reference scan performed at least 30 days after the onset of the initial clinical event is useful in meeting imaging diagnostic criteria for dissemination in time. it should be noted though that a new t2 lesion must be of sufficient size and location to exclude lesions that could have been missed previously for technical reasons of slice orientation, thickness or spacing, tissue contrast, patient motion, or other artifacts. this requires standardized scanning procedures with emphasis on careful repositioning, as well as input from qualified evaluators experienced in ms imaging. with the new revision, there are two ways to show dissemination in time using imaging: (1) detection of gadolinium enhancement at least 3 months after the onset of initial clinical event, if not at the site corresponding to the initial event; or (2) detection of a new t2 lesion if this appears at any time compared with a reference scan done at least 30 days after onset of the initial clinical event. spinal cord lesions can be important in differentiating ms from other white matter diseases; however, the original mcdonald criteria did not provide sufficient guidelines for the use of cord imaging in ms. spinal cord imaging that detects typical ms cord lesions (minimal or no swelling of the cord; clearly hyperintense on t2-weighted imaging; at least 3 mm in size, but less than two vertebral segments long; and occupying only part of the cord cross section) is particularly helpful if brain imaging does not detect dissemination in space in a patient suspected to have ms. for dissemination in space, a spinal cord lesion is equivalent to, and can substitute for, a brain infratentorial lesion, but not for a periventricular or juxtacortical lesion. an enhancing spinal cord lesion is equivalent to an enhancing brain lesion, and an enhancing spinal cord lesion can "count" doubly in fulfilling the criteria (e.g., a single enhancing spinal cord lesion can "count" for an enhancing lesion and an infratentorial lesion). individual cord lesions can contribute together with individual brain lesions to reach the required nine t2 lesions to satisfy the barkhof-tintore criteria (the mri criteria incorporated in the original mcdonald's criteria). the panel recognized that diffuse cord changes may occur in ms, especially in the progressive forms; however, these changes are not sufficiently reliable to allow for their incorporation into the diagnostic criteria at this time. repeat spinal cord imaging in patients without new symptoms of myelitis has a low yield in efforts to demonstrate dissemination of lesions in time. in other words, while it is common to see asymptomatic new brain lesions on repeated scans, new cord lesions generally do result in new neurological symptoms. therefore, repeat cord imaging is recommended only to support an ms diagnosis when there is a clinical reason to suspect a new cord lesion. important changes have been proposed in diagnosing primary progressive ms. these revised criteria stress clinical and imaging (brain or spinal cord) evidence for diagnosis and place less emphasis on csf findings. the new criteria for ppms is as follows: (1) at least 1 year of disease progression (retrospectively or prospectively determined) (2) plus two of the following: (a) positive brain mri (nine t2 lesions or four or more t2 lesions with positive vep), (b) positive spinal cord mri (two focal t2 lesions), (c) positive csf (isoelectric focusing evidence of oligoclonal igg bands or increased igg index, or both). evoked potentials are summed cortical electrical responses to peripheral sensory stimulation that can be used to localize sites of disease and measure conduction velocity along sensory pathways. vep and somatosensory evoked potentials (sseps) may detect subclinical sites of demyelination, thus providing evidence of multifocality. brain stem auditory evoked potentials (baeps) are occasionally informative. more than 90% of persons with a history of on have an abnormal vep, and 85% of cdms patients have abnormalities on veps even when the history of on is absent. slowed conduction is present on ssep in nearly three fourths of patients with cdms. baes are the least sensitive, with abnormalities in less than 50%. mri has largely supplanted the use of evoked potentials in ms because of the greater sensitivity in the diagnosis and the detailed anatomical information it provides. in 2001, the american academy of neurology released practice parameters regarding the usefulness of evoked potential studies in ms. according to these recommendations, veps are considered probably useful (class ii evidence) to identify patients at increased risk for developing clinically definite ms. sseps are possibly useful, whereas the evidence for baeps supporting the diagnosis of cdms is insufficient. management. there is no available prevention or cure for ms. treatments focus on three areas: treating acute exacerbations and hastening their recovery; altering the natural history of ms; and providing symptomatic relief of current symptoms by enhancing physical abilities and preventing or treating complications. a fourth management topic concerns special treatment issues related to pregnancy. acute exacerbations. corticosteroids are the most commonly used treatment for ms, although there have been few studies to address their efficacy. adrenocorticotropic hormone (acth) was shown to speed recovery from an exacerbation but had no effect on the ultimate degree of recovery. because of the unpredictable cortisone response to acth, oral prednisone and later intravenous methylprednisolone became the preferred treatments. the optic neuritis treatment trial verified that intravenous methylprednisolone but not prednisone increased the recovery rate and unexpectedly increased the time to the next relapse, thus delaying the diagnosis of cdms. 54 moreover, the prednisone-treated group had twice as many recurrences. the finding was not replicated in a second study, but it has affected the practice of treating acute ms exacerbations. the current recommendation is to treat disabling attacks with 500 to 1000 mg of intravenous methylprednisolone per day for 3 to 5 days with or without a short tapering dose of oral corticosteroids. according to the practice parameters for steroid treatment of acute on attacks released by the american academy of neurology in 2001, oral prednisone in doses of 1 mg/kg/day has no proven value. higher dose of oral or parenteral methylprednisolone may result in quicker and more thorough recovery of visual function. there is no evidence of long-term benefit for visual function. a study suggested that intravenous steroids may also have long-term effects on disease progression when given regularly. 55 in this study, rrms patients randomized to receive regularly scheduled pulses of iv methylprednisolone (every 4 months for 3 years, then every 6 months for 2 years) demonstrated stability or improvement in disability measures, fewer "t1 black holes," and less brain atrophy than did control patients randomized to receive steroids only with relapses. these findings suggest a possible long-term benefit of pulsed iv methylprednisolone therapy on brain atrophy and disability. this as yet unconfirmed approach to long-term therapy might be considered a reasonable "control arm" in future phase iii trials of experimental therapies. up to one third of patients do not have an adequate recovery after a relapse despite the use of corticosteroids. plasma exchange (plex) alone was found beneficial in a substantial proportion of patients with severe inflammatory demyelinating episodes who had failed to improve following treatment with high-dose iv methylprednisolone. 56, 57 a randomized, sham-controlled, double-blind trial in 22 patients (seven exchanges over 14 days) without concomitant use of immunosuppressants in acute demyelinating events confirmed these findings. 58 moderate or greater clinical improvement was observed in over 42% of participants. a trial of seven plex treatments (alternate days) is a reasonable option for patients who fail to respond to conventional iv methylprednisolone therapy in acute, severe episodes of demyelinating diseases. the response to plex is stongly associated with the histological ms subtype. 59 antibody and complement plays a crucial role in pattern ii lesion formation. in a study of 19 biopsy-proven ms cases by keegan and associates, 10 pattern ii cases showed good response to plex, whereas 9 cases of pattern i or iii did not respond at all to plex. neuromyelitis optica, which is now considered an antibody-mediated condition with a known serological marker, also shows good response to plex: in a study by the mayo group, 60% of nmo patients showed moderate or marked improvement to plex, and an additional 10% showed mild improvement. 60 alteration of the natural course. the primary goal of drug treatment is to alter the natural course of the disease (e.g., reducing the frequency and severity of relapses, preventing the chronic progressive phase, and slowing the progression of disability). the disease activity seen on mri is often used as a secondary outcome, although mri measures currently correlate imperfectly with clinical outcome. knowledge on altering the course of ms is largely restricted to three patient groups, those with clinically isolated syndromes, those with rrms, and those with secondary progressive ms. before we discuss the known data in each of these demyelinating disease categories, it may be worth while to review the use of the most important evidence-based medicine (ebm) statistics that are applied to measure the magnitude of treatment effect. relative risk reduction (rrr) is the metric most commonly cited in publications and promotional materials about clinical trials. the rrr is the degree that the treatment reduced the frequency of the outcome measure (experimental event rate, e.g., relapse, progression) compared with the control treatment (control event rate). the rrr is a ratio, not an absolute number, and is calculated as follows: rrr â¼ ã°control event rate ã� experimental event rateã�= control event rate if the control event rate is low (making the denominator smaller), it will obviously inflate the rrr. an "impressive" 50% rrr may have a low biological significance if the outcome occurs infrequently. therefore, the absolute risk reduction (arr) should be calculated as this corrects for the frequency of the outcome. for most of the approved ms agents, the calculated arr is considerably less than rrr. this metric is usually not cited in reports of clinical trials of disease-modifying agents. to calculate risk reduction, one must have access to the data citing comparisons of proportions (ratios), and this is not always immediately available in publications. another useful measure of treatment effect is the "number needed to treat" (nnt). it is calculated as the inverse of the arr: overall, the nnts for the disease-modifying agents in ms are in the 7 to 14 range for treatment periods of 2 to 3 years. however, these nnts are for outcomes that have limited predictive value for long-term outcomes (e.g., relapse behavior does not precisely predict long-term disability) and the agents are expensive, inconvenient to use, and not without risk. we must also remember that clinical trials typically enroll patients with very restricted eligibility criteria (often a history of considerable recent disease activity or progression), and considerable efforts are in place in trials to optimize compliance with the treatment plan. as such, the nnt experienced in a practice setting (effectiveness) may considerably exceed what was reported in the trial setting. altering the course of a clinically isolated syndrome. when should treatment be initiated in patients with very early demyelinating disease? two recently published multicenter studies have addressed this issue in persons at high risk of developing ms. in the champs study, 61 383 patients with their first episode of presumed demyelinating disease ("clinically isolated syndrome") in the setting of an abnormal, asymptomatic baseline cranial mri scan, were randomized to receive either weekly interferon-b 1a 30 mg im or placebo after an initial course of steroid therapy. this study was terminated early when the primary outcome measure of conversion to "clinically definite ms" (cdms) status was reached in a greater number of placebo-treated patients. these findings were not unexpected given the known effect of interferons on reducing relapse rate but do provide some support for early treatment. the duration of follow-up in this study (71% 1 year, 34% 2 years, 16% 3 years) is insufficient to determine long-term benefit from early intervention, however. it is also clear that the treatment is only partially effective, as 50% of interferon (ifn)-treated patients in the champs trial had clinical or mri evidence of recurrent disease within 18 months of starting treatment. 62 the analysis of treatment effect related to the champs trial reveal a rrr of 38%, an arr of 14.6%, and an nnt of 7 patients over 2 years to prevent one conversion to "clinically definite ms." in a second placebo-controlled study of 309 patients with either monosymptomatic (61%) or multifocal onset (39%) early demyelinating disease, early treatment with interferon-b 1a in an unusually low dose (22 mg subcutaneously once weekly), reduced conversion to cdms (34% versus 45%) at 2 years. 63 again, there is no data on whether these treatments offer long-term benefit. the ebm calculations regarding this trial show an rrr of 24%, and arr of 11%, and an nnt of 9 patients over 2 years in order to prevent one conversion to "clinically definite ms." these two studies provide support for considering early treatment in patients presenting with first attack, in the presence of multiple asymptomatic mri lesions, but further studies are needed to determine whether this approach will provide a prolonged benefit on disease course. it is important to note that these studies do not provide guidance about clinically isolated syndromes that present with a brain mri that is not suggestive of ms (i.e., only one optic nerve lesion, or one brain stem or cord lesion explaining the cis symptoms). we do not recommend that cis patients with fewer than two asymptomatic mri lesions receive treatment with interferons. please see the discussions under "summary of recommendations for the treatment of rrms patients" for further advice on patient counseling and decision making about the use of the disease-modifying medications. altering the course of relapsing-remitting multiple sclerosis b-interferons. interferons are a class of peptides that have antiviral and immunoregulatory functions. both interferon-a and interferon-b are part of the anti-inflammatory t h 2 response. interferon-b 1b (betaseron) was the first drug approved by the u.s. food and drug administration (fda) specifically for the treatment of ms. a large clinical study in rrms patients demonstrated a reduction in the frequency of relapses by about one third with subcutaneous injection every other day. 64 the severity of relapses was also lessened. interferon-b 1b had a striking effect on mri measures of disease activity. the placebo-control group continued to accumulate white matter lesions, whereas patients in the high-dose arm (8 million iu) had stabilization of their mri lesion load. no difference was found in the disability levels, however. side effects include injection site reactions, flu-like symptoms (low-grade fever, myalgias, headache; these lessen in frequency after treatment for a few months), mild liver enzyme elevation, and lymphopenia. depression and attempted suicide were more common in the treated groups. to illustrate the magnitude of treatment effect of the pivotal interferon-b 1b trial, the rrr was 18%, the arr was 15%, and the nnt analysis showed that 7 patients are needed to be treated over 3 years to increase the number of those who were relapse free by one. one particularly disturbing result was the production of neutralizing autoantibodies (nabs) in 38% of patients after 3 years of treatment. not only do patients with these antibodies thereafter fail to respond to this drug, but there is also a concern that nabs may cross-react with natural interferon-b and interfere with its function. all positive sera for nabs seem to cross-react with both interferon-b 1a and 1b. switching from one preparation to the other does not change the pattern of antibody response. 65 the long-term effects of nabs are unknown. recent studies seem to support that nab formation reduces clinical and mri effects although often nab formation subsides with time. there are no firm guidelines for monitoring nab formation. most physicians do not measure nabs but rather change therapies empirically when patients appear to be failing treatment. low titer nabs may be just transient phenomenon related to ifn treatment; persistent high titer nabs on two consecutive tests at least 6 months apart is likely associated with poor treatment response to inf. interferon-b 1a (avonex) has the same amino acid sequence as natural interferon-b and differs from interferon-b 1b by one amino acid as well as by the presence of carbohydrate moieties. once-weekly intramuscular interferon-b 1a has been found to have effects similar to that of interferon-b 1b in reducing the frequency of ms relapses. in addition, a favorable effect on disability was also demonstrated and side effects were less common. in the original interferon-b 1-a intramuscular trial, the primary outcome measure was time to edss progression. the rrr was 37%, the arr was 13%, and the nnt was 8 for 2 years to prevent one patient from developing edss progression. the calculations for "proportion relapse free" show an rrr of 16%, and arr of 12%, and an nnt of 8 over 2 years (8 patients need to be treated for 2 years to increase the number of patients who were relapse free by one). nabs occurred half as often as with interferon-b 1b. interferon-b 1a has been approved by the fda for treatment of "relapsing ms." 66 the "correct" dose of interferon continues to be debated. in a recent placebo-controlled trial, patients randomized to a high dose of interferon-b 1a (44 mg three times per week) did better than those receiving half this weekly dose. both groups outperformed placebo and the high dose seemed to have more effect on relapse severity, hospitalizations, mri activity, and lesion volume accumulation, and possibly on delaying disability in the most severely disabled patients. at the end of the 2 years of follow-up, placebo-treated patients were randomized to 22 or 44 mg subcutaneously three times weekly; patients on active treatment were continued on their original dose. 67 the authors reported a benefit for the higher dose and for those treated for the full 4 years, again suggesting that early treatment and perhaps higher doses of interferons may be beneficial. the primary outcome, however, was relapse count per patient per 4 years and, as such, patients treated early had a significant advantage using this outcome measure. there were trends favoring the higher dose (relapse rate, mri volumes; not for time to first confirmed progression, however). the authors did not make statistical adjustments for multiple comparisons and there were many dropouts in the high-dose groups, making it difficult to draw firm conclusions. again, the answer to the question about the benefit of early treatment can best come from long-term (perhaps 8 to 10 years) studies using "hard outcomes" (e.g., time to progression, major milestones in disability). the ebm calculations based on the "proportion relapse free" data for the original interferon-b 1a (rebif) study show an rrr of 19%, and arr of 16%, and an nnt of 6 over 2 years to increase the number of relapse free by one. relative treatment advantages of interferon-b 1a and 1b have not been clearly established but are under study. 68, 69 a pilot study in rrms patients suggests that interferon-a may also have a therapeutic effect. 70 a study of interferon responders showed that younger patients with frequent relapses, and higher edss scores upon entry may be associated better response. 71 laboratory monitoring of interferon products. it is important to note that even though the interferon products are generally safe to use, they can be associated with potentially harmful adverse reactions. we recommend that every newly starting patient should have a baseline complete blood count, liver function tests, and thyroid-stimulating hormone (tsh) test. the liver function tests and blood count studies should be repeated in 1 week, 1 month, and every 3 months thereafter; the tsh should be repeated every 6 to 12 months. glatiramer acetate. glatiramer acetate (ga) is a synthetic mixture of polypeptides produced by the random combinations of four amino acids that are frequent in mbp. after a preliminary study suggested efficacy, 72 a phase iii randomized, double-blind, placebo-controlled, multicenter trial showed a 29% reduction in relapse rate. 73 the fda has approved this medication for use in rrms. even though this disease-modifying therapy requires daily subcutaneous administration, the side effects are relatively minor compared to the interferons, and patients do not need regular laboratory monitoring (table 48lesions, lesion volumes, and the percentage of new lesions that will evolve into t1 "black holes," although the mri effect may be less pronounced compared to the interferon products, and is not apparent until the agent has been used for at least 6 months. [74] [75] [76] the ebm calculations for ga using the "proportion relapse free" data show an rrr of 10%, arr of 7%, and an nnt of 14 over 2 years to increase the number of relapse free by one. combined azathioprine and interferon-b 1b. a small trial at nih showed significant reduction in the number of contrast-enhancing lesions when azathioprine in an average maintenance dose of 2 mg/kg/day was added to interferon-b 1b in a study of six rrms patients followed for a median period of 15 months. the addition of azathioprine may be considered in "treatment failure" cases, but this study was hampered by the small number of patients, no control subjects, and no blinding. 77 intravenous immunoglobulin. monthly treatment with low-dose (0.15 to 0.2 g/kg) intravenous immunoglobulin (ivig) in rrms patients resulted in fewer and less severe relapses in addition to slowing the accumulation of disability in a single randomized trial. the outcome was similar to that of injectable interferons. 78 this therapy is less accepted in the united states. more studies with larger number of patients and extended follow-up are needed to confirm these limited observations. recent studies have failed to demonstrate that ivig administration reverses long-standing deficits from ms and on. [79] [80] [81] ivig was also recently studied in acute on and failed to demonstrate benefit on any of the outcome measures. 82 natalizumab. in late 2004, natalizumab was approved for the treatment of rrms. 83 natalizumab is a humanized a-4 integrin antibody that inhibits the migration of all leukocytes (except for neutrophils) to target organs. a phase 2 study established 84 that a 300-mg monthly dose reduced the number of gadolinium-enhancing lesions by 90% and the clinical relapse rate by over 50% compared to placebo. this study was followed by the affirm and sentinel phase iii studies. the affirm study enrolled over 900 patients with rrms; none of them had been on other approved immunomodulators for longer than 6 months. the annualized relapse rate at 1 year was reduced from 0.74 in the placebo group to 0.25 in the treated group (66% relative reduction, p < 0.0001). the proportion of relapse-free patients was 76% in the treated group, 53% in the placebo group. the number of enhancing lesions was reduced by 92%, and the number of new or newly enlarging t2 lesions was reduced by 80%. the proportion of patients without clinical and mri activity was 46% in the natalizumab group, and 14% in the placebo group. in the sentinel trial, the combination of intramuscular interferon-b 1a and natalizumab was studied against im interferon-b 1a and placebo in patients who had demonstrated an incomplete response (relapse suppression) to interferon therapy. the ebm calculations of the affirm data based on proportion with relapses suggests an rrr of 49%, an arr of 23%, and an nnt of 4 over 1 year to increase the proportion of relapse free by one. the senti-nel data shows an rrr of 31%, an arr of 17%, and an nnt of 6 over 1 year to increase the proportion relapse free. the original pilot trial data shows a rrr of 50% and an arr of 19%, with an nnt of 5 over 6 months to increase the proportion relapse free. based on these data, the fda granted expedited approval of natalizumab on november 23, 2004. on february 28, 2005 , the medication was voluntarily withdrawn from the market by the sponsor (biogen-elan) after two cases of progressive multifocal leukoencephalopathy (pml) were reported in the sentinel study cohort. 85 both patients were in the combined interferon and natalizumab arm. a third pml case was later identified from one of the phase iii inflammatory bowel trials of this agent. at the time of writing this manuscript, natalizumab is still off the market. the natalizumab story has received significant media attention. several consequences can be drawn from this failure. first, highly potent immunomodulators like natalizumab are best used by specialists in selected cases. a large number of prescriptions were written for natalizumab during its short 3 months on the market, including prescriptions by general practitioners. widespread use of such medication in relatively stable cases of ms is not indicated. second, the combination of potent immunomodulators may result in unpredictable adverse outcomes. many ms experts anticipate that in the future ms therapies will need to be administered in combination to optimize therapeutic benefit. however, the exact effect of such combinations on the highly complex immune system is difficult if not impossible to predict. furthermore, our inability to treat ms more effectively does not stem from the fact that we can not provide powerful immunosuppression, as evidenced by the autologous bone marrow transplantation studies. ms is a complex disease with a prominent inflammatory component; however, increasing evidence suggests that the neurodegenerative component of this illness may be independent of the inflammatory component, and is just as important, if not more important, from the standpoint of long-term disability. third, in chronic diseases such as ms, a short 1-year trial, no matter how convincing the outcome may be, should not be considered sufficient to approve a medication, which will then be used in tens of thousands of patients on a "lifelong" basis. there clearly is a need for new and more effective medications for treating ms; however, clinical trials in chronic conditions are very difficult to sustain. to overcome this, many ms trials use primary mri outcome measures, since inflammation and new lesion formation-related mri markers respond more immediately to treatment; however, these markers do not correlate well with long term disability, as discussed earlier. summary of recommendations for the treatment of rrms patients. when making decisions about starting an ms patients on immunomodulators, several factors must be considered. one must realize that even though there are medications available for relapsing forms of ms, all the currently available therapies are only partially effective, the most reliable data is about short-term relapse rate reduction, and a relapse rate reduction does not necessarily translate into reduction of future disabilities. natural history data clearly suggest that a subset of ms patients will do very well without treatment (see discussion about the olmsted county cohort later); this information can be very useful when deciding about treatment in patients with a 5-or 10-year disease history and minimal disability (edss 2.0). in this patient group, a careful wait and see approach with appropriate monitoring is acceptable. counseling of newly diagnosed ms patients is of crucial importance, and it should usually include family members. most patients have easy access to an abundance of frequently misleading information on the internet, or from relatives and friends with ms. it is important to realize that every case is different; however, through the rational use of natural history data, clinical and mri features of the specific case, and the clear understanding of the available clinical trial data, the clinician should be able to provide customized and relevant advice to patients and families. considering that the treatments are only partially effective, the wishes of an educated patient constitute an important part in the decision-making process. ultimately, the treatment decisions should remain individualized between the patients and their treating physicians, and the physician's role as an information clearinghouse and educator cannot be overemphasized in this process. the three interferon products and ga represent the most commonly used ms immunomodulators in the united states; therefore, it is important to draw some practical conclusions about these agents. by now, several class i studies demonstrate that these agents are effective in reducing the relapse rate in rrms over a 2-to 3-year period; the reduction is roughly 30% with the high-dose interferons and ga. the above-mentioned nnt data are also very useful for the clinician and the well-informed patient when making treatment decisions. there is evidence for a dose-response relationship among the interferon products, mostly from the evidence and incomin studies. the double dose im interferon-b 1a study did not show a dose-response relationship; this may be related to the fact that the increased dose was given with the same frequency as the standard dose. the injectable immunomodulators have incomplete evidence for efficacy in disability-based outcome measures. many of the long-term extension studies suffer from several drawbacks, including open label unblinded design, significant dropout rate, and lack of control subjects; this is especially true for the ga extension data. the currently available few head-to-head comparison studies are also hampered with methodological issues; new comparative studies are under way. overall, these agents remain partially effective in relapsing forms of ms; their long-term effects on reducing the clinically most important feature of ms-disability-still remains unclear. altering the course of secondary progressive ms. within 15 years of onset, almost 60% of rrms patients will enter the secondary progressive phase of the disease. treatment approaches aimed to affect the natural course of disease are available for these patients. interferons. interferon-b 1b may have a beneficial effect on the overall outcome of spms and may also alter mr lesions, 86, 87 but this question remains incompletely answered. in the placebo-controlled european study 88 of inteferon-b 1b in spms, the time to worsening was extended for treated patients. treated patients were less likely to be wheelchair-bound and had fewer hospitalizations. another analysis 89 of this study confirmed the benefits, though the dropout rate in this study was relatively high. the patients who responded best to interferon therapy were those who experienced relapses during their disease course. mri monitoring suggested that the benefit on t2 lesion activity was seen early and persisted into the second half of the second year of treatment. t2 lesion load increased in placebo-but not interferon-treated patients in the first 2 years of treatment. 90 contrasting with these results, in another trial involving patients with spms, 91 both high dose (44 mg) and lower dose (22 mg) interferon-b 1a failed to change the primary outcome of time to disability worsening. positive effects were seen on relapse rate and reduction of mri activity, but the effect on disability did not replicate the european interferon-b 1b report. a combined analysis of the american and european trials concluded that continued relapse activity and more rapid progression over the preceding year (by >1 on the edss scale) are the best predictors of response. 92 ivig in secondary progressive ms. a recent european trial reported that ivig did not have a significant impact on clinical and disability related outcome measures. ivig did reduce the accumulation of brain atrophy in spms, but did not reduce the incidence of blood-brain barrier abnormalities. there was no statistically significant change on magnetization transfer mri measurements; however, a trend for conservation of normal-appearing brain tissue was found. 93 overall recommendations for spms. in general, as the evidence that interferons alter long-term disability is limited and controversial, we generally do not newly start spms patients on interferon products. in a subset of patients still having disabling relapses, interferon therapy may be offered to specifically reduce relapse rate. the data by confavreux and associates, however, suggest that the edss in populations of spms patients continues to progress independent of relapses 94 once a "fixed" baseline level of moderate disability has been reached. therefore, while interferons may reduce the relapse rate in spms, the rate of progression of disability may not be reduced by these treatments. more usually, spms patients are already on an injectable immunomodulator, and the question of whether it is worth continuing the therapy may come up, especially in patients who have a hard time tolerating these medications and feel that the side effects of the medications have a clear negative impact on their overall health. in these cases, we usually allow the patients to stop their medications. just like in the rrms cases, however, patient education about spms trials and realistic expectations about the treatment is a crucial element in the decision-making process. understanding the patient's needs and fears and clarifying potential misconceptions constitute a very important role of the treating neurologist. in those patients who continue their interferon therapy, we must continue to follow them for toxicity and disease activity. please also see the following discussion under mitoxantrone for recommendations on the potential use of that specific agent in spms. mitoxantrone. mitoxantrone (novantrone) is an anthracenedione chemotherapeutic agent licensed . . .for reducing neurologic disability and/or the frequency of clinical relapses in patients with secondary (chronic) progressive, progressive relapsing, or worsening relapsing-remitting multiple sclerosis (i.e. patients whose neurologic status is significantly abnormal between relapses). mitoxantrone is not indicated in treatment of patients with primary progressive multiple sclerosis. significant benefits were observed in a group of spms patients as in a european phase iii study of mitoxantrone. 95 it has also been used in combination with methylprednisolone. 96 several clinical and functional outcome measures were reported to stabilize or improve with every 3-month administration of this intravenous medication. secondary mri outcome measures, including enhancing lesion formation and overall t2 lesion load, were also better in the treated patients. the greatest concern regarding this medication is its cardiac toxicity: the cumulative lifetime maximum dose was established at 140 mg/m 2 . mitoxantrone can induce a seemingly dose-dependent cardiomyopathy, leading to potentially fatal congestive heart failure. we generally avoid exceeding a total lifetime dose of 96 mg/m 2 (8 doses of 12 mg/m 2 ). patients receiving mitoxantrone should also be monitored every 3 months with echocardiograms or muga (multiple gated acquisition) scans to determine the ejection fraction. reduction in the ejection fraction should prompt discontinuation of this therapy. besides the cardiac side effects, mitoxantrone may cause menstrual irregularities or overall ablation of the menstrual cycle, which may be permanent. in a review of the literature, ghalie 97 estimated the risk of mitoxantrone therapyrelated acute leukemia in ms patients at 0.05% to 0.1%; in an international registry of ms patients taking mitoxantrone, the risk of leukemia seems somewhat higher. this therapy has been approved by the fda for treatment of spms, but no peer-reviewed full report of the mims study had been published until 4 years after the initial report in an abstracts form. the study 98 showed a treatment effect in rrms and spms patients with "recent rapid worsening." the study had a high dropout rate and a small sample size. most patients (74%) had relapses in the preceding 2 years, suggesting this cohort mostly includes worsening rrms or prms patients, in whom a positive treatment effect is expected; however, it does not mean that for classic spms patients who no longer have relapses the study outcome is applicable, and it is especially not applicable to ppms cases. the primary outcome measure was a composite score comprised of five clinical measures: change in edss at 2 years; change in ambulation index at 2 years; change in the baseline standardized neurological status at 2 years; number of relapses requiring corticosteroid treatment; and time to first relapse. seventy-seven percent completed 24 months of follow-up. at 24 months, benefit was reported in all five components of the composite measure for both active treatment arms, with the overall greatest benefit noted between placebo and the group receiving mitoxantrone at a dose of 12 mg/m 2 . the magnitude of the effect on edss was rather modest (mean edss change for high-dose mitoxantrone, -0.13 [sd 0.90] versus ã¾0.23 [sd 1.01] in the placebo group). the mri results of the mims trial were published in 2005 and are frankly disappointing. 99 in a subset of 110 patients (out of 194 in the trial overall), the 12 mg/m 2 dose failed to reach a significant difference from placebo as measured by the primary mri outcome (total number of scans with gadoliniumenhancing lesions). the 12 mg/m 2 dose reduced the number of t2-weighted lesions at month 24 (p â¼ 0.027) and showed a trend at month 12 (p â¼ 0.069). the number of active mr lesions showed a trend toward reduction in the 12 mg/m 2 group only at month 24 (p â¼ 0.054). overall, the limited evidence to date supports the conclusion that mitoxantrone reduces relapse frequency and mri evidence for blood-brain barrier disruption in patients with very active ms. the benefit for patients with relapse-independent progression is uncertain at best. from the mims results, one would need to treat 11 patients with secondary progressive multiple sclerosis for 2 years to prevent one person from worsening by 1.0 edss point. this modest benefit must be carefully examined in light of the significant risk for toxicity. therapy of ppms. unfortunately, for classical ppms cases that present with insidious progression of usually myelopathy symptoms, none of the currently available treatments offer any clear benefit. the promise trial, in which over 900 ppms patients were treated with ga, was terminated early owing to lack of effectiveness. the results of this trial have not yet been published. a small study with intramuscular interferon-b 1a was also negative. 100 currently a large trial is under way with rituximab in csf ocb-positive ppms patients. until we clearly understand the pathophysiology of slow progression in ms, it is unlikely that we will find a treatment that has an important impact on this form of ms. symptomatic treatment modalities, including physical and occupational therapy, are very important, yet frequently overlooked in this patient population. other immunomodulator therapies. cyclophosphamide is an alkylating agent that has indiscriminate cytotoxic effects on rapidly dividing cells, including lymphocytes, making it a potent immunosuppressant. several studies have claimed a beneficial effect in both relapsing and progressive patients. because one of the major studies included acth, iv methylprednisolone is sometimes given with the cyclophosphamide. other trials have not found a favorable effect. because of the inconsistent results, high potential for serious side effects, and adverse reactions, including hemorrhagic cystitis and malignancy, cyclophosphamide is not widely used. some centers, however, use cyclophosphamide in patients with aggressive disease in whom more conventional treatments have failed. azathioprine, a purine analog antimetabolite, has marginal efficacy in the treatment of ms. a meta-analysis of all blinded, placebo-controlled studies confirmed a slight benefit of slowed progression and less frequent relapses. 101 the toxicity of azathioprine and its slow onset of action have prevented its widespread use. besides the liver toxicity and hematological effects, the induction of malignancies has been a concern. one retrospective study did not find an increased incidence of cancer in ms patients treated with azathioprine, but this remains a potential risk. methotrexate is a folate antagonist that is effective in rheumatoid arthritis. weekly low-dose oral methotrexate was found to delay upper extremity dysfunction in spms patients, although it had no effect on the more traditional measures of disability, including the expanded disability status scale (edss). 102 the use of cyclosporine in the treatment of ms has been evaluated in three clinical trials, none of which have demonstrated a convincing benefit. in addition, side effects such as hypertension and elevation of creatinine were common. numerous additional therapies have been tested, and many others are undergoing evaluation. the antiherpesvirus drug acyclovir has been shown to reduce relapse frequency in a small prospective trial. total lymphoid irradiation was found to slow the chronic progression of ms, but because this approach precludes the later initiation of immunosuppressant drugs and may be associated with a higher mortality rate, it is not widely used. cladribine is a nucleoside derivative that was found to decrease relapse rate and slow the progression in patients with spms in an initial investigation. 103 the drug is better tolerated than other parenteral immunosuppressants, although bone marrow suppression is a risk. in a more extensive clinical trial, 104 cladribine therapy did not change disability scores, but significant reduction in enhancing lesions and overall t2 lesion burden was observed with higher dose treatment. a study of a small number of patients treated with autologous stem cell transplantation 105 suggested possible clinical stabilization or minor improvement over a 15-month period of follow-up in both secondary and primary progressive ms. the induction chemotherapy (beam regimen) resulted in one fatality in this trial; similar incidences are known in patients undergoing this procedure. the small number of patients and the different methods used (some patients received cd34 ã¾ selected graft) makes the interpretation of this data very difficult. trials with higher number of patients under standardized circumstances are needed to verify the validity of these observations. symptomatic treatment of existing disabilities spasticity. spasticity is common even in patients with only minimal weakness (video 3, spastic gait). it is usually prudent to begin treatment of mild spasticity with a stretching program. a randomized controlled crossover trial 106 of physical therapy (8-week blocks of therapy twice a week, for 45 minutes per session) showed significant benefit on several outcome measures related to improved mobility. no apparent differences were observed between home-based or hospital-based therapy. the addition of an evening dose of benzodiazepine may help relieve extensor spasms and clonus that may interfere with sleep. as spasticity worsens, it becomes necessary to use baclofen. doses should be escalated slowly to prevent the occurrence of overt side effects, and up to 120 mg/day may be required. although baclofen is well tolerated in most patients, limiting side effects such as sedation and increased muscle weakness may occur, and rarely a paradoxical increase in spasticity is noted. liver enzyme elevation and nonconvulsive status epilepticus presenting as encephalopathy have also been reported in association with baclofen. abrupt withdrawal of baclofen may result in hallucinations or seizures, making it necessary to taper doses. despite symptomatic improvement, antispasticity measures may not increase function or independence. in paraplegic patients with severe spasticity and intolerance to the required oral dose, intrathecal baclofen delivered by a subcutaneously implanted pump allows a much smaller dose and is often effective in alleviating intractable spasticity and may lessen urinary urgency. tizanidine seems to be as effective as baclofen, although it may be associated with more fatigue. dantrolene has been used for spasticity, although the therapeutic window is small. fatigue. for treating fatigue, medications are only partially effective. amantadine at 100 mg twice a day is the standard initial treatment, although pemoline 37.5 mg daily is also superior to placebo. a recent, small pilot study by the mayo group suggested that high-dose aspirin (1300 mg/day) may sometimes be effective in the treatment of ms-related fatigue. 107 this finding needs to be confirmed by a second, larger trial, however. the stimulating effects of the selective serotonin reuptake inhibitors may also be somewhat effective in combating ms-related fatigue. modafinil, a medication approved for the treatment of narcolepsy, has also been used with good success. often, however, patients need to limit activities and schedule rest periods. paroxysmal symptoms of ms. paroxysmal symptoms are highly responsive to medical treatment. a small dose of carbamazepine is often very effective. if not tolerated, several alternative medications may be tried, including phenytoin, acetazolamide, baclofen, and gabapentin. in addition, misoprostol has been claimed to be effective in ms-related trigeminal neuralgia. after about 1 month of treatment, a periodic attempt at tapering off these medications is a reasonable approach because these symptoms usually remit. seizures in ms are treated no differently than in non-ms conditions. heat sensitivity. heat sensitivity may require avoidance of precipitating activities, but this depends on the nature of symptoms and the situation in which they occur. if the precipitating activity cannot be avoided, a cooling jacket may be an option. a potassium channel blocker, 4-aminopyridine, improves temperature sensitivity in some patients but occasionally causes seizures or disturbing paresthesias. action tremor. action tremor is a common disabling symptom (video 14, tremor with ataxia). unfortunately, it is often only marginally amenable to medical therapy. clonazepam may offer some relief, but tolerance frequently develops, necessitating increasing doses. isoniazid and carbamazepine have also been found marginally beneficial. one clinical trial showed ondansetron to reduce tremor-related disability. anecdotal reports suggest that gabapentin may be partially effective. improvements in stereotactic neurosurgery have made thalamotomy a legitimate option in those whose disability is mainly due to tremor and not ataxia. cognitive and memory problems. cognitive problems can also be seen in ms patients. these symptoms are generally not very severe; however, in some patients these may be one of their subjectively most bothersome complaints. it is important to make sure that such complaints are not depression related, as mood disorders are otherwise rather common in ms, and may explain the subjective cognitive impairment. while it is not fda approved for the treatment of ms related cognitive dysfunction, in a placebo controlled, randomized, 24-week long study of donepezil in 69 patients, significant improvement was found on the selective reminding test (srt). 108 this improvement was independent of ms subtype, gender, age, reading ability, and baseline srt results. the patients did not improve on other cognitive scales, but they were twice as likely to report cognitive improvement. dysesthetic pain. dysesthetic pains are difficult to control but sometimes respond to tricyclic antidepressants, carbamazepine, or baclofen. gabapentin, tramadol, and duloxetine may also be effective. standard analgesics are not often useful in ms-associated pain, and narcotics should be avoided in the treatment of chronic pain. emotional incontinence may be amenable to a low dose of a tricyclic antidepressant (video 10, dysarthria). symptoms of bladder dysfunction. symptoms of a hyper-reflexic bladder (urgency, frequency, and urge incontinence) are often manageable with anticholinergics such as oxybutynin, propantheline, or imipramine. a flaccid bladder can sometimes be aided by bethanechol, although intermittent self-catheterization is more often needed. symptoms that suggest urinary retention (a feeling of incomplete emptying, frequency, hesitancy, or a need to apply pressure to the lower abdomen to urinate) should prompt evaluation with urinalysis and a post-void residual urine measurement. residuals in excess of 15 ml are abnormal; and if they are above 50 ml, consideration should be given to urological consultation for more thorough investigation and to blood chemistries to determine urea and creatinine levels. detrusor-sphincter dyssynergia, diagnosed by cystometrography, is treated with anticholinergics, sometimes with the addition of an a-1 blocking agent (terazosin) or intermittent catheterization. it is important to reassess bladder function periodically, and residual urine volumes should be monitored if there are any persistent changes in function or symptoms. intermittent catheterization should be considered when post-void residuals reach 100 ml. a chronic indwelling urinary catheter should be avoided if reasonably possible. it is usually not necessary to use antibiotics prophylactically in the prevention of urinary tract infections. urinary calculi may be prevented by acidification of the urine with cranberry juice. bowel dysfunction in ms. constipation can usually be managed with bulk laxatives and stool softeners. more severe cases may require osmotic agents, bowel stimulants, anal stimulation, suppositories, or enemas. bedridden patients may develop fecal impaction unresponsive to these measures and require manual disimpaction. fecal incontinence can be minimized by adherence to a schedule for bowel movements. fiber supplementation may be of some benefit even in these cases. sexual dysfunction in ms. the clinician should determine the precise nature of any sexual dysfunction in patients with ms. physical difficulty from spasticity may be alleviated by premedication with baclofen, and a fast-acting anticholinergic such as oxybutynin may calm urinary urgency. sexual dysfunction should not be automatically attributed to ms. it may be necessary to investigate hormonal levels and to obtain urological or gynecological consultation. manual lubrication with gel is a ready solution to vaginal dryness. erectile dysfunction responds very well to sildenafil. less frequently, vacuum devices, intracavernous injections of papaverine (or combinations of papaverine, prostaglandins, and epinephrine; triple agent), or penile implant are also used in the treatment of erectile dysfunction. thalamotomy or thalamic stimulation may provide some short-term clinical benefit to patients disabled by appendicular cerebellar tremor and ataxia. the benefits on disability and quality of life are much less clear, however, and the early benefits may wain within 1 to 3 years. 109, 110 further studies are needed to clarify how best to select patients for these ablative and stimulation treatment interventions. general recommendations for ms patients. it is advisable for ms patients to attain good health habits, including proper diet and fitness. smoking, excess alcohol intake, and obesity should be avoided. exercise can help maximize function by increasing and maintaining joint mobility, strength, and stamina; may promote improved sleep hygiene; and may reduce the severity of fatigue. physical and occupational therapy can play an important role in regaining independence. canes, walkers, and wheelchairs or scooters may be needed to maintain safe mobility. hand controls can be installed in automobiles for patients with lower extremity dysfunction. in debilitated, immobilized patients, periodic shifts in posture to change weight-bearing regions and air or water mattresses prevent bedsores. passive range of motion exercises prevent contractures. when ventilatory dysfunction occurs, it should be evaluated and activity schedules should be appropriately modified. 111 special considerations during pregnancy. before initiation of any drug in a woman of reproductive age, the potential for teratogenicity must be discussed. in general, immunomodulator therapy should be avoided if one is planning a pregnancy. the treatment of acute exacerbations is unchanged during pregnancy, although one might have a higher threshold for treatment. both corticosteroids and plasma exchange are relatively safe during pregnancy. none of the drugs used to alter the disease course, however, should be used during pregnancy. interferon-b drugs should be stopped 2 to 3 months before planning pregnancy. interferons are associated with an approximately 39% likelihood of abortions. this is close to eight times higher than the usually quoted approximately 5% spontaneous abortion rate in women. interferon-b use is also associated with lower birth weight. 112 the cytotoxic immunosuppressants have teratogenic effects. the effects of many of the other drugs are unknown. it is best if these drugs are stopped several months before a planned pregnancy. prognosis and future perspectives. because most information on the prognosis of ms is reported in terms of the edss, it is important to have some understanding of this scale. the edss is a 10-point scale, with each increase representing worsening symptoms of function. the score is derived from severity scores in each of six systems as well as ambulation and work ability. a score of 0 means no signs or symptoms; 1 to 3 represent mild disability with no or minimal impairment of ambulation; 3.5 to 5.5 refer to moderate disability and impairment of gait; the need for a cane to walk one-half block (100 m) receives a score of 6; an edss of 8 refers to the need for a wheelchair and effective upper extremity function; an edss of 10 refers to death related to ms. ms has a highly variable outcome, ranging from asymptomatic to fulminant with death ensuing in a matter of months. autopsy series have estimated that unsuspected ms may occur in as many as 0.2% of the population. even when symptomatic, ms may cause only nuisance symptoms. benign ms, when defined as unrestricted ambulation or edss of 3 or less 10 years after onset, accounts for about one third of cases. however, many of these patients acquire more disability later. when considering all patients with ms, weinshenker found that 15 years after onset, 80% had edss worse than 3, 50% had reached edss of 6 or more, 10% were at edss 8, and 2% had died. the percentage of patients with initially rrms who develop spms increases steadily with disease duration. 113 at 10 years, 40% to 50% have continual deterioration; after 25 years, approximately 80% have slow progression. in the most recent study published about the olmsted county ms prevalence cohort consisting of 161 patients, the mean change in edss over 10 years was 1 point, and only 20% of patients had a change larger than 2.0 points. 114 eighty-three percent of the patients with mild symptoms (edss < 3.0) were still ambulatory without cane 10 years later. among the patients with an edss of 3 to 5, 51% were using a cane; in the 6 to 7 edds range, 51% were wheelchair bound. strong predictors for the outcome were not identified in this study. population-based studies with complete ascertainment can effectively remove the bias of a referral practice, which is inherently biased towards the more active and more serious cases. these studies also provide some much needed balance to the "heavily skewed for recent disease activity" clinical trial experience. from the most recent extensions to the olmsted county ms cohort studies conducted by the mayo group, several conclusions can be drawn. the number of relapses in the first year of the disease do not predict long term outcome. the time to disability is not influenced by ongoing relapses once patients achieve an apparently permanent degree of moderate disability (edss 3.5). overall, 80% patients who are still classified as rrms into their second decade of disease continue to do well for 15 to 20 years with limited permanent disability. patients doing extremely well (edss 2.0) after 10 years of ms generally do well in the next decade. however, very rarely patients doing well even for 2 to 3 decades may develop severe late disability. we advise that neurologists share these findings with patients who are in periods of prolonged remission during the discussions about the merits of beginning disease-modifying agents. natural history studies have identified several prognostic indicators that predict outcome to a limited extent. factors associated with a better prognosis (slower accumulation of disability, longer time before chronic progression) include young age at onset, female gender, rr course (as opposed to ppms), initial symptoms of sensory impairment or on, first manifestations affecting only one cns region, high degree of recovery from initial bout, longer interval between first and second relapses, low number of relapses in the first 2 years, and less disability at 5 years after onset (both edss and number of systems affected-sensory, motor, sphincter, brain stem, vision, cerebral). despite the indolent nature, a pp course is the worst prognostic factor, with the median time to reach edss 6 of only 6 years, compared with approximately 20 years in rr patients. men and patients with an older age at onset are more likely to have ppms. the survival of ms patients is only slightly below expected. seventy-six percent of patients are alive 25 years after onset, which is 85% of that seen in age-and sexmatched control subjects. 35 ms is rarely the direct cause of death. complications of ms such as pneumonia, pulmonary emboli, aspiration, urosepsis, and decubiti are responsible for 50% of deaths. most of the other deaths are from heart disease, cancer, cerebrovascular disease, and trauma. suicide is the only cause of death that is overrepresented among these cases. the suicide rate among ms patients may be as high as two to seven times that of non-ms persons. neuromyelitis optica (nmo) is an uncommon neurological illness characterized by the occurrence of optic neuritis and myelitis. the names devic's syndrome, devic's disease, and nmo are often used interchangeably, although the first name encompasses all patients who fit the preceding definition and the second and third should only be used to refer to those patients presumed to have a distinct disorder. the term opticospinal ms is often used in the far east to denote patients with exclusive or predominant involvement of optic nerves and spinal cord, encompassing most patients with devic's syndrome. devic's disease (nmo) may be a monophasic illness, or may show a relapsingremitting course. 115 it is the first inflammatory demyelinating disease with a known serum marker, the nmo-igg antibody. pathogenesis and pathophysiology. devic's syndrome may occur with adem, autoimmune disorders (e.g., systemic lupus erythematosus), ms, and possibly viral infections. also, patients with devic's disease may have other coexisting autoimmune conditions. classically, acute spinal cord lesions demonstrate diffuse swelling that extend over several levels or involve nearly the entire cross section of the cord. acutely, there is destruction with dense macrophage infiltration involving white and gray matter, loss of myelin and axons, and lymphocytic cuffing of vessels. in chronic lesions, the cord is atrophic and necrotic, occasionally with cystic degeneration and gliosis. in the absence of perivascular cuffing, these extensive lesions resemble infarctions. the prominent spinal cord swelling in the confines of the restrictive pia presumably may raise intramedullary pressure, leading to the collapse of small parenchymal vessels, further propagating tissue injury. proliferation of vessels with thickened and hyalinized walls similar to that seen after infarction or other extensive injury may occur. 116 less fulminant lesions may coexist and are much more typical of inflammatory demyelination. the optic nerve lesions often involve the chiasm. even though nmo is usually restricted to the optic nerves and spinal cord, one may see classic ms like lesions in up to 10% of cases, and hypothalamic lesions have also been described in approximately 10%. the newly discovered serum marker, nmo-igg has a sensitivity of 73% and specificity of 91%. 117 the discovery of this novel immune marker also clarified that most if not all cases of "opticospinal ms" reported in the japanese literature are also cases of nmo. to the surprise of the ms research community, the antigen is neither myelin nor neuron related: it is the aquaporin-4 water channel, a component of the dystroglycan protein complex located in astrocytic foot processes at the bloodbrain barrier. 11 nmo thus may represent the first example of a novel class of autoimmune channelopathies. epidemiology and risk factors. devic's syndrome occurs in patients of varied ages (range, 1 to 73 years). the mean age at onset of monophasic devic's syndrome is 27, whereas relapsing nmo (see later) tends to occur in an older age group (mean age at onset of 43). monophasic devic's syndrome affects males and females equally, whereas relapsing nmo affects females predominantly (f:m, 3.8:1). one third of patients have a preceding infection within a few weeks of neurological symptom onset. most commonly this is a nonspecific upper respiratory tract infection, flu, or gastroenteritis. the most common specific infections preceding the development of devic's syndrome are chickenpox and pulmonary tuberculosis. devic's syndrome has also followed vaccination for swine flu and mumps. only a few instances of a possible familial occurrence of devic's syndrome have been reported, and in one of these families, a unique mitochondrial mutation was found. devic's syndrome is said to be more common in japan and east asia, although even there it is uncommon (less than 5 per 100,000). three cases have been described in the literature with familial occurrence of devic's disease in the far east. in a genetic study, hla-dpb1*0501 was more frequently associated with "opticospinal ms," whereas hla-dpb1*0301 is the most strongly associated allele with conventional ms in the japanese. symptoms of on and myelitis develop over hours to days and are often preceded or accompanied by headache, nausea, somnolence, fever, or myalgias. continued progression of symptoms over weeks or months occasionally occurs. most patients (greater than 80%) develop bilateral optic neuritis. bitemporal or junctional field deficits, indicating chiasm involvement, are sometimes present early in the course of the on. visual loss is often accompanied by periocular pain, and myelitis onset is sometimes heralded by localized back or radicular pain. lhermitte's sign is common. severe degrees of neurological deficits are usual, and the degree of recovery is variable. approximately 35% of nmo patients have a monophasic illness, 55% develop relapses usually limited to the optic nerves and spinal cord (relapsing nmo or opticospinal ms), and rarely patients have a fulminantly progressive course without relapses or a course typical of ms. 115 according to a study conducted at the mayo clinic, 115 patients with a monophasic course usually presented with rapidly sequential events (median, 5 days) with only moderate recovery. patients showing characteristics of the relapsing form of devic's had a median interval of 166 days between index events, followed within 3 years by clusters of severe relapses isolated to the optic nerves and spinal cord. most relapsing patients developed severe disability in a stepwise manner. approximately one third died from respiratory failure. predictors of a relapsing course in nmo 118 include longer inter-attack intervals (relative risk [rr]: 2.16 per month increase), older age at onset (rr â¼ 1.08 per year increase), female sex (rr â¼ 10.0), and less severe motor impairment with sentinel myelitis event (rr â¼ 0.48 per severity scale point increase). autoimmune disease history (rr â¼ 4.15), higher attack frequency in first 2 years (rr â¼ 1.21 per attack), and better recovery following index myelitis (rr â¼ 1.84 per point) are associated with increased mortality rate. features of nmo distinct from "typical" ms included normal initial brain mri, more than 50 cells/ml in csf with polymorphonuclear predominance, and lesions extending over three or more vertebral segments on spinal cord mri. relapsing nmo is often associated with autoimmune disorders, most commonly systemic lupus erythematosus. these patients also frequently have an elevated erythrocyte sedimentation rate and nonspecific elevation of autoantibodies, including antinuclear antibodies, anti-ds-dna, and antiphospholipid antibodies. tonic spasms and neuropathic lower extremity pain are common sequelae to the spinal cord damage. symptoms referable to brain stem lesions (nystagmus, ophthalmoparesis, and vertigo) can occur in these patients as well. differential diagnosis. the differential diagnoses for devic's syndrome includes ms, adem, pulmonary tuberculosis, and viral infection (especially in the immunocompromised patient). in patients with an apparent affected family member, consideration should be given to mitochondrial disease. relapsing nmo should raise the suspicion for associated autoimmune disorders. because devic's syndrome can occur in persons older than age 60, when an unrelated ischemic optic neuropathy could occur, and because isolated or recurrent myelopathy may precede the on, additional consideration must be given to spinal cord compression, spinal cord tumor, and spinal arteriovenous malformation (avm) or dural fistula. evaluation. imaging is needed to exclude structural lesions and provide information on the pathological process. optic nerve or chiasm enlargement, t2-weighted signal changes, and enhancement may be seen on head mri during the acute phase. increased t2-weighted signal in the medulla is not uncommon and usually represents extension of high cervical lesions. spine mri characteristically shows cord swelling, signal changes, and enhancement extending over at least three levels ( fig. 48-4) . this appearance may resemble a spinal cord tumor, prompting consideration for biopsy. on magnetization transfer (mt) mri, no significant difference was found on normal-appearing white matter of devic's patients and control subjects, whereas ms patients had a significantly lower mt ratio peak and histogram average. 119 t1 hypointense lesions in the cord and linear lesions that cross over more than two segments are more suggestive of devic's disease. an occasional patient may need prone and supine myelography to exclude a spinal dural-based avm. laboratory investigations reveal an elevated erythrocyte sedimentation rate in one third, positive antinuclear antibodies in nearly one half, and occasionally other autoantibodies (e.g., thyroperoxidase antibodies). 39 it is reasonable to exclude syphilis, lyme disease, and human immunodeficiency virus by laboratory testing. in a few patients with the far east variety of devic's disease, hyperprolactinemia was described predominantly with optic nerve involvement. a chest x-ray helps to exclude pulmonary tuberculosis and sarcoidosis. csf examination is an essential part of the evaluation for devic's syndrome, and repeated studies are sometimes necessary to ensure that there is no infection in that the csf findings are sometimes atypical for inflammatory demyelination. a marked pleocytosis is often present, sometimes exceeding 100 cells. moreover, neutrophils are commonly seen in csf and may predominate, a situation virtually unknown in ms. 120 the protein concentration is often very high and in 41% exceeds 100 mg/dl. anti-mog antibodies are the predominant autoantibody detected in csf; anti-mbp or anti-s100b antibodies are less frequently seen. despite the intense inflammatory response, ocbs are conspicuously absent in the majority, being present in fewer than 20% of patients. csf serology for the herpesvirus family (hsv types 1 and 2, vzv, ebv, and cmv) is important, and polymerase chain reaction testing should be done in cases suggestive of viral infection (immunocompromised patients). management. patients with acute or subacute devic's syndrome may respond to corticosteroids (e.g., intravenous methylprednisolone). they may respond to plasma exchange even when intravenous methylprednisolone does not produce significant improvement. attempts at preventing relapses and the subsequent disability are often disappointing even with the use of immunosuppressive agents. the classic injectable immunomodulators used in ms are insufficient to reduce the relapse rate in relapsing nmo. most commonly, a combination of azathioprine and prednisone is used for secondary prevention. other agents including mycophenolate mofetil, ivig, and mitoxantrone have been described to be effective in some cases. a small study of rituximab, a humanized anti-cd20 antibody showed a siginifcant reduction in the relapse rate of 8 patients, making 6 of 8 relapse free. 121 a large multicenter study of rituximab in nmo is in the planning stages. supportive care is important in the management of nmo. these patients are prone to many complications and require measures to prevent deep venous thrombosis and pulmonary embolism, urinary tract infection, decubiti, and contractures. mechanical ventilation may be needed either temporarily or permanently. patients with monophasic devic's syndrome generally have simultaneous or rapid onset of the on and myelitis (interval usually less than 1 month). although some have significant residual disability, many recover remarkably and have little or no permanent deficits. a history of previous vague neurological symptoms or definite demyelinating events is predictive of future relapses, either typical of ms or relapsing nmo. those patients destined for recurrent myelitis and on have a longer interval between the onsets of myelitis and on. the vast majority of patients with relapsing nmo have very aggressive disease with frequent and severe exacerbations and a poor prognosis. adem is a monophasic inflammatory demyelinating disorder that characteristically begins within 6 weeks of an antigenic challenge such as infection or immunization. it occurs more often in the young and causes the rapid development of multifocal or focal neurological deficits. perivenous inflammation, edema, and demyelination are the pathological hallmarks of adem, although these lesions commonly enlarge and coalesce, forming lesions pathologically indistinguishable from ms. moreover, perivascular changes typical of adem are common in patients with ms. there is considerable overlap in the epidemiological, clinical, csf, imaging, and pathological features between adem and ms, often making it difficult to distinguish between the two with reasonable confidence when encountering patients with a single demyelinating event. pathogenesis and pathophysiology. adem closely resembles the experimental allergic encephalomyelitis animal model of ms (eae) both clinically and pathologically, and is most likely due to a transient autoimmune response toward myelin. the occurrence of adem after vaccination with the rabbit spinal cord preparation of rabies virus led to the discovery of eae. infections and non-cnscontaining vaccinations may induce adem by molecular mimicry or by activating autoreactive t-cell clones in a nonspecific manner. lymphocyte reactivity toward mbp has been identified in blood and csf from patients with adem, but its absence in others indicates a role for other antigens. increased peripheral blood g interferon-producing t cells have been described in adem. epidemiology and risk factors. adem can occur at any age but perhaps because of the higher frequency of immunization and exposure to new antigens; it is most common during childhood. unlike ms, both sexes are affected with equal frequency. no association has been noted with pregnancy. adem has been reported to follow a number of different immunizations, usually within 6 weeks, including those for pertussis, diphtheria, measles, mumps, rubella, influenza (postvaccination adem), tetanus, and yellow fever. in addition, there are case reports of adem following hepatitis b vaccination. however, the only epidemiologically and pathologically proved association is with rabies vaccination, which also causes demyelinating peripheral neuropathies. the original pasteur rabies vaccine, prepared in rabbit spinal cord, was associated with an incidence of adem of approximately 1 per 3000 to 1 per 35,000 vaccinations and is no longer in use. a later vaccine, made in duck embryo, which contains little neural tissue, carries a risk for adem of 1 per 25,000 vaccines. the use of human diploid cell lines, which contain no nervous system tissue, for the production of rabies vaccine has virtually eliminated the risk of adem. the association of bee stings with adem has also been reported. parainfectious adem usually follows onset of the infectious illness, often during the recovery phase, but because of the latency between pathogen exposure and illness it may precede clinical symptoms of infection or the two may occur simultaneously. the most commonly reported associated illness is a nonspecific upper respiratory tract infection. there have been a vast number of specific infections associated with adem, such as virus infections (including rubella, mumps, vzv, ebv, cmv, influenza, coxsackievirus, and hepatitis c) and infection with mycoplasma, borrelia burgdorferi, and leptospira. measles carries the highest risk for adem of any infection, occurring in 1 per 400 to 1 per 1000 cases. although adem has been reported in association with measles immunization, the risk is far lower than the risk of acquiring measles and its neurological complications. clinical features and associated disor-ders. a prodrome of headache, low-grade fever, myalgias, and malaise often precedes the onset of adem by a few days. in a german study of 40 cases, 122 the most frequent clinical signs were motor deficit (80%), followed by sensory deficits, brain stem signs, and cerebellar signs. csf findings were variable; normal results were present in up to 20% of patients. oligoclonal bands were positive in over 60%. almost all patients improved during the acute phase of the disease. of the 26 patients with the final diagnosis of adem, 21 had minor or no symptoms, 2 died, the rest had moderate symptoms. compared to ms patients, the adem patients were older, and more often had a preceding infection, clinical signs of brain stem involvement, a higher csf albumin fraction, and infratentorial lesions. neurological symptoms develop rapidly in the acute phase and are commonly associated with encephalopathy, stupor, coma, meningismus, and seizures. peak severity occurs within several days, and recovery may begin soon afterward. occasionally, adem may evolve over a few months and there may be a second clinical deterioration or subacute progression for a time. in these unusual cases, the distinction from ms is difficult. three recent large retrospective series and an accompanying editorial have highlighted that there remain no clinical or laboratory features that accurately allow one to predict which adult or pediatric adem patients will develop. [122] [123] [124] [125] differential diagnosis. one of the primary concerns after a single demyelinating episode is whether other bouts can be expected (e.g., ms). several features may tip the balance toward one or the other, but the proper diagnosis becomes apparent only with time. classically, adem is characterized by the multifocal involvement at onset whereas ms often presents with monosymptomatic deficits such as on. however, adem may cause unifocal symptoms and ms may present with multifocal cns involvement, especially in children. the monosymptomatic deficits caused by adem are more commonly severe, such as bilateral on and complete transverse myelitis. although ocbs occur transiently in about one third of adem cases, their persistence implies a diagnosis of ms. the subsequent disappearance of ocbs, when performed by consistent techniques, is evidence against ms. the mri appearance of these two disorders is often identical, 125 but the presence of basal ganglia or cortical lesions, or large globular white matter lesions, is more frequent in adem. the fulminant development of adem is distinctive but not pathognomonic, because a rare form of ms known as marburg's ms is also rapid in onset and often deadly. the appearance of brain stem, periventricular, and multiple, large cerebral white matter lesions and the presence of ocbs may distinguish marburg's variant from adem. on rare occasions, inflammatory demyelinating lesions may reach a large size and resemble tumors (especially lymphoma) on mri, necessitating biopsy for clarification. there is usually one dominant lesion, but smaller separate lesions may be identifiable. these have been referred to as both adem and ms in the literature. the prognosis for recovery is often quite good, although approximately one third suffer subsequent attacks. some develop typical ms, whereas others have recurring tumor-like lesions. the term multiphasic adem has been used when patients have large recurrences in the same location, and relapsing adem refers to recurrences at different sites. the relationship of these entities with ms is unclear. balo's concentric sclerosis refers to the pathological finding of alternating bands of demyelination and remyelination. these patients typically have large lesions and subacute deficits similar to those described earlier. typical demyelinating lesions commonly coexist, and rarely cdms patients are noted to have similar-appearing lesions. the reason for this peculiar alternating pattern is unknown. schilder's myelinoclastic diffuse sclerosis is another rare condition that may be confused with adem or other demyelinating conditions. this progressive demyelinating disorder usually begins in childhood. the features are often atypical and include dementia, aphasia, homonymous hemianopia, seizures, psychosis, elevated intracranial pressure, and the absence of ocbs. the most characteristic finding is the presence of two large, roughly symmetrical lesions on mri, one in each hemisphere. the diagnosis is made by excluding the known inherited leukodystrophies, especially adrenoleukodystrophy. management. treatment with intravenous methylprednisolone seems to halt progression and allow recovery to begin sooner, just as with ms. plasma exchange can be tried in those with severe deficits and little response to corticosteroids. ivig has also been used successfully according to case reports in the literature. one fulminant case responded to hypothermia only. genetics of multiple sclerosis utility of mri in suspected ms the use of mitoxantrone (novantrone) for the treatment of multiple sclerosis disease modifying therapies in multiple sclerosis natural history of multiple sclerosis a randomized study of two interferon-beta treatments in relapsing-remitting multiple sclerosis cortical lesions and brain atrophy in ms the pathology of multiple sclerosis management of multiple sclerosis: current trials and future options competing interests in multiple sclerosis research diagnostic criteria for multiple sclerosis: 2005 revisions to the "mcdonald criteria immunization and multiple sclerosis: a summary of published evidence and recommendations revised diagnostic criteria for neuromyelitisi optica neuromyelitis optica randomized controlled trials to assess therapies for multiple sclerosis does the history of multiple sclerosis go back as far as the 14th century? the 150-year anniversary of multiple sclerosis: does its early history give an etiological clue? encephalomyelitis accompanied by myelin destruction experimentally produced in monkeys morphological aspects of myelin and myelination t-cell subsets in multiple sclerosis: a comparative study between cell surface antigens and function molecular mimicry in t cellmediated autoimmunity: viral peptides activate human t cell clones specific for myelin basic protein t cells responsive to myelin basic protein in patients with multiple sclerosis the cd4-th1 model for multiple sclerosis: a critical experimental allergic encephalomyelitis: a misleading model of multiple sclerosis igg marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel isolation of herpes simplex virus type 1 during first attack of multiple sclerosis isolation of virus from the spinal fluid of three patients with multiple sclerosis and one with amyotrophic lateral sclerosis plaque-associated expression of human herpesvirus 6 in multiple sclerosis oligodendrocytes and oligodendrocyte/type-2 astrocyte progenitor cells of adult rats are specifically susceptible to the lytic effects of complement in absence of antibody experimental autoimmune encephalomyelitis: immunotherapy with anti-tumor necrosis factor antibodies and soluble tumor necrosis factor receptors evidence for pathogenic heterogeneity in multiple sclerosis transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions cortical demyelination and diffuse white matter injury in multiple sclerosis a genetic basis for familial aggregation in multiple sclerosis. canadian collaborative study group genetics of multiple sclerosis multiple sclerosis: changing times epidemiology of multiple sclerosis in u.s. veterans: v. ancestry and the risk of multiple sclerosis unusual occurrence on a tropical island of multiple sclerosis when is an apparent excess of neurologic cases epidemiologically significant smoking is a risk factor for multiple sclerosis environmental risk factors in multiple sclerosis: causes, triggers, and patient autonomy pregnancy is associated with a lower risk of onset and a better prognosis in multiple sclerosis diagnosis of ms the natural history of recurrent optic neuritis the prevalence of cognitive impairment in a community survey of multiple sclerosis defining the clinical course of multiple sclerosis: results of an international survey. national multiple sclerosis society (usa) advisory committee on clinical trials of new agents in multiple sclerosis epilepsy and multiple sclerosis multiple sclerosis-associated uveitis a reappraisal of the epidemiology of multiple sclerosis in olmsted county optic neuritis: a population-based study in olmsted county the significance of brain magnetic resonance imaging abnormalities at presentation with clinically isolated syndromes suggestive of multiple sclerosis. a 5-year follow-up study interscanner variation in brain mri lesion load measurements in ms: implications for clinical trials the prognostic value of brain mri in clinically isolated syndromes of the cns. a 10-year follow-up a longitudinal study of abnormalities on mri and disability from multiple sclerosis occurrence of a multiple sclerosis-like illness in women who have a leber's hereditary optic neuropathy mitochondrial dna mutation genetic disorders that masquerade as multiple sclerosis new diagnostic criteria for multiple sclerosis: guidelines for research protocols recommended diagnostic criteria for multiple sclerosis: guidelines from the international panel on the diagnosis of multiple sclerosis assessment of mri criteria for a diagnosis of ms neuroimaging in multiple sclerosis the relationship between whole brain volume and disability in multiple sclerosis: a comparison of normalized gray vs. white matter with misclassification correction magnetization transfer ratio histogram analysis of normal-appearing gray matter and normalappearing white matter in multiple sclerosis mri t2 hypointensity of the dentate nucleus is related to ambulatory impairment in multiple sclerosis evidence of early cortical atrophy in ms: relevance to white matter changes and disability csf electrophoresis in one thousand patients early differential diagnosis of multiple sclerosis using a new oligoclonal band test diagnostic criteria for multiple sclerosis: 2005 revisions to the "mcdonald criteria the optic neuritis treatment trial: three-year follow-up results effects of iv methylprednisolone on brain atrophy in relapsing-remitting ms double-blind study of true vs. sham plasma exchange in patients treated with immunosuppression for acute attacks of multiple sclerosis plasmapheresis in acute episodes of fulminant cns inflammatory demyelination a randomized trial of plasma exchange in acute central nervous system inflammatory demyelinating disease relation between humoral pathological changes in multiple sclerosis and response to therapeutic plasma exchange plasma exchange for severe attacks of cns demyelination: predictors of response intramuscular interferon beta-1a therapy initiated during a first demyelinating event in multiple sclerosis. champs study group interferon beta-1a for early multiple sclerosis: champs trial subgroup analyses effect of early interferon treatment on conversion to definite multiple sclerosis: a randomised study interferon beta-1b is effective in relapsing-remitting multiple sclerosis. i. clinical results of a multicenter, randomized, double-blind, placebo-controlled trial. the ifnb multiple sclerosis study group further study on the specificity and incidence of neutralizing antibodies to interferon (ifn) in relapsing remitting multiple sclerosis patients treated with ifn beta-1a or ifn beta-1b intramuscular interferon beta-1a for disease progression in relapsing multiple sclerosis. the multiple sclerosis collaborative research group (mscrg) the long-term safety and tolerability of high-dose interferon beta-1a in relapsing-remitting multiple sclerosis: 4-year data from the prisms study randomised double-blind placebo-controlled study of interferon beta-1a in relapsing/remitting multiple sclerosis. prisms (prevention of relapses and disability by interferon beta-1a subcutaneously in multiple sclerosis) study group magnetic resonance imaging results of the prisms trial: a randomized, double-blind, placebo-controlled study of interferon-beta1a in relapsing-remitting multiple sclerosis. prevention of relapses and disability by interferon-beta1a subcutaneously in multiple sclerosis ingested ifn-alpha: results of a pilot study in relapsing-remitting ms clinical characteristics of responders to interferon therapy for relapsing ms a pilot trial of cop 1 in exacerbating-remitting multiple sclerosis copolymer 1 reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase iii multicenter, double-blind placebocontrolled trial. the copolymer 1 multiple sclerosis study group glatiramer acetate reduces the proportion of new ms lesions evolving into "black holes european/canadian multicenter, double-blind, randomized, placebo-controlled study of the effects of glatiramer acetate on magnetic resonance imaging-measured disease activity and burden in patients with relapsing multiple sclerosis. european/canadian glatiramer acetate study group united states open-label glatiramer acetate extension trial for relapsing multiple sclerosis: mri and clinical correlates. multiple sclerosis study group and the mri analysis center longitudinal mri study: the effects of azathioprine in ms patients refractory to interferon beta-1b the austrian immunoglobulin in ms (aims) study: final analysis a randomized trial of intravenous immunoglobulin in inflammatory demyelinating optic neuritis iv immunoglobulin does not reverse established weakness in ms placebo controlled pilot trial to study the remyelinating potential of intravenous immunoglobulins in multiple sclerosis a double-blind, randomized trial of iv immunoglobulin treatment in acute optic neuritis a controlled trial of natalizumab for relapsing multiple sclerosis progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta-1a for multiple sclerosis t(1) hypointense lesions in secondary progressive multiple sclerosis: effect of interferon beta-1b treatment placebo-controlled multicentre randomised trial of interferon beta-1b in treatment of secondary progressive multiple sclerosis. european study group on interferon beta-1b in secondary progressive ms effect of interferon-beta-1b on magnetic resonance imaging outcomes in secondary progressive multiple sclerosis: results of a european multicenter, randomized, double-blind, placebo-controlled trial. european study group on interferon-beta-1b in secondary progressive multiple sclerosis final analysis of the european multicenter trial on ifnbeta-1b in secondary-progressive ms the effect of interferon beta-1b treatment on mri measures of cerebral atrophy in secondary progressive multiple sclerosis. european study group on interferon beta-1b in secondary progressive multiple sclerosis randomized controlled trial of interferonbeta-1a in secondary progressive ms: mri results interferon beta-1b in secondary progressive ms: a combined analysis of the two trials european study on intravenous immunoglobulin in multiple sclerosis: results of magnetization transfer magnetic resonance imaging analysis relapses and progression of disability in multiple sclerosis mitoxantrone in progressive ms: a placebo controlled, randomized, observer blinded phase iii multi-center study: clinical results therapeutic effect of mitoxantrone combined with methylprednisolone in multiple sclerosis: a randomised multicentre study of active disease using mri and clinical criteria a study of therapy-related acute leukaemia after mitoxantrone therapy for multiple sclerosis mitoxantrone in progressive multiple sclerosis: a placebo-controlled, double-blind, randomised, multicentre trial effect of mitoxantrone on mri in progressive ms: results of the mims trial interferon beta-1a in primary progressive multiple sclerosis overview of azathioprine treatment in multiple sclerosis lowdose (7.5 mg) oral methotrexate reduces the rate of progression in chronic progressive multiple sclerosis cladribine in treatment of chronic progressive multiple sclerosis cladribine and progressive ms: clinical and mri outcomes of a multicenter controlled trial. cladribine mri study group autologous stem cell transplantation in progressive multiple sclerosis-an interim analysis of efficacy controlled randomised crossover trial of the effects of physiotherapy on mobility in chronic multiple sclerosis a randomized controlled crossover trial of aspirin for fatigue in multiple sclerosis donepezil improved memory in multiple sclerosis in a randomized clinical trial stereotactic lesional surgery for the treatment of tremor in multiple sclerosis: a prospective case-controlled study a study of tremor in multiple sclerosis ventilatory dysfunction in multiple sclerosis the reproductive effects of beta interferon therapy in pregnancy: a longitudinal cohort the natural history of multiple sclerosis: a geographically based study. i. clinical course and disability change in ms-related disability in a population-based cohort: a 10-year follow-up study the clinical course of neuromyelitis optica (devic's syndrome) devic's neuromyelitis optica: a clinicopathological study of 8 patients a serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis neuromyelitis optica: clinical predictors of a relapsing course and survival mri and magnetization transfer imaging changes in the brain and cervical cord of patients with devic's neuromyelitis optica clinical, csf, and mri findings in devic's neuromyelitis optica neuromyelitis optica acute disseminated encephalomyelitis: a follow-up study of 40 adult patients adem: distinct disease or part of the ms spectrum clinical and neuroradiologic features of acute disseminated encephalomyelitis in children acute disseminated encephalomyelitis. mri findings and the distinction from multiple sclerosis interactive study questions and referenced videos for this chapter are available on the dvd-rom key: cord-353242-9vy8k6du authors: dhaiban, sarah; al-ani, mena; elemam, noha mousaad; maghazachi, azzam a title: targeting chemokines and chemokine receptors in multiple sclerosis and experimental autoimmune encephalomyelitis date: 2020-09-29 journal: j inflamm res doi: 10.2147/jir.s270872 sha: doc_id: 353242 cord_uid: 9vy8k6du multiple sclerosis (ms) is an immune-mediated and neurodegenerative disorder that results in inflammation and demyelination of the central nervous system (cns). ms symptoms include walking difficulties, visual weakening, as well as learning and memory impairment, thus affecting the quality of the patient’s life. chemokines and chemokine receptors are expressed on the immune cells as well as the cns resident cells. several sets of chemokine receptors and their ligands tend to be pathogenic players in ms, including ccl2, ccl3, ccl4, ccl5, ccl7, ccl8, ccl11, ccl17, ccl19, ccl21, ccl22, cxcl1, cxcl8, cxcl9, cxcl10, cxcl11, and cxcl16. furthermore, current modulatory drugs that are used in the treatment of ms and its animal model, the experimental autoimmune encephalomyelitis (eae), affect the expression of several chemokine and chemokine receptors. in this review, we highlight the pathogenic roles of chemokines and their receptors as well as utilizing them as potential therapeutic targets through selective agents, such as specific antibodies and receptor blockers, or indirectly through ms or eae immunomodulatory drugs. multiple sclerosis (ms) is an immune-mediated and neurodegenerative disease, causing demyelination of the central nervous system (cns) characterized by formation of separated areas of inflammation called ms lesions. 1 there are four key pathological characteristics of ms: (a) inflammation, that is proposed to be the essential cause of most of the events of cns tissue damage; (b) demyelination, the trademark of ms, in which the myelin sheath or the oligodendrocyte cell body is damaged through the inflammatory process; (c) axonal loss or impairment; and (d) gliosis (astrocytic response to cns damage). 1,2 ms is a long-lasting disease that can influence the brain, spinal cord, and the optic nerves. 3 it can cause impairment in vision, balance control, muscle control, and other essential body functions. this could be attributed to the damage of the myelin sheath, the protective layer of the nerve fibers, which adversely affects the communication between the brain and the rest of the body as the message or information conveyed through the nerves may arrive slower or may never arrive. eventually, the nerves themselves may get damaged. over time, the brain may shrink in size because axons are destroyed. 4 symptoms of ms are different, depending on the affected nerves and the severity of the symptoms. in difficult cases, patients with ms lose the ability to walk or talk. 4 most individuals with ms have attacks when the condition gets recognizably worse (relapse), followed by recovery times when symptoms improve (remission). in certain individuals, the illness may progress into secondary stage. 4 there are four main subtypes for ms. the most common subtype is relapsing-remitting ms (rrms), accounting for about 85% of ms patients. it is characterized by periods of recurring symptoms that are followed by remission phase, during which symptoms could partially or totally disappear. 5 the second type is secondary progressive ms (spms) which starts with an initial relapse phase, and that could progress gradually leading to neurological worsening. 5 the third type is primary progressive ms (ppms) characterized by a continuous disease deterioration and no specific relapse phase. 5 finally, the fourth subtype is progressive relapsing ms (prms). in prms, there is a constant deterioration from the initial stage of the disease followed by a relapse phase. 5, 6 a mouse model for ms has been well established which is known as the experimental autoimmune encephalomyelitis (eae). this model was extensively used to discover new therapies for ms. in fact, most, if not all experimental ms drugs must be examined in this mouse model before advancing any of these new drugs into clinical trials. 7 in this review, we will briefly discuss the chemokines and their receptors in ms and eae, as well as the impact of current ms and eae modulatory drugs on the chemokine system. chemokines and their receptors have many roles in immune regulation, whether in physiological or pathological conditions. 8 chemokines are classified either according to the cysteine residues of the ligands (cc, cxc, c, and cx3c), or their function and expression. 9 cc chemokines compose the biggest family containing two adjacent cysteine residues near their n-terminus, while their respective genes are clustered on chromosome 17 in humans. in cxc and cx 3 c chemokine subfamilies, there are one or three additional amino acids between the first two cysteine residues, respectively. most of the cxc chemokines are grouped on chromosome 4 in humans. 9 the xc chemokine is characterized by the absence of one of the first two n-terminal cysteine residues. 10 chemokines are crucial mediators in the recruitment and migration of immune cells to the inflammatory sites. 11 some chemokines can bind to several chemokine receptors, whereas certain chemokine receptors bind to more than one chemokine ligand. chemokines are involved in autoimmune diseases, which include rheumatoid arthritis (ra), graves' disease (gd), systemic lupus erythematosus (sle), and ms, among others. 12, 13 hence, chemokine receptors represent novel targets for treating autoimmune diseases. table 1 demonstrates the recent understanding of the 4 chemokine subfamilies and their receptors (for reviews, please see). [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] role of chemokines in the pathophysiology of ms chemokines and chemokine receptors are expressed in different brain areas not only by immune cells but also by neurons and cns resident cells such as astrocytes and oligodendrocytes. chemokines and their receptors are increasingly expressed in ms patients. 30 the chemokines/chemokine receptors axis expressed in t cells have the capability of recruiting inflammatory cells into cns, which may lead to its destruction. 26, 30 different chemokines play pathogenic roles in ms. these include ccl2, ccl3, ccl4, ccl5, ccl7, ccl8, ccl11, ccl17, ccl19, ccl21, ccl22, cxcl1, cxcl8, cxcl9, cxcl10, cxcl11, and cxcl16. [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] targeting of chemokines and chemokine receptors involved in ms or eae targeting ccr1/ccl3 axis ccr1 and its chemokine ligands are implicated in chronic inflammatory disease. 44 ccl3 (macrophage inflammatory protein-1α, mip-1α) and ccl5 (regulated on activation, normal t cell expressed and secreted, rantes) are the primary ccr1 ligands. other chemokine ligands for ccr1 include ccl6, ccl9, ccl15, and ccl23. 45, 46 one study demonstrated that ccr1 is implicated in the pathogenesis of eae using the ccr1 antagonist bx 471 which showed strong therapeutic effects in eae animals. 47 ccl3 was identified as a macrophage-derived inflammatory mediator 48 and has been shown to be implicated in the pathophysiology of ms. 30 for instance, treatment of eae -secreted by several hematopoietic and non-hematopoietic cells. -induces inflammatory cells recruitment, wound healing, maintaining effector immune response and inhibition of stem cells. -activates cells responsible for bone resorption and bone destruction. -induces nk cell activities. ackr2 ccl4 -mainly secreted by macrophages. -acts as chemoattractant for macrophages, monocytes, nk cells, immature dcs, and coronary endothelial cells. -activates nk cells. ccr5 ackr2 -expressed by t cells, macrophages, synovial fibroblasts, platelets, tubular epithelium, and certain tumor cells. -triggers recruitment of leukocytes into inflammatory sites. -stimulates the activation and proliferation of specific nk cells to produce cc chemokine-activated killer cells. -ccl5 produced by cd8 + t cells inhibits hiv entry into target cells. ccr3 ccr5 ackr2 -produced by macrophages, keratinocytes, fibroblasts, skeletal muscle, vascular smooth muscle cells. -acts as chemotactic for macrophages, monocytes, and t cells. -functions as a cell survival factor. -important for the recruitment of myeloid progenitors to intestinal tumors and enhancement of invasion. -produced by endothelial cells, epithelial cells, eosinophils, fibroblasts, and keratinocytes. -stimulates the migration of macrophages, th2 cells, neutrophils, mast cells, basophils, eosinophils, and neural progenitor cells. -highly expressed at sites of brain injury and is correlated with neurodegeneration and aging. ccr3 ccr5 ccl12 -m1 marker as highly expressed by macrophages and is associated with monocyte-derived macrophage and fibroblast recruitment. -inhibits wound healing. -acts on macrophages, monocytes, and thp-1 cells. -involved in several diseases, including allergic airway inflammation and certain cancers. -activates nk cells. ackr2 ccl18 -secreted primarily by myeloid cells, m2 macrophages, alveolar macrophages, tolerogenic dcs, and keratinocytes. -highly expressed in skin lesions. -induces trafficking of human peripheral blood th2 cells and treg cells. -acts as an antagonist by binding to ccr1-5, leading to inhibition of cellular recruitment and chemotaxis. -inhibits dc apoptosis. ccr7 ackr4 ccrl2 ccl20 -produced by psoriatic keratinocytes and endothelial cells. -induces immune cells' migration. -crucial in mucosal immune surveillance. -inducing the migration of dcs and t cells. -implicated in leukocyte infiltration into tissues such as the cns. -activates nk cells. ccl22 -produced by the thymus and myeloid cells, m2 macrophages, and monocyte-derived dcs. -acts as a chemoattractant for nk cells, monocytes, dcs and antigen-experienced t lymphocytes. -stimulates interactions between treg cells and dcs in the lymph nodes. ackr2 ccl23 -recruits resting t lymphocytes, dcs, and monocytes. -inhibits proliferation of myeloid progenitor cells and promotes angiogenesis. -acts as chemoattractant for osteoclast to promote bone formation. ccl24 -promotes immune cell trafficking and activation. -involved in inflammation and fibrosis of the lung and skin. -acts as chemoattractant for antigen-specific t lymphocytes. -expressed in the brain and upregulated in atopic dermatitis. -participates in a variety of cellular processes, such as dna synthesis, glycolysis, and camp accumulation. -acts as an antimicrobial protein. mice with ccl3 antibodies suppressed eae and prevented the mononuclear cells from accumulating in the cns. 49 however, treatment with met-rantes, the ccr1 and ccr5 antagonist, did not affect leukocyte migration and decreased the severity of chronic-relapsing eae mildly. 50 targeting ccr6/ccl20 axis the binding of ccl20 chemokine ligand to ccr6 has been shown to induce homeostatic and inflammatory functions. elevated ccr6 and ccl20 expression levels were observed in the spinal cord and lymph nodes of eae mice and were associated with the severity of the disease. 51 additionally, ccl20 binding ccr6 induced the trafficking of t helper 17 (th17) cells into the cns through the choroid plexus in the brain of eae mice. 52 ccr6-deficient mice or mice that were treated with a neutralizing anti-ccr6 showed resistance to eae development. 51 also, elevated ccl20 serum levels were found in ms patients. 53 targeting ccrl2/ccl19 axis cc chemokine receptor-like 2 (ccrl2), also known as cram, is a member of the atypical chemokine receptor family and thus is a non-classical seven transmembrane spanning domain receptor. the ligand for ccrl2 is the homeostatic chemokine ccl19. 54 ccrl2 is found in most immune cells such as dendritic cells (dcs), macrophages, neutrophils, lymphocytes, natural killer (nk) cells, and mast cells. 55, 56 it has been shown that ccrl2 is overexpressed in mouse microglia and astrocytes as well as being expressed by infiltrating macrophages in eae. 57, 58 the knockout of ccrl2 in eae mice resulted in defective acquisition of m2 phenotype markers by infiltrating macrophages and altered the m1/m2 ratio. this was responsible for the sustained t cell perivascular infiltration and inflammatory reaction resulting in delayed eae recovery. 59 targeting cxcr3/cxcl10 axis -expressed in the lung as well as other mucosal and endocrine organs. -induces neutrophils recruitment during inflammation. not defined cxcl16 -produced by macrophages and dcs. -regulates immune cells chemotaxis, and induces endothelial cells proliferation, migration, and angiogenesis. cxcr6 cxcl17 -myeloid cell-attracting chemokine. -produced by the airways at normal and inflammatory conditions. possibly gpr35 cx 3 cl1 -expressed by immune and non-immune cells. -induces migration and adhesion of macrophages, nk cells, and t cells. -implicated in multiple inflammatory diseases. expression of cxcr3 on peripheral blood cd4 + lymphocytes correlated with ms relapses. 45 additionally, cxcr3 + t cells levels are increased in rrms patients during the ms attacks. 45 the chemokine cxcl10 is a chemoattractant for activated t cells and nk cells through binding to cxcr3. 62 cxcl10 levels in the cns have been associated with eae development and the recruitment of t cells expressing cxcr3 receptor into the cns. 63 in sjl mice treated with a neutralizing antibody against cxcl10, less accumulation of mononuclear inflammatory cells was observed along with a decreased incidence of eae indicating that cxcl10 could contribute to cns pathology via the recruitment and accumulation of t cells. 63 furthermore, upon elisa analysis of the supernatant fluid collected from the cerebrospinal fluid (csf) of 43 ms patients, elevated levels of cxcl10 were correlated with relapse phase of ms. hence, cxcl10 or its receptor cxcr3 may be therapeutic targets for ms. 64 in contrast, a study in c57bl/6j mice deficient in cxcr3 and cxcl10, found that these mice developed a severe myelin oligodendrocyte glycoprotein (mog)-induced eae disease. 65 targeting cxcl12/cxcr4/cxcr7 axis cxcl12 is continuously expressed in the healthy cns where it is produced mainly by glial cells and neurons. in addition, its receptors cxcr4 and cxcr7 are abundantly expressed in diverse brain area. 66 cxcl12 expression is upregulated in the ms lesions especially in astrocytes, that are likely to attract macrophages and lymphocytes into the cns inflamed areas. 67 cxcr4 is expressed on t and nk cells and has an important role in the homeostasis of the immune system. 68 a study by meiron et al 69 demonstrated that the use of cxcr4 antagonist amd3100, blocked cxcl12-induced production of il-10 completely, thus interfering with the selection of the il-10 producing regulatory t (treg) cells. these results suggest that cxcl12 acts as a regulatory chemokine that redirects the polarization of effector th1 cells into treg cells which in turn suppress the autoimmune responses through il-10 secretion. 69 moreover, treatment of c57bl/6 mice with the amd3100 during eae induction led to increased deteriorating of the disease along with parenchymal infiltration and demyelination. 70 along these findings, another study demonstrated that cxcl12 is up-regulated in spinal cord tissue after injection of mog (which is the glycoprotein responsible for myelination of the nerves), in eae-resistant albino oxford rats. treatment of these rats with cxcl12 antagonist amd3100 rendered them susceptible to eae, indicating that cxcl12 is able to suppress inflammatory responses within the cns. 71 on the other hand, cxcr7 is a non-classical chemokine receptor and was proposed to have more than five natural ligands, although research focused on cxcl11 and cxcl12. 72 it was observed that the main function of cxcr7 is to sequester cxcl12, thus regulating the function of cxcr4. 72 treatment of eae mice with nsc-87877 a shp-2 inhibitor induced the expression of cxcr7 which resulted in decreased t cell trafficking in response to cxcl12. 73 this finding suggests that cxcr7, present on cd8 + t cells, is upregulated as a result of shp-2 inhibition and is negatively regulated in these cells. 73 it is worth mentioning that the initiation of treatment of nsc-87877 at day 0 after immunization blocked the incidence of eae effectively, while that starting from day 13 did not. these observations suggest that nsc-87877 treatment does not change the encephalitogenic response of peripheral t cells but affects their ability to migrate into the cns and cause inflammation. 73 it has also been demonstrated that cxcl12 inhibits the trafficking of infiltrating cells from the perivascular space into the brain parenchyma, thereby limiting inflammation. 74 a study reported loss of cxcl12 from abluminal surfaces of the blood-brain barrier in ms lesions. 74 therefore, treatment with a cxcr7 antagonist ccx771 prevented cxcr7 from sequestering cxcl12, resulting in elevated abluminal levels of cxcl12 and reduced leukocyte infiltration, which ameliorate the severity of eae. 75 targeting cxcl13/cxcr5 axis cxcl13 is a homeostatic chemokine that is continuously expressed in lymphoid tissues, which has a vital role in activating the migration of lymphocytes and antigenpresenting cells. 76, 77 upon binding of cxcl13 to cxcr5, cns inflammation developed. 78 in ms patients, the levels of cxcl13 were found to be significantly increased and consequently, cxcl13 is considered a biomarker for the disease severity. 79, 80 blocking cxcl13 binding to its receptor results in inhibition of b, t follicular helper (tfh), and th17 cell migration and subsequent development of tissue inflammation. the use of anti-human cxcl13 antibody, mab 5261, inhibited cxcl13-induced migration of b cells into secondary lymphoid organs resulting in halting the progression and severity of th17-induced eae in sjl mice. 81 targeting cxcl16/cxcr6 axis the cxc chemokine ligand cxcl16 is the ligand for cxc chemokine receptor 6 (cxcr6). cxcl16 in a soluble form acts as a chemoattractant for activated cd8 + t cells, nkt cells and th1 cells that express cxcr6. 82 cxcl16 is highly expressed in the brain during ms. 83 serum levels of cxcl16 reflect disease activity in ms, suggesting that cxcl16 could be a potential marker of ms disease. 43 moreover, animals treated with anti-cxcl16 antibodies were found to be resistant to the induction of eae. 84 currently, several drugs are used for the treatment of ms or eae, these include: 1) steroids such as methylprednisolone (mp); 2) disease-modifying agents such as glatiramer acetate (ga), 4-methylumbelliferone (4mu) and interferon-beta (ifn-β); 3) immunosuppressants such as mitoxantrone and cladribine; and 4) monoclonal antibodies such as natalizumab and rituximab. [85] [86] [87] [88] some of these modulatory agents were found to have regulatory effects on the chemokines and chemokine receptor levels in ms patients or eae models. 26, 30 in the following section, we focus on the effects of these agents on modifying the expression of chemokines and chemokine receptors. methylprednisolone (mp) is a glucocorticosteroid drug that has been used for the treatment of ms patients. it is an anti-inflammatory drug that inhibits the activation of t cells and their trafficking into the cns while reducing the inflammatory cytokine levels. 89, 90 treatment of ms patients in the active phase with mp decreased the migratory ability of cd4 + ccr5 + t cells. 91 additionally, the level of cxcl10 was reduced in the sera of ms patients after intravenous administration of mp. 92 moreover, the application of mp in vitro and in vivo increased the chemotaxis of monocytes towards ccl2, ccl5, and cx 3 cl1 in ms patients compared to healthy controls. this boost was independent of chemokine receptor levels, suggesting that mp induces monocytes polarization towards the anti-inflammatory form and enhances their trafficking into inflamed cns, where they plausibly suppress the pathogenic immune responses. 93 additionally, a study by sellebjerg et al 94 found that cxcl13 concentrations in csf of ms patients were reduced after treatment with high-dose mp and natalizumab. on the other hand, cxcr1 and cxcr2, which are up-regulated in ms patients, showed a further increase in their expression upon mp treatment. 95 effect of glatiramer acetate (ga) on chemokines/chemokine receptors and nk cell cytolytic activity glatiramer acetate (ga), is an immunomodulating drug used for the treatment of ms patients. 96 this drug increased the levels of the chemokine receptor ccr7 and decreased the levels of ccr5, cxcr3 and cxcr6, on t cells. 31 further, ga activates in vitro human nk cell lysis of dcs. 97 similarly, this drug stimulates nk cell killing of dcs in ga-treated ms patients. 98 in mice, ga reduces the eae clinical score corroborated with enhancing nk cell cytolysis of dcs, hence impeding antigen presentation to autoreactive t cells. 99 ifn-β was the first agent that was approved for treatment of relapsing-remitting ms. 100 ifn-β-1b therapy reduced the rate of clinical exacerbations and the number of lesions in brain magnetic resonance imaging (mri), and it has been established as an effective drug for treatment of rrms patients. 101 treatment with ifn-β reduced the expression of ccr5 on t cells of ms patients, whereas treatment of t cells with ifn-β in vitro, inhibited their expression of this chemokine receptor as well its ligands ccl5 and ccl3. 102 furthermore, the expression of cxcr3 on cd4 + and cd8 + t cells was considerably decreased in rrms patients after treatment with ifn-β. 103 however, ifn-β induced a transient strong increase of cxcl10 level in ms patients. 104 moreover, it was found that in vitro treatment of peripheral blood mononuclear cells with ifn-β-1b lowered the production of cxcl8, cxcl9, cxcl10, ccl2, and ccl7 chemokines. 105 effect of 4-methylumbelliferone on chemokines and chemokine receptors 4-methylumbelliferone (4-mu) is a drug that hinders the synthesis of hyaluronan, which is an extracellular glycosaminoglycan that is present in the pericellular and extracellular matrix of vertebrate tissues and is implicated in autoimmunity. in ms patients, hyaluronan has proinflammatory roles in the cns, as it accumulates in demyelinated ms lesions, inducing antigen presentation by antigen-presenting cells leading to t cell activation and proliferation. 106 4-mu inhibits hyaluronan production in vitro and in vivo. 107, 108 in eae, this drug reduces the polarization of th1 phenotype and increases foxp3 + treg cells that are correlated with the suppression of eae. moreover, 4-mu inhibits the reactive response of astrocytes, immunocompetent resident cells of the cns, and prevents activated t cells from migrating into the cns. 85 additionally, spinal cxcl12 protein levels were raised in non-inflamed cns tissues and in inflamed cns tissues of lipopolysaccharide (lps)-injected mice after the oral 4mu administration. feeding eae mice with 4mu induced protective effects against eae as the disease clinical scores were considerably lower in 4mu-fed mice compared to untreated eae mice. 106 mitoxantrone (novantrone) is an anticancer drug that inhibits the dna topoisomerase ii activity which results in preventing the dna synthesis process, and inhibiting cellular proliferation. 109 this drug was approved for the treatment of rrms, spms, and prms, where it acts as an immunosuppressant inhibiting immune cells proliferation, their antigen presentation and inflammatory cytokines secretion. 110 a study by scott et al 111 showed that intravenous treatment of ms patients with mitoxantrone reduced the relapse rate and delayed the progression of the disease. it has been demonstrated that therapy of ms patients with mitoxantrone resulted in decreased cxcr1 and cxcr2 expression on peripheral blood mononuclear cells, suggesting a pathogenic role for these chemokine receptors in ms that can be inhibited by this drug. 95 another study indicated that in vitro treatment of primary astrocytes with mitoxantrone, inhibited lps-induced production of ccl2 and other inflammatory molecules. 112 on the other hand, it was observed that the chemokine receptor ccr2 expression was significantly upregulated on monocytes of two out of eight mitoxantrone treated patients who had active inflammation when compared to placebo patients who showed no significant changes in the expression of this chemokine receptor. 113 cladribine (2-chlorodeoxyadenosine) is a deoxyadenosine analogue pro-drug that selectively depletes lymphocytes such as b and t cells. it was identified as an anticancer drug for the treatment of b and t cell lymphoid malignancies, but was later used for the treatment of rrms due to its immunosuppressive activity for these immune cells that play a significant role in ms pathogenesis. 114 regarding its effects on the chemokine system of ms patients, it was demonstrated that treatment of rrms patients with cladribine resulted in declined levels of cxcl8 in their csf, and a significant decrease in ccl5 levels in the csf and serum of those patients. 115 effects of natalizumab on chemokines and chemokine receptors natalizumab (tysabri) is a humanized monoclonal antibody that selectively binds to α4 integrins expressed on the surface of human leukocytes. 116 the drug is approved in north america for relapsing ms and in europe for the treatment of rrms. 117 studies in relapsing ms patients indicated that natalizumab markedly reduced the relapse rate, and the accumulation of new brain mri lesions of these patients. 118, 119 in another study, natalizumab suppressed the development of new gadolinium-enhanced lesions in patients with relapsing ms and reduced the rate of brain volume loss and enhanced brain tissue integrity in rrms. 120 it was also demonstrated that rrms patients receiving natalizumab therapy, had significantly reduced levels of plasma pro-inflammatory cytokines and chemokines. after natalizumab treatment, there was a significant decline in the levels of chemokines correlated with th1 chemokines such as cxcl9, cxcl10, and cxcl11, or th2 chemokine ccl22 in the csf, in addition to the expected decline of the proinflammatory cytokines. 121 rituximab is a chimeric monoclonal antibody that binds to the protein cd20 and depletes b cells from the circulation. 122 this drug was initially used to treat lymphomas but currently is being used for the treatment of certain autoimmune diseases. 123 the effect of rituximab results from its regulatory effect on the cell cycle, including apoptosis stimulation, complement-dependent b cell lysis, and antibody-dependent cell-mediated cytotoxicity. 124 recent studies showed that rituximab not only affects b cells but has another possible mechanism of action against cd20 + t cells in ms patients, 125 or in eae mice. 88 several studies indicated the high efficacy of rituximab in relapsing-remitting ms patients. other studies have showed the efficacy of rituximab in treating progressive ms patients. 126,127 treatment of ms patients with rituximab affects chemokine levels since infusion therapy resulted in a significant decrease of cxcl13 and ccl19 in those patients. 128, 129 further, the levels of cxcl8 and cxcl10 chemokines decreased significantly after treatment with rituximab. 130 figure 1 the effects of modulating chemokines and chemokine receptors involved in ms or eae. several studies investigated the roles of chemokines and chemokine receptors on the pathogenesis of multiple sclerosis (ms) and experimental autoimmune encephalomyelitis (eae) through targeting them directly (pink boxes), with specific agents acting as antagonists, receptors blockers, or antibodies. moreover, some current ms or eae drugs could indirectly modulate the expression of these chemokines and chemokine receptors (blue boxes). abbreviations: eae, experimental autoimmune encephalomyelitis; ms, multiple sclerosis. several chemokines and chemokine receptors have been involved in the pathophysiology of ms, some of which have been targeted directly (table 2) or modulated indirectly by immunomodulatory agents and drugs ( figure 1 ). this targeting strategy demonstrates promising future approaches in the treatment of ms and eae. however, due to the promiscuity and the multiple functions of the chemokine system, targeting chemokines and their receptors with selective agents such as antibodies or receptor blockers could present a challenge and might not be effective. 131, 132 on the other hand, the general chemokine blockage might affect the host defense dramatically. 133 moreover, chemotaxis results obtained from studies on eae mouse model may not be fully applicable for ms disease in humans, as sometimes 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authors' laboratory is supported by a grant from terry fox foundation number misc051. the authors report no conflicts of interest in this work. the journal of inflammation research is an international, peerreviewed open-access journal that welcomes laboratory and clinical findings on the molecular basis, cell biology and pharmacology of inflammation including original research, reviews, symposium reports, hypothesis formation and commentaries on: acute/chronic inflammation; mediators of inflammation; cellular processes; molecular mechanisms; pharmacology and novel anti-inflammatory drugs; clinical conditions involving inflammation. the manuscript management system is completely online and includes a very quick and fair peerreview system. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.submit your manuscript here: https://www.dovepress.com/journal-of-inflammation-research-journal key: cord-325624-6anybxnk authors: ireland, derek d. c.; stohlman, stephen a.; hinton, david r.; kapil, parul; silverman, robert h.; atkinson, roscoe a.; bergmann, cornelia c. title: rnase l mediated protection from virus induced demyelination date: 2009-10-02 journal: plos pathog doi: 10.1371/journal.ppat.1000602 sha: doc_id: 325624 cord_uid: 6anybxnk ifn-α/β plays a critical role in limiting viral spread, restricting viral tropism and protecting mice from neurotropic coronavirus infection. however, the ifn-α/β dependent mechanisms underlying innate anti-viral functions within the cns are poorly understood. the role of rnase l in viral encephalomyelitis was explored based on its functions in inhibiting translation, inducing apoptosis, and propagating the ifn-α/β pathway through rna degradation intermediates. infection of rnase l deficient (rl(−/−)) mice with a sub-lethal, demyelinating mouse hepatitis virus variant revealed that the majority of mice succumbed to infection by day 12 p.i. however, rnase l deficiency did not affect overall control of infectious virus, or diminish ifn-α/β expression in the cns. furthermore, increased morbidity and mortality could not be attributed to altered proinflammatory signals or composition of cells infiltrating the cns. the unique phenotype of infected rl(−/−) mice was rather manifested in earlier onset and increased severity of demyelination and axonal damage in brain stem and spinal cord without evidence for enhanced neuronal infection. increased tissue damage coincided with sustained brain stem infection, foci of microglia infection in grey matter, and increased apoptotic cells. these data demonstrate a novel protective role for rnase l in viral induced cns encephalomyelitis, which is not reflected in overall viral control or propagation of ifn-α/β mediated signals. protective function is rather associated with cell type specific and regional restriction of viral replication in grey matter and ameliorated neurodegeneration and demyelination. type i interferon (ifn-a/b) induced anti-viral responses are critical for the initial control of most virus infections. although anti-viral responses are initiated in infected cells, they act in an autocrine and paracrine fashion, antagonizing virus replication in host cells and inducing a protective anti-viral state in neighboring cells. the anti-viral state is distinguished by the expression of numerous ifn stimulated genes (isg), which directly or indirectly interfere with both viral and host rna transcription and translation [1] . many of these pathways can also result in the apoptosis of infected cells [2] . nevertheless, the relative contributions of these various anti-viral effector mechanisms to overall virus control differ with the virus as well as the tissue and cell type infected. the best characterized anti-viral mechanisms are mediated by activation of double-stranded rna-dependent protein kinase (pkr) and the 29-59-oligoadenylatesynthase (oas)/ribonuclease l (rnase l) pathways [3] [4] [5] . upon recognition of double-stranded rna, the oas family of proteins convert atp into unique, unstable 29-59-a oligomers, which are bound by latent rnase l monomers, driving the dimerization and activation of rnase l. activated rnase l cleaves single-stranded rna, 39 of u-a and u-u sites found in both viral and cellular rna. rnase l endoribonuclease activity is thus not limited to viral rna, but also degrades host cell rna, including ribosomal rna. as oas genes are stimulated by ifn-a/b, elevated oas levels enhance the antiviral state in adjacent, non-infected cells responding to ifn-a/b. nevertheless, inappropriate activation of rnase l is counterbalanced by the unstable nature of 29-59a oligomers and the endogenous rnase l inhibitor, rl1 [6] . a novel aspect of the oas/rnase l pathway is the cleavage of host rna releasing free 39-monophosphates that can activate the cytoplasmic rna helicases rig-i and mda-5 [7] . recognition by these pattern recognition receptors initiates ifn-b transcription, similar to the activation mediated by recognition of specific viral rna structures. rnase l thus not only exerts anti-viral effects via rna degradation and apoptosis, but also has the capacity to amplify and prolong expression of anti-viral genes and other isgs. the anti-viral activity of the oas/rnase l system has been studied in several virus infections in vitro and in vivo [3, 4] . protective mechanisms of the oas/rnase l pathway are generally more evident for rna relative to dna virus infections. however, the contribution of rnase l to disease severity and viral control varies significantly depending on the virus and even virus strain studied, presumably reflecting the diverse mediators and targets of these anti-viral mediators. for example, encephalomyocarditis virus (emcv) replication was only modestly increased in rnase l deficient (rl 2/2 ) mouse embryonic fibroblasts, consistent with enhanced susceptibility to infection in vivo. although rnase l prolonged survival, mortality rates of infected rl 2/2 and control mice were similar. furthermore, ifn-b treatment increased the survival time of infected wild-type (wt) and to a lesser extent rl 2/2 mice, indicating that alternate ifn-dependent anti-viral mechanisms act in the absence of rnase l [8] . in contrast, the requirement for rnase l activity to protect from another picornavirus, coxsackievirus b4, was evident by significantly increased mortality rates of infected rl 2/2 compared to wt mice. however, increased mortality was not the result of uncontrolled virus replication, as virus titers were only slightly elevated within pancreatic islet cells in vivo. by contrast, pancreatic islet cells derived from rl 2/2 mice, treated with ifn-a and infected in vitro exhibited increased virus replication and loss of cellular integrity, compared to ifn-a treated rnase l sufficient cells [9] . these results revealed distinct differences in direct antiviral functions of rnase l in vitro and in vivo. cell type specific effects of anti-viral rnase l activity are also clearly evident from studies with west nile virus (wnv). while mouse embryonic fibroblasts display rnase l dependent anti-viral activity [10] , ifn-b treatment revealed no affect of rnase l in reducing wnv replication in either cortical or peripheral motor neurons [11] . nevertheless, a modest effect was observed in bone marrow derived macrophages, consistent with increased viral burden in cd11b + splenocytes derived from rl 2/2 compared to wt mice [11] . furthermore, increased mortality of rl 2/2 mice in response to peripheral wnv infection correlated with increased replication in lymphoid tissue, but not enhanced viral dissemination into the cns. a minor role for rnase l in control of wnv in the cns was supported by similar viral titers in brains as well as kinetics and rates of mortality following intracranial infection of rl 2/2 and wt mice. although viral replication was only transiently increased in spinal cords of intracranially infected rl 2/2 mice, the most prominent effect was increased spread to the spleen and liver [11] . the more critical role of rnase l in limiting wnv spread in peripheral tissues rather than the cns highlights the complexities of oas/rnase l mediated anti-viral mechanisms in vivo. ifn-a/b mediated innate responses are also essential to control virus replication and survival following coronavirus infections [12] [13] [14] [15] . infections in vitro and in vivo induce upregulation of ifn-a/b and anti-viral mediators, including pkr and oas [12, 16, 17] ; however, the participation of these pathways in the anti-viral response to cns infection has not been elucidated. in vitro, the dual liver and neurotropic mhv-a59 strain does not elicit degradation of 18s and 28s ribosomal rna, suggesting the absence of rnase l activation in hela cells [18] . however, administration of an rnase l agonist inhibited replication of the hepatotropic mhv-3 strain in peritoneal macrophages in vitro and in liver in vivo [19] . nevertheless, reduced viral replication via rnase l function in vivo was insufficient to prevent liver necrosis and death. the nonlethal gliotropic jhm strain of mouse hepatitis virus (mhv-jhm) replicates exclusively in the cns following intracranial infection and is controlled by both innate and adaptive responses [20, 21] . cns infection results in a non-lethal encephalitis with immune-mediated demyelination resulting in hind limb paralysis [21] . based on expanded tropism to hippocampal neurons and widespread dissemination within the cns of mice deficient in ifn-a/b signalling (ifnar 2/2 ) [12] , the gliotropic mhv-jhm variant was used to probe an anti-viral role of rnase l within the cns. while the vast majority of wt mice survived, rl 2/2 mice succumbed to infection, albeit delayed compared to ifnar 2/2 mice. although enhanced morbidity suggested a prominent role of rnase l in anti-viral protection, the absence of rnase l neither diminished overall virus control in the cns, nor enhanced neuronal infection. furthermore, rl 2/2 mice did not reveal any impairment in ifn-a/b induction, alterations in proinflammatory cytokines or inflammation compared to wt mice. these data rather reveal a novel role of rnase l in specifically counteracting focal microglia/macrophage infection, thus potentially sustaining microglia mediated neuroprotective effects and ameliorating demyelination. mice deficient in ifn-a/b signalling succumb to an otherwise sub-lethal gliotropic mhv-jhm within 8 days of infection despite functional cd8 t cell responses [12] . uncontrolled viral replication in the absence of ifn-a/b implicated a crucial role of anti-viral innate mechanisms in stemming viral spread within the cns. to elucidate the role of rnase l in cns pathogenesis, gliotropic mhv-jhm infection was examined in rl 2/2 mice [8] . increased susceptibility of rl 2/2 mice was evident by more severe clinical symptoms of acute encephalitis at 9-11 days post infection (p.i.) (fig. 1, top panel) . with the exception of increased severity, onset and progression of clinical symptoms in infected rl 2/2 were similar to those exhibited by infected wt mice. these included initial symptoms of acute viral encephalitis, including: lethargy, dehydration and weight loss, which progressed to diminished hind limb function. furthermore, mortality rates were significantly higher in rl 2/2 mice, with over 90% succumbing to infection by 12 days p.i. (fig. 1, middle panel) , approximately 3-4 days delayed relative to ifnar 2/2 mice [12] . assessment of direct anti-viral functions of rnase l [9, 11] revealed no initial spread of viruses is controlled by type i interferon induced antiviral molecules. early intervention with viral replication is especially critical in central nervous system infections to reduce loss of non-renewable cells and mitigate immune pathology. one of the best characterized anti-viral mechanisms is mediated by ribonuclease l (rnase l). rnase l exerts activity at multiple levels, including degradation of viral and host rna, induction of apoptosis, and propagation of the ifn-a/b pathway. recent studies suggest that rnase l antiviral activity is dependent on the virus, as well as the cell type and tissue infected. this study demonstrates that rnase l protects mice infected with a sub-lethal, demyelinating neurotropic coronavirus by ameliorating encephalitis and morbidity, albeit without affecting control of infectious virus or ifn-a/ b expression. rnase l specifically protected the brain stem from sustained infection and prevented spread of virus to microglia/macrophages located in spinal cord grey matter. the subtle regional alteration in tropism in the absence of rnase l coincided with increased apoptotic cells and earlier onset as well as increased severity of axonal damage and demyelination. the results demonstrate how subtle regional alterations in viral tropism within the cns may severely affect the balance between neuroprotection and neurotoxicity mediated by microglia/macrophages. significant changes of infectious virus in brains of rl 2/2 , compared to wt mice. virus titers were increased less than 0.5 log 10 in rl 2/2 mice (fig. 1, bottom panel) . very modest anti-viral rnase l activity in the cns was confirmed by analysis of viral rna from cns tissue. levels of viral mrna encoding the viral nucleocapsid protein were increased less than 2.4-fold in spinal cords of rl 2/2 compared to wt mice at any time point (table 1) . as liver may constitute an extraneural infection site in immunocompromised mice, viral replication in this tissue was also assessed by real-time pcr. the abundant viral mrna encoding the nucleocapsid protein was detected in liver at very low levels compared to the cns in both groups, albeit only at early time points (table 1) . although viral rna was elevated in livers of rl 2/2 mice at day 7 p.i., clearance by day 10 p.i. suggested hepatitis did not contribute to mortality. necrotic foci were not evident in either mouse group by gross examination. a minor role of rnase l in viral control in vivo was reminiscent of the previously described picornavirus models [8, 9, 11] . in addition to rna degradation, rnase l also induces a multitude of genes in the ifn-a/b pathways during infections with sendai virus and emcv [7] . thus, both self and virus small rna products released by rnase l activate cytoplasmic rna helicases rig-i and mda-5 to optimize and perpetuate anti-viral responses. however, ifn-a and ifn-b expression levels were slightly increased, rather than decreased in brains of mhv-jhm infected rl 2/2 mice relative to wt mice (fig. 2) . furthermore, oas-2, ifit-1, ifit-2, and irf-7, representing prominently activated isgs, were induced to similar, or modestly increased levels in the cns of infected rl 2/2 mice. slightly enhanced ifn-a/b and isg expression was also observed in spinal cords from infected rl 2/2 relative to wt mice (data not shown). consistent with similar virus replication and control in the cns, neither ifn-a/b nor the expression of downstream isgs were impaired at the tissue level. these data suggest that a positive rnase l-dependent feedback loop in propagating ifn-a/b signalling is not activated during mhv-jhm infection in the cns. an unanticipated feature of rl 2/2 mice is delayed tissue rejection in transplant studies [22] , suggesting a role of rnase l in modifying antigen presentation, lymphocyte trafficking or function. to assure that altered pathogenesis was not due to differential inflammatory responses, the cns was examined for extent, composition and localization of infiltrating cells. characterization of cells recruited to the cns using flow cytometry revealed similar total numbers of infiltrating cells and no alterations in their composition (fig. 3 ). unlike ifnar 2/2 mice [12] , neutrophil infiltration was not affected in rl 2/2 mice and macrophages comprised the most prominent population early p.i. in both strains. cd4 + and cd8 + t cells peaked to nearly identical numbers at day 7 p.i. in both groups. furthermore, cns infiltrating cd8 + t cells in both groups were comprised of 50% and 60% virus specific cells at day 7 and 10 pi, respectively, as monitored by d b /s510 class i tetramer staining [23] . similarly, no significant differences in the extent of inflammatory cells were observed by histochemical analysis (data not shown). these data confirm that priming and trafficking of virus specific t cells was unaffected by the loss of rnase l activity, consistent with effective clearance of infectious virus. rnase l did not overtly affect overall cns viral replication; however, it may act in a tissue or cell type specific manner [11] . mhv-jhm initially infects ependymal cells and spreads to astrocytes, microglia/macrophages and predominantly oligodendrocytes, but rarely infects neurons [24] . however, mhv-jhm induced mortality in the absence of ifn-a/b signalling is associated with a dramatically expanded virus tropism to hippocampal neurons [12] . to assess the possibility that rnase l exerts cell type dependent anti-viral effects not apparent from whole tissue homogenates, the distribution of virus infected cells was analysed by immunohistochemistry. sequential analysis demonstrated a limited infection of neurons within the brain with a predominance in the brain stem at day 5 p.i. in both rl 2/2 and wt mice (fig. 4) . glia tropism prevailed in both groups and overall distribution of virus infected cells was similar with occasional neurons still infected at day 7 p.i. (data not shown). by day 10 p.i. virus infected cells declined in cortex and brain stem of both wt and rl 2/2 mice. however, in contrast to wt mice which had cleared virus from brain stem, virus infected cells were sustained in the brain stem of rl 2/2 mice and were predominantly microglial in appearance (fig. 4) . the overall extent and distribution of viral infected cells was thus similar during peak virus replication in brains of rl 2/2 and wt mice, with the exception of sustained infection in brain stem in the absence of rnase l. preliminary experiments indicate that these cells are microglia or macrophage/ monocytes (see below). these data contrast with the infection of ifnar 2/2 mice [12] which show predominant neuronal infection. therefore, preferential infection of neurons could not account for the increased mortality in rl 2/2 mice. the data also indicated that protective rnase l functions are not overtly reflected in early anti-viral activity but are manifested in region specific protection. rnase l activation leads to apoptosis and elimination of infected cells, a process requiring activated caspase 3 [3, 25] . furthermore, ifna/b mediated activation of rnase l also induces apoptosis in non-infected cells [8] . during mhv-jhm infection of wt mice the majority of apoptotic cells are lymphocytes [26] , which are not targets of infection. although oligodendrocyte apoptosis has been linked to the demyelinating process, the demonstration of apoptotic oligodendrocytes during mhv-jhm infection has been elusive [27, 28] . the retention of virus infected cells in brain stem may result in enhanced local t cell activation and apoptosis of either t cells, infected cells, or both. therefore, the frequency of apoptotic cells was examined at day 10 p.i. in contrast to the brain stems of wt mice which contained few apoptotic cells, apoptotic cells were prominent in brain stems of rl 2/2 mice (fig. 4 ) coincident with retention of virus infected cells. although increased focal virus infection associated with substantially increased apoptotic cells presumably dysregulates neuronal function and potentiates tissue damage, there was no evidence that apoptotic cells are preferentially located adjacent to neurons. sustained brain stem infection and apoptosis in rl 2/2 mice coincided with increased morbidity and mortality, despite similar overall control of viral replication. the morbidity in mhv-jhm infected rl 2/2 mice also coincided temporally with the onset of acute primary demyelination in wt mice. rl 2/2 mice were thus examined for enhanced tissue pathology and demyelination. brain stems of infected wt mice revealed minimal demyelination by day 10 p.i. (fig. 5 ). by contrast, demyelination was more severe in brain stems of rl 2/2 mice, correlating with increased numbers of virus infected as well as apoptotic cells. demyelination is associated with a variable degree of axonal damage [29, 30] . primary immune mediated demyelination in the spinal cord during mhv-jhm infection coincides with early axonal degeneration [31, 32] . while axons were largely intact in brain stems of wt mice, axonal integrity within demyelinated lesions was severely compromised in rl 2/2 mice as indicated by a striking decrease in neurofilament and an increase in dystrophic axons within lesions (fig. 5) . in spinal cords of wt mice, demyelination was also minimal at day 7 p.i. and increased by day 10 p.i. (fig. 6) , when control of virus replication is clearly evident (table 1) . by contrast, large focal areas of severe demyelination were already apparent in rl 2/2 mice at day 7 p.i., resembling those in wt mice at day 10 p.i. in addition to the accelerated kinetics of demyelination, the severity of myelin loss was more pronounced in the absence of rnase l at both days 7 and 10 p.i. consistent with the more rapid loss of myelin in infected rl 2/2 mice, quantification of demyelinated areas at day 7 p.i. revealed 1.260.8% demyelination in spinal cords of wt mice and 3.061.7% in rl 2/2 mice. increased demyelination in both brain stem and spinal cord are thus a hallmark of infected rl 2/2 mice, irrespective of overall viral control. apoptotic cell numbers and axonal damage in the spinal cords of rl 2/2 mice were also evaluated to determine an association with increased demyelination. in wt mice, apoptotic cells were undetectable in spinal cord at day 7 p.i. (data not shown), but were prominent in white matter by day 10 p.i. (fig. 7) . by contrast, spinal cords from rl 2/2 mice already exhibited small areas of apoptotic cells at 7 days p.i., albeit only in white matter. however, by day 10 p.i. numerous apoptotic cells were evident in both white matter and grey matter of spinal cords from infected rl 2/2 mice. furthermore, in contrast to brain stems, apoptotic cells were often detected in close proximity to neurons (inset fig. 7) ; however, there . rnase l does not alter the extent of neuronal infection but delays viral clearance from brain stem. viral nucleocapsid antigen detected by immunoperoxidase staining using mab j.3.3 in brains from infected wt and rl 2/2 mice at days 5 and 10 p.i. (top panels: brown chromogen; hematoxylin counterstain). note neuronal infection (arrows) and similar foci of viral antigen in glial cells in brain stem at day 5 p.i. by day 10 p.i. infection is controlled in wt mice, but foci of infected non neuronal cells with glia morphology are sustained in rl 2/2 mice. scale bar = 100 mm. immunohistochemical staining for activated caspase-3 (bottom panels) at day 10 p.i. indicates increased numbers of cells undergoing apoptosis in brain stem of rl 2/2 relative to wt mice. scale bar = 100 mm. doi:10.1371/journal.ppat.1000602.g004 was no evidence for neuronal apoptosis. these data suggest that the absence, rather than presence, of rnase l contributes to enhanced apoptosis in white matter, and subsequently also in grey matter. analysis of axonal integrity revealed that all axons were intact in spinal cords of wt mice consistent with minimal demyelination at day 7 p.i. (fig. 7) . by contrast, a striking decrease in neurofilament and an increase in dystrophic axons within demyelinated lesions demonstrated axonal integrity was already compromised in white matter of rl 2/2 mice by day 7 p.i. at day 10 p.i. the amount of axonal degeneration in wt mice resembled that of rl 2/2 mice at day 7 p.i., consistent with the severity of demyelination. however, by day 10 p.i. spinal cords of rl 2/2 mice showed marked axonal loss located in areas of myelin loss (fig. 7) , similar to results in brain stem. the increased pathological changes in both brain stem and spinal cord thus correlated with the high mortality rates of rl 2/2 mice starting at day 9 p.i. the inability to directly link increased axonal damage with enhanced neuronal infection remains puzzling and supports dysregulation of oligodendrocyte function and/or neuroprotective functions of microglia as contributing factors to the severe pathological phenotype and clinical outcome. increased demyelination in spinal cords has been correlated with the extent of white and grey matter infection in mice infected with heterologous mhv strains [33] . furthermore increased apoptosis, specifically in spinal cord grey matter of rl 2/2 mice supported grey matter infection. increased demyelination as well as apoptosis in grey matter in rl 2/2 mice thus prompted a more detailed investigation of viral antigen distribution in spinal cords, where the demarcation between grey and white matter is linear as opposed to the more complex organization of brain stem. neuronal infection was not observed in spinal cords of either rl 2/2 or wt mice. furthermore, the number of infected cells with oligodendrocyte morphology in the white matter of rl 2/2 mice was similar to wt mice at day 7 p.i. (fig. 8) . however, a prominent difference in viral antigen positive cells was noted in spinal cord grey matter. rl 2/2 mice harboured several foci of infected cells with microglia morphology, whereas viral antigen positive cells were rarely observed in spinal cord grey matter of wt mice (fig. 8) . furthermore, a large number of infected cells in the grey matter of rl 2/2 mice were identified as microglia and/or infiltrating monocytes based on co-localization with iba-1 positive cells (fig. 9 ). foci of iba-1 positive cells co-expressing viral antigen in the grey matter were only observed in rl 2/2 but not wt mice, confirming results obtained by immunohistochemistry (fig. 8) . nevertheless, infected iba-1 positive cells were dispersed throughout the white matter of wt and rl 2/2 mice. similar characterization of the brain stem at day 10 p.i. also identified iba -1 positive cells as prominent cells harbouring sustained viral infection (data not shown). co-stains with the astrocyte specific marker gfap showed no evidence of infected astrocytes in spinal cord grey matter; astrocytes were also only rarely infected in white matter of wt and rl 2/2 mice (data not shown). these data indicate that rnase l mediated antiviral function acts specifically in microglia and/or monocytes. moreover the focal nature of infection in grey matter suggests microglia localization is associated with enhanced susceptibility to infection in the absence of rnase l. to determine whether infection of iba-1 positive cells was biased to microglia or infiltrating monocytes, both cell populations were purified from spinal cords of infected mice by fluorescence activated cell sorting. measurement of viral mrna encoding the nucleocapsid protein revealed that microglia derived from rl 2/2 mice harbored 3.0-fold higher levels of viral rna at day 7 p.i., while the relative levels in monocytes was only increased 1.6-fold relative to infected wt mice (table 2) . however, viral mrna levels decreased in both cell types reaching comparable levels by day 10 p.i. while rna levels for tnf were elevated in rnase l deficient microglia and monocytes by 2-fold and 1.4 fold, respectively, inos levels remained similar relative to wt derived cells (data not shown). ifnc relative to gapdh mrna levels in spinal cord were also modestly increased from 4.660.5 in wt mice to 8.160.3 in rl 2/2 mice at the peak (day 7 p.i.) and declined to 1.460.4 and 1.260.1, respectively, by day 10 p.i., confirming a modest contribution of proinflammatory cytokines to pathogenesis. moderately increased viral rna levels in microglia are thus consistent with foci of microglia infection in spinal cord grey matter not present in wt mice. overall these data demonstrate that accelerated and enhanced demyelination in the absence of rnase l coincided with significant neuronal damage in the absence of expanded neuronal infection. furthermore, the limited extent of increased focal glial infection appeared insufficient to mediate the pathological effect. to assess potential differences in the overall activation state of microglia, the distribution and morphology of iba-1 positive cells was assessed in spinal cord grey matter. microglia in wt mice were activated as demonstrated by their intense staining and ramified phenotype and localized in close proximity to neuronal cell bodies (fig. 10) . by contrast, in infected rl 2/2 mice, fewer microglia were activated based on their iba-1 staining and morphology and did not show preferential association with neurons. whether the absence of rnase l itself or infection of microglia mitigates a neuroprotective function of microglia remains to be explored. the notion that inflammatory insults disrupt neuroprotective functions by microglia is based on microglia mediated neuroprotection in models of injury and lps preconditioning [34] [35] [36] and loss of protection following viral infection [37] . ifn-a/b dependent innate immunity is essential to contain viral spread during most viral infections prior to control by adaptive responses. nevertheless, the in vivo anti-viral effector mechanisms contributing to innate viral control remain poorly understood. the best characterized pathways are mediated by activation of pkr and rnase l [3] [4] [5] . however, in contrast to the conclusive effects of pkr and rnase l deficiency on viral replication in vitro, in vivo studies demonstrate more subtle anti-viral effects [8, 9, 11] . during wnv infection, rnase l deficiency is manifested in profoundly altered morbidity, despite similar viral control after footpad inoculation [11] . ifn-a/b-mediated anti-viral responses are also critical for controlling spread of neurotropic coronavirus within glial cell populations and preventing infection of neurons [12] . based on its activation by numerous rna viruses, rnase l was investigated as a prototypical anti-viral effector molecule in controlling mhv-jhm virus replication in the cns. rnase l deficiency was not sufficient to duplicate uncontrolled replication and the rapid uniform lethality observed in ifnar 2/2 mice [12] . nevertheless, a critical contribution to virus susceptibility was demonstrated by a significant increase in both clinical disease and mortality. mortality was delayed by 3-4 days in rl 2/2 relative to ifnar 2/2 mice, but could not be attributed to uncontrolled viral replication or spread to neurons. in fact, overall virus replication in brains and spinal cords of rl 2/2 mice was only modestly increased and declined with kinetics similar to wt mice. these latter findings were reminiscent of transiently increased viral replication in the cns of rl 2/2 mice following intracerebral wnv inoculation [11] . in addition to direct anti-viral activities, activated rnase l degrades viral and host rna [38, 39] . the cleavage products may in turn activate rig-i/mda-5 cytoplasmic pattern recognition receptors, propagating the expression of ifn-a/b and isgs [7] . while this function of rnase l is indeed evident by reduced ifn-b production in emcv infected rl 2/2 mice [7] , mhv-jhm infection of the cns presented no evidence for this pathway. the negligible affects of rnase l deficiency on overall viral replication [26] . nevertheless, the preferential susceptibility of rnase l deficient microglia/ monocytes to mhv-jhm infection demonstrates that viral rna does activate cellular rna sensors, albeit in a cell type specific manner. this is supported by mda-5 triggered activation of the ifn-a/b pathway in microglia and macrophages infected with mhv-a59 in vitro [17] . whether the apparent inability of gliotropic mhv-jhm to activate rnase l in other cell types in vivo resides in distinct basal oas/rnase l expression levels, activation of distinct oas enzymes, or other rna sensing receptors such as mda-5 has not been elucidated. numerous functions of rnase l, not directly associated with anti-viral activity, may contribute to the increased susceptibility of rl 2/2 mice to mhv-jhm infection. rnase l plays a role in translational inhibition, regulation of mrna stability, apoptosis, proliferation and tumor suppression [25, 40] . for example, rnase l contributes to ifn-a/b mediated apoptosis, as well as homeostasis of thymocytes and splenocytes in young naïve mice [8] . rnase l deficiency also delays tissue graft rejection [22] implicating a defect in t cell function or trafficking. nevertheless, both histochemical and flow cytometric analysis revealed a similar extent and composition of inflammatory cells in the cns of infected rl 2/2 and wt mice. the lack of rnase l mediated alterations in t cells was supported by similar peripheral expansion of virus-specific cd8 t cells and kinetics of viral control. increased morbidity could thus not readily be attributed to altered inflammation or pro-inflammatory signals at the tissue level. neuronal infection by gliotropic mhv-jhm is sparse and only evident early p.i. in wt mice. no evidence for enhanced neuronal infection in rl 2/2 mice thus suggested that the ifn-a/b mediated anti-viral mechanisms in neurons are rnase l independent. this contrasts with a protective role for rnase l in inhibiting hsv-1 replication in neurons of ifn-b treated trigeminal ganglia [41] , and supports virus specific susceptibilities to innate anti-viral immunity. distinguishing features in mhv-jhm infected rl 2/2 mice are the sustained areas of microglia infection in brain stem and focal areas of infected microglia within the spinal cord grey matter. infected cells in spinal cord grey matter are also evident in scid [42] and ifnar 2/2 mice (unpublished observation) following infection with the gliotropic mhv-jhm. in rl 2/2 mice the prominent infected cells in brain stem and spinal cord grey matter were identified as iba-1 positive microglia or infiltrating monocytes, which cannot readily be distinguished by immunohistochemistry. nevertheless the morphology of infected cells in the grey matter was consistent with activated microglia, rather than the monocyte/macrophages with dense cytoplasm found prominently in demyelinating lesions. furthermore, preferential microglia infection was supported by a relatively greater increase of viral rna in microglia relative to monocytes, when comparing rl 2/2 versus wt mice. the prominent location of iba-1 positive cells in grey matter of rl 2/2 mice could not be attributed to the absence of activated microglia in grey matter of wt mice. in fact, microglia in wt mice exhibited a more ramified phenotype surrounding neurons compared to microglia in rl 2/2 infected mice. it remains unclear whether abrogation of the proximal localization of microglia to neurons in rl 2/2 spinal cords reflects an unknown function of an rnase l enhanced neuroprotective effect, or altered function due to infection. surprisingly, both prolonged brain stem infection and enhanced spinal cord grey matter infection was associated with more severe demyelination and axonal damage. overall, the cns pathology characteristic of mhv-jhm infection was accelerated by 3-4 days in rl 2/2 relative to wt mice. although infection of microglia correlated with a subsequent increase in apoptotic cells, apoptotic cells surrounding neurons were only evident in spinal cord, not in brain stem. the inability to detect increased numbers of infected or apoptotic cells in spinal cord white matter is consistent with both an apparent lack of rnase l activation in oligodendrocytes during mhv-jhm infection and paucity of apoptotic oligodendrocytes in wt mice [28] . whether apoptotic cells originated from infected myeloid cells themselves or from lymphocytes migrating to the infected areas and exerting effector function is unclear. preliminary analysis showed no co-localization of activated caspase 3 and iba-1 (data not shown). overall, the neurologic disability and morbidity of the infected rl 2/2 mice appears to result from axonal or neural degeneration, independent of neuronal infection in both brain stem and spinal cord. the enhanced demyelination phenotype is thus distinct from demyelination attributed to enhanced white matter infection by recombinant neuronotropic mhv variants [33] . rnase l deficiency and infection of microglia may contribute to this pathology in several ways. infection in the absence of rnase l may impair neuroprotective effects exerted by microglia under inflammatory conditions [34] [35] [36] . alternatively, enhanced infection of microglia may increase proinflammatory responses resulting not only in enhanced recruitment of t cells and monocytes, but also increased local production of neurotoxic factors such as tnf, nitric oxide, oxidative radicals, and matrix metalloproteases. however, only modest increases in ifnc mrna in spinal cord, as well as minor differences in tnf and inos mrna in microglia/monocytes suggest these effects may only be apparent at a focal level. increased localized t cell effector function, manifested in release of perforin and granzymes, has also been shown to contribute to axonal injury without affecting demyelination [30] . lastly, rnase l deficient and/or infected microglia may perturb normal microglia functions in maintaining neuronal health as indicated by disruption of neuronal autophagy by siv infected microglia [37] . it thus remains to be determined to what extent accelerated and more severe pathology is due to disruption of the neuroprotective functions of microglia and/or from overt activation due to infected cells [43] [44] [45] [46] [47] . independent of a pathogenic effect, it is interesting to note that the absence of rnase l also enhanced macrophage susceptibility to wnv infection [11] . whether this reflects differential activation of oas enzymes or participation of other pattern recognition receptors such as mda-5 in susceptible cell types in vivo remains to be elucidated. in summary, these data highlight a novel role of rnase l in protection from virus induced demyelination. although rnase l did not play an overt anti-viral role as measured by viral replication, it did provide specific protection from focal infection of microglia/macrophages in the cns. rnase l was not crucial in protecting neurons from infection and played no obvious role in protecting oligodendrocytes or astrocytes. furthermore, rnase l deficiency did not alter proinflammatory responses, diminish ifna/b mediated signals, or dampen adaptive immune mediators. the results rather uncover how subtle local alterations in viral tropism may affect the balance between neuroprotection and neurotoxicity mediated by microglia/macrophages. animals, viruses, and clinical scores c57bl/6 mice were purchased from the national cancer institute (nci, fredrick, md). c57bl/6-rl 2/2 mice were bred and housed under pathogen-free conditions in the biological resources unit of the cleveland clinic. all procedures were performed in compliance with protocols approved by the institutional animal care and use committee. mice were infected at 6 weeks of age by intracranial injection with 250 pfu of the gliotropic mhv-jhm variant v2.2-1 [48] in 30 ml endotoxinfree dulbecco's phosphate buffered saline (dpbs). the severity of clinical disease was graded as previously described [48] : 1 = ruffled fur, 2 = slow righting reflex, 3 = loss of righting reflex, 4 = moribund. following intraperitoneal administration of ketamin/xylaxine (100 mg/kg/10 mg/kg), mice were perfused intracardially with 10 ml dpbs. brains, spinal cords, spleens, cervical lymph nodes (cln) and livers were collected and processed as described below. brains were bisected sagittally. one half-brain and whole spinal cord from each mouse were homogenized in ice cold tenbroeck glass grinders in 4 ml or 2 ml of dpbs, respectively. homogenates were clarified by centrifugation for 7 min at 4006g. supernatants were stored at 280uc. virus in supernatants was measured by plaque assay on monolayers of delayed brain tumor (dbt) astrocytoma cells as previously described [48] . cns cells from homogenate pellets were resuspended in rpmi containing 25 mm hepes, adjusted to 30% percoll (pharmacia, upsalla, sweden), underlayed with 1 ml 70% percoll, centrifuged at 8006g for 30 minutes and collected from the 30%/70% percoll interface as previous described [23] . purified cns cells were washed and resuspended in rpmi. cell populations isolated from the brain and spinal cord were phenotyped using four-color flow cytometry. prior to staining, cells were incubated with 1% mouse serum and 1% rat anti-mouse fcciii/iir monoclonal antibody (mab) in fluorescent antibody cell sorting (facs) buffer (0.5% bovine serum albumin in dpbs) for 20 minutes at 4uc to block non-specific binding. cell types were identified using fluorescein isothiocyanate-, phycoerythrin-, peridinin-chlorophyll-protein complex-or allophycocyanin-conjugated anti-mouse mab: ly-6 g (1a8), cd4 (gk1.5), cd8 (53.67) (all from bd biosciences, san diego, ca) and f4/80 (ci:a3-1; serotec, raleigh, nc). virus specific cd8 t cells were identified using h-2d b /s510 mhc class i tetramers as described previously [23] . cells were incubated with antibodies for 30 minutes on ice, washed twice with facs buffer and fixed with 2% paraformaldehyde for 10 minutes on ice. at least 100,000 events were acquired on a facscalibur flow cytometer (bd biosciences, san jose, ca) for subsequent data analysis using flow-jo 7 software (tree star, inc. ashland, or). for fluorescence activated cell sorting of microglia and monocyte populations, spinal cords from eight mice per group were finely minced using a razor blade and dissociated in a 0.25% trypsin solution as described [27, 49] at 37uc for 30 minutes with periodic tituration. trypsin was quenched by addition of rpmi supplemented with 25 mm hepes and 20% new born calf serum. the dissociated cells were washed in rpmi containing 25 mm hepes, 1% fcs, then isolated from the interphase of a 30%/70% percoll gradient as described above. cells were incubated with 1% mouse serum and cd16/32 prior to staining with allophycocyanin-conjugated mab specific for cd45 (30-f11), peridinin-chlorophyll protein-conjugated cd11b (m1/70) (bd biosciences, san diego, ca) and phycoerythrin-conjugated mab specific for f4/80 (ci:a3-1; serotec, raleigh, nc). monocyte/ macrophages and microglia were purified on a facsaria cell sorter (bd biosciences, san diego, ca) based on their respective cd45 hi cd11b + f4/80 + and cd45 lo cd11b + f4/80 + phenotypes. one half-brain and whole spinal cords were fixed with formalin and embedded in paraffin. sections were stained with either hematoxylin and eosin or luxol fast blue as described [50] . distribution of viral antigen was determined by immunoperoxidase staining (vectastain-abc kit; vector laboratory, burlingame, ca) using mab j.3.3, specific for the carboxyl terminus of the viral nucleocapsid protein as primary antibody, biotinylated horse antimouse as secondary antibody, streptavidin-conjugated horse radish peroxidise and 3,39-diaminobenzidine substrate (vector laboratory) [50] . similarly, immunoperoxidase staining for neurofilament used mouse anti-phosphorylated and anti-non-phosphorylated neurofilament mab (smi-31 and smi-32, covance, princeton, nj) and immunoperoxidase staining for microglia/macrophages used anti-ionized calcium-binding adapter molecule-1 antibody (iba-1, abcam, cambridge, ma). apoptotic cells were detected by rabbit anti-activated caspase-3 ab (asp175, cell signalling technology, beverly, ma). sections were scored for inflammation, viral antigen, apoptotic cells, axonal damage and demyelination in a blinded fashion. representative fields-of-view were identified based on average scores of all sections in each experimental group. for calculation of the percentage area of demyelination, sections were digitized and analysed as previously described [27] . to identify infected cell types, spinal cords were embedded in tissuetek o.c.t. compound (andwin scientific, tryon, nc), flash frozen in liquid nitrogen and stored at 280uc. blocks were warmed to 220uc prior to cutting 10 mm sections by cryostat at this temperature. following fixation with 4% paraformaldehyde for 30 minutes at room temperature, non-specific antibody binding was blocked using 1% bovine serum albumin, 5% normal goat serum. infected cells were identified using anti-mhv-jhm j3.3 mab, rabbit anti-glial fibrillary acidic protein antibody (gfap, abcam, cambridge, ma) and rabbit anti-ionized calcium-binding adapter molecule-1 antibody (iba-1, wako, richmond, va) in combination with goat anti-mouse alexa-fluor 488 and goat anti-rabbit alexa-fluor 594-conjugated igg (invitrogen, carlsbad, ca) as secondary antibodies, respectively. sections were mounted with prolong gold antifade mounting media containing 49,6-diamidino-2-phenylindole (invitrogen, carlsbad, ca). imaging of immunofluorescent sections was performed with a leica sp-5 confocal microscope (leica microsystems, bannockburn, il). z-projections of sections were processed by imagej software (national institutes of health, bethesda, md) one-half brains and whole spinal cords were snap frozen in liquid nitrogen and stored at 280uc. frozen brain or spinal cord tissue was homogenized in 2 ml and 1 ml trizol reagent (invitrogen, carlsbad, ca), respectively, in rnase-free tenbroeck glass grinders. rna was purified according to the manufacturer's protocol (invitrogen, carlsbad, ca). briefly, 0.2 ml chloroform/ 1 ml trizol (sigma-aldrich, st. louis, mo) was added to homogenate, mixed and centrifuged at 12,0006g for 15 minutes at 4uc. rna was precipitated from the aqueous phase by addition of isopropyl alcohol and centrifugation at 12,0006g for 10 min at 4uc washed in rnase-free 75% ethanol and resuspended in ultrapure dnase/rnase-free water (invitrogen, carlsbad, ca). cells isolated by facs were immediately resuspended in 400 ml of trizol reagent (invitrogen, carlsbad, ca) and treated as above. isolated total rna was treated with dnase1, using the dna-free kit (applied biosystems, foster city, ca) following the manufacturer's protocol. the concentration and purity of rna was measured by spectrophotometry at 260/280 nm. rna integrity was confirmed by 1.2% formaldehyde-agarose gel electrophoresis. reverse transcription was performed on 2 mg total rna isolated from brain and spinal cord or all total rna isolated from facs sorted cells, primed with 250 ng random hexamers, (invitrogen, carlsbad, ca) using mmlv reverse transcriptase (invitrogen, carlsbad, ca) for 50 minutes at 37uc. real-time pcr was performed using sybr green 26master mix (applied biosystems, foster city, ca) for the following primer sets: interferon-induced protein with tetratricopeptide repeats ( the reaction conditions were: 95uc for 10 minutes, followed by 40 cycles of: denaturation at 95uc for 10 seconds, elongation at 60uc for 30 seconds and annealing at 72uc for 30 seconds. ifn-b1, ifn-a4 and ifn-c levels were determined by real time pcr using abi gene expression assays with 26universal taqman fast master mix (applied biosystems, foster city, ca), using manufacturer default cycling conditions and gapdh expression as an endogenous control. all real-time reactions were run using an abi 7500fast real-time cycler and analysed with 7500fast software (applied biosystems, foster city, ca). data presented are expressed as fold-induction relative to gapdh based on the following formula: (2 (ct(gapdh)-ct(target)) )*1000. students' t-test with equal variance was used to compare rl 2/2 and wt c57bl/6 mice. significant differences between groups are noted by: *, = p#0.05. interferons at age 50: past, current and future impact on biomedicine type 1 interferons and the virus-host relationship: a lesson in detente a scientific journey through the 2-5a/rnase l system viral encounters with 29,59-oligoadenylate synthetase and rnase l during the interferon antiviral response the dsrna protein kinase pkr: virus and cell control cloning and characterization of a rnase l inhibitor. a new component of the interferonregulated 2-5a pathway small self-rna generated by rnase l amplifies antiviral innate immunity interferon action and apoptosis are defective in mice devoid of 29,59-oligoadenylate-dependent rnase l rnase l and double-stranded rna-dependent protein kinase exert complementary roles in islet cell defense during coxsackievirus infection rnase l plays a role in the antiviral response to west nile virus pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd8 t cells interferon and cytokine responses to sars-coronavirus infection control of coronavirus infection through plasmacytoid dendritic cell-derived type i interferon type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection viral induction of central nervous system innate immune responses murine coronavirus mouse hepatitis virus is recognized by mda5 and induces type i interferon in brain macrophages/microglia mouse hepatitis coronavirus a59 nucleocapsid protein is a type i interferon antagonist a 29,59-oligoadenylate analogue inhibits murine hepatitis virus strain 3 (mhv-3) replication in vitro but does not reduce mhv-3-related mortality or induction of procoagulant activity in susceptible mice coronavirus infection of the central nervous system: host-virus stand-off murine coronavirus infection: a paradigm for virus-induced demyelinating disease skin allograft rejection is suppressed in mice lacking the antiviral enzyme, 29,59-oligoadenylate-dependent rnase l inverted immunodominance and impaired cytolytic function of cd8+ t cells during viral persistence in the central nervous system sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv-4) leads to a characteristic distribution of demyelination diverse functions of rnase l and implications in pathology ctl effector function within the central nervous system requires cd4+ t cells inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination differential induction of apoptosis in demyelinating and nondemyelinating infection by mouse hepatitis virus contrasting roles for axonal degeneration in an autoimmune versus viral model of multiple sclerosis: when can axonal injury be beneficial? absence of perforin expression confers axonal protection despite demyelination role of ifn-gamma responsiveness in cd8 tcell-mediated viral clearance and demyelination in coronavirus-infected mice axonal damage is t cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism evidence for synaptic stripping by cortical microglia microglia overexpressing the macrophage colony-stimulating factor receptor are neuroprotective in a microglial-hippocampal organotypic coculture system enhanced cell-to-cell contacts between activated microglia and pyramidal cell dendrites following kainic acid-induced neurotoxicity in the hippocampus disruption of neuronal autophagy by infected microglia results in neurodegeneration interferon action: rna cleavage pattern of a (29-59)oligoadenylate-dependent endonuclease interferon actionsequence specificity of the ppp(a29p)na-dependent ribonuclease rnase l: its biological roles and regulation interferon-beta suppresses herpes simplex virus type 1 replication in trigeminal ganglion cells through an rnase l-dependent pathway memory cd4+ t-cell-mediated protection from lethal coronavirus encephalomyelitis microglia as neuroprotective, immunocompetent cells of the cns molecular and cellular immune mediators of neuroprotection multiple sclerosis: novel perspectives on newly forming lesions experimental models of neuroprotection relevant to multiple sclerosis the role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: hiv-associated dementia, alzheimer disease, and multiple sclerosis pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies induction of class i antigen processing components in oligodendroglia and microglia during viral encephalomyelitis perforin-mediated effector function within the central nervous system requires ifn-gamma-mediated mhc up-regulation the authors thank dr. bruce trapp for helpful discussions and comments on the manuscript. we also thank wen wei and ernesto barron for excellent assistance with the histopathology and jennifer powers for cell purification by facs. conceived and designed the experiments: ddci sas ccb. performed the experiments: ddci pk. analyzed the data: ddci sas drh pk rhs raa ccb. contributed reagents/materials/analysis tools: ccb. wrote the paper: ddci sas drh rhs ccb. key: cord-345339-kyboibtq authors: steiner, israel; nisipianu, puiu; wirguin, itzhak title: infection and the etiology and pathogenesis of multiple sclerosis date: 2001 journal: curr neurol neurosci rep doi: 10.1007/s11910-001-0030-x sha: doc_id: 345339 cord_uid: kyboibtq multiple sclerosis (ms) currently defies clinical and scientific definitions, and carries a prognosis that remains practically unchanged despite many years of intensive research. although the prevailing dogma is that ms is an immune-mediated condition, it fulfills none of the criteria of an autoimmune disease. on the other hand, there is enough significant data to suggest that infectious agents(s) could be involved in either direct damage to the white matter or induce inflammatory responses that secondarily affect the brain. our goal here is to review the data supporting the possibility that infection has a critical role in the disease, examine the list of potential candidates that have been suggested, and outline an approach regarding the potential role of infectious agents in the etiology and pathogenesis of ms. despite years of investigation, no direct evidence has been found to incriminate a specific etiology and pathogenesis for multiple sclerosis (ms). based on meager evidence [1•] and on extrapolation, an autoimmune pathogenesis has been hypothesized. however, we feel that autoimmune pathogenesis is not the likely culprit; instead, we believe that infection is more likely responsible for the damage to the central nervous system (cns). we review the data for an infectious process and examine the list of potential candidates. multiple sclerosis is a chronic, inflammatory white matter disease of the cns that usually affects young adults. successive sets of diagnostic criteria, essentially requiring objective evidence of at least two distinct lesions of cns white matter (the last set almost 20 years old), allude not only to the absence of a pathognomonic test for ms, but also to the lack of adequate evidence for the reliability of the various criteria. of note is the requirement for lack of an alternative diagnosis [2] , a criterion that imposes an element of "diagnosis by exclusion." the white matter is progressively destroyed, leaving some of the victims incapacitated for life. for many years, the leading, and practically dogmatic, hypothesis has been that ms is an autoimmune condition and hence, that its symptoms and progression could be ameliorated by immunomodulating strategies. however, while the field of neuroimmunology has thrived, little progress has been made towards understanding ms. the current diagnostic criteria accommodate a wide range of conditions. indeed, the extremely variable course and prognosis of the disease suggest that what is considered today a homogenous and consistent entity of similar etiology and pathogenesis, is, in fact, a syndrome caused by various etiologies and multiple pathogenic mechanisms [1•] . pierre marie, charcot's favorite student, (but not his successor, as erroneously suggested by several reviews), was the first, at the end of the 19th century, to raise the possibility that ms is caused by an infection [3] . since then, the amount of information in this realm is overwhelming. our goal here is not to quote the copious literature, but to delineate concepts, outline an approach, and provide some food for thought regarding the possible role of infectious agents in the etiology and pathogenesis ms. basically, an infective process can damage the tissue via two major avenues: directly, due to the pathogen present in the tissue during the disease; or indirectly, where the infection is only the trigger, and the damage occurs while the infection has already been cleared and removed from the organism. each alternative has different histologic, serologic, and epidemiologic characteristics. multiple sclerosis lesions (plaques) are multifocal and mainly perivenous [4••] . their distribution, though random enough to merit conflicting generalizations and conclusions, can reflect the route of penetration of infection. the myelin loss, with neurons and axons being relatively spared, which is characteristic of the ms lesion, is associated with mononuclear inflammatory cell infiltrates within active plaques and in perivascular spaces. macrophages and t lymphocytes are present in the center of a new lesion, which also shows reduction in the number of oligodendrocytes, reactive astrocytes, and microglial cells. obviously, these findings may accompany an active ongoing infectious process. the predilection of the inflammation for the white matter is a multiple sclerosis (ms) currently defies clinical and scientific definitions, and carries a prognosis that remains practically unchanged despite many years of intensive research. although the prevailing dogma is that ms is an immune-mediated condition, it fulfills none of the criteria of an autoimmune disease. on the other hand, there is enough significant data to suggest that infectious agents(s) could be involved in either direct damage to the white matter or induce inflammatory responses that secondarily affect the brain. our goal here is to review the data supporting the possibility that infection has a critical role in the disease, examine the list of potential candidates that have been suggested, and outline an approach regarding the potential role of infectious agents in the etiology and pathogenesis of ms. relatively uncommon feature of acute viral infections of the cns, and has been noted mainly in certain chronic viral infections (see following). this discipline has provided compelling evidence for an environmental factor in ms. for example, disease distribution in different geographic regions can be classified according to climate [5•] . although differences in quality of healthcare systems and recording in different countries may temper this finding, it seems that there is a high prevalence (or risk) in temperate countries (northern europe, united states, canada, southern australia, and new zealand), a low prevalence in warm, equatorial regions (the caribbean, mexico) and a medium prevalence in between (southern europe). migration might also induce epidemics. probably the best-studied example is the outbreak of an ms epidemic in the faroe islands following stationing of british troops there during the second world war [6] . migration studies have also suggested that age at the time of migration is important. in many instances, when individuals migrate prior to 15 years of age, they exhibit the prevalence of their new home. if they migrate at a later age, they retain the disease prevalence of their native country. this has been used to argue for the presence of an infectious factor, because there are examples for the critical role of the timing of primary infection in induction of an infectious cns disorder [7] . anecdotal evidence of occurrence of ms in families has been noted for many years [8] . there are few reports of conjugal ms [9] and of ms diagnosed in children of affected parents [10] , but not enough to enable statistical analysis. whether these reports, together with other documentation of ms affecting members of the same community [11] , those who share a profession, a work place, or a residence are more than just coincidental anecdotes is not settled. with no true understanding of the cause of ms, the animal demyelinating disease models abound. there is quite a spectrum of viruses that can produce acute and chronic demyelinating conditions in a variety of experimental animals [12] . these include dna viruses such as the papova virus sv40 (rhesus monkeys) and herpes simplex virus type 1 (hsv-1) and 2 (in mice), rna viruses such as canine distemper virus (mice and hamsters), several corona virus models in mice and monkeys, the murine picorna theiler's virus, and retroviruses such as visna virus in sheep. these models prove the concept that viruses can induce demyelination. interferons for the treatment of multiple sclerosis as of today, three β-interferon preparations are licensed for the treatment of ms. they were shown to have a moderate effect upon disease relapse rate and progression of disability [13] [14] [15] . interferons are naturally occurring species-specific glycoproteins that were first discovered in 1957 because of their antiviral activity, but they also possess a variety of other biologic actions. they belong to the ever-growing group of cytokines that are capable of modulating the immune system. nevertheless, one of their major functions is to combat viral infections. they are in clinical use for treatment of conditions such as infections due to hepatitis b and c viruses, and genital warts caused by papillomavirus. although it has been argued that the β-interferons' effect upon ms is immunomodulatory, the possibility that their impact is directly antiviral is just as likely. viruses can cause a variety of naturally occurring human cns demyelinating diseases. infective and postinfective conditions have been noted: progressive multifocal leukoencephalopathy (pml) and tropical spastic paraparesis are examples of the first, whereas postinfectious encephalomyelitis occurs following diverse infections. although these disorders are clinically distinct from ms, they nevertheless suggest that viruses are capable of inducing demyelinating diseases in the human cns. cerebrospinal fluid (csf) oligoclonal bands are present in the vast majority of ms patients. although attributed to a chronic immune-mediated process, csf oligoclonal bands are also a feature of chronic cns infections [16] . in view of the current dogma of ms as an immune-mediated disorder, the possibility that the disease and/or its relapses are triggered by infections, whereas the eventual tissue damage is due to immune activation, might be more appealing than the concept that ms is a true infective process. the methodology used to examine an association between infection and clinical diseases is both epidemiologic and serologic/immunologic. some studies that examined the association between antecedent infection and occurrence of ms may suggest a sequential relationship. these included unspecified infections [17] and infections with specific agents, such as measles [18] and epstein-barr virus (ebv) [19] . there seems also to be a relationship between an infection and the occurrence of a relapse in a setup of already defined clinical ms [20] . approximately 9% of infections were temporally related to exacerbations, whereas 27% of exacerbations followed infections. this observation was confirmed by a prospective study that followed ms relapses by magnetic resonance imaging (mri) [21] . ms relapses also tended to have a seasonal increase, in association with respiratory infections. immunology/serology can serve to suggest both an ongoing infectious process and/or a previous exposure to a pathogen. antiviral antibodies were examined in the serum and the csf of ms patients. higher titers of antibodies against a large spectrum of viruses were found (but not always confirmed) in ms patients when compared with control patients. the list [12, 22] includes measles, parainfluenza, influenza, varicella zoster, hsv, rubella, ebv, mumps, human t-cell lymphotropic virus (htlv)-1 and 2, and several others. direct proof that a pathogen is responsible for the tissue damage in ms requires isolating the agent from the diseased nervous system and demonstrating that this finding is disease-specific. so far, this task has eluded researchers: no virus or any other infective agent has met these criteria. isolating a pathogen from ms patients' tissues is based on the available technology. for more than half a century, a wide spectrum of laboratory approaches has been used, from cytopathic effect produced in culture by fluids isolated from patients, to viral culture and cocultivation studies, to the present polymerase chain reaction (pcr) amplification and representational difference analysis. a long list of suspected viruses includes rabies, hsv, scrapie agent, parainfluenza, measles, simian virus v, coronavirus, htlv-1, and others. none has withstood tests of reproducibility, specific presence in a significant number of ms patients as compared with control patients, or a reasonable experimental explanation. nevertheless, based on the assumption that we are dealing with a heterogeneous entity, most likely a syndrome, it is possible that the disorder is due to many different pathogens, where in certain individual cases, an isolated pathogen is indeed responsible for the disease. therefore, several or all such pathogens might cause the disease, but would not stand out when examined on a large series of patients. as of today, four pathogens are under scrutiny, and are, therefore, discussed here in more depth. human herpes virus 6 (hhv-6) is a lately discovered dna virus that is responsible for exanthema subitum (roseola infantum) in children; up to 95% of the general population has been exposed to it [23] . it is a lymphotropic and a neurotropic virus that has been shown to infect glial cells in culture [24] . a look at control tissues, pcr amplification of hhv-6 nucleic acids, and representational difference analysis (the technology that enabled to link hhv-8 with kaposi's sarcoma) demonstrates that it resides in the nervous system of the majority of the population [25] . hhv-6 has been linked with a variety of acute and chronic neurologic disorders, such as encephalitis [26] , menin-goencephalitis [27] , and myelopathy [28] under normal or immune-compromised states. a possible association between hhv-6 and ms is suggested by a number of studies. these include studies that find higher levels of anti-hhv-6 antibodies in the sera of patients with ms as compared with control patients [29,30•] , studies that identify hhv-6 dna in a larger percentage of ms patients compared with control patients [30•] (findings which are not confirmed by other groups [31] ), and evidence for hhv-6 gene expression in the brain, in oligodendrocytes, and in plaques of ms patients [25,32•] . the latter findings, however, were not confirmed by other researchers [33] . although not unique to hhv-6, it is important to note that the timing of exposure to the virus corresponds with the exposure time to a possible infectious pathogen in ms. at present, the most intriguing finding is the association of hhv-6 with ms lesions. if confirmed, it should lead to the question whether this is an outcome of the disease (or its treatment, as the immunosuppressive and immunomodulatory therapies may induce viral reactivation with expression of viral gene products), a noncontributory factor, or indeed related to the pathogenesis of the disease. epstein-barr virus is a lymphotrophic herpes virus responsible for infectious mononucleosis that has been associated with a spectrum of neurologic disorders, including certain cns lymphomas. like other herpes viruses, it establishes latent infection and can reactivate later [34] . in several studies, ebv was found to be present or have left evidence of previous exposure in a higher percentage of ms patients compared with control patients [35] . likewise, epidemiologic studies suggest a relationship between a previous ebv infection and ms [19] . it is worth noting that, during acute ebv infections, antibodies that crossreact with neuroglial antigens are produced [36] . however, identification of the pathogen(s) in ms cannot rely on fingerprints alone. it is mandatory to detect presence of viral nucleic acids and/or antigens in the diseased tissue, which should not constitute a technical problem because ebv is already revealed and examined in certain cns lymphomas. we are not aware of any report on the presence or absence of such compounds in brain tissues of ms patients. retroviruses are rna viruses that encode proviral dna integrated into the host cell genome and can be transmitted via the germ line [37] . endogenous retroviral sequences constitute up to 20% of the human genome. the retrovirus visna, found in sheep, was one of the first animal models of a demyelinating disease, and htlv-1, which can cause a human demyelinating chronic myelopathy, was implicated, but not confirmed, as the cause of ms. in the past decade, another, yet unidentified, retrovirus has been traced. activity of the retroviral enzyme reverse transcriptase was identified in tissues and cells from ms patients [38•] , as well as retroviral sequences in the csf [38•] and serum [39] . this has come to be termed msassociated retrovirus (msrv). indeed, endogenous retrovirus can theoretically induce an immune-mediated condition [40] or an infectious process per se. unfortunately, other groups were unable to confirm these findings, and there is a similarity between the identified sequences and those of endogenous retroviruses present in the majority of the population [38•] . a collaboration between a retrovirus and ebv in inducing ms and triggering the relapses has also been suggested [41•] . the hypothesis is based on the observation that herpes viruses can transactivate retroviruses [42] , and on the ability of herpes viruses to reactivate periodically and induce recurrent disease [34] . chlamydia pneumoniae is an obligate, intracellular, gramnegative bacterium known to cause respiratory infections in humans. the organism has also been implicated in a wide variety of disorders ranging from asthma, atherosclerotic vascular disease, and arthritis to guillain-barre syndrome and encephalitis. the possible relation of chlamydia to ms was recently resurrected by a case report of a 24-year-old man who presented with a fulminant mslike disease [43] . his condition deteriorated despite corticosteroid and β-interferon therapy. although treated with cyclophosphamide, csf cultures and pcr analysis turned out to be positive for chlamydia pneumoniae, and prolonged antibiotic therapy was associated with neurologic recovery. this report was followed by analysis of 37 additional patients from the same center [44••] , showing 64% of csf samples culture-positive, and 97% pcr-positive for the organism, (compared with 11% and 18% in control patients, respectively), and 86% with csf anti-chlamydia antibodies. these findings were supported by one group [45] , but not by others [46] . although the discrepancies may be due to lack of uniform standards and technical difficulties, it seems remarkable that a bacterium with distinct morphologic features could have eluded generations of neuropathologists. furthermore, recent studies failed to detect chlamydia dna within brain lesions obtained by biopsy or autopsy of ms patients [47] . thus, although in some geographical areas some ms patients may have a chronic cns chlamydial infection, its cause and effect role in ms is questionable, because the pathogen is known to infect macrophages and could secondarily gain entrance into the cns during ms exacerbations [44••] . many details concerning the mechanism of myelin damage in ms are still obscure, but putative models for activated t-cell penetration of the cns with resultant demyelination have been put forward [48] . thus, injury might be due to a concerted effect of cytokine-and antibody-mediated injury, direct injury to oligodendrocytes by cd4+ and cd8+ t cells, and myelin digestion by macrophages. naturally, a heavy emphasis on the immune response to selfantigens is the premise of these models, but such cascades could still be easily compatible with the presence of microbial agents as the primary inciting events, or as modulatory factors during ongoing inflammation. after 50 years, no breakthrough evidence to support either a pure autoimmune pathogenesis or a direct destructive infection of white matter components has become available. infection could take part in tissue damage via a direct, pml-like infective process. although it might be argued that the attempts to treat ms with intense immunosuppression should have induced exacerbations attributed to enhanced viral proliferation (which they usually did not), there are certain nervous system persistent or latent infections that are not influenced by the host's immune state [34] . at the other end, any intercurrent infection may induce an antimyelin immune response through molecular mimicry (ie, microorganism antigens that resemble myelin antigens that are presented to the immune system under conditions which favor breakdown of self tolerance followed by the myelin being attacked by the newly primed effector cells and autoantibodies) [49] . indeed, microbial epitopes resembling myelin basic protein have been shown to induce experimental autoimmune encephalomyelitis [50••] , and fine specificities of altered epitopes capable of inhibiting the self response are under study. however, guillain-barre syndrome and paraneoplastic disorders in which molecular mimicry is thought to play a central role are either mainly monophasic or subacute progressive disorders, not characterized by frequent relapses. additionally, some viruses may act as superantigens [40] that bind outside the antigen grove, and activate whole classes of t cells, including autoreactive t cells specific for myelin antigen(s), which once activated may cross the blood-brain barrier. there are also possibilities that combine these two extremes. theoretically, latent viral infection of oligodendrocytes (capable of frequent mutations) may occasionally express novel gene products that will be recognized as foreign, and the immune system will eventually mount an inflammatory response with extensive local damage. the relative immune privilege of the cns may support such damage, because single cells harboring the virus will remain undetected and the immune response delayed, enabling tissue alteration to spread locally, causing a more extensive response later. other possibilities are even more hypothetical. for instance, double infections, in which products of one infectious process enhance a nonspecific or specific responses to a second or a latent pathogen, might be also considered. the answers are probably more complex and multifactorial. the infection could be the primary etiology, but an immune-mediated response will play an important pathogenic role. alternatively, if ms is indeed a syndrome, inter-patient differences may account for the difficulty in establishing a unifying hypothesis on ms etiology. this is supported by a recent pathologic study [4••] , where four different demyelination patterns, two showing evidence for a viral-or toxin-mediated oligodendrocytopathy, are suggested. at any rate, non-unifying approaches aimed at splitting the syndrome of ms into distinct entities are probably necessary to unravel the mystery of ms etiology. we have provided the data in favor of an infectious etiology of ms. however, as of today, there is still no evidence to incriminate a specific pathogen. the hunt is still on, and until the causative agent and the destructive scheme are be exposed, the neurologic scientific and clinical community will not give up. multiple sclerosis-in need of critical reappraisal a critical review of the immunologic hypothesis of multiple sclerosis problems of experimental trials of therapy in multiple sclerosis: report by the panel on the evaluation of experimental trials of therapy in multiple sclerosis sclerose en plaques et maladies infectieuses heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination a new look at multiple sclerosis (ms) pathology supporting the nonuniformity of the condition and the concept that ms is a syndrome epidemiologic evidence for multiple sclerosis as an infection epidemiologic studies of measles, measles vaccine, and subacute sclerosing panencephalitis sobre la clinical de la escleroisis multiple conugal multiple sclerosis. immunogenetic characterization and analysis of t-and b-cell reactivity to myelin protein familial and conjugal multiple sclerosis multiple sclerosis in research workers studying swayback in lambs: an updated report the virology of demyelinating diseases interferon beta 1b is effective in relapsing-remitting multiple sclerosis. i. clinical results of a multicenter, randomized, double blind, placebo-controlled trial impact of interferon beta-1a on neurologic disability in relapsing multiple sclerosis. the multiple sclerosis collaborative research group (mscrg) prism (prevention of relapses and disability by interferon b-1a subcutaneously in multiple sclerosis) study group.: randomized double blind placebo controlled study of interferon b-1a in relapsing/remitting multiple sclerosis virological aspects of tropical spastic paraparesis/ htlv-i associated myelopathy and htlv-i infection detels r: a 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the viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. the collaborative research group on multiple sclerosis an important contribution to the concept that a retrovirus might be present in multiple sclerosis patients' nervous system at a higher percentage than in the normal population detection of virionassociated msrv-rna in serum of patients with multiple sclerosis a human endogenous retroviral superantigen as candidate autoimmune gene in type i diabetes the association between multiple sclerosis and infection with epstein-barr virus and retrovirus discussion on the possible combined action of more than one pathogen in inducing central nervous system damage in multiple sclerosis transactivation of human t-cell leukemia virus type 1 by herpes simplex virus type 1 multiple sclerosis associated with chlamydia pneumoniae infection of the cns chlamydia pneumoniae infection of the central nervous system in multiple sclerosis a systematic analysis of multiple sclerosis patients for evidence of a bacterial infection by a group that has first raised the possibility of chlamydia pneumoniae infection in multiple sclerosis evidence for infection with chlamydia pneumoniae in a subgroup of patients with multiple sclerosis failure to detect chlamydia pneumoniae in the central nervous system of patients with ms lack of detectable chlamydia pneumoniae in brain lesions of patients with multiple sclerosis multiple sclerosis more mayhem from molecular mimics microbial epitopes act as altered peptide ligands to prevent experimental autoimmune encephalomyelitis on the possible role of antigens of infective agents in modulating the immune response against nervous system constituents key: cord-355413-ls2nud43 authors: shi, fu-dong; ransohoff, richard m. title: nature killer cells in the central nervous system date: 2010-01-29 journal: natural killer cells doi: 10.1016/b978-0-12-370454-2.00028-4 sha: doc_id: 355413 cord_uid: ls2nud43 natural killer (nk) cells, a prominent component of the innate immune system, are large granular lymphocytes that respond rapidly to a variety of insults via cytokine secretion and cytolytic activity. recently, there has been growing insight into the biological functions of nk cells, in particular into their roles in infection, tumorurveillance and autoimmunity. under these pathophysiological circumstances, nk cells readily home to the central nervous system (cns) tissues to combat infection and presumably to curb progression of tumor. bystander neuronal and/or glial cell damage can occur in this setting. paradoxically, nk cells appear to have an inhibitory role for autoimmune responses within the cns. as in the periphery, nk cells act in concert with t cells and other lymphocytes responsible for cns pathology and immune regulation. insights into the molecular signals and pathways governing the diverse biological effects of nk cells are keys for designing nk cell-based therapy for cns infections, tumor and autoimmune diseases.nk cells readily accumulate in homing to cns tissues under the pathophysiological situations. this process is tightly controlled by a number of chemokines and chemokine receptors. there is ample of evidence that nk cells within the cns contribute to the control of infections and might limit progression of certain tumor. bystander neuronal and/or glial cell damage can occur. in certain autoimmune conditions of the cns, nk cells appear to have an inhibitory role. disassociation of disease-inhibiting versus disease-promoting effects of nk cells is a key to harnessing nk cells for therapeutic purposes. to achieve this goal, a generation of genetic models with selective nk cell deficiency, and development of reagents (antibodies) for visualizing subsets of nk cells in situ will be necessary. cells in human cns tissues is still lacking. this is partly because of the lack of suitable antibodies for staining human nk cells in situ (see chapter 31). visualization of nk cells in mouse brains during experimental autoimmune encephalomyelitis (eae) was achieved using anti-nk1.1 mab (pk136) (hammarberg et al., 2000) . antibodies such as ly49 have been successfully used for staining nk cells in lymphoid organs (o'leary et al., 2006) . suitability of these antibodies for staining cns nk cells needs to be verified in additional studies. although direct visualization of nk cells in cns tissues is technically challenging, there is little doubt that nk cells, as with other types of lymphocytes, enter the cns during inflammatory processes. in fact, it has been reported that nk cells are among the earliest recruited cells during adoptive transfer eae (kerfoot and kubes, 2002; wekerle and fierz, 1985) . chemokine receptors such as ccr2, ccr5, cxcr3, cx3cr1 as well as lysophospholipid sphingosine 1-phosphate (s1p) are involved in the rapid nk-cell mobilization that occurs in inflammatory conditions (ajuebor et al., 2007; hokeness et al., 2005; huang et al., 2006; inngjerdingen et al., 2001; jiang et al., 2004; khan et al., 2006; kveberg et al., 2002; lavergne et al., 2003; martin-fontecha et al., 2004; thapa et al., 2007; wald et al., 2006; walzer et al., 2007; yu et al., 2007) , and several of these chemokine receptors (ccr5, cx3cr1) are directly involved in nk cell homing to the cns (huang et al., 2006; martin-fontecha et al., 2004; thapa et al., 2007) . the biological implication of chemokine-guided homing of nk cells during cns inflammation is discussed in greater detail in the following section. the ability of nk cells to kill various transformed and virus-infected cells raises an important question whether direct nk cell cytolytic effects contribute to the pathogenesis of inflammatory, degenerative and autoimmune disorders of the nervous system. neurodegenerative diseases such as parkinson's disease and alzheimer's disease are characterized by the death of neurons in distinct functional neuron-anatomic systems. multiple sclerosis (ms), on the other hand, is characterized by inflammation and demyelination within the spinal cord and brain, and axonal damage and brain atrophy also occur during the course of disease. the peripheral form of ms is guillain-barré syndrome, which is also characterized by demyelination and cellular infiltrates of the peripheral nervous system. some of these diseases involve immunologic components or reactions, and some have been characterized extensively in the different systems. autoreactive t cells and adaptive immune system components in the pathogenesis in some of these disorders are well characterized. as discussed here, evidence for direct nk cell cytolytic effects is emerging. although the in vivo relevance of a great proportion of these studies needs to be validated, the current data emphasize the importance of nk cells either in direct cytotoxic effects or in collaboration with cells from both innate and adaptive immune systems in the initiation of these neurodegenerative and inflammatory diseases. the nk cell-dependent death of sympathetic neurons resident in the superior cervical ganglia of rats, observed after the exposure to the drug guanethidine (hickey et al., 1992; hougen et al., 1992) , is the first in vivo disorder of the nervous system in which nk cells appear to be the dominant effector cells. the pathogenic mechanism observed appeared to represent a novel type of autoimmune reaction: an exogenously/chemically induced alteration in a specific subset of cells that was suggested to target them for direct nk cell-mediated killing. interestingly, neuronal cells from the peripheral system and the cns appear to have different susceptibility to nk cell killing. ljunggren and colleagues have carried out a series of well-designed studies addressing this puzzling phenomenon. initially, it was demonstrated that nk cells could readily kill syngeneic dorsal root ganglia (drg) neurons by a perforin-dependent mechanism (backstrom et al., 2000 (backstrom et al., , 2003 . ventral spinal cord neurons and hippocampal neurons of the cns were resistant to lysis. the resistance to nk cell-mediated lysis of the latter neurons was not related to protection by mhc class i molecules, since similar  2 -micro-globulin / neurons were equally resistant to lysis (backstrom et al., 2003) . nk cell function is tightly regulated by multiple signals transmitted via inhibitory and activating receptors. the prerequisite for nk cell killing is its activation via signalling from activating receptor ligand pathways. nk cell activation generally appears to be elicited by a distinct set of molecules that have weak homology with mhc class i molecules. the activating receptor nkg2d which differs dramatically from other nkg2 receptor proteins is of particular interest since it, in contrast to other nk cell-activating receptors, is constitutively expressed on nk cells. the endogenous ligand of nkg2d in the mouse was recently identified as retinoic acid early inducible gene-1 (rae-1)-encoded proteins and minor histocompatibility antigen h60 (cerwenka et al., 2000; diefenbach et al., 2000; malarkannan et al., 1998) . differential expression of nkg2d and its ligand on neuronal cells from the peripheral system or the cns appears a key mechanism underlying variable susceptibility to nk lyses. rae-1, the product of which is a ligand for the nk cell-activating receptor nkg2d, was expressed at high levels in the drg neurons. in contrast, rae-1 was expressed only at very low levels in the resistant cns-derived neurons. blocking nk cells with anti-nkg2d antibodies inhibited nk cell-mediated killing of the drg neurons. these findings are important in revealing novel immunopathologic effects of several cns diseases. indeed, progressive motor and sensory neuropathy developed in a patient with chronic nk cell lymphocytosis (cnkl) (noguchi et al., 2005) . a sural nerve biopsy revealed infiltration of nk cells into the nerve fascicles, and demyelinating changes with secondary axonal degeneration. the infiltrating nk cells were adjacent to myelinated fibres, showing damage of schwann cell membranes. treatment with oral prednisolone resulted in rapid improvement of sensory disturbance and weakness with a significant decrease of nk cells in the blood and disappearance of the conduction block. these observations suggest that the infiltrating nk cells may directly damage myelin and schwann cells, thus causing demyelination. since expression of nkg2d ligands is likely regulated by viral infection or transformation, and the inhibitory mhc class i expression is low or absent in the nervous system, it is plausible that a viral infection or transformation could well break the balance of activating/inhibiting activities on nk cells, and nk cell-mediated immune pathology would occur in such circumstances. human oligodendrocytes do not express mhc class ii molecules; thus, direct mhc-restricted injury mediated by myelin-reactive cd4  t cells is less likely to occur. the migration of nk cells to the cns during inflammatory responses and lack of inhibitory signal mhc class i expression within the cns invites prediction that direct cytolytic effects of nk cells contribute to oligodendrocyte damage and demyelination to cns diseases such as ms. antel and colleagues demonstrated using in vitro assays that human oligodendrocytes, as well as other glial elements (astrocytes, microglia), were susceptible to injury mediated by peripheral blood-derived mononuclear cell preparations (mncs), which were enriched for nk cells by depleting cd3  , with or without cd19  cells through the use of either magnetic beads or cell sorting (antel et al., 1998; morse et al., 2001) . cytotoxic effects of the nk cell-enriched effectors were dependant on pre-exposure of these cells to il-2. furthermore, it was found that autologous oligodendrocytes were as susceptible to injury mediated by il-2-activated nk cells as were heterogonous oligodendrocytes. in searching for receptor ligand pathways that control the nk cell and oligodendrocyte interactions, it was found that human adult oligodendrocytes and foetal astrocytes expressed ligands for nkg2d in vitro, whereas neurons, microglia, and adult astrocytes did not (saikali et al., 2007) . disruption of the nkg2d-nkg2d ligand interaction using blocking antibodies significantly inhibited killing of primary human oligodendrocytes mediated by activated human nk cells (saikali et al., 2007) . these results imply that nkg2d-nkg2d ligand interactions can potentially contribute to cytotoxic responses mediated by activated immune effector cells in the inflamed cns, as observed in ms. in the context of tissue injury that occurs in ms, the inflammatory milieu in ms lesions may provide conditions required for nk cell activation, raising the possibility that such effector cells would play a role in the pathogenesis. in addition to direct cytotoxicity, cytokine release by nk cells may also participate in tissue damage as well as in regulating t cell immune responses. interferon-gamma (ifn-) is a pleiotropic cytokine produced by t cells and nk cells that has been implicated as a deleterious factor in ms, the immune-mediated demyelinating disorder. in vitro, purified developing and mature oligodendrocytes die in the presence of ifn- by apoptosis and necrosis, respectively. transgenic expression of plp/socs1 (proteolipid protein regulating suppressor of cytokine signalling 1), brings about diminished oligodendrocyte responsiveness to ifn- attributable to the targeted expression of socs1 in these cells (balabanov et al., 2006) . consequently, oligodendrocytes in the plp/socs1 transgenic mice are protected against the injurious effect of ifn-. although both nk cells and t cells produce ifn-, nk cells are the principal sources of early ifn- production prior to t cell activation. this time kinetic might be particularly relevant for early oligodendrocyte damage during inflammation. efficient early control of viral infections is determined by viral tissue tropism and rate of replication as well as the host's ability to mount an effective immune response. cellular cytotoxicity, in particular, that of nk cells and cytotoxic t cells, is central to the early antiviral immune response. table 28 .1 illustrates several immunedeficient conditions in humans, which stem from mutations affecting nk cells (biron et al., 1989; gilmour et al., 2001; markel et al., 2004; moins-teisserenc et al., 1999) . a number of studies have demonstrated the recruitment and activation of nk cells following infection with a wide range of viruses. however, not all viral infections p a r t i i i are susceptible to nk-mediated clearance, and susceptibility depends upon the effector mechanisms induced. for example, the induction of both cytotoxicity and ifn- production by nk cells following murine cytomegalovirus (cmv) and influenza virus infection results in reduced virally induced disease and enhanced survival. along the same time, deficient ifn- production by nk cells correlates with the absence of an effective innate response to lymphocytic choriomeningitis virus infection. moreover, the organs targeted by viral infection can also influence the participation of nk cells. indeed, it has been shown that the nk response to murine cmv is perforin-dependent within the spleen, whereas production of ifn- is required for viral clearance from the liver. these results indicate that the importance of the nk cell response to viral infection can depend upon multiple factors, including the tissue infected, as well as the effector mechanisms induced. although a number of studies have documented the possible role for nk cells in controlling cns infection with cmv, influenza and other viruses, the following studies provide relatively direct evidence for the importance of nk cells during cns viral infection: theiler's murine encephalomyelitis virus (tmev) is a picornavirus. infection of susceptible mice (sjl) with tmev causes a biphasic disease characterized by grey matter inflammation followed by late chronic demyelination (roos and wollmann, 1984; rosenthal et al., 1986) . after inoculation, cns tmev titres were higher in sjl mice compared with c57bl/10 mice, correlating with a 50% lower nk cell activity in the sjl mice than in the c57bl/10 mice (paya et al., 1989) . clinically, sjl mice are much more susceptible than c57bl/10 mice to tmev. when resistant (c57bl/10) mice were depleted of nk cells using either mab nk1.1 or polyclonal anti-asialo-gm1, tmev induced the development of diffuse encephalitis and meningitis early in the post-infection period. however, the second phase of tmev-induced cns disease (demyelination) was observed only in resistant c57bl/10 mice treated with anti-asialo-gm1. experiments with beige/beige mice of c57bl/10 background showed a mild degree of grey matter inflammation but no demyelination (paya et al., 1989) . nk cells are critical effectors in protecting against tmev-induced grey matter disease, whereas a different population of either nk1.1-nk cells, or other activated lymphocytes, may be critical in resistance or susceptibility to demyelination. in support of the involvement of nk cells during tmev of the cns, another study demonstrated that stressed mice developed clinical signs of encephalitis, thymic atrophy, and adrenal hypertrophy after infection with theiler's virus (welsh et al., 2004) . this syndrome was associated with significantly reduced virus-induced nk cell cytotoxic activity in restrained mice at 1 day post-infection, which may account for the reduced viral clearance from the cns. mouse hepatitis virus (mhv) is a coronavirus that causes infection of the cns (marten et al., 2001; wang et al., 1990) . intracerebral infection of susceptible strains of mice with mhv results in an acute encephalomyelitis followed by a chronic immune-mediated demyelinating disease that is similar in pathology to the human demyelinating disease ms (walsh et al., 2007; zuo et al., 2006) . intracerebral infection of rag1 / mice with a recombinant cxcl10-expressing murine coronavirus (mhv) resulted in protection from disease and increased survival that correlated with a significant increase in recruitment and activation of nk cells within the cns (walsh et al., 2007) . accumulation of nk cells resulted in a reduction in viral titres that was dependant on ifn- secretion (walsh et al., 2007) . these results indicate that the cxcl10-guided nk cell homing to the cns might play a pivotal role in defence following coronavirus infection of the cns. semliki forest virus (sfv) is a positive-stranded rna virus (atkins et al., 1999; smithburn and haddow, 1944) . infection of c57bl/6 mice with sfv leads to pronounced cns cellular infiltration and apoptosis of microglial and neuronal cells (alsharifi et al., 2006) . in this model, nk cells and, to a lesser degree, cytotoxic t cells are major contributors in combating sfv infection. mice lacking the tc cell compartment ( 2 -microglobulin-deficient mice, and thus cd8  t cells) exhibit susceptibility similar to wild-type mice. depletion of nk cells significantly delayed the mean time to death but did not prevent mortality in sfv-infected b6 mice suggesting that cytolytic activity of nk cells is detrimental, while ifn- production is beneficial for recovery from sfv infection (alsharifi et al., 2006) . with greater than 1.6 million americans infected annually (armstrong et al., 2001) , herpes simplex virus type 1 and 2 (hsv-1, hsv-2) are pathogens with a significant impact on public health. typically, infection results in a life-long latent infection of the host (halioua and malkin, 1999; whitley, 2002) . the transmission of hsv-2 in the human population includes asymptomatic shedding of the virus even in the presence of cd8  cytotoxic t lymphocytes and the production of a viral glycoprotein that indirectly elicits nk cell death (bellner et al., 2005; posavad et al., 2000; wald et al., 2000) . in a mouse model of hsv-2 infection, it was shown that mice deficient in ccr5 (ccr5 / ) displayed a significant reduction in cumulative survival following infection in comparison with wild-type hsv-2-infected controls. associated with decreased resistance to viral infection, ccr5 / mice yielded significantly more virus and expressed higher levels of tumour necrosis factor alpha (tnf-), cxcl1, ccl2, ccl3 and ccl5 in the vagina, spinal cord, and/or brain stem than did wild-type mice. in addition, when comparing wildtype hsv-2-infected mice with ccr5 / mice prior to or after infection, there were significantly more nk cells (nk1.1  cd3  ) residing in the brain stem and spleen of infected wild-type mice. functionally, nk activity from cells isolated from the brain stem of hsv-2-infected wild-type mice was greater than that from hsv-2-infected ccr5 / mice. further, antibody-mediated depletion of nk cells resulted in an increase in hsv-2 levels in the vaginal, spinal cord and brain stem tissue of wild-type mice but not ccr5 / mice (kveberg et al., 2002) . collectively, the absence of ccr5 expression significantly impacts the ability of the host to control genital hsv-2 infection, inflammation and spread associated with a specific reduction in nk cell expansion, infiltration and activity in the nervous system. congenital toxoplasmosis poses a public health problem, being capable of causing foetal death and ocular and neurological sequelae in congenitally infected children. congenital infection occurs only when mothers first encounter toxoplasma gondii (t. gondii) during pregnancy (remington et al., 1994; roberts and alexander, 1992) . resistance to t. gondii is mainly mediated by type 1 cytokines, such as ifn- and interleukin 2 (il-2), whereas type 2 cytokines, such as il-4 and il-10, are associated with increased susceptibility to infection (hunter et al., 1996; khan et al., 1994) . susceptibility of the pregnant host to toxoplasmosis may be attributable to a type 2-cytokine bias that is maintained during gestation (shirahata et al., 1992) . cell-mediated immune responses involving cd4 and cd8t cells and nk cells play a protective role in t. gondii primary infection (goldszmid et al., 2007; scharton-kersten et al., 1998; scorza et al., 2003; scott and trinchieri, 1995; subauste et al., 1992) . to clarify the roles of nk cells and ifn- in protection against primary congenital toxoplasmosis, abou-bacar and colleagues (2004) used recombination activating gene 2 knockout (ko) (rag-2 / ) mice, which lack t and b lymphocytes, in comparison with the wild-type balb/c model. rag-2 / mice had a significantly lower risk of foetal toxoplasmosis than balb/c mice. this protection was associated with an increased number of maternal nk cells, ifn- secretion by spleen cells, and decreased parasitemia. in the rag-2 / mice, nk cell depletion increased the rate of foetal infection. these data suggest that a partially protective immunity against congenital toxoplasmosis is achieved owing to the increased number of nk cells in rag-2 / mice (abou-bacar et al., 2004) . protective effect of nk cells was confirmed in another study using the scid model (kang and suzuki, 2001) . the innate immune system plays an instrumental role in generating and directing the adaptive immune responses (shi et al., 2001) . nk cells represent a critical first line of defence against malignant transformation. earlier results by karre and ljunggren demonstrated that nk cells can preferentially kill and reject cells that fail to express 'self ' mhc class i molecules (karre et al., 1986) . these findings led to the formation of the famous 'missing self hypothesis' (ljunggren and karre, 1990) . over the years, the missing self hypothesis has been repeatedly demonstrated in a variety of experimental tumour systems by different groups of investigators, and a number of molecular pathways governing the interactions of nk cell-target cells have been revealed. surveillance against 'missing self ' may thus be one, but not the only function of nk cells (ljunggren and malmberg, 2007) . non-surgical resectable tumours within the cns constitute significant challenges for physicians. furthermore, studies have documented frequent immune system defects in intracranial tumour-bearing patients and an inability of certain lymphocyte subset to mediate antitumour effector functions in the cns. malignant melanoma is notorious for metastasis to discrete locations such as testis and brain. malignant melanoma is the third most common type of cancer that metastasizes to the brain (prins et al., 2006) , which presents clinicians with few treatment options. although nearly a dozen melanoma antigens specifically recognized by t cells have been identified, melanoma cells are still able to avoid immune destruction in most instances. because the generation of an effective antitumour immune response requires both the presence of foreign antigen and a costimulatory molecule or signal, tumour cells displaying tumour antigens may avoid immune detection because of the absence of appropriate costimulation. thus, anti-tumour immune responses might be achieved by more effective local delivery of costimulatory molecules. activation and expansion of nk cells may independently lyse tumour cells, or provide t cells with costimulatory molecules including cytokines, and overall enhance antigen presentation to t cells. several attempts have been made in an effort to use nk cells to target cns melanoma. the specific receptor for il-2 on nk cells allows several approaches to deliver il-2 intrathecally and activate nk cells. ewend and associates carried out a study in c57bl/6 mice that were simultaneously given intracranial injections of tumour and of irradiated b16f10 melanoma cells transduced to secrete il-2 (prins et al., 2006) . il-2 therapy generated antitumour responses capable of extending the survival of animals that received simultaneous intracranial tumour challenges either locally or at distant sites in the brain. non-transduced melanoma cells had little effect. elimination of t-cell and nk subsets using gene ko mice and antibody-depletion techniques demonstrated that nk cells were most important for the initial anti-tumour response, whereas cd4  t cells were not necessary. these studies demonstrate that local il-2 therapy in the brain not only generates an immediate local antitumour immune response, but also establishes long-term immunologic memory capable of eliminating subsequent tumour challenges within and outside of the cns. furthermore, the antitumour response to paracrine il-2 in the brain differed significantly from that in the flank, suggesting that the intrinsic cns cells involved in initiating immunity within the brain have different cytokine requirements from their peripheral counterparts. using the same model, a recent study showed that dcs administration induced dramatic anti-tumour immune protection in cd8 ko mice that were challenged with b16 melanoma both subcutaneously and in the brain (ewend et al., 2000) . the cns anti-tumour immunity was dependant on both cd4  t cells and nk cells. administration of non-ag-loaded, immature dc resulted in significant cd4  t cell and nk cell expansion in the draining lymph nodes at 6 days post-vaccination, which persisted for 2 weeks. finally, ag-loaded dc administration in cd8 ko mice was associated with robust infiltration of cd4  t cells and nk cells into the brain tumour parenchyma (ewend et al., 2000) . glioma cells interfere with anti-tumour immune responses by expressing immune inhibitory cell surface molecules, such as hla-g, or by releasing soluble immunosuppressants such as transforming growth factor (tgf-). they rarely metastasize outside the brain, raising the possibility of immune-mediated control of these cells outside, but not inside, the brain. il-2, as well as growth hormone, is potent in enhancing nk cell activity against glioma both in human trials and in several experimental systems (eisele et al., 2006; hayes et al., 1995; shimizu et al., 2005; wischhusen et al., 2005) . as receptors governing nk cell action and effector functions are being elucidated, more sophisticated means of manipulating nk cells have been generated. as noted above, nkg2d is a powerful, activating nk cell receptor (wischhusen et al., 2005) . accordingly, activating the innate immune system by forcing glioma cells to express danger signals such as nkg2d ligands is a promising strategy of immunotherapy for these tumours. the remaining challenges would be to downregulate hla-e expression on glioma cells and suppress production of tgf- by glioma. both hla-e and tgfbeta can down-regulate nkg2d expression on glioma, which enable these tumour cells to escape nk cell surveillance. various studies have documented the role of nk cells in surveillance and suppression of other type of cns tumours including medulloblastoma (castriconi et al., 2007) , astrocytoma and neuroblastoma (kang et al., 2004) . on the other hand nk cells appear to have little, if any, role in suppressing cns lymphomas (yamasaki et al., 2003) . clearly, cumulative evidence suggests that nk cells play a role in curbing malignant transformation and progression of many primary and metastatic cns tumours (table 28 .2). direct cytotoxic effects and collaboration with t cells and other immune cells are required to achieve these functions (table 28 .2). effective therapies harnessing nk cells will be facilitated through understanding of the molecular signalling pathways that will be governing nk cell activation, expansion and maintenance. specific anatomical factions within the cns should also be considered. furthermore, effort must be taken in suppressing the capacity of certain tumours to down-regulate activating signals and production of inhibitory proteins against nk cells. during cns infection, cytolytic activity of nk cells contributes to elimination of viral and bacterial infected cells and controls the magnitude of inflammation. debris from neuronal and/or glial cell death is taken up by antigen-presenting cells (apcs) and presentation to t cells. cytokine (ifn-) secretion by nk cells increases mhc class ii expression by apc and, thus, favours generation of th1 type of t helper cells. thus, nk cells function not only as the initial line of host defence, but also as fuel to the generation of adaptive immune responses. overall, nk cells are expected to boost immune response within the cns. paradoxically, emerging evidence suggests that nk cells can inhibit cns inflammation and control the magnitude of autoimmunity (table 28 .2). ms is a classic autoimmune disease characterized by extensive cns inflammation and immune-mediated destruction of myelin. consequently, the function of myelin sheaths becomes compromised and neurological impairment occurs. the pathogenesis of ms is mirrored, in part, in eae, which can be induced in susceptible strains of mice with neuron-antigens and complete freund's adjuvant. the roles of nk cells in the pathogenesis of ms and eae have been investigated. in patients with ms, nk cells (cd56 and cd57) are present in the peripheral blood with reduced numbers and cytolytic activity (shibatomi et al., 2001; trinchieri, 1989) . this finding is not unique to ms and similar phenotype of nk cells have been documented in many other types of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, etc (shibatomi et al., 2001) . in parallel, patients with ms and other autoimmune diseases have defective functions of 'regulatory cells', including nkt cells and cd4  cd25  regulatory t cells (treg) (la cava et al., 2006) . a reduced number and/or compromised function of nk cells, nkt cells and treg cells invite a hypothesis that autoimmunity is associated with a result of global defective regulatory cell functions in these patients. this hypothesis has been tested in several eae models. because gene encoding nk cells cannot be targeted by the current technology, several depleting antibodies have been used to study the function of nk cells in vivo. several groups have utilized anti-nk1.1 mab and observed that depletion of nk cells by injecting anti-nk1.1 mab leads to exacerbation of eae (zhang et al., 1997) . apparently, both peripheral and cns nk cells are absent in this experimental system. it is, therefore, not possible to differentiate the role of nk cells in the periphery and in the cns. nk cell homing to cns is controlled by a specific chemokine receptor ligand pathway involving cx3cr1 and fractalkine (cx3cl1). cx3cr1 is expressed almost exclusively by cns glial cells (boehme et al., 2000; cardona et al., 2006) . thus, germ-line deletion of cx3cr1 leads to impaired homing of nk cells to the cns. this model would be ideal in addressing cns inflammation/autoimmunity in relation to nk cells. interestingly, upon immunization, cx3cr1-deficient mice with reduced nk cells in the cns and intact nk cells in the periphery developed their wild-type controls. thus, lack of cns nk cells alone is sufficient to cause exacerbated cns inflammation and autoimmunity (huang et al., 2006) . it is also conceivable that chemokine guided nk cell homing to cns might serve as pathway that can be therapeutically targeted (figure 28.1) . infection is suggested to play a role triggering the initiation of ms in some patients (bendelac and medzhitov, 2002; pulendran and ahmed, 2006; shirahata et al., 1992) . the use of complete freund's adjuvant in the induction of eae may mimic this process (fearon and locksley, 1996) . in these patients or in eae animals, nk cells may contribute to the demyelination through bystander damage while controlling the infection. once infection is controlled, nk cells may function to inhibit the excessive (auto) immune responses elicited by pathogens. the immune system may use nk cell as a versatile regulator to tune its capacity in combating infection and avoiding autoimmunity. the mechanism underlying this unique role for nk cells within the cns during eae is still elusive. a close survey of the literature reveals multiple steps where nk cells can regulate inflammation and intervene in the loss of self-tolerance. importantly, the findings also caution against inferring a similar role for nk cells in all types of autoimmune phenomena or during separate stages of the same disease (flodstrom et al., 2002; yokoyama and plougastel, 2003) . nk cells can both promote and inhibit autoreactive t cells. these possibilities have been extensively reviewed recently (shi and van kaer, 2006) . the specific cns anatomical location, as reflected by diverse cns apcs and multiple antigens may also influence the outcome of autoimmunity. as with eae, it appears that nk cells control t cell proliferation in an antigen non-specific manner, both in the periphery and within the cns (shi, et al., unpublished) . recently, it has been demonstrated that human nk cells kill resting but not activated microglia via nkg2d-and nkp46-mediated recognition (lünemann et al., 2008) . this study emphasizes the potential importance of interactions between nk cells and cns resident apcs. however, whether the regulatory effects of nk cells can be attributed to the action of nk cells on apcs, directly on t cells, or both, is not known and currently under investigation. nk cells readily accumulate in homing to cns tissues under the pathophysiological situations. this process is tightly controlled by a number of chemokines and chemokine receptors. there is ample of evidence that nk cells within the cns contribute to the control of infections and might limit progression of certain tumours. bystander neuronal and/or glial cell damage can occur. in certain autoimmune conditions of the cns, nk cells appear to have an inhibitory role. activation and expansion of nk cells through engaging il-2 receptors on nk cells not only inhibit several cns tumours, but also might slow the progression of ms and other autoimmune diseases (bielekova et al., 2006; li et al., 2005) . furthermore, the ability of ifn- and ifn- to ameliorate ms in humans and ifn- to inhibit eae in mice may reflect the ability of these cytokines to transiently activate nk-dependent regulatory responses. however, because ifn treatment also upregulates qa-1 expression on t cells (ota et al., 2005) , the short duration and usually modest nature of these therapeutic effects may reflect a qa-1-dependent decrease in nk cell activation and associated immunoregulatory activity (lu et al., 2007) . disassociation of disease-inhibiting versus diseasepromoting effects of nk cells is a key to harnessing nk cells for therapeutic purposes. to achieve this goal, a generation of genetic models with selective nk cell deficiency, and development of reagents (antibodies) for visualizing subsets of nk cells in situ will be necessary. optimization of methods to produce nk cells in large quantities for therapeutic usage is also important. clearly, understanding the molecular signals and pathways governing these differential biological effects of nk cells as well as their cross talk with t cells is key to designing nk cell-based therapy for cns infections, tumours and autoimmune diseases. role of nk cells and gamma interferon in transplacental passage of toxoplasma gondii in a mouse model of primary infection ccr5 deficiency drives enhanced natural killer cell trafficking to and activation within the liver in murine t cell-mediated hepatitis nk cell-mediated immunopathology during an acute viral infection of the cns non-mhc-restricted cellmediated lysis of human oligodendrocytes in 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thank dr r. liu, dr w. piao, and dr. dayao and ms. linda swanson for their wonderful assistance in preparation of this chapter. the muscular dystrophy association, the barrow neurological foundation, and the national institutes of health support the authors' work in the laboratories. key: cord-332109-ont0tqpn authors: wei, yufeng; shah, rameen title: substance use disorder in the covid-19 pandemic: a systematic review of vulnerabilities and complications date: 2020-07-18 journal: pharmaceuticals (basel) doi: 10.3390/ph13070155 sha: doc_id: 332109 cord_uid: ont0tqpn as the world endures the coronavirus disease 2019 (covid-19) pandemic, the conditions of 35 million vulnerable individuals struggling with substance use disorders (suds) worldwide have not received sufficient attention for their special health and medical needs. many of these individuals are complicated by underlying health conditions, such as cardiovascular and lung diseases and undermined immune systems. during the pandemic, access to the healthcare systems and support groups is greatly diminished. current research on covid-19 has not addressed the unique challenges facing individuals with suds, including the heightened vulnerability and susceptibility to the disease. in this systematic review, we will discuss the pathogenesis and pathology of covid-19, and highlight potential risk factors and complications to these individuals. we will also provide insights and considerations for covid-19 treatment and prevention in patients with suds. the novel coronavirus 2019, now officially termed as sars-cov-2, causes the coronavirus disease 2019 (covid-19) by infecting the respiratory system [1] . the disease was first detected and reported in wuhan, china, in december 2019 [2] , and has now spread to over 150 countries on all continents except antarctica. according to the world health organization (who) coronavirus disease dashboard, the global tally of coronavirus cases has approached 13.5 million, with a death toll of over 580,000 [3] at the time of writing (15 july 2020). the united states alone has counted over 3.4 million infections and over 131,000 deaths, contributing more than a quarter of both overall infections and deaths globally (john hopkins university coronavirus resource center) [4] . the who characterized the covid-19 as a pandemic on 11 march 2020 [5] . the fatality rate of the disease is particularly high among patients who are older and who have underlying health issues, such as cancer, diabetes, and compromised lung function or lung disease. the united nations (un) reported that 35 million people worldwide suffer from substance use disorders (suds) [6] . in the u.s., the number of individuals experiencing suds is 20.3 million [7] . a large number of individuals with suds have underlying health conditions, particularly cardiovascular and lung diseases and hepatitis c or hiv-1 infections. together with complicated socioeconomic issues, these populations are particularly vulnerable to covid-19 [8] . despite the fact that researchers and clinicians around the world have collected and disseminated tremendous amounts of data on covid-19, we have very little knowledge of the interactions and comorbidity of covid-19 and sud. in this review, we will analyze relevant covid-19 and sud literatures, and highlight the susceptibilities and vulnerabilities for individuals with sud. similar to sars-cov, sars-cov-2 recognizes the angiotensin converting enzyme 2 (ace2) receptor by its s protein and utilizes it for cell entry [20, 22] . the heavily glycosylated s protein triggers virus cell entry by fusing the receptor binding domain (rbd) on the s1 subunit to the host ace2 receptor, engaging the transition of s2 subunit to a stable post-fusion conformation [23] . cryoelectron microscopy (em) structures of the pre-fusion [23] and post-fusion structures [24] of the s protein have been reported. the sars-cov-2 s protein has been shown to have a much higher binding affinity to the ace2 than the sars-cov s protein [23, 25] . the s protein contains 22 n-linked glycans, and the complex glycosylation is likely to play a role in shielding and camouflaging for immune evasion of the virus [26, 27] . the s protein is activated by type ii transmembrane serine protease (tmprss2), a host protease co-expressed with ace2 on the cell surface [24, 28] . in cells not expressing tmprss2, other proteases, such as cathepsin b/l, may activate the s protein and facilitate viral entry [29] . upon cell entry, sars-cov-2 has a similar life cycle and pathogenesis as other β-coronaviruses, including sars-cov and mers-cov [30] . upon ace2 receptor binding, the virus fuses its membrane with the host cell plasma membrane, releasing its genomic rna into the cytoplasm. since the viral rna is similar to the human messenger rna (mrna), it triggers the host ribosome to start translating the viral rna and producing viral proteins. the viral replicase orf is translated into two overlapping polyproteins, pp1a (nsp1-11) and pp1ab (nsp1-16), which require extensive processing. nsp5, the 33.8-kda main viral protease (m pro ), also referred to as the 3-chymotrypsin-like protease (3cl pro ), performs the function by autolytic cleavage of the protease itself, and then subsequently similar to sars-cov, sars-cov-2 recognizes the angiotensin converting enzyme 2 (ace2) receptor by its s protein and utilizes it for cell entry [20, 22] . the heavily glycosylated s protein triggers virus cell entry by fusing the receptor binding domain (rbd) on the s1 subunit to the host ace2 receptor, engaging the transition of s2 subunit to a stable post-fusion conformation [23] . cryo-electron microscopy (em) structures of the pre-fusion [23] and post-fusion structures [24] of the s protein have been reported. the sars-cov-2 s protein has been shown to have a much higher binding affinity to the ace2 than the sars-cov s protein [23, 25] . the s protein contains 22 n-linked glycans, and the complex glycosylation is likely to play a role in shielding and camouflaging for immune evasion of the virus [26, 27] . the s protein is activated by type ii transmembrane serine protease (tmprss2), a host protease co-expressed with ace2 on the cell surface [24, 28] . in cells not expressing tmprss2, other proteases, such as cathepsin b/l, may activate the s protein and facilitate viral entry [29] . upon cell entry, sars-cov-2 has a similar life cycle and pathogenesis as other β-coronaviruses, including sars-cov and mers-cov [30] . upon ace2 receptor binding, the virus fuses its membrane with the host cell plasma membrane, releasing its genomic rna into the cytoplasm. since the viral rna is similar to the human messenger rna (mrna), it triggers the host ribosome to start translating the viral rna and producing viral proteins. the viral replicase orf is translated into two overlapping polyproteins, pp1a (nsp1-11) and pp1ab (nsp1-16), which require extensive processing. nsp5, the 33.8-kda main viral protease (m pro ), also referred to as the 3-chymotrypsin-like protease (3cl pro ), performs the function by autolytic cleavage of the protease itself, and then subsequently digests the polyproteins into 16 non-structural proteins. nsp12, known as the rna-dependent rna polymerase (rdrp), together with nsp7 and nsp8, carries out the critical process of the viral rna synthesis, and is central to the viral replication and transcription cycle. the n-terminal non-structural protein, nsp1, has been shown to bind to the 40s small ribosomal subunit, shutting down all host cell protein production by blocking the mrna entry tunnel. nsp1 binding to ribosomes and blocking host cell translation effectively inhibits type-i interferon (ifn-i)-induced innate immune response by turning off the retinoic acid-inducible gene (rig)-i antiviral sensor [31] . the inhibition of the ifn-i-induced innate immunity allows the assembly of viral particles inside the host cell. the newly produced structural proteins, s, m, and e, are inserted into the endoplasmic reticulum (er) or golgi membrane, while the n protein associates with the newly synthesized viral rna to stabilize the genome. the viral particles are assembled into the er-golgi intermediate compartment (ergic), fuse with the plasma membrane, and bud off the host cell. the released virions will further infect more cells. the functions of other nsps are not fully understood. a comparative structural genomics study revealed a possible functional intra-viral and human-virus interaction network of nsps [32] . recurrent mutations in the sars-cov-2 genome have been identified in some nsps and the s protein, suggesting ongoing adaptations of the coronavirus through transmission [33] . particularly, the d614g mutation in the s protein makes it more stable, and the virus becomes more infectious and transmissible [34, 35] . this mutated virus is the dominant form in europe and north america since march 2020 [36] . underlying medical conditions can put individuals at increased risk for severe illness from covid-19. the comorbid conditions include copd, cardiovascular diseases, other chronical respiratory diseases, diabetes, obesity, and cancer. according to a large-scale study with 72,314 cases conducted by the chinese cdc, case-fatality and mortality rates are significantly increased in patients with comorbid conditions comparing to those with no underlying conditions (table 1 ) [12] . in a study in new york city, the epicenter of the covid-19 pandemic in the u.s., comorbid conditions are highly associated with hospitalization and severity of the illness (table 2 ) [37] . a nationwide case-control study in korea also confirmed that diabetes, hypertension, and chronic respiratory disease, among others, were associated with severe covid-19 [38] . individuals with suds commonly experience respiratory and cardiovascular disorders, including hypertension and copd, and have undermined immune systems, making them particularly vulnerable in covid-19. a significant portion of individuals with suds have underlying medical conditions, and are more likely to be marginalized. according to a recent study in british columbia, canada, among 19,005 individuals who had one or more non-fatal overdose events between 2015 and 2017, 10,649 (56.0%) had a record of receiving social assistance, and 5716 (30.0%) had no fixed address record. these individuals with a history of overdose are more likely to have at least three known chronical conditions associated with covid-19 severity, including chronical pulmonary disease, diabetes, and coronary heart disease, with adjusted odds ratios (ors) to be 2.01, 1.24 and 2.08, respectively, with reference to people without an overdose [39] . during the covid-19 pandemic, risks of abusing substances and addictive behaviors are also increasing. the stress and social isolation associated with the response to covid-19 increases the risk of alcohol abuse and misuse, which is known to suppress immune systems and cause emotional dysregulation [40] . a study in china showed that relapses of alcohol and smoking abuse were prominent (18.7% and 25.3%, respectively), and 32.1% of regular drinkers and 19.7% of regular smokers increased alcohol and cigarette consumption [41] . sars-cov-2 can attack and damage human organs through two major events: direct viral attacks against target organs and abnormal immune responses and inflammation [42] . initial evidence focuses on the damage to the respiratory system and the lung, and is correlated with clinical symptoms of the patients [2, 12, 20, 21] . the identified viral entry receptor, ace2, is abundantly expressed in the epithelial cells along the respiratory tract and the lung alveoli [29, 43, 44] . high level expression of ace2 receptors is also reported in organs and tissues outside the respiratory system, including the heart, kidney, and intestine [45] . therefore, these organs are the potential targets of and could be damaged by sars-cov-2. as mentioned earlier, smoking of tobacco and marijuana directly impairs respiratory system. other substances of abuse can cause cardiovascular diseases, which amplify respiratory and pulmonary complications. we will discuss these complications in the next section. more severely, ace2 is abundantly expressed in vascular endothelial cells [45] . several clinical cases have been reported to indicate direct involvement of vascular endothelial cells in covid-19 pathology at different organs, suggesting that the damages to the lung, heart, kidney, liver, small intestine, and bowel, are actually caused by endotheliitis (endothelialitis). direct viral infection of endothelial cells induces inflammation and inflammatory cell death at the endothelium ( figure 2 ) [46] . comparing lung tissues from deceased patients of acute respiratory distress syndrome (ards) associated with influenza and covid-19, the lungs from covid-19 patients displayed distinctive vascular impairments of the pulmonary vessels. most significantly, viruses were found inside the endothelial cells of the lung tissues from covid-19 patients, which disrupted cell membrane, caused prevalent thrombosis with microangiopathy, and induced elevated intussusceptive angiogenesis [47] . a greater number of ace2+ endothelial cells were found in covid-19 patients, correlating to changes in endothelial morphology, including disruption of endothelial cell junctions, cell swelling, and detachment from the basal membrane. the presence of the sars-cov-2 virus inside the endothelial cells, together with the induced inflammation, may directly contribute to the endothelial injury [46, 47] . although there are no reported cases yet, one potential target of sars-cov-2 infection is the brain microvascular endothelial cell (bmvec). bmvecs line up the microcapillary beds and form the blood-brain barrier (bbb) together with brain astrocytes and pericytes. the bbb prevents pathogens and toxins from trespassing into the brain side. tight-junction (tj) protein complexes, composed of occludin, claudins, junctional adhesion molecules (jams) and membrane-directed scaffolding protein zonulae occludentes (zo), form a physical inter-endothelial barrier that strictly controls migration of molecular and cellular contents from the circulation into the brain ( figure 2 ) [48, 49] . high expression of efflux pumps and stereospecific solute transporters at the endothelium additionally limits molecules from crossing the barrier [50] [51] [52] . the bbb plays an essential role in protecting the brain from pathogen invasion. viral infection of bmvecs could result in endothelial dysfunction, leakage, and even rupture, and is detrimental to the bbb integrity. the damaged bbb allows the virus to migrate into the brain side, and infect neuronal tissues [53] . another possible route of cns invasion could be through invading the peripheral nerve terminals and then entering the cns via trans-synaptic transfer [54] . the first case of meningitis associated with sars-cov-2 has been reported, in which viral rna was detected in the cerebrospinal fluid (csf) of the patient [55] . clinical evidence from wuhan, china showed that more than 1/3 of covid-19 patients had neurological symptoms, and cns involvement was linked to the prognosis and severity of the disease [56, 57] . substances of abuse have been shown to severely compromise the endothelial barrier at the bbb, leading to increased bbb permeability and possibly intensified brain damage in covid-19 ( figure 2 ). pharmaceuticals 2020, 13, x for peer review 6 of 29 dysfunction, leakage, and even rupture, and is detrimental to the bbb integrity. the damaged bbb allows the virus to migrate into the brain side, and infect neuronal tissues [53] . another possible route of cns invasion could be through invading the peripheral nerve terminals and then entering the cns via trans-synaptic transfer [54] . the first case of meningitis associated with sars-cov-2 has been reported, in which viral rna was detected in the cerebrospinal fluid (csf) of the patient [55] . clinical evidence from wuhan, china showed that more than 1/3 of covid-19 patients had neurological symptoms, and cns involvement was linked to the prognosis and severity of the disease [56, 57] . substances of abuse have been shown to severely compromise the endothelial barrier at the bbb, leading to increased bbb permeability and possibly intensified brain damage in covid-19 ( figure 2 ). the other aspect of covid-19 pathology involves abnormal immune responses, which could exaggerate into a cytokine storm [58, 59] . sars-cov-2 dramatically promotes host cell kinase activities, including casein kinase ii (ck2) and p38, and stimulates production of diverse cytokines [60] . evidence has shown that covid-19 elevates proinflammatory cytokines and chemokines, including tumor necrosis factor (tnf)-α, interleukin (il)-1β and il-6, granulocyte-colony stimulating factor (g-csf), interferon γ (ifn-γ)-induced protein-10 (ip-10), monocyte chemoattractant protein-1 (mcp-1), and macrophage inflammatory proteins-1α (mip-1α) [61] [62] [63] . although there has been no reported evidence, it is possible that pattern recognition receptors, such as toll-like receptors (tlr3, tlr7, and tlr8), rig-i, and nucleotide-binding oligomerization domain (nod)-like receptors (nlrp1, nlrp3, and nlrp12), are also activated by covid-19 through innate immunity [64] . it has been reported that il-6 was significantly increased in severe covid-19 cases, and its level was closely correlated with the severity of patients [65] . human bronchial epithelial cells infected with sarsthe other aspect of covid-19 pathology involves abnormal immune responses, which could exaggerate into a cytokine storm [58, 59] . sars-cov-2 dramatically promotes host cell kinase activities, including casein kinase ii (ck2) and p38, and stimulates production of diverse cytokines [60] . evidence has shown that covid-19 elevates proinflammatory cytokines and chemokines, including tumor necrosis factor (tnf)-α, interleukin (il)-1β and il-6, granulocyte-colony stimulating factor (g-csf), interferon γ (ifn-γ)-induced protein-10 (ip-10), monocyte chemoattractant protein-1 (mcp-1), and macrophage inflammatory proteins-1α (mip-1α) [61] [62] [63] . although there has been no reported evidence, it is possible that pattern recognition receptors, such as toll-like receptors (tlr3, tlr7, and tlr8), rig-i, and nucleotide-binding oligomerization domain (nod)-like receptors (nlrp1, nlrp3, and nlrp12), are also activated by covid-19 through innate immunity [64] . it has been reported that il-6 was significantly increased in severe covid-19 cases, and its level was closely correlated with the severity of patients [65] . human bronchial epithelial cells infected with sars-cov-2 showed elevated expression of type i and type iii ifns and il-6 [44] . furthermore, type i interferon, ifn-α, stimulates the expression of ace2, the molecular target of sars-cov-2, in primary human nasal epithelial cells [43] . type iii interferon, ifn-λ, has been shown to disrupt the lung epithelial barrier by direct inhibition of lung epithelial proliferation and repair, contributing to covid-19 pathogenesis in the lower airways [66] . the upregulation of il-6 and other proinflammatory cytokines was also observed in sars cases [67] and influenza infection [68] . substances of abuse can induce high level of expression of proinflammatory cytokines and chemokines in the cns and cause neuroinflammation, which can worsen inflammatory responses in covid-19. details will be discussed in the next section. the bidirectional communication between the brain and the immune system plays a critical role in covid-19 pathogenesis. it has been well established that brain-immune interactions are widespread and significant. for instance, the immune system produces hormones and neurotransmitters [69] [70] [71] , while the anterior pituitary cells in cns produce proinflammatory cytokines, such as il-6 [72] . microglial cells are immune effectors in the cns, which produce and secrete cytokines and neurotrophic or neuron survival factors upon inflammation and injury [73] . for pathogen infections, the innate immunity provides the first line of defense through recognition of pathogen-associated molecular patterns (pamps), initiating nonspecific cellular and humoral responses and rapidly activating nonspecific neural responses, including systemic hormonal responses through the hypothalamic-pituitary-adrenal (hpa) axis ( figure 3 ) [74] . consisting of the hypothalamus of the brain, and endocrine organs, the pituitary and cortex of the adrenal glands, the hpa axis is responsible for systematic inflammation control. the paraventricular nucleus (pvn) of the hypothalamus plays the main governing role of the hpa axis, releasing a wide range of neuropeptides, including the corticotrophin-releasing hormone (crh) and arginin-vasopressin (avp). these neuropeptides reach the anterior pituitary (ap) to activate corticotrope cells to secrete adrenocorticotropic hormone (acth). acth subsequently enables the synthesis and secretion of glucocorticoids in the zona fasciculata of the adrenal cortex through melanocortin type 2 receptors [75] . the physiological feedback loop involves releasing immune mediators and cytokines, such as il-1, il-6, and tumor-necrosis factors (tnfs), by the innate immune system to activate neural responses, which amplify local inflammation to contain and eliminate pathogen invasions. upon pathogen clearance, the brain responds by activating the hpa axis and releasing anti-inflammatory molecules, glucocorticoids, from the adrenal cortex. the release of this final product of the hpa axis sends a signal to the immune system to terminate the inflammatory responses, completing the hormonal negative-feedback loop. glucocorticoids also negatively regulate the hpa axis itself, restoring host homeostasis, including cns and cardiovascular system, as well as metabolic balances. the interplay between the nervous system and the immune system plays a critical role in forming a cohesive and integrated early host response for pathogen clearance through an optimized innate inflammatory response. impairment of the hpa axis by various substances of abuse could render the host highly susceptible to inflammation and even increased mortality from septic shock from exposure to infectious and proinflammatory triggers, including covid-19. inappropriate and excessive cns responses could predispose the host to extreme inflammation, including cytokine storm that has been observed clinically in influenza [68, 76] , sars [77] , and covid-19 [59] . excessive activation of the hpa axis and the release of glucocorticoids by several substances of abuse will suppress the activities of various immune cells, including macrophages, dendritic cells, and t cells, and will inhibit activities of nk cells, b cells, and t cells (figure 3 ) [74] . the immunosuppression reduces antibody production, cytotoxicity, and t cell-mediated immune responses, and is linked to higher incidences of pathogen infections, slowed recovery, and severe disease progression in covid-19. bidirectional communication between the brain and the immune system. the hpa axis: upon activation (cytokines, pathogens, etc.), the hypothalamus in the brain produces crh and avp, activating anterior pituitary, which secretes acth. acth circulates with general blood stream to reach adrenal gland, which synthesizes the anti-inflammatory molecule, glucocorticoids. glucocorticoids suppress the immune system and the expression of proinflammatory cytokines, which concludes the negative feedback and turns off the hpa axis. glucocorticoids suppress the activities of various immune cells, including macrophages, dendritic cells, and t cells, which are responsible for cytokine release. the immunosuppression also involves inhibition of nk cells, b cells, and t cells for reduced cytotoxicity, antibody production, and t cell-mediated immune responses. substances of abuse alter the hpa axis. excessive production of glucocorticoids suppresses immune responses to viral infection, leading to high incidences of infection and severe infection in covid-19. arrows indicate stimulation; blunted arrows indicate inhibition. for individuals with suds, both the infection of vascular endothelial cells and the proinflammatory immune responses of covid-19 could pose severe risks. it has been well studied that substances of abuse can cause irreversible bbb damage [78] and impair the hpa axis and immune responses [75] . to understand the vulnerabilities of suds in covid-19 infection at the molecular level, we will examine the brain-immune interactions and the hpa axis attenuation, neuroinflammation, immunosuppression, and bbb impairment induced by most commonly abused substances in the comorbidity of covid-19. nicotine is available in the form of tobacco, which also contains many other types of chemicals. tobacco smoking is associated with arterial thrombosis and atherosclerosis in the heart, abdomen, figure 3 . bidirectional communication between the brain and the immune system. the hpa axis: upon activation (cytokines, pathogens, etc.), the hypothalamus in the brain produces crh and avp, activating anterior pituitary, which secretes acth. acth circulates with general blood stream to reach adrenal gland, which synthesizes the anti-inflammatory molecule, glucocorticoids. glucocorticoids suppress the immune system and the expression of proinflammatory cytokines, which concludes the negative feedback and turns off the hpa axis. glucocorticoids suppress the activities of various immune cells, including macrophages, dendritic cells, and t cells, which are responsible for cytokine release. the immunosuppression also involves inhibition of nk cells, b cells, and t cells for reduced cytotoxicity, antibody production, and t cell-mediated immune responses. substances of abuse alter the hpa axis. excessive production of glucocorticoids suppresses immune responses to viral infection, leading to high incidences of infection and severe infection in covid-19. arrows indicate stimulation; blunted arrows indicate inhibition. for individuals with suds, both the infection of vascular endothelial cells and the proinflammatory immune responses of covid-19 could pose severe risks. it has been well studied that substances of abuse can cause irreversible bbb damage [78] and impair the hpa axis and immune responses [75] . to understand the vulnerabilities of suds in covid-19 infection at the molecular level, we will examine the brain-immune interactions and the hpa axis attenuation, neuroinflammation, immunosuppression, and bbb impairment induced by most commonly abused substances in the comorbidity of covid-19. nicotine is available in the form of tobacco, which also contains many other types of chemicals. tobacco smoking is associated with arterial thrombosis and atherosclerosis in the heart, abdomen, and neck [79] , and is associated with increased risk of stroke, pulmonary disease, and emphysema [80] . as nicotine damages the lung directly, smoking is one of the leading causes of copd. smokers and copd patients are of high risk in developing severe disease and have a higher mortality rate in covid-19 [13] . smoking can significantly worsen covid-19 progression, with more critical conditions and higher fatality [81] . cigarette smoking increases the number of alveolar macrophages (ams), the innate immune cells in the lung by several fold. these cells secrete lysosomal enzyme, elastase, which can damage connective tissue and parenchymal cells of the lung, one of the contributors to the copd pathogenesis [82] . nicotine is also an important immune modulator. it significantly reduces antibody responses and t-cell proliferation [83] . the immune suppression by nicotine, particularly the decrease in cd8+ t-cells that facilitate the rapid resolution of acute viral infections, increases the susceptibility of smokers for viral infections [84] . analysis of clinical data has indicated that smokers are twice as much as non-smokers to contract the virus, have a more severe disease progression, and have higher mortality rates [85] . nicotine induces bbb leakage and increases bbb permeability by diminishing the expression of tight junction proteins, including occludin, claudin-3, zo-1, and jams [86] [87] [88] [89] . nicotine alters actin cytoskeleton arrangement in the bmvec, which also greatly increases bbb permeability, resulting in a surge of bacterial invasion to the brain [90] . nicotine induces oxidative stress, which can progressively compromise the bbb integrity [91] . nicotine increases gene expression of proinflammatory cytokines, tnfα, il-1β, and il-18, and chemokines, ccl2, ccl8, and cxc3cl1, and suppresses anti-inflammatory factors, bcl6, il-10, and ccl25 in the brain microvessels [92] . nicotine's detrimental effects on the bbb and induced neuroinflammation are serious concerns for covid-19. the history of fermentation production and the use of alcohol can be dated back to 10,000 bc. although light-to-moderate consumption may arguably have positive health benefits, particularly in lowering cardiovascular risks [93, 94] , high dose alcohol can have severe neurotoxic effects and cause dementia [95] . alcohol has been linked to liver damage, inflammation of the pancreas and stomach, and neurodegenerative disorders. high alcohol consumption changes gene expression of immune response genes in the brain region frontal cortex [96] . proinflammatory signaling is also connected to high alcohol intake [97] . binge alcohol (blood alcohol content b.a.c ≥ 0.08%) elevates expression of proinflammatory cytokines, il-1β and il-6, and chemokine ccl-2 (mcp-1) [98] . binge alcohol can also damage various organs, including the gut, liver, and brain [99] , and lead to spleen atrophy in a hippocampus-mediated fashion [100] . as suggested in a comparative risk assessment using the margin of exposure (moe) benchmark, alcohol is considered to have the highest risk of mortality [101] . the alcohol-induced inflammatory cytokine release could expose covid-19 patients to excessive inflammatory responses. the spleen damage by alcohol could weaken the immune response in covid-19 by reducing the production of antibodies and lymphocytes against the virus [102] . alcohol can induce bbb disruption by decreasing the expression of tight junction proteins and increasing mitogen-activated protein kinase (mapk) activities [103] . alcohol may also stimulate inositol 1,4,5-triphosphate receptor (ip3r)-operated intracellular ca 2+ release, activating myosin light chain kinase (mlck). the heightened kinase activities lead to the phosphorylation of cytoskeletal and tight junction proteins, compromising the bbb integrity [104] . alcohol-mediated oxidative stress in bmvec can also activate mlck that alters cytoskeleton and tight junction protein structures, causing bbb leakage [105] . disruption of the bbb associated with chronic alcohol use could increase the possibilities of invading pathogens, including sars-cov-2, to infiltrate the brain. marijuana, also known as cannabis, is the most frequently used illicit substance of abuse in the u.s. currently, 11 states and the district of columbia have legalized the recreational use of marijuana, and 16 states have decriminalized marijuana use and possession. the use of marijuana for medical purposes is legal in 33 states. it is, however, illegal under federal law to use and possess marijuana. it is classified as a schedule i substance by the us drug enforcement administration (dea), indicating a high potential for abuse and no accepted medical use, despite the fact that medical marijuana has been well established [106, 107] . two cannabinoids, ∆ 9 -tetrahydrocannabinol (thc) and cannabidiol (cbd), are the main active ingredients in marijuana. cannabinoids act on the endocannabinoid system (ecs), which is composed of two cannabinoid receptors, cb1, expressed in most neuronal cells, and cb2, expressed predominantly in immune cells [108] . thc is the component that produces psychotropic effects through stimulating the cb1 receptor and mediating the inhibition of neurotransmitter release [109] . cbd is considered to regulate immune response, such as cytokine release, and blood pressure, with little to no psychotropic side effects, as cbd only binds to cb2 but not to cb1 receptors [109] . thc can also activate cb1 receptor in the cardiovascular system, which has been associated with adverse cardiovascular events, including myocardial infarction, cardiomyopathy, arrhythmias, stroke, and cardiac arrest [110] . both thc and cbd are lipophilic, allowing them to readily pass through the bbb and enter the cns. thc is an immunosuppressor. it has been reported to suppress antibody response and t lymphocytes activities. it prevents macrophage and macrophage-like cells, such as microglia, from migrating towards the nodes of microbial invasions. it also suppresses proinflammatory factors and promotes anti-inflammatory activities [111] . thc downregulates proinflammatory cytokines, il-1α, il-1β, and tnfα. by dampening immune responses to invading pathogens, thc could make the host more susceptible to viral infections, such as hiv-1 [111] and possibly covid-19. other harmful effects of marijuana include cardiovascular, cerebrovascular, and neurological complications, such as stroke, cognitive dysfunction, and behavioral problems [112] . some evidence suggested a link between smoking marijuana and risk of copd [113] , one of the major risk factors for covid-19 complications, but the risk is only significant if the individuals also smoked tobacco [114] . on the contrary, a study, which has not been peer-reviewed at the time of preparation of this article, showed the beneficial effect of cbd in preventing covid-19 by modulating ace2 expression and downregulating serine protease tmprss2 [115] . opioids, including illegal drug heroin, synthetic drug fentanyl, prescription pain-killing drugs oxycodone (oxycontin ® ), hydrocodone (vicodin ® ), codeine, and morphine, are effectors on the endocrine system [116] . they act as immune suppressors that impair the function of macrophages, natural killer (nk) cells, and t-cells, and are associated with higher risks of infectious diseases, such as pneumonia [117] . the endogenous opioid system (eos), comprising three naturally occurring opioid peptides (β-endorphins, dynorphins, and enkephalins) and three classes of opioid receptors, µ (mor), δ (dor), and k (kor), are tightly linked to substance abuse and the development of addiction, and are responsible for systemic infection [118] . activation of opioid receptors in the brain stem could lead to respiratory depression and overdose fatality [119] . respiratory depression is a leading factor to hypoxemia in covid-19 complications. morphine desensitizes the hpa axis and inhibits the release of anti-inflammatory glucocorticoids through potentiating proinflammatory cytokine, il-1β [120] . inhibition of endogenous glucocorticoid and activation of il-1β by opioids could significantly increase the severity and inflammatory response of covid-19 in patients with oud. it has been shown that individuals with oud are more susceptible to opportunistic infections [121] , disposing these individuals at a higher chance of contracting the virus in covid-19. cocaine is a potent stimulant, and is the second most abused illicit drug after marijuana. cocaine can be abused in several forms, such as chewing the leaves of erythroxylum coca tree, injecting the water-soluble hydrochloride salt form, and smoking or snorting pure, freebase form, called "crack". one of the most significant pathophysiological effects of cocaine is its cardiotoxicity, which has been well documented and extensively reviewed [122] [123] [124] [125] [126] . cardiac arrhythmias and acute myocardial ischemia or infarction (mi) are the leading causes of cocaine-induced sudden cardiac death. other cardiovascular diseases associated with cocaine include heart failure, cardiomyopathies, aortic dissection, and endocarditis. cocaine blocks voltage-dependent na + and k + channels in the sinoatrial node and the myocardium, depressing cardiovascular contractility [127] . due to its function as an ion channel blocker, cocaine has been effectively used as a local anesthetic [128] . cocaine can also induce binding and opening of l-type ca 2+ channels, causing the influx of ca 2+ in cardiomyocytes and elevation of intracellular ca 2+ concentration. this second messenger pathway may also lead to cardiac arrhythmia [129] . clinically, cocaine increases myocardial oxygen demand by increasing heart rate and hypertension, while it decreases the oxygen supply due to coronary vasoconstriction [130] [131] [132] . cocaine impairs endothelial functions [133] , sensitizes constrictor effects of catecholamines [134] , and causes microvascular diseases and thrombosis [135, 136] . the cocaine-mediated oxygen imbalance can be particularly detrimental in covid-19, in which the coronavirus can cause hypoxemia because of the diminishing of lung capacity. cocaine exerts its effect through binding to three monoamine transporters on nerve terminals: the serotonin transporter (sert), the dopamine transporter (dat), and the norepinephrine transporter (net), with k i of 0.14, 0.64, and 1.6 µm, respectively [137] . upon binding to these transporters, cocaine inhibits the reuptake of the neurotransmitters from the synaptic cleft, leading to prolonged synapses and activation of postsynaptic receptors. cocaine also binds directly to two classes of neurotransmitter receptors, muscarinic acetylcholine and sigma receptors [138] [139] [140] . the interactions with the transporters and the receptors form the molecular basis for cocaine neurotoxicity. cocaine stimulates the hpa axis, increasing the secretion of neuronal peptide crh, which leads to subsequent releasing of β-endorphin and acth. through general circulation, acth reaches the adrenal glands and promotes the biosynthesis of glucocorticoids [141] . cocaine-induced stimulation of the hpa axis and immune suppression can alter antibody formation, lymphocyte subset profile, and lymphocyte proliferation. cocaine suppresses responses to the proinflammatory cytokine, il-6, and dampens cytotoxic activation of macrophages, natural killer cells, and cytotoxic t lymphocytes [142] . due to compromised immune responses, cocaine abusers have considerably high incidences of viral infections, including human immunodeficiency virus (hiv), influenza, and potentially sars-cov-2. cocaine can induce bbb dysfunction, disrupt neurovascular capillaries and basement membrane [143] , and increase bbb permeability [144] [145] [146] . the detrimental effects of cocaine on the bbb are partially attributed to the loss of tight junction protein complexes, including zo-1 and jam-2 [146] [147] [148] [149] . cocaine also increases the expression of matrix metallopeptidase (mmp)-1, which contributes to the rearrangement of the cytoskeleton structure of the basement membrane [147, 148] . the adverse effects of decreased tight junction protein complexes and remodeling of the basement membrane fibers cause the bbb leakage and make it open to peripheral toxins and viruses, including sars-cov-2. an increase in proinflammatory cytokine, tnfα, has also been reported in bmvecs exposed to cocaine [150] , which could be a concern for endothelial health in covid-19. d-amphetamine and its synthetic derivatives, meth and mdma, are addictive psychostimulants associated with neuropsychiatric complications, including deficits in attention, memory, and executive functioning [151, 152] . amphetamines mediate neurodegenerative changes in the brain, including persistent loss of dopamine (da) transporters [153] [154] [155] [156] [157] and receptors [158] , loss of serotonin (5-ht) transporters [159] , and decrease in dopamine and serotonin level and its metabolites [160] [161] [162] . meth has been linked to various cardiac pathologies, including hypertension, tachycardia, and congestive heart failure or cardiomyopathy [16] . clinically, meth abusers commonly showed dilated and poorly contracting left ventricles (lv) and substantially lowered left ventricular ejection fractions (lvef) in comparison to non-users [163] . meth-associated cardiac pathologies are a major cause of pulmonary edema. the reduced lung capacity due to the fluid collection in the lungs and constriction of blood vessels can severely complicate covid-19 symptoms and negative prognosis. amphetamines stimulate the hpa axis and increase the plasma glucocorticoids through a crh-dependent mechanism involving serotonin [164] [165] [166] [167] . the stimulation increases the production of crh and avp in the pvn neurons, which, in turn, activates the production of acth in corticotropic cells in the anterior pituitary gland. acth circulates through the systemic blood stream to reach and activate the adrenal cortex to release glucocorticoids. the hpa axis is an essential component of the response to pathogen infections. however, chronic activation of the hpa axis and markedly increased glucocorticoid due to the recreational use of amphetamine, meth, and mdma can be harmful to the brain. amphetamines modify brain expression of the genes and proteins associated with the hpa axis, including the glucocorticoid receptor (gr) and the mineralocorticoid receptor (mr). the remodeling of brain cells and disruption of the hpa axis are the hallmarks of depression and anxiety/despair states associated with drug use [168, 169] . as mentioned earlier, the disruption of the hpa axis and the associated immune suppression could pose the drug abusers to a higher risk of viral infections. sars-cov-2, known for its high infection rate, will be particularly harmful to individuals with defective immune systems related to the use of addictive drugs. amphetamine-like psychostimulants can trigger inflammatory processes, compromise neurogenesis in the brain, and damage the bbb integrity [170] . meth, for example, is strongly associated with ischemic stroke and hypoxia [171, 172] . binge use of meth causes a sustained reduction in global and cerebral blood flow [173, 174] . meth is also known to damage the central nervous system (cns) by compromising the integrity of the blood-brain barrier (bbb) [175] [176] [177] . meth and mdma can decrease the expression of tight junction proteins, including zo-1, occludin, and claudin-5. these drugs activate microglia and astrocyte to secrete proinflammatory cytokines and chemokines, as well as vasoactive factors, and elevate expression of peptidases, such as mmp-1 and mmp-9, to degrade tight junction proteins and modify bbb basement membrane structure [178, 179] . meth abuse has been shown to increase brain infection of peripheral bacteria and viruses [180] [181] [182] . similarly, mdma has been shown to cause bbb dysfunction with increased bbb permeability [183] , and to excessively activate astrocytes and microglia [184] . it may lead to edema [183] . with bbb dysfunction, it is almost certain that the risks of sars-cov-2 invasion into the brain will be immensely heightened among individuals using or abusing these psychostimulants, and complications in these patients are vastly expected. neuroinflammation induced by meth and mdma also plays a major role in bbb damage and may deteriorate covid-19 conditions. covid-19 patients exhibit abnormal immune responses related to high levels of proinflammatory cytokines, including tnfα and il-6. meth significantly increases the expression of tnfα and il-6 in the hippocampus, frontal cortex, and striatum [185] . the expression of these proinflammatory cytokines is linked to meth-induced microglial activation [186, 187] . mdma also elevates the expression of proinflammatory cytokines, such as il-1β, in the brain [188] . the excessive expression of proinflammatory cytokines in brain tissues could further damage the bbb and cause oxidative stress [78, 189] . neuroinflammation poses a major risk for individuals with covid-19. substances of abuse may lead to covid-19 complication and severity in several ways. smoking tobacco and marijuana could cause direct damage to the respiratory system, such as copd. other substances mostly work through modulating brain and immune functions, including the promotion of proinflammatory factors, suppression of immune responses, and impairment of the bbb. neuroinflammation induced by several substances of abuse and inflammatory activities caused by covid-19 in the peripheral tissues may mutually intensify the adverse effects of one another, leading to negative progression of the disease. with impaired hpa axis and immune imbalance, the patients are highly susceptible to sars-cov-2 infections. compromised bbb may pose a high risk of viral infection in the brain tissue. figure 4 sketches the possible pathological effects of commonly abused substances on various tissues and systems and their connection to covid-19 complications. these adverse effects of these substances on the respiratory system, cardiovascular system, the immune system, and the cns, as well as their relations to the severity and negative prognosis of covid-19, are summarized in table 3 . pharmaceuticals 2020, 13, x for peer review 13 of 29 adverse effects of these substances on the respiratory system, cardiovascular system, the immune system, and the cns, as well as their relations to the severity and negative prognosis of covid-19, are summarized in table 3 . main cause of copd [10, 11, 82] increased severity and mortality [13, 81] immune system immune suppression, decreased cd8+ t-cells [83, 84] higher infection rate [85] increased inflammatory cytokines (tnfα, il-1β, il-18) and chemokines (ccl2, ccl8, and cxc3cl1); decreased anti-inflammatory factors, bcl6, il-10, and ccl25 [92] increased inflammatory cytokines and chemokines, tnfα, il-1β, il-6 [61] [62] [63] cns bbb leakage through loss of tight junction proteins [86] [87] [88] [89] endotheliitis and cns infection [53,55-57] immune system increased proinflammatory cytokines, il-1β and il-6, and chemokine ccl-2 increased inflammatory cytokines and respiratory system main cause of copd [10, 11, 82] increased severity and mortality [13, 81] immune system immune suppression, decreased cd8+ t-cells [83, 84] higher infection rate [85] increased inflammatory cytokines (tnfα, il-1β, il-18) and chemokines (ccl2, ccl8, and cxc3cl1); decreased anti-inflammatory factors, bcl6, il-10, and ccl25 [92] increased inflammatory cytokines and chemokines, tnfα, il-1β, il-6 [61] [62] [63] cns bbb leakage through loss of tight junction proteins [86] [87] [88] [89] endotheliitis and cns infection [53, [55] [56] [57] alcohol increased proinflammatory cytokines, il-1β and il-6, and chemokine ccl-2 [98] increased inflammatory cytokines and chemokines, tnfα, il-1β, il-6 [61] [62] [63] spleen atrophy [100] . impaired production of antibodies and lymphocytes [102] cns increased bbb permeability through cytoskeletal and tight junction remodeling [103] [104] [105] . endotheliitis and cns infection [53, [55] [56] [57] marijuana (thc, cbd) enhanced copd with tobacco [113, 114] increased severity and mortality [13, 81] immune system immunosuppression; reduced antibody response and t lymphocyte activities; reduced migration of macrophage [111] increased infection and reduced viral response and clearance [111] opioids (heroine, fentanyl, morphine) respiratory system respiratory depression [14, 15, 119] increased severity and mortality [14, 15] immune system desensitizing hpa axis; inhibiting glucocorticoid release, increased il-1β; neuroinflammation [120] increased opportunistic infections, excessive inflammatory response [117, 118, 121] cocaine cardiac arrhythmias and acute mi; oxygen imbalance; microvascular diseases and thrombosis [122] [123] [124] [125] [126] [127] [129] [130] [131] [132] increased severity and mortality [12, 37, 38] immune system stimulating hpa axis; immunosuppression; defects in antibody formation, lymphocyte proliferation, macrophage and nk activation [141, 142] high incidence of viral infection [142] cns increased bbb permeability due to loss of tight junction proteins; rearrangement of cytoskeleton structure [143] [144] [145] [146] endotheliitis and cns infection [53, [55] [56] [57] amphetamine, meth, mdma hypertension, tachycardia, and cardiomyopathy leading to pulmonary edema [16, 163] ; ischemic stroke and hypoxia; restricted blood flow [171] [172] [173] [174] increased severity and mortality [12, 37, 38] immune system altered hpa axis, impairing gr and mr expression, immunosuppression [164] [165] [166] [167] increased infection rate, depression, anxiety/despair [168, 169] increased expression of tnfα, il-1β, and il-6; neuroinflammation [185] [186] [187] [188] excessive inflammatory response [61] [62] [63] cns bbb damage due to loss of tight junction protein; edema [175] [176] [177] [178] [179] endotheliitis and cns infection [53, [55] [56] [57] [180] [181] [182] the therapeutic strategies for covid-19 have been focused on repurposing existing drugs against this novel coronavirus [190] [191] [192] . among these repurposed drugs, the antimalaria drugs chloroquine and hydroxychloroquine [193, 194] are extremely controversial, including a retracted study [195] and a terminated solidarity clinical trial by the who [196] . the u.s. food and drug administration (fda) recently revoked its emergency use authorization (eua) to treat covid-19 [197] . other drugs include the anti-hiv drugs lopinavir-ritonavir in combination with ribavirin [198] [199] [200] , tumor chemotherapy drugs, doxorubicin and paclitaxel [201] , traditional herbal medicines [202] , broad spectrum antiviral drug niclosamide [203] , janus-associated kinase (jak) 1 and 2 inhibitor, ruxolitinib [204] , anti-influenza drug favipiravir [205] , antiviral drug remdesivir [206] [207] [208] , and most recently, a commonly used steroid, dexamethasone [209] . many of these drugs primarily target the rdrp (nsp12) or the main protease, m pro . a cryo-em structure of nsp12 in complex with its cofactors nsp7 and nsp8 has been reported [210] . remdesivir, the only proven effective drug against covid-19 so far, is found to potently inhibit rdrp in mers-cov [211] and sars-cov2 [212] . ribavirin and favipiravir also function as rdrp inhibitors. the crystal structure of the m pro with its inhibitor has been recently solved and reported [213, 214] . due to its importance in viral production, and the lack of similar proteins in human cells, the m pro is considered an important drug target in treating covid-19 [213, 215, 216] . niclosamide and the anti-hiv combination drug lopinavir-ritonavir, are inhibitors of the main protease. structure-based design has led to the development of new inhibitors of the m pro with desirable pharmacokinetic properties and low toxicity [217] . the sars-cov-2 entry point, ace2, and the associated protease, tmprss2, are also considered potential drug targets [28] . tremendous research efforts have been put into identifying, isolating, and developing neutralizing antibodies against sars-cov-2. the s protein, which plays a key role in recognizing and binding to the ace2 receptor to gain entry into the host cell, is the main focus for developing neutralizing antibodies and vaccines against sars-cov-2 [24, 25] . neutralizing monoclonal antibodies isolated from convalescent covid-19 patients were found to block the rbd surface of the s protein from binding to ace2, and these antibodies showed effectiveness in reducing viral infection in animal models [218] [219] [220] . the crystal structure of the rbd-bound antibody provided a clear picture on inhibition of viral interaction with ace2 [221] . the neutralizing antibodies isolated from sars-cov patients seemed to cross-neutralize sars-cov-2 [222] . the rbd is not the only site that neutralizing antibodies may block. a monoclonal antibody isolated from convalescent covid-19 patients exhibits high neutralization potency against sars-cov-2. this antibody does not bind to the rbd, but it tightly associates with the n-terminal domain of the s protein [223] . a recombinant antibody fused with the human ace2 extracellular domain displayed desired neutralizing properties in vivo and in mice [224] . single-domain camelid antibodies from a llama were found to cross-react and neutralize mers-cov, sars-cov, and sars-cov-2 s pseudotyped viruses [225] . cocktails of antibodies that simultaneously bind to different epitopes of the rbd may provide more potent neutralizing power and significantly reduce virus escaping through mutations [226] [227] [228] . vaccines are crucial in combating the covid-19 pandemic. the rbd of the s protein is, again, the main target for vaccine development [229] . according to who "draft landscape of covid-19 candidate vaccines", there are 13 candidates currently in clinical trials, and 128 candidates in preclinical stage [230] . these vaccine candidates cover almost all technology platforms, including more traditional non-replicating or replicating viral vector, inactivated or live attenuated virus, recombinant protein subunit, to more recently developed nucleic acid (dna or rna), peptide, and viral-like particle [231] . there have been some encouraging results from clinical trials, including the first mrna vaccine, mrna-1273 [232, 233] , and an adenovirus type-5 (ad-5) vectored vaccine [234] . with the race to therapeutics and vaccines towards covid-19, rarely have the efforts specifically been directed at sud complications in covid-19. as we analyzed earlier, suds complicate and impair the respiratory system directly, and can intensify the severity of the disease through cardiovascular damage and immune abnormality. there are increasing concerns on capillary endothelial damage or endotheliitis by covid-19 and suds, including the harmful effects on the bbb integrity. if the coronavirus is allowed to migrate across the bbb and to infect the brain, long-term neurological degeneracy is expected, and the treatment will be deemed challenging. suds can weaken the immune system, alter and disrupt the hpa axis, and stimulate neuroinflammation with heightened expression of tnfα, il-1β, and il-6 in the cns. anti-tnfα therapy has been used in severe cases of autoimmune inflammatory disease to control inflammation by downregulating il-6 and il-1β [235] . anti-il-6 antibody may also be beneficial in inflammation control [236] . these treatments could be helpful for covid-19 patients with suds. current antiviral drugs are designed to interfere with viral replication or viral protein processing. for example, the most effective drug, remdesivir, which can shorten the recovery time by 30% [206] , inhibits viral rna replication [212] . neutralizing antibodies bind to rbd of the s protein, so the virus cannot bind to ace2. however, clearance of the virus is heavily dependent on the individual's immune system, including activating phagocytes and natural killer cells. as discussed earlier, several substances of abuse, such as nicotine, marijuana, cocaine, and amphetamines, have shown to suppress immune and antibody responses. clinical cases indicated that antibody-secreting cells (ascs), t follicular helper (t fh ) cells, as well as activated cd4+ and cd8+ t-cells, are critical to symptomatic recovery from covid-19 [237] . as a result, sud-induced reduction of t-cell activation will dampen the ability to efficiently clear the virus from the body. for vaccines to work efficiently, robust immune responses are required. cd4+ and cd8+ t-cell responses are essential in protective antiviral immunity by vaccination [238, 239] . due to the immunosuppression and reduction of t-cell responses, individuals with suds may not develop sufficient protective antibodies against the virus. a clinical study suggested that sars-cov-2 specific immunoglobin g (igg) antibodies may last only 2-3 months before a steep decline in both asymptomatic and mildly symptomatic covid-19 patients [240] . considering their substantially undermined immune systems, the protective antibodies in patients with suds are very likely to decline much quicker, making vaccines considerably less effective in these patients. there is a potential risk, although no reported cases yet, that antibodies or vaccines may promote covid-19 pathogenesis through antibody-dependent enhancement (ade). while suds compromise the immune system, virus-specific antibodies are likely to promote viral entry into various immune cells, including monocytes, macrophages, and b cells [241, 242] , which may further deteriorate the immune response towards the virus. covid-19 pandemic poses tremendous challenges for the treatment of suds [243] . the regulatory and policy obstacles for patients with suds are intensified in the times of crisis, making it more difficult for healthcare providers to address the needs of sud patients with the availability of medications. physical and social distancing requirement renders the face-to-face group treatment and mutual support groups inaccessible. these difficulties will disrupt the treatment of patients with suds when these patients start to experience withdrawal symptoms. it has been called for relaxing rules and regulations for these patients to receive treatment, and adopting the model of pharmacy-based addiction care by integrating primary-care and pharmacy prescription, dispense, and management [243] . it is now widely accepted that covid-19 may stay with us for some extended periods. people with mild or no symptoms can transmit the pathogen as effectively as those with severe symptoms [244, 245] . the complications and compromised immune systems associated with suds make drug abusers particularly vulnerable to covid-19. therapeutics and vaccines currently under development do not address the specific concerns and risk factors for these individuals. there are even greater challenges for people with suds under covid-19 pandemic, as they will experience a higher infectious rate, limited access to healthcare system and support groups, inadequate food and housing, and increased likelihood of homelessness and incarceration. they may also face excessive discrimination, and have higher chances of relapses and overdose death. the research community should heed to the challenges and difficulties these individuals may experience in the pandemic, uncover scientific evidence to link covid-19 severity and mortality with substance use, and advance effective treatment and prevention 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immunological assessment of asymptomatic sars-cov-2 infections the potential danger of suboptimal antibody responses in covid-19 antibody-dependent enhancement of viral infections opioid use disorder and the covid 19 pandemic: a call to sustain regulatory easements and further expand access to treatment projecting the transmission dynamics of sars-cov-2 through the postpandemic period this article is an open access article distributed under the terms and conditions of the creative commons attribution we thank ammer abdin, madellyne sanchez, and song zhang, for collecting and analyzing relevant literatures and intellectual discussions for this article. the authors declare no conflict of interest. key: cord-275795-ee7qyw5h authors: monette, anne; mouland, andrew j. title: t lymphocytes as measurable targets of protection and vaccination against viral disorders date: 2018-10-24 journal: int rev cell mol biol doi: 10.1016/bs.ircmb.2018.07.006 sha: doc_id: 275795 cord_uid: ee7qyw5h continuous epidemiological surveillance of existing and emerging viruses and their associated disorders is gaining importance in light of their abilities to cause unpredictable outbreaks as a result of increased travel and vaccination choices by steadily growing and aging populations. close surveillance of outbreaks and herd immunity are also at the forefront, even in industrialized countries, where previously eradicated viruses are now at risk of re-emergence due to instances of strain recombination, contractions in viral vector geographies, and from their potential use as agents of bioterrorism. there is a great need for the rational design of current and future vaccines targeting viruses, with a strong focus on vaccine targeting of adaptive immune effector memory t cells as the gold standard of immunity conferring long-lived protection against a wide variety of pathogens and malignancies. here, we review viruses that have historically caused large outbreaks and severe lethal disorders, including respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic fevers. to observe trends in vaccinology against these viral disorders, we describe viral genetic, replication, transmission, and tropism, host-immune evasion strategies, and the epidemiology and health risks of their associated syndromes. we focus on immunity generated against both natural infection and vaccination, where a steady shift in conferred vaccination immunogenicity is observed from quantifying activated and proliferating, long-lived effector memory t cell subsets, as the prominent biomarkers of long-term immunity against viruses and their associated disorders causing high morbidity and mortality rates. the world was forever changed by the introduction of vaccine against smallpox in the late 1700s, at the time protecting its first 100,000 individuals. this was the first demonstration that a vaccine could successfully eradicate viruses causing disorders and diseases that even when not lethal, still had the potential to cripple both surviving populations and their surrounding geographical economies. since that time, the occurrence of epidemics and outbreaks are now at lower risk, following the introduction of massive vaccination programs able to induce immune system targeting of viruses causing severe disorders affecting distinct geographical locations, and with many epidemiological reports demonstrating long-term efficacy of viral control of non-naïve populations. outbreaks of existing and emerging viral diseases or disorders vary widely in duration and frequency across geographical populations. some can be predicted annually, while others may see decades between outbreaks, therefore driving the continuous epidemiological surveillance of associated infectious disorders, the development and implementation of targeting vaccines, and development of immune-monitoring strategies measuring vaccine efficiency in target populations. despite vaccination-mediated protection of numerous nations documenting complete eradication of the causative agents of viral disorders in endemic populations, there is threat of re-emergence of pandemic proportions of these agents. threats to virus re-emergence are caused by contemporary choices made to not vaccinate children, by strain recombination, by viral spread to naïve populations by world-travelers, migrants, or climate change, causing redistribution of viral vectors, by potential use of these agents in acts of bioterrorism or war upheaval, or by depletion of vaccination stocks required for protection against pandemics. other factors include the increasing and aging global population and increased number of immunocompromised individualsd factors strongly supporting the maintenance of herd immunity against existing viruses, despite lower incidence of outbreaks in industrialized countries. in the event of a re-emergence of previously eradicated viruses or the acquisition of increased pathogenicity by existing viral strains, there is an urgent need for vaccine development strategies that can rapidly and effectively arrest global spread. important advances are continuously being made in vaccine development strategies toward the control of viruses and associated disorders. vaccine design has been modified from the use of attenuated viruses to use of more precise viral protein subunits specifically targeted by t cells. historically, vaccination immunogenicity was documented by measures of serum immunoglobulin (ig) classes and antigen-specific antibodies produced by humoral immunity. more recently, quantification of cellular components of innate immunity at the interface between innate and adaptive immunity are made, in addition to more precise measurements of adaptive immunity. long-term protection achieved by adaptive immunity can be quantified by measuring levels of circulating cytokines, along with specific phenotypic profiles of effector memory, antigen-specific t cells. though both humoral and cellular arms of immunity are integrally linked during the initial induction of immunity against pathogens, these can become disconnected with developing pathology due to their individual needs for survival factors, unequal declines in immune function, and differential cellular lifespans. this loss of correlation between memory t cells and neutralizing antibody responses varies according to different viruses, suggesting that independent time course measures of these separate immune responses are required over time for adequate recording of biomarkers of natural infection and vaccine efficacy or suggesting that t cell status may be most crucial measure of conferred long-term immunity. from the standpoint of fundamental or clinical research, it has become established that the targeted induction of specific pathogen-and tumorclearing effector memory t cell subsets is our endgame armor toward long-term human survival against infectious diseases and cancers. this chapter provides an overview of viruses that have historically caused severe lethal disorders, including those of the respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic types. the features of viruses and associated disorders that we herein describe include viral genetics and replication cycles, transmission modalities, cell and organ tropism, host-immune evasion strategies, associated viral disorders and diseases, and epidemiology. we also report on well-accepted and other important documented instances of viral control by t cells, currently available and successful vaccines, and recorded measures of vaccination immunogenicity. we focus on quantification of vaccine-induced effector memory t cellemediated immunity, representing the gold standard of successful vaccination. just as it is for advances in vaccinology, investigations into the biology of t cells are currently at the forefront of many research fields examining various disorders, diseases, and malignancies not formerly considered to be controlled by immunity. although many viral infections are limited to the upper respiratory tract, it is lower respiratory tract infections (lrti) that most predominantly cause enormous disease burden in children and immunocompromised adults suffering from human immunodeficiency virus (hiv) infection or in patients having received stem cell or solid organ transplants for which immunosuppressive therapies were administered (henrickson et al., 2004; pavia, 2011; kim et al., 2007) . acute lower respiratory illnesses (alris) are a major cause of morbidity and mortality, accounting for approximately 1.6 million deaths, globally, per year (black et al., 2010) . frequently overlapping lrti syndromes include bronchiolitis, asthma exacerbation, wheezing, croup, and pneumonia. although certain specific syndromes can be more precisely associated with infection by specific viruses, syndromes overlaps can complicate diagnosis of these numerous viruses, and quite often, difficulties in differentiating between viral and bacterial pneumonias symptoms can also result in antibiotics being mistakenly prescribed during viral disorders. several viruses are normally, however, considered to be primarily responsible for lrtis, beginning with upper respiratory tract infections, most commonly caused by respiratory viruses that are typically spread from person-to-person by contact with infected respiratory droplets, and including respiratory syncytial virus (rsv), epidemic influenza a and b, h5n1 and h7n9 avian influenza a viruses (iavs), parainfluenza viruses 1 through 4, adenovirus, human metapneumovirus (hmpv), severe acute respiratory syndrome coronavirus, human coronaviruses nl63 and hku1, rhinoviruses, and bocaviruses (pavia, 2011; mahony, 2008; nichols et al., 2008) (table 1) . currently, vaccines for human influenza viruses, human parainfluenza viruses (hpivs), and adenoviruses causing upper and lower respiratory infections are used to control these infections and the resulting propagation of their morbid symptom derivations. human influenza viruses make up three of the five genera of the family orthomyxoviridae and are classified as a, b, and c types, based on their highly conserved matrix protein 1 (m1), membrane matrix protein (m2), and nucleoprotein (np). type a influenza viruses can be further sub-subtyped by the antigenicity of their hemagglutinin (ha) and neuraminidase (na) surface glycoproteins (gps). antigenic drift, caused by point mutations in ha and na and recombination of the ha genes, results in the generation of new strains that can escape pre-existing immunity, causing both the prediction of circulating strains difficult and antigenic mismatch by existing vaccines. approximately 18 ha and 9 na subtypes of influenza a are documented in aquatic birds, representing their natural hosts (i.e., vectors). influenza a h1 and h3 subtypes cocirculate seasonally, and influenza b viruses can only infect humans, via two distinct, seasonally cocirculating, lineages. type c influenza viruses are more rarely documented to infect humans and pigs (berlanda scorza et al., 2016) . influenza viruses cause acute upper and lower respiratory infections, and due to their rapid and unpredictable genetic drift, represent the most likely of pathogens to cause a human pandemics. annually, human influenza viruses have the potential to cause up to 5 million cases of severe illness, with an associated 500,000 deaths worldwide (who_influenza_(seasonal), 2018), causing great economic burden. four influenza pandemics have occurred over the past century, as a consequence of the h1n1 (1918), h2n2 (1957), h3n2 (1968), and h1n1 (1977) variants (palese, 2004) . since the most recent outbreak in 2009, an estimated 200,000 people globally have succumbed to the h1n1 variant of swine origin (dawood et al., 2012) . epithelial cells that are infected with influenza virus produce inflammatory cytokines acting as chemoattractants for homing macrophages and dendritic cells (dc). dcs take up influenza viral particles to trigger their maturation and pursuant migration to the lymph, where they initiate antigen-specific t cell maturation. these influenza-specific effector t cells then enter the respiratory tract to counteract viral titres through cytokine expression and the direct lysis of infected cells, with activated cd8 þ effector cytotoxic t cells (ctls) representing the main constituents of this response by their release of perforins and granzymes, and the engagement of tumor necrosis factor (tnf) receptors (spitaels et al., 2016) . influenza-specific cd4 þ t helper cells can act directly and indirectly in viral clearance, primarily by producing cytokines that induce the functions of b cells and cd8 þ t cells and which have also been reported to directly eliminate infected cells themselves (topham and doherty, 1998; hua et al., 2013) . while preexisting cd8 þ t cell immunity has not yet been demonstrated to prevent infection from occurring, it is hypothesized to be the result of the loss of granzyme expression by memory cd8 þ t cells and populations of iavspecific cd8 þ t cells are still importantly correlated with the control of spread and recovery in healthy populations (grant et al., 2016) . the most currently administered influenza vaccines are inactivated (iv) trivalent (tiv) or quadrivalent formulations containing equal amounts of ha of two influenza a strains (h1n1 and h3n2) and one of two influenza b strains (yamagata and victoria lineage). these are derived from viruses typically grown in fertilized chicken eggs, are mainly focused on eliciting a strainmatched humoral immune responsedrequiring yearly updatesdand are unable to provide protection to all vaccinated individuals. the requirement of memory t cell immunity for long-term protection against influenza virus promotes the development of vaccines that elicit both humoral and cellular immunity: a strategy expected to overcome the inadequacies of current vaccines against influenza and other viruses (spitaels et al., 2016) . there is broad interest in the development of a universal influenza vaccine, considered to be the "holy grail" of influenza vaccine research. this approach is being developed to use virus-infected cell-killing antibodies that produce an antiviral environment; these termed antibody-dependent cellular cytotoxicity (adcc)-mediating antibodies, which are predicted to link innate and adaptive immune responses, and is becoming possible due to new technologies for rapid isolation and characterization of monoclonal antibodies targeting conserved regions of influenza virus, reviewed in jegaskanda et al. (2017) . this approach has been postulated to work, in part, from reports of iavspecific cd8 þ t cells, promoting viral clearance in the absence of neutralizing antibodies, and can also mediate cross-reactive immunity against distinct iavs to drive a rapid recovery from severe influenza disorders (grant et al., 2016) . the induction of infection-permissive immunity is both protective and allows virus-induced cross-reactive immune responses. vaccines targeting the conserved ectodomain of m2 deliver this kind of non-neutralizing immunity since these antibodies rely on fc receptors and innate immune components (el bakkouri et al., 2011) . antagonizing antibodies inhibiting na activity represents another promising strategy, not by blocking viral entry and eliciting sterilizing immunity, but by contributing to immunity against a virus possessing a similar na type (wan et al., 2013) . there is also progress being made in the development of recombinant t celleinducing vaccines, with the most advanced version of this strategy demonstrated by modified vaccinia ankara (mva) viruses expressing influenza virus np and m1 antigens (berthoud et al., 2011) , with vaccinated individuals demonstrating increases in interferon-gamma (ifn-g) expressing cd8 þ t cells and increased protection against influenza infection (antrobus et al., 2012; powell et al., 2013) . co-administration of this mva-based vaccine with tiv formulations results in increased influenza strainespecific antibody responses and the generation of memory t cells that recognize a range of influenza a subtypes (antrobus et al., 2014) . additional research demonstrates production of antigen-specific t cell responses using alternate prime/boost regiments of combinations of vaccination regimens employing recombinant replication-deficient adenovirus or mva, expressing iav np and matrix protein 1 (lambe et al., 2013) . quality and clonal t cell receptor (tcr) characteristics of influenza-specific cd8 þ t cells, in addition to in silico predicted and peptide-based approaches for pools of minimal iav epitopes, are investigated for their induction of cellular immunity and recognition by cd8 þ t cells (reviewed in grant et al., 2016) . hpiv are enveloped negative-sense rna genome viruses of 150e250 nm, belonging to the large and rapidly growing paramyxoviridae family, causing significant human and veterinary disorders (henrickson, 2003) . hpiv is divided into serotypes 1 to 4, with hpiv-1 representing the most common etiologic agent of associated disease, but with hpiv-1, -2, and -3 representing common causative agents of respiratory illness in pediatric, geriatric, and immunocompromised populations (schmidt et al., 2011) . hpivs are a common cause of acute respiratory illness throughout all stages of human life (schomacker et al., 2012) , causing acute respiratory infections in children (cooney et al., 1975) . second, only to human respiratory syncytial virus, hpivs are the major contributors to hospitalization due to alris and global pneumonia mortalities in young children, and up to 80% of children are seropositive for hpivs by the age of 5 years (murphy, 1988; weinberg et al., 2009) . hpiv infections can induce potent humoral and cellular immune responses, including innate immune responses, local and systemic igg and iga responses, and adaptive cd8 þ and cd4 þ t cell responses (gitlin et al., 2010; hou et al., 1992) . though cellular responses can restrict hpiv replication dynamics and clear primary infections, neutralizing antibodies against virus envelope hemagglutininneuraminidase (hn) and fusion (f) gps are required for early infection (suzuki et al., 2001; zhang et al., 2005) and confer long-term protection against hpiv-related disorders (murphy, 1988; spriggs et al., 1987; schmidt et al., 2011) . currently, there is no vaccine to protect against human hpiv infection. progress in the development of hpiv vaccines using reverse genetics for serotypes à1 to à3 has generated several live-attenuated, intranasal hpiv vaccines evaluation in adults and in children, two of which, hpiv-3 are well tolerated in hpiv3-seronegative pediatric populations (schmidt et al., 2011) . ongoing pediatric trials testing live-attenuated hpiv vaccines for hpiv-1 and hpiv-2 predict these will replicate in the upper respiratory tract of infants to induce the full spectrum of humoral and cellular immune responses (karron et al., 2015) . heterologous (i.e., jennerian) vaccine design strategies using the sendai virus (sev) to control infections by hpivs and rsv are discussed in the following section. human respiratory syncytial virus (rsv) types a and b are found within the genus orthopneumovirus, family pneumoviridae, of the order mononegavirales. rsv is an enveloped, spherical virus of w150 nm in diameter, and reaching up to several micrometers in length (gower et al., 2005) . the negative-sense rna genome encodes for outer structural, np, polymerase, ns, transmembrane, and regulatory proteins (griffiths et al., 2017) . rsv is a major cause of alris, resulting in numerous pediatric hospitalizations globally, where by 3 years of age, most children have been exposed and are at risk of developing life-threatening bronchiolitis and pneumonia (glezen et al., 1986; hall et al., 2009; henrickson, 1994) . up to 120,000 infants are hospitalized due to rsv infection in the united states (marks, 1992) , and 34 million episodes of rsv-associated alri in children globally represent at least 3 million cases resulting in hospitalization, and approximately 199,000 associated deaths per year (nair et al., 2010) . a balance of adaptive immune ctls and neutralizing antibodies of the humoral immune response mediate protection and clearance of rsv infection (griffiths et al., 2017) . though neutrophils are the highest proportion of leukocytes found in the airways of those infected with rsv (everard et al., 1994) , and despite observations that natural killer (nk) cells are first to attain infected airways (hussell and openshaw, 1998) , it is cd4 þ helper and cd8 þ ctls that correlate with the early clearance of rsv-infected cells (anderson et al., 1990) . in infants and immunocompromised populations, fatalities resulting from rsv infection are associated with deficiencies in cd4 þ and cytotoxic cd8 þ t cells (hall et al., 1986; welliver et al., 2008) . later in infection, increases in neutralizing antibodies prevent reinfection by opsonizing viral epitopes required for rsv entry and infection, and rsv clearance can be associated with rsv-neutralizing nasal immunoglobulin a (iga) (mcintosh et al., 1978) . more recently, enhanced rsv clearance with reduced disease severity has been associated with vaccine-elicited memory cd8 þ t cells (lee et al., 2012) . while memory cd8 þ t cells can mediate protection against rsv infection, in the absence of antibodies and memory cd4 þ t cells, these cause mortality via systemic proinflammatory cytokine storms and local ifn-g production (stoley et al., 2016; schmidt et al., 2018) . other recent studies however indicate that the cd8 þ t cell response may not be the major determinant of severity of rsv-related pathology (collins and melero, 2011) . there are currently several recombinant rsv subunit vaccines in clinical trials, including the novavax rsv f vaccine representing the most promising candidate for licensing. this rsv f subunit targeting vaccine has been demonstrated to elicit the expression of circulating neutralizing antibodies against rsv (glenn et al., 2016) . toward the development of future adaptive immunity-inducing rsv vaccines, it has been demonstrated that the transfer of airway-resident t cells protect against rsv, where it has been suggested that, to lessen the burden of t cellemediated damage to airways, the induction of lung tissue t cells should be the focus of vaccine development (kinnear et al., 2018) . recently, the sev has been used as a component of a jennerian vaccine model for hpiv-1 and as a backbone for other viruses causing serious lower respiratory infections (lris), including other hpivs, rsv, and hmpv. sev-based vaccines have proven to be effective toward inducing b cell and t cell immune responses, and in the protection from hpiv-1, -2, and -3, and rsv, where they can also be used in combination with other vaccines to primeeboosts or to target one or more than one paramyxovirus pathogen (russell and hurwitz, 2016) . sev is attractive for use in human vaccination because it is a murine pathogen unable to infect humans (bousse et al., 2006) and therefore does not require attenuation as it can never revert to a human pathogenic phenotype (schickli et al., 2012) . another important feature supporting the use of sev as a pan-virus vaccination agent stems from its ability to grow transiently in mammalian cells, accommodating the endogenous expression of antigens with posttranslational modifications matching those of the target antigens and neutralizing epitopes (henrickson et al., 1991) , with endogenous expression of antigens ensuring robust activation of cd8 þ t cells able to destroy antigen-producing cells and terminate virus amplification (york and rock, 1996; russell and hurwitz, 2016) . in murine studies, sev could elicit rapid and durable respiratory mucosa and systemic hpiv-specific b cell and t cell responses (sealy et al., 2010; rudraraju et al., 2011) . clinical testing of human populations infected with rsv and hpivs is underway (adderson et al., 2015) . for a full review of current development of antiviral compounds and vaccine candidates tested against rsv, see costello et al., 2012. 2.4 adenovirus classification, epidemiology, immunology, and vaccinology human adenoviruses (hadvs) are classified in the mastadenovirus genus, containing seven known hadv species, from hadv-a to hadv-g, and with at least 57 unique known human serotypes (buckwalter et al., 2012) . adenoviruses are nonenveloped double-stranded dna viruses ranging from 65 to 80 nm in diameter and are composed of a protein capsid, a np core, and internal proteins. dna homology between hadv subgroups ranges from 48% to 99% (walls et al., 2003) . hadv infection rarely causes serious or fatal illness in immunocompetent individuals but may cause severe disease in immunocompromised, pediatric, and geriatric populations (lynch et al., 2011) . clinical disease symptoms associated with hadvs are dependent on hadv genotypes, with at least 69 recognized, and assigned to subgroups a through g. clinical symptoms include fever, rhinorrhea, pharyngitis, conjunctivitis, gastroenteritis, bronchitis, pneumonia, acute hemorrhagic cystitis, and meningoencephalitis (lynch et al., 2011) . recombination between hadvs are largely responsible for outbreaks of acute febrile respiratory disease in immunocompetent military recruits, where serotypes 4 and type 7 have been documented to account for approximately 60% of these respiratory illnesses (hilleman et al., 1957; dudding et al., 1973) , and are associated to other frequently occurring disorders including upper and lower respiratory illnesses, gastroenteritis, hepatitis, keratoconjunctivitis, meningoencephalitis, cystitis, and myocarditis in these immunocompetent populations, reviewed in lion (2014) . adenoviruses are endemic in pediatric populations (echavarria, 2008) . the incidence of adenovirus infection peaks in infants and children, where, globally, 5%e7% of respiratory tract infections in pediatric patients are ascribed to hadv (ghebremedhin, 2014) . recently, re-emergence of type 7d hadv has caused fatal outbreaks due to severe pneumonia syndromes in children from high-density populations . immune responses to adenovirus infection are dependent on primary sites of inoculation, methods of transmission, viral serotypes, and secretory ig antibody status of the infected host; igas are present in respiratory tract early following infection, and igg2 is present in serum and nasal secretions at later time points, reviewed in walls et al. (2003) . histopathological changes resulting from infection can be divided into two phases: the first phase of immune histopathology predominantly involves nonspecific, cytokine-mediated inflammatory recruitment of monocytes and macrophages, while the second phase involves t cell infiltration (prince et al., 1993) . t cellemediated immunity is believed to be required for hadv recovery from acute infections, and individuals lacking adaptive immunity are found to be at elevated risk of infection, with cd8 þ t cells as primary mediators of response to respiratory viruses, with relatively little contribution by cd4 þ cells (woodland et al., 2001) . however, following adenovirus exposure, cd4 þ t cells have been shown to be responsible for increasing proliferation status of peripheral blood mononuclear cells (pbmc), and cd4 þ t cells represent the major ctl subsets produced and recognize conserved antigens across adenovirus serotypes (flomenberg et al., 1995; regn et al., 2001) . adenoviruses have, however, evolved several hostevasion strategies, including inhibition of apoptosis, responses to ifn-g and tnf-a, and major histocompatibility complex (mhc) class i expression (mahr and gooding, 1999; wold et al., 1994 wold et al., , 1999 . the live, oral adenovirus vaccine was licensed in the 1970s for active immunization toward the prevention of febrile acute respiratory disease in military populations, where it initially reduced adenovirus-associated respiratory illnesses by over five-fold (dudding et al., 1973) . vaccine stock depletion and associated epidemics led to the manufacture of another vaccine in 2011, again denting adenovirus-associated disease burden by approximately 100-fold among recruits within the first 2 years of its introduction (radin et al., 2014) . these oral lyophilized vaccines replicate asymptomatically in the gut, inducing humoral and cell-mediated immunity, to confer longlasting protection from infection (berg et al., 2014) . due to their abilities to induce potent transgene product-specific t-and b-cell responses, adenovirus vectors are explored for use as vaccine carriers against a variety of many other pathogens (chen et al., 2010; harro et al., 2009; hill et al., 2010; radosevic et al., 2007) . alris by respiratory viruses are a major cause of morbidity and mortality, accounting for over 1.5 million deaths globally each year, and are predominantly resulting from human transmission of virus containing respiratory droplets. licensed vaccines are useful against several viruses causing these severe, often lethal, associated disorders. influenza has no borders and causes great economic burden, making it a prominent international concern. its rapid and unpredictable genetic drift causes human pandemics, where it has an annual potential of causing 5 million infections and 500,000 deaths worldwide. influenza vaccinology requiring constant yearly updates has stimulated interest in the development of universal t cell vaccines that can elicit both humoral and cellular immunity, whereby influenza-specific memory cd8 þ t cell responses against a range of influenza subtypes could be induced to clear infection in absence of neutralizing antibodies. rsv causes 34 million alris in children annually, resulting in 3 million hospitalizations and almost 200,000 deaths per year. rsv is cleared by balance of adaptive immune ctls and humoral neutralizing antibody responses, correlating most highly with cd4 þ helper and cd8 þ ctls during natural infections, and with memory cd8 þ and cd4 þ t cells following vaccination, with research endeavours targeting their strengthening by specific induction of lung tissue rsv targeting t cells. second, only to rsv, hpivs cause many ari-and lri-associated mortalities in children. hpivs can induce potent humoral, innate, and adaptive cd8 þ and cd4 þ t cell responses able to restrict their replication and where neutralizing antibodies can confer long-term protection against their associated disorders. hadvs infect both immunocompetent and immunocompromised humans and have been shown to cause up to 60% of respiratory disorders in hospitalized military personnel. despite their having evolved convoluted host-evasion strategies, adaptive t cell immunity against hadvs starts early in diseases phases and is key to recovery from acute natural infection, with its greatest contributions by cytotoxic cd8 þ t cells that require stimulating by cd4 þ t cells for their expansion. vaccines against hadvs induce both humoral and adaptive immunity, including potent transgene virus-specific t-and b-cell responses conferring long-term protection. success from hadv vaccinology has influenced explorations of adenovirus vectors as target carriers for vaccination against numerous other pathogens. diarrheal disorders remain a leading cause of morbidity and mortality worldwide, with these listed in the top five causes of death worldwide, and which are associated with global estimates at 4e6 million deaths per year; reviewed in clark and mckendrick (2004) . the majority of gastric infections are viral in origin, and viral gastroenteritis is one of the most common illnesses in all age groups and an important cause of morbidity in industrialized countries (chang et al., 2003) . the human risk of viral gastroenteritis in the united states alone is at least one per individual per year, with 450,000 adults and 160,000 children hospitalizations recorded and an associated 4000 mortalities per year (mead et al., 1999; mounts et al., 1999) . several viruses are responsible for viral gastroenteritis, where their transmission typically occurs from person-to-person by the oral-fecal route. viruses commonly causing gastroenteritis include rotavirus (rv; causing the most serious gastric disorders), norovirus, astrovirus, adenovirus, and coronavirus-like agents (table 1) . rvs are classified as a genus within the family reoviridae. these are nonenveloped viruses measuring 70 nm in diameter and have inner and outer capsids surrounding their cores containing double-stranded rna viral genomes encoding viral capsid (vp-1 to vp-6, and vp-7; vp4 outer capsid protein mediates virus attachment to cells) (bishop et al., 1973) and nonstructural (nsp-1 to nsp-6) proteins, reviewed in desselberger (2014) . rvs classify into seven serotypes (aeg), based on antigenic properties of the inner capsid vp6 protein, where subtypes aec represent human pathogens and are further subclassified into serotypes within these groups on the basis of differing outer capsid composition (anderson and weber, 2004; wilhelmi et al., 2003) . diarrhea is a major cause of death among children globally (liu et al., 2012) , and rv is the leading cause of severe diarrhea, globally causing an estimated 453,000 deaths in developing countries and 2.3 million pediatric patient hospitalizations parashar et al., 2003) . rv also represents a significant cause of disease in industrialized countries, with greater numbers of hospital admissions reported relative to developing countries (chang et al., 2003) . though group a rv causes the majority of endemic infections and can also lead to significant outbreaks in infant and geriatric populations (villena et al., 2003; marshall et al., 2003) , group b rvs are less common but can also lead to outbreaks and epidemics (sanekata et al., 2003; ahmed et al., 2004) , whereas group c rv is less often observed causing sporadic diseases. of the existing 10 g and eight p rv group a serotypes, g1 to 4, p (kostouros et al., 2003; clark and mckendrick, 2004) . studies of t cell responses to rv infection in humans have reported that most healthy adults and children have circulating rv-specific t cells, with approximately 50% of rv-cd4 þ t cells expressing the intestinal homing receptor a4b7, and with circulating rv-cd4 þ and rv-cd8 þ t cells secreting ifn-g or interleukin (il)-2 (makela et al., 2004; offit et al., 1992; yasukawa et al., 1990; rott et al., 1997; parra et al., 2014) . frequencies of circulating ifn-g þ rv t cells are comparable to those specific for other mucosal respiratory viruses (mesa et al., 2007) , but these often possess profiles of terminally differentiated effector cells that are usually associated to those unable to provide long-term immunity (parra et al., 2014) . (yen et al., 2014) . as in the case of natural neonatal rv infection, fair protection rates are achieved via humoral immunity using these vaccines. though these are unable to protect against rv reinfection, they do offer protection against severe associated clinical symptoms causing patient hospitalization. these vaccines offer both homotypic and heterotypic immunity, and protection often correlates with increases in rv typee specific igg or iga antibodies, reviewed in desselberger and huppertz (2011) . although rv vaccineeinduced humoral immunity substantially decreases disease burden, these vaccination strategies are less effective and difficult to implement in low-income countries requiring them most (patel et al., 2012) . as with natural rv infection, vaccines provide nonsterilizing immunity to children (angel et al., 2007) , where lack of establishment of long-term immunity against rv causes half of children's guardians to be at risk of becoming infected and presenting with severe associated disorders (rodriguez et al., 1987) . this further demonstrates that rv-specific t (rv-t) cells are crucial for the development of overall, long-term, protective immunity against rv (franco et al., 2006; offit et al., 1993) . indeed, in models of rv infection, vaccine-induced protective immune responses are dependent on antiviral cytokine production and by direct killing of rv-infected cells by t cell and b cell adaptive immune subsets (jiang et al., 2008; wen et al., 2016) . in addition, with observations that gut cd4 þ t cells may become tolerogenic or anergic in response to rv infection, stimulating t cells with rv antigen in the presence of il-2, il-12, or r59949, a pharmacological diacylglycerol kinase alpha inhibitor, causes increased pbmc frequencies of rv antigen-specific t effector cells, including rv-cd4 þ tnf-a þ , rv-cd4 þ ifn-g þ , and rv-cd8 þ ifn-g þ cells (parra et al., 2014) . diarrheal disorders cause an annual 4e6 million deaths worldwide. rv is the leading cause of severe diarrhea outbreaks in infant and geriatric populations, with global annual estimates of 453,000 deaths in developing countries and 2.3 million pediatric hospitalizations. most immunocompetent individuals have circulating rv-specific ctls at comparable frequencies to those elicited by other respiratory viruses, but which have terminally differentiated effector profiles rendering them incapable of conferring long-term protection against the reoccurrence of associated disorders. rv vaccineemediated protection from severe disorders is from humoral nonsterilizing immunity unable to protect against reinfection, yet vaccination programs are challenging to implement in countries requiring them the most. rv-specific t cells are key to long-term protection, and vaccineinduced protection is dependent on cytokine production and direct killing of infected cells by t cells. countermeasures against crucial helper cd4 þ t celledeveloping anergic states may assist the development of vaccines conferring long-term protection. an exanthem is a widespread eruptive skin rash that may be associated with fever or other systemic symptoms. more than 50 infectious agents causing exanthems have been identified (cherry, 1983) , where more than 70% of recorded cases of combined fever and widespread rash in pediatric populations were caused by viral infections, relative to the 20% resulting from bacterial infections (goodyear, laidler, price, kenny and harper, 1991) . correct diagnosis of these skin manifestations, resulting from direct inoculation of the infectious agent onto the cutaneous surface, or by dissemination from a distant site, is a main research theme on viral exanthems. this is because, while infections by many viral (i.e., paraviral) exanthems are benign and resolve spontaneously, others may rapidly lead to fatal conditions, reviewed in drago et al. (2017) . thus, special attention in diagnosing even vaccine-preventable viral exanthems must be applied to avoid the arising of serious complications in nonimmune pregnant women and their fetuses from the more harmful classes of viruses causing exanthems (white et al., 2012) . common exanthematous infections are typically caused by transmission of viruses from person-to-person (with exception of alphaviruses having a mosquito vector), and where a multitude of viruses are their causative agents, including rubeola virus, rubella virus, human parvovirus b19, human herpesvirus (hhv) type 6, varicella-zoster virus (vzv), variola, alphaviruses, and molluscum contagiosum virus (table 1) . numerous other exanthematous disorder causing viruses are not covered in this section, including ebola and zika, but which are becoming classified as emerging viral exanthems due to the increasing numbers of at-risk populations and the critical need to classify these diseases to minimize outbreaks and risk to pregnant women and fetuses (keighley et al., 2015) . rubeola, or measles virus (mev), belonging to the morbillivirus genus of the paramyxoviridae family, is a negative-sense rna virus having a nonsegmented genome and a lipid envelope, and measuring up to 250 nm in diameter, reviewed in griffin et al. (2012) . the 16 kb genome encodes eight proteins: the viral envelope is composed of hemagglutinin (h) and fusion (f) gps projecting from the matrix (m) protein lining its interior. the helical nucleocapsid is composed of the rna and nucleocapsid (n) protein packed within the envelope as a coil with the phosphoprotein (p) and large polymerase (l) proteins attached. the two ns proteins, c and v, regulate cellular response to infection and modulate ifn signaling (bellini et al., 1985) . humans are the only natural host of highly contagious mev virus spread by the respiratory route. despite the availability of a safe and efficacious vaccine, measles remains one of the most important viruses causing child morbidity and mortality worldwide (moss and griffin, 2006; wolfson et al., 2009) . infection by mev is associated with up to 10% of mortality rates in african children (grais et al., 2007; nandy et al., 2006) , and with 25% in unvaccinated refugee camp and virus-naive population mortalities (moss, 2007; shanks et al., 2011) . female mortality is a dominant feature disorders resulting from infection (garenne, 1994) , and many acute mortalities from secondary infections resulting from immune suppression induced by mev are also observed (beckford et al., 1985) . mev has a persistent and long latency infection period, often resulting in the development of subacute sclerosing panencephalitis (sspe) in males, causing fatal neurologic disease presenting itself many years following the original infection (bellini et al., 2005) . adaptive cellular immune responses are generally regarded as most important for clearance of mev. children with low plasma ig may recover from mev infection, while those with defects in cellular immunity develop progressive infections (albertyn et al., 2011; mcquaid et al., 1998) . mev-specific antibody and t cell responses coincide with the onset of the rash, whereby rash biopsies of mev-replicating, infected epithelial cells, have high levels of cd4 þ and cd8 þ t cell infiltrates (polack et al., 1999) . cd8 þ t cell subsets appear to be particularly important for control and clearance of infectious mev, where expanded circulating virus-specific ctls are found in the blood of patients suffering rash, and increases in cd8 þ t cells are also found in mev-induced pneumonias (jaye et al., 1998; mongkolsapaya et al., 1999; myou et al., 1993) . in addition, depending on the target tissue and cell type analyzed with regards to mev infection, though differentially rated, both cytotoxicity and ifn production have been implicated as key effector mechanisms for mev clearance (patterson et al., 2002; stubblefield park et al., 2011; finke et al., 1995) , with specific combinations of cd4 þ t cells, cd8 þ t cells, and b cells recorded as required for the control of primary mev infection (tishon et al., 2006) . protection against measles is based on mev-specific humoral, antibodybased, immunity. diagnostically, the current gold standard of protection is via quantification of neutralizing antibodies against the viral hemagglutinin (h) and fusion (f) surface gps (bouche et al., 2002; haralambieva et al., 2011; plotkin, 2010) . mev, however, triggers an aggressive immune response, involving both the humoral and cellular arms of the immune system (moss and griffin, 2012; de vries et al., 2012; buchanan and bonthius, 2012) . once measles has been cleared, it is memory t cells that can provide lifelong immunity against reinfection by mev (bester, 2016) . importantly, during mev infection, immune reactions to other pathogens are suppressed from weeks to years, leading to risk and susceptibility to secondary infections, and which is believed to be a driver of complications and mortality long after measles had been cleared. conversely, this measlesinduced immune "amnesia," sometimes disabling immune memory for up to 3 years, has been suggested to work toward herd protection against other infections and is supported by the association of measles vaccination with lowered mortality rates from other childhood infections (mina et al., 2015) . occasional spontaneous tumor regressions have also been observed to occur during natural measles infection, suggesting that mev infection may be adopted in the generation of safe and effective oncolytic viruses (russell and peng, 2009 ). rubella virus belongs to the togaviridae family and is the sole member of the rubivirus genus. rubella contains a single-stranded, positive-sense rna genome (frey, 1994) , and its viral particles measure between 50 and 85 nm in diameter (oshiro et al., 1969) and have a pleomorphic nucleocapsid surrounded by a host-derived lipid membrane (battisti et al., 2012) . the e1 and e2 rubella protein spikes are anchored to the external layer of the membrane, with membrane-bound e2 proteins bridging rows of e1 proteins, considered as the main immunodominant antigens responsible for controlling receptor-mediated endocytosis (petruzziello et al., 1996; katow and sugiura, 1985) . antibody levels against the neutralizing domain of e1 correlate with protection against rubella virus (mitchell et al., 1996; cordoba et al., 2000; wilson et al., 2006) . rubella virus is spread from person-to-person via the respiratory route and is the causative agent of rubella disease, commonly known as german measles (lambert et al., 2015) . although rarer in the united states, rubella infection remains a major health concern in developing countries (tosh et al., 2009) . although acquired rubella infection is not severe in adults, transplacental transfer of the virus to the developing fetus during maternal viremia can cause devastating consequences of congenital rubella syndrome (crs) (watson et al., 1998) , where more than 100,000 infants worldwide are born with crs each year . common crs symptoms include spontaneous abortion, premature delivery, fetal death, ocular abnormalities, neurological problems, abnormal cardiac development, and deafness (white et al., 2012) . congenital malformations due to crs may be present at birth, while other conditions such as diabetes mellitus, deafness, intellectual disability, and/or subacute encephalitis may develop months to years later (watson et al., 1998; white et al., 2012) . from mass immunization programs, the number of rubella cases has progressively declined and was no longer endemic in the united states as of 2004 but remains endemic in other countries, with a dramatic increase in reported cases the last decade (reef et al., 2011) . recently, africa and asian have seen 20-fold increases in rubella cases, representing a significant proportion of the over 121,000 global cases reported, but where neither of these regions has immunization policies in place to control rubella outbreaks (white et al., 2012) . a recent, 2013 rubella epidemic in japan reporting over 11,000 cases, with at least 13 crs cases (minakami et al., 2014) , has also served to demonstrate that partial vaccination strategies can lead to major outbreaks. in this case, vaccination was only provided to young women, while outbreaks affected the adult male populationsda phenomenon which has also been observed in other countries applying such vaccination strategies (paradowska-stankiewicz et al., 2013; janta et al., 2012) . once measles is cleared, memory t cells can provide lifelong immunity to mev (bester, 2016) , and distinct patterns of cellular immunity to rubella virus are observed and related to the time elapsed following vaccination (lambert et al., 2015) . predominant biomarkers of early cellular measles immunity are characterized by an immunosuppressive phenotype, with increases in il-10 and tnf-a and decreases in ifn-g and proliferative properties of circulating peripheral lymphocytes (pukhalsky et al., 2003) . late immunity is shifted to predominantly proinflammatory cytokine profiles via increased concentrations of il-6, granulocyte-macrophage colony-stimulating factor, and tnf-a, in combination with decreases in il-10 (dhiman et al., 2010) . human leukocyte antigens (hlas), known to play critical roles in immune response to viruses, contribute to the heterogeneity of the immune response to rubella virus as a result of their polymorphic nature, whereby hla class i and ii polymorphisms restrict the available repertoire of rubella antigens presented to t cells and therefore influence the subsequent immune response (mitchell et al., 1996; ou et al., 1994 ou et al., , 1998 . current efforts are placed on deciphering the immunogenetics of antirubella humoral and cell-mediated immune responses, with a focus on better understanding hla polymorphisms toward the development of vaccine candidates that utilize constructs comprised of hla-specific epitopes that can induce immunity across heterogenetic populations, reviewed in lambert et al. (2015) . both natural infection and vaccines induce humoral and cellular immune responses conferring protection against rubella (tosh et al., 2009) . while humoral responses have been conventionally used to measure and record protective immunity in human populations, cellular immune responses are intrinsic to humoral immunity (bautista-lopez et al., 2000; horstmann et al., 1985; ovsyannikova et al., 2004; nepom et al., 1997; vesikari et al., 1975; akaboshi et al., 2001; farzaneh et al., 2003) . since its induction into healthcare systems, immunization with live attenuated rubella virus vaccine has been demonstrated to be safe and effective at preventing infection, crs, and to interrupt endemic rubella transmission (lambert et al., 2015) . the live attenuated rubella vaccine strain ra27/3 has a proven track record for safety and immunogenicity efficacy (hilleman et al., 1968; plotkin, 1979) , where single doses have been demonstrated to potently induce humoral immunity and lifelong protection against infection, and where the vaccine has also been demonstrated to boost previously immunized persons (diaz-ortega et al., 2014) . from their safety and efficacy, use of recombinant rubella vectors has also been tested toward enhancing immune responses against siv and hiv epitopes, where increases in memory b cell repertoires have been observed upon re-exposure to rubella vectors (virnik et al., 2013) . durable hiv-specific cellular immunity has been observed from rubella vector boosting, with cytotoxic antigenspecific responses by central and effector memory cd4 þ and cd8 þ t cell subsets (rosati et al., 2015) . vzv, also known as hhv-3, is a virus of the varicellovirus genus from the herpesviridae family. humans are its only vector (hambleton and , where it specifically infects t cells, epithelial cells, and ganglia (gershon et al., 2015) . vzv viruses have diameters measuring up to 200 nm and are encoded by a linear double-stranded dna genome consisting of approximately 125 kb and encoding at least 70 unique genes, with all but the exception of 6, having homologs in herpes simplex virus (cohen, 2010) . vzv virions are composed of the viral dna, the capsid, the tegument surrounding the capsid, and the envelope surrounding the tegument and which incorporates the major viral gps (arvin, 1996) . during lytic infection phases, vzv produces at least 12 gps expressed on both virions and human cell surfaces. during this process, and which is common to other herpesviruses, gene expression is believed to proceed in an orderly cascade of immediate early genes, early genes, and late genes. during latent vzv infection, gene expression is restricted until reactivation for additional rounds of lytic infection (gershon and gershon, 2010) . vzv has extraordinarily high transmission rates and is highly communicable via the airborne transmission route, with concentrated virus coming from vesicles shedding from skin lesions, leading to cell-free contagious airborne viruses, and as evidenced by the fact that infected children without skin lesions are not contagious (tsolia et al., 1990; chen et al., 2004) . primary vzv infection causes varicella, also commonly known as chickenpox. as cellular immunity to vzv wanes in the elderly and immunocompromised populations, latent vzv becomes reactivated and causes zoster (i.e., shingles, herpes zoster), which is usually associated with chronic pain but also numerous other serious neurological and ocular disorders, as well as multiple visceral and gastrointestinal disorders, including ulcers, hepatitis, and pancreatitis (gershon et al., 2015; gilden et al., 2010) . available antiviral drugs and vaccines against varicella and zoster are safe and effective for treatment and prevention strategies (gershon and gershon, 2010) . varicella is globally endemic and is transmitted year-round, with frequent epidemics occurring every 2 to 3 years. outbreaks most commonly occur in nurseries and schools, in hospitals and other medical institutions, and in refugee camps and military and correctional facilities (izurieta et al., 1997; levy et al., 2003; longfield et al., 1990) . although it can often be a self-limiting disease, varicella can also result in death, where in developed countries, an estimated 5 of 1000 patients are hospitalized with serious complications, with up to three deaths per 100,000 patients (galil et al., 2002; rawson et al., 2001) . complications from varicella requiring hospitalization include bacterial superinfections of the skin, blood, bones, and lungs, as well as encephalitis and hemorrhagic manifestations in pediatric and immunocompromised populations (gershon et al., 2015) . importantly, acquiring vzv during early pregnancy often results in severe congenital defects in 1% of newborns (enders, 1984) . vzv is a great example of success through herd vaccination programs for children, dramatically influencing its epidemiology, and causing 95% declines of hospitalization cases in the united states (gershon et al., 2015) . following its transmission to the respiratory mucosa, vzv proliferates in the oral pharynx, where it infects human tonsillar activated memory cd4 þ t cells and induces their tissue-homing properties (sen et al., 2014) . vzv can be propagated to t cellerich regional lymph nodes for rapid proliferation and is then disseminated by the circulation to infect dermis, epidermis, and other organs (ku et al., 2002 (ku et al., , 2004 . lymphopenia is typically observed in patients during viral incubation, followed by an increase in leukocyte counts, correlating with the onset of rash until the resolution of viremia. during infection, vzv can be recovered from pbmcs in children exhibiting rash (ozaki et al., 1984; koropchak et al., 1992; sawyer et al., 1992) , is extensively observed in thymic lymphocytes (levin, 2014) , and observed in all t cell subsets examined (moffat et al., 1995) . though innate skin immunity can cause delays in multiplication of skin-bound vzv while the adaptive immune system mounts an attack, however, aggressive vzv replication in the skin results in characteristic varicella rash (ku et al., 2004) . high vzv titre-skin vesicles from rash provide cell-free virus for person-to-person transmission (chen et al., 2004) . vzv also latently infects neurons of cranial nerve ganglia, dorsal root ganglia, and enteric and autonomic ganglia (gershon and gershon, 2010) . vzv reactivation causes ganglia to become necrotic and hemorrhagic (head et al., 1997) , with vzv proteins found in neurons and non-neuronal cells, and where this vzv-induced ganglionitis is often also marked by the upregulation of mhc class i and ii proteins associated infiltration of cd4 þ and cd8 þ t cells (schmidbauer et al., 1992; steain et al., 2014; gowrishankar et al., 2010) . before vzv vaccines became available, approximately 30% of infected adults later developed shingles (yawn et al., 2007) . the single dose, lyophilized, live, attenuated vzv vaccine (i.e., zoster vaccine live (zvl), zostavax, merck) is indicated for prevention of latent vzv reactivation leading to shingles in individuals older than 50 years. zvl is licensed in over 55 countries, with 34 million distributed doses globally (willis et al., 2017) , and which has associated efficacy rates of over 50% in all ages tested (oxman et al., 2005; schmader et al., 2012) ; consistent with original clinical trial datasets (tseng et al., 2011; langan et al., 2013; marin et al., 2015) . however, increases in vzv susceptibility have arisen due to increasing aging populations, and in immune-suppressed organ transplant recipients, chemotherapy patients, hiv-infected individuals, and those suffering from chronic illnesses (forbes et al., 2014) . in these patients, earlier exposure to exogenous vzv protects against shingles by boosting cellular immunity (arvin et al., 1983; thomas et al., 2002) . the memory immune response following naturally acquired primary vzv infection is characterized by vzv igg and iga antibodies, as well as vzv-specific cd4 þ and cd8 þ t cells, where vzv-specific igg antibodies bind many vzv proteins and mediate virus neutralization and antibodydependent cytotoxicity, reviewed in arvin (2008). the frequency of vzv-specific memory proliferating t cells is estimated to be approximately one in 40,000 pbmc (hayward et al., 1986) . vsv-specific memory cytotoxic mhc class i-or class ii-restricted t cells producing ifn-g and tnf-a can recognize the vzv ge, gb, gc, gh, gi, ie62, and ie63 proteins and can be found to persist for over 20 years after varicella exposure (jenkins et al., 1998; huang et al., 1992; asanuma et al., 2000; diaz et al., 1989; hayward et al., 1986; sharp et al., 1992; sadzot-delvaux et al., 1997) . the ability of the live attenuated varicella vaccine to elicit vzv-specific igg and t cell immunity in naive hosts was established during its prelicensing clinical evaluations (gershon et al., 1992) , and where, as expected from its design, the magnitude of these vzv-specific immune responses correlated with infectious virus content and with antigen content of individual vaccine formulations (bergen et al., 1990; watson et al., 1993) . importantly, it was later discovered that providing two doses to children resulted in higher igg antibody titres and increased t cell proliferation and where experimental evidence suggesting that memory responses were sustained more effectively from such regimens (watson, 2008) . these observations led to the more recent recommendation of implementing of a two-dose regimen of varicella vaccine for all vaccine recipients (arvin, 2008) . studies of how regimens affect long-term protection by the adaptive t cell immune response to vaccination, as exemplified by vzv vaccination studies, have the potential to modify dosages and timelines to maximize overall and persisting beneficial long-term effects from vaccination against many other viruses. historically, smallpox was a severe human disease caused by the variola virus (varv), which was both highly lethal and highly contagious prior to its eradication from human populations in 1980 (moore et al., 2006) . varv belongs to the genus orthopoxvirus of the family poxviridae, which also includes zoonotic species: vaccinia virus (vacv), monkeypox virus, cowpox virus, and camelpox virus (shchelkunov, 2013) . orthopoxviruses are enveloped, brick-shaped viruses measuring 350 by 270 nm, and containing a double-stranded dna genomes encoding 150e200 genes, and measuring approximately 200 kb (garon et al., 1978) . unlike other dna viruses, these replicate as 'virus factories' in the cytoplasm of infected cells (pauli et al., 2010) . varv encodes approximately 200 proteins, where over 80 of these are found at terminal regions of the genome and are associated with host immune evasion. the origin of smallpox is unknown, but varv is considered to be one of the most deadly diseases of human history, decimating populations to such an extent that it significantly altered the course of human civilizations. smallpox is believed to have first appeared in 10,000 bc in africa, with the oldest credible confirmation found in sanskrit writings from 1500 bc and where smallpox lesions are believed to be observed on the mummified egyptian ruler ramses v (1100 bc) (ristanovic et al., 2016) . prior to its eradication in 1980, varv circulated in the human population for many centuries and repeatedly caused large-scale epidemics. in the 18th century for instance, smallpox caused the death of more than 400,000 europeans per year (babkin and babkina, 2015; smith and mcfadden, 2002) . despite varv eradication from the human population more than two decades ago, fears about its potential re-emergence or the threat of its use as a potential bioterrorism agent have not subsided. this has led to numerous debates concerning the destruction of existing viral stocks, currently maintained in the united states and russia. destruction of these stocks has been postponed for the benefit of further research elucidating varv mechanisms of pathogenesis toward the design of therapeutics as well as on efficacious vaccine strategies that may be required for potential future outbreak (smith and mcfadden, 2002; stone, 2002) . immune-evasion mechanisms by varv are the least understood among the orthopoxviruses due to difficulties of finding an appropriate host animal model (turner and moyer, 2002) , along with limited availability of authentic variola proteins since its eradication (massung et al., 1993) . thus only two variola proteins, namely smallpox inhibitor of complement enzymes (spice) and vaccinia virus complement control protein (vcp), have been characterized and are similar in structure (dunlop et al., 2003) . these viral antigens regulate the human complement system (yadav et al., 2008) , are important for stimulating innate immunity, and also have important features for adaptive immunity, shown to bolster antiviral t cell responses including ifn and cytokine expression (noris and remuzzi, 2013; moss and shisler, 2001) . vacv has been used more extensively for human immunization than any other vaccine and what was employed to provide cross-protection against varv toward smallpox eradication (jacobs et al., 2009 ). the first generation vacv/varv vaccines produced in the 1970s and 1980s (dryvax, apsv, lancyevaxina, l-ivp) contained live vacv , and induced robust humoral immunity is characterized by high antibody titers, neutralizing and opsonizing viral particles, fixing complement, hemagglutination, and antibody-dependent cell cytotoxicity (amanna et al., 2006; panchanathan et al., 2008) . these vaccines have since been observed to generate adaptive immune responses over many concentrations (frey et al., 2002; rock et al., 2006) , including the secretion of effector cytokines (e.g., ifn-g) and the lysing of infected cells (amanna et al., 2006; hammarlund et al., 2003) . most second-generation vaccines created for biodefense contain replication competent viruses (artenstein and grabenstein, 2008) and have comparable efficacies to dryvax. thirdgeneration vaccine formulations using attenuated vacv strains (lc16m8, mva, nyvac, dvvl) have increased safety profiles (artenstein, 2008; kennedy et al., 2009) . proof that adaptive cellular immunity is essential in preventing the spread of varv following immunization and in its generating overall protective immunity against smallpox comes from observations that individuals having t celledeficiency disorders suffered serious and sometimes fatal infections after vaccination, but that agammaglobulinemic children were not at risk of these adverse complications (rock et al., 2006) . varv vaccine induces strong cd4 þ and cd8 þ t cell responses, peaking after immunization and then contracting to provide stable memory t cell populations that remain detectable for decades (amanna et al., 2006; hammarlund et al., 2003) and with memory cd4 þ t cells persisting the longest (amara et al., 2004) . defects in cellular immunity lead to uncontrolled vaccinia infection (lane et al., 1970) , where cd4 þ and cd8 þ t cells are able to prevent mortality of b celledeficient animals infected with vacv (belyakov et al., 2003) and where cd4 þ t cells have the most protective overall effects (xu et al., 2004) , and are essential for optimal ctl function and memory formation (sun and bevan, 2003; kennedy et al., 2009) . vacv-specific cd4 þ and cd8 þ t cells recognize a diverse array of viral proteins, and cd8 þ t cell epitopes are predominantly found in early, non-structural genes and transcription factors (terajima et al., 2008) . cd4 þ t cell epitopes are from late viral products including membrane, structural proteins, and replicative enzymes (jing et al., 2008) , and linkage of b cell and cd4 þ t cell epitopes to varv proteins suggests t helper celleb cell interactions are those required for generation of robust vacv-specific antibody responses (sette et al., 2008) . in humans, varvspecific cd4 þ and cd8 þ t cells have been observed to persist for over 75 years following immunization (rock et al., 2006) . exanthem disorders by viruses represent more than 70% of cases of combined fever and widespread rash in pediatric populations, and their correct diagnosis is especially critical for the distinguishing of benign versus lethal viral strain variations that can cause lifelong morbidities in children born from infected mothers. mev is transmitted via human respiratory routes, and despite vaccine availability, still causes 10% of african children mortalities, and severe risk of sspe-derived fatalities years later in survivors. historically, in common with many other viruses, the gold standard diagnostic of protection is made by quantification of humoral neutralizing antibodies. adaptive cellular immune responses are, however, those most critical for mev clearance, where mev-specific t cell responses coincide with rashes densely infiltrated by cd4 þ and cd8 þ t cells. combinations of cd4 þ t cells, cd8 þ t cells, and b cells control primary infection, where cd8 þ t cells dominate for control and clearance, and memory t cells are able to provide lifelong protection. mev infection induces general longterm immunosuppression leading to vulnerability to other pathogens causing secondary infections, but this immunosuppression is believed, by some, to be simultaneously conferring herd protection and have been observed to induce spontaneous tumor regression. rubella virus infection has progressively declined from immunization programs but continues to be endemic in many countries, as a result of complete absence of or problematic or partial vaccination programs, still causing severe crs cases in 100,000 infants worldwide, per year. while humoral responses are conventionally used to measure protective immunity, it is adaptive immunity that confers protection. both natural infection and vaccination induce humoral and cellular immune responses, where memory t cells can provide lifelong immunity, with presence of cytolytic t cell biomarkers from vaccine-induced immunogenicity. vaccines in development can comprise hla-specific epitopes inducing immunity across heterogenetic populations. since rubella vaccines can boost the previously immunized, their vectors are being investigated for use toward immunization programs for unrelated viruses. vzv has extraordinarily high human transmission rates. primary vzv infection causes varicella, and before vaccination programs were initiated, would re-emerge from declines in adaptive immunity to cause zoster in 30% of in immunocompromised populations to cause the hospitalization of 1 of every 200 and the death of three per 100,000 patients. vzv represents a poster child of herd vaccination programs that led to 95% declines in hospitalization events. vzv infection rates are again on the rise in immunocompromised and immune-suppressed populations. it infects human tonsillar activated memory cd4 þ t cells that home to the lymph to then infect cd4 þ and cd8 þ t cells, followed by a lymphopenia resolved at rash onset. innate immunity controls vzv spread until adaptive immunity develops to fully counter the infection. vsv-specific memory ctls persist 20 years after varicella exposure, and observations that increased t cell proliferation with better-sustained memory responses result from multiple booster doses of vaccine have caused modifications in vaccination programs. smallpox by varv was one of the most deadly diseases in human history, causing more than 400,000 european casualties annually prior to its vaccine-mediated eradication. viral stocks are maintained from the necessity of developing new vaccines to counter potential future re-emergence of varv from natural-or bioterrorism-derived sources. characterized variola proteins amplify and strengthen t cell responses. first-generation vacv vaccine induced robust humoral immunity and adcc, in addition to generating adaptive immune responses marked by cytokines and cell lysis. varv vaccine induces strong initial effector cd4 þ and cd8 þ t cell responses having b cell linkage, then contacting to generate stable memory populations of varv-specific cd4 þ and cd8 þ t cells that can persist for over 75 years. accordingly, second-and third-generation vaccines created for biodefense are designed to stimulate adaptive cellular immunity. globally, liver cancer is the fifth most common of cancers, with an average of 374,000 cases per year, representing 7.2% of all cancers, and with mortality rates reflecting geographic incidence rates. almost 85% of liver cancers occur in developing countries, with over 20 of 100,000 individuals affected by these diseases. hepatocellular carcinoma (hcc) is the most common form of liver cancer, and approximately 80% of cases are associated with chronic infection by hepatitis b virus (hbv) or hepatitis c virus (hcv) (el-serag, 2012) . hepatitis viruses are so named because they display hepatotropism by preferentially infecting hepatocytes to cause liver inflammation, also known as viral hepatitis. infection by hbv and hcv promotes liver cirrhosis in most affected, leading to the development of hcc in up to 30% of patients (fattovich et al., 2004) . approximately 5% of the global population (350e400 million people) are chronically infected with hbv and strong correlations between hbv prevalence and hcc incidence and mortality. chronic hbv infection accounts for approximately 50% of hcc cases in adults and for all hcc cases in children (el-serag, 2012) . hepatitis transmission is from person-to-person contact with infected blood or body secretions or by the fecal-oral route and involving at least five specific viruses, namely hepatitis a, b, c, d, and e viruses (table 1) . infectious viral hepatitis is an important challenge to health worldwide: hepatitis a virus (hav) and hepatitis e virus (hev) are acute and endemic in many low-income countries, usually causing self-limiting hepatitis, whereas hbc and hcv also cause acute illness but usually lead to chronic and progressive liver fibrosis, cirrhosis, and an increased risk of hcc (stanaway et al., 2016) . hbv is controlled in adults but is chronically persistent from neonatal infection (shin et al., 2016) . hepatitis viruses differ in their virology. hbv is an enveloped dna virus that belongs to the hepadnaviridae family. it contains a 3200 bp, partially double-stranded relaxed-circular dna genome that is reverse transcribed via a pregenomic rna intermediate and encodes four overlapping open reading frames, which are translated to produce viral core protein, surface proteins, reverse transcriptase, and hbx (nguyen et al., 2008) . transmission of hbv results from exposure to infectious blood or body fluids containing blood, and hbv can integrate into the human genome, contributing to its genomic instability and ultimately to hcc (zhao et al., 2016) . hcv is also transmitted by infected blood; but unlike hbv, hcv does not integrate into the host genome . hcv is also a positivestranded rna virus but is classified in the hepacivirus genus within the flaviviridae family. its genome is 9.6 kb in length, includes an internal ribosome entry site, and encodes structural and ns proteins. the structural proteins form the viral particle and include the core protein and the envelope gps e1 and e2. the ns proteins include the p7 ion channel, the ns2-3 protease, the ns3 serine protease and rna helicase, the ns4a polypeptide, the ns4b and ns5a proteins, and the ns5b rnadependent rna polymerase (moradpour et al., 2007) . hepatitis d virus (hdv) is also transmitted by contact with infected blood or other body fluids. hdv is an enveloped, negative sense, singlestranded, closed circular rna virus, and requires hbv coinfection for its propagation, where infection with both viruses commonly results in severe liver pathologies. hdv genomic rna of hdv is composed of approximately 1700 bp, packaged with approximately 200 molecules of hepatitis delta antigen to form viral particles. hdv envelope surrounding its genome and hdag protein is composed of the three hbv small, medium, and large hbv hbsag envelope proteins. hdv also does not encode its own replicase or polymerase, and rather utilizes host cellular machineries for its replication (abbas and afzal, 2013) . hav and hev are positive-stranded nonenveloped rna viruses transmitted via the fecal-oral route, and unlike chronically persisting hbv and hcv, are typically cleared after acute infection of immunocompetent individuals (park and rehermann, 2014). hav is a hepatotropic virus belonging to the hepatovirus genus within the picornaviridae family. its genome consists of approximately 7500 bp and encompasses a single open reading frame coding for a single polyprotein, which is post-translationally processed into structural and ns proteins. the structural proteins of hav are divided into the polypeptides vp1, vp2, vp3, and vp4, forming the icosahedral capsid of the virus. ns proteins 2b, 2c, 3a, 3b, 3c, and 3d are involved in rna replication and viral polyprotein processing (martin and lemon, 2006) . hev of the family hepeviridae and genus orthohepevirus has a 7.2 kb genome having three opening reading frames encoding for the viral replicase, the capsid protein, and a small phosphoprotein required for the secretion of viral particles (debing et al., 2016) . hav and hev are waterborne viruses that usually cause acute hepatitis without progressing to chronic liver disease (joon et al., 2015) , where annually, over 100 million cases of hav and 28 million cases of hev infections have been recorded globally (makiala-mandanda et al., 2017) . hev outbreaks are reported in africa nearly every year, with some involving over 10,000 cases (kim et al., 2014) . hav is highly endemic in africa, infecting most children, conferring long-term immunity to reduce serious epidemics (jacobsen, 2014) . hbv, hcv, and hdv can be sexually, parenterally, or vertically transmitted and usually evolve into chronic hepatitis, liver cirrhosis, and hcc causing high morbidity and mortality rates, where globally, over 350 million people are chronically infected with hbv, 150 million with hcv, and 15 million with hdv (kramvis and kew, 2007; hughes et al., 2011; thursz and fontanet, 2014) . superinfection of hbv patients with hdv frequently accelerates the progression of hbv disease to liver cirrhosis, considerably increasing the burden of chronic liver disease (hughes et al., 2011) . hav, hbv and hcv are responsible for the majority of viral hepatitis cases, and there are similarities and differences in immune responses to infections by these three viruses, possibly explaining the distinct disease courses and outcomes of each hepatitis virus infection (shin et al., 2016) . type i and iii ifns, major components of the antiviral innate immune system, induce the expression of ifn-stimulated genes (isgs), observed to be much more highly induced by hcv than hav, and not at all by hbv, indicating that this virus is not recognized by the innate immune system (su et al., 2002; lanford et al., 2011; wieland et al., 2004) . another component of the innate immune system, nk cells, are also believed to be responsible for protection against hcv, where increased nk cells in protected individuals coincide with increased ifn-g and cytotoxicity (shin et al., 2016) . though virus-specific antibodies are produced by all viral hepatitis infections, these have differing roles according to the hepatitis virus infection. hav-specific antibodies with virus-neutralizing activity are induced by natural infection and vaccine immunization and confer lifelong protective immunity (walker et al., 2015; martin and lemon, 2006) . hbv surface antigen hbsag-specific antibodies are induced by infection and immunization with the recombinant protein and have virus-neutralizing activity conferring protective immunity (guidotti and chisari, 2006) . hcv-specific antibodies produced after infection do not offer long-term protection as these do not persist, are subject to loss of neutralizing activity from virus mutation, and are ineffective for cellto-cell hcv transmission (takaki et al., 2000; dowd et al., 2009; timpe et al., 2008) . t cells play critical roles during acute hcv and hbv infections, where robust and multiple epitope-specific cd8 þ t cell responses are assisted by cd4 þ t cells for spontaneous resolution of infection (shin et al., 2016) . this is supported by observations that the depletion of cd4 þ or cd8 þ t cells in chimpanzees delays rapid clearance and recovery from infection by these viruses (grakoui et al., 2003; thimme et al., 2003) . when hcv and hbv infections become chronically persistent, virus-specific t cells become exhausted and functionally impaired. in acute hcv infection, virus-specific t cells are only detected in the blood and liver after 8 weeks postinfection, and their appearance coincides with large declines of virus titres shin et al., 2006 shin et al., , 2011 . hbv virusespecific t cell responses are also important for spontaneous resolution of hbv infection, where their responses are observed to be vigorous, broad, and polyclonal in patients resolving primary infections and where their absences are associated with prolonged infection and delayed viral clearance (chisari et al., 2010; thimme et al., 2003) . cd8 þ t cells also play important roles in hav infection, where these have been observed to target multiple epitopes of hav, despite more recent results suggesting that hav is controlled by virus-specific cd4 þ t cells and not cd8 þ t cells (walker et al., 2015; shin et al., 2016) . finally, hepatitis virus infection results in liver injury, not directly caused by these viruses but rather by immunemediated mechanisms (guidotti and chisari, 2006) . liver injury biomarkers correlate with acute hav, hab, and hac infection (guidotti and chisari, 2006; park and rehermann, 2014; walker et al., 2015) and may result from cytotoxic activity of cd8 þ t cells, believed to induce apoptosis of hepatocytes in close proximity to their targeted cells (guidotti and chisari, 2006) , by il-22 producing th17-differentiated t cells, and by recruitment of nonspecific mononuclear cells by hbvspecific cytokine secreting cd8 þ t cells (iannacone et al., 2007; shin et al., 2016) . effective vaccines controlling hav and hbv have been available for over 2 decades, and an hev vaccine has also been licensed for use in china since 2011 (zhu et al., 2010; stanaway et al., 2016) . neonatal hbv vaccination has proven to be highly effective in inducing protective antibodies and preventing perinatal and horizontal transmission of hbv (lee et al., 1991) . however, observations that hbsag-specific ifn-g-or il-5-secreting pbmcs are absent in many adolescents suggest that booster vaccines should be administered to provide continued hbv immunization (lu et al., 2008) . hav vaccination provides long-term immunity in the general population and in immunocompromised patients infected with hiv (crum-cianflone et al., 2011). there is no existing vaccine for hcv, despite ongoing efforts toward their design and testing for their ability to generate prolonged cellular and humoral immune responses, reviewed in naderi et al. (2014) . in the absence of a vaccine, progress in hcv treatment includes oral treatments achieving cure in most patients, including those previously considered as difficult to treat cases (poordad et al., 2013; lawitz et al., 2013) . liver cancer is the fifth most common cancer, representing 7.2% of all cancers, with 374,000 annual cases from which 20 in 100,000 mortalities occur. hcc is the most common liver cancer, with 80% of cases resulting from chronic infection by hbv or hcv, with a significant 350 and 150 million chronically infected, respectively. in contrast, hav and hev cause acute hepatitis but do not progress to chronic liver diseases, and hdv infection depends on pre-existing hbv infection. hcv induces the expression of type i and iii isgs and nk cells, not at all present from hbv infection unrecognized by innate immunity. virus-specific antibodies are produced by all viral hepatitis infections but have differing roles across infections. robust and multiple epitope-specific cd8 þ t cell responses and dominant but depend on assistance from cd4 þ t cells for resolution of acute hav, hbv, and hcv infections. in chronic infections, cytolytic t cells either cause extensive liver injury to hepatocytes and/or become tolerant and functionally impaired. effective vaccines controlling hav and hbv provide protective antibodies. hav vaccination provides longterm immunity to the immunocompromised, but booster vaccination programs are required for persistence of hbv immunization. no vaccine is licensed for highly variable and rapidly mutating hcv, despite numerous ongoing efforts to generate those which will provide robust cellular and humoral immune responses. historically, the central nervous system (cns) has been considered to be an immunologically privileged site within the body (bailey et al., 2006; galea et al., 2007; engelhardt, 2008; prendergast and anderton, 2009 ). by definition, immunologically privileged sites, also including the brain, cornea, testis, and pregnant uterus, have a reduced or delayed ability to reject foreign tissue grafts compared with conventional sites within the body, such as skin (streilein, 2003; bailey et al., 2006; carson et al., 2006; mrass and weninger, 2006; kaplan and niederkorn, 2007) . though the cns is protected by a highly complex barrier system, a wide variety of viruses still manage to gain access to it and induce diseases. due to their sizes and tissue penetration strategies, the number of cns viral infections outweigh bacterial, fungal, and protozoa cns infections combined (romero and newland, 2003) . following cns infection, inflammatory events can arise in distinct anatomical regions such as the meninges (meningitis), brain (encephalitis), and spinal cord (myelitis) or can also simultaneously arise in multiple regions (meningoencephalitis, encephalomyelitis). for many neurotropic viruses, viral cytopathology plays a major role in cns dysfunction, reviewed in swanson and mcgavern (2015) . virus can breach the protective barriers of the cns in many ways, with the main route mechanism being via the blood, where inhaled or ingested viruses can move past the mucosa to establish infection in secondary lymphoid tissues and later be shed into circulating blood to cause broad systemic infections (swanson and mcgavern, 2015) . the cns parenchyma is protected from a plethora of agents carried in the circulation via an elaborate network called the blood-brain barrier (bbb) and the blood-cerebrospinal fluid barrier (ransohoff et al., 2003) . viruses have evolved and adapted to overcome these barriers (mcgavern and kang, 2011) , where some viruses can infect vascular endothelial cells, permitting direct passage across the bbb into the cns (verma et al., 2009; moses et al., 1993; coyne et al., 2007) . in addition, parts of the cns that are not completely protected by the bbb permit more rapid entry of several viruses (van den pol et al., 1999; wolinsky et al., 1974) . infected hematopoietic cells in circulating blood can also serve as "trojan horses" that can transport undetected virus into the cns (clay et al., 2007; tabor-godwin et al., 2010) . other mechanisms can include systemic viral infections leading to massive systemic inflammation and an ensuing bbb breakdown which opens the floodgates to cns infection by a variety of otherwise restricted infectious agents (arsenio-nunes et al., 1975; eugenin et al., 2006) . there are more than 30 recognized distinct virus strains that cause human neurological disease, and the majority of documented cases are caused by viruses that are transmitted to humans by blood-eating arthropod vectors, also known as arboviruses, that are mainly transmitted by mosquitoes and ticks and include polioviruses (pvs), alphaviruses, mosquito-borne flaviviruses, tick-borne orthobunyaviruses, mosquito-borne mammarenaviruses, and rabies virus (rabv; table 1 ). pv, the causative agent of poliomyelitis, more commonly referred to as polio, is a human enterovirus and member of the family of picornaviridae. typically spherical, nonenveloped picornaviruses range in diameter between 27 and 30 nm and have a positive-strand rna of 7000e9000 nucleotides, translated into a polyprotein (i.e., vp4-vp2-vp3-vp1-2a-2b poliovirus 2c-3a-3b-3c-3d), which yields 11 proteins upon its cleavage by viral proteases. picornavirus replication occurs in the cytoplasm of infected cells in association with intracellular membranes, where virions are released by cell lysis, ultimately killing cells and causing extensive damage to tissues. the host immune response against picornaviruses includes cytokine release, antibody production, and ctl activation, reviewed in dotzauer and kraemer (2012) . the viral genome of pv is a single-stranded rna of approximately 7500 nucleotides, enclosed in a nonenveloped capsid comprising 60 copies of four different polypeptides arranged with icosahedral symmetry (racaniello, 2006) . all three pv serotypes cause paralytic disease, and cd155 is the cellular receptor for all three serotypes, whereby pv interaction with cd155, expressed by many different cell types, leads to a conformational change of the virus particle and the following release of the rna genome into the cellular cytoplasm (mendelsohn et al., 1989; hogle, 2002) . once in the cytoplasm, the viral rna genome is translated, and the production of new infectious virions begins. pv infection results from ingested virus that replicates in the oropharyngeal and intestinal mucosa (sabin and ward, 1941) . from the primary sites of multiplication in the mucosa, pv virus drains into cervical and mesenteric lymph nodes and then into the blood, causing transient viremia symptoms. the person-to-person transmission of pv virus is through the fecal-oral route. once pv is shed in the feces, the majority of the natural human infection ends at this stage with a modest symptoms including sore throat, fever, and malaise (dotzauer and kraemer, 2012) . however, in 1%e2% of pv-infected individuals, the virus gains entry to the cns through neurons at neuromuscular junctions (nmjs) and replicates in motor neurons within the spinal cord, brain stem, or motor cortex and leads to the pv-characteristic flaccid muscle paralysis disorder poliomyelitis (racaniello, 2006; koyuncu et al., 2013) . approximately 10% of these paralytic cases result in death (roush et al., 2007) . following both pv infection and vaccination, neutralizing antibodies are generated to clear the virus, and these can be detected for many years, providing lifelong protection (libbey and fujinami, 2014) . vaccination with the injected inactivated pv vaccine prevents viral spread to the cns, whereas vaccination with the live-attenuated oral pv vaccine protects against infection of the intestinal tract and also prevents person-to-person spread of the virus (nathanson, 2008; griffin, 2010) . pv remains an important cause of neurologic disease as the three live-attenuated vaccine strains are at risk of recombining their genomes to revert to virulent form (griffin, 2010) . pv is endemic in afghanistan, pakistan, india, and nigeria, where political reasons, in part, are the most significant modality toward achieving pv eradication via vaccination. pv can usually be cleared by the adaptive immune response. under conditions of antibody deficiencies in humans, however, continuous fecal shedding of pv contributes to the establishment of persistent infection cycles (martin, 2006; nathanson, 2008; libbey and fujinami, 2014) . though less is known about the roles of adaptive t cell responses in controlling pv infections relative to that of neutralizing antibody responses, it is known that pv-specific cd4 þ t cells are induced in vaccinated individuals, where key epitopes have also been identified (graham et al., 1993; simons et al., 1993) . the induction of pv-specific cd4 þ t cells has been suggested to be the result of stimulating by pv-infected dcs and macrophages (wahid et al., 2005; dotzauer and kraemer, 2012) , where it has been demonstrated that hla class ii presentation remains intact in infected, antigen-presenting cells (apcs), and that cytolytic cd4 þ t cells produce ifn-g to lyse pv-infected cells for virus clearance. pv-specific cytotoxic, ifn-g-secreting cytotoxic cd8 þ t cell responses induced by infected macrophages have also been documented (wahid et al., 2005) , suggesting that both cd4 þ and cd8 þ cytolytic t cells partake in the adaptive immune reaction against pv (dotzauer and kraemer, 2012) . approximately 73 viruses are included in the flavivirus genus of the flaviviridae family of viruses, with 40 of its species associated with dengue, yellow fever (yf), japanese encephalitis (je), tick-borne encephalitis, and west nile encephalitis as the most important arboviruses causing extensive global morbidity and mortality (diamond, 2003) . flaviviruses are enveloped viruses with single-stranded rna genomes that are translated in the cytoplasm to generate a single polyprotein that is then cleaved into structural and ns proteins by virus and host proteases. the various encoded viral proteins assemble to generate the capsid, the envelope for receptor binding, membrane fusion and viral assembly, and the transmembrane proteins (prm) that assist in protein folding and function. the entry of flaviviruses into their target cells is mediated by the interaction of the e gp with host cell surface receptors (perera-lecoin et al., 2013) . flaviviruses are believed to evade the immune system to enter the brain and spinal cord via circulating blood (johnson and mims, 1968) , where these may cross the bbb by passive transport across the endothelium, by active replication in endothelial cells, or by a "trojan horse" mechanism, where virus hides in inflammatory cells during their transit into the brain (solomon and vaughn, 2002) . ifn-dependent and complement system innate immune responses, along with humoral neutralizing antibodies, protect against virus dissemination and spread, reviewed in diamond (2003) . adaptive cellular immunity is also important toward the destruction of infected cells, whereby virus-specific ctls become activated, proliferate, and release inflammatory cytokines following exposure to flavivirus-infected cells (kesson et al., 1987; kurane et al., 1989a; liu et al., 1989; murali-krishna et al., 1996) . there is also evidence that flavivirus replication is enhanced by myeloid cells, as observed for dengue, yf, west nile, tick-borne, and je viruses (diamond, 2003) . the je flavivirus is the leading cause of encephalitis and is amplified by waterfowl and only transmitted to humans by mosquito vectors, with no possibility of human-to-human transmission (erlanger et al., 2009) . pediatric and geriatric populations are at higher risk of infection by je (burchard et al., 2009) . while 99% of je infections remain asymptomatic, je can be devastating in symptomatic patients, causing mortality rates of 30% in these patient populations (batchelor and petersen, 2015) . after approximately 14 days of je incubation, these patients suffer from high fever, chills, headache, myalgia, and confusion, where pediatric patients also have symptoms of gastrointestinal pain, vomiting, and seizures. post-je infection, handicaps from persistent neurological deficits can last a lifetime in up to 50% of these survivors. as there is no treatment against je, the only method of prevention is avoidance of mosquitos and vaccination (batchelor and petersen, 2015) . the four available je vaccines are registered worldwide and used in national immunization programs for different age groups, including inactivated vero cell culture vaccine (je-vc) (ixiaro), inactivated mouse braine derived vaccine (je-mb), a cell cultureederived (primary hamster kidney) live-attenuated vaccine based on the sa 14-14-2 strain manufactured in china, and a live-attenuated chimeric vaccine based on the genes of yf 17d backbone combined with vero cellepropagated sa 14-14-2 strain (imojev) (chen et al., 2015) . t cell responses to je vaccination have been reported, where for sa14-14-2, t cell responses were detected in the majority following vaccination, and these cross-reacted with other flaviviruses (turtle et al., 2017) . je-specific t cell responses are observed in pbmcs isolated from je-infected patients and vaccinated individuals. cd4 þ and cd8 þ t cells directed against structural viral proteins were identified in vaccinated individuals, in contrast to specific cd4 þ and cd8 þ responses against ns or c proteins in infected patients (nathanson and cole, 1971) . these findings indicate that ns proteins, and especially ns3 have important roles in the initiation of t cell responses, as the main target of je-specific t cellemediated immune responses. in addition, cytolytic cd4 þ t cells clones that cross-reactive with other flaviviruses have been generated from individuals immunized with inactivated je vaccine (aihara et al., 1998) . finally, cd4 þ and cd8 þ and th1 t cells are believed to be primary determinants of protection from je infection (kumar et al., 2004a (kumar et al., , 2004b ). viruses from the alphavirus genus are members of the togaviridae family of viruses, a group of enveloped positive-sense rna viruses. these are mosquito-borne viruses causing two major types of human disease. the old world alphavirusesdsindbis, chikungunya, and ross river virusdcause arthritis and arthralgia, while the new world alphavirusesdeastern (eeev), western (weev), and venezuelan equine encephalitis virus (veev)dcause encephalitis (trobaugh and klimstra, 2017) . alphaviruses are small, icosahedral-shaped, enveloped viruses and are approximately 70 nm diameter in size (mancini et al., 2000; morgan et al., 1961; fuller, 1987) . alphavirus virions acquire host cell lipid membranes during viral assembly (fuller, 1987; acheson and tamm, 1967; vogel et al., 1986) , with 80 e1 and e2 viral gps spike protrusions, arranged in an icosahedral pattern embedded within their membranes and interacting with nucleocapsid (fuller, 1987; vogel et al., 1986; owen and kuhn, 1997) . alphavirus single-stranded, positive-sense, rna genomes are 12 kb long and consist of two large open reading frames encoding the ns and structural polyproteins that are subsequently cleaved by both viral and host proteases to create four ns proteins (nsp1 to 4) and five structural proteins (c, e3, e2, 6k, e1) (strauss et al., 1984; hardy and strauss, 1989) ; reviewed in leung et al. (2011) . of major concern are the new world eeev, weev, and veev alphaviruses, which are naturally transmitted by mosquitos, but where veev is also highly infectious via the aerosol route (zacks and paessler, 2010) . precise mechanisms of entry of alphaviruses into the cns remains elusive, however, once in, alphaviruses infect humans and equines neurons, causing neurologic symptoms from mild febrile illness to severe encephalitis resulting in death (ramakrishna et al., 2002; zacks and paessler, 2010) . development of severe encephalitis is believed to result from neuronal cell death from accelerated viral spread and host neuroinflammatory viral responses (paessler et al., 2006 (paessler et al., , 2007 . antibodies are protective against lethal meningoencephalitis when the virus is transmitted by insects, and virus-specific cd4 þ t cells are found to be important for protection from lethal meningoencephalitis from aerosol transmission routes (paessler et al., 2007; yun et al., 2009) ; reviewed in libbey and fujinami (2014) . veev remains an emerging disease threat by natural transmission as well as via its usage as a biological weapon. of the new world alphaviruses, veev is the most important human and equine pathogen, it having caused outbreaks of febrile and neurological disease primarily in latin america during the past century. past outbreaks have lasted several years and have involved up to 100,000 equine and human cases over large geographical regions, with the largest outbreaks on record were from the 1960s, where central colombia saw over 200,000 human cases and an estimated 100,000 equine deaths. more recent outbreaks in mexico and south america are behind the classification of veev as a re-emerging disease (weaver et al., 2004) . because veev can also be developed as a biological weapon amenable to use in warfare or terrorism, current global emphases on biological defenses have renewed interest in its virology (hawley and eitzen, 2001; weaver et al., 2004) . veev infection in humans typically causes nonlethal, incapacitating symptoms including fever, headache, malaise, myalgia, sore throat, and vomiting. up to 4% of rarer cases of cns involvement usually follow acute febrile phases, with associated severities of neurological disease ranging from somnolence and mild confusion, to seizures, ataxia, paralysis, and coma, with mortality rates ranging as high as 35% in infected children and 10% in infected adults (bowen et al., 1976) . veev has also been reported to cause long-term neurological deficits, abortions, and teratogenic effects (de la monte et al., 1985; rivas et al., 1997; weaver et al., 1996) . like veev, though the majority of human infections with eeev are asymptomatic, cns involvement results in severe neurological signs, lesions, and sequelae, with an estimated associated human mortality rate of 75%, and with its neurological manifestations including facial edema, paresis, paralysis, respiratory impairment, altered mental state, and seizures in children, many of these symptoms persisting long-term in surviving patients. in fatal cases of eeev, gross lesions in the brain include edema, meningeal congestion, hemorrhage, and malacia (deresiewicz et al., 1997) . as with veev and eeev, natural human cases of weev typically show an early, flu-like illness with associated fever, malaise, and headache. similar to eeev, weev results in cns involvement in a significant proportion of cases, including symptoms of somnolence, seizures, coma, and motor neuron dysfunction. ninety percent of infants infected with weev have severe cns symptoms (calisher, 1994) . human mortality rates from weev infection range from 3% to 15%, and neurological sequelae may become permanent features in survivors (steele and twenhafel, 2010) . alphavirus expression vectors based on sindbis, semliki forest, and veev have been demonstrated to induce strong cd8 þ t cell responses against their antigens (rayner et al., 2002; lundstrom, 2002 lundstrom, , 2003 riezebos-brilman et al., 2006; schlesinger and dubensky, 1999; polo et al., 2000) . both innate and adaptive immune responses can control viruses targeting cns neurons (griffin, 2003) . viral disruption of the type i ifn signaling pathways interferes with survival from veev, as well as of those infected with sindbis and west nile viruses (ryman et al., 2000; samuel and diamond, 2005; white et al., 2001) . virus-specific antibody responses are critical in limiting viral spread and facilitating clearance of infectious virus from neurons within the brain levine et al., 1991) . both alpha beta (ab) and gamma delta (gd) t cell responses have been demonstrated as being important for the control of veev (paessler et al., 2006) . t cell responses reduce mortality rates by direct killing of infected cells, producing antiviral cytokines and increasing production of virusspecific antibodies (bilzer and stitz, 1994; patterson et al., 2002; shrestha et al., 2006; sitati and diamond, 2006) . veev replicon particles delivered as an adjuvant have been demonstrated to induce activation of cd8 þ t cell responses (thompson et al., 2008) . more recently, t cells have been demonstrated to facilitate recovery from veev-induced encephalomyelitis in absence of antibodies, responsible for dramatic reduction in viral titres in cns, where cd4 þ t cells were the best t cell producers of ifn-g response and were more efficient at controlling veev in cns lesions than cd8 þ t cells, facilitating recovery from severe viral encephalomyelitis (brooke et al., 2010) . commercial equine vaccines marketed in the united states are generated with inactivated tc-83, which produces viremia, fever, and leukopenia in horses but generates robust neutralizing antibodies and veev protection from rechallenge (walton et al., 1972) . u.s. army special immunization programs provide inactivated c-84 to individuals failing to seroconvert in response to tc-83 boosters (pittman et al., 1996) ; however, neither of these vaccines can be shown to completely protect nonhuman primates against aerosol exposure (pratt et al., 1998) . a more stably attenuated veev vaccine candidate called v3526 has been produced, where preclinical testing has demonstrated it to be safe and immunogenic and possibly superior to tc-83 (pratt et al., 2003; hart et al., 2000; ludwig et al., 2001) . adaptive immune pbmc-derived biomarker signatures have been identified and able to efficiently stratify tc-83 vaccinated from naïve or nonresponding individuals (erwincohen et al., 2012) . rabv is the type species of the genus lyssavirus, within the rhabdoviridae family. rhabdoviruses are negative-sense, single-stranded rna viruses having a distinctive bullet-shaped structure. up to 10 viruses of lyssaviruses have the potential to cause rabies in humans. these have a 12,000 nucleotide genome encoding five proteins: nucleoprotein (n), phosphoprotein (p), matrix (m), glycoprotein (g), and rna-dependent-rna polymerase (l) (marston et al., 2007) . rabv causes acute encephalitis in mammals, causing fatality rates of almost 100%. rabv commonly infects many animals, including bats, skunks, foxes, and dogs and can also infect insects and plants. rabv in animal saliva spreads between hosts via bites or scratches. infected animals can survive for years, secreting infectious particles in their saliva, but untreated infection in humans generally results in rapidly fatal acute myeloencephalitis (koyuncu et al., 2013) . rabid dogs are the most important reservoirs for rabv, where dog bites account for more than 99% of human infections. rabv, like all members of lyssaviruses, is neurotropic and infects peripheral nerves close to the primary site of the bite. rabv then rapidly moves by retrograde axonal transport to the dorsal root ganglia where virus replication begins . rabv particles enter axons of motor neurons at the nmj via their binding to nicotinic acetylcholine receptors (e.g., nachr) and neural cell adhesion molecules (ugolini, 2011) . transneuronal rabv spread occurs between synaptically connected neurons, whereby viruses move from postsynaptic to presynaptic neurons. in humans, a relatively long asymptomatic incubation period after initial rabv infection can occur, sometimes lasting up to 1 year, and providing some time for cns infection intervention. however, death almost always ensues after rabv infection reaches the cns, with marked behavioral and neurological symptoms (koyuncu et al., 2013) . once rabv has entered the cns, it rapidly moves to the brain and is associated with an explosive increase in virus replication. initial symptoms include pain or paraesthesia close to the bite site and are often associated with fever, fatigue, and weakness in associated limbs. nonspecific neurological symptoms including headache and anxiety occur days prior to acute encephalitis (morrison and wenzel, 1985) . currently, there are no available therapies against disease symptoms once they develop, and death ensues within a number of days following cns-associated symptoms (jackson et al., 2003) and reviewed in johnson et al. (2010) . rabv replication begins following cns penetration, thereby limiting earlier possible detection of low-level primary antigens in the peripheral circulation. this delays antigen presentation, where antigens later but rapidly drain from the cns to local lymphoid tissues (knopf et al., 1998) . once b cells are stimulated, the next delaying obstacle is re-entry into the cns, but experimental models have demonstrated t and b cell infiltration of dorsal root ganglia, spinal cord, and brain (johnson et al., 2008) , with t cells as the major immune subsets, but where most of these cns-infiltrated t cells have fas-mediated apoptotic phenotypes (baloul and lafon, 2003) . further intrinsic complexities in immune responses are present in the cns, including tight mhc expression regulation (irwin et al., 1999) , and the expression of immunosuppressive factors by neuronal cells. additionally, the bbb remains intact during rabv infection (roy et al., 2007) . numerous studies have suggested that the virus suppresses the adaptive immune response, believed to be in part due to a deficit of adaptive immune effector cell accumulation within the cns due to a virally induced reduction in bbb permeability (libbey and fujinami, 2014; roy et al., 2007) . two rabv vaccines are licensed for human application, the human diploid cell vaccine manufactured by aventis pasteur and the purified chick embryo cell vaccine manufactured by chiron . pre-exposure vaccination given to healthcare personnel, laboratory workers, and travelers to endemic areas causes detectable igm and igg antibodies within a week following exposure, and long-term studies have provided evidence that igg antibodies provide the most effective protection against rabv due to its ability to penetrate tissues, in contrast to igm which cannot penetrate tissues (turner, 1978) . a multifaceted approach for human rabies eradication involving government support, disease awareness, and vaccination of at-risk humans and dogs will be required to achieve the goals of the world health organization in eradication of rabies by 2030 (fooks et al., 2017) . the cns is immunologically privileged and protected by a highly complex barrier system. viruses that have evolved to overcome these barriers can cause cns infections greatly outnumbering those from all bacterial, fungal, and protozoa infections combined. ingested pv multiplies in the oropharyngeal and intestinal mucosa and drains to cervical and mesenteric lymph nodes and then into the blood ahead of penetrating the cns to cause polio, with 10% of cases resulting in death. both neutralizing antibodies and the adaptive immune system can clear pv infection and may provide lifelong protection. vaccination combinations can induce pv-specific cytolytic cd4 þ and cd8 þ t cells for virus clearance, but their coadministration can pose the risk for reversion to virulence by recombination. the je flavivirus is amplified by waterfowl and transmitted to humans by mosquitoes, and while 99% of its infections are asymptomatic, mortality rates in 30% of infected individuals cause associated disorders that leave its survivors a lifetime of associated morbidities. as there is no existing je treatment, prevention involves either avoidance of mosquitoes or vaccination. flaviviruses evade the immune system to cross the bbb by an inflammatory celle mediated "trojan horse" mechanism. je dissemination is limited by innate immune responses, neutralizing antibodies produced by humoral immunity, and by virus-specific ctls. je vaccines are licensed worldwide, and the majority of vaccinated individuals have circulating je-specific cd4 þ and cd8 þ t cells that can cross-react with other flaviviruses. alphaviruses are transmitted by mosquito bites to infect neurons, causing mild to severe encephalitis resulting in death, with past outbreaks numbering in the hundreds of thousands. veev infection causes up to 35% mortality in children, 75% of which involve cns penetration, causing severe long-term neurological disorders. veev is not only a naturally emerging disease threat but is also a highly developed biological weapon amenable to warfare or terrorism due to its aerosol transmission route and associated lethal meningoencephalitis. ifn signaling pathways and ab and gd t cell response from innate and adaptive immunity can control veev targeting of cns, where virus-specific antibody responses are critical in limiting viral spread. in the absence of antibodies, veev replicon particles can induce t cell responses able to induce recovery from veev-induced encephalomyelitis, where cytotoxic cd4 þ t cells control veev in cns lesions. veev vaccines induce robust neutralizing antibodies for protection against rechallenge. tc-83 vaccine responders have circulating pbmc biomarkers, and military programs give boosters of c-84 to those failing to seroconvert. in contrast to these other viruses, rabv replication only begins after cns penetration, as facilitated by depth of bite by its canine vector, thereby limiting possible detection of primary viral antigen in the periphery and resulting in delayed and minimal innate and humoral responses. once rabv-related acute encephalitis symptoms begin, fatality is sure to follow due to absence of cns infiltration by adaptive immune effector cells as a result of virus-induced decreases in bbb permeability. other countermeasures against protection are tight mhc expression regulation and apoptotic phenotypes of bbb-infiltrated t cells. rabv vaccines cause increases in ig, but little is known concerning vaccine-associated adaptive immune responses. viral hemorrhagic fever (vhf) classification originates from the study of hantaviral hemorrhagic fever (hf) and was later extended to include crimeanecongo hf and omsk hf. vhf can results from infection by 23 enveloped rna viruses from four families: flaviviridae, filoviridae, arenaviridae, and bunyaviridae. vhf designation is given to severe febrile illnesses with abnormal vascular regulation and vascular damage (peters and zaki, 2002) . vascular dysregulation occurs early in the course of disease, visible as skin flushing, hypotension, and conjunctival vasodilation, whereby vascular damage with capillary leakage occurs as disease progresses, causing edema and serous effusions of pleural and peritoneal cavities. the terminal phase of vhf, or shock, arises from increased disease severity from combinations of vascular dysregulation and damage from capillary leakage (paessler and walker, 2013) . detailed mechanisms of hemorrhage and plasma leakage during vhf include endothelial injury, activation of the mononuclear phagocytic system, cytokine storm, platelet aggregation and consumption, activation of the coagulation cascade, and insufficiency of coagulation factors from severe hepatic damage (schnittler and feldmann, 2003; chen and cosgriff, 2000) . these mechanisms vary among diseases, cell and organ tropism of causative viruses, and host responses (paessler and walker, 2013) . flaviviruses, filoviruses, arenaviruses, and bunyaviruses are the main causes of hf (table 1) . these viruses continue to propagate as part of the life cycles of primates, bats, rodents, farm animals, mosquitoes, and ticks. infection by these viruses can cause mild vascular instability to fatal shock, with hemorrhage ranging from unnoticeable to life-threatening. pathogenic mechanisms of hfv are diverse and include hepatic necrosis leading to deregulation of coagulation factors, cytokine storm, increased permeability, and complement activation. overall disease severity by these viruses is varied, whereby ebola and marburg hf can cause high fatality rates, whereas yf and dengue infections can be asymptomatic. severe vhf is commonly correlated with ineffective immunity and high viral loads, and severe plasma leakage can occur from viral clearance and fever breaks in dengue hf (dhf). approximately 73 viruses are included in the flavivirus genus of the flaviviridae family of viruses, with 40 of these species associated with dengue, yf, je, tick-borne encephalitis, and west nile encephalitis as the most important arboviruses causing extensive global morbidity and mortality (diamond, 2003) . flaviviruses are enveloped viruses with single-stranded rna genomes that are translated in the cytoplasm to generate a single polyprotein that is then cleaved into structural and ns proteins by virus and host proteases. the various encoded viral proteins include capsid, envelope for receptor binding, membrane fusion and viral assembly, and transmembrane proteins (prm) that assist in protein folding and function. although the precise mechanism is unclear, flaviviruses are believed to evade the immune system to enter the brain and spinal cord via circulating blood (johnson and mims, 1968) , where these may cross the bbb by passive transport across the endothelium, by active replication in endothelial cells, or by a "trojan horse" mechanism utilizing inflammatory cells (solomon and vaughn, 2002) . ifn-dependent and complement system innate immune responses and humoral immunity, producing neutralizing antibodies limit dissemination of infection and protect against viral spread, reviewed in diamond (2003) . cellular immunity is also important toward eradication of infected cells, whereby infection induces the recognition of flavivirusinfected cells by virus-specific ctls, which then become activated, proliferate, and release inflammatory cytokines (kesson et al., 1987; kurane et al., 1989a; liu et al., 1989; murali-krishna et al., 1996) . however, there is also evidence that flavivirus replication is enhanced by myeloid cells and has been observed for dengue, yf, west nile, tick-borne, and je viruses (diamond, 2003) . yf virus (yfv) is important both historically and currently. it was once one of the most globally feared diseases terrorizing africa, europe, and the americas. hundreds of thousands were killed in the americas over a 250-year spandcrippling economies (watson and klimstra, 2017) . yfv is a member of the genus flavivirus of the flaviviridae family and contains a single-stranded rna genome of approximately 11 kb. yfv virions are icosahedral and are composed of nucleocapsid, composed of capsid (c) protein subunits and a surrounding lipid bilayer derived from host membranes. the viral envelope is studded with dimers of envelope (e) and membrane (m) proteins, for a total diameter of approximately 45 nm. as the major component of the virion surface, the e protein is responsible for cell-surface receptor binding, virion assembly, fusion, and immunogenicity. viral proteins are encoded in a single open reading frame and produced as a polyprotein later processed by proteolytic cleavage into structural (c, m, and e) and ns proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5) , reviewed in gardner and ryman (2010) . during most yfv infections, the virus is transmitted by the bite of an infected aedes aegypti mosquito found in urban areas. infected patients often develop severe acute illness hemorrhagic yf disease, with associated symptoms of fever, nausea, vomiting, epigastric pain, hepatitis, jaundice, renal failure, hemorrhage, and shock, with 20%e60% of cases resulting in death (watson and klimstra, 2017) . yf is the prototypical vhf, sharing many pathophysiological features with other viral disorders only associated via similarities in syndromes, but with the exception that yf causes the most severe symptoms of hepatic dysfunction (monath and vasconcelos, 2015) . yfv remains endemic in south american and african countries, with monkeys as its reservoir, causing regular outbreaks of jungle yf, and resulting in as many as 200,000 infections per year causing 30,000 deaths. millions are at risk for infection in africa, where vaccination prevalence is low. the 2016 outbreak in angola serves as an example of yfv traveler-associated spreading to neighboring countries, where it reached as far as china, then naïve for virus (watson and klimstra, 2017) , and representing a prime population for a major outbreak of epic proportions (wasserman et al., 2016) . geographical shifting of mosquito populations to north america is also creating new risk for yfv, dengue, and zika infection of naïve populations (monaghan et al., 2016) . despite the availability of vaccination against yfv since the 1940s, large epidemics have still arisen, with dramatic surges of yfv in africa in the 1960s and the late 1980s, with each reporting over 100,000 cases. recent outbreaks have also affected brazil, paraguay and argentina, uganda, and sudan and ethiopia. immunity is the critical for reducing and eliminating viral infections, but other contributing factors to virus amplification are multifactorial and elusive, including the emergence of new viral strains and prolonged periods of hot and humid weather promoting insect propagation, reviewed in monath and vasconcelos (2015) . fifty-seven million people were vaccinated against yf across africa between 2007 and 2010. five hundred million doses of the live-attenuated yf 17d vaccine, representing the most effective vaccine ever created, have been distributed over the last 50 years (monath and vasconcelos, 2015) . both humoral and cellular immunity elicited by 17d are observed and well characterized, where neutralizing antibodies provide protection, but 17d also provides a robust, long-lived, and polyfunctional adaptive t cell immune response (watson and klimstra, 2017) . neutralizing antibodies remain the accepted correlate of protection against yfv, with 90% or greater of 17d immunized individuals developing neutralizing antibodies (gotuzzo et al., 2013) . 17d also elicits a complex modulation of innate immune cytokines, with elevated levels of plasma ifn-g 15 days postvaccination (neves et al., 2009) . restimulation of innate immune cell cultures of nk cells, neutrophils, and monocytes from 17d vaccinated humans with yf antigen results in the increased production of ifn-g, il-1beta, il-10, il-12, tnf-a, and il-10 (neves et al., 2009; gardner and ryman, 2010; luiza-silva et al., 2011; silva et al., 2011) . since its development, humoral immunity, as a gold standard of general vaccine development, was the most studied aspect of human immunity to 17d. however, recent studies of adaptive t cellemediated immunity to 17d have demonstrated that both cd4 þ and cd8 þ t cells strongly respond to 17d, with activated cd8 þ t cells detected as 3 days after vaccination , and cd4 þ t cells detected several days later kohler et al., 2012; blom et al., 2013) . increased cd8 þ t cell proliferation correlates directly with the levels of virus genomes in plasma, which peaks once virus is eliminated . cd8 þ t cell clones responding to 17d differentiate into central memory and effector memory subpopulations (dewitt et al., 2015) and are still detectable 25 years following vaccination (wieten et al., 2016) . 17d-specific cd8 þ t cells respond to epitopes contained from every protein product generated by the 17d polyprotein, and upon peptide restimulation, these 17d-specific cd8 þ t cells have activated cytotoxic profiles including increased expression of ifn-g, tnf-a, and mip1-b and il-2 granzyme b and cd107a (blom et al., 2013; akondy et al., 2009) but are not exhausted and retain long-lived memory and polyfunctional phenotypes for at least 2 years following 17d rechallenge (akondy et al., 2009) . dengue virus (denv), also a member of the single-stranded positivesense rna viruses from the flaviviridae family, causes visceral and cns disease in humans and is closely related to yfv, where denv fever has often been mistaken for yfv infection. far more serious is dhf, where additional symptoms develop, including hemorrhage and shock, and have mortality rates exceeding 30% if left untreated (rogers et al., 2006) . denv is a spherical, 50-nm virion, comprising of three structural proteins: capsid (c), premembrane and membrane (prm and m), and envelope (e). the e protein directs several critical steps of the viral replication cycle, including engagement with cellular attachment and entry factors, membrane fusion, and virion assembly. denv binds to target cells via glycosaminoglycans, c-type lectins such as dc-sign, the mannose receptor cd206, and immunomodulatory proteins (tim and tam receptors; diamond and pierson, 2015) . thus targets for denv infection include monocytes, macrophages, dcs, mast cells, and possibly hepatocytes and endothelial cells. following its entry into the cellular cytoplasm, the viral genomic 10.7 kb rna is translated into a single polyprotein, later cleaved into three structural and seven ns proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5 ) by viral ns3 and host cell proteases. twenty-five percent of denv infections cause both mild symptoms including dengue fever (df) to more severe and lethal dhf, causing shock via hemorrhagic and capillary leak syndrome. df can be characterized by abrupt onset febrile illness causing headache, severe muscle and joint pain, and rash, whereas dhf is characterized by rapid onset capillary leakage accompanied by significant thrombocytopenia and liver injury (halstead, 2007) . as with yf, denv origins are believed to be that of a sylvatic virus, with a natural life cycle involving multiple mosquito and vertebrate species from asia and africa (diallo et al., 2003) . denv adaptation to human demography is via mosquito vector aedes aegypti, breeding in urban areas (trpis and hausermann, 1986) . cases of denv infection have increased since the 1960s, with an estimated 50 million cases of df, and 500,000 cases of dhf occurring globally every year. there are no cures for denvassociated disorders, and vaccine development has been complicated by antibody-dependent enhancement of future heterotypic infection induced by vaccination (vaughn, 2000; halstead and deen, 2002) . thus avoidance and control of aedes aegypti is the best approach for limiting denv infection (rogers et al., 2006) . adaptive immune cd8 þ t cells vigorously and frequently recognize denv ns3, ns4b, and ns5 proteins, whereas the capsid, envelope, and ns3 proteins are the dominant targets for cd4 þ t cells (simmons et al., 2005; duangchinda et al., 2010; weiskopf et al., 2011 weiskopf et al., , 2013 rivino et al., 2013) . both cd4 þ and cd8 þ t cells are believed to contribute to protection against denv, as denv-specific cd4 þ t and cd8 þ t cells proliferate, produce ifn-g, and lyse target cells, from primary denv infection (kurane et al., 1989b; mathew et al., 1996; gagnon et al., 1999; livingston et al., 1995) . higher frequencies of denv-specific ifn-gproducing t cells are present in children with asymptomatic denv infection (hatch et al., 2011) . both cd4 þ and cd8 þ t cells contribute to protection against denv challenge (yauch et al., 2009 (yauch et al., , 2010 zompi et al., 2012; zellweger et al., 2013) , and hla alleles associated with increased risk of denv severity correlated with weak cd8 þ t cell responses and vice versa, implying a protective role for cd8 þ t cells against severe denv disease in humans (weiskopf et al., 2013) . in 2015, the first dengue vaccine (dengvaxia) was licensed in asian and south american countries for protection against all four denv serotypes, and while it demonstrated an good safety and efficacy in clinical trials, it has recently been withdrawn in the philippines due to its causing elevated disease severity if administered following infection (wichmann et al., 2017) . it has been suggested that failure of this and other live-attenuated tetravalent dengueeyf chimeric virus vaccines (guy et al., 2011) is the result of their lacking the ns proteins ns3, ns4b, and ns5, otherwise dominantly targeted by cd8 þ t cells (simmons et al., 2005; duangchinda et al., 2010; weiskopf et al., 2011 weiskopf et al., , 2013 rivino et al., 2013) , making it critical to accurately assess not only antibody responses but rather t cell responses in the context of denv vaccine development (weiskopf and sette, 2014) . lassa virus (lasv), causing lassa fever (lf), is an enveloped virus with two single-stranded rna segments and is another virus causing hf. lasv is an old world member of the arenavirida family of viruses. the single-stranded arenavirus genome consists of a small (s) and a large (l) rna segment, measuring 3.4 and 7 kb, respectively. the large segment encodes a small zinc-binding (z) protein which regulates transcription, replication, and viral budding, along with the rna polymerase (l). the small segment encodes the np and the two envelope glycoproteins (gp1 and gp2) responsible for cell entry, reviewed in russier et al. (2012) . lasv disorder is endemic in africa and its neighboring countries (safronetz et al., 2010; gunther et al., 2000) , and though infection rates are difficult to quantify due to limited survey infrastructure, classification of its clinical symptoms is common to other diseases. lasv is predicted to be responsible for approximately 300,000 infections and up to 6000 resultant deaths each year (ogbu et al., 2007; mccormick et al., 1987) . transmission to humans is via the rodent host mastomys natalensis (mccormick et al., 1987) . apcs, dcs, and macrophages are believed to be the first cells targeted by lasv infection (baize et al., 2004; mahanty et al., 2003) , which can rapidly speed up dissemination of lasv to multisystem organs due to their widespread physiological distributions in mucosal tissues and skin. due to their ease in motility across various organs and tissues, apcs are believed to the responsible for the spread of lasv for the establishment of systemic infection (hensley et al., 2011) . apc infection results in substantial virus release in the secondary lymphoid organs, the liver, hepatocytes, fibroblasts, and endothelial cells that are subsequently infected. lymphopenia of cd4 þ and cd8 þ t cells, nk cells, and b cells is observed early during disease onset, reviewed in russier et al. (2012) . lasv infection severities range from asymptomatic infection to fatal hf (fisher-hoch et al., 1995) and commonly resulting from other viral infections, nonspecific symptoms beginning several days after infection include fever, headache, arthralgia, myalgia, and severe asthenia. these early symptoms are typically followed by more severe symptoms of pharyngitis, conjunctivitis, cough, abdominal pain, diarrhea, and vomiting. in severely affected patients, cervical and facial edema, hemorrhages, renal and liver failures, and encephalopathy occur, and death follows systemic shock (edington and white, 1972) . survivors of lasv-related disorders have persisting lifelong morbidities and disabling conditions including deafness (cummins et al., 1990) . no vaccine has been licensed against lasv, and ribavirin is the only existing treatment, but is only effective if administered very early after infection and is not available for broad distribution in countries where lasv is endemic (mccormick et al., 1986) . t cells play a crucial role in the outcome of severe lasv infection, which has been associated with defective t cell responses since the very cells responsible for stimulating t cell antigen responses are those infected by the virus. however, t cell responses have been demonstrated to play critical roles in the control of lasv, where strong memory cd4 þ t cell responses directed against lasv np and gp proteins are observed in lasvseropositive healthy individuals from endemic regions (ter meulen et al., 2004) . high serum concentrations of il-8 and cxcl10 chemokines that attract and activate t cells are associated with nonfatal lasv infections (christensen et al., 2006; dufour et al., 2002) and vice versa in fatality cases (mahanty et al., 2001) . the control of acute lf has been correlated with increases in circulating activated cd4 þ and cd8 þ t cells in response to lasv infection or antigen (baize et al., 2009) . in vaccine studies, protection against a lethal lasv rechallenge is associated with the induction of t cell immunity (fisher-hoch et al., 2000; geisbert et al., 2005) . however, in comparison to other viruses, lasv-infected dcs are unable to mount effector t cell responses (pannetier et al., 2011) . human and nonhuman primate studies have demonstrated that lasv np and gp proteins are the main viral antigens recognized by activated t cells (meulen et al., 2004; ter meulen et al., 2000; fisher-hoch et al., 2000; fisher-hoch and mccormick, 2001, 2004; geisbert et al., 2005) , suggesting that vaccines using these proteins to induce long-term memory t cell expansion will best control the spread of lf. ebola virus (ebov) causes a rapidly fatal hf for which there is currently no treatment (muyembe-tamfum et al., 2012; team et al., 2014) . ebov is a member of the filoviridae family, which are filamentous, negative-stranded rna viruses that cause severe human disease. filoviruses viruses are variable, with long filaments measuring 80 nm in diameter and which can reach lengths of up to 1000 nm, with many turns and branches and which have tendency to curve to resemble the number 6. viruses are composed of nucleocapsid, matrix, and envelope proteins, whereby seven genes encode np, the viral proteins vp24-vp30-vp35-vp40, l (polymerase), and the gp (hoenen et al., 2006) , expressed as gp1 and gp2, and regulating virus production and release (mohan et al., 2015) . nps embed the genome in complex with vp30 and vp35 for rna synthesis. vp40 and vp24 proteins are localized in virus matrix space (watanabe et al., 2007; hoenen et al., 2010) . ebov is transmitted to humans via mucosal surfaces, skin injury, and vertical transmission (feldmann and geisbert, 2011) , and with the exception of t cells, can infect almost all human cells using various different attachment mechanisms, reviewed in falasca et al. (2015) . both innate and adaptive immune responses are involved in ebov pathogenesis, where innate immune deregulation involves inhibition of type-i ifn response and perturbation of cytokine signaling, along with impairment of dc and nk cells, and adaptive immune deregulation involves both humoral and cell-mediated immunity (falasca et al., 2015) . because high levels of ebov replication are associated to multiple cell types, its associated systemic dissemination results in a highly complex pathogenesis model, including detrimental immune suppression and hyperactivation, and leading to disordered coagulation and tissue damage, that, in the absence of treatment, results in rapid multiple organ failure and death within days of symptomatic infection (baseler et al., 2017) . for 50 years, ebov and related filoviruses have been repeatedly re-emerging to cause large epidemics of highly fatal hf. ongoing ebov outbreaks in africa has brought this virus to the forefront of research, with over 20,000 reported cases of infection and an associated 8000 deaths (mcelroy et al., 2015) . natural serologic response to ebov infection involves virus-specific igm and igg antibody responses sometimes detected early, but usually later, once symptoms begin rowe et al., 1999) . ebovinfected dcs are impaired in cytokine production required for t cell activation (mahanty et al., 2003) , whereas infected macrophages are unable to mature (bosio et al., 2003) . ebov is classified as an immunosuppressive virus since numerous of its proteins interfere with immune responses by inducing t cell apoptosis, lymphopenia, and absence of antibody responses in fatal cases (basler and amarasinghe, 2009 ). classification of ebov-targeting mechanisms has been compromised by lack of infrastructure for adequate biosafety containment level facilities required to analyze this deadly virus. however, observations of the adaptive t cell immune response have shown that ebov correlates with fatal outcomes by causing aberrant cytokine responses (baize et al., 1999 (baize et al., , 2002 wauquier et al., 2010; villinger et al., 1999; ansari, 2014) , decreased cd4 þ and cd8 þ t cells, and increased apoptotic t cell phenotypes (baize et al., 1999; wauquier et al., 2010; geisbert et al., 2000; bradfute et al., 2010; gupta et al., 2007) . in recent work, ebov induced increased cd4 þ and cd8 þ t cell activation against the viral np, with cd8 þ t cells demonstrating the largest increases in expression of activation and proliferation biomarkers, with sustained activation following ebov clearance and following patient discharge, suggesting continued antigen stimulation after resolution of the disease (mcelroy et al., 2015) . recently, an rvsv-zebov recombinant, replicationecompetent vesicular stomatitis virusebased vaccine expressing a surface gp of zaire ebolavirus, demonstrate a 100% efficacy in preventing ebov disease in contacts and contacts of contacts of recently confirmed cases in guinea, west africa (henao-restrepo, 2017 #550). this vaccine produced rapid innate immune responses after a single dose, suggested to lead to longerterm full protection by providing an essential period of restricted virus replication during the development of specific adaptive responses (marzi, 2015 #551) . crimean-congo hemorrhagic fever virus (cchfv) is a tick-borne virus causing hf resulting in human fatalities. cchfv is a member of the nairoviridae family of viruses from the genus of orthonairovirus and the order of the bunyaviridae viruses. it has a single-stranded, negative-sense rna genome possessing three segments: the large (l), medium (m), and small (s) segments (casals, 1969; clerx et al., 1981) . the l segment encodes the viral rna-dependent rna polymerase responsible for mrna synthesis and rna replication (honig et al., 2004) . the m-segment encodes numerous ns and two structural gps (gn and gc) responsible for cell tropism and attachment and are targets for neutralizing antibodies. the s-segment encodes the viral np binding the rna segments toward formation of ribonucleoprotein complexes (altamura et al., 2007; sanchez et al., 2006) . though hf by cchfv infection in humans is not among the most common viral disorders reported, it remains important because it is fatal in up to 30% of cases (bente et al., 2013; goedhals et al., 2017) . transmission of cchfv to humans occurs through contact with infected animal blood, or ticks, belonging to the genus hyalomma, as its primary vectors and providing transit from one infected human to another (mousavi-jazi et al., 2012) . cchfv human infection involves sudden onset of acute symptoms, including high fever, headache, myalgia, and petechial rash, followed by hemorrhage progressing to multiorgan failure, with leukopenia, thrombocytopenia, and elevated liver enzymes as hallmarks of the overall disorder (begum et al., 1970; sanchez et al., 2006) . outbreak-associated fatality rates are varied but can reach 70% (mousavi-jazi et al., 2012) . there is currently no licensed vaccine, and use of ribavirin as treatment has been investigated but remains controversial (begum et al., 1970; bente et al., 2010) . distribution of cchfv infection and associated disease follows geographical spread of the principal vectors (bente et al., 2013; whitehouse, 2004) . clinical cchfv disorders are described in africa, asia, the middle and east eastern europe, and have recently emerged in other countries including turkey, india, spain, and greece, and with almost 10,000 cases reported in turkey between 2002 and 2015 (maltezou et al., 2010; papa et al., 2008; leblebicioglu et al., 2016) . typically, transient igm and igg antibody responses develop within days following primary cchfv infection and can persist long-term (shepherd et al., 1989; burt et al., 2013) , but where lack thereof usually results in fatality (shepherd et al., 1989) . igm and igg antibodies have however not been correlated with clearance, viral load, or outcomes (duh et al., 2007) , implying that innate and t cell immunity must be critical for viral clearance. neutralizing antibodies also do not cause protection, and nonneutralizing antibodies may assist in antibody-dependent cell-mediated cytotoxicity (bertolotti-ciarlet et al., 2005) . thus, as immune correlates of protection for cchfv are not well documented, vaccine design has aimed at targeting the cchfv np or gps. only an inactivated vaccine is available, and even though the attaining of immunogenicity has required its administration in multiple doses, it was demonstrated to reduce infections and induce both neutralizing antibody responses and t cell responses to np peptides (mousavi-jazi et al., 2012) . another promising approach is the use of modified vacv ankara recombinant vaccine expressing the viral gps, which induce cellular and humoral responses and which are observed to provide protection from lethal disease in mice (buttigieg et al., 2014; dowall et al., 2016) . although t cell responses are known to play a role in protection from and clearance of viral infections, it was only recently that specific cd8 þ t cell epitopes against gn and gc were shown to stimulate ifn-g production, whereby responses were detectable several years after the acute cchfv infection, even in the absence of continued antigenic stimulation, and where ifn-g-producing cd8 þ t cells were confirmed as responsible for providing long-term protection (goedhals et al., 2017) . vhfs causes high fatality rates and are commonly correlated with ineffective immunity and high viral loads. yfv is important for both historical and current reasons. historically, it crippled economies and families by killing hundreds of thousands over 250 years. yfv is transmitted by mosquitos in urban areas, causing as many as 200,000 infections and 30,000 deaths annually. like other mosquito vector viruses, yfv is an existing and re-emerging threat due to increased geological spread by both world travelers and shifting mosquito breeding geographies. flaviviruses evade immunity to enter the brain and spinal cord via circulating blood, crossing the bbb via "trojan horse" mechanism. dissemination of infection and protection against yfv is elicited by ifn-signaling, complement system, and other innate, humoral, and adaptive immune responses. neutralizing antibodies are still the gold standard correlates of protection, but evidence that a robust, long-lived, and polyfunctional adaptive cd4 þ and cd8 þ t cell immune response is what is provided, early after vaccination by the 500 million doses of effective yf 17d vaccine distributed globally. importantly, these are nonexhausted, polyfunctional central memory and effector memory t cell subsets that are detected for over 25 years following vaccination. far more serious is dhf by infection by denv, contributing to either mild or severe hf, with mortality rates exceeding 30% in the untreated. as with yfv, denv uses mosquitos adapted to urban areas as their vectors. fifty million cases of df and 500,000 cases of dhf occur globally each year, with no available cures for its associated disorders and where vaccine development has been complicated by antibody-dependent vaccineinduced enhancement of future heterotypic infections. cytotoxic cd8 þ and cd4 þ t cells, however, vigorously and frequently recognize denv proteins and are believed to contribute protection against primary infection. the denv vaccine dengvaxia has been recently withdrawn, with its failure posited to result from its lack of denv proteins specifically targeted by cd8 þ t cells, and demonstrates that is essential to accurately assess t cell responses in the context of denv development. lasv also causes mild and severe vhf and may cause 300,000 infections and up to 6000 deaths each year. lasv is transmitted to humans by a rodent vector and first targets apc, dc, and macrophages, contributing to rapid multisystem and organ lasv dissemination due to their widespread physiological distributions. lasv also causes lymphopenia of cd4 þ and cd8 þ t cells, nk cells, and b cells, and survivors of lasv-related disorders can be expected to maintain lifelong morbidities. there is no licensed lasv vaccine as of yet, complicated by the fact that the very cells responsible for stimulating t cell antigen responses are those infected by the virus. however, control of acute lf is correlated with increased circulating strong memory cd4 þ and cd8 þ t cells in response to lasv infection or rechallenge and lasv antigen. ebov also causes mass hf. it can infect almost all human cells with exception of lymphocytes and has no known treatment despite documented involvement of innate and adaptive immune responses. classification of ebov targeting mechanisms are compromised by lack of infrastructure for adequate biosafety containment level facilities. ebov causes dcs to be impaired in cytokine production for t cells activation and inhibits macrophages maturation, thus classifying it as an immunosuppressive virus, with fatalities correlating with its induction of lymphopenia the absence of antibody responses. ebov, however, causes increased cd4 þ and cd8 þ t cell activation, with recorded persistence of activated cd8 þ t cells in survivors. the knowledge that cd4 þ and cd8 þ t cell responses are against the ebov np will be useful for the design of efficient targeting vaccines. cchfv also causes hf yielding fatalities in up to 30% of cases. there is no current vaccine, and cchfv has recently reemerged in naïve countries in response to geographical relocation of its primary tick vector. infection by cchfv causes transient ig antibody responses, inversely correlating with sure fatality, but not correlating with clearance, viral load, or outcomes. no protection is granted by neutralizing antibodies either, implying that innate and t cell arms of immunity are critical for viral clearance. with little to no immune correlates of protection for cchfv, vaccine design has aimed at targeting the cchfv np or gps, where immunogenicity requires the administration of multiple doses to induce both neutralizing antibody responses and t cell responses. only recently, specific cd8 þ t cell epitopes against these cchfv proteins have been confirmed to stimulate cytotoxic cd8 þ t cells providing protection in survivors. despite many documented instances of virus eradication from populations by earlier vaccination strategies, there is a continuous imminent risk of outbreaks of pandemic proportions by existing and emerging viruses, as a result of anti-vaccination campaigns, unforeseen viral strain recombination, exposure of naïve populations to viruses by infected world travellers, a rapidly growing and aging and immunocompromised world population, acts of bioterrorism, and use of viruses as biological weapons in war. past strategies in the development of certain vaccines have often been lucky in their efficacies due to their eliciting both humoral and long-lasting adaptive t cell immunity, whereas others have failed and have only induced shorter lived humoral responses that do not always confer long-lasting protection. the precise and likely overlapping mechanisms dictating lifelong immunity conferred by successful vaccines against smallpox, measles, mumps, rubella, polio, and yf are not completely understood. what has become clear however is that innate immunity usually primes adaptive immunity to confer this long-term protection. it is not a lack of existing immune-monitoring methodologies but rather perhaps a lack of their correct implementation in the continued analysis of vaccinated individuals that are hampering the gaining of this important knowledge. therefore it seems there is an urgent need for the standardization of vaccine methodologies adequately measuring effector memory t cell responses and host-immune evasion mechanisms in appropriate animal models and humans and through the use of multiparametric 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republic of the congo. commission de lutte contre les epidemies a kikwit failure to open the blood-brain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection sendai virus as a backbone for vaccines against rsv and other human paramyxoviruses measles virus for cancer therapy immune responses and lassa virus infection alpha/ beta interferon protects adult mice from fatal sindbis virus infection and is an important determinant of cell and tissue tropism the natural history of human poliomyelitis: ii. elimination of the virus recognition of the latency-associated immediate early protein ie63 of varicella-zoster virus by human memory t lymphocytes detection of lassa virus alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by furin-like and ski-1 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antibody forming cells in the diffuse-nalt and lungs of sendai virus-vaccinated cotton rats associate with rapid protection against human parainfluenza virus-type 1 single-cell mass cytometry analysis of human tonsil t cell remodeling by varicella zoster virus selective cd4 þ t cell help for antibody responses to a large viral pathogen: deterministic linkage of specificities extreme mortality after first introduction of measles virus to the polynesian island of rotuma kinetics and viral protein specificity of the cytotoxic t lymphocyte response in healthy adults immunised with live attenuated varicella vaccine an increasing danger of zoonotic orthopoxvirus infections antibody response in crimean-congo hemorrhagic fever virus-induced type i ifn stimulates generation of immunoproteasomes at the site of infection immune responses and immunopathology in acute and chronic viral hepatitis cd8 þ t cells require perforin to clear west nile virus from infected neurons characterization of main 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presentation by the class i major histocompatibility complex fatal community-acquired pneumonia in children caused by re-emergent human adenovirus 7d associated with higher severity of illness and fatality rate cd4 þ t cells provide protection against acute lethal encephalitis caused by venezuelan equine encephalitis virus role of humoral versus cellular responses induced by a protective dengue vaccine candidate infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium genomic and oncogenic preference of hbv integration in hepatocellular carcinoma efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale protection from secondary dengue virus infection in a mouse model reveals the role of serotype cross-reactive b and t cells key: cord-022756-kdgo4rqb authors: nan title: hematopoietic tumors date: 2012-11-28 journal: withrow and macewen's small animal clinical oncology doi: 10.1016/b978-1-4377-2362-5.00032-3 sha: doc_id: 22756 cord_uid: kdgo4rqb nan the lymphomas (malignant lymphoma or lymphosarcoma) are a diverse group of neoplasms that have in common their origin from lymphoreticular cells. they usually arise in lymphoid tissues such as lymph nodes, spleen, and bone marrow; however, they may arise in almost any tissue in the body. although the annual incidence of lymphoma is difficult to predict in the absence of a national canine tumor registry, it is clear that it represents one of the most common neoplasms seen in the dog. the annual incidence has been estimated to range between 13 to 24 per 100,000 dogs at risk. [1] [2] [3] the annual incidence rates at specific ages are estimated to be 1.5 per 100,000 for dogs less than 1.0 year of age and 84 per 100,000 in the 10-to 11-year-old group. lymphoma comprises approximately 7% to 24% of all canine neoplasia and 83% of all canine hematopoietic malignancies. 4, 5 in a review of the veterinary medical data base program (vmdp) at purdue university from 1987 to 1997, the frequency of canine lymphoma patients presented to 20 veterinary institutions increased from 0.75% of total case load to 2.0%, and it appears the frequency is continuing to increase. a similar trend is present in physician-based oncology; non-hodgkin's lymphoma (nhl) represents 5% of all new cancer cases, the fifth leading cause of cancer death, and the second fastest growing cancer in terms of mortality in humans. 6 middle-aged to older (median age of 6 to 9 years) dogs are primarily affected. a decreased risk for lymphoma is reported for intact females. 7 breeds reported to have a higher incidence include boxers, bull mastiffs, basset hounds, st. bernards, scottish terriers, airedales, and bulldogs; breeds at lower risk include dachshunds and pomeranians. 8, 9 etiology the etiology of canine lymphoma is likely multifactorial and largely unknown; however, investigations are currently shedding significant light on the subject. recent advances in molecular cytogenetics (see chapter 1, section a), including gene microarray techniques, have been and are currently being applied to investigations of chromosomal aberrations in dogs with lymphoma. [10] [11] [12] [13] [14] [15] [16] the publication of the canine genome and the commercial availability of canine gene microarrays (genechip canine genome 2.0 array, affymetrix, inc.) have led to advances in our understanding of genetic events occurring in lymphoma. 17 breen's group has documented gain of dog chromosomes 13 and 31 and loss of chromosome 14 as the most common aberrations in a group of 25 cases analyzed. 11 chromosomal aberrations have also been associated with prognosis in dogs with lymphoma. a study of 61 dogs with lymphoma demonstrated a prognostic advantage in dogs with trisomy of chromosome 13 (25% of the dogs studied) as evidenced by increase in duration of first remission and overall survival time. 18 germline and somatic genetic mutations and altered oncogene/tumor suppressor gene expression, epigenetic changes (e.g., dna hypomethylation), signal transduction, and death-pathway alterations (e.g., bcl-2 family) are common in human lymphomas and have been reported in the dog as well (see chapter 1, section a, and chapter 14, section b). these include n-ras, p53, rb, and p16 cyclin-dependent kinase aberrations. [19] [20] [21] [22] [23] [24] additionally, differences in the prevalence of immunophenotypic subtypes of lymphoma among different breeds indicate heritable risks. 25 additionally, telomerase activity (see chapter 2) has been documented in canine lymphoma tissues. [26] [27] [28] the hypothesis that a retrovirus may be involved in the pathogenesis of canine lymphoma has not been confirmed. however, serologic detection of epstein-barr virus infection, linked to some forms of lymphoma in humans, has been documented in dogs with lymphoma and is currently being investigated. 29 in humans, a direct association between helicobacter sp. infections and development of gastric lymphoma has been made. 30 although this has not been definitively shown in dogs, there is evidence of helicobacter sp. infection in laboratory beagle dogs resulting in gastric lymphoid follicle formation that is considered a precursor of mucosa-associated lymphoid tissue (malt) lymphoma in humans. 31 in humans, evidence has accumulated implicating phenoxyacetic acid herbicides, in particular 2, 4-dichlorophenoxyacetic acid (2, 4-d) , in the development of nhl. a published hospital-based casecontrol study of dogs indicated that owners in households with dogs that develop malignant lymphoma applied 2, 4-d herbicides to their lawn and/or employed commercial lawn care companies to treat their yard more frequently than owners of dogs without lymphoma. 32 the risk of canine lymphoma was reported to rise twofold (odds ratio [or] = 1.3) with four or more yearly owner applications of 2, 4-d. the results of this study have come under criticism, and three additional follow-up investigations have not validated assertions of increased risk. [33] [34] [35] in another study, dogs exposed to lawn treatment within 7 days of application were greater than 50 times histopathologically, distinguishing between gi lymphoma and lpe can be difficult. some have suggested that lpe may be a prelymphomatous change in the gi tract. a syndrome of immunoproliferative intestinal disease characterized by lpe has been described in basenjis, which subsequently develop gi lymphoma. 49 in addition, plasma cell-rich areas with heterogeneous lymphomatous infiltration may resemble lesions of lpe. only a few reports specifically identify the immunophenotype of the lymphocyte subpopulations in alimentary lymphoma in dogs. historically, it was presumed that they most likely originate from b cells; however, recent evidence suggests that most gi lymphomas in dogs arise from t cells and often exhibit epitheliotropism. 48, 50 the boxer and shar-pei breeds may be overrepresented in cases of alimentary lymphoma. 50, 51 the mediastinal form of the disease occurs in approximately 5% of cases. 46 this form is characterized by enlargement of the cranial mediastinal lymph nodes, thymus, or both ( figure 32 -2). hypercalcemia is reported to occur in 10% to 40% of dogs with lymphoma and is most common with the mediastinal form. in a study of 37 dogs with lymphoma and hypercalcemia, 16 (43%) had mediastinal lymphoma. 52 the mediastinal form in dogs is most commonly associated with a t-cell phenotype. 53, 54 cutaneous lymphoma can be solitary or more generalized and usually is classified as epitheliotropic (mycosis fungoides) or nonepitheliotropic. 55 canine epitheliotropic cutaneous lymphoma originates from t-cells, [55] [56] [57] [58] [59] [60] similar to its development in humans. in dogs, these more commonly represent cd8 + cells, whereas in humans they are typically cd4 + cells. a rare form of cutaneous t-cell lymphoma, characterized by skin involvement with evidence of peripherally circulating large (15 to 20 µm in diameter) malignant t-cells with folded, grooved nuclei, has been described. in humans, this is referred to as sézary syndrome and has been reported in both dogs and cats. [61] [62] [63] nonepitheliotropic cutaneous lymphomas form single or multiple dermal or subcutaneous nodules or plaques; histologically, they spare the epidermis and papillary dermis and affect the middle and deep portions of the dermis and subcutis. 55 more likely to have urine levels of 2, 4-d at 50 µg/l or higher. 36 the highest concentration was noted 2 days after application. in an environmental case-control study performed in europe, two variables, residency in industrial areas and use of chemicals (defined as paints or solvents) by owners, modestly increased the risk of developing lymphoma; however, no link was found with pesticide use. 37 a weak association between lymphoma in dogs and exposure to strong magnetic fields was observed in a preliminary epidemiologic study. 38 in this hospital-based case-control study, the risk of developing lymphoma categorized into high or very high exposure was increased (odds ratio = 1.8). more thorough studies are necessary to evaluate this association further. proximity to environmental waste was implicated in two european studies; however, it was felt to be a risk indicator rather than a risk factor and would require further case-control investigations. 39, 40 impaired immune function has also been implicated in dogs with lymphoma. immune system alterations in the dog such as immunemediated thrombocytopenia, independent of age and sex, have been associated with a higher risk of subsequently developing lymphoma when compared to the normal population. 41, 42 additional evidence comes from observations in human and feline transplantation patients. in a case-control study of cats undergoing renal transplant, 24% of cases developed cancer (36% of those were lymphoma) while on cyclosporine immunosuppressive therapy compared to 5.1% of control cats, none of which developed lymphoma (or, 6.1; p = 0.001). 43 a case of lymphoma developing in a dog following treatment with cyclosporine also exists. 44 one report suggests an association between the immunodysregulation observed in dogs with atopic dermatitis and the risk of developing epitheliotropic t-cell lymphoma; whether this is associated with the disease or the immunomodulatory treatments commonly applied is unknown. 45 classification of malignant lymphoma in dogs is based on anatomic location, histologic criteria, and immunophenotypic characteristics. the most common anatomic forms of lymphoma, in order of decreasing prevalence, are multicentric, gastrointestinal (gi), mediastinal, and cutaneous forms. 46 primary extranodal forms, which can occur in any location outside the lymphatic system, include the eyes, central nervous system (cns), bone marrow, bladder, heart, and nasal cavity. the pathologic characteristics of the various anatomic classifications will be discussed in this section and clinical characteristics will be described in subsequent sections. eighty-four percent of dogs with lymphoma develop the multicentric form, which is usually characterized by the presence of superficial lymphadenopathy (figure 32-1) . 46 the alimentary form of lymphoma is much less common, accounting for 5% to 7% of all canine lymphomas. this form is reported to be more common in male dogs than female dogs. 6 primary gi lymphoma in dogs may occur focally but more often affects multiple segments, with thickening of the wall, narrowing of the lumen, and frequently mucosal ulceration. 47, 48 histologically, there is infiltration of neoplastic lymphocytes throughout the mucosa and submucosa, with occasional transmural infiltration. liver and local lymph nodes are often secondarily involved. lymphocytic-plasmacytic enteritis (lpe) can be seen adjacent to or distant from the primary tumor. pathologically, some of these neoplasms may resemble plasma cell tumors, and aberrant production of immunoglobulins may occur. species. the national cancer institute (nci) working formulation 76 and the updated kiel system 77 have been adapted to canine tumors with some success. the world health organization (who) also publishes a histologic classification scheme, which uses the revised european american lymphoma (real) system as a basis for defining histologic categories of hematopoietic and lymphoid tumors in domestic animals. 78 this system incorporates anatomic, histologic, and immunophenotypic criteria (b-and t-cell immunophenotype), with the goal of enabling accurate and reproducible diagnosis of specific neoplastic disease entities. this theoretically should assist in better tailoring of treatment protocols, better correlation of prognosis, and better comparative capabilities. 79, 80 ; some of the less common categories in the who system were not represented and are not listed. the who system provides accurate and consistent reproducible diagnostic results similar to the system used in human pathology; accuracy among a group of pathologists examining 300 cases was at 83% agreement, and accuracy in evaluating the six most common diagnoses (80% of the cases) was 87%. 81 clinical studies are needed to correlate the various categories of disease with biologic behavior, response to treatment, and prognosis. preliminary results indicate dogs with indolent lymphoma (e.g., marginal zone lymphoma, follicular lymphoma, b-or t-cell small cell lymphoma, t-cell-rich b-cell lymphoma, and t zone lymphoma) maintain normal activity and appetite levels even during advanced stages of disease and experience long-term survival even with limited or no therapy. [81] [82] [83] [84] the working formulation (wf) was developed to allow investigators to "translate" among the numerous classification systems so that clinical trials could be compared in humans. most of the larger compilations agree that most canine lymphomas are intermediate or high grade; however, diffuse immunoblastic forms appear to predominate in the united states, whereas the follicular hepatosplenic lymphoma is a relatively uncommon, distinct presentation in the dog marked by a lack of significant peripheral lymphadenopathy in the face of hepatic, splenic, and bone marrow infiltration with malignant lymphocytes, usually of t-cell origin. 64, 65 biologically, this form of lymphoma is extremely aggressive and poorly responsive to therapy. in humans the tumor usually is composed of γδt-cells (i.e., t-cells that express the γδt-cell receptor), and this immunophenotype has been confirmed in at least one dog in the veterinary literature. 65 intravascular (angiotropic, angioendotheliomatosis) lymphoma is a distinct form of lymphoma defined as proliferations of neoplastic lymphocytes within the lumen and wall of blood vessels in the absence of a primary extravascular mass or leukemia. it has been reported several times in the veterinary literature, and in most cases it involves the cns and peripheral nervous system (pns), including the eye. [66] [67] [68] [69] [70] [71] the b-cell immunophenotype is most common in humans; however, in most reported cases in dogs, the origin is either t-cell or null cell (neither b-nor t-cell), although one case of a b-cell phenotype has been reported. pulmonary lymphomatoid granulomatosis (plg) is a rare pulmonary infiltrative and/or nodular disorder characterized by a heterogenous accumulation of lymphocytes (both b and t, although some evidence suggests primarily a t-cell origin), neutrophils, plasma cells, and macrophages, often arranged angiocentrically. [72] [73] [74] [75] whether this syndrome is a true lymphoma or a prelymphoma state is debatable. clinical signs are related to respiratory compromise, and various chemotherapeutic protocols have been used with reported results varying from rapid progression to long-term clinical remissions. lymphomas arise from clonal expansion of lymphoid cells with distinctive morphologic and immunophenotypic features. many histologic systems have been used to classify nhl in humans, and some of these have been applied to lymphoma in the dog and other percentage of canine lymphomas (5.3% to 29%) are considered low-grade tumors. high-grade lymphomas occur frequently if diffuse large-cell lymphomas, classified as intermediate grade in the wf, are considered high-grade, as in the updated kiel classification (in which they are labeled as diffuse centroblastic lymphomas). a documented difference exists in the prevalence of the various immunophenotypes based on breed. 25 for example, cocker spaniels and doberman pinschers are more likely to develop b-cell lymphoma, boxers are more likely to have t-cell lymphoma, and golden retrievers appear to have an equal likelihood of b-and t-cell tumors. to be clinically useful, these classification systems in the end must yield information about response to therapy, maintenance of remission, and survival. some studies suggest that the subtypes in the wf can be correlated with survival, and the kiel system may be useful for predicting relapse. 86, 87 in most studies, high-grade lymphomas achieve a complete response (cr) to chemotherapy significantly more often than low-grade tumors. however, dogs with low-grade tumors may live a long time without aggressive chemotherapy. 83, 84 dogs with t-cell lymphomas have shown a lower rate of cr to chemotherapy and shorter remission and survival times than dogs with b-cell tumors (with the exception of lowgrade t-cell subtypes). 53, 54, 86, 88 furthermore, t-cell lymphomas tend to be associated with hypercalcemia. 89, 90 large cell variations predominate in europe. a comparison of european and american classifications is warranted based on this discrepancy. the wf categorizes tumors according to pattern (diffuse or follicular) and cell type (e.g., small cleaved cell, large cell, immunoblastic), but it does not include information about the immunophenotype of the tumor. 76 the wf subtypes are related to the biology of the tumor and patient survival. the updated kiel classification includes the architectural pattern, morphology (centroblastic, centrocytic, or immunoblastic), and immunophenotype (b-cell or t-cell) of the tumor cells. 77 in both systems, the tumors can then be categorized as low-grade, intermediate grade, or highgrade malignancies. low-grade lymphomas composed of small cells with a low mitotic rate typically progress slowly and are associated with long survival times but are ultimately incurable. highgrade lymphomas with a high mitotic rate progress rapidly but are more likely to respond initially to chemotherapy and, in humans, are potentially curable. several features of canine lymphomas become apparent when these classification systems are applied. the most striking difference between canine and human lymphomas is the scarcity of follicular lymphomas in the dog. 79, 80 some diffuse lymphomas in the dog may initially be follicular, but these may progress to the more aggressive, diffuse form by the time of diagnostic biopsy. the most common form of canine lymphoma is diffuse large-cell lymphoma, a highgrade tumor most commonly of b-cell origin. 80, 81, 85 only a small particularly evident in dogs with hypercalcemia of malignancy. dogs may also be presented with clinical signs related to blood dyscrasias secondary to marked tumor infiltration of bone marrow (myelophthisis) or paraneoplastic anemia, thrombocytopenia, or neutropenia. these could include fever, sepsis, anemia, and hemorrhage. diffuse pulmonary infiltration is seen in 27% to 34% of dogs with the multicentric form, as detected by radiographic changes (figure 32-3) . 97 , 98 based on bronchoalveolar lavage, the actual incidence of lung involvement may be higher. 99, 100 in the veterinary literature, 60% to 80% of canine lymphomas are of b-cell origin; t-cell lymphomas account for 10% to 38%; mixed b-and t-cell lymphomas account for as many as 22%; and null cell tumors (i.e., neither b-cell nor t-cell immunoreactive) represent fewer than 5%. 53, 54, [91] [92] [93] the development of monoclonal antibodies to detect specific markers on canine lymphocytes has made immunophenotyping of tumors in dogs routinely available in many commercial laboratories. such techniques can be performed on paraffin-embedded samples, from tissue microarrays, on cytologic specimens obtained by fine-needle aspiration (fna) of lesions, or by flow cytometric analysis of cellular fluid samples (e.g., peripheral blood, effusions) and lesion aspirates. the rappaport classification system, proposed in 1956 for human nhl, describes the architectural pattern (follicular or diffuse) and the cytologic features (well differentiated, poorly differentiated, or histiocytic) of lymphoma. 94, 95 this system has not proved useful in providing prognostic information or in guiding therapy in dogs with lymphoma because of the low number of follicular lymphomas in dogs, the problematic "histiocytic" subgroup, and the failure to account for different morphologic and immunologic cell types. one criticism of the rappaport, kiel, and wf classification systems is that they fail to include extranodal lymphomas as a separate category. the who system does include anatomic location as a factor in determining certain categories. although differences between nodal and extranodal tumors in biologic behavior and prognosis are well recognized, comparative information about the histogenesis of these tumors is lacking. for example, in humans small-cell lymphomas arising from malt are composed of cells with a different immunophenotype than that of other small-cell lymphomas (i.e., malt lymphomas typically are negative for both cd5 and cd10). except for cutaneous lymphoid neoplasms, detailed characterization of extranodal lymphomas in dogs has not been done. although cutaneous lymphoma is a heterogeneous group of neoplasms that includes an epitheliotropic form resembling mycosis fungoides and a nonepitheliotropic form, most cutaneous lymphomas have a t-cell phenotype. 64, 96 to summarize, it is important to determine the histologic grade of canine lymphomas as low (small lymphocytic or centrocytic lymphomas) or intermediate to high (diffuse large cell, centroblastic, and immunoblastic lymphomas) and the architecture as diffuse or follicular. furthermore, determining the immunophenotype of the tumor provides useful information. response rates to chemotherapy are, in general, better in animals with b-cell tumors and intermediate-to high-grade lymphomas. dogs with low-grade lymphomas can have long survival times without aggressive therapy. the clinical signs associated with canine lymphoma are variable and depend on the extent and location of the tumor. multicentric lymphoma, the most common form, is usually distinguished by the presence of generalized painless lymphadenopathy (see figure 32 -1). enlarged lymph nodes are usually painless, rubbery, and discrete and may initially include the mandibular and prescapular nodes. in addition, hepatosplenomegaly and bone marrow involvement occur commonly. most dogs with multicentric lymphoma present without dramatic signs of systemic illness (who substage a) (box 32-1); however, a large array of nonspecific signs such as anorexia, weight loss, vomiting, diarrhea, emaciation, ascites, dyspnea, polydipsia, polyuria, and fever can occur (who substage b). dogs presented with t-cell lymphoma are more likely to have constitutional (i.e., substage b) signs. polydipsia and polyuria are i involvement limited to a single node or lymphoid tissue in a single organ. † ii involvement of many lymph nodes in a regional area (±tonsils). iii generalized lymph node involvement. iv liver and/or spleen involvement (±stage iii). syndrome, characterized by pitting edema of the head, neck, and forelimbs secondary to tumor compression or invasion of the cranial vena cava (figure 32-4) . signs in dogs with extranodal lymphoma depend on the specific organ involved. cutaneous lymphoma is usually generalized or multifocal. [55] [56] [57] tumors occur as nodules, plaques, ulcers, and erythemic or exfoliative dermatitis with focal hypopigmentation and alopecia. epitheliotropic t-cell lymphoma (e.g., mycosis fungoides) typically has a clinical course with three apparent clinical stages. initially, there will be scaling, alopecia, and pruritus ( figure 32 -5, a), which can mimic a variety of other skin conditions. as the disease progresses, the skin becomes more erythematous, dogs with gi or alimentary lymphoma are usually presented with nonspecific gi signs, such as vomiting, diarrhea, weight loss, and malabsorption. 47, 101, 102 mesenteric lymph nodes, spleen, and liver may be involved. the mediastinal form of lymphoma is characterized by enlargement of the cranial mediastinal structures and/or thymus (see figure 32 -2), and clinical signs are associated with the extent of disease with resulting respiratory compromise or polydipsia/ polyuria from hypercalcemia. commonly, dogs are presented with respiratory distress caused by a space-occupying mass and pleural effusion, exercise intolerance, and possibly regurgitation. additionally, dogs with mediastinal lymphoma may present with precaval a c b intravascular lymphoma usually present with signs relative to cns, pns, or ocular involvement. [66] [67] [68] [69] [70] [71] these include paraparesis, ataxia, hyperesthesia, seizures, blindness, lethargy, anorexia, weight loss, diarrhea, polyuria, polydipsia, and intermittent fever. finally, dogs with pure hepatosplenic lymphoma usually are presented with nonspecific signs of lethargy, inappetence, and weakness and often are icteric. 64, 65 the differential diagnosis of lymphadenopathy depends on the dog's travel history (i.e., relative to infectious disease) and the size, consistency, and location of affected lymph nodes. other causes of lymphadenopathy include infections caused by bacteria, viruses, parasites (toxoplasma sp., leishmania sp.), rickettsial organisms (salmon-poisoning, ehrlichia sp.), and fungal agents (blastomyces and histoplasma sp.). the potential for hypercalcemia to accompany systemic fungal diseases may further complicate differentiation from lymphoma. discrete, hard, asymmetric lymph nodes, particularly if they are fixed to underlying tissues, may indicate metastatic tumors such as mast cell tumor or carcinoma. immunemediated diseases (e.g., pemphigus, systemic lupus erythematosus) also may result in mild-to-moderately enlarged lymph nodes. the thickened, ulcerated, and exudative. the final stage is characterized by proliferative plaques and nodules with progressive ulceration (figure 32-5, b) . oral involvement may also occur and this can appear as multicentric erythematous plaque-like lesions or nodules associated with the gum and lips ( figure 32 -5, c). extracutaneous involvement can also occur, most often in the lymph nodes, spleen, liver, and bone marrow. nonepitheliotropic cutaneous lymphomas form single or multiple dermal or subcutaneous nodules or plaques; histologically, they spare the epidermis and papillary dermis and affect the middle and deep portions of the dermis and subcutis. 55 dogs with primary cns lymphoma may be presented with either multifocal or solitary involvement. [103] [104] [105] seizures, paralysis, and paresis may be noted. ocular lymphoma is characterized by infiltration and thickening of the iris, uveitis, hypopyon, hyphema, posterior synechia, and glaucoma. 106, 107 in one study of 94 cases of canine multicentric lymphoma, 37% had ocular changes consistent with lymphoma, and in a series of 102 cases of uveitis in dogs, 17% were secondary to lymphoma. 107 anterior uveitis was most commonly seen in advanced stage of disease (stage v). dogs with • figure 32-5 a, early epitheliotropic cutaneous lymphoma in the scaly, plaque stage in a dog. b, advanced epitheliotropic cutaneous lymphoma in the nodular stage in a dog. c, oral mucosal epitheliotropic cutaneous lymphoma in a dog. c count (cbc), with a differential cell count, including a platelet count; a serum biochemical profile; and urinalysis. optimally, ionized calcium should be measured. ultimately, obtaining tissue or cytologic specimens for a definitive diagnosis is essential. a thorough physical examination should include palpation of all assessable lymph nodes, including a rectal examination; in the authors' experience, a significant proportion of dogs will have rectal polyps consisting of aggregates of neoplastic lymphocytes. inspection of mucous membranes for pallor, icterus, petechiae, and ulceration should be undertaken as these signs may indicate anemia or thrombocytopenia secondary to myelophthisis or immunemediated disease or may be evidence of major organ failure or uremia. abdominal palpation may reveal organomegaly, intestinal wall thickening, or mesenteric lymphadenopathy. the presence of a mediastinal mass and/or pleural effusion can be suspected following thoracic auscultation. an ocular examination, including funduscopic assessment, may reveal abnormalities (e.g., uveitis, retinal hemorrhage, ocular infiltration) in approximately one-third to onehalf of dogs with lymphoma. 107, 111 anemia, the most common lymphoma-related hematologic abnormality, is usually normochromic and normocytic (nonregenerative), consistent with anemia of chronic disease. 108 however, hemorrhagic and hemolytic anemias may also occur, and regenerative anemias may reflect concomitant blood loss or hemolysis. additionally, if significant myelophthisis is present, anemia may be accompanied by thrombocytopenia and leukopenia. 112, 113 in animals with anemia or evidence of bleeding, in addition to a platelet count, a reticulocyte count and coagulation testing may be indicated. thrombocytopenia may be seen in 30% to 50% of cases, but bleeding is seldom a clinical problem. neutrophilia can be seen in 25% to 40% of dogs and lymphocytosis occurs in approximately 20% of affected dogs. 108 circulating atypical lymphocytes may be indicative of bone marrow involvement and leukemia. it is important to differentiate multicentric lymphoma with bone marrow involvement (i.e., stage v disease) from primary lymphoblastic leukemia (see later), as the prognosis for each is entirely different. hypoproteinemia is observed more frequently in animals with alimentary lymphoma. in dogs with a high total protein or evidence of an increased globulin fraction on a chemistry profile, serum proteins may be evaluated by serum electrophoresis. monoclonal gammopathies have been reported to occur in approximately 6% of dogs with lymphoma. 114 serum biochemical abnormalities often reflect the anatomic site involved, as well as paraneoplastic syndromes such as hypercalcemia. in cases of hypercalcemia of unknown origin, lymphoma should always be considered high on the differential disease list, and diagnostic testing directed at this possibility should be undertaken (see chapter 5) . in addition, the presence of hypercalcemia can serve as a biomarker for response to therapy and early recurrence. increased urea nitrogen and creatinine concentrations can occur secondary to renal infiltration with tumor, hypercalcemic nephrosis, or prerenal azotemia from dehydration. increases in liver-specific enzyme activities or bilirubin concentrations may result from hepatic parenchymal infiltration. increased serum globulin concentrations, usually monoclonal, occur infrequently with b-cell lymphoma. urinalysis is part of the minimum database used to assess renal function and the urinary tract. for example, isosthenuria and various differential diseases or conditions that can resemble canine lymphoma are listed in table 32-2. canine lymphoma also may be associated with paraneoplastic syndromes (see chapter 5) . anemia is the most common lymphoma-related paraneoplastic syndrome. 108 paraneoplastic hypercalcemia is also common and is characterized clinically by anorexia, weight loss, muscle weakness, lethargy, polyuria, polydipsia, and rarely cns depression and coma. lymphoma-induced hypercalcemia in most cases results from parathyroid hormonerelated peptide (pthrp), elaborated by neoplastic cells; however, it can also be related to the production of several other humoral factors, including interleukin-1 (il-1), tumor necrosis factor-α (tnf-α), transforming growth factor-β (tgf-β), and vitamin d analogs (e.g., 1,25-dihydroxyvitamin d). 89, 109, 110 as previously discussed, hypercalcemia is most commonly associated with the t-cell immunophenotype. other paraneoplastic syndromes that may be encountered include monoclonal gammopathies, neuropathies, and cancer cachexia. for dogs suspected of having lymphoma, the diagnostic evaluation should include a thorough physical examination; complete blood • variable, depending on organ/system involved *the existence of this disease is controversial; in most cases, the disease has been reclassified as a lymphoid neoplasm. prescapular or popliteal lymph nodes are preferable if also involved. also, lymphoid cells are fragile, and in preparing smears of aspirated material only gentle pressure should be applied in spreading material on the slides. in most cases, a diagnosis of lymphoma can be made on evaluation of fine-needle aspirates of affected lymph nodes or other tissues. typically, most of the cells are large lymphoid cells (>2 times the diameter of a red blood cell [rbc] or larger than neutrophils), and they may have visible nucleoli and basophilic cytoplasm (figure 32-6 , a) or fine chromatin with indistinct nucleoli. because tissue architecture is not maintained in cytologic specimens, effacement of the node or capsular disruption cannot be detected. therefore marked reactive hyperplasia characterized by increased numbers of large lymphoid cells may be difficult to distinguish from lymphoma, and small cell lymphomas may have few cytologic clues that point to malignancy. also, classification of lymphoma, which has been attempted using cytologic appearance and immunophenotypic analysis, 123 into subcategories that make up the low-, intermediate-, and high-grade forms is performed most accurately on histologic sections (discussed previously). proteinuria in the absence of an active sediment may indicate renal disease, and hematuria may result from a hemostatic abnormality. it is important to remember that isosthenuria in azotemic dogs with hypercalcemia is not necessarily indicative of renal disease as the high calcium levels interfere with tubular concentration capabilities through disruption of antidiuretic hormone (adh) control. several abnormalities in serum have been explored as biomarkers of lymphoma in the dog. examples include alpha-fetoprotein, alpha-1 glycoprotein levels, zinc, chromium, iron, endostatin, vascular endothelial growth factor (vegf), lactate dehydrogenase, c-reactive protein haptoglobin, and antioxidants/oxidative stress markers. [115] [116] [117] [118] [119] [120] [121] [122] the clinical, biologic, and prognostic significance of these alterations is yet to be definitively characterized. morphologic examination of the tissue and cells that constitute the tumor is essential to the diagnosis of lymphoma. care should be taken to avoid lymph nodes from reactive areas (e.g., mandibular lymph nodes), unless those nodes are the only ones enlarged; the immunophenotyping is used to determine the type of cells that comprise the tumor, but this technique also can be helpful for making the initial diagnosis. 133-140,155 when a heterogenous population of lymphocytes is expected in a tissue, documentation of a homogeneous population of the same immunophenotype is supportive of a neoplastic process. the immunophenotype of a lymphocyte is identified by determining the expression of molecules specific for b-cells (e.g., cd79a, cd20) and t-cells (e.g., cd3). although tumor cells sometimes have morphologic characteristics that typify a particular immunophenotype, exceptions occur, and morphologic appearance cannot be used as the sole determinant of immunophenotype. for example, in a series of nine high-grade t-cell lymphomas and leukemias in dogs, the cells had a plasmacytoid appearance, typically associated with b-cell lymphoma. 156 similarly, anatomic location does not always predict the immunophenotype. for accurate determination of immunophenotype, antibodies against lymphocyte markers are applied to tissue sections (immunohistochemistry), cytologic specimens (immunocytochemistry), or individual cells in a fluid medium (flow cytometry). flow cytometric evaluation of cells obtained by needle aspiration is also feasible. for t-cells, markers include cd3 (pan t), cd4 (helper t), and cd8 (cytotoxic t); for b-cells, the markers are cd79a ( figure 32-6, b) , cd20, and cd21. increasingly, aberrant expression of cd molecules has been reported in canine lymphoma. in a study of 59 dogs with lymphoma, tumor cells from six dogs were positive for both t-and b-cell markers; however, a clonality assay (see later) revealed clonality either of the t-cell or the immunoglobulin receptor but not both. this indicates that in some cases, the malignant cells may co-express b-and t-cell markers. 93 antibodies against these molecules are used to determine the immunophenotype; however, they also have potential utility as a therapeutic modality if tumor cells could be targeted using these antibodies. assessments of markers of multidrug resistance and apoptotic pathways (e.g., p-glycoprotein, p53, bcl-2 proteins) have been evaluated in dogs with lymphoma. 19,79,142,156a,156b however, their clinical significance and utility await further evaluation. occasionally, diagnosis of lymphoma and differentiation of malignant versus benign proliferation of lymphocytes are not possible based on standard histologic and cytologic criteria. in these cases, advanced molecular analyses may be helpful to confirm a diagnosis. clonality is the hallmark of malignancy; that is, the malignant cell population theoretically should be derived from expansion of a single malignant clone characterized by a particular dna region unique to that tumor. for example, in a dog with t-cell lymphoma, all the malignant cells theoretically should have the same dna sequence for the variable region of the t-cell receptor gene. for accurate histopathologic evaluation, an entire lymph node, including the capsule, should be removed, placed in buffered formalin, and submitted to a pathologist. although needle core biopsies may be satisfactory, it is important to avoid crush artifact or inadequate sample size. most pathologists prefer whole node biopsies because they provide the maximal amount of information. effacement of normal nodal architecture by neoplastic lymphocytes and capsular disruption are characteristic findings (figure 32 -6, c and d). diagnostic ultrasonography and ultrasound-guided fna or needle biopsy have been useful for evaluation of involvement of the liver, spleen, or abdominal lymph nodes. [124] [125] [126] aspiration of ultrasonographically normal splenic tissue is rarely contributory to a diagnosis. 124 if possible, the diagnosis should be made by sampling peripheral nodes, avoiding percutaneous biopsies of the liver and spleen. however, if there is no peripheral node involvement, it is appropriate to biopsy affected tissues in the abdominal cavity. when gi lymphoma is suspected, an open surgical wedge biopsy of the intestine is preferred in most cases to differentiate lymphoma from lymphocytic enteritis. if associated abdominal lymph nodes also appear involved, image-guided biopsies may be associated with less morbidity than intestinal biopsies. multiple samples may be necessary to accurately diagnose segmental disease. endoscopic biopsies may be inadequate as only a superficial specimen is obtained; however, more aggressive endoscopic biopsy techniques combined with more accurate histopathologic, immunophenotypic, and molecular assessments are improving the diagnostic yield of these less invasive techniques. [127] [128] [129] [130] in many dogs with primary gi lymphoma, an inflammatory nonneoplastic infiltrate (i.e., lpe) may be misdiagnosed on biopsy specimens that are too superficial. the application of assays for clonal expansion (e.g., parr-see next section on molecular diagnostic techniques) does not appear as yet to be as accurate for endoscopically derived intestinal biopsies as with other solid lymphoid tumors in dogs. cytologic examination of cerebrospinal fluid (csf), thoracic fluid, or mass aspirates is indicated in animals with cns disease, pleural effusion, or an intrathoracic mass, respectively. in one study of dogs with cns involvement, csf analysis was diagnostic in seven of eight dogs. 103 characteristics of the csf included an increased nucleated cell count in the seven dogs, and 95% to 100% of the cells were atypical lymphocytes. the csf protein concentration was increased in five of the dogs, ranging from 34 to 310 mg/ dl (reference interval: <25 mg/dl). for cutaneous lymphoma, punch biopsies (4 to 8 mm) should be taken from the most representative and infiltrative, but not secondarily infected, skin lesions. application of immunophenotypic and clonality assessments of cutaneous biopsies can aid in differentiating lymphoma from benign lymphocytic lesions. 58, 131, 132 molecular techniques can be used to establish a diagnosis of lymphoma or to further characterize the tumor after the initial diagnosis is made. tissues and cells from peripheral blood, lymph nodes, nonlymphoid sites, and effusions can be analyzed by various molecular means to aid in cases that represent a more difficult diagnostic challenge, particularly in cases where reactive lymphocytosis and lymphoma are both possible based on standard histologic or cytologic assessment. these include histochemical and cytochemical, immunohistochemical and immunocytochemical, flow cytometric, and polymerase chain reaction (pcr) techniques. for example, a bone marrow aspirate or biopsy (from proximal humerus or iliac crest) is recommended for complete staging and prognostication and is indicated in dogs with anemia, lymphocytosis, peripheral lymphocyte atypia, or other peripheral cytopenias. in one study of 53 dogs with lymphoma, 28% had circulating malignant cells and were considered leukemic, whereas bone marrow examination indicated involvement in 57% of the dogs. 162 the presence of a few prolymphocytes and large lymphocytes with nucleoli in the circulation of dogs with lymphoma may indicate bone marrow involvement. it is important to remember these cells also can be seen with gi parasitism, immune-mediated hemolytic anemia, and other immune-mediated and infectious diseases. as discussed previously, tumor cells within the peripheral and bone marrow compartments can also be identified using clonality assays (parr) that are more sensitive than routine microscopic examination in detecting malignant cells; however, the prognostic significance of the knowledge gained with more sensitive staging methodologies is yet to be determined. although bone marrow evaluation may offer prognostically valuable information, it is not necessary to perform the procedure if the client is committed to treat regardless of stage. evaluation of thoracic and abdominal radiographs may be important in determining the extent of internal involvement ( figure 32-7) . approximately 60% to 75% of dogs with multicentric lymphoma have abnormalities on thoracic radiographs, with one-third having evidence of pulmonary infiltrates (see figure 32 -3) and twothirds having thoracic lymphadenopathy (sternal and tracheobronchial lymph nodes [see ) and widening of the cranial mediastinum (see figure 32 -2). 97, 98 pulmonary infiltrates usually are represented by an interstitial and/or alveolar pattern; however, nodules (rarely) and bronchial infiltrates can also occur. 163 pleural effusion may also be present. cranial mediastinal lymphadenopathy is detected in 20% of dogs with lymphoma. 97, 163 abdominal radiographs reveal evidence of involvement of medial iliac (sublumbar) and/or mesenteric lymph node, spleen, or liver in approximately 50% of cases. in the authors' practice, for the typical cases of canine multicentric lymphoma, imaging is limited to thoracic likewise, in a dog with b-cell lymphoma, the tumor cells should have identical dna sequences in the variable region of the immunoglobulin (ig) receptor gene. conversely, in reactive lymphocytosis, the cells are polyclonal for their antigen receptors. using this knowledge, investigators have used pcr technology to amplify the variable regions of the t-cell and immunoglobulin receptor genes to detect the presence of clonal lymphocyte populations in dogs (see figure 8 -4 of chapter 8). these techniques are reviewed in chapter 8 and elsewhere. 150 in physician-based medicine, such assays of clonality are approximately 70% to 90% sensitive and have a false-positive rate of approximately 5%, and recent studies report similar rates in dogs. false-negative and false-positive results can occur with clonality assays. for example, cells from a dog with lymphoma may be negative for clonality if the clonal segment of dna is not detected with the primers used, if the malignant cells are natural killer (nk) cells (rare), or if the malignant cells are present in too low a frequency to be detected. false positives occur rarely in some infectious diseases (e.g., ehrlichiosis and lyme disease). in these cases, a diagnosis should be made only after considering the results of all the diagnostic tests, including histologic/cytologic evaluation, immunophenotyping, and clonality studies in conjunction with signalment and physical examination findings. these molecular techniques, although helpful for diagnosis, could also have utility in detecting early recurrence and in determining more accurate clinical stage and so-called "molecular remission rates" because they are more sensitive than standard cytologic assessment of peripheral blood, bone marrow, or lymph nodes (covered subsequently in section on treatment response). proteomics comprises, simplistically, methodologies that analyze the entire protein component or protein signature of cells (the proteome). protein components of a cell (normal or malignant) change over time with upregulation and downregulation of gene expression in response to varied stimuli (e.g., growth factors, environmental cues). it may therefore be possible to use the field of proteomics to identify serum biomarkers of malignancy (i.e., cancer-specific protein markers) and to further analyze response to therapy or even to predict which therapies are appropriate for an individual patient's tumor. although in its infancy in veterinary oncology, preliminary investigations of the proteome of dogs with lymphoma have been reported 157-160 ; however, they have yet to reach the level of sophistication in which useful output would have an impact on clinical decision making. after a diagnosis has been established, the extent of disease should be determined and categorized by the clinical stage of disease. the who staging system routinely used to stage dogs with lymphoma is presented in box 32-1. most dogs (>80%) are presented in advanced stages (iii to iv). diagnostic imaging and assessment of bone marrow involvement may be indicated for staging. the degree to which thorough staging is implemented depends on whether the result will alter the treatment plan, whether relevant prognostic information is gleamed, and whether the clients need to know the stage prior to initiating (or declining) a treatment plan. additionally, when comparing different treatment protocols with respect to efficacy, consistent and similar staging diagnostics should be used to avoid so-called "stage migration, " which results when one staging methodology is more accurate than another. 161 the impact of stage migration on prognosis should be considered when comparing different published outcomes. radiographs as there is no prognostic difference between dogs with stage iii and iv disease (i.e., liver/spleen involvement), whereas the presence of cranial mediastinal lymphadenopathy is of prognostic significance (see prognosis section). however, if there are clinical signs attributable to abdominal disease or if complete staging is necessary (e.g., for clinical trial inclusion), further imaging of the abdomen is warranted. abdominal ultrasonography can be important for obtaining ultrasound-guided intraabdominal samples for diagnosis. it may also be useful for the diagnosis of gi, abdominal nodal, and hepatosplenic lymphoma. 125 ultrasonographic (including doppler ultrasound) assessment of peripheral lymph nodes has also been explored 126 ; however, its clinical applicability is questionable because cytologic assessment of peripheral nodes is easy, inexpensive, and of higher diagnostic utility. advanced imaging modalities, including computed tomography (ct), magnetic resonance imaging (mri), positron emission tomography (pet), or pet/ct imaging, are becoming more commonplace in veterinary practice and their utility is only now being determined. 164-169 pet/ct imaging is the current standard of care for following and indeed predicting durability of treatment response in human patients with lymphoma, and both [18f]fluorothymidine (18flt) pet/ct and [18f]fluoro-d-glucose (18fdg) pet imaging have been reported in dogs with lymphoma. 164-166 18flt-pet/ct functional and anatomic imaging shows promise for the evaluation of response to cytotoxic chemotherapy in dogs with lymphoma and for predicting relapse before standard clinical and clinicopathologic confirmation (figure 32-8) . the therapeutic approach to a particular patient with lymphoma is determined by the stage and substage of disease, the presence or absence of paraneoplastic disease, the overall physiologic status of the patient, financial and time commitment of the clients, and their level of comfort with respect to likelihood of treatment-related success and/or side effects. without treatment, most dogs with lymphoma will die of their disease in 4 to 6 weeks after diagnosis, although significant variability exists. 114 with few exceptions, canine lymphoma is considered a systemic disease and therefore requires systemic therapy in order to achieve remission and prolong survival. the majority of canine multicentric lymphomas are intermediate to high grade, and, currently, histopathologic and immunophenotypic characterization has not played a significant role in determining the initial treatment protocol. it is hoped that in the near future, sufficient data will emerge to better tailor treatment protocols chosen for dogs with lymphoma based on these and other yet to be characterized parameters. that being said, systemic multiagent chemotherapy continues to be the therapy of choice for canine lymphoma. in general, combination chemotherapy protocols are superior in efficacy to single-agent protocols. single-agent protocols result in lower response rates that are not as durable as combination chemotherapy, which is summarized in table 11 -2 in chapter 11. in rare cases in which lymphoma is limited to one site (especially an extranodal site), the animal can be treated with a local modality such as surgery or radiation therapy (rt) as long as the client and clinician are committed to diligent reevaluation to document subsequent progression to systemic involvement, should it occur. many chemotherapeutic protocols for dogs with lymphoma have been developed over the past 15 to 20 years (table 32-3) . 91, significant limitations arise when comparing efficacy studies in the that is, clinical trials are inherently costly, and because most of the known effective drugs are unregistered off-label human generic (i.e., off patent) drugs, the incentive for pharmaceutical-funded, sufficiently powered, randomized field trials is low, resulting in a general lack of comparative data. despite the plethora of available combination protocols, most are modifications of chop protocols initially designed for human oncologic use, and currently randomized prospective evidence does not exist to clearly recommend one over the other as long as the basic "chop" components are present. chop represents combinations of cyclophosphamide (c), doxorubicin (h, hydroxydaunorubicin), vincristine (o, oncovin), and prednisone (p). conventional chop-based chemotherapy induces remission in approximately 80% to 95% of dogs, with overall median survival times (msts) of 10 to 12 months. approximately 20% to 25% of treated dogs will be alive 2 years after initiation of these protocols (figure 32-9 ). veterinary literature for the various published protocols. few of these studies include sufficient numbers for adequate statistical power and even fewer compare treatment protocols in a randomized prospective fashion. in addition, staging, inclusion, and response criteria vary considerably between reports. therefore evaluations of efficacy among various protocols are subject to substantial bias, making direct comparisons difficult and indeed precarious. a recurring theme in the concluding statement in most of these published protocols is some variation of "prospective randomized trials will be required to confirm these suggestive findings. " in an attempt to better standardize response criteria and outcome reporting of future trials, the veterinary cooperative oncology group (vcog) has recently published response evaluation criteria (v1.0) 169 (see subsequent response evaluation section). the greatest obstacle to the performance of prospective randomized comparative lymphoma trials in veterinary oncology is financial; • ("treatment holidays") are not uncommonly required in individual cases, only a minority of dogs develop significant adverse events requiring hospitalization. 191, 192 studies assessing client perceptions of medical treatment for cancer in general and lymphoma in particular report a positive experience; most owners feel treatment was worthwhile, that it resulted in improvement in the well-being of their pet, and that quality of life during treatment was good. 193, 194 very few clients express regret about treating lymphoma using a multidrug protocol. with lymphoma, the fundamental goals of chemotherapy are to induce a complete durable (>6 months) first remission (termed induction), to reinduce a remission when the tumor recrudesces (or the patient relapses) following achievement of a remission (termed reinduction), and, finally, to induce remissions when the cancer fails to respond to induction or reinduction using drugs not present in the initial protocols (termed rescue). an unanswered question in the treatment of lymphoma has been whether long-term maintenance chemotherapy is useful following an initial course of aggressive induction chemotherapy lasting 6 months or less. long-term maintenance chemotherapy has not been shown to be of significant value in humans with most forms of nhl; however, in humans, the initial induction course of chemotherapy is much more aggressive than that used in veterinary patients. although no randomized prospective studies have been performed to address the therapeutic benefit of long-term maintenance chemotherapy in dogs, most comparisons of dogs treated with chop-based protocols do not show any clear advantage for a maintenance or consolidation phase after induction therapy.* indeed, in most reports, dogs receiving shorter, less costly protocols that do not include a prolonged maintenance phase have comparable remission and progression-free survival (pfs) durations and appear to more readily achieve second remissions when they relapse following completion of chemotherapy than their counterparts receiving long-term maintenance. these data, taken together, suggest that maintenance therapy is not beneficial for most dogs with lymphoma. until well-designed randomized prospective trials indicate otherwise, the author (dmv) prefers protocols that utilize an aggressive induction without maintenance. the most effective, currently available chemotherapeutic agents for canine lymphoma include doxorubicin, l-asparaginase, vincristine, cyclophosphamide, and prednisone-most of which are represented to one degree or another in most first-line multiagent chemotherapy protocols. other drugs that have documented activity are often considered second-line agents and include lomustine, vinblastine, actinomycin-d, mitoxantrone, mustargen, chlorambucil, methotrexate, dacarbazine (dtic), 9-aminocamptothecin, ifosfamide, cytosine arabinoside, and gemcitabine. of these, cytosine arabinoside, 199 ifosfamide, 200 and gemcitabine 201 appear to have only minimal activity. with the exception of doxorubicin, induction therapy with single-agent chemotherapy does not typically result in durable remission durations when compared with standard combination protocols (see table 11 -2, chapter 11). incorporation of other standard cytotoxic drugs with single-agent activity into standard chop-based protocols has not resulted in significant gains, and most are reserved for subsequent rescue settings. response rates and duration of response vary according to the presence or absence of prognostic factors discussed subsequently in the section on prognosis in this chapter. the relative cost of the various protocols to the client depends on the drug(s) selected, the size of the animal, the frequency of administration, and the laboratory tests required to monitor adverse events and response. dogs responding to chemotherapy and undergoing complete remission are usually free of clinical signs associated with lymphoma and subsequently return to a very good quality of life. treating dogs with lymphoma is initially gratifying because a high percentage enjoy a complete response. most dogs tolerate chemotherapy well, and although dose reductions and treatment breaks initiation or at varying times throughout the protocol, several studies suggest this does not result in clinically relevant increases in remission rate, speed of attaining remission, or first-remission duration, and therefore the author reserves its use for rescue situations. 172, 195, 202, 203 if client or other considerations preclude a chop-based protocol, single-agent doxorubicin (30 mg/m 2 , intravenous [iv], every 3 weeks for 5 total treatments) is offered along with a 4-week tapering oral prednisone regimen (same prednisone regimen in box 32-2) as a less aggressive and less costly approach. the expected cr rate will range from 50% to 75%, with an anticipated median survival of 6 to 8 months. 171, 172, 204, 205 the addition of oral cyclophosphamide (50 mg/m 2 daily for 3 days starting on the same day as doxorubicin) to single-agent doxorubicin resulted in a numerically but not statistically superior outcome in a recent randomized trial 205 comparing doxorubicin/prednisone with doxorubicin/cyclophosphamide/ prednisone (pfs of 5.6 months versus 8.2 months, respectively). this trial was only powered to detect a threefold difference in pfs; therefore larger trials should be undertaken to confirm any benefit. if clients are reticent to include iv medications, the author often recommends a protocol of oral lomustine (ccnu; 70 mg/m 2 by mouth [po] every 3 weeks for 5 treatments) and prednisone. this protocol has been associated with short median remissions (40 days) in only one small case series 206 ; however, in the author's experience, a subset of dogs have remained in remission for several months on this protocol when clients decline iv medication. if financial or other client concerns preclude the use of systemic chemotherapy, prednisone alone (2 mg/kg po, daily) will often result in short-lived remissions of approximately 1 to 2 months. in these cases, it is important to educate clients that, should they decide to pursue more aggressive therapy at a later date, dogs receiving single-agent prednisone therapy are more likely to develop multidrug resistance (mdr) and experience shorter remission and survival durations with subsequent combination protocols. [207] [208] [209] this is especially true following long-term prednisone use or in dogs that have experienced a recurrence while receiving prednisone. therefore the earlier that clients opt for more aggressive therapy, the more likely a durable response will result. a cbc should be performed prior to each chemotherapy treatment. a minimum of 1500 neutrophils/µl (some oncologists use a cut-off of 2000 neutrophils/µl) and 50,000 platelets/µl should be present prior to the administration of myelosuppressive chemotherapy. if the neutrophil count is lower than 1500/µl, it is best to wait 5 to 7 days and repeat the cbc; if the neutrophil count has increased to more than 1500 cells/µl, the drug can be safely administered. a caveat to these restrictions is that for dogs presented prior to initiation of chemotherapy with low neutrophil and platelet counts due to bone marrow effacement, myelosuppressive chemotherapy is instituted in the face of cytopenias in order to clear the bone marrow of neoplastic cells and allow hematopoiesis to normalize. in those breeds likely to have mdr1 gene mutations (e.g., collies; see chapter 11) and therefore to be at risk for serious chemotherapeutic toxicity, 210 the author will initiate a chop protocol out of sequence, beginning with non-mdr1-associated drugs, such as cyclophosphamide. this ensures treatment of the lymphoma while allowing sufficient time for analysis of mdr1 gene mutations prior to initiating mdr1 substrate drugs. no specific protocols have been scrutinized for treating dogs that are doublemutant for mdr1; however, if using mdr1 substrate drugs, the author initiates at a 40% dose reduction. subsequent dose modifications (increased or decreased dosage) can be implemented, several factors should be considered and discussed with caregivers on a case-by-case basis when choosing the protocol to be used. these factors include cost, time commitment involved, efficacy, adverse event profiles, and experience of the clinician with the protocols under consideration. it is now clearly established that "standard of care" combination protocols used in dogs with lymphoma are essentially variations of "chop" protocols (see table 32 -3). specific details regarding dose and timing of the chop protocol currently preferred by the author (dmv) are outlined in box 32-2. this protocol does not have a maintenance therapy arm, and all treatments cease at 19 weeks, provided the animal is in complete remission. although several other chop-based protocols include l-asparaginase either at † in dogs <15 kg in body weight, a doxorubicin dose of 1 mg/kg is substituted for 30 mg/m 2 . molecular and biologic markers of minimal disease. advanced functional and anatomic imaging (i.e., pet/ct) are the current standard for assessing treatment response and early relapse of lymphoma in humans and have also been investigated in dogs (see . [164] [165] [166] 168 as this technology becomes available to a broader veterinary population, its clinical application will surely increase. molecular detection of mrd applies clonality and pcr techniques previously discussed in this chapter. beyond diagnostic applications, these techniques have been applied to determine cytoreductive efficacy of various chemotherapeutic drugs and to document and predict early relapse in patients prior to more conventional methods. 215-219 regarding biomarkers of mrd, preliminary investigations have suggested serum lactate dehydrogenase activity, 120 thymidine kinase 1 activity, 220 and serum c-reactive protein 221 may be candidates in the dog. as we become more proficient at defining mrd, the pressing clinical question becomes how we use this information. theoretically, such information could suggest when more aggressive therapy or alternative therapy should be instituted in patients who have not achieved a "molecular remission" or who are undergoing early relapse; however, until we determine what these interventions should be, their clinical utility remains theoretical. eventually, the majority of dogs that achieve a remission will relapse or experience recrudescence of lymphoma. this usually represents the emergence of tumor clones or tumor stem cells 222 (see chapter 2) that are inherently more resistant to chemotherapy than the original tumor, the so-called mdr clones that either were initially drug resistant or became so following exposure to selected chemotherapy agents. evidence suggests that in dogs with recurrent lymphoma, tumor cells are more likely to express the mdr1 gene that encodes the protein transmembrane drug pump often associated with mdr. 156a,156b,225 mdr1 represents only one of the plethora of mechanisms that lead to drug-resistant disease (see chapter 11) . other causes for relapse following chemotherapy include inadequate dosing and frequency of administration of chemotherapy, failure to achieve high concentrations of chemotherapeutic drugs in certain sites such as the cns, and initial treatment with prednisone alone. at the first recurrence of lymphoma, it is recommended that reinduction be attempted first by reintroducing the induction protocol that was initially successful, provided the recurrence occurred temporally far enough from the conclusion of the initial protocol (e.g., ≥2 months) to make reinduction likely. attention must be given to the cumulative dose of doxorubicin that will result from reinduction, and baseline cardiac assessment, the use of cardioprotectants, alternative drug choices, and client education should all be considered. in general, the length of the reinduction will be half that encountered in the initial therapy; however, a subset of animals will enjoy long-term reinductions, especially if the dog completed the initial induction treatment protocol and was currently not receiving chemotherapy for several months when relapse occurred. nearly 80% to 90% reinduction rates can be expected in dogs that have completed chop-based protocols and then relapse while not receiving therapy. 185, 226 the duration of a second chop-based remission in one report was predicted by the duration of the interval between protocols and the duration of the first remission. 226 if reinduction fails or the dog does not respond to the initial induction, the use of so-called "rescue" agents or "rescue" protocols may be attempted. these are single drugs or drug combinations that are typically not found in standard chop protocols and are depending on adverse event levels observed, particularly neutrophil counts at nadir. with some exceptions, multicentric t-cell lymphoma, when compared with multicentric b-cell lymphoma, is associated with similar initial response rates, but significantly lower response durability (e.g., pfs) following chemotherapy (including chop-based protocols).* additionally, the effectiveness of a single treatment of doxorubicin in the treatment of naïve dogs with lymphoma in one retrospective case series suggested a lower initial response rate for t-cell, compared with b-cell, immunophenotypes. 213 this has led many to question whether dogs diagnosed with t-cell lymphoma should be treated with standard chop-based protocols or with alternative protocols. this is a valid question; however, the answer remains elusive because adequately powered randomized controlled trials do not currently exist in the literature to show superiority for an alternate protocol in this scenario. a retrospective study of an l-asparaginase and mopp (m, mechlorethamine; o, oncovin; p, procarbazine; p, prednisone) protocol suggested improvement in pfs in dogs with either confirmed t-cell lymphoma or lymphoma with hypercalcemia and no immunophenotypic classification. 214 however, differences in determining pfs, response evaluation, and study population in this retrospective study did not definitively confirm superiority. 211 further, some have advocated early inclusion of lomustine (ccnu) into protocols for treating multicentric t-cell lymphoma based on moderate success of lomustine-based rescue protocols in dogs failing chop. as yet, no randomized trials have documented superiority with this approach. ultimately, superior protocol development for t-cell lymphoma awaits careful, randomized, prospective trial assessment. until such time, the author prefers to initiate chop-based induction and switch to lomustinebased rescue at the first sign of progression. vcog has recently published response evaluation criteria (v1.0) 169 to standardize reporting of outcome results and comparisons among protocols for peripheral nodal disease. the most important of these outcome measures and the preferred temporal outcome criterion for assessing protocol activity is now considered to be pfs, which is defined as being from the time of treatment initiation until tumor progression or death from any cause. this brings veterinary outcome reporting more in line with human standards. because the majority of dogs with lymphoma eventually experience recurrence following chemotherapy-induced remissions and because methodology for differentiating complete and partial responses is analysis dependent, pfs removes many sources of bias. further, overall survival in published reports invariably includes patients who go on to receive varied rescue protocols that bias the overall result, making it a less comparable outcome. widespread application of these standardized criteria should allow more suitable comparisons in the future. superior methods of detection of minimal residual disease (mrd) or early recurrence have been investigated in dogs with lymphoma and include advanced imaging and detection of despite the plethora of published chemotherapeutic protocols for dogs with lymphoma, it appears we have achieved as much as we can from currently available chemotherapeutics in standard settings. the 12-month median survival "wall" and the 20% to 25% 2-year survival rates have not improved dramatically. further advances in remission and survival durations await the development of new methods of delivering or targeting traditional chemotherapeutic drugs, new generations of chemotherapeutic drugs, or novel nonchemotherapeutic treatment modalities. mechanisms of avoiding or abrogating mdr, enhancing tumor apoptosis (programmed cell death), tumor ablation, and immune-system reconstitution, as well as novel immunomodulatory therapies for lymphoma, are all active areas of investigation in both human and veterinary medicine. drug resistance can be inherent in cancer cells or develop following exposure to selected chemotherapeutic agents and often is associated with increased expression of members of the adenosine triphosphate (atp)-binding cassette (abc) transporter superfamily (e.g., p-glycoprotein pump), many of which efflux various withheld for use in the drug-resistant setting. the most common rescue protocols used in dogs include single-agent use or a combination of actinomycin d, mitoxantrone, doxorubicin (if doxorubicin was not part of the original induction protocol), dacarbazine (dtic), temozolomide, lomustine (ccnu), l-asparaginase, mechlorethamine, vincristine, vinblastine, procarbazine, prednisone, and etoposide. some rescue protocols are easy and convenient single-agent treatments, whereas others are more complicated (and expensive) multiagent protocols, such as mopp. overall rescue response rates of 40% to 90% are reported; however, responses are usually not durable, with median responses of 1.5 to 2.5 months being typical, regardless of the complexity of the protocol. a small (<20%) subset of animals will enjoy longer rescue durations. table 32 -4 provides a summary of canine rescue protocols and published results. 227-238 current published data from rescue protocols do not include sufficient numbers for adequate statistical power nor do they compare protocols in a randomized prospective fashion. therefore evaluations of efficacy among various protocols are subject to substantial bias, making direct comparisons difficult and indeed precarious. choice of a particular rescue protocol should depend on several factors, including cost, time commitment required, efficacy, toxicity, and experience of the clinician with the protocols in question. as the complexity of rescue protocols does not yet appear to be associated with significant gains in rescue durability, the author tends to choose simpler and less costly protocols (e.g., ccnu/l-asparaginase/prednisone) ( table 32-5) . however, the use of multiple varied rescue protocols, switching as needed based on response, continues as long as clients are • nr, not reported. *few of these protocols include sufficient numbers for adequate statistical power and fewer compare treatment protocols in a randomized prospective fashion. in addition, staging, inclusion, and response criteria vary considerably between protocols presented. therefore evaluations of efficacy between the various protocols are subject to bias, making direct comparisons difficult and indeed precarious. † various temporal response endpoints were used, including disease-free interval, time to progression, and progression-free survival. ‡ prednisone often used concurrently. performed. 243,244 although efficacy was established, enhancement of remission or survival durations over equivalent doses of native doxorubicin was not observed. in the past decade, enhanced durability of first remissions in humans with non-hodgkin's b-cell lymphoma has been achieved primarily through the institution of monoclonal antibody (mab)based therapies (so-called r-chop protocols); the "r" refers to rituximab, a recombinant chimeric murine/human antibody directed against the cd20 antigen, a hydrophobic transmembrane protein located on normal pre-b and mature b lymphocytes. following binding, rituximab triggers a host cytotoxic immune response against cd20-positive cells. unfortunately, rituximab does not have therapeutic activity in dogs due to a lack of external recognition of a similar antigen on canine lymphoma cells and the inherent antigenicity of human-derived antibodies in dogs. 245, 246 another immunotherapy approach involved mab-231, a murinederived anticanine mab (igg2a). it mediates antibody-dependent cellular cytotoxicity (addc) and complement-mediated cellular cytotoxicity (cmcc). 247-249 it also prevented outgrowth of canine lymphoma xenografts in nude mice. in a noncontrolled clinical study of 215 dogs treated with chop-based chemotherapy and mab-231, enhanced overall survival was observed; however, the antibody was removed from the commercial market in the mid-1990s without definitive randomized trials being performed. several laboratories throughout the world are currently working to characterize and develop effective mab therapies for use in dogs. several antitumor vaccine approaches have been applied in dogs with lymphoma. a tumor vaccine extract using killed lymphoma cells combined with freund's adjuvant was administered to a small number of dogs after remission induction with combination chemotherapy. 250 prolongation of median survival was noted in the treatment group; however, a subsequent study revealed that prolongation was likely due to the freund's adjuvant. 251 an autologous killed lymphoma tumor cell vaccine has been intralymphatically administered to dogs placed in remission using a combination chemotherapy protocol, and, although modest gains were reported in remission times, no survival advantage was found. 252 an exploratory vaccine study targeting telomerase 253 (see chapter 14, section d) and one using rna-loaded cd40-activated b cells 254 in dogs with lymphoma have also been conducted. these studies involved small numbers of nonrandomized patients and lacked controlled populations for comparison. in a randomized study of 60 dogs with lymphoma comparing chop-based chemotherapy with chopbased chemotherapy and a human granulocyte-macrophage colony-stimulating factor (gm-csf) dna cationic-lipid complexed autologous whole tumor cell vaccine, a small measure of immunomodulation was documented by delayed-type hypersensitivity; however, significant improvement in clinical outcome was not noted. 255 although little well-supported activity is reported to date with these immunomodulatory approaches, our basic understanding of methodologies is expanding. most dogs with lymphoma have multicentric disease and therefore require systemic chemotherapy to effectively treat their disease. chemotherapeutic compounds from cells (see chapter 11). 239 p-glycoprotein is under the control of the mdr1 gene. mdr has been reported in canine lymphoma following exposure to chemotherapy. 156a,225,240 expression levels of mrna encoding the canine mdr1 gene have been characterized in canine cell lines and lymphomas. although expression of mdr1 mrna correlated with in vitro drug sensitivity, it did not correlate with in vivo doxorubicin sensitivity in dogs with lymphoma in this study. additionally, quantitative analysis of mrna for 10 different drug-resistance factors was performed in 23 dogs with lymphoma. 241 these dogs were divided into drug "sensitive" and "resistant" categories based on response to a chop-based protocol; however, significant differences in expression were not observed in this small study. methods of increasing the time that tumor cells are exposed to chemotherapeutics should theoretically enhance tumor killing. these methods could include long-term continuous infusions (impractical in many veterinary situations), increasing the frequency of treatments, or enhancing the circulation time of drugs used. in one study, dogs with lymphoma received lower dose doxorubicin weekly rather than a higher dose every 3 weeks (thereby decreasing c max , which is associated with cardiotoxicity) in order to potentially increase the time of drug exposure. 242 no benefit was noted, and, in fact, remission rates were inferior. studies evaluating pegylated long-circulating doxorubicin-containing liposome drug delivery systems in dogs with lymphoma have also been • 3. increase in alt activity >2× upper limit of normal (or 2× baseline if higher than upper limit of normal at initiation)-institute drug discontinuation and reinstitution/dose reduction depending on normalization of alt. practice. 266,267 because of the high cost, limited accessibility to relatively sophisticated equipment, and management requirements, these types of procedures are limited to preliminary investigations at a few centers. currently, long-term results in significant numbers of treated cases have yet to be presented. in general, the veterinary literature suffers from a paucity of information on treating various extranodal forms of lymphoma in dogs, and our ability to predict outcome is thus limited. in general, it is recommended that, following extensive staging, in those cases where disease is shown to be localized to a solitary site, local therapies (e.g., surgery, local rt) can be used. in contrast, if multiple extranodal sites are involved or they are part of a more generalized process, systemic chemotherapy should be chosen. most dogs with alimentary lymphoma are presented with diffuse involvement of the intestinal tract, and involvement of local lymph nodes and liver is common. chemotherapy in dogs with diffuse disease has been reported to be unrewarding for the most part 47,268,269 ; however, more aggressive chop-based protocols used extensively for multicentric lymphoma in dogs have resulted in durable remissions in a small subset of cases. solitary alimentary lymphoma is rare in the dog; however, if the tumor is localized and can be surgically removed, results (with or without follow-up chemotherapy) can be encouraging. cns lymphoma in dogs usually results from extension of multicentric lymphoma. however, primary cns lymphoma (pcnsl) has been reported. [103] [104] [105] if tumors are localized, local rt should be considered. few studies have reported the use of chemotherapy. in one study, cytosine arabinoside (ara-c) at a dosage of 20 mg/m 2 was given intrathecally; this treatment was combined with systemic chemotherapy and cns radiation. 103 overall, the response rates are low and of short duration (several weeks to months). treatment of cutaneous lymphoma depends on the extent of disease. solitary lesions may be treated with surgical excision or rt. fractionated rt (to a total dose of 30 to 45 gy) has been associated with long-term control. 270 diffuse cutaneous lymphoma is best managed with combination chemotherapy, although the rate and durability of response is generally less than in multicentric lymphoma. the most widely used protocols for epitheliotropic cutaneous t-cell lymphoma include ccnu (60 to 70 mg/m 2 po, every 3 weeks) along with prednisone. 271,272 although response rates approach 80%, median remission is approximately 3 months; occasionally, durable remissions are encountered. the author has added l-asparaginase to this protocol (see table 32 -5), and although anecdotally it appears to improve response, comparative data are not available. sporadic reports of other therapies for cutaneous lymphoma in small numbers of cases include the use of coap (cyclophosphamide, vincristine [oncovin], ara-c, and prednisone), 273 retinoic acid analogs (e.g., accutane, etretinate), 274 l-asparaginase and pegylated l-asparaginase, 275 topical mechlorethamine (mustargen), 276 and recombinant human α-interferon. 277 all of these reports involved small numbers of cases and resulted in limited response rates with short durations. a form of cutaneous lymphocytic infiltration has recently been characterized as an indolent t-cell lymphoma based on clonality. 131 however, surgery has been used to treat solitary lymphoma (early stage i) or solitary extranodal disease. careful staging is necessary in such cases to rule out multicentric involvement prior to treating local disease. the benefit of surgical removal of the spleen in dogs with massive splenomegaly remains unclear. 82, 83, 256 in an older report, 16 dogs with lymphoma underwent splenectomy to remove a massively enlarged spleen and were subsequently treated with chemotherapy. 256 within 6 weeks of splenectomy, 5 of the 16 dogs died of disseminated intravascular coagulation (dic) and sepsis. the remaining 11 dogs (66%) had a cr, and 7 dogs had a mst of 14 months. no staging or histologic information was provided, so the information appears of limited usefulness, although those with follow-up lived approximately 1 year. in two reports of indolent nodular lymphoma of the spleen (marginal zone lymphoma [mzl] and mantle cell lymphoma [mcl]), outcome was available on seven mzl cases, including three cases that did not receive adjuvant chemotherapy after surgery, 82, 83 and only one died of lymphoma following splenectomy. in a recent report of indolent lymphomas, four splenic lymphomas (three mzl and one mcl) underwent splenectomy alone and all survived greater than 1 year with none dying of their primary disease. 84 splenectomy should be considered if the lymphoma is not documented in other sites following thorough staging, if lymphoma is an indolent form histologically, or if splenic rupture has occurred. of note, no control population consisting of dogs that did not undergo splenectomy exists, so the natural history of indolent splenic lymphoma remains uncertain. radiation therapy, although its use is limited in the treatment of lymphoma, may be indicated in selected cases. 257 indications are as follows: 1. curative intent therapy for stage i lymph node and solitary extranodal disease (i.e., nasal, cutaneous, spinal lymphoma). 2. palliation for local disease (e.g., mandibular lymphadenopathy, rectal lymphoma, mediastinal lymphoma where precaval syndrome is present, localized bone involvement). 3. total body radiation combined with bone marrow or stem cell transplantation. 4. whole or staged half-body rt following chemotherapyinduced remissions. in the latter case, staged half-body irradiation sandwiched between chemotherapy cycles or following the attainment of remission by induction chemotherapy has been preliminarily investigated as a form of consolidation or maintenance. 198,258-262 radiation therapy was delivered to either the cranial or the caudal half of the dog's body in 4 to 8 gy fractions, and following a 2-or 4-week rest the other half of the body was irradiated in a similar fashion. although these preliminary investigations were not randomized, they suggest that rt applied when dogs are in either complete or partial remission is safe and warrants further investigation to determine if a significant therapeutic gain can be realized. a pilot study of low-dose (1 gy) single-fraction total body irradiation in seven dogs with relapsed drug-resistant lymphoma, although safely applied, resulted in only partial nondurable (1 to 4 week) remissions. 263 total body irradiation (and/or ablative chemotherapy) for complete or partial bone marrow ablation followed by reconstitution with bone marrow or stem-cell transplant in dogs, although a recognized model in comparative research settings, 264,265 is still in its early phases of development and application in clinical veterinary it is associated with slow progression and long-term survival following corticosteroid management; however, it does have the potential to progress to high-grade lymphoma. the prognosis for dogs with lymphoma is highly variable and depends on a wide variety of factors documented or presumed to affect response to therapy. although rarely curable (<10% of cases), crs and a good quality of life during extended remissions and survival are typical. factors that have been shown to influence treatment response and survival are summarized in tables 32-6* and 32-7. † the two prognostic factors most consistently identified are immunophenotype and who substage (see figure 32 -9). many reports have confirmed that dogs with cd3-immunoreactive tumors (i.e., t-cell derivation) are associated with significantly shorter remission and survival durations. ‡ this holds true primarily for dogs with multicentric lymphoma because the immunophenotype of solitary or extranodal forms of lymphoma has not been thoroughly investigated with respect to prognosis. additionally, it has been shown that dogs with b-cell lymphomas that express lower than normal levels of b5 antigen (expressed in 95% of nonneoplastic lymphocytes) also experience shorter remission and survival durations. 54 recently, low levels of class ii mhc expression on b-cell lymphoma predicted poor outcomes. 279 dogs presented with who substage b disease (i.e., clinically ill) also do poorly when compared with dogs with substage a disease. 53, 86, 91, 181, 278 dogs with stage i and ii disease have a better prognosis than those dogs in more advanced stages (stage iii, iv, and v). histologic grade (subtype) has been found to influence prognosis in some studies; however, our ability to predict outcome based on subtype is still quite limited. dogs with lymphoma classified as intermediate or high grade (large cell, centroblastic, and immunoblastic) tend to respond to chemotherapy but can relapse early. dogs with low-grade lymphomas (small lymphocytic or centrocytic) have a poorer response rate to chemotherapy, yet have a survival advantage over dogs with intermediate-and high-grade lymphomas ( figure 32 -10) in that the disease may be more indolent. several case compilations have documented that dogs with indolent lymphoma (e.g., mzl, mcl, t-zone) experience prolonged survivals, often in the absence of any or aggressive chemotherapy. [82] [83] [84] proliferative assays such as analysis of bromodeoxyuridine (brdu) uptake, ki67 antibody reactivity, and argyrophilic nucleolar organizer region (agnor) indices to measure proliferative activity of tumor cells have been shown to provide prognostic information in dogs treated with combination chemotherapy. results of different studies are contradictory, however. in two trials, dogs having tumors with short doubling times, high agnor frequencies, or high ki67 immunoreactivity had a better prognosis than those with tumors with long doubling times or low agnor frequencies. 53, 142 in other trials, the low-proliferating tumor groups were associated with a better prognosis. 283,284 additionally, in one trial, the proportion of tumor cells undergoing apoptosis was modestly predictive of remission duration. 142 the anatomic site of disease is also of considerable prognostic importance. primary diffuse cutaneous, diffuse gi, hepatosplenic, and primary cns lymphomas tend to be associated with a poor ‡ rights were not granted to include this figure in electronic media. please refer to the printed publication. prognosis. dogs with indolent cutaneous t-cell lymphocytic infiltration experience long-term survivals. 131 sex has been shown to influence prognosis in some studies. 175, 181 neutered females tend to have a better prognosis; male dogs may have a higher incidence of the t-cell phenotype, which may account for the poorer prognosis. reported biomarkers of prognosis, summarized in table 32 -7, include circulating levels of glutathione-s-transferase, thymidine kinase, lactate dehydrogenase, serum c-reactive proteins, and vegf. finally, one report suggests that a history of chronic inflammatory disease of several types predicts likelihood of early relapse. 291 these putative prognostic indicators require further confirmation in larger trials. lymphocytic leukemia is typically defined as proliferation of neoplastic lymphocytes in bone marrow. neoplastic cells usually originate in the bone marrow, but occasionally in the spleen, and may or may not be circulating in the peripheral blood. although our ability to diagnose lymphocytic leukemias using flow cytometric and molecular diagnostic techniques has increased significantly in the past decade, little information on treatment and prognosis is available except for chronic lymphocytic leukemia (cll). differentiating between true leukemia and stage v lymphoma can be difficult and arbitrary and is often based on lack of significant lymphadenopathy, degree of blood and bone marrow involvement, and immunophenotypic characteristics. lymphocytic leukemia is more common than acute myeloid leukemia and myeloproliferative disorders (mpd), but the true incidence is unknown. german shepherd dogs and golden retrievers may be overrepresented. 137,292 lymphocytic leukemia can occur in dogs of any age but typically occurs in middle-aged to older dogs (mean of 7 to 10 years); cll usually occurs in older dogs (mean of 10 years). 137,280,292,293 a significant sex predilection is not reported. as with lymphoma, the etiology of lymphocytic leukemia is for the • acute (large cells with an immature cytologic phenotype). immunophenotypic assessment using flow cytometric and molecular assays can further characterize these two major subtypes; however, some discordance exists in the veterinary literature. three primary subtypes of cll are reported in dogs, based primarily on immunophenotyping 137,280,293 : (1) t-cll, which is the most common form, with cells in the majority of cases being cd8 + granular lymphocytes; (2) b-cll, which is the next most common subtype; and (3) atypical cll, which represents a combination of immunophenotypes (cd3 − , cd8 + ; cd3 + , cd4 − , cd8 − ; cd3 + , cd4 + , cd8 + ; and cd3 + + cd21 + *). this is in contrast to cll in humans, which is primarily a disease of b-cells. in cll, lymphocytes often are indistinguishable morphologically from normal small lymphocytes ( figure 32 most part unknown. genetic factors likely play a role and have been compared between dogs and humans. 16 retroviruses have been implicated in diverse animal species such as cats, cattle, fish, snakes, birds, rodents, nonhuman primates, and humans; however, there is no proven evidence implicating a retroviral cause in dogs. in humans, acute lymphocytic leukemia (all) has been associated with genetic factors and exposure to radiation, benzene, phenylbutazone, and antineoplastic agents. extrapolation of predisposing factors across species is not warranted; in fact, etiologic factors in dogs may be quite different from those for humans given the difference in the predominant immunophenotype of the neoplastic cells (see later). lymphocytic leukemias can be subdivided based on cell size, maturity, genetic aberrations, microrna expression, and immunophenotype.* the simplest classification divides leukemia into two groups: chronic (small cells with a mature cytologic phenotype) and *note that either cd21 or cd79 can be used for assessing b-cell lineage in this context. *references 16, 137, 150, 280, 292-294. chromatin from disintegrated cells also is visible. (wright's stain, ×60 objective.) b, peripheral blood from a dog with chronic lymphocytic leukemia (cll). note the small lymphocytes of normal morphology (smaller than the neutrophil). (wright's stain, ×60 objective.) constitute the remaining fraction. in the t-cell fraction, helper t-cells (cd4 + ) outnumber cytotoxic t-cells (cd8 + ). 296 lymphocytic leukemia should be a consideration if atypical lymphocytes are in circulation, the immunophenotype of the lymphocytes in circulation is homogenous as determined by flow cytometric analysis, a phenotype typically present in low frequency has increased, or if clonality is documented (e.g., by parr analysis). other differential diagnoses for lymphocytosis include infectious diseases, such as chronic ehrlichiosis, postvaccinal responses in young dogs, il-2 administration, and transient physiologic or epinephrine-induced lymphocytosis. in some cases, reactive and neoplastic lymphocytosis are difficult to distinguish. expansion of neoplastic lymphocytes in bone marrow is the hallmark of all and, in most cases, cll. careful examination of peripheral blood and bone marrow by an experienced cytopathologist is important in establishing a diagnosis of lymphoid leukemia; in cases of marked lymphocytosis with atypia, peripheral blood can be used for analysis of immunophenotype and clonality, and examination of bone marrow is not essential. if diagnostic bone marrow cannot be adequately obtained by aspiration, bone marrow core biopsy should be performed. in all, lymphoblasts predominate in the bone marrow and are also present in peripheral blood, and other lineages are decreased. in b-and t-cell cll, the lymphocytes are small mature cells that occur in excessive numbers in bone marrow (≥30% of all nucleated cells) early in the disease. 295 in t-cll, lymphocytes may contain pink granules. infiltration becomes more extensive as the disease slowly progresses, and eventually the neoplastic cells replace normal marrow. a separate clinical staging system has not been developed for lymphoid leukemia. currently, all dogs with leukemia are classified as stage v based on the who staging system for lymphoma as presented in table 32 -2. because of the indolent and often asymptomatic nature of cll, the decision to treat is often based on the clinical and laboratory findings in the individual dog. most oncologists recommend active surveillance (monthly or bimonthly physical examination and cbc) over active therapy for patients when cll is identified incidentally, there are no accompanying clinical signs, and other significant hematologic abnormalities are not identified. if the animal is significantly anemic or thrombocytopenic, is showing evidence of significant lymphadenopathy or hepatosplenomegaly, or has an excessively high lymphocyte count (e.g., >60,000/µl), therapy should be instituted. the definition of "excessively high" varies among oncologists, and a standard has not been established in veterinary medicine. the author (dmv) prefers to base treatment decisions on the presence of significant constitutional signs and peripheral cytopenias. currently, the most effective drug available for treatment of cll is chlorambucil. 295 chlorambucil is given orally at a dose of 0.2 mg/kg or 6 mg/m 2 po once daily for 7 to 14 days; the dose can then be reduced to 0.1 mg/kg or 3 mg/m 2 po daily. for long-term maintenance, a dose of 2.0 mg/m 2 every other day can be used. the dose is adjusted based on clinical response and bone marrow tolerance. oral prednisone is used concurrently with chlorambucil at doses of 1 mg/kg daily for 1 to 2 weeks, then 0.5 mg/kg every other day thereafter. the addition of vincristine or the substitution of cyclophosphamide for chlorambucil has been advocated in animals that do not respond to chlorambucil. origin (cd3 + ,cd4 − , cd8 − , cd21 − ). 137 in general, these cells tend to be intermediate-sized or large cells with moderate amounts of basophilic cytoplasm. perhaps the most distinguishing feature of lymphoblasts is the nuclear chromatin pattern, which typically is more condensed than the chromatin in myeloblasts. lymphoblasts are larger than neutrophils, have a high nuclear : cytoplasmic ratio, and contain blue cytoplasm that in some cases is intensely basophilic (see figure 32 -11). nucleoli, although present, are less prominent in lymphoblasts than in myeloblasts. nevertheless, these cells cannot be distinguished easily from blast cells of other hematopoietic lineages, and identification of lineage-specific markers by immunocytochemical or flow cytometric analysis is required to ascertain the lineage. if the cells express cd34, a stem cell marker, an acute phenotype is implied 137,280 ; however, both myeloid and lymphoid lineages express cd34, and our ability to differentiate all from acute myeloid leukemia (aml) relies on detection of other markers, including t-and b-cell markers and myeloperoxidase, a myeloid marker. dogs with cll are often asymptomatic, but some owners report lethargy and decreased appetite. mild lymphadenopathy and splenomegaly may be present, although late in the disease splenomegaly may be marked. 295 the white blood cell (wbc) count is usually greater than 30,000 cells/µl but can vary from normal to greater than 100,000 cells/µl because of an increase in circulating mature lymphocytes. lymphocytosis is persistent and granulocytes are usually present in normal numbers. other than lymphocytosis, hemograms of dogs with cll tend to have few abnormalities when lymphocytes are less than 30,000/µl. 137, 280, 293 in some dogs, the disease is identified incidentally when the animal is undergoing evaluation for an unrelated problem. mild anemia, neutropenia, and thrombocytopenia are common but may become marked as the disease progresses and lymphocyte counts increase above 30,000/µl. despite the well-differentiated appearance of the lymphocytes in cll, these cells may function abnormally. paraneoplastic syndromes include monoclonal gammopathies, immunemediated hemolytic anemia, pure red cell aplasia, and, rarely, hypercalcemia. 296,297 in one report of 22 dogs with cll, 68% had monoclonal gammopathies (usually igm or iga). 296 the immunophenotypes were not reported, but a monoclonal gammopathy would be more likely to occur in b-cll. dogs with all usually are presented with clinical signs of anorexia, weight loss, and lethargy. splenomegaly is typical and other physical abnormalities may include hemorrhage, lymphadenopathy, and hepatomegaly. 298 infiltration of bone marrow by neoplastic lymphoblasts may be extensive, resulting in significant depression of normal hematopoietic elements or myelophthisis. 137,280,292,298,299 anemia, neutropenia, and thrombocytopenia are typically much more severe than with cll and may become life threatening. infiltration of extramedullary sites such as the cns, bone, and gi tract may also occur and can result in neuropathies, bone pain, and gi signs, respectively. consideration of signalment, history, physical findings, and morphologic appearance and immunophenotype of cells is essential in making an accurate diagnosis. it is helpful to know the profile of lymphocyte subsets in the peripheral blood of normal dogs to determine if a particular subset has expanded. approximately 80% of circulating lymphocytes in normal dogs are t-cells, and about 15% are b-cells. nk cells and double-negative (cd4 − , cd8 − ) t-cells animal, nat cancer inst treatment of cll is primarily palliative with rare complete remissions. owing to the indolent nature of this disease, however, survival times have been in the range of 1 to 3 years with a good quality of life. 295,300 the phenotypic expression of cll is usually stable over months to years. however, the disease may evolve into an acute phase, and some dogs will develop a form of lymphoma that is rapidly progressive and characterized by the presence of pleomorphic immunoblasts; in humans, this is termed richter's syndrome. 301 the prognosis for response to treatment is poor for this form of lymphoma. much of the morbidity in dogs with all results from effacement of bone marrow (myelophthisis) and subsequent life-threatening peripheral cytopenias. neutropenia, thrombocytopenia, and anemia may be severe. patients often require supportive therapy, such as fresh whole-blood transfusions, broad-spectrum antibiotics, fluid therapy, and nutritional support. careful monitoring for sepsis, hemorrhage, and dic is important. specific treatment of all requires aggressive chemotherapy. consistently efficacious protocols for all have not been developed in veterinary medicine, and there are few published reports. chop-based protocols, similar to those used for lymphoma (see table 32 -4), have been used by the author (dmv) for dogs with all; however, responses and durability of response are generally disappointing. the standard of care in humans with acute leukemia generally involves bone marrow ablative treatments with stem cell or marrow replacement, a protocol not generally available in veterinary oncology. in general, cll is a slowly progressive disease, and some animals will not require therapy for some time after diagnosis; one dog was reported to survive almost 2 years without treatment. 302 for those dogs that are treated, normalization of lymphocyte counts can be expected in 70% of cases. in one report of 17 dogs treated with vincristine, chlorambucil, and prednisone, mst was approximately 12 months with an expected 30% survival at 2 years. 295 in larger compilations of cases that include immunophenotypic analysis, treatment protocols were poorly documented, although most received chlorambucil and prednisone; in 43 dogs with follow-up, for dogs with t-cll, b-cll, and atypical cll, median survival was 930, 480, and only 22 days, respectively. 293 in this group of dogs, young age and anemia were also associated with a poor prognosis. in another series with limited treatment information, dogs with cll of a cd8 + immunophenotype that presented with less than 30,000 lymphocytes/µl or greater than 30,000 lymphocytes/µl had median survivals of 1098 and 131 days, respectively. prognosis for dogs with all is generally very poor. in a study of 21 dogs treated with vincristine and prednisone, the dogs achieving complete or partial remission (29%) had a mst of 120 days, and few dogs survived longer than 8 months with that protocol. 298 in one report of 46 cases of all with a cd34 + phenotype, dogs had a median survival of 16 days (ranged from 3 to 128 days), even though the majority received a chop-based treatment protocol. 280 additionally, dogs with b-cell all (cd21 + ) in which the lymphocytes were large cells (forward scatter lymphocyte/forward scatter neutrophil ratio of >0. 58 the lymphomas (malignant lymphoma or lymphosarcoma) are a diverse group of neoplasms that have in common their origin from lymphoreticular cells. they usually arise in lymphoid tissues such as lymph nodes, spleen, and bone marrow; however, they may arise in almost any tissue in the body. lymphoma is one of the most common neoplasms seen in the cat. epidemiologic reports prior to 1990 suggested that lymphoma accounted for 50% to 90% of all hematopoietic tumors in the cat, 1,2 and since hematopoietic tumors (lymphoid and myeloid) represent approximately one-third of all feline tumors, it was estimated lymphoid neoplasia accounted for an incidence of 200 per 100,000 cats at risk. 3 in one series of 400 cats with hematopoietic tumors, 61% had lymphoma and 39% had leukemias and mpds, of which 21% were categorized as undifferentiated leukemias, most likely myeloid in origin. 4 mediastinal form that is not felv associated and represents a younger population (median of 2 years). felv was the most common cause of hematopoietic tumors in the cat in the so-called "felv era" of the 1960s through the 1980s when approximately two-thirds of lymphoma cases were associated with felv antigenemia. several studies have documented the potential molecular means by which felv can result in lymphoid neoplasia (see chapter 1, section c). as one would predict, along with a shift away from felv-associated tumors came a shift away from traditional signalment and relative frequency of anatomic sites. this is also supported outside of north america by similar signalment and anatomic frequency data observed in australia where felv infection is quite rare. 10, 11 the median age of approximately 11 years now reported in north america is considerably higher than the median ages of 4 to 6 years reported in the felv era. 1,2,5-7 the median age of cats within various anatomic tumor groupings has not changed, and anatomic forms traditionally associated with felv such as the mediastinal form still occur in younger, felv antigenemic cats. similarly, the alimentary form occurs most often in older, felvnegative cats. table 32 -8 presents an overview of the characteristics, including felv antigenemic status, of the various anatomic sites of lymphoma in cats. as our ability to interrogate felv associations on a molecular basis has improved (e.g., pcr amplification and fluorescent in-situ hybridization), several reports exist defining the role or potential role of felv in cats with and without felv antigenemia. [12] [13] [14] [15] [16] [17] collectively, these studies indicate felv proviral insertion exists in a significant proportion of feline lymphoma tissues and is more common in those of t-cell origin, particularly the thymic and peripheral lymph node anatomic forms. they also suggest that several common felv integration sites exist. there is also evidence that feline immunodeficiency virus (fiv) infection can increase the incidence of lymphoma in cats. [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] in contrast to the direct role of felv in tumorigenesis, most evidence available felv vaccines appearing in the late 1980s (see the later section on viral etiology). the decline in felv-associated lymphoma was mirrored by a decline in the overall prevalence per year of felv positivity in cats tested as characterized by reports, including the tufts veterinary diagnostic laboratory from 1989 to 1997, 5, 6 and by the louwerens group, who reported a decline in felv association in over 500 cases of lymphoma in cats presenting to the university of california at davis veterinary teaching hospital. 7 in these reports, felv antigenicity declined to represent only 14% to 25% of cats presenting with lymphoma. importantly, louwerens' study revealed that despite a sharp drop in felv-associated lymphoma, the overall prevalence of lymphoma in cats is increasing. the increased prevalence appears due to an increase in the number and relative frequency of the alimentary (and in particular the intestinal) anatomic form of lymphoma in the species. this is supported by an epidemiologic survey of 619 cases of feline intestinal lymphoma; 534 (86%) were from the 20 years following 1985 and only 14% were from cases diagnosed in the 20 years prior to 1985. 8 the true annual incidence rate for lymphoma in cats is currently unknown. with respect to feline pediatric tumors, a study in the united kingdom (n = 233 pathology specimens) found that 73 (31%) represented hematopoietic tumors, of which 51 (70%) were lymphoma-note that felv status was unavailable for this compilation of cases. 9 the typical signalment for cats with lymphoma cannot be uniformly stated as it varies widely based on anatomic site and felv status and therefore will be discussed individually under sitespecific discussions. in general, based on two large compilations (n = 700) of cases in north america, 5, 7 siamese cats appear overrepresented and although a 1.5 : 1 male to female ratio was observed in one, no association with sex or neutering status was observed in the other. in a large compilation of australian cases, male cats and the siamese/oriental breeds were overrepresented, 10 and similar breed findings have been observed in north america, although similar sex predilections have not been found. within the siamese/ oriental breeds, there appears to be a predisposition for a • felv, feline leukemia virus; id, insufficient data; cns, central nervous system. common = >50% of clinical presentations; moderate = 20%-50% of clinical presentations; uncommon = 5%-20% of clinical presentation; rare = <5% of clinical presentations. *data may include overlap or mixing of sites and represents the post-felv era. † as the primary site of presentation, rather than extension or progression. ‡ includes those reported as "intraabdominal" in which intestinal is a documented component. support for this concept. 47 additionally, an association between gastric helicobacter infection and gastric malt lymphoma in cats is suggested in one study, and because this is a recognized syndrome in humans, it warrants further investigation. 47a although no direct evidence exists, a link between diet and the development of intestinal lymphoma in cats has been suggested. 7 support is offered by the relative and absolute increase in the alimentary form of lymphoma in the past 20 years and the fact that several dietary modifications in cat food have occurred in a similar timeframe in response to diseases, such as urinary tract disease. further investigation is warranted to prove or disprove such assertions. lymphoma can be classified based on anatomic location and histologic and immunophenotypic criteria; often, the two are intimately associated because certain histologic and immunophenotypic types are commonly associated with specific anatomic locations necessitating discussions within the individual anatomic categories that follow. the largest compilation of feline cases subjected to rigorous histologic classification was reported by valli and others 48 using the nci wf. who has also published a histologic classification system that uses the real system as a basis for defining histologic categories of hematopoietic tumors of domestic animals. 49 this system incorporates both histologic criteria and immunohistologic criteria (e.g., b-and t-cell immunophenotype). regarding anatomic location, as discussed previously, a profound change in presentation, signalment, felv antigenemia, immunophenotype, and frequency of anatomic sites has occurred in cats with lymphoma in the "post-felv" era (see table 32 -8). because of this shift, characteristics of feline lymphoma discussed in this chapter will be primarily limited to reports collected from cases presenting after 1995. several anatomic classifications exist for lymphoma in the cat, and some categorize the disease as mediastinal, alimentary, multicentric, nodal, leukemic, and individual extranodal forms. others have combined various nodal and extranodal forms into categories of atypical, unclassified, and mixed, and others have combined intestinal, splenic, hepatic, and mesenteric nodal forms into one category termed intraabdominal. some discrepancies in the discussion of frequency will inevitably result from the variations in classification used in the literature. the relative frequency of anatomic forms and their associated immunophenotype may also vary with geographic distribution and may be related to genetic and felv strain differences, as well as prevalence of felv vaccine use. alimentary/gi lymphoma can present as a purely intestinal infiltration or a combination of intestinal, mesenteric lymph nodes and liver involvement. the tumors can be solitary but more commonly diffuse throughout the intestines. some reports limit the alimentary form to gi involvement with or without extension to the liver. lymphoma is the most common tumor type found in the intestines of cats, representing 55% of cases in an epidemiologic survey of 1129 intestinal tumors in the species. 8 the siamese breed is reported at increased risk. 7, 8 while lymphoma may occur in cats of any age, it is primarily a disease of aged cats with a mean age of approximately 13 years for t-cell alimentary lymphoma and 12 years for b-cell lymphoma. 7, 8, [50] [51] [52] no consistent sex bias is noted. anatomically, alimentary lymphoma is nearly 4 times more likely to occur in the small intestine than the large intestine. 52 in a series of colonic points toward an indirect role for fiv secondary to the immunosuppressive effects of the virus. shelton 18 determined that fiv infection alone in cats was associated with a fivefold increased risk for development of lymphoma. coinfection with felv will further potentiate the development of lymphoproliferative disorders. experimentally, cats infected with fiv have developed lymphoma in the kidney, alimentary tract, liver, and multicentric sites. fivassociated lymphoma is more likely that of the b-cell immunophenotype rather than the t-cell predominance associated with felv. it has been suggested that fiv infection may be associated more commonly with alimentary lymphoma of b-cell origin, 28, 29 and this may be related to chronic dysregulation of the immune system or the activation of oncogenic pathways; however, fiv antigenemia was only rarely associated with alimentary lymphoma in other large compilations of cases. 5,30-33 as discussed earlier in section a, recent advances in molecular cytogenetics (see chapter 1, section a, and chapter 8), including gene microarray techniques, have and are currently being applied to investigations of chromosomal aberrations in veterinary species with lymphoma. indeed a predisposition of the oriental cat breeds to develop lymphoma suggests a genetic predisposition and indicates heritable risk. 7,10 altered oncogene/tumor suppressor gene expression, epigenetic changes, signal transduction, and cell deathpathway alterations are common in lymphomas of humans and are likely also involved in the cat. several genetic factors have already been discussed as they relate to felv associations. additionally, n-ras aberrations have been implicated, although they are rare in cats. 34 furthermore, telomerase activity (see chapter 2) has been documented in feline lymphoma tissues. 35, 36 alterations in cellular proliferation and in cell-cycle and death (apoptosis) pathways, in particular the cyclin-dependent kinase cell-cycle regulators and the bcl-2 family of proapoptotic and antiapoptotic governing molecules, have also been implicated in feline lymphoma. [37] [38] [39] evidence for exposure to environmental tobacco smoke (ets) as a risk factor for lymphoma in humans has prompted investigations in cats. in one report, the relative risk of developing lymphoma in cats with any exposure to ets and with 5 or more years of exposure to ets was 2.4 and 3.2, respectively. 40 a large european study documenting an association between proximity of waste management and cancer in dogs failed to show increased risk in cats. 41 immune system alterations in the cat such as those accompanying fiv infection has been implicated in the development of lymphoma. [18] [19] [20] 25 as is the case in immunosuppressed human organ transplantation patients, two reports of immunosuppressed feline renal transplant recipients document increased risk of lymphoma following transplant and associated immunosuppressive therapy. 42, 43 in both studies, nearly 10% of transplanted cats developed de novo malignant lymphoma. although definitive proof is lacking, there is a growing body of indirect evidence to suggest that lymphoma can be associated with the presence of chronic inflammation, which theoretically could be the case with intestinal and nasal lymphoma. in particular, an association has been suggested between intestinal lymphoma and inflammatory bowel disease 7,44-46 ; however, others have not found with malignant pleural effusion and a mediastinal mass present. peripheral blood involvement was present in 10% of cases in one report 59 and 86% in another. 60 affected cats are generally felv/fiv negative. the mediastinal form can involve the thymus, mediastinal, and sternal lymph nodes. pleural effusion is common. in two large compilations, 63% of cats with thymic disease and 17% of cats with pleural effusion were documented as having lymphoma. 62, 63 hypercalcemia occurs frequently with mediastinal lymphoma in dogs but is rare in cats. the majority of cats with mediastinal lymphoma are young (median age 2 to 4 years), felv positive, and the t-cell immunophenotype. 5, 7, [9] [10] [11] the disease is confined to the mediastinum in most cases. 7 there also exists a form of mediastinal lymphoma occurring primarily in young, felv-negative siamese cats that appears to be less biologically aggressive and more responsive to chemotherapy than felv-associated forms. 64 involvement limited to peripheral lymph nodes is unusual in cats with lymphoma, representing approximately 4% to 10% of cases. 5, 7 in contrast, approximately one-quarter of all other anatomic forms of lymphoma have some component of lymph node involvement. one-third of cats with nodal lymphoma are t-cell immunophenotype and felv antigenemic; however, complete categorizations have not occurred in the post-felv era and this may no longer be true. 5, 7, 11, 55 peripheral nodal lymphoma was the most common anatomic form of lymphoma reported in a recent compilation of cases in cats under the age of 1 year, representing a full third of cases in this age group. 9 as lymphoma progresses, bone marrow and hepatic infiltration may develop. an uncommon and distinct form of nodal lymphoma in cats referred to as "hodgkin's-like" lymphoma has been reported. 65, 66 this form typically involves solitary or regional nodes of the head and neck (figure 32-12) and histologically resembles hodgkin's lymphoma in humans. affected cats generally present with enlargement of one or two mandibular or cervical nodes initially, and tumors are immunophenotypically classified as t-cell-rich, b-cell lymphoma. one case each of inguinal node, multicentric nodal, and neoplasia in cats, lymphoma was the second most common malignancy (41%), second only to adenocarcinoma. 33 there is some discordance in the literature regarding the histologic type (primarily cell size: small versus large), immunophenotype, and architecture involved with gi lymphoma. while studies (often older or smaller reports) suggest a majority of b-cell immunophenotypes, 5,53 larger, more recent reports 51, 54, 55 indicate the majority represent mucosal low-grade t-cell immunophenotypes. conversely, the vast majority of b-cell gi lymphomas in cats are large cells and intermediate or high grade. 51, 53 the largest compilation to date (n = 120), by moore and others, 51 classified gi lymphomas based on immunophenotype, then as either mucosal (infiltrate confined to mucosa and lamina propria with minimal submucosal extension) or transmural (significant extension into submucosa and muscularis propria). they then compared infiltration patterns with the who classification scheme, 56 as well as documenting anatomic location, cell size, presence of epitheliotropism, clonality, and outcome data. this information is summarized in table 32-9. of the 120 cases, none tested serologically positive for felv and only 3 for fiv. four cats had concurrent large b-cell lymphoma (stomach, cecum, or colon) and small t-cell lymphoma of the small intestine. topographically, t-cell variants are much more likely to occur in the small intestine (94%) and rarely in the stomach or large intestine. conversely, b-cell variants were often multiple and often occurred simultaneously within the stomach, small intestine, and ileocecocolic junction. the vast majority of t-cell variants were mucosal (equivalent to who enteropathy-associated t-cell lymphoma [who eatcl] type ii), and the vast majority of b-cell tumors were transmural (equivalent to who eatcl type i classification). regarding cell size, nearly all mucosal t-cell tumors were composed of small lymphocytes, and slightly more than half of transmural t-cell and all b-cell variants were composed of larger cells. epitheliotropism is present in approximately 40% of t-cell tumors but is rare in b-cell tumors. other abdominal organ involvement is common, and in one report of 29 cases of low-grade t-cell intestinal lymphoma, liver and mesenteric involvement was documented in 53% and 33% of cases, respectively. 57 hepatic lymphoma can occur concurrently with gi lymphoma or be confined solely to the liver. 52, 58 most are t-cell and clonal or oligoclonal based on pcr analysis. a less common, distinct form of alimentary lymphoma, large granular lymphoma (lgl), also occurs in older (median age 9 to 10 years) cats. 51,53,59-61 these granulated round cell tumors have been termed globule leukocyte tumors, although they are likely variations of the same disease. lgl is characterized by lymphoblasts described as 12 to 20 µm in diameter with a round, clefted, or cerebriform nucleus; variably distinct nucleoli; finely granular to lacey chromatin; and a moderate amount of basophilic granular cytoplasm that was occasionally vacuolated. 59 prominent magenta or azurophilic granules are characteristic (see figure 7 -34, chapter 7). they are granzyme b positive by immunohistochemistry. 51 this population of cells includes cytotoxic t-cells and occasionally nk cell immunophenotypes-most are cd3 + , cd8 + , and cd20 − and have t-cell receptor gene rearrangement. 51, 60 in one report, nearly 60% expressed cd103 (integrin). 60 approximately 10% express neither b-or t-cell markers and are thus classified as nk cells. these nk tumors commonly originate in the small intestine, especially the jejunum, are transmural, often exhibit epitheliotropism, and at least two-thirds present with other organs involved-most with mesenteric lymph node involvement and many with liver, spleen, kidney, peritoneal malignant effusions, and bone marrow infiltration. also, thoracic involvement may occur • figure 32 -12 a cat presented with mandibular lymphadenopathy that was confirmed to be hodgkin's-like lymphoma following histologic assessment. • frequent sequela to renal lymphoma and occurs in 40% to 50% of treated cats. 74 cns lymphoma can be intracranial, spinal, or both. cns lymphoma made up 14% of 110 reported cases of extranodal lymphoma, 68 15% to 31% of intracranial tumors, 75, 76 and 39% of spinal cord tumors, 77 making it one of the most common malignancies encountered in the cns in cats. although some discordance exists in the literature, cats with cns lymphoma are younger (median ages of 4 to 10.5 years reported), and 17% to 50% of cases are felv antigenemic. [76] [77] [78] approximately two-thirds of intracranial cases are part of a multicentric, extracranial process, and approximately 40% of spinal lymphoma cases occur in multiple spinal cord sites with one-third also involving intracranial locations. [76] [77] [78] in a compilation of 160 cases of intracranial tumors in cats, diffuse cerebral and diffuse brainstem involvement was most common for lymphoid malignancies. 76 spinal lesions are usually both extradural and intradural, although they can be limited to one or the other compartment. 77 feline cns lymphoma may be primary but more commonly (approximately 80%) represents a multicentric process (especially renal or bone marrow). 76 ,78 a paucity of information exists on the immunophenotype of cns lymphoma. laryngeal lymphoma made up 10% of 110 cases of extranodal forms in one report and represented 11% of all laryngeal disease in the species. 68, 79 it occurs in older cats (median age 9 years), is not associated with felv, and may be a solitary lesion or occur in the presence of other multicentric sites. no information on immunophenotype is available. cutaneous lymphoma is a rarely encountered anatomic form in the cat. it is usually seen in older cats (median age 10 to 13.5 years), with no sex or breed predominance, and is not associated with felv/fiv. 80, 81 it can be solitary or generalized, often affecting the head and face and is generally a slow chronic disease. two forms of cutaneous lymphoma have been distinguished histologically and immunohistochemically. most reports in the cat are epitheliotropic and consist of t-cells, although unlike the disease in dogs, adnexal structures are often spared. a report of nonepitheliotropic cutaneous lymphoma in cats also found five of six cases to be of t-cell derivation. 82 cutaneous "lymphocytosis, " an uncommon disease histologically resembling well-differentiated lymphoma, was characterized in 23 cats. 83 solitary lesions were most common, and all were composed primarily of t-cells, with two-thirds having some b-cell aggregates. cutaneous lymphocytosis was characterized as a slowly progressive disorder; however, a few cases went on to develop internal organ infiltration. two case reports exist of cats with cutaneous t-cell lymphoma and circulating atypical lymphocytes. 84, 85 the circulating cells were lymphocytes with large, hyperchromatic, grooved nuclei, and one case was immunophenotyped as a cd3/ cd8 population. in humans, cutaneous t-cell lymphoma with circulating malignant cells is termed sézary syndrome. ocular lymphoma was identified in 5 of 110 cases of extranodal lymphoma in one report. 68 in a compilation of 75 cases of intraocular tumors, 15 (20%) were lymphoma (7 b-cell and 4 t-cell). 86 it was presumed but not proved that the majority were part of a systemic multicentric process. the clinical signs associated with feline lymphoma are variable and depend on anatomic location and extent of disease. the alimentary form is most commonly associated with nonspecific signs associated with the intestinal tract. in the more conjunctival involvement have been reported. 66, 67 histologically, lymph nodes can be effaced by either nodular or diffuse small to blastic lymphocytes with characteristic bizarre or multinucleated cells (reed-sternberg-like cells) (figure 32-13 ). no association with felv or fiv has been documented. the most common extranodal sites for lymphoma in cats include nasal (including nasopharyngeal and sinonasal), kidney, cns, laryngeal and tracheal, ocular, retrobulbar, and skin. nasal lymphoma is the most common extranodal lymphoma in cats. 68 it is usually a localized disease; however, 20% have local extension or distant metastasis at necropsy. 69 the majority of nonviral nasal/paranasal disease in cats are neoplasias, and lymphoma represents nearly one-third to half of these cases. [70] [71] [72] it occurs primarily in older (median age 9 to 10 years; range 3 to 17 years) felv/ fiv-negative cats and at least three-quarters are b-cell in origin, although t-cell and mixed b-cell/t-cell immunophenotypes can be seen in approximately 10% to 15% of cases. 5, 68, 69, 73 siamese cats appear overrepresented, and one report 73 observed a 2 : 1 male-tofemale ratio. most are of intermediate-or high-grade histology. 69, 73 epitheliotropism is common if the epithelium is present in the biopsy. renal lymphoma is the second most common form of extranodal lymphoma after the nasal form, occurring in approximately one-third of cases. 68 it can present as primary to kidney lymphoma or occur concurrent with alimentary lymphoma. in more contemporary reports, the median age at presentation is 9 years, although 6% occurred in cats under 1 year of age. 68, 69 the vast majority of cases are not associated with either felv or fiv. the greater median age and lack of felv/fiv association are in contrast to reports compiled prior to the post-felv era; in earlier studies, the median age was approximately 7.5 years, 25% of cases were felv antigenemic, and the majority constituted a b-cell immunophenotype. little contemporary information exists on the immunohistologic classification of renal lymphoma; however, in australia where felv is not a significant problem, most renal lymphoma is b-cell and intermediate to high grade. 11 extension to the cns is a hodgkin's-like nodal lymphoma usually present without overt clinical signs. 65, 66 cats with nasal lymphoma are typically presented with nasal discharge (60% to 85%), sneezing (20% to 70%), upper respiratory noise (stridor, stertor, wheezing; 20% to 60%), facial deformity (0% to 20%), anorexia (10% to 60%), epiphora (10% to 30%), and occasionally increased respiratory effort and coughing. 68, 69, 73 the nasal discharge is usually mucopurulent, although epistaxis is present in up to one-third of cases. regional lymphadenopathy can also occur. the median duration of clinical signs prior to diagnosis is 2 months (range of 1 to 1800 days). cats with renal lymphoma present with signs consistent with renal insufficiency: inappetence, weight loss, and polyuria/ polydipsia. 68, 74 on physical examination, renomegaly (usually bilateral, lumpy, and irregular) is palpated in the majority of cases (figure 32-14) . cats with cns lymphoma can present with constitutional signs (anorexia, lethargy) and signs referring to intracranial lesions, spinal lesions, or both. 68, [75] [76] [77] 91, 92 intracranial signs may include ataxia, altered consciousness, aggression, central blindness, and vestibular abnormalities. in a study of cats with seizures, of those diagnosed with intracranial lesions, 8% were due to lymphoma. 75 clinical signs referring to spinal cord involvement may include paresis or paraplegia (>80%; tetraparesis in 20%), ataxia, pain, and constipation, and nonspecific constitutional signs (e.g., anorexia, lethargy, weight loss) are also common. 77, 92 in cats with spinal cord involvement, neurologic examination may further reveal tetraparesis, lower or upper motor neuron bladder, tail flaccidity, and absent deep pain; approximately one-third of signs will be asymmetric and most refer to thoracolumbar involvement. the neurologic dysfunction may be insidious or progress rapidly. common low-grade small cell forms, weight loss (83% to 100%), vomiting and/or diarrhea (73% to 88%), and anorexia (66%) are the most common findings, and icterus is uncommon (7%). 50, 52, 87 abdominal palpation is abnormal in approximately 70% of cases, with half consisting of intestinal wall thickening and one-third having a palpable mass. clinical signs are usually present for several months (median: 6 months). 87 in contrast, although the lymphoblastic high-grade forms tend to cause similar clinical signs, they progress more rapidly with signs present for days or weeks and are more likely to present with a palpable abdominal mass originating from the gi tract, enlarged mesenteric lymph nodes, or liver. 31, 50, 88 icterus is also more common in large cell forms. hematochezia and tenesmus may also be present if the colon is involved. 33 rarely, cats may present with signs consistent with an acute abdomen due to intestinal obstruction or perforation and concurrent peritonitis. cats with intestinal lgl are presented with anorexia, weight loss, lethargy, and vomiting. 59, 60 a palpable abdominal mass is present in approximately half of lgl cases, and hepatomegaly, splenomegaly, and renomegaly are common. abdominal effusions, pleural effusions, and icterus are observed in less than 10% of cases. the clinical signs associated with the mediastinal form of lymphoma include dyspnea, tachypnea, and a noncompressible anterior mediastinum with dull heart and lung sounds. 89 rarely, a horner's syndrome and precaval syndrome may be observed. pleural effusion is common and characterized by serohemorrhagic to chylous effusion, and in most cases, neoplastic cells (lymphoblasts) are identified. 63, 90 cats with the nodal form of lymphoma present with variable clinical signs depending on the extent of disease; however, they are often depressed and lethargic. peripheral lymphadenopathy, as the only physical finding, is an uncommon presentation. cats with • figure 32-14 a , ventrodorsal projection of a cat with renal lymphoma. massive, bilateral renomegaly is observed. b, necropsy specimens of a cat with bilateral renal lymphoma illustrating the diffuse cortical nature of the disease that is most common. the reader is referred to chapter 8 for a general discussion of flow cytometric analysis and molecular diagnostic techniques, as well as the molecular diagnostic techniques section in section a of this chapter for specific applications to lymphoma. parr applications in cats have been described as being approximately 80% sensitive for the diagnosis of feline lymphoma 96 ; however, assessment of specificity has not been clearly established. clonality assessment tools (e.g., primers) for both ig and t-cell receptor variable region genes have been developed in cats. [97] [98] [99] [100] assessments of tumor proliferation rates (e.g., ki67, pcna, agnor), telomerase activity, and serum protein electrophoresis can also be performed on involved tissues in cats; however, consistent prognostic value across the anatomic, histopathologic, and immunophenotypic variants of lymphoma in cats is not well characterized. if these ancillary assays are helpful with respect to prognosis or diagnosis, they will be discussed in site-specific discussions to follow. thorough staging, including a bone marrow aspiration or biopsy, peripheral lymph node assessment (clinically normal or abnormal nodes), and thoracic and/or abdominal imaging, is indicated when (1) solitary site disease is suspected (in particular, extranodal sites) and a decision between locoregional therapy (i.e., surgery and/or rt) versus systemic therapy (i.e., chemotherapy) is being considered; (2) it provides prognostic information that will help a caregiver make treatment decisions; and (3) complete staging of the extent of disease is required as part of a clinical trial. bone marrow evaluation may be of particular interest if anemia, cellular atypia, and leukopenia are present. a who staging system exists for the cat that is similar to that used in the dog (see box 32-1); however, because of the high incidence of visceral/extranodal involvement in the feline species, a separate staging system has been evaluated and is often used (box 32-3). 101 because lymphoma in cats is more varied with respect to anatomic locations, staging systems are generally less helpful for predicting response. alimentary/gastrointestinal lymphoma the diagnosis of large cell, high-grade alimentary/gi lymphoma is generally less complicated than for the more common low-grade gi type. the former (including lgl) is often diagnosed with physical examination, abdominal imaging (e.g., ultrasound), and cytologic or histologic assessment of needle aspirate or needle biopsy samples from intestinal masses, enlarged mesenteric lymph nodes, or liver because mass lesions and gross lymphadenopathy are more commonly present. if obvious abdominal masses are present on physical examination, transabdominal needle aspiration may be possible without the aid of abdominal imaging. less commonly, abdominal exploration is necessary if lesions are more subtle or not amenable to transabdominal sampling. further staging via thoracic imaging, peripheral lymph node aspiration, and bone marrow assessment may be performed, but rarely contributes prognostic information or alters treatment decisions because the disease is already widespread and systemic therapy is required. in contrast, low-grade, small cell gi lymphoma is more commonly associated with modest (or palpably absent) intestinal thickening without mass effect and is clinically similar if not identical in presentation to benign inflammatory bowel disease (ibd). cytologic assessment alone is often not sufficient for diagnosis; in one study, eight of nine cases in which mesenteric lymph nodes were confirmed histologically as lymphoma, cytologic assessment incorrectly indicated benign lymphoid hyperplasia. 52 the key elements necessary for the diagnosis of low-grade, small cell gi lymphoma signs associated with laryngeal lymphoma in cats most commonly include dyspnea, dysphonia, stridor, gagging or retching, and rarely, coughing. 68, 79 cutaneous lymphoma may be solitary or diffuse with a varied presentation. 80, 83 in decreasing order of likelihood, lesions may include erythematous patches, alopecia, scaling, dermal nodules, or ulcerative plaques. nasal hypopigmentation, miliary dermatitis, and mucosal lesions are rarely observed. peripheral lymphadenopathy may also be present. in most cats, the duration of signs will be prolonged, lasting several months. cats with ocular lymphoma are presented with uveitis or iridial masses, as well as signs related to systemic involvement of disease. 68 all cats with lymphoma, regardless of site, may be presented with nonspecific constitutional signs that may include anorexia, weight loss, lethargy, or depression. secondary bone marrow infiltration may lead to anemia-at least 50% of affected cats have moderateto-severe nonregenerative anemia. signs related to paraneoplastic hypercalcemia (pu/pd) can occur in cats, however, much less commonly than in the dog. in one survey of hypercalcemia in cats, approximately 10% were diagnosed with lymphoma of various anatomic types. 93 for most cats with suspect lymphoma, the diagnostic evaluation should include a baseline assessment consisting of a cbc with differential cell count, platelet count, serum chemistry profile, urinalysis, and retroviral (felv/fiv) screen. serum chemistry profiles can help establish the overall health of the animal, as well as, in some cases, suggest site-specific tumor involvement; for example, increased activities of liver enzymes may indicate hepatic infiltration and increased blood urea nitrogen (bun) and creatinine may indicate renal lymphoma. for cats with alimentary lymphoma, hypoproteinemia and anemia are reported to occur in up to 23% and 76% of cases, respectively. 31, 52, 94 hypercalcemia is rarely seen in cats but has been reported in cats with lymphoma at various anatomic sites. hypoglycemia was reported in approximately one-third of cats with lymphoma in one australian study. 94 in a series of cats with various anatomic forms of lymphoma, serum albumin concentrations were significantly lower and β-globulin concentrations (as measured by protein electrophoresis) were significantly higher than a healthy control population. 95 the use of various imaging modalities in cats with lymphoma depends on the anatomic site and will be discussed in site-specific discussions to follow. cytopathologic or histopathologic evaluation of lymph node or involved organ tissue, procured via needle aspirate cytology (see chapter 7), surgical, endoscopic, or needle-core biopsy (see chapter 9) is required for a definitive diagnosis. fna cytology alone may not be sufficient in some cases, owing to difficulties encountered in distinguishing lymphoma from benign hyperplastic or reactive lymphoid conditions. in such cases, whole lymph node excision and/or involved organ biopsy is preferred because orientation and information regarding invasiveness and architectural abnormalities may be necessary for diagnosis. additionally, involved tissue, needle aspirate, and fluid samples can be further interrogated by various histochemical, immunohistochemical, flow cytometric analysis (e.g., size and immunophenotypic assessment), and molecular techniques (e.g., parr to assess clonality) to further characterize the disease process and refine the diagnosis in equivocal cases. the stomach, liver, spleen, colon, and pancreas, and occasionally, mild effusions are observed. as mentioned previously, although aspirate cytology may be sufficient for diagnosis of large cell intermediate-or high-grade alimentary lymphoma in cats, it is rarely diagnostic for low-grade, small cell intestinal lymphoma, and tissue procurement for histologic and ancillary assessment is required for diagnosis (and differentiation from ibd). the debate still rages as to whether endoscopically obtained tissue is sufficient for diagnosis or if fullthickness tissue procured during laparotomy or laparoscopy is necessary in light of similarities with ibd. 51, 54, 103, 104 as previously discussed, histologic features that help differentiate intestinal lymphoma from ibd include lymphoid infiltration of the intestinal wall beyond the mucosa, epitheliotropism (especially intraepithelial nests and plaques), heterogeneity, and nuclear size of lymphocytes. 51, 54 although the presence of transmural involvement is highly suggestive of lymphoma, the lack of transmural infiltration is not pathognomonic for ibd; transmural infiltration is common with b-cell and large t-cell (including lgl) intestinal lymphoma but is observed in the minority of low-grade t-cell intestinal lymphomas that represent the largest group in cats (see table 32 -9). 51 for these reasons, if the differentiation of lymphoma and ibd is equivocal after standard histopathologic assessment, the addition of immunophenotypic and parr analysis in a stepwise fashion, as proposed by kiupel and others, 54 may be ultimately necessary for a definitive diagnosis. their study of 63 cats with either lymphoma or ibd found that, although standard histopathology was highly specific for diagnosis of lymphoma (99% specific, 72% sensitive), sensitivity was enhanced by the addition of immunophenotypic analysis (99% specific, 78% sensitive) and further enhanced by parr analysis (99% specific, 83% sensitive). for cats with mediastinal lymphoma, diagnostic suspicion may begin with a noncompressible cranial thorax on physical examination and confirmation of a mediastinal mass/pleural effusion on thoracic radiograph. fna of the mass or cytologic evaluation of pleural fluid may be sufficient to establish a diagnosis. in most cats, the finding of a monotonous population of intermediate-or high-grade cells will establish a diagnosis. however, definitive diagnosis of lymphoma in cats with a mediastinal mass and concurrent chylothorax can be challenging. ct appearance may be helpful but generally does not contribute to a definitive diagnosis. if lymphoblasts are not identified in the pleural chylous effusion, then cholesterol and triglyceride concentrations can be measured. 105 in chylous effusions, the pleural fluid triglyceride concentration will be greater than in the serum; however, anorectic cats will have lower triglyceride levels in the pleural fluid. a major differential for mediastinal lymphoma is thymoma. the cytologic features of thymoma can be distinct from lymphoma in many cases, but the diagnosis can be challenging because of a preponderance of small lymphocytes in thymoma. mast cells can also be seen in up to 50% of aspirations from thymomas. the addition of immunophenotypic and clonality assessment may be helpful in equivocal cases. if nasal lymphoma is suspected, advanced imaging (ct, mri), rhinoscopy, and biopsy are usually necessary for diagnosis (see chapter 23, section b). ct or mri is useful to determine the extent of involvement and to help plan biopsy procurement and rt if that treatment option is pursued. ct characteristics associated with sinonasal tumors in cats include the presence of a unilateral or bilateral nasal/sinus mass or fluid, bulla (and differentiation from ibd) include abdominal imaging (usually ultrasound), procurement of tissue for histopathology, and if necessary, assessment of immunophenotype and clonality. abdominal ultrasound will be abnormal in approximately 60% to 90% of cats with low-grade, small cell gi lymphoma. 31, 52, 102, 103 diffuse small intestinal wall thickening is the most common finding; 50% to 70% of cats with lymphoma will have ultrasonic evidence of wall thickening, which predominantly involves the muscularis propria, and submucosa, although mucosal thickening can also occur. focal mural masses are uncommon. mesenteric lymphadenopathy is also common and reported in 45% to 80% of affected cats. these ultrasonographic findings are by no means pathognomonic for lymphoma, however, because 10% to 50% and 15% to 20% of cats with ibd also have ultrasonographic evidence of intestinal wall thickening and lymphadenopathy, respectively. 102, 103 mucosal thickening is more common, and muscularis propria thickening is less common in ibd than lymphoma. less commonly, cats with low-grade intestinal lymphoma will have ultrasonographic abnormalities in other abdominal organs such as stage 1 • a single tumor (extranodal) with regional lymph node involvement • two or more nodal areas on the same side of the diaphragm • two single (extranodal) tumors with or without regional lymph node involvement on the same side of the diaphragm • a resectable primary gastrointestinal tract tumor, usually in the ileocecal area, with or without involvement of associated mesenteric nodes only • two single tumors (extranodal) on opposite sides of the diaphragm • two or more nodal areas above and below the diaphragm • all extensive primary unresectable intraabdominal disease • all paraspinal or epidural tumors, regardless of other tumor site or sites • stages 1-3 with liver and/or spleen involvement yield enough tissue for a cytologic diagnosis. ct or mr also reveals multifocal disease in the majority of cats with intracranial lymphoma. 75 ,76 csf analysis may be helpful but is rarely definitive for lymphoma. one of 11 cats with confirmed spinal lymphoma in one study 77 and 6 of 17 with confirmed intracranial lymphoma in another 76 had evidence of lymphoblasts in the cns, and an increased protein content was commonly found. in cats suspected of cns lymphoma, bone marrow and renal involvement are often present, and cytologic assessment of these or other more accessible organs is generally more easily attainable than from spinal sites. for cats suspected of cutaneous lymphoma, punch biopsies (4 to 8 mm) should be taken from the most representative and infiltrative sites, while avoiding overtly infected skin lesions. immunophenotypic and parr analysis often are helpful in definitive diagnosis. complete staging to rule out systemic disease is also recommended for cats with cutaneous lymphoma because local therapies can be applied in cases of solitary disease. our knowledge base for treating cats with lymphoma is less well established, and outcomes are less predictable than that in dogs, primarily due to the greater variation in histologic type and anatomic location observed in the species. this is further complicated by the plethora of papers that "lump" very small numbers of cases representing multiple anatomic/immunophenotypic and histologic subtypes (e.g., small cell versus large cell variants) together when reporting survival analysis following chemotherapy. this provides only general observations rather than important specific outcome information (i.e., response rate and durability of response) that can vary significantly with respect to anatomic and histologic subtype. in general, canine lymphoma is most commonly intermediate-high grade and nodal, whereas cats more commonly present with gi or extranodal (±nodal extension), small cell, low-grade, and/or indolent forms. as will be discussed subsequently, the author bases most treatment decisions on assessment of whether the individual case represents a low-grade (e.g., indolent, small cell variants) versus an intermediate-or high-grade (e.g., large cell) lymphoma. finally, much of the early work on chemotherapy protocol development for cats with lymphoma occurred during the felv era, and care should be exercised when applying this information in the post-felv era. in general, cats tolerate chemotherapy for lymphoma quite well, most clients are happy with their choice to initiate treatment, and quality of life generally improves following commencement of therapy. 109, 110 the chemotherapeutic agents used most commonly to treat intermediate-or high-grade lymphoma in cats are similar to those used for dogs and humans with lymphoma (see section a in this chapter) and include doxorubicin, vincristine, cyclophosphamide, methotrexate, l-asparaginase, ccnu (lomustine), and prednisone. most combination induction protocols currently employed in cats are modifications of chop protocols initially designed for human oncologic use. 5,110-116 chop represents combinations of cyclophosphamide (c), doxorubicin (h, hydroxydaunorubicin), vincristine (o, oncovin) and prednisone (p). in general, chopbased protocols are appropriate for cats with large cell, intermediate-and high-grade lymphoma involving any anatomic site (e.g., peripheral nodal, mediastinal, and renal forms) but should not be first-line therapy for small cell, low-grade variants. as in the dog (see section a in this chapter), a plethora of modifications are used with chop-based protocols, although virtually no quality comparative data exist to compare outcomes, and as such, the protocol used should be based on cost, ease, client/veterinarian preference, and level of comfort. the current chop-based protocol in use by effusion, and lysis of associated bony structures. 106, 107 a biopsy can be procured either by intranasal procurement (with or without rhinoscopy) or by flushing one hemicavity with a bulb syringe and saline while occluding the contralateral cavity and collecting samples flushed out of the nasopharynx (figure 32-15 ). thorough staging (i.e., regional node assessment, thoracic and abdominal staging, and bone marrow assessment) to ensure the disease is confined to the nasal passages is recommended, if local rt without systemic chemotherapy is being considered. in the case of renal lymphoma, physical examination findings of massive and often bilateral renomegaly will raise the index of suspicion. radiographic appearance is smoothto-irregular renomegaly (see figure 32-14, a) . ultrasonographic imaging usually reveals bilateral (>80%), irregular renomegaly with hypoechoic subcapsular thickening. 108 approximately one-third of cases will have ultrasonographic evidence of other abdominal organ involvement. the disease is usually diffuse throughout the renal cortex (see figure 32-14, b) and transabdominal needle aspirate or core biopsy is diagnostic in most cases. in cats with suspected spinal lymphoma, survey radiographs of the spine will rarely reveal osseous lesions. myelograms, ct, or mri are indicated, and in approximately 75% of the cases, an extradural or intradural mass will be detected. 76, 77, 91, 92 most lesions occur at a thoracolumbar or lumbosacral location, and they are often found in more than one location. image-guided needle aspiration of epidural lesions may used in cats in europe, and one compilation reported similar results to chop. 64 a cop protocol commonly employed in cats is presented in table 32 -11. some studies with relatively few case entries have reported limited activity for doxorubicin as a single agent in cats with lymphoma 118, 119 ; however, larger studies using combination protocols have more consistently reported the addition of doxorubicin as necessary for the attainment of more durable responses. 5, 114 interestingly, in a report of 23 cats having relapsed following cop-based protocols (without doxorubicin), only 22% responded subsequently to doxorubicin-containing rescue therapy. 120 a small number of cats with lymphoma have been treated with single-agent oral ccnu (lomustine) at a dosage range of 30 to 60 mg/m 2 every 3 to 6 weeks. 121, 122 whereas activity was noted, only partial responses were reported. l-asparaginase, which is often included in protocols for lymphoma in cats, has a much shorter asparagine-depleting effect in cats (lost by 7 days) than in dogs and in one study in 13 cats with lymphoma resulted in only a 30% response rate. 123 in general, cats with intermediate-and high-grade lymphoma treated with chop-based or cop protocols do not enjoy the same level of success as dogs. bearing in mind that these reports group together a wide variety of subtypes having dissimilar prognoses (see subsequent site-specific treatment sections), the overall response rates tend to be in the 50% to 80% range with median remission and survival durations of 4 and 6 months, respectively. 5, 64, [110] [111] [112] [113] [114] [115] [116] 124 alimentary/gastrointestinal lymphoma representing the most common presentation for cats with lymphoma, the large majority have the small cell, mucosal, t-cell variant that carries a good prognosis, often with less aggressive chemotherapy protocols (e.g., oral chlorambucil and prednisone). 51, 52, 87, 125 chlorambucil (20 mg/m 2 po, every 2 weeks [preferred by the author] or 2 mg po every other day) and prednisone or prednisolone (initially 1 to 2 mg/kg po daily, reduced to 0.5 to 1.0 mg/kg every other day over several weeks) results in response rates (i.e., resolution of clinical signs) of greater than 90% and median survivals of approximately 2 years or longer. 52, 87, 125 cats who relapse with this protocol often will subsequently respond to alternative alkylators, such as cyclophosphamide or lomustine. 125 anecdotally, many will also respond the author is presented in table 32 -10. this protocol has been used in many cats with various forms of intermediate-and high-grade lymphoma and is generally well tolerated. at present, most canine lymphoma protocols (see section a in this chapter) discontinue chemotherapy by the 25th week, and we have sufficient data to show shorter, maintenance-free protocols are as good if not superior to longer maintenance protocols; however, similar comparative data do not exist in the cat. until such time as evidence to the contrary exists, the author presently recommends discontinuation of chemotherapy at week 25 in cats who have attained a complete remission. doxorubicin alone (25 mg/m 2 , every 3 weeks for 5 total treatments) or palliative prednisone therapy is offered if clients decline more aggressive chop-based therapy. cats are generally less tolerant of doxorubicin than are dogs; therefore a lower dosage (25 mg/m 2 iv or 1 mg/kg iv) is used (see chapter 11) . cardiac toxicity does not appear to be a clinically significant problem in cats, although renal toxicity is more commonly encountered in the species 117 and renal function should be monitored (i.e., serial bun, creatinine, and urine specific gravity) closely prior to and during therapy. the use of cop (i.e., chop without the addition of doxorubicin) is often • nasal cavity following thorough staging (node cytology, thoracic and abdominal imaging, bone marrow aspiration), then rt is the treatment of choice. crs in the order of 75% to 95% are reported, with reports of median survivals following rt of 1.5 to 3 years. 73, 129 cats that do not achieve a cr with rt have a median survival of approximately 4.5 months. total radiation dosage does affect survival durations, and a total dose greater than 32 gy is recommended. 73 the addition of chemotherapy has not been shown to enhance survival for cats with locally confined disease; combinations of rt and chemotherapy result in similar response rates and survival times. 73, 128, 129 chemotherapy (cop-or chop-based protocols) used in the absence of rt is a reasonable alternative, with complete response rates of approximately 75% and median survivals of approximately 2 years reported for cats achieving cr. 68 the author's preference is to initiate systemic chemotherapy only for (1) cases that have confirmed disease beyond the nasal passage, (2) cases that relapsed following rt, or (3) cases in which rt is unavailable or declined. ing treatment for cns lymphoma exist, and although an occasional case experienced durable response to systemic chemotherapy, generally less than 50% will respond and median survivals of 1 to 2 months can be expected. 68, 76, 77 laryngeal/tracheal lymphoma the vast majority of cats with laryngeal or tracheal lymphoma respond to either rt (if localized) or systemic chemotherapy (90% cr to cop-or chop-based protocols) (figure 32-16) . 68 whereas the authors experience is that most have durable responses and survival durations typically approach or exceed 1 year, the only case series (n = 8) reported a median survival of 5.5 months following achievement of a cr. ing the treatment of cutaneous lymphoma or mycosis fungoides in cats 80 ; however, a report of a cr to lomustine exists. 130 cats with a solitary disease could theoretically be treated with surgical excision to vinblastine chemotherapy if they no longer are responsive to alkylators. in contrast, cats with b-cell or large t-cell (including lgl) or small t-cell lymphoma that is transmural typically do not enjoy a durable response to therapy and survivals are much shorter. 31, 51, 59, 60 median survivals range from 45 to 100 days, even in cats treated with more aggressive cop-based protocols. in the author's experience, these variants are more likely to respond to chop-based protocols than chlorambucil/prednisone; however, durable responses occur only in a minority of cases. in particular, lgl appears to carry a grave prognosis 59, 60 ; in 2 compilations of 66 cats with lgl, median survivals of approximately 2 months were reported, including 23 cats receiving either cop or chop-based protocols, which resulted in only a 30% response rate. nutritional support is especially important for cats with gi lymphomas. it may be necessary to place a feeding tube in cats undergoing chemotherapy, particularly if prolonged anorexia is present (see chapter 15, section b). recently, two preliminary studies evaluated rt, either as rescue following recurrence or in addition to chemotherapy for the treatment of intestinal lymphoma in cats. 126, 127 eleven cats (6 small cell, 4 large cell, and 1 lgl) that progressed following chemotherapy received abdominal radiation (8 gy in 2 fractions over 2 days) and resulted in a median survival of 7 months, although numbers were small and 40% were lost to follow-up. 127 a second report of eight cats (seven with large cell lymphoma) underwent 6 weeks of chopbased combination chemotherapy, followed 2 weeks later by whole abdomen radiation consisting of 10 daily 1.5 gy fractions. 126 although three cats died within 3 weeks of rt, five enjoyed durable remissions. these preliminary promising outcomes warrant further investigation. felv-positive cats is generally associated with a poor prognosis, and survival times of approximately 2 to 3 months are expected following chop-or cop-based protocols . 5, 116 in contrast, young felv-negative siamese cats with mediastinal lymphoma experience remission rates approaching 90%, and responses tend to be more durable (median ≈9 months). 64 the treatment choice for peripheral nodal lymphoma in cats depends on whether the individual case represents a low-grade (e.g., indolent, small cell variants) versus an intermediate-or high-grade (e.g., large cell) lymphoma; the latter are best treated with chop-or cop-based protocols and carry a less favorable prognosis, whereas the former generally respond to less aggressive chlorambucil/corticosteroid protocols and enjoy durable responses. less is known regarding the treatment of hodgkin's-like lymphoma involving solitary or regional nodes of the head and neck. 65, 66 clinical outcome following surgical extirpation of the affected node (or nodes if a reasonable number) is often associated with long-term, disease-free intervals and survivals of approximately 1 year, suggesting it is a more indolent form of lymphoma. eventual recurrence in distal nodes following surgical excision is common, and the author currently offers clients the option of adjuvant chlorambucil/corticosteroids following surgery-this theoretically may have benefit; however, insufficient data exist to document a survival advantage with this approach. the use of chemotherapy to treat all has been disappointing. using cop-based protocols, cotter 124 reported a 27% cr rate. cll can be treated with chlorambucil (0.2 mg/kg po or 2 mg/cat qod) and prednisone (1 mg/kg po daily); however, little information exists regarding outcome. as in humans and dogs, if significant clinical signs or profound cytopenias are not present, treatment can be withheld-one cat with cll remained stable without chemotherapy for over a year. 140 the prognoses for acute nonlymphoblastic leukemias are generally very poor, although some exceptions exist in case report form in the historic literature. or rt, although clinical staging is necessary to rule out possible further systemic involvement. for multiple sites, combination chemotherapy may be considered. as previously discussed, the prediction of outcomes in cats with lymphoma is not generalizable due to the wide spectrum of histologic and anatomic subtypes encountered. much has been mentioned in the previous treatment sections, and tables 32-8 and table 32 -9 summarize prognostic parameters for lymphoma in cats. for a complete discussion of leukemias and mpds, including a general discussion of hematopoiesis, etiologies, lineage classification and descriptions, see section c of this chapter. the classification of leukemias in cats is difficult because of the similarity of clinical and pathologic features and the transition, overlap, or mixture of cell types involved. [131] [132] [133] [134] [135] most case-series reports are from the felv era and generally only single case reports exist from the more contemporary post-felv era, which further confuses our understanding of the biology and outcome. for this reason, only a simplistic discussion, primarily relating to the lymphoid leukemias will be presented here and the interested reader is again referred to section c for a general discussion of nonlymphoid leukemia. for cats with suspected leukemia, peripheral blood assessment (e.g., cbc with differential, flow cytometric analysis for size and immunophenotype, and parr [for lymphoid leukemias]), and bone marrow aspiration or biopsy may contribute to a diagnosis. the preferred sites for bone marrow aspiration are the proximal humerus or iliac crest. cats with acute leukemia are likely to have malignant cellular infiltrates in organs other than bone marrow. 134 a bone marrow aspirate with greater than 30% abnormal blast cells is sufficient to make a diagnosis of an acute leukemia. in cats with suspected cll, infiltration of the bone marrow with more than 20% mature lymphocytes helps confirm the diagnosis. all cats with leukemia should be tested for felv/fiv. determining the lineage of some leukemias can be challenging; most can be distinguished from one another by histologic appearance, histochemical stains, or immunohistochemical or flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage (see chapter 8 and section c in this chapter). 131, 133, 136 in addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features. the french-american-british (fab) classification system is considered useful in cats with myelodysplastic syndromes and almost all will be felv antigenemic. 136, 137 lymphoid leukemia all was the most commonly encountered type of leukemia in cats in the felv era; however, it is much less common today. all is characterized by poorly differentiated lymphoblasts and prolymphocytes in blood and bone marrow. approximately 60% to 80% of cats with all were felv positive, and most malignant cells have t-cell immunophenotypes 138 ; however, little information is available in the contemporary literature. cll is rarely reported in cats and is characterized by welldifferentiated, small, mature lymphocytes in peripheral blood and 1 aberrant proliferation of cells with defective maturation and function leads to reduction of normal hematopoiesis and invasion of other tissues. these disorders have been classified based on biologic behavior, degree of cellular differentiation, and lineage of the neoplastic cells (granulocytic, monocytic, erythroid, megakaryocytic, or mixed). newer classification systems in humans have incorporated genetics and molecular genetic analysis; these are currently areas of active investigation in the study of animal leukemias. 2 in 1991 the animal leukemia study group made recommendations for classifying nonlymphoid leukemias in dogs and cats. 3 more recently, the oncology committee of the american college of veterinary pathologists (acvp) has been reexamining criteria for a classification system and spearheading large multiinstitutional studies to validate the criteria. long-term produced experimentally following irradiation. [22] [23] [24] in contrast to mpds in cats, no causative viral agent has been demonstrated in dogs, although retrovirus-like budding particles were observed in the neoplastic cells of a dog with granulocytic leukemia. 25 pathology and natural behavior a review of normal hematopoiesis will aid in understanding the various manifestations of mpds. hematopoiesis is the process of proliferation, differentiation, and maturation of stem cells into terminally differentiated blood cells. a simplified scheme is presented in figure 32 -17. pluripotent stem cells differentiate into either lymphopoietic or hematopoietic multipotent stem cells. 26 under the influence of specific regulatory and microenvironmental factors, multipotent stem cells in bone marrow differentiate into progenitor cells committed to a specific hematopoietic cell line, for example, erythroid, granulocytic-monocytic, or megakaryocytic. maturation results in the production of terminally differentiated blood cellserythrocytes, granulocytes, monocytes, and platelets-that are delivered to the circulation. in some cases, as in the maturation of reticulocytes to erythrocytes, final development may occur in the spleen. proliferation and differentiation of hematopoietic cells are controlled by a group of regulatory growth factors. 26, 27 of these, objectives of these studies are to define molecular lesions, establish prognostic markers, and target effective therapeutic approaches. 4 myeloid neoplasms are uncommon or rare in the dog and occur 10 times less frequently than lymphoproliferative disorders. 5 accurate information about incidence and other epidemiologic information await consistent use of a uniform classification system (see later discussion). there is no known age, breed, or sex predisposition, although in some retrospective studies, large-breed dogs have been overrepresented. [6] [7] [8] [9] [10] [11] [12] [13] [14] in dogs, the etiology of spontaneously occurring leukemia is unknown. it is likely that genetic and environmental factors (including exposure to radiation, drugs, or toxic chemicals) play a role. in humans, acquired chromosomal derangements lead to clonal overgrowth with arrested development. 15 at the end of the last century, chromosomal abnormalities were reported in dogs with aml, chronic myelogenous leukemia (cml), and lymphoid leukemia. 16, 17 however, because karyotyping is difficult to perform in dogs because of the large number and morphologic similarity of their chromosomes and their resistance to banding, defining genetic factors in canine myeloid neoplasms has awaited application of molecular technologies and use of the canine genome map. erythropoietin is the best characterized; it regulates erythroid proliferation and differentiation and is produced in the kidney, where changes in oxygen tension are detected. the myeloid compartment depends on a group of factors, collectively referred to as colonystimulating factors (csfs). these factors act at the level of the committed progenitor cells but also influence the functional capabilities of mature cells. some of these factors have a broad spectrum of activity; others are more restricted in their target cells and actions. csfs are produced in vitro by a multitude of cell types, including monocytes, macrophages, lymphocytes, and endothelial cells, and these cells likely play a role in the production and regulation of these factors in vivo. the gene for thrombopoietin also has been cloned, and it appears that this hormone alone can induce differentiation of megakaryocytes and platelet production. 28 recombinant forms of many of these hormones are increasingly available. clonal disorders of bone marrow include myeloaplasia (usually referred to as aplastic anemia), myelodysplasia, and myeloproliferation. a preleukemic syndrome, characterized by peripheral pancytopenia and bone marrow hyperplasia with maturation arrest, is more correctly termed myelodysplasia because the syndrome does not always progress to overt leukemia. this syndrome has been described in cats, usually in association with felv infection but has only rarely been recognized in dogs. [29] [30] [31] [32] these clonal disorders may be manifested by abnormalities in any or all lineages because hematopoietic cells share a common stem cell. in addition, transformation from one form to another may occur. 33 myeloid neoplasms are classified in several ways. the terms acute and chronic refer to the degree of cellular differentiation of the leukemic cells, but these terms also correlate with the biologic behavior of the neoplasm. 34 disorders resulting from uncontrolled proliferation or decreased apoptosis of cells incapable of maturation lead to the accumulation of poorly differentiated or "blast" cells. these disorders are included under the umbrella term, acute myeloid leukemia (aml). disorders resulting from unregulated proliferation of cells that exhibit progressive, albeit incomplete and defective, maturation lead to the accumulation of differentiated cells. these disorders are termed myeloproliferative neoplasms (mpn) and include polycythemia vera, cml and its variants, essential thrombocythemia, and possibly primary myelofibrosis. myeloid neoplasms are further classified by the lineage of the dominant cell type(s), defined by romanowsky stains, special cytochemical stains, ultrastructural features, flow cytometric analysis, and immunologic cell markers, and they have been classified into subtypes (see later discussion). aml has a more sudden onset and is more aggressive. in both acute and chronic disorders, however, abnormalities in proliferation, maturation, and functional characteristics can occur in any hematopoietic cell line. 1 in addition, normal hematopoiesis is adversely affected. animals with leukemia usually have decreased numbers of circulating normal cells. the pathogenesis of the cytopenias is complex and may result in part from production of inhibitory factors. eventually, neoplastic cells displace normal hematopoietic cells, and this is termed myelophthisis. anemia and thrombocytopenia are particularly common. neutropenia and thrombocytopenia result in infection and hemorrhage, which may be more deleterious to the animal than the primary disease process. aml is rare and is characterized by aberrant proliferation and/or decreased apoptosis of a clone of cells without maturation. this results in accumulation of immature blast cells in bone marrow and peripheral blood (figure 32-18, a to e) . the wbc count is variable and ranges from leukopenia to counts up to 150,000/µl. spleen, liver, and lymph nodes are frequently involved, and other tissues, including tonsils, kidney, heart, and the cns, may be infiltrated as well. there is no characteristic age, and even very young dogs may be affected. 35 the clinical course of these disorders tends to be rapid. production of normal peripheral blood cells is usually diminished or absent, and anemia, neutropenia, and thrombocytopenia are common with infection and hemorrhage occurring as frequent sequelae. occasionally, neoplastic blasts are present in bone marrow but not in peripheral blood. this is termed aleukemic leukemia, whereas subleukemic suggests a normal or decreased wbc count with some neoplastic cells in circulation. in 1985 the animal leukemia study group was formed under the auspices of the american society for veterinary clinical pathology to develop specific morphologic and cytochemical criteria for classifying acute nonlymphocytic leukemias. recognition of specific subtypes of leukemia is required to compile accurate and useful information about prognosis and response to treatment, as well as to compare studies from different sites. in 1991, this group proposed a classification system following adaptation of the french-american-british (fab) system and criteria established by the nci workshop. 3 group members examined blood and bone marrow from 49 dogs and cats with myeloid neoplasms. romanowsky-stained specimens were examined first to identify blast cells and their percentages. lineage specificity was then determined using cytochemical markers. the percentage of blasts and the information about lineage specificity were used in combination to classify disorders as acute undifferentiated leukemia (aul), acute myeloid leukemia (aml, subtypes m1 to m5 and m7), and erythroleukemia with or without erythroid predominance (m6 and m6er). a description of these subtypes is presented in table 32-12. canine karyotyping is difficult, but with advancements in molecular cytogenetic analysis, chromosome painting, and genomic hybridization, aml in dogs can now be analyzed at the base-pair level, 18, 19 and missense mutations in flt3, c-kit, and ras sequences have been identified in dogs with aml, similar to what has been found for human aml. 36 in addition to serving as diagnostic and prognostic markers, cytogenetic lesions may be therapeutic targets. as cytogenetic abnormalities continue to be identified, this information will need to be incorporated into classification schemes. with the exception of acute promyelocytic leukemia or m3, all of these subtypes have been described in dogs. however, because this modified fab system has been adopted only recently, the names given to these disorders in the literature vary considerably. in addition, in the absence of cytochemical staining, immunophenotyping, or electron microscopic evaluation, the specific subtype of leukemia has often been uncertain, making retrospective analysis of epidemiologic information, prognosis, and response to therapy confusing at best. although defining specific subtypes may seem to be an academic exercise owing to the uniformly poor prognosis of acute leukemias, this information is critical to improving the management of these diseases. because of the low incidence of aml, national and international cooperative efforts will be required to accumulate information on the pathogenesis and response to different treatment modalities of specific subtypes. utilization of a uniform classification system is an essential first step. different forms of aml are demonstrated in figure 32 polycythemia vera (pv) is a clonal disorder of stem cells, although whether the defect is in the pluripotent stem cell or the hematopoietic multipotent stem cell is still not clear. in humans, progenitor cells have an increased sensitivity to insulin-like growth factor 1, which stimulates hematopoiesis. 64 it is not known whether this hypersensitivity is the primary defect or is secondary to another gene mutation. in any case, the result is overproduction of red blood cells (rbcs). the disease is rare and must be distinguished from more common causes of polycythemia, including relative and secondary absolute polycythemia (see later discussion). in pv, there is neoplastic proliferation of the erythroid series with terminal differentiation to rbcs. the disease has been reported in dogs that tend to be middle-aged with no breed or sex predilection [65] [66] [67] [68] [69] [70] [71] [72] [73] and is characterized by an increased rbc mass evidenced by an increased packed cell volume (pcv), rbc count, and hemoglobin concentration. the pcv is typically in the range of 65% to 85%. the bone marrow is hyperplastic, although the myeloid : erythroid (m : e) ratio tends to be normal. in contrast to the disease in humans, other cell lines do not appear to be involved, and transformation to other mpns has not been reported. the disease in dogs may be more appropriately termed primary erythrocytosis. in humans, acquired jak2 gene mutations are identified in 90% of patients with primary polycythemia, and recently an identical mutation in the jak2 gene of one of five dogs with primary polycythemia was reported. 74 in dogs, cml is more similar to chronic neutrophilic leukemia, a rare form of mpn in humans, than to cml in humans because it is a neoplastic proliferation of the neutrophil series, although concurrent eosinophilic and basophilic differentiation can occur. cml can occur in dogs of any age. 35, [75] [76] [77] [78] [79] neutrophils and neutrophilic precursors accumulate in bone marrow and peripheral blood as well as in other organs. the peripheral wbc count is usually, but not always, greater than 100,000/µl. both immature and mature neutrophils are present, as demonstrated in figure 32 -18, f. mature forms are usually more numerous, but sometimes an "uneven" left shift is present. signs of dysplasia may be evident, including hypersegmentation, ringed nuclei, and giant forms. eosinophils and basophils may also be increased. the bone marrow is characterized by granulocytic hyperplasia, and morphologic abnormalities may not be present. erythroid and megakaryocytic lines may be affected, resulting in anemia, thrombocytopenia, or less commonly, thrombocytosis. this disorder must be distinguished from severe neutrophilic leukocytosis and "leukemoid reactions" caused by inflammation or immune-mediated diseases. leukemoid reactions can also occur as a paraneoplastic syndrome. in humans with cml, characteristic cytogenetic abnormalities are present in all bone marrow cells, signifying a lesion at the level of an early multipotent stem cell. typically, these individuals have a chromosomal translocation, resulting in the philadelphia chromosome or bcr-abl translocation between chromosomes 9 and 22. 80 the analogous chromosomes in dogs are chromosomes 9 and 26, and bcr-abl mutations have now been reported in three cases of cml in dogs. 2 variants of cml are chronic myelomonocytic leukemia (cmml) and chronic monocytic leukemia (cmol). [81] [82] [83] these diagnoses are made based on the percentage of monocytes in the leukemic cell leukemia (m4).* megakaryoblastic leukemia (m7) also is well recognized in dogs 10,47-58 and may be associated with platelet dysfunction. 51 monocytic leukemias have likely included those with and without monocytic differentiation (m5a and m5b), 11, 59 but in some cases the diagnosis may have been chronic myelomonocytic or chronic monocytic leukemia (see later discussion). there are few reports in dogs of spontaneously occurring erythroleukemia (m6) in which the leukemic cells include myeloblasts, monoblasts, and erythroid elements. [60] [61] [62] auls have uncertain lineages because they are negative for all cytochemical markers. these leukemias should be distinguished from lymphoid leukemias by flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage. 63 in addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features. mpns, previously termed chronic myeloproliferative disorders, are characterized by excessive production of differentiated bone marrow cells, resulting in the accumulation of erythrocytes (polycythemia vera), granulocytes and/or monocytes (cml and its variants), or platelets (essential thrombocythemia). primary myelofibrosis as a clonal disorder of marrow stromal cells, characterized by proliferation of megakaryocytes and granulocytic precursors with accumulation of collagen in bone marrow, has been *references 5-9, 23-34, 37-46. • myelodysplastic syndrome with erythroid predominance out this disorder. however, spurious microcytosis may be reported if a dog has many giant platelets that are counted by an analyzer as small rbcs. 93 microscopic review of the blood film may be helpful in these cases. myelofibrosis primary myelofibrosis has been reported only rarely in dogs and is usually a secondary, or reactive, process. 94, 95 in humans, myelofibrosis is characterized by collagen deposition in bone marrow and increased numbers of megakaryocytes and granulocytic precursors, many of which exhibit morphologic abnormalities. in fact, breakdown of intramedullary megakaryocytes and subsequent release of factors that promote fibroblast proliferation or inhibit collagen breakdown may be the underlying pathogenesis of the fibrosis. 96 focal osteosclerosis is sometimes present. anemia, thrombocytopenia, splenomegaly, and myeloid metaplasia (production of hematopoietic cells outside the bone marrow) are consistent features. in dogs, myelofibrosis occurs secondary to mpds, radiation damage, and congenital hemolytic anemias. [97] [98] [99] [100] in some cases, the inciting cause is unknown (idiopathic myelofibrosis). there may be concurrent marrow necrosis in cases of ehrlichiosis, septicemia, or drug toxicity (estrogens, cephalosporins), and there is speculation that fibroblasts proliferate in response to release of inflammatory mediators associated with the necrosis. 94 myeloid metaplasia has been reported to occur in the liver, spleen, and lung. 100 extramedullary hematopoiesis is ineffective in preventing or correcting the pancytopenia that eventually develops. dysfunction of the hematopoietic system can be manifested by a variety of abnormalities that constitute myelodysplastic syndrome (mds). in dogs, in which the syndrome is rare, there usually are cytopenias in two or three lines in the peripheral blood (anemia, neutropenia, and/or thrombocytopenia). other blood abnormalities can include macrocytic erythrocytes and metarubricytosis. the bone marrow is typically normocellular or hypercellular, and dysplastic changes are evident in several cell lines. if blast cells are present, they make up less than 30% of all nucleated cells, 2 although this threshold is being changed to less than 20%. 4, 20 myelodysplasia is sometimes referred to as preleukemia because, in some cases, it may progress to acute leukemia. [29] [30] [31] based on reported cases, poor prognostic indices include increased percentage of blast cells, cytopenias involving more than one lineage, and cellular atypia. primary mdss are clonal disorders and are considered neoplastic. complex classification schemes for human mds, based on percentages of blasts in bone marrow, cytogenetic analysis, cytopenias, need for transfusions, and other variables, comprise at least nine subtypes; their applicability to veterinary medicine is unknown. 5 three subtypes are proposed for dogs and cats and include mds with excessive blasts (mds-eb), in which blast percentages are greater than 5% and less than 20%, and progression to aml may occur; mds with refractory cytopenia (mds-rc) with blast percentages less than 5% and cytopenias in one or more lineages; and mds with erythroid predominance (mds-er) in which the m : e ratio is less than 1 and prognosis is poor. 4 larger studies are needed to determine the utility of this classification scheme and other potential prognostic indices, such as sex, age, and felv positivity. in addition to accumulating enough cases, another confounding factor to studying and classifying mds is the presence of reversible mdss population. bcr-abl translocation has also been reported in a dog with cmol. 45 in addition to accumulating in bone marrow and peripheral blood, leukemic cells also are found in the red pulp of the spleen, the periportal and sinusoidal areas of the liver, and sometimes lymph nodes. other organs such as the kidney, heart, and lung are less commonly affected. in addition, extramedullary hematopoiesis may be present in the liver and spleen. death is usually due to complications of infection or hemorrhage secondary to neutrophil dysfunction and thrombocytopenia. in some cases, cml may terminate in "blast crisis, " in which there is a transformation from a predominance of well-differentiated granulocytes to excessive numbers of poorly differentiated blast cells in peripheral blood and bone marrow. this phenomenon is well documented in the dog. 75, 76, 78 basophilic and eosinophilic leukemia basophilic leukemia, although rare, has been reported in dogs and is characterized by an increased wbc count with a high proportion of basophils in peripheral blood and bone marrow. [84] [85] [86] hepatosplenomegaly, lymphadenopathy, and thrombocytosis may be present. all the dogs have been anemic. basophilic leukemia should be distinguished from mast cell leukemia (mastocytosis). whether dogs develop eosinophilic leukemia remains in question. reported cases have had high blood eosinophil counts and eosinophilic infiltrates in organs. 87, 88 one dog responded well to treatment with corticosteroids. the distinction between neoplastic proliferation of eosinophils and idiopathic hypereosinophilic syndrome remains elusive. disorders associated with eosinophilia such as parasitism, skin diseases, or diseases of the respiratory and gi tracts should be considered first in an animal with eosinophilia. one distinguishing feature should be clonality, with reactive eosinophilia comprising polyclonal cells and the neoplastic condition arising from a single clone. as clonality assays become more available, this discrepancy may be resolved. in humans, essential thrombocythemia, or primary thrombocytosis, is characterized by platelet counts that are persistently greater than 600,000/µl. there are no blast cells in circulation, and marked megakaryocytic hyperplasia of the bone marrow without myelofibrosis is present. thrombosis and bleeding are the most common sequelae, and most patients have splenomegaly. other mpds, especially pv, should be ruled out, and importantly, there should be no primary disorders associated with reactive thrombocytosis. 89 these include inflammation, hemolytic anemia, iron deficiency anemia, malignancies, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, and splenectomy. in addition, certain drugs such as vincristine can induce thrombocytosis. essential thrombocythemia has been recognized in dogs. 33, [90] [91] [92] [93] in one dog, the platelet count exceeded 4 million/µl and bizarre giant forms with abnormal granulation were present. the bone marrow contained increased numbers of megakaryocytes and megakaryoblasts, but circulating blast cells were not seen. other findings included splenomegaly, gi bleeding, and increased numbers of circulating basophils. causes of secondary or reactive thrombocytosis were ruled out. 90 basophilia was also reported in a more recent case. 92 in another dog, primary thrombocytosis was diagnosed and then progressed to cml. 33 in some cases reported in the literature as essential thrombocythemia, the dogs had microcytic hypochromic anemias. because iron deficiency anemia is associated with reactive or secondary thrombocytosis, care must be taken to rule or thrombocytopenia are usually present. occasionally, neoplastic cells can be found in cerebrospinal fluid in animals with invasion of the cns. smears of aspirates from tissues such as the lymph nodes, spleen, or liver may contain blasts but usually contribute little to the diagnostic work-up. examination of blasts stained with standard romanowsky stains may give clues as to the lineage of the cells (figure 32-18 , a to c and e). in myelomonocytic leukemia, the nuclei of the blasts are usually pleomorphic, with round to lobulated forms. in some cells, the cytoplasm may contain large azurophilic granules or vacuoles. blasts in megakaryocytic leukemia may contain vacuoles and have cytoplasmic blebs. in addition, bizarre macroplatelets may be present. although these distinguishing morphologic features may suggest a definitive diagnosis, cytochemical staining or immunophenotyping are usually required to define the lineage of the blasts. several investigators have reported modification of diagnoses following cytochemical staining. 102, 103 it is especially important to distinguish aml from lymphocytic leukemia in order to provide accurate prognostic information to the owner and institute appropriate therapy. the animal leukemia group has recommended the following diagnostic criteria, summarized in figure 32 -19. 3 using wellprepared romanowsky-stained blood and bone marrow films, a minimum of 200 cells are counted to determine the leukocyte differential in blood and the percentage of blast cells in bone marrow and/or blood. in bone marrow, blast cells are calculated both as a percentage of all nucleated cells (anc) and nonerythroid cells (nec) and are further characterized using cytochemical markers. [102] [103] [104] neutrophil differentiation is identified by positive staining of blasts for peroxidase, sudan black b, and chloracetate esterase. nonspecific esterases (alpha-naphthyl acetate esterase or alpha-naphthyl butyrate esterase), especially if they are inhibited by sodium fluoride, mark monocytes. canine monocytes may also contain a few peroxidase-positive granules. acetylcholinesterase is a marker for megakaryocytes in dogs and cats. in addition, positive immunostaining for von willebrand's factor (factor viii-related antigen) and platelet glycoproteins on the surface of blasts identifies them as megakaryocyte precursors. 10, [49] [50] [51] [52] [53] alkaline phosphatase (ap) only rarely marks normal cells in dogs and cats but is present in blasts cells in acute myeloblastic and myelomonocytic leukemias. however, owing to reports of ap activity in lymphoid leukemias in dogs, its specificity as a marker for myeloid cells is not certain. omega exonuclease is a specific marker for basophils, which are also positive for chloracetate esterase activity. 86 blood and bone marrow differential counts and cytochemical staining should be performed and interpreted by experienced veterinary cytopathologists. if erythroid cells are less than 50% of anc and the blast cells are greater than 30%, a diagnosis of aml or aul is made. if erythroid cells are greater than 50% of anc and the blast cells are greater than 30%, a diagnosis of erythroleukemia (m6) is made. if rubriblasts are a significant proportion of the blast cells, a diagnosis of m6er, or erythroleukemia with erythroid predominance, can be made. it should be noted that in the human aml classification system, the blast threshold has been lowered to 20% and similar recommendations are being made for aml in dogs and cats. in some cases, electron microscopy is required to identify the lineage of the blast cells. for example, megakaryocyte precursors are positive for platelet peroxidase activity and contain demarcation membranes and alpha granules. 49, 53 both of these features are detected at the ultrastructural level. immunophenotyping, used to identify cell lineages in human patients, awaits development of that occur secondary to immune-mediated, infectious, and other diseases in both dogs and cats. dogs with myeloid neoplasms have similar presentations regardless of the specific disease entity, although animals with aml have a more acute onset of illness and a more rapid clinical course. a history of lethargy, inappetence, and weight loss is common. clinical signs include emaciation, persistent fever, pallor, petechiation, hepatosplenomegaly, and, less commonly, lymphadenopathy and enlarged tonsils. shifting leg lameness, ocular lesions, and recurrent infections are also seen. vomiting, diarrhea, dyspnea, and neurologic signs are variable features. serum biochemical analytes may be within the reference intervals but can change if significant organ infiltration occurs. animals with mds may be lethargic and anorectic and have pallor, fever, and hepatosplenomegaly. in pv, dogs often have erythema of mucous membranes owing to the increase in rbc mass. some dogs are polydipsic. in addition, neurologic signs such as disorientation, ataxia, or seizures may be present and are thought to be the result of hyperviscosity or hypervolemia. 69 hepatosplenomegaly is usually absent. peripheral blood abnormalities are consistently found. in addition to the presence of neoplastic cells, other abnormalities, including cytopenias of any lineage, may be present. low numbers of nucleated rbcs are present in the blood of about half the dogs with acute nonlymphocytic leukemia. 3 nonregenerative anemia and thrombocytopenia are present in most cases. anemia is usually normocytic and normochromic, although macrocytic anemia is sometimes present. pathogenic mechanisms include effects of inhibitory factors leading to ineffective hematopoiesis, myelophthisis, immune-mediated anemia secondary to neoplasia, and hemorrhage secondary to thrombocytopenia, platelet dysfunction, or dic. anemia is most severe in aml, although both anemia and thrombocytopenia may be milder in animals with the m5 subtype (acute monocytic leukemia). in myelofibrosis, the anemia is characterized by anisocytosis and poikilocytosis. in addition, pancytopenia and leukoerythroblastosis, in which immature erythroid and myeloid cells are in circulation, may be present. these phenomena probably result from replacement of marrow by fibrous tissue with resultant shearing of red cells and escape of immature cells normally confined to bone marrow. in pv, the pcv is increased, usually in the range of 65% to 85%. the bone marrow is hyperplastic, and the m : e ratio is usually in the normal range. neoplastic cells are often defective functionally. platelet dysfunction has been reported in a dog with acute megakaryoblastic leukemia (m7), 51 and in cml, neutrophils have decreased phagocytic capacity and other abnormalities. one exception to this was a report of cml in a dog in which the neutrophils had enhanced phagocytic capacity and superoxide production. 101 the authors hypothesized that increased synthesis of gm-csf resulted from a lactoferrin deficiency in the neoplastic neutrophils and mediated the enhanced function of these cells. in all cases of myeloid neoplasms, diagnosis depends on examination of peripheral blood and bone marrow. aml is diagnosed on the basis of finding blast cells with clearly visible nucleoli in blood and bone marrow. most dogs with acute leukemia have circulating blasts. these cells may be present in low numbers in peripheral blood, and a careful search of the smear, especially at the feathered edge, should be made. even if blasts are not detected in circulation, indications of bone marrow disease such as nonregenerative anemia increases in these cell types. in order to make a diagnosis of pv, it must first be established that the polycythemia is absolute rather than relative. in relative polycythemias, plasma volume is decreased from hemoconcentration, dehydration, or hypovolemia, and the absolute rbc mass is not increased. splenic contraction can also result in relative polycythemia. absolute polycythemia, in which rbc mass is increased, is usually secondary to tissue hypoxia, causing appropriate increased production of erythropoietin. rarely, erythropoietin may be produced inappropriately by a tumor (e.g., renal cell carcinoma) or in renal disease (pyelonephritis) or localized renal hypoxia. [109] [110] [111] these causes of polycythemia should be eliminated by appropriate laboratory work, thoracic radiographs, arterial blood gas analysis, and renal ultrasonography. in humans with pv, plasma erythropoietin (epo) levels are low. epo levels in dogs with pv tend to be low or low-normal, whereas in animals with secondary absolute polycythemia, the levels are high. 112, 113 samples for determination of epo concentrations should be taken prior to therapeutic phlebotomy used to treat hyperviscosity and, owing to fluctuations in epo levels, should be repeated if results are incongruous with other information. there are no pathognomonic features of cml in dogs, and other common causes for marked leukocytosis with a left shift ("leukemoid reaction") and granulocytic hyperplasia of bone marrow must be eliminated. these include infections, especially pyogenic ones; immune-mediated diseases; and other malignant neoplasms. in cml, maturation sometimes appears disorderly, and there may be variation in the size and shape of neutrophils at the same level of maturation. in addition, neoplastic leukocytes may disintegrate more rapidly and appear vacuolated. 35 because of the invasive nature of cml, biopsy of liver or spleen may also help to distinguish true leukemia from a leukemoid reaction, assuming the animal can tolerate the procedure. if characteristic cytogenetic abnormalities can be found in dogs with cml, this analysis may be helpful. basophilic leukemia is diagnosed by finding excessive numbers of basophils in circulation and in bone marrow. basophilic leukemia must be differentiated from mastocytosis based on the morphology of the cell type present. basophils have a segmented nucleus and variably sized granules, whereas mast cells have a round-to-oval nucleus that may be partially or totally obscured by small, round, metachromatic-staining granules. this distinction is usually easy to make; however, in basophilic leukemia, changes in the morphology of the nucleus and granules make the distinction less clear. 85 essential thrombocythemia has been diagnosed based on finding persistent and excessive thrombocytosis (>600,000/µl) without circulating blast cells and in the absence of another mpd (e.g., pv), myelofibrosis, or disorders known to cause secondary thrombocytosis. 89 these include iron deficiency anemia, chronic inflammatory diseases, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, and absence of a spleen. thrombocytosis is transient in these disorders or abates with resolution of the primary disease. in essential thrombocythemia, platelet morphology may be abnormal, with bizarre giant forms and abnormal granulation. 90 in the bone marrow, megakaryocytic hyperplasia is a consistent feature, and dysplastic changes may be evident in megakaryocytes. 93 spurious hyperkalemia may be present in serum samples from dogs with thrombocytosis from any cause due to the release of potassium from platelets during clot formation. 114 measuring potassium in plasma is recommended in these cases and usually demonstrates a potassium concentration within reference interval. platelet aggregability has been appropriate markers for animal species (see later). increasingly, cytogenetic abnormalities are being identified in animal leukemias; cytogenetic analysis may yield important diagnostic and prognostic information and become a valuable tool for identifying targeted therapeutic approaches. although morphologic and cytochemical analyses have formed the mainstay of cell identification, newer technologies now are routinely used to classify leukemias by using monoclonal antibodies to detect antigens associated with certain cell types. cells can be immunophenotyped using flow cytometric analysis or immunocytochemistry. 20, 63, [105] [106] [107] [108] cells from both acute lymphoid leukemia and aml are positive for cd34. many lymphocyte markers, including cd3, cd4, cd8, cd18, cd21, cd45, cd79, and igg, are available for dogs and can be used to rule out lymphoblastic leukemia in dogs with acute leukemias. 63, 105 other markers include myeloperoxidase (mpo) and cd11b for myeloid cells and cd41 for megakaryoblasts. there is some overlap in expression of these cellular antigens. for example, canine (but not human) granulocytes express cd4. it is best to use a panel of antibodies (similar to using a battery of cytochemical stains) because antigens are often expressed on multiple lineages, and lineage infidelity can occur. these tests have become more valuable with the availability of canine reagents. currently, the acvp oncology committee recommends that the following immunophenotyping panel be done on bone marrow and/ or blood smears to characterize animal leukemias: for b lymphocytes, cd79a; for t lymphocytes, cd3; for myeloid cells, mpo and cd11b; for megakaryoblasts, cd41; for dendritic cells, cd1c; and for acute leukemias, cd34. 20 because of the degree of differentiation of cells in mpn, these disorders must be distinguished from nonneoplastic causes of lymphoma, could be used as maintenance therapy. 9, 118 another protocol that has been used in treating acute myeloblastic leukemia is presented in table 32 -13. regardless of the chemotherapy protocol used, significant bone marrow suppression will develop, and intensive supportive care will be necessary. transfusions of whole blood or platelet-rich plasma may be required to treat anemia and thrombocytopenia, and infection should be managed with aggressive antibiotic therapy. because of the generally poor response, the major thrust of therapy may be to provide palliative supportive care. in treating pv, therapy is directed at reducing rbc mass. the pcv should be reduced to 50% to 60% or by one-sixth of its starting value; phlebotomies should be performed as needed, administering appropriate colloid and crystalloid solutions to replace lost electrolytes; 20 ml of whole blood/kg of body weight can be removed at regular intervals. 67 in humans, phlebotomy continues to be the therapeutic approach used most frequently. radiophosphorus ( 32 p) has been shown to provide long-term control but can only be used in specialized centers. 121 the chemotherapeutic drug of choice is hydroxyurea, an inhibitor of dna synthesis. this drug should be administered at an initial dose of 30 mg/kg for 10 days and then reduced to 15 mg/kg po daily. 69 the major goal of treatment is to maintain the pcv as close to normal as possible. cml is best managed with chemotherapy to control the proliferation of the abnormal cell line and improve the quality of life. hydroxyurea is the most effective agent for treating cml during the chronic phase. 75, 122 the initial dosage is 20 to 25 mg/kg twice daily. treatment with hydroxyurea should continue until the leukocyte count falls to 15,000 to 20,000 cells/µl. 75, 79, 84 then the dosage of hydroxyurea can be reduced by 50% on a daily basis or to 50 mg/ kg given biweekly or triweekly. in humans, the alkylating agent busulfan can be used as an alternative. 123 an effective dosage has not been established in the dog, but following human protocols, 0.1 mg/kg/day po is given until the leukocyte count is reduced to 15,000 to 20,000 cells/µl. variably reported as impaired 90 or enhanced. 93 in the one dog in which it was measured, the plasma thrombopoietin (tpo) concentration was normal. 92 it is unclear whether tpo plays a role in essential thrombocythemia or is suppressed by the high platelet mass. elucidation of the pathogenesis of this disorder should be aided by the recent cloning of the genes for thrombopoietin and its receptor, the proto-oncogene mpl. 115 in mds, abnormalities in two or three cell lines are usually manifested in peripheral blood as neutropenia with or without a left shift, nonregenerative anemia, or thrombocytopenia. other changes include macrocytosis and metarubricytosis. the bone marrow is typically normocellular or hypercellular with an increased m : e ratio, and blasts cells, although increased, constitute less than 20% of nucleated cells; in a report of 13 dogs with primary or secondary mds, in all but one dog the blast cell percentage was less than 20%. 116 dysplastic changes can be detected in any cell line. dyserythropoiesis is characterized by asynchronous maturation of erythroid cells typified by large hemoglobinized cells with immature nuclei (megaloblastic change). if the erythroid component is dominant, the mds is called mds-er (see table 32 -12) . 3, 32 in dysgranulopoiesis, giant neutrophil precursors and abnormalities in nuclear segmentation and cytoplasmic granulation can be seen. finally, dysthrombopoiesis is characterized by giant platelets and micromegakaryocytes. myelofibrosis should be suspected in animals with nonregenerative anemia or pancytopenia, abnormalities in erythrocyte morphology (especially shape), and leukoerythroblastosis. bone marrow aspiration is usually unsuccessful, resulting in a "dry tap. " this necessitates a bone marrow biopsy taken with a jamshidi needle. 117 the specimen is processed for routine histopathologic examination, and if necessary, special stains for fibrous tissue can be used. because myelofibrosis occurs secondary to other diseases of bone marrow such as chronic hemolytic anemia or bone marrow necrosis, the clinician should look for a primary disease process. treatment of acute nonlymphocytic leukemias has been unrewarding to date. however, we have little information on the response of specific subtypes of leukemia to uniform chemotherapeutic protocols, in part due to the rarity of these disease processes and the paucity of cases in the literature. the veterinarian is advised to contact a veterinary oncologist for advice on new protocols and appropriate management of these cases. the therapeutic goal is to eradicate leukemic cells and reestablish normal hematopoiesis. currently, this is best accomplished by cytoreductive chemotherapy, and the agents most commonly utilized include a combination of ara-c plus an anthracycline, such as doxorubicin or cyclophosphamide, vincristine, and prednisone.* in humans, the introduction of cytosine arabinoside has been the single most important development in the therapy of acute nonlymphocytic leukemia. 120 in dogs, ara-c, 100 to 200 mg/m 2 , given by slow infusion (12 to 24 hrs) daily for 3 days and repeated weekly, has been used, as well as several other variations using subcutaneous injections of cytosine (see chapter 11) . doxorubicin, 30 mg/m 2 iv every 2 to 3 weeks, can be administered at intervals alternating with ara-c. if remission is achieved, as evidenced by normalization of the hemogram, the coap protocol (cyclophosphamide, vincristine (oncovin), ara-c, and prednisone), as described for canine • benzene (chronic exposure), and alkylating agents. 130 new classification systems have incorporated genetic mutations, more accurately reflect prognoses, and facilitate use of consistent categorization among institutions. 131 therapeutic modalities under investigation or development include combination chemotherapy, immunotherapy, cytokine therapy, drug-resistance modulators, proapoptotic agents, antiangiogenic factors, signal transduction-active agents, and bone marrow transplantation. the prognosis for mpn is better than for aml. for acute nonlymphocytic leukemias, the prognosis is better for children than adults, with only 10% of adults receiving chemotherapy maintaining remissions for more than 5 years. 130 the spontaneous canine diseases probably occur too infrequently to serve as useful models. myeloid neoplasms have been induced experimentally in the dog by irradiation and transplantation in an attempt to create models for study. many similarities between human and canine myeloid neoplasms exist, and veterinary medicine may benefit from any therapeutic advances made in the human field. despite response to chemotherapy and control for many months, most dogs with cml will eventually enter a terminal phase of their disease. in one study of seven dogs with cml, four underwent terminal phase blast crisis. 75 in humans, blast crisis may be lymphoid or myeloid. 124 in dogs, it is usually difficult to determine the cell of origin. these dogs have a poor prognosis, and the best treatment to consider, if any, would be that listed in table 32-13. it has now been documented that a subset of cml in dogs may be associated with a bcr-abl chromosomal abnormality (the so-called "raleigh chromosome") similar to the "philadelphia chromosome" translocation responsible for a large majority of cml in humans. 2 while imatinib mesylate (gleevec) is known to be an effective therapy for cml in humans, bcr-abl kinase inhibitors have, as yet, not been investigated for this subset of cml in dogs. few cases have been reported, but one dog was treated successfully with a combination chemotherapy protocol that included vincristine, ara-c, cyclophosphamide, and prednisone. 91 treatment is controversial in humans because of the lack of evidence that asymptomatic patients benefit from chemotherapy. patients with thrombosis or bleeding are given cytoreductive therapy. hydroxyurea is the drug of choice for initially controlling the thrombocytosis. 89 there is no standard therapeutic regime for mds. often, humans receive no treatment if the cytopenias do not cause clinical signs. transfusions are given when necessary, and patients with fever are evaluated aggressively to detect infections. growth factors, such as epo, gm-csf, g-csf, and il-3, are sometimes used in patients who require frequent transfusions to increase their blood cell counts and enhance neutrophil function. 125, 126 in one case report, human epo was administered (100 u/kg sq, every 48 hours) to a dog with mds because of profound anemia. the rationale for use of epo was to promote terminal differentiation of dysplastic erythrocytes. the pcv increased from 12% to 34% by day 19 of epo treatment. this dog remained in remission for more than 30 months. 32 other factors that induce differentiation of hematopoietic cells include retinoic acid analogs, 127 1,25 dihydroxyvitamin d3, 128 interferon-α, and conventional chemotherapeutic agents, such as 6-thioguanine and ara-c. 129 the propensity of these factors to enhance progression to leukemia is not known in many cases, but the potential risk exists. in general, the prognosis for animals with mpn is better than for dogs with aml, in which it is grave. the prognosis for pv and cml is guarded, but significant remissions have been achieved with certain therapeutic regimes and careful monitoring. animals commonly survive a year or more. 75, 84 development of blast crisis portends a grave prognosis. the pathophysiology and therapy of nonlymphocytic leukemia in humans are being studied intensively. myeloid neoplasms have been demonstrated to be clonal, with abnormalities evident in all hematopoietic cell lines. leukemogenesis is likely caused by mutation or amplification of proto-oncogenes in a two-step process that initially involves a single cell and is followed by additional chromosomal alterations that may involve oncogenes. 1, 15 these alterations are manifested as cytogenetic abnormalities. environmental factors known to cause leukemia are exposure to high-dose radiation, blurring of the distinction between mm and multicentric noncutaneous emp in cats and these two mrds will be discussed together in this species. although mm represents less than 1% of all malignant tumors in animals, it is responsible for approximately 8% of all hematopoietic tumors and 3.6% of all primary and secondary tumors affecting bone in dogs. 1, 2 in a compilation of bone marrow disorders in dogs (n = 717), mm represented 4.4% and 19.8% of all abnormal samples and neoplastic processes, respectively. 3 further, in a compilation of serum protein electrophoretic samples (n = 147 dogs), mm accounted for 4.3% of abnormal and 28.5% of neoplastic processes encountered, respectively. 4 early studies suggested a male predisposition, 5 although subsequent reports have not supported this. 1, 6 older dogs are affected with an average age of between 8 and 9 years. 1, 5, 6 in one large case series, german shepherd dogs were overrepresented based on the hospital population. 1 the true incidence of mm in the cat is unknown; however, it is a more rare diagnosis than in the dog, representing only 1 of 395 and 4 of 3248 tumors in two large compilations of feline malignancies and 0.9% of all malignancies and 1.9% of hematologic malignancies in another report. 7-9 mm represented 1.4% and 14% of abnormal and malignant serum protein electrophoretic samples, respectively, in a compilation of 155 feline samples. 10 mm occurs in aged cats (median age 12 to 14 years), most commonly in domestic short hairs and no sex predilection has been consistently reported, although a male preponderance may exist. 6, 9, 11, 12 mm has not been associated with corona virus or felv or fiv infections. the etiology of mm is for the most part unknown. genetic predispositions, molecular aberrations (e.g., c-kit), viral infections, chronic immune stimulation, and exposure to carcinogen stimulation have all been suggested as contributing factors. 6,13-18 suggestion of a familial association in cats follows cases reported among siblings. 12 evidence exists that molecular mechanisms of cellular control, including overexpression of cell cycle control components like cyclin d1 (see chapter 2) and receptor tyrosine kinase dysregulation may be involved in canine myeloma and plasma cell tumors. 17, 18 in rodent models, chronic immune stimulation and exposure to implanted silicone gel have been associated with development of mm, 13, 14 as have chronic infections and prolonged hyposensitization therapy in humans. 15 viral aleutian disease of mink results in monoclonal gammopathies in a small percentage of cases. 16 exposure to the agricultural industry, petroleum products, and irradiation are known risk factors for development in humans. [19] [20] [21] additionally, progression of solitary plasma cell tumors to mm has been reported in both dogs and cats, and a single case of a b-cell lymphoma progressing to mm exists in the dog. 22, 23 pathology and natural behavior multiple myeloma is a systemic proliferation of malignant plasma cells or their precursors arising as a clone of a single cell that usually involves multiple bone marrow sites in dogs. in cats, as previously stated, a blurring of the distinction of mm and multicentric noncutaneous emp within the mrd occurs because widespread abdominal organ involvement without significant bone marrow infiltration has been described in a significant proportion of cases in european compilations. 11, 24 because both mm and multicentric noncutaneous emp have a similar clinical course and widespread systemic involvement with hyperglobulinemia in cats, they will be  section d ig-secreting lymphomas and leukemias (including plasma cell leukemia). mm is the most important mrd based on clinical incidence and severity. there appears to be some discordance and below 37° c and require blood collection and clotting to be performed at 37° c prior to serum separation. if whole blood is allowed to clot at temperatures below this, the protein precipitates in the clot and is lost. pure light-chain m component is rare but has been reported in both dogs and cats. 38, 39 the pathology associated with mm is a result of either high levels of circulating m component, organ or bone infiltration with neoplastic cells, or both. associated pathologic conditions include bone disease, bleeding diathesis, hyperviscosity syndrome, renal disease, hypercalcemia, immunodeficiency (and subsequent susceptibility to infections), cytopenias secondary to myelophthisis, and cardiac failure. bone lesions can be isolated, discrete osteolytic lesions (including pathologic fractures) (figure 32-22, a) or diffuse osteopenias, or both (figure 32-23) . approximately one-quarter to two-thirds of dogs with mm have radiographic evidence of bony lysis or diffuse osteoporosis. 1, 5, 6 the incidence of radiographic skeletal lesions in cats varies tremendously within reports, from as few as 8% in european case series to as high as 65% in north american case series. 8, 9, 11, 12, 26 those bones engaged in active hematopoiesis are more commonly affected and include the vertebrae, ribs, pelvis, skull, and proximal or distal long bones. skeletal lesions are rare with igm (waldenström's) macrogammaglobulinemia, in which malignant cells often infiltrate the spleen, liver, and lymph tissue rather than bone. 6, [40] [41] [42] bleeding diathesis can result from one or a combination of events. m components may interfere with coagulation by (1) inhibiting platelet aggregation and the release of platelet factor-3; (2) causing adsorption of minor clotting proteins; (3) generating abnormal fibrin polymerization; and (4) producing a functional decrease in calcium. 6 to very large anaplastic round cells (often referred to as plasmablasts), with a high mitotic index representing early stages of differentiation. 5, 6, 9, 24, 25 binucleate and multinucleate cells are often present (see figure 7 -32, chapter 7). in 16 cats with mm in a north american case series, the majority (83%) of plasma cells were immature and had marked atypia, including increased size, multiple nuclei, clefted nuclei, anisocytosis, anisokaryosis, variable n : c ratios, decreased chromatin density, and variable nucleoli; nearly one-quarter had "flame cell" morphology characterized by peripheral eosinophilic cytoplasmic processes. 9 however, in a european compilation of feline multicentric noncutaneous mrd cases (n = 17), 78% had well-differentiated morphologies. 24 the authors of this latter case series developed a grading system dependent on the percentage of plasmablasts within the neoplastic cells in which well-differentiated, intermediate-grade, and poorly differentiated have less than 15%, 15% to 49%, and 50% or more plasmablasts, respectively. malignant plasma cells typically produce an overabundance of a single type of or component of immunoglobulin, which is referred to as the m component (figure 32-21) . the m component can be represented by any class of the entire immunoglobulin or only a portion of the molecule, such as the light chain (bence jones protein) or heavy chain (heavy chain disease) of the molecule. in the dog, the m component is usually represented by either iga or igg immunoglobulin types in nearly equal incidence, whereas the ratio of igg : iga in cats is approximately 5 : 1 in some reports and approximately 1 : 1 in others. 1, [5] [6] [7] [8] [9] 24, 26 that being said, in the author's (dmv) experience, the vast majority of canine cases are of the iga type. if the m component is the igm type, the term macroglobulinemia (waldenström's) is often applied. several cases of biclonal gammopathy in dogs and cats have been reported. 9, 11, 12, [27] [28] [29] [30] [31] [32] several cases of nonsecretory mm have been reported in dogs. 33, 34 rarely, cryoglobulinemia occurs in dogs with mm and igm macroglobulinemia and has been reported in a cat with igg myeloma. 6, [35] [36] [37] cryoglobulins are paraproteins that are insoluble at temperatures a b nonneoplastic immunoglobulin production. in the case of mm, an unbalanced excess of light-chain products may be produced. light chains are of low molecular weight and are normally filtered by the renal glomerulus, and their presence in urine can result in protein precipitates and subsequent renal tubular injury. the presence of light chains in urine without a concomitant monoclonal spike in serum, although rare, is indicative of pure light-chain disease. 38 tubules become obstructed by large laminated casts containing albumin, immunoglobulin, and light chains. 6, 38, 43, 44 bence jones proteinuria occurs in approximately 25% to 40% of dogs with mm. 1, 5, 6 bence jones proteinuria is reported to occur in approximately 40% of cats with mm/mrd. 9, 11 hypercalcemia is reported in 15% to 20% of dogs with mm and is thought to result primarily from the production of osteoclast-activating factor by neoplastic cells. 1, 6, 54 other factors, including increased levels of various cytokines, tnf, il-1, and il-6 have been implicated in human mm. in two dogs with mm and hypercalcemia, serum elevations in circulating n-terminal pthrp were noted. 55 hypercalcemia may also be exacerbated by associated renal disease. hypercalcemia, initially thought to be a rare event in cats with mm, occurred in 10% to 25% of recently reported cases. 9, 11, 12, 56 susceptibility to infection and immunodeficiency have long been associated with mm and are often the ultimate cause of death in affected animals. 1, 6, 26 infection rates in humans with mm are fifteen times higher than normal and usually represent pneumonia or urinary tract infections. 57 response to vaccination has also been shown to be suppressed in humans with mm. 57 immunoglobulin levels are often severely depressed in affected animals. 6 in addition, leukopenias may be present secondary to myelophthisis. variable cytopenias may be observed in association with mm. a normocytic, normochromic, nonregenerative anemia is encountered in approximately two-thirds of dogs with mm. 1, 5, 6 this can result from marrow infiltration (myelophthisis), blood loss from coagulation disorders, anemia of chronic disease, or increased erythrocyte destruction secondary to high serum viscosity. rare erythrophagocytic forms of mm have also been reported in both dogs and cats and may contribute to anemia. 58, 59 similar factors lead to thrombocytopenia and leukopenia in nearly one-third and onequarter of dogs with mm, respectively. in cats, approximately twothirds, half, and one-third will be anemic, thrombocytopenic, and neutropenic, respectively. 9, 11, 12 cardiac disease if present is usually a result of excessive cardiac workload and myocardial hypoxia secondary to hyperviscosity. 43, 45, 53 myocardial infiltration with amyloid and anemia may be complicating factors. nearly half of cats with mm in one report presented with a cardiac murmur, the etiology of which was not established. 9 three cats with hvs presented with congestive heart failure, murmurs, and echocardiographic signs consistent with hypertrophic cardiomyopathy. 53 clinical signs of mm may be present up to a year prior to diagnosis with a median duration of one month reported in dogs. 1, 9 in one cat, m-component elevations were detected 9 years prior to clinical presentation. in this latter case, the m-component elevation was consistent with monoclonal gammopathy of unknown significance (mgus). mgus (i.e., benign, essential, or idiopathic monoclonal gammopathy) is a benign monoclonal gammopathy that is not associated with osteolysis, bone marrow infiltration, or bence jones proteinuria. mgus has also been reported in dogs. 60, 61 signs of mm can be variable based on the wide range of pathologic effects one-quarter of cats have clinical evidence of hemorrhage. 1, 9, 11, 12 in dogs, nearly half have abnormal prothrombin (pt) and partial thromboplastin (ptt) times. thrombocytopenia may also play a role if bone marrow infiltration is significant (i.e., myelophthisis). hyperviscosity syndrome (hvs) represents one of a constellation of clinicopathologic abnormalities resulting from greatly increased serum viscosity. the magnitude of viscosity changes is related to the type, size, shape, and concentration of the m component in the blood. hvs is more common with igm macroglobulinemias due to the high molecular weight of this class of immunoglobulin. iga-secreting myelomas (usually present as a dimer in the dog), may undergo polymerization resulting in increased serum viscosity. 1, 6, 45 igg-associated hvs can also occur, albeit less frequently. high serum viscosity occurs in approximately 20% of dogs with mm and can result in bleeding diathesis, neurologic signs (e.g., dementia, depression, seizure activity, coma), ophthalmic abnormalities (e.g., dilated and tortuous retinal vessels, retinal hemorrhage [ figure 32 -24], retinal detachment), and increased cardiac workload with the potential for subsequent development of cardiomyopathy.* these consequences are thought to be a result of sludging of blood in small vessels, ineffective delivery of oxygen and nutrients, and coagulation abnormalities. hvs has been reported in cats with igg-, iga-, and igm-secreting tumors. 6, 8, [49] [50] [51] [52] [53] in several of these cases, relative serum viscosity was increased above control ranges. renal disease is present in approximately one-third to one-half of dogs with mm, and azotemia was observed in one-third of cats in one report. 1, 5, 9, 11 the pathogenesis of renal failure is often multifactorial and can ensue as a result of bence jones (light-chain) proteinuria, tumor infiltration into renal tissue, hypercalcemia, amyloidosis, diminished perfusion secondary to hyperviscosity syndrome, dehydration, or ascending urinary tract infections. 1, 6, 43, 44 normally, heavy-and light-chain synthesis is well balanced in serum biochemistry profile, and urinalysis. particular attention should be paid to renal function and serum calcium levels. if clinical hemorrhage is present, a coagulation assessment (e.g., platelet count, pt, ptt) and serum viscosity measurements are indicated. all animals should undergo a careful funduscopic examination. serum electrophoresis and immunoelectrophoresis are performed to determine the presence of a monoclonal m component (see and to categorize the immunoglobulin class involved. heat precipitation and electrophoresis of urine may be performed to determine presence of bence jones proteinuria possible. tables 32-14 and 32-15 list the relative frequencies of clinical signs observed in the dog and cat, respectively, based on a compilation of several reports.* bleeding diathesis is usually represented by epistaxis and gingival bleeding. funduscopic abnormalities may include retinal hemorrhage (see figure 32 -24), venous dilatation with sacculation and tortuosity, retinal detachment, and blindness. † cns signs may include dementia, seizure activity, tremors, and deficiencies in midbrain or brain-stem localizing reflexes secondary to hvs or extreme hypercalcemia. signs reflective of transverse myelopathies secondary to vertebral column infiltration, pathologic fracture, or extradural mass compression can also occur. 1, 6, 35, 62, 63 one case of ataxia and seizure activity in a dog with emp secondary to tumor-associated hypoglycemia has been reported. 64 additionally, paraneoplastic polyneuropathy has been reported in a dog with mm. 65 a history of chronic respiratory infections and persistent fever may also be present in cats. hepatosplenomegaly and renomegaly can occur due to organ infiltration. bleeding diathesis due to hvs is less common in the cat; however, epistaxis, pleural and peritoneal hemorrhagic effusions, retinal hemorrhage, and central neurologic signs have been reported. 6, 8, [49] [50] [51] [52] [53] polydipsia and polyuria can occur secondary to renal disease or hypercalcemia, and dehydration may develop. hindlimb paresis secondary to osteolysis of lumbar vertebral bodies or extradural compression has been reported in cats. 12, 66 diagnosis and staging the diagnosis of mm in dogs usually follows the demonstration of bone marrow plasmacytosis (see , the presence of osteolytic bone lesions (see , and the demonstration of serum or urine myeloma proteins (m component) (see . in the absence of osteolytic bone lesions, a diagnosis can also be made if marrow plasmacytosis is associated with a progressive increase in the m component. in the cat, because the degree of bone marrow infiltration may not be as marked, it has been suggested that consideration of plasma cell morphology and visceral organ infiltration (figure 32 -25) be given in cases with demonstrable m-component disease in the absence of marked (<20%) marrow plasmacytosis. 9, 11, 24 all animals suspected of plasma cell tumors should receive a minimal diagnostic evaluation including a cbc, platelet count, • because commercial urine dipstick methods are not capable of this determination. definitive diagnosis usually follows the performance of a bone marrow aspiration in the dog. a bone marrow core biopsy or multiple aspirations may be necessary due to the possibility of uneven clustering or infiltration of plasma cells in the bone marrow. normal marrow contains less than 5% plasma cells, whereas myelomatous marrow often greatly exceeds this level. current recommendations require more than 20% marrow plasmacytosis to be present, although a 10% cutoff in cats has been recently recommended with special attention to cellular atypia. 9 even the 10% threshold is problematic in cats, and cellular atypia and visceral organ involvement (assessed through needle aspiration cytology or tissue biopsy) should be considered equally important in the species. 9, 11, 24 rarely, biopsy of osteolytic lesions (i.e., jamshidi core biopsy; see chapter 24) is necessary for diagnosis in the dog. in one case of mm in a dog, splenic aspirates were diagnostically helpful. 67 overall frequencies of clinical diagnostic abnormalities for dogs and cats with mm are compiled from published series having at least five cases each and are listed in table 32 -16. histochemical and immunohistochemical analyses of cells or tissues suspected of mrd are more often applied in the case of solitary plasmacytomas or where emp is suspected in the absence of marrow involvement and will be discussed in subsequent sections; however, they have been occasionally useful in the diagnosis of mm. molecular diagnostic techniques for mm have received limited use thus far in veterinary oncology; however, determining clonality of the immunoglobulin heavy chain variable region gene has been performed in feline plasmacytoma and myeloma using parr techniques (see chapter 8), 68 and use of this technology in cases where diagnosis is not straightforward awaits further investigation. the author has used parr analysis both before treatment and after clinical remission in a small number of dogs with mm involved in clinical trials and documented its utility (1) for initial diagnosis and (2) to characterize molecular remission. routine thoracic and abdominal radiographs are recommended in suspected cases. occasionally, bony lesions can be observed in skeletal areas on these standard films, and organomegaly (liver, spleen, kidney) is observed in the majority of cats. 9, 11 abdominal ultrasound is recommended in all cats suspected of mm because this modality reveals involvement of one or more abdominal organs in the majority of cases. 9,11 these include splenomegaly with or without nodules, diffuse hyperechoic hepatomegaly with or without nodules, renomegaly, and iliac lymph node enlargement. in one case series in cats, 85% of organs with ultrasonographic abnormalities were subsequently confirmed to have plasma cell infiltration. 11 skeletal survey radiographs are recommended to determine presence and extent of osteolytic lesions, which may have diagnostic, prognostic, and therapeutic implications. although nuclear scintigraphy (bone scan) for clinical staging of dogs with mm has been performed, due to the predominant osteolytic activity with osteoblastic inactivity present, scans seldom give positive results and are therefore not useful for routine diagnosis. 69 in physician-based oncology, bone mineral density analysis (dual-energy x-ray absorptiometry [dexa] scan) to document osteoporosis, mri scan of bone marrow, and pet/ct are commonly used for staging; however, these modalities have not been applied consistently in the veterinary literature. a clinical staging system for canine mm has been suggested 1 ; however, at present, no prognostic significance has been attributed to it. • disease syndromes other than plasma cell tumors can be associated with monoclonal gammopathies and should be considered in any list of differentials. these include other lymphoreticular tumors (b-cell lymphoma, extramedullary plasmacytoma, chronic and acute b-lymphocytic leukemia), chronic infections (e.g., ehrlichiosis, leishmaniasis, fip), and mgus.* agent may more quickly alleviate systemic effects of the disease. cyclophosphamide is initiated at a dosage of 200 mg/m 2 iv, once, at the same time oral melphalan therapy is started. because cyclophosphamide is less likely to affect thrombocytes, it may be substituted in those patients in which thrombocytopenia has developed secondary to long-term melphalan use. chlorambucil, another alkylating agent, has been used successfully for the treatment of igm macroglobulinemia in dogs at a dosage of 0.2 mg/kg po, once daily. 6, 40 little or no clinical signs of toxicity result from this dosing schedule. chlorambucil has also been used in cats with mrd. 11 lomustine (ccnu), yet another alkylating agent, has been used in a limited number of cats with mm and a partial response has been reported following dosing at 50 mg/m 2 po, every 21 days. 74 evaluation of response to systemic therapy for multiple myeloma is based on improvement in clinical signs, clinicopathologic parameters, and radiographic improvement of skeletal lesions or ultrasonographic improvement of organ involvement. 1, 6, 11 subjective improvement in clinical signs of bone pain, lameness, lethargy, and anorexia should be evident within 3 to 4 weeks following initiation of therapy. objective laboratory improvement, including reduction in serum globulin, immunoglobulin, and calcium, along with normalization of the hemogram, is usually noted within 3 to 6 weeks ( figure 32-26) . radiographic improvement in osteolytic bone lesions may take months and resolution may only be partial. ophthalmic complications (including long-standing retinal detachments) and paraneoplastic neuropathies can be expected to resolve along with tumor mass. 48, 65 in cats responding to chemotherapy, clinical improvement is noted in 2 to 4 weeks and serum protein and radiographic bone abnormalities were greatly improved by 8 weeks. 11, 12 as previously discussed, complete resolution of mm does not generally occur and a good response is defined as a reduction in measured m component (i.e., immunoglobulin or bence jones proteins) of at least 50% of pretreatment values. 6 reduction in serum immunoglobulin levels may lag behind reductions in bence jones proteinuria because the half-lives are 15 to 20 days and 8 to 12 hours, respectively. 75 for routine follow-up, quantification of the increased serum globulin, immunoglobulin, or urine bence jones protein is performed monthly until a good response is noted and then every 2 to 3 months thereafter. repeat bone marrow aspiration or imaging (in the case of visceral disease) for evaluation of plasma cell infiltration may be occasionally necessary. bone marrow reevaluation is particularly prudent when cytopenias develop during chemotherapy, and drug-induced myelosuppression must be differentiated from myelophthisis due to neoplastic marrow recurrence. the long-term control of complications, including hypercalcemia, hvs, bleeding diathesis, renal disease, immunosuppression, ophthalmic complications, and pathologic skeletal fractures, depend on controlling the primary tumor mass. therapy directed more specifically at these complications may, however, be indicated in the short term. if hypercalcemia is marked and significant clinical signs exist, standard therapies, including fluid diureses, with or without pharmacologic agents (e.g., calcitonin), may be indicated (see chapter 5) . moderate hypercalcemia will typically resolve within 2 to 3 days following initiation of melphalan/prednisone chemotherapy. therapy for mm is directed at both the tumor cell mass and the secondary systemic effects they elicit. all diagnostic procedures should be completed before initiating primary therapy to ensure a diagnosis is complete and baseline values are procured for monitoring response. chemotherapy is effective at reducing myeloma cell burden, relieving bone pain, allowing for skeletal healing, and reducing levels of serum immunoglobulins in the majority of dogs with mm and will greatly extend both the quality and quantity of most patients' lives. mm in dogs is initially a gratifying disease to treat for both the clinician and the companion animal owner, although complete elimination of neoplastic myeloma cells is rarely achieved and eventual relapse is to be expected. unlike dogs, only one-half of cats with mm will respond to chemotherapy and most responses will be short-lived; however, several long-term responses (i.e., >1 year) have been reported and treatment should be attempted when educated clients decide on a therapeutic option.* melphalan, an alkylating agent, is the chemotherapeutic of choice for the treatment of multiple myeloma. 1, 6 in the dog, an initial starting dose of 0.1 mg/kg po, once daily for 10 days, is then reduced to 0.05 mg/kg po, once daily continuously. the addition of prednisone therapy is thought to increase the efficacy of melphalan therapy. prednisone is initiated at a dosage of 0.5 mg/kg po, once daily for 10 days, then reduced to 0.5 mg/kg every other day prior to discontinuation after 60 days of therapy. melphalan, however, is continued at 0.05 mg/kg/day until clinical relapse occurs or myelosuppression necessitates a dose reduction. the vast majority of dogs on melphalan and prednisone combination therapy tolerate the regimen well. the most clinically significant toxicity of melphalan is myelosuppression, in particular a delayed thrombocytopenia. cbcs, including platelet counts, should be performed biweekly for 2 months of therapy and monthly thereafter. if significant myelosuppression occurs (usually thrombocytopenia or neutropenia), reduction of the dosage or treatment frequency may be necessary. an alternative pulse-dosing regimen for melphalan (7 mg/m 2 po, daily for 5 consecutive days every 3 weeks) has been used successfully by the author in a small number of cases in which myelosuppression was limiting more conventional continuous low-dose therapy. this pulse-dose regimen is now being used first-line by the author with the caveat that long-term response data are currently lacking. melphalan and prednisone therapy can also be used in cats with multiple myeloma; however, it appears this protocol is more myelosuppressive than in the dog and careful monitoring is required. in the cat, a dosing schedule similar to the dog has been reported 12,26 ; 0.1 mg/kg (approximately 0.5 mg, or one-quarter of a 2 mg tablet) once daily for 10 to 14 days, then every other day until clinical improvement or leukopenia develop. long-term continuous maintenance (0.1 mg/kg, once every 7 days) has been advocated. 12 an alternative protocol advocated in the cat uses melphalan at 2 mg/ m 2 , once every 4 days continuously, and appears to be well tolerated. 11 cyclophosphamide has been used as an alternative alkylating agent or in combination with melphalan in dogs and cats with mm. 1, 6, 11 there is no evidence to suggest it is superior to melphalan therapy. in the author's practice, cyclophosphamide is limited to those cases presenting with severe hypercalcemia or with widespread systemic involvement in which a faster acting alkylating *references 6, 9, 11, 12, 31, 26. water intake at home is important, and occasionally, educating owners in subcutaneous fluid administration is indicated. continued monitoring of renal function is recommended along with follow-up directed at tumor response. patients with mm can be thought of as immunologically impaired. some have recommended prophylactic antibiotic therapy in dogs with mm 6 ; however, in humans, no benefit for this approach over diligent monitoring and aggressive antimicrobial management when indicated has been observed. 43 cidal antimicrobials are preferred over static drugs, and avoidance of nephrotoxic antimicrobials is recommended. pathologic fractures of weight-bearing long bones and vertebrae resulting in spinal cord compression may require immediate surgical intervention in conjunction with systemic chemotherapy. hvs is best treated in the short term by plasmapheresis.* whole blood is collected from the patient and centrifuged to separate plasma from packed cells. packed red cells are resuspended in normal saline or other crystalloid and reinfused into the patient. bleeding diathesis will usually resolve along with hvs; however, platelet-rich plasma transfusions may be necessary in the face of thrombocytopenia. renal impairment may necessitate aggressive fluid therapy in the short term and maintenance of adequate hydration in the long term. careful attention to secondary urinary tract infections and appropriate antimicrobial therapy is indicated. ensuring adequate the prognosis for dogs with mm is good for initial control of tumor and a return to good quality of life. in a group of 60 dogs with mm, approximately 43% achieved a complete remission (i.e., serum immunoglobulins normalized), 49% achieved a partial remission (i.e., immunoglobulins <50% pretreatment values), and only 8% did not respond to melphalan and prednisone chemotherapy. 1 longterm survival is the norm, with a median of 540 days reported (figure 32-27) . the presence of hypercalcemia, bence jones proteinuria, and extensive bony lysis are known negative prognostic indices in the dog. 1 the long-term prognosis for dogs with mm is poor because recurrence of tumor mass and associated clinical signs is expected. eventually, the tumor is no longer responsive to available chemotherapeutics and death follows from renal failure, sepsis, or euthanasia for intractable bone or spinal pain. 1, 6 the prognosis for mm in the cat is not as favorable in the short term as it is in the dog. 6, 9, 11, 12, 26 whereas most cats (approximately 60%) transiently respond to melphalan/prednisone or cop-based protocols, most responses are partial and not durable. typically, cats with mm succumb to their disease within 4 months. 8, 9, 12, 26 however, long-term survivors (>1 year) have been occasionally reported.* in one european case series, seven cats undergoing melphalan or cop-based therapy had a median survival of 9.5 months. 11 one investigator grouped mm in cats into two prognostic categories (table 32 -17) based on criteria known to predict behavior in dogs. 12 although no rigorous statistical analysis was performed on this small group of nine cats, the median survival for cats in "aggressive" and "nonaggressive" categories was 5 days and 387 days, respectively. experience in dogs with igm macroglobulinemia is limited. 6, [40] [41] [42] response to chlorambucil is to be expected, and in nine treated dogs, 77% achieved remission with a median survival of 11 months. 6 orthopedic stabilization of fractures should be undertaken and may be followed with external-beam rt (see . recently, inhibition of osteoclast activity by bisphosphonate drugs has been shown to reduce the incidence and severity of skeletal complications of mm in humans. 69 this class of drugs may hold promise for use in dogs and cats with various skeletal tumors; however, they have not been adequately evaluated in mrd. 78 when mm eventually relapses in dogs and cats undergoing melphalan therapy or in the uncommon case that is initially resistant to alkylating agents, rescue therapy may be attempted. the author has had success with vad, which is a combination of doxorubicin (30 mg/m 2 iv, every 21 days), vincristine (0.7 mg/m 2 iv, days 8 and 15), and dexamethasone sodium phosphate (1.0 mg/kg iv, once a week on days 1, 8, and 15), given in 21-day cycles. whereas most dogs initially respond to this rescue protocol, the duration of response tends to be short, lasting only a few months. high-dose cyclophosphamide (300 mg/m 2 iv, every 7 days) has also been used with limited success as a rescue agent. liposomal doxorubicin has produced a long-term remission in a dog with mm previously resistant to native doxorubicin. 79 mm is ultimately a uniformly fatal disease in most species, including humans, and thus significant effort is being placed on investigational therapies for this disease. currently, bone marrow ablative therapy and marrow or stem cell rescue, thalidomide (and other antiangiogenic therapies), bortezomib (a proteasome inhibitor), arsenic trioxide, the bisphosphonates, and several molecular targeting therapies are under investigation; however, their use in veterinary species is limited or completely absent at present. the promise of molecular targeted therapies is, however, foreshadowed by a case of a dog with mm that was resistant to melphalan, prednisone, and doxorubicin that subsequently achieved a partial response to tyrosine kinase inhibitor therapy (toceranib; see chapter 14, section b) that was maintained for 6 months. 18 rights were not granted to include this figure in electronic media. please refer to the printed publication. to be of low biologic aggressiveness, and most do not recur following surgical excision. 100 conversely, the majority of sops eventually progress to systemic mm; however, the time course from local tumor development to systemic mm may be many months to years. 33, 102 sops have been reported in the dog involving the appendicular skeleton, as well as the zygomatic arch, and ribs. 33 sops are less common in cats, and fewer reports exist in the literature. 11, 22, [103] [104] [105] [106] [107] they occur in older cats (mean ages 9 to 14 years), with no significant sex predilection. the skin is the most common site; however, other sites include the oral cavity, eye, gi tract, liver, subcutaneous tissues, and brain. reports exist of cutaneous emp in cats that progressed to systemic mrd. 11, 22, 105 clinical signs associated with sops relate to the location of involvement, or in those rare cases with high levels of m component, hvs may occur. most cutaneous plasmacytomas are solitary, smooth, raised pink nodules from 1 to 2 cm in diameter (see figure 32 -28), although tumors as large as 10 cm have been reported. combining large series, greater than 95% occur as solitary masses and less than 1% occur as part of a systemic mm process. 17, [80] [81] [82] [83] [84] [85] [86] 95, 96 cutaneous and oral emps usually have a benign course with no related clinical signs. gi emp, however, typically presents with relatively nonspecific signs, which may suggest alimentary involvement. colorectal plasmacytomas usually present with rectal bleeding, hematochezia, tenesmus, and rectal prolapse. 100 one case of ataxia and seizure activity in a dog with emp secondary to tumor-associated hypoglycemia has been reported. 64 sop is usually associated with pain and lameness if the appendicular skeleton is affected or neurologic signs if vertebral bodies are involved. the diagnosis of sop and emp usually requires tissue biopsy or fna for diagnosis. cells making up solitary plasmacytic tumors in both cats and dogs have been histologically classified into mature, hyaline, cleaved, asynchronous, monomorphous blastic and solitary collections of monoclonal plasmacytic tumors can originate in soft tissues or bone and are referred to as extramedullary plasmacytoma (emp) and solitary osseous plasmacytoma (sop), respectively. the systemic, multicentric, biologically aggressive emp syndrome encountered in cats 11, 24 has been discussed in the mm section and will not be included in this discussion. a number of large case compilations of cutaneous plasmacytoma have been reported in the dog. 17, [80] [81] [82] [83] [84] [85] [86] the most common locations for emp in the dog are cutaneous (86%; figure 32 -28), the mucous membranes of the oral cavity and lips (9%; figure 32 -29) , and the rectum and colon (4%). the skin of the limbs and head (including the ears) are most frequently reported cutaneous sites. other sites accounted for only 1% of the remaining cases and can include stomach, spleen, genitalia, eye, uterus, liver, larynx, trachea, third eyelid, sinonasal cavity, and intracranial sites. [87] [88] [89] [90] [91] [92] [93] [94] the american cocker spaniel, english cocker spaniel, and west highland white terrier (and perhaps yorkshire terriers, boxers, german shepherds, and airedale terriers) are at increased risk for developing plasmacytomas and the median age of affected dogs is 9 to 10 years of age. 86 cutaneous and oral emp in dogs are typically benign tumors that are highly amenable to local therapy. there exists, however, a rare form of multiple cutaneous plasmacytomas in dogs that is part of a more generalized biologically aggressive mm process. 95, 96 the natural behavior of noncutaneous/nonoral emp appears to be somewhat more aggressive in the dog. gi emp have been reported on a number of occasions in the veterinary literature, including the esophagus, 91 stomach, 97, 98 and small 99 and large intestine. [98] [99] [100] [101] metastasis to associated lymph nodes is more common in these cases; however, bone marrow involvement and monoclonal gammopathies are less commonly encountered. colorectal emps tend • • figure 32 -28 a cutaneous plasmacytoma on the limb of a dog. cases of sop in cats were recently reported; one was treated with external-beam rt and one managed with melphalan chemotherapy and both enjoyed durable remissions of greater than 4 years. 114 similarly, emp of the gi tract in humans are treated most commonly by surgical excision and thorough staging of disease. systemic therapy is not initiated unless systemic involvement is documented. systemic chemotherapy has been used following gastric emp in a cat; however, the utility of adjuvant therapy in the species is unknown. 115 long-term follow-up of patients with sop is indicated in order to recognize both recurrence of disease and systemic spread. careful attention is given to serum globulin levels, bone pain, and radiographic appearance of bone healing in cases of sop. restaging of disease, including bone marrow evaluation, is indicated if systemic spread is suspected. prognosis for solitary plasma cell tumors is generally good. cutaneous and mucocutaneous plasmacytomas are usually cured following surgical excision. 17, 86, 112 in large compilations of cases in dogs, the local recurrence rate was approximately 5%, and nodal or distant metastasis occurred in only 7 of 349 cases (2%). 17, [80] [81] [82] 86 new cutaneous plasmacytomas at sites distant from the primary developed in less than 2% of cases. neither tumor cell proliferation rate (as measured by ki67 immunohistochemistry) in the dog nor histopathologic grading in dogs and cats were prognostic in large compilations of cases, although it has been suggested that the polymorphous-blastic and plasmablastic type may act more aggressively in the dog and cat. 17, 24, 86 the presence of amyloid and overexpression of cyclin d1 (prognostic in human plasmacytomas) were not shown to be of prognostic value in dogs. 17 dogs with emp of the alimentary tract and other abdominal organs (e.g., liver, uterus) treated by surgical excision alone or in combination with systemic chemotherapy (if metastasis is present) can enjoy longterm survival in the majority of cases.* in a compilation of nine dogs with colorectal plasmacytoma, two dogs had local recurrence at 5 and 8 months following surgery, and the overall median survival was 15 months following surgery alone. 100 dna ploidy and c-myc oncoprotein expression in biopsy samples were determined to be prognostic for emps in dogs; however, those that were malignant were all from noncutaneous sites (i.e., lymph node, colon, spleen). therefore location appears to be as predictive. 116 as previously discussed, the majority of cases of sop will eventually develop systemic disease; however, long disease-free periods usually precede the event. the prognosis in cats is less well-defined 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polycythaemia vera in a bitch polycythemia vera in a dog polycythemia vera in dogs polycythemia vera in a dog diagnosis of canine primary polycythemia and management with hydroxy-urea cytogenetic analysis of leukemic cells in the dog: a report of 10 cases and a review of the literature trisomy 1 in a canine acute leukemia indicating the pathogenetic importance of polysomy 1 in leukemias of the dog canine cytogenetics-from band to base pair update on genomics in veterinary oncology classification of myeloid neoplasms: a comparative review working with canine chromosomes: current recommendations for karyotype description erythroblastic malignancy in a beagle irradiation-induced erythroleukemia and myelogenous leukemia in the beagle dog: hematology and ultrastructure acute monocytic leukemia in an irradiated beagle retrovirus-like particles associated with myeloproliferative disease in the dog hemopoietic stem cells, progenitor cells, and cytokines the hemopoietic colony stimulating factors cloning and expression of murine thrombopoietin cdna and stimulation of platelet production in vivo preleukemic syndrome in a dog clinicopathologic aspects of acute leukemias in the dog myelodysplastic syndrome in two dogs use of human recombinant erythropoietin and prednisone for treatment of myelodysplastic syndrome with erythroid predominance in a dog thrombocytosis associated with a myeloproliferative disorder in a dog myeloproliferative disease in the dog and cat: definition, aetiology and classification the leukemia complex ras, flt3, and c-kit mutations in immunophenotyped canine leukemias acute myelomonocytic leukemia in a dog acute myelomonocytic leukemia in a dog clinical-pathological findings and cytochemical characterizations of myelomonocytic leukaemia in 5 dogs acute myelomonocytic leukemia in a dog tumors of the lymphoid and hemopoietic tissues pathogenesis of myelofibrosis: role of ineffective megakaryopoiesis and megakaryocyte components megakaryozytenleukose beim hund development of a myeloproliferative disorder in beagles continuously exposed to 90 sr pyruvate kinase deficiency anemia with terminal myelofibrosis and osteosclerosis in a beagle myelofibrosis in the dog: three case reports enhanced granulocyte function in a case of chronic granulocytic leukemia in a dog cytochemical reactions in cells from leukemic dogs cytochemical characterization of leukemic cells from 20 dogs clinical diagnosis and management of acute nonlymphoid leukemias and chronic myeloproliferative disorders monoclonal antibodies that define canine homologues of human cd antigens: summary of the first international canine leukocyte antigen workshop (claw) hematologic abnormalities and flow cytometric immunophenotyping results in dogs with hematopoietic neoplasia: 210 cases identification of acute myeloid leukemia in dogs using flow cytometry with myeloperoxidase, mac387, and a canine neutrophil-specific antibody flow cytometric patterns in blood from dogs with non-neoplastic and neoplastic hematologic diseases using double labeling for cd18 and cd45 inappropriate erythropoietin production from a renal carcinoma in a dog with polycythemia renal carcinoma associated with secondary polycythemia in a dog secondary polycythemia associated with renal disease in the dog: two case reports and review of literature serum erythropoietin concentrations measured by radioimmunoassay in normal, polycythemic, and anemic dogs and cats serum erythropoietin concentrations in polycythemic and anemic dogs. proceedings of the 9th annual vet med forum (acvim) factitious hyperkalemia in dogs with thrombocytosis thrombopoietin: the primary regulator of platelet production cytologic evaluation of primary and secondary myelodysplastic syndromes in the dog how to collect diagnostic bone marrow samples kruth sa: polycythemia vera and glomerulonephritis in a dog greydanus-van-der-putten swm: polycythaemia vera in a dog treated by repeated phlebotomies polycythaemia vera in a dog polycythaemia vera in a dog identification of jak2 mutations in canine primary polycythemia chronic myelogenous leukemia in the dog blastic crisis in chronic myelogenous leukaemia in a dog chronic myelogenous leukaemia with meningeal infiltration in a dog chronic granulocytic leukaemia in a dog with associated bacterial endocarditis, thrombocytopenia and preretinal and retinal hemorrhages chronic granulocytic leukemia in a dog chronic myelogenous leukemia and related disorders bcr-abl translocation in a dog with chronic monocytic leukemia extreme monocytosis in a dog with chronic monocytic leukaemia a dog with myelodysplastic syndrome: chronic myelomonocytic leukemia treatment of basophilic leukemia in a dog basophilic leukemia in a dog basophilic leukemia in a dog eosinophilic leukemoid reaction in a dog chronic granulocytic leukaemia/eosinophilic leukaemia in a dog essential thrombocythemia probable essential thrombocythemia in a dog successful treatment of suspected essential thrombocythemia in the dog essential thrombocythemia in a dog: case report and literature review diagnostic and hematologic features of probable essential thrombocythemia in two dogs a review of myelofibrosis in dogs a retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital myeloproliferative disease in the dog and cat: clinical presentations, diagnosis and treatment current chemotherapeutic treatment approaches to the management of previously untreated adults with de novo acute myelogenous leukemia radiophosphorus ( 32 p) treatment of bone marrow disorders in dogs: 11 cases (1970-1987) enzymes and random synthetics busulfan versus hydroxyurea in long-term therapy of chronic myelogenous leukemia blast crisis of chronic granulocytic leukemia: morphologic variants and therapeutic implications myelodysplastic syndromes (clonal cytopenias and oligoblastic myelogenous leukemia) treatment of myelodysplastic syndromes with hematopoietic growth factors treatment of myelodysplastic syndromes with all-trans retinoic acid sustained haematological response to high dose oral alfacalcidol in patients with myelodysplastic syndrome treatment for the myelodysplastic syndromes acute myelogenous leukemia the 2008 revision of the world health organization (who) classification of the myeloid neoplasms and acute leukemia: rationale and important changes myeloma-related disorders in cats commonly present as extramedullary neoplasms in contrast to myeloma in human patients: 24 cases with clinical follow-up multiple myelomas in cats • figure 32-25 necropsy specimen of a spleen from a cat with multiple myeloma showing diffuse plasma cell infiltration serum protein electrophoresis in 147 dogs multiple myeloma in the dog diagnosis and management of monoclonal gammopathies a retrospective study of 395 feline neoplasms tumors and tumor like lesions multiple myeloma in 16 cats: a retrospective study serum protein electrophoresis in 155 cats myeloma-related disorders in cats commonly present as extramedullary neoplasms in contrast to myeloma in human patients: 24 cases with clinical follow-up multiple myelomas in cats a resume of the current status of the development of plasma cell tumors in mice induction of plasmacytomas with silicone gel in genetically susceptible strains of mice multiple myeloma and prolonged stimulation of res the development of myeloma-like condition in mink with aleutian disease clinico-pathological aspects of canine cutaneous and mucocutaneous plasmacytomas phase i doseescalating study of su11654, a small molecule receptor tyrosine kinase inhibitor, in dogs with spontaneous malignancies multiple myeloma and family history of cancer multiple myeloma: a case control study a case-control study of multiple myeloma in whites: chronic antigenic stimulation, occupation and drug use progression of a solitary, malignant cutaneous plasma-cell tumour to multiple myeloma in a cat evolution of a b-cell lymphoma to multiple myeloma after chemotherapy histopathologic, immunohistochemical, and cytologic analysis of feline myelomarelated disorders: further evidence for primary extramedullary development in the cat histological classification of hematopoietic tumors of domestic animals multiple myeloma in the cat biclonal gammopathy in a dog with myeloma and cutaneous lymphoma immunoglobulin a and immunoglobulin g biclonal gammopathy in a dog with multiple myeloma prognostic factors for multiple myeloma in the dog primary and secondary bone tumors in the dog a retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital treatment of three cats with hyperviscosity syndrome and congestive heart failure using plasmapheresis bone destruction and hypercalcemia in plasma cell myeloma parathyroid hormone (pth)-related protein, pth, and 1,25-dihydroxyvitamin d in dogs with cancer associated hypercalcemia hypercalcemia in two cats with multiple myeloma infections complicating multiple myeloma and chronic lymphocytic leukemia erythrophagocytic multiple myeloma in a cat phagocytic plasmacytoma in a dog a benign hypergammaglobulinemia mimicking plasma cell myeloma idiopathic monoclonal (iga) gammopathy in a dog cervical cord compression as a neurologic complication in an igg multiple myeloma in a dog vertebral plasma cell tumors in 8 dogs hypoglycemia and polyclonal gammopathy in a dog with plasma cell dyscrasia multiple myeloma with associated polyneuropathy in a german shepherd dog plasma cell sarcoma in a cat fine-needle aspiration of the spleen as an aid in the diagnosis of splenomegaly characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of b-cell neoplasia plasma cell neoplasms use of plasmapheresis and chemotherapy for treatment of monoclonal gammopathy associated with ehrlichia canis infection in a dog monoclonal gammopathy associated with naturally occurring canine ehrlichiosis hyperviscosity syndrome associated with lymphocytic leukemia in three dogs monoclonal gammopathy in a dog with visceral leishmaniasis hematological toxicity and therapeutic efficacy of lomustine in 20 tumor-bearing cats: critical assessment of a practical dosing regimen the treatment of multiple myeloma therapeutic plasmapheresis multiple myeloma in a cat bisphosphonates and cancer biclonal gammopathy associated with immunoglobulin a in a dog with multiple myeloma monoclonal gammopathies in the dog: a retrospective study of 18 cases (1986-1999) and literature review multiple myeloma in cats: variable presentation with different immunoglobulin isotypes in two cats detection of biclonal gammopathy by capillary zone electrophoresis in a cat and a dog with plasma cell neoplasia nonsecretory multiple myeloma in two dogs monoclonal gammopathy without hyperglobulinemia in 2 dogs with iga secretory neoplasm neurologic complications of iga multiple myeloma associated with cryoglobulinemia in a dog monoclonal cryoglobulinemia with macroglobulinemia in a dog monoclonal immunoglobulin g cryoglobulinemia and multiple myeloma in a domestic shorthair cat light-chain myeloma in a dog light-chain multiple myeloma in a cat different biological behaviour of waldenstrom macroglobulinemia in two dogs macroglobulinemia in the dog, the canine analogue of gamma m monoclonal gammopathy macroglobulinemia with hyperviscosity syndrome in a dog plasma cell tumors plasma cell neoplasms serum hyperviscosity syndrome associated with iga multiple myeloma in two dogs ocular lesions in a dog with hyperviscosity secondary to an iga myeloma blindness in a dog with iga-forming myeloma ophthalmic disease as the presenting complaint in five dogs with multiple myeloma immunoglobulin a myeloma in a cat with pleural effusion and serum hyperviscosity hyperviscosity syndrome with igm monoclonal gammopathy and hepatic plasmacytoid lymphosarcoma in a cat serum hyperviscosity syndrome associated with multiple myeloma in two cats serum hyperviscosity syndrome associated with igg myeloma in a cat primary igg secreting plasma cell tumor in the gastrointestinal tract of a dog colorectal plasmacytomas: a retrospective study of nine dogs metastatic extramedullary plasmacytoma of the colon and rectum in a dog solitary plasmacytomas of bone and extramedullary plasmacytomas histopathologic and immunophenotypic characterization of extramedullary plasmacytomas in nine cats intraocular extramedullary plasmacytoma in a cat extramedullary plasmacytoma and immunoglobulin-associated amyloidosis in a cat immunohistochemical staining of neoplastic and inflammatory plasma cell lesions in feline tissues immunoglobulin-producing tumours in dogs and cats identification of immunoglobulin light chains in canine extramedullary plasmacytomas by thioflavine t and immunohistochemistry immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors immunohistochemical detection of multiple myeloma 1/interferon regulatory factor 4 (mum1/irf-4) in canine plasmacytoma: comparison with cd79a and cd20 imaging diagnosis-fdg-pet/ct of a canine splenic plasma cell tumor survival data for canine oral extramedullary plasmacytoma: a retrospective analysis use of strontium-90 plesiotherapy for the treatment of a lingual plasmacytoma in a dog solitary plasmacytoma of bone in two successfully treated cats gastric extramedullary plasmacytoma in a cat analysis of dna aneuploidy and c-myc oncoprotein content of canine plasma cell tumors using flow cytometry response to liposomeencapsulated doxorubicin (tlc d-99) in a dog with myeloma extramedullary plasmacytomas in dogs: results of surgical excision in 131 cases mucocutaneous plasmacytomas in dogs: 75 cases cutaneous plasmacytomas in dogs: a morphologic and immunohistochemical study cutaneous plasmacytomas with amyloid in six dogs primary cutaneous plasmacytomas in the dog and cat an immunohistochemical study of canine extramedullary plasma cell tumours prognostic value of histopathological grading in canine extramedullary plasmacytomas extramedullary laryngeal plasmacytoma in a dog solitary extramedullary plasmacytoma of the canine larynx extramedullary plasmacytoma of the third eyelid gland in a dog sinonasal plasmacytoma in a cat solitary intracerebral plasmacytoma in a dog: microscopic, immunohistochemical, and molecular features extramedullary plasmacytoma in the trachea of a dog uterine extramedullary plasmacytoma in a dog a primary hepatic plasma cell tumor in a dog anaplastic atypical myeloma with extensive cutaneous involvement in a dog immunoglobulin a multiple myeloma with cutaneous involvement in a dog esophageal plasmacytoma in a dog extramedullary plasmacytoma of the gastrointestinal tract in two dogs for gi emp (including colorectal emp), endoscopic evaluation of the entire gi tract is recommended. a single case report of the use of pet/ct imaging for extramedullary splenic plasmacytoma in a dog exists; however, its utility remains unknown. 111 cutaneous and oral plasma cell tumors in the dog are almost always benign and carry an excellent prognosis following conservative surgical excision. 17, [81] [82] [83] [84] [85] [86] 100, 112 emps of the trachea, liver, and uterus have also been reported in dogs, and all had a benign course following local resection. [92] [93] [94] successful therapy with melphalan and prednisone has been rarely applied for a local recurrence or incomplete margins in dogs and cats. rt has been used infrequently for cases that are nonsurgical, including the application of strontium-90 plesiotherapy for lingual plasmacytoma in a dog. 113 surgery is recommended in combination with radiotherapy for those cases of sop in which the lesion results in an unstable, long bone fracture (see , or the patient is nonambulatory from neurologic compromise resulting from a vertebral body sop. in the latter case, spinal cord decompression, mass excision, and possibly spinal stabilization may be necessary. 63 radiotherapy can be used alone (i.e., without surgery) in those cases where fractures are stable, as a palliative measure for bone pain, or in the case of vertebral sop if the patient is ambulatory and stable. good local control is usually achieved; however, most go on to develop systemic multiple myeloma. 33, 63, 102 sop of the axial skeleton can be managed by excision or radiotherapy alone. there is controversy as to whether systemic chemotherapy should be initiated at the time of local therapy for sop when systemic involvement is not documented. systemic spread may not occur for many months to even years beyond primary sop diagnosis in humans and dogs, and studies in humans reveal no benefit derived from initiation of systemic chemotherapy prior to documentation of subsequent systemic spread. 44, 63 two polymorphous blastic cell types; however, no prognostic significance has been observed following classification, although it has been suggested that the polymorphous-blastic type may act more aggressively in the dog. 17,86,103 a different classification was proposed for emp in cats based on percentage of plasmablasts (see previous section), and some prognostic importance has been documented. 24 in the case of poorly differentiated plasmacytic tumors, immunohistochemical studies, directed at detecting immunoglobulin, light-and heavy-chains, mm-1/interferon regulatory factor-4 (mum1/irf4), and thioflavin t, may be helpful in differentiation from other round cell tumors. 33, 85, 86, [106] [107] [108] [109] [110] immunoreactivity has been demonstrated for canine igg f(ab) 2 and vimentin. 82 a variant characterized by an igg-reactive amyloid interspersed with the neoplastic cells has also been described. 83 a panel of mabs (recognizing tryptase, chymase, serotonin, cd1a, cd3, cd79a, cd18, mhc class ii) in association with a histochemical stain (naphthol as-d chloroacetate) has been advocated for use on formalin-fixed, paraffin-embedded sections of cutaneous round cell tumors to help classify poorly differentiated round cell tumors (mast cell tumors, histiocytomas, lymphomas, and plasmacytomas). 109 additionally, clonality of the immunoglobulin heavy chain variable region gene can be performed in plasmacytomas and myelomas using pcr technology, and this may have some diagnostic utility in difficult cases.it is important to thoroughly stage dogs and cats with plasmacytomas that are at higher risk for systemic spread if contemplating local or locoregional therapy without systemic therapy. this should include bone marrow aspiration, serum electrophoresis, abdominal ultrasound, and skeletal survey radiographs to ensure the disease is confined to a local site prior to initiation of therapy. this is most important in cases of sop and gi emp due to their relatively high metastatic rate and less important for cutaneous, oral, and colorectal plasmacytomas because of their more typical benign behavior. key: cord-331268-kzy33hdb authors: lynch, sharon g.; rose, john w. title: multiple sclerosis date: 1996-01-31 journal: disease-a-month doi: 10.1016/s0011-5029(96)90012-7 sha: doc_id: 331268 cord_uid: kzy33hdb abstract multiple sclerosis is a chronic disease that begins in late adolescence or adulthood. it is highly variable in its expression and severity. it is believed to be autoimmune in nature. the cause is unknown; both genetic and environmental factors have been implicated in the pathogenesis. ms generally presents with the acute or subacute onset of neurologic abnormalities that may wax and wane over many years. diagnosis is generally made by means of observation of the clinical course in conjunction with a neurologic examination and laboratory tests. these tests may include magnetic resonance imaging of the head and spine, lumbar puncture, and evoked potentials. treatment is based on general supportive care, the use of corticosteroids for relapses, and symptomatic management of ongoing problems. the frequency of relapses can be reduced with interferon-β (betaseron). copolymer 1 and interferon-β la are being evaluated by the u.s. food and drug administration for approval for use for reduction in the frequency of relapses in relapsing-remitting ms. treatment of chronic progression is often attempted with immunosuppressive agents such as corticosteroids, azathioprine, and cyclophosphamide. use of other agents is being investigated. multiple sclerosis (ms) is a chronic disease of the central nervous system that typically begins in late adolescence or early adulthood. the cause is unknown, although the disease is believed to be autoimmune in nature. both genetic and environmental factors have been implicated in the pathogenesis of ms. a viral cause has been postulated, but no single virus has been confirmed to be associated with ms. the pathologic features of ms include the presence of demyelinating areas in the white matter of the brain with perivascular in-flammation and relative sparing of the axons. plaques are commonly found in the periventricular areas of the cerebral hemispheres, in the optic nerves, the brainstem, the cerebellum, and the spinal cord. the presence of inflammation in ms plaques and the presence of oligoclonal immunoglobulin bands suggest an autoimmune basis of the disease. characterization of the inflammatory cells in the plaques and in the cerebrospinal fluid has revealed a predominance of t cells. this finding has focused a great deal of attention on the trimolecular complex, which consists of the major histocompatibility complex, the t-cell receptor, and the antigen. consistent associations with dr2, drbl501, dqb602, and the dw2 haplotypes have been identified in white persons. studies of restricted use of specific t-cell receptor regions in the immune process have not revealed a specific receptor in this disease. the antigen remains unknown, although many investigators are working with myelin basic protein and other proteins associated with myelin. two animal models, experimental allergic encephalomyelitis and theiler murine encephalomyelitis, are valuable in testing experimental immunotherapies and other aspects of autoimmune mediated demyelination. ms generally appears with the acute or subacute onset of neurologic abnormalities that may wax and wane over many years. common early symptoms include numbness, double vision, paraparesis, monoparesis, bladder control problems, optic neuritis, ataxia, or tremor. common ongoing symptoms include those just mentioned, vertigo, increasing spasticity, depression, emotional lability, gait abnormalities, fatigue, dysarthria, quadriparesis, constipation, incoordination, fatigue, and pain. diagnosis is made by means of observation of the clinical course in conjunction with the neurologic examination and laboratory tests. magnetic resonance imaging of the head and spine can be valuable in the evaluation of suspected ms. the presence of an elevated immunoglobulin g (igg) index or oligoclonal bands in the spinal fluid also can be helpful. evoked potentials can help confirm subclinical involvement of the eyes, vestibular function, or sensory tracts. the differential diagnosis of ms includes other demyelinating syndromes, particularly the monophasic syndromes, such as postinfectious encephalomyelitis, postinfectious transverse myelitis, and isolated optic neuritis. some infectious diseases, such as lyme disease, syphilis, and htlv-1 myelopathy, can be confused with ms. other autoimmune conditions, such as systemic lupus erythematosus, behcet's syndrome, sarcoidosis, and sjogren's syndrome, can cause symptoms similar to those of ms. some leukodystrophies and hereditary degenerative syndromes can be confused with ms. ms is often classified by its clinical course. benign ms is charac-dm, january terized by mild intermittent relapses with nearly complete resolution. relapsing-remitting ms is the most common form of the disease. it is characterized by episodes of acute or subacute neurologic dysfunction followed by periods of improvement and stabilization. secondary progressive ms begins with a relapsing-remitting course, but the disease gradually worsens, causing slow accumulation of neurologic signs and symptoms. ms that never has a relapsing-remitting course but begins with a slow progression of signs and symptoms is classified as primary progressive ms. treatment of ms is based on the progression of an individual case. general health measures include exercise, physical and occupational therapy, a balanced diet, and aggressive treatment of fever and overheating. treatment of relapses is recommended for moderate to severe relapses. corticosteroids are choice of treatment of relapses. steroids should be used with caution because of the large number of side effects associated with long-term use. the frequency of relapses can be reduced with interferon-i3lb (betaseron). copolymer 1 and interferon43la are being evaluated by the u.s. food and drug administration for approval for use in reduction of the frequency of relapses in relapsing-remitting ms. these drugs soon may be available for clinical use. treatment of chronic progression is often attempted with immunosuppressive agents such as corticosteroids, azathioprine, methotrexate, and cyclophosphamide (cytoxan). other agents under investigation are cladribine and intravenous immunoglobulin. symptomatic treatment of the chronic symptoms of ms is important. treatment of symptoms can help patients remain functional and comfortable even with relatively severe chronic problems. fatigue can be treated with rest breaks during the day, exercise, and energy-conservation techniques. medications that may help are amantadine hydrochloride and pemoline. spasticity is a severe problem that causes contractures, pain, insomnia, and increased fatigue. it can be treated conservatively with physical therapy, particularly stretching exercises. baclofen and diazepam can also be useful and are often used alone or in combination. in patients with severe spasticity, baclofen can be administered with an intrathecal pump. urinary dysfunction is a common problem. a urologist usually is needed to define the type of dysfunction present. a hypertonic, spastic bladder can be treated with anticholinergic agents. a hypotonic bladder may require intermittent or long-term catheterization. detrusor-sphincter dyssynergia may require a combination of anticholinergic agents and intermittent catheterization. urinary retention, which causes frequent bladder infections, may require acidification of the urine or long-term administration of antibiotics. patients with severe retention may require urinary diversion. sexual dysfunction often requires a multidisciplinary approach, including counseling, modification of sexual techniques, medication, or prosthetic devices for men. tremor can be a severe, intractable problem. medications include clonazepam, propranolol, acetazolamide, or diazepam. emotional problems are common in patients with ms. emotional lability may respond to tricyclic antidepressant medications. depression is treated with antidepressant agents and counseling. pain is a prominent concern in many patients with ms. dysesthetic pain can often be managed with tricyclic antidepressants, carbamazepine, phenytoin (dilantin), or valproic acid. musculoskeletal pain is treated with antiinflammatory medications and physical therapy. cognitive dysfunction can be a disabling and distressing component of ms. documentation with neuropsychiatric testing may be helpful in managing these problems. current investigations of ms center on the concept of autoimmunity, possibly mediated by a viral illness. studies designed to define the role of the immune system in ms may be useful. medications designed to reduce a specific autoimmune response and medications that assist in stimulation of remyelination or improvements in quality of life are being developed. over the past few years, great strides have been made in understanding the role of the immune system, in improving diagnostic capabilities, and in managing the problems associated with ms. as this trend continues, we may have more diverse and effective therapies for the management of ms. table 1 . jean martin charcot's description of the clinical and pathologic features of ms is the foundation of our knowledge of the disease.l the historical aspects of ms are reviewed in previous publications. 2s3 we are now entering a new phase of understanding brought about by careful clinical trials and the capability of monitoring the disorder with longitudinal magnetic resonance imaging (mri). in an individual patient, ms can be described by means of clinical observation. current concepts of the clinical courses, their relative frequencies, and mri characteristics of ms are portrayed in table 2 . investigations with mri have changed the concept of ms by demonstrating more evidence of disease activity than is expected from clinical examination. disease activity, as measured with mri, is particularly high among patients with chronic progressive disease. 4 the acute lesions of ms can now be demonstrated with gadolinium-enhanced mri. the initial event is associated with local disruption of the blood-brain barrier (fig. 1) . as the abnormality evolves, increased signal intensity becomes evident on t2-weighted images (figs. 2 and 3). the lesion may grow larger over a few days and then the areas of high signal intensity may begin to recede. over time, the lesions may completely resolve on tzweighted images. with each relapse, which is defined by new or newly enhancing lesions on mr images, the older areas of involvement may be reactivated. reactivation is associated with the development of permanent lesions on mr images. 5 clinical correlation is frequently observed with areas of contrast enhancement or abnormal signal intensity in the cerebellum, brainstem, or spinal cord. abnormalities in the cerebral hemispheres are frequently periventricular in distribution and only occasionally correlate with specific symptoms or signs.6,7 the accumulation of lesions in the frontal lobes is associated with a decline in memory.8 in addition, a change in the number of lesions on cranial mr images correlates with a change in overall clinical status as measured with standard scales.g observations made with mri are having a marked impact on both our basic knowledge of ms and on therapeutic trialsjo mri studies will provide considerable insight into the natural history of the disease and will be an excellent independent variable in future clinical trials. traditionally ms is thought to have a relatively high incidence in the northernmost latitudes of the northern hemisphere.l* this theory is based on the incidence of the disease in scandinavia and the northern united states. a similar association is documented in the southern hemisphere in australia and new zealand. these observations are supplemented by data from m igration studies, which demonstrate a relation between age at m igration and assumption of disease risk for the location. risk is conferred by exposure to an environcertain populations are susceptible to ms, and certain populations are resistant to ms. for example, lapps in scandinavia have a very low incidence of ms, even though they reside predominantly in the far northern latitudes. in north america the disease is infrequent among hutterites and native americans. ms is uncommon in japan. 13 the incidence of the disease in first-degree relatives of patients with ms is 20 times that of the general population,14 suggesting that genetic factors influence disease expression. the results of populatidn-based studies of twins offer evidence that environmental and genetic factors contribute to the development of ms. these investigations show that the concordance in monozygotic twins is greater than 30%. it is less than 5% in dizygotic twinp suggesting that although genetic factors are important, environmental exposure also is important for disease expression. it is now commonly accepted that multiple genes influence autoimmune diseases in both animals and human beings.16 therefore polygenic inheritance is postulated for ms. like other autoimmune diseases, ms is more frequent in women, with a ratio of 2:l. the pathologic features of multiple sclerosis were first described by charcot,l who recognized plaques in.the white matter (scleroses en plaque) during pathologic examination of brain sections. these plaques were demonstrated to lack myelin and to contain perivascular inflammation. these features were established as the pathologic hallmarks of ms. the following discussion centers on the typical findings in ms. comparisons are made between ms and other forms of inflammatory demyelinating disease. the distribution of plaques within the white matter is restricted to the central nervous system (cns). plaques are found frequently in a periventricular distribution in the cerebral hemispheres. some of these plaques may be associated with the distribution of terminal veins.17j8 plaques may occur anywhere within the white matter. when plaques are near the cortex, sparing of the subcortical myelinated fibers is often observed. plaques adjacent to gray matter may at times spread into the gray matter, including the cortex and deeper nuclei. plaques are frequently found in the white matter of the optic nerves, the brainstem, the cerebellum, and the spinal cord. plaques in these locations more frequently correlate with symptoms. within a plaque, axons are frequently preserved. 18 the evolution of a plaque is not known. mri investigations show that the blood-brain barrier is locally disrupted at the onset of symptoms. pathologists disagree as to whether demyelination precedes inflammation or is secondary to inflammation. at present the latter view predominates. in acute plaques, the inflammatory response of lymphocytes, plasma cells, and macrophages is capable of producing or augmenting demyelination by direct and indirect mechanisms. the inflammatory response is predominantly perivenular, with a lesser response at the edges of or within plaques. the macrophages associated with acute plaques characteristically contain myelin fragments or myelin breakdown products.lg lymphocytes may contribute to the pathologic process by means of direct or indirect pathways. direct mechanisms include antibodyand cell-mediated immunity. t-cell-mediated reactivity is favored because most inflammatory cells are t cells. indirect mechanisms include the secretion of lymphokines and cytokines. the ability of molecules such as tumor necrosis factor to damage myelin or oligodendrocytes is the focus of ongoing research.20 cytokines may influence macrophage activation, stimulating the phagocytosis of myelin. in addition, the release of heat shock proteins may result in stimulation of ty6 cells, resulting in increased cytotoxicity. the cns lesions of ms can be classified as early active, active, inactive, early remyelinating, and late remyelinating, according to histologic criteria. the features of these lesions are detailed in table 3 . studies of oligodendrocytes early in the course of ms have demonstrated relative preservation of these cells in some patients,z1,22 and remyelination is possible in these patients. other patients have a striking loss of oligodendrocytes, making remyelination unlikely. these differences may reflect the severity of the injury at a specific site of demyelination, or they may indicate that the pathogenesis of demyelination varies among patients with ms. this may imply that an autoimmune basis for ms has long been suspected because of the inflammation in the cns and the presence of oligoclonal bands in the cerebrospinal fluid (csf). the inflammatory response is primarily lymphocytic and mononuclear.2~3 the predominance of t cells among the lymphocytes has led investigators to evaluate the role of the t-cell receptor and its recognition of antigen combined with major histocompatibility antigens (mhc). this has been named the trimolecular complex.23 the t-cell receptor recognizes antigen in the context of the mhc molecule. in the case of mhc class ii molecules such as drz, the antigen fragments are bound in a cleft, which is presented to the tcell receptor for recognition. with regard to the components of the 18 in the context of the trimolecular complex, it is important to note that ms has been associated with certain mhc or human leukocyte antigen (hla) markers. a consistent observation is the association of dr2, drbl501, dqb602, and the du2 haplotype with ms.31 different hla associations are reported within ethnic groups. the mhc molecules may contribute to genetic susceptibility to the disorder, but they are only one of a number of factors that confer risk for the disease.32j3 the presence of oligoclonal bands in the csf of patients with ms is frequently observed (fig. 4) . these abnormal immunoglobulins are identified in a high percentage of patients with clinically definite ms, and they are present in approximately 60% of patients at the clinical onset of the disease. 34 the oligoclonal bands in ms are of unknown specificity. small percentages may bind to known viral antigens in some patients. consistent binding of these antibodies to specific viral polypeptides or viral oligopeptides with homology to myelin components has yet to be demonstrated. the oligoclonal bands are not specific to ms and can be observed in patients with cns infections such as syphilis, subacute sclerosing panencephalitis, viral encephalitis, or meningitis. 35, 36 if the infection is self-limited, the oligoclonal bands may be a transitory abnormality. in comparison, chronic infections of the cns are associated with persistence of the oligoclonal bands. in these settings, the antibodies that compose the oligoclonal bands have pathogen specificity. oligoclonal bands can be observed in patients with autoimmune diseases such as systemic lupus erythematosus. the probability that an environmental factor is involved in the pathogenesis of ms has stimulated interest in a viral cause. although viral isolates are reported from the cns of patients with ms,37-3g there are no consistent observations. attempts to detect viral nucleic acids by means of in situ hybridization and polymerase chain reaction (pcr) are in progress. these techniques are extremely sensitive and require rigorous controls. careful confirmation of any future viral isolates or viral nucleic acids by multiple laboratories is required. [40] [41] [42] [43] [44] [45] [46] recent studies of tropical spastic paraparesis demonstrate that the retrovirus human t-cell lymphotropic virus type i (htlv-i) is involved in the pathogenesis of this disorder, which shares some clinical features with ms.3ga it is clear, however, that htlv-i is not a pathogen in ms. 40 there remains the possibility that a retrovirus or endogenous retrovirus could contribute to the pathogenesis of ms. there is considerable interest in the possibility that exposure to a virus may lead to an immunopathologic condition that results in ms. of particular note are investigations that demonstrate the potential of molecular mimicry to produce autoimmunity. the term molecular mimicry arises from the demonstration of shared homology between normal human myelin proteins and viral polypeptides. if an immune response is mounted to such a viral epitope, then it may be perpetuated by exposure of the shared region on the normal human protein. in ms, homology between myelin antigens and viral peptides is established. thus this mechanism could result in cns demyelination after viral infection. autoimmunity could also result from superantigenic stimulation of t cells by viral or bacterial proteins. superantigens are capable of binding to specific t-cell receptor proteins, producing nonspecific stimulation of relatively large numbers of t cells, which might cause clonal expansion of t cells reactive to myelin or oligodendrocyte antigens.47s48 animal mqdels of demyelinating disease cns demyelination associated with inflammation is present in animal models of experimental allergic encephalomyelitis (eae) and theiler murine encephalomyelitis (tme). these models provide an opportunity for the investigation of autoimmune and virus-associated disease, respectively. eae is an autoimmune disease of the cns and a model for immunotherapy. a cd4+ t-cell population specific for a myelin antigen, either mbp or proteolipid protein, is required for initiation eae. eae and ms share characteristics that include cns demyelination, perivascular t cells, association with mhc class ii antigens, and possibly restricted tcr v-gene utilization.4g the murine adoptive transfer model has another important feature of ms: the chronic relapsing clinical course.4g this clinical course is useful for investigations of the immune response and immunotherapy not only during onset of the disease but also during relapse. the pathologic features of this murine transfer model are inflammation and prominent demyelination.4g-51 eae is not associated with an environmental factor. the tme model of immune-mediated demyelination is of particular interest because it has important parallels with postinfectious encephalomyelitis and ms. in this model, antecedent mild or even subclinical viral encephalitis is followed by a period of quiescence and the eventual onset of demyelination. 50 the virus is persistent during the demyelinating phase of the disease. this implies that either low-level expression of viral polypeptides or immunologic cross-reactivity between virus and myelin antigens is crucial for initiating demyelination. the demyelination in the tme virus model is mediated by t lymphocytes. these t cells may have viral specificity but produce demyelination. this mechanism would be relevant to ms if the suspected environmental factor were one or several viruses. as in ms and eae, t cells appear to initiate immunemediated demyelination in tme.!~~z~~ experimental immunotherapies are evaluated in these animal models and provide a basis for clinical trials in human beings. examples of these investigational treatments include cytokine transforming growth factor-i3, (tgf131,53 lymphokine-toxin,54 anti-t-cell receptor vb-specific monoclonal antibody,55,56 t-cell vaccination,57 blocking peptides, anti-adhesion molecule specific monoclonal antibodies,5g and nitric oxide synthetase inhibition. 60 these experimental models provide an invaluable resource for the study of immunotherapy. although these experimental models are not likely to mirror the pathogenesis of ms, they are extremely useful in the study of cns inflammation and demyelination. ms is primarily a disease of young adults. most patients report their first symptoms between the ages of 20 and 45 years. the disorder rarely appears before the age of 15 years or after the age of 50 years, although it has been reported to occur in both children and the elderly. the symptoms of ms in children are essentially the same as those in adults; ataxia, numbness, and visual disturbance are the most common presenting symptoms. in elderly persons, a progressive onset is more common. ms is characterized by episodes of neurologic dysfunction, followed by periods of stabilization or remission. symptoms, once they appear, may partially or completely resolve or may be permanent. these episodes tend to develop over hours or days. sometimes the symptoms occur with almost strokelike suddenness, or they may develop slowly over a few weeks. once the symptoms have developed, resolution generally occurs over weeks or months. certain signs and symptoms are more common in the early stages of ms. these include numbness, double vision, monoparesis, paraparesis, bladder control problems, optic neuritis, ataxia, or tremor (table 1) . 22 dm, january 1996 numbness can be difficult to evaluate. numbness that suggests early ms includes an ascending numbness beginning at the feet. this may be a sign of transverse myelitis. hemiparesthesia, bilateral hand numbness, and dysesthesia in both hands, both feet, or on one side of the body, also are early symptoms of ms. the numbness is usually present for days, weeks, or months. many patients describe numbness or paresthesia with no objective abnormalities. if objective sensory abnormalities occur, they are more commonly reduction of vibration, proprioception, or stereognosis rather than pain or fever. the diplopia that occurs with ms is frequently partial or complete internuclear ophthalmoplegia, which is often bilateral. a small percentage of patients have sixth nerve palsy" or, more rarely, third or fourth nerve palsy. ww sometimes monocular diplopia is a symptom of optic neuritis. optic neuritis is usually characterized by monocular blurred vision, sometimes with scotomata and often with alteration of color vision. retroorbital pain or headache is common in patients with active optic neuritis. 63 the pain may intensify with eye movement. motor weakness is usually accompanied by upper motor neuron signs, such as hyperreflexia or the babinski sign. paraparesis is the most common early symptom, but the weakness also can occur as hemiparesis or monoparesis. spas.ticity can be a later manifestation. signs and symptoms that commonly occur as ms progresses include vertigo, tremor, incoordination, increasing spasticity, depression, mood swings, cognitive abnormalities, impotence or other sexual dysfunction, weakness, lhermitte's sign, gait abnormalities, constipation, urinary incontinence, optic nerve pallor, fatigue, quadriparesis, dysarthria, loss of upper extremity coordination, and dysesthetic pain (table 1) . uncommon but important problems include seizures, atypical facial pain or tic douloureux (trigeminal neuralgia), bowel incontinence, swallowing problems, hearing loss, and dystonia. bell's palsy is sometimes seen in patients with ms (table 1) . the classic course of ms is one of intermittent neurologic signs and symptoms over many years. as time progresses, chronic problems accumulate. the amount of total disability varies from patient to patient. after a number of years, a patient's condition may stabilize permanently, but this does not always occur. d&f, january 1996 23 subtypes of disease ms can be divided into subtypes according to the course of the disease. there is a continuum among the various subtypes, and the disease in some patients does not fit into a pattern. benign ms accounts for 10% to 20% of cases and occurs more often in young women. in this type of ms, symptoms are mild and often sensory. resolution of neurologic problems is nearly complete. over the years, these patients rarely experience considerable disability. relapsing-remitting ms is the most common form of the disease. it is characterized by episodes of neurologic dysfunction [variably called exacerbations, relapses, or attacks) followed by periods of improvement and stabilization (called remissions). during a remission, not all symptoms resolve completely. the patient may be left with permanent disabilities, which may vary in severity. the condition of 30% to 50% of patients with an initial relapsingremitting course begins to worsen gradually over time, and neurologic signs and symptoms accumulate. this form of the disease is classified as secondary chronic progressive ms or relapsing-progressive ms. the latter term is also used to describe disease in patients who have sudden deteriorations in a stepwise manner without clinically significant recovery. primaryprogressive ms occurs in 10% to 20% of patients. disease in these patients begins with a slow progression of neurologic deficits with no history of relapse and may also have periods of stabilization or subacute worsening. common problems that appear and gradually worsen with time include spastic paraparesis, cerebellar ataxia, and urinary incontinence. clinical findings although no neurologic findings are pathognomonic for ms, certain abnormalities found during a physical examination can be helpful in providing a clue to the diagnosis of ms. these include internuclear ophthalmoplegia, which is rarely seen in other diseases and is especially rare in young adults. hyperreflexia and the babinski sign are common in early ms. optic nerve pallor can provide a clue to subclinical or resolved optic neuritis. altered color vision in one eye and a marcus-gunn pupil also are signs of optic neuritis. nystagmus is a common finding in patients with ms. many types of nystagmus are identified, including pendular nystagmus, small-amplitude nystagmus, or gaze-evoke nystagmus.63a65a66 absent abdominal reflexes in a slender patient who has not undergone an abdominal operation may be a helpful sign. a mild intention tremor with or without past-pointing is also an early sign, as is a positive romberg sign or difficulty with balance with 24 divz,january 1996 tandem gait. subtle motor weakness or spasticity may also be found. loss of vibratory or proprioceptive sensation in the lower extremities is common early in the course of the disease. ms should be strongly suspected in young or middle-aged adults who describe symptoms consistent with the lhermitte sign in the absence of obvious cervical cord abnormalities. the lhermitte sign consists of paresthesia or an electric shock-like sensation that radiates up the head or down the spine on neck flexion or extension. other important abnormalities are gait disturbances, persistent binocular double vision when looking in a particular direction, or a history of optic neuritis or transverse myelitis. fatigue and depression are not criteria for the diagnosis of ms. no laboratory test is universally diagnostic for ms. certain studies can be helpful in confirming the presence of separation of lesions in space and time. examination of the csf can be valuable for two reasons. first, the pattern of csf findings can help confirm the presence of demyelinating disease. the protein level is often slightly elevated but is rarely greater than 0.1 g/l unless the patient is experiencing a severe exacerbation, particularly optic neuritis or transverse myelitis. a modest elevation in cell count, generally less than 50/mm3, is seen in some patients. the cell pattern usually consists mostly of mononuclear cells. if more sophisticated testing is conducted, most cells can be identified as t lymphocytes. qualitative analysis of proteins can be helpful in suggesting the diagnosis of ms. at electrophoresis oligoclonal immunoglobulin bands can be identified in the csf but not in the serum of many patients with ms 34,67 (fig. 4) . the igg index, a comparison between igg levels in the csf and igg levels in the serum, is elevated in many patients with ms. 68,6g although these findings suggest ms, they also are found in other diseases, most commonly other inflammatory diseases of the cns. these diseases include lyme disease, systemic lupus erythematosus, progressive multifocal leukoencephalopathy, encephalitis, and subacute sclerosing panencephalitis. 35 the ver is abnormal in approximately 70% of patients with ms, regardless of whether there is a history of optic neuritis.70 a slowed ploo in a patient without a history of optic neuritis can be paraclinical evidence of a second lesion and can be used to confirm a diagnosis of ms (fig. 5) .6g the baer is more difficult to interpret than the ver and is abnormal in approximately 30% of patients with ms. in the baer, five 26 d&z, january 1996 consecutive waves are identified; these are numbered i-v the wave interval i-iii is considered the peripheral system. abnormalities in this wave suggest a lesion in the peripheral auditory nerve. the wave interval iii-v is generated from the central hearing areas in the brainstem. slowing in this area suggests a brainstem lesion. abnormalities in waves iii-v are seen in approximately 30% of patients with ms. 70 the sser is a technically more difficult study than the other responses, but it is useful for identification of slowed central conduction in the sensory pathway in the spinal cord and brain. the sser is abnormal in approximately 80% of patients with definite ms. 70 the sser also is useful in the identification of peripheral lesions, suggesting that peripheral neuropathy rather than a central lesion is the cause of numbness. the development of mri has been extremely important in both making the diagnosis of ms and helping researchers understand the dynamics of ms in patients with the disease. mri findings should be interpreted with caution, however. abnormal mri findings alone are not sufficient to confirm a diagnosis of ms without clinical evidence. 71, 72 in patients with ms, patchy areas of abnormal white matter are seen on t%weighted and spin-echo images. these are most commonly found in the cerebral hemispheres in the periventricular areas. in some patients, however, lesions also are identified in the brainstem and cerebellum. mri also helps identify lesions in the cervical and thoracic spinal cord. gadolinium enhancement can be seen around some lesions, particularly if a patient is having an exacerbation or fairly rapid chronic progression. gadolinium enhancement is considered a sign of an active lesion. (table 4 ).76-7g mr images should be interpreted with caution, particularly in patients with chronic illness of any kind or in patients older than 50 years. fazekas et a1.75 attempted to differentiate the mr images of healthy persons older than 50 years from those of patients with ms. they identified the following three criteria for the diagnosis of ms: lesions abutting the lateral ventricles, lesion diameter greater than 0.6 cm, and lesions present in the posterior fossa. if two of the three criteria were met, the specificity for ms was 88% and the sensitivity was 100%. a follow-up study in which 1500 consecutive mris were examined yielded a sensitivity of 81% and a specificity of 96%" these criteria may be useful in the interpretation of mri findings in some patients, but they should be used with caution for patients with other diseases that can affect mri, such as hypertension and diabetes mellitus. patients with those diseases were excluded from the study by fazekas et al. the size and area of the lesions present on mr images correlate poorly with the patient's disability. 4,81 many patients with large lesions on mr images have minor clinical findings, whereas some patients with small lesions have severe disability. one area in which mri may indicate the severity of the problem is in the cognitive status of the patient. an increase in the area of the lesions in the cerebral hemispheres or thinning of the corpus callosum may correlate with poor cognitive function. the presence of lesions in the spinal cord does not correlate with disease severity. a recent study in which body coil imaging was used showed that 74% of patients with ms had lesions in the spinal cord that were identified by this technique. 82 although the presence of lesions and the area and number of lesions did not correlate with a patient's level of disability, the presence of spinal cord atrophy did correlate with greater disability. 82 patients with partial or complete transverse myelitis who subsequently are found to have ms often have lesions on mr images that correspond to the level indicated by symptoms and the level of neurologic findings (simnad v, rose jw, manuscript in preparation). the use of mri for the follow-up evaluation of ms has become an integral part of research into the course of the disease. however, because mri findings do not correlate with a patient's clinical condition, new abnormalities on mr images in the absence of clinical worsening should not be treated as an exacerbation of the disease. new abnormalities can, however, indicate that the disease remains active. mri should be repeated in patients in whom the diagnosis has not been confirmed or in patients who have new symptoms that suggest a second disease. as the choice of treatments of ms increases, monitoring of disease activity may become useful in determining the course of treatment. optic neuritis is often seen as a first demyelinating episode in patients with ms. the diagnosis of ms should be considered in patients with optic neuritis, and a careful history and examination should be performed to exclude other neurologic abnormalities. however, many patients who have a single episode of optic neuritis never have other demyelinating episodes. one study of 60 patientsgo found that ms developed in 74% of women and 34% of men within 15 years of an attack of optic neuritis. transverse myelitis, inflammation of an area of the spinal cord causing ascending weakness and numbness up to the level of the lesion, can also be seen as the initial demyelinating event in ms. 88 other causes include infectious, postinfectious, and postvaccinal demyelination. 81 sometimes the cause is never determined. when transverse myelitis occurs, an imflammatory lesion can be identified on mri images of the cervical or thoracic spinal cord. estimates of the risk of ms after an isolated episode of transverse myelitis range from 50% to 80%.g1-g3 im'z, january 1996 29 the use of the cranial mr images in patients with optic neuritis or transverse myelitis may be helpful in predicting which patients are more likely to have additional problems. one prospective study identified patients with a single demyelinating episode such as optic neuritis or transverse myelitis. patients with abnormal mri findings at the time of the first episode had a 65% risk of a second episode within 5 years. patients with normal mri findings at the time of the first episode had a 5% risk of development of a second lesion in 5 years.g4 a syndrome in which optic neuritis and transverse myelitis develop with no other demyelinating events is called devic's neuromyelitis optica. in this disorder, cranial mri findings remain normal. this is considered a monophasic illness-both abnormalities occur within a year of each other, and patients may never have another demyelinating event. this is a rare syndrome.g5 the following characteristics are associated with a favorable prognosis: (1) female sex, (2) early age at onset, (3) onset of symptoms referable to a single neurologic system, (4) substantial recovery from relapses, (5) early symptoms of numbness rather than corticospinal or cerebellar symptoms. unfavorable prognosis is associated with chronic progressive disease (either primary or secondary), older age at onset, and male sex.g6-g8 dlagnostic criteria because of the difficulties involved in the diagnosis of ms, several criteria have been published to standardize the terms used to describe the certainty of the diagnosis. the two primary sets of criteria are those of poser et a1.6g and shumacher et a1. 83 the poser criteria are more recent and are summarized in table 5 . it is important to remember that no abnormality should be used as a criterion if it can be explained by another medical problem. other conditions may commonly be confused with ms and should be considered in the differential diagnosis. the differential diagnosis depends in part on the clinical and laboratory findings in an individual patient. postinfectious encephalomyelitis is a subacute syndrome, possibly caused by an autoimmune response to a viral infection. patients with this illness experience the acute or subacute onset of confusion, disorientation, gait abnormalities, loss of bowel or bladder control, weakness, or other symptoms. abnormalities in the white matter can be seen with mri, and evidence of inflammation frequently is seen in the cse the patient's condition may or may not return to normal; recovery may take months or even years. 84 lyme disease is a prominent concern and appears to be a cause of intermittent neurologic events, 85 the most common of which is bell's palsy. encephalomyelitis may develop, with vague symptoms of numbness, fatigue, and memory deficit. abnormalities in the white matter may be seen with mri, and csf findings may resemble those in ms, including mild leukocytosis and oligoclonal bands. patients may have a history of a tick bite, a rash, or recent arthralgia. lyme titers or a lyme pcr in the blood or csf may be helpful to these patients. 85 systemic lupus erythematosus is a well-known syndrome that may cause transverse myelitis, strokes, encephalopathy, and optic abnormalities. clues to the differential diagnosis are systemic abnormalities such as hematuria or leukopenia, arthritis, or an elevated antinuclear antibody titer, erythrocyte sedimentation rate, or other blood measurement. sometimes both systemic lupus erythematosus and ms occur in the same patient. primary cns vasculitis can cause a syndrome similar to ms. differentiating features include prominent headaches, confusion, and sudden strokelike episodes. an elevated erythrocyte sedimentation rate may be present in some patients, as may an elevated csf protein level. patients may have an abnormal cerebral angiogram. bi-opsy of the temporal lobe or meninges may be helpful in the diagnosis of this syndrome. 77 the htlv-i, a retrovirus, causes a syndrome known as tropical spastic paraparesis or htlv-i-associated myelopathy. it may cause progressive spastic paraparesis or generalized white matter disease. htlv-i is relatively rare in the united states but is present in some patients who have resided around the caribbean sea. 86 behqet's syndrome can cause mri findings identical to those in ms. cardinal features of behqet's syndrome include oral ulcers, genital ulcers, and uveitis. variable features include involvement of the skin, eyes, joints, lungs, intestines, and heart and venous thrombosis. neuropsychiatric symptoms, including quadriparesis, pseudobulbar palsy, cranial neuropathy, cerebellar ataxia, peripheral neuropathic lesions, or cerebral venous thrombosis may be present.7g,87 sarcoidosis and sjggren's syndrome are autoimmune diseases that may show lesions on mr images that resemble those of ms. meningeal enhancement is a clue to cns sarcoidosis. a chest radiograph may show granulomatous lesions suggestive of systemic sarcoidosis. although igg levels are raised in the csf of patients with cns sarcoidosis, oligoclonal bands are found in some patients. csf angiotensin-converting enzyme determination may be used to further differentiate cns sarcoidosis from ms.78 vitamin b deficiency and syphilis can cause posterior column abnormali& and dementia. tests for these problems should be performed when a patient with these symptoms is seen. certain leukodystrophies may appear in adulthood. these include adrenal leukodystrophy, krabbe's disease, and metachromatic leukodystrophy. mri findings in these diseases show large areas in which no normal white matter is present. female carriers of the adrenal leukodystrophy gene may have an ms-like syndrome.88~8g hereditary degenerative syndromes, such as familial spastic paraparesis, olivopontocerebellar degeneration, and spinocerebellar degeneration, may be confused with ms, particularly with primary progressive ms. in these diseases, mr images may be normal or may show atrophy of the brainstem, spinal cord, or cerebellum. the csf is normal in these patients. studies support the concept that exercise is beneficial for the patients with ms.ggjoo simple measures such as walking, using an exercise bicycle, and swimming may be of considerable value. exercise should be performed in a cool environment whenever possible to prevent heat-associated transient declines in neurologic function. swimming and water aerobics in pools that are not overly heated are particularly valuable, because the patient is cooled while exercising. physical and occupational therapy are often invaluable for maintenance or improvement of neurologic function. bracing disabled portions of limbs, particularly the ankle, provides considerable benefit. exercise regimens tailored to the patient may help to maintain or improve strength, range of motion, and mobility. devices that provide assistance with walking can be important in reducing the risk of falls, allowing for greater independence and increased activity. other assistive devices can be helpful in reducing fatigue and increasing independent activity. careful consultation with a specialist in rehabilitative medicine can assist the patient with management of work and daily activities.l"o it is advisable for persons with ms to maintain a balanced diet. weight control is a prominent concern. overweight patients with motor, sensory, or coordination deficits that impair ambulation are at particular risk of falls, which may result in serious injuries, including fractures. patients who are overweight and whose strength is decreased lose any reserve strength they may have because of their weight. some patients with ms lose weight and require dietary supplementation. patients with dysphagia may require feeding tubes to help prevent aspiration pneumonia. although various diets have been advocated for ms, there are no substantial data from controlled trials to support the assertions. as a general health measure, it is commonly suggested that patients with ms restrict cholesterol and fat in the diet. diets that meet the requirements of the american heart association are likely to be useful, because most patients with ms live into middle age and beyond. pregnancy is a concern among young women with ms. many studies of the effect of pregnancy on ms have been undertaken. an increased risk of exacerbations in the first 3 months postpartum has been reported.lol-la4 however, the risk of exacerbations during pregnancy appears to be unchanged or slightly reduced.lo5 overall longterm disability does not appear to be altered by pregnancy.lo4j05 the increased relapse rate seen during the postpartum period has been postulated to be caused by an increase in immune tolerance during pregnancy, followed by a return to normal in the postpartum dm, january 1996 33 period. it has also been postulated that the relapses are secondary to the decrease in the level of female hormones after parturition.lo*-lo3 in addition to the physical effects of pregnancy, another major concern is the care of an infant or child by a person with physical problems. persons with ms need to consider carefully whether they can handle the additional work of caring for a child. persons with chronic physical problems may need special provisions, such as extra assistance in the home or special equipment. the physician should discuss pregnancy, delivery, and child care with women of childbearing age. increased core temperature, whether due to heat exposure or to a febrile response, may lead to a transient increase in neurologic symptoms.lo6 if the event is due to heat exposure, the patient simply needs to rest in a cool environment and await recovery. if an infection is responsible, the source of the infection should be determined and treated. an antipyretic medication such as acetaminophen can then be administered. many patients with ms are susceptible to urinary tract infections and may not have clinical manifestations of the infection. in some patients this is due to impaired sensory capabilities, and some patients have chronic urinary symptoms that may not change substantially with an infection. one study of ms exacerbations pointed to an association with antecedent infection.lo7 if a patient has persistent worsening after an infection that has been appropriately treated and resolved, steroid therapy should be considered in the event the infection recurs. a relapse is considered to be the onset of new neurologic symptoms or marked worsening of old symptoms lasting longer than 24 hours. certain conditions may mimic an exacerbation and should be ruled out or treated before steroid therapy is considered. these include fever, infection (commonly urinary tract infection or viral illness), overheating, fatigue, severe emotional stress, or the effects of medications such as baclofen, which can increase weakness. if these problems are appropriately treated, the patient's condition usually improves. mild relapses may be best treated without steroid therapy. the symptoms include a mild numbness, mild changes in bladder function, mild optic neuritis (visual acuity better than 20/40), slight increase in spasticity, or a dysesthetic pain syndrome. any new abnormality that does not change a person's ability to perform his or her usual daily activities may not require steroid therapy. in these patients, rest is sometimes helpful. patients with more severe worsen-ing may benefit from steroid therapy. the symptoms include gait disturbances, severe numbness or paresthesia, moderate to severe paresis, moderate or severe optic neuritis, severe vertigo, or marked impairment of eye movement. it is often appropriate for the physician to observe the patient for a few days before making a decision about the use of steroids. standard therapy for many years, immunosuppression with corticotropin (acth) or steroids has been used in the treatment of the exacerbations of ms. the primary effect of these agents is to shorten the duration of an attack, and no benefit has been proven in the overall outcome from an attack. steroids should not be given until an abnormality resolves because this may never occur. acth was the first immunosuppressant to be widely used in ms.lo8 although it is still given to some patients who respond well to the medication, acth has been largely supplanted by other steroids, most commonly prednisone and methylprednisolone. many different regimens have been used. a typical regimen is 80 units by intravenous or intramuscular injection once a day for 10 days. prednisone is commonly used for mild or moderate exacerbations of ms. although low doses do not appear to have any effect on an exacerbation, larger doses do appear to shorten the duration of an ms attack.log there is no standard treatment regimen; a dose of at least 1 mg/kg per day is commonly recommended and should be continued for 7 to 10 days. our regimen is 80 mg once a day by mouth for 10 days, then tapered by 20 mg every 3 days. other regimens range from 10 days to 6 weeks or longer. methylprednisolone with sodium succinate (solu-medrol) is often used in the treatment of severe relapses, or when the patient's condition continues to worsen after several days of high-dose prednisone.'lo typical dosages range from 500 to 1000 mg/day and last from 3 to 14 days. a typical dose is 250 mg in 250 ml of 5% dextrose in water over 45 minutes every 6 hours to a total of 16 doses. another is 500 mg in 250 ml of 5% dextrose in water over 45 minutes every 12 hours for 10 doses. an oral prednisone taper over about 10 days to 2 weeks may be used afterward. one study of optic neuritis suggested that high-dose methylprednisolone produces more favorable results than oral prednisone for patients with poor visual acuity. this study showed only a faster recovery time; follow-up examinations at 1 year did not show any difference in final outcome."l the study involved patients who did not necessarily have a diagnosis of ms. however, a follow-up evaluation with patients in whom ms subsequently developed did suggest that the methylprednisolone-treated group had a longer time interval to the development of a second demyelinating event than dm,january 1996 3.5 those who received prednisone or placebo.l12 for this reason, some neurologists believe that all attacks of ms should be treated with intravenous methylprednisolone. the side effects of steroids are well known. these include nonspecific immunosuppression leading to opportunistic infections, induction of hyperglycemia, fluid retention, hypertension, emotional abnormalities, hypokalemia, peptic ulcers, occasional aseptic necrosis of the femoral head or other bones, and demineralization of bone. chronic use may lead to cataracts, osteoporosis, muscle wasting, hypertension, diabetes, increased susceptibility to infections, and a cushingoid appearance. steroids should be used with caution. we have found the,following precautions helpful: administration of calcium and possibly vitamin d during the administration of steroids and restriction of foods with a high sugar or sodium content. we encourage our patients to eat foods rich in potassium, such as bananas, orange juice, and tomatoes. patients who experience indigestion may benefit from the use of histamine blockers such as ranitidine. some patients may need sedation with diazepam or other agents because of severe mood swings, anxiety, or sleeplessness. patients who receive high doses of methylprednisolone should be observed for hypertension, electrolyte imbalance, and hyperglycemia. these problems should be treated appropriately. occasional psychiatric symptoms, including depression, psychosis, and severe anxiety, may necessitate cessation of steroid therapy. betaseron, a recombinant interferon-& has been approved by the u.s. food and drug administration (fda) for use in ambulatory patients with relapsing-remitting ms. this approval followed a 2year, controlled, double-blind study that showed in patients treated with 8 million units of betaseron administered subcutaneously every other day the relapse rate was reduced to 0.84 relapse per year compared with 1.27 relapses per year in patients given placebo.l13 an mri study performed with the same population revealed fewer new lesions in the treatment group than in the control group.lo the drug did not improve ongoing symptoms. the study was limited to patients with relapsing-remitting disease, and the findings should not be extrapolated to patients with chronic progressive disease. a study of the use of betaseron by patients with chronic progressive ms is planned. patients whose condition is stable would not benefit from the use of betaseron. there are problems with the use of betaseron. although the drug may be helpful in patients with frequent relapses, it does have seri-ous side effects. almost all patients experience local reactions at the site of injection, and some patients have had tissue necrosis at injection sites. the injection site must be changed regularly to reduce the likelihood of ulceration. many patients have a flulike reaction, which may include fever, chills, malaise, and myalgia. this reaction resolves with time and commonly lasts only a few months; however, it may last as long as a year. these symptoms can be partially controlled with acetaminophen or ibuprofen. liver function studies may show abnormalities, and leukopenia may be present. fatigue and emotional disturbances have been reported. our patients have experienced episodes of acute depression and anxiety, and one patient had an episode of uncontrollable rage. depression may necessitate temporary or permanent cessation of betaseron treatment. however, antidepressants, such as fluoxetine, sertraline, and paroxetine hydrochloride, may help counteract the depression. in a few cases, ms appears to worsen when the patient is taking betaseron. acute weakness develops in some patients with the first few injections. this is not always associated with fever and may resolve with time. menstrual irregularities have been reported, and betaseron cannot be used during pregnancy. some patients tolerate the medication better if the full dose is titrated up over approximately 1 month. periodic blood tests to check for leukopenia and abnormal liver function are suggested. clinical trials of other preparations of interferon-a and interferon-13 are nearing completion. one clinical trial involved administration of a weekly intramuscular injection of interferon4la. the results suggested that this drug reduces the likelihood of progression in patients with early disease. 114 a phase iii clinical trial of another investigational agent, copolymer 1, has been completed. this drug appears promising in reducing relapses and has a good safety profile.115j16 these agents will likely be available in the near future, pending fda approval. although most treatment aimed at chronic progression remains experimental, the use of intermittent intravenous methylprednisolone has become a common practice. most commonly, patients who experience subacute worsening may respond to a course of highdose solu-medrol similar to that given for a severe relapse. the condition of some patients appears to stabilize, at least temporarily, with this course of therapy. some patients with progressive disease may respond to a single dose of 1000 mg of solu-medrol in 250 ml of 5% dextrose in water given over 1 hour once a month for 6 to 12 months. subsequent treatments may be given every 6 to 8 weeks. azathioprine has been used for the treatment of chronic progres-sion with some success. studies have shown a modest benefit of azathioprine, primarily in stabilizing the condition of some patients.l17j18 patients who take this drug should be examined for leukopenia or hepatotoxicity. about 15% of patients are unable to tolerate azathioprine because of fever, rash, or nausea. patients with continued progression during therapy with azathioprine or solu-medrol may benefit from combined therapy. cyclosporine was evaluated in a multicenter clinical trial and was found to have modest clinical benefit.llg the prolonged use of cyclosporine in patients with chronic progressive ms was complicated by side effects, principally nephrotoxicity and hypertension. the use of cyclophosphamide in the treatment of chronic progressive ms is controversia1.120-122 the results of clinical trials of this agent in chronic progressive ms are contradictory. the drug may have use in rapidly progressive ms that does not respond to steroid therapy. further investigation with mri and neuropsychological testing and careful clinical assessment should resolve the controversy. a number of promising phase iii clinical trials of therapeutic agents for relapsing-remitting ms are being conducted. for two of these agents, the 2-year placebo-controlled phase has been completed. these are an interferon-& given once a week by intramuscular injection, and copolymer 1. both drugs reduce the frequency of relapses and favorably influence disability. the interferon-l3 is identical to human interferon-8 and differs from betaseron in that it has the sequence of amino acids and glycosylation of human interferon.l14 the results of a review of the safety profile of this drug compared with that of betaseron will be of considerable interest. copolymer 1 appears to have activity similar to that of betaseron with regard to reduction of relapses in ms.l15,116 the side-effect profile appears to be favorable compared with that of betaseron. laboratory investigations demonstrate additive effects of copolymer 1 and interferon-l3 in vitro. because the drugs theoretically act through different mechanisms, combined therapy might be possible. because of the results of a pilot study, oral myelin is being tested in a phase iii clinical trial. lz3 in the pilot trial, the efficacy of the drug was observed in only a subgroup of patients (dr2-negative men). two pilot studies of the use of methotrexate for ms have been performed.124j25 methotrexate in low doses is used for the treatment of rheumatoid arthritis, psoriasis, and crohn's disease. similar therapy may be of benefit to patients with advanced ms.lz5 a phase iii controlled trial and dose response testing will be of considerable interest. methotrexate should be used in clinical settings that allow careful neurologic and laboratory follow-up evaluation. 38 dm, january 1996 cladribine by intravenous administration appears to alter the progression of ms. lz6 the drug has relatively selective toxicity for lymphocytes; however, the side effects can be substantial. additional studies to evaluate dose and route of administration are being initiated. the clinical effects of repeated dosage with this medication also require study. immunoglobulin therapy may be useful in ms; however, controlled trials of intravenous immunoglobulin (ivig) must be completed.lz7 this therapy may be useful in relapsing disease and can be considered for patients with both ms and diabetes. ivig therapy is not necessarily benign and can be responsible for the transmission of viral hepatitis. several clinical trials of monoclonal antibodies are in progress. a number of monoclonal antibodies with specificities for either lymphocytes or adhesion molecules are being subjected to initial trials in human beings. a monoclonal antibody that appears to lower lymphocytes and have an appreciable effect on the lesions of patients with ms as seen on mr images is being studied.lz8 ,%klptomtic therapy one of the most important aspects of the treatment of ms is helping patients manage their ongoing symptoms. because of the chronic nature of the problems associated with ms, medication and adjustments in lifestyle are used to help patients cope with their disabilities. table 6 gives a summary of possible symptomatic treatments. fatigue can be disabling in patients with ms. it is described in different ways by different patients. the classic description of fatigue is increased weakness with exercise or as the day progresses. the patient may walk fairly well in the morning but need a cane or walker by afternoon. other descriptions include sudden attacks of sleepiness or excessive chronic sleepiness, even though the patient has had enough sleep at night.lzg patients who describe fatigue should be questioned closely about their sleep habits and other symptoms of depression. many patients with fatigue may have poor sleep habits or insomnia, which lead to daytime fatigue. depression is a common problem in patients with ms.130 if the fatigue is a product of depression, treatment of the depression should be helpful. fatigue is sometimes managed without medication. patients may respond to one or two brief (15 to 30 minutes) naps during the day. if this is not helpful or not possible, amantadine may be given to help control the problem. the mechanism of action of amantadine is not known, but it is helpful in approximately 40% of patients.lzg side effects, such as dizziness, headaches, nervousness, or edema, may limit the usefulness of the drug. pemoline is a cns stimulant that may be helpful in some patients.131 it should be used in low doses and should generally be given early in the day because it may cause insomnia. anxiety and anorexia are other problems that may occur with this drug. liver function studies should be performed periodically to monitor for hepatotoxicity. fluoxetine (prozac) may be helpful both to increase energy and to treat depression.*32 40 d&z, january 1996 vertigo vertigo can be an intractable and disabling problem. vertigo can occur in sudden spells that last a few minutes, or it can be chronic and last for hours. some physical therapy techniques involve habituation exercises to help with vertigo. medications that may be helpful include meclizine, promethazine hydrochloride, and low-dose diazepam. oscillopsia may occasionally respond to clonazepam or baclofen. vertigo with nausea and vomiting may respond to metoclopramide. spasticity can appear in many different ways. it may be seen at direct examination as a "catch" in the muscles with passive rapid movement of the limbs, or it may cause severe stiffness or rigidity. some patients may have severe spasms of the affected limb, which may be precipitated by movement or occur at night. these are most common in the lower limbs and may be either flexor or extensor spasms. the spasms can be quite painful. primary treatment of spasticity includes physical therapy with stretching exercises, combined with medication. baclofen is the most commonly used drug for spasticity, although its mechanism of action is not known. the dose of baclofen should be low when treatment begins and should be titrated slowly and carefully. patients who take an overdose of baclofen experience weakness. the dose of baclofen is extremely variable-some patients with only moderate spasticity tolerate high doses, whereas others with severe spasticity tolerate only low doses. other limiting side effects include drowsiness, confusion, and nausea. use of baclofen should not be discontinued abruptly but should be tapered over a few weeks.132 *133 diazepam in combination with baclofen may be helpful for patients with severe spasticity or those who cannot tolerate high doses of baclofen but need to control spasticity. diazepam can be used alone for spasticity, but it is not as effective as baclofen.133 diazepam can be particularly helpful for flexor or extensor spasms at night. dantrolene has limited value because of its hepatotoxicity and the weakness that accompanies the muscle-relaxant effect. it may be helpful in intractable cases of spasticity. the baclofen pump was developed for use in patients with intractable spasticity. 134 this device is an intrathecal pump with a subcutaneous reservoir of baclofen that administers continuous doses of baclofen directly into the spinal canal. this method of administration can be effective. with the lower dose delivered directly to the spinal cord, patients seem to have fewer side effects than with other routes of administration. dose levels can be programmed to change throughout the day, so patients with problems that are worse during the night or another part of the day can take increased doses of the drug during those times. tizanidine is an agent used outside the united states for spasticity. 135 it is being studied in the united states and may become available in the near future. other agents that may be useful in the treatment of spasticity include carbamazepine, phenytoin sodium, methocarbamol, and cyclobenzaprine hydrochloride. clonidine patches may be used for adjunctive therapy in patients with persisting spasms who are taking other drugs. spastic dysarthria is an uncommon symptom in ms. speech is hesitant and stuttering, and breath control is difficult. baclofen sometimes is helpful in this condition. bladder dysfunction is an extremely common problem in ms. examination of postvoid residual urine volume and urodynamic testing are extremely important in delineating the causes of bladder dysfunction. other urologic examinations, such as cystoscopy, may help eliminate mechanical problems as the cause of urinary dysfunction. consultation with a urologist skilled in the evaluation of neurologic dysfunction of the bladder is essential to the best therapeutic outcome. the most common problem is a spastic bladder. this is a small, hyperactive bladder. symptoms of this type of bladder dysfunction are urgency, increased frequency, and incontinence in which the bladder empties completely with brief warning. this condition can be treated with anticholinergic agents such as oxybutynin or propantheline .136j37 sometimes baclofen or amitriptyline can be of use in the control of this problem (table 7) . detrusor-external sphincter dyssynergia is a common problem. in this syndrome, the bladder attempts to empty, but the urethra remains closed. symptoms may be urgency and hesitancy, double voiding, and increased frequency with a feeling of incomplete emptying. anticholinergic or tricyclic agents alone may be of help with this syndrome, but more commonly a combination of anticholinergic drugs and intermittent catheterization is needed to control the problem. 137 the patient performs self-catheterization two to four times a day. a flaccid bladder is less common than the other types of bladder dysfunction. this is an enlarged bladder that empties poorly. symptoms include hesitancy, double voiding, a feeling of incomplete emptying, and dribbling incontinence. untreated urinary retention can result in hydronephrosis. urecholine can be of use in a few patients. frequently, however, a schedule of intermittent selfcatheterization may be needed (table 7) . patients with flaccid bladder or sphincter dyssynergia may have frequent urinary tract infections. acidifying agents such as hippuric acid or vitamin c may be useful in the prevention of infections.136 longterm administration of antibiotics should be avoided to reduce the risk z patients with severe bladder problems that are unresponsive to noninvasive therapy may require a chronic indwelling catheter or urinary diversion. these techniques may be required by patients who cannot perform intermittent self-catheterization. sexual dysfunction is common in both men and women with ms. women often report decreased sensation, lack of vaginal lubrication, difficulty achieving orgasm, or painful muscle spasms in the legs or pelvis during intercourse. men report diminished sensation and difficulty in achieving or maintaining an erection or experiencing orgasm. there is no simple answer to the sexual problems that occur with ms. a multidisciplinary approach is needed in which the physical and psychological aspects of sexual problems are considered. for women, treatment of muscle spasms with medications for spasticity may allow intercourse with less pain. techniques to increase vaginal and clitoral stimulation may help women experience orgasm. other methods of increasing arousal may be helpful. men are interviewed to determine whether there are other causes of erectile dysfunction. medications that may affect erectile function should be eliminated if possible. yohimbine, an a-2-adrenergic receptor antagonist, can sometimes help restore function in a patient with borderline function .138 other methods, including papaverine or phentolamine injections, a vacuum erectile device, or a penile prosthesis, may be considered.137 inappropriate affect can be a problem in patients with ms. many patients have severe mood swings that can affect both their work and their social relationships. low-dose amitriptyline or another tricyclic antidepressant is frequently helpful in controlling mood swings.13g depression is a common problem in ms.130j32,140 the suicide rate among persons with ms is estimated to be 7.5 times that of the healthy population. 130 whether the depression is a primary symptom of ms or a situational problem is not known. physicians should be alert to the possibility of depression in their patients. full-dose antidepressant medications and psychological counseling may be beneficial. tremor can be a limiting factor in many patients with ms. treatment with medications is frequently unsuccessful. agents that may be useful include clonazepam, acetazolamide, propranolol, primidone, and diazepam. 132 isoniazid has been reported to be helpful in some patients. 141 we have found clonazepam to be the most helpful of these agents in our patients, but treatment may be limited by drowsiness. a common misconception is that pain is not a symptom in patients with ms. the truth is that pain is often a problem and may be a prominent concern for patients with ms.142 this can be a primary factor in the disease, or it can be a consequence of disability associated with the disease. much of the pain reported with ms is musculoskeletal and is related to abnormal use of muscles and joints. for example, patients who use a wheelchair may experience wrist, shoulder, or elbow pain from manipulating the wheelchair. patients with paraparesis or ataxia may experience back or leg pain from poor posture and balance when walking. these problems should be treated with antiinflammatory medications and physical therapy. primary ms pain is often dysesthetic.14z the patient describes a burning sensation or perhaps even electric shock-like pain. this pain can be in any location, but it is most commonly in the lower extremities. some patients experience tic douloureux or atypical facial pain. this primary pain may be controlled with tricyclic antidepressants, phenytoin, or carbamazepine.142 in patients with refractory pain, valproic acid can be tried. 13z headaches can become a problem in patients with ms. it is not known whether these headaches are caused by ms or are a separate problem. both tension and migraine headaches are common, and treatment is similar to the treatment of headaches in patients who do not have ms. retro-orbital pain is frequently observed in patients with optic neuritis. these patients may require steroid therapy. spasticity and muscle spasms can cause severe pain. treatment of the spasticity helps the pain. many patients with ms experience cognitive abnormalities. unlike the dementia of alzheimer's disease, the cognitive deficits seem to be more scattered and tend to be retrieval deficits rather than memory loss. 143 patients can have substantial cognitive difficulties but still have normal mini-mental state examination findings. neuropsychological studies have shown that as many as 40% of patients may have some cognitive difficulties.143 these difficulties can be important in terms of disability and ability to cope with illness. only a minority of patients have severe cognitive abnormalities. mr images in patients with cognitive problems tend to show a larger number and size of lesions in the white matter of the cerebral hemispheres. frontal lesions are more common in patients with cognitive difficulties.* the corpus callosum may be thinner than normal, as seen on sagittal images.8 patients with cognitive problems should undergo careful neuropsychiatric testing. sometimes depression or anxiety can be contributing factors in these symptoms. the minnesota multiphasic personality index or the beck depression scale in conjunction with cognitive testing may be helpful in differentiating emotional problems from structural cognitive deficits. proper treatment of the anxiety or depression may lead to improved cognitive function. recognition of the areas and degree of cognitive difficulty in patients with ms may be helpful in the care of the patients. patients may be able to learn ways of working around a problem. problems with a job may be related to cognitive problems, and ways of altering the job may be found. patients may become disabled from working because of these problems. this testing also may help the family understand the need for helping the patient deal with problems that have become too difficult to handle alone. cognitive rehabilitation techniques are being tested for patients with ms in some centers. further investigation is needed to evaluate the efficacy of these techniques. careful assessment of the patient's abilities and disabilities is crucial for proper management. in many patients, chronic symptoms cannot be prevented. symptomatic therapies are often effective for alleviating the afflictions produced by ms and for allowing the patients to live a productive and comfortable life. the cause of ms is unknown. theories revolve around the idea that the disease is either autoimmune or virus-mediated. it is still reasonable to question which pathologic feature is the inciting event. much research is focused on the t cell and potential mechanisms by which these cells could initiate ms. hla associations are found in many populations; however, hla markers are neither necessary nor sufficient to confer disease susceptibility, and other factors that confer disease susceptibility are being sought. at this time there is no confirmed evidence of a viral cause of ms. investigations with in situ hybridization and pcr technology are being conducted in an attempt to identify viral nucleic acids in the cns. perhaps these techniques will assist in unraveling the pathogenesis of ms. an intriguing possibility is that molecular mimicry may be re-sponsible for the initial generation of autoreactive lymphocytes. this mechanism involves exposure to viral or bacterial antigens, which generates an immunologic response that consists of reactive t-cell populations. because t cells cross-react with myelin peptides, a potential for demyelination exists. this theoretic mechanism is known to cause demyelination in rabbits. 144 an interesting investigation of human mbp-reactive t cells demonstrates that mbp-specific t-cell clones can recognize multiple viral polypeptides presented by dr2 or dql mhc antigens. 145 this would imply that ms could be generated by exposure to any one of a number of antigenic stimuli, such as influenza viruses or herpesviruses or even bacterial antigens. selected activated t-cell populations that enter the cns could then recognize a myelin epitope and initiate the autoimmune response, which would persist long after the inciting infection was cleared. recent investigation with mr spectroscopy demonstrates that white matter outside ms plaques may be abnorma1.14" these findings may signify that there is a fundamental abnormality in the white matter. whether these findings are secondary to genetic, biochemical, autoimmune, or viral factors remains to be determined. despite the deficiencies in our understanding of disease pathogenesis, therapy for ms has advanced. phase iii clinical trials with interferon-8 and copolymer 1 have demonstrated modest but definite benefit. the mechanisms by which these drugs favorably influence the clinical course of ms remain to be elucidated. recent studies of chemotherapeutic agents suggest that control of chronic progressive disease may be a real possibility. future clinical trials will attempt to define the efficacy of and parameters for these therapies. another question that remains unanswered is whether the use of multiple-drug therapy might be beneficial in the treatment of ms. for example, combined therapy with interferon-i3lb and copolymer 1 may produce more benefit than either drug alone. in chronic progressive disease, the use of solu-medrol in combination with another immunosuppressant such as azathioprine or methotrexate also should be explored. remyelination is another topic of interest for future research. research is being conducted into the use of ivig as a remyelinating agent. in addition, oligodendrocyte transplant experiments are being conducted in canine modes and may eventually be used for human patients. research involving medications to improve the symptoms that limit the lives of many patients with ms is ongoing and should continue. 4-amino-pyridine and 2,3-diamino-pyridine are being studied as agents that may improve conduction through poorly myelinated areas. these agents may reduce double vision, improve strength, and possibly reduce tremor. more research is needed to evaluate these dm,january 1996 47 and other compounds that may improve the quality of life of many patients with ms. although the cause of ms remains a mystery, important advances have been made in the understanding and treatment of ms in the past few years. as this trend continues, we may have more diverse and effective therapies to offer patients with ms in the years to come. lectures on the diseases of the nervous system multiple sclerosis multiple sclerosis benign versus chronic progressive multiple sclerosis: magnetic resonance imaging features neuroimaging in multiple sclerosis acute vith cranial nerve dysfunction in multiple sclerosis genetic epidemiology of multiple sclerosis: a survey genetics of multiple sclerosis a population-based study of multiple sclerosis in twins: update genetic analysis of autoimmune type i diabetes mellitus in mice demyelinating diseases oligodendrocytes in the early course of multiple sclerosis allen iv pathology of multiple sclerosis tumor necrosis factor identified in multiple sclerosis brain histopathology and the bloodcerebrospinal fluid barrier in multiple sclerosis multiple sclerosis: oligodendroglia survival and proliferation in an active established lesion isolation of myelin basic proteinreactive t-cell lines from normal human blood assessment of antigenic determinants for the human t cell response against myelin basic protein using overlapping synthetic peptides heterogeneity of the t-cell receptor beta gene rearrangements generated in myelin basic proteinspecific t-cell clones isolated from a patient with multiple sclerosis a myelin basic protein peptide is recognized by cytotoxic t cells in the context of four hla-dr types associated with multiple sclerosis t and b cell responses to myelin-oligodendrocyte glycoprotein in multiple sclerosis antibodies to myelin-oligodendrocyte glycoprotein in cerebrospinal fluid from patients with multiple sclerosis and controls multiple sclerosis: cells secreting antibodies against myelin-associated glycoprotein are present in cerebrospinal fluid multiple sclerosis is associated with genes within or close to the hla-dr-dq subregion on a normal dr15, dq6, du2 haplotype the molecular and genetic basis of neurological disease linkage strategies for genetically complex traits. part 1. multilocus models multiple sclerosis: diagnostic usefulness of cerebrospinal fluid immunoglobulin abnormalities in the guillain-barre syndrome relationship between measles virus-specific antibody activities and oligoclonal igg in the central nervous system of patients with subacute sclerosing panencephalitis and multiple sclerosis multiple sclerosis: relationship to a retrovirus? multiple sclerosis and human tcell lymphotrophic retroviruses amplification and molecular cloning of htlv-i sequences from dna of multiple sclerosis patients htlv1 and tropical spastic paraparesis the g and brahic m analysis of human tlymphotrophic virus sequences in multiple sclerosis tissue serologic studies of ms patients, controls, and patients with other neurologic diseases: antibodies to htlv i human t lymphotrophic virus type i may not be associated with multiple sclerosis in japan detection of human t-cell lymphoma virus type i dna and antigen in spinal fluid and blood of patients with chronic progressive myelopathy detection of coronavirus rna and antigen in multiple sclerosis brain bacterial toxin superantigens activate human t lymphocytes reactive with myelin autoantigens v-beta specific stimulation of human t cells by staphylococcal toxins the t lymphocyte in experimental allergic encephalomyelitis adoptive transfer of myelin basic proteinsensitized t cells produces chronic relapsing demyelination disease in mice adoptive transfer of experimental allergic encephalomyelitis in sjl/j mice after in vitro activation of lymph node cells by myelin basic protein: requirement for lyt-1+2-t lymphocytes pathogenesis of theiler's murine encephalomyelitis virus cellular immunity in chronic theiler's virus central nervous system infection characterization of theiler's murine encephalomyelitis virus (tmev)-specific delayed hypersensitivity response in tmev-induced demyelinating disease, correlation with clinical signs prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 1 chimeric cytotoxin il2-pe40 inhibits relapsing experimental allergic encephalomyelitis limited heterogeneity of tcell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention experimental allergic encephalomyelitis by t cell receptor v-beta-specific antibodies immunization with a synthetic t-cell receptor v-region peptide protects against experimental autoimmune encephalomyelitis prevention of experimental encephalomyelitis with peptides that block interaction of t cells with mhc proteins prevention of experimental autoimmune encephalomyelitis by antibodies against alpha, beta, integrin aminoguanidine, an inhibitor of inducible nitric oxide synthase, ameliorates experimental autoimmune encephalomyelitis in sjl mice isolated pupil-sparing third nerve palsy as the presenting sign of multiple sclerosis the ocular manifestations of multiple sclerosis. part 2. abnormalities of eye movements optic neuritis and ischemic optic neuropathy: overlapping clinical profiles mcalpine's multiple sclerosis nystagmus in multiple sclerosis acquired pendular nystagmus in multiple sclerosis: clinical observations and the role of optic neuropathy cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report formulas for the quantitation of intrathecal igg production: their validity in the presence of blood-brain barrier damage and their utility in multiple sclerosis new diagnostic criteria for multiple sclerosis: guidelines for research protocols trimodal evoked potentials compared with magnetic resonance imaging in the diagnosis of multiple sclerosis contribution of mri to the diagnosis of multiple sclerosis national multiple sclerosis society working group on neuroimaging for the medical advisory board the role of i\tmr imaging in the assessment of ms and isolated neurological lesions fazekas e magnetic resonance signal abnormalities in asymptomatic individuals: their incidence and functional correlates criteria for an increased specificity of mri interpretation in elderly subjects with suspected multiple sclerosis nuclear magnetic resonance image white matter lesions and risk factors for stroke in normal individuals mri brain scanning in patients with vasculitis: differentiation from multiple sclerosis magnetic resonance imaging in central nervous system sarcoidosis mri findings in neuro-behcet's disease assessment of mri criteria for a diagnosis of ms patterns of disease activity in multiple sclerosis: a clinical and magnetic resonance imaging study spinal cord mri using multi-array coils and fast spin-echo. part 2. findings in multiple sclerosis problems of experimental trials of therapy in multiple sclerosis: report by the panel on evaluation of experimental trials of therapy in multiple sclerosis influenza1 encephalopathy and post-influenza1 encephalitis lyme disease: recommendations for diagnosis and treatment human t-lymphocyte virus type i antibodies in the serum of patients with tropical spastic paraparesis in the seychelles the diagnosis of childhood neurodegenerative disorders presenting as dementia in adults textbook of child neurology risk of developing multiple sclerosis after uncomplicated optic neuritis: a long-term prospective study transverse myelitis: retrospective analysis of 33 cases, with differentiation of cases associated with multiple sclerosis and parainfectious events long-term follow-up of acute partial transverse myelopathy early risk of multiple sclerosis following isolated acute syndromes of the brainstem and spinal cord prognostic significance of brain mri at presentation with a clinically isolated syndrome suggestive of ms: a five-year follow-up study neuromyelitis optica and schilder's myelinoclastic diffuse sclerosis prognostic factors in a multiple sclerosis incidence cohort with twenty-five years of follow-up multiple sclerosis: early prognostic guidelines studies on the natural history of multiple sclerosis: eight early prognostic features of the later course of the illness cardiovascular testing and exercise prescription in multiple sclerosis patients multiple sclerosis. part 1. common physical disabilities and rehabilitation multiple sclerosis andgestation pregnancy and multiple sclerosis: a longitudinal study of 125 remittent patients pregnancy and multiple sclerosis: a prospective study pregnancy and multiple sclerosis the effect of pregnancy in multiple sclerosis the effects of induced hyperthermia on patients with multiple sclerosis clinical viral infections and multiple sclerosis multiple sclerosis: treatment of acute exacerbations with corticotropin (acth) use of oral corticosteroids in the treatment of multiple sclerosis: a double-blind study a double-blind controlled trial of high dose methylprednisolone in patients with multiple sclerosis: part 1. clinical effects a randomized, controlled trial of corticosteroids in the treatment of acute optic neuritis the effect of corticosteroids for acute optic neuritis on the subsequent development of multiple sclerosis ifnb multiple sclerosis study group. interferon beta-lb is effective in relapsing-remitting multiple sclerosis results of a phase iii trial of intramuscular recombinant beta interferon as treatment for multiple sclerosis experimental therapy of relapsing-remitting multiple sclerosis with copolymer-l clinical experience with cop-l in multiple sclerosis clinical experience with azathioprine: the pros azathioprine in multiple sclerosis: the cons efficacy and toxicity of cyclosporine in chronic progressive multiple sclerosis: a randomized, double-blind, placebo-controlled clinical trial intensive immunosuppression in progressive multiple sclerosis: a randomized, three-arm study of highdose intravenous cyclophosphamide, plasma exchange, and acth experience with cyclophosphamide in multiple sclerosis: the cons intermittent cyclophosphamide pulse therapy in progressive multiple sclerosis: final report of the northeast cooperative multiple sclerosis treatment group double-blind pilot trial of oral tolerization with myelin antigens in multiple sclerosis meydrech ee low dose oral methotrexate treatment of multiple sclerosis: a pilot study low-dose (7.5 mg) oral methotrexate reduces the rate of progression in chronic progressive multiple sclerosis cladribine in treatment of chronic progressive multiple sclerosis open controlled therapeutic trial of intravenous immune globulin in relapsing-remitting multiple sclerosis preliminary evidence from magnetic resonance imaging for reduction in disease activity after lymphocyte depletion in multiple sclerosis amantadine therapy for fatigue in multiple sclerosis depression and multiple sclerosis a double-blind, randomized crossover trial of pemoline in fatigue associated with multiple sclerosis new advances in symptom management in multiple sclerosis antispasticity drugs: mechanisms of action intrathecal baclofen for spasticity of spinal origin: seven years of experience safety and efficacy of tizanidine in therapy of spasticity secondary to multiple sclerosis management of bladder dysfunction in multiple sclerosis urological and sexual problems in multiple sclerosis the role of yohimbine for the treatment of erectile impotence treatment of pathologic laughing and weeping with amitriptyline suicide in the medical patient a controlled trial of isoniazid therapy for action tremor in multiple sclerosis pain syndromes in multiple sclerosis fujinami rs, oldstone mba. amino acid homology between the encephalitogenic site of myelin basic protein and virus: a mechanism for autoimmunity molecular mimicry in t cell-mediated autoimmunity: viral peptides activate human t cell clones specific for myelin basic protein fdg-pet, mri and nmr spectroscopy of normal appearing white matter (nawm) in multiple sclerosis key: cord-311908-sgdq6j6x authors: atkins, g. j.; mcquaid, s.; morris‐downes, m. m.; galbraith, s. e.; amor, s.; cosby, s. l.; sheahan, b. j. title: transient virus infection and multiple sclerosis date: 2000-09-28 journal: rev med virol doi: 10.1002/1099-1654(200009/10)10:5<291::aid-rmv278>3.0.co;2-u sha: doc_id: 311908 cord_uid: sgdq6j6x multiple sclerosis (ms) is a chronic, demyelinating disease of the cns in which autoimmunity to myelin plays a role in pathogenesis. the epidemiology of ms indicates that it may be triggered by a virus infection before the age of adolescence, but attempts to associate a specific virus with ms have produced equivocal results. many studies of the aetiology of ms have postulated that a persistent virus infection is involved, but transient virus infection may provide a plausible alternative mechanism that could explain many of the inconsistencies in ms research. the most studied animal model of ms is chronic relapsing experimental autoimmune encephalomyelitis (creae), which is induced in susceptible animals following injection of myelin components. while creae cannot provide information on the initiating factor for ms, it may mimic disease processes occurring after an initial trigger that may involve transient virus infection. the disease process may comprise separate triggering and relapse phases. the triggering phase may involve sensitisation to myelin antigens as a result of damage to oligodendrocytes or molecular mimicry. the relapse phase could be similar to creae, or alternatively relapses may be induced by further transient virus infections which may not involve infection of the cns, but which may involve the recrudescence of anti‐myelin autoimmunity. although current vaccines have a high degree of biosafety, it is suggested that the measles‐mumps‐rubella vaccine in particular could be modified to obviate any possibility of triggering anti‐myelin autoimmunity. copyright © 2000 john wiley & sons, ltd. the possible involvement of viruses in the aetiology of multiple sclerosis (ms) is a subject that has created much controversy. from epidemiological studies based on immigration data and the occurrence of clusters, it has been suggested that an environmental factor(s) triggers ms before the age of adolescence, while symptoms of the disease are not observed until years later. it is also apparent that there is a genetic susceptibility to ms, so a hypothesis for the aetiology of ms formulated on this evidence is that it is a disease triggered by an environmental factor in genetically susceptible individuals during childhood [1±8] . circumstantial evidence suggests that the environmental factor in ms could be a virus [7±14] . several human and animal virus infections are characterised by the cns demyelination that is also characteristic of ms (table 1) . these include subacute sclerosing panencephalitis (sspe), caused by a persistent measles virus infection, and human t cell lymphotropic virus-i (htlv-i)associated myelopathy, which is a slowly progressive neurological disease characterised by in¯ammatory in®ltrates and demyelination in the cns, and is caused by an exogenous retrovirus. many viruses have been implicated in ms itself [13, 14] (table 2 ), including measles virus and more recently ebv, [15] human herpesvirus 6 (hhv6) [16] and human endogenous retroviruses [17] . there is also evidence to suggest that some of the relapses which are characteristic of most ms cases are preceded by virus infections [18±20] . for example, a recent report demonstrated a signi®cantly higher exacerbation rate in ms patients following in¯uenza virus infection, suggesting that in¯uenza infection may trigger relapses [21] . evidence for the involvement of virus infection in ms also comes from several animal diseases in which persistent virus infection gives rise to demyelination [10,11,22±24] . while these studies point to a viral aetiology of ms, no virus has yet been de®nitely associated with this disease despite intensive effort. one possible explanation for the equivocal results obtained for identi®cation of viruses common uncharacterised infections transient associated with ms is that, while the disease is triggered by a virus infection, the subsequent course of the disease may not require virus persistence. it is probable that virus persistence does occur in some cases, but it may or may not be relevant to the progression of the disease. it is also possible that a number of different viruses, rather than one speci®c type, may act as a trigger for ms in susceptible individuals. another important factor that requires consideration is the heterogeneity in the detailed neuropathology and clinical disease progression in ms, suggesting that the disease may not have a single aetiology. it is possible that primarily progressive ms and relapsing remitting ms are two distinct disease entities [25] . ms may in fact be divided into at least ®ve disease subtypes on the basis of detailed analysis of neuropathology [26, 27] , and it is possible that only some of these may have viral involvement. the main assumption that has been made in considering the possible viral aetiology of ms is that a persistent infection by one virus is involved. here we review the evidence for transient virus infection, with a variety of agents, as an alternative to persistent infection in the aetiology of ms. in an attempt to understand the aetiology of ms, animal models have been used. the most studied autoimmune model of ms is experimental autoimmune encephalomyelitis (eae), which may be induced in a number of animal species by immunisation with myelin antigens or transfer of myelin-reactive class ii restricted cd4+ t lymphocytes [10] . the type of eae induced is dependent on the immunisation protocol, animal strain and antigen used. in some cases the protocol results in an acute monophasic disease and in others a chronic relapsing disease (creae) occurs. creae resembles ms because animals develop accumulating neurological de®cit following each disease phase. eae is characterised by in¯ammatory in®ltration of the cns associated with lesions of demyelination, and the topography of lesions within the cns is dependent on the nature and origin of the autoantigen [28] . the extent of demyelination varies with the protocol used but is more pronounced when myelin-speci®c antibodies are injected following transfer of mbp-speci®c t cells or at the onset of spinal cord homogenate-induced disease [29, 30] . the possible role of anti-myelin antibodies is supported by the ®ndings of myelin reactive antibodies in the blood of ms patients, and recently autoantibodies directed to myelin basic protein (mbp) and myelin oligodendrocyte protein (mog) peptides have been detected within lesions of demyelination in the cns of ms patients [31] . while antibodies may be involved in the development of demyelination, many other immunological mechanisms, including the release of in¯ammatory mediators of disease such as tnf-a and reactive oxygen species, or the action of cytotoxic t-lymphocytes, may play a role in the myelin damage [32] . the chronicity of the disease both in creae and possibly ms may be due to the perpetuation of autoimmune responses by determinant spreading. this process occurs as a result of myelin damage in the initial phase of disease, when new myelin antigens are released. the release of such antigens may subsequently induce new t-cell speci®cities, some of which may be pathogenic [33] . in contrast to eae are the virus-induced models, in which both natural and experimental infections of animals result in widespread demyelination and neurological de®cit. the naturally occurring diseases include canine distemper virus of dogs, and visna virus infection of sheep, both of which have been used to study the mechanism of virusinduced demyelination as models for ms. laboratory virus infections of mice and rats have also been used as models to understand the processes of virus-induced demyelination. these experimental models include murine coronavirus, where persistent virus infection induces an eae-like disease [22, 34] . myelin reactive t cells are also observed following experimental infection of rats with measles virus and some of these t cell lines are encephalitogenic, supporting the hypothesis that a virus trigger can lead to autoimmunity and myelin damage [35±37]. another virus model of ms is theiler's murine encephalomyelitis virus (tmev) infection of mice, which produces a chronic demyelinating disease associated with virus persistence [10, 11, 22, 23, 38] . autoimmune responses to myelin antigens are observed following infection with tmev, and while they may not play a major role in the initiation of demyelination, autoimmunity to myelin components probably contributes to lesion progression in chronically diseased animals [23] . an alternative model of virus-induced demyelination is semliki forest virus (sfv) infection of mice [39, 40] . sfv induces acute immune-mediated demyelination in mice, which is repaired and does not recur in most mouse strains. the initial immune-mediated demyelination induced by sfv infection may be due to targeting of sfv-infected oligodendrocytes by cytotoxic t-cells. it is clear, however, that t-cell responses to mbp [41±43] and to a range of encephalitogenic myelin epitopes (m. morris-downes and s. amor, unpublished data) are also induced. a mog peptide showing sequence similarity to the sfv e2 envelope protein, as well as the e2 peptide itself, has been shown to induce creae in mice, and it has been suggested that this molecular mimicry may contribute to sfv-induced demyelination [44] . in most mouse strains, for example in balb/c mice, myelin repair is complete by 3 months after avirulent sfv infection. however, in sjl mice, small lesions of demyelination persist up to a year after infection and some of these lesions show active in¯ammatory demyelination. long-term lesions do not correlate in individual mice with persistence of the virus genome, but do correlate with expression of the cytokines tnf-a or ifn-c [45, 46] . expression of these cytokines in the cns is undetectable in balb/c mice by 3 months after infection, but continues in most sjl mice beyond a year after infection [45] . sjl mice, which are susceptible to eae, appear to have a defect in innate suppression of immune responses, which is associated with secretion of lower levels of the regulatory cytokine tgf-b [41] . prior infection with sfv predisposes mice to eae, even in strains that are normally resistant to eae, and infection with sfv has been shown to exacerbate eae [42, 47, 48] . it is clear that the demyelination characteristic of sfv infection is immune-mediated and it appears that this may be triggered by virus infection of oligodendrocytes early in infection. such infection may induce damage to the infected cells and the subsequent release of myelin antigens by the induction of oligodendrocyte cell death [49] . sfv-infected oligodendrocytes could also be targeted by cd8+ t-lymphocytes [50, 51] or alternatively, uninfected oligodendrocytes and/or myelin in the vicinity of aggressive immune responses may be damaged by the action of cytokines such as tnf-a [43, 51] . either way, normally sequestered myelin antigens are likely to be presented to the immune system to induce autoimmunity to myelin. a possible mechanism whereby recrudescence of immunity in the cns could be triggered by a virus infection is suggested by a study on in¯uenza virus infection of mice. following an initial cns infection, subsequent infections led to the long-term persistence of activated cytotoxic t-lymphocytes in the brain parenchyma, long after the infection was cleared [52] . another study that does not involve virus infection, but may nonetheless provide information concerning the possible viral aetiology of ms, concerns the exacerbation of brain damage following eae induction. if a brain cryolesion is in¯icted on rats after induction of eae by injection of myelin components, eae lesions are enhanced [53] . this may be consistent with the proposal that the brain damage caused by virus infection could enhance eae in a manner that is not speci®c to the virus. evidence for the involvement of speci®c viruses in ms comes from serological studies and direct virus detection. it was found many years ago that antibody levels to several common viruses are elevated in ms patients, but it is not clear whether this is related to the cause of ms or an epiphenomenon. many viruses have been detected in cns autopsy tissue from ms patients [13±17] ( table 2 ); many such claims have either been retracted or have not been con®rmed by other workers. the inconsistent detection of viruses in cns autopsy tissue from ms patients is illustrated by a study in which brain samples from 8 cases of ms and 56 controls were examined by in situ hybridisation [54] . samples were examined for genomic rna sequences of measles virus, canine distemper virus, rubella virus and simian virus 5, all viruses that have been implicated in ms. positive hybridisation was detected in two of the ms cases for measles virus only, but was also detected in one of the controls. the latter was a neurological control that was a case of disseminated cmv infection, but which showed demyelination that is uncharacteristic of cmv infection [54] . in subsequent investigation, the number of ms cases found to be positive for measles virus rna has increased to 4 out of 14 cases examined (s. mcquaid and l. cosby, unpublished data). in another study, measles virus rna could not be detected by rt-pcr in peripheral blood leucocytes from ms patients [55] . measles, mumps and rubella virus rna has been shown to be absent from brain autopsy tissue from ms patients by rt-pcr [56] . these and other studies indicate that most cases of ms are not associated with persistent virus infection, at least with the viruses tested, but a minority of cases may be associated with the persistence of measles virus in the cns. more recently attention has focused on hhv6 [16] , ebv [57] and the ms-associated retroviruses [17] . while causal associations have yet to be de®nitely made with ms, it is possible that infection with a single virus alone is insuf®cient for the development of ms and dual infections such as retrovirus and ebv are required [15] . some positive evidence is available, however, linking transient virus infection with ms or exacerbations of ms. common virus infections, identi®ed by their symptoms rather than by virus detection, have been associated with exacerbations of ms [18, 20] . apart from post-vaccination encephalitis, rabies vaccination has been associated with a disease that is indistinguishable from ms at autopsy [12] , and it has been shown that the semple vaccine strain of rabies virus can induce anti-myelin autoimmunity [58] . recent studies have suggested that both in¯uenza infection and in¯uenza vaccination are associated with an increased frequency of exacerbations of ms [21] . like the evidence for viruses in ms, the data are controversial, since other studies have failed to ®nd an association between exacerbations of ms and in¯uenza vaccination [59] . a summary of possible mechanisms for the involvement of transient virus infections in stim-ulating anti-myelin autoimmunity is shown in figure 1 . it is possible that sensitisation to myelin antigens may follow an initial trigger involving a virus infection. subsequently, recrudescence of anti-myelin autoimmunity could occur during relapse phases induced by further transient virus infections. the anti-myelin autoimmunity may only produce a threshold of damage to myelin, resulting in clinical signs and symptoms, in a minority of patients with a genetic susceptibility to ms. this may involve an inability to suppress anti-myelin autoimmunity. the initial phase could result in damage to myelin and the release of normally sequestered myelin antigens, which would then prime the immune system to induce anti-myelin autoimmunity. in the case of a virus infection, this initial phase could involve damage to oligodendrocytes. such damage could be caused by direct infection of oligodendrocytes and the induction of cell an alternative mechanism of priming of the immune system to myelin antigens may be molecular mimicry. many bacterial and viral proteins share sequence and structural homologies with known autoantigens. several viruses share part of the sequence of myelin proteins in their genome [60, 61] , and a number of peptides based on these sequences activated myelin-speci®c t cell clones [61] . sequence similarity between mbp and hepatitis b virus exists, and this peptide sequence induced eae in rabbits [62] ; also, a rubella peptide shows sequence similarity to myelin proteolipid protein [63] . a mechanism similar to molecular mimicry may be the incorporation of self-antigens into the envelope of budding viruses leading to autosensitisation. if vesicular stomatitis virus, a model enveloped virus, is grown in mbp-expressing cell cultures it is highly ef®cient in triggering t-cell responses to mbp, both in vitro and in vivo [64] . thus, viruses could act as presenters of host antigen to the immune system. this initial triggering phase could be followed by a relapse phase in which reactivation of myelin-speci®c t-cells already present in the cns occurs, or alternatively myelin-reactive immunity is induced outside the cns and in®ltration of the brain parenchyma by lymphocytes and other components of the immune system occurs. in either case, such activation may be a result of a transient virus infection which compromises the blood±brain barrier and leads to further in¯ux of cells and soluble components [65, 66] . a possible mechanism for the recrudescence of anti-myelin autoimmunity, as well as those mentioned above for the triggering phase, may be the activation of myelin-reactive t-cells by viral superantigens. such activation relies on the preexistence of myelin reactive t cells but does not occur via the classical t-cell receptor : peptide : mhc interaction. instead it involves the polyclonal activation of t cells through the vb element of the t cell receptor. it has been demonstrated that eae relapses may be induced with staphylococcal superantigens, although injection of the same superantigens into naive mice failed to induce eae [67] . it is feasible that viruses may also act in this way, as has been shown for murine mammary tumour virus [68] . if the proposed triggering mechanism is correct, then de®nitive evidence for it could not be obtained from ms autopsy or biopsy tissue, because the myelin damage resulting in the triggering event probably occurred some time previously and only the consequences of this would be evident. also, if the damage were a result of virus infections, it may not be speci®c to any one virus, as at least in experimental infection many infections may give rise to a similar pathology, suggesting that any virus (including non-neurotropic viruses) that induces myelin sensitisation could act as a trigger. with regard to the activity of known human viruses in the induction of myelin damage, there is evidence that virus infections associated with cns demyelination can cause damage to oligodendrocytes. measles virus has been implicated in ms [54] , and virus infection of oligodendrocytes is seen in cases of sspe [69] , which is a rare human demyelinating disease caused by a persistent infection with measles virus (figure 2a) . similarly, progressive multifocal leucoencephalopathy (pml) is a rare human demyelinating disease caused by persistent infection with a human papovavirus. this virus has also been implicated in ms [70] , and virus infection of oligodendrocytes is seen in this disease (figure 2b) . in the sfv model, infection of oligodendrocytes occurs early in infection, and is followed by acute immune-mediated demyelination [71, 72] (figure 2e, f) . virus infection of oligodendrocytes by sfv is also seen in rat mixed glial cell cultures [73] (figure 2c, d) . the damage to oligodendrocytes induced by sfv infection may be due to induction of programmed cell death or apoptosis, since this is induced in cultured rat oligodendrocytes infected by sfv [74] . however, it has also been proposed that some mature oligodendrocytes in the animal survive sfv infection, and are capable of carrying out the remyelination observed later in the disease [75] . such virus-infected oligodendrocytes could be targeted by cd8+ t-lymphocytes [49] , which would also result in myelin damage. rubella virus, a togavirus like sfv, and another virus that has been implicated in ms [76] , shows tropism for oligodendrocytes in rat mixed glial cell cultures [77] . in canine distemper, which is a disease of dogs caused by a virus that has also been implicated in ms in the past [13] , damage to oligodendrocytes also occurs, although this does not appear to be associated with productive virus infection, which mainly occurs in astrocytes [78, 79] . lymphoproliferative responses to mbp have been detected in patients with encephalitis after measles virus, vzv or rubella virus infection, and after rabies vaccination [80] . in the case of measles virus, a t cell-mediated response to mbp was induced following intracerebral infection of rats; such sensitised t-cells showed no crossreactivity to measles virus, but could induce eae by adoptive transfer to naõ ève recipients. also, a normally non-encephalitogenic mbp peptide induced eae in measles virus-infected rats [37] . this is further evidence that virus infection of the cns can sensitise to subsequent autoimmune demyelination. the chronic immune-mediated demyelination that occurs in ms could be maintained by either of two mechanisms following sensitisation of the immune system to myelin antigens after the initial trigger. one mechanism could be similar to creae. another possibility is that the relapses characteristic of most cases of ms could be induced by further transient virus infections, which may not necessarily involve infection of the cns. the clinical evidence that transient virus infections could be implicated in relapses of ms is mentioned above; there is also indirect evidence that such a mechanism could operate. many virus infections lead to the production of pro-in¯ammatory cytokines such tnf-a or ifn-c. infection of sjl mice with sfv results in the expression of these cytokines in the cns for more than a year after the initial infection [45] . it is known that administration of ifn-c to ms patients results in exacerbation of the disease [81] . also, the demyelination in measles virus encephalitis cannot be associated with the presence of measles virus antigen in the cns at time of death, although it is not clear whether virus infection of the cns occurred earlier in the course of the disease [82] . it is possible that administration of ifn-b, which is a treatment currently used for ms with partial success [83] , could ameliorate the effects of some transient infections that may trigger exacerbations of ms. however, adminis-tration of ifn-b may also have an immunomodulatory effect [84] . it is possible that virus infection could induce secretion of pro-in¯ammatory cytokines that could penetrate the cns parenchyma from the blood and lead to the recrudescence of anti-myelin autoimmunity by reactivation of previously primed t-cells. it is also possible that such myelin-reactive t-cells could penetrate the brain parenchyma following damage to the blood±brain barrier caused by transient virus infection [66] , or following the secretion of pro-in¯ammatory cytokines and/or chemokines by cells within the cns (astrocytes and microglia as well as lymphocytes) [85] . if such a mechanism were to operate, it may not be virus-speci®c, but may be induced by many different transient infections. much of the evidence cited to support the idea that ms is associated with a speci®c persistent virus infection of the cns has been challenged, or shown to be incorrect. it is possible that there are still unknown viruses which infect the cns and which may contribute to ms pathogenesis. current candidates for persistent virus infection associated with the aetiology of ms include hhv6, ebv and human endogenous retroviruses. whether these viruses are indeed involved in the pathogenesis of at least some cases of ms is still unknown. however, ms may comprise a group of diseases of different aetiologies: persistent virus infection may represent one such aetiology, and the mechanisms triggered by transient infection described here may represent another. since the circumstantial evidence that ms is associated with a virus infection has not been refuted, it seems reasonable to consider the possibility that most cases of ms are not associated with a persistent virus infection, but that other mechanisms involving transient virus infection could operate. if ms is induced and/or exacerbated by transient virus infection, there are several corollaries that may explain the equivocal results obtained for the association of viruses with ms. firstly, it would not be possible in most cases to detect virus in autopsy tissue from ms patients, or in biopsy samples taken after the initial triggering phase. secondly, virus infections need not be speci®c, and it is possible that a range of viruses with common properties could be involved in either the triggering or maintenance phases. thirdly, it is probable that, if viruses are involved in triggering and/or maintaining ms, that these are common viruses that only have this effect in a minority of genetically susceptible individuals. if transient virus infection is involved in the triggering or maintenance of the antimyelin autoimmunity that is a risk factor for ms, the role of vaccination must be considered. of the common diseases for which vaccines are available, measles, mumps and rubella viruses have all been implicated in ms [9, 13, 14] . there is con¯icting evidence as to whether in¯uenza or hepatitis b vaccination can trigger cns in¯ammation [21, 59, 86, 87] . however, possible mechanisms of induction of cns in¯ammation may be different for hepatitis b and in¯uenza vaccines, which are non-replicative, compared with the measlesmumps-rubella (mmr) vaccine, which is a cocktail of three replicating attenuated viruses. wildtype measles, mumps and rubella viruses are all known to infect the cns, and the urabe am 9 mumps vaccine strain, now no longer used as a vaccine component, caused cns symptoms (aseptic meningitis) in a small minority of recipients [88±90] , showing that it is neurotropic. in developed countries, the mmr vaccine is routinely given to children to prevent disease caused by measles, mumps and rubella virus infections [89] . although it is clear that the incidence of ms has not been signi®cantly affected by the introduction of the vaccine, this could be explained if either the wild-type infections or the vaccine could trigger one type of ms in a minority of individuals. the mechanism by which vaccines may induce cns in¯ammation could be but one of a number of different mechanisms operating as an ms trigger. the hypothesis that vaccination could trigger ms is consistent with current knowledge of the epidemiology of ms, in particular the hypothesis deduced from studies of the epidemiology of ms, that it is a disease triggered by an environmental factor exerting its effect in a minority of genetically susceptible individuals before the age of adolescence. in the uk, rubella vaccination was introduced in 1970 for girls only but was extended to both sexes in 1988 when the mmr vaccine was introduced [89] . recently, peripheral neuropathy has been associated with rubella vaccination in which anti-mbp reactivity was prominent [91] . however, as has been suggested for in¯uenza [21] , vaccination may be less likely to trigger ms or ms exacerbations than the virulent infection. vaccination has been the most successful method of controlling virus diseases [92] , and current evidence indicates that the bene®ts of vaccination outweigh any disadvantages, for which there is no conclusive evidence at present. thus there is no justi®cation for the discontinuation of vaccination either in the general population or for ms patients. however, although current vaccines have a high degree of biosafety, a small risk associated with vaccination, which is much less than that associated with the wild-type infection, cannot be excluded. the evidence available at present does not indicate that vaccination can trigger ms and/or exacerbations of ms, but this possibility warrants further investigation of vaccines, particularly the mmr vaccine. in particular, the ability of vaccine strains to stimulate anti-myelin autoimmunity in a manner similar to wild-type virus strains, and as a combination vaccine, should be investigated. it is possible that the more rational design of vaccines, based on recombinant dna technology, could improve them and circumvent any possible problems which may be associated with current vaccines. it may be possible to replace live virus vaccines with engineered virus vaccines expressing only desired protective epitopes. the use of new genetically manipulated vaccines will create its own controversy, and the use of such vaccines can only be justi®ed if they are demonstrably more effective and have greater biosafety than conventional vaccines currently in use. of particular concern is a report that the plasmid backbone used to construct naked dna vaccines, which have been suggested as replacements for current vaccines [93] , has been shown to potentiate eae. the mechanism appears to be the induction of th1-promoting cytokines [94] . however, dna vaccines have poor immunogenicity and persist for long periods in the host tissue [95] (m. m. morris-downes and g. j. atkins, unpublished results). other types of prototype recombinant vaccines include naked rna vaccines and recombinant suicide particles based on the sfv genome [40, 96] . such vaccines stimulate immune responses more ef®ciently than naked dna vaccines [97] , and induce apoptosis which may lead to the removal of the vaccine from inoculated tissue [98] . this may lead to a 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measles, mumps and rubella vaccines peripheral neuropathy associated with anti-myelin basic protein antibodies in a woman vaccinated with rubella virus vaccine defending vaccines from the enemy within dna immunization exacerbation of viral and autoimmune animal models for multiple sclerosis by bacterial dna dna vaccines with a kick vaccination with recombinant suicidal dna/rna recombinant semliki forest virus particles expressing louping ill virus antigens induce a better protective response than plasmid-based dna vaccines or an inactivated whole particle vaccine the semliki forest virus vector induces p53-independent apoptosis this review was greatly in¯uenced by a workshop on the infectious aetiology of multiple sclerosis held in brighton, uk in august 1999 by the us and uk multiple sclerosis societies; we are very grateful to the organisers. key: cord-015352-2d02eq3y authors: nan title: espr 2017 date: 2017-04-26 journal: pediatr radiol doi: 10.1007/s00247-017-3820-2 sha: doc_id: 15352 cord_uid: 2d02eq3y nan prof. michael riccabona undertook his medical school & university training at the university of innsbruck, tirol, austria, completing his md at karl franzens university in graz, austria. following an internship in neurology, surgery and internal medicine he then specialized in paediatrics at the dept. of paediatrics, university hospital in graz. there he took charge of the paediatric radiology and sonography sections at university hospital in graz, as associate professor of paediatrics. in 1993 he additionally started to specialise in radiology, becoming associate professor of radiology in 1998 -then taking charge of the subsection of paediatric sonography at the dept. of radiology, university hospital in graz, where in march 2006 he was appointed full univ. prof at the medical university graz, austria. he has a distinguished academic career and written over 200 papers, more than 50 chapters and several textbooks, and is a very popular international speaker, delivering numerous lectures at many high profile scientific meetings. he is an active member of several reputable international societies, has been chair of the paediatric ultrasound section of the austrian ultrasound society since 2003 , and president of the society of german speaking paediatric radiologists (2010) (2011) (2012) (2013) (2014) (2015) (2016) . he is a constant source of inspiration within his subspecialist areas of interest in ultrasound and abdominal radiology in children. he has been course director at several important meetings and served as president of espr in graz in 2015 and as lead of the paediatric subcommittee at ecr in 2012. he has provided inspirational leadership as chair of the espr task force on uroradiology since 2002writing state of the art guidelines and procedural recommendations to facilitate standardised best practice for imaging within paediatric uroradiology. he has been a reviewer for many international journals. he has on-going active roles in postgraduate education for medical colleagues from eastern europe, including basic ultrasound education and refresher courses, and workshops. michael riccabona is very deserving of honorary membership of espr, which reflects his seminal role within paediatric radiology in europe and his tireless dedication working for the good of children. & to discuss how to optimally adapt the various imaging techniques minimising radiation exposure and risks during diagnostic imaging in children. & to consider common restrictions, challenges, and possible solutions in paediatric radiology within the different settings in different countries, regions, continents and clinical scenarios -discussing all these aspects with colleagues, and to mingle with experts from all over the world learning from each other and fostering networking in paediatric radiology to try to grant optimal imaging for all children. an application for the 53 rd annual meeting as well as for the 39 th post graduate course has been submitted to the eaccme® for cme accreditation of this event. the eaccme is an institution of the uems (www.uems.net). the number of cme points will be announced at the espr 2017 congress website. each medical specialist should only claim those hours of credit that he/she actually spent in the educational activity. certificates of attendance will be available in the espr myuserarea after the meeting. the answers right away is: yes we can and we constantly have to. it is part of human nature to recognize problems and to find solutions. we define ideals but we also face reality. being aware of the gap in between we are constantly driven to improve. this overview will highlight some milestones and disputes throughout the 100 years of development in the use of plain x-ray imaging. it will hint on scientific literature and sources of information. and it will hint on some swiss contributions as the espr meeting 2017 will be held in davos, switzerland. fighting the glow not the fire -122 years of x-ray imaging development and improvement: in the beginning of the clinical use of x-ray imaging, there was great enthusiasm in its potential without knowing the unfavorable dangers of uncontrolled use of x-ray. the dangers were recognized and the 'beast' was tamed and domesticated. in respect to radiation protection, the most significant achievements took place in the first half of that development. throughout the recent decades, some further considerable steps in dose reduction took place mainly by the improvement of film-screen systems and the recent introduction of computed (cr) and digitized radiography (dr). even if the early computerized x-ray imaging brought a slight increase in patient doses (which was overcompensated later on by direct digital radiography) a whole new world of further advantages launched the digital era in which we live today. as we all know new dangers arose with these techniques as the uncontrolled distribution of images and thereby confidential patient data over hospital departments and across borders throughout. also, the risk of an evitable overexposure in digital radiography is a significant issue. throughout the process of taming the radiation and controlling it, today's doses are attained within the lowest range of the danger scale. this range still is perceived as a black box within which we do not exactly know which concept reflects the potential harm best. the linear no-threshold model [lnt] is acknowledged as the concept which most reliably supports the idea of radiation protection. other concepts partly oppose the linear idea and question the relevance of that dose range because there lie so much greater health benefits in the appropriate use of diagnostic x-rays. several scientists even propagate the idea that very low levels are producing health benefits instead of physical harm [hormesis model]. nevertheless, the lnt model is widely accepted as the most helpful in the context of diagnostic radiology. some recent studies were able to support the idea of potential harm at very low dose levels as they were able to prove the induction of attributable cancers in the pediatric age group. so today we are fighting the glow, not the fire. as trained medical professionals we are fully aware of the fact that there is only little potential harm to the patient by using x-rays in the current state of the art. but on the other hand, we also have to be aware of the fact that our patients and their parents still fear the fire. one of our main tasks, therefore, is to explain the risks and benefits to the patients and their advocates and to educate the public. developed straight from the first radiographic technique's digital radiography today is state-of-the-art in plain 2d-imaging. throughout the last decade, it has replaced cr and conventional radiography in many institutions. in the united states of america, one of the most developed healthcare systems, healthcare authorities propagate incentives to abolish cr and older imaging systems by making them financially unattractive. the market is fully concentrated on the spread of dr systems. momentarily there are no real milestones but many refinements of existing systems such as tomosynthesis, dual energy subtraction and advanced auto-stitching, fluoroscopy capability, basic angiography applications and 3-d cone-beam ct images are made available. combinations of these features can be found in some recently designed x-ray machines. grid-less imaging software can reduce patient dose significantly. concerning detectors, there are cr retrofit systems which will support the easy upgrade of existing systems to dr capability. wireless detectors with large internal storage, different sizes and high resolutions of 100 microns are available. dr is becoming a part of the system in the current era of full digitalization of our lives and big data, digital radiology is a cornerstone of our healthcare systems. ris and pacs as part of integrated healthcare (ihs) systems are widely disseminated. at the next step, all accessible data will be used for analyses. the major vendors of imaging systems, as well as pacs suppliers and independent companies, offer readymade software tools for reports and evaluations of all kind. the doses from different x-ray sources can be screened internally and be used for optimization purposes. they also can be sent to remote servers for dose monitoring, comparison and optimization in multi-hospital health care provider settings or to comprehensive databases like the american college of radiology dose index registry, cancer registries, or for central billing. has everything been invented? many technologies have been declared dead before a new transformation appeared. this was the case for example with single slice ct before the invention of spiral ct by willi kalender, germany and peter vock, switzerland in 1988. often the plain x-ray image was meant to be needless or redundant as newer technologies like ct or mri approached. but it still is of value because of different reasons as the low dose, high availability, well known and easy interpretation to name a few. there are some new and sophisticated techniques on the way like the "smart x-ray source" which uses coherent beams of x-rays from an array of micronsized point sources, developed by scientists at the massachusetts institute of technology (mit). the developers promise less radiation, less weight of the equipment and a far better soft tissue resolution. another promising approach is phase contrast x-ray imaging which has the potential to reduce the dose up to 1/100 of the actual value. it also has its strengths in additional soft tissue information as recent experimental publications show (paul scherrer institute switzerland) e. g. in functional evaluation of lung fluid (munich, germany). functional imaging of the lungs can also be achieved without any radiation as the development of the known concept of electrical impedance tomography highlights. this functional imaging method usually is not within the modality spectrum of radiologists. dose control and reduction -local -regional -international the most effective measures to achieve significant dose reductions in your own department are still the same strategies which are based on the "eternal rules" as we know them from our teachers: avoiding unnecessary exposures by strictly controlling the appropriateness of a referral. justification is a shared responsibility between radiologists and clinicians. there are many tools available for justification like the appropriateness criteria, guidelines or rules (like wrist or ankle rules) of several national societies and different study groups. the process of optimization is mainly in the hands of the technicians. as many studies show, the proper collimation still has the greatest effect on dose reduction. other important factors are the positioning of the patient and the shielding of radiosensitive organs which are not relevant for image interpretation so that they may be covered by lead shields. the proper use of the grid in bigger children can now partially be replaced by software solutions. in digital radiography, a profound knowledge of postprocessing possibilities is mandatory as well as the active control of the exposure indices. dose limitation procedures should be regularly checked in a team-based approach to avoid overexposure by less experienced staff or "exposure creep". existing standards should be actively used to guarantee a constant satisfactory image quality. in 2011, the image gently campaign released a safety checklist for performing digital radiography examinations on pediatric patients which is easily applicable to every radiology service. organizational improvements: at regional and national level, efforts should be made to check for best practice use in the departments and to compare and discuss imaging strategies. the establishment of national and international dose reference levels helps to keep the overall doses low and to protect the population from unnecessary overexposure. the pidrl project prepared the "european diagnostic reference levels for pediatric imaging" as part of the eurosafe project. momentarily the results of pidrl-workgroup are harmonized with international organizations. the european guidelines on drls for paediatric imaging can be accessed as a preliminary final for workshop drafts on the internet. on a worldwide basis, the world health organization has published a fundamental information brochure concerning radiation risks and the communication of health professionals and patients. health care professionals have a shared responsibility for communicating risks and benefits of imaging procedures to patients, especially in the case of pediatric patients. the document "communicating radiation risks in paediatric imaging-information to support health care discussions about benefit and risk" is intended to serve as a tool for health care providers, to communicate known or potential radiation risks associated with pediatric imaging procedures and to support risk-benefit dialogue in health care settings. as said before we are fighting the glow, not the fire. the paper of the swiss pediatric oncology group stirred a broad discussion. among other issues, there was a question if it shouldn't be a logical consequence to transfer kids from areas with higher background radiation to safer areas. the author's answers were clear: that swiss health authorities better concentrate their efforts more effectively and with greater benefit for more people by supporting prevention "toward modifiable environmental factors leading to larger numbers of deaths from several causes, such as exposure to radon, air pollution, and second-hand tobacco smoke". this leads to the conclusion that we as medical radiological professionals do have the obligation to make every effort to prevent our patients and personnel from harm of the usage or non-usage of radiation. as health specialists, we also should support the fields of prevention with broad mass effects as far as we have the opportunity. and as human beings, we are summoned to do so in respect to other beings, to our environment and to the resources we all share. radiation protection and quality improvement is just a small part of it all, but it is our field -and 'yes we can'. "communicating radiation risks in paediatric imaging. freely available at the who homepage." computed tomography: are we doing enough? e. sorantin; graz/at summary: already in 1912 the alara principle was publishedbut the implementation is still far from complete. according to the surveys of the ec tender project "pidrl -european diagnostic reference levels for paediatric imaging" the most frequent computed tomography (ct) examinations in children are, in descending order, head/neck, chest and abdomen thus counting for about 75% of all pediatric ct's. therefore it makes sense to optimize these examinations first. surveys of the "international atomic energy agency (iaea)" in 40 countries have shown, there is considerable lack of organizationeg in about 50% of facilities protocols for children were missing, indication based protocols available only in 57%, ctdi values for head and chest two to five times of those for adults. all of these simple facts indicate we are not doing enough for radiation protection in pediatric ct. actions to lower dose in ct can be categorized in organisational, optimization and alternatives. the interdisciplinary implementation of international guidelines for ct in minor head trauma with trauma surgeons could serve as an example of organisational actions. for dose optimization knowledge about dose relevant factors according the "imaging chain" is mandatory as well as adjusting kv to pediatric needs. dose influence on image quality must be known, by exploiting the fact, that, if all ct parameters are kept constant but hte slice thickness is just halve there must be an increase in noisein particular about two times more. therefore if a standard examination is reconstructed at half slice thickness and image quality is still appropriate the amount of waste radiation is in the range of 100%. therefore if the next examination will be reduced with eg 20% mas setting less will be for sure in appropriate quality and the process can be started again. after a couple of examinations the optimal dose will be reached. thus the "half slice thickness" approach is easy to do, does not need special equipment or human resources and will help to find the appropriate dose. the third point is alternatives -ultrasound and mri being the candidates in the first row. new, radiation free, techniques like electrical impedance tomography and others are already developed and can be expected to be release soon. take home points: & we are not doing enough for ct dose savingeven more than 100 years after release of alara principle & dose saving actions can be categorized inthe subtasks organisation, optimization and alternatives & "the half slice thickness approach" is an easy to do technique to elaborate the optimal dose on an particular ct machine. prenatal thoracic mr l. alamo; lausanne/ch the generalization of screening us has considerably increased the detection of congenital anomalies in utero. in the last years, important technological advances and especially, the development of fast heavily t2-weighted sequences has led to an increasing use of prenatal mri as additional diagnostic imaging method. mri is increasingly used for evaluation of thoracic pathology, including tumours and vascular malformations as well as anomalies of the diaphragm, the lungs and more recently, even of the foetal heart: -thoracic tumours and vascular malformations: the diagnosis of a congenital tumor during pregnancy involves a tremendous emotional impact for a family. the most frequently observed thoracic tumours are teratoma, myocardial rhabdomyoma and exceptionally, pleuropulmonary blastomas. mri may provide relevant additional information concerning the origin of the lesion and its real anatomical extent. -diaphragmatic pathology: congenital hernia is by far the most commonly reported foetal diaphragmatic anomaly. the large field of view and the multiplanar possibilities of mri may help to clarify the position of the herniated organs and to evaluate the severity of lung hypoplasia, considered the most important parameter for predicting outcome. other rare pathologies include eventration, paralysis and diaphragmatic lung sequestrations. -lung anomalies: congenital lung abnormalities are a heterogeneous group of pathologies consisting of isolated bronchopulmonary or vascular anomalies or a combination of both of them. congenital pulmonary airway malformation, bronchopulmonary sequestration and bronchial atresia are the most often observed pathologies but they present significant overlap imaging findings. mri allows accurate information concerning the location and extension of the lesion and the volume of the normal and abnormal lung. -heart pathology: the evaluation of the foetal heart remains extremely difficult because of its small size and high rate of battements. the unpredictable foetal motions during data acquisition and the absence of a foetal ecg signal to synchronize data acquisition are additional problems. in the last years, different approaches have been made to overcome these challenges. radiologists should know the typical imaging findings of the thoracic pathology most often observed in foetuses. prenatal mri may provide additional relevant information in a wide spectrum of congenital thoracic anomalies, but in general, it should only be performed if it is considered that additional results might influence the management of the pregnancy and/or the therapeutic approach. therefore, it is important to know the right indications for mri and to recognize the limits of the method. interruptions during embryogenesis of the muellerian or wolffian ducts result in various, potentially complex genitourinary abnormalities of a wide spectrum or combinations. multiple imaging modalities are employed to evaluate patients with these abnormalities. ultrasound is the frontline imaging modality. mr imaging is mostly reserved for complex cases and may incorporate an mr urography, too. other imaging modalities are less frequently used or provide only ancillary information. this presentation will demonstrate the utility of ultrasound and mr imaging, in particular, in the routine diagnostic imaging of patients with the wide spectrum of muellerian and wolffian duct abnormalities. & mr imaging is reserved for more complex cases. neonatal hepatic tumors and vascular malformations d. pariente, s. franchi-abella; le kremlin bicêtre/fr neonatal hepatic tumours and vascular malformations are rare but imaging plays a key role in diagnosis and treatment. the most frequent hepatic tumour is haemangioma (fig1) which often is asymptomatic but may be complicated by cardiac failure, coagulopathy or compartment syndrome. the differential diagnosis mainly includes hepatoblastoma, hematoma (fig2), abscess, mesenchymal hamartoma, choriocarcinoma in the solitary form and metastatic neuroblastoma, cirrhosis, neonatal leukemia in the multifocal form. pertinent biological data are alpha-fetoprotein (but level may be normally high in neonate), betahcg, and urinary catechol amines. hepatic vascular malformations are rarer and include intra or extra porto-systemic shunts (pss), arterio-portal fistula or complex mixed forms. intrahepatic pss may be associated with haemangioma and regress in most cases rapidly (fig3). on the contrary the extrahepatic pss which are located below the portal vein, should be urgently closed to avoid occurrence of agenesis of the portal vein. the best imaging modality is us which must be performed with high frequency probes and colour doppler to identify hepatic vessels and assess patency, direction of flow, abnormal communication. mri and ct with contrast injection may also be useful. hepatic mass in a 12 do neonate with increased crp and afp. us showing a hyperechoic mass with thrombosis of the left portal vein (black arrow) and a track (white) extending to the mass: hematoma due to malposition of an umbilical vein catheter. hemangioma of antenatal diagnosis on d1. the mass is composed of a large anterior vascular lake corresponding to a porto-systemic shunt and a tissular hyperechoic part. the infant has remained asymptomatic. intrahepatic porto-systemic shunt between the left portal branch of segment 2 (white arrow) and the left hepatic vein (black arrow) in a neonate. at 3 months of age this shunt has completely resolved. haemangioma is the most frequent hepatic tumour in the neonate and is often asymptomatic with spontaneous resolution. levels of alphafetoprotein are physiologically high in the neonate, and can be misleading. hepatic hematoma can be secondary to traumatic delivery, to coagulation disorders or to umbilical vein catheterization. intrahepatic porto-hepatic shunts are the most frequent vascular malformations and regress in most of the cases in the first year of life. us with colour doppler remains the best imaging modality in the neonatal period. imaging in crohn disease: state of the art in diagnosis, prognosis and followup n. colavolpe, a. aschero, b. bourliere-najean, c. roman, f. khachab, h. pico, m. kheiri, g. gorincour, c. desvignes, p. petit; marseille/fr summary: during the past years the inflammatory bowed diseases (ibd) have increase in frequency (1). less than twenty-five percent of them occur in children of less than 18 years (2) and crohn's disease (cd) is twice as frequent than ulcerative colitis (uc) in the pediatric age group. specific phenotypic and genotypic subtype of ibd occur in younger children. early onset (eo) pediatric ibd (before 5 years of age) represent 11% of childhood ibd (3) . uc and undetermined colitis are more frequent in this age group. eo cd showed a more frequent isolated colonic and upper gastrointestinal involvement than later-onset disease where locations are predominantly colic and terminal ileum later on childhood. some pediatric ibd specificities exist than can interfere with the imaging findings: -cd can be limited to the terminal ileum or to the colon in up to 20% of children (4) . isolated jejunal involvement is reported to occur in 5-6% of children. this location is more frequent in the youngest and is more at risk of complicated course of disease (2) . for auvin et al. (3) the small bowel is involved in 80% of cases with less involvement of the terminal ileum than in the adult population. -uc: the classical contiguous alteration of the bowel wall from the rectum to the caecum is inconstant. a macroscopic rectal sparing is reported from 5 et 30% and the absence of continuous disease from rectum to caecum (caecal patch) described in 2% of children. transmural inflammation may be present in severe form as well as terminal ileitis without granulomata (backwash ileitis) (2) . in order to assess these pathologies, and more specifically cd small bowel locations which are difficulty explored by others modalities, small bowel follow-through, barium enema, ultrasound, computed tomography and mr imaging have been widely used. among them, mr-enterography has gained worldwide acceptance due to multiple factors including: a high contrast resolution, a multiplanar ability, an absence of radiation, the possibility to explore in the same exploration the whole bowel and the extra-bowel diseases (perianal fistulae, sacroiliac joint, biliary tract), the ability to compare of side by side consecutive studies in a reproducible manner, a more easily understood exploration by the clinicians than ultrasound, and first of all for its performances. in order to technically harmonize this exploration a recent consensus statements on mre protocol has been published by the esgar and the espr societies (5) . preparation: -depending on their age children must not have solid oral intake from 2 to 6 hours prior to the examination to reduce bowel wall motility. morning mr appointment is more favorable for this purpose. no gasless fluid restriction is recommended but is reabsorbed too quickly to distend enough the small bowel. none hyperosmolar non absorbable solution is superior to another. its ingestion must start 45 to 60 minutes prior to mre. the recommended volume is 20 ml/kg with a maximum up to 25 ml/kg. explanations long before the mre concerning the importance of such absorption and the use of a refreshed product mixed with aromatized flavors will facilitate the child's participation. -the use of spasmolytic agents is optional. however, there are recommended in adults by multiple societies including esgar (5) , the society of abdominal radiology (6) and the acr (www.acr.org/ quality-safety/standards-guidelines). but, mre without antiperistaltic agents result has reached a high diagnostic confidence and excellent agreement with ct enterography for the presence of cd (7) . if used, they need to be administered immediately prior to motion sensitive sequence (t1w dynamic enhanced sequences). if the pictures obtain with these medications are of better quality, there is no evidence that they change the final diagnosis and the children's therapeutic management (8) . the use of these products increase the length of the exploration and their side effects are frequent (nausea > vomiting) which balance their visual benefice (8) . if a spasmolytic agent is used, the recommended first line spasmolytic agent is i.v. hyoscine butylbromide (0.5mg/kg i.v). the recommended second line agent is i.v. glucagon, 0.5mg (<24.9kg) and 1mg (>24.9kg), given as a slow infusion with i.v. saline at an infusion rate at 1ml/s. -no rectal enema is needed. -exploration can be performed either at 1.5tesla or 3testla. more chemical shift and susceptibility artifacts are present with the latter. prone position has been demonstrated to allow better small bowel distension than the supine one with reduction of the peristaltism but without better lesion detections (9, 10) . large multi-elements coils are needed to cover with high resolution from the perineum up to the left colonic flexure. sequences: both morphologic steady state free precession gradient echo and 2d -t2-weighted images are needed in the axial and coronal planes. fat saturation in one of this plane is recommended and maximal slice thickness of 5 mm is required. nowadays, non-enhanced then enhanced 3d t1-fat saturation weighted sequences are mandatory. slice thickness does not exceed 3 mm. enhance sequence need to be acquired at the portal phase of injection. however, in recent studies the need for gadolinium has been questioned when dwi is added to the morphologic sequences. dwi sequences have been considered optional (5,6) but we consider their place essential in pediatric practice. they must be done with high b values, from 600 up to 800 in the coronal and axial planes with 5 to 8 mm contiguous cut in free breathing. axial plane is less prone to artefact than the coronal plane. interestingly enough shenoy et al (11) report in 27 pediatric patients that dwi does not perform as well as standard mre for detection of active crohn disease but the combination of dwi and mre increases imaging accuracy for determining disease activity compared with either technique alone. seo et al (12) in 44 young adults said that dwi mre was noninferior to contrast-enhanced mre for the evaluation of inflammation in cd. based on the exploration of 130 cd adult and pediatric population, dwi proved to be efficient and would avoid gadolinium injection (14) . sirin et al. (14) report in 37 children that dwi revealed lesions that were not detectable with mre done with gadolinium injection. finally, respectively dubron et al. (15) in 48 children and neubauer et al (16) in 33 children and young adults demonstrate better performance of dwi than gadolinium enhanced imaging. like the existing mr protocols for suspected appendicitis (17) it will not be surprising to see fast mr ibd explorations becoming an alternative to emergency us as already proposed (16) . this fast mr limited to a morphologic t2 sequence in two planes associated with dwi sequences will allow a positive diagnosis and the ibd work up. apart from bowel obstruction and its spontaneous bowel distension one of the limiting factors will be the need for an oral water agent uptake in a potential surgical patient. however, it has been published in the adult literature than an oral or rectal preparation was not necessary to rule out uc (18) nor a cd (19) . the other limiting factor is the length of exploration. mre can be shorten especially if the patient's positioning is easy to do (dorsal decubitus) (20) and if there is no need for injection, either for spasmolotytic agent and for gadolinium chelates. the suppression of the iv line, the absence of potential side effects (nausea, vomiting) of paralytic agents and the decreased of repeated long apneas with no loss of significant information will be strong progresses toward the holy grail. -positive diagnosis, disease activity, prognosis and follow-up: mre has a better accuracy to detect inflammation for the small bowel than for the colon (21) . one of its goal is to try to accurately identify features of active inflammation vs fibrotic disease. this is of paramount importance since the former may respond to medical treatment and the latter may need surgical resection. however, inflammation and fibrosis are associated within the same bowel segment and progress in a parallel way making the goal difficult to reach (22) (23) (24) . in their study based on the analyze of 20 children operated for cd strictures, barkmeier et al. (24) report than strictures demonstrating >3 cm upstream dilatation with associated feces sign were highly associated with transmural fibrosis. the most severely fibrotic strictures were associated to the greatest amount of inflammation and there was no significant correlation between stricture length, mural thickness, degree of post-contrast enhancement (arterial and delayed venous phases), diffusion-weighted imaging apparent diffusion coefficient, pattern of post-contrast enhancement, or normalized t2-weighted signal intensity and histological fibrosis or inflammation scores. however, correlation with histological specimens of cd done on a other series s312 (2017) 47 (suppl 2):s297-s pediatr radiol demonstrated that the enhancement ratio of the wall is positively correlated with disease chronicity due to a possible increasing microvessels permeability and inversely correlated to acute disease (25). on the other hand, several authors have tried to correlate the adc values to cd activity. fibrotic tissue does not restrict diffusion and presents a decrease of signal at high b values and high adc values whether acute inflammation shows decrease adc values. variable thresholds from 1.6 x10 -3 mm 2 /s to 2.4 x10 -3 mm 2 /s have been proposed to separate active vs non active disease (21) . however, others authors have reported low adc value of fibrosis compare to histology (26). even if promising results have been published with high correlation with the crohn disease endoscopic index of severity (13) , adc measurements are associated with sever limitation factors including sample size overlap between the bowel wall and its atmosphere, lack of reproducibility between mri-units and mri-vendors, non-standardized sequence b-values parameters (21) . two mre scores are available to quantify the activity of cd. one is using gadolinium injection (27) and the other dwi (13) . due to the complexity of the formula, both are difficult to use in daily practice and have not been evaluated in paediatric practice. interestingly enough if a simplify mre paediatric protocol appears to become a reality, us stays a good imaging challenger and (28). in a recent meta-analysis, based on adult and pediatric series, calabrese et al (29) reported that bowel us showed 79.7% sensitivity and 96.7% specificity for the diagnosis of suspected cd, and 89% sensitivity and 94.3% specificity for initial assessment in established patients with cd. bowel us identified ileal cd with 92.7% sensitivity, 88.2% specificity, and colon cd with 81.8% sensitivity, 95.3% specificity, with lower accuracy for detecting proximal lesions. the absence of abnormal thickness wall would have a negative predictive value, high enough to exclude the need for further exploration, especially when cd is concerned (30,31). concordance between us and mre have been variably reported from excellent (32) to just correct (33). rosembaum and al (22) report that the us findings present in children operated for cd include: bowel wall thickness above 4.3 mm (mean, 6.1 mm) and an increased frequency of loss of mural stratification and fibrofatty proliferation. others us technologies are used in children to better approach the disease activity. it includes, hydrosonograpy using specific oral agents (mannitol, sorbitol, polyethylene glycol, etc…), contrast-enhanced ultrasound and dynamic contrast-enhanced ultrasound (nowadays, contrast agent is offlabel in children) (34) and elastography (35). their enthusiastic results and their efficiency to assess disease activity need to be confirmed (36). in conclusion, as we suspected 7 years ago (37), mre has dramatically modified our approach of pediatric ibd especially when considering its orientation toward a less invasive exploration and the increasing importance of dwi imaging. a cost benefice between mre and us remains to be done on this increasing disease. heterotaxy and isomerism c. lapierre; montreal/ca summary: objectives: to review the classification of visceroatrial situs to describe the associated cardiac and non-cardiac anomalies to illustrate typical findings in fetuses, neonates and children to discuss the surgical consideration and the long-term follow-up in these patients abstract: by definition, the type of situs is determined by the relationship between the atria and the adjacent organs. anatomically, the atrial chamber differentiation is based on the morphologic aspect of the atrial appendages, earlike extensions of the atria. three types of situs exist: solitus (normal), inversus (mirror image) and ambiguus. a single type of situs is present in a patient. when the situs is neither solitus nor inversus, it is referred to as situs ambiguus or heterotaxy. heterotaxy may manifest with various abnormal visceroatrial configurations that are associated with cardiac (in 90-100% of cases) and extracardiac anomalies such as splenic abnormalities, biliary atresia and intestinal malrotation. two subsets of situs ambiguus are well-recognized: right isomerism (asplenia) and left isomerism (polysplenia). in heterotaxy, the venoatrial connections are frequently abnormal. left isomerism is usually indicated by bilateral bilobed lungs, interruption of the ivc and multiple spleens. the more likely found cardiac anomalies are: pulmonary or aortic stenosis, isolated atrial and ventricular septal defects, cardiac arrhythmia due to sinus node dysfunction as well as pulmonary veins that drain into both the right and the left atria. in the presence of right isomerism, bilateral trilobed lungs, a large symmetric liver, and absence of the spleen are frequently observed. at the cardiac level, patients are more likely to have a common atrioventricular defect, a double outlet right ventricle and pulmonary stenosis. total anomaly of the pulmonary venous return and absence of coronary sinus will always be present in right isomerism. heterotaxy can be diagnosed with high accuracy by prenatal echography. a diagnosis should be suggested in the presence of congenital heart disease, visceroatrial heterotaxy and interruption of inferior vena cava with azygos continuation for left isomerism or abnormally closed juxtaposition of inferior vena cava and descending aorta in right isomerism. the mortality in fetuses is high in the presence of heart block and hydrops whereas the cardiac anomalies influence the long-term outcome. as discussed in the literature, the clinical outcomes and long-term prognosis in these patients are relatively poor when compared with non-heterotaxy patients. the risk factors are cardiac (underlying anatomy and arrhythmia risk) and non-cardiac. based on the cardiac anatomy, one of the main determinants is left versus right isomerism. with right isomerism, the cardiac malformation is more severe and an univentricular correction is more frequent. another predictor of mortality is pulmonary vein stenosis/obstruction. whatever the severity of cardiac lesions, the postoperative or discharge mortality is higher in patients with heterotaxy. prenatal diagnosis seems not improve the survival. extracardiac anomalies also contribute to the increased morbidity and mortality. three of the more challenging entities are respiratory, immunologic and gastrointestinal. recurrent respiratory infections, failed extubation or chronic respiratory failure are frequently observed in patients with heterotaxy. recent studies revealed an association between heterotaxy and primary ciliary dyskinesia which can explain the increased postoperative respiratory complications. the spleen is important for the bacterial clearance. patients with asplenia or polysplenia are thought to have "functional asplenia". so, they are at risk for sepsis and severe bacterial infection. the incidence of intestinal malrotation is high, approximately 40% to 90%. observation versus prophylactic ladd procedure and screening for asymptomatic intestinal malrotation are a growing area of debate. the trend seems to go along conservative management and surveillance of malrotation. bronchopulmonary malformations, such as congenital pulmonary airway malformation (cpam), bronchopulmonary sequestration (bps), and congenital lobar emphysema (currently known as congenital lobar overinflation [clo] ), are common congenital lung diseases. these conditions are detected prenatally, usually in the second trimester, in countries where obstetric sonography is routinely performed. the malformations are seen as hyperechoic images with respect to normal fetal lung parenchyma, with a mass effect and homogenous appearance or with coexisting cysts. the lesions usually decrease in size along gestation. a residual mass is seen on postnatal chest radiography, the first imaging technique performed, in only 40% of cases. cpam and bps are predominantly located in the posterior lower chest and can be identified postnatally on ultrasound using a small vector probe and a subcostal and subxiphoid approach. potential feeding arteries can be visualized using color or power doppler. based on clinical and sonographic findings, the differential diagnosis between congenital lung malformations and tumors such as neuroblastoma, type i pleuropulmonary blastoma, and myofibroblastic tumor will be discussed. postnatal management and imaging of newborns with congenital lung malformations is controversial, particularly in asymptomatic patients (approximately 80% of cases). chest radiography is mandatory at birth and chest ultrasound is also recommended to confirm the prenatal diagnosis. computed tomography (ct) or magnetic resonance imaging (mri) using angiographic techniques should be performed some months (8 months) after birth in asymptomatic patients. these techniques are also recommended in symptomatic newborns and before surgery to characterize the arterial supply and venous drainage in cpam and bps, as ultrasound is limited in this regard. in premature infants, sonography complements radiography in the study of prematurity-related lung diseases such as respiratory distress syndrome and its pulmonary complications (eg, pneumothorax), in predicting bronchopulmonary dysplasia, and in diagnosing transient tachypnea of the newborn when clinical and radiographic features are inconclusive. the main ultrasound finding in these conditions is visualization of numerous "b-lines", vertical narrow-based hyperechoic bands extending from the pleural surface to the end of the field of view, representing what is currently known as "sonographic interstitial syndrome". b-lines are artifacts originating from variations in the air-fluid relationship of the lung and are better seen using high-frequency linear probes . use of sonography for follow-up of these patients will reduce the number of the chest plain films performed, and therefore, the amount of radiation exposure in this vulnerable population. for proper interpretation of the sonographic findings in these conditions, the radiologist should be familiar with current related terms, such as lines a, lines b, comet tail artifact, interstitial-alveolar syndrome, septal syndrome, and white lung. trauma is the leading cause of mortality and morbidity in children after the first year of life. motor vehicle accidents are the leading cause of death from unintentional injury in children up to the age of 15. of these cases, the abdomen is the fourth most commonly injured area. in pediatric patients non-operative management of these injuries predominate, hence the importance of early radiologic assessment for appropriate clinical follow-up. anatomically, compared to adults, childrens' abdomens are more square, less muscular and with less intraperitoneal and subcutaneous fat to absorb impact. the diaphragm is more horizontal causing downward displacement of the liver and the spleen outside the protective casement of the ribs. the pelvis is smaller and hence the bladder is displaced upward, also resulting in more vulnerability to this organ. the organ surface area is larger in children and they have a smaller body mass-hence more force applied per-unit of body surface area. the ribs are flexible, and although we see fewer rib fractures, this results in more internal damage. physiologically, children maintain hemodynamic stability longer, often presenting with only mild tachycardia, even when in severe hemodynamic shock. decrease in blood pressure may not be evident before the loss of 30% blood volume. nevertheless, bleeding is less severe and operative intervention is rarely performed. mechanics of blunt abdominal trauma include organ compression from seat belt injury with the presence of erythema, ecchymossis or abrasion on the abdominal wall increasing the likelihood of internal organ injury (55% likelihood of injury). other common mechanisms include pedestrian-car collisions( 4% with intra-abdominal injuries), falls (4% with intra-abdominal injuries), or handle bar injuries (54% with intraabdominal injuries). after the child arrives in the hospital, a trauma algorithm is initiated. generally, for the unstable patient, algorithms are similar and require a rapid atls protocol, followed by a fast ultrasound to confirm free fluid prior to operation. in stable patients, institutional algorithms vary greatly between countries and in different centers. some rely solely on mechanism to determine the need for fast vs ct (not complete ultrasound), others will rely on clinical exam (in a conscious patient with reliable exam) and blood work to determine the need for imaging (ct or us) and others may chose to perform an initial us and complete the exam with a constrastenhanced us during work hours. in the literature many management prediction rules exist based on the history, physical examination, mechanism of injury and are supplemented by blood work and/or intial imaging. most are based on retrospective reviews, with only a few controlled clinical trials. however, the validity of these studies is limited because of different populations, institutional policies and variable radiological practices in terms of when imaging is performed, which modalities are most beneficial and which are less valuable, all the while, considering the utilization of the least irradiating techniques. a representative sample of such algorithms will be discussed. routine and extensive initial trauma panels are not required according to a number of studies. abdominal ultrasound and urinalysis together have been found to confirm 98% of all intra-abdominal injures, in some studies. serial haemoglobins/hematocrit is valuable for determining ongoing s315 (2017) 47 (suppl 2):s297-s pediatr radiol blood loss and assists clinical surveillance. electrolyte abnormalities are uncommon in children unless severe shock is present (metabolic acidosis). liver function tests are elevated in most cases of blunt abdominal trauma, hence, are often performed for its high sensitivity, to avoid ct if the liver panel is negative. imaging, however, is needed for grading of the potential liver injury if the liver panel is positive. abdominal xray is not useful in blunt abdominal trauma, and is usually normal. ultrasound has an important role in the pediatric community, as a sensitive and non-irradiating modality. however, this sensitivity is dependent on the type of ultrasound performed (fast vs. complete abdominal ultrasound vs. contrast-enhanced ultrasound) but also on the qualifications and experience of the performing physician. a meta-analysis of fast in pediatrics demonstrates that it has a sensitivity of 66% (grade i-ii evidence) for identifying hemoperitoneum. a negative fast is not sufficient to rule out intra-abdominal trauma. one prospective observational trial demonstrasted that 34 % of patients without free fluid on fast (performed by formally trained pediatric truama surgeons demonstrated at least grade iii liver or splenic injuries on ct). we know that pediatric ultrasound is operator-dependent, and generally an ultrasound performed by the skillful hand of a pediatric radiologist is more sensitive than that performed by surgeons or by adult radiologists. furthermore, we know that the benefits of contrast-enhanced ultrasound in pediatric trauma exist-highly accurate in visualising lesions, hence avoiding non-contributive ct imaging, however, the feasibility of providing 24-hr contrast-enhanced ultrasound by a qualified radiologist is resource intesive: both structurally and with respect to personnel. published indications for abdominal ct in stable pediatric patients included suspected mechanism of blunt abdominal trauma, significant fluid resusitation without apparant blood loss, hemoglobin <100mg/l without obvious blood loss, multisystem trauma and unreliable abdominal exam. one series with 1500 children undergoing ct for blunt abdominal trauma demonstrate postive findings in 388 (26%), of which all solid organ injuries and 96% of hollow viscus injuries were identified on ct. however, ct has its limitations: it was found to identify gastrointestinal perforation in only 47% of patients with known perforation, but with findings of free fluid, wall thickening and/or bowel dilatation. it is also less accurate in identifying pancreatic trauma, with normal scans in 33-53% of children with pancreatic trauma. again, findings of pancreatic trauma can be non-specific: free fluid or, less commonly, thickening of the gerota's fascia, presence of mesenteric fluid or of fluid between the pancreas and the superior mesenteric vein. when and where to perform ct depends on the imaging algorithms established by individual centers. generally, unstable patients with very high grade visceral injuries are taken to surgery. the stable patients are treated with non-operative management. the literature on angiographic embolization in pediatric blunt trauma is limited to case series that demonstrate a limited utility in hemodynamically stable patients with ongoing blood loss or for the definitive treatment of traumatic pseudoanevryms. a dialogue with the interventional radiologist is imperative in such cases. common imaging findings and pitfalls will be illustrated with case examples. in conclusion, a child's anatomy and physiology must be taken into account when determing the level of urgency and appropriate imaging work-up in blunt abdominal trauma. imaging of these patients cannot follow a standard algorithm as institutions vary with respect to types of personel, training, frequency of trauma, emergency department trauma protocols and availability of an in-house pediatric radiologist. ultrasound and ct have their advantages and disadvantages with associated pitfalls that the pediatric radiologist must recognize to provide an optimal diagnostic workup with minium irradiation. take home points: a child's anatomy and physiology must be taken into account when determing the level of urgency and appropriate imaging work-up in blunt abdominal trauma. imaging of pediatric abdominal trauma cannot follow a standard algorithm as institutions vary with respect to types of personel, training, frequency of trauma, emergency department trauma protocols and availability of a pediatric radiologist. ultrasound and ct have their advantages and disadvantages with associated pitfalls that the pediatric radiologist must recognize to provide an optimal diagnostic workup with minium irradiation. sport injuries d. jaramillo; miami, fl/us the growing skeleton has unique vulnerabilities to acute and chronic injuries due to sports. the practice of intensive sports during puberty and adolescence has led to a great increase in the incidence of sportsrelated injuries. during the growth spurt of early adolescence, the physis becomes weak, and is the site of fractures and avulsions (particularly in the apophyses) and of physeal widening due to repeated stresses, such as the wrist in gymnasts or the proximal humerus of baseball pitchers. both lesions can result in growth arrest. the chondro-osseous junctions of the ossifying epiphyses and apophyses are also vulnerable to avulsions, and the avulsed fragment may be entirely cartilaginous and not visible radiographically (such as in the patellar sleeve fracture). repeated trauma to epiphyses or round bones can lead to osteonecrosis (panner's disease) but more often to osteochondritis dissecans (ocd). in adolescents, ocd occurs most frequently in the medial femoral condyle, the capitellum of the elbow and the talar dome. juvenile ocd has a better prognosis than the adult form. when the skeleton begins to mature, there are fractures unique to partially closing physes such as the triplane and tillaux fracture. some sturctures have propensity to unique injuries during adolescence. a stress on the anterior cruciate ligament (acl) can lead to a tibial eminence avulsion in puberty ( figure) , an incomplete acl tear in early adolescence or a complete acl tear later. meniscal tears are almost always vertical and often involve large meniscal fragments that can flip. patellar dislocations often result in osteochondral injuries. this review will cover the main types of sports-related injuries and the imaging modalities used to diagnose them. 11 year-old with pain and popping sensation during a fall on a football game. ap radiograph is normal & it is important to take into account the specific sport in order to anticipate subtle injuries that may be difficult to detect. a. c. offiah; sheffield/uk the radiographs obtained when inflicted injury is suspected are collectively termed the "skeletal survey". a full skeletal survey should be performed in all children below 2 years of age in whom abuse is suspected. the investigation is not complete until follow-up skeletal imaging has been performed in the 11 to 14 days following the initial survey. children below one year of age should also receive a ct brain. neurological imaging in older children will depend on the clinical scenario. ct chest/abdomen is indicated when visceral injury is suspected. in terms of imaging in suspected abuse, espr has adopted the rcr guidelines. in the absence of a history of significant trauma, fractures highly specific for abuse in pre-ambulatory children include rib, metaphyseal and diaphyseal fractures. simple linear skull fractures have a relatively low specificity for abuse. the combination of subdural haemorrhage, retinal haemorrhage and diffuse cerebral oedema/encephalopathy (the so-called, "triad") suggests shaking. whereas the presence of a skull fracture implies impact. visceral injury often results from direct blunt trauma and may therefore be accompanied by anterior and/or costochondral rib fractures. the posterior rib arcs are protected by soft tissue and posterior rib fractures result from compressive/squeezing forces rather than direct trauma. the dating of fractures has a subjective element and it is more important to recognise that fractures are in different stages of healing, rather than to assign a definite age/age range to the injuries. the major differential diagnoses are accidental trauma and osteogenesis imperfecta. if rickets is the cause of the fractures, then radiology and/or biochemistry will show evidence of rickets. a low vitamin d level, in the absence of rachitic features, is not the cause of fractures. close liaison between radiologists and paediatricians is vital and any siblings/children in the same household who are below 2 years of age should also receive a skeletal survey. remember that the presence of injury does not always mean abuse and that the absence of injury does not always exclude abuse. scoliosis may be primitive, structural, particularly during adolescence; during this period, careful follow-up is mandatory, because worsening is frequent. clinical examination with evaluation of a hump (gibbosity) with a scoliometer is mandatory, with also neurological assessment. beside radiography, additional tools have been developed to avoid xray exposure: "spinal mouse", back surface topography systems, ultrasound and other computer-assisted systems. but scoliosis can also be secondary, and imaging is important to find a cause and adapt management. among the etiologies, radiologist must recognize spine malformations, dysplastic and neuromuscular scoliosis. in addition, scoliosis may also be in relation with a primitive lesion, tumor-related or not, whether the initial disease could be within the spinal canal, spinal or paravertebral. imaging studies lies first on pa and lateral full spine x-rays, if possible with a low dose device (flat panel, slot-scanning system), keeping in mind that follow-up with repetitive exposures may be necessary. reproducible measures of different curvatures help to assess the overall static spine and the importance of scoliosis with cobb angle. the assessment of axial rotation can be obtained through 3d simulations, with frontal and axial views (see figure) . morphologic evaluation of the s317 (2017) 47 (suppl 2):s297-s pediatr radiol spine is mandatory: if a secondary scoliosis is suspected, the research to etiology needs to perform ct or mri, depending on the clinical signs and the results of plain x rays evaluation. similarly, these explorations are useful in the preoperative assessment when surgical treatment is necessary. girl scoliosis, pa and lateral views with eos®, 3d simulation, coronal and axial views take home points: clinical evaluation is always the first step in subject with suspected scoliosis radiation burning is quite low with new devices, but repetitive exposures for follow-up need to carefully respect justification for x-rays exposures new tools are available to appreciate 3d spinal deformation and evaluate prognosis and surgical procedures ct and/or mri are useful in presurgical assessment and to look for etiologies in suspected secondary scoliosis malformations of the spine and spinal cord a. rossi; genoa/it summary: embryology and classification: spinal cord development occurs through three consecutive periods: (i) gastrulation (2 nd gestational week): the embryonic disk is converted from a bilaminar into a trilaminar arrangement, with formation of the intervening mesoderm; the notochord is laid down along the midline, identifying the craniocaudal embryonic axis; (ii) primary neurulation (18 th -27 th day): under the induction of the notochord, the midline ectoderm specializes into neural ectoderm. the initially flat neural plate progressively bends and folds until it fuses in the midline to form the neural tube. the primary neural tube produces the uppermost 9/10 of spinal cord; (iii) secondary neurulation (28 th -48 th day): a secondary neural tube is laid down caudad to the termination of the primary neural tube. retrogressive differentiation of the secondary neural tube results in the tip of the conus medullaris and filum terminale. defects in one of these three embryological steps produce spinal dysraphisms, characterized by anomalous differentiation and fusion of dorsal midline structures. spinal dysraphisms may be categorized clinically in two subsets: open and closed spinal dysraphisms. in open spinal dysraphisms (osd) the placode (non-neurulated neural tissue) is exposed to the environment through a cutaneous defect along the child's back. osd include myelomeningocele, myelocele, hemimyelomeningocele and hemimyelocele, and are associated with a chiari ii malformation. myelomeningocele is by far the most common of these forms; the placode protrudes through a posterior defect and is elevated above the skin surface due to concurrent dilatation of the subarachnoid spaces. closed spinal dysraphisms (csd) are covered by intact skin, although cutaneous stigmata usually indicate their presence. two subsets may be identified based on whether a subcutaneous mass is present. csd with tumefaction comprise lipomas with dural defect (lipomyelocele and lipomyelomeningocele), meningocele, and myelocystocele. lipomas with dural defect are more common; they are differentiated from one another based on the position of the cord-lipoma interface, that lies within the spinal canal in lipomyelocele, and outside the spinal canal (ie, into a meningocele) in lipomyelomeningocele. csd without tumefaction comprise complex dysraphic states (ranging from complete dorsal enteric fistula to neurenteric cysts, diastematomyelia, dermal sinuses, caudal agenesis, and spinal segmental dysgenesis), bony spina bifida, tight filum terminale, filar and intradural lipomas, and persisting terminal ventricle. the most complicated forms (complex dysraphic states), including diastematomyelia, caudal regression, and segmental spinal dysgenesis) are related to faulty gastrulation. diastematomyelia (literally, split cord) is caused by failure of midline notochordal integration, resulting into two separate hemineural plates. caudal agenesis and segmental spinal dysgenesis are related to defective notochordal formation, characterized by absence or hypoplasia of a segment of the notochord, in turn resulting into absence or hypoplasia of a corresponding segment of the spinal cord. functional neuroimaging of cns is a fast advancing field with frequent new developments in scanner's hardware, protocols, clinical indications, and post-processing techniques. for radiation safety reasons in the case of children, functional neuroimaging is mostly based on mr techniques especially designed to focus on the assessment of functional tissue characteristics, such as neuronal activity (fmri),, metabolism (mrs) and perfusion (dsc perfusion, asl). pediatric coils with multiple elements, multiple slice excitation, 3d spectroscopy, 3d asl, reduced fov (zoom) and improved motion compensation techniques are important tools available to meet the permanent challenges of pediatric mr functional imaging: fast motionless acquisitions and increased resolution. functional mri (fmri) reveals brain activation during performance of behavioral tasks, based on the blood oxygen level dependent (bold) mri signal, which is modulated by neural activity via a process of neurovascular coupling. for children, especially of younger age unable to follow a task, resting-state fmri (rfmri) can be performed and correlates brain areas with similar spontaneous fluctuations in the bold signalthereby enabling estimates of 'functional connectivity.' main clinical applications of fmri are the delineation of eloquent cortex near a space-occupying lesion and the determination of the "dominant hemisphere" for language. intense research is conducted in the areas of language organization and development, brain plasticity, and neurobehavioral disorders (e.g. adhd). magnetic resonance spectroscopy (mrs) is a noninvasive mr technique, that detects intracellular metabolites, and may provide neuroimaging biomarkers of normal biological and pathological processes or response to a therapeutic intervention. although the main field of application of mrs is the brain tumors, it has also been of particular (2017) 47 (suppl 2):s297-s pediatr radiol usefulness in assessing ischemic or traumatic brain injury and neurometabolic disorders. perfusion mr imaging methods detect signal changes that accompany the passage of a tracer through the cerebrovascular system. a less invasive approach is arterial spin labeling (asl) that uses arterial water as an endogenous tracer to measure cbf and thus it is more suitable for pediatric studies. mr perfusion is applied in the evaluation of brain tumors, neurological diseases and developmental disorders. functional neuroimaging clinical applications are expected to expand greatly in the future due to the increasing availability of their techniques, as well as the continuous advancements in the field of pediatric research. good knowledge of these techniques will become more necessary for an effective clinical practice and will enhance the role of radiology in the healthcare system. functional neuroimaging advanced techniques based on mri allow us to study complex cns processes such as cerebral perfusion (dsc, asl), metabolic activity (mrs) and brain activation (fmri). functional neuroimaging techniques already have significant clinical pediatric applications and assisted by recent advances in mr technology are expected to become even more powerful in the near future. kidney: perfusion, excretion, obstruction k. darge; philadelphia/us the functional imaging of the urinary tract entails the evaluation of the renal perfusion and excretion. in this complex process the sites of the main abnormalities could be pre-renal, renal parenchymal, renal pelvicalyceal or post-renal or even a combination at different sites. functional mr urography (fmru) is an advanced tool that not only allows the exquisite morphological depiction of the urinary tract, but also makes it possible to generate comprehensive functional data. these provide information about the function of the kidney as well as the excretion of urine from the renal parenchyma into the pelvicalyces and ureter. the functional results are mainly divided into two groups: 1. transit timesthese are recorded in minutes and a side comparison gives idea how much time it takes for the contrast to go through the renal parenchymathe longer the more abnormal in general. 2. differential renal functionsthese can be based on the enhanced renal parenchymal volume or the patalk number generated from this area and provides in percentage the split renal function. this presentation will discuss in detail the functional aspect of mr urography and demonstrate its utility in routine pediatric uroradiologic imaging. in chronic childhood lung disease (e.g. cystic fibrosis) global pulmonary function tests (pft) can be normal although lung damage is already present. moreover, in comparison to imaging, pft is challenging in young children. thus, cross-sectional imaging became more important in the past two decades. regarding morphological evaluation, multidetector computed tomography (mdct) serves as the most sensitive and reproducible modality. for functional evaluation perfusion/ventilation scintigraphy remains the reference standard. although the individual radiation burden by a single chest ct has decreased significantly in the past, radiation doses can cumulate considerably when repeated examinations are performed in a longterm follow-up. pulmonary mri exists as an alternative method, especially for paediatric patients. however, standard h+mr sequences do not demonstrate small airway disease due to inherent limitations of low signal and rapid t2* signal decay of lung tissue. for comprehensive diagnosis, functional mri offers the unique possibility to measure regional ventilation and perfusion, and mapping relaxation times and diffusion. focussing on research applications, a variety of methods are available for these purposes. in this context, ventilation imaging using inert fluorinated gas indicates to overcome the limitations of the expensive setting necessary for imaging with hyperpolarized noble gasses. regarding lung perfusion, dynamic contrast-enhanced mri (dce-mri) is the most established method in clinical practice. however, especially in children, techniques that are completely non-invasive and do not require i.v.-contrast agents administration or gas inhalation could be promising to achieve broad acceptance. concerning non-invasive methods, ventilation can be assessed by sequences with ultra-short echo times (ute), perfusion by arterial-spin-labeling (asl) and both by fourier decomposition mri (fd-mri). in conclusion, pulmonary mri offers both, the assessment of morphology and the unique possibility to measure regional ventilation and perfusion, and mapping relaxation times and diffusion. new mr techniques that are completely non-invasive are now available. however, further scientific evaluation is needed. ibd and related arthropaties d. jaramillo; miami, fl/us musculoskeletal diseases affect about 5% of patients with crohn's disease and are the most frequent extra-intestinal manifestation of inflammatory disease. the articular manifestations of inflammatory bowel disease (ibd) are one of the seronegative arthritides, although they have a lower incidence of hla -b27 than other seronegative arthritis such as ankylosing spondylitis. there are manifestations in the joints of the extremities, and findings in the pelvis, especially in the sacro-iliac joints, and spine. involvement of the extremities occurs in about 10% of patients with ibd related arthropathies, are more common with crohn's disease, and can have either manifestations related olygoarticular jia, or can have symmetrical involvement of smaller joints. the axial manifestations include ankylosing spondylitis and sacro-iliitis. sacroilliitis is typically bilateral (figure) and often has radiographic as well as mri abnormalities. enthesitis, tenosynovitis and dactylitis can occur with ibd just as they occur with other arthritides. it is important to differentiate ibd related arthritis from septic arthritis due to extension of an enteric fistula. deceased bone mineral density is a common finding in inflammatory disease. it occurs as a combination of malabsorption of vitamin d due to intestinal involvement and the effects of therapy, particularly corticosteroids. insufficiency fractures of the spine, sacrum and extremities can mimic the symptoms of arthritis. finally, ibd can be associated with chronic non-bacterial osteomyelitis, although this association is relatively rare. this review will illustrate several of the skeletal manifestations of ibd, focusing on the arthropathies. juvenile idiopathic arthritis (jia) can be defined as an arthritis of unknown cause occuring in children younger than 16 years and of at least 6 weeks duration. juvenile spondyloarthritis (jspa) is a subset of jia and is characterized by enthesitis (inflammation at the attachment of tendons, ligaments and the joint capsule), arthritis and an increased risk of axial disease. there is also a strong association with human leukocyte antigen b27. jspa accounts for approximately 10-15% of juveniles arthritis cases in europe and is the most common form of juvenile arthritis in asia. the condition is associated with significant long-term morbidity, high health-care costs and poorer outcomes compared with other forms of juvenile arthritis as well as its adult counterpart. up to 40% of patients continue to be at risk of developing ankylosing spondylitis (as) during the disease course. recognizing spondyloarthritis (spa) in children is challenging, particularly early in the course of disease, as the signs and symptoms at disease onset differ from those seen in adults. jspa typically presents with hip and lower limb arthritis in conjunction with enthesitis. inflammatory back pain as a presenting symptom is less common. as a consequence, jspa may be missed or confused with other juvenile arthritides and patients often experience prolonged delays in diagnosis. currently there is no single diagnostic or classification system that is representative of the jspa population. according to the international league of associations for rheumatology (ilar) classification system, most childhood spa's are classified as enthesitis-related arthritis (era), psoriatic arthritis or undifferentiated arthritis. recent studies indicate that there are two clinical phenotypes of era: those with early axial disease often associated with hip arthritis in addition to peripheral arthritis; and those who follow a more peripheral disease course with arthritis and enthesitis and do not develop axial disease. the ilar classification system places patients with both axial and peripheral involvement into the era subtype, and does not specifically address children who meet the criteria for as. the correct approach to the classification of era is uncertain, and this issue is confusing to both pediatric and adult rheumatologists. unlike other categories of juvenile arthritis, jspa affects boys more often than girls, and peak age of onset is early adolescence. enthesitis is a defining characteristic of jspa. it is more common and affects more sites in the paediatric population compared with the adult one. the most commonly tender entheses are the insertions of the patellar ligament at the inferior patella, plantar fascia at the calcaneus, and the achilles tendon. arthritis in jspa is typically asymmetrical, oligoarticular (< 5 joints) and involves predominantely the weightbearing joints. isolated hip joint arthritis may be the presenting feature and predicts early axial disease. involvement of the small toe joints is common in jspa but rare in other forms of jia. midfoot arthritis and tarsitis (inflammation of the intertarsal bones, overlying tendons, entheses and soft tissue) is highly suggestive of spondyloarthritis. in adults, inflammatory back pain typically heralds the onset of sacroiliitis, whereas children seldom present with symptoms of axial disease. however, according to several studies, sacroilitis can be asymptomatic in jspa and only detectable by imaging. other axial manifestations in jspa are inflammation of the lumbar apophyseal joints and interspinous ligaments, corner lesions of the spine and other sites of axial enthesitis-osteitis including the various ligamentous and muscular attachments of the pelvis. extraarticular manifestations of jspa are highly associated with axial disease and include acute anterior uveitis, bowel inflammation, psoriasis, and cardiac disease. clinical diagnosis of jspa can be difficult and the role of imaging may be more critical than in adult disease. the major goal of imaging in jspa is to identify children with early signs of axial disease, as this group is at the greatest risk for progression to as. the presence of axial disease in spa has also major implications for treatment decisions, since traditional firstline therapies appear to have minimal effectiveness in the management of axial inflammation. in addition, recent studies in adults suggest that earlier initiation of biologic agents (anti-tnfs) may slow radiographic progression. x-rays are not sensitive to acute inflammatory changes and will only show advanced disease in the sacroiliac joints. for these reasons plain radiographs are not useful in children or adolescents. ultrasound is a non-invasive, non-ionizing and relatively inexpensive technique that can be performed in a clinical setting. it is emerging as a valid diagnostic tool in spa and can be used to visualize peripheral synovitis, tendonitis and enthesitis, but the method is heavily operator-dependent and there does not yet exist a clear definition for the diagnosis and grading of enthesitis in children. secondary changes (calcifications, enthesophytes) have been observed much less in children compared with adults. there is a need for better consensus on abnormal ultrasonographic findings that define enthesitis lesions and standardization of methods. magnetic resonance imaging (mri) is a radiation free and sensitive imaging modality for detection of synovitis as well as cartilage and bone destruction. mri of the sacroiliac joints is increasingly obtained for early detection of inflammatory changes, as it shows active inflammatory (bone marrow edema, osteitis, enthesitis and capsulitis) and structural (erosions, subchondral sclerosis, subchondral fatty change and bony ankylosis) lesions of sacroiliitis long before radiographic changes become evident. in adults, mri has become the gold standard imaging modality for detecting arthritis and enthesitis. consensus definitions of lesions indicating pathology on mri are now incorporated into diagnostic criteria for adult with spa. in children and adolescents there is no gold standard mri technique and it is therefore not clearly defined whether changes s320 (2017) 47 (suppl 2):s297-s pediatr radiol seen in the sacroiliac joints are pathologic or part of normal maturation in the growing skeleton. the use of contrast enhanced imaging for the detection of active sacroiliitis on mri in jspa is a major controversy. synovial enhancement can be detected without accompanying bone marrow edema in children, and it can be argued that contrast should be administered in order not to miss the diagnosis. some authors argue that contrast administration does not change or add substantially to the mri findings made on non-enhanced scans. certainly, given the risks associated with gadolinium administration, contrast should be used with caution. perhaps the use of contrast agents should be limited to selected cases when high stir signal in the joint is the only finding in order to confirm the presence of synovitis, and when the differential diagnosis includes etiologies such as infection or tumor. the development of new mri techniques has made it possible to perform whole body mri scans (wbmri) that allow assessment of the full range of affected entheses and joints. there is limited data on the utility of wbmri in the pediatric population. it is worth noting that edema-like changes seen in the marrow of healthy children is an important potential pitfall to consider during interpretation and further studies are required in order to establish specific reference standards for mri of the pediatric skeleton. diffusion-weighted imaging (dwi) offers a new approach to detect inflammation. inflammation produces an increase in the apparent diffusion coefficient (adc) of water molecules in affected tissues. several studies in adults and a few recent studies in children have demonstrated that adc is elevated in sacroilitis versus controls and that diffusion scores correlates well with stir images. dwi is promising as a potential biomarker of disease activity in jia and presents a novel approach to contrast-free imaging of synovitis. however, further studies are needed before it can be implemented in clinical practice. jspa is distinct from adult spa and manifests more frequently as peripheral arthritis and enthesitis. symptoms involving the spine and sacroiliac joints often occures later in this population. clinical diagnosis of jspa can be difficult, and imaging therefore plays an important role in the diagnostic workup of disease. identifying early signs of axial disease has major implications for treatment decisions and mri of the sacroiliac joints is increasingly obtained for early detection of inflammatory changes. however, mri criteria for sacroilitis in children are lacking. a major controversy in imaging of sacroilitis in jspa is the use of contrast, as children can have sacroilitis without accompanying bone-marrow edema. dwi presents a novel approach to contrast-free imaging of synovitis but further studies are needed before it can be used in clinical practice.wbmri has been shown to be more sensitive than clinical examination in the assessment of both disease activity and extent, but there is limited data on wbmri in children. normal variants in the growing skeleton may mimic pathologic changes and potentially cause overdiagnosing and -staging of disease. hence, there is an urgent need to establish specific reference standards for mri of the pediatric skeleton and to develop a gold standard mri technique for the axial skeleton in children and adolescents. juvenile idiopathic arthritis o. olsen; london/uk summary: juvenile idiopathic arthritis (jia) is common (about 1:1,000 children). diagnosis and classification are based on clinical criteria. these criteria are in flux depending on 1) contemporaneous knowledge about aetiology and 2) available treatment options. radiology has currently no role in establishing the diagnosis. the clinical classification rests on whether the child has few joints affected (oligo jia), many joints (poly jia), has a condition similar to adult spondyloarthritis (entesitis-related arthritis) or other clinical presentations (systemic-onset jia, psoriatic jia, etc). radiology can potentially assess expressions of jia, such as synovitis, tenosynovitis, systemic manifestations and permanent damage caused by inflammation. it is therefore thought to play a part in gauging the disease activity. the clinical care aims at optimising the child's everyday function, reducing acute symptoms (pain, swelling, joint restriction), allowing normal growth, minimising long-term sequelae (joint deformity) and minimising adverse effects of medical treatment. medical treatment in jia is systemic (immuno-modulation) and local (steroid injection to joints and tendon sheaths). both modes of therapy may to some degree be guided by imaging. however, there currently is no evidence that any form of whole-body imaging is efficacious for guiding treatment. this means that, in principle, indication for imaging should be 1) specific clinical questions, e.g. uncertainty regarding active inflammation at specific sites, or 2) a high pre-test likelihood of inflammation at a site which is difficult to assess clinically and where imaging offers reasonable accuracy. one example of the latter are the temporo-mandibular joints where destruction is frequently seen at an early stage, often without prior symptomatic warning. there is one fundamental challenge for imaging research in jia: what is the reference standard? for lack of anything better, a standardised clinical examination is often used as 'ground truth'. the dilemma is obvious. if clinical examination is reliable and accurate, then why bother with imaging? but we think imaging offers an improvement, then we cannot use an inferior method to set the standard. this problem is not unique to jia. as is often the case, radiology in jia is all about: knowing your clinicians (i.e. the pretest likelihood for disease) being technically eloquent (e.g. using high-resolution us probes, not delaying post-contrast mri acquisitions) knowing what is normal (e.g. normal undulations in the articular surface, focal bone marrow signal variation) not being dogmatic about individual observations or measurements interpreting your findings in a clinical context the lecture will demonstrate similarities and differences among joints and modalities in children with variable-severity jia. the following points will be made: focal areas in the bone marrow with high signal (t2) and corresponding enhancement are often seen in healthy children. in isolation, these do not signify active inflammation. active synovitis in children often is not associated with (much) effusion the combination of synovial thickening with hyperaemia (us)/abnormal contrast enhancement (mri) and surrounding softtissue swelling suggests active inflammation, however there is (yet) no established system for quantifying hyperaemia/enhancement focal pits in the carpal bones do not represent erosions unless there is an associated cartilage defect radiographs are useful for detection of destructive abnormality in mri, scan fairly soon after injecting contrast. gadolinium physiologically leaks into the synovial fluid making it difficult to delineate the synovium a few differential diagnoses to keep in mind when there is mass-like swelling within or adjacent to a joint: vascular and neoplastic lesion, pigmented villonodular synovitis, synovial chondromatosis, lipoma arborescens. synovial inflammation is not always primary. even when there is an established diagnosis of jia, do consider that it may be secondary to biomechanical abnormality (erosion, osteochondral lesion, deformity). focal areas in the bone marrow with high signal (t2) and corresponding enhancement are often seen in healthy children. in isolation, these do not signify active inflammation. active synovitis in children often is not associated with (much) effusion the combination of synovial thickening with hyperaemia (us)/abnormal contrast enhancement (mri) and surrounding soft-tissue swelling suggests active inflammation, however there is (yet) no established system for quantifying hyperaemia/enhancement focal pits in the carpal s321 (2017) 47 (suppl 2):s297-s pediatr radiol bones do not represent erosions unless there is an associated cartilage defect radiographs are useful for detection of destructive abnormality in mri, scan fairly soon after injecting contrast. gadolinium physiologically leaks into the synovial fluid making it difficult to delineate the synovium pulmonary manifestation of connective tissue disorders c. m. owens; london/uk summary: connective tissue diseases are an important cause of morbidity and mortality in children with very varied presentations. nomenclature is confusing and a more appropriate descriptive term would be "multisystem inflammatory disorder +/-autoimmunity". it is important for the radiologist to be aware of the protean radiological appearances and clinical manifestations. take home points: different patterns of diffuse lung disease (eg, desquamative interstitial pneumonia, non specific interstitial pneumonia, lymphocytic interstitial pneumonia, organising pneumonia, diffuse alveolar damage) may be present in several forms of collagen vascular disease, (and indeed other rheumatological conditions such as jia) including scleroderma, systemic lupus erythematosis, juvenile dermato and polymyositis, sjogren's syndrome and mixed connective tissue disease. these will be discussed in detail with illustrations for thin section high resolution ct with histopathological correlation. the clinical presentation, prognosis and response to therapy vary depending on the histological pattern of diffuse lung disease, as well as on the underlying collagen vascular disease. whole body imaging in children: sonography, ct, mri, nuclear medicine -what and when? r. a. nievelstein; utrecht/nl there are several (benign and malignant) disease processes in children that frequently involve more than one organ system or body region. diagnostic imaging of children with such multifocal or multisystem diseases has been quite challenging, often requiring a combination of different imaging techniques for a whole body coverage. the recent technical developments in computed tomography (ct), magnetic resonance imaging (mri) and nuclear medicine (nm) have changed the role of imaging in these children revolutionary. in the past, imaging techniques have been mainly used as a tool to detect the cause of illness and to assess the extent of disease spread before, during and after therapy (i.e. structural imaging). but nowadays, it has also become possible to use imaging techniques to gain information on the biological behavior of diseases before and during therapy (i.e. functional imaging). plain radiography, ultrasonography (us) and computed tomography (ct) have been the structural imaging techniques of choice for many decades, more recently supplemented by functional imaging techniques like single-photon emission tomography (spect) and positron emission tomography (pet). a major disadvantage of most of these techniques is the use of ionizing radiation, which may be associated with induction of second cancers later during life. this small but not negligible health risk is of particular concern in children as their tissues are more radiosensitive than adults and they have more years ahead in which cancerous changes might occur. that is why there is an increasing interest in the use of alternative imaging techniques that do not use ionizing radiation. with mri it is nowadays possible to acquire images with a high spatial resolution and excellent soft tissue contrast throughout the body, which makes it an ideal radiation-free tool for the detection of pathology, especially in soft tissue, parenchymal and bone marrow locations. moreover, recent technological advances have resulted in fast diagnostic sequences for whole-body mr imaging (wb-mri), including functional techniques such as diffusion weighted imaging (dwi). as a result, wb-mri has become a clinically feasible imaging modality for diagnosis and follow-up of multifocal and multisystem diseases in children. in this scope, the recent development of integrated pet/mri systems is very interesting, combining the superior structural imaging of mri with the functional (molecular) information of both imaging techniques while decreasing the radiation dose. traditionally, whole body imaging techniques have been mainly used for oncological indications, such as staging of malignancies, and monitoring of the effectiveness of therapy. however, whole-body imaging techniques are increasingly used for the diagnostic imaging of other benign multisystem diseases and indications, including chronic recurrent multifocal osteomyelitis (crmo), rheumatological diseases, neuromuscular diseases, neurofibromatosis type 1, generalized vascular malformations, multifocal osteonecrosis after intensive chemotherapy, fever of unknown origin, and post-mortem imaging. finally, these imaging techniques may be used for the screening of children with a cancer predisposition syndrome. during this lecture, imaging protocols and indications of the different whole body imaging techniques will be discussed with a focus on their clinical application in children with benign and malignant multifocal or multisystem diseases. (2017) 47 (suppl 2):s297-s pediatr radiol appearances is important for any radiologist involved in child imaging, because we have an important role in characterizing the lesions and guiding purposeful and minimally invasive but successful diagnostic procedures. most head and neck masses in children are benign and have an inflammatory, infective, vascular or congenital cause (cf. special presentation on vascular malformations). malignant lesions are less common, however, early diagnosis is paramount as many of these cancers are readily treatable and often curable. differential diagnosis is guided by patient age, clinical presentation, tumour localisation, and imaging characteristics. while some masses such as (epi-)dermoids, fibromatosis colli and swollen lymph nodes including atypical mycobacterial infections (mott) may be readily diagnosed by clinical inspection und ultrasound, others present special diagnostic challenges. fibromatosis, for example, is a benign lesion with an often complex and potentially destructive local spread. some malignant lesions tend to be localised such as the embryonal rhabdomyosarcoma, while others may be part of a systemic disease such as lymphoma and langerhans cell histiocytosis (lchc). in case of a suspected malignancy, patients should be referred to a specialized centre which will be able to provide the full spectrum of multidisciplinary evaluation and treatment according to the guidelines of an international oncology study group. this is also important for image guided or surgical biopsies as long term outcome and survival of many of the young patients are directly associated with these initial diagnostic and therapeutic strategies. with its excellent spatial resolution in the near field, ultrasound is the method of choice for all superficial masses. an experienced paediatric radiologist will be able to identify most of the benign lesions and in other cases will be able to guide further diagnostic decisions. tumours in the midline require thorough workup to exclude an encephalocele or a dermal sinus with connection to the intracranial space. high resolution mri is required if such an extension cannot be ruled out by ultrasound or if a tumour is larger than the transducer's scan area. soft tissue tumours in the deeper parts of face and neck as well as tumours of osseous origin are also best delineated by mri. in lesions adjacent to the skull base contrast enhanced and fat saturated mr images with high spatial resolution are of utmost importance to completely depict the tumour's extension through the foramina and along the meninges (fig. 1) . ct can provide additional information on the involvement of osseous structures. embryonal rhabdomyosarcoma. high resolution mri with fat saturation after contrast injection depicts the tumour's extension through the foramen ovale (long arrow) and along the meninges (short arrows). skull base and face lesions are less frequent in children than in adults. symptoms may be subtle or unspecific. depending on their localization, clinical findings may be common (nasal obstruction, otitis…) or more disturbing (cranial nerves palsies, exophthalmos, vision loss …). clinical history and physical examination findings are important to reduce the spectrum of differential diagnosis, but imaging data are the key features to determine the nature of these lesions. ct and mri play an important role in diagnosis, treatment survey and surgery planning of skull base and face lesions. skull base and face bone lesions are either intrinsic lesions of the bone or secondary to soft-tissue tumors or pseudo tumors invasion. this lecture will focus on bone intrinsic lesions, and include soft-tissue and pseudo tumors only as differential diagnoses. computed tomography plays the role for skull base and face of plain radiograph for long bones. therefore, the same semiology may be used to determine if the lesion is slowly or rapidly growing, aggressive or looks benign. helical ct allows reconstructions with both soft-tissue and bone algorithms as well as multiplanar reformations. it gives a good visualization of the anatomy of the skull base and allows a good depiction of the bone architecture. ct is first used for the initial work up of the disease but also for surgery and therapeutic planning (endoscopic sinus surgery with navigation). however, ct analysis may be challenging in children due to growth changes: normal process of pneumatization according to age, sutures not yet fused has to be recognized. some variations in pneumatization must not be mistaken for pathology: asymmetrical pneumatization of the petrous apex and arrested pneumatization of the sphenoid mimicking intraosseous lesion are the most common. both ct and mr imaging are complementary: most preferably, contrast-enhanced mr is associated with non-contrast high resolution ct. mri allows a good delineation of bone involvement of skull base lesion due to bone marrow changes, whether ct can fail to detect subtle extension within the bone. in addition to t1 and t2 weighted sequences, the use of specific sequences and/or techniques such as fat-saturation, diffusion, dynamic-contrast-enhanced sequences, and mr angiography helps to characterize the lesions. t1 spin echo sequence is mandatory to appreciate bone marrow infiltration in adults and older children. but when red bone marrow has not yet be replaced by fatty bone marrow, in young children, this can be challenging. it is useful to know the bone marrow fatty conversion of the skull base chronology. cranial mr can also be associated to whole body mr to look for multifocal or metastatic disease. epidemiologic data concerning bone tumors of the skull base are scarce due the rarity of these lesions. they can be classified according to their location within anterior, middle or posterior cranial fossa or classified according to their origin: osteogenic (osteoblastoma, osteoma, osteosarcoma...), chondrogenic (chondroma, chondrosarcoma), fibrous ( fibrous dysplasia, fibro-s323 (2017) 47 (suppl 2):s297-s pediatr radiol osseous lesions..), notochord (chordoma), hematopoietic (leukemia, histiocytosis ), vascular (hemangioma), neuro ectodermic ( ewing sarcoma) or unknown origin (aneurysmal cyst, giant cell tumor). the aim of this presentation is to draw attention to skull base growth changes that can mimic pathology and to describe the imaging specificities of the most common bone tumors of the skull base and face in children. because conflicted nomenclature can cause confusion, accurate diagnosis and classification of these anomalies is important for proper clinical evaluation and management. many of these patients require multidisciplinary care, consequently the usage of a correct nomenclature across all disciplines is a sine qua non. the international society for the study of vascular anomalies (issva) classification, updated in 2014, offers a comprehensive classification accepted by many subspecialities. this approach/ classification has facilitated correct communication for all medical subspecialties involved in the care of these complex vascular anomalies. pediatric radiologists play a critical role in evaluating these patients since the majority present during childhood. in this presentation, we present a state of the art mri imaging protocol with exemplary cases of the most common types of vascular anomalies in the pediatric trunk and extremities, using the current issva classification. in addition, we discuss the common syndromes associated with vascular anomalies such as klippel-trenaunay and lumbar syndrome. genetic skeletal disorders (gsd's) are a heterogeneous group of syndromes characterized by an intrinsic abnormality in growth and (re-)modeling of cartilage and bone. a large sub-group of gsd's may have additional involvement of other structures/organs beside the skeleton, such as the central nervous system (cns). cns abnormalities have an important role in long-term prognosis of children with gsd's and should consequently not be missed. sensitive and specific identification of cns lesions while evaluating a child with a gsd requires a detailed knowledge of the possible associated cns abnormalities. here, we will present and discuss a pattern-recognition approach for identifying relevant neuroimaging findings in gsd's guided by the obvious skeletal manifestations of gsd. in particular, we will discuss which cns findings should be ruled out for the various gsd. to facilitate this diagnostic approach the multiple gsd are classified based on the pattern of skeletal involvement (1. abnormal metaphysis or epiphysis, 2) abnormal size/number of bones, 3) abnormal shape of bones and joints, and 4) abnormal dynamic or structural changes). skeletal involvement is defined in accordance with online mendelian inheritance in man. the spectrum of co-existing cns involvement is extracted from an extensive literature search. selected examples will be shown based on prevalence of the diseases and significance of the cns involvement. cns involvement is common in gsd's. a wide spectrum of morphological abnormalities is associated with gsd's. early diagnosis of cns involvement is important in the management of children with gsd's. this pattern-recognition approach aims to assist and guide physicians in the diagnostic work-up of cns involvement in children with gsd's and their management. not infrequently the correct radiological differentiation of skeletal and/or central nervous system findings secondary to non-accidental injury versus inherited genetic and/or metabolic disorders may be challenging. imaging findings may be non-specific, can result in incorrect diagnosis and subsequently inadequate patient management or initiation of faulty treatment. the diagnostic work-up of children suspected of non-accidental injury or genetic/metabolic disorders requires a multi-disciplinary approach involving many key players including physicans of various disciplines, nurses, psychologists, social workers and many more. a proper and detailed medical history and physical examination of the patient, collection of the relevant family history, a metabolic and genetic work up, a detailed interview of care givers, friends and family are essential for the correct and comprehensive evaluation of imaging findings. in the current session, various exemplary and possibly confusing cases will be interactively discussed with the audience by a panel of experts (susan blaser, thierry a.g.m. huisman and andrea superti-furga). goal is to offer a case based approach to challenging patients with discussion of the best diagnostic approach including differential considerations. the zikv is transmitted mainly by the bite of female aedes aegypti and aedes albopictus mosquitoes. other forms of transmission, including through sexual intercourse, blood transfusion, and neonatal, are currently under evaluation, although more elements are still needed to assess the real importance of these transmission routes 4 . the course of the zikv infection is self-limited. so far, no specific symptoms have been attributed to the disease, and a wide variety of manifestations ranging from absent to mild symptoms (in 25% of cases) have been described. when symptoms are present, they may lead to a misdiagnosis of other bacterial and viral infections, especially other arboviroses in endemic areas. the most frequently reported symptoms are mild fever, cutaneous rash, fatigue, arthralgia/myalgia, and conjunctivitis. dizziness, malaise, edema of the extremities, anorexia, retro orbital pain, photophobia, gastrointestinal disorders, sore throat, cough, sweating, and lymphadenopathy have also been reported. infection by the zikv in adults may be associated with autoimmune complications such as guillain-barré syndrome 9 . the laboratory diagnosis of zikv infection is based on the demonstration of the virus in the urine and blood using real-time reverse transcription polymerase chain reaction (rt-pcr). the main limitation of this diagnostic method is a false-negative result after the viremia is resolved. the serological diagnosis of the disease is limited due to cross-reactivity of the zikv with other viruses of the flavivirus genus, especially those causing dengue and chikungunya. physicians should be aware of this fact when the diagnosis of zikv infection relies solely on serological results. the diagnosis is also possible by igm measurement in serum, urine, or cerebrospinal fluid using enzyme-linked immunosorbent assay (elisa) 9 . the prevention against zikv infection is similar to that of other arboviroses, including vector control and mosquito bite prevention. the first major zikv epidemics were reported in the french polynesia in 2013 and 2015. at that time, some neurological changes were observed in neonates of infected pregnant women but were not associated with a maternal-fetal transmission of the virus. the growing increase in the number of cases and the severity of the infection specific to this subpopulation then led to the evidence of a congenital disease 9 . in brazil, the situation became alarming with the report of a high number of infected individuals in the second half of 2015 2, 5 . the brazilian ministry of health attributed to congenital zikv infection the 20-fold increase in cases of neonatal microcephaly in the northeastern part of the country, particularly in the state of pernambuco. this led the world health organization (who) to declare the zikv infection a "public health emergency of international concern" in february 2016 6 . the main challenge for radiologists practicing in regions of endemic zikv infection is to become familiarized with findings of congenital zikv infection in perinatal imaging studies; this is particularly important for the prenatal screening of pregnant women 3, 6 . the diagnosis of zikv infection in the fetus by neuroimaging is based on prenatal ultrasound (us), especially in the third trimester, and complemented with magnetic resonance imaging (mri). postnatal imaging was obtained by transfontanellar us, ct or mri. the main imaging findings on ct are microcephaly, an exuberant external occipital protuberance, rectification of the frontonasal angle, and a redundant scalp skin. three-dimensional (3d) reconstruction of al skull permits a better evaluation of these findings and enhances the parents' understanding of the disease. moreover, ct scan data may yield a 3d virtual physical model that can maybe obtained from ct scan data and printed onto using thermoplastic acrylonitrile butadiene styrene 7 . the aim of this study was to describe the perinatal imaging findings in cases of congenital zikv infection. we studied 18 mothers diagnosed with zikv infection from october 2015 to november 2016. they had all presented a maculopapular rash and fever during the first or second trimester of pregnancy, and their neonates presented neurological defects that were attributed to intrauterine transmission of the zikv. the maternal diagnosis of zikv infection was confirmed by serology (n=4) or rt-pcr (n=14). all patients were torch (toxoplasma, rubella, cytomegalovirus, herpes simplex) negative. prenatal us was performed every 3 weeks after the first imaging findings, and fetal mri was obtained in all cases. microcephaly was considered present when the infant's head circumference was two standard deviations below the mean value for age and sex or below the second percentile. postnatal imaging follow-up was obtained in all cases by transfontanellar us, ct or mri. we found several cns malformations, including lissencephaly, pachygyria and/or polymicrogyria, cerebral atrophy (panel 1), enlarged cisterna magna with abnormalities of the corpus callosum, ventriculomegaly, brainstem hypoplasia, malformation of the cortical development, and cortical and/or periventricular calcifications mainly in the junction between the cortical and subcortical white matter (panel 2). the skull of the infants had a collapsed appearance, with overlapping sutures and redundant skinfolds (panel 3). craniofacial disproportion was easily identifiable, and arthrogryposis was identified in one case. similar neurological findings were observed in the infected patients and seemed to differ from findings of other infectious diseases. the finding of microcephaly in neonates with congenital zikv infection seems to be only the tip of the iceberg, as several cns malformations have been identified in connection with the disease. in brazil, a spectrum of imaging findings associated with congenital zikv infection has been observed. such findings are useful in helping radiologists to identify suspected cases of the disease. panel 1: prenatal ultrasound (37 weeks) shows calcifications (arrows) and microcephaly. axial and sagittal t2 shows relative smoothness of the brain surface (arrows) and assymmetric colpocephaly. panel 2:ax t1-wi multiple cortical-subcortical fronto-parietal hyperintense foci (arrows) and markedly hypointense on swi. sagittal t2: dysgenesis of the corpus callosum, with dilation of the posterior horns of the lateral ventricles (colpocephaly). pre-and postnatal imaging in zika virus: where are we? early insights into zika's microcephaly physiopathology, from the epicenter of the outbreak: a case for teratogenic apoptosis of central nervous system. p. jungmann; recife/br early insights into zika's microcephaly physiopathology, from the epicenter of the outbreak: a case for teratogenic apoptosis of central nervous system. in mid-october 2015, intense interaction among surgical pathology and fetal medicine specialists from university of pernambuco was only focused on the dramatic and non explained ultrasonographic (us) findings and hopelessness due to lack of explanations on the odd us discoveries on the first gestational cases of zika's microcephaly. this is the field of our history of a physiopathological hypothesis on zika virus (zikv) related microcephaly when it first struck pernambuco state (pe), northeast brazil, the place that has been at the front line of the global response to the microcephaly and responsible for a large amount of data from affected children. the outbreak onset came with a sudden increase in microcephalic newborns being reported in pe state from august 2015 (panel, fig. 1) . zikv was previously thought to cause a relatively mild disease, but was recently accepted to lead to severe and diverse neurologic conditions in s329 (2017) 47 (suppl 2):s297-s pediatr radiol some children born from infected mothers and in adults 1 . the scientific community is actively trying to uncover the extent of these disorders but little has been reported on the early days of the outbreak when doctors were approaching the unknown. while evidence that zikv is related to microcephaly in newborns is accumulating, the mechanisms of how the virus affects the fetus is still uncertain. in the outbreak onset we had to face daunting challenges to search the cause of microcephaly and the emotional toll on the families. we took a very early approach from 9 microcephalic fetuses on gestation and 46 microcephalic babies on clinical follow-up from different pe areas, evaluated between 15 october and 17 december 2015 in oswaldo cruz hospital, to propose the early physiopathologic hypothesis that, a viral-related brain developmental disruption could be the basic neuropathogenesis in zikv babies instead of a direct injurious process due to viral insult followed by active inflammation. the eight pregnant women were all in the 3 rd gestational trimester and had had normal us follow-ups till week 20 th . crucially, we were facing a temporal-geographic association of cases presenting an unanticipated pattern of us alterations. because of their late alarming findings they were re-examined and the us scans revealed sudden encephalic alterations after 28 th gestational week. such devastating us clustering images were not seen here before, but are now considered as part of the "congenital zika syndrome". we observed late appearing severe dysmorphic encephalic changes in 9 out of 9 fetuses, including small skull, small brain, sub arachnoidal space enlargement, ventricular dilation, brain calcifications of varied shape and distribution, inclined frontal bone, progressive decline of head growth potential, early fontanels closure and redundant scalp (panel, figs. 2a, 2b). we had no clues on the causes and mechanisms responsible for this phenotype of severe alterations. thus, we had no explanation to offer to patients, in particular, or to the medical community. both as physicians and human beings, we were committed straightaway to continue the study of these victims of an unknown medical tragedy, engaging our expertise in fetal imaging and immunopathology. from beginning october, the first microcephalic babies were referred to the upe pediatric infectology service for initial investigation. strikingly, the newborns exhibited "healthy" appearance, excluded the microcephaly itself and motor sequels. we then looked for csf analysis of the microcephalic babies. for that, we obtained from dr. patricia travassos, a csf specialist at upe, a cohort of 120 csf samples that have been studied for signs of meningitis or encephalitis. about 87% (42 cases) of the csf analyzed looked normal for any signs of central nervous system ongoing inflammatory responses (panel, fig. 3 ). the babies had been examined by outpatient clinic dr angela rocha, from the upe hospital infectology reference center that have stated that although small, the babies were near to full term gestation (36-39 weeks gestation), had good apgar scores and variable degrees of microcephaly and neurologic impairments, i.e. contractures, spasms, irritability and in some retinal macular atrophy. during the follow-up, the babies were cared at home, breastfeeding, gaining weight and having routine vaccines. none of them expressed signs of ongoing inflammatory reaction in the cns (panel, fig. 4 ) or alterations on peripheral blood count and other routine laboratory tests up to 3 months of age. despite the striking neurological phenotype, 80% of the babies were negative for torch agents, no deaths were recorded. furthermore only in january 2016, the first evidence associating zikv to microcephaly from rt-pcr test on amniotic fluid was reported 2 . astonishingly, a particular kind of physiopathological process linked to fetal brain development was arising without clinical manifestation of inflammatory reactions or necrotic processes in these babies. unfortunately, no necroscopic samples of affected brain tissues were available to us to monitor the presence of putative neural dysgenesis and the very nature of brain calcifications background offering histological support for our hypothesis. nevertheless, with this restricted dataset we hypothesized that whatever the etiologic agent involved in these cases, its physiopathologic mechanism must trigger the cellular death programthe apoptotic process -at a particular development window on the cns, assuming clinically that the agent was not encephaalitogenic but silently tertogenic. if not, the clinical outcome of affected babies would not be so mild as far as signs of inflammation on cns was concerned. consequently, the inflammation-free clinical status of patients suggested us that a massive enhanced apoptotic cell death during the window of telencephalic expansion was the most probable physiopathologic process operating this microcephaly phenotype, with no direct direct lytic brain lesion or significant necrosis due to usual injury. furthermore, knowles and penn 3 stated that this window is very active to select the "fittest" neural cells by a constitutive apoptotic pathway. we so hypothetized that during this developmental time window, the "fit or not fit" status of the rapid, transient amplifying neural progenitors cells facing zikv, would heavily shift the selective process toward the self-elimination of virusbearing cells through apoptotic pathways. thus, zikv-enhanced constitutive apoptotic mechanisms would lead a massive loss of developing telencephalic neuronal precursors and, consequently, provoking losses of dividing cells and the arrest of further brain development. this could be particularly inferred by the absence of the characteristic morphology of late stages structures of neocortex, according with our us images of zikv microcephalics in gestation. similar processo could also be inferred to neurocrest derivatives as deformities in the viscercrany always accompany the cephalic malformation. our initial understandings based on clinical examination on the field, when no specific laboratory test, necroscopic data or experimental evidence on the disease causality were available, conducted our physiopathological approach to the "apoptosis hypothesis" for zika microcephaly that is now gaining strong support. in february, mlarkar et al 4 showed clear connection between zikv and microcephaly, presenting cns histopathologic analyses, revealing remnants of neural germinative matrix, intense gliosis, alterations in cortical ribbon, calcifications in gray and white matters without associated necrosis, encephalitis or meningitis and the presence of the virus, further supporting neurodevelopmental arrest. similar results were showed by driggers et al 5 . the ct scans from microcephalic babies from hazin at al 6 , have added details of brain development arrest with no radiological signs of brain destruction or active inflammation. finally, experimental models have provided a body of evidence f or neuroprogenitors permissiveness to zikv and viralinduced apoptotic process. tang et al 7 demonstrated by icq that the zikv infection of cortical neural progenitors attenuates their growth and increases caspase-3 activation, calling for an apoptotic process. this finding was corroborated by the up regulations of caspase-3 genes by rna sequencing. nowakowsky et al 8 demonstrated that zikv may hijack axl protein as an entryway to infection. interestingly, axl is highly abundant on the surface of neural stem cells but not on differentiated neurons in the developing brain. recently, cugola et al 9 demonstrated that zikv was able to cause cns congenital brain dysgenesis upon vertical transmission in mice. in parallel, human brain organoids infected by zikv show a reduction of proliferative zones and disrupted cortical layers, so targeting cortical progenitors and inducing apoptotic cell death with impaired development. for babies born with zikv-related microcephaly, the many expected consequences besides the evolving congenital neurosequels, are the unanticipated pattern of persistence of zikv in cns host cells, unsafe maintenance of neuron genome stability on remaining arrested populations, implying risks for brain tumors, risks for impaired adult type neuron wiring and neuron survival in an affected neuronal circuitry. in brief, evolve life with a wide vulnerable brain. the outbreak of zikv in the americas will eventually decline as herd immunity increases, but the world remains at risk of further waves of infection in affected countries and spread into new territories 1 . while experimental studies will be carried out to fully understand the pathophysiology of zikv infection in the developing fetus, our findings provide a coherent and testable physiopathological hypothesis for cns teratogenic phenotype linked to zikv congenital infection, which may be critical for the clinical care of pregnant mothers and their babies before and after birth. take home points: fetal dysmorphisms detected by ultrassonographic and mri images in congenital zika syndrome are late findings, usually after the 20 th gestational week and requires acurate analyses. clinically, zika's virus microcephaly is an infectiuos congenital condition that is not encephalitogenic but primarily teratogenic on the nervous system. the most important process leading to zika's virus microcephaly is pathologically induced apoptosis in telencephalic neuroprecursosrs cells and neurocrest precurssors cells. viral induced autophagy and low antiviral responses during the fetal period are linked to zika virus persistence in the central nervous system of affected new borns and babies a. vossough; philadelphia/us summary: susceptibility-weighted imaging (swi) has proven to be a valuable mr imaging sequence in a variety of applications. pediatric imaging has also immensely benefitted from this technique. in this presentation we will review pediatric neuroimaging applications in trauma, arterial and venous vascular disorders, hypoxic-anoxic injury, congenital malformations, congenital heart disorders, neoplasms, and pediatric degenerative disease. use of swi in pediatrics other than demonstrating hemorrhage and calcification will be reviewed. challenges in the clinical use of swi in pediatrics, interpretive pitfalls, and sources of clinical misinterpretation of swi will also be explored. we will also briefly present ongoing research and clinical use of swi in pediatrics and potentials for future collaborative investigations. & swi is highly sensitive in detection of susceptibility effects on mri. & in many cases, but not all, swi processing can differentiate between calclium and blood products. & quantitation information can also be obtained from swi with further processing. state of the art imaging of the single ventricle d.m. biko; philadelphia/us there are many congenital heart defects that result in a functional single ventricle. this may be functional or anatomical as a result of a dysfunctional valve or absent or ineffective pumping chamber. the repair of single ventricle physiology most often involves a staged reconstruction due to changing physiology ultimately resulting in a total cavopulomonary connection or fontan procedure. to appropriately image the single ventricle throughout its stages of palliation, familiarity with the physiology of the various steps in surgical palliation of the single ventricle is essential although echocardiography is a mainstay of cardiac imaging, cross sectional imaging has a vital role in the evaluation of the single ventricle. the role of ct angiography is mostly for anatomic evaluation. although it is fast and has high spatial resolution for evaluation of vasculature, ct has lower temporal resolution than mri and is unable to quantify flow. ventricular performance along with quantification of flow can be performed with mri. systemic to pulmonary collateral flow, which has been shown to result in adverse outcomes after fontan, can be quantified. valvular insufficiency and myocardial scarring can also be assessed. additionally, high anatomic vascular detail can be obtained with mri, particularly with the recent investigational use of the blood pool agent ferumoxytol. mri also has the ability to assess the lymphatics either through non-contrast t2 weighted imaging and/or dynamic contrast mr lymphangiography as lymphatic pathology may play a role in postsurgical hemodynamics in single ventricle patients. this lecture will focus on the use of ct and mri in the evaluation of the single ventricle particularly concentrating on the developing use of mri for anatomic and physiologic assessment. take home points: in single ventricle physiology, there is only one effective pumping chamber. familiarity with the physiology of the various steps in surgical palliation of the single ventricle is essential in imaging this disorder ct angiography provides high anatomic detail but limited in its assessment of physiology since it cannot quantify flow and has lower temporal resolution than mri. mri can evaluate ventricular performance, quantify flow and valvular insufficiency, and assess myocardial scarring. high anatomic vascular detail can also be obtained with mri particularly with the emerging investigational use of ferumoxytol. with non-contrast t2 weighted mri and/or dynamic contrast mr lymphangiography, lymphatic evaluation can be performed which may play a role in post-surgical hemodynamics in single ventricle patients. neuroimaging in head trauma m. argyropoulou, g. alexiou; ioannina/gr summary: objective: head trauma in children is one of the most common reasons for visiting emergency department. however, only a small portion of patients will have a traumatic brain injury. patients with moderate or severe head trauma should undergo ct scan, however, a debate exists for the indication and yield of neuroimaging for minor head trauma. we performed a systematic literature review on the accuracy of symptoms and signs in children with minor head trauma in order to identify those with severe intracranial injuries. materials: a systematic literature search of medline (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) was performed to identify studies assessing the diagnosis of intracranial injuries in children. the authors independently performed critical appraisal and data extraction. results: we identified studies that evaluated the performance of findings for detecting intracranial injury using the reference standard of neuroimaging or follow-up examination. mechanism of injury, multiple vomiting episodes and decline in gcs score were more commonly associated with severe intracranial injury on ct. normal variations in the amount of joint fluid, ganglion cysts, bone marrow edema, and bony depressions that resemble erosions are frequent in the wrists of children. the results of a follow-up of a healthy cohort aged 6-17 will be presented. the cohort was examined twice with mr of the wrist, and the second time also with a cartilage sequence for better visualization of the bony depressions. knowledge of these normal variations is important because they can resemble disease. bone marrow edema, joint fluid more than 2 mm, and bony depressions that can resemble erosion are frequent findings in the normal wrist. take home points: bone marrow edema, joint fluid more than 2 mm, and bony depressions that can resemble erosion are frequent findings in the normal wrist. these findings can not be attributed to dissease without additional findings of synovitis. a cartilage sequence can be of use in the differentiation between true erosions and bony depressions. mri scoring of the wrist in patients with jia-current status and future perspectives c. nusman; amsterdam/nl the wrist is a frequently affected joint in patients with juvenile idiopathic arthritis. due to recent improvements in treatment strategies, permanent damage is not that common anymore. also, imaging has been playing a key role in monitoring the disease activity in the wrist of jia patients. the past years lots of efforts have been made to improve the assessment of acute and permanent changes of the jia wrist. requisites and recommendations for the mri protocol to use for of the jia wrist are available in literature. currently, the features of scoring the jia wrist are synovitis, tenosynovitis, bone marrow edema and bone erosions. the repeatability of the above-mentioned scoring features proved to be acceptable. recent studies showed that the appearance of the wrist in healthy children can mimic pathology. therefore, construct validity of the scoring features needs to be assessed by comparing wrists of healthy children with the wrists of jia patients. & construct validity of the scoring features needs to be assessed by comparing wrists of healthy children with the wrists of jia patients a novel radiographic scoring system for permanent hip involvement l. tanturri de horatio 1 , p.l. di paolo 1 , s.c. shelmerdine 2 , p. toma 1 , k. rosendahl 3 ; 1 rome/it, 2 london/uk, 3 approximately 20-50% of children with jia, particularly those with systemic onset disease, will have hip-involvement within 1-6 years after disease onset. as scoring systems for radiographic changes in children with hip involvement are lacking, we aimed to examine the reliability of potential markers and suggest a novel scoring system. a set of 75 hip-radiographs from 75 children with jia and clinical hipinvolvement: 59 seen at the outpatient clinic at great ormond street hospital (gosh), london, and 16 seen at ospedale pediatrico bambino gesù, rome, was used. all hip radiographs were scored in a blinded fashion, once by an experienced paediatric radiologist and a paediatric radiologist with minor experience in musculo-skeletal imaging in rome, and twice by an experienced radiologist and a research fellow in bergen/ london. radiographic findings suggestive of 1) destructive change (bone erosion, flattening of the femoral head, squaring of the femoral head contour, presence of sclerosis, joint space height, and 2) growth abnormality (length and width of the femoral neck, varus/valgus deformity, the ccd angle and the trochanteric-femoral head height) were assessed. assessment of erosions of the femoral head, femoral neck and the acetabulum showed moderate to good agreement for the same reader. the inter-reader agreement was, however lower. there was a high to moderate (2017) 47 (suppl 2):s297-s pediatr radiol agreement for the assessment of femoral head flattening using the mose' circle. the measurements of femoral neck length and width, the ccd and trochanteric-femoral head lengths were precise, with 95% limits of agreements within 10-15% of the mean. we have identified a set of relative robust radiographic findings suggestive of growth abnormalities and destructive change in children with hip-jia, and suggested a novel scoring system. x-ray of a 21 years-old jia patient with severe chronic hip involvement. x-ray of a 6 years-old boy with growth abnormalities on hips (bilateral coxa magna). in jia hip involvement is often a predictor of a severe disease course. radiographic findings vary according to mode of onset and age: in younger children the initial findings may be developmental rather than destructive while children with later onset jia may have destruction/narrowed joint space as the first feature. several of the commonly used radiographic findings for chronic hipchange are inaccurate. we have identified a set of relative robust radiographic findings suggestive of growth abnormalities and destructive changes in children with hip-jia, and suggested a novel scoring system. bone age assessment -statement from the msk task force k. rosendahl; bergen/no summary: age assessment is an important, yet complex and challenging issue that authorities may need to perform to determine whether an individual is an adult or a child in circumstances where their age is unknown. there is currently no method which can identify the exact age of an individual and there are concerns about the invasiveness and accuracy of the methods in use, namely analysis of documentary evidence, interviews, physical or other form of medical examination such as imaging. the main imaging methods include carpal, collar bone and dental examinations. whilst many countries make use of these methods they do not apply them in the same way and often use different combinations and/or order. one of the main reasons for this is the fact that age assessment procedures remain to a large extent determined by national legislation, with procedures evolving through national jurisprudence (ref.: european asylum support office (easo age assessment practice in europe)). the ethical and legal aspects of using bone age to determine age will be addressed in a statement from the msk task force. the ethical and legal aspects of using bone age to determine age will be addressed in a statement from the msk task force. & the application of drls should be the responsibility of all providers of x-ray imaging. this means that drls should also be applied to imaging performed outside the radiology department. & the physical quantity used to establish drls should be an easily measurable quantity, usually directly obtainable from the x-ray equipment console, obtained either by manual recording or preferably by automatic recording and analysis. organ doses and effective dose are not considered feasible as a drl quantity because these cannot be easily determined. the ultimate mission of eurosafe imaging is to support and strengthen medical radiation protection across europe following a holistic, inclusive approach. most common imaging procedures in children and their contribution to collective dose e. sorantin 1 , c. granata 2 ; 1 graz/at, 2 genova/it summary: several countries have released "diagnostic reference levels (drl)" for imaging procedures using ionizing radiation. unfortunately those drl differ in types of procedures and granularity as well as information about the proportion of pediatric patients within the different examinations are sparse. therefore an more evidence based approach seems to be feasable -meaning releasing drl first for frequent and radiation burdened examinations. therefore a survey within europe was conducted and a questionnaire was sent to key persons of the european society of pediatric radiology (www.espr.org) as well to members of a large academic, interdisciplinary, international network within the ceepus programme (central european exchange programm for university studieswww.ceepus.info). alltogether 33 centers were contacted and an response was received from 18 (54.5.%). from one center only frequencies for interventional radiology was sent. plain films: most frequent procedures are extremities (48.5%), followed by chest films (31.4%) -both account together for more than ¾. flouroscopy: voding cysto urethrography (vcu) 37.2%, followed by upper gastro intestinal (gi) series with 32.1% -again representing 2/3 of those examinations. computed tomography: head & neck 46.8%, chest 27.8%, abdomen 13.7% -together almost 90%. interventional radiology and cardiac interventions: only limited data available and procedures quite hardly standardize and comparable. it seems adviseable, that only a few procedures are suitable for drl like peripheral insertion of vascular lines, occlusion of ductus arteriosus botalli or stent implantation for coarctation. in order to estimate the contribution to the relative collective dose all values were normalized to a chest xrays (1.0) and the following numbers could be calculated: abdominal plain film 0.1, skull 0.01, ct head 2.6, ct chest 10.2, ct abdomen 4.5. the most frequent imaging procedures using on ionizing radiation are: in plain films extremities and chest xrays in flouroscopy vcu and upper gi series in ct head, chest and abdomen therefore eu wide drl should be released for those examinations. as it could be expected chest ct is the main contributor to the collective dose. since the espr abdominal (gi and gu) imaging task force has changed its name and agenda, extending from initially only genitourinary queries to also other abdominal imaging topics, new projects have been added such as for example imaging in anorectal and cloacal malformations, imaging in paediatric inflammatory bowel disease (ibd, a joint project with esgar), or paediatric abdominal ceus applications. results of these new projects will be presented in the upcoming talkshoping that again (as the last procedural recommendations and proposed imaging algorithms) our proposals and recommendations will help to standardise paediatric imaging, to reduce radiation burden, and to facilitate comparable imaging data for future research. other topics in this session are a proposal for a more standardised approach to gastrointestinal ultrasonography, and considerations on gadolinium applications in children in the light of new observations (i.e., gadolinium deposit in tissue even in children with normal renal function). the work goes ononly achievable with active participation of interested and competent members. many interesting topics for either recommendations or joint research are on the list such as addressing late decompensating pujo or specific imaging needs in ibd in early childhood; other new ones may be proposed by any task force member. thus all espr members are invited to join the group, work with us and share their expertise. (2017) 47 (suppl 2):s297-s pediatr radiol contrast enhanced us in childhood -applications in children: literature review and results from the questionnaire c. bruno; verona/it in adults, following the characterization of focal liver lesions, several applications of contrast-enhanced ultrasound (ceus) have emerged in the last two decades, since second-generation contrast agents have been introduced and approved for use in most european countries. from many points of view, children represent an ideal population for ceus, because of the absence of radiation exposure and of need of sedation. moreover, due to the small body size many anatomical targets in children can be adequately explored with high-frequency ultrasound, obtaining images with higher spatial resolution than in adults. however, to date comparatively few data on pediatric ceus are available. although very rare and usually mild, possible adverse effects of contrast agents probably limit their use in many centers. in addition, the intravenous administration of ultrasound contrast agents in children is still off-label in europe, which makes informed consent necessary in every case. finally, for unclear reasons information on this topic does not flow easily. & from the comparison between the data available, similar or better results are likely to be obtained with ceus in children than in adults, and some specific pediatric indications might be proposed. imaging in ibd-joint recommendation statement with esgar f.e. avni 1 , m. napolitano 2 , p. petit 3 ; 1 brussels/be, 2 milan/it, 3 marseille/fr the first joint esgar/espr consensus statement on the technical performance of cross-sectional small bowel and colonic imaging (1) objective: to develop guidelines describing a standardized approach to patient preparation and acquisition protocols for magnetic resonance imaging (mri), computed tomography (ct) and ultrasound (us) of the small bowel and colon, with an emphasis on imaging inflammatory bowel disease. methods: an expert consensus committee of 13 members from the european society of gastrointestinal and abdominal radiology (esgar) and european society of paediatric radiology (espr) undertook a six-stage modified delphi process, including a detailed literature review, to create a series of consensus statements concerning patient preparation, imaging hardware and image acquisition protocols in pediatric and adult patients. the delphi process is constructed as follow: step 1 questionnaire construction to includes all contents relevant to the guideline and set up of working groups; step 2 questionnaire completed by all committee member, step 3 literature search; step 4 draft consensus produced by each wg based on the literature review and questionnaire responses; step 5 committee members indicate agreement or otherwise for each individual draft consensus; step 6 acceptance of agreed statements (more than 80% of members), face to face meeting to modify statements without agreement. committee members indicate agreement or otherwise for each modified consensus statement and final consensus statements. the questionnaire was split into four broad topics, each of them treated by a subgroup including in each of them a pediatric radiologist: (1) patient preparation for mre/mr enteroclysis/cte/ct enteroclysis, (2) mre/ mr enteroclysis technique and sequence selection, (3) cte/ct enteroclysis technique, and (4) enteric us patient preparation and technique. after an extensive literature research each member were instructed to always base their statements on the retrieved literature wherever possible, and to this end graded the strength of retrieved relevant publications from i (high) to v (low) using the criteria of the oxford centre for evidence based medicine (2) during their review process. if no relevant literature was available for a particular item, members used expert opinion to construct the consensus statements. the pediatric guidelines were based on the opinion of 3 pediatric radiologists and 4 adult radiologists who have experience in pediatric practice. & it is recommended that children aged 6-9 should not eat any solid & it is recommended that the use of a spasmolytic agent is optional. unlike adult practice, the use of spasmolytic prior to mre is considered optional in paediatric patients and use is likely dependent on the age of the patient, with older children more likely to tolerate spasmolytic injection. there are data supporting the benefits of glucagon on image quality, at the expense of prolonged imaging time and precipitation of nausea in just under half of paediatric patients (3, 4) . however, high diagnostic accuracy can also be achieved without spasmolytic (5) . & it is recommended that children aged over 9years should be nil by mouth for carbonated and milk beverages for 4-6h. ingestion of still water or non-carbonated fruit juice is recommended. & it is recommended that for dedicated colonic evaluation, a standard protocol without specific modification is used. & use of a spasmolytic agent is not recommended. & the use of i.v. us contrast is not recommended. & it is recommended that scan coverage should include an abdominal and pelvic examination, including the liver. there are no specific recommendations as to the use of hydro us in the paediatric patient as practice is not well developed. if oral contrast is given prior to us, it would seem sensible to follow the recommendations for mre in the paediatric population & it is recommended that if ct scanning is used in the paediatric population, no specific preparation is usually required although administration of positive oral contrast could be considered; for example, prior to percutaneous drainage of abscesses. limitations: there is little evidence in the literature to ascertain all these proposals. the recommendations were mainly based on expert opinion. no recommendations have been proposed for children before 6 years of age. especially the benefice of mre under sedation (6) in the younger compare to us doppler need to be explored. contrast media application is essential for a number of mri studies in children. there is some evidence that gadolinium-based contrast agents (gbca) are well tolerated in infants and children. the risk of adverse reaction is no higher in children than in adults. there are only few data available about pharamakokinetics in children, especially for the use of gbca in neonates. age-adapted reference values of the glomerular filtration rate (gfr) have to be used to identify children with a potential risk. in the past few years there was some attention toward the potential cellular toxicity of gadolinium and its role in the development of nephrogenic systemic fibrosis (nsf). there were only few children identified with proof of nsf. but, particulary renal insufficiency, poor hydration, acidosis and inflammation increase the risk for nsf. because the cases of nsf have been observed with linear componds the guidelines (2017) 47 (suppl 2):s297-s pediatr radiol from the esur and the espr and others propose to avoid linear compounds and to prefer macrocyclic gbca. in the past year several studies have described observations about possible gadolinium retention in the brain; hyperintense brain structures in native t1 weighted sequences were verified -globus pallidum and dentate nucleaus -also in children undergoing multiple mri examinations with gbca application. so, repeated mr investigations within a short time should be avoided -the cumulative dose of gbca should be recorded. consider all these points, the benefit of a contrast-enhanced study should be weighted against the potential risks before administering a gbca for each child separately. but, never deny a child an indicated cemri study. use single dose application (0.05-0.1 ml/kg body weight), improve renal function and hydration, balance acidosis -and ask your pediatric nephrologist íf necessary. gadolinium-based contrast agents are safe. macrocyclic compounds should be used in children. avoid contrast media in neonates and be careful in infants. identify risk factors. avoid repetivite application. procedural recommendation: how to perform pediatric gastrointestinal us m.l. lobo 1 , m. riccabona 2 ; 1 lisbon/pt, 2 graz/at summary: ultrasound (us) is the first imaging modality applied in the investigation of abdominal complaints in children, and an increasingly valuable imaging tool in the assessment of the gastrointestinal (gi) tract in neonates, infants and children. a comprehensive us examination is a critical first-step to optimize the potential of us diagnostic yield in many paediatric gi conditions. using proper high resolution transducers and graded compression technique is an essential part of gi us examination. a methodical and systematic analysis is crucial to facilitate a thorough evaluation of the bowel segments as complete as possible: follow bowel in a cross section, complete by longitudinal and oblique views. for some bowel sections filling is essentialsuch as stomach for gastroesophageal reflux and pyloric function, and distensibility and size of the colon by enema (e.g. for query microcolon). modern us methods are valuable, but not a pre-requisite. proper documentation of abnormal size of the gi tract segments, their luminal content, peristalsis, bowel wall characteristics and its surroundings, as well as local tenderness should be noted. a proposal for recommendation on how to perform paediatric gastrointestinal us will be presented for public discussion. & careful and dedicated us examination is crucial to obtain maximum anatomic and functional information in many gastrointestinal disorders in children. & systematic and methodical analysis helps to assess the bowel as complete as possible. & satndardization of us technique is essential to optimize us diagnostic capabilities and to allow for comparable examinations wich is essential to improve future evidence-based knowledge. hominid evo-devo: reconstructing the evolution of human development c. zollikofer; zurich/ch from an evolutionary biologist's perspective, modern humans represent the only surviving species of a group of highly specialized "bipedal great apes". they evolved more than seven million years ago in africa and managed to spread over the entire globe. in this talk, i will trace the history of our species with an emphasis on key developmental innovations that underlie major evolutionary innovations. why are we born with brains that have the size of adult great ape brains? and why do we grow up so slowly and get so old? i will highlight how advanced biomedical imaging methods help addressing these questions, and show how combined fossil, clinical and great ape data yield surprising insights into the evolution of our development. to present our experience with innovative imaging in pediatric interventional radiology. imaging technologies presented will include: 1. use of bubble contrast (lumeson) for indicatons including; complex pleural effusion and abdominal collection assessment pre and post therapy, primary g tube placement, renal perfusion pre and post rena artery angioplasty, vascular patency during central venous line placement, vascular malformation therapy and biliary tube assessment. 2. intravascular us (ivus) in arterial intervention pre and post angioplasty and venous thrombolysis intervention. 3. optical coherence tomography pilot study assesssment for renal artery intervention -validation in normal subjects. currently this imagng which uses laser light technology to assess vascular mural detail at the micron level, is only validated in coronary artery intervention in adults. 4. mr overlay -a technology that fuses mr imaging with low dose fluoroscopy and can faciltate biopsy of mr positive/ct negative lesions in the ir suite. focus will be on bone lesion biopsy and vascular malformation therapy. critical structures to be avoided can be outlined on the mr and transposed onto the fluoroscopic image during biopsy. in our experience this technology has promise in the pediatric setting with significant dose reduction when compared to ct. 5. mr fusion and i guide fusion technology enables an mr positive/ct negative lesion that would require ct guided imaging to be biopsed, using low dose c -arm ct, with fusion of the ct and mri images performed using landmarks, facilitating fluoroscopically guided biopsy in the ir suite. critical landmarks/structures to be avoided can be outlined on the ct or mr and transposed onto the fluoroscopic image during biopsy path planning and orchestration. focus will be on bone lesion biopsy. 6. color parametric flow related imaging in vascular interventionthis software enables time to peak opacification of arterial or venous contrast to be color coded in time and can provide adjunctive information for assessent of perfusion change during vascular intervention such as renal artery angioplasty, dialysis access intervention and cerebral embolization. 7. mr guided intervention -this focus will be on the initial development of an mr interventional program and our initial experience with mr arthrography. discussion will also involve the use of this modality for vascular malformation sclerotherapy and other msk interventions such as biopsy and nerve injections. 8. high frequency us imaging-focus will be on the use of a 40mhz us probe in the ir suite for various indications including visualization of smaller targets such as neonatal central venous access, superficial vascular malformation therapy and thyroid fine needle biopsy. 1. participants will become more familiar with exisitng and emerging innovative imaging technologies for pediatric intervention. participants will learn about the various indications and limitations of these technologies. 3. participants will gain insight into the process of introducing new imaging modalities into their pediatric interventional practice. increasing evidence supports the notion that autism spectrum disorder is associated with anomalies of brain function and connectivity. it is also evident that there are atypicalities in development/maturation of brain systems. particular promise arises from findings of atypical electrophysiology -indexing brain neuronal activity in real time. in particular, this talk will address a characteristic electrophsyiologic signature of delayed auditory evoked response latency (at~100ms). this, and related timing anomalies, have been proposed as biomarkers for asd -with candidate use for diagnosis, prognosis, stratification and therapy monitoring. progress along each of these axes will be discussed. however, to justify the term "biomarker", we demonstrate converging evidence from spectrally-edited (megapress) mrs and diffusion-mri. mrs offers insights into neurotransmitter levels, especially gaba and glutamate, imbalance of which may be associated with anomalous electrophysiologic oscillations in the gamma band. diffusion offers insights into the white matter of the brain (auditory pathway will be illustrated) and an interpretation of diffusion parameters as an index of central conduction velocity will be offered. combining these mechanistic measures with the spectrospatio-temporal capabilities of magnetoencephalography (meg), this talk will present a state of the art review of multimodal biomarker development in asd. take home points: meg captures brain activity in space and time as well as showing sensitivity to activity at different frequencies (where, when and what) delays in cortical neuronal response latency are evidence in asd atypical coupling between diffusion evidence of conduction velocity and timing of cortical responses in shown in asd oscillatory activity is atypical in asd (elevated "noise", decreased "synchrony") diminished inhibitory neurotransmitter (gaba) levels are shown in asd disturbance of teh typical coupling between gaba and gamma-band oscillations in development leads to anomalous adult oscillatory activity (taken to index local circuit function). multimodal and longitudinal approaches may be required to tackjle the complex and heterogeneous landscape of asd the paediatric radiologist can play an important role in establishing vascular access in paediatric patients ranging from neonates to teenagers. a breadth of knowledge and skills are needed to deal with changing body morphology and varied pathology in this age range. some of the skills particular to performing and managing vascular access in children will be discussed. different devices which can be placed, their indications, advantages and disadvantages will be reviewed. choice of access vessel is important in children, because there are known long term complication such as central venous stenosis and thrombosis, which can have a huge impact for future venous procedures or potential creation of an arteriovenous fistula of the arm for dialysis. preserving venous access sites is a (2017) 47 (suppl 2):s297-s pediatr radiol key responsibility especially in children with complex medical and surgical co-morbidities. because vascular access in children has associated morbidity it's important to manage and maintain devices that are placed. the risk of infection when repairing or exchanging a broken line will be highlighted. image guided biopsy is a very frequent procedure in pediatric patients. they range from random organ parenchyma for the diagnosis of medical disease up to tumor biopsies for histopathology analysis. different imaging modalities can be used for guidance as well as different biopsy devices and needles. ultrasound guidance is the most common modality used for this purpose in the pediatric population. the success of this procedure depends on multiple factors: from pain control up to choosing the correct device and area to sample. the radiologist performing the procedure also needs to be familiar with the potential complications of the intervention, how to prevent them and how to manage them. the intention is to perform the safer procedure as possible, obtaining the best quality of sample. the goal of this lecture is to present in a didactic way technical tips to perform safe and effective image guided pediatric biopsies, which may be applicable to different groups of operators, ranging from general pediatric radiologists performing occasional biopsies up to pediatric interventional radiologists. the objectives will be: to identify the safest approach to different types of biopsies; to describe ways to obtain the better quality of sample as possible; to demonstrate the use of different approaches in challenging clinical scenarios; to illustrate new devices currently used in specific applications; to discuss potential complications and its management and to show imaging modality integration applied to biopsy planning an performance. image guided biopsy is a frequent procedure in pediatric patients. a pre-procedure planning is fundamental in the success of the intervention. the operator must be aware of the aims of the biopsy and based on this choose the best approach, device and site for sampling. preparation and competency to manage complications is mandatory. pediatric interventional oncology: big cases in little people m. heran; vancouver/ca summary: the pediatric patient presents unique challenges in diagnosis and management of oncologic disorders. interventional radiology (ir) has a prominent role in the care of these children, with improvements in imaging and equipment offering better and safer options to traditional diagnostic and therapeutic procedures. as cancer can involve any organ system, consultations to the ir service can involve any part of the body, and can be non-vascular and vascular, simple and complex. the most common ir procedures in the pediatric oncology patient are enteric tube placement/change, vascular access, and percutaneous image-guided tissue/organ biopsy. however, with the explosion of interventional oncology in the adult setting, the variety and complexity of ir in pediatric oncology has begun to increase as well. ir techniques, such as thermal ablation, transarterial pharmacotherapy, and preoperative embolization, are now increasingly discussed in multi-disciplinary conferences as complementary or primary modes of treatment of oncologic disorders or related diseases/complications. however, although the principles of these diagnostic and therapeutic ir procedures remain essentially the same in their translation from adults to children, well recognized differences in pediatric physiology and metabolism, as well as the range in weight, size, and age of children, result in a practical question of "how do we do this?" the aim of this presentation is to provide an overview of the role of ir in the pediatric oncology patient, and to highlight areas of research and innovation. vascular anomalies encompass a spectrum of disorders including vascular tumours and vascular malformations. incorrect nomenclature and misdiagnoses resulting in inappropriate treatment are commonly experienced by patients with vascular anomalies. the currently accepted method for classification of vascular anomalies is straightforward and clinically relevant. vascular malformations can be divided into high flow lesions such as arteriovenous malformation or low flow lesions such as venous or lymphatic malformations. in children, a diagnosis can often be made with the history, examination and ultrasound. the classification of vascular anomalies will be briefly reviewed with examples of commonly encountered pathologies. a multidisciplinary team approach to the management of these conditions is vital. paediatric radiologists can play a key role not only in diagnosis but also in management, principally by injection sclerotherapy of low flow lesions and embolization of the much rarer arteriovenous malformation. many sclerotherapy agents are available with sodium tetradecyl sulphate the most commonly used for venous malformations and doxycycline for lymphatic malformations. different sclerotherapy agents have different characteristics and uses which will be covered. symptomatic relief is often achieved with treatment but multiple treatment episodes may be needed to achieve the desired outcome. ensuring the child and family understand this is vital to ensure they are satisfied with the management of the condition. contrast media is commonly used during imaging in children whatever their age and whatever the pathologic conditions. still, youngest patients are vulnerable and unstable. therefore, in neonates and infants the use of s339 (2017) 47 (suppl 2):s297-s pediatr radiol contrast media should be carefully evaluated and customized putting in balance the risk versus the benefit of its use. when using contrast media in neonates and infants, several features should be highlighted: -prematures and neonates have rather immature kidneys and some contrast media might be difficult harmful -the thyroid gland in prematures may be (transitorily) depressed by iodinated contrast media -the use of high osmolar contrast may induce a fluid shift and dehydration especially in premature and neonates -most contrast media are used off label; almost none has obtained the authorization to be used in neonates. -there are very few studies evaluating the short and long term adverse reactions in neonates and infants below the age of two. fortunately these reactions seem very rare in these age groups. -using contrast extends the duration of the examination and the need for sedation different types of techniques will potentially need ingestion, instillation or injection of contrast media: 1) opacification of the entire gi tract pre-and post-operatively 2) retrograde uretro-cystography 3) contrast enhanced ct 4) contrast enhanced mr imaging 5) contrast enhanced us 6) angiography furthermore, different types of contrast media can be used to achieve these purposes 1) barium (sulfate) 2) iodinated water-soluble contrast media (hyper-, iso-or hypoosmolar) remarks regarding opacification of the upper gi tract: -the upper or lower gi tract should be opacified using water soluble contrast in the immediate postoperative period or whenever a bowel perforation is suspected. -air can be used to confirm esophageal atresia and duodenal atresia -barium should be preferred in case of t-e fistula -either barium or water soluble iodinate contrast can be used in order to opacify (sub)obstructed upper gi tract remarks regarding the opacification of the lower gi tract -iodinated iso/hypo osmolar contrast should be used to opacify the colon in case of obstruction -a higher osmolarity iodinated contrast can be used in case of suspected meconium ileus or plug; still this contrast should be used diluted and under close clinical surveillance and adequate hydration. -in some more specific cases, for instance whenever hirschprung disease or a stenosis post nectotizing enterocolitis are suspected, barium enema can be used remarks regarding ct scan -contrast enhancement may help for the global assessment of various pathologies especially in case of cardio-vascular malformations or for the evaluation of abdominal masses. any iodinate contrast among those available is acceptable in neonates. higher osmolality contrast allows to inject a lower volume -injected volumes of 1.5 ml/kg seem adequate using 22-24 gauge needles -power injectors are acceptable as long as adequate catheters can be used -allergic or side effects are very rare and should be managed similarly to adults. remarks regarding mr imaging -the use of gd chelates in neonates remains controversial as there is no data available on the long term effects of gd injected so early in life -gd should be used only when enhancement may provide additional information compared to the non-enhanced study (cns infections, tumors, cardiovascular imaging, abdominal tumors, uro-mr imaging...) -only gd with low nsf risk should be used -gd should not be used in children with renal failure remarks regarding contrast enhanced us -little is known about the use of ce-us in neonates -indications seem equal to older children -there are very few side or allergic effects -doses suggested are 0.1 ml/year of age children present varied histological types of brain tumours. it's now possible to combine different information and image techniques to improve the diagnosis of paediatric brain tumours. the multimodal approach has increased the diagnostic specificity and permits, in most cases, the pre-operative differentiation between low and grade tumours. children with low grade lesions, and in particular the less accessible tumours, would benefit the most from avoiding biopsy. in addition, preoperative spinal mri evaluation to rule out drop metastases should be performed in patients with suspected high grade tumours. in general paediatric brain tumours are less necrotic, i.e. aggressive tumours in paediatric patients tend to be more hypercellular and homogeneous. because of its ready availability and speed, computed tomography (2017) 47 (suppl 2):s297-s pediatr radiol (ct) is the first investigation generally performed for a suspected brain tumour. ct can rule out haemorrhage or calcifications, but can also be used to evaluate tumour cellularity. a hyperdense tumour on ct reflects hypercellularity and is very often high grade. medulloblastoma are, for example, typically hyperdense on ct scans and paediatric low-grade astrocytomas are almost always hypodense. mri plays a major role in the evaluation of brain tumours. in conventional mri, the "general aspect" is the single most important parameter in predicting high-grade tumours in children. the same does not hold true for low-grade tumours, of which only 67% can be predicted using the general aspect. in our previous study, hyperintensity on t2-w and the lack of diffusion changes were the most important single parameters with 83% positive prediction. embryonic tumours, such as medulloblastoma or pnet have high tumour cellularity with consequent very low adc and hypo/isointense t2 compared to the cortex. adc values derived from dwi have been shown to be decrease in highly cellular tumours. adc values cannot reliably be used in individual cases due to the substantial overlap between tumour types previously described in the literature. nevertheless, adc has a higher predictive value in children and increases the accuracy of preoperative differentiation between low grade and high grade paediatric tumours. the cut-off values for differentiating between low and high grade paediatric brain tumours are 0.7 x 10 3 mm 2 /s and 1.0 x 10 3 mm 2 /s for minimum adc and average adc values, respectively. perfusion with relative cerebral blood volume (rcbv) is considered a marker of angiogenesis and is helpful in distinguishing high and low grade tumours. however, perfusion can be difficult to perform in small children; small catheters with manual injection are therefore used in such cases (or, as an alternative, arterial labelling). it should however be taken into account that choroid plexus tumours can have high rcbvs resulting from highly leaky capillaries. mr spectroscopy (mrs) shows the metabolic profile of the tumour. high grade tumours show elevated choline (cho) -reflecting increase in cell membrane turnover -and decreased n-acetylaspartate (naa), which represents a neuronal marker. the absolute values of the mrs peaks are not used by us; we favour to normalize the signal intensities of metabolites to their values in contralateral brain tissue. mrs is helpful not only as guidance for stereotactic biopsy (cho hot spot) but also for determining whether the tumour is high or low grade. as a rule of thumb, a 200% increase of cho when compared to the contralateral brain tissue is highly suggestive of a high-grade tumour. however, in children, increased cho levels can also be found in pilocytic astrocytoma; in this case the typical aspect with cystic component and location can suggest the diagnosis, despite the mrs result. therefore, in children, high cho levels do not necessarily imply the presence of a malignant tumour. task based functional mri (fmri) can be used for pre-operative localization of the eloquent cortex together with the identification of the language and somatomotor function. in the future, small children who are unable to cooperate will probably profit from resting-state fmri. pet mri has the advantage of integrating structural mr imaging with physiologic pet. take home points: take home points although the histology of paediatric brain tumours is diverse, their general morphological aspect on mri has a very high diagnostic reliability. unlike adult grade iv brain tumours, malignant paediatric brain tumours are less necrotic, but are highly cellular with high nuclear-to-cytoplasmic ratios. adding information on signal intensities on t2w and dwi further increases the diagnostic accuracy of conventional mri. the solid areas of high-grade tumours are iso-or hypointense on t2w and hyperintense on dwi, whereas low-grade tumours show inverse signal characteristics. advanced mr techniques (perfusion and spectroscopy) provide important biological information which can be used to correctly identify grading (high vs. low) and to guide biopsy. in children high cho levels, although suggestive, do not necessarily mean a malignant tumour. experience with central review of paediatric renal tumours g. khanna; st louis/us summary: central imaging review of pediatric renal tumors has been performed in children's oncology group since 2006. to date, more than 5500 cases of pediatric renal tumors have been centrally reviewed real time by the study radiologists. the mean time for central review was <8 days. discrepancies between local and central risk stratification were identified for detection of bilateral disease and pulmonary metastasis. in addition, central archiving of images has created a rich repository of cases for future research. the role of imaging in detection of key diagnostic features in pediatric renal tumors will be reviewed. the diagnostic performance of imaging for staging, detection of vascular invasion and tumor rupture will be discussed. real time central review of imaging is feasible in pediatric oncology wilms tumor remains the most common pediatric renal malignancy, followed by renal cell carcinoma cystic nephroma typically presents as a bosniak 3 lesion, and has high association with dicer-1 mutations is there a role for dwi in nephroblastoma? a.s. littooij; utrecht/nl wilms tumour or nephroblastoma is the most common malignant renal tumour in children. ultrasound is usually the first line investigation. mri of the abdomen is often performed to further delineate the tumor and its surroundings. the addition of diffusion-weighted imaging (dwi) to the standard mri protocol may enable subtype characterisation and allows assessing treatment response beyond necrosis and volume change. overall, the survival rate in patients with nephroblastoma is relatively good and the current focus is on finding biomarkers to further improve outcomes while reducing therapy-related side effects in these children. therefore, identifying low-or high-risk type nephroblastoma might be relevant for treatment planning. diffuse anaplastic nephroblastoma and extensive blastema in residual tumour after preoperative chemotherapy may require more intensive treatment. the limited available literature suggest a linear relation between adc values and subtypes nephroblastoma at histopathology. furthermore, the addition of dwi to the standard mri protocol may detect lesions (e.g. nephrogenic rests of nephroblastomatosis) that remain undetected at post contrast t1-weighted images. unfortunately, there is a considerable heterogeneity in acquisition techniques and methods of adc measurements. nephroblastoma often contains areas of necrosis and/or hemorrhage that can demonstrate very low adc values and consequently mimic highly cellular portions of tumours. therefore these areas should be excluded from further analysis. this lecture will highlight the potential additional benefit and limitations of dwi in children presenting with renal tumour. significantly lower radiation exposure even in comparison to low-dose pet/ct, (b) the higher diagnostic accuracy as compared to pet/ct even when using diagnostic contrast-enhanced ct, (c) the unique possibility to combine distinct mr-inherent contrasts (e.g. dwi) with specific pettracers (e.g. 64cu-labeled antibody imaging) for the evaluation of novel targeted therapies, and (d) the opportunity to stage local and systemic tumour burden within a single and highly resolved examination. on the other hand, many circumstances are challenging the extensive use of pet/mri in children. in general, the availability of pet/mri systems is low, particularly for children. thus, only a few sites in europe have experience with this technique in children, and therefore the generated scientific evidence is limited. moreover, whole-body-mri is still not a broadly adopted method for the combined assessment of local disease extent and whole-body staging, potentially replacing other whole-body modalities like the bone scan. in this context, especially the detection of pulmonary metastases is biased also against pet/mri. finally harmonized sequence protocols and specific recommendations for trace dosage are not available for pet/mri. in conclusion, further efforts are needed to keep the promises of pet-mri in the daily practice. common artefacts in paediatric mri-how to recognise, avoid or take advantage of them c. kellenberger; zurich/ch summary: while mri is a robust and radiation free imaging technique for assessing anatomy and pathology of most tissues and organs throughout the body, it is inherently prone to artefacts as no other imaging modality is. mri artefacts may impair image quality potentially leading to difficulties or errors in interpretation, but in some instances can contribute diagnostic information. main sources of image degradation are motion, disturbances of the local magnetic field and other factors inherent to image acquisition. strategies to reduce effects from various kinds of motion and adjustment of sequence parameters for eliminating artefacts will be discussed. & understanding the origin and effects of artefacts encountered in paediatric mri is essential for modification of mri protocols, so that artefacts and associated errors can be avoided. & for safely and successfully imaging children with implants and devices, the composition, location and functionality of the foreign body needs to be known. injuries to the central nervous system in abusive head trauma are responsible for the primary cause of morbidity and mortality in infants. neuroradiology has an important role in diagnosis but also in depicting injury and extent of brain damage of poor outcome. computerized tomography (ct) and magnetic resonance imaging (mri) are the primary imaging techniques. ct is usually performed in the acute phase while mri is performed the following days after injury. some injuries are better identified on mri such as diffuse axonal injury and cerebral edema with susceptibility and diffusion weighted images. abusive head trauma (aht) is the primary cause of morbidity and mortality in infancy, especially during the first year of life. aht is clinically characterized by a triad consisting of subdural hematoma, retinal hemorrhage and encephalopathy caused by brain swelling (1). the most common mechanism responsible for brain damage is thought to be caused by whiplash shaking injury explaining that abusive head trauma is also referred as shaken baby syndrome. impaction, compression and penetrating injury are also possible mechanisms as well as strangulation. however because of the variability of types and severity of injury, clinical symptoms vary from subtle to severe such as alteration of consciousness or coma (2) . the most common symptoms include vomiting, seizure, lethargy, poor feeding and apnea of which vomiting and respiratory pauses are non-specific (3). poor feeding, irritability or lethargy is also nonspecific signs. however apnea and/or retinal hemorrhages seen in children with brain injury are strongly associated with inflicted trauma (4) . in contrast to acute injury some children may manifest with increased head circumference related to chronic subdural hematomas. neuroimaging is therefore playing a crucial role to assess infants and children with a suspicion of abusive head trauma. computerized tomography (ct) and magnetic resonance imaging (mri) are the primary imaging techniques. ct is performed for the initial evaluation in cases with acute symptoms to look for hemorrhagic intracranial injury as subdural hematoma. mri is more often performed in the following days to further evaluate brain injury and to look for spine and spinal cord damage (5, 6) or in the presence of normal or equivocal ct findings (7) . however brain mri may be the first option in children presenting with increased head circumference. recently the study from flom et al showed the high sensitivity of mri for intracranial hemorrhage in well appearing infants at risk for abusive head trauma suggesting mri as a screening tool with 3 pulse sequences (axial t2, axial gradient recalled echo and coronal t1 weighted inversion recovery) (8) . ct is generally performed without intravenous contrast injection with 3d volume rendering (vr) reconstructions for identification of fractures. in some cases postcontrast images are also obtained specially to rule out deep venous thrombosis especially when children present with nonspecific clinical symptoms. mri protocol should include axial t2, t2* or susceptibility weighted images, coronal t1 images, diffusion or diffusion tensor images, and postcontrast 3dt1 images including mip reconstructions to evaluate the venous structures. mr venography can also be performed. susceptibility-weighted images are usually preferred because they allow the depiction of smaller hemorrhagic dai lesions and greater number of lesions compared to gre t2 (9) . it was also reported by colbert et al (10) that the presence of micro-hemorrhages alone was useful for outcome prediction in abusive head trauma with significant poor long-term outcome. the sensitivity and specificity of microhemorrhages was also higher than the other clinical (such as retinal hemorrhages and glasgow coma scale score) and other imaging findings for prediction of outcome. diffusion tensor imaging (dti) measurements were reported in abusive head trauma by imagawa et al: decreased axial diffusivity related to axonal injury with consequent reduced mean diffusivity did correlate with poor outcomes (11) . magnetic resonance spectroscopy (mrs) is usually not part of the standard protocol. however aaen et al (12) showed that n-acetylaspartate/creatine and/or nacetylaspartate/choline ratios were decreased significantly in the corpus callosum, frontal white matter, parieto-occipital white matter, and parietooccipital gray matter in children with poor outcomes. this study mentioned above also reported that the prediction of outcome was accurate in 100% of patients by using a logistic regression model that include age, initial glasgow coma scale score, presence of retinal hemorrhage, lactate on mrs, and mean total n-acetylaspartate/creatine. functional mri, (2017) 47 (suppl 2):s297-s pediatr radiol volumetry may be performed in long-term follow up of victims of child abuse. physical abuse is associated with altered emotion with greater activation in the salience network in response to negative stimuli, that includes amygdala, thalamus, putamen and anterior insula (13) . increased responsiveness of the right amygdala to fearful and angry faces (negative stimuli) and structural changes as reduced hippocampal volume, are reported by dannlowski et al (14) . impaired attention was also reported in patients with childhood abuse (15) with reduced activation during attention tasks in the left hemispheric ventral and dorsolateral prefrontal regions. intracranial injuries include extracerebral hemorrhages and parenchymal damage as brain swelling and ischemia, venous infarction, diffuse axonal injury, contusions and intraparenchymal hematomas (7, 16) . extracerebral hemorrhages subdural hematoma is a characteristic finding of inflicted traumatic brain injury, is generally multifocal and most commonly seen along the posterior interhemispheric scissure, over de convexities at the vertex level and/ or in the posterior fossa (17) (18) (19) . subdural hematomas are most likely bilateral but may be unilateral. all locations are related to disruption of bridging veins. the identification of bridging vein rupture allows the diagnosis of traumatism in relation to acceleration/deceleration, rotational and shearing forces due to violent shaking (20) . a mixed density appearance of subdural hematomas is frequent but is also seen in accidental traumatic brain injury (21) (22) (23) . indeed this feature is often present in the very early hours following trauma and is thought to be secondary to early sedimentation of blood clots and supernatant serum. tubular high density is often seen on non-contrast ct over the convexities in abusive head trauma. this ct feature is related to a clot secondary to venous disruption (24, 25) that can end up in thrombophlebitis. this tubular high density was reported more recently as tadpole sign (26) and lollipop sign (27) respectively seen in 40 and 44% of abusive head trauma. this appearance is strongly associated to inflicted trauma and much less frequent in accidental trauma (3 out of 83 cases (3,6%) of accidental trauma in our experience). associated venous infarction is reported in 12% of cases of abusive head trauma (24) and often located in the parieto-occipital region, unilaterally at the site of venous disruption of bridging veins. subdural hemorrhages, when multiple, in the convexity and interhemispheric, or in the posterior fossa were found significantly associated with abusive head trauma in the meta-analysis reported by kemp et al (28). in addition subdural hematoma, cerebral ischemia, skull fracture, retinal hemorrhage and intracranial injury were significantly associated with abusive head trauma in the review from piteau et al (29). subarachnoid hemorrhages (sah) and epidural hematomas are also found in inflicted trauma and are not considered discriminant-imaging features. however epidural hemorrhages, isolated skull fracture and scalp swelling were reported as significantly associated with accidental traumatic brain injury (29). sah in shaking injury is usually caused by tears of the vessels within the pia and arachnoid predominantly in the interhemispheric fissure and high convexity (7). parenchymal injury parenchymal injury include brain swelling and ischemia, venous infarction (discussed above), diffuse axonal injury related to rotationallyinduced shear-strain injury with different inertia for grey and white matter due to their different specific gravities, contusions seen in deceleration trauma with friction between the skull and brain, and in blunt trauma and intraparenchymal hematomas related to lacerated vessels. brain swelling/ischemia may be related to increased blood volume (congestive swelling), increased presence of water in the nervous tissue, and the combination of both. increased water in the nervous tissue may manifest as vasogenic edema located in the white matter due to extravasation of plasma like fluid related to incompetent blood-brain-barrier and as cytotoxic edema located in the grey matter, related to ionic imbalance. cerebral edema can be recognize on ct within the 12 hours following injury as loss of gray-white matter differentiation and decreased attenuation of grey and white matter. cytotoxic and vasogenic edema are better characterized on mri with diffusion-weighted imaging. brain swelling and edema occur early after trauma with consequent underestimation of subdural hematoma. therefore imaging should be repeated (ct or mri) especially when neurologic symptoms change rapidly. brain swelling/ edema may also involve the posterior fossa and is better identified on brain mri. two frequent patterns have been reported in abusive head trauma (24). diffuse supratentorial brain swelling (infarction) involving the cortex and white matter was reported in 39% of cases and is considered as severe hypoxic-ischemic injury with poor outcome (30). watershed infarction was reported in 36% of cases and considered a less severe form of hypoxia-ischemia. apparent diffusion coefficient (adc) values are strongly associated with poor neurodevelopmental outcomes in the acute phase (within 4 days) especially basal ganglia, thalamus, brainstem, cerebral cortex, cerebellar vermis, cerebellar cortex and mean total brain (31). during the early phase up to 1 month adc values in fewer regions (basal ganglia, thalamus, brainstem and corpus callosum) were associated with poor outcome. when patients with and without parenchymal lesions are compared, the detection of diffuse lesions during the first 3 months as well as beyond 3 months is significantly associated with severe developmental outcome (32). late mri (beyond 3 months after injury) also showed that recovery depends on the extent of brain damage. patients with diffuse lesions show more severe motor and intellectual impairments and are more likely to have blindness and epilepsy than patients with focal or hemispheric lesions (32). diffuse axonal injury (dai) is related to shear-strain injury of small medullary veins and was reported in 6% of cases of abusive head trauma (24). it is encountered in trauma with sudden acceleration-deceleration associated with rotational angular forces and in shaking-impact trauma. the lesions may be hemorrhagic or non hemorrhagic (related to axonal swelling). dai is most often located in the subcortical white matter at the gray-white matter junction, corpus callosum, basal ganglia, brainstem and internal capsule. if the lesions are large enough and hemorrhagic dai may be seen on ct. however dai is usually better identified on mri with susceptibility and diffusion weighted images. the detection of changes in the basal ganglia or brainstem during the first 3 days as well as during the first month after injury is significantly associated with poor long-term outcome in survivors (30). the presence of intraparenchymal brain micro-haemorrhages detected on swi in children with abusive head trauma correlates with significantly poor long-term neurologic outcome (10) contusion is also reported in abusive head trauma and is seen in blunt trauma with impact with or without contrecoup contusion. contusions are located at the surface of the brain (crest of gyri) and may be pial and haemorrhagic (disruption of cortical arteries). they are also found in the frontal and temporal regions related to impact of the brain on the roof of the orbit, middle cranial fossa and sphenoid wing. white matter tears are also seen in the frontal and temporal area related to the vulnerability of unmyelinated and soft white matter in infants. skull fractures are seen in blunt impact and are less frequent than long bones and rib fractures in non-accidental trauma. the most common site is the parietal bone (because of bulging of parietal bones below 1 year of age). the fracture may be linear as in accidental trauma. radiologic features significant for inflicted trauma are multiple fractures, bilateral fractures and fractures that cross suture lines (28, 33). focal underlying brain damage can be seen such as subdural hematoma and hemorrhagic contusion. hypoxic-ischemic encephalopathy is seen in strangulation injury with involvement of the territories of the internal carotid artery related to their anatomic vulnerability. neuroradiology (ct and mr) is crucial for the diagnosis of trauma, to predict outcome when showing edema and hypoxic-ischemic injury. this presentation will present an update on post mortem mri (pmmr) with relevance to clinical developments over the last 2 years. in particular, reference will be made to diagnostic accuracy of pmmr across different body parts, the current limitations of post mortem mr, and protocol development at different field strengths. imaging correlates of post mortem interval are also being investigated. maceration (autolysis within intrauterine fluid) and perimortem hypoxic brain changes caused difficulties in image interpretation, which more advanced and quantitative techniques may be able to address. jawad take home points: below 500g, 1.5-t pmmr shows a significant reduction in diagnostic yield, compared with conventional autopsy, and therefore its clinical usefulness in this setting will depend on individual circumstances. 3t pmmr performs better than 1.5 t particularly <20 weeks gestation, and particularly for the chest, heart and abdomen. diffusion characteristics in different fetal brain areas are multifactorial, with maceration the strongest predictor in most areas. international pm ct protocols c.y. gerrard 1 , o.j. arthurs 2 ; 1 albuquerque, nm/us, 2 london/uk the european society of pediatric radiology (espr) taskforce and the international society of forensic radiology and imaging (isfri) pediatric working group have combined efforts to establish best practice standards for performing perinatal and pediatric post mortem computed tomography (pmct) examinations. use of pmct in the investigation of pediatric death has increased significantly in the past decade. due to quick acquisition times and the ability to acquire thin slice, high detailed images of the whole body, (2017) 47 (suppl 2):s297-s pediatr radiol many hospitals and forensic institutes have implemented pmct into daily practice. however, there lack an overall standardization of how cases are triaged and the acquisition methods when comparing institutes using pmct. in an effort to address inconsistencies in acquisition parameters, post processing, and case selection, pmct protocols were compiled from international institutes and centres currently performing pediatric imaging. this paper will describe both the uniform and divergent elements of image acquisition and procedural uses identified among the participating centres. the outcome is to provide a single source of information that can guide already established and new centres on the best practice standards for implementing pediatric pmct. take home points: describe how pediatric post mortem computed tomography (pmct) has increased in utility over the past decade. identify the differences in acquisition methods for clinical computed tomography versus post mortem computed tomography. discuss the overall consensus of case triage and scan acquisitions when comparing institutes in aggregate. provide comprehensive statement of best practice standards for pediatric pmct. post mortem imaging research: updates and future proposals o.j. arthurs; london/uk paediatric and perinatal post mortem imaging is a new and rapidly growing field, and the post mortem imaging taskforce was founded in graz at espr 2015. the pmi taskforce aims to help reach consensus and guidance regarding imaging protocols and the potential yield of post mortem ultrasound, ct and mr. the key priorities are the themes of collaboration, image acquisition, best practice guidelines, training and education, raising awareness and access to imaging. this presentation will give updates on the latest developments in perinatal and paediatric imaging, with particular focus on where the pmi taskforce can help. in particular, protocol development is underway, and the espr meeting acts as an opportunity for collaborative working and network development, to facilitate best clinical practice and welcome new members. arthurs oj et al., espr post mortem imaging task force: where we begin. pediatr radiol (2016) ; 46: 1363 -1369 take home points: post mortem imaging is an exciting sub-specialty which requires a combination of in depth fetal medicine, perinatal autopsy and pediatric imaging knowledge to help shape and grow the clinical and research arena. dedicated personnel have an opportunity to create the evidence-based behind a growing clinical service, with clear benefits to patients, families and referring clinicians. abstracts appear as submitted to the online submission system and have not been checked for correctness and completeness. sequences, are an emerging tool for evaluating intracranial vessel disease. improved survival due to emended treatment protocols results in an increasing number of long-term medulloblastoma survivors who experience delayed treatment effects. microbleedings, developement of cavernomas, vasculitis and atherosclerotic lesions are cerebrovascular structures affecting sequelae of the applied radiochemotherapy. this study evaluates radiation-induced intracranial vascular changes. twenty-two long-term pediatric medulloblastoma survivors (mean age 25.8 years, range 10-53 years; mean years after primary radiochemotherapy 16.3 years, range 1-45 years) underwent mri. the scan protocol included precontrast 3-dimensional time of flight (tof)magnetic resonance angiography (mra), precontrast 2d t1-and 2d t2-vwisequences and postcontrast 2d t1-vwi-sequences of the medium and large intracranial arteries. vessel wall thickening, contrast enhancement and luminal narrowing were analyzed. additionally precontrast t1-, t2-swi and t1-weighted images of the supra-and infratentorial brain were acquired. results: vwi-sequences: vessel wall changes could be found in 12 (54%) and 14 patients (63%) of the right and left ica, respectively. for the ba 4 (18%) patients revealed vessel wall changes; for the left and right va 2 (9%) patients were detected with vessel wall changes, respectively. in the tof angiography no alteration of the ica, ba or vas could be identified. in total vessel wall changes for the vertebrobasilar system and the icas could be found in 16 (72%) patients. swi-sequences: all patients (100%) revealed swi lesions, the smallest lesion measuring less than 2 mm, the biggest up to 5 mm. sixteen patients (72%) were presented with lesions > 4 mm, suspicious for cavernomas. to ensure quality of life in long term childhood medulloblastoma survivors, monitoring of long-term effects, like vascular changes after rct is gaining in importance. high resolution mri, including swi and vwisequences could be used here for. this study images, asymptomatic vessel wall alterations in former childhood medulloblastoma patients through vwi sequences and micro bleedings through swi sequences. vessel wall alterations, revealing rct induced arteriosclerosis, can lead to symptomatic intracranial stenosis which is associated with ischemia, furthermore micro bleedings and cavernomas can lead to intracranial hemorrhage. however further studies are needed to standardize mri sequence protocols to ensure a high standard follow up protocol, detecting clinically still asymptomatic vascular changes. fast "black-bone" mr imaging in evaluation of craniofacial abnormalities: comparison with high resolution ct z. habib, a. talib, c. parks, s. avula, l.j. abernethy; liverpool/uk to evaluate the feasibility and diagnostic value of a fast field echo, "black bone" mri sequence in children with craniofacial abnormalities. a fast "black bone" mri sequence has been used in addition to standard brain mri in 16 children (mean age 17 months, age range 3 months to 5 years and 5 months) referred to the supra-regional craniofacial surgery unit at alder hey children's hospital, liverpool, uk. a subgroup of 10 of these patients with complex craniofacial abnormalities additionally had high resolution volume ct performed at the same visit. "black bone" mr imaging was performed on philips ingenia 3t and 1.5t scanners, using a 3d fast field echo sequence (tr=8.3 ms, te=4.6 ms, flip angle 5 0 ). this sequence can be performed with an acquisition time of less than 2 minutes. the "black bone" sequences were assessed for accuracy in evaluating the patency of the sagittal, coronal and lambdoid sutures, and, where applicable, were compared with high resolution ct. the fast "black bone" mri sequence was shown to be technically feasible in all cases. the resultant images successfully demonstrated both patent sutures, which were confidently seen, and prematurely fused sutures which were confidently not seen. visualisation of patent sutures was found to be further enhanced by the use of minimum intensity projection. in the subgroup of patients with complex craniofacial abnormalities, comparison with high resolution volume ct confirmed good sensitivity for patency of cranial sutures. there was complete agreement in 50 out of 50 sutures assessed. the "black bone" mr images were also found to produce good-quality surface-rendered images and were also suitable for 3-d printing of models for pre-operative planning. fast "black-bone" mri has proven to be technically feasible and to demonstrate cranial suture patency with good agreement with high resolution ct. additionally "black-bone" mri can be used to produce good quality surface-rendered images and 3-d printed models for surgical planning. main symptom of mucopolysaccharidosis type iva (mps iva) is progressive systemic skeletal dysplasia. this is routinely monitored by cerebral and spinal mri. the vascular system is generally not in the primary focus of interest. in our population of mps iva patients we observed vessel shape alterations of the vertebrobasilar arteries, which has not been described before materials: mri-datasets of 26 patients with mps iva acquired between 2008 and 2015 were eligible for retrospective analysis of the vertebrobasilar arteries. the vessel length and angle of the basilar artery (ba) and both vertebral arteries (va) were analyzed. a deflection angle between 90°and 130°in the vessel course was defined as tortuosity, less than 90°as kinking. the results were compared to an matched control group of 23 patients not suffering from mps. the deflection angle [°] of the va and ba was significantly decreased in the majority (85%) of mps iva patients (fig. 1) mps iva is associated with significantly increased tortuosity of vertebrobasilar arteries. therefore the vascular system of mps iva patients should be monitored on routinely basis, as vessel shape alterations had been associated with dissections, leading to a higher risk of cerebrovascular events. in the pediatric population, intraspinal cysts (arachnoid or neurenteric cysts) are rare lesions mainly located in the thoracic region, whose acute onset is not well described in the literature. (2017) 47 (suppl 2):s297-s pediatr radiol we present a series of four children seen in the last two years as spinal cord emergencies and discuss the clinical aspects, imaging diagnosis, and management approaches, particularly in the emergency setting. a comparison of our cases with those reported in the literature is also provided. as in other types of spinal cord lesions, mr imaging is the diagnostic procedure of choice, because of its potential to demonstrate the exact location and extent of the cyst and its relationship to the spinal cord, valuable information for planning surgical treatment. this is a retrospective review of 4 cases of pediatric intraspinal cyst occurring in 4 boys and 1 girl, aged 2 to 6 years, treated at our institution between 2014 and 2016. onset was sudden in all cases and mimicked transverse myelitis or infarction. all our affected patients had no preceding history of trauma and presented with signs of spinal cord compression-back pain and less commonly abdominal pain-followed by weakness. all patients underwent emergent mr imaging, including t1, t2, t2*, 3d ciss, diffusion imaging and enhanced t1 sequences, mainly in the sagittal and axial planes. in each sequence, mr imaging showed a well-defined cystic lesion with signal intensity similar to cerebrospinal fluid, and secondary spinal cord compression that was severe in most cases. blood remnants were not visualized within or around the arachnoid cyst in any patient, which correlated with the absence of trauma antecedents. three of the four cysts were located in an anterior position relative to the spinal cord, and only one was located posteriorly; this latter had an associated subdural effusion. none of our patients had an associated neural tube defect. all patients were urgently treated with cyst wall fenestration or resection. the symptoms improved in all except one patient, whose symptoms did not abate, but ceased to progress. a prompt emergent diagnosis with mr imaging is important, as the symptoms can resolve if surgical treatment is performed before the spinal cord becomes irreversibly damaged. urgent surgery is essential in these cases, particularly if progressive neurological dysfunction develops over the course of spinal cord compression. the outcome following surgical fenestration or excision is excellent in most cases. nevertheless, a long-term imaging follow-up is recommended to detect possible recurrence. the objective of this study was to evaluate the usefulness of multiparametric quantitative mri model for myelination quantification in children. twenty-two children (age range: 9-5,400 days) were scanned with multiparametric quantitative mri. total volume of myelin water fraction (mwf) (msum), the percentage of msum within the whole brain parenchyma (mbpv), and the percentage of msum within intracranial volume (micv) were obtained. mwf values of brain regions were acquired by drawing regions of interests. the values were fitted to representative models of myelin maturation. spatiotemporal pattern of mwf mapping was visually assessed. values of msum, mbpv, and micv well fitted to a developmental model of myelination. mwf of brain regions well fitted to a developmental model with high r 2 values: pons (r 2 =74.6), middle cerebeller peduncle (r 2 =75.5), genu of corpus callosum (r 2 =93.6), splenium of corpus callosum (r 2 =79.7), thalamus (r 2 =85.8), frontal white matter (wm) (r 2 =95.7), parietal wm (r 2 =95.1), temporal wm (r 2 =93.7), occipital wm (r 2 =94.5), and centrum semiovale (r 2 =82.1). mwf mapping followed the known spatiotemporal pattern of myelination. multiparametric quantitative mri is a useful tool for mwf quantification in children. retinoblastoma is the most common intraocular tumour of childhood. it is a highly malignant. retinoblastoma is curable. if detected while still confined to the globe and if there are no metastatic risk factors, the child will nearly always survive following appropriate treatment. our aim is to assess diagnostic accuracy of preoperatively performed magnetic resonance (mr) imaging for detection of tumor extent in patients with histopathologically proved retinoblastoma. local ethics committee approval and informed consent were required for reviewing of patients' images and records. fifty-eight eyes in 30 girls and 27 boys with retinoblastoma (mean age at diagnosis was 23 months ±18.9) were reviewed on unenhanced t1wi, t2wi, and gadolinium-enhanced t1-weighted mri with and without fat suppression. mri parameters such as anterior chamber hyperintensity, involvement of choroid, ciliary body, optic nerve, sclera, orbital fat, and pineal gland were determined. maximum tumor diameter was measured and correlated to metastatic risk factors. imaging and pathologic findings were compared. choroidal invasion was suspected with mr imaging in 47/58 eyes; findings were false-positive in 6 eyes and false-negative in two (accuracy, 86.2%; sensitivity, 95.3%; specificity, 60%). mr imaging findings were true-positive in 10 of 17 eyes with proved prelaminar optic nerve invasion (60% sensitivity) and false-positive in 7 (82.9% specificity, 75.8% accuracy). postlaminar optic nerve invasion was correctly detected in 23 eyes; 4 eyes were false positive, in other 4 eyes, this metastatic risk factor was missed (accuracy, 86.2%; sensitivity, 85.2%; specificity, 87%). of nine eyes with histologically proven scleral invasion, 5 eyes were true positive . in the other 4 eyes, scleral involvement was missed on mri (accuracy, 93%; sensitivity, 55.6%; specificity, 100%).extraocular fat invasion was suspected on mri in 5/58 eyes. of these, findings were truly positive in 4 eyes (80%) and in 1 eye (20%) was incorrect (false positive) (accuracy, 98.3%; sensitivity, 100%; specificity, 98%).anterior chamber hyperintensity on t1-weighted mr images obtained after contrast agent administration correlated well with main mri and histolopathology findings. tumor size (assessed in our study by the maximum diameter in mm) was statistically associated with postlaminar optic nerve invasion (ρ=.002) and choroidal invasion (ρ=.007). mr imaging shows promising role for tumor staging and detection of metastatic risk factors. tumor diameter, measured with mr imaging, is associated with postlaminar optic nerve and choroidal involvement. patterns of the cortical watershed continuum of term gestation hypoxic ischaemic injurythe "wish-bone sign" a. chacko 1 , s. andronikou 2 , s. vedajallam 1 , j. thai 2 ; 1 east london/za, 2 bristol/uk objective: background partial-prolonged term hypoxic ischaemic injury (hii) involves the cortical and subcortical watershed zones of the brain, which are visually difficult to conceive. new innovative methods of demonstrating watershed cortical atrophy using flattened maps of the brain surface gives added insight into distribution of the watershed zone by demonstrating the entire brain surface. aim determining and validating patterns of hii sustained at birth in term infants using cross-sectional mri and the innovative mercator and scroll map views of cortical surface anatomy, to define the distribution of the watershed zones in children with partial-prolonged injury. one hundred paediatric mri brain scans with an mri and clinical diagnosis of chronic term hypoxic injury were read by 3 radiologists independently. all sites of abnormality were recorded and patterns classified. (2017) 47 (suppl 2):s297-s pediatr radiol patients with partial-prolonged and combined patterns were evaluated using mercator and scroll map reconstructions, generating schematics of the watershed zone. predominant patterns of disease were partial-prolonged and acuteprofound types. the watershed zone was demonstrated, on the derived maps, representing a continuum of involvement in the shape of a 'wish-bone' extending bilateral from frontal lobes to posterior parietal lobes in band-like fashion along the para-falcine cortex and intersected by another band of atrophy in the peri-rolandic regions extending along peri-sylvian cortices. this is defined in schematics as a visual aid. predominant patterns of injury in term hypoxic ischaemic injury are described and quantified, with the 'wish-bone sign' introduced to describe the typical distribution pattern of partial-prolonged hii in the watershed zone. correlation of brain edema degree and biochemical parameters in pediatric posterior reversible encephalopathy syndrome with hematologic/oncologic diseases t. akbas 1 , s. ulus 2 , b. karagun 1 , t. arpaci 1 , c. kalayci 2 , b. antmen 1 ; 1 adana/tr, 2 istanbul/tr posterior reversible encephalopathy syndrome (pres) often associated with hypertension is characterized by typical transient parietooccipital predominantly brain edema on magnetic resonance imaging (mri) with neurological symptoms such as seizures, headache and visual disturbances. even if endothelial dysfunction, increased blood-brain barrier permeability and hyper-hypoperfusion remain as controversial mechanisms to explain, the pathophysiology of pres is unremain. the aim of our study was to investigate the correlation between brain edema degree on mri and serum biochemical parameters such as lactate dehydrogenase (ldh), albumin (alb), creatinine, uric acid (ua) and urea. a total of 27 pediatric hematology and oncology patients (19 male, 8 female, aged 3-17, mean age: 11 years 6 months) diagnosed with pres during treatment and after hematopoietic stem cell transplantation (hsct) were included in this retrospective study. underlying diseases were beta thalassemia (n:14), aplastic anemia (n:4), acute lymphoblastic leukemia (n:4), acute myeloid leukemia (n:3), lymphoid leukemia (n:1) and burkitt's lymphoma (n:1). pres was seen after undergoing hsct in 21 patients. the brain edema degree according to specified anatomical regions on fluid attenuation inversion recovery (flair) mri sequence was scored by two radiologists blinded to patients' records. the levels of serum biochemical parameters at onset of symptoms were correlated with score of brain edema degree on mri. serum ldh concentration was statistically correlated with the score of brain edema degree (spearman's rho correlation, r=0.459, p=0.016). no relationship was found between other biochemical parameters and the score of brain edema degree. our results suggest that increased serum ldh as a marker of endothelial dysfunction is the main biomarker for development of brain edema in pediatric pres patients under treatment and after hsct with underlying hematologic and oncologic diseases. objective: gadolinium based contrast agents (gbcas) have been associated with increasing signal intensities in deep brain nuclei on unenhanced t1-weighted brain imaging. until now, most studies have been performed in adults, while results on pediatric patients are sparse. therefore, the aim of this study was to evaluate if there is any difference between signs of gadolinium retention in pediatric and adult patients. in this irb-approved, single center retrospective study, we extracted all patients with at least 5 contrast-enhanced mris archived on pacs between 2009-16. all patients with gadobenate dimeglumine only enhanced mris were reviewed. seventy-six pediatric patients with the most injections and 86 adult patients with the most injections were included in the final evaluation. therapies were documented. t1 signal intensity measurements for the initial and last unenhanced brain mris were performed for dentate nucleus, pons, globus pallidus and thalamus. signal intensity ratios for dentate-to-pons (dnp) and globus pallidus-to-thalamus (gpt) were calculated and correlated with number of injections and time interval as well as therapy. differences between adults and pediatrics were assessed. mean age for the pediatric group was 9.3 years compared to 47.7 years in the adults. no significant difference was found for gender distribution (47 vs. 43% females) and follow up time (3.1 vs. 3 years). there was no difference concerning the signal intensities on first and last mri in children and adults (p=0.81/0.84, respectively). for each additional year of follow-up the change in ratio increases by 0.016 for adults but only 0.002 for peds (p=0.002). comparing therapies, in children a statistically significant difference between patients with and without former radiation was found (p<0.001) while there was no difference in adult patients with and without therapy (p=0.65). children and adults show a similar increase in t1 signal in deep brain nuclei ascribed to gadolinium deposition. in children, radiation and chemotherapy) seem to have a higher influence on gadolinium deposition. this correlation cannot be found in our adult cohort, indicating therapies have no (additional) influence. kearns-sayre syndrome (kss) is a rare mitochondrial dna-deletion syndrome characterized by early onset (<20 years), progressive external ophthalmoplegia and pigmentary retinopathy, often associated with cerebellar ataxia, muscle weakness, bilateral sensorineural hearing loss and cardiomyopathy. pyramidal symptoms may be present in kss, but they are poorly reported in the literature. through this case series, we aim to evaluate the concordance with the imaging patterns proposed by literature, correlating them with clinical and laboratory data, and to investigate possible microstructural damage with diffusion tensor imaging (dti) and magnetic resonance spectroscopy (mrs). we evaluated eight patients (8-19 years of age) with genetically confirmed diagnosis of kss. all pts. were studied with 3t/1.5t mri. in 5/8 pts. the study was completed by mrs and in 7/8 by dti imaging with reconstruction of cortico-spinal tracts (cst) using a 2-rois approach. a t-test comparative study between mean fractional anisotropy (fa) of cst in the 7 kss patients with dti and a group of 4 healthy controls was performed. cst reconstruction in a patient suffering from kss (images a-c), compared to an healthy control (images d-f). the dti study showed significantly reduced fa values, pointing out a possible microstructural damage. the disease showed an mr pattern of mixed white and gray matter signal abnormality, with periventricular and/or subcortical white matter hyperintense lesions, which in 1/8 patient presented a "tigroid pattern" (fig.2 ) three patients displayed a disease extension to the cervical spinal cord. (fig.2 ) dwi images demonstrated restricted diffusivity in almost all lesions (fig.3) , with persistence of low adc values. mrs study documented a high lactate peak in 2/8 pts. and a naa reduction in 5/8 pts; an increment of gsh was noted in one patient (fig.3) . the t-test comparative study of cst showed a significant reduction of mean fa value in kss patients compared to healthy controls (p= 0,004). involvement of the spinal cord (a-c, yellow arrows). comorbidity was suspected in "a" (myelitis). below (d-f): pale nuclei (d, green arrows) and subcortical white matter (e) alterations. right image displays the "tigroid pattern" (purple arrow). mrs showing the presence of a gsh peak, which may suggest an augmented antioxidative activity within the encephalic tissue. below: dwi hyperintensity in many regions of the brain in patients suffering from kss, due to diffusion resctriction. the integration of neuroimaging with clinical data can implement the diagnosis of mitochondrial diseases such as kss. according to our experience, comorbidities can delay the achievement of a correct diagnosis. the finding of an altered signal in the spinal cord of 2/8 pts. may suggest a new possible localization of the disease, while in one patient was referable to myelitis (fig.2, a) the evidence of a "tigroid patter" in should be taken in count in the differential diagnosis with lysosomal disorders. the presence of a prominent gsh peak may represent an augmented antioxidant activity, which may correlate with a more favorable outcome. an involvment of cst can be speculated even if pyramidal symptoms are poorly represented in kss. remotely distractible, magnetically controlled growing rod (mcgr, fig. 1 ) system has been developed to allow for gradual lengthening on an outpatient basis. this allows for safe spinal lengthening with continuous neurologic monitoring and real-time feedback by the patient. this study aims to evaluate retrospectively our ultrasound (us) geometric method and his accuracy compared with the plain radiograph (gold standard) for assessing mcgr distractions. this is a retrospective study that includes patients with early-onset scoliosis undergoing multiple consecutive distractions after mcgr implant. the rods length was measured for with us, for each distraction (3-months interval), and compared with plain radiograph follow-up (1-year interval). all patients included were treated with dual-rod systems. distraction length was monitored by a senior radiologist with us at each visit, one rod at a (2017) 47 (suppl 2):s297-s pediatr radiol time, before and after magnetic lengthening, with our geometric measurement method (fig.2) . low-dose upright two-projections radiograph were taken immediately after surgery and at 1-year intervals and measured by two radiologists (1 and 10 years of experience respectfully) (fig.3) . we compared measurements with the wilcoxon signed-rank test. from january 2014 to october 2016, a total of 5 patients (4 females and 1 male), which diagnoses included mitochondrial encephalopathy syndrome (n=1), spina bifida (n=1), ataxia of unknown cause (n=1), juvenile idiopathic scoliosis (n=1) and trisomy 8 (n=1), with a mean of 10 distractions per patient (standard deviation [sd] ±1,2), were recruited. fifty distractions for each system (95 measurements in total) were performed, targeting different lengths of distraction (from -2.0 mm to +4.6 mm) on each occasion. a total of 21 sets of plain radiographs were taken. from these, 18 sets of data points were used for correlation analysis. the mean distracted length per year on plain radiographs was 10,1 mm (sd ± 3,8 mm) and the mean distracted length on us per 6-months interval was 4,5 mm (sd ± 2,9 mm). excellent correlation was observed between radiographic and ultrasound measurements. in particular, correlation between rx measurements and ultrasound was excellent both for junior (0.001<p<0.005, w=70) and senior radiologist (p<0.001, w=28,5). there were no rod's breaches identified neither in ultrasound or radiographs in this series. the measured changes in rod length between the two imaging modalities were highly correlated. thus, ultrasonography is at least as accurate as radiographs in measuring changes in rod length. our geometric measurement method adds reproducibility to an effective, non-ionizing and lowcost procedure for monitoring distraction of mcgr in children population. refuting child abuse denialistshealing rickets is clearly different from child abuse metaphyseal fracture a.e. oestreich; cincinnati/us objective: child abuse denialists, who testify against the diagnosis of child abuse, often claim that radiographic findings generally recognized by pediatric radiologists as likely due to abusive trauma are instead a consequence of rickets, including healing rickets. in particular, bucket handle transverse bony density at metaphyses are declared by some denialists to be identical to the pattern of healing rickets. i demonstrate by reviewing the details of these different patterns that this is an incorrect declaration. additionally, concave distal ulna margins alone are not a sign of rickets. rickets is radiologically distinct from classic metaphyseal fractures. i reviewed medicolegal documents and published articles by several well known physicians who claim that certain children suspected of having suffered child abuse instead demonstrate rachitic bone disease. their statements and images in support of their opinions are critically reviewed and contrasted with known examples of rickets, including healing rickets. at the physeal region of long bones in rickets, the zone of provisional calcification (zpc) is not calcified and the normal metaphyseal collar shaftward from that zone is not maintained. in metaphyseal fractures from abuse, the zpc and collar are maintained. in early healing rickets, the zone of provisional calcification calcifies first, giving a transverse calcification separated from the already ossified metaphysis. this calcification is different from a bucket handle fracture pattern without signs of rickets. moreover, concave distal margin of the ulna without other signs of rickets is seen in about 20% of normal infants. repeatedly, denialists have testified against the diagnosis of child abuse, claiming rickets in the absence of radiographic evidence -i critique one well known child abuse is a serious diagnosisclaiming by denialists of an alternate diagnosis, such as rickets, when radiographically no rickets is present, should be refuted. a clear understanding of the differences between abuse fractures and rickets is obligatory. when no rickets is seen at other growth sites, including seeing the presence of zones of provisional calcification, rickets is not present. simultaneous multi-slice turbo spin echo: a 2-fold time-saving alternative to conventional tse in mr imaging of the pediatric knee s. benali, a. gholipour, p.r. johnston, s.d. bixby; boston/us objective: simultaneous multi-slice (sms) is a novel iteration of parallel imaging in mri that reduces acquisition time at least two-fold over conventional turbo spin echo (tse) acquisitions. the aim of this study is to determine whether 2-dimensional (2d) sms tse can replace conventional 2d tse in imaging of the knee in pediatric patients without compromising diagnostic quality. this was an irb-approved retrospective study. subjects included all pediatric patients referred for routine 3-tesla mri of the knee between october through december 2016. mri examination consisted of institutional basic protocol with both traditional t2 tse and sms t2 tse in axial and sagittal planes with fat suppression. two anonymized study folders were created for each subject, one containing all basic sequences (basic exam), and one in which t2 tse sequences were substituted with t2 sms sequences (sms exam). two pediatric radiologists independently and blindly reviewed each study and noted presence or absence of 15 pre-determined abnormalities (table 1) . discrepancies between radiologists were settled by consensus. qualitative assessment was performed using a likert scale to evaluate edge blurring, contrast resolution, image noise, and flow artifact. thirty-five subjects comprised the study population (mean age 14.6 years, range 8-20, 26 female, 9 male). diagnostic performance was assessed by comparing findings (table 1) from basic exam to sms exam based on absolute agreement and cohen's kappa statistic. for all findings, the proportion of absolute agreements between basic and sms exam was >0.96 for reader 1, > 0.88 for reader 2, and 1.00 for consensus between readers. kappas for consensus reads were 1.00 on all 15 structures (p< 0.001, lower 95% confidence limit >0.97). for reader 1, kappas were 1.00 for 14/15 structures (p< 0.001) and 0.00 for pcl. for reader 2, kappas were 1.00 for 14/15 structures (p< 0.001) and 0.45 for cartilage defects. paired t-test was used to compare mean likert scores for image quality characteristics. for both readers, sms was preferred for flow artifacts whereas tse was preferred for the three remaining image quality characteristics (p< 0.05). our primary assessment suggests that sms t2 tse is comparable to standard tse in terms of diagnostic performance in the evaluation of the pediatric knee despite modest decrease in overall image quality. the 2-fold decreased acquisition time of sms is a significant advantage which is felt to offset the mild decrease in image quality, particularly as it increases the likelihood that children will tolerate the examination without motion. mri for sacroiliitis in children: panel findings and inter-observer evaluation using standardised reporting k.e. orr 1 , m.j. bramham 1 , s. andronikou 2 ; 1 plymouth/uk, 2 bristol/uk there is little evidence regarding mri for diagnosing sacroiliitis in children with juvenile idiopathic arthritis (jia). the limited literature presents varied opinions but no published recommendations for standardisation of reporting. axial disease in jia responds poorly to conventional first-line treatments but identifying these children using history and examination findings is unreliable. standardised mri reporting (2017) 47 (suppl 2):s297-s pediatr radiol may improve diagnosis and selection of patients in whom newer biologic treatments are indicated. the aim was to use a standardised reporting proforma based on published definitions for recording mri findings in suspected sacroiliitis to evaluate inter-observer agreement and determine the reliability of findings according to specific sequences. ninety-nine sacroiliac joint mris (198 joints) were included, 80 were initial examinations and 19 were follow-up mris. the age range was between 6.6 and 20.3 years (mean age 15.4 years). three readers retrospectively reported all 99 mris using the standardised proforma. 'reader 1' was the study group panel while readers 2 and 3 were specialist paediatric radiology consultants working in the united kingdom. readers were blinded to additional clinical information and other imaging. inter-reader variation was evaluated for the presence of bone marrow oedema, erosions, effusions, ankylosis, sclerosis and enhancement, as well as the presence or absence of sacroiliitis. the quality of mri examinations was evaluated, including presence and adequacy of sequences performed and alignment of the coronal/oblique studies. mri findings were correlated with clinical details and final diagnosis. there is significant variability in sacroiliac joint mri protocols. refinement of these to include only necessary sequences based on inter-reader reliability and reinforcement of good positioning will improve reporting and result in universal standardisation. there is inconsistency in current reporting practice of sacroiliac joint mri in children but increasingly, clinicians rely on imaging to select patients with sacroiliitis and guide appropriate treatment. using a standardised reporting proforma may improve the quality and consistency of reporting. ultrasound-guided steroid tendon sheath injections in juvenile idiopathic arthritis s. peters, d.a. parra; toronto/ca objective: juvenile idiopathic arthritis (jia) is the most common chronic rheumatic disease in childhood. tenosynovitis is one of the manifestations of jia, which can explain the absence of response to treatment when adjacent joints are injected. steroid injection is one of the treatment options for tenosynovitis and it has been shown to be effective in the literature. utilizing ultrasound (us) guidance for injections into tendon sheaths has shown clinical advantage to conventional blind injections in the adult rheumatoid arthritis population. the aims of this study are to: (a) identify tendon sheaths most commonly treated in our patient population with jia referred for steroid injections; (b) describe technical aspects of the procedure; (c) characterize sonographic appearance of tenosynovitis in jia; (d) assess agreement between clinical request and sites injected. this was a 10 year single-center retrospective study ( may 2006 -april 2016 in which we recruited patients with jia referred by rheumatology for us-guided tendon sheath injections. we collected patient demographics, clinical assessment information, sonographic appearance of the tendons and technical aspects of the intervention from the procedure records. we collected data from 308 visits of 244 patients (75% female, mean age 9 years 8 months) with a total of 926 injections. the ankle region was most commonly injected (85%), specifically the tendon sheaths of tibialis posterior (22%), peroneus longus (20%) and brevis (20%). 63% of the procedures were performed under general anesthesia and triamcinolone hexacetonide was used in 97% of the injections. an "out of plane" approach was used in 86% of the interventions and the 15 mhz "hockey stick" us probe was preferred for guidance (86%). we found 2 minor intra-procedure complications without sequelae. the majority of treated sites (92%) showed peritendinous fluid and sheath thickening on us. other findings were increased color-doppler signal and echogenic peritendinous fluid. a strong agreement between clinical request and sites injected was observed and most patients required one visit (78%). us-guided tendon sheath injections are used frequently to treat patients with jia. it is a safe intervention with a high technical success rate. the ankle region, specifically the medial compartment, is the area most commonly injected in this cohort of patients. the most common sonographic finding is peritendinous fluid and sheath thickening. these findings might assist radiologists and rheumatologists to characterize and more effectively manage tenosynovitis in patients with jia. to evaluate the accuracy of the software for automatic bone age (ba) estimation based on deep learning technique, and to validate the feasibility of this system in clinical practice. the software for automatic ba estimation was developed based on deep learning technique using 18,940 left hand radiographs and estimated ba of each radiograph based on greulich-pyle method. ba estimation was done for left hand radiographs of 100 consecutive patients (9 months -17 years; 42 boys and 58 girls) in three methods: (1) ai bone age (assessed by the software), (2) ai-assisted ba (assessed by two radiologists with the assistance of the software), (3) gp atlas-assisted ba (assessed by two radiologists with only gp atlas but the software). the reference ba was determined by two radiologists by consensus. the accuracy of the estimated ba by each method was assessed using concordance rate (%), pearson's correlation analysis, the root mean square error (rmse), and bland-altman plot. reading time for ba estimation by each method was evaluated. ai bone age showed 61% of concordance rate, and a significant correlation with reference ba (r2=0.986, p<0.05). the bland-altman plot of agreement between the reference ba and ai bone age showed the mean difference of -0.20 years (95% limit of agreement, ±1.22 years). rmse was 0.42 years. in reviewer 1, concordance rates were same between both gp atlasassisted ba and ai-assisted ba (72%), and rmse of ai-assisted ba (0.20) was slightly lower than that of gp atlas-assisted ba s353 (2017) 47 (suppl 2):s297-s pediatr radiol (0.23). in reviewer 2, concordance rate was slightly higher in aiassisted ba (60%) than gp atlas-assisted ba (58%), and rmse was almost the same (0.54 in ai-assisted ba, 0.55 in gp atlasassisted ba). the reading time was reduced 20.0% in reviewer 1 and 62.7% in reviewer 2. the software for automatic ba estimation based on deep learning technique showed high accuracy and may enhance work efficiency in ba estimation by allowing radiologists to save reading time and to improve accuracy. temporomandibular joint mri findings in adolescents with primary disk displacement in comparison to those in juvenile idiopathic arthritis j. bucheli, d. ettlin, c. kellenberger; zurich/ch to investigate potential differences of morphology and degree of inflammation in temporomandibular joints (tmjs) affected by primary anterior disk displacement (add) and juvenile idiopathic arthritis (jia). in 18 adolescents (15 female, age 15 ± 2 y), contrast enhanced magnetic resonance images (fig. a) of tmjs with add were retrospectively compared to those of age-and gender-matched controls with jia. morphology of articular disk and bony structures were described. osseous deformity and inflammation were qualitatively scored with progressive 4-grade scales and compared between groups with mann-whitney-u test. mandibular ramus length, measured on gradient echo minimum intensity projection images (fig. b) , was compared between groups and to normal values with independent samples t-test. in the add-group, 31/36 disks were dislocated anteriorly and showed thickening of the posterior band (27/31). in contrast, tmj disks of jia patients were mainly flattened (n=23) and/or centrally perforated (n=12) and rarely dislocated (n=1). tmjs with add showed similar overall grades of inflammation (p=0.39) and osseous deformation (p=0.53) as tmjs in the jia group. while erosions were frequent in both groups (add 25/31; jia 32/36, p=0.55), the mandibular condyle (p<0.001) and glenoid fossa (p<0.001) were less flattened in tmjs with add. in add tmjs, bone marrow oedema was less frequent (p=0.001) and grades of joint enhancement slightly lower (p=0.03), but presence of synovial thickening (p=0.43) and degree of effusion (p=0.87) were not significantly different between groups. mandibular ramus length was not significantly different (p=0.72) between groups, but in both groups clearly decreased compared to mean normal values (p<0.0001). articular disks in tmjs affected by jia are rarely dislocated. surprisingly, tmjs with primary add show considerable inflammatory change including condylar erosions. still, chronic systemic inflammation in jia joints results in considerable higher deformity of the mandibular condyle and the temporal joint surface. observation of the mostly preserved normal shape of the temporal bone may help differentiating primary add from jia. retrospective magnetic resonance imaging (mri) study of 49 consecutive jia patients (35 female, median age 14 y) with at least two consecutive tmj mri examinations ≥ 2y apart and no csi. degree of tmj inflammation was determined on t2-weighted and contrast-enhanced t1weighted fast spin echo images (fig. a) , and degree of osseous deformity on gradient echo images (fig. b) by progressive 4-grade scales (0-3). change of respective grades was assessed with wilcoxon test. mandibular growth was determined by ramus length change and compared to normal values. over a median period of 3.4 y (interquartile range, 2.4 -4.6 y), degree of tmj inflammation improved (p<0.001) with decrease in frequency of grade 3 (4.1% to 0%) and grade 2 (19.4% to 4.1%). inflammatory grades improved both in patients with (n=39, p=0.007) and without (n=10, p=0.02) systemic disease modifying medication. the degree of osseous deformation slightly improved (p=0.04), with decrease in frequency of grade 3 (5.1% to 3.1%) and grade 2 (9.2% to 6.1%), and increase of grade 0 (48% to 54.1%). overall growth rates of mandibular ramus (median, 1.3 mm/y) were not significantly different from normal growth rates (p=0.27) (fig. c) . growth rates of tmjs from patients only receiving non-steroidal anti-inflammatory drugs (median, 1.25mm/y) were not significantly different (p=0.9) compared to patients treated with systemic disease modifying drugs (median, 1.35mm/y). in patients with systemic treatment of jia, both the degree of tmj inflammation and osseous deformity as seen on mri improved at midterm follow-up. normal growth of the mandibular ramus was maintained. these results are in contrast to those from an earlier cohort treated with csi, in which on average deformities deteriorated and growth was impaired. objective: pediatric ileocolic intussusception, ici, is a common abdominal condition for which pediatric radiologists are asked to attempt emergency pneumatic reduction. because of the high success and low complication rates of pneumatic reductions, radiologists are able to make several attempts at reduction in stable patients if the initial enema attempt is unsuccessful. we have observed patients with successful reductions with rather long periods between initial symptoms of ici and performance of the air enema. we hypothesize that successful pneumatic reduction rates are independent of length of symptoms and in stable patients, repeated reduction attempts can be performed with the expectation of successful reduction. we performed an irb-approved retrospective review of all ici with a pneumatic reduction attempt between 2008-2016 at xxx. clinical, imaging and surgical data was reviewed. time to enema was defined as the time from first symptom to first air enema attempt. linear and second order polynomial statistical analysis was performed to assess the relationship between time to enema and enema outcome. results: 66 ici were identified in 61 patients. air enema was successful in 46 ici, 77%. the mean time to enema was 37.5 hours, range 4-168 hours with sd of 42.2 hours for successfully reduced ici and 35.1 hours, range 2-336 hours with sd of 53.9 hours for unsuccessfully reduced ici. surgical resection was required in 4 patients with ischemic bowel including one with an irreducible meckel's diverticulum as lead point. there was no correlation between time to enema and successful reduction, fig 1. no patient with a successful pneumatic reduction of a ici required subsequent bowel resection. conclusions: air enema for ici can be safely performed despite prolonged time to enema with the anticipation of a successful reduction. the lack of correlation of pneumatic reducibility and time to enema suggests that in surgically cleared patients with ici, the pneumatic reduction attempt may not be a true emergency and that repeated attempts at reduction are safe. additionally, though our numbers are small, they suggest that an ici is reducible or not from the beginning and do not "become irreducible" with prolongation of the time to enema. evaluation of splenic stiffness measurements for the diagnosis and the follow-up of portal stenosis after paediatric liver transplantation c. escalard 1 , a. dabadie 2 , s. chapeliere 1 , d. pariente 1 , c. adamsbaum 1 , s. franchi 1 ; 1 le kremlin-bicêtre, paris/fr, 2 la timone, marseille/fr to report our preliminary findings about the role of splenic and hepatic supersonic shear-wave elastography (sswe) in the diagnosis and followup after treatment of portal stenosis in paediatric liver graft recipients. all paediatric liver recipients with portal stenosis treated by the interventional radiology procedure, and who underwent splenic and hepatic sswe pre and post interventional procedures, were retrospectively reviewed. demographics, data about the portal stenosis (delay post transplantation, clinical presentation, initial radiological findings, hemoglobin and platelet counts), ir procedure performed, clinical and ultrasonographic follow-up and spleen stiffness pre and post ir procedure were collected. four patients were included, median age 6,5 years (range 0,9 months to 8 years) and median delay post transplantation 3,9 years (range 1 month to 4.5 years). two patients presented with anemia, associated in one case with progressive splenomegaly. one patient had liver test abnormalities, and one had decreased portal flow found on systematic doppler followup. spleen stiffness was elevated pre-procedure in all 4 patients, from 36 to 65 kpa (normal <20 kpa), and liver stiffness was normal or mildly elevated in all. portal stenosis was successfully treated by ir in 3 patients. spleen stiffness decreased rapidly, ranging from 38 to 53% (figure 1) . however, the size of the spleen remained unchanged. in the last patient, angioplasty of the portal stenosis failed leading to portal thrombosis. spleen stiffness increased on the subsequent ultrasound ( figure 2 ). mr elastography (mre) is a novel imaging technique that provides a non-invasive evaluation of liver fibrosis. the standard sequence used for this purpose on a siemens scanner has been gradient echo (gre). we also implemented echo planar imaging (epi) available as a work-in-progress (wip). our aim is to compare the liver elastogram values between gre and epi in children. after consent from both research and referred clinical subjects, a dedicated mre of the liver was performed on a 3t mr scanner (magnetom® skyra, siemens) with a pediatric mechanical driver over the right upper quadrant. an axial t2 blade with fat saturation, coronal t1 vibe dixon and axial diffusion weighted imaging (dwi) were obtained. elastograms were obtained using both standard gre and epi, in the axial plane. for the gre sequence, 5 different slices were selected and each scanned sequentially. the epi sequence incorporated 5 different slices in just one series. images were post-processed placing regions-of-interest (roi) and measuring the stiffness in kilopascals (kpa). for each sequence and each slice the mean stiffness and then the average of the means was calculated. a spleen elastogram was simultaneously generated, without changing the mechanical driver location, and the mean stiffness was also calculated. increased stiffness was defined as >2.9 kpa in the liver and >3.6 kpa in the spleen. we focused on a technical comparison between the sequences without clinical or histological correlation of findings. we included 15 subjects that had elastogram measurements of liver and 11 of them spleen stiffness on both gre and epi sequences. mean liver stiffness on gre was 2.4 (sd+/-0.71) and on epi was 2.8 (sd+/-1.04), with a pearson's correlation of r= 0.92 (p<0.001). increased liver stiffness was found in 4/15 (26.6%) of the cases in gre and 9/15 (60%) of the cases in epi. mean spleen stiffness on gre was 3.9 (sd+/-1.39) and on epi was 4.8 (sd +/-1.45) with a pearson's correlation of r=0.69 (p= 0.01). epi reported consistently higher values than gre in both liver and spleen stiffness. our preliminary data shows a moderate to high correlation between gre and epi sequences; however, the epi values were higher in both liver and spleen. in the future, larger studies are needed to validate these thresholds and patterns among different sequences. were also reviewed if done. patient's medical & surgical treatment, and clinical progress were also reviewed. active telephone follow-up 3 days after cevus was performed. results: 120 patients giving a total of 240 pelviureteric units were referred for vus study during the study period, with age ranging from 1 month to 7 years old. no contrast-related complication was encountered. except 2 cases with failed catheterization, 108 were investigations of urinary tract infection (uti), antenatal hydronephrosis and congenital anomalies etc., and remaining 10 were follow up studies of known reflux. of all cases of uti, 36 refluxing units were picked up by vus, ranging from grade i to v. of the 36 refluxing units diagnosed by cevus, 17 were missed on mcu, among which 12 were high grade refluxes (grade iii to v) requiring treatment; whereas cevus only missed one grade i refluxing unit detected by mcu. besides, one grade iv refluxing unit identified on vus was under graded by mcu to grade i. regarding patient outcomes, one patient with mcu-missed refluxing unit presented with breakthrough uti on follow up. two refluxing units that were missed on mcu but detected on cevus demonstrated scarring on dmsa. conclusion: cevus is shown to be more sensitive in detecting vesicoureteric reflux than mcu. the fact that mcu-missed refluxes detected by cevus were associated with breakthrough urinary tract infection and scarring on dmsa indicated that the extra sensitivity brought by cevus did translate to clinical significance. difficulty in visualizing low-grade reflux is a potential limitation of this technique. with favourable diagnostic performance and safety profile, cevus can be further applied in this community in the era of radiation reduction. percutaneous transbiliary needle or forceps biopsy in hepatic masses with biliary dilatation a. dabadie 1 , s. franchi 2 , d. pariente 2 ; 1 la timone, marseille/fr, 2 le kremlin-bicêtre, paris/fr hepatic masses with biliary dilatation are rare in children and mainly include rhabdomyosarcoma of the biliary ducts, but also other masses or pseudo-masses compressing the hepatic hilum. in these patients histological diagnosis of the lesion as well as temporary biliary drainage are warranted. the objective of this study is to report our experience in percutaneous transbiliary biopsy performed simultaneously and using the same access as the percutaneous biliary drainage in children with hepatic mass obstructing the biliary ducts. children presenting with a hepatic mass causing biliary obstruction, with need for biliary drainage, were considered candidates for percutaneous transbiliary biopsy of the lesion performed at the same time. the biopsy was performed under ultrasound guidance, through a sheath introduced in the dilated biliary system, using a semi-automatic 16 gauge needle or the transluminal biliary biopsy forceps set (cook medical, bloomington, usa). between 2009 and 2016, four patients were included, three females and one male, median age 5.5 years (range 2.5-11.5). all presented with jaundice and were diagnosed with a hepatic mass with secondary biliary obstruction. percutaneous transbiliary biopsy was performed in all 4 patients using the 16 gauge needle. in one patient, the biopsy did not demonstrate any tumoral cells and a second biopsy was performed using the forceps device through the same biliary access. the samples deemed adequate for analysis by the pathology department in all patients, however the samples were larger when using the needle. a retrospective -prospective study included 62 patients of both sexes (13,72 +/-3,17 y), in a two-year span. patients were divided into two groups according to the used diagnostic method (positivegroup a on us and a1 on mri, with intestine mural thickness above 3mm, and negativegroup b on us and b1 on mri, with mural thickness below 3mm). overall sensitivity and specificity of us and mri in diagnosing ibd was calculated in comparison to pathohistological (ph) findings. us examination showed an average intestinal mural thickness of 4.93 ±1.39mm and 2.7+0.18mm in group a (28 patients) and group b (34 patients) respectively. mri examination showed an average intestinal mural thickness of 6.50±1.45mm and 2,8+0,16mm in group a1 (27 patients) and group b1 (35 patients) respectively. out of 28 patients from group a, 15 (52%) had irregular mural architecture, contrary to group b in which mural architecture irregularities have not been observed. in groups a1 and b1 14 (51.9%) and 2 (5.7%) patients had irregular mural architecture respectively. average length of affected intestinal segment on us and mri was 103mm and 105mm respectively. five patients from group a and four from group a1 had signs of fibrosis. color doppler showed hyperemia in 17 and 22 patients of group a and a1 respectively. transmural signs of inflammation were found in 59% of patients on us, and 61.3% of patients on mri. average longer diameter of mesentery lymph nodes measured by us and mri was 13.29 ±3.74mm and 12.7±5.68mm, respectively. overall sensitivity of us and mri was 88.4% and 92.31% respectively. both us and mri showed a specificity of 88%. us and mri are reliable and compatible methods in diagnosing ibd, with mri being slightly more accurate. us is an extremely valuable and widely available imaging modality in every-day clinical work, both in diagnosing and follow-up of therapy effects in children with ibd. findings in percutaneous transhepatic cholecysto-cholangiography in neonates and young infants presenting with conjugated hyperbilirubinemia d.a. parra, s. peters, j. amaral; toronto/ca objective: conjugated hyperbilirubinemia is a concerning finding in neonates and young infants, biliary atresia (ba) being one of the main diagnostic considerations. ba is a rare disease characterized by fibrosis of the biliary tree. the obliteration of the biliary system leads to cholestasis and ultimately liver parenchymal injury, cirrhosis and death. an early diagnosis of ba along with a kasai portoenterostomy operation significantly improves the long-term prognosis. percutaneous transhepatic cholecysto-cholangiography (ptcc) is one of the options described in the diagnostic algorithm of ba. the aims of this study are to: (a) describe ptcc findings in patients with conjugated hyperbilirubinemia; (b) identify the abnormal patterns encountered that justify further investigations; (c) analyze technical aspects of the procedure. this is a 16 year single-center retrospective study (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) in which we recruited patients with the diagnosis of cholestasis (less than 6 months old) referred for ptcc. we collected patient demographics, clinical information, findings in ptcc, post-procedure management and long term clinical outcome. eigthy-nine patients were referred for ptcc in the study period. the procedure was technically feasible and successfully performed in 73 patients (68% male, mean age 2.2 months). forty-one had a pre-procedure hida scan suggestive of ba. fifty-nine patients had an ultrasound-guided biopsy in conjunction with the ptcc and in all of them the cholangiography was performed through a needle placed using ultrasound guidance in the gallbladder. 53% (39) of the patients had a normal ptcc. abnormal patterns encountered were: 1) variable degrees of hypoplastic bile ducts seen in 25%; 2) atretic gallbladder without demonstration of communication with bile ducts seen in 18%; and 3) gallbladder communication with a cystic structure not communicated with the biliary ducts (cystic biliary atresia) seen in 4%. the most common diagnosis in the abnormal group was ba (71%). alagille's syndrome, alpha-1 antitrypsin deficiency and progressive familial intrahepatic cholestasis were other diagnoses in this group. no complications related to the procedure were observed. ptcc is a safe and effective option in the diagnostic algorithm of patients presenting with cholestasis early in life. visualization of the gallbladder is fundamental to perform the procedure. the majority of studies were normal in our patient population preventing further invasive investigations. three types of abnormal ptcc patters were encountered, with ba being the most common diagnosis in this group of patients. to evaluate the additive role of shear wave elastography in the sonographic distinction of biliary atresia from other causes of neonatal/ infantile cholestatic liver disease. neonates and infants with clinical and biochemical diagnosis of cholestatic jaundice were enrolled in our study after obtaining informed written consent from the parents. grey scale, doppler and shear wave elastographic findings were recorded after 4 hours of fasting using aixplorer® ultrasound system (supersonic imagine, aix en provence, france). sedation was not needed during the study. for obtaining elastographic values, linear transducer (4-15hz) was used and after image stabilization a q-box measuring 3mm was placed in the most homogenous vessel free area. the mean of three elastographic values were recorded. hida scan, liver biopsy, intra-operative cholangiogram and histopathological evaluation of resected specimens was done wherever feasible and clinically indicated. the prospectively obtained elastographic values were retrospectively evaluated. eleven of 25 patients included in our study were proven to be biliary atresia (ba) by intra operative cholangiogram and histopathological reports. the diagnosis in the remaining 14 patients included other causes of infantile cholestatic jaundice like infantile choledochal cyst, neonatal idiopathic hepatitis, progressive familial intrahepatic cholestasis, abernathy malformation, cmv hepatitis etc. the elastographic values of ba and non-ba patients were compared. six of 25 infants were younger than 60 days which included four patients with ba and their elastographic values (18.75±2.9kpa) were significantly different from that of non-biliary atresia (7±1kpa) in the same age group (p value <0.05). similarly, for patients aged >60 days also we had a significant difference (p value <0.05) in elastographic stiffness between ba (45.7±11kpa; n=7) and non-ba (19.4±3.6kpa; n=12) groups. the mean echogenic area anterior to right portal vein (earpv) was 4.45 ±0.84mm in ba and 1.47±0.36mm in non-ba group (p value <0.05). the mean gall bladder (gb) length was 1.82±0.26 mm in biliary atresia group in contrast to 3.2±0.26mm in the rest (p value <0.05). the roc plot for earpv and gb length gave a youden index cut off value of >2.7mm (sensitivity 72.7 & specificity 71.4%) and <3.32 cm (sensitivity 100 & specificity 42.9%) respectively. infants with biliary atresia have a significantly higher elastographic value when compared to age matched patients with other causes of neonatal cholestasis. we expect to validate the findings in our ongoing study with a larger sample size. to retrospectively define in a large pediatric population the association between testicular microlithiasis and testicular neoplasia. retrospective multicenter study of scrotal ultrasounds performed between january 2000 and april 2014 in subjects <18 years of age. all unique subject scrotal ultrasound reports from each institution were reviewed for mention of microlithiasis. for subjects with serial exams, the most recent exam performed was included in the analysis. all exams mentioning microlithiasis were reviewed by site-specific investigators to confirm the presence of ≥5 punctate calcifications in the testicle on a single image. the presence of testicular germ cell and stromal tumors were determined for subjects with and without microlithiasis through review of institutional pathology and imaging databases. the risk of testicular neoplasia in the context of microlithiasis was expressed in terms of odds ratios with (a-or) and without adjustment (u-or) for fixed study site (institution) effects by logistic regression. the study population included 37,863 unique subjects with confirmed microlithiasis in 1,097 (2.9%). mean subject age was 11.1±4.7 years for subjects with microlithiasis and 9.1±5.9 years for subjects without (p<0.0001). one hundred thirty-nine subjects (0. this large, multicenter study confirms that there is a significant, strong association between testicular microlithiasis and testicular neoplasia, particularly malignant germ cell tumors. children with microlithiasis have approximately 22x greater odds of having a malignant germ cell tumor than children without microlithiasis. this reinforces the need for a large prospective study assessing the risk of developing testicular neoplasia in children with incidentally identified diffuse microlithiasis. do adc-values reflect renal function or obstruction in children with uretero-pelvic-junction obstruction? p. grehten, a.c. eichenberger, c. kellenberger; zurich/ch the use of diffusion weighted imaging (dwi) in renal mri is increasing. in adults as well as in infants a positive linear correlation between adcvalues and glomerular filtration rate has been demonstrated. the aim of our study was to assess whether renal dwi can provide information on the grade of urinary tract obstruction or renal function in children with uretero-pelvic-junction (upj)-obstruction. retrospective analysis of 19 children (age 3.1+/-4.5y) with unilateral upj-obstruction who underwent pre-and postoperative mri at 1.5t and 6 normal controls (age 6.6+/-4.0y). functional mr-urography and multiple b-value dwi were part of the mr-protocol. renal adc-values were correlated to measures of obstruction and function, and compared between obstructed and non-obstructed kidneys and between pre-and postoperative studies. no correlation was found between mean parenchymal, cortical or medullary adc-values and calyceal transit time (ctt), renal transit time (rtt) and measures of differential renal function (%parenchymal s359 (2017) 47 (suppl 2):s297-s pediatr radiol volume, vdrf, pdrf). there was moderate correlation with absolute parenchymal volume and total kidney volume, and low correlation with pelvic volume. adc-values showed high correlation with age and patient's weight. adc-values normalized for age or weight showed low correlation with rtt and ctt, but no correlation with functional measures. adc-values were not significantly different between obstructed and contralateral normal kidneys (p=0.2-0.9) or between pre-and postoperative studies (p=0.3-1). renal adc is dependent on age and weight in young children and does not correlate with differential renal function. for assessing urinary tract obstruction with adc normative values need to be established. to determine the level of knowledge and awareness of medical staff, medical students and parents concerning possible risks associated with ionizing radiation. a prospective study has been conducted at children's hospital, center for adult's radiology, and medical faculty, by filling out two anonymous questionnaires (questionnaire 1medical staff and medical students, questionnaire 2parents of the children exposed to x-ray based procedures), and it included 254 participants. statistical analysis was performed using the spss 21.0. the majority of examinees assessed their knowledge about ionizing radiation as moderate. knowledge level was statistically significantly higher only in the group of medical students who passed the course of radiology, in comparison to the group of those who have not attended the course yet. only 45% of radiologists and up to 37.5% of pediatricians, pediatric surgeons and anesthesiologists are informed about "image gently" campaign. up to 80% of radiologists, and up to 22% of clinicians, both specialists and residents, are aware of alara principle. over 60% of medical doctors think that diagnostic radiology procedures are very often performed unnecessarily among children, while only 12.5% of parents share this opinion. most of the radiologists and clinicians consider it necessary to inform parents about potentially harmful effects of ionizing radiation, but even though 60-80% of clinicians claim they do inform parents in every-day clinical practice, over 70% of parents affirm that they had never been informed about effects of ionizing radiation before diagnostic procedures were performed on their children. only 26% of pediatric surgeons and pediatricians, but 72.7% of radiologist and 60% of anesthesiologists are concerned that informing parents about ionizing radiation would cause problems in every-day work. nearly 71% of parents claimed that they would not refuse to expose their child to x-ray based diagnostic procedure, after the given information about potential harmful effects. over 70% of radiologists and less than 50% of pediatric surgeons and pediatricians support the initiative to calculate the total effective dose child was exposed to during hospitalization, and place it on the discharge list. between 50% and 85% of pediatricians and pediatric surgeons greatly underestimated the effective doses in ct and fluoroscopy procedures. there are 58-100% of clinicians who are aware that ct increases the risk of carcinoma development. this study showed that general knowledge about ionizing radiation, potential risks and effective doses in pediatric population is poor, and that organized education is required. fluoroscopy in pediatric radiology -how important is an individual impact to radiation exposure of children? j. lovrenski, i. varga; novi sad/rs to determine whether there are differences between different pediatric radiologists and radiology residents in exposure of pediatric population to ionizing radiation during fluoroscopy procedures. a retrospective study has been conducted at the regional children's hospital, and included all the diagnostic fluoroscopy examinations performed within a one-year period. the fluoroscopic data along with the names of pediatric radiologists/radiology residents performing these examinations were retrieved from the evidentiary notebooks, and included: dose-area product (dap), skin dose, and fluoroscopy time. there were 4 radiologists (r1-r4), and 4 radiology residents (r5-r8) involved in fluoroscopic examinations. we found all the fluoroscopic findings in the hospital's data base, which enabled a differentiation between positive and negative findings. statistical analysis was performed using the spss 21.0. a p-value less than 0.05 was considered statistically significant. a total of 191 fluoroscopy procedures in children (mean age 4,5y, 107 males and 84 females) have been performed within a one-year period, most of which were voiding cystourethrograms (vcug) -93, and an upper gastrointestinal (gi) series -79 examinations. radiology residents and radiologists carried out 82 and 109 examinations respectively. duration of fluoroscopy procedures performed by residents (av. 32.5s) was statistically significantly shorter in comparison with duration of fluoroscopy examinations performed by radiologists (av. 53s). dap and skin dose did not show statistically significant difference between these two groups, as well as the number of positive and negative fluoroscopic findings in groups of examinations performed by radiologists and radiology residents. mean dap value ranged from 0.82μgym 2 (r2) to 7.5μgym 2 (r7) when performing vcugs, and from 1.35μgym 2 (r2) to 4.79μgym 2 (r1) for upper gi series. mean skin dose ranged from 14.33mgy (r2) to 151.48mgy (r3) for vcugs, and from 28.66mgy (r2) to 120.54mgy (r4) for upper gi series. mean fluoroscopy time ranged from 11.76s (r2) to 48.5s (r3) for vcug, and from 26.22s (r2) to 125.75s (r3) for upper gi series. statistically significant difference was shown only between radiologists r2 and r3 for dap and skin dose values in performing vcug, and for fluoroscopy time in performing an upper gi series. for all examinations dap and skin dose were statistically significantly higher in the group of positive fluoroscopic findings. this study has shown that exposure of children to ionizing radiation during fluoroscopy procedures significantly depends on radiologist/ radiology resident and the nature of fluoroscopic finding. to evaluate image quality and radiation exposure of non-contrast pediatric chest ct with automated tube voltage selection (atvs), in combination with automated tube current modulation (atcm). non-contrast chest ct scans of 160 children (91 male and 69 female; mean age, 8.7 ± 5.4 years) were analysed retrospectively with regard to radiation exposure and image quality before and after the implementation of an automated tube voltage selection. correlations of volume ct dose index (ctdi vol ) and the effective diameter (edm), before and after the implementation of atvs were compared, and confidence intervals related to the change in correlations with and without atvs were determined using fisher's z-transformation. image quality was assessed by mean signal-difference-tonoise ratios (snrs) in the aorta and in the left principal bronchus with the independent samples t-test. subjective image quality was rated by two pediatric radiologists and a general radiologist on a 10point scale. agreement between the readers was assessed using weighted kappa coefficients. a p<0.05 were considered significant. automated tube voltage selection, in combination with an automated tube current modulation, resulted in optimization of scan protocols, homogeneity of image quality, and reduction of radiation exposure for pediatric patients. advantages and disadvantages of cone beam ct for pediatric interventions l. dance, r.b. towbin, d. aria, c. schaefer, r. kaye; phoenix/us objective: illustrate the advantages and disadvantages of cone beam ct (cbct) as an alternative to conventional ct guidance and an adjunct to angiography. there is a steep learning curve to optimize utilization of cbct. we found that cbct reliably identifies high-contrast lesions. however, the lower dose and decreased penetration of cbct resulted in poorer visualization of low-contrast lesions. also cbct can be degraded by streak artifact from hardware or dense contrast. the relatively narrow field of view can be restrictive for peripherally located lesions in larger patients. however, the anatomic display is adequate for guidance in most instances. these findings are illustrated in a series of cbct-guided cases including pulmonary nodule localization, osteoid osteoma ablation, abc sclerotherapy, renal av fistula embolization, and liver lesion biopsy. the advent of cbct as an adjunct modality in the ir suite has significantly decreased the use of conventional ct guidance and significantly decreased the radiation dose in children. we have found cbct to be a practice changer. the aim of this study is to review our local drl in pediatric fluoroscopy and to compare them to values proposed by pidrl guidelines and recent international litterature. data were prospectively collected on consecutive procedures (750 total) performed from january 2016 to december 2016 on 2 different fluoroscopy units (siemens iconos r200, luminos drf). of each procedure patients data (name, weight and birth date), examination-data (kind of procedure, date, dap [cgy*cm2], total fluoroscopy time, number of images) were recorded. data from micturating-cystourethrography(mcu), barium meal/swallow(bs) and most commonly performed procedures were divided into 4 weight-groups (<10kg,10-15kg,15-30kg,30-60kg) and of each one 75th-percentile was calculated. data were compared to europeandrl and recent literature data (by age:newborn,1-,5-,10years old). weight-groups are considered a representative sample if at least 20-patients per procedure-type and per patient-group are included. our local-drl for mcu are 7(<10kg), 10(10-15kg), 24(15-30kg) and 57(30-60kg). they results to be lower than pidrls values (30, 70, 80, 75) but higher if compared to a previous local survey of 2014 (4, 9, 18, 29) . bs data are 9(<10kg), 32(10-15kg), 28(15-30kg); these data are lower than that of a previous local survey of 2014 (23, 34, 68) . the update of local-drl is helpful in daily practice to identify (and solve) critical issues such as incorrect technique or poor practice with new flat-panel equipment. pidrl guidelines: a review of local drl for pediatric head, thorax and abdomen ct in a italian referral center a. magistrelli, v. cannatà, e. genovese, m. cirillo, r. lombardi, p. toma; rome/it the aim of this study is to review our local drl in pediatric ct and to compare them to values proposed by pidrl guidelines and recent international litterature. data were prospectively collected on consecutive procedures (347 total) performed from january 2016 to june 2016 on a somatom definition flash siemens. of each procedure patients data (name, weight and birth date), examination-data (kind of procedure, clincal question, date, ctdivol16/32 and dlp16/32) were recorded. ctdivo/dlp16 from head ct were divided into 4 age-groups (<4weeks,4weeks-1y,1-6y,≥6y) and of each one 75th-percentile was calculated. ctdivol/dlp32 from thorax (chest, cardiovascular ct angiography) and abdomen+pelvis ct examination were divided into 5 weightgroups (<10kg,10-15kg,15-30kg,30-60kg,>60kg) and of each one 75th-percentile was calculated. data were compared to europeandrl and recent literature data. weight-groups are considered a representative sample if at least 20patients per procedure-type and per patient-group are included. our local drl are substantially lower than that proposed by pidrl guidelines. specifically ctdivol/dlp32 for chest ct are 1/22(<5kg), 1,52/42(5-15kg), 1,83/56(15-30kg), 2,99/113(30-60kg), 6,07/ 239(>60kg) respectively. for cardiovascular ct angiography are 0,71/ 15(<5kg), 1,01/21(5-15kg), 2,44/36(15-30kg), 2,87/90(30-60kg), 13,72/311(>60kg). while for abdomen+pelvis ct are 1,9/47(<5kg), 2,68/83(5-15kg), 3,25/131(15-30kg), 7,77/320(30-60kg), 11,21/ 532(>60kg). data for trunk sere not collected. for head ct local drl are higher in age-group 0 and 1 but lower in other age-group if compared to routine head ct pidrl ones. the update of local-drl allowed us to identify (and solve) some critical issues such as incorrect technique. drl-curve in optimization of pediatric body ct r. seuri 1 , p. laarne 2 , a. nikkola-sihto 3 , k. nygaard bolstad 4 , m.s. perhomaa 5 , a. thilander klang 6 , k. rosendahl 4 , j. ruohonen 3 , e. tyrvainen 7 ; 1 helsinki/fi, 2 tampere/fi, 3 seinäjoki/fi, 4 bergen/no, 5 oulu/fi, 6 gothenburg/se, 7 kuopio/fi objective: diagnostic reference levels (drls) in medical imaging represent valuable tools to study dose optimization in clinical practice. this is particularly important in pediatric computed tomography (ct) as the number of the examinations in many institutions is low. drls are typically given as a percentile point, usually as 75% or 3 rd quartile of the observed distribution of patient dose. in pediatric practice drls are often given for each age-or weight group separately. we present continuous drl-curve as a feasible way to compare dose levels in pediatric body ct. during 2016-2017 a selected group of nordic hospitals collected dose values (ct dose index by volume, ctdi vol , and dose-length product, dlp) from pediatric body ct examinations on children aged 5-16 years. the dose values were imported into a dynamic excel table, previously established by the radiation and nuclear safety authority in finland, stuk (fig 1) . the stuk-table includes a graphic presentation of a continuous drl-curve presented as a function of body weight, and the program automatically calculates a dose curve and compares it to the established reference level (fig 2) . the dose values were easily exported to the excel tables, and the graphic presentation and comparison with an established drl-curve was clear and readily understandable for both radiologists and radiographers. in some of the institutions included in the present study, the weight of the patient was not recorded routinely. this represents a challenge for the use of the drl-curves provided by stuk. the drl-curves provided by stuk were feasible for clinical practice. the automatic calculation of the dose curve and graphic presentation were helpful to interpret the results. the drl-curve also allows relevant comparison even with a smaller number of patients. fifty randomly selected ct chest studies performed over 10 years to assess diffuse lung disease were included in the study sample (25 females, 25 males; mean age 9.9 years + 6.6 years), comprising 9 disorders. two pediatric radiologists and a pediatric radiology fellow blinded to the results of the cts evaluated four subsets of complete chest cts (3 slices, every third slice, every other slice, and all images below the thyroid) and compared the subsets with the entire chest ct, interpreted as the control. accuracy of evaluating the primary diagnosis and determination if significant diagnoses were missed in the reduced slice ct subsets were rendered. we assume linear distribution of dose across the anatomy to estimate dose reduction on reduced slice subsets. most significant findings were present on all reduced slice ct subsets. all relevant findings were present in 100% of subthyroid, 96% of every other slice, 86% of every 3 rd slice, and 50% of 3 regional slice subsets respectively. excluded findings included small foci of ground glass opacity, consolidation, focal mosaic attenuation, and linear parenchymal bands; peribronchial thickening, dextrocardia vs dextropositioning, tree-in-bud opacities, extent of mild bronchiectasis. with the exception of consolidation in 1 of the studies, these findings were not thought to inhibit diagnostic assessment. the underlying diagnosis was correctly identified in most of the subsets: 100% subthyroid and every other slice, 90% every 3 rd slice, and 80% of 3 regional slice subsets. dose is significantly decreased by using any of these methods. while some findings are excluded with increasing gaps between slices, equivalent diagnostic information can be provided on reduced slice ct and can serve as a viable strategy to reduce lifetime radiation dose to children and young adults with diffuse lung disease imaged for routine follow-up. as findings are missed with larger gaps, this strategy should be used with caution in patients presenting with acute symptoms 3. to extrapolate the significance of early diagnosis which will compliment to treatment planning and management. case presentation: types a and b niemann-pick disease are lysosomal storage disorders that result from deficient acid sphingomyelinase activity and lead to the accumulation of sphingomyelin, primarily in tissues of the reticuloendothelial system. type b niemann-pick disease manifestations are hepatosplenomegaly, excess bleeding and bruising, growth retardation, and recurrent respiratory infections. features of hrct include thickened peribronchovascular and interlobular septal thickening, ground-glass opacities. the intermixed regions could be characterized as showing crazy paving, although this is not the predominant pattern. type b niemann-pick disease should be added to the list of clinical entities that can demonstrate crazy paving. our patient is a sevenyear old girl, presented with dry cough and fever. physical examination revealed hepato splenomegaly. radiological work up included abdominal ultrasound examination, which showed mild hepatosplenomegaly. chest radiography revealed diffuse reticulonodular infiltration in both lungs. chest hrct was done for more comprehensive evaluation which showed multilobar bilateral peribronchovascular interstitial thickening and interlobular septal thickening with ground-glass opacities and crazy paving appearance. no honeycomb pattern was seen. no sizable pulmonary nodule or sizable mediastinal lymphadenopathy was seen. no pleural effusion was seen. finding were indicating extensive pulmonary intestitial disease. a corroborative analysis along with lab tests and genetic studies revealed the diagnosis of type b niemann pick disease. 2. the spectra of hrct features including crazy paving pattern may be encountered; though not frequent. hence should be included in the differential diagnosis of crazy paving pattern. blast from the past: lemierre's syndrome in adolescents with sore throat o. kvist; stockholm/se a minor ailment such as a sore throat could prove to be a severe disorder known as lemierre's syndrome. this syndrome mostly affects previously healthy adolescents and young adults and in its classical form should meet four diagnostic criteria; primary infection of the oropharynx, septicemia, clinical-or radiographic evidence of thrombosis of the internal jugular vein (ijv) plus secondary metastatic abscesses. the infection is caused by fusobacterium necrophorum, a species of obligate anaerobe bacteria forming part of the normal human flora. the syndrome should be suspected in any patient with pharyngitis, cervicalgia and pulmonary symptoms. the incidence of lemierre's syndrome decreased dramatically after the introduction of antibiotics but has, of unknown reasons, increased over the past 15 years. we will present four patients diagnosed with lemierre's syndrome in our department during the last 8 years. the purpose of this case report is to raise awareness of this "forgotten disease". of the four patients diagnosed with lemierre's syndrome two fulfilled all 4 criteria while two fulfilled 3 out of 4. (table 1 ). the first two presented at the emergency department with one week's history of a sore throat, left sided cervical lymphadenopathy, erythematous tonsils, leukocytosis and elevated crp. in both cases the clinical condition deteriorated and they were referred to the icu. one developed ards and required initiation of ecmo. in both patients, chest ct revealed multiple pulmonary consolidations with cavitations, findings consistent with septic emboli (image 1,2 and 3). incidentally ct-neck revealed thrombosis in the left ejvand ijv (image 4). ultrasound of the neck veins confirmed the finding (image 5 and 6). blood cultures taken on admission later proved positive to f. necrophorum. the third and fourth case, with similar clinical histories but with a less aggressive development, had positive blood cultures but no thrombosis and vice versa. (table 1 and all four patients recovered and could be discharged with oral antibiotics and anticoagulants. unique teaching points: in conclusion, lemierre's syndrome is less common today thanks to antibiotics but may still occur in previously healthy adolescents and may lead to a fatal outcome. the pediatric radiologist should be aware of typical findings like septic emboli in the lungs and thrombosis in the ijv. unicameral bone cyst associated with secondary aneurysmal bone cyst of clavicle i. dasic, g.j. djuricic, s. ducic; belgrade/rs objective: aneurysmal bone cyst (abc) accounts for 2,5 % of all bone tumors. they are benign but locally destructive lesion of the bone characterized by presence of spongy or multiloculated cystic tissue filled with blood. abcs are metaphyseal, excentric, bulging, fluid-filled and multicameral, and may develop in all bones of the skeleton. most common locations include the proximal humerus, distal femur, proximal tibia, and spine. clavicle is a very rare site for aneurysmal bone cyst with only few cases reported in literature. a 10-year-old boy reported to the university children's hospital for detailed examination of swelling of right shoulder. 2-3 days before admission parents noticed tumefaction of right shoulder. there was no history of trauma or fever. physical examination revealed tumefaction of the right shoulder, in projection of acromial end of clavicle, measuring approximately 8x8cm, which was tender and fixed. the swelling was not hot to the touch, and there was no skin discoloration over that area. regional lymph nodes were not palpable. (fig. 1a) x-ray revealed osteolytic, expansible lesion in the lateral end of clavicle and there was no pathological fracture. (fig. 1b) laboratory analyzes were within normal limits. blood cultures remained sterile. chest x ray and abdominal ultrasound were normal. computed tomography (ct) revealed a thinwalled multiloculate lesion in lateral end of right clavicle. (fig. 2a) there was no extension in the soft tissues on magnetic resonance imaging (mri). mri shows the multiloculate cavities and fluid levels. (fig. 2b) . the open biopsy was done. histopathological examination confirmed the secondary aneurysmal bone cyst on the field of simple bone cyst of clavicle. the clavicle is an uncommon site for bone tumors. review of literature shows clavicle accounts for less than 1 % of all bone tumors. the patient with an aneurysmal bone cyst generally presents with pain and swelling, which may vary in duration from weeks to several years. up to 8 % of bone tumors occur in less than 20 years of age with peak incidence in second decade. radiologically, lesion is lytic and may have a soap-bubble appearance with ballooned distension of the periosteum. the differential diagnosis for aneurysmal bone cyst include giant cell tumor, chondromyxoid fibroma and telangiectatic osteosarcoma. distinction from telangiectatic osteosarcoma is difficult because the conditions have overlapping clinical and radiologic features. the differentiation is made from the histologic features. imaging of glomus tumor of liver in a child (case report) n. tewattanarat, j. srinakarind, j. wongwiwatchai, p. komvilaisak, s. areemit, p. ungarereevittaya, p. intarawichian; khonkaen/th objective: glomus tumors occur preferentially in subcutaneous tissue of fingers and toes, but extremely rare in visceral organs. most cases of the tumors are diagnosed in adults. several cases of glomus tumors in liver have been reported in adults. a literature review, no case of glomus tumor in liver in children was published. therefore, we present clinical, imaging findings of the first case of pediatric patient with glomus tumor in liver and also histopathological features. a previously healthy 11 year-old-girl was admitted with a twoweek history of progressive dyspnea on exertion and vomiting. family history was unremarkable. physical examination revealed hypertension and smooth and firm mass at epigastrium. systolic apical murmur on heart examination was noted. liver function test (2017) 47 (suppl 2):s297-s pediatr radiol showed elevated cholesterol (396 mg/dl). other laboratory tests (complete blood count, blood chemistry, renal and liver function test, coagulation test, hepatitis profiles and alpha-fetoprotein) were within normal limits. echocardiogram found mitral and tricuspid regurgitation and poor left ventricular systolic dysfunction. abdominal mri demonstrated a 12-cm well-defined exophytic hypervascular mass with intratumoral hemorrhage at segment 3/4b of the liver. there were no other suspicious lesions in other organs. the biopsy was done and revealed glomus tumor. patient underwent preoperative embolization and the liver mass revealed decreased size to 8-cm after 1-month follow up with ultrasound. after that, exploratory laparotomy with left lateral segmentectomy was performed. the pathological results showed dilated vascular channels surrounded by uniform neoplastic cells, uniform with round nuclei, fine chromatin, inconspicuous nucleoli, and pale eosinophilic cytoplasm, and well-defined cytoplasmic border. no mitotic figures and necrosis are identified. immunohistochemical (ihc) staining of tumor was positive for cd34, smooth muscle actin (sma) and h-caldesmon. others ihc including ae1/ae3, heppar1, cd31, desmin and myogenin were negative. from these findings, the tumor was finally diagnosed as glomus tumor of uncertain malignant potential due to deep location and large size. primary glomus tumor is a rare entity of liver tumor diagnosed in children. however, it should be considered in the differential diagnosis of a hypervascular liver mass. most of these tumors are benign, however tumor in liver have malignant potential due to deep seated position. therefore, tumor removal with pre-operative embolization should be considered. brain mri in a pediatric patient with linear scleroderma en coup de sabre m. mortilla, a. rosati, e. canale, c. filippi; florence/it objective: linear scleroderma "en coup de sabre" (ecds) is a rare subset of localized scleroderma. affected individuals typically have a characteristic atrophic skin lesion involving the fronto-parietal scalp. the disease usually has a benign course but rare neurologic symptoms can be seen associated: the most common described is epilepsy. intracranial mri findings described in the literature include: focal brain atrophy, calcifications and t2-hyperintense white matter lesions that may demonstrate contrast enhancement. white matter lesions and calcifications are found in the cerebral hemisphere ipsilateral to the skin abnormality. in the literature only a few pediatric cases have been described. a 4yrs. old girl was hospitalized at our institution for evaluation of a lesion of the frontal skin associated to a history of febrile seizures and mri alterations. she presented febrile seizures at the age of 2 on april 2013. on january 2014 parents noted a frontal cutaneous lesion that was defined as "linear scleroderma, port-wine stain type". on november 2014 she performed an mri at another institution showing a diffuse white matter alteration in the left emisphere with focal lesions with high susceptibility and mild contrast enhancement. she was addressed to immunosuppresive therapy with steroids and methotrexate, with steroids stopped after 6 months. a clinical cutaneous improvement was noted. on july 2016 a second mri showed a worsening of the findings. we describe a case of a little girl with ecds with no neurologic deficits or symptoms that shows extensive and progressive neuroradiologic alterations. only a few pediatric cases have been described, but it has to be known that also in absence of symptoms, patients with linear scleroderma should be screened with mri to look for cns involvement in this immune disease. brain mri can also be used to monitor the progression of the disease and the response to therapy. mals is a vascular compression syndrome which symptoms can overlap chronic functional abdominal pain. in mals the proximal part of the celiac artery is compressed by the too low located median arcuate ligament during expiration resulting in hemodynamically significant symptoms. we report two cases with mals diagnosed primarily by ultrasonography. case 1 18-year-old girl was admitted to tartu university children's clinic (tucc) due to recurrent acute epigastric pain episodes with nausea and loss of appetite during 7 years. previous analyses were normal, abdominal uss and gastroscopy did not show any abnormalities. she was referred to paediatric radiology department for doppler us (dus) which showed narrowed proximal celiac artery (ca) with turbulent flow, increased peak-systolic and end-diastolic velocities on deep inspiration and expiration, and positive ca deflexion angle on expiration. superior mesenteric artery (sma) was markedly widened, indicating possible collateral blood-supply due to severe ca stenosis. according us findings mals was suspected. abdominal mra showed proximal ca kinking, stenosis and poststenotic dilatation and confirmed diagnosis. during dsa collateral blood-supply from sma via pancreaticoduodenale arcade (pda) was seen. laparoscopic release of mal resulted in relief of patient's symptoms, she has been pain-free for two years. case 2 16-year-old girl applied to tucc due to recurrent abdominal pain episodes for 2-3 years. usually, pain occurred 2-3 times per week about 15 minutes after the start of intense cycling training or competitions, and passed about 5 minutes resting in squat position. mild mid-epigastric bruit was audible at physical examination. dus showed two-fold increase in expiratory peak-systolic and enddiastolic blood flow velocities compared to inspiratory velocities which indicated to the hemodynamically significant worsening of ca compression by mal during expiration. mra showed proximal ca compression, upward angulation and poststenotic dilatation. preoperative ct-angiography depicted collateral supply via pda. during laparoscopic surgery ca was released by transecting mal and surrounding fibrous tissue. after surgery the girl has been pain-free for one year except single pain episode during intense competition. the diagnosis of median arcuate ligament syndrome should be considered in patients with postprandial abdominal pain that does not have other clearly established etiology. colour doppler us should be the first choice imaging method. to confirm diagnosis in pediatric patients abdominal mra is preferred in our institution, but as mra may still have a tendency to movement artifacts and inadequate spatial resolution for smaller blood vessels, in these two cases mra was followed by cta or dsa. understand the unique predilection of infantile malignancies to metastasize and present as skin-based masses, most commonly lymphoma/leukemia. case presentation: an otherwise healthy 37 day old male presented to dermatology with a pedunculated, friable red glabellar mass (centered between the eyes). first noticed as a flat, bluish lesion at 10 days, its subsequent rapid growth led to an emergency department visit where dermatology diagnosed a hemangioma and initiated propranolol treatment. despite this, the mass continued to grow rapidly, encroaching upon the patient's right eye. the patient was admitted for further workup. an elevated beta hcg, anemia (7.4 mg/dl), and thrombocytopenia (92,000) suggested an alternate diagnosis. an mri and ultrasound led to a percutaneous biopsy; pathology was consistent with choriocarcinoma. pet ct found fdg-avid glabellar, liver and lung lesions. maternal and placental testing was negative for choriocarcinoma. ultrasound demonstrates a hypoechoic hypervascular mass. mri brain demonstrates cutaneous confinement of the solid avidly enhancing glabellar mass. ct shows a peripherally enhancing liver mass with a masslike area of consolidation in the right lung. initial pet/ct demonstrated fdg avid liver and lung metastases with a small focus of residual activity at the glabella consistent with incomplete resection. follow-up pet/ct showed astoundingly rapid re-growth of the glabellar mass and enlargement of the hepatic and pulmonary masses just 12 days later demonstrating the extremely aggressive nature of this cancer. 3month follow-up pet/ct showed significantly decreased size and activity of the metastases consistent with a treatment response. in a series of 208 infants with cutaneous metastases, the following diseases presented with cutaneous involvement (ordered most to least common): leukemia, langerhans cell histiocytosis, neuroblastoma, rhabdoid tumor, rhabdomyosarcoma, primitive neuroectodermal tumor, choriocarcinoma, and adrenocortical carcinoma. pathology slides (2017) 47 (suppl 2):s297-s pediatr radiol unique teaching points: considered one of the fastest growing tumors, infantile choriocarcinoma classically presents with hepatomegaly, anemia, failure to thrive, and precocious puberty between 0 days and 5 months of life. left untreated, the disease is usually fatal within 3 weeks of presentation. a solitary cutaneous metastasis can be mistaken for infantile hemangioma both clinically and radiographically. atypical mri appearance is one important clue that can suggest an alternative diagnosis. pet/ct may be useful for staging and follow-up. a rare case of ovarian juvenile granulosa cell tumor associated with ollier's disease -generalised mesodermal dysplasia p. joshi; pune/in to demonstrate a rare case of mesodermal dysplasia -association of ovarian granulosa cell tumour with enchondromatosis case presentation: two year 8 month old girl presented with precocious puberty i.e thelarche. left hand radiograph showed the radiological age corresponding to chronological age, suggestive of peripheral precious puberty. the patient subsequently underwent a sonography which revealed a pelvic mass probably arising from the right ovary ? sex cord stromal tumour. a mri of the abdomen and pelvis confirmed the pelvic mass and revealed multiple bone lesions in the right hemipelvis -on the side of the tumour she was later operated. hpe of pelvis mass revealed juvenile granulosa cell tumour. ultrasound pelvis images reveal a solid pelvic mass, probably ovarian in etiology mri pelvis also reveals multiple bone lesions unique teaching points: the aim of the poster is to create awareness about this association. the bone lesions should not be mistaken for metastasis juvenile granulosa cell tumour of the ovary (jgct) is a well-known sexcord stromal ovarian neoplasm. ollier's disease is a rare, non hereditary mesosermal dysplasia consisting of multiple enchondromas. the association of granulosa call tumour with asymmetric ipsilateral hemiskeletal distribution may indicate generalised mesodermal dysplasia as there is also association of jgct with maffucci's syndrome, other dysplastic conditions such as microcephaly, facial asymmetry,' and potter's syndrome. review of literature showed 11 previous cases of juvenile granulosa cell tumor associated with enchondromatosis, three associated with maffucci's syndrome, and the rest with ollier's disease goldbloom's syndrome is a paediatric idiopathic disease characterized by transient bone marrow oedema with recurrent crisis of bone pain, periosteal hyperostosis, fever, increased inflammatory markers and dysproteinaemia. a case series of wbmr studies in goldbloom's syndrome is reported and differential diagnosis discussed. case presentation: a 9-year-old female girl was admitted to our paediatric department because of daily crisis of bone pain of the lower limbs, associated with fever spikes, limping and nocturnal awakenings. no history of trauma was reported. laboratory tests showed mild anaemia (hb 8.2 g/dl), thrombocytosis (plt 680000/mmc), increased inflammatory markers (ers 75 mm/h, crp 7 mg/dl), high streptolysine o and dnase-b antibody levels (aso 4280 iu/ml and adn-b 6310 ui/ml, respectively). throat swab was positive for group a β-haemolytic streptococcus (gas). unusual dysproteinaemia, characterized by hypoalbuminemia (2.8 g/dl) with increased a1, a2 and g globulinaemia, was noted. x-ray examinations of both legs resulted normal. wbmri showed markedly delineated, high and homogeneous hyper-hypointensity respectively in stir/t1 of the distal tibialperoneal meta-diaphysis of both legs (fig1a,b). distal metaphysis of femur, humerus, radius-ulna and proximal tibia were also homogeneously mildly hyperintense on stir sequences bilaterally (fig1a). bone biopsy revealed signs of chronic inflammation. infectious and neoplastic diseases were ruled out and the diagnosis of gs with dysproteinaemia seemed conceivable. steroid treatment was started in association with indomethacin, leading to a prompt resolution of the clinical picture within a few days. the follow-up stir total body mri, performed after 10 months, showed the complete resolution of bone oedema. (fig2 a,b) the sock sign is a pathognomonic whole-body magnetic resonance imaging (wbmri) feature of goldbloom's syndrome (gs).it is a well marked, symmetric, homogeneous and high bone marrow hyperintensity, localized both at the distal tibial and peroneal meta-diaphysis, which looks like a pair of socks. objective: left ventricle hypoplasia is generally thought as a part of hypoplastic left ventricle syndrome or aortic hypoplasia. it is estimated that about 15-20 ml/m2 left ventricle volume is needed in order to support systemic circulation. less than that volume generally precludes biventricular repair. however conditions associated with severe preload decrease such as total anomolous pulmonary venous return (tapvr) should be considered in the differential diagnosis. tapvr presenting as hypoplastic left ventricle syndrome is presented in this study. six month old female patient admitted to emergency service with symptoms of fever, dyspnea and coughing. emergency staff started intravenous antibiotic theraphy and from medical records learned that she has been followed for partial anomolous pulmonary venous return (papvr) and atrial septal defect (asd). lung x-rays revealed pulmonary edema. echocardiography was performed and revealed very small left ventricle, papvr and 13 mm wide asd. ecg gated cardiac ct was requested with the prediagnosis of hypoplastic left ventricle syndrome. ct images revealed dilated right cavities, very small left ventricle, pulmonary edema, tapvd and peritoneal fluid plus hepatomegaly. we then retrospectively searched our archive and found she was diagnosed as papvr when she was 10 days old. all the cavities that time, were normal sized. according to these we confirmed our diagnosis as tapvr and hypoplastic appearing cavities due to reduced preload and right chamber dilatation due pulmonary overcirculation. surgical team decided to perform corrective operation and they confirmed our diagnosis unique teaching points: small left ventricle cavity in an infant need not to be due to intrinsic hypoplasia. whenever we experience such a situtation we should search for other reasons of pseudohypoplasia in order to give a chance for corrective surgery instead of palliative procesures. we present a case report of kimura disease, a rare benign chronic inflammatory disease that involves the deep subcutaneous tissues and lymph nodes of the head and neck. we report the case of a thirteen year old male who presented with a right sided facial mass which had been present for two years but had enlarged rapidly in the preceding three months. us and mr were interpreted locally as an arteriovenous malformation. review of these examinations and catheter angiography performed at this quaternary referral centre favoured a vascular tumour. subsequent percutaneous biopsy demonstrated angiolymphoid hyperplasia with eosinophilia and blood tests showed a serum eosinophilia, consistent with kimura disease. us shows a mass consisting of scattered heterogenous foci within the fat with multiple large feeding vessels. contrast enhanced mri demonstrated a solid, homogenously enhancing, mass with multiple vascular flow voids from the right external carotid artery branches. catheter angiography showed tumour blood supply from branches of the right transverse facial artery and distal right ima. the dominant supply arose superficially from the transverse facial artery. kimura disease is a rare chronic inflammatory disorder of unknown aetiology that involves the deep subcutaneous tissues and lymph nodes of the head and neck region, most common in asian men in the third decade and sporadic in the non-asian population. the histopathological and biochemical characteristics are eosinophilic lymphfolliculoid granuloma, increased eosinophils in the peripheral blood and increased ige levels. whilst ultrasound and mri are effective imaging modalities, imaging alone does not allow confident differentiation from malignant lesions and biopsy is necessitated. kimura disease has a benign indolent course with an excellent prognosis following surgical excision although local recurrence has been reported. increased naa: is it surely canavan disease? e. varga, p. barsi, g. rudas; budapest/hu leukodystrophies are a group of rare genetic, metabolic diseases that affect the central nervous system, mainly the brain. each type of them is caused by a specific gene abnormality that leads to abnormal development or destruction of the white matter of the brain. the differential diagnosis are made on the basis of clinical and neuroradiological signs. there are some diseases which show typical changes on mr spectroscopy. we present a case of a 12 year-old boy, who has been investigated due to somatomental retardation and muscle dystrophy since his six months of age. his perinatal period was normal except of a nystagmus visible from his birth. the child has muscle dystrophy, spastic quadriparesis, contractures, scoliosis, truncal hypotonia and ataxia and mental retardation. we started examinations to find out the background pathology of his idiopathic encephalo-myopathy. the brain mri showed a bilateral, symmetrical white matter signal alteration, which referred to some kind of metabolic (2017) 47 (suppl 2):s297-s pediatr radiol disease. the mr spectroscopy revealed decreased cholin and increased naa levels, which are typical of canavan disease. despite of this, the clinical aspects and the location of the involved brain areas were more typical of pelizaeus-merzbacher disease (pmd). the pmd is a genetic disorder, which is originated of the mutation of the proteolipid protein gene (plp1) located on long arm of x-chromosome (xq21-22) . this gene has an impact on growth of the myelin sheath. various types of mutations (deletion, duplication, point mutation, insertion) of plp1 gene lead to various severity of clinical picture. all form of mutations show decreased naa level on spectroscopy, except the duplication of plp1 gene. in connection with our case, we present briefly the clinical and neuroradiological differences between the two entities. magnetic resonance imaging findings in medium-chain acyl-coenzyme a dehydrogenase (mcad) deficiency l. talamanca, d. narese, m.c. rossi espagnet, l. pasquini, d. longo; rome/it we report serial brain magnetic resonance (mri) in a patient with medium-chain acyl-coenzyme a dehydrogenase (mcad) deficiency who developed acute encephalopathy. a 12-months-old girl was admitted in the emergency department of our hospital with sudden onset of acute encephalopathy with drowsiness. baseline laboratory investigations revealed severe hypoglycemia, hyperammonemia, hyperchloremic metabolic acidosis and hyperuricemia. the patient was treated with glucose solution infusion that resulted in a gradual resolution of symptoms. the first brain mri, performed within 6 hours of onset of symptoms showed bilateral symmetric restricted diffusion on diffusion-weighted imaging (dwi) in the middle cerebellar peduncle, nucleus caudatus, putamen and periventricular white matter; the adc map showed reduced diffusivity (fig 1) . the second mri, at 72 hours after the onset, revealed bilateral and symmetric hyperintensity on t2-weighted images in the middle cerebellar peduncle, nucleus caudatus, putamen and periventricular white matter. dwi showed restricted diffusion in both globus pallidus (fig 2) . a single voxel h-mrs study performed by placing a roi in the right nucleus lenticularis revealed increased values of gaba and glutamine (fig 3) . a further mri was performed 4 weeks after the first neuroimaging and indicated widespread atrophy and the appearance of a hyperintense signal in t2-wi in both globus pallidus while dwi did not reveal any remarkable signal abnormality. single-voxel mrs of the same region showed a normalization of gaba and glutamine values. brain mri showed bilateral symmetric restricted diffusion on diffusionweighted imaging (dwi) in the middle cerebellar peduncle, nucleus caudatus, putamen and periventricular white matter; the adc map showed reduced diffusivity the second mri, at 72 hours after the onset, revealed bilateral symmetric restricted diffusion on diffusion-weighted imaging (dwi) in both globus pallidus. a single voxel h-mrs study performed by placing a roi in the right nucleus lenticularis revealed increased values of gaba and glutamine. mcad is an enzyme of the mitochondrial b-oxidation of fatty acids, an essential source of energy for cells during stress. mcad deficiency is the most common genetic disorder of fatty acid oxidation. the clinical manifestation of the disorder is typically precipitated by stress due to fasting, vomiting, fever or muscular exertion and occurs in the majority of cases before the age of 2 with the onset of acute hypoketotic hypoglycemia. clinical features of this decompensated state include seizures and lethargy proceeding to coma and death in the absence of prompt treatment with intravenous dextrose infusion. mcad deficiency usually appears in an acute form and has high morbidity and mortality rates; early diagnosis is therefore extremely important in order to promptly begin treatment and obtain a complete recovery from symptoms. mr can play a significant role in the early diagnosis of the decompensated state of the disease; in our case dwi revealed the presence of lesions with a bilateral symmetric topographic distribution that strongly suggested a metabolic disease leading to acute encephalopathy. a full-term male neonate (3 days old) with external perineal anomalies was referred to our hospital. the physical perineal examination revealed a bifid scrotum containing palpable testis and a normal configured penis located at the bottom of the bifid scrotum. two soft masses of 3 and 2 cm respectively, divided from a cutaneous notch, were located below the bifid scrotum and on the right of the midline. the rear biggest mass was normal epithelized, instead the other one was a rugged pigmented mass, which resembled the scrotum (figure 1 ). there were no additional abnormalities of the external genitalia. the other peduncolar mass, located between the right scrotum and the posterior mass, had fluid content. a mild hydrocele in the right scrotum and a sliding testis on the left side were also revealed. us examination showed a hyperechoic solid tissue, corresponding to the rear biggest perineal mass. the other peduncolar mass, located between the right scrotum and the posterior mass, had fluid content (figure 2) . a mild hydrocele in the right scrotum and a sliding testis on the left side were also revealed. mri also confirmed two perineal peduncolar masses: the biggest and posterior one, was made up by homogeneous fatty matter without contrast-enhancement after intravenous gadolinium injection (figure 3 ). the patient underwent excision of perineal masses and no complications occurred in the surgery. the histopathological examination of the perineal masses revealed two areas with different histological features: the first one was characterized by the presence of smooth muscle bundles dispersed in the dermal collagen, instead the other contiguous area showed abundant mature adipose tissue in the deep dermis and hypodermis ( figure 5 ). at last the rugged swelling mass was definitively diagnosed as as without testis tissue inside, and the rear mass was diagnosed as lipoma. the physical perineal examination revealed a bifid scrotum containing palpable testis. two soft masses of 3 and 2 cm respectively, divided from a cutaneous notch, were located below the bifid scrotum. us examination showed a hyperechoic solid tissue, corresponding to the rear biggest perineal mass. the other peduncolar mass, located between the right scrotum and the posterior mass, had fluid content mri confirmed the presence of two perineal peduncolar masses: the biggest and posterior one, was made up by homogeneous fatty matter without contrast-enhancement after intravenous gadolinium injection. neonates presenting with perineal masses are uncommon. these anomalies can occur isolated or more rarely in combination with other abnormalities such as uro-genital or ano-rectal anomalies or with contiguous subcutaneous tumors. when perineal masses are found, with prenatal diagnosis or during a newborn physical examination, it is important to look for any associated congenital anomalies or subcutaneous tumors by using imaging. to describe and emphasize the significance of the "half-moon" sign in pelvic mri. a 13-year-old adolescent, karate athlete, was submitted with left hip pain, decreased range of movement and asymmetry in thigh circumference. markers for infection or inflammation were negative. frog-leg radiograph was negative for hip effusion, slipped epiphysis and equivocal for a left trochanteric abnormality. mri demonstrated a half-moon pattern of bone marrow edema at the left intertrochanteric area and at the major trochanter, surrounding an apophyseal low-intensity lesion. ap radiograph and limited ct confirmed the presence of a lytic lesion with sclerotic margins, containing calcified chondroid matrix. chondroblastoma was histologically confirmed following excision. mri, coronal stir sequence, demonstrates semilunar-shaped hyperintense area abutting the growth plate and the cortex of the femoral neck, consistent with the half-moon sign. note edema surrounding an apophyseal low-intensity lesion and soft-tissue edema. ct confirms a typical apophyseal lesion with sclerotic margins containing chondroid matrix. unique teaching points: "half-moon" sign refers to a semilunar shape of bone marrow edema at the intertrochanteric area of the hip with its base located at the cortex of the femoral neck. this distribution differs from the distribution of edema in metaphyseal and metaphyseal-equivalent osteomyelitis. "half-moon" sign has been described in patients with stress fractures and osteoid osteomas. to our knowledge, this is the first case of chondroblastoma exhibiting this sign. whenever the "half-moon" pattern of edema is identified at pelvic mri scans, a thorough search for an occult fracture line or a nidus corresponding to an osteoid osteoma or a chondroblastoma is mandatory. mr elastography (mre) is a noninvasive imaging technique that quantitatively measures liver stiffness and provides an estimate of the degree of fibrosis. our aim was to evaluate the feasibility of performing mre using both gradient echo (gre) and echo planar (epi) sequences on siemens scanners. a dedicated mre of the liver was performed on a 3t mr scanner (magnetom® skyra, siemens) with a pediatric mechanical (2017) 47 (suppl 2):s297-s pediatr radiol driver (courtesy of mayo clinic) over the right upper quadrant. an axial t2 blade with fat saturation, a coronal t1 vibe dixon and axial diffusion weighted imaging (dwi) were obtained. elastograms were obtained using both an axial standard gre and a works in-progress (wip) epi sequence. for the gre sequence, 5 different slices were selected and each scanned sequentially. the epi sequence incorporated 5 different slices in just one series. images were post-processed placing regions-of-interest (roi) and measuring the stiffness in kilopascals (kpa). for each sequence and each slice the stiffness mean was measured and then the average of the means was obtained. a spleen elastogram was simultaneously generated, without changing the mechanical driver location, and mean stiffness was also calculated. based on cutoffs in the literature, values were considered abnormal if liver stiffness >2.9 kpa and spleen stiffness >3.6 kpa. our initial experience shows that mre is feasible on siemens scanners using both gre and epi sequences. epi sequences are a promising addition to standard gre. prone versus supine ultrasound positioning for evaluation of urinary tract dilation (utd) in children c. maya 1 , y. gorfu 2 , e. dunn 1 , k. darge 1 , s. back 1 ; 1 philadelphia/us, 2 addis ababa/et objective: ultrasound (us) is used in the initial evaluation and surveillance of utd in children. utd classification systems, including the 2014 multidisciplinary consensus, assess anterior-posterior renal pelvic diameter (aprpd) and calyceal dilation. there is currently no consensus regarding optimal patient positioning-prone versus supine-during us assessment of utd. this study was performed to determine if there is a significant difference in the measurement of the aprpd, presence of calyceal dilation, or resulting utd consensus score obtained between supine and prone positions. two raters retrospectively reviewed renal bladder ultrasounds of patients with utd of one or both kidneys. technically adequate ultrasound examinations of orthotopic kidneys that were imaged in both supine and prone positions were included. those with renal anomalies or prior surgery were excluded. aprpd measurements, as well as central and peripheral calyceal dilation, were documented in both prone and supine positions. a postnatal utd consensus score was assigned to each kidney based only on these features. 146 kidneys (77 left) in 89 subjects had utd in either the supine or prone position. mean age was 0.41 years (range: 0.01 -1.97 y). female to male ratio was 1:3 (21/68). the interclass correlation (icc) of the aprpd between raters was 0.88 and 0.87 in the supine and prone positions respectively (ps<0.001). central calyceal dilation was found in 97/146 supine kidneys and 102/146 prone kidneys by rater 1 and 94/146 supine and 99/146 prone kidneys by rater 2 (kappa 0.92). peripheral calyceal dilation was found in 54/146 supine kidneys and 65/146 prone kidneys by rater 1 and 50/146 supine kidneys and 62/146 prone kidneys by rater 2 (kappa 0.89). as such the results are presented as one. the aprpd tended to be greater when prone with a strong correlation between prone and supine measurements (0.92, p<0.001). the mean difference between supine and prone aprpd was 1.1 mm (p< 2.2). in 15 kidneys, calyceal dilation was seen in the prone position and not supine while 1 kidney had central calyceal dilation only when supine. the utd score differed between supine and prone in 13/149 kidneys, with all but one higher when prone. in 10 other kidneys, the aprpd differed between positions however concurrent calyceal dilation resulted in no change in utd class. as a screening tool, performing ultrasounds in the prone position may help identify more kidneys with utd. further research is needed to determine if these differences are clinically significant. during the evaluation of magnetic resonance enterography (mre), diffusion restriction (dr) has been utilized as a marker for bowel inflammation, but in our practice we commonly see dr in otherwise normal segments of jejunum. the purpose of this article is to assess the dr in normal loops of jejunum on mre and to determine if there is a correlation between dr and luminal distention, age, magnet field strength, and bowel segment location. a retrospective analysis of subjects with a normal mre and normal clinical work up (based on available clinical history, endoscopy reports, serum white blood cell count and inflammatory markers, and stool samples) was performed. the abdomen was divided into 4 quadrants. if available, 2 loops of jejunum were randomly chosen in each quadrant. two radiologists independently evaluated these same loops of jejunum for the following: luminal distension, wall thickness, and enhancement pattern. additionally, the loops were then evaluated for the presence or absence of dr. inter-rater reliability was determined. disagreement was resolved by consensus. presence or absence of dr was correlated with luminal distension, age, magnet field strength (1.5 versus 3 tesla), and abdominal quadrant. one hundred ninety-seven loops of jejunum were evaluated in 39 patients. not all subjects had jejunal loops in all quadrants. sixteen subjects (41%) had jejunal loops with dr for a total of 29 loops. one loop had increased wall thickness and another increased enhancement but both did not demonstrate dr. no other loops demonstrate increased enhancement or wall thickening. for the presence or absence of dr, inter-rater reliability was fair (kappa=0.39). there was no correlation between the presence/ absence of dr in relation to luminal distension, age, magnet field strength, or quadrant location. of the 16 subjects who had a single loop with dr, a 2 nd loop with dr was found in 50%. 14 year old who presented with nausea. mr enterography demonstrates no bowel thickening or abnormal enhancement. a. coronal haste demonstrates the craniocaudal position of the axial diffusion sequence for reference (line). 14 year old who presented with nausea. mr enterography demonstrates no bowel thickening or abnormal enhancement. b. axial diffusion weighted seqeunce (b=800) shows diffusion restriction within loops of jejunum (arrow) within the anterior abdomen. 14 year old who presented with nausea. mr enterography demonstrates no bowel thickening or abnormal enhancement. c. corresponding adc map demonstrates low signal within the jejunal wall consistent with diffusion restriction (arrow). diffusion restriction in normal loops of jejunum on mre was present in 41% of patients. if dr is seen in an otherwise normal segment of jejunum, this can be considered non-pathologic. a patient with a loop of jejunum with dr is likely to have an additional loop of jejunum demonstrating dr. there is no correlation with dr of normal jejunum with luminal distension, magnet field strength, or patient age. our data may help reduce overestimation of disease burden when clinically applied. imaging findings in the newborn with meconium peritonitis that require surgery p. caro dominguez 1 , a. zani 2 , a. daneman 2 ; 1 cordoba/es, 2 toronto/ca objective: meconium peritonitis is a rare condition caused by an in-utero bowel perforation resulting in spillage of meconium into the peritoneal cavity and subsequent calcification. the role of prenatal and postnatal imaging is to identify infants who require surgery. the aim of this study was to evaluate the role of postnatal imaging in meconium peritonitis and to correlate the radiologic and sonographic patterns with the need for surgery. imaging studies in infants with meconium peritonitis performed between 1999 and 2014 at our institution were reviewed separately by a pediatric radiologist, a pediatric radiology fellow and a pediatric surgeon. patients were divided in a surgical and a non-surgical group. clinical, surgical and pathology reports were reviewed to validate the diagnosis. statistical analysis: comparisons between sonographic and radiographic findings and patterns in the surgical and non-surgical groups were performed using unpaired t-test and chi-square. during the study period, there were 37 infants with meconium peritonitis managed at our institution. in the 23 (62%) who needed surgery, the most frequent surgical findings were idiopathic perforation, jejunal and ileal atresia. ultrasound identified more cases with hepatic calcifications, meconium pseudocyst, ascites and pneumoperitoneum than radiography and radiography more cases of small bowel obstruction. ascites identified with ultrasound (p=0.01) [fig 1] and bowel obstruction [fig 2] diagnosed either with ultrasound (p=0.04) or radiography (p=0.01) were associated with the need for surgical intervention. one third of children with meconium pseudocysts (4/12) [fig 3] , did not require surgery. diffuse peritoneal or hepatic calcifications as an isolated postnatal finding were not associated with the need for surgery. both radiography and ultrasonography give valuable information to the surgeon to take the decision for surgery. dilatation of bowel loops and ascites detected postnatally with radiography and/or ultrasound require surgical intervention in children with meconium peritonitis. interestingly, a large proportion of infants with meconium peritonitis can be managed conservatively. 0.17 -17.4) . those included had complete fmru analysis, dti (b=0 and b=400, 20 directions), and upjo configuration in at least 1 kidney. cases with motion artifact (n=9), post-pyeloplasty (n=3) or duplex collecting systems (n=3) were excluded. pelvicalyceal dilation grade (pcd), corticomedullary differentiation (cmd), and functional parameters were included. pyeloplasty following fmru was recorded. dti tractography was reconstructed using a fractional anisotropy (fa) and an angle threshold of 0.10 and 55°, respectively (figure 1) . user-defined regions-of-interest (roi) of the renal parenchyma, excluding the collecting system, were drawn to quantify dti parameters: mean fa, apparent diffusion coefficient (adc), tract length and tract volume. the relationships between dti quantitative parameters and fmru parameters were analyzed. age and adc (roi) (p<0.01, r 2 =0.36), tract volume (p<0.01, r 2 =0.77) and tract length (p<0.01, r 2 =0.60) were positively correlated. age and fa (roi) (p<0.01, r 2 =0.46) were negatively correlated. there was a correlation between fmru parenchymal volume and tract volume (p<0.01, r 2 =0.80), but median volumes were higher on dti (tractography=98.5 cm 3 vs. fmru=60.52 cm 3 ; p<0.01). of the 16 children, 12 had pyeloplasty, 1 had nephrectomy, 2 were managed conservatively and 1 was lost to follow-up. fa was significantly lower in kidneys that went on to have pyeloplasty in comparison to those without pyeloplasty, but the 95%ci and the iqr overlapped (table 1) . the adc, tract length and tract volume were similar between these groups (table 1) . there was no difference between the adc of fa values in kidneys with and without pcd or cmd (p>0.11). linear hierarchical regressions controlling the age did not show a significant relation between adc and cortical or renal transit times (p>0.68), but lower fa values were related to a higher renal transit time (p<0.01, r 2 =0.103). table 1 . quantitative dti parameters between kidneys with and without pyeloplasty following fmru. renal adc, fa, tract volume and tract length change with age but tractography overestimates renal parenchymal volume. there was a tendency towards a lower fa in kidneys that went on to pyeloplasty. otherwise, none of the quantitative parameters evaluated in this study differentiated degrees of upjo. echo-enhanced voiding urosonography (eevus) has become an important imaging tool in urodiagnostics; however, it has been observed that during eevus the premature destruction of ultrasound contrast agent microbubbles might occur. the purpose of this study was to evaluate the possible causes of contrast vanishing during investigations and propose the protocol to avoid false negative results. eevus was performed in 163 children from april to december 2016. sonovue mixed with saline solution in a plastic bottle is applied by continuous flow through the urine catheter. the collected data according to the protocol in this prospective study was completed in 105 children, aged from 2 weeks to 10.25 years. the protocol included general patient information, indication for eevus, duration of eevus in minutes, and the presence of vesicoureteric reflux. extensive data about sonovue were recorded: charge number, expiration date, time since opening, amount of initially administered contrast (ml sonovue/ml saline solution), grading of the initial contrast opacification of the bladder, the need for immediate readministration of contrast (dose), grading of contrast opacification during examination, and the need for readministration of contrast later in the course of the examination (dose). in addition, the data regarding bladder (ratio real/predicted bladder volume, wall thickness, ureter dilatation), saline solution, the size of urine catheter (french), and the type of antibiotic prophylaxis were collected. child observation included grading of crying and muscle stiffness. normal contrast opacification of urinary tract during examination was found in 87/105 children, while in 18/105 (17.2%) the contrast opacification was insufficient. in 12/18 (72.2%) microbubble destruction occurred during the first minute, in 4 (22.2%) in 5 minutes, and in 1 in 13 minutes after the beginning of contrast administration. the reason for unsatisfactory contrast opacification at the beginning of the eevus is probably due to small urine catheter size (25% of children with fr6 catheter had insufficient opacification compared to 13.3% with fr8 in whole cohort), time since the contrast is opened (more than 3 hours in 4 children), and insufficient bladder emptying at the beginning of the procedure. the reason for microbubble destruction later in the course of the examination is bladder overfilling in combination with increased muscle stiffness and strong crying, which led to increased bladder pressure. there was no correlation between the type of antibiotics and microbubble destruction. we should be aware of possible false negative vur results during eevus caused by premature microbubble destruction. patients with fontan-type palliation of univentricular congenital heart disease have elevated central venous pressure due to their passive pulmonary flow. the altered circulation has a negative impact on several visceral organs, and these patients have chronic liver congestion. they are at risk of developing hepatic fibrosis and cirrhosis with potential malignant transformation. these changes can occur from only a few years after fontan palliation, making early detection and grading of major importance. the patchy pattern of hepatic changes makes liver biopsy an unreliable diagnostic tool. magnetic resonance imaging (mri) t1 mapping has been suggested as a technique for non-invasive assessment and quantification of hepatic fibrosis/cirrhosis. the aim of this study was to compare two different t1 mapping sequences of the liver in adolescents with fontan palliation, and in healthy controls. materials: 15 adolescents (15-17 y) with fontan circulation and 7 young healthy adults (18-24 y) were included as a part of an ongoing national population-based study. all underwent mri (1.5 tesla) pre-and post-gadolinium contrast, including two types of t1 mapping of the liver. a 3d t1 volumetric interpolated breath-hold examination (3d vibe) sequence with dual flips with b1 correction and a modified look-locker inversion recovery (molli) sequence. t1 relaxation times (ms) were measured by placing five standardized circular regions of interest (roi) in the mid-section of the liver and one in the spleen (fig 1) . statistical analysis was performed comparing measurements pre-and post-contrast, between sequences, and patients and controls. there was a significant difference in the measurements between molli and 3d vibe with increased values for the latter. within each sequence there were small, but significant regional differences in relaxation times (table 1). the same pattern was seen in pre-and post-contrast images in both groups. there were significantly increased native t1 times on both sequences in all regions in the fontan group as compared to the controls, but not post contrast. t1 relaxation times differ between the t1 mapping sequences, molli and 3d vibe, pre-and post-contrast. t1 mapping of the liver revealed significantly increased native t1 times in adolescents with fontan palliation compared to healthy slightly older controls. these findings suggest hepatic fibrosis/cirrhosis, but may also represent a component of congestion. diagnostic accuracy of ultrasound, computed tomography and wedge portography in the work-up for mesenterico-rex bypass in children with extrahepatic portal hypertension s. toso, r. breguet, m. annoshiravani, s. terraz; geneva/ch to identify the diagnostic accuracy of ultrasound (us), computed tomography (ct) scan and portography (wedge hepatic vein portography or direct portography) in the pre-operative work-up of mesenterico-rex bypass performed for extrahepatic portal hypertension in children. we conducted a retrospective analysis of pre-operative imaging for mesenterico-rex bypass in our tertiary hospital over the last 12 years. we analyzed all patients between the ages of 0-16 years, with extrahepatic portal hypertension necessitating surgical treatment that underwent us, ct and portography. three reviewers independently analysed the patency of the left portal vein, mesenteric vein, splenic vein and the presence of communication between the left and right portal vein on preoperative imaging with correlation to surgical findings. statistical analysis of diagnostic accuracy was performed. eleven patients underwent mesenterico-rex bypass for portal hypertension secondary to portal vein thrombosis. two patients had partial liver transplant. ct with ultrasound correlation was sufficient in responding to the preoperative criteria in 72% (8/11) cases. portography was useful in the 27% (3/11) cases where ct could not respond to preoperative criteria, in particular the presence of left-right communication. there was good inter-rater correlation for each modality and good correlation of findings between modalities. in the majority of cases the use of ultrasound and ct is sufficient for preoperative planning for mesentrico-rex bypass. portography is mandatory in cases with large intra-hepatic cavernoma, where the left-right communication could not be confirmed on ct. contemporaneous clinical data was reviewed where available, and a clinical decision on disease severity and activity on a likert scale made with and without imaging. fifty-three patients underwent mre and bowel us in the specified timeframe (29 male; median age 13.02 years, range 4-16 years). twenty patients had sufficient contemporaneous clinical information to be analysed. inter-observer variability for the imaging scores was assessed using bland-altman plots. where variability was beyond pre-determined limits, the studies were consensus reviewed. mean scores were used for the studies within accepted limits of variability. there was no significant difference between total mre and us scores (wilcoxon signed-rank test z=1.13, p=0.26). at the bowel segment level, there was no significant difference between the mre and us segment scores for the ileum and terminal ileum (wilcoxon-signed rank test, z=0.72, p= 0.472), but significant differences were present between the imaging scores for other bowel segments, with mre identifying more abnormalities. there is a significant positive correlation between mre and clinical consensus scores (spearman's rho=0.598, p=0.0053) and between us and clinical consensus scores (spearman's rho =0.657, p=0.0016). imaging caused a refinement to the original clinical assessment in 8 of the 20 cases, with jejunal and ileal disease the most common reason for 'upgrading' a score and absence of any detectable abnormality on us and mre the most common reason for 'downgrading' a score. we found good agreement between mre and us for total patient imaging scores, ileal and terminal ileal scores. both mre and us scores correlated well with the gold standard clinical consensus, with imaging altering the original clinical decision in 40% of cases. although us detected fewer abnormalities than mr, it correlates marginally better with the clinical consensus, suggesting it is at least equally clinically valuable. background: differentiating between acute osteomyelitis (om) and acute bone infarct (bi) in children with sickle cell disease (scd) is a challenge for clinicians and radiologists, particularly when blood cultures are negative. although bone aspiration is the gold standard test for om diagnosis, it is an invasive procedure and infrequently performed. magnetic resonance imaging (mri) has shown a potential role in differentiating between acute bi and acute om. the goal of this case series is to evaluate the utility of fluid signal on unenhanced fat-suppressed (fs) t1-weighted mr sequence in distinguishing acute bi and om in children with scd. methods: we reviewed a total of 22 children with scd admitted with long bone pain during the one -year study period 2015-2016 attributed to either an acute bi or an acute om. twelve of 22 patients with available bone aspiration, blood culture, and mri data were evaluated for fluid signal, marrow signal and other criteria. of 12 patients, nine patients were diagnosed as acute bi and two patients had acute om and one with coexisting bi and om. the diagnosis was based on the fluid signal on t1 unenhanced t1 fs mr images as compared to aspiration cytology in which eight of nine patients with bi had hyperintense fluid signal on non-contrast t1 fs mr images while one of two patients with om demonstrated hypointense fluid signal. the last patient was diagnosed as a probable coexisting lesion (om&bi) based on a giant well demarcated hypointense marrow signal with an extraosseous hyperintense fluid signal. in acute bi, an abnormal hyperintense subperiosteal or paraosteal fluid signal is frequently observed on unenhanced t1-fs weighted images. this finding was present in the majority of cases in our study population regardless of age, sex or site in the appendicular skeleton. mri fluid signal characteristic on unenhanced t1 fs shows promise as a criterion to differentiate between acute bi and om. role of mri to assess skeletal age in pediatric celiac disease s. bernardo, m. martino, a. laghi, e. tomei; rome/it objective: coeliac children are often subject to weight loss and lower somatic growth rate, compared to healthy children of the same age. the purpose of this study was to asses the feasibility of magnetic resonance imaging (mri) of the hand and the wrist to assess skeletal age and growth delay. we enrolled in our study 39 coeliac children (13 males and 26 females) affected by histological proven coeliac disease, with a chronological age ranged between 5 years and 1 month and 16 years and 4 months (mean age of 10years, +/3 years and 8 months standard deviation). a single mri sequence (t13d se, acquisition time: 1 minute 31 seconds) of the hand and wrist in coronal plane was performed of each patient to estimate the skeletal age. patients' data were compared with a population of normal subjects. the preliminary results showed a delay in skeletal age in children affected by coeliac disease in 85,7% of the simple study, with a delay of maturity of 0.83 years (+/-2,2 years of sd). only 3 children showed advance mri skeletal age when compared to normal subjects. mri of hand/wrist to assess skeletal age may be considered as a reliable indicator of somatic growth. mri, without radiation exposure, can be an used as a diagnostic tool in skeletal delay. it could play an important role in the follow up of coeliac children, after glutenfree diet. the prevalence of metaphyseal injury and its mimickers in otherwise healthy children under two years of age p. eide, å. djuve, r.e. gjøsaeter, k.f. forseth, a. nøttveit, c. brudvik, k. rosendahl; bergen/no objective: metaphyseal lesions in infants and toddlers are believed to have a high specificity for inflicted injury, however, normal metaphyseal irregularities may mimic pathology and lead to overdiagnosis. during the period 2010-2015 all children between 0 and 2 years, seen at the a&e department in bergen (bergen legevakt) due to an injury, and who had radiographs taken, were included. data on previous injury, age, sex and injury mechanism were drawn from the medical notes and pacs archive. all radiographs were reviewed by two researchers and an experienced paediatric radiologist, registering the following: number, site and type of fractures, signs of healing (yes, no), bone structure (normal, pathological) and metaphyseal appearances (shape (normal, metaphyseal collar, metaphyseal irregularity), injury). the study was approved by the institutional review board. six hundred one children (293 girls) between 2 and 24 months of age (mean 17.8 months) were included, of whom 218 (109 girls) had a total of 275 fractures. one hundred eight of the fractures (39.3%) involved the forearm, followed by leg-fractures (59/275, 21.5%) and fractures to the clavicle (37/275, 13.5%). one epiphyseal separation and one metaphyseal lesion (without a history of trauma) were seen. one thousand three hundred twenty metaphysis were analysed, of which 212 (16.1 %) were defined as either irregular (105/1320, 8.0 %) or demonstrating a metaphyseal collar (107/1320, 8.1 %). metaphyseal lesions with a history of trauma did not occur in otherwise healthy neonates and infants under 2 years of age, indicating that this type of fracture has a particular mechanism. metaphyseal irregularities are frequent, particularly around the knee, and should not be mistaken for clms to evaluate whether mri might be used for age estimation, based on greulich-pyle (gp) atlas criteria. 1.5tesla mri of the left hand was conducted in 60 adolescents, and subjectively evaluated by two blinded radiologists. for sequence optimization, coronal mri sequences (t1 tirm, t1 vibe-3d-we, and t1 se) and a left hand x-ray were compared in ten patients (eight male, two female; mean age, 13.5 years). the ages of 50 healthy volunteers (17 s375 (2017) 47 (suppl 2):s297-s pediatr radiol male, 33 female; mean age, 15 years) were assessed from coronal t1 vibe-3d-we. bland-altman plots, intraclass correlation coefficients (icc), and logistic regression models were calculated. coronal t1 vibe-3d-we achieved the best image quality. the correlation between estimated patients' ages on x-ray and mri was high. icc showed high inter-observer agreement (0.95 for x-ray, 0.97 for mri). the estimated age of the healthy volunteers tended to be older than their chronological age. the probability of overestimation was higher in girls than in boys. coronal t1 vibe-3d-we of the left hand is feasible for skeletal age estimation by gp criteria with a high readers' agreement. the likelihood of overestimation of healthy children makes it necessary to develop a new hand atlas representing changes since the 1950s. to assess the relationship between the radiographic findings of metabolic bone disease (mbd) and serum biochemical markers in preterm infants. preterm infants in our neonatal intensive care unit between january 2014 and september 2016 were included. two readers retrospectively reviewed the wrist radiography for grading according to mbd severity. we recorded the levels of alkaline phosphatase (alp) and phosphorous (p) immediately after birth, on the same day of the first wrist radiography (alp-s, p-s), the highest alp levels before the first wrist radiography (alp-hb) and during follow-up (alp-h), and the lowest p levels before the first wrist radiography (p-lb) and during follow-up (p-l). patients were subdivided into four groups according to mbd severity determined by wrist radiography for the first analysis, and were divided into two groups according to mbd presence for the second analysis. one-way analysis of variance with a tukey multiple comparison and the student's t-test were used for statistical comparisons in the two analyses, respectively. a receiver operator characteristic (roc) curve was constructed to determine the optimal cut-off values of the biochemical markers for the radiological prediction of mbd. of the 159 patients, 94, 39, 19, and 7 infants were classified into grades 0 1, 2, and 3, respectively. in the first analysis, alp-s, alp-hb, and alp-h were significantly different between grades 0-1 and 2-3 (all p<0.001). plb was significantly different between grades 0 and 2 (p=0.001) and p-l was significantly different between grades 0 and 2 or 3 (p<0.001 or p=0.001). in the second analysis, alp-s, alp-hb, alp-h, p-s, p-lb, and p-l were all significantly different between the two groups (p<0.001). the roc curve of alp-h showed the largest area under the curve values (0.752, 95% confidence interval=0.676-0.828; p=0.039) for detection of a radiographic change. the optimal cut-off value of alp-h was 473.5 u/l, and the sensitivity and specificity were 81.5% and 47.9%, respectively. the first wrist radiography was obtained at 8.3 ± 5.1 weeks after birth, and alp-h was measured at 6.9 ± 5.3 weeks after birth. the cut-off value of alp for the prediction of abnormal radiological changes in wrist radiography was determined to be was 473.5 u/l. our findings indicate that the highest alp level at around 6.9 weeks after birth could be a valuable predictor of radiological mbd in preterm infants, including those with very low and extremely low birth weights. quantitative grading of tmj synovitis in children with jia-influence of mr-coil, timing after contrast-injection and location of measurements on joint-to-muscle enhancement ratio a. hamardzumyan schmid, c. kellenberger; zurich/ch objective: assessment of signal intensity ratio between joint space and longus capitis muscle on contrast-enhanced mri has been proposed as reliable method across different mr-scanners and protocols for grading temporomandibular joint (tmj) arthritis in juvenile idiopathic arthritis (jia) with a cut-off of 1.55 for diagnosing synovitis. the aim of this study was to investigate potential influences on such enhancement ratios (er). retrospective evaluation of 14 contrast-enhanced mr-studies of 28 tmjs in 7 girls with jia (age 11.3±4.3y) obtained at two occasions with two different coils on a 1.5t scanner. joint-to-muscle er were calculated from signal intensity measurements in different joint compartments, muscles and sequences obtained with varying delay after contrast-injection, and compared with paired sample t-test. er of tmjs without synovitis (n=10) and tmjs with synovitis (n=18), determined by qualitative criteria, were compared to er reported in the literature. superior and inferior joint space to longus capitis muscle er for normal tmjs (2.2±0.9; 2.6±0.7 respectively) exceeded 1.55 in all but one case (figure) and for tmjs with synovitis (3.3±1.1, 3.8 ±1.1) were significantly higher than in 211 cases with synovitis from the literature (2.5±0.8, p≤0001). the same er were higher when obtained with dual-ring coil (3.7±1.1; 4.1±0.9) than with multichannel surface coil (2.2±0.7; 2.6±0.8; p≤0.0002). while er to longus capitis muscle were higher than those to pterygoideus muscle for both coils (p≤0.008), er to pterygoideus muscle did not differ between coils (p>0.2). not considering the timing of the scan, er to pterygoideus muscle were highest in the inferior joint space (1.65 ±0.63), followed by the anterior joint recess (1.52±0.44) and superior joint space (1.49±0.62). comparing images acquired immediately after contrast injection to later images (median delay 11min, range 5-15min), pterygoideus muscle er in the superior (1.2±0.4 to 1.8 ±0.7) and inferior (1.2±0.3 to 2.0±0.6) joint space increased substantially (p<0.0001), while er in anterior recess showed no significant increase (1.5±0.4 to 1.6±0.5, p=0.18). conclusion: joint-to-muscle er are clearly dependent on 1) the signal profile of the mr coil with muscles located further away from the coil providing higher er, 2) the time of image acquisition after contrast-injection with later obtained images providing higher er, and 3) where the joint signal intensity is measured. as these factors need to be accounted for, the described normal and pathologic ranges of joint to longus capitis muscle er cannot be generalised for every mr-system and imaging protocol. integration of 3d c-arm ct images with navigational software provides real-time fluoroscopic guidance during percutaneous interventions in the interventional radiology (ir) suite. a trajectory, drawn from skin entry point to the target lesion on the 3d c-arm ct data, is overlaid on intraprocedural fluoroscopy for real-time needle guidance. this study describes our experience with syngo iguide (siemens) needle guidance software in a range of clinical applications in the pediatric ir suite, including technical success, radiation dose and procedure time. in this irb approved study, all percutaneous interventions performed in the ir suite using syngo iguide over a 3-year period were included. cases were classified by procedure type; for each type, mean effective radiation dose (msv) was estimated using pcxmc program (v2.0.1.3, stuk) and procedure times were evaluated. forty-five patients (25 male, 20 female; mean age: 11±6 years) underwent iguide-assisted interventions including: bone biopsies -35/45 (22 pelvic, 7 lumbar, and 6 lower extremity), intra-articular steroid injections -5/45 (4 sacroiliac, and 1 temporomandibular joint), lumbar punctures -2/45, percutaneous catheter placements -2/45 (cecostomy, and chest tube placement) and bone biopsy with radiofrequency (rf) ablation -1/45. iguide was used in particular for the cecostomy procedure due to high sub-hepatic cecal pole position, and in the chest tube procedure due to the presence of loculated pneumothoraces. all procedures were technically successful. the diagnostic bone biopsy rate was 97.7%. the mean estimated dose and procedure times for each procedure type are listed in table 1 . sonography of neonatal spine (sus) is a simple, non-invasive, quick, relatively inexpensive method to evaluate lumbar spine anomalies in infants less than 4 months of age. unossified posterior neural arches allow beam penetration to obtain high-resolution images of the intra-spinal contents. sus is carried out at the bedside, does not utilize radiation & requires no sedation. linear array transducers with extended field-of-view permit diagnostic sensitivity equal to mri. factors affecting mri resolution like patient movement, pulsation & vascular flow do not affect sus. we use sus as first-line screening test in neonates with lumbosacral cutaneous stigmata & spinal dysraphism (sd) associated syndromes. this was a prospective study approved by the institutional ethics committee. thirty five children (age range of 5 to 15 years) with clinically suspected and complicated pulmonary tb were enrolled in the study. lung mri and ct scan was performed in all the patients. the sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of lung mri in detection of radiological findings that were considered highly suggestive or diagnostic for tb, were calculated, with ct as the standard of reference. lung mri performed equivalent to ct in detection of pleural effusion, mediastinal/hilar lymphadenopathy and lung cavitation with sensitivity and specificity of 100%. agreement between ct and mri in detection of each finding was almost perfect (k: 0.8-1). lung mri was found to be comparable to ct scan for detecting various radiological abnormalities which were highly suggestive for tuberculosis. being a radiation free imaging modality, it has the potential, particularly in children, to replace chest radiographs and ct scan in the coming years. to evaluate differences of myocardial strain assessed by feature tracking using ssfp cardiac mri sequences between pectus excavatum (pe) patients and healthy volunteers. in this prospective study, cardiac mri was performed in 10 pe patients (with a pathologic haller-index above 3.25) and 10 healthy volunteers (5 males and females, respectively; age range 13-30 years) including short-and long-axis cine-ssfp sequences on a 3t scanner. post-examination analysis included standard cardiac volumetry with measurements of the biventricular ejection fractions (ef). additionally, manual biventricular contouring by an experienced radiologist, and subsequent automated strain assessment using dedicated software (circle cvi 42 ®) was performed. longitudinal, radial, and circumferential peak systolic strain and strain rates were analyzed for both ventricles. left-ventricular ef was normal in all patients. five pe patients had a normal right-ventricular ef, in 5 pe patients rvef was slightly impaired (40-44%), all healthy volunteers had a normal rvef. compared with healthy volunteers, pe patients showed a significantly higher apical left-ventricular strain (radial: 53±16.8 vs. 26±8%, p<0.001; circumferential: -23.7±4.8 vs. -15.3±3%, p=0.001) and strain rate (radial: 3.76±1.05 vs 1.71±0.33s -1 , p<0.001; circumferential: -1.92±0.51 vs. -1.08±0.48s -1 , p=0.002). mid right-ventricular strain (radial: 14.4±6.5 vs. 8.2±2.6%, p=0.019; circumferential: -9.7±3.3 vs. -6.4±1.5%, p=0.019) and strain rate (radial: 1.07±0.45 vs. 0.63±0.24s -1 , p=0.015; circumferential: -0.79±0.27 vs. -0.5 ±0.14s -1 , p=0.011), as well as apical right-ventricular strain (radial: 25.6±8.6 vs. 16.5±8.6%, p=0.009; circumferential: -15.9±3.8 vs. -11.1±4%, p=0.019) and circumferential strain rate (-1±0.26 vs. -0.72±0.24s -1 , p=0.029) were also significantly higher in pe patients than in healthy volunteers. left-and especially right-ventricular radial and circumferential strain and strain rate increased from the bases to apices in pe patients. longitudinal strain and strain rate did not differ significantly between pe patients and healthy volunteers. myocardial strain assessed by cardiac mri differs significantly between pe patients and healthy volunteers. as the chest wall deformity usually leads to a compression of the basal parts of the ventricles, higher values of myocardial strain in the mid and apical ventricles in pe patients might indicate a compensation mechanism to enhance especially right ventricular output against sternal compression. to determine the normal range of the haller index (hi) value, and its dependence on the age, sex, and respiratory phase. evaluate the possibility of reduction of the effective dose (ed) of ionizing radiation using a single-slice ct scan technique. the retrospective-prospective study included 165 patients (av. 12y, sd 5y). it consisted of 3 parts. the prospective study included evaluation of ct scans performed by single-slice technique in 30 patients with pectus excavatum both in inspiratory and expiratory phase, without topogram. hi was measured in each patient in both respiratory phases. in retrospective study, 100 ct scans of the chest in children without pectus excavatum were analyzed to determine normal range of hi values depending on the age (0-5y, 5-10y, 10-15y, 15-18y) and gender. the retrospective study also included the analysis of another 30 ct scans in patients who were operated or diagnosed with pectus excavatum. in the latter group of patients the average value of ed of ionizing radiation was calculated, and the values were compared with the average ed obtained using low-dose ct examinations applied in the new protocol (single-slice technique). the normal value of hi was 2.23±0.32. a significant positive correlation between age and value of hi was found. older patients had higher hi (0-5y: 2.01±0.29, 15-18y: 2.35±0.33). results of mann-whitney test did not demonstrate any difference between gender in the observed group, however girls had generally higher hi in all age groups. in the group of patients who were operated/diagnosed with pectus excavatum, hi was 3.34±0.88. the average value of hi in inspirium in children with diagnosed deformity was 2.69±0.76, while in expirium it was 3.49±1.19. only 3/32 (9%) patients had hi value over 3.25 (a boundary value for surgical treatment) during inspirium, while 13/32 (41%) patients had it in expirium, which showed statistically significant difference (p=0.012). single-slice ct technique during the inspiratory and expiratory phase showed average ed of 0.02msv, which is an equivalent of 1 chest xray. it reduced ed more than 20 times in comparison with low-dose whole chest ct. the value of haller index increases with the age and in expiratory phase. we propose the single-slice ct technique without topogram in expiratory phase, as a sufficient and reliable technique in evaluation of haller index and preoperative preparation. mps iva is a lysosomal storage disorder caused by a deficiency of nacetylgalactosamine-sulfatase. main symptom is a systemic skeletal dysplasia. affection of the vascular system has not been described yet. our goal is the analysis of the vascular system in patients with mps iva, based on the example of the aorta. in a retrospective study, 32 patients with mps iva were included. the aorta in its course from 4 th thoracic vertebrae to 10 th was analyzed on the basis of 49 craniospinal mr and 4 ct examinations. to describe the course of the aorta, the area around the vertebral body was devided into 5 equal parts (fig.1) . high buckled arteries in relation to the length of the affected aortal part were indicated as aortal kinking, and a moderate twist in relation to the length of the affected aortal part as aortal tortuosity. results: twelve of 32 patients had an aortal kinking, 10 of 32 patients an aortal tortuosity, 4 of these had moderate and 3 strongly tortuous aortae. seven patients had a normal aortal course, 4 couldn't be analyzed. one patient revealed both, aortal kinking and tortuosity. this study reveals the occurrence of aortic tortuosity in patients with mps iva. we suggest that this complication could be due to glycosaminoglycane deposition in the aortic intima, which may be s379 (2017) 47 (suppl 2):s297-s pediatr radiol associated with an increased vulnerability of the vascular wall. we conclude that the examination of the vascular system should be included in regular follow-up protocols. lung ultrasound in the diagnosis and follow-up of pneumonia in children -is it really as reliable as chest x-ray? s. balj-barbir, j. lovrenski, s. petrović; novi sad/rs to investigate the role of lung ultrasound (lus) both in the diagnosis and follow-up of pneumonia in children. a prospective study was carried out in the regional children's hospital, and included 130 children (av. 2.9y, sd 2.93y) with clinically suspected pneumonia, in whom initial lus and subsequent chest x-ray (cxr) were performed within 24h. the final diagnosis of pneumonia at discharge was used as a reference test to determine the reliability of lus, cxr, clinical and laboratory findings in the diagnosis of pneumonia. children with pneumonia formed a study group, while the control group consisted of children without diagnosed pneumonia. lus finding of subpleural lung consolidation was considered a diagnostic sign for pneumonia. the children with lus signs of pneumonia were followed-up until complete resolution of the lus findings. there were from one to five follow-up lus examinations performed. a final diagnosis of pneumonia was confirmed in 105/130 (80.8%) patients, and 77/105 (73.33%) were hospitalized (av. 10.84, sd 6.19 hospital days). in diagnosis of pneumonia lus, cxr, auscultation, elevated crp, and tachypnea showed sensitivity of 94.3%, 93.3%, 79%, 80% and 32.38%, and specificity of 100%, 92%, 56%, 48% and 100% respectively. lus detected lung consolidations in 99 of 105 children with final diagnosis of pneumonia, and in 84/99 patients lus showed air-bronchogram (figures 1, 2) . lus was superior to cxr in the detection of lung consolidations smaller than 15mm. interstitial lung changes were detected by lus in 50/105 (47.62%) patients, and by cxr in 21/105 (20%). lus and cxr detected pleural effusion in 24/105 (22.86%) and 14/105 (13.33%) patients respectively. mcnemar's test showed no statistically significant difference, and cohen's kappa coefficient showed almost perfect agreement (0.864) between us diagnosis of pneumonia and final diagnosis of pneumonia. during the follow-ups, moderate to substantial agreement between lus and clinical evaluation of the course of the disease was obtained (k=0.406-0.621). in children with complete clinical and incomplete us regression of pneumonia, consolidations of less than 15mm were the most prevalent finding. the average time period until complete resolution of the lus findings was 16.3±10.24 days. children with us detected pulmonary consolidations larger than 50mm were statistically significantly longer hospitalized than others. lung ultrasound in the diagnosis of pneumonia in children is just as reliable as radiography, and should be included in the standard diagnostic protocol. the latest uk nice guidelines for childhood tb contact screening require that a chest x-ray (cxr) be requested only when mantoux or igra testing is positive or if there is a documented reason e.g. clinical concern. nice clarifies the role of cxr in determining treatment choice. we aimed to review cxr referral and treatment in the current climate of european migrant screening. retrospective review of 148 paediatric referrals to the infectious diseases clinic for tb contact screening of whom 125 had cxrs, from october 2009 to august 2015 and correlation with the medical notes. a panel of 3 paediatric radiologists independently interpreted radiographs in the clinical context of tb contact screening and a majority decision was reached. of 148 patients referred to the infectious diseases unit, 125 underwent cxr in addition to a mantoux and igra test. of those 125 cxr's, 20 were reported as having features of pulmonary tb but only 16/20 (80%) were treated as active tb. eighteen of the 105 (17%) cxr's which were reported as having no features of pulmonary tb, were treated as active tb. of those 18, only 6/18 (33.3%) had a clearly documented reason. review of the 125 radiographs (mean age 14 years) by the panel of radiologists noted that all were of readable quality, 20 radiographs were in keeping with a diagnosis of tb, 6 were inconclusive and 99 were normal. the diagnosis of tb was based on lymphadenopathy in 19 and (2017) 47 (suppl 2):s297-s pediatr radiol milliary nodules in 1. parenchymal abnormality was seen in 8 patients [one was the milliary] and effusion was seen in 3. this correlated well with the initial radiology reports of duty radiologists. of the 125 who underwent cxr, referral information was available in 120.109 (90.8%) of these had been appropriately referred because of a +ve mantoux/igra. only 8 out of 11 (72.7%) of those who had cxr despite a -ve mantoux or igra, had a documented reason. according to nice guidelines, 20% of cxr reported positive for tb were not treated for active tb. this may represent a lack of clarity regarding the definition of 'latent tb'. furthermore, only a third of the 17% of patients who received treatment despite negative radiographs had a documented reason. the current migrant crisis requires clarity of x-ray definitions of latent tb to avoid the 20% under-treatment and 17% overtreatment identified in our population. is there really no cardiac problem for performing sports ö.i̇. koska, p. bayindir, h. alper; izmir/tr objective: sudden death in young is a rare condition excluding known anomalies and sudden infant death sydrome; but its consequences are devastating because they are so unexpected. %25 of them occur in a context of sports event. everyday parents of millions of children admit hospitals in order to get permission for participating in sports events. and after physical examination and ecg, physicians are expected to decide such an important issue. however there are a number of silent reasons that may lead to child to sudden death. altough we don't perform ct scans for such indications, we have encountered several cases with abnormalities that can lead to sudden child death and while reporting an examination, awareness of these devastating conditions may be usefull. we searched our database from 01.01.2016 to 01.01.2017 in order to see how often we diagnosed such a silent reason from the ct images that are performed for some reasons. as our center is a tertiary center we have performed 452 cardiac ct examinations in that period that are mainly for excluding or defining complex cardiac anomalies. in order to prepare a pictorial review of unexpected but ct detectable sudden cardiac death reasons, we excluded congenital heart diseases (namely obstructive, shunting or complex anomalies) and ecg detectable arrhytmic diseases. the non arrhytmic, non traumatic reasons for sudden cardiac death excluding congenital heart diseases in the papers are: hypertrophic cardiomyopathy (cmp) (%36), some coronary artery path and origin anomalies (mainly abnormal left coronary artery from pulmonary artery (alcapa), and interarterial path)(%24), increased cardiac mass (%10), dilated cmp (%9), marfan disease (%6), myocarditis (%3), ischemic heart disease (%2). we detected 18 examinations according to our inclusion criteria and selected one cases of each; rca path anomaly, alcapa, dilated cmp, hypertrophic cmp, subaortic discrete membrane and increased cardiac mass for presentation although sudden cardiac death is rare in young children it is a so devastating condition that it must be taken into account for every situation. awareness of silent conditions and active search of them may protect professionals from medicolegal issues and unpleasent results. to describe the spectrum of chest ct scan findings of pulmonary involvement in childhood langerhans cell histiocytosis (lch) and propose a simple scoring system to evaluate the profusion and distribution of the main lung lesions. one hundred forty-six chest ct scans of the 48 pediatric patients with pulmonary lch enrolled in the french national database for lch until april 2016, could be retrospectively and independently reviewed by 3 pediatric radiologists. for each ct scan a semi-quantitative analysis was performed for nodular opacities and cystic abnormalities. the chest was divided in 6 fields (upper, medium and lower field of the left and the right lung) and for each field, both for the nodules and the cysts the score was 0=no lesion, 1= lesions involving up to 25% of the parenchyma, 2=25-50%, 3= 50-75% and 4=more than 75%. of 29 patients evaluated at diagnosis, 18 patients (62%) presented with nodules, 13 patients (45%) presented with cysts and 6 patients (21%) presented a combination of both nodular and cystic lesions. on the initial ct scan, median nodules total score was 1 and median cysts total score was 0. during subsequent ct scans almost the same percentage of patients with nodules (30 patients, 62 %) was found but we observed an increase number of patients with cysts (30 patients, 62 %), median nodules total score was 1 and median cysts total score was 2. the distribution of nodules and cysts was symmetric in the upper, medium and lower fields with an involvement of costo-phrenic angles in 68% of the cases. patients with pneumothorax (13 patients, 27%) had a higher cysts median score (10) than patients without pneumothorax (1). we found alveolar condensation in 12 patients (25%). none of them showed signs of infection at bal examination or any improvement after a treatment with a standard antibiotic therapy while they did show regression under the lch standard regimen of chemotherapy. we proposed a score for semiquantitative analysis of distribution and profusion of nodular and cystic lesions on chest ct scans that can be a useful tool in pediatric population to monitor lung involvement. we found a significant correlation between pneumothorax and a high cysts median score. alveolar condensation could be considered as a possible manifestation of plch in children. lung bases involvement was found in 68% of the cases, representing an important different imaging feature from adult plch. high resolution computed tomography for chronic small airway disease in hiv infected adolescents a.-m. du plessis 1 , s. andronikou 2 , h. zar 1 ; 1 cape town/za, 2 bristol/uk early treatment with antiretroviral therapy (art) and decline in infected infants due to prevention of mother-to-child transmission has resulted in an increase in the population of hiv-infected adolescents. pulmonary disease is common among them. cxr is considered insensitive and terminology inconsistent. therefore, despite concerns related to radiation dose in paediatric patients, high resolution computed tomography (hrct) is the modality of choice for the evaluation of small and large airway pathology, prominent in chronic pulmonary disease. hrct findings are used for prognosis, treatment decisions and defining anatomic extent of bronchiectasis for surgical intervention. the aim of this paper is to demonstrate the spectrum, frequency and extent of airway pathology in hiv-infected adolescents using hrct. a nested sub study was undertaken within the cape town adolescent antiretroviral cohort (ctaac); a prospective, descriptive cohort study of 520 hiv-infected adolescents on art and 120 age matched hiv-s381 (2017) 47 (suppl 2):s297-s pediatr radiol negative controls. hrct was performed on 100 patients who demonstrated abnormal lung function (defined by forced expiratory volume in 1 second (fev1) of <85% and/or low lung diffusion capacity (dlco)). single phase, contrasted multi-detector volume acquisitions were performed from the thoracic inlet to the diaphragms at full inspiration and image data postprocessed to yield thin (0,6mm) and thicker slice images (5mm). three 1mm slices at 3 cm intervals were performed in full expiration. three radiologists interpreted the c t scans independently, with strict imaging definitions, and a majority decision was generated for each finding. ages of patients ranged from between 10 to 17 years with a mean of 14,2. there were 53 females and 47 males with a ratio of 1:0,8. bronchiolitis obliterans was seen in 70% of patients and bronchiectasis was demonstrated in 39%, 12% of which was classified as severe (involving either an entire lobe or more than 50% of at least 2 lobes). there was an absence of lymphadenopathy (a sign of primary tuberculosis (tb)), lymphocytic interstitial pneumonitis (lip) and post tuberculous apical architechtural distortion. miliary tb was identified in a single patient. ground glass was seen in 10% and consolidation in 13%. the majority of hiv infected adolescents with poor lung function demonstrated bronchiolitis obliterans strongly emphasizing the use of hrct for confirming small airways disease. hrct was also useful for demonstrating extent of associated bronchiectasis in 39%. hrct allows classification of patients into those with diffuse small airways disease requiring medical management and those with local disease requiring surgery. background: chronic recurrent multifocal osteomyelitis (crmo) is an autoinflammatory paediatric non-infectious bone disease. presenting symptoms are non-specific, prolonging diagnosis, and leading to deformity, morbidity and unnecessary procedures. imaging is critical to diagnosis, with whole-body mri (wbmri) commonly used in all stages of care. in our institution, a baseline whole-body coronal stir sequence is routinely obtained. aim: to determine lesion distribution and extent on baseline wbmri via retrospective panel review of all patients clinically diagnosed with crmo, and to determine any patterns of involvement that could facilitate earlier radiological diagnosis. method: all patients diagnosed with crmo since december 2009 using published bristol criteria were identified and baseline whole body mris reviewed. the reviewing radiologists were blinded to the original report and previous investigations. each mri was reviewed for focal lesions consistent with crmo. the extent of metaphyseal and epiphyseal lesions was categorized into involvement of thirds of the width of the structure. the wbmri of forty children (28 girls, 12 boys), averaging 12 years (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) were reviewed by the panel using a majority decision rule. three hundred three lesions were recorded, averaging 7.6 lesions (0-26). the tibia was most affected (83 lesions), most commonly the distal tibial metaphysis (25 lesions in 16 patients, 9 bilateral). rib, metatarsals and distal femoral epiphyseals lesions were common. humeral, hand and skull lesions were few. complete metaphyseal involvement, the 'smouldering physis', was most prevalent within the proximal and distal tibial metaphyses. although ranked seventh, the reportedly more common clavicular lesions were the site of the most florid lesions, demonstrating bone expansion and periosteal reactions. two clear patterns of involvement emerged. in patients with clavicular lesions, fewer overall lesions were observed (average 6.4), mainly affecting the axial skeleton and feet. patients with tibial involvement had a higher number of overall lesions (average 9.3), but few lesions outside the lower limbs. only four patients had a both clavicular and tibial lesions. twelve vertebral lesions and four cases of spodylo-discitis were identified; two at t5/6 level, one at t6/7 level, and one involving both t5/6 and t6/7. our series of 40 cases of crmo with baseline wbmri, one of the largest in the published literature, identifies the common sites that should be interrogated for involvement. this study also demonstrates potential as-yet undescribed patterns of skeletal involvement that can be used to aid radiological diagnosis and highlights a non-infective cause for spondylo-discitis. hepatic hemangiomas (hh) are the most common benign vascular tumors encountered in the pediatric population. two main types have been described congenital and infantile, which both display distinct clinical courses and biological features. hemangioendothelioma in differential diagnosis of hh is controversy. recent literature suggests that congenital hepatic hemangiomas present in a focal form, whereas infantile hepatic hemangiomas present in either a multifocal or diffuse form. the goal of this study is to evaluate the features associated with focal and multifocal hh. the records of 45 patients diagnosed with a hepatic hemangioma at a tertiary pediatric hospital from 1994 to 2015 were reviewed. we divided our series into 2 groups: focal or multifocal including diffuse form. clinical endpoints are: age of diagnosis, presence of cutaneous hemangioma, alpha-fetoprotein, thrombocytopenia, cardiac insufficiency. imaging endpoints were echogenicity on us (hypoechoic, hyperechoic or mixed), vessels density on color doppler (<2; 2-5, >5 cm 2 ). presence of calcifications, venous lakes, visible vessels and aortic tapering were assessed on us, ct-scan, mr and angio. treatment and outcome were analyzed. univariate and bivariate analysis were done. this study included 24 focal (9m, 15f) and 21 multifocal (17m, 28f) hh. antenatal diagnosis was done in 6 focal and 1 multifocal hh. focal lesions were associated with the presence of cutaneous hemangiomas (p<0.001) and calcifications (p<0.05). no other variable was significant. conservative management was decided in 12 focal and 11 multifocal hh. steroids (focal: 6, multifocal: 3), steroid-interferon (focal: 1, multifocal: 3), propranolol-steroid (focal:4, multifocal: 4) and surgery in one focal form. complete regression was observed in most lesions (focal: n=19, multifocal n=20), whereas incomplete regression <50% was observed in 4 patients (focal: n=3, multifocal: n=1) and 3 patients in the focal group with the pathology diagnosis of hemangioendothelioma. hepatic hemangiomas demonstrate a wide range of radiological features, with important overlaps in focal, multifocal or diffuse forms. focal and multifocal hh can be seen in congenital hemangioma, infantile hemangioma or hemangioendothelioma. the association of cutaneous infantile hemangioma in the focal group confirmed that the focal lesion can be seen in infantile hemangioma. however, calcifications are more frequent in focal hh which is described in congenital hemangioma or hemangioendothelioma. (2017) 47 (suppl 2):s297-s pediatr radiol symptomatic and asymptomatic meckel's diverticulum in the pediatric population -a retrospective analysis of imaging findings with histopathologic correlation n. abu ata, r. cytter-kuint, j. bar-ziv, i. hadas-halpern; jerusalem/il objective: though meckel's diverticulum (md) is a relatively common gastrointestinal anomaly, many of the symptomatic and most of the asymptomatic md's are often missed on abdominal imaging. our purpose is to describe the radiologic appearance of symptomatic and asymptomatic md in the pediatric population and to correlate the radiologic findings with histopathology. a retrospective analysis of all children diagnosed with md between 1/2004-10/2016 and had relevant imaging (n=38) was done. imaging studies-ultrasound (us), computed tomography (ct) and magnetic resonance imaging (mri) were retrospectively reviewed and evaluated for visualization of md in symptomatic and asymptomatic patients. findings were compared with the preoperative radiology report and the pathology specimen. symptomatic group (n=24): mean age 8.6±6 years, nineteen males. md presented with abdominal pain in 9 patients, small bowel obstruction (sbo) in 6 patients, gastrointestinal bleeding or anemia in 5 patients and intussusception in 2 cases. md was identified in 13 preoperative reports (54.16%) and retrospectively identified in 4 more cases (overall 17 patients, 70.1%). in 7 cases, an inflamed or perforated md were found. in 4 cases, mucosal lining resembling gastric folds was seen. inverted meckel and prominent tissue surrounding the diverticula were seen in 2 patients. in a single case md was misdiagnosed as a duplication cyst. asymptomatic group (n=14): mean age 10.9±6, eight males. md was not mentioned in any of the original reports and only 3 md's were identified retrospectively (21.4%). no mucosal abnormality or irregularity were noted. histopathology: ectopic gastric mucosa was found in 16/24 (66.67%) of the symptomatic patients vs. 2/14 (14.29%) in the asymptomatic group. all 4 patients with sonographic appearance of gastric mucosa had gastric mucosa in pathology (specificity-100%, positive predictive value-100%). md has a variety of radiologic appearances. it can be detected in most of the symptomatic patients but is almost undetectable in asymptomatic patients. heterotopic gastric mucosa is more common in the symptomatic group. inflamed gastric mucosa may have a typical appearance resembling a small stomach, a sign that was not described before and has both high specificity and high positive predictive value for gastric mucosa within a meckel diverticulum. preliminary results on dna damage from ct irradiation in pediatric patients i. dilevska, e. nagy, w. schwinger, e. sorantin; graz/at the increased radiation sensitivity in children, compared to adults, is a well-recognised fact in the pediatric radiology community. the high-dose irradiation induced dna damage has been well established, however the dosages that are clinically used in everyday ct procedures are so low, that it remains unclear how this severely affects the dna and can induce cancer in the long run. the aim of this study is to assess the effects of lowdose ionizing radiation from ct in children by establishing a standardization curve ranging from the high to the low, medically significant ctdi values. this is done by measuring the phosphorylation of the h2ax histone (γh2ax), which is considered a biomarker for quantification of radiation induced dna double-strand breaks (ddsbs). the detection of the γh2ax histone was done by two methods: immunofluorescence microscopy (im), which is an established method for detection and quantification of this histone and the new, promising flow cytometry technique (facs). for this study, 35 leucocyte samples ("buffy coats") were provided by the local transfusion department and these samples were irradiated with a clinical ct scanner (aqilionone, tmse) with values ranging from 2153,6 to 0,16 mgy ctdi. afterwards the samples were processed with both methods. for the immunofluorescence, twostep immunostaining was used with two different antibodies and the cell and foci counting were done on an olympus xc10 microscope, while the facs staining was done with a one-step antibody and the samples were measured on a navios flow cytometer (beckman coulter). comparable results were detected with both methods, with a good correlation between the facs and im, with a linear incline (r 2 >0.95) in the high and in the low dosages from 0,16 to 5.0 mgy ctdi. however, in the samples irradiated with doses below 5.0 mgy ctdi, there seems to be less phosphorylated h2ax than in the native samples. two possible explanations arise: a) low dose irradiation initiates repair that extends to ddsbs occurring naturally or b) low dose irradiation doesn´t cause phosphorylation of this histone, but affects other dna damage and repair pathways. the preliminary findings show that the facs analysis can be used as a valid replacement method for the labor-intensive if method in the higher dosages. however more analysis should be done to establish its accuracy in the lower regions since underlying mechanisms are not clear yet. a. turkaj 1 , g. cicero 2 , e. sorantin 1 , r. coroiu 3 ; 1 graz/at, 2 mesina/it, 3 cluj-napoca/ro there is only little information available regarding imaging procedures in the trauma setting of pediatric patients. such data can serve as a rational basis for running pediatric trauma units. the purpose of the paper is to investigate the number, types and distribution of imaging procedures in a tertiary pediatric trauma center serving children of about 1.2 million inhabitants with approximately 225.000 children. all trauma-caused admission to the emergency room and their imaging procedures were analyzed retrospectively occurring within a period of 15 months. a cohort of 263 patients (m:f=1.9:1) were analyzed. patients were grouped according to age into the following categories: neonates, infants, middle childhood, early adolescence, late adolescence. imaging procedures were classified into plain films, us, ct and mri. furthermore, the time of admission was noted and categorized in time slots 07:00-15:00, 15:00-20:00, 20:00-24:00 and 24:00-07:00. referral cause was divided in domestic accidents, motor-scooter-bicycle accidents, car accidents, sport injuries, falls from height and others. the average age was 9.5 ± 5.4 years, aligned in the following age-groups neonates 13 (5%), infants 68 (26%), middle childhood 42 (16%), early adolescence 58 (22%), late adolescence 82 (31%). a total of 364 imaging procedures were performed, of which 210 plain films (58%), us 81 (22%), ct 69 (19%) and mri 4 (1%). there was a statistically significant difference of imaging procedures due to age in particular in us and ct. regarding the timeslots: 07:00-15:00 ;111 patients (42%), 15:00-20:00; 118 patients (45%), 20:00-00:00; 31 patients (12%), 00:00-07:00;3 patients (1%). domestic accidents were the leading referral cause with 82 cases (31%) prevailed age groups were infants and middle childhoods corresponding for more than 75%. motorscooter/bicycle accidents accounted for 51 cases (19%) of which most were early and late adolescence (more than 85%), s383 (2017) 47 (suppl 2):s297-s pediatr radiol sport's accidents 38 (14.4%) equally shared among middle childhood, early and late adolescences. car accidents 37 (14.0%) cases and fall from height 22 (8,3%) did not show any prevalence according to the age groups. for the first time detailed data about imaging procedures at the emergency room for pediatric patients are now available. over half of the admissions (55%) occur outside regular work hours thus representing a challenge for the staff in duty and this fact should be considered in working schedules. due to strict interdisciplinary developed diagnostic pathways the number of ct examinations was reasonable low. head ct in a regional children's hospital without mri -effective doses and justification of clinical indications j. lovrenski, n. milenković; novi sad/rs to calculate effective doses (ed) for pediatric head computed tomography (ct), to determine the most common referral diagnoses, and the share of normal and pathological ct findings. a retrospectiveprospective study comprised all the children with performed ct examination (16-slice scanner) within a one-year period. pediatric ct protocols were used. the values of ed for head cts were calculated based on the two different models (shrimpton's and icrp publication 103). average ed for different age groups was expressed as the number of chest x-rays (cxrs) (1cxr 0.02msv). the most common non-traumatic referral diagnoses for head cts were determined, as well as percentage and type of pathological ct findings. a share of pathological ct findings was also determined for trauma as a referral diagnosis. head cts were represented with 649 (71%) in total number of 924 ct examinations within a one-year period. the different calculation models have shown the difference in ed values of up to 20.5%. eds for head cts were equivalent of 66 (15 years of age and older) to 140 (younger than 3 months of age) cxrs for one sequence of scanning. the most common non-traumatic referral diagnoses for head cts were: loss of consciousness, epilepsy, headache, convulsions, and vertigo. in this group of 273 patients, 86% of completely normal ct findings were found. pathological findings in this group consisted of the patients with the most common non-traumatic referral diagnoses were as follows: cortical atrophy (15 patients), arachnoid cyst (10), ischemia (6), porencephalic cyst (3), agenesis of the corpus callosum (1), chiari malformation -type i (1), open-lip schizencephaly (1), and tumor of the posterior cranial fossa (1). most common incidental, extracerebral pathology discovered included sinusitis and otomastoiditis. in patients with trauma as referral diagnosis, the share of pathological ct findings was 59.5%. it is necessary to get clinicians familiar with the extent of ionizing radiation that children are exposed to during the head ct examinations. a more careful selection of children for head cts is necessary in an every-day clinical practice, especially for patients with non-traumatic referral diagnoses. diffuse and symmetric diffusion restriction involving the white matter of the brain in patients with neonatal seizures j.-y. hwang 1 , y.j. lee 2 , y.-w. kim 2 ; 1 yangsan-si, gyeongsangnam-do/ kr, 2 yangsan-si/kr this study aimed to evaluate magnetic resonance (mr) imaging findings in patients with neonatal seizures focused on the diffuse white matter lesions on diffusion weighted image (dwi) in addition to clinical manifestations. a total of 55 neonates aged less than 1-week old underwent brain mr imaging for evaluation of neonatal seizures between november 2008 and december 2016. among them, 12 patients showed diffuse and symmetric pattern of high signal intensity on dwi. clinical, laboratory, and mr images were analyzed retrospectively. nine patients were males and three patients were females. patient age was 5.2 ± 0.8 days (range, 3-6 days). all the patients were born at full term. the most frequent month of the hospital visit was march (n=4) and january (n=3). eight patients showed generalized clonic seizure and four patients showed partial clonic seizure. stool viral test was performed in nine patients. among them, five patients were positive for the rotavirus and one patient was positive for the astrovirus. nine patients underwent cerebrospinal fluid analysis, however, all showed negative results. mr imaging was performed at 2.2 ± 1.6 days after onset of seizures. diffuse and symmetric diffusion restriction were distributed along the cerebral white matter tracts and both thalami (fig 1) accompanied with high signal intensity on either t2-weighted images or on the fluid-attenuated inversion recovery (flair) sequence. multiple foci of high signal intensity on t1-weighted images at the centrum semiovale that was affected on dwi were also observed. follow-up period was 11.3 ± 9.6 months (range, 1.8 -30.9 months) and developmental delay was encountered in three patients. six patients underwent follow-up mr imaging at the age of 8.4 ± 4.2 months (median, 8.5 months; range, 3.7-13 months). five patients showed volume loss in cerebral white matter on both sides of the brain and four patients showed high signal intensity of the periventricular white matter on either t2weighted images or flair sequences (fig 2) . myelination delay was not observed in follow-up mr images. diffuse and symmetric diffusion restriction involving the cerebral white matters can be seen in patients with neonatal seizures on mr imaging. our study shows that rotavirus is commonly encountered, but not exclusively detected in these patients. nevertheless, viral infection-associated encephalopathy should be considered when a patient is presented with characteristic clinical and mr findings. whole body mri on diagnosis and follow-up of neurofibromatosis type 1 d. grassi, v. tostes, e. caran, h.m. lederman; sao paulo/br demonstrate that whole body mri is effective on showing neurofibromatosis type 1 involvement of different regions of the body not known by the clinicians. review of 41 patients with neurofibromatosis type 1 (nf1) who underwent whole body mri throughout their follow-up with the majority of them had only brain and spine imaging studies. it was possible to demonstrate that whole body mri provides an overview of nf1 systemic manifestations and neurofibroma's extension beyond the clinic expectation. despite being rare, sarcomatous degeneration was suspected when there was any difference on the characteristics of the neurofibromas. whole body overview where its possible to see the neurofibroma's extension in right cervical region, scoliosis and multiple plexiform neurofibromas. only the biggest neurofibroma was detected by clinical exam. however it is possible to identify two others neurofibromas. whole body view of multiple plexiform neurofibromas. whole body mri is a radiation-free exam and it is useful on the diagnosis of nf1 and on patient's follow-up. it provides an overview of the systemic s385 (2017) 47 (suppl 2):s297-s pediatr radiol involvement and neurofibroma's extension beyond the clinical expectation. during patients follow up, it could also show tumor's characteristics modification, which was considered as a possible sarcomatous degeneration. accuracy of non-radiologists and lay-persons for identifying children with cerebral cortical atrophy from 'mercator map' curved reconstructions of the brain s. vedajallam 1 , a. chacko 1 , s. andronikou 2 , e. simpson 2 , j. thai 2 ; 1 east london/za, 2 bristol/uk objective: background: communication of cortical brain atrophy in children with term hypoxic ischaemic injury (hii) to parents and the legal fraternity contesting compensation rights can be very difficult using text and standard cross-sectional images. when demonstrating the cortex in hii, a single image of the brain surface, much like the way a map of the earth is derived from a globe, can be generated from curved reconstruction of coronal magnetic resonance imaging (mri) scans i.e. a mercator map. lay people's ability to identify abnormal scans from such maps without prior training requires evaluation before routine use. aim: to determine the sensitivity and specificity of lay people in detecting abnormal brain scans through review of mercator flat-earth maps of the brain, without prior training. ten mercator map images were provided to 100 participants with a distribution of 5 hii, 1 cortical dysplasia and 4 reported normal. participants were required to identify abnormal scans. sensitivity and specificity overall and for sub-groups were derived by averaging true positives and negatives; false positives and negatives. the results show a strong ability for lay-people to identify normal versus abnormal mri brain studies using mercator maps. the sensitivity and specificity in this group is 67% and 73% respectively. non-radiologist physicians and radiographers performed slightly better than lay people as expected. radiologists of course had very high sensitivity and specificity of 86% and 100%. the mercator map is therefore a viable tool in the communication of complex mr imaging to the lay-person. safety and efficacy of sphenopalatine ganglion blockade in childreninitial experience l. dance, c. schaefer, d. aria, r. kaye, r.b. towbin; phoenix/us objective: sphenopalatine ganglion (spg) blockade is known to be a safe and effective migraine headache treatment among adults. this paper will report the initial experience in the pediatric population with spg blockade. one hundred thirty-three procedures were performed in 85 patients ages 7 to 18 from february through november 2015. pre-intervention headache scores were recorded on a scale of 1 to 10. the procedure was performed supine with neck in hyperextension. anesthesia of the bilateral nares was accomplished with lidocaine spray and gel. contrast was injected using a sphenocath confirming catheter position. 4% lidocaine was injected. patients remained supine with neck in hyperextension for 10 minutes. post-intervention headache scores were recorded. mean pre-treatment score of 5.55 decreased to 3.28 post-treatment (δ2.27, 95% ci 1.34-3.20, p<0.0001). there were no complications. spg blockade is a safe and effective treatment for migraine headaches in children which results in decreased reliance on intravenous drug therapy. orbital masses represent a spectrum of benign and malignant lesions in children that can be challenging to diagnose and treat. imaging plays an important role in diagnosis, due to a potentially limited clinical examination and risks associated with biopsy. mr imaging is a powerful tool for imaging the orbit, due to the excellent tissue contrast it provides. yet conventional mri has a limitation in discriminate the benign from malignant lesions. diffusion-weighted imaging (dwi) is non-invasive rapid technique uses the water diffusibility to produce contrast among different kinds of tissues. our propose was to assess the role of dwi and calculated apparent diffusion coefficient (adc) values in characterization of the pediatric orbital masses regarding benignancy or malignancy. one hundred and thirty patients with recently diagnosed orbital masses and who underwent preoperative conventional mri and dwi were included in this study. the orbit was divided into six compartments: the eye globe, retroocular fat, optic nerve, lacrimal system, bony boundaries and extra-ocular muscles. the average adc obtained from each tumor was compared with the histopathological diagnosis determined from subsequent surgical sample. seventy girls and sixty boys with orbital masses were included in this study. their age was ranged from 1 month to 18 years. the globe is the seat of lesions in 43/130 cases, optic nerve in 27/130 case. seven cases have lesions in the lacrimal gland. forty-five of cases was diagnosed as having benign masses & 85 of cases have malignant lesions. there is a statistically significant difference between the mean adc value of the benign lesions (1.39±0.52 x10 -3 mm 2 /s) and the mean adc value of the malignant lesions (0.69±0.22 x10 -3 mm 2 /s) (p<0.001). the optimal adc cutoff value that was determined for discrimination between these lesions is: 1.075 x10 -3 mm 2 /s), with sensitivity of 97.14% and specificity of 75%. using conventional mri alone in predicting benign and malignant lesions has the sensitivity of 76% and specificity of 91% with 94% positive predictive value and 67% negative predicative value. combining dwi and conventional mri has increased accuracy, as the sensitivity and specificity were 95%, 86% respectively with 93% positive predictive value and 90% negative predicative value. (2017) 47 (suppl 2):s297-s pediatr radiol adc values provide an accurate, sensitive, fast, and non-invasive mean of characterization of pediatric orbital tumors. a priori tumor characterization is useful in timing and treatment planning for orbital tumors. utiliy of resting state fmri in children for preoperative language mapping l.-m. leiber, m. delion, a. ter minassian; angers/fr to assess if resting state fmri is able to detect language eloquent areas in childrens. six children, from 8 to 15 years old suffering from brain lesions were enrolled in this study. they underwent mri with one 3dt1 morphology session and three 10 minutes fmri sessions, including one resting state fmri and two language task induced activity fmri sessions. analysis was performed using a generalized linear model for the first one and a spatial independent component analysis approach for the two others. language maps were compared with cortical mapping obtained by intraoperative direct stimulation. language network was identified systematically by resting state session but not by task induced activity sessions. moreover, in two of the six patients, resting state fmri was able to detect eloquent areas found during intraoperative cortical mapping that were not present in task induced activity sessions. resting state fmri appears superior to task induced activity fmri in detecting language eloquent areas. is sclerotherapy an effective treatment option for ranulas or thyroglossal duct cysts in children? d. aria, l. dance, c. schaefer, r. kaye, r.b. towbin; phoenix/us to assess the utility of sclerotherapy in the treatment of ranulas and thyroglossal duct cysts materials: from 2015-2016, 8 patients varying in age from 16 months to 32 years were referred to the ir department for sclerotherapy. of the 8 patients, 6 had a diagnosis of ranula while 2 had the diagnosis of thyroglossal duct cyst by either mr, ct, or us. sclerotherapy treatments were performed with standard sclerosing agents, i.e. sotradecol 3% foam, absolute ethanol, and bleomycin. in the subset of patients with ranulas, sclerotherapy was commonly performed in accordance with salivary (submandibular and/or sublingual) gland botox injection or ethanol ablation. 22-gauge or 5f sheathed needles were used for us-guided access to the lesions, with ranula sclerotherapy being performed after placement of side-hole drainage catheters (5-10f) due to their increased viscosity. the preferred sclerosing agent was injected with dwell times ranging from 15 mins to 3 hours. salivary gland injection/ablation was performed under usguidance using a 22-gauge needle with volume injection targeted centrally within the gland or in the portion of the gland abutting the ranula. after treatment, all patients were scheduled for follow-up ultrasounds at a minimum of 8 weeks to assess lesion response or residual disease. a total of 23 sclerotherapy treatments were performed. of the 8 patients, 2 were lost to follow-up after single sessions for ranula and thyroglossal duct cyst. the other 6 patients all had follow-up ultrasounds after each of the remaining 21 sclerotherapy sessions. four of these patients showed initial improvement with either decreased size of lesion or lesion resolution while the other 2 showed no improvement with either stable or increased size on initial follow-up. the 4 patients who initially showed promising response unfortunately had recurrence on follow-up imaging and ultimately, demonstrated no favorable response to sclerotherapy after subsequent treatments regardless of whether treatment was combined with ethanol/botox salivary gland injection. in summary, all 6 patients who were successfully followed show no appreciable response to treatment for ranula or thyroglossal duct cyst. despite the emergence of clinical requests for sclerotherapy of ranulas and thyroglossal duct cysts, in our case series, sclerotherapy has not proven to be an effective treatment option using our current drug regimen. role of the susceptibility-weighted imaging (swi) in the neuroimaging of term newborns g. rudas, e. varga, p. barsi, l. kozák, ü. méder; budapest/hu objective: susceptibility-weighted imaging (swi) was introduced in the neonatal neuroimaging only a few years ago. we can find only a few publications about its advantages and disadvantages. according to our experience, swi is extremely useful not only for detecting bleedings but for the diagnosis of other diseases as well. during the last year we had 164 mri examinations on term newborns (0-14 days of life) and the swi gave additional information in 54 cases. we used a 3t philips insignia scanner. in the case of the questionable hypoxic-ischemic-encephalopathy (19 cases) and the metabolic diseases (5 cases) we could find increased signal intensity in the cortex; in the case of stroke we could find the thrombus itself in 6 cases; the avm were much clearer using the swi in 3 cases; at the pvl in 3 cases we could visualize the cysts better using swi; in the case of congenital heart disease (11 cases) and in the case of sinus thrombosis (4) we could find microbleedings and/or dilated veins; in 3 cases the position of the lateral ventricle drain or shunt was much clearer using the swi. the swi gave important additional information in 54/164 (33%). the swi is a strongly recommended new sequence at the mri examination of the term newborns' brain. a disadvantage of swi is that it requires ca. three minutes examination time (in contrast to t2* which is only 1 minute long). mechanical birth-related trauma: imaging of the "accidents of birth" a. chaturvedi, j.g. blickman; rochester/us objective: 1. to discuss definition, incidence and risk factors leading to "mechanical birth-related trauma" and compare these with existing literature. offer an organ-system based classification scheme encompassing the varied manifestations of birth-related trauma and describing the implications on care decisions. materials: the hospital imaging department database was searched for neonates who presented with history of difficult/traumatic birth at our obstetric center between january 1, 2013-june 30, 2016. search software used was primordial customised radiology solutions, san mateo, ca. the search terms used were "macrosomia", "shoulder dystocia", "instrumental delivery", "malpresentation", "cephalopelvic disproportion", "forceps" and "vacuum". initial and follow-up imaging and clinical data on these neonates was reviewed and compiled by two board-certified pediatric radiologists. the relevant literature was reviewed and findings compared. organ-system based classification scheme for birth-realted trauma. in our study, mechanical trauma of birth was seen to manifest within different organ systems, which have been listed below in the order of occurrence within our sample. injuries to the skull (sutural overlap, dents and fractures), scalp hemorrhages (subgaleal hematoma, cephalhematoma, caput). intracranial intraand extra-axial hemorrhages (subdural, subarachnoid, epidural, intraparenchymal). clavicle fractures neonatal brachial plexus injury. sternocleidomastoid hematomas. adrenal hemorrhages. cervical spinal cord contusions. schematic diagram depicting intra-and extracranial hemorrhages by location. 6-year-old with history of calvarial fracture at birth-fracture did not heal but enlarged secondary to leptomeningeal entrapment at the fracture sitean entity called "growing fracture" or "leptomeningeal cyst". multiple newborn organ systems can be injured from mechanical trauma of birth. our numbers compare favourably with the existing literature. mechanical birth-related trauma can occur simultaneously with hypoxic-ischemic birth injury. although most of these injuries spontaneously and completely resolve, long-term complications can be seen in some cases. few of these injuries are life-threatening. imaging plays a crucial role in diagnosis and follow-up, and can assist in decision making as well as in counselling the parents. ewing sarcoma of tibia in an infant girl a. seehofnerova, j. skotáková, i. červinková; brno/cz objective: ewing sarcoma (es) is the second most common primary bone malignancy in children. it histologically originates from neuroectodermal tissue and consists of small round blue cells. ewing tumour family is very close to primitive neuroectoderm tumour (pnet) family with diverse stage of differentiation, ewing sarcoma being less differentiated. approximately 50 % of the cases occur between ten and twenty years of age with slightly higher prevalence in male gender. nine-month-old caucasian girl presented to local surgery department after she had wedged her lower leg in a bed. the right lower leg was swollen and painful. she was initially diagnosed with a ligament injury and underwent standard treatment. oedema gradually disappeared, but swelling and pain increased after three weeks. she also suffered from a fever of 38.1°c (100.58 o f). at that point x-ray of her right lower leg was performed with report describing pathologically changed structure of tibia and she was referred to our university centre. (2017) 47 (suppl 2):s297-s pediatr radiol we made a second reading of the plain film, reporting sclerotic heterogeneous bone structure of the right tibial diaphysis and distal metaphysis, onion-like periosteal reaction with sunburst spiculation and cortical bone destruction. her laboratory results were: crp 7.2 mg/l, ld 6.2 μkat/l, nse 44.3 μg/l, ferritin 18 μg/l. crp has been raising for a week to 25 mg/ l, then decreased to normal level. differential diagnosis was established as a primary bone malignancy (especially es) or, less likely, an osteomyelitis. mri revealed pathological signal of bone marrow of diaphysis of the whole tibia with cortical scalloping and periosteal spiculated apposition. epiphyses were spared. dorsal cortical bone was interrupted with extraosseal spread of the process. intraosseal part enhanced heterogeneously, whereas extraosseal component enhanced almost homogeneously after contrast medium administration. total size of the tumour was assessed as 97x23.5x20 mm (22.8 ml) . adjacent muscles were oedematous with post-contrast enhancement. there were also few enlarged lymph nodes in popliteal region. results from the biopsy confirmed ews with positive ews/fli1 gene. tumour was assessed as a localized disease, enneking iib. patient underwent chemotherapy according to aews1031doc protocol and a knee-exarticulation with no traces of tumour in resection lines. nowadays she is in the first complete remission. x-ray: ap view mri: etw1_tse postcontrast, sagittal view, pre-treatment mri: etw1_tse postcontrast, sagittal view, after initial treatment unique teaching points: ewing sarcoma belongs to common primary bone tumours in children but is a very rare unit in infants. despite the age predilection it is necessary to consider this diagnosis even in children younger than one year of age. scimitar syndrome together with pulmonary sequestration and horseshoe lung: congenital pulmonary venolobar syndrome b.e. derinkuyu, h.n. özcan, y. tasci-yildiz, h g. cınar, u.a. orun; ankara/tr objective: congenital pulmonary venolobar syndrome (cpvls) comprises of a spectrum of pulmonary developmental anomalies. the main components of cpvls are hypogenetic lung partial anomalous pulmonary venous return, absence of pulmonary artery, pulmonary sequestration, systemic arterialization of lung, absence of inferior vena cava. minor components of cpvls include tracheal trifurcation, eventration and partial absence of the diaphragm, phrenic cyst, horseshoe lung, esophageal and gastric lung, anomalous superior vena cava, and absence of the pericardium. in this case presentation, we present a baby with scimitar syndrome, pulmonary sequestration, horseshoe lung and right aberran subclavian artery. a 3 month-old girl was admitted to our hospital with the suspicion of scimitar syndrome from a different hospital. she did not have any symptoms. the physical examination was unremarkable. on plain radiograph, the baby had dextrocardia. there was a doubtful tubular structure with the shape of scimitar and a nodular radioopacity behind the heart (figure 1 ). transthoracic echocardiography demonstrated the dextrocardia, atrial septal defect and the right pulmonary artery hypoplasia. afterwards, the ct angiography was done for confirmation of scimitar syndrome and other accompanying abnormalities. on the ct angiography, there was a partial anomalous pulmonary venous return to the suprahepatic inferior vena cava known as scimitar syndrome. besides this, there was a right pulmonary extralobar sequestration in the lung base. the arterial supply was arising from the celiac trunk, while the venous drainage was going directly to the inferior vena cava. therefore, the right lung was hypoplastic of which the tongue of the right pulmonary parenchyma passing between the aorta and heart, appearing confluent with the left lung in a horseshoe configuration. there was dextrocardia and right aberran s389 (2017) 47 (suppl 2):s297-s pediatr radiol subclavian artery. the patient was subjected to catheterization and angiography for treatment. on plain radiograph, the baby had dextrocardia. there was a doubtful tubular structure with the shape of scimitar and a nodular radioopacity behind the heart unique teaching points: the term cpvls is an umbrella to a group of pulmonary parenchymal and vascular anomalies that may present in combination. mdct is a helpful diagnostic tool in the preoperative evaluation for delineation of the components of this syndrome. congenital pulmonary venolobar syndrome refers to a wide spectrum of pulmonary developmental anomalies that may appear single or in combination. the main components of congenital pulmonary venolobar syndrome are hypogenetic lung (including lobar agenesis, aplasia, or hypoplasia), partial anomalous pulmonary venous return, absence of pulmonary artery, pulmonary sequestration, systemic arterialization of lung, absence of inferior vena cava, and accessory diaphragm. in this case presentation, we describe a child with scimitar syndrome, bilateral sequestration, hypogenetic lung (single lobed left lung) and right aberran subclavian artery. an 8 year-old syrian girl was admitted to our hospital with the history of heart defect. she did not have syncope or ciyanosis whereas she has easy fatigue and palpitation. on plain radiograph the anomalous draining vein was seen as a tubular structure paralleling the right heart border in the shape of a turkish sword ("scimitar") ( figure 1) . transthoracic echocardiography demonstrated the scimitar vein as well as large patent ductus arteriosus (pda), atrial septal defect and left pulmonary hypoplasia. afterwards, the ct angiography was done for confirmation of scimitar syndrome and other accompanying abnormalities. on the ct angiography, there was a partial anomalous pulmonary venous return to the suprahepatic inferior vena cava known as scimitar syndrome. besides this, there was a bilateral intralobar pulmonary sequestration in the lung bases. the arterial supply of the right side was arising from the celiac trunk, while the left side feeding artery was originating directly from the descending aorta. therefore, the left lung had a single lobe with single pulmonary vein draining to left atrium. there was a large pda and right aberran subclavian artery. the patient was subjected to catheterization and angiography for treatment. the right sided anomalous draining pulmonary vein and the feeding artery of the right sequestration were closed in the first session. the procedure was completed without any complication. afterwards, the closure of the feeding artery of the left pulmonary sequestration and the pda were planned in the next sessions. on plain radiograph the anomalous draining vein was seen as a tubular structure paralleling the right heart border in the shape of a turkish sword ("scimitar") unique teaching points: congenital pulmonary venolobar syndrome comprises a heterogeneous group of uncommon abnormalities that may occur in combination. diagnosis of congenital pulmonary venolobar syndrome can be confirmed by ct angiography that allows detailed evaluation of vascular, tracheobronchial, and pulmonary parenchymal abnormalities with a single short, noninvasive procedure. neck infection disclosing diagnosis of congenital fourth branchial arc anomaly in a girl h.n. özcan, z. aycan, b. ardıclı, m. haliloglu; ankara/tr objective: congenital branchial arc anomalies are rare entities. herein, we describe the imaging findings of acute suppurative infection of the neck caused by fourth branchial fistula in a child. case presentation: an 11-year-old girl presented to our pediatric emergency department with fever, left sided neck swelling and redness. her complaints were started five days ago. on her physical examination, there was a 4x4 cm, stiff, painful mass lesion with redness on the left side of the neck. blood count and thyroid function tests were in normal range; however, c-reactive protein level and erythrocyte sedimentation rate were elevated. neck ultrasonography revealed diffuse soft tissue swelling, a hypoechoic mass consistent with abscess in the left thyroid lobe and perithyroid tissue. the left lobe of the thyroid gland had poorly defined margin and a focus of air. contrast-enhanced neck mr imaging demonstrated an abscess in the left thyroid and perithyroid tissue ( figure 1) and enhancement of the soft tissue plane around the left pyriform fossa (figure 2 ). barium swallow revealed the sinus tract originating from the left pyriform sinus apex. the patient was operated after antibiotic treatment and sinus tract was surgically excised. the aim of this report is to describe three cases of right kidney wilms' tumor with cavoatrial tumor extension, referred to our institution between january and september 2016. case presentation: three children, two girls (3 and 7 years old) and one boy (2 years old) were admitted at the emergency service with cardiac failure symptoms; the latter had also liver failure. echocardiography showed right atrial thrombus in all three patients, as an extension of massive obstructive thrombosis of the inferior vena cava (ivc). abdominal ultrasonography revealed in all patients a right renal mass, associated to right renal vein thrombosis that extended to the ivc and to the right hepatic vein. contrast enhenced computed tomography confirmed findings. patients were treated primarily with chemotherapy before surgery, with partial regression of the thrombus in two patients and no response in one. (2017) 47 (suppl 2):s297-s pediatr radiol unique teaching points: wilms' tumor is the most common renal malignancy in children and its intravascular extension is a well-recognized event. incidence of tumor extension to inferior vena cava (ivc) is reported to be of 2-5% and intra-atrial extension of 0,2-1,2%. it occurs most commonly in tumors located in the right kidney (probably due to the shorter path of the right renal vein compared to the left one). this complication does not directly influence the prognosis of malignancy, but the degree of intravascular extension determines technical surgical strategy and increases difficulty of the surgical procedure, especially when there is intracardiac involvement, which increases morbidity. several classifications have been proposed in the adult age group, but due to the similarity of the degree of intraoperative difficulty, the same classifications are used in children. pritchett et al. (1986) described the relation between thrombus and hepatic vessels: level i -intrahepatic intravascular extension; level iiintrahepatic extension; and level iii -suprahepatic or atrial extension. staehler et al. (1987) proposed a different classification that was posteriorly modified and detailed by daum (1994) : stage i -small extension (thrombus size within ivc <5 cm); stage iilarge thrombus (> 5 cm within the ivc), but still below the hepatic vessels; stage iii -thrombus extending to the level and above the hepatic vessels; stage iv -intra-atrial thrombus. a 4 year old boy presented with a soft tissue mass in his forearm which appeared to have grown quickly in size over a period of three to four months. physical examination demonstrated a welldefined mass in the dorsal aspect of the forearm with no pulsatile bruit. intial differentials included a vascular anomaly or a sarcomatous lesion. the patient proceeded to have an ultrasound examination which revealed a very well-defined heterogenous subcutaneous mass, mostly solid in substance. the lesion measured 3.5 cm x 1.6 cm x 3.5 cm (transverse x length x depth). there was no evidence of muscle invasion. prominent internal arterial vascularisation was demonstrated and the mass was classed as inderminate in nature. subsequent mr findings demonstrated a mass with t1 signal isointense to muscle, hyperintense t2 signal and marked homogenous enhancement. small foci of intralesional t1 hyperintensity and larger areas of t2* gradient hypointensity were noted, in keeping with small areas of intralesional blood. vessels were seen to extend from the subcutaneous fat into the lesion. the mass slightly distorted the underlying extensor muscles and tendons of the forearm but there was no deep extension across the fascia. findings deemed the lesion to be more malignant in nature. the patient underwent incisional biopsy and histological findings confirmed a diagnosis of angiomatous fibrous histiocytoma. these tumours are rare soft tissue tumours which most commonly occur in children, adolescents and young adults. while it is rare, there is a potential for local recurrence and metastasis. therefore, it is essential to identify these tumours where possible or at least consider them as a differential for a soft tissue mass in a child. the surgeon commented that the imaging findings and report were essential in making the initial decision about whether to perform an incisional or excisional biopsy as the best treatment for the tumour is wide surgical excision with clearance of margins. unique teaching points: angiomatous fibrous histiocytomas are rare lesions with potential for recurrence and metastasis and therefore should be identified and managed appropriately as a malignant tumour. they are often confused as soft tissue haemangiomas or complex haematomas. it is very important to be aware of the presentation and imaging findings, remembering this form of tumour as a key differential for a soft tissue mass. nasopharyngeal anlage tumor in a neonate with the initial presentation of respiratory difficulty: correlation between imaging and clinicopathologic findings p.-s. tsai, d.-c. lin, s.-l. shih; taipei/tw the etiologies of nasal or nasopharygeal obstruction are variable in neonates. the respiratory symptoms are varied in these cases. mass lesions in nasal cavity or nasopharynx are extremely rare during the neonatal period. however, we must keep it in mind when respiratory problems occur in the neonatal period. here, we report a case presenting with sleep apnea resulting from nasal obstruction by a rare benign salivary anlage tumor in nasopharynx and discuss the imaging findings as well as clinicopathologic characteristics. the 28-day-old female infant had loud breathing sound, slow feeding and sleep apnea since birth. nasal endoscope and laryngoscope disclosed a polypoid tumor in nasopharyngeal cavity with a stalk connecting with posterior nasal septum. further magnetic resonance imaging (mri) revealed a lobulated mass about 1.6 cm in greatest diameter occupying posterior nasal cavity to the nasopharynx that was intermediate signal intensity on t1-weighted/t2-weighted images and heterogeneous gadolinium enhancement. the patient then received endoscopic resection. the tumor was shown locating in nasopharyngeal cavity and having a stalk from posterior nasal septum, partially occluding the choanae as well. resected tissue fragments displayed tan and whitish in color grossly. microscopic examination demonstrated duct-like structures and mesenchymal elements in a nodular pattern which are typical features of salivary gland anlage tumor. until now, there is no tumor recurrence for four years. unique teaching points: "salivary gland anlage tumor (sgat)" was firstly introduced in a report by dehner et al in 1994 . the tumor that has histologic resemblance to the developing salivary gland, is believed to be a hamartoma originating from minor salivary gland rather than a true neoplasm. congenital sgat displays male predilection and is a rare cause of neonatal airway obstruction. the mass is usual in the midline and attached to the posterior nasal septum or posterior nasopharygeal wall by a delicate pedicle. favorable results with simple excision are obtained. once massrelated airway obstruction is established, further examination with computed tomography (ct) or mri is helpful in anatomic evaluation, size measurement, characteristics definition and intracranial involvement. if mass induced airway obstruction is suspected in a neonate and sgat is considered based on imaging studies, invasive procedure should be careful due to the potential of tumor dislodgement from its fine pedicle resulting in complete airway obstruction. the association of intussusception with malrotation is referred to as waugh syndrome. [1] malrotation occurs in approximately 1 in 500 live births. [2] the incidence of malrotation amongchildren with intussusception is 40%. we hereby present a case report of waugh's syndrome associated with midgut volvulus. case presentation: a 5 month old male child reported to the emergency department with the clinical history of vomiting, abdominal distension, bloody mucoid stools and incessant cry. routine blood examination revealed hb: 12.9 gm%, tlc: 14500/cu mm, plt-2. 9lac/cu. mm. ultrasound (us) examination was performed and it revealed dilated fluid-filled small bowel loops with moderate amount of free fluid, right iliac fossa showed bowel within bowel appearance suggestive of target/pseudo kidney sign of bowel intussusception. no pathologic lead point was identified. transverse ultrasound image through the upper abdomen showed superior mesenteric vein noted to the left of the superior mesenteric artery hence malrotation should be considered. in view of surgical emergency non contrast enhanced ct was done and axial image showed target/sausage shaped soft tissue density mass it had alternating areas of low and high attenuation due to bowel wall and mesentry. on emergency laparotomy patient was found to have intestinal malrotation with duodenojejunal junction on the right of the midline and 180 mid gut volvulus in clockwise direction. intussusception with terminal ileum (gangrenous), caecum, appendix, whole of ascending colon, transverse colon were telescoping into descending and sigmoid colon. the volvulus was derotated and the in tussusceptum was reduced. the gangrenous terminal ileum and appendix was resected and ladd's procedure was done, a diverting ileostomy was created. the patient recovered uneventfully after which an ileo-colonic anastomosis was created transverse ultrasound shows a mass with a swirled appearance of alternating hypoechoic and hyperechoic "bowel-within-bowel" appearance (target sign) unique teaching points: on ultrasonography multiple, concentric, target like appearance of wall layers of invaginated segments (target sign) on axial scan, as well as pseudokidney sign (sandwich sign) on longitudinal scans were accepted as diagnostic criteria for intussusception. [8] it can assess the relative positions of the smv and sma which are mostly abnormal in malrotation. upper gastrointestinal contrast study is the imaging reference standard for diagnosis of malrotation with or without volvulus. abnormal position of the duodeno-jejunal junction. spiral, "corkscrew" or z-shaped course of the distal duodenum and proximal jejunum, and location of the proximal jejunum in the right abdomen. [2] a high degree of clinical suspicion and radiologist's awareness of this entity is helpful in guiding the surgeons towards diagnosis and prevention of morbidity and mortality. a rare case of epidermal naevus syndrome p. joshi; pune/in to acquaint the radiologists with the entity of epidermal nevus syndromes (enss) which are a group of rare complex disorders characterized by the presence of skin lesions known as epidermal nevi associated with additional extra-cutaneous abnormalities, most often affecting the brain, eye and skeletal systems case presentation: this one and a half year old child was referred to us for neuroimaging. he had multiple hairy naevi over his face, limbs including the palms, since birth, associated with blackish discolouration of his entire trunk. unique teaching points: epidermal nevi are overgrowths of structures and tissue of the epidermis, the outermost layer of the skin. the different types of epidermal nevi can vary in size, number, location, distribution and appearance. neurological abnormalities that can be associated with enss can include seizures, cognitive impairment, developmental delays and paralysis of one side of the body (hemiparesis). skeletal abnormalities can include abnormal curvature of the spine, the term "epidermal nevus syndrome" has generated significant controversy and confusion in the medical literature. originally, the term was used to denote a disorder that was actually several different disorders erroneously grouped together. in the recent past, the term was used to denote a specific disorder now known as schimmelpenning syndrome. however, the term epidermal nevus syndrome could be correctly applied to several different disorders. therefore, the umbrella term "epidermal nevus syndromes" now represents a group of distinct disorders that have in common the presence of one of the various types of epidermal nevi. however, there is so far no general agreement how to classify the types of this diverse group of disorders, adding to the confusion within the medical literature. these disorders are quite different from one another and are not "variants" of each other as is sometimes mistakenly stated in the medical literature. in the future, as the genetic molecular basis of these disorders is better understood, the classification may change or expand. bilateral axillary lump in a newborn diagnosed as hematoma h.n. özcan, u. aydingoz, m. haliloglu; ankara/tr objective: most birth traumas are self-limiting and have a favorable outcome. injuries to the infant that result from mechanical forces during the birth process are not uncommon. they occur most commonly on head and neck after vaginal breech delivery. however, soft tissue hematomas can be rarely seen after caesarian section (c/s). herein, we describe imaging findings of a newborn with bilateral axillary lump diagnosed as hematoma. case presentation: a 31-year-old woman was admitted to an outside hospital at 38 weeks' gestation for c/s due to prior caesarean operation. it was her fourth pregnancy (g4 p2). the pregnancy was unremarkable and she had normal ultrasounds at gestation. there was no history of trauma or fall during antenatal period. according to the c/s reports, the process of operation was uneventful any undue prolongation and without having used any other instrumentation. the weight of the female baby was 3.1kg at birth. on the 3 rd postnatal day, her mother noticed a left axillary swelling, then admitted to a tertiary children's hospital. her physical examination revealed, bilateral axillary asymmetry with a fluctuant, nontender swellings. there was no redness or discoloration of the skin. there was no clinical feature suggestive of trauma or bleeding diathesis. a superficial ultrasonography showed solid heterogeneous, hyperechogenic masses 38x35 mm in the left axillary region and 15x13 mm in the right side. doppler study did not reveal any flow in the masses. contrast enhanced mr imaging demonstrated, bilateral axillary mass lesions with fluid levels and smooth contours (figure 1 and 2) . t1w images demonstrated hyperintense component suggesting hemorrhage. after the administration of the gadolinium-based contrast material, lesions showed peripheral enhancement (figure 3) . a diagnosis of hematoma was entertained. the child was managed non-operatively. she was monitored clinically and radiologically. follow-up ultrasounds scan revealed significant regression of the swellings. unique teaching points: soft tissue hematomas can be rarely seen in newborns. the formation of axillary hematoma on the background of c/s is a rare complication, which, to the best of our knowledge, has not been previously reported. ultrasonography and mr imaging readily depicts hematoma and aids in the differential diagnosis. colorectal carcinoma (crc) is extremely rare in pediatric age, with an estimated annual incidence of approximately 1 case per million individuals. the majority of reported cases occur in adolescence, while the incidence is further lower for children under 10 years. the distribution between males and females is not equal, with higher prevalence in males (ratio of 2:1). the etiology in children is unclear as these tumors are often sporadic and not linked to a preexisting adenomatous polyp, unlike adults. predisposing factors such as familial polyposis of the colon, other polyposis syndromes, ulcerative colitis and familial multiple cancer syndromes were reported in 10 % of cases. advanced stage at diagnosis, aggressive histologic subtypes (poorly differentiated, signet ring and mucinous adenocarcinoma) and poor survival are the hallmarks of pediatric crc. case presentation: a 15-year-old male presented with a history of dyspeptic symptoms (recurrent epigastric-right flank colic pain and heartburn) for the last eight months, without evidence of irregular bowel function. after a prior diagnosis of esophagitis secondary to a gastroesophageal reflux disease, physical and laboratory examinations revealed anorexia, progressive body weight loss, microcytic iron deficiency anemia and positive fecal occult blood test. during an emergency access, abdominal ultrasound identified rounded target liver lesions and circumferential heterogeneous mural thickening of the ascending colon. contrast-enhanced computed tomography scan (cect) demonstrated a marked circumferential wall thickening of the ascending colon and cecum with a longitudinal extension of 85 mm and thickness of 22 mm; the mass contained lowdensity areas and calcifications. furthermore 5 hypovascular hepatic lesions along with lymph node metastases containing calcifications were identified. no lung metastases were found. histopathological analysis confirmed the diagnosis of metastatic colon adenocarcinoma. after chemo-and radio-therapy, only the hepatic lesions showed reduction in size and number. the patient subsequently underwent right hemicolectomy. one month after surgery he is in a rigorous follow-up through ultrasonographic evaluation of pleural effusion and ascites and cect. unique teaching points: crc, although rare, should be suspected in children presenting with unexplained persistent abdominal pain, progressive body weight loss and positive fecal occult blood test. ultrasound imaging can be appropriate in the preliminary detection of abnormal bowel wall thickening, lymph node and liver metastases; cect is mandatory to confirm the radiological diagnosis and complete the staging. to increase awareness of this rare syndrome and its varied presentation in order to facilitate its early diagnosis and treatment to prevent poor prognostic outcomes. case presentation: lemierre syndrome is a rare disease characterized by an initial infection of the head and neck leading to the development of a septic thrombophlebitis which has a propensity to spread and involve the jugular and facial veins. this progressive infection then leads to the development of metastatic septic emboli to the respiratory tract. we present the case of a 7 year old boy who attended with a 1 week history of fever and a cough. initial imaging on admission demonstrated a large left sided hydropneumothorax with multiple cavitating lesions throughout the lung parenchyma in addition to thrombosis of some of the segmental pulmonary veins. the hydropneumothorax was surgically drained and the patient was transferred to the paediatric intensive care unit after further deterioration with the development of a broncho-pleural fistula. following a short course of antibiotics there was no clinical or radiological improvement and sputum cultures grew coliform organisms which raised suspicion for a more distant source. when pus was noted to be discharging from the left ear, a contrast enhanced ct of the head and neck revealed a left mastoiditis with multiple cerebral abscesses and occlusive thrombi in the left jugular vein, transverse venous sinus, sagittal and straight sinuses. following this diagnosis antibiotic therapy was modified and targeted at anaerobes, which was vital in assisting the patients recovery and successful discharge home. unique teaching points: clasically the majority of lemierres syndrome begins in the oropharynxinvolving the palantine tonsils and peritonsillar tissue often presenting with fever, sore throat and neck pain. our case demonstrates an atypical presentation with sepsis and respiratory symptoms as a result of the septic emboli which delayed diagnosis. we have learnt from this case the importance of considering lemierres syndrome in patients presenting with signs of a respiratory infectionin particular cavitating pulmonary lesions-that have not improved with conventional therapy and to have a low threshold to investigate the head and neck as a potential source of infection. when the working hypothesis of meningitis could not help e. kovacs 1 , n. pinter 2 , g. balázs 1 , a. machovitsch 1 , a. arany 1 , z. liptai 1 , l. fonyad 1 , p. benke 1 ; 1 budapest/hu, 2 amherst/us objective: neuroinfection still represents a diagnostic challenge in the everyday practice, where clinical evaluation, imaging, laboratory and pathological workup and treatment goes hand in hand under the pressure of time. we summarized a case in which, despite the extensive multilateral collaboration the battle was lost, to bring attention to the possible causes. a two year old, previously healthy female was taken to the emergency department for altered state of consciousness and fever. she also suffered from gingivitis. the unconscious child underwent an emergency ct scan: hydrocephalus with signs of raised intraventricular pressure was detected. subsequently mri of the head and spine was performed, and showed signs of diffuse leptomeningeal enhancement with basal predominance. multiple dwi restricted parenchymal lesions with basal predominance were also found. repeated csf and blood tests did not reveal any causative organism, although the gradually increasing crp suggested infection. two weeks after the onset of symptoms a follow up mri study showed extensive cerebral and spinal swelling with no focal lesion. the child passed away three days later due to cardiac failure. autopsy and neuropathological evaluation could not reveal the cause of the disease, which was identified only weeks after the child died, by culturing sputum and csf. unique teaching points: an overview of the clinical and radiological presentation of meningitis basilaris is carried out. attention is given to the circumstances, when tuberculotic infection should be suspected, and antituberculotic treatment should be started, even before the confirmation of the presence of mycobacteria can be obtained. to describe the clinical, laboratory and mri findings of chronic nonbacterial osteomyelitis(cno) in a patient with a negative radiograph and emphasize useful imaging findings, including an unusual radial pattern of edema in both femoral heads. case presentation: a 14-year-old adolescent, was referred with progressive debilitating hip pain and inability to walk since 5 days, that was unsuccessfully treated with non-steroidal anti-inflammatory drugs. during hospitalization he developed fever up to 38 ο with normal full blood count and smear, elevated esr (95mm/h) and crp (12.8 mg/dl), positive serologic markers for streptococcus (asto) and ebv and received antibiotics with relative good response. blood cultures did not grow any pathogens, the rest of serology was negative for acute infection, tuberculin skin test was negative and immunological investigation was unremarkable. pelvic radiographs were negative. mri showed a symmetric pattern of bone marrow involvement around both triradiate cartillages, at both femoral heads and (2017) 47 (suppl 2):s297-s pediatr radiol major trochanters. complementary evaluation of tibial areas with a limited protocol disclosed asymptomatic involvement of tibial epiphyses and apophyses. a radial pattern of edema was seen at the femoral heads with alternating stripes of involved and uninvolved areas. clinical course and imaging appearances were highly suggestive of cno. rapid clinical improvement occurred during hospitalization while a repeat mri 6 months later showed complete resolution of hip findings and the patient was free of any symptoms or signs. coronal stir sequence at presentation showing the radial pattern of bone marrow edema (arrowheads) alternating with stripes of normal marrow (*) at both femoral epiphyses. note hyperinense edema (arrows) around triradial cartillages. coronal stir sequence showing the predilection of bone marrow edema symmetrically around triradial cartillages (arrows) and at major and minor trochanters (arrowheads). coronal stir sequence at 6-months follow-up shows resolution of edema. unique teaching points: cno is a not well known chronic autoinflammatory bone disorder affecting primarily children and adolescents. positive serology for streptococcus or other infectious agents has been previously reported as in our case and may be a triggering factor. striking mri findings with a negative radiograph may occur at initial stages. symmetrical distribution of non-specific bone marrow edema around epiphyses and apophyses is highly suggestive of the diagnosis in the appropriate clinical setting and following exclusion of suppurative bone infections as well as bone or hematologic malignancies. the radial pattern of edema in our patient is unusual and considered to comply with the direction of main trabecular systems in femoral heads. in 26/31 chest cts, 103 nodules (median size 2.0 mm) were detected. display mode a with 5 mm mip yielded the best interreader variability (κ=0.294) and the highest sensitivity (73.9%) compared to mode b and c (κ=0.218, sensitivity 61.2% and κ=0.238, sensitivity 59.3%, respectively). perifissural nodules were detected in all subgroups. conclusion: mip improves the detection of pulmonary nodules in chest cts of young children, but overall interreader agreement is only fair. nodules, including perifissural nodules, occur in children with and without malignancy. images were subsequently read and interpreted by board-certified radiologists and nuclear medicine physicians in communal reading. in case of identifying suspicious lesions in cect additional imaging (mri) or biopsy was performed. compared to pet/ct employing only low dose ct (ldct), the use of cect resulted in the identification of 19 additional suspicious lesions in 13 patients. furthermore the use of cect allowed us to qualify 3 lesions as benign/ physiologic which in pet/ldct were identified as suspicious and 16 lesions suspect for metastases or tumor. in those 37 patients who received combined integrated fdg pet/ct including both ldct and cect the ctdi ranged in between 1,3-15.5 mgy (n= 10.2 mgy) and the dose length product (dlp) ranged in between 82.4-2464 mgy *cm (n= 812.6 mgy *cm) specificity was significantly higher combining pet and ct compared to stand-alone ct and pet. our study showed that the acquisition of cect in combined integrated pet/ct leads to an increased specificity and thus represents an essential component of a good fdg pet/ct in pediatric oncology. in assessment of lymph nodes, inflammatory foci and liver lesions diagnostic contrast enhanced ct is essential. comparison of the detectability of ubos in neurofibromatosis type i patients with proton density-weighted and flair sequences in 3t mri l. porto, s. lescher, n. hillenbrand; frankfurt/de objective: neurofibromatosis type 1 (nf1) is an autosomal-dominant congenital disease. in nf1 patients, significant numbers of so-called unidentified bright objects (ubos) can be found in brain imaging, with predilection sites at the basal ganglia and the dentate nucleus. ubos seem to develop at a very early age, contrary to other criteria leading to diagnosis. the detection of ubos might therefore prove helpful in the early diagnosis of nf1, complementing the clinical diagnosis based on criteria of the "national institutes of health consensus development conference". the aim of the study was to investigate whether the detectability of ubos increases at 3t by comparing proton density-weighted images (pdw) with fluid-attenuated inversion recovery (flair) sequences. a total of 14 nf1 patients (7 male, 7 female, between 8 and 26 years old, mean age 15.4 years) were examined by a 3t magnetic resonance scanner. the presence of ubos was evaluated on pd-w and flair images by 4 evaluators (2 experienced neuroradiologists, 1 junior radiologist and 1 student in his final year). detectability was rated by a three-point scoring system for dedicated regions: lesions which were "well defined/detectable", "suspicious" or "detected after a second look". the wilcoxon signed-rank test was used for comparisons between the raters. the level of significance was p<0.05. significantly more lesions were marked as "well defined/detectable" in the pd-w sequence compared to flair (p<0,001 for all four evaluators together, as well as for each evaluator separately). in particular, pd-w proved to be superior for detecting ubos located in the medulla oblongata (p=0,001) dentate nucleus (p=0,002) and hippocampal region (p=0,007), regardless of the level of the raters' experience. this is the first study that compares flair and pd-w at t3 for the diagnosis of ubos in nf1. significantly more ubos are detected in the pd-w compared to flair sequences, especially for the infratentorial regions. as ubos occur at very early stages of the disease in patients with suspected nf1, pd-w might aid an early diagnosis in these patients. assessment of radiation doses from diagnostic imaging in the followup of paediatric oncology patients p. logan 1 , r. harbron 2 , k. mchugh 1 ; 1 london/uk, 2 newcastle-upon-tyne/uk objective: previous literature (1,2) has suggested paediatric oncology patients accumulate a large radiation burden as a consequence of routine diagnostic imaging examinations during therapy. we retrospectively looked at the effective doses from routine ct and nuclear medicine in three cohorts of children, namely patients with hepatoblastoma, wilms' tumours and rhabdomyosarcoma (rms). of note, in our centre we rely on repeated mris of the primary site for many tumours. effective doses (e), in millisieverts (msv), were estimated using the ncict dose estimation tool (lee et al 2015) , based on details specific to each procedure: patient age, scan region, scanner type and ct dose index (ctdi -an indicator of radiation exposure recorded at the time of each scan). doses for general radiography were estimated using pcxmc v2.0 monte carlo simulations, assuming standard exposure factors and field size. there were 59 patients in total (18 hepatoblastoma, 21 wilms', 20 rms). there were 33 boys. the mean age was 3 years 2 months (ranging from 14 days -11 years 10 months). the mean and median cumulative effective doses from ct for the whole cohort were 7.88 msv and 4.24 msv respectively. four patients in the wilms' cohort had a dmsa nuclear scintigram (0.7 -1.0 msv), no hepatoblastoma patient had any nuclear medicine imaging, and 16 patients with rms received a bone scan (3 -3.5 msv) or a pet scan (approximately 8msv). cumulative radiation doses from routine radiological investigations in paediatric oncology can be kept in a much lower range than reported in the literature (1,2). in our institution, the followup of solid intra-abdominal tumours with mri, with additional ct or nuclear medicine only when clinically justified, has resulted in a significantly low radiation exposure in these patient cohorts. mri of the primary tumour site should be implemented as a replacement for ct imaging when there is no significant detriment to the diagnostic information obtained. (2017) 47 (suppl 2):s297-s pediatr radiol mri-based evaluation of multiorgan iron overload is a predictor of adverse outcomes in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation f. zennaro 1 , d. zanon 2 , r. simeone 2 , g. boz 2 , f. degrassi 2 , m. gregori 2 , g. schillani 2 , c. boyer 1 , n. maximova 2 ; 1 nice/fr, 2 trieste/it objective: iron overload is associated with poor clinical outcomes in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). although the effects of hepatic and cardiac siderosis on patient outcomes have been extensively studied, less is known about the effects of siderosis in other organs. the medical records of 44 consecutive pediatric patients who underwent allogeneic hsct in our institute from 2011 to 2015 were retrospectively reviewed. mri was used to measure iron concentrations in the liver, spleen, pancreas and bone. these patients were divided into two groups, 18 with non-elevated (<100 μmol/g; group 1) and 26 with elevated (>100 μmol/g; group 2) liver iron concentration (lic) at baseline. in group 1, only two patients had normal iron concentrations in all organs. none of the patients of group 2 presented with pathological iron concentrations in only two organs. comparisons of baseline data with results of the first follow-up mri performed 1-6 months after hsct, showed a general worsening of iron accumulation. in group 1, none of the patients showed complete absence of iron overload in a single organ. in group 2, none of the patients showed a total absence of siderosis involving fewer than three organs. this study confirms the correlations between iron overload and the risks of transplant-related complications, such as transplant related mortality, sinusoidal obstruction syndrome, infections, pancreatic insufficiency, and metabolic syndrome, in transplant recipients with systemic siderosis. another important finding of this study was the close correlations between pre-transplant bic and times to neutrophil and platelet engraftment (p<0.001 each). (15), ganglioneuromas (gn, 2) and ganglioneuroblastoma (gnb, 1), examined by 3t mri were retrospectively grouped according to tumor entity, risk factors (bone marrow metastasis, mycn amplification or 1p36 deletion) and therapeutic regime (observation versus chemotherapy). dw (b values 0, 400 and 800) and conventional mri images (t2, t1 pre and post contrast) were analyzed for tumor size, relative si-and absolute adc-values at baseline (base; no therapy), and after 3 (fu1) and 12 (fu2) months. adc values in nb were lower than in gnb and gn (0.75*10 -3 mm 2 /s versus 1.28*10 -3 mm 2 /s; p<0.05). there was a tendency towards lower adc values in tumors with risk factors (n=6) versus no risk factors (n=7) at baseline, which did not reach statistical significance (p=0.08). during follow-up shrinkage of tumor volume was noted (baseline 1206 ml, fu1 159 ml, fu2 51 ml; p<0.05 baseline vs. fu1; p=0.08 baseline vs. fu2). in the observation group, tumor adc values rose without relapse (0.89*10 -3 to 1.07 *10 -3 mm 2 /s). only in eventually relapsing tumors adc values tended to decrease further (0.95*10 -3 to 0.71*10 -3 mm 2 /s, p=0.17), despite initial reduction in tumor size. to establish inter and intra-observer variability in the radiological detection and assessment of pulmonary nodules at diagnosis in children with wilms tumours. a test set of ct thoraxes at diagnosis from 15 patients enrolled in the multicentre 'improving population outcomes of renal tumours of childhood' (import) study in the uk were assessed. five radiologists (3 chest, 2 paediatric) from 5 different centres (4 uk, 1 netherlands) completed a scoring sheet for nodule assessment on the same studies on two occasions, 6 months apart. the readers were blinded to patient respiratory symptoms, the original radiology reports and also that they were scoring identical cases. descriptive statistics, modified bland altman graph and fleiss kappa scores were used for statistical assessment. in total, 93 different pulmonary nodules across the 15 ct thoraces at both rounds were scored by at least one reader. 81 (87%) were seen by at least one reader in round 1 and 81 (87%) in round 2, 69 (85.2%) nodules were seen by at least one reader in both rounds. only 20 (21%) nodules were scored by all 5 readers in round 1, 16 (17%) by all 5 readers in round 2, and 14 (7%) nodules by all 5 readers in both rounds. of the 20 nodules seen in the first round, 11 were measured to be >5mm in at least one dimension and of these, 7 were classified as malignant by all 5 readers. the limits of agreement for mean difference in nodule size in anterior-posterior, transverse and longitudinal measurements were ±1.85mm, ±1.76mm and ±1.88mm respectively. the fleiss kappa scores ranked from poor to fair agreement for nodule border smoothness (0.03), nodule shape (0.2), solidity (0.2) and impression of malignancy (0.4). within the same readers for both rounds, nodule detection rates of agreement were between 60.7-82.9%. the average intra-reader percentage of observed agreements for nodule border smoothness, shape, solidity and impression of malignancy were 84.2%,63.9%, 90.6% and 84.8% respectively. conclusion: detection and characterisation of pulmonary nodules on ct thorax shows both intra-and inter-observer variability. this has important implications for the interpretation of metastatic disease at presentation. fever without a focus is defined as febrile illness without an initial obvious cause or localizing signs. our aim is to assess the diagnostic value of whole-body mri (wb-mri) in the diagnostic work-up of children with fever without a focus. we retrospectively searched for subjects who underwent wb-mri for fever without a focus. a total of 29 children (m=17, f=12), mean age 6.1 years (range: 0.08 -17.61) were included. 8/29 (27.6%) subjects were immunosuppressed and 6/29 (20.7%) subjects were hospitalized at onset of fever. the reference standard was based on positive cultures, biopsy or surgery. when this was not possible, a probable diagnosis was made based on clinical follow-up or serology. initially, the wb-mri images were reviewed independently by 2 pediatric radiologists blinded to all clinical information. at the end of each case the final diagnosis and the diagnostic category (5 categories: a. normal, b. infection, c. oncologic, d. rheumatologic, e. miscellaneous) was recorded. this was followed by a consensus read for comparison with the reference standard. for statistical analyses all subjects were treated as fever without a focus. results: reference standard: the diagnostic category of the reference standard was as follows: infectious 12/29 (44.8%), oncologic 1/29 (3.4%), rheumatologic 8/29 (27.6%), miscellaneous. 1/29 (3.4%). even after extensive work-up in 7/29 (20.7%) no clear cause for the fever was found table 1 . wb-mri: wb-mri diagnosed the cause of fever without a focus in 7/29 subjects (24.1%) ( table 1 ). in 2 subjects (6.8%) wb-mri results were falsely positive (1 jia and 1 myositis), and in the remaining 20 subjects no imaging findings compatible a cause of febrile disease were found. interobserver agreement was fair (kappa 0.53). in children with fever without a focus wb-mri provided the diagnosis in in almost a quarter of the cases. given the multiplicity of causes of fever without a focus, some of them not possible to visualize on mr imaging, wb-mri may be considered in routine imaging practice when evaluating pediatric patients with fever without a focus. to compare linear measurement/volume to direct volumetric measurements using 3 dimensional(3d) post-processing software. for this irb approved study initial diagnostic ct or mr exams in 100 patients(11mo-20yr) with solid tumors were reviewed by 11 radiologists and 3 technologists. radiologists recorded measurements in 3 axes in their routine method, described tumor shape (sphere, ellipse, cone) and surface texture (smooth, almost smooth, or mildly, moderately, markedly irregular). three technologists individually, and 3 radiologists by consensus, used 3d processing software (intellispace portal, philips, cleveland, oh) to directly measure tumor volume. tumor volume (v) was calculated from linear measuments using the following equations: sphere v=4/3πr 3 , ellipsoid v=8πr 2 or 16πr 2 , conicalv=2πr 2 or 4πr 2 , and cuboid v=(xyz). inter-reader variability in tumor measurement in all tumors and for tumors divided by surface characteristics was assessed amongst radiologists and technologists, and radiologist consensus using coefficient of variation (cov). tumor shape analysis was reported as 14 sphere, 84 ellipse, 2 cone, and surface texture 8 smooth, 10 almost smooth, 32 mildly irregular, 31 moderately irregular, 19 markedly irregular. inter-reader variability of as much as 1,119cc above to 383cc below the mean tumor volume was found when using radiologist determined linear measurements, with standard deviation (sd), range 0.65-413. inter-reader variability amongst technologist derived volumes was considerably less, range 102cc above to 90cc below the mean, with sd, range 0.03-97. cov analysis shows a greater degree of variation in tumor volume calculated from linear measurements [smooth(7%), almost smooth(12%), mildly(20%), moderately(18%), markedly(27%) irregular] than direct volume determination [smooth(5%), almost smooth(6%), mildly(3%), moderately(3%), markedly(5%) irregular]. variation was significant only for tumor with irregular surface texture [smooth (p=0.26), almost smooth (p=0.23), mildly (p=0.003), moderately (p=0.001), or markedly (p=0.002) irregular]. variation in linear/volume measurements in very irregular tumors. light blue=middle 50% tumor volume measurements by pediatric radiologists. whiskers mark limits of range. ▲♦ • markers =measurements by technologists. note broad degree of variation. (2017) 47 (suppl 2):s297-s pediatr radiol variation in linear/volume measurements in almost smooth tumors. light blue=middle 50% tumor volume measurements by pediatric radiologists. whiskers mark limits of range. ▲♦ • markers =measurements by technologists. note narrow degree of variation. both graphs show the same informationthe % relative variation in tumor volume measurements determined by 3 dimensional linear measurements (11 pediatric radiologists) v. volumetric processing (technologists & consensus group). radiologist generated measurements are subjective and unreliable. variation in measurement technique leads to differences in calculated tumor volume which significantly over or under estimate volume in tumors with irregular texture and is not significant in smooth tumors. quail-quantitative mri-based evaluation of pancreatic iron overload in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation f. zennaro 1 , m. gregori 1 , f. degrassi 1 , e. cattaruzzi 2 , y. diascorn 3 , c. boyer 3 , n. maximova 1 ; 1 trieste/it, 2 muggia/it, 3 nice/fr objective: iron overload (io) is a relatively common but often neglected transplantrelated complication and has been associated with poor prognosis in patients undergoing allogeneic hsct for hemato-oncological disease. pancreatic io is frequent among patients with transfusion-dependent anemias, but is uncommon among patients with hematologic malignancies. the causes of pancreatic io and the potential effects of pancreas iron deposits on transplant outcomes or on the risk of developing significant late effects in long-term hsct survivors have not been yet determined. our institute routinely uses magnetic resonance imaging (mri) with various gradient-recalled-echo (gre) sequences to quantitatively measure the iron concentration in abdominal parenchymal organs in all pediatric patients before and after allogeneic hsct. this study retrospectively analyzes the correlations of pancreas io with the type of conditioning regimen and pretransplant liver iron concentration (lic) in 50 pediatric patients who underwent allogeneic hsct in our transplant unit over the last 5 years. we enrolled 50 patients, age 1-17 years. pre-transplant mean lic was 147,55 μmol/g (normal values 36 μmol/g). 3 (6%) patients had mild liver io and 25 (50%) had moderate or severe io. pretransplant mean pancreatic iron concentration (pic) was 29,59 μmol/g, whose only 4 (8%) had mild pancreatic io and none had severe io. post-transplant mean lic was 162,6 μmol/g, only one patient had mild liver io but 36 patients (72%) had moderate or severe io. post-transplant mean pic was 66,22 μmol/g, 12 (22%) patients had moderate or severe io. mean pre-transplant pancreatic volume was 33,41 cm 3 , while mean post-transplant pancreatic volume (evaluated 30 days after transplantation) was 25,41 cm 3 . 11 (91,7%, p<0,001) patients with post-transplant moderate or severe pancreatic io underwent tbi-based conditioning. mean reduction of pancreas volume in tbi group was 12,02 cm 3 (p< 0,001). no pancreatic volume reduction was observed in chemotherapy-based group. all patients with pancreatic io have had exocrine pancreatic insufficiency and 9 (83,3%) patients have had metabolic syndrome. volume reduction well correlate (mean 49,8%, p<0,0001) with pancreatic io. this study confirms that pancreatic iron overload is not so rare in patients with hematologic malignancy underwent allogeneic hsct, with increased risk of metabolic syndrome and total deficit of exocrine pancreatic activity, but not of endocrine activity. iron overload monitoring allows for chelation therapy optimization. mr is fast, reproducible and more reliable compared to serum ferritin and transfusional history and allows a multi organ evaluation. pulmonary tb is common in south africa, with many children affected. diagnosis can be challenging and chest x-ray remains fundamental for diagnosis. interpretation is difficult and shown to have wide inter-reader variability. no study however has compared cxr findings and interreader agreement between ambulatory and hospitalised patients. this study compares the frequency of cxr changes, as well as interreader agreement in ambulatory compared to hospitalised children with suspected tb. from nolungile clinic and red cross children's hospital respectively was done. each sample contained 50% proven tb and 50% negative controls. two paediatric radiologists and one paediatrician served as blinded, independent readers for the database using standardised ticksheets. our study demonstrated no significant difference in lymphadenopathy, but an increase in parenchymal change in the hospitalised group. we otherwise showed similar results to literature regarding finding frequency, but poor inter-observer agreement. if the least expert reader were removed, results were comparable with available literature. this highlights the need for development and study of explicit cxr criteria for lymphadenopathy to improve the value of cxr for paediatric tb in all settings. lung ultrasound in pediatric pneumonia -why is it necessary to use the additional trans-abdominal approach? j. lovrenski; novi sad/rs objective: to emphasize the need of lung ultrasound (lus) technique modification, which enables detection of pneumonia in children not visualized by using solely the standard trans-thoracic approach. a prospective study was carried out in the regional children's hospital, and comprised a 2-year period. the inclusion criterion was us finding of pneumonia detected by trans-abdominal, and not with trans-thoracic approach. lus examinations were performed using a combined, trans-abdominal and trans-thoracic approach. longitudinal, transversal (intercostal), and oblique sections were used. trans-abdominal examination included transhepatic and trans-splenic approach. the ultrasound probe was angulated from the most anterior to the most posterior sections while examining the lung bases by trans-abdominal approach. a pneumonia-positive lus finding included subpleural consolidation with air-bronchogram, or with an adjacent area of interstitial/ alveolar-interstitial edema. lus was always performed before the other diagnostic modalities (chest x-ray (cxr) and computed tomography (ct)), if they were indicated by pediatrician or radiologist. within a 2-year period in 14 children (mean age 3.9y, sd 2.3y) the pneumonic focus was discovered using the trans-abdominal approach, while the trans-thoracic approach showed a normal lus pattern. all the children had the clinical symptoms of pneumonia (fever and cough, with or without dyspnea/tachypnea). the auscultatory finding was positive in 4 children. cxr was performed in three children, showed a right-sided pneumonia in two, and was negative in one patient. one child had a contrastenhanced chest ct, which confirmed a left-sided pulmonary base abscess detected during lus examination by trans-splenic approach only (figures 1, 2) . apart from pulmonary symptoms, there has not been any other associated diseases found, apart from otitis media in two children. each child responded to the antibiotics treatment with resolution of infection and us signs of pneumonia. in this oral presentation we will explain and give anatomical and technical reasons for pneumonia-positive us findings within lung bases, that remained undetected by the trans-thoracic approach. left-sided abscess abutted on the spleen (s), and was detected by trans-splenic us approach. it did not contact the pleural surface approachable by trans-thoracic ultrasound (black semi-lunar mark). l-liver. conclusion: trans-abdominal (trans-hepatic and trans-splenic) approach should become an inseparable part of each lus examination, along with a standard trans-thoracic approach. this modification of technique is expected to result in a further increase of lus sensitivity in diagnosing pneumonia. is thoracic ultrasound really competitive to computed tomography in children -a two-year retrospective study j. lovrenski, k. antolović; novi sad/rs to compare diagnostic accuracy of thoracic ultrasound (us) and computed tomography (ct) in children. a retrospective study was conducted in the regional children's hospital, and comprised a 2-year period. the inclusion criteria were: chest ct performed within 24h after the us examination of thorax, and us and ct examinations in the same patient performed by different pediatric radiologists. all us examinations were performed using a combined transabdominal-transthoracic approach. ct examinations were done (2017) 47 (suppl 2):s297-s pediatr radiol according to the body mass based pediatric ct protocols. each hemithorax was analyzed separately in terms of comparison between us and ct findings. statistical analysis included the calculation of sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of ultrasound in diagnosis of pulmonary pathological entities. out of 176 children with chest ct, 21 of them (mean age 8,2y, sd 5,6y) fulfilled the criteria to enter the study group. lung us showed sensitivity, specificity, ppv and npv in diagnosis of pleural effusion: 100%, 96,4%, 91%, 100%; lung consolidation: 100%, 100%, 100%, 100%; lung abscess: 75%, 100%, 100%, 97%; and interstitial lung disease: 57%, 100%, 100%, 91%, respectively. within 3 hemithoraces multiseptation of pleural effusion was observed by us only. air bronchogram within lung consolidation was observed in 5 hemithoraces both by us and ct examinations. necrotic areas within pulmonary consolidations were detected by us in 4 hemithoraces, which was later confirmed by ct examination. lung abscesses were diagnosed in 3 hemithoraces by both us and ct. two small lung abscesses filled with air (1 hemithorax) and bronchiectasis (2 hemithoraces) were detected only by ct examinations. other pathological findings detected both by us and ct examinations were: congenital pulmonary airway malformation (cpam) (1 hemithorax), pulmonary sequestration (1 hemithorax), partial pneumothorax (2 hemithoraces), hidropneumotorax (1 hemithorax), inflamed pneumatocele (1 hemithorax), hydatid cyst (1 hemithorax), pericardial effusion (2 patients), soft tissue masses of thoracic wall with initial bone destruction (2 patients), and lymphomas (2 patients) (figures 1-3) . in one patient us and ct revealed cysts and an extremely dilated bronchus within lung consolidation (pathohistological finding: cpam type 2 combined with subsegmental bronchial atresia, and extensive bronchopneumonia). us examination, unlike ct, could not differentiate between eventration of the left hemidiaphragm and diaphragmatic hernia in one patient. to determine and compare the accuracy of frontal cxrs alone and 'combination frontal-lateral' set of cxrs for diagnosing lymphadenopathy in children with tb using patients with confirmed tb and controls without tb, and to compare findings in hiv-infected and hiv-uninfected children. a total of 172 children (ie: 88 children with gene xpert confirmed tb and 84 control patients admitted with lower respiratory tract infections), which were part of a larger south african study, who had both frontal and lateral cxrs, were included. three qualified radiologists read the cxrs in 2 separate sittings one month apart (one for the frontal x-ray alone and one for the 'combination frontal-lateral' cxrs) for the presence of lymphadenopathy. odds ratios and 95% confidence intervals were calculated to determine the presence of lymphadenopathy using a consensus reading on the frontal cxr and frontal-lateral cxr combination according to the final diagnosis of tb. inter reader agreement was determined using the kappa statistic. lymphadenopathy was reported in 86 (50%) patients on the frontal cxr alone and in 143 (83%) patients on the frontal-lateral cxr combination. 52 (60%) of the 86 patients with lymphadenopathy on the frontal cxr alone were gene xpert positive versus 72 (50%) of the 143 patients with lymphadenopathy on the frontal-lateral cxr combination. in all patients, the consensus reading using a frontal-lateral cxr combination resulted in a 5-fold increase (or 4,9; 95% ci 2, 4) in calling lymphadenopathy compared to using a frontal cxr only in the gene xpert positive group, the consensus reading using a frontal and lateral cxr combination resulted in a 3 fold increase (or 3,1; 95% ci 1,5-6,6) in calling lymphadenopathy compared to a frontal cxr only. overall inter reader agreement for all 3 readers when evaluating for lymphadenopathy was 'fair' on both the frontal cxr (k= 0,2088) and the frontal-lateral cxr combination (k=0,273). the addition of a lateral view to the standard frontal cxr increased the rate of calling lymphadenopathy. however, the accuracy of diagnosing lymphadenopathy on chest x-ray as a marker for tb was poor. this poor accuracy was further hampered by only 'fair' inter reader agreement for the presence of lymphadenopathy on chest x-ray. dynamic 4d ct imaging in children has significant advantages over routine ct scanning, bronchography and bronchoscopy for diagnosing trachebronchomalacia because it can be performed during free breathing without anaesthesia or invasive airwayaccess.itcanalsodemonstratevascularcausesoftracheo-bronchomalaciain the same sitting. the technique is currently performed in 1 paediatric center in the uk. we aimed to report pitfalls encountered while setting up a dynamic 4d ct imaging service for children and report the findings of studies performed. materials: dynamic 4d ctscanning was introduced after installation of a large array (320 slice) ct scanner, applications specialist training and review of the literature. imaging parameters in use by greenberg and colleagues (arkansas children's hospital, usa) were applied. referral indications, pitfalls encountered, quality of scanning and imaging findings/diagnoses were reviewed and enumerated. results: nineteen paediatric dynamic 4d ct scans (9 females, 10 males; 11 days -3 years 9 months; mean 12 months) were performed over 15 months. the first 4 studies were performed without ivi contrast due to lack of experience and 13 subsequent studies were performed with contrast ( figure 1 major pitfalls included initial failure to perform contrasted studies for simultaneous evaluation of vessels, initial failure to withdraw the endotracheal tube, patient motion under care of nurses and clinicians, failure to appreciate the value of imaging the full lung volume while trying to keep dose length product to a minimum and failure to appreciate that collapse of the airway is often in the ap plane and not appreciated on coronal slab projections -rotating 3d volume rendered images is a requirement ( figure 3 ). additional obstacles were initial clinician and radiologist lack of support after early failures and colleague concerns regarding the radiation dose. objective: diagnosis of pulmonary tuberculosis (ptb) in children relies on chest radiography, however there is wide inter-observer agreement in detecting lymphadenopathy, the hallmark of ptb. paediatric airways are pliable, thus detection of airway compression may be a more objective criterion for the presence of lymphadenopathy. thus the objctive was to assess the usefulness of airway compression on chest radiographs for diagnosis of ptb in children. chest radiographs of children admitted to red cross children's hospital with suspected ptb were read by two readers according to a standardised format and a 3 rd when there was disagreement. radiographs of children with definite ptb were compared to those with lower respiratory tract infection (lrti) from another cause. the prevalence and location of airway compression was evaluated. findings were correlated with hiv status and age. inter-observer agreement was assessed using kappa statistic. 13 .9% in older children (or 1.7; 95%ci 1.00-3.00). no association with airway compression and hiv infection was found. inter-observer agreement ranged from 0.0-0.4. eighteen-month-old male patient diagnosed with ptb; hiv negative. majority agreement of airway compression at lmb indicative of lymphadenopathy. left upper lobe opacity is in keeping with a ghon focus. there is a strong association between airway compression on chest radiographs and confirmed ptb, particularly in infants, irrespective of hiv status. however, clinical use is limited by poor inter-observer agreement. paediatric ultrasound-guided biopsies in a tertiary oncology centre: five years experience n. parvizi, m. smedley, s. chakraborty; oxford/uk objective: histological diagnosis is almost always essential to guide appropriate therapy for children diagnosed with cancer. tissue can either be obtained by surgical/open or image-guided percutaneous biopsy. the aim of this study is to assess the safety and diagnostic accuracy of ultrasound-guided biopsies in a tertiary oncology referral centre. a retrospective analysis of clinical data, imaging findings and histological diagnosis of patients aged 0 to 18 years between january 2010 and december 2015 was carried out. a total of 113 ultrasound-guided biopsies were performed in our institution on 110 patients. most of the biopsies were performed in theatre with the patient under general anesthetic and with an 18-gauge spring-loaded core biopsy needle with a minimum of two cores per patient. in 99% of lesions the needle biopsy was diagnostic. the single nondiagnostic case did not have sufficient material to make a full diagnosis and a surgical biopsy was required. eighty-two of the biopsied lesions were malignant and 31 were benign. in no cases was a repeat biopsy required. the vast majority of the biopsies were performed within one week of request with over half performed within 3 days. all biopsies were performed without complication and in the majority of cases the patients were discharged the same day or following an overnight stay. ultrasound-guided percutaneous biopsy is an accurate and safe technique in order to acquire tissue from suspected malignant lesions in children. these can be performed instead of or in addition to open biopsy and will often result in a shorter hospital admission and recovery time. the role of imaging in the diagnosis of thymoma in paediatric patients with myasthenia gravis j. adu, t. a. watson; london/uk thymomas are exceedingly rare tumours in the paediatric age group, with only very few cases having been reported in the literature. thymomas are commonly associated with myasthenia gravis (mg), with thymectomy being potentially curative. ct is the mainstay imaging modality for thymoma diagnosis in the adult population. while, chemical shift mr imaging can be helpful to distinguish thymoma from other anterior mediastinal abnormalities. currently, there is no consensus on the imaging pathway for children with mg with suspected thymoma. our aim is to review the imaging of patients who were referred to our institution for management of mg, and suggest an imaging pathway in cases where thymoma is suspected. we performed a retrospective search of the local pacs system of cases between 2000 and 2016 using the search terms "thymoma" and "myasthenia gravis" in the clinical indication for the study and the body of the final report. forty-three cases were identified using the search criteria. eight cases were excluded owing to an absence of cardiothoracic imaging. 20/35 of all cases (57%) had chest x-rays (cxr's), of these 17/20 (85%) were normal. the three remaining patients who had abnormal cxr's went on to have ct scans, which confirmed an anterior mediastinal mass (amm) in all three cases. 26/35 of all cases (74%) had cross-sectional imaging (mri 15/26 cases, ct 11/26 cases). of those, 21/26 of cases (81%) had normal studies. specifically, all 15 mri studies (100% of cases) were normal, while only 5/15 ct scans (66%) demonstrated an anterior mediastinal abnormality. 12/35 of all cases (34%) had both cxr and cross sectional studies. 9/12 of these cases (75%) had a normal ct or mri. in the remaining three cases, the amm was clearly demonstrated on both cxr and the crosssectional imaging. in our series, radiography, ct and mri studies were normal in the vast majority of cases. however, given that thymectomy is potentially s405 (2017) 47 (suppl 2):s297-s pediatr radiol curative, it is appreciated that clinicians may still be keen to radiologically investigate paediatric patients with myasthenia gravis. cxr is not an efficacious imaging modality in this context, as patients with a normal cxr may be falsely negative, and patients with an abnormal cxr may undergo cross-sectional imaging regardless. we propose that mri should be used as first line investigation for patients in this population. this approach will negate the need for ionizing radiation, maximize diagnostic yield, and facilitate surgical planning if deemed clinically appropriate. increased risk of venous thrombosis of the arm with multiple peripherally inserted central catheters insertion in paediatric patients r. gnannt 1 , n. waespe 2 , j. donnellan 2 , k. liu 2 , l. brandao 2 , b. connolly 2 ; 1 zurich/ch, 2 toronto/ca objective: peripherally inserted central catheters (piccs) are associated with superficial and deep venous thrombosis of the arm. the impact on the incidence of developing a thrombosis of the arm when inserting a subsequent picc remains unclear. the purpose of this study was to analyze the incidence of deep, upper limb thrombosis of repeated upper limb piccs in children. the study population included all patients who underwent their first successful arm picc insertion between january 2010 and december 2015. subsequent ipsilateral arm piccs were included in the analysis. patients were followed until march 2016 or until any alternative central venous line insertion (jugular, femoral, saphenous or umbilical vein lines -because of their thrombogenic effect). for each picc insertion the following data were collected: date of insertion and removal, weight of the patient, type of picc (1.9fr, 2.6fr, 3fr, 4fr, 5fr), left or right arm, and vein cannulated (basilic, brachial, cephalic). all symptomatic deep and superficial thrombosis of the arm were correlated with the picc database. four thousand one hundred thirty-eight piccs were inserted. applying inclusion and exclusion criteria, 1955 piccs remained for analysis. first, 2 nd , 3 rd , and 4 th picc insertions in the same arm were identified in 1773, 146, 30 and 6 patients, respectively. in total there were 57 upper body deep symptomatic thrombotic events diagnosed with ultrasound. a 1 st , 2 nd , 3 rd , and 4 th picc insertion was associated with 46/1773 (incidence 2.6%), 6/146 (4.1%), 4/30 (13.3%), and 1/6 (16.7%) thrombotic events, respectively. an increasing hazard ratio was seen with higher numbers of picc insertions, which was significant when comparing the 1 st with the 3 rd picc insertion in the same arm (hr 3.9, ci95%1.3-11.4, p=0.01). after excluding any confounder, double lumen piccs were associated with a significantly higher risk of thrombosis than single lumen (or 5.3, ci 1.2 -23.4, p=0.026). repetitive picc insertions in the same arm are associated with an increased risk of thrombosis. double lumen piccs are associated with a higher risk of thrombosis compared to single lumen lines. diagnostic performance of lung ultrasound for the detection of community acquired pneumonia in children j.a.m. stadler 1 , s. androunikou 2 , h. zar 3 ; 1 paarl/za, 2 bristol/uk, 3 cape town/za objective: chest radiographs (cxr) are considered the first line imaging modality when investigating cases of suspected community acquired pneumonia (cap) in children. however, cxr interpretation is limited by moderate sensitivity and specificity and poor inter-and intra-rater reliability and expose children to potentially harmful ionizing radiation. point-of-care lung ultrasound (lus) has been proposed as alternative to cxr for diagnosis of pneumonia in children and some published data suggest accuracy and reliability as good as or better than cxr. most of these studies however, were performed in in-hospital settings creating a bias for selceting more severe disease and consequently more overt radiological findings. the mean age of children in most of these studies were also well above one year, while the highest incidence and risk of complicated pneumonia occurs during the first year of life. the purpose of our study was to assess the diagnostic performance of lus for the diagnosis of pneumonia in both hospitalised and non-hospitalised children in an age group representative of the most at risk segment of the population. we performed a lus on 147 children who presented with clinical signs consistent with the who case definition for childhood pneumonia. one hundred of these patients also had chest radiographs performed as part of routine clinical care. inter-rater reliability (irr) between a general practitioner and an expert paediatric radiologist were assessed for the interpretation of lus findings consistent with pneumonia. where radiographs were available concordance between lus and cxr findings of pneumonia were also assessed. results: seventy-four hospitalised and 73 non-hospitalised clinically defined pneumonia cases were included with a median age of 10.3 years. our general practitioner reported lus findings consistent with pneumonia in 77/147 (52%) compared with 66/147 (45%) by the paediatric radiologist. substantial overall agreement between the reporters was found with an overall agreement proportion of 0.79 and kappa=0.62. agreement for the presence of lung consolidation or for a normal scan was also substantial with kappa of 0.67 and 0.68 respectively. agreement on the finding of interstitial syndrome was moderate with kappa=0.45. agreement was higher in hospitalised than in non-hospitalised cases with kappa of 0.62 and 0.56 for the respective categories. results showing concordance between lus and cxr findings are pending. conclusion: lus shows substantial irr for the diagnosis of pneumonia in children. irr are higher for the detection of consolidation or for no pathology than for interstitial syndrome. irr also appears to be lower in clinically less severe disease. 'white-out' on plain chest radiograph-a late presentation of congenital diaphragmatic hernia a. fagan 1 , c. stewart 2 , k. halliday 3 , s. rao 2 , d.t. chang kwok 2 ; 1 peterborough/uk, 2 lincoln/uk, 3 objective: awareness of the limitations of plain radiograph and computed tomography in diagnosis of late presentation of congenital diaphragmatic hernia. case presentation: a 2 year old boy presented with a 6 day history of pyrexia, vomiting and respiratory distress. he was haemodynamically stable, and had no audible air entry over his upper left thorax with occasional wheeze over the left base. he had bronchiolitis previously but did not require ventilatory support. he was otherwise well with unremarkable antenatal scans. initial chest x-ray showed a large air collection with fluid or soft tissue density within the left hemi-thorax and mediastinal shift to the right. repeat x-ray (figure 1 ) demonstrated the nasogastric tube below the diaphragm. complicated pneumonia was suspected but as the findings were atypical a non-contrast ct was performed. this was interpreted as showing a large hydropneumothorax. (figure 2) . a chest drain was inserted which drained only a small volume of fluid, and a repeat chest film showed no change. ct chest and abdomen with oral and intravenous contrast revealed a bochdalek diaphragmatic hernia (figure 3) . fortunately the chest drain had not entered the herniated stomach. the hernia was surgically corrected and the child recovered well. (2017) 47 (suppl 2):s297-s pediatr radiol bochdalek is the most common congenital diaphragmatic hernia (cdh). it is often diagnosed on prenatal ultrasound, with mri used for confirmation. cdh which is not diagnosed in the perinatal period may be asymptomatic and imaging findings can be confusing. postnatal x-ray typically shows an opacified hemi-thorax with or without gas bubbles. there can be mass effect with mediastinal shift. the position of an ng tube can be helpful in localising the stomach, but in this case the infradiaphragmatic position of the tube gave false reassurance. in neonates, the position of an umbilical venous catheter may demonstrate abnormal location of the liver. computed tomography generally demonstrates a posterolateral defect (foramen of bochdalek), which is located on the left in 80% of cases. ct is useful for excluding lung masses or bronchopulmonary foregut malformations, which may appear similar to cdh on x-ray. ct can also identify anatomical abnormalities associated with cdh. late presenting cdh is often misdiagnosed as pleural effusion or pneumothorax. there are other case reports published where chest drains were inserted before cdh was diagnosed. it is important to keep cdh in mind as a potential cause of unilateral hemithorax opacification, even in previously asymptomatic older children. ct with oral contrast can be useful in diagnosis. ovarian tuberculosis with peritoneal dissemination mimicking ovarian tumor with peritoneal seeding d. grassi, v. tostes, a. duarte, s. abib, h.m. lederman; sao paulo/br consider tuberculosis (tb) as a differential diagnosis whenever the case enrolls in an endemic region. case presentation: female, 13 years old adolescent, who presents with abdominal pain and weight loss. abdominal sonography was performed in a public family practice location and bilateral ovarian masses were detected. she was referred to an oncology pediatric facility for further investigation. abdominal mri and chest ct were performed where dissemination through the peritoneal and mesenteric lymph nodes could be detected; chest ct was normal. the patient underwent surgical intervention for diagnosis and on pathology the findings in the bilateral ovarian masses were secondary to tb involvement. sonography showing bilateral pelvic masses. t2-weighted coronal overview bilateral ovarian masses. unique teaching points: whenever a case enrolls in an endemic region of tuberculosis, it is important to consider it as a possible differential diagnosis. in this case, the initial presentation mimicked ovarian tumor with mesenteric seeding. however, only after surgical approach was possible to diagnose ovarian tuberculosis with mesenteric lymph nodes and peritoneal involvement. retrospectively, patient's uncle was discovered as having pulmonary tb. langerhans'-cell histiocytosis with thoracic involvement in infant and young child: ct findings s.-l. shih 1 , k. tsai 1 , w. huang 2 , f.-s. yang 1 ; 1 taipei/tw, 2 taitung/tw the purpose of the study was to evaluate the ct changes of thorax in the patients with langerhans'-cell histiocytosis. the 3-month-old female infant presented with generalized hemorrhagic macular rash over the skin for 2 months. the laboratory findings showed hemoglobin 7.8gm/dl (normal 11.0~16.0gm/dl). the chest radiograph showed bilateral reticulonodular infiltration. high-resolution computed tomography (hrct) of chest showed multiple cystic-like lesions (1-6mm) in the right middle and bilateral lower lobes. the pathological report was langerhans'-cell histiocytosis after skin biopsy from upper chest. then she was on scheduled chemotherapy. she was in remission after one-year treatment. the 1y10m-old girl presented with fever for 3 months. the physical examination revealed hemorrhagic-macular rash over the skin in the anterior chest wall and hepatosplenomegaly. the laboratory findings revealed albumin 2.5g/dl (normal 3.8-4.7g/dl) and hemoglobin 7.0g/dl (normal 11.0-16.0g/dl). hrct of chest showed multiple cystic-like lesions (1-6mm) in the bilateral lower lobes with left pleural effusion as well as multiple osteolytic lesions in the vertebral bodies of t7, t8, t11 and t12. the pathological report was langerhans'-cell histiocytosis after skin biopy from anterior chest wall. then she was on scheduled chemotherapy. she was doing well 10 years after treatment. the 1y6m-old girl presented with yellowish discoloration of skin for one month. the laboratory findings revealed direct/total bilirubin 4.4/7.9mg/dl (normal 0.1-0.5/0.3-1.2mg/dl), got 223iu/l (24-46iu/l) and gpt 220iu/l (12-27iu/l). the chest radiograph revealed enlargement of upper mediastinum. the ct scan of chest and upper abdomen showed punctuate calcification with heterogeneous enhancement in the upper mediastinum and several minute cysts in the lower lobes of lung (hrct) as well as dilatation of bilateral intrahepatic bile ducts in the liver. the pathological report was langerhans'-cell histiocytosis after biopsy from thymus and liver. then she was on scheduled chemotherapy and got initial response. unique teaching points: langerhans'-cell histiocytosis affecting the lungs and thymus may be in isolation or as part of a multiorgan disease. the pulmonary changes on ct scan may not have corresponding respiratory symptoms. ct scan of thorax may have multiple minute cysts (1-6mm) in the lungs, pleural effusion, calcification in the thymus and osteolytic lesions in the thoracic spine. case of fungal infection of the soft tissue in a child with acute myeloid leukemia (ultrasound aspects of diagnosis) i. begun, s. kondaurova; minsk region/by objective: early diagnosis of fungal infections of the tissues is essential for a successful and complete recovery. we describe a clinical case of fungal infection of the soft tissue in a child with acute myeloid leukemia (aml). ultrasound were made for the characteristics of the structural changes in the area of interest to perform biopsies followed by bacteriological culture studies. case presentation: patient k., 13 years old, diagnosed with aml, from which after a course of induction chemotherapy with neutropenia about 3 weeks on the skin of the foreskin appeared removable hard white coating. cultures of plaque it possible to establish the presence of fungi of the genus trichosporon spp. after 4 days, there were hyperemia, compaction and ulceration of the glans penis, which led to extensive tissue defects. with help ultrasound were determined the structural deformation of the glans penis with the pronounced around changed tissues vascularization. after 7 days in the rear surface projection of the left thigh and the lateral surface of the left calf were defined erythematous papules which progressed to ulceration with central black scab. by standard ultrasound were visualized: subcutaneous nodal education oval 1,0х0,4 sm on hip and echogenic skin thickened portion having an average degree of severity of dorsal acoustic shadow on the lower leg (weakening of the signal behind scab). in cultures of biopsies of subcutaneous foci were revealed fungi of the genus trichosporon spp too. the patient received the combination treatment (intravenous liposomal amphotericin b and surgical rehabilitation of lesions of glans and corpus cavernosum of penis). after the stabilization of patient state the treatment of the underlying disease was continued. unique teaching points: for some patients, lesions of superficial tissues may be the only sign of systemic fungal infections, and rapid recognition of these lesions may contribute to early diagnosis and treatment. ultrasound examination in such a situation naturally becomes an main imaging tool and by choice method. the scanning high-resolution of foci of the thigh of the patient k. in grayscale made possibility to determine the configuration consisting of the central echogenic focus surrounded by a hypoechoic rim (fig. 1) with peripheral changes in the type of "infiltrative" according by the active fungal infection at the exit of cytopenia. duplex and triplex ultrasound scanning were indicating to the perifocal vascularization with low level vascular resistance around of the affected area (see fig.2-3) . to increase knowledge and awareness of rare cases and diseases in order to be able to better manage and treat patients in the future. case presentation: an 3-month-old female was presented to our hospital with abdominal distention that increased in the past 3 months associated with low-grade fever, loss of weight and mild respiratory distress. abdominal ultrasonography revealed an enlarged liver with multifocal hypoechoic lesions scattered all over the liver (fig 1) . a ct scan with iv contrast (mri was not available at that time in our district) revealed severe hepatomegaly with the presence of multiple, variable in size, hepatic hypodense lesions which had peripheral (ring) enhancement after contrast injection in the arterial phase (fig 2) . progressive centripetal filling in portal phase is seen and in the delayed images many of the lesions were completely filled (fig 3) . reduction in the aortic caliber (mid-aortic syndrome) below the level of celiac branch was noted. a diagnosis of hemangioendothelioma was made although liver biopsy was not done due to fear of hemorrhage. alternative diagnosis to infantile hemangioendothelioma in this age group is hepatoblastoma, mesenchymal hamartoma and metastatic neuroblastoma. the patient was transferred to another city to a hospital with pediatric oncology department for follow up and treatment. unfortunately the lack of experience and knowledge of such rare cases led to mismanagement and delayed treatment and after less than 2 months the patient was brought back to our hospital to the pediatric icu due to deterioration of her status due to congestive heart failure. unfortunately the patient died shortly afterwards. hemangioendothelioma is twice as common in girls and can have complications due to high output chf secondary to arteriovenous shunting hemangioendotheliomas tend to involute spontaneously without therapy over a course of months to years. they are followed with sequential ultrasounds. medical therapy is reserved for severely symptomatic lesions (e.g. anemia, consumptive coagulopathy, high-output chf) and includes high-dose steroids or alpha-interferon. in cases of failed medical management, surgical resection should be performed. if partial hepatectomy is not technically achievable, transarterial embolization should be used either as definitive therapy or as a temporizing measure until liver transplantation is possible. the sad outcome of this case was mainly due to mismanagment due to lack of medical experience and knowledge of such rare cases so we suggest that such rare cases should be catalogued in a national data bank for future consultation and teaching purposes. fatal outcome of acute gastric dilatation causing acute abdomen compartment syndrome in a child: a case review c.s. yoon; seoul/kr to describe and review presumed acute abdominal compartment syndrome in a child. case presentation: a 3 years and 5months old boy was admitted to emergency room due to abdominal distention. he suffered abdominal pain and vomited since yesterday after lunch. on physical examination, his abdomen was rigid and distended. body temperature is 37.2°c. the white cell count was increased (24,140/μl). esr is 25 mm/hr and c-reactive protein was 123.4 mg/l. creatinine was increased (1.24mg/dl). amylase and lipase were increased (164 u/l and 104 u/l respectively). prothrombin time was prolonged (15.5 sec). plain abdomen radiograph shows markedly distended stomach with air-fluid level (fig.1) . first trial of nasogastric tube insertion was failed due to kinking of tube at gastroesophageal junction. contrast-enhanced abdomen ctscan shows marked distensionofstomachwithlargeamountoffoodmaterialsandintraluminalairwith prominent external compression in the duodenal 3rd-4th junction. esophageal air distention is also markedly noted with l-tube insertion. no opacification of large vessel with contrast media, without contrast enhancement of spleen, pancreas and left kidney is noted (fig.2) . prob. markedly compressed and poorly defined lower abdominal aorta with faintly visible both common iliac arteries and femoral arteries. after ctscan, nasogastric tube exchange was performed due to poor drainage of gastric fluid. about 700cc of gastric fluid was drained. however, sudden cardiac arrest of the patient was developed. although vigorous cardiopulmonary resuscitation was performed, the patient was died. (2017) 47 (suppl 2):s297-s pediatr radiol unique teaching points: acute abdomen compartment syndrome is a very serious and lifethreatening disease. as soon as possible, rapid diagnosis and adequate treatment are necessary for good prognosis. delayed diagnosis and treatment may result in fatal outcome. pleuroperitoneal fistula in a pediatric patient with primary hyperoxaluria type 1 w.p. chu; hang hau/hk to illustrate the imaging features of pleuroperitoneal fistula in a pediatric patient suffering from primary hyperoxaluria type 1 case presentation: an 11-year-old girl with the history of primary hyperoxaluria type 1 was repeatedly admitted to the hospital for recurrent right pleural effusion despite chest drain insertion. the right pleural fluid was transudative in nature and the microbiological cultures for bacteria and mycobacterial species were negative. the radiographic examination [ figure 1 ] showed moderate right pleural effusion a n d f e a t u r e s o f o x a l o s i s i n c l u d i n g b i l a t e r a l c o r t i c a l nephrocalcinosis and generalized increased in bone sclerosis. delayed planar images of the peritoneal scinitigraphy [ figure 2 ] obtained 3 and 5 hours after injection of technetium-99 m suphlur colloid found diffuse tracer activity at the right hemithorax, suggestive of pleuro-peritoneal fistula. the patient subsequently required thoracoscopy and surgical decortication at the right hemithorax and renal transplantation. primary hyperoxaluria is due to defective glyoxylate metabolism and results in increased synthesis of oxalic acid. cortical nephrocalcinosis and diffusely increased bone sclerosis are characteristic radiographic features. pleuroperitoneal fistula is unusual in patients without peritoneal dialysis. possible cause in this patient is increased intra-abdominal pressure related to portal hypertension and cirrhosis. osteosarcoma with pulmonary intra-arterial tumor embolism metastasis a. alzaher, f. alzaher; dammam/sa objective: osteosarcoma rarely invade the veins and small number of cases has been reported with venous invasion at the presentation. however, to our knowledge, no case has been reported with venous invasion and isolated distal metastasis as intra-arterial pulmonary embolisms. we are presenting a case of pediatric pelvic osteosarcoma with venous invasion and pulmonary arterial tumor embolisms as isolated distant metastasis at the presentation. the purpose of this case report is to describe the rare presentation of distant metastasis as isolated pulmonary arterial embolism that might be overlooked radiological. additionally, such tumor embolism might cause respiratory symptoms and differentiating tumor emblism from pulmonary thromboembolism is crucial to avoid the unnecessary anticoagulation. case presentation: fourteen year old boy who presented with 3 months history of right hip and lower limb pain after trauma. this was associated with lower limb swelling. the plain radiography showed right pelvic iliac bone aggressive mass, along with lobulated, soft-tissue components, extensive areas of osseous matrix, and malignant periosteal reaction. the patient could not tolerate the mri and ct scan was performed and it showed that the mass was invading the right external and internal iliac vein with imaging appearance was most consistent with osteosarcoma. patient staging was then carried on with mri under anesthesia and chest, abdomen and pelvic ct scan. the unenhanced and iv contrast enhanced chest ct scan showed multiple beaded expansion of sub segmental pulmonary arteries with soft tissue destinies and calcification suggestive of intra-arterial pulmonary tumor embolisms. there was no isolated pulmonary nodule or any other site of distant metastasis. unique teaching points: we present this case to increase the awareness of isolated intra-arterial pulmonary tumor embolisms as osteosarcoma metastasis especially with the present of venous invasion. additionally, such condition might be with respiratory symptoms and differentiating the tumor embolism from pulmonary thromboembolism is crucial to avoid the unnecessary anticoagulation. case presentation: a 14-year old boy with acute myelodysplastic syndrome presented with recurrent, acute severe anemia (hemoglobin 68 g/dl) and melena. his past history was significant for bone marrow transplant twice followed by graft-versus-host-disease of intestines, bilateral lung transplants for bronchiolitis obliterans, renal failure, scleroderma and acute pancreatitis. ct angiography performed previously did not identify active extravasation. several days before, upper gi endoscopy had demonstrated ulceration of the greater curvature of the gastric wall that was initially treated with epinephrine injection and surgical clip placement. at the time of referral, endoscopic interventions were unsuccessful leading to progressive clinical deterioration. a decision was taken to proceed to angiography to isolate the arterial source of hemorrhage, with an intention to embolize, if feasible. catheter angiography via transfemoral 4fr access revealed a left gastric artery pseudoaneurysm with active extravasation into the gastric lumen through the ulcer. after selecting the feeding pedicle of the left gastric artery with a microcatheter, the pseudoaneurysm was embolized using 40% nbca in lipiodol, resulting in complete angiographic obliteration of the bleeding source. on repeat cbc 6 hours post-procedure, the hemoglobin had increased from 80 to 115 g/dl. the patient remained hemodynamically stable in the intensive care unit. there is no evidence of bleeding recurrence 25 days later. unique teaching points: catheter angiography can define the bleeding source with greater accuracy than cta in children. there should be a low threshold to perform catheter angiography, with an intention to proceed to treatment. nbca embolization is a feasible and effective option for treatment of acute gi bleeding in children. case presentation: an infant born by cesarean section at 38 weeks of gestation, after nonreassuring cardiotocoghraphy, with meconium aspiration at birth, severe hepatocellular failure with hyperbilirubinemia, signs of hemorrhage, edema, ascites, hypoglycemia, increased ferritin values, and lactic acidosis was referred for ultrasound and magnetic resonance. both examinations showed signs of liver cirrhosis with portal hypertension; in addition, on t2-weighted images and gradient-echo images, the signal intensity of the liver and the pancreas was lower than that of the spleen and skeletal muscle, a finding consistent with abnormal iron deposition in those organs. a biopsy of the lower lip confirmed the diagnosis of neonatal hemochromatosis. unique teaching points: although the diagnosis may be suspected clinically, it must be confirmed by demonstrating the generalized iron overload affecting, among other organs, the salivary glands, liver and pancreas, with sparing of the reticuloendothelial system. the underlying cause may be associated with an an alloimmune mechanism; thus, intravenous immunoglobulin during gestation is administered in selected cases to prevent the severity of neonatal hemochromatosis. diagnosis is then crucial not only for management of the affected infant, but also for prevention in the future offspring. fishing for the answer -a rare case of paediatric exogenous lipoid pneumonia secondary to fish oil aspiration h. moodley, d. white, g.d. baker; johannesburg/za objective: lipoid pneumonia is a rare condition caused by the intrapulmonary accumulation of endogenous or exogenous fat containing substances. in the acute exogenous form secondary to aspiration of oil, it is important to make the diagnosis and remove the causative agent to prevent or arrest the progression of pulmonary fibrosis. radiopathological findings usually prompt the diagnosis, as aspiration of mineral oils is usually unnoticed due to the lack of reactive airway symptoms and patients present with vague chronic respiratory symptoms. case presentation: we present the clinical, radiological and pathological correlation of exogenous lipoid pneumonia in a 4-month-old male patient with recurrent respiratory tract infections. a ct chest demonstrated an extensive crazy paving pattern of the dependent lung segments bilaterally. the lung biopsy findings of occasional intra -alveolar macrophages with larger (2017) 47 (suppl 2):s297-s pediatr radiol foamy cytoplasmic vacuoles, raised the possibility of an exogenous lipoid pneumonia secondary to aspiration. on further history, the patient was found to have been fed fish oil by his mother, confirming the diagnosis. unique teaching points: the rare diagnosis of exogenous lipoid pneumonia can be confirmed on ct chest by measuring the hounsfield units in the most hyperdense components of consolidation (typically -150 to -30 hu). histopathological confirmation can be obtained provided that the specimens are not embedded in paraffin. the possible role of visual evaluation of dwibs in childhood renal masses based on our five cases e. varga, g. rudas; budapest/hu objective: nowadays, the diffusion-weighted mri has a great importance not only in the differential diagnosis and follow-ups of childhood renal tumors, but in the early detection of recurrence of the disease as well. the dwibs with appropriate b-values and the adc calculation can be helpful in distinguishing between benign and malignant processes. however, the adc calculation is a time consuming method and in addition, there are cases when we cannot use this technique, but we can still apply the visual evaluation of diffusion. case presentation: between 2013-2016, we had 5 cases in which the visual assessment of dwibs was the best method which helped to make the appropriate therapeutic decisions. left kidney of an infant with nephroblastomatosis was removed because of an arising wilms tumor. 2,5 years later, in the contralateral kidney, a small area of diffusion restriction appeared on the dwibs in one of the cystic residual lesions, but the anatomic sequences haven't showed any changes comparing with the previous examinations. in another patient with beckwidt-wiedemann syndrome, the follow-up ultrasound examination showed a little bulging of the surface of the left kidney. accordingly, the mri showed a barely distinguishable nodule, but the dwibs referred to a wilms tumor. in a 17-month-old child, more nodules were visible in both kidney on the dwibs than on other sequences. with the help of visual evaluation of dwibs, we were able to detect the malignant lesion easily and quickly, among a lot of cystic and solid nodules of the kidneys in a seven years old patient with sclerosis tuberosa. an 8-month-old infant was followed with a benign cystic renal disease and a new small solid nodule was found on the last ultrasound examination. instead, the visual assessment of dwibs indicated a multilocular cystic wilms' tumor. unique teaching points: the diffusion-weighted mri is suitable for differentiate benign and malignant renal lesions in children. the dwibs (with appropriate b-values) and the adc calculation are very sensitive methods in pediatric oncoradiology. the adc calculation is a long process andas our cases demonstrated -we cannot apply in every cases. the visual evaluation of dwibs is a time saving method which is spared from limitations of adc histogram-based assessment, so it may become very useful in the everyday practice. we can use it in the differential diagnosis and follow-ups of childhood renal tumors and we can detect the recurrence of the malgnancy very early and easily. mr urography in a 9-years-old female with unusual urinary dribbling m.c. terranova, c. tudisca, d. narese, g. li voti, s. salerno; palermo/it objective: congenital anomalies of kidney and urinary tract (cakut) occurs in up to 3.2% of infants, and clinically they can range from asymptomatic patients, in which anomaly is detected incidentally even in adulthood, to ante-natal or post-natal mortality due to bilateral kidney agenesis or acute renal failure. dmsa renal scintigraphy is considered gold standard, for evaluation of those cases electable for surgery, in order to assess renal function, depict and locate ectopic kidney and guide surgical management, but has the important limit of radiation exposure and may undetect poorly functional renal moieties. the advent of modern magnetic resonance technics proven to be able to assess anatomical malformations and renal function, overcoming the limits of dmsa scintigraphy, may be used as a valid alternative, especially in vulnerable pediatric population. we herein describe a case of a young girl with small left renal bud and ectopic ureter, draining in vagina, discovered by mr and undetected by previous dsma scintigraphy. case presentation: a 9 years old girl was referred for continuous urinary dribbling, after starting toilet training, with normal bladder voiding pattern, unrelated to any physical and psychological events, and no history of urinary tract infections. physical examination revealed vaginal septa and micturition training was practiced, with no symptoms improvement. abdominal us study was performed, reporting empty left renal fossa and hypertrophic right kidney; no ectopic kidney nor sign of urine stasis or other urogenital anomalies were detected, and dmsa renal scintigraphy was planned. it depicted only normal right kidney radionuclide uptake but no evidence of left renal or ectopic renal tissues activity. patient then underwent mr evaluation for suspected genito urinary malformation, that revealed a small cystic formation, with a slight cortex, at the level of the iv lumbar vertebra -that represented the left immature renal bud -supplied by a short fluid-filled tubular structure, located postero-medially to the bladder -that configured the left ectopic ureter, draining in left vaginal wall. bladder was normal, and regularly connected with the right orthotopic ureter (fig 1) . pre-surgical cystoscopy and vaginoscopy, followed by left ascending urethrogram were performed, confirmed previous mr findings, and patient underwent successfull laparoscopic left nephron-ureterectomy. unique teaching points: mr urography has proven to be a rapid, safe, radiation free, systematic diagnostic tool especially in the evaluation of poorly functioning renal systems, and of collecting system, bladder and ureteral abnormalities, overcoming the limits of conventional imaging technics agenesis of the dorsal pancreas: case report c. lanza, g. pieroni, l. amici, a. giovagnoni; ancona/it objective: agenesis of the dorsal pancreas (adp) is a rare malformation. since 1911 and until 2008, 53 cases have been reported. majority of the patients with this anomaly are asymptomatic or associated with abdominal pain, hyperglycemia, diabetes mellitus, and acute or chronic pancreatitis. case presentation: we present a case report of a 11-year-old girl with adp, diagnosed incidentally during radiological evaluation for abdominal pain. she was hospitalized in the pediatric department for recurrent abdominal pain for the past 10 months. there was no history of nausea, vomiting or trauma. biochemical investigations showed a normal random serum glucose, serum amylase levels slightly increased (149 u/l; reference value 28-100 u/l) and slightly elevated serum pancreatic lipase levels (138 u/l; reference value 22-51 u/l). the day after serum amylase levels decresed up to 80 u/l and lipase levels to 78 u/l. us revealed increased -size pancreatic head with normal contour and echotexture with no parenchymal calcification or duct dilatation. the body and the tail of the pancreas were poorly visualized. mr imaging examinations revealed only a partial visualization of the pancreas: the pancreatic head and the uncinate process were visualized with defined margins with peripancreatic fat stranding, but the distal neck, body, and tail of the pancreas were absent. on mrcp, the dorsal pancreatic duct of santorini and the minor duodenal papilla could not be visualized. the ventral pancreatic duct of wirsung and the common bile duct were normal and clearly visualized. these findings were compatible with complete adp, eliminating the need for ercp. unique teaching points: the clinical presentation of dpa varies greatly ranging from incidental detection on x-ray, surgery or autopsy through to the development of a ductal adenocarcinoma of the pancreas. abdominal pain and diabetes are the most frequent clinical manifestations reflecting exocrine and endocrine insufficiency as most of the islands of langerhans are located in the tail of the pancreas. there have also been reports of an increase in the size of the remnant pancreas and recurrent acute pancreatitis as a form of presentation. diagnosis requires confirmation of the absence of the neck, body and tail of the pancreas and duct of wirsung using endoscopic retrograde cholangiopancreatography (ercp) or mrcp. one hundred four mr images of foetal cns with a us suspicion of acc were retrospectively reviewed. foetal mri was performed at 1.5 t magnetom avanto (siemens, erlangen, germany) without motherfoetal sedation. polymicrogyria, lissencephaly, schizencephaly, subependymal heterotopias and migration disorders were evaluated. cortical findings were compared to three types of acc (complete agenesis, partial agenesis and hypoplasia). genetic tests were collected. postnatal mri or foetopsy for diagnostic confirmation were collected. on 104 foetuses, fetal mri was able to detect cortical malformations in 32 cases even in early gestational ages (<24gw). the mean gestational weeks (gw) at mr diagnosis was 26 (range: 22-36gw). mr imaging found 13/32 polymicrogyria, 7/32 lissencephaly, 5/32 schizencephaly, 4/32 subependymal heterotopias and 3/32 neuronal migration disorders. 22/32 had complete acc, 4/32 had partial acc and 6/32 had cc hypoplasia. statistically significant correlations (p<0.005) between complete acc, focal polymicrogyria and cortical dysmorphism affecting frontal lobes were found. fetal cns mri can detect cortical development malformations in complex acc, providing further information for the clinician to assess the severity of perinatal outcome. mri is a useful tool in improving obstetrical genetic prenatal counselling to predict pregnancy and foetal prognosis. clinical signs of the neonatal lymphatic flow disorder (nlfd) are a combination of the congenital chylothorax, chylous ascites and body edema. it can present as neonatal chylothorax (nc), neonatal chylous ascites, or congenital lymphatic dysplasia (cld). the prenatal appearance of lymphangiectasia has been described as nutmeg lung. the purpose of this study is to describe prenatal and postnatal imaging features and outcomes of neonates with nlfd. materials: this is a retrospective case series of neonates in our institution that had pre-and postnatal lymphatic imaging and nlfd. all patients had prenatal imaging (fetal mri and us) and underwent postnatal dynamic contrast mr lymphangiography (dcmrl) with a three-dimensional (3d) t2 space. conventional lymphangiography (cl) when performed was also reviewed. six patients with nlfd were identified (3 with nc and 3 with cld). one patient had congenital heart disease. nutmeg lung was seen in all patients on fetal mri and 4 patients on fetal us. 5/6 patients had pleural effusions, 2/6 had ascites and 1/6 had body wall edema prenatally. postnatal mri with 3d t2 space revealed soft tissue edema in the upper chest and neck (5/6 patients), mediastinal edema (5/6 patients), interstitial lung edema (6/6 patients), retroperitoneal edema (5/6 patients), and ascites (6/6 patients). dcmrl demonstrated lymphatic flow to the pleural space (5/6 patients) and to the abdominal cavity (1/6 patients) and dermal backflow (2/6 patients). cl was performed in 4 patients, all of which had collateral lymphatic flow to the lung. lymphatic intervention was performed in 3 patients, lipiodol injection for 2 patients with nc and thoracic duct embolization (tde) for 1 patient with cld. mean hospital duration in the first 4 months of life was 51 days (range 5-113) for nc and 105 days (range 75-120) for cld. all 3 patients with cld died after 4 months of age due to respiratory distress including the patient that had tde and both with findings of dermal backflow. the pleural effusions in the 2 patients with nc resolved post lipiodol injection and in the other patient with nc it resolved with conservative therapy. conclusion: nlfd is a disorder that can be recognized on prenatal and postnatal imaging. in this small series, nutmeg lung was present in all patients with nlfd and may be easier to recognize with fetal mr than us. dermal backflow on dcmrl suggests a poor prognosis. both prenatal and postnatal imaging may guide treatment and interventions in nlfd. fetal mri and postnatal ct scans of prenatally diagnosed bpms from 10 patients with available histology were analyzed retrospectively. the fetal mri and ct images were reviewed by two radiologists blinded to histological findings. specific diagnosis was assigned based on predetermined criteria. the accuracy of fetal mri was evaluated. the agreement rate in fetal mri diagnosis between two radiologists was 100 %. an overlap of 80% in fetal mri and histopathological diagnosis was reached. when comparing fetal mri and postnatal ct examinations, the agreement of the results was also 80%. the least matching histological diagnosis was bronchopulmonary sequestration (bps). fetal mri is very accurate in characterizing the bpm spectrum and provides important information on lesion type and structure when compared with histology. with relatively small number of patients high correlation between prenatal mri and postnatal ct was reached. therefore, further investigation with more patients is needed. we hypotethise that fetal mri in late pregnancy could in the future replace early (neonatal) ct examinations if fetal mri provides sufficient inforfmation for clinical management. real time virtual sonography: a new integrated approach for the evaluation of fetal cerebral pathologies? s. bernardo, a. antonelli, v. vinci, m. saldari, c. catalano, l. manganaro; rome/it objective: real-time virtual sonography (rvs) is a new technique that uses magnetic navigation and computer software for the synchronized display of real-time us and multiplanar reconstruction mri images. the purpose of this study was to evaluate the feasibility and ability of rvs to assess the main cerebral pathologies in fetuses with suspected us anomalies. materials: this is a prospective study. fusion imaging (hitachi hi vision ascendus) was offered to 35 patients undergone fetal mri for a us suspicion of cerebral pathology. the mri image dataset acquired was loaded into the fusion system using a cd support and displayed together with the us image. both sets of images were then manually synchronized and images were registered. the possibility to record the images in a video format allowed, however, the possibility to re-evaluated the examination. results: rvs was technically possible in all cases. data registration, matching and fusion imaging were performed in 25 minutes at the beginning and in less than 15-20 minutes after practice. the ability of rvs imaging to assess the main anatomical sites and fetal anomalies was evaluated and compared with standard us and mri images. the principal application of rvs was the study of midline, cerebral gyration and vascular malformations because it also allowed adding a real time doppler signal on mri images. fusion imaging helped the diagnosis in 25%. in the 25/ 35 cases of encephalic pathology, fusion imaging improved the diagnosis; in the other cases mri was superior to us even using the rvs. this is a preliminary study on the feasibility and practical use of a fetal mri-us real-time fusion imaging. both techniques are complementary but still independent and the retrospective synthesis of these exams allows optimal analysis of fetal cerebral anomalies. this technique has many advantages especially on the pedagogic plan. however, rvs is currently limited to the research area. role of foetal mri in the evaluation of ischaemic-haemorrhagic lesions of the foetal brain s. bernardo, a. antonelli, v. vinci, m. saldari, c. catalano, l. manganaro; rome/it the aim of this study was to define the role of fetal magnetic resonance imaging in the evaluation of cerebral ischaemic-haemorrhagic lesions and the extension of parenchymal injuries. from september 2010 to december 2016 we performed 271 fetal mri of cerebral region in foetuses with suspected abnormalities on ultrasound or cmv infection and toxoplasma serum conversion. fetal mri was performed with a 1.5-t magnet system without materno-fetal sedation. fetal mri detected ischaemic-haemorrhagic lesions in 14/271 fetuses, revealing a 5% pathology incidence. mri confirmed the diagnosis in 3/14 cases with us suspect of ischaemic-haemorrhagic lesions associated with ventriculomegaly. in 1/14 cases with us findings of cerebellar haemorrhage, mri confirmed and provided additional information regarding the parenchymal ischaemic injury. in 8/14 cases with us suspect of ventriculomegaly (n=3), corpus callosum agenesis (2), cerebellar vermis hypoplasia (1), holoprosencephaly (1), spina bifida (1) mri detected ischaemic and haemorrhagic lesions unidentified at us examination. in 2/14 fetuses with us suspicion of intracerebral tissue space-occupying lesion, mri modified the diagnosis to extra-axial haematoma associated with dural sinus malformation. results were compared to fetopsy or after-birth follow up. fetal mri is an additional imaging modality in the diagnosis of cerebral ischaemic-haemorrhagic lesions and it is useful in providing further information on the extension of parenchyma injury and associated abnormalities and in improving delivery management. the contribution of mid-trimester virtual autopsy with mr imaging a. d'hondt, n. d'haene, j. rommens, m. cassart, f.e. avni; brussels/be the aim of the study was to assess the potential contribution of fetal virtopsy (post-mortem mr imaging (pm-mri)) in the second trimester of pregnancy. during a one-year period, post-mortem mr imaging (pm-mri) was performed in all fetuses who died in utero or whose pregnancy was interrupted due to major malformations. the study was performed in agreement with the local ethical committee. fetuses of <26 weeks that underwent obstetrical ultrasound and pm-mr were included. mr imaging examination was performed on a 1.5 tesla magnet with a standardized protocol. the findings on pm-mri were compared to obstetrical sonographic findings (and to pathology when available). we have analyzed separately the findings in the central nervous system and those in the rest of the fetus (chest, abdomen and skeleton). the results were classified in three categories according to the diagnostic accuracy: ultrasound>pm-mri, ultrasound=pm-mri and pm-mri>ultrasound. the us and pm-mri data of ten fetuses were analyzed. their gestational age ranged from 17.6-26 weeks and their bodyweight ranged from 160-930g. for the cns malformation: pm-mri offered a better diagnostic accuracy than us in 7 cases (70%) (e.g. agenesis of the corpus callosum and holoprosencephaly). in 3 cases (30%) us offered the same information than pm-mri. there was no case where us was more accurate than pm-mri. for the rest of the body malformations: pm-mri offered a better diagnostic accuracy in 5 cases (50%) (e.g. heterotaxy anomalies or vertebral segmentation anomalies). in 3 cases (30%), us offered the same information as pm-mri. there were 2 cases (20%) where us showed major malformations that were not diagnosed on the pm-mri (two cases of cardiac malformation). post mortem mr imaging is more accurate than obstetrical ultrasound in detecting major malformations in the cns as well as in the rest of the body. the present exceptions are cardiac malformations. the examination offers an easy evaluation of the deceased fetus. it provides, in most cases, important additional information. diffusion coefficient and perfusion fraction parameters correlate with gestational age in normal human in vivo placenta: a preliminary study a. antonelli, m. guerreri, s. bernardo, s. capuani, c. catalano, l. manganaro; rome/it to investigate the potential of diffusion parameters derived from a biexponential analysis as marker to evaluate the perfusion quality of normal in vivo placenta. eighteen normal pregnancies, fulfilling the study inclusion criteria, have been analysed at 1.5 t magnetom avanto (siemens, erlangen, germany) without mother-foetal sedation. dw imaging was collected using seven b values: 0, 50, 100, 150, 400, 700, 1000 (s/mm 2 ). three regions of interest (rois) have been considered -central (c), peripheral (p) and umbilical (u) regions. a bi-exponential model was used to obtain perfusion fraction (f), pseudo-perfusion (d*) and apparent diffusion (d) coefficients. pearson test was performed to investigate correlation between diffusion parameters and gestation weeks (gw), body mass index (bmi) and basal glycaemia (bg). the average values on all rois were d=1.41±0.16•10 -3 (mm 2 /s), d*=1.81 ±1.28•10 -2 (mm 2 /s), f=3.28±0.18•10 -1 , in good agreement with the literature. in the c roi, a positive correlation (p<0.04) was observed between f and gw. after 30 gw in the p roi a positive correlation between f and gw (p<0.05) and a negative correlation between d and gw (p<0.0001) were found. no correlation was found between d, d*, f, bmi and bg. conclusion: the f increase reflects normal placenta perfusion physiology. on the other hand, the decrease of d highlights placental parenchyma maturation becoming more fibrotic during late gestational age. bi-exponential model provides more and useful information about placental morphological changes compared to mono-exponential diffusion model. to demonstrate the diagnostic value of fetal mri in the detection of fetal central nervous system (cns) impairment in prenatally echocardiographic diagnosed congenital heart diseases. we retrospectively examined 24 fetuses between 19 gestational weeks and 33 gestational weeks who performed a fetal mri in our institution after a second-line ultrasonography, between april 2010 and october 2015. fetal heart and cns studies were performed with a 1.5 tesla magnet (siemens magnetm avanto) without maternal sedation. prenatal findings were compared to fetopsy results, fetal mri after 30 gw or postnatal mri. in our sample of 24 cases, 7/24 had interatrial septal defect (iasd),intervertricular septal defect (ivsd), and atrioventricular canal defect (cavc), 6/24 had cardiac rhabdomyomas, 3/24 had hypoplastic left heart syndrome and hypoplastic aorta, 2/24 had transposition of the great vessels, 2/24 had fallot tetralogy, 2/24 had aorta coartation and 2/24 had intracardiac masses of uncertain significance. magnetic resonance imaging was able to detect cns impairment: we recognize 11/24 corpus callosum (cc) dysgenesis (4/13 cc hypoplasia, 4/13 complete cc agenesis, 3/13 partial cc agenesis), 7/24 ventriculomegalies or hydrocephalus, 3/24 subtentorial anomalies (dandy-walker, vermian hypoplasia and vermian malrotation) and 3/24 gyration anomalies. due to the high risk of cns involvement in prenatal congenital heart diseases, it is essential to suggest an mri study of the evolving fetal brain especially in complexes forms that suggest a syndromic background. fetal mri of the cns is mandatory in the study of congenital heart disease due to the high rate of encephalic anomalies associated, particularly in iasd, ivsd and cavc. first experiences and diagnostic utility of micro-ct for fetal autopsy j.c. hutchinson 1 , x. kang 2 , s.c. shelmerdine 3 , m. cannie 2 , v. segers 2 , n. sebire 3 , j. jani 2 , o.j. arthurs 3 ; 1 newcastle upon tyne/uk, 2 brussels/ be, 3 london/uk perinatal autopsy remains poorly accepted by parents, despite yielding information that affects the management of future pregnancies in around 30% of cases. microcomputed tomography (micro-ct) has shown promising results in the examination of ex-vivo fetal organs, and may provide diagnostic imaging in cases where traditional autopsy is challenging, and s416 (2017) 47 (suppl 2):s297-s pediatr radiol existing post mortem imaging techniques (ct and mri) provide insufficient diagnostic resolution. our objective was to examine whole fetuses non-invasively using micro-ct, and compare the findings with standard autopsy as the gold standard. in this ethically approved double blinded study, terminated fetuses or miscarriages underwent iodinated micro-ct examination followed by conventional autopsy. images were acquired using a nikon xth225st microfocus-ct scanner with individual specimen image optimisation. forty indices normally assessed at perinatal autopsy were evaluated for each imaging dataset by two independent reporters and a consensus report produced. autopsies were performed blinded to the imaging findings by one of two perinatal pathologists. we examined 8 fetuses, with a gestational age range of 11-16 gestational weeks. 36/320 indices were non-diagnostic (11%), but there was agreement for 271/284 diagnostic indices (overall concordance of 95.4% (95% ci 92.3, 97.3%). in seven out of eight fetuses (87.5%), the same final diagnosis was made following micro-ct examination and autopsy examination; in one case, micro-ct was non-diagnostic. ten false negatives indices included a vsd, laryngeal anomaly, ambiguous genitalia and incomplete bowel rotation, none of which changed the overall diagnosis. three apparent false positives on micro ct were a cloacal anomaly, incidental cystic neck lesion and thymic atrophy, which were not detected at autopsy. micro-ct of early gestation whole fetuses can provide highly accurate datasets with three-dimensional renderings of complex disease processes. this approach confirms the potential of this technology for non-invasive examination of small fetuses. investigation of perinatal body organ diffusion-weighted post mortem mri s.c. shelmerdine 1 , m. cheryl 2 , j.c. hutchinson 1 , n. sebire 1 , o.j. arthurs 1 ; 1 london/uk, 2 southampton/uk objective: diffusion weighted magnetic resonance imaging (dwi) uses water molecule diffusion to generate mr contrast images, and can reveal microstructural or functional changes in tissues, quantified by measuring the apparent diffusion coefficient (adc). the application of dwi to the post mortem setting is appealing as it does not require the administration of an exogenous contrast agent. a recent pilot study of 15 paediatric cases suggested that lung adc values at pm mri may be a useful marker of post mortem interval (time since death; pmi) which has forensic relevance, but other body organs have not been comprehensively evaluated. the aim of this study was therefore to evaluate the relationship between pmi and body organ adc values in a larger cohort of subjects across a wider gestational range in the setting of perinatal death. whole body perinatal postmortem mri with dwi sequences were performed at 1.5t, with b values of 0, 500, 1000 mm 2 /s. mean adc values were calculated from regions of interest (rois) placed in the lungs, myocardium, spleen, renal cortex, liver and psoas muscle. the values were measured by two independent readers, correlated against gestational age and post mortem interval (pmi) using the pearson product-moment correlation coefficient. bland-altman plots were created, and the limits of agreement used to assess the inter-observer agreement of mean adc values. results: eighty fetal deaths and stillbirths were imaged with mean gestational age 31.5 weeks (range: 20 -41 weeks). the mean pmi was 8.9 days (range 2-20 days). there was a weakly positive correlation between pmi and mean lung adc (r 2 =0.03) and spleen adc (r 2 =0.08). no correlation was found with between adc and pmi for the other body organs. there was reasonable inter-observer agreement between the two readers, with mean adc difference 11.8 mm 2 /s (+/-135.1 mm 2 /s). perinatal lung and splenic adc values show a mild increase with increasing pmi. together with other imaging parameters, this may be useful to evaluate organ-specific changes which occur in the post mortem period, particularly in a forensic setting. further research is needed to understand the organ-specific changes which occur in the post-mortem period. usefulness of combined grey-scale and color doppler ultrasonography(us) findings in the evaluation of acute pyelonephritis in children k. lee, j.h. lee; anyang/kr objective: us diagnosis of apn in children can give a valuable information to the clinicians for the early treatment. but the problem of us in the diagnosis of apn is wide range of sensitivity, which is 11-69%. the purpose of this presentation is to evaluate the usefulness of grey-scale us and color doppler us in the diagnosis of acute pyelonephritis in children. from march 2007 to february 2014, 154 children(308 kidneys), 108 boys and 46 girls, aged 2 weeks to 9 years (mean age, 7.7 months) underwent kidney us as an initial diagnostic tool for acute pyelonephritis and follow up dmsa scintigraphy within a week. criteria for acute pyelonephritis on grey-scale image were focal/diffusely increased/decreased echogenicity or loss of corticomedullary differentiation. on color doppler sonography, the criterion was decreased color flow. we classified the us diagnosis of apn into 4 categories. definite, suggestive, possible and normal. when above two grey-scale us criteria and color doppler us criterion are seen, we classified it as 'definite'. when one of greyscale us and color doppler us finding are seen, it was classified as 'suggestive' of apn. 'possible' apn was abnormal finding either on grey-scale or color doppler us. 'normal' was no abnormal findings on grey-scale and color doppler us. we compared above findings with dmsa scan, which is considered as gold standard for diagnosing apn. statistical analysis was performed on all 308 kidneys. the overall sensitivity of our study was 68%(88/129) and specificity was 72%(123/177). the positive predictive value for each definite, suggestive, possible groups were 86%, 60%, and 38% respectively. the negative predictive value for normal group was 75%, which means the false ppv was 25%. the p-value of the definite and suggestive was statistically significant, but the possible was statistically insignificant. in the diagnosis of apn in children, abnormal us finding either on greyscale or color doppler us is not optimal. abnormal us findings both grey-scale us and color doppler us showed good association with dmsa scan and statistically significant. combined grey-scale and color doppler us findings can give a more reliable information in the diagnosis of apn in children. the greater degree of gastric and/or duodenal wall thickening and increased echogenicity are helpful sonographic features in differentiating congenital duodenal anomalies from malrotation. our findings confirm the superiority of us vs ugi for evaluation of duodenal obstruction in neonates and evaluation of gastric and duodenal wall must be added to the constellation of other features to be assessed on us examinations. a measure of renal morphology as an indicator for potential renal failure a.c. eichenberger, p. grehten, c. kellenberger; zurich/ch this study introduces a measure of renal morphology, herein labelled split renal volume (srv), that should be applied as an indicator for potential renal failure and eventual surgical treatment of obstructive uropathy in children. current practice applies dynamic contrast enhanced functional renal imaging (fri) with complex post-processing methods. fri generates a measure of split renal function (srf). reduced values of srf under 45% are currently considered to be an indicator for surgical treatment. this retrospective study compares the accuracy of srv with the accuracy of srf as methods for assessing potential renal failure. materials: srv is a quotient of volumetric measurements. total renal volume is described by the sum of parenchymal volume and intra-renal collecting system volume. srv is designated in this study as the quotient of two ratios: first, the ratio of total renal volume to parenchymal volume of the left kidney; and second, the ratio of total renal volume to parenchymal volume of the right kidney. twenty-two children were studied: 16 (age 3.1±4.5y) with unilateral asymptomatic intrinsic uretero-pelvic-junction obstruction (upjo), and 6 normal controls (age 6.6±4.0y). all subjects underwent mr urography at 1.5t, which provided estimates of srf and srv for each of the 44 examined kidneys. the sensitivity and specificity of both srf and srv for predicting surgical management were determined by comparing the indicators with an expert review panel's decision to operate. the panel was blinded to values of srv. results: when a cut-off value of 45% for srf was used, the resultant sensitivity and specificity of srf for the detection of kidneys at risk were found to be 44% and 86%. the values of srv ranged between 0.3 and 3.5. it was found that a value greater than 1.1 indicated kidneys at risk. when the cut-off value of 1.1 for srv was used, the resultant sensitivity and specificity of srv for the detection of kidneys at risk were both 100%. in this small population, srv proved to be 100% accurate and is superior to srf for detecting kidneys at risk of failure due to obstruction. routine application of srv promises to simplify mr urography by obviating dynamic contrast enhanced imaging studies. further prospective studies are necessary in order to select an optimal cut-off value of srv. factors that can distort the dj flexure mimicking malrotation v. bhalla 1 , s. mohan 2 , k.a. bradshaw 2 , m. thyagarajan 2 ; 1 stoke-on-trent/uk, 2 birmingham/uk to highlight the varied radiological appearances and position of the duodenal-jejunal flexure in children and to discuss its importance in assessing for malrotation materials: retrospective analysis of the multiple fluoroscopic examinations performed in the assessment for malrotation over the past 5 years in a busy tertiary centre results: the classic position of the dj flexure is to the left of left pedicle of l1 and at the level of the duodenal bulb on frontal views and posterior (retroperitoneal) on lateral views. however variations of the normal location can appear, particularly on frontal views, in the upper gi series that can mimic malrotation which has shown to be more common in neonates. we present cases with examples to illustrate the variability in position due to various causes and its implications in the diagnosis of malrotation and volvulus. our case mix includes patients with excessively distended stomachs, large bowel obstruction, renal pelvic dilatation, repeated naso-jejunal and gastro-jejunal tube insertion and in patients post liver transplantation. malrotation and its assessment have serious management and prognostic implications. this presentation demonstrates that the imaging features can be varied, and knowledge about factors distorting the position of the dj flexure is vital in the accurate management of neonates presenting with bilious vomits. retrospective study of prospectively collected data performed at a single tertiary paediatric institution over a 16.5 year period. a total of 1000 consecutive patients, aged <18 years, were reviewed who underwent native renal biopsy. all biopsies were performed within the interventional radiology department. all patients had renal disease requiring a renal biopsy for diagnosis. outcome measures include technical success, early and late complications and the adequacy of histological samples. in addition, age, body weight, glomeruli number, histological data, number of cores, size of the biopsy needle, use of co-axial needle and the rate of tract embolisation/plugging were recorded. results: from september 1999 to april 2016, 1000 patients (mean age: 10.07 years +/-4.65; range 0.14 -18.0 years) underwent native renal biopsy. one hundred ninety-one patients were <5 years of age. nine hundred forty-six patients (94.6%) had a biopsy of the right kidney, 53 patients (5.3%) had a biopsy of the left kidney and 1 patient (0.1%) had a biopsy of a horseshoe kidney. five hundred fifteen patients were female (51.5%). seven hundred sixtynine patients (76.9%) had the procedure performed under general anaes-thetic and 227 of patients (22.7%) had the procedure performed under local anaesthetic (+/-sedation/entonox). mean number of passes of the core biopsy needle through the renal capsule was 2.7. a 16 gauge core biopsy needle was used in 100% of the patients. 81.9% of the patients had three or less passes of the biopsy needle though the renal capsule. the overall complication rate was 2.6% (n= 26). 1.2% (n= 12) of patients had a non-diagnostic biopsy. fifty-five patients underwent a post biopsy ultrasound due to clinical concerns. twenty patients developed perinephric haematoma (19 were treated conservatively; one underwent embolisation and subsequent nephrectomy). four patients developed arteriovenous fistulas. two patients developed post procedure infections (one at the skin site and one a perinephric collection). histology results were reviewed in all patients. the mean number of glomeruli obtained was 26.97 (range 2-87). glomerulonephritis was the most common histological diagnosis (n=464; 46.4%) conclusion: renal biopsy is an extremely useful diagnostic tool for renal disease. there is no published data of this size assessing the outcome of native renal biopsies in the paediatric population. jr usa 1979 a. lassrich germany 1979 j. sauvegrain france 1982 c. fauré france 1982 a. giedion switzerland 1983 e. willich germany 1984 r. astley united kingdom ringertz sweden 1994 d.g. shaw united kingdom 1996 r. lebowitz usa 1996 b. lombay hungary pena spain gold medallists london/united kingdom the dutch group of paediatric radiologists, the hague/the netherlands 1981 g. stake ringertz (espr) & d. kirks (spr) chicago/united states future espr meeting italy european courses of paediatric radiology (ecpr) genoa/italy (neuroradiology) 2000 r.fotter, graz/austria (abdomen) brussels/belgium (thorax) 2006 j-n. dacher paediatric musculoskeletal imaging) references: 1. 2014-stellungnahme-lnt-modell.pdf [internet]. [zitiert 2 an evaluation of paediatric projection radiography in ireland contrast imaging -> application -dectris background ionizing radiation and the risk of childhood cancer: a census-based nationwide cohort study best practices in digital radiography communicating radiation risks in paediatric imaging kinderradiologie-besonderheiten des strahlenschutzes diagnostic imaging and ionizing radiation -canadian nuclear safety commission epidemiology without biology: false paradigms, unfounded assumptions, and specious statistics in radiation science (with commentaries by inge schmitz-feuerhake and christopher busby and a reply by the authors) european guidelines for ap/pa chest x-rays: routinely satisfiable in a paediatric radiology division? eurosafe imaging together -for patient safety image gently campaign back to basics initiative: ten steps to help manage radiation dose in pediatric digital radiography hostens j, u. a. in-vivo dark-field and phase-contrast x-ray imaging safety commission cn. linear-non-threshold model optimisation of paediatric chest radiography optimizing digital radiography of children radiation exposure in diagnostic imaging: wisdom and prudence, but still a lot to understand radiation shielding for diagnostic radiology strahlenhygienische aspekte bei der röntgenuntersuchung des thorax the image gently pediatric digital radiography safety checklist: tools for improving pediatric radiography the standardized exposure index for digital radiography: an opportunity for optimization of radiation dose to the pediatric population gastroenterology and radiology records were searched to identify ibd patients with colonic strictures. all patients underwent an mre within 3 months of colonoscopy. the following colonic parameters were evaluated: bowel wall thickening with luminal narrowing, pre-stenotic bowel dilatation, bowel wall enhancement, and diffusion restriction (if performed). colonoscopy and operative notes were correlated. results: fourteen patients met the inclusion criteria, one with 2 colonic strictures. bowel wall thickening with luminal narrowing at the site of the reported stricture was present in all cases. pre-stenotic bowel dilatation (>3.0 cm) proximal to the reported stricture was present in 11/15 cases. using luminal narrowing and prestenotic dilatation as criteria for diagnosis of a colonic stricture, 11/15 cases were therefore positive on mre. when comparing to colonoscopy, mre diagnosed colonic strictures in 8/12 cases (67%). in the six patients who had surgery, mre accurately diagnosed colonic strictures in 5/6 cases (83%). conclusion: mre is not the primary modality for colonic evaluation, yet diagnosing colonic pathology on mre, particularly strictures, may be beneficial for the referring gastroenterologist in the assessment of these patients. potential strictures on colonoscopy did not agree with mre in all cases, but when correlating with surgery 83% of colonic strictures were accurately diagnosed in a small subset. although mre is not optimized for the evaluation of the colon, colonic strictures can be recongnized in children with crohn's disease.disorders of sexual differentiations in neonates: standardized sonographic evaluation and proposal of a reading grid h. lerisson, e. amzallag -bellenger, f.e. avni, m. cartigny; lille/fr to propose a systematic and structured sonographic approach in neonates with disorders of sexual differentiation (dsd) materials: review of the us pelvic, external genital and adrenal findings in 20 consecutive patients with clinical suspicion of dsd evaluated in the neonatal period. the us survey included: the uterus (absent or visible -with or without hormonal impregnation), the vagina (absent or present (complete or partial)), the gonads (ovaries, testis or unsetermineddysgenetic ) as well as the adrenals (normal, too small or enlarged). the us conclusions were correlated with the endocrinological and genetical work-up of each patient results: twenty cases of dsd have been included us had correctly identified the presence of a uterus in 11 patients. there was one false positive case; 6 among the 11 patients did not show the physiological hormonal impregnation. the 5 vaginal anomalies were correctly evaluated. the gonads were defined correctly as normal testis in 6 patients, normal ovaries in 4 and dysgenetic gonads in 4. they could not be visualized in 6 patients. adrenals were considered normal in 17 patients (one false negative), hypertrophied in 2 and small in one patient. to compare hepatic 2d shear wave elastography (2d swe) in children between free-breathing and breath-hold conditions, in terms of measurement agreement and time expenditure. a cohort of 57 children (12.7±4.3 years) who underwent standardized 2d swe between may and october 2015 were retrospectively evaluated. liver elastograms were obtained under free-breathing and breath-hold conditions and time expenditure was measured. median stiffness, interquartile range (iqr), and iqr/median ratio were calculated based on 12, six, and three elastograms. results were compared using pearson correlation coefficient, intraclass correlation coefficient (icc), bland-altman analysis, and student's t. median liver stiffness under free-breathing and breath-hold conditions correlated strongly (7.22±4.5kpa vs. 7.21±4.11kpa; r=0.97, p<0.001). time to acquire 12 elastograms with free-breathing was lower than that with breath-holding (79.3±32.5sec vs. 143.7±51.8sec, p<0.001). results for median liver stiffness based of 12, six, and three elastograms demonstrated very high agreement for free-breathing (icc 0.993) and for breath-hold conditions (icc 0.994). hepatic 2d swe performed with free-breathing yields results similar to the breath-hold condition. with a substantially lower time requirement, which can be further reduced by lowering the number of elastograms, the free-breathing technique may be suitable for infants and less cooperative children not capable of breath-holding. abstract: pelvi-ureteric junction obstruction (pujo), classified into intrinsic and estrinsic is one of the most frequent urological diseases affecting the pediatric population. extrinsic causes include the presence of crossing vessels, kinks or adhesions. in cases with extrinsic obstruction of puj, colour doppler ultrasound (cd-us) can detect the presence of crossing vessels. in presence of crossing vessels pyeloplasty or vascular hitch can be performed. the aim of the study is to analyze the sensitivity of cd-us and magnetic resonance urography (mru) in visualizing crossing vessels in extrinsic pediatric hydronephrosis in order to decide the correct diagnostic pathway and evaluate in the pre-operative phase which surgical technique and approach (open, laparoscopic or robotic) is the ideal to be performed. a retrospective review of medical records for patients who underwent surgical treatment for hydronephrosis from august 2006 to february 2016 was performed. a descriptive statistical analysis was performed. the presence of crossing vessels at surgery was considered the gold standard. the sensitivity was calculated for both the imaging techniques as a measure of accuracy, evaluating the ratio between the positive cases divided by the those with aberrant vessels identified at surgery. results 220 clinical charts were reviewed. crossing vessels identified at surgery were 73 (33,2% of pujo). the median age was higher in the group with crossing vessels compared to the group without crossing vessels (p< 0,0001). the sensitivity of cd-us was higher compared to mru (93,3% vs 71,7%). before the surgical time knowing which technique and approach have to be managed in hydronephrotic patients with crossing vessels could be very important. according to our preliminary datacollection cd-us has got a higher sensitivity and could be the gold standard technique. study limitations include the absence of specificity, positive and negative predictive values. in the future it could be useful to perform a double blind trial in which children with moderate-severe hydronephrosis will be subjected to both imaging techniques to evaluate not only the sensitivity, but also the specificity, the positive predictive value and the negative predictive value conclusion: conclusions in the pre-operative phase, cd-us could be sufficient for the surgeon to discern between pujo with the presence or the absence of crossing vessels, as it has a higher sensitivity and lower costs compared to mru.urosonography -nonradiant alternative for voiding cystourethrography o.m. fufezan, c.a. asavoaie, s. tatar; cluj-napoca/ro voiding cystourethrography (vcug) was considered the gold standard in the diagnosis and monitoring of vesicoureteric reflux (vur). this method is invasive due to the radiation exposure. in the present the diagnosis of vur can also be established by contrast ultrasound examination, also known as voiding urosnography (vus). the authors will present the role of vus in the diagnosis and grading of the vur and the role of patient position in the detection of vur. the infants and children with congenital anomalies of the urinary tract and/or urinary tract infection have been evaluated with vus. iatrogenic vur, neurogenic bladder and urogenital sinus anomalies were excluded. the presence and the degree of the vur were evaluated. vus has been performed using a protocol similar to the one used for vcug. in conditions of sterile urine, 0.5 ml sonovue and saline solution have been introduced into the bladder until voiding started. the patients were examined both in a supine and an upright position and the following structures have been scanned: urinary bladder, distal part of the ureters and both pelvicaliceal systems during bladder filling, during and after voiding. the visualisation of the ultrasound contrast agent in the upper urinary tract represented a positive vur diagnosis. the grading of the vur has been established based on the same criteria as in vcug. sixty five patients (130 renal units), ages between 2 weeks and 17 years were evaluated (median age ± sd: 3 years ± 4 years and 2 months) through vus. vcug was performed in 6 patients in a maximum of 48 hours after vus. vur has been identified in 35 patients (40.7% renal units). a high vur grade (iv-v) was identified in 19.2% of renal units. for the patients investigated with both methods, the results were concordant in 4 patients. in two patients vur has not been identified by vcug, but was detected during vus. the upright position (in addition to decubitus) revealed vur in 3 renal units in which the reflux was not detected in decubitus. conclusion: vus is extremely useful and reliable in diagnosing and grading vur in pediatrics. the changing of the patient position during examination can improve vur detection.new sonographic features useful in differentiating congenital duodenal anomalies from malrotation: gastric and duodenal wall thickening and hyperechogenicity p. caro dominguez 1 , s. hameed 2 , a. zani 3 , r. moineddin 3 , o.m. navarro kunstmann 3 , a. daneman 3 ; 1 cordoba/es, 2 london/uk, 3 toronto/ca the clinical and plain radiographic differentiation of congenital duodenal anomalies (atresia, web, stenosis) and intestinal malrotation is not always clear. although sonography has been documented as an important diagnostic tool to differentiate these two entities, its role is still not widely appreciated. the purpose of this study was to assess the sonographic features of the gastric and duodenal wall in a large series of neonates with congenital duodenal obstruction as these have not been reported previously. neonates who had surgically proven congenital duodenal anomalies or malrotation were identified from the surgical database in a tertiary pediatric hospital in a period of 15 years (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) . those with an ultrasound performed within 48 hours of surgery were included in the study. imaging was retrospectively and independently reviewed by two readers in chronological order blinded to final diagnosis. a wall thickness of ≥ 2 mm of a distended loop was considered abnormal. hyperechogenicity was recorded when the wall of the stomach or duodenum was brighter than liver or splenic parenchyma. imaging findings in the group with congenital duodenal anomalies was compared to the group with malrotation using fisher's exact test. one hundred eight neonates were included in the study, 40 with a congenital duodenal anomaly, 49 with malrotation (36 with volvulus) and 19 with both. ugi was performed in 61 neonates who had us. the correct diagnosis was provided only by us in 24 of these 61 newborns (39%), only by ugi in 5 (8%), by both in 26 (43%) and by neither in 6 (10%). ugi was performed in 21 children with malrotation and volvulus, eight were diagnosed only by us, four only by ugi and nine by both. the gastric and/or duodenal wall was significantly thicker and more hyperechoic in neonates with congenital duodenal anomalies than those with malrotation (p<0.0001) [fig 1, table 1 ]. conversely an abnormal relationship between the superior mesenteric artery and vein, abnormal position of the third part of the duodenum and the whirlpool sign were found more commonly in neonates with malrotation than those with congenital anomalies (p<0.0001). key: cord-022659-chwk2bs4 authors: nan title: abstracts: poster session date: 2004-10-08 journal: ann neurol doi: 10.1002/ana.410320224 sha: doc_id: 22659 cord_uid: chwk2bs4 nan an immune etiology has been postulated for acute cerebellar ataxia of childhood (acac) since it frequently follows viral infections. we analyzed serum and cerebrospinal fluid (csf) from 6 acac patients for antibody cross-reacting with cerebellar neurons. serum and csf were obtained within 7 days of onset of pancerebellar ataxia from subjects aged 3.5 to 11 years. varicella infection preceded 4 cases. results of enhanced cranial ct scans were normal; csf demonstrated 2-122 cells/mm3 with sterile cultures. serial dilutions from 1 : 20 of serum and undiluted csf were screened for antineuronal antibody by indirect immunofluorescence (iif) using frozen, unfixed normal human cerebellum. serum (1 : 400) was examined further for antineural antibody by western immunoblotting using purified cerebellar neuronal extracts as antigen. serum from age-matched, neurologically normal pediatric inpatients served as the control group for iif and immunoblot experiments. in acac patients, no antineuronal immunoreactivity was observed by iif. immunoblots demonstrated no consistent pattern of immunoreaction when comparing acac to controls, though 1 patient exhibited distinct bands at 200 kd (neurofilament protein) and 54 kd. although antecedent infection suggests an immune etiology for acac, our preliminary results do not support a humoral mechanism for this disorder. ingrid taff; joseph zito, robert gould, and steven pavlakis, great neck and manhasset, ny in a 20-month period we studied 69 patients between the ages of 7 weeks and 2 1 years with magnetic resonance angiography (mra). studies were performed on a 1.5t magnet (siemens magnetom sp) with a circular polarized head coil. a three-dimensional time-of-flight technique was utilized. occasionally, images were obtained after gadopenetate dimeglumine infusion. two-dimensional projection images were calculated using a maximum intensity projection algorithm and recorded on laser film. sixty-seven patients also had routine mri. a sampling of vascular lesions was demonstrated. nineteen patients had clinical and mri evidence of stroke. mra revealed intracranial vascular occlusion in 2 patients, diminished focal cerebral flow in the affected area in 4, and generalized ipsilateral underdeveloped cerebral circulation in 4. a moya-moya vascular pattern was found in 4 and sickle-cell vasculopathy was found in 1 patient. seven mras were normal. seventeen vascular hamartomas were demonstrated including 2 vene of galen malformations, 7 arteriovascular malformations, and 8 venous angiomas. three aneurysms were found. thirty-one mras were normal. we find m u to be a valuable adjunct to routine mr imaging in the evaluation of pediatric patients with potential cerebrovascular disease. it demonstrates a spectrum of pathology, is noninvasive, and allows for serial follow-up examinations. angiography in pediatric cerebrovascular disease p4. thalamic change in acute encephalopathy of adult rats. twelve weeks after grafting, clinical and histological studies were performed. we developed a protocol for evaluating functional deficits that follow spinal cord injury in the rat. the survival, growth, differentiation, and parenchymal integration of the graft were documented histologically on semi-thin section. animals that received the transplants demonstrated qualitative and quantitative improvements in several parameters of locomotion. donor tissue integrated most often with the host spinal cord at interfaces with host gray matter; however, some implants also exhibited sites of fusion with damaged host white matter. we suggest embryonic rat spinal cord transplantation may be a useful treatment of spinal cord injury and a possible therapeutic strategy in human spinal cord injury and amyotrophic lateral sclerosis. the basic neuropathophysiology of hemineglect after unilateral cerebral lesions is still not clear. one theory holds that degraded perceptual processing occurs in the damaged hemisphere due to intrahemispheric deficits. another holds interhemispheric interaction at fault, with the intact hemisphere actively inhibiting spatial cognitive processes in the damaged one. we tested 11 adult macaca fascicuhris with acute neglect on a task in which the whole visual surround was restricted to 15 degrees from central fixation, and a second in which an opaque lens occluded the eye either ipsilateral (ipsi) or contralateral (contra) to the lesion. using paired t tests, in the first task there were no differences in reaction time to the ipsi and contralesional hemifields. in the second, there was no change in extent of the ipsilesional field (obtained with the contralesional eye occluded), as compared to its extent without occlusion. the contralesional field, however, improved significantly ( p < .03) with the ipsilesional eye occluded. since reducing sensory input to both hemispheres leads to no worsening of hemineglect, but reducing sensory input to the intact hemisphere alone leads to improvement of hemineglect, we conclude that adverse interhemispheric interactions play a major role in the pathophysiology of hemineglect. we assessed the sensitivity and applicability of a new, cornbined cognitive and mood screening battery for multiple sclerosis (ms). sixty consecutive, untreated clinically active ms patients, 30 each relapsing-remitting and chronic progressive, underwent the battery and head mri upon entering 2 concurrent treatment trials. the battery combines the faust-fogel brief cognitive screen and visual analogue dysphoria scale, both previously validated in other neurological diseases. cognitive domains tested were immediate and delayed sentence and word-pair recall, verbal fluency, and conflicting response suppression. patients marked ''usual mood" along a "happy-sad" cartoon continuum. relapsing patients were program and abstracts, american neurological association 235 younger (32.8 vs 41.2 yr mean), with shorter ms durations (6.4 vs 11.3 yr), and had lower kurtzke disability scores (1.7 vs 5.8). modified qualitative mri grading (lesion burden, confluence, localization) was compared. half as many relapsing patients (43% vs 83%) scored ''abnormal'' cognitively, despite similar "sadness" rates (20% vs 23%). subjective dys-~ phoria in both groups correlated with denser periventricular lesion burdens. the battery was well tolerated and easily administered within 10 minutes without special equipment. this combined cognitive and mood screening battery is sensitive and convenient for clinically active ms. alternate forms of the battery are needed for repeatability. questionnaires may be reliable and valid supplements to laboratory tests for brain-damaged patients, as they can be applied to situations for which laboratory testing is not possible. we investigated the usefulness of informant-based data in alzheimer's disease (ad) by comparing caregivers' subjective evaluations of 83 probable a d patients' performance on an abbreviated version of the memory self-report questionnaire to objective evaluations derived from an extensive battery of neuropsychological tests and to clinicians' evaluations. similar information was obtained from 39 healthy agematched controls. caregivers' subjective appraisals of patients' memory correlated significantly with objective measures of secondary memory, with all cognitive variables, measures of activities of daily living, and clinicians' evaluations of dementia staging. scores were independent of clinical indicators of depression. the abbreviated memory questionnaire showed good reliability, internal consistency, and external validity. its positive predictive value is 63.5 and its negative predictive value is close to 100%. results suggest that (1) informant-based questionnaires may be useful for obtaining valid information on cognitive ability outside of laboratory settings; (2) the scale reflected more than just memory functions; and (3) the scale may be promising for screening cognitive difficulties in epidemiological or clinical settings. although neglect along the horizontal dimensions of extrapersonal space is well recognized, there are only a limited number of observations documenting neglect along the vertical and radial spatial dimensions. we report an investigation of neglect along the 3 principal dimensions of extrapersonal space in a patient with bilateral mesial temporo-occipital infarctions. neglect was assessed by asking the patient and controls to bisect lines of 4 lengths oriented in 3 directions with respect to the body: horizontal, vertical, and radial. our patient showed significant neglect of upper vertical and far radial space, as well as neglect of left hemispace. his line bisection errors were consistently in a direction opposite the slight directional biases shown by controls for all 3 line orientations ( p < .05). the magnitude of the patient's bisection errors increased by moving the lines toward the neglected sectors of 3-dimensional space. neglect of upper vertical and far radial space was also evident on line cancellation tasks. our results suggest that following focal brain injury, neglect may be observed along all 3 dimensions of extrapersonal space. these findings provide further empirical support for functional specialization within inferior and mesial temporooccipital regions for attending to upper vertical and far visual space (previc, 1990) . p12. posterior cortical atrophy: degenerative disease with primary visuospatial and visuosemantic deficits a. kertesz, m . polk, and a. kirk, london, ontario, and saskatoon, saskatchewan, canada posterior cortical atrophy is a recent, and heidenhahn's disease is an old, label for a miscellaneous group of patients with imaging or pathological and clinical evidence of visuocognitive deficits and cortical atrophy localized to the posterior cortex. the extent of this cortical localization and the nature of the pathological findings are not fully agreed upon, but spongiform degeneration and alzheimer pathology have been described. detailed examination of patients who are representative of the problem and have uniquely specific deficits is presented. one patient had visual associative agnosia, prosopagnosia, and transcortical sensory aphasia. lexicosemantic experiments of categorization, word retrieval, and comprehension of auditory and visual stimuli showed a specific impairment of visuoverbal semantics. a striking preservation of phonological, orthographic and visual structural input, and intercategory dissociations was demonstrated. consistency of errors argued for specific loss of semantic knowledge. another patient with apraxia, primary visuospatial deficit, agraphia, and amnesia at the beginning had predominantly right-sided posterior cortical atrophy, demonstrating further fractionation of the entity and the striking specificity of visuospatial function. the behavioral specification of degenerative disease is clinically and theoretically important. permanent neurological deficits after ischemic stroke are mainly determined by the location and size of the infarct. clinical recovery also depends on the functional state of adjacent brain tissue, where both neuronal loss and deactivation without gross morphological damage may affect flow and metabolism to a varying degree (g. mies et al, stroke 1983; 1422-27) , and where the ability to respond to stimulation by appropriate neuronal recruitment may be impaired. therefore, degree of resting hypometabolism and of responsiveness to functional activation may provide a measure of prognosis. in 26 patients (age 60.7 10.9 yr) with aphasia consequent to ischemic stroke of the dominant hemisphere, regional cerebral metabolic rate of glucose (rcmrgi) was measured at rest and in 17 of them also during spontaneous speech, using positron emission tomography (pet) of 2-(f18)-fluoro-2-deoxy-~-glucose (fdg). the pet study and a standardized neuropsychological test battery to assess the main aspects of language were performed around the fourteenth day after the stroke, and the language functions were assessed again 3 to 5 months later. performances in various dimensions of language 2 weeks and 3 to 5 months after stroke were related to rcmrgl in topographically meaningful areas at rest and during activation using wilcoxon-rank program and abstracts, american neurological association 237 sum test and multiple regression analysis. severity of aphasia was assessed by the token test, which showed a bimodal distribution to slight and severe, and a lower representation of moderate cases. global and all regional cmrgl at rest and during activation were significantly correlated to scores in token test at first and second examination, with the highest correlation coefficients ( -0.7 to -0.61) for broca's, wernicke's, and left temporoparietal regions. for performance after 3 to 5 months, the relationships were still significant with lower coefficients. verbal fluency also was correlated to kmrgi, but with lower coefficients that slightly increased for the recovery state. language performance at different stages in the course after ischemic stroke was significantly related (r = 0.84 for token test, r = 0.93 for verbal fluency). however, there exists a high variability in recovery that may be explained by stepwise regression of metabolic values. significant effects were observed only for cmrgl of the left hemisphere outside the infarct (partial r' = 0.21) at rest and for cmrgl within the infarct (0.27), the contralat-era1 mirror region (0.16), and broca's region (0.17) during activation, with a sum of all partial weight factors of 0.46 at rest and 0.72 during activation. our results furnish 2 indicators for recovery of aphasia: the resting metabolism of the left hemisphere outside the infarct, and the activated metabolism in residual tissue within the infarct and in languagerelated areas. although the hemispheric metabolism at rest might be related to neuronal loss and thereby to the brain's reserve capacity, the extent of metabolic activation indicates neuronal recruitment and the capability of neuronal networks for functional recovery. heparin therapy for acute myocardial infarction: the timi-i1 pilot and randomized trial combined experience m . a. sloan, t . r. price, m . l. tevrin, and s. forman for the timi investigators, baltimore, m d of 3,924 myocardial infarction (mi) patients treated with rt-pa and heparin, 29 (0.7%) developed ischemic cerebral infarcts (ci). all ci patients had detailed neurological evaluations and 27 (939%) had c t scans. age range was 40 to 74 years (mean 60 yr), 25 were male, and 22 were caucasian. electrocardiographic location of mi was anterior in 22 (76%) and nonanterior in 7 (249%). six cis occurred within 6 hours; 1 between 6 and 12 hours; 2 between 12 and 24 hours; 4 between 24 and 48 hours; 13 during the second week; and 3 others distributed over the 4 weeks after study entry. six of 29 cis did not involve cerebral cortex; 9 (319%) had multiple cis. of 24 cis thought to be embolic in origin, 17 had at least 1 cardiac abnormality (mural clot, wall motion abnormality, aneurysm, or transient atrial fibrillation) known to be associated more specifically with embolism than just the diagnosis of myocardial infarction. eight of 27 (30%) with ct scans had hemorrhagic conversion of varying degrees. the time of occurrence and sites of ci after rt-pa and heparin therapy for acute mi are similar to those reported in the prethrombolytic area. nancy futrell andjeanne m. riddle, detroit, m i photochemical irradiation of the carotid artery of rats has been used to induce endothelial damage, producing a nonocc h i v e thrombus (that apparently embolizes spontaneously) thrombi and emboli and multiple cerebral infarcts. evidence for embolism generally has a presumptive component. to document further that cerebral infarcts in this model are indeed due to embolism, we studied the ultrastructure of the carotid thrombi and the presumed cerebral emboli using scanning and transmission electron microscopy (sem, tem). the right carotid artery of 9 wistar rats was irradiated with a laser (632 nm, 200 mw/cm2, 15 min) following the injection of the photosensitizing dye photofrin 11, 12.5 mgikg. rats were sacrificed from 1 to 24 hours later. endothelial damage with formation of a fragmenting thrombus, composed mainly of platelets and erythrocytes (with no fibrin in most areas), was present in the carotid arteries of all rats by sem. sem was done on 36 cerebral vessels, 3 1 containing peripheral blood elements, with single (1) and aggregated (6) platelets (causing occlusion in 3), single (10) and aggregated (10) erythrocytes (without occlusion), and single (1) and aggregated (3) leukocytes (without occlusion). tem demonstrated that the platelet aggregates did not adhere to the cerebral endothelium. the endothelial surface of all cerebral vessels was normal, which provided additional evidence that the mechanism of cerebral infarction in this model is embolism. model of repetitive ischemia: this effect is significantly enhanced when combined with mild hypothermia ashfaq shuaib and elisabeth sechocka, saskatoon, saskatchewan. canada there is considerable evidence that glutamate release resulting in activation of postsynaptic receptors (especially nmethyhaspartate) is a major mechanism of ischemic neuronal injury. in vivo experiments have shown that a more severe release of glutamate may be responsible for the excessive damage seen with repeated ischemic insults. we have shown that in cell cultures the effect of brief repeated insults is more severe than a single insult of similar duration. in the present study, we tested the protective effects of cgs-19755 in a cell culture model of single ischemic and multiple-insult paradigm. in the multiple-insult paradigm, in some cultures cgs-19755 was combined with mild hypothermia to see if this would offer additional protection. cgs-19755 offered a dose-dependent protection in cell cultures exposed to a single ischemic insult. cgs-19755 was protective to cultures exposed to repeated ischemic insults. the protective effects were enhanced significantly when they were combined with hypothermia, resulting in almost complete protection of the cultures. the combination of therapies appears to be a valuable strategy in neuronal protection during cerebral ischemia. toby i. gropen, i . prohovnik, t . k. tatemirhi, z. sharif; and m. hirano, new york, n y although a rare syndrome, mitochondria1 encephalomyopathy, lactic acidosis, and stroke (melas) may offer a unique insight into stroke mechanisms. we report novel observations in a patient with melas studied with serial and quantitative cerebral perfusion after stroke using """tc-ceretec spect and '3ixe rcbf. a 24-year-old man with melas presented with left-sided headache, generalized seizures, fluent aphasia, and right hemianopia. serial ct and mri showed infarction of the posterior left hemisphere in a multiterritorial distribution. spect performed 15 days after stroke showed 20 to 30% greater flow in the infarct than in normal brain, which reversed 109 days after stroke. quantitative rcbf (m2 isi, reflecting mostly gray matter), when corpatients died. we recommend ct evaluation in all patients who have a seizure or lose consciousness during the peripar-tum period. despite intensive management, mortality is high. seizures should not be attributed to eclampsia without careful neurological assessment. when prenatal care is sought, women should be counseled about the dangers of cocaine to themselves as well as to their babies. changes in circulating blood volume following stephan a. mayer, matthew e. fink, laura lenniban, louise m. klebanoff; auis beckford, lsak prohounik, william young, and robert a. solomon, new york, n y reduction of blood volume (bv) has been implicated as a risk factor for delayed cerebral ischemia (dci) due to vasospasm after aneurysmal subarachnoid hemorrhage (sah). volume expansion guided by target filling pressures has gained popularity as a means of preventing or reversing dci; however, the adequacy of central venous pressure (cvp) as a reflection of bv in this setting remains unclear. we measured bv and cvp concurrently in 10 patients (5 males, 5 females; mean age 57 yr) 1 day after craniotomy (mean 3.5 days after sah) and an average of 5.5 days later. the mean bv (mllkg) measured using chromium5'-labeled red blood cells (rbcs) fell from 69.7 to 55.3 (normal range 55-80), a reduction of 21% ( p = .05, paired student's t test). despite this, mean cvp (mm hg) remained unchanged (7.1 vs 7.9). similar reductions of plasma volume (2 1%) and rbc volume (24%) accounted for no change in mean hematocrit (33.2 vs 33.5). bv fell 25.7% among grade iiiliv patients (n = 5 ) compared to 14.7% among grade 1/11 patients (n = 5). a moderate correlation between bv and cvp ( r = .48, p = .16) was found only with the first set of measurements. time-related alterations in venous capacitance, myocardial contractility, or systemic vascular resistance may explain our findings. axel rosengart, louis r. caplan, michael s. pessin, atherostenosis of the extracranial vertebral artery (ec-va) has rarely been studied systematically in series of patients with acute vertebrobasilar strokes or transient ischemic attacks. we identified by conventional angiography and neuroimaging (ct, mri, ultrasound, mr angiography) 45 patients with ec-va disease among 154 patients with posterior circulation ischemia. patients with cardiac sources of emboli were excluded. the probable etiologic mechanisms were: group a: vertebral artery origin (vao) atherostenosis with embolism-19 patients; group b: vao atherostenosis with hemodynamic spells-7 patients; group c: 1 patient with vao atherostenosis and both intra-arterial embolism and hernodynamic spells; group d: ec-va dissection-8 patients (6 unilateral, 2 bilateral); 1 patient had perioperative compromise of the ec-va with presumed intra-arterial embolism; group e: vao disease in addition to other distal vascular lesions-9 patients (2 with intracranial va and 7 with basilar artery [baf occlusive disease). ec-va disease is not always benign. vao atherostenosis and dissection of the ec-va are sources of intra-arterial emboli. hemodynamicrelated ischemia occurs with bilateral and unilateral va disease but is often transient. vao atherostenosis is often accompanied by severe occlusive disease of the intracranial va and ba. program and abstracts, american neurological association 239 sebastian e. ameriso, vicky l. y. wong, andvas gruber, hidemi ishii, and mark fisher, los angeles and la jolla, c a , and kanagawa, japan hemostasis abnormalities are associated with ischemic stroke. these changes typically are demonstrated in antecubital venous blood samples and may not necessarily represent changes within the vasculature of the brain. the purpose of this study was to identify potential differences in hemostatic profile from samples of cranial versus noncranial venous sites in patients with acute ischemic stroke. eight patients were studied within 7 days of acute brain infarction. some patients were studied on 2 separate days. blood was drawn from the external jugular vein and immediately thereafter from an antecubital vein without the use of tourniquet. we measured hematocrit, leukocyte count, platelet count, fibrin d-dimer (cross-linked fibrin fragment), plasminogen activator inhibitor-1 (pai-1, an important antifibrinolytic protein), and anticoagulant proteins thrombomodulin and activated protein c. a jugular-to-antecubital ratio was calculated for each paired blood sampling. thirteen paired samples were obtained from the eight patients. external jugular-to-antecubital ratios (mean to-antecubitat ratio for pal 1 was significantly different from 1 ( p < 0.05), with higher concentrations in jugular samples. in conclusion, levels of hemostatic proteins measured from cranial venous blood may differ from antecubital samples in patients with acute ischemic stroke. in animal models of transient cerebral ischemia, the effects of repetitive insults are more severe than a single ischemic episode of similar duration. we used the cell culture model of ischemia to determine if the effects of repetitive ischemia are similarly more severe in this model of ischemia. for cell culture, we used fetal mice cortical astrocytic and postnatal cerebellar (glutamatergic) granular neurons and cerebral gamma-aminobutyric acid (gaba)ergic cells. lactic dehydrogenase (ldh) (activity per gram protein) release in the medium was used as a measure of cellular damage. compared to a single insult, there was a large increase in ldh release during repetitive ischemia in astrocytes (233 vs 7 5 , p < 0.02) and granular cells (129 vs 41, p < 0.001) (highly significant) and a modest (but significant) increase in the cortical neurons (11.6 vs 7.5 p = 0.05). the demonstration that repetitive ischemia produces more severe damage in cell culture would suggest that the mechanisms are not predominantly vascular. cell culture could prove useful to study the mechanisms of neuronal damage with repetitive ischemia. we studied the spontaneous recovery of neurological function after acute ischemic stroke using a standardized stroke nih stroke scale scale ( n i h stroke scale) to assess the extent of improvement, differences in stroke types, and early predictors of later outcome. we performed serial neurological assessments on admission; 24, 48, and 72 hours after admission; and 7 to 10 days and >30 days after admission. twenty-six patients had presumed embolic occlusion of the middle cerebral artery (mca) and 14 had a clinical diagnosis of lacune. admission score was better in the lacune group compared to the mca group. the mean scores for all patients improved by the 7to 10-day and the >30-day examination, but the degree of improvement was greater in the mca group than in the lacune group at >30 days ( p < 0.004). the degree of change at 7 to 10 days correlated with the change in score at 24 hours ( r = 0.45, p < 0.05) and 48 hours ( r = 0.86, p < 0.05). most patients improve after acute ischemic stroke, but to variable degrees and at different rates. david w. desmond, thomas k. tatemichi, miguel figueroa, dew'itt t . cross, and yaakov stern, new yovk, n y to investigate the effects of lacunar infarction (li) on cognitive function, we examined 57 li patients 3 months after stroke (age = 71.6 ? 9.0 yr; education = 9.2 2 4.7 yr) and 241 stroke-free nondemented control subjects (age = 70.6 k 6.5 yr; education = 12.4 k 4.5 yr) with a battery of neuropsychological tests. li was defined as a presenting infarct of 5 2 cc and a mean volume of any additional subcortical infarctions of 5 2 cc on ct scan. using multiple regression analyses, with significance set at p < .01 to minimize the risk of type i error, we considered the role of li as a correlate of performance in multiple cognitive domains. controlling for the effects of demographic factors, vascular risk factors, alcohol use, and depression within the multivariate models, li was a significant independent correlate of deficits in memory ((3 = -.40, p = .0002), verbal (p = -.28, p = .0025), visuospatial (p = -.51, p < .oool), abstract reasoning (p = -.31, p = .0029), and attentional skills (p = -.50, p < .0001). we further investigated the effects of infarct number, volume, and location, as well as atrophy, on global cognitive function within the li group. the only significant independent correlate of global cognitive performance was a preponderance of left-hemisphere infarctions (p = -.07, p = ,0454). these results suggest that li may produce dysfunction in multiple cognitive domains, particularly when the left hemisphere is differentially involved. p30. increased intracranial atherosclerotic stroke in hispanics and blacks from northern manhattan ralph l. sacco, christina zamanillo, t . shi, andj. p. mobr, new york, ny intracranial atherosclerosis has been found to be more frequent in blacks compared to whites, whereas hispanics have rarely been characterized. among 2 10 consecutive patients from northern manhattan over age 39 hospitalized at the presbyterian hospital from 1990 to 1991, cerebral infarction occurred in 32 whites, 84 blacks, and 74 hispanics. all patients had at least one c t scan, 96% had duplex doppler, 94% transcranial doppler, and 12% angiography. strokes were classified as atherosclerotic (ath), cardioembolism, lacunar, and as infarcts of undetermined cause. ath was further subdivided into extracranial (eath) or intracranial (iath) . overall, the frequency of ath was similar in the three racelethnic groups (white 2096, black 16'96, hispanic 2096) . the distribution of the atherosclerosis, however, was different in whites compared to blacks and hispanics. whites had more eath stroke than blacks and hispanics (white 17%, black 7 % , hispanic lo%), while iath was similar in blacks and hispanics and greater than in whites (white 394, black 895, hispanic 10%). nonwhites have more iath stroke than whites. the similarity in the distribution of atherosclerosis between blacks and hispanics argues for shared environmental risk factors, rather than genetic differences. ethnic differences in stroke risk factors may help explain differences in infarct subtype. we studied 10 mild to moderate alzheimer's disease (ad) patients with a series of "0 water bolus positron emission tomographic (pet) activation studies, and compared them to similar studies in 10 age-matched normal controls. for each group, pet images were mapped onto the subjects' mri scan, and results of a particular activation condition were averaged across the group. naming a series of pictures (line drawings of animals) minus counting abstract designs as a baseline produced strong activation of the anterior cingulate gyrus only in the ad group. silent reading of words minus viewing a baseline series of "xs" similarly showed strong activation of the anterior cingulate gyms in the ad subjects but not the normals. naming 1 block (activation condition) of 80% unnamed pictures, minus a second block (baseline) of easily named pictures, demonstrated much greater cingulate activation in the ad patients, for naming of the more difficult pictures. we conclude that this cingulate activation may reflect the greater involvement of an attentional network (of which the anterior cingulate is a part) in tasks requiring a higher degree of "mental work" on the part of ad patients. dementia in alzheimer's disease j. w. pettegrew, k. panchalingam, w. e. klunk, and r. j alzheimer's disease (ad) predominantly affects the brain, resulting in the loss of multiple cognitive abilities. some studies suggest the membranes of peripheral cells are involved in the disease. to investigate erythrocyte membrane molecular dynamics in ad patients and age-matched controls, we investigated erythrocyte membrane molecular motion at the surface (fluorescamine), aqueous-hydrocarbon interface (dppe-ans), and hydrocarbon core (1219j-as; ppc-dph) by steady-state fluorescence anisotropy measurements of 16 probable ad patients (5 males; 11 females) and 20 (1 1 males; 9 females) age-matched controls. cognitive function was assessed by the mini-mental, mattis, and blessed scales. we found that intergroup comparisons revealed decreased motion at the surface ( p = 0.001) and aqueous-hydrocarbon interface ( p = 0.03) and increased motion in the hydrocarbon core ( p = 0.01) of the moderately to severely impaired ad patients compared to the controls. in the ad patients, there were significant correlations between decreasing membrane surface motion and worsening blessed scores (males p = 0.05; r = 0.7; femalesp = 0.04; r = 0.5). these findings suggest that molecules are being produced in the brain of ad patients that gain access to the circulation. these molecules insert into the erythrocyte membrane and secondarily alter erythrocyte membrane molecular motion. the production of these molecules correlates with the dementia and could contribute to the molecular pathophysiology of the disease. parkinson's disease (pd) and alzheimer's disease (ad) are 2 common disorders of old age and may therefore coexist. the prognosis in demented pd patients is poor and early recognition of such cases is therefore desirable. the objective of this study was to identify characteristics that distinguish pd + ad from pd patients during early stage. all patients were clinically evaluated over a 22-year period . clinical diagnosis of dementia was made only when unequivocal clinical evidence of progressive decline in memory and cognitive function was documented, and pathological diagnosis of ad and pd was made using standard criteria. twentysix patients who had only pd or pd + ad were identified; 20 had no dementia and at autopsy had pd. six patients had clinical evidence of parkinsonism and dementia and at autopsy had 2 distinct pathological findings-pd and ad. these 6 cases could be classified as having simultaneous or sequential evolution of pd + ad. those with sequential onset had pd before age 65 years but were inexplicably functionally disabled early on, whereas those with simultaneous onset manifested pd after age 65 years. pd + ad patients had rapid disease progression, shorter survival, poorer drug response, and more side effects of levodopa than pd patients. to study the prevalence ofwhite matter lesions in the general elderly population, and to investigate whether white matter lesions were relatively frequent in subjects with classic vascular risk factors and with hemostatic risk factors, magnetic resonance scans were obtained of 11 1 participants, aged 65 to 85 years, of the rotterdam elderly study. the subjects for the imaging study were a random sample from the general population, stratified by age and gender. t2-weighted images were obtained in the axial plane. white matter lesions were considered present when moderate or severe periventricular hyperintensities or when more than 5 small focal lesions or focal confluent lesions were found. overall, 27% of subjects had white matter lesions. the prevalence and severity of le-program and abstracts, american neurological association 241 sions increased with age. history of stroke or myocardial infarction, presence of peripheral arterial disease, factor viic activity, and fibrinogen level were each significantly and independently associated with the presence of white matter lesions. significant relations with actual systolic as well as diastolic blood pressure, with a history of hypertension, and with plasma cholesterol were observed only for subjects between 65 and 74 years. this study suggests that white matter lesions in the elderly may be related not only to the classic cardiovascular risk factors. but also to hemostatic factors. joan m. swearer, paula nelligan, hanno muelher, beatrice woodward, and david drachman, worcester, ma although behavioral disturbances occur frequently in alzheimer's disease and other dementing disorders, little is known about the factors that predict their development or predispose to their occurrence. in the present study we examined 2 sets of possible predictive/predisposing factors retrospectively for behavioral disturbances in 35 mildly to severely demented, community-dwelling patients. the factors examined included: individual distinguishing features (age, gender, age of onset, premorbid personality traits, prior psychiatric history) and dementia severity (dependence in activities of daily living [adls) and self-care, duration of dementia, global disease severity). spearman correlations and t tests were used to assess the relative influence of these factors on the occurrence of 3 types of aberrant behaviors: aggressive behaviors, disordered ideation, and motor abnormalities. forty percent of the patients exhibited aggressive behaviors, 77% exhibited disordered ideation, and 63% had motor abnormalities. neither a prior history of psychiatric disorders nor premorbid personality traits were associated with the occurrence of the target behaviors. dependence in adls and self-care and greater global severity were associated ( p < .01) with the frequency and severity of aggressive behaviors, disordered ideation, and motor abnormalities. these results suggest that severity of dementia is a consistent and reliable factor in the development of aberrant behaviors, whereas preexisting personality traits are not. dementia of the alzheimer t y p e w. j. burke, a. ranno, w. h . roccafrte, s. p. wengel. b. l. bayer, and n . k. willcockson, omaha, ne l-deprenyl is an irreversible inhibitor of mao-b that has been reported to cause modest improvements in short-term memory and behavioral symptoms in persons with dementia of the alzheimer type (dat). thirty-eight subjects meeting research criteria for mild d a t were enrolled in a placebo-controlled, double-blind trial of r-deprenyl at a dose of 5 mg twice a day. subjects underwent extensive clinical and neuropsychological assessments at entry, and at 2 and 8 months. after 8 months, subjects taking both l-deprenyl and placebo showed a significant decline in their scores on the mini-mental state examination, the clinical dementia rating (cdr) scale, and the sum-of-boxes score derived from the cdr. when the change in scores on these clinical measures was examined across the 2 groups, there was no significant difference. there were no significant differences within or between groups on several behavioral measures including the brief psychiatric rating scale and the cornell rating scale for depression in dementia. neuropsychological testing demonstrated no significant differences berween groups based on mean score change. l-deprenyl did not affect cognition or behavioral symptoms of dat in this 8-month study. k. marder, m-x. tang, r. ottman, l. cote, y. stern, and r. mayeux, new york, n y the etiology of dementia in parkinson's disease (pd) is probably multifactorial but there may be a shared susceptibility for p d and alzheimer's disease (ad). reliable risk factor interviews were conducted with informants of 15 1 nondemented p d patients (pd-d) and 65 demented p d patients (pd + d) enrolled in a longitudinal community study of pd. p d + d were older (78.9 yr) than pd-d (71.4 yr) and had later age at onset of motor signs (71.1 yr) than pd-d (64.8 yr) ( p < ,001). the frequency of smoking, alcohol use, head injury, and family history (fh) of pd did not differ but fh of ad was significantly more frequent in the pd + d group (or 2.25, ci 1.01-5.01). using stepwise logistic regression, only age of onset of motor signs 2 6 5 (or 2.86), education <8 years (or 2.53), and the interaction of age of onset of motor signs and fh of a d (or 3.49) were independent predictors of dementia in pd. to address variable years at risk for development of dementia, life table analysis revealed the cumulative risk of a d to age 90 in first-degree relatives of p d + d was .312, and .119 in pd-d relatives ( p < .05). cox proportional hazards analysis controlling for the differences in ages of the relatives of both groups yielded a rate ratio of 2.06 (ci 1.2-3.7) for the development of a d among p d + d compared to pd-d relatives. we conclude that a genetic susceptibility to a d may raise the risk for dementia in patients with pd. differentiated from alzheimer's disease? john c. mowis, elizabeth grant, rita canfield, eugene rubin, and daniel mckeel, jr, st louis. mo vascular dementia (vd) is believed to account for 20 to 30% of all us cases of dementia; however, pathologically confirmed cases are quite rare. this discrepancy suggests that current diagnostic criteria lead to the clinical overdiagnosis of vd. twenty v d subjects (mean age 78.5 yr; 9 men, 11 women) were diagnosed solely on the basis of the presence of dementia, a history of stroke(s), and a documented relationship of stroke to onset andlor course of dementia; ischemic scores (is) and neuroradiographic findings were not used for diagnosis. compared with 89 subjects (mean age 75.2 yr; 34 men, 55 women) with dementia of the alzheimer type (dat), there were no significant group differences for comparable clinical dementia rating stages of dementia for measures of language, activities of daily living, or general cognition. the vd group scored significantly higher than the dat group on the modified is (f [91, 64] = 138.2, p < ,0001). all 27 autopsied d a t subjects had verified alzheimer's disease (ad); 8 also had cerebral infarctions. the 3 autopsied v d subjects had 168, 160, and 55 cc of brain tissue affected by stroke; 1 (168 cc) also satisfied histological criteria for ad. we conclude that (1) the clinical features of vd and a d overlap considerably; (2) diagnostic criteria based on the temporal association of stroke with dementia may have predictive value for vd; and (3) the frequent coexistence of a d and strokes indicates that refinement of criteria is needed to distinguish "mixed" and "pure" vd. clinicopathological correlation remains essential for any study of putative vd. left-handedness has been proposed as a marker for decreased survival in the general population, but possible effects of handedness on longevity in alzheimer's disease (ad) have not been examined. we hypothesized that left-handed ad patients would evince more rapid deterioration and therefore die at an earlier age than right-handed patients. subjects were 103 demented patients consecutively confirmed at autopsy to meet nincds-adrda criteria for "definite" ad. handedness was determined from structured interviews with primary caregivers and validated for most subjects with the edinburgh inventory of handedness. age at onset of dementia symptoms retrospectively determined by caregivers was used to calculate the duration of illness at the time of death. because of reported gender differences with regard to longevity, we first partialled out effects of gender before using hierarchical regression procedures to test the hypothesis. four of 46 men and 2 of 57 women with definite ad were left-handed. the mean age at onset did not differ significantly between handedness groups (f [ l,loo] = .82), but the mean duration of symptoms ( alterations in the optical properties of brain can be used to detect pathological changes in patients with alzheimer's disease (ad). using time-resolved spectroscopy (trs) and phase-modulation spectroscopy (pms), we measured the absorption (ua) coefficient, scattering (us) coefficient, and mean photon pathlength (pl) of red light directed through the base of the frontal lobes of 28 patients with ad and 11 age-matched control subjects. the measured values and the asymmetry index (ai) (an indication of the symmetry of the measurements between the left and right side of the brain) were correlated with the severity of disease as determined by mini-mental state score. there were significant differences between the ad and control group for ua, us, pl, and the standard deviation of ai. there was no correlation between the mms score and ua, us, or pl. however, the highest asymmetry index values were seen in moderately impaired patients , which suggests that the asymmetrical nature of the pathological process detected by optical spectroscopy is most marked during this stage of the illness. this noninvasive technique may provide a convenient method to detect and monitor the pathological changes that occur in the brain of patients with ad. disease p41. memory impairment in very mild alzheimer's a memory impairment is often the earliest indication of alzheimer's disease (ad). we investigated 3 components of disease learning and recall to determine which aspect of memory function is impaired the earliest in incipient ad. using the mayo clinic alzheimer's disease patient registry, which is a longitudinal prospective project on ad and normal aging, we identified 34 patients with very mild ad (i.e., with a mini-mental state score of 24 or greater) and 34 age-and sex-matched controls. we assessed performance on 2 memory measures: the rey auditory verbal learning test and the buschke free and cued selective reminding test (fcsrt). the 3 parameters evaluated included a measure of acquisition, total learning over trials (tl), and delayed recall (dr). on the fcsrt, an index of facilitation of performance with semantic cues (sc) was assessed. results indicated that all 3 indices, tl., dr, and sc, were capable of separating the mild ad group from the controls ( p < 0,001). using a linear discriminant analysis with stepwise variable entry, the measure that assessed the patient's ability to use semantic cues (sc) was the most sensitive parameter for separating the 2 groups ( f = 15.38, p < 0.0002), and the acquisition parameter (tl) was also useful at adding some additional predictive power (f = 8.48, p < 0.005). the delayed recall measure, however, did not add anything to the previous 2 measures. it appears that very early ad can be detected using appropriately structured memory tasks, and these procedures can be helpful in identifying at-risk individuals. alzheimer's disease (ad) has an insidious onset that is difficult to date reliably. we developed a standardized interview to provide objective criteria for dating the onset of 7 different symptoms (memory complainr, performance problems, language deficits, disorientation, depression, behavior problems, and psychosis), yielding an estimated disease onset date. inrerrater reliability (icc = 0.99;p < ,001) and interinformant reliability (icc = 3 5 ; p < .001) for the onset of first symptom was high. interrater agreement for the order in which symptoms appeared was high (icc = 0.72-0.98) as was interinformant reliability for all symptoms except memory complaint. the interview was administered to 2 16 patients with ad. mean estimate duration of illness was 4.26 years k 3.47 years and correlated significanrly with problems in instrumental activities of daily living. sixty-six percent had memory complaint and 45% had performance problems as their initial symptom. this technique provides a reliable characterization of disease onset. longitudinal studies will determine if particular onset symptoms differentially predict disease progression. the purpose of this study was to determine whether there is an excess of white matter disease (wmd) in alzheimer's disease (ad). brun and englund (1986) reported an excess of wmd in brains of patients with ad vs age-matched controls. there have been reports both confirming (bowen et al, 1990; fazekas et al, 1990) and refuting (leys, 1990) these findings using ct and mri in patients with clinically diagnosed ad. postmortem t2-weighted mri scans and program and abstracts, american neurological association 243 neuropathology were graded on 9 brains of pathologically confirmed ad subjects and 6 brains of age-matched neuropathologically normal controls. white matter lesions were scored on a 0 to 3 scale (none, mild, moderate, severe) separately for periventricular (pvl) and deep white matter (dwm) areas in mri scans and lux01 fast blue (lfb)-stained brain sections. correlations between mri and neuropathology were good ( r = 0.61 for pvl, r = 0.66 for dwm). pvl scores were higher in ad than in normal subjects on mri (ad: 2.2 l 0.9 vs controls: 0.2 rfr 0.3; p < .005). on pathology the difference in scores did not reach significance (ad: 1.3 2 0.7 vs controls: 0.5 * 0.4; p = 0.2). similarly, dwm scores were higher in ad subjects than normals on mri, but not neuropathology. in conclusion, ad brains have a significant excess of wmd on mri compared to controls. although the pvl and dwm scores for pathological sections are not different in the 2 groups, mri is much more sensitive than lfb-stained sections for wmd. thirteen autopsy cases of progressive supranuclear palsy (psp) were investigated for clinical-neuropathological correlations and heterogeneity. we reviewed clinical records of 11 men and 2 women aged 61 to 89 years (mean age 72 yr) with disease duration ranging from 4 to 12 years. most patients had classic features of psp including ophthalmoplegia, postural instability, and extrapyramidal signs. dementia was eventually observed in 9 of the 13 patients (67%). six of 9 patients (67 %) on whom adequate initial documentation was available presented with memory loss or behavior change. five of the 13 patients (395%), including 4 with an initial presentation of memory loss, were diagnosed clinically as having alzheimer's disease (ad) rather than psp; neuropathological diagnoses in these cases varied: i had combined ad-psp; 1 had ad-psp combined with parkinson's disease (pd) changes; 1 had psp-pd; and 2 had "pure" psp. the 2 patients with concomitant pd changes showed lewy bodies in the substantia nigra, locus coeruleus, nucleus basalis, and neocortex. the remaining 8 patients were clinically diagnosed as having psp; neuropathological diagnoses in these cases included 4 with "pure" psp and 4 with psp that also met neuropathological criteria for ad (psp-ad). these findings emphasize the clinical and neuropathological heterogeneity in psp. the neuropsychological battery developed for the consortium to establish a registry for alzheimer's disease (cerad) is currently used in many research studies to index the cognitive impairments of alzheimer's disease. in spite of its widespread use, normative informarion on the battery, important for interpretation of performance, has not been available. we report norms for the cerad battery based on a large sample of elderly control subjects (n = 398; white men and women; ages 50-89 yr) enrolled in the national study of cerad. performance on the neuropsychological measures was examined separately for subjects with high (2 12 yr) and low (< 12 yr) education. distribution of scores and basic descriptive information (means and sd) for each measure were determined. significant age and sex effects were observed on most cognitive measures in the highly educated group. in contrast, no significant age effects were observed in the low education group. effect of sex was not explored in this group due to the limited sample size (n = 48). further exploration of cerad performance in normal controls from underrepresented groups including minorities, residents of rural communities, and individuals with low education is in progress. intraneuronal inclusions of cytoskeletal proteins appear in several neurological diseases; for example, the neurofibrillary tangles of alzheimer's disease contain a cytoskeletal protein, tau. because the previously described slowing of axonal transport in aged animals might lead to accumulation of cytoskeletal proteins in nerve-cell bodies and axons, we assessed the abundance of 2 major cytoskeletal proteins in brain tracts of rats at age 4 months or 25 months. immunoassay was performed with monoclonal antibodies to alpha and beta tubulin and to nf-l (the core neurofilament protein) by published methods. samples were dissected in a standardized fashion and 2-mrn pieces of the following tracts were assayed: optic nerve, corticospinal tract (medulla), superior cerebellar peduncle, l5 dorsal root, and l5 ventral root. between 4 months and 25 months, the nf-l content approximately doubled in each brain site. tubulin substantially increased at of aged rats all sites except the fimbria-fornix. in contrast, tubulin did not change in the spinal roots. nf-l increased slightly in the ventral but not the dorsal root. this tendency of senescent brain neurons to accumulate cytoskeletal proteins in their axoplasm may predispose them to formation of intraneuronal inclusions in various degenerative diseases. we performed a prospective study of preoperative magnetic resonance imaging (mri) in 13 consecutive patients with intractable partial epilepsy who underwent a stereotactic resection of an extrahippocampal temporal lobe foreign-tissue lesion, "lesionectomy," between june 1986 and january 199 1. interpretation of the mri studies was performed by an investigator blinded to the presurgical evaluation, surgical outcome, and pathology. hippocampal formation (hf) atrophy was assessed using mri-based volumetry (n = 10) and visual grading of the h f (n = 13). mri-detected hf atrophy has been shown to be a reliable marker of moderate to severe mesial temporal sclerosis (mts) (cascino gd, et al, ann neurol 1991; 30:31-36 evidence from experimental animals indicates that endogenously produced platelet-derived growth factor (pdgf) is an important regulator of glial proliferation and differentiation. because of the striking degree of glial proliferation in chronic epilepsy, we sought to determine whether cultured glia from human epilepsy tissue would be responsive to pdgf. the effects of pdgf on dna synthesis, proliferation, and relative distribution of a2b5(+) glia were studied in a cell culture derived from temporal lobe white matter of adult epilepsy lobectomy tissue. by immunocytochemistry, glial fibrillary acidic protein (gfap) was detectable in 90% of cells in untreated or 4-day pdgf-treated (10 ng/ml) cultures, which confirmed their astrocytic nature. in contrast, a2b5( +) cells increased from 10 to 30% in untreated cultures to 75% after pdgf treatment, which suggested that type 2 astrocytes (a2b5[ + 1, gfap[ +]) had been elicited. dna synthesis of cells resembling oligodendrocyte-type 2 astrocyte (0-2a) progenitors occurred within 24 hours after program and abstracts, american neurological association 245 pdgf treatment as evidenced by nuclear incorporation of brdu in bipolar a2b5( +) cells. these studies demonstrate that expansion of adult human gfap( +) astrocyte populations is sensitive to regulation by pdgf. further, these data imply the existence of pdgf-responsive 0-2a progenitor cells in astrocyte-rich cultures derived from human epilepsy tissue. ( we have recorded vagal and esophageal-evoked potentials after electrical (e) and balloon (b) stimulation in 10 epileptic patients who had vagal stimulators for the control of intractable epilepsy and the results were compared with esophagealevoked potentials in 10 healthy controls and 3 diabetic patients. the vagal and esophageal-evoked potentials showed similar configurations with 3 major positive and negative potentials. the amplitudes of the responses habituated rapidly over 30 trials at 2 per second up to 1 per 6 seconds. latencies were shorter from the upper esophagus (20 cm above lower esophageal sphinctereles]) cf. lower esophagus ( 5 cm above les) yielding conduction velocities of 5 to 7 meters per second but conduction was significantly slower in the diabetics. the vagal-evoked potentials have validated the use of esophageal-evoked potentials as a practical method of assessment of the integrity and speed of conduction in vagal afferent pathways in man. ronald e. kramer and neil l. rosenberg, englewood. co seizure disorders were analyzed in patients in whom toluene was the sole or major drug of abuse. toluene abuse is increasing; therefore, physicians should gain experience with its neuropathological and ciinical sequelae. a retrospective chart review found 15 patients meeting criteria. the average patient age was 28 years; abuse onset averaged 16.7 years; abuse averaged 10.7 years; and 11 patients were male. ten were daily, 3 were weekly, and 2 were intermittent users. seizures occurred in 2. one suffered a single generalized tonic-clonic seizure without recurrence and without treatment. his we characterized the clinical dose-response curves for relief of parkinsonism and production of dyskinesias as a function of plasma levodopa and 3-0-methyldopa levels in 6 patients with parkinson's disease (pd) and fluctuating responses to oral levodopa/carbidopa. dose response to graded intravenous levodopa was measured after overnight drug withdrawal on 2 occasions, first after chronic, intermittent oral levodopa/ carbidopa and second after 3 to 5 days of continuous intravenous levodopa. continuous intravenous levodopa shifted the dyskinesia dose-response curve to the right, and reduced maximum dyskinesia activity, but did not significantly alter dose response for relief of parkinsonism. improvement in dyskinesia was apparent by the second day of continuous levodopa, during which ratios of plasma dopa/3-0-methyldopa remained constant. our results support the hypothesis that relief of parkinsonism and production of dyskinesias occur by separate mechanisms. continuous dopamine-mimetic therapy should be sought as a therapeutic goal for advanced pd. dopa-responsive dystonia (drd) is a distinct subset of idiopathic dystonia with diurnal fl uctuation and a dramatically beneficial response to l-dopa. it has hitherto been considered an autosomal-dominant disease with reduced penetration (mckusick no. 12823) . we studied an arabic family of 67 members with d r d spanning 6 generations. we examined 43 members and 6 of their spouses. l-dopa was withheld for 24 hours from patients in treatment. five family members had generalized dystonia with diurnal auctuation ( i male, 4 in an arabic family females). dystonia started between the age of 2 and 7 years with gait difficulty and involvement of the legs. mri, eeg, evoked potentials, and screening for wilson's disease were negative. an excellent response to l-dopa was noted in all 5 patients with continued long-term clinical stability for as long as 17 years. the 5 patients were the products of 2 consanguineous marriages, and their 7 siblings were normal. the patients were descendants of the same great-great-grandparents. this pedigree suggests an autosomal-recessive type of inheritance. we believe this is the first report of d r d with an autosomal-recessive type of inheritance. a. achiron, m . gornish, h . goldberg, i . ziv, r. djaldetti, y. zoldan, h. smka, and e. mekzmed, petah tiqva, israel freezing gait is an incapacitating symptom that occurs often in advanced parkinson's disease and also in other neurological disorders, eg., multiinfarct state, multisystem atrophies, and normotensive hydrocephalus. we evaluated, videotaped, and rated 18 patients (15 men, age 74 k 6,60-82 yr) who developed pure progressive freezing gait during 2.5 2 1.9, 0.5-6 years. severity was mild in 4 with sudden motor blocks mainly when confronted with obstacles; moderate in 9 with gait arrests upon any attempt to initiate walking and changing direction, requiring a walking stick or partial external assistance; and severe in 5 with total inability to start walking, requiring a walker, massive assistance, or a wheelchair. in all, freezing was associated with postural instability. they could mimic normal gait when seated or lying prone and could overcome arrests by the "walking over lines" maneuver. neurological examination was otherwise normal with no signs of dementia, parkinsonism, or pseudobulbar palsy. ischemic risk factors including ischemic heart disease, hypertension, and diabetes occurred in 7 and previous strokes in 6. brain c t and mri were normal or showed mild cortical atrophy in 12 and putative lacunae in only 6 patients. none responded to levodopa or dopamine agonists. progressive pure freezing gait should be recognized as a separate nonparkinsonian neurological entity. it may be due to degenerative or ischemic non-nigral brainstem lesions. we describe 19 patients with causalgia and dystonia, triggered by peripheral injuries in 15 patients and occurring spontaneously in 4 patients. the injury was often trivial. the mean age at presentation was 28.6 years. the legs were affected in 12 patients, and the arm was affected in the remaining 7 patients. all had burning pain, allodynia, and hyperpathia, along with vasomotor, sudomotor, and trophic changes. all developed typical dystonic muscle spasms in the affected part. the spasms typically were sustained, producing a fixed dystonic posture, in contrast to the mobile spasms characteristic of idiopathic torsion dystonia. dystonia always followed the causalgia and was painful. there was spread of the causalgia and of the dystonia from its initial site both in the affected limb and to other extremities, the latter in a hemiplegic, transverse, and triplegic distribution. all forms of conventional treatment failed to relieve either the pain or the dystonia. we suggest that functional changes in the corticobasal ganglia-thalamic system are responsible for this painful dystonic syndrome. program and abstracts, american neurological association 247 holiday for parkinson's disease: a controlled clinical trial r. kurlan, c . m . tanner, c. g. goetz, j . sutton, p. carvey, c. deeley, l. cui, c. itvine, and m . mcdemzott, rochester, ny, chicago, il, and san jose, c a the efficacy and mechanisms of levodopa (ld) drug holiday for parkinson's disease (pd) remain controversial. we performed a double-blind, randomized study with 11 advanced pd patients (5 men, 6 women; aged 52-79 yr) with entry criteria of inadequate response to ld plus dose-limiting ld-induced side effects (dyskinesias, hallucinations, and confusion). subjects were assigned to: (1) 100% placebo for ld (complete drug holiday) or (2) 50% ld and 50% placebo for ld (50% drug reduction) for 6 days. after subsequent open-label ld dose optimization, subjects were followed to end point (defined as the time when entry criteria were again satisfied or a maximum of 1 year). median survival time to end point was not significantly different for the complete drug holiday (182 days) and 509% drug reduction (100 days) groups ( p = 3 5 ) . aspiration pneumonia occurred in 2 complete drug holiday patients and no significant morbidity occurred with drug reduction. after a 100-mg dose of ld, clinical and pharmacological responses were no different before and after drug holiday ( p = .75) or reduction ( p = 254). subject to the limitations of our small sample size, we conclude that complete drug holiday is associated with greater morbidity and confers no major advantage over 50% drug reduction. we found no evidence of significant alterations of pharmacokinetic or pharmacodynamic properties of ld after drug holiday or reduction. ( (pc) of the substantia nigra on t2-weighted images. the narrowing of the pc signal has been attributed either to atrophy of the pc or to increased deposition of iron in this region. we have studied details of iron distribution in the midbrain of 8 formalin-fixed human brains by scanning pixe analysis. three p d brains, 1 juvenile pd brain, and 4 control (amyotrophic lateral sclerosis) brains were studied. in the controls, the iron content of the pars reticulata (pr) was almost equal to that of the red nucleus (rn), but that of the pc was less than 50% of the pr. in parkinsonian brains, the iron content in the pc was significantly higher than in the controls. the iron content ratios of pc/pr and pc/rn in parkinsonian brains were significantly higher than in the controls. these findings suggest that iron deposition increases in the pc of parkinsonian brains. the difference in the pattern of iron content corresponds to the intensity profile pattern noted on mri. mri findings in parkinsonian patients may reflect a change in the iron distribution pattern. (jankovic and brin, nejm, 1991) . such resistance within 2 exposures is not likely due to toxin antibody formation. of 61 cd patients evaluated per protocol receiving 2 or more botox tx under multichannel emg monitoring, 7 (11.5%) showed no benefit after the first and second tx, despite mild neck weakness on static muscle testing and, in some instances, emg signs of denervation. the 54 responders (16 men, 38 women) had younger age onset cd (mean 45 yr vs 60 yr), but similar duration (mean 10 yr vs 9 yr) compared to the 1 male and 6 female nonresponders (in contrast to jankovic and schwartz, arch neurol, 1991) . both groups had similar degrees of severity and tx dose (mean 200 iu, range 112.5-300). although disparate group size prohibits statistical analysis, some interesting comparisons include: nonresponders were more apt to have extranuchal dystonic sites (72% vs 33%), antecollis (29% vs 4%), dark eyes (57% vs 19%), women with elevated antinuclear antibody titer (67% vs 24%), history of intracranial operation (29% vs o%), significant for age focal mri abnormality (5 of 6 vs 3 of 19). history of cervical operation (6 patients) did not limit responsiveness. rates of prior remission, perinatal stress, antecedent trauma, left-handedness, and family history of movement disorder were similar for both groups. antecollis presents problems for optimizing tx to affected muscles, but central mechanisms may play a role in why some patients with focal dystonia do not improve with botox tx from the outset. enrico fazzini, new york, n y with parkinson's disease does deprenyl have a symptomatic effect on patients with untreated and l-dopa-treated parkinson's disease (pd)? once deprenyl is started, how long is it before another medication is needed to control symptoms of continued disease progression? there has been controversy over whether deprenyl has effects on delaying disease progression (nejm 1989; 321:1364) and/or in alleviating the symptoms of pd. one hundred seventy-five patients already taking l-dopa (group 1) and 33 patients who had never taken l-dopa (group 2) were treated with deprenyl 10 mg/day. unified pd rating scale (updrs) scores were measured before and after deprenyl. patients were followed until pd symptoms progressed to the point of requiring additional medication. one hundred eighteen of 175 (67%) patients in group 1 (reduced updrs mean activities of daily living [adl) 11 to 9, motor [mtr] 30 to 22) and 29/33 (88%) patients in group 2 (reduced updrs mean adl 11 to 6, mtr 26 to 20) reported symptomatic benefit. an average of 12 months' duration was found in both groups before further medication adjustments were needed. deprenyl provides symptomatic benefit for an average of 12 months in the majority of patients with p d regardless of whether or not they are being treated with l-dopa. yasuo iwasaki, masao kinoshita, toshiya shojima, and ken ikeda, tokyo, japan, and cleveland, oh in parkinsonian patients we measured fasting plasma amino acids in 30 parkinson's disease patients and 30 controls matched for age and sex. all patients were receiving l-dopa and they were free of any medications other than l-dopa. normal controls were free of any medication. there were no differences in diets between patients and controls. fasting blood specimens were collected in heparinized tubes and immediately were centrifuged at 20,000 g for 10 minutes. analysis of plasma amino acids was performed by automated ion-exchange chromatography with lithium-based buffer and an amino-acid analyzer. parkinsonian patients had significant elevations of aspartate, glutamate, and glycine. the other amino acids were not significantly different from those in controls. n o correlation between severity or activity and degree of abnormality in plasma level of amino acids in patients was established. we conclude that excitatory amino-acid metabolism is altered in patients with parkinson's disease. bonnie e. levin, rachel tomer, and william weiner, miami, fl disease there is evidence linking obsessive-compulsive symptoms (ocs) to basal ganglia dysfunction. we investigated the presence and severity of ocs in a sample of 44 patients with an unequivocal diagnosis of idiopathic parkinson's disease (pd) using the leyton obsessional inventory. ocs was found in the majority of the patients, with 24 (55%) scoring above the normative cutoff for the symptom score and 32 (73%) scoring above the normative cutoff for the trait score. when severity of oc symptoms was correlated with a battery of neuropsychological measures, significant relationships were observed between ocs and a preponderance of tests associated with right-hemisphere functions. these findings were observed especially on those tests with a strong frontal lobe component (block design: r = -.50; embedded figures: r = -.534; set shifting: r = -.42; and perseverative responses: r = .58; p < .005 for all measures). in all cases, the more severe oc symptoms, the poorer the performance. a similar trend was observed between the leyton trait scores and the cognitive measures. these findings suggest that ocs is present in a subgroup of pd patients, which may reflect greater compromise of right-hemisphere basal ganglia-frontal lobe pathways. intraclass correlations (iccs) were calculated for the total motor score and for each individual sign. results indicated excellent agreement (icc > .7) for the total motor score, resting tremor, gait, arising from a chair, and speeded, repetitive movements; good agreement (icc > .5) for rigidity, action tremor, posture, postural stability, and bradykinesia; and poor agreement (icc < .2) for speech and facial mobility. a factor analysis was then performed on updrs motor scores for 146 pd patients from a community-dwelling cohort. three factors were extracted by principal components analysis with subsequent varimax rotation, accounting for 63.5% of the total variance: factor 1-balance and stability (posture, postural stability, gait, arising from a chair, and bradykinesia); factor 2-rigidity and motor speed (rigidity, speech, facial mobility, rapid alternating movements, leg agility, hand movements, and finger tapping); factor 3-tremor (resting and action). these results indicate that the updrs motor examination is reliable between raters and measures the cardinal signs of pd. an open pilot study was performed to evaluate the efficacy of botulinum a toxin (botox) injections for disabling hand tremors. a previous report on the use of botox for hand tremors suggested that it was helpful, but relied on subjective clinical rating scales. the extent of normal clinical fluctuations or a placebo response could not be determined. to investigate these issues more objectively, 7 patients with parkinson's disease and 1 with essential tremor with refractory hand tremors underwent electromyographically guided intramuscular injections of botox into wrist flexors and extensors. patients without great medication-related tremor fluctuations were selected. results before and after botox were determined by comparing (1) patient perceptions of functional improvement, (2) clinical assessments using the unified pd rating scale for tremor and the webster rating scales, and (3) physiological measurements using accelerometric analysis of hand tremors of tremor frequency, amplitude, and waveform characteristics. all patients reported some improvement, ranging from mild to marked with a mean of 2.6 on a 0 to 4 (4 = marked) global rating scale. however, only 3/8 patients showed a significant improvement in the clinical rating scales, confirmed by >50% reduction in tremor amplitudes. these findings show that most patients reported improvement not confirmed by the clinical or physiological measures. efficacy of botox injections for tremors is implied, but controlled trials are needed before this procedure can be generally recommended. christopher g. goetz and glenn t . stebbins, chicago, i l we tested whether hallucinations, motor disability, and cognitive decline were risk factors for nursing home placement in advanced parkinson's disease (pd) and whether these effects were independent or synergistic. between 1987 and 1991, we identified 11 patients admitted to long-term nursing homes. using case control methodology, we matched each for age, pd duration, and sex with 2 control pd patients remaining at home. parkinsonism was assessed by the motor and activities of daily living subscales of the unified p d rating scale (updrs); hallucinations and dementia were determined by scores 2 2 on the thought disorder and intellectual impairment items of the updrs. tests of synergy were based on a mantel-hentel model. hallucinations were a significant risk factor with odds ratio = 18.0, x2 = 17.0, p < ,001. motor impairment alone and cognitive impairment alone were not significant risk factors for nursing home placement (x2 for motor severity = ,00084, p > .05, and x2 for cognitive impairment = .0023, p > .05). furthermore, combined odds ratios for hallucinationslmotor severity and hallucinations/cognitive impairment showed no synergy of effect (x2 <1.0 for both,p > .05). of the 3 variables studied, hallucinatory behavior is the most prominent and independent risk factor for nursing home placement in these patients; the data suggest that aggressive control of hallucinations may be warranted to prevent nursing home admission. temperature-sensitive paramyotonia congenita phenotype* louis j . ptacek, philip mcmanis, hzrbert kwiecinski, alfred george, robert barchi, launce gouw. and mark leppert, salt lake city, u t , rochester, mn, warsaw, poland, and philadelphia, pa the periodic paralyses are a group of autosomal-dominant muscle diseases sharing a common feature of episodic paralysis. in one form, paramyotonia congenita (pc), the paralysis is temperature-sensitive, usually occurring with muscle cooling. electrophysiological studies of muscle from patients with pc have revealed temperature-dependent alterations in sodium channel (nach) function. this observation led to the identification of 2 distinct mutations in an s4 segment of a skeletal muscle nach in 3 unrelated pc families. we describe the use of the single-strand conformation polymorphism (sscp) technique to define a third allele specific to pc patients in an additional family. this aberrant pattern, though distinct from the first 2, occurs in the same exon of this nach gene. sequencing is currently underway to define the molecular alteration causing this aberrant pattern. two additional families with the pc phenotype have been sampled and do not demonstrate these 3 sscp variants. we are currently searching for new mutations in these 2 families to define further the molecular heterogeneity of this temperature-sensitive pc phenotype. parag mehta and roger w. kukz, brooklyn, n y abnormal accumulation of calcium (ca) in myofibers is thought to play a role in pathogenic myonecrosis. attempts at reducing intracellular ca content with ca channel blockers in duchenne muscular dystrophy (dmd) have been clinically unsuccessful. dantrolene, however, which acts at the sarcoplasmic reticulum to inhibit ca release from intracellular stores, has produced dramatic reductions in serum creatine kinase (ck) in dystrophic mice and more recently in dmd. we investigated the effect of low-dose dantrolene in a group of 20 patients with limb girdle dystrophy (lgd), dmd, and other myopathic disorders. all subjects received dantrolene in incrementing doses from 25 to 100 mg daily over a 6to 12-week period. mean baseline ck was compared to ck with dantrolene treatment. dramatic reductions in serum ck levels averaging 50% were seen at 50to 100-mg doses in lgd patients (8). dmd patients (3) and patients with other myopathies (9) showed a similar but less dramatic reduction in ck. three of the weakest patients complained of increased fatigue while taking 100 mg, which suggests that higher-dose dantrolene may confound longer-term trials assessing clinical muscle strength and function. dantrolene in dosages well below conventional antispastic doses has a dramatic effect on serum ck and possibly myofiber necrosis in lgd, other dystrophies, and other muscle disorders. p77. antigen-specific therapy in myasthenia gravis: myasthenia gravis (mg) is mediated by anti-acetylcholine receptor (achr) antibodies, believed to be t-cell dependent, and antigen-specific therapy would be preferable to current nonspecific immunosuppression. exposing mouse t-cell clones to mhc class i1 molecules complexed with relevant antigen on planar membranes induced proliferative unresponsiveness (quill and schwartz, 1987) , and soluble mhc class i1 molecules complexed with myelin basic protein (mbp) peptide resulted in unresponsiveness of specific tlymphocyte clones in vitro (sharma et al, 1991) . we have used our well-defined dr4-restricted t-cell clone (ong et a [, 1991 ) isolated from an mg patient and specific for p138-167 of the achr alpha subunit. overnight incubation of these t cells with a soluble p138-167 : dr4 complex substantially inhibited the subsequent response to challenge with soluble antigen and presenting cells. in contrast, antigen response after preincubation with dr4 complexed to an irrelevant peptide (mbp 1-14), soluble dr4 alone, or an experimental approach studied in vitro p138-167 alone (at equimolar concentrations) did not differ appreciably from that in untreated cells. the p138-167:dr4 complex had no effect on other non-achrspecific cell lines/clones. these results suggest that the use of soluble mhc-peptide complexes may be an approach to selective immunotherapy in mg patients. p78. high-dose intravenous immunoglobulin in the shawke a. soueidan and marinos c. dalakas, bethesda, md inclusion body myositis (ibm) is a severe disabling inflammatory myopathy with characteristic clinical and histological features. it is commonly suspected when a patient with presumed polymyositis does not respond to available immunotherapies. the need for an effective treatment in patients with ibm prompted the present pilot study using high-dose intravenous immunoglobulin (hd-ivig), an apparently effective immunomodulating agent in several autoimmune neuromuscular disorders. we treated 4 patients with muscle biopsy-proven ibm with up to 2 monthly infusions of 2 gml kg ivig. after the first infusion, 3 of the 4 patients showed definite functional improvement consisting of independent ambulation, fewer falls, and increased ability to lift weights. the muscle strength of the proximal and less atrophic muscle groups improved by one grade mrc scale (from 4 to 5), whereas the distal and atrophic muscles remained unchanged. the improvement, sustained up to 7 months, was greater in patients with the most severe endomysial inflammation. we conclude that hd-ivig may be the first promising agent that can improve the strength of certain muscle groups in patients with ibm. because ivig is prohibitively expensive, the present encouraging results warrant a large-scale controlled therapeutic study. hays, and n . lutov, new york, n y , and milan, italy anti-myelin-associated glycoprotein (mag) antibodies from patients with neuropathy cross-react with the glycolipid 3-sulfated glucuronyl paragloboside (sgpg). among 24 patients tested by enzyme-linked immunosorbent assay and western blot, 15 had highly elevated antibody titers (56,400) to both mag and sgpg, 2 had highly elevated titers to mag alone, and 7 had highly elevated titers to only sgpg. immunostaining of normal nerve myelin by the antibodies correlated better with anti-mag than anti-sgpg activity. twenty-one of the patients, including patients in all 3 groups, had predominantly sensory or sensorimotor neuropathy, and biopsy specimens revealed deposits of igm and complement on affected myelin sheaths. three patients presented with motor syndromes, all with antibodies specific for sgpg; 1 had a predominantly motor demyelinating neuropathy, 1 had upper and lower motor neuron signs and peripheral neuropathy, and 1 had amyotrophic lateral sclerosis confirmed post mortem. all 3 had deposits of complement on peripheral nerve myelin sheaths. these studies suggest the following: (1) that anti-mag or sgpg antibodies may differ in their fine specificities and biological activities, (2) that anti-sgpg antibodies also may occur in motor neuron diseases, complicating the clinical presentation, and (3) that both mag and sgpg should be used as antigens in testing for autoantibody activity in peripheral neuropathy. david b. williams, john steele, ulla-katrina craig, sandra bryant, peter o'brien, and leonard kurland, newcastle, new south wales, australia, mangilao, guam, and rochester, m n continuing surveillance of neurodegenerative diseases in the mariana islands reveals changes in frequency and clinical characteristics since the 1950s that resemble those in other known western pacific foci (kii peninsula, japan, and irian jaya, new guinea). recent surveys of patients 50 years and older were conducted on rota, tinian, and yigo, guam. possible cases of dementia, parkinsonism, and amyotrophic lateral sclerosis (als) were identified by local trained personnel using a questionnaire, world health organization neurology test, and cognitive screening. those who failed the screening were examined by a neurologist. in the small populations of rota and tinian, there were no definite cases of als compared to i to 3 cases present in previous surveys. the high prevalence of parkinsonism-dementia complex (pdc) was unchanged and dementia was increased compared to earlier surveys. in yigo, als and pdc continue to be prevalent; however, the als patients are predominantly long-term survivors (> 10-year disease duration). in areas of previous high prevalence of als/pdc, dementia (as pdc) was associated with extrapyramidal signs, whereas in areas of previously low prevalence of als/pdc, dementia alone, possibly of alzheimer type, predominated. these observations help to confirm previous reports of changing clinical patterns, but suggest that the geographic distribution of the (presumed) environmental etiological agent for als/pdc remains stable after almost 40 years. p. v . fragokz, 0. frongillo, m. michisanti, g. antonini. derangements of the cardiac conducting system are the most common features of heart involvement in myotonic dystrophy (md). in view of chis 65 patients with various grades of md (43 males and 22 females, mean age 37 yr) underwent 12-lead ecg and holter monitoring. in 31 patients (48%), almost all with a severe grade of md, 1 or more conduction defects were found: first-degree atrioventricular block (i-avb) in 21 cases, second-degree avb in 1 case, right bundle branch block in 3 cases, left anterior hemiblock in 2 cases, left bundle branch block in 2 cases, and trifascicular block in 1 case (pacemaker implanted). an 18-year-old boy had a chronic atrial fibrillation with slow ventricular rate; he died suddenly while awaiting electrophysiological study. thirty-seven patients were followed over a mean period of 40 months (range 11-75 mo). a 54-year-old woman experienced a myocardial infarction and was excluded from subsequent considerations. conduction defects de novo appeared in 6 patients: i-avb in 5 and i-avb plus 11-avb (mobitz i and i1 type) in 1. nine patients, all with i-avb at initial evaluation, showed deterioration of their defects' conduction: a bifascicular block was observed in 6 cases and a trifascicular block in 3 cases (pacemaker implanted). conduction defects may run a malignant course in md, mainly in patients with more severe grades of the neuromuscular disease; thus, a close cardiological evaluation is mandatory for a proper therapeutic approach in single cases. hiroshi mitsumoto, surest5 kumar, kevy h. levin, robert w. shields, jr, michelle secic, asa j . wilbourn, and rajendra g . desai, cleveland, oh, and santa ana, ca twenty patients with amyotrophic lateral sclerosis (als) entered a pretreatment study with monthly quantitative isometric muscle strength tests (4 muscles in each extremity) and quantitative tufts scales including vital capacity, bulbar diadochokinetic rate, timed water drinking, timed rising from a chair, and timed walking 5 meters. after 4 to 6 months of pretreatment observation, the patients received 400 mg/kg intravenous immunoglobulin (ivig) (gamimune-n, miles) every month for up to 12 months. during the study period, 5 patients died and 2 patients withdrew from the study. the slope of each variable's changes over time before and after the ivig treatment were compared statistically. none of the quantitative scales showed significant change with ivig. however, the slope of the upper extremity muscle strength revealed improvement with ivig treatment ( p = .002 and .013, right and left, respectively). when all extremities were combined, the slope was also significantly improved with ivig ( p = .035). lower extremity muscle strength alone showed similar trends but no statistical significance. electrophysiological and immunological data were also analyzed. our results warrant a double-blind, controlled study with ivig for the treatment of als. leber's hereditary optic neuropathy (lhon) is a mitochondrial disorder with predominantly optic nerve abnormality. it can be associated with dystonia, ataxia, encephalopathy, cardiac abnormalities, and other less well-characterized neurological syndromes. we describe 2 members of a family with lhon with a slowly progressive motor polyneuropathy. a 22-year-old man and his 11-year-old sister have had mild motor impairment since early childhood. a gait disorder and distal muscle weakness became evident at puberty. the girl, but not the man, also has blindness, distal numbness, type i diabetes mellitus, and short stature. physical examination showed in both: bilateral foot drop, limb hyperreflexia but absent ankle reflexes, distal sensory loss, and a slight brownish scaly skin discoloration over the forearms. only the girl had clonus and optic nerve atrophy. the man had peripapillary telangiectasias. the jaw jerk was normal in both. motor nerve conductions in both showed absent tibia1 and peroneal responses, whereas other motor and sensory nerve conductions were normal. emg revealed denervation, more so distally. muscle biopsy findings showed recent denervation and previous denervation followed by reinnervation. n o raggedred fibers were observed with the modified trichrome and sdh stains. brain mri was normal in both. cerebrospinal fluid in the girl was normal. blood samples from both patients and maternally related family members revealed a mitochondrial dna point mutation at position 11778 (d. c. wallace). in this family, only 1 male had lhon; 3 females had lhon and 2 others, including the patients' mother, were asymptomatic carriers. this association of chronic motor neuropathy and hyperreflexia with lhon appears to be a distinct syndrome. the pathogenic mechanism by which the mitochondrial dna defect causes the neuropathy (and other neurological deficits) requires analysis. duchennelbecker muscular dystrophy henry j . kaminski, mazen al-hakim, r. john leigh, bashar katirji, and robert l. ruff; cleveland, oh fast-twitch extremity muscle fibers are preferentially affected in duchenne/becker muscular dystrophy (dbmd). since saccades are thought to be mediated by fast-twitch fibers, saccadic velocities would be expected to be decreased among these patients. to investigate involvement of extraocular muscle (eom) by dbmd, we studied with infrared oculography 3 patients who were wheelchair-bound and able to perform only minimal activities of daily living. saccades were slightly slowed but were within 95% confidence limits of normal. all 3 patients showed square wave jerk movements (swj). in 2 patients, the frequency of the swj exceeded that of normal subjects, which suggested central nervous system dysfunction. clinical neuroophrhalmological examination of 7 other dbmd patients was normal. this investigation is the first study of ocular motility in dbmd and demonstrates that eom function is relatively preserved even in far advanced patients. eom is composed of a heterogenous mix of fiber types that differ in anatomical and physiological characteristics from extremity muscle. study of eom in dbmd may prove to be useful in understanding why some muscles are resistant to dbmd and in characterizing properties that limit muscle degeneration. (supported by nih grants ey09186, ey067 17, the department of veterans affairs, and the evenor armington fund.) since patients with myotonic dystrophy (mtd) exhibit a marked resistance to insulin effect on glucose uptake and an impaired handling of the insulin-sensitive amino acids, it is possible that muscle wasting in mtd may reflect a derangement of insulin action on muscle protein metabolism. increased muscle protein breakdown in mtd would be expected if the normal inhibitory effect of insulin on protein catabolism is impaired. the forearm perfusion technique combined with measurements of 3-methylhistidine (3-mh) arteriovenous (a-v) differences by high-performance liquid chromatography provides a unique method to investigate skeletal muscle myofibrillar protein degradation in vivo. we studied 3-mh (a-v) and efflux from the forearm muscles in 8 men moderately affected with mtd and 10 normal men. efflux values (q) were calculated as the product of 3-mh (a-v) times forearm plasma flow measured by the indicator dilution technique. forearm 3-mh release (estimated as {a-v} or q) of mtd patients did not differ significantly from normal controls. we conclude that myofibrillar degradation is not increased in mtd even when measured in a muscle compartment selectively affected by wasting. the possibility of an impaired anabolic action of insulin in mtd has yet to be determined. to study the relationship between the ragged red fibers (rrf) and age, we have reviewed 500 muscle biopsy specimens prospectively. patients with well-established mitochondrial myopathy syndrome and with myopathies known to produce secondary rrf were excluded. the number of ragged red fibers (rrf) was counted under 40 x (lpf) magnification. rrf were identified by the modified trichrome and sdh stain. for the final analysis, the sdh staining was used. the frequency of rrf was analyzed in relation to patients' ages. the frequency of cases with more than 1 rrf increased with aging: 0% in the first decade, 16% in the fourth decade, and 55% in the eighth decade. the frequency of cases with more than 10 rrf also increased with aging: 0% in the first three decades, 4% in the fourth decade, and 19% in the eighth decade. of 25 patients with well-established mitochondrial myopathy syndrome, 24 had more than 10 rrf under lpf. the number of rrf in muscle increases with aging, indicating that mitochondrial activity in muscle is affected by aging. this finding may complicate the diagnostic criteria of mitochondrial myopathy in older individuals. ten rrf under lpf seems to be a reasonable cutoff point for the diagnosis of mitochondrial myopathy. schwartz-jampel, a rare autosomal-recessive syndrome characterized by short stature, myotonia, skeletal abnormalities, and peculiar facies, was reported by aberfeld in 1965. the same sibship was earlier reported by schwartz and jampel in 1962 with emphasis on blepharophimosis. as of this writing about 30 cases have been reported in the literature. most of the features of this syndrome are believed to be secondary to primary muscle disease. several peripheral electrophysiological studies showing features of myotonia have been reported. we describe 4 patients with schwartz-jampel syndrome showing evidence of central conduction disturbance documented by somatosensory-evoked potentials (seps). median nerve seps showed normal latencies to erb's point and n-13 in all. interpeak latencies between n13 and n19 were prolonged in 3 with complete block in 1. emg showed typical myotonic discharges in all. motor nerve conduction velocities, visual and brainstem auditory-evoked potentials, ct, and mri were normal in all. seps in the parents were normal. we believe this is the first report documenting evidence of central nervous system (cns) involvement in schwartz-jampel syndrome. schwartz-jampel syndrome and myotonic dystrophy may have similar cns ''lesion'' as sep abnormalities also have been shown in myotonic dystrophy. epidemiological studies have associated consumption of certain batches of l-tryptophan (lt) with development of the eosinophilia-myalgia syndrome (ems). 1,1 '-ethyledenebisltryptophan) (ebt or peak e), a derivative of lt, is a trace contaminant associated with implicated batches of lt. three female lewis rats received ebt, 4 mg per 100 gm daily, by intraperitoneal injection. four control rats received unimplicated lt. n o peripheral eosinophilia, rash, or weakness were observed in either group. one rat from each group died during the experiment (control-bowel infarct; ebt-death under anesthetic). after 132 days, forelimb and hindlimb muscles of the remaining animals were frozen and fixed for program and abstracts, american neurological association 253 ultrastructural and histological studies. two ebt rats had a myopathy involving soleus with a perimysial infiltrate containing lymphocytes, macrophages and sparse eosinophils, and necrotic fibers; the other showed few necrotic fibers in gastrocnemius. occasional eosinophils were seen in fascia in both animals given ebt but not in controls. fiber-type specific quantitative analysis of the microvasculature showed no decrease in the capillary index. ultrastructural examination revealed an increase in the size of microvessels in ebt animals. no denervation or reinnervation were demonstrated. the perimysial inflammation replicates an important feature of human ems and supports the epidemiological evidence that ebt is the causative agent of the disease. britta ostermeyer-shoaib, bernard m. patten, and tetsuo ashizawa, houston, t x five women (patients 1-5) developed motor neuron disease (mnd) 10 years (range 2-23 yr) after receiving silicone gel-filled breast implants. at explant in 4 patients, 2 had both and 2 had the left implant ruptured with silicone spilled into tissue. one woman (patient 6) developed amyotrophic lateral sclerosis (als) 5 years after numerous injections of free silicone into her face. biceps muscle biopsy specimens in all 6 showed neurogenic atrophy. patient 1 developed als with bulbar involvement and died 7 years later of respiratory failure. she had anti-gm1 antibodies and autopsy findings confirmed the diagnosis of typical als. patient 2 developed als, but also fatigue, myalgia, arthralgia, and skin rash. she had anti-gm 1 antibodies, antisilicone antibodies, positive antinuclear antibodies (ana), and decreased serum igg, iga, and c3, but increased igm and creatine phosphokinase (cpk) and chronic inflammation was revealed in muscle biopsy specimens. patient 3 developed als, but also had hair loss, skin rash, fatigue, headache, sjogren's syndrome, and positive ana. patient 4 developed als, but also had myalgia and arthralgia. patient 5 developed lower mnd, but also fevers, arthralgia, and joint stiffness. she had anti-gm1 antibodies, positive ana, antimyelin antibodies, and decreased serum igg and iga with chronic inflammation shown in nerve biopsy findings. patient 6 developed a steroidresponsive and steroid-dependent als with bulbar involvement. she had a monoclonal gammopathy in the cerebrospinal fluid and increased cpk. we suggest that silicone acts as an adjuvant that damages motor neurons via an indirect autoimmune mechanism. implants and silicone injections into the face p90. silicone adjuvant breast disease: more forty-five women developed mixed sensory-motor neuropathy (35), motor neuron disease (5), multiple sclerosis (2), multiple sclerosis-like syndrome (2), or myasthenia gravis (1) 7 years (range 1 mo-23 yr) after receiving silicone-gel breast implants (38), saline-filled silicone-covered breast implants (6), or direct injections of silicone into the breast (1). most patients had, in addition, severe fatigability, myalgia, arthralgia, morning stiffness, skin rash, lymphadenopathy, sjogren's syndrome, and short-term memory problems. laboratory results revealed in most of the women decreased or increased serum immunoglobulins, autoantibodies, a serum monoclonal gammopathy, or oligoclonal bands in cerebrospinal fluid. at explantation in 34, 14 had both and 7 had 1 implant neurological cases ruptured. biopsy of the fibrous implant capsule in most patients showed foreign-body giant cells containing refractile material consistent with silicone whether or not the elastomer shell was ruptured, indicating silicone bleed. the major finding on surd nerve biopsy was loss of myelinated fibers, on biceps muscle biopsy was neurogenic atrophy, and on pectoralis muscle biopsy was myositis with vasculitis and free silicone in some. we suggest that silicone may provoke damage to nerve and muscle, probably indirectly promoting autoimmunity. james f. howard, jr, m . kathleen donovan. and m. susan tucker, chapel hill, nc urinary symptoms of urgency and incontinence have been reported only rarely in patients with myasthenia gravis (mg) and then most often in association with myasthenic crisis. we report the case of a 31-year-old woman who in december 1987 had the onset of chest pain and was found to have a lymphocytic thymoma. in june 1989 she developed urinary incontinence, was found to have an open bladder neck. and underwent a suspension procedure for stress incontinence in january 1990. eight months later she developed exertional fatigue and a diagnosis of m g was made. in july 1991 there was a recurrence of urinary incontinence. these symptoms clustered toward the end of the day and at trough mestinon dose. neuro-urophysiological studies demonstrated her previous open bladder neck, the inability to sustain a pelvic floor contraction, and increased bladder wall contraction. singlefiber electromyography (sfemg) recordings from the anal sphincter demonstrated a mean consecutive difference (mcd) of 88 ysec, and 11% of fiber pairs had impulse blocking while recordings in the extensor digitorium communis muscle were normal. following a course of plasma exchange, there was significant clinical improvement with a reduction in the frequency of urinary incontinence, and improvement in anal sphincter sfemg studies (mcd, 60 ysec with no blocking). this case demonstrates that in those myasthenic patients with predisposing bladder outlet dysfunction, urinary incontinence may be a manifestation of worsening mg. diana m. escolar, mohamed eldaly, and jaime rich, boston, m a debate still exists as to the role of antibody versus celmediated factors in the pathogenesis of guillain-barre syndrome (gbs). we describe a patient with increased proportion of circulating t cells and a t-cell lymphoma who developed gbs and responded to intravenous immunoglobulin (ivig). a 57-year-old man with t-cell lymphoma drveloped gbs by clinical, nerve conduction, and cerebrospinal fluid criteria. he had an elevated proportion of t cells and markedly reduced b cells with a normal cd4/cd8 ratio. he responded rapidly to ivig, with return of nearly normal motor function in 1 week. three weeks later, he relapsed and his vital capacity dropped. ivig was again administered and within 24 hours he nearly recovered. a third relapse, 2 1 days later, again responded to ivig. he has remained asymptomatic with ivig maintenance. the role of t-cell lymphocytes in initiating experimental autoimmune neuritis has been shown by adoptive transfer experiments. this patient with t-cell neoplasia may represent an analogous model in hu-guillain-barre syndrome mans supporting the role of t-cell (cell-mediated) autoimmunity in the pathogenesis of gbs. ivig therapy may act primarily by inhibiting the t-cell-mediated attack on myelin. p93. sympathetic skin response: age effect it is frequently stated that the sympathetic skin response (ssr) can be elicited in all normal subjects, but the age of the investigated population usually is not considered to be a significant factor. we have examined the ssr in the upper and lower limbs of 100 normal subjects, aged 20 to 88 years (5 5 of them males). the ssr was elicitable in the lower limbs in all subjects under the age of 60 years and in the upper limbs in all subjects younger than 70 years. in contrast, it could be elicited in the lower limbs in only 345% and in the upper limbs in 655% of octogenarians. the amplitude of the response, though highly variable, showed a remarkable decline with age, both in the upper ( p < 0.0001, r = 0.51) and in the lower ( p < 0.0001, r = 0.65) limbs. these results indicate that age affects both the elicitability and the amplitude of the ssr. this has to be taken into consideration when evaluating the autonomic function in the elderly. we reviewed records of patients appearing to have motor neuron disease (mnd) to whom we recommended immunosuppression over 4 years (17 of 2 10 m n d patients). atypical findings engendered hope rhat they might have treatable neuropathy. electrophysiological studies were mainly consistent with mnd, but also showed conduction block or other evidence of relatively mild peripheral nerve disease in 12. sensory symptoms were present in 7 ; 1 or more reduced or absent deep tendon reflexes in 6; elevated cerebrospinal fluid protein in 8; and nonspecific abnormalities on sural nerve biopsies in 6 of 7. anti-gm1 levels were measured in 6 (including 1 who improved), but none was significantly elevated. immunosuppression included cyclophosphamide (12 patients); prednisone (10); plasma exchange (4); intravenous gammaglobulin (1); cyclosporine (1); and total lymphoid irradiation (1). seven treated patients died, 6 worsened, 1 remained stable for 2 years, and 1 improved. two patients declined treatment. one died and the other did not worsen in 3 years. we conclude that immunosuppression by our methods is, at best, rarely effective in atypical mnd. we studied serum antiglycolipid antibodies by enzymelinked immunosorbent assay in 11 patients with typical miller fisher syndrome (mfs), 6 patients with atypical mfs who were lacking in some of the 3 cardinal signs, 4 patients with guillain-barrc syndrome (gbs) with ophthalmoplegia, 20 patients with gbs without ophthalmoplegia, 20 patients with multiple sclerosis (ms), and 17 patients with other immunological disorders (oid) including systemic lupus erythematosus, polymyositis, and mixed connective tissue disorder. all patients with typical mfs had increased activity of igg antibody against ganglioside g q l b in the early phase, and it reduced with time. such anti-gqlb igg activity also was detected in 5 of the 6 patients with atypical mfs and in 3 of the 4 patients with gbs with ophthalmoplegia. in atypical mfs, the only patient without increased anti-gq1 b igg activity demonstrated normal eye movement with ptosis, whereas eye movement was impaired in the other 5 patients. no patients with gbs without ophthalmoplegia, ms, or oid had increased anti-gqlb igg activity. these findings suggest the close association between increased anti-gql b igg activity and impaired eye movement in mfs and gbs. serum anti-gqlb igg activity possibly plays a role in impaired eye movement in mfs. our goal is to develop an assay that can be used to monitor a relevant immune effect of interferon p (ifnp) in multiple sclerosis (ms) patients during the course of ifnp immuno-' therapy, since recombinant ifnp is being tested in multicenter clinical trials. this report extends our prior studies of the inhibitory effect of ifnp on t-cell activation. peripheral blood mononuclear cells (pbls) from 11 healthy donors and 10 clinically stable ms patients were studied. pbl cultures were stimulated with cona, mab to cd3, or with the phorbol ester pma in the presence of the calcium ionophore ionomycin. parallel cultures were studied in the presence of ifnp, 100 uiml. t-cell activation was monitored by determining the percent cells positive for il-2 receptor (il-2r) using facs analysis, or with a sensitive enzyme-linked immunosorbent assay for ifny. ifnp markedly inhibited il-2r expression induced by cona, by mab to cd3, or by pma and ionomycin, which activate t cells via different pathways. the results suggest that ifnp inhibits t-cell activation by actions independent of membrane receptors. we observed significant inhibition of cona-induced t-cell il-2r expression in both ms patients (20.6% inhibition, p < 0.05) and controls (26.8% inhibition, p < 0.05). there was no significant difference in percent inhibition between ms and controls, but there was more variance among the ms patients. variability of biological effects of ifnp on t cells may relate to differential therapeutic responses to exogenously administered ifnp in ms patients. preliminary experiments suggested that ifnp inhibited ifn gamma secretion by pbl stimulated with cona. ifnp inhibits a number of events associated with t-cell activation, in both normal and ms t cells. response to ifnp appears more variable in the ms cases. immunological monitoring of t-cell activation in patients receiving ifnp may assist in understanding the observed therapeutic responses and planning clinical protocols. myelin destruction is associated with many central nervous system disorders, such as multiple sclerosis, head trauma, and ischemic injury. inflammatory cells, monocytes/macrophages, and polymorphonuclear leukocytes (pmn) may me-program and abstracts, american neurological association 255 diate myelin injury, and lipid peroxidation may be an important mechanism. inhibiting myelin oxidation could have substantial benefit, so we evaluated the ability of a 21-minosteroid, u74500a, to inhibit myelin oxidation by monocytes and pmn. fresh rat brain myelin was harvested by multiple sucrose gradient, ultracentrifugation steps, and the final product was confirmed to be pure myelin by sds-page electrophoresis. human monocytes or pmn were obtained from 5 healthy, unmedicated volunteers by gradient separation techniques. monocytes (1.47 x lo6 cells/ml) and pmn (3.40 x lo6 cells/ml), myelin (460 pg protein/ml), and lipopolysaccharide (100 pg/ml) were incubated for 16 hours with or without 100 wm u74500a in 1-ml wells. myelin oxidation was evaluated by a thiobarbituric acid reactive substance assay for production of malondialdehyde (nmol/ ml). myelin oxidation by monocytes was 1.80 +-0.35 (mean 2 standard error of mean) without u74500a and was reduced to 0.76 ? 0.15 by 100 pm u74500a ( p < .005). pmn-mediated myelin oxidation was 1.89 _t 0.44 without drug and 0.64 ? 0.10 with drug ( p < 0.02). these results demonstrate that u74500a markedly inhibits monocyte-and pmn-mediated myelin oxidation and suggest that the 21aminosteroids may help disorders associated with inflammatory cell-induced myelin injury. microglia cells participate in the pathological reactions of the cns to multiple insults including trauma, inflammation, and neuronal degeneration. functional roles for these cells could include mediating tissue in jury, promoting repair, or modulating immune responses. with regard to the latter, we have observed that the majority of adult human-derived microglia express major histocompatibility complex (mhc) class 11 molecules under basal culture conditions, in contrast to astrocytes derived from the same surgical biopsy specimens. all morphological subtypes of the microglia (ameboid, bipolar, and ramified) expressed mhc class i1 molecules, indicating a discordance between morphology and mhc antigen expression as markers of microglia activation. the microglia actively ingest myelin constituents, as assessed using fluorescein-labeled myelin basic protein and laser confocal microscopy. autologous t cells (e') freshly isolated from the systemic blood and cocultured with candida antigen underwent active proliferation in the presence of 1 to 10% microglia, indicating the functional capacity of the microglia to serve as antigen-presenting cells. y-interferon augmented both mhc class 11 expression and functional antigen-presenting capacity. these results indicate the potential of the adult human microglia to promote immune reactivity within the cns. protein (mbp) neutralize anti-mbp purified from multiple sclerosis cerebrospinal fluid active phases of multiple sclerosis (ms) are associated with increased titers of intrathecally produced antimyelin basic protein (anti-mbp). anti-mbp can be purified by antigenspecific affinity chromatography from csf igg of patients with acute relapses of ms. eighteen synthetic peptides of human myelin basic protein (h-mbp) containing between 8 and 25 amino-acid residues and covering the entire length of the molecule were synthesized by the fmoc method. purified anti-mbp was reacted with increasing amounts of h-mbp as well as each of the 18 peptides in an initial liquid phase assay, and subsequently titers of f anti-mbp in all resulting mixtures were measured by a solid-phase radioimmunoassay . purified anti-mbp was neutralized by h-mbp and 6 of the 18 synthetic peptides containing overall residues corresponding to 61 to 106 of h-mbp. the remaining 12 synthetic peptides covering both the amino and carboxyl terminals of h-mbp did not significantly react with purified anti-mbp from these patients. in conclusion, anti-mbp purified from csf of ms patients has affinity for epitopes located between residues 61 and 106 of h-mbp. in a double-blind study involving 70 patients, we recently demonstrated that 4-aminopyridine (4-ap) is superior to placebo in the treatment of multiple sclerosis (ms) (ann neurol, in press). the related agent 3,4-diaminopyridine (dap) also appears to be effective. to enable a preliminary comparison, 14 patients, who in our previous study had not benefitted from 4-ap, were now treated (4 wk) with dap (up to 1.0 mg/kg/day) for 4 weeks in an open-label fashion. instruments for assessment and registration of side effects were the same as in the previous trial. the optimal dose of dap was 56.4 mg/day compared to 30.7 mg/day for 4-ap. significant changes in the edss (1.0 point or more) were not found, whereas significant improvements in neurophysiological parameters were found (no difference between 4-ap and dap, allp > 0.05). subjective side effects during 4-ap (8 patients) mainly suggested cns-function disturbance (dizziness and gait disturbance) and during dap (10 patients) mainly suggested peripheral nervous system-function disturbance (paresthesias). systemic tolerability clearly was diminished for dap compared to 4-ap, with 2 patients withdrawing because of severe gastric complaints and 1 developing liver function abnormalities. these data suggest that 4-ap is more valuable than dap in the treatment of ms. cy clophosphamide/methylprednisolone therapy in multiple sclerosis multiple sclerosis (ms) is a presumed autoimmune disease in which various forms of immunotherapy have been attempted. mri studies show the disease to be more chronically active than is clinically evident, thus a single treatment is unlikely to provide lasting benefit. recently, the northeast cooperative treatment group found that pulse cyclophosphamide (700 mg/m2 every other month for 2 years) slows progressive ms. we initiated a pilot study to determine the effect of a more intensive and prolonged pulse therapy regimen in both progressive and earlier stages of the disease. pulse therapy was given after induction with either iv cyclophosphamide/corticotropin (600 mg/m2 x 5 over 8 days) or iv methylprednisolone (1 gm x 7 over 7 days). patients received a single iv dose of cyclophosphamide (800-1,400 mg/m2) adjusted to produce leukopenia plus 1 gram of iv methylprednisolone monthly for a year, every 6 weeks for the next year, and every 2 months in the third year. another group received pulse methylprednisolone without cyclophosphamide. as of this writing, 128 patients have been treated, of which 24 have completed 3 years. interim analysis shows that patients treated with pulse methylprednisolone were more likely to become treatment failures than those treated with pulse cyclophosphamide/methylprednisolone (78% vs 45%) independent of induction therapy. withdrawal due to toxicity, however, was higher in the pulse cyclophospharnide group. current regimens involve methylprednisolone induction alone followed by pulse cyclophosphamide/methylprednisolone, analogous to lupus nephritis pulse therapy. this regimen can be given solely on an outpatient basis, does not cause alopecia, and is more amenable for use in earlier stages of the disease. to determine the incidence of pathologically confirmed malignancy in multiple sclerosis (ms) patients in funded clinical trials of cyclophosphamide (ctx) and of azathioprine (aza), data were collected longitudinally using telephone interviews, written questionnaires, physical examinations, and medical records for ctx and aza patients from a community in-hospital and out-patient ms clinic in fargo, nd. in the ctx study (goodkin et al, arch neurol 1987; 44: 823-4327) , 5 1 clinically definite (cd), chronic progressive ms patients were enrolled. twenty-four were controls and 27 received a mean induction dose of 6.08 grams. fourteen of the induced patients then received boosters every other month for 24 months resulting in a mean total dose of 15.92 grams. no malignancies were detected. in the aza study (goodkin et al, neurology 1991; 41:20-25) , 54 c d relapsing ms patients participated. twenty-five were controls and 29 received 3 mgtkg of aza daily by mouth adjusted to maintain a white blood count of greater than 3,50o/cmm for 2 years (mean dose 2.6 mg/kg). two of 29 aza patients developed resectable skin cancers: 1 basal cell (bcc) at 18 months and i squarnous cell (scc) at 43 months. the incidence of developing a bcc or scc was not significantly increased after initiating therapies as compared to the untreated ms controls (aza: fisher exact testp value = 0.49). no other malignancy has been detected during 46 to 100 months of clinical follow-up. allergic encephalomyelitis paula dore-dufly, ruth washington, and robert h. swanborg, detroit, m i postcapillary endothelium at sites of inflammation undergoes many changes referred to as activation. activated endothelial cells (ec) exhibit increased surface expression of immunorelevant proteins (icam-1; ncam, elam, and mhc class i and class i1 antigens [ags)). the sequence of events that characterizes ec activation may be important in susceptibility, induction, and perpetuation of experimental allergic encephalomyelitis (eae). in this study we examine expression of ec activation antigens in central nervous system (cns) microvessels in response to interferon gamma (ifn-y). cns microvessels from sjl and bio.s mice were incubated for 18 hours in ifn-y (500 u/ml), fixed, permeabilized, and then stained with an antibody that recognizes class i, class i1 mhc antigens, icam-1, and factor viii. relative fluorescence intensity was determined using a laser cytometer. results indicate that microvessels from all strains tested expressed no detectable icam-1 and class i1 ags. little class i antigen and transferrin receptors were expressed. upon stimulation with ifn-y, sjl microvessels exhibited increased surface expression of all ec activation ags. b1o.s microvessels exhibited icam-1 and class i mhc but mhc class i1 ags were not upregulated. results indicate that there are strain differences in the ec response to ifn-y. resistance of b1o.s mouse ec to activation by ifn may be a factor in decreased susceptibility or induction of eae, or both. protein-t-cell line-mediated experimental to investigate a possible pathogenic role of interferongamma (ifn-y) in experimental allergic encephalomyelitis (eae), an immunocytochemical study was undertaken to localize this cytokine in the spinal cord of lewis rats in which eae was produced by adoptive transfer of myelin basic protein-specific t cells. one pm-thick cryosections of spinal cord were labeled with monoclonal antibodies (mab) db-1 and db-12 recognizing different epitopes of rat ifn-y. in the spinal cord of naive rats, mab db-i, but not db-12, stained processes of astrocytes, suggesting that astrocytes contain a protein with an epitope cross-reacting with ifn-y. in rats with at-eae, numerous ifn-y-positive cells stained with both mab db-1-and db-12-positive cells were present from days 3 to 7 after cell transfer and had disappeared on day 10. at day 2 they entered the spinal cords predominantly through subpial vessels. ifn-y-positive cells could be identified as w3/13+ leukocytes as well as ed1-positive macrophages. as in naive rats, astrocytes in at-eae were labeled only with mab db-1, but not db-12. we never observed labeling of motor neurons with these mab. the transient presence of ifn-y in the rat spinal cord at the onset of at-eae suggests a pathogenic role of this cytokine in acute immune-mediated demyelination of the cns probably as a local stimulus for expression of mhc class i1 antigens and adhesion molecules, as well as for the release of tnf-a and toxic oxygen radicals from macrophages and microglia. immune responses to stress or heat shock proteins are implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis (ms). we examined the hypothesis that antigens in myelin cross-reacted with stress protein antigens. two techniques were used: immunocytochemistry and western blotting. frozen histological sections were prepared from normal human central and peripheral nervous system tissues. sections were incubated with 4 murine monoclonal antibodies to different mycobacterial stress proteins. antibody binding was determined using avidin-biotin complexed antimurine antibody linked to alkaline phosphatase. program and abstracts, american neurological association 257 a monoclonal antibody to the stress protein sp65 from m . leprae strongly stained both central and peripheral nervous system myelin. no myelin staining was noted with antibodies to sp70 or sp17. proteins from purified central and peripheral nervous system myelin were separated by sds-page. western blots were prepared using a monoclonal antibody to sp65 and a polyvalent rabbit antibody to myelin basic protein (mbp) as primary antibodies. antibody binding was determined using antimurine or antirabbit igg antibody coupled to alkaline phosphatase. strong staining of central but not peripheral mbp by the anti-sp65 antibody was observed. the rabbit anti-mbp antibody stained both central and peripheral nervous system mbp. the presence of antigenic epitopes shared by a stress protein and the potential autoantigen, mbp, supports the hypothesis that immune responses to stress proteins may be involved in the pathogenesis of presumed autoimmune diseases such as ms. (1). of 5 patients with borderline or positive csf lyme titer, 3 received parenteral ceftriaxone. all had later relapses consistent with ms. one patient received a month of oral doxycycline. the fifth patient had a negative western blot and was not treated. we conclude that an incidental borderline or positive lyme serology in an ms patient is unlikely to indicate neurological lyme disease. borderline serologies should be documented to rise on later testing; positive serologies should be confirmed by retest in a different laboratory or by western blot. csf abnormalities suggestive of neurological lyme disease (pleocytosis, protein elevation, intrathecal lyme antibodies) are distinct from those suggestive of ms (ogb, elevated igg index, mbp). in such patients antibiotic treatment may be appropriate, but will not alter disease course. when designing and analyzing therapeutic trials for multiple sclerosis (ms), investigators commonly compare the proportion of patients in the experimental and control groups who worsen one or more steps on the disability status scale (dss) or expanded dss during the study (typically 2 years' duration). however, the intervals between the scores in the dss may not be equal. it may be easier to change by one or more steps in the lower end of the scale (e.g., dss = 1-3) than in the midportion of the scale (e.g., dss = 4-6). to evaluate this possibility, we compared the proportion of patients who worsened by one or more steps in the 2 years after entering our program with dss = 3 or with dss = 6. fifty-one percent (29157) natural history data may be useful for designing therapeutic trials for multiple sclerosis (ms). since 1971, we have collected such data in a standard format on patients in the ucla multiple sclerosis research and treatment program. t o describe the course in our group, we have performed survival analysis (kaplan-meier) of patients with 3 or more assessments who entered the clinic with disability status scale (dss) scores of 1 to 7 (n = 395). an increase of one or more steps in the dss score persisting for more than 3 months defines worsening. median times to worsening for a dds at entry of 1 to 5 were approximately 2 years (range 1.7-2.2); but were 4.6 years for dss 6 and 3.6 years for dss 7. for those starting at dss 6 (n = 129), only 13% worsened by 1 year, 26% by 2 years, and 39% by 3 years. the percent worsening when starting at dss 3 (n = 61) were 41%, 48%, and 54%, respectively. these variable rates of worsening (i.e., time spent at each starting level) influence therapeutic trial design. including patients with dss 6 or 7 will increase the sample size and study duration. for testing nontoxic agents, we recommend enrolling patients with dss 1 to 5. for more toxic treatments, we suggest dss 3 to 5. (partially supported by usphs grant ns 087 1 1, the conrad n. hilton foundation, and various donors.) pi 12. cholinergic antagonists and p-adrenergic agonists inhibit experimental allergic encephalomyelitis in an additive manner mark a. jensen, avertano noronha, and bawy g. w. amason, chicago, il lymphoid organs receive a sympathetic (sns) and possibly a parasympathetic innervation. lymphocytes express padrenergic and cholinergic receptors and thus are sensitive to regulation by these neurotransmitters. the severity of experimental allergic encephalomyelitis (eae) is increased in sns-ablated animals. local parasympathectomy decreases plaque-forming responses in submandibular nodes. p,-adrenergic receptors are upregulated on cds t cells in progressive multiple sclerosis, as are m,-muscarinic acetylcholine receptors on cd4 t cells. we examined the effect of isoproterenol, a p-adrenergic agonist, and scopolamine, a cholinergic antagonist, on the course of eae in lewis rats. eae was induced by injection of 0.1 ml of incomplete freund's ad juvant containing guinea pig spinal cord (25% wlv) and m. tubercdosis (3 mgiml) in one hind footpad. scopolamine (6.6 mglkg, twice daily) and/or isoproterenol(o.15 mglkg, twice daily) or saline were injected subcutaneously starting on the day of immunization. scopolamine or isoproterenol alone reduced severity ( p < 0.05, t test) and duration ( p < 0.05, t test) of disease compared to controls. the combination of scopolamine and isoproterenol further reduced disease severity compared to either agent alone ( p < 0.05, x2 test), suggesting an additive protective effect of cholinergic antagonists and p-adrenergic agonists in eae. antigen-presenting cells a . conrad, v. sanders, p. schmid, and w. w . tourtellotte, los angeles, c a tissue is cryopreserved by the method of tourtellotte; this procedure minimizes or eliminates ice artifacts and preserves surface protein markers. the dissected plaques are lightly fixed in 5% paraformaldehyde and then suspended in 30% sucrose. blocks are mounted in oct, cryosectioned at 20 p,m, and picked up on gelatinized slides. the activity of plaques is determined by the presence or absence of myelin debris (antimyelin basic protein stain or lux01 fast blue) or presence or absence of neutral lipids indicating myelin digestion as seen by oil red 0 (oro) staining and the presence or absence of a class i1 major histocompatibility complex antigen (evidence for an antigen-presenting cell) on macrophages or microglia as seen by immunocytochemically staining for hla-dr (hb104 atcc). the following is our classification of the activity of multiple sclerosis (ms) plaques. type i, the most active, is defined as an area of hypercellularity, positive for hla-dr with no o r 0 staining or staining for myelin debris. type 11, or active, is defined as an area of hla-dr-positive cells that stain mildly with o r 0 at the plaque edge but positive for myelin debris; evidence for demyelinating activity <72 hours (prineas; raine). there is an inner area of plump cells that are positive for hla-dr and oro. type 111, or modestly active, is defined as a "shelf" of plump hla-dr-positive cells at the edge loaded with program and abstracts, american neurological association 259 oro-stained neutral lipid but a paucity of or no myelin debris. a region of hypocellularity is observed at the center of plaque. type iv, least active or inactive, is defined as scattered hla-dr-positive cells at plaque edge with little or no o r 0 staining and no evidence of myelin debris. the frequency of plaque types was determined from a random sample of 97 ms tissue blocks from 23 patients who died of ms. ten percent of the plaques were type i; 20%: were type 11; 209% were type 111. the majority of plaques (505%) were inactive (type iv). accordingly, it is necessary to classify the demyelinating activity of ms plaques for protocols designed to investigate etiopathogenesis. do these results suggest that whatever causes ms can be eradicated in half of the demyelinating areas by the time of death? p114. localization of g d l b ganglioside antigen in the human peripheral nervous system susumu kusunoki, atsuro chiba, tadashi tai, and lchiro kanazawa, tokyo, japan serum antibodies against ganglioside gm 1 and/or g d l b frequently are detected in autoimmune neuropathies such as rnultifocal motor neuropathy, igm paraproteinemic neuropathy, and guillain-barre syndrome. some of them bind to gm1 or g d l b monospecifically but the others cross-react with both of the antigens. to investigate respective localizations of gm1 and g d l b antigens in the human peripheral nervous system (pns), immunohistochemical study of dorsal root ganglia (drg), dorsal roots, ventral roots, and sympathetic ganglia (sg), which were obtained from human autopsy specimens, was performed by using 2 mouse monoclonal antibodies, each monospecific to gm1 and gdlb. ggr12, monospecific to gd1 b, immunostained nerve cell somas and axons of drg, sg, and dorsal and ventral roots. ggrl2 also recognized some myelin, including paranodal areas. however, gmb16, monospecific to gm1, did not bind to either neuron or myelin. thus, serum anti-gdlb antibody can bind to neurons and some myelin in the human pns. further study is necessary to identify the localization of gm 1 antigen, because previous biochemical studies have shown that gm1 is also present in the human pns. the purpose of this study was to compare t4 and t8 lymphocytes in migraine patients versus controls, and a review of the literature was done. fifty-six migraine patients, aged 18 to 53, were tested, as were 19 controls. the t4 :t8 ratio in migraine patients was 2 : 1 1, compared to 1 : 60 for controls. suppressors (t8) were reduced from 918 in controls to 482 in migraine patients. helpers (t4) decreased from 1,436 in controls to 985 in migraine patients, and total ?' lymphocytes decreased from 2,513 to 1,698. previous studies have revealed similar differences in t lymphocytes, with higher t4 : t8 ratios being found in both migraine and tension-type headache patients. in food-induced migraine, an increase in circulating immune complexes was noted, with increased t4 levels. differences in serotonin binding to mononuclear cells have been noted in migraine patients. a decreased sensitivity of the lymphocyte beta-adrenergic receptor in migraine patients was suggested by one study. after lymphocyte incubation with il-2, the natural cytotoxic response is augmented in cluster patients. the percentage of lymphocytes expressing receptors for il-2 was decreased in cluster patients in a fur-review of the literature ther study. this il-2 receptor defect was independent of whether the clusters were present. there is a loss of highaffinity binding sites for serotonin on lymphocytes in both episodic tension and chronic tension headaches. we used positron emission tomography (pet) to measure local cerebral blood flow in 6 volunteer subjects while they performed tasks of memory-guided saccades and a visual fixation control. tasks were performed continuously for 70 seconds during emission scans, after bolus injection of h2lso. eye movements were verified with electrooculography. areas of significant increase in regional blood flow between tasks were matched to three-dimensional reconstructions of brain magnetic resonance images of each subject. compared to the visual fixation control, saccade tasks evoked bilateral activation in the posterior superior parietal lobule with extension into the inferior parietal lobule. activation was also seen bilaterally in an area of frontal cortex immediately rostra1 to the precentral gyrus extending from the superior frontal gyrus to the inferior frontal sulcus. the increased blood flow in the posterior parietal cortex most likely corresponded to enhancement of visual attention required during saccades, while that in the frontal lobe, which included the frontal eye fields, indicated activation for saccade motor output. pamela blake, alexander s. mark, martin kolsky, and jorge kattah, washington, dc fifty patients with third cranial nerve (cn) palsy underwent precontrast and postcontrast mri to assess the utility of this study in this clinical context. mri demonstrated an appropriate lesion in 32 cases. six patients had brainstem lesions (2 infarcts, 1 mass lesion, 1 cryptic vascular malformation, 1 hemorrhagic shearing injury, 1 with compound q toxicity). lesions of the cisternal segment of the nerve were present in 12 patients (4 aneurysms, 4 lymphomas, 1 ophthalmoplegic migraine, 1 viral meningitis, 1 coccidiodomycosis, 1 nerve avulsion), with enhancement of this segment in 7 patients. fourteen patients had cavernous sinus lesions (2 lymphomas, 2 nasopharyngeal carcinoma, 4 tolosa-hunt syndrome, 2 cavernous carotid aneurysms, 3 pituitary apoplexy, 1 aspergillosis). eighteen patients, all with history of diabetes or vascular disease, had normal mri results, suggesting microvascular infarction of c n 111. in patients with cn i11 palsy, mri can detect the presence of brainstem or cavernous sinus lesions and often can suggest their cause. mri with contrast enhancement can demonstrate involvement of the cisternal segment of cn i11 in patients with inflammatory or infiltrative processes that previously could not be radiographically demonstrated. our study suggests microvascular infarction does not cause nerve enhancement on contrast-enhanced mri. we describe 2 patients with acute hyperglycemic, hyperosmolal, nonketotic stupor who had ocular flutter or opsoclonus clinically. these are the fourth and fifth adult patients reported with the acute onset of stupor and opsoclonus; all patients had nonketotic hyperglycemia and hyperosmolality (rapid eye movement sleep also causes stupor and saccadic eye movements). three of the 5 patients had myoclonic jerks in addition to opsoclonus. in the 5, opsoclonus began when glucose and osmolality acutely increased, and completely resolved when glucose and osmolality became normal, showing that opsoclonus is a specific and reversible effect of the metabolic disorder and implying that either acute hyperglycemia or hyperosmolality directly causes opsoclonus. since acute hyperosmolality caused by nacl or sucrose can cause a similar syndrome in experimental animals (trans am neurol assoc 1962; 87:33-36) and infants, hyperosmolality is probably more important. opsoclonus is thought to be due to abnormal activity of saccadic "burst" cells in the pons. acute hyperosmolality may cause spontaneous saccades by disinhibiting burst cells from normal "pause" cell inhibition or by directly activating burst cells. the combination of acute deterioration in mental status and either ocular flutter or opsoclonus should suggest acute hyperosmolality, in particular, nonketotic hyperglycemia. neural cell adhesion molecule (n-cam) and the related cell adhesion molecule l1 play significant roles in axon outgrowth and mediation of cell-cell contact in development, and after peripheral nervous system injury. to understand the role of these molecules after injury in the adult cns, we studied alterations in n-cam and l1 in the rat brain's response to entorhinal cortex (erc) lesion. post lesion, reactive synaptogenesis and axonal sprouting follow a well-defined temporal course in restoring the synaptic density of the dederented outer two-thirds of the hippocampal dentate gyrus molecular layer (ml) to near prelesion levels. we found striking regionand lamina-specific staining of n-cam and l1 in the normal hippocampus and marked alterations in these molecules after injury. embryonic n-cam, present in high amounts during development but expressed at very low levels in the adult hippocampus, was massively re-expressed in the denervated zone; the embryonic form was still heavily expressed 60 days later, when synapse number returned to >so% of prelesion levels. l1 staining, normally evenly distributed through the ml of the dentate, was completely lost in the outer ml, which is denervated by the erc lesion. this staining had not returned by 60 days after lesion. neural cell adhesion molecules play a role in specificity of neural connectivity both in development and after injury. in reactive synaptogenesis and axonal sprouting after injury, both ontogenetic (the reexpression of embryonic epitopes) as well as uniquely adult sequences of repair are utilized. following hemispherectom y" a. pascual-leone, h . t. chugani, l. g. cohen, j . p . brasil-neto, e. m . wassermann, j . and m. hallett, betbesh, md, and los angeles, ca we studied 7 subjects, aged 18 months to 32 years, who underwent hemispherectomy 6 months to 25 years earlier for intractable epilepsy. all had a spastic hemiparesis contralateral to the resected hemisphere, which was present presurgically. we used focal transcranial magnetic stimulation to map the areas of the preserved hemisphere targeting the abductor pollicis brevis (apb), the biceps, and the deltoid ipsilaterally and contralaterally. in 2 subjects who had hemispherectomy after age 10, the same area targeted ipsilateral and contralateral muscles. in the remaining 5 subjects, who were more functional, 2 areas targeted ipsilateral muscles. one area coincided with the contralateral representation, but stimulation induced motor-evoked potentials (meps) of lower amplitude and longer latency in the ipsilateral muscles. the other area, 2 to 4 cm anterolaterally, targeted exclusively ipsilateral muscles and stimulation induced meps of normal amplitude and latency. this separate ipsilateral representation was more distinct in subjects studied a long time after the hemispherectomy and in those who were younger at the time of the operation. these results show evidence of motor reorganization after hemispherectomy. better motor function is associated with topographically differentiated ipsilat-era1 and contralateral representations, which may depend on age at the time of hemispherectomy and the time since then. cynthia l. comella, glenn t . stebbins, nancy brown-toms, and christopher g. goetz, chicago, il in a single-blind, crossover study, we evaluated the effect of an intensive outpatient physical rehabilitation program (re-hab) on the severity of parkinson's disease (pd). the re-hab program consisted of 3 2-hour sessions per week for 4 weeks. sixteen patients completed 2 phases, a rehab phase and a control phase, separated by 6 months. the order of participation in each phase was randomized. all patients were evaluated using the unified pd rating scale with subscales for mentation (ment), activities of daily living (adl), and motor function (mot) by an investigator blinded to the rehab phase of the patient. all patients were evaluated immediately before and after each phase. pd medications were not changed during any phase. following the re-hab phase, there was significant improvement in adl score (pre-rehab 12, post-rehab 8, p = .005 wilcoxon) and in mot score (pre-rehab 26, post-rehab 2 1, p = ,008 wilcoxon), but no change in ment score. after the con-trol phase, there was no significant change in any outcome measure. this is the first controlled, crossover study of an intensive rehab program in pd. it demonstrates that re-hab improves objective motor function and adl scores. sensitive and quantitative measurements of leg weakness, one of the most common deficits in multiple sclerosis (ms) patients, can be made using specialized extremity testing equipment. to determine whether such determinations could be used in clinical trials, 6 ms patients with leg weakness that had been stable for at least 2 months were evaluated every 60 days over a 4-month period. each testing session was carried out at the same time of day and medication dosages and schedules were kept constant (only 1 patient was taking baclofen and his dosing remained constant). quadriceps and hamstrings strengths were measured in isometric contraction in a mechanical testing apparatus (kincom). variability for each series of determinations was expressed as the standard deviation of the mean as a percent of the mean. the overall variability then was expressed as the mean of the individual variabilities. the mean variability for the strength determinations over 4 months was 5.74 k 5.3896, and only one series of determinations out of 28 had greater than 20% variability. these results suggest that quantitative strength determinations might be useful in clinical trials, and early experience in trials of aminopyridines will be discussed. polymyositis (pm) is an inflammatory myopathy of unknown cause, but the accumulating data strongly suggest an autoim-mune pathogenesis. the histological picture is of muscle fiber necrosis and inflammation, whereas in postviral fatigue syndrome (pfs), a disorder characterized by severe fatigue with myalgia and psychiatric symptoms, the histological picture of muscle is essentially normal. enteroviruses have been implicated on epidemiological and serological studies in both. we have used the polymerase chain reaction (pcr) and an enteroviral-specific probe and found persistent enteroviral genomic material in both pm and pfs muscle biopsy specimens. furthermore, we used a radiolabeled full-length cdna probe derived from coxsackie b1 in an in situ technique to look for viral d n a in pcr-positive cases. coxsackie genome was clearly identifiable in the muscle biopsy specimens of patients with pm but negative in pcr enteroviral-positive cases of pfs. the virus excites an inflammatory reaction only in pm. a murine animal model for pfs developed in our laboratory showed positive muscle pcr using enterovirus probes and a conspicuous increase in interleukin 6 within the brain. these results provide major clues in the search for the etiology of these two puzzling disorders. human t-lymphotropic virus type i (htlv-i) is a cause of adult t-cell leukemia and tropical spastic paraparesis. in a specific population of iranian jews originating from the city of mashad, there is a high incidence of htlv-i infection (1 1.5%) and associated t-cell leukemia. we evaluated the incidence of possible correlation between htlv-i infection and spastic paraparesis in israeli mashadi-born jews. we have examined 41 mashadi-born immigrants in a mashadi community center (16 men, 25 women, mean age 66 t_ 13.7 yr) and 40 non-mashadi iranian-born jews. blood samples were tested for htlv-i antibodies by particle agglutination test. the polymerase chain reaction (pcr) was used to amplify htlv-i sequences of d n a from peripheral blood mononuclear cells. twelve mashadi-born immigrants (295%) were seropositive for htlv-i. in 10 of those serologically positive for htlv-i (83%), neurological examination revealed spastic paraparesis of varying severity. none of the non-mashadi iranian jews were seropositive for htlv-i or had clinical signs of spastic paraparesis. these results support other studies of htlv-i-associated myelopathy. the high incidence of htlv-i-associated spastic paraparesis in the mashadi community might be related to their unique history of a high rate of intermarriage among members of this ethnically segregated group. further epidemiological studies are underway to evaluate the incidence of htlv-i in mashadi families as well as in mashadi-originating jews born in israel, to identify whether infection might be genetically transmitted. lymphotropic virus t y p e i-associated acute t-cell leukemia/lymphoma william j . harrington, jr, william a. sheremata, susan snodgrass, and mark raven, miami, fl human t-cell lymphotropic virus type i (htlv-i)-associated acute t-cell leukernia/lymphoma (atl) is thought to produce important c n s disease infrequently. we wish to correct this impression by presenting neurological findings in 15 patients seen between 1989 and the present. all had positive western blots to htlv with polymerase chain reaction confirmation of htlv-i infection. only 2 had a concomitant human immunodeficiency virus infection. all were black, aged 31 to 86 years, 7 were men and 8 women. all but 3 americans came from the caribbean nations of haiti (51, dominican republic (l), jamaica (6), and trinidad (1). sexual transmission was the risk factor for htlv-i for all except for 1 intravenous drug user. all patients were systemically ill. three had preceding neurological abnormality (1, 8 months, and 14 yr) and 8 had major neurological disease concomitantly. two of these had tumor masses demonstrated in brain and 1 in the spinal canal. one had a minor facial sensory abnormality; only 3 had no deficits. we conclude that cns disease is commonly associated with atl, and that in the us atl occurs principally in caribbean natives. a. nath, v . hartloper, and m . furer, winni$eg, manitoba, canada microglia and astrocyte cultures were established from human fetal brain. microglia were infected with human immunodeficiency virus (hiv) strains, hiv,, or hiv,,,, resulting in a rising titer of p24 antigen in the supernatants. multinucleated giant-cell formation, vacuolar changes, and rising levels of lactate dehydrogenase in the supernatants were seen, indicating a cytopathic infection of microglia. astrocytes were infected with free virus or cocultivated with an hiv-infected lymphocyte cell line (hut-78). after a productive phase (rising titers of p24 antigen and detection of hiv antigens by immunocytochemistry), the cells went into a latent phase where hiv could be detected only by dna polymerase chain reaction. a 10-fold increase in astrocytes staining for hiv antigens was seen after cocultivation with hut-78 cells. lymphocytes adhered to astrocytes by 3 hours of cocultivation. no adhesion was seen to microglia. fusion of plasma membranes was seen on electron microscopy. infected astrocytes did not show cytopathic or morphological changes. cell-to-cell contact may be important in viral transmission to astrocytes. hszao-huei chen, steven b. stein, wing kong, and raymond p. roos, chicago, i l one of our goals is tq delineate molecular determinants for disease phenotypes produced by theiler's virus (tv), a mouse picornavirus. the identification of these genes and gene products may clarify viral pathogenesis and also lead to the identification of genes that are important in normal cns function and nonviral cns disease. members of the gdvii subgroup of tv cause an acute, fatal neuronal infection, whereas members of the to subgroup are less neurovirulent and produce a demyelinating persistent infection. our studies of infectious tv cdna clones have demonstrated that the gdvii 1 b(vp2)-2c segment is critical for neurovirulence. several other areas of the genome, including the 5' untranslated region (s'utr), also affect tmev-induced disease. the 5'utr of poliovirus, another picornavirus, has a critical role in paralysis; this effect on neurovirulence is believed to result from an altered translational efficiency related to bind-region ing of neural cell proteins. tv 5'utr has an unusual predicted secondary structure, even for picornaviruses. our studies demonstrate that the tv 5'utr affects translational efficiency and has a distinctive protein-binding pattern. investigations of the tv 5'utr may clarify features of translational regulation of cns genes in general. p129. coronaviruses infect primate brain from g a y f. cabirac, ronald s. murray, galen cai, kristen hoel, and kenneth soike, englewood, co, and covington, la recently, we described finding coronavirus (cv) rna and antigen in active demyelinating plaques of multiple sclerosis (ms) brain tissue (murray et al, ann neurol, in press). molecular analysis showed the cv rna to be more closely related to murine cvs than to human cvs. we then demonstrated that, following intracerebral inoculation, the murine cv jhm and the putative ms isolate cv-sd could infect and cause demyelination in primate brain (murray et al, virology, in press). we now have data showing that murine cv can infect primate brain following intranasal or intravenous routes of inoculation. standard virology, histopathology, and the molecular analysis of viral cytotropism will be presented. we conclude that cvs related to murine cvs can infect primate cns from peripheral routes and warrant consideration as potential human pathogens. probes (gag, pol, or enw). eleven were white and 9 were black. a history of transfusion was obtained in 8, and sexual risk of transmission was present in 8 but not in 4 others. fulminant disease occurred in 4 transfused men 3 months to 4 years later but such disease was only seen in 1 woman (in 1 month). in contrast, 2 of 3 women with multiple sexual partners had rapid progressive disease. the male-to-female ratio was 4 : 4 for transfusion association and 5:3 for those at sexual risk but 0:4 for unknown risk, transfusion is an important risk for tsp/ham and the diagnosis must be considered in all gait problems, regardless of the "diagnosis."transfusion association should decrease with regular testing of blood donors, but this will not affect the risk of sexual transmission. spastic paraparedhtlv-i-associated myelopathy (tsp/ ham). it is unclear why htlv-i infection causes atl in some individuals and tsplham in others. differences in the genome of viral isolates or immunological and host factors have been hypothesized to play a role in disease expression. at present there are no animal models of tsplham and no suitable animal models of htlv-i infection that can address these issues. we transplanted severe combined immunodeficient (scid) mice with peripheral blood mononuclear cells (pbm) from tsplham patients in an attempt to produce htlv-i disease. two weeks after transplantation of pbm from 2 tsplham patients, we detected anti-htlv-i igg in serum samples of 7 of 8 scid mice by enzyme immunoassay using sonicated whole virus. five weeks after transplantation, we detected anti-htlv-i igg to p19, p24, gp46, or gp61168 in serum samples of 5 of 7 scid mice by immunoblot of disrupted virus. these findings suggest that scid mice may be valuable in the study of molecular and pathological determinants of htlv-i-induced disease. theiler's virus produces an encephalomyelitis in susceptible mice. during the course of the disease, specific regions in the cns become infected. compared to the immunocompetent mouse, the nude mouse provides a useful model where viral dissemination can be studied in the absence of functional t lymphocytes and antibodies. we investigated the distribution and spread of the da strain of theiler's virus in the cns of nude mice. by immunohistochemistry, the hippocampus, arnygdaloid nuclei, entorhinal cortex, cingulate cortex, thalamus (anteroventral nuclei), midbrain, and spinal cord all contained viral antigens by 2 weeks after infection. in addition, the olfactory nuclei, mamillary body, hypothalamus, basal ganglia, and nucleus raphe dorsalis often were involved. in the brain, the limbic system was the site commonly infected by theiler's virus. the time course of virus dissemination varied depending on the site of initial virus infection, though the final distribution of virus was the same. olfactory bulb injection, which is a direct inoculation into the olfactory pathway, resulted in more rapid spread than did cortex injection. we demonstrated the constant presence of viral antigen in the limbic system and a different kinetics of viral dissemination between the two different routes of intracerebral inoculations. these results suggest that limbic structures and their connections are important to the dissemination of theiler's virus. 1-associated myelopathy in a northwest native indian d. foti, d. werke, g. dekaban, g. p. a . rice, andj. oger, vancouver, bc, and london, ontario, canada a 59-year-old indian of the oweekeno tribe was admitted for investigation of a myelopathy. initially symptomatic with midthoracic radicular pain, she developed progressive spastic paraparesis over 1 year with mild sensory symptoms and urinary retention. mri of the cord and brain were normal. csf showed elevated protein (930 mgll), 13 lymphocytes, and weak oligoclonal banding with evidence of intrathecal igg synthesis. antibodies to human t-lymphotropic virus type i (htlv-i) were positive by enzyme-linked immunosorbent assay and western blot on both serum and csf. presence of htlv-i was demonstrated by polymerase chain reaction of blood lymphocytes. htlv-i-associated myelopathy, or tropical spastic paraparesis, is endemic in southern japan, the caribbean basin, and several tropical islands but has not been reported in natives of northwest canada. this patient's only risk factors for htlv-i infection were 3 blood transfusions 30 years previously. ongoing familial and epidemiological studies as well as virus sequencing should indicate if this case represents an indigenous or an imported infection. caused by herpes simplex virus type 1 lawy blankensbz) and herbert 0. newton, columbus, oh myeloradiculitis occasionally occurs secondary to herpes simplex virus type 2 (hsv2) infection, but rarely has been reported after herpes simplex virus type 1 (hsv1) infection without encephalitis. we describe a 30-year-old man who developed cervical myelopathy and radiculitis, never developed symptoms of encephalitis, and had positive hsvl spinal fluid cultures. h e initially developed extremity weakness and incoordination, numbness, paresthesias, and neck pain. evaluation was negative except for mri results, which showed a high-signal lesion centrally within the cervical cord. the weakness, sensory loss, and radicular pain progressed over several months. subsequent mri showed extension of the high-signal abnormality and mild enlargement of the cervical cord. symptoms stabilized briefly with dexamethasone but soon worsened, and were accompanied by paroxysmal kinesiogenic dystonic episodes of his arms and right leg. repeat evaluation was unrevealing except for the spinal fluid, which grew out hsv1. the patient was treated with dilantin and a i-month course of intravenous acyclovir, with slow improvement of neurological status and resolution of the dystonic episodes. this case illustrates that hsvl can cause a myelopathy with a subacute and protracted course, requiring serial was more frequent in the middle-aged group ( p < 0.0001, p = 0.001). history of hypertension, previous strokes, diabetes mellitus, and antithrombotic treatment was similar, as were sex ratio, qualifying event type (transient ischemic attack or nondisabling stroke), and angiographic features; stenosis severity in either side and presence of ulceration were all similar. in elderly patients, ecad is associated with symptomatic cardiac disease (scad or af), whereas in middleaged patients it is associated with precursors of generalized atherosclerosis (smoking and hyperlipidemia). to identify the anatomic factors correlating with dementia in patients with lacunar infarctions, we examined digitized ct data on 58 elderly patients (mean age = 71.6 yr; education = 7 yr) who presented with acute lacunar infarction. dementia was diagnosed in 13 patients (22.4%) based on neuropsychological tests given 3 months after stroke onset. the following ct variables were assessed: infarct location and number, total infarct volume, brain parenchymal and csf areas at 3 levels, and width of the frontal horn, third ventricle (tvw), and lateral ventricle plus their ratio to the intracranial width. atrophy and leukoaraiosis were rated semiquantitatively using a standard scoring method. in the group overall, mean infarct volume was 0.64 cc and mean infarct number was 3.8. in univariate analyses, dementia was significantly related to infarct volume, number, tvw, bilaterality, and infarct predominance on the left side, but not to leukoaraiosis or atrophy. in a regression model adjusting for demographic factors, the ct variables correlating independently with dementia status were infarct number (p = 0.37, p = .01) and the presence of left cerebral infarcts (p = 14.17, p = .002). we conclude that the most important ct variables related to dementia in lacunar stroke are lesion multiplicity and the presence of lesions on the left side. brain ischemia results in potassium (k+)-induced voltageregulated presynaptic calcium (ca") accumulation, which may contribute directly to neuronal in jury presynaptically, and also promote excessive release of excitatory neurotransmitters leading to cell damage postsynaptically. k+-induced depolarization of brain synaptosomes may be used as an in vitro model to study the therapeutic potential of pharmacological agents to alter ischemia-induced presynaptic ca2 + accumulation. we preincubated gerbil cerebral cortical synaptosomes in r56865 (janssen pharmaceutical) at concentrations of to lo-' m, subsequently depolarized the synaptosomes with k + , at concentrations of 5 to 6 0 mm, and measured intrasynaptosomal ca2' ([ca2+]) with the fluorescent indicator fura 2. r56865 had no effect on ecaz'i in nondepolarized synaptosomes, but significantly (<.01, student t ) depressed depolarization-induced {ca2+] at to lo-' m in a dose-dependent fashion. the greater the degree of depolarization employed, the greater degree of depression in [ca2+] was seen at each dose of r56865. since r56865 had no effect on [ca2+] when utilizing ca2+-free incubation media, it was demonstrated that r56865 prevents depolaritation-induced [ca2 ' ] increase by blocking voltage-regulated influx. because r56865 appears to block voltage-regulated presynaptic ca2' accumulation, it should be further evaluated as a potential therapeutic agent after cerebral ischemia. sneddon's syndrome (ss) is a focal and diffuse arthropathy affecting mainly the vascular wall of the skin and cerebral arteries. etiology is not determined. we studied 37 patients with ss (27 females, 12 males), aged 15 to 56 years. clinical, radiological, and immunological studies were done. all patients developed cerebrovascular disorders: ischemic stroke in 82%, transient ischemic attack (tia) in 64%, and ischemic stroke and tia in 46%. cerebral scan showed small and medium (less than 3 cm) ischemic lesions in 77%. these lesions were superficial in the cerebral cortex (57%); deeper in the centrum semiovalis, internal capsule, and basal ganglia (18%); and both superficial and deeper (25%). ultrasound and angiogram studies revealed obstruction of intracranial arteries in 30%, and of extracranial arteries in 3%. partial or complete improvement of cerebrovascular symptoms was observed in 81%. immunological studies showed increased content of b-cell lymphocytes ( p < 0.05), increased levels of igm ( p < o.oool), and circulating immunocomplexes ( p < 0.0001). anticardiolipin antibodies were increased (58%) and lupus anticoagulant detected (5 1%). cerebrovascular disorders in ss that lead to small ischemic cortical lesions have a good prognosis. pathogenesis of this syndrome may be related to antiphospholipid antibodies. the cheiro-oral syndrome is characterized by pure sensory deficit limited to the hand and mouth. this syndrome has been described in diverse lesions of the parietal operculum and brainstem, but occurs most commonly due to lesions of the contralateral thalamus, and suggests a humuncular representation of sensation in the ventral posterior lateral (vpl) and ventral posterior medial (vpm) nuclei. we describe a 54-year-old hypertensive woman who presented with sudden onset of numbness of the left side of her body. examination revealed a left hemisensory deficit to all modalities that spared the hand and peri-oral regions. cat scan and mri demonstrated an acute infarction of the posterolateral right thalamus, with a rim of preservation adjacent to the internal capsule. pure hemisensory loss with sparing of the hand and mouth ("inverse cheiro-oral syndrome") has not been reported previously, and complements previously published studies of the cheiro-oral syndrome in demonstrating somatotopic sensory representation in the thalamus. to examine the relationship between depression and dementia after stroke, we administered the 17-item hamilton depression rating scale (hdrs) and neuropsychological tests to 237 elderly patients 3 months after ischemic stroke. using dsm-111-r criteria, we found dementia in 57 (24.1%). hdrs score was 5.0 -t 4.6 overall, and higher in demented compared to nondemented patients (6.2 2 5.0 vs 4.6 t 4.5, p = 0.025). the frequency of depression (total hdrs score >11) was also higher with dementia (17.5% vs 7.8%, p = 0.03). however, demented patients did not differ from nondemented patients on ratings of depressed mood. instead, hdrs items for psychomotor retardation, reduced work activities, and impaired insight best distinguished the 2 groups. in multiple regression analysis, stroke severity (p = 0.23, p = 0.0003) was the most important correlate of hdrs score; dementia status was not correlated independently. mean scores on mini-mental state examination and neuropsychological tests assessing memory, orientation, verbal, spatial, attentional, and abstract reasoning skills did not differ by depression status. although weakly related to intellectual impairment, hdrs score in our stroke sample was most importantly associated with stroke severity. higher scores on hdrs in demented stroke patients may be explained by physical and cognitive symptoms that are expected with dementia. these findings do not support a causal link between depression and dementia, and argue against the importance of "depressive pseudodementia" as an explanation for intellectual decline after stroke. to investigate the pathophysiological mechanism of the white matter lesions in progressive subcortical vascular encephalopathy (psve) of binswanger type, we measured regional water partition coefficient (pc) (reflecting water content) and effective p h (pht) (weighted average of intra-and extracellular ph) with dynamic positron emission tomographic technique using 0-15 co,, o,, and c-11 co,. si-multaneously, regional cerebral blood flow (rcbf) and regional cerebral metabolic rate of oxygen (rcmro,) were evaluated. eight subjects (3 normal, 3 psve, 2 multiple infarction) were examined. in the white matter lesions in psve, corresponding to high-intensity areas in t2-weighted images of mr, the pc increased and pht was unchanged or decreased, whereas both rcbf and rcmroz declined. the ratio of white matter pc to gray matter pc was 0.92 in psve and 0.87 in normals. in the frontal gray matter, the pc decreased in psve, whereas rcbf and rcmroz also decreased. these results suggest that tissue water content increases in the white matter lesions in psve reflecting the edematous status of the damaged regions. elevated pht with high pc may reflect the increase of extracellular water of the tissue. measurement of pc and pht provides useful information about the pathogenesis of psve. stroke has been the second most common cause of death in korea but its risk factors (rfs) have not been studied intensively, so the rfs or causes were investigated prospectively in 1,507 consecutive stroke patients who were admitted to the chungnam national university hospital, taejon, korea, between 1989 and 1991. they included 775 cases of cerebral ischemia (ci) (51.4%), 467 cases of intracerebral hemorrhage (ich) (31.0%), and 265 cases of subarachnoid hemorrhage (sah) (17.6%). control data were obtained from 209 healthy spouses of the patients. multivariate analyses showed that hypertension (ht) was the strongest rf for all stroke types and was followed by old age, diabetes mellitus (dm), smoking, high-density lipoprotein cholesterol, and fibrinogen. the rfs for atherothrombotic ci included old age, ht, dm, smoking, fibrinogen, and cholesterol. for lacunar infarcts, ht, dm, old age, fibrinogen, alcohol abuse, smoking, and female sex were the significant rfs. ht was the cause of ich in 347 (74.3%) and alcohol abuse (44, 9.4%) and vascular anomalies (32, 6.9%) followed. most alcoholassociated ichs occurred characteristically in the posterior fossa. frequencies of hospital admission of ich patients correlated positively with diurnal variation of temperature ( r = 0.5229, p < 0.001). aneurysmal rupture was the cause of sah in 222 patients (83.8%). three major sites of 247 aneurysms identified on cerebral angiograms were the anterior communicating artery (83, 33.6%), the middle cerebral artery (70, 28.3%), and the posterior communicating artery (56,22.6%). thirty-nine patients (14.7%) had no identifiable cause of sah. in korea curvilinear subinsular lesions have been noted on ct but the underlying pathology is unknown. we reviewed the cranial mr scans (1.5 tesla ge machine) of 49 serial patients over the age of 60 who had no cause for mr hyperintensities (hi) other than age or vascular risk factors. seven (14%) had linear subinsular h i (sihi). four of 7 (57%) patients with sihi and 4 of 42 (12%) without sihi had marked periventricular h i compatible with subcortical arteriosclerotic encephalopathy (pvhi) ( p < 0.05). patients with sihi were older (75.3 yr) than patients without sihi (69.1 yr) ( p < 0.05). four of 7 (57%) patients with sihi had hypertension program and abstracts, american neurological association 267 or vascular risk factors. a further patient with diabetes, hypertension, and the opercular syndrome (bilateral facial, pharyngeal, and lingual weakness) had subinsular lesions on ct. the brain arteries were injected postmortem with a lead/ gelatin suspension, and mr of the whole brain and x-ray films of the brain slices were taken. bilateral sihi were seen on mr and corresponded with linear cavitary infarction pathologically, which on microangiograms was found to lie in the border-zone between cortical and basal penetrating arteries. we conclude that sihi are associated with older age and pvhi, and can be due to infarction in a deep watershed territory and can be associated with clinical deficits. hemodilution-treatment results in 12 consecutive cases james l. frey, phoenix, az because precipitous neurological deterioration occurred during blood pressure reduction in a seminal case of lacunar infarction, 11 subsequent patients with partial or evolving lacunar deficits were treated with hemodilution and blood pressure nonintervention to test the hypothesis that lacunar strokes represent perfusion failure. isovolemic hemodilution was performed using hetastarch with target hematocrit of 30 to 33. results of pretreatment ct brain scans, carotid ultrasound, and echocardiograms were normal. nine patients recovered normal neurological function, and 2 regained complete functional independence in close temporal correlation with hemodilution. mri brain scans demonstrated appropriate single white matter lesions in 9 cases. no specific risk factor combination could be identified. n o patient has had recurrent stroke in follow-up from 12 to 30 months. response to hemodilution suggests a hemodynamic pathophysiology. successful treatment requires (1 } blood pressure nonintervention and (2) hemodilution prior to severe clinical deterioration. the hemodynamic classification for the carotid-cavernous sinus fistula (ccf) is important for the implication of prognosis and therapy, but satisfactory objective criteria for such differentiation is still lacking. retrospectively, we studied the application of extracranial duplex sonography in 9 cases of ccf with emphasis on the hemodynamic parameters of resistivity index and flow volume. a correlation was made with the angiographic findings in an attempt to evolve an objective hemodynamic classification by this noninvasive method. the alterations in membrane metabolism and structure could be the primary etiological event in alzheimer's disease (ad) that results in the clinical and neuropathological findings. to investigate in vivo brain membrane phospholipid and highenergy phosphate metabolism in probable ad patients and control subjects, the building blocks (pme) and breakdown products (pde) of membranes and the high-energy phosphates pcr and atp were measured noninvasively by in vivo brain 31p mrs in 11 probable ad patients ( 5 males; 6 females) and 18 controls (9 males; 9 females). all subjects were assessed by mini-mental, mattis, and blessed scales. we found that, at clinical onset, ad females had elevated pme ( p = 0.004), decreased pcr ( p = 0.001), and decreased atp ( p = 0.03). similar changes were not seen in ad males at clinical onset, but the severely demented ad males had increased atp ( p = 0.05). correlation analysis for the ad patients revealed that increasing dementia was associated with decreasing pme ( p = 0.05; r = 0.4), increasing pde ( p = 0.08; r = 0.4), increasing pcr ( p = 0.05; r = 0.4), and increasing atp ( p = 0.02; y = 0.5). similar metaboliccognitive correlations were not seen in the controls. these results demonstrate alterations in membrane and energy metabolism at the earliest clinical stages of ad. increasing dementia correlates with markers of membrane degeneration and decreased utilization of high-energy phosphates. both of these findings suggest the dementia in ad is secondary to membrane changes resulting in synapse loss. alzheimer's disease: postmortem mri and histological correlates fen-lei f. chang, j. e. purisi, c. r. jack+ jr, and r. c. petersen, rochester, mn hippocampal atrophy, defined by mri-derived volumetric measurement, has been useful in differentiating alzheimer's disease (ad) patients from normals. since several studies have demonstrated anatomical and functional gradients along the rostral-caudal (r-c) axis of the hippocampus, it is tempting to speculate on the existence of a differential distribution of morphometric changes along this axis. the hippocampi from 19 patients with clinically and pathologically confirmed ad were studied by postmortem mri and by reconstruction of serial histological sections. there was good correspondence between these two methods. the r-c length of the hippocampus in ad was preserved compared to normal. this length was longer on the left, both in normals and ad. the adassociated atrophy was due primarily to the reduction of the coronal cross-sectional area of the hippocampus. with increasing hippocampal atrophy, volume reduction was more prominent in the rostral area (pes hippocampus). this nonuniform reduction in volume may be associated with different connectivity patterns between rostral and caudal hippocampus. the purpose of this study was to determine the neuropathological validity of nincds-adrda criteria (nac) for probable and possible ad. mckhann et al (1985) provided clinical criteria for the categories probable ad and possible ad. the validity of these categories has not yet been reported. a retrospective, blinded evaluation of the complete neurological history, examination, neuroimaging, laboratory, and psychometric data was done for 68 subjects from the state of florida brain bank. pathological classification, which was blind to clinical diagnoses, was in 3 categories: pure ad, ad + , and other dementias. ninety-three percent of probable ad (n = 42), whereas only 75% of possible ad (n = 26) patients, had ad or a d + ( p = 0.1). pure ad was found in 62% of probable ad and 38% of possible ad patients ( p = 0.1). pathological evidence of coexisting parkinson's disease was present in 14% of all ad brains. these results suggest differential predictive power of nac for probable and possible ad, as suggested by the labels. nac are, therefore, usefui for research and clinical purposes. our prior study of falls in the elderly had shown significantly more white matter low attenuation (wmla) on ct scan among fallers than nonfallers, but no association between wmla and cognitive impairment among nondemented subjects. as these subjects were followed over time, it appeared that fallers were becoming demented at a more rapid rate than nonfallers. as a result, we analyzed the relationship between wmla and rate of change on the blessed test of information, memory, and concentration (bimc) in a combined cohort of 144 subjects from the falls study and from a longitudinal study of dementia and normal aging. we selected 61 of these 144 subjects whose initial bimc score was less than 25 (i.e., not already severely impaired) and who had at least 4 yearly bimc evaluations. ct scans were scored on an 8-point ordinal scale for hemispheric wmla. linear regression was used to summarize the rate of change for each subjects' test scores. the rate of change on bimc was 2.3 points per year with standard error (se) of 0.2 among subjects who eventually became demented; nondemented subjects declined at 0.4 points per year with se of 0.2. multiple regression analysis was performed with initial bimc score and wmla as the independent variables and rate of change of bimc as the dependent variable. wmla accounted for 16% of the variance (partial r = 0.396, t = 3.26, p < .05). these data suggest that elderly subjects with wmla may be at increased risk for rapid cognitive decline. such as g proteins and adenylate cyclase. the activities of adenylate cyclase, and of the g protein-associated enzyme activity, low-km glutamyl transpeptidase (gtpase), were assayed in membranes prepared from the postmortem brains of 8 alzheimer-diseased and 8 age-matched control subjects. both basal and fluoroaluminate-stimulated adenylate cyclase activities were significantly reduced in ad frontal cortex compared to control subjects ( p < 0.01; two-tailed student's t test). in addition, a significant, though smaller, reduction in basal gtpase activity also was detected in ad frontal cortex ( p < 0.05). in contrast, no significant change in the activity of either enzyme was detected in the hippocampus. the stimulation of gtpase activity by muscarinic and gababreceptor agonists was not altered significantly by the presence of alzheimer's disease. however, the degree of stimulation was much lower in human tissue compared to that observed using fresh rat brain, suggesting that the ability of receptors to activate g proteins declines post mortem. these results suggest that alzheimer's disease causes alterations in some key components involved in signal transduction. the association of lobar hemorrhage (lh) with cerebral amyloid angiopathy (caa) and that of caa with alzheimer's disease (ad) are well known. to determine how frequently lh and caa occur in ad, we reviewed 13 patients with cerebral or cerebellar hemorrhage and caa. five patients were treated surgically, none was demented, 2 died, and 1 came to autopsy. eight patients died of lh and came to autopsy. of the 9 autopsied patients, 3 had ad, both clinically and neuropathologically. they comprised 1.2% of 258 cases of autopsy-confirmed ad in our laboratory. none of the other patients were known to be demented. five had senile plaques, with or without neurofibrillary tangles, in the hippocampus, and occasional senile plaques in the cortex, but none met the consortium for establishing a registry for alzheimer's disease neuropathological criteria for ad. ad patients were 70, 85, and 85 years old and the ages of the 10 nondemented patients ranged from 63 to 87 (mean = 73.1 yr). seven were younger than 75 years of age. despite the frequent occurrence of caa in ad, we found that lh was uncommon. in addition, most of our patients with lh and caa were not demented and tended to be younger than ad patients with lh. these results indicate that the hf is involved primarily in acquisition or leaning processes and less so in retrieval of previously learned information. these findings relate to memory and structural brain changes found in normal aging. alzheimer's disease y . stern, l. stricks, g. alexander, 1. prohovnik, and there is an inverse relationship between parietotemporal cerebral blood flow and years of education in alzheimer's disease (ad) patients matched for clinical severity, which suggests delayed clinical manifestation of ad in patients with higher education (stern et al, soc neurosci abs 1771). we classified the lifetime primary occupations of 5 1 ad patients using the dictionary of occupational titles of the us department of labor and derived 6 factor scores describing intellectual, interpersonal, and physical job demands. after controlling for age, education, age at onset, illness duration, and dementia severity (mental status and activities of daily living), relative perfusion in the parietotemporal region (assessed using 133-xenon inhalation) showed significant correlations with job complexity (pl: r = -.36, p < .008) and interpersonal (p3: r = -.44, p < .001) factor scores. in a stepwise multiple regression, job complexity and interpersonal skills increased explained parietotemporal flow variance by 7.5% ( f = 4.7, p < .05) over that explained by demo-graphic and severity indices; physical demands then accounted for another 11.5% of the variance ( f = 8.4, p < .01). we conclude that occupational demands, similar to but independent of education, may provide a reserve that delays the clinical expression of ad. richard mayeux and ming-xin tang, new york, n y risk factors for alzheimer's disease (ad) were collected from 223 patients with ad and 278 healthy elderly controls in an urban community population consisting of 3 ethnic groups: black, hispanic, and white. advanced age (>80 yr) (or = 10.7; 75% ci 6.2-19.1) and head injury with loss of consciousness (or = 2.70; 1.2-7.3) were associated with ad, controlling for all known putative risk factors. factors such as low education (<8 yr) (or = 1.5; 0.8-2.6) and family history of ad (or = 1.7; 0.7-3.0) were not found to be significantly related to ad. head injury occurred in 15.7% of the patients and 6.?% of controls. most (70%) head injuries in the patients with ad occurred after age 50, prior to disease onset. in controls with head injury, 5592 had experienced a head injury before age 50. the duration of unconsciousness was consistently longer in patients with ad than in the controls. the overall effect in each ethnic group was similar (or,, 2.5; 1.3-4.8). these results confirm and strengthen the previously described putative relationship between head injury and ad. we also conclude that both the severity and the timing of the head injury as well as the frequency of head injury in the population at risk may be important factors in understanding the causal relationship between head injury and ad. the purpose of this study was to determine how native language affects the cutoff scores in screening tests for dementia. there is a paucity of data o n this issue at present. screening tests used in the study were: folstein mini-mental state (mms); clockdrawing (clock); preparing a letter for mailing (mail); 4-item grocery list (list); and hamilton depression scale (ham). the subjects included 175 (74 demented) native english speakers (eng) and 72 (22 demented) native spanish speakers (spa) with memory complaints. diagnosis of dementia was determined by neurological, neuropsychological, and psychiatric evaluation. age and gender were unrelated to mms. in spa only, education was positively related to mms. ham scores were higher in deand 0.77 (spa); c d and list did not add discriminative power to mms for eng but did so for list in spa. mms discriminates between demented and nondemented far better in english than in spanish speakers. only mail adds discriminative power to mms. a. heyman, g. fillenbaum, s. mirra, and participating cerad neuropathologists, durham, nc, and atlanta, ga the clinical diagnosis of alzheimer's disease (ad) has become more accurate in recent years due to the application of specific clinical criteria, wider use of neuroimaging procedures, and greater expertise among physicians. we report the frequency of clinical misdiagnosis of ad among 52 patients who at autopsy were found to meet the rigorous clinical diagnostic criteria imposed by the consortium to establish a registry for alzheimer's disease (cerad) study. these patients included 31 men and 21 women (mean ages 78 and 76 yr, respectively) who were among the group of 95 patients who died in 13 cerad medical centers in the us between 1987 and 1991. the clinical diagnosis of ad was neuropathologically confirmed in 47 (88.5%) of the 52 cases. of these 47 cases, varying degrees of concomitant cerebrovascular disease were present in 26% and coexisting parkinson's disease changes were found in 19%. in 4 of the 5 patients without neuropathological evidence of ad, the diagnoses were: lobar atrophy, diffuse lewy body disease, nonspecific neurodegenerative changes, and mesocorticolimbic dementia, respectively. the fifth patient showed no morphological abnormalities. on the basis of these results, it would appear that application of strict diagnostic criteria, as well as the use of brain scans and detailed clinical and neuropsychological tests by experienced clinicians, cannot yet distinguish some types of primary degenerative dementias from alzheimer's disease. we designed a scale that measures an underexplored facet of functional decline in alzheimer's disease (ad): the patient's dependence on others for supervising or performing activities. two hundred twenty-three informants for patients with mild ad (clinical dementia rating [cdr] = 1 for 200; cdr = 2 for 23) were interviewed and dependence was staged from 0 to 5. interrater reliability was assessed by separate interviews of 20 informants; agreement was 100% for dependence stage. dependence stage differed significantly at the 2 cdr levels (chi square = 41.0, p < .01) and correlated significantly with modified mini-mental state (mmms) ( r = -.30, p < .001) and blessed dementia rating scale-part 2 (bdrs) ( y = .41, p < .001). seventy-eight percent of patients in a health-related facility were at stage 3 or higher vs 25% of patients living at home. in a multiple regression model, both the bdrs and dependence scale accounted for unique portions of the variance in mmms, suggesting that they assess unique aspects of functional ability. dependence increased significantly in 118 patients retested at 1 year. we conclude that the dependence scale is reliable and relates to both disease severity and progression. formal assessment of dependence should prove useful for studies of the natural history of ad as well as for clinical trials. cortical-basal ganglionic degeneration (cbgd) is a disorder characterized by an asymmetrical akinetic-rigid syndrome and cortical signs such as apraxia, alien limb phenomena, and cortical sensory loss. dementia has been present in many cases, but always as a late manifestation. we report 2 cases pathologically consistent with cbgd, presenting as primary degenerative dementia, fulfilling nincds-adrda criteria for probable alzheimer's disease (ad). the first patient presented with changes in memory and personality, language dysfunction, decreased verbal output, and shuffling gait. follow-up examinations over 3 years showed progressive dementia, wide-based gait, and frequent falls. the second patient presented with complaints of memory loss. neuropsychological examinations showed progressive deficits in memory, attention, calculations, and visuospatial functioning. no movement disorder developed over 6 years of follow-up. at autopsy, both patients had typical changes of cbgd and lacked pathological features of ad. definitive diagnosis of cbgd rests on both clinical and pathological criteria. cbgd should be considered in the differential diagnosis of patients with dementia resembling that found in ad, especially if extrapyramidal signs are present. a quick, easily administered and scored test for praxis is desirable in evaluation of neurological patients. during 2 months in 1991, each patient seen in the outpatient setting by the principal investigator (e. k.) received 1 of 2 praxis screening batteries. thirty-six patients were tested with a short battery. eleven had normal cognition based on neurological history, examination, and a short test of mental status. twenty-five had cognitive decline (cd). handedness, male/ female ratio, education, and mean age were similar in both groups. mean time of completion of the test was 102 2 10 seconds in normals and 136 * 41 seconds in patients with cd. total scores (maximum 50) were 47.6 * 1.8 in the normals and 41.2 2 9 in the cognitively declined patients. twenty-five other patients, 12 with cd and 13 with normal cognition, were tested with a longer battery containing oral/ facial, upper and lower limb, axial, sequential, and imitation subtests (maximum score 110). normals completed the battery in 297 i 56, and cognitively declined patients completed the battery in 359 ? 53 seconds. of the various subtests, tests of sequential praxis were performed most poorly by patients with cd: 8.3 * 1.8 in normals vs 4.9 * 3.0 in patients with cd. oral/facial praxis was least affected by cd: olivopontocerebellar atrophy (opca) is generally understood to be a nondementing neurodegenerative disorder affecting the cerebellum, lower brainstem, and spinal cord. one of us (s. k.) recently reported that postmortem cerebral cortex from patients with dominantly inherited opca shows a widespread reduction of cholinergic markers similar to that observed in alzheimer's disease (ad). we were interested to determine the status of other neurotransmitter systems in opca postmortem cerebral cortex. samples of frontal, parietal, temporal, and occipital cortex were dissected from 10 confirmed cases of opca and 11 age-matched controls. after processing, neuropeptide levels were measured by radioimmunoassay. concentrations of somatostatin were significantly reduced by 42 to 58% in 3 of the 4 cortical areas of opca brain that were examined. the area that was spared was the inferior temporal gyrus, a region in which somatostatin levels are markedly reduced in ad. levels of neuropeptide y were normal in all 4 areas, while concentrations of cholecystokinin, vasoactive intestinal polypeptide, and substance p were significantly increased in 2 of the 4 areas. these data show widespread neuropeptide changes in the cerebral cortex of opca postmortem brain. in contrast to cholinergic markers, the pattern of neuropeptide changes is different from what is observed in ad. it has been postulated that demise of the corticomotoneuron is the initial event in amyotrophic lateral sclerosis (als) and that the anterior horn cell dies as the result of antegrade glutamatergic excitotoxicity (muscle nerve 1992;15:219). excitability of the corticomotoneuronal system can be tested by measuring threshold-to-cortical magnetic stimulation and the motor-evoked potential (mep)/compound muscle action potential (cmap) ratio, which estimates the number of corticornotoneurons stimulated. cortical threshold and mep/ cmap ratio were measured in 39 patients early in the course of als. the mean time interval from onset of first symptoms was 8.5 months. mean threshold and mepicmap ratio measured 64.2 f 16.6% and 22.0 rfr 21.2%, respectively. in 12 (30.8%) patients, threshold was paradoxically low (<55%, mean 49.1 +. 4.3%) and in 7 (17.9%) patients there was no response. there was a significant ( r z = 0.698) inverse power relationship between cortical threshold and mep/cmap ratio given by 84.1 x mep/cmap-'j. six months later, 7/24 (29.2%) patients still had low thresholds but the mean mep/ cmap ratio had dropped to 24.9 ? 24.5% and in 33.3% there was no response. we conclude that early in als the corticomotoneuronal pathways are abnormally excitable. this may explain early cramping and fasciculation, which characteristically diminishes as als progresses. one of the distinct clinical features in patients with amyotrophic lateral sclerosis (als) is loss of elasticity of skin. however, little is known concerning the biochemical nature of skin elastin in als. in our study, 2 cross-links unique to elastin, desmosine and isodesmosine, were measured and compared in skin tissue (left upper arm) from 10 patients with als and from 7 age-matched controls. the contents of desmosine and isodesmosine were decreased significantly ( p < 0.01 and p < 0.01, respectively) in patients with als (mean * sd, 0.94 ? 0.33 and 0.74 * 0.30 nmol/mg dry weight; range 0.36-1.51 and 0.27-1.33 nmol/mg dry weight, respectively) as compared with those of controls (mean 2 sd, 1.43 * 0.30and 1.17 2 0.21;range 1.11-2.03 and 0.92-1.52, respectively), and were negatively and significantly associated with duration of illness in patients with with amyotrophic lateral sclerosis als ( r = -0.77, p < 0.01, and r = -0.65, p < 0.05, respectively). the ratio of desmosine and isodesmosine was constant (1 : 3) in all samples analyzed. the decline in skin desmosine and isodesmosine is more rapid in als than in normal aging. thus, cross-linking of skin elastin is affected in als. (supported in part by n i h grants de 18522, de 11233, de 08611, ar 19969, ar 30587, and nasa grant nag-2-181.) pl69. natural history of amyotrophic (1) collection of large numbers of twin pairs in disease of low prevalence is difficult. to circumvent this, we devised a new approach termed the "death discordant twin pair" method. eleven thousand deaths from motor neuron disease (mnd) were extracted from the office of population censuses and surveys during 1979 to 1789. birth indexes from 1900 onward were searched for possible twins. for each twin so identified (13 1 pairs), the national health service central registry located the relevant family practitioner committee and thence the co-twin's general practitioner. the search produced: (1) 53 living co-twins; (2) 5 embarked; (3) 60 dying as adults or infants; (4) 3 not mnd; and (5) validity of the accuracy, sensitivity, and specificity of the world federation of neurology (wfn) subcommittee on motor neuron disease working group criteria for the clinical diagnosis of amyotrophic lateral sclerosis (als) has been tested against neuropathological criteria in 36 autopsied patients (neurology, in press). integration of clinical and electrodiagnostic data to meet wfn criteria for possible, probable, and definite als was studied in this samegroup. patients received 1.7 ifr 1.1 (mean * standard deviation) electromyograms (emgs) per patient and included 1.7 2 0.9 emg levels (bulbar, cervical, thoracic, lumbar) per patient. proportionately fewer emgs were performed as the level of diagnostic certainty at presentation increased: suspected (34.3%), possible (31.4%), probable (25.7%), definite (8.6%). in only 4.8% of all patients studied did the first emg alone change the level of diagnostic certainty of the diagnosis of als. only 16.7% of patients presenting with suspected als alone or with possible or probable als were associated with a change in level of diagnostic certainty following 1 or more emgs. however, although increasing the number of emgs performed per patient may be associated with an increasing chance of increasing the level of diagnostic certainty (22 emgdpatient = 33.3%; 2 3 emgdpatient = 60.0%), selection of the level of emg analysis was more crucial. the "el escorial" criteria emphasize the importance of emg evidence of lower motor neuron involvement in a limb with clinical upper motor neuron signs. our analysis of emg studies in autopsy-confirmed als patients suggests that complete evaluation of bulbar and thoracic levels for lower motor neuron changes and complete evaluation of motor unit recruitment patterns are important for the integration of emg data with clinical data in the application of the "el escorial" criteria for the diagnosis of possible, probable, and definite als. sibships on guam annette grefe, john steele, linda flares, and stephen waving, birmingham, al, and umatac and mangikao, guam reports in the 1950s indicated that 40% of guamanian chamorro patients with amyotrophic lateral sclerosis (als) gave october 20 a positive family history. subsequent investigators have inferred that purely genetic factors are not responsible for guamanian als or its clinical variant, parkinsonism-dementia complex (pdc). we report the first 4 chamorro sibships selected in an ongoing study of 17 1 familial aggregations. the basis for the initial selection was that the youngest patient in the sibship had als. age of onset was 30 to 46 years (mean 35 yr). fourteen of 23 persons in these sibships were affected. others in the sibship developed pdc, progressive supranuclear palsy, or pure dementia later in life (mean 61 yr, range 50-74 yr). these cases developed 14 to 47 years (mean 25 yr) after onset of disease in the first sibling. the age at onset of the first case and the intervals between earliest and latest cases in such sibships may help to determine minimal and maximal latency (i.e., interval between exposure to an exogenous agent and onset of symptoms). our observations suggest that exposure may have occurred early (before age 30) with varying and often long latency (up to 47 yr). we also find that variability of clinical expression may be correlated with the age at onset. concentrating on the study of familial cases on guam may enhance the identification of the etiologic agent(s) of this prevalent and tragic disorder. the spinocerebellar ataxias are an uncommon group of genetic disorders that have been well characterized in north america and europe. information concerning these conditions in africa and other parts is scant. to address this problem, a large-scale survey has been undertaken in the cape province of south africa. in this investigation, more than 500 persons in 18 affected families have been appraised and investigated and phenotypic features have been analyzed in detail. linkage studies have been undertaken in 4 families with similar phenotypes in which the condition was transmitted as an autosomal-dominant trait. human lymphocyte antigen (hla) typing was carried out on 79 members of the 4 families and linkage analysis was undertaken using the liped program to analyze the data with a correction factor for age of onset. maximum lod scores were: family a: 4.13 (0 = 0.005); family b: 0.6 (0 = 0.001); family c: 0.50 (0 = 0.30); family d: 0.0 (0 = 0.50). these results indicate linkage to hla in 1 out of 4 families. these findings provide support for the concept of genetic heterogeneity in these phenotypically homogeneous families. pcr typing with the reportedly more closely linked d6s89 locus is now being undertaken in these south african families. h.-p. hartung, g. f. hoffmann, and g. becker, wunburg, heidelberg, germany recently, l-2-hydroxyglutaric acidemia has been described as a novel metabolic disorder in 6 children. we report the occurrence of this disease in adults. one of 2 brothers developed at the age of 10 an abnormal gait and dysarthria; in the other 1, clumsiness and walking delay were noted at age 3. symptoms progressed and at the time of admission, when the patients were 33 and 39 years, neurological examination revealed a spastic ataxic gait, limb ataxia, dysmetria, dysarthria, dystonic posturing, and mental retardation. ct and mr imaging revealed subcortical white matter changes with loss of arcuate fibers, folial atrophy, and leakage in the cerebellar vermis, as well as atrophic changes in the cerebellar hemi-acidemia program and abstracts, american neurological association 273 spheres. on biochemical screening, highly elevated concentrations of l-2-hydroxyglutaric acid were found in csf, plasma, and urine. the pathological accumulation of l-2hydroxyglutaric acid in these 2 adults, along with the clinical picture characterized by cerebellar, extrapyramidal, and pyramidal symptoms and oligophrenia, and the neuroradiological findings of severe loss of myelinated arcuate fibers in subcortical white matter, conform with what previously has been described in the few neuropediatric cases. the biochemical abnormality underlying accumulation of this organic acid remains elusive. this study was undertaken to differentiate primarily affected areas from functionally suppressed areas due to remote effect in aphasic patients with focal brain degeneration, using an activation method with 0-15 water, in comparison with [sf]2-fluoro-2-deoxy-~-g~ucose (fdg) positron emission tomographic examination at rest. the subjects were 2 patients with slowly progressive aphasia showing contrasted clinical symptoms (nonfluent type vs fluent type). regional cerebral metabolic rate of glucose (cmrglu) was measured with intravenous injection of 130mbq of f-18 fdg at rest. regional cerebral blood flow (cbf) was measured with intravenous bolus injection of 1.5gbq of 0-15 water in 3 different conditions: at rest, under repetition tasks, and under naming tasks. changes of cbf were evaluated between a resting condition and task-performing conditions. regional cmrglu was decreased focally in both broca's and wernicke's areas similarly in these cases in spite of the difference in clinical symptoms, whereas the patterns of regional cbf changes were different. the fluent patient showed prominent activation in the bilateral frontal areas on repetition tasks. the nonfluent patient showed, whereas the fluent patient did not show, focal activation in the left occipitoparietal area on a naming task. evaluation with an activation method can provide detailed information about the pathophysiological process and the location of primary lesion. defective complex i activity has been linked to huntington's disease (hd) and parkinson's disease (pd). intrastriatal injection of inhibitors of complex i reproduces the pathological features of hd, and the neurotoxin mpp+ kills dopaminergic neurons by inhibiting complex i. defects in complex i have been reported in hd and pd, but the distribution of this enzyme in the brain is unknown. to map complex i in brain quantitatively, we developed an assay using e3h)dihydrorotenone to label the enzyme in tissue sections. this high-affinity binding is saturable and is displaceable by rote-in brain none and mpp+. using 100 pm rotenone to define nonspecific binding, more than 95% of binding is specific. highest levels of binding are found in kidney, followed by myocardium. moderate levels of complex i are seen in striated muscle and some brain regions. within the brain, binding varies more than 20-fold and is heaviest in the cerebellar molecular layer and dentate gyrus. lower levels of binding are found in cortex and striatum and very low levels are located in substantia nigra. this assay may help to clarify the role of complex i in neurodegenerative disorders. ( in 1988, 4 patients with medically intractable parkinson's disease underwent autologous adrenal medullary-to-caudate transplants at the university of california-los angeles (ucla). these persons had been followed for several years before operation at 3-or 4-month intervals. at each visit, their disability had been rated on the quantified ucla scale and the hoehn and yahr stage of disease. these provided a longitudinal assessment of the progression of disease in each patient, which could be compared to the rate of progression in the cohort of cases followed at ucla for 15 years. in addition, the unified parkinson's disease rating scale provided supplementary data for the immediate preoperative and subsequent postoperative evaluations. the hours "off" also were recorded for the preoperative and postoperative periods. after operation, the same evaluations were performed by the neurologist who previously had cared for the patients. the longitudinal postoperative data revealed that at 1 to 4 months after operation, all 4 patients improved. after 4 years, 3 continue to be less disabled than their preoperative baselines. the progression of their disease, while still evident, is nonetheless proceeding at a slower rate than before transplant. the fourth patient had brief improvement shortly after operation, but then rapidly worsened to his previous level. his disease has continued to progress at a rapid pace, unchanged from progression before operation. individuals at risk for huntington's disease h . p. h . kremer, w. shtybel, b. snow, c. clark, j . theilmann, m . r. hayden, and w. in huntington's disease (hd), caudate hypometabolism as demonstrated by positron emission tomography (pet) is a well-established feature in symptomatic patients. in individuals at risk, however, conflicting findings are reported. since 1984 we have performed 61 pet scans in 44 asymptomatic persons at risk for hd (age 27-76 yr) . linkage analysis with 9 independent dna probes or subsequent evolution to clinical hd (in 5 patients) allowed a risk estimate for 35 subjects. twenty were considered to be at increased risk (?85%), 14 at decreased risk (<15%), and in 1 individual no modification could be given. nine subjects were not tested. a pet scan was considered abnormal if either the caudate/thalamus ratio or the caudate/whole-brain ratio of rcmrglu was more than these criteria, 8 scans in 6 individuals were abnormal. none had received a decreased risk. comparison of the ratios of increased risk, decreased risk, unmodified risk, and control subjects failed to show statistically significant differences (analysis of variance). follow-up of 3 persons with an abnormal scan showed conversion to symptomatic status within 5 years after the first abnormal scan. this result suggests that an abnormal pet scan in a person at risk for h d heralds the onset of choreic movements. robitaille, m . el-awar, b. clark, l. scbut, m. ball, l. young, r. currier, and k. sbannak, toronto, ontario, and montreal, quebec, canada; pittsburgh, pa, minneapolis, mn, portkmd, or, and jackson, ms we measured the levels of dopamine in striatum of 14 patients with end-stage dominantly inherited olivopontocerebellar atrophy (opca). on average, dopamine levels were reduced in putamen ( -5396, as compared with controls), caudate ( -35%), and nucleus accumbens ( -31%). however, individual patient values showed a wide variation (normal to -9996), indicating that striatal dopamine loss is a common, but not constant feature of opca. seven patients had marked putamen dopamine loss (-62 to -8195) but without corresponding severe substantia nigra cell damage; this suggests a "dying-back'' phenomenon in which nerve terminal loss precedes cell-body degeneration. in this regard, opca may offer the possibility of examining nigrostriatal dopamine neuronal degeneration at an early stage. although 2 patients were found to have severe nigral cell loss with near total ( -95 to -99%) striatal dopamine loss, none had depression in parkinson's disease (pd) has been correlated with low cerebrospinal fluid (csf) levels of 5-hydroxyindoleacetic acid (5-hiaa). l-dopa may precipitate or exacerbate depression in 30% of pd patients. to determine if l-dopa affects 5-hydroxytryptamine (5ht) metabolism, 8 patients were studied. four had pd. of these, 2 were taking l-dopa and both were depressed. the diagnoses in the remaining 4 were progressive supranuclear palsy (psp), striatonigral degeneration (snd), normal pressure hydrocephalus, and pseudoseizures with depression. neuropsychological examinations and lps of all 8 patients were done. three patients were started on l-dopa (the 2 previously untreated pd patients and the psp patient) and retested 3 days later. one pd patient started on l-dopa became depressed. mood in the others was unchanged. csf was analyzed for 5ht and 5-hiaa. 5ht could not be detected in the csf of patients who were not taking l-dopa, but was easily detectable in all of the patients who were taking l-dopa. 5-hiaa levels were low in the untreated pd patients, and also in the patients with psp, snd, and pseudoseizures with depression. 5-hiaa levels were even lower in the l-dopa-treated patients. the ratio of 5-hiaa:5ht (an index of 5ht turnover) was lowest in the l-dopa-treated pd patients who were depressed. low csf 5-hiaa in untreated pd may reflect depletion of brain 5ht. l-dopa may induce depression by inhibiting 5ht turnover in 5ht-depleted brain. p18 1. ventroposterolateral medial pallidotomy in the e. fazzani, m. dogali, a. ben;, d. eidelberg, j. gianutsos, t. kay, b. newman, s. loftus, d. samehon, and l. laitinen, new york, n y , and stockholm, sweden in patients with parkinson's disease (pd), as a consequence of low dopamine there exists an increase in inhibitory output from the globus pallidus. ten patients (5 men and 5 women) with pd received unilateral (6 right, 4 left) ventroposterolateral medial globus pallidotomies (vplmp). the average patient age was 61 years (range 55-73 yr), and the average duration of disease was 14 years (range 6-24 yr). patients fluctuated between "on" chorea and "off" parkinsonism. hoehn and yahr stage "on" was i1 in 7 , 111 in 3; and "off" was i11 in 5, iv in 2, and v in 3 patients. unified pd rating scale (updrs) score averages 12 hours off medicines (12hom) were activities of daily living (adl): 20 and motor (mtr): 40 preoperatively (preop). capit score averages preop 12hom were pronation-supination (ps): 3 1 seconds (s), finger tap (ft): 31s, board (b): 39s for the most affected contralateral side, and gait: 57s (2 patients could not walk). re-examination was done 2 to 5 days after pallidotomy. updrs scores 12hom decreased an average of 60%. three patients had major bilateral improvement in bradykinesia. rest tremor, prominent in 3 patients, also was diminished. capit scores 12hom decreased to ps: 19s, ft: 20s, b: 22s; the average gait of the 8 patients who could walk preop improved to 36s. there were no side effects. vplmp leads to an immediate overall significant improvement in patients with pd. s. kish, y . there is a growing interest in the genetic aspects of parkinson's disease and other basal ganglia disorders. we have studied 3 families whose ancestors immigrated to north america from contiguous regions of northern germany and southern denmark. the pedigrees contain 77, 206, and 376 individuals spanning 6, 7, and 8 generations with 7,6, and 9 affected members, respectively. autosomal-dominant inheritance is clearly present in 2 families and probable in the third. typical levodopa-responsive parkinsonism with bradykinesia, rigidity, resting tremor, and impaired postural reflexes uniformly develops in affected individuals from all 3 families. n o downgaze impairment, pyramidal signs, sensory disturbances, cerebellar dysfunction, or orthostatic blood pressure changes have been observed. dementia, however, has developed in a few elderly individuals, especially in 1 family. laboratory studies are normal. mri shows moderately enlarged ventricles and cortical atrophy. 6-fd positron emission tomography demonstrated reduced striatal uptake in 1 examined patient and normal uptake in l individual at risk. autopsy of only 1 subject has been performed (in 1975). brain weight was 1,380 grams and there were no obvious gross abnormaliprogram and abstracts, american neurological association 275 tuesday, october 20 ties, but microscopic examination was limited. further research on these 3 families is planned. electrophysiological study s. maurri, m . cincotta, a. ragazzoni, g. descisciolo, and f. barontini, florence, ltah many neurophysiological examinations were conducted of a 32-year-old woman with familial mirror movements. n o other neurological abnormalities were detected. examination included voluntary electromyographic (emg) activity from various muscles, f-wave as well as short-and long-latency reflex responses of the thenar muscles from electrical stimulation of the median nerve, mapping of motor-evoked potentials (meps) to transcranial magnetic stimulation, movement-related cortical potentials (mrcps), and somatosensory-evoked potentials (seps). emg documented mirror activity in the upper limbs, most marked in the muscles of both hands. onset latency of emg activity in response to an auditory stimulus was identical in active and mirror muscle. long-latency responses from median nerve stimulation were recorded on contralateral as well as ipsilateral thenar muscles. short-latency reflexes and f wave were strictly ipsilateral. unilateral scalp magnetic stimulation evoked bilateral responses at similar latencies in the thenar muscles; midline scalp stimulation activated no responses. scalp distribution of median nerve seps and of mrcps associated with self-paced thumb abduction were normal. our findings suggest that congenital inherited mirror movements in otherwise normal subjects can be generated by corticospinal fibers pro jecting to ipsilateral motoneurons of the spinal cord. we have previously identified dysphagia and constipation (both slow transit and defecatory dysfunction types) as common gastrointestinal (gi) problems in parkinson's disease (pd). since apomorphine has been shown to be capable of terminating off-periods when injected subcutaneously, we have evaluated the effects of apomorphine injection on objective parameters of dysphagia and bowel dysfunction in pd patients. nine subjects underwent the following battery of studies to characterize their pd features, swallowing, and bowel function: gi assessment survey, unified pd rating scale, videoesophagram, colon transit study, defecography, and anorectal manometry. specific abnormalities on the studies were noted and the most abnormal study was repeated after subcutaneous administration of 6 mg apomorphine. all individuals were pretreated with domperidone 20 mg four times daily for 3 days prior to apomorphine administration. improvement in both esophageal motility and deglutition was noted in the 1 individual in whom videoesophagram was repeated. for the other 8 patients, defecography or anorectal manometry was performed. significant improvement in specific parameters was demonstrated after apomorphine administration, but 2 individuals experienced syncope during radiographic procedures. we conclude that subcutaneous apomorphine administration holds promise as a potential therapeutic approach to dysphagia and, especially, bowel dysfunction in pd, but that further investigation and refinement are necessary. asymmetrical effect of unilateral thalamotomy or subthalamotomy on tremor in parkinson's disease nico diedericb, christopber g. goetz, glenn t . stebbins, harold l. klawans, k. nittner, a. kozrlosakis, p. sanker, and v . strum, cologne, germany, and chicago, il in the past, stereotactic operation was a regular treatment for unilateral tremor in parkinson's disease (pd). however, follow-up studies were usually short term and always unblinded. we examined 17 pd patients in long-term follow-up (mean 10.9 yr after operation) who underwent unilateral thalamotomy for parkinsonian tremor. we used videotapes and the unified parkinson's disease rating scale to blindly compare tremor ipsilateral and contralateral to the side of operation. since the patients were specifically selected for stereotactic operation because of asymmetric tremor, we reasoned that a sign of long-term efficacy would be current postoperative reversal of tremor side predominance. upper extremity tremor was significantly better contralateral to the side of operation compared to the ipsilateral side ( z = 3.29; p < 0.023). for the lower extremities the difference was not statistically significant. in chronic follow-up, stereotactic operation improved the absolute magnitude of arm tremor or ameliorated its rate of progression. since asymmetric bradykinesia and dyskinesia were not prerequisites for the choice of surgical side, we cannot make any conclusion about longterm impact of operation on these features. subtle extrapyramidal signs resembles that of patients with parkinson's disease m . richards, k. marder, l. cote, y . stern, and r. mayeux, new york, n y to investigate the relationship between extrapyramidal signs (eps) and cognition, eps severity and neuropsychological function were assessed in 307 normal elderly individuals and 130 nondemented patients with idiopathic parkinson's disease (pd) from a community-dwelling cohort in new york city. multivariate analysis of variance (manova) indicated poorer neuropsychological performance ( p = .001) in pd patients on verbal memory, orientation, verbal fluency, visuomotor construction, and psychomotor speed, but not naming, abstract reasoning, or matching. controlling for eps severity abolished these differences. one hundred fourteen (37%) of the normal individuals had subtle eps (mostly postural abnormality, bradykinesia, or rigidity) but no identifiable neurological disorder. manova indicated poorer neuropsychological test performance ( j = ,001) in these individuals than in normals without eps on verbal memory, orientation, abstract reasoning, naming, verbal fluency, matching, and psychomotor speed but not visuomotor construction. we conclude that: (1) cognitive impairment in pd is specifically associated with eps, and (2) a similar association occurs in individuals with subtle eps but no neurological disorder. whether this represents a preclinical stage of pd or ad is yet to be determined. disease in olmsted county, minnesota emre kokmen, fatma sibel ozekmekci, c. mary beard, and peter c. o'brien, rochester, m n , and istanbd, turkey there have been many studies of prevalence of huntington's disease (hd) in diverse populations around the world. to study the incidence, we took advantage of the availability of detailed health care records for the population of olmsted county, mn, from mayo clinic, its affiliated hospitals, olmsted medical group, county hospital, state hospital, records of solo practitioners, nursing homes, death certificates, and autopsy records. we reviewed all records with a diagnosis of hd, huntington's chorea, chorea major, and chorea otherwise unidentified, and sought evidence for progressive chorea, progressive cognitive and/or behavioral dysfunction, and family history compatible with autosomal-dominant inheritance with onset of symptoms in the period between january 1, 1950, and december 31, 1989 , while the patient lived in the geographic boundaries of olmsted county. we found 3 males and 6 females who met these criteria. average annual incidence rate (age/sex adjusted to 1960 us white population) for hd for this 40-year period was 0.3 cases/ year/ 100,000 population. we also estimated prevalence by taking account of in-migration, out-migration, and deaths. the agelsex adjusted (1960) prevalence for 1-1-60 was 7.51 100,000 and for 1-1-90 it was 2.4/100,000. the small number of cases caused the instability of the prevalence rates, but our rates are similar to rates reported in other populations. alton e . btyant, 111, l. breeden hollis, john a. hamjian, john d. wooten, ill, and francis 0. walker, nc we described 4 patients with unusual episodic movement disorders and normal diagnostic work-ups: a 34-year-old woman who had recurrent episodes of tonic jaw deviation and forced right-eye closure; a 30-year-old woman who developed unexplained pain and subsequent spells of tonic inversion of the left leg; a 61-year-old man who presented with a bizarre episodic right-arm tremor; and a 15-year-old woman who experienced intermittent abdominal undulations. examination of the affected body part provoked or enhanced symptoms in all patients. using suggestion and placebo activation in the form of a medicated patch, intravenous saline, or cervical massage, we first induced and then aborted typical episodes of their abnormal movements. postinduction discussions of the procedure led to a marked reduction in the frequency of attacks in 2 patients. activation procedures are useful in diagnosing psychogenic disorders because they demonstrate that situational, not medical, factors govern the expression of the abnormal behavior. we speculate that patients who are refractory to simple suggestion may respond to induction because it offers the potential of validating their symptoms. as in the case of psychogenic respiratory distress or pseudoseizures, positive induction can assist in counseling and symptom control. had alzheimer's disease with parkinsonism (adp), 1 had essential tremor (et), 1 had cerebellar tremor in multiple sclerosis (ms), and 1 had tardive dyskinesia (td). clozapine was used either to treat psychosis (20 pd, 2 adp, 3 dys, 1 td) or tremor (1 5 pd, 1 et, 1 ms). two pd patients were retrospective analysis of 38 patients counted twice, 1 who was treated for psychosis and then tremor and 1 who was treated on 2 separate occasions for psychosis with different responses. all 3 dys patients improved, 2 with complete resolution of their dystonia on changing antipsychotic drugs. the patients with et (75 mg) and ms (25 mg) improved mildly but sedation and clumsiness caused drug discontinuation in the ms patient. one adp patient (112.5 mg) responded well and the other became sedated and confused (25 mg). the p d responses for psychosis at a dose range of 6.25 to 400 mg daily were good (5), very good (2), and excellent (8), whereas 4 were intolerant. pd tremor responses were good (51, very good (4), excellent (2), and poor (3) at doses of 12.5 to 100 mg daily. one patient died of unrelated causes shortly after initiation of the drug. adverse effects included sedation, weight gain, hypersalivation, fainting, clumsiness, transient granulocytopenia, and "spasms" necessitating discontinuation in 9 patients (7 pd, l td, and l ms). tourette's syndrome and attention-deficit disorder patients s. m. silverstein, p. g. coma, d. palumbo, l. west, and r. kurlan, rochester, n y impaired attention is a common comorbid behavioral feature of tourette's syndrome (ts) and a key clinical feature of attention-deficit hyperactivity disorder (adhd). however, the pattern of attentional impairments reported in adhd has not been observed in ts. we therefore compared 17 ts patients (9 male, 8 female; mean age 32 ? 11 yr), 17 adhd patients (8 male, 9 female; 36 * 12 yr), and 17 normal controls (8 male, 9 female; 3 1 ? 9 yr) on specific neuropsychological (np) and computer-administered tasks of attentional ability. adhd, but not ts, subjects performed significantly worse than controls on the n p tasks (digit symbol, perceptual speed) and had a trend toward poorer performance on a computerized measure of attention. however, both the adhd and ts groups had significantly greater test performance variability on some, but not all, tasks and had more subjects with deviant scores. among ts patients, higher scores on an obsessive-compulsive disorder (ocd) inventory and a greater number of adhd symptoms correlated significantly with poorer performance on the attentional tasks. moreover, ts patients with observed tics during testing had greater attentional impairment than those without tics. these results suggest that: (1) many adult ts patients do not have impaired attention; (2) attentional impairment in ts differs from that observed in adhd; and (3) attentional impairment in ts is associated with the full neurobehavioral spectrum of ts (i.e., tics, ocd, and adhd). frank r. sharp, cathleen miller, thomas rando, and steven greenberg, san francisco and palo alto, c a six patients are described with choreoathetoid movements and marked proprioceptive sensory loss. one patient had a traumatic injury to the right parietal cortex that produced severe proprioceptive sensory loss and choreoathetosis in the left arm. another patient had a left thalamic infarction that resulted in profound proprioceptive sensory loss and chorea on the right side of the body. two patients had cervical spinal cord disease, proprioceptive sensory loss, and diffuse choreoathetosis. another patient had dorsal root ganglionitis associ-a hypothesis program and abstracts, american neurological association 277 ated with small-cell lung carcinoma that produced diffuse loss of all sensory modalities and chorea. the last patient had an ulnar sensory neuropathy and choreic movements of the fifth finger. lesions anywhere along the pathway that transmits limb proprioception may cause pseudochoreoathetosis. furthermore, choreoathetosis without sensory loss caused by focal lesions of striatum may occur because of disruption of cortical proprioceptive inputs to striatum-perhaps explaining why most focal lesions of striatum do not produce chorea. sporadic inclusion-body myositis (s-ibm) and autosomalrecessive hereditary inclusion-bod y myositis (h-ibm) are of unknown cause and pathogenesis. in both there are muscle fibers with rimmed vacuoles containing 15to 21-nm cytoplasmic tubulofilaments (ctfs) and denervation atrophy; in s-ibm, but not h-ibm, there is a varying degree of inflammation. vacuolated fibers contain ubiquitinated inclusions (askanas 1991) and congo-red positivity indicating amyloid (mendell 199 1). because immunoreactive p-amyloid precursor protein (app) and p-amyloid protein (p-ap) are constituents of ubiquitinated senile plaques in alzheimer's disease (ad) brain, we studied immunolocalization of app and p-ap fibers in ibm muscle using antibodies against: (1) non-p-ap fragments of app, viz. (a) c-terminus (residue 676-695 courtesy d. selkoe) and (b) n-terminus (residue 45-62 courtesy b. frangione and d. levartovsky); (2) p-ap (sequence 8-17, courtesy g. glenner, and sequence 1-40 courtesy d. selkoe); (3) ub (chemicon). in 13 of 13 ibm patients, including one h-ibm, 100% of the vacuolated muscle fibers contained large or several small app and p-ap immunoreactive (ir) inclusions, which by double-labeling fluorescence were closely colocalized with each other and with ub-ir. none of 18 control muscle biopsy specimens (including 7 polymyositis) contained app-ir, p-ap-ir, or ub-ir inclusions characteristic of ibm. control experiments utilizing omitted, replaced, or absorbed primary antisera were negative. p-ap, a product of proteolytic cleavage of app, is receiving attention regarding the pathogenesis of ad. our study provides (1) the first demonstration of app and p-ap accumulations in abnormal human muscle, and (2) raises the possibility that in ibm muscle and ad brain rhey may form from similar cellular events. jeffrrey d. rothstein, lin jin, and ralph kuncl, baltimore, m d the pathogenesis of motor neuron death in amyotrophic lateral sclerosis (als) is unknown. accumulating evidence suggests that the disease is characterized neurochemically by a derangement in the control of neurotransmitter glutamate metabolism: csf levels of glutamate and aspartate are ele-of motor neuron degeneration vated and their high-affinity transporter is defective in brain and spinal cord. inefficient glutamate transport, and subsequent chronic increase in extracellular glutamate, could be responsible for selective motor neuron death. to test the hypothesis that chronic defects in glutamate uptake can produce motor neuron toxicity, we developed a tissue culture model employing organotypic rat spinal cord maintained under conditions of chronic glutamate uptake inhibition. slices (300 pm) of lumbar spinal cord from 8-to 14-day-old rat pups were cultured on millicell membranes. chronic uptake inhibition was produced by culturing tissue in the presence of threohydroxyaspartate (tha) or pyrrolidine-dicarboxylic acid, both known to be specific inhibitors of glutamate transport. tha produced chronic elevation of glutamate in the medium and produced motor neuron toxicity after 25 to 30 days in culture using 100 pm tha, and after 18 days using 500 pm tha, as determined by assay of tissue choline acetyltransferase (chat) activity and by histological analysis of 1-micron plastic sections. motor neuron toxicity was completely blocked by the non-n-methybaspartate (nmda) antagonists cnqx or nbqx, but not by the nmda antagonist mk-801. this model demonstrates that the chronic loss of glutamate transport in als can produce motor neuron degeneration and that motor neurons appear to be susceptible to non-nmda-mediated glutamate toxicity. guillain last year, we described a distinct acute paralytic syndrome in children and young adults from northern china and differentiated it from guillain-barre syndrome (gbs) by epidemiological, clinical, and nerve conduction (nc) features. to distinguish chinese paralytic syndrome (cps) from gbs more clearly, we measured nc in 21 cps patients (mean 8 yr, range 1.5-35 yr) and in 21 gbs patients from johns hopkins (mean 14 yr, range 5-24 yr). sensory nc was normal in all (n = 62) but 1 nerve of cps patients, whereas sensory nc was frequently abnormal in gbs patients: median nerve, 63%; ulnar nerve, 87%; and sural nerve, 21%. motor n c also differed between the groups. in all nerves, distal latency (dl) was significantly longer in gbs than in cps. for example, in the median nerve, mean dl was 8.6 ms (se 1) in gbs and 2.8 ms (0.2) in cps ( p < 0.005). motor conduction velocity was significantly reduced in gbs median and ulnar nerves compared with cps nerves. f-wave latency was significantly longer in gbs median nerves than in cps nerves. these data support the distinction both clinically and electrodiagnostically between cps and north american gbs. the use of n c may be especially important in field epidemiological studies in separating the 2 disorders. clinical manifestations and gene analysis of the first japanese kindred yoshihide sunada, teruo shimizu, lchiro kanazawa, and toru mannen, tokyo, japan familial amyloidotic polyneuropathy type iv (fap iv) has been clustered in the finnish population and only a few cases have been reported from the netherlands. denmark, and united states. we describe the first japanese family with fap iv. the family originates from nagano prefecture, a mountainous district in the middle part of japan, and has no relationship to the finnish population. this family has 42 members in 3 generations, and 14 individuals are affected with slowly progressive cranial neuropathy and corneal lattice dystrophy. the genetic trait is autosomal-dominant. polarizing microscopy and immunohistochemistry show abundant amyloid deposits reactive to an anti-gelsolin monoclonal antibody. direct sequence analysis of a dna fragment spanning codon 187 of the plasmagelsolin cdna from the propositus, and restriction analysis using a modified pcr from other family members demonstrate a single base substitution, g to a at the first base of codon 187, which is identical to the mutation of finnish fap iv. this suggests that the mutation causes the fap iv phenotype regardless of ethnic background. gene expression focal puncture injury has been used as a model to study degenerative and regenerative responses of skeletal muscle. previous studies have demonstrated the ultrastructural and metabolic effects of muscle injury. however, the early genomic response to focal injury is presently unknown. we asked whether the immediate early genes (iegs) or early response genes-~$268, c-jun, nur77, and junb-are responsive to muscle injury. these iegs encode transcription factors and are expressed rapidly after cell-surface stimulation. we have previously shown that surgical denervation and neural stimulation of muscle induced differential patterns of ieg expression. in this study, we produced injury of mouse gastrocnemius muscle by injection of 20 c1.1 of normal saline. we used the contralateral (uninjected) muscle as a control and examined the milna levels of each of these 4 iegs. we found that ~$ 2 6 8 and junb levels were increased at 1 and 4 hours and returned to basal levels by 24 hours. in contrast, mrna levels of nur77 and cjun remained unchanged. this pattern of ieg response is distinct from that seen after muscle stimulation or denervation. the selectivity of this pattern suggests that ieg expression may play a role in the response of muscle to injury. amyotrophic lateral sclerosis (als) is a degenerative disease that leads to the restricted loss of motor neurons (mn). the reason for the selective death of mn remains unknown. we hypothesize that mn-enriched or mn-specific genes are important for normal m n function and that their disturbance may play a role in the pathogenesis of als. we have produced clonal hybrid cells derived from embryonic and neonatal spinal cord m n for the study of m n gene properties. some of these hybrid mn clones express traits typical of mn, such as high levels of choline acetyltransferase enzyme activity and message, glycine receptor message, and neurofilament and neural cell adhesion molecule proteins. we are using molecular techniques to identify novel mn-enriched or mn-specific genes in these cells. with this strategy, we have identified several cdna clones preferentially expressed in mn hybrid cells but not in the parental neuroblastoma cells by differential hybridization of an embryonic mn hybrid cdna phage library. we are extending these observations by performing subtraction hybridization experiments. these results suggest that mn-enriched or mn-specific genes can be identified, and may lead to a greater understanding of the etiology of als. there is a syndrome of slowly progressive, mid-adult-onset fasciculating progressive muscular atrophy (pma) affecting upper more than lower limbs, without bulbar or corticospinal signs, more often in males, associated with igm monoclonal gammopathy, and no nerve conduction block. two such men, ages (a) 59 years and (b) 64 years, duration 11 and 3 and one-half years, csf protein 28 and 60, had failed to achieve sustained improvement with: prednisone, cyclophosphamide, total-body irradiation, and multiple lymphoplasmaphereses in a; and interferon alpha 2a in b. intravenous immunoglobulin (ivig), 0.4 gm/kg/day, has provided dramatic benefit, sustained and increasing for >5 and >4 months to date. (there is a continuing base of depotestosterone, 200 mg weekly, which initially alone provided very minimal improvement.) strength increase was evident at 1 and 3 days after the first course of 5 daily ivig infusions. it further increased for 2 to 2 and one-half weeks after treatment, and then began to diminish. repeat 5-day treatment 3 weeks after the first course resulted in summated improvement, now sustained and enhanced by an average of 1 treatment per week. quantitated strength testing by a blinded observer has shown a 2-fold to >loo-fold gradually increasing muscle function in all limbs. patient a regained the ability to feed himself, get out of a chair, walk unaided, and go up steps; quantitated hip flexors increased 2-and 20-fold. patient b regained the ability to feed himself, take care of personal toilet needs, walk securely, and drive 500 miles; quantitated hip flexors increased 5-fold, and biceps flexions increased from 0 with no weight to > 130 reps while holding 10-pound weights. bukhara jews: a new cluster with typical oculopharyngeal muscular dystrophy (opmd) is a rare, late-onset myopathy with autosomal-dominant inheritance. its ultrastructural hallmark is the finding in muscle fibers of intranuclear tubular filaments of 8.5-nm outer diameter. most opmd cases were described among french canadians; in france, the homeland of their ancestors, the prevalence is 1/200,000 (brunet et al, 1990) . in israel's central area live approximately 40,000 jews who have immigrated from the bukhara and samarkand regions in uzbekistan. they represent a homogeneous ethnic group with its own language and community life. among them we have identified opmd in 15 families (55 affected individuals). the inheritance, clinical, electrophysiological, and histological features of these pa-tients are similar to those described in other pdts of the world, with typical intranuclear inclusions seen on electron microscopy. the minimal estimated prevalence of opmd in this population is approximately 1 : 7 5 0 . this cluster of opmd among bukhara jews is the second largest in the world. because many bukharian families are large, they may be suitable for linkage genetic studies. human muscle during ontogenesis e . scarpini, g. conti, p. l. baron, and g. myoblast transfer has been proposed recently as a possible therapy for duchenne muscular dystrophy patients. because immune rejection can represent a major problem in myoblast implantation, immunological characteristics of human muscle should be investigated. previous studies showed that human muscle cells cultured in vitro can constitutively express human lymphocyte antigen (hla) class i, but not hla class 11. furthermore, human y-interferon induces the surface expression of hla class i1 on mononuclear myoblasts, but not on multinucleated myotubes. however, whether the cells produce and present the antigen by themselves or take this material from the environment, where it could be released by infiltrative cells, is not yet clear. in this study, we analyzed hla molecules at the protein level by immunocytochemistry with monoclonal antibodies against different hla-dr epitopes and hla-abc molecules on frozen serial sections of human muscle during development and at the adult stage. human muscle infiltration by macrophages and monocytesmacrophages also were studied with m718and leum3specific monoclonal antibodies at the same stages of development. our results show that during muscle development and maturation, hla-dr and hla-abc antibodies do not label muscle fibers but some m718-and leum3-positive cells within the muscle. these data can be useful to understand the role of infiltrating monocytes-macrophages in the muscle immune response. mounting evidence suggests that excitotoxicity, mediated via the glutamate receptor, is involved in the pathogenesis of amyotrophic lateral sclerosis (als), as well as in other neurological diseases. we therefore initiated an open label, phasen i trial of highdose dextromethorphan (dm), a noncompetitive, selective n-methybaspartate antagonist, in als. patients began with 2 mg/kg/day, divided into 4 doses, and incrementally escalated their medication to 10 mg/kg/day or their maximum tolerable dose. thirteen patients, all extensive metabolizers of dm, were enrolled. total daily doses ranged from 4.75 to 10 mg/kg. major side effects were lightheadedness (8), slurred speech (7), and fatigue (6). no biochemical, hematological, or neuropsychiatric abnormalities occurred after up to 6 months of maximal therapy, except for depression in 1 patient. plasma kinetics of dextrorphan (dt) (the major metabolite of dm) were studied after an acute oral dose of 2.5 mg/kg dm. median elimination halflife was 2.5 hours. plasma dt concentration peaked at a median of 2 hours, with a median cmax of 14.4 pm. median amyotrophic lateral sclerosis cerebrospinal fluid/plasma dt ratio was 9.796. this study demonstrates the feasibility of long-term, high-dose dm therapy. we are now conducting a phase i1 study of highdose dm in als, designed to assess its efficacy. polyneuropathy: a chronic inflammatory demyelinating polyradiculoneuropathy variant? d. cros, k. h. chiappa, s. patel, and s. gominak, boston, ma, we describe 4 patients (3 men, 1 woman) with a pure, adultonset sensory neuropathy. the course was chronic in all cases. three patients had a relapsing-remitting course over 2 to 22 years with several attacks every year; the onset was gradual and followed by a plateau in the fourth patient. all patients had positive and negative sensory symptoms, and 2 had positive motor symptoms (fasciculations). in all patients, muscle power was normal at the time of peak deficit. all were areflexic and had large fiber sensory deficits, and 3 patients had sensory ataxia. three patients had elevated csf protein, whereas the csf was normal in 1 patient. mri demonstrated marked thickening of the lumbosacral spinal roots in 1 patient. motor conduction studies were normal in all patients, and mild f-response abnormalities were noted in 2. neurophysiological investigations of the sensory pathways were abnormal in all. three patients had several studies over a 2-year period. sensory nerve action potentials were unobtainable in 2 patients, and normal in the others. median and tibial somatosensory-evoked potentials showed conduction slowing consistent with demyelinating lesions affecting the peripheral sensory pathways, either globally or focally in the proximal segments. two patients appeared to respond to plasma exchange or intravenous immunoglobulin therapy, or both. glial fibrillary acidic protein cells in experimental motoneuron disease raul n . mandler, pam c. allgood, and james a. wallace, albuquerque, nm neuronal degeneration in human and animal motoneuron disease has been emphasized, but glial phenotype alterations have not been studied as extensively. we carried out a developmental and topographic study of astrocyte expression in the wobbler mouse model of motoneuron disease. wobbler mice and normal littermates were studied at 3, 4 , 5 , and 6 weeks of postnatal development. anesthetized animals were perfused intracardially with paraformaldehyde. spinal cords were dissected and landmarks were identified carefully for systematic study. sections were stained with monoclonal antibodies against glial fibrillary acidic protein (gfap) neurofilament and neuron-specific enolase. cell quantitation was done with video-enhancing microscopy. in symptomatic animals, marked increases in gfap staining were found in rostral and caudal spinal cord areas. quantitation studies revealed a 15to 20-fold increase in gfap+ cells in the wobbler. we conclude that gfap+ cells are markedly increased in the wobbler mouse at cervical, thoracic, and lumbar areas. this cell may also be relevant in motoneuron disease pathogenesis. ( indirect evidence suggests that polio virus may persist in the human cns years after initial infection and may be a cause for the post-polio syndrome. to evaluate whether the polio virus genome can be detected in the cns of patients with previous polio infection, we identified 11 patients who had died with autopsy findings and clinical history consistent with poliomyelitis. rna was extracted from paraffin-embedded sections of brain or spinal cord and subjected to reverse transcription followed by dna amplification by polymerase chain reaction (rt-pcr) using primers specific for heat shock protein 70 (hsp70) and a conserved region of the polio viruses. hsp70 mrna could be detected in all specimens, indicating that amplifiable rna had been isolated. in no specimens could polio virus rna be detected. this study suggests that polio virus does not persist in the human cns in quantities detectable by the sensitive pcr method. with cmt type 11, seen at mayo clinic rochester between 1981 and 1991, for the frequency of selective calf weakness in cmt type 11, the form of cmt most similar clinically to distal sma. anterior compartment weakness exceeded calf weakness in 49 patients (74%); anterior and posterior involvement was equal in 16 (24%). calfweakness exceeded anterior compartment weakness in 1 patient (2%). selective calf weakness in distal sma thus helps distinguish this disorder from cmt type 11, and similarly from distal sma with weakness resembling cmt, in that we are unaware of the 2 distributions in distal sma occurring in the same family. given the possibility of genetic heterogeneity, linkage studies of distal sma probably should include patient selection criteria such that the distribution of leg muscle weakness is homogeneous. p206. conjugal amyotrophic lateral sclerosis: amyotrophic lateral sclerosis (als) is a sporadic neurodegenerative disorder of unknown cause. unusual cases may provide etiologic clues. we report a married couple, both of clue to etiology? whom developed als in 1 year. the couple grew up in southeastern pennsylvania and attended the same schools. they married after high school and have 2 healthy children. in september 1990, a 38-year-old woman noted right-hand weakness and associated fasciculations that progressed to the entire right upper extremity. by january 1991, the lower extremities were asymmetrically weak and fasciculating. she then developed left-arm weakness, d ysarthria, dysphagia, and emotional incontinence. she had hyperreflexia and bilateral extensor plantar responses. then, in may 1991, her husband, aged 38, noted difficulty whistling, which progressed to frank dysarthria. later, he developed dysphagia, emotional incontinence, and weakness, wasting, and fasciculations in the upper extremities. hyperactive gag, jaw, and limb reflexes were present. in both, electrodiagnostic testing revealed widespread evidence of lower motor neuron degeneration. numerous laboratory tests were normal. although these cases may represent a chance association, the development of als in a young husband and wife suggests a possible environmental cause. the authors welcome suggestions about these cases from the neurological community. conduction block in demyelinating neuropathies usually is assessed from differences in the sizes of surface-recorded maximum m-potentials evoked by supramaximal stimulation at successively more proximal sites along the course of motor nerves. as the maximum m-potential is comprised of many bitriphasic surface-recorded motor unit action potentials (muaps), differences in the relative latencies between muaps may lead to phase cancellations, reducing the m-potential size and rendering any quantitative assessment of the extent of conduction block relative to phase cancellation difficult. cooling a muscle (not the nerve), however, by as much as 15â°c increases the negative peak durations of muaps by as much as 2 to 3 times and moves the point at which maximum phase cancellation might occur to some theoretical point well proximal to the spinal roots. in 5 cases of guillain-barre syndrome (gbs) studied to date, cooling produced little change in percent reductions in m-potential negative peak areas between successively more proximal sites of stimulation. this finding suggests that "true" conduction block rather than interpotential phase cancellation best explains reductions in m-potential size at successively more proximal sites of stimulation in gbs. associated with trimethoprim-sul famethoxazole rare cases of primarily motor polyneuropathy have been associated with the use of sulfonamides. the incidence of polyneuropathy has diminished substantially with the abandonment of earlier methylated compounds. we describe 2 patients who developed allergic phenomena, including a skin rash and debilitating, painful sensory and autonomic polyneuropathy within days of receiving trimethoprimsulfamethoxazole. in 1 patient, examination revealed resting tachycardia, marked blood pressure orthostasis and near-program and abstracts, american neurological association 281 syncope, hyporeffexia, urinary incontinence, and reduced sensation distally in the lower extremities. his cerebrospinal fluid was acellular with a protein of 141 mg/dl. the other patient showed a resting tachycardia, sluggish pupils, reduced distal vibration perception, and hyperpathia of hands and feet. conventional nerve conduction studies demonstrated normal motor results in both patients, absent or reduced sensory amplitudes in the first patient, and normal sensory results in the second. autonomic studies identified profound abnormalities in testing of sympathetic skin potentials, sinus arrhythmia, and valsalva's ratio. in both cases, nerve biopsy was not performed for fear of exacerbating the patient's hyperpathia. subsequent hemodynamic and electrophysiological testing showed improvement in autonomic function, paralleling the patients' clinical amelioration. although uncommon, a painful, sensory and autonomic, partially reversible polyneuropathy may develop after the use of trimethoprim-sulfamethoxazole. the remote effects of botulinum a toxin injections into vocalis muscles for treatment of focal laryngeal dystonia were investigated using single-fiber electromyography (sfemg). botulinum a toxin injections have been proven effective therapy for various dystonic disorders including focal laryngeal dystonia, blepharospasm, and torticollis. previous sfemg studies have demonstrated remote effects of the toxin in noninjected muscles after treatment for both blepharospasm and torticollis. these effects include an increase in fiber density, mean jitter (mcd), and percentage of fiber pairs with increased jitter. other researchers have postulated that the distant effects of this toxin may be related in part to the dose of botulinum toxin injected. to investigate this hypothesis we have studied patients treated for focal laryngeal dystonia because the amounts of toxin required are 1/25th to 1/1ooth of the doses used to treat other dystonias. using electromyographic (emg) guidance, bilateral injections of 2.5 or 5.0 mouse units of botulinum a toxin were injected into each vocalis muscle of 11 patients. each patient had significant improvement in phonotory function within 48 hours after injections and have been followed serially (usually within 3 weeks and again at 2 mo) after injections, with sfemg recordings of the left extensor digitorum communis and sternocleidomastoid muscles. five patients have had more than 1 series of injections over the 10 months since we began this study. sfemg studies have revealed no significant change in the fiber density, mean mcd, or percent of fiber pairs with normal jitter in either muscle. in conclusion, our studies support the hypothesis that the presence of remote effects of botulinum toxin may be related, in part, to the amount of toxin used. the c57bl/6/01a mouse exhibits the remarkable characteristic of prolonged survival of axons separated from their cell bodies (slow wallerian degeneration). previous work has demonstrated that the axon itself is responsible for the phenotype of prolonged survival. we investigated whether the lack of rapid axonal degeneration after axotomy in this substrain is due to an inability to break down cytoskeletal components, a process that is normally accomplished by activation of intrinsic calcium proteases. segments of desheathed sciatic nerves from normal and ola mice were incubated for 2 hours under conditions that disrupt the axolemma (freeze/ thaw or in 1 % triton x-loo), allowing external calcium free access to axoplasm. nerves were analyzed by western blot for neurofilament (nf) proteins and by electron microscopy. in high-calcium media (1 mm caci,), nf immunoreactivity was lost and axoplasm was reduced to watery debris in both substrains, whereas in egta-buffered media, axoplasm was preserved. these results demonstrate that calcium-activated proteases are present and can be activated in ola nerves. the defect in these mice that allows for prolonged survival of transected axons is likely in the mechanism for calcium entry into the distal stump. the mechanism by which the analogue of adrenocorticotrophic hormone, acta4-9, prevents cisplatinum (cp) neurotoxicity is unknown. murine n1e. 115 neuroblastoma cells and neural crest-derived, squirrel fish erythrophore cells tuesday, have similar vesicular transport mechanisms to human neural cells. they were used to study the effects of cp and acth4, on cellular transport. differentiated n 1e. 1 15 cells were treated 1 hour prior to observation with serum-free media (sfm, control); sfm/cp 5 pg/ml; or sfm/cp 5 pg/ml and 100 ng/ml acth4-,. organelle transport was studied (7 neurites and 100-140 organelles per condition) using computer-enhanced video microscopy. mean fast anterograde (1.00 k 0.07 pmsec-' vs 1.65 * 0.07 pmsec-') and retrograde (0.67 2 0.04 pmsec-' vs 1.19 +-0.05 pmsec-') transport were decreased in cp-treated compared to control cells ( p < 0.0001). in cp/acth4,-treated cells, mean anterograde (1.46 +_ 0.07 pmsec-') and retrograde (1.13 2 0.04 pmsec-') velocities were greater than in cp cells ( p < 0.0001). velocities in control and cp/acth4, cells were not statistically different. erythrophore pigment granule transport was observed in a blinded study, using similar techniques. mean aggregation velocity was greater in control (1.99 k 0.09 msec-') and cp/acth4, (1.97 f 0.07 msec-')-treated cells compared to cp (1.43 * 0.07 msec-') cells ( p < 0.0001). incubation with cp for 1 or 72 hours affected velocities equally, but acute exposure was more easily reversed by control or acth,, containing media. there is striking inhibition by cp in cross-species models of organelle transport. this can be prevented by acth4,. erythrophores allow future study of individual transport components. neurotrophin to investigate signal transduction pathways involved in neurite growth, the cytoplasmic regions of p7sngfr, the common neurotrophin receptor monomer, were searched for a motif analogous to the predicted secondary structure of the tetradecapeptide mastoparan. potential sequences were modeled using a semi-empirical molecular mechanical force field approach. the sequence rat ~7 5~~~ 367-379 represents a highly conserved amphiphilic domain predicted to be involved in neurotrophin signal transduction via g-protein mechanisms. to test this prediction, peptides containing sequences homologous to p7 5ngfr 367-3 79 were examined for effects on trophic factor-induced survival/differentiation responses of rat pc12 pheochromocytoma cells, chick embryo drg neurons, and chick embryo ciliary neurons. a peptide identical to ~7 5~~~ 367-379 accelerated the neurite growth response to nerve growth factor (ngf) of pc12 cells and drg neurons in a time frame that paralleled uptake into cells, but mastoparan did not influence ngf-mediated neurite growth. millimolar mg+ + and benzalkonium chloride, known to block the actions of mastoparan, blocked the effect of the peptide on ngf-mediated neurite growth by pc12 cells. peptides mutated to alter cationic amino acid relationships or amphiphilicity were less effective than the peptide in accelerating ngf-mediated neurite growth. these observations complement and extend evidence suggesting a pivotal role of ~7 5~~~ in ngf-mediated signal transduction. these studies complement and extend evidence suggesting a pivotal role of p7sngfr in neurotrophin signal transduction and evidence that activation of intracellular signalling processes involving specific g-protein mechanisms are involved in neurotrophin-mediated neurite growth. crushing the hypoglossal nerve causes hypoglossal motor neurons to decrease expression of choline acetyltransferase (chat) and begin expressing p75ngfr, the low-affinity ngf receptor. these changes are evident within 3 days after the injury and continue for several weeks. inhibition of axonal transport by vincristine applied to uninjured nerves causes loss of chat expression without induction of ~7 5~~" . we sought to determine if topical vincristine would alter p7sngf' expression after nerve injury. hypoglossal nerves were surgically exposed unilaterally in anesthetized rats and crushed. one week later, rats were reanesthetized and the same nerves were re-exposed. vincristine or saline was applied at the crush sites by soaking a strip of cotronoid wrapped around the nerves. one week later, rats were anesthetized and perfused with aldehydes. frozen sections from the brainstems were stained by indirect immunoperoxidase to demonstrate chat and ~7 5~~~' . saline-treated controls showed decreased chat and abundant ~7 5~~" in hypoglossal motor neurons ipsilateral to the crush injury. vincristine-treated animals showed no chat and no p7tngf'. we interpret these results as indicating that a signal originating from the injury site maintains ~7 5~~" expression after nerve injury. catecholamines have been reported to be toxic to embryonic-derived rat neurons and glia via the formation of reactive oxygen species (rosenberg, 1988) . we tried to determine whether oligodendrocytes (ol) from adult 6-month-old rat brain are similarly susceptible. toxicity to ol was examined using light microscopy and galactocerebroside immunohistochemistry where the relative number of surviving ol and their extent of process formation were graded. five days of exposure to norepinephrine (ne) and epinephrine (epi) at 10 and 100 fm produced significant toxicity ( p < 0.05, analysis of variance [anova)) to adult rat ol; this toxicity was evident by 24 hours of exposure. treatment with catalase (50 p,g/ml), a free-radical scavenger enzyme, completely prevented the toxicity of catecholamines. to ascertain whether astrocytes, which have free-radical scavenging capacity, could prevent the catecholamine-induced injury to ol, rat ol were seeded on neonatal rat astrocytes. under such conditions, the toxicity of n e and epi was reduced significantly ( p < 0.05, anova). these findings suggest that impairment of this protective function of astrocytes may render ol and its myelin membrane susceptible to free-radical-mediated damage. lyme neuroborreliosis is an increasingly prevalent disorder, but the diagnosis generally has been indirect. thus, the pres-ence of other manifestations of the infection, consistent findings on neurological exam or lumbar puncture, or presence of csf antibody have been used rather than direct isolation or identification of the organism from the csf. we have previously developed polymerase chain reaction with hybridization (pcr/h) to identify borrelia burgdorferi in the blood and organs of infected mice, and found that the assay was equivalent or, in some cases, preferable to culture (ann neurol 1991; 30:302) . the assay used for primers oligos derived from a sequence of genomic b. burgdovfri d n a expressed on a plasmid by rosa and schwan. a two-stage nested pcr was performed on csf samples in which the d n a was isolated in a variety of ways. pcr products were subsequently hybridized with a digoxigenin-labeled internal probe by slotblot hybridization. the sensitivity of the assay was excellent, being <o. 1 fg of purified b. burgdoorfevi dnaiml of csf, and 1 to 10 spirochetes per ml of csf when "spiked" csf was tested. the assay then was applied to a battery of csfs stored over the past 15 years in patients with definite, probable, and possible lyme neuroborreliosis. the lack of pcr/h positivity in some patients with definite and probable disease signifies that the spirochete does not reside in the lumbar csf in all patients with lyme neuroborreliosis, and may be concentrated in the meninges or parenchyma. progenitor cell line on transplantation into the adult murine brain t . kitagwcbi, d.-h. chui, and t . tabira, tokyo, japan several multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into primary culture cells of the septum of embryonic murine brains (the retroviral vector, kindly provided by d r c. l. cepko, boston, ma). two of those cell lines, when transplanted back into 6to 8-week-old mice by stereotaxic operation, integrated into the septum in a nontumorigenic manner. the implanted cells, which had been labeled with pkh26 dye, could be identified in animals up to at least 10 weeks after transplantation. immunocytochemical analysis demonstrated that the transplanted cells were seen assuming a round morphology and no processes. they did not stain with any glial or neuronal markers except for a2b5 and hnk-i, in the same way as in vitro. interestingly, after 10 weeks of implantation, the cells were located discretely in the transplanted site and displayed a small and round morphology with fine processes. most of the transplanted cells showed immunoreactivity with monoclonal antibodies directed against neuronal markers such as neurofilament and map2. these data indicate that the immortalized cell lines might be useful in characterizing factors that regulate cell type differentiation in the mammalian nervous system. (supported by a grant from the science and technology agency of japan.) related to serum response factor in human brain and muscle dana leifer, dimitri krainc, racbael neve, roger bveitbavt, and stuart a. lipton, boston, m a we have identified cdna clones for a novel transcription factor that is expressed at high levels in human fetal brain and skeletal muscle, but not in a variety of other tissues, as demonstrated by northern blotting. sequence analysis indicates that the clones appear to have 2 alternatively spliced forms and have extensive homology with human serum re-sponse factor (srf) and a family of related transcription factors that has representatives throughout eukaryotic evolution in species from yeast to man. srf is thought to have a role in regulation of immediate early genes and of muscle-specific genes. in gel mobility-shift assays, the srf-like proteins coded for by our clones bind specifically to the myocytespecific enhancer binding factor-2 (mef-2) regulatory element, a dna sequence that has so far been found to be functionally important in the promoter and enhancer regions of a variety of muscle-specific genes. moreover, our srf-like proteins specifically activate transcription of reporter genes containing the mef-2 element. given the abundant expression of our clones in human brain as well as in muscle, our results suggest that these proteins may have a significant role in development not only of muscle but also of brain. a host of quantitative eeg studies, most of which employed variations of the fast-fourier transform, have been made on isolated clinical entities such as the epilepsies, metabolic syndromes, and sleep with varying degrees of satisfactory correlations. using a time domain wave-by-wave computer program that simulated the logical approach of the clinical electroencephalographer, we have studied more than 300 unselected routine neurological clinic and ward patients. the presenting problems ranged from headache to major cerebral involvement. employing the dichotomy of normal and abnormal, the computer-read eegs agreed with the routine eeg, the clinical manifestations (including imaging techniques), and neuropathological findings in 90% of all cases. this high level of correlation, which approached that achieved by welltrained eegers reading the same records, was obtained by solely utilizing the background eeg activity. such results became possible only when the computer program was so organized that the "raw" computed and original oscillograph records were made to match closely. the diagnosis, with topographically diagramed localization, was printed automatically as the final step of the program. our data strongly suggest that routine computer reading of the human eeg is feasible, reliable, and potentially useful. protein accumulation in damaged neurites and microglial cells abnormal accumulation or abnormal processing of amyloid precursor protein (app), or both, may be critical to the development of p-amyloid protein (bap) deposits. however, alzheimer's disease (ad) has another pathological hallmark, which is the appearance of neurofibrillary tangles. the question is, therefore, to clarify the possible relationship between altered app metabolism and neurofibrillary degeneration. one classic method for inducing neurofibrillary tangles is the administration of aluminum salts. although these tangles are morphologically different than those seen in ad brain, this phenomenon has given rise to the hypothesis that aluminum might be an environmental factor in the causation of ad. this theory is controversial, but the animal aluminum model remains one of the few ways that long-lasting neuroskeletal changes can be induced. to explore the effects of aluminum salts on app metabolism, we examined app immunodistribution in rat brain injected intraventricularly or intrastriatally with aici,. we found a long-lasting accumulation of app in affected neurites, as well as in activated microglia/macrophages. abnormal neurites also showed argentophilic changes, neurofilament accumulation, and alz-50 immunoreactivity. the results support the hypothesis that interruption of axoplasmic flow by aluminum salts, as by other means, can lead to both app accumulation and neuroskeletal alterations. p223. nondemyelinating conduction slowing of myelinated fibers due to inactivation of n a channel takanori yokota, yukinobu saito, and tadashi miyatake, tokyo, japan in 4 patients with acute or chronic inflammatory demyelinating polyradiculoneuropathy, the marked improvement of motor-nerve conduction velocity without change of amplitude or configuration of the compound muscle action potentials (cmaps) was observed in 3 days. to investigate the mechanism of the rapid improvement of nerve conduction, the influence of na channel blockade on nerve conduction was studied. in 4 healthy volunteers, the recovery of the cmap and sensory nerve action potential (snap) of median nerve stimulation were recorded after the intravenous infusion of 100 mg of lidocaine. after the loading, cmap and snap were reduced rapidly in amplitude and were slowed in latency. after the recovery of amplitude of cmap and snap, the nerve conduction velocities were improved gradually in 2 hours with the amplitudes and configurations of cmap and snap unchanged. this indicated that myelinated nerve conduction velocity could be slowed by the inactivation of na channels without demyelination or conduction block. the prolongation of rising time in depolarization of axonal membrane potential should be one of the mechanisms of this conduction slowing due to inactivation of na channels. paraneoplastic cerebellar degeneration (pcd) in gynecological and breast cancers frequently is accompanied by an autoantibody response (type i or "anti-yo") reacting with 34and 62-kd cytoplasmic antigens of cerebellar purkinje cells. sakai et al, using a cerebellar library (stratagene), recently have isolated a cdna clone expressing a 52-kd protein recognized by igg from a patient with pcd and uterine carcinoma (sakai et al, ann neurol 1990; 28:692) . this clone is believed to share some homology with the message encoding the 62-kd protein recognized by type i sera. because pcd17 was isolated using serum from a single patient, however, it is not known whether the expressed protein of pcd17 is recognized uniformly by all patients with type i antibody response. e. coli strain xl1-blue transformed with pwr590-2 containing the snabiihindiii fragment of pcd17, pwrsbo-pcdl?sn, was induced using isopropylthiogalactoside. bacterial lysates were electrophoresed on 10% sds-page gels and analyzed by western immunoblot program and abstracts, american neurological association 285 methods using sera and csf from patients with pcd associated with gynecological and breast malignancies who exhibited type i antibody response. the 52-kd expression product was labeled by 818 sera and 414 csf samples tested from antibody-positive patients but was not labeled by sera or csfs from controls. our studies demonstrate that the 52-kd protein expressed by pcd17 contains epitopes that are consistently recognized by both systemically and intrathecally produced antibodies from patients with pcd accompanying gynecological and breast malignancies. tax01 is a novel antitumor drug that has demonstrated impressive clinical activity in breast, ovarian, and lung cancers. although sensory neuropath y has been described secondary to both taxol alone and taxol-cisplatin combination, detailed neurophysiological and pathological studies have not been described. nineteen patients with solid tumors were treated with combinations of taxol (135-350 mg/m2), cisplatin (75-100 mg/m2), and granulocyte colony-stimulating factor (g-csf) (5 pglkglday). sequential neurological examinations, nerve conduction studies (ncs), and quantitative vibration testing (qst) were performed during and after treatment. eighteen of 19 patients developed sensorimotor neuropathy and 3 developed myopathy. thirteen were symptomatic with numbness or weakness and 17 had abnormal results on neuromuscular examination. ncs showed absent or prolonged h-reflexes (14), reduced peroneal motor amplitude (14), reduced sural sensory amplitude (13), and myopathic motor unit potentials in proximal muscles (3). qst results were abnormal in 7 of 15 tested patients. muscle biopsy specimen in a patient with myopathy showed features of a toxic myopathy. the neuropathy was worse with higher cumulative dosages of taxol (>600 mg), with higher single doses of taxol(>300 mg/m2), or with a preexisting neuropathy. we conclude that sensorimotor neuropathy and myopathy are dose-limiting neurotoxicities of combined cisplatin and taxol use, now that neutropenia can be controlled with neuropathy and myopathy g-csf. central nervous system lymphoma casilda balmacedz and lisa deangelis, new york, ny ally periventricular and may seed the csf by direct growth through the ependyma. we reviewed the csf profile of 83 non-acquired immunodeficiency syndrome (aids) patients (pts) with pcnsl. all pts had lumbar puncture (lp) and 46 had multiple samples from an ommaya reservoir. definite lm involvement was identified with a positive csf cytology, lymphomatous lm infiltration on a surgical specimen, or mri with gadolinium showing lm tumor. probable lm lymphoma was diagnosed in pts with suspicious or atypical csf cytology. there were 41 women and 42 men with a median age of 57 (range 20-81 yr). at diagnosis, mean white blood cell count was 49/mm3 (range 0-5,5 10); mean lumbar csf protein was 103 mgldl (range 3-1,893); and mean ventricular csf protein was 32 mgldl (range 4-265). glucose was always normal. nineteen of 57 pts sampled had oligoclonal bands, and 42/65 had elevated pz microglobulin. at diagnosis 43 (52%) had an abnormal csf cytology: 22 positive, 16 suspicious, and 5 atypical. one pt had pathological infiltration of the lm and 1 had lm tumor on spine mri for a total of 45 (54%) pts with definite or probable lm lymphoma. in 43 pts with abnormal cytology, the abnormality was found in 30143 (70%) lps and 32/55 (58%) of the ommaya and ventricular specimens. in 8 pts the lumbar cytology was the only abnormal specimen despite multiple ventricular samples, and in 10 only the ventricular csf was abnormal. thirty-six of 83 (43%) pts developed recurrent tumor afrer treatment. forty-two percent (15136) of all patients with relapse had lm recurrence. lm recurrence was accompanied by brain recurrence in 9 pts, systemic in 1, ocular in 1, both systemic and ocular in 1, and isolated in 3. at diagnosis 59/83 patients received treatment directed against the lm. of these, 8 (14%) had meningeal recurrence whereas 7 of the 24 (29%) patients who did not receive this treatment had lm recurrence. lm involvement by pcnsl is frequent, may be missed on a single csf sample, and requires specific therapy at diagnosis. sixty-three solid cancer patients with a single brain metastasis were prospectively randomized for neurosurgery and radiotherapy combined (arm 1) or radiotherapy alone (arm 2). they were stratified for lung or nonlung cancer and for active versus stable or absent extracranial disease. world health organization performance status was 9. age, sex, performance status, and location of brain metastasis were divided evenly over both groups. one-month mortality was 7% in arm 1 and 6% in arm 2. median survival of 11 months after combination therapy was significantly better compared to 6 months after irradiation alone ( p < .04). it made no difference whether they had lung or nonlung cancer. the largest difference between both treatment arms was observed in patients with stable or absent extracranial disease (12 vs 7 mo, p < .02). when systemic disease activity was present, median survival was 5 months irrespective of treatment arm. functional independent survival was 1 to 2 months shorter than overall survival and was significantly better for patients with stable extracranial disease after combined therapy. multivariate analysis showed that age was also an independent prognostic factor. patients older than 60 years had a hazard ratio for dying of 2.8 (p. 004). we will detail the type and pattern of neurological complications in t-cell non-hodgkin's lymphoma (nhl), and review how they differ from those associated with b-cell nhl, and the lymphomas in general. this study is the first step in a process to characterize these tumors to determine if special staging or cns prophylaxis are indicated in any of the subtypes of t-cell lymphoma. we recently have encountered 5 women with breast cancer and an unusual sensorimotor neuropathy. the neuropathy was the major clinical problem. in 4 women the initial symptom was severe itching, 3 generalized and 1 first localized to the involved breast and then generalized. all developed distal extremity numbness and burning that very slowly progressed proximally, and in 2 became generalized. four complained of painful muscle cramps in the extremities ( 4 ) and jaw (1). all had mild extremity weakness, distal (5) and proximal ( 1 ) . three women developed symptoms up to 21 months prior to cancer diagnosis, 1 shortly after diagnosis, and 1 5 years after diagnosis. four women had disease confined to the breast and regional lymph nodes, and 1 had metastatic disease in remission. although annoying, symptoms were generally not disabling. three women stabilized or had slight improve-associated with breast cancer ment with cancer treatment, and 2 continue to gradually progress while in cancer remission; 1 required a cane to ambulate after 9 years due to sensory ataxia. one who developed cancer relapse had concurrent neurological relapse. one woman treated with high-dose immunoglobulin did not improve. none had significant weight loss. laboratory abnormalities included elevated erythrocyte sedimentation rate (28-60) in 3, antinuclear antibody 1:40 in 1, csf with lymphocytic pleocytosis (7-14 white blood cellslmm) in 3/ 4 and elevated protein (5 1-97 mg/dl) in 414 available. emgi ncv showed mild sensory-to-motor polyneuropathy in 314 available. none had detectable antibodies against peripheral nerve or dorsal root ganglia. the etiology of sensorimotor neuropathy in these patients is unknown, but it may represent a distinct paraneoplastic syndrome that can herald the onset of, and parallel the course of breast cancer. our objective was to determine whether ceramide induces differentiation of anaplastic glioma cells. sphingomyelin hydrolysis resulting in ceramide production has been linked to differentiation of leukemia cells. t9 rat anaplastic glioma cells, seeded at 2 x lo4 cells per well, were grown in serum-a glioma cell line program and abstracts, american neurological association 287 free media. on day 4, cells were photographed, scored for processes longer than one cell body, and counted. c2 ceramide changed plump cells to flattened cells with many long processes: ceramide treatment increased the percentage of cells with processes from 22 2 4% (sd) to 49 * 12% (n = 4, p < 0.01). control cells grew to 6.3 x lo5 cells/well 2 0.6 x lo5 cells; ceramide-treated cells grew to 1.6 x lo5 ? 0.6 x los (n = 4, p < 0.0005). although one ceramide analog reproduced the c2 ceramide-associated changes, the optical isomer of this analog did not, demonstrating stereospecificity. cerarnide did not decrease cell viability by trypan blue. ceramide inhibits proliferation and induces process formation in a glioma cell line, causing it to assume a more differentiated phenotype. cerarnide or its analogs represent possible future therapeutic agents that would inhibit the growth and affect differentiation of anaplastic gliomas. [bashir, mod pathol 1990; 3(4) :429-434)). this is similar to the pattern seen in ebv-infected human b cells and unlike the uniform latent infection seen in burkitt's lymphoma. we tested the hypothesis that long-term passaging of ebv-immortalized human b cells in immunodeficient mice leads to emergence of a uniform nonlytic pattern of ebv infection associated with appearance of the malignant profile. ebv-infected normal human b cells were serially passaged intracerebrally in severe combined immunodeficient cb17 mice (5 x 10' cells per mouse, 5 mice per passage for a total of 10 passages). frozen mouse brain sections from each passage were stained with vca antibody (ebv lytic cycle) and hybridized with biotinylated bamh1-w sequence of ebv. all injected animals developed tumors as previously described (bashir, lab invest 1991; 65(6):702-709) . tumor cells continued to express vca and showed latent and lytic hybridization patterns with bamh,-w after 10 passages despite exhibiting monoclonality (surface immunoglobulins) and random chromosomal changes. lytic infection of immortalized b cells with ebv is stable, resembling brain lymphomas in aids, and unlike the latent infection seen in burkitt's lymphoma. a previously healthy 73-year-old man developed diplopia and incapacitating, diffuse weakness over a period of 6 weeks. examination showed a "one-and-a-half'' syndrome of horizontal gaze paresis, patchy severe weakness with atrophy and fasciculations, absent tendon reflexes in the legs and right biceps, and decreased vibration sense in the feet. csf contained a mild pleocytosis, elevated protein, and oligoclonal igg bands. electrophysiological testing indicated a generalized sensorimotor axonal neuropathy with diffuse denervation. small-cell lung carcinoma was diagnosed by bronchoscopy. prednisone produced mild subjective improvement. chemotherapy was begun but the patient developed fatal septicemia. serum was negative for anti-hu or anti-gm1 with paraneoplastic encephalomyeloneuritis antibodies. serum and csf contained high titers of igg antibodies reacting specifically with a protein antigen of approximately 130 kd in immunoblots of human cerebral cortical neuronal nuclei or of human purkinje cells. this pattern of autoantibody reactivity was not present in sera from any of 37 other patients with small-cell lung carcinoma, 17 of whom had paraneoplastic encephalomyeloneuritis and anti-hu antibodies, nor was it present in many patients with other neurological disorders. the patient's serum has been used to probe a human cerebellum expression library and to isolate a cdna clone that is being characterized. acute encephalopathy is the problem in 17% of neurology consultations reported at mskcc. we studied 94 patients (5 1 prospectively and 43 retrospectively) to determine clinical findings, causes, and outcome. fifty-five were women and 39 were men, and the average age was 63 years. all patients had cancer: lung (20%), gastrointestinal tract (19%), breast (12%), and others (49%), forty-two patients (45%) were delirious on admission and delirium developed an average of 12 days later in 55%. encephalopathy occurred postoperatively in 24%. symptoms included confusion (92%), lethargy (59%), agitation (5 1 %), hallucinations (29%), and seizures (10%). signs included deficits in attention (44%), memory (385%), language (15%), lateralizing signs (50%), and asterixis (33%). the average mini-mental status test (mms) score was 12 (30 = normal). a single cause for delirium was found in only 4% of patients with an average of 4 etiologies per patient. metabolic abnormalities were found in 89% of patients, and were a primary cause in 44%; disseminated intravascular coagulation contributed to delirium in 11%. cns metastases were found in 32% and were a major cause of delirium in all. fifty percent of the patients had fever/ systemic infection, but sepsis was present in only 4%; only 1 patient had cns infection. medication contributed to delirium in 97% but was a primary cause in only 29%. the 30-day mortality rate was 3 1 % and delirium improved in 68% (average mms = 23). patients with cancer have multiple, potentially treatable causes of delirium. delirium is associated with a high death rate, though patients generally improve. radiation therapy for brain tumors d. w. dodick, b. mokri, k. k. unni, g. m. miller, and e. g. shaw, rochester, m n osteosarcoma in a previously normal bone is a rare but recognized remote effect of radiation therapy. any bone in the field of radiation can be affected. involvement of cranial bones is exceedingly rare. we could identify only 4 patients (2 men and 2 women) with postirradiation osteosarcoma of the calvarium seen at the mayo clinic over a 50-year period, from 1931 to 1991. all had received radiation for brain tumor, osteosarcoma had appeared in the field of radiation in all, the interval from radiation therapy to the appearance of sarcoma ranged from 7 to 23 years, and diagnosis of sarcoma was confirmed histologically in all cases. the patients' age at the diagnosis of the brain tumor ranged from 18 to 41 years. the nature of the brain tumor was unverified in 2 cases, was a low-grade ependymoma in the third case, and a pilocystic astrocytoma in the fourth case. one patient is still alive 12 months after the diagnosis of the sarcoma. she received chemotherapy and subsequently underwent resection of the osteosarcoma. one patient died postoperatively after partial resection of the sarcoma. the other 2 patients died 7 months and 15 months after the diagnosis of the osteosarcoma despite additional radiation therapy in the former and aggressive chemotherapy in the latter. element-la2 fusion gene as a potential marker of neural tumor differentiation lawrence recht, chiffon wu, and louis j. degennam, worcester, ma inducing cancers to differentiate into more benign differentiated tumors represents a novel oncological strategy. to establish a model that would permit assessment of this phenomenon at a molecular level, we created and have partially characterized a murine neuroblastoma line that has been stably transfected with a synthetic fusion gene containing the promoter element of the rodent synapsin i gene (synapsin i regulatory element isre)). in vivo, this promoter directs the neuron-specific expression of the synapsin i gene in normal adults. the gene also is expressed in varying amounts in neuronal tumors including neuroblastoma. in the synthetic fusion gene, the sre has been linked to the lacz gene that encodes bacterial p-galactosidase. a simple histochemical assay for p-galactosidase therefore provides a specific marker of the expression of the fusion gene. our preliminary experiments as of this writing have shown that it is possible to detect p-galactosidase activity in the transfected neuroblastoma cells both in vitro and in transplanted tumors. it appears possible therefore that this transfected neuroblastoma cell line can provide a useful model system with which to assess the effects of differentiation therapies. larger lesions (>18 cm2), and larger midline shifts (>4 mm). twenty-eight of 36 (78%) patients with prior has had bt has compared to 25 of 75 (33%) patients without prior has. in 8 patients, their bt h a was similar to their previous ha but was more frequent or severe. we conclude that has in bt patients are common but usually not severe. nausea, vomiting, an abnormal neurological examination, or a change in prior headaches warrant further investigation. cairncross and macdonald showed that procarbazine, lomustine, and vincristine (pcv) are effective for recurrent anaplastic oligodendrogliomas (ao) (ann neurol 1988; 23:360-364) . pcv now has a major role in management of all forms of oligodendrogliomas (0), but the biological basis for this response is unknown. to evaluate one subset of possibilities, we studied 14 patients (6 ao, 8 0) with a ct method that permits measurement of blood-to-tissue transport (kj, tumor-to-blood transport (k2), and vascular volume (v,) (ann neurol 1991; 30:581-587) . pcv was used to treat 11 of the 14 patients. k, (~1 grr-' min-') values were highly variable for whole tumor, ranging from 1.49 to 14.37 (mean 4.87 k 4.17) with no difference between a 0 and 0. k2 and v, were also highly variable. k, of tumor-free brain was 1.40 to 2.69 (mean 1.89 ? 0.50). in comparison to malignant astrocytomas, which have a mean k, in the range of 21.0 with some as high as 33.9, a 0 and 0 appear to be much less permeable. this suggests that the efficacy of pcv may be due to factors other than capillary transport, such as tumor-cell sensitivity. frameless stereotactic localizer gene h. barnett, donald w. komos, and charles p . steiner, cleveland, oh extent of tumor resection has been shown to correlate with prognosis in malignant gliomas. although frame-based stereotactic techniques can provide information regarding tumor margin, they are often unwieldy and require expensive and elaborate computing systems. a frameless stereotactic neurosurgical localizing system was designed that overcomes these liabilities. this armless, frameless, stereotactic pointing device provides real-time three-dimensional localization information during operation. in addition to assisting in placement of a trephine craniotomy, it allows volumetric resection of the tumor with virtually complete excision of even large irregularly shaped tumors. mean error on localizing a point in space using this system has proven to be less than 2 mm. a technical description of the system as well as surgical results are presented. to investigate the effect of age on response rate to chemotherapy and time to progression (ttp) and death ('itd) in patients with recurrent astrocytomas and malignant astrocytomas, we reviewed case records and scans of 143 patients who received chemotherapy at the university of michigan with bischloroethylnitrosourea or procarbazine. three age groups were studied: (1) <39 yr (n = 64); (2) 40-60 yr (n = 56); (3) >60 yr (n = 23). tumors were grouped as grade 2r+ 3 (recurrent grade 2 plus grade 3, n = 72) or grade 4 (n = 7 1). serial computed tomographic or magnetic resonance scans were analyzed in a blinded fashion and graded as progressive disease (pd), stable disease (sd), or partial response (pr, >25% decrease in size). the pr rates for the 3 age groups were 58%/32%/0% for grade 2r+ 3 tumors ( p = 0.023) and 40%/10%/5% for grade 4 tumors ( p = 0.011). median 'itp was 3112316 weeks for grade 2 r + 3 and 221 16/10 weeks for grade 4. median ltd was 53/48/19 weeks for grade 2r+ 3 and 36/31/24 weeks for grade 4. we conclude that age is an important prognostic factor with respect to likelihood of response to chemotherapy, duration of response, and survival irrespective of grade. cheryl p. harris and kurt a. jaeckle, salt lake city, ut intravascular malignant lymphomatosis (iml), a b-cell lymphoma confined to small venules and capillaries, often presents with neurological symptoms. this disease is uniformly fatal (5-month mean survival); no successful treatment has been identified. we observed marked reproducible neurological improvement after plasmapheresis in a 43-year-old woman with iml. presenting with a cauda equina syndrome, she progressed over 1 year with neurological, hepatic, and hematological disease. persistent laboratory abnormalities included a high sedimentation rate (140 mm/hr), coagulopathy, hemolytic anemia, and elevated liver enzymes. extensive evaluations for infectious, autoimmune, and neoplastic processes, including bone marrow examination, were inconclusive. because of neurological progression, empiric therapy with high-dose steroids followed by cyclophosphamide was initiated without response. plasmapheresis (250 ml/kg in 6 exchanges) effected resolution of encephalopathy and normalization of the coagulopathy and sedimentation rate. neurological progression recurred within 2 weeks of pheresis; 6 repetitive courses reproduced neurological response. finally, progressive dementia ensued, and a decision was made to cease pheresis; the patient died 5 days later, 16 months after presentation. autopsy disclosed diffuse intravascular cd-20 positive malignant lymphoma cells in small vessels of all organs. although the mechanism is unknown, the serendipitous discovery of response to plasmapheresis in this patient warrants further consideration. morphine is an effective analgesic in the rat after injection into a number of discrete brainstem regions, including the periaqueductal gray (pag), the locus coeruleus (lc), and the nucleus raphe magnus (nrm). early work with morphine gray and the nucleus raphe magnus established the existence of synergy between the brainstem and the spinal cord in rats. more recently, studies from our laboratory revealed synergy between two brainstem structures, the pag and the lc. in the current study, we explored the analgesic interactions between the pag and the nrm using indwelling cannulae. first, we established morphine dose-response curves and calculated the ed,, independently in the pag (1.9 pg) and the nrm (2.5 pg). we then simultaneously injected various morphine doses into both regions. injecting morphine at 1 pg into either the pag or the nrm did not elevate tailflick latencies above baseline values. however, administered into both regions simultaneously, the 2 1-pg doses produced an 80% maximal response, corresponding to more than a threefold increase of baseline latencies. a fixed morphine dose of 1 pg in the pag shifted the morphine dose-response curve fivefold in the nrm (ed,, 0.5 pg), whereas a fixed nrm dose of 1 fg shifted morphine's dose-response in the pag approximately twofold. together, these results clearly show synergistic interactions for morphine between the pag and the nrm. the presence of synergistic interactions between brainstem nuclei as well as between the brainstem and the spinal cord underscores the complexity of opioid analgesic systems. p244. the glutamate uptake inhibitor ~-trans-2,4-pyrrolidine dicarboxylate is neurotoxic in neonatal rat brain john d. e. barks and faye s. siloerstein, ann arbor, m i important evidence of the neurotoxicity of endogenous glutamate (glu) in mammalian brain was provided by the observation that dl-threo-3-hydroxyaspartate, a high-affinity glutamate uptake (hagu) inhibitor, was neurotoxic in adult rodent striatum (j neurochem 1985; 44:247) ; however, the absence of neurotoxicity in neonatal brain was interpreted as evidence that immaturity of glutamatergic innervation limited the potential role of endogenous glu as a neurotoxin in the immature brain. yet, considerable data provide indirect support for the hypothesis that glu can be neurotoxic at this stage. to resolve this issue, we assessed the neurotoxicity of a novel, selective hagu inhibitor, ~-trans-2,4-pyrrolidine dicarboxylate (l-pdc) (j med chem 1991;34:717), in postnatal day (pnd) 7 rats (n = 8). l-pdc (ph 7.4) was stereotaxically injected into right anterior striatum (str) (568 nrnol, n = 2) or through dorsal hippocampus into posterior str (568 nmol, n = 4; 150 nmol, n = 2). animals were killed 5 days later, and neuropathology was assessed in cresyl violet-stained sections. after anterior injections, focal neuronal necrosis was evident in dorsal str; high-dose posterior injections caused prominent hippocampal lesions with pyramidal layer thinning and focal necrosis in dorsal thalamus, while 150 nmol produced small foci of pyramidal cell loss. in both groups, focal cortical necrosis and callosal cysts were apparent adjacent to the injection track. l-pdc-induced brain injury provides direct support for the hypothesis that endogenous glu may be neurotoxic in the developing brain. (sax et al, ann neurol 1991; 30:311a) . the signs and symptoms of 7 of these patients lessened for 12 to 22 months. furthermore, a patient with a severe oral-lingual biting dyskinesia improved when taking doses up to 300 milligrams per day for 4 months. we noted no-significant adverse reactions, although 4 patients had mild but labile elevations in sgop and sgpt. one patient receiving opioid analgesics for pain inadvertently failed to discontinue the naltrexone but noted no reduction in the pain-alleviating effects of the analgesic. although hyperkinesia, especially of midline functions, as well as quality of life improved for the hd patients, their cognitive deficits remained unaffected. these observations suggest that chronic naltrexone is a safe and effective agent to treat chorea, dysphagia, and oral dyskinesia in hd for periods longer than a year. furthermore, they indicate that naltrexone can be effective in ameliorating oral-lingual biting tardive dyskinesia. these findings support our previous hypothesis that endogenous opioids play a role in rhe modulation of the dopamine system in hyperkinetic stereotypic movement disorders. a. jon stoessl, elizabeth szczutkmuski, and hanna fydvszak, london, ontario, canada we previously have demonstrated (psychopharmacology 1989; 98:372-379 ) that intraperitoneally (ip) administered cholecystokinin (cck)-8s suppresses vacuous chewing mouth movements (vcms, a putative mode1 of tardive dyskinesia) in rats exposed to chronic neuroleptics. as cck is not thought to cross the blood-brain barrier in significant amounts, its site of action in this paradigm is unclear. other behavioral and neurochemical effects of ip cck are blocked by vagotomy. male sprague-dawley rats were administered fluphenazine decanoate (flu; 25 mg/kg im) or its vehicle every 3 weeks for approximately 20 weeks. cck (10, 20, or 50 ng intracerebroventricularly) had no effect on neuroleptic-induced vcms. another group of neuroleptic-treated rats was subjected to bilateral subdiaphragmatic vagotomy or a sham procedure. cck-8s (10, 30, or 50 pg/kg intraperitoneally) suppressed neuroleptic-induced vcms in shamoperated animals, which confirmed our previous results. in vagotomited animals, chronic flu failed to induce vcms and cck was without effect in vehicle-or neuroleptictreated animals. these data suggest that the effects of cck on flu-induced vcms may be mediated peripherally, and that vagal pathways may be important for generating this response. (supported by the ontario mental health foundation and the ontario ministry of health.) p247. excitotoxic amino acids are not involved in dopaminergic neurotoxicity of m p t p eldad mehmed, jutta rosenthal, and avinoam reches, petah tiqva, tel aviv, and jerusalem, israel the dopaminergic (da) neurotoxicity of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp) is mediated via its oxidation in cns to mpp + , which enters da neurons and poisons mitochondrial complex i. da neuronal damage induced by direct nigral mpp+ injection is prevented by pretreatment with n-methyl-d-aspartate receptor antagonists, which suggests that excitatory amino acids are involved in mptp toxicity. since local mpp + application may produce nonselective nigral damage, we examined whether excitotoxins have a role in toxicity of systemically administered mptp. c57 black mice were injected intraperitoneally, once, with mptp.hci(40 mg/kg) and decapitated 5 days later. groups of animals underwent the following pretreatments: (i) decortication 1 week prior to mftp; (2) intracerebroventricular injections of the excitatory amino acid receptor antagonists 2-amino-phosphonoheptanoate and d-glutamyl-glycine; and (3) intraperitoneal injections of the calcium channel antagonists nimodipine, diltiazem, and flunarizine 30 minutes prior to mptp. mptp produced marked striatal da depletions. decortication, destroying glutamatergic corticostriatal projections, intracerebral amino acid receptor antagonists, and systemic calcium channel antagonists did not protect mice against mptp toxicity; mptp-induced striatal da decreases were similar to those given the neurotoxin alone. this study suggests that excitotoxins are not involved in the mechanism of mptp toxicity. scopolamine-induced cognitive deficits k . j . meador, m . e. allen, p. franke, e. e. moore, and d. w. loring, augusta, ga a unique neurophysiological role for thiamine in cholinergic systems has been suggested. total thiamine content in cholinergic nerve terminals is comparable to that of acetylcholine, and the phosphorylation state of thiamine changes with release of acetylcholine. thiamine binds to nicotinic receptors and may exhibit anticholinesterase activity. based on these observations, we investigated the effects of pharmacological doses of thiamine on the cognitive deficits induced by the anticholinergic scopolamine in 13 healthy young adults using a randomized, double-blind, placebo-conrrolled, double crossover design. cognitive tests included the p3 eventrelated potential and free recall memory for a verbal paragraph. conditions included baseline (bl), thiamine 5 gm by mouth and scopolamine .007 mg/kg intramuscularly (b1 + scop), and lactose by mouth and scopolamine (plac + scop). testing was performed 3 hours post thiamine or placebo, and 1.5 hours post scopolamine. thiamine significantly reduced the adverse effects of scopolamine on p3 latency (f [i, 121 = 11.84, p 5 .005) and percent recall memory ( f el, 12) = 10.62, p 5 .007). means (+sd) and p3 latency (ms) were bl = 335 (24), b1 + scop = 360 (29), and plac neurol 1990; 27: 186-192) . we previously reported that cff improved in 11 of 17 ms patients (65%) given el-970 orally in divided daily doses ranging from 7.5 to 52.5 mg (stefoski et al, neurology 1991; 41:1344 -1348 . subsequent analysis revealed that in 14 of the 17 patients cff improvements and reversals followed in a phase-locked trend the rising and falling serum concentrations of el-970, including 3 patients whose changes remained below the 15% increase needed to qualify for the improved category. these results resemble the phase-locked effects of temperature on neurological function in ms. the cff changes, because they so closely reflect variations in serum concentration, suggest that el-970 tissue levels closely follow those in serum and that el-970 rapidly crosses the blood-brain barrier. efficacy of el-970 in ms is also predicted to have a close relationship to serum levels. p250. development of a n internal standard detectable by proton and phosphorus-3 1 nmr and hplc w. e. klunk, k. panchalingam, r. j . mcclure, and j . w. pettegrnu, pittsburgh, pa comparison of quantitative results from different analytical techniques can prove difficult due to the peculiarities of the particular techniques and the lack of a common standard applicable to all of the techniques. recently, both in vivo and in vitro nuclear magnetic resonance (nmr) have been applied to the quantification of a large variety of metabolites. although nmr can be applied to the study of living tissue, the question arises of how this technique compares with more traditional techniques such as high-pressure liquid chromatography (hplc). although this question can be addressed partly by studying perchloric acid (pca) extracts, it is difficult to directly compare this in vitro nmr data with results from hplc. to address this question, we have developed an internal standard that can be quantified directly by in vitro phosphorus-3 1 nmr, proton nmr, and by 9-fluorenylmethyl chloroformate (fmoc) derivatization followed by separation by hplc. a variety of aminophosphonic acids were studied by 3'p nmr, 'h nmr, and hplc. promising compounds were added to pca extracts of human brain. the optimal compound was found to be 3-aminopropylphosphonic acid (app, ho3p-ch2-ch,-ch2-nh2). app is easily detectable by all 3 techniques, falls well outside of the region of interest in phosphorus-31 nmr, and is well resolved by proton nmr at 500 mhz. it is easily separable from the phosphomonoesters and phosphodiesters observable by hplc and from the amino acids that occur in brain in significant concentrations. app appears to be a useful internal standard in the study of phosphorus and amino acid metabolites by in vitro 31p nmr, 'h nmr, and hplc. theories of sentence comprehension hypothesize at least a grammatical component that establishes the relationship among words in a sentence, and a semantic component that determines the meanings of these words. we used positron emission tomography (pet) to quantify regional cerebral blood flow (rcbf) in 12 neurologically intact subjects during their detection of a letter target, a grammatical target, or a semantic target in the same written sentences. a mixedmodel analysis of variance (anova) revealed significant main effects for region (f e30, 210) = 19.32; p < .001), condition (f 12, 143 = 3 . 9 6 ;~ < .05), and a significant region times condition interaction ( f {go, 4203 = 1.46; p < .05), but there were no differences between individual subjects. subsequent anovas revealed increased rcbf in a unique set of brain regions during the subjects' response to a grammatical probe when compared to their response to a letter probe of the same sentences. a unique distribution of rcbf also distinguished response to the semantic probe from response to the grammatical probe and the letter probe. other brain regions apparently contributed to performance for several activation conditions. these findings support the hypothesized dissociation of specific linguistic components based on their unique cerebral topographical representation, and that a distributed network of brain regions subserves sentence comprehension. cerebral music processing-a comparison study of musically trained and naive individuals louis s. russo, jr, jachonville, fl we performed topographical mapping of brain electrical activity in 6 right-handed symphony musicians and 5 righthanded, musically naive individuals during various musical tasks; namely, listening to solo piano music, silent singing of familiar music, and silent reading of unfamiliar music. fast-fourier transform ( f r ) of electrocortical activity was carried out during task performance and the eyes-open resting state. data were analyzed using a computer-assisted model. increases in regional beta activity of greater than 3 standard deviations from the resting state were considered significant of activation. during audition, the musicians showed activation in the right posterior parietotemporal region; the naive showed no change from the resting state. during silent singing, the musicians showed bitemporal activation, r > l; the naive showed activation in the right mid-and posteriortemporal regions alone. during silent sight reading, the musicians showed a major activation in both temporal regions, l > > r, the naive showed only a marginal change in the posterior temporal-occipital regions. these data suggest that music processing is primarily a right cerebral function in untrained individuals and a bilateral function in musicians. musicians, in contrast to the naive, show progressively more left brain activation as task complexity increases. the present study analyzes language profiles in 10 patients who presented with primary progressive aphasia (ppa) without global dementia for at least 2 years. language and cognitive impairment were evaluated using the western aphasia battery (wab) and the mattis dementia rating scale (drs). expressive language disability with reduced speech fluency and anomia, but preserved language comprehension and nonverbal cognition, were typical features in early stages. spontaneous speech was significantly more impaired in ppa than in anomic aphasia after left-hemisphere stroke and in language impairment in probable alzheimer's disease (ad) ( p = 0.0004). the profile of aphasia suggests that ppa tends to affect anterior parts of the language-dominant cortex first. neuroimaging generally showed mild to moderate brain atrophy. in 2 patients atrophy involved especially the left frontal progressive aphasia cortex. follow-up examinations that were done in 7 patients 1 or several years after the first assessment revealed continuous, most often rapid deterioration of language impairment. two patients died 3 and 4 years after the onset of ppa. neuropathological examination showed ad in 1 patient and pick's disease in the other patient. beverly clarke, adrian upton, markad kamath, and helene griff;., hamilton, ontario, canada eight patients implanted with a cyberonics neurocybernetic prosthesis model 100 to stimulate the vagus nerve were assessed for changes in cognitive performance. the patients had complex partial seizures for more than 10 years, with more than 6 per month. patients were 34 years * 7.8 sd old. cognitive evaluation included response time to a randomized light signal appearing on a switch box (test a); test b, in which the signal appeared bilaterally; and test c, in which a response to the signal was required while the patient simultaneously ignored a second signal. data were collected and analyzed using an apple i1 e computer and switch pad. all patients were taking therapeutic levels of 3 anticonvulsant medications and dosages were constant. testing occurred 10 times during a day preoperatively (day l), 2 weeks postoperatively with the stimulator on (day 2), and 3 months after turn-on (day 3). patients were randomized into high-and low-frequency stimulation groups (hfg and lfg). hfg parameters were 30 hz, so0 msec pulse width (pw), and lfg 1 hz, 130 msec pw. examiners were blinded as to group. student's t-test analyses of mean differences between groups and individual measurements showed a significant difference between hfg and lfg for test c ( p < 0.05). lfg showed a significant improvement for tests a, b, c between day 1 and 2, and for test c between day 2 and 3. no group effect was seen between day 1 and 3 in the lfg. individual measurements showed improvement for test b ( p < 0.05) for the hfg between day 1 and 2, test b ( p < 0.0s) between day 2 and 3, and tests a and b ( p < 0.01) and ( p < 0.05) day 1 vs 1. the lfg group improved between days 1 and 2 and 2 and 3. between day 1 and day 3, the lfg showed improvement only for test b ( p < 0.001). chronic stimulation of the vagus nerve improves cognitive function in epileptic patients and this improvement is more marked with low-frequency stimulation. a. guidotti, and j. d. rothstein, bologna, itah, washington, dc, and baltimore, m d we reported idiopathic recurring stupor (irs) in a patient with stuporous episodes without known causes and reversed by flumazenil, a specific benzodiazepine (bz) antagonist. ictal plasmdcerebrospinal fluid (csf) showed increased bz-like activity (ann neurol, in press). recently, an endogenous bzreceptor ligand (endozepine [ez]) has been purified from mammalian brain with properties similar to diazepam. it acts like diazepam to potentiate gamma-aminobutyric acidmediated postsynaptic inhibition. we hypothesized that irs might be due to an excess of this substance. irs was diagnosed in 2 patients, 41 and 58 years old, who had recurring stupor or coma episodes lasting hours to days. ictal brain ct/mri, kidney, liver, heart, blood glucose, ammonia, and osmolality were normal. eeg showed fast 13-hz background activity while the patients were unreactive to stimuli, reversed by flumazenil. ictal serum or csf revealed an enormous increase of the ez in both patients, with levels as high as 400 nm, compared to 5 to 10 nm in control serum/csf. interictal csf or serum in irs contained ez levels similar to control csf and serum. irs may be due to excess ez. the cause for increased ez is unknown. parkinson's disease: evidence from word learning murray grossman, jenger mickanin, barbara schaefr, kris onishi, matthew b. stern, steven gollomp, and howard hurtig, philadelphia, pa several reports have suggested that patients with parkinson's disease (pd) have intellectual impairments in several domains such as memory, but few studies have explored difficulties in language processing. we investigated the ability of 20 nondemented pd patients with mild motor impairments to learn about the grammatical and semantic information represented in a new verb. the new verb was presented to patients in a sentence-picture matching context, and we probed their recall of the verb 10 minutes later. a sentence judgment task assessed grammatical knowledge by asking patients to judge the new verb, known verbs, and pseudowords used appropriately or incorrectly in a sentence. we found that 65% of pd patients were significantly impaired in their grammatical appreciation of the new verb (f [i, 331 = 17.03;p < 0.003). this was not related to their motor disorder or neuropsychological performance. a picture classification task used pictures illustrating specific aspects of the new word's meaning to evaluate semantic knowledge. pd patients were as accurate as controls at deciding whether a picture illustrated the meaning of the new verb (f (1, 331 = 0.lo;p > 0.10). only 1 pd patient (5%) had difficulty sorting pictures. selective difficulty recalling only grammatical aspects of a new word suggests that the word learning impairment in pd cannot be entirely explained by poor memory. instead, in agreement with other recent findings, pd patients may be impaired in some aspect of grammatical processing in language. we discuss the hypothesis that defects in the frontocaudate axis in pd underlie this impairment. neuropsychological performance on both verbal and nonverbal tasks is reported to differ between healthy men and women. some of these cognitive differences are postulated to reflect differences in interhemispheric and intrahemispheric cerebral organization. our preliminary study indicated that women with alzheimer's disease (ad) performed worse than men on a composite neuropsychological battery, even after effects of potentially confounding variables were considered (buckwalter et al, j clin exp neuropsychol 1992; 14:23) . to explore further the nature of gender-associated differences in ad, we analyzed data from a verbal and a nonverbal task (the boston naming test and drawings from the spatial quantitative battery supplement to the boston diagnostic aphasia examination) for 22 men and 23 women who met nincds-adrda criteria for "probable" ad. prior to ana-grip strength 9 kg [range 1.5-20 kg]). the electrophysiological examination showed improvement with reversal of conduction block in 3 patients and was unchanged in 4. an apparent response to the placebo was seen in 3 patients. improvement after ivig therapy was maintained for variable durations (3-20 wk) and reoccurred with subsequent infusions. an equally effective response was documented after infusion of a single ivig dose of 1 gm/kg. we conclude that ivig therapy is effective in some patients with cidp, even after long duration of illness. the best responses were observed in patients with recent relapse. a single high-dose treatment may be equally effective. the object of this study was to examine whether borrelia burgdorjeri antigens could be detected in csf in the absence of detectable antibodies to b. burgdorjeri (the etiological agent of lyme disease). osp a is a 31-kd antigen that is specific for 8. duvgdorferi osp a was probed using western (immuno) blot and specific mouse monoclonal antibodies. polyclonal lyme antibodies were detected in csf using standard micro enzyme-linked immunosorbent assay. seven patients had osp a in csf without detectable lyme antibodies. there were 3 men and 4 women aged 20 to 58 years (mean 38 yr). disease duration ranged from 2 weeks to 8 years. neurological syndromes included confusion with acute flulike illness, optic neuritis, hemiparesis with inflammatory brain lesion, encephalitis, headache with erythema migrans, bilateral facial nerve palsies, and encephalomyeloradiculitis. three patients had csf abnormalities. in 4 patients csf parameters were otherwise completely normal. possible explanations for undetectable csf lyme antibodies included early infection (3 patients), prior antibiotics (2 patients), and prior steroids plus antibiotics (1 patient). in 1 patient there was no obvious explanation. we conclude that osp a, a specific antigen of b. burgdorferi, may be present in csf without a detectable humoral response. the diagnosis of neurological infection with b. bwgdorjeri should not require a positive csf serology. mollaret's meningitis: demonstration by polymerase chain reaction a 29-year-old man was seen in september 1991 for his fourth episode of aseptic meningitis over a 4-year period. the episodes conformed to criteria for mollaret's meningitis as published by bruyn, straathof, and raymakers, and subsequently by others; they lasted about 1 week, and were characterized by fever, headache, meningismus, lymphocytic pleocytosis, elevated protein in cerebrospinal fluid (csf), and spontaneous resolution without residua. extensive prior evaluations had failed to uncover a cause. the patient was otherwise well and neither he nor his wife had any history of sexually transmitted diseases. suspicion of herpes simplex virus (hsv) arose due to a single transient, raised skin rash several weeks earlier that failed to yield virus on culture. the patient was treated with acyclovir, which resulted in rapid resolution of symptoms. though culture and immunological studies of csf and blood again were unrevealing, polymerase chain reaction (pcr) studies of csf confirmed the presence of hsv type 2. we suspect that herpes simplex virus is a more common cause of recurrent aseptic meningitis than current culture and immunological techniques would suggest. pcr offers increased diagnostic sensitivity for neurotropic viruses and should be considered in patients with recurrent meningitis of cryptic etiology. differentiation by inorganic lead: a role for protein kinase c j. lutewa, j. p. bressler, r. r. indurti, l. belloni-olivi, and g. w . goldstein, baltimore, m d microvascular endothelial function in developing brain is altered by inorganic lead. this may result from changes in protein kinase c (pkc) modulation. we examined the effects of inorganic lead on an in vitro model of neural endothelial differentiation. astroglial-induced endothelial differentiation into capillary-like structures was inhibited by lead acetate with 50% maximal inhibition occurring at 0.5 pm lead. inhibition was independent of effects on cell viability or growth. we examined the effects of lead on cellular pkc pools under conditions that inhibited capillary-like structure formation. membranous pkc increased in c6 astroglial and neural endothelial cells after exposure to lead acetate. exposing c6 cells to 5 p,m lead for 16 hours increased membranous pkc by 150% as determined by immunoblotting. membranous pkc increased in response to as little as 50 nm lead and saturated at 1 pm. phorbol esters were used to determine if pkc modulation was mechanistically related to lead's inhibition of capillary-like structure formation. 12-myristate 13-acetate (10 nm) inhibited endothelial differentiation by 65 * 5%, whereas 4-alpha-phorbol 12,13didecanoate was without effect. these findings demonstrate that inorganic lead may induce cerebral microvessel dysfunction by interfering with pkc modulation in microvascular endothelial or perivascular astroglial cells. w. f. brown, b. v . watson, j . garland, g . c. ebers, and n . desai, london, ontario, canada gait difficulties in multiple sclerosis (ms) are commonly accompanied by fatigue and dyspnea. possible explanations for the latter include weakness andlor dyssynergia of the respiratory muscles, including possible abnormalities in central pathways regulating respiration. this study examined central and peripheral motor conduction to the diaphragm in 15 ms patients whose gait was notably labored and accompanied by breathlessness. peripheral conduction was assessed by measuring the latency and size of the surface-recorded diaphragmatic maximum m-potential responses to supramaximal stimulation of the phrenic nerve in the neck, and central motor conduction by comparable measurements in response to magnetoelectrical stimulation over the vertex. peripheral motor conduction was normal. the most striking abnormalities were in central motor conduction. cortical stimulus-evoked diaphragmatic responses were absent on both sides in 3 patients, and unilaterally in l patient, whereas in 5 others the latency of the cortical stimulus-evoked response was increased and the size clearly reduced and entirely normal in only 3 patients. these studies show that central conduction program and abstracts, american neurological association 295 to the diaphragm is commonly abnormal and may play a role in the fatigue and dyspnea experienced by ms patients. six men (40-53 yr) developed myelopathy that progressed slowly over several months and was characterized by asymmetrical, incomplete spinal cord syndrome manifested at the sensory level at the trunk, mild spastic paraparesis, and urinary incontinence. the spinal cord lesions at appropriate levels were recognized by mri as enhancing lesions in 4 of the men. coxiella burnetii infection was confirmed in the blood of all patients by immunofluorescence microscopic assay (ifa) and transmission electron microscopy (tem). in 2 patients, we detected c. burnetti by tem and ifa using csf of the patients inoculated onto fresh peripheral blood lymphocytes. four patients who were treated with appropriate antibiotics responded with either partial resolution of symptoms or arrest of further neurological progression. in 3 patients the lesion was shown on mri to have decreased in size. in summary, we report 6 cases of transverse myelopathy associated with c. barnetti infection. this is the first report, to our knowledge, of coxiella-related chronic myelopathy. we present a series of patients with different labyrinthine lesions diagnosed by mri. twelve patients with sensorineural hearing loss were studied by gadolinium-enhanced mri, including 3-mm contiguous t1-weighted images through the labyrinth. ten patients had enhancement of the cochlea or vestibule, or both. all patients with cochlear enhancement had severe neural sensory hearing loss. all patients with vestibular enhancement had severe vestibular symptoms. the patients' final diagnosis included viral labyrinthitis (3 patients), syphilitic labyrinthitis (2 patients), bacterial labyrinthitis (1 patient), and vestibular neuromas (3 patients). one patient had an acoustic neuroma extending in the basal turn of the cochlea. the enhancement in patients with vestibular neuromas was brighter and there was slight mass effect in comparison with the patients with inflammatory labyrinthine lesions. one patient had hemorrhage within the vestibule from an adjacent temporal bone hemangioma. one patient with ct-proven cochlear otosclerosis had pericochlear areas of enhancement on gadolinium mri. mri can diagnose a variety of labyrinthine lesions that correlate very well with the patient's clinical symptoms. gadolinium should be used routinely in patients with suspected labyrinthine disease. the diagnosis of meniere's disease and endolymphatic hydrops remains a diagnosis of exclusion. few radiographic findings have been correlated with the clinical symptoms of this entity. we describe 9 patients with symptoms of hearing loss or vertigo, or both, who demonstrated enhancement of the endolymphatic sac on gadolinium-enhanced mri. no enhancement was noted in a series of 20 controls with no symptoms of hearing loss and vertigo. enhancement in the brain correlates with inflammatory or neoplastic conditions. we thus can speculate that enhancement of the endolymphatic sac reflects an inflammatory process in this location that may interfere with the normal resorption of indulin and secondary hydrops. in addition to excluding an acoustic neuroma and a labyrinthine schwannoma (which clinically may be confused with meniere's disease), contrast-enhanced mri may provide objective evidence in favor of labyrinthine hydrops. it was intermittent (62%) but progressive. the ha was mild to moderate in severity; it was the worst symptom in only 24 (459%) and the first symptom in 32 (60%) patients. has were worse in the morning in 19 (36%) and interfered with sleep in 17 (32%) patients. unlike true tension-type has, bt has were worse with bending over in 17 (32%), with valsalva's maneuver in 12 (23%), and nausea or vomiting were present in 21 (40%) patients. an abnormal neurological exam was found in 38 (72%) patients with has and 43 (74%) patients without has lyzing the effects of gender, we used a hierarchical regression procedure to control for possible effects of subject age, education, age at onset of dementia symptoms, dementia duration, and family history of dementia. significant gender effects were found for the verbal task ( p < 0.005) (mean boston naming test score of 2 1.4 for women and 34.4 for men), but not for the drawing task. we conclude that verbal abilities are more severely affected in women than in men with ad, a difference that may in part reflect premorbid gender-associated differences in cerebral hemispheric organization. hemispatial placement is known to affect line bisection in patients with neglect. whereas placing stimuli in neglected space increases bisection error, placing stimuli in nonneglected space attenuates error. the effects of hemispatial placement on line bisection were examined in 4 patients with chronic neglect (over 5 months after stroke). all patients had large (frontotemporoparietal), unilateral, right-hemisphere lesions. each patient bisected lines of different lengths (24, 26, 28 , and 30 cm) in 3 hemispatial conditions (30 cm left of midline, midline, and 30 cm right of midline). like previous reports, when patients bisected lines in left hemispace, a consistent (20/24 trials) left-sided neglect was observed (2.6 cm). however, when lines were bisected in center space, misbisections occurred on either side of the midline; and, unlike previous studies, when lines were bisected in right hemispace, a consistent (20/24 trials) right-sided neglect was observed (2.0 cm). the magnitude and directional consistency of line bisection errors were significant. neither visual field defects nor limitations in reaching accounted for the results. recovery in chronic neglect may involve a realignment of limited attentional resources favoring the body's midline. consequently, performance in both hemispatial fields can be biased toward midline, resulting in neglect of opposite directions. despite agreement that depression is the most common neuropsychiatric symptom associated with multiple sclerosis (ms), many aspects of this emotional change are unclear. one of the more controversial issues concerns the relationship between severity of ms and depression. this relationship is used to evaluate whether depression is an integral or reactive symptom of ms. examination of this relationship is complicated by the presumed overlap between somatic features of depressive and neurological symptoms in ms. to clarify this situation, we examined the relationship between severity of ms and 4 categories of depressive symptoms using the beck depression inventory (bdi). eighty-nine patients and 47 normal controls were examined. for certain comparisons, patients were classified as mild (extended disability status scale of 0-2) or moderatelsevere (3) (4) (5) (6) (7) (8) . results indicated that total bdi scores and the depressive symptom categories (mood, self-reproach, vegetative, and somatic features) were elevated in patients with ms, but the extent of these elevations was not related to severity of disease. these results suggest that depression in ms is not a simple reaction to physical disability. furthermore, clinical examination of depressive symptoms is straightforward and not confounded by severity of ms. neurological involvement in wegener's granulomatosis was studied in 324 consecutive patients diagnosed at the mayo clinic. one hundred and nine patients (34%) had neurological involvement. peripheral neuropathy was seen in 53 (16.4%), cranial neuropathy in 2 1, external ophthalmoplegia in 16, cerebrovascular events in 13, seizures in 10, and miscellaneous involvement in 25. the mean age and sex ratio did not differ in those with or without neurological involvement. among the patients with peripheral neuropathy, 42 had multiple mononeuropathy, 6 had distal symmetric polyneuropathy, and 5 had unclassified peripheral neuropathy. multiple mononeuropathy was one of the major presenting symptoms in 8 patients. kidney involvement was significantly higher in the patients with peripheral neuropathy compared to those without it (p < 0.001). among the cranial nerves, the second, sixth, and seventh nerves were affected most frequently. multiple cranial nerves were affected in 8 patients. unusual neurological manifestations among the miscellaneous group included spastic paraparesis, temporal arteritis, homer's syndrome, and papilledema. this is the first comprehensive study on the frequency and distribution of neurological involvement in wegener's granulomatosis. chronic inflammatory demyelinating polyneuropathy: a double-blind placebo-controlled crossover study treatment with high-dose intravenous human immunoglobulin (ivig) has been reported to be beneficial in some patients with chronic inflammatory demyelinating polyneuropathy (cidp), yet most observations have been nonblinded. we examined the effect of ivig therapy in 10 patients (6 men, 4 women) with cidp in a double-blind, placebo-controlled crossover study. disease was chronic progressive (n = 5 ) or chronic relapsing (n = 5) and of variable duration (4 mo to 2 1 yr). the diagnosis was confirmed by electrophysiological (10) and nerve biopsy (7) examinations. the trial consisted of two 28-day periods each. patients were randomly treated with ivig (0.4 mg/kg/day) or placebo on 5 consecutive days and followed. function was assessed by a quantitative neurological disability score, functional grade, grip strength measurement, and electrophysiological examinations at the beginning and end of each treatment period. with ivig therapy, significant improvement was documented in 7 / 10 patients (improvement in neurological disability score mean key: cord-030371-wp6xmaqe authors: kubota, kazuo; ogawa, mikako; ji, bin; watabe, tadashi; zhang, ming-rong; suzuki, hiromi; sawada, makoto; nishi, kodai; kudo, takashi title: basic science of pet imaging for inflammatory diseases date: 2019-12-21 journal: pet/ct for inflammatory diseases doi: 10.1007/978-981-15-0810-3_1 sha: doc_id: 30371 cord_uid: wp6xmaqe fdg-pet/ct has recently emerged as a useful tool for the evaluation of inflammatory diseases too, in addition to that of malignant diseases. the imaging is based on active glucose utilization by inflammatory tissue. autoradiography studies have demonstrated high fdg uptake in macrophages, granulocytes, fibroblasts, and granulation tissue. especially, activated macrophages are responsible for the elevated fdg uptake in some types of inflammation. according to one study, after activation by lipopolysaccharide of cultured macrophages, the [(14)c]2dg uptake by the cells doubled, reaching the level seen in glioblastoma cells. in activated macrophages, increase in the expression of total glut1 and redistributions from the intracellular compartments toward the cell surface have been reported. in one rheumatoid arthritis model, following stimulation by hypoxia or tnf-α, the highest elevation of the [(3)h]fdg uptake was observed in the fibroblasts, followed by that in macrophages and neutrophils. as the fundamental mechanism of elevated glucose uptake in both cancer cells and inflammatory cells, activation of glucose metabolism as an adaptive response to a hypoxic environment has been reported, with transcription factor hif-1α playing a key role. inflammatory cells and cancer cells seem to share the same molecular mechanism of elevated glucose metabolism, lending support to the notion of usefulness of fdgpet/ct for the evaluation of inflammatory diseases, besides cancer. fluorine-18-labeled 2-deoxy-2-fluoro-glucose (fdg) is used as a radiopharmaceutical in pet for evaluating glucose metabolism, and accumulates in malignant tissues because of the enhanced glucose utilization by neoplastic cells. because of the increased metabolic demand for glucose, elevated activity of hexokinase and elevated expression of glucose transporter have been shown in tumor tissues [1] . various applications of fdg-pet have been extensively studied in the field of clinical oncology, and the imaging modality, now used worldwide, is recognized as a powerful diagnostic modality for cancer [2, 3] . in 1989, elevated fdg uptake was reported in two patients with abdominal abscesses [4] . this was followed by reports of fdg uptake in brain abscess [5] , tuberculosis, aspergillosis, sarcoidosis, and so on. in addition, early postoperative scarring and early inflammatory reactions after radiotherapy were also reported to show increased fdg uptake. thus, elevation in glucose metabolism is not only specific for cancer but also seen in inflammation. because fdg uptake is seen in benign inflammatory diseases as well, the accuracy of fdg-pet for the diagnosis of cancer is not 100% [6] . although the metabolic fate of fdg is well known, its cellular distribution within tumors or sites of inflammation has not yet been described. to clarify the mechanisms of fdg uptake by inflammatory and tumor tissues, we performed autoradiographic studies. to demonstrate the cellular localization of fdg and [ 3 h]2dg uptake by tumors in vivo, c3h/ he mice with subcutaneously transplanted fm3a tumors were studied 1 h after intravenous injection of fdg or [ 3 h]2dg. newly formed granulation tissue around tumors and macrophages, which had massively infiltrated the marginal areas surrounding the necrotic areas of the tumor, showed a higher fdg uptake than the viable tumor cells. a maximum of 24% of the glucose utilization was derived from the non-neoplastic tissues in these tumors ( fig. 1.1) . the strong accumulation of fdg in these tumors was thought to represent both the high metabolic activity of the viable tumor cells and that of the tumor-associated inflammatory cells, especially activated macrophages. these results indicate that not only glucose uptake by the tumor cells but also that by non-neoplastic cellular elements which appear in association with the growth or necrosis of tumor cells should be considered for a precise analysis of fdg uptake in tumors, especially after radiotherapy ( fig. 1. 2) [7, 8] . fdg uptake by inflammatory tissues was investigated by yamada et al. [9] . a rat model of chemically induced inflammation using turpentine oil was used. a time-course study of the fdg tissue distribution showed that the uptake of fdg in inflammatory tissue increased gradually until 60 min, followed by a steady decrease thereafter. a longitudinal study showed that the uptake increased progressively after the start of inflammation, peaking at 4 days after the inoculation, and then gradually decreased ( fig. 1.3) . these findings suggested that fdg uptake may reach a maximum during the subacute phase of inflammation, and then slightly decreases during the chronic phase of inflammation. an autoradiography study showed a high fdg uptake in the abscess wall, consisting of an inflammatory cell layer and granulation tissue. at the cellular level, the highest radioactivity was found in the marginal zone that contained young fibroblasts, endothelial cells of vessels, and phagocytes consisting of neutrophils and macrophages, followed by that in the neutrophil layer and the granulation tissue layer ( fig. 1.4) . fdg uptake by inflammatory tissue seems to represent the activity of immune cells and fibroblasts mobilized to the lesion. mochizuki et al. [10] compared the fdg uptake by experimental hepatoma and by tissue with experimental infection with staphylococcus aureus. the expressions of glut1 and glut3 were also studied in both the tumor and infected tissue by immunostaining. uptake by the tumor tissue was significantly higher than that by the infected tissue. both the tumor and infected tissue showed strong expression of glut1 and glut 3, although the expression level of glut1 was significantly higher in the tumor than in the infected tissue. glut1 may be responsible for fdg uptake in both tumor tissue and infected tissue. zhao et al. [11] compared the two models of inflammation: bcg-induced granuloma simulating sarcoidosis and turpentine oil-induced inflammation. fdg uptake by the granuloma was significantly higher than that by the tissue with chemical-induced inflammation. their results explained the very high fdg uptake in sarcoidosis, equivalent to the uptake in cancer. however, the main objective of their study was not to compare the models of inflammation, but to compare the uptakes of 14 c-methionine, 18 f-fluorothymidine, and fdg. 14 c-methionine showed the best results for discrimination between tumor and inflammation. deichen et al. [12] studied the uptake of fdg in isolated human monocyte-macrophages (hmms) in vitro. fdg uptake by the hmms increased significantly with the duration of culture, the percent id/100 μg being 7.5% ± 0.9% (% id/100 μg) on day 14. stimulation by lipopolysaccharide further enhanced the fdg uptake in the hmms by a factor of 2. radio-thin layer chromatography of intracellular metabolites revealed that the fdg was trapped by the hmms mainly as fdg-6-phosphate and fdg-1,6-diphosphate. fdg uptake by the hmms was almost equal to that by human glioblastoma and pancreatic carcinoma cells. malide et al. [13] reported the changes in the subcellular localization of the glucose transporter proteins glut-1, glut-3, and glut-5 as the human monocytes differentiated into macrophages in culture, and the effects of the activating agents n-formyl-methionyl-leucylphenylalanine (fmlp) and phorbol myristate acetate (pma). western blot analysis demonstrated progressively increasing glut-1 expression, rapidly decreasing glut-3 expression, and a delayed increase of the glut-5 expression during the differentiation process. confocal microscopy revealed that each isoform exhibited a unique subcellular distribution and cell-activation response. glut-1 was localized primarily at the cell surface, but was also detected in the perinuclear region, in a pattern characteristic of recycling endosomes. activation with fmlp induced similar glut-1 and glut-5 redistributions from the intracellular compartments toward the cell surface. addition of pma elicited a similar translocation of glut-1, but glut-5 was redistributed from the plasma membrane to a distinct intracellular compartment that appeared connected to the cell surface. these results suggest specific subcellular targeting of each transporter isoform and differential regulation of their trafficking pathways in cultured macrophages. not only macrophages but also neutrophil granulocytes play a key role in the pathogenesis of various types of inflammation. jones et al. [14] studied [ 3 h]dg uptake in neutrophils isolated from human peripheral blood. they found elevated uptake of [ 3 h]dg by neutrophils, both after priming with tumor necrotic factor-alpha (tnf-α) and after activation with fmlp. it was not correlated with the respiratory burst or secretory activity, but may reflect the polarization and migrational status of these cells. their study suggested that as far as neutrophils are concerned, priming is the cellular event predominantly responsible for the fdg uptake. ishimori et al. [15] studied immunocompetent balb/c mice and nude mice administered an intravenous injection of 10 mg/kg of concanavalin a (con a). after the injection, the con a-activated lymphocytes actively took up fdg both in vitro and in vivo, and fdg specifically accumulated in the tissues showing con a-mediated acute inflammation in the immunocompetent mice. rheumatoid arthritis (ra) is an autoimmune disorder of unknown etiology and is characterized by systematic, symmetric, and erosive synovitis. ra synovitis is characterized by massive leukocyte infiltration, proliferative synovial membranes, and neovascularization, which give rise to a synovial proliferative fibrovascular tissue known as a pannus. formation of pannus is directly responsible for the cartilage and bone destruction [16] . matsui et al. [17] reported the mechanism of fdg accumulation in ra in vivo using a murine collagen-induced arthritis model, as well as [ 3 h] fdg uptake in vitro using various cell types. they showed that fdg accumulation increased with the progression of joint swelling. fdg uptake began in the area of inflammatory cell infiltration and synovial cell hyperplasia, and the areas showing strong fdg uptake often coincided with the areas where mixed cellular patterns of macrophages and fibroblasts as well as bone destruction by mature osteoclasts were visible. these findings indicated that fdg accumulation reflected the characteristic changes of pathological progression, such as pannus formation and bone destruction. based on the findings of in vitro experiments, matsui et al. suggested that the cell types responsible for fdg uptake were mostly proliferating fibroblasts, with lesser contributions from activated macrophages. fdg uptake by fibroblasts was enhanced in the presence of cytokine stimulation and hypoxia within a joint. hypoxia is a known feature of the microenvironment in inflamed joints [18] . recently, garcia-carbonell et al. directly compared fibroblast-like synoviocytes (fls) from ra patients and osteoarthritis patients [19] . in vitro experiments have shown that the fls from ra patients were dependent on glycolysis rather than on oxidative phosphorylation, and this difference was more pronounced than that for the fls from osteoarthritis patients. the expression of glut-1 messenger rna was correlated with the functions of the fls from the ra patients. these studies showed the importance of fibroblasts in the inflammation in ra. recently, the processes, at the molecular level, occurring in association with hypoxia and glucose metabolism within tumor and inflammatory tissues have been described. cramer et al. reported that activation of hypoxia-inducible factor one-alpha (hif-1α) is essential for myeloid cell infiltration and activation in vivo [20] . they showed that hif-1α is essential for regulation of the glycolytic capacity of myeloid cells; when hif-1α is knocked out, the cellular atp pool decreases drastically. this metabolic defect results in a profound impairment of myeloid cell aggregation, motility, invasiveness, and bacterial killing at sites of inflammation where the tissue environment is hypoxic. thus, hif-1α has a direct regulatory effect on both the survival and functioning of cells in inflammatory microenvironments. furthermore, the enhanced glycolysis in activated macrophages results in elevation of the fdg uptake. regarding the molecular mechanisms responsible for the regulation of glycolysis, hif-1α affects both glucose transporter and hexokinase expressions in tumors and inflamed tissues. the significance of hypoxia in tumor pathophysiology has also been described by denko [21] . usually, proliferation of cancer cells is faster than the growth of capillaries, resulting in inadequate perfusion and hypoxia within the tumor. activation of hif-1α in the hypoxic tumor shifts the energy metabolism from oxidative phosphorylation to anaerobic glycolysis, saves oxygen, and avoids massive cell death. as a result of the elevated glucose metabolism, fdg-pet serves as a useful imaging modality for cancer patients. dominant inflammatory cells activated and responsible for fdg uptake in inflammatory diseases may differ depending upon the disease. all inflammatory cells and cancer cells seem to share the same molecular mechanism of elevated glucose metabolism, lending support to the idea of the usefulness of fdgpet/ct also for the evaluation of inflammatory diseases, besides cancer. although a guideline [22] has been proposed, use of fdgpet/ct for inflammatory diseases is still limited. i hope that this book will help spread knowledge about the application of fdgpet/ct for inflammatory diseases to improve patient management, representing an advance in the field of medicine. atherosclerotic plaques are classified into two types: stable and vulnerable. vulnerable plaques are easy to rupture and may cause stroke and heart attack. therefore, the prompt detection and treatment of vulnerable plaques, prior to the manifestation of symptoms, is of crucial importance. vulnerable plaques are characterized by a lipid-rich atheromatous core, which is infiltrated by macrophages. there is a correlation between the number of infiltrating macrophages and the severity of symptoms in acute myocardial infarction [23, 24] . in contrast, infiltration by macrophages is rarely observed in stable plaques. macrophages are active in energy metabolism. therefore, fdg-pet is a promising tool for the detection of vulnerable plaques, depending on the extent of macrophage infiltration. thus far, numerous clinical and nonclinical studies have investigated the use of fdg for the imaging of vulnerable plaques. in 1994, kubota et al. demonstrated that fdg accumulated more in macrophages than in tumor cells [25] . therefore, it is assumed that vulnerable plaques can be detected depending on the rate of macrophage infiltration in the plaque. in the early 2000s, several clinical studies suggested that it is possible to detect atherosclerosis, according to the level of inflammation [26] [27] [28] . notably, through histological analysis of symptomatic carotid artery plaques, rudd et al. reported the co-localization of [ 3 h]fdg and macrophages [27] . the investigators examined the relationship between the degree of macrophage infiltration and accumulation of fdg in watanabe heritable hyperlipidemic (whhl) rabbits-an arteriosclerotic animal model-, and explored the possibility of detection of vulnerable plaques using fdg-pet [29] . the atherosclerotic plaques were detected by fdg-pet ( fig. 1.5 ). in whhl rabbits, thickening of the intima and infiltration by macrophages was observed in all individuals. we examined the degree of macrophage filtration via histological analysis. the results showed that the fdg uptake and number of macrophages in the atherosclerotic lesions were strongly correlated ( fig. 1.6 ). however, there was no correlation between the degree of macrophage infiltration and that of intimal thickening. these results suggested that macrophages are responsible for the accumulation of fdg in atherosclerotic lesions, and vulnerable plaques may be detected through fdg-pet. studies in humans have shown that the uptake of fdg into vulnerable plaques correlates with the infiltration by macrophages [30, 31] . immunohistochemical staining of macrophage marker cd68 revealed a strong correlation with fdg accumulation. it has also been reported that the incorporation of matrix metalloproteinase-1-an enzyme involved in the destabilization of plaques-and fdg are strongly related [32] . tahara et al. reported correlations between the accumulation of fdg and waist circumference, hypertension, insulin resistance, lowering of high-density lipoprotein cholesterol, etc. [33] . a recent study showed a correlation of fdg accumulation with c-reactive protein [34] . the results of these studies indicate the prospect for the use of fdg-pet in arteriosclerosis-related diseases. foam cell formation is responsible for the vulnerability of plaques. macrophages are recruited from blood monocytes that enter through the endothelium ( fig. 1.7) . scavenger receptors on macrophages mediate the uptake of oxidized low-density lipoprotein (ldl) and cause the accumulation of ldl-derived cholesterol and foam cell formation. foam cells release several proteases and cytokines, which lead to rupture of the plaques. we evaluated the effects of foam cell formation on the uptake of fdg [35] . macrophages were isolated from the peritoneum cavity of mice, and foam cells recent studies have described that polarization of macrophages affects the development of atherosclerosis and rupture of plaques. classically activated m1 macrophages are considered to possess the most proatherogenic phenotype and promote the destabilization of atherosclerotic plaques. in contrast, alternatively activated m2 macrophages possess anti-inflammatory properties and stimulate reparative processes, which lead to the stabilization of atherosclerotic plaques [37, 38] (fig. 1.8) . therefore, the detection of m1 macrophages may assist in predicting cardiovascular events with greater accuracy. in our investigation, m1 macrophages showed a 2.6-fold increased uptake of [ 3 h] fdg versus m2 macrophages [39] . glucose transporter (glut)-1 and glut-3, which are major isoforms of a glucose transporter in macrophages, were significantly upregulated in m1 macrophages versus m2 macrophages. to date, numerous drugs have been developed for the treatment of atherosclerosis. the therapeutic effects of these agents are usually monitored by determining the levels of lipids in the blood. however, lipid-lowering therapy does not always lead to the stabilization of vulnerable plaques. several statins may effectively reduce the levels of cholesterol in the plasma; however, they do not decrease the degree of macrophage infiltration [40, 41] . thus, merely monitoring the levels of lipids in the plasma is not sufficient to determine the therapeutic effect of drugs. macrophage infiltration plays an essential role in the rupture of plaques. therefore, the administration of pharmacological therapy that reduces macrophage infiltration is required to stabilize the vulnerable plaques. considering the individual differences in the stabilization of plaques induced by such drugs, monitoring the therapeutic effect in each individual plaque is important to accurately assess the effect. using probucol as a therapeutic drug in whhl rabbits, we investigated the usefulness of fdg-pet for the monitoring of therapies that target vascular inflammation. in a number of animal studies, the lipid-lowering effect of probucol was moderate, and the drug did not decrease the levels of cholesterol in the plasma [42, 43] . however, probucol can reduce the macrophage-rich plaques even in advanced atherosclerotic lesions [43] . in the present study, the uptake of fdg was significantly decreased in the probucol group versus the pretreatment period ( fig. 1.9 ). however, there was no difference in the levels of cholesterol in the plasma between the probucol and control groups [44] . a large number of macrophages was observed at the initiation of the study. nevertheless, treatment with probucol for 6 months resulted in diminished macrophage infiltration ( fig. 1.10) . notably, the ratio of the intima to the whole cross-sectional area was not affected by treatment with probucol. hence, the observed decrease in the uptake of fdg was attributed to the decrease in the number of infiltrating macrophages. collectively, these studies showed that the therapeutic effect of probucol was successfully monitored by fdg-pet independently of the cholesterol-lowering effect. these results demonstrated the usefulness of fdg-pet for drug development, and numerous clinical studies have been performed [45] [46] [47] [48] [49] . tahara et al. conducted one of the initial clinical trials, showing that the therapeutic effect of simvastatin was successfully monitored by fdg-pet [50] . in 2011, the results of the first clinical multicenter study investigating the efficacy of dalcetrapib against atherosclerotic disease via a novel noninvasive multimodality imaging technique (dal-plaque) were reported. of note, fdg-pet was used to evaluate the level of vascular inflammation [51] . moreover, in the dal-plaque study, the relationship between serum inflammatory biomarkers and plaque inflammation was also assessed using fdg-pet [52] . recently, several pet imaging tracers-apart from fdghave been reported for the imaging of vulnerable plaques. [60] [61] [62] [63] . recently, the β-amyloid imaging agent [ 18 f]flutemetamol showed specific accumulation in human carotid plaques, especially in amyloid betapositive areas [64] . among these tracers, [ 18 f]naf is considered a promising probe for the detection of atherosclerosis, and several comparison studies with fdg have been performed. the results of these studies showed that the area of accumulation differs between fdg and [ 18 f]naf [65] [66] [67] . the accumulation of [ 18 f]naf in atherosclerotic plaques depends on the level of calcification [68] . the [ 18 f]naf-positive area does not completely match with the ct-positive area, and it is thought that [ 18 f]naf accumulates in the early stage of calcification (i.e., microcalcification) [60, 69] . since target molecules are different among these tracers, comparison study should be important to elucidate the specificity of these tracers for the different stages of atherosclerosis progression. this may assist in understanding the mechanism of plaque progression and provide important insight into therapy aimed toward the stabilization of plaques. abstract neuroinflammation is a general event in acute and chronic neurodegenerative disorders. based on the critical role of neuroinflammation characterized by glial activation in neuropathogenesis, in vivo imaging with positron emission tomography (pet) is required in clinical and preclinical studies for the purposes of elucidation of pathogenesis and novel treatment development, because it is commonly available in human and experimental animal models. as a most widely used imaging biomarker for neuroinflammation, 18 kda translocator protein (tspo) imaging has been performed in a large number clinical and preclinical studies. neuropathologyassociated tspo induction has been generally detected in various neurodegenerative animal models with acute and chronic neuroinflammation. however, studies with human subjects showed confusing results likely due to ineffectiveness of the tracers used or impediment of non-microglial tspo expression in human diseased brains. based on the above reasons, recently, alternative molecular targets for microglia imaging instead of tspo have been proposed. colony-stimulating factor 1 receptor (csf1r) is a promising candidate because of its highly specific expression in microglia in the central nervous system (cns). purinergic receptors p2x7r and p2y12r have been proposed as imaging biomarkers of m1 and m2 phenotype microglia, respectively. several pet tracers for these non-tspo biomarkers have been developed, and some of them showed positive results in neuroinflammation is characterized by glial activation, which would lead to an increase in the inflammatory factor level in the cns. microglia are a collection of glial cells that act as the first and main form of active immune defense in the cns. accordingly, its activation is considered to trigger neuroinflammation, and pet tracers bound to active microglia are used for imaging of neuroinflammation. therefore, molecules exclusively expressed in microglia have the potential to be molecular targets for neuroinflammation. because imaging with pet is a commonly available technology for human subjects and experimental animal models, numerous pet tracers have been developed for visualization of microglia in living brains. moreover, increasing evidence has indicated that microglial activation is heterogeneous, and can be categorized into two opposite types: pro-inflammatory (m1) and anti-inflammatory (m2) microglial phenotypes. pet imaging tracers that bind to either general or phenotypespecific microglia are required for studies of neurodegenerative disorders. pet imaging in animal models is an indispensable step for the development and clinical application of pet tracers, as it will provide predictive evidence for tracer utility in human subjects. more importantly, it can easily provide a direct comparison between pet images and histopathology, which is usually difficult in human studies. pet imaging in animal models would supplement the weak points of human studies and provide interpretation for imaging in human subjects. response to neural injury. pet tracers with high affinity for tspo are the most widely used for neuroinflammation imaging. tracers have been developed for tspo imaging for several decades, including typical first-generation tracer 11 c-pk11195 and second-generation tracer 11 c-daa1106 [70] . major parts of these tracers enable the visualization of microglial activation in acute neuroinflammation models triggered by acute events like traumatic brain injury, ischemia, and excitotoxic damages. a longitudinal pet imaging study has shown a continuing increased tspo pet tracer uptake in a mouse model of mesial temporal lobe epilepsy induced by unilaterally intracranial kainic acid injection. the radioactivity uptake reached a peak at 7 days, mostly related to microglial activation. after 14 days, reactive astrocytes provided a major binding site for tspo tracer [71] . the finding of phase-dependent tspo expression in subtypes of glial cells might contribute to the identification of optimal treatment windows in further clinical studies [71] . chronic neuroinflammation is usually observed in many chronic neurodegenerative disorders such as alzheimer's disease (ad), amyotrophic lateral sclerosis (als), and non-ad tauopathies. most animal models of chronic neurodegenerative disorders are genetically modified mouse models. authors have clearly demonstrated glial activation in response to accumulation of amyloid aggregates and treatment with the aid of tspo-pet, indicating the utility of neuroinflammation imaging in monitoring of pathogenesis ( fig. 1.11 ). amyloid accumulation is the earliest pathological change in the brain with ad, followed by tau aggregation and glial activation. glial activation is predominantly concentrated around neuritic plaques, not diffuse plaques [72] . more clinical studies have reported increased tspo tracer accumulation in temporoparietal, entorhinal, and cingulate cortex rather than frontal cortex with rich diffuse plaques and poor neuritic plaques, partially supporting such observation, while a considerable number of studies failed to detect an increase in tspo non-tg representative merged coronal brain images of t2-weighted mr, tspo-pet and merged images showed that tracer binding was greatly increased in transgenic mice (tg) of app and tauopathy models mimicking amyloid (22-month-old) and tau (12-month-old) pathologies, respectively, compared with respective age-matched non-transgenic littermate mice (non-tg). no obvious brain atrophy was observed in hippocampi of app model as well as non-tg mice (white arrows), while tauopathy model showed overtly atrophic hippocampi (white arrowheads). red arrowheads indicated enlarged ventricles due to the hippocampal atrophy. unpublished data expression level [73] . the amyloid plaques accumulated in the amyloid precursor protein (app) model are dense-core plaques, around which there are abundant activated glial cells and tspo expression, indicating a similar glial response between neuritic and dense core plaques. however, immunohistochemical analysis showed that active astrocytes have provided the majority of binding sites for tspo tracer in the app model [74] , while microglia around neuritic plaques expressed abundant tspo [75] . this distinction between species concerning glial characteristics might be attributable to neurodegenerative tau pathologies, which exist in ad but are uncommon in the app model. in vivo tspo imaging also demonstrated increased tspo expression accompanied by tau-related atrophic brain in another model of ad mimicking the tau-related pathologies ( fig. 1.11 ) [72, 76] . considering that predominant microglial tspo expression and age-dependent brain atrophy in these tauopathy models mimicking tau-related pathologies are consistent with the condition in ad patient brains, neuroinflammation imaging in tauopathy models might well reflect the actual situation of pathologies in human subjects. based on the fact that in vivo imaging for amyloid and tau, as well as tspo have been available for clinical and preclinical studies, multiple tracer imaging enables the clarification of the mutual relationship between these ad-related pathologies. for example, longitudinal in vivo imagings with tspo-pet ( 18 f-fedaa1106) and amyloid-pet ( 11 c-pib) have captured the increased level of neuroinflammation triggered by aβ immunotherapy, which greatly reduced amyloid accumulation in an identical app model over the course of treatment ( fig. 1.12 ) [77] . tspo-pet in combination with tau-pet ( 11 c-pbb3) in two tauopathy models showed that neuroinflammation was induced by two different tau aggregates (pbb3-positive and -negative) and thereby accelerated tau-mediated neuron damage via a distinct molecular mechanism [78] . these studies provide experimental evidence for neuroinflammation-targeting therapeutic strategy. similarly, tspo tracer signals were also increased in lesioned brain regions in a mouse model of als, being consistent with microglial immunostaining [79] . in contrast, the results from parkinson's disease (pd) animal models look more complicated. pd animal models are relatively easy to prepare by treating the animals with neurotoxins such as methamphetamine and 6-hydroxydompaine (6-ohda). methamphetamine-induced degeneration of dopaminergic neuron terminals did not trigger tspo-positive gliosis [74] , while 6-ohda induced degeneration of dopaminergic neurons in either striatum or substantia nigra and increased the tspo tracer signal [80] . interestingly, most of the clinical pet studies found no increase in the level of tspo expression in the striatal regions of pd patients [73, 81, 82] despite severe dopaminergic degeneration in these regions, supporting the proposal that tspo imaging is inapplicable to monitoring the degeneration of nonmyelinated nerve [74] . quantitative pet studies require that the input function, representing cumulative availability of authentic radiotracer in arterial plasma, is measured in case of the absence of ideal reference tissue. because of the constitutive expression of tspo in the whole brain, blood collection is needed for calculating the binding potential. however, this usually faces technical difficulties in the actual operation for the small mouse body. so the brain regions without pathological changes, such as cerebellum in rtg4510 mouse [76, 78] or striatum in ps19 mouse [72] , are used as reference tissue when reference-tissue models are used. although it will lead to underestimation of the binding potential due to constitutive low-level tspo expression in reference regions, such analysis can provide relatively stable results compared to the percentage of injection dose, which is greatly affected by body weight, metabolic capacity, or other factors. another issue is a concern for the partial volume effect due to the small volume of the region of interesting (roi) in the case of mouse. this makes it difficult to correctly measure the radioactivity in certain subregions of mouse brain. the use of rat and nonhuman primate models is an alternative, but selectable neurodegenerative models are limited compared to mouse, as most models with pathologies of chronic neurodegenerative disorders, such as ad, als, and non-ad tauopathies [83, 84] , are genetically modified murine models. despite the fact that tspo-pet successfully captured neuroinflammation in various animal models with acute and chronic neuroinflammation, the results in patients with chronic neuroinflammation seemed more complicated [73, 81] . one of the reasons is that a tspo polymorphism (rs6971) greatly influences the affinity of a large majority of tspo tracers in a tracer-dependent manner [85] . another possible reason is the differing cellular distribution of tspo expression. recent studies have shown that tspo expression is not limited to microglia. other brain components, for example, astrocytes and vascular endothelial cells, also express tspo. some studies have demonstrated dominant induction of tspo expression in active astrocytes in a neuropathological type-dependent and phase-dependent manner [71, 74] . vascular expression of tspo is also a factor that disturbs microglial tspo imaging. although no study has quantitatively measured the influence of vascular tspo on tracer binding, inclusion of the additional vascular component in the modified reference-tissue model amplified the binding potential in ad more than in control by decreasing tracer binding to the vasculature in the disease cohort or was better correlated to brain tspo mrna [86, 87] . as few studies have focused on changes in vascular tspo expression in diseased brains, determining which is responsible for the changes in tspo tracer binding in diseased brains is a complicated issue. further studies in combination with postmortem analyses or microglial tspo-specific or vascular tspo-specific knockout animals are desirable. csf1r is a novel imaging biomarker for microglia by its exclusive expression in microglia in the cns [88] . recently, horti et al. reported a newly developed pet tracer, 11 c-cppc, for csf1r imaging. 11 c-cppc showed high initial brain uptake and rapid washout from brain in normal rodent brain, and increased accumulation in inflammatory brains of rodent and nonhuman primate models including lps-injection and ad mouse models. in vitro autoradiogram showed higher binding in postmortem brains with ad compared to healthy control [88] . these results support the concept that csf1r is a suitable molecular target for microglia imaging. however, 11 c-cppc binding is increased in whole brain regions, and is not limited to regions with lesions as exemplified by cerebellum in ad model and intracranial lps injection model, and immunohistochemistry for active microglia and csf1r expression to support imaging results was not provided. another reported csf1r imaging tracer, 11 c-az683, showed high affinity for csf1r (ki = 8 nm) and >250-fold selectivity over other kinases tested, but low brain uptake in rodents and nonhuman primates limited its utility in living brains [89] . further examinations and improvements will be required for their clinical application. it is still unclear which microglia phenotypes predominantly express csf1r because of the lack of data from immunohistochemical and biochemical analyses of the various neurodegenerative models. p2x7r and p2y12r were considered to be predominantly expressed in m1 and m2 microglia phenotypes, respectively [90] . in vitro autoradiographic analysis with 11 c-smw139 showed high specific binding to viral vectormediated human p2x7r expressed in rat brain, but no increase in binding was detected in postmortem brain with ad compared to healthy control [91] . specific binding of 11 c-gsk1482160 is also detectable in p2x7roverexpressing samples under in vitro condition [92] . however, more evidence is required for both of the above two p2x7r tracers to prove their utilities in the living inflammatory brain. in vivo imaging with 18 f-jnj-64413739 with high affinity and selectivity for p2x7r showed significant increase in tracer accumulation in the living lpsinduced neuroinflammatory brain, indicating its leading status among the current p2x7r tracers, while further work is needed to verify its utility in other types of neuroinflammation [93] [94] [95] . villa et al. recently developed a carbon-11-labeled p2y12r-binding compound. in vitro and ex vivo experiments with this radioactive compound showed increased binding in brain sections of mice treated with anti-inflammatory stimuli and decreased binding to brain sections of a murine stroke model and of a stroke patient [96] . immunohistochemistry and immunoblotting also supported the viewpoints that p2y12r is a biomarker for m2 phenotype microglia [90, 96] . abstract neuroinflammation is defined as inflammatory responses in the brain and spinal cord, and microglia and astrocytes are the mainly involved cells. translocator protein (tspo) has been the major target used in positron emission tomography (pet) to detect neuroinflammation with glial activation. in a preclinical study conducted in small animal models of brain ischemia and traumatic brain injury, a second-generation tspo tracer [ 18 f]dpa-714 pet was used for the evaluation of glial activation; the tspo uptake was validated by comparison with the immunohistochemical findings of co-staining for tspo and a microglial/macrophage or astrocyte maker. tspo expression has also been reported in a model of alzheimer's disease, where tspopositive cells with two opposing roles were observed: neuroprotective astrocytes for reversible neuronal injury and detrimental microglia for irreversible neuronal injury. recently, new targets have been identified in microglia, namely, cyclooxygenase-1 (cox-1) and purinergic receptor p2x7, which have been shown to be specifically expressed in the microglia. pet imaging targeting glial cells is a promising modality to characterize and monitor neuroinflammation longitudinally and with quantitative accuracy. the findings of preclinical models need to be confirmed in clinical studies beyond the limitation of animal model and the species differences between rodents and humans. keywords: neuroinflammation, glial activation, translocator protein, microglia, astrocyte neuroinflammation is defined as inflammatory responses in the brain and spinal cord [97] . it is observed in various types of brain diseases, including brain ischemia, traumatic brain injury, neurodegenerative disorders (alzheimer's disease, parkinson's disease, etc.), and neuropsychiatric disorders (schizophrenia, autism spectrum disorder, etc.) [98] [99] [100] [101] . in the central nervous system, two major glial cells are involved in the process of neuroinflammation, namely, microglia and astrocytes [99] . to evaluate neuroinflammation in vivo, translocator protein (tspo) has been the major target used in pet for the detection of neuroinflammation with glial activation [102] . in glial cells, especially microglia, expression of tspo is minimal in the resting state, but upregulated in the activated state [103] . in previous studies, the first-generation tspo-pet tracer, [ 11 c]pk-11195, was mainly used to evaluate glial activation in small animals as well as humans. however, the evaluation using [ 11 c]pk-11195 was found to be highly limited by the high nonspecific binding of the tracer [104, 105] ; therefore, over the last 10 years, second-generation tspo-pet tracers, such as [ 11 c]dpa-713 and [ 18 f]dpa-714, have been used because of their higher specific binding [104, 105] . although the binding affinity in humans has been shown to vary due to polymorphism, tspo pet has been employed as an effective tool to visualize and quantify the degree of neuroinflammation associated with glial activation in preclinical studies conducted using animal models, including rodents [106] . however, attention needs to be paid to the inflammatory cells taking up tspo, as tspo expression is not only observed in activated microglia/macrophages but also in reactive astrocytes [107] . for preclinical studies using small animals, the middle cerebral artery occlusion (maco) model was frequently used owing to its ease of handling for reperfusion and the abundance of accumulated evidence [108, 109] . martin et al. [111] . thus, tspo pet can also be used for monitoring the response to immunomodulatory therapy after brain ischemia. to evaluate traumatic brain injury (tbi) in small animals, a model of controlled cortical impact with a pneumatic impact device has been used for its high reproducibility and low mortality [112] . we reported two peaks of [ 18 f]dpa-714 uptake in this model, namely, on day 7 in the cortical area and on day 21 in the thalamus after induction of the focal brain injury [99] . the enhanced thalamic uptake was sustained until 14 weeks after induction of the tbi (fig. 1.14) . immunohistochemical analysis on day 7 revealed tspo staining mainly co-localized with amoeboid cd11b-positive microglia/macrophages, and weakly with gfap-positive astrocytes in the cortex; however, tspo immunoreactivity was scarcely observed in the thalamus. giemsa staining on day 7 revealed mononuclear cells, such as phagocytic macrophages, in the cortical area, although these cells were not detected in the ipsilateral thalamus. in the macrophage depletion study performed 1 week post-injury, the tspo uptake was decreased in the cortex, but enhanced in the thalamus, suggesting that the cortical uptake was mainly attributable to uptake by phagocytic macrophages recruited from the bloodstream, while the thalamic uptake was most likely attributable to uptake by resident microglia. electron microscopy at 4 weeks post-tbi revealed morphologically activated microglia surrounding the degenerated axons and poorly myelinated neurons in the ipsilateral thalamus, indicating activation of the resident microglia and damage to the remaining neurons. tspo expression was mainly evident in the activated microglia in the thalamus at 6 weeks postinjury. thus, by using [ 18 f]dpa-714 pet, inflammatory migration could be detected from the cortex to the thalamus after focal brain injury. in addition, we also showed that depletion of myeloid-derived suppressor cells (mdscs) was associated with enhanced cortical tspo uptake on day 7 after the induction of focal brain injury, by both immunohistochemistry and cellular phenotype analysis [113] . this finding indicated that mdscs may have strong immunosuppressive characteristics and limit inflammation and facilitate wound healing and recovery. postmortem analyses of the brains of patients with alzheimer's disease (ad) have revealed neuroinflammatory changes, namely, accumulation of activated microglia and astrocytes, around senile plaques and fibrillary tau lesions [114] . in this study, diffuse plaques in the brains of these patients were enveloped by a small number of iba-1positive microglia not expressing tspo, while numerous microglia expressing both iba-1 and tspo were found to be in close contact with neuritic plaques. thus, microglia expressing tspo were recruited to fibrillar tau inclusions, but not to aβ deposits unaccompanied by tau pathology. jin et al. reported that tspo expression was observed, mainly in the astrocytes, in amyloid precursor protein 23 (app23) transgenic (tg) mice which show minimal neuronal loss, while tspo-positive microglia were observed in the tangle-like tau lesions of tau tg mice which show remarkable neuronal loss. they concluded that tspo-positive astrocytes have protective roles against reversible neuronal injury, while tspo-positive microglia have detrimental effects for irreversible neuronal injury [115] . we evaluated tspo expression in the app23 tg mice by immunohistochemistry ( fig. 1.15 ). tspo immunoreactivity was observed around the amyloid beta (aβ) plaques and in reactive astrocytes (gfap-positive shukuri et al. demonstrated that the expression of cyclooxygenase-1 (cox-1) in activated microglia and macrophages during neuroinflammation can be visualized by pet using [ 11 c]ketoprofen methyl ester (ktp-me), with immunohistochemical confirmation [117] . one advantage of [ 11 c]ktp-me pet as compared to tspo pet is that cox-1 is expressed only in activated microglia and not in astrocytes. they also reported that expression of cox-1 could be detected in 16-to 24-month-old app23 tg mice, in accordance with the time of histopathologic appearance of abundant aβ plaques and activated microglia [118] . they also reported the possibility of treating ad by inhibition of cox-1 activity, as a treatment target, with nonsteroidal anti-inflammatory drugs (nsaids). besides tspo, pet imaging of [ 11 c]ktp-me could also be a useful tool for monitoring cox-1 activity in activated microglia in neuroinflammation. imamoto et al. showed that glial activation could be quantitatively imaged in the spinal cord of a rat model of neuropathic pain using [ 11 c]pk11195 pet [119] . it was suggested that high-resolution pet using tspo-specific radioligands is useful for imaging to assess the role of glial activation, including neuroinflammatory processes, in the spinal cord. belolli et al. reported increased enhanced tspo uptake in a mouse model of multiple sclerosis induced by experimental autoimmune encephalomyelitis (eae), with neuropathological confirmation of activated microglia [120] . they concluded that combined use of tspo-pet and mri could provide complementary evidence of the ongoing disease process. evaluation of neuroinflammation by pet in neuropsychiatric disorders, such as schizophrenia and autism spectrum disorder, has been limited to clinical studies, possibly due to the difficulty in establishing appropriate animal models mimicking the pathological condition of the patients. as mentioned in sect. 1.4.4, tspo-positive glial cells have both neurotoxic and neuroprotective effects. two phenotypes of microglia have been recognized, namely, neurotoxic "m1" and neuroprotective "m2," although it cannot be clearly separated as m1 or m2 [121] . tspo-positive microglia that accelerate tau deposition and neuronal deterioration are likely to be m1-polarized. when considering the treatment strategy for neurodegenerative disorders, it is important to detect the detrimental glial cells to suppress their function. expression of the p2x7 receptor (purinergic receptor) has been reported to be observed in m1-polarized microglia, which could be a promising target for the detection of deleterious neuroinflammation [122] . in contrast, expression of the p2y12 receptor has been reported to be observed in the m2-like microglia [123] . recently, a pet radioligand for the p2x7 receptor was synthesized and its efficacy was evaluated in preclinical models. finally, reproducible and dosedependent receptor occupancy studies with a p2x7 receptor antagonist were performed using [ 18 f]jnj-54175446 in rhesus monkeys and humans [124, 125] . the usefulness of pet radioligands for the p2x7 receptor is not only limited to the diagnosis of neuroinflammation. [ 18 f]pttp, which is also a radioligand targeting the p2x7 receptor, has been shown to be potentially useful for the quantification of peripheral inflammation targeting macrophages and to distinguish inflammation from certain solid tumors [126] . other targets in microglia, such as sphingosine-1-phosphate receptor 1 (s1p1) or receptor for advanced glycation end products (rage), are also promising, but only in the preliminary stage of investigation at this moment [127] . findings of pet imaging in animal models of neuroinflammation are summarized in this article. in preclinical studies, the pathology can be confirmed by immunohistochemistry or cellular phenotype analysis, as well as by comparison of the pet results. pet imaging targeting tspo, cox-1, or p2x7 is a promising modality to characterize and monitor neuroinflammation longitudinally and with quantitative accuracy. however, it needs to be borne in mind that preclinical models do not always reflect the pathologies of the heterogeneous characteristics of patients in clinical practice. in addition, we should consider the species differences between rodents and humans. any findings in preclinical models should be confirmed in clinical situations, which is one of the goals of translational research. translocator protein (18 kda; tspo) is the critical component of a multimeric 140-200-kda complex located in the outer mitochondrial membrane and enriched in the outer/inner mitochondrial membrane contact sites [128] . tspo has broad functions related to the regulation of cholesterol transport, steroid hormone synthesis, porphyrin and heme transport, apoptosis, cell proliferation, anion transport, mitochondrial function regulation, immunomodulation, and inflammation [128] . tspo is widely distributed in the peripheral tissues, and the mrna levels of tspo are high in the adrenal glands, kidneys, spleen, skeletal muscle, heart, and lungs, and are low in the liver and brain. tspo expression in blood cells has also been reported, in which phagocytic cells, including monocytes and polymorphonuclear neutrophils, show significantly higher tspo expression than lymphocytes. it has been demonstrated that tspo expression increases in inflammatory cells during the occurrence and progression of inflammation. thus, tspo has become a useful biomarker for monitoring inflammation using pet with a tspospecific radiotracer [128] . since 2002, we have developed more than 10 pet radiotracers for the imaging of tspo in preclinical studies and have translated four new tspo radiotracers to clinical studies in our institute: [ [137] [138] [139] . here, the author reviews their results from pet with [ 18 f]fedac to noninvasively visualize tspo in the peripheral tissues [140] and monitor various inflammatory diseases, such as lung inflammation [141] , nonalcoholic fatty liver disease [142] , liver damage [143] , liver fibrosis [144] , multiple sclerosis [145] , and rheumatic arthritis [146, 147] , in animal models. using small-animal pet with [ 18 f]fedac, we performed tspo imaging to quantify tspo density in rat peripheral tissues, including heart, lungs, and kidneys [140] . pet study showed that the uptake of radioactivity was highly distributed in the lungs, heart, and kidneys, and the tspo-enriched tissues could be clearly visualized after the injection of [ 18 f]fedac ( fig. 1.16) . the kinetics of this radiotracer in the tissues was moderate, which is suitable for determining the in vivo binding parameters and receptor occupancy. the b max values of tspo in the heart, lung, and kidney were 393, 141, and 158 pmol/ml, respectively ( tspo is highly expressed on the bronchial and bronchiole epithelium, submucosal glands in intrapulmonary bronchi, pneumocytes and alveolar macrophages in human lungs. we performed pet imaging of lung inflammation with [ 18 f] fedac and determined cellular sources that enrich tspo in the lung [141] . we prepared an acute lung injury model by using intratracheal administration of lipopolysaccharide (lps) to rats. pet with [ 18 f]fedac demonstrated that, in response to lps treatment, the uptake of radioactivity increased in the lungs as the inflammation progressed ( fig. 1.17 ). pretreatment with a tspo-selective unlabeled ligand pk11195 significantly decreased the lung uptake of [ 18 f]fedac because of competitive binding to tspo. tspo expression was elevated in the inflamed lung section and its level responded to the [ 18 f]fedac uptake and severity of inflammation. the presence of tspo was examined in the lung tissue using western blotting and immunohistochemical assays. the increase of tspo expression was mainly found in the neutrophils and macrophages of inflamed lungs ( fig. 1.18 ). through this study, we demonstrated that pet with [ 18 f] fedac is a useful tool for imaging tspo expression and evaluating the progression of lung inflammation. mitochondrial dysfunction is responsible for liver damage and disease progression in nonalcoholic fatty liver disease (nafld). tspo, as a mitochondrial transmembrane protein, plays important roles in regulating mitochondrial function. we conducted pet with [ 18 f]fedac to explore if tspo could be used as an imaging biomarker of noninvasive diagnosis and staging of nafld [142] . pet with [ 18 f] fedac, ct, autoradiography, histopathology, and gene analysis were performed to evaluate and quantify tspo levels and nafld progression in methionine and cholinedeficient diet-fed mice. the uptake of [ 18 f]fedac increased with disease progression from simple steatosis to nonalcoholic steatohepatitis (nash) (fig. 1.19) . a strong correlation was observed between [ 18 f]fedac uptake ratio and nafld activity score in the liver. the specific binding of [ 18 f]fedac to tspo in the nafld livers was demonstrated by competition experiments with the unlabeled tspo-selective pk11195. autoradiography and histopathology supported these pet imaging results. further, there were greater mrna levels of the functional macromolecular signaling complex composed of tspo, compared to controls (fig. 1.20) . through this study, we demonstrated that tspo expression increased in nafld and was strongly correlated with nafld progression. tspo as a specific molecular imaging biomarker could provide a novel tool for noninvasive, reliable, and quantitative diagnosis and staging of nafld. liver damage induced by drug toxicity is an important problem for both medical doctors and patients. in this study, we noninvasively visualized acute liver damage using pet with [ 18 including tspo (blue) and cd11b (orange). preferential localization of tspo indicated in the liver lesion sites, cd11b/tspo active macrophages and lymphocytes increased in the mcd livers (scale bars = 500 μm). * * p < 0.01 sources enriching tspo expression in the liver [143] . a mild acute liver damage model was prepared by a single intraperitoneal injection of cycloheximide (chx) to rats. treatment with chx induced apoptosis and necrotic changes in hepatocytes with a slight neutrophil infiltration. the uptake of radioactivity in the rats' livers was measured with pet after injection of [ 18 f]fedac. pet with [ 18 f] fedac showed that the uptake of radioactivity increased in livers treated with chx, compared to the controls ( fig. 1.21) . the mrna expression of tspo was increased in the damaged livers compared to the controls, and the tspo level was correlated with the [ 18 f]fedac uptake and severity of damage. tspo expression in the damaged liver sections was mainly found in macrophages (kupffer cells) and neutrophils, but not in hepatocytes ( fig. 1.22) . the increase in expression of tspo mrna was induced by an increase in the numbers of macrophages and neutrophils with tspo in the damaged livers. through this study, we demonstrated that pet imaging with [ 18 f]fedac provided visible evidence that mild liver damage occurs through the enhanced tspo signal in inflammatory cells, and that this method is a useful diagnostic tool in the early stages of acute liver damage. liver fibrosis is the wound healing response to chronic liver injury, which is caused by various factors. in this study, we evaluated the utility of tspo as a molecular imaging biomarker for monitoring the progression of liver fibrosis in response to cirrhosis [144] . to induce cirrhosis, carbon tetrachloride (ccl 4 ) was administered to rats and the subsequent liver fibrosis was assessed. pet with [ 18 f]fedac was used to noninvasively visualize liver fibrosis in vivo. pet scanning, immunohistochemical staining, ex vivo autoradiography, and quantitative reverse-transcription polymerase chain reaction were performed to elucidate the relationships among radioactivity uptake, tspo level, and cellular source enriching tspo expression in damaged livers. pet with [ 18 f]fedac showed that the uptake of radioactivity in livers significantly increased after 2, 4, 6, and 8 weeks of ccl 4 treatment ( fig. 1.23 ). immunohistochemical analysis demonstrated that tspo was primarily expressed in macrophages and liver stellate cells (hscs). tspo-expressing macrophages and hscs increased with the progression of liver fibrosis (fig. 1.24) . the distribution of radioactivity from [ 18 f]fedac was strongly correlated with tspo expression, and tspo mrna levels increased with the severity of liver damage. through this study, we demonstrated that tspo is a useful molecular imaging biomarker for monitoring the progression of liver fibrosis in response to cirrhosis with pet. imaging modalities have the potential to monitor inflammation in order to guide treatment before significant functional impairment or irreversible cellular damage occurs in multiple sclerosis (ms). in this study, [ 11 c]dac, a carbon-11labeled tspo radiotracer derived from [ 18 f]fedac, was used to evaluate neuroinflammation and quantify the therapeutic effects of experimental autoimmune encephalomyelitis (eae), an animal model of ms [145] . pet with [ 11 c] dac was used to visually assess neuroinflammation in eae by measuring tspo expression in the spinal cords; there was the maximal uptake on day 11 and 20 eae rats with significant inflammatory cell infiltration, compared to controls, day 0 and 60 eae rats ( fig. 1.25) . biodistribution studies and in vitro autoradiography confirmed the pet imaging results. doubling immunohistochemical studies demonstrated the infiltration and expansion of cd4+ t cells and cd11b+ microglia; cd68+ macrophages caused the increase in tspo levels visualized by [ 11 c]dac-pet. furthermore, mrna level analysis of the cytokines using quantitative reversetranscription polymerase chain reaction revealed that tspo+/cd4 t cells, tspo+ microglia, and tspo+ macrophages in eae spinal cords were activated and secreted multiple pro-inflammation cytokines, which mediated the inflammation lesions of eae. the eae rats treated with an immunosuppressive agent fty720 exhibited an absence of inflammatory cell infiltrates and displayed a faint radioactive signal, compared to the high accumulation in untreated eae rats ( fig. 1.26) . through this study, we demonstrated that [ 11 c]dac-pet imaging is a useful tool for noninvasively monitoring the neuroinflammation response and evaluating therapeutic interventions in eae. rheumatoid arthritis (ra) is a chronic disease characterized by systemic inflammation that results in the destruction of multiple articular cartilages and bones. activated macrophages have been known to play important roles in the pathogenesis of rheumatoid arthritis (ra). cheon et al. evaluated the feasibility of [ 18 f]fedac-pet in a murine ra model [146] . in the collagen-induced arthritis (cia) mice, joint swelling was apparent on day 26 after the first immunization, and the condition worsened by day 37. in these mice, the mrna and protein expression of the tspo increased in activated macrophages. the uptake of [ 18 f]fedac in activated macrophages was greater compared to the uptake in nonactivated cells, which was inhibited by pk11195. the [ 18 f]fedac uptake by arthritic joints increased early on (day 23), whereas [ 18 f] fdg uptake did not ( fig. 1.27 ). however, [ 18 f]fdg uptake by arthritic joints markedly increased at later stages (day 37) to a higher level than [ 18 f]fedac uptake. the [ 18 f]fedac uptake correlated weakly with the summed severity score, whereas the [ 18 f]fdg uptake correlated strongly with the summed severity score. histologic sections of arthritic joints demonstrated an influx of macrophages compared to that in normal joints ( fig. 1.28) . moreover, these indicated that pet imaging using [ 18 f]fedac may be used as a predictor of the therapeutic effects caused by biological disease-modifying antirheumatic drugs that have anti-inflammatory actions to inhibit activated macrophages [147] . [ 18 f]fedac-pet is a powerful tool for visualizing tspo and monitoring the progress of various inflammatory diseases of the peripheral system. the present studies will contribute to determination of the pathogenic progress and will be of use in evaluating the therapeutic effects of antiinflammatory drugs. in future studies, we will perform or are performing pet imaging with [ 18 f]fedac or other tspo radiotracers to detect various inflammation in humans. hiromi suzuki and makoto sawada abstract at present, common diseases of cns were often chronic and progressive which could affect the patient's quality of life seriously. but the pathogenesis for most of them had not been fully understood and was no effective prevention or treatment. certain pathological injury could cause cellular inflammatory response. microglia, the first activated effector cells, played an important role in the immune defense process. microglia had a two-way effect. on the one hand, activated microglia phagocytosed damaged cell debris and removed antigenic substances, and could release cytotoxic factors which would aggravate the injury of neuron. on the other hand, activated microglia may accumulate around damaged neurons and might induce neurotrophin-dependent protective activity. therefore, to study the mechanism of microglia in the diseases of cns and limit their effects on neuronal injury may help to retard the procession of some neurodegenerative diseases such as alzheimer's disease and parkinson's disease cause neuroinflammation including expression of mhc antigens and production of inflammatory cytokines. however, the condition changes greatly in relation to the onset and the formation of a pathological condition. microglia, macrophage-like cells in the central nervous system (cns), are multifunctional cells; they play important roles not only in the neuro-inflammation but also in the development, differentiation, and maintenance of neural cells via their phagocytic activity and production of enzymes, cytokines, and trophic factors [148] . microglial cells show a rather uniform distribution of cell numbers throughout the brain with only minor population in some brain regions. their in situ morphologies, however, may vary markedly from elongated forms observed in apposition with neuronal fibers to spherical cell bodies with sometimes extremely elaborated branching. human fetal microglia display heterogeneity in phenotype identified by cd68 [149] . this heterogeneity gave rise to the hypothesis that these cells are differentially conditioned by their microenvironment and, therefore, also display specific patterns of differential gene expression. for example, mrnas of tnfalpha, cd4, and fc gamma receptor ii are differentially expressed in microglia isolated from different area of the brain [150] . it has been reported that production of il-12 [151] and histamine [152] are different in the subtypes of microglia. furthermore, the subtypes of microglia show distinct acidification profiles by treatment with pepstatin a [153] and selective clearance of oligomeric beta-amyloid peptide [154] . the distinct phenotypes in activated forms of microglia might result from this heterogeneity. although the origin of microglia is still controversial, resident microglia is thought to be of mesodermal origin, being derived from a bone marrow precursor cell [155] . these cells or a lineage related to monocytes and macrophages may invade the cns at an early embryonic stage to give rise eventually to typical process-bearing resident microglia [155] . like macrophages in other tissues, the growth and activation of microglia is regulated by macrophage colony-stimulating factor (m-csf) [156] . however, unlike macrophages, microglia are observed in macrophagedeficient mice [157] [158] [159] , suggesting the possibility of the presence of m-csf-independent populations. despite the postulate that microglia arise from blood monocytes, microglia appear to display phenotypes differing from macrophages. in vivo, microglia do not normally express mhc class i and ii [160] , cd14 [161] , or scavenger receptor i and ii [162] . their appearance takes a ramified form [160] . in contrast, monocytes and macrophages express these surface antigens absent in microglia and their shape is ameboidal [160] . like their in vivo counterparts, isolated microglia also differ from macrophages. although cultured microglia are ameboidal, they can transform to a ramified form [163] . microglial cell proliferation is induced by conditioned medium from astrocyte-enriched cultures [163] . microglial production of interleukin (il)-5 is stimulated by bioactive phorbol ester but not by interferon-γ, while the opposite is there for macrophages [164] . microglia do not constitutively express il-2 receptors under normal culture conditions, as do monocytes and macrophages [165] . microglia express these receptors only after stimulation such as with lipopolysaccharide (lps) [165] . il-2 increases viability and proliferation of lipopolysaccharide-stimulated microglia [165] , while macrophages respond to il-2 by expressing il-1, il-6, and tumor necrosis factor α mrnas, peroxide production, and microbicidal and tumoricidal activity [166, 167] . lps, cyclosporin a, and fk506 downregulate expression of cd4 in microglia, but not in macrophages [168] . finally, bone marrow chimera experiments have shown that resident microglia form highly stable pool of cns cells displaying very little exchange with the bone marrow compartment [169] [170] [171] . these controversial observations suggest that microglia in the brain may be different from macrophages. recently, we identified two distinct subpopulations of microglia in the normal mouse brain: type i and type ii. both types had similar phagocytic activity, but they showed distinct phenotypes such as cell-surface markers, mrna expression, and growth factor dependency. type i, but not type ii, microglia expressed the hallmarks of microglia such as cd14 and c3b receptor, and production of interleukin 1β. type ii microglia expresse surface markers for immature bone marrow cells, such as antigens against er-mp12 and er-mp20 antibodies, which are not detected in mature monocyte/macrophages. in addition, it also express onetenth amount of mature cell markers compared to monocytes/macrophages. therefore, type ii microglia may arise from progenitor cells similar to immature bone marrow cells which migrate to the cns during the early developmental stages then differentiate in the cns; monocytedifferentiation out of bone marrow like extrathymic differentiation of t-cells [172] . this suggests that the two distinct type of microglia arise from distinct origins. in addition, type i microglia exhibited m-csf-dependent growth, while the type ii were m-csf-independent. a previous report suggested that alveolar macrophages are gm-csf-but not m-csf-dependent [173] . these observations indicate that there are two distinct populations of microglia which are probably of different origins and have different functions in the brain. the presence of oxidative stress and inflammatory activity is one of the significant pathological features of neuroinflammatory diseases [174, 175] . it has been shown that the levels of cytokines such as tumor necrosis factor (tnf)-α, interleukin (il)-1β, and interferon (ifn)-γ are elevated in the substantia nigra of patients with parkinson's disease [176] . since microglia are a principal source of these cytokines, the data support microglial involvement in the pathogenesis of neuroinflammatory diseases. however, the role of activated microglia is controversial. for example, the characteristic pathological features of the parkinson's disease brain are a selective and progressive loss of dopamine neurons of the substantia nigra and their terminals in the caudate-putamen, along with focal accumulation of activated microglia in the substantia nigra and caudate-putamen. the traditional view is that microglia act merely as scavengers and their activation is secondary to the neuronal damage. however, activated microglia have been observed in the limbic system and neocortex, where there are few or no degenerating neurons, in significantly higher numbers in pd brains than in brains from normal controls [177] . activation of microglia has also been identified in the substantia nigra and/or striatum of parkinsonian animal models, such as l-methyl-4-phenyl-1,2,3,6tetrahydropyridine (mptp)-induced parkinsonism [178, 179] . the link between microglia activation and selective neuronal vulnerability has led many researchers to suggest that microglia activity participates in neuronal demise. in this respect, microglial cytotoxicity may contribute to or even promote neuronal damage. activated microglia are capable of releasing numerous cytotoxic agents, including proteolytic enzymes, cytokines, complement proteins, reactive oxygen intermediates, nmda-like toxins, and nitric oxide [148] . in fact, β-amyloid, the senile plaquederived component found in alzheimer's disease, appears to elicit neurotoxic responses through the activation of microglia [180] . however, this suggestion relies solely on in vitro data and as yet no evidence has been presented that indicates that activated microglia destroy neurons under in vivo conditions. recently we showed that highly activated microglia treated with lps may have neurotrophic potential toward dopamine neurons in neonatal mice administered mptp [181] . tyrosine hydroxylase activity and the levels of dopamine and dihydroxyphenylacetic acid (dopac), as well as those of the pro-inflammatory cytokines il-1β and il-6, were elevated in the midbrain of lps-and mptp-treated neonatal mice. the viability of dopamine (a9) neurons was preserved in neonatal mice of the lps-mptp group compared with the mptp group. in contrast, the viability of these neurons in aged mice dropped significantly. these results may suggest that activated microglia show different phenotypes; i.e., microglia activated by lps in the neonatal brain may be neuroprotective for dopaminergic neurons in mptp-treated mice, whereas in aged mice they may be neurotoxic for dopaminergic neurons. the dissociation between injury-induced microglial activation and neuronal degeneration in tnf receptor and colony-stimulating factor 1 (csf-1) knockout mice suggests that microglial activation is not a determinate event in dopaminergic neuronal damage in brain. furthermore, there is a growing body of evidence that microglia may play a beneficial role in ischemia by secreting factors (growth factors or cytokines) that promote survival of neurons. therefore, activated microglia may produce not only neurotoxic effects but also neuroprotective ones, depending upon their environmental situation. we have reported that exogenous microglia enter the brain parenchyma through the blood-brain barrier and migrate to ischemic hippocampal lesions when they are injected into the circulation. by applying this brain-targeted delivery technique, we investigated the effect of exogenous microglia on ischemic pyramidal neurons [182] . to this end, we isolated microglia from neonatal mixed brain cultures, labeled them with a fluorescent dye pkh26, and injected them into the artery of mongolian gerbils subjected to ischemia reperfusion neuronal injury. pkh26-labeled microglia migrated to the ischemic hippocampal lesion and increased the survival of neurons, even when the cells were injected 24 h after the ischemic insult. furthermore, stimulation of isolated microglia with ifn-γ enhanced the neuroprotective effect on the ischemic neurons. microglia also protected ischemia-induced learning disability. as migratory microglia increased the expression of brain-derived neurotrophic factor (bdnf) and glial cell linederived neurotrophic factor (gdnf) in the ischemic hippocampus, they might induce neurotrophin-dependent protective activity in damaged neurons. these results represent the first experimental demonstration of neurotrophic effects of microglia on transient global ischemia in vivo. since peripherally injected microglia exhibit specific affinity for ischemic brain lesions and protect against ischemic neuronal injury in the present model, we suggest that microglia may have the potential to be used as a candidate for cell therapy for cns repair following transitory global ischemia and other neurodegenerative diseases. nef, a multifunctional hiv protein, activates the vav/rac/ p21-activated kinase (pak) signaling pathway. given the potential role of this pathway in the activation of the phagocyte nadph oxidase, we have investigated the effect of the hiv-1 nef protein on microglia superoxide production and toxicity for neurons [183] . microglia were transduced with lentiviral expression vectors to produce a high level of nef protein. expression of nef did not activate the nadph oxidase by itself, but led to a massive enhancement of the responses to a variety of stimuli (ca2+ ionophore, formyl peptide, endotoxin) and induction to produce a large amount of superoxide. these effects were not caused by upregulation of phagocyte nadph oxidase subunits. nef mutants lacking motifs involved in the interaction with plasma membrane and/or other cytosolic proteins failed to reproduce the effects of wild-type nef, suggesting involvement of a certain signaling pathway in microglia for their trophic-toxic control. our results suggest a key role for rac activation in the priming for microglia toxic activation, which is enhanced by nef introduction in the nontoxic form of microglia. rac activation is not sufficient to induce the toxic form of microglia accompanied by stimulation of the phagocyte nadph oxidase; however, it markedly enhances the nadph oxidase response to other stimuli and might be involved in the trophic-toxic control of microglia. cytokines are polypeptidic soluble factors that control the growth and differentiation of cells involved in immune and hematopoietic systems. these factors were initially considered to act on target cells in a cell-type and stage-specific manner. however, it has been shown that their biological actions are pleiotropic, complementary, and counteractive; each of them exerts multiple effects on different cells, and different factors can act on the same cell populations to induce similar or opposite effects. moreover, production of several cytokines is controlled by other cytokines in a stimulatory or inhibitory manner, and, in some cases, acts as a cascade. these complex cytokine actions are therefore referred to as a cytokine network. recent evidence suggests that bidirectional communication occurs between cells of the nervous and immune systems. the basis for this communication is the release of cytokines by immune component cells, as well as hormone products of the neuroendocrine system. cells resident within the cns can synthesize, secrete, and respond to inflammatory cytokines not only contributing to the responses to injury or immunological challenge within the cns but also regulating their own growth and differentiation potential. there are many similarities of cytokine actions between the immune system and the cns. however, there are also several unique modes of cytokine action in the cns (i.e., their target cells, inducing functions on the target cells such as other cytokine production, major histocompatibility complex (mhc) antigen induction, and cell growth control, and inducing agents of their production). therefore, the actions and communication of cytokines in the cns are designated as the cns cytokine network [184] . microglia are one of the most important cells in the cns cytokine network. they produce a variety of cytokines, as well as their proliferation and other functions are regulated by cytokines. astrocytes are another cell population important in the cns cytokine network. there are many similar aspects of cytokine production and responses between microglia and astrocytes; however, the roles in the cns cytokine network may differ between these cells; microglia may have an important function in the removal of dead cells or their remnants by phagocytosis in brain injury, while astrocytes provide structural and environmental support for neurons. there are no clear indications of the difference between the roles of microglia and astrocytes in the cytokine production and response. some of these cytokines control the growth, differentiation, and activation of cells in the cns, and some modulate growth factor production, mhc antigen expression, and morphological changes. their biological functions are mediated by specific receptors, some of which are expressed in the cns, and cellular localization of cytokine receptors in the cns has been demonstrated. neuronal cells and oligodendrocytes as well as microglia and astrocytes expressed several cytokine receptors indicating that certain functions of these cells may also be controlled by some cytokines. however, to determine the involvement of cytokines in controlling the functions and development of cns cells requires elucidating the functional relationships of cytokines and these cells. there are two aspects of roles of the cns cytokine network, one of which is regulation of growth and differentiation of neural cells, and production of cytokines in the cns. another aspect of the cns cytokine network is its interaction with the immune system. since the majority of cytokines are produced by immune cells, it is possible that immune cells may also control the growth and function of cns cells. therefore, the control of microglial growth by cytokines is an important method of regulation of cns cells by peripheral immune cells. in addition to this, cytokines produced in the cns can control immune cell functions. these communications may be important for brain-immune interactions, and some aspects may play crucial roles in certain pathological conditions such as aids-dementia complex and multiple sclerosis as well as several neurological diseases. pet imaging has recently gained wide use not only in clinical stages but also in basic studies. small-animal imaging uses the same technology as clinical imaging. clinical research and problems can thus be introduced to preclinical research directly using small animals. conversely, applying results obtained from preclinical studies to clinical imaging is also possible. imaging has bridged the gap between basic studies and clinical research, and has become widely recognized as a tool for translational research. in traditional animal experiments, relatively high numbers of animals are often required. one reason is that animals are sacrificed to remove organs or tissues for the purposes of observation and measurement. another is the influence of individual differences. in particular, the four elements of pharmacokinetics (absorption, distribution, metabolism, and excretion) are known to be strongly affected by individual differences. the number of samples must therefore be increased for accurate statistical processing. on the other hand, preclinical imaging experiments allow use of the same individual, performing the study and repeatedly acquiring data without killing the individual. this means that applying human imaging techniques to a small animal allows follow-up of changes over time in disease models in the same individual, clarifying changes according to the disease condition more precisely, as in daily practice in the clinical field. in addition, preclinical imaging is an effective technique not only for providing biological information but also for reducing the number of experimental animals. in the area of research into infectious diseases, evaluation of pathogen infection, tracking disease state, and observation over time of individuals are very important, because infectious and inflammatory diseases change with the passage of time. for example, diseases may pass through stages of infection, pathogenesis, ingravescence, remission, and healing. when those stages of infectious disease are studied by sacrificing the animals, the number of animals required to obtain meaningful results will become extremely large due to the large interindi-vidual variance of animals. small animal pet imaging has therefore gained attention as a useful experimental method. to conduct imaging studies using infected model animals, a special laboratory is required. this is because researchers must be protected not only from radiation exposure but also from the risk of infection. biosafety level (bsl) is a classification depending on the degree of pathogen risk. table 1 .2 shows representative pathogens sorted by bsl from the guidelines of the national institute of infectious disease (niid). when dealing with high-risk pathogens, researchers need to use a biocontainment laboratory conforming to the world health organization (who) guidelines. when using imaging systems, measures must also be taken to avoid contamination by biohazards, such as installation in separated safety rooms, prevention of cross-contamination using sealed animal tubes, and so on [185] . in addition, researchers need to wear a tyvek coverall, mask, rubber gloves and face guard ( fig. 1.29 ). given the restrictions as described above, only limited numbers of facilities around the world are capable of performing imaging studies for infectious diseases, and relatively few studies have been conducted. in 2012, a small-animal imaging system was introduced to the bsl-3 section of the radioisotope center at nagasaki university, making this the only facility in which researchers could perform imaging of infectious diseases requiring a high bsl in japan. since then, various infectious diseases imaging studies have been conducted. hayasaka et al. reported an fdg-pet study using mice infected with severe fever with thrombocytopenia syndrome (sfts) virus, which is categorized as bsl-3. sfts is a tickborne infection that causes severe inflammation in the gastrointestinal tract [186] . in that study, sftsv-infected mice were administered about 10 mbq of fdg intravenously via the tail vain. pet acquisition was performed for 30 min from 30 min after fdg injection. fdg uptake was found along the course of the intestine in the sfts-infected mouse ( fig. 1.30 ) [187] . pet studies on sfts have also been performed using 68 ga tracer. fuchigami et al. tried to visualize sfts with 68 ga-citrate [188] . generally, 67 ga-citrate is a radiotracer used for single-photon computed tomography (spect), and is widely used to detect tumors [189, 190] , inflammation, and infectious diseases [190, 191] . since 68 ga-citrate is a pet tracer behaving exactly the same as 67 ga-citrate, targets can be imaged quantitatively and with higher sensitivity. in that study, sftsv-infected mice were administered about 5 mbq of 68 ga-citrate intravenously via the tail vain. pet acquisition was performed for 1 h, starting 2 h after 68 ga-citrate injection. as shown in fig. 1.31 , high accumulation of 68 ga-citrate in the gastrointestinal tract was observed in sftsv-infected mice, and the accumulation pattern of 68 ga-citrate resembled that of fdg. in addition, 68 ga-citrate was used for pet imaging of leishmaniasis [188] . leishmaniasis is a tropical disease caused by leishmania parasites categorized as bsl-2, and leishmania leads to strong localized inflammation [192] . leishmania parasite-infected mice were administered about 4 mbq of 68 ga-citrate intravenously via the tail vein. pet acquisition was performed for 1 h, starting 2 h after 68 ga-citrate injection. as shown in fig. 1.32 , inflammation site-specific accumulation of 68 ga-citrate was observed [188] . the examples given here are only a small selection of many studies underway at nagasaki university. infectious disease studies are more diverse than tumor studies, because infectious diseases have numerous diseases classified as tropical infections and emerging infectious diseases, and many issues have not been elucidated for each disease. fdg and 68 ga-citrate used in the studies introduced this section are widely used general radiotracers that are easily obtained. even without using special or unorthodox radiotracers, new knowledge can be obtained from preclinical pet studies. the pathological condition of the infected animal will change depending on species differences and the infected pathogen. some models only have about 3 days between infection and death. when conducting imaging research into infectious diseases, investigation of the survival rate of model animals as a preliminary experiment and preparation of a survival curve is desirable. also, breeding of model animals requires prevention of infection with other infectious diseases. experiments using infected animals thus require some additional caution, as previously mentioned. in conclusion, small animal imaging using 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mice: an imaging study with positron emission tomography tracer (1)(8) f-fluoromethylcholine evaluation and comparison of 11c-choline uptake and calcification in aortic and common carotid arterial walls with combined pet/ct carotid artery plaque uptake of (11)c-pk11195 inversely correlates with circulating monocytes and classical cd14(++)cd16(−) monocytes expressing hla-dr imaging intraplaque inflammation in carotid atherosclerosis with 11c-pk11195 positron emission tomography/computed tomography measurement of (68)ga-dotatoc uptake in the thoracic aorta and its correlation with cardiovascular risk 64cu-dotatate for noninvasive assessment of atherosclerosis in large arteries and its correlation with risk factors: head-to-head comparison with 68ga-dotatoc in 60 patients noninvasive assessment of hypoxia in rabbit advanced atherosclerosis using (1)(8)f-fluoromisonidazole positron emission tomographic imaging correlation of fluorine 18-labeled sodium fluoride uptake and arterial calcification on whole-body pet/ct in cancer patients predictive value of (18)f-sodium fluoride positron emission tomography in detecting high-risk coronary artery disease in combination with computed tomography in search of the vulnerable patient or the vulnerable plaque: (18)f-sodium fluoride positron emission tomography for cardiovascular risk stratification 18)f-sodium fluoride positron emission tomography for molecular imaging of coronary atherosclerosis based on computed tomography analysis amyloid-targeting pet tracer [(18)f]flutemetamol accumulates in atherosclerotic plaques 18)f-naf and (18)f-fdg as molecular probes in the evaluation of atherosclerosis association between osteogenesis and inflammation during the progression of calcified plaque evaluated by (18)f-fluoride and (18)f-fdg new insight of functional molecular imaging into the atheroma biology: 18f-naf and 18f-fdg in symptomatic and asymptomatic carotid plaques after recent cva. preliminary results quantification of temporal changes in calcium score in active atherosclerotic plaque in major vessels by (18)f-sodium fluoride pet/ct identifying active vascular microcalcification by (18)f-sodium fluoride positron emission tomography recent progress in the development of tspo pet ligands for neuroinflammation imaging in neurological diseases longitudinal imaging of microglia-astrocyte activation in mouse mesial temporal lobe epilepsy with tspo pet to identify the best therapeutic time windows in vivo positron emission tomographic imaging of glial responses to amyloid-beta and tau pathologies in mouse models of alzheimer's disease and related disorders translocator protein-18 kda (tspo) positron emission tomography (pet) imaging and its clinical impact in neurodegenerative diseases imaging of peripheral benzodiazepine receptor expression as biomarkers of detrimental versus beneficial glial responses in mouse models of alzheimer's and other cns pathologies distinct binding of amyloid imaging ligands to unique amyloid-beta deposited in the presubiculum of alzheimer's disease in vivo visualization of tau accumulation, microglial activation, and brain atrophy in a mouse model of tauopathy rtg4510 longitudinal, quantitative assessment of amyloid, neuroinflammation, and anti-amyloid treatment in a living mouse model of alzheimer's disease enabled by positron emission tomography comparative in vitro and in vivo quantifications of pathologic tau deposits and their association with neurodegeneration in tauopathy mouse models imaging of brain tspo expression in a mouse model of amyotrophic lateral sclerosis with (18) f-dpa-714 and micro-pet/ct 11c]pbr28 pet imaging is sensitive to neuroinflammation in the aged rat tspo imaging in parkinsonian disorders pet imaging of [(11)c]pbr28 in parkinson's disease patients does not indicate increased binding to tspo despite reduced dopamine transporter binding animal models of amyotrophic lateral sclerosis: a comparison of model validity practical considerations for choosing a mouse model of alzheimer's disease an 18-kda translocator protein (tspo) polymorphism explains differences in binding affinity of the pet radioligand pbr28 kinetic modeling without accounting for the vascular component impairs the quantification of [(11)c]pbr28 brain pet data novel reference region model reveals increased microglial and reduced vascular binding of 11c-(r)-pk11195 in patients with alzheimer's disease pet imaging of microglia by targeting macrophage colony-stimulating factor 1 receptor (csf1r) synthesis and initial in vivo evaluation of [(11) c]az683-a novel pet radiotracer for colony stimulating factor 1 receptor (csf1r) purinergic receptors p2y12r and p2x7r: potential targets for pet imaging of microglia phenotypes in multiple sclerosis identification of the allosteric p2x7 receptor antagonist [(11)c]smw139 as a pet tracer of microglial activation characterization of (11)c-gsk1482160 for targeting the p2x7 receptor as a biomarker for neuroinflammation pet imaging of the p2x7 ion channel with a novel tracer [(18)f]jnj-64413739 in a rat model of neuroinflammation preclinical evaluation and non-human primate receptor occupancy study of (18)f-jnj-64413739, a novel pet radioligand for p2x7 receptors f-jnj-64413739, a novel pet ligand for the p2x7 ion channel: radiation dosimetry, kinetic modeling, testretest variability and occupancy of the p2x7 antagonist jnj-54175446 identification of new molecular targets for pet imaging of the microglial anti-inflammatory activation state neuroinflammation: the devil is in the details inflammatory responses in brain ischemia inflammatory projections after focal brain injury trigger neuronal network disruption: an (18) f-dpa714 pet study in mice role of neuroinflammation in neurodegenerative diseases (review) bridging autism spectrum disorders and schizophrenia through inflammation and biomarkers-pre-clinical and clinical investigations imaging microglial activation with tspo pet: lighting up neurologic diseases? comparison of [ 11 c]-(r)-pk 11195 and [ 11 c]pbr28, two radioligands for translocator protein (18 kda) in human and monkey: implications for positron emission tomographic imaging of this inflammation biomarker 11 c-dpa-713 has much greater specific binding to translocator protein 18 kda (tspo) in human brain than 11 c-( r)-pk11195 as new pet tracers for tspo: a comparison with [ 11 c]-(r)-pk11195 in a rat model of herpes encephalitis an 18-kda translocator protein (tspo) polymorphism explains differences in binding affinity of the pet radioligand pbr28 reactive astrocytes overexpress tspo and are detected by tspo positron emission tomography imaging evaluation of recanalization of occluded middle cerebral artery and restored cerebral blood flow in rats with transient brain ischemia: a combination study of digital subtraction angiography and 15 o-water positron emission tomography experimental studies of ischemic brain edema. a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemia area evaluation of the pbr/ tspo radioligand [(18)f]dpa-714 in a rat model of focal cerebral ischemia reduced pbr/tspo expression after minocycline treatment in a rat model of focal cerebral ischemia: a pet study using temporal changes in cell marker expression and cellular infiltration in a controlled cortical impact model in adult male c57bl/6 mice myeloid-derived suppressor cells infiltrate the brain and suppress neuroinflammation in a mouse model of focal traumatic brain injury in vivo positron emission tomographic imaging of glial responses to amyloid-beta and tau pathologies in mouse models of alzheimer's disease and related disorders imaging of peripheral benzodiazepine receptor expression as biomarkers of detrimental versus beneficial glial responses in mouse models of alzheimer's and other cns pathologies longitudinal pet-mri reveals beta-amyloid deposition and rcbf dynamics and connects vascular amyloidosis to quantitative loss of perfusion in vivo expression of cyclooxygenase-1 in activated microglia and macrophages during neuroinflammation visualized by pet with 11c-ketoprofen methyl ester detection of cyclooxygenase-1 in activated microglia during amyloid plaque progression: pet studies in alzheimer's disease model mice 11c]pk11195 pet imaging of spinal glial activation after nerve injury in rats )f-vc701-pet and mri in the in vivo neuroinflammation assessment of a mouse model of multiple sclerosis in vivo imaging of neuroinflammation in alzheimer's disease p2x7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of alzheimer's disease p2y12 expression and function in alternatively activated human microglia preclinical evaluation and non-human primate receptor occupancy study of (18)f-jnj-64413739, a novel pet radioligand for p2x7 receptors 18)f-jnj-64413739, a novel pet ligand for the p2x7 ion channel: radiation dosimetry, kinetic modeling, test-retest variability and occupancy of the p2x7 antagonist jnj-54175446 p2x7 radioligand (18)f-pttp for the differentiation of lung tumor and inflammation emerging pet radiotracers and targets for imaging of neuroinflammation in neurodegenerative diseases: outlook beyond tspo translocator protein (18 kda): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function c]daa1106: radiosynthesis and in vivo binding to peripheral benzodiazepine receptors in mouse brain novel peripheral benzodiazepine receptor ligand [ 11 c]daa1106 for pet: an imaging tool for glial cells in the brain quantitative analysis for estimating binding potential of the peripheral benzodiazepine receptor with [ 11 c]daa1106 18 f]fmdaa1106 and [ 18 f]fedaa1106: two positron-emitter labeled ligands for peripheral benzodiazepine receptor (pbr) development of a new radioligand, n-(5-fluoro-2-phenoxyphenyl)-n-(2-[ 18 f] fluoroethyl-5-methoxybenzyl)acetamide, for pet imaging of peripheral benzodiazepine receptor in primate brain quantitative analyses of 18 f-fedaa1106 binding to peripheral benzodiazepine receptors in living human brain 11 c-ac-5216: a novel pet ligand for peripheral benzodiazepine receptors in the primate brain quantitative analysis of peripheral benzodiazepine receptor in the human brain using pet with 11 c-ac-5216 18 f]feac and [ 18 f] fedac: two novel positron emission tomography ligands for peripheral-type benzodiazepine receptor in the brain 18 f-feac and 18 f-fedac: pet of the monkey brain and imaging of translocator protein (18 kda) in the infarcted rat brain efficient radiosynthesis and non-clinical safety tests of the tspo radioprobe [ 18 f] fedac: prerequisites for clinical application in vivo imaging and quantitative analysis of tspo in rat peripheral tissues using smallanimal pet with [ 18 f]fedac pet imaging of lung inflammation with [ 18 f]fedac, a radioligand for translocator protein (18 kda) translocator protein (18 kda), a potential molecular imaging biomarker for non-invasively distinguishing non-alcoholic fatty liver disease visualization of acute liver damage induced by cycloheximide in rats using pet with [ 18 f]fedac, a radiotracer for translocator protein (18 kda) utility of translocator protein (18 kda) as a molecular imaging biomarker to monitor the progression of liver fibrosis 11 c]dac-pet for noninvasively monitoring neuroinflammation and immunosuppressive therapy efficacy in rat experimental autoimmune encephalomyelitis model 18 f-fedac as a targeting agent for activated macrophages in dba/1 mice with collagen-induced arthritis: comparison with 18 f-fdg in vivo imaging of activated macrophages by 18 f-fedac, a tspo targeting pet ligand, in the use of biologic disease-modifying anti-rheumatic drugs (bdmards) microglia and neurodegeneration: the role of systemic inflammation microglia in the human fetal spinal cord; patterns of distribution, morphology and phenotype production of interleukin-12 and expression of its receptors by murine microglia histamine production by cultured microglial cells of the mouse pepstatin a induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines interleukin-4-induced selective clearance of oligomeric betaamyloid peptide1-42 by rat primary type-2 microglia the origin and nature of ramified and amoeboid microglia: a historical review and current concepts activation and proliferation of the isolated microglia by colony stimulating factor-1 and possible involvement of protein kinase c identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse the murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene microglia: intrinsic immuneffector cell of the brain cd14: cell surface receptor and differentiation marker macrophage scavenger receptors morphological transformation of microglia in vitro production of interleukin-5 by mouse astrocytes and microglia in culture induction of functional interleukin-2 receptor in mouse microglia il-2 induction of il-1 beta mrna expression in monocytes monocyte interleukin-2-receptor gene expression and interleukin-2 augmentation of microbicidal activity down regulation of cd4 expression in cultured microglia by immunosuppressants and lipopolysaccharide bone marrow derived elements and resident microglia in brain inflammation absence of donor-type major histocompatibility complex class i antigen-bearing microglia in the rat central nervous system of radiation bone marrow chimeras immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to ia-positive cells with dendritic morphology selection of intraepithelial lymphocytes with cd8 alpha/alpha co-receptors by self-antigen in the murine gut granulocyte-macrophage colony-stimulating factor promotes the proliferation of human alveolar macrophages in vitro nigral dopaminergic cell loss in vitamin e deficient rats cellular and molecular mechanisms of parkinson's disease: neurotoxins, causative genes, and inflammatory cytokines role of cytokines in inflammatory process in parkinson's disease distribution of major histocompatibility complex class il-positive microglia and cytokine profile of parkinson's disease brains blockade of microglial activation is neuroprotective in the 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of parkinson disease nadph oxidase mediates oxidative stress in the 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine model of parkinson disease amyloid-beta peptides induce several chemokine mrna expressions in the primary microglia and ra2 cell line via pi3k/akt and/or erk pathway activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with 1-methyl-4-phenyl 1,2,3,6-tetra-hydropyridine neuroprotective effect of exogenous microglia in global brain ischemia the hiv-1 nef protein and phagocyte nadph oxidase activation cytokine network in the central nervous system and its roles in growth and differentiation of glial and neuronal cells validation of an animal isolation imaging chamber for use in animal biosafety level-3 containment severe fever with thrombocytopenia syndrome and its pathogen sftsv 18f-fdg pet imaging for identifying the dynamics of intestinal disease caused by sftsv infection in a mouse model development of a (68) ge/(68)ga generator system using polysaccharide polymers and its application in pet imaging of tropical infectious diseases ga-67 citrate imaging in tumors of the genito-urinary tract: report of cooperative study pediatric solid tumors: evaluation by gallium-67 spect studies mechanism of gallium-67 accumulation in inflammatory lesions cell biology and immunology of leishmania acknowledgements i would like to thank my collaborators: dr. sanae hosomi (department of traumatology and acute critical medicine) for the tbi study and dr. naoyuki sato (department of clinical gene therapy) for the ad study. the author thanks mr. kumata, ms. hatori, dr. xie, dr. yanamoto, and dr. yamasaki in my group for their fruitful experiments and continuous outputs which contributed to this review. the authors are grateful to the american chemical society (acs) for allowing reuse of some images (figs. 1.31 and 1.32) and a direct link to the acs article: https://pubs.acs. org/doi/abs/10.1021/acsomega.7b00147. key: cord-026031-hnf5vayd authors: ford, richard b.; mazzaferro, elisa m. title: emergency care date: 2009-05-21 journal: kirk and bistner's handbook of veterinary procedures and emergency treatment doi: 10.1016/b0-72-160138-3/50002-3 sha: doc_id: 26031 cord_uid: hnf5vayd nan in the event that you suspect peritonitis and have a negative tap with abdominal paracentesis, a diagnostic peritoneal lavage can be performed. to perform abdominal paracentesis, follow this procedure: 1. place the patient in left lateral recumbency and clip a 4-to 6-inch square with the umbilicus in the center. 2. aseptically scrub the clipped area with antimicrobial scrub solution. 3. wearing gloves, insert a 22-or 20-gauge needle or over-the-needle catheter in four quadrants: cranial and to the right, cranial and to the left, caudal and to the right, and caudal and to the left of the umbilicus. as you insert the needle or catheter, gently twist the needle to push any abdominal organs away from the tip of the needle. local anesthesia typically is not required for this procedure, although a light sedative or analgesic may be necessary if severe abdominal pain is present. in some cases, fluid will flow freely from one or more of the needles. if not, gently aspirate with a 3-to 6-ml syringe or aspirate with the patient in a standing position. avoid changing positions with needles in place because iatrogenic puncture of intraabdominal organs may occur. 4. save any fluid collected in sterile red-and lavender-topped tubes for cytologic and biochemical analyses and bacterial culture. monitor hemorrhagic fluid carefully for the presence of clots. normally, hemorrhagic effusions rapidly become defibrinated and do not clot. clot formation can occur in the presence of ongoing active hemorrhage or may be due to the iatrogenic puncture of organs such as the spleen or liver. if abdominal paracentesis is negative, a diagnostic peritoneal lavage can be performed. peritoneal dialysis kits are commercially available but are fairly expensive and often impractical. to perform a diagnostic peritoneal lavage, follow this procedure: 1. clip and aseptically scrub the ventral abdomen as described previously. 2. wearing sterile gloves, cut multiple side ports in a 16-or 18-gauge over-the needle catheter. use care to not cut more than 50% of the circumference of the catheter, or else the catheter will become weakened and potentially can break off in the patient's abdomen. 3. insert the catheter into the peritoneal cavity caudal and to the right of the umbilicus, directing the catheter dorsally and caudally. 4. infuse 10 to 20 ml of sterile lactated ringer's solution or 0.9% saline solution that has been warmed to the patient's body temperature. during the instillation of fluid into the peritoneal cavity, watch closely for signs of respiratory distress because an increase in intraabdominal pressure can impair diaphragmatic excursions and respiratory function. 5. remove the catheter. 6. in ambulatory patients, walk the patient around while massaging the abdomen to distribute the fluid throughout the abdominal cavity. in nonambulatory patients, gently roll the patient from side to side. 7. next, aseptically scrub the patient's ventral abdomen again, and perform an abdominal paracentesis as described previously. save collected fluid for culture and cytologic analyses; however, biochemical analyses may be artifactually decreased because of dilution. remember that you likely will retrieve only a small portion of the fluid that you instilled. during the early stage of repair, granulation tissue, some exudate, and minor epithelialization is observed. place a nonadherent bandage with some antibacterial properties (petroleum or nitrofurazone-impregnated gauze) or absorbent material (foam sponge, hydrogel, or hydrocolloid dressing) in direct contact with the wound to minimize disruption of the granulation tissue bed. next, place an absorbent intermediate layer, followed by a porous outer layer, as previously described. granulation tissue can grow through gauze mesh or adhere to foam sponges and can be ripped away at the time of bandage removal. hemorrhage and disruption of the granulation tissue bed can occur. later in the repair process, granulation tissue can exude sanguineous drainage and have some epithelialization. a late nonadherent bandage is required. the contact layer should be some form of nonadherent dressing, foam sponge, hydrogel, or hydrocolloid substance. the intermediate layer and outer layers should be absorbent material and porous tape, respectively. with nonadherent dressings, wounds with viscous exudates may not be absorbed well. this may be advantageous and enhance epithelialization, provided that complications do not occur. infection, exuberant granulation tissue, or adherence of absorbent materials to the wound may occur and delay the healing process. moist healing is a newer concept of wound management in which wound exudates are allowed to stay in contact with the wound. in the absence of infection a moist wound heals faster and has enzymatic activity as a result of macrophage and polymorphonuclear cell breakdown. enzymatic degradation or "autolytic debridement" of the wound occurs. moist wounds tend to promote neutrophil and macrophage chemotaxis and bacterial phagocytosis better than use of wet-to-dry bandages. a potential complication and disadvantage of moist healing, however, is the development of bacterial colonization, folliculitis, and trauma to wound edges that can occur because of the continuously moist environment. use surfactant-type solutions (constant clens; kendall, mansfield, massachusetts) for initial wound cleansing and debridement. use occlusive dressings for rapid enzymatic debridement with bactericidal properties to aid in wound healing. bandage wet necrotic wounds with a dressing premoistened with hypertonic saline (curasalt [kendall] , 20% saline) to clean and debride the wounds. hypertonic saline functions to desiccate necrotic tissue and bacteria to debride the infected wound. remove and replace the hypertonic saline bandage every 24 to 48 hours. next, place gauze impregnated with antibacterial agents (kerlix amd [kendall] ) over the wound in the bandage layer to act as a barrier to bacterial colonization. if the wound is initially dry or has minimal exudate and is not obviously contaminated or infected, place amorphous gels of water, glycerin, and a polymer (curafil [kendall] ) over the wound to promote moisture and proteolytic healing. discontinue moisture gels such as curafil once the dry wound has become moist. finally, the final stage of moist healing helps to promote the development of a healthy granulation tissue bed. use calcium alginate dressings (curasorb or curasorb zn with zinc [kendall] ) in noninfected wounds with a moderate amount of drainage. alginate gels promote rapid development of a granulation tissue bed and epithelialization. foam dressings also can be applied to exudative wounds after a healthy granulation bed has formed. change foam dressings at least once every 4 to 7 days. for closed wounds without any drainage, such as a laceration that has been repaired surgically, a simple bandage with a nonadherent contact layer (telfa pad [kendall] , for example), intermediate layer of absorbent material, and an outer porous layer (elastikon, vetrap) can 1 be placed to prevent wound contamination during healing. the nonadherent pad will not stick to the wound and cause patient discomfort. because there usually is minimal drainage from the wound, the function of the intermediate layer is more protective than absorptive. any small amount will be absorbed into the intermediate layer of the bandage. it is important in any bandage to place the tape strips or "stirrups" on the patient's limb and then overlap in the bandage, to prevent the bandage from slipping. place the intermediate and tertiary layers loosely around the limb, starting distally and working proximally, with some overlap with each consecutive layer. this method prevents excessive pressure and potential to impair venous drainage. leave the toenails of the third and fourth digits exposed, whenever possible, to allow daily examination of the bandage to determine whether the bandage is impairing venous drainage. if the bandage is too tight and constricting or impeding vascular flow, the toes will become swollen and spread apart. when placed and maintained properly (e.g., the bandage does not get wet), there usually are relatively few complications observed with this type of bandage. in some cases, it is necessary to cover a wound in which a penrose drain has been placed to allow drainage. in many cases, there is a considerable amount of drainage from the drain and underlying soft tissues. the function of the bandage is to help obliterate dead space created by the wound itself, absorb the fluid that drains from the wound and that will contaminate the environment, and prevent external wicking of material from the external environment into the wound. when the bandage is removed, the clinician can examine the amount and type of material that has drained from the wound in order to determine when the drain should be removed. when placing a bandage over a draining wound, the contact layer should be a commercially available nonadherent dressing and several layers of absorbent wide-mesh gauze placed directly over the drain at the distal end of the incision. overlay the layers of gauze with a thick layer of absorbent intermediate dressing to absorb fluid that drains from the wound. if the gauze and intermediate layers are not thick or absorbent enough, there is a potential for the drainage fluid to reach the outer layer of the bandage and provide a source of wicking of bacteria from the external environment into the wound, leading to infection. some wounds such as lacerations have minor bleeding or hemorrhage that require an immediate bandage until definitive care can be provided. to create a pressure bandage, place a nonadherent dressing immediately in contact with the wound, followed by a thick layer of absorbent material, topped by a layer of elastic bandage material such as elastikon or vetrap. unlike the bandage for a closed wound, the top tertiary outer layer should be wrapped with some tension and even pressure around the limb, starting from the distal extremity (toes) and working proximally. the pressure bandage serves to control hemorrhage but should not be left on for long periods. pressure bandages that have been left on for too long can impair nerve function and lead to tissue necrosis and slough. therefore, pressure bandages should be used in the hospital only, so that the patient can be observed closely. if hemorrhage through the bandage occurs, place another bandage over the first until the wound can be repaired definitively. removal of the first bandage will only disrupt any clot that has formed and cause additional hemorrhage to occur. fractures require immediate immobilization to prevent additional patient discomfort and further trauma to the soft tissues of the affected limb. as with all bandages, a contact layer, intermediate layer, and outer layer should be used. place the contact layer in accordance 1 with any type of wound present. the intermediate layer should be thick absorbent material, followed by a top layer of elastic bandage material. an example is to place a telfa pad over a wound in an open distal radius-ulna fracture, followed by a thick layer of cotton gauze cast padding, followed by an elastic layer of kling (johnson & johnson medical, arlington, texas) , pulling each layer tightly over the previous layer with some overlap until the resultant bandage can be "thumped" with the clinician's thumb and forefinger and sound like a ripe watermelon. the bandage should be smooth with consecutive layers of even pressure on the limb, starting distally and working proximally. leave the toenails of the third and fourth digits exposed to monitor for impaired venous drainage that would suggest that the bandage is too tight and needs to be replaced. finally, place a top layer of vetrap or elastikon over the intermediary layer to protect it from becoming contaminated. if the bandage is used with a compound or open fracture, drainage may be impaired and actually lead to enhanced risk of wound infection. bandages placed for initial fracture immobilization are temporary until definitive fracture repair can be performed once the patient's cardiovascular and respiratory status are stable. wounds with exuberant granulation tissue must be handled carefully so as to not disrupt the healing process but to keep an overabundance of tissue from forming that will impair epithelialization. to bandage a wound with exuberant granulation tissue, place a corticosteroid-containing ointment on the wound, followed by a nonadherent contact layer. the corticosteroid will help control the exuberant growth of granulation tissue. next, carefully wrap an absorbent material over the contact layer, followed by careful placement of and overlay of elastic bandage material to place some pressure on the wound. leave the toenails of the third and fourth digits exposed so that circulation can be monitored several times daily. bandages that are too tight must be removed immediately to prevent damage to neuronal tissue and impaired vascularization, tissue necrosis, and slough. because wound drainage may be impaired, there is a risk of infection. gaping wounds or those that have undermined in between layers of subcutaneous tissue and fascia should be bandaged with a pressure bandage to help obliterate dead space and prevent seroma formation. an example of a wound that may require this type of bandage is removal of an infiltrative lipoma on the lateral or ventral thorax. use caution when placing pressure bandages around the thorax or cervical region because bandages placed too tightly may impair adequate ventilation. to place a pressure bandage and obliterate dead space, place a nonadherent contact layer over the wound. usually, a drain is placed in the wound, so place a large amount of wide-mesh gauze at the distal end of the drain to absorb any wound exudate or drainage. place several layers of absorbent material over the site to further absorb any drainage. place a layer of elastic cotton such as kling carefully but firmly over the dead space to cause enough pressure to control drainage. place at least two fingers in between the animal's thorax and the bandage to ensure that the bandage is not too tight. in many cases, the bandage should be placed once the animal has recovered from surgery and is able to stand. if the bandage is placed while the animal is still anesthetized and recumbent, there is a tendency for the bandage to be too tight. finally, the tertiary layer should be an elastic material such as elastikon or vetrap. many wounds require a pressure relief bandage to prevent contact with the external environment. wounds that may require pressure relief for healing include decubitus ulcers, pressure bandage or cast ulcers, impending ulcer areas (such as the ileum or ischium of recumbent or cachexic patients), and surgical repair sites of ulcerated areas. pressure relief bandages can be of two basic varieties: modified doughnut bandage and doughnut-shaped bandage. to create a cup or clamshell splint, follow this procedure (figures 1-7 to 1-11): 1. place a nonadherent contact layer directly over the wound. 2. place stirrups of tape in contact with the skin of the dog, to be placed over the intermediate layer and prevent the bandage from slipping. 3. place a fairly thick layer of absorbent intermediate bandage material over the contact layer such that the bandage is well-padded. pull the tape stirrups and secure them to the intermediate layer. 4. place a length of cast material that has been rolled to the appropriate length, such that the cast material is cupped around the patient's paw, and lies adjacent to the caudal aspect of the limb to the level of the carpus or tarsus. in the case of a clamshell splint, place a layer of cast material on the cranial and caudal aspect of the paw and conform it in place. 5. take the length of cast padding and soak it in warm water after it has been rolled to the appropriate length. wring out the pad, and secure/conform it to the caudal (or cranial and caudal, in the case of a clamshell splint) aspect of the distal limb and paw. 6. secure the cast material in place with a layer of elastic cotton gauze (kling). 7. secure the bandage in place with a snug layer of elastikon or vetrap. short or long splints made of cast material can be incorporated into a soft padded bandage to provide extra support of a limb above and below a fracture site. for a caudal or lateral splint to be effective, it must be incorporated for at least one joint above any fracture site to prevent a fulcrum effect and further disruption or damage to underlying soft tissue structures. a short lateral or caudal splint is used for fractures and luxations of the distal metacarpus, metatarsus, carpus, and tarsus. to place a short lateral or caudal splint, follow this procedure: 1. secure a contact layer as determined by the presence or absence of any wound in the area. 2. place tape stirrups on the distal extremity to be secured later to the intermediate bandage layer and to prevent slipping of the bandage distally. 3. place layers of roll cotton from the toes to the level of the mid tibia/fibula or mid radius/ulna. place the layers with even tension, with some overlap of each consecutive layer, moving distally to proximally on the limb. 4. secure the short caudal or lateral splint and conform it to the distal extremity to the level of the toes and proximally to the level of the mid tibia/fibula or mid radius/ulna. 5. secure the lateral or caudal splint to the limb with another outer layer of elastic cotton (kling). 6. cover the entire bandage and splint with an outer tertiary layer of vetrap or elastikon. make sure that the toenails of the third and fourth digits remain visible to allow daily evaluation of circulation. long lateral or caudal splints are used to immobilize fractures of the tibia/fibula and radius/ulna. the splints are fashioned as directed for short splints but extend proximally to the level of the axilla and inguinal regions to immobilize above the fracture site. â�¢ packed cell volume drops rapidly to less than 20% in the dog and less than 12% to 15% in the cat â�¢ acute loss of more than 30% of blood volume (30 ml/kg in dog, 20 ml/kg in cat) â�¢ clinical signs of lethargy, collapse, hypotension, tachycardia, tachypnea (acute or chronic blood loss) â�¢ ongoing hemorrhage is present â�¢ poor response to crystalloid and colloid infusion â�¢ life-threatening hemorrhage caused by thrombocytopenia or thrombocytopathia â�¢ surgical intervention is necessary in a patient with severe thrombocytopenia or thrombocytopathia plasma support â�¢ life-threatening hemorrhage with decreased coagulation factor activity â�¢ severe inflammation (pancreatitis, systemic inflammatory response syndrome) â�¢ replenish antithrombin (disseminated intravascular coagulation, protein-losing enteropathy or nephropathy) â�¢ surgery is necessary in a patient with decreased coagulation factor activity â�¢ severe hypoproteinemia is present; to partially replenish albumin, globulin, and clotting factors type a cats typically possess weak anti-b antibodies of igg and igm subtypes. transfusion of type b blood into a type a cat will result in milder clinical signs of reaction and a markedly decreased survival half-life of the infused rbcs to just 2 days. because type ab cats possess both moieties on their cell surface, they lack naturally occurring alloantibodies; transfusion of type a blood into a type ab cat can be performed safely if a type ab donor is not available. the life span of an rbc from a type-specific transfusion into a cat is approximately 33 days. . indications for fresh whole blood transfusion include disorders of hemostasis and coagulopathies including disseminated intravascular coagulation, von willebrand's disease, and hemophilia. fresh whole blood and platelet-rich plasma also can be administered in cases of severe thrombocytopenia and thrombocytopathia. stored whole blood and packed rbcs can be administered in patients with anemia. if pcv drops to below 10% or if rapid hemorrhage causes the pcv to drop below 20% in the dog or less than 12% to 1 *indicates that this must be done for each donor being tested. minor crossmatch* 2. obtain a crossmatch segment from blood bank refrigerator for each donor to be crossmatched, or use an edta tube of donor's blood. make sure tubes are labeled prop-erly. 3. collect 2 ml of blood from recipient and place in an edta tube. centrifuge blood for 5 minutes. 4. extract blood from donor tubing. centrifuge blood for 5 minutes. use a separate pipette for each transfer because cross-contamination can occur. 5. pipette plasma off of donor and recipient cells and place in tubes labeled dp and rp, respectively. 6. place 125 âµl of donor and recipient cells in tubes labeled dr and rr, respectively. 7. add 2.5 ml 0.9% sodium chloride solution from wash bottle to each red blood cell (rbc) tube, using some force to cause cells to mix. 8. centrifuge rbc suspension for 2 minutes. 9. discard supernatant and resuspend rbcs with 0.9% sodium chloride from wash bottle. 10 . repeat steps 8 and 9 for a total of three washes. 11. place 2 drops of donor rbc suspension and 2 drops of recipient plasma in tube labeled ma (this is the major crossmatch). 12. place 2 drops of donor plasma and 2 drops recipient rbc suspension in tube labeled mi (this is the minor crossmatch). 13. prepare control tubes by placing 2 drops donor plasma with 2 drops donor rbc suspension (this is the donor control); and place 2 drops recipient plasma with 2 drops recipient rbc suspension (this is the recipient control). 14. incubate major and minor crossmatches and control tubes at room temperature for 15 minutes. 15. centrifuge all tubes for 1 minute. 16. read tubes using an agglutination viewer. 17. check for agglutination and/or hemolysis. 18. score agglutination with the following scoring scale: 4+ one solid clump of cells 3+ several large clumps of cells 2+ medium-sized clumps of cells with a clear background 1+ hemolysis, no clumping of cells neg = negative for hemolysis; negative for clumping of red blood cells fresh whole blood coagulopathy with active hemorrhage (disseminated intravascular coagulation, thrombocytopenia; massive acute hemorrhage; no stored blood available) stored whole blood massive acute or ongoing hemorrhage; hypovolemic shock caused by hemorrhage that is unresponsive to conventional crystalloid and colloid fluid therapy; unavailability of equipment required to prepare blood components packed red blood cells nonregenerative anemia, immune-mediated hemolytic anemia, correction of anemia before surgery, acute or chronic blood loss fresh frozen plasma factor depletion associated with active hemorrhage (congenital: von willebrand's factor, hemophilia a, hemophilia b; acquired: vitamin k antagonist, rodenticide intoxication, dic); acute or chronic hypoproteinemia (burns, wound exudates, body cavity effusion; hepatic, renal, or gastrointestinal loss); colostrum replacement in neonates frozen plasma acute plasma or protein loss; chronic hypoproteinemia; (contains stable colostrum replacement in neonates; hemophilia b and clotting factors) selected clotting factor deficiencies platelet-rich plasma* thrombocytopenia with active hemorrhage (immune-mediated thrombocytopenia, dic); platelet function abnormality (congenital: thrombasthenia in bassett hounds; acquired: nsaids, other drugs) cryoprecipitate congenital factor deficiencies (routine or before surgery): (concentration of factor hemophilia a, hemophilia b, von willebrand's disease, viii, von willebrand's hypofibrinogenemia; acquired factor deficiencies factor, and fibrinogen) *must be purchased because logistically one cannot obtain enough blood simultaneously to provide a significant amount of platelets; platelets infused have a very short (<2 hours) half-life. dic, disseminated intravascular coagulation; nsaids, nonsteroidal antiinflammatory drugs. universal donor (e.g., should be administered whenever possible. because there is no universal donor in the cat and because cats possess naturally occurring alloantibodies, all cat blood should be typed and crossmatched before any transfusion. if fresh whole blood is not available, a hemoglobin-based oxygen carrier (oxyglobin, 2 to 7 ml/kg iv) can be administered until blood products become available. table 1 -4 indicates blood component dose and administration rates. blood products should be warmed slowly to 37â°c before administering them to the patient. blood warmer units are available for use in veterinary medicine to facilitate rapid transfusion without decreasing patient body temperature (thermal angel; enstill medical technologies, inc., dallas, texas). red blood cell and plasma products should be administered in a blood administration set containing a 170-âµm in-line filter. smaller in-line filters (20 âµm) also can be used in cases in which extremely small volumes are to be administered. blood products should be administered over a period of 4 hours, whenever possible, according to guidelines set by the american association of blood banks. the volume of blood components required to achieve a specific increment in the patient's pcv depends largely on whether whole blood or packed rbcs are transfused and whole blood 20 ml/kg will increase max rate: 22 ml/kg/ max: 22 ml/kg/ volume by 10% 24 hours hour packed red 10 ml/kg will increase critically ill blood cells volume by 10% patients (e.g., cardiac failure or renal failure): 3-4 ml/kg/hour fresh frozen 10 ml/kg body mass (repeat 4-10 ml/minute or use rates as for plasma in 2-3 days or in 3-5 days whole blood (infuse within 4-6 hours) or until bleeding stops); monitor act, aptt, and pt before and 1 hour after transfusion cryoprecipitate general: 1 unit/10 kg/12 hours 4-10 ml/minute or use rates as for whole or until bleeding stops blood (infuse within 4-6 hours) hemophilia a: 12-20 units factor viii/kg; 1 unit of cryoprecipitate contains approximately 125 units of factor viii platelet-rich 1 unit/10 kg (1 unit of 2 ml/minute plasma platelet-rich plasma will check platelet count before and 1 hour increase platelet count after transfusion 1 hour after transfusion by 10,000/âµl) whether there is ongoing hemorrhage or rbc destruction. because the pcv of packed rbcs is unusually high (80% for greyhound blood), a smaller total volume is required than whole blood to achieve a comparable increase in the patient's pcv. in general, 10 ml/kg of packed rbcs or 20 ml/kg whole blood will raise the recipient's pcv by 10%. the "rule of ones" states that 1 ml per 1 lb of whole blood will raise the pcv by 1%. if the patient's pcv does not raise by the amount anticipated by the foregoing calculation(s), causes of ongoing hemorrhage or destruction should be considered. the goal of red blood component therapy is to raise the pcv to 25% to 30% in dogs and 15% to 20% in cats. if an animal is hypovolemic and whole blood is administered, the fluid is redistributed into the extravascular compartment within 24 hours of transfusion. this will result in a secondary rise in the pcv 24 hours after the transfusion in addition to the initial rise 1 to 2 hours after the rbc transfusion is complete. the volume of plasma transfused depends largely on the patient's need. in general, plasma transfusion should not exceed more than 22 ml/kg during a 24-hour period for normovolemic animals. thaw plasma at room temperature, or place it in a ziplock freezer bag and run under cool (not warm) water until thawed. then administer the plasma through a blood administration set that contains an in-line blood filter or through a standard driptype administration set with a detachable in-line blood administration filter. the average rate of plasma infusion in a normovolemic patient should not exceed 22 ml/kg/hour. in acute need situations, plasma can be delivered at rates up to 5 to 6 ml/kg/minute. for patients with cardiac insufficiency or other circulatory problems, plasma infusion rates should not exceed 5 ml/kg/hour. plasma or other blood products should not be mixed with or used in the same infusion line as calcium-containing fluids, including lactated ringer's solution, calcium chloride, or calcium gluconate. the safest fluid to mix with any blood product is 0.9% sodium chloride. administer fresh frozen plasma, frozen plasma, and cryoprecipitate at a volume of 10 ml/kg until bleeding is controlled or source of ongoing albumin loss ceases. the goal of plasma transfusion therapy is to raise the albumin to a minimum of 2.0 g/dl or until bleeding stops as in the case of coagulopathies. monitor the patient to ensure that bleeding has stopped, coagulation profiles (act, aptt, and pt) have normalized, hypovolemia has stabilized, and/or total protein is normalizing, which are indications for discontinuing ongoing transfusion therapy. plasma cryoprecipitate can be purchased or manufactured through the partial thawing and then centrifugation of fresh frozen plasma. cryoprecipitate contains concentrated quantities of vwf, factor viii, and fibrinogen and is indicated in severe forms of von willebrand's disease and hemophilia a (factor viii deficiency). platelet-rich plasma must be purchased from a commercial source. one unit of fresh whole blood contains 2000 to 5000 platelets. the viability of the platelets contained in the fresh whole blood is short-lived, just 1 to 2 hours after transfusion into the recipient. because platelet-rich plasma is difficult to obtain, animals with severe thrombocytopenia or thrombocytopathia should be treated with immunomodulating therapies and the administration of fresh frozen plasma. in dogs, blood and plasma transfusions can be administered intravenously or intraosseously. the cephalic, lateral saphenous, medial saphenous, and jugular veins are used most commonly. fill the recipient set so that the blood in the drip chamber covers the filter (normal 170-âµm filter). with small amounts of blood (50 ml) or critically ill patients, use a 40-âµm filter. avoid latex filters for plasma and cryoprecipitate administration. blood can 30 1 emergency care be administered at variable rates, but the routine figure of 4 to 5 ml/minute often is used. normovolemic animals can receive blood at 22 ml/kg/day. dogs in heart failure should receive infusions at no more than 4 ml/kg/hour. volume is given as needed. to calculate the approximate volume of blood needed to raise hematocrit levels, use the following formula for the dog: anticoagulated blood volume (ml) = body mass (kg) ã� 90 ã� pcv desired â�� pcv of recipient pcv of donor in anticoagulant an alternative formula is the following: 2.2 ã� recipient body mass (kg) ã� 30 (dog) ã� pcv desired â�� pcv of recipient pcv of donor in anticoagulant surgical emergencies and shock may require several times this volume within a short period. if greater than 25% of the patient's blood volume is lost, supplementation with colloids, crystalloids, and blood products is indicated for fluid replacement. one volume of whole blood achieves the same increase in plasma as two to three volumes of plasma. if the patient's blood type is unknown and type a-negative whole blood is not available, any dog blood can be administered to a dog in acute need if the dog has never had a transfusion before. if mismatched blood is given, the patient will become sensitized, and after 5 days, destruction of the donor rbcs will begin. in addition, any subsequent mismatched transfusions may cause an immediate reaction (usually mild) and rapid destruction of the transfused rbcs. the clinical signs of a transfusion reaction typically only are seen when type a blood is administered to a type a-negative recipient that has been sensitized previously. incompatible blood transfusions to breeding females can result in isoimmunization and in hemolytic disease in the puppies. the a-negative bitch that receives a transfusion with a-positive and that produces a litter from an a-positive stud can have puppies with neonatal isoerythrolysis. cats with severe anemia in need of a blood transfusion are typically extremely depressed, lethargic, and anorexic. the stress of restraint and handling can push these critically ill patients over the edge and cause them to die. extreme gentleness and care are mandatory in restraint and handling. the critically ill cat should be cradled in a towel or blanket. supplemental flow-by or mask oxygen should be administered, whenever possible, although it may not be clinically helpful until oxygen-carrying capacity is replenished with infusion of rbcs or hemoglobin. blood can be administered by way of cephalic, medial saphenous, or the jugular vein. intramedullary infusion is also possible, if vascular access cannot be accomplished. the average 2-to 4-kg cat can accept 40 to 60 ml of whole blood injected intravenously over a period of 30 to 60 minutes. administer filtered blood at a rate of 5 to 10 ml/kg/hour. the following formula can be used to estimate the volume of blood required for transfusion in a cat: anticoagulated blood volume (ml) = body mass (kg) ã� 70 ã� pcv desired â�� pcv of recipient pcv of donor in anticoagulant the exact overall incidence and clinical significance of transfusion reactions in veterinary medicine are unknown. several studies have been performed that document the incidence of transfusion reactions in dogs and cats. overall, the incidence of transfusion reactions in dogs and cats is 2.5% and 2%, respectively. transfusion reactions can be immune-mediated and non-immune-mediated and can happen immediately or can be delayed until after a transfusion. acute reactions usually occur within minutes to hours of the onset of transfusion but may occur up to 48 hours after the transfusion has been stopped. acute immunologic reactions include hemolysis and acute hypersensitivity including rbcs, platelets, and leukocytes. signs of a delayed immunologic reaction include hemolysis, purpura, immunosuppression, and neonatal isoerythrolysis. acute nonimmunologic reactions include donor cell hemolysis before onset of transfusion, circulatory volume overload, bacterial contamination, citrate toxicity with clinical signs of hypocalcemia, coagulopathies, hyperammonemia, hypothermia, air embolism, acidosis, and pulmonary microembolism. delayed nonimmunologic reactions include the transmission and development of infectious diseases and hemosiderosis. clinical signs of a transfusion reaction typically depend on the amount of blood transfused, the type and amount of antibody involved in the reaction, and whether the recipient has had previous sensitization. monitoring the patient carefully during the transfusion period is essential in recognizing early signs of a transfusion reaction, including those that may become life threatening. a general guideline for patient monitoring is first to start the transfusion slowly during the first 15 minutes. monitor temperature, pulse, and respiration every 15 minutes for the first hour, 1 hour after the end of the transfusion, and every 12 hours minimally thereafter. also obtain a pcv immediately before the transfusion, 1 hour after the transfusion has been stopped, and every 12 hours thereafter. monitor coagulation parameters such as an act and platelet count at least daily in patients requiring transfusion therapy. the most common documented clinical signs of a transfusion reaction include pyrexia, urticaria, salivation/ptyalism, nausea, chills, and vomiting. other clinical signs of a transfusion reaction may include tachycardia, tremors, collapse, dyspnea, weakness, hypotension, collapse, and seizures. severe intravascular hemolytic reactions may occur within minutes of the start of the transfusion, causing hemoglobinemia, hemoglobinuria, disseminated intravascular coagulation, and clinical signs of shock. extravascular hemolytic reactions typically occur later and will result in hyperbilirubinemia and bilirubinuria. pretreatment of patients to help decrease the risk of a transfusion reaction remains controversial, and in most cases, pretreatment with glucocorticoids and antihistamines is ineffective at preventing intravascular hemolysis and other reactions should they occur. the most important component of preventing a transfusion reaction is to screen each recipient carefully and process the donor component therapy carefully before the administration of any blood products. treatment of a transfusion reaction depends on its severity. in all cases, stop the transfusion immediately when clinical signs of a reaction occur. in most cases, discontinuation of the transfusion and administration of drugs to stop the hypersensitivity reaction will be sufficient. once the medications have taken effect, restart the transfusion slowly and monitor the patient carefully for further signs of reaction. in more severe cases in which a patient's cardiovascular or respiratory system become compromised and hypotension, tachycardia, or tachypnea occurs, immediately discontinue the transfusion and administer diphenhydramine (1 mg/kg im), dexamethasone-sodium phosphate (0.25 to 0.5 mg/kg iv), and epinephrine to the patient. the patient should have a urinary catheter and central venous catheter placed for measurement of urine output and central venous pressures. aggressive fluid therapy may be necessary to avoid renal insufficiency or renal damage associated with severe intravascular hemolysis. overhydration with subsequent pulmonary edema generally can be managed with supplemental oxygen administration and intravenous or intramuscular administration of furosemide (2 to 4 mg/kg). plasma products with or without heparin can be administered for disseminated intravascular coagulation. the hbocs can be stored at room temperature and have a relatively long shelf life compared with red blood component products. the hbocs function to carry oxygen through the blood and can diffuse oxygen past areas of poor tissue perfusion. an additional characteristic of hbocs is as a potent colloid, serving to maintain fluid within the vascular space. for this reason, hbocs must be used with caution in euvolemic patients and patients with cardiovascular insufficiency. central venous pressure (cvp) measures the hydrostatic pressure in the anterior vena cava and is influenced by vascular fluid volume, vascular tone, function of the right side of the heart, and changes in intrathoracic pressure during the respiratory cycle. the cvp is not a true measure of blood volume but is used to gauge fluid therapy as a method of determining how effectively the heart can pump the fluid that is being delivered to it. thus the cvp reflects the interaction of the vascular fluid volume, vascular tone, and cardiac function. measure cvp in any patient with acute circulatory failure, large volume fluid diuresis (i.e., toxin or oliguric or anuric renal failure), fluid in-and-out monitoring, and cardiac dysfunction. the placement of central venous catheters and thus cvp measurements is contraindicated in patients with known coagulopathies including hypercoagulable states. to perform cvp monitoring, place a central venous catheter in the right or left jugular vein. in cats and small dogs, however, a long catheter placed in the lateral or medial saphenous vein can be used for trends in cvp monitoring. first, assemble the equipment necessary for jugular catheter (see vascular access techniques for how to place a jugular or saphenous long catheter) and cvp monitoring (box 1-7). after placing the jugular catheter, take a lateral thoracic radiograph to ensure that the tip of the catheter sits just outside of the right atrium for proper cvp measurements (see to establish an intravenous catheter for cvp, follow this procedure: 1. assemble the cvp setup such that the male end of a length of sterile intravenous catheter extension tubing is inserted into the t port of the jugular or medial/lateral saphenous catheter. make sure to flush the length of tubing with sterile saline before connecting it to the patient to avoid iatrogenic air embolism. 2. next, insert the male end of a three-way stopcock into the female end of the extension tubing. 3. attach a 20-ml syringe filled with heparinized sterile 0.9% saline to one of the female ports of the three-way stopcock and either a manometer or a second length of intravenous extension tubing attached to a metric ruler. 4. lay the patient in lateral or sternal recumbancy. 5. turn the stopcock off to the manometer/ruler and on to the patient. infuse a small amount of heparinized saline through the catheter to flush the catheter. 6. next, turn the stopcock off to the patient and on to the manometer. gently flush the manometer or length of extension tubing with heparinized saline from the syringe. use care not to agitate the fluid and create air bubbles within the line or manometer that will artifactually change the cvp measured. 7. next, lower the 0 cm point on the manometer or ruler to the level of the patient's manubrium (if the patient is in lateral recumbancy) or the point of the elbow (if the patient is in sternal recumbancy). 8. turn the stopcock off to the syringe, and allow the fluid column to equilibrate with the patient's intravascular volume. once the fluid column stops falling and the level rises and falls with the patient's heartbeat, measure the number adjacent to the bottom of the meniscus of the fluid column. this is the cvp in centimeters of water (see figure 1 -4). 9. repeat the measurement several times with the patient in the same position to make sure that none of the values has been increased or decreased artifactually in error. alternately, attach the central catheter to a pressure transducer and perform electronic monitoring of cvp. there is no absolute value for normal cvp. the normal cvp for small animal patients is 0 to 5 cm h 2 o. values less than zero are associated with absolute or relative hypovolemia. values of 5 to 10 cm h 2 o are borderline hypervolemia, and values greater than 10 cm h 2 o suggest intravascular volume overload. values greater than 15 cm h 2 o may be correlated with congestive heart failure and the development of pulmonary edema. in individual patients, the trend in change in cvp is more important than absolute values. as a rule of thumb, when using cvp measurements to gauge fluid therapy and avoid vascular and pulmonary overload, the cvp should not increase by more than 5 cm h 2 o in any 24-hour period. if an abrupt increase in cvp is found, repeat the measurement to make sure that the elevated value was not obtained in error. if the value truly has increased dramatically, temporarily discontinue fluid therapy and consider administration of a diuretic. delaforcade am, rozanski ea: central venous pressure and arterial blood pressure measurements, vet clin north am small anim pract 31 (6) the diagnosis of intracellular fluid deficit is difficult and is based more on the presence of hypernatremia or hyperosmolality than on clinical signs. an intracellular fluid deficit is expected when free water loss by insensible losses and vomiting, diarrhea, or urine is not matched by free water intake. consideration of the location of the patient's fluid deficit, history of vomiting and diarrhea, no visible clinical signs of deficit 4% dry mucous membranes, mild skin tenting 5% increased skin tenting, dry mucous membranes, mild tachycardia, normal pulse* 7% increased skin tenting, dry mucous membranes, tachycardia, weak pulse pressure 10% increased skin tenting, dry corneas, dry mucous membranes, 12% elevated or decreased heart rate, poor pulse quality, altered level of consciousness* the respiratory system further contributes to acid-base status by changes in the elimination of carbon dioxide. hyperventilation decreases the blood pco 2 and causes a respiratory alkalosis. hypoventilation increases the blood pco 2 and causes a respiratory acidosis. depending on the altitude, the pco 2 in dogs can range from 32 to 44 mm hg. in cats, normal is 28 to 32 mm hg. venous pco 2 values are 33 to 50 mm hg in dogs and 33 to 45 mm hg in cats. use a systematic approach whenever attempting to interpret a patient's acid-base status. ideally, obtain an arterial blood sample so that you can monitor the patient's oxygenation and ventilation. once an arterial blood sample has been obtained, follow these steps: 1. determine whether the blood sample is arterial or venous by looking at the oxygen saturation (sao 2 ). the sao 2 should be greater than 90% if the sample is truly arterial, although it can be as low as 80% if a patient has severe hypoxemia. 2. consider the patient's ph. if the ph is outside of the normal range, an acid-base disturbance is present. if the ph is within the normal range, an acid-base disturbance may or may not be present. if the ph is low, the patient is acidotic. if the ph is high, the patient is alkalotic. 3. next, look at the base excess or deficit. if the base excess is increased, the patient has higher than normal bicarbonate. if there is a base deficit, the patient may have a low bicarbonate or increase in unmeasured anions (e.g., lactic acid or ketoacids). 4. next, look at the bicarbonate. if the ph is low and the bicarbonate is low, the patient has a metabolic acidosis. if the ph is high and the bicarbonate is elevated, the patient has a metabolic alkalosis. 5. next, look at the paco 2 . if the patient's ph is low and the paco 2 is elevated, the patient has a respiratory acidosis. if the patient's ph is high and the paco 2 is low, the patient has a respiratory alkalosis. 6. finally, if you are interested in the patient's oxygenation, look at the pao 2 . normal pao 2 is greater than 80 mm hg. the metabolic acidosis early in renal failure may be hyperchloremic and later may convert to typical increased anion gap acidosis. 7. next, you must determine whether the disorders present are primary disorders or an expected compensation for disorders in the opposing system. for example, is the patient retaining bicarbonate (metabolic alkalosis) because of carbon dioxide retention (respiratory acidosis)? use the chart in table 1 -6 to evaluate whether the appropriate degree of compensation is occurring. if the adaptive response falls within the expected range, a simple acid-base disorder is present. if the response falls outside of the expected range, a mixed acid-base disorder is likely present. 8. finally, you must determine whether the patient's acid-base disturbance is compatible with the history and physical examination findings. if the acid-base disturbance does not fit with the patient's history and physical examination abnormalities, question the results of the blood gas analyses and possibly repeat them. the most desirable method of assessing the acid-base status of an animal is with a blood gas analyzer. arterial samples are preferred over venous samples, with heparin used as an anticoagulant (table 1-7) . potassium primarily is located in the intracellular fluid compartment. serum potassium is regulated by the actions of the sodium-potassium-adenosinetriphosphatase pump on cellular membranes, including those of the renal tubular epithelium. inorganic metabolic acidosis artifactually can raise serum potassium levels because of redistribution of extracellular potassium in exchange for intracellular hydrogen ion movement in an attempt to correct serum ph. metabolic acidosis potassium is one of the major players in the maintenance of resting membrane potentials of excitable tissue, including neurons and cardiac myocytes. changes in serum potassium can affect cardiac conduction adversely. hyperkalemia lowers the resting membrane potential and makes cardiac cells, particularly those of the atria, more susceptible to depolarization. characteristic signs of severe hyperkalemia that can be observed on an ecg rhythm strip include an absence of p waves, widened qrs complexes, and tall tented or spiked t waves. further increases in serum potassium can be associated with bradycardia, ventricular fibrillation, and cardiac asystole (death). treatment of hyperkalemia consists of administration of insulin (0.25 to 0.5 units/kg, iv regular insulin) and dextrose (1 g dextrose per unit of insulin administered, followed by 2.5% dextrose iv cri to prevent hypoglycemia), calcium (2 to 10 ml of 10% calcium gluconate administered iv slowly to effect), or sodium bicarbonate (1 meq/kg, iv slowly). insulin plus dextrose and bicarbonate therapy help drive the potassium intracellularly, whereas calcium antagonizes the effect of hyperkalemia on the myocardial cells. all of the treatments work within minutes, although the effects are relatively short-lived (20 minutes to 1 hour) unless the cause of the hyperkalemia is identified and treated appropriately (box 1-10). dilution of serum potassium also results from restoring intravascular fluid volume and correcting metabolic acidosis, in most cases. treatment with a fluid that does not contain potassium (preferably 0.9% sodium chloride) is recommended. hypokalemia elevates the resting membrane potential and results in cellular hyperpolarization. hypokalemia may be associated with ventricular dysrhythmias, but the ecg changes are not as characteristic as those observed with hyperkalemia. causes of hypokalemia include renal losses, anorexia, gastrointestinal loss (vomiting, diarrhea), intravenous fluid diuresis, loop diuretics, and postobstructive diuresis (box 1-11). if the serum potassium concentration is known, potassium supplementation in the form of potassium chloride or potassium phosphate can be added to the patient's intravenous fluids. correct serum potassium levels less than 3.0 meq/l or greater than 6.0 meq/l. potassium rates should not exceed 0.5 meq/kg/hour (table 1 -8) . metabolic acidosis from bicarbonate depletion often corrects itself with volume restoration in most small animal patients. patients with moderate to severe metabolic acidosis may benefit from bicarbonate supplementation therapy. the metabolic contribution to acid-base balance is identified by measuring the total carbon dioxide concentration or calculating the bicarbonate concentration. if these measurements are not available, the degree of expected metabolic acidosis can be estimated subjectively by the severity of underlying disease that often contributes to metabolic acidosis: hypovolemic or traumatic shock, septic shock, diabetic ketoacidosis, or oliguric/anuric renal failure. if the metabolic acidosis is estimated to be mild, moderate, or severe, add sodium bicarbonate at 1, 3, and 5 meq/kg body mass, respectively. patients with diabetic ketoacidosis may not require bicarbonate administration once volume replacement and perfusion is restored, and the ketoacids are metabolized to bicarbonate. if the bicarbonate measurement of base deficit is known, the following formula can be used as a gauge for bicarbonate supplementation: base deficit ã� 0.3 = body mass (kg) = meq bicarbonate to administer osmolality osmolality is measured by freezing point depression or a vapor pressure osmometer, or it may be calculated by the following formula: mosm/kg = 2[(na + ) + (k + )] + bun/2.8 + glucose/18 where sodium and potassium are measured in milliequivalents, and bun and glucose are measured in milligrams per deciliter. osmolalities less than 260 mosm/kg or greater 38 1 emergency care than 360 mosm/kg are serious enough to warrant therapy. the difference between the measured osmolality and the calculated osmolality (the osmolal gap) should be less than 10 mosm/kg. if the osmolal gap is greater than 20 mosm/kg, consider the presence of unmeasured anions such as ethylene glycol metabolites. the volume of extracellular fluid is determined by the total body sodium content, whereas the osmolality and sodium concentration are determined by water balance. serum sodium concentration is an indication of the amount of sodium relative to water in the extracellular fluid and provides no direct information about the total body sodium content. unlikely to cause hyperkalemia in presence of normal renal function unless iatrogenic (e.g., continuous infusion of potassium-containing fluids at an excessively rapid rate) acute mineral acidosis (e.g., hydrochloric acid or ammonium chloride) insulin deficiency (e.g., diabetic ketoacidosis) acute tumor lysis syndrome reperfusion of extremities after aortic thromboembolism in cats with cardiomyopathy hyperkalemic periodic paralysis (one case report in a pit bull) mild hyperkalemia after exercise in dogs with induced hypothyroidism infusion of lysine or arginine in total parenteral nutrition solutions nonspecific î²-blockers (e.g., propranolol)* cardiac glycosides (e.g., digoxin)* urethral obstruction ruptured bladder anuric or oliguric renal failure hypoadrenocorticism selected gastrointestinal disease (e.g., trichuriasis, salmonellosis, or perforated duodenal ulcer) late pregnancy in greyhound dogs (mechanism unknown but affected dogs had gastrointestinal fluid loss) chylothorax with repeated pleural fluid drainage hyporeninemic hypoaldosteronism â�  angiotensin-converting enzyme inhibitors (e.g., enalapril)* angiotensin receptor blockers (e.g., losartan)* cyclosporine and tacrolimus* potassium-sparing diuretics (e.g., spironolactone, amiloride, and triamterene)* nonsteroidal antiinflammatory drugs* heparin* trimethoprim* from dibartola sp: fluid, electrolyte and acid-base disorders in small animal practice, st louis, 2005, saunders. *likely to cause hyperkalemia only in conjunction with other contributing factors (e.g., other drugs, decreased renal function, or concurrent administration of potassium supplements). â�  not well documented in veterinary medicine. if refractory hypokalemia is present, supplement magnesium at 0.75 meq/kg/day for 24 hours. alone unlikely to cause hypokalemia unless diet is aberrant administration of potassium-free (e.g., 0.9% sodium chloride or 5% dextrose in water) or potassium-deficient fluids (e.g., lactated ringer's solution over several days) bentonite clay ingestion (e.g., cat litter) alkalemia insulin/glucose-containing fluids catecholamines hypothermia hypokalemic periodic paralysis (burmese cats) albuterol overdosage patients with hyponatremia or hypernatremia may have decreased, normal, or increased total body sodium content (boxes 1-12 and [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] ). an increased serum sodium concentration implies hyperosmolality, whereas a decrease in serum sodium concentration usually, but not always, implies hypoosmolality. the severity of clinical signs of hypernatremia and hyponatremia is related primarily to the rapidity of the onset of the change rather than to the magnitude of the associated plasma hyperosmolality or hypoosmolality. clinical signs of neurologic disturbances include disorientation, ataxia, and seizures, and coma may occur at serum sodium concentrations less than 120 meq/l or greater than 170 meq/l in dogs. therapy of hypernatremia or hyponatremia with fluid containing low or higher concentrations of sodium should proceed with caution, for rapid changes (decreases or increases) of serum sodium and osmolality can cause rapid changes in the intracellular and extracellular fluid flux, leading to intracellular dehydration or edema, even though the serum sodium has not been returned to normal. a rule of thumb is to not raise or lower the serum sodium by more than 15 meq/l during any one 24-hour period. restoration of the serum sodium concentration over a period of 48 to 72 hours is better. in almost all circumstances, an animal will correct its sodium balance with simple fluid restoration. if severe hypernatremia exists that suggests a free water deficit, however, the free water deficit should be calculated from the following formula: hypernatremia can be corrected slowly with 0.45% sodium chloride plus 2.5% dextrose, 5% dextrose in water, or lactated ringer's solution (sodium content: 130 meq/l). correct hyponatremia initially with 0.9% sodium chloride. sodium is balanced predominantly by chloride and bicarbonate. the difference between these concentrations, (na , has been called the anion gap. the normal anion gap is between 12 and 25 meq/l. when the anion gap exceeds 25, consider the possibility of an accumulation of unmeasured anions (e.g., lactate, ketoacids, phosphate, sulfate, ethylene glycol metabolites, and salicylate). abnormalities in the anion gap may be helpful in determining the cause of metabolic acidosis (boxes 1-14 and 1-15). the colloid oncotic pressure of blood is associated primarily with large-molecular-weight colloidal substances in circulation. the major player in maintaining intravascular and interstitial oncotic pressure, the water-retaining property of each fluid compartment, is albumin. albumin contributes roughly 80% to the colloidal oncotic pressure of blood. the majority of albumin is located within the interstitial space. hypoalbuminemia can result from increased loss in the form of protein-losing enteropathy or nephropathy and wound exudates, or it may be due to lack of hepatic albumin synthesis. serum albumin pools are in a constant flux with interstitial albumin. once interstitial albumin pools become depleted from replenishing serum albumin, serum albumin levels can continue to decrease, which can lead to a decrease in colloidal oncotic pressure. serum albumin less than 2.0 g/dl has been associated with inadequate intravascular fluid retention and the development of peripheral edema and third spacing of fluid. oncotic pressure can be restored with the use of artificial or synthetic colloids or natural colloids (see colloids). maintenance fluid requirements have been extrapolated from the formulas used to calculate a patient's daily metabolic energy requirements because it takes 1 ml of water to metabolize 1 kcal of energy (table 1 -9) . the patient's daily metabolic water (fluid) requirements can be calculated by the following formula: administration of an isotonic crystalloid fluid for maintenance requirements often can produce iatrogenic hypokalemia. in most cases, supplemental potassium must be added to prevent hypokalemia resulting from inappetance, kalliuresis, and supplementation with isotonic crystalloid fluids. the most reliable method of determining the degree of fluid deficit is by weighing the animal and calculating acute weight loss. acute weight loss in a patient with volume loss in the form of vomiting, feces, wound exudates, and urine is due to fluid loss and not loss of muscle or fat. lean body mass normally is not gained or lost rapidly enough to cause major changes in body weight. one milliliter of water weighs approximately 1 g. this fact allows calculation of the patient's fluid deficit, if ongoing losses can be measured. when a patient first presents, however, the body weight before a fluid deficit has occurred rarely is known. instead, one must rely on subjective measures of dehydration to estimate the patient's percent dehydration and to calculate the volume of fluid required to rehydrate the patient over the next 24 hours. to calculate the volume deficit, use the following formula: body mass (kg) ã� (% dehydration) ã� 1000 = fluid deficit (ml) the patient's fluid deficit must be added to the daily maintenance fluid requirements and administered over a 24-hour period. ongoing losses can be determined by measuring urine output, weighing the patient at least 2 to 3 times a day, and measuring the volume or weight of vomitus or diarrhea. a crystalloid fluid contains crystals of salts with a composition similar to that of the extracellular fluid space and can be used to maintain daily fluid requirements and replace fluid deficits or ongoing fluid losses (table 110) . metabolic, acid-base, and electrolyte imbalances also can be treated with isotonic fluids with or without supplemental electrolytes and buffers. depending on the patient's clinical condition, choose the specific isotonic crystalloid fluid to replace and maintain the patient's acid-base and electrolyte status ( table 1-11) . crystalloid fluids are readily available, are relatively inexpensive, and can be administered safely in large volumes to patients with no preexisting cardiac or renal disease or cerebral edema. following infusion, approximately 80% of the volume of a crystalloid fluid infused will redistribute to the interstitial fluid compartment. as such, crystalloid fluids alone are ineffective for ongoing intravascular volume depletion when given as a bolus. the crystalloid fluid bolus must be followed by a constant rate infusion, taking into consideration the patient's daily maintenance fluid requirements and ongoing fluid losses. administration of a large volume of crystalloid fluids can cause dilutional anemia and coagulopathies. *30 ã� bw kg + 70 = kcal/day = ml/day. note: this formula will slightly underestimate the requirements for patients that are less than 2 kg and will slightly overestimate the requirements for patients greater than 70 kg. retain fluid in the vascular space, the volume of crystalloid fluid infused (maintenance + deficit + ongoing losses) should be decreased by 25% to 50% to avoid vascular volume overload. two major classes of colloids exist: natural and synthetic. natural colloids (whole blood, packed rbcs, plasma) are discussed elsewhere in this text. concentrated human albumin is a natural purified colloid that recently has become more popular in the treatment of advanced hypoalbuminemia and hypoproteinemia and will be discussed here. synthetic colloids are starch polymers and include dextrans and hetastarch. concentrated human albumin is available as a 5% or 25% solution. the 5% solution has an osmolality similar to that of serum (308 mosm/l), whereas the 25% solution is hyperoncotic (1500 mosm/l). a 25% albumin solution draws fluid from the interstitial space into the intravascular space. concentrated albumin solutions often are used to restore circulating volume when synthetic colloids are not available. albumin not only is important at maintaining the colloidal oncotic pressure of blood but also serves as a valuable free-radical scavenger and carrier of drugs and hormones necessary for normal tissue function and healing. albumin levels less than 2.0 g/dl have been associated with increased morbidity and mortality. concentrated human albumin solutions can be administered as an effective method of restoring interstitial and serum albumin concentrations in situations of acute and chronic hypoalbuminemia. albumin (25%) is available in 50-and 100-ml vials and is more cost-efficient as an albumin replacement than procurement and administration of fresh frozen plasma. recommended albumin infusion rates are 2 to 5 ml/kg over 4 hours, after pretreatment with diphenhydramine. although concentrated human albumin is structurally similar to canine albumin, closely monitor the patient for signs of allergic reaction during and after the infusion. dextran-70 is a synthetic high-molecular-weight polysaccharide (sucrose polymer) with a molecular weight of 70,000 d. particles less than 50,000 d, are cleared rapidly by the kidneys, whereas larger particles are cleared more slowly by the hepatic reticuloendothelial system. dextran-70 can coat platelets and inhibit platelet function and so must be used with caution in patients with known coagulopathies. the total daily dosage should not exceed 40 ml/kg/day. hetastarch (hydroxyethyl starch) is a large-molecular-weight amylopectin polymer, has molecules with a molecular weight that exceeds 100,000 d, and has an average half-life of 24 to 36 hours in circulation. hetastarch can bind with vwf and cause prolongation of the act and aptt; however, it does not cause a coagulopathy. recommended rates of hetastarch infusion are 5-to 10-ml incremental boluses for the treatment of hypotension and 20 to 30 ml/kg/day as a constant rate infusion for maintenance of colloidal oncotic pressure. many are the acceptable ways to administer the fluids prescribed for each patient based on the degree of dehydration, estimation of ongoing losses, ability to tolerate oral fluid, and metabolic, acid-base, and electrolyte derangements. administer the fluids in a manner that is best for the patient and most appropriate for the practice. to determine the rate of intravenous fluid infusion, take the total volume of fluids that have been prescribed and divide the total volume by the total number of hours in a day that intravenous fluids can be delivered safely and monitored. the safest and most accurate way to deliver intravenous fluids, particularly in extremely small animals or those with congestive heart failure, is through an intravenous fluid pump. fluid should not be administered intravenously if the patient cannot be monitored to make sure that the fluids are being delivered at a safe rate and that the fluid line has not become disconnected. supplement fluids over as many hours as possible to allow the patient as much time as possible to redistribute and fully utilize the fluids administered. fluids administered too quickly can cause a diuresis to occur, such that the majority of the fluids administered will be excreted in the urine. if time is limited or if extra time is needed for safe administration of fluids, consider using a combination of intravenously and subcutaneously 46 1 emergency care administered fluids. intravenous is the preferred route of administration of fluids in any patient with dehydration and hypovolemia. as intravascular volume depletion occurs, reflex peripheral vasoconstriction occurs to restore core perfusion. the subcutaneous tissue are not perfused well and therefore fluids administered subcutaneously will not be absorbed well into the interstitial and intravascular spaces. subcutaneously administered fluids can be absorbed slowly and delivered effectively in the management of mild interstitial dehydration and in the treatment of renal insufficiency. subcutaneously administered fluids should never take the place of intravenously administered fluids in a hypovolemic patient or one with severe interstitial dehydration. intramedullary (intraosseous) infusion works well in small patients in which vascular access cannot be established. shock doses of fluids and other substances, including blood products, can be administered under pressure through an intraosseous cannula. because of the inherent discomfort and risk of osteomyelitis with intraosseous infusion, establish vascular access as soon as possible. the safest and most efficient method of intravenous fluid infusion is through a fluid pump. in cases in which a fluid pump is unavailable, infusion by gravity feed is the next option. infusion sets from various manufacturers have calibrated drip chambers such that a specific number of drops will equal 1 ml of fluid. fluid rates can be calculated based on the number of drops that fall into the drip chamber per minute: fluid volume to be infused (ml) = ml/hour number of hours available many pediatric drip sets deliver 60 drops/ml, such that milliliters/hour equals drops/ minute. carefully record fluid orders so that the volume to be administered is recorded as milliliters/hour, milliliters/day, and drops/minute. this will allow personnel to detect major discrepancies and calculation errors more readily. the volume actually delivered should be recorded in the record by nursing personnel. all additives should be listed clearly on the bottle on a piece of adhesive tape or a special label manufactured for this purpose. a strip of adhesive tape also can be attached to the bottle and marked appropriately to provide a quick visualization of the estimate of volume delivered. includes a large-bore flexible orogastric lavage tube, permanent marker or white tape, lubricating jelly, warm water, two large buckets, a roll of 2-inch white tape, and a manual lavage pump. to perform the orogastric lavage, follow this procedure: 1. place all animals under general anesthesia with a cuffed endotracheal tube in place to protect the airway and prevent aspiration of gastric contents into the lungs. 2. place a roll of 2-inch white tape into the animal's mouth, and secure the tape around the muzzle. you will insert the tube through the hole in the center of the roll of tape. 3. next, place the distal end of the tube at the level of the last rib, directly adjacent to the animal's thorax and abdomen. measure the length of the tube from the most distal end to the point where it comes out of the mouth, and label this location on the tube with a permanent marker or piece of white tape. 4. lubricate the distal portion of the tube, and gently insert it through the roll of tape in the animal's mouth. 5. gently push the tube down the esophagus. palpate the tube within the esophagus. two tubes should be palpable, the orogastric tube, and the patient's trachea. push the tube down into the stomach. you can verify location by blowing into the proximal end of the tube and simultaneously auscultating the stomach for borborygmi. 6. insert the manual pump to the proximal end of the tube, and instill the warm water. alternate instilling water with removal of fluid and gastric debris by gravity. repeat the process until the efflux fluid is clear of any debris. 7. save fluid from the gastric efflux fluid for toxicologic analyses. hackett tb: emergency approach to intoxications, clin tech small anim pract 15 (2):82-87, 2000. hypoxia, or inadequate tissue oxygenation, is the primary reason for supplemental oxygen therapy. major causes of hypoxia include hypoventilation, ventilation-perfusion mismatch, physiologic or right-to-left cardiac shunt, diffusion impairment, and decreased fraction of inspired oxygen (table 1-12) . inadequate tissue perfusion caused by low cardiac output or vascular obstruction also can result in circulatory hypoxia. finally, histiocytic hypoxia results from inability of cells to use oxygen that is delivered to them. this form of hypoxia can be observed with various toxin ingestions (bromethalin, cyanide) and in septic shock. a patient's oxygenation status can be monitored invasively by drawing of arterial blood gas samples or noninvasively through pulse oximetry, in most cases (see acid-base physiology and pulse oximetry). inspired air at sea level has a po 2 of 150 mm hg. as the air travels through the upper respiratory system to the level of the alveolus, the po 2 drops to 100 mm hg. tissue oxygen saturation in a normal healthy animal is 95 mm hg. after oxygen has been delivered to the tissues, the oxygen left in the venous system (pvo 2 ) is approximately 40 mm hg. normally, oxygen diffuses across the alveolar capillary membrane and binds reversibly with hemoglobin in rbcs. a small amount of oxygen is carried in an unbound diffusible form in the plasma. when an animal has an adequate amount of hemoglobin and hemoglobin becomes fully saturated while breathing room air, supplemental oxygen administration will only increase the sao 2 a small amount. the unbound form of oxygen dissolved in plasma will increase. if, however, inadequate hemoglobin saturation is obtained by breathing room air, as in a case of pneumonia or pulmonary edema, for example, breathing a higher fraction of inspired oxygen (fio 2 ) will improve bound and unbound hemoglobin levels. the formula for calculating oxygen content of arterial blood is as follows: where cao 2 is the arterial oxygen content, 1.34 is the amount of oxygen that can be carried by hemoglobin (hb), sao 2 is the hemoglobin saturation, and 0.003 ã� pao 2 is the amount of oxygen dissolved (unbound) in plasma. dissolved oxygen actually contributes little to the total amount of oxygen carried in the arterial blood, and the majority depends on the amount or availability of hemoglobin and the ability of the body (ph and respiratory status) to saturate the hemoglobin at the level of the alveoli. oxygen therapy is indicated whenever hypoxia is present. the underlying cause of the hypoxia also must be identified and treated, for chronic, lifelong oxygen therapy is rarely feasible in veterinary patients. if hemoglobin levels are low due to anemia, oxygen supplementation must occur along with rbc transfusions to increase hemoglobin mass. whenever possible, use arterial blood gas analyses or pulse oximetry to gauge a patient's response to oxygen therapy and to determine when an animal can be weaned from supplemental oxygen. the goal of oxygen therapy is to increase the amount of oxygen bound to hemoglobin in arterial blood. oxygen supplementation can be by hood, oxygen cage or tent, nasal or nasopharyngeal catheter, or tracheal tube. in rare cases, administration of oxygen with mechanical ventilation may be indicated. administration of supplemental oxygen to patients with chronic hypoxia is sometimes necessary but also dangerous. with chronic hypoxia the patient develops a chronic respiratory acidosis (elevated paco 2 ) and depends almost entirely on the hypoxic ventilatory drive to breathe. administration of supplemental oxygen increases pao 2 and may inhibit the central respiratory drive, leading to hypoventilation and possibly respiratory arrest. therefore, closely monitor animals with chronic hypoxia that are treated with supplemental oxygen. oxygen hoods can be purchased from commercial sources or can be manufactured in the hospital using a rigid elizabethan collar, tape, and plastic wrap. to make an oxygen hood, place several lengths of plastic wrap over the front of the elizabethan collar and tape them in place. leave the ventral third of the collar open to allow moisture and heat to dissipate and carbon dioxide to be eliminated. place a length of flexible oxygen tubing under the patient's collar into the front of the hood, and run humidified oxygen at a rate of 50 to 100 ml/kg/minute. animals may become overheated with an oxygen hood in place. carefully monitor the patient's temperature so that iatrogenic hyperthermia does not occur. commercially available plexiglass oxygen cages can be purchased from a variety of manufacturers. the best units include a mechanical thermostatically controlled compressor cooling unit, a circulatory fan, nebulizers or humidifiers to moisten the air, and a carbon dioxide absorber. alternately, a pediatric (infant) incubator can be purchased from hospital supply sources, and humidified oxygen can be run into the cage at 2 to 10 l/minute (depending on the size of the cage). high flow rates may be required to eliminate nitrogen and carbon dioxide from the cage. in most cases, the fio 2 inside the cage reaches 40% to 50% using this technique. disadvantages of using an oxygen cage are high consumption/ use of oxygen, rapid decrease in the fio 2 within the cage whenever the cage must be opened for patient treatments, lack of immediate access to the patient, and potential for iatrogenic hyperthermia. one of the most common methods for oxygen supplementation in dogs is nasal or nasopharyngeal oxygen catheters: 1. to place a nasal or nasopharyngeal catheter, obtain a red rubber catheter (8f to 12f, depending on the size of the patient). a. for nasal oxygen supplementation, measure the distal tip of the catheter from the medial canthus of the eye to the tip of the nose. b. for nasopharyngeal oxygen supplementation, measure the catheter from the ramus of the mandible to the tip of the nose. 2. mark the tube length at the tip of the nose with a permanent marker. 3. instill topical anesthetic such as proparacaine (0.5%) or lidocaine (2%) into the nostril before placement. 4. place a stay suture adjacent to (lateral aspect) the nostril while the topical anesthetic is taking effect. 5. lubricate the tip of the tube with sterile lubricant. 6. gently insert the tube into the ventral medial aspect of the nostril to the level made with the permanent marker. if you are inserting the tube into the nasopharynx, push the nasal meatus dorsally while simultaneously pushing the lateral aspect of the nostril medially to direct the tube into the ventral nasal meatus and avoid the cribriform plate. 7. once the tube has been inserted to the appropriate length, hold the tube in place with your fingers adjacent to the nostril, and suture the tube to the stay suture. if the tube is removed, you can cut the suture around the tube and leave the stay suture in place for later use, if necessary. 8. suture or staple the rest of the tube dorsally over the nose and in between the eyes to the top of the head, or laterally along the zygomatic arch. 9. attach the tube to a length of flexible oxygen tubing, and provide humidified oxygen at 50 to 100 ml/kg/minute. 10. secure an elizabethan collar around the patient's head to prevent the patient from scratching at the tube and removing it. the rule of 60s states that if a patient's pao 2 is less than 60 mm hg, or if the paco 2 is 60 mm hg, mechanical ventilation should be considered. for mechanical ventilation, anesthetize the patient and intubate the patient with an endotracheal tube. alternately, a temporary tracheostomy can be performed and the patient can be maintained on a plane of light to heavy sedation and ventilated through the tracheostomy site. this method, a noninvasive means of determining oxygenation is through the use of pulse oximetry. a pulse oximeter uses different wavelengths of light to distinguish characteristic differences in the properties of the different molecules in a fluid or gas mixture, in this case, oxygenated (oxyhemoglobin) and deoxygenated hemoglobin (deoxyhemoglobin) in pulsatile blood. the process is termed pulse oximetry. oxyhemoglobin and deoxyhemoglobin are different molecules that absorb and reflect different wavelengths of light. oxyhemoglobin absorbs light in the infrared spectrum, allowing wavelengths of light in the red spectrum to transmit through it. conversely, deoxyhemoglobin absorbs wavelengths of the red spectrum and allows wavelengths in the infrared spectrum to transmit through the molecule. the spectrophotometer in the pulse oximeter transmits light in the red (660 nanometers) and infrared (920 nanometers) spectra. the different wavelengths of light are transmitted across a pulsatile vascular bed and are detected by a photodetector on the other side. the photodetector processes the amount of light of varying wavelengths that reaches it, then transmits an electrical current to a processor that calculates the difference in the amount of light originally transmitted and the amount of light of similar wavelength that actually reaches the photodetector. the difference in each reflects the amount of light absorbed in the pulsatile blood and can be used to calculate the amount or ratio of oxyhemoglobin to deoxyhemoglobin in circulation, or the functional hemoglobin saturation by the formula: where hbo 2 is oxygenated hemoglobin, and hb is deoxygenated hemoglobin. four molecules of oxygen reversibly bind to hemoglobin for transport to the tissues. carbon monoxide similarly binds to hemoglobin and forms carboxyhemoglobin, a molecule that is detected similarly as oxygenated hemoglobin. thus sao 2 as detected by a pulse oximeter is not reliable if carboxyhemoglobin is present. in most cases, pulse oximetry or sao 2 corresponds reliably to the oxyhemoglobin dissociation curve. oxygen saturation greater than 90% corresponds to a pao 2 greater than 60 mm hg. above this value, large changes in pao 2 are reflected in relatively small changes in sao 2 , making pulse oximetry a relatively insensitive method of determining oxygenation status when pao 2 is normal. because pulse oximetry measures oxygenated versus nonoxygenated hemoglobin in pulsatile blood flow, it is fairly unreliable when severe vasoconstriction, hypothermia, shivering or trembling, or excessive patient movement are present. additionally, increased ambient lighting and the presence of methemoglobin or carboxyhemoglobin also can cause artifactual changes in the sao 2 , and thus the measurement is not reliable or accurate. most pulse oximeters also display a waveform and the patient's heart rate. if the photodetector does not detect a good quality signal, the waveform will not be normal, and the heart rate displayed on the monitor will not correlate with the patient's actual heart rate. the efficiency of ventilation is evaluated using the paco 2 value on an arterial blood gas sample. alternatively, a noninvasive method to determine end-tidal carbon dioxide is through use of a capnograph. the science of capnometry uses a spectrophotometer to measure carbon dioxide levels in exhaled gas. the capnometer is placed in the expiratory limb of an anesthetic circuit. a sample of exhaled gas is aliquoted from the breath, and an infrared light source is passed across the sample. a photodetector on the other side of the sample flow measures the amount or concentration of carbon dioxide in the sample of expired gas. the calculated value is displayed as end-tidal carbon dioxide. this value also can be displayed as a waveform. when placed in graphic form, a waveform known as a capnograph is displayed throughout the ventilatory cycle. normally, at the onset of exhalation, the gas exhaled into the expiratory limb of the tubing comes from the upper airway or physiologic dead space and contains relatively little carbon dioxide. as exhalation continues, a steep uphill slope occurs as more carbon dioxide is exhaled from the bronchial tree. near the end of exhalation, the capnogram reaches a plateau, which most accurately reflects the carbon dioxide level at the level of the alveolus. because carbon dioxide diffuses across the alveolar basement membrane so rapidly, this reflects arterial carbon dioxide levels. if a plateau is not reached and notching of the waveform occurs, check the system for leaks. if the baseline waveform does not reach zero, the patient may be rebreathing carbon dioxide or may be tachypneic, causing physiologic positive end-expiratory pressure. the soda-sorb in the system should be replaced if it has expired. conversely, low end-tidal carbon dioxide may be associated with a decrease in perfusion or blood flow. decreased perfusion can be associated with low end-tidal carbon dioxide values, particularly during cardiopulmonary cerebral resuscitation. end-tidal carbon dioxide levels are one of the most accurate predictors of the efficacy of cardiopulmonary cerebral resuscitation and patient outcome. additionally, the difference between arterial carbon dioxide levels (paco 2 ) and end-tidal carbon dioxide can be used to calculate dead-space ventilation. increases in the difference also occur with poor lung perfusion and pulmonary diffusion impairment. thoracocentesis refers to the aspiration of fluid or air from within the pleural space. thoracocentesis may be diagnostic to determine whether air or fluid is present and to characterize the nature of the fluid obtained. thoracocentesis also can be therapeutic when removing large volumes of air or fluid to allow pulmonary reexpansion and correction of hypoxemia and orthopnea. to perform thoracocentesis, follow this procedure: 1. first, assemble the equipment necessary (box 1-16). 2. next, clip a 10-cm square in the center of the patient's thorax on both sides. 3. aseptically scrub the clipped area. 4. ideally, thoracocentesis should be performed within the seventh to ninth intercostal space. rather than count rib spaces in an emergent situation, visualize the thoracic cage as a box, and the clipped area as a box within the box. you will insert your needle or catheter in the center of the box and then direct the bevel of the needle dorsally or ventrally to penetrate pockets of fluid or air present. 5. attach the needle or catheter hub to the length of intravenous extension tubing. attach the female port of the intravenous extension tubing to the male port of the three-way stopcock. attach the male port of the 60-ml syringe to one of the female ports of the three-way stopcock. the apparatus is now assembled for use. 6. insert the needle through the intercostal space such that the bevel of the needle initially is directed downward. 7. next, push down on the hub of the needle such that the needle becomes parallel with the thoracic wall. by moving the hub of the needle in a clockwise or counterclockwise manner, the bevel of the needle will move within the thoracic cavity to penetrate pockets of air or fluid. in general, air is located dorsally and fluid is located more ventrally, although this does not always occur. 8. aspirate air or fluid. save any fluid obtained for cytologic and biochemical analyses and bacterial culture and susceptibility testing. in cases of pneumothorax, if the thoracocentesis needs to be repeated more than 3 times, consider using a thoracostomy tube. place a thoracostomy tube in cases of pneumothorax whenever negative suction cannot be obtained or repeated accumulation of air requires multiple thoracocentesis procedures. thoracostomy tubes also can be placed to drain rapidly accumulating pleural effusion and for the medical management of pyothorax. before attempting thoracostomy tube placement, make sure that all necessary supplies are assembled (box 1-17; table 1-13) . to place a thoracostomy tube, follow this procedure: 1. lay the patient in lateral recumbency. 2. clip the patient's entire lateral thorax. 3. aseptically scrub the lateral thorax. 4. palpate the tenth intercostal space. 5. have an assistant pull the patient's skin cranially and ventrally toward the point of the elbow. this will facilitate creating a subcutaneous tunnel around the thoracostomy tube. 6. draw up 2 mg/kg 2% lidocaine (1 mg/kg for cats) along with a small amount of sodium bicarbonate to take away some of the sting. 7. insert the needle at the dorsal aspect of the tenth intercostal space and to the seventh intercostal space. inject the lidocaine into the seventh intercostal space at the point where the trocarized thoracic drainage catheter will penetrate into the thoracic cavity. slowly infuse the lidocaine as you withdraw the needle to create an anesthetized tunnel through which to insert the catheter. 8. while the local anesthetic is taking effect, remove the trocar from the catheter and cut the proximal end of the catheter with a mayo scissors to facilitate adaptation with the christmas tree adapter. 9. attach the christmas tree adapter to the three-way stopcock and the three-way stopcock to a length of intravenous extension tubing and the 60-ml syringe so that the apparatus can be attached immediately to the thoracostomy tube after placement. 10. aseptically scrub the lateral thorax a second time and then drape it with sterile huck towels secured with towel clamps. 11. wearing sterile gloves, make a small stab incision at the dorsal aspect of the tenth intercostal space. 12. insert the trocar back into the thoracostomy drainage tube. insert the trocar and tube into the incision. tunnel the tube cranially for approximately 3 intercostal spaces while an assistant simultaneously pulls the skin cranially and ventrally toward the point of the elbow. 13. at the seventh intercostal space, direct the trocar and catheter perpendicular to the thorax. grasp the catheter apparatus at the base adjacent to the thorax to prevent the trocar from going too far into the thorax. 14. place the palm of your dominant hand over the end of the trocar, and push the trocar and catheter into the thoracic cavity, throwing your weight into the placement in a swift motion, not by banging the butt of your hand on the end of the stylette. for small individuals, standing on a stool, or kneeling over the patient on the triage table can create leverage and make this process easier. the tube will enter the thorax with a pop. 15. gently push the catheter off of the stylette, and remove the stylette. 16. immediately attach the christmas tree adapter and have an assistant start to withdraw air or fluid while you secure the tube in place. 17. first, place a horizontal mattress suture around the tube to cinch the skin securely to the tube. use care to not penetrate the tube with your needle and suture. 18. next, place a purse-string suture around the tube at the tube entrance site. leave the ends of the suture long, so that you can create a finger-trap suture to the tube, holding the tube in place. 19. place a large square of antimicrobial-impregnated adhesive tape over the tube for further security and sterility. 20. if antimicrobial adhesive is not available, place a gauze pad 4 ã� 4 inches square over the tube, and then wrap the tube to the thorax with cotton roll gauze and elastikon adhesive tape. 21. draw the location of the tube on the bandage to prevent cutting it with subsequent bandage changes. an alternate technique to use if a trocar thoracic drainage catheter is not available is the following: 1. prepare the lateral thorax and infuse local lidocaine anesthetic as listed before. 2. make a small stab incision with a no. 10 scalpel blade, as listed before. 3. obtain the appropriately sized red rubber catheter and cut multiple side ports in the distal end of the catheter, taking care to not cut more than 50% of the circumference of the diameter of the tube. 4. insert a rigid, long urinary catheter into the red rubber catheter to make the catheter more rigid during insertion into the pleural space. 5. grasp the distal end of the catheter(s) in the teeth of a large carmalt. tunnel a metzenbaum scissors under the skin to the seventh intercostal space and make a puncture through the intercostal space. 6. remove the metzenbaum scissors, and then tunnel the carmalt and red rubber tube under the skin to the hole created in the seventh intercostal space with the metzenbaum scissors. 7. insert the tips of the carmalt and the red rubber catheter through the hole, and then open the teeth of the carmalt. 8. push the red rubber catheter cranially into the pleural cavity. 9. remove the carmalt and the rigid urinary catheter, and immediately attach the suction apparatus. secure the red rubber catheter in place as listed before. placement of a temporary tracheostomy can be lifesaving to relieve upper respiratory tract obstruction, to facilitate removal of airway secretions, to decrease dead space ventilation, to provide a route of inhalant anesthesia during maxillofacial surgery, and to facilitate mechanical ventilation. in an emergent situation in which asphyxiation is imminent and endotracheal intubation is not possible, any cutting instrument placed into the trachea distal to the point of obstruction can be used. to perform a slash tracheostomy, quickly clip the fur and scrub the skin over the third tracheal ring. make a small cut in the trachea with a no. 11 scalpel blade, and insert a firm tube, such as a syringe casing. alternately, insertion of a 22-gauge needle attached to intravenous extension tubing and adapted with a 1-ml syringe case to attach to a humidified oxygen source also temporarily can relieve obstruction until a temporary tracheostomy can be performed. in less emergent situations, place the patient under general anesthesia and intubate the patient. assemble all the equipment necessary before starting the temporary tracheostomy procedure (box 1-18). to perform a tracheostomy, follow this procedure: 1. place the patient in dorsal recumbency. 2. clip the ventral cervical region from the level of the ramus of the mandible caudally to the thoracic inlet and dorsally to midline. 3. aseptically scrub the clipped area, and then drape with sterile huck towels secured with towel clamps. 4. make a 3-cm ventral midline skin incision over the third to sixth tracheal rings, perpendicular to the trachea. 5. bluntly dissect through the sternohyoid muscles to the level of the trachea. 6. carefully pick up the fascia overlying the trachea and cut it away with a metzenbaum scissors. 7. place two stay sutures through/around adjacent tracheal rings. 8. incise in between trachea rings with a no. 11 scalpel blade. take care to not cut more than 50% of the circumference of the trachea. 9. using the stay sutures, pull the edges of the tracheal incision apart, and insert the tracheostomy tube. the shiley tube contains an internal obturator to facilitate placement into the tracheal lumen. remove the obturator, and then insert the inner cannula, which can be removed for cleaning as needed. 10. once the tube is in place, secure the tube around the neck with a length of sterile umbilical tape. postoperative care of the tracheostomy tube is as important as the procedure itself. because the tracheostomy tube essentially bypasses the protective effects of the upper respiratory system, one of the most important aspects of tracheostomy tube care and maintenance is to maintain sterility at all times. any oxygen source should be humidified with sterile water or saline to prevent drying of the respiratory mucosa. if supplemental oxygen is not required, instill 2 to 3 ml of sterile saline every 1 to 2 hours to moisten the mucosa. wearing sterile gloves, remove the internal tube and place it in a sterile bowl filled with sterile hydrogen peroxide and to be cleaned every 4 hours (or more frequently as necessary). if a shiley tube is not available, apply suction to the internal lumen of the tracheostomy tube every 1 to 2 hours (or more frequently as needed) with a sterile 12f red rubber catheter attached to a vacuum pump to remove any mucus or other debris that potentially could plug the tube. unless the patient demonstrates clinical signs of fever or infection, the prophylactic use of antibiotics is discouraged because of the risk of causing a resistant infection. after the temporary tracheostomy is no longer necessary, remove the tube and sutures, and leave the wound to heal by second intention. primary closure of the wounds could predispose the patient to subcutaneous emphysema and infection. baker gd: trans-tracheal oxygen therapy in dogs with severe respiratory compromise due to tick (i. holocyclus) toxicity, aust vet pract 34 (2) urohydropulsion is a therapeutic procedure for removal of uroliths from the urethra of the male dog. the technique works best if the animal is heavily sedated or is placed under general anesthesia (figure 1-12) . to perform urohydropulsion, follow this procedure: 1. place the animal in lateral recumbency. 2. clip the fur from the distal portion of the prepuce. 3. aseptically scrub the prepuce and flush the prepuce with 12 to 20 ml of antimicrobial flush solution. 4. have an assistant who is wearing gloves retract the penis from the prepuce. 5. while wearing sterile gloves, lubricate the tip of a rigid urinary catheter as for urethral catheterization. 6. gently insert the tip of the catheter into the urethra until you meet the resistance of the obstruction. 7. pinch the tip of the penis around the catheter. 8. have an assistant insert a gloved lubricated finger into the patient's rectum and press ventrally on the floor of the rectum to obstruct the pelvic urethra. 9. attach a 60-ml syringe filled with sterile saline into proximal tip of the catheter. 10. quickly inject fluid into the catheter and alternate compression and relaxation on the pelvic urethra such that the urethra dilates and suddenly releases the pressure, causing dislodgement of the stone. small stones may be ejected from the tip of the urethra, whereas larger stones may be retropulsed back into the urinary bladder to be removed surgically at a later time. the type of catheter that you choose for vascular access depends largely on the size and species of the patient, the fragility of the vessels to be catheterized, the proposed length of time that the catheter will be in place, the type and viscosity of the fluid or drug to be administered, the rate of fluid flow desired, and whether multiple repeated blood samples will be required (table 1-14) . a variety of over-the-needle, through-the-needle, and over-the-wire catheters are available for placement in a variety of vessels, including the jugular, cephalic, accessory cephalic, medial saphenous, lateral saphenous, dorsal pedal artery, and femoral artery. one of the most important aspects of proper catheter placement and maintenance is to maintain cleanliness at all times. the patient's urine, feces, saliva, and vomit are common sources of contamination of the catheter site. before placing a peripheral or central catheter in any patient, consider the patient's physical status including whether vomiting, diarrhea, excessive urination, or seizures. in a patient with an oral mass that is drooling excessively or a patient that is vomiting, peripheral cephalic catheterization may not be the most appropriate, to prevent contamination. conversely, in a patient with excessive urination or diarrhea, a lateral or medial saphenous catheter is likely to become contaminated quickly. whenever one places or handles a catheter or intravenous infusion line, the person should wash the hands carefully and wear gloves to prevent contamination of the intravenous catheter and fluid lines. one of the most common sources of catheter contamination in veterinary hospitals is through caretakers' hands. in emergent situations, placement of a catheter may be necessary under less than ideal circumstances. remove those catheters as soon as the patient is more stable, and place a second catheter using aseptic techniques. in general, once the location of the catheter has been decided, set up all equipment necessary for catheter placement before starting to handle and restrain the patient. lists the equipment needed for most types of catheter placement. after setting up all of the supplies needed, clip the fur over the site of catheter placement. make sure to clip all excess fur and long feathers away from the catheter site, to prevent contamination. for catheter placement in limbs, clip the fur circumferentially around the site of catheter placement to facilitate adherence of the tape to the limb and to facilitate catheter removal with minimal discomfort at a later date. next, aseptically scrub the catheter site with an antimicrobial scrub solution such as hibiclens. the site is now ready for catheter insertion. consider using a central venous catheter whenever multiple repeated blood samples will need to be collected from a patient during the hospital stay. central venous catheters also can be used for cvp measurement, administration of hyperoncotic solutions such as parenteral nutrition, and administration of crystalloid and colloid fluids, anesthesia, and other injectable drugs (figures 1-13 and 1-14) . to place a jugular central venous catheter, place the patient in lateral recumbancy and extend the head and neck such that the jugular furrow is straight. clip the fur from the ramus of the mandible caudally to the thoracic inlet and dorsally and ventrally to midline. wipe the clipped area with gauze 4 ã� 4-inch squares to remove any loose fur and other debris. aseptically scrub the clipped area with an antimicrobial cleanser. venocaths (abbott laboratories) are a through-the-needle catheter that is contained within a sterile sleeve for placement. alternately, other over-the-wire central venous catheters can be placed by the seldinger technique. sterility must be maintained at all times, regardless of the type of catheter placed. wearing sterile gloves, drape the site of catheter placement with sterile drapes, and occlude the jugular vein at the level of the thoracic inlet. pull the clear ring and wings of emergency diagnostic and therapeutic procedures 59 1 figure 1 -13: lateral thoracic radiograph of a central venous catheter. note that the tip of the catheter is inserted in its proper location, just outside of the right atrium. the catheter cover down toward the catheter itself to expose the needle. remove the guard off of the needle. lift the skin over the proposed site of catheter insertion and insert the needle under the skin, with the bevel of the needle facing up. next, reocclude the vessel and pull the skin tight over the vessel to prevent movement of the vessel as you attempt to insert the needle. in some cases, it may be difficult actually to see the vessel in obese patients. if you cannot visualize or palpate the needle, gently bounce the needle over the vessel with the bevel up. the vessel will bounce in place slightly, allowing a brief moment of visualization to facilitate catheter placement. once the vessel has been isolated and visualized, insert the needle into the vessel at a 15-to 30-degree angle. watch closely for a flash of blood in the catheter. when blood is observed, insert the needle a small distance farther, and then push the catheter and stylette into the vessel for the entire length, until the catheter and stylette can be secured in the catheter hub. if the catheter cannot be inserted fully into the vessel for its entire length, the tip of the needle may not be within the entire lumen, the catheter may be directed perivascularly, and the catheter may be caught at the thoracic flexure and may be moving into one of the tributaries that feeds the forelimb. extend the patient's head and neck, and lift the forelimb up to help facilitate placement. do not force the catheter in because the catheter potentially can form a knot and will need to be removed surgically. remove the needle from the vessel, and have an assistant place several 4 ã� 4-inch gauze squares over the site of catheter placement with some pressure to control hemorrhage. secure the catheter hub into the needle guard, and remove the stylette from the catheter. immediately insert a 3-to 6-ml syringe of heparinized saline and flush the catheter and draw back. if you are in the correct place, you will be able to draw blood from the catheter. to secure the catheter in place, tear a length of 1-inch white tape that will wrap around the patient's neck. pull a small length of the catheter out of the jugular vein to make a semicircle. the semicircle should be approximately 1 /2 inch in diameter. let the length of catheter lie on the skin, and then place 4 ã� 4-inch gauze squares impregnated with antimicrobial ointment over the site of catheter insertion. secure the proximal end of white tape around the white and blue pieces of the catheter, and wrap the tape around the patient's neck so that the tape adheres to the skin and fur. repeat the process by securing the gauze to the skin with two additional lengths of white tape, starting to secure the gauze in place by first wrapping the tape dorsally over the patient's neck, rather than under the patient's neck. in between each piece of tape and bandage layer, make sure that the catheter flushes and draws back freely, or else occlusion can occur. gently wrap layers of cotton roll gauze, kling, and elastikon or vetrap over the catheter. secure a male adapter or t port that has been flushed with heparinized saline, and then label the catheter with the size and length of catheter, date of catheter placement, and initials of the person who placed the catheter. the catheter is ready for use. monitor the catheter site daily for erythema, drainage, vessel thickening, or pain upon infusion. if any of these signs occur, or if the patient develops a fever of unknown origin, remove the catheter, culture the catheter tip aseptically, and replace the catheter in a different location. as long as the catheter is functional without complications, the catheter can remain in place. central catheters also can be placed via the seldinger or over-the-wire technique. a number of companies manufacture kits that contain the supplies necessary for over-the-wire catheter placement. each kit minimally should contain an over-the-needle catheter to place into the vessel, a long wire to insert through the original catheter placed, a vascular dilator to dilate the hole in the vessel created by the first catheter, and a long catheter to place into the vessel over the wire. additional accessories can include a paper drape, sterile gauze, a scalpel blade, local anesthetic, 22-gauge needles, and 3-or 6-ml syringes. restrain the patient and prepare the jugular furrow aseptically as for the percutaneous through-the-needle catheter placement. the person placing the catheter should wear sterile gloves throughout the process to maintain sterility. pick up the skin over the site of catheter placement, and insert a small bleb of local anesthetic through the skin. the local anesthetic should not be injected into the underlying vessel (figure 1-15) . make a small nick into the skin through the local anesthetic with a no. 10 or no. 11 scalpel blade. use care to avoid lacerating the underlying vessel. next, occlude the jugular vein as previously described, and insert the over-the-needle catheter into the vessel. watch for a flash of blood in the catheter hub. remove the stylette from the catheter. next, insert the long wire into the catheter and into the vessel (figures 1-16 and 1-17) . never let go of the wire. remove the catheter, and place the vascular dilator over the wire and into the vessel (figure 1-18) . gently twist to place the dilator into the vessel a short distance, creating a larger hole in the vessel. the vessel will bleed more after creating a larger hole. remove the vascular dilator, and leave the wire in place within the vessel. insert the long catheter over the wire into the vessel (figure 1-19) . push the catheter into the vessel to the catheter hub (figure 1-20) . slowly thread the wire through a proximal port in the catheter. once the catheter is in place, remove the wire, and suture the catheter in place to the skin with nonabsorbable suture. cover the catheter site with sterile gauze and antimicrobial ointment, cotton roll bandaging material, gauze, and kling or vetrap. flush the catheter with heparinized saline solution, and then use the catheter for infusion of parenteral nutrition, blood products, crystalloid and colloid fluids, medications, and frequent blood sample collection. examine the catheter site daily for evidence of infection or thrombophlebitis. the catheter can remain in place as long as it functions and no complications occur. place the patient in sternal recumbency as for cephalic venipuncture. clip the antebrachium circumferentially, and wipe the area clean of any loose fur and debris (figure 1-21) . aseptically scrub the clipped area, and have an assistant occlude the cephalic vein at the crook of the elbow. the person placing the catheter should grasp the distal carpus with the nondominant hand and insert the over-the-needle catheter into the vessel at a 15-to 30-degree angle ( figure 1-22) . watch for a flash of blood in the catheter hub, and then gently push the catheter off of the stylette (figure 1-23) . have the assistant occlude the vessel over the catheter to prevent backflow. flush the catheter with heparinized saline solution. make sure that the skin and catheter hub are clean and dry to ensure that the tape adheres to the catheter hub and skin. secure a length of 1 /2-inch white tape tightly around the catheter and then around the limb. make sure that the catheter hub does not "spin" in the tape, or else the catheter will fall out. next, secure a second length of 1-inch adhesive tape under the catheter and around the limb and catheter hub (figure 1-24 ). this piece of tape helps to stabilize the catheter in place. finally, place a flushed t port or male adapter in the catheter hub and secure to the limb with white tape. make sure that the tape is adhered to the skin securely, but not so tightly as to impede venous outflow (figure 1-25) . the catheter site can be covered with a cotton ball impregnated with antimicrobial ointment and layers of bandage material. label all catheters with the date of placement, the type and gauge of catheter inserted, and the initials of the person who placed the catheter. the femoral artery can be catheterized for placement of an indwelling arterial catheter. indwelling arterial catheters can be used for continuous invasive arterial blood pressure monitoring and for procurement of arterial blood samples. place the patient in lateral recumbancy, and tape the down leg in an extended position. clip the fur over the femoral artery and aseptically scrub the clipped area. palpate the femoral artery as it courses distally on the medial surface of the femur and anterior to the pectineus muscle. make a small nick incision over the proposed site of catheter placement using the bevel of an 18-gauge needle. place a long over-the-needle catheter through the nick in the skin and direct it toward the palpable pulse. place the tip of the catheter so that the needle tip rests in the subcutaneous tissue between the artery and the palpating index finger. advance the needle steeply at a 30-degree angle to secure the superficial wall of the vessel and then the deep wall of the vessel. the spontaneous flow of blood in the catheter hub ensures that the catheter is 1 figure 1 -25: catheter is taped in place with a t-port. situated in the lumen of the artery. feed the catheter off of the stylette, and cover the hub with a catheter cap. flush the catheter with sterile heparinized saline solution, and then secure it in place. some persons simply tape the catheter in place with pieces of 1 /2-and 1-inch adhesive tape. others use a "butterfly" piece of tape around the catheter hub and suture or glue the tape to the adjacent skin for added security. the dorsal pedal artery commonly is used for catheter placement. to place a dorsal pedal arterial catheter, place the patient in lateral recumbency. clip the fur over the dorsal pedal artery, and then aseptically scrub the clipped area. tape the distal limb so that the leg is twisted slightly medially for better exposure of the vessel, or the person placing the arterial catheter can manipulate the limb into the appropriate position. palpate the dorsal pedal pulse as it courses dorsally over the tarsus. place an over-the-needle catheter percutaneously at a 15-to 30-degree angle, threading the tip of the needle carefully toward the pulse. advance the needle in short, blunt movements, and watch the catheter hub closely for a flash of pulsating blood that signifies penetration into the lumen of the artery. then thread the catheter off of the stylette, and cover the catheter hub with a catheter cap. secure the catheter in place with lengths of 1 /2-and 1-inch adhesive tape as with any other intravenous catheter, and then flush it with heparinized saline solution every 2 to 4 hours. any vessel that can be catheterized percutaneously also can be catheterized with surgical cutdown. restrain the patient and clip and aseptically scrub the limb or jugular vein as for a percutaneous catheterization procedure. block the area for catheter placement with a local anesthetic before cutting the skin over the vessel with a no. 11 scalpel blade. while wearing sterile gloves, pick up the skin and incise the skin over the vessel. direct the sharp edge of the blade upward to avoid lacerating the underlying vessel. using blunt dissection, push the underlying subcutaneous fat and perivascular fascia away from the vessel with a mosquito hemostat. make sure that all tissue is removed from the vessel. using the mosquito hemostat, place two stay sutures of absorbable suture under the vessel. elevate the vessel until it is parallel with the incision, and gently insert the catheter and stylette into the vessel. secure the stay sutures loosely around the catheter. suture the skin over the catheter site with nonabsorbable suture, and then tape and bandage the catheter in place as for percutaneous placement. remove catheters placed surgically as soon as possible and exchange them for a percutaneously placed catheter to avoid infection and thrombophlebitis. the most important aspect of catheter maintenance is to maintain cleanliness and sterility at all times. an indwelling catheter can remain in place for as long as it is functional and no complications occur. change the bandage whenever it becomes wet or soiled to prevent wicking of bacteria and debris from the environment into the vessel. check the bandages and catheter sites at least once a day for signs of thrombophlebitis: erythema, vessel hardening or ropiness, pain upon injection or infusion, and discharge. also closely examine the tissue around and proximal and distal to the catheter. swelling of the paw can signify that the catheter tape and bandage are too tight and are occluding venous outflow. swelling above the catheter site is characteristic of perivascular leakage of fluid and may signify that the catheter is no longer within the lumen of the vessel. remove the catheter if it is no longer functional, if there is pain or resistance upon infusion, if there is unexplained fever or leukocytosis, or if there is evidence of cellulitis, thrombophlebitis, or catheter-related bacteremia or septicemia. aseptically culture the tip of the indwelling catheter for bacteria. animals should wear elizabethan collars or other forms of restraint if they lick or chew at the catheter or bandage. catheter patency may be maintained with constant fluid infusion or by intermittent flushing with heparinized saline (1000 units of unfractionated heparin per 250 to 500 ml of saline) every 6 hours. flush arterial catheters more frequently (every 2 hours). disconnect intravenous connections only when absolutely necessary. wear gloves whenever handling the catheter or connections. label all fluid lines and elevate them off of the floor to prevent contamination. date each fluid line and replace it once every 24 to 36 hours. if an intravenous catheter cannot be placed because of small patient size, hypovolemia, hypothermia, or severe hypotension, needles can be placed into the marrow cavity of the femur, humerus, and tibia for intraosseous infusion of fluids, drugs, and blood products. this technique is particularly useful in small kittens and puppies and in exotic species. contraindications to intraosseous infusion is in avian species (which have air in their bones), fractures, and sepsis, because osteomyelitis can develop. an intraosseous catheter is relatively easy to place and maintain but can cause patient discomfort and so should be changed to an intravenous catheter as soon as vascular access becomes possible. to place an intraosseous catheter, clip and aseptically scrub the fur over the proposed site of catheter placement. the easiest place for intraosseous placement is in the intertrochanteric fossa of the femur. inject a small amount of a local anesthetic through the skin and into the periosteum where the trocar or needle will be inserted. place the patient in lateral recumbency, and grasp the leg in between your fingers, with the stifle braced against the palm of your hand. push the stifle toward the abdomen (medially) to abduct the proximal femur away from the body. this will shift the sciatic nerve out of the way of catheter placement. insert the tip of the needle through the skin and into the intertrochanteric fossa. gently push with a simultaneous twisting motion, pushing the needle parallel with the shaft of the femur, toward your palm. you may feel a pop or decreased resistance as the needle enters the marrow cavity. gently flush the needle with heparinized saline. if the needle is plugged with bone debris, remove the needle and replace it with a fresh needle of the same type and size in the hole that you have created. a spinal needle with an internal stylette also can be placed. the stylette will prevent the needle from becoming clogged with bone debris during insertion. secure the hub of the needle with a butterfly length of white adhesive tape and then suture it to the skin to keep the catheter in place. the catheter is now ready for use. the patient should wear an elizabethan collar to prevent disruption or removal of the catheter. the intraosseous catheter can be maintained as any peripheral catheter, with frequent flushing and daily evaluation of the catheter site. the definition of pain has been debated philosophically over the ages and has changed as knowledge has increased. pain is defined as an unpleasant sensory or emotional experience associated with actual or perceived tissue damage. until recognition of a noxious stimulus occurs in the cerebral cortex, no response or adaptation results. rational management of pain requires an understanding of the underlying mechanisms involved in pain and an appreciation of how analgesic agents interact to disrupt pain mechanisms. multiple factors and causes produce pain in human beings and domestic animal species. the causes of pain, psychological and physical, may derive from many different mechanisms within emergency medicine, among them trauma, infectious disease, neglect, environmental stress, surgery, and acute decompensation of chronic medical conditions. the two major classes of pain are acute and chronic pain. box 1-20 gives specific categories and causes of pain. the pain sensing and response system can be divided into the following categories: nociceptors, which detect and filter the intensity of the noxious stimuli; primary afferent nerves, which transmit impulses to the central nervous system (cns); ascending tracts, which are part of the dorsal horn and the spinal cord that conveys stimuli to higher centers in the brain; higher centers, which are involved in pain discrimination, some memory, and motor control; and modulating or descending systems, which are a means of processing, memorizing, and modifying incoming impulses. current analgesic therapies may inhibit afferent nociceptive transmission within the brain and spinal cord; directly interrupt neural impulse conduction through the dorsal horn, primary afferent nerves, or dorsal root ganglion; or prevent the nociceptor sensitization that accompanies initial pain and inflammation. the physiologic aspects of pain are believed to be produced by the transmission, transduction, and integration of initial nerve endings, peripheral neuronal input, and ascending afferent nerves via the thalamus to the cerebral cortex. ascending afferent nerves to the limbic system are believed to be responsible for the emotional aspects of pain. there are several classification schemes for different types of pain. acute pain, such as that which results from trauma, surgery, or infectious agents, is abrupt in onset, relatively short in duration, and may be alleviated easily by analgesics. in contrast, chronic pain is a long-standing physical disorder or emotional distress that is slow in onset and difficult to treat. both types of pain can be classified further based on site of origin. somatic pain arises from superficial skin, subcutaneous tissue, body wall, or appendages. visceral pain arises from abdominal or thoracic viscera and primarily is associated with serosal irritation. analgesia, then, is the loss of pain without the loss of consciousness. this is in contrast to anesthesia, which is the loss of sensation in the whole body or a part of the body with the loss of consciousness or at least depression of the cns. untreated pain causes immediate changes in the neurohormonal axis, which in turn causes restlessness, agitation, increased heart and respiratory rates, fever, and blood pressure fluctuations, all of which are detrimental to the healing of the animal. a catabolic state is created as a result of increased secretion of catabolic hormones and decreased secretion of anabolic hormones. the net effect the majority of neurohormonal changes produce is an increase in the secretion of catabolic hormones. hyperglycemia is produced and may persist because of production of glucagon and relative lack of insulin. lipolytic activity is stimulated by cortisol, catecholamines, and growth hormone. cardiorespiratory effects of pain include increased cardiac output, vasoconstriction, hypoxemia, and hyperventilation. protein catabolism is a common occurrence and major concern regarding healing. pain associated with inflammation causes increase in tissue and blood levels of prostaglandins and cytokines, both of which promote protein catabolism indirectly by increasing the energy expenditure of the body. powerful evidence indicates that local anesthetic, sympathetic agonist, and opioid neural blockade may produce a modification of the responses to these physiologic changes. variable reduction in plasma cortisol, growth hormone, antidiuretic hormone, î²-endorphin, aldosterone, epinephrine, norepinephrine, and renin is based on the anesthetic technique and the drugs selected. prophylactic administration of analgesics blunts the response before it occurs; analgesics administered following perception or pain are not as effective, and higher doses are generally necessary to achieve an equivalent level of analgesia. effective pain control can be achieved only when the signs of pain can be assessed effectively, reliably, and regularly. the experience of pain is unique to each individual, which makes pain assessment difficult, especially in traumatized and critical patients. most attempts to assess clinical pain use behavioral observations and interactive variables in addition to assessment of physiologic responses such as heart rate and respiratory rate, blood pressure, and temperature. but many factors can influence the processing and outward projection of pain, including altered environments, species differences, withinspecies variations (age, breed, sex), and the type, severity, and chronicity of pain. within-species differences (age, breed, and sex) further complicate the pain assessment. most notable is that different breeds of dogs act differently when confronted with pain or fear. labrador retrievers tend to be stoic, whereas greyhounds and teacup breeds tend to react with a heightened state of arousal around even the simplest of procedures (e.g., subcutaneous injections and nail trims). the individual character and temperament of the animal further influences its response. pediatric and neonatal animals seem to have a lower threshold for pain and anxiety than older animals. in any species, the duration and type of pain make it more (acute) or less (chronic) likely to be expressed or exhibited outwardly. unfamiliarity with normal behaviors typical of a particular species or breed makes recognition of their painful behaviors and responses impossible. the definition and recognition of pain in an individual animal is challenging. because of all the differences discussed, there is no straight line from insult, albeit actual or perceived, to degree of pain experienced. nor is there a formula for treating "x" type of pain with "y" type of analgesic. a goal of analgesia is to treat all animals with analgesic drugs and modalities as preemptively as possible and using a multimodal approach. use analgesic treatment as a tool for diagnosis of pain in the event that recognition of these phenomena is difficult for the patient. in other words, with countless drugs and treatment modalities available, analgesic administration should never be withheld in an animal, even if pain is questionable. it is important to remember that no behavior or physiologic variable in and of itself is pathognomonic for pain. interactive and unprovoked (noninteractive) behavior assessments and trending of physiologic data are useful to determine the pain in an individual animal. this is known as pain scoring. baseline observations, especially those observations from someone who has known the animal well, can be helpful to serial behavior and pain assessments. pain scoring systems have been developed and are reviewed elsewhere; the purposes of these systems are to evaluate and to help guide diagnostic and analgesic treatments (table 1 -15) . regardless of the scale or method used to assess pain, the caregiver must recognize the limitations of the scale. if in doubt of whether pain is present or not, analgesic therapy should be used as a diagnostic tool. classic behaviors associated with pain in dogs and cats include abnormal postures, gaits, movements, and behaviors (boxes 1-21 and . stoicism is the apparent apathy and pain: assessment, prevention, and management 71 indifference in the presence of pain and is perhaps the no. 1 sign of ineffective pain relief or persistent pain in many animals, because so many display apathy and classically normal physiologic parameters even in the face of severe distress, overt suffering, or blatant trauma and illness. the absence of normal behaviors is also a clinical sign of pain, even when abnormal behaviors are not observed. acute pain results in many of the aforementioned behavioral and physiologic signs, but chronic pain in small animals is an entirely different and distinct entity. chronic pain is often present in the absence of obvious tissue pathology and changes in physical demeanor. again, the severity of the pain may not correlate with the severity of any pathologic condition that may or may not be present. chronic pain, especially if insidious in onset (cancer, dental, or degenerative pain), may well go unnoticed in dogs and cats, even by family members or intermittent caregivers. inappetance, lack of activity, panting in a species classically designed to be nose breathers, decreased interest in surroundings, different activity patterns, and abnormal postures are just a few signs of chronic pain in cats and dogs. cats are a species that in particular are exemplary in their abilities to hide chronic pain. they will exhibit marked familial withdrawal, finding secluded areas where they may remain for days to weeks when they experience acute and chronic pain. when deciding on a pain management protocol for a patient, always perform a thorough physical examination and include a pain score assessment before injury and pain has occurred, whenever possible. form a problem list to guide your choice of anesthesia and analgesia. for example, using a nonsteroidal antiinflammatory drug (nsaid) in an animal with renal failure would not be wise. remember to account for current medications that the patient may be taking that may augment or interfere with the analgesic or anesthetic drugs. use multimodal techniques and regional therapy and drugs to target pain at different sites before it occurs. once a strategy is decided upon, frequently reassess the patient and tailor the protocol to meet each patient's response and needs. drug therapy (in particular, opioids with or without î± 2 -agonists) is a cornerstone for acute pain treatment and surgical preemptive pain prevention. however, local anesthetics delivered epidurally, via perineural or plexus injection, intraarticular or trigger point injection, are also effective analgesics for acute and chronic forms of pain and inflammation. the nsaids that classically have been reserved for treatment of more chronic or persistent pain states now are being used regularly for treatment of acute and perioperative pain once blood pressure, coagulation, and gastrointestinal parameters have been normalized. an opioid is any natural or synthetic drug that is derived from the poppy, which interacts with opiate receptors identified on cell membranes. the drugs from this class constitute the most effective means of controlling acute, perioperative, and chronic pain in human and veterinary medicine (table 1 -16) . their physiologic effects result from the interaction with one or more of at least five endogenous opioid receptors (âµ, ï�, î´, îµ, and îº). âµ-receptor agonists are noted for their ability to produce profound analgesia with mild sedation. these drugs diminish "wind-up," the hyperexcitable state resulting from an afferent volley of nociceptive impulses. they elevate the pain threshold and are used preemptively to prevent acute pain. as a class, opioids cause cns depression with their intense analgesia. dose-related respiratory depression reflects diminished response to carbon dioxide levels. cardiac depression is secondary only to bradycardia and is more likely with certain opioids such as morphine and oxymorphone. narcotics produce few if any clinically significant cardiovascular effects in dogs and cats; they are considered cardiac soothing or sparing. because opioids increase intracranial and intraocular pressure, use them more cautiously in patients with severe cranial trauma and or ocular lesions. opioids directly stimulate the chemoreceptor trigger zone and may cause nausea and vomiting. most opioids depress the cough reflex via a central mechanism; this may be helpful in patients recovering from endotracheal intubation irritation. a key characteristic of opioids that makes them desirable for use in emergency and critical care situations is their reversibility. antagonists block or reverse the effect of agonists by combining with receptors and producing minimal or no effects. administer all reversal agents, such as naloxone and naltrexone, slowly if given intravenously and to effect. î± î± 2 -agonists as a class of drugs, î± 2 -agonists warrant special attention because most members of the group possess potent analgesic power at doses that are capable of causing sedation, cns depression, cardiovascular depression, and even general anesthetic states. originally developed for antihypertensive use, î± 2 -agonists quickly have attained sedative analgesic status in veterinary medicine (table 1 -17) . like the opioids, î± 2 -agonists produce their effects by aggravating î±-adrenergic receptors in the cns and periphery. 1 emergency care among them cyclooxygenase-1 (cox-1), the major constitutive enzyme primarily involved in normal physiologic functions, and cox-2, the enzyme responsible for most of the hyperalgesia and pain responses experienced after tissue injury or trauma. some nsaids inhibit cyclooxygenase and lipoxygenase activity. most of the currently available oral and parenteral nsaids for small animal medicine and surgery target the cyclooxygenase pathways predominantly, although one (tepoxalin) is thought to inhibit both pathways. inhibition of cox-1 and cox-2 can inhibit the protective effects and impair platelet aggregation and lead to gastrointestinal ulceration. there are definite contraindications and relative contraindications for the use of nsaids. nonsteroidal antiinflammatory drugs should not be administered to patients with renal or hepatic insufficiency, dehydration, hypotension or conditions that are associated with low circulating volume (congestive heart failure, unregulated anesthesia, shock), or evidence of ulcerative gastrointestinal disease. trauma patients should be stabilized completely regarding vascular volume, tone, and pressure before the use of nsaids. patients receiving concurrent administration of other nsaids or corticosteroids, or those considered to be cushingoid, should be evaluated carefully for an adequate "washout" period (time of clearance of drug from the system) before use of an nsaid or before switching nsaids. patients with coagulopathies, particularly those that are caused by platelet number or function defects or those caused by factor deficiencies, and patients with severe, uncontrolled asthma or other bronchial disease are probably not the patients in which to use nsaids. other advice is that nsaids not be administered to pregnant patients or to females attempting to become pregnant because cox-2 induction is necessary for ovulation and subsequent implantation of the embryo. the administration of nsaids should be considered only in the well-hydrated, normotensive dog or cat with normal renal or hepatic function, with no hemostatic abnormalities, and no concurrent steroid administration. nonsteroidal antiinflammatory drugs can be used in many settings of acute and chronic pain and inflammation. among these are the use in well-stabilized musculoskeletal trauma and surgical pain, osteoarthritis management, meningitis, mastitis, animal bite and other wound healing, mammary or transitional cell carcinoma, epithelial (dental, oral, urethral) inflammation, ophthalmologic procedures, and dermatologic or otic disease. whereas opioids seem to have an immediate analgesic effect when administered, most nsaids will take up to 30 minutes for their effect to be recognized. as such, most perioperative or acute nsaids use is part of a balanced pain management scheme, one that uses narcotics and local anesthetic techniques. nonsteroidal antiinflammatory drugs are devoid of many of the side effects of narcotic administration; namely, decreased gastrointestinal motility, altered sensorium, nausea/vomition, and sedation. nonsteroidal antiinflammatory drugs are also devoid of many of the side effects of steroid administration; namely, suppression of the pituitary adrenal axis. the toxic effects of salicylates in cats are well documented. cats are susceptible because of slow clearance and dose-dependent elimination because of deficient glucuronidation in this species. because of this, the dose and the dosing interval of most commonly used nsaids need to be altered in order for these drugs to be used. cats that have been given canine doses of nsaids (twice daily or even once daily repetitively) may show hyperthermia, hemorrhagic or ulcerative gastritis, kidney and liver injury, hyperthermia, respiratory alkalosis, and metabolic acidosis. acute and chronic toxicities of nsaids have been reported in cats, especially after repeat once daily dosing. ketoprofen, flunixin, aspirin, carprofen, and meloxicam have been administered safely to cats, although like most antibiotics and other medications, they are not approved and licensed for use in cats. an important note, though, is that dosing intervals ranging from 48 to 96 hours have been used, and antithrombotic effects often can be achieved at much lower doses than those required to treat fevers and inflammation. i recommend the use of no loading doses, minimum 48-hour dosing intervals, and assurance of adequate circulating blood volume, blood pressure, and renal function. because many of the nsaids are used off-label in cats, it is imperative that the clinician carefully calculate the dose, modify the dosing interval, and communicate this information to the client before dispensing the drug. even drugs that come in liquid form (meloxicam), if administered to cats via box-labeled directions used for dogs, will be given in near toxic doses. to worsen the misunderstanding about dosages for cats, drops from manufacturer's bottles often are calibrated drops; when these same liquids are transferred into pharmacy syringes for drop administration, the calibration of course is lost, and the animal potentially is overdosed. a more accurate method of dispensing and administering oral nsaids in cats is to calculate the dose in milligrams and determine the exact number of milliliters to administer, rather than use the drop method. ketamine classically was considered a dissociative anesthetic, but it also has potent activity as an n-methyl-d-aspartate (nmda) receptor antagonist. this receptor located in the cns mediates windup and central sensitization (a pathway from acute to chronic pain). blockade of this receptor with microdoses of ketamine results in the ability to provide body surface, somatic, and skin analgesia with potentially lower doses of opioids and î±-agonists. loading doses of 0.5 to 2 mg/kg are used intravenously with continuous rate infusions of 2 to 20 âµg/kg/minute. in and of itself, this drug possesses little to no analgesic ability and indeed in high doses alone often can aggravate, sensitize, or excite the animal in subacute or acute pain. amantadine is another nmda blocker that has been used for its antiviral and parkinson's stabilizing effects. amantadine has been used for neuropathic pain in human beings but is only available in an oral form. suggested starting doses for cats and dogs range from 3 to 10 mg/kg po daily. when the drug is given orally and intravenously, patients are unlikely to develop behavioral or cardiorespiratory effects with ketamine or amantadine. tramadol is an analgesic that possesses weak opioid âµ-agonist activity and norepinephrine and serotonin reuptake inhibition. tramadol is useful for mild to moderate pain in small animals. although the parent compound has very weak opioid activity, the metabolites have excellent binding affinity for the âµ-receptor. tramadol has been used for perisurgical pain control when given orally in cats and dogs at a dose of 1 to 10 mg/kg po sid to bid. cats appear to require only once daily dosing. regardless of its affinity for the opioid receptors, the true mechanism of action of tramadol in companion animals remains largely unknown. gabapentin is a synthetic analog of î³-aminobutyric acid (gaba). originally introduced as an antiepileptic drug, the mechanism of action of gabapentin remains somewhat unclear in veterinary medicine. the drug is among a number of commonly used antiepileptic medications used to treat central pain in human beings. the rationale for use is the ability of the drugs to suppress discharge in pathologically altered neurons. gabapentin does this through calcium channel modulation without binding to glutamate receptors. chronic, burning, neuropathic, and lancinating pain in small animals responds well to 1 to 10 mg/kg po daily. local anesthetic agents are the major class used as a peripheral-acting analgesic ( table 1 -19) . local anesthetics block the transmission of pain impulses at the peripheral nerve nociceptor regions. local anesthetics may be used to block peripheral nerves or inhibit nerve "zones" using regional techniques. although all local anesthetics are capable of providing pain relief, agents with a longer duration of action are preferred for pain management purposes. bupivacaine is an example of a long-acting local anesthetic drug that is used along with lidocaine for long-acting pain relief. a single dose of bupivacaine injected at a local site will provide local anesthesia and analgesia for 6 to 10 hours. when lidocaine is administered as an intravenous constant rate infusion (50 to 75 âµg/kg/minute in dogs, 1 to 10 âµg/kg/minute in cats) is effective in the treatment of chronic neuropathic pain and periosteal and peritoneal pain (e.g., pancreatitis). mexiletine, an oral sodium channel blocker, can be used as an alternative to injectable lidocaine for provision of background analgesia. many drugs (table 1 -20) are used in combination with opioids, î± 2 -agonists, and ketamine to provide anxiolysis and sedation. injection of local anesthetic solution into the connective tissue surrounding a particular nerve produces loss of sensation (sensory blockade) and/or paralysis (motor nerve blockade) in the region supplied by the nerve. local anesthetics also may be administered epidurally, intrathoracically, intraperitoneally, and intraarticularly. lidocaine and bupivacaine are the most commonly administered local anesthetics. lidocaine provides for quick, short-acting sensory and motor impairment. bupivacaine provides for later-onset, longerlasting desensitization without motor impairment. combinations of the two agents diluted with saline are used frequently to provide for quick-onset analgesia that lasts between 4 and 6 hours in most patients. adding narcotic and/or î± 2 agent often maximizes the analgesia and increases the pain-free interval to 8 to 18 hours. epinephrine and preservative-free solutions are recommended. precision placement of anesthetic close to nerves, roots, or plexuses is improved with the use of a stimulating nerve locator. cats seem to be more sensitive to the effects of local anesthetics; as such the lower ends of most dosing ranges are used for blockades in this species. unlike most instances of general anesthesia, during which the animal is rendered unconscious and nerve transmission is decreased by virtue of cns depression, local and regional techniques block the initiation of noxious signals, thereby effectively preventing pain from entering the cns. this is an effective means of not only preventing initial pain but also reducing the changes that take place in the dorsal horn of the spinal cord, spinothalamic tracts, limbic and reticular activating centers, and cortex. frequently, the neurohormonal response that is stimulated in pain and stress is blunted as well. overall, the patient has fewer local and systemic adverse effects of pain, disease processes are minimized, chronic pain states are unlikely, and outcome is improved. regional techniques are best used as part of an analgesic regimen that consists of their continuous administration, narcotics, î±-agonists, anxiolytics, and good nursing. lidocaine can be added to sterile lubricant in a one-to-one concentration to provide decreased sensation for urinary catheterization, nasal catheter insertion, minor road burn analgesia, and pyotraumatic dermatitis analgesia. proparacaine is a topical anesthetic useful for corneal or scleral injuries. local anesthetics can be used to infiltrate areas of damage or surgery by using long-term continuous drainage catheters and small, portable infusion pumps. this is an effective means of providing days of analgesia for massive surgical or traumatic soft tissue injury. even without the catheter, incisional or regional soft tissue blocking using a combination of 1 to 2 mg/kg lidocaine and 0.5 to 2 mg/kg bupivacaine diluted with equal volume of saline and 1:9 with sodium bicarbonate is effective for infiltrating large areas of injury. administration of local anesthetic drugs around the infraorbital, maxillary, ophthalmic mental, and alveolar nerves can provide excellent analgesia for dental, orofacial, and ophthalmic trauma and surgical procedures. each nerve may be desensitized by injecting 0.1 to 0.3 ml of a 2% lidocaine hydrochloride solution and 0.1 to 0.3 ml of 0.5% bupivacaine solution using a 1.2-to 2.5-cm, 22-to 25-gauge needle. precise placement perineurally versus intraneurally (neuroma formation common) is enhanced by using catheters in the foramen versus needle administration. always perform aspiration before administration to rule out intravascular injection of agents. this block is used to provide analgesia for thoracic, lower cervical, cranial abdominal, and diaphragmatic pain. following aseptic preparation, place a small through-the-needle (20-to 22-gauge) catheter in the thoracic cavity between the seventh and ninth intercostal space on the midlateral aspect of the thorax. aseptically mix a 0.5 to 1 mg/kg lidocaine and a 0.2 to 0.5 mg/kg bupivacaine dose with volume of saline equal to the volume of bupivacaine, and slowly inject it over a period of 2 to 5 minutes following aspiration to ensure that no intravascular injection occurs. depending on where the lesion is, position the patient to allow the intrapleural infusion to "coat" the area. most effective is positioning the patient in dorsal recumbency for several minutes following the block to make sure local anesthetic occupies the paravertebral gutters and hence the spinal nerve roots. the block should be repeated every 3 hours in dogs and every 8 to 12 hours in cats. secure the catheter to the skin surface for repetitive administration. administration of local anesthetic around the brachial plexus provides excellent analgesia for forelimb surgery, particularly that distal to the shoulder, and amputations. nerve locator-guided techniques are much more accurate and successful than blind placement of local anesthetic; however, even the latter is useful. to administer a brachial plexus blockade, follow this procedure: 1. aseptically prepare a small area of skin over the point of the shoulder. 2. insert a 22-gauge, 1 1 /2-to 3-inch spinal needle medial to the shoulder joint, axial to the lesser tubercle, and advance it caudally, medial to the body of the scapula, and toward the costochondral junction of the first rib. aspirate first before injection to make sure that intravenous injection does not occur. 3. inject one third of the volume of local anesthetic mix, and then slowly withdraw the needle and fan dorsally and ventrally while infusing the remaining fluid. 4. local anesthetic doses are similar to those for intrapleural blockade. epidural analgesia refers to the injection of an opioid, a phencyclidine, an î±-agonist, or an nsaid into the epidural space. epidural anesthesia refers to the injection of a local anesthetic. in most patients a combination of the two is used. epidural analgesia and anesthesia are used for a variety of acute and chronic surgical pain or traumatically induced pain in the pelvis, tail, perineum, hind limbs, abdomen, and thorax (table 1 -21) . procedures in which epidural analgesia and anesthesia are useful include forelimb and hind limb amputation, tail or perineal procedures, cesarean sections, diaphragmatic hernia repair, pancreatitis, peritonitis, and intervertebral disk disease. epidural blocks performed using opioids or bupivacaine will not result in hind limb paresis or decreased urinary or anal tone (incontinence), unlike lidocaine or mepivicaine epidural blocks. morphine is one of the most useful opioids for administration in the epidural space because of its slow systemic absorption. epidural catheters used for the instillation of drugs through constant rate infusion or intermittent injection can be placed in dogs and cats. routinely placed at the lumbosacral junction, these catheters are used with cocktails including preservative-free morphine, bupivacaine, medetomidine, and ketamine. extremely effective for preventing windup pain in the peritoneal cavity or caudal half of the body, the catheters may be maintained if placed aseptically for 7 to 14 days. to provide epidural analgesia or anesthesia, follow this procedure: 1. position the animal in lateral or sternal recumbency. 2. clip and aseptically scrub over the lumbosacral site. 3. palpate the craniodorsal-most extent of the wings of the ileum bilaterally and draw an imaginary line through them to envision the spine of l7 located immediately behind the imaginary line. 4. advance a 20-to 22-gauge, 1 1 /2-to 3-inch spinal or epidural needle through the skin just caudal to the spine of l7. 5. the needle will lose resistance as it is introduced into the epidural space. drop saline into the hub of the needle, and the saline will be pulled into the epidural space as the needle enters. discrete intercostal nerve blocks can provide effective analgesia for traumatic or postsurgical pain. identify the area of the injury, and infiltrate three segments on either side of the injury with analgesic. to perform an intercostal nerve block, follow this procedure: 1. clip and aseptically scrub the dorsal and ventral third of the chest wall. 2. palpate the intercostal space as far dorsally as possible. 3. use a 25-gauge, 0.625-inch needle at the caudolateral aspect of the affected rib segments and those cranial and caudal. 4. direct the tip of the needle caudally such that the tip of the needle "drops" off of the caudal rib. (this places the needle tip in proximity to the neuromuscular bundle that contains the intercostal nerve that runs in a groove on the caudomedial surface of the rib.) 5. aspirate to confirm that the drug will not go intravenously. 6. inject while slowly withdrawing the needle. inject 0.5 to 1.0 ml at each site, depending on the size of the animal. gaynor js, an acute condition in the abdomen is defined as the sudden onset of abdominal discomfort or pain caused by a variety of conditions involving intraabdominal organs. many animals have the primary complaint of lethargy, anorexia, ptyalism, vomiting, retching, diarrhea, hematochezia, crying out, moaning, or abnormal postures. abnormal postures can include generalized rigidity, walking tenderly or as if "on eggshells," or a prayer position in which the front limbs are lowered to the ground while the hind end remains standing. in some cases, it may be difficult initially to distinguish between true abdominal pain or referred pain from intervertebral disk disease. rapid progression and decompensation of the patient's cardiovascular status can lead to stupor, coma, and death in the most extreme cases, making rapid assessment, treatment, and definitive care extremely challenging. often the patient's signalment and history can increase the index of suspicion for a particular disease process. a thorough history often is overlooked or postponed in the initial stages of resuscitation of the patient with acute abdominal pain. often, asking the same question in a variety of methods can elicit an answer from the client that may lead to the source of the problem and the reason for acute abdominal pain. important questions to ask the client include the following: â�¢ what is your chief complaint or reason that you brought your animal in on emergency? â�¢ when did the signs first start, or when was your animal last normal? â�¢ do you think that the signs have been the same, better, or getting worse? â�¢ does your animal have any ongoing or past medical problems? â�¢ have similar signs occurred in the past? â�¢ does your animal have access to any known toxins, or does he or she run loose unattended? as with any other emergency, the clinician must follow the abcs of therapy, treating the most life-threatening problems first. first, perform a perfunctory physical examination. examination of the abdomen ideally should be performed last, in case inciting a painful stimulus precludes you from evaluating other organ systems more thoroughly. briefly observe the patient from a distance. are there any abnormal postures? is there respiratory distress? is the animal ambulatory, and if so, do you observe any gait abnormalities? do you observe any ptyalism or attempts to vomit? auscultate the patient's thorax for crackles that may signify aspiration pneumonia resulting from vomiting. examine the patient's mucous membrane color and capillary refill time, heart rate, heart rhythm, and pulse quality. many patients in pain have tachycardia that may or may not be accompanied by dysrhythmias. if a patient's heart rate is inappropriately bradycardic, consider hypoadrenocorticism, whipworm infestation, or urinary obstruction or trauma as a cause of hyperkalemia. assess the patient's hydration status by evaluating skin turgor, mucous membrane dryness, and whether the eyes appear sunken in their orbits. a brief neurologic examination should consist of whether the patient is actively having a seizure, or whether mental dullness, stupor, coma, or nystagmus are present. posture and spinal reflexes can assist in making a diagnosis of intervertebral disk disease versus abdominal pain. perform a rectal examination to evaluate for the presence of hematochezia or melena. finally, examination of the abdomen should proceed first with superficial and then deeper palpation. visually inspect the abdomen for the presence of external masses, bruising, or penetrating injuries. reddish discoloration of the periumbilical area often is associated with the presence of intraabdominal hemorrhage. it may be necessary to shave the fur to inspect the skin and underlying structures visually for bruising and ecchymoses. auscultate the abdomen for the presence or absence of borborygmi to characterize gut sounds. next, perform percussion and ballottement to evaluate for the presence of a gas-distended viscus or peritoneal effusion. finally, perform first superficial and then deep palpation of all quadrants of the abdomen, noting abnormal enlargement, masses, or whether focal pain is elicited in any one area. once the physical examination has been performed, implement initial therapy in the form of analgesia, fluid resuscitation, and antibiotics. treatment for any patient with an acute condition in the abdomen and shock is to treat the underlying cause, maintain tissue oxygen delivery, and prevent end-organ damage and failure. a more complete description of shock and oxygen delivery is given in the section on shock. 1 emergency care the administration of analgesic agents to any patient with acute abdominal pain is one of the most important therapies in the initial stages of case management. many patients with acute abdominal pain are clinically dehydrated or are in hypovolemic shock because of hemorrhage. careful titration of intravenous crystalloid and colloid fluids including blood products is necessary based on the patient's perfusion parameters including heart rate, capillary refill time, blood pressure, urine output, and pcv. fluid therapy also should be based on the most likely differential diagnoses, with specific fluid types administered according to the primary disease process. in dogs, a shock volume of fluids is calculated based on the total blood volume of 90 ml/kg/hour. in cats, shock fluid rate is based on plasma volume of 44 ml/kg/hour. in most cases, any crystalloid fluid can be administered at an initial volume of one fourth of a calculated shock dose and then titrated according to whether the patient's cardiovascular status responds favorably or not. in cases of an acute condition in the abdomen from known or suspected hypoadrenocorticism, severe whipworm infestation, or urinary tract obstruction or rupture, 0.9% sodium chloride fluid without added potassium is the fluid of choice. when hemorrhage is present, the administration of whole blood or packed rbcs may be indicated if the patient has clinical signs of anemia and shows clinical signs of lethargy, tachypnea, and weakness. fresh frozen plasma is indicated in cases of hemorrhage resulting from vitamin k antagonist rodenticide intoxication or hepatic failure or in cases of suspected disseminated intravascular coagulation (dic). a more thorough description of fluid therapy is given under the sections on shock and fluid therapy. the empiric use of broad-spectrum antibiotics is warranted in cases of suspected sepsis or peritonitis as a cause of acute abdominal pain. ampicillin sulbactam (22 mg/kg iv q6-8h) and enrofloxacin (10 mg/kg once daily) are the combination treatment of choice to cover gram-negative, gram-positive, aerobic, and anaerobic infections. alternative therapies include a second-generation cephalosporin such as cefotetan (30 mg/kg iv tid) or cefoxitin (22 mg/kg iv tid) or added anaerobic coverage with metronidazole (10 to 20 mg/kg iv tid). tissue oxygen delivery depends on a number of factors, including arterial oxygen content and cardiac output. if an animal has had vomiting and subsequent aspiration pneumonitis, treatment of hypoxemia with supplemental oxygen in the form of nasal, nasopharyngeal, hood, or transtracheal oxygen administration is important (see oxygen supplementation under emergency diagnostic and therapeutic procedures). perform a complete blood count in all cases of acute abdominal pain to determine if lifethreatening infection or coagulopathy including dic is present. in cases of sepsis, infection, or severe nonseptic inflammation, the white blood cell count may be normal, elevated, or low. examine a peripheral blood smear for the presence of toxic neutrophils, eosinophils, atypical lymphocytes, nucleated rbcs, platelet estimate, anisocytosis, and blood parasites. a falling pcv in the face of rbc transfusion suggests ongoing hemorrhage. perform a biochemistry panel to evaluate organ system function. azotemia with elevated bun and creatinine may be associated with prerenal dehydration, impaired renal function, or postrenal obstruction or leakage. the bun also can be elevated when gastrointestinal hemorrhage is present. serum amylase may be elevated with decreased renal function or in cases of pancreatitis. a normal serum amylase, however, does not rule out pancreatitis as a source of abdominal pain. serum lipase may be elevated with gastrointestinal inflammation or pancreatitis. like amylase, a normal serum lipase does not rule out pancreatitis. total bilirubin, alkaline phosphatase, and alanine transaminase may be elevated with primary cholestatic or hepatocellular diseases or may be due to extrahepatic causes including sepsis. obtain a urinalysis via cystocentesis whenever possible, except in cases of suspected pyometra or transitional cell carcinoma. azotemia in the presence of a nonconcentrated (isosthenuric or hyposthenuric) urine suggests primary renal disease. secondary causes of apparent renal azotemia and lack of concentrating ability also occur in cases of hypoadrenocorticism and gram-negative sepsis. renal tubular casts may be present in cases of acute renal ischemia or toxic insult to the kidneys. bacteriuria and pyuria may be present with infection and inflammation. when a urinalysis is obtained via free catch or urethral catheterization, the presence of bacteriuria or pyuria also may be associated with pyometra, vaginitis, or prostatitis/prostatic abscess. serum lactate is a biochemical indicator of decreased organ perfusion, decreased oxygen delivery or extraction, and end-organ anaerobic glycolysis. elevated serum lactate greater than 6 mmol/l has been associated with increased morbidity and need for gastric resection in cases of gdv and increased patient morbidity and mortality in other disease processes. rising serum lactate in the face of adequate fluid resuscitation is a negative prognostic sign. obtain abdominal radiographs as one of the first diagnostic tests when deciding whether to pursue medical or surgical management. the presence of gdv, linear foreign body, pneumoperitoneum, pyometra, or splenic torsion warrants immediate surgical intervention. if a loss of abdominal detail occurs because of peritoneal effusion, perform additional diagnostic tests including abdominal paracentesis (abdominocentesis) and abdominal ultrasound to determine the cause of the peritoneal effusion. abdominal ultrasonography is often useful in place of or in addition to abdominal radiographs. the sensitivity of abdominal ultrasonography is largely operator dependent. indications for immediate surgical intervention include loss of blood flow to an organ, linear bunching or placation of the intestinal tract, intussusception, pancreatic phlegmon or abscess, a fluid-filled uterus suggestive of pyometra, gastrointestinal obstruction, intraluminal gastrointestinal foreign body, dilated bile duct, or gallbladder mucocele, or gas within the wall of the stomach or gallbladder (emphysematous cholecystitis). the presence of peritoneal fluid alone does not warrant immediate surgical intervention without cytologic and biochemical evaluation of the fluid present. see also abdominal paracentesis and diagnostic peritoneal lavage. abdominal paracentesis (abdominocentesis) often is the deciding factor in whether to perform immediate surgery. abdominocentesis is a sensitive technique for detecting peritoneal effusion when more than 6 ml/kg of fluid is present within the abdominal cavity. abdominal effusion collected should be saved for bacterial culture and evaluated biochemically and cytologically based on your index of suspicion of the primary disease process. if creatinine, urea nitrogen (bun) or potassium is elevated compared with that of serum, uroabdomen is present. elevated abdominal fluid lipase or amylase compared with serum supports a diagnosis of pancreatitis. elevated lactate compared with serum lactate or an abdominal fluid glucose less than 50 mg/dl is highly sensitive and specific for bacterial/ septic peritonitis. the presence of bile pigment or bacteria is supportive of bile and septic peritonitis, respectively. free fibers in abdominal fluid along with clinical signs of abdominal pain strongly support gastrointestinal perforation, and immediate surgical exploration is required. text continued on p. 93 the following are clinical conditions, patient signalment, common history, physical examination, and characteristic findings of various diagnostic tests. a blank column next to a condition indicates no specific signalment, history, physical examination, or diagnostic test characteristic for a particular disease process. lack of contiguity of body wall surgical ( medical unless perforation present present c-shaped abnormal gas pattern with plication on radiographs surgical (immediate) dilation of bowel cranial to foreign object, radiopaque object in surgical (immediate) stomach or intestines, hypochloremic metabolic acidosis on bloodwork if pyloric outflow obstruction is present elevated or decreased wbc; foreign material, wbcs and medical unless perforation bacteria on abdominal fluid, elevated lactate and decreased present glucose on abdominal fluid target shaped soft tissue density on abdominal u/s, soft tissue surgical (immediate): density with gas dilation cranially on abdominal radiographs medical management of primary cause colonic distension with hard feces on radiographs medical increased or decreased wbc, septic abdominal effusion surgical (immediate) elevated t bili, alt, alk phos, and wbc hypoechoic hepatic medical after biopsy parenchyma on ultasound hepatomegaly elevated t bili, alt, alk phos, and wbc hyperechoic foci in surgical (immediate) gallbladder or sludge on u/s, free gas in wall of gall bladder abdominal effusion, bile pigment in effusion surgical (immediate) elevated t bili, alk phos, alt surgical (immediate) elevated or decreased wbc, elevated t bili, alk phos and surgical (immediate) alt, free gas in hepatic parenchyma on rads, hypoechoic mass with hyperechoic material in hepatic parenchyma on u/s heteroechoic liver with hyperechoic center on ultrasound surgical (immediate) mixed echogenic mass on ultrasound, soft tissue mass surgical (immediate or density on radiographs, elevated alk phos, alt, delayed) t bili, hypoglycemia pain-cont'd elevated t bili, alk phos, alt, amylase and/or lipase, elevated medical in most cases or decreased wbc, hypocalcemia, focal loss of detail in right unless abscess or cranial quadrant on radiographs hypo-to hyperechoic phlegmon is present pancreas with hyperechoic peri-pancreatic fat on ultrasound, abdominal and/or pleural effusion on radiographs and ultrasound pancreatic soft tissue mass effect on radiographs and surgical if mass identified, ultrasound, elevated amylase and lipase, hypoglycemia, otherwise medical elevated serum insulin management of hypoglycemia splenomegaly on radiographs, hyperechoic spleen with no surgical (immediate) blood flow on ultrasound soft tissue mass effect and loss of abdominal detail on surgical (immediate) radiographs, cavitated mass with abdominal effusion on u/s hyperechoic spleen with no blood flow on abdominal u/s, surgical (immediate) abdominal effusion, thrombocytopenia loss of abdominal detail on radiographs, peritoneal effusion medical unless refractory on u/s, hemoabdomen on abdominocentesis hypotension diagnosis based primarily on clinical signs medical fracture of the os penis on radiographs largely medical unless urethral tear diagnosis based primarily on clinical signs medical, although prepuce may need to be incised to allow replacement of penis into sheath prostatomegaly on radiographs and ultrasound hypoechoic medical prostate on u/s, pyuria and bacteriuria and u/a prostatomegaly on radiographs and ultrasound hypo-to surgical (delayed) hyperechoic prostate on u/s, bacteriuria and pyuria on u/a prostatomegaly on radiographs and ultrasound, prostatic medical/surgical mineralization on radiographs and ultrasound hypoechoic kidneys on u/s, pyuria on u/a, elevated wbc, medical azotemia pyuria, bacteriuria on u/a medical pyelectasia in abdominal u/s, azotemia surgical (immediate) renomegaly on radiographs, azotemia renal mass on u/s, renomegaly on radiographs surgical (immediate) renal mass on u/s, azotemia, lack of renal blood flow surgical (delayed) on u/s calculi in renal pelvis on radiographs and ultrasound, azotemia medical unless both kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, medical unless both azotemia kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, surgical (delayed until fluid or soft tissue density on u/s, azotemia electrolyte stabilization) diagnosis largely based on physical examination medical unless cannot pass findings urethral catheter azotemia, no peritoneal effusion, lack of urine output or surgical (delayed until outflow with ureteral catheterization, double contrast electrolyte stabilization) cystourethrogram indicated transitional cellular casts on u/a, hematuria, mass effect or surgical and medical thickened irregular urethra on ultrasound or management cystourethrogram hypoechoic swollen testicle on testicular ultrasound surgical (immediate) fluid or gas-filled tubular structure on abdominal ultrasound or surgical (immediate) abdominal radiographs soft tissue tubular structure on radiographs, fluid-filled uterus surgical ( in the event of a negative abdominocentesis, but peritoneal effusion or bile or gastrointestinal perforation are suspected, perform a diagnostic peritoneal lavage. peritoneal dialysis kits are commercially available but are often expensive and impractical (see p. 6). animals that have acute abdominal pain can be divided into three broad categories, depending on the primary cause of pain and the initial definitive treatment (table 1-24) . some diseases warrant a nonsurgical, medical approach to case management. other conditions require immediate surgery following rapid stabilization. other conditions initially can be managed medically until the patient is hemodynamically more stable and then may or may not require surgical intervention at a later time. specific management of each disease entity is listed under its own subheading. box 1-23 lists specific indications for exploratory laparotomy. the best means to explore the abdominal cavity accurately and thoroughly is to open the abdomen on midline from the level of the xyphoid process caudally to the pubis for full exposure and then to evaluate all organs in every quadrant in a systematic manner. address specific problems such as gastric or splenic torsion, enteroplication, and foreign body removal, and then copiously lavage the abdomen with warmed sterile saline solution. suction the saline solution thoroughly from the peritoneal cavity so as to not impair macrophage function. in cases of septic peritonitis, the abdomen may be left open, or a drain may be placed for further suction and lavage. the routine use of antibiotics in irrigation solutions is contraindicated because the antibiotics can irritate the peritoneum and delay healing. when the abdominal cavity is left open, secure sterile laparotomy towels and water-impermeable dressings over the abdominal wound with umbilical tape, and then change these daily or as strike-through occurs. open abdomen cases are often effusive and require meticulous evaluation and management of electrolyte imbalances and hypoalbuminemia. the abdomen can be closed and/or the abdominal drain removed when the volume of the effusion decreases, when bacteria are no longer present, and when the neutrophils become more healthy in appearance. bischoff mg: radiographic techniques and interpretation of the acute abdomen, clin tech small anim pract 18 (1) anaphylactic shock occurs as an immediate hypersensitivity reaction to a variety of inciting stimuli (box 1-24). in animals, the most naturally occurring anaphylactic reaction results from wasp or bee stings. most other reactions occur as a result of an abnormal sensitivity to items used in making medical diagnoses or treatment. during an anaphylactic reaction, activation of c5a and the complement system results in vascular smooth muscle dilation and the release of a cascade of inflammatory mediators, including histamine, slow-reacting substance of anaphylaxis, serotonin, heparin, acetylcholine, and bradykinin. clinical signs associated with anaphylaxis differ between dogs and cats. in dogs, clinical signs may include restlessness, vomiting, diarrhea, hematochezia, circulatory collapse, coma, and death. in cats, clinical signs often are associated with respiratory system abnormalities. clinical signs may include ptyalism, pruritus, vomiting, incoordination, bronchoconstriction, pulmonary edema and hemorrhage, laryngeal edema, collapse, and death. the most important steps to remember in any emergency is to follow the abcs of airway, breathing, and circulation. first, establish an airway through endotracheal intubation or emergency tracheostomy, if necessary. concurrently, an assistant should establish vascular or intraosseous access to administer drugs and fluids (box 1-25). the patient should be hospitalized until complete resolution of clinical signs. after initial stabilization and treatment, it is important to maintain vascular access and continue intravenous fluid therapy until the patient is no longer hypotensive, and vomiting and diarrhea have resolved. in cases of fulminant pulmonary hemorrhage and edema, administer supplemental oxygen until the patient is no longer hypoxemic or orthopneic on room air. normalize and maintain blood pressure using positive inotropes (dobutamine, 3-10 âµg/kg/ minute cri) or pressors (dopamine, 3 to 10 âµg/kg/minute iv cri; see shock). if bloodtinged vomitus or diarrhea has been observed, administer antibiotics to decrease the risk of bacterial translocation and sepsis (cefoxitin, 22 mg/kg iv tid; metronidazole, 10 mg/kg iv tid). also consider using gastroprotectant drugs (famotidine, 0.5 to 1.0 mg/kg iv; ranitidine, 0.5 to 2.0 mg/kg po, iv, im bid; sucralfate, 0.25 to 1.0 g po tid; omeprazole, 0.7 to 1.0 mg/kg po sid). a second and less serious form of allergic reaction is manifested as angioneurotic edema and urticaria. in most cases, clinical signs develop within 20 minutes of an inciting allergen. although this type of reaction causes patient discomfort, it rarely poses a life-threatening problem. most animals have mild to severe swelling of the maxilla and periorbital regions. the facial edema also may be accompanied by mild to severe generalized urticaria. some animals may paw at their face, rub at their eyes, or have vomiting or diarrhea. the treatment for angioneurotic edema involves suppressing the immune response by administration of short-acting glucocorticoid drugs and blocking the actions of histamine by the synergistic use of histamine 1 and histamine 2 receptor blockers (box 1-26). in some cases, the inciting cause is a known recent vaccination or insect sting. many times, however, the inciting cause is not known and is likely an exposure to a stinging insect or arachnid. differential diagnoses for acute facial swelling and/or urticaria include acetaminophen toxicity (cats), anterior caval syndrome, lymphadenitis, vasculitis, hypoalbuminemia, and contact dermatitis. observe animals that have presented for angioneurotic edema for a minimum of 20 to 30 minutes after injection of the short-acting glucocorticoids and antihistamines. monitor blood pressure to make sure that the patient does not have concurrent anaphylaxis and hypotension. after partial or complete resolution of clinical signs, the animal can be discharged to its owner for observation. in dogs, mild vomiting or diarrhea may occur within 1 to 2 days after this type of reaction. wherever possible, exposure to the inciting allergen should be avoided. â�¢ administer short-acting glucocorticoid: complications observed while a patient is under anesthesia can be divided into two broad categories: (1) those related to equipment malfunction or human error and (2) the patient's physiologic response to the cardiorespiratory effects of the anesthetic drugs. careful observation of the patient and familiarity with anesthetic equipment, drug protocols, and monitoring equipment is necessary for the safest anesthesia to occur. despite this, however, anesthetic-related complications are frequent and need to be recognized and treated appropriately. many anesthetic drugs have a dose-dependent depressive effect on the respiratory system and cause a decrease in respiratory rate and tidal volume, leading to hypoventilation. respiratory rate alone is not a reliable indicator of the patient's oxygenation and ventilatory status. the respiratory tidal volume can be measured with a wright's respirometer. perform pulse oximetry and capnography as noninvasive measures of the patient's oxygenation and ventilation. ventilation can be impaired as a result of anesthetic drugs, patient position, pneumothorax, pleural effusion (chylothorax, hemothorax, pyothorax), equipment malfunction, rebreathing of carbon dioxide, thoracic wall injury, or alveolar fluid (pulmonary edema, hemorrhage, or pneumonia). problems such as a diaphragmatic hernia, gdv, or gravid uterus can impede diaphragmatic excursions once the patient is placed on its back and can lead to impaired ventilation. the work of breathing also may be increased because of increased resistance of the anesthesia circuit and increased dead space ventilation. this is particularly important in small toy breeds. clinical signs of inadequate ventilation and respiratory complications include abnormal respiratory pattern, sudden changes in heart rate, cardiac dysrhythmias, cyanosis, and cardiopulmonary arrest. end-tidal carbon dioxide, or capnography, gives a graphic display of adequacy of ventilation. rapid decreases in end-tidal carbon dioxide can be caused by disconnection or obstruction of the patient's endotracheal tube or poor perfusion, namely, cardiopulmonary arrest (see capnometry [end-tidal carbon dioxide monitoring]). postoperatively, hypoventilation can occur because of the residual effects of the anesthetic drugs, hypothermia, overventilation during intraoperative support, surgical techniques that compromise ventilation (thoracotomy, cervical disk surgery, atlantooccipital stabilization), postoperative bandaging of the abdomen or thorax, ventilatory muscle fatigue, or injury to the cns. cardiac output is a function of heart rate and stroke volume. factors that influence stroke volume include vascular and cardiac preload, cardiac afterload, and cardiac contractility. the patient's cardiac output can be affected adversely by the negative inotropic and chronotropic and vasodilatory effects of anesthetic drugs, all leading to hypotension. 96 1 emergency care bradycardia, tachycardia, cardiac dysrhythmias, and vascular dilation can lead to hypotension and inadequate organ perfusion. table 1 -25 lists the normal heart rate and blood pressure in dogs and cats. bradycardia is defined as a heart rate below normal values. many anesthetic drugs can cause bradycardia. causes of bradycardia include the use of narcotics or î± 2 -agonist drugs, deep plane of anesthesia, increased vagal tone, hypothermia, and hypoxia. table 1 -26 lists the causes of bradycardia and the necessary immediate action or treatment. tachycardia is defined as a heart rate above normal values. common causes of tachycardia include vasodilation, drugs, inadequate anesthetic depth and perceived pain, hypercapnia, hypoxemia, hypotension, shock, or hyperthermia. table 1 -27 lists the causes and immediate action or treatment for tachycardia. hypotension is defined as physiologically low blood pressure (mean arterial pressure less than 65 mm hg). a mean arterial blood pressure less than 60 mm hg can result in inadequate tissue perfusion and oxygen delivery. the coronary arteries are perfused during diastole. inadequate diastolic blood pressure, less than 40 mm hg, can cause decreased coronary artery perfusion and myocardial hypoxemia that can predispose the heart to dysrhythmias. causes of perianesthetic hypotension include peripheral vasodilation by anesthetic drugs, bradycardia or tachyarrhythmias, hypothermia, inadequate cardiac preload from vasodilation or hemorrhage, decreased venous return from patient position or surgical manipulation of viscera, and decreased cardiac contractility. electrocardiogram monitoring is useful for the early detection of cardiac dysrhythmias during the perianesthetic period. clinical signs of cardiac dysrhythmias include irregular pulse rate or pressure, abnormal or irregular heart sounds, pallor, cyanosis, hypotension, and an abnormal ecg tracing. remember that the single best method of detecting cardiac 98 1 emergency care vagolytic drugs atropine allow time for the drug to wear off. glycopyrrolate allow time for the drug to wear off. sympathomimetic drugs epinephrine allow time for the drug to wear off; administer a î²-blocker; turn off infusion. isoproterenol administer a î²-blocker. turn off infusion; administer a î²-blocker. allow time for drug to wear off. inadequate anesthetic depth increase anesthetic depth. hypercapnia increase ventilation (assisted ventilation). hypoxemia increase gas flow and oxygenation. hypotension decrease anesthetic depth; administer an intravenous crystalloid or colloid bolus, positive inotrope drug, positive chronotrope drug, or pressor. hyperthermia apply ambient or active cooling measures; administer dantrolene sodium if malignant hyperthermia is suspected. hypothermia provide ambient rewarming. hypocalcemia * administer calcium chloride (10 mg/kg iv) or calcium gluconate (23 mg/kg). decrease vaporizer setting/anesthetic depth. reverse with opioids or a 2 -agonists. vasodilation administer an intravenous crystalloid bolus (10 ml/kg). administer an intravenous colloid bolus (5 ml/kg). administer a pressor (epinephrine, phenylephrine dysrhythmias is with your fingertips (palpate a pulse or apex heartbeat) and ears (auscultate the heart). confirm the dysrhythmia by auscultating the heart rate and rhythm, identify the p waves and the qrs complexes, and evaluate the relationship between the p waves and qrs complexes. is there a p wave for every qrs, and a qrs for every p wave? during anesthesia, fluid, acid-base, and electrolyte imbalances can predispose the patient to dysrhythmias. sympathetic and parasympathetic stimulation, including the time of intubation, can predispose the patient to dysrhythmias. if the patient's plane of anesthesia is too light, perception of pain can cause catecholamine release, sensitizing the myocardium to ectopic beats. atrioventricular blockade can be induced with the administration of î± 2 -agonist medications, including xylazine and medetomidine. thiobarbiturates (thiopental) can induce ventricular ectopy and bigeminy. although these dysrhythmias may not be harmful in the awake patient, anesthetized patients are at a particular risk of dysrhythmia-induced hypotension. carefully monitor and treat all dysrhythmias (see cardiac dysrhythmias). box 1-27 lists steps to take to prevent perianesthetic dysrhythmias. awakening during anesthesia can occur and can be caused by equipment failure and simply, although no one likes to admit it, human error. table 1 -29 lists causes of arousal during anesthesia and appropriate immediate actions. awaken patient, and administer dantrolene arousal (e.g., malignant hyperthermia) sodium. â�¢ stabilize acid-base and electrolyte balance before anesthetic induction, whenever possible. â�¢ rehydrate patient before anesthetic induction. â�¢ select anesthetic agents appropriate for the particular patient. â�¢ be aware of the effects of the drugs on the myocardium. â�¢ ensure adequate anesthetic depth and oxygenation before anesthetic induction. â�¢ ensure ventilatory support during anesthesia. â�¢ monitor heart rate, rhythm, blood pressure, pulse oximetry, and capnometry during anesthesia. â�¢ ensure adequate anesthetic depth before surgical stimulation. â�¢ avoid surgical manipulation to the heart or great vessels, whenever possible. â�¢ avoid changes in perianesthetic depth. â�¢ avoid hypothermia. delayed recovery can be caused by a number of factors, including excessive anesthetic depth, hypothermia, residual action of narcotics or tranquilizers, delayed metabolism of anesthetic drugs, hypoglycemia, hypocalcemia, hemorrhage, and breed or animal predisposition. careful monitoring of the patient's blood pressure, acid-base and electrolyte status, anesthetic depth, pcv, and vascular volume intraoperatively and taking care with supportive measures to prevent abnormalities can hasten anesthetic recovery and avoid postoperative complications. gaynor the presentation of a patient with a bleeding disorder often is a diagnostic challenge for the veterinary practitioner (boxes 1-28 and 1-29). in general, abnormal bleeding can be caused by five major categories: (1) vascular trauma, (2) circulating inhibitors of coagulation heparin fibrin degradation products development of spontaneous deep hematomas, unusually prolonged bleeding after traumatic injury, bleeding at multiple sites throughout the body involving multiple organ systems, delayed onset of severe hemorrhage after bleeding, and an inability on the practitioner's part to find an organic cause of bleeding. the signalment, history, clinical signs, and results of coagulation often can aid in making a rapid diagnosis of the primary cause of the disorder and in the selection of appropriate case management. when taking a history, ask the following important questions: â�¢ what is the nature of the bleeding? â�¢ what sites are affected? â�¢ how long has the bleeding been going on? â�¢ has your animal had any previous or similar episodes? â�¢ is there any possibility of any toxin exposure? â�¢ if so, when and how much did your animal consume? â�¢ is there any possibility of trauma? â�¢ does your animal run loose outdoors unattended? â�¢ have you ever traveled, and if so, where? â�¢ has your animal been on any medications recently or currently? â�¢ has your animal been vaccinated recently? â�¢ have any known relatives of your animal had any bleeding disorders? â�¢ are there any other abnormal signs that you have seen? abnormalities found on physical examination may aid in determining whether the hemorrhage is localized or generalized (i.e., bleeding from a venipuncture site versus bleeding diathesis). note whether the clinical signs are associated with a platelet problem and superficial hemorrhage or whether deep bleeding can be associated with abnormalities of the coagulation cascade. also, make an attempt to identify any concurrent illness that can predispose the patient to a bleeding disorder (i.e., pancreatitis, snakebite, sepsis, immunemediated hemolytic anemia, or severe trauma and crush or burn injury). abnormalities associated with coagulopathies include petechiae and ecchymoses, epistaxis, gingival bleeding, hematuria, hemarthrosis, melena, and hemorrhagic cavity (pleural and peritoneal or retroperitoneal) effusions. disseminated intravascular coagulation is a complex syndrome that results from the inappropriate activation of the clotting cascade, leading to disruption of the normal balance between thrombosis and fibrinolysis. the formation of diffuse microthrombi with concurrent consumption of platelets and activated clotting factors leads to end-organ thrombosis with various degrees of clinical hemorrhage. in animals, dic always results from some other pathologic process, including various forms of neoplasia, crush and heat-induced injury, sepsis, inflammation, and immune-mediated disorders (box 1-30). the pathophysiologic mechanisms involved in dic include vascular endothelial damage, activation and consumption of platelets, release of tissue procoagulants, and consumption of endogenous anticoagulants. because dic always results from some other disease process, diagnosis of dic is based on a number of criteria when evaluating various coagulation tests, peripheral blood smears, platelet count, and end products of thrombosis and fibrinolysis. there is no one definitive criterion for the diagnosis of dic (box 1-31). thrombocytopenia occurs as platelets are consumed during thrombosis. it is important to remember that trends in decline in platelet numbers are just as important as thrombocytopenia when making the diagnosis. in some cases the platelet count still may be within the normal reference range but has significantly decreased in the last 24 hours. early in dic the procoagulant cascade dominates, with hypercoagulability. activated clotting time, aptt, and pt may be rapid and shorter than normal. in most cases, we do not recognize the hypercoagulable state in our critically ill patients. later in dic, as platelets and activated clotting factors become consumed, the act, aptt, and pt become prolonged. antithrombin, a natural anticoagulant, also becomes consumed, and antithrombin levels decline. antithrombin levels can be measured at commercial laboratories and in some large veterinary institutions. the end products of thrombosis and subsequent fibrinolysis also can be measured. fibrinogen levels may decline, although this test is not sensitive or specific for dic. fibrin degradation (split) products also become elevated. fibrin degradation products are normally cleared by the liver, and these also become elevated in cases of hepatic failure because of lack of clearance. more recently, cageside d-dimer tests have become available to measure the breakdown product of cross-linked fibrin as a more sensitive and specific monitor of dic. management of dic first involves treating the primary underlying cause. by the time dic becomes evident, rapid and aggressive treatment is necessary. if you are suspicious of dic in any patient with a disease known to incite dic, then ideally, you should begin treatment before the hemostatic abnormalities start to occur for the best possible prognosis. treatment involves replacement of clotting factors and antithrombin and prevention of further clot formation. to replenish clotting factors and antithrombin, administer fresh whole blood or fresh frozen plasma. heparin requires antithrombin as a cofactor to inactivate thrombin and other activated coagulation factors. administer heparin (50 to 100 units/kg sq q6-8h of unfractionated heparin; or fractionated enoxaparin [lovenox], 1 mg/kg sq bid). aspirin (5 mg/kg po bid in dogs; every third day in cats) also can be administered to prevent platelet adhesion. management of dic also involves the rule of twenty monitoring and case management to maintain end-organ perfusion and oxygen delivery (see the rule of 20). hemophilia a is a sex-liked recessive trait that is carried by females and manifested in males. female hemophiliacs can occur when a hemophiliac male is bred with a carrier female. hemophilia a has been reported in cats and a number of dog breeds, including miniature schnauzer, saint bernard, miniature poodle, shetland sheepdog, english and irish setters, labrador retriever, german shepherd, collie, weimaraner, greyhound, chihuahua, english bulldog, samoyed, and vizsla. mild to moderate internal or external bleeding can occur. clinical signs of umbilical cord bleeding can become apparent in some animals shortly after weaning. gingival hemorrhage, hemarthrosis, gastrointestinal hemorrhage, and hematomas may occur. clotting profiles in animals with factor viii deficiency include prolonged aptt and act. the pt and buccal mucosa bleeding time are normal. affected animals have low factor viii activity but normal to high levels of factor viii-related antigen. carrier females can be detected by low (30% to 60% of normal) factor viii activity and normal to elevated levels of factor vii-related antigen. von willebrand's disease is a deficiency or defect in von willebrand's protein. a number of variants of the disease have been described: von willebrand's disease type i is associated with a defect in factor viir/protein concentration, and von willebrand's disease type ii is associated with a defect in viiir:vwf. type i von willebrand's disease is most common in veterinary medicine. von willebrand's disease has been identified in more than 29 breeds of dogs, with an incidence that varies from 10% to 60% depending on the breed of origin. affected breeds include doberman pinchers, german shepherd dogs, scottish terriers and standard manchester terriers, golden retrievers, chesapeake bay retrievers, miniature schnauzers, and pembroke welsh corgis. two forms of genetic expression occur: (1) autosomal recessive disease in which homozygous von willebrand's disease individuals have a bleeding disorder, whereas heterozygous individuals carry the trait but are clinically normal. the second variant of genetic expression involves an autosomal dominant disease with incomplete expression such that heterozygous individuals are affected carriers and homozygous individuals are severely affected. von willebrand's disease has high morbidity, but fortunately a low mortality. dogs with 30% or less than normal vwf tend to hemorrhage. platelet counts are normal, but bleeding times can be prolonged. the aptt can be slightly prolonged when factor viii is less than 50% of normal. routine screening tests are nondiagnostic for this disease, although in a predisposed breed with a normal platelet count, a prolonged buccal mucosa bleeding time strongly supports a diagnosis of von willebrand's disease. documentation of clinical bleeding with low or undetectable levels of factor viii antigen or platelet-related activities of vwf support a diagnosis of von willebrand's disease. recessive animals have zero vwf:antigen (a subunit of factor iii); heterozygotes have 15% to 60% of normal. in the incompletely dominant form, levels of vwf antigen are reduced (less than 7% to 60%). clinical signs in affected animals include epistaxis, hematuria, diarrhea with melena, penile bleeding, lameness, hemarthrosis, hematoma formation, and excessive bleeding with routine procedures such as nail trimming, ear cropping, tail docking, surgical procedures (spay, neuter), and lacerations. estrous and postpartum bleeding may be prolonged. a dna test to detect carriers of the vwf gene is available through vetgen (ann arbor, michigan) and michigan state university. patients with von willebrand's disease should avoid drugs known to affect platelet function adversely (sulfonamide, ampicillin, chloramphenicol, antihistamines, theophylline, phenothiazine tranquilizers, heparin, and estrogen). hemophilia b is an x-linked recessive trait that occurs with less frequency that hemophilia a. the disease has been reported in scottish terriers, shetland and old english sheepdogs, saint bernards, cocker spaniels, alaskan malamutes, labrador retrievers, bichon frises, airdale terriers, and british shorthair cats. carrier females have low (40% to 60% of normal) factor ix activity. clinical signs are more severe than for hemophilia a. congenital deficiencies of factor vii have been reported as an autosomal, incompletely dominant characteristic in beagles. heterozygotes have 50% factor vii deficiency. bleeding tends to be mild. the pt is prolonged in affected individuals. factor x deficiency has been documented in cocker spaniels and resembles fading-puppy syndrome in newborn dogs. internal or umbilical bleeding can occur, and affected dogs typically die. bleeding may be mild in adult dogs. in severe cases, factor x levels are reduced to 20% of normal; in mild cases, factor x levels are 20% to 70% of normal. factor xii deficiency has been documented as an inherited autosomal recessive trait in domestic cats. heterozygotes can be detected because they have a partial deficiency (50% of normal) of factor xii. homozygote cats have less than 2% factor xii activity. deficiency of hageman factor usually does not result in bleeding or other disorders. factor xi deficiency is an autosomal disease that has been documented in kerry blue terriers, great pyrenees, and english springer spaniels. in affected individuals, protracted bleeding may be observed. homozygotes have low factor xi activity (< 20% of normal), and heterozygotes have 40% to 60% of normal. the management of congenital defects of hemostasis typically involves replenishing the clotting factor that is present. usually, this can be accomplished in the form of fresh frozen plasma transfusion (20 ml/kg). if anemia is present because of severe hemorrhage, fresh whole blood or packed rbcs also can be administered. recent research has investigated the use of recombinant gene therapy in the treatment of specific factor deficiencies in dogs; however, the therapy is not yet available for use in clinical practice. in cases of von willebrand's disease, administration of fresh frozen plasma (10 to 20 ml/kg) or cryoprecipitate (1 unit/10 kg body mass) provides vwf, factor viii, and fibrinogen. doses can be repeated until hemorrhage ceases. 1-desamino-8-d-arginine vasopressin (ddavp) also can be administered (1 âµg/kg sc or iv diluted in 0.9% saline given over 10 to 20 minutes) to the donor and patient to increase the release of stored vwf from endothelial cells. a fresh whole blood transfusion can be obtained from the donor and immediately administered to the patient, or spun down and the fresh plasma administered if rbcs are not needed. administer a dose of ddavp to any affected dog before initiating any elective surgical procedures. a supply of fresh frozen plasma and rbcs should be on hand, should uncontrolled hemorrhage occur. platelets are essential to normal blood coagulation. after a vessel is damaged, release of vasoactive amines causes vasoconstriction and sluggish flow of blood in an attempt to squelch hemorrhage. platelets become activated by platelet activating factor, and attach to the damaged vascular endothelium. normal platelet adhesion depends on mediators such as calcium, fibrinogen, vwf:antigen, and a portion of factor viii. after adhesion, the platelets undergo primary aggregation and release a variety of chemical mediators including adenosine diphosphate, prostaglandins, serotonin, epinephrine, thromboplastin, and thromboxane a that promote secondary aggregation and contraction. platelet abnormalities can include decreased platelet production (thrombocytopenia), decreased platelet function (thrombocytopathia), increased platelet destruction, increased platelet consumption, and platelet sequestration. thrombocytopathia refers to platelet function abnormalities. alterations in platelet function can affect platelet adhesion, aggregation, or release of vasoactive substances that help form a stable clot (box 1-32). in von willebrand's disease there is a deficiency in vwf:antigen that results in altered platelet adhesion. vascular purpuras are reported and have been seen in collagen abnormalities such as ehlers-danlos syndrome, which can be inherited as an autosomal dominant trait with complete penetrance and has been recognized in german shepherd dogs, dachshunds, saint bernards, and labrador retrievers. thrombasthenic thrombopathia is a hereditary autosomal dominant abnormality that has been described in otterhounds, foxhounds and scottish terriers. in this condition, platelets do not aggregate normally in response to adenosine diphosphate and thrombin stimulation. evaluation of platelet function is based on a total platelet count, buccal mucosa bleeding time, and thromboelastography. platelet function defects (thrombocytopenia and thrombocytopathia) can affect both sexes. clinical signs can resemble von willebrand's disease. in most cases, buccal mucosa bleeding time will be prolonged, but platelet count and clotting tests will be normal. platelet count can be decreased because of problems with production, increased consumption, sequestration, or destruction. causes of accelerated platelet destruction are typically immune-mediated autoantibodies, drug antibodies, infection, and isoimmune destruction. consumption and sequestration usually are caused by dic, vasculitis, microangiopathic hemolytic anemia, severe vascular injury, hemolytic uremic syndrome, and gram-negative septicemia. primary thrombocytopenia with no known cause has been called idiopathic thrombocytic purpura. in approximately 80% of the cases, thrombocytopenia is associated with immune-mediated destruction caused by immune-mediated hemolytic anemia, systemic lupus erythematosus, rheumatoid arthritis, dic, and diseases that affect the bone marrow. in systemic lupus erythematosus, 20% to 30% of the affected dogs have concurrent idiopathic thrombocytic purpura. when immune-mediated hemolytic anemia and idiopathic thrombocytic purpura are present in the same patient, the disease is called evans syndrome. pf-3 is a non-complement-fixing antibody that is produced in the spleen and affects peripheral and bone marrow platelets and megakaryocytes. antibodies directed against platelets are usually of the igg subtype in animals. antiplatelet antibodies can be measured by a pf-3 release test. platelet counts with immune-mediated destruction typically are less than 50,000 platelets/âµl. infectious causes of thrombocytopenia include ehrlichia canis, anaplasma phagocytophilum (formerly, ehrlichia equi), and rickettsia rickettsii (rocky mountain spotted fever). primary immune-mediated thrombocytopenia has an unknown cause and most frequently is seen in middle-to older-aged female dogs. breed predispositions include cocker spaniels, german shepherd dogs, poodles (toy, miniature, standard), and old english sheepdogs. thrombocytopenia usually is manifested as petechiae, ecchymoses of skin and mucous membranes, hyphema, gingival and conjunctival bleeding, hematuria, melena, and epistaxis. to make a diagnosis of idiopathic thrombocytic purpura, measure the severity of thrombocytopenia (< 50,000 platelets/âµl), analyze the peripheral blood smear for evidence of platelet fragmentation or microthrombocytosis, normal to increased numbers of megakaryocytes in the bone marrow, detection of antiplatelet antibody, increased platelet counts after starting glucocorticoid therapy, and elimination of other causes of thrombocytopenia. if tick-borne illnesses are suspected, antibody titers for e. canis, a. phagocytophilum (formerly e. equi), and r. rickettsii should be performed. treatment of immune-mediated thrombocytopenia involves suppression of the immune system to stop the immune-mediated destruction and to stimulate platelet release from the bone marrow. traditionally, the gold standard to suppress the immune system is to use glucocorticoids (prednisone or prednisolone, 2 to 4 mg/kg po bid divided, or dexamethasone, 0.1 to 0.3 mg/kg iv or po q12h). more recently human serum immunoglobulin (igg) also has been used (0.2 to 0.5 g/kg iv in saline over 8 hours; pretreat with 1 mg/kg diphenhydramine 15 minutes before starting infusion). vincristine (0.5 mg/m 2 iv once) can stimulate the release of platelets from the bone marrow if megakaryocytic precursors are present; however, the platelets released may be immature and potentially nonfunctional. treatment with fresh whole blood or packed rbcs is appropriate if anemia is present; however, unless specific platelet-rich plasma has been purchased from a blood bank, fresh whole blood contains relatively few platelets, which are shortlived (2 hours) and will not effectively raise the platelet count at all. finally, long-term therapy is usually in the form of azathioprine (2 mg/kg po once daily, tapered to 1 mg/kg daily to every other day after 1 week) and cyclosporine (10 to 25 mg/kg po divided). if a tickborne illness is suspected, administer doxycycline (5 to 10 mg/kg po bid) for 4 weeks or if titers come back negative. thrombocytopenia also can occur in the cat. causes for thrombocytopenia in cats include infections (29%), neoplasia (20%), cardiac disease (7%), primary immune-mediated disease (2%), and unknown causes (20%). in one study of cats with feline leukemia and myeloproliferative disease, 44% of cases had thrombocytopenia. warfarin and coumarin derivatives are the major class of rodenticides used in the united states. vitamin k antagonist rodenticides inhibit the epoxidase reaction and deplete active vitamin k, causing a depletion of vitamin k-dependent coagulation factors (ii, vii, ix, x) within 24 hours to 1 week of ingestion, depending on the ingested dose. affected animals can spontaneously hemorrhage anywhere in the body. clinical signs can include hemoptysis, respiratory difficulty, cough, gingival bleeding, epistaxis, hematuria, hyphema, conjunctival bleeding, petechiae and ecchymoses, cavity hemorrhage (pleural, peritoneal, retroperitoneal) with acute weakness, lethargy or collapse, hemarthrosis with lameness, deep muscle bleeds, and intracranial or spinal cord hemorrhage. diagnosis of vitamin k antagonism includes prolonged pt. a pivka (protein induced by vitamin k absence or antagonism) test also can be performed, if possible. treatment of vitamin k antagonist rodenticide intoxication and other causes of vitamin k deficiency involves supplementation with vitamin k 1 (phytonadione, 5 mg/kg sq once with 25-gauge needle in multiple sites, and then 2.5 mg/kg po bid to tid for 30 days). never administer injections of vitamin k intramuscularly, because of the risk of causing deep muscle hematomas, or intravenously, because of the risk of anaphylaxis. the pt should be rechecked 2 days after the last vitamin k capsule is administered, for some of the secondgeneration warfarin derivates are fat-soluble, and treatment may be required for an additional 2 weeks. act, activated clotting time; aptt, activated partial thromboplastin time; bmbt, buccal mucosa bleeding time; fdp, fibrin degradation products; n, normal; pt, prothrombin time. thermal burns are fortunately a relatively infrequent occurrence in veterinary patients. box 1-33 lists various causes of malicious and accidental burns. the location of the burn is also important in assessing its severity and potential to lose function. burns on the perineum, feet, face, and ears are considered to be the most severe because of loss of function and severe pain. often the severity of thermal injury is difficult to assess in animals because hair coat potentially can mask clinical signs and because the thermal injury can continue after the animal has been removed from the heat source. the skin cools slowly and warms slowly, considerations that become important when initiating therapy for burns. the severity of thermal injury is associated with the temperature to which the animal is exposed, the duration of contact, and the ability of the tissue to dissipate heat. the tissue closest to the heat source undergoes necrosis and has decreased blood flow. the severity of thermal burn injury is associated directly with the temperature to which the animal is exposed, the percentage of total body surface area affected, the thickness of injured tissue, and whether underlying complications with other body systems occur. prognosis largely depends on the total body surface area affected (table 1-31) . superficial partial thickness, or first-degree, burns offer the most favorable prognosis. the affected epidermis initially appears erythematous and then quickly desquamates within 3 to 6 days. in most cases, fur grows back without leaving a scar. deep partial thickness, or second-degree, burns involve the epidermis and dermis and are associated with subcutaneous edema, inflammation, and pain. deep partial thickness burns heal from deeper adnexal tissues and from the wound edges and are associated with an increased chance of scarring and depigmentation. the most severe type is known as full thickness, or third-degree, burns, in which thermal injury destroys the entire thickness of the skin and forms an eschar. thrombosis of superficial and deeper skin vasculature and gangrene occurs. treatment involves sequential wound debridement. healing occurs by second intention and reepithelialization or by wound reconstruction. in most cases, scarring is extensive in affected areas. burns greater than 20% of total body surface area will have systemic effects, including impaired cardiovascular function, pulmonary dysfunction, and impaired immune function. burned tissue, with capillary damage, has increased permeability. the release of inflammatory cytokines, oxygen-derived free radical species, prostaglandins, leukotrienes, 108 1 emergency care histamine, serotonin, and kinins results in increased vascular permeability and leakage of plasma proteins into the interstitium and extravascular space. at the time of presentation, first examine the patient and ascertain whether airway obstruction, impaired ventilatory function, circulatory shock, or pain are present. if necessary, establish an airway with endotracheal intubation or emergency tracheostomy. next, cool the burned area(s) with topical cool water. use care to avoid overcooling and iatrogenic hypothermia. the best approach is to cool only one portion of the patient's body at a time, then dry, and repeat the process for all affected areas to avoid overcooling and iatrogenic hypothermia. establish vascular access and administer appropriate and judicious analgesic drugs and intravenous fluid therapy. whenever possible, avoid placing a catheter through an area of burned or damaged skin. in the early stages of burn injury, shock doses of intravenous crystalloid fluids usually are not required. later, however, as severe tissue exudation occurs, protein and fluid losses can become extensive, requiring aggressive crystalloid and colloid support to treat hypovolemia and hypoproteinemia. flush the eyes with sterile saline and examine behind the third eyelids for any particulate matter. stain the corneas to make sure that superficial corneal burns are not present. treat superficial corneal burns with triple antibiotic ophthalmic ointment. next, assess the total body surface area affected, as this will gauge prognosis. depending on the extent of the damage, decide whether the burn is superficial and local therapy is indicated or whether more severe injuries exist that may involve systemic therapy or possibly euthanasia. in most cases the diagnoses of thermal burns are based on a clinical history of being in a house fire, clothes dryer, or under a heating lamp. too frequently, however, thermal burns become apparent days after an elective surgical procedure in which the patient was placed on a faulty heating pad rather than a circulating warm water or warm air blanket. superficial burns appear as singed fur with desquamating, easily epilated hair. this condition also can resemble a superficial or deeper dermatophytosis if history is unknown. other differential diagnoses include immune-mediated vasculitis or erythema multiforme. unless the superficial dermis is blistered, it may be difficult to distinguish between a thermal burn, chemical burn, or electrical burn if the trauma went unnoticed. management of burn injury largely depends on the depth of injury and the total body surface area affected. partial thickness burns and those affecting less than 15% of the total body surface area will require support in the form of antibiotic ointment and systemic analgesic drugs. burns affecting greater than 15% of total body surface area or deep thickness burns require more aggressive therapy. central venous catheters can be placed to administer crystalloid and colloid fluids, parenteral nutrition if necessary, antibiotics, and analgesic drugs. monitor perfusion parameters closely, including heart rate, blood pressure, capillary refill time, and urine output. respiratory function can be impaired because of concurrent smoke inhalation, thermal damage to the upper airways and alveoli, and carboxyhemoglobin or methemoglobin intoxication. respiratory function also can be impaired because of burn injury to the skin around the thoracic cage. thoracic radiographs may reveal patchy interstitial to alveolar infiltrates associated with pulmonary edema, pneumonia, and atelectasis. bronchoscopy often reveals edema, inflammation, particulate matter, and ulceration of the tracheobronchial tree. in some cases, upper airway inflammation is so severe that an emergency tracheostomy must be performed to treat airway obstruction. administer supplemental humidified oxygen at 50 to 100 ml/kg/minute via endotracheal tube, tracheostomy, nasal or intratracheal tube, or hood oxygen if respiratory function and hypoxemia are present. perform blood work including a hematocrit, albumin, bun, creatinine, and glucose at the time of presentation. monitor serum electrolytes, albumin, and colloid oncotic pressure closely because derangements can be severe as burns become exudative. the goal of fluid therapy in the burn patient is to establish and maintain intravascular and interstitial fluid volume, normalize electrolyte and acid-base status, and maintain serum albumin and oncotic pressure. in the first 24 hours following burn injury, direct fluid therapy to maintaining the patient's metabolic fluid requirements. crystalloid fluids in the form of normosol-r, plasmalyte-m, or lactated ringer's solution can be administered according to the patient's electrolyte and acid-base status (see fluid therapy). monitor urine output, and keep it at 1 to 2 ml/kg/hour. avoid overhydration in the early stages of burn injury. in affected burn patients, calculate the amount of fluid that should be administered over a 24-hour period from the formula 1 â�� 4 ml/kg ã� percent total body surface area. administer half of this calculated dose over the first 8 hours and then the remaining half over the next 16 hours. in cats, administer only 50% to 75% of this calculated volume. to administer this volume and also avoid fluid overload is often difficult in critically ill patients with pulmonary involvement associated with smoke inhalation injury. avoid colloids in the first 6 hours after burn injury. monitor the patient closely for serous nasal discharge, chemosis, and rales that may signify pulmonary edema. as burns become exudative, weigh the patient at least twice daily. infused fluid should equal fluid output in the form of urine and wound exudates. acute weight loss signifies acute fluid loss and that crystalloid fluid infusion should be more aggressive. ideally, keep the patient's serum albumin equal to or greater than 2.0 g/dl and total protein between 4.0 and 6.5 g/dl using a combination of fresh frozen plasma or concentrated human albumin. adjunct colloidal support can be provided with synthetic colloids including hetastarch or hbocs. keep serum potassium within 3.5 to 4.5 meq/l using potassium chloride or potassium phosphate supplementation. if potassium supplementation exceeds 80 to 100 meq/l and the patient continues to have severe refractory hypokalemia, administer magnesium chloride (0.75 meq/kg/day) to enhance potassium retention. if anemia occurs, administer packed rbcs or whole blood (see blood component therapy). lavage wounds daily with lactated ringer's solution or 0.9% sodium chloride solution. place wet-to-dry bandages or bandages soaked in silver sulfadiazine or nitrofurazone ointment over the wounds. depending on the thickness of the burn, epilation and eschar formation and separation may take 2 to 10 days. at each bandage change, debride devitalized tissue to normal tissue. perform staged partial or total escharectomy, and leave the wound to heal by second intention or by reconstruction using skin advancement flaps or grafts. maintain meticulous sterility at all times, given that burn patients are at high risk for infection. administer broad-spectrum antibiotics including cefazolin and enrofloxacin. perform wound culture if a resistant bacterial infection is suspected. the most common cause of electrical injury is associated with an animal chewing on low-voltage alternating current electrical cords in the household. damage is caused by the current flowing through the path of least resistance, causing heat and thrombosis of vessels and neurons. in some cases, the owner witnesses the event. in other cases, the owner presents the patient because of vague nonspecific signs, and characteristic abnormalities on physical examination support a diagnosis of electrocution. burns on the face, paws, commissures of the mouth, tongue, and soft palate may be present. electrocution causes a massive release of catecholamines and can predispose the patient to noncardiogenic pulmonary edema within 36 hours of the incident. clinical signs may be isolated to the pulmonary system, including orthopnea, pulmonary crackles, and cyanosis. assess the patient's lips, tongue, soft palate, gingivae, and commissures of the mouth. early after electrocution, the wound may appear small and white, black, or yellow. later, the wound may become larger as tissue sloughs because of damaged vascular supply. assess the patient's respiratory status. auscultate the lungs to determine whether pulmonary crackles 110 1 emergency care are present. if the patient is stable, thoracic radiographs may demonstrate an interstitial to alveolar lung pattern in the dorsocaudal lung fields. measure the patient's heart rate, blood pressure, oxygenation as determined by pulse oximetry or arterial blood gas and urine output. immediate treatment consists of judicious use of analgesics for the burn injury, antibiotics (cefazolin, 22 mg/kg q8h; cephalexin, 22 mg/kg q8h), and humidified supplemental oxygen (50 to 100 ml/kg/minute). direct fluid therapy at providing the patient's metabolic fluid requirements. because of the risk of development of noncardiogenic pulmonary edema, avoid overzealous administration of crystalloid fluids. differential diagnoses for the patient with electrical burn injury and electrocution include chemical or thermal burn, immune-mediated glossitis, cardiogenic pulmonary edema, and pneumonia. management of the patient with electrical burn injury and electrocution primarily involves the administration of analgesic agents, supplemental humidified oxygen, and topical treatment of electrical burns. the noncardiogenic pulmonary edema is typically unresponsive to diuretics (i.e., furosemide), bronchodilators (i.e., aminophylline), and splanchnic vascular dilators (i.e., low-dose morphine). the use of glucocorticoids has no proven benefit and may impair respiratory immune function and is therefore contraindicated. oral burns may require debridement and advancement flaps if large defects or oronasal fistulas develop. if oral injury is severe, place an esophagostomy or percutaneous gastrostomy tube to ensure adequate nutrition during the healing process. if an animal survives the initial electrocution, prognosis is generally favorable with aggressive supportive care. chemical burns are associated with a number of inciting causes, including oxidizing agents, reducing agents, corrosive chemicals, protoplasmic poisons, desiccants, and vesicants. the treatment for chemical burns differs slightly from that for thermal burns, so it remains important to investigate the cause of the burn when providing initial treatment, whenever possible. at the scene, advise the owner to wrap the patient in a clean towel for transport. chilling can be avoided by then wrapping the patient in a second or third blanket. placement of ointments by well-doers should be avoided. encourage immediate transport to the nearest triage facility. the first and foremost consideration when treating a patient with chemical burn is to remove the animal from the inciting cause or offending agent. make no attempt to neutralize alkaline or acid substances because the procedure potentially could cause an exothermic reaction, leading to thermal injury in addition to the chemical injury. remove collars or leashes that may act as tourniquets or constricting devices. flush affected areas with copious amounts of cool water for several minutes, not cooling more than 10% to 20% of the body at any one time to prevent iatrogenic hypothermia. support breathing by extending the patient's head and neck. carefully clip the fur over affected areas for further evaluation of the extent of the injury. lavage exposed eyes with sterile saline, and stain the cornea to evaluate for any corneal burns. debride any wounds carefully, knowing that the full extent of the wound may not manifest itself for several days. then cover the wounds with antibiotic burn ointment such as silver sulfadiazine and an occlusive dressing. without a history of exposure, the differential diagnosis for any chemical burn includes thermal burn, necrotizing vasculitis, erythema multiforme, or superficial or deep pyoderma. contact local or national animal poison control regarding whether to attempt neutralization. perform daily bandage changes with staged debridement as the full extent of the wound manifests itself. place antimicrobial ointment and silver sulfadiazine ointment over the wound to prevent infection. the routine use of antibiotics may promote the development of a resistant bacterial infection. first-generation cephalosporin can be administered. if a more serious infection develops, perform culture and susceptibility testing to direct appropriate antibiotic therapy. the wound can heal by second intention or may require reconstructive repair for definitive closure. the primary cause of radiation injury in small animal patients is radiation therapy for neoplastic conditions. the goal of radiation therapy is to kill neoplastic cells. an unfortunate side effect is damage to adjacent normal tissue that results in necrosis, fibrosis, and impaired circulation to the affected area. radiation burns result in dermatitis, mucositis, impaired surgical wound healing, and chronic nonhealing wounds. in many cases, the degree of secondary radiation injury to normal tissue can be prevented or decreased with careful radiation planning and mapping of the radiation field, such that radiation exposure to normal tissue is limited to the smallest extent possible. with the advent of three-dimensional imaging modalities such as computed tomography (ct) and magnetic resonance imaging (mri), this has become more routine in veterinary oncology to date. radiation injury can be early and appear at the later stage of the course of radiation therapy. late effects can be delayed and occur 6 months to years after treatment. the degree of radiation injury is categorized based on the depth of tissue affected. first-degree changes cause cutaneous erythema. second-degree changes cause superficial desquamation. thirddegree changes cause deeper moist desquamation, and fourth-degree changes are associated with complete dermal destruction and ulceration. during the early stages of radiation injury, affected tissues may appear erythematous and edematous. wound exudates may be moist, or the skin may appear dry and scaly with desquamation or ulceration. later, the area may scar and depigment or may have induration, atrophy, telangiectasia, keratosis, and decreased adnexal structures. treatment for radiation dermatitis is to irrigate the area with warmed saline and to protect the area from self-mutilation. no-bite, or elizabethan, collars or loose clothing can be used to protect the area for patient-induced injury. mucositis can be treated with topical green tea baths and the administration of an oral solution of l-glutamine powder (4 g/m 2 ). local irrigation of xylocaine or lidocaine viscous jelly can be used in dogs but should be avoided in cats because of the risk of inducing hemolytic anemia and neurotoxicity. topical and systemic antibiotics (cephalexin, 22 mg/kg po tid) also can be administered. avoid antibiotics that can be sensitized by radiation (i.e., metronidazole). because most radiation burns are associated with a known exposure to radiation therapy, the cause of the patient's injury usually is known. if an animal presents to you with a scar, however, differential diagnoses may include nasal planum solar dermatitis, pemphigus foliaceus, discoid lupus, superficial necrolytic dermatitis, superficial or deep pyoderma, chemical burn, or thermal burn. treatment of radiation injury involves making the patient as comfortable as possible with analgesic drugs, prevention of self-mutilation, and staged debridement techniques. wounds can heal by second intention or may require reconstructive surgery. distress syndrome (ards), and anesthetic agents. the acute onset of bradycardia, change in mucous membrane color and capillary refill time, change in respiratory pattern, and change in mentation are signs of possible deterioration and impending cardiopulmonary arrest. the diagnosis of cardiopulmonary arrest is based on the absence of effective ventilation, severe cyanosis, absence of a palpable pulse or apex heartbeat, absence of heart sounds, and ecg evidence of asystole or other nonperfusing rhythm such as electricalmechanical dissociation (aka pulseless electrical activity) or ventricular fibrillation. the goals of cpcr are to obtain airway access, provide artificial ventilation and supplemental oxygen, implement cardiac compressions and cardiovascular support, recognize and treat dysrhythmias and arrhythmias, and provide stabilization and treatment for cardiovascular, pulmonary, and cerebral function in the event of a successful resuscitation. even with aggressive treatment and management, the overall success of cpcr is less than 5% in critically ill or traumatized patients and 20% to 30% in anesthetized patients. basic life support involves rapid intubation to gain airway access, artificial ventilation, and cardiac compressions to promote blood flow and delivery of oxygen to the brain and other important tissues (figure 1-26 ). perform the abcs or cabs of cpcr, where a is airway, b is breathing, and c is compression and circulation. recently, the paradigm has shifted to cabs. while a team member is grabbing an endotracheal tube, clearing the airway of foreign debris, and establishing airway access through endotracheal intubation, a second person starts external cardiac compressions to deliver oxygen that is in the bloodstream to the vital organs. the patient should be positioned in dorsal (> 7 kg) or lateral (< 7 kg) recumbency for external cardiac compressions. approximately 80 to 120 external compressions should be performed over the patient's sternum. a team member should palpate for a peripheral pulse to determine whether cardiac compressions are actually effective. if a peripheral pulse cannot be palpated for every chest compression, change the patient's position and have a larger individual perform compressions, or initiate open-chest cardiac resuscitation. once the patient is intubated, tie in the endotracheal tube and attach it to an oxygen source (anesthetic machine or mechanical ventilator or ambu bag) for artificial ventilation. the oxygen flow rate should be 150 ml/kg/minute. give two long breaths, and then 12 to 16 breaths per minute. simultaneous ventilation with thoracic compression increases the pressure difference in the thorax and allows more forward flow of oxygenated blood through the great vessels into the periphery. if possible, a third team member can initiate interposed abdominal compressions, compressing the abdomen when the thoracic cage is relaxed, to improve forward flow. if only one person is available to perform the thoracic compressions and ventilation, give two breaths for every 15 compressions (i.e., 15 thoracic compressions followed by two long breaths, and then start thoracic compressions again). the jen chung maneuver can be performed by placing a 25-to 22-gauge hypodermic needle through the skin of the nasal philtrum and twisting the needle into the periosteum to stimulate respirations. this maneuver appears to work better in cats than dogs at return to spontaneous respiration. advanced life support during cpcr involves ecg, pulse oximetry and capnometry monitoring, administration of drugs, and the administration of intravenous fluids (in select cases). most of the drugs used during cpcr can be administered directly into the lungs from the endotracheal tube (intratracheal tube). therefore, only in select instances is it necessary to establish vascular or intraosseous access during cpcr (figure 1-27) . if an animal experiences cardiopulmonary arrest because of extreme hemorrhage or hypovolemia, inappropriate vasodilation caused by sepsis or systemic inflammation, or vasodilation resulting from anesthesia, the administration of shock volumes (90 ml/kg/hour in dogs and 44 ml/kg/hour in cats) is appropriate. if a patient is euvolemic and experiences cardiopulmonary arrest, however, an increase in circulating fluid volume actually can impair coronary artery perfusion by increasing diastolic arterial blood pressure and is asystole is one of the most common rhythm disturbances that causes cardiac arrest in small animal patients. one of the most important things to do when the ecg looks like asystole is to make sure that the ecg monitor is working properly and that all ecg leads are attached properly to the patient. if asystole is truly present, reverse any opiate, î± 2 -agonist, or benzodiazepine drugs with their appropriate reversal agents. lowdose epinephrine (0.02 to 0.04 mg/kg diluted with 5 ml sterile saline) can be administered directly into the endotracheal tube via a rigid or red rubber catheter. if vascular access is available, epinephrine (0.02 to 0.04 mg/kg) can be administered intravenously. no drug should ever be administered directly into the heart by intracardiac injection. unless the heart is in the veterinarian's hand during open-chest cpcr, intracardiac injection is risky and potentially could lacerate a coronary artery or cause the myocardium to become more irritable and refractory to other therapies, if a drug is delivered into the myocardium and not into the ventricle. for these reasons, intracardiac injections are contraindicated. administer atropine (0.4 mg/kg iv, io, or 0.4 mg/kg it) immediately after the epinephrine. atropine, a vagolytic drug, serves to decrease tonic vagal inhibition of the sinoatrial and atrioventricular node and increase heart rate. administer atropine and epinephrine every 2 to 5 minutes during asystole while cardiac compressions, interposed abdominal compressions, and artificial ventilation are continued. although discontinuation of thoracic compressions can decrease the chance of success during cpcr, you must intermittently evaluate the ecg monitor for any rhythm change that may require different drug therapies. if the cardiac arrest was not witnessed or more than 2 to 5 minutes have passed without successful return to a perfusing rhythm, perform open-chest cpcr, if the client wishes. administer sodium bicarbonate (1 to 2 meq/kg iv) every 10 to 15 minutes during cpcr. sodium bicarbonate is the only drug used in cpcr that should not be administered intratracheally because of inactivation of pulmonary surfactant. electrical-mechanical dissociation also is known as pulseless electrical activity and is an electrical rhythm that may look wide and bizarre and irregular with no associated mechanical contraction of the ventricles. the rhythm can appear different from patient to patient. electrical-mechanical dissociation is one of the more common nonperfusing rhythms observed during cardiopulmonary arrest in small animal patients (figure 1-28) . when electrical-mechanical dissociation is identified, first confirm the rhythm and proceed with cpcr as previously described. electrical-mechanical dissociation is thought to be associated with high doses of endogenous endorphins and high vagal tone. the treatment of choice for electrical-mechanical dissociation is high-dose atropine (4 mg/kg iv, it [10 times the normal dose]) and naloxone hydrochloride (0.03 mg/kg iv, io, it). administer epinephrine (0.02 to 0.04 mg/kg diluted in 5 ml sterile 0.9% saline it). if the rhythm does not change within 2 minutes, consider open-chest cardiac massage. ventricular fibrillation can be coarse (figure 1-29) . patients with coarse ventricular fibrillation are easier to defibrillate than those with fine defibrillation. if ventricular fibrillation is identified, initiate cpcr as described previously (figure 1-30) . if an electrical defibrillator is available, administer 5 j/kg of direct current externally. when a patient in cardiopulmonary arrest is attached to ecg leads, it is important to use contact electrode paste, water-soluble gel such as ky jelly, or water, rather than any form of alcohol. electrical defibrillation of a patient who has alcohol on the ecg leads can lead to fire and thermal burns. reverse any opioid, î± 2 -agonist, and phenothiazine drugs that have been administered to the patient. if fine ventricular fibrillation is identified, administer epinephrine 1 figure 1 -28: electrical-mechanical dissociation (emd), also known as pulseless electrical activity (pea). the complexes often appear wide and bizarre without a palpable apex beat or functional contraction of the heart. this is just one example of emd, as many shapes and complexes may be observed. organized according to whether an electrical defibrillator is available. after each intervention step, the ecg should be reevaluated and the next step initiated if v-fib is still seen. if a new arrhythmia develops, the appropriate therapy for that rhythm should be inititated. if a sinus rhythm is seen with a palpable apex beat, postresuscitation measures should be implemented. perform open-chest cpcr immediately if a pathologic condition exists that prevents enough of a change in intrathoracic pressure that closed-chest cpcr will not be effective in promoting forward blood flow (box . to perform open-chest cpcr, place the patient in right lateral recumbency. clip a wide strip of fur over the left fifth to seventh intercostal space and quickly aseptically scrub over the clipped area. using a no. 10 scalpel blade, incise over the fifth intercostal space through the skin and subcutaneous tissue to the level of the intercostal muscles. with a mayo scissors, make a blunt stab incision through the intercostal muscles in the left sixth intercostal space. make sure that the person who is breathing for the patient deflates the lungs as you make the stab incision to avoid iatrogenic lung puncture. after the stab incision, open the tips of the mayo scissors and quickly open the muscle dorsally and ventrally to the sternum with a sliding motion. avoid the internal thoracic artery at the sternum and the intercostal arteries at the caudal aspect of each rib. cut the rib adjacent to the sternum and push it behind the rib in front of and at the caudal aspect of the incision to allow more room and better visualization if a rib spreading retractor is not available. visualize the heart in the pericardial sac. visualize the phrenic nerve, and incise the pericardium just ventral to the phrenic nerve. make sure to not cut the phrenic nerve. grasp the heart in your hand(s) and gently squeeze it from apex to base, allowing time for the ventricle to fill before the next "contraction." if the heart does not seem to be filling, administer fluids intravenously or directly into the right atrium. the descending aorta can be cross-clamped with a rummel tourniquet or red rubber catheter to improve perfusion to the brain and heart. postresuscitation care and monitoring (prolonged life support) postresuscitation care involves careful monitoring and management of the adverse effects of hypoxia and reperfusion injury on the brain and other vital organs. the first 4 hours after an arrest are most critical, because this is the time period in which an animal is most likely to rearrest unless the underlying cause of the initial arrest has been determine and treated (table 1 -32) . until an animal is adequately ventilating on its own, artificial ventilation by manual bagging or attaching the patient to a mechanical ventilator with supplemental oxygen must continue. the efficacy of oxygenation and ventilation can be monitored using a wright's respirometer, pulse oximetry, capnometry, and arterial blood . once an animal is extubated, administer supplemental oxygen (50 to 100 ml/ kg/minute) (see oxygen supplementation). the brain is sensitive to ischemia and reperfusion injury. the effects of cellular hypoxia and reperfusion include the development of oxygen-derived free radical species that contribute to cerebral edema. administer mannitol (0.5 to 1 g/kg iv over 5 to 10 minutes), followed by furosemide (1 mg/kg iv) 20 minutes later, to all patients that have experienced cardiopulmonary arrest and have had successful resuscitation. mannitol and furosemide work synergistically to decrease cerebral edema formation and scavenge oxygen-derived free radical species. the combination of cardiac arrest, myocardial ischemia and acidosis, and external or internal cardiac compressions often make the myocardium irritable and predisposed to dysrhythmias following successful cpcr. start lidocaine (1 to 2 mg/kg iv, followed by 50 to 100 âµg/kg/minute iv cri) in all patients following successful resuscitative efforts. monitor the ecg continuously for the presence of cardiac dysrhythmias and recurrence of nonperfusing rhythms. perform direct or indirect blood pressure monitoring. if a patient's systolic blood pressure is less than 80 mm hg, diastolic pressure is less than 40 mm hg, or mean arterial blood pressure is less than 60 mm hg, administer positive inotropic drugs (dobutamine, 1 to 20 âµg/kg/minute) and pressor agents (epinephrine, 0.02 to 0.04 mg/kg iv, io, it) to improve cardiac contractility, cardiac output, and core organ perfusion. the kidneys are sensitive to decreased perfusion and cellular hypoxia. place a urinary catheter and monitor urine output. in a euvolemic patient, normal urine output should be no less than 1 to 2 ml/kg/hour. if urine output is low, administer low-dose dopamine (3 to 5 âµg/kg/minute iv cri) in an attempt to dilate afferent renal vessels and improve renal perfusion. maintain acid-base and electrolyte status within normal reference ranges. monitor serum lactate as a rough indicator of organ perfusion and cellular oxygen extraction. the presence of elevated or rising serum lactate in the face of aggressive cardiorespiratory and cerebral support makes prognosis less favorable. cole sg, otto cm, hughes d: cardiopulmonary cerebral resuscitation: a clinical practice review part i, j vet emerg crit care 12 (4) immediate action depends largely on recognition of the primary or secondary cause of the dysrhythmia and treating the dysrhythmia and underlying cause. diagnosis of cardiac dysrhythmias is based on physical examination findings of abnormal thoracic/cardiac auscultation, the presence of abnormal pulse rhythm and quality, and recognition of ecg abnormalities. the ecg is critical to the accurate diagnosis of dysrhythmias. ventricular dysrhythmias arise from ectopic foci in the ventricles that cause the wave of depolarization to spread from cell to cell rather than spread through fast-conducting tissue. this causes the qrs complex to appear wide and bizarre, unless the ectopic focus originates close to the atrioventricular node high in the ventricle. other ecg features of ventricular dysrhythmias include a t wave polarity that is opposite to the qrs complex and nonrelated p waves. ventricular dysrhythmias may manifest as isolated ventricular premature complexes, couplets, or triplets; bigeminy; or ventricular tachycardia. relatively slow ventricular tachycardia is known as an idioventricular rhythm and is not as hemodynamically significant as faster ventricular tachycardia. idioventricular rhythm usually is less than 130 beats per minute and may alternate spontaneously with sinus arrhythmias (figures 1-31 to . supraventricular dysrhythmias arise from ectopic foci in the atria and are commonly associated with atrial dilatation and structural heart disease such as advanced acquired or congenital heart disease, cardiomyopathies, cardiac neoplasia, or advanced heartworm disease. occasionally, supraventricular dysrhythmias may be associated with respiratory or other systemic illness. sustained supraventricular tachycardia in the absence of underlying structural heart or systemic disease is disturbing and should alert the clinician that an accessory pathway conduction disturbance may be present, particularly in labrador retrievers. supraventricular dysrhythmias can manifest as isolated premature complexes (atrial premature complexes or contractions), sustained or paroxysmal supraventricular tachycardia (atrial tachycardia), or atrial fibrillation or flutter. in the dog, atrial fibrillation most commonly is associated with dilative cardiomyopathy. rarely and primarily in giant breed dogs, lone atrial fibrillation can occur with no underlying heart disease. atrial fibrillation and the resultant sustained elevation in ventricular rate are presumed to progress to dilative cardiomyopathy in such breeds. by comparison, atrial fibrillation is relatively uncommon in cats because of the small size of their atria but is associated most commonly with hypertrophic and restrictive cardiomyopathy. the ecg is critical to the diagnosis of a supraventricular dysrhythmia. the ecg usually demonstrates a normal appearance to the qrs complex unless aberrant conduction occurs in the ventricles, in which case the qrs can be wide but still originate from above the atrioventricular node. in most cases of a supraventricular dysrhythmia, some evidence of atrial activity including p waves, atrial flutter, or atrial fibrillation is apparent. in some cases, it may be difficult to diagnose the exact rhythm without slowing the rate down mechanically or through pharmacologic intervention. once a rhythm diagnosis is made, appropriate treatment strategies can be implemented (figures 1-35 and 1-36 ). treatment of ventricular dysrhythmias largely depends on the number of ectopic foci discharging, the rate and character of the dysrhythmia, and whether the presence of the abnormal beats is of adverse hemodynamic consequence, including risk of sudden death. many ventricular dysrhythmias, including slow idioventricular rhythms, ventricular bigeminy, or intermittent ventricular premature complexes, do not warrant antiarrhythmic therapy unless the patient is hypotensive and the dysrhythmia is thought to be contributing to the hypotension. in such cases, correction of the underlying disease process including hypoxia, pain, or anxiety often alleviates or decreases the incidence of the dysrhythmia. more serious ventricular dysrhythmias that warrant antiarrhythmic therapy (table 1 -33) include sustained ventricular tachycardia (>160 beats/minute in dogs; >220 beats/minute in cats), multifocal ventricular premature complexes originating from more than one place in the ventricles, and the presence of r-on-t phenomena where the t wave of the preceding complex is superimposed on the qrs of the next complex with no return to isoelectric shelf in between complexes. treat these ventricular dysrhythmias immediately and aggressively. in dogs, the mainstay of emergency treatment for ventricular dysrhythmias is lidocaine therapy. administer lidocaine (1 to 2 mg/kg iv bolus) over a period of 5 minutes to prevent the adverse side effects of seizures or vomiting. the bolus can be repeated an additional 3 times (total dose 8 mg/kg) over 15 minutes, or the patient can be placed on a constant rate infusion (50 to 100 âµg/kg/minute) if control of ventricular tachycardia is accomplished. also correct the patient's magnesium and potassium deficiencies to maximize the success of lidocaine therapy in the treatment of ventricular tachycardia. procainamide (4 mg/kg iv slowly over 3 to 5 minutes) also can be used to control ventricular tachycardia. if procainamide is successful at controlling ventricular tachycardia, administer it as a constant rate infusion (25 to 40 âµg/kg/minute). side effects of procainamide include vomiting, diarrhea, and hypotension. chronic oral therapy may or may not be necessary in the treatment of acute ventricular tachycardia. the decision to continue antiarrhythmic therapy depends on the underlying disease process and the expectation of persistent arrhythmogenesis of the underlying disease process. oral antiarrhythmic therapy is warranted in cases in which a serious ventricular dysrhythmia is recognized but the animal does not require hospitalization, such as the syncopal boxer with intermittent ventricular dysrhythmias and no evidence of structural heart disease. it deserves emphasis that asymptomatic, low-grade ventricular dysrhythmias probably do not require treatment. if maintenance therapy for ventricular dysrhythmias is needed, use an oral drug based on the underlying disease process, clinical familiarity, class of drug, dosing frequency, owner compliance, concurrent medications, cost, and potential adverse side effects. in the cat the mainstay of antiarrhythmic therapy is the use of a î²-adrenergic antagonist. in the acute management of ventricular dysrhythmias in cases of hypertrophic, restrictive, or unclassified cardiomyopathies, consider using injectable esmolol (0.05 to 1.0 mg/kg iv slowly to effect) or propranolol (0.02 to 0.06 mg/kg iv slowly to effect), particularly if the dysrhythmia results from hyperthyroidism. for chronic oral ventricular antiarrhythmic therapy in cats, propranolol (2.5 to 5.0 mg po per cat q8h) or atenolol (6.25 to 12.5 mg po per cat q12-24h) can be used. the decision to treat supraventricular dysrhythmias depends on the ventricular rate and the hemodynamic consequences of the dysrhythmia. for intermittent isolated atrial 124 1 emergency care procainamide 10-20 mg/kg po q6-8h tocainide* 10-20 mg/kg po q8h sotalol 40-120 mg per dog q12h (start low, then titrate up to effect) mexiletine 5-8 mg/kg po q8h atenolol 0.25-1.0 mg/kg po q12-24h (start low, titrate upward to effect) *do not use for longer than 2 weeks because of idiosyncratic blindness. premature contractions, couplets, and triplets, usually no treatment is required. when the ventricular rate exceeds 180 beats/minute, diastolic filling time is shortened, causing the heart to not fill adequately. the consequence is decreased cardiac output and decreased coronary artery perfusion. the goal of therapy is rhythm control or, in most cases, rate control. in cases of atrial fibrillation and congestive heart failure, conversion to a normal sinus rhythm rarely can be achieved, although electrocardioversion or pharmacoconversion can be attempted. in the dog a vagal maneuver can be attempted by pressing on the eyeballs or massaging the carotid body. for sustained supraventricular tachycardia, diltiazem (0.25 mg/kg iv), esmolol (0.05 to 0.1, titrated upward to a cumulative dose of 0.5 mg/kg iv), or propranolol (0.04 to 0.1 mg/kg iv slowly to effect) can be administered in an attempt to slow the ventricular rate in emergent situations. administer oral diltiazem (0.5 mg/kg po q8h), diltiazem (dilacor-xr) (1.5 to 6 mg/kg po q12-24h), propranolol (0.1 to 0.2 mg/kg tid, titrated up to a maximum of 0.5 mg/kg po q8h), atenolol (0.25 to 1 mg/kg q12-24h), or digoxin (0.005 to 0.01 mg/kg bid or 0.22 mg/m 2 for dogs greater than 15 kg). in the cat a vagal maneuver can be attempted by ocular or carotid massage. (diltiazem [dilacor] 30 to 60 po q12-24h), propranolol (2.5 to 10 mg/kg q12-24h), or atenolol (6.25 mg q12-24h) also can be administered. if structural heart disease is present, treat pulmonary edema and start angiotensin-converting enzyme inhibitor therapy. table 1 -34 summarizes the drugs used in the management of supraventricular dysrhythmias. severe bradycardia often results from systemic disease, drug therapy, anesthetic agents, or hypothermia and thus rarely requires specific therapy except to treat or reverse the underlying mechanisms promoting bradycardia. hemodynamically significant bradyarrhythmias that must be treated include atrial standstill, atrioventricular block, and sick sinus syndrome. atrial standstill most commonly is associated with hyperkalemia and is seen most often in urinary obstruction, renal failure, urinary trauma with uroabdomen, and hypoadrenocorticism. characteristic ecg abnormalities observed in atrial standstill are an absence of p waves, widened qrs complexes, and tall spiked t waves (figure 1-37 ). the treatment for hyperkalemia-induced atrial standstill is to correct the underlying cause and to drive potassium intracellularly and protect the myocardium from the adverse effects of hyperkalemia. regular insulin (0.25 to 0.5 units/kg iv) followed by dextrose (1 g/unit insulin iv, followed by 2.5% dextrose cri to prevent hypoglycemia) or sodium bicarbonate (1 meq/kg iv) can be administered to drive potassium intracellularly. calcium gluconate (0.5 ml/kg of 20% solution iv over 5 minutes) also can be administered as a cardioprotective drug until the cause of hyperkalemia has been identified and resolved. also administer sodium chloride fluids (0.9% sodium chloride iv) to promote kaliuresis. less commonly, atrial standstill is associated with atrial cardiomyopathy or silent atrium syndrome. persistent atrial standstill has been recognized without electrolyte abnormalities in the english springer spaniel and the siamese cat. short-term therapy for persistent atrial standstill includes atropine (0.04 mg/kg sq) until definitive treatment by implantation of a cardiac pacemaker can be performed. complete or third-degree atrioventricular block or high-grade symptomatic seconddegree atrioventricular block can be hemodynamically significant when ventricular rates are less than 60 beats/minute in the dog. classic clinical signs include weakness, exercise intolerance, lethargy, anorexia, syncope, and occasionally seizures. advanced atrioventricular block usually is caused by advanced idiopathic degeneration of the atrioventricular node. less commonly, atrioventricular block has been associated with digoxin toxicity, magnesium oversupplementation, cardiomyopathy, endocarditis, or infectious myocarditis (lyme disease). an accurate diagnosis is made based on the ecg findings of nonconducted p waves with ventricular escape beats. first-and second-degree atrioventricular block may not be hemodynamically significant and therefore may not require therapy. initially treat third-degree (complete) or symptomatic high-grade second-degree atrioventricular block (<60 beats/minute) with atropine (0.04 mg/kg sq or im). perform a follow-up ecg in 15 to 20 minutes. atropine is rarely successful in treating complete atrioventricular block. also attempt treatment with isoproterenol (0.04 to 0.08 âµg/kg/minute iv cri or 0.4 mg in 250 ml 5% dextrose in water iv slowly), a pure î²-agonist. definitive treatment requires permanent pacemaker implantation. consultation with a veterinary cardiologist who implants pacemakers is suggested. never attempt to convert or treat the observed ventricular escape beats with lidocaine ( figure 1-38) . sick sinus syndrome most commonly is recognized in the miniature schnauzer, although any dog can be affected. sick sinus syndrome usually results from idiopathic degeneration of the sinus node in the dog. in the cat, sinus node degeneration usually is associated with cardiomyopathy. dysfunction of the sinus node may manifest as marked bradycardia with periods of sinus arrest followed by junctional or ventricular escape complexes. a variant of sick sinus syndrome is the presence of severe bradycardia followed by periods of supraventricular tachycardia, often termed bradycardia-tachycardia syndrome. the most common clinical signs are syncope, exercise intolerance, and lethargy. in cats, hypertrophic cardiomyopathy is the most common form of acquired cardiac disease observed. congestive heart failure resulting from hypertrophic cardiomyopathy can occur in animals as young as 6 to 10 months of age. hypertrophic cardiomyopathy is characterized by stiff, noncompliant ventricles that do not relax during diastole, causing an increase in left atrial pressures and left atrial enlargement. other cardiomyopathies, including unclassified, restrictive, and dilated, are less common but also can occur in the cat. cats often develop acute exacerbation of clinical signs because of stress or arterial embolization. the rapid diagnosis of chf often is made on owner history, signalment, and physical examination findings (box 1-36). typical physical examination findings include a cardiac murmur or gallop dysrhythmia, abnormal breath sounds, respiratory difficulty and orthopnea, tachycardia, weak pulse quality, cool peripheral extremities, and pale or cyanotic mucous membrane. initiate immediate treatment based on physical examination findings and index of suspicion. in some cases, it is difficult to distinguish between chf and feline lower airway disease (asthma) without performing thoracic radiographs. let the animal rest and become stabilized before attempting any stressful procedures, including thoracic radiographs. immediate treatment consists of administering supplemental oxygen, decreasing circulating fluid volume with furosemide, dilating pulmonary and splanchnic capacitance vessels with topical nitroglycerine and morphine, and alleviating patient anxiety and stress (box 1-37). primary differential diagnoses are made based primarily on the patient's breed, age, clinical signs, history, and physical examination abnormalities. the most common differential diagnoses in a patient with chf are cardiac abnormalities and respiratory disease (chronic bronchitis [asthma], pulmonary hypertension, cor pulmonale, neoplasia). postpone diagnostic tests in any patient with suspected chf until the immediate treatments have taken effect and the patient is cardiovascularly more stable. in most cases, lateral and dorsoventral thoracic radiographs are one of the most important diagnostic tools in helping make a diagnosis of chf. increased perihilar interstitial to alveolar infiltrates are characteristic of pulmonary edema. left atrial enlargement may be observed as a "backpack" sign at the caudal cardiac waist. cardiomegaly of the right or left side also may 128 be present in cases of valvular insufficiency. in cats, increased sternal contact and a classic valentine-shaped heart may be observed in cases of hypertrophic cardiomyopathy. perform a vertebral heart score (sum) to measure cardiac size and determine whether cardiomegaly is present (box 1-38). also obtain arterial blood pressure and ecg readings to determine whether hypotension and dysrhythmias are present. atrial fibrillation, ventricular premature contractions, and supraventricular tachycardia are common rhythm disturbances that can affect cardiac output adversely and influence treatment choices. the echocardiogram is a useful noninvasive and nonstressful method to determine the degree of cardiac disease present. the echocardiogram is largely user-dependent. the quality of the study is based on the experience of the operator and the quality of the ultrasound machine. echocardiography can be a useful tool in making a diagnosis of pericardial effusion, dilated or hypertrophic cardiomyopathy, cardiac neoplasia, and endocarditis. the medical management of chf is designed to improve cardiac output and relieve clinical signs. the immediate goal of therapy is to reduce abnormal fluid accumulation and provide adequate cardiac output by increasing contractility, decreasing preload and ventricular afterload, and/or normalizing cardiac dysrhythmias. strict cage rest is of utmost importance when managing a patient with chf. after initial administration of furosemide, morphine, oxygen, and nitroglycerine paste, clinical signs of respiratory distress should show improvement within 30 minutes. if no improvement is observed, administer repeated doses of furosemide. reevaluate severe cases that are refractory to this standard treatment protocol. vasodilation should be the next step in the management of refractory cases, provided that a normal blood pressure is present. sodium nitroprusside is a potent balanced vasodilator that should be administered (1 to 10 âµg/kg/minute iv cri), taking care to monitor blood pressure continuously because severe vasodilation and hypotension can occur. the goal of nitroprusside therapy is to maintain a mean arterial blood pressure of 60 mm hg. sodium nitroprusside should not be considered in cases of refractory chf with severe hypotension. for more long-term management of chf, the use of angiotensin-converting enzyme (ace) inhibitors including enalapril (0.5 mg/kg po q12-24h), benazepril (0.5 mg/kg po q24h), and lisinopril (0.5 mg/kg po q24h) have become the mainstay of therapy to reduce sodium and fluid retention and decrease afterload. start angiotensin-converting enzyme inhibition as soon as a patient is able to tolerate oral medications. dobutamine (2.5 to 10 âµg/kg/minute cri diluted in 5% dextrose in water) can be administered to improve cardiac contractility, particularly in cases of dilated cardiomyopathy. at low doses, dobutamine, primarily a î²-adrenergic agonist, will improve cardiac output with minimal effects on heart rate. dobutamine must be given as a constant rate infusion with careful, continuous ecg monitoring. despite minimal effects on heart rate, emergency management of specific conditions 129 the vertebral heart sum can be calculated by performing the following steps: 1. measure the long axis of the heart from the apex to the carina on the lateral view and mark the distance on a sheet of paper. 2. measure the length of the long axis of the heart in terms of vertebral bodies, starting by counting caudally from the fourth thoracic vertebra; count the number of vertebrae that are covered by the length of the long axis of the heart. 3. measure the short axis of the heart at the caudal vena cava, perpendicular to the long axis of the heart. 4. count the number of thoracic vertebrae covered by the short axis of the heart, starting at t4. 5. add the two numbers together to yield the vertebral heart sum; a vertebral heart sum greater than 10.5 is consistent with cardiomegaly. sinus tachycardia or ventricular dysrhythmias may develop during infusion. cats are more sensitive to the effects of dobutamine than dogs. monitor carefully for seizures and facial twitching. digoxin is a cardiac glycoside that acts as a positive inotrope and negative chronotrope in the long-term management of chf. digoxin has a long (24 hours in dogs, and 60 hours in cats) half-life and so has minimal use in the emergency management of chf. in chronic management of chf resulting from dilated cardiomyopathy or advanced mitral disease, however, digoxin is extremely useful. oral digitalization protocols have been developed but are risky in that dysrhythmias and severe gastrointestinal side effects can occur. cats with chf often have fulminant pulmonary edema, pleural effusion, arterial thromboembolism, or some combination of all three. if the pleural effusion is significant, perform therapeutic thoracocentesis to relieve pulmonary atelectasis and improve oxygenation. once the diagnosis and initial management of chf has been made, formulate a plan for continued management and monitoring. tailor the therapeutic plan to the patient based on the cause of the chf, the presence of concurrent diseases, and response to therapy. an important and often overlooked part of the successful emergency management of chf is the open communication with the owner regarding the owner's emotional and financial commitment for immediate and long-term management to ensure appropriate quality of life for each patient. pathophysiology and treatment, vet j 162 (3) caval syndrome resulting from severe heartworm disease is caused by the rapid maturation of a large quantity of adult worms in the right atrium and cranial and caudal venae cavae. most cases of caval syndrome occur in regions of the world where heartworm disease is highly endemic and dogs spend a large portion of time living outdoors. caval syndrome is recognized by the following clinical signs and results of biochemical analyses: acute renal and hepatic failure, enlarged right atrium and posterior vena cava, ascites, hemoglobinuria, anemia, acute collapse, respiratory distress, dic, jugular pulses, circulating microfilariae, and sometimes tricuspid insufficiency. immediate action in cases of caval syndrome in dogs involves immediate stabilization of the cardiovascular and respiratory systems with supplemental oxygen, furosemide (4 mg/kg iv), and careful crystalloid fluid infusion. diagnosis of caval syndrome is based on clinical signs of cardiogenic shock with right ventricular heart failure, intravascular hemolysis, and renal and hepatic failure. thoracic radiographs reveal cardiomegaly of the right side and enlarged tortuous pulmonary arteries. a right axis deviation may be seen on ecg tracings. clinicopathologic changes observed include azotemia, inflammatory leukogram, regenerative anemia, eosinophilia, elevated hepatocellular enzyme activities, hemoglobinuria, and proteinuria. circulating microfilariae may be observed on peripheral blood smears or in the buffy coat of microhematocrit tubes. heart worm antigen tests will be strongly positive. echocardiographic changes include visualization of a large number of heartworms in the right atrium, pulmonary arteries, and vena cava, tricuspid insufficiency, and right atrial and ventricular enlargement. treatment involves surgical removal of as many of the adult heartworms as possible from the right jugular vein and right atrium. glucocorticosteroids are recommended to decrease inflammation and microangiopathic disease associated with heartworm infection. for more long-term management, administer adulticide therapy several weeks following surgery, followed by routine microfilaricide therapy and then prophylaxis. calvert pericardial effusion often develops as a consequence of neoplasia in the older dog and cat. the most common types of neoplasia that affect the heart and pericardium include hemangiosarcoma, chemodectoma, mesothelioma, and metastatic neoplasia. more rarely, other causes of pericardial effusion include benign idiopathic pericardial effusion, coagulopathy, left atrial rupture in dogs with chronic mitral valvular insufficiency, infection, or pericardial cysts. regardless of the cause of the effusion, the development of pericardial tamponade adversely affects cardiac output. cardiac output is a function of heart rate and stroke volume. stroke volume depends on cardiac preload. the presence of pericardial effusion can impede venous return to the heart and thus adversely affect preload. in addition, as preload decreases, heart rate reflexively increases in an attempt to maintain normal cardiac output. as heart rate increases more than 160 beats/minute, diastolic filling is impaired further, and cardiac output further declines. animals with pericardial effusion often demonstrate the classic signs of hypovolemic or cardiogenic shock: anorexia, weakness, lethargy, cyanosis, cool peripheral extremities, tachycardia, weak thready pulses, hypotension, and collapse. physical examination abnormalities may include muffled heart sounds, thready femoral pulses, pulsus paradoxus, jugular venous distention, weakness, tachycardia, cyanosis, and tachypnea. electrocardiogram findings may include low amplitude qrs complexes (<0.5 mv), sinus tachycardia, ventricular dysrhythmias, or electrical alternans (figure 1-39) . thoracic radiographs often demonstrate a globoid cardiac silhouette, although the cardiac silhouette rarely may appear normal with concurrent clinical signs of cardiogenic shock in cases of acute hemorrhage. in such cases the removal of even small amounts of pericardial effusion by pericardiocentesis can increase cardiac output exponentially and alleviate clinical signs (table 1-35) . unless an animal is dying before your eyes, ideally perform an echocardiogram to attempt to determine whether a right atrial, right auricular, or heart base mass is present before pericardiocentesis. before attempting pericardiocentesis, assemble all of the required supplies (box 1-39) . to perform pericardiocentesis, follow this procedure: 1. place the patient in sternal or lateral recumbency. 2. attach ecg leads to monitor the patient for dysrhythmias during the procedure. 3. clip a 6-cm square caudal to the right elbow over the fifth to seventh intercostal space. 4. aseptically scrub the clipped area, and infuse 1 to 2 mg/kg of 2% lidocaine mixed with a small amount of sodium bicarbonate just dorsal to the sternum at the sixth intercostal space. bury the needle to the hub, and inject the lidocaine as you withdraw the needle. 5. while the local anesthetic is taking effect, assemble the intravenous extension tubing, three-way stopcock, and 60-ml syringe. 6. wearing sterile gloves, make a small nick incision in the skin to decrease drag on the needle and catheter during insertion. 7. slowly insert the needle and catheter, watching for a flash of blood in the hub of the needle, and simultaneously watching for cardiac dysrhythmias on the ecg monitor. 8. once a flash of blood is observed in the hub of the needle, advance the catheter off of the stylette further into the pericardial sac, and remove the stylette. 9. attach the length of intravenous extension tubing to the catheter, and have an assistant withdraw the fluid slowly. 10. place a small amount of fluid in a red-topped tube, and watch for clots. clot formation could signify that you have penetrated the right ventricle inadvertently or that active hemorrhage is occurring. withdraw as much of the fluid as possible, and then remove the catheter. monitor the patient closely for fluid reaccumulation and recurrence of clinical signs of cardiogenic shock. less rd, bright jm, orton ec: intrapericardial cyst causing cardiac tamponade in a cat, j am anim hosp assoc 36 (2) foreign bodies within the ear canal (e.g., foxtails) can present as emergencies because of acute inflammation and pressure necrosis of the tissue of the external auditory meatus causing pain and discomfort. clinical signs may be limited to incessant head shaking or scratching of the ear canal. complete examination of the ear canal and removal of any foreign body often requires administration of a short-acting anesthetic agent. once the animal has been restrained sufficiently and placed under anesthesia, carefully examine the ear canal and remove any foreign material with an alligator forceps. stimulation of the ear canal can cause awakening after removal of all debris and detritus, gently wipe the internal and external ear canal with a sterile gauze. place a topical antimicrobial-antifungal-steroid ointment such as otomax in the ear every 8 to 12 hours. if pain and discomfort is severe, systemically effective opioids or nsaids may be required. otitis externa is a common emergency that causes excessive head shaking, scratching, and purulent malodorous aural discharge. clean the ear canal with an irrigating solution such as epiotic and wipe it clean of debris. perform a complete aural examination to determine whether a foreign body or tumor is present and whether the tympanic membrane is intact. heat-fix any discharge and examine it cytologically for bacteria and fungal organisms. following careful cleansing, instill a topical antibiotic-antifungal-steroid ointment. in severe cases in which the ear canal has scarred and closed down with chronicity, consider administering systemically effective antibiotics (cephalexin, 22 mg/kg po tid) and antifungal agents (ketoconazole, 10 mg/kg po q12h) instead of topical therapy. systemically effective steroids (prednisone or prednisolone, 0.5 mg/kg po q12h) may be indicated in cases of severe inflammation to decrease pruritus and patient discomfort. presentation of a patient with otitis interna often is characterized by torticollis, head tilt, nystagmus, circling to the affected side, or rolling. fever, pain, vomiting, and severe depression may accompany clinical signs. most cases of severe otitis interna are accompanied by severe otitis media. both conditions must be treated simultaneously. the most common causes of otitis interna are staphylococcus aureus, pseudomonas, escherichia coli, or proteus spp. otitis interna can develop by infection spreading across the tympanic membrane, through the eustachian tubes, or by hematogenous spread from the blood supply to the middle ear. in most cases of otitis media, the tympanic membrane is ruptured. perform a culture and susceptibility test of the debris behind the tympanic membrane and within the aural canal. carefully clean the external ear canal. medicate with a topical combination antibiotic, antifungal, and antibiotic ointment. administer high-dose antibiotics (cephalexin, 22 mg/kg po q8h, or enrofloxacin, 10 to 20 mg/kg po q24h). if the tympanic membrane is not ruptured but appears swollen and erythematous, a myringotomy may need to be performed. if clinical signs of otitis media persist despite topical and systemic therapy, radiographic or ct/mri examination of the tympanic bullae may be required. chronic shaking of the head and ears or aural trauma (bite wounds) causes disruption of the blood vessels and leads to the development of unilateral or bilateral aural hematomas. aural hematomas are clinically significant because they cause patient discomfort and are often due to the presence of some other underlying problem such as otitis externa, atopy, or aural foreign bodies. acute swelling of the external ear pinna with fluid is characteristic of an aural hematoma. in some cases, swelling can be so severe that the hematoma breaks open, bathing the patient and external living environment in blood. when a patient has an aural hematoma, investigate the underlying cause. perform a complete aural examination to determine whether an aural foreign body, otitis externa, or atopy are present. carefully examine and gently clean the inner ear canal. treat underlying causes. management of an aural hematoma involves draining the hemorrhagic fluid from the aural tissue and tacking the skin down in multiple places to prevent reaccumulation of fluid until the secondary cause is resolved. many techniques have been described to surgically tack down the skin overlying the hematoma. after the animal has been placed under general anesthesia, lance the hematoma down the middle with a scalpel blade and remove the fluid and blood clot. tack down the skin with multiple through-and-through interrupted or mattress sutures through the ear. some clinicians prefer to suture through and attach a sponge or length of x-ray film to the front and back of the ear for stabilization and support. more recently, a laser can be used to drill holes in the hematoma and tack the skin down in multiple areas. compress the ear against the head with a compression bandage, whenever possible, for 5 to 7 days after the initial surgery, and then recheck the ear. the patient must wear an elizabethan collar until the surgical wound and hematoma heal to prevent selfmutilation. also systemically treat underlying causative factors such as otitis externa with antibiotics, antifungals, and steroids as indicated. investigate and treat other underlying causes such as hypothyroidism or allergies. bass electrocution usually is observed in young animals after they have chewed on an electric cord. other causes of electrocution include use of defective electrical equipment or being struck by lightning. electric current passing through the body can produce severe dysrhythmias, including supraventricular or ventricular tachycardia and first-and thirddegree atrioventricular block. the electric current also can produce tissue destruction from heat and electrothermal burns. electrocution also commonly results in noncardiogenic pulmonary edema caused by massive catecholamine release and increase in pulmonary vascular pressures during the event. ventricular fibrillation can occur, although that depends on the intensity and path of the electrical current and duration of contact. clinical signs of electrocution include acute onset of respiratory distress with moist rales, and localized necrosis or thermal burns of the lips and tongue. often the skin at the commissures of the mouth appears white or yellow and firm to the touch. muscle fasciculations, loss of consciousness, and ventricular fibrillation may occur. thoracic radiographs often reveal an increased interstitial to alveolar lung pattern in the dorsocaudal lung fields. noncardiogenic pulmonary edema can develop up to 24 to 36 hours after the initial incident. the first 24 hours are most critical for the patient, and then prognosis improves. the most important aspect in the treatment of the patient with noncardiogenic pulmonary edema is to minimize stress and to provide supplemental oxygen, with positive pressure ventilation, when necessary. although treatment with vasodilators (low-dose morphine) and diuretics (furosemide) can be attempted, noncardiogenic pulmonary edema is typically resistant to vasodilator and diuretic therapy. positive inotropes and pressor drugs may be necessary to treat shock and hypotension. opioid drugs (morphine, hydromorphone, oxymorphone) may be useful in controlling anxiety until the pulmonary edema resolves. administer broad-spectrum antibiotics (cefazolin; amoxicillin and clavulanic acid [clavamox]) to treat thermal burns. use analgesic drugs to control patient discomfort. if thermal burns are extensive and prohibit adequate food intake, place a feeding tube as soon as the patient's cardiovascular and respiratory function are stable and the patient can tolerate anesthesia. prolapse of the uterus occurs in the immediate postparturient period in the bitch and queen. excessive straining during or after parturition causes the uterus to prolapse caudally through the vagina and vulva. immediate intervention is necessary. examine the bitch or queen for a retained fetus. treatment consists of general anesthesia to replace the prolapsed tissue. if the uterus is edematous, physical replacement may be difficult or impossible. application of a hypertonic solution such as hypertonic (7%) saline or dextrose (50%) to the exposed endometrium can help shrink the tissue. that, combined with gentle massage to stimulate uterine contraction and involution and lubrication with sterile lubricating jelly, can aid in replacement of the organ into its proper place. to ensure proper placement in the abdominal cavity and to prevent recurrence, perform an exploratory laparotomy and hysteropexy. postoperatively, administer oxytocin (5 to 20 units im) to cause uterine contraction. if the uterus contracts, it is usually not necessary to suture the vulva. administer antibiotics postoperatively. recurrence is uncommon, even with subsequent pregnancies. if the tissue is damaged or too edematous to replace or if the tissue is devitalized, traumatized or necrotic, perform an ovariohysterectomy. in some instances, replacement of the damaged tissue is not necessary before removal. pyometra occurs in dogs and cats. the disease process occurs as a result of infection overlying cystic endometrial hyperplasia under the constant influence of progesterone. during the 2-month luteal phase after estrus or following copulation, artificial insemination, or administration of hormones (particularly estradiol or progesterone), the myometrium becomes relaxed and favors a quiescent environment for bacterial proliferation. clinical signs of pyometra are associated with the presence of bacterial endotoxin and sepsis. early, affected animals become lethargic and anorectic. polyuria with secondary polydipsia is often present because of the influence of bacterial endotoxin on renal tubular concentration. if the cervix is open, purulent or mucoid vaginal discharge may be observed. later in the course of pyometra, vomiting, diarrhea, and progressive debilitation resulting from sepsis occur. diagnosis is based on clinical signs in an intact queen or bitch and radiographic or ultrasonographic evidence of a fluid-filled tubular density in the ventrocaudal abdomen, adjacent to the urinary bladder (figures 1-40 and 1-41) . treatment of open and closed pyometra is correction of fluid and electrolyte abnormalities, administration of broad-spectrum antibiotics, and ovariohysterectomy. close pyometra is a life-threatening septic condition. open pyometra also can become life-threatening and so should be treated aggressively. in closed pyometra, conservative medical therapy is not advised. administration of prostaglandins and oxytocin do not reliably cause the cervix 136 1 to open and can result in ascending infection from the uterus into the abdomen or uterine rupture, both of which can result in severe peritonitis. for animals with an open pyometra, ovariohysterectomy is the most reliable treatment for chronic cystic endometrial hyperplasia. although less successful than ovariohysterectomy, medical therapy may be attempted in breeding bitches as an alternative to surgery. the most widely used medical therapy in the breeding queen and bitch is administration of prostaglandin f 2î± . this drug has not been approved for use in the queen or bitch in the united states. to proceed with medical management of pyometra, first determine the size of the uterus. start the patient on antibiotic therapy (ampicillin, 22 mg/kg iv q6h, or enrofloxacin, 10 mg/kg po q24h). administer the prostaglandin f 2î± (250 âµg/kg sq q24h) for 2 to 7 days until the size of the uterus approaches normal. measure serum progesterone concentrations if the bitch is in diestrus. as the corpus luteum degrades under the influence of prostaglandin f 2î± , serum progesterone levels will decline. prostaglandin f 2î± is an abortifacient and thus should not be administered to the pregnant bitch or queen. clinical signs of a reaction to prostaglandin f 2î± can occur within 5 to 60 minutes in the bitch and can last for as long as 20 minutes. clinical signs of a reaction include restlessness, hypersalivation, panting, vomiting, defecation, abdominal pain, fever, and vocalization. in a very ill animal, death can occur. the efficacy of prostaglandin f 2î± is limited and may require more than one treatment. the bitch should be bred on the next heat cycle and then spayed because progressive cystic endometrial hyperplasia will continue to occur. acute metritis is an acute bacterial infection of the uterus that typically occurs within 1 to 2 weeks after parturition. the most common organism observed in metritis is e. coli ascending from the vulva and vaginal vault. sepsis can progress rapidly. clinical signs of acute metritis include inability to nurse puppies, anorexia, lethargy, foul-smelling purulentsanguineous vaginal discharge, vomiting, or acute collapse. physical examination may reveal fever, dehydration, and a turgid distended uterus. septic inflammation will be observed on vaginal cytologic examination. an enlarged uterus can be observed with abdominal radiographs and ultrasonography. treatment of acute metritis is directed at restoring hydration status with intravenous fluids and treating the infection with antibiotics. because the primary cause of metritis is e. coli infection, start enrofloxacin (10 mg/kg iv or po once daily) therapy. as soon as the patient's cardiovascular status is stable enough for anesthesia, perform an ovariohysterectomy. if the patient is not critical and is a valuable breeding bitch, medical therapy can be attempted. medical management of acute bacterial metritis includes administration of oxytocin (5 to 10 units q3h for three treatments) or administration of prostaglandin f 2î± (250 âµg/kg/day for 2 to 5 days) to evacuate the uterine exudate and increase uterine blood flow. either drug should be used concurrently with antibiotics. rupture of the gravid uterus is rare in cats and dogs but has been reported. uterine rupture may occur as a consequence of parturition or result from blunt abdominal trauma. feti expelled into the abdominal cavity may be resorbed but more commonly cause the development of peritonitis. if fetal circulation is not disrupted, the fetus actually may live to term. uterine rupture is an acute surgical emergency. an ovariohysterectomy with removal of the extrauterine puppies and membranes is recommended. if only one horn of the uterus is affected, a unilateral ovariohysterectomy can be performed to salvage the remaining unaffected puppies and preserve the breeding potential for the valuable bitch. if uterine rupture occurs because of pyometra, peritonitis is likely, and copious peritoneal lavage should be performed at the time of surgery. the patient should be placed on 7 to 14 days of antibiotic therapy (amoxicillin or amoxicillin and clavulanic acid [clavamox] with enrofloxacin). vaginal prolapse occurs from excessive proliferation and hyperplasia of vaginal tissue while under the influence of estrogen during proestrus (figure 1-42) . the hyperplastic tissue usually recedes during diestrus but reappears with subsequent heat cycles. vaginal prolapse can be confused with vaginal neoplasia. the former condition occurs primarily in younger animals, whereas the latter condition occurs primarily in older animals. treatment for vaginal hyperplasia or prolapse generally is not required if the tissue remains within the vagina. the proliferation can lead to dysuria or anuria, however. in some cases, the tissue becomes 138 1 emergency care dried out and devitalized or becomes traumatized by the animal. such extreme cases warrant immediate surgical intervention. the treatment for vaginal prolapse consists of ovariohysterectomy to remove the influence of estrogen, placement of an indwelling urinary catheter if the patient is dysuric, and protection of the hyperplastic tissue until it recedes on its own. although surgical resection of the hyperplastic tissue has been recommended, excessive hemorrhage after removal can occur, and so the procedure should not be attempted. the patient should wear an elizabethan collar at all times to prevent selfmutilation. administer broad-spectrum antibiotics for a minimum of 7 to 14 days or until the hyperplastic tissue recedes. keep the tissue clean with saline solution. dystocia, or difficult birth, can occur in the dog and cat but is more common in the dog. a diagnosis of dystocia is made based on the time of onset of visible labor and the time in which the last puppy or no puppy has been born, the intensity and timing of contractions, the timing of when the amniotic membranes first appear, the condition of the bitch, and the timing of gestation. causes of dystocia can be maternal or fetal and include primary or secondary uterine inertia, narrowing of the pelvic canal, hypocalcemia, psychological disturbances, or uterine torsion. maternal-fetal disproportion, or large fetus size in relation to the bitch or queen, also can result in dystocia (box 1-40). obtain an abdominal radiograph for all cases of suspected dystocia at the time of presentation to determine the size of the fetus, presentation of the fetus (both anterior or posterior presentation can be normal in the bitch or queen, but fetal malpositioning can cause dystocia), and whether there is radiographic evidence of a uterine rupture or torsion. if maternal-fetal disproportion, uterine torsion, or uterine rupture is observed, take the patient immediately to surgery. if the puppies or kittens are in a normal position for birth, medical management can be attempted. clip the perineum and aseptically scrub it. wearing sterile gloves, insert a lubricated finger into the vagina and palpate the cervix. massage (or "feather") the dorsal wall of the vagina to stimulate contractions. place an intravenous catheter, and administer oxytocin (2 to 20 units im), repeating up to 3 times at 30-minute intervals. in some cases, hypoglycemia or hypocalcemia can contribute to uterine inertia. administration of a calciumcontaining solution (lactated ringer's solution) with 2.5% dextrose is advised. alternately, administer 10% calcium gluconate (100 mg/5 kg iv slowly). if labor has not progressed after 1 hour, immediately perform a cesarean section. uterine torsion is an uncommon emergency seen in the gravid and nongravid uterus and has been reported in dogs and cats. the onset of clinical signs of abdominal pain and straining as if to whelp/queen or defecate is usually acute and constitutes a surgical emergency. in some cases, there may have been a history of delivery of a live or dead fetus. vaginal discharge may or may not be present. radiographs or ultrasound examination reveal a fluid-filled or air-filled tubular density in the ventral abdomen. treatment consists of placing an intravenous catheter, stabilizing the patient's cardiovascular status with intravenous fluids and sometimes blood products, and performing an immediate ovariohysterectomy. if there are viable feti, the uterus should be delivered en mass and the puppies or kittens delivered. the expulsion of one or more fetus before term is known as spontaneous abortion. in dogs and cats, it is possible to expel or abort one or more fetuses and still carry viable fetuses to term and deliver normally. clinical signs of spontaneous abortion include vaginal discharge and abdominal contractions. in some cases, the fetus is found, or there may be evidence of fetal membranes or remnants. causes of spontaneous abortion in dogs include brucella canis, herpesvirus, coronavirus, and toxoplasmosis. in cats, herpesvirus, coronavirus, and feline leukemia virus can cause spontaneous abortion. in both species, trauma, hormonal factors, environmental pathogens, drugs, and fetal factors also can result in spontaneous abortion. the safest method of pregnancy termination in the bitch or queen is by performing an ovariohysterectomy. oral diethylstilbesterol is not an effective mechanism of pregnancy termination in the bitch. a so-called mismating shot, an injection of estradiol cypionate (0.02 mg/lb im) is effective at causing termination of an early pregnancy but can be associated with severe side effects, including bone marrow suppression and pyometra. estradiol cypionate is not approved for use in the bitch or queen and is not recommended. prostaglandin f 2î± is a natural abortifacient in the bitch if treatment is started within 5 days of cytologic evidence of diestrus (noncornified epithelium on a vaginal smear). the prostaglandin f 2î± causes lysis of the corpora lutea and a rapid decline in progesterone concentration. the prostaglandin f 2î± is administered for a total of eight injections (250 âµg/kg q12h for 4 days), along with atropine (100 to 500 âµg/kg sq). side effects can occur within 5 to 40 minutes of injection and include restlessness, panting, salivation, abdominal pain, urination, vomiting, and diarrhea. walking the patient for 20 to 30 minutes after each treatment sometimes decreases the intensity of the reactions. bitches in the first half of the pregnancy often resorb the embryos. if prostaglandin f 2î± is administered in the second half of the pregnancy, the fetuses are aborted within 5 to 7 days of treatment. measure serum progesterone concentrations at the end of treatment to ensure complete lysis of the corpus luteum. prostaglandin f 2î± is not approved for pregnancy termination in the bitch. in cats, prostaglandin f 2î± can terminate pregnancy after day 4 of gestation. prostaglandin f 2î± should be used only in healthy queens (100 to 250 âµg/kg sq q24h for 2 days). side effects in the queen are similar to those observed in the bitch but typically have a shorter duration (2 to 20 minutes). prostaglandin f 2î± is not approved for use in cats in the united states. the use of prostaglandin f 2î± does not preclude breeding and pregnancy at a later date. biddle d, macintire dk: obstetrical emergencies, clin tech small anim pract 15 (2) in the dog and cat the majority of injuries to the scrotum are associated with animal fights or shearing and abrasive injuries sustained in accidents involving automobiles. scrotal injuries should be categorized as superficial or penetrating. treatment of superficial injuries to the scrotum includes cleaning the wound with dilute antimicrobial cleanser and drying it. administer antiinflammatory doses of steroids (prednisolone, 0.5 to 1.0 mg/kg po q12-24h) or nsaids (carprofen, 2.2 mg/kg po q12h in dogs) for the first several days after scrotal injury to prevent or treat edema. administer topical antibiotic ointment until the wound heals. in most cases, place an elizabethan collar to prevent self-mutilation. prognosis is generally favorable; however, semen quality may be affected for months after injury because of scrotal swelling and increased scrotal temperature. penetrating injuries to the scrotum are more serious and are associated with severe swelling and infection. surgically explore and debride penetrating scrotal wounds. administer systemically effective antibiotics and analgesics. in extreme cases, particularly those that involve the testicle, consider castration and scrotal ablation. scrotal dermatitis is common in intact male dogs and can be associated with direct physical injury, self-infliction from licking, chemical irritation, burns, or contact dermatitis. in affected animals, the scrotum can become extremely inflamed, swollen, and painful. if left untreated, pyogranulomatous dermatitis can develop. make an attempt to determine whether an underlying systemic illness is present that could predispose the animal to scrotal dermatitis. widespread vasculitis with scrotal edema, pain, fever, and dermatitis has been associated with rickettsia rickettsii (rocky mountain spotted fever) infection. brucella canis also has been associated with scrotal irritation and dermatitis. if scrotal dermatitis follows from an infectious cause, empiric use of glucocorticosteroids potentially can make the condition worse by suppressing immune function. empiric treatment with antibiotics also potentially can confound making an accurate diagnosis. treatment of scrotal dermatitis is to eliminate predisposing causes, if possible. place an elizabethan collar at all times to prevent self-mutilation. bathe the scrotum with a mild antimicrobial soap and dry it to remove any offending chemical irritants. topical medications including tar shampoo, tetracaine, neomycin, and petroleum can cause further irritation and are contraindicated. use oral or parenteral administration of glucocorticosteroids or nsaids to control discomfort and inflammation. scrotal hernias occur when the contents of the abdomen (intestines, fat, mesentery, omentum) protrude through the inguinal ring into the scrotal sac. like inguinal hernias, scrotal definitive therapy for a scrotal hernia involves exploratory laparotomy and surgical reduction of the contents of the hernia, surgical correction of the rent in the inguinal ring, and castration. trauma to the epididymis or testicle can cause testicular pain and swelling of one or both testes. treat penetrating trauma to the testicle by castration to prevent infection and selfmutilation. administer oral antibiotics (amoxicillin or amoxicillin-clavulanate) for 7 to 10 days after the injury. nonpenetrating injuries to the scrotum and testicle rarely may cause acute testicular hemorrhage or hydrocele formation. palpation of the affected area often reveals a peritesticular, soft, compliant area. treatment consists of cool compresses on the scrotum and testicle and administration of antiinflammatory doses of glucocorticosteroids or nsaids. if the swelling does not resolve spontaneously in 5 to 7 days, consider surgical exploration and drainage. increased scrotal temperature and testicular inflammation can affect semen quality for months after the initial incident. testicular torsion, or torsion of the spermatic cord, causes rotation of the testicle, ultimately causing obstruction to venous drainage. testicular torsion often is associated with a neoplastic mass of a retained testicle within the abdomen but also can be observed with nonneoplastic testes located within the scrotum. the predominant clinical signs are pain, stiff stilted gait, and the presence of an abnormally swollen testicle (if located within the scrotum). if an intraabdominal testicular torsion is present, pain, lethargy, anorexia, and vomiting can occur (see acute condition in the abdomen). an intraabdominal mass may be palpable. perform an abdominal or testicular ultrasound, preferably with color flow doppler to evaluate perfusion to the testicle. treatment involves surgical removal of the involved testes. bacterial infections of the testicle or epididymis most commonly are caused by ascending infections of the normal bacterial flora of the prepuce or urethra. common inhabitants include escherichia coli, staphylococcus aureus, streptococcus spp., and mycobacterium canis. brucella canis and r. rickettsii are also capable of causing orchitis and epididymitis in the dog. clinical signs of orchitis or epididymitis include testicular enlargement, stiff stilted gait, and reluctance to walk. physical examination often reveals a fever and self-induced trauma to the scrotum from licking or chewing at the inflamed area. collect a semen sample by ejaculation, and culture it to identify the causative organism. alternately, collect samples by needle aspiration of the affected organ(s) and test serologically for b. canis. treatment of infectious orchitis involves a minimum of 3 to 4 weeks of specific antimicrobial therapy, based on culture and susceptibility testing, whenever possible. if a bacterial culture cannot be obtained, initiate fluoroquinolone therapy (enrofloxacin, 10 mg/kg po q24h). doxycycline (5 mg/kg po bid for 7 days) has been shown to suppress but not eradicate b. canis infection. testicular inflammation and increased temperature can affect sperm quality for months after infection. the most common causes of acute prostatitis are associated with acute bacterial infection (e. coli, proteus spp., pseudomonas spp., and mycoplasma spp.). less common causes include fungal infection (blastomyces dermatitidis) or anaerobic bacterial infection. acute prostatitis is characterized by fever, caudal abdominal pain, lethargy, anorexia, blood in the ejaculate, hematuria, dyschezia, and occasionally stranguria or dysuria. the patient often appears painful and depressed and may be dehydrated on physical examination. symmetric or asymmetric prostatomegaly and prostate pain may be evident on rectal palpation. in severely affected dogs, clinical signs of tachycardia, hyperemic or injected mucous membranes, bounding pulses, lethargy, dehydration, and fever may be present because of sepsis. death can occur within 2 days if a prostatic abscess ruptures. diagnosis of acute prostatitis is confirmed based on the presenting clinical signs, neutrophilic leukocytosis (with or without a left shift), and positive urine culture results. prostatic samples may be obtained from the prostatic portion of the ejaculate, prostatic massage, urethral discharge, urine, or (less commonly) prostatic aspirate. although semen samples can yield positive bacterial cultures, dogs with acute prostatitis are often unwilling to ejaculate. radiography may reveal an enlarged prostate, but this alone does not confirm the diagnosis of prostatitis. an abdominal ultrasound often reveals prostatic abscessation and allows for the collection of samples from the affected area(s) via prostatic aspirate. aspiration of the affected tissue potentially can wick infection into periprostatic tracks. cytologic examination of the patient's ejaculate or prostatic wash from a dog with acute prostatitis reveals numerous inflammatory cells and may contain bacterial organisms. the treatment of a patient with acute prostatitis is directed at correcting dysuria and constipation associated with prostatic enlargement. enrofloxaxin (10 mg/kg po sid) can penetrate the inflamed prostatic tissue and is effective in treating gram-negative and mycoplasma spp. infections. ciprofloxacin does not appear to penetrate prostatic tissue as readily. alternatives to enrofloxacin therapy are trimethoprim-sulfamethoxazole (30 mg/kg po q12h) or chloramphenicol (25-50 mg/kg po q8h) for a minimum of 2 to 3 weeks. castration is recommended because benign prostatic hyperplasia may be a predisposing factor in the development of acute prostatitis. do not perform castration until the patient has been on antibiotic therapy for a minimum of 7 days, to prevent the surgical complication of schirrous cords. finasteride (proscar, 1 mg/kg po q24h), an antiandrogen 5î±-reductase inhibitor, may help reduce the size of prostatic tissue until the effects of castration are observed. if a prostatic abscess is present, perform marsupialization, surgical drainage, or ultrasonographic drainage. surgical therapy is associated with a large incidence of complications, including incontinence, chronic drainage from fistulas and stomas, septic shock, and death. fracture of the os penis is an uncommon condition encountered in male dogs. os penis fractures can occur with minimal soft tissue damage but cause hematuria and dysuria. on physical examination, urethral obstruction and crepitus in the penis are found. a lateral abdominal radiograph is usually sufficient to document the fracture. treatment consists of conservative therapy, in most cases, and consists primarily of analgesia administration. if the urethra also is damaged, place a urethral catheter for 5 to 7 days to allow the urethral mucosa to heal. fractures of the os penis that are comminuted or severe enough to cause urethral obstruction require open reduction and fixation, partial penile amputation, or antescrotal (prescrotal) urethrostomy. lacerations of the penis cause significant bleeding because of the extensive vascular supply to the penis. dogs and cats tend to lick penile lacerations and prevent adequate clot formation. sedation or general anesthesia often is required to evaluate and treat the laceration. after sedation or general anesthesia, place a urinary catheter and examine the penis under a stream of cold water. small lacerations can be managed with cold compresses and one to several absorbable sutures. extensive suturing usually is not required. prevent erection by isolating the patient from females in estrus or allowing excitement or excessive activity. place an elizabethan collar to prevent self-mutilation. initiate systemic antibiotic therapy to prevent infection. the inability to withdraw the penis into the prepuce in male dogs or cats is known as paraphimosis. paraphimosis usually develops following an erection in young male dogs and in 144 1 emergency care older dogs after coitus. mucosal edema, hemorrhage, self-mutilation, and necrosis requiring penile amputation can occur if left untreated. treatment consists of applying cold water to the penis and reducing edema with application of an osmotic substance such as sugar. examine the base of the penis for hair rings that can prevent retraction of the penis into the prepuce. rinse the penis carefully with cold water and lubricate it with sterile lubricant and replace it into the prepuce. if the penis cannot be reduced easily into the prepuce, anesthetize the patient and make a small incision at the lateral aspect of the preputial opening. replace the penis and close the incision with absorbable suture. place a purse-string suture and leave it in place for several days to prevent recurrence. instill topical antimicrobial ointment with steroids into the prepuce several times a day. in severe cases, a urinary catheter may need to be placed to prevent urethral obstruction, until penile swelling and edema resolve. place an elizabethan collar to prevent excessive licking during the healing process. prolapse of the distal urethra is a condition usually confined to intact male english bulldogs, although isolated incidences also have been reported in yorkshire and boston terriers. the exact cause of this condition is unknown but usually is associated with a condition that causes increased intraabdominal pressure or urethral straining, including sexual excitement, coughing, vomiting, obstructed airway or brachycephalic airway syndrome, urethral calculi, genitourinary tract infection, and masturbation. the urethral prolapse usually appears as a mushroom-tip congested, irritated mass at the end of the penis that may or may not bleed (figure 1-44) . in some cases, bleeding occurs or worsens with sexual excitement. clinical signs associated with the prolapsed urethra include excessive licking of the prepuce, stranguria, and preputial bleeding. once the mass is observed, other differential diagnoses include transmissible venereal tumor, urethral polyp, trauma, urethritis, and neoplasia. in most cases, however, the prolapse occurs in intact young dogs, making neoplastic conditions less likely. treatment for urethral prolapse should occur at the time of diagnosis to prevent selfinduced trauma and infection. immediate therapy includes manual reduction of the prolapsed tissue and placement of a purse-string suture around an indwelling urinary catheter. the purse-string suture can remain in place for up to 5 days until definitive repair. until the time of surgery, place an elizabethan collar on the patient to prevent self-mutilation. several forms of surgical correction have been described. in some cases, surgical resection of the prolapsed tissue with apposition of the urethral and penile mucosa can be attempted. more recently, a technique involving placement of several mattress sutures to reduce and secure the prolapsed tissue has been described. recurrence of prolapse can occur with either technique, particularly if the inciting event recurs. because there may be a genetic predisposition in this breed and because the prolapse can recur with sexual excitement, neutering should strongly be recommended. local freezing or frostbite most commonly affects the peripheral tissues of the ears, tail, paws, and genitalia that are sparsely covered with fur, are poorly vascularized, and may have been traumatized previously by cold. clinical signs of frostbite are paleness and appearance of a blanched pink to white discoloration to the skin. the skin also may appear black and necrotic. immediate treatment consists of slowly rewarming the affected area with moist heat at 29.5â° c (85â°f) or by immersion in warm water baths. analgesics may be required to alleviate patient discomfort. carefully dry the injured areas and protect them from further trauma. the use of prophylactic antibiotics is controversial because it can promote resistant bacterial infection. use of antibiotics should be based on the presence of infection. treatments that are ineffective and may be harmful include rubbing the affected areas, pressure bandages, and ointments. corticosteroids can decrease cellular immunity and promote infection and are therefore contraindicated. many frostbitten areas that appear nonviable can regain function gradually. use care when removing areas of necrotic tissue. affected areas may take several days to a week before fully manifesting areas of demarcation between healthy viable and necrotic nonviable tissue. chilling of the entire body from exposure or immersion in extremely cold water results in a decrease in core body temperature and physiologic processes that become irreversible when the body temperature falls below 24â°c (75â°f). mild hypothermia can be 32â°to 37â°c, moderate hypothermia from 28â°to 32â°c, and severe hypothermia below 28â°c. the duration of exposure and the general condition of the animal influences its ability to survive. clinical signs and consequences associated with hypothermia include shivering, vasoconstriction, mental depression, hypotension, sinus bradycardia, hypoventilation with decreased respiratory rate, increased blood viscosity, muscle stiffness, atrial and ventricular irritability, decreased level of consciousness, decreased oxygen consumption, metabolic (lactic) acidosis, respiratory acidosis, and coagulopathies including dic. if the animal is breathing, administer warm, humidified oxygen at 4 to 10 breaths per minute. if the animal is not breathing or is severely hypoventilating, endotracheal intubation with mechanical ventilation may be necessary. place an intravenous catheter and infuse warmed crystalloid fluids. if the blood glucose is less than 60 mg/dl, add supplemental dextrose (2.5%) to the crystalloid fluids. monitor the core body temperature and ecg closely. rewarming should occur in the form of external circulating warm water blankets, radiant heat, and circulating warm air blankets (bair hugger). never use a heating pad, to avoid iatrogenic thermal burn injury. severe hypothermia may require core rewarming in the form of intraperitoneal fluids (10 to 20 ml/kg of lactated ringer's solution warmed to 39.4â°c [103â°f]). place a temporary peritoneal dialysis catheter, and repeat the dialysis every 30 minutes until the patient's body temperature reaches 36.6â°to 37.7â°c (98â°to 100â°f). the body temperature should rise slowly, ideally no more than 1â°f per hour. because the response of the body to drugs is unpredictable, avoid administering drugs whenever possible, until the body temperature returns to normal. complications observed during rewarming include dic, cardiac dysrhythmias including cardiac arrest, pneumonia, pulmonary edema, cns edema, ards, and renal failure. heat stroke and heat-induced illness in dogs can be associated with excessive exertion, exposure to high environmental temperatures, stress, and other factors that cause an inability to dissipate heat. brachycephalic breeds, obesity, laryngeal paralysis, and older animals with cardiovascular disease can be particularly affected. hyperthermia is defined as a rectal temperature of 41â°to 43â°c (105â°to 110â°f). clinical signs of hyperthermia include congested hyperemic mucous membranes, tachycardia, and panting. more severe clinical signs include collapse (heat prostration), ataxia, vomiting, diarrhea, hypersalivation, muscle tremors, loss of consciousness, and seizures. heat-induced illness can affect all major organ systems in the body because of denaturation of cellular proteins and enzyme activities, inappropriate shunting of blood, hypotension, decreased oxygen delivery, and lactic acidosis. cardiac dysrhythmias, interstitial and intracellular dehydration, intravascular hypovolemia, central nervous dysfunction, slough of gastrointestinal mucosa, oliguria, and coagulopathies can be seen as organ function declines. excessive panting can result in respiratory alkalosis. poor tissue perfusion results in a metabolic acidosis. loss of water in excess of solutes such as sodium and chloride can lead to a free water deficit and severe hypernatremia. a marked increase in pcv occurs because of the free water loss. severe abnormalities in electrolytes and ph can lead to cerebral edema and death. treatment goals for the patient with heat-induced illness are to lower the core body temperature and support cardiovascular, respiratory, renal, gastrointestinal, neurologic, and hepatic functions. at the scene the veterinarian or caretaker can spray the animal with tepid (not cold) water. immersion in cold water or ice baths is absolutely contraindicated. cold water and ice will cause extreme peripheral vasoconstriction, inhibiting the patient's ability to dissipate heat through conductive and convective cooling mechanisms. as a result, core body temperature will continue to rise despite the good intentions of well-doers at the scene. animals that present to the veterinarian that have been cooled to the point of hypothermia have a worse prognosis. once the animal has presented to the veterinarian, the goal is to cool the animal's body temperature with towels soaked in tepid water, cool intravenous fluids, and fans until the temperature has decreased to 103â°f. organ system monitoring and support is based on the severity and duration of the heat stroke and the ability of the body to compensate and respond to treatment. management of the patient with heat-induced illness involves prompt aggressive cooling without being overzealous and creating iatrogenic hypothermia. administer cool intravenous crystalloid fluids to replenish volume and interstitial hydration and correct the patient's acid-base and electrolyte abnormalities. management consists of rule of twenty monitoring (see rule of 20), taking care to evaluate, restore, and maintain a normal cardiac rhythm, blood pressure, urine output, and mentation. administer antibiotics if there are any signs of gastrointestinal bleeding that will predispose the patient to bacterial translocation. monitor baseline chemistry tests including a complete blood count, biochemical panel, platelet count, coagulation tests, and urinalysis. treat coagulopathies including dic aggressively and promptly (see also disseminated intravascular coagulation). severe changes in mentation including stupor or coma worsen a patient's prognosis. following initial therapy, monitor the patient for a minimum of 24 to 48 hours for secondary organ damage, including renal failure, myoglobinuria, cerebral edema, and dic. dogs that are going to die of heat-induced illness usually die within the first 24 hours. animals that survive longer than 24 hours have a more favorable prognosis. immediate treatment consists of cooling the patient with cooling measures as for hyperthermia and heat-induced illness (see the previous discussion), and eliminating the cause (i.e., exertion, anesthesia, or neuromuscular blockers such as succinylcholine). if the patient is under general anesthesia, hyperventilate the patient to help eliminate carbon dioxide and respiratory acidosis. administer dantrolene sodium (1 to 2 mg/kg iv) to stabilize the sarcoplasmic reticulum and decrease its permeability to calcium. animals with malignant hyperthermia should avoid any predisposing factors, including exertion, hyperthermia, and anesthesia. after an episode of malignant hyperthermia, administer crystalloid fluids intravenously to aid in the elimination of myoglobin. monitor renal function closely for myoglobinuria and pigment damage to the renal tubular epithelium. monitor and correct acid-base and electrolyte changes. walters jm: hyperthermia. in wingfield we, editor: the veterinary icu book, jackson, wyo, 2001, teton newmedia. sometimes it is difficult to assess whether an animal has been bitten by a poisonous or nonpoisonous snake. in colorado, the bull snake closely resembles the prairie rattlesnake. both snakes make similar noise and can be alarming if noticed on a hike or in the backyard. whenever possible, identify the offending reptile but never risk being bitten. know what types of venomous creatures are in the geographic area of the practice. if an animal has been bitten by a nonpoisonous snake, usually the bite marks are small with multiple small tooth punctures, and the bite is relatively nonpainful. usually local reaction is negligible. however, large boas or pythons also can inflict large crushing injuries that can cause severe trauma, including bony fractures. treatment for a nonpoisonous snakebite involves clipping the bite wound and carefully cleaning the area with antimicrobial scrub solution. broad-spectrum antibiotics (e.g., amoxicillin-clavulanate, 16.25 mg/kg po q12h) are indicated because of the extensive bacterial flora in the mouths of snakes. monitor all snakebite victims for a minimum of 8 hours after the incident, particularly when the species of the offending reptile is in question. if clinical signs of envenomation occur, modify the patient's treatment appropriately and aggressively. the two major groups of venomous snakes in north america are the pit viper and the coral snake. all venomous snakes are dangerous. the severity of any given bite depends on the toxicity of the venom, the amount of venom injected, the site of envenomation, the size of the animal bitten, and the time from bite/envenomation to seeking appropriate medical intervention. the majority of reptile envenomations in the united states are inflicted by pit vipers, including the water moccasin (cottonmouth), copperhead, and numerous species of rattlesnakes. pit vipers are characterized by a deep pit located between the eye and nostril, elliptic pupils, and retractable front fangs (figure 1-45) . localized clinical signs of pit viper envenomation may include the presence of bleeding puncture wounds, local edema close to puncture wounds, immediate severe pain or collapse, edema, petechiae, and ecchymosis with subsequent tissue necrosis. systemic signs of pit viper envenomation may include hypotension, shock, coagulopathies, lethargy, weakness, muscle fasciculations, lymphangitis, rhabdomyolysis, and neurologic signs including respiratory depression and seizures. neurologic signs largely are associated with envenomation emergency management of specific conditions 149 by the mojave and canebrake rattlesnakes, although a potent neurotoxin, mojave toxin a, also has been identified in other subspecies of rattlesnake. clinical signs of envenomation may take several hours to appear. hospitalize all suspected victims and monitor them for a minimum of 24 hours. the severity of envenomation cannot be judged solely on the basis of local tissue reaction. first aid measures by animal caretakers do little to prevent further envenomation. the most important aspect of initiating therapy is to transport the animal to the nearest veterinary emergency facility. to determine whether an animal has been envenomated by a pit viper, examine a peripheral blood smear for the presence of echinocytes. echinocytes will appear within 15 minutes of envenomation and may disappear within 48 hours. other treatment should be initiated as rapidly and aggressively as possible, although controversy exists whether some therapies are warranted. the mainstay of therapy is to improve tissue perfusion with intravenous crystalloid fluids, prevent pain with judicious use of analgesic drugs, and when necessary, reverse or negate the effects of the venom with antivenin. because pit viper venom consists of multiple fractions, treat each envenomation as a complex poisoning. obtain vascular access and administer intravenous crystalloid fluids (one fourth of a calculated shock dose) according to the patient's perfusion parameters of heart rate, blood pressure, and capillary refill time (see also shock and fluid therapy). opioid analgesics are potent and should be administered at the time of presentation. (see also pharmacologic means to analgesia: major analgesics). diphenhydramine (0.5 to 1 mg/kg im or iv) also can be administered to decrease the effects of histamine. famotidine, a histamine 1 receptor antagonist, also can be administered (0.5 to 1 mg/kg iv) to work synergistically with diphenhydramine. although antihistamines have no effect on the venom per se, they may have an effect on the tissue reaction to the venom and may prevent an adverse reaction to antivenin. the use of glucocorticosteroids is controversial. glucocorticosteroids (dexamethasone sodium phosphate [dex-sp], 0.25 to 0.5 mg/kg iv) may stabilize cellular membranes and inhibit phospholipase, an active component of some pit viper toxins. polyvalent antivenin is necessary in many cases of pit viper envenomation, except in most cases of prairie rattlesnake (crotalus viridis viridis) envenomation in colorado. a recent study demonstrated no difference in outcome with or without the use of antivenin in cases of prairie rattlesnake envenomation. clinically, however, patients that receive antivenin are more comfortable and leave the hospital sooner than those that do not receive antivenin. the exact dose of antivenin is unknown in small animal patients. administer a dose of at least 1 vial of antivenin to neutralize circulating venom. mix antivenin with a swirling, rather than a shaking motion, to prevent foaming. mix the antivenin with a 250-ml bag of 0.9% saline, and then administer it slowly over a period of 4 hours. pretreat animals with diphenhydramine (0.5 to 1 mg/kg im) before the administration of antivenin, and then monitor the animal closely for clinical signs of angioneurotic edema, urticaria, tachyarrhythmias, vomiting, diarrhea, and weakness during the infusion. administration of antivenin into the bite site is relatively contraindicated and ineffective because uptake is delayed, and systemic effects are the more life-threatening. management of pit viper envenomation largely involves maintenance of normal tissue perfusion with intravenous fluids, decreasing patient discomfort with analgesia, and negating circulating venom with antivenin. hydrotherapy to the affected bite site with tepid water is often soothing to the patient. the empiric use of antibiotics is controversial but is recommended because of the favorable environment created by a snakebite (i.e., impregnation of superficial gram-positive bacteria and gram-negative bacteria from the mouth of the snake into a site of edematous necrotic tissue). administer amoxicillin-clavulanate (16.25 mg/kg po q12h, or cephalexin, 22 mg/kg po q8h). also consider administration of nsaids (carprofen, 2.2 mg/kg po q12h). monitor the patient closely for signs of local tissue necrosis and the development of thrombocytopenia and coagulopathies including dic (see management of disseminated intravascular coagulation). treat coagulopathies aggressively to prevent end-organ damage. coral snakes are characterized by brightly colored bands encircling the body, with red and black separated by yellow. "red on black, friend of jack; red on yellow, kill a fellow." types of coral snakes include the eastern coral, texas coral, and sonoran coral snakes. clinical signs of coral snake envenomation may include small puncture wounds, transient initial pain, muscle fasciculations, weakness, difficulty swallowing/dysphagia, ascending lower motor neuron paralysis, miotic pinpoint pupils, bulbar paralysis, respiratory collapse, and severe hemolysis. clinical signs may be delayed for as long as 18 hours after the initial bite. immediate treatment with antivenin is necessary in cases of coral snake envenomation before the clinical signs become apparent, whenever possible. support respiration during paralysis with mechanical ventilation. secure the patient's airway with a cuffed endotracheal tube to prevent aspiration pneumonia. clinical signs will progress rapidly once they develop. rapid administration with antivenin is the mainstay of therapy in suspected coral snake envenomation. respiratory and cardiovascular support should occur with mechanical ventilation and intravenous crystalloid fluids. keep the patient warm and dry in a quiet place. turn the patient every 4 to 6 hours to prevent atelectasis and decubitus ulcer formation. maintain cleanliness using a urinary catheter and closed urinary collection system. perform passive range of motion and deep muscle massage to prevent disuse atrophy of limb muscles and function. treat aspiration pneumonia aggressively with broad-spectrum antibiotics (ampicillin, 22 mg/kg iv q6h, with enrofloxacin, 10 mg/kg iv q24h, and then change to oral once tolerated and the patient is able to swallow) for 2 weeks past the resolution of radiographic signs of pneumonia, intravenous fluids, and nebulization with sterile saline and coupage chest physiotherapy. several weeks may elapse before a complete recovery. the adult black widow spider (latrodectus spp.) can be recognized by a red to orange hourglass-shaped marking on the underside of a globous, shiny, black abdomen. the immature female can be recognized by a colorful pattern of red, brown, and beige on the dorsal surface of the abdomen. adult and immature females are equally capable of envenomation. the male is unable to penetrate the skin because of its small size. black widow spiders are found throughout the united states and canada. black widow spider venom is neurotoxic and acts presynaptically, releasing large amounts of acetylcholine and norepinephrine. there appears to be a seasonal variation in the potency of the venom, lowest in the spring and highest in the fall. in dogs, envenomation results in hyperesthesia, muscle fasciculations, and hypertension. muscle rigidity without tenderness is characteristic. affected animals may demonstrate clinical signs of acute abdominal pain. tonic-clonic convulsions may occur but are rare. in cats, paralytic signs predominate and appear early as a ascending lower motor neuron paralysis. increased salivation, vomiting, and diarrhea may occur. serum biochemistry profiles often reveal significant elevations in creatine kinase and hypocalcemia. myoglobinemia and myoglobinuria can occur because of extreme muscle damage. management of black widow spider envenomation should be aggressive in the cat and dog, particularly when the exposure is known. in many cases, however, the diagnosis is made based on clinical signs, biochemical abnormalities, and lack of other apparent cause. antivenin (one vial) is available and should be administered after pretreatment with diphenhydramine. if antivenin is unavailable, administer a slow infusion of calcium-containing fluid such as lactated ringer's solution with calcium gluconate while carefully monitoring the patient's ecg. the small brown nonaggressive spider is characterized by a violin-shaped marking on the cephalothorax. the neck of the violin points toward the abdomen. brown spiders are found primarily in the southern half of the united states but have been documented as far north as michigan. the venom of the brown spider has a potent dermatonecrolytic effect and starts with a classic bull's-eye lesion. the lesion then develops into an indolent ulcer into dependent tissues promoted by complement fixation and influx of neutrophils into the affected area. the ulcer can take months to heal and often leaves a disfiguring scar. systemic reactions are rare but can include hemolysis, fever, thrombocytopenia, weakness, and joint pain. fatalities are possible. immediate management of an animal with brown spider envenomation is difficult because there is no specific antidote and because clinical signs may be delayed until necrosis of the skin and underlying tissues becomes apparent through the patient's fur 7 to 14 days after the initial bite. dapsone has been recommended at a dose of 1 mg/kg for 14 days. surgical excision of the ulcer may be helpful if performed in the early stages of wound appearance. glucocorticosteroids may be of some benefit if used within 48 hours of the bite. the ulcer should be left to heal by second intention. deep ulcers should be treated with antibiotics. bufo toad species (b. marinus, aka cane toad, marine toad, giant toad; and the colorado river toad or sonoran desert toad b. alvarius) can be associated with severe cardiac and neurotoxicity if an animal licks its skin. the severity of toxicity depends largely on the size of the dog. toxins in the cane toad, b. marinus, include catecholamines and vasoactive substances (epinephrine, norepinephrine, serotonin, dopamine) and bufo toxins (bufagins, bufotoxin, and bufotenine), the mechanism of which is similar to cardiac glycosides. clinical signs can range from ptyalism, weakness, ataxia, extensor rigidity, opisthotonus, and collapse to seizures. clinical signs associated with b. alvarius toxicity are limited largely to cardiac dysrhythmias, ataxia, and salivation. the animal should have its mouth rinsed out thoroughly with tap water even before presentation to the veterinarian. if the animal is unconscious or actively seizing and cannot protect its airway, flushing the mouth is contraindicated. once an animal presents to the veterinarian, the veterinarian should place an intravenous catheter and monitor the patient's ecg and blood pressure. attempt seizure control with diazepam (0.5 mg/kg iv) or pentobarbital (2 to 8 mg/kg iv to effect). ventricular dysrhythmias can be controlled first with esmolol (0.1 mg/kg). if esmolol is ineffective, administer a longer-acting parenteral î²-antagonist such as propranolol (0.05 mg/kg iv). ventricular tachycardia also can be treated with lidocaine (1 to 2 mg/kg iv, followed by 50 to 100 âµg/kg/minute iv cri). case management largely depends on supportive care and treating clinical signs as they occur. monitor baseline acid-base and electrolyte balance because severe metabolic acidosis may occur that should be treated with intravenous fluids and sodium bicarbonate (0.25 to 1 meq/kg iv). monitor ecg, blood pressure, and mentation changes closely. control seizures and cardiac dysrhythmias. eubig pa: bufo species intoxication: big toad, big problem, vet med 96 (8) lizards of the family hemodermatidae are the only two poisonous lizards in the world. they are found in the southwestern united states and mexico. the venom glands are located on either side of the lower jaw. because these lizards are typically lethargic and nonaggressive, bite wounds are rare. the lizards have grooved teeth that introduce the venom with a chewing motion as the lizard holds tenaciously to the victim. the majority of affected dogs are bitten on the upper lip, which is very painful. there are no proven first aid measures for bites from gila monsters or mexican bearded lizards. the lizard can be disengaged by inserting a prying instrument in between the jaws 1 and pushing at the back of the mouth. the teeth of the lizard are brittle and break off in the wound. topical irrigation with lidocaine and probing with a needle will aid in finding and removing the teeth from the victim. bite wounds will bleed excessively. irrigate wounds with sterile saline or lactated ringer's solution, and place compression on the affected area until bleeding ceases. monitor the patient for hypotension. establish intravenous access, and administer intravenous fluids according to the patient's perfusion parameters. antibiotic therapy is indicated because of the bacteria in the lizard's mouth. because no antidote is available, treatment is supportive according to patient signs. the majority of musculoskeletal emergencies are the result of external trauma, most commonly from motor vehicle accidents. blunt trauma invokes injury to multiple organ systems as a rule, rather than an exception. because of this, massive musculoskeletal injuries are assigned a relatively low priority during the initial triage and treatment of a traumatized animal. perform a rapid primary survey and institute any lifesaving emergency therapies. adhere to a crash plan or the abcs of resuscitation (see initial emergency examination, management, and triage). although musculoskeletal injuries are assigned a relatively lower priority, the degree of recovery from these injuries and financial obligation for fracture repair sometimes becomes a critical factor in a client's decision whether to pursue further therapy. one of the most important deciding factors is the long-term prognosis for the patient to have a good quality of life following fracture repair. the initial management of musculoskeletal injuries is important in ensuring the best chance for maximal recovery with minimal complications after definitive surgical fracture repair. this is particularly important for open fractures, spinal cord compromise, multiple fractures, open joints, articular fractures, physeal fractures, and concomitant ligamentous or neurologic compromise (box 1-41). immediately after the initial primary survey of a patient, perform a more thorough examination, including an orthopedic examination. multiple injuries often are observed in the patient that falls from height (e.g., "high-rise syndrome"), motor vehicle accidents, gunshot wounds, and encounters with other animals (e.g., "big-dog-little-dog"). address the most life threatening injuries, and palliate musculoskeletal injuries until more definitive repair can be attempted when the patient is more stable. in animals with the history of potential for multiple injuries, search thoroughly and meticulously for areas of injury to the spinal column, extremities, and for small puncture wounds. helpful signs that can provide a clue as to an underlying injury include swelling, bruising, abnormal motion, and crepitus (caused by subcutaneous emphysema or bony fracture). if the patient is alert, look for areas of tenderness or pain. in unconscious or depressed patients, reexamine the patient after the patient becomes more mentally alert. injuries often are missed during the initial examination in obtunded patients because of the early response and attenuation of pain. unconscious or immobile patients must have radiographic examination of the spinal column following stabilization and support. palpate the skull carefully for obvious depressions or crepitus that may be associated with a skull fracture. localization of the injury can be determined by motion in abnormal locations, swelling caused by hemorrhage or edema, pain during gentle movement or palpation, deformity, angular change, or a significant increase or decrease in normal range of motion of bones and joints. perform a rectal examination in all cases to palpate for pelvic fractures and displacement. once the diagnosis of a fracture or luxation has been confirmed, look for any evidence of skin lacerations or punctures near the fracture site. in long-haired breeds, clipping the fur near the fracture site often is necessary to perform a thorough examination of the area. if any wounds are found, the fracture is classified as an open fracture until proven otherwise. in some cases, the open fracture is obvious, with a large section of bone fragment protruding through the skin. in other cases, the puncture wound may be subtle, with only a small amount of blood or pinpoint hole in the skin surface. characteristics observed with open fractures include bone penetration, fat droplets or marrow elements in blood coming from the wound, subcutaneous emphysema on radiographs, and lacerations in the area of a fracture. protect the patient from further injury or contamination of wounds. excessive palpation to intentionally produce crepitus is inappropriate because it causes severe patient discomfort and has the potential to cause severe soft tissue and neurologic injury at the fracture site. sedation and analgesia aids in making the examination more comfortable for the patient and allows localization of the injury and comparison with the opposite extremity. higher-quality radiographs can be performed to determine the extent of the injury when the animal is sedated adequately and pain is controlled. sedate the patient judiciously with analgesic drugs. opioid drugs work well for orthopedic pain, produce minimal cardiorespiratory depression, and can be reversed with naloxone if necessary. handle the fracture site gently to avoid causing further pain and soft tissue injury at the fracture site. rough or careless handling of a fracture site can cause a closed fracture to penetrate through the skin and become an open fracture. cover open fractures immediately to prevent contamination of the fracture with nosocomial infection from the hospital. administer a first-generation cephalosporin (cephalexin, 22 mg/kg po q8h, or cefazolin, 22 mg/kg iv q8h). the bandage also serves to control hemorrhage and prevent desiccation of the bones and surrounding soft tissue structures. leave the initial bandages in place until the patient's cardiorespiratory status has been determined to be stable and more definitive wound management can occur in a clean, preferably sterile location. examine the neurologic status and cardiovascular status of the limb before and after treatment. determine the vascular status of the limb by checking the color and temperature of the limb, the state of distal pulses, and the degree of bleeding from a cut nail bed. in patients with severe cardiovascular compromise and hypotension caused by hemorrhagic shock, the viability of the limb may be in question until the cardiovascular status and blood pressure are normalized. reduction of the fracture or straightening of gross deformities may return normal vascularity to the limb. when checking neurologic status, examine for motor and sensory function to the limb. swelling may increase pressure on the nerves as they run through osteofascial compartments, resulting in decreased sensory or motor function, or neurapraxia. diminished function often returns to normal once the swelling subsides. serial physical examinations in the patient and response to initial stabilization therapy can lead to a higher index of suspicion that more occult injuries are present, such as a diaphragmatic hernia, perforated bowel, lacerated liver or spleen, or uroabdomen. to prevent ongoing trauma, reduce any fracture and then stabilize the site above and below the fracture. a modified robert jones splint or bandage often works well for fractures emergency management of specific conditions 155 involving the distal extremities. fractures of the humerus or femur are difficult to immobilize without the use of spica or over-the-hip coaptation splints to prevent mobility. inappropriate bandaging of humerus or femur fractures can result in a fulcrum effect and worsen the soft tissue and neurologic injuries. further displacement of vertebral bodies or luxations can cause cord compression or laceration such that return to function becomes impossible. immediately place any patient with a suspected spinal injury on a flat surface, and tape down the animal to prevent further movement until the spine has been cleared by a minimum or two orthogonal radiographic views (lateral and ventrodorsal views performed as a cross-table x-ray technique). wounds associated with musculoskeletal trauma are common and include injury to the bones, joints, tendons, and surrounding musculature (box 1-42). major problems associated with these cases are the presence of soft tissue trauma that makes wound closure hazardous or impossible, because of the risk of infection. chronic deep infection of traumatized wounds can cause delayed healing and sequestrum to develop, particularly if there is avascular bone or cartilage within the wound. in the early management of an open fracture, the areas should be splinted without pulling any exposed bone back into the soft tissue. the wound should not be probed or soaked, as nosocomial bacteria and other external contaminants can be introduced into the wound, leading to severe infection. because of the risk of actually causing infection, probing, flushing, or replacing tissues back into the wound should be performed at the time of formal debridement when the patient is physiologically stable. immediate bactericidal antibiotic therapy with a first-generation cephalosporin should be started immediately to obtain adequate concentrations of antibiotics at the fracture site. the duration of antibiotic therapy should ideally be limited to 2-3 days to prevent the risk of superinfection. treatment of open musculoskeletal injury involves three considerations: initial inspection and wound debridement, stabilization and repair, and wound bandaging. 156 1 emergency care when associated with a fracture, wound is created from the inside out by penetration of bone fragments through the skin or from a low-energy gunshot. simple or comminuted fracture pattern good stability of the two main bone segments treatment and prognosis are good and similar to those of a closed injury if wound is debrided and stabilized within 6 to 8 hours. when associated with a fracture, wound is created from the outside in. major deep injury with considerable soft tissue stripping from bone and muscle damage simple or comminuted fracture pattern prognosis is good if wound is debrided within 6 hours of injury and provided rigid stabilization with a bone plate or external fixator. results from major external force severe damage and necrosis of skin, subcutaneous tissue, muscle, nerve, bone, tendon, and arteries soft tissue damage may vary from crush injury to shearing injury associated with bite wounds or low-speed automobile accidents. requires immediate and delayed sequential debridement and rigid external fixation can require prolonged healing times guarded prognosis initial inspection and wound debridement include the following steps: 1. after the patient's cardiovascular status has been stabilized and it has been determined that it can withstand anesthesia, place the animal under general anesthesia and remove the temporary splint. 2. keeping the wound covered, shave the surrounding fur. 3. remove the covering and then place sterile lubricant jelly over the wound. shave the fur to the edges of the wound margin. 4. wash away any entrapped fur and the lubricant jelly. 5. complete an antiseptic scrub of the surrounding skin. 6. if the wound is a small puncture (e.g., gunshot pellets or bites), probe the wound with a sterile hemostat. do a thorough debridement if tissues deep to the hole are cavitated. if not deep, create a hole for drainage. 7. flush the wound with a physiologic solution (lactated ringer's solution is preferred). 8. debride the wound from outward to inward. cut away damaged areas of skin and deeper tissues to open up underlying cavitations and tissue injury. 9. continuously irrigate with warm physiologic solution (lactated ringer's solution is preferred). the stream must be strong enough to flush debris out of the bottom of the wound. to accomplish this, attach a 20-gauge needle to a 35-ml syringe (will deliver 7 psi). excise any obviously devitalized tissue. 10. do not remove any bone fragments that are firmly attached to soft tissue. do not cut into healthy soft tissue to find bullet or bone fragments, unless the bullet can cause injury to joints or nerve tissue. 11. do a primary repair of tendons and nerves if the wound is type i and recent (within 8 hours of the initial injury). if the wound is too severe or if there is obvious infection, tag the ends of the tendons and nerves for later repair. it is best to stabilize and repair open fractures as soon as the patient's cardiovascular and respiratory status can tolerate general anesthesia, provided that adequate stabilization is possible. if this is not possible because of the level of experience of the surgeon or the lack of necessary equipment, it is best to perform wound management and place a temporary splint until definitive repair can be performed. wound bandaging is discussed in the section on bandaging techniques. structural injuries to the joints are common and can involve both ligaments and articular cartilage injuries. cartilage does not heal well; therefore, injuries involving articular cartilage can lead to a significant loss of function and degenerative joint disease (osteoarthritis). cartilage injuries that are superficial evoke a short-lived enzymatic and metabolic response that does not stimulate enough cellular growth to repair the defect. superficial lesions remain as defects but do not progress to chondromalacia or osteoarthritis. deep cartilage lacerations that extend to subchondral bone produce an exuberant healing response from the cells of the underlying cartilage. in many cases, this material undergoes degeneration and leads to osteoarthritis. impact injuries to surface cartilage can cause chondrocyte and underlying bone injury. these lesions rapidly progress to osteoarthritis; however, they may be totally or partially reversible. treatment of grade i injuries requires short-term coaptation splints and has a good prognosis. grade ii injuries require surgical treatment with a suture stent and consistent postoperative coaptation splints to heal and maintain good function. healing of grade iii injuries often is a problem, and suture stents or surgical reapproximation may be indicated. failure to immobilize joints that are frequently flexed (elbow and stifle) can result in late complications of ligament repair. ligamentous injuries of joints, particularly the collateral ligaments of the stifle, elbow, and hock, and carpal hyperextension injuries are commonly missed and may require surgical fixation, including arthrodesis (box 1-43). fractures in immature animals differ from those in adults in that young puppies and kittens have a great ability to remodel bone. remodeling is dependent on the age of the patient and the location of the fracture. the younger the puppy or kitten and the closer the fracture to the epiphysis or growth plate, the greater the potential for remodeling and the development of angular limb deformities. remodeling occurs more effectively in longlimbed breeds of dogs than in short-limbed breeds. fractures through the growth plate of immature animals may potentially cause angular limb deformities, joint dislocations or incongruity, and osteoarthritis. this form of injury is commonly observed in the distal ulnar growth plate and the proximal and distal radial growth plates. high-rise syndrome in cats is seen in cats that fall from a height usually greater than 30 feet. it occurs most frequently in high-rise buildings in urban areas where cats lie on window ledges and suddenly fall out the window. the most common lesions observed in cats that fall from heights are thoracic injuries (rib and sternal fractures, pneumothorax, and pulmonary contusions) and facial and oral trauma (lip avulsions, mandibular symphyseal fractures, fractures of the hard palate, and maxillary fractures). limb and spinal cord fractures and luxations, radius and ulna fractures, abdominal trauma, urinary tract trauma, and diaphragmatic hernias are also common. the injuries sustained are often found in combination, rather than as an isolated injury of one area of the body. follow the mnemonic a crash plan when managing a cat suffering from high-rise syndrome, treating the animal immediately for shock. following cardiovascular and respiratory stabilization, evaluate thoracic and abdominal radiographs, including those of the spine. evaluate the bladder closely, making sure that the cat is able to urinate effectively. examine the hard palate, maxilla, and mandibular symphysis for fractures. palpate the pelvis and carefully manipulate all limbs to examine for fractures or ligamentous injuries. finally, perform a complete neurologic examination. patients that fall less than five stories often have a more guarded prognosis than patients that fall from higher levels. sometimes the owner witnesses the ingestion of a foreign body during play, such as throwing a stick or fetching a ball. cats tend to play with string or thread that becomes caught around the base of the tongue. in many cases, however, ingestion of the foreign object is not witnessed, and diagnosis is made based on clinical signs and physical examination. foreign bodies lodged in the oral cavity often cause irritation and discomfort, including difficulty breathing and difficulty swallowing. often, an animal paws at its mouth in an attempt to dislodge a stick or bones wedged across the roof of the mouth. irritation, inability to close the mouth, and blockage of the orpharynx can result in excessive drooling. the saliva may appear blood-tinged due to concurrent soft tissue trauma (figs 1-46 and 1-47) . obstruction of the glottis by a foreign body (e.g., tennis ball or toy) can result in cyanosis secondary to an obstructed airway and hypoxemia. in many cases, the object is small enough to enter the larynx but too large to be expelled. if a foreign object is lodged in the mouth for more than several days, halitosis and purulent discharge may be present. many animals are anxious at the time of presentation and may require sedation or a light plane of anesthesia to remove the foreign object. the animal may bite personnel and may have bitten the owner during his or her attempt to remove the object from the mouth en route to the hospital. propofol (47 mg/kg iv) or a combination of propofol with diazepam (0.5-1 mg/kg iv) is an excellent combination for a light plane of anesthesia. exercise caution when anesthetizing a patient with a ball lodged in the airway, as further compromise of respiratory function may occur and cause worsening of the hypoxemia. before inducing anesthesia, assemble all supplies necessary to remove the object. make sure that rigid towel clamps, sponge forceps, and bone forceps are on hand, because the foreign object is often very slippery with saliva. hemostats and carmalts may slip and not be useful in the removal of the foreign object. place a peripheral intravenous catheter to secure vascular access prior to anesthetic induction. have available the supplies necessary for an emergency tracheostomy, if the foreign object cannot be removed by usual methods. induce a light plane of anesthesia and then grasp the object with the sponge forceps or towel clamps, and extract. monitor the cardiorespiratory status of the animal at all times during the extraction process. if you are unable to remove the object, and if severe respiratory distress, including cyanosis, bradycardia, or ventricular dysrhythmias, develop, perform a tracheostomy distal to the site of obstruction. once the foreign body has been removed, administer supplemental flow-by oxygen until the animal awakens. if laryngeal edema or stridor on inspiration is present, administer a dose of dexamethasone sodium phosphate (0.25 mg/kg iv, im, sq) to decrease inflammation. the patient should be carefully monitored for 24 hours, because noncardiogenic pulmonary edema can develop secondary to airway obstruction. esophageal foreign bodies pose a serious medical emergency. it is helpful if the owner witnessed ingestion of the object and noted rapid onset of clinical signs. in many cases, however, ingestion is not witnessed, and the diagnosis must be made based on clinical signs, thoracic radiographs, and results of a barium swallow. the most common clinical signs are excessive salivation with drooling, gulping, and regurgitation after eating. many animals will make repeated swallowing motions. some animals exhibit a rigid "sawhorse" stance, with reluctance to move immediately after foreign body ingestion and esophageal entrapment. after completing a physical examination, evaluate cervical and thoracic radiographs to determine the location of the esophageal obstruction. esophageal foreign objects are lodged most commonly at the base of the heart, the carina, or just orad to the lower esophageal sphincter. if the object has been lodged for several days, pleural effusion and pneumomediastinum may be present secondary to esophageal perforation. endoscopy is useful for both diagnosis and removal of the foreign object; however, it is invasive and requires general anesthesia ( fig. 1-48) . remove foreign objects lodged in the esophagus with a rigid or flexible endoscope after the patient has been placed under general anesthesia. evaluate the integrity of the esophagus both before and after removal of the material because focal perforation or pressure necrosis can be present. necrosis of the mucosa and submucosa of the esophagus often leads to stricture formation or perforation. attempt to retrieve the object with a flexible fiberoptic endoscope if available. rigid tube endoscopy can also be performed. in many cases, smooth objects that cannot be easily grasped can be pushed into the stomach and allowed to dissolve or may be removed by gastrotomy. if the foreign body is firmly lodged in the esophagus and cannot be pulled or pushed into the stomach, or if perforation has already occurred, the prognosis for return to function without strictures is not favorable. in such cases, referral to a surgical specialist is recommended for esophagostomy or esophageal resection. after removal of the object, carefully examine the esophagus and then administer gastroprotectant agents (famotidine, 0.5 mg/kg po bid; sucralfate slurry, 0.5-1.0 g/dog) for a minimum of 5 to 7 days. to rest the esophagus, the patient should receive nothing per os (npo) for 24 to 48 hours. if esophageal irritation or erosion is moderate to severe, a percutaneous gastrotomy tube should be placed for feeding until the esophagus heals. perform repeat endoscopy every 7 days to evaluate the healing process and to determine whether stricture formation is occurring. persistent vomiting immediately or soon after eating is often associated with a gastric foreign body. in some cases, the owner knows that the patient has ingested a foreign body of some kind. in other cases, continued vomiting despite lack of response to conservative treatment (npo, antiemetics, gastroprotectant drugs) prompts further diagnostic procedures, including abdominal radiographs and bloodwork. obstruction to gastric outflow and vomiting of hydrochloric acid often cause a hypochloremic metabolic acidosis. radiopaque gastric foreign bodies may be observed on plain films. radiolucent cloth material may require a barium series to delineate the shape and location of the foreign body ( fig. 1-49) . treatment consists of removal with flexible endoscopy or a simple gastrotomy. most animals with uncomplicated gastric foreign bodies are relatively healthy, but any metabolic and electrolyte abnormalities should be corrected prior to anesthesia and surgery. small intestinal obstruction can be caused by foreign bodies, tumors, intussusception, volvulus, or strangulation within hernias. regardless of the cause, clinical signs of small intestinal obstruction depend on the location and degree of obstruction, and whether the bowel has perforated. clinical signs associated with a high small intestinal obstruction are usually more severe and more rapid in onset compared with partial or complete obstruction of the jejunum or ileum. complete obstructions that allow no fluid or chyme to pass are worse than partial obstructions, which can cause intermittent clinical signs interspersed with periods of normality (table 1 -36). the most common clinical signs associated with a complete small intestinal obstruction are anorexia, vomiting, lethargy, depression, dehydration, and sometimes abdominal pain. early clinical signs may be limited to anorexia and depression, making a diagnosis challenging unless the owner has a suspicion that the animal ingested some kind of foreign object. obstructions cranial to the common bile duct and pancreatic papillae lead to vomiting of gastric contents, namely hydrochloric acid, and a hypochloremic metabolic alkalosis. obstructions caudal to the common bile duct and pancreatic papillae result in loss of other electrolytes and sometimes mixed acid-base disorders. eventually, all animals with small intestinal obstruction vomit and have fluid loss into dilated segments of bowel, leading to dehydration and electrolyte abnormalities. increased luminal pressure causes decreased lymphatic drainage and bowel edema. the bowel wall eventually becomes ischemic and may rupture. linear foreign bodies should be suspected in any vomiting patient, particularly cats. string or thread often is looped around the base of the tongue and can be visualized in many cases by a thorough oral examination. to look properly under the tongue, grasp the top of the animal's head with one hand, and pull the lower jaw open with the index finger of the opposite hand while pushing up the thumb simultaneously on the tongue in between the intermandibular space. thread and string can be observed lying along the ventral aspect of the tongue. in some cases, if a linear foreign body is lodged very caudally, it cannot be visualized without heavy sedation or anesthesia. linear foreign bodies eventually cause bowel obstruction and perforation of the intestines along the mesenteric border. the foreign material (e.g., string, thread, cloth, pantyhose) becomes lodged proximally, and the intestines become plicated as the body attempts to push the material caudally through the intestines ( fig. 1-50) . continued peristalsis eventually causes a sawing motion of the material and perforation of the mesenteric border of the intestines. once peritonitis occurs, the prognosis is less favorable unless prompt and aggressive treatment is initiated. reevaluate any patient that does not respond to conservative symptomatic therapy, performing a complete blood count, serum biochemical panel (including electrolytes), and abdominal radiographs. intestinal masses may be palpable on physical examination and are often associated with signs of discomfort or pain when palpating over the mass. radiography and abdominal ultrasound are the most useful diagnostic aids. plain radiographs may be diagnostic when the foreign object is radiodense or there is characteristic dilation or plication of bowel loops. as a rule of thumb, the width of a loop of small bowel should be no larger than twice the width of a rib. diagnosis of small intestinal obstruction or ileus can be based on the appearance of stacking loops of dilated bowel. comparison of the width of the bowel with the width of a rib is often performed. with mild dilation, the bowel width is three to four times the rib width; with extensive dilation, five to six times the rib width ( fig.1-51) . in cases of linear foreign bodies, c-areas (comma-shaped areas) of gas trapped in the plicated bowel will appear stacked on one another. blunt, wedge-shaped areas of gas or square linear areas of gas adjacent to a distended bowel loop are characteristic of a foreign body lodged in the intestine. contrast radiography is indicated when confirmation of the suspected diagnosis is necessary and ultrasonography is not available. contrast material may outline the object or abruptly stop orad to the obstruction. the definitive treatment of any type of small intestinal foreign body is surgical removal. linear foreign bodies sometimes pass, but they should never be left untreated in a patient that is demonstrating clinical signs of inappetence, vomiting, lethargy, and dehydration. the timing of surgery is critical because the risk of intestinal perforation increases with time. prior to surgery, correct any acid-base and electrolyte abnormalities with intravenous fluid therapy. administer broad-spectrum antibiotics. perform an enterotomy or intestinal resection and anastomosis as soon as possible once the patient's acid-base and electrolyte status have been corrected. clinical signs of a foreign body in the large bowel are usually nonexistent. in most cases, if a foreign object has passed successfully through the small bowel, it will pass through the large bowel without incident unless bowel perforation and peritonitis occur. penetrating foreign bodies such as needles often cause localized or generalized peritonitis, abdominal pain, and fever. hematochezia may be present if the foreign object causes abrasion of the rectal mucosa. symptomatic patients should have abdominal radiographs performed. colonoscopy or exploratory laparotomy should be performed if survey radiographs are suggestive of a large intestinal obstruction or perforation. in most cases, large intestinal foreign bodies will pass without incident. surgery is required to treat perforations, peritonitis, or abscesses. 164 1 emergency care 1 figure 1 -51: after 60 minutes, the barium has stopped moving and has reached a blunt, intraluminal intestinal foreign body. note that barium appears wedge-shaped or square at the site of the foreign body. foreign bodies in the rectum and anus often are the result of ingestion of bones, wood material, needles, and thread, or malicious external insertion. often the material can pass through the entire gastrointestinal tract and then get stuck in the anal ring. clinical signs include hematochezia and dyschezia with straining to defecate. diagnosis is made by visual examination of the item in the anus, or by careful digital palpation after heavy sedation or short-acting general anesthesia. radiography is helpful in locating needles that have penetrated the rectum and lodged in the perirectal or perinatal tissues. treatment consists of careful removal of the needle digitally or surgically. intussusception is the acute invagination of one segment of bowel (the intussusceptum) into another (the intussuscipiens). the proximal segment always invaginates into the distal segment of bowel. intussusception most commonly occurs in puppies and kittens less than 1 year of age but can occur in an animal of any age with hypermotility of the small bowel, gastrointestinal parasites, and severe viral or bacterial enteritis. intussusception occurs primarily in the small bowel in the jejunum, ileum, and ileocolic junction. clinical signs include vomiting, abdominal discomfort, and hemorrhagic diarrhea. usually, hemorrhagic diarrhea is the first noticeable sign, and in puppies, may be due to parvoviral enteritis, with secondary intussusception. usually, the obstruction is partial with mild clinical signs. more serious clinical signs develop as the obstruction becomes more complete. differential diagnoses include hemorrhagic gastroenteritis, parvoviral enteritis, gastrointestinal parasites, intestinal foreign body, bacterial enteritis, and other causes of vomiting and diarrhea. the diagnosis of intussusception is often made based on palpation of a sausage-shaped firm, tubular structure in the abdomen accompanied by clinical signs and abdominal pain. plain radiographs may demonstrate segmental or generalized dilated segments of bowel, depending on the duration of the problem. ultrasonographs of the palpable mass resemble the layers of an onion, with hyperechoic intestinal walls separated by less echogenic edema. treatment consists of correction of the patient's acid-base and electrolyte abnormalities with intravenous fluids and surgical reduction or removal of the intussusception with resection and anastomosis. although enteroplication has been suggested, the technique has fallen out of favor because of the increased risk of later obstruction. the primary cause of intestinal inflammation and hypermotility must be identified and corrected. gastric dilatation can occur with or without volvulus in the dog. gastric dilatationvolvulus (gdv) occurs primarily in large-and giant-breed dogs with deep chests, such as the great dane, labrador retriever, saint bernard, german shepherd dog, gordon and irish setters, standard poodle, bernese mountain dog, and bassett hound. the risk of gdv increases with age; however, it can be seen in dogs as young as 4 months. deep, narrow-chested breeds are more likely to develop gdv than dogs with broader chests. the overall mortality for surgically treated gastric dilatation-volvulus ranges from 10% to 18%, with most deaths occurring in patients that required splenectomy and partial gastrectomy. clinical signs of gdv include abdominal distention, unproductive vomiting or retching, lethargy, weakness, sometimes straining to defecate, and collapse. the owner may think that the animal is vomiting productively because of the white foamy froth (saliva) that is not able to pass into the twisted stomach. in some cases, there is a history of the dog's being fed a large meal or consuming a large quantity of water prior to the onset of clinical signs. instruct the owner of any patient with a predisposition for and clinical signs of gdv to transport the animal to the nearest veterinary facility immediately. physical examination often reveals a distended abdomen with a tympanic area on auscultation. in dogs with very deep chests, it may be difficult to appreciate abdominal distention if the stomach is tucked up under the rib cage. depending on the stage of shock, the patient may have sinus tachycardia with bounding pulses, cardiac dysrhythmias with pulse deficits, or bradycardia. the mucous membranes may appear red and injected or pale with a prolonged capillary refill time. the patient may appear anxious and attempt to retch unproductively. if the patient is nonambulatory at the time of presentation, the prognosis is more guarded. the definitive diagnosis of gdv is based on clinical signs, physical examination findings, and radiographic appearance of gas distention of the gastric fundus with dorsocranial displacement of the pylorus and duodenum (the so-called "double-bubble" or "popeye arm" sign) ( fig.1-52) . in simple gastric dilatation without volvulus, there is gas distention of the stomach with anatomy appearing normal on radiography. with "food bloat," or gastric distention from overconsumption of food, ingesta is visible in the distended stomach ( fig. 1-53) . as soon as a patient presents with a possible gdv, place a large-bore intravenous catheter in the cephalic vein(s) and assess the patient's ecg, blood pressure, heart rate, capillary refill time, and respiratory function. obtain blood samples for a complete blood count, serum biochemistry profile, immediate lactate measurement, and coagulation tests before taking any radiographs. rapidly infuse a colloid (hetastarch or oxyglobin, 5 ml/kg iv bolus) along with shock volumes of a crystalloid fluid (up to 90 ml/kg/hour) (see section on shock). monitor perfusion parameters (heart rate, blood pressure, capillary refill time, and ecg) and titrate fluid therapy according to the patient's response. the use of short-acting glucocorticosteroids is controversial. glucocorticosteroids may help stabilize cellular membranes and decrease the mechanisms of ischemia-reperfusion injury, but no detailed studies have proved them to be beneficial versus not using glucocorticosteroids in the patient with gdv. attempt gastric decompression, either with placement of an orogastric tube or by trocharization. to place an orogastric tube, position the distal end of the tube at the level of the patient's last rib ( fig. 1-54 ) and place it adjacent to the animal's thorax; then put a piece of tape around the tube where it comes out of the mouth, once it is in place. put a roll of 2-inch tape in the patient's mouth behind the canine teeth and then secure the roll in place by taping the mouth closed around the roll of tape. lubricate the tube with lubricating jelly and slowly insert the tube through the center of the roll of tape into the stomach. the passing of the tube does not rule out volvulus. in some cases, the front legs of the patient need to be elevated, and the caudal aspect of the patient lowered (front legs standing on a table with back legs on the ground) to allow gravity to pull the stomach down to allow the tube to pass. once the tube has been passed, air within the stomach is relieved, and the stomach can be lavaged. the presence of gastric mucosa or blood in the efflux from the tube makes the prognosis more guarded. if an orogastric tube cannot be passed, clip and aseptically scrub the patient's lateral abdomen and then insert 16-gauge over-the-needle catheter. "pinging" the animal's side with simultaneous auscultation allows determination of the location that is most tympanic-that is, the proper location for catheter insertion. once intravenous fluids have been started in the animal, take a right lateral abdominal radiograph to document gdv. if no volvulus is present, the owner may elect for more conservative care, and the animal should be monitored in the hospital for a minimum of 24 hours. because some cases of gdv intermittently twist and untwist, the owner should be cautioned that although the stomach is not twisted at that moment, a volvulus can occur at any time. if radiographs demonstrate food bloat, induce emesis (apomorphine, 0.04 mg/kg iv) or perform orogastric lavage under general anesthesia. documentation of gastric dilatation-volvulus constitutes a surgical emergency. 1 figure 1 -53: example of "food bloat" with severe gastric distention caused by overconsump-following diagnosis of gdv, continue administration of intravenous fluids. serum lactate measurements greater than 6.0 mmol/l are associated with an increased risk of gastric necrosis, requirement for partial gastrectomy, and increased mortality. administer fresh frozen plasma (20 ml/kg) to patients with thrombocytopenia or prolonged pt, activated partial thromboplastin time (aptt), or activated clotting time (act). cardiac dysrhythmias, particularly ventricular dysrhythmias, are common in cases of gdv and are thought to occur secondary to ischemia and proinflammatory cytokines released during volvulus and reperfusion. lidocaine (1-2 mg/kg followed by 50 mcg/kg/minute iv cri) can be used to treat cardiac dysrhythmias preemptively that are associated with ischemia-reperfusion injury, or administration can be started when ventricular dysrhythmias are present. correct any electrolyte abnormalities, including hypokalemia and hypomagnesemia. the use of nonsteroidal antiinflammatory drugs (flunixin meglumine, carprofen, ketoprofen) that can potentially decrease renal perfusion and predispose to gastric ulcers is absolutely contraindicated. administer analgesic drugs (fentanyl, 2 âµ/kg iv bolus, followed by 3-20 âµ/kg/hour iv cri; or hydromorphone, 0.1 mg/kg iv) before anesthetic induction. after carrying out a balanced anesthesia protocol, the patient should be taken immediately to surgery for gastric derotation and gastropexy. postoperatively, assess the patient's ecg, blood pressure, platelet count, coagulation parameters, and gastric function (see section on rule of twenty). if no resection is required, the animal can be given small amounts of water beginning 12 hours after surgery. depending on the severity of the patient's condition, small amounts of a bland diet can be offered 12 to 24 hours postoperatively. continute supportive care with analgesia and crystalloid fluids until the patient is able to tolerate oral analgesic drugs (tramadol, 1-3 mg/kg po q8-12h). once the patient is ambulatory and able to eat and drink on its own, it can be released from the hospital; instruct the owner to feed the animal multiple small meals throughout the day for the first week. when the intestines twist around the root of the mesentery, a small intestinal or mesenteric volvulus occurs. the problem is most common in the young german shepherd dog, although it has been observed in other large and giant breeds. predisposing factors include pancreatic atrophy, gastrointestinal disease, trauma, and splenectomy. clinical signs of mesenteric volvulus include vomiting, hemorrhagic diarrhea, bowel distention, acute onset of clinical signs of shock, abdominal pain, brick-red mucous membranes (septicemia), and sudden death. diagnosis is based on an index of suspicion and the presence of clinical signs in a predisposed breed. plain radiographs often reveal grossly distended loops of bowel in a palisade gas pattern. in some dogs, multiple, tear-drop-shaped, gas-filled loops appear to rise from a focal point in the abdomen. usually, massive distention of the entire small bowel is observed ( fig. 1-55) . the presence of pneumoperitoneum or lack of abdominal detail secondary to the presence of abdominal fluid is characteristic of bowel perforation and peritonitis. in a patient with mesenteric volvulus, immediate aggressive action is necessary for the animal to have any chance of survival. treatment consists of massive volumes of iv crystalloid and colloid fluids (see section on iv therapy), broad-spectrum antibiotics (ampicillin, 22 mg/kg iv qid, with enrofloxacin, 10 mg/kg iv once daily), and surgical correction of the bowel. because of the massive release of proinflammatory cytokines, bacterial translocation, and ischemia, treatment for shock is of paramount importance (see sections on rule of twenty and shock). prognosis for any patient with mesenteric volvulus is poor. obstipation (obstructive constipation) is most common in the older cat. in cases of simple constipation, rehydrating the animal with intravenous fluids and stool softeners is often volvulus. this consistutes an immediate surgical emergency, and the prognosis is often poor. this condition is most common in young german shepherd dogs, but can be observed in any breed. sufficient for it to regain the ability to have a bowel movement. obstipation, however, is caused by adynamic ileus of the large bowel that eventually leads to megacolon. affected cats usually are anorectic, lethargic, and extremely dehydrated. treatment consists of rehydration with intravenous crystalloid fluids, correction of electrolyte abnormalities, enemas, and promotility agents such as cisapride (0.5 mg/kg po q8-24h). the use of phosphate enemas in cats is absolutely contraindicated because of the risk of causing acute, fatal hyperphosphatemia. in many cases, the patient should be placed under general anesthesia and manual deobstipation is performed with warm water soapy enemas and a gloved finger to relieve and disimpact the rectum. stool softeners such as lactulose and docusate stool sofener (dss) may also be used. predisposing causes of obstipation such as narrowing of the pelvic canal, perineal hernia, and tumors should be ruled out. adenocarcinoma is the most common neoplasm of the gastrointestinal tract that causes partial to complete obstruction. adenocarcinomas tend to be annular and constricting, and they may cause progressive obstruction of the lumen of the small or large bowel. siamese cats tend to have adenocarcinomas in the small intestine, whereas in dogs, the tumor tends to occur in the large intestine. clinical signs of adenocarcinoma are both acute and chronic and consist of anorexia, weight loss, and progressive vomiting that occur over weeks to months. effusion may be present if metastasis to peritoneal surfaces has occurred. diagnosis is based on clinical signs and physical examination findings of a palpable abdominal mass, radiographic evidence of an abdominal mass and small or large intestinal obstruction, or ultrasonographic evidence of an intestinal mass. treatment consists of surgical resection of the affected bowel segment. the prognosis for long-term survival (10-12 months) is good if the mass is completely resected and if other clinical signs of cachexia or metastasis are observed at the time of diagnosis. median survival is 15 to 30 weeks if metastasis to lymph nodes, liver, or the peritoneum are absent at the time of diagnosis. in dogs, the prognosis is more guarded. leiomyoma and leiomyosarcoma are tumors that can cause partial or complete obstruction of the bowel. clinical signs are often referred to progressive anemia, including weakness, lethargy, inappetence, and melena. hypoglycemia can be observed as a paraneoplastic syndrome, or due to sepsis and peritonitis secondary to bowel perforation. leiomyomas are most commonly observed at the ceco-colic junction or in the cecum. surgical resection and anastomosis is usually curative, and has a favorable prognosis. incarceration of a loop of bowel into congenital or acquired defects in the body wall can cause small bowel obstruction. pregnant females and young animals with congenital hernias are most at risk. rarely, older animals with perineal hernias and animals of any age with traumatic hernias can be affected. clinical signs are consistent with a small intestinal obstruction: anorexia, vomiting, lethargy, abdominal pain, and weakness. diagnosis is often made based on physical examination of a reducible or nonreducible mass in the body wall. hernias whose contents are reducible are usually asymptomatic. treatment consists of supportive care and rehydration, administration of broad-spectrum antibiotics, and surgical correction of the body wall hernia. in some cases, intestinal resection and anastomosis of the affected area is necessary when bowel ischemia occurs. the potential for bowel perforation should be suspected whenever there is any penetrating injury (knife, gunshot wound, bite wound, stick impalement) of the abdomen. injuries that result in bowel ischemia and rupture can also occur secondary to nonpenetrating blunt 170 1 emergency care trauma or shear forces (e.g., big dog-little dog/cat). perforation of the stomach and small and large intestines can occur with use of nonsteroidal antiinflammatory drugs. diagnosis of bowel perforation first depends on the alertness to the possibility that the bowel may have been perforated or penetrated. as a general rule, all penetrating injuries of the abdomen should be investigated by exploratory laparotomy. diagnostic peritoneal lavage (dpl) can be performed; however, early after penetrating injury of the bowel, dpl may be negative or nondiagnostic until peritonitis develops. whenever any patient with blunt or penetrating abdominal trauma does not respond to initial fluid therapy, or responds and then deteriorates, the index of suspicion for bowel injury should be raised. the findings of pneumoperitoneum on abdominal radiographs or of intracellular bacteria, extracellular bacteria, bile pigment, bowel contents, and cloudy appearance of fluid obtained by abdominocentesis or diagnostic peritoneal lavage fluid (see sections on abdominocentesis and diagnostic peritoneal lavage) warrant immediate surgical exploration. treatment largely consists of stabilizing the patient's cardiovascular and electrolyte status with intravenous fluids, administration of broad-spectrum antibiotics, and definitive surgical exploration and repair of injured structures. prolapse of the rectum is observed most frequently secondary to parasitism and gastrointestinal viral infections in young puppies and kittens with chronic diarrhea. older animals with rectal prolapse often have an underlying problem such as a tumor or mucosal lesion that causes straining and dyschezia. the diagnosis of a rectal prolapse is made based on physical examination findings. the diagnosis of rectal prolapse is sometimes difficult to distinguish from small intestinal intussusception. in rare cases, the intussusception can invaginate through the large bowel, rectum, and anus. the two entities are distinguished from one another by inserting a lubricated thermometer or blunt probe into the cul-de-sac formed by the junction of the prolapsed mucosa and mucocutaneous junction at the anal ring. inability to insert the probe or thermometer indicates that the rectal mucosa is prolapsed. passage of the probe signifies that the prolapsed segment is actually the intussusceptum. treatment can be performed easily if the prolapse is acute and the rectal mucosa is not too irritated or edematous. the presence of severely necrotic tissue warrants surgical intervention. to reduce an acute rectal prolapse, after placing the patient under general anesthesia, lubricate the prolapsed tissue and gently push it back into the rectum, using a lubricated syringe or syringe casing. apply a loose purse-string suture, leaving it in place for a minimum of 48 hours. de-worm the patient and administer stool softeners. if a rectal prolapse cannot be reduced, or if the tissue is nonviable, surgical intervention is warranted. in patients in which viable tissue does not stay reduced with a purse-string suture, a colopexy can be performed during a laparotomy. first, place tension on the colon to reduce the prolapse, and then suture the colon to the peritoneum of the lateral abdominal wall with two to three rows of 2-0 or 3-0 monofilament suture material. if the prolapsed tissue is nonviable, it must be amputated. place four stay sutures at 90-degree intervals through the wall of the prolapse at the mucocutaneous junction. resect the prolapse distal to the stay sutures and then reestablish the rectal continuity by suturing the seromuscular layers together in one circumferential line and the mucosal layers together in the other. replace the suture incision into the anal canal. following surgery, de-worm the patient and administer a stool softener and analgesic drugs. avoid using thermometers or other probes in the immediate postoperative period because they may disrupt suture lines. acute gastritis may be associated with a variety of clinical conditions, including oral hemorrhage, ingestion of highly fermentable nondigestable foods or garbage, toxins, foreign bodies, renal or hepatic failure, inflammatory bowel disease, and bacterial and viral infections. diarrhea often accompanies or follows acute gastritis. hemorrhagic gastroenteritis often occurs as a shock-like syndrome with a rapidly rising hematocrit level. clinical signs of gastritis include depression, lethargy, anterior abdominal pain, excessive water consumption, vomiting, and dehydration. differential diagnosis of acute gastritis includes pancreatitis, hepatic or renal failure, gastrointestinal obstruction, and toxicities (box 1-44). the diagnosis is often a diagnosis of exclusion of other causes (see preceding text). a careful and thorough examination of the vomitus may be helpful in arriving at a diagnosis. a complete blood count, serum biochemistry profile including amylase and lipase, parvovirus test (in young puppies), fecal flotation and cytology, abdominal radiographs (plain and/or contrast studies), and abdominal ultrasound may be warranted to rule out other causes of acute vomiting. while diagnostic tests are being performed, treatment consists of withholding all food and water for a minimum of 24 hours. after calculating the patient's degree of dehydration, administer a balanced crystalloid fluid to normalize acid-base and electrolyte status. control vomiting with antiemetics such as metoclopramide, prochlorperazine, chlorpromazine, dolasetron, and ondansetron (table 1-37). if vomiting is accompanied by diarrhea, administer broad-spectrum antibiotics (cefazolin, 22 mg/kg iv q8h, with metronidazole, 10 mg/kg iv q8h; or ampicillin, 22 mg/kg iv q6h, with enrofloxacin, 10 mg/kg iv q24h) to decrease the risk of bacterial translocation and bacteremia/septicemia. although antacids (famotidine, ranitidine, cimetidine) do not have a direct antiemetic effect, their use can decrease gastric acidity and esophageal irritation during vomiting. if gastritis is secondary to uremia or nonsteroidal antiinflammatory drug use, administer gastroprotectant and antiemetic drugs (ranitidine, 1 mg/kg po q12h; sucralfate, 0.25-1 g/dog po q8h; or omeprazole (0.5-1 mg/kg po q24h) to decrease acid secretion and coat areas of gastric ulceration (table 1 -37) . once food and water can be tolerated, the patient can be placed on an oral diet and medications, and intravenous fluids can be discontinued. do not use until a gastrointestinal obstruction has been ruled out. hemorrhagic gastroenteritis (hge) is an acute onset of severe hemorrhagic vomiting and diarrhea most commonly observed in young small-breed dogs (e.g., poodles, miniature dachshunds, miniature schnauzers) 2 to 4 years of age. clinical signs develop rapidly and include vomiting and fetid diarrhea with hemorrhage, often strawberry jam-like in appearance. the hematocrit can rise from 55% to 75%. often, the animal is extremely hypovolemic but has no apparent signs of abdominal pain. there is no known cause of hge, although clostridium perfringens, escherichia coli, campylobacter, and viral infections have been suggested but not consistently confirmed. other differential diagnoses of of hematemesis and hemorrhagic diarrhea include coronavirus, parvovirus, vascular stasis, sepsis, hepatic cirrhosis with portal hypertension, and other causes of severe shock. immediate treatment consists of placement of a large-bore intravenous catheter and replenishment of intravascular fluid volume with crystalloid fluids (up to 90 ml/kg/hour), while carefully monitoring the patient's hematocrit and total protein. administer broad-spectrum antibiotics (ampicillin, 22 mg/kg iv q6h, and enrofloxacin 10 mg/kg iv q24h) because of the high risk of bacterial translocation and sepsis. control vomiting with antiemetic drugs. monitor the patient's platelet count and coagulation tests for impending disseminated intravascular coagulation (dic), and administer fresh frozen plasma and heparin, as needed (see section on disseminated intravascular coagulation). when vomiting has ceased for 24 hours, offer the animal small amounts of water, and then a bland diet (e.g., boiled chicken and rice or boiled ground beef and rice mixed with low-fat cottage cheese). pancreatitis occurs most frequently in dogs but can occur in cats as well. in dogs, the onset of pancreatitis is sometimes preceded by ingestion of a fatty meal or the administration of drugs (e.g., potassium bromide or glucocorticoids). glucocorticoids can increase the viscosity of pancreatic secretions and induce ductal proliferation, resulting in narrowing and obstruction of the lumen of the pancreatic duct. pancreatitis can also occur following blunt or penetrating abdominal trauma, high duodenal obstruction causing outflow obstruction of the pancreatic papilla, pancreatic ischemia, duodenal reflux, biliary disease, and hyperadrenocorticism. in cats, acute necrotizing pancreatitis is associated with anorexia, lethargy, hyperglycemia, icterus, and sometimes acute death. chronic pancreatitis is more common in cats and results in intermittent vomiting, anorexia, weight loss, and lethargy. predisposing causes of chronic pancreatitis in cats include pancreatic flukes, viral infection, hepatic lipidosis, drugs, organophosphate toxicity, and toxoplasmosis. clinical signs of acute pancreatitis include sudden severe vomiting, abdominal pain, and lethargy. depending on the severity of pancreatic inflammation, depression, hypotension, and systemic inflammatory response syndrome (sirs) may be present. subacute cases may have minimal clinical signs. severe pancreatic edema can result in vascular changes and ischemia that perpetuates severe inflammation. hypovolemic shock and dic can also decrease pancreatic perfusion. severe pancreatic edema, autolysis, and ischemia lead to pancreatic necrosis. duodenal irritation is manifested as both vomiting and diarrhea. pain may be localized to the right upper abdominal quadrant or may be generalized if peripancreatic saponification occurs. differential diagnosis of pancreatitis is the same as for any other cause of vomiting. complications that occur in patients with severe pancreatitis include dehydration, acidbase and electrolyte abnormalities, hyperlipemia, hypotension, and localized peritonitis. hepatic necrosis, lipidosis, congestion, and abnormal architecture can develop. inflammatory mediators (bradykinin, phospholipase a, elastase, myocardial depressant factor, and bacterial endotoxins) stimulate the inflammatory cascade and can lead to sirs, with severe hypotension, clotting system activation, and dic. electrolyte imbalances and hypovolemia secondary to vomiting all can lead to multiple organ dysfunction syndrome (mods), and ultimately, death. if a patient survives an episode of acute pancreatitis, long-term sequelae can include diabetes mellitus. monitor patients with recurrent pancreatitis for clinical signs of polyuria, polydipsia, polyphagia, hyperglycemia, and glucosuria. the diagnosis of pancreatitis is based on the presence of clinical signs (which may be absent in cats), laboratory findings, and ultrasonographic evidence of pancreatic edema and increased peripancreatic echogenicity. serum biochemistry analyses can sometimes support a diagnosis of pancreatitis; however, serum amylase and lipase are often unreliable indicators of pancreatitis, depending on the chronicity of the process in the individual patient. both serum amylase and lipase are excreted in the urine. impaired renal clearance/ function can cause artifactual elevations of serum amylase and lipase in the absence of pancreatic inflammation. furthermore, serum lipase levels can be elevated as a result of gastrointestinal obstruction (e.g., foreign body). early in the course of the disease, levels can be two to six times normal, but they may decrease to within normal ranges at the time of presentation to the veterinarian. the transient nature of amylase elevation makes this test difficult to interpret, and it is not highly sensitive if a normal value is found. lipase levels also increase later in the course of the disease. amylase and lipase should be tested concurrently with the rest of the biochemistry profile. other changes often observed are elevations in bun and creatinine levels secondary to dehydration and prerenal azotemia, hyperglycemia, and hyperlipemia. hypocalcemia can occur secondary to peripancreatic fat saponification, and its presence warrants a more negative prognosis. a more specific measure is pancreatic lipase immunoreactivity, which becomes elevated in dogs and cats with pancreatitis. this test, combined with ultrasonographic or computed tomography evidence of pancreatitis, is the most sensitive and specific test available for making an accurate diagnosis. however, because the results of this test take time to obtain, animals must be treated in the meantime. abdominal effusion or fluid from diagnostic peritoneal lavage can be compared with serum amylase and lipase activity. abdominal lipase and amylase concentrations in the fluid greater than that in the peripheral blood are characteristic of chemical peritonitis associated with pancreatitis. wbc counts greater than 1000 cells/mm 3 , the presence of bacteria, toxic neutrophils, glucose levels less than 50 mg/dl, or lactate levels greater than that of serum are characteristic of septic peritonitis, and immediate exploratory laparotomy is warranted. if a biopsy sample obtained during laparotomy does not demonstrate inflammation, but this does not rule out pancreatitis, because disease can be focal in nature and yet cause severe clinical signs. abdominal radiographs may sometimes reveal a loss of abdominal detail or a ground glass appearance in the right upper quadrant. pancreatic edema and duodenal irritation can displace the gastric axis toward the left, toward the left with dorsomedial displacement of the proximal duodenum (the so-called "backwards 7" or "shepherd's crook" sign). ultrasonography and ct are more sensitive in making a diagnosis of pancreatitis. treatment of pancreatitis is largely supportive in nature and is designed to correct hypovolemia and electrolyte imbalances, prevent or reverse shock, maintain vital organ perfusion, alleviate discomfort and pain, and prevent vomiting (see section on rule of twenty). when treating pancreatitis in dogs, all food and water should be restricted. however, food should not be withheld from cats with chronic pancreatitis. give fresh frozen plasma to replenish alpha-2-macroglobulins. administer antiemetics such as chlorpromazine (use with caution in a hypovolemic or hypotensive patient), dolasetron, ondansetron, or metoclopramide to prevent or control vomiting. analgesic drugs can be provided in the form of constant rate infusion (fentanyl, 3-7 âµ/kg/hour iv cri, and lidocaine, 30-50 âµ/kg/minute iv cri), intrapleural injection (lidocaine, 1-2 mg/kg q8h), or intermittent parenteral injections (morphine, 0.25-1 mg/kg sq, im; hydromorphone, 0.1 mg/kg im or sq). because the pancreas must be rested, consider using parenteral nutrition. acute hepatic failure may be associated with toxins, adverse reaction to prescription medication, and bacterial or viral infections. the most frequent clinical signs observed in a patient with acute hepatic failure are anorexia, lethargy, vomiting, icterus, bleeding, and cns depression or seizures (associated with hepatic encephalopathy). differential diagnosis and causes of acute hepatic failure are listed in box 1-45. diagnosis of acute hepatic failure is based on clinical signs and biochemical evidence of hepatocellular (ast, alt) and cholestatic (alk phos, t bili, ggt) enzyme elevations. ultrasonography may be helpful in distinguishing the architecture of the liver, but unless a mass or abscess is present, cannot provide a specific diagnosis of the cause of the hepatic damage. management of the patient with acute hepatic failure includes correction of dehydration and acid-base and electrolyte abnormalities, as shown in the following list: â�¢ hypoalbuminemia: plasma or concentrated albumin. plasma also is an excellent source of clotting factors that can become depleted. â�¢ clotting abnormalities: vitamin k 1 (2.5 mg/kg sq or po q8-12h) to â�¢ severe anemia: fresh or stored blood â�¢ gastric hemorrhage: gastroprotectant drugs (omeprazole, ranitidine, famotidine, cimetidine, sucralfate) â�¢ hypoglycemia: dextrose supplementation (2.5%-5%) â�¢ hepatic failure, particularly when hypoglycemia is present: broad-spectrum antibiotics (ampicillin 22 mg/kg iv q6h; with enrofloxacin, 5 mg/kg iv q24h) â�¢ hepatic encephalopathy: lactulose or betadine enemas â�¢ cerebral edema: mannitol (0.5-1.0 g/kg iv over 10 to 15 minutes) followed by furosemide (1 mg/kg iv 20 minutes later). deterioration of clinical signs may signify the development of cerebral edema. applewhite aa, cornell kk, selcer ba: diagnosis and treatment of intussusception in dogs. comp cont educ pract vet 24 (2) often, systemic hypertension is diagnosed when the animal is seen by the veterinarian because of some other clinical sign, such as acute blindness, retinal detachment, hyphema, epistaxis, and cns signs following intracranial hemorrhage. diagnosis of systemic hypertension is often difficult in the absence of clinical signs and without performing invasive or noninvasive blood pressure monitoring. normal blood pressure (bp) measurements in dogs and cats are listed in table 1-38. hypertension is defined as a consistent elevation in systolic bp >200 mm hg, consistent diastolic bp >110 mm hg, and consistent mean arterial blood pressure >130 mm hg. the effects of systemic hypertension include left ventricular hypertrophy, cerebrovascular accident, renal vascular injury, optic nerve edema, hyphema, retinal vascular tortuosity, retinal hemorrhage, retinal detachment, vomiting, neurologic defects, coma, and excessive bleeding from cut surfaces. 176 1 emergency care dog 100-160 80-120 90-120 cat 120-150 70-130 100-150 patients with systemic hypertension should have a thorough diagnostic work-up to determine the underlying cause. although uncommon, hypertensive emergencies can occur with pheochromocytoma, acute renal failure, and acute glomerulonephritis. sodium nitroprusside (1-10 âµ/kg/minute iv cri) or diltiazem (0.3-0.5 mg/kg iv given slowly over 10 minutes, followed by 15 âµ/kg/minute) can be used to treat systemic hypertension. with the use of sodium nitroprusside or diltiazem, monitor carefully for hypotension. diagnosis is based on consistent elevations in systolic, diastolic, and/or mean arterial bp. because many of the clinical signs associated with systemic hypertension involve hemorrhage into some closed cavity, other causes of hemorrhage, such as vasculitis, thrombocytopenia, thrombocytopathia, and hepatic or renal failure, should be investigated (see section on coagulation disorders). diagnostic testing is based on clinical signs and index of suspicion for an underlying disease and may include a complete blood count; urinalysis; urine protein:creatinine ratio; acth stimulation test; thoracic and abdominal radiographs; thoracic and abdominal ultrasound; tick serology; brain ct or mri; and assays of serum electrolytes, aldosterone concentration, t4, endogenous tsh, plasma catecholamine, and growth hormone. management of systemic hypertension involves treatment of the primary underlying disorder, whenever possible. long-term adjunctive management includes sodium restriction in the form of cooked or prescription diets to decrease fluid retention. obese animals should be placed on dietary restrictions and undergo a weight reduction program. thiazide and loop diuretics may be used to decrease sodium retention and circulating blood volume. alpha-and beta-adrenergic blockers may be used, but they are largely ineffective as monotherapeutic agents for treating hypertension. calcium channel blockers and angiotensin-converting enzyme (ace) inhibitors are the mainstay of therapy in the treatment of hypertension in dogs and cats ( diabetic ketoacidosis (dka) is a potentially fatal and terminal consequence of unregulated insulin deficiency and possible glucagon excess. in the absence of insulin, unregulated lipolysis results in the beta-hydroxylation of fatty acids by abnormal hepatic metabolism. as a result, ketoacids-namely, acetoacetic acid, beta-hydroxybutyric acid, and acetoneare produced. early in the course of the disease, patients exhibit clinical signs associated with diabetes mellitus: weight loss, polyuria, polyphagia, and polydipsia. later, as ketoacids stimulate the chemoreceptor trigger zone, vomiting and dehydration occur, with resulting hypovolemia, hypotension, severe depression, abdominal pain, oliguria, and coma. at the time of presentation, often a strong odor of ketones (acetone) is present on the patient's breath. physical examination often reveals dehydration, severe depression or coma, and hypovolemic shock. in extreme cases, the patient exhibits a slow, deep kussmaul respiratory pattern in an attempt to blow off excess co 2 to compensate for the metabolic acidosis. a serum biochemistry profile and complete blood count often reveal prerenal azotemia, severe hyperglycemia (blood glucose >400 mg/dl), hyperosmolarity (>330 mosm/kg), lipemia, hypernatremia (sodium >145 meq/l), elevated hepatocellular and cholestatic enzyme activities, high anion gap, and metabolic acidosis. although a whole body potassium deficit is usually present, the serum potassium may appear artifactually elevated in response to metabolic acidosis. with severe metabolic acidosis, potassium moves extracellularly in exchange for a hydrogen ion. phosphorus too moves intracellularly in response to acidosis, and serum phosphorus is usually decreased. hypophosphatemia >2 mg/dl can result in intravascular hemolysis. urinalysis often reveals 4+ glucosuria, ketonuria, and a specific gravity of 1.030 or greater. the urine of all diabetic animals should be cultured to rule out a urinary tract infection or pyelonephritis. treatment of a patient with dka presents a therapeutic challenge. treatment is aimed at providing adequate insulin to normalize cellular glucose metabolism, correcting acidbase and electrolyte imbalances, rehydration and restoration of perfusion, correcting acidosis, providing carbohydrate sources for utilization during insulin administration, and identifying any precipitating cause of the dka. obtain blood samples for a complete blood count, and serum biochemistry electrolyte profiles. whenever possible, insert a central venous catheter for fluid infusion and procurement of repeat blood samples. calculate the patient's dehydration deficit and maintenance fluid requirements and give appropriate fluid and electrolytes over a period of 24 hours. it is advisable to rehydrate patients with severe hyperosmolarity for a minimum of 6 hours before starting insulin administration. use a balanced electrolyte solution (e.g., plasmalyte-m, normosol-r, lactated ringer's solution) or 0.9% saline solution for maintenance and rehydration. balanced electrolyte solutions contain small amounts of potassium and bicarbonate precursors that aid in the treatment of metabolic acidosis. treat animals with severe metabolic acidosis with an hco 3 â�� >11 meq/l or a ph <7.1 with supplemental bicarbonate (0.25-0.5 meq/kg). add supplemental dextrose to the patient's fluids as a carbohydrate source during insulin infusion. both insulin and carbohydrates are necessary for the proper metabolism of ketone bodies in patients with dka. the rate and type of fluid and amount of dextrose supplementation will change according to the patient's blood glucose concentration. serum potassium will drop rapidly as the metabolic acidosis is corrected with fluid and insulin administration. measure serum potassium every 8 hours, if possible, and supplement accordingly (see section on fluid therapy for chart of potassium supplementation). if the patient's potassium requirement exceeds 100 meq/l, or if the rate of potassium infusion approaches 0.5 meq/ kg/hour in the face of continued hypokalemia, magnesium should be supplemented. magnesium is required as a cofactor for many enzymatic processes and for normal function of the na,k-atpase pump. hypomagnesemia is a common electrolyte disturbance in many forms of critical illness. replenishing magnesium (mgcl 2 , 0.75 meq/kg/day iv cri) often helps to correct the refractory hypokalemia observed in patients with dka. patients with hypophosphatemia that approaches 2.0 mmol/l should receive potassium phosphate (0.01-0.03 mmol/kg/hour iv cri). when providing potassium phosphate supplementation, be aware of the additional potassium added to the patient's fluids, so as to not exceed recommended rates of potassium infusion. to determine the amount of potassium chloride (kcl) to add along with potassium phosphate (kpo 4 ), use the following formula: meq k + derived from kcl = total meq of k + to be administered over 24 hours â�� meq in which k + is derived from kpo 4 clinical signs of severe hypophosphatemia include muscle weakness, rhabdomyolysis, intravascular hemolysis, and decreased cerebral function that can lead to depression, stupor, seizures, or coma. regular insulin can be administered either im or as a constant rate infusion in the treatment of patients with dka. subcutaneous insulin should not be administered. because of the severe dehydration present in most patients with dka, subcutaneous insulin is poorly absorbed and is not effective until hydration has been restored. in the low-dose intravenous method, place regular insulin (1.1 units/kg for a cat, and 2.2 units/kg for a dog) in 250 ml of 0.9% saline solution. run 50 ml of this mixture through the intravenous line to allow the insulin to adsorb to the plastic tubing. administer the patient's insulin fluid rate according to blood glucose levels ( table 1 -40) . adjust the patient's total fluid volume according to changes in the insulin fluid rate as necessary. in many cases, multiple bags of fluids are necessary because they must be changed when fluctuations in blood glucose concentrations occur in response to therapy. infusion of the insulin mixture should be in a separate intravenous catheter. to replenish hydration, use a second intravenous line for the more rapid infusion of non-insulin-containing fluids. to administer the regular insulin im, first give 0.22 unit/kg im and then re-check the patient's blood glucose every hour. additional injections of regular insulin (0.11 unit/kg other fluid type (ml/hour) >250 10 0.9% nacl 200-250 7 0.45% nacl + 2.5% dextrose 150-200 5 0.45% nacl + 2.5% dextrose 100-150 5 0.45% nacl + 2.5% dextrose <100 0 0.45% nacl + 5% dextrose im) should be administered based on the patient's response to subsequent injections. once the patient's blood glucose falls to 200 to 250 mg/dl, add 2.5% to 5% dextrose to the fluids to maintain the blood glucose concentration at 200 to 300 mg/dl. continue intramuscular injection of regular insulin (0.1-0.4 unit/kg q4-6h) until the patient is rehydrated, no longer vomiting, and able to tolerate oral fluids and food without vomiting. even in patients with intramuscular regular insulin therapy, a central venous catheter should be placed for frequent blood sample collection. as the patient begins to respond to therapy, monitor electrolytes, glucose, and acid-base status carefully. hypokalemia, hypophosphatemia, and hypomagnesemia can occur. when the patient's hydration and acid-base status has normalized and the patient is able to tolerate oral food and water, a longer-acting insulin can be administered as for treatment of a patient with uncomplicated diabetes. extreme hyperosmolarity can result in a coma, if uncorrected. in patients with diabetes mellitus, hyperglycemia and hypernatremia secondary to osmotic diuresis and free water loss can lead to severe hyperosmolarity. in dogs, normal serum osmolality is <300 mosm/l of serum. hyperosmolarity is expected when serum osmolality is >340 mosm/l. if equipment for determining serum osmolarity is not available, osmolarity can be calculated by the following formula: osm/l = 2(na + k) + (glucose/18) + (bun/2.8) patients with severe dehydration, hyperglycemia, hypernatremia, and azotemia may experience cerebral edema without ketonemia. treatment is directed solely at rehydrating the patient and slowly reducing blood glucose levels using a hypotonic solution such as 0.45% nacl + 2.5% dextrose or 5% dextrose in water (d 5 w). after the initial rehydration period, administer potassium supplementation conservatively. red blood cells and the brain absolutely depend on the oxidation of glucose for energy. hypoglycemia can be caused by various systemic abnormalities that can be related to intestinal malabsorption of nutrients, impaired hepatic glycogenolysis or gluconeogenesis, and inadequate peripheral utilization of glucose. clinical signs of hypoglycemia are extremely variable and can include weakness, tremors, nervousness, polyphagia, ataxia, tachycardia, muscle twitching, incoordination, visual disturbances, and generalized seizures. clinical signs typically occur when serum glucose levels are <60 mg/dl. the combination of the clinical signs listed previously, documentation of low serum glucose, and alleviation of clinical signs upon glucose administration is known as whipple's triad. whenever a patient presents with hypoglycemia, consider the following important factors: the age of onset, the nature of the hypoglycemic episode (transient, persisent, or recurrent) , and the pattern based on the patient's history . treatment of hypoglycemia is directed at providing glucose supplementation and determining any underlying cause. administer supplemental dextrose (25%-50% dextrose, 2-5 ml/kg iv; or 10% dextrose, 20 ml/kg po) as quickly as possible. do not attempt oral glucose supplementation in any patient having a seizure or if the airway cannot be protected. administer intravenous fluids (e.g., normosol-r, lactated ringer's solution, 0.9% saline solution) with 2.5%-5% supplemental dextrose until the patient is eating and able to maintain euglycemia without supplementation. in some cases (e.g., insulinoma), eating or administration of supplemental dextrose can promote insulin secretion and exacerbate clinical signs and hypoglycemia. in cases of refractory hypoglycemia secondary to iatrogenic insulin overdose, glucagon (50 mg/kg iv bolus, then 10-40 ng/kg/minute iv cri) can also be administered along with supplemental dextrose. to make a glucagon infusion of 1000 ng/ml, reconstitute 1 ml (1 mg/ml) of glucagon according to the manufacturer's instructions and add this amount to 1000 ml of 0.9% saline solution. 1 emergency care the diagnosis of eclampsia (puerperal tetany) is often made on the basis of history and clinical signs. clinical signs can become evident when total calcium decreases to <8.0 mg/dl in dogs and <7.0 mg/dl in cats. the disease is often observed in small, excitable dogs, and stress may play a complicating role in the etiology. in most bitches, the disease manifests itself 1 to 3 weeks after parturition. in some cases, however, clinical signs can develop before parturition occurs. hypophosphatemia may accompany hypocalcemia. clinical signs of hypocalcemia include muscle tremors or fasciculations, panting, restlessness, aggression, hypersensitivity, disorientation, muscle cramping, hyperthermia, stiff gait, seizures, tachycardia, a prolonged qt interval on ecg, polydipsia, polyuria, and respiratory arrest. treatment of eclampsia consists of slow, cautious calcium supplementation (10% calcium gluconate, 0.15 mg/kg iv over 30 minutes). severe refractory tetanus can be controlled with intravenous diazepam. supportive care includes intravenous fluid administration and cooling (see section on hyperthermia and heat-induced illness). instruct the owner to give the patient oral calcium supplements (e.g., 1 to 2 tablets of tums bid-tid) after discharge from the hospital. also instruct the owner about how to wean the puppies, allowing the bitch to dry up, in order to prevent recurrence. recurrence with subsequent pregnancies is common, particularly in patients that receive calcium supplementation during gestation (table 1-41) . hypercalcemia can occur from a variety of causes. the gosh darn it mnemonic can be used to remember the various causes of hypercalcemia in small animal patients (box 1-47) . the gastrointestinal, renal, and nervous systems are most commonly affected, particularly when serum total calcium rises above 16.0 mg/dl. clinical signs of severe hypercalcemia include muscle weakness, vomiting, seizures, and coma. ecg abnormalities include prolonged pr interval, rapid qt interval, and ventricular fibrillation. the most serious clinical signs are often seen when hypercalcemia is observed in combination with hyperphosphatemia or hypokalemia. pay special attention to the "calcium ã� phosphorus product." if this product exceeds 70, dystrophic calcification can occur, leading to renal failure. renal complications include polyuria, polydipsia, dehydration, and loss of renal tubular concentrating ability. renal blood flow and the glomerular filtration rate (gfr) are impaired when serum total calcium exceeds 20 mg/dl. the extent, location, and number of renal tubular injuries are the main factors in determining whether renal damage secondary to hypercalcemia is reversible or irreversible. emergency therapy of hypercalcemia is warranted when severe renal compromise, cardiac dysfunction, or neurologic abnormalities are present, or if no clinical signs occur but the calcium ã� phosphorus product exceeds 70. the treatment of choice is correction of the underlying cause of hypercalcemia, whenever possible. in some cases, the results of diagnostic tests take time, and emergency therapy should be initiated immediately, before a definitive cause of the hypercalcemia is found. emergency management of hypercalcemia consists of reduction of serum calcium levels. administer intravenous fluids (0.9% saline solution) to expand extracellular fluid volume and promote calciuresis. to promote diuresis, initial intravenous fluid rates should approach two to three times maintenance levels (120-180 ml/kg/day). potassium supplementation may be required to prevent iatrogenic hypokalemia. administration of a loop diuretic such as furosemide (2-5 mg/kg iv) will promote calcium excretion. calcitonin (4 iu/kg im q12h for cats and 8 iu/kg im q24h for dogs) can be administered to decrease serum calcium levels. in severe refractory hypercalcemia secondary to cholecalciferol toxicity, more aggressive calcitonin therapy (4-7 iu/kg sq q6-8h) can be attempted. side effects of calcitonin treatment include vomiting and diarrhea. alternatively, bisphosphonates (pamidronate, 1.02-2.0 mg/kg iv) are useful in rapidly reducing serum calcium concentrations. glucocorticosteroids reduce calcium release from the bone, decrease intestinal absorption of calcium, and promote renal calcium excretion. administer glucocorticosteroids only after the underlying cause of hypercalcemia has been determined and appropriate therapy started. because many forms of neoplasia can result in hypercalcemia as a paraneoplastic syndrome, empiric use of glucocorticosteroids can induce multiple drug resistance, making the tumor refractory to the effects of chemotherapeutic agents. hypoadrenocorticism is most commonly observed in young to middle-aged female dogs, but it can occur in animals of any age, gender, and breed. clinical signs, which are referable to deficiency in glucocorticoid (cortisol) and mineralocorticoid (aldosterone) hormones, may develop slowly over time, leading to a waxing and waning course; acute clinical signs occur when >90% of the adrenal functional reserve has been destroyed. in such cases, complete adrenocortical collapse can result in an addisonian crisis. lack of aldosterone causes a lack of renal sodium and water retention, and impaired potassium excretion. the most significant clinical signs associated with hypoadrenocorticism are depression, lethargy, weakness, anorexia, shaking, shivering, vomiting, diarrhea, weight loss, abdominal pain, weakness, hypotension, dehydration, and inappropriate bradycardia (box 1-48) . the diagnosis of hypoadrenocorticism is made based on the patient's clinical signs in combination with electrolyte abnormalities that include hyperkalemia, hyponatremia, and hypochloremia. serum sodium concentration (115-130 meq/l) is often greatly reduced, and serum potassium is elevated (>6.0 meq/l). a sodium:potassium ratio of <27 is characteristic of hypoadrenocorticism, although not exactly pathognomonic. electrocardiographic changes associated with hyperkalemia include inappropriate bradycardia, absence of p waves, elevated spiked t waves, and widened qrs complexes. other more variable bloodwork abnormalities include a lack of a stress leukogram, eosinophilia, hypoglycemia, hyperphosphatemia, hypercalcemia, azotemia, and hypocholesterolemia. a definitive diagnosis of hypoadrenocorticism is based on an adrenocorticotropic hormone (acth) stimulation test. in patients with hypoadrenocorticism, baseline cortisol levels are usually low, with a lack of appropriate cortisol release after administration of acth analogue. rarely, animals with "atypical" hypoadrenocorticism lose glucocorticoid secreting ability from the zona fasciculata, but retain mineralocorticoid secretory ability from the zona glomerulosa. atypical addisonian patients have normal serum electrolytes but still have clinical signs of vomiting, diarrhea, weakness, lethargy, inappetence, muscle wasting, and weight loss. the diagnosis is more difficult in such cases because of the presence of normal electrolytes. an acth stimulation test should be considered, particularly in predisposed breeds. treatment of hypoadrenocorticism includes placement of a large-bore intravenous catheter, infusion of intravenous crystalloid fluids (0.9% saline solution), and replenishment of glucocorticoid and mineralocorticoid hormones. administer dexamethasone or dexamethasone-sodium phosphate (0.5-1.0 mg/kg iv). dexamethasone will not interfere with the acth stimulation test, unlike other longer-acting steroids (e.g., prednisolone, methylprednisolone sodium succinate, triamcinolone). depending on the severity of the patient's condition, consider monitoring using the rule of twenty. administer antiemetics and gastroprotectant drugs to treat nausea, vomiting, and hematemesis. give the patient broad-spectrum antibiotics (ampicillin, 22 mg/kg iv q6h) if hematochezia or hemorrhagic diarrhea is present. if severe gastrointestinal blood loss occurs, whole blood, packed red blood cells, or fresh frozen plasma may be required. control hypoglycemia with 2.5%-5.0% dextrose. use sodium bicarbonate, regular insulin with dextrose, or calcium gluconate to correct severe hyperkalemia with atrial standstill (see section on atrial standstill). chronic therapy for hypoadrenocorticism consists of mineralocorticoid and glucocorticosteroids supplementation for the rest of the animal's life. mineralocorticoid supplementation can be in the form of desoxycorticosterone pivalate (docp) (2.2 mg/kg im) or fludrocortisone acetate (0.1 mg/2.5-5 kg body weight daily). fludrocortisone acetate possesses both mineralocorticoid and glucocorticoid activities and can be used as the sole daily treatment of hypoadrenocorticism. (because fludrocortisone is poorly absorbed in some dogs, it may not completely normalize electrolyte abnormalities in these animals.) docp is primarily a mineralocorticoid. give supplemental glucocorticosteroids in the form of prednis(ol)one (1-0.25 mg/kg/day). in dogs, iatrogenic hypoadrenocorticism can be caused by abrupt discontinuation of glucocorticosteroid treatment. long-term glucocorticosteroid supplementation can downregulate the pituitary gland's excretion of endogenous acth and the zona fasciculata's ability to excrete cortisol. however, the zona glomerulosa's ability to secrete aldosterone does not appear to be affected. clinical signs of iatrogenic hypoadrenocorticism include inability to compensate for stress, weakness, lethargy, vomiting, diarrhea, and collapse. treatment of iatrogenic hypoadrenocorticism is the same as for naturally occurring disease. following immediate emergency treatment, the patient should be weaned slowly from exogenous glucocorticosteroid supplementation. severe hyperthyroidism can manifest as a medical emergency as a result of hypermetabolism. clinical signs in affected cats with severe thyrotoxicosis include fever, severe tachycardia (heart rate >240 bpm), vomiting, hypertension, congestive heart failure with pulmonary edema, and fulminant collapse. clinical signs typically are manifested as an end-stage of chronic debilitation associated with hyperthyroidism and are often preceded by polyphagia, weight loss, cardiac murmur, polyuria/polydipsia (pu/pd), vomiting, and diarrhea. treatment of thyrotoxicosis includes antagonizing the adrenergic activity by administration of a beta-adrenergic blocker (esmolol, (25-50 âµ/kg/minute, or propranolol, 0.02 mg/ kg/hour). administration of glucocorticosteroids (dexamethasone, 1 mg/kg) may inhibit the conversion of thyroxine (t 4 ) to the active form triiodothyronine (t 3 ) and decrease peripheral tissue responsiveness to t 3 , effectively blocking its effects. correct hypoglycemia with supplemental dextrose (2.5%). use care to avoid overhydration in a patient with cardiac failure or insufficiency. start the patient on methimazole as quickly as possible and consider the use of radioactive iodine therapy. to maintain cerebral perfusion pressure, blood pressure must be normalized. if other concurrent injuries are suspected (e.g., pulmonary contusions), administer synthetic colloid fluids (dextran-70, 5-10 ml/kg iv, or hetastarch, 5-10 ml/kg iv) to normalize blood pressure. although the use of colloids is controversial because of their potential to leak into the calvarium, the benefits of reestablishing cerebral perfusion far outweigh the risks of their use. hypertonic saline (7.5% nacl, 3-5 ml/kg iv) can also be administered over 10 to 15 minutes to expand intravascular volume. maintain blood glucose within normal reference ranges whenever possible, because hyperglycemia is a negative prognostic indicator in cases of head trauma. if tremors or seizures cause hyperthermia or increased metabolism, active cooling of the patient is warranted (see sections on hyperthermia and heat-induced injury). all patients with head trauma should receive care and monitoring based on the rule of twenty (see section on rule of twenty). examine the patient's level of consciousness, response to various stimuli, pupil size and reactivity to light, physiologic nystagmus, and cranial nerve deficits. in dogs, damage to the midbrain often produces coma and decerebrate rigidity. initial consciousness followed by a unconsciousness or stupor usually involves an injury to the brainstem. brainstem lesions can be caused by compressive skull fractures, extradural or subdural hematomas, or herniation through the foramen magnum from cerebral edema (box 1-49) . the patient's pupil size and response to light can be used to localize a diagnosis and give a rough prognosis for severity of disease and possibility for return to function. pupils can be normal in size, mydriatic, or miotic. whenever a pupil appears miotic, direct ocular 186 1 emergency care unconscious with no response to noxious stimuli injury with uveitis or secondary miosis due to brachial plexus injury should be ruled out. the eyes should always be examined to rule out ocular trauma. in a patient with head trauma, a change from dilated to constricted to normal pupil size is suggestive of improvement in clinical function. bilateral mydriatic pupils that are unresponsive to light in an unconscious animal are a grave prognostic sign and usually indicate an irreversible severe midbrain contusion. bilateral miotic pupils with normal nystagmus and ocular movements are associated with diffuse cerebral or diencephalic lesions. miotic pupils that become mydriatic indicate a progressive midbrain lesion with a poor prognosis. unilateral, slowly progressive pupillary abnormalities in the absence of direct ocular injury are characteristic of brainstem compression or herniation caused by progressive brain swelling. asymmetric pupils are seen in patients with rostral brainstem lesions and can change rapidly. unresponsive pupils that are seen in the midposition occur with brainstem lesions that extend into the medulla and are a grave sign. visual deficits are common with intracranial injury. lesions that are less severe and limited to the cerebrum produce contralateral menace deficits with normal pupillary light response. bilateral cerebral edema can cause blindness with a normal response to light if the midbrain is not disturbed. a patient that is severely depressed and recumbent may not respond to menacing gestures, even when visual pathways are intact. ocular, optic tract, optic nerve, or optic chiasm lesions can interfere with vision and the pupillary light response. brainstem contusion and cerebral edema may produce blindness and dilated unresponsive pupils due to disturbance of the oculomotor area. examine all cranial nerves carefully. cranial nerve abnormalities can indicate direct contusion or laceration of the neurons in the brainstem or where they exit the skull. cranial nerves that are initially normal then later lose function indicate a progressively expanding lesion. when specific cranial nerve deficits are present, the prognosis is considered guarded. clinical signs such as rolling to one side, torticollis, head tilt, and abnormal nystagmus are usually associated with petrosal bone or cerebellomedullary lesions that produce vestibular neuron dysfunction. fractures of the petrosal temporal bone often cause hemorrhage and cerebrospinal fluid (csf) leak from the external ear canal. if the lesion is limited to the membranous labyrinth, the loss of balance will be toward the injured side and the quick phase of the nystagmus will be toward the injured side. normal physiologic nystagmus requires that the pathway is between the peripheral vestibular neurons and the pontomedullary vestibular nuclei to the nuclei of the cranial nerves that innervate the extraocular muscles (iii, iv, vi). severe brainstem lesions disrupt this pathway. disruption of the pathway is manifested as an inability to produce normal physiologic nystagmus by moving the patient's head from side to side. in patients with severe central nervous system depression, this reflex may not be observed. next, assess postural changes and motor function abilities. a loss of the normal oculocephalic ("dolls-eye") reflex is an early sign of brainstem hemorrhage and a late sign of brainstem compression and herniation. any intracranial injury may be accompanied by a concurrent cervical spinal cord injury. handle animals with such injuries with extreme care to avoid causing further damage. whenever there is uncertainty whether a spinal cord lesion exists, strap the patient down to a flat surface and obtain radiographs of the spine. at least two orthogonal views may be required to see fractures; however, do not manipulate the patient until radiography has been completed. crosstable views, in which the bucky is turned perpendicular to the patient's spine, with a radiograph plate secured behind the patient, may be required to minimize patient motion. in patients with cerebral lesions, hemiparesis usually resolves within 1 to 3 days. evaluation of cranial nerve function at frequent intervals may reveal an initial injury or a progressively expanding lesion in the brain. signs of vestibular disorientation, marked head tilt, and abnormal nystagmus occur with contusions of the membranous labyrinth and fracture of the petrous temporal bone. hemorrhage and cerebrospinal fluid otorrhea may be visible from the external ear canal. rolling movements indicate an injury to the cerebellar-medullary vestibular system. respiratory dysfunction and abnormal respiratory patterns are sometimes observed with severe head injury. lesions of the diencephalon produce cheyne-stokes respirations, in which the patient takes progressively larger and larger breaths, pauses, then takes progressively smaller and smaller breaths. mesencephalic lesions cause hyperventilation and can result in respiratory alkalosis. medullary lesions result in a choppy, irregular respiratory pattern. clinical signs of respiratory dysfunction in the absence of primary respiratory damage indicate a guarded prognosis. after injury, seizures may be associated with intracranial hemorrhage, trauma, or an expanding intracranial mass lesion. immediately begin medical therapy to control the seizure. administer diazepam (0.5 mg/kg iv or 0.1-0.5 mg/kg/hour iv cri) to treat seizures. if diazepam is not effective in combination with other treatments to control intracranial edema, consider giving pentobarbital . loading doses of phenobarbital (16-20 mg/kg iv divided into 4 or 5 doses, given every 20 to 30 minutes) may be beneficial in preventing further seizures. severe refractory seizures or decreased mentation may be associated with cerebral edema and increased intracranial pressure. mannitol, an osmotic diuretic, is effective at reducing cerebral edema (0.5-1.0 g/kg iv over 10 to 15 minutes). mannitol also acts as a free radical scavenger that can inhibit the effects of cerebral ischemia-reperfusion injury. mannitol works synergistically with furosemide (1 mg/kg iv given 20 minutes after the mannitol infusion). corticosteroids have not been demonstrated to be beneficial in the treatment of head trauma and may induce hyperglycemia. hyperglycemia has been shown to be a negative prognostic indicator in cases of head trauma. also, glucocorticoids can suppress immune system function and impair wound healing. because of the known risks and lack of known benefits of glucocorticosteroids, their use in treatment of head trauma is contraindicated. the prognosis for any patient with severe head trauma is guarded. management of head trauma patients may include intense nursing care for a period of weeks to months, depending on the presence and extent of concurrent injuries. if progressive loss of consciousness occurs, surgery for decompression of compressive skull injuries should be considered. the most common injury associated with head trauma in small animals is a contusion with hemorrhage in the midbrain and pons. subdural or extradural hemorrhage with space-occupying blood clots is uncommon. diagnostic tests of head trauma may include skull radiographs, ct, and mri of the brain. special studies can help detect edema and hemorrhage in the brain and brainstem, and aid in making an accurate diagnosis and prognosis. a cerebrospinal fluid tap is contraindicated in patients with head trauma because of the risk of causing a rapid decrease in intracranial pressure and brainstem herniation. if a compressive skull fracture is present, the patient should be stabilized for surgery to remove the compression. surgery to alleviate increased intracranial pressure is rarely performed in veterinary medicine because of the poor prognosis and results. in some cases, when a lesion can be localized to one area, 1-to 2-cm burr holes can be placed through the skull over the affected area of the cerebrum, exposing the underlying brain tissue. blood clots can be removed through the holes. the bone flap may or may not be replaced, depending on the surgeon's preference and the degree of brain swelling. spinal cord injuries may be associated with trauma, disk rupture, fractures, and dislocation of the spinal column. proceed with caution when moving a patient with suspected spinal cord injury. avoid flexion, extension, and torsion of the vertebral column. all animals that are unconscious following a traumatic event should be considered to have cervical or thoracolumbar spinal injury until proved otherwise by radiography, ct, or mri. the animal should be moved onto a flat surface (e.g., board, door, window, picture frame) and taped down to prevent motion and further displacement of vertebrae. sedation with analgesics or tranquilizers may be necessary to keep the animal immobile and to minimize patient motion. whenever possible, avoid the use of narcotics in patients with head trauma because of the risk of increasing intracranial pressure. as in other emergencies, the abcs 188 1 emergency care should be evaluated, and the patient treated for shock, hemorrhage, and respiratory compromise. once the cardiovascular and respiratory systems have been evaluated and stabilized, a more thorough neurologic examination can be performed. protrusion of an intervertebral disk indicates that the disk is bulging into the vertebral canal as a result of dorsal shifting of the nuclear pulposus disk material. disk extrusion refers to the rupture of the outer disk membrane and extrusion of the nuclear material into the vertebral column. in dogs and cats, there are 36 intervertebral disks that potentially can cause a problem. chondrodystrophic breeds of dogs are predisposed to endochondral ossification and include the dachshund, shih tzu, french bulldog, bassett hound, welsh corgis, american spaniel, beagle, lhasa apso, and pekingese. initial examination of the patient with suspected intervertebral disk disease includes identifying the neuroanatomic location of the lesion based on clinical signs and neurologic deficits and then establishing a prognosis. the neurologic examination should be carried out without excessive manipulation of the animal. the presence of pain, edema, hemorrhage, or a visible deformity may localize an area of vertebral injury. once an area of suspected lesion is localized based on physical examination findings, take radiographs to establish a diagnosis and to institute therapy. in most cases, the animal must receive a short-acting anesthestic for proper radiographic technique and to prevent further injury. lateral and crosstable ventrodorsal (vd) or dorsoventral (dv) radiographs require less manipulation of the animal compared with traditional vd and dv projections. myelography is often required to delineate the location of the herniated disk material. prognosis in spinal cord injury depends on the extent of the injury and the reversibility of the damage. perception of noxious stimuli, or the presence of "deep pain," by the animal when the stimulus is applied caudal to the level of the lesion is a good sign. to apply a noxious stimulus, apply firm pressure to a toe on one of the rear limbs using a thick hemostat or a pair of pliers. flexion or withdrawl of the limb is simply a local spinal reflex, and should not be perceived as a positive response to or patient perception of the noxious stimulus. turning of the head, vocalization, dilation of the pupils, change in respiratory rate or character, or attempts to bite are behaviors that are more consistent with perception of the noxious stimulus. absence of perception of the noxious stimulus ("loss of deep pain") is a very poor prognosis for return to function. focal lesions are usually associated with vertebral fractures and displacement of the vertebral canal. focal lesions in one or more of the spinal cord segments from t 3 to t 4 can cause complete dysfunction of the injured tissue as a result of concussion, contusion, or laceration. the degree of structural damage cannot be determined from the neurologic signs alone. transverse focal lesions result in paraplegia, with intact pelvic limb spinal reflexes and analgesia of the limbs and body caudal to the lesion. clinical signs in patients with spinal injury are summarized in table 1 -43. carefully evaluate the cardiovascular and respiratory status of patients with spinal injuries. immediately address specific injuries such as pneumothorax, pulmonary contusions, hypovolemic shock, and open wounds. if there is palpable or radiographic evidence of a vertebral lesion causing compressive injury, surgery is the treatment of choice unless the displacement has compromised most or all of the vertebral canal. displacements through 50% to 100% of the vertebral canal are associated with a poor prognosis, particularly if deep pain is absent caudal to the lesion. in the absence of a radiographic lesion and in the presence of continued neurologic deficits, an mri or ct scan or myelography is warranted to localize a potentially correctable lesion. surgical exploration can be considered: with the objectives of providing spinal cord decompression by hemilaminectomy or laminectomy with removal of disk material or blood clots, realign and stabilize the vertebral column, and perform a meningotomy, if necessary. place the patient on a backboard or other rigid surface, taped down for transport and sedated, to be transported to a surgical specialist. the presence of worsening or ascending clinical signs may signify ascending-descending myelomalacia and is characteristic of a very poor prognosis.in acute spinal trauma, the use of glucocorticoids has been the mainstay of therapy; however, controversy exists about whether they actually offer any benefit. traditional glucocorticosteroid therapy is listed in box 1-50. more recently, the use of propylene glycol has proved to be beneficial in the treatment of acute traumatic herniated disk. high-dose glucocorticoids should only be used for the first 48 hours after initial injury. side effects of glucocorticosteroid therapy include gastric and intestinal ulceration. the prophylactic use of gastroprotectant drugs will not prevent gastrointestinal ulcer formation; however, if signs of gastrointestinal ulcer are present, institute gastroprotectant therapy. management of the patient with spinal cord injury includes aggressive nursing care and physical therapy. many patients with spinal cord injury have little to no control over bladder function, which results in chronic dribbling or retention of urine and overdistention of the urinary bladder with overflow incontinence. urinary bladder retention can lead to urinary tract infection, bladder atony, and overflow incontinence. manual expression of the bladder several times a day may be enough to keep the bladder empty. alternatively, place a urinary catheter to maintain patient cleanliness and to keep the bladder decompressed. (see section 5 on urinary catheterization). paralytic ileus and fecal retention are frequent complications of spinal cord injury. to help prevent constipation, provide highly digestable foods and maintain the patient's hydration with oral and intravenous fluids. mild enemas or stool softeners can also be used to treat fecal retention. to prevent decubital ulcer formation, turn the patient every 4 to 6 hours, and use clean, dry, soft padded bedding. apply deep muscle massage and passive range of motion exercises to prevent disuse atrophy of the muscles and dependent edema. the radial nerve innervates the extensor muscles of the elbow, carpus, and digits. the radial nerve also supplies sensory innervation to the distal craniolateral surface of the forearm and the dorsal surface of the forepaw. injuries to the radial nerve at the level of the elbow 190 1 emergency care cranial to c6 spastic tetraplegia or tetraparesis hyperreflexive all four limbs severe injury can result in death from respiratory failure. c6-t2 tetraparesis or tetraplegia depressed thoracic limb spinal reflexes (lower motor neuron) hyperreflexive pelvic limbs (upper motor neuron) t1-t3 horner' syndrome (prolapsed nictitans, enophthalmos, and miosis) t3-l3 schiff-sherrington syndrome (extensor rigidity of thoracic limbs, flaccid paralysis with atonia, areflexia, and analgesia of pelvic limbs) result in an inability to extend the carpus and digits. as a result, the animal walks and bears weight on the dorsal surface of the paw. there is also loss of cutaneous sensation, which leads to paw injury. injuries to the radial nerve above the elbow (in the shoulder area) results in an inability to extend the elbow and bear weight on the affected limb. it can take weeks before the full extent of the injury and any return to function are manifested. the animal may need to be placed in a carpal flexion sling or have eventual amputation if distal limb injury or self-mutilation occurs. the sciatic nerve primarily innervates the caudal thigh muscles that flex the stifle and extend the hip. the tibial branch of the sciatic nerve innervates the caudal leg muscles that extend the tarsus and flex the digits. the tibial nerve provides the sole cutaneous sensory innervation to the plantar aspect of the paw and digits. the peroneal branch of the sciatic nerve provides the sole sensory cutaneous innervation to the dorsal surface of the paw ( table 1 -44) . sciatic nerve injury may occur with pelvic fractures, particularly those that involve the body of the ileum at the greater ischiatic notch, or with sacroiliac luxations that contuse the l6 and l7 spinal nerves that pass ventral to the sacrum to contribute to the sciatic nerve. with sciatic nerve injury, there is decreased stifle flexion and overflexion of the hock (tibial nerve), and the animal walks on the dorsal surface of the paw (peroneal nerve). clinical signs of tibial or peroneal damage are seen with femur fractures or with inadvertent injection of drugs into the caudal thigh muscles. the femoral nerve innervates the extensor muscles of the stifle. the saphenous branch of the femoral nerve provides the sole cutaneous innervation to an area on the medial distal thigh, the leg, and the paw. the femoral nerve is protected by muscles and is rarely injured in pelvic fractures. clinical signs of femoral nerve injury are inability to support weight on the pelvic limb, absence of a patellar reflex, and analgesia in the area of cutaneous innervation. coma is complete loss of consciousness, with no response to noxious stimuli. in some animals that present in a coma or stuporous state, the immediate cause will be apparent. in other cases, however, a careful and thorough diagnostic work-up must be performed. a coma scale devised to assist in the clinical evaluation of the comatose patient is shown in table 1 -45. whenever an animal presents in a comatose state, immediately secure the emergency management of specific conditions 191 c6-t2 nerve roots radial nerve paralysis musculocutaneous nerve inability to flex the elbow axillary or thoracodorsal dropped elbow nerve median and ulnar nerves loss of cutaneous sensation on the caudal surface of the forearm and palmar and lateral surfaces of the paw; inability to flex the carpus and digits c8-t1 nerve roots radial, median, or ulnar nerve injury c6-c7 nerve roots musculocutaneous, suprascapular, and axillary injury c7-t3 horner's syndrome (miosis, enophthalmos, and prolapsed nictitans) airway by placing an endotracheal tube (see section on endotracheal intubation). if necessary, provide respiratory assistance, or at a minimum, supplemental oxygen. control existing hemorrhage and treat shock, if present. take a careful and thorough history from the owner. make careful note of any seizure, trauma, or toxin exposure, and whether prior episodes of coma have ever occurred. perform a careful physical examination, taking note of the patient's temperature, pulse, and respiration. an elevated temperature may suggest the presence of systemic infection, such as pneumonia or hepatitis, or a brain lesion with loss of hypothalamic thermoregulatory control. very high temperatures associated with shock and coma are often observed in animals with heat stroke (see section on heat stroke and heat-induced illness). circulatory collapse or barbiturate overdose can produce coma and hypothermia. abnormal respiratory patterns also may be observed in a comatose patient. hypoventilation may occur with elevated intracranial pressure or barbiturate overdose. rapid respiratory rate may be associated with pneumonia, metabolic acidosis (dka, uremia), or brainstem injury. examine the skin for any bruises or external trauma. examine the mucous membranes and make note of color and capillary refill time. icterus with petechiae or ecchymotic hemorrhage in a comatose patient may be associated with end-stage hepatic failure and hepatic encephalopathy. smell the patient's breath for the odor of ketones that may signify dka or end-stage hepatic failure. motor activity 6 normal gait, normal spinal reflexes hemiparesis, tetraparesis, or decerebrate activity 5 recumbent, intermittent extensor rigidity 4 recumbent, constant extensor rigidity 3 recumbent, constant extensor rigidity with opisthotonus 2 recumbent, hypotonia of muscles, depressed or absent spinal reflexes 1 normal papillary reflexes and oculocephalic reflexes 6 slow pupillary light reflexes and normal to reduced oculocephalic reflexes 5 bilateral unresponsive miosis with normal to reduced oculocephalic reflexes 4 pinpoint pupils with reduced to absent oculocephalic reflexes 3 unilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes 2 bilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes 1 occasional periods of alertness and responsive to environment 6 depression of delirium, capable of responding to environment but response 5 may be inappropriate semicomatose, responsive to visual stimuli 4 semicomatose, responsive to auditory stimuli 3 semicomatose, responsive only to repeated noxious stimuli 2 comatose, unresponsive to repeated noxious stimuli 1 *neurologic function is assessed for each of the three categories and a grade of 1 to 6 is assigned according to the descriptions for each grade. the total score is the sum of the three category scores. this scale is designed to assist the clinician in evaluating the neurologic status of the craniocerebral trauma patient. as a guideline and according to clinical impressions, a consistent total score of 3 to 8 represents a grave prognosis, 9 to 14 a poor to guarded prognosis, and 15 to 18 a good prognosis. (modified from the glasgow coma scale used in humans.) from shores a: craniocerebral trauma. in kirk rw, ed: current veterinary therapy x. small animal practice. philadelphia, wb saunders, 1989, p 849. finally, conduct a complete neurologic evaluation. the presence of asymmetric neurologic signs may suggest an intracranial mass lesion (e.g., hemorrhage, neoplasia, injury). usually, toxicities or metabolic disturbances (e.g., dka, hepatic encephalopathy) cause symmetric clinical signs of neurologic dysfunction, with cerebral signs predominating. in hepatic encephalopathy, pupils are usually normal in size and responsive to light. in toxicities, the pupils are abnormal in size and may be unresponsive to light. obtain a complete blood count, serum biochemistry profile, urinalysis, and specific tests for glucosuria and ketonuria. findings of a drastically elevated blood glucose with glucosuria, ketonuria, and high specific gravity are characteristic of dka. fever and uremic encephalopathy are characterized by severe azotemia with a low urine specific gravity. if barbiturate intoxication is suspected, save urine for later toxin analysis. evaluate urine sediment for calcium oxalate crystalluria that may indicate ethylene glycol toxicity. calculate plasma osmolality (see following section) to check for nonketotic hyperosmolar diabetes mellitus. elevated blood ammonia levels may be associated with hepatic encephalopathy. in uncontrolled diabetes mellitus, hyperosmolarity can result in clinical signs of disorientation, prostration, and coma. plasma osmolarity can be calculated from the formula: mosm/l = 2(na + k) + (glucose/18) + (bun/2.8) clinical signs of hyperosmolarity can occur when the plasma osmolarity exceeds 340 mosm/l. treatment of dka or nonketotic hyperosmolar syndrome is aimed at reducing ketoacid production, stimulating carbohydrate utilization, and impeding peripheral release of fatty acids. the treatment of choice is rehydration and provision of supplemental regular insulin and a carbohydrate source (see section on diabetic ketoacidosis). during ketosis, insulin resistance may be present. slow rehydration with 0.9% saline solution or other balanced crystalloid fluids (e.g., normosol-r, plasmalyte-m, lactated ringer's solution), should occur, with the goal of rehydration over 24 to 48 hours. too rapid rehydration can result in cerebral edema and exacerbation of clinical signs. hepatic encephalopathy (he) is characterized by an abnormal mental state associated with severe hepatic insufficiency. the most common cause of he is congenital or acquired c o m a portosystemic shunts. acute hepatic destruction can also be caused by toxins, drugs, or infectious causes. the treatment of he is considered a medical emergency (table 1 -46) . absorption of ammonia and other nitrogenous substances from the gastrointestinal tract is thought to be one of the complicating factors in he. prevent absorption of ammonia and other nitrogenous substances from the gastrointestinal tract by restricting dietary protein to 15% to 20% for dogs, and to 30% to 35% (on a dry matter basis) for cats. dietary protein should be from a nonanimal plant source (e.g., soybean) whenever possible. caloric requirements are met with lipids and carbohydrates. also prescribe cleansing enemas to rid the colon of residual material, and antibiotic therapy to reduce gastrointestinal tract bacteria. neomycin (15 mg/kg q6h) can be administered as a retention enema. metronidazole (7.5 mg/kg po, q8-12h) or amoxicillin-clavulanate (16.25 mg po q12h) can also be administered. administer lactulose (2.5-5.0 ml q8h for cats; 2.5-15 ml q8h for dogs) to trap ammonia in the colon to prevent absorption (table 1 -46) . administer lactulose orally to an alert animal, or as a retention enema to a comatose animal. if lactulose is not available, betadine retention enemas will change colonic ph and prevent ammonia absorption. a side effect of lactulose administration (po) is soft to diarrheic stool. a seizure is a transient disturbance of brain function that is sudden in onset, ceases spontaneously, and has a tendency to recur, depending on the cause. most seizures are generalized and result in a loss of consciousness and severe involuntary contraction of the skeletal muscles, resulting in tonic-clonic limb activity and opisthotonus. mastication, salivation, urination, and defecation are common. partial (petit mal) seizures range from limited limb activity, facial muscle twitching, and episodic behavioral abnormalities to brief loss of consciousness. similar clinical signs also can occur with syncopal episodes. conduct a careful cardiac examination in any patient with a history of petit mal seizures. seizures of any form constitute a medical emergency, particularly when they occur in clusters, or as status epilepticus. most seizures are of short duration and may have subsided by the time the animal is presented for treatment. whenever a seizure occurs, however, it is important that the animal does not inadvertently injure itself or a bystander. it is important to evaluate whether the patient has a coexisting disease that can predispose it to seizures, such as hepatic failure, uremia, diabetes mellitus, hypoglycemia, toxin exposure, insulin-secreting tumors, and thiamine deficiency. many toxins are responsible for clinical signs of tremors or seizures (see section on poisons and toxins). treatment of a primary disease entity can help control seizures, in some cases, provided that the underlying cause is investigated and treated. status epilepticus, a state of continuous uncontrolled seizure activity, is a medical emergency. when an animal is in a state of status epilepticus, immediately place a lateral or medial saphenous intravenous catheter and administer diazepam (0.5 mg/kg iv) to help control the seizure. in most cases, the seizure must be controlled before a diagnostic workup is attempted. whenever possible, however, blood samples should be collected before administration of any anticonvulsant agent because of the risk of incorrect test results. for example, the propylene glycol carrier in diazepam can cause a false-positive ethylene glycol test using an in-house testing kit. whenever possible, check blood glucose levels, particularly in young puppies or kittens, to evaluate and treat hypoglycemia as a cause of seizures. if hypoglycemia exists, administer 25% dextrose (1 g/kg iv). if diazepam partially controls the status epilepticus, administer a constant rate infusion (0.1 mg/kg/hour in 5% dextrose in water). diazepam is sensitive to light, and the bag and infusion line must be covered to prevent degradation of the drug. if diazepam fails to control status epilepticus, give pentobarbital (3-25 mg/kg iv to effect). the animal's airway should be intubated and protected while the patient is kept in the drug-induced coma. protracted cases of seizures may require mannitol and furosemide therapy to treat cerebral edema. administer intravenous fluids (balanced crystalloid at maintenance doses [see section on intravenous fluid therapy]). the patient should be turned every 4 to 6 hours to 194 1 emergency care prevent atelectasis. insert a urinary catheter for cleanliness, and place the animal on soft dry padded bedding to prevent decubital ulcer formation. depending on the length of time that the patient is rendered unconscious, apply passive range of motion exercises and deep muscle massage to prevent disuse atrophy of the muscles and dependent or disuse edema. monitor the patient's oxygenation and ventilation status by arterial blood gas measurement or pulse oximetry and capnometry (see section 5 on blood gas, pulse oximetry, and capnometry). administer supplemental oxygen to any patient that is hypoxemic secondary to hypoventilation or other causes. severe refractory seizures can result in the development of neurogenic pulmonary edema. lubricate the animal's eyes every 4 hours to prevent drying out and corneal abrasions. depending on the cause of the seizure, administer phenobarbital at a loading dose of 16 to 20 mg/kg iv given in four to five injections, every 20 to 30 minutes; make sure that the patient is rousable in between injections). seizures in cats often are associated with structural brain disease. the occurrence of partial focal seizures is unequivocally associated with a focal cerebral lesion and acquired structural brain disease. an initial high frequency of seizures is also a strong indication that structural brain disease is present. seizure activity in cats may occur as mild generalized seizures or complex partial seizures and may be associated with systemic disorders such as feline infectious peritonitis virus, toxoplasmosis, cryptococcus infection, lymphosarcoma, meningiomas, ischemic encephalopathy, and thiamine deficiency. thiamine deficiency in the cat can be a medical emergency characterized by dilated pupils, ataxic gait, cerebellar tremor, abnormal oculocephalic reflex, and seizures. treatment consists of administration of thiamine (50 mg/day) for three days. steffen f, grasmueck s: propofol for treatment of refractory seizures in dogs and a cat with intracranial disorders. j small anim pract 41 (11) (1997) (1998) (1999) . j am vet med assoc 218 (7): [1124] [1125] [1126] [1127] [1128] [1129] 2001 . an ocular emergency is any serious condition that causes or threatens to cause severe pain, deformity, or loss of vision. treat ocular emergencies immediately, within 1 to several hours after the emergency, whenever possible (box 1-51, 1-52). to assess the location and degree of ocular injury, perform a complete ocular examination. in some cases, short-acting sedation or general anesthesia in conjunction with topical local anesthetic may be necessary to perform the examination, because of patient discomfort and blepharospasm. the equipment listed in box 1-53 may be necessary and may be invaluable in making an accurate diagnosis. to perform a systematic and thorough ocular examination, first obtain a history from the owner. has there been any prior incident of ocular disease? is there any history of trauma or known chemical irritant or exposure? did the owner attempt any irrigation or medical techniques prior to presentation? when was the problem first noticed? has it changed at all since the owner noticed the problem? after a history has been obtained, examine the patient's eyes for discharge, blepharospasm, or photophobia. if any discharge is present, note its color and consistency. do not attempt to force the eyelids open if the patient is in extreme discomfort. administer a short-acting sedative and topical local anesthetic such as 0.5% proparacaine. note the position of the globe within its orbit. if the eye is exophthalmic, strabismus and protrusion of the third eyelid are often visible. exposure keratitis may be present. in cases of retrobulbar or zygomatic salivary gland inflammation, the patient will resist opening the mouth and exhibit signs of discomfort or pain. note any swelling, contusions, abrasions, or lacerations of the eyelids. note whether the lids are able to close completely and cover the cornea. if a laceration of the lid is present, determine the depth of the laceration. palpate the orbit for fractures, swelling, pain, crepitus, and cellulitis. examine the cornea and sclera for penetrating injury or foreign material. the use of lid retractors or small forceps can be very helpful in these cases. if a wound appears to penetrate completely into the globe, look for loss of uveal tissue, lens, or vitreous. do not put any pressure on the globe, because intraocular herniation may result. examine the conjunctiva for hemorrhage, chemosis, lacerations, and foreign bodies. examine the superior and inferior conjunctival cul-de-sacs for foreign material. in such cases, placement of a topical anesthetic and use of a moistened cotton swab is invaluable to sweep the conjunctival fornix to pick up foreign bodies. use a small, fine-tipped forceps to retract the third eyelid away from the globe and examine behind the third eyelid for foreign bodies. next, examine the cornea for opacities, ulcers, foreign bodies, abrasions, or lacerations. place a small amount of fluroescein stain mixed with sterile water or saline on the dorsal sclera. close the eye to disperse the stain over the surface of the cornea, then flush gently with sterile saline irrigation. examine the cornea again for any defects. a linear defect perpendicular to the long axis of the eye should alert the clinician to investigate the conjunctiva for dystechia. record the pupil size, shape, and response to light (both direct and consensual). examine the anterior chamber and note its depth and whether hyphema or aqueous flare are present. is the lens clear and is it in the normal position? lens luxation can cause the lens tissue to touch the cornea and cause acute corneal edema. measure intraocular pressure with a schiotz tonometer or tonopen. finally, dilate the pupil and examine the posterior chamber using a direct or indirect ophthalmoscope to look for intraocular hemorrhage, retinal hemorrhage, retinal detachment, tortuous retinal vessels, optic neuritis, and inflammation. the basic surgical instruments listed in box 1-54 may be useful in the treatment of ocular lacerations and other ophthalmic injuries: bite wounds and automobile trauma commonly cause lacerations and abrasions of the lid margins. the lids can be considered to be two-layer structures, with the anterior composed of the skin and orbicularis muscle and the posterior layer composed of the tarsus and conjunctiva. the openings of the meibomian glands in the lid margin form the approximate line separating the lids into anterior and posterior segments. splitting the lid into these two segments facilitates the use of sliding skin flaps to close wound defects, if necessary. clean and thoroughly but gently irrigate the wound with sterile saline solution before attempting any lid laceration repair. use sterile saline solution to irrigate the wound and conjunctiva. a 1% povidone-iodine scrub can be used on the skin, taking care to avoid getting any scrub material in the soft tissues of the eye. drape the eye with an adhesive ocular drape, if possible, to prevent further wound contamination. trim the ragged wound edges, but be very conservative with tissue debridement. leave as much tissue as possible to insure proper wound contracture with minimal lid deformity. close a small lid wound with a figure-of-eight or two-layered simple interrupted suture of absorbable suture material or nylon in the skin. the lid margins must be absolutely apposed to prevent postoperative lid notching. direct blunt trauma to the eye can cause severe ecchymosis because of the excellent vascular supply of the eyelids. other associated ocular injuries such as orbital hemorrhage, proptosis, and corneal laceration may also occur. trauma, allergic reactions, inflammation of the sebaceous glands (hordeolum), thrombocytopenia, and vitamin k antagonist rodenticide intoxication can all cause ecchymoses of the lids. treat eyelid ecchymoses initially with cool compresses, followed by warm compresses. resorption of blood can occur from 3 to 10 days after the initial insult. ocular allergies respond well to topical application (dexamethasone ophthalmic ointment q6-8h) and systemic administration of glucocorticosteroids, along with cool compresses. in order to fully assess the conjunctiva for abnormalities, it may be necessary to carefully dissect it away from the underlying sclera. when performing this dissection, do not place undue pressure on the globe because of the risk of herniation of the intraocular contents through a scleral wound. repair large conjunctival lacerations with 6-0 absorbable sutures, using an interrupted or continuous pattern. carefully approximate the margins of the conjunctiva to prevent formation of inclusion cysts. when large areas of the conjunctiva have been damaged, advancement flaps may be required to close the defect. subconjunctival hemorrhage is a common sequela of head trauma, and it may also be observed in various coagulopathies. by itself, it is not a serious problem but may signify severe underlying intraocular damage. a complete ocular examination is indicated. other causes of subconjunctival hemorrhage include thrombocytopenia, autoimmune hemolytic anemia, hemophilia, leptospirosis, vitamin k antagonist rodenticide intoxication, severe systemic infection or inflammation, and prolonged labor (dystocia). uncomplicated subconjunctival hemorrhage usually clears on its own within 14 days. if the conjunctiva is exposed because of swelling and hemorrhage, administer a topical protective triple antibiotic ophthalmic ointment every 6 to 8 hours until the conjunctival hemorrhage resolves. toxic, acid, and alkaline chemical injuries to the eye can sometimes occur. the severity of the injury caused by ocular burns depends on the concentration, type, and ph of the chemical and on the duration of exposure. weak acids do not penetrate biologic tissue very well. the hydrogen ion precipitates the protein upon contact and therefore provides some protection to the corneal stroma and intraocular contents. precipitation of corneal proteins produces a ground-glass appearance in the cornea. alkaline solutions and very strong acids penetrate tissues rapidly, causing saponification of the plasma membrane, denaturation of collagen, and vascular thrombosis within the conjunctiva, episclera, and anterior uvea. severe pain, blepharospasm, and photophobia are produced by exposure of free nerve endings in the corneal epithelium and conjunctiva. severe alkaline burns cause an increase in intraocular pressure. intraocular prostaglandins are released, and the intraocular aqueous ph increases, producing changes in the blood-aqueous barrier and secondary uveitis. uveitis with anterior synechia formation, eventual chronic glaucoma, phthisis, secondary cataract, and corneal perforation can occur. healing of the corneal epithelium is usually accomplished by neovascularization and sliding and increased mitosis of the corneal epithelium. severe stromal burns within the cornea heal by degradation and removal of necrotic debris, followed by replacement of the collagen matrix and corneal epithelial cells. the release of collagenase, endopeptidase, and cathepsins from polymorphonuclear cells serves to cause further corneal breakdown. in severe cases, only pmns may be present, and fibroblasts may never invade the corneal stroma. all chemical burns should be washed copiously with any clean aqueous solution available. if any sticky paste or powder is adherent to the conjunctival sac, remove it with moist cotton swabs and irrigation. begin mydriasis and cycloplegia by topical application of 1% atropine ophthalmic drops or ointment. start antibiotic therapy with triple antibiotic ophthalmic ointment or gentocin ointment every 6 to 8 hours. treat secondary glaucomas with topical carbonic anhydrase inhibitors. to avoid fibrinous adhesions and symblepharon formation, keep the conjunctival cul-de-sacs free of proteinaceous exudate that can form adhesions. analgesics are required for pain. oral nonsteroidal antiinflammatory agents such as carprofen, ketoprofen, meloxicam, or aspirin are recommended. persistent epithelial erosions may require a conjunctival flap left in place for 3 to 4 weeks or placement of a topical collagen shield (contact lens). topical antibiotics, mydriatics, and lubricants (lacrilube or puralube ointment) should also be used. strong acid or alkali burns can result in severe corneal stromal loss. in the past, topical n-acetylcysteine (10% mucomyst) has been recommended. this treatment is very painful. other treatments are also available, such as ethylenediaminetetraacetic acid (edta) (0.2 m solution) and patient serum to inhibit mammalian collagenase activity. to prepare patient serum, obtain 10 to 12 ml of whole blood from the patient. spin it down in a serum separator tube after a clot forms and then place the serum in a red-topped tube on the patient's cage. (the contents of the tube are viable for 4 days without refrigeration.) apply the serum topically to the affected eye every 1 to 2 hours. avoid using topical steroids because they inhibit fibroblast formation and corneal healing. in severe cases, if conjunctival swelling and chemosis also are present, antiinflammatory doses of oral steroids can be administered short-term. oral steroids and nonsteroidal antiinflammatory drugs should never be administered to the patient concurrently, because of the risk of gastrointestinal ulcer and perforation. corneal abrasions are associated with severe pain, blepharospasm, lacrimation, and photophobia. animals with such intense pain are often difficult to examine until analgesia has been administered. topical use of proparacaine (0.5% proparacaine hydrochloride) is usually sufficient to permit relaxation of the eyelids so that the eye can be examined. using a focal source of illumination and an eye loupe, examine the cornea, inferior and superior conjunctival fornixes, and medial aspect of the nictitans for foreign bodies. place a sterile drop of saline on a fluorescein-impregnated strip and touch the superior conjunctiva once to allow the stain to spread onto the surface of the eye. irrigate the eye to remove excess stain and then examine the corneal surface for any areas of stain uptake. if an area of the cornea persistently remains green, there is damage to the corneal epithelium in that area. initial treatment consists of application of a topical mydriatic (1 drop of 1% atropine in affected eye q12h) to prevent anterior synechiae and improve cycloplegia. triple antibiotic ointment is the treatment of choice (a 1 /4-inch strip in the affected eye q8h) until the ulcer heals. in some cases, nonhealing ulcers (e.g., boxer ulcer, indolent ulcer) form in which the epithelial growth does not adhere to the underlying cornea. gently debride the loose edges 1 of the ulcer/erosion with a cotton swab and topical anesthesia. more severe cases in which only minimal healing has occurred after 7 days of treatment require grid keratectomy, in which a 25-gauge needle is used to gently scratch the surface of the abrasion or ulcer in the form of a grid to promote neovascularization. apply a topical anesthetic before performing the procedure. a collagen contact lens also may be required to promote wound healing. all corneal abrasions should be reevaluated in 48 hours, and then every 4 to 7 days thereafter until they have healed. acute infectious keratitis secondary to bacterial infection is characterized by mucopurulent ocular discharge, rapidly progressing epithelial and corneal stromal loss, inflammatory cellular infiltrates into the corneal stroma, and secondary uveitis, often with hypopyon formation. confirmation of infectious keratitis is based on corneal scrapings and a positive gram stain. initial treatment for bacterial keratitis consists of systemic antibiotics and topical ciprofloxacin (0.3% eyedrops or ointment). penetrating injuries through the cornea may result in prolapse of intraocular contents. frequently, pieces of uveal tissue or fibrin effectively but temporarily seal the defect and permit the anterior chamber to re-form. avoid manipulation of these wounds until the animal has been anesthetized, as struggling or excitement can promote loss or dislodgement of the temporary seal and cause the intraocular contents to be extruded. superficial corneal lacerations need not be sutured and can be treated the same as a superficial corneal ulcer or abrasion. if the laceration penetrates more than 50% the thickness of the cornea, or extends more than 3 to 4 mm, it should be sutured. when placing sutures in the cornea, it is helpful to use magnification. referral to a veterinary ophthalmologist is advised. if a veterinary ophthalmologist is not available, use 7-0 or 8-0 silk, collagen, or nylon sutures on a micropoint spatula-type needle. use a simple interrupted suture pattern and leave the sutures in place for a minimum of 3 weeks. because many corneal lacerations are jagged and corneal edema forms, most of the wound edges cannot be tightly juxtaposed. in such cases, pull a conjunctival flap across the wound to prevent leakage of aqueous fluid. never suture through the full thickness of the cornea; rather, the suture should pass through the mid-third of the cornea. following closure of the corneal wound, the anterior chamber must be re-formed to prevent anterior synechia formation with secondary glaucoma. taking care to avoid iris injury, use a 25-or 26-gauge needle to insert sterile saline at the limbus. any defect in the suture line will be apparent because of leakage of the fluid from the site and should be repaired. incarceration of uveal tissue in corneal wounds is a difficult surgical problem. persistent incarceration of uveal tissue can result in development of a chronic wick in the cornea, a shallow anterior chamber, chronic irritation, edema, vascularization of the cornea, and intraocular infection that can lead to panophthalmitis. referral to a veterinary ophthalmologist is strongly recommended. the most common foreign bodies associated with ocular injuries in small animals are birdshot, bb pellets, and glass. the site of intraocular penetration of the foreign bodies may be obscured by the eyelids. a foreign body entering the eye may penetrate the cornea and fall into the anterior chamber or become lodged in the iris. foreign bodies may occasionally penetrate the lens capsule, producing cataracts. some metallic high-speed foreign bodies may penetrate the cornea, iris, and lens to lodge in the posterior wall of the eye or vitreous chamber. direct visualization of a foreign body is the best means of localization. examination of the eye with an indirect ophthalmoscope or biomicroscope (if available) is invaluable for locating foreign bodies. indirect visualization of the ocular foreign body can also be achieved through radiographic techniques. three separate views should be obtained to determine the plane of location of the foreign object. ct or mri may prove useful, although scatter from the foreign body may make it difficult to directly visualize with these techniques. ocular ultrasound is perhaps the most useful and refined radiographic technique for locating intraocular foreign bodies. before removing any foreign body from the eye, the risk and surgical danger of removing it must be weighed against the risks of leaving it in place. metallic foreign bodies in the anterior chamber are much easier to remove than nonmagnetic ones. attempted removal of foreign objects from the vitreous chamber of the eye has consistently produced poor results. for the best chance of recovery, ocular foreign bodies should be removed by a veterinary ophthalmologist whenever possible. blunt trauma to the globe can result in luxation or subluxation of the lens. the subluxated lens may move anteriorly and make the anterior chamber more shallow. trembling of the iris (iridodonesis) may be noticed when the lens is subluxated. in complete luxation, the lens may fall totally into the anterior chamber and obstruct aqueous outflow, causing secondary glaucoma. alternatively, the lens may be lost into the vitreous cavity. luxation of the lens is almost always associated with rupture of the hyaloid membrane and herniation of the vitreous through the pupillary space. emergency surgery for lens luxation is required if the lens is entirely within the anterior chamber or incarcerated within the pupil, causing a secondary pupillary block glaucoma. acute elevation in intraocular pressure can cause vision loss within 48 hours; thus, lens removal should be accomplished as quickly as possible. referral to a veterinary ophthalmologist is recommended. severe trauma to the globe or a direct blow to the head can result in retinal or vitreous hemorrhage. there may be large areas of subretinal or intraretinal hemorrhage. subretinal hemorrhage assumes a discrete globular form, and the blood appears reddish-blue in color. the retina is detached at the site of hemorrhage. superficial retinal hemorrhage may assume a flame-shaped appearance, and preretinal or vitreous hemorrhage assumes a bright-red amorphous appearance, obliterating the underlying retinal architecture. retinal and vitreous hemorrhage secondary to trauma usually resorbs spontaneously over a 2-to 3-week period. unfortunately, vitreous hemorrhage, as it organizes, can produce vitreous traction bands that eventually produce retinal detachment. expulsive choroid hemorrhage can occur at the time of injury and usually leads to retinal detachment, severe visual impairment, and total loss of vision. treatment of vitreal and retinal hemorrhage includes rest and correction of factors that may predispose to intraocular hemorrhage. more complicated cases may require vitrectomy performed by a veterinary ophthalmologist. hyphema refers to blood in the anterior chamber of the eye. the most common traumatic cause of hyphema is an automobile accident. hyphema may also present because of penetrating ocular wounds and coagulopathies. blood within the eye may come from the anterior or posterior uveal tract. trauma to the eye may result in iridodialysis or a tearing of the iris at its root, permitting excessive bleeding from the iris and ciliary body. usually, simple hyphema resolves spontaneously in 7 to 10 days and does not cause vision loss. loss of vision following bleeding into the anterior chamber is associated with secondary ocular injuries such as glaucoma, traumatic iritis, cataract, retinal detachment, endophthalmitis, and corneal scarring. treatment of hyphema must be individualized, but there are severe general principles of treatment. first, stop ongoing hemorrhage and prevent further bleeding whenever possible. this may involve correction of the underlying cause, if a coagulopathy is present. next, aid in the elimination of blood from the anterior chamber, control secondary glaucoma, and treat associated injuries, including traumatic iritis. finally, detect and treat any late complications of glaucoma. in most cases of traumatic hyphema, little can be done to arrest or prevent ongoing hemorrhage. it is best to restrict the animal's activity and prohibit exertion. rebleeding can occur within 5 days, and intraocular pressure must be monitored closely. after 5 to 7 days, the blood in the anterior chamber will change color from a bright red to bluish-black ("eight-ball hemorrhage"). if total hyphema persists and intraocular pressure rises despite therapy, surgical intervention by a veterinary ophthalmologist may be necessary. the primary route of escape of rbcs from the anterior chamber is via the anterior drainage angle. iris absorption and phagocytosis play a minor role in the removal of blood from the anterior chamber. because of the associated traumatic iritis in hyphema, topical administration of a glucocorticoid (1% dexamethasone drops or 1% prednisolone drops) is advised to control anterior chamber inflammation. a cycloplegic agent (1% atropine) should also be used. the formation of fibrin in the anterior chamber of the eye secondary to hemorrhage can produce adhesions of the iris and secondary glaucoma (see section on glaucoma secondary to hyphema) by blocking the trabecular network. hyphema secondary to retinal detachment (collie ectasia syndrome) and end-stage glaucoma are extremely difficult to treat medically and have a poor prognosis. proptosis of the globe is common secondary to trauma, particularly in brachycephalic breeds. proptosis of the globe in dolichocephalic breeds requires a greater degree of initiating contusion than the brachycephalic breeds because the orbits are so much deeper. therefore, secondary damage to the eye and cns associated with proptosis of the globe may be greater in the collie or greyhound than in the pug. when proptosis occurs, carefully evaluate the cardiovascular system for evidence of hypovolemic or hemorrhagic shock. examine the respiratory and neurologic systems. be sure to establish an airway and treat shock, if present. control hemorrhage and stabilize the cardiovascular system before attempting to replace the globe within its orbit or perform enucleation. during the initial management of the cardiovascular and respiratory systems, the eye should be covered with an ophthalmic grade ointment or sponges soaked in sterile saline to prevent the globe from drying out. proptosis of the globe can be associated with serious intraocular problems including iritis, chorioretinitis, retinal detachment, lens luxation, and avulsion of the optic nerve. stain the surface of the eye with fluorescein to look for topical abrasions or ulcers. carefully examine the sclera, cornea, and conjunctiva for penetrating injuries that may allow aqueous leakage. evaluate the size, location, and response to light of the pupil. a reactive pupil is better than a mydriatic fixed pupil. topical administration of a mydriatic (atropine 1%) to prevent persistent miosis and synechia formation is indicated, along with topical and oral antibiotics and oral analgesic therapy. reposition the proptosed globe with the patient under general anesthesia. make a lateral canthotomy incision to widen the palpebral fissure. lavage the globe with sterile saline irrigation to remove any external debris. place a copious amount of triple antibiotic ophthalmic ointment on the surface of the eye and then gently press the globe into the orbit using the flat side of a scalpel handle or a moistened sterile surgical sponge. do not probe the retro-orbital space with a needle or attempt to reduce intraocular pressure by paracentesis. when the globe is replaced in the orbit, close the lateral canthotomy incision with simple interrupted sutures. place three non-penetrating mattress sutures in the lid margins but do not draw them together. tighten the lid sutures through small pieces of a red rubber catheter or length of intravenous extension tubing to prevent the sutures from causing lid necrosis. leave the medial canthus of the eye open in order to allow topical treatment. postoperative treatment is directed at preventing further iritis and preventing infection. administer systemic broad-spectrum antibiotics (clavamox, 16.25 mg/kg po bid) and analgesic drugs. apply topical triple antibiotic ophthalmic ointment ( 1 /4 inch in affected eye q6-8h) and atropine (1% in affected eye q12h) to prevent infection, cycloplegia, and anterior synechiae. antiinflammatory doses of systemic steroids can also be added to the treatment 202 if severe periorbital inflammation is present. systemic steroids should never be used in conjunction with nonsteroidal antiinflammatory drugs, because of the risk of gastrointestinal ulceration and perforation. the sutures should remain in place for a minimum of 3 weeks. after this time, remove the sutures and inspect the globe. if proptosis recurs, repeat the treatment. following proptosis, strabismus is common secondary to periorbital muscle injury. even after extensive treatment, vision in the eye may still be lost. nonvisual eyes can remain in place, but phthisis may develop. carbonic anhydrase inhibitors such as acetazolamide and dichlorphenamide decrease aqueous secretion and may effectively reduce intraocular pressure if the trabecular outflow is still functioning at 40% of its capacity. an eye with a poorly functional trabecular outflow system will respond poorly to therapy with carbonic anhydrase inhibitors. osmotic agents such as mannitol or glycerol may be helpful in controlling glaucoma secondary to hyphema. reduction in vitreous chamber size can make the anterior chamber deeper and may allow increased aqueous outflow. evacuation of blood or blood clots from the anterior chamber is not advisable unless the glaucoma cannot be controlled medically or there is no indication after a prolonged period of time that blood is being resorbed. tissue plasminogen activator (t-pa) has proved to be useful in may be helpful in lysing blood clots and preventing excessive fibrin formation. the t-pa is reconstituted to make a solution of 250 âµ/ml, which is then frozen at â��70â°c in 0.5-ml aliquots. the thawed, warmed reconstituted t-pa is injected into the anterior chamber. blind probing of the anterior chamber of the eye and surgical intervention in an attempt to remove blood clots can cause serious complications such as rebleeding, lens luxation, iris damage, and damage to the corneal epithelium, and therefore is not advised. acute glaucoma is a rise in intraocular pressure that is not compatible with normal vision. glaucoma may present as early acute congestive or noncongestive glaucoma, or as end-stage disease. cardinal signs of glaucoma are a sudden onset of pain, photophobia, lacrimation, deep episcleral vascular engorgement, edematous insensitive cornea, shallow anterior chamber depth, dilated unresponsive pupil, loss of visual acuity, and buphthalmia. intraocular pressure usually exceeds 40 mm hg but may be normal or only slightly increased if glaucoma is secondary to anterior uveitis. most forms of clinical glaucoma in dogs are secondary to some other intraocular problem. primary glaucoma is recognized in some breeds, including the bassett hound, cocker spaniel, samoyed, bouvier des flandres, and some terrier breeds either from goniodysgenesis or a predisposition to lens luxation. other common causes of acute glaucoma are anterior uveitis and intumescent lens secondary to rapid cataract development, particularly in dogs with diabetes mellitus. treatment involves investigation of the underlying cause of the sudden rise in intraocular pressure and rapid reduction in intraocular pressure. permanent visual impairment is often associated with chronically buphthalmic globes or the presence of rippling or striae formation on the cornea. referral to a veterinary ophthalmologist is recommended. if the eye is still visual and not buphthalmic, the prognosis is favorable, depending on the cause of the acute glaucoma. treatment to reduce intraocular pressure consists of improving aqueous outflow, reducing intraocular volume with osmotic agents, and reducing aqueous formation (table 1 -47). the use of topical mydriatic agents in acute glaucoma is contraindicated because of the risk of making lens luxation or anterior uveitis worse. referral to a veterinary ophthalmologist for emergency surgery is indicated in cases of iris bombe, intumescent lens, or lens subluxation. administer osmotic agents to reduce the size of the vitreous body and the amount of aqueous. osmotic agents create an osmotic gradient between the intraocular fluids and the emergency management of specific conditions 203 vascular bed, thus allowing osmotic removal of fluid independent of the aqueous inflow and outflow systems. if no other treatments are available, oral glycerol (50%, 0.6 ml/kg or 1.4 g/kg) can be used to effectively reduce intraocular pressure. an adverse side effect of oral glycerol treatment is protracted vomiting. do not use glycerol in a diabetic patient. mannitol (1-2 g/kg iv over 1 hour) also effectively reduces intraocular pressure but does not cause vomiting. carbonic anhydrase inhibitors can be used to reduce intraocular volume by reducing aqueous production. oral administration of dichlorphenamide, methazolamide, and acetazolamide (2-4 mg/kg) is usually not very effective alone in reducing aqueous volume and intraocular pressure and also can cause metabolic acidosis. topical carbonic anhydrase inhibitors appear to be more effective (dorzolamide, trusopt) when used in conjunction with topical beta-blockers (timolol, 0.25% or 0.5% solution q8h). the most effective treatment for acute pressure reduction is use of a topical prostaglandin inhibitor (latanaprost). usually just one or two drops effectively reduces intraocular pressure in the emergency stages, until the patient can be referred to a veterinary ophthalmologist the following day. many clinical conditions that are presented as emergencies may be due in part or wholly to the presence of a neoplasm. paraneoplastic signs are summarized in table 1 -48. prompt identification of the neoplasia combined with knowledge of treatment, expected response to therapy, and long-term prognosis can aid owners and practitioners in making appropriate treatment decisions. hemorrhage or effusion can occur in any body cavity as a result of the presence of benign or malignant tumors. tumors secrete anticoagulants to allow angiogenesis to grow unchecked. hemorrhage often occurs as a result of rupture of a neoplasm or invasion of a neoplasm into a major vascular structure. effusion may be the result of direct fluid production by the mass or may be due to obstruction of lymphatic or venous flow. hemorrhagic effusions in the abdominal cavity occur most commonly with neoplastic masses of the spleen or liver. the most common causes are hemangiosarcoma and hepatocellular carcinoma. clinical signs associated with acute abdominal hemorrhage, regardless of the cause, are related to hypovolemic shock and decreased perfusion and include pale mucous membranes, tachycardia, anemia, lethargy, and acute collapse. treatment for abdominal hemorrhage includes placement of a large-bore peripheral cephalic catheter and starting one fourth of a shock dose (90 ml/kg/hour for dogs, and 44 ml/kg/hour for cats) of intravenous crystalloid fluids, taking care to carefully monitor perfusion parameters of heart rate, capillary refill time, mucous membrane color, and blood pressure. administer intravenous colloids such as dextran-70, hetastarch, and oxyglobin (5-10 ml/kg iv bolus) to restore intravascular volume and normotension. treat severe anemia with whole blood or packed rbcs to improve oxygen-carrying capacity and oxygen delivery (see sections on transfusion medicine and treatment of shock). confirm the presence of hemoabdomen abdominocentesis (see section on abdominocentesis). the presence of nonclotting hemorrhagic effusion is consistent with free blood. packed cell volume of the fluid is usually the same or higher than that of the peripheral blood. an abdominal compression bandage can be placed while further diagnostics are being performed. in cases of acute hemoabdomen, obtain right lateral, left lateral, and ventrodorsal or dorsoventral thoracic radiographs to help rule out obvious metastasis. monitor the patient's ecg and correct dysrhythmias as necessary (see section on cardiac dysrhythmias). surgery is indicated once the patient is stabilized. in some cases, hemorrhage is so severe that the patient should be taken immediately to surgery. when recommending surgery for a hemorrhaging intraabdominal mass, it is important to discuss likely diagnoses and long-term prognosis with the owner. hemangiosarcoma usually involves the spleen or liver or both. the presence of free abdominal hemorrhage is associated with a malignant tumor in 80% of cases. even when free abdominal hemorrhage is not present, the tumor is malignant in 50% of cases. approximately 66% (two thirds) of masses in the spleen are malignant (hemangiosarcoma, lymphoma, mast cell tumor, malignant fibrous histiocytoma, leiomyosarcoma, fibrosarcoma), and approximately one third are benign (hematoma, hemangioma). hepatocellular carcinoma usually affects one liver lobe (usually the left), and surgery is the treatment of choice. with complete surgical excision, median survival in dogs is longer than 300 days. if diffuse disease is observed at the time of surgery, the prognosis is poor. nonhemorrhagic effusions are associated with mesothelioma, lymphoma, carcinomatosis, or any mass that causes vascular or lymphatic obstruction. clinical signs of respiratory distress and abdominal distention with nonhemorrhagic effusions are usually slowly progressive in onset and not as severe as those observed with hemorrhage. treatment is usually aimed at identification of the underlying cause. obtain a fluid sample via thoracocentesis or abdominocentesis. to obtain further cells for cytologic evaluation, aspirate fluid from the thoracic or abdominal mass with ultrasound guidance. cytologic evaluation of the fluid will often elucidate the causative tumor type. an abdominal ultrasound can determine the degree of metastasis. perform therapeutic abdominocentesis or thoracocentesis if the effusion is causing respiratory difficulty. rapid re-accumulation of the fluid potentially can cause hypoproteinemia and hypovolemic shock. mesothelioma is a rare tumor most commonly observed in urban environments. in humans, mesothelioma has been associated with exposure to asbestos. it is sometimes difficult to differentiate between reactive mesothelial cells and malignant mesothelial cells. treatment is aimed at controlling the neoplastic effusion. intracavitary cisplatin has been demonstrated to slow rates of fluid re-accumulation, but is largely a palliative therapy. lymphoma is another tumor type that can cause thoracic or abdominal effusion. cytologic evaluation of the fluid usually reveals abundant lymphoblasts. treatment with multiagent chemotherapy protocols, with or without adjunctive radiation therapy, can prevent tumor remission and stop fluid accumulation. carcinomatosis occurs as a result of diffuse seeding of the abdominal cavity with malignant carcinomas and has a poor prognosis. carcinomatosis may occur de novo or from 1 metastasis of a primary tumor. treatment consists of fluid removal when respiratory difficulty occurs, with or without intracavitary cisplatin as a palliative measure. cisplatin should never be used in cats due to fatal acute pulmonary edema. clinical signs of hemorrhagic thoracic effusion include acute respiratory distress, anemia, hypovolemic or cardiogenic shock, and collapse. hemorrhagic thoracic effusions are rare in association with neoplastic effusions. a notable exception is intrathoracic hemorrhage in young dogs with osteosarcoma of the rib. hemorrhage can result when a primary lung tumor erodes through a vessel. hemangiosarcoma of the lungs or right auricular area can also result in hemorrhagic thoracic effusion. in many cases, hemorrhage may be confined to the pericardial sac with a right auricular mass, causing a globoid cardiac silhouette on thoracic radiographs. treatment consists of pericardiocentesis (see section on pericardial effusion and pericardiocentesis) and placement of a pericardial window, or the mass may be removed if it is in the right auricular appendage and resectable. although surgery can resolve clinical signs of right-sided heart failure, metastatic disease often develops soon afterward. nonhemorrhagic thoracic effusion is more common than hemorrhagic thoracic effusion, and is caused most commonly by mesothelioma, lymphoma, carcinomatosis, and thymoma. clinical signs develop gradually and include respiratory difficulty, cyanosis, and cough. supplemental oxygen should be administered. in many cases, thoracocentesis can be therapeutic and diagnostic. obtain thoracic radiographs both before and after thoracocentesis to determine whether a mass effect is present. following identification of a cause, definitive therapy can be instituted. mesotheliomas are rare and are associated with diffuse serosal disease. they are more common in dogs than in cats. effusions caused by mesotheliomas can affect the pleural or pericardial cavities. treatment is directed at removing effusion fluid and controlling reaccumulation with use of intracavitary platinum compounds, carboplatin, and cisplatin can be used in dogs. (cisplatin and carboplatin should never be used in cats.) chemical or physical pleurodesis may be helpful in controlling reaccumulation of fluid, but it is very painful in small animal patients. thoracic effusion secondary to lymphoma often is associated with an anterior mediastinal mass. t-cell lymphoma is the most common type of mediastinal mass observed in dogs. b-cell lymphoma is associated with a decreased response to chemotherapy and shorter survival times. treatment consists of combination chemotherapy with or without radiation therapy to decrease mass size. carcinomatosis is a diffuse disease of the pleural cavity that often is a result of metastasis from a primary pulmonary carcinoma or mammary adenocarcinoma. treatment is similar to that for mesothelioma and is aimed at controlling the effusion and delaying its recurrence. thymomas have been documented in both dogs and cats. dogs most commonly present with a cough, while cats present with clinical signs of respiratory distress and a restrictive respiratory pattern associated with the presence of pleural effusion. an anterior mediastinal mass is often observed on thoracic radiographs. in some cases, the pleural effusion must be drained via thoracocentesis before a mass is visible. ultrasound-guided aspiration and cytologic evaluation of the mass reveal a malignant epithelial tumor with small lymphocytes and mast cells. prognosis is good if the tumor can be completely excised. treatment consists of surgical removal with or without presurgical radiation therapy to shrink the mass. paraneoplastic syndromes of myasthenia gravis have been documented in dogs with thymomas. if megaesophagus or aspiration pneumonia is present, the prognosis is more guarded because of the high rate of complications. obstructive lesions affecting the urinary tract can be extramural (intra-abdominal, pelvic, or retroperitoneal) or intramural (urethral, bladder, or urethral wall) . transitional cell 1 carcinoma is the most common type of bladder tumor observed in dogs. prostatic adenocarcinoma, or neoplasia of the sublumbar lymph nodes (lymphoma, adenocarcinoma from apocrine gland adenocarcinoma), also can cause urethral obstruction. treatment is aimed at relieving the obstruction and then attempting to identify the cause of the disease. to alleviate the obstruction, pass a urinary catheter whenever possible. perform cystocentesis only as a last resort because of the risk of seeding the peritoneal cavity with tumor cells if transitional cell carcinoma is the cause of the obstruction. institute supportive therapy including intravenous fluids and correction of electrolyte abnormalities. plain radiographs may reveal a mass lesion or may not be helpful without double contrast cystography. abdominal ultrasound is more sensitive in identifying a mass lesion in the urinary bladder. masses in the pelvic urethra are difficult to visualize with ultrasonography. double contrast cystourethrography is preferred. once the patient is stabilized, biopsy or surgery is indicated to identify the cause of the mass and attempt resection. urine tests for transitional cell carcinoma are available for identification of transitional cell carcinoma in the dog. complete surgical excision of transitional cell carcinoma or removal of benign tumors of the urinary bladder yields a favorable prognosis. poorer prognosis is seen with incomplete excision. many transitional cell carcinomas are located in the trigone region of the bladder and cannot be completely excised. the nonsteroidal antiinflammatory drug piroxicam is helpful in alleviating clinical signs for a reported 7-month median survival. in some dogs, cisplatin and carboplatin may delay recurrence of transitional cell carcinoma. tumors of the prostate gland are always malignant and occur with equal frequency in castrated and uncastrated male dogs. diagnosis of prostatic tumors is based on ultrasonographic evidence of a mass effect or prostatomegaly and on transrectal or transabdominal aspiration or biopsy. surgery, chemotherapy, and radiation therapy generally are unrewarding over the long term, although palliative radiation therapy may relieve clinical signs for 2 to 6 months. luminal tumors of the gastrointestinal tract typically cause obstruction, with slowly progressive clinical signs including vomiting, inappetence, and weight loss, or with acute severe protracted vomiting. extraluminal obstructive lesions usually arise from adhesions, or strangulation may occur, resulting in obstruction. perforation of the mass through the gastric or intestinal wall can cause peritonitis. treatment consists of initial stabilization and rehydration, evaluation for evidence of metastasis, and surgical resection of the affected area in cases of adenocarcinoma, leiomyoma, leiomyosarcoma, and obstructive or perforated lymphoma. gastric and intestinal adenocarcinoma are the most common gastrointestinal tumors observed in dogs. affected animals typically have a history of anorexia, weight loss, and vomiting. obtain an abdominal ultrasound before performing any surgery. fine needle aspirates of the mass and adjacent lymph nodes are usually diagnostic and can determine whether there is local metastasis. many tumors are not resectable, and metastasis occurs in approximately 70% of cases. dogs with smaller tumors that can be resected typically have longer survival times. leiomyosarcomas occur in the intestines of dogs, and carry a more favorable prognosis than adenocarcinoma if the mass can be completely resected. with complete resection, the average survival time is longer than 1 year. the paraneoplastic syndrome of hypoglycemia has been observed with this tumor type. gastrointestinal lymphoma is the most common tumor of the gastrointestinal tract observed in cats. in comparison, it is relatively rare in dogs. unless there is complete obstruction or perforation of the gastrointestinal tract, surgical treatment for gastrointestinal lymphoma is not indicated. rather, multiple chemotherapy drugs are used in combination to achieve remission and resolution of the clinical signs of anorexia, weight loss, and vomiting. treatment responses unfortunately are poor. mast cell tumors of the gastrointestinal tract typically are manifested as gastrointestinal ulceration and hemorrhage in up to 83% of patients. the gastrointestinal hemorrhage that occurs with mast cell tumors results from increased acid secretion as a result of histamine receptor stimulation. treatment consists of histamine or proton pump inhibition (ranitidine, famotidine, cimetidine, or omeprazole). bowel perforation is a rare complication. many chemotherapy agents exert their effects on rapidly dividing normal and neoplastic cells. normal tissues that are commonly affected include the bone marrow, gastrointestinal tract, skin and hair follicles, and reproductive organs. some drugs have unique organspecific toxicities that must be monitored. knowledge and recognition of the expected type and onset of complications can alleviate their severity by rapid treatment, when complications occur (see table 1 -48) . neutropenia is the most common bone marrow toxicity observed secondary to chemotherapy in small animal patients (table 1 -49) . in most cases, the neutropenia is dose-dependent. the nadir, or lowest neutrophil count, is typically observed 5 to 10 days after chemotherapy treatment. once the nadir occurs, bone marrow recovery is observed, with an increase in circulating neutrophils within 36 to 72 hours (table 1 -49) . treatment of myelosuppression is largely supportive to treat or prevent sepsis. prophylactic antibiotics are recommended in the afebrile patient with a neutrophil count <2000/âµl. acceptable antibiotics include trimethoprim-sulfa and amoxicillin-clavulanate. granulocyte-colony stimulating factor (g-csf) (e.g., neupogen) is a recombinant human product that stimulates the release of neutrophils from the bone marrow, and its use shortens the recovery time following myelosuppressive drug therapy. disadvantages of g-csf include antibody production in response to the drug within 4 weeks of use and its high cost. to prevent ongoing neutropenia, subsequent chemotherapy dosages should be decreased by 25%, and the interval in between treatments increased. whenever possible, overlap of myelosuppressive drugs should be avoided. acute gastrointestinal toxicity can occur within 6 to 12 hours after administration of cisplatin and actinomycin d. in many cases, pretreatment with the antiemetics metoclopramide, butorphanol, chlorpromazine, dolasetron or ondansetron can prevent chemotherapyinduced nausea and vomiting. vomiting can also occur as a delayed side effect 3 to 5 days after treatment with doxorubicin (adriamycin), actinomycin d, methotrexate, and cytoxan. in delayed reactions, vomiting and diarrhea are caused by damage to intestinal crypt cells. treatment consists of administration of antiemetics, intravenous fluids, and a bland highly digestible diet. doxorubicin also can cause hemorrhagic colitis within 5 to 7 days of administration. treatment includes a bland diet, metronidazole, and tylosin tartrate (tylan powder). 1 emergency care mild to none not observed vincristine (low-dose), l-asparaginase, glucocorticosteroids moderate 7-10 days melphalan, cisplatin, mitoxantrone, actinomycin d severe 7-10 days doxorubicin, cyclophosphamide, vinblastine 1 paralytic ileus can be observed 2 to 5 days after administration of vincristine. this side effect is more common in humans than animals and can be treated with metoclopramide once a gastrointestinal obstruction has been ruled out. cardiotoxicity doxorubicin (adriamycin) causes a dose-dependent dilative cardiomyopathy when the cumulative dose reaches 100 to 150 mg/m 2 . in many cases, however, clinical signs do not occur until the cumulative dose is 240 mg/m 2 . the myocardial lesions are irreversible. treatment of cardiac dysrhythmias is dependent on the type of dysrhythmia (see section on treatment of dysrhythmias). discontinue doxorubicin and administer diuretics and positive inotropic therapy for dilative cardiomyopathy in order to delay the progression of congestive heart failure (see sections on treatment of congestive heart failure). if abnormalities are shown on electrocardiography performed before beginning therapy, substitute liposome-encapsulated doxorubicin or mitoxantrone substituted in the chemotherapy protocol. cardioprotectant drugs such as vitamin e, selenium, and n-acetyl cysteine have shown some promise in the prevention of doxorubicin-induced cardiotoxicity. cyclophosphamide can cause a sterile hemorrhagic cystitis. damage to the urinary bladder mucosa and vessels is caused by the toxic metabolite acrolein. clinical signs of sterile hemorrhagic cystitis include a history of cyclophosphamide administration, stranguria, hematuria, and pollakiuria. treatment for sterile hemorrhagic cystitis is discontinuation of the drug, treatment of any underlying urinary tract infection with antibiotic therapy based on susceptibility testing, and intravesicle drug administration. in extremely refractory cases, surgical debridement and cauterization of the bladder mucosa may be necessary. prevention of sterile hemorrhagic cystitis includes emptying the bladder frequently and administering the drug in the morning. concurrent administration of prednisone can induce polyuria and polydipsia. if sterile hemorrhagic cystitis occurs, chlorambucil can be substituted as a chemotherapeutic agent. anaphylactic reactions have been observed with the administration of l-asparaginase, adriamycin, etoposide, and paclitaxel. the risk of anaphylaxis increases with repeated administration, although in some animals anaphylaxis will occur on the first exposure to the drug. treatment consists of administration of epinephrine, diphenhydramine, famotidine, and glucocorticosteroids, as with any other life-threatening allergic reaction (see section on treatment of allergic reactions). to decrease the risk of an adverse reaction, give diphenhydramine (2.2 mg/kg im) 15 to 30 minutes before drug administration. slowing the rate of intravenous infusion also can decrease the chance of an anaphylactic reaction. cisplatin can cause a fatal irreversible pulmonary edema in cats, even at low dosages. 5-fluorouracil (5-fu) can cause a severe neurotoxicity in cats that results in ataxia and seizures. never use cisplatin or 5-fu in cats. poisoning cases benefit from a rapid, organized approach. key points in this approach are giving appropriate advice over the telephone, being able to access information sources, and providing appropriate treatment. there are only a few classes of poisons that account for the majority of toxicities reported in dogs and cats. every veterinarian should develop a familiarity with the clinical management of rodenticide and insecticide toxicity and be prepared with antidotes on hand. beyond the most common toxins, the spectrum of possibilities is endless, and the veterinarian must rely on appropriate information resources. it is important to have available a comprehensive source of pharmaceutical and plant identification resources. remarkably, considering the myriad of potentially toxic substances to which an animal can be exposed, relatively few specific antidotes are commonly used in veterinary medicine. because of the lack of specific antidotes, the veterinarian must treat each toxicity with general methods of poison management, applying basic critical care in the treatment of specific clinical signs associated with the poison exposure or toxicity. the adage "treat the patient, not the poison" often comes into play when the exact toxic substance is unknown, or has no specific antidote. before an animal arrives, the staff should be prepared to ask specific questions over the phone, and provide initial advice for clients, particularly if the animal lives some distance from the hospital (box 1-55.) it is important to have access to a database of information on toxic substances. thousands of potentially toxic substances are available on the market today. the american society for the prevention of cruelty to animals (aspca) animal poison control center provides direct access to veterinary toxicologists 24 hours a day, 365 days a year. for additional information, call the nearest veterinary school or emergency center (box 1-56). also, see section 6 for a table of emergency hotlines. check your local telephone book for a poison control center listing under emergency numbers, usually found on the front cover. although these numbers are for human poisonings, they have access to extensive poison and toxin databases and can potentially provide useful information for veterinarians, particularly regarding antidotal substances suitable for out of the ordinary toxins and human medications. information on the toxic ingredients in thousands of medications, insecticides, pesticides, and other registered commercial products has been confidentially placed by the government in these poison control centers. as new products are marketed, information regarding toxin ingredients is forwarded to the centers. various e-mail discussion lists can serve as an informative resource for practitioners, but access generally requires an initial subscription and may have the disadvantage of delayed 1 *do not keep the client on the telephone for too long. lengthy histories can be performed once the animal is at your hospital and you have started to initiate treatment. â�  hair dressing products sometimes have hydrogen peroxide as a 30% w/v; this concentration is not suitable for induction of emesis. is your animal breathing or does it have respiratory difficulty? what is the color of the gums or tongue? is your animal able to walk? is there any vomiting, diarrhea, trembling, or seizures? does it appear lethargic or hyperactive? what is the substance that your animal ingested (was exposed to)? did you witness the ingestion or exposure? how much did the animal consume? how long ago was the exposure? was the substance swallowed, or is it on the animal's skin or eyes? how is the patient acting? how long has the animal been acting that way? or when was the last time you saw your animal act normally? 2. first aid instructions for the client: induce vomiting at home and save the vomitus. never induce vomiting if the patient is depressed, appears comatose, or is actively seizing. if the animal has ingested a caustic substance (strong alkali or acids) or a petroleum-based product (kerosene or turpentine), never recommend induction of emesis. hydrogen peroxide (3% w/v â�  ) 5 ml = 1 tsp/10 lb of body weight can repeat once if no vomiting occurs after 10 minutes 3. remind the owner to bring a sample of the toxin and the vomitus in with the patient. 4. advise the owner to transport the patient as rapidly as possible to the nearest veterinary hospital. 1 response times. they are useful for ideas on standard and long-term therapy, but not emergency stabilization. an exception to this is the veterinary interactive network (vin), which posts message board communications. previous communications from veterinarians who treated a case with the same poison/toxin can be accessed with a subscription. many manufacturers operate an information service about their products. if the product label or name is available, check for a telephone number that may route you to a specialist. there are six essential steps in treating toxicities: 1. performing a physical examination 2. stabilizing the patient's vital signs 3. taking a thorough history 4. preventing continued absorption of the toxin 5. administering specific antidotes when available 6. facilitating clearance or metabolism of the absorbed toxin it is most important to provide symptomatic and supportive care both during and following emergency treatment. immediately on presentation, perform a brief but thorough physical examination. obtain a minimum database as well as serum, urine, or orogastric lavage samples for later toxicologic analyses. it is important at this time to systematically evaluate the patient's physical status, focusing particularly on the toxins most common to a particular geographic location and the organ systems most commonly affected by toxins in veterinary medicinenamely, the neurologic and gastrointestinal tracts. a checklist is useful when performing a complete physical examination (box 1-57). the minimium database includes a urine sample, packed cell volume, total protein, serum urea, and serum glucose. the information obtained from these simple cage-side tests is useful for determining dehydration, hemoconcentration, azotemia (renal or prerenal), and hypo-or hyperglycemia. when appropriate, obtain samples for serum biochemistry profiles, serum electrolytes, blood gases, serum osmolality, a complete hemogram, and coagulation profiles. samples of serum, urine, and any vomitus or orogastric lavage contents should be collected and saved for later toxicologic analyses as required later. stabilization of vital signs includes four major goals of treatment: maintain respiration, maintain cardiovascular function, control cns excitation, and control body temperature. in any patient with clinical signs of respiratory distress or respiratory dysfunction, supplemental oxygen should be administered via flow-by, oxygen hood, oxygen cage, nasal, nasopharyngeal, or transtracheal oxygen sources. ventilatory assistance may be necessary. irritant or corrosive substances can cause damage to the oropharyngeal mucosa to such an extent that airway obstruction occurs. when necessary, a temporary tracheostomy should be performed. arterial blood gases, pulse oximetry, and capnometry may be required to monitor oxygenation and ventilation. at the time of presentation, immediately place an intravenous catheter for administration of intravenous fluids, inotropes, antiarrhythmics, and antidotes, if necessary. the initial fluid of choice is a balanced crystalloid solution such as normosol-r, plasmalyte-m, or lactated ringer's solution. fluid therapy can later be changed based on the patient's acidbase and electrolyte status. some toxins can cause severe dysrhythmias and hyper-or hypotension. monitor blood pressure and perform ecg and correct any abnormalities according to standard therapy (see sections on hypotension and cardiac dysrhythmias). what is the pupil size? what is the pupil reactivity to light? is the ocular examination normal? what is the sensitivity to light or sound? nose: is it moist, dry, bubbling, or frothy, or caked with dirt? throat: are there any characteristic odors on the breath? are there any traces of foreign material on the tongue or in the crevices of the teeth or gums? are there petechiae or ecchymosis on the gums or bleeding from the gumline? what is the mucous membrane color? is it normal and pink, or dark red (injected), pale, or icteric? what is the capillary refill time? is it fast, normal, or slow? what is the patient's heart rate? are there any pulse deficits or dysrhythmias auscultated? what is the patient's blood pressure? what is the quality of the femoral pulse? is it synchronous with the heart rate, or are there dropped pulses? is the pulse bounding, normal, thready, or not palpable? what is the patient's electrocardiogram? what is the patient's respiratory rate? what is the patient's respiratory character? is it normal, fast, shallow, or labored? what do you hear on thoracic auscultation? do you hear harsh airway sounds or pulmonary crackles? what is the patient's rectal temperature? is there excessive salivation? is there evidence of vomiting or diarrhea? is abdominal palpation painful? do the intestinal loops feel normal, or are they fluid-filled or gas-filled? what is the color and consistency of the feces? is there a palpable urinary bladder? is there urine production? what is the color of the urine? peripheral lymph nodes should be normal in poisonings. some toxins cause hemolysis, methemoglobinemia, heinz body anemia, and coagulopathies. whole blood, fresh frozen plasma, packed rbcs, or hemoglobin-based oxygen carriers should be available and used if necessary. treat methemoglobinemia with a combination of ascorbic acid and n-acetylcysteine. many toxins affect the cns, producing clinical signs of excitation and/or seizures. diazepam is the drug of choice for most but not all seizures and tremors. if an animal has cns excitation secondary to the ingestion of selective norepinephrine reuptake inhibitors, avoid using diazepam, as it can potentially exacerbate clinical signs. muscle relaxants such as guaifenesin or methocarbamol may be required to control muscle spasm and tremors associated with some toxicities. consider animals that are in status epilepticus because of toxin exposure at high risk. such patients may not require the full dose of anesthetics or sedatives for seizure control. give phenobarbital (16-20 mg/kg iv) or pentobarbital (3-25 mg/kg iv to effect) for longer-term management of seizures. core body temperature can easily increase or decrease secondary to increased muscle activity or coma. animals may present as hypo-or hyperthermic, depending on the toxin ingested and the stage of toxicity. manage hypothermia with circulating hot water or hot air blankets, or place bubble wrap or saran wrap around the animal's peripheral extremities. manage hyperthermia by placing lukewarm wet towels on the patient until the rectal temperature has decreased to 39.5â°c (103â°f). (see section on of hyperthermia and heat-induced illness). if sedatives or anesthetics have been used, initial hyperthermia may initially resolve due to hypothalamic loss of thermoregulatory control, cool water bathing should not be performed. when the patient is first presented to the veterinarian, have the owner complete a toxicologic history form (figure 1-56) while the animal is being initially assessed and vital signs are being stabilized. when initial stabilization of vital signs has been accomplished, the veterinarian can discuss the patient's history with the owner. in urgent situations, the veterinarian should obtain a brief history as an initial procedure (box 1-58). knowing when the animal was last seen as normal provides a time frame in which the toxic substance was most likely accessed, allowing differential diagnoses to be ranked in some order of probability by rate of onset. in eliciting a history from the owner about the animal's access to poisons, it is important not to take anything for granted. many owners do not realize how poisonous some substances can be, such as insecticide products, garbage, cleaning chemicals, and over-the-counter drugs commonly used by humans. many owners will deny that an animal could have ingested anything that might be toxic, not wanting to believe that the source of the toxin is within their household or property, particularly if recreational drug exposure is suspected. it is useful to phrase questions in a neutral fashion-for example, "is such-and-such present on the premises?" rather than "could the dog have eaten such-and-such?" if recreational drug exposure is suspected, another way to question the owners is to ask whether they have had any guests in their house recently that may have had such-and-such (e.g., marijuana, cocaine, methamphetamine). this approach serves to minimize the suggestion of any bias or preconceptions. when questioning an owner about recent events, it is useful to realize and acknowledge that disruption in the household routine is a distinct factor in the occurrence accidents, including poisonings. examples of such disruptive events include moving from the house, family member is ill or in the hospital, and renovations or recent construction. while these events are occurring, the safeguards followed by a normally careful owner may be disrupted. often, doors or gates may be left open, animals may be outside instead of inside (or vice versa), and inexperienced people may be pet-sitters. once owners are made aware of the importance of assessing such risks, they are often able to provide insight into otherwise baffling circumstances. various methods can be used to remove toxins from the gastrointestinal tract, including emesis, orogastric lavage, cathartics, and enemas. adsorbents, ion exchange resins, or 1 precipitating or chelating agents may be used. removal of a toxic substance from the body surface may be necessary, depending on the toxin.the use of both emesis and orogastric lavage is less and less frequent in human medicine because of the risk of aspiration pneumonia and doubts about their efficacy. currently, management of poisonings in human medicine relies heavily on the use of activated charcoal combined with sorbitol as a cathartic, when appropriate, and supportive critical care. it should be emphasized, however, that the majority of poisonings in humans are due to drug overdoses (illicit or otherwise) (which have a relatively small volume and rapid absorption), for which this treatment is appropriate. furthermore, adoption of the approach rests on the availability of a hospital intensive care infrastructure, which is not always available in veterinary practice. induce emesis if the animal's physiology and neurologic status are stable (i.e., does not have respiratory depression or is not actively seizing, obtunded, unable to swallow or protect its airway). do not administer the same emetic more than twice. if the emetic doesn't work after two doses, give a different emetic or perform orogastric lavage under general anesthesia. emetics are strictly contraindicated for toxicity from petroleum-based products and corrosives because of the risk of aspiration pneumonia and further esophageal damage. emetics may also be of little value if poisons with antiemetic properties have been ingested, such as benzodiazepines, tricyclic antidepressants, and marijuana (table 1-50) . various emetics traditionally have been recommended for use in veterinary medicine. many have fallen out of favor because of the risk of causing adverse consequences and side effects. apomorphine (0.04 mg/kg iv or in the conjunctival sac) remains the standard but is less useful in certain situations in which the poison causes cns excitation or stimulation. it is ineffective in cats. other emetics include xylazine and hydrogen peroxide. do not use table salt because of the risk of severe oropharyngeal irritation and hypernatremia. do not use mustard powder or dishwashing liquid detergent because of the risk of severe oropharyngeal, esophageal, and gastric irritation. orogastric lavage is described in detail in the section on emergency procedures gastric lavage is contraindicated in treatment of toxicity from petroleum-based compounds and acid/alkali ingestion. the procedure can be messy but is very effective if performed within 1 to 2 hours of ingestion of the poison. to prevent aspiration, the patient should be placed under general anesthesia. keep the animal's head lowered during the procedure to prevent aspiration of stomach contents into the trachea. it is sometimes helpful to put the animal in both right and left lateral recumbency to allow complete emptying of gastric contents. repeat the procedure until the fluid runs clear from the stomach. in some cases in which solid material has been ingested, this process can take a long time, so be prepared with a large volume of warm water. following successful evacuation and lavage, administer a slurry of activated charcoal through the orogastric tube before removing it. keep the endotracheal tube cuffed and in place until the animal is semi-conscious, is starting the fight the tube, and is visibly able to swallow and protect its airway. â�¢ when was the animal last seen as normal? â�¢ what clinical signs developed? â�¢ how fast did the clinical signs develop? â�¢ when was the onset of clinical signs? â�¢ what is the animal's activity level? â�¢ does the animal have access to any poisonous substances? â�¢ this includes known toxins or chemicals, over-the-counter or prescription medications (including the owner's), and recreational drugs. enemas are useful to facilitate the action of cathartics and in cases in which the poison is a solid material (e.g., compost, snail bait, garbage) (box 1-59). it is best to use just lukewarm water. commercially available phosphate enema solutions can cause severe electrolyte disturbances (hyperphosphatemia, hyponatremia, hypocalcemia, and hypomagnesemia) and acid-base abnormalities (metabolic acidosis); therefore, they are absolutely contraindicated in small animal patients. use nonsterile nonspermicidal water-soluble lubricants (k-y jelly) old intravenous fluid bag enema bag 60-to 120-ml syringe fluid warm water, with or without hand or liquid dish soap the fluid volume required depends on the size of the animal and the state of its lower gastrointestinal tract. as with orogastric lavage, continue the procedure until the water runs clear. if difficulty is encountered emptying the lower gastrointestinal tract, repeat the enema in 1 or 2 hours, rather than be overzealous on the first attempt. cathartics are useful for hastening gastrointestinal elimination of toxins, and they are particularly useful for elimination of most solid toxicants (e.g., compost, garbage, snail baits). cathartics can be used in conjunction with activated charcoal. do not use magnesium-based cathartics in patients with cns depression, because hypermagnesemia can worsen this disorder and also cause cardiac rhythm disturbances (table 1-51) . activated charcoal (1-4 ml/kg) is the safest and to date the most effective adsorbent for the treatment of ingested toxins. activated charcoal can be administered after emesis or orogastric lavage or can be administered as the sole treatment. various preparations are available on the market, including dry powder, compressed tablets, granules, liquid suspensions, and concentrated paste preparations. commercially available products are relatively inexpensive and should be used whenever possible for ease of administration. vegetableorigin activated charcoal is the most efficient adsorbent and binds compounds with weak, nonionic bonds. some preparations are combined with sorbitol to provide simultaneous administration of an adsorbent and a cathartic; this combination has been shown to be most efficacious. repeated administration of activated charcoal every 4 to 6 hours has been shown to be beneficial in the management of a toxin that undergoes enterohepatic recirculation. administration of an oily cathartic or mixing the activated charcoal with food only serves to reduce the absorptive surface of the activated charcoal and therefore is not recommended. in general, substances that are very soluble and are rapidly absorbed are not well adsorbed by activated charcoal, including alkalis, nitrates, mineral acids, ethanol, methanol, ferrous sulfate, ammonia, and cyanide. kaolin and bentonite are clays that have been used as adsorbents. both are usually less effective than activated charcoal. however, they are reported to be better adsorbents than activated charcoal for the herbicide paraquat. ion exchange resins can ionically bind certain drugs or toxins. cholestyramine is one such resin, commonly used in human medicine to bind intestinal bile acids and thereby decrease cholesterol absorption. its application in toxicology extends to the absorption of fat-soluble toxins such as organochlorine and certain acidic compounds such as digitalis. ion exchange resins also have been used to delay or reduce the absorption of phenylbutazone, warfarin, chlorothiazide, tetracycline, phenobarbital, and thyroid preparations. precipitating, chelating, and diluting agents precipitating, chelating, and diluting agents are used primarily in the management of heavy metal intoxications, such as alkaloids or oxalates. they work by binding preferentially to the metal ion and creating a more soluble complex that is amenable to renal excretion. those chelating agents in common usage are calcium edta, deferoxamine, and d-penicillamine. calcium edta and deferoxamine should both be on hand in the veterinary hospital because they are necessary to treat zinc and iron toxicity, respectively, both of which have a short window of opportunity for therapeutic intervention. d-penicillamine has a wide application for a number of metal toxicities but tends to be used for long-term chronic therapy because it can be administered orally. various agents used for nonspecific dilution of toxins, including milk of magnesia and egg whites, although old-fashioned, still have wide application in many cases in which low-grade irritants have been ingested. bathing the animal is an important aspect of treatment for topical exposures to toxins such as insecticidal products, petroleum-based products, and aromatic oils. bathing an animal is not an innocuous procedure. to avoid hypothermia and shock, use warm water at all times. actively dry the animal to further minimize the risk of hypothermia. when bathing the animal, use rubber gloves and a plastic apron to avoid exposure to noxious agents. in most cases, a mild dishwashing soap is appropriate. medicated or antibacterial shampoos are less appropriate in this situation. for petroleum-based products in particular, dawn dishwashing liquid that "cuts the grease" works well to remove the oils. if dawn is not available, mechanics' hand cleaners or coconut oil-based soaps can be used instead. as a general principle, best results are obtained by barely wetting the patient's fur until the detergent is worked well into the fur, keeping the amount of water to a minimum until ready for the rinse. oil-based paint is best removed by clipping rather than by attempting removal with solvents, because solvents are also toxic. to remove powder products, brush and vacuum the animal before bathing it to eliminate further toxic exposure. with caustic alkaline or acidic products, the primary treatment is to dilute and flush the skin with warm water; do not attempt neutralization. neutralization can cause an exothermic reaction that causes further damage to the underlying tissues. eliminating poison from the eyes for ocular exposures, irrigate the eyes for a minimum of 20 to 30 minutes with warm (body temperature) tap water or warmed 0.9% sterile saline solution. the use of neutralizing substances is not recommended because of the risk of causing further ocular damage. following adequate irrigation, treat chemical burns of the eyes with lubricating ointments and possibly a temporary tarsorrhaphy. atropine may be indicated as a cycloplegic agent. systemic nonsteroidal antiinflammatory drugs can be used to control patient discomfort. daily follow-up examinations are required because epithelial damage may be delayed, especially with alkali burns, and it is difficult to predict the final extent of ocular damage. topical glucocorticosteroids are contraindicated if the corneal epithelium is not intact. if severe conjunctival swelling is present with a corneal ulcer, parenteral glucocorticosteroids can be administered to help alleviate inflammation, but nonsteroidal antiinflammatory drugs should not be used simultaneously due to the risk of gastrointestinal ulceration or perforation. whenever possible, administer specific antidotes to negate the effects of the toxin and prevent conversion of the substance to the toxic metabolite. three categories of agents are used in the management of poisonings. the first category is specific antidotes. unfortunately, few specific antidotes are available for use in veterinary medicine. some "classic" toxins and antidotes are now considered to be rare, such as curare and physostigmine, thallium and prussian blue, and fluoride and calcium borogluconate. these and a few others have been omitted from the table. the second, broader category of antidotes includes those drugs used in the symptomatic management of clinical signs, which are part of our routine veterinary stock. drugs such as atropine, sedatives, steroids, antiarrhythmics, and beta-blockers fall into this category. the third category comprises nonspecific decontaminants such as activated charcoal, cathartics, and emetics. these were discussed previously. many patients benefit from efforts to enhance clearance or metabolism of the absorbed toxins. some specific therapies have been developed for this purpose, including 4-methylpyrazole for ethylene glycol toxicity and specific antibodies such as digibind (digoxin immune fab [ovine]) for digitalis toxicity. other strategies are aimed at promoting renal excretion. renal excretion strategies include diuresis, ion trapping, and peritoneal dialysis or hemodialysis (see section on peritoneal dialysis). diuresis and ion trapping are applicable to a large number of toxins and are discussed here in more detail. other toxins respond to urine acidification and urine alkalinization. enhancing renal excretion of substances is most useful for those organic substances that are present in significant concentrations in the plasma. substances that are non-ionic and lipid-soluble, such as certain herbicides, are likely to be less affected by attempts to promote rapid renal elimination. before starting diuresis or ion trapping, intravenous fluid therapy should be adequate as determined by normal central venous pressure, urine output, and mean arterial blood pressure. if any of these values are less than normal, use other measures to ensure adequate renal perfusion, including but not limited to a constant rate infusion of dopamine. simple fluid diuresis can influence the excretion of certain substances. the use of mannitol as an osmotic diuretic may reduce the passive reabsorption of some toxic substances in the proximal renal convoluted tubule by reducing water reabsorption. dextrose (50%) can be used as an osmotic diuretic. furosemide can be used to promote diuresis, but again, there is no substitute for intravenous fluid therapy. the use of mannitol, dextrose, and furosemide is contraindicated in hypotensive or hypovolemic patients. take care to avoid causing dehydration with any diuretic; central venous pressure monitoring is strongly recommended. ion trapping is based on the principle that ionized substances do not cross renal tubular membranes easily, and are not well reabsorbed. if the urinary ph can be changed so that the toxin's chemical equilibrium shifts to its ionized form, then that toxin can be "trapped" in the urine and excreted. alkaline urine favors the ionization of acidic compounds, and acidic urine favors the ionization of alkaline compounds. those toxins that are amenable to ion trapping are mostly weak acids and weak bases. ammonium chloride can be used to promote urinary acidification. contraindications to the use of ammonium chloride include a preexisting metabolic acidosis, hepatic or renal insufficiency, and hemolysis or rhabdomyolysis leading to hemoglobinuria or myoglobinuria. signs of ammonia intoxication include cns depression and coma. when performing urine acidification, frequently check the serum potassium concentration and urine ph. urine alkalinization can be performed with use of sodium bicarbonate. contraindications to the use of sodium bicarbonate include metabolic alkalosis (particularly with concurrent use of furosemide), hypocalcemia, and hypokalemia. as with urine acidification, monitor the serum potassium concentration and urine ph frequently. the major steps in management of poisonings discussed here must be accompanied by application of the fundamentals of critical care. respiratory and cardiovascular support have been discussed previously. renal and gastrointestinal function and analgesia are particularly important in the management of the poisoning patient. maintenance of renal perfusion is a priority in the poisoning patient. fluid, electrolyte, and acid-base balance must be controlled and be accurate. poisoning patients are at particularly high risk for renal damage and acute renal failure, whether by primary toxic insult to the renal parenchyma or by acute or prolonged renal hypoperfusion. for this reason, a protocol that aims at preventing oliguria and ensuing renal failure is one of the therapeutic strategies that should be routinely employed. this protocol is described in box 1-60. gastrointestinal protectant drugs may be indicated for the management of those poisons that are gastrointestinal irritants or ulcerogenic. commonly used gastroprotectant drugs include cimetidine, ranitidine, famotidine, omeprazole, sucralfate, and misoprostol. antiemetics may be used to suppress intractable vomiting. metoclopramide is commonly used, and it is the drug of choice for centrally mediated nausea. antiemetics that work by different mechanisms can be used in combination as necessary. examples are dopamine 2-receptor antagonists such as prochlorperazine, 5-hydroxytryptamine antagonists such as ondansetron and dolasetron, and h-1 receptor antagonists such as diphenhydramine and meclizine. analgesics are more appropriate to treat poisonings than once thought. common effects of poisons including severe gastroenteritis and topical burns or ulcerations may warrant the use of analgesics. longer-acting analgesics such as morphine, hydromorphone, and buprenorphine are particularly useful. nutritional support may be necessary in the form of enteral or parenteral feeding in patients that have esophageal or gastric damage or that need to be sedated for long periods of time. endoscopy may be useful in assessing the degree of esophageal and gastric damage, particularly after ingestion of caustic substances. introduction: acetaminophen (paracetamol) is the active ingredient in tylenol and many over-thecounter cold products. acetaminophen is converted to n-acetyl-p-benzoquinonimine in the liver, a toxic substance that can cause oxidative injury of red blood cells and hepatocytes. clinical signs of acetaminophen toxicity include respiratory distress from lack of oxygen-carrying capacity, cyanosis, methemoglobinemia (chocolate-brown appearance of the blood and mucous membranes), lethargy, vomiting, and facial and paw swelling (cats). the toxic dose of acetaminophen is >100 mg/kg for dogs, and 50 mg/kg for cats. treatment of acetaminophen toxicity includes induction of emesis or orogastric lavage if the substance has been ingested within 30 minutes. activated charcoal should also be administered. in cases of severe anemia, give supplemental oxygen along with a packed rbc transfusion. administer intravenous fluids to maintain renal and hepatic perfusion. n-acetylcysteine, vitamin c, and cimetidine are the treatments of choice for methemoglobinemia in patients with acetaminophen toxicity. introduction: hydrochloric, nitric, and phosphoric acids cause chemical burns through contact with the skin and/or eyes. localized superficial coagulative necrosis occurs upon contact. usually, the patient's skin is painful to the touch or the animal may lick or chew at an irritated area that is not visible under the haircoat. if the chemical is swallowed, do not induce emesis or perform orogastric lavage, because of the risk of worsening esophageal irritation. rinse the patient's skin and eyes with warm water or warm saline for a minimum of 1 /2 hour. use analgesics and treat corneal ulcers (see section on corneal ulcers) as required. do not attempt chemical neutralization, because of the risk of causing an exothermic reaction and worsening tissue injury. aflatoxin (aspergillus flavus) is found in moldy feed grains. clinical signs of toxicity occur after ingestion and include vomiting, diarrhea, and acute hepatitis; abortion may occur in pregnant bitches. treatment of suspected aflatoxin ingestion consists of gastric decontamination, administration of activated charcoal, intravenous fluids, and hepatic supportive care (s-adenosyl methionine [same], milk thistle). drinking (ethanol), rubbing (isopropyl), and methyl (methanol) alcohols can be harmful if ingested (4.1 to 8.0 g/kg po). all cause disruption of neuronal membrane structure, impaired motor coordination, cns excitation followed by depression, and stupor that can lead to cardiac and respiratory arrest, depending on the amount ingested. affected animals may appear excited and then ataxic and lethargic. contact or inhalant injury can occur, causing dermal irritation and cutaneous hyperemia. methanol also can cause hepatotoxicity. 1 and diarrhea result from muscarinic overload. nicotinic overload produces muscle tremors. toxicity can result in seizures, coma, and death. 1 and cause severe irritation and corrosion of the mucous membranes and skin. some compounds also can cause clinical signs similar to those observed with anticholinesterase compounds, including muscle tremors, seizures, paralysis, and coma. methemoglobinemia can occur. 1 signs of ethylene glycol intoxication and renal impairment or failure, a negative test for the presence of calcium oxalate crystalluria means that there is no more ethylene glycol in the patient's serum because it has all been metabolized. cats are very sensitive to the toxic effects of ethylene glycol. in many cases, cat may have ingested a toxic dose, but because the sensitivity of the assay is low, test results will be negative. lack of treatment can result in death. there are three phases of ethylene glycol intoxication. in the first 1 to12 hours after ingestion (stage i), the patient may appear lethargic, disoriented, and ataxic. in stage ii (12 to 24 hours following ingestion), the patient improves and appears clinically normal. in stage iii (24 to 72 hours following ingestion), the patient demonstrates clinical signs of renal failure (polyuria and polydipsia) that progress to uremic renal failure (vomiting, lethargy, oral ulceration). finally, seizures, coma, and death occur. crosses, old english sheepdogs, and some terriers. clinical signs of ivermectin toxicity include vomiting, ataxia, hypersalivation, agitation, tremors, hyperactivity, hyperthermia, hypoventilation, coma, seizures, signs of circulatory shock, bradycardia, and death. clinical signs often occur within 2 to 24 hours after ingestion or iatrogenic overdose. blood ivermectin levels can be measured, but diagnosis is often made based on clinical signs and knowledge of exposure in predisposed breeds. there is no known antidote. the clinical course can be prolonged for weeks to months before recovery occurs. to treat known exposure, induce emesis or perform orogastric lavage if the substance was ingested was within 1 hour of presentation and the patient is not symptomatic. administer activated charcoal. control seizures with phenobarbital, pentobarbital, or propofol administered as intermittent boluses or as a constant rate infusion. diazepam, which potentially can worsen central nervous stimulation, is contraindicated. administer intravenous fluids to maintain perfusion and hydration, and treat hyperthermia. supportive care may be necessary, including supplemental oxygen (or mechanical ventilation, if necessary), frequent turning of the patient and passive range-of-motion exercises, placement of a urinary catheter to maintain patient cleanliness and monitor urine output, lubrication of the eyes, and parenteral nutrition (see section on rule of twenty). specific antidotes used to treat ivermectin toxicity include physostigmine and picrotoxin. physostigmine therapy was beneficial in some patients for a short period; picrotoxin caused severe violent seizures and therefore should be avoided. introduction d-limonene and linalool are components of citrus oil extracts used in some flea control products. the toxic dose is unknown, but cats appear to be very sensitive to exposure. clinical signs of toxicity include hypersalivation, muscle tremors, ataxia, and hypothermia. treatment of d-limonene and linalool exposure includes treatment of hypothermia, administration of activated charcoal to prevent further absorption, and careful, thorough bathing to prevent further dermal exposure. lead is ubiquitous, and is found in some paints, car batteries, fishing equipment/ sinkers, and plumbing materials. lead can be toxic at doses of 3 mg/kg. if more than than 10-25 mg/kg of lead is ingested, death can occur. lead causes toxicity by inhibiting sulfur-containing enzymes, leading to increased rbc fragility, and cns damage. clinical signs of hyperexcitability, dementia, vocalization, seizures, and lower motor neuron polyneuropathy can occur. affected animals may appear blind, or vomiting, anorexia, and constipation or diarrhea may occur. if lead toxicity is suspected, blood and urine lead levels can be measured. treatment of lead toxicity is supportive and is directed at treatment of clinical signs. control seizures with diazepam or phenobarbital. if cerebral edema is present, administer mannitol (0.5-1.0 g/kg iv), followed by furosemide (1 mg/kg iv 20 minutes after mannitol). sodium or magnesium sulfate should be administered as a cathartic. initiate chelation therapy with dimercaprol, penicillamine, or calcium edta. if a lead object is identified in the gastrointestinal tract on radiographs, remove the object using endoscopy or exploratory laparotomy. 1 hyperthermia, that occurs within 15 -30 minutes of ingestion. diarrhea and convulsions can develop. if hyperthermia is severe, renal failure secondary to myoglobinuria and disseminated intravascular coagulation can result. delayed hepatic failure has been described days after initial recovery. if metaldehyde toxicosis is suspected, analysis of urine, serum, and stomach contents is warranted. to treat metaldehyde toxicity, procure and maintain a patent airway and control cns excitation and muscle tremors. if an animal has just ingested the metaldehyde and is not symptomatic, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by administration of one dose of activated charcoal. administer intravenous fluids to control hyperthermia, prevent dehydration, and correct acid-base and electrolyte abnormalities. methocarbamol is the treatment of choice to control muscle tremors. diazepam can be used to control seizures if they occur. introduction mushroom ingestion most commonly causes activation of the autonomic nervous system, resulting in tremors, agitation, restlessness, hyperexcitability, and seizures. in some cases slud (salivation, lacrimation, urination, and defecation) is seen. some mushrooms (amanita spp.) also can cause hepatocellular toxicity. clinical signs include vomiting, anorexia, lethargy, and progressive icterus. 1 hemoglobinuria and pigment damage of the renal tubular epithelium. heinz bodies may be observed on cytologic evaluation of the peripheral blood smear. 1 paint in a sorbitol or glycerol carrier. when large quantities of these osmotically active sugars are ingested, osmotic shifts of fluid cause a sudden onset of neurologic or gastrointestinal signs, including ataxia, seizures, and osmotic diarrhea caused by massive fluid shifts into the gastrointestinal tract. the loss of water in excess of solute can result in hypernatremia, a free water deficit, and increased serum osmolality. following orogastric lavage, treatment of ingestion includes administering warm water enemas to help speed the movement of the paintballs through the gastrointestinal tract. do not administer activated charcoal (usually in a propylene glycol carrier), because the compound's cathartic action will pull more fluid into the gastrointestinal tract. baseline electrolytes should be obtained and then carefully monitored. if severe hypernatremia develops, administer hypotonic solutions such as 0.45% nacl + 2.5% dextrose or 5% dextrose in water after calculating the patient's free water deficit. because of the large volume of fluid loss, intravenous fluid rates may seem excessive but are necessary to normalize acid-base, electrolyte, and hydration status. in most cases, these patients can survive if the problem is recognized promptly and corrected with careful electrolyte monitoring, aggressive decontamination strategies, and intravenous fluid support. introduction paraquat, a dipyridyl compound, is the active ingredient in some herbicides. the ld 50 of paraquat is 25-50 mg/kg. paraquat initially causes cns excitation. it also causes production of oxygen-derived free radical species in the lungs, that can lead to the development of acute respiratory distress syndrome. initial clinical signs include vomiting, diarrhea, and seizures. within 2 to 3 days, clinical signs associated with severe respiratory distress and acute respiratory distress syndrome (ards) can develop, leading to death. chronic effects include pulmonary fibrosis, if the patient survives the initial toxicity period. the prognosis for paraquat toxicity is generally unfavorable. to treat paraquat ingestion, remove the toxin from the gastrointestinal tract as rapidly as possible after ingestion. there are no known antidotes. if the compound was ingested within the past hour and the animal is able to protect its airway, induce emesis. otherwise, perform orogastric lavage. activated charcoal is not as effective as clay or bentonite adsorbents for removing this particular toxin. early in the course of paraquat toxicity, oxygen therapy is contraindicated because of the risk of producing oxygen-derived free radical species. later, oxygen therapy, including mechanical ventilation, is necessary if ards develops. experimentally, free radical scavengers (n-acetyl cysteine, vitamin c, vitamin e, same) have been shown to be useful in preventing damage caused by oxygen-derived free radical species. hemoperfusion may be useful in eliminating the toxin, if it is performed early in the course of toxicity. pennyroyal oil is an herbal flea control compound that contains menthofuran as its toxic compound. menthofuran is hepatotoxic and may cause gastrointestinal hemorrhage and coagulopathies. to treat toxicity, administer a cathartic and activated charcoal and antiemetic and gastroprotectant drugs, and thoroughly bathe the animal to prevent further dermal exposure. petroleum distillates: see fuels phenobarbital: see barbiturates phenylcyclidine (angel dust) introduction phenylcyclidine (angel dust) is an illicit recreational drug that causes both cns depression and excitation, decreased cardiac output, and hypotension. to treat phenylcyclidine toxicity, place an intravenous catheter, and administer intravenous fluids and antiarrhythmic drugs to maintain organ perfusion. administer supplemental oxygen, and administer diazepam to control seizures. urine alkalinization can help eliminate the compound. phenylephrine is an î±-adrenergic agonist in many over-the-counter decongestant preparations. clinical signs of intoxication include mydriasis, tachypnea, agitation, hyperactivity, and abnormal flybiting and staring behavior. tachycardia, bradycardia, hypertension, hyperthermia, and seizures can occur. to treat phenylephrine toxicity, place an intravenous catheter and give intravenous fluids to maintain hydration, promote diuresis, and treat hyperthermia. administer prazosin or sodium nitroprusside to treat hypertension, antiarrhythmic drugs as necessary, and diazepam to control seizures. phenylpropanolamine has both î±and î²-adrenergic agonist effects, and is used primarily in the treatment of urinary incontinence in dogs. the drug was taken off of the market for use in humans because of the risk of stroke. clinical signs of phenylpropanolamine intoxication include hyperactivity, hyperthermia, mydriasis, tachyarrhythmias or bradycardia, hypertension, agitation, and seizures. to treat toxicity, administer prazosin or nitroprusside to control hypertension, a betablocker (esmolol, propranolol, atenolol) to control tachyarrhythmias, diazepam to control seizures, and intravenous fluids to maintain hydration and promote diuresis. urine acidification may aid in facilitating excretion. if bradycardia occurs, do not use atropine. pseudoephedrine is an î±and î²-adrenergic agonist that is a component of many over-thecounter decongestants and is used in the manufacture of crystal methamphetamine. clinical signs of toxicity include severe restlessness, tremors, mydriasis, agitation, hyperthermia, tachyarrhythmias or bradycardia, hypertension, and seizures. to treat toxicity, administer activated charcoal, intravenous fluids to promote diuresis and treat hyperthermia, chlorpromazine to combat î±-adrenergic effects, a beta-blocker (propranolol, esmolol, atenolol) to treat î²-adrenergic effects, and cyproheptadine (per rectum) to combat serotoninergic effects. piperazine is a gaba agonist, and causes cervical and truncal ataxia, tremors, seizures, coma, and death. salt used for thawing ice commonly contains calcium chloride, a compound that has a moderate toxic potential. calcium chloride produces strong local irritation and can cause gastroenteritis and gastrointestinal ulcers if ingested. respiratory emergencies consist of any problem that impairs delivery of oxygen to the level of the alveoli or diffusion of oxygen across the alveolar capillary membrane into the pulmonary capillary network. decreased respiratory rate or tidal volume can result in hypoxia and buildup of carbon dioxide, or hypercarbia, leading to respiratory acidosis. conditions most frequently encountered result in airflow obstruction, prevention of normal lung expansion, interference with pulmonary gas exchange (ventilation-perfusion mismatch), and alterations of pulmonary circulation. evaluation of the patient with respiratory distress is often challenging, because the most minimal stress can cause rapid deterioration, or even death in critical cases. careful observation of the patient from a distance often allows the clinician to determine the severity of respiratory distress and localize the lesion based on the patient's respiratory pattern and effort. animals in respiratory distress often have a rapid respiratory rate (>30 breaths per minute). as respiratory distress progresses, the patient may appear anxious and start openmouth breathing. the animal often develops an orthopneic posture, characterized by neck extension, open-mouthed breathing, and elbows abducted or pulled away from the body. cyanosis of the mucous membranes often indicates extreme decompensation. clinical signs of respiratory distress can develop acutely, or from decompensation of a more chronic problem that was preceded by a cough, noisy respirations, or exercise intolerance. localization of the cause of respiratory distress is essential to successful case management. in any patient with clinical signs of respiratory distress, the differential diagnosis should include primary pulmonary parenchymal disease, airway disease, thoracic cage disorders, congestive heart failure, dyshemoglobinemias (carbon monoxide, methemoglobin), and anemia. careful observation of the patient's respiratory pattern can aid in making a diagnosis of upper airway disease/obstruction, primary pulmonary parenchymal disease, pleural space disease, and abnormalities of the thoracic cage. it is often helpful to rest a hand on the patient and breathe along with the patient's effort, to confirm the periods of inhalation and exhalation. the pharynx, larynx, and extrathoracic trachea comprise the upper airway. obstructive lesions are associated with a marked inspiratory wheeze or stridor and slow deep inspiratory effort. auscultation of the larynx and trachea may reveal more subtle obstructions of normal air flow. stridor can usually be auscultated without the use of a stethoscope. lung sounds are usually normal. the neck should be carefully palpated for a mass lesion, tracheal collapse, and subcutaneous emphysema. subcutaneous emphysema suggests tracheal damage or collapse secondary to severe trauma. in some cases, there is a history of voice, or bark, change secondary to laryngeal dysfunction. differential diagnosis is usually based on the patient's signalment, history, and index of suspicion of a particular disease process. differential diagnoses of upper airway obstruction are listed in box 1-61. diseases of the pleural space often are associated with a restrictive respiratory pattern. inspiratory efforts are short, rapid, and shallow, and there is often a marked abdominal push. the pattern has been referred to as a choppy "dysynchronous" respiratory pattern. depending on the disease present, lung sounds may be muffled ventrally and enhanced dorsally. percussion of the thorax reveals decreased resonance if fluid is present. increased resonance is present with pneumothorax. decreased compressibility of the anterior thorax may be present with an anterior mediastinal mass lesion, particularly in cats and ferrets. a pneumothorax or diaphragmatic hernia is commonly associated with evidence of trauma, with or without rib fractures. respiratory distress due to hemothorax may be exacerbated by anemia. differential diagnoses for patients with evidence of pleural cavity disease include pneumothorax, diaphragmatic hernia, neoplasia, and various types of pleural effusion. primary pulmonary parenchymal disease can involve the intrathoracic airways, alveoli, interstitial space, and pulmonary vasculature. a rapid, shallow, restrictive respiratory pattern may be observed with a marked push on exhalation, particularly with obstructive airway disease such as chronic bronchitis (asthma) in cats. crackles or wheezes are heard on thoracic auscultation. differential diagnoses for pulmonary parenchymal disease include cardiogenic and noncardiogenic pulmonary edema, pneumonia, feline bronchitis (asthma), pulmonary contusion, aspiration pneumonitis, pulmonary thromboembolism, neoplasia, infection (bacterial, fungal, protozoal, viral) , and/or chronic bronchitis. other abnormal respiratory patterns may be evident, and warrant further consideration. tachypnea present in the absence of other signs of respiratory distress can be a normal response to nonrespiratory problems, including pain, hyperthermia, and stress. a restrictive respiratory pattern with minimal thoracic excursions can be associated with diseases of neuromuscular function, including ascending polyradiculoneuritis, botulism, and tick paralysis. if adequate ventilation cannot be maintained by the patient, mechanical ventilation may be indicated. kussmaul respiration manifests as very slow, very deep respirations when a metabolic acidosis is present. this type of respiratory pattern typically is observed in patients with severe diabetic ketoacidosis and renal failure in a compensatory attempt to blow off carbon dioxide. cheyne-stokes respiration is usually observed with a defect in the central respiratory control center. the classic pattern of cheyne-stokes respiration is normal or hyperventilation followed by a period of apnea or hypoventilation. in cases of lower cervical cord damage or damage to the central respiratory control center in the cns, the diaphragm alone may assume most of the ventilatory movement. with diaphragmatic fatigue, severe hypoventilation and resultant hypoxemia may require mechanical ventilation. immediate management of any patient in respiratory distress is to minimize stress at all costs. relatively benign procedures such as radiography or intravenous catheter placement can be fatal in patients with severe respiratory compromise. stabilization should always precede further diagnostic evaluation. in some cases, sedation may be required before performing any diagnostics, to prevent further stress. all patients should receive some form of supplemental oxygen, either by mask, cage, or flow-by techniques. in cases in which a severe pneumothorax or pleural effusion is suspected, perform therapeutic and diagnostic thoracocentesis bilaterally to allow lung re-expansion and alleviate respiratory distress, whenever possible. if thoracocentesis alone is not effective at maintaining lung re-expansion, place a thoracostomy tube (particularly in cases of tension pneumothorax). if hypovolemic/ hemorrhagic shock is present, initiate treatment while stabilizing the respiratory system (see section on shock). if an animal is suspected of having an upper airway obstruction, reestablish airflow. in cases of laryngeal paralysis, tracheal collapse, and brachycephalic airway syndrome, sedation is often very useful in alleviating the distress of airway obstruction. in cases of laryngeal collapse, however, sedation may make the condition worse. if laryngeal edema is severe, administer a dose of short-acting glucocorticosteroids (dexamethasone sodium phosphate) to decrease laryngeal inflammation and edema. if a foreign body is lodged in the pharynx, perform the heimlich maneuver by thrusting bluntly several times on the patient's sternum. objects such as balls or bones may be small enough to enter the larynx but too large to be expelled, and will require rapid-acting general anesthesia to facilitate dislodgement and removal. if the obstruction cannot be removed, bypassing the obstruction with an endotracheal tube or temporary tracheostomy should be considered. in an emergency, a temporary transtracheal oxygen catheter can quickly be placed in the following manner. connect a 20-or 22-gauge needle to a length of intravenous extension tubing and a 3-ml syringe. place the male connector of the syringe into the female portion of the extension tubing. cut off the syringe plunger and connect the resulting blunt end to a length of flexible tubing attached to a humidified oxygen source. run the oxygen at 10 l/minute to provide adequate oxygenation until a tracheostomy can be performed. (see sections on oxygen supplementation and tracheostomy). once the animal's condition has been stabilized, specific diagnostic tests, including arterial blood gas analyses, thoracic radiographs, and/or transtracheal wash, can be performed, depending on the patient's condition and needs. specific therapies for management of upper airway obstruction, pleural space disease, and pulmonary disease are discussed next. upper airway obstruction can occur as a result of intraluminal or extraluminal mass lesions or foreign bodies in the oropharynx (abscess, neoplasia), laryngeal paralysis, trauma, and anatomic abnormalities. clinical signs of an upper airway obstruction are associated with an animal's extreme efforts to inhale air past the obstruction. marked negative pressure occurs in the extrathoracic airways and can cause worsening of clinical signs. mucosal edema and inflammation further worsen the obstruction. therapy for upper airway obstruction is aimed at breaking the cycle of anxiety and respiratory distress. administer the anxiolytic tranquilizer acepromazine (0.02-0.05 mg/kg iv, im, sq) to decrease patient anxiety. many animals develop hyperthermia from increased respiratory effort and extreme anxiety. implement cooling measures in the form of cool intravenous fluids and wet towels soaked in tepid water placed over the animal (see section on hyperthermia). administer supplemental oxygen in a manner that is least stressful for the animal. short-acting glucocorticosteroids can also be administered (dexamethasone sodium phosphate, 0.25 mg/kg iv, sq, im) to decrease edema and inflammation. if the airway obstruction is severe and there is no response to initial measures to alleviate anxiety and decrease inflammation, establish control of ventilation by placement of an endotracheal tube (see section on endotracheal intubation), tracheal oxygen catheter, or temporary tracheostomy. to obtain airway control, administer a rapid-acting anesthetic (propofol, 4-7 mg/kg iv to effect), and intubate with a temporary tracheostomy. an intratracheal oxygen catheter can be placed with sedation and/or a local anesthetic (see technique for transtracheal wash). laryngeal paralysis is a congenital or acquired condition that occurs primarily in largebreed dogs secondary to denervation of the arytenoid cartilages by the recurrent laryngeal nerve. congenital laryngeal paralysis occurs in the bouvier des flandres, siberian husky, and bull terrier. acquired laryngeal paralysis occurs in labrador retrievers, saint bernards, and irish setters. acquired laryngeal paralysis can be idiopathic, acquired secondary to trauma to the recurrent laryngeal nerve, or can be a component of systemic neuromuscular disease. although rare, this condition also occurs in cats. with dysfunction of the recurrent laryngeal nerve, the intrinsic laryngeal muscles atrophy and degenerate. as a result, the vocal folds and arytenoid cartilage move in a paramedian position within the airway and fail to abduct during inhalation, causing airway obstruction. laryngeal paralysis can be partial or complete, unilateral or bilateral. in many cases, a change in bark is noted prior to the development of clinical signs of respiratory distress or exercise intolerance. when a patient presents with severe inspiratory stridor (with or without hyperthermia) initiate stabilization with anxiolytic tranquilizers, supplemental oxygen, and cooling measures. once the patient's condition has been stabilized, definitive measures to accurately document and assess the patient's airway should be considered. place the patient under very heavy sedation with short-acting barbiturates or propofol (4-7 mg/kg iv) and observe the arytenoid cartilages closely in all phases of respiration. administer just enough drug to allow careful examination without getting bitten. if the arytenoid cartilages do not abduct during inhalation, administer dopram (doxapram hydrochloride, 1-5 mg/kg iv) to stimulate respiration. absent or paradoxical laryngeal motion (closed during inspiration and open during exhalation) is characteristic of laryngeal paralysis. correction of the defect involves documentation and treatment of any underlying disorder and surgical repair of the area to open the airway. partial laryngectomy, arytenoid lateralization ("tie-back" surgery), or removal of the vocal folds has been used with some success. aspiration pneumonitis is common following these procedures. brachycephalic airway syndrome is associated with a series of anatomic abnormalities that collectively increase resistance to airflow. affected animals typically have stenotic nares, an elongated soft palate, and a hypoplastic trachea. components of the syndrome can occur alone or in combination. in severe cases, laryngeal saccular edema and eversion, and eventual pharyngeal collapse, can occur secondary to the severe increase in intrathoracic airway pressure required to overcome the resistance of the upper airways. specific airway anomalies can be identified with general anesthesia and laryngoscopy. severe respiratory distress should be treated as discussed previously. treatment requires surgical correction of the anatomic abnormalities. in animals with laryngeal collapse, surgical correction may not be possible, and a permanent tracheostomy may be required. because an elongated soft palate and stenotic nares can be identified before the onset of clinical signs, surgical correction to improve airflow when the animal is young may decrease the negative intra-thoracic pressure necessary to move air past these obstructions. the chronic consequences of everted laryngeal saccules and laryngeal collapse potentially can be prevented. tracheal collapse is common in middle-aged and older toy and small-breed dogs. the owner typically reports a chronic cough that is readily induced by excitement or palpation of the trachea. the cough often sounds like a "goose honk." diagnostic confirmation is obtained by lateral radiography or fluoroscopy of the cervical and thoracic trachea during all phases of respiration. acute decompensation is uncommon but does occur, particularly with excitement, exercise, and increased environmental temperatures or ambient humidity. therapy of the patient with acute respiratory distress secondary to tracheal collapse includes sedation, administration of supplemental oxygen, and provision of cooling measures to treat hyperthermia. cough suppressants (hydrocodone bitartrate-homatropine methylbromide, 0.25 mg/kg po q8-12h, or butorphanol, 0.5 mg/kg po q6-12h) are useful. tracheal collapse is a dynamic process that usually involves both the upper and lower airways. because of this, bypassing the obstruction is often difficult. tracheal stents have been 256 1 emergency care 1 used with limited success in combination with treatment of chronic lower airway disease. crush or bite injuries to the neck can result in fractures or avulsion of the laryngeal or tracheal cartilages. bypassing the obstructed area may be necessary until the patient is stable and can undergo surgical correction of the injury. if there is avulsion of the cranial trachea, it may be difficult to intubate the patient. a long, rigid urinary catheter can be inserted past the area of avulsion into the distal segment, and an endotracheal tube passed over the rigid catheter, to establish a secure airway. neck injury can also result in damage to the recurrent laryngeal nerve and laryngeal paralysis. foreign bodies can lodge in the nasal cavity, pharynx, larynx, and distal trachea. signs of foreign bodies in the nares include acute sneezing and pawing at or rubbing the muzzle on the ground. if the object is not removed, sneezing continues and a chronic nasal discharge develops. respiratory distress is uncommon, but the foreign body is severely irritating. pharyngeal and tracheal foreign bodies can cause severe obstruction to airflow and respiratory distress. diagnosis of a foreign body is based on the patient history, physical examination findings, and thoracic or cervical radiographs. smaller foreign bodies lodged in the distal airways may not be apparent radiographically but can cause pulmonary atelectasis. foreign bodies of the nose or pharynx can often be removed with an alligator forceps with the patient under anesthesia. if removal is not possible with a forceps, flushing the nasal cavity from cranial to caudal (pack the back of the mouth with gauze to prevent aspiration) can sometimes dislodge the foreign material into the gauze packing. rhinoscopy may be necessary. if an endoscope is not available, an otoscope can be used. foreign objects lodged in the trachea can be small and function like a ball valve during inhalation and exhalation, causing episodic hypoxia and collapse. when attempting to remove these objects, suspend the patient with its head down. remove the object with an alligator forceps, using a laryngoscope to aid in visualization. foreign bodies lodged in the trachea or bronchi require removal with endoscopic assistance. nasopharyngeal polyps (in cats, tumors, obstructive laryngitis, granulomas, abscesses, and cysts) can cause upper airway obstruction. clinical signs are usually gradual in onset. the lesions can be identified through careful laryngoscopic examination performed with the patient under general anesthesia. the nasopharynx above the soft palpate should always be included in the examination. pedunculated masses and cysts are excised at the time of evaluation. biopsy of diffusely infiltrative masses is indicated for histologic examination and prognosis. it is impossible to distinguish obstructive laryngitis from neoplasia based on gross appearance alone. whenever possible, material should be collected from abscesses and granulomas for cytologic evaluation and bacterial culture. extraluminal masses impinge on and slowly compress the upper airways, resulting in slow progression of clinical signs. masses are usually identified by palpation of the neck. enlarged mandibular lymph nodes, thyroid tumors, and other neoplasms may be present. diagnosis is usually based on a combination of radiography and ultrasonography. ct and/or mri are helpful in identifying the full extent and invasiveness of the lesion. definitive diagnosis is made with a fine-needle aspirate or biopsy. many thyroid tumors bleed excessively. the inside of each side of the hemithorax is covered in parietal pleura. the lung lobes are covered in visceral pleura. the two surfaces are in close contact with each other, and are contiguous at the hilum under normal circumstances. pneumothorax refers to free air within the pleural space, accumulating in between the parietal and visceral pleura. the term pleural effusion refers to fluid accumulation in that area but does not reflect the amount or type of fluid present. the mediastinal reflections of the pleura typically are thin in dogs and cats, and usually, but not always, connect. bilateral involvement of pneumothorax or pleural effusion is common. both pneumothorax and pleural effusion compromise the lungs' ability to expand and result in hypoxia and respiratory distress. pneumothorax can be classified as open versus closed, simple versus complicated, and tension. an open pneumothorax communicates with the external environment through a rent in the thoracic wall. a closed pneumothorax results from tears in the visceral pleura but does not communicate with the outside. a tension pneumothorax occurs as a result of a tear in the lung or chest wall that creates a flap valve, such that air is allowed to leave the lung and accumulate in the pleural space during inhalation, and closes to seal off exit of air from the pleural space during exhalation. tension pneumothorax can cause rapid decline in cardiopulmonary status and death if not recognized and treated immediately. a simple pneumothorax is one that can be controlled with a simple thoracocentesis. complicated pneumothorax involves repeated accumulation of air, requiring placement of a thoracic drainage catheter. in many cases, pneumothorax develops as a result of trauma. spontaneous pneumothorax occurs with rupture of cavitary lesions of the lung that may be congenital or acquired as a result of prior trauma, heartworm disease, airway disease (emphysema), paragonimiasis, neoplasia, or lung abscess. pneumothorax also rarely occurs as a result of esophageal tears or esophageal foreign bodies. rapid circulatory and respiratory compromise following traumatic pneumothorax can develop as a result of open or tension pneumothorax, rib fractures, airway obstruction, pulmonary contusions, hemothorax, cardiac dysrhythmias, cardiac tamponade, and hypovolemic shock. any patient that is rapidly decompensating after a traumatic episode must be quickly assessed, and emergency therapy initiated (see section on immediate management of trauma, a crash plan). diagnosis of pneumothorax is usually made based on a history of trauma, a rapid, shallow, restrictive respiratory pattern, and muffled heart and lung sounds on thoracic auscultation. the clinical signs and history alone should prompt the clinician to perform a bilateral diagnostic and therapeutic thoracocentesis before taking thoracic radiographs (see section on thoracocentesis). the stress of handling the patient for radiography can be deadly in severe cases of pneumothorax. although the mediastinum on both sides of the thorax connects, it is necessary to perform thoracocentesis on both sides to ensure maximal removal of free air in the pleural space and allow maximal lung expansion. if negative pressure cannot be obtained, or if the patient rapidly reaccumulates air, place a thoracostomy tube connected to continuous suction. (see section on thoracostomy tube placement). treat all penetrating wounds to the thorax as open sucking chest wounds unless proved otherwise. to "close" an open sucking chest wound, clip the fur around the wound as quickly as possible, and place sterile lubricant jelly or antimicrobial ointment circumferentially around the wound. cut a sterile glove to provide a covering. place the covering over the wound, making sure to cover all of the sterile lubricant, thus creating a seal to close the wound temporarily from the external environment. evaluate the patient's thorax via thoracocentesis while placing a thoracostomy tube. once the patient is stable, the open chest wound can be surgically explored, lavaged, and definitively corrected. all animals with open chest wounds should receive antibiotics (first-generation cephalosporin) to prevent infection. following stabilization, radiographs can be taken and evaluated. pneumothorax is confirmed by evidence of elevation of the cardiac silhouette above the sternum, increased density of the pulmonary parenchymal tissue, free air in between the parietal and visceral 1 pleura (making the outline of the lungs visible), and absence of pulmonary vascular structures in the periphery. parenchymal lesions within the lungs are best identified after as much air as possible has been removed from the thorax. obtain left and right lateral and ventrodorsal or dorsoventral views. a standing lateral view may reveal air-or fluid-filled cavitary masses. if underlying pulmonary disease is suspected as a cause of spontaneous pneumothorax, a transtracheal wash, fecal flotation, and heartworm test may be indicated. treatment of pneumothorax includes immediate bilateral thoracocentesis, covering of any open chest wounds, administration of supplemental oxygen, and placement of a thoracostomy tube if negative pressure cannot be obtained or if air rapidly reaccumulates. serial radiography, ct, or mri should be performed in dogs with spontaneous pneumothorax, because the condition can be associated with generalized pulmonary parenchymal disease. strict cage rest is required until air stops accumulating and the thoracostomy tube can be removed. the patient's chest tube should be aspirated every 4 hours after discontinuing continuous suction. if no air reaccumulates after 24 hours, the chest tube can be removed. exercise restriction is indicated for a minimum of 1 week. if bullae or mass lesions are present, exploratory thoracotomy should be considered as a diagnostic and potentially therapeutic option for long-term management in prevention of recurrence. pleural fluid cytologic analysis is indicated for all patients with pleural effusion before administration of antibiotics. the general term pleural effusion means a collection of fluid in the space between the parietal and visceral pleura but does not indicate what kind or how much fluid is present. clinical signs associated with pleural effusion depend on how much fluid is present, and how rapidly the fluid has accumulated. clinical signs associated with pleural effusion include respiratory distress, reluctance to lie down, labored breathing with an abdominal component on exhalation, cough, and lethargy. auscultation of the thorax may reveal muffled heart and lung sounds ventrally and increased lung sounds dorsally, although pockets of fluid may be present, depending on the chronicity of the effusion. percussion of the thorax may reveal decreased resonance. in stable patients, the presence of pleural effusion can be confirmed radiographically. radiographic confirmation of the pleural effusion should include right and left lateral and dorsoventral or ventrodorsal views. a handling or standing lateral view should be obtained if an anterior mediastinal mass is suspected. the standing lateral view will allow the fluid to collect in the costophrenic recess. in patients with respiratory distress, muffled heart and lung sounds, and suspicion of pleural effusion, thoracocentesis should be performed immediately. thoracocentesis can be both therapeutic and diagnostic. radiography is contraindicated because the procedure can cause undue stress and exacerbation of clinical signs in an unstable patient. pleural effusion can cause severe respiratory distress, and can be the result of a number of factors that must be considered when implementing an appropriate treatment plan. pathology of the pleura is almost always a secondary process except for primary bacterial pleuritis and pleural mesotheliomas. causes of pleural effusion in the cat and dog include pyothorax, feline infectious peritonitis, congestive heart failure, chylothorax, heartworm disease, hemothorax, hypoalbuminemia, lung lobe torsions, neoplasia, diaphragmatic hernia, and pancreatitis (box 1-62). in stable animals, diagnosis of pleural effusion can be made based â�¢ imbalance of transpleural or hydrostatic or protein osmotic forces â�¢ change in membrane permeability â�¢ decrease in rate of fluid reabsorption â�¢ combination of foregoing mechanisms on thoracic radiography or ultrasound. thoracic radiographs can show whether the pleural effusion is unilateral or bilateral. effusions in dogs and cats are usually bilateral. the lung parenchyma and the cardiac silhouette cannot be fully evaluated until most of the fluid has been evacuated from the pleural cavity. following thoracocentesis, radiography should be performed with left and right lateral and ventrodorsal or dorsoventral views. in cases of suspected heart failure, echocardiography also is necessary. pleural fluid cytologic analysis is indicated for all patients with pleural effusion. collect specimens before administering antibiotics, whenever possible, because treatment with antibiotics can make a septic condition (pyothorax) appear nonseptic. the remainder of the diagnostic workup and treatment is based on the type of fluid present (table 1 -52). the fluid may be a transudate, nonseptic exudate, septic exudate, chylous, hemorrhagic, or neoplastic. ultrasonographic evaluation of the thorax can be helpful in identifying intrathoracic masses, diaphragmatic hernias, lung lobe torsions, and cardiac abnormalities. unlike radiography, ultrasonography is facilitated by the presence of fluid in the pleural space. pyothorax refers to a septic effusion of the pleural cavity. the infection is generally the result of a combination of aerobic and anaerobic bacteria. rarely, fungal organisms are present. the source of the underlying organisms is rarely identified, particularly in cats, but can be caused by penetrating wounds through the chest wall, esophagus, migrating foreign bodies (especially grass awns), or primary lung infections. the most common organisms associated with pyothorax in the cat are pasteurella, bacteroides, and fusobacterium. fever is often present in addition to clinical signs of pleural effusion. septic shock is ununcommon. diagnosis of pyothorax is made based on cytologic analysis and the demonstration of intracellular and extracellular bacteria, toxin neutrophils and macrophages, and sometimes the presence of sulfur granules. gram stains of the fluid can assist in the initial identification of some organisms. bacterial cultures are indicated for bacteria identification and antibiotic susceptibility testing. administration of antibiotics before cytologic evaluation can cause a septic effusion to appear nonseptic. emergency treatment for pyothorax involves placement of an intravenous catheter, intravenous fluids to treat hypovolemic shock, and broad-spectrum antibiotics (ampicillin, 22 mg/kg iv q6h, and enrofloxacin, 10 mg/kg iv q24h). chloramphenicol also is an appropriate antibiotic to use for penetration into pockets of fluid. administration of a beta-lactam antibiotic (ampicillin or amoxicillin) with a beta-lactamase inhibitor (amoxicillin clavulanate or ampicillin sulbactam) is helpful in achieving better coverage of bacteroides spp. treatment of pyothorax differs in the cat and dog. in the cat, placement of one or two thoracic drainage catheters is recommended to allow continuous drainage of the intrathoracic abscess. inadequate drainage can result in treatment failure. fluid should be evaluated and the pleural cavity lavaged with 10 ml/kg of warmed 0.9% saline or lactated ringer's solution every 8 hours. approximately 75% of the infused volume should be recovered after each lavage. in dogs, or in cats with refractory pyothorax, perform an exploratory thoracotomy to remove any nidus of infection. rarely a foreign body is visible that can be removed at the time of surgery, but this finding is rare. antibiotics are indicated for a minimum of 6 to 8 weeks after removal of the thoracostomy tube. early diagnosis and aggressive treatment result in a good prognosis in the majority of patients with pyothorax. in cats, clinical signs of ptyalism and hypothermia at the time of presentation worsen the prognosis. chylothorax refers to the abnormal accumulation of chyle (lymphatic fluid) in the pleural cavity. the cisterna chili is the dilated collection pool of lymphatic ducts in the abdomen that accumulate chyle prior to entry into the thoracic duct located within the thoracic cavity. the thoracic duct enters the thorax at the aortic hiatus. numerous tributaries or collateral ducts exist. the functions of the lymphatic vessels collectively serve to deliver triglycerides and fat-soluble vitamins into the peripheral vascular circulation. damage of the thoracic duct or lymphatic system or obstruction to lymphatic flow can result in the development of chylous effusion in the pleural or peritoneal space. it is difficult to identify chylous effusions based on their milky appearance alone. to identify a chylous effusion versus a pseudochylous effusion, the triglyceride and cholesterol levels of the fluid must be compared with those of peripheral blood. chylous effusions have a higher triglyceride and lower cholesterol levels than peripheral blood. pseudochylous effusions have a higher cholesterol and lower triglyceride levels than peripheral blood. disease processes that can result in chylous effusions are listed in the box 1-63. clinical signs associated with chylous effusion are typical of any pleural effusion and of the disease process that caused the effusion. weight loss may be evident, depending on the chronicity of the process. the diagnosis is made based on thoracocentesis, cytology, and biochemical evaluation of the fluid (i.e., triglyceride and cholesterol levels). the fluid often appears milky or bloodtinged but can be clear if the patient has significant anorexia. typical cytologic characteristics are listed in table 1 -52. lymphangiography can be used to confirm trauma to the thoracic duct, but this is usually not necessary unless surgical ligation is going to be attempted. the diagnostic evaluation must also attempt to identify an underlying cause. therapy for chylothorax is difficult and primarily involves documentation and treatment of the underlying cause. if an underlying cause is not found, treatment is largely supportive and consists of intermittent thoracocentesis to drain the fluid as it accumulates and causes respiratory dysfunction, nutritional support, and maintenance of fluid balance. a variety of surgical techniques, including ligation of the thoracic duct, pleural-peritoneal shunts, and pleurodesis, have been attempted but have had limited success. most recently, the combination of thoracic duct ligation with subtotal pericardectomy has been shown to improve surgical success rates in the treatment of chylothorax. rutin, a bioflavinoid, has been used with limited success in the treatment of idiopathic chylothorax in cats. prognosis in many cases of chylothorax is guarded. extensive hemorrhage into the pleural cavity can cause fulminant respiratory distress due to sudden hypovolemia and anemia and interference with lung expansion. hemothorax typically is associated with trauma, systemic coagulopathy, lung lobe torsions, and erosive lesions within the thorax (usually neoplasia). diagnosis of hemothorax involves obtaining a fluid sample via thoracocentesis. hemorrhagic effusion must be differentiated from systemic blood inadvertently collected during the thoracocentesis procedure. unless the hemorrhage is peracute, fluid in cases of hemothorax is rapidly defibrinated and will not clot, has a packed cell volume less than that of venous blood, contains rbcs and macrophages. hemorrhagic effusions also usually contain a disproportionately higher number of white blood cells compared with peripheral blood. hemothorax commonly is the sole clinical sign observed in animals with vitamin k antagonist rodenticide intoxication and systemic coagulopathy. whenever an animal presents with signs of a hemorrhagic pleural effusion, perform coagulation testing immediately to determine whether a coagulopathy exists. the prothrombin time test is fast and can be performed as a cage-side test (see section on coagulopathy). therapy for hemorrhagic pleural effusions should address the blood and fluid loss. administer intravenous crystalloid fluids and rbc products (see section on transfusion therapy). when necessary, administer coagulation factors in the form of fresh whole blood or fresh frozen plasma, along with vitamin k 1 (5 mg/kg sq in multiple sites with a 25-gauge needle). if severe respiratory distress is present, evacuate the blood within the pleural space via thoracocentesis until clinical signs of respiratory distress resolve. fluid that remains aids in the recovery of the patient, because rbcs and proteins eventually will be reabsorbed. autotransfusion can be performed to salvage blood and reinfuse it into the anemic patient. in cases of neoplastic or traumatic uncontrollable hemorrhagic effusions, surgical exploration of the thorax is warranted. diaphragmatic hernia, or a rent in the diaphragm, can result in the protrusion of abdominal organs into the thoracic cavity and impair pulmonary expansion. organs that are commonly herniated into the thorax include the liver, stomach, and small intestines. diaphragmatic hernia usually is secondary to trauma but can occur as a congenital anomaly. in cases of trauma, rib fractures, pulmonary contusions, traumatic myocarditis, hemothorax, and shock are also often present concurrently with diaphragmatic hernia. respiratory distress can be caused by any one or a combination of the above lesions. animals with prior or chronic diaphragmatic hernias may have minimal clinical signs despite the presence of abdominal organs within the thorax. clinical signs of acute or severe diaphragmatic hernia include respiratory distress, cyanosis, and shock. a diagnosis of diaphragmatic hernia is made based on the patient's history (traumatic event), clinical signs, and radiographs. in some cases, ultrasonography or contrast peritoneography is necessary to confirm the diagnosis. contrast radiographs may show the presence of the stomach or intestines within the thorax following oral administration of barium. never administer barium directly into the peritoneal cavity or in cases of suspected gastrointestinal rupture. treatment of a patient with a diaphragmatic hernia includes cardiovascular and respiratory system stabilization before attempting surgical repair of the diaphragm. if the stomach is within the thorax, or if the patient's respiratory distress cannot be alleviated with medical management alone, immediate surgery is necessary. if the respiratory distress is minimal and the stomach is not located within the thorax, surgery can be postponed until the patient is a more stable anesthetic candidate. at the time of surgery, the abdominal organs are replaced into the abdominal cavity, and the rent in the diaphragm is closed. air must be evacuated from the thorax following closure of the diaphragm. if chronic diaphragmatic hernia is repaired, the complication of reexpansion pulmonary edema can occur. cardiac injury is a common complication secondary to blunt thoracic trauma. in most cases, cardiac injury is manifested as arrhythmias, including multiple premature ventricular contractions, ventricular tachycardia, st segment depression or elevation secondary to myocardial hypoxemia, and atrial fibrillation (see section on cardiac emergencies). myocardial infarction and cardiac failure can occur. careful and repeated assessments of the patient's blood pressure and ecg tracing should be a part of any diagnostic work-up for a patient that has sustained blunt thoracic trauma. rib fractures are associated with localized pain and painful respiratory movements. radiographs are helpful to confirm the diagnosis. careful palpation may reveal crepitus and instability of the fractured ribs. common problems associated with rib fractures 264 1 emergency care include pulmonary contusions, pericardial laceration, traumatic myocarditis, diaphragmatic hernia, and splenic laceration or rupture. a flail segment results from rib fractures of more than three adjacent ribs that produce a "floating segment" of the chest wall. the flail segment moves paradoxically with respiration-that is, it moves inward during inhalation and outward during exhalation. respiratory distress is associated with the pain caused by the fractures and the presence of traumatic underlying pulmonary pathology. therapy for rib fractures and flail chest includes administration of supplemental oxygen, treatment of pneumothorax or diaphragmatic hernia, and administration of systemic and local anesthesia to alleviate the discomfort associated with the fractures. although controversial, positioning the patient with the flail segment up may reduce pain and improve ventilation. avoid the use of chest wraps, which do nothing to stabilize the flail segment and can further impair respiratory excursions. following administration of a systemic analgesic, administer a local anesthetic at the dorsocaudal and ventrocaudal segment of each fractured rib, and in one rib in front of and behind the flail segment. often, pulmonary function will improve once the pain associated with rib fractures has been adequately treated. in rare cases in which the flail segment involves five or more ribs, surgical stabilization may be necessary. single rib fractures or smaller flail segments are allowed to heal on their own. feline bronchitis has a variety of names (bronchial asthma, asthma, acute bronchitis, allergic bronchitis, chronic asthmatic bronchitis, feline lower airway disease) and refers to the acute onset of respiratory distress secondary to narrowing of the bronchi. cats may present with an acute onset of severe restrictive respiratory pattern associated with lower airway obstruction. acute bronchitis in cats typically has an inflammatory component in the lower airways, resulting in acute bronchoconstriction, excessive mucus production, and inflammatory exudates. in cats with chronic bronchitis, there may be damage of the bronchial epithelium and fibrosis of the airways. these patients often have a history if intermittent exacerbation of clinical signs, intermittent cough, and periods of normality throughout the year. because there appears to be an allergic or inflammatory component in feline bronchitis, clinical signs can be acutely exacerbated by stress and the presence of aerosolized particles such as perfume, smoke, and carpet powders. causes of feline bronchitis include heartworm disease, parasitic infestation (lungworms), and (rarely) bacterial infection. on presentation, the patient should be placed in an oxygen cage and allowed to rest while being observed from a distance. postpone performing stressful diagnostic procedures until the patient's respiratory status has been stabilized. after careful thoracic auscultation, administer a short-acting bronchodilator (terbutaline, 0.01 mg/kg sq or im) along with a glucocorticosteroid (dexamethasone sodium phosphate 1 mg/kg im, sq, iv) to alleviate immediate bronchospasm and airway inflammation. clinical signs of feline bronchitis are characterized by a short, rapid respiratory pattern with prolonged expiration with an abdominal push. wheezes may be heard on thoracic auscultation. in some cases, no abnormalities are found on auscultation, but become acutely worse when the patient is stimulated to cough by tracheal palpation. radiographs may reveal a hyperinflated lung field with bronchial markings and caudal displacement of the diaphragm. in some cases, consolidation of the right middle lung lobe is present. a complete blood count and serum biochemistry profile can be performed, but results usually are unrewarding. in endemic areas, a heartworm test is warranted. fecal examination 1 by flotation and the baermann technique is helpful in ruling out lungworms and other parasites. bronchoalveolar lavage or transtracheal wash is useful for cytologic and bacterial examination. long-term management of feline bronchitis includes isolation from environmental exposure to potential allergens (litter dust, perfumes, smoke, incense, carpet powders) and treatment of bronchoconstriction and inflammation with a combination of oral and inhaled glucocorticosteroids and bronchodilators (table 1 -53). antibiotic therapy is contraindicated unless a pure culture of a pathogen is documented. oral therapy with steroids and bronchodilators should be used for a minimum of 4 weeks after an acute exacerbation and then gradually decreased to the lowest dose possible to alleviate clinical signs. metered dose inhalers are now available (aerokat.com) for administration of inhaled bronchodilators and steroids. fluticasone (flovent, 100 mcg/puff ) can be administered initially every 12 hours for 1 week and then decreased to once daily, in most cases. inhaled glucocorticosteroids are not absorbed systemically, and therefore patients do not develop the adverse side effects sometimes documented with oral glucocorticosteroid administration. because it takes time for glucocorticosteroids to reach peak effects in the lungs, administration of inhaled glucocorticosteroids should overlap with oral prednisolone administration for 5 to 7 days. treatment of pulmonary contusions is supportive. administer supplemental oxygen in a manner that is least stressful for the animal. arterial blood gas analysis or pulse oximetry can determine the degree of hypoxemia and monitor the response to therapy. intravenous fluids should be administered with caution to avoid exacerbating pulmonary hemorrhage or fluid accumulation in the alveoli. treat other conditions associated with the traumatic event. possible complications of pulmonary contusions are rare but include bacterial infection, abscessation, lung lobe consolidation, and the development of cavitary lesions. the routine use of antibiotics or steroids in cases of pulmonary contusions is contraindicated unless external wounds are present. empiric antibiotic use without evidence of external injury or known infection can potentially increase the risk of a resistant bacterial infection. steroids have been shown to decrease pulmonary alveolar macrophage function and impair wound healing and are contraindicated. aspiration pneumonia can occur in animals as a result of abnormal laryngeal or pharyngeal protective mechanisms or can be secondary to vomiting during states of altered mentation, including anesthesia, recovery from anesthesia, and sleep. megaesophagus, systemic polyneuropathy, myasthenia gravis, and localized oropharyngeal defects such as cleft palate can increase the risk of developing aspiration pneumonitis. iatrogenic causes of aspiration pneumonia include improper placement of nasogastric feeding tubes, overly aggressive force-feeding, and oral administration of drugs. aspiration of contents into the airways can cause mechanical airway obstruction, bronchoconstriction, chemical damage to the alveoli, and infection. severe inflammation and airway edema are common. pulmonary hemorrhage and necrosis can occur. diagnosis of aspiration pneumonia is based on clinical signs of pulmonary parenchymal disease, a history consistent with vomiting or other predisposing causes, and thoracic radiographs demonstrating a bronchointerstitial to alveolar pulmonary infiltrate. the most common site is the right middle lung lobe, although the pneumonia can occur anywhere, depending on the position of the patient at the time of aspiration. a transtracheal wash or bronchoalveolar lavage is useful for bacterial culture and susceptibility testing. treatment of aspiration pneumonia includes antibiotic therapy for the infection, administration of supplemental oxygen, and loosening the debris in the airways. administer intravenous fluids to maintain hydration. nebulization with sterile saline and chest physiotherapy (coupage) should be performed at least every 8 hours. antibiotics to consider in the treatment of aspiration pneumonia include ampicillin/enrofloxacin, amoxicillinclavulanate, ampicillin-sulbactam, trimethoprim sulfa, and chloramphenicol. the use of glucocorticosteroids is absolutely contraindicated. continue antibiotic therapy for a minimum of 2 weeks after the resolution of radiographic signs of pneumonia. pulmonary edema arises from the accumulation of fluid in the pulmonary interstitial alveolar spaces, and airways. ventilation-perfusion abnormalities result in hypoxia. pulmonary edema can be caused by increased pulmonary vasculature hydrostatic pressure, decreased pulmonary oncotic pressure, obstruction of lymphatic drainage, or increased capillary permeability. multiple factors can occur simultaneously. the most common cause of edema is increased pulmonary hydrostatic pressure resulting from left-sided congestive heart failure. decreased plasma oncotic pressure with albumin <1.5 g/dl can also result in accumulation of fluid in the pulmonary parenchyma. overzealous intravenous crystalloid fluid administration can result in dilution of serum oncotic pressure and vascular overload. obstruction of lymphatic drainage is usually caused by neoplasia. other causes of pulmonary edema include pulmonary thromboembolic disease, severe upper airway obstruction (noncardiogenic pulmonary edema), seizures, and head trauma. increased capillary permeability is associated with a variety of diseases that cause severe inflammation (systemic inflammatory response syndrome). the resultant pulmonary edema contains a high amount of protein and is known as acute respiratory 1 distress syndrome (ards). ards can be associated with pulmonary or extrapulmonary causes, including direct lung injury from trauma, aspiration pneumonia, sepsis, pancreatitis, smoke inhalation, oxygen toxicity, electrocution, and immune-mediated hemolytic anemia with disseminated intravascular coagulation. diagnosis of pulmonary edema is made based on clinical signs of respiratory distress and the presence of crackles on thoracic auscultation. in severe cases, cyanosis and fulminant blood-tinged frothy edema fluid may be present in the mouth and nostrils. immediate management includes administration of furosemide (4-8 mg/kg iv, im) and supplemental oxygen. sedation with low-dose morphine sulfate (0.025-0.1 mg/kg iv) is helpful in dilating the splanchnic capacitance vasculature and relieving anxiety for the patient. if fluid overload is suspected secondary to intravenous fluid administration, fluids should be discontinued. severely hypoalbuminemic patients should receive concentrated human albumin (2 ml/kg of a 25% solution) or fresh frozen plasma. furosemide as a constant rate infusion (0.66-0.1 mg/kg/hour) also can dilate the pulmonary vasculature and decrease fluid accumulation in cases of ards. following initial stabilization of the patient, thoracic radiographs and an echocardiogram should be assessed to determine cardiac side, pulmonary vascular size, and cardiac contractility. further diagnostic testing may be required to determine other underlying causes of pulmonary edema. heart failure is managed with vasodilators, diuretics, oxygen, and sometimes positive inotropes. treatment ultimately consists of administration of supplemental oxygen, minimal stress and patient handling, and judicious use of diuretics. in cases of cardiogenic pulmonary edema, administer furosemide (4-8 mg/kg iv, im) every 30 to 60 minutes until the patient loses 7% of its body weight. positive inotropic and antiarrhythmic therapy may be necessary to improve cardiac contractility and control dysrhythmias. the clinician should determine whether the cause of the pulmonary edema is secondary to congestive heart failure with pulmonary vascular overload, volume overload, hypoalbuminemia, or increased permeability (ards). pulmonary edema secondary to ards typically is refractory to supplemental oxygen and diuretic therapy. in many cases, mechanical ventilation should be considered. a diagnosis of pulmonary thromboembolism (pte) is difficult to make and is based on clinical signs of respiratory distress consistent with pte, lack of other causes of hypoxemia, a high index of suspicion in susceptible animals, the presence of a condition associated with pte, and radiographic findings. virchow's triad consists of vascular endothelial injury, sluggish blood flow with increased vascular stasis, and a hypercoagulable state as predisposing factors for thromboembolic disease. clinical conditions that predispose an animal to pte include hyperadrenocorticism, disseminated intravascular coagulation (dic), catheterization of blood vessels, bacterial endocarditis, protein-losing nephropathy or enteropathy, hyperviscosity syndromes, heat-induced illness, pancreatitis, diabetes mellitus, inflammatory bowel disease, and immune-mediated hemolytic anemia. definitive diagnosis requires angiography or a lung perfusion scan. clinical signs associated with pte include an acute onset of tachypnea, tachycardia, orthopnea, and cyanosis. if the embolism is large, the patient may respond poorly to supplemental oxygen administration. pulmonary hypertension can cause a split second heart sound on cardiac auscultation. in some cases, a normal thoracic radiograph is present in the face of severe respiratory distress. this is a classic finding in cases of pte. potential radiographic abnormalities include dilated, tortuous, or blunted pulmonary arteries; wedge-shaped opacities in the lungs distal to an obstructed artery; and interstitial to alveolar infiltrates. the right heart may be enlarged. echocardiography can show right heart enlargement, tricuspid regurgitation, pulmonary hypertension, and evidence of underlying cardiac disease, possibly with clots in the atria. measurement of antithrombin (at) and d-dimer levels can be useful in the identification of hypercoagulable states, including dic. treatment of any patient with at deficiency or dic includes replenishment of at and clotting factors in the form of fresh frozen plasma. treatment of pte includes therapy for cardiovascular shock, oxygen supplementation, and thrombolytic therapy (see section on thromboembolic therapy). for short-term treatment, administer heparin (heparin sodium, 200-300 units/kg sq once, followed by 100 units/kg q8h of unfractionated heparin; or fractionated heparin). thrombolytic therapy may include tissue plasminogen activator, streptokinase, or urokinase. long-term therapy with low molecular weight heparin or warfarin may be required to prevent further thromboembolic events. ideally, management should include treatment and elimination of the underlying disease. smoke inhalation commonly occurs when an animal is trapped in a burning building. the most severe respiratory complications of smoke inhalation are seen in animals that are close enough to the flames to also sustain burn injuries (see section on burn injury). at the scene, many animals are unconscious from the effects of hypoxia, hypercapnia, carbon monoxide intoxication, and hydrogen cyanide gases that accumulate in a fire. carbon monoxide produces hypoxia by avidly binding to and displacing oxygen binding to hemoglobin, resulting in severe impairment of oxygen-carrying capacity. the percentage of carboxyhemoglobin in peripheral blood depends on the amount or carbon monoxide in inhaled gases and the length of time of exposure. clinical signs of carbon monoxide intoxication include cyanosis, nausea, vomiting, collapse, respiratory failure, loss of consciousness, and death. smoke inhalation of superheated particles also causes damage to the upper airways and respiratory tree. the larynx can become severely edematous and obstruct inspiration. emergency endotracheal intubation, tracheal oxygen, or tracheostomy tube may be required in the initial resuscitation of the patient, depending on the extent of airway edema. inhalation of noxious gases and particles can cause damage to the terminal respiratory bronchioles. specific noxious gases that can cause alveolar damage include combustible particles from plastic, rubber, and other synthetic products. pulmonary edema, bacterial infection, and ards can result. in any case of smoke inhalation, the first and foremost treatment is to get the animal away from the source of the flames and smoke and administer supplemental oxygen at the scene. at the time of presentation, carefully examine the animal's eyes, mouth, and oropharynx suction soot and debris from the mouth and upper airways. evaluate the patient's respiratory rate, rhythm, and pulmonary sounds. arterial blood gases should be analyzed with co-oximetry to evaluate the pao 2 and carboxyhemoglobin concentrations. evaluation of sao 2 by pulse oximetry is not accurate in cases of smoke inhalation, as the pao 2 may appear normal, even when large quantities of carboxyhemoglobin are present. radiographs are helpful in determining the extent of pulmonary involvement, although radiographic signs may lag behind the appearance of clinical respiratory abnormalities by 16 to 24 hours. bronchoscopy and bronchoalveolar lavage provide a more thorough and accurate evaluation of the respiratory tree; however, these procedures should be performed only in patients whose cardiovascular and respiratory status is stable. management of the patient with smoke inhalation includes maintaining a patent airway, administration of supplemental oxygen, correction of hypoxemia and acid-base abnormalities, preventing infection, and treating thermal burns (see section on burn injury). if severe laryngeal edema is present, a temporary tracheostomy may be necessary to allow adequate oxygenation and ventilation. glucocorticosteroids should not be empirically used in the treatment of smoke inhalation, because of the risk of decreasing pulmonary alveolar macrophage function and increasing the potential for infection. in cases of severe laryngeal edema, however, glucocorticosteroids may be necessary to decrease edema and inflammation. the use of empiric antibiotics is contraindicated unless clinical signs of deterioration and bacterial pneumonia develop. epistaxis can be caused by facial trauma, a foreign body, bacterial or fungal rhinitis, neoplasia, coagulopathies, and systemic hypertension. acute, severe bilateral hemorrhage without wounds have been classified in several ways according their degree of tissue integrity, etiologic force, degree of contamination and duration, and degree of contamination and infection (table 1 -54) . there are also unique causes of wounds such as burns, psychogenic dermatoses, frostbite, decubital ulcers, and snake bite. the animal should be transported to the nearest veterinary facility for definitive care. the wound should be covered or packed with dry gauze or clean linen to protect the wound, and to prevent further hemorrhage and contamination. if an open fracture is present, the limb should be splinted without placing the exposed bone back into the wound. replacing the exposed bone fragment back through the skin wound can cause further damage to underlying soft tissue structures and increase the degree of contamination of deeper tissues. if a spinal fracture is suspected, the patient should be transported on a stable flat surface to prevent further spinal mobilization and neurologic injury. at the time of presentation, first refer to the abcs of trauma, taking care to evaluate and stabilize the patient's cardiovascular and respiratory status. after a complete physical examination and history, ancillary diagnostic techniques can be performed if the patient is hemodynamically stable (see section on triage, assessment, and treatment of emergencies). initially, every patient with superficial wound should receive some degree of analgesia and an injection of a first-generation cephalosporin, preferably within 3 hours of the injury. evaluate the wound after the patient's cardiovascular and respiratory status have been stabilized. always cover an open wound before taking an animal to the hospital to prevent a nosocomial infection. evaluate limb wounds for neural, vascular, and orthopedic abnormalities. carefully examine the structures deep to the superficial wounds. when there has been a delay in assessment of the wound, obtain samples for culture and antimicrobial susceptibility testing. if the wound is older and obviously infected, a gram stain can help guide appropriate antimicrobial therapy pending results of culture 1 and susceptibility testing. place a support bandage saturated with a water-soluble antibiotic ointment or nonirritating antimicrobial solution (e.g., 0.05% chlorhexidine, if bone or joint tissue is not exposed) around the wound. in addition to a first-generation cephalosporin, other appropriate antibiotic choices include amoxicillin-clavulanate, trimethoprim-sulfadiazine, amoxicillin, and ampicillin. if gram-negative flora are present, administer enrofloxacin. administer the antibiotics of choice for a minimum of 7 days unless a change of antibiotic therapy is indicated. at the time of wound cleansing or definitive wound repair, the patient should be placed under general anesthesia with endotracheal intubation, unless the procedure will be brief (i.e., less than 10 minutes). in such cases, a short-acting anesthetic combination open lacerations or skin loss closed crushing injuries and contusions etiologic force abrasion loss of epidermis and portions of dermis, usually caused by shearing between two compressive surfaces avulsion tearing of tissue from its attachment because of forces similar to those causing abrasion but of a greater magnitude incision wound created by a sharp object; wound edges are smooth and there is minimal trauma in the surrounding tissues laceration irregular wound caused by tearing of tissue with variable damage to the superficial and underlying tissue puncture penetrating wound caused by a missile or sharp object; superficial damage may be minimal; damage to deeper structures may be considerable; contamination by fur and bacteria with subsequent infection is common class i 0-6 hours with minimal contamination class ii 6-12 hours with significant contamination class iii >12 hours with gross contamination (analgesia + propofol, analgesia + ketamine/diazepam) can be administered to effect. heavy sedation with infiltration of a local anesthetic may also be appropriate for very small wounds, depending on the location of the wound and temperament of the patient. protect the wound by packing it with sterile gauze sponges soaked in sterile saline, or with watersoluble lubricating gel such as k-y jelly. clip the fur surrounding the wound, moving from the inner edge of the wound outward, to help prevent wound contamination with fur or other debris. scrub the wound and surrounding skin with an antimicrobial soap and solution such as dilute chlorhexidine until the area is free of all gross debris. gross debris within the wound itself can be flushed using a 30-ml syringe filled with sterile saline or lactated ringer's solution and an 18-gauge needle. pressure-lavage systems are also available for use, if desired. grossly contaminated wounds can be rinsed first with warm tap water to eliminate gross contamination, and then prepared as just described. debride the wound, removing skin and other soft tissue that is not obviously viable. obviously viable and questionable tissue should remain, and the wound left open for frequent reassessment on a daily basis. remove any dark or white segments of skin. questionable skin edges may or not regain viability and should be left in place for 48 hours, so the wound can fully reveal itself. excise grossly contaminated areas of fat and underlying fascia. blood vessels that are actively bleeding should be ligated to control hemorrhage, if collateral circulation is present. if nerve bundles are ligated cleanly in a clean wound, the nerve edges should be reapposed and anastomosed. if gross contamination is present, however, definitive neurologic repair should be delayed until healthy tissue is present. excise contaminated muscle until healthy bleeding tissue is present. anastamoe tendon lacerations if the wound is clean and not grossly contaminated. if gross contamination is present, the tendon can be temporarily anastomosed and a splint placed on the limb until definitive repair of healthy tissue is possible. thoroughly lavage open wounds to a joint with sterile saline or lactated ringer's solution. infusion of chlorhexidine or povidone-iodine solution into the joint can cause a decrease in cartilage repair and is contraindicated. smooth sharp edges and remove any obvious fragments. whenever possible, the joint capsule and ligaments should be partially or completely closed. after removing bullets and metal fragments, the subcutaneous tissue and skin should be left open to heal by second intention, or should be partially closed with a drain. the joint should then be immobilized. injuries and exposed bone should be carefully lavaged, taking care to remove any gross debris without pushing the debris further into the bone and wound. the bone should be covered with a moist dressing and stabilized until definitive fracture repair can be made. this type of injury typically is seen with shearing injuries of the distal extremities caused by interaction with slow-moving vehicles. perform wet-to-dry or enzymatic debridement until a healthy granulation bed is present. if large areas of contamination are present (e.g., necrotizing fasciitis), en bloc debridement may be necessary. en bloc debridement consists of complete excision of badly infected wounds without entering the wound cavity, to prevent systemic infection. this technique should be used only if there is sufficient skin and soft tissue to allow later closure and it can be performed without damaging any major nerves, tendons, or blood vessels. open wounds often are managed by second intention healing, delayed primary closure, or secondary closure. see section on wound management and bandaging for a more complete discussion on the use of various bandaging materials in the treatment of open wounds. if an animal is presented very shortly after a wound has occurred and there is minimal contamination and trauma, the wound can be closed after induction of anesthesia and 1 careful preparation of the wound and surrounding tissues. close any dead space under the skin with absorbable suture material in an interrupted suture pattern. avoid incising major blood vessels or nerves. close the subcutaneous tissues with absorbable suture material in an interrupted or continuous suture pattern. take care that there is not too much tension on the wound, or else surgical dehiscence will occur with patient movement. close the skin with nonabsorbable suture or surgical staples (2-0 to 4-0) . if there is any doubt at the time of repair about tissue status or inability to close all dead space, place a passive drain (penrose drain) so that the proximal end of the drain is anchored in the proximal aspect of the wound with a suture(s). leave the ends long so that the suture can be accurately identified at the time of drain removal. pass the suture through the skin, through the drain, and out the other side of the skin. place the rest of the drain into the wound and then secure it at the most ventral portion of the wound or exit hole in the most dependent area of the body, to allow drainage and prevent seroma formation. close the subcutaneous tissue over the drain before skin closure. during wound closure, be sure to not incorporate the subcutaneous or skin sutures into the drain, or it will not be possible to remove the drain without reopening the wound. bandage the area to prevent contamination. the drain can be removed once drainage is minimal (usually 3 to 5 days). active drains can be constructed or purchased; their use is indicated in wounds that are free of material that can plug the drain. to construct a small suction drain, remove the female portion or catheter hub at the end of a butterfly catheter. fenestrate the tubing so that there are multiple side holes, taking care to avoid making the holes larger than 50% of the circumference of the tubing. place the tubing into the wound via a small stab incision distal to the wound. use a purse-string suture around the tubing to facilitate a tight seal and prevent the tubing from exiting the wound. following wound closure, insert the butterfly needle into a 5-to 10-ml evacuated blood collection tube to allow fluid to drain into the tube. incorporate the tube into the bandage, and replace it when it becomes full. alternatively, the butterfly portion of the system can be removed and the tube fenestrated as described previously. place the tube into the wound and suture it in place to create a tight seal. secure the catheter hub to a syringe in which the plunger has been drawn back slightly to create suction. insert a metal pin or 16-to 18-gauge needle through the plunger at the top of the barrel to hold it at the desired level. incorporate the suction apparatus into the bandage and replace it when it becomes full. delayed primary closure should be considered when there is heavy contamination, purulent exudate, residual necrotic debris, skin tension, edema and erythema, and lymphangitis. delayed primary closure usually is made 3 to 5 days after the initial wound infliction and open wound management has been performed. once healthy tissue is observed, the skin edges should be debrided and the wound closed as with primary closure. secondary wound closure should be considered when infection and tissue trauma necessitate open wound management for more than 5 days. secondary wound closure is performed after the development of a healthy granulation bed. this technique also is useful when a wound has dehisced and has formed granulation tissue. if the wound edges can be manipulated into apposition and if epithelialization has not begun, the wound can be cleansed and the wound edges apposed and sutured. this is known as early secondary closure. late secondary closure should be performed whenever there is a considerable amount of granulation tissue, the edges of the wound cannot be manipulated into position, and epithelialization has already started. in such cases, the wound should be cleaned, and the skin edges debrided to remove the epithelium. the remaining wound edges are then sutured over the granulation tissue ( shock is defined as a state of inadequate circulating volume and inability to meet cellular oxygen demands. there are three types of shock: hypovolemic, cardiogenic, and septic. early recognition of the type of shock present is crucial in the successful clinical management of shock syndrome. tissue oxygen delivery is based on cardiac output and arterial oxygen concentration. knowledge of the components of normal oxygen delivery is essential to the treatment of shock in the critical patient. improper handling of animal during further tissue and neurologic damage may occur transport (e.g., improper limb or spine immobilization). inadequate assessment of animal's animal's condition may worsen or animal may general condition or wounded tissues succumb; tissue injuries may be overlooked. inadequate wound protection during further wound contamination may occur at assessment, resuscitation, or veterinary facility. stabilization procedures inadequate wound protection while further wound contamination with fur and preparing the surrounding area debris may occur. insufficient wound lavage wound infection may occur. hydrogen peroxide wound lavage lavage offers little bactericidal activity and contributes to irritation of tissues and delayed healing. lavage has short residual activity and absorption with large wound. overly aggressive initial layered debridement may result in the removal of viable debridement tissue. en bloc debridement debridement results in removal of large amounts of tissue and a large defect for closure. use of drains potential exists for bacteria to ascend along the drain, for drain removal by the animal or breakage of the drain, and for possible tissue emphysema with air being sucked under the skin with patient movement. tube-type drains drains may cause postoperative discomfort; fenestrations may become occluded to stop intraluminal drainage. deeply placed sutures in the presence drain may be incorporated into the repair and of a drain prevent drain removal. active drains high negative pressure may cause tissue injury; highly productive wounds may necessitate changing the evacuated blood tubes several times a day with constructed drains. oxygen delivery (do 2 ) = cardiac output (q) ã� arterial oxygen content (cao 2 ) where q = heart rate ã� stroke volume. stroke volume is affected by preload, afterload, and cardiac contractility. where hb = hemoglobin concentration, sao 2 = oxygen saturation, and pao 2 = arterial partial pressure of oxygen in mm hg. thus, factors that can adversely affect oxygen delivery include inadequate preload or loss of circulating volume, severe peripheral vasoconstriction and increased afterload, depressed cardiac contractility, tachycardia and decreased diastolic filling, cardiac dysrhythmias, inadequate circulating hemoglobin, and inadequate oxygen saturation of hemoglobin. during septic shock, enzymatic dysfunction and decreased cellular uptake and utilization of oxygen also contribute to anaerobic glycolysis. an inadequate circulating volume may develop secondary to maldistribution of available blood volume (traumatic, septic, and cardiogenic origin) or as a result of absolute hypovolemia (whole blood or loss of extracellular fluid). normally, the animal compensates by (1) splenic and vascular constriction to translocated blood from venous capacitance vessels to central arterial circulation, (2) arteriolar constriction to help maintain diastolic blood pressure and tissue perfusion, and (3) an increase in heart rate to help maintain cardiac output. arteriolar vasoconstrictions support perfusion to the brain and heart at the expense of other visceral organs. if vasoconstriction is severe enough to interfere with delivery of adequate tissue oxygen for a sufficient period of time, the animal may die. hypovolemic shock can result from acute hemorrhage or from severe fluid loss from vomiting, diarrhea, or third spacing of fluids. early in shock, baroreceptors in the carotid body and aortic arch sense a decrease in wall stretch from a decrease in circulating fluid volume. tonic inhibition of sympathetic tone via vagal stimulation is diminished, and heart rate and contractility increase and peripheral vessels constrict to compensate for the decrease in cardiac output. the compensatory mechanisms protect and support blood supply to the brain and heart at the expense of peripheral organ perfusion. this is called early compensatory shock. early compensatory shock is characterized by tachycardia, normal to fast capillary refill time, tachypnea, and normothermia. as shock progresses, the body loses its ability to compensate for ongoing fluid losses. early decompensatory shock is characterized by tachycardia, tachypnea, delayed capillary refill time, normotension to hypotension, and a fall in body temperature. end-stage decompensatory shock is characterized by bradycardia, markedly prolonged capillary refill time, hypothermia, and hypotension. aggressive treatment is necessary for any hope of a favorable outcome. septic shock should be considered in any patient with a known infection, recent instrumentation that could potentially introduce infection (indwelling intravenous or urinary catheter, surgery or penetrating injury), disorders or medical therapy that can compromise immune function (diabetes mellitus, immunodeficiency virus, parvovirus or feline panleukopenia virus infection, stress, malnutrition, glucocorticoids, chemotherapy). the presence of bacteria, viruses or rickettsiae, protozoa, or fungal organisms in the blood constitutes septicemia. septic shock is characterized by the presence of sepsis and refractory hypotension that is unresponsive to standard aggressive fluid therapy and inotropic or pressor support. septic shock and other causes of inflammation can lead to systemic inflammatory response syndrome (sirs). in animals, the presence of two or more of the criteria in table 1 -56 in the presence of suspected inflammation or sepsis constitutes sirs (table 1 -56). clinical signs associated with sepsis may be vague and nonspecific, including weakness, lethargy, vomiting, and diarrhea. cough and pulmonary crackles may be associated with pneumonia. decreased lung sounds may be associated with pyothorax. abdominal pain and fluid may be associated with septic peritonitis. vaginal discharge may or may not be present in patients with pyometra. diagnostic tests should include a white blood cell count, serum biochemical profile, coagulation tests, thoracic and abdominal radiographs, and urinalysis. the white blood cell count in a septic patient that is appropriately responding to the infection will be elevated with a left-shifted neutrophilia and leukocytosis. a degenerative left shift, in which leukopenia with elevated band neutrophils suggests an overwhelming infection. biochemical analyses may demonstrate hypoglycemia and nonspecific hepatocellular and cholestatic enzyme elevations. in the most severe cases, metabolic (lactic) acidosis, coagulopathies, and end-organ failure, including anuria and ards, may be present. cardiogenic shock occurs as a result of cardiac output inadequate to meet cellular oxygen demands. cardiogenic shock is associated with primary cardiomyopathies, cardiac dysrhythmias, pericardial fluid, and pericardial fibrosis. abnormalities seen on physical examination often are similar to those seen in other categories of shock, but they can also include cardiac murmurs, dysrhythmias, pulmonary rales, bloody frothy pulmonary edema fluid from the nares or mouth, orthopnea, and cyanosis. it is important to distinguish the primary cause of shock before implementing treatment (table 1-57) , whenever possible, because treatment for a suspected ruptured hemangiosarcoma differs markedly from the treatment for end-stage dilatative cardiomyopathy. the patient's clinical signs may be similar and include a peritoneal fluid wave, but the treatment for hypovolemia can dramatically worsen the congestive heart failure secondary to dilatative cardiomyopathy. when a patient presents with some form of shock, immediate vascular access is of paramount importance. place a large-bore peripheral or central venous catheter for the infusion of crystalloid or colloid fluids, blood component therapy, and drugs. monitor the patient's cardiopulmonary status (by ecg), blood pressure, oxygen saturation (as determined by pulse oximetry or arterial blood gas analyses), hematocrit, bun, and glucose. ancillary diagnostics, including thoracic and abdominal radiography, urinalysis, serum biochemistry profile, coagulation tests, complete blood count, abdominal ultrasound, and echocardiography, should be performed as determined by the individual patient's needs and the type of shock. the following list, called the "rule of twenty," is a guideline for case management of the shock patient. consideration of each aspect of the rule of twenty on a daily basis ensures temperature <100â°f or >103.5â°f <100â°f or >103.5â°f heart rate >120 beats/minute in dogs <140 or >250 beats/minute in cats respiratory rate >20 breaths/minute or paco 2 >40 breaths/minute or paco 2 <32 mm hg <32 mm hg white blood cell >18,000 cells/âµl 19,000 cells/âµl count or <4000 cells/âµl o r <5000 cells/ml or >10% bands or >10% bands 1 that major organ systems are not overlooked. the list also provides a means to integrate and relate changes in different organ systems functions with one another.* the treatment of hypovolemic and septic shock requires the placement of large-bore intravenous catheters in peripheral and central veins. if vascular access cannot be obtained percutaneously or by cutdown methods, intraosseous catheterization should be considered. once vascular access is achieved, rapidly administer large volumes of crystalloid or colloid fluids. as a rule of thumb, administer 1 /4 of a calculated shock dose of fluids-that is, 1 /4 ã� (90 ml/kg/hour) in dogs and 1 /4 ã� (44 ml/kg/hour) in cats) of a balanced crystalloid fluid ( normosol-r, plasmalyte-m, lactated ringer's solution, or 0.9% sterile saline). reassess the patient's perfusion parameters (heart rate, capillary refill time, blood pressure, urine output) on a continual basis to direct further fluid therapy. synthetic colloid fluids (hetastarch, dextran 70, or oxyglobin) can also be administered in the initial resuscitation from shock. a guideline is to administer 5 to 10 ml/kg of hetastarch or dextran as a bolus over 10 to 15 minutes and then reassess perfusion parameters. hypertonic saline (0.7% nacl, 4 ml/kg) can be used in cases of hemorrhagic shock to temporarily restore intravascular fluid volume by drawing fluid from the interstitial space. because this type of fluid resuscitation is short-lived, hypertonic saline should always be used with another crystalloid or colloid fluid, and it should not be used in patients with interstitial dehydration. if hemorrhagic shock is present, the goal should be to return a patient's blood pressure to normal (not supraphysiologic) levels (i.e., systolic pressure 90-100 mm hg, diastolic pressure >40 mm hg, and mean arterial pressure â�¥60 mm hg) to avoid iatrogenically causing clots to fall off and hemorrhage to re-start. in critically ill patients, fluid loss can be measured in the form of urine, vomit, diarrhea, body cavity effusions, and wound exudates. additionally, insensible losses (those that cannot be readily measured from sweat, panting, and cellular metabolism) constitute 20 ml/kg/ day. measurement of fluid "ins and outs" in conjunction with the patient's central venous pressure, hematocrit, albumin, and colloid oncotic pressure can help guide fluid therapy (see also section on fluid therapy). maintenance of normotension is necessary for adequate oxygen delivery to meet cellular energy demands. blood pressure can be measured using direct arterial catheterization, or through indirect means such as doppler plesthymography or oscillometric methods. the systolic pressure should remain at or greater than 90-100 mm hg at all times. the diastolic pressure is very important, too, as it constitutes two thirds of the mean arterial pressure; it must be greater than 40 mm hg for coronary artery perfusion. the mean arterial pressure should be greater than 60 mm hg for adequate tissue perfusion. if fluid resuscitation and pain management are not adequate in restoring blood pressure to normal, vasoactive drugs including positive inotropes and pressors should be considered (table 1 -58). in cases of cardiogenic shock, vasodilator drugs (table 1 -59) can be used to decrease vascular resistance and afterload. low-dose morphine (0.05 mg/kg, iv, im) dilates splanchnic vessels and helps reduce pulmonary edema. furosemide (1 mg/kg/hour) also can dilate pulmonary vasculature and potentially reduce edema fluid formation in cases of ards. cardiac output is a function of both heart rate and stroke volume. stroke volume or (the amount of blood that the ventricle pumps in 1 minute) is affected by preload, afterload, and contractility. during hypovolemic shock, there is a fall in cardiac preload due to a decrease in circulating blood volume. during septic and cardiogenic shock, there is a decrease in contractility secondary to inherent defects of the myocardium or due to the negative inotropic effects of inflammatory cytokines such as tnf-alpha, myocardial depressant factor, il-1, and il-10 released during sepsis and systemic inflammation. afterload also may be increased because of the compensatory mechanisms and neurohumoral activation of the renin-angiotensin-aldosterone axis in hypovolemic or cardiogenic shock. as heart rate increases to compensate for a decline in cardiac output, myocardial oxygen demand increases and diastolic filling time becomes shorter. because the coronary arteries are perfused during diastole, coronary perfusion can be impaired, and myocardial lactic acidosis can develop, causing a further decline in contractility. in addition to lactic acidosis, acid-base and electrolyte abnormalities, inflammatory cytokines, direct bruising of the myocardium from trauma, and areas of ischemia can further predispose the patient to ventricular or atrial dysrhythmias. cardiac dysrhythmias should be controlled whenever possible. treatment of bradycardia should be directed at treating the underlying cause. administer anticholinergic drugs such as atropine (0.04 mg/kg im) or glycopyrrolate (0.02 mg/kg im) as necessary. in cases of third-degree or complete atrioventricular (av) block, administer a pure betaagonist such as isoproterenol (0.04-0.08 âµg/kg/minute iv cri, or 0.4 mg in 250 ml of 5% dextrose in water iv slowly). perform passive rewarming if the patient is hypothermic. receptor activity dosage (iv) dopamine da 1 , da 2 , î± +++ , 5-25 âµg/kg/minute (blood pressure support)* î² +++ 1-5 âµg/kg/minute (renal afferent diuresis) dobutamine î± + , î² +++ 3-20 âµg/kg/minute* (blood pressure support, positive inotrope) norepinephrine î± +++ , î² + 0.05-0.3 mg/kg/minute; 0.01-0.02 mg/kg phenylephrine î± +++ , î² 0 0.05-0.2 mg/kg epinephrine î± +++ , î² +++ 0.02-0.5 mg/kg, 0.05-0.2 mg/kg/minute +++, strong receptor activity; 0, no receptor activity; +, weak receptor activity. *monitor for tachyarrhythmias at higher doses. correct any underlying electrolyte abnormalities such as hyperkalemia and hypo-and hypermagnesemia. treat ventricular dysrhythmias such as multifocal premature ventricular contractions (pvcs), sustained ventricular tachycardia >160 beats per minute, and r on t phenomenon (the t wave of the preceding beat occurs superimposed on the qrs complex of the next beat, and there is no return to isoelectric shelf), or if runs of ventricular tachycardia cause a drop in blood pressure. intravenous lidocaine and procainamide are the first drugs of choice for ventricular dysrhythmias. supraventricular tachycardia can impair cardiac output by impairing diastolic filling time. control supraventricular dysrhythmias with calcium channel blockers, beta-adrenergic blockers, or quinidine (table 1-60) . (disorientation); is 1 minute; 2 minutes) light sensitive and must be covered in foil and not kept for longer than 4 hours 1 albumin can decrease as a result of loss from the gastrointestinal tract, urinary system, and wound exudates, or into body cavity effusions. albumin synthesis can decrease during various forms of shock due to a preferential increase in hepatic acute phase protein synthesis. serum albumin contributes 80% of the colloid oncotic pressure of blood, in addition to its important roles as a free radical scavenger at sites of inflammation and as a drug and hormone carrier. albumin levels <2.0 g/dl have been associated with an increase in morbidity and mortality in human and veterinary patients. administer fresh frozen plasma (20 ml/kg) or concentrated human albumin (2 ml/kg of 25% solution) to maintain serum albumin â�¥2.0 g/dl. additional oncotic support can be in the form of synthetic colloids, as indicated. colloid oncotic pressure within the intravascular and interstitial spaces contributes to fluid flux. oncotic pressure can be measured with a colloid osmometer. normal oncotic pressure is 15 mm hg. in cases of sepsis and sirs, increased vascular permeability increases the tendency for leakage of fluids into the interstitial spaces. colloids that can be administered until the source of albumin loss resolves include the synthetic colloids hetastarch and dextran 70 (20-30 ml/kg/day), synthetic hemoglobin-based oxygen carriers (oxyglobin, 3-7 ml/kg/day), concentrated human albumin (25% albumin, 2 ml/kg), and plasma (20 ml/kg). oxygenation and ventilation can be evaluated by arterial blood gas analysis or by the noninvasive means of pulse oximetry and capnometry (see sections on pulse oximetry and capnometry). oxygen delivery can be impaired in cases of hypovolemic shock because of hemorrhage and anemia, and thus a decrease in functional capacity to carry oxygen, and is not to be used for more than 2 weeks due to idiosyncratic blindness. in cases of cardiogenic shock as a result of impaired ability to saturate hemoglobin due to pulmonary edema in the lungs, or decrease in cardiac output. in septic shock, decreases in cardiac output due to inflammatory cytokines and a decrease in cellular oxygen extraction can lead to lactic acidosis. increased cellular metabolism and decreases in respiratory function can lead to respiratory acidosis as co 2 increases. administer supplemental oxygen as flow-by, nasal or nasopharyngeal catheter, oxygen hood, or oxygen cage. supplemental oxygen should be humidified, and delivered at 50-100 ml/kg/minute. if oxygenation and ventilation are so impaired that the pao 2 remains <60 mm hg with the patient on supplemental oxygen, a paco 2 >60 mm hg, or severe respiratory fatigue, develops, and mechanical ventilation should be considered. glucose is a necessary fuel source for red blood cells and neuronal tissues, and serum glucose should be maintained within normal reference ranges. glucose supplementation can be administered as 2.5-5% solutions in crystalloid fluids, or in parenteral and enteral nutrition products. arterial and venous ph can be measured by performing blood gas analyses. decrease in tissue perfusion, impaired oxygen delivery, and decreased oxygen extraction in the various forms of shock can lead to anaerobic metabolism and metabolic acidosis. in most cases, improving tissue perfusion and oxygen delivery with crystalloid and colloid fluids, supplemental oxygen, and inotropic drugs will help normalize metabolic acidosis. serial measurements of serum lactate (normal, <2.5 mmol/l) can be used as a guide to evaluate the tissue response to fluid resuscitative efforts. serum electrolytes often become severely deranged in shock states. serum potassium, magnesium, sodium, chloride, and total and ionized calcium should be maintained within normal reference ranges. if metabolic acidosis is severe, sodium bicarbonate can be administered by calculating the formula base deficit ã� 0.3 ã� body weight in kg = meq bicarbonate to administer because iatrogenic metabolic alkalosis can occur, a conservative approach is to administer 1 /4 of the calculated dose and then recheck the patient's ph and bicarbonate levels. if the base excess is unknown, sodium bicarbonate can be administered in incremental doses of 1 meq/kg until the ph is above 7.2. complications associated with bicarbonate therapy include iatrogenic hypocalcemia, metabolic alkalosis, paradoxical cerebrospinal fluid acidosis, hypotension, restlessness, and death. massive trauma, neoplasia, sepsis, and systemic inflammation can all lead to coagulation abnormalities, including disseminated intravascular coagulation (dic). cage-side coagulation monitors are available for daily measurement of prothrombin time (pt), activated partial thromboplastin time (aptt), and platelet counts. fibrin degradation products (fibrin split products) become elevated in dic, trauma, hepatic disease, and surgery. coagulation proteins (clotting factors) and antithrombin often are lost with other proteins in hypoproteinemia or are consumed when microclots are formed and then dissolved. antithrombin levels can be measured by commercial laboratories. antithrombin and clotting factors can be replenished in the form of fresh frozen plasma transfusions. a more sensitive and specific test for dic is the detection of d-dimers, which can be measured by commercial laboratories. treatment for dic involves treatment and resolution of the underlying disease and administration of antithrombin and clotting factors in the form of fresh frozen plasma (20 ml/kg) and heparin (unfractionated, 50-100 units/kg sq tid; fractionated [lovenox], 1 mg/kg sq bid). monitor the patient for changes in mental status, including stupor, coma, decreased ability to swallow and protect the airway, and seizures. elevation of the patient's head can help to protect the airway and decrease the risk of increased intracranial pressure. serum glucose should be maintained within normal levels to prevent hypoglycemia-induced seizures. one of the major components of oxygen delivery is the binding to hemoglobin. packed cell volume must be kept above 20-30% for adequate cellular oxygen delivery. acid-base status can adversely affect oxygen offloading at the tissue level if metabolic or respiratory alkalosis is present. oxygen-carrying capacity and hemoglobin levels can be increased with administration of rbc component therapy or with hemoglobin-based oxygen carriers. monitoring of renal function includes daily measurement of bun, creatinine, and urine output. normal urine output in a hydrated euvolemic patient is 1-2 ml/kg/hour. fluid ins and outs should be measured in cases of suspected oliguria or anuria. in patients with oliguria or anuria, furosemide can be administered as a bolus (4-8 mg/kg) or by constant rate infusion (cri)(0.66-1 mg/kg/hour). mannitol should also be administered (0.5-1 g/kg over 10 to 15 minutes). dopamine (1-5 âµg/kg/minute cri) can be administered to dilate renal afferent vessels and improve urine output. the patient's white blood cell count may be elevated, normal, or decreased, depending on the type of shock. the decision to administer antibiotics should be made on a daily basis. superficial or deep staphylococcus or streptococcus infection usually can be treated with a first-generation cephalosporin (cefazolin, 22 mg/kg iv tid). if a known source of infection is present, administer a broad-spectrum antibiotic (cefoxitin, 22 mg/kg iv tid; ampicillin, 22 mg/kg qid, or enrofloxacin, 5-10 mg/kg once daily) pending results of culture and susceptibility testing. if broader anaerobic coverage is required, metronidazole (10 mg/kg iv tid) should be considered. gentamicin (3-5 mg/kg iv once daily) is a good choice for gram-negative sepsis, provided that the patient is well hydrated and has normal renal function. ideally, patients receiving any aminoglycoside antibiotic should have a daily urinalysis to check for renal tubular casts that signify renal damage. in dogs, the gut is the shock organ. impaired gastrointestinal motility and vomiting should aggressively be treated with antiemetics and promotility drugs (dolasetron, 0.6 mg/kg iv once daily, and metoclopramide, 1-2 mg/kg/day iv cri). metoclopramide is contraindicated in cases of suspected gastrointestinal obstruction. histamine-receptor blockers such as famotidine (0.5 mg/kg bid iv) and ranitidine (0.5 to 2 mg/kg iv bid, tid) or proton-pump inhibitors (omeprazole, 0.5-1 mg/kg po once daily) can be administered for esophagitis. administer sucralfate (0.25-1 g po tid) to treat gastric ulceration. if the gastrointestinal barrier function is diminished due to poor perfusion, infection, or inflammation, administer broad-spectrum antibiotics such as ampicillin (22 mg/kg iv qid) to prevent gastrointestinal bacterial translocation. the course of drug therapy should be reviewd daily and the patient should be monitored for potential drug interactions. for example, metoclopramide and dopamine, working at the same receptor, can effectively negate the effects of each other. cimetidine, a cytochrome p450 enzyme inhibitor, can decrease the metabolism of some drugs. drugs that are avidly protein-bound may have an increase in unbound fraction with concurrent hypoalbuminemia or when hypoalbuminemia is present. decreased renal function may impair the renal clearance of some drugs, requiring increased dosing interval or decreased dose. nutrition is of utmost importance in any critically ill patient. patients with septic shock may become hypermetabolic and require supraphysiologic nutrient caloric requirements, while others may actually become hypometabolic. enteral nutrition is preferred, whenever possible, because enterocytes undergo atrophy without luminal nutrient stimulation. a variety of enteral feeding tubes can be placed, depending on what portion of the gut is functional, to provide enteral nutrition in an inappetent patient. loss of gastrointestinal mucosal barrier function may predispose the patients to the development of bacterial translocation and may contribute to sepsis. if enteral nutrition is impossible because of protracted vomiting or gastrointestinal resection, glucose, lipid, and amino acid products are available that can be administered parenterally to meet nutrient needs until the gastrointestinal tract is functioning and the patient can be transitioned to enteral nutrition. assessment of pain in animals in shock can be challenging. pain can result in the release of catecholamines and glucocounterregulatory hormones that can impair nutrient assimilation and lead to negative nitrogen balance, impaired wound healing, and immunocompromise. in any animal determined to be in pain, analgesic drugs should be administered to control pain and discomfort at all times. opioids are cardiovascularly friendly, and their effects can easily be reversed with naloxone if adverse effects such as hypotension and hypoventilation occur. if the patient is nonambulatory, rotate the animal from side to side every 4 to 6 hours to prevent lung atelectasis. passive range-of-motion exercises and deep muscle massage should be performed to increase tissue perfusion, decrease dependent edema, and prevent disuse atrophy. animals should be kept completely dry on soft, padded bedding to prevent the development of decubital ulcers. all bandages, wound sites, and catheter sites should be checked daily for the presence of swelling, erythema, and pain. soiled bandages should be changed to prevent strike-through and contamination of the underlying catheter or wound. hospitalization can be a stressful experience for patient and client alike. allowing brief visits and walks outside in the fresh air can improve a patient's temperament and decrease stress. the preemptive use of analgesic drugs on a regular schedule (not prn) should be used to prevent pain before it occurs. pain decreases the patient's ability to sleep. lack of sleep can promote further stress and impaired wound healing. the use of glucocorticosteroids and antiprostaglandins in shock therapy remains a topic of wide controversy. although the use of these agents potentially may stabilize membranes, decrease the absorption of endotoxin, and decrease prostaglandin release, the routine use of glucocorticosteroids and antiprostaglandins can decrease renal perfusion and gastrointestinal blood flow, promoting gastrointestinal ulceration and impaired renal function. the administration of supraphysiologic levels of glucocorticosteroids in patients in any type of shock can increase sodium and water retention, depress cellular immune function, and impair wound healing. in clinical studies of small animal patients, the routine use of glucocorticosteroids and antiprostaglandins has not demonstrated definite improved survival. the risks of therapy do outweigh the anecdotal reported benefits, and therefore the empiric use of glucocorticosteroids and antiprostaglandins in any shock patient is urinary tract emergencies azotemia azotemia occurs when 75% or more of the nephrons are nonfunctional. the magnitude of the azotemia alone cannot be used to determine whether the azotemia is prerenal, renal, or postrenal in origin, or whether the disease process is acute or chronic, reversible or irreversible, progressive or nonprogressive. before beginning treatment for azotemia, the location or cause of the azotemia must be identified. take a thorough history and then perform a physical examination. obtain blood and urine samples before initiating fluid therapy, for accurate assessment of the location of the azotemia. for example, an azotemic animal with a history of vomiting and diarrhea that appears clinically dehydrated on physical examination, normally should have a concentrated urine specific gravity (>1.045) reflecting the attempt to conserve fluid. if this level is found, the azotemia is much less likely to be renal in origin, and the azotemia will likely resolve after rehydration. if, however, the urine specific gravity is isosthenuric or hyposthenuric (1.007-1.015) in the presence of azotemia and dehydration, primary intrinsic renal insufficiency is likely present. if the azotemia resolves with fluid therapy, the patient has prerenal and primary renal disease. if the azotemia does not resolve after rehydration, the patient has prerenal and primary renal failure. dogs with hypoadrenocorticism can have both prerenal and primary renal disease secondary to the lack of mineralocorticoid (aldosterone) influence on the renal collecting duct and renal interstitial medullary gradient. medullary washout can occur, causing isosthenuric urine in the presence of dehydration from vomiting and diarrhea. the patient often has azotemia due to fluid loss (dehydration and urinary loss) and gastric or intestinal hemorrhage (elevated bun). the prerenal component will resolve with treatment with glucocorticoids and crystalloid fluids, but the renal component may take several weeks to resolve, until the medullary concentration gradient is reestablished with the treatment and influence of mineralocorticoids. drugs such as corticosteroids and diuretics can influence renal tubular uptake and excretion of fluid, and cause a prerenal azotemia and isosthenuric urine in the absence of primary renal disease. treatment of azotemia includes calculation of the patient's dehydration estimate and maintenance fluid volumes, and administering that volume over the course of 24 hours. identify and treat underlying causes of prerenal azotemia (shock, vomiting, diarrhea). monitor urine output closely. once a patient is euvolemic, oliguria is defined as urine output <1-2 ml/kg/hour. urine output should return to normal in patients with prerenal azotemia as rehydration occurs. if a patient remains oliguric after rehydration, consider the possibility of oliguric acute intrinsic renal failure, and administer additional fluid therapy based on the patient's urine output, body weight, central venous pressure, and response to other medical therapies. prerenal azotemia is caused by conditions that decrease renal perfusion, including hypovolemic shock, severe dehydration, hypoadrenocorticism, congestive heart failure, cardiac tamponade, cardiac dysrhythmias, and hypotension. once renal perfusion is restored, the kidneys can resume normal function. glomerular filtration rate decreases when the mean arterial blood pressure falls to less than 80 mm hg in a patient with normal renal autoregulation. renal autoregulation can be impaired in some diseases. passive reabsorption of urea from the renal tubules can occur during states of low tubular flow (dehydration, hypotension) even if glomerular filtration is not decreased. if renal hypoperfusion is not quickly restored, the condition can progress from prerenal disease to acute intrinsic renal failure. prerenal and renal azotemia can coexist in animals with primary renal disease, as a result of vomiting and ongoing polyuria in the absence of any oral fluid intake. the treatment of prerenal azotemia consists of rehydration, antiemetic therapy, and treatment of the underlying cause of vomiting, diarrhea, or third spacing of fluids. acute intrinsic renal failure is characterized by an abrupt decline in renal function to the extent that azotemia and an inability to regulate solute and fluid balance. patients with acute intrinsic renal failure may be oliguric or polyuric, depending on the cause and state of renal failure. in small animals, the most common causes of acute intrinsic renal failure are renal ischemia and toxins. there are three phases of acute intrinsic renal failure: induction, maintenance, and recovery. during the induction phase, some insult (ischemia or toxin) to the kidneys occurs, leading to a defective concentrating mechanism, decreased renal clearance of nitrogenous waste (azotemia), and polyuria or oliguria. if treatment is initiated during the induction phase, progression to the maintenance phase potentially can be stopped. as the induction phase progresses, there is worsening of the urine-concentrating ability and azotemia. renal tubular epithelial cells and renal tubular casts can be seen on examination of the urine sediment. glucosuria may be present. the maintenance phase of acute intrinsic renal failure occurs after a critical amount of irreversible nephron injury. correction of the azotemia and removal of the cause of the problem do not result in return to normal function. in patients with oliguria, the extent of nephron damage is greater than that observed in patients with polyuria. the maintenance phase may last for several weeks to months. recovery of renal function may or may not occur, depending on the extent of injury. the most serious complications (overhydration and hyperkalemia) are observed in patients with oliguria. the recovery phase occurs with sufficient healing of damaged nephrons. azotemia may resolve, but concentrating defects may remain. if the patient was oliguric in the maintenance phase, a marked diuresis develops during the recovery phase that may be accompanied by fluid and electrolyte losses. this phase may last for weeks to months. treatment of acute intrinsic renal failure consists of determining the cause and ruling out obstruction or uroabdomen whenever possible. a careful history can sometimes determine whether there has been exposure to nephrotoxic drugs, chemicals, or food items. if ingestion or exposure to a toxic drug, chemical, or food occurred recently (within 2 to 4 hours), induce emesis with apomorphine (0.04 mg/kg iv). next, administer activated charcoal either orally or via stomach tube, to prevent further absorption of the toxin. obtain blood and urine samples for toxicologic analysis (e.g., ethylene glycol) and to determine whether azotemia or abnormalities in the urine sediment exist. (see section on ethylene glycol, grapes and raisins, and nonsteroidal antiinflammatory drugs). obtain a complete blood count, biochemical profile, and urinalysis to determine the presence of signs of chronic renal failure, including polyuria, polydipsia, and nonregenerative anemia. radiographs and abdominal ultrasound can help in determining the chronicity of renal failure. normal renal size is 2.5-3.5 times the length of l2 in dogs and 2.4-3.0 times the length of l2 in cats. monitor the patient's body weight at least twice a day to avoid overhydration. also monitor urine output; normal output is 1-2 ml/kg/hour. in cases of polyuric renal failure, massive fluid and electrolyte losses can occur. place a urinary catheter for patient cleanliness and to facilitate urine quantitation. measure fluid ins and outs (see section on fluid therapy). after the patient has been rehydrated, the amount of fluids administered should equal maintenance and insensible needs plus the volume of urine produced each day. if a urinary catheter cannot be placed or maintained, serial body weight measurements and central venous pressure should be used to monitor the patient's fluid balance and prevent overhydration. if the patient is oliguric (urine output <1-2 ml/kg/hour), pharmacologic intervention is necessary to increase urine output. first, administer furosemide (2-4 mg/kg or 0.66 mg/kg/hour iv cri). repeat bolus doses of furosemide if there is no response to initial treatment. if necessary, administer low-dose dopamine (3-5 âµg/kg/minute iv cri) to increase renal afferent dilatation and renal perfusion. dopamine and furosemide may be synergistic if administered together. if dopamine and furosemide therapy is ineffective, administer mannitol (0.25-0.5 g/kg iv) once only. if polyuria is present, management is 1 simplified because of the decreased risk of overhydration. if oliguria cannot be reversed, monitor the central venous pessure, body weight, and respiratory rate and effort, auscultate for crackles, and examine the patient carefully for signs of chemosis and the presence of serous nasal discharge. correct hyperkalemia with sodium bicarbonate (0.25-1.0 meq/kg iv) or with insulin (0.25 units/kg) plus dextrose (1 g/unit of insulin iv, followed by 2.5% dextrose iv cri). treat severe metabolic acidosis (ph <7.2 or hco 3 â�� <12 meq/l) with sodium bicarbonate. if anuria develops or oliguria is irreversible despite this therapy, begin peritoneal dialysis. obtain a renal biopsy to establish a diagnosis and prognosis (see section on renal biopsy). administer gastroprotectant drugs and antiemetics to control nausea and vomiting. if possible, avoid the use of nephrotoxic drugs and general anesthesia. initiate nutritional support in the form of an enteral feeding tube or parenteral nutrition as early as possible. once the patient enters the recovery phase, diuresis may occur that can lead to dehydration and electrolyte imbalances (hyponatremia, hypokalemia). dehydration and electrolyte imbalances can be treated with parenteral fluid and electrolyte supplementation. postrenal azotemia is primarily caused by urethral obstruction or leakage from the urinary tract into the abdomen (uroabdomen). complete urinary tract obstruction and uroabdomen are both ultimately fatal within 3 to 5 days if left untreated. in dogs, the most common causes of urethral obstruction are urinary (urethral) calculi or tumors of the urinary bladder or urethra. in male cats, feline urologic syndrome (fus) is the most common cause of urethral obstruction, although there has been an increased incidence of urethral calculi observed in recent years. a ruptured urinary bladder is the most common cause of uroabdomen and is usually secondary to blunt trauma. clinical signs of urinary tract obstruction include dysuria, hematuria, inability to urinate or initiate an adequate stream of urine, and a distended painful urinary bladder. late in the course of obstructive disease, clinical signs referable to uremia and azotemia (vomiting, oral ulcers, hematemesis, dehydration, lethargy, and anorexia) occur. the initial goal of treatment of urinary tract obstruction is to relieve the obstruction. in male dogs, a lubricated catheter can be inserted past the area of obstruction with the animal under heavy sedation or general anesthesia (see section on urohydropulsion). depending on the chronicity of the obstruction, serum electrolytes should be measured;an ecg should be obtained before administering any anesthetic drugs, because of the cardiotoxic effects of hyperkalemia (see section on atrial standstill). correct fluid, electrolyte, and acid-base abnormalities. if a urinary catheter cannot be placed, perform cystocentesis only as a last resort, because of the risk of urinary bladder rupture. definitive treatment includes identification and treatment of the underlying cause (tumor versus urinary calculi). in most cases, surgical intervention is necessary. if an unresectable tumor is present, a low-profile permanent cystostomy tube can be placed, if the owner desires. administration of piroxicam (feldene, 0.3 mg/kg po q24-48h) with or without chemotherapy may shrink the tumor mass and delay the progression of clinical signs. a complete discussion of this disorder is beyond the scope of this text (see additional reading for other sources of information). feline lower urinary tract disease can cause urethral obstruction, particularly in male cats. clinical signs include stranguria, dribbling of small amounts of urine, lethargy, inappetence, and vomiting. often, owners call with the primary complaint of constipation, because the cat is making frequent trips to the litterbox and straining. cases with a duration of obstruction <36 hours are considered uncomplicated; those with a duration >36 hours are complicated. treatment of urethral obstruction includes stabilizing and normalizing the patient's electrolyte status, induction of sedation or general anesthesia, and relieving the obstruction. obtain blood samples for analysis of electrolyte abnormalities. treat hyperkalemia (k + > 6.0 meq/l) with sodium bicarbonate (0.25-1.0 meq/kg iv), regular insulin (0.25 unit/ kg iv) plus dextrose (1 g//unit of insulin iv), followed by 2.5% dextrose iv cri to prevent hypoglycemia; or calcium gluconate (0.2 ml/kg 10% iv slowly). administer non-potassiumcontaining intravenous fluids in 0.9% saline solution. obtain an ecg to detect atrial standstill (see section on atrial standstill). in some cases, a urethral plug is visible at the tip of the penis. the urethral plug can sometimes be manually extracted or massaged from the penis, and the obstruction temporarily relieved. in such cases, it is still necessary to pass a urethral catheter to flush sediment from the urethra and urinary bladder. unless a patient is obtunded, administer an anesthetic such as ketamine, atropine, or propofol (4-7 mg/kg iv) with diazepam iv for patient comfort and muscle relaxation. once the patient is under anesthesia or heavily sedated, urinary catheterization should be performed. in some cases, it will be difficult to advance the catheter. lubricate a closedended tomcat catheter and pass the tip into the distal urethra. fill a 12-ml syringe with sterile saline and sterile lubricant and connect the syringe to the hub of the catheter. pulse the fluid into the catheter as you gently move the catheter tip back and forth against the urethral obstruction. when the catheter has been passed into the urinary bladder, obtain a urine sample for urinalysis. drain the bladder and flush with sterile saline solution until the urine efflux appears clear. remove the tomcat catheter and insert a 3-5 fr red rubber tube or argyle infant feeding catheter into the urethra for urine collection and quantitation. secure the urinary catheter to prepuce with a butterfly strip of 1-inch adhesive tape secured around the catheter and then sutured to either side of the prepuce. the catheter should be connected to a closed urinary collection system for cleanliness and to reduce the risk of ascending bacterial infection. an elizabethan collar should be placed at all times to prevent the patient from damaging or removing the catheter. when the urethral obstruction has been relieved and the catheter placed, continue intravenous fluid diuresis to alleviate postrenal azotemia. monitor the urine for bacteria and other sediment. in some cases, postobstructive diuresis can be severe. carefully monitor fluid ins and outs, along with body weight, to maintain adequate hydration and perfusion. remove the urinary catheter can be removed after 24 to 48 hours. palpate the bladder frequently to make sure that the patient is voiding normally and to detect the recurrence of obstruction. in patients with severe penile or urethral trauma or edema, administer a short-acting steroid (dexamethasone sodium phosphate, 0.25 mg/kg iv, im, sq). at the time of initial diagnosis and again at the time of discharge, the clients need to be instructed about the long-term management of feline lower urinary tract disease at home, and informed of the risks and consequences of recurrence. uroabdomen can occur from trauma or leakage from the kidneys, ureter, or urinary bladder. clinical signs of uroabdomen (azotemia, uremia, hyperkalemia) can also occur secondary to third spacing of urine and leakage into muscular tissue from a ruptured urethra. in most cases, urinary bladder trauma and rupture are secondary to blunt trauma. abdominocentesis should be performed in any animal with suspected blunt abdominal trauma, and any fluid obtained should be analyzed for creatinine or potassium and compared with the patient's serum levels. an abdominal effusion that has a low packed cell volume and a potassium or creatinine level greater than that of the patient's serum is consistent with the diagnosis of uroabdomen. uroabdomen is not a surgical emergency. however, medical management consists of placement of a temporary abdominal drainage catheter into the abdomen, to facilitate removal of urine from the peritoneal cavity. to place the catheter, position the patient in dorsal or lateral recumbency, shave the ventral abdomen, as for any exploratory laparotomy. aseptically scrub the clipped area, and instill a local anesthestic (lidocaine, 1-2 mg/kg) caudal and to the right of the umbilicus, through the skin, subcutaneous tissues, and rectus 290 1 emergency care clinical differentiation of acute necrotizing from chronic nonsuppurative pancreatitis in cats: 63 cases acute pancreatitis in dogs mesenteric volvulus in the dog: a retrospective study of 12 cases incidence and prognostic value of low plasma ionized calcium concentration in cats with pancreatitis: 46 cases (1996-1998) review of feline pancreatitis. part 2: clinical signs, diagnosis and treatment gastric dilatation-volvulus syndrome in dogs diagnostic approach to acute pancreatitis pathophysiology of organ failure in severe acute pancreatitis in dogs washabau rj: gastrointestinal motility disorders and gastrointestinal prokinetic therapy watson pt: exocrine pancreatic insufficiency as an end-stage of pancreatitis in 4 dogs clinical signs, underlying cause, and outcome in cats with seizures: 17 cases fibrocartilaginous embolism in 75 dogs: clinical findings and factors influencing the recovery rate kirk's current veterinary therapy xiii intervertebral disc extrusion in six cats medical management of acute spinal cord disease risk factors for recurrence of clinical signs associated with thoracolumbar intervertebral disk herniation in dogs: 229 cases intervertebral disk disease in 10 cats long-term functional outcome of dogs with severe injuries of the thoracolumbar spinal cord: 87 cases canine status epilepticus: a retrospective study of 50 cases risk factors for development of status epilepticus in dogs with idiopathic epilepsy and effects of status epilepticus on outcome and survival time: 32 cases (1990-1996) skills laboratory part i: performing a neurologic examination skills laboratory part ii: interpreting the results of the neurologic examination accuracy of localization of cervical intervertebral disk extrusion or protrusion using survey radiography in dogs medical and surgical management of the glaucoma patient the feline glaucomas: 82 cases (1995-1999) the canine glaucomas traumatic ocular protrusion in dogs and cats: 84 cases traumatic glaucoma in a dog ocular and orbital porcupine quills in the dog: a review and case series hyphema: pathophysiologic considerations. comp cont educ pract vet van der woerdt a: the treatment of acute glaucoma in dogs and cats administer crystalloid intravenous fluids at maintenance rates using a balanced electrolyte solution perform urinary catheterization and collection to monitor urine output monitor serum urea nitrogen and creatinine every 12 hours treat oliguria, defined as a drop in urine output to less than 1 ml/kg/hour ml/kg) bolus start dopamine at 3 to 5 âµg/kg/minute if no response to crystalloid/colloid bolus occurs within 30 minutes consider mannitol (0.5 to 1 g/kg iv) administration if no response to dopamine occurs within 30 minutes consider furosemide (4 to 8 mg/kg iv, or 0.66 to 1 mg/kg/hour iv cri) if no response to dopamine or mannitol occurs in 30 to 60 minutes if no response to furosemide, peritoneal dialysis or hemodialysis is indicated immediately, particularly if anuria is present administered with caution, because of the risk of exacerbating increased capillary permeability and causing pulmonary edema. animal patients. chlorphenoxy derivatives exert their toxic effects by an unknown mechanism, and cause clinical signs of gastroenteritis and muscle rigidity severe anemia should be treated with packed rbcs or hemoglobin-based oxygen carriers handbook of small animal toxicology and poisonings macadamia nut toxicosis in dogs the recognition and treatment of the intermediate syndrome of organophosphate poisoning in a dog acute renal failure in four dogs after raisin or grape ingestion pleural effusion in cats pulmonary function, ventilator management, and outcome of dogs with thoracic trauma and pulmonary contusions: 10 cases (1994-1998) acute lung injury and acute respiratory distress syndrome smoke exposure in cats: 22 cases (1986-1997) smoke exposure in dogs: 27 cases (1988-1997) thoracic duct ligation and pericardectomy for treatment of idiopathic chylothorax use of intraluminal nitinol stents in the treatment of tracheal collapse in a dog clinical approach to epistaxis the veterinary icu book. teton newmedia radiographic diagnosis of diaphragmatic hernia: review of 60 cases in dogs and cats tracheal collapse: diagnosis and medical and surgical management acute respiratory distress syndrome brachycephalic syndrome in dogs outcome and postoperative complications in dogs undergoing surgical treatment of laryngeal paralysis: 140 cases (1985-1998) full recovery following delayed neurologic signs after smoke inhalation in a dog aspiration pneumonitis the veterinary icu book. teton newmedia allergic airway disease canine pleural and mediastinal effusion, a retrospective study of 81 cases suggested strategies for ventilatory management in veterinary patients with acute respiratory distress syndrome laryngeal and tracheal disorders the veterinary icu book. teton newmedia medical and surgical treatment of pyothorax in dogs: 26 cases traumatic diaphragmatic hernia in cats: 34 cases canine pyothorax: clinical presentation, diagnosis, and treatment canine pyothorax: pleural anatomy and pathophysiology treatment of chronic pleural effusion with pleuroperitoneal shunt in dogs: 14 cases (1985-1999) effects of doxapram hydrochloride on laryngeal function of normal dogs and dogs with naturally occurring laryngeal paralysis an overview of positive pressure ventilation risk factors, prognostic indicators, and outcome of pyothorax in cats: 80 cases (1986-1999) use of percutaneous arterial embolization for the treatment of intractable epistaxis in 3 dogs systemic inflammatory response syndrome, sepsis, and multiple organ dysfunction cardiogenic shock and cardiac arrest hemostatic changes in dogs with naturally occurring sepsis multiple organ dysfunction syndrome in humans and dogs increased lactate concentrations in ill and injured dogs the role of albumin in health and disease pathophysiologic characteristics of hypovolemic shock usefulness of systemic inflammatory response syndrome criteria as an index for prognosis judgement current principles and application of d-dimer analysis in small animal practice choosing fluids in traumatic hypovolemic shock: the role of crystalloids, colloids and hypertonic saline colloid and crystalloid resuscitation thromboembolic disease: predispositions and management marks sl: systemic arterial thromboembolism retrospective study of streptokinase administration in 46 cats with arterial thromboembolism feline arterial thromboembolism: an update arterial thromboembolism in cats: acute crises in 127 cases (1992-2001) and long-term management with low-dose aspirin in 24 cases cut multiple holes in the side of a 14-16 fr red rubber tube or thoracic drainage catheter, using care not to make the cut wider than 50% of the circumference of the tube. insert the catheter into the abdominal cavity in a dorsal caudal direction. make sure that all incisions within the abdomen. secure the tube by placing a pursestring suture around the tube entrance site in the abdominal musculature with absorbable suture material. close the dead space in the subcutaneous tissues with absorbable suture. close the skin around the tube with another purse-string suture secured using a finger-trap technique. connect the tube to a closed urinary collection system and bandage the catheter to the abdomen. the tube can remain in place until the patient retrospective evaluation of acute renal failure in dogs uroabdomen in dogs and cats drug-induced nephrotoxicity: recognition and prevention peritoneal dialysis in emergency and critical care acute renal failure caused by lily ingestion in six cats early diagnosis of renal disease and renal failure acute renal failure in four dogs after raisin or grape ingestion disorders of the feline lower urinary tract the use of a low-profile cystostomy tube to relieve urethral obstruction in a dog renal biopsy: methods and interpretation feline idiopathic cystitis: current understanding of pathophysiology and management today's problem when did you first notice that something was wrong with your pet? when was the last time you noticed your pet act normally? what was the first abnormal sign noticed? what other conditions have developed and what are they? how soon did other signs develop? have the signs become better or worse since you first saw them? what is the name of the product? do you have the container with you today? is it a liquid concentrate, dilute spray, or solid? how long ago do you think that your pet was exposed to the poison? where do you think it happened? do you have any over-the-counter or prescription medications that your animal may have had access to? did you give any medications to your animal? is there any possibility of recreational drug exposure?your pet's recent activity did your pet eat this morning or last night? what is he/she normally fed? is there a chance that your pet may have gotten into the garbage? have you fed table scraps or anything new recently? if so, what? has your pet been off your property in the last 24-48 hours? does your pet run loose unattended? has your pet had any antiflea/tick medication within the last week?your pet's environment is your animal kept inside or outside of the house? is your pet kept in a fenced-in yard or allowed to run loose unattended? does your pet have access to neighboring properties (even for a short time)? where has your pet been in the last 24 hours? has your pet traveled outside of your immediate geographic location? if so, when? has your pet been to rural areas in the last week? has there been any gardening work recently? does your pet have access to a compost pile? any fertilizers or weed killer used in the last week? any construction work or renovation recently? any mouse or rat poison in your house, yard, or garage? any cleaning products used inside or outside the house within the last 48 hours? if so, which? have you changed your radiator fluid or does a car leak antifreeze? induce and maintain a patent airway and stabilize the patient's cardiovascular and respiratory status. control cns excitation with diazepam, if necessary, and control the patient's body temperature (both hypo-and hyperthermia) . induce vomiting if the patient is alert and can protect its airway; otherwise, perform orogastric lavage with the patient under general anesthesia with a cuffed endotracheal tube in place. alcohols do not bind well with activated charcoal. treat dermal exposure by bathing the area with warm water. introduction: if ingested, sodium or potassium hydroxide can cause severe contact dermatitis or irritation of the gastrointestinal tract. esophageal burns and full-thickness coagulative necrosis can occur. if an animal ingests a caustic alkali substance, feed the animal four egg whites mixed with 1 quart of warmed water. perform endoscopy within 24 hours to evaluate the extent of injury and to place a feeding tube, in severe cases. do not induce emesis , and do not perform orogastric lavage, because of the risk of worsening esophageal irritation. in cases of contact exposure to the skin or eyes, rinse the exposed area with warm water baths for at least 30 minutes. administer gastroprotectant, antiemetic, and analgesic drugs as necessary. avoid neutralization, which can cause a hyperthermic reaction and worsen injury to the skin and gastrointestinal tract. amitraz is the active ingredient in ascaricides and anti-tick and anti-mite products such as mitaban and taktic. the toxic dose is 10 to20 mg/kg. amitraz exerts its toxic effects by causing î±-adrenergic stimulation, and causes clinical signs similar to those observed with administration of xylazine: bradycardia, cns depression, ataxia, hypotension, hyperglycemia, hypothermia, cyanotic mucous membranes, polyuria, mydriasis, and emesis. a coma can develop. treatment of amitraz intoxication includes cardiovascular support with intravenous crystalloid fluids and induction of emesis in asymptomatic animals. if clinical signs are present, orogastric lavage may be required. many toxic compounds are impregnated in a collar form. if the patient has ingested a collar and does not vomit it, it should be removed using endoscopy or gastrotomy. administer activated charcoal to prevent or delay absorption of the toxic compound. yohimbine or atepamizole, both î±-adrenergic antagonists, are the treatment(s) of choice to reverse the clinical signs of toxicity. avoid the use of atropine, because it can potentially increase the viscosity of respiratory secretions and cause gastrointestinal ileus, thus promoting increased absorption of the toxic compound. ammonium hydroxide, or cleaning ammonia, can be caustic at high concentrations (see alkalis/caustics) and cause severe injury to the respiratory system if inhaled. pulmonary edema or pneumonia can occur, resulting in respiratory distress. ingestion of ammonia can cause severe irritation to the gastrointestinal tract and cause vomiting and esophageal injury. if ammonia is ingested, administer a dilute solution of egg white.administer gastroprotectant, antiemetic, and analgesic drugs as necessary. if pneumonia or pulmonary edema occurs secondary to aspiration of ammonia into the airways and alveolar spaces, treatment is largely supportive with supplemental oxygen administration, antibiotics, fluid therapy, and mechanical ventilation as necessary. diuretics may or may not be useful in the treatment of pulmonary edema secondary to ammonia inhalation. amphetamines cause cns excitation due to neurosynaptic stimulation, resulting in hypersensitivity to noise and motion, agitation, tremors, vomiting, diarrhea, and seizures. clinical signs of amphetamine toxicity include muscle tremors, tachyarrhythmias, mydriasis, ptyalism, and hyperthermia. amphetamines are rapidly absorbed from the gastrointestinal tract. treatment includes administration of intravenous fluids to maintain hydration and renal perfusion and correction of hyperthermia. administer sedative drugs such as chlorpromazine to control agitation and tremors, and diazepam to control seizures. urinary acidification can promote excretion and prevent reabsorption from the urinary bladder. in severe cases, treat cerebral edema with a combination of mannitol followed by furosemide to control increased intracranial pressure.antifreeze: see ethylene glycol antihistamines introduction antihistamines (loratadine, diphenhydramine, doxylamine, clemastine, meclizine, dimenhydrinate, chlorpheniramine, cyclizine, terfenadine, hydroxyzine) are available as over-thecounter and prescription allergy and anti-motion sickness products. clinical signs of antihistamine toxicity include restlessness, nausea, vomiting, agitation, seizures, hyperthermia, and tachyarrhythmias. treatment of antihistamine intoxication is largely symptomatic and supportive, as there is no known antidote. if ingestion is recent (within 1 to 2 hours) and the patient is not actively seizing and can protect its airway, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. monitor the patient's heart rate, rhythm, and blood pressure. treat cardiac arrhythmias, if present, with appropriate therapies (see section on cardiac dysrhythmias). administer cooling measures and intravenous fluids to treat hyperthermia. a constant rate infusion of guaifenasin can be used to control muscle tremors. introduction î±-naphthylthiourea (antu) is manufactured as a white or blue-gray powder. the toxic dose in dogs is 10-40 mg/kg, and in cats is 75-100 mg/kg. younger dogs appear to be more resistant to its toxic effects. antu usually causes profound emesis and increased capillary permeability that eventually leads to pulmonary edema. treatment of antu toxicity includes respiratory support. mechanical ventilation may be required in severe cases of pulmonary edema. if an animal does not vomit, orogastric lavage should be performed. administer gastrointestinal protectant, antiemetic, and analgesic drugs. cardiovascular support in the form of intravenous crystalloids should be arsenic introduction inorganic arsenic (arsenic trioxide, sodium arsenite, sodium arsenate) is the active ingredient in many herbicides, defoliants, and insecticides, including ant killers. the toxic dose of sodium arsenate is 100-150 mg/kg; that of sodium arsenite is 1-25 mg/kg. sodium arsenite is less toxic, although cats are very susceptible. arsenic compounds interfere with cellular respiration by combining with sulfhydryl enzymes. clinical signs of toxicity include severe gastroenteritis, muscle weakness, capillary damage, hypotension, renal failure, seizures, and death. in many cases, clinical signs are acute in onset. treatment of arsenic toxicity involves procuring and maintaining a patent airway. administer intravenous crystalloid fluids to correct hypotension and hypovolemia, and normalize acidbase and electrolyte balance. if no clinical signs are present and if the compound was ingested within 2 hours, induce emesis. if clinical signs are present, perform orogastric lavage followed by administration of activated charcoal. if dermal exposure has occurred, throughly bathe the animal to prevent further absorption. dimercaprol (bal, 3-4 mg/kg im q8h) can be administered as a chelating agent. n-acetylcysteine (mucomyst) (for cats, 140-240 mg/kg po iv, then 70 mg/kg po iv q6h for 3 days; for dogs, 280 mg/kg po or iv, then 140 mg/kg po iv q4h for 3 days) has been shown to decrease arsenic toxicity in rats. aspirin causes inhibition of the production of prostaglandins, a high anion gap metabolic acidosis, gastrointestinal ulceration, hypophosphatemia, and decreased platelet aggregation when ingested in high quantities (>50 mg/kg/24 hours in dogs; >25 mg/kg/24 hours in cats). clinical signs of aspirin toxicity include tachypnea, vomiting, anorexia, lethargy, hematemesis, and melena. treatment of aspirin toxicity is largely supportive. if the ingestion was recent (within the last hour), induce emesis or perform orogastric lavage followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration and correct acid-base abnormalities. administer synthetic prostaglandin analogues (misoprostol), gastroprotectant drugs, and antiemetics. alkalinization of the urine can enhance excretion. introduction baclofen is a gaba agonist centrally acting muscle relaxant. clinical signs of toxicity include vomiting, ataxia, vocalization, disorientation, seizures, hypoventilation, coma, and apnea. clinical signs can occur at doses as low as 1.3 mg/kg. treatment of baclofen ingestion includes induction of emesis if the animal is asymptomatic. otherwise, perform orogastric lavage. emesis or orogastric lavage should be followed by administration of activated charcoal. perform intravenous crystalloid fluid diuresis to promote elimination of the toxin, maintain renal perfusion, and normalize body temperature. supplemental oxygen or mechanical ventilation may be required for hypoventilation or apnea. if seizures occur, avoid the use of diazepam, which is a gaba agonist and can potentially worsen clinical signs. control seizures with intravenous introduction î²-adrenergic agonists, including terbutaline, albuterol (salbutamol), and metaproterenol, are commonly used in inhaled form for the treatment of asthma. animals commonly are exposed to the compounds after chewing on their owners' inhalers. clinical signs of î²-adrenergic stimulation include tachycardia, muscle tremors, and agitation. severe hypokalemia can occur. treatment of î²-adrenergic agonist intoxication includes treatment with beta-blockers (propranolol, esmolol, atenolol), intravenous fluids, and intravenous potassium supplementation. diazepam or acepromazine may be administered for sedation and muscle relaxation. introduction barbiturates such as phenobarbital are gaba agonists and induce cns depression. clinical signs of barbiturate overdose or toxicity include weakness, lethargy, hypotension, hypoventilation, stupor, coma, and death. treatment of barbiturate toxicity includes maintenance and support of the cardiovascular and respiratory systems. if clinical signs are absent and the patient can protect its airway, induce emesis followed by repeated doses of activated charcoal. perform orogastric lavage if emesis is contraindicated. administer supplemental oxygen if hypoventilation occurs. some animals may require mechanical ventilation. administer intravenous fluids to control perfusion and blood pressure. positive inotropic drugs may be required if dosedependent decrease in cardiac output and blood pressure occurs. alkalinization of the urine and peritoneal dialysis can be performed to enhance excretion and elimination. hemodialysis should be considered in severe cases, if available. automotive and dry cell batteries contain sulfuric acid that can be irritating on contact with the eyes, skin, and gastrointestinal tract. button batteries, which contain sodium or potassium hydroxide, cause contact irritation if chewed. to treat exposure, rinse the eyes and skin with copious amounts of warm tap water or sterile saline solution for a minimum of 30 minutes. if ingestion occurred, administer gastroprotectant and antiemetic drugs. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of aspiration pneumonia and worsening esophageal irritation. no attempt should be made at performing neutralization because of the risk of causing an exothermic reaction and worsening tissue damage. administer analgesics to control discomfort. benzoyl peroxide is the active ingredient in many over-the-counter acne preparations. ingestion can result in production of hydrogen peroxide, gastroenteritis, and gastric dilatation. topical exposure can cause dermal irritation and blistering. if an animal has ingested benzoyl peroxide, do not induce emesis, because of the risk of worsening esophageal irritation. instead, perform orogastric lavage. administer gastroprotectant and antiemetic medications and closely observe the patient observed for signs of gastric dilatation.bismuth subsalicylate (pepto-bismol): see aspirin bleach, chlorine (sodium hypochlorite) introduction sodium hypochlorite is available in dilute (3%-6%) or concentrated (50% industrial strength or swimming pool) solutions for a variety of purposes. sodium hypochlorite can cause severe contact irritation and tissue destruction, depending on the concentration. affected animals may have a bleached haircoat. treatment of exposure includes dilution with copious amounts of warm water or saline baths and ocular lavage. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of causing further esophageal irritation. to treat ingestion, give the animal milk or large amounts of water, in combination with gastroprotectant and antiemetic drugs, to dilute the contents in the stomach. administration of sodium bicarbonate or milk of magnesia is no longer recommended. nonchlorine bleaches (sodium peroxide or sodium perborate) have a moderate toxic potential if ingested. sodium peroxide can cause gastric distention. sodium perborate can cause severe gastric irritation, with vomiting and diarrhea; renal damage and cns excitation followed by depression can occur, depending on the amount ingested. to treat dermal or ocular exposure, rinse the skin or eyes with copious amounts of warm tap water or sterile saline for a minimum of 30 minutes; treat ocular injuries as necessary, if corneal burns have occurred. if the bleach has been ingested, do induce emesis and perform orogastric lavage. administer milk of magnesia (2-3 ml/kg). boric acid is the active ingredient in many ant and roach killers. the toxic ingredient (in amounts of 1-3 g/kg) can cause clinical signs in dogs by an unknown mechanism. clinical signs include vomiting (blue-green vomitus), blue-green stools, renal damage, and cns excitation and depression. treatment of boric acid or borate ingestion includes gastric decontamination with induction of emesis or orogastric lavage, followed by administration of a cathartic to hasten elimination. activated charcoal is not useful to treat ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. clostridium botulinum endospores can be found in carrion, food, garbage, and the environment. ingestion of endospores and c. botulinum endotoxin rarely can cause generalized neuromuscular blockade of spinal and cranial nerves, resulting in miosis, anisocoria, lower motor neuron weakness, and paralysis. respiratory paralysis, megaesophagus, and aspiration pneumonia can occur. clinical signs usually develop within 6 days of ingestion. differential diagnosis includes acute polyradiculoneuritis (coonhound paralysis), bromethalin intoxication, and tick paralysis. treatment of botulism is largely supportive; although an antitoxin exists, it often is of no benefit. treatment may include administration of intravenous fluids, frequent turning of the patient and passive range-of-motion exercises to prevent disuse muscle atrophy, and supplemental oxygen administration or mechanical ventilation. administer amoxicillin, ampicillin, or metronidazole. recovery may be prolonged, up to 3 to 4 weeks in some cases. bromethalin is the active ingredient in some brands of mouse and rat poisons. it usually is packaged as 0.01% bromethalin in green or tan pellets, and packaged in 16 -42.5 g place packs. the toxic dose for dogs is 116.7 g/kg, and for cats 3 g/kg. bromethalin causes toxicity by uncoupling of oxidative phosphorylation. an acute syndrome of vomiting, tremors, extensor rigidity, and seizures occurs within 24 hours of ingestion of high doses. delayed clinical signs occur within 3 to 7 days of ingestion of a lower dose and include posterior paresis progressing to ascending paralysis, cns depression, and coma. treatment of known bromethalin ingestion includes induction of emesis or orogastric lavage, and repeated doses of activated charcoal every 4 to 6 hours for 3 days, because bromethalin undergoes enterohepatic recirculation. supportive care includes intravenous fluids, anticonvulsants, muscle relaxants (methocarbamol up to 220 mg/kg/day iv to effect), frequent turning of the patient, and passive range-of-motion exercises. supplemental oxygen and /or mechanical ventilation may be required in patients with coma and severe hypoventilation. administer mannitol (0.5-1 g/kg) in conjunction with furosemide (1 mg/kg iv) if cerebral edema is suspected. the majority of caffeine toxicities occur in dogs that ingest coffee beans. caffeine causes phosphodiesterase inhibition, and can cause cardiac tachyarrhythmias, cns stimulation (hyperexcitability and seizures), diuresis, gastric ulcers, vomiting, and diarrhea. muscle tremors and seizures can occur, resulting in severe hyperthermia. treatment of caffeine toxicity is largely symptomatic and supportive, as there is no known antidote. if clinical signs are not apparent and the patient is able to protect its airway, induce emesis. alternatively, orogastric lavage can be performed, followed by administration of activated charcoal. administer diazepam to control seizures. administer betaadrenergic blockers (e.g., esmolol, propranolol, atenolol) to control tachyarrhythmias. give intravenous fluids to maintain hydration and correct hyperthermia. the patient should be walked frequently or have a urinary catheter placed to prevent reabsorption of the toxin from the urinary bladder. carbamate compounds are found in agricultural and home insecticide products. examples of carbamates include carbofuran, aldicarb, propoxur, carbaryl, and methiocarb. the toxic dose of each compound varies. carbamate compounds function by causing acetylcholinesterase inhibition. toxic amounts cause cns excitation, muscarinic acetylcholine overload, and slud (salivation, lacrimation, urination, and defecation). miosis, vomiting, treatment of carbamate intoxication includes maintaining an airway and, if necessary, artificial ventilation. administer intravenous crystalloid fluids to control the patient's hydration, blood pressure, and temperature. cooling measures may be warranted. induce emesis if the substance was ingested within 60 minutes and the animal is asymptomatic. give repeated doses of activated charcoal if the animal can swallow and protect its airway. control seizures with diazepam (0.5 mg/kg iv). bathe the patient thoroughly. atropine (0.2 mg/kg iv) is useful in controlling some of the muscarinic signs associated with the toxicity. pralidoxime hydrochloride (2-pam) is not useful in cases of carbamate intoxication. control muscle tremors with methocarbamol (up to 220 mg/kg iv) or guaifenesin. in humans, ingestion or inhalation of 3-5 ml of carbon tetrachloride can be fatal. clinical signs of carbon tetrachloride toxicity include vomiting and diarrhea, then progressive respiratory and central nervous system depression. ventricular dysrhythmias and hepatorenal damage ensue. the prognosis is grave. treatment of carbon tetrachloride inhalation includes procurement and maintenance of a patent airway with supplemental oxygen, and cardiovascular support. to treat ingestion, administer activated charcoal, and give intravenous fluids to maintain hydration and support renal function. chlorinated hydrocarbons include ddt, methoxychlor, lindane, dieldrin, aldrin, chlordane, chlordecone, perthane, toxaphene, heptachlor, mirex, and endosulfan. the toxic dose of each compound varies. chlorinated hydrocarbons exert their toxic effects by an unknown mechanism, and can be absorbed through the skin and the gastrointestinal tract. clinical signs are similar to those observed in organophosphate toxicity: cns excitation, seizures, slud, (salivation, lacrimation, urination, defecation), excessive bronchial secretions, vomiting, diarrhea, muscle tremors, and respiratory paralysis. secondary toxicity from toxic metabolites can cause renal and hepatic failure. chronic exposure may cause anorexia, vomiting, weight loss, tremors, seizures, and hepatic failure. the clinical course can be prolonged in small animal patients. treatment of chlorinated hydrocarbon toxicity is largely supportive in nature, as there is no known antidote. procure and maintain the patient's airway. normalize the body temperature to prevent hyperthermia. if the substance was just ingested and the patient is not demonstrating any clinical signs, induce emesis. if the patient is symptomatic, perform orogastric lavage followed by activated charcoal administration. bathe the patient thoroughly in cases of topical exposure. administer intravenous crystalloid fluids to maintain hydration. these compounds do not appear to be amenable to fluid diuresis. introduction: chlorphenoxy derivatives are found in 2,4-d, 2,4,5-t, mcpa, mcpp, and silvex. the ld 50 of 2,4-d is 100 mg/kg; however, the toxic dose appears to be much lower in small treatment treatment of chlorphenoxy derivative toxicity is largely supportive in nature, as there is no known antidote. secure the patient's airway and administer supplemental oxygen, as necessary. control cns excitation with diazepam (0.5 mg/kg iv). intravenous crystalloid fluid diuresis and urinary alkalinization can promote elimination. administer gastroprotectant and antiemetic drugs, as needed. the toxic effects of chocolate are related to theobromine. various types of chocolate have different concentrations of theobromine and thus can cause clinical signs of toxicity with ingestion of varying amounts of chocolate, depending on the type. the toxic dose of theobromine is 100-150 mg/kg in dogs. milk chocolate contains 44 mg/oz (154 mg/100 g) of chocolate, and has a low toxic potential. semisweet chocolate contains 150 mg/oz (528 mg/100 g), and baking chocolate contains 390 mg/oz (1365 mg/100 g). semisweet and baking chocolate, being the most concentrated, have a moderate to severe toxic potential, even in large dogs.clinical signs of theobromine intoxication are associated with phosphodiesterase inhibition and include cns stimulation (tremors, anxiety, seizures), myocardial stimulation (tachycardia and tachyarrhythmias), diuresis, and (at very high doses) gastrointestinal ulceration. with treatment, the condition of most dogs returns to normal within 12 to 24 hours (t1 /2 = 17.5 hours in dogs). potential side effects include gastroenteritis and pancreatitis due to the fat content of the chocolate. treatment of chocolate toxicity includes obtaining and maintaining a protected airway (if necessary), intravenous fluid diuresis, induction of emesis or orogastric lavage followed by administration of repeated doses of activated charcoal, and placement of a urinary catheter to prevent reabsorption of the toxin from the urinary bladder. cholecalciferol rodenticide ingestion can lead to increased intestinal and renal reabsorption of calcium, causing an increase in serum calcium and dystrophic mineralization of the kidneys and liver at 2-3 mg/kg. clinical signs include lethargy, anorexia, vomiting, constipation, and renal pain within 2 to 3 days of ingestion. seizures, muscle twitching, and central nervous system depression may be observed at very high doses. as renal failure progresses, polyuria, polydipsia, vomiting/hematemesis, uremic oral ulcers, and melena may be observed. if the compound was ingested recently (within 2 to 4 hours) induce emesis or perform orogastric lavage, followed by administration of activated charcoal. check the patient's serum calcium once daily for three days following ingestion. if clinical signs of toxicity or hypercalcemia are present, decrease serum calcium with loop diuretics (furosemide, 2-5 mg/kg po or iv q12h) and glucocorticosteroids (prednisone or prednisolone, 2-3 mg/kg po bid) to promote renal calcium excretion. in severe cases, salmon calcitonin (4-6 iu/kg sc q2-12h in dogs) or bisphosphonate compounds may be required. correct acid-base abnormalities with intravenous crystalloid fluid diuresis and sodium bicarbonate, if necessary. (see section on hypercalcemia.) denture cleaners contain sodium perborate as the active compound. sodium perborate can cause severe direct irritation of the mucous membranes and may also act as a cns depressant. clinical signs are similar to those seen if bleach or boric acid compound is ingested, namely vomiting, diarrhea, cns excitation then depression, and renal failure. treatment for ingestion of denture cleaner includes gastric decontamination along with induction of emesis or orogastric lavage and administration of a cathartic to hasten elimination. activated charcoal is not useful for treatment of ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. deodorants are usually composed of aluminum chloride and aluminum chlorohydrate. both have a moderate potential for toxicity. ingestion of deodorant compounds can cause oral irritation or necrosis, gastroenteritis, and nephrosis. treatment of deodorant ingestion includes orogastric lavage, and administration of antiemetic and gastroprotectant drugs. introduction anionic detergents include sulfonated or phosphorylated forms of benzene. dishwashing liquid is an example of an anionic detergent that can be toxic at doses of 1 -5 g/kg. anionic detergents cause significant mucosal damage and edema, gastrointestinal irritation, cns depression, seizures, and possible hemolysis. ocular exposure can cause corneal ulcers and edema. treatment of anionic detergent exposure is largely symptomatic, as there is no known antidote. to treat topical toxicity, flush the patient's eyes and skin with warmed tap water or 0.9% saline solution for a minimum of 30 minutes, taking care to avoid hypothermia. to treat ingestion, feed the patient milk and large amounts of water to dilute the toxin. do not induce emesis, because of the risk of worsening esophageal irritation. to dilute the toxin, perform orogastric lavage, followed by administration of activated charcoal. closely monitor the patient's respiratory status, because oropharyngeal edema can be severe. if necessary, perform endotracheal intubation in cases of airway obstruction. monitor the patient for signs of intravascular hemolysis. administer intravenous crystalloid fluids to maintain hydration until the patient is able to tolerate oral fluids. cationic detergents and disinfectants include quaternary ammonia compounds, isopropyl alcohol, and isopropanol. quaternary ammonia compounds have a serious toxic potential treatment treatment of cationic detergent exposure includes careful bathing and ocular rinsing of the patient for a minimum of 30 minutes, taking care to avoid hypotension. secure the patient's airway and monitor the patient's respiratory status. administer supplemental oxygen, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation. give milk or large amounts of water orally, as tolerated by the patient, to dilute the toxin. nonionic detergents include alkyl and aryl polyether sulfates, alcohols, and sulfonates; alkyl phenol; polyethylene glycol; and phenol compounds. phenols are particularly toxic in cats and puppies. clinical signs of exposure include severe gastroenteritis and topical irritation. some compounds can be metabolized to glycolic and oxalic acid, causing renal damage similar to that observed with ethylene glycol toxicity. topical and ocular exposure should be treated with careful bathing or ocular irrigation for at least 30 minutes. administer activated charcoal to prevent absorption of the compound. as tolerated, give dilute milk or straight tap water orally to dilute the compound. administer antiemetic and gastroprotectant drugs to control vomiting and decrease gastrointestinal irritation. administer intravenous crystalloid fluids to maintain hydration and decrease the potential for renal tubular damage. monitor the patient's acid-base and electrolyte status and correct any abnormalities with appropriate intravenous fluid therapy. introduction diclone (phigone) is a dipyridyl compound that is a cns depressant. the ld 50 in rats is 25-50 mg/kg. dichlone reacts with thiol enzymes to cause methemoglobinemia and hepatorenal damage. to treat dichlone ingestion, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. procure and maintain a patent airway. perform intravenous fluid diuresis to maintain renal perfusion. n-acetylcysteine may be useful in the treatment of methemoglobinemia. diethyltoluamide (deet) is the active ingredient in many insect repellants (e.g., off, cutters, hartz blockade). the mechanism of action of deet is not fully understood, but it acts as a lipophilic neurotoxin within 5 to 10 minutes of exposure. cats appear to be particularly sensitive to deet. a lethal dermal dose is 1.8 g/kg; if ingested, the lethal dose is much less. the toxic dose of dermal exposure in dogs is 7 g/kg. clinical signs of toxicity include aimless gazing, hypersalivation, chewing motions, and muscle tremors that progress to seizures. recumbency and death can occur within 30 minutes of exposure at high doses. treatment of deet toxicity is largely supportive, as there are no known antidotes. procure and maintain a patent airway and perform mechanical ventilation, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to control hydration and treat hypotension, as necessary. treat seizures with diazepam (0.5 mg/kg iv) or phenobarbital. because of the rapid onset of clinical signs, induction of emesis is contraindicated. perform orogastric lavage if the compound was ingested within the last 2 hours. administer multiple repeated doses of activated charcoal. cooling measures should be implemented to control hyperthermia. if dermal exposure has occurred, bathe the patient thoroughly to avoid further exposure and absorption. diquat is a dipyridyl compound that is the active ingredient in some herbicide compounds. the ld 50 of diquat is 25-50 mg/kg. like paraquat, diquat induces its toxic effects by causing the production of oxygen-derived free radical species. clinical signs of diquat intoxication include anorexia, vomiting, diarrhea, and acute renal failure. massive dehydration and electrolyte imbalances can occur as a result of fluid loss into the gastrointestinal tract. treatment of diquat intoxication is similar to that for paraquat ingestion. if the animal had ingested diquat within 1 hour of presentation, induce emesis. in clinical cases, orogastric lavage may be required. both emesis and orogastric lavage should be followed by administration of kaolin or bentonite as an adsorbent, rather than activated charcoal. place an intravenous catheter and administer crystalloid fluids to restore volume status and maintain renal perfusion. monitor urine output. if oliguria or anuria occurs, treatment with mannitol, furosemide, and dopamine may be considered. ecstasy (3,4-methylenedioxymethylamphetamine; mdma) is a recreational drug used by humans. ecstasy causes release of serotonin. clinical signs of intoxication are related to the serotonin syndrome (excitation, hyperthermia, tremors, and hypertension), and seizures may be observed. a urine drug screening test can be used to detect the presence of mdma. treatment of ecstasy intoxication is largely supportive, as there is no known antidote. administer intravenous fluids to maintain hydration, correct acid-base status, and treat hyperthermia. serotonin antagonist drugs (cyproheptadine) can be dissolved and administered per rectum to alleviate clinical signs. intravenous propranolol has additional antiserotonin effects. administer diazepam (0.5-2 mg/kg iv) to control seizures. if cerebral edema is suspected, administer mannitol, followed by furosemide. ethylene glycol is most commonly found in antifreeze solutions but is also in some paints, photography developer solutions, and windshield wiper fluid. ethylene glycol in itself is only minimally toxic. however, when it is metabolized to glycolate, glyoxal, glyoxylate, and oxalate, the metabolites cause an increased anion gap metabolic acidosis and precipitation of calcium oxalate crystals in the renal tubules, renal failure, and (ultimately) death.the toxic dose in dogs is 6.6 ml/kg, and in cats is 1.5 ml/kg. the toxin is absorbed quite readily from the gastrointestinal tract and can be detected in the patient's serum within an hour of ingestion. colorimetric tests that can be performed in most veterinary hospitals can detect larger quantities of ethylene glycol in the patient's serum. in a dog with clinical treatment begin treatment of known ethylene glycol ingestion immediately. induce emesis or perform orogastric lavage and adminiser repeated doses of activated charcoal. place an intravenous catheter and perform crystalloid fluid diuresis with a known antidote. the treatment of choice for dogs is administration of 4-methylpyrrazole (4-mp), which directly inhibits alcohol dehydrogenase, thus preventing the conversion of ethylene glycol to its toxic metabolites. the dose for dogs is 20 mg/kg initially, followed by 15 mg/kg at 12 and 24 hours and 5 mg/kg at 36 hours. 4-mp has been used experimentally at 6.25 times the recommended dose for dogs. in cats, treatment with 4-mp is effective if it is administered within the first 3 hours of ingestion.cats will demonstrate signs of sedation and hypothermia with this treatment. if 4-mp is not available, administer ethanol (600 mg/kg iv loading dose, followed by 100 mg/ kg/hour), or as a 20% solution (for dogs, 5.5 ml/kg iv q4h for five treatments, then q 6h for five more treatments; for cats, 5 ml/kg q8h for four treatments). grain alcohol (190 proof) contains approximately 715 mg/ml of ethanol. antiemetics and gastroprotective agents should be considered. urinary alkalinization and peritoneal dialysis may enhance the elimination of ethylene glycol and its metabolites. many fertilizers are on the market, and may be composed of urea or ammonium salts, phosphates, nitrates, potash, and metal salts. fertilizers have a moderate toxic potential, depending on the type and amount ingested. clinical signs of fertilizer ingestion include vomiting, diarrhea, metabolic acidosis, and diuresis. nitrates or nitrites can cause formation of methemoglobin and chocolate-brown blood. electrolyte disturbances include hyperkalemia, hyperphosphatemia, hyperammonemia, and hyperosmolality. treatment of fertilizer ingestion includes cardiovascular support, and administration of milk or a mixture of egg whites and water, followed by induction of emesis or orogastric lavage. correct electrolyte abnormalities as they occur (see section on hyperkalemia). administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to control hydration and maintain blood pressure. n-acetylcysteine may be useful if methemoglobinemia is present. fipronil is the active ingredient in frontline, a flea control product. fipronil exerts its effects by gaba antagonism and can cause cns excitation. treatment of fiprinol toxicity includes treatment of cns excitation, treatment of hyperthermia by cooling measures, and administration of activated charcoal. fire extinguisher fluid contains chlorobromomethane or methyl bromide, both of which have a serious toxic potential. dermal or ocular irritation can occur. if ingested, the compounds can be converted to methanol, and cause high anion gap metabolic acidosis, cns excitation and depression, aspiration pneumonitis, and hepatorenal damage. to treat ocular or dermal exposure to fire extinguisher fluids, flush the eyes or skin with warmed tap water or 0.9% saline solution for a minimum of 30 minutes. do not induce emesis or perform orogastric lavage to treat ingestion, because of the risk of causing severe aspiration pneumonitis. gastroprotectant and antiemetic drugs may be used, if indicated. administer intravenous fluids to maintain hydration and renal perfusion. supplemental oxygen or mechanical ventilation may be required in severe cases of aspiration pneumonitis. fireplace colors contain salts of heavy metals-namely, copper rubidium, cesium, lead, arsenic, antimony, barium, selenium, and zinc, all of which have moderate toxic potential, depending on the amount ingested and the size of the patient. clinical signs are largely associated with gastrointestinal irritation (vomiting, diarrhea, anorexia). zinc toxicity can cause intravascular hemolysis and hepatorenal damage. to treat ingestion of fireplace colors, administer cathartics and activated charcoal and gastroprotectant and antiemetic drugs. place an intravenous catheter for intravenous crystalloid fluid administration to maintain hydration and renal perfusion. specific chelating agents may be useful in hastening elimination of the heavy metals. fireworks contain oxidizing agents (nitrates and chlorates) and metals (mercury, copper, strontium, barium, and phosphorus). ingestion of fireworks can cause hemorrhagic gastroenteritis and methemoglobinemia. to treat firework ingestion, induce emesis or perform orogastric lavage and administer activated charcoal. administer specific chelating drugs if the amount and type of metal are known, and administer gastroprotectant and antiemetic drugs. if methemoglobinemia occurs, administer n-acetylcysteine; a blood transfusion may be necessary. introduction fuels such as barbecue lighter fluid, gasoline, kerosene, and oils (mineral, fuel, lubricating) are petroleum distillate products that have a low toxic potential if ingested but can cause severe aspiration pneumonitis if as little as 1 ml is inhaled into the tracheobronchial tree. cns depression, mucosal damage, hepatorenal insufficiency, seizures, and corneal irritation can occur. if fuels are ingested, administer gastroprotectant and antiemetics drugs. do not induce emesis or perform orogastric lavage, because of the risk of aspiration pneumonia. to treat topical exposure, rinse the skin and eyes copiously with warm tap water or 0.9% saline solution. administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to maintain hydration and treat acid-base and electrolyte abnormalities. children's glue contains polyvinyl acetate, which has a very low toxic potential. if inhaled, the compound can cause pneumonitis. treatment of polyvinyl acetate should be performed as clinical signs of pneumonitis (increased respiratory effort, cough, lethargy, respiratory distress) occur. introduction superglue contains methyl-2-cyanoacrylate, a compound that can cause severe dermal irritation on contact. do not induce emesis. do not bathe the animal, and do not apply other compounds (acetone, turpentine) in an attempt to remove the glue from the skin. the fur can be shaved, using care to avoid damaging the underlying skin. the affected area should be allowed to exfoliate naturally. glyophosate is a herbicide found in roundup and kleenup. if applied properly, the product has a very low toxic potential. clinical signs of toxicity include dermal and gastric irritation, including dermal erythema, anorexia, and vomiting. cns depression can occur. treatment includes thorough bathing in cases of dermal exposure, and induction of emesis or orogastric lavage followed by administration of activated charcoal. administer antiemetic and gastroprotectant drugs as necessary. administer intravenous crystalloid fluids to prevent dehydration secondary to vomiting. even small amounts of grapes and raisins can be toxic to dogs. the mechanism of toxicity remains unknown. clinical signs occur within 24 hours of ingestion of raisins or grapes, and include vomiting, anorexia, lethargy, and diarrhea (often with visible raisins or grapes in the fecal matter). within 48 hours, dogs demonstrate signs of acute renal failure (polyuria, polydipsia, vomiting) that can progress to anuria. to treat known ingestion of raisins or grapes, induce emesis or perform orogastric lavage, followed by repeated doses of activated charcoal. if clinical signs of vomiting and diarrhea are present, administer intravenous fluids and monitor urine output. aggressive intravenous fluid therapy, in conjunction with maintenance of renal perfusion, is necessary. in cases of anuric renal failure, dopamine, furosemide, and mannitol can be useful in increasing urine output. peritoneal or hemodialysis may be necessary in cases of severe oliguric or anuric renal failure. calcium channel blockers such as amlodipine and diltiazem can be used to treat systemic hypertension. supportive care includes treatment of hyperkalemia, and administration of gastroprotectant and antiemetic drugs and (if the animal is eating) phosphate binders. aromatic hydrocarbons include phenols, cresols, toluene, and naphthalene. all have a moderate toxic potential if ingested. toxicities associated with ingestion of aromatic hydrocarbons include cns depression, hepatorenal damage, muscle tremors, pneumonia, methemoglobinemia, and intravascular hemolysis. if an aromatic hydrocarbon is ingested, do not induce emesis, because of the risk of aspiration pneumonia. a dilute milk solution or water can be administered to dilute the compound. perform orogastric lavage. carefully monitor the patient's respiratory and cardiovascular status. administer supplemental oxygen if aspiration pneumonia is present. to treat topical exposure, thoroughly rinse the eyes and skin with copious amounts of warm tap water or 0.9% saline solution. imidacloprid is the compound used in the flea product advantage. clinical signs of toxicity are related to nicotinic cholinergic stimulation, causing neuromuscular excitation followed by collapse. the compound may induce respiratory paralysis. to treat imidacloprid toxicity, procure and maintain a patent airway with supplemental oxygen administration. control cns excitation with diazepam, phenobarbital, or propofol. administer enemas to hasten gastrointestinal elimination, and administer activated charcoal. bathe the animal thoroughly to prevent further dermal absorption. closely monitor the patient's oxygenation and ventilation status. if severe hypoventilation or respiratory paralysis occurs, initiate mechanical ventilation. iron and iron salts can cause severe gastroenteritis, myocardial toxicity, and hepatic damage if high enough doses are ingested. lawn fertilizers are a common source of iron salts. treatment of ingestion of iron and iron salts includes cardiovascular support in the form of intravenous fluids and antiarrhythmic drugs, as needed. induce emesis or perform orogastric lavage for gastric decontamination. a cathartic can be administered to promote elimination from the gastrointestinal tract. antiemetic and gastroprotectant drugs should be administered to prevent nausea and vomiting. in some cases, radiographs can aid in making a diagnosis of whether the compound was actually ingested. iron toxicity can be treated with the chelating agent deferoxamine. ivermectin is a gaba agonist that is used in commercial heartworm prevention and antihelminthic compounds and can be toxic in predisposed breeds, including collies, collie loperamide is an opioid derivative that is used to treat diarrhea. clinical signs of loperamide intoxication include constipation, ataxia, nausea, and sedation. induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. naloxone may be beneficial in the temporary reversal of ataxia and sedation. ingestion of macadamia nuts can cause clinical signs of vomiting, ataxia, and ascending paralysis in dogs. the toxic principle in macadamia nuts is unknown. there is no known antidote. treatment consists of supportive care, including administration of intravenous fluids and antiemetics and placement of a urinary catheter for patient cleanliness. clinical signs resolve in most cases within 72 hours. marijuana is a hallucinogen that can cause cns depression, ataxia, mydriasis, increased sensitivity to motion or sound, salivation, and tremors. along with these findings, a classic clinical sign is the sudden onset of dribbling urine. urine can be tested with drug test kits for tetrahydrocannabinoid (thc), the toxic compound in marijuana. there is no known antidote for marijuana toxicity; therefore, treatment is largely symptomatic. place an intravenous catheter and administer intravenous fluids to support hydration. administer atropine if severe bradycardia exists. induction of emesis can be attempted but because of the antiemetic effects of thc, is usually unsuccessful. orogastric lavage can be performed, followed by repeated doses of activated charcoal. clinical signs usually resolve within 12 to 16 hours. introduction "strike anywhere" matches, safety matches, and the striking surface of matchbook covers contain iron phosphorus or potassium chlorate. both compounds have a low toxic potential but can cause clinical signs of gastroenteritis and methemoglobinemia if large quantities are ingested. treatment of match and matchbook ingestion includes gastric decontamination with induction of emesis or orogastric lavage and administration of activated charcoal and a cathartic. if methemoglobinemia occurs, administer n-acetylcysteine, intravenous fluids, and supplemental oxygen. metaldehyde is the active ingredient in most brands of snail bait. the exact mechanism of toxicity is unknown but may involve inhibition of gaba channels. clinical signs associated with metaldehyde toxicity include severe muscle tremors, cns excitation, and treatment treatment of mushroom toxicity is largely supportive. if the mushroom was ingested within the last 2 hours, induce emesis or perform orogastric lavage and then administer activated charcoal. symptomatic treatment includes intravenous fluids to promote diuresis and treat hyperthermia and skeletal muscle relaxants to control tremors and seizures (methocarbamol, diazepam). if amanita ingestion is suspected, administer hepatoprotectant agents including milk thistle. mycotoxins from penicillium spp. are found in moldy foods, cream cheese, and nuts. clinical signs of intoxication include tremors, agitation, hyperesthesia, and seizures. if tremorigenic mycotoxin toxicity is suspected, a sample of the patient's serum and gastric contents or vomitus can be submitted to the michigan state university veterinary toxicology laboratory for tremorigen assay. there is no known antidote. perform orogastric lavage, followed by administration of activated charcoal. control tremors and seizures with methocarbamol, diazepam, phenobarbital, or pentobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. in cases in which cerebral edema is suspected secondary to severe refractory seizures, administer intravenous mannitol and furosemide. naphthalene is the active ingredient in mothballs and has a high toxic potential. clinical signs associated with naphthalene toxicity include vomiting, methemoglobinemia, cns stimulation, seizures, and hepatic toxicity. a complete blood count often reveals heinz bodies and anemia. do not induce emesis if naphthalene ingestion is suspected. if the ingestion was within 1 hour of presentation, perform orogastric lavage. control seizures with diazepam or phenobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. n-acetylcysteine can play a role in the treatment of methemoglobinemia. a packed rbc transfusion may be necessary if anemia is severe. observe the patient for clinical signs associated with hepatitis. nicotine toxicity occurs in animals as the result of ingestion of cigarettes, nicotine-containing gum, and some insecticides. nicotine stimulates autonomic ganglia at low doses, and blocks autonomic ganglia and the neuromuscular junction at high doses. absorption after ingestion is rapid. clinical signs include hyperexcitability and slud (salivation, lacrimation, urination, and defecation). muscle tremors, respiratory muscle fatigue or hypoventilation, tachyarrhythmias, seizures, coma, and death can occur. if the patient presents within 1 hour of ingestion and has no clinical signs, induce emesis, followed by administration of repeated doses of activated charcoal. in patients with clinical signs of toxicity, perform orogastric lavage. administer intravenous fluids to maintain hydration and promote diuresis, and treat hyperthermia. administer atropine to treat cholinergic symptoms. urinary acidification can promote nicotine excretion. nonsteroidal antiinflammatory drugs (nsaids) include ibuprofen, ketoprofen, carprofen, diclofenac, naproxen, celecoxib, valdecoxib, rofecoxib, and deracoxib. nsaids cause inhibition of prostaglandin synthesis, leading to gastrointestinal ulceration, renal failure and hepatotoxicity. ibuprofen toxicity has been associated with seizures in dogs, cats, and ferrets. the toxic dose varies with the specific compound ingested. to treat nsaid toxicity, induce emesis or perform orogastric lavage, followed by administration of multiple repeated doses of activated charcoal. place an intravenous catheter for crystalloid fluid diuresis to maintain renal perfusion. administer the synthetic prostaglandin analogue misoprostol to help maintain gastric and renal perfusion. control seizures, if present, with intravenous diazepam. administer gastroprotectant and antiemetic drugs to control vomiting and gastrointestinal hemorrhage. continue intravenous fluid diuresis for a minimum of 48 hours, with frequent monitoring of the patient's bun and creatinine. when the bun and creatinine levels are normal or have plateaued for 24 hours, slowly decrease fluid diuresis 25% per day until maintenance levels are restored. onions, garlic, and chives contain sulfoxide compounds that can cause oxidative damage of rbcs, leading to heinz body anemia, methemoglobinemia, and intravascular hemolysis. clinical signs of toxicity include weakness, lethargy, tachypnea, tachycardia, and pale mucous membranes. vomiting and diarrhea can occur. intravascular hemolysis can cause treatment treatment of onion, chive, and garlic toxicity includes administration of intravenous fluid diuresis, and induction of emesis or orogastric lavage, followed by administration of activated charcoal and a cathartic. in cases of severe anemia, packed rbc transfusion or administration of a hemoglobin-based oxygen carrier should be considered. opiate drugs include heroin, morphine, oxymorphone, fentanyl, meperidine, and codeine. opiate compounds bind to specific opioid receptors throughout the body and produce clinical signs of miosis or mydriasis (cats), and cns excitation, followed by ataxia and cns depression, leading to stupor and coma. hypoventilation, bradycardia, hypoxia, and cyanosis can occur. to treat known overdose or ingestion of an opiate compound, induce emesis (in asymptomatic animals) or perform orogastric lavage, followed by administration of activated charcoal. administer intravenous fluids and supplemental oxygen to support the cardiovascular and respiratory systems. mechanical ventilation may be necessary until hypoventilation resolves. administer repeated doses of naloxone as a specific antidote to reverse clinical signs of narcosis and hypoventilation. if seizures are present (meperidine toxicity), administer diazepam. organophosphate compounds traditionally are used in flea control products and insecticides. common examples of organophosphates include chlorpyrifos, coumaphos, diazinon, dichlorvos, and malathion. the toxic dose varies, depending on the particular compound and individual animal sensitivity. organophosphate toxicity causes acetylcholinesterase inhibition, resulting in clinical signs of cns stimulation, including tremors and seizures. muscarinic acetylcholine overload causes the classic slud signs of salivation, lacrimation, urination, and defecation. miosis, excessive bronchial secretions, muscle tremors, and respiratory paralysis can occur. an intermediate syndrome of generalized weakness, hypoventilation, and eventual paralysis with ventral cervical ventroflexion that may require mechanical ventilation has been described. if organophosphate toxicity is suspected, whole-blood acetylcholinesterase activity can be measured and will be low. treatment of toxicity includes careful and thorough bathing in cases of dermal exposure and, if the substance was ingested, gastric decontamination with induction of emesis or orogastric lavage, followed by administration of activated charcoal, and administration of the antidote pralidoxime hydrochloride . atropine can help control the muscarinic clinical signs. supportive care in the form of cooling measures, intravenous crystalloid fluids, and supplemental oxygen or mechanical ventilation may be required, depending on the severity of clinical signs. introduction ingestion of large amounts of paintballs can cause neurologic signs, electrolyte abnormalities, and occasionally death. paintballs are gelatin capsules that contain multiple colors of if ingestion was recent and if no clinical signs of toxicity are present, induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal. there is no known antidote. treatment includes supportive care in the form of intravenous fluids and administration of phenobarbital or methocarbamol to control seizures and tremors. diazepam, a gaba agonist, is contraindicated, because it can potentially worsen clinical signs. urine acidification may hasten elimination. clinical signs can last from 3 to 5 days. pyrethrin and pyrethroid compounds are extracted from chrysanthemums, and include allethrin, decamethrin, tralomethrin, fenpropanthrin, pallethrin, sumethrin, permethrin, tetramethrin, cyfluthrin, and resemethrin. the oral toxicity is fairly low; however, the compounds can be significantly harmful if inhaled or applied to the skin. pyrethrin and pyrethroid compounds cause depolarization and blockade of nerve membrane potentials, causing clinical signs of tremors, seizures, respiratory distress, and paralysis. contact dermatitis can occur. to distinguish between pyrethrin/pyrethroid toxicity and organophosphate toxicity, acetylcholinesterase levels should be obtained; they will be normal if pyrethrins are the cause of the animal's clinical signs. treatment of toxicity is supportive, as there is no known antidote. carefully bathe the animal in lukewarm water to prevent further oral and dermal exposure. both hyperthermia and hypothermia can worsen clinical signs. administer activated charcoal to decrease enterohepatic recirculation. atropine may control clinical signs of excessive salivation. to control muscle tremors, administer methocarbamol to effect. administer diazepam or phenobarbital to control seizures, as necessary. rotenone is used as a common garden and delousing insecticide. fish and birds are very susceptible to rotenone toxicity. rotenone inhibits mitochondrial electron transport. clinical signs of tissue irritation and hypoglycemia can occur after topical or oral exposure. if the compound is inhaled, cns depression and seizures can occur. to treat toxicity, perform orogastric lavage, followed by administration of a cathartic and activated charcoal. bathe the animal carefully to prevent further dermal exposure and further ingestion. administer diazepam or phenobarbital to control seizures. the prognosis generally is guarded. treatment of ingestion includes dilution with milk, water, or egg whites. perform orogastric lavage, followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration. administer antiemetic and gastroprotectant drugs to treat gastroenteritis and vomiting.shampoos, nonmedicated: see detergents, nonionic shampoos, selenium sulfide introduction selenium sulfide shampoos (e.g., selsun blue) have a low toxic potential, and primarily cause gastroenteritis. treatment of ingestion includes dilution with water, milk, or egg whites and administration of activated charcoal. carefully and thoroughly rinse the skin and eyes to prevent further exposure. administer antiemetic and gastroprotectant drugs in cases of severe gastroenteritis. zinc-based (zinc pyridinethione) anti-dandruff shampoos have a serious toxic potential if ingested or if ocular exposure occurs. gastrointestinal irritation, retinal detachment, progressive blindness, and exudative chorioretinitis can occur. treatment of ingestion includes gastric decontamination. induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal.to treat ocular exposure, thoroughly rinse the patient's eyes for a minimum of 30 minutes. carefully monitor the animal for clinical signs of blindness. implement intravenous fluid to maintain hydration and renal perfusion in cases of severe gastroenteritis. silver polish contains the alkali substance sodium carbonate and cyanide salts, and has a serious toxic potential. ingestion results in rapid onset of vomiting and possibly cyanide toxicity. to treat ingestion, monitor and maintain the patient's respiration and cardiovascular status and administer intravenous crystalloid fluids. induce emesis, followed by administration of activated charcoal. administer sodium nitrite or sodium thiosulfate iv for cyanide toxicity. bath soap (bar soap) usually has low toxic potential and causes mild gastroenteritis with vomiting if ingested. to treat ingestion, include dilution with water, administration of intravenous fluids to maintain hydration, and administration of antiemetic and gastroprotectant drugs to treat gastroenteritis. sodium fluoroacetate is a colorless, odorless, tasteless compound that causes uncoupling of oxidative phosphorylation. the toxic dose in dogs and cats is 0.05-1.0 mg/kg. clinical signs of toxicity include cns excitation, seizures, and coma secondary to cerebral edema. the prognosis is guarded. to treat toxicity, procure and maintain a patent airway, monitor and stabilize the cardiovascular status, and control hyperthermia. perform orogastric lavage, followed by administration of activated charcoal. if clinical signs are not present at the time of presentation, induce emesis. administer intravenous fluids and supplemental oxygen, as necessary. strattera (atomoxetine hydrochloride) is a selective norepinephrine reuptake inhibitor used in the treatment of attention deficit hyperactivity disorder (adhd) in humans. peak serum concentrations occur in dogs within 3 to 4 hours of ingestion, with a peak half-life at 4 to 5 hours following ingestion. clinical signs of toxicity include cardiac tachyarrhythmias, hypertension, disorientation, agitation, trembling, tremors, and hyperthermia. treatment of intoxication is largely symptomatic and supportive in nature. first, induce emesis if the patient is conscious and has an intact gag reflex. orogastric lavage can also be performed. administer one dose of activated charcoal to prevent further absorption of the compound from the gastrointestinal tract. identify cardiac dysrhythmias and treat accordingly. control hypertension with sodium nitroprusside or diltiazem as a constant rate infusion. administer acepromazine or chlorpromazine to control agitation. do not use diazepam, because it can potentially worsen clinical signs. administer intravenous fluids to maintain hydration and promote diuresis. strychnine is the active ingredient in pesticides used to control rodents and other vermin. the toxic dose in dogs is 0.75 mg/kg, and in cats is 2 mg/kg. strychnine antagonizes spinal inhibitory neurotransmitters and causes severe muscle tremors, muscle rigidity, and seizures. clinical signs are stimulated or exacerbated by noise, touch, light, and sound. mydriasis, hyperthermia, and respiratory paralysis can occur. if strychnine toxicity is suspected, gastric contents should be collected and saved for analysis. if the animal is asymptomatic at the time of presentation, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by the administration of activated charcoal. administer intravenous crystalloid fluids to support the cardiovascular system, aid in cooling measures, and improve renal diuresis. treat cns stimulation with methocarbamol, diazepam, or phenobarbital. the animal should have cotton packed in its ears to prevent noise stimulation, and should be placed in a quiet, dark room. treatment of ingestion includes dilution with milk of magnesia or water, administration of antiemetic and gastroprotectant drugs, and administration of intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation.sunscreen: see zinc and zinc oxide suntan lotion: see shampoos, zinc-based, and alcohols tar: see fuels tea tree oil (melaleuca oil) introduction tea tree (melaleuca) oil is an herbal-origin flea-control product. the toxic principles in tea tree oil are monoterpenes, which produce clinical signs of neuromuscular weakness, and ataxia. treatment of tea tree oil toxicity includes administration of cathartics and activated charcoal to prevent further absorption. carefully bathe the animal to prevent further dermal exposure. tetanus spores from clostridium tetani organisms are ubiquitous in the soil and feces, particularly in barnyards. cases have been reported in dogs after tooth eruption and after abdominal surgeries performed with cold sterilization packs. anaerobic wound infections can contain tetanus spores. the neurotoxin from c. tetani inhibits spinal inhibitory neurons, causing motor neuron excitation. extensor muscle rigidity ("sawhorse stance"), erect ears, and risus sardonicus (a sardonic grin) are characteristic features of tetanus. administer tetanus antitoxin if toxin has not already been bound in the cns. to eliminate the source of the toxin (e.g., abscess), open and debride all wounds. intravenous administration of ampicillin or penicillin g is the treatment of choice for tetanus. supportive care in the form of skeletal muscle relaxants, intravenous fluids and parenteral nutrition, and nursing care to prevent decubitus ulcer formation is required. in extreme cases, mechanical ventilation may be necessary. triazene compounds include atrazine, prometone, and monuron (telvar). the toxic mechanism of triazene compounds is unknown. clinical signs of toxicity include salivation, ataxia, hyporeflexia, contact dermatitis, hepatorenal damage, muscle spasms, respiratory difficulty, and death. treatment of triazene exposure includes cardiovascular and renal support in the form of intravenous crystalloid fluids, inotropic drugs, and antiarrhythmic agents, as necessary. if the exposure is recent, induce emesis. perform orogastric lavage in animals that cannot protect the airway. emesis and orogastric lavage should be followed by the administration of activated charcoal and a cathartic. carefully bathe the patient to prevent further dermal absorption. a variety of tricyclic antidepressants are available for use in both humans and animals, including amitriptyline, amoxapine, desipramine, doxepine, fluoxetine (prozac), fluvoxamine (luvox), imipramine, nortriptyline, paroxetine (paxil), protriptyline, sertraline (zoloft), and trimipramine. selective serotonin reuptake inhibitors (ssris) are rapidly absorbed from the digestive tract, with peak serum concentrations occurring 2 to 8 hours after ingestion. the elimination half-life for each drug differs in dogs, but typically last 16 to 24 hours. ssris inhibit the reuptake of serotonin, causing serotonin to accumulate in the brain. this can cause "serotonin syndrome," characterized by trembling, seizures, hyperthermia, ptyalism or hypersalivation, cramping or abdominal pain, vomiting, and diarrhea. other clinical signs of ssri intoxication include depression, tremors, bradycardia, tachyarrhythmias, and anorexia. any animal that has ingested an ssri should be promptly treated and carefully observed for at least 72 hours for side effects. the treatment of suspected ssri intoxication involves gastric decontamination if the patient is not depressed and has an intact gag reflex. perform orogastric lavage and administer activated charcoal to prevent further toxin absorption and hasten elimination from the gastrointestinal tract. treat other clinical signs symptomatically. administer intravenous diazepam to control seizures. treat tachyarrhythmias according to type. administer methocarbamol to control muscle tremors. cyproheptadine (1 mg/kg), a serotonin antagonist, can be dissolved in water and administered per rectum. vitamin k antagonist rodenticides, which are commonly found in pelleted or block form, inhibit the activation of the vitamin k-dependent coagulation factors ii, vii, ix, and x. clinical signs of hemorrhage occur within 2 to 7 days of exposure. hemorrhage can occur anywhere in the body, and can be manifested as petechiation of the skin or mucous membranes, hemorrhagic sclera, epistaxis, pulmonary parenchymal or pleural hemorrhage, gastrointestinal hemorrhage, pericardial hemorrhage, hematuria, retroperitoneal hemorrhage, hemarthrosis, and central nervous system hemorrhage. clinical signs include respiratory distress, cough, bleeding from the gums or into the eyes, ataxia, paresis, paralysis, seizures, hematuria, joint swelling, lameness, lethargy, weakness, inappetence, and collapse.diagnosis is made based on clinical signs and a prolonged activated clotting time, or prothrombin time. the pivka (proteins induced by vitamin k antagonism) test may be helpful but usually cannot be performed in-house. slight thrombocytopenia may be present secondary to hemorrhage; however, blood levels usually do not reach the critical level of <50,000 platelets/âµl to cause clinical signs of hemorrhage. in some cases, severe stressinduced hyperglycemia and glucosuria may be present but resolves within 24 hours. if the rodenticide was ingested within the last 2 hours, induce emesis. alternatively, orogastric lavage can be performed in an uncooperative patient. both emesis and orogastric lavage should be followed by administration of activated charcoal. the stomach contents can be submitted for analysis. following successful treatment, administer oral vitamin k for 30 days after the exposure; or a check prothrombin time 2 days after gastric decontamination. if the prothrombin time is prolonged, administer fresh frozen plasma and vitamin k.if the prothrombin time is normal, gastric decontamination was successful, and no further treatment is necessary.if an animal presents with clinical signs of intoxication, administer activated clotting factors in the form of fresh frozen plasma (20 ml/kg), and vitamin k 1 (5 mg/kg sq in multiple sites with a 24-gauge needle). packed rbcs or fresh whole blood may be required if the patient is also anemic. supportive care in the form of supplemental oxygen may be necessary in cases of pulmonary or pleural hemorrhage. following initial therapy and discharge, the patient should receive vitamin k 1 (2.5 mg/kg po q8-2h for 30 days), and prothrombin time should be checked 2 days after the last vitamin k capsule is administered. in some cases, depending on the type of anticoagulant ingested, an additional 2 weeks of vitamin k1 therapy may be required. xylitol is a sugar alcohol that, when ingested by humans, does not cause a significant increase in blood glucose, and therefore does not stimulate insulin release from the human pancreas. in dogs, however, xylitol causes a massive rapid and dose-dependent release of insulin from pancreatic beta-cells. following insulin release, clinically significant hypoglycemia can develop, followed by signs of vomiting, weakness, ataxia, mental depression, hypokalemia, hypoglycemic seizures, and coma. clinical signs associated with xylitol ingestion can be seen within 30 minutes of ingestion and can last for more than 12 hours, even with aggressive treatment. known xylitol ingestion should be treated as for other toxin ingestion. if no neurologic abnormalities exist at the time the patient is seen, induce emesis, followed by administration of activated charcoal. it remains unknown at this time whether activated charcoal actually delays or prevents the absorption of xylitol from the canine gastrointestinal tract. if clinical signs have already developed, perform orogastric lavage and gastric decontamination. blood glucose concentrations should be analyzed and maintained with supplemental dextrose as a constant rate infusion (2.5%-5%) until normoglycemia can be maintained with multiple frequent small meals. hypokalemia may develop because it is driven intracellularly by the actions of insulin. treat hypokalemia with supplemental potassium chloride by infusion, not to exceed 0.5 meq/kg/hour. pennies minted in the u.s. after 1982 contain large amounts of zinc rather than copper. other sources of zinc include zinc oxide ointment and hardware such as that found in metal bird cages. zinc toxicity causes intravascular hemolysis, anemia, gastroenteritis, and renal failure. if zinc toxicity is suspected, take an abdominal radiograph to document the presence of the metal in the stomach or intestines. (if zinc-containing ointment was ingested, this will not be visible on radiographs.) induce emesis or perform orogastric lavage, depending on the size of the object ingested. often, small objects such as pennies can be retrieved using endoscopy or surgical gastrotomy/enterotomy. always take an additional radiograph after the removal procedure to ensure that all objects have been successfully removed. administer intravenous fluids to maintain renal perfusion and promote fluid diuresis. administer gastroprotectant and antiemetic drugs. chelation therapy with succimer, calcium edta, dimercaprol, or penicillamine may be necessary. do not administer pulmonary contusions are a common sequela of blunt traumatic injury. a contusion basically is a bruise characterized by edema, hemorrhage, and vascular injury. contusions may be present at the time of presentation or can develop over the first 24 hours after injury. a diagnosis of pulmonary contusion can be made based on auscultation of pulmonary crackles, presence of respiratory distress, and the presence of patchy interstitial to alveolar infiltrates on thoracic radiographs. radiographic signs can lag behind the development of clinical signs of respiratory distress and hypoxemia by 24 hours. in most cases, cage rest is sufficient to temporarily diminish blood loss. sedation (acepromazine, 0.02-0.05 mg/kg iv, im, sq) may be helpful in alleviating anxiety and decreasing blood pressure. the hypotensive effects of acepromazine are potentially harmful if severe blood loss has occurred. if evidence of hypovolemia is present (see section on hypovolemic shock), intravenous fluid resuscitation should be administered. rapid assessment of clotting ability, with a platelet count estimate and clotting profile (act or aptt and pt), should be performed. if epistaxis secondary to vitamin k antagonist rodenticide intoxication is suspected, administer vitamin k 1 and fresh frozen plasma or fresh whole blood.persistent hemorrhage from a nasal disorder can be treated with dilute epinephrine (1:100,000) into the nasal cavity with the nose pointed toward the ceiling to promote vasoconstriction. if this fails, the animal can be anesthetized, and the nasal cavity packed with gauze, and the caudal oropharynx and external nares covered with umbilical tape to control hemorrhage. a rhinoscopy should be performed to determine the cause of ongoing hemorrhage. continued excessive hemorrhage can be controlled with ligation of the carotid artery on the side of the hemorrhage, or with percutaneous arterial embolization. systemic thromboembolism is most commonly recognized in cats with cardiomyopathies (hypertrophic, restrictive, unclassified, and dilatative) but can also occur in dogs with hyperadrenocorticism, disseminated intravascular coagulation (dic), systemic inflammatory response syndrome (sirs), protein-losing enteropathy and nephropathy, and tumors affecting the aorta and vena cava. thrombosis occurs through a complex series of mechanisms when the components of virchow's triad (hypercoaguable state, sluggish blood flow, and vascular endothelial injury or damage) are present. in cats, blood flow through a severely stretched left atrium is a predisposing factor to the development of clots and thromboembolism.the most common site of embolism is the aortic bifurcation, or "saddle thrombus." other, less common locations of thromboembolism include the forelimbs, kidneys, gastrointestinal tract, and cerebrum. diagnosis usually is made based on clinical signs of cool extremities, the presence of a cardiac murmur or gallop rhythm, auscultation of pulmonary crackles resulting from pulmonary edema, acute pain or paralysis of one or more peripheral extremities, respiratory distress, and pain and lack of a palpable pulse in affected limbs. the affected nailbeds and paw pads are cyanotic, and nails do not bleed when cut with a nail clipper.client education is one of the most important aspects of emergency management of the patient with thromboembolic disease. concurrent congestive heart failure (chf) occurs in 40% to 60% of cats with arterial thromboembolism. more than 70% of cats are euthanized during the initial thromboembolic event because of the poor long-term prognosis and the high risk of recurrence within days to months after the initial event, even with aggressive therapy. although the long-term prognosis varies from 2 months to 2 years after initial diagnosis and treatment, in the majority of cats thromboembolic disease recurs within 9 months. rectal temperature hypothermia and bradycardia on presentation are negative prognostic indicators.immediate treatment of a patient with chf and thromboembolic disease involves management of the chf with furosemide, oxygen, and vasodilators (nitroglycerine paste, morphine, nitroprusside). additional management includes analgesia (butorphanol, 0.1-0.4 mg/kg iv, im) and prevention of further clot formation. aspirin (10 mg/kg po q48h) is beneficial bcause of its antiplatelet effects. heparin works in conjunction with antithrombin to prevent further clot formation (100-200 units/kg iv, followed by 250-300 units/kg sq q8h in cats, and 100-200 units/kg sq q8h in dogs). acepromazine can cause peripheral vasodilation and decreased afterload but also can promote hypotension in a patient with concurrent chf. acepromazine (0.01-00.02 mg/kg sq) should be used with extreme caution, if at all.thrombolytic therapy can also be attempted, but in most cases is not without risk, and may be cost-prohibitive for many clients. streptokinase (90,000 units iv over 30 minutes and then 45,000 units/hour iv cri for 3 hours) was administered with some success in cats; however, many died of hyperkalemia or other complications during the infusion. tissue plasminogen activator (0.25-1 mg /kg/hour iv cri, up to 10 mg/kg total dose, to effect) has been used with some success but is cost-prohibitive for most clients. side effects of thrombolytic therapy include hyperkalemia with reperfusion and hemorrhage.in cats, the primary cause of arterial thromboembolism is cardiomyopathy. once an animal is determined to be stable enough for diagnostic procedures, lateral and dv thoracic radiographs and an echocardiogram should be performed. ultrasound of the distal aorta and renal arteries should also be performed to determine the location of the clot and help establish the prognosis.other diagnostic procedures to evaluate the presence and cause of thromboembolism include a complete blood count, serum biochemistry profile, urinalysis (to rule out proteinlosing nephropathy), urine protein:creatinine ratio, antithrombin levels, acth stimulation test (to rule out hyperadrenocorticism), heartworm antigen test (in dogs), thyroid profile (to rule out hyperthyroidism in cats, and hypothyroidism in dogs), thoracic radiographs, arterial blood gas analyses, coagulation tests, and coombs' test. selective and nonselective angiography can also be performed to determine the exact location of the thrombus.long-term management of thromboembolism involves management of the underlying disease process and preventing further clot formation. begin therapy with heparin until the aptt becomes prolonged 1.5 times; then administer warfarin (0.06-0.09 mg/kg/day). monitoring therapy based on prothrombin time and the international normalized ratio (inr, 2.0-4.0) is recommended. low-dose aspirin (5-10 mg/kg q48h) also has been recommended. physical therapy with warm water bathing, deep muscle massage, and passive range-of-motion exercises should be performed until the patient regains motor function. future therapy may involve the use of platelet receptor antagonists to prevent platelet activation and adhesion. key: cord-023026-2r84ndzv authors: nan title: posters date: 2013-06-14 journal: glia doi: 10.1002/glia.22530 sha: doc_id: 23026 cord_uid: 2r84ndzv nan university of colorado school of medicine, aurora, united states oligodendrocyte progenitor cells (opcs) migrate long distances before differentiating and myelinating axons. it is well established that both short and long-range soluble guidance factors, as well as contact with the surrounding extracellular matrix (ecm) and neighboring cells, can modulate opc migration and differentiation. however, the influence of cell-matrix interactions on opc responses to guidance molecules is less clear. previous in vitro studies of opc migration in response to semaphorins and netrin-1 yielded inconsistent results, but these studies were performed on a variety of ecm substrates, i.e., explants, collagen, poly-d-lysine. in the current study, we systematically studied opc migration in response to guidance factors on several different ecm substrates, to better understand the influence of ecm interactions on opc responses to chemotropic molecules. understanding the regulation of opc migration and differentiation has important implications for the treatment of demyelinating diseases such as multiple sclerosis (ms). for example, semaphorins 3a and 3f are expressed in active, but not chronic, ms lesions, and in demyelination/remyelination studies in animal models, semaphorins influence opc migration into lesions, affecting the rate at which remyelination occurs. in addition, aberrant expression of ecm ligands occurs in and around ms lesions. we performed live imaging experiments of opc migration on different ecm substrates in response to gradients of chemotropic molecules. these studies are combined with ihc, co-ip, western blot and rt-pcr analyses to determine the effects of ecm substrates on opc migration and signaling pathway responses to chemotropic molecules. our preliminarily results suggest that ecm substrate can regulate both the direction and rate of opc migration. semaphorin 3a is repulsive to opcs on laminin 1, while on fibronectin opc migration remains uniform in the presence of semaphorin 3a. since laminin 1 is the ligand for a6b1 integrin heterodimers, while opc migration on fibronectin is mediated by avb3 integrins, the repulsive effect of semaphorin 3a appears likely to function through b1 integrin. thus, signaling through the neuropilin-plexin receptor complex that underlies the repulsive effects of semaphorin 3a on opc migration may be modulated by interactions with b1 integrins. x. bai university of saarland, homburg, germany secondary injury processes after acute brain trauma involve activation of different cell types like astrocytes, oligodendrocytes and microglia cells. a complex and yet not completely understood sequence of cellular responses initiate functional recovery after the neurodegeneration process. by using double-transgenic mice gcpb (gfap-egfp 3 plp-dsred1) mice, we were able to identify a particular type of activated glia expressing astro-and oligodendroglial properties simultaneously (ao cells) that transiently appeared after three types of acute cortical injuries (stab wound injury, pial vessel disruption and middle cerebral artery occlusion). ao cells could be labeled by oligodendrocyte precursor cell (opc) markers olig2 and sox10, but not for markers of mature astrocytes or neurons. two-photon live imaging of gcpb mice revealed that ao cells originated from plp-dsred-positive oligodendrocyte lineage cells. electrophysiological inspection of ao cells in acutely isolated brain slices revealed the expression of delayed rectifying, voltage gated k 1 currents, a typical property of ng2 glia. therefore, we classified ao cells as opcs. to follow the fate of ao cells which disappeared about 15 days after the injury, we generated gfap-n-cre 3 plp-c-cre 3 rosa26eyfp triple transgenic (crec) mice. under non-injury conditions, few recombined cells were observed. however, numerous recombined cells appeared after 1 week of stab wound injury adjacent to the lesion site, and lasted over 8 weeks. we found that 55-70% of recombined cells were gfap-positive, 18-29% of them were pdgfra-positive and 19-30% were positive for the oligodendrocyte marker gstp. in line with that, about 46% of newly generated gfap-positive astrocytes were observed from ng2-creert2 x rosa26tdtomato mice after 1 week of stab wound injury. these results provide a strong evidence for opcs giving rise to astrocytes after acute cortical injuries. to further understand the molecular mechanism, we performed intra-cerebral injection of bone morphogenetic protein 4 (bmp4, known to promote astrocyte, but blocking oligodendrocyte differentiation) into gcpb and crec mice. bmp4, but not leukemia inhibitory factor (lif), significantly increased the number of ao cells as well as astrocytes in the respective mice. in conclusion, ng2/opcs display strong potential to differentiate into astrocytes after acute cortical injury. for instance, sox9 and sox10 have a crucial role in opc specification and differentiation, respectively. using microarray analysis of oligodendrocyte lineage cells, we previously identified sox17 as a major regulator of oligodendrocyte development (sohn et al., 2006) . in opc cultures, sox17 expression was maximal at developmental corresponding to cell cycle exit and onset of differentiation. these findings, suggest that sox17 has critical functions in the controlling of opc cell cycle exit and differentiation. in order to decipher the functional role of sox17 in oligodendrocyte development, we generated a tet-sox17:sox10 rtta double transgenic mouse line. this mouse strain allows a sox17 overexpression specifically in sox101 cells in a doxycycline dependant manner (tet-on system). in the cns of dox-treated transgenic animals, we showed that sox17 overexpression was specifically targeted in sox101 oligodendroglial cells. in the developing cns, sox17 overexpression was targeted in pdgfra1/nkx2.21 opcs and in olig21/cc11 differentiated oligodendrocytes. to assess the effect of sox17 gain-of-function on oligodendrocyte development and myelination, we analysed opc proliferation, apoptosis and differentiation, after doxycycline induction from e12.5 to p15. in the embryonic spinal cord, we showed that overexpression of sox17 did not modify cell proliferation or the number of pdgfra1 opcs. these data strongly suggest that sox17 is not involved in early steps of oligodendrocyte development. interestingly, we found that sox17 gain-of-function drastically reduces the density of cc11 mature oligodendrocytes at postnatal stages, correlating with a delay in myelination in transgenic mouse spinal cords. altogether, our data reveal critical functions of sox17 in oligodendrocyte lineage progression and myelination. supports: arsep and nmss (usa). m.f is a phd fellow of the french mesr (ed3c). m. rocha, p. fernandes, a.c. manhães, p.c. barradas, f. tenorio universidade do estado rio de janeiro, instituto de biologia, rio de janeiro, brazil nutrient restriction during the perinatal period exerts a profound influence on brain development. previous studies using an undernutrition protocol (0% protein diet) during the first half of the lactation period in rats showed that the offspring presented an altered feeding pattern, which reflected a metabolic imprinting effect on feeding behavior. there is a delay in the pattern of npy expression in the arcuateparaventricular (arc-pvn) pathway, reflecting an effect on the development of the hypothalamic circuitry, leading to metabolic imprinting. here we studied the effects of undernutrition on glial differentiation using the same model. we used rats from 5 to 30 postnatal (p) days of age whose dams were either fed a 0% protein diet (dg) or a normoprotein diet (cg) from p1 to p10. we assessed ki67, vimentin, gfap, ed-1 and cnpase distribution in hypothalamic nuclei using immunohistochemistry. our results showed impairment in cell proliferation, more evident in the pvn and in the lateral hypothalamus (lh), where dg animals showed a lower number of ki-67positive (ki671) cells at p5 when compared to cg. however, at p10 and p15, the number of ki671 cells was significantly increased in dg. from p20 onwards, staining intensity remained relatively stable and similar in all groups. dg animals showed a delay in astroglial differentiation, presenting a decrease in gfap expression and a peak of vimentin expression at p10 and p15 accompanied by an increase in vimentin/ki671 cells. an increase in the number of ed1 positive cells next to the third ventricle was also shown at the same ages in dg. cnpase staining was very faint in most nuclei and did not show any obvious difference in dg animals. our results suggest that glial differentiation is delayed in dg, which may contribute to the changes in hypothalamic circuitry that causes metabolic imprinting. recent studies have demonstrated that neural precursor cells (npcs) residing in the adult subventricular zone also exhibit the capacity to generate new oligodendrocytes following experimental demyelination. the relative capacities of these two cell types to contribute to oligodendrogenesis in this context remain largely unexplored. we have adopted transgenic approaches that enable either the specific labeling of npcs or oligodendrocytes and the interrogation of their subsequent fate within the corpus callosi of mice subjected to central demyelination induced by cuprizone. adult nestin-creer t2 : r26r-eyfp mice were administered tamoxifen (0.3g/kg/day) for 4 days by oral gavage, inducing the permanent expression of yellow fluorescent protein (yfp) in npcs and their progeny. mice were subsequently fed 0.2% cuprizone for 6 weeks followed by 6 weeks recovery on normal food to enable the analysis of remyelination. expression of yfp and other cellular markers was examined immunohistochemically to determine the fate and migratory potential of npcs in the remyelinating corpus callosum (cc). rostrocaudal analyses of cc in the cuprizonechallenged mice demonstrated that approximately 60% of yfp 1 npcs commit to an oligodendroglial fate. there was robust recruitment of npc-derived oligodendroglial cells, with a 14-fold increase in yfp 1 sox10 1 cells compared to unchallenged mice. most of these cells were mature oligodendrocytes (yfp 1 cc1 1 ) and their density in the remyelinating cc was highest adjacent to the dorsolateral corner of svz and decreased proportionally with distance in the mediolateral axis. on the other hand, fate mapping of opcs using pdgfracreer t2 : r26r-eyfp mice revealed that oligodendrocytes derived from parenchymal opcs were distributed in a converse manner to svz-derived oligodendrocytes. in addition, we found that, independent of the cellular source, many oligodendrocytes repopulated the corpus callosum as linear arrays of clonally derived cells, in a manner that recapitulated the postnatal development of these structures. these results demonstrate that lineage determination and maturation of oligodendroglia from npcs occurs efficiently in situ. we also reveal that remyelination occurs in a coordinated way, most likely secondary to interactions between a restricted pool of axons and a sentinel opc induced to proliferate in situ. play an important role in developmental oligodendrogenesis and myelination and mounting evidence from rodent models of demyelination suggest that they also promote remyelination. the therapeutic application of thyroid hormone to demyelinating disorders is limited by the potential for cardiac side effects mediated by thyroid hormone receptor (thr) a signaling. here we report that gc-1, a thyromimetic with selective thrb1 action promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. in addition, we used pdgfar-creer;rosa26-eyfp double-transgenic mice to examine the effect of gc-1 on the fate of oligodendrocyte progenitor cells and find that treatment with gc-1 during developmental myelination promotes oligodendrogenesis in vivo. these results indicate that a b receptor selective thyromimetic can enhance ol differentiation in vitro and during developmental myelination and warrants further study in demyelinating models. the olfactory epithelial layer contains multipotent horizontal basal cells (hbcs) that differentiate into olfactory sensory neurons. we generated olfactory sphere (os) cells in cultures that were derived from adult rat olfactory mucosa. fluorescence-activated cell sorting and immunofluorescence analyses showed that os cells were derived from hbcs. os cells underwent neuronal and glial differentiation in vitro. to examine multipotent differentiation in vivo, os cells were transplanted into injured rat spinal cords. the transplanted cells integrated into host tissue and underwent glial differentiation. our data showed the stem cell properties of hbcs. oligodendrocyte progenitor cells (opcs) comprise the main cycling cell population of the cns parenchyma during the early postnatal period and at adult stages. however, the molecular mechanisms implicated in opc divisions are still by large obscure. with the aims to unveil i) whether opc divisions exploit the same molecular machinery of neuronal precursors, and ii) whether distinct opc subsets exist with different cell division machineries, we focused on a mutant mouse where citron-kinase, a crucial regulator of cytokinesis in neuronal precursors, is germinally ablated (cit-k ko). these mice have severe cns developmental abnormalities, reduction of selected neuronal populations due to apoptosis triggered by defective divisions, display ataxia, epileptic seizures and die by the third postnatal week. interestingly, they were reported to have myelin defects, suggesting that the cit-ko affects oligodendroglial cells. notably, already early after birth we found a 2-fold decrease in opc density in both the cortical grey (gm) and white matter (wm), compared to wild-type mice (wt). here, most opcs were hypertrophic and multinucleated, indicating a cytokinesis failure after nuclear division. at later ages, opcs progressively disappeared from the cortical gm, while in both wm and ventral areas of the telencephalon (i.e. striatum, hypothalamus), uni-and multi-nucleated opcs could be detected, although at lower densities compared to wt. these data suggests opc heterogeneity in both cit-k requirement for cytokinesis and susceptibility to death. however, even where normal uninucleated opcs persisted, we could not find pre-myelinating or myelinating cells, in line with additional differentiation defects. to discriminate the contribution of cell-autonomous vs. environmental factors in differentiation impairment, we performed homochronic grafts of wt fluorescently tagged (gfp1) cells into perinatal cit-ko mice. strikingly, gfp1 opcs invaded the whole cit-k ko brain and actively divided. however, they hardly ever differentiated into more mature cells at difference with cells grafted in the wt, indicating that altered environmental signals contribute to myelination defects in cit-k ko. in conclusion, our results show that cit-k is involved also in glial cell divisions and suggest distinct cit-k requirement and proneness to death of dorsal and ventral opcs. additionally, cit-k ablation results in pervasive nervous tissue alterations affecting opc maturation. further studies will clarify the molecular mechanisms underlying these alterations. knowledge of how these cells act in repair is lacking, partly due to a limited understanding of their behaviour in normal developmental processes. radial glia cells are a unique population of neural progenitors that have recently been suggested to function in neurogenesis in the cerebral cortex. however, many questions still remain regarding their specific role in the developing spinal cord. using an organotypic spinal cord slice culture model and two-photon microscopy we directly visualized radial glial cell behaviour in the developing spinal cord. 400 mm slices of rat embryonic spinal cord (e12-e16) were cultured in order to preserve the 3d cytoarchitecture of the cellular environment. using electroporation, slices were transfected with a brain-lipid binding protein (blbp) driven gfp expression plasmid, which specifically labels endogenous radial glial cells. slice cultures were then imaged using two-photon time-lapse microscopy, allowing for high spatially and temporally resolved 4d imaging. we were able to follow individual gfp expressing radial glial cells over time (24 hours to 6 days) and characterized precise morphological changes. at early developmental ages, gfp expressing cells initially exhibited a radial morphology with the soma located in the ventricular zone and processes extending out to the pial surface. over time, many cells developed a multipolar morphology and migrated away from the lumen, primarily using somal translocation. using immunohistochemistry we identified populations of transfected cells which also expressed gfap, representing the differentiation of blbp-expressing radial glia cells into astrocytes. we also found that radial glial cells differentiate into oligodendrocytes (olig2 and cnpase positive) but found little evidence for gfp co-localization with neuronal markers (neun, dcx, biiitubulin). using this model we can directly observe complex developmental behaviours of specific cell populations and elucidate the relevant genetic regulation and molecular mechanisms that govern spinal cord development. by understanding these intricate events, we will gain insight into how a simple tube of undifferentiated neuroepithelial cells generates different cell populations that will eventually develop into the adult spinal cord. methods: we used the new cre-expressing mouse strain cx 3 cr1 creer to express a diphtheria toxin receptor (dtr) specifically on microglia/macrophages. in these mice, administration of tamoxifen leads to cre-mediated excision of a loxp flanked transcriptional stop element in both macrophages and microglia, thus allowing for the expression of the dtr as well as an yfp reporter. due to their fast turnover macrophages are replaced by unaffected precursors whereas microglia persist in the modified stage. these mice were used for in vivo and ex vivo analysis of microglia by facs and histology. results: our optimized protocol with two tamoxifen injections at the age of 2 weeks resulted in 80-90% of reporter-positive microglia in adulthood, whereas all peripheral macrophages were reporter-negative. with additional three successive dt injections we could achieve a microglia ablation efficiency of 80%. interestingly, histological as well as facs analysis revealed 50% of microglia repopulation already 4 days after depletion. two weeks after depletion microglia numbers were even higher compared to unaffected controls. on day 6, we found a cd45.2 high ly6c 1 population, indicating replenishment by peripheral monocytes. in bm chimera experiments, where peripheral cells were labeled with a congenic marker, we obtained striking evidence showing that almost all of the repopulating cells are of peripheral origin. conclusions: after depletion microglia were rapidly repopulated, which emphasizes their critical role in brain homeostasis and function. even though ms has an onset in young patients, it persists throughout life and progresses along adulthood and aging. therefore, mscs from aged individuals should be tested for their pro-oligodendrogenic activity before moving into the clinical trials. this is insofar of clinical relevance, since the brain's capacity for self-repair and remyelination strongly declines with aging. thus, the aim of this study was to determine whether mscs from aged individuals maintain their known prooligodendrogenic activity. mscs were obtained from bone marrow of young (2 months) as well as old (17-20 months) rats and analyzed their stem cell properties as well as their pro-oligodendrogenic activity. as expected, mscs derived from aged bone marrow presented diminished stem cell properties and differentiation capacity. to test the msc prooligodendrogenic activity, npcs were incubated with msc conditioned medium. surprisingly, soluble factors derived from old rats mscs maintain their ability to induce an increase of mbp-expressing oligodendrocytes on npcs. moreover, aged mscs conserve their oligodendrogenic activity also on npcs derived from old donors (20 months). these results indicate that mscs keep their oligodendrogenic activity regardless of age and, therefore, this study provides a rationale for clinical trials of autologous msc transplantation in ms therapy. 6 munich cluster for systems neurology (synergy), munich, germany astrocytes fulfill essential functions under physiological conditions and are involved in various beneficial and adverse functions after brain injury (reviewed by sofroniew 2009). in order to improve functional recovery after injury, it is essential to delineate the beneficial from adverse effects. however, little is yet to know which extent distinct sets of astrocytes perform distinct functions or whether all astrocytes behave in a uniform manner. live imaging of astrocytes after stab wound injury in the adult cerebral cortex in vivo revealed a striking heterogeneity of astrocyte behaviour with distinct subsets extending long processes towards the site of injury, others proliferating and yet other subtypes undergoing only hypertrophy. most strikingly, the vast majority of astrocytes proliferating were located with their somata directly at blood vessels (capillaries or post-capillary vessels). this close proximity was confirmed at ultrastructural level and identified as juxtavascular positions, sometimes adjacent to pericytes located in the perivascular space. this novel population of reactive astrocytes at juxtavascular positions is of particular interest, as the capacity of astrocytes to reactivate proliferation ( cell stem cell, in press). therefore we examined whether this population of juxtavascular astrocytes would also preferentially proliferate in even more severe injury conditions, such as 1 hour of middle cerebral artery occlusion (mcao). the proliferation of juxtavasular astrocyte was analyzed immunohistochemically in fixed brain sections co-stained for s100b/gfap/ki67/cd31 at different time points after injury. at 3 days post injury, 18% of all astrocytes were actively dividing (ki671). strikingly the majority of these was again located at juxtavascular positions (68%), suggesting a general bias of astrocytes at this position to proliferate after injury, while astrocytes further distant from blood vessels virtually fail to divide. we shall further present data from conditional knock-out mice with reduced proliferative activity of juxtavascular astrocytes in order to elucidate the function of this highly specific astrocyte population and their proliferative reaction after injury. a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom the generation of oligodendrocytes (ols) from their precursors (opcs) occurs throughout adult life and is particularly important following demyelinating insults, such as occurs in multiple sclerosis (ms). glycogen synthase kinase 3-b (gsk3b) acts as a constitutively active negative regulator of multiple pathways that regulate proliferation, survival and differentiation of opcs. lithium is a potent inhibitor of gsk-3 and is used for the treatment of bipolar disorder, but in addition is neuroprotective in a wide range of diseases, including stroke, amyotrophic lateral sclerosis, and alzheimer's disease. lithium inhibits gsk-3 by binding directly to the enzyme's magnesium-sensitive site and indirectly by enhancing its phosphorylation. notably, by inhibiting gsk3b, lithium acts as a wnt mimetic to promote b-catenin-dependent transcriptional events, including enhancement of genes involved in apoptotic inhibition. here, we have examined the effects of lithium on oligodendrocytes in the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act (1986) . transgenic mice aged postnatal day (p) in the developing and adult cns multipotent neural stem cells reside in distinct niches. specific carbohydrates and glycoproteins are expressed in these niche microenvironments that are important regulators of cell fate determination and stem cell maintenance. in previous studies we described lewisx (lex) glycan as a specific marker of neural stem precursor cells (nspcs) in developing brain and showed it to be involved in nspcs migration and proliferation. using lex glycosylation as a biomarker we aimed to identify the lex carrier glycoproteins which could have a functional relevance for cns development. in addition to already known lex carriers we have found lrp1 as new major lex carrier and for the first time showed it's expression in nspcs and developing brain. lrp1 is essential for the normal neuronal function in adult cns, whereas the role of lrp1 in development is still unclear, mainly due to the lethality of the lrp1 knock-out in mice. in the current study we investigated the basic properties of lrp1 knock-out nspcs created by means of cre-loxp mediated recombination. the elimination of lrp1 in vitro was induced by the addition of cell permeable cre-recombinase to nspcs derived from embryonic brain of lrp1flox/flox mice. the functional status of lrp1-deficient cells was subsequently studied using proliferation, migration and differentiation assays. lrp1 knock-out cells maintained as neurospheres, retained the ability to migrate and differentiate. interestingly, lrp1 knock-out nspcs generated 3-times less opcs in comparison to lrp1wt/wt cells. this suggests that lrp1 is involved in the control of the oligodendroglial differentiation. astrocytes are multifaceted cells in the adult central nervous system and react to pathology of the adult brain with a finely graded continuum of morphological and behavioral changes. given that astrocytes in healthy adult brain rarely divide outside the stem cell niches, but activate both proliferation as well as a stem cell potential after injury (for review see robel et al., 2011) , it is important to understand their exact mode of cell division. indeed, a defining hallmark of a stem cell is the capacity to self-renew -often by asymmetric cell division. therefore we set out to image reactive astrocyte divisions in vitro by developing a primary culture system for reactive astrocytes obtained from the somatosensory cortex of adult mouse at 5 days after stab wound injury. continuous live imaging and single cell tracking (costa et al., 2008 (costa et al., , 2011 allowed us observing the mode of cell division mode as well the cell fate of their immediate progeny. results of our study reveal (i) that reactive astrocytes can undergo multiple rounds of cell division, (ii) the shh signaling has a profound influence on this behavior, (iii) reactive astrocytes divide with distinct modes of cell division, which can be influenced by the local microenvironment. taken together, this new culture system allows close examination of signals on the mode and multitude of reactive astrocyte cell divisions and provides a great tool to identify signals derived from the extracellular matrix, other cell types or as diffusible signals. interestingly, even without injury temporary depletion of proliferating opcs leads to behavioral deficits which will be described. to identify the molecular mechanisms by which ng2 1 cells may react and influence other cells after brain injury, we isolated sox10-gfp 1 cells by facs either from the intact or the lesioned cerebral cortex at three days after stab wound injury. comparative genome-wide expression profiling revealed exciting candidates for opc function in the physiological and pathological brain. oligodendrocytes are the myelin forming cells of the central nervous system (cns) that enable fast salutatory signalling transduction and mediate trophic support and protection of axons. oligodendrocytes originate from a population of precursors cells (oligodendrocyte precursor cells, opcs) that are formed at distinct developmental stages. the biological process leading to opc differentiation involves a cascade of molecular events that eventually constitute the complex and multifaceted cellular program that determines the generation of mature oligodendrocytes. to identify the earliest detectable transcriptional correlates of differentiation we used a microarray approach investigating changes in gene expression occurring in primary rat opcs within the first 12 hours after purification. amongst the most regulated factors that were detected was hairless (hr), a nuclear receptor co-repressor that has been associated with thyroid hormone signalling in the neonatal cns. validation of the microarray results by rt-qpcr, western blot and immunochemistry confirmed a rapid increase of hr expression during opc differentiation in media containing thyroid hormone. in the absence of thyroid hormone, hr expression was suppressed and associated with impaired opc differentiation. similarly, rnai mediated gene silencing of hr induced an impairment of opc differentiation demonstrating an important functional role of hr in the differentiation program. thyroid hormones regulate hr expression via the thyroid hormone receptors thra and thrb as rnai-mediated gene silencing of each of the receptors resulted in a decrease in hr expression. hr is known to negatively regulate rar-related orphan receptor alpha (rora) and vitamin d receptor (vdr). however, rnai-mediated gene silencing of rora and vdr did not change the course of opc differentiation. interestingly, an interaction between hr and histone de-acetylases (hdacs), of which hdac1 and 2 are known regulators of opc differentiation, has been noted in the past. using an in situ protein-protein interaction assay we were able to demonstrate a direct interaction of hr with hdac1 and hdac2 in differentiating opcs. moreover, chip qpcr analysis revealed hr-hdac1 recruitment to the opc differentiation inhibitor hes5. taken together these data show that thyroid hormone promotes opc differentiation via hr by modulating hdac1 and 2 function. as iodine deficiency results in hypothyriodism our results give an example of a developmental disease that is triggered by environmental factors (the absence of iodine) causing epigenetic dysregulation. c. stagni, a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom astrocytes respond to all forms of cns insults by reactive astrogliosis, which involves changes in their molecular expression and morphology, and in most severe cases glial scar formation. reactive astrogliosis is potentially neuroprotective, but can also act as chemical and physical barrier to axon growth and repair. however, mechanisms underlying this process are still poorly understood. the enzyme glycogen synthase kinase 3-b (gsk-3b) is a key regulator of many signalling pathways involved in cell proliferation, survival and differentiation, including wnt signalling. lithium is a potent inhibitor of gsk-3 and acts as a wnt mimetic to promote b-catenin-dependent transcriptional events. wnt signalling is altered following cns injury and in a range of neurodegenerative diseases, such as stroke, amyotrophic lateral sclerosis, and alzheimer's disease. notably, lithium is neuroprotective in these same diseases. here, we have examined the effects of wnt3a and lithium on astrocytes of the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act (1986). we used transgenic mice aged postnatal day (p) 35 inwhich gfap drives the expression of egfp. optic nerves (ons) were isolated with the retina intact and maintained in organotypic culture for 3 days in vitro (div), in control medium, or medium containing lithium chloride, or wnt3a. both agents resulted in a significant increase in the number of astrocytes, compared to controls (p < 0.05, unrelated t-tests), but lithium and wnts3a had markedly divergent effects on astrocyte morphology. in the presence of lithium, astrocytes became highly polarised, extending long thin processes that traversed the nerve perpendicularly to the long axis. in contrast, wnt3a treatment resulted in the generation of morphologically simple astrocytes, with short, fine branching processes that extended radially from a centrally located cell body. genome wide microarray analysis indicated wnt3a and lithium differentially altered chondroitin sulphate proteoglycans (cspgs), a family of extracellular matrix molecules highly expressed at cns injury sites. further experiments are required to define the molecular phenotypes of the novel astrocytes generated by these treatments, but the results provide evidence that wnt signalling regulates astrogenesis in situ and is therefore a potential molecular target to modulate astrogliosis. the contrasting effects of lithium suggest it is acting via other pathways, which require further study. studies of mouse schwann cell culture. the proliferation of scs is regulated by axonal signals and several growth factors. growth factors that are essential for rodent sc survival in culture include heregulin-b1 and forskolin. in the case of rat scs, these factors are also sufficient to allow a robust proliferation. in contrast, the proliferation of mousederived scs in culture requires the presence of other growth factors. we studied effect of several factors known to be expressed in scs after nerve injury on mouse sc proliferation. these included fibroblast growth factor type 2 (fgf), pituitary extract (pe), epidermal growth factor, platelet-derived growth factor bb, and transforming growth factor b1. we found that heregulin-b1 and forskolin are essential for mouse sc to survive in culture, fgf and pe were the best combination of growth factors promoting the mouse sc proliferation, and that neuronal axons provide effective mitogenic environment for mouse scs. the expression of the large myelin-associated glycoprotein in pns was studied in scs cultured alone and in cocultures of scs and drgns. when schwann cells are alone without axons, expression of the cytoplasmic domain of l-mag is seen in the nucleus of schwann cells. when schwann cells make contact with neuronal axons, the location of the expression shifts out of the nucleus to the perinuclear and cytoplasmic regions; the extracellular domain is not visible. until the schwann cells initiate the myelination programme. these results support the hypothesis that l-mag might have role in the regulation of the myelination programme in the pns. it has been postulated that trophic support provided by either stem cells or precursors is actuallyone of the most beneficial effects in the cell-based therapies. to address this issue, a comparison between the neuroprotective properties of opcs and the stem cells derived from warton jelly was carried out in the co-culture system with ischemically injured organotypic hippocampal slices. those two cell types were selected in respect of the advancement in their differentiation process. while the mesenchymal stem cells residing in warthon jelly are still pluripotent, the opcs are already the glia-committed precursors. to evaluate their neuroprotective properties, we set -up an ex vivo co-culture system of either stem cells or opcs isolated from neonatal rat brain with organotypic hippocampal slices subjected to a short oxygenglucose deprivation (ogd). the cell death, as well as the number of the newborn cells has been quantified in slices co-cultured for one week. a microarray analysis of a broad spectrum of trophic factors and cytokines expressed by opcs was performed for the purpose of finding the factor(s) contributing to the observed effect. three of them were selected for the subsequent blocking experiments with specificl antibodies to verify their influence on the injured tissue. the results obtained in the study prove that both cell types secrete soluble factors and are potent in exerting the neuroprotective effect in a paracrine-like manner, promoting cell survival and proliferation in damaged hippocampal slices. in conclusion, the observed advantageous qualities of stem cells and oligodendroglia-biased progenitors significantly contribute to anticipating a clinical improvement in cell replacement therapies. ng2 is a type i transmembrane glycoprotein and also known as chondroitin sulphate proteoglycan 4 (cspg4). in the central nervous system ng2-expressing cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes in the developing white matter. for temporally-controlled gene targeting of ng2 glia in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of cre recombinase (creert2) was inserted into the ng2 locus by homologous recombination. here, we investigated the differentiation potential of ng2 glia at different developmental stages of the forebrain. cre recombinase activity was induced at embryonic day 17.5, postnatal day 0 (p0), p3, p8, p30 and p60 by intraperitoneal tamoxifen injections into novel tgh(ng2-creert2) mice crossbred to rosa26-tdtomato and rosa26-eyfp reporter lines that helped to identify ng2 glia and its progeny. recombination and cell identification were determined 10 to 60 days after the first tamoxifen injection. induction of recombination at embryonic stages revealed that embryonic ng21 cells mainly generated more ng2 glia and oligodendrocytes, however, a significant number of astrocytes could be detected as well. recombined cells were predominantly restricted to the ventral brain. in contrast, after tamoxifen injections at p0 and p3 recombined cells were widely found in all brain regions, thereby suggesting the presence of a distinct subpopulation of ng21 cells after birth. interestingly, only very few recombined astrocytes could be found, which are probably derived from few remaining embryonic ng2 glia. starting from p8, ng2 glia stopped generating astrocytes, with the progeny largely restricted to the oligodendrocyte lineage. however, consistently, we found recombined neun1 cells with the morphology of neurons in the ventral cortex after administration of tamoxifen at p8. even more of such cells were present in the whole cortex when cre activity was induced in adult mice. the morphology, the presence of neun immunoreactivity and our electrophysiological characterization that demonstrated bona fide action potentials clearly identify these cells as functional neurons. our results suggest that ng21 cells display a broad differentiation potential that appears to be highly age-dependent during development. brdu-assay) and the number of surviving cells (by counting dapi-stained nuclei). after we had observed, that the choice of the basal medium (neurobasal, rpmi or dmem) exerts a pronounced effect on opc proliferation we here investigated the effect of cell density on opc proliferation. starting from a small seeding density (10.000 cells/cm 2 ) a tenfold increase in seeding density only resulted in a fourfold augmentation of the density of surviving cells after 7 days in culture. using plating densities of 5.000; 20.000 and 80.000 cells/cm 2 we observed, that an increase in seeding density led to a decrease in the proliferation rate, as tested with a brduassay and monitored after 3 days in culture. similarly, the percentage of a2b5-positive, immature, cells was lower, the higher the cell density. we then tested whether this finding is due to proliferation inhibiting factors secreted in cultures with high seeding densities and whether the remaining microglia population could be involved. to this aim we seeded opcs at low density (5.000 cells/cm 2 ) and cultivated them with the medium supernatant of cultures with high density (80.000 cells/ cm 2 ). furthermore we seeded opcs in low density (5.000 cells/cm 2 ) and co-cultivated them with an added density of microglia as counted in cultures of high density (80.000 cells/cm 2 ). the resulting cell counts after 3 days showed, that the percentage of brdu positive cells after cultivation with the supernatant was significantly reduced compared with control cultures, although the number of a2b5-positive cells was not influenced by this treatment. the addition of microglia to the cultures lead to a strong reduction of surviving cells, even though the percentage of brdu and a2b5 positive cells were not altered. so apoptosis, necrosis or phagocytosis might be responsible for the reduction of surviving cells. our findings suggest that the reduction of surviving opcs in high density cultures is due to secreted factors inhibiting opc proliferation and that microglia could be involved in this effect. in neuropathological studies, it is important to detect microglial cells; however, a specific microglia marker has yet to be determined. [methods] in this study, we examined and compared the usefulness of various microglia markers, including human glucose transporter-5 (glut-5), mhc class ii, cd68, and iba-1. [results] all of the microglia markers consistently stained for microglia in paraffin sections fixed with formalin. cd68 staining of microglia in the gray matter was not as well defined as the other microglia markers. moreover, cd68 did not clearly stain the microglial processes. with regard to mhc class ii, it was difficult to observe the microglial processes in the white matter. in contrast, both glut-5 and iba-1 clearly stained for microglia in the gray and white matter, and provided detailed staining of microglial processes. although it has previously been shown that perivascular cells are negative for glut-5, we found perivascular macrophages to be glut-5 positive. [conclusions] nevertheless, glut-5 and iba-1 may be considered as markers suitable for routine histopathological staining procedures. neurons and glia of the enteric nervous system (ens) originate from a pool of sox101 progenitors. in addition to being a scaffold for enteric neurons, enteric glial cells (egcs) have been suggested to function in mucosal integrity, neuroprotection, adult neurogenesis, neuro-immune interactions and synaptic transmission. currently, diverse glial populations are known to reside within the ens. however, whether, specific functions are carried out by specialized glial subtypes, with characteristic morphologies and molecular markers and within specific locations in the gut remains elusive. to address this question we used a repertoire of genetic tools, immunostaining and imaging techniques. for high single-cell resolution study of egcs, we mosaic analysis with double markers (madm) in conjunction with sox10-cre. we analyzed adult gut tissue to characterize the different glial populations based on morphology, position in the ens and their relationship with the intestinal epithelial and vascular system. moreover, we used reporter mice (sox10-icreer t2 ; r26r-yfp and gfap-icreer t2 ; r26r-yfp) to quantify the expression of three glial markers -gfap, s100b and sox10 in myenteric plexus (mp) preparations of adult mice. our investigation on madm-mp preparations, revealed three subtypes of enteric glia in the mp of adult mice. type i and type ii glia, present in the primary plexus of mouse ens have been previously defined. in addition, we characterized a third subtype in the extraganglionic space of the mp of adult mice, at present termed as type iii-extraganglionic glia. furthermore, our analyses using 3d-imaging, on madm-gut sections allowed us to characterize the relationship of enteric glia located within the mucosa, with the intestinal epithelium and vascular system of the villi. our marker expression analysis showed that while the majority of glial cells co-expressed gfap and s100b, a large fraction of glia within the myenteric ganglia was positive for either s100b ($30%) or gfap ($10%). we also found that s100b 1 /gfapglia were abundant outside the ganglia. most glial cells co-expressed sox10 with gfap and s100b yet a significant proportion ($10%) of mainly extra-ganglionic glia (type iii) expressed only sox10. surprisingly, $5% of the cells that were s100band sox10displayed high gfap expression. use of gfap-icreer t2 ; r26r-yfp mice showed that about $30% of yfp-labelled cells did not express gfap, implying dynamic regulation of gfap expression. overall, our high resolution studies reveal extensive heterogeneity of enteric glial cells in terms of morphology, position and expression of molecular markers suggesting differential functional roles. our future experiments will address whether different physiological roles of egcs can be assigned to specific subtypes. university of washington, seattle, united states m€ uller glia are the major type of glia in the retina, spanning its entire thickness. it is known that bmp signaling promotes astroglial differentiation in cortex, but its role in m€ uller glial development in the retina has not been studied. the purpose of this study was to investigate the role of bmp-smad1/5/8 signaling in m€ uller glial development. c57bl/6 mouse retinas were collected at various ages between postnatal day 0 (p0) and p21 in order to analyze the expression and activation of smad1/5/8. smad1/5/8 was transiently but robustly activated in the inner nuclear layer, including developing m€ uller glia between p5-p8. the timing of this transient activation of smad1/5/8 corresponds with the timing of m€ uller glial differentiation and maturation. to test the hypothesis that activation of bmp-smad1/5/8 signaling is essential for m€ uller glial development, retinas were explanted at p3 or p6 and treated with bmp4 or a bmp inhibitor dorsomorphin (dm) for 5 days in order to activate or inhibit smad1/5/8, respectively. when p3 and p6 retinal explants were treated with dm for 5 days in vitro, there was a significant reduction in m€ uller glial markers (cralbp, gs, sox9, and sox2). smad1/5/8 activation and cralbp expression were also significantly attenuated in vivo 1 day after intraocular injection of a bmp inhibitor dm at p5. our data suggest that activation of bmp-smad1/5/8 signaling is necessary for the development of m€ uller glia. this work was supported by 1r01ey021482 to tar. huazhong university of science and technology, wuhan, china rho-associated kinase (rock) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. although its effects on astrocyte proliferation have not been well characterized, rock has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. in the present study, our aim was to investigate the effect of rock inhibition by [(1)-(r)trans-4-(1-aminoethyl)-n-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] (y27632) on proliferation and dna synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. western blots showed that treatment of astrocytes with y27632 increased their expression of cyclin d1, cdk4, and cyclin e, thereby causing cell cycle progression. furthermore, y27632-induced astrocyte proliferation was mediated through the extracellular-signalregulated kinase signaling cascade. these results indicate the importance of rock in astrocyte proliferation. here we used cell transplantation experiments to determine to which extent this differential behavior is regulated by environmental signals differing between the wm and gm, or intrinsic fate determinants restricted to one or the other cell population. these experiments indicate that the wm promotes differentiation of opcs to mature oligodendrocytes, as the rate of differentiation of gm cells increased when exposed to this environment. interestingly, cells derived from the wm retained their higher differentiation rate also in a less supportive environment like the gm. these results reveal not only important environmental differences for opc maturation, but also support the concept of intrinsic heterogeneity among adult opcs, persisting even when exposed to more inhibitory environments. thus, this work provides the basis to identify molecular pathways regulated by distinct niche/environmental signals and involved in the heterogeneity of adult opcs. multiple sclerosis (ms) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system (cns) characterized by inflammation, which leads to formation of demyelinating areas due to loss of oligodendrocytes, astrogliosis and, finally, axonal degeneration. different studies have suggested that 3 0 -5 0 -cyclic adenosine monophosphate (camp) levels might play an important role in neuroprotection and neuroinflammatory response so the modulation of this nucleotide intracellular levels might control the neuroinflammatory pathological process and, consequently, to delay the progression of ms. intracellular camp levels depend on its synthesis by adenylyl cyclases, and on its degradation by cyclic nucleotide 3 0 ,5 0 -phosphodiesterases (pdes). specific inhibitors for the different isoforms of pdes family, particularly camp-specific pde, emerge as putative treatments of this kind of disease. pde7 has emerged as a new therapeutic target, not only for a variety of immunological and immunodeficiency conditions to alleviate chronic inflammation, but also for several neurodegenerative disorders, including ms, in which both immune system and cns are implicated. in the present work, we have detected the expression of pde7 in oligodendrocyte precursor cells (opcs) isolated from cerebral cortex of p0 and p15 mice and from adult human samples derived from neurosurgery of epilepsy. opcs are abundant in the mice and human cns during development but they also represent 5-7% of the total number of cells in the adult cns, where they serve as a source of new oligodendrocytes to replace those which die. however, in ms these endogenous opcs are not able to effectively replace dead oligodendrocytes. in vitro, we have demonstrated that two newly-developed pde7 selective inhibitors (tc 3.6 and vp 1.15) favour opc survival and differentiation towards mature oligodendrocytes, being both more effective than the commercial pde7 inhibitor brl-50481. erk intracellular pathway has been identified as a key in these cellular processes related with the camp/pka/creb pathway. moreover, we have observed that both specific inhibitors (vp1.15 and tc3.6) increase the differentiation of human opcs. these findings, combined with the already known anti-inflammatory effect carried out by these pde7 inhibitors, point them as potential therapeutic agents to treat ms. the knowledge of factors that affect the biology of murine opcs and even more, human opcs, are critical for possible remyelination in this kind of pathologies. their role in cns remyelination has not been fully investigated. previous studies using genetic fate mapping techniques revealed their recruitment into injured spinal cord and differentiation into astrocytes and oligodendrocytes. in this study, we used aninducible cre-floxed-stop-rosa26-yfp mediated lineage tracing strategy to characterize the fate of foxj1 expressing ependymal cells during remyelination of toxin-induced demyelination. we found that in unlesioned spinal cord, foxj1 labelled ependymal cells. however, it also labeled a population of cells in the spinal roots with immnochemical features of fibroblasts. following lysolecithin-induced demyelination in the dorsal funiculus, there were few yfp expressing cells in the lesion area at 5 and 14 days post lesion (dpl) indicating that the ependymal cells were not recruited in demyelinated area. however, in ventral white matter lesions, which were adjacent to damaged nerve roots, many cells were detected expressing yfp. the distribution of yfp positive cells overlapped that of remyelinating schwann cells and a number of yfp expressing cells colocalised with mature schwann cell marker periaxin. these data revealed a foxj1 expressing population in peripheral nerve which migrates into demyelinated lesions andcontributes to cns remyelinaiton. the generation of myelinating cells from multipotential neural stem cells in the cns requires the initiation of specific gene expression programs in oligodendrocytes (ols). we reasoned that micrornas (mirnas) could play an important role in this process by regulating genes crucial for ol development. here we identified mir-7a as one of the highly enriched mirnas in oligodendrocyte precursor cells (opcs), overexpression of which in either neural progenitor cells (npcs) or embryonic mouse cortex promoted the generation of ol lineage cells. blocking the function of mir-7a in differentiating npcs led to a reduction in ol number and an expansion of neuronal populations simultaneously. we also found that overexpression of this mirna in purified opc cultures promoted cell proliferation and inhibited further maturation. in addition, mir-7a might exert the effects just mentioned partially by directly repressing proneuronal differentiation factors including pax6 and neurod4, or prool genes involved in oligodendrocyte maturation. these results suggest that mirna pathway is essential in determining cell fate commitment for ols and thus providing a new strategy for modulating this process in ol loss diseases. a. guzman de la fuente 1 , j. huang 1 new sheaths can be regenerated following the recruitment of adult neural progenitor cells (opc) into areas of injury and their differentiation into myelinating oligodendrocytes. although this regenerative process (called remyelination) occurs efficiently in the early stages of multiple sclerosis, its efficiency gradually declines leaving axons demyelinated and vulnerable to degeneration. several signalling pathways are involved in regulating opc differentiation and subsequent myelination. understanding their mechanisms is necessary to design regenerative therapies to overcome remyelination failure. we have identified rxrc as a key positive regulator of opc differentiation and remyelination (huang et al., 2011). rxrc is a nuclear receptor forms a heterodimer with other nuclear receptors to activate its downstream signalling cascades. here we describe rxrc binding partners expressed in both opc and oligodendrocytes, and the binding of vdr, rarb and pparc to rxrc within oligodendrocyte lineage cells by coimmunoprecipitation. vdr-rxrc heterodimer is necessary for the oligodendrocyte differentiation. treatment of oligodendrocytes with vdr antagonist inhibits opc differentiation and abrogates the effect of 9-cis retinoic acid, an rxrc agonist, in opc differentiation. our data suggests a model in which rxrc is an essential anchor for different nuclear receptors, including vdr, with a shifting and complex rxrc signaling in oligodendrocyte lineage cells. cleavage of type i membrane proteins by a-and c-secretases to yield biologically active fragments is best exemplified for the evolutionarily conserved protein notch. here we show that the protein ng2, a type i membrane protein which has homologues in mouse (ng2) and drosophila (kontiki) and is expressed by multiple immature cell types including glia, is processed in a similar fashion to notch. in both mouse and drosophila this generates a major 290 kd shedded ectodomain, a c-terminal fragment (ctf) and a small intracellular domain (icd). the ng2 icd is present after overexpression in cells of biochemical isolates of nuclear fractions, and can be visualized in the nucleus by immunofluorescence of cultured murine glial cells. in glial nuclei of developing drosophila larva, a striking intranuclear staining of the kontiki icd is observed. levels of expression of ng2 mrna and protein in mouse glial cells are regulated by the neurotransmitter glutamate and overexpression of cleaved fragments modulates protein expression in murine glial cells. these results demonstrate that cleavage of ng2 takes place in mouse and drosophila glia and that this generates fragments with signaling properties. modulation of ng2 expression by glutamate provides a way for neuronal activity to regulate ng2 signalling. our goal is to understand the glial signaling that controls inflammatory responses in the central nervous system (cns). this glial response can play both a detrimental and beneficial role. toll like receptor (tlr) signaling is implicated in responses to pathogens or endogenous signals. tlr signaling mediates immune response by inducing cytokines, including type i interferons (ifn). ifn-beta, a member of the type i ifn family, is a first-line therapeutic for multiple sclerosis. type i ifns signal through a common receptor, ifnar. the aim of the present study was to investigate the in vivo response of microglia and astrocytes to cns administration of tlr ligand/agonist, and to examine whether this response involves type i ifn signaling. mice were administered tlr ligands/agonists by injection to the cisterna magna. we analyzed leukocyte entry to the cns by flow cytometry, and their localization by immunostaining. the induction of ifn-beta was examined by in vivo imaging of an ifn-beta reporter mouse. astrocytes and microglia were sorted by facs and gene expression was measured using quantitative real time-pcr. injection of ligands/agonists for tlr2, 3 and 4 resulted in infiltration of cd451 leukocytes after 18 hrs, most strongly by tlr2. immunostaining showed parenchymal localization of infiltrating cells in cerebellum. facs sorted astrocytes expressed equivalent levels of tlr3 mrna to microglia but lower levels of tlr2 or 4. astrocytes were induced by tlr3 signaling to express interferon regulatory factor 7, which regulates the induction of type i ifn. the induction of ifnbeta in cns in response to tlr3 signaling was verified in ifn-beta reporter mice. tlr2, 3 and 4 signaling led to increased levels of mrna for glial cxcl10. together these results suggest the involvement of type i ifn signaling. however, unlike cxcl10 gene expression, that was dependent on ifnar signaling, tlr2-induced leukocyte infiltration was not affected in ifnar deficient mice. these studies point to a role for tlr signaling in the innate glial response that regulates cns inflammation. microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. here we investigated the regulatory function of the signaling protein phosphoinositide 3-kinasec (pi3kc) in phagocytosis of bacteria and zymosan particles by mouse brain microglia in vitro and in vivo. using genetic and pharmacological approaches our data revealed pi3kc as an essential mediator of microglial phagocytosis. unexpectedly, microglia expressing lipid kinase deficient mutant pi3kc exhibited similar phagocytosis as wild-type cells. detailed analysis disclosed pi3kc dependent stimulation of camp phosphodiesterase as a crucial mediator of phagocytosis. both stimulation of protein kinase a and epac were shown to convey inhibitory effects of camp on phagocytosis. together our findings indicate pi3kc-dependent suppression of camp signaling as an indispensible regulatory element of microglial phagocytosis. v. kovaleva, e. berezhnaya, m. rudkovskii, a. uzdensky southern federal university, rostov-on-don, russian federation photodynamic therapy (pdt) is currently used in oncology, particularly, in treatment of brain tumors. the possible role of no-mediated signaling in photodynamic injury and protection of neurons and surrounding glial cells (gc) was studied. crayfish stretch receptor consisting of a single neuron enveloped by gc was photosensitized with alumophthalocyanine photosens (10 nm, 30 min incubation) and irradiated with laser diode (670 nm, 0.4 w/cm2). application of no generators notably 10 mm sodium nitroprusside and 10 mm nonoate reduced pdtinduced necrosis of gc and showed the same tendency in neuronal necrosis. nonoate significantly increased pdt-induced apoptosis of gc. inhibitor of neuronal no-synthase l-name (1mm) significantly increased the percentage of necrotic glial cells after pdt but did not influence neuronal necrosis. this confirmed the anti-necrotic effect of no in glial cells and involvement of neuronal no synthase in protection of glial cells. 1 mm l-name and 1 mm l-nna (another inhibitor of neuronal no-synthase) as well as 50 lm s-methhylisothioharnstoff sulfat (inhibitor of inducible no synthase) protected gc from pdtinduced apoptosis. therefore no may be involved in pdt-induced apoptosis of glial cells. inhibition of no-activated protein kinase g with 10 lm kt5823 decreased the percentage of necrotic glial cells but not neurons. therefore protein kinase g appeared to be involved in pdtinduced necrosis of gc, possibly independently on no. kt5823 also increased the level of apoptosis of gc indicating the anti-apoptotic role of protein kinase g. thus no is involved in regulation of pdt-induced necrosis of neurons and glial cells as well as in apoptosis of glial cells. g. tyzack, t. cymes, n. lau, a. lakatos university of cambridge, cambridge, united kingdom background and question. emerging evidence suggests that signalling by damaged neurones leads to reactive astrocyte response that may have a fundamental impact on synaptic reorganisation. soluble factors, such as cytokines, have been described to induce such astrocyte activation. however, rapid contact dependent signalling between damaged neurones and astrocytes is also a possibility. in light of recent findings of ephb1 upregulation on injured neurons closely surrounded by reactive astrocytes, we hypothesized that ephb1 can signal to ephrin-b1 expressing astrocytes. methods. to test this in simplified in vitro assays, purified astrocyte cultures were treated with clustered-ephb1-fc, and the characteristic features of glial activation, such as cytoskeletal protein expression, stat3 phosphorylation, and p-stat3 nuclear transfer were monitored. results. we have demonstrated that 1) astrocytes express ephrin-b1, 2) ephb1 triggers a two-fold increase in gfap and ezrin expression, 3) ephb1 induces both stat3 phosphorylation and nuclear transfer by a three-fold value, and 4) all effects were significantly diminished by knocking down ephrin-b1 in astrocytes. conclusions. our data show that ephb1 induces astrocyte activation by triggering ephrinb1-mediated reverse signalling via stat3 activation. this work therefore suggests a potential novel route in neuron-astrocyte communication after injury. a further characterisation of this signalling pathway may provide a better understanding of mechanisms underlying glial activation and its role in plasticity. n. kramann 1 , r. pf€ ortner 1 , u.-k. hanisch 1 , k. hagemeier 2 , t. kuhlmann 2 , w. br€ uck 1 , c. wegner 1 1 university medical center g€ ottingen, department of neuropathology, g€ ottingen, germany 2 university hospital m€ unster, institute of neuropathology, m€ unster, germany introduction: laquinimod (laq) is an oral well-tolerated molecule that has been shown to reduce brain atrophy, disability progression and relapse rate in patients with relapsing-remitting multiple sclerosis. recent experimental data indicate that laq minimizes demyelination, inflammation and axonal damage in mice with cuprizone challenge. aim: since astrocytic nfjb activation plays a crucial role in cuprizone-induced demyelination, we investigated the effects of laq on cns cells in vitro and in vivo. methods: in vitro experiments used primary cells to test effects of laq on oligodendroglial survival as well as on cytokine secretion and nfjb activation in astrocytes and microglia. primary mouse astrocytes and microglia were pre-treated with 0, 0.25 and 2.5 mm laq and stimulated by pro-inflammatory cytokines. a dual-luciferase reporter assay was used to measure nfjb activity. for in vivo experiments, 10-week-old male c57bl/6j mice were challenged with 0.25% cuprizone and treated daily with laq (25 mg/ kg) or water. to assess astrocytic and microglial nfkb activation in vivo, nuclear translocation of nfjb p65 was assessed in astrocytes and microglia by double immunofluorescence with antibodies against p65 and iba1 or gfap. results: in vitro, astrocytic but not microglial nfjb activation was markedly reduced by laq as evidenced by nfjb reporter assay. in astrocytes, pre-treatment with 0.25 and 2.5 mm laq significantly reduced the induced nfjb activity after tnfa stimulation compared to stimulated controls. pre-treatment with 2.5 mm laq also significantly reduced nfjb activation after stimulation with the combination of il-1b and ifnc compared to untreated stimulated controls. oligodendroglial viability and survival were not affected by laq treatment. to confirm the in vivo relevance of these findings, we also examined astrocytic and microglial p65 translocation in mice with and without laq treatment after cuprizone challenge. the proportion of astrocytes with nuclear p65 immunoreactivity was significantly reduced in laqtreated mice (14.0% 6 0.9%) compared to untreated controls (25.8% 6 1.1%). microglia did not display marked translocation of nfjb p65 after cuprizone challenge. conclusion: our data indicate that laq prevents cuprizoneinduced demyelination by attenuating astrocytic nfjb activation. in vitro, laq reduced the astrocytic nfjb activation by up to 46% as evidenced by nfjb reporter assay. similar quantitative findings were obtained in vivo when laq treatment also led to a 46% reduction of astrocytes with nfjb activation, as evidenced by nuclear translocation of p65. these findings suggest that targeting the astrocytic nfjb pathway might have therapeutic effects in demyelinating cns disorders. . we asked whether brain-derived neurotrophic factor (bdnf), known to be highly expressed in a circadian manner in scn, might be involved in the mechanisms governing these astroglial rearrangements. using an analog-sensitive kinase allele murine model (trkb f616a ) and semi-quantitative electron microscopy, we found that the pharmacological blockade of the tropomyosin-related kinase receptor type b (trkb), the high affinity receptor of bdnf, abolished the day/night changes in glial coverage of vip dendrites. the bdnf/trkb signaling pathway therefore exerts a permissive role on the ultrastructural rearrangements that occur in scn across the day/night cycle. in contrast, the extent of glial coverage of non-vip dendrites was not different at daytime and nighttime in trkb f616a mice submitted to trkb inactivation or not receiving any pharmacological treatment. considering the well-established role of bdnf in the clock's photic synchronization process, these dataprovide strong support to the view that the daily astroglial rearrangements in scn could be involved in the photic entrainment of circadian rhythms. above all, they highly suggest that they are necessary for the modulatory action of bdnf on the scn responses to light. features of this form of neurodegenerative ataxia, including a striking reduction in lifespan when polyq atrophins are expressed in the glia. a sensible hypothesis is that affected glia will act non-autonomously on neurons as in other neurodegenerative pathologies. this is an emerging field that yields new and important therapeutic possibilities. we now wish to elucidate the mechanisms through which degenerating glia impairs organism functions, and identify genes involved cell-autonomously and non-autonomously in generating this aspect of the pathology. first, we investigated the impact of glial polyq atrophin on lifespan. using different glia marker, we have demonstrated that the expression of polyq atrophin in glia, or specifically in astrocyte-like cortex glia, has a dramatic effect on drosophila lifespan while brood brain barrier glia do not have any phenotype. we will then investigate the effect of other subtypes of glia looking at lifespan and motility to identify the relevance of each subtype in drpla. second, to identify new regulators of gliopathology we will conduct unbiased genetic screens for mutations in neurons that modify the lifespan of drpla flies, expressing polyq atrophin within the glia. our studies will be paramount in deciphering the complex glia-neurons interactions in ataxias and may be beneficial for several ataxias. they will open up new avenues for therapies aimed at targeting glianeuron interactions during disease progression. in alzheimer's disease, microglial clearance of beta-amyloid (ab) is considered neuroprotective. whether ab uptake regulates microglial cell responsiveness, and whether macrophages participate, remains unclear. here we investigated whether in vivo phagocytosis of endogenously-produced ab disturbs the turnover of microglia and macrophages by changing rates of cellular proliferation and apoptosis. using app swe /ps1 de9 tg mice, we show that plaque-associated microglia and cd11b 1 cd45 high macrophages bind and ingest endogenously-produced ab in vivo. plaqueassociated microglia and cd45 1 leukocyte-like cells were also observed in brains from patients with alzheimer's disease. phagocytic microglia and macrophages had a reduced rate of proliferation compared to abcells. instead, high proportions of apoptotic annexin v 1 microglia and macrophages were found in aged app/ps1 tg mice. phagocytic microglia and macrophages had greater caspase activity and increased p53 expression after in vivo uptake of ab, than cells that did not phagocytose ab. in vitro, we found that ab uptake induced caspase activation in microglia, whereas blocking ab uptake triggered apoptosis-induced cell death in macrophages. our data demonstrate that ab uptake impairs microglial/ macrophage viability and responsiveness. amyloid clearance may fail as phagocytic microglia and macrophages undergo ab-induced apoptosis. f. michetti, s. ceccariglia, a. d'altocolle, f. pizzolante, m. barba, a. delf a, c. gangitano universit a cattolica del s. cuore, rome, italy trimethyltin (tmt) is a neurotoxicant producing neuronal degeneration in the mammalian central nervous system , especially in the hippocampus (for reviews, 1,2). magnetic resonance imaging (mri) investigation in tmt-treated rats has evidenced dilation of lateral ventricles, possibly correlated to alterations in blood brain barrier permeability. we have investigated in the hippocampus and cortex of rats the expression of aquaporin 4 (aqp4), a glial water channel protein which is regarded to play a role in brain oedematous conditions, to explore the molecular mechanisms involved in the phenomenon. tmt-treated aqp4 expression was tested both by real-time pcr and western blotting analysis in hippocampus and cortex homogenates. to confirm molecular results and visualize the aqp4 cell distribution, double-label immunofluorescence for aqp4 and gfap was performed. real-time pcr and western blotting data show a significant upregulation of aqp4 starting from 14 days of tmt treatment in the hippocampus and, at a lower degree, in the cortex. accordingly, the immunofluorescence shows an intense astrogliosis and aqp4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting from 14 days after intoxication. aqp4 immunolabelling was localized in astrocytic end-feet encircling the blood vessels, as expected. the study of the rhodamine b fluorescent tracer, intraperitoneally administered, also revealed an intense vascular reaction, characterized by hypertrophic vessels with abnormal course and dimensions in the brain of tmt-treated rats, indicating a vascular involvement in the tmt-induced neurodegenerative processes. aqp4 over-expression and the concurrent astrogliosis occurring in the brain of tmt-treated rats might be candidated to play a role in alterations of vascular permeability and brain oedema formation evidenced by mri studies. amyotrophic lateral sclerosis (als) is a neurodegenerative disease affecting predominantly motoneurons but with a particular pathological role of non-neuronal cells. previous research suggested that increased concentration of extracellular potassium could lead to motoneuron dysfunction. the na 1 /k 1 -atpase should regulate the homeostasis of potassium ions and thus help maintain regular neuronal activity. this homeostasis may be regulated by na 1 /k 1 -atpase in both neurons and astrocytes. in order to reveal the possible role of the na 1 /k 1 -atpase in als pathology, we explored the expression of the alpha1 catalytic subunit of na 1 /k 1 -atpase in neurons and astrocytes from different brain regions of the sod1 g93a rat model of als. labeling immunofluorescently the alpha1 isoform of the na 1 /k 1 -atpase revealed a decreased signal in the trigeminal nucleus and cerebellum of the als rat. double immunofluorecence with neun for neurons showed that the reduced alpha1 was on the neuronal surface in the trigeminal nucleus. in addition, the alpha1 ring-like staining of cerebellar granule neurons was lower in als as compared to the wild type rat. in comparison to neurons, astrocytes from the wild type rat were characterized with a lower expression of alpha1. in the examined brain regions we did not observe a prominent difference in alpha1 expression between astrocytes of wild type and als rats. based on the findings of this study the prominent reduction of na 1 /k 1 -atpase level observed in neurons and not in astrocytes could contribute to further understaning of impaired ion homeostasis affecting proper neuronal physiology in als. the role of glial na 1 /k 1 -atpase needs to be further studied with markers of other alpha subunit isoforms. although commonly used as nutritional supplements, excessive intake of bcaas might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of bcaa catabolism such as maple syrup urine disease (msud). recent evidence indicates that bcaas induce excitotoxicity through mechanisms that require the presence of astrocytes. in the present study, we evaluated the effects of bcaas on microglia, the main immune cells of the brain. as an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high bcaa medium (h-bcaa). we show that h-bcaa microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the m2 state, with enhanced il-10 expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. we suggest that such an intermediate m1/m2 phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. in addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which bcaas might turn into toxicants and facilitate neurodegeneration. the proteolipid protein gene (plp1), located on the x-chromosome, undergoes alternative splicing to produce the most abundant proteins of central nervous system myelin: plp and dm20. related to the extent of myelin defect, mutations in the plp1 gene in humans are associated with a spectrum of x-linked disorders, from the severe pelizaeus-merzbacher disease (pmd), to the mild form spastic paraplegia type 2 (spg2). phenotype-genotype correlation exists, large duplications of plp1 gene are predominantly found in pmd and null mutations in spg2. plp is almost 100% conserved across mammalian species, and the large spectrum of phenotype severity is also found plp transgenic mice. experiments on these models have contributed to our understanding in the pathophysiology of plp related disorders. in one hand, mice with extra copies of the plp1 gene (1plptg mice) have neurological symptoms and cns pathology similar to those found in pmd patients while mice with plp1 gene inactivation (plp null mice) share similarities with spg2 patients. then, these two mouse models represent very useful tools to evaluate the efficacy of a therapeutic strategy for the severe and the mild form of the plp related disorders on the neuropathological and behavioral aspects. olesoxime (tro19622), a cholesterol-like small molecule, has been shown to rescue motor neurons from cell death and to promote axonal regeneration both in vitro and in vivo. more recently, olesoxime has also been shown to promote central remyelination by accelerating oligodendrocyte maturation both in vitro and in vivo. altogether, these results strongly suggest that olesoxime may be a potential drug candidate for the treatment of white matter neurodegenerative disorders and notably to plp related disorders. here we treated 1plptg mice with olesoxime for 2 months starting at birth, as well as plp null mice from 3 to 12 months of age (presymptomatic treatment) or from 9 to 15 months of age (symptomatic treatment). behavioral and neuropathological analysis revealed that olesoxime failed to correct the development of symptoms in mice modeling the severe form of plp related disorders. on the contrary, treatment of plp null mice with olesoxime resulted in a 3-month delay in the onset of abnormal behaviors related to anxiety and in the slowing of central nerve conduction. these beneficial effects are observed only when the drug is administered before disease onset. treatment with olesoxime also reduces the effect of aging on rotarod performance decline in both wt and plp null old mice. altogether these results suggest that olesoxime could be of interest as a treatment to delay the onset of symptoms in plp related disorders provided that patients suffer from a mild form of the disease and are treated before the establishment of symptoms. objectives: glial cells play an important role in amyloid-beta (aß) clearance, however little is known about the cellular routes involved in internalization of different aß aggregation forms by adult human microglia and astrocytes. therefore, we evaluated the uptake mechanism of aß 1-42 by primary human adult microglia and astrocytes isolated from brain tissue of non-demented control and ad cases. methods: cells were preincubated for 1 hour with different inhibitors, before exposure to fluorescence labelled (fam) oligomeric and fibrillar forms of synthetic aß. the number of amyloid positive cells was quantified by flow cytometry. inhibitors of various pathways tested, include cytochalasin b (inhibits actin polymerization; general endocytosis inhibitor), nocadozole (inhibits tubulin depolymerization; general endocytosis inhibitor) and fucoidan (scavenger receptor inhibitor). results: cytochalasin b inhibited aß fibril uptake (90% reduction, p < 0.01) and to a lesser extent oligomer (50% reduction, p < 0.05) uptake in microglia, whereas no effect was seen on aß uptake by astrocytes. in contrast, nocodazole was found to inhibit aß fibril uptake (54% reduction, p < 0.05) and oligomer (77% reduction, p < 0.01) uptake in astrocytes, whereas no effect was seen on aß uptake by the microglia. interestingly, fucoidan prevented aß uptake in both astrocytes (oligomers:86% (p < 0.01); fibrils: 79% (p 5 0.01) and microglia (oligomers:67% (p < 0.001); fibrils: 79% (p < 0.05)). conclusions: these data indicate that both human astrocytes and microglia use scavenger receptors to internalize aß oligomers and fibrils. whether different types of scavenger receptors are involved in aß uptake by astrocytes and microglia will be further investigated. tel-aviv university, tel aviv, israel alzheimer's disease (ad) accounts for the majority of cases of dementia worldwide, affecting over 50% of the population over 85. the disease is characterized by a progressive memory loss, cognitive deterioration, behavioral disorders, deposit of beta-amyloid (ab) peptides, neurofibrillary tangles, reactive astrocytosis and activation of microglia cells. mutations in the genes that encode app (amyloid precursor protein) and components of the proteases that generate amyloid beta cause familial forms of ad. microglia, the resident immune cells of the central nervous system (cns) were suggested to play both beneficial and harmful roles in the course of the disease. therefore, modulating the microglial response is being considered as a promising approach for the treatment of this disease. we have recently shown using in vitro and in vivo systems that the ectoenzyme cd38, regulates microglial activation. in view of the significant role of activated microglia in ad and the important role played by cd38 in microglial activation, we hypothesized that cd38 deficiency would affect the course of the disease. in order to test this hypothesis, we implicated the app swe ps1de9 transgenic mouse model for ad, and compared the cognitive performance (as evident by behavioral studies) of aged app swe ps1de9 mice, to app swe ps1de9 cd38 -/mice. in addition, abload, accumulation of microglia and astrocytes was assessed in the brains of these mice. our preliminary results show that cd38 deficiency has a neuroprotective role in this ad mouse model as indicated by improvement in cognitive performance and dramatic reduction in ab load. oxidative stress induced by reactive oxygen species (ros) is associated with various pathological conditions, including aging, neurodegenerative disorders, traumatic and ischemic insults. the mechanism by which the gap junction protein connexin43 (cx43) contributes to oxidative stress-induced cell death or the "rescue" of the dying cells is still unclear. cx43 is highly expressed in astrocytes. we have previously shown that astrocytic cx43 is critical for neuroprotection during ischemic insults in vivo. to investigate the protective effect of cx43 in oxidative stress pathways, we induced oxidative stress in wild-type and cx43 knockout astrocytes using hydrogen peroxide (h 2 o 2 ). cx43 knockout astrocytes exhibited increased h 2 o 2 -induced cell death as measured by the loss of membrane integrity using the dye rhodamine b dextran, supporting a cell protective effect of cx43; this effect was not observed in wild-type astrocytes. to examine the involvement of cx43 in h 2 o 2 -induced cell death, we studied the expression and distribution of cx43 in response to h 2 o 2 in wild-type astrocytes. h 2 o 2 treatment altered the expression level and phosphorylation of cx43. this was accompanied with changes in cx43 distribution and gap junction plaque formation. hemichannel activity was measured using dye uptake. h 2 o 2 (0.7 mm) increased dye uptake, an effect that was blocked by gap26 (syntethic mimetic peptide, 200 mm) or carbenoxolone (100 mm), two connexin hemichannels blockers. however, h 2 o 2 treatment did not result in any measureable gap junction activity in cx43 knockout cells as measured by a scrape-loading dye transfer assay. these findings suggests that gap junction activity contributes to cx43-mediated protection in response to ros. one of the receptors found on microglia is the triggering receptor expressed on myeloid cells 2 (trem2), which signals intracellularly via an adapter molecule referred to as tyro protein tyrosine kinase-binding protein (tyrobp, known also as dap12). in humans, loss of function of either trem2 or tyrobp leads to nasu-hakola disease, which is characterized by neuroinflammation and bone cysts. by using a trem2 knock-out (ko) mouse line, we provided further insights on the trem2 function in neurodegenerative diseases. first, we observed mild signs of dopaminergic neurodegeneration and a slight increase in microglial iba1-immunoreactivity in different brain regions of 3 months trem2 ko mice compared with the wild type (wt) control animals. however, the transcription levels of inflammatory cytokines (tnfa, il1b) or inducible nitric oxide synthase (inos) were unaffected in mice of different ages (3, 9 or 12 months). to shorten the time required for neurodegeneration to occur, we have injected the mice intraperitoneally with lipopolysaccharides (lps) on four consecutive days and analyzed the dopaminergic neurodegeneration in the substantia nigra after a three weeks period. while the quantification of dopaminergic neurons in the substantia nigra showed a slight decrease in number for wt animals, a higher loss was recorded in the trem2 ko mice after systemic lps challenge. concomitantly, significant increase of microglia activation was detected in the lpstreated trem2 ko mice compared with lps-treated wt animals. our results are pointing towards a neuroprotective effect of the microglial trem2 receptor under chronic and systemic inflammatory conditions. increasing evidence suggest that brain energy metabolism is severely altered in multiple sclerosis patients, which may contribute to the process of neurodegeneration. imaging studies reveal a disturbed cerebral perfusion and glucose uptake and metabolism in ms patients. moreover mitochondrial dysfunction is recognized to play a crucial role in ms pathology. to date little is known about the distribution of glucose transporters (gluts), key molecules for the cellular uptake of glucose, in human brain and their role in the pathogenesis of ms. therefore, the aim of this study was to provide a complete overview of the distribution and expression levels of glucose transporters in control and ms brain. our data so far show that mrna levels of glut1, 3 and 4 as well as their regulators hypoxia inducible factor (hif)-1a, hif-2a and peroxisome proliferator-activated receptor gamma coactivator (pgc)-1a, are increased in normal appearing white matter (nawm) of ms patients compared to controls. immunohistochemical analysis revealed that brain endothelial cells not only express glut1 but also glut3 and glut4. glut 1, 3 and 4 are present in resting microglia and highly expressed in activated microglia and macrophages in active ms lesions. interestingly reactive astrocytes expressed increased levels of glut1 and glut4 compared to control, in addition glut3 positive astrocyes were observed in ms brain. axons expressed high levels of glut3 in active ms lesions, which was evidently reduced in chronic lesions. strikingly, all glucose transporters studied were down regulated in chronic ms lesions. together, these data emphasize the extent to which glucose metabolism appears to be affected in all stages of ms. a better understanding of these metabolic changes, especially in the nawm and chronic stage of the disease, is essential to develop strategies to improve neuronal survival and to gain more insight in the mechanisms underlying lesion formation. pericytes are vascular mural cells which are enriched within the capillary basement membranes of the brain. recent studies have provided evidence that pericytes regulate the blood-brain barrier (bbb) and modulate cerebral blood flow. neurovascular dysfunction and bbb damage are hallmarks of alzheimer's disease (ad), but the role of pericytes in ad pathogenesis is still unclear. we have examined post-mortem samples of middle frontal gyrus in collaboration with the university of cambridge after ethical approval by the local irb. ten cases with neither history of neurological disorder nor evidence of neuropathology were compared with 10 ad cases provided by the neurological foundation of the new zealand human brain bank. we performed immunostainings for laminin and plateletderived growth factor receptor (pdgfr)-b which mark vessels and pericytes, respectively. we also stained for ab 1-42 for evidence of amyloid pathology. quantification of pericyte and vessel density was performed using stereological methods, which are considered as gold standard in morphometric quantification. the spaceballs method was used to measure the density of capillaries. pericyte density was assessed using the optical fractionator. the age of the subjects did not differ between the control and ad groups (76,2 6 4,0 vs. 75,4 6 5,5 years). the cortex of ad cases contained more capillary vessels than controls (mean 6 sd: 308 6 40 vs. 365 6 41 mm/mm 3 ; p < 0,01; fig. 1a ). the capillary density increased in correlation to the total plaque load (r 5 0,58; p < 0,001). in contrast, we could not observe a significant difference in the number of pericytes per mm capillary length between control and ad cases (8,56 6 1,21 vs. 8,17 6 0,94 cells/mm; p 5 0,44; fig. 1b ). relative to tissue volume, the cortex of ad individuals showed a tendency to contain more pericytes than controls (2624 6 406 vs. 2985 6 588 cells/mm 3 ; p 5 0,13; fig. 1c ). this is the first study using state-of-the-art techniques to investigate the relationship between capillaries and pericytes in ad brains. impaired clearance of b amyloid across the pericyte-regulated bbb and deficient microvascular regulation of blood flow may contribute causally to ad pathogenesis. in consequence, loss of pericytes could contribute to disease progression. however, our results show that ad cortical pathology is characterized by an increase in the density of capillary vessels without deficit in pericyte coverage. thus, they do not substantiate the notion that pericyte loss is a significative feature of ad. m. noack, j. leyk, c. richter-landsberg carl von ossietzky universit€ at oldenburg, oldenburg, germany histone deacetylase 6 (hdac6), a member of the class ii hdacs, is a unique cytoplasmic a-tubulin deacetylase that interacts with the microtubule (mt)-associated protein tau. in healthy human brain six tau isoforms are expressed generated by alternative mrna splicing. these contain either three (3r-tau) or four (4r-tau) microtubule binding repeats regulating mt assembly and stability. tau hyperphosphorylation impairs its mt-binding activity. tau deposits in nerve cells and glia are the characteristic hallmark of a number of neurodegenerative diseases termed tauopathies. in progressive supranuclear palsy and corticobasal degeneration, pathogenic glial cell inclusions are observable in oligodendrocytes, the myelin forming cells of the cns. hdac6 plays an important role in the degradation and accumulation of misfolded proteins by promoting transport of aggregated proteins to the aggresome, localized at the microtubule-organizing center where protein aggregates are deposited and processed by autophagy. the present study was undertaken to elucidate the functional significance of hdac6 in oligodendrocytes and its role in pathological protein aggregate formation. towards this cultured rat brain oligodendrocytes and oln-t40 cells, an oligodendroglial cell line expressing the longest human tau isoform, were used. treatment of primary oligodendrocytes with tubastatin a (tst), a selective inhibitor of the tubulin deacetylation activity of hdac6, caused a significant increase in the level of acetylated tubulin, as determined by indirect immunofluorescence and immunoblot procedure. mts appeared more bundled and stabilized. inhibition of hdac6 attenuated the phosphorylation of tau at the phf-1-epitope (serine 396/ 404) and its enhancement at the 12e8-epitope (serine 262, located within the microtubule binding repeat region 1). furthermore, altered protein levels of tau isoforms containing either three or four mt binding repeats were observed. while 3r-isoforms were reduced, 4r-isoforms were increased in primary oligodendrocytes suggesting an impact on microtubule binding ability. hdac6 knockdown in oln-t40 cells revealed no alteration in tau phosphorylation at phf-1-epitope, but also augmented phosphorylation at the 12e8-epitope. cells were morphologically damaged and mt organization was disturbed. the data indicate that hdca6 modulates tau expression and phosphorylation, its inhibition leads to the accumulation of 4r-tau which is also prominent in tauopathies with oligodendroglial pathology. to test the hypothesis, that these cells are part of a possible compensatory mechanism to cope with the 6-ohda-induced loss of dopaminergic neurons, we studied the expression of tyrosine hydroxylase (th), the rate-limiting enzyme in the catecholamine synthesis pathway, in the cortex of 6-ohda-lesioned animals. performing immuncytochemistry at 4d after the lesion we demonstrated a 21-fold increase in the number of th-positive (th1somata in rat cortex following 6-ohda injection) compared to sham-lesioned animals. th1somata in the parietal cortex were restricted to the layers ii-v with most of the cells being bipolar. combining th immuncytochemistry with classical nissl stain yielded complete congruency. a small fraction of th1cells co-expressed calretinin thus pointing to an interneuron affiliation. virtually none of the th1cells expressed other markers of interneurons (calcium binding proteins, neuropeptides) or the glial markers gfap and nestin. in contrast, we found a co-localization of th with markers of glial progenitor cells (sox2 and s100b) and with psa-ncam, which has been shown to be expressed in immature, but not recently generated cortical neurons in cortical layer ii of the cat (varea et al.; front. neurosci. 5: 17 2011). taken together, these findings indicate that 6-ohda lesions might induce a glial de-differentiation followed by a fate re-direction towards neuronal committed cells. future studies have to be performed to confirm this and to find out if the th1cells are capable of dopamine synthesis. objective: using the mouse optic nerve (mon) wm model, we tested whether hydrogen in drinking water reduced functional wm ischemic injury, and if it reduced cellular lipid peroxidation and mitochondrial dysfunction including mitochondrial and nuclear dna oxidation. methods: functional integrity of mon was determined by quantitatively monitoring the area of mon compound action potential (cap) before, during and after a standardized 60 min period of oxygen and glucose deprivation (ogd), the ischemia equivalent ex vivo. nuclear 8-oxoguanine (nu8-oxog) was used as a marker of oxidative dna damage. results: a 60 min period of ogd caused prompt loss of the cap, followed by an average 20% recovery. in mice that had received hydrogen-containing drinking water for 7-10 days, the cap area did not disappear during ischemia and recovered to a significantly greater extent during reperfusion (normal oxygen and glucose levels). immunostaining of axonal neurofilament by smi-31 showed significant protection in mons from mice drinking hydrogen-water. accumulation of nu8-oxog was observed mainly in oligodendrocytes after ogd. the levels of 8-oxog after ogd were significantly reduced in optic nerves from hydrogen-water drinking mice. the 'protection' induced by 7-10 days of hydrogen-water drinking lasted several days. conclusions: our results show that several days of hydrogen exposure reduced the extent of cns wm irreversible injury associated with ogd. the importance of these observations is that oligodendrocytes may play an important role in the protective effect against ischemic injury. these observations raise intriguing therapeutic options. amyloid-b (ab) deposits in the brain extracellular space (ecs) and neurofibrillary tangles are typical features of alzheimer's disease (ad). both affections are expressed in triple transgenic (3xtg-ad) mice, making them a useful model for studying the pathophysiology of ad. in these mice, significant changes in astroglial morphology appear in the limbic brain structures [1, 2] , which may affect the diffusion of neuroactive substances through the ecs. using the real-time iontophoretic method, we determined the ecs volume fraction a (a 5 ecs volume / total tissue volume) and the geometrical factor tortuosity k (k 2 5 free / apparent diffusion coefficient) in the ca1 region of the hippocampus, dentate gyrus (dg) and prelimbic cortex (plc) in brain slices obtained from 10-month-old (10m) and 20month-old (20m) 3xtg-ad mice and age-matched wild-type (wt) controls. to study potential diffusion anisotropy, the measurements were performed along 3 orthogonal axes: medio-lateral, rostro-caudal and ventro-dorsal. astroglial morphology and the presence of ab were assessed using immunohistochemical staining for gfap and ab. we found no anisotropy in the ca1 region, dg, or plc, thus the data for each structure along the three orthogonal axes were pooled. the ecs diffusion parameters measured in the ca1 are shown in table 1. in the ca1 of 10m mice, there was no significant difference in the ecs diffusion parameters between wt and 3xtg-ad mice. in 20m mice, a decreased by 9% in wt but increased by 27% in 3xtg-ad mice in comparison with the values found in the younger animals. during aging, there was also a significant decrease in k in wt but not in the 3xtg-ad mice. a similar pattern of changes in the ecs diffusion parameters during aging was also observed in the dg and plc. unlike in the ca1, we found significant differences in young animals: a was lower in the dg and k was higher in the dg and plc of 3xtg-ad mice than in wt mice. in 20m 3xtg-ad mice, ab deposits accompanied with astroglial atrophy or hypertrophy were observed. we suggest that the amyloid load in 3xtg-ad mice and the subsequent prevailing astroglial atrophy affect the normal aging process by inducing an increase in the ecs volume during aging instead of the normal decrease. this leads to a larger ecs space in aged 3xtg-ad mice than in age-matched wt mice, which may alter the efficacy of extrasynaptic as well as synaptic transmission and contribute to the impaired cognitive functions in ad. the study was supported by the grants: gacr p304/11/0184, gacr p304/12/g069. significant differences between 10m and 20m mice of the same group are marked with *, differences between wt and 3xtg-ad mice of the same age are marked with 1. sequence or any treatment. however growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. to better characterize the time course of pathological events in an animal model that recapitulates human als symptoms, we investigated functional and cellular characteristics of hsod1 g93a mice. we have evaluated locomotor function of hsod1 g93a mice through dynamic walking patterns and spontaneous motor activity analysis. we detected early functional deficits that redefine symptoms onset at 60 days of age, i.e. 20 days earlier than previously described. moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. to tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. we show that (1) decrease in neuromuscular junction's number correlates with motor impairment, (2) astrocytes number is not altered at pre-and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and (3) microglia modifications precede disease onset. at pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge. we are now establishing the transcriptomic profile of microglia over the course of the disease. in conclusion, precise motor analysis updates the onset of the disease in hsod1 g93a mice and allows locomotor monitoring until the end stage of the disease. early functional deficits coincide with alterations of neuromuscular junctions. importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. this finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets. in order to gain more knowledge about the disease pathophysiology, two mouse models for vwm are developed in our lab. co-cultures between astrocytes (wt and vwm) and wt oligodendrocyte progenitor cells (opcs) are done to investigate the effect of astrocytes on opc maturation. furthermore, induced pluripotent stem cells (ipscs) from both vwm and wt mice are compared in their glial differentiation efficiency in vitro and in vivo. the astrocyte-opc co-cultures show that vwm astrocytes inhibit opc maturation. this suggest that astrocytes might have a crucial role in the development of the oligodendrocyte and myelin pathology in vwm. currently our research focuses on rescuing the maturation inhibition in vitro, which can aid the development of stem cell therapy for vwm. particularly in response to b-amyloid. using the appps1 alzheimer's disease mouse model, we observed the production of the common interleukin (il)212 and il-23 subunit p40 by microglia. genetic ablation of various il-12/il-23 signaling molecules, of which deficiency in p40 had the strongest effect, resulted in a drastic decrease in cerebral amyloid burden. although deletion of il-12/il-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in reduction of cerebral b-amyloid in appps1 mice. furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced soluble ab species and reversed cognitive deficits in aged appps1 mice. our results suggest that inhibition of il-12/il-23 signaling reduces cerebral amyloidosis and cognitive dysfunction, and may pose a novel potential pharmacological target to combat alzheimer's disease. charit e -universit€ atsmedizin berlin, berlin, germany question: microglia are attracted to and surround b-amyloid deposits, the main hallmark of alzheimer's disease (ad), suggesting a role for these cells in disease pathogenesis. however, recent in vivo data using the cd11b-hsvtk system for microglial depletion suggests that resident microglia are not sufficiently capable of restricting and clearing bamyloid plaques. to underpin the cd11b-hsvtk system and shed more light into the biological mechanistics of microglial depletion mediated by ganciclovir (gcv), we aim to intravitally monitor the depletion process. methods: we are using cd11b-hsvtk transgenic mice crossed to fractalkine-gfp 1/mice harboring green fluorescent microglia. a chronic cranial window is implanted onto the head of the offsprings followed by depletion of microglia and subsequent in vivo two-photon (2p) imaging. as 2p imaging is able to visualize the upper 300 mm of the cortex, a new topical depletion approach of cd11b-hsvtk 1 microglia was established. here, gcv is applied directly onto the brain surface in the area of the cranial window through a catheter. results: manipulation of the brain skull by installation of the cranial window and placing a catheter for topic gcv application resulted in strong depletion of microglia up to 85% in fractalkine-gfp 1/mice crossed to cd11b-hsvtk mice. conclusions: taken together, we established a minimal invasive technique for visualization of the microglia depletion process in cd11b-hsvtk mice using intravital 2p microscopy. these tools will allow the underpinning of the cd11b-hsvtk system as well as the study of relevant biological questions involving resident and peripheral derived microglia in ad. (icrea) , barcelona, spain x-linked adrenoleukodystrophy (x-ald) is a metabolic genetic disorder of the central nervous system characterized by demyelination in brain and/or axonopathy in the spinal cords, adrenal insufficiency and accumulation of very long-chain fatty acids (vlcfa) in plasma and tissues. the disease is caused by inactivation of the abcd1 transporter with the consequence production of oxidative stress mediators and damage. here we aimed to determine: i) the existence of endoplasmic reticulum (er) stress and the ensuing unfolded-protein response (upr) in brains and fibroblasts from x-ald patients, and in the x-ald mouse model (the abcd1 null mouse); and ii) whether the early and rampant oxidative stress formerly identified accounts for the er stress. indeed, here we report signs of er stress and upr response in human and mouse tissues. a common feature is the activation of atf6 and lower amounts of ire1, two er transducers constituting the core signature of upr in x-ald. chaperone grp78 and grp94 expression, by contrast, differ between variants and stages of the disease. we highlight how chaperones are down-regulated in x-ald mice at 12 months of age, pointing to selective depletion of upr-related factors overtime. treatment of the mouse model with a combination of antioxidants reversed the initial activation of transducers and chaperones, indicating that oxidative stress caused er stress. altogether, we postulate that oxidative-stress elicited er stress occurs in x-ald, and that a defective upr may contribute to axonopathy progression. thus, potentiation of the upr may be a therapeutic avenue in x-ald. representing opc-like cells with the potential to differentiate upon growth factor withdrawal and addition of triiodothyronine (t3) into premyelinating oligodendrocytes. upon differentiation, significant fewer cg4_wt-a-syn cells express the myelin basic protein (mbp; see figure a) as a marker for terminal differentiation and additionally, mrna levels were remarkably reduced compared to control cells (see figure b ). as mrna levels of the myelin protein proteolipid protein 1 (plp1) as well as the major myelin-gene regulatory factor (mrf) are also reduced in differentiated cg4_wt-a-syn cells (see figure b) we hypothesized that the process of differentiation is delayed upon a-syn expression. using various differentiation-promoting agents targeting distinct maturation-associated pathways we aimed to dissect the a-synmediated effect on differentiation in more detail and evaluate potential disease-modifying approaches for this devastating neurodegenerative disorder. methods: two nutritionally distinct groups of male newborn ratswell-nourished (w; n 5 26) and malnourished (m; n 5 33) were treated from the 5th to the 24th day of postnatal life with daily intraperitoneal injections of saline (sal) or cona (sigma-aldrich) at doses of 1 mg/kg (group w-l1 and m-l1) or 10 mg/kg (groups w-l10 and m-l10). two groups of "naive" (non-injected) pups were used as additional controls. when the pups reached adult age (90-120 days), under ip anesthesia (1 g/kg urethane 1 40 mg/kg chloralose) they were submitted to the csd recording (ecog and slow potential change) in two points of the parietal cortical surface. csd was triggered every 20 minutes by applying 2% kcl for 1 min at a point in the frontal cortex. this study was approved by the ethics committee of our university, case no.: 23076.031272/2010-53. results: compared to w-sal controls (mean 6 sd velocity in mm/ min 5 3.41 6 0.10), m-sal rats presented significantly higher velocities (4.26 6 0.16; p < 0.05). in both nutritional conditions l1 and l10 treated groups presented significantly lower csd velocities (3.12 6 0.14 and 2.82 6 0.18 for the w-l1 and w-l10, respectively, and 3.74 6 0.13 and 3.25 6 0.16 for the m-l1 and m-l10). this effect was dose-dependent, and was greater in the m-condition. the na€ ıve-and salgroups had similar csd velocities. conclusions: the lectin cona administered early in life to rats decelerated csd in a dose-dependent manner at adulthood, suggesting a long-lasting effect. the largest relative reduction was observed in the m group, suggesting modulation of the effect by the nutritional status. the involvement of glial cells in this effect is discussed. the endocannabinoid system is of growing interest as a therapeutic target in neurological diseases. the two major endocannabinoids 2arachidonoylglycerol (2-ag) and n-arachidonoyl ethanolamine (anandamide, aea), are the endogenous ligands for the cannabinoid receptors 1 (cb 1 ) and 2 (cb 2 ). cb 1 is mainly expressed in the brain and associated with neuroprotective effects, whereas cb 2 is primarily found in hematopoietic cells and associated with anti-inflammatory effects. in neurodegenerative diseases, e. g. amyotrophic lateral sclerosis (als) and parkinson's disease (pd), an increased cb 2 expression on microglia has been reported and is therefore an interesting therapeutic target. furthermore, an increase of endocannabinoid levels occurs in the affected neuronal tissues. stimulating this disease-related increase in endocannabinoids might thus be of therapeutic interest. in our project, we evaluated the novel, highly selective inhibitor kml29 of an enzyme central to the degradation of the endocannabinoid 2-ag, the monoacylglycerol lipase [1] . an in vitro monoacylglycerol lipase inhibitor screening assay for the comparison of the utilized inhibitor with others was performed. additionally, behavioral experiments including body temperature measurement, pain threshold measurement and motor activity analysis in healthy control mice were performed. furthermore, endocannabinoid system baseline levels of healthy control mice vs. sod1 (superoxide dismutase 1) mice as an als mouse model were compared at the gene expression and protein level. in vitro inhibition of the monoacylglycerol lipase by the recently published compound kml29 resulted in lower ic 50 values and therefore indicated a higher potency of kml29 when compared to the other monoacylglycerol lipase inhibitors jzl184 and cpc12 applied in the assay. in vivo administration of the reported monoacylglycerol lipase inhibitor kml29 resulted in significant decrease in body temperature, increase in pain threshold in b6sjlf1/j mice as well as in impairments in the nocturnal motor activity of c57bl/6j mice when compared to vehicle-treated mice. in summary, our data show a comparatively high potency in vitro and significant dose-dependent effects on physiological parameters in vivo of the monoacylglycerol lipase inhibitor kml29 and therefore lay the foundation for a future therapeutic study targeting neuroinflammation in mouse models of als and pd. glaz/apod protects against oxidative stress and promotes axon regeneration after injury, but its mechanism of action is unknown. we hypothesize that glaz/apod modulates membrane dynamics and oxidation. to contrast this hypothesis we study glaz protective role on the polyq-based spinocerebellar ataxia type i (sca1) model in drosophila. human polyq-ataxin1 expression in fly photoreceptors triggers glaz up-regulation. overexpressing glaz in photoreceptors or in retinal support cells rescues neurodegeneration. when expressed in retinal support cells, the glaz rescue is dependent on lipocalin-receptor expression by photoreceptor neurons, and the glaz-gfp fusion protein co-localizes with photoreceptor markers, suggesting the existence of receptor-mediated endocytosis of glaz. upon neurodegeneration, oxidative stress and induction of autophagy coexist. to test whether the glaz beneficial effects are mediated by the modulation of autophagy we quantified atg8a expression and monitored the accumulation of ubiquitinated proteins and p62. overexpression of glaz reduces atg8a induction, but concurrently reduces the accumulation of ubiquitinated proteins and p62. upon autophagy stimulation by rapamycin treatment, the levels of sca1-dependent accumulation of ubiquitinated proteins and p62 are further reduced by glaz. in addition, glaz decreases the sca1-dependent induction of gsts1, a sca1 genetic modifier contributing to the clearance of lipid peroxides. our data support that glaz enters the degenerating neurons by a receptor-mediated mechanism and promotes the resolution of autophagy, increasing its flow and helping to clear polyq-induced protein aggregates. moreover, the beneficial effects of glaz are linked to lipid peroxide clearance, either directly or through receptor-mediated modulation of protective gene networks. micinn(bfu2011-23978); jcyl(va180a11-2); fund. rodr ıguez-pascual. we investigated the effect of three different intensities of running exercises on the survival of sod1 g93a mice. at the early-symptomatic stage (p60), males were isolated and randomly assigned to 5 conditions: 2 sedentary groups ("sedentary" and "sedentary treadmill" placed on the inert treadmill), and 3 different training intensity groups (5 cm/s, 10 cm/s and 21 cm/s; 15 min/day, 5 days/week). we first demonstrated that an appropriate "control" of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an 11.6% increase in survival in the "sedentary treadmill" group. moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. in a second step, we showed that when compared with the proper control, all three runningbased training did not modify lifespan of the animals, but resulted in glial and motoneuronal changes. conclusions/significance: we demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of sod1 g93a . using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of als mice but induce cellular modifications. our results highlight the critical importance of the control of the environment in als studies and may explain discrepancy in the literature regarding the effect of exercise in als. alzheimer's disease is the most prevalent neurodegenerative disorder, characterized by occurrence of senile plaques, neurofibrillary tangles and aberrant function of classical neurotransmitters and peptide messengers, such as neuropeptides and growth factors. in the last years a role of astrocytes in mediating and amplifying the progression of neurodegenerative disorders has been proposed. moreover, a growing body of evidence has shown that astrocytes can release non-peptide and peptide transmitters to influence neuronal development, function and plasticity. here we analyzed the impact of the main component of senile plaques, the amyloid-b (ab), on two key components of peptide vesicles, carboxypeptidase e (cpe) and secretogranin iii (sgiii), in astrocytes in vitro and in vivo. we consistently located cpe and sgiii proteins in cultured and in situ astrocytes. traffic and secretion of astroglial cpe and sgiii were analyzed under basal and stimulated conditions in primary cultures. we found that exposure of astrocytes to ab1-42 markedly impairs expression, traffic and secretion of glial cpe and sgiii. protein levels were decreased in the media, but increased into the cells, by ab1-42 incubations. the effect of ab on cpe and sgiii in glial cells was also investigated in vivo using the amyloid-forming transgenic mice appswe/ps1de9. an aberrant accumulation of cpe and sgiii was found in numerous activated-astrocytes surrounding senile plaques through all the cns of aged transgenic mice. taken together, the present study shows that ab dramatically alters expression, traffic and secretion of cpe and sgiii. because cpe and sgiii are essential in the process and targeting of neuropeptides and growth factors, an involvement of the astroglial secretory pathway in the pathology of alzheimer's disease is suggested. glia cytokine that is produced subsequent to h-i, that collaborates with other cytokines to stimulate the production of astrocytes from subventricular zone glial progenitors. the specific goal of this study was to evaluate gliogenesis after h-i when the tgfb1 receptor alk5 is antagonized in the vanucci p6 h-i rat model. by q-pcr, the relative amount of tgfb1 mrna peaked 7 days after h-i. therefore, sb-505124, and alk5 antagonist, was given intraperitoneally (i.p.) 7 days after the injury to a group of rats while another group received pbs. animals were sacrificed at different time points and brain samples processed for western blot analysis. validating the importance of alk-5 signaling, sb505124 reduced the levels of phosphorylated smad 2/3 that increased after h-i. in another study, sb-50512 was administered i.p. from p10 to p15 and animals sacrificed at p20 for immunohistochemistry for gfap, iba-1, gstpi and mbp. gfap and iba-1 positive cells were dramatically increased in the injured striatum and corpus callosum after h-i and mbp staining was decreased. by contrast, both the extent of injury and the degree of reactive gliosis was decreased by sb-505124 treatment. altogether, our results indicate that sb-505124 inhibits alk5 signaling in the damaged brain. furthermore, sb-505124 administration decreases the extent of microgliosis and astrogliosis while preserving myelination in the damaged brain after neonatal h-i. supported by nih r01 hd052064 and a grant from the leducq foundation awarded to swl. [2] . despite efforts to improve air quality, dep persists as a problem and the share of diesel engined passenger cars in western europe is increasing steadily. honey bees are an important pollinator of food crops and wild flowering plants. they not only contribute to food security by providing pollination services of an enormous economic value but also play an important ecological role. over the last five years, bee keepers worldwide have reported a decline in honey bee populations. the reasons for this decline remain unknown, but it is thought to be multi-factorial [3] . honey bee colonies are confronted with a number of stressors, for example infection with the mite varroa destructor or exposure to pesticides. to forage successfully honey bees rely on their learning and memory abilities. our research aims to establish whether dep might be a factor contributing to declines in honey bee populations through impairment of healthy cns function. methods: we have investigated the impact of dep exposure on the learning abilities of the honey bee using the proboscis extension reflex and classical conditioning. western blotting and histology were used to determine regional expression levels of stress proteins in the brain in response to an acute diesel exhaust exposure. we are examining the morphological appearance of glial cells to investigate the impact of dep in the honey bee cns of dep exposed, and control, forager bees. results and conclusions: we hypothesize that dep acts as a stressor in the brain of the honey bee causing changes in glial cells that are detectable using morphological and molecular techniques -similar to microglial activation or immunological responses of astroglia in the mammalian brain. this work will further our understanding of how dep acts on the brain and how it might impact on the health of the animal. current results indicate that diesel exhaust pollution impacts on the neurobiology of the honey bee and this may contribute to decline by impairing cns functions. hepatic encephalopathy (he) is a serious neurological disorder caused by liver failure. the prime candidate responsible for he pathology is an increased ammonium concentration; and following acute liver failure, ammonium can reach levels of up to 5 mm in the brain. astrocytes are a major target of ammonium toxicity and earlier studies suggested that this toxicity includes a disturbance in intracellular calcium signaling. in the present study, we analyzed the effect of acute hyperammonia on the intracellular calcium concentration of astrocytes in tissue slices of mouse brain, using quantitative intracellular calcium imaging with the indicator dye fura-2. astrocytes were identified by staining with the vital dye sr101; cerebellar bergmann glial cells were identified based on their morphology. in all brain regions studied, the hippocampal ca1 area, somatosensoric cortex, and cerebellar cortex, bath perfusion with 5 mm ammonium for 30 min induced a small, but persistent elevation in intracellular calcium, averaging about 50 nm. in addition, a fast and transient increase by on average 100 nm that lasted about 10 minutes was seen in a small percentage of astrocytes in hippocampal and cortical slices at the onset of ammonium perfusion. this transient increase was never observed in cerebellar bergmann glial cells. both the transient, as well as the plateau phase of ammonium-induced calcium changes were unaltered in the presence of ttx, indicating that they are independent from action potential generation by neurons. furthermore, ammonium-induced calcium increases largely persisted upon removal of extracellular calcium, indicating that they are caused by release from intracellular stores. taken together, our experiments demonstrate that ammonium evokes complex changes in the intracellular calcium concentration of astrocytes in the intact tissue, which differ both between cells in one preparation as well as between different brain regions. because of the central role of astrocyte calcium in gliotransmission, the observed ammonium-induced calcium dysbalance might result in disturbed neuronglia interaction and contribute to the pathology of he. it has been shown in a number of animal models that microglia in the degenerating brain are primed to show exaggerated cytokine responses to subsequent stimulation with toll-like receptors agonists such as lps and poly i:c. it is not clear whether the degenerating brain shows similarly exaggerated responses to pro-inflammatory cytokines. in the current study we hypothesised that glial cells in the hippocampus of animals with chronic neurodegenerative disease (me7 prion disease) would display abnormal responses to central cytokine challenges. in normal animals it has previously been established that intracerebral cytokine challenges elicit specific pathways, i.e. il-1b a cxcl-1 a neutrophil recruitment or tnfa a ccl-2 a monocyte recruitment. unilateral 1ll doses of tnfa (300ng/ll), il-1b (10ng/ll) or saline were administered intrahippocampally via pulled glass microcapillary, in normal (nbh) and me7 mice. these animals were terminally perfused for formalin fixation and paraffin embedding at 2, 24 and 72 hours post challenge. at 2 hours post challenge me7 microglia produced il-1b following either il-1b or tnfa challenge while nbh microglia did not. furthermore, there was very robust nuclear localisation of the nfkb subunit p65 in the astrocyte population and this was associated with very marked astrocytic synthesis of the chemokines cxcl-1 and ccl-2 in response to both cytokine challenges in me7 animals. conversely, very limited expression of these chemokines was apparent in nbh animals similarly challenged. thus astrocytes are the primary chemokine synthesizing brain cell and in the prion-diseased brain are they primed to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines. this abnormal pattern of chemokine expression is predicted to have consequences for leukocyte infiltration and the ramifications of an altered cellular infiltration pathway for the degenerating brain will be discussed. these findings suggested that the presence of mutated htt in astrocytes alters glial glutamate transport capacity early in the disease process and may contribute to excitotoxicity. to decipher whether astrocytic mutated htt expression was directly associated with msns dysfunction, we investigated the functional properties of individual msns in proximity of mutated htt expressing astrocytes in acute slices. results from whole-cell patch clamp technique showed that msns surrounded (0 -100 microns distance) by astrocytes expressing mutated htt vs. astrocytes expressing normal htt did not differ with regard to passive membrane properties (input resistance, resting membrane potential), action potential firing rates nor spontaneous excitatory postsynaptic current properties (averaged amplitude and frequency). thus, astrocytic expression of mutated htt does not lead to readily apparent functional consequences in msns in these conditions. our data show that 11 month old tg mice displayed a significant increase of ab-plaques in the brain, compared to wt mice. furthermore, we show that the a1c subunit colocalizes with 90% of the abpositive plaques. the cellular expression of a1c was predominantly found in activated gfap1 astroglia around plaques. qpcr profiles of the hippocampus revealed expression of the majority of calcium channel subunits, which was stable during aging. the auxiliary beta4 subunit was expressed in these mice but did not co-localize with a1c1 astroglia around plaques. in cultures of primary astrocytes, ab(42) slightly enhanced the a1c mrna expression after 3 days. we are currently underway to characterize this expression in more detail. in summary, our data provide evidence for a largely stable expression of most ltcc subunits in alzheimer tg mice during aging. finally, the expression of a1c but not beta4 is upregulated in activated astrocytes located around ab-plaques and ab(42) may directly induce astroglial ca v 1.2 calcium channels. this study was supported by the sonderforschungsbereich sfb f4405-b19 and f4406-b19 of the austrian science funds. increasing evidence indicate that both physical and cognitive stimulation induce plastic changes in the brain, such as increase in synaptic and spine densities or neurogenesis, and counteract b amyloid (ab) pathology recovering cognitive function. in the present study we, for the first time, demonstrate the effects of psychostimulation on glial fibrillary acid protein (gfap) architecture in the dentate gyrus (dg) of 3xtg-ad, a well-established mouse model of ad. starting from 3 months of age, 3xtg-ad and their corresponding controls (non-tg) were housed either in the presence of a running wheel (run) or in the enriched environment (enr) for 9 months. as reported previously (olabarria et al., 2010), in standard housing, 3xtg-ad animals showed marked atrophy of gfap-positive profiles, evidenced by significant reduction in gfap surface area, by 39%, and volume, by 42%, compared with non-tg mice. however, physical and cognitive stimulation affected astrocyte morphology by increasing their cytoskeleton surface area and volume, both in non-tg and in 3xtg-ad mice. the surface area of gfap-ir astrocytes increased by 145% and 154% in 3xtg-ad mice housed in run and enr, respectively, compared with transgenic mice kept in standard housing. in addition, the cell volume of gfap-ir astrocytes was higher by 169% and 199% in 3xtg-ad mice housed in run and enr, respectively. the hypertrophy was also evidenced by an increase in the surface area and the volume of astroglial somata and processes in both non-tg and 3xtg-ad mice. further correlation analysis between 3xtg-ad and their corresponding control mice housed under the same conditions, revealed that run and enr not only reversed the genotype-induced astrocytic atrophy, but even induced a hypertrophy in 3xtg-ad mice, with gfap positive profiles rising to the levels of non-tg mice. thus our study indicates that long-term exercise and enriched environment restore and improve astroglial plasticity in the context of adlike pathology, which may recover cognitive functions by compensating disease-associated degeneration of neuronal-glial network. methods: intraperitoneal administration of wa at a dosage of 4mg/ kg of body weight was initiated from postnatal day 40 (p40) till end stage in sod1g93a mice, from 9 months till end stage in sod1g37r mice and from 6 months of age in tdp43a315t mice. results: wa was able to improve the survival by more than a week in sod1g93a and by two weeks in sod1g37r familial mouse model. in addition wa administration also conferred significant neuroprotection in tdp43a315t transgenic mice model. beneficial effects of wa in sod1g93a mice model was accompanied by reduction in loss of motor neurons and also by reduction in level of misfolded sod1 protein in the spinal cord as detected by immunoprecipitation with antibody specific to misfolded sod1. wa was found to be an inducer of heat shock protein 27 (hsp-27), which could possibly explain reduced level of misfolded sod1 and increased neuroprotection in wa treated mice. moreover real-time imaging with the use of biophotonic sod1g93a transgenic mice carrying luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine gap-43 promoter revealed that wa was able to reduce neuronal stress at post symptomatic stage. conclusion: taken together, our results suggest that wa may represent a promising therapeutic drug for treatment of als. the temporal lobe epilepsy (tle) is the most common form of epilepsy that originates from the hippocampus and then propagates to other limbic areas such as the amygdala and entorhinal cortex. the pathological feature associated with tle is hippocampal sclerosis which is characterized by atrophy, induration, gliosis and loss of neurons in ca1, ca3 and the dentate hilar regions. some animal models, albeit do not exactly match the complex etiologies identified in humans, are found to recapitulate most of the pathological features observed in tle. there is evidence that administration of kainic acid can cause seizures in the ca3 region of the hippocampus that can lead to loss of neurons and astrogliosis characteristic of tle, but the underlying mechanisms associated with the degeneration of neurons remain unclear. since lysosomal enzymes, cathepsins b and d, can have important roles in the loss of neurons in a variety of experimental conditions, we evaluated their potential roles along with the insulin-like growth factor-ii (igf-ii) receptor, which is involved in the intracellular transport of these enzymes, in the kainic acid treated rats. our results clearly showed that systemic administration of kainic acid evoked severe loss of neurons along with hypertrophy of astrocytes and microglia in the hippocampal region of the adult rat brain. the levels and expression of cathepsins b and d as well as igf-ii receptor increased progressively with time in the hippocampus of kainic acid treated rats compared to control rats. the activity of both cathepsins b and d was also found to be enhanced in the hippocampus of treated rats. our double labelling studies revealed that expression of both cathepsins were initially increased in the pyramidal neurons and then decreased with time. this was accompanied by the expression of immunoreactive igf-ii receptors as well as cathepsins b and d in a subset of gfap-labelled activated astrocytes and iba1-labelled microglia in kainic acid treated rats. these results, taken together, suggest that enhanced levels/expression and activity of lysosomal enzymes may have a role in the loss of neurons observed in kainic acid treated rats. in addition, increased ng2 glia immunoreactivity and myelin loss were found specifically in the gray matter of patients' motor cortex and spinal cord ventral horn. furthermore, oligodendroglial specific monocarboxylate transporter 1 expression is decreased not only in als mouse models but also in patients. given that oligodendrocytes not only facilitate saltatory conduction of action potentials by myelinating axons, but also support neuronal functions metabolically through monocarboxylate transporter 1 (mct1), the injury to oligodendroglia in als may have pathogenic consequences. methods. to further investigate whether oligodendrocytes play a role in als development, we selectively removed mutant human sod1 (g37r) from postnatal ng21 cells in pdgfar-creer;loxsod1(g37r) mice. results. the administration of 4-hydorxytamoxifen (4ht) caused significant decrease in the expression of mutant human sod1 transgene in ng2 glia as determined by quantitative pcr analysis. we found that excision of mutant hsod1 from ng2 glia dramatically delayed disease onset and early disease, and significantly prolonged animal survival. in addition, at disease onset stage, activated astroglial and microglial responses were delayed in the animals received 4ht treatment. moreover, the removal of mutant sod1 helped to preserve mct1 expression at disease onset. conclusions. these data indicate that expression of mutant sod1 in ng21 cells and their oligodendrocyte progeny has a deleterious effect on motor neuron survival, and suggest that a key negative consequence of mutant sod1 expression in oligodendrocytes is to diminish their capacity to provide metabolic support to neurons. recent studies suggest that innate immunity might be also involved in the pathogenesis of neurodegenerative diseases, cerebral ischemia and brain injury. although the recent works demonstrated that adaptive immune system is involved in the motor neuron disease process, the role of innate immune system in motor neuron disease was not fully investigated. to assess the contribution of innate immunity in the pathogenesis of amyotrophic lateral sclerosis (als), the gene expression profile of lumbar spinal cord from symptomatic mutant sod1 mice was obtained by microarray approach and subsequent pathway analysis indicated the involvement of innate immune pathway. next, to test the role of innate immunity in the pathogenesis of als, sod1 g93a als model mice were mated with myd88 and trif (tir domain-containing adaptor inducing ifnb) deficient mice. myd88 and trif are the essential adaptor proteins for toll-like receptor mediated signaling pathway. as compared with sod1 g93a mice, myd88/trif double-deficient and trif-deficient sod1 g93a mice exhibited the substantially shorter survival times with accelerated disease progression. the disease duration was shortened by 50% in trif-deficient sod1 g93a mice. in contrast, elimination of myd88 in sod1 g93a mice showed marginal effect in survival time. in addition, the expression levels of pro-inflammatory chemokines, ccl5 and cxcl-10 were significantly suppressed in the spinal cord of trifdeficient sod1 g93a mice, as compared with sod1 g93a mice. to determine the cell type in which trif-dependent pathway contributes to the production of these chemokines, we examined the expression of poster abstracts s75 glia chemokines in lps-stimulated primary microglia or astrocyte derived from trif-deficient or myd88-deficient mice. trif-dependent induction of these chemokines was observed only in lps-stimulated microglia. moreover, we found that infiltration of t-lymphocytes and other immune cells were significantly decreased in the spinal cord of symptomatic trif-deficient sod1 g93a mice. these results suggest that the basal level of trif-dependent innate immune activation of microglia is beneficial to slow disease progression of als models through the maintenance of pro-inflammatory chemokines and the infiltration of the immune cells to the spinal cords. the detailed analyses to clarify the role of these infiltrating immune cells are underway. alzheimer's disease (ad), the most common age-dependent neurodegenerative disorder, causes a chronically progressive decline in cognitive functions. there is growing evidence that glial changes are early involved in this pathology. pdapp mouse, a well-defined model of ad, accumulates toxic soluble and deposited ab, derived from proteolytic processing of the amyloid precursor protein (app) and develops adlike synaptic deficits and cognitive impairment. on the other hand, autophagy has been associated to the neurodegenerative process, in particular with the clearance of aggregation-prone proteins like ab. the amyloid plaques, mainly located in cortex and hippocampus, are closely surrounded by reactive and hypertrophic gfap1 astrocytes. the aim of this work was to evaluate the potential astrocyte autophagic activity during the progression of ad in pdapp mice from 5 to 20 months (m) of age. lc3ii/i ratio was studied by western blot. amyloid deposit load, stained with congo red, exhibited a continuing rise according to age in transgenic mice. the markers gfap and lc3 were analyzed by immunofluorescence and confocal microscopy on hippocampal sections showing an increasing colocalization that reached the top at 14 m, where 52.5 6 9.9% of plaque associated gfap cells were lc31. at 20 m, this proportion was lower. conversely, the subpopulation of astrocytes located far from ab deposits were gfap1/lc3-, besides a decreased cell volume compared with control mice astrocytes. additionally, the density of astroglial cells in the stratum radiatum diminished with aging (5 compared to 14 m, total gfap1 cells, p < 0.05) along with higher plaque load, in a more prominently manner than in control mice. our results clearly show that 1) astroglia is strongly affected during ad progression: two different morphologic subpopulations -close to or far from deposits-are distinguished in the hippocampus. aging correlates with a glial density reduction; 2) a gradual increase of autophagic activity in plaque associated-astrocytes is verified by the first time, suggesting a contribution to ab clearance until 14 m in pdapp mice, with a significant decay after this age. it also shows widespread neuron loss and gliosis in the brain. interestingly, in cstb-/-mice pronounced microglial activation has been detected in selected brain areas already in presymptomatic mice, preceding astrocytosis and neuronal death. in this study, we examine the microglial contribution to the neuronal dysfunction and death in epm1. we aim to characterize the functional properties of cstb-/-microglia and their effects on the survival of cstb-/-neurons. our results show that the cstb mrna level in cultured wild type mouse microglia analyzed by real time quantitative pcr is high in relation to primary astrocytes or neurons. a gene-expression profiling of microglia from cstb-/-mice and from wild type mice has been obtained by using the affymetrix mouse exon 1.0 st array and reveals a down-regulation of interferon-regulated pathways in cstb-/-microglia. the stimulation of primary wild type mouse microglia with the endotoxin lipopolysaccharide (lps) up-regulates cstb mrna expression measured by real time quantitative pcr. cstb-/-microglia show an altered inflammatory response to the stimulation with lps by increased secretion of chemokines demonstrated by cytokine array analyses. moreover, the release of nitric oxide from cstb-/-microglia measured by griess assay is elevated. our data indicate an altered response of primary cstb-/-microglia to inflammatory stimuli, which may contribute to neurodegeneration in epm1. the data provide a basis for further detailed studies on the pathophysiology and therapy of this devastating disease. activation of astrocytes and microglia surrounding amyloid extracellular plaques in the brain is a hallmark of alzheimer's disease (ad). increasing evidence suggests that chronic neuroinflammation and elevated levels of several cytokines play important roles in ad development. beside astro-and microgliosis, demyelination and oligodendrogenesis can be observed in human patients as well as in murine models of ad. oligodendrocyte progenitor cells, also termed ng2 cells because they express the neuron/glial antigen 2 (ng2) proteoglycan, comprise about 5% of total cell number of the adult brain and are the main proliferating cells present. ng2 cells receive gabaergic and glutamatergic synaptic input and can secrete pro-and antiinflammatory substances upon activation by microglia-derived cytokines. overall, ng2 cells are highly reactive cells and one of the first cells which respond to changes in the cns. however, their role during ad pathology is still uncompletely characterized. using immunohistochemistry, we have analyzed their expression pattern in a transgenic murine model of ad (coexpressing a mutated form of the amyloid precursor protein (app) and the presenilin1(ps1) gene). we focused on the spatial arrangement of ng2 cells in the hippocampus of transgenic mice at 3, 7 and 10 months of age and age-matched controls (n 5 4/ time point). our goal is to provide an anatomical and structural basis for elucidating the role of ng2 cells in ad. the first results indicate an uniform distribution of ng2 cells in the hippocampus of app/ps1 mice, with an accumulation at the sites of plaque deposits in close association with microglia. ng2 cells are normaly absent from the pyramidal and granular cell layers. however, when plaques deposits were found at these principal cell layers, ng2 cells could be seen surrounding them, suggesting an active migration. changes in ng2 cell phenotype were noted from 3 months of age on. like microglia, ng2 cells assume an activated morphology, as in a more "bushy" appearance. in numerous plaques, ng2 immunoreactivity was strongly increased, mostly in the intermingled processes that encircle the core of the plaques. whether these activated ng2 cells proliferate and differentiate into oligodendrocytes or communicate with activated microglia in the plaque microenvironment remains to be investigated. further, levels of the glutamatergic ampa receptor expression will be evaluated. ultimately we aim at understanding how ng2 cells and microglia interact in ad, which could be usefull in the development of novel therapies. accumulating evidence supports an increasing role of astrocytes in the initiation or progression of a variety of neuropathological conditions. in alzheimer's disease (ad), numerous data indicate that astrocyte properties are modified with potential deleterious effects on neurons. accordingly, we have described changes in the expression of astroglial connexins, the gap junction channel and hemichannel forming proteins, in the vicinity of amyloid-b (ab) plaques in brains from ad patients and murine models of ad. also ab peptide was shown to trigger astroglial cx43 hemichannel activation in culture and in acute slices leading to neuronal degeneration. hence, we have investigated hemichannel function of astroglial connexins in 8-9 months old app swe /ps1 de9 mice that exhibit ab plaques in the cortex and hippocampus. ethidium bromide (etbr) uptake performed in acute brain hemisphere slices was used as an index of hemichannel activation and was quantified in astrocytes identified by gfap immunostaining. in app swe /ps1 de9 mice, compared to wildtype mice of the same age, an increased uptake of etbr was detected in the overall population of astrocytes. this uptake was blocked by carbenoxolone and lanthanum ions, indicating connexin hemichannel involvement. such activation may be linked to the increase in resting astroglial [ca 21 ] i described in this mouse model. interestingly, etbr uptake was higher in reactive astrocytes contacting ab plaques than in non-reactive astrocytes located far from (! 50 mm) these deposits. we are currently investigating their respective pharmacological profiles. moreover, in app swe /ps1 de9 mice knock-out for cx30 generated in our facility, etbr uptake was comparable to that observed in app swe /ps1 de9 mice, suggesting that cx43 is the major contributor to the hemichannel activity observed. altogether, these results indicate that neuroglial interactions could be affected by astroglial hemichannel activation and could account for neuronal alterations or death observed in neurodegenerative diseases. since it is well established that amyloid plaques are associated with activated astrocytes we asked whether concentrations of the astrocyte-derived proteins glial fibrillary acidic protein (gfap) and s100b in human csf might serve as additional biomarkers. to functionally link the role of astrocytes to mechanisms involved in memory formation, we asked whether the amount of gfap-positive astrocytes correlates to the level of ltp in the hippocampus of aged ad transgenic mice. we used elisa kits for the quantitative analysis of gfap (ibl-international, hamburg, germany) and s100b (ibl). in a mixed disease cohort (n 5 52 diseased patients, n 5 16 controls), we correlated standard biomarkers (abeta and tau-protein) and gliaderived proteins. furthermore we compared mean levels between groups of ad patients (n 5 36) and healthy control patients (n 5 35) and correlated levels of astroglial proteins in the csf and cognitive performance (mmst values). in transgenic (app/ps1 transgenic mice), aged (10-15 months) mice (n 5 10), we performed ltp experiments at the schaffer collateral synapses in the ca1 region of the hippocampus. the number of gfap-positive astrocytes was quantified contralaterally. results: in the mixed cohort, we found a significant correlation of t-tau and gfap levels in csf (r 5 0.261, pbetween the s100b levels and the patients cognitive performance as measured by the mmst values (r 5 -0,42) as well as between the gfap levels and the mmst values (r 5 -0,38). when correlating the number of astrocytes in the ca1 region to the level of ltp, 30 min after induction, we found a significant correlation of the number of astrocytes per mm 3 to the level of ltp (r 5 0,648, p conclusions: the astrocyte-derived proteins gfap and s100b can be reliably detected in human csf. measurement of astroglial biomarkers might allow an improved pathobiological staging of ad, also since astrocytes appear to play a functional role in memory formation in the context of an ad-like pathology in mice. retinitis pigmentosa is a group of inherited disorders affecting photoreceptors or retinal pigment epithelium (rpe) that leads to progressive loss of vision. the pde6b rd1 mouse model is a very well-known animal model for retinal degeneration, in which rods carry a mutation in b subunit of rod cgmp-phosphodiesterease. glial cell line-derived neurotrophic factor (gdnf) has already been shown to rescue morphology as well as function of rod cells in pde6b rd1 mouse. this effect was indirect, through stimulation of m€ uller glial cells (rmg). to better understand the neuroprotective effect of gdnf, primary retinal rmg were stimulated in vitro with gdnf and the secreted proteome was analyzed by proteome profiler arrays. among others, cyr61/ccn1, a member of ccn family, was found strongly induced upon rmg stimulation with gdnf. when applied directly to medium, cyr61 significantly reduced photoreceptor death in organotypic ex vivo cultures of pde6b rd1 retinas. in order to identify the target cells of cyr61 we treated, arpe19 and mio-m1 cell lines with cyr61, and observed an increase in phosphorylation of akt and erk1/2 signaling molecules. these results suggest that stimulation of rpe and rmg cells may have a protective influence on photoreceptor survival in organotypic ex vivo conditions. we postulate cyr61 as a novel potential candidate for future therapeutic approaches in neurodegenerative retinal disorders. since astrocytes react to multiple physiological and pathological stimuli in the microenvironment of the brain, they could be mediators for gene x environment interactions in the pathogenesis of psychiatric disorders. altered gene/protein expression and regressive changes have been reported for astrocytes in post-mortem studies of psychiatric disorders, i.e. schizophrenia (scz), bipolar disorder (bp) and autism spectrum disorders (asd). by contrast, no genetic link to astrocytes has emerged from the extensive genomic analysis of psychiatric disorders. genetic findings mostly relate to neurons for disorders rooted in neurodevelopment (asd, scz). it is timely to perform a formal analysis of genomic information emerging for psychiatric disorders for a contribution of genes expressed by astrocytes. methods: we generated a meta-list of genes highly expressed in rodent astrocytes using published gene lists and literature mining. datasets were prepared for candidate genes and genes from gwas in attention deficit/hyperactivity disorder (adhd), asd, bp and scz, by using literature and databases (szgene, sfari base, dbgap). datasets for copy number variations (cnvs) associated with neurodevelopmental delay were also assembled. then the overlap between the meta-list of genes highly expressed in astrocytes and gene datasets for psychiatric disease was determined. overlapping genes were pooled and subjected to david bioinformatics analysis. results: the meta-list of genes highly expressed in astrocytes contained n 5 2,680 genes (13.4% of the genome). datasets for risk genes in psychiatric disorders showed random overlap with the meta-list (adhd 18%; asd 13%; bp 12%; scz 13%). cnvs related to neurodevelopment were not enriched for astrocytic genes (8%). when the overlapping genes were pooled from all disorders, a small set of shared astrocytic genes emerged (n 5 23). cntnap2, nrxn1 and sdc2 were linked by david analysis (p 5 0.013). conclusions: this correlative analysis makes it unlikely that genes highly expressed in astrocytes make a major contribution to the genetic risk in four major psychiatric disorders. no genetic link was found between astrocytes and neurodevelopmental delay. changes in gene/ protein expression and pathology in astrocytes in the adult brain in scz and bp may be explained by chronic neuronal dysfunction, chronic medication or acute changes. the neuronal ceroid lipofuscinoses (ncls, batten disease) are a group of autosomal recessively inherited lysosomal storage disorders affecting children and young adults, each of which is caused by a mutation in a different gene. a common feature across all ncls is the early, localised glial activation that occurs long before the onset of neuron loss. this glial activation appears to be an accurate predictor of which neuron populations are vulnerable and will subsequently die. both astrocytes and microglia express the ncl gene products and it is likely that normal glial cell function may be compromised. given the close functional relationship between neurons and glial cells it is possible that glial activation and/or dysfunction may affect neuronal health. we have begun to explore the biology of astrocytes and microglia in the juvenile form of ncl using primary cultures from cln3 -/mice. defects in both astrocytes and microglia, including altered protein secretion profiles and impaired morphological and proliferative responses were apparent. co-culturing with mutant microglia and astrocytes influenced the survival and morphology of wild type neurons, with more profound effects upon cln3 -/neurons. intriguingly, these effects were largely reversed by substituting wild type glia, which rescue mutant neurons. a pilot study has provided similar evidence for glial dysfunction in infantile ncl, including and altered protein secretion and response to stimulation. these changes appear to be different from those observed in juvenile ncl and we are in the process of exploring whether the glial cell phenotypes discovered so far, and their impact upon neurons are specific to the different forms of the disease. the accumulation of aggregated proteins in nerve cells and glia underlie the pathogenesis of many neurodegenerative diseases. glial pathology is characteristically observed in tauopathies, such as corticobasal degeneration and progressive supranuclear palsy, and alexander disease, a primary disease of astrocytes caused by mutations in the gfap (glial fibrillary acidic protein) gene. irreversibly damaged proteins can be degraded by the proteasomal system. for proteasomal degradation they are covalently linked to ubiquitin in a three-step enzymatic pathway (e1, ubiquitin-activating enzyme; e2, ubiquitin-conjugating enzyme; and e3 ubiquitin ligases). the polyubiquitin chain needs to be removed before the translocation of the substrates to the lumen of the proteasome. this is achieved by deubiquitinating enzymes (dubs), which oppose the functions of e3-ligases and assist in targeting substrates to specific pathways. to assess whether impairment of dubs may contribute to age-related neurodegenerative diseases and the regulation of cell death and survival, we have subjected primary cultures of rat brain astrocytes to pr-619, a dub inhibitor with broad specificity. immunoblot analysis demonstrates that after a 18h treatment, pr-619 caused the induction of heat shock proteins, hsp70 and hsp32, and an increase in p62, which is considered a cargo receptor for ubiquitinated proteins and a common constituent of ubiquitinated protein inclusions. as analyzed by indirect immunofluorescence, protein aggregates positively stained by antibodies against ubiquitin and p62 assembled in the perinuclear region at the microtubule organizing center (mtoc). these aggregates were surrounded by a cage-like network of gfap intermediate filaments, thus resembling aggresomes. using mitotracker and lysotracker staining procedures, the fluorescent images demonstrate that after dub inhibition lysosomes and mitochondria are recruited to the mtoc. this process was dependent on an intact microtubule network, since it was interrupted after treatment with nocodazole, a microtubule destabilization drug. furthermore, pr-619 caused mitochondrial fragmentation which may be a stress response to enable the removal of damaged mitochondria by mitophagy. in conclusion, dub inhibition impairs the cellular architecture, leads to protein aggregate formation and mitochondrial alterations in astrocytes. our data sustain the hypothesis that an imbalance in dub activities may contribute to the pathogenesis of neurodegenerative diseases. shown that the collapsin response mediator protein 2 (crmp-2), which play a significant physiological role in neuronal cell bodies and axons within the cns, is phosphorylated during the neurodegenerative phase of the inflammatory disease. methods: we investigated the limitation of axonal degeneration by transducing retinal ganglion neurons with a phosphorylation mutant of crmp-2 utilising an intraocular adeno-associated virus 2 (aav2) delivery system in eae-induced mice. the other group of mice injected with aav2 consisting of the green flourescent protein reporter only (aav2-gfp) with eae was used as a control (n 5 8 per each group). results: we showed substantial preservation of axons of the optic nerve in the eae-induced mice injected with the aav2 carrying the crmp-2 phospho-mutant compared ($10-fold difference) with those mice injected with aav2-gfp. the aav2-gfp transduced axons showed significant degeneration during the peak stage of eae. conclusion: our data suggest that phosphorylation of crmp-2 may be a central mechanism that governs axonal degeneration during inflammatory demyelination of the cns (as occurs in ms) and inhibition of phosphorylation of crmp-2 may be of therapeutic potential for the progressive phase of the disease in ms patients. y. shinozaki, s. koizumi univ. of yamanashi, yamanashi, japan atp, a major gliotransmitter integrating neuron-glia networks, is known to be released or leaked from injured cells. in the cns, atp dramatically changes glial phenotypesand could affect pathogenesis of brain disorders. however, whether such glial responses facilitate or rather inhibit the diseases is still under debate. to address this issue,we studied the neuroprotective role of atp/purinergic signaling using in vivo stab injury model on mouse cerebral cortex. without injury or the contralateral side of the injured brain exhibited no fluoro-jade(fj)-positive neuronal death, cd45 1 infiltrating leukocytes or gfap 1 reactive astrocytes. three days after injury, fj signals, cd45 1 cells and reactive astrocytes were increased in the ipsilateral side of the brain. the cd45 1 cells were enclosed by reactive astrocytes-formed glial scar. administration of an atp-degrading enzyme apyrase, a broad p2 receptor antagonist ppads, and a p2y 1 receptor antagonist mrs2179 significantly reduced the stabincreased fj signals. immunohistochemical analysis revealed p2y 1 receptor was expressed in gfap 1 astrocytes. p2y 1 receptor knockout (p2y 1 ko) mice also exhibited reduced fj signals and infiltration of cd45 1 cells. they exhibited higher level of gfap expression, more remarkable hypertrophy of astrocytes and tighter glial scar formation than wild type mice did. the accelerated glial scar formation reduced cd45 1 cell number and inhibition of astrocytes by fluorocitrate increased cd45 1 cell number. although glial scar formation was accelerated, no enhanced proliferation was observed. we then analyzed the mechanism of enhanced glial scar formation and neuroprotection using in vitro scratch-wound model. in cultured astrocytes, the scratch induced a transient increase in extracellular atp, which was followed by decrease in extracellular atp mainly due to an increase in ectoatpase activity. suppression of purinergic signaling by either apyrase, ppads or mrs2179 enhanced scratchevoked astrocytic migration rather than proliferation. neither activation nor inhibition of p2y 1 receptors in cultured cortical neurons affected the scratch-induced damages. our data suggest that the decrease in atp/ p2y 1 receptor-mediated signal should be a trigger that transforms astrocytes into migratory phenotype, which forms tighter glial scar structure thereby suppressing neuronal death. background: oligodendrocyte damage and loss are key features of multiple sclerosis (ms) pathology and oligodendrocytes appear to be particularly vulnerable to ros. in vitro studies showed that ros induce cell death and prevent the differentiation of oligodendrocyte precursor cells (opcs) into mature myelin-producing oligodendrocytes. hence, a potential therapeutic strategy to protect these cells from ros-mediated damage is urgently needed. here we investigated the efficacy of several compounds that are able to boost antioxidant enzyme production, including monomethyl fumarate (mmf), tert-butylhydroquinone (tbhq), sulforaphane (sfn) and protandim. these compounds are thought to exert their protective function via activation of the nuclear-factor-e2-related factor-2 (nrf2) transcriptional pathway, which is involved in the production of antioxidant enzymes necessary for oxidative stress defense. methods: primary rat oligodendrocytes were treated with different concentrations of mmf, tbhq, sfn and protandim. expression of antioxidant enzymes were analyzed by pcr and western blot analyses. to study the beneficial effects of the different nrf2 activators, oligodendrocytes were first incubated with nrf2 activators and subsequently exposed to various concentrations of hydrogen peroxide. oligodendrocyte cell survival was measured by a live/dead cell viability assay. results: sfn, mmf and protandim are well-tolerated and induce nrf2driven antioxidant enzyme production in oligodendrocytes. protandim was the most potent compound with regard to antioxidant enzyme induction and protected oligodendrocytes against ros-induced cytotoxicity. conclusions: our findings indicate that several nrf2 activators are able to induce antioxidant enzyme production in oligodendrocytes. interestingly, protandim, a dietary supplement consisting of herbal ingredients, was the most potent protector of primary rat oligodendrocytes. in future experiments we will determine whether protandim can also promote the differentiation of opcs under oxidative stress and test the clinical efficacy of protandim in an experimental animal model for ms. the arising of the "tripartite synapse" concept and the discovery of the astrocytic excitability have been highlighting astrocytes as active elements concerning information flow in the brain. however, the impact of such remarkable features in complex brain function is still under-explored mostly due to the difficulty of studying neuron-astrocyte interactions in vivo. the aim of this work was to use an in vivo model of astrocytic dysfunction and investigate the impact of this treatment in complex cognitive functions. the rationale consisted in studying behavioral performance that relies on the prefrontal cortex, such as behavioral flexibility and working memory in an animal model of astrocytic dysfunction. for that purpose, we used a pharmacological model in which wistar-han rats were subjected to bilateral intracranial injections of aminoadipate in the prelimbic portion of the medial prefrontal cortex to cause astrocyte depletion specifically in this region, mimicking pathological states such as depression, in which marked decreases of gfappositive cells are observed. this animal model was tested for its cognitive abilities by the attentional set-shifting task (asst) and water maze-based tests. a clear impairment of cognitive function was observed in the animals treated with aminoadipate. a detailed morphological and histological analysis showed that along with the astrocytic lesion, neurons were also affected at the site of lesion. this seems to contribute to the cognitive decline observed in this model and may explain similar observations under pathological states. a. sternotte, e. hermans universit e catholique de louvain, brussels, belgium amyotrophic lateral sclerosis (als) is an adult disease characterized by a selective loss of motor neurons, resulting in progressive paralysis and death within 3-5 years. in several inherited cases, the disease is caused by point mutations in the gene encoding for superoxide dismutase 1 (sod1) which promotes its aggregation, leading to biochemical damages, including mitochondrial dysfunction. alteration of mitochondrial membrane potential and/or respiratory chain leads to atp depletion and these metabolic dysfunctions could contribute to motor neuron death in patients and animal models of als. in motor-neurons, the combination of altered axonal transport with mitochondrial dysfunction likely leads to considerable decrease in atp levels at distant neuromuscular junctions. commonly know as the fuel gauge of mammalian cells, amp-activated protein kinase (ampk) is a key enzyme in the control of cellular atp and energy homeostasis. in this study, the implication of ampk in the progression of als was specifically investigated by measuring the expression and activity of this enzyme in the spinal cord of mice overexpressing the mutated human sod1 (hsod1 g93a ), a commonly used experimental model of als. no consistent modification of the enzyme was detected in spinal cord samples throughout the progression of als. indeed, such experiment did not discriminate between cell types present in the tissue. hence, a more detailed characterization of ampk in cultured astrocytes derived from als animals revealed a robust induction of the enzyme activity, which correlates with decreased atp levels in these cells. in a second part of our study, we have investigated the consequences of ablating ampk in the mouse model of als by breeding ampk knock out mice with hsod1 g93a mice. while we did not detect any difference in the lifespan of ampk(-/-)/hsod1 g93a mice as compared to hsod1 g93a mice, we observed substantial alterations in the progression of the disease in selected behavioural tests. in particular, analysis of the gait (catwalk) which enables early detection of muscle weakness revealed that the onset was delayed by up to 30 days while the progression of the disease after onset was accelerated. further studies will be conducted to establish the importance of atp-in the disease, and to validate this enzyme as a putative pharmacological target in als and in other neurodegenerative diseases involving mitochondrial dysfunction. institute of neuroscience, sibs, cas, shanghai, china ng2 glia is the fourth type of neuroglia in vertebrate nervous system, which also known as oligodendrocyte progenitor cell (opc) in a developmental point of view. in adult brain, ng2 glia has a small cell body with highly branched extensions and represents a new type of glial cell differing from other glial cell types such as astrocyte, microglia and mature oligodendrocytes. published studies by others have shown that ng2 glia is able to promptly respond to brain injury and activated in several neurodegenerative disease animal models including 6-ohdainduced rat parkinson's disease model. however, the pattern and function of these activated ng2 glia remain largely unknown. here, we performed a time-course study of ng2 glia activation in mptp mouse model of parkinson's disease using immunohistochemistry. it was found that ng2 glia was activated as early as 24 hours after the last mptp injection in the substantia nigra (sn) of mptp-treated mice. this temporal character is very similar to those of microglia, indicating ng2 glia respond to mptp challenges very quickly. the activation of ng2 glia became stronger over time and reached the peak at about 5 days after the final mptp injection. then, the extent of ng2 glia activation declined gradually and diminished 10 days after mptp injection. interestingly, we found that prominent ng2 glia activation could only be observed in the substantia nigra. the dorsolateral striatum which is the projection target of nigral dopaminergic neurons, however, was devoid of ng2 glia activation in all the time-points examined. in summary, we characterized the ng2 glia activation in mptp mouse model of parkinson's disease. these data suggest that activated ng2 glia may play a role in the degenerative process of dopaminergic neurons in pd. hallmarks of cns inflammation, including microglial and astrocyte activation, are a feature of post-mortem tissue from amyotrophic lateral sclerosis (als) patients and in transgenic mice overexpressing mutant superoxide dismutase-1 (sod1 g93a ). administration of glucocorticoids does not significantly alter disease progression, but this may reflect poor cns delivery. here, we sought to discover whether cns-targeted liposomally-packaged glucocorticoid would inhibit the cns inflammatory response and reduce motor neuron loss. sod1 g93a mice were treated with saline, free methylprednisolone (mp, 4mg/kg/week) or glutathione pegylated liposomal mp (2b3-201, 4mg/kg/week) and compared to saline treated wild-type animals. animals were treated weekly with intravenous injections for 9 weeks from 60 days of age. animal weights and motor behaviour were monitored during this period. at the end of the experimental paradigm (116 days) mice were imaged using t 2 -weighted mri for brainstem pathology, and brain and spinal cord tissue was collected for histological analysis. all sod1 g93a animals showed a significant decrease in motor performance compared to baseline from $100 days. sod1 g93a animals showed a significant increase in signal intensity on t 2 weighted mr images, which may reflect the combination of neuronal vacuolation and glial activation in these motor nuclei. treatment with 2b3-201, but not free mp, significantly reduced t 2 hyperintensity, which correlated with significantly reduced histopathological manifestations in the brainstem motor nuclei, but not the spinal cord. interestingly, there was a significant reduction in astrogliosis but not microglial activation following treatment with 2b3-201. the results of this work indicated that the cns-targeted anti-inflammatory agent 2b3-201 has therapeutic potential in als. the toxic effects of new antiretroviral drugs on the central nervous system (cns) are unclear. because these drugs penetrate the brain even at low concentrations, it becomes crucial to determine the doses which can be toxic for the cns resident cells. moreover, after the recent introduction into clinical practice, it is unclear whether the efficacy of the antiretroviral drugs of new generation may also derive from their ability to exert extravirological effects on factors responsible for the development of hiv brain injury, e.g. matrix metalloproteinases (mmps). objective: to investigate on the toxicity of four different antiretroviral drugs and their ability to modulate the expression of gelatinase b (mmp-9) in astrocyte cultures. methods: primary cultures of rat astrocytes were activated by exposure to 10 mg/ml lipopolysaccaride (lps) (positive control) and simultaneously treated for 20 h with increasing doses (1-5-10-25. 50 mm) of: efavirenz (efv); darunavir (drv); maraviroc (mvc) or raltegravir (ral). mmp-9 mrna expression was assessed by rt-pcr. quantitative determination of mmp-9 expression was done by computerized scanning densitometry. single drug toxicity was assessed by the mtt test. each drug was considered toxic at the concentration able to induce a percentage of cell survival above 60%. results: the treatment with antiretroviral drugs inhibited mmp-9 mrna expression in lps-activated astrocytes in a dose-dependent manner. in particular, a statistically significant inhibition of mmp-9 expression was observed when astrocytes were treated with 25mm efv (54% of inhibition) or with 50 mm ral (43% of inhibition). as assessed by the mtt test, the toxicity of the antiretrovirals ranges from 10 and 50 mm. in particular, efv was toxic for astrocytes at the concentration of 25 mm, mvc at 10 mm, while drv and ral were toxic at the concentration of 50mm. conclusions: the present results indicate that efv and ral directly inhibit mmp-9 expression in lps-activated astrocytes with mechanisms that are independent from their antiviral activity. the toxic doses of antiretrovirals are much higher than those found in the csf of hiv-positive patients. our results highlight some beneficial/deleterious extra-viral effects of the antiretroviral drugs that may be useful to improve the development of new therapeutic strategies for the management of hiv infection. we used a parahippocampal kainate injection mouse model to induce mtle-hs and to study early expression patterns of kir4.1 and apq4 after se. mice were sacrificed 24 h, 72 h and 7d after kainate injection. immunhistochemistry of the hippocampus was performed to assess kir4.1 and aqp4 expression, and gfap staining was used for identification of astrocytes. gfap expression was increased compared to saline injected controls, suggesting that kainate injection induces astrogliosis. comparison of kir4.1 and aqp4 staining showed a similar change in the expression pattern. our data indicate a drop in kir4.1 and aqp4 expression within 24 hrs after se followed by an upregulation within the first week. altered expression of kir4.1 and aqp4 could contribute to dysregulation of potassium and water homeostasis and play a role in the early phase of epileptogenesis. c. l€ o€ ov, a. erlandsson uppsala university, neuroscience/neurosurgery, uppsala, sweden we have previously shown that astrocytes effectively engulf dead cells after trauma both in vitro and in vivo, but that they store the ingested material rather than degrade it. compared to macrophages, which degrade engulfed, dead cells within hours, our data show that astrocytes store the ingested material for weeks before the degradation is completed. to further study the routes of degradation and the possibilities to speed up this process we have used a cell culture model where uvtreated, dead cells are added to stem cell-derived astrocytes. by labeling the dead cells with the ph-sensitive dye phrodo prior to the engulfment, we demonstrate that the ph in the astrocytic phago-lysosomes is higher than in professional phagocytes. interestingly, lamp1 and lamp2, which are involved in the phago-lysosome fusion, are both highly expressed in the astrocytes, particularly around the ingested material. dendritic cells are known to express the inhibitory protein rab27a that slow down the degradation in order to preserve antigen for presentation. rab27a prolongs the actin coating around the phagosomes, which physically inhibit the phago-lysosome fusion, and interacts with nox-2, which leads to an increased consumption of protons. western blot analysis shows a high expression of rab27a in our cell cultures which may explain the slow degradation. moreover, dead cells in astrocytes are surrounded by actin rings for a long period of time after the ingestion. to counteract this, the astrocytes were treated with 0.1 or 1 mm of the actin inhibitor latrunculin b. the number of actin rings were significantly lower in the cultures treated with 1 mm of latrunculin b compared to controls, but the intensity of phrodo was unaltered indicating that the prolonged degradation may be due to a higher ph in the lysosomes rather than a delayed actin coating. next we will investigate the effect of rab27a expression on the degradation process by performing sirna experiments. one of the promising strategies for the treatment of spinal cord injury is the transplantation of glial cells obtained from the olfactory system; olfactory ensheathing cells (oecs). effective proliferation and migration of oecs are essential for optimizing clinical applications and there is a need for identification of small molecules that regulate glial cell biology. curcumin is a natural polyphenol compound found in the spice turmeric, which is known for its neuro-protective properties and has been reported to have an effect on neurogenesis and nerve regeneration. however, the effect of curcumin on oecs has not been determined. we have examined the effect of curcumin on oecs at the cellular level using fluorescence and timelapse microscopy. oecs were purified from s100b-dsred transgenic mice in which oecs express the fluorescent protein dsred. different treatments: (i) control medium, (ii) curcumin (0.5 to 20 mm), (iii) g5 commercial growth factor, or (iv) a combination of g5 and curcumin were used to determine the effect of curcumin on oecs proliferation and migration. cell proliferation was quantified at two different times by cell counting and mts proliferation assay. timelapse microscopy was used to visualize changes in cell morphology and cell migration. cell branching, number and area of lamellipodia and speed of migration per cell were measured for each treatment. we found that lower concentrations of curcumin (0.5 and 1 mm) increase oec proliferation. as well, combination of g5 and curcumin showed the highest proliferative effect on oecs suggesting a possible synergistic relation between g5 and curcumin. live cell imaging analysis showed that curcumin increased the number and area of lamellipodia resulting in faster migration of oecs. these results suggest that curcumin can regulate the proliferation, morphology and migration of oecs which could improve the therapeutic use of oecs for spinal cord injury repair. methods: the dams were divided in four groups and had received 50 ml of nonalcoholic or alcoholic beer solution -control, vehicle (nonalcoholic solution), etoh 5% (nonalcoholic solution 1 5%v.v ethanol) or etoh 10% (nonalcoholic solution 1 10%v.v ethanol) during gestational day 1 up to weaning (postnatal day 22). male offspring (30 -35 days old) were sacrificed and hippocampal acute slices were prepared. we analyzed gfap content, s100b and glutamate uptake as astrocyte parameters. we tested the animals in the plus-maze discriminative avoidance task to check anxiety and memory. results: gfap content decreased after pnee for etoh 5% and etoh 10% treatment (f 3,59 5 17.75, p < 0.0001). interestingly, we found that only the etoh 10% treatment showed an increase of hippocampal s100b content (t 5 2.38, p 5 0.02) and in the csf (f 3,18 5 3.249, p 5 0.0462), but no difference in secretion. glutamate uptake was significantly decreased for etoh 10% group (f 3,20 5 4.069, p 5 0.0208). to perform the plus-maze discriminative avoidance task we selected pups from control, vehicle and etoh 10% treatment on pnd 30. two-way anova revealed significant effects of treatment (f 2,46 5 4.32, p 5 0.019), arm type (f 1,46 5 103.9, p < 0.0001) and treatment x arm type interaction (f 2,46 5 6.66, p < 0.01). in the test session, only a significant prenatal treatment group effect (f 2,46 5 6.44 p < 0.01). bonferroni's post-hoc test revealed that only control group presented significantly less time in the enclosed aversive arm than nonaversive one. conclusions: in this study we showed that pnee with moderate doses could alter the structure and functional astrocytes balance. thus, high levels of s100b are indicated in some neurodegenerative disorders. taken this data together we could demonstrate that moderate ethanol exposure can be harmful to fetal brain. aging has been associated to neuroinflammation in the central nervous system, however it is not known whether microglial changes induced by aging are affected by early effects, such as litter size and sedentary life style. in addition, the lateral septum has been recognized by its complementary role in the memory processing for tasks of object recognition for identity and spatial location. in the present report, we investigated whether aging cognitive decline and microglial morphological changes in the lateral septal region are influenced by changing litter size early in life and by a sedentary life style. to assess these questions, wistar rats suckled in litters of either six or 12 pups per dam were raised sedentarily in groups of 2 up to 3, from the 21 st post-natal day onwards. at 4 (young adult) or 23 (aged) months-old, half of the sedentary rats underwent progressive daily treadmill exercise for five weeks, while the others remained sedentary. after performing the tests of recognition for spatial localization and object identity all of the animals were sacrificed and their brains were processed for selective microglia/macrophages immunolabeling with anti-iba-1 antibodies. a representative sample of the immunolabeled cells in the lateral septum was analyzed after three-dimensional reconstruction with neurolucida software (microbright field inc.) and morphological features of each cell were quantified by neuroexplorer (microbright field inc.). it was found that sedentary life style of wistar rats maintained in standard laboratory cages is associated with spatial memory deficits in both mature and aged subjects no matter the litter size, and that exercise decreased these effects in aged subjects raised in small but not in large litters. on the other hand, all sedentary aged rats, despite of the litter size, presented impairment of object recognition memory for identity, and exercise decreased this effect in animals from both large and small litter size groups. microglial morphological analysis revealed that cell soma area, perimeter and branches volume seem to be more intensely affected by aging and that these changes are mainly associated with animals raised in large litters. furthermore, it was observed important shrinkage and thickening of the microglial branches in aged individuals on a higher proportion in the sedentary group suckled in large litters. taken together the results suggest that litter size and sedentary life style may affect object recognition and spatial memories in both adult and aged rats and that exercise minimizes these effects on animals suckled in small but not larger litters. in addition, we suggest that these changes are related to distinct effects on the soma and branching patterns of septal microglia from young and aged subjects. huntington disease (hd) is a dominant inherited neurodegenerative disorder caused by an unstable expansion of a cag repeat within the gene encoding for huntingtin protein (htt). this mutation induces formation of a poly-glutamine tract in the mutant htt (mhtt) protein that prones its aggregation into the cells. hd is characterized by an initial and massive degeneration of the medium spiny neurons in the striatum with later neuronal loss in cortex, globus pallidus, and other structures leading to motor and cognitive symptoms. alterations of energy metabolism contribute to hd pathogenesis but the mechanisms responsible for these alterations are not well understood. a recent pet study provided evidence for a selective impairment of striatal glycolytic and not oxidative metabolism of pre-symptomatic hd patients. in the brain, glycolysis is predominantly an astrocytic metabolic process, whereas oxidative metabolism is primarily neuronal raising the question that metabolic defects in hd could originates from astrocytes. the purpose of our study was to characterize (1) the metabolic alterations in vivo in a transgenic mouse model of hd (bachd mice) and (2) the respective roles of astrocytes and neurons in metabolic defects occurring in hd using an in vitro strategy. bachd mice express the human full length htt protein with 97 glutamine repetitions under the human htt promoter into a bacterial artificial chromosome. bachd mice present a slow disease progression with motor deficits occurring at 6 months and progressive neuronal aggregates from 12 months, reflecting human pathology. first, we performed [14c]-2-deoxyglucose autoradiography experiments in vivo to assess energy metabolism in 14 months bachd (n 5 6) and wt (n 5 6) mice. we performed a 3d analysis of the whole brain glucose uptake without any regional a priori. using statistical parametric mapping (spm), a voxel-wise statistical analysis approach, we showed that compared to age-matched wt mice, 14 months bachd mice present hypometabolism in the striatum, like pre symptomatic hd patients, and also in the hippocampus. hypermetabolism was also detected in the hypothalamus. second, we performed an in vitro study to dissect out the cellular mechanisms responsible for these metabolic changes. by using cell insert, we realized neurons/astrocytes co-culture without any physical contact. this culture system allowed us to grow neurons in presence of wt or bachd astrocytes. we showed that both bachd and wt neurons have a decreased [14c]-2-deoxyglucose uptake when cultured in presence of bachd astrocytes but not wt astrocytes. these results collectively suggest that energy defects in bachd mice are due to noncell autonomous mechanisms by which astrocytes expressing mhtt induce neuronal metabolic dysfunction. naag and naa are abundant metabolites in the brain which can be utilised by oligodendrocytes to make myelin. but elevated levels of naag and naa are associated with myelin loss in the leukodystophies canavan and pelizaeus-merzbacher-like disease, whereas lower levels of naa are observed in brain tissue of ms patients. currently, the physiological function of these two metabolites as well as their role in white matter pathology is unknown. naag and naa can also act as signalling molecules as they can affect glutamate receptors. oligodendrocyte precursor cells (opcs) express nmda receptors and activation of these receptors may be important for their differentiation and efficient myelination. in disease opcs are recruited to demyelinating lesions where they proliferate and differentiate into new myelinating oligodendrocytes. we therefore investigated the effects of both physiological and pathological concentrations of naa and naag on opc biology in vitro. we used calcium imaging to see whether naag or naa can induce intracellular calcium changes in opcs as they are known to act on neuronal nmda receptors. 1mm naa or 1mm naag evoked intracellular calcium changes in opcs (df[340/380] 44.6 6 0.7 3 10 23 and 9.0 6 3 3 10 23 respectively), but only one third of the cells responded which corresponds to the proportion of nmda responsive cells in the cultures. however, not all opcs that responded to nmda did also respond to naag or naa. therefore, it seems that the naa and naag evoked calcium raise is via an nmda receptor independent pathway. naag and naa evoked a small but significant increase in intracellular calcium in opcs, which even after prolonged application of pathological levels of naa and naag did not affect the cells' viability as propidium iodide staining of opcs showed no significant difference in viability. over 90% of the opcs survived 4hrs application of 1mm naa and 1mm naag. naag and naa are produced and released by neurons but it is not known how opcs take them up from the environment and how it affects them. we therefore addressed which possible transporters opcs express and the effect naa and naag have on opcs' proliferation and differentiation. it is unlikely that naa and naag even at high concentrations have a detrimental effect on opcs. perhaps these high concentrations of naa and naag found in canavan and pelizaeus-merzbacher-like disease indicate rather opcs' deficiency to transport and utilise naa and naag for energy and myelin formation. vanishing white matter (vwm) disease is a severe leukoencephalopathy, classically starting in childhood. the disease is characterized by neurological symptoms, including uncoordinated movements and balance disturbances which are slowly progressive. episodes of rapid deterioration are triggered by febrile infections and minor head trauma, which can lead to coma. a treatment is unavailable and eventually all patients die within a few years upon diagnosis. the disease is caused by mutations in any of the five genes encoding the subunits of the eukaryotic translation initiation factor 2b (eif2b). although this protein has an important function in all cells, mainly the cells in the brain white matter (astrocytes and oligodendrocytes) are affected by these mutations. pathologically the brain shows cystic degeneration with meager gliosis, dysmorphic astrocytes, lack of myelin and an increased number of oligodendrocyte and astrocyte progenitor cells. we have developed two mouse models for vwm to further unravel the disease mechanisms at molecular level and to develop and test possible treatments. the 2b4 mouse line is homozygous for a mutation in the eif2b4 gene, encoding the eif2bd subunit, and the 2b5 mouse line is homozygous for a mutation in the eif2b5 gene, encoding the eif2be subunit. both mutations have been described in human patients and are associated with a severe form of vwm. preliminary data show that these mice models recapitulate the clinical and neuropathological phenotype observed in vwm patients. pathologically, the central nervous system white matter is vacuolated and shows lack of myelin while astrocytes are dysmorphic and immature. these transgenic mice appear to be a representative model for vwm. microglial cell lines were differentiated from ips cells and represent in vitro models for human microglia. rt-pcr and flow cytometry analysis of ipsdm showed gene transcription and protein expression of cd33 respectively. the protein expression was increased by breaking the cis-interacting sialic acids on the microglial glycocalyx using sialidases. cd33-fc fusion protein was designed and purified via the fc tag to study binding patterns of cd33 to various cell types expressing different glycocalyx structures. additionally, ipsdm exhibiting either an over expression or knockdown of cd33 will be used to determine protein functions. furthermore, preliminary immunohistochemical analysis of control brain tissue compared to ad patients showed increased cd33 protein expression in cd68 positive cells, in the latter. in summary, data show that cd33 is expressed by human microglial cell line enabling further characteristic and functional analysis of the protein. furthermore, preliminary analysis on primary brain tissue corroborates the association of cd33 -expressed on microglia -to lateonset ad. the neurotrophins are essential for peripheral nervous system development and myelination. we have previously demonstrated that the neurotrophin bdnf exerts contrasting influences upon myelinationacting through neuronal p75 ntr to enhance myelination, but inhibiting it via neuronal trkb. recently we have generated a small peptide called cyclo-dpakkr that structurally mimics the region of bdnf that binds p75 ntr . here we aim to investigate whether utilising cyclo-dpakkr to selectively target p75 ntr is an approach that could exert a unified promyelinating response. like bdnf, cyclo-dpakkr promoted myelination of ngf-dependent neurons in vitro, an effect dependent on the neuronal expression of p75 ntr . importantly, cyclo-dpakkr significantly enhanced the myelination of bdnf-dependent neurons in vitro, whereas bdnf inhibited it. local injection of cyclo-dpakkr adjacent to the neonatal sciatic nerve in vivo significantly enhanced myelin protein expression and increased the number of myelinated axons. we found that injection of cyclo-dpakkr also significantly upregulated the expression of neuregulin 1 type-iii, a key factor required to induce peripheral myelination. thus, our data demonstrate that cyclo-dpakkr promotes peripheral myelination in vitro and in vivo. to investigate whether cyclo-dpakkr promoted the remyelination of peripheral neurons, we utilized the ean, a rodent model of peripheral demyelinating neuropathy. our data show that administration of cyclo-dpakkr significantly delayed the disease onset and reduced clinical severity in ean. this improved disease phenotype is supported by reduced demyelination, as cyclo-dpakkr substantially reduced the extent of myelin damage in myelinated peripheral nerves subjected to ean. collectively, our data demonstrate that using cyclo-dpakkr to selectively target p75 ntr promotes peripheral myelin development, and is effective at ameliorating ean disease and protecting against myelin damage. our findings suggest that selective targeting of p75 ntr is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases introduction huntington's disease (hd) is a neurodegenerative disease characterised by huntingtin protein misfolding and the intra-cellular accumulation of mutant huntingtin (mhtt). accumulation of mhtt is most pronounced in neurons and is thought to underpin neuronal dysfunction. oligodendrocytes have been shown to accumulate mhtt aggregates, which may contribute to cellular dysfunction in these cells. evidence from diffusion tensor mri (dt-mri) studies shows that white-matter changes are amongst the earliest pre-symptomatic changes observed in hd. these findings have been taken to implicate axonal and or glial aberrations prior to disease onset. methods we have used a variety of techniques, which include, emulsion in situ hybridisation, whole brain sub-fractionation, immunohistochemistry, meso scale cytokine assay, and western blots. results our investigations using the r6/2 mouse model show that mhtt aggregates result in a specific down-regulation of the small heat shock protein (shsp) hspb5 (alpha-b-crystallin). localisation of hspb5 by whole brain sub-fractionation shows that hspb5 is enriched in the myelin fraction. emulsion in situ hybridisation further shows localises hspb5 in oligodendrocytes. detergent extraction of myelin fractions show differential hspb5 partitioning between soluble and insoluble fractions. cumulatively, these findings suggest that hspb5 is constitutively expressed in oligodendrocytes and is present in two distinct pools. recent studies implicate hspb5 as being a negative downregulator of inflammation. as such down-regulation of the shsp in r6/ 2 mice may promote increased oligodendrocyte vulnerability to mhtt cytotoxicity. to investigate the role of hspb5 as a negative regulator of inflammation, we obtained transgenic hspb5 knockout mice. the knockout mice are viable and show very little phenotypic difference against littermate controls. we have analysed the inflammatory profiles of these mice using the meso scale. to validate our findings in the r6/2 mice, we are currently investigating human tissue from distinct vonsattel stages of disease, to see if hspb5 downregulation is also observed in the human disease. conclusion as hspb5 has several critical cellular roles, its downregulation may compromise oligodendrocyte structure and function, which ultimately has negative consequences for neurons. it is therefore worthwhile to investigate it's specific role in the cns. the myelin sheath is attached to the axon through multiprotein complexes that form the axon-glial interactions. they consist of cell adhesion molecules and voltage gated ion channels and divide the axon into distinct domains: the node of ranvier, the paranode, the juxtaparanode and the internode. this molecular organization is crucial since recent studies identified distinct perinodal proteins as autoantigens in ms patients: nodal and paranodal neurofascin isoforms and juxtaparanodal tag-1/contactin-2. tag-1 is a cell adhesion molecule expressed both by axons and glial cells. in the adult nervous system, tag-1 organizes the juxtaparanodal domain of the fiber, where it interacts with caspr2 and the potassium channels (vgkcs). question: in this study we have analyzed the alterations of the juxtaparanodal proteins caspr2, tag-1 and vgkcs in relation to paranodal caspr and nodal sodium channels upon the onset and progression of experimental autoimmune encephalomyelitis (eae) and in the cuprizone model of toxic demyelination. methods: we immunohistochemically and biochemically analyzed both models of cns demyelination. results-conclusions: our results show a higher paranodal susceptibility to disruption compared to juxtaparanodes in eae. as eae progresses there is subsequent compensatory efforts for myelinated fiber restoration that include paranodal protein re-clustering and increased heminodal formation, without subsequent juxtaparanodal protein aggregation. in contrast, juxtaparanodal organization was observed only when remyelination occured in the cuprizone model of toxic demyelination. supported by: imbb-forth scholarship, manassaki scholarship (university of crete), the project "myelintag" (593) is implemented under the "aristeia" action of the "operational programme edu-cation and lifelong learning" and is co-funded by the european social fund (esf) and national resources. multiple sclerosis (ms) is a chronic disease of the human central nervous system that is characterized by focal lesions with inflammation, infiltration of immune cells, demyelination and axonal damage. activation of cannabinoid cb 1 /cb 2 receptors is considered a potential therapeutic strategy for the treatment of ms, based on the evidence that exogenous cannabinoid agonists exert neuroprotective and immunosuppressive effects in experimental models of the disease. nevertheless, the therapeutic use of synthetic and/or plant-derived agonists acting on brain cannabinoid receptors is limited by the possible adverse responses related to memory and learning impairment. an alternative approach that could avoid this limitation consists of enhancing the concentration of the main endocannabinoids (aea, 2-ag) by increasing their synthesis or decreasing their degradation. the main objective of this study was to analyze the effects of jzl184, a selective inhibitor of 2-ag hydrolyzing enzyme monoacylglycerol lipase (magl), in the chronic eae model of ms. mice were treated daily with jzl184 (8 mg/ kg and 32 mg/kg) or vehicle from onset of the motor symptoms to the end of the experiment. comparison of the motor score curves indicated that both doses of jzl184 ameliorated the deficits observed in vehicletreated mice during the disease course. nevertheless, the beneficial effect of the 32 mg/kg dose was no longer evident in mice scored at 40 dpi. chronic treatment with 8 mg/kg jzl184 reduced the conduction latency of the corticospinal tract measured at the end of the experiment, as well as the number and size of inflammatory lesions in spinal cord, whereas chronic administration of the high dose of the magl inhibitor had no effect on these parameters. importantly, treatment with the 32 mg/kg dose of jzl184 reduced the coupling ability of cannabinoid receptors to g i/o proteins, measured by [ 35 s]gtpcs autoradiography, in all the brain areas analyzed. finally, incubation with jzl184 prevented cytotoxicity by activation of ampa-type glutamate receptors in oligodendrocyte precursors in vitro. this protective effect of the jzl184 was blocked by the cb 1 receptor antagonist am281. our findings point to the involvement of cb 1 receptors in the in vivo therapeutic effects of jzl184, and suggest that chronic administration of magl inhibitors may be a promising strategy for the treatment demyelinating disorders in which oligodendrocyte excitotoxicity plays an important role as mechanism of white matter damage. under disease conditions, connexins can also release these signaling molecules into the extracellular milieu through hemichannels. since astrocytes are involved in the disease progression of als, we are exploring if abnormal gj activity can serve as a potential mechanism for the reported astrocyte-mediated toxicity. we used the sod1 g93a mutation as a als mouse model to address the role of connexins in als. we observed that at the end stage of the disease, spinal cords of sod1 g93a mice exhibited a significant increase in expression of connexin 43 protein compared to their spinal cords of control mice. this increase in cx43 co-localizes primarily to astrocytes in the gray matter of the spinal cord. importantly, human post-mortem tissue from als patients compared to control patients exhibited a significant increase in cx43 rna and protein levels in the motor cortex and a trend for increase in cx43 in the cervical spinal cord. this is an important finding in pursuing cx43 as a potential candidate contributing to astrocyte-mediated toxicity in als. in addition, when astrocytes were isolated from wt and sod1 g93a mice, endogenous levels of cx43 rna and protein were elevated in sod1 g93a mice compared to the wt derived astrocytes. we are currently investigating if functional properties of astrocytes from sod1 g93a mice are altered in addition to biochemical changes. in vitro studies using cx43 specific blockers to assess neuroprotection are also being conducted in co-cultures of neurons and astrocytes. these findings will be novel and an important step towards harnessing therapeutic strategies for als. the pdz protein omi/htra2 is localized in the intermembrane space of mitochondria. it is involved in mitochondrial homeostasis and may act as a chaperone. mutations in the serine protease domain of omi/ htra2 (mnd2 mice), leads to massive loss of striatal neurons and neuromuscular disorders in mice confirming the protective function within mitochondria. upon cellular stress, omi/htra2 is translocated from mitochondria into the cytosol when the mitochondrial outer membrane is permeabilised. in the cytosol, omi/htra2 blocks the action of the inhibitors of apoptosis proteins (iaps) by competitive binding or by degradation via the protease activity. this leads to caspase activation and apoptosis induction. omi/htra2 also regulates apoptosis in a caspaseindependent manner via its protease activity; recent studies have defined multiple substrates of the serine protease. in a yeast twohybrid screen we identified the ng2 proteoglycan as a binding partner of omi/htra2. ng2 is expressed by oligodendrocyte progenitor cells (opc). in the presence of hydrogen peroxide, which is a model of cellular oxidative stress, omi/htra2 is released from mitochondria and can bind to ng2 in opcs. binding of ng2 to omi/htra2 via the pdz domain may thus be a way to sequester or regulate the serine protease. oligodendrocyte-lineage cells are known to be particularly sensitive to cellular stress and white matter diseases of new borns is a major pathology in situations where premature infants are exposed to unphysiological oxygen concentrations. it has been recently shown that glioma cells often express the ng2 protein which may play a protective role. our results indicate that the interaction between omi and ng2 may help protect opcs from cellular stress. microglia are the immunocompetent cells of the cns that continuously survey the extracellular milieu for foreign antigens and play an instrumental role in brain inflammation. in contrast, astrocytes extend highly ramified processes between neurons and synapses and play a vital role in regulating brain homeostasis, synaptic function and plasticity. interestingly, both microglia and astrocytes adopt a specialized 'activated' or 'reactive' state when exposed to adverse brain conditions. the changes in their molecular profiles include the release a diversity of proteins such as pro-and anti-inflammatory cytokines and extracellular matrix molecules that could impact each other's behavior and regulate the properties of nearby neurons. in alzheimer's disease (ad), activated microglia and reactive astrocytes are detected around plaques in mouse models of ad and human ad tissues. however, exactly how these glial types contribute to the onset or progression of ad still remains an open question. to gain a better understanding of the bidirectional communication and response of microglia and astrocytes in the development of ad-like symptomatology, we investigated the properties of these cells types in the crnd8 transgenic ad mouse model by applying confocal imaging and western blot analysis. crnd8 is an early-onset model of ad-like symptomatology showing ab deposits present at 2 months of age and neuritic pathology by 5 months. our results show complex spatio-temporal changes in the interaction of microglia and astrocytes around ab plaques during the progression of the disease in both cortex and hippocampus. we found that subtle rearrangements of microglial morphology and astrocyte reactivity were detected as early as 1 month when ab plaque deposition is absent. by 2-3 months, low numbers of activated microglia and reactive astrocytes begin to surround small ab deposits. the establishment of complex 'reactive glial domains (rgds)' can be observed at 4 months, when amoeboid microglia fully encompass large ab plaques and become surrounded by reactive astrocytes forming an elaborate outer shell-like structure. intriguingly, after 6 months, the domains become progressively disrupted due to the reorganization and recruitment of additional microglia and astrocytes. we also observed gradated inflammatory phenotypes of glial cells. these are first restricted to rgds in the early and middle stages of ad but spread locally in late stages when disorganized rgds are observed. interestingly, neuronal dystrophy was correlated with the severity of the inflammation. we conclude that communication between microglia and astrocytes may be a key process in the ability of the brain to surmount a reparative response to early neurodegenerative conditions but may be disrupted or become overwhelmed in later stages of ad. supported by alzheimer society of canada (db) and by cihr (kkm and rq). poster topic 04 extracellular matrix and cell adhesion molecules opcs exist in mature rodent and human central nervous system (cns; around 5-7% of the total cells) and constitute an interesting source regenerative therapies in demyelinating diseases, like multiple sclerosis (ms). different molecules are involved in opc morphofunctional development during embryogenesis and postnatal stages, and some of them are upregulated in ms lesions, suggesting their involvement in the pathogenesis of the disease. this is de case of anosmin-1, an extracellular matrix glycoprotein coded by the kal1 gene and responsible for the x-linked form of kallmann syndrome. the best known mechanism of action of anosmin-1 seems to be mediated through the interaction of this protein with fibroblast growth factor receptor 1 (fgfr1) and the modulation of the activation of this receptor by fgf2. this protein participates in the adhesion, migration and differentiation of various cell types in the cns; among others, anosmin-1 promotes the adhesion of neurons, neurite outgrowth, axonal guidance and branching of different cns projection neurons, as well as having a role in the migration of different types of neuronal precursors, immortalized gnrh-producing neurons and embryonic opcs. in addition, previous results of our group also suggest a role of anosmin-1 in demyelinated lesions from ms patients and point to the feasible pharmacological and genetic manipulation of the fgf2/fgfr-1/anosmin-1 system on endogenous and/or exogenous opcs in demyelinating lesions. in the present work we studied the functional implications of anosmin-1 and fgf-2 on neurobiology (cell death, proliferation, motility, migration) of postnatal/adult murine opcs isolated from cerebral cortex (p0, p15, p60) and of opcs isolated from adult human biopsies, using both in vivo (immunohistochemistry) and in vitro (chemotaxis chambers, migration, brdu uptake, tunel, immunocytochemistry) techniques. these results would be useful for the design of effective neuroreparative therapies in ms and other demyelinating diseases. funded we generated ipsc lines from fibroblasts isolated from pitx3-gfp knock-in mice which allowed facs-purification of mature midbrain da neurons upon in vitro differentiation based on the expression of the mature da marker pitx3. subsequently, mouse pitx3-gfp knock-in ips cells were transduced with lentiviral vectors containing genes encoding for hl1 and for stx, the enzyme that add psa onto ncam. next they were differentiated in vitro into da neurons. the effects of hl1 and psa-ncam on differentiation, survival and outgrowth of ipsc-derived da neuron were analyzed in vitro and in vivo. indeed, we were able to obtain a pure suspension of mature ipscderived da neurons upon in vitro differentiation and fac sorting. transfection of pitx3-gfp knock-in ips cells with hl1 or stx did not affect the pluripotent potential of these cells and did not interfere with the process of dopaminergic differentiation itself. we continued to establish the beneficial effect of l1 and psa-ncam expression on survival and outgrowth of ipsc-derived da neurons in vitro and after grafting in the striatum of parkinsonian rats. vascular endothelial growth factor (vegf) is a major regulator of neurovascular remodeling following brain injury. while much have been learnt about vegf functions on target endothelial cells, little is known about its intracellular trafficking, mode of secretion and matrix interactions in "source" cells, i.e. mainly reactive astrocytes. here, we generated a vegf::gfp fusion protein to follow the distribution of vegf165 during its trafficking in primary astrocytes and cos7 cells. we found that vegf::gfp forms dimers and gets glycosylated similarly to wild type, and as expected, the molecule follows the endoplasmic reticulum-golgi pathway. however, its post-golgi trafficking suggests a unique route with features that do not conform the classical constitutive secretory pathway: the secretion of vegf165 occurs even at 19 c and shows a ca21-and pkc-induced increase. we investigated the distribution of vegf in polarized primary astrocytes in an in vitro scratch wound assay. in these cells, vegf::gfp appeared to follow a vectorial distribution and accumulated behind the leading edge. the accumulation appeared to be on the extracellular surface, moreover, ultrastructural immunogold labeling revealed that extracellular vegf::gfp remains associated with discrete areas of the cell membrane and is accumulated in caveolae as well as in microvesicular shedding elements. this particular localization corresponds to fibrillar adhesions (fb), where vegf::gfp is co-localized with fibronectin. we also observed that vegf::gfp is targeted to adherens junctions between astrocytes, however, at these sites vegf::gfp was not co-localized with fibronectin. finally, we show in frap experiments that integrin turnover is decreased in vegf associated fbs, which raises the possibility of an autocrine/paracrine regulation of astrocytic functions. together, these findings have strong implications for understanding focal coordination of angiogenesis and vascular remodeling by astroglia derived vegf. after invading the embryonic cns, microglia migrate extensively to occupy their final positions. however, the cellular and molecular mechanisms of microglial migration are unknown. therefore, this study aims to elucidate the microglial migration behavior, cellular interaction partners and ecm-integrin interactions essential for migration in the developing neocortex. methods: immunohistochemical stainings and immunogold labeling for transmission electron microscopy were carried out on fixed brain tissue of c57bl/6 cx3cr1 1/egfp mice embryos (embryonic day (e) 10.5-17.5). multicolor facs analyses were performed on homogenized age-pooled cx3cr1 1/egfp embryonic cortici. microglial migration was recorded in cx3cr1 1/egfp -hgfap-cfp acute brain slices using multiphoton time-lapse excitation and analyzed using mtrackj in imagej. results: laminin (lm) is expressed as punctuate dots near the pia and in the ventricular zone at e10.5 and disappears at e15.5 to remain only in blood vessels and the pia. fibronectin (fn) is present as aggregates from e10.5 until e17.5. both ecm proteins follow the course of radial cells. preliminary data indicate a transient upregulation of the lm receptor (lmr) on e15.5 and e16.5. in addition, the percentage of microglia expressing the fn receptor (fnr) tends to decrease during development. ex vivo time-lapse recordings show that embryonic microglia are dynamic cells that migrate in random patterns. these cells seem to make brief contacts with the processes of radial glia. conclusions: embryonic microglia express laminin and fibronectin receptors and possibly change the expression level during development. they are dynamic cells that migrate in random patterns, and possibly make occasional contact with radial glia. knowledge about ecm-integrin interactions during microglial migration could reveal targets for intervention in pathological settings, such as multiple sclerosis and alzheimer's disease. multiple sclerosis (ms) is a chronic demyelinating disease of the central nervous system in which astroglial cells become hypertrophic, produce various extracellular matrix (ecm) proteins which become aggregated when secreted. these aggregated ecm proteins contribute to a non-permissive environment for repair. tissue transglutaminase (tg2) expression is enhanced in astrocytes in ms lesions. this enzyme is well-known for protein cross-linking capacities. moreover, tg2 can act as a co-receptor for b-integrins to bind to ecm proteins, thereby mediating cell adhesion processes. in this study we questioned whether astrocyte-derived tg2 affects ecm protein production and/or aggregation, and if astrocyte-derived tg2 mediates cell adhesion onto various ecm proteins. primary rat astrocytes were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg2. alternatively, primary rat astrocytes were transduced with a lentiviral vector expressing scrambled shrna or tg2 shrna to knockdown endogenous rat tg2. the rat primary astrocytes overexpressing human tg2 showed an increased production of fibronectin and collagen v. conversely, knocking-down tg2 showed a decrease in fibronectin and collagen production. interestingly, overexpression of tg2 resulted in enhanced aggregation of extracellular laminin. also, human astrocytoma cells (u373) were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg2 and allowed to adhere onto ecmcoated wells. astrocytoma cells overexpressing human tg2 showed increased adhesion onto laminin-, and fibronectin-coated wells. we conclude that astrocyte-derived tg2 affects production/aggregation of fibronectin and laminin. moreover, the astrocyte-derived tg2mediated elevated production/aggregation of fibronectin and laminin coincides with more adherence of astrocytes onto these ecm proteins. we hypothesize that tg2-mediated enhanced production/aggregation of fibronectin and laminin is related to increased astrocyte adhesion which together could contribute to the non-permissive environment for repair in the central nervous system of ms patients. a. tripathi, z. parikh, p. pillai the m.s. university of baroda, vadodara, india background: oligodendrocytes originate as progenitor cells (opcs) in discrete areas of the developing brain which undergoes a complex and precisely timed program of proliferation, migration, differentiation, and myelination in cns. during migration it interacts with its surrounding environment through integrins. purpose: to evaluate the interactions between integrins and growth factors (gfrs) that promote the molecular events such as proliferation, cytoskeletal reorganization and migration in oligodendrocytes leading to oligodendrocyte mediated myelination. methods: cortical cells were prepared from p0 rat cerebral cortex following standard protocols. immunocytochemistry (icc), western blot (wb), immunoprecipitation (ip), migration assay and real-time rt-pcr analysis were performed. for dissecting out the roles of different intracellular signalling proteins in the regulation of events downstream of receptor activation, use of pharmacological inhibitors was carried out. results: tnfa, pdgfa and estradiol significantly increased cell proliferation and cell processes. data also suggest differential effects of extracellular matrix (ecm) on signalling component activation. significant increase in erk expression was found following gfrs-integrin interactions. ecm proteins bind to integrin family members and mediate bidirectional signalling between the cellular interior and the external environment through the activation of focal adhesion complexes. conclusion: integrin switching, oligodendrocyte proliferation, cytoskeletal reorganization and differentiation profile is highly influenced by ecm and gfrs. integrin governs the signalling cascades underlying oligodendrocyte differentiation and myelination. this study was performed in strict accordance with the recommendations for the care and use of laboratory animals. all the protocols were duly approved by the institutional ethical committee, the m.s. university of baroda. topography, roughness and mechanical properties of micro environment are crucial parameters influencing cell adhesion/motility, morphology and mechanics as well as the development of cells e.g. stem/progenitor cells, and neuronal cells [1] [2] [3] [4] [5] . atomic force microscopy is a powerful tool not only to study the morphology in terms of high resolution imaging and roughness measurements, but also to map mechanical and adhesive properties of the sample/cells and tissues. combining these remarkable abilities with advanced optical microscopy allows for extensive characterization of biomaterials and tissue slices [6] [7] [8] . however, commercially existing technical solutions are either time consuming, or only limited practicable for soft, sticky, or fragile samples. therefore, we developed a new force curve based afm mode -quantitative imaging (qi tm ). the novel qi tip movement algorithm prevents lateral forces and controls the vertical forces for nondestructive imaging. to demonstrate the performance and flexibility qi tm mode we investigated topography, adhesion properties, and young's modulus local distribution of living dorsal root ganglion cells. a specialized platform -cellhesion v r -has been developed to run single cell force spectroscopy (scfs) with a need for long-range cellsurface and cell-cell binding experiments with up to 100 microns pulling length. using single cell force spectroscopy (scfc), cell-cell adhesion can be quantified [e.g. 3], and the contribution of different components e.g. from the extra cellular matrix, can be assessed [9] . a new technical solution will be demonstrated if a cantilever attached cell can be transferred through the liquid-air interface. this approach allows to measure the adhesion of the same individual single cell on different materials within different dishes as it will presented for cho cell adhesion on plastic as well as albumin coated surfaces. we want to present the progress in automatic data processing and display to investigate topography, young's modulus and local adhesion phenomena on transparent and non-transparent biomaterials like titanium, peptide functionalized surface or cell layers, and tissue slices. l. vargova 1 , m. cicanic 1,2 , e. sykova 1,2 1 charles university, 2nd faculty of medicine, prague, czech republic 2 institute of experimental medicine, v.v.i., department of neuroscience, prague, czech republic bral-2 is a link protein that is localized in distinct brain regions, where it stabilizes the perineuronal nets of the extracellular matrix (ecm). our previous studies showed that qualitative and quantitative ecm changes, might evoke changes in the diffusion properties of the extracellular space (ecs), thus affecting the movement of neuroactive substances in the cns. in the current study, we determined by the real-time iontophoretic method the extracellular space volume fraction a (a 5 ecs volume / total tissue volume) and the geometrical factor tortuosity k (k 2 5 free / apparent diffusion coefficient) in coronal brain slices obtained from the sensorimotor cortex and the ventral posteromedial thalamic nucleus of sixteen-month-old bral-2 deficient mice (bral-2 -/-), age-matched bral-2 1/1 controls and young adult (5-month-old) bral-2 1/1 controls. the diffusion parameters in the cortex of young adult bral-2 1/1 mice were: a 5 0.21 6 0.01 (mean 6 s. e. m.) and k 5 1.48 6 0.02, n 5 6, n 5 5 (n -number of slices, n -number of animals). in the thalamus, there was a smaller a and a larger k (0.16 6 0.01 and 1.53 6 0.01, respectively, n 5 26, n 5 10). in young adult mice, we found no significant differences in the ecs diffusion parameters between bral2 positive and negative mice in either the cortex or the thalamus. in the cortex of aged bral-21/1 mice we found a smaller a (0.18 6 0.01, n 5 10, n 5 7) than in the cortex of young adult mice. during aging, there was no significant difference in the thalamic diffusion properties in wild-type animals, but in the thalamus of aged bral-2 -/-mice, a significantly decreased (0.13 6 0.01, n 5 28, n 5 11) in comparison to the younger bral2-/-animals as well as to age-matched controls. immunohistochemical analysis of the ecm in the ventral posteromedial thalamic nucleus revealed no positivity for bral2 protein in either group of bral2-/-mice. in the youger mice, bral deficiency was associated with a small attenuation in the positivity of brevican and another link protein, ctrl1; however, in aged bral2-/-animals, brevican and ctrl1 positivity was increased in comparison with the age-matched controls. in contrast to the cortex, we found no decrease in a in the thalamus of aged wt animals, suggesting that the process of aging may differ between the cortex and the thalamus. a decrease in the ecs volume fraction in the thalamus of bral-2 -/-aged mice may result from a disruption of the perineuronal nets and rearrangements of the molecular assembly, which seem to differ in young adult and aged animals. here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of camp pathways. a bulk of astroglial transcripts were differentially regulated by camp signaling. camp analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. moreover, genes typically activated in reactive cells, such as immunological mediators and scar components, were repressed by camp. gene set enrichment analysis and evaluation in situ of gene expression in astrocytes in different states showed that camp signaling conferred a mature and in vivo-like transcriptional profile to cultured astrocytes. these results indicate that camp signaling is a key pathway restricting developmental and activation features of astrocytes and promoting their maturation. a positive modulation of camp signaling is suggested to suppress the mechanisms of activation driven by pathological situations and to promote the physiologically normal state of differentiated astrocytes. question: microglia, the innate immune system of the central nervous system, are crucial to maintain homeostasis by cleaning debris and attacking stressors. in the ageing brain, increased numbers of microglia with ameboid morphology and increased inflammatory cytokine levels are reported. the altered microglial phenotype in the ageing brain is characterized by hyperresponsive to immune stimuli, which is called immune priming. immune priming could be a potential cause of age-associated neurodegeneration. the aim of this study is to characterize the immune pathways and biological processes that are altered in ageing-induced immune primed microglia and to find genes potential drivers of this process. design/methods: the effect of ageing on microglia has been addressed in naturally aged mice (24 months old) and in ercc1 transgenic mouse model in which accelerated ageing is induced by dna damage accumulation. from each of these models a pure population of microglia was isolated. microarray technology was used to obtain information about the genome wide gene expression profile. weighted gene co-expression network analysis (wgcna), uses correlational structures to find clusters of genes to make optimal use of a dataset in biological interpretation. results: using weighted gene co-expression network analysis, a very robust microglial immune priming gene module was discovered. in this module, there is a significant enrichment of the complement system, antigen presentation, interferon type-1 signaling and phagocytosis-machinery. the average expression of this module is stronger up-regulated in ercc1 transgenic than in normally physiologically aged mice, supporting the notion that ercc1 is characterized by excessive immune priming. conclusion: microglial priming which occurs in normal ageing or in accelerated ageing models is marked by increased expression of several immune signaling pathways. methods: we investigated three single nucleotide polymorphisms (snps) (rs1042713 (arg16gly), rs1042714 (gln27gly) and rs1042711) and five haplotypes in adrb2 to explore whether these polymorphisms were associated with ms progression in 436 well phenotyped ms patients. genotyping was done using illumina bead microarray and missing genotypes were imputed using beagle. results: a trend towards a decreased frequency of rs1042714c allele (27gln) was in people with progressive ms than in people with relapsing remitting ms. this was seen in both sp-ms (p (uncorrected) 5 0.0072) and in pp-ms (v 2 5 6.85, p (uncorrected) 5 0.0325). however, those p values did not reach statistical significance after correction for multiple testing. neither haplotypes nor snps were significantly associated with altered msss, age of onset, time between fist and second relapse, processing speed (sdt) or brain atrophy. conclusion: we found no evidence that polymorphisms in adrb2 influence severity in multiple sclerosis. here we examine the expression of these brain-specific pgc-1a isoforms in neurons and glia cells. we characterize the abundance of the different isoforms of pgc-1a in primary murine cell cultures of neurons, glia cells, including oligodendrocytes, astrocytes and microglia under control conditions and metabolic stress. we show that pgc-1a is differentially expressed in the different cell types, e.g. neurons and oligodendrocytes express much higher levels of the novel brain-specific isoforms of pgc-1a. the different expression levels might reflect the different metabolic needs of the different cell types. s. marques, g. castelo-branco karolinska institutet, stockholm, sweden oligodendrocytes (ol) are neuroepithelium-derived cells that insulate neuronal axons through myelin-containing membranes, essential for axonal integrity and impulse transmission. defects in this process are present in several diseases, including the highly debilitating multiple sclerosis (ms). the process of remyelination can be promoted by ol precursor cells (opc) endogenously and occurs during a short window of time at earlier stages of ms, ultimately failing as the disease evolve. the understanding of development and differentiation regulation of ol is essential to clarify the reasons behind the defective myelination during pathologies and to open the pave to new remyelinating cell therapies. opcs start to be specified early during embryogenesis but regardless of the origin, their terminal differentiation and functional maturation occurs only at post-natal stages. in this project, we are characterizing the transcriptome of opcs during development, which will allow us to better characterize the heterogeneity of these cells and point out what might be the key factors involved in transition between states. recent studies suggest that non-coding rnas can interact with proteins complexes containing chromatin modifying enzymes and transcription factors, regulating their activity, acting as scaffolds and/or recruiting them to specific loci in the genome. therefore, we are investigating their role in opcs by rna interference and how they might modulate the function of key transcription factors. neural stem cells (nscs) represent a potentially valuable resource when considering repair strategies for cns damage. unlike their embryonic counterparts, adult neural stem cells (anscs) in the two neurogenic niches have an astrocyte-like identity, raising the question of what regulates this ansc state. some early postnatal astrocytes in the parenchyma also retain some nsc-like characteristics and in vitro display self-renewal and multipotency, though these properties are lost following in vivo maturation. interestingly, following injury, adult astrocytes can become reactive and reacquire nsc-like properties, whilst others can generate new neurons through forced expression of specific transcription factors. we are aiming to elucidate the gene regulatory networks underlying specific nsc and astrocyte states and transitions between them using transcriptomic and epigenomic tools alongside computational methods in different nsc and astrocyte populations. we will describe our latest findings from an in vitro system that aims to model immature versus mature astrocytes derived from a common progenitor. from microarray data, we have identified candidate genes and pathways involved in regulating astrocyte potential versus differentiation, including a role for specific pro-inflammatory signalling. we will also describe the associated epigenetic signatures that accompany cell state and discuss the implications of our latest findings. microglia are the cns immune-related cells and associated with the pathogenesis of several types of neurological diseases. following peripheral nerve injury (pni), spinal microglia become activated and have crucial roles in generating neuropathic pain. we have previously shown that interferon regulatory factor-8 (irf8), upregulated in spinal microglia after pni, regulates gene expressions that switch to a reactive phenotype. here we identified irf5 as a crucial factor of reactive microglia that works cooperatively with irf8. we found that pni increased expression of irf5 in the spinal cord, the expression of which was restricted to microglia. forced expression of irf8 in cultured microglia markedly increased expression of irf5 in a manner that depended on its ability to bind dna. furthermore, irf8-deficient mice failed to increase the expression of irf5 in spinal microglia after pni, indicating irf8-dependent expression of irf5 in microglia in vivo. interestingly, upregulation of p2x4 receptor (p2x4r; an atp-gated channel crucial for producing neuropathic pain) by forced expression of irf8 in cultured microglia was markedly suppressed by knockdown of irf5 expression. in a model of neuropathic pain, suppressing upregulated irf5 protein by spinal administration of sirna targeting irf5 alleviated pain hypersensitivity. altogether, the irf8-irf5 axis drives a program of gene expression that promotes p2x4r hi microglia gating neuropathic pain. astrocytes are the most abundant glial cell type. they express a range of neurotransmitter receptors localized along their processes that cover synapses and constitute "microdomains" for signalling pathways. to investigate astroglial purinergic p2y1 and glutamatergic nmda receptors we generated knockout mice in which gene recombination of "floxed" receptors was controlled by an astrocyte-specific and tamoxifen-inducible cre recombinase (glast-creert2). here, we present data on the dna recombination efficiencies of glast-creert2 x floxed p2y1 mice and glast-creert2 x floxed glun1 mice in different brain regions (cerebellum, hippocampus, brainstem, optic nerve and cortex). recombination is determined by quantitative real-time pcr of genomic dna using primers across the loxp sequences flanking exon 1 of the p2ry1 gene and exon 11-21 of the grin1 gene, respectively. dna recombination was induced in young adult mice by intraperitoneal tamoxifen injection for three consecutive days and analyzed three weeks later. primers were designed such that reduction of the pcr signal reveals increasing recombination (i.e. gene excision and knockout) compared to control. the degree of reduction in the investigated brain regions is expected to be similar in both mouse lines; allowing conclusions (1) about the reliability of the glast-creert2/loxp system in general (i.e. recombination efficiency at different chromosomal locations), and (2) gives a percentage of glastpositive cells (predominantly astrocytes) in the given brain area. first data demonstrate 30% recombination in the cortex, which is well in line with the percentage of astrocytes in this brain region. these results could be confirmed by using a reporter mouse line expressing the red fluorescent protein tdtomato (r26-tdtomato) and using immunohistochemistry with astrocyte markers. in conclusion, the combination of quantitative real-time pcr analysis of gene recombination and cell-specific cre mouse lines represents a reliable approach to reveal the percentage of a given cell type in a given tissue region. the transcription factor creb is protective in injury and neurodegeneration, but the contribution of neuronal vs astrocytes is unknown. we have generated a mouse with targeted over-expression of a constitutively active creb (vp16-creb) in astrocytes by crossing teto-vp16 and tta-gfap mice. vp16-creb/gfap mice show expression of vp16 in astrocytes -as detected by immunohistochemistry -restricted to cerebellum, hippocampus and outer layers of cortex. we determined the role of astrocytic creb in the outcome of brain injury by subjecting wild type (wt) and vp16-creb/gfap (tg) mice to a focal cryolesion (c) in the frontal cortex. at 3 days post-lesion, wt mice presented a necrotic region filled with macrophages (labeled with lectin) surrounded by a rim of tissue with robust gliosis (labeled with gfap). tg mice showed reduced macrophage infiltration as compared with wt mice. a dna array analysis of the damaged zone (agilent) has revealed differentially expressed genes in paired comparisons. wtc/wt: 13,984; wtc/tgc: 5,821; tgc-t:913; tg/wt:0 (statistical linear model in limma., p values computed by empirical bayes moderated t-statistics at 0.05). there were 159 significantly enriched kegg pathways regulated by the cryolesion, according to gsea and hypergeometric tests set at p < 0.05. vp16-creb had an effect on 127 of the pathways. upon increasing the analysis stringency by setting the cut-off at p < 0.0001, the number of significantly enriched kegg pathways regulated by vp16-creb was reduced to 16. we selected 10 of these pathways related to 5 functions: energy metabolism, inflammation, structural reorganization, genomic stability and apoptosis, which we define as the core signature of the vp16-creb-elicited change. the differential expression of representative genes related to this signature was validated by real-time pcr. thus, vp16-creb rescued the decreased expression of enzymes regulating energy metabolism, while decreasing the expression of pathways related to apoptosis, extracellular matrix and inflammation. the latter is consistent with the decreased macrophage infiltration detected by immunohistochemistry in tg mice. we posit that vp16-creb protects the brain against bionergetic failure and secondary damage due to macrophage infiltration, while rendering the tissue more conducive to neurite regeneration by reducing extracellular matrix components. the study supports the notion of astrocytes as therapeutic targets in brain injury. furthermore, ca 21 /cn/nfat dependent mmp3 expression was confirmed in pure astrocyte cultures derived from neural stem cells (ast-nsc), demonstrating that the induced mmp3 expression occurs in astrocytes, and not microglial cells. in an in vivo stab-wound model of brain injury, mmp3 expression was detected in nfatc3-positive scarforming astrocytes. since [ca 21 ] i increase is an early event in most brain injuries, these data support an important role for ca 21 /cn/ nfat-induced astrocyte mmp3 expression in the early neuroinflammatory response. understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain. the posterior hypothalamus (ph) is crucial for maintaining wakefulness whereas the anterior hypothalamus (ah) constitutes a sleep-promoting region. consequently, the energy needs of ah and ph should probably change throughout the sleep-wake cycle. the glycogen mobilization altogether with the astrocyte-neuron lactate shuttle (anls) likely constitutes two major mechanisms by which astrocytes ensure the neurometabolic coupling. therefore, we hypothesized that astrocytes of ah and ph may display differences in genes expression involved in these mechanisms during the sleep-wake cycle. to investigate this, we measured the gene expression levels in astrocytes obtained from transgenic mice expressing the fluorescent protein egfp under the control of the astrocyte-specific gene gfap promoter. mice were sacrificed at four time, zt0, zt6, zt12 and zt18 (zt0 corresponding to light-on). after brain dissection, "punches" of ah and ph were micro-dissected from slices. cell suspensions were obtained from a pool of 3 samples by enzymatic digestion and cell trituration. then, gfp-positive cells, which were highly enriched in astrocytes, were sorted by facs. total rna was extracted from these cells and gene expression levels were assayed by qrt-pcr and normalized by cyclophylin mrna levels. seven genes related to anls were tested: the alpha 2 sub-unit of the na-k atpase (atp1a2), the two sub-unit of the lactate dehydrogenase (ldha and ldhb), the glutamate transporters (glast and glt1) and two monocarboxylate transporters (mct1 and mct4). levels of mrna encoding genes related to glycogen metabolism such as ptg, the glycogen synthase (gs) and the glycogen phosphorylase (gphos) were also measured results showed that the levels of mrna encoding ldha, mct1 and mct4 expressed significant circadian variations which can reach 200% for mct4 at zt0, compared to zt6 levels in ah. the glt1 mrna levels as well as mct1 and mct4 mrna levels also displayed significant circadian variations in ph. levels of expression of genes encoding the gphos, gs and ptg did not display major regional circadian variation. this study shows that astrocytes displayed some transcriptional regulation in hypothalamic areas involved in the sleep-wake cycle. the difference in the pattern of expression of anls related genes in ah and ph, such as glt1, suggests that neuro-metabolic coupling capacity of each structure might be different and might play a functional role in the sleep-wake regulation. , are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter 1 (glt-1) with significantly increased levels of glt-1 in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. to study glt-1 regulation in more detail, we generated a family of transgenic mice using increasing lengths of the 5 0 non-coding region of the glt-1 gene controlling expression of tdtomato. these mice were crossed with full length bac-glt-1-egfp mice to compare astrocytic expression of the reporter genes. interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an 8.3 kb eaat2 promoter fragment mouse with the bac-glt-1 mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat2 expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato1/gfp1 and tdtomato-/gfp1 astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax6 and kir4.1 were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir4.1 mrna was noted to be selectively enriched in cortical tdtomato1/gfp1 cells, compared to gfp1 only cells. it suggests kir4.1 may serve as an astroglial subtype marker. . we have hypothesized that microglial c/ebpb is a potential target to attenuate the neurotoxic effects of neuroinflammation. in order to test this hypothesis in vivo, c/ebpb knockout mice are not suitable because they show multiple phenotypic alterations as a result of the widespread expression of c/ ebpb. mice with specific c/ebpb deletion in microglia would be a better model. unlike gfap promoter for astrocytes, there is not a gold standard promoter for cre expression in microglia. cd11b, cx3cr1 or lysozime m (lysm), among other promoters, have been used to this end. we have generated lysm-cre/c/ebpb fl/fl mice with two main aims: 1) to validate the use of lysm as a suitable promoter for microglial gene deletion by cre-lox recombination 2) to analyze the effects of the absence of microglial c/ebpb in vitro and in vivo lysm-cre/c/ebpb fl/fl mice which were fertile and viable and did not show the female infertility and high perinatal mortality described in c/ ebpb knockout mice. western blot experiments revealed the presence of c/ebpb in protein extracts from wild-type but not from lysm-cre/c/ ebpb fl/fl microglial cultures. immunocytochemistry experiments confirmed this observation. thus, c/ebpb immunoreactivity was present in all cells in wild-type microglial cultures and it was absent in all microglial cells in lysm-cre/c/ebpb fl/fl microglial cultures. the microglial specificity of lysm-cre recombination was demonstrated in primary mixed glial cultures. whereas in wild-type primary mixed glial cultures c/ebpb immunoreactivity was observed both in astrocytes and microglia, in lysm-cre/c/ebpb fl/fl mixed glial cultures it was detected in astrocytes, but never in microglia,. to assess the functional outcome of microglial c/ebpb deficiency, we analyzed lps1ifncinduced no production and we observed a marked decrease in lysm-cre/c/ebpb fl/fl microglial cultures. this decrease was also observed in mixed glial cultures in fitting with the microglial origin of no production in this model. in summary, these findings show that lysm-cre promoter causes recombination in 100% of microglial cells in primary culture and it is therefore suitable for microglial-specific gene deletion in vitro. experiments are in progress to determine the degree and specifity of microglial c/ebpb deletion in vivo in these mice. supported by grants pi10/378 and pi12/00709, instituto de salud carlos iii, spain. to study the functional role of cpeb3, transgenic mice overexpressing cpeb3 in astrocytes were generated. cpeb3 overexpression without the endogenous 3 0 untranslated region (utr) led to the downregulation of astrocytic connexins (cx43 & cx30). consistently, in the hippocampus of cpeb3 overexpressing mice, intercellular gap junction coupling was strongly impaired. cpeb3 overexpression also led to downregulation of glutamate transporter 1 (glt-1) and glutamine synthetase (gs), key players involved in extracellular glutamate clearance. we have now raised mice overexpressing cpeb3 with the endogenous 3 0 utr. in these mice, we observed the downregulation of cx43, cx30, glt-1 and gs. since interastrocytic coupling and astrocytic glt-1 and gs activities are downregulated in epilepsy patients with hippocampal sclerosis, we hypothesize that upregulation of cpebs in astrocytes may contribute to the pathogenesis of epilepsy by causing astrocyte dysfunction. to study the influence of the p53 gene in the structural and quantitative characteristics of astrocytes in the mice retina methods: adult mice of the c57bl/6 strain (12 months old) were distributed into two groups: 1) mice with two extra copies of p53 ("super p53"; n 5 6) and 2) wild-type p53 age-matched control, as the control group (wt; n 5 6). retinas were immunohistochemically processed with gfap. gfap1 astrocytes were quantified. results: in comparison con wt: i) retinal-astrocyte distribution followed established patterns; however, morphological changes were seen through the retinas in relation to p53 availability: astrocytes were more robust and secondary processes were more evident; ii) astroglial density was significantly higher in the sp53 retinas, both in the whole-retina (p < 0,01 student's t-test) and in the intermediate and peripheral concentric areas of the retina (p < 0.05 student's t-test). conclusion: the astrocyte changes in the sp53 retinas might improve the resistance of the retinal cells against oxidative stress and its downstream signalling pathways. however, neuronal damage caused due to excessive microglial activation is a well known phenomenon in neurodegenerative diseases. mirnas are novel regulators of gene function, controlling several biological processes. recent reports show altered mirna expression in immune-mediated pathologies, suggesting that mirnas have roles in modulating immune responses. thus, the present study was initiated to identify micrornas and their target mrnas which can modulate microglial inflammatory responses. a pilot study was designed to understand the role of mirna-200b in modulating microglia-mediated immune response as this was found to be localized in the microglia. recently, mirna 200b has been shown to target several proteins including c-jun, the substrate of jnk mapk (mitogen-activated protein kinase) which mediates the release of proinflammatory cytokines in activated microglia. qrt-pcr revealed the decrease of mir-200b expression level in activated bv2 microglia. loss-of-function and gain-of-function studies confirmed c-jun to be the target of mir-200b in microglia. overexpression of mir-200b in activated microglia resulted in a decrease in c-jun and jnk expression and activity, thereby dysregulating the mapk-jnk pathway and proinflammatory cytokines. further, treatment of neuronal cells, mn9d with conditioned medium obtained from activated microglial cells, resulted in increased inflammatorymediated cell death upon knockdown of mir-200b. overexpression of mirna-200b also reduced the phagocytic ability in activated microglia. taken together, these results demonstrate the important role of mir-200b in modulating the mapk pathway via c-jun which in turn affects different aspects of the inflammatory process accompanying microglia activation including cytokine response, no production, phagocytosis and neuronal cell death. thus, mir-200b may prove to be a useful target for developing therapeutic strategies to control microglial mediated inflammation in neurodegenerative diseases. further in order to understand the global mirna changes contributing to microglial activation, a global mirna microarray was carried out using control, lps-activated and amyloid b-activated primary microglia. this screen identified several families of mirnas differentially expressed between the different treatment groups enabling us to analyze the mirna signature in activated microglia. national university of singapore, singapore, singapore an inflammatory process in the brain as a result of an injury or infection is mediated by the microglia, the prime immune effector cells of the central nervous system (cns). microglia have also been shown to display chronic inflammatory responses leading to neurodegenerative disorders. small ubiquitin-like modifier-1 (sumo-1) is a post-translational protein modifier, which has been shown to be associated with nuclear factor kappa b (nfjb)-mediated inflammatory pathway. in this study, we showed the expression of sumo-1 in microglia and characterized the roles of sumo-1 in activated microglia. we found that sumo-1 is specifically expressed in the amoeboid microglial cells of the early postnatal rat brain. secondly, lipopolysaccharide-mediated activation of microglia led to a significant increase in the expression as well the nuclear translocation of sumo-1 in microglia in vitro. in order to establish a function for sumo-1 in activated microglia, we sought to silence its expression using sirna-mediated gene knockdown. sumo-1 knockdown in lps-treated bv-2 microglia in vitro subsequently showed the decreased nuclear translocation of nfjb and expression of tumour necrosis factor-alpha (tnf-a) level. further, we performed an in silico screening to identify the targets of sumo-1 in the nfjb pathway. our study thus, suggests that sumo-1 regulates nfjb translocation into the nucleus for the transcription of pro-inflammatory genes including tnf-a in activated microglia. microglia are the main resident immunological cells the cns. in the healthy brain, microglial cells are in a surveillance state whereas upon rupture of cns homeostasis they enter activated states. in ischemic stroke, activated microglia is one of the most important cellular components of post-stroke neuroinflammation, which mainly occurs in and around the area of the infarct. microglia activation is characterized by morphological alterations, functional and transcriptional remodeling, which account for the acquisition of immune phenotypes. purinergic receptors are known to play important roles in microglia activation, and p2x4r have been suggested as potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain diseases. in the present study, we have developed an experimental approach based on laser micro-capture to isolate microglia from the penumbra area at different post-lesion times (from 4h to 7 days post-lesion). the repertoire of genes expressed by microglial cells was determined through cdna microarray analysis. with this approach we have been able to determine clusters of genes that are co-regulated thus revealing the time course of microglia activation in this model. in addition, we have compared the transcriptional remodeling of microglia in wild-type and p2x4-deficient mice. our results suggest that wild-type and p2x4r ko mice present specific transcriptional profiles, which only partially overlapped. thus our data suggest that p2x4r play significant roles in the regulation of microglial cells functions. z. chen 1 , m. ghosh 2 , r. sattler 1 , m. robinson 2 , j. rothstein 1 1 johns hopkins university, baltimore, united states 2 university of pennsylvania, philadelphia, united states question: astrocytes, the most abundant cell type in the central nervous system (cns), are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter 1 (glt-1) with significantly increased levels of glt-1 in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. methods: to study glt-1 regulation in more detail, we generated a family of transgenic mice using increasing lengths of the 5 0 non-coding region of the glt-1 gene controlling expression of tdtomato. these mice were crossed with full length bac-glt-1-egfp mice to compare astrocytic expression of the reporter genes. results: interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an 8.3 kb eaat2 promoter fragment mouse with the bac-glt-1 mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat2 expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato1/gfp1 and tdtomato-/gfp1 astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax6 and kir4.1 were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir4.1 mrna was noted to be selectively enriched in cortical tdtomato1/ gfp1 cells, compared to gfp1 only cells. it suggests kir4.1 may serve as an astroglial subtype marker. conclusions: our data clearly suggest the existence of molecular subtypes of astrocytes. factor expressed by activated astrocytes and microglia. studies with non-glial cells have shown that c/ebpd is able to regulate the expression of proinflammatory genes. however, the role of c/ebpd in neuroinflammation has not been established. we have here analyzed the effects of c/ebpd absence on glial activation in vitro and in vivo. in primary mixed glial and microglial-enriched cultures the expression of the pro-inflammatory genes nos-2, cox-2 and il-6 induced by lps1ifnc, but not by lps alone, was attenuated in the absence of c/ebpd. chip experiments revealed binding of c/ebpd to the promoters of these genes in activated, but not in control, mixed glial cultures. in neuronal-microglial co-cultures the neurotoxicity elicited by lps1ifnc-activated microglia was completely abolished when microglial cells were devoid of c/ebpd suggesting that this transcription factor plays a key regulatory role of the expression of potentially detrimental proinflammatory genes by activated microglia. to analyze the role of c/ebpd in neuroinflammation in vivo mice were treated systemically with lps (100 ug/mouse; i.p.). this treatment induced a marked increase in c/ebpd mrna and protein brain levels. double immunofluorescence experiments showed the presence of c/ebpd in astrocytes and microglia in lps-treated mouse brain. in c/ ebpd -/-mice, systemic lps-induced brain expression of nos-2, tnfa, il-1b and il-6 was attenuated. finally, in mouse experimental autoimmune encephalitis (eae) c/ebpd was upregulated in the spinal cord and the severity of eae symptoms was reduced in c/ebpd -/-mice. altogether these findings demonstrate that c/ebpd plays a major role in the regulation of proinflammatory gene expression in neuroinflammation and point to microglial c/ebpd inhibition as a novel strategy to reduce the detrimental effects of neuroinflammation. supported by pi10/378 and pi12/709 from instituto de salud carlos iii, spain gene targeting strategies have become a powerful technology for elucidating mammalian gene function. the recently generated knockout (ko)-first strategy produces a knockout at the rna processing level and also allows for the generation of conditional ko alleles by combining flp/frt and cre/loxp systems, thereby providing high flexibility in gene manipulation. however, this multipurpose ko-first cassette might produce hypomorphic rather than complete knockouts if the rna processing module is bypassed. moreover, the generation of a conditional phenotype is also dependent on specific activity of cre recombinase. here, we report the use of an efficient molecular biological approach to test pannexin1 (panx1) mrna expression in global and conditional panx1 ko mice derived from the ko-first mouse line, panx1 tm1a(komp)wtsi . using qrt-pcr, we demonstrate that tissues from wild-type mice show a range of panx1 mrna expression levels, with highest expression in trigeminal ganglia, bladder and spleen. unexpectedly, we found that in mice homozygous for the ko-first allele, panx1 mrna expression is not abolished but reduced by 70% compared to that of wild-type tissues. thus, panx1 ko-first mice present a hypomorphic phenotype. crosses of panx1 ko-first with flp deleter mice generated panx1 f/f mice. further crosses of the later mice with mgfap-cre or nfh-cre mice were used to generate astrocyte-and neuronal-specific panx1 deletions, respectively. a high incidence of ectopic cre expression was found in offspring of both types of conditional panx1 ko mice. our study demonstrates that panx1 expression levels in the global and conditional panx1 ko mice derived from ko-first mouse lines must be carefully characterized to ensure modulation of panx1 gene expression. the precise quantitation of panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of panx1 under physiological and pathological conditions. agarwal, e. hughes, d. bergles johns hopkins university, baltimore, united states astrocytes elevate intracellular ca21 in response to stimulation of metabotropic receptors. these ca21 transients are linked to processes as diverse as synaptic plasticity and functional hyperemia, but the relevance of this signaling remains uncertain. although most studies of ca21 signaling in astrocytes have focused on somatic events, anatomical and functional studies indicate that ca21 signaling in astrocytes may be compartmentalized into functionally isolated "microdomains." to facilitate analysis of ca21 signaling, we generated transgenic mice in which the cytosolic and membrane anchored variant of genetically encoded calcium indicator gcamp3, referred as cgcamp3 and mgcamp3 respectively, can be expressed conditionally in a cell specific manner. a ubiquitous cag promoter is used to control cgcamp3 and mgcamp3 expression, and a "stopper" sequence flanked by loxp sites was placed upstream of the coding sequence, preventing expression until cre excises this dna. these constructs were targeted to rosa26 locus to ensure widespread expression. to evaluate whether cgcamp3 and mgcamp3 were expressed at levels sufficient to resolve ca21 transients, we bred r26-lsl-cgcamp3 and r26-lsl-mgcamp3 mice to glast-creer mice. double transgenic offspring were injected with tamoxifen at 3 weeks of age and analyzed 2-3 weeks later. histology revealed that cgcamp3 and mgcamp3 were expressed in astrocytes throughout the brain. astrocytes in acute cortical slices exhibited large amplitude, spontaneous increases in both cgcamp3 and mgcamp3 fluorescence. the signal to noise ratio of the fluorescence intensity was greatly improved in astyrocytes expressing mgcamp3, partially due to the ability of mgcamp3 to better resolve near membrane ca21 flux. ca21 transients were typically restricted to small regions of an individual astrocyte, consistent with ca21 elevations within microdomains. spontaneous ca21 transients were occasionally observed in the soma of astrocytes expressing cgcamp3 but not mgcamp3, and were not coincident with activity in the processes, suggesting that these regions are functionally uncoupled. to determine if gcamp3 expression is sufficient to visualize ca21 signaling in vivo, we made cranial windows over the somatosensory cortex and performed 2-photon imaging. microdomain ca21 transients were prominent in cortical astrocytes, indicating that these mice provide new opportunities for understanding the significance of this form of signaling. in the trigeminal ganglion (tg), satellite glial cells (sgcs) surround neurons and are able to release substances that may affect sensory transmission through the tg and contribute to mechanisms underlying craniofacial pain. the aims of this study were 1) to investigate whether sgcs could be provoked to release glu in vitro and 2) to study the in vivo response properties of trigeminal afferent fibres upon artificial elevation of glu concentration in the tg. methods: confocal microscopy was used to assess glu content in and the expression of excitatory amino acid transporter 1 (eaat1) and eaat2 by sgcs of the tg. glu release was investigated in vitro, where sgcs were isolated from the tg of 9 adult male sprague-dawley (sd) rats and treated with control medium or medium containing 10 mm kcl together with 0, 0.1, 1, 10 mm of the eaat1/2 inhibitor tfb-tboa. in vivo electrophysiology experiments were conducted in 6 additional anaesthetized adult male sd rats to determine the effect of repeated intraganglionic injections of glu (500 mm, 3 ml 3 2, 30 min apart) on the response properties of 6 tg neurons that innervated either the temporalis or masseter muscle. cumulative afferent discharge was calculated as the sum of action potentials (ap) 10 min post injection subtracted from the sum of ap 10 min prior to injection. an electronic von-frey hair was used to measure afferent mechanical threshold (mt) at 1 min intervals for 10 min prior to the first and again 20 min after the second microinjection of glu. results: most sgcs were found to be glu positive and express eaat1 and eaat2. treatment with 10 mm kcl resulted in a $2-fold increase in glu concentration, compared to control (10.6 6 1.1 vs. 4.8 6 0.1 mm, respectively; p 5 0.038). treatment with 1 mm tfb-tboa significantly reduced the glu concentration to 5.8 6 1.4 mm (p 5 0.047), which was further lowered to 3.0 6 0.8 mm when 10 mm was applied (p 5 0.001). cumulative afferent discharge evoked in tg neurons by the second injection of glu (20 6 9 ap) was not significantly different from that evoked by the initial injection (21 6 10 ap; p 5 0.92). glu injections significantly reduced muscle afferent mt (baseline: 35 6 19 g) by 30 6 3% (p 5 0.03). these results indicate that glu can be released from sgcs through eaats, and that increased glu concentrations in the tg excite neurons and induce a state of peripheral sensitization. this may hypothetically be a mechanism, whereby activated tg sgcs could contribute to e.g. migraine headache. in an accompanying poster, we present data demonstrating that in cultured neurons l-lactate induces plasticity-related genes expression through a nmda-dependent mechanism (i.e. arc, zif268 and c-fos). here, we describe the effect of l-lactate on neuronal excitability by performing electrophysiological recordings of cultured cortical neurons. we observed that application of l-lactate (10 mm) triggers an inward current with an amplitude of 20.55 1/-0.1 na and slow kinetics, with a peak reached at 189 1/-55s (called ilac). the membrane current decreases gradually to reach a plateau, with a mean inward current of 20.13 1/-0.04 na persisting up to 780s (13 minutes) after addition of l-lactate (called ipyr). in order to assess l-lactate specificity on the generation of these currents l-pyruvate, another monocarboxylate, as well as d-lactate, the non-metabolized enantiomer of l-lactate) were tested. in contrast to l-lactate, l-pyruvate (10 mm) induced a low-amplitude sustained current (20.07 1/-0.03 na) similar to the late-phase slow current evoked by l-lactate (i.e ipyr), but no current similar to ilac. dlactate on its side did not elicit any specific current. pharmacological characterization of ilac and ipyr currents demonstrate that ilac is generated by nmda receptors (as it is blocked by mk801) and relies on active nmda receptors (as it is blocked by either glutamate or glycine binding sites inhibitors). in contrast, generation of ipyr by both l-lactate and lpyruvate is prevented by diazoxide, a katp opener, whereas ilac was unaffected by the treatment. in order to determine the importance of katp for plasticity-related gene expression, the katp closer glibenclamide (200 um) was applied to neurons and mrna gene expression of arc and zif268 quantified. results obtained demonstrate that arc and zif268 mrna expression are unaffected by glibenclamide. as a whole this set of data demonstrates that l-lactate generates two types of inward currents in neurons i.e. nmda-dependent (ilac) or katp-dependent (ipyr). moreover, they highlight the nmda-dependent ilac current as the key mediator, in contrast to katp, of l-lactate-induced plasticity gene expression. this is further sustained by the observation that l-pyruvate does not reproduce the effect of l-lactate on gene expression (see accompanying poster). this set of results provides novel insights into the mechanisms of action of l-lactate as an inducer of plasticity gene expression, and an important signaling molecule for neuronal plasticity. the reliance on drg neuron co-culture for sclcs fate commitment stands as a major hurdle for the therapeutic application of this finding. thus, we aim to unravel the mechanisms underlying sclcs fate commitment in order to develop a drg neuron-free condition for generating fate committed schwann cells. we hypothesised that the switch is brought about by the membrane bound neuregulin (nrg), nrg 1 type iii, but not the other soluble isoforms of neuregulin. as our results show that sclcs treated with soluble neuregulin did not show significant changes in morphology nor marker expression when compared with untreated sclcs. nrg 1 type iii expression was observed on purified drg neurons by immunocytochemistry, western blot analysis as well as reverse transcription pcr for the mrna. mammalian expression constructs for nrg 1 type iii were made by transfection into human embryonic kidney cells, hek 293t, and mouse embryonic fibroblasts (mef) with the aim of generating surrogate cell types for co-culture with sclcs to pursue cell-specific effects of nrg 1 type iii on differentiation of sclcs. the findings promise a way for generating fate committed schwann cells for autologous transplantation for recovery after nerve injury. (2) the co 2 /hco 3 buffer system. the latter being an open buffer system, because biomembranes are usually permeable to co 2 , and, in addition, most cells express hco 3 transporting membrane proteins. the enzyme family of carbonic anhydrases (cas) catalyses the reversible reaction from co 2 and h 2 o to hco 3 and h 1 , and thereby modulates the intracellular h 1 buffer dynamics.we have performed calibrated in situ live-cell imaging with the proton-sensitive fluophore bcecf in glial cells and neurons of acute cerebellar slices of wild-type and knockout mice. the studies were used to quantify the intracellular buffer capacity and to dissect the contribution of the intracellular caii and the extracelluar caiv by comparing intracellular h 1 shifts in glial cells and neurons from wild-type mice and mice deficient in their caii or caiv gene. we found that only one proton in 400000 is unbound and thereby chemically active, and that about 50% of this buffer capacity is mediated by the co 2 /hco 3 buffer system and the other 50% by intrinsic buffers. the rate of co 2 -induced change in intracellular h 1 concentration was increased by intracellular caii in both glial cells and neurons. the extracellular caiv on the other hand affected primarily neurons, but also showed unexpected intracellular activity (schneider et al. 2013, pnas 110). the rates of acidification and alkalinisation in wild-type and knockout mice were significantly reduced in the presence of the ca blocker 6-ethoxy-2-benzothiazolsulfonamid (10 mm). we confirmed ca protein expression by western blot and could also show that the expression of the remaining ca is not upregulated. these results provide new insights into cellular proton-coupled processes in acute neural tissue, which shows some new complex roles of carbonic anhydrases in shaping proton dynamics in cells and tissues. objective: it is becoming increasingly evident that inflammatory mechanisms promote neuronal hyper-synchronization and epileptogenesis following brain insults. our recent studies identified serum albumin as a key player in the inflammatory cascade leading to epilepsy, at least partially through the activation of tgf-b signaling pathway in astrocytes. in this study we set out to explore the mechanisms by which tgf-b1 induces glial activation and epileptogenesis. methods: primary cultures of microglia and astrocytes were treated with tgf-b1 and gene expression analysis along with protein quantification were performed by quantitative pcr and elisa. electrophysiological response to il-6 was obtained in acute brain slices (field potential recordings) and in-vivo (sub-dural corticoencephalography) following to a continuous administration of il-6 into the lateral ventricle for 7 days. results: in glia cultures tgf-b1 induced early and rapid up-regulation of il-6 at both mrna and protein levels whereas the upregulation of other pro-inflammatory cytokines such as il-1b and tnf-a was milder and at a later time point. notably, smad2/3-dependent tgf-b1 signaling induced the expression of il-6 primarily in astrocytes and to a significantly lesser extent in microglia. il-6 was sufficient to induce epileptiform activity in acute brain slices and recurrent seizures within 3-4 days a following continuous intracerebroventricular injection of il-6. conclusions: we thus suggest astrocytic release of il-6 as a potential key mechanism in epileptogenesis. okayama univ., dept. of brain science, okayama, japan dopamine transporter (dat) is expressed not only in catecholaminergic neurons but also in astrocytes. we previously showed that repeated l-dopa treatment markedly induced expression of dat and apparent dopamine (da)-immunoreactivity in the reactive astrocytes in the striatum of animal models of parkinson's disease. it is thought that astrocytes can uptake both l-dopa and da via neutral amino acid transporter lat and dat, respectively. therefore, uptake and metabolism of l-dopa and da in the striatal astrocytes may influence their availability in dopaminergic system of parkinsonian patients. to clarify uptake and metabolism of l-dopa and da in striatal astrocytes, the contents of l-dopa, da and their metabolites were measured after the l-dopa/da treatment using primary cultured astrocytes. first, we revealed the expression of neutral amino acid transporter lat and aromatic amino acid decarboxylase (aadc) in the striatal astrocytes. the level of l-dopa in astrocytes was markedly increased after 4-hr l-dopa exposure, but da was not detected in the astrocytes 4 or 8 hr after the treatment. on the other hand, the da treatment for 4 hr increased levels of da and its metabolites, especially dopac. these results indicate that uptaken da into astrocytes is rapidly metabolized and that uptaken l-dopa never be converted to da in astrocytes. furthermore, level of uptaken l-dopa in cultured striatal astrocytes was rapidly decreased after removing extracellular l-dopa. taken together, the present results suggest that striatal astrocytes act as a reservoir of l-dopa to uptake or release l-dopa depending on the extracellular l-dopa concentration, but have less ability to convert l-dopa to dopamine. s. limmer, c. kl€ ambt universit€ at m€ unster, m€ unster, germany neuronal function consumes a large amount of energy and thus the brain requires a constant but regulated supply of metabolites. in invertebrates, such as drosophila, the nervous system is floating in the hemolymph and all metabolites that enter the brain have to be transported across the blood brain barrier (bbb). as in primitive vertebrates, the drosophila bbb is generated by glia. a layer of so-called subperineurial glial cells completely encapsulates the nervous system and thus, all metabolites reach the neurons en route through glia. the main energy supply of the inner organs of drosophila is provided by trehalose that is found in high concentrations in the hemolymph. trehalose is a nonreducing disaccharide, in which two dglucose units are linked via a a,a-1,1-glycosidic bond. the uptake of trehalose is mediated by two trehalose transporters, which are members of the slc2a gene family. the subperineurial glial cells then either secrete c 6 sugars to the neurons -or alternatively supply energy to neurons by secreting c 3 metabolites as described for the bee retina. to discriminate between these possibilities we have followed a number of different approaches. the relevance of trehalose transporters in the different cell types has been determined by mutant analysis and rnai studies. furthermore, we have studied the role of all other putative sugar and monocarboxylate transporters in glial cells by rnai knockdown. to determine whether c 6 or c 3 carbohydrates are secreted by glial cells we suppressed the expression of core enzymes regulating glycolysis or components of the respiratory chain in either glial cells or neurons. moreover, we have ablated mitochondria specifically in glia or in neurons through expression of a restriction enzyme targeted to mitochondria. we will summarize our results and discuss approaches towards identifying neuronal signals that control glial carbohydrate secretion during energy homeostasis of the brain. microglia have long been known to respond to virtually any insult to the central nervous system. they quickly migrate to the site of the insult, and play many important roles, such as barricading the injury site, phagocytosing debris, and releasing cytokines. the role of microglia in the normal brain, however, has only recently begun to be appreciated. with the advent of in vivo imaging, microglia have been shown to be constantly surveying their environment with rapid extensions and retractions of their processes. subsequent studies have shown that these 'resting' microglia (now known as 'surveying microglia') are indeed active throughout development and adulthood. during development, microglia have been shown to be involved in synaptic monitoring and pruning as well as synaptic stripping after injury. thus far, many studies have focused on the role of microglia at the synapse. however, we report here, for the first time, that in the cortex of normal adult rodents, a small percentage of microglia are specifically associated with the axon initial segment (ais). the ais is characterized by a high density of voltage-gated ion channels and plays an important role in initiation of the action potential and maintenance of neuronal polarity. this interaction seems to be limited to the 'surveying' phenotype of microglia and much less frequent in 'activated' microglia. this overlap of processes appears early in development and continues throughout adulthood although the function of microglia at the ais is still currently unknown. x. liu 1 , j.-m. petit 2,3 , p.j. magistretti 2,3 , c. giaume 1 1 cirb, collège de france, paris, france 2 lndc, brain mind institute, epfl, lausanne, switzerland 3 centre de neurosciences psychiatriques, chuv, prilly, switzerland astrocytes act as modulators of neuronal activity through mechanisms such as neurotransmitter uptake, 'gliotransmitters' release, and metabolic supply. recently, astrocytes were shown to modulate sleep by vesicular release of atp. besides the cellular mechanism, we are interested in the network behavior of astrocytes in sleep-wake cycle. indeed, astrocytes form networks of communicating cells via gap junction channels constituted by connexins (cxs). recently, we have demonstrated that astroglial networking is regulated by neuronal activity and that neuronal activity is affected by deletion of the two main astroglial cxs (cx43, cx30). to investigate how astroglial networks behave in sleep-wake cycle, we used pharmacological treatments and sleep deprivation to manipulate sleep-wake status of mice and determined whether astroglial networks would change in response. astroglial networks in acute cortical slices were revealed by "dye coupling", i.e. by loading of a patch-clamped astrocyte with sulforhodamine b that diffuses into neighboring astrocytes through gap junction channels. also, cxs expression at mrna and protein levels was measured as an index of connections among astrocytes. the results are as follows. first, i.p. injection of modafinil, a potent wakefulness-promoting drug, increased both mrna and protein expression of cx 30 in the mouse cortex. superfusion of modafinil also enhanced dye coupling among astrocytes in acute coronal slices of the mouse somatosensory cortex. this effect was abolished by ttx treatment, which demonstrates neuronal activity is necessary for the effect of modafinil. in contrast, c-hydroxybutyric acid (ghb), a sleep-promoting agent, and propofol, a general anesthetic, decreased astroglial coupling in cortical slices. interestingly, the effect of ghb was not affected by ttx, suggesting a different pathway of action from modafinil. next, we used a "gentle" sleep deprivation model to sleep deprive mice for 6 hours from the onset of the light period. as a result, mrna of cx30, but not cx43, was increased in both the cortex and hippocampus. finally, dye coupling in cortical astrocytes was enhanced in mice after sleep deprivation compared to mice after normal sleep. this increase in dye coupling, however, was not observed in slices from cx30 knock out mice after the same sleep deprivation treatment. altogether these results indicate that astroglial networks are bidirectionally regulated by perturbations in sleep-wake cycle and that cx 30 is the major cx sensitive to such perturbations. in neurons, synaptic and extrasynaptic gaba a receptors (gaba a rs) differ in their subunit composition, conferring them distinct functional and pharmacological properties. oligodendrocyte precursor cells, also called ng2 cells, are contacted by bona fide neuronal gabaergic synapses. however, we recently showed a synaptic to extrasynaptic switch in the mode of transmission between gabaergic interneurons and ng2 cells during postnatal development of the somatosensory cortex. we therefore hypothesized that the postnatal switch of gabaergic transmission in ng2 cells is accompanied by changes in the expression of gaba a r subunits. to test for this hypothesis, we stimulated neuronal fibers to evoke gaba a r-mediated responses in ng2 cells recorded in acute slices of the somatosensory cortex of ng2-dsred transgenic mice. the effect of zolpidem and a5ia on evoked gabaergic responses reveals the predominance of functional a1-and a5-containing gaba a receptors, respectively, at interneuron-ng2 cell synapses early in development. however, the expression level of a5 decreases when responses rely exclusively in extrasynaptic transmission at more mature developmental stages. more importantly, specific pharmacology for the c2 subunit, a crucial molecular component for the clustering of gaba a receptors at postsynaptic sites, demonstrated a down-regulation of this subunit in ng2 cells prior to the complete loss of gabaergic synaptic activity. in keeping with the synaptic nature of the c2 subunit in neurons, this molecular change in ng2 cells correlates with the switch from synaptic to extrasynaptic transmission. to corroborate these functional data, we performed single cell multiplex rt-pcr of a1-5, b1-3, c1-3 and d subunit mrnas in ng2 cells of the second and fourth postnatal weeks. despite the large heterogeneity of subunit mrna expression at both developmental stages, we found that the number of cells expressing c2 mrnas dramatically decreases in the fourth postnatal week. in conclusion, the expression loss of the c2 subunit is an important molecular determinant impacting the change of transmission modes between interneurons and ng2 cells during cortical development. financial support: frc, anr blanche, arsep, dfg (sfb/tr3) and eu (fp7-202167 neuroglia). university of rochester, medical center, rochester, united states synaptic plasticity is a critical process throughout the lifespan for achieving and maintaining normal and efficient operation of the nervous system. while much is known about the functional and structural changes that occur at the synapse during synaptic plasticity, the mechanisms implementing these changes are poorly understood. despite being classically characterized as immune cells, microglia have recently been shown to play a role in normal brain function by restructuring and removing synapses. given the novelty of this role, few details are known about how microglia behave to implement such a role during periods of plasticity. to characterize the changes in microglial behavior during ocular dominance plasticity in the developing visual cortex, we examined microglial morphology in fixed brain sections as well as in vivo using two-photon microscopy. for analysis of microglia in fixed sections, c57/bl6 mice were monocularly deprived for either 12 hours, 1, 2, 4, or 7 days during the visual critical period (p23-p30). fixed coronal sections were stained with iba1, a specific marker for microglia, imaged on a confocal microscope and analyzed using imagej. microglial density decreased rapidly (within 12 hours) and remained low for the 7 day deprivation period. characterization of microglial morphology revealed an increase in the complexity of the microglial process arbor after 12 hours, which steadily decreased back to baseline by 7 days. this suggests that microglia do undergo a rapid change in morphology during plastic events that is reminiscent but distinct from the traditional activation pattern observed during pathological events. for analysis of microglia in vivo, heterozygous cx3cr1/gfp (jung 2000) mice were monocularly deprived directly preceding the first imaging session, the binocular segment of primary visual cortex was imaged, and the same cortical area was re-imaged 2 and 4 days later. while no change in microglial density was observed, analysis of microglial process motility showed a decrease in motility after periods of monocular deprivation. these results, combined with the observed morphological changes, show that microglia undergo behavioral changes after a period of monocular deprivation that are inconsistent with pathological activation, suggesting that microglia take on different roles and activities during normal brain function. g. baltazar, j. oliveira, t. roxo, c. fonseca faculty of health sciences, cics-ubi, covilhã, portugal neuroinflammation is a pathological hallmark in patients and experimental models of parkinson's disease. both present the classical features of inflammation, with evidence of an uncontrolled process. moreover, microglia may become activated early in the disease process and remain primed, responding strongly to subsequent stimuli, and thereby enhancing inflammation-induced oxidative stress and cytokinedependent toxicity in vulnerable neuronal populations. we have previously demonstrated that glial cell line-derived neurotrophic factor (gdnf), released by ventral midbrain astrocyte cultures, potently prevents microglial activation induced by a pro-inflammatory agent. therefore, gdnf is an important mediator in the astrocytemicroglia crosstalk keeping microglia in a resting state. however, in the brain, microglia and astrocytes are not alone and other cell types, e.g. neurons, may interfere and influence this astrocytic control of inflammation. therefore, it is important to investigate if the presence of neurons alters gdnf-mediated astrocytic control of microglial activity. in this work we aimed at investigating whether the presence of neurons, injured or not, changes the ability of astrocyte-derived gdnf to prevent microglial activation. for that purpose, we used primary cultures of ventral midbrain astrocytes and microglia, as well as neuronastrocyte mixed cultures from the same brain region. neuron-astrocyte cocultures were exposed to vehicle (control) or to the da neurotoxin mpp 1 . the media conditioned by control and mpp 1 -challenged neuron-astrocyte mixed cultures, or by astrocyte cultures, was collected and transferred to ventral midbrain microglia cultures, which were then exposed to the pro-inflammatory agent lipopolysaccharide. the effect of conditioned media obtained from the different cell culture systems on microglial activity was determined by analyzing the production of nitric oxide by the griess reaction and of the phagocytic activity by determining the engulfment of fluorescent microspheres by fluorescence microscopy. university of lausanne, lausanne, switzerland lactate is produced by astrocytes and used by neurons as metabolic substrate during activity. recent evidence indicates that lactate may not only serve as energy substrate but also as modulator of glutamatergic or gabaergic neurotransmission. we tested this hypothesis using primary cultures of cortical neurons obtained from wild type and gad67-gfp knock-in mice (c57bl/6). neuronal excitability was monitored by electrophysiological recordings and calcium imaging using the fluorescent probe fluo-4 am. spontaneous action potentials and rhythmic calcium transients were present in more than 50% of cultured neurons, which were either gabaergic or glutamatergic cells. in the presence of 5mm glucose, l-lactate application reversibly diminished calcium transient frequency in a concentration-dependent manner in both cell types (glutamatergic neurons ic50 4.23 6 1.9mm and gabaergic neurons ic50 4.18 6 2.8mm). to test whether lactate effects were dependent on metabolism, we applied the closely related substrate pyruvate (5mm) or switched to different glucose concentrations (0.5 or 10mm). none of these conditions altered the calcium transient frequency. in contrast, the application of d-lactate, thought not to be metabolized by neurons, decreased the spiking activity in the same concentration range as l-lactate (ic50 4.58 6 1.2mm). we determined that d-lactate was taken up by neurons, however more than two-fold less efficiently than l-lactate. these results suggest that the mechanisms of lactate sensitivity are not purely metabolic. since d-lactate has the same potency as l-lactate but it is less internalized, it is likely that lactate acts as an extracellular ligand that triggers the reduction of neuronal activity. this hypothesis is currently under investigation. this modulating effect of lactate represents a novel role for the compound that has to be taken into account in the context of the interactions between astrocytes and neurons. ben-gurion university, ber-sheva, israel microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network, a process which appears to contribute to the integrity, maintenance and functioning of the brain. since microglia are long-lived, they are subjected to senescence processes which may severely compromise their function with age. here we used a digital tool for the quantitative morphometric characterization of fine cortical microglia structures in mice. we thus followed the morphological changes microglia underwent with aging and with the progression of alzheimer's-like disease. compared with microglia in young mice, microglia in old mice are less ramified and possess less branches and fine processes, a phenomenon which was associated with increased expression of pro-inflammatory genes. notably, a similar microglial pathology appeared 6-12 months earlier in mouse models of alzheimer's disease (ad). we thus demonstrate that in addition to promoting neurotoxic inflammation, amyloid plaques attract the microglia and modify their structure. this, in turn, causes a severe microglial process deficiency, which possibly results in compromised neuronal function and repair. blood-borne glucose is the main energy substrate for the brain under physiological conditions. however, the question remains unresolved if the two main cell types in the brain -neurons and astrocytes -consume glucose upon their individual needs or if glucose is mainly taken up by one cell group and glycolytically degraded to energy-rich substrates (e.g. lactate), which are then shuttled to the other cell group. a novel genetically encoded fret glucose sensor (bittner et al., 2010, 2011) together with two-photon microscopy now provides for the first time the opportunity to determine intracellular glucose concentrations in single cells in the intact organism. cortical microinjections of an adenoviral vector (aav9 and aav6) coding for the sensor flii 12 pglu600md6 with the promoter sgfap for astrocytes and synapsin for neurons were performed in mice and chronic windows were implanted above the somatosensory cortex. after two weeks, specific expression in astrocytes and neurons was observed. the ratiometric fret signal in both cell types increased after intraperitoneal glucose injection and decreased after insulin application demonstrating the functionality of the construct in vivo. the astrocytic sensor displayed a large linear range at blood glucose levels ranging from 3 to 20 mmol/l. in a second set of experiments, we examined the dynamic behavior of cellular glucose concentrations upon increased brain activity. during electrical hindpaw stimulation 21% of examined astrocytes showed a 0.5% decrease of the fret signal, whereas neuronal glucose levels did not exhibit activity-dependent fluctuations. to be able to draw conclusions about metabolic rates of substrate oxidation we recently started with experiments where cellular glucose uptake was transiently inhibited using different blockers of glucose transport. drugs were applied systemically and intracortically. we could demonstrate this principle on first measurements on astrocytes. glucose fret sensors allow measurements of the dynamics of glucose concentrations of single neurons and astrocytes in the intact organism. measurements of cellular glucose transients during transport blockade hold the potential to compare glycolytic rates of neurons and astrocytes. this tool may substantially contribute to uncover the complex organization of brain energy metabolism. neuron-glial communication in the cns is fundamentally important for many brain processes including synaptic transmission and plasticity. perisynaptic astrocytic processes (pap) contact excitatory synapses, forming tripartite structures with neurons. in the hippocampus, the morphology of pap has been shown to remodel rapidly and continuously but the mechanisms and roles of this form of structural plasticity remain unknown. this study investigated the physiological mechanisms driving pap movements and their role during long-term potentiation (ltp). pap and spines contacts were labeled by viral gene delivery of farnesylated fluorescent proteins in hippocampal slice cultures and imaged by confocal microscopy. electron-microscopy of infected slices confirmed that pap-synapse morphology is preserved in organotypic cultures. pap movements adjacent to dendritic spines were evaluated with an index of motility (mi). increasing neuronal activity by schaffer collaterals (sc) stimulation elevated pap mi and this was prevented by both ttx and mglur inhibition, suggesting that neuronal activity modulates pap movements. we next investigated whether intracellular calcium (ca 21 i ) in astrocytes influenced pap motility. bapta-am bulk-loading specifically chelated ca 21 i in astrocytes and reduced pap movements. when exogenous gq-coupled receptors mrga1 or mrgc11 were specifically targeted to astrocytes, their agonist fmrfa induced ca 21 i increases and accelerated pap motility. moreover, fmrfa delivery at the synaptic level by two-photons flash photolysis was sufficient to elevate pap mi. we then investigated pap dynamics in relation to ltp. application of theta-burst sc stimulation initially increased pap motility, but then resulted 30min later in a decrease of pap motility. interestingly, the reduction of pap movements correlated with both spine enlargement and increased pap's spine coverage. to assess the consequences of these changes, we then specifically triggered elevation of pap movements at the synaptic level by flash photolysis. spine stability analysis 24 hour after uncaging revealed that spines in contact with activated paps were more stable than others. this study suggests that excitatory synapses control the motility of surrounding paps through the triggering of neurotransmitter-evoked astrocytic ca 21 i elevations. increased pap motility seems to be necessary to elevate their coverage of the synapse during ltp, leading to higher synapse stability. astrocyte reactivity occurs in response to many pathological brain situations. reactive astrocytes display morphological and functional changes that could result in concentration variations of some brain metabolites localized both in astrocytes and in neurons. such changes could be detected by magnetic resonance spectroscopy (mrs), allowing non-invasive monitoring of astrocyte reactivity and thereby neuronal dysfunction in vivo. one of the major peak detected by mrs in the brain corresponds to neuronal metabolite n-acetyl-aspartate (naa). although its function is unclear, it is considered as a biomarker for neurodegeneration and a decrease in its concentration is interpreted as neuronal death or dysfunction. however, there is not clear evidence that other cell types, specifically astroglia, could not contribute to change in naa levels in the brain. in this study, we used a rat model of astrocyte activation by stereotaxic injections of lentiviral vectors encoding for either the cytokine ciliary neurotrophic factor (lenti-cntf) into the right striatum or betagalactosidase (lenti-lacz) into the left striatum, as a control. mrs data were acquired on a 7t magnet from two voxels placed over each injected striatum. a diffusion-weighted laser sequence with echo time te 5 40 ms was used. concentrations were measured using lcmodel software for total n-acetyl-aspartate (naa 1 naag), myo-inositol (ins), total choline (cho), glutamate and taurine relative to total creatine. lenti-cntf injection promotes a sustained, extensive and selective activation of astrocytes, as evidenced by overexpression of gfap and vimentin and cellular hypertrophy (fig.1) . importantly, neurons displayed unaltered morphological, molecular and electrophysiological features, as described previously (escartin et al., 2006; beurrier et al., 2010). mrs evidences changes in metabolite concentration: in the lenti-cntf injected striatum, levels of the astrocyte metabolites ins and cho were increased, whereas levels of the neuronal metabolite naa and glutamate were decreased. increased levels of ins and cho are generally found associated with astrocyte reactivity, however, the decrease in naa is unexpected given that neurons are not dysfunctional with cntf. to understand how astrocyte reactivity can influence naa levels, we are currently characterizing the molecular changes occurring in the naa metabolism using quantitative pcr, biochemistry, histology and hplc. our data suggest that reactive astrocytes alone result in alterations of metabolite concentrations detectable by nmr. although the mechanisms by which activated astrocytes regulate neuronal metabolism remain to be elucidated, our work challenges the mrs dogma that decreased neuronal metabolites can be used as a biomarker for neurodegeneration. microglial phagocytosis is a vital phenomenon for the clearance of damaged and death cells or infectious agents in a context of brain injury or infection, respectively. in addition, microglia can boost synaptic plasticity by the phagocytosis of axon terminals and dendritic spines. therefore, it is crucial to better understand the mechanisms involved in microglia clearance in order to devise new strategies to promote an efficient brain repair. recently, we showed that histamine modulates microglia motility and cytokines release. in this work, we aimed to investigate the role of this molecule and its receptors in microgliainduced inflammation by evaluating microglial phagocytic activity and reactive oxygen species (ros) production. for that purpose, an iggopsonized latex bead assay was performed in n9 murine microglial cell lines exposed to lipopolysaccharide (lps) or histamine 1, 10 and 100 mm. we showed that histamine significantly stimulated phagocytosis of opsonized latex beads via h1 receptor activation. this effect was accompanied by the rearrangement of cytoskeleton analyzed by phaloidin and acetylated tubulin expression and cellular distribution. the phagocytic activity was significantly reduced after pretreatment with apocynin, a putative napdh oxidase (nox) inhibitor. all nox isoforms were expressed by microglial cells, but only the expression of nox1 was increased by treatment of histamine. rac1, an important subunit for the activation of nox1, was also activated by histamine. in addition, histamine induced ros production via h1 and h4 receptor activation. in conclusion, histamine plays an important role in the regulation of microglial phagocytosis, which is mediated by nox1 and rac1 activation. multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal damage, involving both white (wm) and gray matter (gm) areas. furthermore, cortical pathology correlates with ms progression and cognitive dysfunction. we previously found altered expression of gap junction (gj) proteins connexin32 (cx32) and cx47 in oligodendrocytes and cx43 in astrocytes in wm lesions and normal appearing wm (nawm) in brain samples from ms patients, and in a mouse model of experimental autoimmune encephalomyelitis (eae), implicating uncoupling of myelinating cells as an important aspect of ms pathology. here we studied cx32 and cx47 and their astrocytic partners cx30 and cx43 in and around cortical lesions and the normal appearing cortex (nacx) in ms samples and non-ms controls. we examined their expression by real-time pcr, quantitative immunoblot and immunohistochemichal analysis. compared to non-ms controls, expression levels of cx32 and cx47 mrna were reduced within lesions whereas cx30 and cx43 were increased. in nacx, all four connexins were upregulated with cx47 being significantly increased. immunoblot analysis of ms cortex revealed reduced cx32 and increased cx43 levels, while cx30 and cx47 showed no significant change. immunohistochemical analysis showed severely reduced cx47 gj plaques within lesions with gradual increase toward nacx. in contrast to nawm, cx47 gj plaques were not increased in nacx. both astrocytic gj proteins were increased in ms, but in distinct patterns: cx30 gj plaque counts were higher within lesions and perilesional areas. cx43 was increased in ms cortex compared to non-ms controls, but in contrast to nawm, cx43 gj numbers were higher in nacx than in lesions. similar alterations were found in the eae mice, in which cx32 and cx47 gj counts were reduced whereas cx43 and cx30 gjs were increased. our results indicate that oligodentrocytic as well as astrocytic gjs are affected not only in ms wm but also in the gm. loss of oligodendrocyte gjs and in parallel increase of astrocytic gjs reflecting astrogliosis occurs throughout the ms brain and may negatively affect the prospects of repair and remyelination, worsening disease progression. acknowledgements: this project was funded by the cyprus research promotion foundation (grants access/0308/11 and health/bios/ 0308/01 to kak) and by cyprus telethon (2010-14 grant to kak). post-mortem human brain tissues were kindly provided by the uk multiple sclerosis society tissue bank, imperial college london, funded by the uk multiple sclerosis society. the mainly astroglialocated enzyme glutamine synthetase is required to synthesize glutamine from the re-uptake of either glutamate or gaba. thereby, this enzyme significantly contributes to the control of brain glutamate and gaba levels. there is good evidence that both neurotransmitter systems are dysregulated in depression. hence, we decided to study the cellular expression of this enzyme in subjects with major depression and bipolar disorder. material and methods: we investigated the numerical density of glutamine synthetase immunoreactive astroglial cells in different brain areas (prefrontal cortex, anterior insula) of 14 subjects with major depression, 15 subjects with bipolar disorder and 19 matched control cases. results and discussion: we found that compared with controls in cases with major depression there was a significant reduction of the density of immunostained glial cells in all brain regions studied, whereas bipolar cases showed a normal expression of the enzyme. our findings point to a profound disturbance of the glutamateglutamine-gaba cycle in major depression, which is in good agreement with neuroimaging observations and molecular biologic data of others. m.-c. marx, d. billups, b. billups university of cambridge, cambridge, united kingdom perisynaptic astrocytes are thought to play a key role in the recycling pathway of glutamate by supplying the precursor glutamine to the presynaptic terminal. using fluorescent ph i measurements of astrocytes and direct patch-clamp recordings from the calyx of held synapse in rat brain slices, we have investigated the transport mechanisms that may mediate this release of glutamine from astrocytes in situ and assessed the role of glutamine in maintaining glutamatergic neurotransmission. glutamine transporter currents were elicited by puff application of 10 mm glutamine from a nearby pipette. for imaging astrocytic ph i , hpts was included in the patch pipette. system a (sa) transporter function was probed using the substrate inhibitors meaib, alanine and serine, while system n (sn) function was isolated using its unique ability to transport li 1 in place of na 1 . histidine was used as a substrate inhibitor of both sa and sn. glutamine-induced membrane currents i gln (220.0 6 2.4 pa, n 5 21) were blocked by bath applied alanine (10 mm) and meaib (20 mm), as well as substitution of external na 1 with li 1 . fluorescent ph i measurement revealed an alkalinisation during puff application of glutamine. while bath application of histidine (20 mm) abolished the ph change, meaib and serine (20 mm) only attenuated it. most of the ph change (63 6 11%; n 5 7) was insensitive to the substitution of na 1 with li 1 . in presynaptic terminals, puff application of glutamate did not induce a membrane current indicating a lack of direct glutamate uptake; however, puff application of glutamine resulted in a i gln which was eliminated by the removal of external na 1 and by meaib. miniature epscs recorded from postsynaptic neurons (238.8 6 3.5 pa) were inhibited by meaib (228.6 6 3.4 pa; n 5 5) following turnover of the presynaptic glutamate pool. single epscs were unaffected by meaib indicating no direct effect on postsynaptic ampa receptors. together, these data identify two glutamine transport systems within astrocytes. properties of the glial i gln indicate expression of sa while ph imaging reveals a non-electrogenic glutamine transporter with properties identifying it as sn. sn is known to be readily reversible under physiological conditions and we propose that this mediates the astrocytic glutamine release mechanism within the glutamate-glutamine cycle. furthermore, system a mediates glutamine transport in glutamatergic nerve terminals and is important in maintaining the supply of glutamate for excitatory neurotransmission during periods of high frequency stimulation. a. briens, m. schwalm, f. docagne, d. vivien inserm u919, caen, france astrocytes are key players in the brain, cooperating with neurons to modulate crucial processes. in particular, they are able to uptake and release neurotransmitters and neuromodulators to control their synaptic level. in our study, we hypothesize that astrocytes can regulate some effects of the serine protease plasmin in the cns through a mechanism of uptake. conversion of the zymogen plasminogen to the serine protease plasmin by the activators (tpa or upa) is the basis of fibrinolysis in the vasculature. but cerebral functions for the plasminogen activator/plasmin system have recently arisen. it has been shown that plasmin was essential to activate the brain derived neurotrophic factor (bdnf) in its mature form, promoting long-term hippocampal plasticity. in alzheimer's disease, plasmin plays a protective role by participating in beta-amyloid clearance. plasmin can also lead to neuronal injury by degrading extra-cellular matrix proteins. in animal models of multiple sclerosis, plasmin can also help removing intraparenchymal deposits of fibrin, thus limiting axonal damage. these data suggest that a system of regulation of plasmin effects in the brain is necessary. in this study, we showed that astrocytes can actively and specifically uptake plasminogen and plasmin in a time-dependent and a dose-dependent manner. this mechanism involves clathrin pits formation in astrocytes and the lysine-binding site of plasminogen/plasmin. plasminogen and plasmin, following endocytosis are found in early endosomes, late endosomes, lysosomes and recycling endosomes. finally, we noticed that astrocytes uptake plasmin more efficiently than plasminogen. this study brings out a mechanism of glial uptake of plasminogen and plasmin. this could control their extra-cellular levels and regulate their effects within the brain. further investigations should determine whether this mechanism can modulate plasminogen activation and synaptic plasmin activity. besides, plasminogen activators (tpa) and cerebral substrates of plasmin (pro-bdnf) are also taken up by astrocytes. a link could thus exist between these processes to regulate the amount of synaptic mature bdnf. this would reveal a cross-talk between neurons, releasing precursors in the synapse (pro-bdnf and plasminogen) and astrocytes, regulating their activation and their clearance. understanding and modulating plasmin uptake could be very important in pathological conditions where the plasminogen/plasmin system is involved, such as alzheimer's disease, multiple sclerosis or stroke. healthy brain aging is characterized by neuronal loss and cognitive decline being inflammation a major causative factor. microglial activation is considered to be a major driver for the neuropathological findings in alzheimer's disease (ad). we evaluated whether young vs. aged microglia responded differently to ab soluble oligomers (abo) and to conditioned media from abo-treated hippocampal neurons. microglia from 1 day cd1 pups were incubated for 24 h at 2 (young) and 16 (old) days in vitro (div) with abo (ab1-42 at 50 and 1000 nm). in parallel, e16 mice hippocampal neurons were treated with abo at 4 (young) and 18 (old) div for 24 h, and the incubation media used as conditioned media to treat microglia, at respective div, for further 24h. cell migration (boyden chamber), extracellular content in glutamate (commercial kit), and activity of matrix metalloproteinases (mmp)22 and 29 (gelatin zymography) were assessed. an increased migration towards abo and atp was observed in young microglia but almost undetectable in aged cells (p < 0.01). interestingly, microglia exposure to conditioned media from 1000 nm abotreated neurons markedly decreased both young and old microglia migration, while treatment with media from 50 nm abo-treated neurons enhanced migration, mainly in the young microglia (p < 0.05). concerning glutamate, aged cells released less than young ones upon abo treatment (p < 0.01). in addition, when exposed to neuronal conditioned media, only the young microglia were able to exert a neuroprotective role by reducing the media glutamate content. curiously, while abo promoted a microglia release of mmp-9, independently of cell age, it only induced mmp-2 secretion in old cells in an abo concentrationdependent manner (p < 0.05 for 1000 nm abo). in contrast, conditioned media from abo-treated neurons elicited mmp-2 release only from young microglia. together, our data point to a loss of microglia function towards abo with ageing and are unique in suggesting that senescent microglia may release high levels of mmp-2 in ad. in addition, our results indicate that abo-treated neurons enhance the release of mmp-2 even by young microglia. whether this finding may contribute to enhance inflammatory stress and the onset of ad will be the aim of further studies. human immunodefeciency virus-1 (hiv-1) productively infects microglia within the central nervous system (cns). viral replication within microglia and the resulting immune response and neurotoxicity leads to significant cognitive impairments not limited to dementia, neurocognitive abnormalities, and memory deficits. combination antiretroviral therapy reduces the cognitive impairments associated with hiv-1, but the regulatory hiv-1 tat protein is still produced in the cns during treatment, even in the absence of productive infection. our laboratory analyzes the effect of tat on the murine cns by stereotactically injected into cortex, which mimics in part the neuroinflammation and neurodegeneration characteristic of hiv-1 associated neurocognitive disorders (hands). additionally, in vitro tat-treated bv-2 microglial cells exhibit activation associated with increased cytokine expression and phagocytosis. mixed lineage kinase 3 (mlk3) has recently been implicated in the tat-mediated activation of microglia. mlk3 activity is increased by the presence of tat protein, which results in microglial activation and increased production of inflammatory cytokines. conversely, the brain penetrable, small molecule, new chemical entity urmc-099 inhibits the mlk3 pathway. we hypothesize that urmc-099 attenuates tat-induced microglial activation as measured by microglia process length, phagocytosis assay and cytokine expression. in vitro application of urmc-099 decreased tatinduced production of ccl2 and il-6, microglia process length, and phagocytosis of charged and uncharged beads in bv-2 microglia cells. these data, in aggregate, strongly support a role for the mlk3 pathway in neuroinflammation and suggest that inhibition of this pathway with urmc-099 may have significant anti-inflammatory effects in an inflammatory cns milieu. in conclusion, this work will advance our understanding of mlk3 activity and may provide a viable drug therapy for hands. the contributions of glial cells to the modulation of adult synaptic plasticity are becoming increasingly more appreciated. astrocytes in the supraoptic nucleus (son) of the hypothalamus demonstrate prominent temporary structural changes during physiological events such as dehydration and lactation. such structural remodeling is characterized by a reduction in the astrocytic coverage of oxytocin neurons and their synapses. these astrocyte morphological changes also contribute to the observed functional changes in synaptic activity during dehydration and lactation. in order to elucidate the underlying mechanisms regulating the astrocytic contributions to synaptic plasticity we have established an astrocyte protein expression profile for structural changes in the son during lactation and hyperosmotic challenge. for this, son from virgin, lactating, and hyperosmotic rats were collected from acute hypothalamic slices and analyzed by mass spectrometry to identify proteins differentially regulated by the changes in synaptic structure induced by lactation and hyper-osmolarity. our mass spectrometry data analysis has placed particular focus on regulation of astrocyte-specific proteins and the roles of these proteins in molecular pathways known to be involved in son plasticity, such as cytoskeleton remodeling, cell adhesion, and cell signaling. gene ontology analysis revealed over representation of pathways known to be highly active in astrocytes including metabolism and antioxidant activity. promising target proteins for future functional studies will be discussed. here we elucidate a novel merlin isoform 2 (merlin-iso2)-specific function in maintaining axonal integrity and propose that reduced axonal nf2 gene dosage leads to nf2-associated polyneuropathy. we identify a merlin-iso2-dependent complex corresponding to activation of the gtpase rhoa, enabling downstream rho-associated kinase to promote neurofilament heavy chain phosphorylation. in vivo, specifically merlin-iso2 knockout mice exhibit impaired locomotor capacities, delayed sensory reactions, and electrophysiological signs of axonal neuropathy. sciatic nerves from these mice and sural nerve biopsies from nf2 patients show reduced nf-h phosphorylation, decreased interfilament spacings and irregular-shaped axons. a. catenaccio, p. silva, f. court pontificia universidad cat olica de chile, santiago, chile axonal degeneration is an active process involved in a variety of neurodegenerative conditions triggered by diverse stimuli. this degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. we have recently shown that axonal degeneration triggered by distinct mechanical and toxic damage is dependent on the activation of the mitochondrial permeability transition pore (mptp), which probably represents a central execution program of axonal degeneration, upon which several pathways converge (barrientos et. al., journal of neuroscience 2011). nevertheless, the participation of other cellular types in this degenerative process has been largely overlooked. after axonal damage in the peripheral nervous system (pns), schwann cells enveloping axons have been regarded as responsible for clearing degenerating axonal debris and to mount an inflammatory response for tissue clearance by invading macrophages. nevertheless, a possible function of glial cells in modulating axonal degeneration has not been addressed so far. here we explored the possibility that glial cells actively participates not only in the clearance of degenerating axons, but directly in the axonal degeneration program during wallerian degeneration. we found that axonal injury activates an early response of schwann cells that results in the fragmentation of their associated axons by actin-rich cytoplasmic domains of schwann cells known as schmidt-lanterman incisures (sli). this first step of schwann cell fragmentation of axons is dependent on the erk signaling pathway, wich control the dedifferentiation program initiated in schwann cells after nerve injury (napoli et. al., neuron 2012). in the axonal compartment, injury triggers a parallel cell autonomous degenerating program dependent on mitochondrial mptp activation, which is cell non-autonomously assisted by schwann cells through the activation of a cell extrinsic pathway of axonal destruction by tnf-alpha secretion. we showed for first that time that axonal degeneration in the pns proceeds by axonal autonomous mechanisms, as well as cell non-autonomous ones dependent on scs. therefore, effective targets for neuroprotection should consider not only the axonal compartment, but also the associated glial cell. glutamate is the major excitatory neurotransmitter in the brain. its concentration is the synaptic area is highly regulated in order to maintain point-to-point transmission and to prevent excitotoxicity associated with chronic activation of glutamate receptors. as there is no endogenous extracellular enzyme to degrade glutamate, the clearance of glutamate is ensured by transporters located on the surface of astrocytes. among these surface transporters glt-1 ensures almost 90% of glutamate uptake at synapses. it is widely accepted that many receptors and transporters on the surface of neurons and glia display surface trafficking properties and are not simply static proteins. the basic surface trafficking properties of glutamate receptors at the synapse have been studied in depth and have allowed us to understand many important trafficking events that occur during synaptic plasticity. the action of glt-1 is fundamentally important in synaptic communication. surface trafficking of this transporter may play a pivotal role in the spatial and temporal properties of glutamate diffusion at the synapse. however, it is still unknown whether glt-1 is dynamically regulated on the surface of astrocytes. thus we set out to uncover the surface trafficking of glt-1 in astrocytes by using a combination of high-resolution imaging, such as single particle (quantum dot [qd]) tracking, molecular biology and electrophysiological approaches. here we have demonstrated that glt-1 is indeed subject to surface trafficking on astrocytes. using pharmacological approaches we have shown that this diffusion is subject to regulation by the activity of the transporter itself as well as neuronal activity. furthermore, we have demonstrated that the speed of glt-1 diffusion is greatly reduced at excitatory synapses. this leads us to the conclusion that glt-1 surface diffusion is highly regulated at the synapse where it can effectively carry out its role as the principal glutamate transporter at the synapse. finally, to elucidate the physiological role of glt-1 surface diffusion, we have carried out electrophysiological experiments in which glt-1 trafficking is effectively reduced using a cross-link (x-link) protocol. we observed that a reduction in the surface trafficking of glt-1 resulted in an increase of neuronal excitability. thus glt-1 surface trafficking on astrocytes has an implicit role in regulating basal neuronal activity through glutamate uptake at the synapse. o. chever, e. dossi, n. rouach collège de france, paris, france astrocytes play crucial roles in brain physiology by dynamic interactions with neurons. they form plastic and extensive networks mediated by gap junction channels. it has recently been shown that astroglial networks limit neuronal network activity. however, it is currently unclear how astroglial networks influence neuronal excitability and population activity. to investigate how astroglial assembly regulates neuronal network activity, we performed electrophysiological recordings in hippocampal slices (ca3 and ca1 areas) from mice with disconnected astrocytes, in which both astroglial gap junction forming proteins, connexin 30 (cx30) and connexin 43 (cx43), are knocked out (gfap-cre cx30 -/-cx43 fl/fl , dko). synchronized excitatory discharges were recorded in an acute pharmacological model of epileptic-like activity. in this model, spontaneous bursting discharges are characterized by a sharp and large amplitude shift in field potential, generated by a major depolarization and firing of all putative neurons. pyramidal cells depolarized up to 40 mv during seconds and such depolarization is followed by a long-lasting undershoot of the membrane potential. we found that the frequency of bursting activity in dko hippocampal slices is drastically increased. however, the duration of neuronal depolarizing bursts is severely reduced. furthermore, pyramidal neurons are more depolarized due to an increase of synaptic bombardment. this suggests that increased synaptic background promoting resting membrane potential depolarization facilitates triggering of neuronal bursts, but compromises the strength of synchronized events. to investigate local dynamics of bursts generation, we performed multielectrodes arrays recordings in the ca3 area of the hippocampus. in slices from dko mice, bursting activity in the ca3 region is generated within a more restricted area, indicating that the number of neurons to be recruited is highly decreased. altogether, these results indicate that gap junction-mediated astroglial networks strengthen the coordination of neurons during synchronized events. acutely loaded hypoxia increases ventilation, and increased ventilation does not immediately return to the pre-hypoxic level, but persists for a while after resuming room air inhalation. this phenomenon is referred to as short term potentiation or plasticity of breathing, and is thought to contribute to the stabilization of ventilation. although extensive studies have been conducted to clarify the cellular mechanism of respiratory plasticity focusing on neurons and synapses, its precise mechanism remains unknown. on the basis of the recent discovery that astrocytes are actively involved in neural plasticity in various brain regions, we hypothesized that the post-hypoxic potentiation of breathing is mediated by astrocytes. we used unanaesthetized unrestrained adult mice. ventilatory parameters (respiratory frequency, tidal volume and minute ventilation) were measured by whole body plethysmography. to test the hypoxic response, mice breathed room air, hypoxic gas (7% o 2 , 93% n 2 ), and again room air. we also analyzed the hypercapnic response that was conducted under a hyperoxic condition to eliminate the influence of the carotid body. to test the hypercapnic response, mice breathed pure o 2 , hyperoxic hypercapnic gas (95% o 2 , 5% co 2 ), and again pure o 2 . after recording of the hypoxic and hypercapnic responses with their recovery time courses, arundic acid (ono-2506) was injected intraperitoneally. arundic acid is a compound that suppresses the function of astrocytes through the inhibition of s100b production in astrocytes. arundic acid did not affect ventilatory augmentation induced by either hypoxia or hypercapnia. however, arundic acid suppressed the short term potentiation induced by hypoxia (but not by hypercapnia) in a dose-dependent fashion. high dose arundic acid completely eliminated hypoxia-induced short term potentiation. these results suggest that the post-hypoxic potentiation of breathing is mediated by astrocytes with the involvement of s100b. the mechanism of hypercapnia-induced potentiation is different from that of hypoxia. we propose a model to explain the cellular mechanism of post-hypoxic potentiation: hypoxia stimulates the respiratory neuronal network in the brainstem through the excitation of the carotid body. neurotransmitters spilled from excited neuronal synapses activate nearby astrocytes. the activation of astrocytes is sustained, and these astrocytes persistently excite respiratory neuronal network by releasing gliotransmitters. monosynaptic c-fibre evoked excitatory postsynaptic currents (epscs) were recorded in lamina i neurons using whole-cell patchclamp in rat transverse spinal cord dorsal-root slice preparations. spontaneous epscs were recorded simultaneously. in all experiments bicuculline and strychnine were added to the bath solution to block gaba a -and glycine-mediated synaptic currents. the superfusion of spinal cord slices in vitro with fkn results in a rapid facilitation of evoked epscs in spinal lamina i neurons receiving monosynaptic input from primary afferent c-fibres. both the microglial cell inhibitor minocycline and a fkn neutralising antibody, completely prevented fkn-induced changes in synaptic transmission. in addition, the inclusion of the ca 21 chelator bapta or the nmda receptor antagonist mk801 in the pipette solution completely prevented fkn-induced enhancement of synaptic strength, suggesting a post-synaptic ca 21 -dependent mechanism of ltp induction. analysis of paired-pulse ratio (ppr) indicated that fkn-induced facilitation of synaptic strength is accompanied by a decrease in ppr suggesting a pre-synaptic mechanism of ltp expression. in support of this finding, fkn increases the number of spontaneous epscs recorded from lamina i neurons. these data indicate that stimulation of microglial cells in order to induce a pro-nociceptive activity state is sufficient for facilitation of synaptic strength within the dorsal horn. both pre-and post-synaptic mechanisms contribute to fkn-induced facilitation of synaptic strength. this work was funded by a wellcome trust flexible travel fellowship to akc. the organization and connectivity of the developing neocortex remains largely unresolved. the establishment of neuronal microcircuits is a complex process that partly depends on the diversity of neuronal cell types and their ultimate role in the mature brain. this complexity is increased by the existence of bona fide synaptic connections between neurons and oligodendrocyte precursor cells, also called ng2 cells, which properties and function remains largely unknown. to unravel the physiological characteristics of gabaergic synapses between interneurons and ng2 cells, we performed paired recordings in the developing somatosensory cortex. experiments were carried out during the second postnatal week in vgat-venus;ng2-dsred double transgenic mice that allowed us to identify interneurons (venus 1 ) and opcs (dsred 1 ) in acute slices. our results demonstrated that ng2 cells are synaptically contacted by fast-spiking (fsi) and non-fast-spiking (nfsi) interneurons. however, a predominant input was received by fsi, a class of interneuron that constitutes only a minor population in the second postnatal week. on the contrary, only a small proportion of the abundant nfsi were connected to ng2 cells. all the connections showed paired-pulse depression, low probability of response, and preliminary analyses suggest that each interneuron form a single synaptic contact onto ng2 cells. paired recordings, extensively used to examine the synaptic properties of neuronal microcircuits, may open a new perspective to understand in detail the mechanisms of gaba release and signaling between interneurons and ng2 cells in the healthy and pathological developing brain. first, we characterized a microglial cell line obtained from cd1 mouse cortex (n9), in order to clarify the mechanisms involved in the activation pattern of these cells. for that, we incubated n9 microglia with 300 ng/ml of lipopolysaccharide (lps) for 24 h. morphology (anti-iba1 staining), phagocytic ability (fluorescent latex beads) and chemotaxis to atp (boyden chamber) were evaluated. we observed that n9 microglia is ramified in control conditions and after lps treatment they change to an amoeboid morphology, which is accompanied by increased phagocytosis and decreased migratory ability, indicating that lps induces activation of n9 cell line. next, we investigated the role of the released factors from a mn-like cell line expressing human sod1 with g93a mutation (nsc-34/ hsod1g93a) on microglia functions. nsc-34 expressing human sod1 wt were used as control. thus, n9 microglia were incubated for 4 and 24 h with nsc-34 conditioned media that had been collected at 1 and 4 days of differentiation (div) and reactivity tests were performed as above. although after 4 h of incubation no significant changes were observed, data evidenced that incubation for 24 h with nsc-34/ hsod1g93a conditioned media collected at 4 div changed the bipolar morphology of the n9 microglia into an active amoeboid state, decreased phagocytic ability (22%, p < 0.05) and induced apoptosis (50%) (fig.1) . moreover, microglia have shown to not be attracted by the released factors from degenerating mn, as determined by the chemotaxis assay. together, our results evidence that released factors from nsc-34/ hsod1g93a degenerating cells although inducing microglia morphological activation trigger a reduction in cell phagocytic ability and loss of viability, suggesting a role for microglia along als progression. organotypic explants culture has a pivotal role in studying the complex structure of neuronal tissue. although embryonic tissue explants and slices are used to obtain long term culture, most applications such as drug testing require adult tissue for reliable results. in this study, we show that nanostructured metal-oxide arrays can be employed to yield long-term organotypic culture of adult neuronal tissue explants as demonstrated for the adult guinea pig retina and adult murine brain slices. even after 14 days of culture, the thickness and the layered structure of the retina were well maintained. quantitatively, the number of nuclei within the inner and outer nuclear layers was comparable to freshly isolated retinae and no significant change in the densities of the nuclei within the layers was observed. the outer plexiform layer, as the site of synaptic connection between photoreceptors and neurons, was still well preserved. moreover, the thickness of the photoreceptor layer was conserved, indicating that the inner and the outer segments of the photoreceptors survived well during two weeks culture. this is hardly observed in long-term cultures since the outer segments were cultured without retinal pigment epithelium. concerning the adult murine brain slices from neo-cortex, after 9 days of culture, the neurofilaments and cell nuclei were still well preserved and the neuronal network displayed the typical arrangement of long axons. however, preservation of the tissue depends strongly on the geometry of the nanostructured array surface such as roughness and spacing, whereby organotypic brain slice culture requires different geometries than retina culture. since organotypic culture of adult tissue could only be obtained for about 7 days with conventional techniques, our new substrate material could be the breakthrough for in vitro studies on tissue regeneration, neuroplasticity, as well as drug testing. the intrinsic optical signal (ios) is widely used for mapping neuronal activity in afferent activated brain areas. ios evoked by afferent stimulation in vitro is generally attributed to glial cell swelling via activation of na 1 /k 1 /clcotransporter that follows postsynaptic activation. despite the phenomenon is known for decades, the relative contribution of different cell types and the underlying molecular mechanisms have not been disclosed in detail. our goal was to explore the glial and neuronal correlates underlying in vitro ios genesis. we characterized ios initiated by schaffer collateral stimulation in the rat hippocampal slice using simultaneous high-frequency imaging and field potential recordings. we used a 464-element photodiode-array device (pda) that enables ios detection with 0.6 ms time resolution, making it achievable to align optical and electrophysiological signals. ios was primarily observed in the proximal region of the stratum radiatum and stratum pyramidale of the hippocampus. ios was decreased by blockade of neuronal activity by voltage-gated na 1 channel inhibitor tetrodotoxin and was significantly enhanced by suppressing inhibitory signaling with the gaba a antagonist picrotoxin. we found that ios was predominantly initiated by postsynaptic glutamate receptor activation and progressed by the activation of astroglial glutamate transporters and mg 21 -independent astroglial nmda receptors. in agreement with previous studies, furosemide, the blocker of both the neuronal k 1 / clcotransporter kcc2 and the glial na 1 /k 1 /clcotransporter nkcc1 decreased the ios amplitude. to decide which cotransporter is responsible for this effect of furosemide, we applied selective blockers of nkcc1 and kcc2, bumetanide and (1)[r]-dioa, respectively. we evidenced, that nkcc1 did not contribute to the ios generation, in contrast to previous suggestions. enhancement and slight inhibition of ios through anion and volume-regulated anion channels, respectively, were also depicted. major players of ios mechanisms disclosed by high-frequency ios imaging imply that spatiotemporal ios reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. our model may help to better interpret in vivo ios and support diagnosis in the future. question: a poor x3 polyunsaturated fatty acids (x3 pufa) status, favored by the low x3/x6 ratio in western diets, seems to contribute to cognitive decline in the elderly, but mechanistic evidence is lacking. we therefore explored the impact of x3 status on the evolution of glutamatergic transmission, astroglial regulation and neurogenesis in the hippocampus during ageing in rats. these processes are involved in memory formation and their dysregulation participates to the agerelated brain damage leading to cognitive decline. methods: we have compared 6 groups of rats aged 6 to 22 months fed x3-deficient, x3/x6-balanced, or x3 (fish oil) supplemented diets: young x3 balanced (yb), deficient (yd) or supplemented (ys), and old x3 balanced (ob), deficient (od) or supplemented (os) rats. we have evaluated synaptic efficacy and plasticity (electrophysiological recording), astroglial regulations (glutamate uptake and gfap expression), neuronal markers (glutamate transporters and receptors), neurogenesis (proliferation of neuronal precursors in the sub granular zone), and analyzed brain fatty acids composition. results: dietary modulation of x3 intakes efficiently modified the incorporation of docosahexaenoic acid (dha, the main x3 in cell membranes) in brain (250% deficient vs balanced, 110% supplemented vs balanced). ageing induced a 35% reduction of synaptic efficacy due to decreased pre-synaptic glutamate release, and a 30% decrease in the astroglial glutamate uptake associated to a marked astrogliosis (1100% gfap). x3 deficiency further decreased these hallmarks of ageing (od vs ob rats: 235% synaptic efficacy, 215% glutamate uptake, 130% gfap). on the opposite, x3 supplementation increased synaptic efficacy (125% os vs od) and seems to abolish astrogliosis (os vs ys : no change in gfap). neurogenesis was altered by x3 deficiency but not by supplementation. conclusion: our results characterize some specific age-related alterations of the glutamatergic synapse in the hippocampus that are aggravated by a dietary deficit in x3 and attenuated by x3 supplementation. methods: sgcs were isolated from the trigeminal ganglion (tg) of adult male wistar rats (n 5 9). animals were deeply anaesthetized and both tg were extracted and digested first in 5 mg/ml collagenase for 15 min and then in 0.125% trypsin containing dnase for 10 min. in initial experiments, isolated sgcs were treated with control medium or medium containing 16.5, 33, or 66 mm cis for 8h. in the subsequent experiments, sgcs were pretreated for 24h with control medium or medium containing 100 mm ibu or 1 mm skf. afterwards, sgcs were stimulated for 8 hours with medium containing 66 mm cis. pge 2 concentration was determined by elisa. results: cis caused a concentration-dependent increase in pge 2 release from sgcs. treatment with 33 mm cis significantly increased pge 2 concentration to 354.8 6 87.3 pg/ml, compared with control 211.3 6 39.0 pg/ml (p 5 0.018). pge 2 concentration was further increased to 464.1 6 77.0 pg/ml (p 5 0.002) following 66 mm cis. pretreatment with ibu significantly lowered the pge 2 concentration compared with cis-stimulated sgcs (197. 5 6 85.2 pg/ml vs. 415.8 6 143.8 pg/ml, respectively; p 5 0.05). a more pronounced inhibitory effect was observed following skf pretreatment, where it reduced the pge 2 concentration to 19.7 6 1.8 pg/ml (p 5 0.003, compared with cis-stimulated sgcs). conclusion: these findings suggest that cis contributes to an inflammatory response in the tg by provoking release of pge 2 from sgcs, which may lead to development or maintenance of peripheral sensitization involved in cipn. the data also suggest that treatment with agents that target pge 2 release cascade may prove beneficial for preventing development or maintenance of cipn. functional alterations in synaptic contacts have often been described as a hallmark of major depressive disorder (mdd). antidepressants (ads) have been shown to restore neuronal circuits through an enhancement of synaptogenesis in some regions of the brain. nevertheless, the underlying mechanisms are still unclear. glia cells have been since long acknowledged as active partners of neurons in orchestrating molecular signals crucial for the proper arrangement of neuronal circuits in the developing and adult brain. therefore, understanding how both neurons and astrocytes respond to antidepressants (ad) is of high interest. using rat c6 glioma cells and primary cultures of astrocytes and neurons, we showed a time-dependent cell-autonomous modulation of the erk/mapk signalling pathway after acute treatment with various classes of ads in both cell types. specifically, glia cells responded to ads with the simultaneous and fast activation (after 10 min treatment) of both erk1/2, in contrast to treatment with antipsychotics or mood stabilizers. this activation induced 48 hrs later an increased release of gdnf, a factor involved in synapse formation and axonal wiring, which was mapk-dependent. on the contrary, hippocampal neurons showed a reduction in erk activity that correlated with neuronal activity inhibition after short term (10 min) ads administration, as demonstrated by quantification of c-fos expression after kcl stimulation. interestingly, this inhibitory effect was reversible after long term (48 hrs) ads treatment. to further identify whether gdnf released from astrocytes treated with ads might be responsible for long term changes in neuronal synapses, we examined how ads influenced synaptic densities in neurons alone or co-cultured with astrocytes. strikingly, we found that the number of synapses was reduced at 48hrs after ad treatments, but only in the presence of intact astrocytes and not in cultures of neurons alone or neurons treated with astrocyte-conditioned media. this effect was reversed after 120 hrs treatment and rescued by the soluble form of the specific gdnf receptor gfralpha1, but not by gdnf alone. moreover, live imaging of astrocytes showed dramatic morphologic changes of their processes in response to ads that might underlie the observed synaptic remodelling. our analysis of changes occurring at the neuronglia interface upon ad treatments might elucidate novel mechanisms of ads action that may open the avenue for unravelling the role of astrocytes in psychiatric diseases and implement pharmacologic treatment regimens for mdd patients. here we extend these findings showing that the neurotrophin nt-3, which also belongs to the putative factors secreted from glial cells after t3-stimulation, also increases the navd in neuron enriched cultures. the effects of nt-3 and fgf-2 are, however, not additive. antibodies against nt-3 potentiated the effects of fgf-2, suggesting that by converging signal cascades nt-3 could reduce the effect of fgf-2. in neuron-glia mixed cultures antibodies against nt-3 enhanced the t3-induced up-regulation of navd, suggesting, that a co-secretion of nt-3 from glial cells could limit the effects of fgf-2. in a second series of experiments we investigated, whether the well-known effect of t3 on the expression of na 1 /k 1 -atpases, thought to contribute to the effect of thyroid hormone on metabolism, is also influenced by glial cells. we found, that the effect of t3 on 3 [h]ouabain-binding and on the expression of na 1 / k 1 -atpase alpha2 subunits detected by western-blots was highest in neuron-mixed cultures, suggesting that a neuron-glia interaction is also involved in the t3 effects on na 1 /k 1 -atpase expression. in the healthy brain microglia engage in bi-directional interactions with neurons and other brain cells, which is crucial for maintaining normal physiology and cognitive function of the brain. disturbances in homeostasis and cell-cell communication may predispose to age-related dysfunction and neurodegenerative disease. for decades it has been speculated that microglia exhibit phenotypic diversity allowing specialisation to a variety of brain microenvironments. previous observations of regional variations in microglial densities and morphologies support the existence of microglial heterogeneity. however it is clear that a more comprehensive understanding, in particular of the molecular basis of microglial phenotypic and functional diversity on a global scale, is required. the limitation of recent gene expression studies is the use of mixed whole brain or dissected mixed cell extracts which preclude the detection of cell-specific gene expression profiles. thus, our aim was to validate an efficient extraction process for microglia, ideally minimising the impact on the resting state. the method developed is based on existing procedures and was refined to ensure consistency and to maximise cell yield and purity. the extraction of highly pure microglia as a single cell type was achieved by a two-step isolation technique using density-gradient and magnetic bead separation. purity between 90-95% was verified by flow cytometry and immunohistochemistry. quantitative pcr also demonstrated expression of specific microglial genes indicating that isolated cells are microglia. cytokine assays suggest that no or only minimal activation of cells was induced by the isolation. hence, the microglia retained their ability to respond to immunogenic stimuli and produce il-1b as a key inflammatory cytokine. overall, the purification procedure was shown to achieve consistent high purities and retain crucial functional properties. thus, the extracted microglia are likely to adequately represent the real in vivo state and enable the study of microglial phenotypes and functionality in the healthy and ageing whole brain and accordingly brain regions. early diversity experiments showed region-specific microglia phenotypes in the brain of healthy and immune challenged animals as well as differences between unchallenged and challenged animals. question: activation of nmda receptors increase the respiratory frequency in mammals. astrocytes are in intimate contact with neurons, specially in glutamatergic synapses, and are able to sense neuronal activity, react to, and even influence nearby neurons. furthermore, they can sense changes in h 1 or pco 2 , and in response to these stimuli, they can release molecules like atp and d-serine, which will activate neuronal circuits. then d-serine, an agonist of the glycine site of the nmda receptor, can affect the respiratory rhythm during hypercapnia. our aim was to study the probable role of d-serine in the modulation of the respiratory rhythm in mice neonates. methods: fictive respiration was recorded with suction electrodes from c4-c5 ventral roots in "en bloc" (brainstem-spinal cord) preparations from p0-p3 cf1 mice. superfusion was done with artificial cerebrospinal fluid equilibrated with o2:co2 5 95%: 5%, (ph 7.4,28 c) containing different d-serine concentrations (0.1-100 mm) in presence or absence of d-amino acid oxidase (daao), which degrades d-serine. in addition, hypercarbic acidosis (switching equilibration from 5 to 10% co2, changing ph from 7.4 to 7.2) was done in absence or presence of daao. results: application of d-serine increased the frequency of the respiratory rhythm in a concentration-dependent way. application of dao attenuated the effects of d-serine application. likewise, daao reduced slightly the effect of hypercarbia on the respiratory rhythm. conclusions: our preliminary experiments indicate that d-serine is a potent agent increasing the respiratory rhythm frequency in in vitro neonatal preparations, and likely, in association with atp, is mediating the respiratory response to hypercarbia. there, we showed that mac-2 positive microglia accumulate within the motoneuron area and phagocyted apoptotic bodies at the onset of motoneuron developmental cell death (from embryonic day 12.5-e12.5). motoneuron developmental cell death and microglia accumulation in the motoneuron area increased at e13.5, decreased at e14.5 and disappeared at e15.5. since it was supposed that microglia promote developmental cell death in the developing central nervous system, at least in vitro, we determined to what extent it was also the case in the embryonic spinal cord in vivo. to address this issue, we analysed in vivo the progression of developmental neuronal death in the spinal cord of transgenic mouse embryos lacking microglia (pu.1-ko mice). as opposed to what is observed in the sc of wild type mouse embryo, we found that activated caspase-3 staining was detectable before e12.5 in pu.1-ko mouse embryos. in pu.1-ko mouse embryos, activated caspase-3 staining was not restricted to the motoneuron area, as several positive cells are also present in the dorsal region of the spinal cord and in the progenitor zone. at e13.5, activated caspase-3 staining was 10 times greater in the spinal cord ventral area of pu.1-ko mouse embryos when compared to wild type littermates. moreover, and opposed to what occurs in wild type animals, caspase-3 staining in the motoneuron area of pu.1-ko mouse embryos increased at e14.5 and persisted at e15.5, being likely partly due to the accumulation of apoptotic bodies in the absence of microglia. our results suggest that, in addition to their phagocytic role, embryonic microglia are likely to protect neuron from death at the onset of developmental cell death and to promote survival of progenitor-like cells in the embryonic sc in vivo. accordingly, microglia could have an opposite effect on cell survival in the embryonic sc when compared to postnatal snc developmental stages and pathological conditions in the adult. we recently demonstrated a key role of the a-secretase tace, also known as adam17, in peripheral nervous system (pns) myelination by tace-mediated cleavage and subsequent inhibition of neuregulin1 (nrg1) type iii activity. unlike the pns in which nrg1 type iii is an essential instructive signal for myelination, oligodendrocyte (ol) development and myelination in the central nervous system (cns) are likely controlled by several growth factors some of which undergo cleavage by secretases. to study the role of tace on opc development, immunopanned a2b51 opcs were cultured in proliferating or differentiating medium and treated with a soluble form of tace (rhtace). when ols were scored according to their morphology, we observed enhanced opc differentiation upon addition of rhtace to proliferating opcs. addition of rhtace to differentiating opcs also increased the number of ols with a complex morphology and already presenting membrane sheets, suggesting that tace might promote opc differentiation. to further investigate the role of neuronal tace in cns, we used an in vitro ol myelinating coculture system. when cocultured with tace null drg neurons, rat wild type opcs never myelinate, suggesting that neuronal tace might control opc differentiation. in agreement, preliminary ultrastructural analyses of transgenic mice lacking tace in cns neurons showed hypomyelination and signs of myelin degeneration. accordingly, conditional transgenic mice lacking tace in ol are normally myelinated. these studies suggest that in the cns the a-secretase tace promotes opc differentiation and might regulate cns myelination. a better understanding of the role of tace will provide novel insights into the mechanism regulating cns myelination. in the excitatory network glutamate is released in the synaptic cleft during synaptic activity. glutamate clearance by astrocytes is mainly performed by na 1 -dependent glutamate transporters. the na 1 load during glutamate uptake evokes an activation of the na 1 /k 1 atpase, which dramatically increases energy demands in astrocytes. astrocytes are also involved in the regulation of extracellular potassium (k e 1 ) which significantly increases during the repolarization of excitatory neurons. in this study we evaluated how these fundamental functions of astrocytes coexist and if they interact. we investigated the impact of altering k e 1 concentrations on glutamate transporter activity measuring intracellular sodium (na i 1 ) using the na 1 sensitive fluorescent dye asante sodium green loaded in cultured astrocytes. we observed that glutamate uptake caused a reversible rise in na i 1 , which was tightly modulated in amplitude, slope and recovery rate by k e 1 levels. in order to evaluate the impact of physiologically relevant k e 1 fluctuations on the kinetics of the glutamate transporter the na 1 /k 1 atpase was inhibited. therefore we found that low k e 1 evoked a significantly faster influx of na 1 , whereas high k e 1 markedly slowed down glutamate capture, indicating that k e 1 directly modulates the kinetics of glutamate transporters. we then investigated the energy demands of astrocytes caused by k e 1 alterations. atp hydrolysis was indirectly measured through the changes in intracellular free mg 21 using the mg 21 sensitive fluorescent probe magnesium green. we found that low k e 1 led to higher energy demands of astrocytes during glutamate uptake. overall these results indicate that k e 1 acts as a negative feedback on glutamate uptake in astrocytes. the physiological consequences of these findings have to be considered in the context of k 1 buffering and of maintenance of neurotransmitter and energy homeostasis. recovery was significantly improved by nf fractions, as well as tubulin (tub), added after lpc removal: compared to cultures treated with lpc alone the number of olp (a2b51 cells), and their proliferation increased significantly, as well as the number of differentiated (cnp1) and mature (mbp1) ol. moreover, nf and tub protected ol from lpc toxicity when added at the time of lpc treatment: they increased olp proliferation, as well as the number of cnp1 and mbp1 ol significantly, compared to cultures treated only with lpc. on the contrary irrelevant proteins (actin, and skin proteins) were ineffective, demonstrating the specificity of the cytoskeleton proteins effects. importantly, nf and tub increase olp survival and proliferation when challenged with lpc, without delaying differentiation and maturation. we hypothesize that release of nf and tub following axonal damage in ms could participate in the regulation of remyelination through this process. this putative phenomenon might be complexified by alterations of axon protein expression, or of proteolysis, known to occur in ms models. purpose: microglial proliferation is commonly accepted as a hallmark of glial activation. an automatic method that allows assessing the number of microglial cells to analyze the proliferative microglial behavior in a laser-induced ocular hypertension (oht) model has been developed. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n 5 6) and lasered (n 5 6). retinal wholemounts were immunostained with anti-iba1 and images of iba-11 microglias were recorded with a fluorescence microscope. new algorithms of segmentation and control of distances were developed in matlab to obtain the number of iba-1 microglial cells. automatic results were compared with those obtain by direct human observation of the samples. results: the automatic method detected the number of iba-11 cells in the inner and outer plexiform layers of the retina in both, na€ ıve and oht-retinas. the number of cells present in the samples was not an obstacle for the program to run properly. the time required for counting iba-11 cells decreased from the human guide to the program based counting method from days to one hour. results show a strong correlation between both automatic and manual method (pearson correlation test, r 5 0.979; p 5 0.000 and r 5 0.942; p 5 0.000 for outer and inner plexiform layer respectively) indicating the consistency of the automatic counting. conclusions: a new, reliable and quick algorithm was developed with matlab to obtain the number of iba-11 microglial cells and also cellular density maps through retinal wholemounts in both na€ ıve and other proliferative conditions (i.e. glaucoma). due to this new automatic method, a bigger set of images or samples could be included in future studies to analyze the behavior of microglial cells. we are interested to understand whether scs in the peripheral nerves of mammals express functional ionotropic glutamate receptors, and whether scs use these receptors for communication with axons. to start addressing this question, we established a preparation of mouse live sciatic nerve slices employing "know-how" of live brain slices technique. using this preparation, we performed whole-cell patch-clamp recordings of scs at different stages of development (embryonic and early postnatal). we included a fluorescent dye into the pipette solution in order to perform post-recording morphological analysis of single scs. first, we recorded current responses of scs to a series of depolarizing voltage steps, aiming to test whether all scs in the nerve are electrophysiologically akin. we found that mouse scs differ in their passive membrane properties and expression of voltage-gated k1 channels: depending on the age, 3 to 4 cell types could be distinguished. post-recording morphological characterization of the recorded cells showed that scs in the mouse sciatic nerve differ in their size, as well as in the number and length of their processes. next, we used fast pressureinduced application of glutamate to investigate whether scs possess ionotropic glutamate receptors, and whether electrophysiologically distinct sc types differ in their expression of glutamate receptors. application of 1 mm glutamate caused an inward current in at least two types of scs, and preliminary analysis revealed a peak current amplitude in the range of 14-250 pa (n 5 18). glutamate-induced currents in scs were blocked by gyki53655, indicating that mouse scs carry ionotropic glutamate receptors of ampa/kainate type. currently we are studying which subunits of ampa receptors are present in scs, and how these receptors get activated in situ. although cx32 is expressed by myelinating schwann cells in the peripheral nerves, patients with cmt1x develop relatively early axonal degeneration, which correlates best with chronic disability. we found in previous studies of gjb1-null mice, a model of cmt1x, that the diameter of myelinated axons was progressively reduced at the age of 2 months compared to wild type (wt) littermates, resulting from progressive dephosphorylation of axonal neurofilaments and increased packing density. fast axonal transport was slower in distal axons of mutant compared to wt animals, with impaired mobility of synaptic vesicleassociated proteins and accumulation of beta-amyloid precursor protein and tau. how cx32-formed gjs are involved in the regulation of axonal cytoskeleton dynamics remains unclear. methods-here we investigated the signaling mechanisms regulating axonal cytoskeleton focusing on major kinases and phosphatases involved in neurofilament phosphorylation by immunohistochemistry and immunoblot. in addition we examined the localization of axonal mitochondrial, which also depend on axonal transport. we focused on nerve pathology in 2 month-old gjb1-null mice, when demyelination and inflammation is minimal, in order to identify the direct effects of gj loss on the axon. results-our results show that erk1/2 kinase and pp2a phosphatase were localized in non-compact myelin areas, including the schmidt-lantermann incisures and paranodes, where gjs are normally formed by cx32. another kinase, cdk5, was more diffusely localized along the axon. biochemical analysis showed altered amounts of erk1/ 2, pp2a and cdk5 as well as their upstream activators in gjb1-null nerves. the pp2a inhibitor phapi was also localized in non-compact myelin areas and significantly increased in gjb1-null nerves. assessment of axonal mitochondria by porin immunostaining showed significantly increased density in gjb1-null nerves, consistent with slower axonal transport. conclusion-our findings clarify some mechanisms of early axonal pathology that are independent of demyelination in this mouse model of cmt1x. several components of the signaling pathway regulating axonal cytoskeleton and axonal transport appear to be functionally related to gjs at the non-compact myelin sheath. loss of myelin gjs results in impaired regulation of axonal cytoskeleton, energy supply, and axonal transport. we have recently discovered that oligodendrocytes secrete exosomes containing a distinct set of proteins as well as mrna and microrna. intriguingly, oligodendroglial exosome release is stimulated by the neurotransmitter glutamate indicating that neuronal electrical activity controls glial exosome release. in this study we examined the role of exosomes in neuron-glia communication and its implications in glial support. results: to analyze the transfer of oligodendroglial exosomes to neurons, we exposed cultured cortical neurons to fluorescently labeled oligodendroglial exosomes. indeed, cultured cortical neurons internalized and accumulated oligodendroglial exosomes in the neuronal cell soma in a time-dependent manner. addition of endocytosis inhibitors or expression of dominant negative dynamin interfered with neuronal exosome internalization indicating that exosome uptake is mediated by clathrin-dependent endocytosis. furthermore, neuronal internalization of exosomes resulted in functional retrieval of exosomal cargo in vitro and in vivo upon stereotactic injection of exosomes. to investigate the influence of oligodendroglial exosomes on neuronal gene expression we performed a microarray screen of neuronal mrnas after exosome exposure. interesting candidates were further validated by qrt-pcr. conclusion: taken together, our results represent a proof of principle of exosome transmission from oligodendrocytes to neurons suggesting a new route of horizontal transfer in the cns. astrocytes are essential for the regulation of neuronal excitability, synaptic plasticity, and the vascular coupling of brain metabolism. unlike neurons, they are electrically silent, but produce a complex repertoire of ca 21 events that coordinate their major functions. however, the canonical cellular mechanism for ca 21 integration in astrocytes is unknown. using cultured astocytes and astrocytes in brain slices expressing ca 21 sensor, gcamp2, we report a candidate principle for ca 21 integration in single astrocytes. analysis of the spatiotemporal dynamics of ca 21 signaling in cultured astrocytes demonstrated that ca 21 event distribution can be described by a power law. we show that metabotropic glutamate receptor activation or changes in extracellular ca 21 regulate the power law exponent. these findings demonstrate scale-free ca 21 dynamics in astrocytes that can provide a potential computational theory for brain operation and metabolism. the biogenesis of the axon-myelin unit is controlled by reciprocal interactions between oligodendrocytes and neurons, which continue throughout life. oligodendrocytes secrete endosome-derived microvesicles termed exosomes, implicated in intercellular communication. these exosomes carry a specific set of proteins as well as rna species. here, we show that the neurotransmitter glutamate stimulates exosome secretion from oligodendrocytes by provoking ca 21 entry through oligodendroglial nmda and ampa receptors. furthermore, active neurons evoke exosome release from oligodendrocytes, indicating that neuronal activity controls oligodendroglial exosome release. in turn, neurons internalize oligodendroglial exosomes and functionally retrieve the exosomal cargo. thus, oligodendrocytes may influence the neuronal metabolism by an exosome-dependent transfer of bioactive substances to neurons. axonal degeneration resulting from lack of glial support occurs in plp and cnp deficient mice. intriguingly, both proteins are components of oligodendroglial exosomes. a proteomic approach revealed that amount and composition of exosomes derived from plp -/and cnp -/oligodendrocytes are altered, supporting the hypothesis that disturbed intercellular transfer of substances by exosomes may contribute to axonal degeneration in these mouse models. a. shahraz, j. kopatz, h. neumann university of bonn, bonn, germany microglial cells are the resident macrophages of the brain, which are derived from the embryonic haematopoiesis of the yolk sac. recently, we established a protocol for in vitro differentiation of pluripotent stem cells into microglial precursors by mimicking the embryonic haematopoiesis in a neural microenvironment. we now succeeded to produce microglia from human induced pluripotent stem (ips) cells. these human microglial cells were responsive to amyloid-b by increasing reactive oxygen species (ros) production. in addition, we produced primitive neural stem cells (pnsc) from ips cells that differentiated with suitable growth factors towards neurons. in human microglia-neuron co-culture, amyloid-b treatment resulted in shorter neural branches length compare to the control group. furthermore, results showed that the inhibitory human lineage specific receptor sialic acid binding immunoglobulin-like lectin-11 (siglec-11) was expressed on human microglial lines. we are now studying the neuroprotective role of siglec-11 in the amyloid-b-mediated microglia-neuron co-culture model. by expressing the human-lineage specific receptors, these microglial cells will be a suitable model to investigate these receptors in a human microglianeurons co-culture system.x purpose: organotypic retinal culture is a useful tool to perform preclinical drug testing, in which cellular interactions as well as tissue threedimensionality are preserved. the present study aimed to provide a detailed characterization of the morphologic changes of macro and microglial cells in this culture system. methods: retinas were isolated from 7 days old crl: cd(sd) rats with the retinal pigment epithelium attached as described previously (arango-gonzalez et al. 2010). explants were cultured for 4, 10 and 14 days (div4, div10 and div14). glial cell populations were characterized by immunostaining cryosections and wholemounts of age-matched control and cultured retinas using specific antibodies against gfap, vimentin and cd11-b. results: in div4 and div10 cultures, gfap-positive astrocytes were more robust and not homogeneously distributed as observed in vivo: in some retinal areas the astrocytic network was thicker and in other regions astrocytes were sparsely distributed. at div14 only few thinned gfap-positive astrocytes remained. gfap immunoreactivity of m€ uller cells was up-regulated in culture. however, no major difference was observed in vimentin staining between both, in vivo and in vitro retinas. m€ uller cells extended processes from the inner to the outer limiting membrane were observed for all examined retinas. microglial cells stained with cd11-b at div4 and div10 had somas more robust than in vivo and cell processes were thicker and retracted. same cells at div14 exhibited mainly a rounded morphology. conclusions: progressive morphological changes were observed in glial cell populations in retina explants. these changes include reactive macrogliosis, rearranged astrocytic distribution and microglial activation. since glial response is a hallmark of several retinal diseases, our organotypic retinal culture is a valuable resource for future investigations in retinal degenerative processes and therapy. it is widely recognized that grouped housing in enriched environment (enr) impacts the animals' brain structure and function. for instance, rats reared in enr exhibit enhanced neurogenesis in the dentate gyrus, improved hippocampus-dependent spatial memory task performance, and spine density increases of ca3 and ca1 pyramidal neurons. we found that that enr housing for four weeks after weaning induces an enhancement in gamma band local field potential oscillation power and an increase in spine density in the right ca1 stratum radiatum of long-evans rats. the mean spine size, as assessed by serial measurements of postsynaptic density areas, did not change substantially by laterality or rearing condition. one standing question is how glial processes are organized in enr treated rats. unlike cerebellar parallel fiber synapses, the astrocytic coverage of a hippocampal synapse varies from synapse to synapse. our preliminary electron microscopic observation suggests that right ca1 str. radiatum excitatory synapses tend to have larger degrees of astrocytic coverage in enr treated rats than singly caged rats.we are currently making morphometric measurements to quantitatively assess the peri-synaptic glial changes. it is becoming more and more evident that proper functioning of the neuron-astrocyte-microglia triad is fundamental for the functional organization of the brain, and thus it is of the utmost importance to better characterize their interactions in physiological and pathological processes. we recently demonstrated, in rat models of normal brain aging and lps-induced acute inflammation, that astrocytes and microglia actively collaborate in the clearance of apoptotic neurons and neuronal debris associated with programmed cell death. here we studied the interactions between neurons, microglia and astrocytes within the ca1 region of the hippocampus after bilateral common carotid artery occlusion (bccao) in the rat, a valid model of chronic cerebral hypoperfusion which leads to persistent ischemic conditions and ultimately to neuronal death. male wistar rats were subjected to permanent bccao. a group of rats was infused into the jugular vein with dipyridamole (7 days, 4 mg/kg/day by an osmotic minipump). sham-operated animals were used as controls. three months after bccao, immunohistochemical studies were performed on brain coronal slices, focussing on the hippocampal ca1 region. using an antibody against glial fibrillary acidic protein (gfap) no astrogliosis was detected. we found a significant increase in the number of total microglia, visualized using the iba1 antibody, in bccao-treated rats in comparison to the sham group (1 18%, p < 0.01, one way anova and newman-keuls) and this effect was completely reverted by dipyridamole (p < 0.01, one way anova and newman-keuls). as exemplified in figure 1 , in the ca1 and str. radiatum of bccao-treated rats, many neurons (anti-neun antibody, red) showing signs of degeneration were closely apposed to and infiltrated by astrocyte branches (anti-gfap antibody, green) which appeared to be bisecting the cell body into cellular debris, and microglia cells (anti-iba1, antibody, blue) were actively phagocytosing the damaged neurons. this finding is consistent with the scavenging activity of microglia upon dying neurons or debris, a possible mechanism that prevents further injury to neighboring neurons. it will be interesting to investigate which intercellular communication mechanisms allow the recruitment and activation of different glial cells in a well-organized reciprocal interaction to scavenge the damaged neurons and to verify the mechanisms of the protective effects of dipyridamole found in this chronic cerebral ischemic model. astrocytes are glial cells which provide important metabolic support to neurons, actively tune synaptic activity and influence brain microcirculation [1] . one of the key processes which sustain astrocyte communication with neighbouring cells is regulated exocytosis mediating the release of gliotransmitters (peptides, amino acids, atp), delivery of membrane transporters, channels and other molecules to the plasma membrane. the exocytic gliotransmitter release is thought to be associated with snare complexes. these consist of four a-helices where one of these is contributed by syntaxin-1, one by synaptobrevin-2 (sb2) and two by snap-23 in astrocytes. out of these, sb2 is a trans-membrane protein present on the secretory vesicle [2] . however, the subvesicular nano-architecture and the number of sb2 molecules per vesicle are unclear. moreover, it is also unknown how many sb2 molecules are involved in the fusion between the vesicle and the plasma membrane in astrocytes. to study single vesicles and their association with sb2 in live astrocytes, we generated a ph-sensitive indicator yellow synapto-phluorin (ysph) as a marker for sb2 and as a functional readout for monitoring the properties of fusion pores, which are formed following the merger between the vesicle and plasma membranes. in an acidic environment ysph is non-fluorescent and becomes fluorescent upon alkalinisation, permitting the study of fusion of vesicles with the membrane during gliotransmitter release [3] . our preliminary results, obtained by confocal fluorescence microscopy (with the resolution limit of about 200 nm) and by a super-resolution microscopic technique structured illumination microscopy (sim; with the resolution limit of about 120 nm) show that the new probe (i.e. ysph) efficiently reports the fusion pore establishment in astrocytes and the configuration of sb2 molecules in a vesicle. the number of schwann cells is fitted to the axonal length in the peripheral nerves. this setting is lost when tumorigenic stimuli induce uncontrolled schwann cell proliferation, generating tumours such us neurofibromas and schwannomas. schwann cells proliferate as well during wallerian degeneration. in both cases proliferation is finally arrested. we show that in neurofibroma, the induction of jmjd3 removes trimethyl groups on lysine-27 of histone-h3 and epigenetically activates the ink4a/arf-locus, forcing schwann cells towards replicative senescence. remarkably, loss of function of this mechanism allows unrestricted proliferation, inducing malignant transformation of neurofibromas. interestingly, our data suggest that in injured nerves, schwann cells epigenetically activate the same locus and go as well into the senescence program. indeed, when this pathway is genetically blocked, cell proliferation results increased after nerve injury. we postulate that the ink4a/arf-locus is expressed as part of a physiological response that prevents uncontrolled proliferation of the de-differentiated schwann cells generated during nerve regeneration, a response that is also activated to avoid overproliferation after tumorigenic stimuli in the peripheral nervous system. university of rochester medical center, rochester, united states synaptic plasticity is critical for normal neurodevelopment and proper circuit function in the adult nervous system. recent evidence suggests that microglia, classically studied in neuroinflammation, play critical roles in neurodevelopment and synaptic plasticity. however, the mechanisms driving these roles are not well understood. to start elucidating the molecular players involved in microglial communication with neurons during plasticity, we decided to first explore the role of purinergic signaling in this process. while purinergic signaling has been implicated in microglial behaviors, studies have primarily focused on neuroinflammatory roles. however, noninflamed microglia highly express the purinergic receptor, p2y12, which has known functions in microglial chemotaxis in early inflammation and can stimulate the release of cytokines implicated in synaptic plasticity. thus, we posited that purinergic signaling could contribute to the microglial motility that underlies synapse surveillance in the non-inflamed brain. this in turn may be critical for microglial roles in synaptic refinement during development. to explore this possibility, we took advantage of the p2y12 knock-out (ko) mouse and examined the morphology and motility of microglia in the visual cortex as compared to microglia in wildtype mice. first, we used immunohistochemistry for the microglia specific ionized calcium-binding adapter (iba-1) protein to stain microglia in fixed sections. we then used confocal imaging and scholl analysis to investigate the complexity of the microglial processes. we also used 2-photon microscopy to monitor microglial motility in p2y12 ko mice by crossing them with cx3cr1-gfp knock-in mice that allow visualization of microglia including fine processes in vivo. our preliminary evidence suggests that p2y12 disruption alters basal microglial morphology and behavior making microglia less complex and altering their motility patterns. we are now investigating whether microglia-synapse interactions, as well as functional plasticity elicited by visual experience are altered in p2y12 ko mice. our studies will expand our understanding of emerging roles for microglia in neurodevelopment and will pioneer new non-pathological roles for microglial purinergic signaling. a. dvorzhak, a. wojtowicz, r. grantyn charit e -university medicine, berlin, germany in neurodegenerative diseases, the afflicted brain is both an important object of study and an opportunity to characterize a given cellular interaction from a pathophysiological perspective. this dual approach is particularly advantageous when human disease is based on a monogenetic defect and an appropriate animal model becomes available for detailed investigation, as in case of z_q175_ki (q175), a new knock-in mouse expressing a mutant form of murine huntingtin. electrophysiological recordings of gabaergic unitary ipscs from striatal output neurons (sons) in sagittal slices from wild-type and homozygous q175 challenged the current viewpoint that gabaergic transmission is enhanced in the hd striatum. quantal analysis in combination with high frequency stimulation and paired pulse tests revealed that synaptic gaba release is in fact tonically suppressed resulting in disinhibition of striatal output activity (dvorzhak et al j physiol 2013). the underlying mechanism involves a retrograde endocannabinoid signalling pathway linking postsynaptic mglur5 with presynaptic cb1 and gaba release. in addition to this deficit, z-q175_ki homozygotes exhibited a significant reduction of tonic inhibition via extrasynaptic gaba(a) receptors. using pharmacological tools to alter the ratio of ambient glutamate and gaba concentrations made clear that the hd-related depression of synaptic and extrasynaptic gabaergic actions depends on the state of both the astrocytic glutamate transporter glt-1 and the gaba transporter gat-3. in support of h eja and colleagues, our imaging experiments suggest that in normal striatal astrocytes gat-3 is in a releasing mode and driven by the activity of glt-1. however, the latter activity is much lower in the hd striatum. sbfi recordings of glt-1-related na transients and patch clamp recordings of glt-1 transporter currents revealed a marked reduction (less than 50% of wt level) in sr-labelled astrocytes from symptomatic hd mice. together, our results suggest that the deficiency of astrocytic glutamate uptake might represent a pathophysiological key mechanism underlying the observed disinhibition in the striatum of mice affected by huntington's disease. nodes of ranvier are highly enriched in voltage-gated sodium channels (nav), allowing rapid action potential propagation on myelinated axons. cell adhesion molecules (neurofascin-186 (nfasc186) and contactin) and scaffold proteins (ankyring and bivspectrin), which are also enriched at the nodes, have a critical role in assembly and/or stabilization of na v clusters. oligodendrocytes have been described to induce nav channel clustering at nodes in the central nervous system (cns); however, the nature of the signal(s) involved in nodal formation remains mostly unknown in the cns. to gain insight into cns node assembly, we developed hippocampal neuronal cultures from e18 rat embryos. during the first week, as expected, na v channel accumulation was detected at the axon initial segment (ais), co-localized with ankyring and nfasc186, whereas expression along the axon was diffuse and hardly visible. in contrast, at 14 days in vitro (div), regularly spaced clusters of na v channels, ankyring and nfasc186 were detected along the axon. the percentage of hippocampal axons with clusters increased from 4.1 6 3.1% at 14 div, to 15.8 6 3.5% and 17.167.1% at 17 and 21 div, respectively (mean 6 sd). importantly, these clusters were formed in the absence of myelin and no accumulation of the paranodal protein caspr was detected at this stage. to study the role of extrinsic versus intrinsic pro-aggregating factors, we analyzed nearly pure hippocampal neuronal cultures. these purified neurons still formed ais, while only few axons formed na v clusters. interestingly, when these purified neurons were co-cultured with oligodendrocytes or with conditioned medium from oligodendrocytes, the percentage of neurons with nascent nodes was greatly enhanced. taken together, these results confirm that some glial soluble factor(s) might be involved in na v channels clustering, as previously shown for retinal neurons (kaplan et al., 2001). furthermore, nascent nodes were detected on gabaergic neurons, but not on glutamatergic hippocampal neurons, thus arguing also for the role of intrinsic neuronal factors in their establishment. using hippocampal sections from gad67-gfp mice, we showed that some gabaergic interneurons were myelinated in vivo. moreover, we observed that nascent node formation prior to myelination also takes place in vivo, strengthening physiological relevance. the electrophysiological properties of neurons with nascent nodes will be studied. microglia in the degenerating or aging brain are primed by aspects of pathology to produce exaggerated pro-inflammatory responses to subsequent central and systemic inflammatory insults. however, the mechanisms by which microglia become primed, rather than adopting an m1 phenotype, are not clear. triggers for priming may include altered expression of complement, recognition of amyloid or neuronal/synaptic debris for phagocytosis, decreased engagement of microglial regulatory molecules such as cd200r, trem2 or fractalkine receptor. there is also evidence that loss of basal neurotransmitter tone may contribute to this state. microglia are known to express the nicotinic cholinergic a 7 receptor, which has been shown to influence macrophage and microglial reactivity. in the current study we performed limited lesions of the basal forebrain cholinergic system using the murine-p75-saporin immunotoxin (mu-p75-sap), to investigate the degree to which loss of cholinergic tone predisposes the microglia to subsequent inflammatory stimulation and to assess the consequences of this for cognitive function in the vulnerable brain. intracerebroventricular injection of mu-p75-sap (0.08 lg) depleted cholinergic neurons in the basal forebrain and decreased cholinergic innervation of the hippocampus, but left performance on hippocampal-dependent reference and working memory tasks relatively intact. however, decreased cholinergic innervation of the hippocampus conferred increased susceptibility to cognitive deficits induced by systemic lps (100 lg/kg) 40 days after lesioning, but microglia were not primed 40 days after 20% lesions. to investigate the regional, temporal and neurochemical basis of this we induced more severe lesions of the basal forebrain (using 1.2 lg mu-p-75 sap) and assessed microglial priming at 14 or 40 days post-lesion. these data demonstrate that microglia are primed by low dose challenge at 14 but not 40 days and, when more robust lesions are induced, priming remains even at 40 days. these responses are observed at the site of injury (medial septum) but more profoundly at the site of denervation, the hippocampus. indicating that neuronal injury and, particularly, loss of cholinergic tone have a profound effect on the reactivity of the microglia to subsequent inflammatory challenge. these data expand our knowledge of microglial priming and have clear implications for the interaction of cholinergic tone and inflammation in cognitive dysfunction such as dementia and delirium in which cholinergic dysfunction is implicated. ). however, glucose may also be metabolized to glycogen or oxidized to co 2 , so it is not obvious that the stimulation of glut1 and hexokinase leads to lactate release. the objective of this work was to investigate whether lactate may be released by cultured mouse astrocytes within the time frame of the interstitial lactate rise observed in vivo. a first approach with an enzymatic assay showed a significant increase in extracellular lactate after 1 minute of exposure to 12 mm k 1 or 50 lm glutamate. to obtain better time resolution we developed a "lactate sniffer", a hek293 cell that expresses the fret lactate nanosensor laconic (san mart ın et al., plos one 2013). seeded on top of an astrocytic monolayer, the sniffer cell responded within 10 seconds of exposure to elevated k 1 12 mm and a similar response was elicited by 3 mm ba 21 . the response to k 1 and ba 21 supports a role for the na 1 /bicarbonate co-transporter nbce1, previously shown to mediate the stimulation of astrocytic glycolysis (ruminot et al., j. neurosci., 2011). the sniffer cell detected an equally fast increase in extracellular lactate when the culture was exposed to 50 lm glutamate. previous work had shown that glutamate does not stimulate glucose consumption in the short-term (bittner et al., j. neurosci. 2011), and therefore the rapid release of lactate came as a surprise. this phenomenon may relate to glycogen mobilization or perhaps to inhibition of astrocytic oxygen consumption (azarias et al., j. neurosci. 2011). k 1 , ba 21 and glutamate did not produce detectable effects on the sniffer cell in the absence of astrocytes. the rapid release of lactate by astrocytes exposed to k 1 and glutamate supports a role for these cells in the fast increase in interstitial lactate concentration that accompanies synaptic activity. ] i ) are regularly occurring as a result of uptake of glutamate from the synaptic space. glutamate uptake occurs via the glutamate/na 1 co-transporters glast and glt-1, where one glutamate is accompanied by 3 na 1 and 1 h 1 in exchange for 1 k 1 . the salt pump, na,k-atpase (nka), is responsible for the maintenance of the trans-membrane na 1 gradient by exporting 3 na 1 and importing 2 k 1 for each atp. nka is dose-dependently inhibited by ouabain. astrocytes express two isoforms of the catalytic nka a subunit, the ubiquitous a1 and in the cns the glia-specific a2. the isoforms differ with regard to na 1 affinity, which is lower for a2 and with regard to ouabain affinity, which is higher for a2 than for a1. although a2 mutations give rise to neurological symptoms -familial hemiplegic migraine type 2, the relative roles of the a1 and a2 isoforms in astrocytes are still incompletely understood. we hypothesized that the low na affinity of a2 makes it more suitable to cope with transient large increases in na i . to test this, we compared the effect of 200 mm glutamate for 10 min on real time changes of [na 1 ] i in primary astrocytes, which, following transient transfection, predominantly expressed either the a1 or the a2 isoform. in a1 cells glutamate caused a significantly larger increase in [na 1 ] i and longer recovery time to base line [na 1 ] i than in cells expressing a2. glutamate uptake dependence on either a isoform was determined by aspartate uptake in astrocytes expressing endogenous a1 and a2. in the presence of ouabain concentrations that selectively inhibit a2, we found a modest (1.9mm) increase in [na 1 ] i , accompanied by a disproportionately large decrease (24%) in glutamate uptake. it has been suggested that astrocyte glutamate transporters are clustered in microdomains where they may interact with nka. in gst pull down assays on rat brain we found that both glt-1 and glast interacted with the 1st intracellular loop of both a1 and a2, but the interaction was significantly stronger for the a2 isoform. no interaction was found with other segments of the a molecule. the data indicate that the nka a2 isoform, which has a more restricted expression than a1, plays a specific role for the interaction with the glutamate transporters and for sodium homeostasis in astrocytes. this specificity may be attributed to low sodium affinity of a2 and to the relatively high capacity of a2 to interact with the astrocyte glutamate transporters. ozone, a major component of air pollution, has considerable impact on public health. besides its well described inflammatory and dysfunction effects on the respiratory tract, there is accumulating evidence indicating that ozone exposure also affects brain functions. however, the mechanisms through which ozone exerts toxic effects on the cns remain poorly understood. we previously showed that in addition to lung inflammation,ozone exposure caused a neuronal activation in the dorsolateral regions of the rat nucleus tractus solitarius (nts, a sensory nucleus involved in visceral information processing) overlapping terminal fields of lung primary afferent running in the vagus nerves to investigate this hypothesis, we used electron microscopy and immunoblot techniques. in ozone-exposed animals, the astrocytic coverage of nts glutamatergic synapses is increased while the astrocyte volume fraction and the astrocyte membrane densities are not significantly increased. moreover, the expression of specific astrocyte markers gfap, s100beta and ezrin did not change in ozone-exposed animals. altogether, our results indicate that ozone inhalation induces glial plasticity, which is restricted to the peri-synaptic coverage. . we recorded pp-gc synaptic activity after eae induction via adoptive transfert (at-eae) and found that mepsc frequency was increased compared to control animals. moreover synaptic alteration in at-eae mice depended on astrocytic tnfr1 because the effect was absent in tnfr1-/-mice but reappeared upon tnfr1 re-expression in astrocytes. nr2b receptors are also necessary because in vivo ifenprodil injection prevented synaptic alteration. overall, our study reveals a specific mechanism responsible for hippocampal synaptic dysfunction possibly relevant for cognitive impairment in ms. research supported by snsf grant 31003a-140999 and nccr "synapsy" to av. we patched ng2 cells in the hippocampal ca1 region of ng2-dsred mice (8-20 days old) and investigated in current clamp mode how sodium and potassium channels affected synaptic depolarizations. epsps and ipsps were evoked from a resting membrane potential of 285mv by injecting mock epsc or ipsc waveforms derived from miniature currents. epsps which depolarized ng2 cells to more than 245 mv were increasingly shortened by vgcs. at 216 6 1 mv, corresponding to a quantal content of 640, the half width of epsps was shortened to 47 6 3% while the amplitude was hardly altered. application of ttx, 4-ap and tea showed that most of the observed shaping of epsps was primarily due to activation of a-type k1 current rectifying the depolarizing input. na1 currents only slightly ($10%) amplified epsp input, but this effect was outcompeted by the dampening effect contributed by a-type current ($15%). tea was almost without effect on epsps. due to their slower time course ipsps were very differently shaped by vgcs: while recruitment of vgcs by mock ipsps started at a similar threshold voltage, the maximal suppression of ipsp amplitude was much stronger. when depolarizing ng2 cells to 235 6 1 mv, corresponding to a quantal content of 235, ipsp amplitudes were suppressed to 52 6 4% and the duration of ipsps was not shortened but increased to 193 6 5%. a-type k1 current was the primary contributor for rectifying as well. interestingly, if a-type k1 current was blocked, ipsps with a quantal content of 105 unmasked a small ttx-sensitive spikelike depolarization of 20 6 2 mv (duration: 7 6 1 ms). for comparison, while synaptic responses to simultaneous release of 100 glutamatergic vesicles were hardly altered by vgcs, the synaptic response of 100 gabaergic vesicles was substantially low-pass filtered by a-type potassium currents. altogether, our results suggest that vgcs in ng2 cells normalize the synaptic strength of excitatory and inhibitory inputs and support their differentiation by ng2 cells through further emphasizing their pre-existing kinetic differences. to explore the involvement of this pathway in pain and analyze its impact separately in sensory neurons and sgcs, we tested pharmacological inhibitors and transgenic mice in an orofacial pain model. transient inflammation in the submandibular region of mice was induced using complete freund's adjuvant (cfa) and tactile sensitivity was quantified using von frey filaments. although inflammation was resolved by 28 days after injection, tactile hypersensitivity persisted in wildtype mice for at least 53 days, consistent with chronic post-inflammatory pain. tactile hypersensitivity in mice was reversed by systemic injection of the panx1/gap junction blockers mefloquine or carbenoxolone at 7 days (peak inflammation) and at 28 days after cfa-injection, suggesting the contribution of these channels to tactile hypersensitivity. in dissociated trigeminal ganglia (tg), which innervate the submandibular skin, bzatp-induced yopro uptake into neurons and glia was prevented by mefloquine, demonstrating the functional presence of the p2x 7 r-panx1 complex in tg. moreover, tg of cfa-injected mice showed more atp release and immunostaining of tg revealed higher expression of panx1 at 1 week after cfa injection compared to controls. development of hypersensitivity was prevented in p2x 7 r-null and in panx1-null mice, and was attenuated in mice with glia-specific deletion of panx1 (gfapcre-panx1f/f) but not with neuron-specific deletion (mnfhcre-panx1f/f), emphasizing the importance of glial panx1 signaling in pain. because p2x 7 r and panx1 both have a key role in the immune response by activating the inflammasome, we are currently dissecting the relative importance of neuron-glial communication compared to inflammatory responses in the development and maintenance of orofacial pain. overall, our results show that the p2x 7 r-panx1 complex likely plays a major role in signaling events contributing to tactile hypersensitivity. synaptic plasticity is central to the process of learning and memory and is accompanied by strengthening of existing synapses and/or formation of new synapses. numerous studies have investigated the molecular mechanisms of learning and memory with neurons as the primary interest. given the tight coupling between synaptic activity and brain metabolism, it seems reasonable to assume that synaptic plasticity changes occurring at the synaptic level may also occur at the metabolic level in order to keep up with the augmented demand at the synapse. in this study we investigated the importance of genes involved in brain energy metabolism, and in particular those involved in neuronglia metabolic coupling during fear-motivated inhibitory avoidance learning. using ( 14 c) 2-deoxyglucose (2-dg) technique we first defined the brain areas that are involved in this learning process. context-dependent avoidance behavior was tested in c57bl/6 mice using the step-through inhibitory avoidance paradigm (ia). regional brain metabolic activity, as measured by the 2dg uptake, was quantified in several brain regions. brain metabolic mapping revealed increased glucose utilization in hippocampus, amygdala [(basolateral complex (bla) and the central nucleus (cea)], and anterior cingulate cortex and mammillary bodies. quantitative mrna levels were assessed in dorsal hippocampal tissue at different time points following ia learning. results obtained demonstrate that learning modulates the expression pattern of genes involved in brain energy metabolism, i.e. glycogen synthesis and degradation, pyruvate metabolism, the pentose phosphate shunt and the astrocyte-neuron lactate shuttle (anls) in a time dependent manner. we found a late-phase of enhanced dorsal hippocampal gene expression, particularly for anls related genes, including monocarboxylate transporter 1 (mct1). furthermore, we found that mice deficient in mct1 transporter display impaired long term memory in the inhibitory avoidance and spatial learning in morris water maze. these observations indicate that metabolic adaptation shown by neuron-glia metabolic coupling might be a relevant phenomenon following learning in conjunction with synaptic plasticity. in order to systematically as well as functionally analyse rmg reactivity to retinal degeneration and thus explore the rmg-derived signalling molecules in depth, we aim at comprehensively profiling proteomewide cellular responses to stimuli. we thus developed a proteomics approach screening specifically for cell surface and membrane proteins as well as secreted protein expression profiles and in addition monitor quantitative changes induced by prototype inflammatory inducer lipopolysaccharide lps. this workflow utilises sugar residues of cell surface proteins on intact rmg which are mildly oxidized and then covalently coupled to biotin. biotinylated proteins are affinity purified and glycosylated peptides are specifically released by pngasef followed by protein identification and quantification by label-free lc-msms. this workflow resulted in the identification of more than 500 proteins on cell surfaces complemented by more than 700 proteins identified in the rmg secretome. bioinformatic analysis allocates 75% of the cell surface proteins to be truly membrane or extracellular matrix proteins. this comprehensive cell surfaceome includes transmembrane receptors, transporters, adhesion molecules, signalling molecules and proteases, including 18 cd markers, 18 integrins, 41 solute carriers, two ephrins, five ephrin receptors and six plexins. treatment of cells with lps results in a highly reproducible significant shift of cell surface proteome with upregulation of 36 proteins and downregulation of 13 proteins. among those lps-induced cell surface expression changes are proteins that suggest an active role of these glial cells in inflammatory processes. the specific changes will be discussed in detail. cell surface biotinylation on glycosyl-residues in combination with label-free lc-msms is a sensitive and reproducible method to profile cell surface proteomics and sheds light on the biological properties of cells. background: neuronal activity alters calcium ion (ca 21 ) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. to examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol 1,4,5-trisphosphate absorbent, "ip 3 sponge", attenuates astrocytic ca 21 signaling. results: attenuated ca 21 activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal ca1 region in these animals. the decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced n-methyl-d-aspartate receptor currents in ca1 pyramidal neurons in response to burst stimulation. these mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. conclusions: our findings suggest that ip 3 -mediated astrocytic ca 21 signaling correlates with the formation of functional tripartite synapses in the hippocampus. they are the only non-neuronal cell type in the brain that receives synaptic input from neurons. ng2-cells exhibit a variety of ca 21 -signalling pathways, which might be triggered by pre-synaptic neuronal activity. however, no global increase in the intracellular ca 21 -concentration ([ca 21 ] i ) was detected in ng2-cells employing the minimal stimulation technique at neuron-glial synapses. therefore we assume that post-synaptic ca 21 -microdomains might be activated under physiological conditions. these ca 21 -signals are locally restricted to the plasma membrane. membrane bound ca 21 -sensors with a high signal-to-noise ratio, such as lck-gcamp3, are best suited for optimal visualisation of ca 21 -microdomains. here, we set out to express lck-gcamp3 in ng2cells together with the cytosolic reporter dye dsred. we generated transgenic mice which express a bidirectional promoter encoding both proteins under the control of a tet-off system. this strategy allows an exclusive expression of the transgenes only in the presence of a tet-responsive transcriptional activator (tta). the construct was successfully cloned and expressed in hek293 cells. as expected, lck-gcamp3 was located only at the inner plasma membrane while dsred was expressed all-over the cytosol. functionality of the ca 21 -sensor was tested in vitro. elevation of [ca 21 ] i reliably increased the fluorescence intensity of lck-gcamp3. after zygote injection seven positive founder animals could be identified and will be crossbred with a ng2-tta mouse line. taken together, the presented construct appears suitable for functional imaging of ca 21 -microdomains in post-synaptic ng2-cells. according to the "lactate shuttle" hypothesis, glycogen stored in astrocytes can be converted to l-lactate and exported to neurones as preferred energy substrate. we use optogenetics to investigate signalling between astrocytes and noradrenergic (naergic) neurones of the locus coeruleus (lc). to selectively activate g s -protein-mediated signalling in astrocytes, we generated a viral vector to express a chimera of rhodopsin and b 2 -adrenoceptor (optob 2 ar). we confirmed that this construct activates camp-mediated signalling in astrocytes. in cultured astrocytes, excitation of optob2ar resulted in acidification as detected using snarf-5 indicator, and this acidification could be blocked by pre-incubation with an inhibitor of glycogen breakdown 1,4-dideoxy-1,4-imino-d-arabinitol (dab), suggesting that the acidification was due to build-up of l-lactate. fast scan cyclic voltammetry (fcv) was used to measure noradrenaline (na) release in organotypic slices cut at the level of the lc in which astrocytes were expressing optob 2 ar. light stimulation of astrocytes powerfully induced release of na which could be prevented by pre-incubation with dab or application of d-lactate. bath application of l-lactate (!100 mm) in absence of optogenetic stimulation triggered the release of na. in patch clamp experiments, l-lactate depolarised lc neurones and evoked vigorous firing of action potentials. these experiments suggest that activation of g s -protein-coupled receptors in astrocytes could potentiate the release of na from lc neurones via l-lactate. the cellular and molecular mechanisms of this signalling mechanism are currently under investigation. supported by the british heart foundation. rgcs. in addition to this, the organization of astrocytes in the retina of a rat model of glaucoma was found to be significantly different to those of a healthy retina. these alterations in glial cell morphology may reflect changes in their relationship with rgcs and could signify profound changes in their secretome, which may influence rgc survival. in this study we investigated labeling of astrocytes by sr101 in acute slices from the ventro-lateral medulla and the hippocampus of transgenic mice expressing egfp under the control of an astrocyte-specific promoter. while sr101 efficiently labeled egfp-expressing astrocytes in hippocampus, we found that sr101-staining was very weak in the ventro-lateral medulla and rather unspecific. thus, sr101 is not a reliable marker for brainstem astrocytes. although carbenoxolone decreased the labeling of astrocytes in the hippocampus significantly, mefloquine which blocks pannexin and connexin hemichannels, was unable to prevent sr101 uptake in hippocampal astrocytes. time-lapse 2-photon imaging revealed that in hippocampus both astrocytes and neurons showed temporary sr101-loading. in astrocytes the rise of sr101 fluorescence was slower as compared to egfp-negative cells and sr101 was quickly removed from non-astrocytic cells during washout, while it was retained in astrocytes. in brainstem astrocytes, however, only a very weak and transient sr101-labeling was observed. to test if sr101 is actively removed from astrocytes in the brainstem, we applied mk-571 to block the multi-drug resistance transporters mrp-1. we did not observe any increase of sr101-labeling in brainstem astrocytes. in contrast, astrocytic sr101 labeling was significantly reduced by substrates of organic anion transport, probenecid, estron-3sulfate and dehydroepiandrosterone sulfate suggesting that sr101 is actively transported into hippocampal astrocytes by an organic anion transporting polypeptide (oatp). additionally, the data suggest that astrocytes modulate extracellular concentration of neurosteroids in the hippocampus. as an alternative approach we have used scanning electron microscopy (sem) to obtain high-resolution back scattered images (bsi) of serial ultrathin sections over a wide-area. in tem the size of the specimen is limited to less than 1 mm 2 , whereas in sem it can be expanded to 20-30 mm 2 , allowing us to mount and examine more than 100 serial ultrathin sections on the same stage, collecting 3d information about many nodal structures simultaneously. here we demonstrate a variety of nodal architectures assembled from bsi-sem (su8010,hitachi). in our preliminary study using this technique, the pattern of cellular processes approaching the perinodal axolemma is quite different among axons even in only four nodes examined in optic nerve. in the present study, we analyzed perinodal elements at 25 whole nodes in rat optic nerves and reconfirmed this variety of perinodal glial elements include the followings: 1) perinodal space with extracellular matrix (20-100%); 2) closely abutting astrocytic processes (0-80%) ; 3) unidentified glial cell, presumable ng21 glial processes (0-50%), but could not find 4) pre-synapse-like pseudo-neuronal processes, which was observed at the perinode in the cns grey matter (cerebral cortex) in the previous study. additionally, we found unexpected structures, small teardrop-like protrusion(s) of axolemma at 18 out of 25 nodes examined (72%). in 16 nodes out of these 18 nodes (88.9%), glial elements encircled or densely contacted with the teardrop protrusions. a typical case of 3d reconstructed image is shown in the figure. nodal axon (ax, red), unidentified glial process (yellow; ug, presumable ng2 cell) and teardrop-like protrusion (asterisks) are demonstrated. outer part of the protrusion from the position indicated two arrows is encircled by the process of ug cell. based on these observations, we propose that some glial cells, including astrocytes and/or ng2 cells, might contribute to remove wasted membranous elements from the axon at the node of ranvier, which is a new type of neuron-glial interaction. to understand what makes this ssc network more synchronous and excitable, we performed a morphological study of neurons and astrocytes by estimating densities of neun and s100-positive cells respectively. although we found similar densities of neurons and astrocytes between gaers and non-epileptic controls, the thickness of the ssc was 30% smaller in gaers. western blotting quantification of gfap, another astrocytic marker, was significantly higher at p15-p17, as compared with non-epileptic control. moreover, gfap-immunolabeling suggested that these astrocytes were reactive. we hypothesized that these modifications are linked with an alteration of astrocytic and/or neuronal physiological calcium excitability that could lead to the occurrence of epileptic seizures. to better evaluate the role of astrocytes and neurons networks during the development of ssc between p15 and p25, we used two-photon microscopy imaging coupled with eeg recording in animal under anesthesia and neuroleptanalgesia. we first analyzed neuropile, representing ascendant projection of deeper cortical layers, astrocytes and neurons calcium activities. preliminary results obtained from animals under isoflurane anesthesia showed similar neuropile activities. astrocytic and neuronal activities were too weak to highlight differences between gaers and non-epileptic control. this is likely due to the mode of anesthesia known to strongly decrease astrocyte calcium signaling and neuronal synchronization. current experiments are therefore performed using neuroleptanalgesia know to allow the occurrence of swd. the expected data should lead to a better understanding of neuron-astrocyte interactions during brain development and the mechanism underlying epileptogenesis in a genetic model of epilepsy. purpose: to study the effects of laser-induced ocular hypertension (oht) in the astrocytes of contralateral eyes two weeks after lasering. methods: adult swiss mice were divided into two groups: na€ ıve (n 5 6) and lasered (n 5 6). retinal whole-mounts were immunostained with antibodies against gfap. the gfap-labelled retinal area (gfap-ra) and the number of astrocytes were quantified. results: in comparison with na€ ıve: i) astrocytes were more robust in contralateral eyes. in oht-eyes, the astrocyte population was not homogeneous, given that astrocytes displaying only primary processes coexisted with astrocytes in which primary and secondary processes could be recognized, the former having less intense gfap-ir (p<0.001). the mean percentage of astrocytes in which only primary processes could be detected was 37.8%; ii) gfap-ra was increased in contralateral (p<0.05) and decreased in oht-eyes (p <0.001); iii) the mean intensity of gfap-ir was higher in oht-eyes (p<0.01), and the percentage of the retinal area occupied by gfap1 cells with higher intensity levels was increased in contralateral (p 5 0.05) and in oht-eyes (p<0.01); iv) the astrocyte number did not differ significantly among the eyes analyzed. however, in oht-eyes the number of astrocytes in which primary and secondary processes could be observed were decreased (p<0.01). conclusions: two weeks of laser-induced oht caused changes in the gfap-labelled retinal area but not in astrocyte number in both, contralateral and oht-eyes. on the basis of the astroglial changes detected in the present work, the use of the contralateral eye as an internal control in experimental model unilateral oht should be reconsidered. astrocytes show a high level of functional and morphological heterogeneity and are involved in many aspects of neural function, e.g., formation of the blood-brain barrier, regulation of ion homeostasis, synaptogenesis, and synaptic plasticity. despite the diversity of this cell type, the various astrocyte subpopulations are not well characterized, which is mainly due to the lack of markers that would allow for classification of astrocyte subsets. our study aimed at phenotyping of astrocyte subpopulations based on the expression of cell surface markers, which are associated with certain functions. we used two novel monoclonal antibodies, acsa-1 and acsa-2 (acsa: astrocyte cell surface antigen), directed against extracellular epitopes of astrocyte-specific cell surface markers to identify astrocyte subtypes. the acsa-1 antibody was generated by immunization of glast1 knockout mice and specifically detects the astrocyte-specific l-glutamate/l-aspartate transporter glast (eaat1, slc1a3), whereas the acsa-2 antibody results from an immunization of rats with astrocytes isolated from gfap-egfp transgenic mice. a mass spectrometric approach for the ligand-based identification of the acsa-2 antigen pointed to a number of candidates, which have to be validated by further experiments. the antibodies acsa-1 and acsa-2 were carefully analyzed by costaining experiments with commonly used intracellular astrocyte markers. we found that both antibodies specifically detect astrocytes in the developing and adult central nervous system. in contrast to antibodies against intracellular markers, such as gfap, s100ß, or glutamine synthetase, acsa-1 and acsa-2 can be used to detect and isolate living astrocytes, which enables further analysis and culture of the separated cells. flow cytometric analysis of acsa-1 and acsa-2 antigen expression on cells from different brain regions, as well as immunohistochemical analysis, revealed differences in the expression patterns. this allowed us to define different astrocyte subtypes especially in the cerebellum and olfactory bulb of the neonatal mouse brain. future work will focus on the identification of additional astrocytespecific cell surface proteins for a comprehensive classification of astrocyte subtypes based on cell surface marker expression or expression patterns. the reciprocal communication between neuronal and glial cells represents the key component of the immunosurveillance system of the brain. it has been proposed that the neuro-glial interaction is impaired in human alzheimer's disease. in this study we focus on neuronal "on" and "off" signalling molecules in the transgenic rat model for alzheimer's disease. using enzyme-linked immunosorbent assay we determined the level of cd47, cx3cl1 ("off" signalling molecules) and mmp3, trem2 ("on" signalling molecules) in brain homogenates. our results demonstrated significantly up-regulated level of cd47 (p < 0,001) in our ad rat transgenic animal model. on the other hand, quantification of mmp3 level revealed significant decrease of mmp3 expression (p < 0,001) in tested transgenic animals. previous studies showed that cd47 and mmp3 are predominantly expressed on neuronal cells in cns where cd47 functions normally as a marker of "self" to protect intact body component or "don't eat me" molecule which protect cd47-expresing cells from phagocytosis. our results indicate that overexpression of cd47 on neuronal cells expressing pathologically modified truncated tau protein can be used as a neuroprotective mechanism potentiating spread and accumulation of pathological modified tau protein in neurons affected by ad pathology which in final stage support progression of the developing disease. in conclusion, we showed, that pathologically modified tau protein can modulate specific "on and off" signalling molecules and thus affects neuron-glia interaction in ad. field-excitatory post-synaptic potentials (fepsp) were recorded from the ca1 area of hippocampal slices prepared from wistar rats (3-5 weeks old) as before (fontinha et al., 2008) . after obtaining a stable value of the fepsp slope for at least 30 min, ltp was induced by delivery of 3 trains of 3 pulses at 100hz, each train being separated by 200 ms. ltp magnitude (% increase of fepsp) was evaluated 50-60 min after induction. ltp magnitude in control conditions was 16 6 5.9%, whereas in a second independent pathway of the same slices but in the presence of bdnf (20ng/ml) it was 39 6 4.7% (n 5 7; p <0.05). ltp was completely abolished when hippocampal slices were superfused with the gliotoxin fluorocitrate (fc, 200 mm), which selectively reduces the astrocytic metabolism decreasing intracellular ca 21 signalling and the release of gliotransmitters. interestingly, in presence of fc, the facilitatory action bdnf (20 ng/ml) upon ltp was completely prevented. noteworthy, when fc (200 mm) treated slices were superfused with the selective adenosine a 2a receptor agonist, cgs21680 (30nm), the facilitatory action of bdnf (20 ng/ml) upon ltp was rescued (n 5 5; p < 0.05), so that dltp caused by bdnf (ltp in the presence of bdnf -ltp in the absence of bdnf in two independent pathways of the same slices) in fc 1 cgs 21680 treated slices (19%) was similar to that observed in control slices (23% in the absence of fc and cgs 21680). the results suggest that the facilitatory action of bdnf upon ltp is controlled by astrocytes, and that this role of astrocytes results from their contribution to the extracellular accumulation of adenosine allowing a 2a receptor activation to gate plasticity actions of bdnf. several studies demonstrated the ability of astrocytes to sense, respond to and regulate neuronal function. among the many functions of glial proteins, glutamate (glu) and gaba transporters play important roles in balancing excitatory and inhibitory signals in the brain. here we show that astrocytes regulate the tonic inhibition of neurons by the concerted action of glu and gaba transporters, thereby protecting both neurons and glial cells from overactivation. we demonstrate that the uptake of glutamate triggers the reverse function of glial gat-2/3 transporters by elevating the intracellular na 1 concentration in astrocytes. the released gaba significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. we also describe the source of the releasing gaba that is synthesized by an alternative pathway from polyamines. moreover, in the low-[mg 21 ] model of epilepsy, we show that blockade of the glial glu/gaba exchange mechanism increases the duration of seizure-like events and also results in increased activity of astrocytes, demonstrating the neuroprotective impact of the mechanism. finally, we show that the released glial gaba modulates the power of gamma range oscillation in vivo, suggesting that the glu/gaba exchange mechanism is also functioning in the intact hippocampus under physiological conditions. revealing this novel molecular mechanism by which astrocytes provide an adjustable, in situ negative feedback on the excitability of neurons is expected to broaden our understanding about the regulation of neuronal activity by astrocytes and may open up new targets for the treatments of pathological conditions, such as epilepsy or ischemia. chronic stress is well recognised to decrease the number of gfap1 astrocytes within the prefrontal cortex (pfc). recent research, however, has suggested that our understanding of how stress alters astrocytes may be incomplete. specifically, chronic stress has been shown to induce a unique form of microglial remodelling, but it is not yet clear whether astrocytes also undergo similar structural modifications. such alterations may be significant given the role of astrocytes in modulating synaptic function. accordingly, in the current study we have examined changes in astrocyte morphology following exposure to chronic stress in adult rats, using three-dimensional digital reconstructions of astrocytes. our analysis indicated that chronic stress produced profound atrophy of astrocyte process length, branching and volume. we additionally examined changes in astrocyte-specific s100b, which is both a putative astrocyte marker, and a protein whose expression is associated with astrocyte distress. while we found that s100b levels were increased by stress, this increase was not correlated with atrophy. we further established that while chronic stress was associated with a decrease in astrocyte numbers when gfap labelling was used as a marker, we could find no evidence of a decrease in the total number of cells, based on nissl staining, or in the number of s100b1 cells. this finding suggests that chronic stress may not actually reduce astrocyte numbers and may instead selectively decrease gfap expression. together, these results provide a significantly more elaborate picture of how chronic stress alters the pfc. the drosophila nervous system is ensheathed by several types of glial cells, one of which, namely the subperineurial glia, forms pleated septate junctions (psj) to build a tight blood-brain barrier (bbb). hence, nutrients from the surrounding hemolymph must cross these glial cells to reach the neurons. the amount of nutrients entering the nervous system is likely to be tightly controlled through neuron-glia communication to ensure optimal metabolic supply while avoiding disturbance of the extracellular homeostasis. we aim to elucidate signals involved in this regulatory interaction. to this end, an rnai-based screen in glia of adult flies is performed using the gal4/gal80 ts system. we analyze the intake of blue-dyed food by photometric measurement. since impairment of the metabolic supply of the brain should severely affect the organism, compensatory changes in feeding behavior are expected. for instance, knockdown of nutrient transporters specifically in glia would then induce excessive feeding in normally nurtured individuals. the first set of rnai lines screened includes genes that have previously been found to be essential in glia for survival or locomotion of the animal. furthermore it comprises metabolic enzymes, all carbohydrate transporters, neuropeptides and their corresponding receptors. candidate genes will be further characterized regarding their role in controlling the energy homeostasis of the nervous system. the discovery that activation of non-neuronal cns microglia plays a causal role in spinal processing of nociceptive signaling has shed new light on the processes underlying neuropathic pain facilitation. however, there remains much uncertainty as to the necessary contribution of microglia to enhanced pain states. we aim to define the particular role of microglia for the initiation of neuropathic pain and by answering this question also learn if the function of peripheral myeloid cells is distinct or redundant in this process. methods: to specifically investigate spinal microglia and peripheral macrophages in the pathogenesis of neuropathic pain, we model chronic pain by performing partial ligature of the sciatic nerve in cd11b-hsvtk mice engrafted with gfp bone marrow (gfp>cd11b-hsvtk). cd11b-hsvtk 1/mice allow the exchange with peripherally-derived, gfp 1 macrophages upon central depletion of endogenous cd11b 1 microglia. following this depletion/ repopulation paradigm, behavioral analyses of mechanical and thermal allodynia are conducted. results: we established a selective tool to exchange cns parenchymal microglia with peripheral gfp 1 myeloid cells. in chronic pain tests for mechanical and thermal hyperalgesia, gfp>cd11b-hsvtk 1/mice show considerable decreases in paw withdrawal thresholds in response to mechanical, but not thermal stimuli ipsilateral to the injury, indicating distinct roles of microglia and macrophages in the facilitation of thermal hyperalgesia. conclusions: we identified a differential contribution of resident spinal microglia vs. peripheral myeloid cells in the development of neuropathic pain in gfp>cd11b-hsvtk chimeras. future studies aim to examine the exact mechanisms underlying the distinction between these two populations. a deeper understanding of the processes involved in the compartmentation of brain energy metabolism is of major interest. not only to enwrap pathomechanisms of brain diseases associated with metabolic deficits but also for a more accurate interpretation of functional neuroimaging signals which often use metabolic signals as surrogate marker for brain activity (e.g. fdg pet). large amount of knowledge in the field has been obtained from small animal neuroimaging studies. but due to their invasiveness they often need anesthesia. anesthetics heavily alter physiological read-outs. the aim of this study was 1.) to test if metabolic imaging using two-photon microscopy (2pm) and genetically encoded glucose sensors is feasible in the awake, head-fixed mouse and 2.) to examine the effects of isoflurane anesthesia on the signals. in two mice, a recombinant adeno-associated virus as a carrier for the genetically encoded glucose sensor flii 12 pglu600md6 was injected into the whisker barrel cortex. by using an astrocyte-specific promoter (short gfap) astrocytic expression was ensured. in addition, a chronic window and a head post were implanted. behavioral training started a few days after surgery. animals had to learn to tolerate head fixation without spontaneous motor activity. within a single trial animals were trained not to move for 10 seconds (imaging period) to receive a water reward. animals were subjected to water restriction during the behavioral training. animals were trained for three weeks before 2pm imaging started. animals learned to tolerate head fixation up to 90 minutes allowing acquisition of up to 400 trials per session. upon oral glucose administration (per single water reward $1 mg glucose was administered) fret signal started to increase within 5 minutes and went up to a maximum of 8%. surprisingly, isoflurane anesthesia led to an even higher increase of the signal (mean increase of 12%) compared to the awake state. in some experiments the barrel cortex was activated by vibrotactile single whisker deflections generated by a piezo bending actuator. fret signal did not show a difference between baseline and stimulated condition (1.133 6 0.3 vs. 1.136 6 0.29). in conclusion, the presented experiments demonstrate for the first time the feasibility of awake 2pm imaging for metabolic measurements on a single cell level. the signal increase upon peroral glucose administration unambiguously supports the functionality of the sensor. isoflurane exhibits a large effect on intracellular astrocytic glucose concentration distinctly revealing the need for experiments in awake animals for unbiased results. future experiments will now be expanded to neuronal glucose sensors to enable comparisons between astrocytes and neurons. our previous studies indicate that neuronal electrical activity controls glial exosome release resulting in subsequent neuronal uptake and functional retrieval of the exosomal content. proteomic analysis of oligodendroglial exosomes revealed a list of candidates with potential neurotrophic action in neurons. to elucidate the impact of oligodendroglial exosomes on the neuronal metabolism, we analyzed the metabolic activity of neurons after co-culture with oligodendrocytes or direct treatment with exosomes. methods: neurons were subjected to different stress paradigms such as oxidative stress, hypoxia, and nutrient deprivation. neuronal vitality was assessed by mtt assay and the mitochondrial membrane potential was visualized by staining with mitocapture. results: neurons grown under optimal conditions were not affected by the presence of exosomes. intriguingly, when neurons were subjected to stress (oxidative stress, nutrient deprivation, oxygen-glucose deprivation) their metabolic activity was significantly increased in the presence of exosomes. when challenged with oxidative stress prior to exosome treatment, neurons were not able to recover. mitocapture staining demonstrated that oligodendroglial exosomes prevent the breakdown of the mitochondrial membrane in nutrient-deprived neurons. conclusions: our results indicate that exosome supply is protective for neurons but is unlikely to support their recovery. we suggest that oligodendroglial exosomes carry neuroprotective substances, which protect neurons from stress. interestingly, this effect of lactate on synaptic plasticity mechanisms was not fully mimicked by glucose, suggesting that the effect was not only related to supporting the potential increase in energy demands related to plasticity, but that l-lactate could act as a signaling molecule for the regulation of expression of plasticity-related genes. we have followed up on these observations and show that l-lactate significantly stimulates mrna expression of key immediate early genes (iegs), in a time-and concentration-dependent manner, in cultured neurons. following one hour of treatment with 20 mm l-lactate, arc, zif268 and c-fos mrna levels were increased by 5.2, 3.7 and 8.2 folds, respectively. in addition, the increased iegs mrna expression levels induced by l-lactate are correlated at the protein level with respectively 5.5, 4.0 and 3.2 fold increase compared to control values at the same time point. these effects are specific for llactate, since d-lactate (non-metabolized enantiomer of l-lactate), l-pyruvate and d-glucose (at equicaloric concentrations) have no effect on gene expression. interestingly, we observed that, in similar culture conditions, l-lactate-induced iegs expression is only observed in neurons but not in astrocytes, implying a cell specific effect. characterization of the underlying molecular mechanisms of l-lactate action on iegs expression demonstrate the involvement of nmda receptors. finally, we obtained evidence that such regulatory mechanisms of ieg expression also operate in vivo as direct intracortical injections of l-lactate (10 mm) into the somatosensory-motor cortex areas resulted, within 1 hour of application, in a significant stimulation of arc, zif268 and c-fos expression (by 61 6 13%, 46 6 8% and 60 6 12%, respectively) as compared to d-lactate (10 mm)-injected contralaterally areas. taken together these findings demonstrate that l-lactate acts as a direct signaling molecule which regulates neuronal plasticity-related iegs expression both in vitro and in vivo. interestingly, the underlying mechanism of action of l-lactate involves nmda receptors.this set of observations therefore reveals a novel signaling role of lactate which may represent a likely mechanism to account for the role of astrocyticderived l-lactate on ltm formation. neuronal activity in the brain is associated with a transient increase in the extracellular k 1 concentration. the excess k 1 is removed from the extracellular space, primarily by the surrounding glial cells, leading to an intracellular accumulation of k 1 via mechanisms not fully identified and/or quantified. post-stimulus recovery of [k 1 ] o has been proposed to be dependent on kir4.1-mediated spatial buffering and/or to na 1 /k 1 -atpase activity. to resolve the molecular mechanisms involved in k 1 clearance, we initially determined the contribution from the different k 1 -transporting mechanisms present in primary culture of rat astrocytes and their k 0.5 for k 1 . the na 1 /k 1 /2clcotransporter, nkcc1, increased its activity within a physiological concentration range of k 1 , while the na 1 / k 1 -atpase saturated at much lower concentrations. consequently, a concentration-dependent switch occurs between the two mechanisms of k 1 uptake in cultured astrocytes. in addition, nkcc1 was capable of mediating robust k 1 -induced astrocytic cell swelling. thus, nkcc1 could potentially act as a molecular mechanism responsible for clearance of the stimulus-evoked rise in [k 1 ] o and the associated shrinkage of the extracellular space. to determine the contribution of each of the three molecular mechanisms to k 1 -clearance in native brain tissue, we used rat hippocampal brain slices and employed ion-sensitive microelectrodes in association with high-frequency electrical stimulation as well as focal appliances of k 1 by ionophoresis. inhibition of kir4.1 (100 um bacl 2 ) or nkcc1 (10 um bumetanide) failed to show a significant effect on the rate of k 1 removal from the extracellular space. in contrast, inhibition of the a2 and a3 isoforms of the na 1 /k 1 -atpase significantly delayed post-stimulus recovery of [k 1 ] o and this delay was further potentiated by additional inhibition of the a1 isoform. the na 1 /k 1 atpase emerged as the primary factor responsible for stimulus-evoked k 1 -clearance with no evidence in favor of kir4.1 and nkcc1 involvement. further characterization of the different na 1 /k 1 -atpase isoforms, by heterologous expression in xenopus laevis oocytes, revealed that the a1 isoform reached its maximal activity already at resting k 1 concentrations, hinting at a "housekeeping" function. the a2 subunit displayed voltage-sensitivity and increased turnover rate along physiologically relevant increments in extracellular k 1 concentration. these a2-related features may render this astrocyte-specific subunit variant specifically geared for post-stimulus recovery of [ as a model of ischemic injury, and sham-operated mice were used as controls. we isolated gfp 1 cells from the dorsal part of the lv of sham-operated-and post-ischemic brains and employed a neurosphereforming assay in order to estimate their ability to proliferate and selfrenew. furthermore, we followed their immunocytochemical and electrophysiological properties during in vitro differentiation and compared the differentiation potential of gfp 1 cells isolated from the controls to those isolated from post-ischemic brains. the gfp 1 cells isolated from the dorsal part of the lv of controls formed neurospheres and differentiated only into a glial phenotype. the gfp 1 cells isolated from the dorsal part of the lv of post-ischemic brains were able to form neurospheres as well; however, besides their differentiation into a glial phenotype, they also gave rise to cells with the properties of neuronal precursors. we also performed in situ immunohistochemical/electrophysiological analyses of gfp 1 cells in the adult brain of controls and those after mcao. in situ analyses revealed that gfp 1 cells expressed the phenotype of adult nscs or neuroblasts in controls or following ischemia and that their number was increased in both hemispheres following mcao. compared to controls, we found that the number of gfp/doublecortin-positive cells significantly increased in the dorsal part of the lv as well as the number of gfp 1 cells in the olfactory bulb, where they probably differentiated into calretinin 1 interneurons. our results indicate that gfp 1 cells with an active mdach1 gene in the dorsal part of the lv play an important role in the increased production of neuroblasts after injury and that this process is also enhanced in the contralateral hemisphere. collectively, our results reveal the involvement of the mdach1 gene in adult neurogenesis. cells expressing this gene exhibit the properties of adult nscs or neuroblasts and respond to mcao by enhanced neurogenesis. experimental cerebral ischemia leads to activation of microglia and astrocytes, which subsequently release pro-and anti-inflammatory factors. furthermore, during the earliest periods of neuronal damage, large quantities of dopamine are released, which may exacerbate the vulnerability of the lesioned tissue. on the other hand, delayed treatment with levodopa has been proven beneficial in stroke patients, suggesting a more complex effect of dopamine. recent studies on astrocytic involvement in neuroinflammation have shown a remarkable modulation of the inflammatory response over the dopamine d2 receptor (d2r). after stroke, reactive astrocytes in the astroglial scar were found to be immunopositive for d2r. in this study, we report the expression of d2r in activated microglia, both after experimental stroke in vivo and after oxygen-glucose deprivation (ogd), an in vitro model of ischemia. using immunohistological stains, we analyzed the expression of d2r in iba1 positive cells 1, 3, 5, 7, and 14 days after middle cerebral artery occlusion (mcao; n 5 2-4 mice/time point). for each animal, >4 pictures were taken from the ischemic core as well as from the contralateral, non-lesioned cortex from 3 sections. a total of >300 (control) and >500 (ischemic core) iba11 cells were analyzed for each time point. in the ischemic core, $25% of cells immunopositive for iba1 expressed d2r in contrast to <1% in control areas. a strong depletion of d2r immunoreactivity was observed in the ischemic striatum, accompanied by an increase in d2r expression in the adjoining cortex and an elevated presence and activation of microglia. for in vitro experiments, we used fluorescence activated cell sorting to isolate cd45 low /cd111 microglial cells. no significant d2r expression at mrna level could be detected by rt-pcr in this population, while we were able to detect d2r in non-microglial cells. interestingly, cultured primary mouse microglia expressed low quantities of d2r, as revealed by western blotting and rt-pcr, suggesting that culturing is sufficient to induce d2r in microglial cells. upon in vitro activation of primary microglia through 60 minutes of ogd, we found a 1.5 fold increase in d2r expression after 24 hours (n 5 4). furthermore, treatment of primary microglia with the selective d2r agonist pramipexole showed a dose-dependent tendency to increase lipopolysaccharideinduced tnfa release (n 5 5). our studies indicate that activated microglia express a functional d2r, which may contribute to the pro-inflammatory reaction of microglia after ischemia. white matter (wm) is injured in most strokes and axonal injury and dysfunction contribute to disability associated with clinical deficits. in young wm, the damage from ischemic injury involves the sequence of energy depletion (ionic pathway), excessive glutamate release (excitotoxicity), generation of reactive oxygen species and oxidative stress (oxidative pathway). in older wm the injury is mediated by ca21-independent excitotoxicity due to an earlier and more robust glutamate release. because excitotoxicity leads to oxidative stress in wm we investigated whether blocking nitric oxide synthase (nos) activity before or after a period of oxygen glucose deprivation (ogd) promoted axon function in an age-dependent manner. acutely isolated optic nerves from young and old (1 and 12 month) swiss webster (sw) or c57bl/6 (bl6) mice were used to ascertain quantitative measurements of wm function and structure. to support a biological basis for nos inhibitor nitro-l-arginine methyl ester (l-name) action in themonpreparation, we evaluated the expression and localization of brain nos (bnos) using immunohistochemistry in conjunction with confocal imaging. the expression of bnos co-localized with gfap (1) astrocyte nuclei, cytoplasm, end-feet as well as nf-200 (1) axons. the pattern of bnos expression paralleled astrocyte morphology with age and became more punctate in appearance. evoked compound action potentials (caps) recovered to 21.8 6 2.8% (n 5 18) after 60 min of oxygen glucose deprivation (ogd) in young bl6 mons. pretreatment of mons with l-name (200 mm) promotedcaprecovery to 69.4 6 11.3% (n 5 8) compared to ogd while caps recovered to 39.9 6 4.4% (n 5 11) when l-name was applied after the end of ogd. pretreatment of mons from 1 or 12 month old sw with l-name improvedcaprecovery to 49.6 6 3.7% (n 5 8, vs ogd 21.3 6 3.7%, n 5 12) or to 51.1 6 9.3% (n 5 7, vs ogd 5.7 61.7%, n 5 8) respectively. l-name application after the end of ogd failed to promote aging axon function (8.8 6 3.5%, n 5 6). changes in nos activity help unveil agedependent oxidative injury mechanisms in ischemic white matter. question: cerebral endothelial cells have been reported to exert a protective effect against brain damage. does transplantation of healthy endothelial cells have a beneficial effect on the outcome of ischemic brain damage? methods: we injected endothelin-1 (et-1) into the rat internal capsule to induce lacunar infarction. seven days after et-1 injection, microvascular endothelial cells (mvecs) prepared from adult rat cerebral cortices were transplanted into the internal capsule. meningeal cells prepared from adult rat cerebra or 0.2% bovine serum albumin-hank's balanced salt solution were injected as controls. two weeks later, the footprint test and histochemical analysis were performed. results: we found that mvec transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (p <0.01) and induced remyelination (p<0.01) compared with the control groups. also the inflammatory response was repressed by mvec transplantation, judging from fewer ed-1-positive activated microglial cells in the mvec-transplanted group than in the other groups. conclusions: elucidation of the mechanisms by which mvecs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia. objective: though lactate is a known neuroprotective agent, its mechanism of action is not known. we hypothesized that lactate can provide neuroprotection by activating trek channels. the effect of lactate on the electrophysiology of trek channels was studied both in an in vitro brain slice model, after blocking the activity of other ion channels and in a heterologous expression system. results: whole cell patch clamp experiments, on ca1 stratum radiatum astrocytes, in acute hippocampal slices, significantly increased trek channel activity by 23.3 6 6.4% and hyperpolarized their resting membrane potential by 5 6 0.6 mv, on bath application of 30 mm lactate. comparison of rectification index at negative potentials between control and 30 mm lactate treatment showed no significant difference, suggesting that the conductance activated by lactate is outward rectifying. lactate-evoked increase in trek channel activity was reversibly inhibited by 200 mm quinine, a potent inhibitor of trek channels. further, lactate was unable to increase trek channel activity after disruption of intracellular lactate uptake with 2 mm a-cyano-4-hydroxy cinnamate, a blocker of monocarboxylate transporter. this was confirmed by inside-out patch clamp experiments on human trek1 channel expressed in hek293 cells. conclusion: the experimental observations suggest uptake of lactate by astrocytes to activate trek channels intracellularly. regenerative responses occurring after different types of postnatal brain injury often involve expansion of an endogenous neural progenitor cell pool, but the molecular mechanisms underlying this process are still poorly understood. we studied expansion of the glial progenitor cell pool in a mouse model of neonatal hypoxia (hx) that reproduces the hallmarks of perinatal brain injury in infants, including diffused white matter injury (dwmi). we have previously shown that hx causes proliferation of wm oligodendrocyte progenitor cells (opcs), and that this process is regulated by the cyclin-dependent kinase 2 (cdk2) pathway. our analysis in wm in vivo and in cultured cells demonstrated: i) higher expression of cdk2, cycline, prb806/811 and e2f1 in wm opcs after hx; ii) activation of the cdk2 pathway after hx, and iii) reduced hx-induced opc proliferation in cdk2-null mutant mice. here, we studied the upstream molecular mechanisms that regulate activity of the cdk2 signaling pathway resulting in opc proliferation after hx. we show that sirtuin 1 (sirt1) histone deacetylase activity is a major regulator of cdk2 signaling and of the regenerative response of opcs after hx. under hypoxic conditions, sirt1 is upregulated in wm, and interacts with both rb and cdk2. patterns of posttranslational modifications of cdk2 or sirt1 proteins after silencing either of these genes in cultured cells from normoxic and hypoxic wm indicate that cdk2 is a substrate for deacetylation by sirt1, and that sirt1 is phosphorylated by cdk2. also, sirt1 causes rb deacetylation resulting in dissociation of e2f1 from the rb/e2f1 complex, which ultimately leads to higher opc proliferation. knockdown of sirt1 in hypoxic wm cell cultures suppresses opc proliferation. finally, inhibition of sirt1 activity by sirtinol accelerates opc differentiation to mature oligodendrocytes, and reduces the number of opcs. we are currently analyzing the effects of hx on opc proliferation and differentiation in the wm of sirt1-null mice. our results provide evidence that sirt1 is an essential regulator of the cdk2-dependent regenerative response observed in wm opcs after hx, and that inhibition of sirt1 activity facilitates oligodendrocyte regeneration from opcs, which might ultimately promote wm recovery after hx. supported by nih r01ns045702, p01ns062686 and iddrc p30hd40677. gonadal steroid hormones reveal a potential neuroprotective role in acute brain ischemia. in rat in vivo studies using the transient occlusion of the middle cerebral artery as ischemic stroke model, we have shown that 17ß-estradiol and progesterone reduce the infarct area by more than 70% given 1h after the onset of stroke, prevent neuronal death and behavioral deficits. an intriguing aspect of this study was that the application of steroid hormones reduced the number of microglia and microglia-related markers in the penumbra during the first 24 h after the onset of stroke. besides microglia, penumbral astroglia coevally appeared as cellular target for steroid hormones by reducing the expression of proinflammatory-and molecules necessary for the attraction and activation of microglia and lymphocytes. this suggests that protective steroid hormones influence glia cell cross-talk and activity during early damaging conditions in the lesioned brain site. by using an in vitro hypoxic approach, we have now demonstrated that microglia express steroid hormone receptors and directly respond to ischemic conditions and steroid hormone treatment by changing expression and secretion of inflammatory compounds, trans-signaling factors and by modulating phagocytotic activity. this clearly shows that microglia is an important direct target for steroid hormones but also indirectly influenced via cross-talk by adjacent astrocytes at the damaged brain site. this generally points at an extensive bidirectional crosstalk between both glial cell types to convey steroid-mediated neuroprotection in ischemic brain tissue. in summary, the balance of inflammatory astrogliamicroglia interactions within the injured brain tissue during an early phase of ischemic destruction is a pivotal step for tissue repair. cannabinoids are considered as key regulators in the pathology of various diseases, including ischemic stroke. we previously demonstrated that post-ischemic treatment of two naturally occurring phenylpropanoids, trans-and cis-hinokiresinols (hrs) significantly reduced ischemic injury in rats subjected to middle cerebral artery occlusion (mcao) . in studies to screen their cellular targets, we found that hrs selectively bind to both type 1 and type 2 cannabinoid receptors (cb1r and cb2r). in the cb1r reporter gene assay, both hrs demonstrated antagonistic activity. in cb2r-overexpressing u 2 os cell lines, both hrs increased forskolininduced camp accumulation, suggesting their inverse agonism. since camp induces expression of heme oxyagenase-1 (ho-1), a well-known antioxidant stress protein, we further investigated the effect of hinokiresinols on ho-1 expression in mixed cortical neuron/glia cultures. trans-hr increased astroglial expression of ho-1 greater than cis-hr did. a selective ho-1 inhibitor, tin protoporphyrin ix significantly reduces the neuroprotective effect of trans-hr in cortical cultures exposed to oxygenglucose deprivation. in addition, both hrs reduced the number of ed-1immunopositive cells (i.e., active microglia and macrophages) in ischemic lesions. also, both hrs significantly reduced microglial migration induced by an endocannabinoid, 2-arachidonoylglycerol. the present study suggests that hrs reduce ischemic injury via regulation of glial cbrs. , the main anaplerotic enzyme in the brain, is predominantly located in astrocytes. in the adult brain, ischemia is known to reduce pyruvate carboxylation. moreover, reperfusion after ischemia leads to production of reactive oxygen species (ros). the pentose phosphate pathway (ppp) is, by making nadph necessary for the regeneration of reduced glutathione, a major pathway in the protection against ros. however, astrocytic and neuronal metabolic function in the neonatal brain after hypoxic-ischemic brain injury (hi) remains to be explored. methods: hi was induced in 7-day-old rats by unilateral severing of the carotid artery followed by 2 hours recovery and subsequent exposure to hypoxia (8%o 2 ) for 90 min. 30 min after end of hypoxia (the early reperfusion phase), rats were injected with [1,2-13 c]glucose and decapitated 30 min later. one group was sham-operated, and not exposed to hypoxia, thus reflecting normal metabolism in the neonate. extracts of ipsilateral hemispheres from hi and sham animals were analysed with 1 h-and 13 c-nmr spectroscopy. results: the sham animals had similar glutamine content, but lower amounts of 13 c labelled glutamine compared to corresponding values from adult rats, reflecting a generally lower rate of glucose metabolism in the neonatal astrocytes. however, approximately equal amounts of 13 c labelled isotopomers of glutamine were derived from pc and pyruvate dehydrogenase (pdh), resulting in a relatively high pc/pdh-ratio compared the same ratio in adults. following hi and reperfusion, a reduction in glucose metabolism and mitochondrial metabolism was seen by increased glucose and reduced incorporation of 13 c labelling in glutamate, glutamine and aspartate via pc and pdh. however, the pc/pdh-ratio in glutamine was similar in hi and sham. total mean values of glutamate, glutamine and aspartate were decreased, but only the latter to a significant degree. labelling in lactate via the ppp was reduced following hi. conclusion: the low labelling of lactate via the ppp indicates that the flux through the ppp was in fact reduced in the early reperfusion phase after hi. moreover, mitochondrial metabolism of pyruvate from glucose was reduced in both astrocytes and neurons. impaired anaplerosis in astrocytes was confirmed by lower amounts of aspartate. however, the proportionally similar decrease in 13 c labelling in glutamate and glutamine via pc, and the maintained pc/pdh-ratio implies that astrocytes continue to provide metabolic support for the neurons during the early reperfusion phase. ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (gdnf). ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons and it is necessary for the postnatal maintenance of dopaminergic neurons. ret expression has been as well demonstrated on microglia and several evidence indicate that gdnf regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. here we demonstrated that isolectin ib4, commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of ret both on the surface of living nih3t3 fibroblasts cells stably transfected with ret than in adult rat brain sections as revealed by immunoblotting. further, confocal immunofluorescence analysis demonstrated a clear overlap staining between pret and ib4 labeling in primary microglia cultures as well as in adult rat sections obtained from control or postischemic brain after permanent middle artery occlusion (pmcao). interestingly, ib4 staining identified activated or amoeboid retexpressing microglia under ischemic conditions. collectively, our data indicate ret receptor as one of the ib4-reactive glycoconjugate accounting for the ib4 stain in microglia under physiological and ischemic conditions. astrocytes can adapt to lower oxygen tensions by enhancing their glycolytic rate of energy production. in this case, adequate expression of monocarboxylate transporters (mcts) may be essential to sustain prominent lactate efflux and prevent glycolysis inhibition. here we show that oxygen level is an important factor controlling mct4 expression in primary cultures of mouse cortical astrocytes. indeed, while mct4 is clearly expressed by astrocytes in vivo, its basal in vitro expression in primary cultures of mouse cortical astrocytes is undetectable under classical culture conditions (95% air, 5%co 2 ). exposing astrocytes to more physiological brain oxygen tensions (hypoxic chamber flushed with 1%o 2 /5%co 2 /94%n 2 , leading to a concentration of dissolved oxygen in the culture medium of 40-60mmhg) restored mct4 expression. mct4 mrna expression became detectable after 12 hours under lower oxygen tension and was still present after 10 days with a maximum at 36 hours of incubation. this effect was paralleled by the appearance of the mct4 protein which reached a plateau between 48 hours and 96 hours, with a further enhancement at 7 and 10 days of incubation. this induction was specific for mct4 since mct1 expression was not altered. lactate release was significantly enhanced after 48 hours of incubation and further increased up to 10 days compared to astrocytes maintained under atmospheric conditions. treatment with dimethyloxalylglycine (dmog), a compound that mimics hypoxia by stabilizing the hypoxia-inducible factor 1 alpha (hif-1a) , produced a similar effect. both mct4 mrna and protein induction reached their maxima when astrocytes were treated with a concentration of dmog ranging between 1 to 2.5mm, which is also the range for greatest hif-1a expression. again, no effect was detectable on mct1 expression. transfecting astrocyte cultures with a sirna against hif-1a significantly reduced the effect of lower oxygen tension on mct4 expression. our results suggest that 1) under physiological conditions, changes in local oxygenation might regulate the capacity of astrocytes to supply lactate to neighboring cells via notably alteration in mct4 expression 2) under pathological conditions, mct4 may be upregulated as a consequence of hypoxia and contribute to neuroprotection by favouring the release and accumulation of lactate in the extracellular space. indeed, this process might give rise to the beneficial effect of lactate for neurons observed during the recovery from hypoxic/ischemic conditions in vitro and in vivo. early onset dementia and. the loss-of-function of trem2 or its coreceptor dap12 is responsible for the recessively inherited nasu-hakola disease (also known as plosl). the brains of plosl affected patients show strong microglial activation in the cerebral white matter. reports demonstrate that trem2-mediated phagocytic function of microglia is required for debris clearance and cns tissue homeostasis. perinatal brain injury is the underlying etiology for a host of developmental disabilities that includes spastic motor deficits and cognitive, behavioral and learning difficulties. hence, our aim is to characterize the expression of trem2 in the control of neuroinflammation following hypoxia/ ischemia (hi) in the newborn brain. we performed hi brain damage in postnatal day 7 (p7) c57/bl6 mice by permanent left carotid occlusion and litters were exposed to 8% of oxygen balanced with nitrogen for 50 minutes in a hypoxic chamber with controlled humidity and temperature maintained at 37 c. pups were then returned to their dam until sacrifice. the age-matched controls and samples from 3 hours to 7 days after hypoxia were collected and processed for immunohistochemistry. in control animals, trem2 expression was observed in the corpus callosum and in the subventricular zone at p7 that almost disappeared at p10. following hi, an increase in trem2 staining was observed in the corpus callosum, hippocampus, caudate-putamen, fimbria, cortex and thalamus in the ipsilateral (il) hemisphere, following a similar regional pattern of microglia activation. the increased expression of trem2 was observed from 24 hours to 7 days post-hypoxia. trem2 colocalized mainly with microglia markers, such as iba-1 and cd68. oligodendrocyte expression of trem2 was also analyzed. in conclusion, hi produced an increase in trem2 expression on the il damaged regions. these results suggest that the modulation of trem2 might be a possible target for damage control in neonatal brain. increasing evidence shows that astrocytes play a critical role in neuronal protection during an ischemic insult. in the vertebrate retina, most of astrocytic functions are executed by m€ uller glial cells, the major macroglial cell type in this tissue. the aim of this study was to evaluate the role of m€ uller glia in the onset and progression of retinal ganglion cell (rgc) degeneration after acute ischemic injury in the mouse eye in vivo. here, we used the selective gliotoxin fluorocitrate (fc) in order to transiently impair m€ uller glial metabolism at different time points during and after induction of an acute retinal ischemia/reperfusion by means of elevated intraocular pressure (iop). the effect of fc (3 mm) on metabolism was determined by measuring retinal glutamine levels, aconitase activity and mitochondrial membrane depolarization after intravitreal injection of the gliotoxin. retinal ischemia was unilaterally induced by elevating iop for 45 min. fc was intravitreally injected 4 h before onset of ischemia and after 6, 12 or 24h of reperfusion. saline was injected in the contralateral eye and served as control. surviving rgcs were quantified by means of confocal scanning microscopy and automated cell counting-based analysis on retinal wholemounts 7 days after lesion. fc treatment reduced glutamine levels to 35-38% as compared to control 4-6 h after injection. inhibition was completely reversed 24 h after injection. aconitase activity was reduced to $30% from control levels 4 h after fc injection. mitochondrial membrane potential was reduced by 50-70% as indicated by reduced intensity of the specific stain mitotracker red cmxros. one week after ischemia, only 40% of rgcs survived as compared to unlesioned controls. impairment of m€ uller glial metabolism during ischemia further reduced rgcs by 15% as compared to ischemia alone. fc treatment did not significantly modify rgcs survival 6 and 12 h after lesion. results support the notion for a critical neuroprotective role of m€ uller glial cells during ischemia. in the acute phase following ischemia, however, m€ uller glia inhibition does not further affect rgcs survival. further research is required to establish the time point for this switch in m€ uller glial function and the underlying mechanisms. extract has been widely investigated in animal models and clinical studies for various diseases including ischemic stroke. its major metabolite, cordycepin, has been reported to act as a selective a3 adenosine receptor agonist and to protect neurons against ischemic injury. however, their underlying mechanisms remain unclear. in the present study, we report that the standardized c. militaris extract, wib-801c, which contains cordycepin 8% of total dry weight of the extract, markedly reduced ischemic injury by inhibiting post-ischemic inflammatory responses. postischemic treatment with wib-801c (orally administered twice at 3 and 8 h after onset of mcao) significantly reduced infarct size and edema in rats subjected to transient middle cerebral artery occlusion (mcao, 1.5 h) and subsequent reperfusion (22 h). wib-801c also significantly improved integrity of glial cells in ischemic lesions, as evidenced by reduced white matter degeneration and loss of blood-brain barrier. importantly, wib-801c significantly ameliorated neurological deficits in not only transient but also in permanent stroke models. moreover, wib-801csignificantly improved long-term survival of mcao rats over 30 days. as we previously reported with selective a3 receptor agonists introduction: cd200 & cd200r are immune inhibitory molecules that have been shown to be involved in inducing immune tolerance and in contributing to immune privileged status of the cns. the developing brain exhibits distinct morphological as well as physiological characteristics determining a peculiar response to injury showing an aggravated susceptibility to excitotoxicity and pro-inflammatory cytokines, along with an exacerbated inflammatory response. previous studies from our group have described the expression of cd200-cd200r in brain during development showing a distinct pattern of expression in the cortex and the hippocampus. hence, the aim of this study is phenotypic characterization of cd200r1 microglia/macrophages and cd2001 neuronal cells following hypoxia/ischemia (h/i) in neonatal mice brain. wild-type c57/bl6 mice postnatal (p) day 1,3,5,7,10,14,21 and adult were used for developmental studies. p7 mice was used for h/i injury that was administered using vannucci model modified for neonatal mice (8% o 2 , 55 min) and samples were collected 3h, 12h, 24h, 48h, 72h & 7 days after hypoxia for immunofluorescence staining. results: phenotypic characterization of cd200r1 microglia/macrophages showed them to express markers of pro-or anti-inflammatory phenotype in a temporal fashion post lesion. they expressed m2 phenotype as observed by colocalization with cd2061 cells throughout the time points studied but a subpopulation of cd200r1 cells exhibited m1 phenotype as exhibited by mhcii1, cd861 cells from 24-48 hrs post lesion. calretinin (cr) used for the characterization of cd2001 neurons showed that most cr1 neurons were cd2001 especially the interneurons in the innermolecular layer of hippocampus, layer i of the neocortex and the hilus at most age groups studied. after h/i, cd200 immunolabeling was increased in the hippocampal fissure until 7 days post-lesion. this followed a change in cd200r1 microglial cells described above. to conclude, a balance between m1/m2 phenotype of cd200r1 microglia/macrophages and expression of cd200 by cr1 cells are involved in evolution of h/i induced brain injury in neonatal mice. objective: the cytokine interleukin-1 (il-1) and its naturally occurring receptor antagonist (il-1ra) play a key role in determining neuronal cell death and survival in focal cerebral ischemia. the objective of this study was to determine the cellular production of il-1/il-1ra, and to test the neuroprotective potential of post-surgically injected il-1ra overexpressing bone marrow (bm) cells in a mouse model of focal cerebral ischemia. materials and methods: c57bl/6 mice were injected i.v. with bm cells isolated from sil-1ra overexpressing mice, 30 min after permanent middle cerebral artery occlusion (pmcao). physiological parameters and behaviour were recorded post-surgically, infarct sizes were estimated, and microglial-leukocyte expression of il-1/il-1ra was analyzed by flowcytometry, in situ hybridization and immunohistochemistry. results: we identify microglia, and not recruited leukocytes as the major producers of il-1ra after pmcao in mice, and we show by using il-1ra knock out mice that microglial-produced il-1ra is neuroprotective. we report that the sil-1ra produced by the post-surgically injected bm cells, potentiates the neuroprotective effect of microglialderived il-1ra, at both 24 h and 5 days, which is consistent with behavioural improvement at 5 days, and detection of recruited bm cells in the ischemic area 1.5 h after pmcao. interestingly, we also find that the sil-1ra overexpressing bm cells stimulate microglial production of il-1ra 6 h after pmcao, at which time both il-1a and il-1b is upregulated. conclusion: our results provide proof of principle that increasing the production of il-1ra by recruited bm cells can counteract the effect of il-1a/b, increase neuronal survival and improve motor function alone or through induction of microglial-produced il-1ra after pmcao in mice. bogomoletz institute of physiology, kyiv, ukraine short-term oxygen-glucose deprivation (ogd) is known to activate excitatory glutamatergic synapses, induce long-term potentiation and result in neuronal and synaptical ultrastructural remodelling in organitypic cultured hippocampal slices (ochs). in this work we studied changes in glial synaptic coverage following 30 min ogd episode using 3d reconstruction of ochs confocal and electron microscopic images. propidium iodide (pi) and mitotracker orange cmtmros (mto) were used to estimate cell viability and their mitochondrial potential. astroglial and microglial cells were identified using immunohistochemical staining with anti-gfap and anti-iba-1 antibodies. the study demonstrated that astroglial and microglial cells retain viability (there was no significant increase in the amount of pi-labbelled cells). 3d reconstruction revealed significant increase in excitatory synapses glial coverage as early as 1 hour following ogd accompanied by a significant increase in number of astroglial and microglial processes. the deprivation also resulted in gradual increase of mitochondrial potential from 4 hr to 24 hr in both astroglial and microglial cells. it was previously shown that pyramidal neurons under the same conditions lose mitochondrial potential and became pi-positive after 4 and 24 reoxygenetion, indicating damage of cytoplasmic membrane, the effect prevented by application of nmda receptor antagonist d-ap5. thus, unlike neurons astroglial and microglial cells are ogd-resistant and demonstrate marked mitochondrial activation -a process that might be involved in maintaining neuronal function during oxygen-glucose deficiency. using a microfluidic high throughput qpcr platform, we measured the expression of 47 selected genes encoding astrocytic and polydendrocytic markers, ion channels, transporters and receptors that participate in maintaining k 1 and glutamate homeostasis in each of 292 individual gfap/egfp-positive glial cells. in this study we show how the expression of these selected genes changes during development (postnatal days 10, 20, 30 and 50) as well as 3, 7 and 14 days after middle cerebral artery occlusion (mcao). self-organizing maps and principal component analyses divided the cells according to their similarity in gene expression into three subpopulations of astrocytes present within the first 10-50 days of postnatal development (p10-p50). the first subpopulation comprises immature glia mainly from p10, characterized by the high transcriptional activity of all of the studied genes, including polydendrocytic markers. the second subpopulation is dominated by cells from p20 and displayed low transcript levels of all of the studied genes. the third subpopulation represents mature astrocytes mainly from p30 and p50. three, seven and fourteen days after ischemia (d3, d7, d14), additional astrocytic subpopulations appear: resting astroglia (mainly from p50 and d3), transcriptionally active early reactive astroglia (mainly from d7) expressing the mrna of polydendrocytic markers, and, presumably, permanent reactive astroglia (solely from d14). following focal cerebral ischemia, reactive astrocytes undergo pronounced changes in their expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and markers of reactive astrocytes and polydendrocytes. from the data obtained from single cell pcr analysis, we calculated the spearman correlation coefficients between pairs of genes, which revealed interesting positive gene expression correlations with polydendrocytic markers (gria2-4, grik1-5, grin3a, kcnj16, kcnk2 and kcnk10 genes). this study was further supplemented by an immunohistochemical analysis of nmda receptor subunits and pdgfar, which confirmed that changes in gene expressions correlate with immunodetected proteins. ga cr 13-02154s, astf 110-2011, gauk 604212. reactive astrogliosis is often regarded as universal type of astroglial response to various forms of injuries. among its most characteristic features one may include: glial fibrillary acidic protein (gfap) up-regulation, cellular hypertrophy and proliferation and gliotic scar formation. an important element of glial cells response is their ability of de-differentiation, which is related with their re-gaining of immunological and molecular properties characteristic for earlier developmental forms. the origin of cells proliferating in response to various types of brain injury is still a subject of debate. among others it is related to the pathomechanism of lesion, affected brain structure and developmental stage of the nervous system. aim: in our study we aimed to assess the differences of de-differentiation and proliferation potential of astroglia localized in the cerebral cortex and striatum to the transient focal brain ischemia. material and methods: transient focal brain ischemia was evoked in 20 adult male wistar rats by placement of the monofilament surgical thread into the internal carotid artery and occlusion of the middle cerebral artery for 1h. the postoperative survival period was up to 6 weeks. immunocytochemical double-staining for astrocytic marker (gfap), developmental (pax6) and proliferative (ki-67) markers was performed and subsequent qualitative and quantitative study was done by means of confocal microscopy. results: double-labeled gfap/pax6 and gfap/ki67 reactive astroglial cells were revealed in the cerebral cortex and striatum of the ischemic region, starting from 24h after initiating the ischemia. the apparent difference in the intensity of morphological changes, quantity of de-differentiating and proliferating astroglial cells was observed between ischemic cerebral cortex and striatum. intensity of astroglial response and proliferative potential were much higher in the striatum than in the cerebral cortex. summary and conclusion: apparent difference in proliferative response between the cerebral cortex and striatum may be consequence of metabolic and molecular differences between astroglial cells populating two mentioned above structures. it may be also consequence of uneven decrease of cerebral blood flow and resulting different metabolic conditions influencing astroglial function in various fragments of ischemic area. reassuming, the differentiated potential of astroglial proliferative response to ischemic changes in different brain regions must be taken into account while analyzing clinical consequences of cerebral infarcts of different localization. triggering receptor expressed on myeloid cells-2 (trem2) is a microglial surface receptor involved in phagocytosis. clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. the role of trem2 following stroke is currently unclear. as an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). quantitative pcr (qpcr) revealed a greatly increased transcription of trem2 after stroke ($10fold). we subsequently analyzed the expression of proinflammatory cytokines, chemokines and their receptors in trem2knockout (trem2-ko) mice via qpcr. microglial activation (cd68, iba1) and cd3-positive t-cell invasion were analyzed via qpcr and immunohistochemistry. functional consequences of trem2 knockout were assessed by infarct volumetry. the acute inflammatory response (12 h reperfusion) was very similar between trem2-ko mice and their littermate controls. however, in the sub-acute phase (7 d reperfusion) following stroke, trem2-ko mice showed a decreased transcription of pro-inflammatory cytokines tnfa, il-1a and il-1b, associated with a reduced microglial activity (cd68, iba1). furthermore, trem2-ko mice showed a reduced transcription of chemokines ccl2 (mcp1), ccl3 (mip1a) and the chemokine receptor cx3cr1, followed by a diminished invasion of cd3-positive t-cells. no effect on the lesion size was observed. conclusions although we initially expected an exaggerated pro-inflammatory response following ablation of trem2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in trem2-ko mice. we therefore conclude that trem2 appears to sustain a distinct inflammatory response after stroke. metabotropic glutamate receptors (mglurs) are seven transmembrane domain g-protein-coupled receptors for l-glutamate, the main excitatory neurotransmitter in the mammalian brain. in addition, glutamatemediated excitotoxicity plays a central role in mediating neuron and oligodendrocyte death during ischemic episodes. in vitro, developing oligodendrocytes (ols) in particular have been shown to be highly vulnerable to excitotoxicity and oxidative stress. notably, activation of group 1 mglurs has been shown to attenuate ol excitotoxicity in vitro and in brain slices has been shown to reduce ischemia-mediated impairment of astrocytes. here, we have examined the functional expression of mglurs and their role in acute ischemic injury in developing cns white matter of the mouse optic nerve. calcium imaging experiments were performed in optic nerves from p9-13 wild-type mice. optic nerves were isolated intact, loaded with fluo-4 am and visualized using a zeiss lsm 5 pascal confocal microscope; images were collected in optical z-sections and changes in fluorescence intensity were measured. the application of mglur agonists acpd and dhpg showed that group i mglur mediate a rise in glial [ca 21 ] i . the action of the group i mglur antagonist aida versus acpd indicated that the rise in [ca 21 ] i also involves group ii (and possibly group iii) mglur. immunohistochemistry confirmed glial expression of group i subunit mglur5 and group ii subunits mglur2 and mglur3 in the optic nerve, although further analysis of cellular localization is required. to examine the role of mglurs in white matter ischemia, optic nerves from p8-9 mice in which fluorescent reporters are driven by astrocyte (gfap-egfp) or oligodendrocyte (sox10-egfp) genes were isolated intact into artificial cerebrospinal fluid (acsf) and subjected to oxygen glucose deprivation (ogd) for 1 hour. activation of group i or group ii mglur with the agonists acpd, dhpg or ly379268 protected astrocytes and oligodendrocytes from ischemic cell death. further experiments are required to determine the mechanisms involved, but these findings demonstrate functional expression of mglur in optic nerve glia and indicate that activation of mglur is a possible therapeutic strategy to protect against glial damage in response to ischemia. the cerebral cortex is one of the most often affected areas after stroke, but there are no studies comparing the outcome in different cortical regions after focal ischemia. the aim of this investigation was to evaluate the patterns of glial activation, tissue degeneration and neuronal loss in different survival times following cortical ischemia. focal ischemia was induced by stereotaxic microinjections of endothelin-1 (et-1) into the somatosensory, motor and association cortices of adult rats (n 5 45). the control animals were injected with the same volume of sterile saline (n 5 27). the animals were perfused at 1, 3 and 7 days after ischemia. gross histopathology was evaluated using cresyl violet staining. 20 lm sections were submitted to immunohistochemistry for astrocytes (anti-gfap), activated microglia/macrophages (anti-ed1) and microglia (anti-iba1 and ed1). tissue loss and glial activation were more intense in the somatosensory cortex than in other cortical areas. the motor cortex was the second more affected area. the association cortex was the less damaged area, which was confirmed by quantitative analysis (anova-tukey). the results suggest that an ischemic lesion of the same intensity induces a differential pattern of tissue loss and neuroinflammation, depending on the cortical area, and that the primary sensory and motor areas are more susceptible to ischemia than association areas. tlr4 is required for ipc-induced neuroprotection. in order to elucidate the mechanisms by which microglial tlr4 contributes to ipc, we carried out cell-targeted genomic analyses specifically on microglia exposed to either ischemic conditions in vitro or ipc in vivo. for our in vitro model, we exposed wt and tlr4 -/mouse primary microglia to hypoxic/hypoglycemic conditions for 24 hours. for our in vivo model, we carried out transient middle cerebral artery occlusion (mcao) or sham surgery as our ipc pulse on wt and tlr4 -/adult male mice. three days later mice were sacrificed and microglia acutely isolated from ipsilateral cortex using magnetic affinity chromatography cell separation and ex vivo flow cytometry. microarray analysis was carried out on rna from both in vitro and in vivo microglia. results from both datasets identified robust expression of type 1 and/or type 3 interferon (ifn)-stimulated genes (isgs) as the predominant transcriptomal feature of ischemia-exposed microglia. in vitro, we found that 25 out of the 60 (40%) individual genes that were significantly up-regulated >2fold by hypoxia/hypoglycemia in wt, but not tlr4 -/-, microglia were isgs including mx1, oas2, irf7 and ccl5. promoter analysis demonstrated that the top four most active transcription factors were isgf3, irf2, irf1 and irf7; all ifn regulatory factors (irfs). a similar pattern emerged from the in vivo dataset with multiple irfs again among the most activated transcription factors in sorted microglia from ipcexposed mice. however, unlike the isg response in vitro, the isg response from the in vivo dataset was not tlr4-dependent. the ifn family of cytokines is recognized as a key component of the innate immune response to infection and other types of injury. the role of microglial ifn-signaling in ipc is unknown. these results suggest that broad activation of isgs may be a key feature of the microglial response to ischemic conditions. astrocytes respond to central nervous system (cns) injury by the formation of a glial scar that develops within few days after insult and is characterized by astrocyte proliferation and cellular hypertrophy. reactive astrocytes change their immunohistochemical profile, such as increasing expression of gfap, nestin and vimentin; however, they also markedly alter the expression of ion channels, receptors and transporters, which participate in the maintenance of ionic-, water-and neurotransmitter homeostasis, such as k 1 channels, aquaporins or glutamate transporters. here, we aimed to characterize the gene expression profile and functional properties of reactive astrocytes 5 weeks after global cerebral ischemia (gci) or focal cerebral ischemia (fci) with a specific focus on k 1 channels or non-specific cationic channels. gci was induced in adult rats by bilateral, 15-minute common carotid artery occlusion combined with low oxygen, while fci was induced in adult egfp/gfap mice by permanent middle cerebral artery occlusion. using the patch-clamp, we investigated the membrane properties of astrocytes in situ 5 weeks after gci in the rat hippocampal ca1 region and 5 weeks after fci in the mouse cortex. regardless of the type of ischemic injury, astrocytes from both cns regions depolarized their membrane potential by $14 mv 5 weeks after ischemia. when compared to astrocytes from the non-injured ca1 region or the cortex, the post-ischemic astrocytes displayed large hyperpolarizationactivated time-and voltage-dependent, non-inactivating inward currents, the current density of which increased 3-fold in response to gci or fci. in addition, these non-specific cationic channels were sensitive to zd7288 and to a low extracellular na 1 concentration, suggesting that they may belong to the family of hyperpolarization-activated cyclic nucleotide-gated (hcn) channels. we have taken advantage of fluorescently labeled astrocytes in egfp/gfap mice and isolated egfp 1 cells by facs from non-injured as well as ischemic regions and performed gene expression profiling using single-cell rt-qpcr. our data revealed that cortical astrocytes after fci express, besides the increased expression of gfap, gfapd, aqp1 and 9, extremely high levels of hcn1-3 transcripts that encode hcn 1-3 channels. moreover, our immunohistochemical analyses confirmed the presence of hcn1-3 in reactive astrocytes, especially hcn1, 2 and 3 channels. in summary, our results show that reactive astrocytes from post-ischemic tissue express hcn channels and that their activity might significantly contribute to na 1 influx, possibly maintaining atpase function and/or contributing to intracellular na 1 -dependent events, such as glutamate transporter or na 1 /ca 21 exchanger reversal. ga cr 13-02154s, p304/12/g069 and gauk 383711. the optimal operation of electrogenic astrocytic transporters and exchangers for some well-defined astrocyte brain homeostatic functions depends on the presence of k 1 channels in the cell membranes and the hyperpolarized membrane potential. our previous study showed that astrocytes functionally express two-pore domain k 1 channel trek-1, which helps to set the negative resting membrane potential. however, the roles of trek-1 on astrocytic function under normal and ischemic conditions remain unclear. in this study, we investigated the effects of trek-1 activity on astrocytic function and neuronal death under ischemic conditions. in an in vitro hypoxia model with astrocytes culture, trek-1 immunoreactivity was up-regulated after hypoxia. suppression of trek-1 activity inhibited the glutamate clearance capability, enhanced the inflammatory secretion of astrocytes derived s100b and led to increased neuronal apoptosis after ischemic insult. after middle cerebral artery occlusion induced focal ischemia in rats, the trek-1 expression was significantly increased which correlated with reactive astrogliosis. application of lineonic acid, a trek-1 agonist which could potentiate the astrocytic conductance and hyperpolarization membrane potential, up-regulated the expression of astrocytic glutamate transporter glt-1, inhibited the micro-glial inflammatory response and attenuated neuronal damage. our results suggest that trek-1 activity is involved in astrocytic function and neuronal survival. this would provide evidence showing astrocytic trek-1 involvement in ischemia pathology which may serve as a potential therapeutic target in stroke. oligodendrocytes are the myelinating cells of the central nervous system (cns). they are responsible for ensheathing myelin layers around axons, which contributes to improve conduction of neuronal impulses and additionally provides axonal support. oligodendrocytes are generated by oligodendrocyte precursor cells (opcs), a self-renewing and proliferating adult stem/precursor cell population in the cns. this process occurs not only during developmental myelination but also mediates cns remyelination, a regenerative process that restores myelin sheaths to demyelinated axons. although energy metabolism in the oligodendrocyte lineage is thought to be very active to support the lipid production specific to this cell type, it is still poorly characterized. as a first step towards a detailed characterization of the energy metabolism associated with specific lineage stages we investigated how glucose -the main cerebral energy substrate -is metabolized at an immature opc stage and in differentiated oligodendrocytes. primary cultures of opcs and oligodendrocytes were incubated with medium containing [1,6-13 c]glucose for 4h and metabolites in both cell extracts and culture medium were collected and analysed for excess % 13 c enrichment using gas-chromatography-mass spectrometry (gc-ms) as well as for total amounts using hplc. the significant 13 c-enrichment in citrate, malate, and aspartate in cell extracts indicate that oligodendrocytes oxidize glucose-derived metabolites extensively in the tricarboxylic acid (tca) cycle. moreover, they release 13 c-labelled aspartate and glutamate to the extracellular medium, although a significant consumption of aspartate, glutamate and glutamine from the medium was also observed. enrichment of extracellular lactate and increased alanine synthesis in oligodendrocytes as compared to opcs suggests that the rate of aerobic glycolysis is increased in immature as compared to differentiated cells. in conclusion, these data confirm that both opcs and oligodendrocytes are highly metabolically active and, in addition to extensively oxidizing glucose, synthesize and release distinct metabolites. we anticipate that further studies on this subject will contribute to a better understanding of the physiological role of oligodendrocytes in the cns and the role of glycolysis and mitochondrial metabolism during their maturation. schwann cells in the peripheral and oligodendrocytes in the central nervous system require a tremendous amount of lipids to be capable of producing all cell membranes forming the myelin structure. furthermore, when compared to other cells, myelin membrane presents an extremely high lipid content ($80% of dry weight) coupled to an unique lipid composition. the importance of myelin lipid composition towards myelin formation and maintenance is highlighted by the frequent appearance of myelin defects in lipid-metabolism human disorders. one of the main represented class of lipids in myelin are phospholipids, of which fatty acids (fas) represents the building blocks. most cells preferentially uptake circulating free fas. however, cells under increased membrane production, e.g. highly proliferating cells, can endogenously produce them through the enzyme fatty acid synthase (fasn), which synthesize palmitate. in these cells, fasn is transcriptionally augmented via the mtorc1 pathway. thus, we hypothesized that due to their increased lipid demand myelinating cells would require fasn activity towards myelin production. the functional role of fasn in the metabolic control of myelin membrane lipid composition and myelination is unidentified. thus, we addressed it experimentally by conditionally deleting fasn in myelinating cells in vivo. we show that endogenous fas biosynthesis in myelinating cells is essential to maintain myelin membrane lipid homeostasis. furthermore, maintaining a correct lipid homeostasis is critical for myelination. thus, we analyzed whether fasn is required to allow palmitoylation of myelin proteins and their targeting to the myelin membrane. in addition, we show that interfering with myelin fas homeostasis results in the activation of systemic compensating metabolic loops promoting fas uptake, although not sufficient to preserve efficient myelination. taken together our data demonstrate that in myelinating cells fas homeostasis is under the synergistic control of endogenous metabolic fas production via fasn and systemic functional regulation of free fas uptake. most importantly, maintaining a correct homeostasis of fa-derived lipids in myelin is critical for myelination. homeostasis and signaling, respectively (1). in mammals, two cytosolic and two mainly mitochondrial located glutaredoxins have been identified (2). we already described an essential role for the formation of a functional neuronal network during embryonic development of one of these proteins, the vertebrate specific glutaredoxin 2 (3). here, we will focus on the contribution of glutaredoxin 2 on basic cellular functions of oligodendrocytes, the myelinating cells of the central nervous system, under physiological and pathophysiological situations, e.g. multiple sclerosis, which is characterized by inflammatory axonal demyelination (4). oligodendrocyte progenitors and mature oligodendrocytes are very sensitive against oxidative stress induced by release of nitric oxide by activated microglia (5) . using a2b5 positive glia restricted progenitor cells as well as organotypic cerebellar slice cultures, we found that increased levels of glutaredoxin 2 protect against cell death induced by either activated microglia or s-nitrosoglutathione, a physiological nitric oxide donor. under physiological conditions elevated levels of glutaredoxin 2 increased migration of a2b5 positive cells nearly three fold. western blot analyses, quantitative real time pcr, as well as immunocytochemistry revealed that glutaredoxin 2 inhibited further differentiation of ng2 positive oligodendrocyte progenitor cells. in summary, our data indicate the importance of specific thiol redox signaling during cellular protection and function in this type of glia cells. current multiple sclerosis therapeutics act as immunomodulators and/ or immunosuppressors. this strategy is largely ineffective at preventing progression of the disease. significant benefits to multiple sclerosis patients might be seen with a therapeutic agent which induces remyelination, however, to date no such agents have been developed. using in vitro and in vivo models we have identified nefiracetam as a potential remyelinating therapeutic. to study remyelination in vitro, hippocampal organotypic cultures were exposed to lysophospatidylcholine (lpc) for 18hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for 24hrs of this recovery period. in all cases immunofluorescent labelling of myelin basic protein (mbp) was used as a measure of myelin. nefiracetam was shown to increase mbp expression, relative to untreated controls, accelerating recovery from lpc. two different models were used to investigate the efficacy of nefiracetam in vivo; an inflammatory model, experimental autoimmune encephalomyelitis (eae), and a toxin induced model of demyelination, achieved through the use of cuprizone chow. nefiracetam treatment did not alleviate the symptoms of eae. however, nefiracetam did restore the amount of myelin in cuprizone exposed animals, compared to saline-treated controls. in combination, these observations suggest that nefiracetam is capable of accelerating remyelination through a mechanism of action which is independent of immunomodulation. we conclude that nefiracetam encourages remyelination both in vitro and in vivo. these pro-myelin properties identify nefiracetam as a potential therapeutic for demyelinating disorders. university of naples "federico", neuroscience, naples, italy changes in intracellular [ca 21 ] i levels have been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (opcs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes. in the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (ncx) family members ncx1, ncx2, and ncx3, play a role in oligodendrocyte maturation. functional studies, as well as mrna and protein expression analyses, revealed that ncx1 and ncx3, but not ncx2, were divergently modulated during opc differentiation into oligodendrocyte phenotype. in fact, while ncx1 was down-regulated, ncx3 was strongly up-regulated during the oligodendrocyte development. the importance of calcium signaling mediated by ncx3 during oligodendrocyte maturation was supported by several findings. indeed, whereas the knocking down of the ncx3 isoform in opcs prevented the up-regulation of the myelin protein markers cnpase and mbp, its overexpression induced an up-regulation of cnpase and mbp. furthermore, ncx3 knock-out mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in mbp expression and by the accompanying increase in opcs number. collectively, our findings indicate that calcium signaling mediated by ncx3 plays a crucial role in oligodendrocyte maturation and myelin formation. to explore the role of kif13b in myelination in vivo, we generated conditional knock-out mice with kif13b specific ablation in either schwann cells or oligodendrocytes. we found that kif13bfl/fl p0cre mouse nerves are hypomyelinated with reduced myelin thickness, thus confirming in vitro data. in the pns, axonal neuregulin (nrg) 1 type iii is one of the main signaling promoting myelination and remyelination after injury. nrg1 type iii binds to erbb2/erbb3 receptors in schwann cells, which signal through a plethora of downstream signaling pathways also including the pi3k/akt complex. the pi3k/akt signaling pathway is attenuated by the pten phosphatase, which dephosphorylates ptdins(3,4,5)p 3 (s. goebbels et al., 2010). dlg1 is believed to interact with pten and potentiate its ability to dephosphorylate ptdins(3,4,5)p 3 , thus negatively modulating the akt-mtor pathway (l. cotter et al, 2010). as kif13b interacts with dlg1 in schwann cells, we investigated the pi3k/akt pathway in kif13b-null nerves and surprisingly, we found that dlg1 expression levels are increased whereas akt phosphorylation is decreased in mutant nerves. our findings suggest that kif13b negatively regulates dlg1 activity and acts as a promoter of myelination in the pns. on the contrary, when kif13b is lost specifically in oligodendrocytes, myelin sheaths are thicker. at the molecular level, the hypermyelination is consistent with increased akt phosphorylation. we are currently investigating the molecular mechanisms at the basis of the kif13b/dlg1 interaction in both schwann cells and oligodendrocytes. we have identified the nootropic nefiracetam as a potential novel remyelinating therapeutic using in vivo and in vitro models of demyelination. in this study we investigate the mechanism of action of nefiracetam in the context of remyelination as regulated by wnt/b-catenin signalling and spontaneous activity. to investigate whether nefiracetam acts by modulating wnt signalling, organotypic hippocampal cultures were exposed to nefiracetam or the wnt/b-catenin inhibitor cardamonin. both increased immunofluorescent staining for the opc marker ng2. myelin formation, as assessed by mbp staining, was enhanced by nefiracetam but was not affected by cardamonin. thus, modifying wnt signalling appears to impact opc development without effecting differentiation, suggesting the pro-myelinating effects of nefiracetam involve an additional mechanism. we next investigated the effect of nefircatam on opc differentiation using purified opc cultures. nefiracetam increased the ol:opc ratio, as assessed by immunofluorescent staining for mbp and ng2 respectively, suggesting nefiracetam is capable of promoting ol maturation through a direct action on opcs. finally, we investigated the effect of nefiracetam on spontaneous activity. organotypic hippocampal cultures were exposed to lysophospatidylcholine (lpc) for 18hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for 24hrs of the recovery period. spontaneous calcium activity was then measured using fluo4-am. cultures not exposed to lpc displayed low levels of activity which were unaffected by nefiracetam. lpc exposure caused an increase in activity which was enhanced by nefiracetam. additionally, in these cultures, lpc decreased mbp staining compared to control while nefiracetam post-lpc caused a return to control levels. these data suggest increased activity may be an adaptive response related to myelin repair which is enhanced by nefiracetam. together, these findings suggest a complex mechanism of action for nefiracetam as a remyelinating agent involving modulation of wnt signalling, spontaneous activity and opc differentiation. in an assay in which cortical oligodendrocytes ensheath dorsal root ganglion cells 3 we found that nrg and nmda receptors interact to regulate myelination. without nrg, blocking nmda receptors had no effect on myelination. adding nrg increased myelination at all timepoints analysed (2-5 weeks), suggesting that nrg did not merely accelerate myelination. strikingly, nrg led to the majority of myelination becoming dependent on activation of nmda receptors and action potential activity. nrg's effect was associated with a 4-fold increase in nmda receptor current in oligodendrocyte lineage cells. thus, nrg switches myelination from a default programme, which is independent of neuronal activity, to a mechanism that is regulated by glutamate released from active axons. the effects of nrg were associated with an increase in the phosphorylation of akt and the transcription factor creb, but not associated with changes in the phosphorylation of erk. these data reveal a function for oligodendrocyte nmda receptors. the absence of nrg in multiple sclerosis lesions, and enhanced remyelination by added nrg, suggest a role for neuregulin/nmda receptor dependent remyelination after pathology. low-density lipoprotein receptor-related proteins (lrps) are endocytic receptors that internalize several ligands and are involved in lipoprotein homeostasis in adult brain. lrp-2, also known as megalin, is involved in the development of the central nervous system (cns) and in the transport of molecules across the blood brain barrier (bbb). in a previous work, our group revealed that megalin is selectively involved in sonic hedgehog-mediated effects on oligodendrocyte precursor cell migration and proliferation during the development of the cns. although there are some reports describing the presence of lrps in acute lesions of multiple sclerosis (ms) patients and pointing to an important role of shh in the pathogenesis of this disease, there are not data about the expression of megalin in ms tissue. in the present study, we analyse the distribution and the cellular characterisation of megalin in samples of human brain from ms patients to elucidate the role of this receptor in the pathogenesis of demyelination. changes in megalin distribution parallel the different ms histopathological lesions: we detected megalin in active demyelinating lesions and in the periplaque of chronic-active lesions, areas where remyelination spontaneously occurs. on the contrary, megalin was absent in the demyelinated region of chronic lesions. in addition, we observed the up-regulation of megalin in a subpopulation of perivascular astrocytes within the normal appearing grey matter (nagm) of ms patients. moreover, megalin was up-regulated in blood vessels within active lesions, in the periplaque of chronic-active lesions and in the nagm. in parallel, we also analysed the bbb profile to determine structural and functional alterations. altogether, we suggest that megalin is an important receptor expressed in diverse areas of the cns of ms patients representing different aspects of ms development: i) a potential target to improve remyelination; ii) an indicator of a functional rather than a structural disruption of the bbb. therapeutic strategies to modulate specific lrps would be useful to effectively treat ms. bdnf is known to promote central nervous system myelination both in vitro and in vivo. adopting in vitro myelination assays, we identified that bdnf acts through oligodendroglial-expressed trkb receptors to promote myelination. this was verified in vivo, as deletion of trkb in mature oligodendrocytes (in trkb fl/fl mbp-cre mice) resulted in a hypomyelinating phenotype during development. question: here we investigate the consequence of trkb deletion in oligodendrocyte progenitor cells (in trkb fl/fl cnpase-cre mice) and the signalling pathways downstream of trkb that promote myelination. methods and results: trkb fl/fl cnpase-cre mice were born in mendelian ratios and were phenotypically indistinguishable from littermate control mice. surprisingly, analysis of myelinated axonal tracts in the spinal cord and optic nerve revealed normal myelin development. in addition, the number of cells in the oligodendroglial lineage was the same as control mice during postnatal development. these data indicate that the timing of trkb deletion in the oligodendroglial lineage is critical, as deletion in opcs results in no significant phenotype, whereas deletion in mature oligodendrocytes results in a hypomyelinating phenotype. these data suggest that opcs are able to compensate for the loss of trkb and myelinate normally in the absence of bdnf signalling. our in vitro data have shown that the promyelinating influence of bdnf strongly correlates with erk1/2 activation. here we show that overexpression of erk2 in opcs exerts a more significant promyelinating effect than erk1. as erk1/2 are known to exert some of their effects through direct transcription factor phosphorylation, we have undertaken an in silico analysis of myelin-specific transcription factors and identified several that contain potential erk1/2 binding domains and phosphorylation sites. co-immunoprecipitation experiments show an interaction between erk1/2 and these transcription factors. investigation into the nature of these interactions and their functional consequences are ongoing. conclusions: this work suggests a novel role for erk1/2 signalling within oligodendrocytes that regulates cns myelination, possibly via activation of myelin-specific transcription factors. as erk1/2 is activated by multiple receptors, other receptors could potentially compensate for the loss of trkb in opcs, resulting in a normally myelinated cns in vivo. myelin is vulnerable to impairment by genetic, toxic, and immunological triggers, but the molecular basis for many aspects of myelin pathology has remained unknown. to identify molecules required for the structural integrity of central nervous system myelin we have systematically analyzed its protein composition by quantitative proteome analysis. based on the high abundance of filament-forming septins in myelin, we hypothesized that septin filaments may constitute a cytoskeletal membrane cortex in myelin. we find that septin filaments localize to the non-compact adaxonal myelin compartment, which is considered relevant for the metabolic maintenance and the turnover of myelin. for functional analysis, we generated mice lacking the most abundant septin of myelin, sept8, from myelinating glia. our results indicate that sept2, sept4, sept7 and sept9 multimerize with sept8 and that this interaction is essential for their incorporation into myelin. myelin lacking septin filaments display pathogenic outfoldings emerging from adaxonal myelin. we propose that this phenotype reflects that myelin septins provide long-term structural integrity to the adaxonal compartment of cns myelin. here, we used primary opc cultures to investigate the mechanisms underlying gpr17 endogenous regulation. this is quite important since gpr17 is upregulated at injury sites in both a demyelinating in vivo model (boda et al., glia, 2011) and in multiple sclerosis (ms) patients (chen et al., nat neurosci 2009), which, could, in turn, cause defective myelination. in line with these findings, we have recently shown that gpr17 forced over-expression at late differentiation stages, obtained by transfecting cultured opcs with a gfp-gpr17 fusion vector, indeed impairs terminal cell maturation. this suggests that the receptor needs to be down-regulated/ desensitized to allow opc terminal maturation. physiologically, gpr17 downregulation may occur through agonist-induced receptor phosphorylation via g-protein coupled receptor kinases (grks) (daniele et al., j pharm exp ther, 2011; frantangeli et al., j biol chem, 2013), which are, in turn, controlled by mtor kinases, and are altered in ms. our in vitro data show that rapamycin, an inhibitor of mtor kinase, reduces grk2 levels, with parallel increases in gpr17 expression and strong impairment of opc maturation. globally, these data suggest that dysregulation of these interconnected pathways leading to aberrant gpr17 overexpression may prematurely block opc maturation. analysis of gpr17 in vivo in ms models will confirm if the receptor is dysregulated during disease development and will help finding ways to re-establish myelin repair in demyelinating diseases. loss of central nervous system myelin, and the failure of remyelination by oligodendrocytes, contributes to the functional impairment that characterizes neurodegenerative disorders and demyelinating diseases such as multiple sclerosis (ms). since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting (re)myelination are pivotal in precluding progression of disease. although the reasons for remyelination failure are likely multifactorial, one of the major impediments to successful remyelination is the altered lesion microenvironment. our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure. here, we aim at elucidating whether exogenous gangliosides, i.e., cell surface lipids with a potential to modulate fibronectin-cell surface interactions, could overcome fibronectin-mediated inhibition of myelination. of the gangliosides tested, only addition of gd1a, perturbed adherence of oligodendrocyte progenitor cells to fibronectin, but not to poly-l-lysine, without affecting cell viability. gd1a slightly increased growth factor-mediated proliferation on fibronectin, but in the presence of the lipid migration of oligodendrocyte progenitor cells on fibronectin was reduced. furthermore, addition of gd1a increased myelin-like membrane formation on (aggregated) fibronectin, whereas gm1 reduced oligodendrocyte differentiation, as reflected by a decrease in the number of mbp positive cells. most interestingly, gd1a counteracted the myelination inhibiting effect of aggregated fibronectin in co-cultures of dorsal root ganglion neurons and oligodendrocytes. also, gd1a enhanced remyelination in demyelinated cerebellar slice cultures that contained fibronectin aggregates. taken together, gd1a is able to overcome the inhibiting effect of aggregated fibronectin in remyelination. given the persistent presence of these aggregates in ms lesions, gd1a might therefore act as a potentially novel molecular tool to selectively modulate the impeding signaling environment, apparently detrimental to remyelination. its mechanism of action and exploration of gd1a's potential in the treatment of ms, are currently investigated. in the peripheral nervous system (pns) it is secreted by schwann cells and fibroblasts and its expression is strongly induced upon injury. we are interested in the function of apod in the molecular events that take place after a pns injury. we have studied in vivo the process of wallerian degeneration following sciatic nerve crush and we have assayed ex vivo the phagocytosis of labelled myelin from wt and apod-ko mice by flow cytometry. the analysis of cellular processes and protein or mrna expression of a group of signalling molecules induced by injury shows that the lack of apod results in an exacerbated mcp-1 and tnfa-dependent macrophage recruitment. at the injury site, free aa decreases in the wt. lack of apod results in higher basal levels and a stronger injurytriggered depletion of aa. control by apod of the availability of aa to produce the lipid mediators involved in macrophage recruitment is therefore a key mechanism that conditions both wallerian degeneration and injury resolution later on. on the other hand, the phagocytosis of apod-ko cns myelin is less efficient than the phagocytosis of wt myelin. these results indicate that there are also genotype-dependent differences in myelin composition and/or in the interaction between myelin and macrophages. an electron microscopy analysis of crude myelin preparations reveals that the cns myelin from apod-ko mice has abnormal periodicity and shows defective myelin compaction. lipid analysis of apod-ko myelin shows altered phospholipid composition, particularly in phosphoinositid species. a study of the function of apod in the myelination process is currently underway, our results demonstrate that apod function is relevant for both, myelin membrane properties that influence myelin-macrophage interactions, and the control of the lipid-mediated signalling events controlling the extent of macrophage recruitment. adhesion g protein-coupled receptors (ad-gpcrs) are a subgroup of gpcrs that facilitate cell-cell and cell-matrix interaction. structurally, they differ from other gpcrs by the presence of an extremely larger extracellular n-terminal region that is separated from the 7-transmembrane region by a gpcr autoproteolysis-inducing (gain) domain that in most ad-gpcrs facilitates an auto-catalytic process to produce an nand c-terminal fragments during the maturation process. gpr56 is a member of the ad-gpcr family and its mutations are responsible for a human brain malformation known as bilateral frontoparietal polymicrogyria (bfpp). individuals with bfpp present with changes in the white matter tracts in the form of hypomyelination, in addition to cortical malformation. here, we show that gpr56 knockout mice and zebrafish exhibit a reduction of myelin-specific protein expression, fewer myelinated axons and decreased number of mature oligodendrocytes in the central nervous system (cns). during cortical development, gpr56 functions together with its ligand collagen iii to regulate neuronal migration and cortical lamination. however, it is not clear whether collagen iii is also the ligand of gpr56 during cns myelination. gpr56 has a very long and poorly characterized n-terminal fragment, allowing for the possibility of multiple binding partners that mediate cell-extracellular matrix interactions. indeed, gpr56 also binds to tissue transglutaminase (tg2) in melanoma cells. we have begun to study the possible role of collagen iii and tg2 in oligodendrocyte development and cns myelination. the likelihood of these two proteins as being the ligand(s) of gpr56 during oligodendroglial development will be discussed as will our ongoing work to define the role of gpr56 in cns myelination. proper control of cell cycle, proliferation and differentiation is fundamental to regulate oligodendrocyte (ol) development and recruitment of oligodendrocyte precursors (opc) after brain damage. failure in this process results in severe dysmyelinating disorders and defective brain repair. the molecular machinery that couples differentiation to proliferation withdrawal plays therefore a critical role in brain development and repair and is regulated by multiple extracellular cues including extracellular matrix (ecm) and growth factor derived signals. however, how these signals are integrated into ol to control cell cycle progression and differentiation is only partially understood. we are investigating the role of jun activating binding protein 1 (jab1), an intracellular signalling molecule, shuttling between nucleus and cytoplasm, involved in the control of cell proliferation, survival and gene transcription. recent evidence showed that jab1 function is regulated by ecm and growth factors. we demonstrated that jab1 is expressed in glial cells in peripheral and central nervous system (cns), and conditional inactivation of jab1 in schwann cells results in dysmyelinating neuropathy. here we present preliminary data showing that jab1 plays a role in cns development. mice with conditional inactivation of jab1 in ol, by cnp-cre tansgene, show cns hypomyelination, including corpus callosum, optic nerve and spinal cord. concomitant fiber degeneration was observed. our preliminary data suggest that jab1 expression in ol plays a role in cns myelination and possibility axonal survival. c. gonsior, j. trotter university of mainz, mainz, germany myelin basic protein (mbp), a major component of central nervous system (cns) myelin, is essential for oligodendroglial function and myelination in the cns and its expression is tightly regulated in time and space. the mrna of mbp is sorted to cytoplasmic rna granules and transported up to the distal processes of oligodendrocytes in a translationally silenced state. we and others could show that, dependent on axonal signals, activation of the non-receptor tyrosine kinase fyn leads to a release of translational inhibition allowing synthesis of mbp protein at the axon-glial contact site. a key factor in this pathway is the fyn target and rna-binding protein heterogeneous nuclear ribonucleoprotein (hnrnp) a2, that recognizes the a2 response element (a2re) in the mbp 3 0 utr and orchestrates the dynamics of the mbp mrna granule. moreover, we identified hnrnp f as an additional target of fyn kinase present in these ribonucleoprotein complexes and contributing to posttranscriptional regulation of mbp. cell stress conditions, as present in various disease states such as multiple sclerosis, may result in the formation of cytoplasmic rna-containing stress granules (sgs), potentially influencing the fate of myelin transcripts including their translation.mbp mrna was shown to be sorted to these cytoplasmic foci. we here identified the hnrnps a2 and f to be associated with oligodendroglial sgs. de-regulation of such factors involved in mbp mrna metabolism could affect stress dependent synthesis of mbp and thus (re-)myelination and myelin maintenance. we performed the experiments in biozzi abh mice known to develop a chronic relapsing-remitting form of eae following immunization with myelin oligodendrocyte glycoprotein. our findings suggest that in demyelinating lesions, myelin layers are pulled off from the myelin sheath and form bulb-like structures that we call "myelinosomes". we can detect the formation of such structures in acute and in chronic stages in the animal model, as well in biopsies from multiple sclerosis patients. we are currently investigating the molecular interactions that underlie the formation of myelinosomes to gather further insight into the mechanisms that underlie immue-mediated demyelination. adenosine is a potent neuromodulator which can be released from most cells and its extracellular concentration increases dramatically after brain injury. it acts on four g protein-coupled receptors, a 1 , a 2a , a 2b and a 3, and their activation is involved in myelination as well as in apoptosis linked to neurodegenerative diseases. in this study, we initially found that rat oligodendrocytes in vitro express all four adenosine receptor subtypes, adenosine transporters ent1 and ent2 and the adenosine degrading enzymes adenosine deaminase and adenosine kinase. activation of adenosine receptors caused mitochondrial dysfunction and oligodendroglial apoptosis. notably, selective activation of adenosine receptors revealed that a 3 receptors are the main subtype mediating oligodendrocyte death. thus, a 3 receptor agonist 2-ci-ib-meca caused a robust increase in ros levels, mitochondrial membrane depolarization and caspase-dependent apoptosis. in turn, selective a 3 receptor activation induced mapk modifications compatible with cellular damage, which was prevented by the selective antagonist mrs1220. the modulation of g protein and adenylate cyclase suggested that 2-ci-ib-meca acts via camp-pka. moreover, selective activation of a 3 receptor triggered intracellular calcium mobilization via plc pathway. finally, we used cerebellar organotypic slices and optic nerve ex vivo to investigate adenosinemediated oligodendroglial death in more integral preparations. consistent with data in dissociated cultures, a 3 receptor activation induced a significant damage in the optic nerve as well as in the cerebellum white matter, an effect that was prevented by caffeine, an adenosine receptor blocker. together, these results indicate that adenosine can trigger oligodendrocyte death via activation of a 3 receptors, and suggest that this mechanism may contribute to the etiology of demyelinating diseases. question-one of the commonest forms of inherited neuropathy, xlinked charcot marie-tooth disease (cmt1x) leads to progressive distal muscle weakness and sensory loss. the disease is caused by a large number of mutations in the gjb1 gene encoding the gap junction protein connexin32 (cx32). cx32 is expressed by schwann cells in peripheral nerves and forms important channels of communication between the layers of the non-compact myelin sheath and is necessary for the homeostasis and survival of the myelinating cells and the axon. transgenic disease models generated in our lab as well as clinical-genetic analysis of large number of patients have demonstrated that most cmt1x mutations result in loss of cx32 function and failure to form gap junctions. cx32 ko mice offer a well characterized and relevant model of the disease to study future therapies for cmt1x, for which gene replacement may be an effective treatment strategy. methods-for this purpose, we have generated a novel 3 rd generation lentiviral vector to allow gene delivery to peripheral nerves. in this vector, the cx32 open reading frame is placed under the control of cell-specific promoter for schwann cells, the rat mpz/p0 promoter. the expression cassette also includes a green fluorescent protein (egfp) as a marker. we have successfully produced lentiviral vector particles and delivered them to the sciatic nerves of 2-month old wild type (wt) and cx32 ko mice, immediately distal to the sciatic notch. the expression of egfp on wt background and of cx32 on ko background was determined by western blot analysis and immunohistochemistry of teased nerve fibers at different time points after injection. results-our initial studies in wt mice (n 5 15) show that more than 60% of schwann cells from different nerve fascicles expressed egfp as far as 5 mm distal to the injection site. expression started a week after the intraneural injection and remained stable for up to 3 months. when the lentiviral construct was injected into the sciatic nerves of cx32 ko mice (n 5 5), virally delivered cx32 was expressed and was properly localized at the non-compact myelin areas including the paranodal regions and incisures of myelinated fibers, where gap junctions are normally formed. poster abstracts s139 glia conclusions-thus, we have generated lentiviral vector that leads to efficient gene expression specifically in schwann cells. these results indicate that gene replacement therapy is feasible and may lead to correction of the gap junction loss in myelinating cells of the pns. perinatal inflammation is intensively discussed to have a long-term impact on various cell populations and on the development of autoimmune diseases such as multiple sclerosis (ms) in adulthood. in order to determine long-term effects of perinatal inflammation on the course of demyelination in adulthood we mimic a bacterial inflammation by exposure to lipopolysaccharide (lps) of either pregnant or newborn mice. demyelination as a "second hit" was induced by feeding adult mice with the oligodendrocyte toxin cuprizone (bis-cyclohexanone oxaldihydrazone). a single prenatal lps injection at e13.5 did not affect the course of demyelination in adulthood. in contrast, serial postnatal lps injections lead to a delayed demyelination and a reduced number of activated microglia in the corpus callosum, suggesting a long-lasting effect of perinatal inflammation on the function of microglia. surprisingly, mice exposed to lps after birth showed an enhancement of early remyelination accompanied by an increased number of newly differentiated oligodendrocytes. furthermore, independent of cuprizone administration the perinatal lps injections seem to induce a long-term impact on the blood-brain barrier (bbb) by decreasing the number of claudin-5 positive vessels. in summary, perinatal inflammation mimicked by the administration of lps has long-lasting effects on the bbb, microglia activation, and remyelination. question: epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. however, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging and we were wondering whether methylation of histones is involved in this process. methods: we describe the function of ezh2 in primary schwann cells and drg cocultures using immunohistochemistry, transfection and lentiviral transduction, shrna, qrt-pcr analysis, morphological analysis, western blotting and chromatin immunoprecipitation. results: here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog 2 (ezh2) gene, on schwann cell differentiation. in sciatic nerves, ezh2 expression was found in schwann cells and to peak perinatally. suppression of ezh2 expression in cultured primary rat schwann cells reduced the length of cell processes. these morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p57kip2, which we have recently identified as an intrinsic inhibitory regulator of schwann cell maturation. in addition, we show that ezh2 suppression in dorsal root ganglion cocultures interferes with in vitro myelination. chromatin immunoprecipitation analysis revealed binding of ezh2 at the p57kip2 promoter and reduction of histone h3k27 trimethylation upon gene suppression. ezh2 suppression-dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p57kip2, indicating that p57kip2 is a downstream effector of ezh2. furthermore, we describe hes5 as transcriptional repressor of myelin genes in schwann cells, which was induced upon ezh2 suppression and downregulated in p57kip2-suppressed schwann cells. conclusions: we have identified a molecular link between histone methylation and control of schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed. previous studies, performed primarily in cell culture, suggest that electrical activity may regulate multiple stages of oligodendrocyte development including proliferation, migration, initial wrapping and myelination. in this study, we have examined the requirement for electrical activity in oligodendrocyte development in vivo using zebrafish as a model system. results: we find that electrical activity is not required for proliferation, migration, or the initiation of axon wrapping. rather, our data indicate a specific role for electrical activity in oligodendrocyte differentiation by regulating a subset of myelin genes, including myelin basic protein (mbp). silencing electrical activity with tetrodotoxin reduced mrna expression of mbp, as assessed by rna in situ hybridization and quantitative rt-pcr. in contrast, myelin protein zero, claudin k, claudin 11a, ugt8, and 36k levels were not regulated by electrical activity. ongoing experiments are aimed to uncover the mechanism of activity-dependent mbp mrna expression during oligodendrocyte differentiation. conclusions: taken together, these data indicate that electrical activity can modulate myelin gene expression in vivo but do not support the notion that electrical activity drives key axon-glia messengers that are required for the initial stages of myelination. (fancy et al., 2011) . eight-week-old lgals3-/-and wild type (wt) mice were fed a diet containing 0.2% cpz w/w during 6 weeks, after which cpz was withdrawn in order to evaluate remyelination 2 weeks after. according to our results, cpz-induced demyelination in lgals3-/mice showed an exacerbated astrocytic and microglial response as compared to wt littermates. electron microscopy showed a significant lack of myelinated axons in lgals3-/-mice as compared to controls. furthermore, the few myelinated axons present were nearly 50% less myelinated than those of controls and were found to be collapsed. remarkably, the remyelination process seemed to be faster in lgals3-/-mice than in wt. remyelinated lgals3-/-mice showed a higher myelin basic protein (mbp) recovery rate as compared to their controls. flow cytometry assays showed a sharper microglial response in lgals3-/-mice, which was supported by an exacerbated number of cd11b1 and cd451 cells. however, electron microscopy images from remyelinated lgals3-/-animals showed, again, collapsed axons with a defective myelin wrap, as compared to wt mice showing normal axons without any relevant myelin wrap disruption. behavioral performance observed during cpz treatment recovery correlates with alterations in the morphological studies, which show that neither lgals3-/-nor wt mice reach basal myelination levels. we have shown that new oligodendrocytes (ols) continue to be generated in healthy adult mice after 8 months of age, even in the optic nerves, which are considered to be completely myelinated already [1] . this raises the question of whether the adult-born ols and myelin are engaged in remodeling existing myelin (e.g. intercalating among the existing myelin sheaths), or replacing ols that die in use. to ask whether ols die and are replaced during healthy adulthood we generated transgenic mice that express icreer t2 under the transcriptional control of the myelin basic protein promoter (mbp6-icreer t2 ). the $6kb mbp upstream fragment that we used is reported to direct transcription to ols but not to either ol precursors (ops) or schwann cells [2] . we confirmed that our mbp transgene targets cre recombination specifically to differentiated ols by crossing to ros26-yfp conditional reporters; when tamoxifen was administered to double-transgenic offspring, essentially all yfp 1 cells in the cns were also cc1 1 . when mbp6-icreer t2 is crossed to tau-mgfp conditional reporter mice (mgfp is membrane-tethered) we can visualize differentiated ols and their myelin internodes in white and grey matter, allowing us to follow the fates of myelinating ols for extended periods of time after tamoxifen labeling. following tamoxifen administration to mbp6-icre t2 : tau-mgfp mice on postnatal day 60 (p60) or p120, a large proportion of ols became gfp-labelled in the corpus callosum, optic nerve and spinal cord. examining these mice at increasing times post-tamoxifen will reveal if and how the number and fraction of mgfp-labeled ols/ internodes changes with age -whether there is net loss or gain of ols or whether new ol synthesis is balanced by the rate of ol death (i.e. there is ol turnover). our preliminary results indicate that new ol synthesis exceeds ol death, so the function of adult ol genesis is not simply to replace dying ols. this study aims to identify protein networks that participate in peripheral myelin formation and maintenance. we use a cell culture system, where c57bl/6j mouse embryos (e13.5) are used for the isolation of dorsal root ganglia neuron explants. explants are cultured in matrigel, and endogenous schwann cells appear in the culture within a few days. myelin formation is induced by adding myelination-promoting substances to the culture medium. immunocytochemistry is used to evaluate the myelination efficiency and the developmental timetable of myelination in culture. electron microscopy is used to study structural features of myelin in culture and to compare its ultrastructural features with those of myelin in situ. rna microarray analysis is performed to study gene expression changes during myelin development in culture. the rna expression data indicates the changes in the rna expression levels, but it does not give information about the amount of final protein product, their posttranslational modifications or stability. we combine the rna expression analysis with a proteomics approach. proteins are extracted for 2-dimensional electrophoresis analysis at the same time points, and protein spots exhibiting time-dependent changes in their expression levels are analyzed by mass spectrometry. we show that mouse dorsal root ganglion explant cultures produce substantial amounts of mature myelin in 3 weeks after the induction of myelination. ultrastructural analysis shows that myelin in our cell culture system has the same structural characteristics as those observed in peripheral nerves in situ. rna microarray analysis reveals changes in myelin-, nervous system-and schwann cell-related genes. the most significant changes in gene expression occur at the induction of myelination. at the protein level, peaks of specific proteins can be observed at the first and the last time points, which complement the results obtained using microarray data. we have developed a reproducible and efficient method to study molecular changes in myelination at the proteomic and genomic levels. this approach will be exploited further for the characterization of the molecular networks involved in the myelination process. results: our findings show that primary rat schwann cells respond to ivig stimulation with altered cell morphologies accompanied by an accelerated growth of cellular protrusions in early stages of the differentiation process. stimulation with ivig was also found to reduce cellular proliferation rates without affecting cell survival. furthermore, we observed that ivig treatment transiently boosts myelin gene expression of immature cells, whereas myelin gene expression of differentiating schwann cells is promoted on a long-term scale. importantly, myelin gene expression responses cannot be detected upon application of igg1 control antibodies (herceptin, synagis, avastin). in addition, we could demonstrate that primary rat schwann cells express the cd64 fc receptor and that in differentiating schwann cells cd64 levels were significantly increased. on the other hand, we could also reveal a specific binding of the human ivig on the schwann cell surface. conclusions: we therefore conclude that schwann cells are not only able to respond to but also specifically bind immunoglobulins and that ivig stimulation can promote their differentiation. here, we demonstrate that cnp deficient mice exhibit alterations in mechanical and thermal sensation. histological analyses of cnp deficient mutants reveal loss of sensory axons when quantified in the dorsal root and the predominantly sensory saphenous nerve. in contrast ventral roots remain unaltered. moreover, electron microscopical assessments of saphenous nerves display a hypermyelination of small to mid-sized caliber axons and an overrepresentation of the smallest fibers. the myelin pathology of sensory axons is reflected by alterations of sensory nerve conduction velocities. thus, our data demonstrate that, contrary to findings in the central nervous system, cnp has an impact on myelination and is especially important for the integrity of small to mid-sized caliber axons within the sensory peripheral nervous system. to unravel the pathogenetic mechanism of the s63del mutation, we generated transgenic mice that express p0s63del protein and manifest a demyelinating neuropathy similar to cmt1b. p0s63del is a misfolded protein retained in the er, where it activates a chronic unfolded protein response (upr). the upr is a cellular response to er stress, aimed to reduce the load of abnormal proteins by attenuating protein translation, increasing the er folding capacity and stimulating protein degradation. among the degradation strategies upregulated by the upr is the er-associated-degradation (erad) pathway. in erad, proteins destined for degradation are retrotranslocated from the er to the cytosol, ubiquitylated and degraded by the proteasome. microarray analysis of s63del nerves revealed that several erad genes, such as derlins, are strongly upregulated and derlin-3 expression returns towards normal in association with the amelioration of demyelination produced by ablation of chop, indicating a possible involvement of this pathway in cmt1b neuropathy. derlins elicit particular interest for exerting a protective role in several er stress-mediated diseases. in s63del nerves, derlins co-immunoprecipitate with p0s63del protein and, finally, derlin-1 overexpression reduces the level of er stress in cos7 cells expressing p0s63del protein, suggesting a positive role in the modulation of p0s63del protein retrotranslocation. these observations indicate that derlins may be part of the schwann cell adaptive response that attempts to cope with chronic er stress by modulating p0s63del clearance. we are now in the process of investigating the role of derlins, in the pathophysiology of pns myelination using in vitro, ex vivo and in vivo approaches. during the myelination process, the oligodendrocyte extends processes to and wraps around multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. this is one unique example of how cells in the central nervous system establish asymmetry. transport of mrna and local regulation of protein synthesis represent one mechanism by which such cellular asymmetry can be generated and the different requirements for myelin sheath at each axo-glia interaction can be controlled. the mrna of a key myelin sheath protein, myelin basic protein (mbp), is known to be transported into the oligodendrocyte processes, and a tight temporal and spatial control of mbp mrna translation is thought to be required for normal myelination. the molecular basis for the tight control of mrna transport and translation is the assembly of large mrna-protein complexes. prior work has identified a number of proteins that interact with the mbp mrna, including hnrnp-a2, hnrnp-k and hnrnp-e1. however, it is currently unknown how these protein functions together to prevent translation during transport. it is also unknown how targeting of the mbp mrna to the myelin sheets occurs. to delineate the precise role of the individual binding protein in regulating mbp mrna translation, we have identified regulatory elements within the 3 0 utr of the mbp mrna involved in translational inhibition, and we have identified binding sites in the mrna for hnrnp-k and hnrnp-e1. furthermore, we have analyzed the effect of sirna-mediated knockdown and overexpression of these proteins in primary oligodendrocytes. together, our results allow us to propose a model, in which the individual mrna binding proteins are assigned distinct roles for correct spatial and temporal expression of mbp. remarkably, this manipulation was sufficient to achieve both the expansion of oligodendrocyte precursor cells (opc) and the de novo myelination of a large fraction of enlarged parallel fiber axons in the cerebellar molecular layer, independent of axonal neuregulin-1 expression. by laser-guided microdissection and gene expression profiling of cerebellar gc, we identified neuronal factors that promote opc proliferation and myelin synthesis in vitro. these findings suggest that oligodendrocytes and their precursors respond to multiple 'instructive' signals expressed by cns neurons in vivo, but only on axons with diameters >0.2 lm that became 'permissive' for myelination. l. marziali, p. franco, j. pasquini university of buenos aires, caba, argentina promyelinating effects of both apo-transferrin (atf) and thyroid hormone (th) on rat brain are well documented. th effects are mediated by nuclear receptors which act as transcription factors and regulate different cell processes. previous results from our laboratory showed that th promotes the commitment of oligodendrocyte progenitors (opcs) to mature myelinating oligodendrocytes (olg). on the other hand, atf is able to promote the commitment of neural stem cells (nsc) to cells of the oligodendroglial linage and favors olg maturation after an intracranial injection. our previous demonstration that th administration is able to up-regulate tf mrna expression leads us to hypothesize that both factors converge in the control of oligodendrogenesis. to test if the combined effects of both atf and th are required for proper myelination in the rat brain, studies were done at p10 and p20 for each experimental condition. a hyperthyroid state was rendered by daily subcutaneous th administration from the day of birth (hyper). hypothyroidism was achieved by giving propylthiouracil to the mother from gestational day 18 to the end of the experiment (hypo). half of the hypo pups received an intracranial injection of atf at postnatal day 3 (hypo1atf). a set of euthyroid animals received an intracranial injection of atf at postnatal day 3 (atf). control groups received an intracranial injection or subcutaneous saline solution (c). at p10, hyper animals showed an up-regulation of 55% in tf mrna expression and 87% in protein levels as compared to controls. hypo showed a decrease of 25% in tf mrna levels. this effect was not affected by exogenous administration of atf. at p20, hyper showed no differences in the expression of tf mrna or protein levels as compared to controls. hypo showed a decrease of 25% in tf mrna and a 40% decrease in protein levels. this effect was not affected by exogenous administration of atf. no differences were observed in the expression of insulin growth factor i (igf-i) or igf-i receptor between groups at p10 or p20. immunohistochemical analyses were performed at p20 in all groups usingspecific markers anti caii, anti mbp, anti rip and pdgfra. our conclusion is that, in a hypothyroid state, tf is not able to produce olg and myelin maturation at the corpus callosum, which indicates that th is necessary for atf action. however, the finding that hyperthyroid animals showed a significant increase in tf mrna strongly suggests that tf could be involved in th effects. new experiments are carrying out in order to knock down the tf gen to probe this last piece of evidence. h. yigit, l. meyer, c. gonsior, p. hoch-kraft, j. trotter johannes gutenberg university, mainz, germany myelination is the prerequisite for fast saltatory conductance of action potentials. in the central nervous system, the myelin sheath is produced by oligodendrocytes which extend processes and wrap the axons. this structure is subsequently compacted by the help of certain proteins. of major importance here is the myelin basic protein (mbp) which interacts with the cytoplasmic leaflet of the plasma membrane. mbp is a highly basic protein and binds to the negatively charged plasma membrane with high affinity. it has 6 isoforms in mice produced by alternative splicing. the mostly studied 4 isoforms are 14 kd, 17 kd, 18,5 kd and 21,5 kd. interestingly, exon-ii containing 17 kd and 21,5 kd isoforms are actively transported into the nucleus while 14 kd and 18,5 kd isoforms are rather localized to the distal part of the plasma membrane. transport to the nucleus depends on temperature and energy. recently, it was reported that exon-ii contains a non-canonical py nuclear localization signal. as a cytoplasmic protein, mbp is essential for proper myelination. furthemore mbp is supposed to have multiple additional functions, e.g. in calcium homeostasis and cytoskeleton dynamics. it was also shown that the expression level of mbp exon-ii containing is changed in schizophrenia patients. selectively transport of exon-ii containing isoforms to the nucleus raises the question of its nuclear functions which have not been elucidated yet. this study aims to unravel the role of exon-ii containing mbps in the nucleus. gene expression profiling of the microdissected svz regions revealed that wnt-responsive genes were enriched in the postnatal dorsal svz compared to the lateral svz. we performed inhibition of gsk3b by intraventricular infusion of ara to initiate wnt-signalling. several approaches confirmed activation of the wnt-signalling pathway in the dorsal svz as well as within ops. thus, gene expression profiles of wnt-target genes of the dorsal svz indicated that axin2, lef1, fzdl1 and tcf4, but not those other pathways, were dramatically up-regulated. furthermore, nuclear b-catenin was transiently up-regulated in the dorsal svz including in a large population of sox10-egfp expressing cells following pharmacological wnt-activation. gene expression profiling and immunostainings revealed that the densities of nscs, cycling progenitors and subsequent op differentiation in the dorsal svz were augmented. in parallel, we genetically manipulated wnt-signalling in stem and progenitor cells of the dorsal wall of the lateral ventricle. targeted dorsal svz electroporation of cre-gfp plasmids in floxed stabilised b-catenin (gain-of-function) and floxed b-catenin (loss-of-function) transgenic mouse lines were performed and resulted in a phenocopy of the results observed following ara-intraventricular infusion. in summary, our findings illustrate that wnt-signalling endogenously persists in the dorsal svz after birth. pharmacological as well as genetic interventions reveal an important role of this signalling pathway in the regulation of op genesis during the period of myelination. in the cerebellum the generation of neurons and glia and the relationships between these two lineages are poorly understood. all the cerebellar neuronal phenotypes derive from two distinct embryonic germinal neuroepithelia that are restricted regarding the neuronal cell fate: the rombic lip (rl) gives rise to the glutamatergic neurons, whereas the ventricular zone (vz) generates all gabaergic neurons. in particular, different types of inhibitory interneurons are generated during the postnatal life from a common pool of progenitors expressing the transcription factor pax2. on the other hand, the glial development is relatively unexplored. part of the oligodendrocytes derives from extracerebellar sources, whereas some of them derive from endogenous precursors. astrocytes, instead, may derive from progenitors that delaminate from the vz and continue to divide in the prospective white matter (pwm) up to postnatal life. given the vn origin of both astrocytes and interneurons, we wondered whether a lineage relationship exists between these two cell populations and whether they share the same bipotent progenitor. in order to address these issues, we performed a genetic fate mapping study of glastcreert2 mice. we showed that both cerebellar gabaergic interneurons and astrocytes derive from proliferating glast 1 precursors. in particular, we found that these progenitors are able to produce interneurons at earlier developmental phases, whereas after p6 they appear restricted to the glial lineage. proliferating glast 1 cells that may produce interneurons comprise bergmann glia and pwm progenitors. however, when recombination was specifically induced in bergmann glia, we found that these cells were not neurogenic. therefore, we concluded that glast 1 progenitors reside in the pwm. to get further insight in the behaviour of these precursors, we injected in the pwm a gfp-containing lentiviral vector with a specific tropism for astroglial cells. some days after, infected cells generated pax2 1 -interneurons that were able to mature into interneurons of different cortical layers. moreover, in vitro and in vivo clonal analysis of pwm derivatives indicates that the pwm is neurogenic and both astrocytes and interneurons may derive from a common progenitor. evidence is accumulating that neurogenesis in the subventricular zone (svz) is boosted after trauma or ischemia, also through the interaction with surrounding parenchyma or niche cells. nevertheless, the number of newborn neurons that survive and integrate in the damaged areas is negligible, suggesting a non-permissive environment. thus, understanding the complex signaling network guiding neuroblast generation/survival could help identifying strategies to limit negative inputs and promote regeneration. extracellular nucleotides (ents) are among the hypothetical modulators of svz cell functions, especially under pathological conditions where their concentrations raise several folds and they contribute to reactive astrogliosis. few literature data have recently pointed for a role of the p2y 1 receptor subtype (one of the 8 known g protein-coupled nucleotide receptors) in controlling the proliferation and differentiative potential of svz cells. thus, we tested the ability of adpbs, a stable p2y 1 agonist, to modulate stem cell properties in the adult brain, with a focus on the possible modulatory effects exerted by reactive astrocytes. the administration of adpbs in the lateral ventricle of adult mice led to reactive astrogliosis in the surrounding brain parenchima, and to a massive reaction of gfapexpressing precursors and astrocytes in the svz. also proliferation was increased, paralleled by a significant expansion of the population of mash11 transit-amplifying cells and of doublecortin1 neuroblasts. thanks to the conditional glast::creert2 yfp mouse model, we also demonstrated that adpbs promoted the proliferation of glast-expressing progenitors in the neurogenic niche, and sustained their progression towards the generation of rapidly dividing transit-amplifying cells. in vitro the nucleotide analog increased the proliferation of svz cells grown as neurospheres, and their differentiation towards neurons, fully confirming in vivo data. interestingly, a significant enhancement in neurosphere generation was detected when svz cells were initially grown in the supernatant of astrocytes exposed to adpbs, and then shifted to normal medium. this suggests that adpbs stimulates the release of yet-to-be identified astrocytic mediator(s) whose removal from the culture medium boosted proliferation of svz cells. our results further strengthen the notion that the purinergic system is a key regulator of the neurogenic potential of svz cells, both directly and through the involvement of reactive astrocytes. cambridge stem cell institute, cambridge, united kingdom oligodendrocytes, one of the principal glial cell-types of the central nervous system, are critical for neuronal function and represent the primary target of many neurological diseases, including multiple sclerosis and leukodystrophies. despite the recent progress in stem cell research and the possibility to generate various neural cell types in vitro, the derivation of oligodendrocytes from human pluripotent stem cells remains inefficient and unreliable. we employed a new experimental approach termed "direct cellular reprogrammng" which was already succesfully applied for the generation of different types of neurons and neural stem cells. aided by bioinformatic filtering we have generated a pool of seventeen candidate reprogramming factors that could potentially convert fibroblasts directly into oligodendrocyte lineage cells. combinatorial testing of the candidate factors in overexpression experiments has revealed a combination of just two transcription factors that was sufficient to convert mouse embryonic fibroblasts directly into o4-expressing oligodendrocyte precursors. supplementation of additional factors and multicistronic gene delivery strategies render the reprogramming process more efficient and widen the applicability of this protocol. cellular reprogramming offers a new tool for the generation of oligodendrocyte precursors. this approach, that circumvents the protracted specification and differentiation process inherent to the oligodendrocyte lineage will foster the quest for a culture system of human oligodendrocyte lineage cells. transplanted are derived from progenitors within the intestine. to gain further insight in possible stem cell niches the compelling evidence that mouse enteric glia can also be neuronal precursors was related to human tissue. human colon from infants with hirschsprung's disease was investigated with a panel of glial and stem cell markers. tissue samples from infants with hirschsprung's disease were investigated with glial and stem cell markers along the gut axis. the tissue samples from ganglionic, aganglionic and transient segments were immuonstained either for s100/nestin, s100/p75, gfap/nestin and gfap/p75. in all samples investigated, nestin positive ganglia could be found, even in the distal parts with a severe hypo-or aganglionosis. beside nestin positive cells in all segments, there were also different expression pattern glial markers within the ganglia, indicating that distinct phenotypes of glia cells could be found. neural and glial precursor cells are present in the ganglionic as well as in the hypoganglionic segments of hirschsprung's colon, suggesting that these cells might be suitable for ncsc generation and further transplantation. the subventricular zone (svz) of the lateral ventricle, the largest adult stem cell niche, has a striking pinwheel organization specific to regions of adult neurogenesis. the pinwheel's core contains the apical endings of b1 cells and in its periphery two types of ependymal cells: multiciliated (e1) and biciliated (e2) cells. previous studies showed that svz cells are reactivated in response to demyelination. however, the mechanisms inducing svz reactivation in response to inflammatory demyelination such as in multiple sclerosis (ms) are poorly understood. we hypothesize that b1 and e, which are in direct contact with csf through cilia, may play a crucial role in initiation of svz reactivation. in the present study, we examined the svz reactivation in mice in which we targeted an ms-like pathology to the forebrain white matter (t-eae). animals were immunized with subclinical doses of myelin oligodendrocyte protein and after 20 days, received a stereotaxic injection of a cytokine cocktail consisting of tumour necrosis factor (tnfa) and interferon (ifn c) into the corpus callosum. to obtain an en face view of the ventricular surface, we dissected whole mounts of lateral ventricle of control and eae animals at 3, 7, 14 and 28 days post-eae induction (dpi). we investigated by immunohistochemistry whether and how the pinwheel architecture of the svz is modified and ependymal cells are reactivated during disease course of eae. in svz niche of t-eae animals we found that the number of b1 cells increased and peaked at 7 dpi compared to control. the number of e2 cells and pinwheels decreased and then reached control level at 7 dpi. while the number of e1 cells remained steady, their surface area increased at 3dpi and then returned to normal size over time. in addition, the number of their ciliary basal bodies increased during eae time courses, and e1 cells highly expressed gfap in eae animals compared to controls. thus, the findings of our ongoing research suggest that neuroinflammation induces some changes in cell number and morphology of svz niche as well as reactivation of ependymal cells, which altogether may contribute to svz reactivation mechanisms. further study will provide detailed information on the homotypic and heterotypic interactions between pinwheel cells in response to t-eae. to test whether hypothalamic tanycytes act as bona fide neural stem/ progenitor cells in vivo, we analysed their immumoprofile and proliferative potential, and lineage-traced them in vivo. for this, we exploited our earlier findings that in the adult hypothalamus, fibroblast growth factor 10 (fgf10) is expressed exclusively by tanycytes (hajihosseini et al. 2008 mcn 37: 857-68) , a radial glial-cell like population that lines the floor and ventral walls of the third ventricle. using a line of fgf10-lacz reporter mice, we found that fgf101 beta tanycytes express a panel of neural/stem progenitor markers such as nestin, musashi1, blbp and sox2. fgf101 tanycytes also incorporated brdu under cumulative brdu-labelling paradigms, and formed neurospheres in vitro. to directly test the potential of fgf101ve tanycytes, we lineage-traced them at p28 and p70 by activating the constitutive expression of the marker gene, tomato-dsred, in these cells and their descendants. this was achieved by applying tamoxifen to fgf10-creer-t2::rosa26r-tomato double transgenic mice. after short survival time-points, tomato was detected exclusively in tanycytes, whilst with longer survival time-point, tomato1 cells emerged in the neighbouring parenhcyma. the majority of these were neun1 neurons with fine arborizations. we noted that the newly-generated neurons become associated mainly with hypothalamic nuclei that regulate appetite and energy-balance. moreover, they respond to appetite/energy balance regulating signals such as acute food deprivation and leptin administration. our findings establish fgf101 beta-tanycytes as a putative population of neural stem/ progenitor cells that generate appetite/energy balance regulating neurons in the postnatal and adult hypothalamus. we used an inducible transgenic mouse line (hgfap-creert2) to conditionally label and fate map putative gfap(1) nscs populations in the adult svz and spinal cord (sc), and compare their self-renewal and multipotential properties. we evidence that a population of gfap 1 cells that share the morphology and antigenic properties of svz-nscs reside in the dorsal and ventral aspects of the central canal (cc) throughout the spinal cord. these cells are non-proliferative in the intact spinal cord, but incorporate edu, an s-phase marker following spinal cord injury. multipotent yfp-expressing neurospheres (i.e. which derive from recombined gfap-expressing cells) were successfully obtained from both the intact and injured spinal cord, confirming their early gfap origin. notably, only spheres isolated from the injured spinal cord revealed long-term self-renewal properties resembling those of svz neurospheres. altogether, this work highlights discrepancies in the identity of nscs that reside in distinct regions of the adult cns. our observations however reconcile divergent views on the location and identity of spinal cord-nscs by showing a population of multipotent gfap 1 progenitors with limited self-renewal properties co-existing alongside with multipotent, self-renewing ependymal cells within the spinal cord central canal. during development neural precursors (nps) both divide, to expand the cell population, and produce many different kinds of neurons and glia. this balance appears to be regulated by par complex proteins, which polarize neural precursors and can thereby direct daughter cells for different fates. how par complex proteins are regulated to appropriately polarize nps remains unknown. in recent years, regulation of gene function by micrornas has emerged as an important mechanism during development. using bioinformatics we identified the polarity gene pard3 as a candidate target for microrna219 (mir-219). mir-219 is specifically expressed in the developing central nervous system (cns) of vertebrates and mir-219-deficient zebrafish embryos have a deficit of oligodendrocytes, the myelinating glial cells of the cns. because a disruption in polarity could affect the types of cell divisions that nps undergo, thus altering the balance of cell types that arise, we hypothesize that neural precursor maintenance is regulated by modulation of polarity cues through mir-219. by using an in vitro reporter assay we found that mir-219 can downregulate expression of a luciferase gene fused to the pard3 3 0 utr. reduction of pard3 function in zebrafish embryos suppressed the oligodendrocyte phenotype resulting from loss of mir-219 function and injection of a morpholino oligonucleotide designed to block binding of mir-219 to its pard3 target sequence phenocopied mir-219 knock down. together, these data provide strong evidence that pard3 is a functionally relevant in vivo target of mir-219. to further investigate the role of mir-219 function we tested expression of np, radial glial and neuronal markers in mir-219-deficient embryos. the number of cells expressing sox2, a marker of nps and neural stem cells, was significantly increased upon mir-219 knockdown. concomitantly, mir-219-deficient embryos had a dramatic deficit of radial glia and late born neurons. these data provide evidence for a new mechanism of np regulation, in which mir-219 regulates pard3 levels, thereby regulating the transition of dividing neural precursors to differentiated neurons and glia. in demyelinating diseases such as multiple sclerosis myelin repair activities based on recruitment, activation and differentiation of resident progenitor and stem cells can be observed. however, the overall degree of successful remyelination is limited. it is therefore of considerable interest to understand oligodendroglial precursor cell (opc) homeostasis and maturation processes in order to develop remyelination therapies. mesenchymal stem cells (msc) were shown to exert positive immunomodulatory effects, to reduce demyelination, to increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. we here addressed whether msc secreted factors can influence primary opcs in a myelin non-permissive environment. methods: to this end we analyzed cellular morphologies, expression and regulation of key genes/proteins involved in oligodendroglial cell fate and maturation upon incubation with mesenchymal stem cell conditioned medium. results: this demonstrated that msc derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. accelerated maturation featured elevated levels of 2 0 , 3 0 -cyclic nucleotide 3 0 -phosphodiesterase and myelin basic protein expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as id2 and id4. conclusions: we thus conclude that besides the previously established immunomodulatory and neuroprotective roles of mscs these cells can also positively influence oligodendrogenesis in the adult central nervous system. in this study, we evaluated the effects of epo and the epo splicing variant (vepo) on murine neurogenesis ex vivo. for this purpose, we analyzed the survival of cultured neural stem cells (nscs) either exposed transiently to recombinant protein, or transduced with lentiviral vectors to achieve stable protein expression. in comparison to non-exposed controls, recombinant epo and vepo enhanced survival of nscs ex vivo (epo: 150 6 25%; vepo: 250 6 60% survival). nscs transduced with pcl20-mscv-epo-and pcl20-mscv-vepo showed also higher viability ex vivo compared to nontransduced nscs (epo: 182 6 4% vepo: 131 6 4% survival). furthermore, pcl20-mscv-epo, but not pcl20-mscv-vepo, increased the proportion of differentiated neurons when compared to non-transduced controls. thus, stable expressed vepo may have rather an effect on nsc survival than on nsc differentiation. our results demonstrate that vepos are neuroprotective in vitro. vepo may serve as therapeutic protein for treatment of neurodegenerative disease associated with impaired neurogenesis such as huntington disease. in the subependymal zone (sez) cytogenic niche of the adult mouse brain neural stem cells drive the continuous generation of new cells, mostly of olfactory bulb interneurons, but also of cells of the oligodendroglial lineage. previous reports have shown that newly-born cells exit the sez and migrate to the adjacent corpus callosum (cc) where they differentiate into oligodendroglial cells. however, the real contribution of these niche-derived oligodendrocyte progenitor cells and oligodendrocytes (nopcs and noligos) to the oligodendroglial population of the cc has not been assessed so far. in this study we used the hgfap-cre ert2 3 rosa26-eyfp double transgenic mice in order to label specifically the progeny of sez-located adult neural stem cells. we found that cells expressing markers of opcs, such as pdgfra and olig2, are constantly generated in the sez and migrate to the proximal fraction of the cc. interestingly though, these cells do not remain in the cc for more than 15 days with their contribution to the total population of oligodedroglial cells remaining stably below 2% even in the ageing brain. moreover, immunostaining for markers of cell cycle revealed that a higher fraction of nopcs undergoes mitosis, when compared with local opcs; hence, they constitute almost 25% of proliferating opcs of the cc. notably, during their transient presence in the cc, nopcs express markers of maturing oligodendrocytes and are incorporated in established local cell structures. when the cc is challenged with a focal demyelinating insult the local and the niche-derived oligodendroglial machineries exhibit differential cell kinetics, nopcs increase their contribution to the total pool of oligodendroglial cells; however, their presence remains transient. in the human and mouse retina m€ uller glia (mg) are well known to undergo gliosis in all major types of retinal diseases -which sometimes may even lead to scar formation due to proliferative gliosis. some studies suggest that in the mouse retina mg derived neuronal regeneration can be stimulated, but only to a very limited extent. here, we started to find out, if conditional immortalization might stimulate mg derived proliferative gliosis and /or neuronal regeneration. others and we reported that in the juvenile mouse retina, after retinogenesis is finished, some m€ uller glia shift from a quiescent differentiated state into a proliferative state upon damage of retinal explants ex vivo. we now observed that this process is differentially inducible by mitogens like egf (epidermal growth factor). edu (s-phase marker) pulse chase experiments revealed a tremendous increase in mg proliferation within the first two days, a peak of edu positive cells at day 4 (14 122 sem, n 5 4) and a massive decrease until day 6 (4 120.4 sem, n 5 4) per 400 mm of a central retina section. next, we used transgenic mice with tightly and temporally controlled expression of the proto-oncogene sv40 large t-antigen (csv40lt). it is well reported that csv40lt binds several proteins including the tumor suppressor p53 and retinoblastoma and bypasses cell cycle checkpoints. induction of the csv40lt for 6 days ex vivo led to an overall increase in proliferation compared to control. the number of edu1 cells was 8.5 fold increased (csv40lt: 23 126 sem, n 5 4; control: 3 120.16 sem, n 5 4; p our results so far suggest that induction of csv40lt not only overcomes the proliferative restriction of m€ uller glia but also maintains its progeny in the cell cycle over extended period of time. surprisingly, major parts of the generated cell progeny formed gliotic cell clusters, which were all located within the boundaries of the retina. further, we present data of successful m€ uller glia isolation at high purity by fac-sorting. in our current and future work we study the m€ uller glia and its derived progeny to find out the underlying mechanisms that enable neuronal regeneration and prevent gliotic scar formation. in mammals, regeneration capacity of the central nervous system (cns) is considered too low to recover the neurological functions after traumatic injury. in fishes and amphibians, however, the spontaneous regeneration is vigorous enough to complete the anatomical reconstruction and functional recovery even after severe damage of cns, in which the ependymal cells play the central role to exhibit proliferation, epithelial-mesodermal transition, cell migration, support of regenerating axons and neurogenesis to contribute to recovery from injury. these positive roles of the ependymal cells observed in fish and amphibian damaged cns may be good candidates for treating mammalian cns injury. in this study, we attempted to control the activity of the ependymal cells in the adult rodent spinal cord by virus-mediated gene transfer for imitating the reactions that are observed in the ependymal cells of fish and amphibian damaged cns. we introduced the gene known to be expressed in both the ependymal cells and neural stem cells (nscs) and found the proliferation of ependymal cells. in this case, the ependymal cells expressed glial fibrillary acific protein suggesting that this gene regulates proliferation and differentiation into astrocytes. in addition, introduction of another gene, also known to be expressed in the ependymal cells and nscs, caused cell proliferation without differentiation tendency. these findings indicate that the cell fate of the ependymal cells in the adult rodent spinal cord can be modulated by artificial intervention. future studies will clarify whether cell fate modification in the ependymal cells can contribute to anatomical reconstruction and functional recovery in the adult mammalian damaged spinal cord. methods neural stem cells are isolated from different parts of the human gastrointestinal tract, and also from different compartments (muscle, submucous and mucous layer) using enzymatical digestion approaches. generated neurospheres were screened for neuronal, glial, neural stem cell and pluripotency markers, prior and after differentiation. additional experiments were performed where neurospheres were either co-cultured with tissue blocks from different parts of the brain, or transplanted into brain slices from the rat. results neurospheres could be generated from all areas investigated and differentiated on glas coverslips. nestin and musashi-1 could be found in all neurospheres. moreover sox-10, nanog and oct-4 could be found in the differentiating cultures. after differentiation tubulin and pgp 9.5 positive neurons could be found. transplantation experiments showed that cells from the transplanted spheres migrated into the brain slices and differentiated into neurons or glial cells. neurospheres co-cultured with tissue blocks from cortex, cerebellum or hippocampus showed a significant higher outgrowth than neurospheres cultured without any brain tissue. conclusions in general, the ens is a suitable tissue for the isolation of neural stem cells in the individual patient and might so be an appropriate source for autologous neural stem cells. neurogenesis. at the end of embryogenesis, oligodendrocytes and astrocytes appear during the process of gliogenesis. although in the last decades diverse extrinsic and intrinsic factors involved in these developmental processes were identified, our understanding of detailed mechanisms is still comparatively low. in order to increase the knowledge about the cellular and molecular mechanisms that underlie central nervous system development, transfection of nscs showed to be a promising method to understand the function of specific genes and proteins. to overcome the well-known difficulties to transfect cells of the central nervous system, we improved the electroporation conditions to achieve high efficiency transfection and survival rates for embryonic and adult nscs isolated from mouse forebrain structures. one day after electroporation transfection and viability rates of around 80% were achieved in murine nscs (bertram et al. j neurosci meth 2012). due to our interest in differentiation and maturation of oligodendrocytes we are currently improving the transfection conditions of oligodendrocyte precursor cells (opcs), since a sufficient protocol for the transfection of opcs derived from neonatal rat cortices is currently not established. the opcs are isolated from rat cortical tissue by a shaking protocol and transfected with the 4d-nucleofector (lonza). when we used the same pulse protocol that successfully works on nscs, oligodendrocytes were sensitive to the transfection process, and many cells died or did not undergo normal differentiation. this demonstrates that other pulses are needed to electroporate opcs. in cooperation with lonza we tested several recommended pulses to increase the transfection and survival rates of opcs. a dramatic increase of the viability rate has already been achieved. in further experiments the pulse protocol will be improved to obtain transfection rates above 20%. by establishing this transfection protocol we want to accomplish transfection and viability rates comparable to murine nscs. this improved protocol for the transfection of sensitive opcs will allow for reliable studies of gene function by over-expression or knockdown experiments, and will enhance our understanding of cell biological mechanisms important for oligodendrocyte development. recent studies demonstrate that astroglial cells isolated from non-neurogenic brain regions have the potential to be reprogrammed into functional neurons through forced expression of transcription factors known to instruct neurogenesis. based on our previous studies on the potential of the neurogenic gene cend1 in directing neural stem/precursor cells (nsc) to exit the cell cycle and acquire a neuronal phenotype, in parallel with evidence demonstrating activation of cend1 expression by genes of the neurogenin family, we explored the combined effect of cend1 and neurogenin-2 on their reprogramming potential on postnatal cortical astrocytes. to this end, forced expression of either cend1, neurogenin-2 or both, resulted in an important increase of two morphologically distinct subpopulations of gfapcells with elongated morphology, that strongly expressed the radial glial marker glutamate transporter glt-1. further characterization revealed that a subpopulation of these cells differentiates towards the neuronal lineage, as they were exhibiting a differentiated neuronal morphology and expressed b-iii tubulin, as well as neuronal subtype-specific markers, including gaba and th. further analysis using long time live cell imaging and brdu cumulative labeling revealed that the majority of cend1-transduced astrocytes undergo 1-2 cell divisions before differentiating to gabaergic neurons, whereas most ngn2 1 astrocytes directly transdifferentiate giving rise to th 1 neurons. additionally, only in the doubletransduced cultures, cend1 1 /ngn2 1 astrocytes seemed to take a step back forming colonies of small round glast 1 /nestin 1 cells, detected 24h following transduction. a day later, these colonies grew as three-dimensional spheres of high proliferative potential attached to the culture dish. when 'astrospheres' were isolated and cultured under nsc conditions, they grew as neurospheres and were propagated for over ten passages. importantly, as soon as 'astrospheres' were cultured in the absence of growth factors, they differentiated into neurons, astrocytes and oligodendrocytes, implying that they possess neural stem cell properties. at the moment we are performing electrophysiology recordings to assess the functionality of cend1derived neurons in vitro, while studies are in progress to explore the potential role of cend1 and ngn2 on astrocytic reprogramming in vivo following cortical brain injury. average membrane potential (v m ) was 277 mv and input resistance (ir) was 92 mx, while dcx 1 or map2 1 cells expressing fast activating outwardly rectifying k 1 currents (ka), and delayed outwardly rectifying k 1 currents (kdr) were defined by a v m of 272 mv and high values of ir (1943 mx). ng2 1 cells with a complex current pattern expressed inwardly rectifying k 1 (kir) currents, in addition to kdr and ka currents, and their v m was 276 mv and ir was 383 mx. the electrophysiological analysis revealed that the inhibition of the wnt signaling pathway significantly increased the incidence of cells with a complex current pattern, while the number of cells with the marked expression of outwardly rectifying k 1 currents declined. wnt signaling inhibition also resulted in increased kir and decreased ka current amplitudes. 4oht-treated cells were also hyperpolarized when compared to controls. immunocytochemistry using antibodies against map2 and dcx showed that neuronal progenitors in 4oht-treated cultures were less developed, having fewer and less branched processes. in summary, our data imply that the canonical wnt pathway in the neonatal svz promotes nsc differentiation into cells with neuronal characteristics. gacr p303/12/0855; p304/12/g069. adult neurogenesis plays a critical role in the overall health of, and repair processes occurring within the nervous system. while a great deal of information has been gained about the elements of the neurogenic niche and the factors that mediate neurogenesis, many unanswered questions remain. specifically, studies suggest that microglia have a significant impact on adult neurogenesis, but no unifying theory exists to explain their exact role in this process. in order to determine if microglia play a pivotal role in adult hippocampal neurogenesis and to examine the mechanisms through which microglia exert their effects on the hippocampal neurogenic niche, we employed a transgenic mouse model that allows for the selective ablation of microglia in the central nervous system (cns), the cd11b-herpes simplex virus thymidine kinase (cd11b-hsvtk) mouse. using this system, we removed microglia from the adult hippocampal neurogenic niche and evaluated the effects of this manipulation on neurogenesis and hippocampal homeostasis, including immunohistochemical analyses of stem cell survival and proliferation, as well as analyses of neuroblast development, migration and electrophysiological activity. taken together, these studies help to provide a better understanding of the factors governing adult neurogenesis and further our knowledge about the increasingly diverse role of microglia in the cns. the current assumption is that ependymal damage, caused by viral infections may result in reactive gliosis in the subventricular zone (svz), leading to sgns. however, as the svz is currently recognized as one of the neurogenic niches in the adult human brain, and svz astrocytes have been identified as neural stem cells (nscs), the assumption of sgns being reactive astrocytes has to be reconsidered. therefore, we have immunophenotyped the cell types present in these structures in the brains of individuals of various ages. in addition, we assessed nodule incidence in the svz in cases infected with hiv, where cells in the svz are likely to be to be affected. we found sgns in almost 90% of all cases, independent of age or hiv infection. we determined that cells in the sgns express adult nsc markers, rather than markers for reactive astrocytes. we hypothesized that direct contact of the nscs with cerebrospinal fluid (csf) may induce nodule formation. indeed, we could show that human ventricular csf stimulated the proliferation of human nscs in culture. from our data, we conclude that sgns most likely are formed upon ependymal damage in response to exposure to csf and represent local expansions of the svz. . in experimental murine model of demyelination, the repair capacity is robust and sufficient, even if it might decrease with aging. one major therapeutic goal in ms is to favour endogenous myelin repair, to prevent neurodegeneration and disability progression. using a transcriptomic approach on pure populations of opcs, we try to identify mechanisms specifically involved in opcs activation. we set up a method to isolate a purified population of opcs, by fluorescent-activated cell sorting from pdgfar::gfp mouse brains. we created the gene expression profile of neonatal opcs, adult opcs and adult oligodendrocytes (ols) in control conditions and of adult opcs in cuprizone-induced demyelinating conditions. we analyzed the transcriptomic profile of adult opcs, neo-natal opcs and ols, and identified more than 1000 differentially expressed genes. after demyelination, we identified more than 2000 adult-opc-specific genes that are either up-or down-regulated compared to control conditions. preliminary analysis suggests that adult opcs are more "mature" than neonatal opcs and partially revert to a more immature profile after demyelination. bio-informatic analysis is ongoing, with the perspective of identifying key candidates for myelin repair capacity, then develop functional experiments to validate their involvement in remyelination. in parallel, we plan to compare the transcriptomic profile of adult opcs from aged versus young mice, to get insight into age-related decrease of repair efficacy. question: after damage, astrocytes are activated and exert specific roles for central nervous system (cns) repair. among these, astrocytes within the injury site may represent a novel source of cells with stem cell potential. in this study, we used sedimentation field-flow fractionation (sdfff) method to rapidly sort well preserved astrocyte subpopulations and we identified stem cell properties within one of these subpopulations. methods: cells were collected from newborn rat cortex, separated mechanically and subjected to sdfff. cell fractions obtained were analyzed by immunocytofluorescence using antibodies against specific epitopes: glial fibrillary acidic protein (gfap), o4, b iii tubulin, and cd68, labelling respectively astrocytes, oligodendrocytes, neurons, and microglial cells. then, proteomic analysis was performed on astrocyte subpopulations and their behaviour in culture was analyzed, particularly under culture conditions usually allowing neurosphere development. results: three main fractions (f1, f2, and f3) were isolated and compared with the total eluted population (tp). after one week in vitro, tp and f2 derived cell cultures contained respectively 74.5% and 11.9% of gfap expressing astrocytes while this percentage was extremely high in f1 and f3 derived cell cultures (95.6% and 98.0% respectively). f3 derived cells displayed higher migratory capacities than those of f1. proteomic analysis of f1 and f3 derived cells indicated a high expression of vimentin in f3 derived cells. in the presence of epidermal growth factor (egf), f3 derived cells formed neurospheres containing cells expressing gfap and nestin (figure) that, when placed in appropriate conditions, were able to generate the three major cell types of the cns (neurons, astrocytes and oligendrocytes). moreover, colony-forming cell assay in a collagen-containing semi-solid matrix allowed in the presence of egf the formation of large colonies. in addition, 7 days after cortical lesion in adult rats, cells presenting similar stem properties were obtained after sdfff cell sorting. conclusions. our study demonstrates that sdfff enables the isolation of almost pure astrocyte subpopulations after one week in vitro. in addition, after sdfff cell sorting, we isolated an astrocyte subpopulation expressing vimentin and displaying the self-renewal and multipotency properties of neural stem cells. our data support that sdfff is an efficient tool to explore the different astrocyte subpopulations of the cns, particularly those involved in cns repair. experimental studies have shown that loss of myelin results in axonal loss and disability. so, finding of an expandable and autologous source of myelin-forming cells to enhance remyelination is required. induced pluripotent stem cell-derived neural progenitor cells (ipsc-npcs) have been developed recently. the remyelination potential and safety of these cells still remained to be well-addressed. the main goal of this study is to characterize mouse ips-npcs in vitro and in vivo after transplantation. we used embryonic mouse neural progenitor cells (mnpcs) as control and characterized mouse ips-npcs for their expression of major markers of immature or mature cells by immunostaining. rt-pcr was performed to analyze expression of nestin, olig2, b3-tubulin, olig1, sox10, and nkx2.2 mrnas. furthermore, to investigate the fate of these cells in vivo, we injected the lysolecithin to induce focal demyelination in the spinal cord of adult nude or shiverer mice. we transplanted cells in the lesion site 2 days after demyelination. animals were sacrificed 2 days, 1, 2, 6 and 8 weeks post transplantation to assess survival and differentiation of the grafted cells. our preliminary results showed that mips-npcs similar to mnpcs were nestin1, ki671, olig21, b3-tubulin1 but o4-and map5-. mips-npcs, were more proliferative than mnpc. moreover, mips-npc expressed all mrna transcripts except sox10. a first line of mips-npcs, was transplanted in the newborn shiverer:rag forebrain or the demyelinated spinal cord of immunosuppressed shiverer mice with a constant failure in terms of cell survival beyond 2 days of transplantation, highlighting the fragility of some of the reprogrammed cell lines. cells of a second line of mips-npcs were transplanted in nude mice to be sacrificed at 1, 2 and 6 weeks. an additional serie of transplantation was performed in the demyelinated shi:rag spinal cord. data at 1 and 2 weeks indicate excellent survival and integration of the mips-npc at both time points. some of the grafted cells expressed gfap, olig2 and ki67 (few only) and so far, no tumors were observed at these timepoints. immunohistochemistry with other markers and analysis of 6 weeks post transplantation animals, are in progress. our preliminary results show that although mips-npcs have very similar immature phenotype of mnpcs in vitro, they may have different survival capacities in vivo. future studies will reveal whether transplanted cells which showed short-term survival after grafting are capable of differentiation into myelin-forming cells. . however these putative stem cells have only been partly characterized in vitro and their therapeutic potential for cns repair has not been addressed. we cultured adult mouse drg cells in sphere-forming conditions. in parallel, we derived spheres from adult spinal cord of the same animals and compared their properties. this last tissue contains well-described stem/progenitor cells whose repair capacities have already been proven for spinal cord injury but not for primary demyelination. in vitro, regardless of their origins, all tissues generated self-renewable spheres up to 10 passages. rt-pcr and immunocytochemistry revealed that sphere-forming cells actively proliferate and express neural stem cells markers. drg spheres were multipotent: as they differentiated into schwann cells, neurons and myofibroblasts, whereas spinal cord spheres differentiated into astrocytes, neurons and oligodendrocytes. we then compared the integration, differentiation potential and remyelination capacity of primary sphereforming cells isolated from actin-gfp transgenic mice after their engraftment in two distinct environments: the adult nude lpc-demyelinated spinal cord and the developing newborn shiverer forebrain. both cell types survived but with distinct integration and differentiation patterns. in the mbp deficient mutant, spinal cord cells exhibit a substantial tropism toward white matter tracts and differentiated mainly into myelinating oligodendrocytes while drg cells were largely distributed in the parenchyma in close association with blood vessels and differentiated in vascular smooth muscle cells. surprisingly, after focal demyelination, in the adult spinal cord, drg cells migrated extensively towards the lesion and differentiated into remyelinating schwann cells whereas adult spinal cord cells migrated less efficiently, and gave rise to oligodendrocytes and a large population of astrocytes. our study reveals that both cell types present different but promising potential for cns remyelination. funding by ela foudation, dim nerf, arsep foundation. ischemia-induced progenitor cell proliferation is a prominent example of the adult mammalian brain's ability to regenerate injured tissue resulting from pathophysiological processes. a key locus of regeneration is the neurogenic niche located in the subgranular zone of the dentate gyrus (sgz). within the sgz, radial glia-like progenitor cells generate neural precursor cells which migrate into the granule cell layer (gcl) of the dentate gyrus, mature, and integrate into the hippocampal synaptic network. in order to better understand and ultimately exploit the cell signaling mechanisms that regulate ischemia-induced proliferation in the sgz, we tested the role of the p42/44 mitogen-activated protein kinase (mapk) cascade effector ribosomal s6 kinase (rsk) in this process. using the endothelin-1 ischemia model in c57bl/6 mice, we report that intrahippocampal cerebral ischemia triggered rsk activation in sgz progenitor cells. further, microinjection of a rsk inhibitor significantly reduced ischemia-induced sgz progenitor cell proliferation. using the neurosphere assay, we also show that sgz-and subventricular zone (svz)-derived adult neural stem cells (nsc) exhibit a significant reduction in proliferation in the presence of rsk and mapk inhibitors. taken together, these data indicate that rsk functions as a cell-autonomous regulator of ischemia-induced progenitor cell proliferation. here we demonstrated that the inpcs not only possess npc-specific marker genes, but also have qualitites of primary brain-derived npcs (wt-npcs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. moreover, the mature neurons derived from inpcs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. importantly, besides survival, the transplanted inpcs exhibit the migratory property responding to inflammatory stimulation in vivo. these results suggest that directly reprogrammed inpcs closely resemble wt-npcs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (incs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders. the pathogenesis of neurodegenerative disorders, including human immunodeficiency virus (hiv)21 associated neurocognitive disorders (hand), is exacerbated by an imbalance between metalloproteinases (mmps) and their inhibitors, tissue inhibitors of metalloproteinases (timps). as the timps exhibit diverse non-classical functions including anti-apoptotic effects, the induction of timp-1 in neuroinflammatory conditions likely serves multiple roles in addition to modulating mmp activity. our work demonstrates differential timp-1 expression in acute versus chronic activation of astrocytes and hiv-1 associated dementia (had) brain tissues. the timp-1 promoter harbors five consensus ccaat boxes. ccaat enhancer binding protein (c/ebp)b levels were elevated in brain specimens from hiv-1 patients and this transcription factor regulated astrocyte timp-1 expression. hiv-relevant stimuli increased c/ebpb expression in human astrocytes and localized the factor to the nucleus. overexpressing c/ebpb in astrocytes increased timp-1 promoter activity, mrna and protein expression, while knockdown of c/ebpb decreased timp-1 mrna and protein expression. erk1/2 activation is critical for il-1b-mediated astrocyte timp-1 expression and p38k activation contributes to il-1b-induced astrocyte timp-1 and c/ebpb expression. these data suggest that erk1/2 signals downstream of c/ ebpb to facilitate il-1b-induced astrocyte timp-1 expression. the role of astrocyte-timp-1 as a neurotrophic factor was examined. interestingly, cotreatment with timp-1 protected neurons from apoptosis and reversed neuronal morphological changes induced by staurosporine (sts) and hiv-1 ada virus. further, the anti-apoptotic effect was specific to timp-1 and partially independent of mmp-inhibition. additionally, timp-1 modulated the bcl-2 family of proteins and inhibited opening of mitochondrial permeability transition pores induced by hiv-1 or sts. together, these findings describe a novel function, regulatory mechanism and direct role of astrocyte-timp-1 in neuroprotection suggesting its therapeutic potential in had and possibly in other neurodegenerative diseases. mounting evidence and our pervious study has also confirmed that the axon outgrowth inhibition receptor ngr was also expressed on microglia, and regulated cell adhesion and migration behavior in vitro. however, microglia has been proposed to play a pivotal role in the neuroinflammation through expressing a range of proinflammatory enzymes and proinflammatory cytokines under pathological stimulus. these findings initiate the possibility of nogo/ngr signal on mediating neural inflammation processes during cns injury beside neurite outgrowth inhibition. in the present study, we found that nogo-p4, a 25-aa core inhibitory peptide sequence of nogo-66, was able to induce microglia expressing cox-2, inos, as well as releasing proinflammatory cytokines including il-1b, no and pge2, which could been reversed by nep1-40 or pi-plc treatment. microglia stimulated with nogo-p4 increased the phosphorylation lever of signal transducer and activator of transcription 3 (stat3). the activation of stat3 further mediated the promotion of nogo-p4 on microglia expression proinflammatory factors. furthermore, administration nep1-40 was capable to attenuate the inflammation response in a mouse spinal cord compression injury model by reducing the expression of several proinflammatory mediators, as well as attenuating the activation of stat3. taken together, our results demonstrated, for the first time, that nogo-66 may directly take part in cns inflammation process by influencing microglia expressing proinflammatory factors, which was related to ngr /stat3 signal pathway. these results imply that the interaction of nogo-66 with ngr expressed on microglia may participate in diverse cns diseases related to neural inflammation, including trauma, stroke, and neurodegeneration diseases, etc. our results indicate that ccl-1 is one of the key molecules in pathogenesis and ccl-1/ccr-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain. demyelination plays a central role in the pathophysiology of multiple sclerosis (ms) not only because it disrupts nerve conduction, but also because of effects that compromise axonal survival. there is a growing consensus that any effective treatment for ms must include a component that restores or enhances remyelination by endogenous oligodendrocyte progenitor cells (opc). however achieving this goal requires a far better understanding of why remyelination fails in the first place. we now report that fgf9 is selectively up regulated in demyelinating ms lesions and acts to inhibit (re)myelination in vitro. to explore the mechanism involved we compared the activity of fgf9 to that of fgf2, another member of the fgf family with well characterised effects on opc differentiation and myelination. fgf2 acts directly on opc to stimulate proliferation, while at the same restricting their differentiation into mature oligodendrocytes. in contrast, fgf9 is neither an opc mitogen, nor is it able to inhibit opc differentiation directly. however, opc differentiation and myelination in myelinating cultures were inhibited by supernatants harvested from fgf9 treated astrocytes, even in the presence of an excess of fgf9 neutralising antibody. microarray studies were performed to identify astrocyte derived factors responsible for this inhibitory effect. this approach identified several potential candidates that were significantly up regulated by fgf9 in astrocytes. these included lif, a member of the gp130/il6 cytokine family that is generally described as having pro-myelinating effects, although our own studies indicate when present in excess relative to other gp130/il6 family members it will inhibit myelination in vitro. our experiments demonstrate that fgf9-mediated signal transduction in astrocytes disrupts their ability to maintain a growth factor milieu that can support myelination. been shown that inflammation is primarily mediated by macrophages that are recruited to the tissue. similarly, a recent study demonstrated reactive microgliosis in the hypothalamus of mice fed a high-fat diet. in the cns, however, the relevance of microglial activation in response to a high fat diet remains unclear. we aim to determine if microglia activation occurring in response to overnutrition is a cause or consequence of dysregulation of energy homeostasis and obesity. to determine if the absence of microglia (and therefore microglia-mediated inflammation) may influence the metabolic response to a high fat diet, we used a transgenic mouse model, the cd11b-hsvtk mouse (tk), which allows for an inducible, specific ablation of microglia in response to treatment with the nucleoside analog, gancilovir (gcv). mice were treated with gcv for 4 weeks during which time they were maintained on a diet high in fat (60%) or a respective control diet. body weight, food intake, locomotor activity and energy expenditure were measured and compared to wildtype mice. analyses revealed alterations in the metabolic phenotype of mice fed both hfd and control diet in the absence of microglia cells. our findings point towards a physiological role of microglia in energy homeostasis, the study of which will be the focus of further experiments. interleukin-10 (il-10), a cytokine classically related with anti-inflammatory and protective functions in the central nervous system (cns), has been reported to be produced by astrocytes and microglia in different neurodegenerative and neuroinflammatory conditions. in order to study the specific role of il-10 in the cns, we have created a new transgenic mouse with astrocyte-targeted production of il-10 (gfap-il10tg). the aim of this study was to evaluate if production of this cytokine in the cns has any effect on the phenotype of glial cells. for this purpose, coronal cryostat free-floating sections from the brain of both adult transgenic mice and their corresponding wild-type (wt) littermates, were processed for the study of astrocytes using gfap immunohistochemistry and microglia using antibodies against iba1 and several markers commonly related to the activated phenotype of these microglial cells, such as cd16/32 (fc receptor), f4/80, cd11b, cd206, cd150 and mhc-ii. our results showed that astrocyte-targeted production of il-10 induces an increase in the area covered by gfap immunolabelling. microglial cells in these gfap-il10tg animals displayed also morphological changes in specific areas of the brain, including the hippocampus, cortex and cerebellum, characterized by coarser processes and a notable enlargement of the cell body. distinctively, in the hippocampus, microglial cells adopted an elongated morphology parallel to the dendrites of pyramidal neurons. in addition, in the aforementioned areas, microglia exhibited a statistically significant increase in iba1, cd16/32, f4/80 and cd11b markers and "de novo" expression of cd150, whereas no detectable levels of either cd206 or mhc-ii was found. in conclusion, these results indicate that in specific areas, glial cells are influenced by the presence of astrocyte-targeted il-10. further studies are necessary to evaluate whether after injury these transgenic animals will present changes in the microglial and astrocyte activation that can influence the evolution of the lesion. networks. brain-resident macrophages, microglial cells were considered ''resting,'' but it has recently become evident that they actively sense neuronal microenvironment with their motile and ramified processes. they develop specific activities (cytotoxic, neuroprotective or phagocytic) depending on brain molecular context. for example, they contribute directly to the development of neuronal networks by eliminating or maintaining synapses in an activity-dependent manner. morphofunctional plasticity of microglial cells is finely tuned in order to keep brain homeostasis and to ensure such developmental activity. like macrophages at the periphery, microglial cells indeed adopt m1 or m2 phenotypes, distinguishable through their pattern of cytokine expression and of specific cell surface markers, a m1 phenotype being more pro-inflammatory while a m2 phenotype is more related to phagocytosis. linoleic acid (la, 12:2n-6) and alpha-linolenic acid (ala, 18:3n-3) are essential fatty acids that cannot be synthesized de novo by mammals and have to be provided through the diet. they are precursors of arachidonic acid (aa; 20:4n-6) and docosahexaenoic acid (dha; 22:6n-3) respectively which are key structural components of brain cell membrane phospholipids and precursors of bioactive lipid messengers involved in the regulation of inflammation and microglial activity. most aa and dha accumulate in the brain during the perinatal period via placenta and milk. we previously demonstrated that depleting the diet in n-3 pufas from the first day of gestation alters some neuronal functions of the offspring at adulthood. we here hypothesized that this is due to an alteration of microglial mrophofunctional activity in the developing brain. to test this hypothesis, mice were submitted to a diet deficient or not in ala throughout gestation and lactation. microglial morphology, phenotypes and functions were determined in the brain of pups at post-natal day 21 (pnd21). we show that dha levels is decreased in membrane phospholipids of microglia. in addition, microglial cells from mice fed with an ala deficient diet present a drastic increase of the cd36 marker expression combined to a dysregulated cytokine/chemokine expression profile and an altered phagocytic activity. all together, our results show that dietary n-3 pufas deficiency is likely to impair brain innate immune system activity at pnd21. the outcome of such microglial impairment on brain function is discussed. in recent years, one of the most promising therapeutic means for ad was thought to be immunization with specific antibodies against ab. several therapeutic antibodies were developed, however, most of the clinical trials were canceled due to unexpected deaths and neuroinflammatory responses in some patients. the causes and mechanisms of such responses are currently only partially understood. we have recently raised monoclonal antibodies against oligomeric forms of ab and were investigating whether these antibodies would prevent the neurotoxicity of ab oligomers in primary neuronal-glial cerebellar granule cell (cgc) cultures. antibodies were not directly toxic to cgcs when applied at concentrations relevant to concentrations reported to be in blood serum. however, surprisingly, the antibodies when in complexes with ab oligomers or firbrils dramatically increased the neurotoxicity of ab. similar effects were also observed with antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein vp1, human metapneumovirus nucleocapsid protein and measles virus nucleocapsid protein strongly potentiated the neurotoxicity of their antigens. the neurotoxicity of antibody-oligomeric antigen complexes was abolished by removal of the fc region from the monoclonal antibodies or by the removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. in conclusion, we find that immune complexes formed by ab oligomers or other oligomeric antigens and their specific monoclonal antibodies can cause neuronal death in primary neuronal-glial cultures via fc-dependent microglial activation. the results suggest that therapies resulting in antibodies to oligomeric ab or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation. additionally, if endogenous antibodies contribute to neuronal loss in ad or viral encephalitis, suppression of microglial activation may be therapeutic. antidepressants have often been recommended for potential treatment of chronic pain, especially for neuropathic pain caused by nerve damage. it is known that nerve injury-induced activation of microglia and astroglia can be a cause for the development of neuropathic pain. recent data indicate that abnormal neuronal activity mediated by glia activation may influence the level of neuroimmune signaling molecules in chronic pain. however, it remains unclear whether the mechanism underlying antidepressant effects is related to glia activation. this study investigated the potential pain-relieving properties of milnacipran and venlafaxine, selective serotonin/noradrenaline reuptake inhibitors (snri), in neuropathic pain and attempted to resolve how these antidepressants affect the factors synthesized by the activated glia in neuropathy. chronic constriction injury (cci) was performed in wistar rats by loose ligation of the sciatic nerve. after a single intraperitoneal (i.p.) injection of antidepressants, the responses to mechanical and thermal stimuli in neuropathic rats were evaluated 7 days after cci by the von frey test and the cold plate test, respectively. to confirm the influence of antidepressants on microglia and astroglia, western blot analysis of iba-1 (microglia marker) and gfap (astroglia marker) in the spinal cord and dorsal root ganglions (drg) was performed. the experiments were carried out according to the iasp and local bioethics committee rules. our findings confirmed that the administration of milnacipran (20 mg/kg) and venlafaxine (10 mg/kg) elicited antiallodynic and antihyperalgesic effects in cci-exposed rats. western blot analysis showed that milnacipran elevated the level of iba-1 protein in the spinal cord (but not in drg) in cci-exposed rats. venlafaxine decreased the cci-induced iba-1 protein level in the spinal cord and in drg. we observed no changes in gfap in the spinal cord and drg after milnacipran and venlafaxine administration. these results provide a substantial evidence that both used antidepressants belonging to snri attenuated allodynia and hyperalgesia in an animal model of neuropathic pain, although their effect on microglial cells and astrocytes was different, namely, milnacipran increased the microglia activation, in contrast to venlafaxine. this effect can be explained by comparing the effects of these drugs during their repeated application, and relevant experiments are currently in progress. myeloid cells can respond to intra-and extracellular danger/stress signals by inflammasome-mediated activation. this process consists of two steps: toll-like receptor-induced production of pro-il-1b followed by inflammasome-mediated cleavage and secretion of bioactive il-1b. inflammasome-mediated activation is strictly regulated by expression of regulatory proteins and by inhibitory processes like autophagy. recent studies describe that microglia are markedly different from other myeloid cells. they originate from a separate progenitor, are chronically exposed to the neural microenvironment and their activation may be regulated differently. the objective of this study was to determine the expression profile of inflammasome components in primary microglia and to compare inflammasome-mediated microglial activation and its regulation with other myeloid cells. primary microglia, bone marrow-and blood-derived macrophages (bmdms) of adult rhesus macaques of the same donor were profiled for all nod-like receptors (nlrs), inflammasome-associated adaptor proteins, caspases, and regulatory proteins by qpcr. inflammasome function and kinetics were assessed by exposing lps-primed microglia to atp, silica or msu (danger-associated molecular patterns: damps) followed by measuring the transcription of pro-il-1b-encoding mrna and the secretion of il-1b. primary microglia expressed nlrs (nalp1-3, nod1/3/4, aim2, ipaf, naip), adaptor proteins (asc), caspases (1/3-5/7/8), and regulatory proteins (a20), and the expression profile closely resembled that of bmdms. priming of microglia with lps induced high levels of pro-il-1b-encoding mrna, but il-1b protein was only secreted in response to subsequent stimulation with various damps. interestingly, in lpsprimed bmdms the window for inflammasome-mediated activation was 4-8 hours, while lps-primed microglia remained sensitive for inflammasome-mediated activation for at least 20 hours. in conclusion, primary microglia express multiple inflammasome components closely resembling the expression profile of bmdms and they can be induced to form functional inflammasomes. importantly, primed microglia remain sensitive to inflammasome-mediated activation for much longer than other macrophages. we will present data on whether this reflects a difference in negative regulation of the inflammasome, or differences in autophagy or apoptosis sensitivity. objective: the susceptibility of the aging brain to neurodegenerative diseases may in part be attributed to the intrinsic senescence of the microglia. cellular aging or senescence is linked to telomere shortening/dysfunction. evidence is accumulating that aging microglia show morphological changes, referred to as microglial dystrophy, which may be accompanied by accumulation of the iron-binding protein, ferritin. here, we investigated the effect of telomere shortening on microglial morphology, numbers, and activation state in telomerase deficient (terc -/ -) mice, a model of premature aging due to shortened telomeres. methods: male terc 1/1 mice and 3 rd generation terc -/mice, which show a clear aging phenotype, were perfusion-fixed with 4% paraformaldehyde. brains were processed into 60 lm thick horizontal sections. every fourth section was used for stereological estimation of cd11b 1 microglia in the molecular layer of the dentate gyrus by the optical fractionator method. sections were also stained for the microglial markers iba-1 to monitor microglial morphology, cd45 and cd68 to study microglial activation, and ferritin to study microglial aging. further sections were stained for the apoptosis marker cleaved poly (adp-ribose) polymerase (cparp). results: unlike resting microglia in littermate terc 1/1 mice, which display thin, finely branched processes, microglia in terc -/mice exhibited partial retraction and hypertrophy of processes. microglial cd45 and cd68 immunoreactivity were similar in terc 1/1 and terc -/mice. absolute numbers of mac-1 1 microglia were significantly reduced in young and old terc -/versus terc 1/1 mice. an increased microglial density in old terc -/versus terc 1/1 mice could be attributed to a volume reduction of the dentate gyrus. we also observed an increased number of cparp 1 cells in old terc -/versus terc 1/1 mice. this increase was most prominent in the sub-granular zone, an active site of neurogenesis, but also occurred in the molecular layer of the dentate gyrus and might reflect microglial apoptosis. we observed no difference in ferritin staining in terc -/versus terc 1/1 mice. conclusion: our findings suggest that telomere shortening impairs microglial capacity for self-renewal. surprisingly, changes in microglial morphology in terc -/mice occurred without differences in cd45 and cd68 expression. ongoing studies will show if increased numbers of microglia in terc -/mice co-express cparp or ferritin as evidence of increased microglial aging. tetrahydrocannabivarin (d9thcv) and cannabidivarin (cbdv). we have developed a series of new cannabinoid quinones, among them the cbg quinone (vce-003) that shows pparg and cb2 receptors agonism. in addition we have found that vce-003 activates the nrf2/are pathway in neuronal cell lines. in the present study, we investigated the therapeutic potential of vce-003 in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis (ms) by immunization con mog 33-55 . vce-003 (5mg/kg ip, daily) was administered to susceptible c57/bl6 mice at the onset of symptomatology. clinical score and weights of mice were daily recorded until the day of sacrifice (28 days post-immunization). vce-003 treatment delayed the onset of disease and ameliorated the symptomatology. histological analysis of spinal cord of eae mice treated with vce-003 showed decreased microglia reactivity and reduced cellular infiltrates, in particular cd4 1 t lymphocytes. double labeling with neurofilament and the myelin protein, rip indicated that vce-003 diminished the axonal damage. demyelination was evaluated by luxol fast blue labeling. changes in the expression of several cytokines, adhesion molecules and nrf2-dependent genes were determined by qrt-pcr. the implication of pparg and cb2 receptors in the beneficial effects of vce-003 in the eae model of ms is being investigated by using specific receptors antagonists. taken together our results support the potential of vce-003 for the treatment of ms and other chronic inflammatory diseases. we found that the lipid molecule lactosylceramide (laccer), is upregulated in ms patients. to better understand the role of laccer in ms, we used an animal model of spms, and found that laccer synthase (b4galt6) is upregulated during disease progression by astrocytes, and that its inhibition halts the progressive phase of the disease, attenuating cytokine and chemokine production by the astrocytes, and reducing the recruitment of inflammatory ly6c high monocytes to the cns. moreover, it also skewed the microglia and monocytes towards a m2 phenotype, and shifted the astrocyte phenotype to support re-myelination and axonal growth. in vitro experiments, using primary cells demonstrated that the inhibition of laccer synthesis directly inhibits cytokines and chemokines production in astrocytes. importantly, similar results were also obtained with human astrocytes, reinforcing the biological importance of our findings. in summary, we identified a novel lipid-signaling pathway that promotes the activation of local cns immunity and neurodegeneration in experimental spms and is a potential therapeutic target for spms. converging clinical studies report an increased prevalence of comorbid neuropsychiatric symptoms, particularly major depressive disorders, in a number of conditions (e.g., aging, obesity) and diseases (e.g., atherosclerosis, congestive heart failure, rheumatoid arthritis) sharing inflammation as a common denominator. by using an experimental approach in mice exposed to innate immune system (iss) stimulation, we demonstrated that induction of depressive-like behavior is mediated by cytokine-induced brain activation of a tryptophan-catabolizing enzyme, the indoleamine 2,3-dioxygenase (ido). in the metabolic syndrome (mets), a widely accepted concept that identifies a cluster of individual risk factors for type 2 diabetes and cardiovascular disease (including obesity, metabolic dysregulations and basal low-grade inflammation), neuropsychiatric symptoms emerge as significant factors for aggravation of the disease and related outcomes. recently, we demonstrated in a model of mets, the diabetic and obese db/db mice that cognitive alterations and increased anxiety-like behavior are related to hippocampal inflammation. on the other hand, depressive-like behavior is not affected by basal inflammation displayed by db/db mice. given the data linking increased depressive-like behavior and ido activation by cytokines in conditions of iss stimulation, the question arises as to whether depressive-like behavior is increased in those conditions in db/db mice. to answer this question, we measured in db/db mice and in their healthy db/1 littermates the effect of a lipopolysaccharide (lps) challenge (5 mg/mouse, ip) on behavioral reactivity in a depression test (the forced swim test, fst), plasma levels and hippocampus expression of inflammatory cytokines and related neuronal targets, and brain ido activity. plasma levels and hippocampus expression of il-1b, il-6, tnfa and il-10 are similarly increased 2h after lps in both db/1 and db/db mice. as expected, brain ido activity and duration of immobility in the fst are increased in lps-treated db/1 mice 24h after lps. on the contrary, induction of brain ido activity is significantly blunted in db/ db mice compared to their db/1 counterparts and no increase of depressive-like behavior is observed. moreover, some data suggest an impairment of the neuroimmune interactions in db/db mice. we need now to understand how obesity and related neurobiological alterations impair ido activation by cytokines and their consequences in terms of vulnerability to infections in mets. in this study, we tested it in a collagenase-induced mouse intracerebral hemorrhage (ich) model using tlr2 knock-out (ko) mice. to induce ich, collagenase or blood was injected into the right caudate putamen in 8-10 week old male mice. tlr2 expression was upregulated in the ipsilateral hemorrhagic tissues of the collagenase-injected mice. brain injury volume and neurological deficits following ich were reduced in the tlr2 ko mice as compared to the wild type (wt) mice. heterologous blood-transfer experiments show that tlr2 signaling in the brain-resident cells, but not leukocytes, contributes to the injury. in our study to elucidate underlying mechanisms, we found that damage in the blood-brain barrier (bbb) integrity following the ich was attenuated in the tlr2 ko mice compared to the wt mice, which may be due to reduced mmp-9 activation in brain astrocyte. the reduced bbb damages accompanies with reduced neutrophil infiltration and proinflammatory gene expression the injured brain parenchyma, which may account for the attenuated brain damage in the tlr2 ko mice after ich. conclusively, these data demonstrate that tlr2 contributes to brain injury following ich by compromising bbb through activating mmp-9 in brain. the pathological relevance of this autoantibody response is unknown, but it is generally assumed that it exacerbates demyelination via activation of complement and/or cell meditated effector mechanisms. however in a majority of cases the mog-specific autoantibody titre is far lower than that required to induce widespread demyelination and exacerbate disease severity in animal models of ms. to explore the possibility mog-specific antibodies may play other more subtle roles in disease pathogenesis we investigated possible effects in myelinating cultures derived from embryonic rat spinal cord; a model system that allows us to explore antibody-dependent effects in the absence of exogenous complement and effector cells. myelinating cultures were treated continuously with monoclonal antibodies specific for either mog (clone z2, igg2a), sulphatide (clone o4, igm), proteolipid protein (plp) (clone o10, igm), or an appropriate isotype control from 18 days in vitro onwards. in the absence of an exogenous source of complement none of these antibodies induced demyelination, but by 24 div had all had a significant inhibitory effect on myelination compared to cultures treated with appropriate isotype control antibodies. to investigate possible mechanisms contributing to this inhibitory effect qpcr arrays were used to determine if this complement-independent effect on myelination was associated changes in expression of immune mediators. unexpectedly we found recognition of antigens exposed at the surface of the myelin sheath induced a rapid increase in expression of three chemokines known to be involved in recruitment of effector t cells into the cns. within 24 hours of adding antibody to the cultures expression of ccl5, ccl20 and cxcl11 increased by at least three orders of magnitude and then declined to baseline over the following five days despite continuous presence of antibody. this transient increase in mrna transcripts for these cytokines resulted in sustained protein synthesis and secretion of biological active products as demonstrated by analysis of culture supernatants. these results challenge the traditional view that myelin-specific autoantibodies contribute to the pathogenesis of diseases such as ms and adem by virtue of their ability to initiate immune-mediated demyelination. we now demonstrate that even in the absence of recruited immune effector mechanisms myelin-specific autoantibodies not only inhibit myelination but trigger secretion of chemokines predicted to trigger or exacerbate inflammation within the cns. the blood-brain barrier (bbb), comprising specialized brain endothelial cells, is crucial in maintaining a controlled environment within the central nervous system (cns) to safeguard normal neuronal function. in multiple sclerosis (ms) the function of the bbb is disturbed, leading to uncontrolled influx of metabolites and immune cells and neuroinflammation. therefore, restoring the function of the bbb may be a novel therapeutic tool to halt disease progression or new lesion formation. we previously showed the importance of the vitamin a metabolite retinoic acid (ra) in bbb function during cns development 1 , where fetal astrocyte-derived ra is needed to induce bbb function during cns embryogenesis. considering the possible involvement of developmental pathways that could regenerate the disrupted bbb, we investigated the possible role for ra in the protection or reinstatement of disrupted bbb function. we show that ra counteracts the deleterious effects of inflammatory cytokines such as tumour necrosis factor (tnf)-a and interferon (ifn)c on bbb function in vitro. moreover, ra diminishes the induction of inflammation-related genes in human brain endothelial cells, and induces general immune quiescence in resting brain endothelium. our preliminary data as well as a recently described study in the cuprizone animal model for demyelination suggest that ra synthesis in the cns is increased under inflammatory conditions. the impact of cns-derived ra on the inflamed bbb, as well as the cellular source is currently under investigation. insight into the ra-synthetic pathway and its protective effect on the bbb may lead to the development of novel therapies which are aimed to restore bbb function and reduce the inflammatory cascade in ms lesions. tissue transglutaminase (tg2) is a multifunctional enzyme whose expression and activity is enhanced during inflammatory processes. we previously observed that the expression of tg2 is increased in monocytes in lesions in post-mortem material of ms patients. moreover, tg2 activity and expression is enhanced in chronic-relapsing experimental autoimmune encephalomyelitis (cr-eae) in rats. in this same ms model, pharmacological inhibition of tg2 activity dramatically reduced clinical symptoms and attenuated the influx of monocytes. in the present study we question whether tg2 plays a role in mouse eae, as transgenic mice have to be used for subsequent in vivo imaging of leukocytes/monocytes during eae. methods and results: eae was induced in tg2 -/mice and littermate wildtype mice (c57bl/6) using mog 35-55 . during the early phase of disease (day 14-15), the clinical symptoms observed were significantly less severe in tg2 -/compared to the wildtype mice. moreover, the maximal clinical score was significantly lower in the knockout mice. there was no difference in the day of onset of disease symptoms between the two groups. secondly, we aimed at visualizing leukocytes/monocytes in the spinal cord of lysm-gfp and cd11c-yfp mice suffering from mog 35-55induced eae using intravital video microscopy. a permanent window was fixed on top of the spinal cord of mice to allow reimaging of the same field of view in the same mouse over the disease course. in a pilot experiment we observed numerous gfp positive cells in the white matter around the imaged blood vessel in the spinal cord of a lysm-gfp eae mouse. although equally present, this was less prominent in a cd11c-yfp mouse suffering from eae. subsequent immunohistochemical analysis revealed that various cell types had infiltrated the spinal of cord of both mice. outlook: to further address the role of tg2 in the infiltration of leukocytes/monocytes into the spinal cord of eae mice in vivo, specific inhibitors for tg2 activity will be administered to the transgenic mice and leukocytes/monocytes will be visualized using intravital video microscopy. z. muneer 1 , c. wiesinger 1 , g. regelsberger 2 , j. berger 1 , s. forss-petter 1 1 medical universty of vienna, center for brain research, vienna, austria 2 medical university of vienna, institute of neurology, vienna, austria x-linked adrenoleukodystrophy (x-ald) is the most frequently occurring peroxisomal neurodegenerative disorder and is often associated with cerebral demyelination and inflammation. x-ald is caused by mutations in the atp-binding cassette sub-family d member 1 (abcd1) gene encoding adrenoleukodystrophy protein (aldp), which is a transporter for coa esters of very long-chain fatty acids (vlcfa) across the peroxisomal membrane. adrenoleukodystrophy related protein (aldrp), another member of the abcd sub-family, encoded by abcd2, is the closest homologue of abcd1. upon over-expression, abcd2 has been shown to compensate for abcd1 deficiency in vitro and in vivo. several lines of evidence imply an important role of microglia/macrophages in the disease progression. here we used mouse peritoneal macrophages (mpmu) to correlate the gene expression levels of candidate modifiers with x-ald associated defects, like accumulation of vlcfa and decreased peroxisomal b-oxidation, in abcd1 and abcd2 single ko mutants as well as in abcd1/abcd2 double deficient (doko) mice. by quantitative rt-pcr analysis, in mpmu from wild type mice the abcd2 mrna was present at about half the level of the abcd1 mrna. abcd1 ko mpmu showed elevated accumulation of vlcfa (c26:0) compared to the mpmu from wild type mice as measured by gc-ms. the vlcfa levels of abcd2 ko mpmu were similar to wild type amounts. interestingly, there was much higher accumulation of vlcfa in the mpmu from doko mice. there were no differences in the mrna expression level of the elovl1 gene, encoding the enzyme catalyzing the elongation step of vlcfa biosynthesis. peroxisomal boxidation activity was decreased in abcd1 ko mpmu compared to wild type level. this defect was strongly enhanced in mpmu from abcd1/ abcd2 doko mice. these results show that the increased accumulation of vlcfa in mpmu from doko mice as compared to abcd1 ko mice was due to a more severe defect in peroxisomal b-oxidation in doko mpmu. we conclude that a substantial expression of abcd2 mrna prevents a more severe metabolic phenotype in abcd1 deficient mouse peritoneal macrophages. this study also supports the validity of abcd2 up-regulation as a potential therapeutic target in x-ald. j. claude, b. linnartz-gerlach, h. neumann reconstructive neurobiology, bonn, germany microglia have innate immune receptors recognizing pathogens and disease-associated molecular patterns but also molecules that could sense the intact tissue. a subfamily of these receptors is the inhibitory signaling sialic acid-binding immunoglobulin-like lectin (siglec) group including siglec-e that has an immunoreceptor tyrosine-based inhibitory motif (itim) in the cytoplasmic tail to suppress activatory microglial signals. in this study, we used primary and stem cell-derived microglia that were modified by lentiviral vectors. here we show that siglec-e is expressed on microglia and is up-regulated following interferon-c (ifnc) treatment. we performed lentiviral knock-down and overexpression of siglec-e. lentiviral overexpression of siglec-e decreased, while knock-down increased the phagocytosis of neural debris and its associated reactive oxygen burst. the extracellular domain of siglec-e linked to a fc-part of immunoglobulin bound to the sialic acid residues of the neuronal glycocalyx. therefore, we co-cultured these modified microglia with primary hippocampal neurons. overexpression and knock-down of siglec-e showed an increase and decrease in relative neurite-length, respectively. the neuroprotective effect of siglec-e was abrogated after removal of the sialic acid residues on the neuronal glycocalyx. treatment with the anti-oxidant trolox abolished the neurotoxic effect of the siglec-e knock-down on neurite length. in summary, our data suggests an immunomodulatory function of siglec-e on microglia which leads to a neuroprotective phenotype by decreasing the production of reactive oxygen species and a reduced phagocytosis rate of neural debris. a. nadjar, c. madore, a. sere, a. aubert, s. lay e university of bordeaux 2, bordeaux, france many epidemiological studies have linked maternal exposure to infections during pregnancy to later development of cognitive disorders in the descendants. these alterations are likely to originate from a generalized neuroinflammation in the fetal brain following activation of the maternal immune system. this neuroinflammatory response relies on microglial cells activation. these latter normally contribute to the development of neuronal networks especially by eliminating/maintaining synapses, a phenomenon also known as synaptic stripping, in optimal conditions of brain development. neonatal inflammation may alter the synaptic stripping capacity of microglial cells. thus, targeting this persistent inflammatory microglial activation could represent an original and promising strategy to improve cognitive performances in the descendants. omega-3 are known as immunomodulators and target microglial cells. we thus hypothesized that enriching the diet with omega-3 from the first day of gestation may impair the development of neurological deficits at adulthood, through limitation of microglial inflammatory response in favor of its synaptic stripping activity. to characterize the beneficial effects of omega-3 on microglial activity, pregnant mice were fed with an omega-3-deficient diet, or a balanced omega-3/ omega-6 diet or supplemented with omega-3 and received a peripheral injection of either lps or saline at e18 of gestation. using the golgi method, we first evaluated the morphology of dendrites and quantified the dendritic spines density as a measure of neuronal networks maturation in the hippocampus. we found that a prenatal exposure to lps increased the number of immature spines and this was reversed by an omega-3 supplemented diet. to correlate these data with alterations of microglia-neurons interactions, we took advantage of the cx3cr1-gfp mice and injected them with a neuronspecific lentivirus containing dsred protein, in the hippocampus, 7 days prior to two-photon experiments. we focused on microglia general dynamic and microglia-neuron physical interactions and found an alteration in microglial behavior and microglia-neurons interactions in pups from lps-treated females. these alterations were reversed by an omega-3 supplemented diet. overall, our data show that alterations of microglia-neurons interactions in the developing brain may explain the neurological disorders observed in the descendants of females with prenatal inflammation. these deleterious effects may be prevented by nutritional strategies. , which is characterized by a relapsing phase with inflammatory cell infiltrates and a remitting period, where patients partially recover. among the different cell types involved in the necessary immunomodulation to allow the relapsing-to-remitting transition, the role of myeloid-derived suppressor cells (mdscs) gains importance. mdscs form a heterogenic population of immature myeloid cells that is able to suppress the inflammatory response. this cells act, among other mechanisms, through arginase-i (arg-i) activity on tcells. a previous study of our group showed that arg-i 1 -mdscs transiently enter the spinal cord of eae mice and takes part in the immune response control by inducing t cell apoptosis around the peak of the clinical score. therefore, changes in the mdsc population during eae should modify the evolution of the disease. the retinoid acid family molecules are used for the treatment of different leukaemia due to their role as mdsc differentiation factors into diverse cell populations, abolishing t cell immunosuppression. am80, a synthetic analogue of the retinoic acid with a higher bioavailability and less side effects than the natural ones, has controversial actions on eae depending on its administration period. in this work, we administered am80 specifically in the critical moment for immune modulation (around the maximum clinical score). drug administration affected mdsc population and clearly worsened the eae course. our results point to endogenous strengthening of mdsc population as a new and promising therapeutic strategy to treat ms by speeding up the transition from the relapsing to the remitting period. sildenafil induces cgmp accumulation by pde5 inhibition. we have demonstrated that sildenafil decreases microgliosis and astrogliosis and proinflammatory cytokines expression in a demyelination model. however, little is known about mechanisms of sildenafil neuroprotection. since nfjb plays an important role in the regulation of glial activation, we examine the hypothesis that nfkb is part of the mechanism underlying the sildenafil neuroprotective effects. five male mice (c57bl/6), 6 weeks-old, were used per group. the groups received for four weeks: 0.2% cuprizone (cpz) mixed into the chow; cpz into the chow plus sildenafil (viagra v r , pfizer, 25 mg/kg) in the drinking water, starting concomitantly (sild-t0) or fifteen days (sild-t15) after initiation of cpz; controls received pure chow/water. cerebella were processed for western blotting and immunofluorescence (if). results showed that cpz increased gfap expression compared to control (astrocytes activation). sild-t0 (but not sild-t15) significantly decreased gfap compared to cpz group. in controls, microglia showed resting phenotype, with thin and branched processes positive to iba1/nfjbp65 (inactive fraction) double labeling. cpz treatment decreased inactive-nfjb expression, indicating that this transcription factor was activated. in agreement, ikba (nfjb inactivating protein) was also decreased. if showed increase of iba-1 expression, indicating microglia activation. sild-t0 strongly increased the nfjbp65 and its inhibitory protein, ikba, suggesting inactivation of nfjb. if showed decreasing in iba-1 labeling, suggesting inactive microglia. the delayed treatment (sild-t15) did not decrease iba-1 or increase inactive-nfjb and ikba expression. in conclusion, treatment with sildenafil concomitant with cpz exposure prevents micro-and astrogliosis in mice, possibly through ikba-nfjb signaling pathway. sildenafil may be suitable to improve neuroinflammatory/neurodegenerative diseases treatment. financial support: cnpq, capes, facepe, fapesp. mt-1&2 levels have been found to be increased in several human neurodegenerative diseases including alzheimer's disease (ad). moreover, mt112 are also upregulated in different ad mouse models, for example tg2576 mice, which show a significant up-regulation of these proteins in the vicinity of the amyloid plaques. in the present study we generated a double transgenic mouse line that develops ad-like pathology in addition to having an overexpresion of mt1, in order to determine the role of mts in different aspects of ad pathology. the results show that the overexpression of mt1 does affect the mortality rate of the tg2576 and control mice in a gender-dependent manner and partially reverses the behavioural phenotype of young (4-5 months) tg2576 mice, reducing the exploratory activity and improving the learning process; in contrast, mt1 does not cause relevant changes in deambulations and anxiety. on the other hand, the amyloid cascade and neuroinflammation is increased in the hippocampus of old (15-16 months) apptgmt mice, but the lower level of gliosis in the hippocampus of young male apptgmt mice suggests that the overexpression of mt1 is capable of reducing inflamatory response well before amyloid plaques are formed but not afterwards. further molecular and immunohistochemistry analyses are underway to give further insight into the role of mts in amyloidosis and neuroinflammation in this ad mouse line. exposure to prenatal inflammation is a risk factor for neurodevelopmental and neurobehavioural abnormalities that manifest in later life in disorders such as autism, schizophrenia and seizure development. the amygdaloid complex is composed of more than 10 nuclei with subdivisions that have different cytoarchitectonic, chemoarchitectonic, and projection characteristics and that are responsible for the processing of higher functions such as memory consolidation and emotional conditioning. it is also known that the basolateral nucleus of the amygdala is implicated in seizure propagation and initiation. seizure onset later in life may be linked to abnormal development of the amygdalaoid complex. neuropeptide y (npy) and its receptors are known to be involved in a number of important functions such as feeding behaviours, anxiety and seizure modulation. npy y1 and y2 receptors are the most abundant in the brain and are expressed at different stages of development. during foetal development different brain regions are populated by neurones and glia at different times and the interface between mother and foetus plays a crucial role in the development process. lipopolysaccharide (lps) induced maternal inflammation is a known animal model for studying the inflammatory effects on the developing foetus. in this study we are investigating the effects of an acute prenatal systemic insult using the lps model on amygdala morphology and structure. embryonic day 12 (e12) c57bl6j pregnant mice receive an intraperionteal injection of lps (50 mg/kg) or 0.9% sterile saline. embryos at e12, e16, e18 and pups at p1, p7 p14 and p40 are taken and used to study the developing hippocampus and amygdala. for each age and treatment group foetal brains, maternal brains, maternal serum and placentas are collected. incidence of preterm birth, resorption rate, litter size and weights are also assessed. immunohistochemistry, western blot and rt-pcr are then used to assess the expression of neuronal and glial markers in the amygdala and surrounding limbic structures. specifically alterations in expression, activation and distribution of npy and its receptors, microglia and astrocytes in the developing amygdala and hippocampus at different stages following lps treatment are being investigated. it is hoped that this study will further enhance our understanding of how the maternal environment influences brain development and, if disturbed at a critical time in the development of the amygdala, may predispose individuals to amygdala related disorders in later life. in vivo imaging of transgenic fluorescent mice in the cns by twophoton laser-scanning microscopy (2p-lsm) has become a powerful tool in neuroscience. for in vivo imaging of the cortex a craniotomy of the skull has to be made. in addition to anaesthesia and analgesia treatment, we occasionally apply anti-inflammatory drugs to prevent immunological activity that can obscure the images. as anti-inflammatory drug we used carprofen ((rs)22-(6-chloro-9h-carbazol-2-yl)propanoic acid), a known cyclooxygenase-2 (cox-2) inhibitor. adult cx3cr1egfp mice in which microglia are labeled by expression of the green fluorescent protein egfp were treated with a single dose of carprofen or with vehicle 12 hours before the craniotomy. in all mice we exposed the right somato-sensory cortex, and quantified the microglial response to a laser-induced micro-lesion. this micro-lesion was caused by increasing the power of the laser for 1 second in the middle of the region of interest. results -conclusions unexpectedly, we observed that carprofen reduced the microglial process motility significantly. the speed with which the processes approached the lesion dropped from 0.5 mm per minute in the untreated mice to 0.2 mm per minute in the treated mice. a molecular understanding of microglia response in inflammatory processes and how anti-inflammatory drugs modify normal microglia response will provide a strong impact in developing treatment strategies for diseases with strong inflammatory components. resting and activated (lipopolysaccharide, lps) cultured cstb-/-and control microglia were analyzed for cytokine production, chemotaxis and phagocytosis. microglia extracted directly from the mouse brains were studied for expression of mhc ii and m1-m2 polarization using flow cytometry. mouse brain cortex (p14) was analysed for m1-m2 microglial phenotypes and the presence of other immunological cells from the periphery. moreover, we have checked myeloid cell population in bone marrow and spleen of p14 animals as well as cytokines' level in blood serum. our results from cultured microglia show that secretion of proinflammatory chemokines is elevated by cstb-/-microglia. also, cstb-/microglia are chemotactically more active compared to the controls whereas their phagocytic activity is decreased. cstb-deficient microglia show decreased expression of mhc ii on the cell surface indicating reduced antigen presentation. activated cstb-/-microglia directly extracted from the brain has predominantly proinflammatory m1 phenotype. cstb-/-mice display inflammatory changes in the peripheral tissues at their early postnatal period. we have registered high concentrations of proinflammatory chemokines and cytokines in blood serum of p14 cstb-/-pups, but no difference in amount of neutrophils and lymphocytes between brains of wild-type and knockout mice. also, in bone marrow and spleen amount of granulocytes is enhanced in cstb-/mice during early postnatal period as well as level of granulocyte macrophage colony-stimulating factor in their blood serum. our results suggest a role for cystatin b in regulation of immune response and that cstb-deficiency is associated with early inflammatory processes both in the brain and the peripheral tissues. therefore, we consider that epm1 should be treated as a disorder combining neuropathological and immunological features. the contribution of glial cells in the pathophysiology of epilepsy is increasingly valued. furthermore, clinical and experimental evidence suggests a direct relationship between epileptic activity and cns inflammation, which is characterized by accumulation, activation and proliferation of microglia and astrocytes. concomitant glia-mediated mechanisms of action of several aeds have been proposed. however, their direct effects on glial cells, especially microglia, have jet been hardly investigated. we aimed to investigate the influence of commonly used aeds on the glial viability and microglial in-/ activation state in a physiological and inflammatory modified in-vitro astroglia/ microglia coculture model. methods:primary astrocytic cultures were prepared from brains of postnatal (p0-p2) wistar rats and cocultured with a physiological amount of 5% (m5), as well as 30% (m30) microglia in order to mimic inflammatory conditions. cocultures were treated for 24 hours with valproic acid (vpa), carbamazepine (cbz), phenytoin (phe) and gabapentine (gbt) with a concentration of 10, 25, 50 and 100 mg/ml. viability and proliferation was measured using the tetrazolium (mtt) assay. the microglial activation state was determined by immunocytochemical labeling using a monoclonal antibody to the ed1 marker. results:m5 and m30 cocultures showed a dose-dependent, significant reduction in glial viability after incubation with phe and cbz. furthermore, low doses of vpa led to highly significant microglial activation in m5 cocultures. however, cbz significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia in the inflammatory modified m30 cocultures. conclusion: cns inflammation is characterized by a disturbance of glial cell functions. strong microglial activation, a typical hallmark of inflammation, was induced by vpa in m5 and continued in m30 cocultures. with regard to the direct relation between cns inflammation and seizures, vpa seems to be unsuitable to reduce inflammatory condition. the reverse effect was achieved after cbz. we noticed significant microglial inactivation, after incubation of the m30 cocultures. as it has been demonstrated for levetiracetam before, we assume a beneficial therapeutic effect of anti-epileptic drugs (aed) with an antiinflammatory glial potential in epileptic patients with persistent inflammation. microglial cell activation due to homeostatic imbalances of external and/or internal etiology implies, among others, secretion of proinflammatory cytokines. the fact that microglial activation, inflammation and neurodegeneration often coexist, suggests that proinflammatory cytokines might induce and/or enhance neuronal damage. we investigated the neurotoxic impact of microglial activation in organotypic hippocampal slice cultures by exposing them to the toll-like receptor 4 ligand, lipopolysaccharide (lps), for 72 hours. microglial activation was characterized by unbiased estimation of their number (stereology) and quantification of soma and branching morphology (neurolucida v r -based cell reconstructions). moreover, proinflammatory cytokine and nitrite levels in the culture supernatant were estimated by elisa and griess. the neurotoxic impact was assessed by morphology (nissl staining, fluoro-jade v r b) and extracellular electrophysiological recordings of spontaneous and evoked neuronal activity in the hippocampal ca3 subregion. lps exposure induced microglial population expansion, morphological changes, such as process thickening and somatic enlargement, as well as substantial secretion of proinflammatory cytokines (tnfa, il6) and nitrite accumulation in the culture medium. however, these changes did not coincide with neurodegeneration and were associated with only minor effects on neuronal function, as demonstrated by the amplitude of evoked neuronal responses and short-term plasticity properties. we conclude that, in contrast to what has been observed in vivo, chronic microglial activation by lps is not sufficient to drive neuronal death and dysfunction in organotypic hippocampal slice cultures. the neonatal brain is particularly susceptible to oxidative stress. our group has previously shown that following hypoxic-ischemic injury, hydrogen peroxide (h 2 o 2 ) levels rise significantly particularly in the neonatal brain and are sustained for up to 24 hours. this rapidly accumulated h 2 o 2 is detrimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause extensive injury. to date, there is limited literature on the effects of increased h 2 o 2 levels, particularly on microglial cells, which have been extensively indicated in the mediation of ensuing injury. here we describe the effects of a continuous exposure of microglia to h 2 o 2 , as generated using the glucose oxidase-catalase (gox-cat) system. this system allows us to generate and continuously maintain for up to 24h pathophysiological levels of h 2 o 2 >1 um. microglial cultures were derived from the p1 mouse brain and exposed to either bolus concentrations of h 2 o 2 [1-100 mm] or varying concentrations of gox-cat for varying lengths of time. conditioned medium was collected from cells at 4, 18 and 24h of treatment and analyzed for secreted cytokine levels using the 25-plex cytokine microbead array kit. treated cell extracts were processed for protein and fixed cells were labeled with m1 and m2a phenotype markers. continuous exposure to very low levels of h 2 o 2 can produce 10-20-fold higher (over control) pro-inflammatory cytokine protein levels of il5, il6, il12p40, il12p70, il15, il17 and ifng and at least a 100-fold higher level of il1a, il1b, il7 and tnfa by 18h. anti-inflammatory cytokine (il4, il10 and il13) and chemokine (rantes, g-csf and gm-csf) protein levels were also increased by 18h. interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. low, continuous h 2 o 2 promoted a predominantly m2a microglial phenotype by 24h. we conclude that studying continuous exposure effects will help delineate the various specific effects h 2 o 2 can have on microglial cells. these specific effects can then be used to clarify when and how microglial cell responses can be beneficial or detrimental following injury in the immature brain. we have previously shown that natural (15-deoxy-d 12,14 prostaglandin j 2 , 15d) and synthetic (pioglitazone) agonists of peroxisome proliferator activated receptor-c (ppar) potentiate intrinsic cellular mechanisms protecting oligodendrocyte (ol) progenitors (ops) from oxidative insults and promote their differentiation to ols. in addition, ppar-c agonists potentiate mitochondrial activities, as the mitochondrial respiratory chain activity and the regulation of cytoplasmic ca21 waves, which are known to be crucial for ol differentiation. in the present study we sought to investigate whether ppar-c agonists can protect ol cultures from conditions causing mitochondrial stress. first, to specifically induce a mitochondrial impairment, we used the complex i inhibitor rotenone. as expected rotenone, at concentration not affecting cell viability, significantly inhibited ol differentiation, as indicated by the reduced number of cells expressing specific markers of differentiation (o 4 and o 1 ). in ppar-c agonist-treated ols the inhibitory effects of rotenone were significantly attenuated, suggesting a protective effect of the agonists against the mitochondrial toxin. we next examined a condition mimicking inflammatory stress by challenging op cultures with tnf-a, an inflammatory cytokine known to retard the differentiating program of ops. in parallel with the expected reduction of the percentage of o 4 and o 1 positive cells, the cytokine induced a significant reduction of mitochondrial membrane potential (mmp), suggesting an impairment of the mitochondrial functions. the simultaneous treatment with tnf-a and ppar-c agonists (15d or pioglitazone) significantly reverted both tnf-a dependent reduction of ol differentiation and mmp. at the molecular level, we found that in op cultures, ppar-c agonists increased the expression of the uncoupling protein-2 (ucp-2), a mitochondrial protein known to contribute to the protection of mitochondria against oxidative stress. these findings suggest that ppar-c agonists protect ols and promote myelination through several mechanisms, including those involving mitochondrial functions. our studies support the therapeutic potential of ppar-c agonists in brain diseases in which mitochondrial alteration, oxidative stress and demyelination occur and point to the need to better understand the role of ppar-c and its agonists in ol biology. it is characterized by lesions of demyelination and inflammation, which can be classified according to the activation state of the microglia/macrophages. well before any myelin and blood-brain barrier damage clusters of activated microglia are noticeable. these clusters are considered to be the first stage of lesion formation and therefore called 'pre-active lesions. however, they do not always develop into demyelinating lesions. here we postulate that stressed oligodendrocytes play an crucial role in pre-active lesion formation, by producing factors that will attract and activate microglia to either a more pro-inflammatory (m1) or antiinflammatory (m2) activation status. this can contribute to the amount of damage to the environment and further lesion formation. this study sets out to identify the best method to mimic this early lesion formation in vitro. first, human primary isolated microglia are stimulated to either a m1 or m2 phenotype and characterized by marker expression and cytokine profile. oligodendrocytes are isolated out of the human brain. medium of (stressed) oligodendrocytes will be used as attractant as well as activator for microglia. by immunohistochemical staining the activation status of microglia in pre-active lesions will be determined. preliminary data reveal that human microglia are able to be skewed towards a pro-and anti-inflammatory phenotype. first experiments of primary human oligodendrocyte cultures, showed pure oligodendrocytes. thus far our data support our hypothesis, however further research is required to make our conclusions more robust. question: although cell transplantation is increasingly suggested to be beneficial for the treatment of various neurodegenerative diseases, the therapeutic application of such intervention is currently hindered by the limited knowledge regarding central nervous system (cns) transplantation immunology. in this study, we aimed to investigate the early post transplantation innate immune events following grafting of autologous mesenchymal stromal cells (msc) in the cns of immune competent mice. methods and results: first, the survival of grafted luciferase/egfpexpressing msc (msc-luc/egfp) was demonstrated to be stable from on day 3 post implantation using in vivo bioluminescence imaging (bli), which was further confirmed by quantitative histological analysis of msc-luc/egfp graft survival. additional histological analyses at week 1 and week 2 post grafting revealed the appearance of (i) graftsurrounding/-invading iba11 microglia and (ii) graft-surrounding gfap1 astrocytes, as compared to day 0 post grafting. while the density of graft-surrounding astrocytes and microglia did not change between week 1 and week 2 post grafting, the density of graft-invading microglia significantly decreased between week 1 and week 2 post implantation. however, despite the observed decrease in microglial density within the graft site, additional phenotypic analysis of graftinvading microglia, based on cd11b-and mhcii-expression, revealed > 50% of graft-invading microglia at week 2 post implantation to display an activated status. although microglial expression of cd11b and mhcii is already suggestive for a pro-inflammatory m1-oriented phenotype, the latter was further confirmed by: (i) the expression of nos2 by microglia within the graft site, and (ii) the absence of arginase 1-expression, an enzyme known to suppress no activity in m2oriented microglia, on graft-surrounding and -invading microglia. conclusions: in summary, we here provide a detailed phenotypic analysis of post transplantation innate immune events in the cns of mice, and warrant that such intervention is associated with an m1-oriented microglia response and severe astrogliosis. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). cd300f is a novel immunoreceptor that dampens inflammatory reactions in experimental autoimmune encephalitis (eae) and diverse allergy models. we have analyzed the function of cd300f immunoreceptor in an nmda excitotoxicity model and in a traumatic brain injury model (tbi). for this purpose, we determined the pattern of expression of both cd300f and its putative ligand(s) in the cns, as well as the neuroprotective role of cd300f after both acute brain injuries. first, we used a human and rat cd300f-ig soluble protein to show by confocal microscopy the presence of endogenous ligand(s) for this receptor mainly in cns white matter oligodendrocytes and on the surface of oligodendrocytes, fibrous astrocytes and neurons in vitro. interestingly, when we analyzed the in vitro expression pattern of rcd300f in brain cells by q-pcr and immunohistochemistry, in addition to the expected expression in microglial cells, we detected expression of cd300f in oligodendrocytes and neurons. in vivo, after a tbi, cd300f is expressed at early timepoints (3d) in small round macrophages, and at later time-points (14d) in neurons of the penumbra. q-pcr studies showed significant upregulation of the mrna for rcd300f at 7 and 14d after tbi. interestingly, a delayed (4 hours after the lesion) intraparenchymal injections of a non-viral modular recombinant gene therapy vector termed nlsct overexpressing hcd300f induced a decrease in the lesion volume 3d after nmda injection or tbi when compared to control gfp over-expression. in addition, the over-expression of hcd300f decreased the long-term sensitive functional deficits observed by the "sticky tape" test. in order to validate these results with the endogenous molecule, we cloned the rat ortholog of cd300f protein. on-going positron emission tomography (pet) studies using 2-[ 18 f]-fluoro-2-desoxiglucosa ( 18 f-fdg) are being used to evaluate the long-term functional recovery after tbi. the overexpression of rcd300f receptor had a comparable neuroprotective effect as the human molecule after nmda injection. these data suggest that cd300f may be important in neuron-glia inflammatory and trophic interactions. in addition, our results suggest that delayed cd300f overexpression by means of a modular recombinant gene therapy may constitute an interesting therapeutic strategy for acute brain injuries. depending on the type of signals produced after an inflammatory event, microglia develops specific activities, including cytotoxic, neuroprotective or phagocytic activities in order to come back to brain homeostasis. as described for macrophages, microglia can adopt m1 (proinflammatory) or m2 (phagocytic) phenotypes distinguishable through their pattern of factors expression and specific cell surface markers. interestingly, polyunsaturated fatty acids (pufas) are precursors of bioactive lipid messengers involved in the regulation of inflammation and in particular, docosahexanoic acid (dha, 22:6n-3) an n-3 pufa, presents anti-inflammatory properties. the aim of our study was thus to investigate the effect of an increase of n-3 pufas on the inflammatory response and cognitive abilities after a peripheral immune challenge. to increase n-3 pufas, we took advantage of transgenic mice carrying the fat-1 gene from the roundworm caenorhabditis elegans. this fat-1 transgenic mouse is capable of producing n-3 fatty acids from the n-6 type endogenously, eliminating confounding factors of the diet. this conversion leads to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in tissues, including the brain. to induce a neuroinflammation, we injected mice intraperitoneally with lipopolysaccharide (lps), components of gram negative bacteria walls. we first studied the microglial phenotype 24 h after lps injection and found that microglia in fat-1 mice presented a significant increase of m2 phenotype markers. we then measured cytokine expression in the hippocampus after a lps challenge and found a significant decrease in il-1b mrna in fat-1mice compared to wildtype littermates. in terms of hippocampal memories abilities, only control mice presented a deficit in the y-maze task whereas fat-1 mice were able to perform the test correctly. our results indicate that 24 h after a lps injection, fat-1 mice presented a decreased of the inflammatory response and normal hippocampal memory abilities compared to wild-type littermates. glia integrity and homeostasis as they have prominent role in blood brain barrier formation and maintenance, as well as in active regulation of an immune response. however, astrocytes are affected in neuroinflammation, when their protective roles might be suppressed and when they might be shifted towards pro-inflammatory phenotype. experimental autoimmune encephalomyelitis (eae) is a widely used model of autoimmune neuroinflammation. chemokine cxcl12 produced by astrocytes and endothelial cells has profound anti-inflammatory and anti-encephalitogenic role in eae. nitric oxide (no) produced by inducible no synthase (inos) within the cns is considered as disease promoting, while interleukin (il)-10 as protective in eae. the aim of this work was to investigate effects of no and il-10 on cxcl12 gene expression in astrocytes. methods: astrocytes were stimulated with supernatant collected from concanavalin a-stimulated splenocytes (conasn) and simultaneously exposed to no donor, sodium-nitroprusside (snp) or anti-il-10 antibody. also, astrocytes were stimulated with ifn-c1il-17 in the absence or presence of il-10. peritoneal macrophages isolated from healthy rats were co-cultivated with astrocytes. after 24h incubation period cxcl12 gene expression in astrocytes was measured by rt-qpcr. phosphorylation of p38 mapk and nf-jb was assessed by immunoblot in astrocytes exposed to snp. results: we found that no released from snp or macrophages significantly reduced cxcl12 gene expression in astrocytes. this reduction was mediated by inhibition of p38 mapk activation, but not of nf-jb signaling. on the contrary, il-10 stimulated and anti-il-10 antibody suppressed cxcl12 gene expression in astrocytes. conclusions: these results imply that expression of cxcl12 in astrocytes is inversely regulated by no and il-10 in the central nervous system affected by inflammation. having in mind, profound regulatory role of cxcl12 in neuroinflammation, these data contribute to our understanding of pro-and anti-inflammatory nature of no and il-10, respectively. in the presence of pathological insults, they acquire reactive phenotypes aimed at re-establishing brain homeostasis and minimizing neuronal damage. however, reactive microglia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect. consequently, the progression and resolution of microglial activation have to be tightly controlled to avoid detrimental secondary effects. signals arising from neuronal cells play an important role in the activation state of microglial cells. among them, inhibitory mechanisms, such as neuronal cd200 and microglial cd200r1 interaction, keep the proinflammatory phenotype of microglia under control. alterations in the expression of cd200 and cd200r1 have been described in pathological conditions, and the modulation of cd200-cd200r signalling could be an interesting target to be considered in therapeutic approaches against neuroinflammation occurring in neurodegenerative diseases. little is known on the molecular mechanisms involved in the regulation of cd200 and cd200r1 expression. the aim of the present work is to study the possible modulation of cd200 and cd200r1 by ppar-c agonists, and the involvement of cd200-cd200r1 in the anti-inflammatory and neuroprotective effects of ppar-c activation. we have used mouse microglial, mixed glial and neuronal cell cultures, as well as neuron-microglia co-cultures. cd200r1 expression was detected in microglial cells, and it was decreased in response to pro-inflammatory stimuli such as lps/ifn-c. the ppar-c agonist 15-deoxy-d 12, 14 -prostaglandin j 2 (15d-pgj 2 ) abrogated the inflammatory response in lps/ ifn-c-treated microglial cells and prevented cd200r1 expression inhibition. cd200 expression was detected in neuronal cultures and, at a lesser extent, in astrocytes in mixed glial cultures. lps/ifn-c-treatment did not modify cd200 expression in neuronal cultures, but it induced an increase in mixed glial cultures. this increase was inhibited by 15d-pgj 2 pre-treatment. 15d-pgj2 also protected against lps/ifnc-induced neurotoxicity in neuron-microglia co-cultures, but this effect was abolished when cd200-cd200r1 interaction was interrupted using an anti-cd200r1 blocking antibody. these results show that ppar-c agonists modulate cd200 and cd200r1 expression in reactive glial cells, and that cd200-cd200r1 interaction is necessary for the neuroprotective effect of ppar-c agonists. to gain insight into the contribution of the cwbc pathway to remyelination in ms, we analyzed the expression pattern of tcf7l2, a downstream target of the cwbc pathway and coactivatior of b-catenin in ms lesions. tcf7l2 was expressed in ol and astrocytes in a subset of early ms lesions, but was also observed in tissue samples from patients with inflammatory, non-demyelinating diseases. in contrast, in chronic lesions, no expression of tcf7l2 was detected. by wb we found slightly increased b-catenin levels in ms lesions compared to normal appearing white matter. aspirin (ass), a non-steroidal anti-inflammatory drug, attenuates bcatenin signaling activity by inhibition of protein phosphatase 2a (pp2a) via increased phosphorylation. therefore, we determined the effect of ass on ol differentiation and myelin gene expression (mge). as a control for the cwbc pathway activation or inhibition the selective cwbc pathway inhibitor (icg-001) was chosen. icg-001 binds specifically to cbp and causes a disruption of the b-catenin/cbp interaction. exposure of ol to 0.3 mm icg-001 for 48 hours increased mge and had a positive effect on ol differentiation. in contrast, ass had no positive effect on process formation and did not promote mge in primary murine ol. our results suggest that icg-001, but not ass increases the expression of myelin genes. further experiments are required to determine the functional role of the cwbc pathway for ol differentiation and remyelination in ol and ms. (braak et al., 2003) . furthermore, neuroinflammation, i.e. activation of microglial cells in the substantia nigra (sn), has been widely implicated in pd progression (mcgeer et al., 1988) . in the present study, we question whether microglial activation occurs in brain regions beside the sn which are affected in pd patients. methods: to this end, we studied microglial activation and protein pathology in the ob and hc of clinically and neuropathologically verified pd patients (braak 4-6) and control subjects (braak 0). human post-mortem formaldehyde-fixed material of pd patients included sn (n 5 13), ob (n 5 9) and hc (n 5 12), and material from age-matched control subjects without neurological deficits included sn (n 5 10),ob(n 5 6), and hc (n 5 8). results: the presence of a-syn pathology concentrated in the anterior olfactory nucleus of the ob and in the ca1-2 region of the hc. furthermore, using cd68 as a microglial marker, we observed a significant increase in the number of immunopositive microglial cells, with an activated, amoeboid morphology, in the sn as well as in the ob and hc of pd patients compared to control subjects. co-localization studies indicated that these activated microglia were found in the proximity of a-syn inclusion bodies and neurites, but did not co-localize suggesting that the microglial cells do not actively phagocytose a-syn. conclusion: we conclude that microglial activation occurs in other brain regions beside the substantia nigra of pd patients. the functional role of the microglial cells in those brain regions and their contribution to pd pathology remains to be established. in the present study, we question whether ccl2 and cx3cl1 and its receptors are present in hippocampal lesions of ms patients. to this end, semi-quantitative rt-pcr was performed on cdna transcribed from rna isolated from hippocampi of ms patients and control subjects. moreover, immunohistochemical analysis of ccl2, cx3cl1 and its receptors ccr2 and cx3cr1 was performed on post-mortem, formalin-fixed hippocampal lesions (plp -) from ms patients and on hippocampal material (plp 1 ) from control subjects. ccl2 and ccr2 mrna was significantly enhanced in demyelinated ms hippocampal homogenates compared to non-demyelinated and control hippocampal homogenates. cx3cr1 mrna was significantly increased in demyelinated ms hippocampal homogenates compared tot non-demyelinated hippocampal homogenates, but cx3cl1 mrna levels showed no difference between groups. ccl2, ccr2, cx3cl1 and cx3cr1 immunoreactivity was present mainly in white matter of control and ms hippocampi and was clearly enhanced in hippocampal grey (cornu amonis) and white (stratum lacunosum, stratum radiatum, alveus) matter lesions. the intensity of the ccl2 and cx3cl1 immunoreactivity was most pronounced when active lesions were present. co-localization studies indicated that ccl2 and cx3cl1 are present in astrocytes, whereas cx3cr1 and ccr2 are present in or on microglial cells. we conclude that the chemokines ccl2 and cx3cl1 and their respective receptors are enhanced in hippocampal ms lesions. as no clear influx of leukocytes is observed in the grey matter lesions, we propose that astrocyte-derived ccl2 and cx3cl1, via interaction with their receptors, affect microglial activation and/or function thereby contributing to neuronal dysfunction in the hippocampus as seen in ms patients. , is a 17 kda cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and in inflammatory reactions. in central nervous system (cns), iba1 is a sensitive marker associated to activated microglia and is upregulated following neuronal death or brain lesions. iba1-like factors have been described in several metazoan and share a well conserved amino acid primary structure throughout evolution suggesting a common, functional role. the medicinal leech hirudo medicinalis is able to regenerate its cns after injury, leading to a complete functional repair. similarly to vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. we investigated the expression of hirudo iba1 to track the activation state of leech microglial cells involved in nerve repair events. results: we recently identified a gene, named hmiba1, coding a 17.5 kda protein showing high similarity with vertebrate iba1 factor. quantitative rt-pcr analyses showed that hmiba1 is constitutively expressed in cultured nerve chains. a weak down regulation was observed in the days following experimental injury. gene transcripts rise back to basal level one week later. cultured nerve chains stimulated with atp shows a significant increase of hmiba1 transcript 6 hours after treatment. immunoblot analysis, performed with anti-hmiba1 polyclonal antibodies, revealed an immunopositive band at the predicted size. the presence of hmiba1 protein in na€ ıve and experimentally challenged tissues was evaluated by immunohistochemistry. the protein is constitutively present in spread, stellar shaped microglial cells, distributed in connective fibers and in segmental ganglia. a few hours after experimental injury of cns, the amount of immunopositive microglial cells increases at the lesion site and at the cut end of nerve fibers until. the amount of hmiba11 cells in connectives rapidly increases in atp treated nerve chains. this augmentation is visible in ganglia microglia and in connective fibers, where cells located between axon fibers display an elongated and stretched shape. conclusion: hmiba1 is a good marker of activated microglia. like in vertebrates, the atp induces its expression in leech cns. also if the functional role of hmiba1 has to be further elucidated, this molecule appears as a good activation marker of microglia and an interesting tool to study and follow the activity of such cells during nerve repair in leech. hypertension is the single most important risk factor for cardiovascular disease. despite significant advancements, 20-30% of all hypertensives remain resistant to all current available pharmacotherapy. these patients exhibit elevated sympathetic drive, increased norepinephrine spillover, and dampened parasympathetic drive. the dysfunctional autonomic nervous system of these resistant patients indicates a neurogenic origin for their pharmacotherapy resistance. previous studies have proposed that neuroinflammation in the autonomic brain regions plays an important role in neurogenic hypertension. this coupled with the emerging interest in microglia led us to hypothesize that activation of microglial cells in the brain could be a critical event in the initiation and establishment of hypertension pathophysiology. we have previously established that chronic low dose angiotensin ii (ang ii) induced hypertension involves activation of microglia, and increase in proinflammatory cytokines and reactive oxygen species (ros) in cardioregulatory brain areas, such as the paraventricular nucleus of the hypothalamus (pvn). intracerebroventricular (icv) delivery of minocycline, an anti-inflammatory antibiotic that inhibits microglia activation, decreased activated microglia, inhibited increase in cytokines and ros, and attenuated hypertension. in addition, inhibition of brain mitochondrial ros attenuated hypertension, microglial activation, and the overexpression of brain pro-inflammatory cytokines. a time-course experiment demonstrated that there was a significant increase in activated microglia before the increase in blood pressure was detectable by radiotelemetry. furthermore, the origin of microglia in established hypertension appears to be both from resident and bone marrow derivation. these observations indicate that the activation of microglia is a critical player in the development of neurogenic hypertension. they suggest that activation of microglia in cardioregulatory regions of the brain is an early occurrence that may initiates a cascade of signaling events leading to increase sympathetic nerve activity and initiation of hypertension. this research is supported by nih grant (r37 hl 33610 to mkr). our results demonstrate significant inflammatory activation of the neurons and their sgc not only in drg associated but also non-associated with injured nerve. a distinct expression of cytokines and their receptors was identified in sgc surrounding largesized drg neurons. significant inflammatory reactions of sgc were found in all drg of pac-treated rats. moreover, inflammatory activation sgc was also observed in drg of sham-operated rats indicating other kinds of trigger than traumatic nerve injury. the nerve injury and pac-treatment also triggered socs3 expression in sgc to control stat3 activation. inflammatory activation of sgc significantly contributes to ectopic activation of the drg neurons not only associated with injured nerve, and is involved in npp induction. aging is associated with reduced function and degenerative changes of the central nervous system (cns). increasing evidence suggests that changes in microglia cells, i.e. the resident macrophages of the cns, contribute to the age-related deterioration of the cns. the most prominent age-related change of microglia concerns enhanced sensitivity to proinflammatory stimuli of microglia in mice, rats and primates, called priming. we have addressed this issue in ercc1 mutant mice, ercc1 d/mice, a progeroid, dna repair deficient mouse model that displays features of accelerated aging in multiple tissues including the cns. in aged ercc1 d/mice, microglia showed increased proliferation and enhanced immune function including expression of cytokines and antigen presentation molecules, increased phagocytosis and production of reactive oxygen species (ros). this microglial functionality was found to be surprisingly hyper-responsive to immune stimuli indicative of microglia priming. transcriptome analysis revealed an expression pattern that characterizes microglia priming, featuring genes associated with increased antigen pattern recognition and antigen presentation and phagocytic-, adhesion-and chemotactic ability. in mice where the ercc1related dna repair deficit was targeted to forebrain neurons, microglia priming was restricted to forebrain areas, suggesting that the dna damage-induced changes in neurons provided the signals leading to microglial immune priming. acidosis is a clinical consequence of many major diseases. non-infectious diseases are increasing in prevalence and many have been shown to have an inflammatory component, which in the absence of infection, will be sterile. the nlrp3 inflammasome, a multimeric protein complex which induces processing of il-1b into its active form, is a well established mediator of sterile inflammation and is known to drive the worsening of brain injury in numerous experimental disease paradigms. we sought to investigate whether acidic conditions, typical of a disease environment, affected il-1b processing in primary mouse glial cells. mixed glial cultures were grown from wild type and nlrp3 knockout mice. culture media was reduced to ph6.2 and il-1b release was measured following addition of activators of the nlrp3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) with or without pre-treatment with caspase-1 or cathepsin d inhibitors (yvad-cho or pepstatin a respectively). subsequently, il-1b release was measured following addition of lactic acid with or without pre-treatment with the above inhibitors. at ph6.2, activators of the nlrp3 inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) induced the release of il-1b from mixed glial cultures. the il-1b released at this low ph was 20kda in size in addition to the mature 17kda il-1b. this 20kda il-1b release was maintained in nlrp3 deficient cells and was not significantly altered by pre-treatment with the caspase-1 inhibitor yvad-cho. lactic acid, itself released during disease and a common cause of acidosis, also induced the release of 20kda il-1b from mouse glial cultures. as with the nlrp3 activators under acidic conditions, the lactic acid-induced 20kda il-1b release was maintained in nlrp3 knock-out cultures and with pre-treatment with yvad-cho. pre-treatment with the cathepsin d inhibitor pepstatin a, however, significantly reduced the 20kda il-1b released with the nlrp3 activators and lactic acid. here we show that under disease relevant conditions (low ph), 20kda il-1b is released from mouse glial cultures and this 20kda il-1b is independent of the classical nlrp3 inflammasome/caspase-1 pathway and likely mediated by cathepsin d. further investigation of this caspase-1-independent il-1b pathway may in future provide novel targets for the treatment of inflammatory disease. the phoneutria nigriventer (ctenidae-araneaeomorpha) spider venom (pnv) causes reactive gliosis, neuroinflammation and blood-brain barrier (bbb) impairment. nitric oxide(no)-soluble guanylate cyclase(sgc)-cgmp has been implicated in pnv-induced cavernosal relaxation. we investigate whether no-sgc-cgmp signaling acts on astrocytes/microglia activation and neuroinflammation induced by pnv. male wistar rats (6-8-week-old) were pre-treated (i.p.) with sgc inhibitor (odq, 10 mg/kg), nnos inhibitor (7-nitroindazole-7ni, 40 mg/kg), no donor (nitroprussiate-ntp, 3 mg/kg) or pde5 inhibitor (sildenafil, 10 mg/kg). after 30 minutes, the venom (0.85 mg/kg) was injected in the tail vein (i.v.). saline (i.v.), pnv alone or dmso (i.p) followed by pnv were used as controls. one hour after injection, cerebella were processed for immunofluorescence or western blotting. pnv increased gfap, iba-1, ifn-c and sgc, compared to saline control, indicating astrocytes and microglia activation, neuroinflammation and sgc-cgmp involvement. the sgc inhibition by odq intensified the pnv effects, increasing even more gfap, iba-1 and ifn-c levels. this indicates that sgc-cgmp production can be inhibited by venom. in agreement, the cgmp accumulation by sildenafil inhibited the pnv effects, decreasing gfap, iba-1, ifn-c and sgc. the increase of sgc by venom could result from a feedback mechanism, when sgc activity is inhibited and its expression is increased. 7ni and ntp pre-treatment did not show difference compared to venom alone, suggesting that no per se is not involved in the inflammatory effects of venom. however, it is not discarded no and sgs-cgmp coupling in the mediation of pnv effects. we suggest that pnv induces glial reaction and neuroinflammation by sgc-cgmp inhibition. the study gives evidence that sgc-cgmp, coupled or not to no signaling, mediates pnv cerebellar effects including the bbb impairment. pnv can be a useful tool for studies on the mechanisms involved in glial regulation. cnpq/fapesp/faepex support. j. engele, m. puchert, v. € odemis university of leipzig, leipzig, germany it is currently believed that cxcl12 predominantly signals through cxcr4. in addition, it is assumed that the alternate cxcl12 receptor, cxcr7, represents a non-classical g protein-coupled receptor which primarily acts as a modulator of the function of cxcr4. discrepant from this view, we demonstrated recently that in primary rodent astrocytes and human glioma cell lines, sdf-1 exclusively signals through cxcr7 by a g protein-dependent mechanism. we now provide evidence that cxcr4 is essential for cxcl11/i-tac signalling in primary rodent astrocytes and some human glioma cells. treatment of cultured rat astrocytes with cxcl11 for 10 min resulted in the dose-dependent activation (phosphorylation) of erk1/2 and akt with maximum activation in the presence of 100 ng/ml of the chemokine. cxcl-11-dependent activation of both signalling molecules persisted in astrocytes in which expression of the established receptors for cxcl11, cxcr3 and cxcr7, were inhibited by rnai. however, cxcl11-dependent activation of erk and akt was abrogated following sirna-mediated inhibition of cxcr4. likewise, cxcl11 failed to activate erk and akt in cortical astrocytes cultured from a cxcr4-/-transgenic mouse line. moreover, similar to primary astrocytes cxcl11 activated erk and akt in the human glioma cell line, a767. again activation of both signalling molecules was abrogated following rnai-mediated inhibition of cxcr4. cxcl11-dependent activation of erk and akt further remained undetectable in a non cxcr4-expressing subpopulation of a767 cells, previously isolated by flow cytometry. together, these findings unravel a unique processing of cxcl11 and cxcl12 signalling in primary astrocytes which is preserved at least in some malignant astroglial cells. long-lasting activation of gfap-positive astrocytes occurs in brain tissue exposed to injuries that promote epilepsy development. studies in experimental models of epilepsy showed that activated astrocytes release various molecules, such as proinflammatory cytokines and danger signals, that play a role in seizures generation and recurrence. these activated cells also loose their ability to buffer extracellular k 1 and glutamate. this set of evidence suggests, therefore, that astrocytes may contribute to epileptogenesis (i.e. the post-injury phase prodromal to generation of spontaneous seizures), thus representing a putative biomarker of epilepsy. to test this hypothesis, we set up an in vivo longitudinal study using 1 h-magnetic resonance spectroscopy (mrs) to measure the hippocampal levels of metabolites that could reflect the extent and the duration of astrocytes activation after an epileptogenic brain injury. status epilepticus (se), which provokes epilepsy, was induced by pilocarpine in adult male rats. 1 h-mrs measurements were done in the hippocampus every 24 h for 7 d post-se, thus encompassing the epileptogenesis phase, and in chronic epileptic rats using a 7 tesla bruker biospec. spectra were processed and analysed using jmrui and tarquin freeware softwares. we studied changes in myo-inositol (mins) and glutathione (gsh), which reflect astrocytes activation. we found a progressive (2-fold, pwhich was maintained in epileptic rats. immunohistochemical analysis (ihc) of s100ß and gfap confirmed the concomitant activation of astrocytes in separate timematched groups of rats. gsh levels during epileptogenesis showed a negative correlation with the frequency of spontaneous seizures which developed after se. a negative correlation was also found between gsh and mins levels during epileptogenesis and the extent of neurodegeneration in hippocampus of epileptic rats. ihc done in epileptic rats at the end of the mrs experiments, showed that hippocampal s100ß levels positively correlated with spontaneous seizure frequency. since this is a soluble protein, further investigations of its csf and blood levels are warrented. our mrs findings show that gsh levels during epileptogenesis could serve as a predictive biomarker of the ensuing seizure frequency (thus of epilepsy severity) and, together with mins levels, also predict the extent of neuronal cell loss which develops following se in epileptic tissue. notably, ihc-detected s100ß levels in the epileptic tissue also reflect seizure frequency. these findings highlight the potential use of serial 1 h-mrs analysis of astrocyte activation for predicting the severity of epilepsy and the extent of neuropathology in the clinical setting. interleukin-6 (il-6) is a highly plurifunctional cytokine, with many pleitropic actions, considered one of the main cytokines controlling the immune system and coordinating it with the nervous and endocrine systems. il-6 is produced in multiple cell types in the cns, and in turn many cells do respond to it. it is therefore important to ascertain which the contribution of each cell type is in the overall role of il-6 during both physiological and pathological conditions. astrocytes are major responders to il-6 as well as one of the main cns producers of il-6. for this work we used astrocytary il-6 ko (ast-il6 ko) mice, which we already proved to have an important role in physiological conditions (like body weight control and exploratory/ locomotion behavior), in order to test astrocytary il-6 role during a neuroinflammation situation. for this purpose, we induced either an extensively used animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (eae), or a traumatic brain injury (cryolesion) in our mice. regarding eae, results indicate that lack of astrocytary il-6 delays clinical course of eae and ameliorate eae symptomatology in ast-il6 ko respect littermate controls in a gender-dependent way. further immunohistochemistry analyses confirm a decreased number of cellular and lymphocytes infiltrates and lesser demyelination, angiogenesis and gliosis in spinal cord of ast-il6 ko animals. regarding traumatic injury, astrocytary lack of il-6 facilitates microgliosis, lymphocytes infiltration and a faster decrease of the injured area. all these results are likely to bring some answers to astrocytesecreted il-6 involvement in neuroinflammation pathways and eae pathogenesis. interleukin-10 (il-10) is a counterregulatory cytokine that plays an important role in controlling inflammatory and immune reactions. in the central nervous system (cns), production of il-10 has been demonstrated in activated astrocytes and microglia after different types of injuries. the specific role played by il-10 inmodulating glial responses is however still unclear. hence, the objective of this study was to evaluate the effects of local astrocyte-targeted production of il-10 on glial reactivity using the axonal anterograde degeneration paradigm. for this purpose, unilateral perforant pathway transection (ppt) was performed on adult gfap-il10 transgenic (tg) animals and their corresponding wild types (wt) littermates. at 3 and 7 days post-lesion (dpl), animals were intracardially perfused with 4% of paraformaldehyde, brains frozen and parallel free-floating sections processed for glial analysis using immunohistochemistry for iba-1 (microglia) and gfap (astrocytes). our results showed that in comparison with wt animals, microglial cells in the dennervated dentate gyrus molecular layer of gfap-il10tg showed differential reactivity changes. after lesion, astrocytic reactivity presented a higher hypertrophy in gfap-il10tg animals when compared with their corresponding wt littermates. in conclusion, this study demonstrated that local production of il-10 inthe cns can modify the glial response associated with ppt. further studies are warranted to evaluate if these alterations in microglial and astrocytic reactivity have any repercussions for the evolution of the lesion and the associated axonal sprouting. astrocyte and microglia become reactive under many brain pathological conditions, making this process of neuroinflammation a surrogate marker of neuronal dysfunction. several studies have reported that reactive microglia overexpress the translocator protein 18kda (tspo, formerly known as peripheral benzodiazepine receptor). positron emission tomography (pet) using radioligands of tspo is thus considered as a potent technique to detect reactive microglia in situ. however, it is still controversial whether tspo pet imaging is also able to monitor reactive astrocytes in situ. this is an important question as reactive astrocytes and reactive microglia play very different roles in brain physiology and could impact disease progression in opposite ways. to address this question, we used a model of selective astrocyte activation through unilateral lentiviral gene transfer of the cytokine ciliary neurotrophic factor (lenti-cntf) in the rat striatum. cntf induced an extensive activation of astrocytes, which overexpressed gfap and became hypertrophic. microglia, on the contrary, displayed a minimal increase in the expression of markers of reactivity. cntf-activated astrocytes overexpressed tspo at the mrna and protein levels. pet imaging experiments demonstrated a significant and specific binding of two tspo radioligands [ 18 f]dpa-714 and [ 11 c]ssr180575 in the lenti-cntf-injected striatum. we show that reactive astrocytes can be monitored by tspo-pet imaging in the rat brain. this technique is thus well suited to monitor reactive microglia as well as reactive astrocytes, the two cell types involved in neuroinflammation, but would not allow their discrimination in situ. chronicity might establish a neuroinflammatory ganglion profile with inflammatory cells contributing to the hypersensitive phenotype. we first investigated whether, in trigeminal sensory ganglia, cytokines such as tnfa might contribute to a local inflammatory phenotype of a transgenic mouse model of familial hemiplegic migraine type-1 (fhm-1, cacna1a r192q knock-in mice). with respect to wild-type, r192q ki trigeminal ganglia were enriched in activated macrophages and expressed higher mrna levels of il1b, il6, il10 and tnfa cytokines and the mcp-1. functional consequences of crosstalk between macrophages and sensory neurons were studied in primary ganglia cultures, where larger release of soluble factors and larger currents mediated by pain-transducing atp-gated p2x3 receptors were found. consistently, we observed that, following lps injection, tnfa expression and macrophage occurrence were significantly higher in r192q knock-in ganglia with respect to wild-type ganglia. our data suggest that, complex cellular and molecular environment of sensory ganglia could support a new tissue phenotype compatible with a neuroinflammatory profile. we propose that, in selected patients, this condition might contribute to pain pathophysiology through release of soluble mediators, including tnfa and atp that may modulate the crosstalk between sensory neurons and resident glia, underlying the sensitisation process. cultures of astrocyte/microglia are stimulated with ifn-gamma and then infected with tachyzoites of neospora caninum they release nitric oxide (no) that controls parasite proliferation. in order of elucidating the immune response of cns against this parasite, this study investigated the participation of inducible nitric oxide synthase (inos) in the control of its proliferation in co-cultures of neuron-glia obtained from rat brain. the cells were stimulated with ifn-gamma (100 iu/ml-24h) then supplemented with l-ng-nitroarginine methyl ester/l-name, an inhibitor of inos (1.5 mm/ml -60 min) before infection with tachyzoites of n. caninum (1:1 parasite:cell). after 72h, in the cultures supplemented with l-name, it was verified, a reduction of tachyzoites number in 55.7% (control cells) and 59.8% (ifn-y stimulated cells). however, in infected co-cultures, it was not observed any release of no, even when cells where modulated by ifn-g. additionally, in cultures infected and treated with l-name, the levels of il-10 were increased in six fold (non stimulated) and nine fold (ifn-g stimulated). these data suggest that no should not participate as a mediator of n.caninum control in neuron/glia co-culture but, the regulatory pattern of immune response must play a role in its down modulation regulating the inflammatory mediators released during the cns infection, perhaps to protect neurons. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n 5 6) and lasered (n 5 6). anti-iba1 was used in retinal whole-mounts immunolabelled to analyze the distribution, morphology, density and arbor area of iba11 microglial cells. results: in na€ ıve, contralateral and lasered eyes, iba11 microglias were distributed throughout the retina in the: subretinal space (ss), outer plexiform layer (opl), inner plexiform layer (ipl), nerve-fibre layer (nfl) and ganglion-cell layer (gcl). in na€ ıve eyes, iba11 microglias: i) had rounded bodies with fewer and shorter processes in the ss; ii) exhibited a branched morphology emanating from small cell bodies in the opl and ipl; iii) were less ramified and were related with the retinal vessels in the nfl and in the gcl. by contrast, the morphological features exhibited by iba11 cells in contralateral and oht-eyes differed from na€ ıve: i) somas were more robust; ii) processes were thicker and more branched in contralateral eyes and thicker and retracted in oht-eyes. in oht-eyes and contralateral untreated eyes the iba11 microglial number was increased in comparison with na€ ıve (p < 0.001 and p < 0.05 respectively, t-test). in comparison to na€ ıve retinas the iba-11 cell arbor in the opl and ipl decreased in both oht-eyes (p < 0.001 and p < 0.001 respectively, t-test) and in the contralateral untreated eyes (p < 0.05 and p < 0.001 respectively, t-test). conclusions: two weeks of laser induced-oht triggered microglial retinal changes suggestive of activation in the contralateral untreated and oht-eyes. the microglial activation in contralateral eyes could be related to the immune response. this behaviour in contralateral untreated eyes, lead us to suggest that the use of contralateral eyes as internal controls in experimental unilateral oht, should be reconsidered. interleukin-6 (il-6) is a pleiotropic cytokine involved in inflammatory and non-inflammatory responses. among the latter, it participates in the regulation of body weight and metabolism. il-6-deficient mice develop mature onset obesity and have higher blood glucose, as well as impaired glucose tolerance and elevated blood leptin levels, suggesting that il-6 participates in suppressing adiposity in mice. moreover, transgenic mice with astrocyte-targeted production of il-6 challenged with a high-fat diet are resistant to high-fat diet-induced obesity, highlighting the role of centrally produced il-6 in the regulation of body weight. in this context, we hypothesize that mice lacking il-6 produced by astrocytes (ast-il6ko) will be more prone to develop obesity. we therefore challenged ast-il6ko mice (obtained with the cre-lox technology) with a high fat diet (58% kcal from fat) for 17 weeks and compared their body weight and food intake with those of mice fed a standard diet (18% kcal from fat). on weeks 14 and 15, the metabolic status was evaluated by an insulin tolerance test (itt) and an oral glucose tolerance test (ogtt), respectively. regarding body weight gain, we see a clear increase in ast-il6ko females on a high-fat diet in comparison to their controls with no apparent difference in food intake, which is also already apparent in the control diet. in males a similar tendency is observed. part of this difference can be attributed to the heavier subcutaneous white adipose tissue depots (relative to body weight) in ast-il6ko mice (fig 1) . insulin and oral glucose tolerance are compromised in mice fed a high-fat diet, but no differences between genotypes are observed. taken together, these results indicate that centrally produced il-6 has indeed a major role in the regulation of body weight, affecting subcutaneous white adipose tissue, but without significantly altering peripheral glucose metabolism. we are currently working on assessing hypothalamic neuropeptides with in situ hybridization to study the effects of astrocytic il-6 deficiency at the central level. minocycline is an agent with pleiotropic properties that targets multiple proteins and cellular processes associated with development of neuropathic pain, including inhibition of the injury-induced glia activation. the aim of our study was to examine the effects of minocycline on the injury-induced changes in immune factors and on morphine effectiveness in a rat model of neuropathic pain. chronic constriction injury (cci) to the sciatic nerve in rats was performed according to bennett and xie (1988) and 2 behavioral tests were conducted to measure allodynia (von frey test) and hyperalgesia (cold plate test). minocycline was administered intraperitoneally 16h and 1h before cci and then twice daily for 7 days. the studies were performed using competitive rt-pcr in the spinal cord and drg from control, cci-exposed and minocycline-treated cciexposed rats. morphine was administrated i.t. (chronic catheterization according to yaksh and rudy, 1976) and ip 1h after the last minocycline injection. the experiments were carried out according to iasp rules (zimmermann, 1983) . repeated administration of minocycline attenuated allodynia and hyperalgesia when measured 7 days after cci. minocycline downregulated the spinal and drg level of several immune factors: c1q, mmp-9, mmp-2, il-1beta, il-6, il-18, nos1 and nos2 which were increased in consequence of the injury. moreover, chronic administration of minocycline improved the response to i.t. and i.p. morphine injections. our results demonstrate that minocycline reduces the level of pro-inflammatory factors and increases the effectiveness of morphine, which may have clinical significance for enhancing analgesic effects of opioids in neuropathic pain. to date, there is no standardized simple model available to investigate the biology of human microglia. the aim of this study was to establish a new in vitro microglia model using blood-derived precursor cells. for that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of a mixture of cytokines and chemokines (m-csf, gm-csf, ngf and ccl2) to generate monocyte-derived microglia (m-mg). monocyte-derived dendritic cells (m-dc) were also generated as a control population using gm-csf and il-4. the human microglia cell line hmc3 was used as control. m-mg were clearly different in morphology, phenotype and function from m-dc, but shared many properties with hmc3 cells. m-mg acquired a ramified morphology with primary and secondary processes, comparable to hmc3. they expressed very low levels of cd45, cd14 and hla-dr, cd11b and cd11c; but a distinct pattern of chemokine receptors, including ccr1, ccr2, ccr3, ccr4, ccr5, cxcr1, cxcr3, cx3cr1. similar to hmc3, under non-activated condition, the m-mg secreted of il-8 and il-6. in comparison with m-dc, m-mg displayed lower t-lymphocyte stimulatory capacity, as well as lower phagocytosis activity. in summary, we have established a new protocol for the generation of human monocyte-derived microglia, which is is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. this model will certainly be very helpful for future studies investigating the biology and pathology of human microglia. w. schaafsma university of groningen, neuroscience, section medical physiology, groningen, netherlands background: microglia are the innate immune cells of the cns. like other tissue macrophages, they can activate and commit to distinct reactive phenotypes in response to tissue damage and infections. however, the microglial response to tissue damage may change over age and by past experience. these changes in activation patterns may be caused by epigenetic modifications which in turn can result in changes in gene expression of, for example inflammatory cytokines. a well studied condition with an altered responsiveness of innate immune cells is 'endotoxin tolerance' (et). innate immune cells that are pre-exposed to endotoxins are dampened in their inflammatory response upon re-exposure to these endotoxins. while et is well described for peripheral macrophages/monocytes, very little is known about et in relation to microglia. objective: in our experiments we set out to investigate the presence of et in microglia and if an 'epigenetic' memory is involved. design/methods: in our in vitro experiments, microglia cells were stimulated with lps (100 ng/ml). naive microglia only received one lps stimulation at the end of culture, pre-stimulated microglia received an 24h pre-stimulation of lps (100 ng/ml) and a second stimulation 5 days later. in vivo, male c57bl/6 mice received an i.p. injection with either pbs or lps (1 mg/kg) and 4 weeks later again an i.p. injection with pbs or lps. transcription levels and secretion of il1-b and tnf-a were assessed by use of quantitative pcr and elisa. in order to answer if there is a long lasting epigenetic memory involved in this tolerance we used chromatin immune-precipitation (chip), to investigate changes in covalent histon tail modifications associated with either permissive or repressed chromatin. results : in vitro, we show a 'tolerant' phenotype in microglia which receive a second lps stimulation after 5 days. a reduced expression level of pro-inflammatory genes il1-b and tnf-a and reduced secretion of tnf-a were observed in response to a second lps challenge. furthermore, with in vivo experiments we showed that mice which received a second lps injection after 4 weeks, showed a dampened response in their microglia inflammatory reaction at the level of il1-b and tnf-a gene transcription. in both in vitro and in vivo experiments we showed a corresponding pattern for histon tail modifications in the enrichment of activation marks h3k4me3 and ach3 at the il1-b and tnf-a promotors. we are the first to show a long lasting 'tolerant' phenotype in mouse microglia in vitro and in vivo and the involvement of epigenetic changes at the level of histon modifications. morphological criteria and dual immunofluorescent labelling confirmed expression of er stress protein in neurons, astrocytes, microglia or oligodendrocytes. an intriguing finding was localisation of crt to the rim of oro-positive myelin fragments and the 'patchy' nature of crt staining seen when tissue was dual-labelled with crt and gfap or iba1. these results are the first demonstration of significantly higher levels of crt in rodent eae. chop and p-eif2a data has also not been reported in rat eae. this study highlights the potential importance of er stress in inflammatory demyelination. the authors acknowledge support from the irish research council and from the foundation office of nui, galway. (3). considering the responsible signaling pathways regulating adult neurogenesis (4), we observed differential regulation of wnt members in the hippocampus on transcriptional level, e.g. wnt dependent transcription factors (axin2, tcf4) and ligands (wnt3, 5a, 8b and 11), as well as on translational level represented by the wnt signaling cascade (active-bcatenin, phospo-glycogen synthase kinase 3b) during acute and chronic states of disease. by using in vitro studies in primary hippocampal cultures, we further show the role of transforming growth factorbb1 (tgfb1), a key cytokine early involved during eae (5), in regulation of wnt ligand expression and wnt signaling activity. furthermore we are able to visualize the connection between tgfb1 and wnt signaling by using the axin2-lacz mouse, a wnt reporter mouse, for functional and histological investigation of the hippocampus. taken together our results suggest a cross talk of inflammation and wnt signaling for dysregulated hippocampal neurogenesis. interleukin-6 (il-6) is a cytokine with major regulating effects of the inflammatory response. moreover, il-6 is a neuropoietin that has neurotrophic effects related to neuronal survival and protection. to establish the importance of il-6 produced only in the central nervous system, we have generated mice producing il-6 essentially only in the brain by crossing gfap-il6 mice (transgenic mice with astrocyte-targeted production of il-6) with il-6 ko mice (il-6-deficient). we studied the inflammatory response in a traumatic brain injury model, cryolesion, after 3 and 10 days post-lesion gfap-il6-il6 ko mice, comparing them with appropriate controls. in basal conditions wt and il-6 ko mice showed a similar phenotype. this was also the case for gfap-il6 and gfap-il6-il-6 ko mice, which showed prominent astrogliosis, microgliosis, increased recruitment of t lymphocytes and vascularisation compared with the other two groups. in response to cryolesion of the cortex an increased astrogliosis, microgliosis, recruitment of t lymphocytes and vascularisation was observed. il-6 deficiency produced an altered inflammatory response, in a time-and gender-dependent manner. this study with il-6 ko and gfap-il6 transgenic mice indicates that during an acute neuropathological insult such as traumatic brain injury il-6, from either the brain or the periphery, has an important role on the inflammatory response. increasing evidence suggests that iron accumulation in the brain might contribute to neurodegeneration. iron is a potential source of free radicals as it can catalyze the production of hydroxyl radicals under oxidative conditions. this can lead to amplification of tissue injury caused by oxidative damage. we thus characterized iron storage within the central nervous system (cns) of animal models of different neurodegenerative diseases. we examined animals with acute inflammation mediated by cd8 or cd4 positive t cells and animals suffering from t cell and antibody mediated chronic inflammation due to active immunization. further, we studied lps induced lesions which represent cns disease caused by the innate immune system. similarly, we characterized oxidative damage in the different models for inflammation. we did not find evidence for the presence of oxidized phospholipids or oxidized dna in the experimental lesions, which is in contrast to our results of ms lesions. none of these models showed iron accumulation in glial cells comparable to what is seen in ms tissue. however, some iron positive microglia and perivascular macrophages were observed in acute and chronic models. in preliminary experiments we found evidence that the presence of iron exacerbates h 2 o 2 induced cell death in purified glial cells in vitro. as a next step, we wanted to study a possible amplification of oxidative damage and neurodegeneration by iron in a more elaborate system. for this purpose we treated myelinating spinal cord cultures with iron chloride (fecl3) or ferritin. we observed iron loading in microglia in both experimental setups. moreover we found a selective decrease of microglia in iron chloride treated cultures but not after ferritin application. we did not find iron loaded oligodendrocytes in this in vitro model. iron accumulation is only minimal compared to the human brain in all the tested animal models. these observations necessitate the search for additional animal models mimicking the human situation more closely. objectives: microglia are a brain resident population of immune cells, involved in homeostatic surveillance of the brain parenchyma, with high importance in the regulation of inflammatory processes after injury. there are unresolved questions regarding microglia origin, and their similarities to peripheral macrophage populations. our objective was to compare the capacity of microglia and bone marrow derived macrophages (bmdms) to adopt different phenotypes, and how this influenced cell death after brain injury. methods: we compared the phenotype of bmdms and microglia (both bv2 microglial cell line and primary microglia) after treatment with well known polarising agents. lipopolysaccharide (lps) was used as an inducer of the classical m1 phenotype and il-4 was used to induce an alternative m2 phenotype. microglia or bmdms, of different phenotypes, were added to hippocampal organotypic slices (hosc) subjected to oxygen glucose deprivation (ogd) as an in vitro model of brain injury. results: bmdms and microglia (bv2 and primary microglia) both adopt m1 or m2 phenotypes after treatment with lps or il-4 respectively. however, addition of polarised microglia or bmdms onto hosc resulted in different outcomes. addition of activated bmdms, of either m1 or m2 phenotypes, led to cell death in control hosc and increased death when combined with ogd. on the other hand, addition of bv2-microglia did not induce any cell death in control hosc, and were protective after ogd, except for m1microglia which were toxic. endogenous microglia within the hosc were also able to adopt different phenotypes and exert neuroprotection after il-4 treatment. these ex vivo data correlated with the in vivo situation where we observed increases in microglial activation early after experimental stroke, although the vast majority of these activated cells did not express markers of the m1 phenotype. conclusions: this study highlights functional differences between macrophages and microglia, especially in response to brain injury. although microglia, like peripheral macrophages, can adopt different demyelination, thereby creating an unfavorable environment for remyelination. exploring efficient ways to prevent or overcome tlr-induced fibronectin aggregation by astrocytes might pave the way for new approaches in remyelination-targeted therapeutic strategies in ms. to study the tlr2 response in-vivo, sod1 g93a mice were crossed with the tlr2-luc-gfp reporter mice, a transgenic mouse bearing the dual reporter system luciferase and green fluorescent protein under the transcriptional control of the murine tlr2 promoter. double transgenic mice and littermates controls were monitored longitudinally using in-vivo biophotonic/bioluminescent imaging to measure microglial activation over the course of the disease. surprisingly, this analysis did not reveal an increase of activation in sod1 g93a mice as the disease progressed, with only weak signal coming from olfactory bulb and brain area. however, quantification of this signal suggested a lower level of tlr2 activation in the sod1 g93a mice compared to wild-type littermates. to further study the microglial impairment observed in the sod1 g93a mice, these transgenic mice were given lps with i.p. injections at 5 mg/kg and were followed for 48h by bioluminescence imaging, both at a pre-symptomatic age (60 days) and closer to paralysis (115 days). the level of tlr2 activation was lower in the sod1 g93a mice than the wild-type littermates, both at 60 days and 115 days. immunostaining of olfactory bulbs reveal the same phenomenon, which is less iba1 and less tlr2-positive cells in the sod1 g93a tissue. mrna analysis by in-situ hybridization confirms a similar pattern of microglial response. in a parallel study, primary microglial cell culture, derived from adult mice (60days) were similarly stimulated with lps to further understand the altered sod1 mutant microglia response. here again, sod1 cells appears less responsive to lps stimulation as observed in our mice studies. these combined results suggest that sod1 g93a microglial cells might have a pre-symptomatic suboptimal immune response. the role of reactive glia in the etiology and progress of neurological diseases is still unknown. reactive glia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect, highlighting the relevance of a strict control of the progression and resolution of glial activation. under some circumstances glial activation does not resolve, but it exacerbates or becomes chronic resulting in detrimental secondary effects. it is necessary to develop strategies to halt the negative outcome of glial activation in these situations, by controlling the pro-inflammatory phenotype of reactive glial cells and potentiating their beneficial effects. we studied the temporal pattern of inflammatory reaction in spinal cord and brain regions in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis. special attention was paid to the involvement of members of the c/ebp family of transcription factors, which regulate the expression of pro-inflammatory genes in reactive glia, and cd200, cd200r1 and trem-2, membrane-associated inhibitors of the pro-inflammatory response in resting/surveillant microglia. mice were scored for signs of eae for up to 28 days postimmunization (dpi). eae symptoms were present from 10 dpi, reaching score peak at 14 dpi. in the spinal cord, we observed a sustained increase in c/ebpa and a transient increase in c/ebpb and c/ebpd expression peaking at 14 dpi. cd200 expression decreased at 9 dpi, remaining below control values at 28 dpi. cd200r1 and trem-2 expression increased at 14 dpi, thereafter this effect progressively attenuated. no alterations were observed in the brain. an acute increase in the expression of pro-inflammatory genes (il-1b, il-6, tnf-a, inos, cox2) occurred in the spinal cord at 14 dpi. our results suggest that cd200 expression decreases before onset of eae symptoms resulting in downregulation of the microglia inhibitory signal mediated by cd200-cd200r1, which would facilitate the proinflammatory response observed after onset of the eae symptoms. the increase in cd200r1 expression observed after the onset of eae symptoms suggests a concomitant intend of microglia/macrophages to resolve the inflammatory response. the transient increase in c/ebpb and c/ebpd expression could be related to the acute increase in proinflammatory gene expression, while the sustained increases of c/ebpa expression and trem-2 could be involved in the resolution of the inflammatory response. recently, we showed a beneficial effect of myelin reactive t cells on oligodendrocyte precursor cell differentiation in zones of axonal degeneration in the hippocampal dentate gyrus, which mirrors grey matter injury in multiple sclerosis. this effect was associated with a marked expression of t-cell cytokines, such as interferon-c and interleukin-17, enhanced microglial clearance of myelin debris, and an enhanced sprouting of calretinergic axons. to gain better insight in which cytokines and signaling pathways are elevated in response to the t cell enhanced regenerative responses, we performed a mrna microarray study, in which the transcript profile in hippocampi from mice with adoptively transferred proteolipid protein specific t cells combined with an axonal lesion were compared to the transcript profile from mice with axonal lesion. thereby we identified 1446 transcripts, which were differently expressed. we identified the interleukin-1 (il-1) signaling pathway as a potential target by performing pathway analysis using david and amigo databases. we discovered that il-1a, il-1b and il-1 receptor antagonist (ra) mrna as well as the il-1 signaling pathway were highly upregulated in response to myelin reactive t cells. in order to determine if il-1b mrna was translated into protein, we performed immunohistochemical stainings on tissues from mice with 2 days post lesion survival. expression of il-1b was observed in the molecular layer of t cell infiltrated, but not in t cell na€ ıve mice with axonal lesion, suggesting that myelin reactive t cells stimulate il-1b protein expression. ongoing studies will determine the expression of il-1a and il-1ra protein and determine the cellular expression of the il-1 signaling pathway in t-cell infiltrated versus non-t cell infiltrated mice with axonal lesion. understanding the regulation of the expression of il-1a, il-1b and il-1ra in t cell compared to non-t cell infiltrated cns may lead to a better understanding of the functional consequences of inflammatory responses in the cns. when io were challenged with kainate in the presence of conditioned medium (cm) from astrocytes, susceptibility to kainate-induced cell death was reduced, but cm from l-ap4-treated astrocytes reverted kainate toxicity. in contrast, treatment with cm from control or lpsactivated microglia, either untreated or pre-exposed to l-ap4, was not able to affect kainate-induced io cell death. to establish whether mglur4 activation could modulate the antigen presenting activity of microglia, the bv2 microglia cell line was used. treatment with l-ap4 (50 lm/48 h) did not affect changes in morphology induced by activation with lps (0.1 lg/ml/48 h), but significantly reduced the percentage of cells expressing mhc class ii, as detected by flow cytometry. these data suggest that during neuroinflammation, activation of mglur4 directly on io or through astrocytes and microglia may contribute to a protective effect resulting in survival of the oligodendrocyte lineage. mirnas are small non-coding rna molecules that modulate gene expression at a post-transcriptional level and their role in the regulation of biological processes makes them an emerging class of therapeutic targets. dysregulation of mirna networks has been linked to neurodegeneration and immune dysfunctions and has become a research focus in the context of alzheimer's disease (ad). in this work, we demonstrate that mir-155 expression is increased in the brain of 33trg ad transgenic mice, prior to senile plaque formation, and co-localized with intraneuronal app accumulation in the cortex and hippocampus. in view of the pro-inflammatory functions of mir-155 and aiming at clarifying its role in ad, we evaluated the levels of mir-155 in ab-stressed astrocytes and microglia cultures, two brain-related cell types involved in neuroinflammation. we show that mir-155 expression levels are increased in astrocytes and microglia following activation with ab fibrils but not with ab oligomers. these findings correlate with the observed decrease in socs-1 expression, a mir-155 validated target, and with the increase in the production of tnf-a, il-1b and il-6 by these cells. importantly, we show that mir-155 expression is regulated by c-jun, since silencing of this transcription factor led to the reduction of mir-155 levels following astrocyte and microglia exposure to ab fibrils or lps. c-jun was also found to be upregulated in the 33trg ad transgenic model since early ages, which can help to explain the observed early increase in mir-155 expression. overall, our results demonstrate the important role of mir-155 in ad and show that silencing of c-jun in glial cells may constitute an interesting and promising anti-inflammatory therapeutic strategy towards this disease, by targeting mir-155-mediated inflammatory responses. here we demonstrate that tgfb is an important endogenous factor promoting quiescence of microglia in vitro. inhibition of microglial tgfb signalling resulted in a microglia polarisation towards the classical activation state. moreover, tgfb is able to significantly decrease ifnc-induced classical activation of primary microglia and to enhance il4-induced alternative microglia activation. we further provide evidence that il4-mediated alternative microglia activation is dependent on active tgfb signalling. these results demonstrate the essential functions of tgfb as an important regulator of microglia activation states and further suggest that tgfb might be an interesting therapeutical molecule to modulate microglial functions during development and progression of neurodegenerative diseases. we found that young mice lost weight only in the first 24h after infection, whereas aged mice exhibited a biphasic pattern of weight loss, with the second phase of weight loss beginning 1 week after infection and continuing for approximately 2 1 =2 weeks. aged mice also demonstrated a co-ordination and balance deficit in the static rod test 1 week after infection compared to uninfected or 3 week post infection aged mice, whereas young mice were unimpaired at any timepoint. s. typhimurium infection caused a significant, progressive reduction in open field rearing activity at 1 week and 3 weeks after infection in both young and aged mice. a similar but not significant trend was apparent in novel object performance. forced swim test data showed a trend towards depressive behaviour at 1 week after infection, but not at 3 weeks. analysis of pro-inflammatory mediators in the hippocampus did not indicate significantly upregulated il-1b, tnf-a, cox-1 or cox-2 transcript at any timepoint, suggesting that microglia might not play a significant role in mediating these behavioural effects, or that the changes in these molecules were too subtle to reliably detect using qpcr in the hippocampus. the effects of ageing on s. typhimurium induced weight and co-ordination changes may be a result of regional differences in the sensitivity of microglia to peripheral infection within the cns or increased sensitivity to activation of neuronal circuits involved in weight loss or co-ordination with age. we have studied microglial cells with anti-iba1 and anti-cd68 immunohistochemistry in the brains of 18 subjects with pretangle neuropathology ranging from 4 to 51 years of age. regions examined included the locus coeruleus and surrounding brainstem areas, entorhinal cortex, hippocampal formation, and other parts of the mesial temporal lobe. ten of the subjects (median age 34) showed severe neuroinflammation secondary to infectious disease (hiv, sepsis, toxoplasmosis) and cns trauma, while 8 subjects (median age 32.5) died from non-infectious conditions and lacked neuroinflammatory changes (controls). we also examined two 52-year-old subjects with down syndrome both of which showed braak stage vi neurofibrillary degeneration, where one had comorbid sepsis and the other one did not. pretangle neuropathology was staged 1a and 1b in all 18 subjects, i.e. they exhibited primarily subcortical tau pathology in the locus coeruleus and only sporadic lesions in the transentorhinal cortex. our findings show that rampant microglial activation in the ten subjects with infections and trauma was coincident with the same level of tau pathology as seen in the 8 control subjects, demonstrating that neuroinflammation does not initiate or exacerbate neurofibrillary degeneration. in addition, our findings show that the extent of neurofibrillary pathology seen in down syndrome is unrelated to neuroinflammation since the down subject without comorbid sepsis revealed a complete absence of microglial activation. overall we conclude that neuroinflammation is not causally involved in the development of neurofibrillary degeneration which is most characteristically associated with alzheimer's disease dementia. it has been suggested that peripheral infection/inflammation may play a role in the onset or development of not only neurodegenerative diseases but also depression, autism and chronic fatigue syndrome through inflammatory responses in the brain. to investigate the role of microglia in this hypothesis we used a model of neuroinflammation induced by an intraperitoneal (i.p.) injection of the toll-like receptor 3 (tlr3) agonist, polyinosinic:polycytidylic acid (poly i:c) in rats. as we reported previously an injection of poly i:c (i.p) decreased the daily amounts of spontaneous running wheel activity to $60% of the preinjection levels until day 7. microglia were morphologically activated in the prefrontal cortex (pfc) until 72 hrs after the injection of poly i:c. pretreatment with minocycline for the 3 consecutive days (40 mg/kg/ day) blocked the poly i:c-induced decrease in the running wheel activity as well as microglial activation. quantitative analysis of mrna levels demonstrated that interferon-a (ifn-a) increased in the pfc on both days 1 and 7. we found in the present study that poly i:c injection induced increases in mrnas relevant to tlr-signaling such as tlr3, interferon regulatory factor 3 (irf3) and irf7 in the pfc. furthermore, direct application of poly i:c to the primary cultured microglia also induced an enhanced expression of ifn-a, tlr3, irf3 and irf7. it has been reported that permeability of blood-brain barrier is increased after poly i:c injection (wang et al., 2004) . therefore, it is possible that the peripheral poly i:c enters into the brain to induce neuroinflammation by activating microglia. interestingly, intracerebroventricular (i.c.v.) injection of primary cultured microglia activated by poly i:c, but not by lipopolysaccharide, effectively produced a significant decrease in spontaneous activity in rats. taken together, these findings suggest that microglial activation is important for poly i:cinduced neuroinflammation through inducing tlr3-related genes. roles of each gene in the behavioral changes will be further studied. results: microglia produced nanogram levels of pgrn (10 fold higher in microglia than in astrocytes), and pgrn release from microglia was suppressed by the tlr ligands or il-1/ifnc, but increased by il-4 or il-13. unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of slpi, none were detected in microglial cultures. we also identified mmp-12 as a pgrn proteolytic enzyme, and slpi as an inhibitor of mmp-12-induced pgrn proteolysis in human microglia. conclusions: our results establish microglia as a significant source of pgrn. mmp-12 and slpi are modulators of microglial pgrn proteolysis. negative and positive regulation of microglial pgrn release by the proinflammatory/th1 and the th2 stimuli, respectively, suggests a fundamentally different aspect of pgrn regulation compared to other known microglial activation products. microglial pgrn appears to function as an endogenous modulator of innate immune responses. at pathological condition such as injury or ischemia to the central nervous system (cns), microglial cells and astrocytes become activate and inflammation occurs like proliferate, migrate and secrete some proinflammatory cytokines, il-1ß, tnf-a or il-6. however, it is unclear that the reason why inflammation is induced in the cns, and whether or not it is necessary for the brain during recovery from pathological situation. to know the molecular mechanisms for inflammation occurs in activated glial cells, we made a stab wound mouse model to the brain. we performed microarray analysis for rna extracted from the brain tissue around stab wound site compared among day after 0, 1, 3 and 7. it revealed that most of the genes from top 20 genes at high expression level around lesion site were concerned in immunological or inflammatory functions. we successfully identified and focused on to osteopontin (opn), which is an inducer of pro-inflammatory cytokine production expressed not only in reactive microglial cells but also in reactive astrocytes around the injured cns. furthermore, we also found receptors against opn functioning in the injured brain. however, functional role of opn in reactive astrocytes is not yet clarified. to analyze the central role of opn in reactive astrocytes, we examined primary culture of astrocytes from opn-deficient (opn/ko) mice and found that the morphology of these cells was unusual. by the stimulation with lipopolysaccharide to the primary culture of astrocytes from opn/ko mice, some of the pro-inflammatory cytokine expression was altered. moreover, reactivity of astrocytes caused by stab wound to the brain was examined using analogue of nucleic acid, bromo-deoxy-uridine (brdu), and clarified that it was decreased in opn/ko mice. from these results, we conclude that opn might play a major role in the activation of astrocytes under inflammation occurred to the cns. here we determined the influence of a hfd on eae. we show that feeding mice with a hfd exacerbates eae severity through the induction of the brain renin angiotensin system (ras), resulting in an increased immune cell infiltration into the cns, oxidative stress and th1 cytokines. moreover, peripheral inflammation was boosted upon a hfd due to a ras independent mechanism, indicating that a hfd exacerbates neuroinflammation in various manners. these data suggest that nutritional modulation may have an important influence on the course of ms and other neuroinflammatory conditions. the brain's immune privilege has been attributed to a lack of dendritic cells (dc) within its parenchyma and the adjacent meninges implying maintenance of antigens rather than their presentation in lymphoid organs (lo). using cd11c-gfp mice, we have recently reported the existence of a cd11c/iba-1/cd11b 1 -population in the juxtavascular parenchyma which extent their processes into the glia limitans, an ideal position for antigen-presentation. therefore, we phenotypically compared them to the cd11c 1 /cd45 1 population (all isolated with the same protocol used for brain cd11c 1 cells) from lung, liver and spleen in healthy mice using 7-color flow cytometry. we found unique, sitespecific expression patterns of f4/80, cd80, cd86, cx3cr1, ccr2, flt3 and mhc-ii. as described before, two different cd45 1 -populations (cd45 high and cd45 int ) in the brain can be separated, whereas liver, lung and spleen exhibit a more homogeneous cd45 high population. a higher percentage of the brain's cd45 1 /cd11c 1 -cells were f4/ 80-positive compared to spleen, liver and lung, but expressed it at lower levels. within the cd45 int /cd11c 1 -population most cells expressed the microglia marker cx3cr1 and ccr2 low (marker for inflammatory, motile cells). most importantly, compared to spleen and liver, cd45 int /cd11c 1 -cells from the brain almost completely lacked mhc-ii expression and cd45 high /cd11c 1 -cells from the brain have a lower percentage of mhc-ii 1 -cells. since the cd45 int cells are widely regarded as the resident, intraparenchymal microglial population and cd45 high as dcs and perivascular macrophages, our data confirm the view that a small subpopulation of microglia (cx3cr1 high , f4/ 80 1 ,ccr2 low ) share established immune phenotypical characteristics of dcs (cd11c 1 ). additionally we showed that intraparenchymal cd11c 1 microglia are unique in their low expression of mhc-ii. their weak expression of cd80 is in line with a tolerogenic phenotype. methods: we performed a series of experiments, where we examined the expression of pro-inflammatory factors in resident microglia and infiltrating macrophages after fluorescence-activated cell sorting (facs) at different eae stages. in order to properly discriminate the two cell types we used a set of markers where resident microglia were defined as cd11b 1 cd45 int ly6c -, and infiltrating macrophages cd11b 1 cd45 hi ly6c 1 . using this protocol we have analyzed the rna expression levels of mhcii (cd74, h2-aa), co-stimulatory molecules (cd80, cd86, cd40, cd274), pro-inflammatory genes (il-1b, tnf-a) and inflammasome-regulated cytokines in these immune cells with quantitative pcr. followed by analysis of mhcii, cd80 and cd40 at the protein level with flow cytometry. results: our results indicate that infiltrating macrophages have a pronounced activated (cd74 and h2-aa) and pro-inflammatory phenotype (il-1b and tnf-a) at rna level. this is supported with an increase of the expression of mhcii and the key co-stimulatory molecules cd80 and cd40 at rna as well as protein level. in contrast, microglia display a decrease in the expression of the co-stimulatory molecules cd80 and cd86 at the rna level and do not up-regulate expression of il-1b and tnf-a during the progression of eae. conclusion: our data suggest that upon eae, infiltrating macrophages display an inflammatory profile whereas microglia are only mildly immune activated. background: age-related hearing loss (presbycusis) affects half of people by the age of 75. it has a large impact on quality of life and is associated with accelerated cognitive decline. however, presbycusis is not an inevitable process of aging, suggesting that its progression could be minimised. hearing relies on sound vibrations from the middle ear to elicit movement of the basilar membrane in the cochlea, resulting in excitation of hair cells. spiral ganglion neurons relay the inner hair cell signals to the central auditory system via the vestibulocochlear nerve. sounds are integrated in the central auditory pathway and then processed by cognitive areas of the brain. degeneration of cochlear structures, including hair cells and spiral ganglion cells occurs in presbycusis. in the central auditory system there are alterations in neuronal plasticity and neurodegeneration may occur. chronic inflammation has previously been correlated with agerelated hearing loss in humans, implicating immune cells in the progression of presbycusis. microglia within the central nervous system are susceptible to priming by neurodegeneration or aging, both factors in presbycusis. it is feasible that microglia in the cochlea, an organ with similar immune privilege as the brain, could also become primed. priming increases the chance of microglia becoming activated by subsequent inflammatory events, such as 'flu, which may cause them to contribute to neurodegeneration in the cochlea and central auditory system. hypothesis: our working hypothesis is that as age-related hearing loss progresses, microglia in the cochlea and central auditory pathway will become primed. we predict that systemic inflammation will cause primed microglia to be activated to a pro-inflammatory damaging phenotype, which will increase the rate of cochlea and auditory pathway degeneration and hence hearing loss progression. methods: c57bl/6j mice are genetically predisposed to develop age-related hearing loss. young and middle-aged mice will be challenged with lps or saline (control) and microglial phenotype assessed in the cochlea and central auditory system. neurodegeneration will be evaluated at corresponding points in the cochlea and auditory pathway using molecular techniques. conclusion: if our hypothesis is proved this would suggest that rigorous control of infection in older individuals would reduce progression of hearing loss by minimising degeneration in the auditory system. this receptor triggers the response via both myd88-and trif-dependent signalling pathways, leading to production of cyto-and chemokines and thereby alarming and attracting the immune cells of the periphery to invade the brain. however, tlr4 is only fully functional in complex with several co/receptors, such as cd14. even though cd14 has been traditionally considered simply as a lps affinity provider, recent data indicate that cd14 is also necessary for the endocytosis of tlr4, which links tlr4 to intracellular signalling via the adapter protein trif. our latest data reveal the critical role of this receptor for the profile as well as the magnitude of microglial cyto/chemokine production in response to diverse lps variants. cd14 increases the sensitivity towards lps in a cell type-specific manner, making microglia far more sensitive to lps than bone marrow and peritoneal macrophages. while striatal applications of low lps doses reveal less incoming neutrophils into a brain of cd14ko mice, as compared to wildtypes, high doses lps lead to an excessive neutrophil infiltration. this phenomenon is well correlating with the observations in vitro, where cd14 absence in microglia stimulated with high doses lps causes an excessive production of selected chemokines, with the highest impact on the neutrophil chemoatractant cxcl1. these regulatory activities of cd14 require its membrane insertion and prolonged functionality, pointing to an involvement of signalling. in order to reveal candidates for such signalling mechanisms, we tested the role of syk and plc. even though these enzymes were described to play a role in cd14-dependent signalling in dendritic cells, they have no contribution in microglia, further pointing to a cell typespecific organization of the cd14/tlr4 complex in microglia. importantly, we identified receptor systems that impose influences on cd14 expression itself, which would thereby determine the extent and impact of a cd14-mediated regulation of tlr4 functions. supported by the dfg (for1336). in neurodegenerative disease misfolded proteins and cues released by dying cells can attract immune cells by chemotaxis and alter their phenotype. to better understand and differentiate between the effect of these cues, we aim to elucidate the spatiotemporal immunological events in the brain in vivo. zebrafish are an excellent model system to study leukocytes in vivo, and we previously showed by live imaging how engulfment occurs in the developing zebrafish brain (van ham et al., curr biol 2012). here we use genetically targeted cell ablation to specifically induce cell death of target brain cells. we subsequently track dying cells and immune cells by single and multiphoton 4d imaging using fluorescent reporter genes. to analyse immunological infiltrate and resident cells in an unbiased fashion, we use large-scale electron microscopy (em) of complete zebrafish cross-sections in parallel as a complementary approach. we find that within a day after onset of cell death multiple types of phagocytic cells, including microglia, accumulate in areas where cell death occurs, engulfing dying cells. granulocytes, do not migrate towards these dying cells and are not found inside the brain at these stages. we also find total numbers of immune cells, microglia in particular, are increased. in addition to microglia, we find phagocytic peripheral cells to be involved in clearance of dying cells. our findings provide an initial in vivo characterization of the immune response to programmed cell death in the brain, indicating involvement of resident and peripheral immune cells. we show spatiotemporal recruitment of these different immune cell types, and proliferation of microglia. the latter together with the recruitment of peripheral leukocytes are likely triggered to increase capacity to dispose of dying cells efficiently. our results indicate zebrafish provides a model to tease apart basic dynamic properties of immune maintenance of the diseased brain in vivo. our current studies focus on identifying the sequence of immunological events in resolving brain injury in vivo and how this is controlled, to ultimately help identify which of these aspects are harmful during brain damage or promote tissue recovery. in humans, lif and osm both signal through the lif receptor (lifr), however osm can also activate its specific receptor, osmr. lifr signaling promotes the survival of glial cells and neurons, and is thought to limit the development of pathogenic t helper 17 cells while promoting differentiation of protective regulatory t cells. osmr signaling is also neuroprotective, however the effects on immune cells are not yet elucidated. in this study, the immunomodulatory effects of lifr and osmr signaling in humans are investigated. we determined which immune cells express the receptors for lif and osm in healthy donors and compared this to the expression levels in ms patients. we found that in blood of healthy donors approximately half of the monocytes express the lifr and osmr, while 5-10% of the t cells and b cells express the receptors. more importantly, in untreated ms patients higher numbers of t cells and b cells express both the lifr and osmr as compared to healthy controls and treated ms patients. available treatments suppress the immune system, resulting in less activated t and b cells, which could explain the lower receptor expression. indeed, we measured the receptor expression on t cells and b cells after activation and demonstrated this strongly induces expression of both receptors. currently, we are investigating the effect of lif and osm on proliferation, cytokine production and antigen presentation of the different immune cell subsets. as blood circulating immune subsets of ms patients have a higher expression of lif and osm receptors, patients show increased susceptibility for modulation by these cytokines. thus, their immunoregulating properties together with the protection of glial cells and neurons indicates that these cytokines are promising candidates for the treatment of ms and other neuroinflammatory diseases. interleukin-6 (il-6) and interleukin-10 (il-10) are key cytokines with an important role in the regulation of the inflammatory and immune responses. in the central nervous system (cns), increased expression of both il-6 and il-10 occurs in a wide range of pathological conditions. meanwhile il-6 is recognized as a cytokine with a dual role acting as a pro-inflammatory or anti-inflammatory signal inducing glial activation, il-10 is well known by its ability to counteract the inflammatory and immune reactions. the objective of the present study was to evaluate the effects of local production of either il-6 or il-10 on the microglial response and the neuronal degeneration induced by facial nerve axotomy, a sterile neuronal injury model. to accomplish that, we used two transgenic mice, gfap-il6tg and gfap-il10tg, which express either il-6 or il-10 under the gfap promoter on astrocytes, i.e. a local production within the cns. unilateral facial nerve 1mm resection was performed, one set of transgenic and wild-type (wt) axotomized animals were sacrificed at 3, 7, 14, 21 and 28 days post injury (dpi) and cryostat free-floating sections were processed for immunohistochemical analysis. another set of animals were sacrificed at 21dpi to study neuronal survival. our results showed that, at 21 days after facial nerve axotomy, in comparison with the usual neuronal death observed in wt, selective cns il-6 production had a detrimental effect on neuronal survival, whereas, il-10 production was able to reduce neuronal death. these effects on neuronal survival correlated with changes in the expression of different molecules associated with microglial activation such as iba1, cd11b, cd16/32, mhc class ii, and some integrins like osteopontin and its receptors (cd44 and a5), along the different time points after injury. remarkably, when compared with their wt littermates, cd11b expression was higher on gfap-il10tg mice, whereas remains lower on gfap-il6tg animals along all the time-points analyzed. similarly, cd44 expression, which increases after axotomy in wt mice, was higher on gfap-il10tg mice at 14 dpi and 28 dpi, whereas no induction of this molecule was detected on the gfap-il6tg axotomized animals. in conclusion, our results indicate that astrocyte-targeted il-6 and il-10 production has a direct impact on neuronal survival, glial activation and integrin mediated signaling, producing a specific outcome of facial nerve axotomy. methods: we used bv-2 cells, a murine microglial cell line. in a first experiment aimed at determining the time course of the induction of expression of the lipoxygenases and receptors, they were incubated with lps. in a second experiment aimed at determining the effects of the resolvins and lipoxin on the production of il-10, il-1b, il-6 and tnfalpha, they were firstly incubated with lxa4, rvd1 and rve1 and then incubated with lps. results: our results indicated that lps only enhanced the expression of alx, the receptor for rve1 and lxa4 ( i2) (p < 0.05). the expression of the 15-lipoxygenase was also affected by lps (p < 0.01) and varied during the time course, reaching a maximum after 6h of incubation (p < 0.05). the production of the proinflammatory cytokines decreased after 18h of incubation: il-1b with rvd1 (100 nm, p < 0.001) and rve1 (10 nm, p < 0.01); il-6 with rve1 (1 and 10 nm, p < 0.05 and p < 0.001); tnfalpha with rve1 (1 and 10 nm, p < 0.001 and p < 0.01). the production of the anti-inflammatory cytokine il-10 increased only when cells were incubated with lxa4 after 6h (1 and 10 nm, p < 0.01). conclusions: these results suggested a potential role of pufa-derivates in the resolution of inflammation. methods: the expression of a panel of m1 and m2 markers on human monocyte derived m1 and m2 macrophages was analyzed using flow cytometry. this revealed that cd40 and mannose receptor (mr) were the most distinctive markers for m1 and m2 macrophages, respectively. we next examined the activation status of macrophages/microglia in ms and control tissue using a panel of m1 and m2 markers. results: our data show that m1 markers, were abundantly expressed by microglia in normal appearing white matter and by activated microglia/macrophages throughout active demyelinating ms lesions. m2 markers, mr and cd163, were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. double stainings using anti-cd40 and anti-mr revealed that 70% of the results and conclusions: the aim of the present study was to evaluate the specific binding of [ 3 h]pk11195, a compound targeting tspo, to human tissue sections from patients with ms, and to correlate these findings with presence of tspo1 microglia in different types of lesions. specific binding of [ 3 h]pk11195 was evaluated using autoradiography, and histological analysis was performed on tissue sections to evaluate lesion type, tspo expression and presence of microglia. there was a clear correlation between level of specific binding of [ 3 h]pk11195 and lesion type, such that there was an increased binding to tissue with active or chronic-active lesions compared to control tissue or tissue with chronic lesions. this was concomitant with an increased tspo1 expression and increased presence of microglia in tissue sections with active or chronic-active lesions. in conclusion, specific binding of [ 3 h]pk11195 is correlated with lesion type, tspo expression and presence of microglia in tissue sections from ms patients. being astrocytes key regulators of microglial cell activation. during inflammatory response, glial cells secrete cytokines such as tnfa, il1b and tgfb, as well as nitric oxide (no), and activate phagocytosis, chemotaxis, increased expression of receptors that bind cytokines and inflammatory ligands. among ligand receptors are induced pattern recognition receptors such as scavenger receptor (srs), including class a receptor (sra), expressed by astrocytes and microglia. we will evaluate if sra has active roles in ab clearance and inflammatory activation of glia. methods: we evaluated the role played by sra in glial cell activation, and the regulatory capacity of astrocytes upon microglia in vitro, assessing production of no griess assay and cytokines by elisa; phagocytosis and activation of activity identity markers of signaling pathways by western blot. results: in primary glial cultures from wild type (wt) and knockout mice (ko) for sra, it was found that lps-stimulated sra ko astrocytes release 50% more no than sra wt astrocytes. these differences were associated with an extended activation of erk signaling pathway. in contrast, evaluation of lps-induced cytokines production, only wt astrocytes released il1b, whereas production of tnfa and tgfb reached similar levels in wt and sra ko animals. the abolition of il1b release by sra ko astrocytes appeared to be associated to the inhibition of activation of jnk and p38 signaling, and to the delayed activation of ijb/nfjb pathway in response to lps. microglia from sra ko mice also showed several differences in response to lps stimulation compared with their wt counterparts. they failed to show activation-associated morphological changes and were unable to secrete il1b, although they showed an increased release of no. in terms of activation pattern, sra ko microglia showed increased expression of mhc-ii, indicating that sra could be involved in a mechanism inhibiting mhc-ii expression that has not been previously documented. conclusions: our results suggest that sra participates in the activation of specific inflammatory responses by astrocytes and microglia, and the activation of signaling pathways involved in the production of soluble molecules, being also needed for astrocytes in order to be able to modulate microglia activation. microglia are the resident macrophages of the brain and the first responders to disturbances of brain homeostasis. they exist in one of two broadly defined states, a rather inaptly named "resting state" in which they exhibit a ramified morphology and, upon tissue disturbance, an "activated state" in which they exhibit an amoeboid morphology. the characterization as "resting" is in stark contrast with the frantic morphological changes that ramified microglia undergo in brain slices and in vivo, constantly extending and retracting processes. while the purinergic receptor p2y12 has been identified as a key receptor via which microglial processes are guided to a source of atp or to the site of an injury, the signals and pathways guiding microglial processes in the healthy tissue remain elusive. here, by imaging microglia in acute hippocampal brain slices, we assessed the baseline and targeted motility of microglial processes in the presence of different pharmacological agents targeting purinergic signaling. we found that while bath application of a p2y12 specific blocker alone (psb-0739 at 500 nm) or of a p2y1 specific blocker alone (mrs2179 at 25 mm) did not affect the baseline motility of microglial processes, bath application of a p2y1/12/13 blocker (mrs2211 at 25 mm) strongly reduced the baseline motility of microglia, leading them to retract most of their processes. moreover, a strong rebound of motility was observed after washing out mrs2211 that did not lead to microglial activation within the time window of the experiment (up to 90 minutes). in experiments measuring the chemotactic response of microglia to a pipette containing 1 mm of atp inserted into the slice, we found that chemotaxis was completely abolished by psb-0739 (bath applied at 2 mm) but not by mrs2211 (20 mm). finally, no chemotaxis was observed in response to a pipette containing udp-glucose (5 mm), an agonist of the p2y14 receptor. these results confirm that p2y12 alone controls the targeted chemotactic response of microglial processes to atp, with no discernable involvement of p2y1, 13 or 14, but suggest that p2y1, 12 and 13 might collectively play a role in controlling the baseline motility of microglial processes in the healthy tissue. how the different p2y receptors interact with each other to control microglial movements remains to be determined. supported by an eu marie curie fellowship, the wellcome trust and the erc. the ubiquitin-proteasome-system (ups) plays a central role in degradation and clearance of short lived regulatory as well as misfolded or damaged proteins. upon stimulation by interferons (ifn) the catalytic subunits b1, b2 and b5 of the 20s proteasomes (s-proteasomes) can be replaced with b1i/lmp2, b2i/mecl-1 and b5i/lmp7 subunits, giving rise to the catalytically active immuno-proteasomes (i-proteasomes), respectively. since i-proteasomes play an important role in clearance of oxidant-damaged proteins that preferentially accumulate upon inflammatory stimulation, and many neurodegenerative diseases, including alzheimer's disease (ad) and parkinson's disease (pd) are associated with a chronic inflammatory reaction, we tested the impact of i-proteasome deficiency on the pathogenesis and progression of ad and pd. while appps1 mice, a model for ad associated extracellular plaque pathology, exhibited increased levels of i-proteasome subunits in the brain, genetic ablation of the b5i/lmp7 subunit, which significantly impairs i-proteasome function, resulted in a reduction of cerebral plaque burden, indicating a disease exacerbating role of iproteasomes in ad. in contrast, transgenic alpha-synuclein (tgasn) mice, a mouse model for intracellular alpha-synuclein pathology associated with pd, crossed to b5i/lmp7 subunit deficient mice, highlight an increase of aggregated alpha-synuclein accompanied by worsening of pathology in tgasn mice lacking functional i-proteasomes, suggesting a protective role of i-proteasomes for intracellular amyloid pathologies. future studies will aim at resolving the mechanistic underpinnings of the seemingly opposing effects of i-proteasome deficiency on the pathogenesis and progression of extracellular and intracellular protein aggregation to aid better understanding and guide novel therapeutic opportunities for neurodegenerative diseases. in the most severe form x-linked adrenoleukodystrophy (x-ald) is a fatal neurodegenerative disorder with inflammatory demyelination of the brain caused by a deficiency of the adrenoleukodystrophy protein (aldp), encoded by the abcd1 gene. aldp, a member of the abc transporter subfamily d, is located in the peroxisomal membrane and transports very long-chain fatty acids (vlcfa) as coa-esters for degradation into the peroxisome. currently, the only curative therapies are allogeneic hematopoietic cell transplantation (hct) or genetically corrected autologous hematopoietic cd34 1 stem cell therapy (hsct). the success probably relies on the settlement of microglia and perivascular macrophages derived from the myelo-monocytic donor cells in the brain of the patient. therefore, we explored the phenotypes of the major immune cell types derived from the cd34 1 stem cell. immune cells from peripheral blood of controls and x-ald patients were isolated by magnetic activated cell sorting (macs). purity of cell isolations was verified by flow cytometry. in purified cells qrt-pcr analysis was used to determine mrna levels of the abcd transporters and gc-ms was used for the quantification of vlcfa levels, a diagnostic marker. the three peroxisomal abc transporters were differentially expressed in cd34 1 derived cell types of healthy controls: abcd1 and abcd2 mrna were inversely expressed in all cell types, whereas abcd3 was equally distributed. abcd2, the closest homolog of abcd1 could be expected to compensate for the loss of abcd1 function. however, the expression pattern of abcd2 was unchanged in x-ald patients; accordingly, cell types lacking abcd2 mrna (e.g. monocytes) displayed the most severe biochemical phenotype concerning vlcfa catabolism, whereas cells with substantial abcd2 expression (e.g. t-lymphocytes) were barely affected. thus, not all investigated immune cells present an intrinsic metabolic defect. therefore, we propose that the beneficial effect of hct and hsct may rely on the replacement of those cells lacking sufficient abcd2 expression in abcd1 deficiency. in addition, these findings support the concept that abcd2 is a target gene for pharmacological induction, as an alternative treatment strategy, to rescue vlcfa metabolism and possibly halt the inflammation in x-ald patients. interleukin-33 (il-33) is a cytokine that has important functions in inflammatory and autoimmune diseases. little is known, however, about il-33 in the brain. we analyzed expression of il-33 in the mouse brain during embryonic and postnatal development. being undetected until late embryogenesis, il-33 was highly expressed during the first two weeks of postnatal life, after which expression gradually decreased and was then absent from the normal adult brain. astrocytes and oligodendrocyte precursors expressed il-33, but not neural progenitors or neurons. the vast majority of il-33 positive cells displayed nuclear staining. apart from its function as a pro-inflammatory cytokine, a role for nuclear il-33, potentially in transcriptional regulation, is also emerging. the important role of neuro-inflammation in traumatic brain injury is being increasingly recognized, and local cytokine production can mediate the inflammatory response. because of its potential for attracting inflammatory cells we analyzed il-33 expression in a cortical contusion infarction model (cci). we found that il-33 expression was induced by injury, with a peak of expression three days after cci, and at six days the levels declined. il-33 positive cells showed an overlap with astrocytic and oligodendrocytic lineage markers, but not with neurons, neural progenitors, endothelial cells or microglia/macrophages. a similar time course for il-33 expression in human brain trauma was found, using cerebral microdialysis, which allows continuous sampling of the parenchymal concentrations of molecules over several days in vivo. the peak of released il-33 for the patient analyzed was 4 days post trauma. il-33 binds to a heterodimeric receptor composed of st2 and il-1racp. following injury of st2 knockout mice we found a general reduction of inflammatory cells at the site of injury, compared to wild type animals, and there were fewer microglia at the trauma site of mice lacking il-33 receptors. our data show that il-33 expression is under tight regulation in the normal brain, but is induced by traumatic injury where is important for attracting inflammatory cells. the small heat shock protein alpha b-crystallin (cryab) is expressed by oligodendrocytes (ol) and astrocytes at several stages of multiple sclerosis (ms) lesions. cryab has been shown to be protective and therapeutic in the eae model of ms, possibly due to its anti-apoptotic functions and regulation of inflammatory pathways. in this study, we further investigated the role of cryab in de-and remyelination in vivo, applying the cuprizone model of demyelination to cryab -/mice. surprisingly, we found that cuprizone-induced lesions are significantly smaller, less severe and less inflammatory in cryab -/mice, compared to wild type (wt) controls. staining for microglia and astrocytes revealed that astrocytes are the main inflammatory cell type that is affected by cryab expression: lesions in cryab -/mice display less reactive astrogliosis than wt controls. conversely, although less severe, lesions in cryab -/mice also contain fewer oligodendrocyte progenitor cells (opc) than in wt mice. in addition, our data suggest that remyelination is less efficient in cryab -/mice than in wt controls and that remyelinated areas in cryab -/mice contain fewer ol than remyelinated areas in wt mice. together, we hypothesize that cryab, due to its anti-apoptotic actions, mediates protection in both astrocytes and ol, enabling on one hand reactive astrogliosis, which contributes to demyelination, and on the other hand ol survival, facilitating remyelination. additional in vitro experiments support this hypothesis, revealing that cryab -/astrocytes are less reactive than wt astrocytes and that sirna-mediated knockdown of cryab affects ol survival. interestingly, analyzing phosphorylation patterns of cryab in cuprizone lesions revealed that cryab is differentially phosphorylated in astrocytes and ol. furthermore, we found that cryab is differentially phosphorylated in astrocytes in active demyelinating ms lesions, indicating that phosphorylation of the protein might underlie its (pathogenic) role in active demyelination. these paradoxical findings not only suggest a role for cryab as a potential target in a regenerative approach for ms treatment, but also point to a possible pivotal role for reactive astrogliosis in cuprizoneinduced demyelination and, more importantly, in early ms pathology. in a comprehensive immunohistochemical survey, we compared cortical ms lesions to those of inflammatory (tuberculous meningitis, rasmussen's encephalitis, b-cell lymphoma, and meningitis) and neurodegenerative (alzheimer's disease) diseases. although complex and fulminant immune responses were seen in many disease cases, primary demyelination was only detected in ms. in search of ms-specific molecular mechanisms, we performed whole-genome microarrays on micro-dissected archival formalin-fixed paraffin-embedded material. apart from cortical ms lesions, we also included brain tissue from patients suffering from tuberculous meningitis (inflammatory control) and alzheimer's disease (neurodegenerative control). additionally, control cases without brain pathology were included. using a restrictive cut-off, we identified 301 genes differentially expressed in ms lesions. more than 80% of these candidate genes could be assigned to t-cell mediated inflammation, microglia activation, oxidative stress, tissue injury, dna damage/repair as well as remyelination and regenerative processes. subsequent neuropathological analysis confirmed that significantly more oxidatively damaged neurons (stained positive for oxidized phospholipids) were present in cortical ms lesions than in any other examined disease. tunel stainings for the detection of dna strand breaks showed that neurons and oligodendrocytes with tunel-positive nuclei were most abundant in ms lesions containing zones of active demyelination and tissue injury. interestingly, immunohistochemical stainings for radical producing enzymes revealed that oxidative stress-mediated tissue damage in cortical ms lesions seems to be driven by nadph oxidases rather than by nitric oxide synthases. taken together, we were able to show that ms-specific primary demyelination is driven by mechanisms of tissue injury that differ from any other investigated inflammatory and neurodegenerative disease. among these mechanisms, oxidative tissue injury seems to play a major role. f. labombarda 1 , s. gonzalez 1 , i. jure 2 , a. de nicola 1 1 ibyme/conicet/uba, buenos aires, argentina 2 ibyme/coni-cet, buenos aires, argentina reactive gliosis and inflammatory mediators are implicated in demyelination and secondary damage after spinal cord injury (sci). we have previously reported that after sci, short-term progesterone treatment (3 days) stimulates oligodendrocyte precursor cells proliferation and decreases reactive gliosis, whereas chronic treatment (21 days) differenciates oligodendrocytes precursor cells into mature oligodendrocytes and enhances remyelination. presently, we further studied whether progesterone was able to modulate the inflammatory reaction and cytokine production by astrocytes and microglial cells. thus, the timecourse mrna expression of pro-inflammatory cytokines (il1b, tnfa and il-6) and pro-inflammatory enzymes (cox-2 and inos) was studied by pcr in real time. results showed that the highest increase in cytokine and pro-inflammatory enzymes mrna production occurred in rat spinal cord 6 h after sci. progesterone treatment significantly decreased the early rise of proinflammatory mediators mrnas at 6h. as progesterone action in spinal cord involves multiple mechanisms, the role played by the classical progesterone receptor (pr) on the progesterone inhibitory effects on cytokines, was assessed in prko mice. in agreement with data obtained in the rat model, sci strongly stimulated tnfa, il1b and il-6 mrna levels in spinal cord of both wild type and prko mice. however, whereas progesterone treatment inhibited the mrnas of cytokines in wild type mice 6h after sci, it was ineffective in prko mice, involving pr in the inhibition of cytokines production. finally, we measured astrocyte and microglial cells density by immunohistochemsitry after 6 h of progesterone treatment. the steroid administration significantly decreases gfap1 and ox-421 cells, which correlated with cytokine and pro-inflammatory enzymes inhibition. we conclude that progesterone attenuates reactive gliosis and inflammatory reaction, probably reducing the secondary spinal cord damage and favouring remyelination. in the steady state they are rarely observed in the cns parenchyma. however, during cns inflammation they cross the blood-brain barrier where together with microglia (cns-resident apc) they can present antigen to infiltrating t cells. the goal of this study was to compare two subpopulations of microglia: cd11c-and cd11c1 with brain dc in terms of promoting different t cell responses. two subpopulations of microglia: (cd11c-cd45dim cd11b1) and (cd11c1 cd45dim cd11b1) as well as (cd11c1 cd45hi) bdc have been sorted from the cns of c57bl/6 mice subjected to eae. sorted cells were used either for ex vivo proliferation and cytokine assays or for qrt-pcr. our results show that bdc and cd11c1 microglia are similar with regard to ability to induce proliferative t cell response from both primed and na€ ıve t cells. nevertheless, they differ in their cytokine profile and ability to promote cytokine release by t cells. our findings suggest that different subtypes of apc in the cns promote different immune responses. this work has been supported by lundbeckfonden. melanocortin 4 receptor (mc4r) is predominantly expressed in the brain and it is the only mcr expressed in astrocytes. our previous results showed that a-melanocyte-stimulating hormone (a-msh) the anti-inflammatory action is mediated by mc4r in astrocytes and in the hypothalamus of male rats and that these effects may lead to neuroprotection. we have already demonstrated that ndp-msh (an a-msh analogue) increased brain-derived neurotrophic factor (bdnf) expression through the camp-pka-creb pathway in astrocytes. in the present study we examined the participation of mitogen activated protein kinases (mapk) and phosphatidylinosotol-3 kinase (pi3k)-akt pathways in mc4r signaling in astrocytes. we also investigated the effect of a-msh on bdnf expression in vivo in brains of male rats.we preincubated cultured rat primary astrocytes with mapk (p38, jnk and erk) and pi3k-pdk1-akt inhibitors 15 min before addition of 1 mm ndp-msh for 1 h. we found that ndp-msh-stimulating effect on bdnf expression assayed by qrt-pcr was abolished only in the presence of erk and pi3k inhibitors. accordingly, levels of phospho-erk1/2 determined by western blot were increased by ndp-msh whereas phospho-akt levels were not modified at 30 min. ndp-mshinduced erk1/2 activation was decreased by adenylate cyclase and pi3k inhibitors but not by a pka inhibitor, suggesting a camp and pi3k involvement in this effect. we also investigated if a-msh was able to induced bdnf expression in vivo. for that purpose we injected male rats with a-msh (0.5 mg/kg, ip) or vehicle (saline) once and sacrificed them after 3 h. rats were also injected twice daily and for two days and killed 48 h after the first injection. we observed that bdnf mrna levels increased after a-msh treatment at 3h in the hypothalamus whereas in the cortex bdnf expression was not modified. at 48 h bdnf expression was not modified by a-msh. we showed by immunohistochemistry that mc4r and bdnf co-localizes with neurons and astrocytes in the brain. since melanocortins have anti-inflammatory and neuroprotective effects in the brain, the mechanisms described in astroglia help understand mc4r action. our results indicate that melanocortins induce bdnf expression in astrocytes through erk and pi3k, suggesting that this effect could be involved in the neuroprotective actions of melanocortins. the fact that bdnf expression also increases in vivo in the hypothalamus reinforce this idea. methods: we analyzed microglia activation and the role of nmda receptors in this process using the microglia cell line bv2 and cortical slice cultures. the in vivo analysis was performed in two models: first, in odtr mice that express the diphtheria toxin receptor specifically in oligodendrocytes (odcs) which allows induced killing of odcs (locatelli et al., 2012) and second in mice that were immunized with mog/ cfa to induce eae. results: the proinflammatory cytokine il-17a plays a pivotal role in the pathogenesis of ms and eae. we showed il-17a activates microglia in vitro. therefore, we activated the microglia cell line bv2 and cortical slice cultures with il-17a to and subsequently treated the cultures with the nmda receptor antagonists mk801 and ap5 to block nmda receptor signaling. this treatment inhibited il-17a-mediated activation of microglia and in particular ros production, proliferation and migration. additionally, we detected increased secretion of il-6 and g-csf. furthermore, il-17a enhanced activation of the nmda receptor through phosphorylation leading to higher influx of calcium upon ligand binding. to further analyze nmda receptor-depended microglia activation we used two different mouse models that provoke different mechanisms of microglia activation. whereas in eae a strong inflammatory process activates microglia, in odtr mice, activation occurs due to induced odc death. interestingly, in the context of eae microglia activation was clearly diminished when mice were treated with mk801 whereas inhibition of the nmda receptor had no effect on microglia activation in the odtr model. in line with the in vivo data we show that inhibition of microglia activation takes place only when the cells were stimulated with il-17a and not upon stimulation with lps. conclusions: our findings show that nmda receptors are not involved in general microglia activation but rather play a role in microglia activation in an inflammatory context with specific cytokines such as il-17a. aims of the study: to investigate whether cx3cl1-cx3cr1 signaling contributes to microglia migratio00ctivation and subsequent astrogliosis following msc transplantation in the cns. methods: first, in order to allow localization of grafted msc in vivo, wt c57bl/6 msc were transduced with a lentiviral vector encoding the blue fluorescent protein (bfp). next, bfp1 msc were injected into the cns (striatum) of cx3cr1 1/-(n 5 10) and cx3cr1 -/-(n 5 7) transgenic mice. these mice have respectively one or both copies of the cx3cr1 gene replaced by the egfp reporter gene. at 10 days posttransplantation, histological analyses were performed to determine iba11 microglia and s100b1 astrocytes within and surrounding the bfp1 msc graft site. astrogliosis was determined based on staining for gfap. cx3cr1 expression was evaluated based on egfp expression. all histological evaluations were quantified using tissuequest and/or imagej cell analysis software. results: (i) bfp1 msc graft survival is observed in both cx3cr1 -/and cx3cr1 1/mice; (ii) microglia migration towards grafted msc occurs independent of functional cx3cr1-cx3cl1 signaling; (iii) down-regulation of egfp gene-expression (and thus also cx3cr1 gene expression) is observed in both cx3cr1 -/and cx3cr1 1/mice on msc graft-invading microglia, but not on msc graft-surrounding microglia; (iv) the absence of functional cx3cr1-cx3cl1 signaling in cx3cr1 -/mice results in significantly less astrogliosis around the msc graft site, as compared to the msc graft site in cx3cr1 1/mice. conclusions: here we identified cx3cr1-cx3cl1 signaling as a potential target to modulate and/or control astroglial scarring following (stem) cell grafting in the cns. the latter is of significant importance as grafted cells can only functionally interact with (injured) brain tissue in the absence of a graft-surrounding astroglial scar. this accumulation is known to be essential for the usual sprouting of injured axons and leads to a functional nerve repair. because of the insignificant infiltration of macrophages in the injured leech cns, we investigate the chemotactic mechanisms of the recruitment of only resident microglial cells in order to explore in fine the crosstalk between damaged neurons and activated microglia leading to the leech cns repair. methods: in vitro and ex vivo chemotactic assays were performed by using recombinant form of hmc1q on microglial cells which were pre-incubated or not with anti-cc1qr or anti-gc1qr antibodies. then, affinity purification analyses using biotinylated c1q were performed from leech microglial cell protein extracts. finally, immunohistochemistry analyses were located the production of newly characterized c1q receptors in microglial cells. results: we demonstrated that rhmc1q contributes to the recruitment of some leech microglial cells. chemotaxis assays showed that the blocking of respective receptors, homologous to human gc1qr and cc1qr, inhibits the hmc1q-dependent recruitment. importantly, affinity purification analyses demonstrated the interaction between both receptors and hmc1q. finally, those receptors were located on distinct microglial subsets. conclusion: this work is used to specify the activation processes of only resident microglial cells. the results show the importance of hmc1q in the microglial accumulation leading to the nerve repair. in h. medicinalis, hmc1q activity is driven through two different receptors which are not present on the same cells suggesting that hmc1q activates two distinct microglial cell subpopulations. the question whether blood-borne immune cells are infiltrating brain areas afflicted with neurodegeneration in ad and other tauopathies yielded contradictory results. some authors showed that the chronic neuroinflammation in ad was provided almost exclusively by resident cns cells without any apparent influx of leukocytes from the blood while others reported that hematopoietic cells can enter the brain in alzheimer's disease and may contribute to an increased inflammatory burden. in order to address this issue we have used two independent transgenic lines expressing human misfolded truncated tau in two different genetic background (wistar and shr). we have shown that tau neurodegeneration can induce inflammatory responses mediated by reactive microglia in both transgenic lines, however the microglial responses showed a striking difference between the lines. we have identified two significantly different inflammatory responses to the same inducer. moreover, we found that the genetic background significantly modified the molecular cascade influencing leukocyte influx into the brain. in both transgenic lines, the majority of the blood cells entering the brain belonged to antigen presenting cell family -monocytes and dendritic cells. at the proteomic level we found striking differences between the lines. while in the wistar transgenic line we observed significant increase of cytokine-induced neutrophil chemoattractant-1, in shr transgenic line, tissue metalloproteinase 3 was significantly reduced. these findings suggest that blood cell infiltration of the brain affected by tau neurodegeneration is modified by genetic background. this inter-individual variability should be taken into the consideration in the development of novel drugs with anti-inflammatory properties. acknowledgement: this work was supported by axon neuroscience and research grants vega 2/0161/11, 2/0205/11, 2/0193/11, apvv 0200-11, apvv 0206-11 and structural fund 26240220046. charit e -universit€ atsmedizin, berlin, germany toll-like receptors (tlrs) are key molecules of the innate and adaptive immune response in vertebrates. the original protein toll in drosophila melanogaster regulates both host defense and morphogenesis during development. tlrs recognize host-and pathogen-derived stimuli. single-stranded rna is sensed by tlr7 localized to the endolysosomal compartment of immune cells. we found that both extracellular viral rna and host-derived micro-rnas induce cell-autonomous and microglia-mediated neuronal cell death through the endosomal receptor tlr7 in vitro and in vivo. however, the exact intracellular signalling cascade and the cellular mechanisms through which tlr7 leads to tissue injury in this context remained unclear. unc93b1 is a molecule specifically involved in trafficking of nucleotide-sensing tlrs, such as tlr7, in immune cells of both humans and mice. unc93b1 physically interacts with this receptor in the endoplasmic reticulum (er), and the function of the membrane protein unc93b1 is to deliver the nucleotidesensing receptors from the er to endolysosomes. making use of real-time pcr, immunocytochemistry, and histochemistry we systematically examined the expression of unc93b1 in the murine cns. whereas a distinct expression for this molecule was observed in microglia and astrocytes, expression of unc93b1 in cultured neurons was negligible. however, expression of this molecule was detected in cortical neurons of brain sections. moreover, unc93b1 was strongly regulated during different embryonic, postnatal, and adult stages of the developing mouse brain. neurons of various brain regions were identified as the main cell type expressing unc93b1 in the developing brain. taken together, our data reveal a specific expression pattern of unc93b1 in the cns, in particular in a developmental context, and lay foundation for further investigation of the pathophysiological significance of this molecule for both injurious and developmental processes in the central nervous system of vertebrates. has benefited from a number of studies that precisely depicted different types of plaques in white or grey matter areas. also, both inflammation and diffuse axonal loss in the normal appearing white matter (nawm) were extensively described. however, little attention was given to the so-called periplaque, which is usually defined as a partially demyelinated ribbon of tissue surrounding the plaque. whether periplaques correspond to expanding lesions, sites of ongoing remyelination or areas of tract degeneration remains uncertain. methods: in this context, our study aimed to bring quantitative insights to the neuropathology of ms periplaques in the spinal cord of primary or secondary progressive ms patients. a neuropathological quantitative assessment of inflammation, axonal loss and myelin loss was performed concurrently in the periplaques, plaques and normal-appearing white matter (nawm) of cervical spinal cord samples from 16 patients with progressive ms. results: periplaques formed large areas of incomplete myelin loss that extended distant away from the border of plaques. axonal loss was quantitatively similar in plaques and periplaques but signs of axonal dystrophy were predominantly observed in plaques. surprisingly, axons that remained myelinated in periplaques presented a thicker myelin sheath than myelinated axons of the nawm. inflammation in the periplaque was mainly characterized by an accumulation of macrophages/microglia that were closely apposed to myelin sheaths but exerted poor phagocytic activity. finally, we found that neuropathological features of periplaques were overall disconnected from that of plaques with regard to size, shape, inflammation and axonal integrity. conclusions: our work indicates that in ms spinal cords, periplaques correspond to demyelinating rather than remyelinating lesions. it further suggests that in progressive forms of ms, periplaques are likely to impact significantly on neurological disability. finally, we propose that tract degeneration might not be the only cause of periplaque extension and that slowly-expanding demyelination, temporally remote from plaque formation, might occur in periplaques. molecular results will be presented that support these hypotheses. cell-adhesion between endothelial cells at the blood-brain barrier (bbb) makes the endothelium impermeable to blood-derivatives and immune cells. to establish and maintain this barrier during development, adulthood, and during disease, brain endo-thelial cells must develop and sustain these strong adhesive contacts, through expression of tight junction molecules. however, we do not know whether netrin supports inter-endothelial cell adhesion at the blood-brain barrier. given this, we hypothesize that netrin tightens the bbb during development, adulthood, and protects it during disease. methods: to test this, we used both human adult primary brainderived endothelial cells and newborn netrin-1 knockout mice and evaluated netrin's effect on inter-endothelial cell adhesion and barrier permeability. we also assessed netrins' therapeutic potential to maintain the barrier and limit immune cell infiltration into the central nervous system (cns) during experimental autoimmune encephalomyelitis (eae). results: our results demonstrate that brain endothelial cells express netrins. they help to form a tighter bbb during development. they also maintain and protect the adult barrier by increasing the expression of endothelial junction molecules, thus promoting inter-endothelial adhesion and reducing protein leakage across the barrier. netrins also reduce bbb breakdown and diminish initial myeloid cell infiltration into the brain and spinal cord during eae, which delays disease onset and ameliorates disease severity. discussion: we conclude that netrins enhance bbb stability, and protects the bbb during neuroinflammatory disease. microglial cells were shown to play an important role in many diseases of the central nervous system, not least due to their capability to become activated upon signs of brain injury. they migrate to lesion sites, proliferate, release cytokines and chemokines, and phagocytose cells or cellular debris. therefore, the microglial cytoskeleton has to be highly rearrangeable. one major element of the contractile system in nonmuscle cells is nonmuscle myosin ii (nm ii). as the role of nm ii in glial cells is poorly characterized, it is an aim of this project to study nm ii-dependent functions in microglia. we found that inhibition of nm ii by blebbistatin, which is a highly selective inhibitor of nm ii adenosine phosphatase, prevents any morphological shaping in freshly plated microglia and leads to functional deficits during migration and phagocytosis of fluorescence-labeled beads. using confocal microscopy, we could find diffuse expression of nm ii in resting microglia in vitro, whereas activation with bacterial lipopolysaccharide (lps) led to perinuclear accumulation of nm ii protein. in an activated state, microglial cells release pro-inflammatory cytokines and reactive oxygen species; interestingly, in the presence of blebbistatin the release of nitric oxide (no) as well as rearrangement of nm ii was prevented. taken together, these results illustrate a key role for nm ii in microglial shaping and functioning. ongoing studies will improve our knowledge on spatial and functional aspects of the actomyosin complex during demyelinating processes and open targets for development of therapeutic strategies towards remyelination in the cns. prolonged activation of the nucleus basalis of meynert (nbm), the primary source of cholinergic projection to the cerebral cortex has been reported to cause significant increases in cerebral cortical blood flow (cbf) in rodents. while the nbm also gives rise to gabaergic and glutamatergic projections to the cerebral cortex, the nbm-driven increase of cbf has been described to be dependent in part on muscarinic acetylcholine receptors. lately, several groups reported that astrocytes modulate local cbf via intracellular ca 21 signaling. considering that cortical astrocytes express machrs and in vivo activation of the nbm leads to machr-dependent ca 21 surges in astrocytes, cholinergic modulation of cbf via astrocytic ca 21 surges is conceivable. we report here that a single train stimulation of the nbm (stnbm; 100 hz, 0.5 s, 200 ma) induces a biphasic (a rapid increase, followed by an overshooting slower decrease that goes back to baseline within a minute) laser doppler flowmetry (ldf) response in the somatosensory cortex. moreover, the stnbm-induced ldf response was sensitive to the machr antagonist atropine. stnbm with a weaker stimulation intensity (50 ma) induces a transient decrease. surprisingly, we find that ip3r2 knockout mice, which lack cytosolic ca 21 surges in astrocytes, show similar ldf responses to stnbm. a.-c. boulay, c. giaume, m. cohen-salmon cirb, paris, france astrocytic endfeet are specialized processes, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. one of the most striking properties of astrocyte endfeet is their enrichment in the gap junction proteins connexin (cx) 43 and cx30 allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. however, the role of these proteins at the gliovascular interface is not fully understood. we have recently demonstrated that astroglial perivascular cxs expression starts after birth during bloodbrain barrier maturation and controls its integrity (ezan et al. cx30 d/d purified vessels showed essentially a strong upregulation (fold change > 12) of sgcg. this gene encodes gamma-sarcoglycan, a membrane glycoprotein associated with the dystrophin-dystroglycan complex which is essential for muscle integrity and is impaired in duchenne muscular dystrophy (petrof et al., 1993) . at the gliovascular interface, the dystrophin-dystroglycan complex anchors astrocyte endfeet to the basal lamina (nico et al., 2010) , and is crucial to the bbb integrity. however, expression of sgcg and its role in the brain has until now only been poorly documented. moreover, we found an upregulation of s100a8 and s100a9 (fold change 6,3 and 5,5, respectively) encoding the calgranulin a and b, two proteins present in neutrophils and monocytes, and involved in their migration to inflammatory sites, attachment to endothelial cells and extravasation (ryckman et al., 2003) . altogether, our results suggest that cx30 might modify the dystrophin-dystroglycan complex thereby influencing blood-brain barrier properties, and control the attachement as well as the transmigration of leukocytes through the endothelium, thus contributing to the regulation of inflammatory processes in the brain. the blood-brain barrier is supported by the perivascular glia. a crucial event during the gliogenesis is the replacement of radial glia by astrocytes. immunohistochemical staining of nestin visualized the first gliovascular connections after e16, and by e20 the radial glial endfeet cover the vessels completely. by e20 numerous nestin immunoreactive astrocyte-like cells participated in it. in parallel, nestin immunoreactivity also appeared in the wall of the vessels. by about p10 both radial glia and nestin immunopositive astrocytes disappeared and gave place to astrocytes immunoreactive to s100 protein and glutamine synthetase, markers of mature astrocytes. in the radial glia these markers were not found, and they colocalized with nestin only scarcely. the s100 and glutamine synthetase immunoreactive cells appeared at first in the middle zone of the pallium about p2, and their population gradually extended to both the pial surface and the deep cortical zones, colonizing the whole thickness by p8, in parallel with the disappearance of nestin immunoreactive glial elements, either radial or astrocytic ones. at p6 gfap immunoreactive cells were still confined to the most superficial and deepest cortical zones, so showed no colocalization with s100 and glutamine synthetase, unlike that found after p8. whether this glial 'takeover' results in a transitory increase of leakage of the blood-brain barrier, it remains to be answered. it seems however, to underly that cerebrovascular fibronectin and laminin immunoreactivities disappear at first from the middle zone of the developing cortex and only later from the superficial and deep ones, between p2 and p10. according to krum and rosenstein (1991) disappearance of these immunoreactivities refers to the fusion of the glial and vascular basal laminae, the maturation of the definite gliovascular connections. surrounded by astroglial scars that inhibit such growth. little is known about how astrocytes assemble to form this scar, and a deeper understanding of the cellular and molecular mechanisms that control this process is needed if this goal is to be achieved. in vitro data suggest that certain bioactive molecules can alter astrocyte morphology into a more growth supporting state. other molecules have been shown to promote axon growth. clinical application of many of these molecules would require neurosurgical delivery directly into the spinal cord. injectable biomaterials have the potential to serve as depots and scaffolds for in vivo delivery of bioactive molecules. we are developing di-block co-polypeptide hydrogels (dch) as fully synthetic biomaterials that could safely and easily be injected into specific sites after sci to deliver molecules in order (i) to understand better, and (ii) to manipulate, the mechanisms that control glial scar formation. we have previously shown that dch are biocompatible after injection into brain or spinal cord and selfassemble into structures with finely controllable properties. our current work shows that dch depots can deliver diffusible bioactive growth factors that influence local neurons or astrocytes in predictable ways and form gradients that are effective up to several mm away from depots. we are now testing the ability of dch depots to deliver specific growth factors that will manipulate scar-forming cells when dch are injected at clinically realistic sub-acute times after sci. to investigate the molecular phenotype of esdm, we performed a whole transcriptome analysis of esdm in comparison to primary cultured microglia, flow cytometry-sorted microglia, different myeloid cells, t cells, astrocyte-and neuron-enriched cultures. while being distinct from t cells, neurons and astrocytes, esdm were closest related to primary cultured microglia followed by flow cytometry-sorted microglia and other myeloid cells. esdm and primary cultured microglia showed a strong overlap in their transcriptome by only having 143 gene transcripts differentially expressed. most of these differentially expressed genes were immune-related genes that were up-regulated in primary cultured microglia. this indicates that esdm were in a less activated state rather reflecting the in vivo biology of these cells. flow cytometry analysis of cell surface markers selected from the microarray confirmed the close relationship between esdm and primary cultured microglia. esdm resemble primary microglia not only in respect to surface marker expression and functional properties, but also display a molecular phenotype similar to primary microglia. our data suggest that esdm are a reliable tool for the replacement of primary microglia. spinal cord injury (sci) is a devastating condition which usually leads to paralysis. this outcome of injury in most cases is permanent due to the cascade of immunological factors that create an anti-repair environment around the site of injury and due to the limited capacity of central nervous system (cns) axons to regenerate, creates a daunting challenge to establish an effective therapy. from many years of research it is apparent that a combination of therapies would be the best course of action for treatment of sci; this however greatly increase the animals cohorts and time to establish the most effective course of treatment. for this reason we have developed an in vitro model of spinal cord injury as a moderate throughput screen, which is in accordance with the 3r's. this culture consist of dissociated mixed rat embryonic spinal cord cells plated onto a monolayer of astrocytes this is essential for the spinal cord cells, obtained from rats of embryonic day 15, which then develop into a carpet of neurites that over time become myelinated forming internodes of myelin interspaced with nodes of ranvier. this mixed cns culture was utilized using a scalpel blade to axotomise the axons and a persistently neurite-free area is created which is accompanied by many features of sci, including demyelination and reduced neurite density adjacent to the lesion and the presence of and reactive astrocytes. these finding are congruent with changes seen in vivo after spinal injury. data will be presented regarding the effects from proven and novel pharmacological agents on neurite outgrowth and myelination looking to understand the mechanism in which they act. as well as modifications of the culture system to incorporate methods to align axons and isolate cell bodies from their processes for a more detailed analysis of regeneration. the role of increased activity of astrocyte networks in structural synaptic plasticity following remote axonal injury is unexplored. we set out to investigate the efficiency of synaptic rearrangements in models where astrocyte reactivity is selectively diminished by the inhibition of the main cytokine pathway in transgenic mice (tg) in comparison to that observed in wild type mice (wt). first, synapse densities were compared in the facial nuclei two weeks after unilateral facial nerve transection by synaptotagmin-1/psd-95 immunolabelling and ultrastructural analysis. we found that the recovery of synapse density was decreased by 40% in tg mice compared to wt mice, and that the reduction in synaptic density correlates with a relative loss of neuronal coverage by reactive astrocyte end-feet in the same conditions. specifically, we observed a 30% reduction in excitatory synapse density in the tg mice. second, we developed an organotypic entorinho-hippocampal slice culture system in which astrocyte activation and synaptic plasticity could be more closely monitored in real time. this was assisted by two-photon microscopy, using glial ca 21 -imaging and dendritic spine motility analysis after perforant pathway transection. in summary, we demonstrated that full astrocyte activation is necessary for partial structural recovery of synapses after axonal injury. our work, using simplified models, may help experimental approaches aimed at optimizing adaptive plasticity in cns injuries. pathological loss of myelin in diseases like multiple sclerosis (ms) is usually followed by a phenomenon of remyelination, in which oligodendrocytes synthesize new myelin sheaths to envelope exposed around axons in the adult central nervous system (cns). the importance of remyelination arises from the fact that this process not only restores saltatory conduction, but also protects axons from different insults and thus limits clinical disability in demyelinating diseases. in this scenario, the mammalian subventricular zone (svz) has garnered attention as a potential source of replacement cells after injury. this zone harbours stem cells and supports long-distance migration, and is activated in ms patients to promote gliogenesis. although ng2 1 precursor cells are the first to react to demyelination and show the highest proliferation rate in comparison with other cells, the relative contribution of the svz with respect to the inflammatory demyelination induced by the theiler's virus infection and oligodendrogenesis has never been addressed. in the present study, we have investigated the behavior of the svz in theiler's murine encephalomyelitis virus -induced demyelinating disease (tmev-idd). we report that in this viral model for ms, there is a preclinical phase of the disease with demyelination in the corpus callosum that is followed later by an attempt of remyelination. this phase is accompanied by an activation of the svz with no apparent activation of ng2 1 precursors, and a strong and clear staingalectins (gals) are a family of soluble lectins that, when binding to b-galactosides, form multivalent complexes with glycoconjugates of the cellular surface and induce a modulation of intracellular signaling pathways for differentiation and survival. recently, galectin-1 has been described as a microglial deactivator which prevents neurodegeneration and promotes neuroprotection in an eae model. however, its action at neuronal level is poorly understood. spinal cord injury (sci) also remains a major challenge to neurological research, as several factors such as extracellular matrix molecules inhibit axonal regeneration. among them, semaphorine 3a (sema3a) contributes to the inhibition of axonal regeneration by acting on microtubules and actin cytoskeleton. neuropilin-1 (nrp-1) is a neuronal receptor whose binding of sema3a generates repulsion of axonal growth. in addition, nrp-1 has been described as a target of gal-1 in non-neuronal systems. the aim of this work was to study the role of gal-1 intraumatic sci, considering that sema3a expression is "turned on" in sci and prevents axonal regeneration via nrp-1. gal-1 knockout mice (lgals1 -/-) were submitted to a full traumatic sci at thoracic level (t9-t10) and treated with different concentrations of recombinant gal-1. we demonstrated that lgals1 -/mice treated with gal-1 have a dose-dependent functional recovery of motility as early as 6 days post-treatment, which is structurally associated to neuronal repopulation and high axonal regeneration. these effects were only observed in animals treated with gal-1 with dimerizing capacity, but not in mice treated with a mutant gal-1, only present in a monomeric form. in addition, we observed that microglial response was "turned off" in gal-1-treated mice. moreover, exogenous dimeric gal-1 bound to the surface of injured motoneurons and promoted the spread of nrp-1. 3d confocal microscopy reconstruction showed gal-1 and nrp-1 spatial proximity. to confirm this result, we carried out immunoprecipitation assays, which allowed us to determine that only the dimeric form of gal-1 interacted with nrp-1 and induced a decrease in the levels of sema-3a bound to nrp-1. our results show that gal-1-based treatments combine its neuronal effect on nrp-1 with its deactivating effect on microglia, which leads to a better restorative process after medullar lesion. the findings presented here support the potential of dimeric gal-1 as a therapeutic agent for human sci patients. theophylline and caffeine. it depresses activation of microglial cells and astrocytes which is associated with neuronal damage during inflammation and hipoxia. it is largely known that ethidium bromide (eb) injection into the cns induces local oligodendroglial and astrocytic death, resulting in primary demyelination, blood-brain barrier disruption and schwann cell invasion, and thus serving as an experimental demyelinating model in animals. the aim of this study was to evaluate if prop had the capacity of affecting glial cell behaviour during the process of demyelination and remyelination following gliotoxic injury with eb. wistar rats were divided into 2 groups: i-rats injected with 10 microlitres of 0.1% eb into the cisterna pontina and treated with prop; ii-rats injected with eb and not treated with the xanthine. prop (agener) treatment was done using 12.5 mg/kg/ day by intraperitonial route during all experimental period. the rats were euthanized from 7 to 31 days after eb injection and brainstem sections were collected and processed for light and transmission electron microscopy studies. results from both groups were compared by using a semi-quantitative method developed for documenting in semithin sections the extent and nature of remyelination in gliotoxic lesions. in general terms, by 7-11 days following gliotoxic lesion, the center of the lesion was expanded and filled with huge amounts of myelin debris among foamy macrophages and demyelinated axons. no astrocytic processes were found in this site and some lymphocytes were seen in the neuropil and around blood vessels. the most prominent feature of the 15 th day was the initial association at peripheral locations between naked axons and remyelinating cells. schwann cells were associated with one or multiple demyelinated axons or already forming thin myelin lamellae around single axons in astrocytic-free areas. oligodendrocytes began to form thin myelin sheaths in areas completely or partially filled with astrocytic prolongations. by 21-31 days, it became evident that rats treated with prop presented a small improve in the repair process when compared to untreated animals, the latter presenting greater amounts of myelin-derived membranes in the central area and a lesser extension of oligodendrocyte remyelination, but without any apparent difference regarding to schwann cells. by 31 days results showed that prop administration following eb injection seemed to stimulate oligodendroglial remyelination (mean remyelination scores of 3. peripheral axotomy results in a disconnection of the neuronal cell body from the target organ. a complex rearrangement of the nerve microenvironment, the so called wallerian degeneration, takes place distally to the lesion and involves schwann cell proliferation as well as macrophage infiltration. the process results in the formation of the bands of b€ ungner which guide the axonal sprouts throughout the nerve distal stump. although this process has been extensively studied, a thorough understanding of the three-dimensional nerve tissue reorganization is not completely known. such knowledge may in turn lead to more effective strategies aiming at the repair of peripheral nerves following transection or crushing. in this sense, the tetrahydrofuran (thf)-based tissue-clearing technique has been adapted for sciatic nerves of mice and used for subsequent observation to 2p-lsm. transgenic mice that express fluorescent proteins in neurons, astrocytes, schwann cells and microglia/macrophages as well as double and triple hybrids were used. additionally, by immunohistochemical analysis, it was possible to observe the structure of the nerve after injury by the expression of neurofilament and s100b, in non fluorescent mice. also, increased non neural cell responsiveness after injury was studied with anti-p75 ntr immunostaining. transmission electron microscopy allowed the evaluation of the ultrastructure of the nerve microenvironment, as well as the regenerative process by 3, 7 and 14 days after injury. microscopic examination of cleared tissues of transgenic mice through 2p-lsm consisted in comparing the different cell type interaction before and after injury, with particular interest on the schwann cells, macrophages and growing axons. question: mass spectrometry (ms) has become nowadays an essential tool for the detection and identification of biomolecules in various contexts of biological problematic. ms can be performed from all types of biological materials ranging from biological fluids to tissues. end of 90's it was shown that with certain instrumentation such as those equipped with a maldi ion source it was possible to record ms spectra directly in situ from tissue sections. more, point to point automated acquisition of maldi ms spectra from a whole tissue section was shown to allow for reconstruction of the 2d images of biomolecules contained in the section distribution through ion density maps (caprioli rm et al., anal. chem. 1997). methods: maldi ms imaging (maldi msi) is a molecular imaging modality that presents several advantages including possible imaging of hundreds of biomolecules in one step acquisition and the untargeted character of the method allowing to study molecules without any prerequisite knowledge or use of probes. results: over the past decade maldi msi has demonstrated its potentiality in terms of applications for biology and clinics (franck j et al., mol. cell. proteomics. 2009). maldi msi can be use to study both endogenous molecules namely metabolites, lipids, peptides and proteins or exogenous ones such as drugs or xeniobiotics and was applied to various problematic. understanding of physiological or physiopathological processes requires knowledge on the identity and localization of molecules within a tissue. maldi msi reseals a clear potential to answer this objective and give a unique contribution. indeed, more recently maldi msi has gained new strategies for identification of molecules in situ on tissues. in particular, many efforts were given to develop confident identification of proteins from tissue sections. conclusion: here we will review this novel technology and discuss some of its current and potential contributions for neurosciences application (wisztorski m et al., dev. neurobiol. 2008). this will be illustrated by presentation of the investigation of invertebrate neuroimmune response as well as vertebrate neuropathological disorders using either fresh frozen or formalin fixed paraffin embedded tissues. the sphingosine 1-phosphate (s1p) signaling pathway is known to influence astroglial reactivity in experimental autoimmune encephalomyelitis and the synthetic s1p analog fty720 has been shown to provide neuroprotection in experimental models of acute stroke. however, the effects of a manipulation of s1p-s1p1 signalling at later time points after experimental stroke have not yet been investigated. we examined whether a relatively late initiation of a fty720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors. we induced photothrombotic stroke (pt) in adult c57bl/6j mice and allowed them to recover for three days. starting on post-stroke day 3, mice were treated with fty720 (1 mg/kg b.i.d.) for 5 days. behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, taqman v r analysis and immunofluorescence with especial emphasis on markers of astroglial reactivity. fty720 treatment results in a significantly better functional outcome persisting up to day 31 after pt. this is accompanied by a significant decrease in gfap-immunoreactivity (-ir) and an increase in psdsize. however no change in gfap-mrna, gs-ir or cs56-ir was observed. while fty720-treatment leads to a decrease in s1p concentration, it increases vegf-mrna in the periinfarct cortex. the initiation of fty720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors. due to the restrictions in regenerative capacity of the brain the tissue most severely damaged after traumatic brain injury (tbi) will end up as a fluid filled cavity. presently, it is an impossibility to effectively restore lost brain tissue, but ongoing research on the stimulation of endogenous neural stem cells has increased the hope to viably and functionally repopulate the injured parenchyma. however, it is crucial that possible therapies can show a long-term effect on both regeneration and functional recovery to be of clinical interest. in this study the enhanced induction of neurogenesis in rats after experimental tbi was evaluated three or six weeks after injury. severe focal tbi was performed using the controlled cortical impact model after which a combination therapy of intracerebroventricular administration of epidermal growth factor (egf) for seven days followed by deposition of extracellular matrix scaffold, containing vascular endothelial growth factor, into the cortical cavity. the treatment was devised to accomplish an optimal effect on the stem cell regeneration. the animals with six weeks survival time were functionally evaluated using the morris water maze (mwm). before sacrifice the animals were injected with bromodeoxyuridine (brdu) to identify newly generate cells. sections were made and immunohistochemically double stained for brdu and cell type markers for neurons, astrocytes and blood vessels. the sections were micrographed and analyzed for hemispheric tissue loss as well as number of new astrocytes, neurons and blood vessels. three weeks after injury there was a significant treatment effect as shown by an increase in neuronal and astrocytic regeneration. however, after six weeks there was no difference in the number of newly generated neurons and astrocytes. evaluation of tissue loss and spatial learning in the mwm corroborated that the treatment had no effect at the later time point. the results clearly show the importance of longterm studies in rodents to ensure that a promising effect on tissue regeneration and functional outcome is not merely temporary. astrocyte activation to extracellular matrix (ecm) -producing cells is inhibitory to regeneration in cns injury or disease. here we show that the cleaved neurotrophin receptor p75 ntr controls transforming growth factor (tgf)-beta-mediated astrocyte activation via regulation of nucleocytoplasmic shuttling of smad2. loss of p75 ntr completely rescues tgf-beta-induced hydrocephaly, astrocyte activation, and ecm production in gfap-tgf-beta transgenic mice. nuclear fractionation of tgfbeta-treated p75 ntr knock out astrocytes revealed reduced nuclear phosphorylated smad2 than their wt counterparts. in accordance to p75 ntr depletion, inhibition of tgf-beta-induced gamma-secretasemediated p75 ntr cleavage also reduces nuclear accumulation of smad2 and the secretion of proteoglycans that inhibit neurite outgrowth. taken together, our results identify regulated intramembrane cleavage of p75 ntr as a mechanism for astrocyte activation resulting in the regulation of tgf-beta signaling and scar formation in the diseased brain. the reaction of glial cells toward brain injury comprises a proliferative response with some reactive astrocytes even reacquiring stem cell potential as indicated by forming long-term self-renewing and mutlipotent neurospheres (for review: robel et al., 2011). here we addressed first the question to which extent the type of injury selectively affects the proliferative response of ng2 glia and astrocytes and to which extent the proliferative reaction of astrocytes may be linked to the stem cell response. interestingly, while both astrocytes and ng2 glia mounted a profound proliferative response after acute invasive injury, such as stab wound and mcao, non-invasive injury models, such as amyloid plaque deposition or widespread neuronal death could not activate the proliferation of these macroglial cells, while microglia proliferated actively in all these lesion models. intriguingly, the proliferative response of astrocytes correlated to the activation of their potential to form self-renewing, multipotent neurospheres with a steep decline with age. we then set-out to determine the signals regulation reactive astrocyte proliferation and neurosphere formation after invasive injury and demonstrate that invasive lesions result in entrance of sonic hedgehog (shh) into the brain from extraneural sources, such as the cerebro-spinal fluid, and activate astrocyte proliferation and neurosphere formation. this response can be blocked by systemic injection of the shh-signaling antagonist cyclopamine as well as inducible astrocyte-specific deletion of the receptor smoothened by glast creert2 . interestingly, shh-agonists can boost the proliferative response of astrocytes in vivo and their subsequent neurosphere-forming capacity, thereby providing a new approach how to activate this response in conditions where it is limited, as e.g. in age or noninvasive injury conditions. taken together, our work highlighted for the first time differences in the proliferative and stem cell response of reactive astrocytes in different injury paradigms and identified the shh signaling pathway as a key signal in this response. tumor necrosis factor (tnf) is a pleotrophic cytokine involved in normal brain function and inflammation, apoptosis, and other responses that occur following injury and disease. there is a considerable amount of confusion as to whether or not tnf activation is beneficial or detrimental following injury to the cns. genetic studies in mice have suggested that inflammation in disease models involves soluble tnf (soltnf) and that maintenance of innate immune function involves transmembrane tnf (tmtnf). these findings suggest that the outcome of selective pharmacologic inhibition of tnf may depend on whether the pharmacologic intervention is targeted towards soltnf or tmtnf. therefore, we took advantage of a dominant-negative inhibitor of soltnf, xpro195, that selectively inhibits soltnf and compared the effect of this inhibitor to a more widely used igg1 fc-tnf receptor (tnfr) 2 fusion protein etanercept, an inhibitor of both soltnf and tmtnf, that has been a successful treatment strategy for several autoimmune diseases. however, etanercept has been shown to display severe side effects by increasing the risk of infections. female c57bl/6 mice were subjected to a spinal cord injury at t9 level. mice were divided into three groups, receiving etanercept, xpro1595, or saline. the drug was delivered directly onto the lesion site using alzet mini osmotic pumps for 3 days starting immediately after introduction of the lesion. mice were allowed to survive 35 days. functional evaluation was performed using the basso mouse scale, rung walk, open field test and hargreaves test. in contrast to etanercept, xpro1595 significantly ameliorated recovery of hind limb function (evaluated by motor recovery score) compared to saline when administered continuously to the lesioned cord, whereas s.c. injections every 3 days for 8 weeks following sci had no effect. our study suggests that selective inhibition of soltnf is beneficial following sci when administered directly to the contused spinal cord and raises the possibility that selective inhibition of soltnf may represent a new therapeutic strategy for the treatment of sci without compromising the innate immune response. here, we show that expression of oncostatin m (osm) is upregulated after spinal cord injury (sci). to reveal the relevance of increased osm signaling in the pathophysiology of sci, osm was applied locally after hemisection. acute osm-treatment significantly improved locomotor recovery after mild and severe sci. delayed osm-treatment in the chronic phase was ineffective. improved recovery in osm-treated mice was associated with a reduced lesion size, less astrogliosis and diminished neuroinflammation. the underlying mechanism involves neuroprotective effects of osm. furthermore, osm promoted nerve fiber regeneration both in vitro and in vivo. thus, local production of osm after sci is an endogenous response that limits cns lesion propagation and promotes recovery. the university of western australia, crawley, australia following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess ca 21 a likely trigger for loss of neurons, compact myelin and function. treatment of secondary degeneration by limiting excess ca 21 entry into cells using inhibitors of specific ca 21 channels showed promise in preclinical studies, but clinical trials were disappointing and combinatorial approaches are acknowledged as necessary. we assessed efficacy of every possible combination of three ca 21 channel inhibitors at reducing secondary degeneration 3 months after partial optic nerve (on) transection in rat. we used lomerizine to inhibit voltage gated ca 21 channels (for 3 months); oxidised adenosine-triphosphate (oxatp) to inhibit purinergic p2x 7 receptors and/ or 2-[7-(1h-imidazol-1-yl)26-nitro-2,3-dioxo-1,2,3,4-tetrahydro quinoxalin-1-yl]acetic acid (inq) to inhibit ca 21 permeable a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (ampa) receptors (both for the first 2 weeks after injury). only the three ca 21 channel inhibitors delivered in combination completely preserved visual function to levels not different from normal animals. in order to determine the mechanism/s by which the three inhibitors delivered in combination preserved function, we assessed neuroprotection, nerve swelling, myelin compaction and node/paranode complexes. preservation of retinal ganglion cell (rgc) somata and axons is unlikely to have accounted for the full recovery of function as only partial neuroprotection of rgcs vulnerable to secondary degeneration was achieved. a range of the ca 21 channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration, with no one agent selectively effective. each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin and decreased the proportion of axons with partially decompacted myelin, indicating the importance of sustained control of ca 21 flux via voltage gated ca 21 channels for prevention of chronic myelin decompaction. nevertheless, limiting decompaction of myelin or formation of atypical node/ paranode complexes was not sufficient for preservation of function in our model. it is likely that the additional prevention of the lengthening of the paranodal gap that was only achieved by treatment with the three ca 21 channel inhibitors in combination was an important effect that contributed to the associated preservation of visual function by this combinatorial treatment strategy. to generate myelinating oligodendrocytes opcs undergo a distinct series of morphological and molecular changes. these are regulated by a combination of extrinsic and intrinsic factors, which have only been partially been revealed so far. in the present study we identify a novel signaling mechanism as potent negative regulator of opc maturation. ephrin b3, a member of the b-class family of ephrins, inhibits opc process formation and blocks the formation of mature oligodendrocytes in vitro. the presence of ephrinb3 results in activation rhoa and pkca signalling and deactivation of fak kinase in opcs in vitro. moreover, epha4 rtk, a receptor responsive to ephrinb3 in opcs, is also expressed by opcs in demyelinating lesions. infusion of recombinant ephrin b3 into demyelinating lesions results in a failure of remyelination caused by an inhibition of opc maturation. in contrast, antibody-mediated masking of ephrin b3 epitopes in vitro as well as in demyelinating lesions of aged rats promotes opc differentiation and accelerates myelin regeneration. our results demonstrate an important role of ephrin b3 for the process of remyelination. after peripheral nerve injury, axons rapidly degenerate and start regrowing only after a few days. full functional recovery, however, is rare. a progressive loss of intact axons also determines the clinical phenotype of chronic demyelinating peripheral neuropathies, such as charcot marie tooth disease type 1a (cmt1a). cmt1a is the commonest inherited neuropathy, caused by a duplication of the peripheral myelin protein 22kda (pmp22) gene. we demonstrate that the transgenic neuronal overexpression of the growth factor neuregulin-1 type i enhances axonal regeneration and functional recovery after experimental acute peripheral nerve injury. similarly, neuregulin-1 type i overexpression preserves axonal loss and improves nerve function in a mouse model for cmt1a. importantly, neuregulin-1 type i induced axonal preservation in cmt1a mice was independent of myelination, as myelin sheath thickness was not altered. we conclude that axonal recovery and preservation relies on common supportive factors and that paracrine neuregulin-1 type i constitutes a beneficial signal in peripheral nerve disorders. g. szab o university of tuebingen, tuebingen, germany question: the enormous capacity for repair of the cns after lesion is one of the most amazing property of lower vertebrates. in the mammalian central nervous system (cns) the astrocytes reveal the so-called orthogonal arrays of particles (oaps); the clusterization of the aqp4 molecules at the astrocytic endfeet. in lower vertebrates these structures are lacking. in the case of the amphibia -anolis carolinensis-the caudal spinal cord has the capability for regeneration after loss of the tail, an ability which might be in relation with the presence of tight junctions (tj) between the glial cells like the olfactory ensheating cells act in mammalian olfactory system through the lifespan. the growing axons of the fish optic nerve are embraced by astrocytes which are also interconnected by tjs. tj connected glial cells could contribute to a growth-supportive microenvironment which might reinforce our hypothesis namely the mutual exclusion of aqp4 occurrence in astrocytes and regenerative growth of nerve fiber. methods: we performed immunocyto-and immunohystochemical staining, western blot, freeze-fracture electron microscopy and pcr on cultured aqp4 knockout and wildtype astrocytes and also on formalin fixed paraffin embedded tissue sections regarding junctional proteins, such as occludin, claudin 1,3,5. result: in the light of our experiments, there is no mutual exclusiveness between the presence of aqp4 and tight junctions in astrocytes, at least in mammals. further investigations need to be completed. glutamate and electrical activity influence opc differentiation and myelination in normal development. opcs receive synaptic input from unmyelinated axons, possibly to initiate myelination. both opcs and mature oligodendrocytes respond to glutamate via ampa and nmda receptors. activation of glutamate receptors in vitro increase opc migration but decreases proliferation. here we examine the role of glutamate signalling in remyelination following experimental demyelination in the adult rodent cns. we voltage-clamped opcs in brain-slices of adult rat cerebellar peduncle containing focal areas of primary demyelination and post-identified these by ng2-immunolabelling. recruited opcs mainly expressed ampa receptors at the peak of the opcs proliferation (5-10 days postlesion), as over 90% of the glutamate evoked current was blocked with nbqx (ampa/kainate receptor antagonist) and unaffected by ap5 (nmda receptor antagonist). the demyelinated axons continued to propagate action potentials with latencies similar to those in unmyelinated axons during development. critically, the demyelinated axons established synapses with opcs expressing voltage-gated sodium currents. these synaptic inputs had identical decay times to those recorded during developmental myelination. pharmacological inhibition of neuronal activity or glutamate signalling decreased remyelination efficiency by impairing differentiation. these results indicate that 1) glutamate currents are mainly mediated via ampa and kainate receptors during the opc recruitment 2) opcs establish synaptic contact with demyelinated axons and 3) the neuronal activity is essential for remyelination. together, these data suggest that demyelinated axons remain active and communicate with opcs through glutamate release during the regenerative process to guide their remyelination. there has been much recent interest in the use of intraocular stem cell transplantation to slow or reverse visual loss in glaucoma. however, migration and integration of stem cells into the retina is limited by reactive gliosis, a major barrier to retinal engraftment. our aim was to identify glial modulators of low toxicity and measure their potential to facilitate stem cell entry into the host retina. an explant organotypic culture system was used as a stem cell transplantation model and screening tool for candidate drugs. gfp1 mouse mesenchymal stem cells (mmscs) were co-cultured on the surface of the explants for 4 days.the microglial contribution to mscinduced reactive gliosis was investigated by assessing microglial activation and proliferation using immunohistochemistry for f4/80 and edu. distribution of chondroitin sulphate proteoglycans (cspgs) and their role in the barrier was also explored by immunostaining for chondroitin sulphate (cs-56) and by chondroitinase abc(chabc) treatment. stat3 inhibitor vi and jak1 inhibitor were used to suppress glial fibrillary acidic protein (gfap) expression. the efficacy of each drug to overcome the barrier was evaluated by immunohistochemistry for the glial markers gfap. mmsc integration was assessed by simultaneous immunostaining for gfp. cell viability after drug treatment was investigated by measuring the preservation of retinal thickness and by immunostaining for the neuronal marker neun and for caspase3. co-culture of retinal explants with mmscs caused a 1.5 6 0.4 fold increase in gfap expression. microglial activation and proliferation was not markedly affected by the presence of grafted mmscs on retinal surface. cspgs, highly expressed in the outer retina but only moderately in the inner retina, do not contribute substantially to the barrierto inner retinal engraftment. in contrast, interfering with the jak/stat pathway successfully reduced gfap expression by 60% 6 10% compared to control and facilitated mmscs integration into the host tissue. no toxic effect on cell viability was detected. on the contrary, over the 4 days ex-vivo culture, stat3 inhibitor vi treatment resulted in a neuroprotective effect on the outer nuclear layer, whose thickness (50.84 6 4.4 mm) is better preserved compared to control (38.4 6 9.0 mm) the results show that microglia and cspgs do not play a dominant role in the barrier to retinal engraftment, but the jak/stat pathway represents a promising pharmacological target.targeting modulators of glia reactivity to promote stem cell integration in the host tissue may facilitate stem cell therapy in glaucoma and other neurodegenerative diseases. oecs possess many characteristics favorable for the repair of sci including the secretion of growth and survival factors, the expression of cell adhesion molecules and the ability to integrate with astrocytes. oecs have also been reported to promote exceptional outgrowth of severed axons across the inhibitory environment of the lesion scar. the unique combination of regenerative properties suggests oecs would be beneficial for the repair of ventral spinal root avulsion injuries. after ventral root avulsion anterior horn neurons often fail to regenerate axons back into the surgically re-implanted nerve root resulting in poor functional recovery. to evaluate the efficacy of oec transplants for the repair of ventral root avulsions, the ability of oecs to promote neurite outgrowth from spinal cord neurons was investigated in a spinal cord organotypic co-culture. the integration of oecs with the spinal cord tissue was also examined by confocal fluorescence microscopy. oecs were harvested from adult sprauge dawley (sd) rats, cultured for 14 days and transfected with gfp. slices of cervical spinal cord were prepared from p8 sd pups and cultured on collagen coated membrane inserts. each slice was surrounded by a collagen gel with or without the addition of gfp oecs and glial derived neurotrophic factor (gdnf). our results indicate that oecs alone and in combination with gdnf have a positive effect on neurite outgrowth and morphology. the aim of this study was to obtain an hydrogel for the controlled delivery of vascular endothelial growth factor (vegf-a165). hydrogels are a class of liquid-gel biomaterials with high water content, typically composed of cross-linked, three-dimensional hydrophilic water-soluble polymer networks. the advantages of injectable hydrogel systems are: biocompatibility, biodegradability, adjustable shape, low cost and similarity to the extracellular matrix; moreover, they can be used as growth factor and cell delivery systems for tissue engineering applications. agarose/gelatin (a/gl) hydrogel was cross-linked with genepin. rheological measurements show that a/gl hydrogel can be injected through a syringe into the inner cavity of a nerve guide. in vitro assays performed to evaluate adhesion, proliferation, viability and migrationusing a fibroblast cell line (nih3t3), neonatal olfactory bulb ensheathing cells (nobec) and a schwann cell line (rt4-d6p2t)show that hydrogel allows cell adhesion, proliferation, viability and migration. vegf-a165 is a potent angiogenic factor and angiogenesis has long been recognized as an important and necessary step during tissue repair. vegf is known to have a positive effect on schwann cell proliferation and migration, neuron survival and outgrowth of regenerating nerve fibers. the hydrogel preparation protocol was optimized to be functionally efficient at physiological conditions, allowing the loading of vegf without the risk of its denaturation. different amounts of vegf (50-100-200 ng/ml) were incorporated in the a/gl hydrogel and the releasing rate in vitro up to 65 days was quantified by elisa . released vegf bioactivity was validated in human umbilical vein endothelial cells (huvec) and in nobec by evaluating phosphorylation of vegf-receptor 2 (vegfr2), akt and erk1-2. moreover, dorsal root ganglia explants were cultured on hydrogel containing different amounts of vegf, and an increased neurite outgrowth was quantitatively assessed. these results demonstrate that vegf can be successfully incorporated and bioactive released from a/gl hydrogel inducing vegfr2 activation and neurite outgrowth. in vivo test are ongoing on adult rats: one centimetre lesions on medial nerve were performed, followed by implantation of porous polye-caprolactone tubes filled with a/gl hydrogel containing vegf-a165. functional tests and histological analysis will be carried out to evaluate peripheral nerve regeneration. .spinal cord injury (sci) often leads to death of oligodendrocytes and considerable demyelination. demyelination of axons compromises signal conduction, and contributes to the functional deficits following sci. enhancing remyelination may serve to restore conduction and likely prevents axonal degeneration. here we focussed on a potential therapeutic intervention to promote remyelination, the transplantation of human oligodendrocytes precursor cells (opc)s capable of differentiating into mature oligodendrocytes and remyelinating denuded axons. human platelet derived growth factor (pdgf)-responsive neural precursor (prp) cells were isolated from the fetal forebrain and had been previously shown to differentiate into mature oligodendrocytes with a higher efficiency in vitro than traditional egf/fgf responsive neural stem cells (chojnacki and weiss 2004; deleyrolle et al. 2006 ). therefore, we tested the potential of human prps to differentiate into new oligodendrocytes and remyelinate axons in a rodent thoracic contusion spinal cord injury model. one week following injury, human prps were transplanted rostral and caudal to the lesion site. subsequent histological examination at two, seventeen and forty-two days post-transplantation demonstrated that human prps survived and integrated well into host tissue, particularly when nk cells were depleted. transplanted human prp's labelled with the mature oligodendrocyte marker apc, and 45% of the cells colabelled with the oligodendroglial marker olig2. astrocyte differentiation was also observed, suggesting human prps function as a multipotent glial progenitor following sci. human-specific immunoreactive processes were seen in close association with axons expressing the myelin protein mbp, providing evidence of remyelination by the transplanted cells. overall, these findings indicate that human prps are a source of new oligodendrocytes capable of producing myelin in response to spinal cord injury. remyelination is a regenerative process during which demyelinated axons are enwrapped by newly formed myelin sheaths. in the central nervous system (cns), this process takes place in two stages: first, during the recruitment stage, oligodendrocyte precursor cells (opcs) divide, migrate and engage the demyelinated axons. then, during the differentiation stage, opcs differentiate to myelin-forming oligodendrocytes. under pathological conditions such as multiple sclerosis (ms), remyelination often fails and as a consequence chronic demyelinated areas accumulate in ms. post mortem evidence suggests that opc differentiation is often impaired in ms patients. however, the reason why opc differentiation fails in ms is not completely understood. it has been proposed that ms lesions contain factors that act as specific inhibitors of opc differentiation. amongst them, myelin associated inhibitors play an important role. we have demonstrated that the inhibitory effect of myelin protein extracts (mpe) is mediated by activation of signalling cascades including pkca-marcks in vitro. furthermore, modulation of pkca has been shown to rescue opc differentiation in the presence of inhibitory mpe. here we demonstrate that tamoxifen, a drug that is used in various clinical settings because of its pkc-inhibitory activity, is able to promote opc differentiation in vitro. in a series of in vivo experiments, we tested the hypothesis that inhibition of pkca by tamoxifen promotes cns remyelination. demyelination was induced in the caudal cerebellar peduncle (ccp) of young-adult sprague-dawley rats by the administration of ethidium bromide. animals received daily doses of tamoxifen. although tamoxifen treatment did not induce changes in the number of opcs expressing immature markers (ng2 and pdgfr-a), a significant increase in the number of cells expressing mature marker of oligodendrocytes (cc1/olig2 and plp) was detected. furthermore, analysis of resin-embedded tissue demonstrated that tamoxifen significantly promotes remyelination in the cns when compared to controls (mann-whitney's test, p < 0.05). although further investigations are required to elucidate the mechanisms by which tamoxifen exerts cns remyelination, our results demonstrate that administration of tamoxifen represents a potent and clinically accessible strategy to promote endogenous myelin regeneration. induced pluripotent stem (ips) cell technology now allows development of autologous oligodendrocyte precursor cells for putative remyelination cell therapy for multiple sclerosis (ms). we have successfully developed mouse and human ips cells and converted these into oligodendrocyte precursor cells (opcs) that can differentiate into fully mature oligodendrocytes. furthermore, we have shown that (intracerebral) implantation of ips-derived opcs in mouse ms models enhances remyelination.before implantation-induced remyelination therapy with autologous ips-derived opc can proceed to a clinical trial for remyelination cell therapy in ms patients, we have to provide solid evidence for long term efficacy and to establish the safety thus ultimately excluding contamination of teratogenic (pluripotent) stem cells. these issues are best examined in a nonhuman primate model. the biomedical primate research center (bprc), rijswijk has developed a welldocumented experimental autoimmune encephalomyelitis (eae) model in marmoset monkeys that most closely approximates the pathology of ms in humans. in this animal model we will verify that intrathecally administered ips-derived opcs migrate to and remyelinate eae lesions and determine their long-term fate.we have developed an efficient protocol to induce marmoset ips cells from skin fibroblasts. for that we have used a lentiviral polycistronic construct, containing the four (human) reprogramming genes oct4, klf4, sox2 and cmyc, that is excisable using cre-recombinase. the pluripotent nature of the marmoset ips cells was established based on the endogenous expression of pluripotency transcription factors and the ability to differentiate in-vitro (via embryoid bodies) and in-vivo (subcutaneous injection for teratoma formation) into all the different tissues of the three germ layers. presently we are developing protocols for the differentiation of these marmoset ips cells into an oligodendrocyte cell lineage. published protocols have been modified for production of opcs for research purposes, and produced cells are used for characterization of opcs. ultimately the aim is to define an opc population with optimal myelination capacity. results and discussion: currently, human oligodendrocytes and opcs can be produced from pluripotent stem cells. these cells are routinely characterized in protein expression level, and methods for more comprehensive characterization are under development. produced opcs can be purified with fluorescence-activated cell sorting based on ng2 protein expression in order to study if this cell population is heterogeneous in terms of myelination capacity. conclusions: oligodendrocytes and opcs can be produced from human pluripotent stem cells. studies are ongoing to determine the molecular characteristics and myelination capacity of the differentiated and purified opc-population. remyelination, the regenerative process in which myelin is restored to demyelinated axons, is carried by oligodendrocyte precursor cells (opcs). in a previous study we have shown that oligodendrocyte precursor cells are multipotent and they can differentiate into oligodendrocytes as well as to schwann cells and occasionally astrocytes in the remyelinating lesions. we observed that schwann cells derived from opcs in the central nervous system occupied almost exclusively tissue around blood vessels in astrocyte deficient areas. therefore, we postulated the occurrence of specific niches or microenvironments that modulate opc fate. the aim of present study was to identify the factors and their downstream effectors that significantly discriminate vascular and nonvascular niche and determine the fate of oligodendrocyte precursor cells. we microdissected tissue from vascular and non-vascular regions of lesion areas at 6, 10 and 14 days after bilateral stereotaxic injection of ethidium bromide into the brain white matter of adult rats and performed microarrays to profile the whole transcriptome of both niches separately. this approach allowed us to distinguish between mrnas that are enriched within two separate environmental niches within the same lesion area. the study identified 138 differentially expressed transcripts. comparative bioinformatic analysis of global gene expression combined with signalling transduction pathway structure and genes function analysis revealed specific candidate signaling pathways differentially expressed in local peri-vascular niche compared to nonvascular one. results of rt-pcr-based validation of microarray data have proven that expression gradient of bmp4, bmp7, their antagonist sostdc1, as well as wnt2b, wnt6, dhh and scube1 act as the major regulators of opcs fate in the vascular niche. moreover, we observed that blood vessels undergo substantial regeneration in the course of remyelination and postulated the important role of blood vessels reconstruction in creating the specific microenvironment of neurovascular niche during tissue regeneration. our analysis shown that unique properties of tissue around blood vessels differ from the rest of the lesion which can be one of the factors favoring opcs alternative differentiation in this area of lesion. . however, key aspects of dynamic behavior, such as cell migration or process orientation and motility, can only be monitored by live imaging. to elucidate these key aspects of opc behavior after injury, we used repetitive in vivo two-photon laser scanning microscopy (2plsm) to follow ng2 1 -cells after smaller and larger stab wound injuries in the mouse somatosensory cortex. we therefore used the bac transgenic mouse line sox10 icreert2 (simon et al., 2012) crossed to the inducible reporter line cag-gfp (nakamura et al., 2006) selectively labeling cells of the oligodendrocyte lineage. gfp 1 opcs were imaged through a cranial window at the day of injury and at different time points thereafter. identification of the same cells was possible based on blood vessels labeled by rhodamine dextran as stable landmarks. live imaging revealed that the majority of opcs reacts within 2 days after injury with hypertrophy (enlarged soma and processes), polarization (elongation and concentration of processes towards the injury site), directed migration towards the injury site (defined as movement of the cell body for at least 10 mm) and proliferation (defined by a single cell being replaced by 2 or even more daughter cells). only a small proportion of cells within $500 mm of the injury did not react in any detectable manner. we noted that polarization and hypertrophy occur rather fast after inflicting the injury, while proliferation peaks 4 days after injury. taken together, the live observation of opcs reacting to stab wound injury supports the concept of opc heterogeneity and reveals new insights into the functional role of these cells after injury: the fast process orientation towards the injury site implies a contribution to wound closure and their substantial proliferation sometimes for several rounds amplifies the number of ng2 1 -cells surrounding the injury site with implications for scar formation. the aim of this study was to create a non-invasive tool for cell visualization and cell population tracking that can be used in long term studies. methods: this study was performed using human pluripotent stem cell lines derived in institute of biomedical technology (former regea), university of tampere, finland. cells were differentiated to neural cells using neural differentiation protocol described earlier by lappalainen and colleagues. fluorescent probes used in this study were 1) 5chloromethylfluorescein diacetate (celltracker green cmfda, ct) and 2) 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindodicarbocyanine perchlorate (did). cell viability and proliferation of the labeled populations was briefly studied. also, the labeled cells were characterized using immunocytochemistry and neuronal functionality was studied using micro electrode array (mea). results: during labeling optimization a wide range of different labeling parameters was tested for both probes to obtain long term labeling. most suitable parameters for human neural cells were 10 mm ct with 72 hours incubation time and 10 mm did with 2 hours incubation time. with these concentrations the labeling was visible up to 4 weeks and had no statistical effect on cell viability. ct labeling was not affecting to cell proliferation but with did slight decrease in proliferation was seen in long term culture. labeled populations contained both map-2 positive neurons and gfap-positive astrocytes. also, the ct or did labeled population and their mixed-cultures expressed normal spontaneous network activity when cultured on mea-platform. conclusions: this study indicated that fluorescent probes ct and did are suitable for long-term labeling of human derived neural cultures in vitro. the labeling had no negative effects on cell behavior with the exception of did labeled cells having slight decrease on cell proliferation. importantly, the labeled neural networks were electrically functional. axonal regeneration after spinal cord injury (sci) is limited mainly due to the presence of an inhibitory microenvironment, especially reactive astrogliosis and glial scar formation. overcoming this inhibitory barrier of reactive astrocytes and inhibitory substances making the microenvironment of the damaged neurons permissive for axonal regrowth might be crucial for cns repair. single-dose irradiation has been proved to be neuroprotective in cns trauma through elimination of reactive astrocytes, however, the irradiation conditions with singledose protocols are not in clinical use. in the present study, we aimed to investigate whether low-dose-fractionated irradiation, which is currently being used widely for clinical treatment of several tumor types, could regulate cell cycle elements of reactive astrocytes just like its use in cancers, inhibit glial scar formation, attenuate astrogliosis-mediated inhibition of axonal regeneration and facilitate recovery of motor function after spinal cord hemi-section in beagle dogs. as expected, our results demonstrated that low-dose-fractionated irradiation reduced reactive astrogliosis, attenuated astrogliosis-associated neural injuries, ameliorated microenvironment of axonal regeneration, and benefited recovery of the animals subjected to spinal cord trauma. accordingly, irradiation might be a promising therapeutic strategy targeting at excessive astrogliosis for sci in the near future. mesenchymal stem cell transplantation has been proven to have beneficial effects in various degenerative diseases, including demyelinating models. both in our lab as well as others have shown that they express a number of trophic factors that are capable of inducing remyelination, mainly by activating nearby oligodendrocyte progenitors towards mature oligodendrocytes that in turn remyelinate the damaged area. however, this effect was only observed locally, in the area surrounding the graft, thus in order to achieve general remyelination in various brain structures simultaneously, we decided to perform bone marrow-derived mesenchymal stem cell injections into the lateral ventricles. in this manner, the cells may attach to various areas such as the hippocampus, corpus callosum and fornix, among others, all of which are demyelinated. previous to the graft, the cells were incubated with iron nanoparticles. this way, it is possible to track in vivo the grafted cells by magnetic resonance imaging (mri). as a result, the cells were observed at different time points (0-15-30-60-90 days) by mri. also, the demyelinated areas can also be visualized and myelin quantified using image analysis software. this allows the quantification myelin density in the same individual mice at different moments before and after transplantation. j. muenzel 1,2 , c. becker 2 , t. becker 2 , a. williams 1 1 university of edinburgh, centre for regenerative medicine, edinburgh, united kingdom 2 university of edinburgh, centre for neuroregeneration, edinburgh, united kingdom central nervous system (cns) remyelination is important for the restoration and protection of proper nervous system function in demyelinating diseases such as multiple sclerosis but is poorly understood, and inefficient in humans. in contrast, zebrafish are able to regenerate many tissues very efficiently. as zebrafish are being increasingly used to study myelination, we aimed to develop and characterise an adult zebrafish model of cns de-and remyelination, to answer the question if remyelination is also very efficient, to describe the biology and to identify similarities and differences with mammalian remyelination. this then may allow us to better understand why zebrafish have a high regenerative capacity compared to rodents and humans. previous studies in teleost fishes have used optic nerve crush as a paradigm of myelin injury to investigate myelin repair. this, however, involves the de novo myelination of regenerated axons, rather than deand remyelination of the same axons. lysophosphatidylcholine (lpc) is a detergent-like toxin, which is widely used in rodent models to demyelinate axons. we administer lpc to the optic nerve of adult zebrafish and observe less myelin by both immunoreactivity and electron microscopy at the lesion site at 8 days post lesion (dpl) and microglia activation along the optic pathway. immunoreactivity and electron microscopy suggest complete regeneration of myelin at 28 dpl. we cannot identify any axonal injury in this model. in young zebrafish (aged 4-6 months), the myelin thickness of remyelinated fibres shows no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. however, in old fish (aged 18 1 months), although the axon diameter is the same as pre-lesion and in young animals, , remyelinated fibres have thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. we believe that this new zebrafish model of cns remyelination can be added to the suite of current models to better understand the remyelination process, why it fails, and to test for compounds to improve it, with the added benefit that zebrafish are rapid breeders, transgenesis is easy and there is a high potential for live imaging. although often described as a hard-wired component of the vertebrate body, the nervous system is a plastic and considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity of its core components. unlike the cns, the pns is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that underlie this phenomenon. peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. they are ensheathed by myelinating or non-myelintaing schwann cell glia, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. this structure forms from centrally-derived glial cells and serves as a protective barrier that is essential for nerve function. following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris, regenerate and re-innervate targets. schwann cells have been shown to play an important role in this process by trans-differentiating, proliferating, clearing debris, and guiding re-growing axons, but less is known about the potential role of perineurial glia. to investigate the role of perineurial cells in pns regeneration, we have developed an injury response assay that uses a micropoint laser to create injuries along the motor nerves in live transgenic zebrafish. time-lapse imaging of injured nerves reveals that perineurial glia rapidly respond to nerve injury and extend processes toward the injury zone. this is in contrast to schwann cells, which we observe orienting towards the distal stump where they engulf and clear axonal debris. these data demonstrate that perineurial glia respond immediately to motor nerve injuries in a manner distinct from schwann cells, and future work is aimed at defining the molecular mechanisms that mediate the cellular responses of perineurial glia and schwann cells, as well as determining if developmental paradigms are recapitulated in these glial populations during the regenerative process. we have previously shown that in schwann cells the transcription factor c-jun acts as a master regulator of wallerian degeneration and is required for successful repair following nerve injury (arthur-farraj et al., methods. we here investigate on the release of the gliotransmitter glutamate from superfused isolated purified glial processes (gliosomes) obtained from adult rat cerebellum. intracellular ca 21 levels were measured by a microfluorimetric technique. immunocytochemical confocal and western blot analysis were also carried on cerebellar gliosomes. results. confocal and western blot analysis confirmed that cerebellar gfap-positive gliosomes are a purified preparation of glia processes. more then 80% of gliosomes were positive for brain-specific lipid binding protein (blbp), indicating that they derived from bergmann cells. by measuring the release of glutamate we obtained evidence for the presence of glutamate release-facilitatory ampa receptors on the bergmann glial processes. in fact, ampa evoked [ 3 h]d-aspartate or endogenous glutamate release which was abolished in ca 21 -free medium; effectiveness of the selective inhibitor naphthylacetyl spermine indicated the involvement of ca 21 -permeable, glua2-lacking ampa receptors. ca 21 imaging confirmed the presence of ca 21 -permeable, glua2lacking ampa receptors on bergmann glia processes. activation of the glua2-lacking ampa receptors triggered vesicular exocytotic glutamate release: inhibition of vesicular loading by the vesicular glutamate transporter (vglut) inhibitors rose bengal or trypan blue, or by the h 1 -atpase inhibitor bafilomycin a1 prevented the ampa-evoked glutamate release (see figure) . confocal analysis confirmed that blbpand gfap-positive processes expressed vglut1 and vglut2. conclusions. we here report evidence that: 1. functional processes of bergmann cells are recovered in purified preparation of adult rat cerebellar gliosomes; 2. ampa receptors located on bergmann glia processes show features of ca 21 permeable, glua2-lacking ampa receptors; 3. activation of the ampa receptors evokes ca 21 entry in isolated bergmann glia processes and ca 21 -dependent vesicular glutamate release from the processes. activation of ca 21 -permeable, glua2lacking ampa receptors coupled to vesicular glutamate release might represent a mechanism by which bergmann glia processes modulate synaptic transmission at parallel fibers/purkinje cell synapses. the formation of brain edema, which accompanies ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to extracellular k 1 elevation and neurotransmitter accumulation in the extracellular space. an increased uptake of these osmotically active substances, predominantly provided by astrocytes, is accompanied by intracellular water accumulation via aquaporin-4 (aqp4). previously, it has been shown that the removal of perivascular aqp4 via the deletion of alpha-syntrophin, a protein responsible for anchoring aqp4 on the astrocytic membrane, delays edema formation and k 1 clearance (amiry-moghaddam et al.,2003, pnas 11;100(23):13615-20). therefore, we aimed to elucidate the impact of alpha-syntrophin deletion on astrocyte volume changes in the cortex during pathological states, such as hypoosmotic stress or oxygen-glucose deprivation (ogd), using three-dimensional (3d) confocal morphometry in situ. in addition, single cell rt-qpcr profiling was carried out to reveal possible differences in the expression profiles of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. in order to visualize individual astrocytes that lack alpha-syntrophin, double transgenic mice were generated by crossbreeding gfap/egfp mice ( . 3d-confocal morphometry revealed that alpha-syntrophin deletion results in significantly smaller/slower astrocyte swelling when induced by 20 min hypoosmotic stress (210 mosm), 30 min ogd or by high extracellular k 1 (50 mm), while alphasyntrophin deletion had no effect on the astrocytic shrinkage evoked by hyperosmotic stress (350 mosm). the volume recovery of cortical astrocytes from gfap/egfp/alpha-syntrophin knockout mice was significantly slower following their exposure to hypo-or hyperosmotic stress, whereas no differences were found in astrocyte volume recovery following ogd or after their exposure to high extracellular k 1 . compared to the cortical astrocytes of gfap/egfp mice, single cell rt-qpcr analyses revealed that astrocytes from gfap/egfp/alpha-syntrophin knockout mice express higher mrna levels for two-pore domain k 1 channels (twik-1, task-2, task-3), inwardly or outwardly rectifying k 1 channels (kir3.1, kir5.1, kv1.3, kv1.6) and chloride channels (clc1,clc4), while mrna expression for the glutamate transporter glt-1 is lower. in summary, the deletion of alpha-syntrophin slowed down astrocyte swelling during hypoosmotic stress, ogd or high k 1 ; however, it also resulted in alterations in astrocytic gene expression profiles. supported by grants ga cr 13-02154s and p304/12/g069 from the gacr. microglia undergo a process of activation in any type of pathology which is controlled by many factors such as cytokines, chemokines or growth factors, but also by neurotransmitters. we found that a subpopulation (16%) of freshly isolated microglial cells from the adult brain respond to the muscarinic acetylcholine receptor agonist carbachol with a ca 21 response, indicating the expression of functional receptors. the carbachol-sensitive population increased in microglia/ brain macrophages isolated from the tissue of mouse models for stroke (57%) and alzheimer's disease (26%), but not for glioma and multiple sclerosis. microglia cultured from adult and neonatal brain contained a similar carbachol-sensitive sub-population (17% and 10%) which was increased by treatment with interferon-c to 60% within 12 hours, and was sensitive to blockers of protein synthesis. carbachol was a chemoattractant for microglia and decreased phagocytosis activity, indicating that microglia are a functionally heterogeneous population. t13-05a astrocytes and s1p receptors the family of sphingosine 1-phosphate receptors (s1prs) are g protein-coupled comprising five subtypes (s1p1r-s1p5r). these receptors are expressed in cells of the immune, cardiovascular, and central nervous systems (cns), in addition to others. s1prs play important roles in celular proliferation, differentiation, survival and migration. recently, the immunomodulatory drug, gilenya v r has been approved as the first oral therapy for multiple sclerosis (ms), after proving efficacious in clinical trials. the active ingredient of gilenya v r is the phosphorylated compound fty720 (pfty720), which is a potent agonist on all s1prs, except s1p2rs. pfty720 has been suggested to work as a 'functional antagonist' causing s1p1r internalisation in lymphocytes, thus limiting t cell auto-immunity. in addition to regulating the immune system, the lipophilic nature of the pro-drug fty720 allows it to readily cross the blood-brain-barrier (bbb) where it may also activate s1prs expressed on both neurons and glia. here, the role of s1prs in the cns was investigated, by specifically investigating their role in astrocyte function. a range of methods were used to examine the effects of s1pr activation and their roles in astrocytes, including: (i) s1p1r subtype trafficking, (ii) transient and continued intracelular signalling, (iii) astrocyte cell migration, (iv) release of pro-inflammatory cytokines, (v) oligodendrocyte cell differentiation and survival and (vi) myelination state in brain slice cultures. the data showed that activation of the s1p1r subtype leads to (i) its internalisation and (ii) continued signalling in astrocytes, and that s1p1rs were found to (iii) promote astrocyte migration, (iv) limit release of pro-inflammatory cytokines, (v) promote oligodendrocyte survival and (vi) limit demyelination. these studies demonstrate a role for s1p1rs in regulating astrocyte function and suggest their use a drug targets for neuroinflammtory and neurodegenerative disorders. in particular, both astrocytes and oligodendrocytes express kir4.1, which are essential for setting their strongly negative rmp, as well as the maintenance of [k 1 ] o following neuronal activity. notably, strong expression of the kir7.1 subtype was determined during a whole genome microarray analysis of the mouse optic nerve, a typical cns white matter that contains mainly astrocytes and oligodendrocytes. the kir7.1 subunit is known to be involved in potassium transport in epithelial cells and has been identified in cerebellar purkinje neurons, but has not been reported in glia. here, we examined functional expression of kir7.1 in mouse brain and optic nerve. rt-pcr and western blot confirmed expression of kir7.1 mrna and protein expression in the brain and optic nerve. positive kir7.1 immunostaining was observed in astrocytes and oligodendrocytes in brain sections and in optic nerve explant cultures and the results indicated a developmental increase in kir7.1 expression. in astrocytes, kir7.1 was localised to perivascular end-feet, supporting a potential role in k 1 regulation. in oligodendrocytes, kir7.1 were localised to cell somata, suggesting a developmental role in setting their rmp. in addition, oxygen and glucose deprivation (ogd) experiments were performed on isolated intact optic nerves from mice aged p10 and examined for the effects of the kir7.1 channel blocker vu590. after 60 min ogd, blockade of kir7.1 resulted in increased cell death of optic nerve glia, measured using propidium iodide (pi) labelling, from 80 6 30 in controls to 189 6 49 in vu590 (n 5 4 nerves per group, 6 fov per nerve; p < 0.001, anova). our results demonstrate expression of kir7.1 in glial cells, and indicate they are important in protecting glial cells from ischemic damage. is an eight transmembrane domain protein highly expressed in brain. human mutations in mlc1 lead to the rare genetic disorder "megalencephalic leukoencephalopathy with subcortical cyst" (mlc). characteristic for the disease are the onset of macrocephaly within the first year of life, and a slowly progressive loss of motor skills with epilepsy and cognitive decline. at the cellular level, countless fluid-filled vacuoles occur within myelin sheaths surrounding axons and in astrocytic endfeet. in cell culture models, mlc1 mutations are associated with defects in chloride currents and cell volume regulation. thus far, the expression and function of mlc1 in intact murine and human brain are still controversial. to understand the role and developmental expression of mcl1 in the brain, we have developed a mlc1 mutant null mouse model carrying an egfp reporter gene under the mlc1 promotor. we now show the exclusive expression of mlc1 in astrocytes throughout the brain with specifically higher expression around blood vessels, at the sub-ventricular zone and at the glia limitans. the functional loss of mlc1 in mutant mice recapitulates in part the human mlc disease. ko mice show macrocephaly with high brain wet weight. water filled vacuoles develop in the white matter of the cerebrum and in large fiber tracts of the brainstem. by contrast, the heterozygous loss of mlc1 has no consequences on myelin structural integrity. both heterozygous and homozygous mlc1 ko mice have dysmorphic peri-vascular and peri-ventricular astrocytes. in contrast to humans, ko mice have no motor deficits, but are hyperactive and show an anxiety-like behavior. in the mlc1 ko mouse, a dysfunction of an astrocytic protein causes loss of myelin structural integrity leading to vacuolating myelinopathy. therefore, the mlc mutant mouse could be a key model to study the astrocytic involvement in brain water and ion homeostasis. gaba activated slowly desensitizing responses in ng2 cells which were mimicked by muscimol and inhibited by bicuculline. to elucidate the subunit composition of the receptors we tested its pharmacological properties. co-application of pentobarbital, benzodiazipines and zolpidem all significantly increased the gaba responses. the presence of small tonic currents indicated the presence of extrasynaptic gaba a receptors. to further analyze the subunit expression, single cell transcript analysis was performed subsequent to functional characterization of ng2 cells. the subunits a1, a2, b3, c1 and c2 were most abundantly expressed, matching properties resulting from pharmacological characterization. importantly, lack of the c2 subunit conferred a high zn 21 sensitivity to the gaba a receptors of ng2 cells. to determine the effect of gaba a receptor activation on membrane potential, perforated patch recordings were performed. in the current-clamp mode, gaba depolarized the cells to about 230 mv, indicating a higher intracellular clconcentration (about 50 mm) than previously reported. gaba-induced depolarization in ng2 cells might trigger ca 21 influx through voltage-activated ca 21 channels. schwann cells (sc) play important roles in the development and regeneration of the peripheral nervous system (pns) following injury. several molecules such as neurosteroids and neurotransmitters have been suggested as potential pharmacological targets in regulating sc physiology and regenerative potential. nevertheless, the slow growth rate and difficulties in harvesting limit sc applications in regenerative medicine. adipose-derived stem cells (asc) can be differentiated into a sc-like phenotype (dasc) sharing morphological and functional properties in the present study, we analysed changes in glutamate transporter expression and function following a mechanical lesion in organotypic slice cultures of the mouse hippocampus using immunohistochemistry, western blots and dynamic imaging. the lesion was positioned perpendicular to the stratum pyramidale in the ca1 area and comprised the entire hippocampus proper. after three-six days, a glial scar had formed along the lesion site. activated astrocytes in close proximity (100-150 mm) to the lesion ("scar cells") directed long, palisading gfap-positive processes towards the lesion, had significantly swollen somata and lost their ability to take up sr101. furthermore, some exhibited distinct clustering of glast and glt-1 immunoreactivity. scar cells showed greatly diminished increases in intracellular sodium in response to application of d-aspartate, an agonist for glutamate transporters. astrocytes in the periphery to the lesion, in contrast, maintained their ability to take up sr101 and showed only slight upregulation of gfap, as well as less swollen cell bodies. cells in the periphery displayed only marginal changes in glutamate transporter immunoreactivity and unaltered amplitudes of sodium changes in response to d-aspartate. taken together, our data show that mild astrogliosis in the periphery of a mechanical lesion is not accompanied by a significant change in glial glutamate uptake capacity. at the scar itself, a strong clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in glutamate uptake. transport of base equivalent (hco 3 -) across the membrane is a crucial process to maintain the intracellular and extracellular proton concentrations within a narrow physiological range. the electrogenic sodium-bicarbonate cotransporter (nbce1, slc4 a4) is a major base transporter in eukaryotes and expressed in many tissues, especially in epithelial cells. nbce1 may transport significant amounts of bicarbonate into and out of the cell. in the brain, nbce1 is predominantly expressed in glial cells and operates with a transport stoichiometry of 1na:2hco 3 -. we have studied the bicarbonate sensitivity of nbce1, in primary cultured mouse cortical astrocytes (c57bl6/n) using live cell fluorescence imaging with confocal microscope and bcecf-am as a proton sensitive fluorescence probe. we have also used the primary cortical astrocyte culture from nbce1-ko mouse to compare the nbce1-mediated processes in wt astrocyte culture. bicarbonate dose response protocols suggest that nbce1 has a very high affinity for bicarbonate (k m <1 mm). due to this high affinity for bicarbonate, nbce1 can sensitize even residual bicarbonate concentrations of 150-300 lm present in nominally bicarbonate-free extracellular solutions (buffered with hepes). the removal of residual bicarbonate by saturating extracellular buffer (hepes) with 100% o 2 reduced these nbce1-mediated intracellular proton changes significantly. in astrocytes of nbce1-ko mice, cytosolic h 1 changes could not be detected at hco 3 concentrations lower than 3 mm. it has been reported that astrocytic nbce1 activity mediates the activation of astrocytic glycolysis by a mechanism dependent of intracellular bicarbonate and ph (ruminot et al 2011 j neurosci 40: 64-71). using a genetically encoded fret-based glucose nanosensor, we show that glucose metabolism can be activated at low millimolar bicarbonate concentration, which was largely absent in astrocytes from nbce1-ko. our results demonstrate the highest bicarbonate sensitivity yet described in animal cells, mediated by nbce1 in cortical astrocytes. nbce1 may function as a bicarbonate sensor in these cells, e.g. for modulating energy metabolism. supported by the deutsche forschungsgemeinschaft de 231/24-1. inward currents which were significantly blocked by the l-type calcium channel antagonist nimodipine. to better investigate the role of l-type calcium channels in ng2 glia at different developmental stages of the cns, we take advantage of the inducible cre-lox system by breeding homozygous cav1.2 floxed mice and cav1.3 flexed mice with ng2-creert2 knock-in mice. we are currently investigating how the removal of cav1.2/1.3 alters proliferation, migration and survival of ng2 glia. a particular focus lies on the analysis of behavioral abnormalities generated by cav1.2/1.3-deficient ng2 glia. chronic neuropathic pain is a frequent consequence of spinal cord injury (sci). yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. previous studies have demonstrated that sci results in excessive atp release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. we sought to assess the role of connexin 43 (cx43) as a mediator of cns inflammation and chronic pain. to determine the extent of cx43 involvement in chronic pain, a weight-drop sci was performed on transgenic mice with cx43/cx30 deletions. sci induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after 4 weeks and was maintained after 8 weeks. notably, sciinduced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with cx43/cx30 deletions, but fully developed in transgenic mice with only cx30 deletion. sci-induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with cx43/cx30 deletions, when compared with littermate controls. in comparison, a standard regimen of post-sci treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than cx43 deletion. these findings suggest cx43 is critically linked to the development of central neuropathic pain following acute sci. since cx43/cx30 is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain. mast cells (mcs) are immune cells that reside in normal brain tissue. they store in granules an array of pro-inflammatory mediators, which are rapidly released to the extracellular milieu upon activation through an extracellular ca 21 -dependent mechanism. neurotoxic amyloid peptides are known to induce mcs degranulation but the membrane transduction mechanism remains unknown. here, the possible role of pannexin 1 hemichannel (panx1 hc) known to be permeable to ca 21 was studied. objective: since ca 21 influx is essential for mcs degranulation, we evaluated if the neurotoxic amyloid fragment ab 25-35 peptide promotes mcs degranulation via activation of panx1 hcs. methods: primary na€ ıve mcs were differentiated from bone marrow precursors of wild type (wt) and panx1 knock out (panx1 -/-) c57bl/6 mice using il-3 present in wehi3 conditioned medium. the presence of panxs 1, 2 and 3 mrnas presence was evaluated by rt-pcr analyses. the hc activity was assessed using the 4',6-diamidino-2-phenylindole (dapi) uptake assays and measurements of membrane current using whole cell patch clamp while intracellular ca 21 signal was evaluated using fura-2am. degranulation was assessed by quantification of extracellular histamine as well as release of toluidine blue (tb) from tb preloaded mcs from wt and panx1 -/mice. results: only panx1 mrna was detected in wt mcs. stimulation with ab 25-35 but not ab 35-25 peptide induced histamine and tb release. it also enhanced dapi uptake and total membrane current in wt mcs. these responses were drastically reduced in wt mcs pretreated with 10 mm carbenoxolone and 200 mm 10 panx1, blockers of panx hcs and were absent in panx1 -/-mcs. in wt mcs treated with ab 25-35 increase the intracellular ca 21 signal and was drastically prevented by 10 panx1, absence of extracellular divalent cations and in panx1 -/-mcs. conclusion: these findings indicate that mcs express functional panx1 hcs, which are essential for ca 21 influx required for the degranulation response induced by ab 25-35 . thus, inhibition of panx1 hcs might avoid spreading of mc-dependent inflammatory mediators released during alzheimer's disease progression. gliomas are the most frequent primitive cns tumors and are thought to derive from astrocytes or from neural progenitors/stem cells. however, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. tgf alpha is frequently over-expressed in the early stages of glioma progression. sharif & al (oncogene. 2007 (19):2695-706) previously demonstrated that prolonged exposure of normal astrocytes to tgf alpha is sufficient to trigger their reversion to a neural progenitor-like state. when astrocytes de-differentiated with tgf alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. our study aims to identify and characterize the protein signature of those in vitro transformed cells in an attempt to understand their neoplastic behavior and the effect of transformation on metabolic processes. this involves a global proteomic analysis using the 2d-dige methodology and a study of the metabolism of these cells (carbon source, lactate, glucose and glutamine use, ros metabolism…) by 1hand 13c-nmr nmr quantification of metabolites. such approaches are expected to provide information allowing understanding the metabolic reprogrammation that occurred during transformation. the comparative 2d-dige proteomic analysis of normal and transformed astrocytes shows that during transformation, the cells increase their expression of glycolytic enzymes, thus acquiring the ability to use aerobic glycolysis (warburg effect). moreover, the transformed cells reduce their capacity for tricarboxylic acid oxidation and for neurotransmitters (glutamate and gaba) metabolism. ingenuity pathway analysis indicates major effects on carbohydrates, amino acids and nucleotides metabolic components. using enzymatic activity measurements and the detection of protein isoforms by 2d-western blot and zymography, we document a change in expression and activity of various isoenzymes that may be responsible for those metabolic reprogrammations. r.e. k€ alin, a. jarczewski, f. apel, r. monk, s. kraft, j. radke, f.l. heppner charit e -universit€ atsmedizin berlin, neuropathology, berlin, germany question: glioma are the most frequent malignant primary tumors of the brain. glioma survival and growth is determined by the interaction with brain parenchyma, which includes intense tumor angiogenesis. this process is supported by glioma-invading myeloid (gim) cells, namely brain resident microglia or brain-invading macrophages. depletion of gim cells leads to a significant decrease in glioma volume. in a previous study, we established the novel neuroendocrine hormone apelin as an angiogenic factor and described its upregulation in human glioblastoma multiforme. our aim is thus to study the role of apelin signaling in tumor angiogenesis but also its possible effect on brain monocytes for the regulation of glioma growth. methods: to investigate apelin signaling during gliomagenesis we took advantage of an orthotopic mouse model for tumor formation. intracerebral xenotransplantation of the human glioma cell line u87mg, expressing lentiviral control or apelin shrna, allows us to study the specific contribution of glioma-derived apelin to gliomagenesis. to further dissect a putative immune function of apelin, we used the isogenic mouse glioma cell line gl261 for intracerebral implantation into immunocompetent mice lacking (apelin-ko) or overexpressing apelin (apelin-tg). results: loss-of-apelin expression in u87mg cells resulted in a reduced glioma volume and attenuated the formation of the xenograft vasculature. gl261 implants in apelin-ko mice produced a similar phenotype. in addition, invasion of glm cells was significantly reduced. interestingly, expression analysis showed an upregulation of the apelin receptor apj in brain myeloid cells making them competent to respond to glioma-derived apelin. conclusions: our findings demonstrate that apelin plays an important role in tumor angiogenesis and glioma growth. we show here that both, glioma cell-derived apelin and apelin expressed by the tumor neovasculature are contributing to tumor angiogenesis, gim invasion and glioma growth. we anticipate that our study will provide insights whether apelin signaling may serve as a future novel target for antiangiogenic tumor therapy. tumor infiltrating microglia/macrophages (tims) constitute the largest population of infiltrating cells in glioblastoma (gbm), the most aggressive brain tumor. data from the clinic and from experimental work performed in murine and human models indicate that tims play a significant role in gbm biology as they support proliferation, migration and invasion of tumor cells. evidence is amounting that tumor cells actually polarize tims towards this m2-like, tumor-supportive phenotype. we showed that toll-like receptor 3 ligand reverses this m2-into a m1-like phenotype. pre-activated, m1-like polarized tims incubated with spheroids of gbm cells reduced migration, killed and phagocytosed tumor cells over a 15 day-period, indicating a sustained m1 activation of tims in absence of exogenously added stimuli. in order to analyse in more detail tims-gbm cells interactions, we have undertaken two approaches. we use spheroids of cells (tumor with/without tims) embedded in collagen matrix, in which tims can be implanted, as an experimental model. this three-dimensional in-vitro system is well suited for a qualitative and quantitative monitoring of cell proliferation, death, migration and invasion. data experimentally generated are then implemented in a mathematical model that is, to our knowledge, the first one proposed to take into account tumor cells and tims in order to simulate gbm progressive behaviour. in a first step, the capacity of spheroids made of murine glioma cells to invade collagen matrices was evaluated in absence and presence of microglia. invasion was monitored by photography of the spheroids and images were processed with photoshop cs5 software. in order to achieve a precise determination of invasion, we chose to measure the diameter of the core plus the invasive rim as a read out of expansion rate. untreated microglia promoted growth and invasion of tumor cells. data generated through the proposed in-vitro system were comparable to and reproduced the insilico simulations obtained with the mathematical model, hence validating our mathematical and experimental approaches. we currently evaluate the rate of proliferation and death of tumor cells in various settings that modulate tims polarization, using flow cytometry and confocal (live) imaging. data contributed by these two approaches should facilitate the delineation of a predictive model for tumor progression in a tims-enriched microenvironment with possible therapeutic fallouts. sialic-acid-binding immunoglobulin-like lectins (siglecs) are type 1 membrane proteins displaying an amino-terminal immunoglobulin-like variable (v-set ig-like) domain that binds sialic acid and variable numbers of immunoglobulin-like constant region type 2 (c2-set ig-like) domains. siglec-h is a recently identified mouse-specific cd33-related siglec that signals via the itam linked adaptor protein dap12. so far the function of siglec-h and its ligand are unknown. in this project, we demonstrate gene transcription and protein expression of siglec-h by mouse microglia after interferon-c (ifn-c) treatment or polarization into a m1-subtype. targeting of beads to siglec-h by specific antibodies triggered the phagocytic uptake of the beads by the microglia. a siglec-h fc fusion protein generated by our laboratory selectively bound to cells with an altered glycocalyx, particularly glioma cells, but not to normal control cells. m1-polarized microglia phagocytosed glioma cells and also limited their proliferation in a co-culture system. interestingly, no signs of apoptosis were observed before phagocytosis of the glioma cells by siglec-h expressing microglia. phagocytosis of glioma cells was reduced after shrna mediated siglec-h knock down in microglia. furthermore, microglial clearance of glioma in vitro was dependent on the interaction of siglec-h with its corresponding adaptor protein dap12. our data show that m1-polarized microglial cells can engulf glioma cells via a dap12-mediated and siglec-h dependent uptake. glioblastomas (gbm) are the most common brain tumors in humans. although advances in the chemotherapeutic management of glioblastoma have been made, almost 80% of the patients die within the first 2 years after diagnosis. on the basis of these observations, the identification of new molecules that can counteract the gbm growth and invasiveness appears relevant. previously, we have demostrated that m2 muscarinic receptor agonist arecaidine inhibited cell proliferation in a time and dose-dependent manner and induced a severe apoptosis both in two glioma stable cell lines (u251 and u87) and in primary cultures derived from human biopsies. in order to clarify the mechanisms causing the decreased cell proliferation, we have evaluated the ability of m2 receptor activation to counteract the notch-1 and egfr pathways. the analysis by real time pcr and western blot have demonstrated that, in both cell lines, m2 receptor caused a decreased expression of egfr and notch-1 and its ligands (e.g. delta-1, jagged-1 and 22), suggesting that the decreased cell proliferation may dependent on altered expression and activity of these pathways. although facs analysis have showed that arecaidine induced an arrest of cell cycle progression, we observed, in both cell lines, a significant increase of dividing cells already after 24 hours of treatment. in particular, the number of metaphases increased significantly, while the percentage of anaphases diminished. furthermore we observed in both cell lines, a dis-regulation of mitotic spindle assembly, misalignment of chromosomes and the presence of multipolar spindles. moreover, due to prolonged activation of the promethaphase/methaphase mitotic checkpoint, chromosomes appeared more condensed in cells treated with arecaidine. facs analysis and immunocitochemistry using anti-histone c-h2ax, a marker of double strand breaks, have also demonstrated that arecaidine treatment induced dna damage. on the other hand, m2 agonist induced also oxidative stress, followed by an increased expression of the enzyme superoxide dismutase and sirtuins (sirt1 and sirt2). in conclusion, the data obtained suggest that the activation of m2 receptor induces cytostatic and cytotoxic effects in glioblastoma cell lines, opening new therapeutic perspectives for this receptor in glioblastoma therapy. univdersidade federal do rio de janeiro, rio de janeiro, brazil glioblastoma (gbm) is one of the most aggressive human cancers. despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. there is now a growing awareness that the main limitations in understanding and successfully treating gbm might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacitybrain tumor stem cells, which could represent a therapeutic target. in addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of gbm therapies targeting microglial cells. finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. further, extensive effort is being dedicated to characterize the molecular basis of gbm resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. we show that a non-cytotoxic concentration of equinatoxin ii (eqtx-ii), a pore-forming toxin from the sea anemone actinia equina, potentiates the cytotoxicity induced by temozolomide (tmz), a first-line gbm treatment, and to etoposide (vp-16), a second-or third-line gbm treatment. we also suggest that this effect is selective to gbm cells and occurs via pi3k/akt pathway inhibition. finally, magnetic resonance imaging (mri) revealed that a non-cytotoxic concentration of eqtx-ii potentiates the vp-16-induced inhibition of gbm growth in vivo. these combination therapies constitute a new and potentially valuable tool for gbm treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir-302-367, which induces the exit of gsc from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, 2012). we postulated that such a drastic change in the cell phenotype should be accompanied with metabolic alterations that could be instrumental in the loss of functional properties of gscs induced by the micro-rna cluster. metabolome profiling by mass-spectrometry of gscs and gsc-mir-302-367 pointed to changes in the gaba synthesis pathway (see abstract el-habr et al). remarkably, exposure of na€ ıve-gsc to metabolites of the gaba synthesis pathway found to be overproduced in gsc-mir-302-367 reproduced at least in part the effects of mir-302-367, including inhibition of clonality, down-regulated expression of selfrenewal markers, and loss of self-renewal properties. the metabolites studied acted by interfering with progression of the cell cycle and the nuclear localization of transcription factors crucial for maintenance of gsc stem-like properties. studies assaying the effects of the metabolites on gsc tumor-initiating properties are under way. these results demonstrate that metabolic regulations can participate in the control of gsc properties, and opens novel paths in therapeutic targeting of glioblastoma. *lgd and eae contributed equally. glioblastomas are the most frequent and aggressive form of adult primary brain tumors. glioblastoma stem cells (gscs) are thought to play key roles in the development and resistance of the tumor to existing radio-and chemotherapies. our previous results showed that the cluster of micrornas, mirna-302-367, induces loss of gsc stem-like and tumor-inducing properties (fareh et al. 2012). to further understand the molecular pathways controlling the peculiar properties of gscs, we sought for metabolic changes likely to accompany the drastic change in cell phenotype induced by mir-302-367 expression. we measured the intracellular and secreted levels of 271 metabolites by mass spectrometry. reconstitution of the metabolomes revealed significant and coordinated changes in components of the krebs cycle, and the glutamine/glutamate neuropeptide metabolic pathway that suggested altered turnover of the gaba synthesis pathway. exon array hybridization and western blot analysis, revealed changes in expression of several enzymes of the gaba synthesis pathway in mir-302-367-gscs. of note, none of the analyzed enzyme transcripts appeared as a direct target of mir-302-367. our results could be integrated in a complex multistep schema compatible with enhanced turnover of the gaba synthesis pathways, resulting in decreased cell gaba levels and increased levels of gaba by-products, as observed in mir-302-367 gscs. further studies showed that these metabolic changes participate in the induced loss of gsc properties (see abstract by dubois et al). * eae and lgd contributed equally colonization of the brain by carcinoma cells is barely understood, in particular when considering interactions with the host tissue. we recently demonstrated that microglia assist carcinoma cell invasion. current findings indicate that this is part of a danger response of the entire brain tissue. in the brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia as well as astrocytes comparable to that seen at the interface of human cerebral carcinoma metastases. this response tries to protect the brain from intrusion of benign epithelial cells by inducing apoptosis. however, this is ineffective against malignant cells which did not undergo apoptosis and actually exploited this reaction to invade instead. gene expression and functional analyses revealed that c-x-c chemokine receptor type 4 (cxcr4) and wnt signaling are involved in this process. furthermore, cxcr4 regulates the recruitment of microglia towards brain injury in a zebrafish model and was expressed in human stroke patients, suggesting a conserved role of cxcr4 in various danger reactions. we propose that glia-assisted malignant invasion takes advantage of the physiological two-stage glial defense reaction. carcinoma cells escape the second step of cell killing and misuse the damage response to colonize the brain. university of tuebingen, tuebingen, germany aquaporin-4 (aqp4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. in contrast, most cultivated glioma cell lines do not express aqp4, and primary cell cultures of human glioblastoma lose it during the first passages. accordingly, in two glioma cell lines of the rat (c6 cells and rg2 cells) and in sma mouse glioma cell lines we found no aqp4 expression. this let us consider the possibility that aqp4 expression depends on brain microenvironment. aqp4 negative rat glioma cells were implanted into rat brain. within two weeks, a tumor developed. aqp4 staining of the tumor cells was positive. however, if the identical cells were implanted into the rat's flank, they did not express aqp4. in contrast to the normal brain, where aqp4 staining is polarized in the astrocytic endfoot membranes, the aqp4 staining in c6 and rg2 tumors was distributed over the whole glioma cell as in human glioblastoma. we conclude that the micro-environment is crucial for aqp4expression in brain and brain-tumor. glioblastomas (gbm) are infiltrative and highly vascularised brain tumors containing a subpopulation of multipotent stem-like cells. here we questioned whether notch pathway activation in these cells influenced the extent of tumoral vascularisation and dissemination. we used two cd133 1 sox2 1 gbm stem-like cultures generating highly infiltrative but poorly vascularised tumors upon intracranial grafts. the use of a hes5 promoter reporter construct indicated that the notch pathway was only active in a small fraction of cells. sustained notch activation in these cultures, using an activated form of the notch-1 receptor (nicd), induced a profound alteration of their morphology, phenotype and properties. a severe decline of their migration and proliferation rates was observed in vitro and in vivo. intracranial and subcutaneous transplantation revealed that nicd triggered an extensive vascularisation of tumoral cells indicated by the presence of wellformed vessels with large lumens. close interactions between gbm cells and host endothelial cells were readily observed but no evidence for endothelial transdifferentiation was found either in vitro or in vivo. microarray and proteomic analyses showed that nicd induced the expression of vascular adhesion proteins (icam-1, vcam-1, itga9), proangiogenic cytokines (plgf, il-8, hb-egf), metalloproteinase (mmp-9), a-smooth muscle actin and dll4, a notch ligand essential for vascularisation. this was associated with a remarkable transcription factor switch whereby cells became klf9 1 snai2 1 while ascl1, olig2, and sox2 were reduced or lost. thus in addition to its well-described role in vascular development and remodelling, the notch pathway is also central in controlling proliferation, migration and vascularisation of gbm-stem like cells. t04-04b extracellular space diffusion parameters in the mouse thalamus in bral2 knock-out mice retinal wholemounts were immunostained with antibodies against gfap, iba-1 and mhc-ii. results: in the na€ ıve retinas, weak constitutive mhc-ii expression was scarcely found in some iba-11 microglial cells and rarely in gfap1 astrocytes. only a small dendritiform subpopulation of iba-11 cells, located in the juxtapapillary area and in the marginal region of the retina, had a strong mhc-ii immunoreaction. in comparison with na€ ıve both, in contralateral and in oht-eyes: i) gfap was upregulated in m€ uller cells and microglia was activated; ii) mhc-ii was upregulated on macroglia and microglia. in microglia, it was similarly expressed in contralateral and ohteyes. by contrast, in macroglia, mhc-ii upregulation was observed mainly in astrocytes in contralateral eyes and in m€ uller cells in oht-eyes conclusions: both, contralateral and oht-eyes had macro-and microglial retinal changes in mhc-ii expression after two weeks of laser-induced oht approximately 70% of the nmo patients harbor antibodies against the water-channel aquaporin-4, aqp4-igg (seropositive nmo), expressed mainly by perivascular astrocytes. however, some patients diagnosed with nmo don't have aqp4-igg (seronegative nmo). the aim of this work is to compare in vitro the neuroinflammatory profile of igg from seropositive (igg-nmo1) and seronegative (igg-nmo-) nmo patients. pool of purified igg from igg-nmo1 and igg-nmo-patients fulfilling diagnostic criteria of 2006, and from multiple sclerosis (igg-ms) patients and healthy controls (igg-hc) were include in the study. mixed glial cell cultures of astrocytes (40%), oligodendrocytes (40%) and microglia (15%) were obtained from spinal cord of postnatal c57bl6/j mice. after 21 days in vitro, cells were treated with the igg pools for 12 hours for rt-pcr, and 24 hours for western blot and immunocytofluorescence (if) igg-nmo1 caused a stronger upregulation of c/ebpb mrna and nos2 protein than igg-ms and igg-nmo-. all these data show that igg-nmo1 and igg-nmo-induce a different proinflammatory glial activation. the proinflammatory pattern of igg-nmo-is quite similar to that induced by igg-ms patients. these findings raise the question if igg-nmo-could be a different disease. such as gdnf, artemin, bdnf and sonic hedgehog, adhesion molecules including p75ntr, n-cadherin and n-cam and the transcription factor olig1. c-jun also controls the structure of the regeneration tracks. in schwann cell c-jun null mice, the initial rate of axonal regeneration after injury is impeded and long term recovery of function measured by footprint analysis, toe pinching , von frey hairs, and the hargreaves test is not seen 10 weeks after injury. there is widespread death of both large and small drg neurons and although spinal cord motor neurons survive there is widespread failure of both sensory and motor neurons to reinnervate target muscles united states mutations in the neurofibromatosis 2 (nf2) gene cause neurofibromatosis type 2 (nf2) characterized by formation of multiple schwannomas and meningiomas. nf2 encodes a tumor suppressor protein called merlin. loss of function of merlin is associated with an increased level of active rac and p21-activated kinases (pak). the lim domain kinases (limk1 and 2) are rac-pak substrates and modulate actin dynamics and cytoskeletal organization by phosphorylating cofilin, an actin severing and depolymerizing agent acknowledgements: this work was supported by grant ncn 2011/03/b/ nz4/00042 and statutory funds. phenotypes, their functions are fundamentally different in a model of brain injury. these data provide new insights into the protective role of microglia after brain injury. . although the pathogenesis is still not clear, activation of the immune system at some point of the disease process plays a crucial role. it is known that myelin-specific autoreactive t-cells and monocytes cross the blood-brain barrier (bbb) and migrate into the cns. within the cns, these cells secrete proinflammatory cytokines which stimulate local glial cells to produce inflammatory factors, including reactive oxygen species that contribute to demyelination and ultimately axonal loss. the destruction of the bbb and the influx of monocytes and t-cells from the circulation are key events in the progression of ms pathology. tissue transglutaminase (tg2) is a multifunctional enzyme that has been shown to play a role in monocyte/macrophage adhesion and migration onto extra cellular matrix proteins in vitro. this binding occurs via interaction with bintegrins. previously, we observed the appearance of tg2 in monocytes in active human post-mortem ms lesions in the cns.in the present study we question whether tg2 expression is regulated in primary human monocytes by inflammatory mediators and whether it is modulated in ms patients. methods: to this end, tg2 in monocytes was detected by semiquantitative rt-pcr.results and conclusions: our preliminary results show that activation of primary human monocytes with lps or lps1ifnc results in a time-dependent increase in tg2 mrna whereas the expression of factor xiiia (another member of the transglutaminase family) remains stable over time. moreover, the tg2 mrna level is higher in unstimulated monocytes derived from ms patients compared to control subjects while factor xiiia mrna level remains unaffected.these initial data are promising with respect to a possible role for monocyte-derived tg2 being involved in adhesion to and migration across the bbb under inflammatory conditions (e.g. multiple sclerosis). this hypothesis has not been proven yet. in addition, by increasing the number of patients, a consistent difference in tg2 expression in monocytes between ms patients and control subjects may point to tg2 as a novel biomarker for disease. an important aspect of chronic neurodegenerative diseases, such as alzheimer's, parkinson's, huntington's and prion disease, is the generation of an innate inflammatory response within the central nervous system (cns). microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and morphological activation. using a laboratory model of chronic neurodegeneration (me7 murine model of prion disease), we studied the time-course and regulation of microglial proliferation. our results show that resident microglial cells have an increased proliferation rate during the development of the disease, leading to a significant increase in the population, without a contribution from circulating cells. microglial proliferation is differentially regulated in diverse regions of the cns, pointing to a heterogeneous development of the pathology. we have identified novel molecular regulators of the proliferative response, and addressed the significance of the contribution of microglial cells to the pathological course of the disease by modifying their proliferation. we also found a correlation of our results with the scenario present in chronic human neurodegenerative conditions variant creutzfeldt-jakob disease (vcjd) and alzheimer's disease. our results demonstrate that microglial proliferation is an important feature of the evolution of chronic neurodegenerative disease, with direct implications for understanding the contribution of the cns innate immune response to disease progression. here, we examined the underlying mechanism of fibronectin aggregation and address whether inflammatory mediators, such as toll like receptor (tlr) agonists or pro-inflammatory cytokines induce fibronectin aggregation by astrocytes. our findings reveal that only tlr3 and 24 agonists, including the endogenous tlr3 agonist stathmin, increased stable fibronectin aggregation. interestingly, only white matter-derived astrocytes displayed fibronectin aggregation whereas in cortical astrocyte cultures fibronectin remained unaffected, suggesting regional differences in the functional response of astrocytes. furthermore, in rat cerebellar slice cultures, the tlr3 agonists only induce aggregation of fibronectin after lysolecitin-induced demyelination. in this manner, efficient remyelination is impeded and, consequentially, progressive neuronal decline occurs.taken together, tlr3 and 24 agonists promote fibronectin aggregation by primary rat astrocytes. since tlr3 on astrocytes and stathmin are both upregulated in ms lesions, stathmin, as an endogenous tlr3 agonist, might play a role in inducing fibronectin aggregation after c-jun reprograms schwann cells of injured nerves to generate a repair cell essential for regeneration. neuron. 75(4): 633-47). in this study we identify a novel role for c-jun in the activation of notch signalling in the denervated schwann cell. we find that c-jun is required to activate notch signalling, leading to upregulation of the bhlh protein hes1. hes1 then plays two functions in the denervated cell, promoting myelin breakdown and acting as part of a negative feedback loop to reduce c-jun levels. as a result of this, ablating notch signalling specifically in schwann cells acts to increase c-jun levels. we show that this upregulation of schwann cell c-jun accelerates axon outgrowth, target re-innervation and remyelination by generating a cell, which promotes faster than normal functional recovery. these results identify novel functional links between the c-jun and notch signalling pathways. they also show that not only is schwann cell c-jun necessary for successful nerve regeneration, but that nerve repair can be improved by enhancing normal c-jun signalling. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). after injury of the peripheral nervous system (pns), schwann cells and macrophages phagocyte myelin debris in wallerian degeneration in order to regenerate axons to distal targets. the immunoreceptor cd300f is normally expressed in the myeloid line and in nervous system in microglia, oligodendrocytes and neurons under certain conditions. however, little is known about the cd300f ligands. by using a cd300f-fc fusion protein we have analyzed the expression of the cd300f ligands in the pns. moreover, we have also analyzed the possible role of the cd300f immunoreceptor in peripheral nerve regeneration by blocking the interaction between cd300f and its ligands with the same fusion protein. thy1-yfp-h mice sciatic nerves were injected with cd300f-fc, control migg2a or pbs immediately before a crush injury. the results show that cd300f ligand is expressed in schwann cells. moreover, after a sciatic nerve injury, animals injected with the cd300f-fc protein show a lower number of yfp-positive fibers growing into the tibial nerve after 10 days post-lesion (dpl) than control groups. moreover, the cd300f-fc group shows a higher number of macrophages and cd206-positive cells at 10 dpl when compared to control groups. we do not see these differences in axon regeneration and macrophage infiltration after 28 dpl. we have also evaluated the mrna expression of the pro-inflammatory cytokine il-1b at 24 hours after crush and injection of the fusion protein. q-pcr shows an up-regulation of the mrna in control groups and a lower mrna expression level in the cd300f-fc protein group. together these results show that the pair cd300f receptor and ligand is implicated in some aspects of wallerian degeneration and nerve regeneration such as the modulation of both the influx and phenotype of macrophages. in response to the central nervous system (cns) injury, hematopoetic cells migrate to the lesion where they can potentially contribute to tissue regeneration. following cns injury, these cells can secrete a plethora of immunomodulating cytokines and/or pro-regenerative growth factors as well as phagocyte proinflammatory tissue debris. nevertheless, the role of primitive hematopoetic stem/progenitor cells (hspc) -derived from bone marrow -to support cns regeneration has not been studied in detail. therefore, the aim of the present study was to examine characteristics of hspc in vitro as well as to test proregenerative potential of these cells to support cns repair in vivo.highly pure population of primitive hematopoetic stem/progenitor cells was isolated from the bone marrow and the cns with fluorescence activated cell sorting (facs). the number of hspc in the cns lesion was significantly increased compared to intact cns tissue. sorted-bone marrow hspc were co-cultured with primary astrocytes to examine their phenotype according to the presence of specific antigens and gene expression as well as to test their phagocytosis potential. in the presence of astrocytes, bone marrow-derived hspc differentiated in vitro into microglia-like cells expressing specific myeloid/microglia markers and showing high ability to ingest fluorescent microbeads. the in vivo potential of hpsc for supporting regeneration was examined by their transplantation into the cns lesion.results of our study demonstrated that primitive hematopoetic stem/progenitor cells are the source of microglia-like cells which can support regeneration in the central nervous system. when the brain or spinal cord is injured, glial cells in the damaged area undergo complex changes resulting in the formation of the glial scar. whether the scar is beneficial and detrimental to recovery remains controversial. the signals that initiate the formation of the glial scar are unknown. because both canonical and non-canonical wnts are increased after spinal cord injury (sci), we examined the role of canonical wnt signaling in the glial reactions to cns injury. to disrupt b-catenin-dependent wnt signaling specifically in opcs, we created transgenic mice carrying an opc-specific conditionally deleted bcatenin gene. after moderate contusion injuries to the thoracic spinal cord of control mice, opcs proliferate and accumulate in the penumbra region surrounding the injury epicenter. in the b-catenin-depleted mice, there is reduced proliferation and accumulation of opcs after sci, reduced accumulation of activated microglia/macrophages and reduced astrocyte hypertrophy. using a crushed optic nerve model, we show that these reduced glial reactions create an environment that is permissive for axonal regeneration. these results suggest that canonical wnt signaling in glia after cns injury is necessary for the formation of the glial scar and they identify wnt signaling as a new therapeutic target for promoting axon regeneration. the crucial role of central nervous system (cns) glial cells in the integrity and physiology of neuronal networks, is well known. however, little is known about glial cells in the peripheral nervous system (pns). one of the main glial cell types in the pns is the perineuronal satellite glial cells (sgcs), that surround drg neurons in an envelope-like fashion. despite their abundance in peripheral nerves, having stem cells properties and playing a role in neuropathic pain, there is limited information on their physiology. although sgcs have similarities to astrocytes in terms of purinergic-and gap junction-mediated signaling, their membrane properties and ionotrophic glutamate receptor expression are more or less unknown. our aim was therefore to characterize the membrane properties and glutamatergic ion channel expression of sgcs. we performed patchclamp experiments on sgcs wrapped around sensory neurons in the rat dorsal root ganglion (drg) in vitro. whole-cell recordings revealed that sgcs are slightly hyperpolarized in comparison to the neurons they ensheath, with a resting membrane potential of approximately $80mv and a high input resistance (&gt;1gx). moreover, upon membrane depolarization no detectable voltage-gated na 1 , ca 21 or k 1 currents were detected. extracellular application of glutamate agonist, kainic acid, n-methyl-d-aspartic acid (nmda) and 2-amino-3-(3hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (ampa) during the recording, did not evoke any response from the cells, indicating their lack of ionotropic glutamatergic receptor (iglur) expression. double immunocytochemistry on lucifer yellow (ly) filled scgs revealed that they express the scip/oct6 transcription factor, which is also expressed by promyelinating schwann cells.drg ganglion sgcs differ from cns perineuronal cells as they lack glutamatergic receptors, but are similar to astrocytes as they have no voltage-gated ion channels, are gap-junctionally coupled and express glutamine synthetase. thus, we are currently addressing if sgcs respond to neuronal activity in a similar manner to astrocytes in the cns with the use of calcium imaging. these studies will provide further insights into the physiological role of sgcs in the pns. multiple sclerosis (ms), a chronic neuroinflammatory disease with presumed autoimmune etiology, is the most common demyelinating disorder of the central nervous system among young adults. progressive neurological impairment of the patients are associated with accumulation of characteristic brain lesions characterized by inflammation, demyelination, gliosis and axonal damage. demyelination and loss of oligodendrocytes contributes to axonal loss and permanent neurological deficits. remyelination occurs but is limited; one contributing factor is the incapability of oligodendrocyte precursor cells (opc) to differentiate and form new myelin sheaths. k 1 channels are not only known to predominantly set and maintain the membrane potential but also to be important regulators of various cell functions like proliferation and maturation. specific regulation of these cellular functions is based on high channel diversity and their ability to form heteromers as well as alternate mrna splicing and posttranslational modifications. so far the expression and function of potassium channels in cells of the oligodendroglial lineage has not been clearly identified. previous studies indicate that an unspecific blockade of k 1 channels in opc suppresses maturation as well as proliferation. moreover, for one distinct channel (k ir 4.1) a functional relevance has been shown, as deficiency of kir4.1 leads to altered opc maturation and hypomyelination in mice. here, we elucidate the expression and functional relevance of k 1 channels in oligodendrocytes. our first electrophysiological recordings from primary murine opcs revealed the presence of different k 1 channels with both inward and outward rectification. in future experiments, we aim at identifying k 1 channel subtypes that specifically regulate opc cell functions and might influence cell maturation under neuroinflammatory conditions. thereby, we want to offer new insights in the functional role of k 1 channels in opcs/oligodendrocytes and contribute to novel therapeutic strategies for the treatment of ms. aqp4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. aqp4 has been described as an important entry and exit site for water during formation of brain edema and regulation of aqp4 is therefore of broad interest. phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. protein kinase (pk)-dependent phosphorylation of ser 111 has been reported to increase the water permeability of aqp4 expressed in an astrocytic cell line. this possibility was, however, questioned based on the crystal structure of the human aqp4. our study aimed to resolve if ser 111 was indeed a site involved in phosphorylation-mediated gating of aqp4. the water permeability of aqp4-expressing xenopus oocytes was not altered by a range of activators and inhibitors of pkg and pka. mutation of ser 111 to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of aqp4. pkg activation had no effect on the water permeability of aqp4 in primary cultures of rat astrocytes. molecular dynamics simulations of a phosphorylation of aqp4.ser 111 recorded no phosphorylation-induced change in water permeability. a phospho-specific antibody, exclusively recognizing aqp4 when phosphorylated on ser 111 , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. thus, our data indicate a lack of phosphorylation of ser 111 and of phosphorylation-dependent gating of aqp4. undocked connexons may form open hemichannels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules with masses lower than 1 kda. a range of physiologically relevant molecules, smaller than the assumed molecular cut-off of 1 kda, have therefore been proposed to permeate connexin 43 (cx43) in its hemichannel configuration. however, the permeability profile of cx43 hemichannels remains unresolved as does the molecular substrate for hemichannel activity in astrocytes. exposure of cx43-expressing xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. in spite thereof, a range of smaller biological molecules, such as water, glutamate, lactate, glucose, and atp, did not gain detectable access through the pore. in contrast, permeability of glutamate, glucose and atp was observed in oocytes expressing the other major astrocytic connexin, cx30. exposure to low divalent cation solutions also induced a robust membrane conductance in cx30-expressing oocytes but none in cx43-expressing oocytes. expression of cx43 in c6 glioma cells failed to induce hemichannel activity. our results thus call for caution when assigning molecular identity to astrocytic channel activity and when interpreting hemichannel-mediated dye-uptake as equal to permeability of physiologically relevant molecules. atp-gated p2x4 receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. p2y6 metabotropic g q -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by udp. it has been reported recently that expression of both p2x4 and p2y6 is upregulated in activated microglia following nerve injury. we show here, in resting as well as lps-activated primary microglia, that p2y6 decreases p2x4-mediated calcium entry and inhibits the dilation of p2x4 channels into a large-conductance pore measured with a yo-pro-1 uptake assay. furthermore, p2y6 activation modulates the atp-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype.reconstituting the p2x4-p2y6 interaction in recombinant systems shows that p2y6 activation decreases p2x4 current amplitude, activation and desensitization rates, and reduces p2x4 channel permeability to the large cation nmdg 1 . phospholipase c-mediated hydrolysis of the phosphoinositide pi(4,5)p 2 , a necessary cofactor for p2x4 channel function, underlies this inhibitory crosstalk. as extracellular levels of both atp and udp are increased in the spinal cord following nerve injury, the control of p2x4 activity by p2y6 might play a critical role in regulating neuropathic pain-inducing microglial responses. with sc, thus representing a valid sc alternative. we have previously shown that dasc express c-aminobutyric-acid (gaba) receptors (i.e. gaba-a and gaba-b receptors), which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitter systems in asc.in this study we investigated the expression of purinergic receptors in dasc. using rt-pcr, western blot analyses and immunohistochemistry we have demonstrated that asc express p2x 3 , p2x 4 and p2x 7 purinoceptors. interestingly, differentiation of asc towards glial phenotype was accompanied by up-regulation of p2x 4 and p2x 7 receptors. using ca 21imaging techniques, we have shown that stimulation of purinoceptors with adenosine-5 0 -triphosphate (atp) results in intracellular ca 21 signals, indication functional activity of these receptors.. moreover, we have shown that the increase of intracellular ca 21 leads to sc death, an effect that can be prevented using specific p2x 4 or p2x 7 antagonists.altogether, these results show, for the first time, the presence of functional purinergic receptors in sc-like derived from asc and their link with critical physiological processes such as cellular death and survival. the presence of these novel pharmacological targets in dasc might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dasc for peripheral nerve repair. university of freiburg, freiburg, germany intracellular ph homeostasis is a vital function shared by all cells and is mainly regulated by the coordinated action of several acid-base transporters. in the brain, ph homeostasis is of particular importance since changes of extracellular ph (pho) are events associated with both physiological conditions and pathological states in brain function. transient pho changes usually accompany physiological processes, including neuronal activity, and astrocytes respond to a rise in extracellular k 1 with plasma membrane depolarization and intracellular alkalinization. on the other hand, loss of brain ph homeostasis can lead to severe pathological conditions and significant changes of pho have been observed during seizures and spreading depression.in the present study, we sought to investigate regulation mechanisms of the electrogenic sodium/bicarbonate cotranspoter 1, nbce1, and of the vacuolar h 1 -atpase (v-atpase) in primary hippocampal astrocytes following extracellular acid-base changes, following neuronal activity, and using the 4-aminopyridine (4-ap) model of epilepsy in vitro.we show that extracellular acidosis, but not extracellular alkalosis, increased the number of activated astrocytes. our data also show differential regulation of acid-base transporters in the hippocampal glial cells during extracellular acid-base changes. intracellular ph measurements revealed a crucial role for v-atpase in regulation of intracellular ph, a process accompanied by increased membrane expression of the transporter, as assessed by immunofluorescence and surface biotinylation. nbce1-b is the nh2-terminal variant of nbce1 predominantly expressed in glial cells and is transcriptionally regulated followed neuronal activity or treatment of the cells with 4-ap.these data propose transcriptional and post-translational modification of acid-base transporters as putative regulatory mechanisms in astrocytes to cope with changes of extracellular ph serving the maintenance of intracellular ph homeostasis. these astroglial networks fulfil a variety of functions in the brain, e.g. potassium buffering and metabolite transport. in this study we compared glial networks in different brain regions to investigate site specific effects on network formation. we combined electrophysiology and immunohistochemstry with semi-quantitative rt-pcr and western blot analysis to investigate connexin expression, gap junction coupling and antigen profiles. experiments were performed in wild type and transgenic mice with glia specific fluorescence labelling as well as in cx30ko mice. astrocytes were investigated between postnatal days 12-60. gap junction networks in the ca1 region of the hippocampus and the ventrobasal thalamus show abundant coupling in hgfap-egfp mice. intriguingly, we found significant coupling between oligodendrocytes and astrocytes in the thalamus, while in the hippocampus panglial coupling was less abundant. other glial cells did not participate in the networks. we also found that a fluorescent glucose analog, 2-nbdg, propagates through the thalamic panglial network. the function of these panglial networks remains largely unclear.in heterozygous cx43-ecfpki mice, deletion of one allele of the major hippocampal cx43 significantly reduced the number of coupled astrocytes only in the hippocampus, while the thalamic networks remained unchanged. sr101 labelling of astrocytes and subsequent 2p microscopy identified a significant subset of thalamic sr1011 cells lacking cx43-ecfp expression. sr101 did not label oligodendrocytes as analysed in plp-gfp mice. semi-quantitative rt-pcr and western blot analysis revealed stronger expression of cx30 in thalamic nuclei while cx43 levels were higher in the hippocampus. this indicates a minor role for cx43 in gap junction coupling of astrocytes in the thalamus. consistent with these findings, the thalamus of cx30ko mice displayed a strong decrease in astrocytic cell coupling compared to wild type littermates.together, these results indicate that thalamic astrocytes differ in various aspects from their counterpart in other brain regions and support the emerging concept of astrocyte heterogeneity. the mechanism of secondary damage spread after brain trauma remains unresolved. in this work, we redirected the attention to astrocytic communication pathways. using an in vitro trauma model that consists of a scratch injury applied to a rat astrocyte monolayer, we found a significant induction of connexin43 hemichannel activity, demonstrated by ethidium uptake, in regions distal from the injury ($17 mm away and maximal $1 h after injury). this response was abolished by two connexin hemichannel blockers, la 31 and the peptide gap26. in addition, the trauma-induced increase in hemichannel activity was prevented by inhibition of purinergic p2 receptors. the scratch-induced increase in hemichannel activity was absent in astrocytes from cx43 knockout mice. this activity took place with a singular spatial distribution, since cells located at $17 mm away from the scratch gained connexin hemichannel activity. however, the functional state of gap junction channels (dye coupling) was not significantly affected in the same locations. the connexin43 hemichannel activity was also enhanced by the acute extracellular application of 60 mm k 1 . the increase in hemichannel activity was correlated with an increment in apoptotic cells, measured by immunofluorescence to annexin v at 24 h post-trauma, which was totally prevented by gap26 peptide. these findings could open a new approach to prevent or reduce the secondary cell damage due to brain trauma. l. schlosser, a. scheller, f. kirchhoff institute of physiology, saarland university, homburg, germany current research suggests that astrocytes represent heterogeneous neuroglial populations in different regions of the brain. this is particularly evident when the expression pattern of various transmitter receptors is analyzed. hippocampal astrocytes, for example, do not express ampa receptors, while cerebellar bergmann glia is loaded. functional analysis of glial receptors requires dedicated efforts for each distinct brain region and developmental age. unfortunately, the molecular identification using antibody approaches is often hampered by either poor antibody quality or abundant receptor expression on adjacent neuronal membranes. therefore, we are using cre/lox-mediated gene ablation for proper functional analysis.here, we are focusing on the characterization of astrocyte-specific gene deletion of the gaba b1 receptor. metabotropic gaba b receptors consist of two subunits gaba b1 and gaba b2 forming a heteromeric receptor complex of which gaba b1 is essential. in contrast to neurons, activation of astroglial gaba b receptors leads to transient rises of intracellular ca 21 . the functional impact of these glial gaba b -receptors is largely unknown.to address the gaba b receptor function in astrocytes we took advantage of the cre/lox system and crossbred glast-creert2 knockin mice with floxed gaba b1 receptor mice. first immunohistochemical analysis reveals a selective deletion of gaba b1 on a majority of astroglial processes. functional studies addressing the role of these receptors in astroglial ca 21 signaling are in progress. purinergic signaling is the most diverse system in astrocytes to communicate with other glial cells and neurons. astrocytes express a variety of p2x (ionotropic) and p2y (metabotropic) receptors. especially in case of brain injury, atp levels and receptor expression on astrocytes are increased. atp is immediately degraded to adp, amp and adenosine. these nucleotides/nucleosides act back on the preferred purinoreceptor expressed on glial and neuronal cells. in the last decade researchers tried to evaluate the functional impact of astrocytic purinoreceptors on the complex information flux at the tripartite synapse. a prominent role has been suggested for the p2y1 receptor subtype. we generated conditional mouse mutants to investigate the function of p2y1 receptors in astrocytes in vivo and crossbred mice carrying the floxed p2y1 gene with mice that express the inducible dna recombinase (creert2) under the control of the glast (l-glutamate/ l-aspartate transporter) locus. ablation of the p2y1 receptor is induced in development and adulthood by intraperitoneal tamoxifen injections. successful recombination of the targeted p2y1 receptor is evaluated by qrt-pcr on genomic dna and mrna usually 21 days after tamoxifen application. at the genomic level we find $25% and $50% recombination in astrocytes of the cerebellum and hippocampus, respectively. similar recombination frequencies are observed in young (p11) or adult mice (6-10 weeks). when looking at the transcript level, the mrna is reduced to 57% in the cerebellum of adult mice and to 76% in the hippocampus. in young mice we determined a reduction to 40% and 42% mrna expression in the cerebellum and hippocampus, respectively. given the high level of p2y1 expression on other cells of the brain these findings indicate a successful astrocyte-specific knockout. further fluorescence-activated cell sorting (facs) of recombined cells expressing the red fluorescent protein td-tomato will be used to verify the knockout. the potential function of these astroglial receptors in neuronal networks will be as well investigated using histological (em and light microscopy), twophoton ca 21 imaging in situ and in vivo and behavioral approaches. we recently showed that they invade the cortex at 11.5 days of embryonic age (e11.5). they first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. the absence of the expression of mac-2 and mhc ii suggest these cells have a na€ ıve/quiscent phenotype during embryonic development of the cortex. besides immunohistochemical markers is the presence of different k 1 channels on microglia also an indication of their activation stage. however most studies have been conducted on postnatal and adult microglial cells. therefore we aimed at determining the electrophysiological activation phenotype of these embryonic microglia.at the age of e13.5 and e15.5, microglial cells display a small inward rectifying k 1 current and this independent of their location in the embryonic cerebral cortex and their cell morphology. these cells also express functional p2x receptors, which based on the profile of the response are most probably p2x7 receptors. time-lapse analysis showed that embryonic microglia are highly dynamic cells. suggesting that these cells are already very active during fetal development. rapid extracellular removal of glutamate, the major excitatory neurotransmitter in the cns, is essential for normal brain function. this task is primarily accomplished by the action of the sodium-dependent, high-affinity transporters glast and glt-1 (rodent analogous of eaat1 and eaat2), which are mainly expressed by astrocytes. impairment or failure of glast and glt-1 plays an important role in many pathological conditions. question: pelizaeus-merzbacher-like disease (pmld) is a hypomyelinating leukodystrophy caused by mutations in the gjc2 gene encoding the gap junction (gj) protein connexin47 (cx47). cx47 is expressed in oligodendrocytes and forms most gj channels to astrocytes and to other oligodendrocytes. since pmld-associated gjc2 mutations cause loss of cx47 function, gene replacement strategies may be promising for developing future treatments. a mouse model for plmd, the cx32/ cx47 double knockout (ko), is characterized by early onset of severe leukodystrophy at 4 weeks of age leading to death by 6 weeks, offering the possibility to test therapies. the aim of the present study was to generate a lentiviral vector to allow gene delivery specifically to oligodendrocytes, and to examine the transduction efficacy, distribution, duration and levels of egfp reporter gene and cx47 expression throughout the cns, in order to establish a method for oligodendrocyte-targeted gene therapy.methods: the expression cassette with the cx47 gene along with the ires-egfp as a reporter gene under the control of oligodendrocyte-specific cnp promoter was cloned into the lentiviral vector pcclsin.ppt.hpgk.gfp.pre. mock vectors lacking the cx47 gene were also generated as controls. viral particles were produced to high titers and purified, before injection. lentiviral vectors were delivered into the brain of wild type (wt) and cx47ko mice at postnatal day 1 (p1), intraventricularly and in the stratum radiatum of the dorsal hippocampus. egfp and cx47 expression was assessed using immunochemistry and immunoblot analysis at different time points post injection.results: we found widespread expression of virally delivered egfp in different brain regions colocalizing with oligodendrocyte markers (cc1 and olig2). the expression of egfp was detected from p15 until 3 months post-injection. on average 20.3 6 2.56% of oligodendrocytes were egfp positive, with highest rates in the subventricular zone (29.3 6 6.31%, n 5 6) and olfactory bulb (19.9 6 4.36%, n 5 8) and lower rates in the cortex (16.1 6 2.60%, n 5 6) and corpus callosum (12.3 6 3.40%, n 5 3). in cx47ko brain expression of virally delivered cx47 was also found after p21 with formation of gj plaques in a subpopulation of oligodendrocytes.conclusions: our results show that neonatal lentiviral gene delivery may result in stable and widespread cns expression targeted to oligodendrocytes by using cell specific promoters. thus, gene therapy approaches using lentiviral vectors may be feasible for future treatment of leukodystrophy and should be further studied. recent reports identified mrna coding for different cav subtypes like 1.2 and 1.3 in hippocampal ng2 glia. using acute brain slices prepared from developing and mature ng2-eyfp mice, we also found that all ng2 glia upon depolarization in different brain regions elicited a. grimaldi, g. d'alessandro, c. lauro, m. catalano, c. limatola sapienza university of rome, rome, italy glioblastoma (gbm) is one of the most aggressive tumor of the central nervous system, being characterized by a great invasiveness and a very low survival rate of patients. this work wants to better define the role of tumor microenvironment in the modulation of tumor invasiveness. it is known that tumor migration and invasiveness can be modulated by the chemokine cxcl12 and its receptor cxcr4. the expression of both these proteins has been demonstrated in various human gbm cell lines and it has been shown that, blocking this pathway, tumor cells greatly reduce their invasive ability. we have recently demonstrated that an important molecule directly implicated in tumor migration is the intermediate conductance calcium-activated potassium channel (kca3.1). given the expression of these channels also in other cell types infiltrating the tumor mass, like microglia, we investigated the effect of kca3.1 inhibition on microglia-glioblastoma interaction. preliminary data indicate that kca3.1 activity on microglia is involved in the movement of these cells toward the tumor mass. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in human glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir-302-367, which induces the exit of gscs from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, 2012) . to elucidate the gene networks that govern gsc exit from their stem state, we used chip-seq (chromatin immunoprecipitation followed by next generation dna sequencing), to identify gene loci showing enrichment of the active (h3k4me3) and repressive (h3k27me3) epigenetic marks in gsc-mir-302-367 as compared to na€ ıve gscs. functional significance of the chip-seq results was evaluated with confrontation of the chip data with the transcriptomes of the corresponding cells derived from exon array hybridization. western blot analysis showed that global h3k4me3 and h3k27me3 expression levels were similar in gsc and gsc-mir-302-367. chip-seq analysis revealed similar gross number of gene loci associated with h3k4me3 or h3k27me3 (13000 and 5000 genes, respectively) in either cell type. interestingly, 16% (2381 genes) of the analyzed genes exhibited a change in either h3k4me3 or h3k27me3, or both in mir-302-367-gscs as compared to na€ ıve gscs. these changes resulted into a transcript variation in 14% of the cases (332/2381) considering as significant an increase or a decrease of at least 2-fold and the 77% of these genes translated into congruent alterations in the corresponding transcript levels.analysis of the functional significance of the changes observed using functional annotation databases identified novel gene networks, likely to participate in the regulation of gsc properties. studies under way focus on members of these networks specifically activated in mir-302-367 gscs that might alter gsc dialog with their microenvironment. malignant gliomas are the most frequent primary tumors of the brain with poor clinical prognosis. infiltrating peripheral macrophages and resident microglia contribute significantly to the tumor mass. we have previously shown that microglia as the intrinsic immune competent brain cells promote glioma expansion by up-regulating metalloprotease mt1-mmp through toll-like receptor (tlr) and its adaptor protein myd88. in this study we identified tlr2, as the main tlr controlling mt1-mmp expression and pro-tumorigenic signaling in microglia. glioma-derived soluble factors and synthetic tlr specific ligands induced mt1-mmp expression in microglia from wt mice but not tlr2-/-mice. by using the organotypic brain slice model, we found that tumor expansion depended on both parenchymal tlr2 expression and the presence of microglia. tlr2 is also highly expressed in human gliomas and inversely correlates with patient survival. in search for an endogenous tlr2 ligand released from glioma cells, we screened glioma conditioned medium (gcm) by mass spectrometry for endogenous tlr2 agonists produced by glioma cells and found versican, an extracellular matrix proteoglycan and a reported ligand of tlr2. to examine if versican is the factor mediating the glioma-microglia interaction, we silenced versican expression in gl261 cells. primary microglia were then stimulated with gcm from versican sirna and non-target sirna transfected gl261 cells and microglial mt1-mmp expression was analyzed by real-time pcr. an almost 3-fold up-regulation in microglial mt1-mmp expression was observed using gcm from non-target transfectants while the expression was reduced with gcm from versican knock-down gl261 transfectants. our results show that tlr2 activation is an essential part of the signaling cascade employed by glioma cells to convert microglia into a pro-tumorigenic phenotype and tlr2 thus may be a novel target for glioma therapies. two ipsdms were used to determine their responses in the presence of glioblastoma cells. using lc-ms/ms method, proteomic profiles were generated and compared between ipsdms exposed to human glioblastoma cells and ipsdms exposed to normal human astrocytes as control.results: immunolabeling of the two ipsdm cell lines showed specific expression of microglial markers such as iba-1, cd11b, and cd68. the comparative proteomic analyses indicated that microglia exposed to glioblastoma cells showed differential protein expressions relevant for cytoskeletal activity, energy production, and cell survival compared to control.conclusion: this is the first study showing the proteomes of human induced pluripotent stem cell-derived microglia exposed to glioblastoma cells, and the findings could provide further insight of the interaction between microglia and glioblastoma. key: cord-257167-rz4r5sj7 authors: nan title: abstracts for the 29th annual meeting of the japan neuroscience society (neuroscience2006) date: 2006-12-31 journal: neuroscience research doi: 10.1016/j.neures.2006.04.004 sha: doc_id: 257167 cord_uid: rz4r5sj7 nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns19640. sl2-1-1 frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl3-1-1 neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif1a/kif1bbeta), nmda type(kif17) and ampa type(kif5s) glutamate receptors and mrnas(kif5s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least 42 rna related proteins such as hn rnp-u, staufen, and fmrps. kif2a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif3 suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif4 controls the activity-dependent survival of postmitotic neurons by regulating parp-1 activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award 2-1 what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in 1-3 months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v1). after complete ablation of v1, the monkeys recover performance of visually guided saccades toward the blind field in 2 months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi 17023055, kakenhi 15029261 sy1-1-01-2 distinct roles of two 7-pass transmembrane cadherins in neurite growth control tadashi uemura 1,6 , yasuyuki shima 1,6 , keisuke sehara 2 , manabu nakayama 3 , shinya kawaguchi 4 , mikio hoshino 5 , yoichi nabeshima 5 , tomoo hirano 4,6 1 graduate school of biostudies, kyoto university, kyoto, japan; 2 school of science, japan; 3 kazusa dna research center at chiba, japan; 4 graduate school of science, japan; 5 graduate school of medicine, japan; 6 crest, jst, japan drosophila flamingo (fmi) and mammalian celsr1-3, which are 7pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian 7-pass transmembrane cadherins celsr2 and celsr3 are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr2 enhanced neurite growth whereas celsr3 suppressed it. our result suggested that celsr2 had a stronger activity as g-protein coupled receptor than celsr3 did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of 7-pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin-1 is preferentially localized in axons; and nectin-3, in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin-1 and dendritic nectin-3 is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi 14104026 sy1-1-01-5 lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa 1 , fumikazu suto 2 1 nagoya university, nagoya, japan; 2 institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca3 pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema6a is expressed in ca3 pyramidal cells and principally suppresses invasion of mossy fibers to ca3. second, the repulsive signal of sema6a is mediated by plexin-a4 expressing in mossy fibers. third, the repulsive activities of sema6a are attenuated by plexin-a2 in the proximal dendritic segments of ca3 pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on (1) working memory maintenance and manipulation, (2) rule representation and task-switching, and (3) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy1-1-09-5 towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy1-1-09-6 neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of −25 genes on chromosome 7q11.23, has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi (17023501) sy1-1-17-3 a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total 21 full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and 18 k songbird cdna array. we used the arrays to reveal a set of 33 genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. -1-17-4 stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca1 pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi 17680033 sy1-2-02-4 role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using 2-photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., 2006) . furthermore, in response to single spine stimulation using 2-photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration (50 ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by 20 ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy1-2-10-3 basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼20 hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy1-2-10-4 coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f-18 fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos7 cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d1 receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad-1 found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca 2+ sensing protein was activated. synchronies of sv fusion are maintained by a ca 2+ sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori 21st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy1-3-11-3 sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim1. thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka 1 , takeshi baba 1 , sumiko mochida 2 , yoshimi takai 1 1 department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; 2 depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca 2+ -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab3 system, munc-13, munc-18, doc-2, and tomosyn. we have previously reported that tomosyn, a syntaxin-1-binding protein, works as a molecular clamp that controls free syntaxin-1 availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin-1, resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca 2+ sensor for neurotransmitter release. however, the physiological contributions of its ca 2+ -binding domains (c 2 a and c 2 b) are still unclear. we have studied the roles of aspartate (asp) residues in the c 2 b ca 2+ -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell 79, p. 717) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca 2+ binding to the c 2 b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca 2+ binding to the c 2 b because mutation in the second asp inhibits ca 2+ binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin-3 (nt-3) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt-3 and its low-affinity receptor p75 genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c270t in the 5 noncoding region, bdnf-linked polymorphic region, and val66met), three polymorphisms of the nt-3 gene (g-3004a, microsatellite in intron 1, and gly-63glu) and a missene polymorphism of the p75 gene (ser205leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p75 in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val66met) in the bdnf gene resulting in a prodomain substitution at position 66 from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps10 protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns052819 sy1-3-19-6 processing of bdnf and brain disorders masami kojima 1,2 1 aist, osaka, japan; 2 sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p75 neurotrophin receptor (p75ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); 372 (t/g) and 378 (t/g) of the human bdnf gene, causing amino acid substitution (r125m and r127l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy1-4-04-1 pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣1 leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣1, the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv-1 vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il2ss+fgf-2, and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi (14657350) sy1-4-04-4 hgf gene therapy for the treatment of spinal cord injury masaya nakamura 1 , akio iwanami 1 , kazuya kitamura 1,2 , yoshiaki toyama 1 , hideyuki okano 2 1 department of ophthalmology surgery, keio university, tokyo, japan; 2 department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th10 level in adult rat 3 days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. 1 or 6 weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap43+fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., 1989) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., 1999) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., 1999 . nature neuroscience. nakamura et al., 1989 . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first 1-2 months after stroke, suggesting the presence of a critical period. in the cat, illert et al. (1977) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c3-c4 segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. (1) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c3-c4 propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. (2) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca 2+ responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l1cam(l1) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l1. therefore, these syndromes have been reclassified as l1 syndrome. we performed a nation-wide l1gene analysis and identified li gene mutations in 35 families with l1 syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l1-mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l1 syndrome. research funds: kakenhi (16390424), (16689025) sy1-5-05-4 rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein 2 gene (mecp2) located at xq28 is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp2 was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like 5 gene (cdkl5) manifest as atypical rtt. the mutations of mecp2 are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in 6-ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase-9 are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp150, suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca3, and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣1or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣1and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context 2-4 days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr1, nr2a, 2b, 2c), glycine transporter 1, and eaac1, by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase 6 ␣ -subunit (pde6c), a component of cone-type pde. since genetic mutations of pde6c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde6 in visual functions. shinichi nakagawa 1 , masatoshi takeichi 2,3 , fumi kubo 1,3 1 nakagawa initiative research unit, riken, wako, japan; 2 riken cdb, kobe, japan; 3 department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt2b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt2b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt2b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi (16570184) sy1-6-06-3 identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea-1 to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea-1 during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea-1 double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea-1-positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea-1 have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx2 and crx play critical roles in retinal photoreceptor development. while otx2 is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi (16790070, 16027257, 17024061) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf19 found in the mesoderm. we find that although fgf19 can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf3 and is actually inhibited by fgf19. for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy1-6-06-6 how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel2, encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel2 in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel2 promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel2 which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein 57e was identified as the gene responsible for this behavioral difference among the species. obp57e forms a gene cluster with obp57d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp57d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp57d/e region obtained from 30 species phylogenetically located between d. melanogaster and d. pseudoobscula. sy1-6-14-4 an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi 1,2 , akinori nishi 1,2 , toshihiko shiroishi 1,2 , tsuyoshi koide 1,2 1 sokendai, kanagawa, japan; 2 national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c57bl/6 (b6) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b6 and msm, and those strains have the same genetic background as b6 except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b6-17msm, which have msm chromosome 17, showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi (16.12064) sy1-6-14-5 cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome 2 (capsq1) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv1) was impaired in kjr neurons in contrast to that of c57bl/6j mice. by searching the candidate genes, eh domain-containing four (ehd4), a trp-binding scaffold protein encoding gene was found. ehd4 binds to c-terminal of trpv1. three mutations were found in ehd4 of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv1. this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy1-6-14-6 comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than 45 different strains of mutant mice with the battery. surprisingly, more than 90% of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least 28 genes and 12 loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt4f caused by prx, and scan1 caused by tdp1. sy1-6-22-2 gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm1, who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm1 can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm2/gd2 synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, 2004) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa1) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi (17109015) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo-1 is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo-1 mediates the inhibition of axonal growth as a component of the ngr1/p75/lingo-1 and ngr1/troy/lingo-1 signaling complex. inhibition of endogenous lingo-1 by soluble lingo-1 or dominant negative lingo-1 can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo-1 treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo-1 treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo-1 mediates the inhibition of differentiation and myelination. loss of lingo-1 function using dominant negative lingo-1, lingo-1 rnai, or soluble lingo-1 or lingo-1 knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo-1 led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo-1 in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a 2d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc005787-01a1 sy1-7-15-2 neuronal avalanches reveal neuronal wirings of layer 2/3 cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi (17700318) sy1-7-15-3 spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi 1 , jean-pascal pfister 2 , kazuyuki aihara 1,3 , wulfram gerstner 2 1 department of complexity science and engineering, university of tokyo, japan; 2 school of computer and communication science & bmi, epfl, japan; 3 aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows 03j11691 and sci. res. on priority areas 17022012 from mext of japan, and swiss natl. sci. found. 200020-108097/1 sy1-7-15-4 timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as 200-300 ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi 1 , ikue otaki 1 , saya suzuki 1 , yasunobu yasoshima 2 , kazuto kobayashi 2 1 laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; 2 institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin-2 receptor ␣ subunit (hil-2r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil-2r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. -8-16-5 integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy1-8-24-1 molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj-1 is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are (1) leukemia in patients exposed with atomic bomb during the iind world war, and (2) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, (3) many genetic/familial diseases show clinical symptoms at the middle or old age. (4) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. (5) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp58, which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp58 gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp58 mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼100 drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc0/+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi3k/akt signaling, which results in decreased gsk3 activity, has also been shown to ameliorate ami. both pka and gsk3 phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk3 at young ages, declining igf levels during aging may increase gsk3 activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk3 inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk3 causes neuronal dysfunction during normal aging. research funds: kakenhi sy1-8-24-5 molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p53 pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, 5-1-1 tsukiji, chuo-ku, tokyo 104-0045, japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy1-8-24-7 identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue 1,2 1 department of geriatric medicine, university of tokyo hospital, tokyo, japan; 2 research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of 14-3-3 sigma, a negative cell cycle regulator that causes g2 arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr2d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka 1 , hossein esteky 2 , kiani roozbeh 2 , tadashi sugihara 1 , gang wang 3 1 riken brain science institute, wako, saitama, japan; 2 institute for studies in theoretical physics and mathematics, tehran, iran; 3 kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of 670 inferotemporal cells to 1100 object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to 60 • rotation. research funds: kakenhi 17022047 alexander thiele 1 , gene stoner 2 , louise s. delicato 1 , mark roberts 1 1 university of newcastle upon tyne, uk; 2 the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v1 while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy2-1-01-3 context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy2-1-01-4 effects of task demands on color processing in area te of the monkey hidehiko komatsu 1,2 , kowa koida 1 1 national institute for physiological sciences, okazaki, japan; 2 sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development1. monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception2-4. here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy2-2-02-1 dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui 1 , hirotaka sakamoto 2 , katsunori sasahara 1 , hanako shikimi 1 , kazuyoshi ukena 1 , mitsuhiro kawata 2 1 laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; 2 department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi (15207007 and 16086206 to kt) sy2-2-02-2 roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi (17330151, 17051001) sy2-2-02-3 sex steroid receptor function in sexual behavior shigeaki kato 1,2 , takashi sato 1 , takahiro matsumoto 1,2 1 imcb, university of tokyo, tokyo, japan; 2 erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., 2003) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., 2004) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., 2006) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., 2003 . pnas usa 100, 9416. sato, t., et al., 2004 . pnas, usa 101, 1673 . shiina, h., et al., 2006 research funds: probrain sy2-2-02-4 annexin 1: a mediator of cell-cell communication in the neuroendocrine system julia buckingham 1 , helen christian 2 , john morris 2 1 imperial college london, uk; 2 department of human anatomy and genetics, university of oxford, uk annexin 1 (anxa1) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa1 to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca1. the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa1 receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori 1 , yuki takayanagi 2 , masahide yoshida 1 , yoshiyuki kasahara 1 , masaki kawamata 1 1 graduate school of agricultural science, tohoku university, sendai, japan; 2 department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at 5 • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) (14360046) sy2-2-08-1 cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f5); f5 activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m1). local field potential activity in f5 and m1 is also tuned to grasp, and there is strong beta coherence between f5 and m1, indicating reciprocal transmission of information. this is also seen in synaptic responses of m1 neurones to stimulation of f5 (and vice-versa). single pulse stimulation in f5 strongly modulates corticospinal outputs from m1 through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy2-2-08-2 where tactile signals are ordered in time shigeru kitazawa 1,2 1 department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; 2 crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by 30 ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., 1.5 s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy2-2-08-3 decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo 1,2 1 biology/cns, california institute of technology, pasadena, ca, usa; 2 jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. 122-channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha (8) (9) (10) (11) (12) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy2-2-08-4 why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust (061140) sy2-2-08-5 recognizing self actions through externalized eyes atsushi iriki 1,2 1 symbolic cognitive development, riken brain science institute, saitama, japan; 2 cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut1, existing mostly in cortical pyramidal cells, and vglut2, found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut1-expressing terminals (n = 289), but were found in-10% of spines innervated also by vglut2-expressing terminals (n = 444). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy2-3-03-2 gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼44-fold decrease in the potassium-chloride co-transporter 2 (kcc2) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer 2/3 pyramidal cells in visual cortex share common excitatory inputs from layer 4 and from within layer 2/3, when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer 2/3 cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi (17023026,17500208) sy2-3-03-5 local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer 1 of rat visual cortex and layer 2/3 of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer 1, axondescending cells receive excitatory input predominantly from layer 2/3 while neurogliaform cells receive stronger input from deeper layers. layer 2/3 neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer 2/3, while the cr− martinotti cells also receive strong excitation from layer 4. irregular-spiking basket cells also receive strong excitatory input from layers 2/3 and 4, but they often have a gap at the top of layer 4, with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers 2/3 and 4, and only weak input from deeper layers. research funds: nih sy2-3-03-6 physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers (30-50) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r03 ey015273 , mcknight neuroscience of brain disorders award sy2-3-09-1 axon guidance mediated by localized ca 2+ signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca 2+ signals. its turning direction depends on the occurrence of ca 2+ -induced ca 2+ release (cicr) through the ryanodine receptor type 3 (ryr3). the activity of ryr3 is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr3, that serves as a key regulator of axon guidance. attractive ca 2+ signals facilitate intracellular membrane transport to the leading front and subsequent vamp2-mediated exocytosis on the side with elevated ca 2+ . in contrast, repulsive ca 2+ signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp2-mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy2-3-09-2 molecular mechanisms for signaling through plexin-a1 hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema3a acts as a guidance cue for axons through a receptor complex comprising neuropilin-1 as the ligand-binding subunit and plexin-a1 as the signal-transducing subunit. the ferm domain-containing gef, farp2, associates directly with plexin-a1. sema3a induces the dissociation of farp2 from plexin-a1, resulting in activation of farp2's rac gef activity, rnd1 recruitment to plexin-a1 and down regulation of r-ras. simultaneously, the ferm domain of farp2 sequesters pipki␥661 from talin, thereby inhibiting its kinase activity. these activities are necessary for sema3a-mediated repulsion of outgrowing axons. plexin-a1 also functions as a ligand binding receptor of a transmembrane semaphorin, sema6d and contributes to cardiac morphogenesis. sema6d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a1 forms a receptor complex with off-track in the ventricle or with vegf receptor type 2 in the conotruncal segment, which are responsible for the effects of sema6d on the respective regions. research funds: crest sy2-3-09-3 axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins3a (sema3a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema3a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema3a stimulated the transport of neuropilin-1::egfp, plexin-a4::egfp, and fyn::egfp, which are the components of sema3a signaling, but not neuropilin-2::egfp. interestingly, sema3a accelerated the anterograde transport of trka::egfp. these facts suggest that sema3a selectively facilitates the transport of its own signaling components and that sema3a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein-2 (crmp-2) is enriched in growing axon, and play a crucial role in axon specification. crmp-2 interacts with tubulin dimers to promote microtubule-assembly, and binds to sra-1, an effector of rac1 to regulate wave-dependent reorganization of actin filaments. crmp-2 links kinesin-1 to tubulin dimmers and sra-1, and participates in the kinesin-1-dependent transport of tubulin dimmers and the sra-1/wave complex to growing axons. we also found that the par-6/par-3 complex and the ras/pi3-kinase/akt/gsk-3b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk-3b and inactivates its kinase activity downstream of ras/pi3-kinase, thereby increasing non-phosphorylated active crmp-2 which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy2-3-09-5 regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki 1,2 , takashi abe 1,2,3 , shigeyuki kan 1,4 , satoru miyauchi 1,5 1 national institute of information and communications technology, kobe, japan; 2 japan society for the promotion of science, tokyo, japan; 3 graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; 4 kyushu institute of technology, kitakyushu, japan; 5 crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o2-and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p2x4 receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p2x4 receptors produce a reduction of the neuropathic pain behaviour (2003. nature 424, 778-783) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p2x4 causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury (2005 ( . nature 438, 1017 ( -1021 . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi (15209051) sy2-5-05-4 pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the 18 kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk11195, which labeled with carbon-11 can be used for positron emission tomography (pet). using 11c-(r)-pk11195 pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy2-5-10-1 application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past 25 years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy2-5-10-3 brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality (2005 . neuropsychobiology 52, 45), aging (2004 . neuroimage 22, 1715 , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, 2006) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia (2004. biol. psychiatry 55, 501; 2006. neuroimage 29, 172) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every 0.1s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy2-5-10-5 clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy2-5-10-6 measurement of language related brain activities during recovery from aphasia eiju watanabe 1 , yumiko muroi 2 , chizuru nakajima 2 1 department of neurosurgery, jichi medical university, tochigi, japan; 2 department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured 17 aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the 1990s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in 21st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy2-6-06-3 neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy2-6-06-4 neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy2-6-06-5 neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy2-6-06-6 bridging neuroscience and public: neuroethics in cultural contexts osamu sakura 1,2 1 interfaculty initiative in information studies, university of tokyo, tokyo, japan; 2 research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura 1,2 , akira midorikawa 3 , yoshiki kaneoke 4 , shinichi koyama 1 , masato taira 5 , argye hillis 6 1 showa university school of medicine, japan; 2 crest, jst, saitama, japan; 3 national institute of neuroscience, ncnp, tokyo, japan; 4 national institute for physiological sciences, okazaki, japan; 5 nihon university, tokyo, japan; 6 johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi (17022035), crest sy2-6-11-2 impairment of surface perception in patients with ventral occipital damages shinichi koyama 1 , mitsuru kawamura 1 , akira midorikawa 2 , yoshiki kaneoke 3 , masato taira 4 , argye hillis 5 1 showa university, tokyo, japan; 2 national institute of neuroscience, ncnp, tokyo, japan; 3 national institute for physiological sciences, okazaki, japan; 4 nihon university, tokyo, japan; 5 johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient 1 was a 61-year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory 1970) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient 2 was a 63-year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy2-6-11-3 how do pictorial cues influence 3d information processing in the parietal association cortex? masato taira 1,2 1 arish, nihon university, tokyo, japan; 2 department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for 3d perception. basically, it is thought that the parietal association cortex processes 3d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of 3d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of 3d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in 3d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence 3d information processing in the parietal association cortex. research funds: kakenhi 17022038 sy2-6-11-4 case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa 1 1 national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy2-6-11-5 neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v5+ for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi (15500221) sy2-6-11-6 neural correlates of visual attention argye hillis 1 , mitsuru kawamura 2 , akira midorikawa 3 , yoshiki kaneoke 4 , shinichi koyama 2 , masato taira 5 1 johns hopkins university, usa; 2 showa university, tokyo, japan; 3 national institute of neuroscience, ncnp, tokyo, japan; 4 national institute for physiological sciences, okazaki, japan; 5 nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp2 gene, encoding methyl-cpg binding protein 2, cause rtt. mecp2 acts as a transcriptional silencer. abnormal expression of some genes due to mecp2 dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp2 mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp2-downstream genes. using mecp2-null mice (mecp2 −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp2 directly regulates expression of insulin-like growth factor binding protein 3 (igfbp3) gene in human and mouse brains. pathological features of mecp2 −/y have the similarity of igfbp3 transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp3 due to lack of mecp2 may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements-1 or l1 (line-1) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute 45% of the mammalian genome with l1 elements alone representing 20%. the fact that l1 can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l1 on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy2-7-07-6 notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima 1 , jun kohyma 1 , tetsuya taga 2 , masakazu namihira 1 1 grad. sch. biol. sci., naist, ikoma, japan; 2 inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat3. a cpg dinucleotide in the stat3 recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat3 to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat3 binding element in the gfap gene promoter. sy2-8-12-1 neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. (1) which type of brain infarction will be a future target of this therapeutic approach? (2) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? (3) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy2-8-12-2 gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that (1) motor function may recover after minor injury to the primary motor cortex, (2) this recovery is, at least in part, associated with reorganization of cortical motor representation, (3) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and (4) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to 50 repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy3-1-01-3 neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji 1 , toshiya sato 2 , mitsunori yamada 3 1 department of neurology, the university of tokyo, tokyo, japan; 2 center for bioresource-based researches, japan; 3 department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with 129 cag repeats. the q129 mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy3-1-06-1 control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi 17390086, 17023061 sy3-1-06-2 mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima 1,2 1 dept. of anat., keio univ. sch. of med., tokyo, japan; 2 inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day 12.5 mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca 2+ signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca 2+ elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca 2+ and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp1, rho gtpase, mapk and pi 3 k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy3-2-02-1 membranes, water and diffusion denis j. le bihan shfj/cea, france among 1905 einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid 1980s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy3-2-02-2 diffusion tensor fiber tractography using a 3tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display 3d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded 3-t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on 3-t tractography compared to 1.5 t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using 3-t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of 14 patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy3-2-02-4 anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to 15% of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor (12-60 mg/kg) into male wistar rats (from 5 days to 3 weeks of age) significantly caused hyperactivity at 4-5 weeks old. immunohistochemical analyses of the brain tissues at 8 weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy3-2-07-3 non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around 1 mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around 1 mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about 150 -200 meters from a base station. another remarkable observation in our studies is the fact that a significant (p < 0.002) neuronal damage is seen in rat brains 50 days after a 2 hour exposure to gsm at sar values 200, 20 and 2 mw/kg. we have followed up this observation in a study where 96 animals were sacrificed 14 and 28 days respectively after an exposure for 2 hours to gsm mobile phone electromagnetic fields at sar values 200, 20, 2, 0.2 and 0 (controls) mw/kg. significant neuronal damage is seen after 28 days and albumin leakage after 14. our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after 28 and 50 days. sy3-2-07-4 the mitochondrial toxin 3-nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs 2210, france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin 3-nitropropionic acid (3np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of 3np toxicity and show that 3np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by 3np. in summary, the 3np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of 6-ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at 5 days of age showed behavioral hyperactivity at 4-5 weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of 6-ohda-induced neurotoxicity, using pc12 cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of 6-ohda. we also characterized the oxidation of cellular proteins by 6-ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by 6-ohda, ros and p-quinone. sy3-2-07-6 environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥2 subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥2 subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥2 subunit-containing receptors in gh3 and hek293 cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥2 subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi (16109010) sy3-3-03-2 involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer 5 pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca 2+ rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k252a at 200 nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k252a application was started soon after hfs or when k252a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi (17300101) sy3-3-03-3 autocrine mglur1 activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb1 receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur1 on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur1-gq/11 transduction pathway as well as being initiated by direct mglur1 activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur1 activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur1 activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy3-3-03-4 regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi 1,2 , tomoo hirano 1,2 1 dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca 2+ -activated protease calpain or by disturbance of association of gaba a receptor ␥2 subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy3-3-08-1 contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than 50 years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the 50th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy3-3-08-2 detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai 1,2 , susumu takahashi 2 1 department of psychology, kyoto university, kyoto, japan; 2 crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl-1s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi (17022036) sy3-3-08-5 impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto 619-0237, japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy3-4-04-1 tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad65 the amount of the tonic inhibition is reduced in ca1 hippocampal interneurons, while unchanged in pyramidal cells. sy3-4-04-2 modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca1 region yoshihiko yamazaki 1 , yasukazu hozumi 2 , kenya kaneko 1 , satoshi fujii 1 , hiroshi kato 1 1 dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; 2 dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca1 region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a1 receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca1 region. research funds: kakenhi (15082201) sy3-4-04-3 carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of 30 m carbachol induces beta oscillations which occur intermittently with the interval of 20-30 s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is 16.9 ± 0.9 hz (mean ± s.e.m.). the oscillations are induced via muscarinic m1, 3, 4 receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations (1-3 hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that (1) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, (2) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca3 and ca1 neurons discharged during both up and down states, (3) gamma and fast (ripple) oscillations were observed in hippocampal ca1 area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: (1) most of the mprf neurons were active during rem sleep; (2) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; (3) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi (17605001) sy3-5-09-1 clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) 18:6 h and ld 6:18 h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per1 and per2 rhythms suggest that light-on resets per1 rhythms in both light conditions, while per2 rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per1 promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld 18:6. the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per1 and per2 showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy3-5-09-3 genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified 169 genes that fluctuated between day and night in the livers, 100 lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose-6-phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other 100 genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy3-5-09-5 neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy3-6-10-1 a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e)10.5 to e14.5, the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy3-6-10-2 live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi (17024040) sy3-6-10-3 visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol 1,4,5-trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy3-6-10-4 rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦2 (glur␦2) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦2 into glur␦2-null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦2 lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi 16650071 sy3-6-10-5 gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi (17300100) sy3-7-11-1 physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi (17022042) sy3-7-11-2 deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid-20th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy3-7-11-3 firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no.454) sy3-7-11-5 characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama 1,2 1 department of neurological surgery, nihon university school of medicine, tokyo, japan; 2 division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate (49.4 ± 31.7 hz, n = 27) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca 2+ -atpase isoform 2 that is located on the 6th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that (1) d2-type dopamine receptors are downregulated presynaptically in the striatum, and (2) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy3-8-05-2 metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr54 (2001. nature 411, 613) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr54 impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat (2005. endocrinology 146, 4431) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca 2+ imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi 15370032 sy3-8-05-5 metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy3-8-12-1 molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi 17510039, 17390060 sy3-8-12-2 thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of 15 isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp1c1 is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t4 and reverse t3, but the transport activity of t3 is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp1a4, the transport activities of t4 and t3 are similar, is also expressed. the uptake of t4 and t3 by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t4 and t3. research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy3-8-12-3 alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi 1 , masaki takahashi 1 , yasuhiro yoshikawa 2 , tomoko tashiro 1 1 department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; 2 department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to 4-hydroxy-2,2 ,3 ,4 ,5pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to 4-hydroxy-2,2 ,3,4 ,5,5 ,6-heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites (2005. dev. brain res. 154, 259) . koibuchi et al. (2004) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. (2004) detected some pcb and oh-pcb congeners in human csf, of which 4oh-cb187 is the highest concentration. to determine its effects on developing neurons, we examined 4oh-cb187 in rodent cerebellar culture cells. interestingly, 4oh-cb187 promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or 5-htp to examine the relevance of serotoninergic function. the treatment of 5-htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., 2005) . reference kawano et al., 2005. j. neurosci. res. 80, 191-202. research funds: kakenhi 16500234 os2a-8-03 neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama 1 , kazumi takahashi 2 , yukihiko kayama 2 1 cluster of science and technology, fukushima university, fukushima, japan; 2 department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic (5ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the 5ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣1 receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os2a-8-04 correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi 1,2 , kazuyoshi hirano 2,3 , yukimasa muraishi 2,3 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, japan; 3 secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l2) and grey matter volume among students aged 13-17, who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l2. os2a-8-05 grammar center activation in honorification judgment of japanese sentences kanako momo 1,2 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f3op/f3t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os2a-8-06 top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui 1,2,3 , kimitaka kaga 2 , kuniyoshi l. sakai 1,3 1 dept. of basic science, univ. of tokyo, tokyo, japan; 2 dept. of otolaryngology, univ. of tokyo school of medicine, japan; 3 crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m140) (neurosci. res. 52, s61). in a subsequent study, we confirmed that m140 was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m140 was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m140 elicited by the original songs and that by the modified ones. however, while m140 without the deviation showed no significant effect of lateralization, m140 with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os2a-8-07 cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto 1,2 , kuniyoshi l. sakai 1,2 1 department of basic science, university of tokyo, tokyo, japan; 2 crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in 16 sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os2a-8-08 hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima 1,2 , naoki fukui 3 , kuniyoshi l. sakai 1,2 1 dept. of basic science, univ. of tokyo, komaba, japan; 2 crest, jst; 3 dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as 130 ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os2a-8-09 individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext (14002005), jsps research fellowship (1611149) os2a-8-10 brain activity of happy facial recognition in mother-daughter relationship jun shinozaki 1 , nobukatsu sawamoto 1 , toshiya murai 2 , takashi hanakawa 1 , hidenao fukuyama 1 1 hbrc, kyoto univ. grad. sch. of med. kyoto, japan; 2 neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os2a-8-11 anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto 1,2 , atsushi maki 1,2 , kuniyoshi l. sakai 2,3 1 advanced research laboratory, hitachi, ltd., tokyo, japan; 2 crest, japan science and technology agency, saitama, japan; 3 dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f3t/f3o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f3t and f3o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (−54, 27, 21) , (−39, 3, 42) , and (−51, 27, −6) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os2a-8-12 effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada 1 , sunao iwaki 1 , mitsuo tonoike 2 1 aist, osaka, japan; 2 chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = 4), and with those from an inner semantic category (middle, d = 2), while others were not changed (normal, d = 0). peak amplitudes of waveforms of the root mean square values on the components of 170 ms (f (2/38) = 4.92, p = 0.013) and 220 ms (f (2/38) = 5.23, p = 0.0098) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the 170 and 220 ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os2p-2-02 mechanisms of p2y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono 1 , xiaoming zhu 1 , takashi tominaga 2 , fumihito saitow 3 , shiro konishi 1,2 1 waseda-olympus bioscience research institute, singapore, singapore; 2 department of neurophysiology, tokushima bunri university, kagawa, japan; 3 department of pharmacology, nippon medical school, tokyo, japan postsynaptic p2y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and 5-15 min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were 160 ± 9.9% and 94 ± 6.1% of the controls, respectively. pretreatment with the protein kinase a inhibitor h-89, but not the calmodulin kinase ii inhibitor kn-62, completely abolished the atp-induced ipsc enhancement. os2p-2-03 activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip1 isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca 2+ permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca 2+ influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein 1 (arip1). activin-induced nmdar activation persists more than 24 h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os2p-2-04 roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr177 by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr177 phosphorylation following ltp induction in rat hippocampal ca1 region. like camkii activation following ltp (fukunaga et al., 1993) , the increased camki-thr177 phosphorylation remained elevated at least for 60 min after ltp induction. the increased camki-thr177 phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca1 region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto-609 significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca1 region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing (5 ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing (20 ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi (17021037) os2p-2-06 mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p6) rats that had received a single brdu injection at p5 were substituted with the corresponding region of entorhino-hippocampal slices from p6 rats. after five days in vitro, newborn gcs, detected by brdu and prox1, migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os2p-3-02 cdk5 is required for neuroblast migration in the adult mouse brain yuki hirota 1,2,6 , toshio ohshima 3 , takuji iwasato 4 , ashok b. kulkarni 5 , hideyuki okano 2,6 , kazunobu sawamoto 1,2,6 1 bridgestone lab. keio univ., tokyo, japan; 2 dept. physiol., keio univ., tokyo, japan; 3 dev. neurobiol. riken, tokyo, japan; 4 behavioral gen. riken, tokyo, japan; 5 nih, bethesda, usa; 6 sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase 5 (cdk5) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk5 mice to emx1cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk5 has a cell autonomous function in neuroblast migration in the adult brain. os2p-3-03 colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi 1 , wu sheng-xi 2 , yuchio yanagawa 3,4 , kunihiko obata 5 , nobuaki tamamaki 1 1 kumamoto univ., kumamoto, japan; 2 fourth military medical univ., xi'an, people's republic of china; 3 gunma univ., maebashi, japan; 4 sokendai, hayama, japan; 5 riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng2, plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad67-gfp knock-in mouse neocortex at e18-p0 by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t7 rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s100-␤ expression in the gabaergic neuron progenitors. os2p-3-04 role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi 1,2,3 , masanori sakaguchi 3 , toru yamashita 2,3,6 , yuko fujita 3 , yukiko gotoh 4 , arturo alvarez-buylla 5 , takeshi kawase 1 , hideyuki okano 3 , kazunobu sawamoto 2,3 1 neurosurgery, keio univ. sch. med., tokyo, japan; 2 bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; 3 physiol., keio univ. sch. med., tokyo, japan; 4 inst. mol. cell biosciences, univ. tokyo, tokyo, japan; 5 neurosurgery, ucsf, san francisco, usa; 6 neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt2b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt2b. to examine the molecular interactions between c-tsk and wnt2b, we co-electroporated them into the optic vesicle at stage 9-10 chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt2b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt2b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) 9 in developing cortical neurons. an expression vector of gfp-tagged hdac9 was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac9 was primarily localized in nuclear until 1 week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac9 contributes to neuronal differentiation in cortical development. research funds: kakenhi (16700286) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., 2004) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum 2.45 ns) was measured. in high-voltage electron microscopy (hitachi1250m, nips, 2005 , gap junctions (average size 0.86 m long) were present in contacts. in confocal laser-scanning imaging, connexin36 localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be 5180/m 2 and a single channel conductance is 15 ps, the conductance of each junction would be 45 ns. the presence of seven junctions between a pair will lead to estimate a total junction of 315 ns. the measured conductance could allow to estimate a fraction of open channels as 0.8%. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi (17023014), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost 2000 genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os2p-4-04 multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a 60 channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer 4 stimuli and propagated horizontally in the layer 2/3. in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer 4 stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap5 + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os2p-4-05 presence of two phases in the sensitive period of orientation plasticity shigeru tanaka 1 , toshiki tani 1 , kazunori o'hashi 1,2 , jerome ribot 1 1 brain science institute, riken, saitama, japan; 2 graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after 2 weeks of goggle rearing starting around the age of 3 weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of 5 weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os2p-4-06 residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida 1,2,3 , tadashi isa 1,2,3 1 dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; 2 sch. life sci., grad. univ. adv. stud., hayama, japan; 3 crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v1 lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield (80-90% correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v1 lesion is unlike normal, near-threshold vision. research funds: kakenhi 13854029, kakenhi 16700343 and crest, jst os2p-4-07 comparison of the angle representation in macaque visual areas v1 and v2 minami ito 1,2 , hidehiko komatsu 1,2 1 national institute for physiological sciences, okazaki, japan; 2 the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v2 neurons has angle selectivity. here, we studied the angle selectivity of area v1, which is the major source of inputs to area v2. we conducted single-unit recordings from the superficial layer of area v1, while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v1 neurons responded mainly to sharp angles (30 • ), straight lines (180 • ) or right corners (90 • ), but not to intermediate angles (60 • or 120 • angle width). this contrasted with area v2, where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v1 neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v1 neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v2. os2p-4-08 firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to 10 • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os2p-4-10 integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v1, we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os2p-4-12 probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner 1,2 , pei sun 2 , keiji tanaka 2 , david j. heeger 1 , kang cheng 2 1 department of psychology and center for neural science, new york university, usa; 2 laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri (0.75 mm × 0.75 mm × 3 mm voxels) in human v1. we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t2*-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih (1f32ey016260-01) os2p-4-13 relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations (18-350 ms; isi = 1 s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase (350-550 ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase (70-270 ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼50% of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os2p-4-15 the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe 1 , kazuto terai 2 , osamu hieda 3 , shigeru kinoshita 2 1 dept. of ophthal., kyoto second red cross hospital, kyoto, japan; 2 dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; 3 baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from 0.4 to 1.2. the logmar average was 0.31. eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was 0.03, improved from 0.50. the residual five eyes showed less or no improvement. the change of logmar average was from 0.15 to 0.08. conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os2p-5-01 the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada 1 , hideomi tanaka 1,2 , satomi nakayama 1 , miki iwasaki 1,2 , hitoshi okamoto 1,2 1 laboratory for developmental gene regulation, bsi, riken, japan; 2 crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) 4 and migrate tangentially to r6 near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr2 and frizzled3a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled3a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os2p-5-02 disrupted-in-schizophrenia 1 (disc1) regulates the transport of the nudel/lis1 complex to axons via direct interaction with kinesin-1 shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc1 is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc1. disc1 is reported to interact with nudel, which forms a complex with lis1. although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc1 remain largely unknown. here we report that disc1 interacted with kinesin-1, a motor protein of anterograde axonal transport. kinesin-1 interacted with the nudel/lis1 complex through disc1, and these molecules accumulated at the distal part of axons. the knockdown of disc1 by rnai of disc1 induced the delocalization of nudel and lis1 from the axons and reduced axonal growth. the knockdown of kinesin-1 induced the delocalization of disc1 from the axons. taken together, these results indicate that disc1 links kinesin-1 to the nudel/lis1 complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os2p-5-03 role of a novel collapsin response mediator protein-2 interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein 1 (slp1) can mediate the vesicle transport. slp1 is known to associate with rab27. we found that slp1 associates with collapsin response mediator protein-2 (crmp-2), which is a key regulator of axon formation. slp1 could form the trimeric complex with rab27b and crmp-2, and also associate with kinesin-1 through crmp-2. slp1 is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp1 functions as a mediator of recruitment of certain receptor depending on crmp-2 and kinesin-1. os2p-5-04 absolute quantification of mdr1a, mrp1, mrp4 and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie 1,2 , yuki katsukura 1,2 , sumio ohtsuki 1,2 , xiao-kun cai 1,2 , tetsuya terasaki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr1a, mrp1, mrp4, and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between 10 and 1000 fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr1a, mrp4, bcrp were 3.9, 2.7 and 4.8 fmol/g, respectively, while it of mrp1 was under detection limit of standard curve. this quantitative profile suggests that mrp4 and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr1a. os2p-5-05 dominant expression of claudin-5 in highly purified brain capillary endothelial cells sumio ohtsuki 1,2 , hirofumi yamaguchi 1 , saori sato 1 , tmoko asashima 1,2 , tetsuya terasaki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam-1) and magnetic cell sorting. mrna expression of caludin-1-23 was measured by real-time rt-pcr. claudin-5 showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin-1 and -12 were 0.0027% and 0.20% of that of claudin-5. claudin-5 mrna was concentrated in the purified bcecs, while claudin-1 and -12 mrna in the purified bcecs were lower than that in the whole brain. rat claudin-5 mrna was also concentrated in rat brain capillary fraction, but claudin-12 mrna did not. these results suggest that claudin-5 is a dominant tjs protein in bcecs, and expression of claudin-1 and -12, which was reported as tj protein in bcecs, are not restricted in bcecs. os2p-5-06 effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad 1 , a. pauzi m. yusof 1 , p.d. mourad 2 , m. bainbridge 3 , s. ab ghani 1 1 universiti sains malaysia; 2 university of washington, seattle, usa; 3 brody school of medicine, east carolina university, nc, usa h 2 o 2 is the main peroxides produced in mammalian cells that consume o 2 . the main source of h 2 o 2 in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h 2 o 2 towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h 2 o 2 microsensor, we investigated the level of h 2 o 2 in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h 2 o 2 level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h 2 o 2 was injected into the brain. fatality occurs when the injected h 2 o 2 exceeds 250 m. these finding showed that the altered paracellular permeability in the presence of h 2 o 2 is caused by a series of events that happen one after another. research funds: short term grants 304pkimia633134 and 304pfar-masi670008 os2p-6-01 somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t9-11) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t9-11 spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os2p-6-02 responses of renal sympathetic nerve activity and sodium excretion to 3 days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the 7 days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions (0, 50, 154, 308 meq./l nacl) over 3 days. the sodium loading with 308 meq./l nacl suppressed rsna significantly and then it gradually recovered while either 0 meq./l nacl or 154 meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os2p-6-03 cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku 1 , haruko masumiya 1 , yasumasa okada 2 1 dept. physiol., hyogo col. med., nishinomiya, japan; 2 dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c4 ventral root (c4vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as 3d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: (1) a continuous column extending from the para-facial region to the pre-bötzinger complex, and (2) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c4vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os2p-6-04 slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca 2+ -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi 16590180, 16086210 os2p-6-05 the effect of music to sex hormones of elderly person hajime fukui 1 , kumiko toyoshima 2 , kiyoto kuda 1 , katsuhiko iguchi 3 1 nara univ. of edu., nara, japan; 2 grad. school of human sciences, osaka univ. japan; 3 nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and 36 females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a 30-kda protein band in csf of 100% (n = 5) of confirmed and 90% (n = 138) of suspected tbm patients out of 153 tbm patients. the 30-kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv3804c (ag85a) and rv1886c (ag 85b) and one host derived protein as the components of the tbm specific 30-kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q 10 , vitamin d 3 and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os2p-7-02 involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh 1 , boon chock 2 , dennis l. murphy 3 , chuang c. chiueh 4 1 dept. applied pharmacol., univ. toayama, toyama, japan; 2 lab. bioch., nhlbi, nih, md, usa; 3 lab. clin. sci., nimh, nih, md, usa; 4 cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy5y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk1/2 and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy5y cells. consistently, trx increased the expression of mnsod and bcl-2 supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl-2 proteins for protecting against mpp + -induced neurotoxicity. os2p-7-03 pgd 2 induces neuronal apoptosis via 15d-12,14 -pgj 2 tatsurou yagami 1 , noboru okamura 2 , toshiyuki sakaeda 2 1 facul. health care sci., himeji dokkyo univ., himeji, japan; 2 kobe univ. grad. sch. med., japan prostaglandin d 2 (pgd 2 ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd 2 induced neuronal apoptosis in rat cortical cultures. however, a pgd 2 receptor blocker did not suppress neurotoxicity of pgd 2 . little pgd 2 receptor was detected, suggesting an involvement of pgd 2 metabolites in the apoptosis. among pgd 2 metabolites, 15-deoxy-12,14 -prostaglandin j 2 (15d-12,14 -pgj 2 ) caused neuronal apoptosis most potently and rapidly. although 15d-12,14 -pgj 2 is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that 15d-12,14 -pgj 2 induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ 3 h]15d-12,14 -pgj 2 (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd 2 induced apoptosis via 15d-12,14 -pgj 2 in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the 15d-12,14 -pgj 2 -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c6 to t1). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi 17022043 os2p-7-06 compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than 20 longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi (17022027) os2p-7-09 timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae 1 , xiaofeng lu 1,2 , yusuke uchida 1 , toshimitsu takahashi 1,2 , shigeru kitazawa 1,2 1 dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; 2 crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of 16 targets following a gosignal given at a random timing between 500 and 800 ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: (1) those that showed buildup activity during the delay period until the go-signal, and (2) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, 30-200 h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (>9 ms) than larvae (>80 hpf, 3 ms). m-cell activity monitored with confocal ca 2+ imaging during the tail flip (>80 hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (<25%) after 100 hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca 2+ release through ryanodine receptors, triggered by glutamate receptor-mediated ca 2+ influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os2p-8-02 distribution of voltage-gated calcium channel ␣ 2 ␦-4 mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka 570-8506, japan the ␣ 2 ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ 1 subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ 2 ␦-1 to ␣ 2 ␦-4. the distributions of ␣ 2 ␦-1, -2, -3 mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ 2 ␦-4 remains to be determined. we cloned ␣ 2 ␦-4 cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ 2 ␦-4 mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ 2 ␦-4 mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ 2 ␦-4 subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about 34 glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about 6 glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os2p-8-05 hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb334867) depressed the excitation, while bicuculline depressed the inhibition. h 1 or h 2 histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os3a-5-01 activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c57 bl/6 mice. by recording field epsp (fepsp) along 80 m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at 7-8 div, then the amplitudes of fepsps in the ventral side began to decrease at 9-10 div, and dominated in the dorsal area at 14 div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at 7-9 div but the axons terminals in the ventral area were eliminated until 14 div. this synapse elimination from the ventral side was blocked by apv application from 6 div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until 7 div. the number of those ventral axons is reduced before 14 div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co2 incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at 6 div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during 7-11 div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os3a-5-06 transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os3a-5-07 segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r3 receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa2 in r2 is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa2 expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa2 function resulted in selective loss of eph4 expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa2 dependent cues are required to determine the territory for whisker representation in r3 and the assembly of a somatosensory circuit. os3a-5-08 the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p7 and those in the venrtolateral (vl) area were eliminated from p8 to p10 and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p12 and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p12, reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p14 some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os3a-5-09 proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi (17023030), kakenhi (15300107) os3a-6-01 the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine 1 department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m1, s1, sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms (1, 5, 10 hz, 500 stimulations) was compared in 20 dp patients. the pain relief was evaluated with visual analogue scale. high frequency (5, 10 hz) rtms of m1 was the only effective stimulation for treating intractable pain in 10 of 20 patients (50%). the pain relief continued for 3 h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m1. os3a-6-02 involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p2x 3 r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p2x 1,3,2/3 rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p2x 1,2,3,5,1/5,4/5 r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p2x 3 rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p2x 3 r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased 7 days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os3a-6-04 the cantor-like patterns in rat hippocampal ca1 pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca1. this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca1 pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca1 pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after 35 days of age, before which they had been reared with song tutors from birth. when the noise stopped at 102-200 days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization (30 days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d90 of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before 80 days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, 2005) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, 2005) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada (1996) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca1 hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca1. they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca3. this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca3, may be encoded in ca1 in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca1, using 2-compartment model of neuron, and we found the cantor coding of information of time series in the model ca1. it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca1 region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in 15 patients with chronic ischemia due to diverse vascular occlusive diseases (n = 12) and moyamoya disease (n = 3). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in 8 patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os3a-7-02 hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata 1,2 , heng zhao 2 , gary steinberg 2 1 department of neurology, brain research institute, niigata university, niigata, japan; 2 department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia (30 • c) reduced infarct size by 84%. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < 0.05). p-␦pkc (t505) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s643) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, 4-vessle occlusion was induced. fluo-3/am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu-21, yokogawa, japan) equipped with 10× objective lens] was inserted into the brain. 4-vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo-3 increased up to 115%, and persistently increased up to 130% during 20-min reperfusion, indicating the long-lasting ca 2+ increase in the ca1 region. in contrast, in the frontal cortex, 10-min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca1 region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca 2+ in situ. research funds: kakenhi #16390407, #16047212 os3a-7-04 reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto 1 , h. watanabe 1 , y. kumon 1 , t. ohnishi 1 , chi ii 2 , y. imai 2 , j. tanaka 2 1 dept. neurosurgery, ehime university, japan; 2 dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within 36 h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b4 and cd11b recognized with ox42 antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba1+ mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba1+ amoeboid-shaped mg started to accumulate 3 days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os3a-7-05 insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki 1,2 , chihiro tamaki 1 , sumio ohtsuki 1,2 1 graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein 1 (lrp-1) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp-1 expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤(1-40), which reached 1.6-fold greater uptake than non-treated control after 10 min treatment. increase of the hepatic uptake of a␤(1-40) by insulin was concentration dependent (ec 50 = 230 pm), and was completely suppressed by rap (2 m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp-1 to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os3a-7-06 mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used 1321n1 astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of 1321n1 cells was inhibited by an inhibitor of ip 3 receptors, 2-aminoethoxydiphenyl borate (2-apb) or an endoplasmic reticulum ca 2+ -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca 2+ was inhibited by 2-apb and cpa. moreover, removal of external ca 2+ accelerated the reversal of thrombin effects. these results suggest that refilling of ca 2+ store by ca 2+ entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age 38 and a 25-year-clinical course. the brain showed severe atrophy (630 g). microscopic examination disclosed that all telencephalic neocortices had nft of more than 100 and sp of more than 10. all limbic cortices and nuclei had nft of more than 100 and sp of more than 10. although there was no sp, various numbers of nft were observed in the following structures: claustrum 85, caudate 9, globus pallidus 6, hypothalamus 22, meynert's nucleus 4, thalamus 74, substantia nigra 6, central gray 12, locus ceruleus 6, purkinje cells 0, posterior root ganglion 0, adrenal medulla 3. this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os3a-7-09 transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with 32plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv4 is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv4 was expressed in soma, dendrites and synapses in the neurons. since trpv4 was firstly cloned as an osmotically activated channel, we hypothesized trpv4 might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv4 expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv4-deficient neurons at 37 • c, and found rmp in wild type was more depolarized by approximately 5 mv than rmp in trpv4-deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv4-deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv4 is involved in regulation of both neural activity and spine motility in hippocampus. os3p-2-04 a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at 100 locations per 200 ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by 2-photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os3p-2-05 d1-like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a 1 receptors (adora1) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora1 in the ng decreased by >90% of that in the non-treated ng, being not accompanied by a change in mrna level for a 2a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase-3 activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf1 in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf1 blockers such as brefeldin a, exo1, and arf1 n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf1 and subsequent pld may regulate the k + -current response induced by da. os3p-3-05 p250gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa 1 , toshihiko kuriu 2 , ayako m. watabe 3 , toshiya manabe 3 , shigeo okabe 2 , tadashi yamamoto 1 1 div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; 2 dept. of cell biol., tokyo medical and dental univ., tokyo, japan; 3 div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p250gap, which interacts with the nr2b subunit of nmdar. within neurons, p250gap was highly concentrated in the postsynaptic density and co-localized with nr2b and an actin-binding protein, cortactin. p250gap promoted gtp hydrolysis of cdc42 and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p250gap. when over-expressed in dissociated neuron, p250gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p250gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os3p-3-06 non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os3p-4-01 spatial frequency tuning of disparity-selective neurons in macaque v4 hironori kumano 1 , seiji tanabe 2 , ichiro fujita 2 1 grad. sch. of engineering science, osaka univ., osaka, japan; 2 grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v4 of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional (2-d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v4 neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using 2-d filtered noise stereograms. the disparity tuning of most v4 neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v4 neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os3p-4-02 predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v4 hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v4 of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v4 are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v4 while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v4 neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v4 neurons. the results suggest that v4 neurons are involved in mediating stereoacuity. os3p-4-03 the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field 2-d ternary dynamic random noise, generally refreshed every 26-40 ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v1/v2 neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v1, and especially areas beyond. research funds: mext(13041033), jsps(13308048), coe21 os3p-4-06 analysis of center-surround organization of v1 neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area 17 (v1) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of 52 cells analyzed, 80% showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ±90 degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os3p-4-07 spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki 1 , izumi ohzawa 1,2 1 grad. school of eng. sci., osaka univ., japan; 2 grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial 2nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with 51 × 51 dots was presented over an area 2 to 4 times larger than the rf of a complex cell. for each dot location, a 2nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of 2nd-order kernels at all locations. 2nd-order kernels had roughly comparable extent as the rf, and contained 3.3 subregions on average (n = 53). among 35 complex cells, whose rf envelopes were elongated, 16 cells exhibited the horizontal elongation. research funds: mext(13041033), jsps(13308048), coe21 os3p-4-08 orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito 1 , osamu sadakane 1 , masahiro okamoto 2 , hironobu osaki 3 , hiromichi sato 1 1 grad. sch. med., osaka univ., osaka, japan; 2 grad. sch. front. biosci., osaka univ., osaka, japan; 3 med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about 19% lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that 97% of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os3p-4-09 temporal dynamics of suppressive receptive field surround in cat v1 satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v1), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes (500 ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v1 cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (<100 ms) but not in middle (100-200 ms) and late responses (200-500 ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from 2.5 deg (early response) to 5 deg (middle response) and 7.5 deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi (15500259) os3p-4-10 spatial-frequency dependent surround suppression in cat v1 ayako ishikawa 1 , satoshi shimegi 2 , hiroyuki kida 3 , hiromichi sato 2 1 grad. sch. front. biosci., osaka univ., osaka, japan; 2 grad. sch. med., osaka univ., japan; 3 grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v1. we used a stationary flash (duration, 500 ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus (50 ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = 0) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os3p-4-11 contrast-dependency of spatial summation property in cat v1 and lgn masahiro okamoto 1 , tomoyuki naito 2 , osamu sadakane 2 , hiromichi sato 2 1 grad. sch. front. biosci., osaka univ., japan; 2 grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v1) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v1 and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by 0.76 and 0.68 times in v1 and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v1, the strength of surround suppression did not affected by stimulus contrast on average. we conclude that 1) rog model describes the surround suppression better than dog model both in v1 and lgn, 2) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida 1 , satoshi shimegi 2 , ayako ishikawa 3 , hiroshi sakamoto 3 , hiromichi sato 2 1 grad. sch. eng. sci., osaka univ., japan; 2 grad. sch. med. sci., osaka univ., japan; 3 grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v1), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v1 neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into 8 flanks (45 • step), and stationary stimulated with an annulus, oppositely-faced flanks (2-fk) or a flank (1-fk) stimulus. the durations of stimulus presentation were 500 ms for crf and 50 ms for srf stimulation. localized srf stimulation with either 2-fk or 1-fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for 1-fk stimulation throughout the crf response. however, 2-fk stimulation of end position to crf had strong and long-lasting suppression on responses during 80-200 ms after onset. os3p-4-13 temporal-frequency dependency of receptive field size and surround suppression in lgn and v1 osamu sadakane 1 , tomoyuki naito 1 , hironobu osaki 2 , masahiro okamoto 3 , hiromichi sato 1 1 grad. sch. med., osaka univ., japan; 2 med. sch., osaka univ., japan; 3 grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v1) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v1 neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v1 neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v1 neurons, suggesting that the subcortical tf tuning property contributes to that in v1. ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. 2001; yang and maunsell 2004) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across 70 daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using 4.7 t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of 50 m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around 1%) was smaller than that of bold (>2%), the temporal profile showed a constant precedence of the diffusion signal by 2.4s. os3p-5-03 new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os3p-5-04 spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international 10-20 system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within 1 cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad (1.33), approaching unity (∼1.02) for oh, and in between for control (1.14). statistical analysis was performed and significant differences were observed for these groups (p < 0.01). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p38 mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p38 mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: (1) resting, (2) staring at a standing human object, (3) thinking of standing, (4) standing with eyes open, (5) standing with eyes closed. regional cbf data analyzed by spm2 were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os3p-6-03 potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono 1,2 , eiji shigetomi 2 , kiyoharu inoue 1 , fusao kato 2 1 dept. neurol., jikei univ., sch. med., tokyo, japan; 2 lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os3p-6-04 effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan 3,4-methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin (5-ht) release and cause degeneration of 5-htergic nerve terminals via 5-ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the 5-htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on 5-ht release and 5-htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant 5-htergic neurons around the raphe nuclei. treatment with a 5-htergic neurotoxin 5,7-dihydroxytryptamine dramatically reduced the tissue contents of 5-ht and its metabolite, which was blocked by a selective 5-ht reuptake inhibitor. mdma and meth (0.1-1000 m) increased 5-ht release, and reduced the tissue contents of 5-ht and its metabolite at higher doses. the mesencephalic slice culture including the 5-htergic raphe nuclei may be useful to examine the mechanisms underlying 5-htergic neurotoxic effect of mdma in vitro. os3p-6-05 studies on drug dependence (rept. 421): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os3p-6-06 prostaglandin d 2 is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d 2 , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp1 receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd 2 by hpgds and that astrocytes expressed both dp1 and dp2 receptors and were activated by pgd 2 . we found that gliosis and demyelination were well suppressed in hpgds-or dp1-null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd 2 is a key molecule of neuroinflammation involved in the demyelination. research funds: 09670806, 12558078 os3p-7-01 on a sodium channel distribution enabling high frequency signal processing go ashida 1,2 , kousuke abe 2 , kazuo funabiki 1 1 grad. sch. medicine, kyoto univ., kyoto, japan; 2 grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to 8 khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below 1khz, while that in the node does up to 10 khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated 140 nl cells from 16 owls and achieved intracellular recordings in 38 of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = 58 ± 17 mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, 13 vision impaired (7 blind and 6 low vision) and 15 sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os3p-8-01 autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada 1 , runa araya 1 , naoto kitamura 1 , yuji mishinsa 2 1 yamada unit, riken bsi, saitama, japan; 2 nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr1a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr1a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr1a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba1-positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os3p-8-04 fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita 1,2 , hideomi tanaka 1,2 , sachiko tsuruoka 3 , hironori wada 1,2 , hitoshi okamoto 1,2 1 riken bsi, wako, japan; 2 jst crest, kawaguchi, japan; 3 riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose 4,6 dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi (17024013) os3p-8-06 initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd95-homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin2 (fas2) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas2 genetically expressed on a muscle, we found fas2 accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas2 is crucial for the postsynaptic localization of fas2. in addition, postsynaptic localization of a scaffolding protein dlg, psd95-homolog, and glutamate receptors was impaired in fas2 mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor 2a (mef2a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef2a that is sumoylated at lys403 promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef2a at ser408 and thereby promoted a switch from sumoylation to acetylation at lys403, leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns4102, ag11085 ps1a-a002 characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., 1999) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified 1152 randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., 1999. mol. brain res., 72, 147-157. research funds: kakenhi (17500252) ps1a-a003 the distribution of snap-25 protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap-25 derived from exon 5 splicing are expressed in brain. we generated two specific antibodies for snap-25a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap-25a and b during early postnatal period. snap-25b was low at the birth and increased remarkably thereafter. by the contrast, snap-25a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap-25b but not snap-25a was recovered in raft-enriched fractions of triton x-100-treated lp1 membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap-25b was widely distributed throughout brain, whereas, snap-25a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap-25 protein are regulated differently in isoformspecific manners, and snap-25a and snap-25b play different functional roles in brain. ps1a-a004 erc(elks/rab6ip2/cast) regulates syaptic short-term plasticity by recruiting bmunc13-2 to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain (2001. mol. brain res. 89, 20-28) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos7 cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt-1, are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt-1, the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt-1(-/-) mice, and a novel antagonist of glial glutamate transporters, (2s,3s)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately 20%) of functional transporters, glast and/or glt-1, is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat-4 gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat-4 mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat-4 vglut. a mutation in avr-14 glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr-14 functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps1a-a008 biochemical characterisation of the vesicular glutamate transporter 1 stephan schenck, shigeo takamori department of neurology and neurological science, 21st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut1 and vglut2 are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut1 and vglut2 has been found. to further characterise the specific properties of the transporters we make use of the vglut1-ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut1 by yeast two-hybrid screening and found endophilin a1. as expected for the interacting molecule to vglut1, endophilin a1 was typically localized to vglut1-positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in 60∼90 min, followed by spontaneously recovered after120 min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of 14c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) 2 was demonstrated in the lactate medium. results suggested that increased mct2 expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu 1 , kazuhito tomizawa 1 , shuang liang 2 , iori ohmori 1 , teiichi nishiki 1 , kohji takei 2 , hideki matsui 1 1 dept. of physiol., okayama univ., okayama, japan; 2 dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca 2+ -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos-7 cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately (27th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm1-43 and fm4-64, and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca2+ channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin 1a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin 1a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin 1a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca 2+ channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca 2+ influx and epscs, suggesting that exocytotic machinery downstream of ca 2+ influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca 2+ or g-proteins. ps1a-b017 spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb1 cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca 2+ channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap-2 subunit can directly bind with synprint site. this interaction was ca 2+ -dependent, being weaker at concentrations higher than 200 nm. in contrast, the interaction of synaptotagmin with synprint was optimal at 100 m ca 2+ , being weaker at lower or higher concentrations. the binding domain of synprint for ap-2 and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca2+ channel synprint site makes ca 2+ -dependent interactions with ap-2 and synaptotagmin thereby contributing to vesicular endocytosis. ps1a-b019 acl-4, an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl-1˜14, and generated their deletion mutants. the mutants of acl-4 gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat-3 mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl-4 expression, suggesting that certain phospholipid species produced by acl-4 are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku 1 , hussam jourdi 1 , akiyoshi kakita 2 , tadasato nagano 1 , hitoshi takahashi 3 , nobuyuki takei 1 , hiroyuki nawa 1 1 dept. mol. neurobiol., brain res. inst., niigata univ., japan; 2 brain resource center, brain res. inst., niigata univ., japan; 3 dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb1) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip1 and sap97. moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb1 receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima 1 , eriko miura 2 , keiko matsuda 1 , tetsuro kondo 1,3 , masahiko watanabe 2 , michisuke yuzaki 1 1 dept. physiol., sch. med., keio univ., tokyo, japan; 2 dept. anatomy, hokkaido univ., sch. med., sapporo, japan; 3 mol. neurophysiol., aist, tsukuba, japan cbln1 is a member of the c1q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln1 is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln2-4, are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln1, cbln2 and cbln4 were secreted as glycoproteins, whereas cbln3 was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd-95 and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd-95. more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd-95 at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf4/ra175 (ra175), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca2+independent homophilic trans-cell adhesion activity. ra175 participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra175-/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra175 interacted with par-3 being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par-3 was colocalized in the cell adherent region of p19 embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra175+/+ testis, whereas it was undetectable in the spermatid of the ra175−/− testis. ra175 and jam-c were localized around the head portion of spermatid and ra175 deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra175 and par-3. research funds: 12324122 izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd-95 clusters in cultured hippocampal interneurons. higher density of dendritic psd-95 clusters was observed in interneurons at 11 div. at 18 div, this difference was less prominent, mainly due to >3-fold increase of psds in excitatory neurons. psd-95-gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at 11 div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd-95-gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps1a-c025 two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in 3-7div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac1 in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p2x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, 2001; shigetomi & kato, 2003) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites (2 s, 4-micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p2x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi (17300123) ps1a-c027 ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of 2d-and 3d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps1a-c028 integrative spike dynamics of rat ca1 neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca1 stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca1 circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur6 connects with syntenin at the dendrites and mglur7 with pick1 at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur7 that was known to be ectopicly expressed in the dendrites of mglur6-deficient on cone bipolar cells. we observed no pick1 but only syntenin at the mglur6-deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur7 immunopositivity. these findings suggest that mglur7 connects with syntenin in place of mglur6 that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca 2+ -permeable channels. trpv1, expressed in sensory neurons, is activated by noxious heat above 42 • c, whereas trpv 4, expressed in keratinocytes, is sensitive to moderate temperatures (>34 • c). here we examined the role of trpv1 and 4 in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to 55 • c did not induce the change in bt, desensitization of trpv1 with capsaicin resulted in the increase in bt. on the other hand, in trpv4-knockout mouse, moderate thermal radiation (>43 • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv1 and 4 respectively. research funds: kakenhi (17657052) ps1a-c032 generation and biochemical analysis of a glur␣2 knockout mouse hirotsugu azechi 1 , manabu abe 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, niigata, japan; 2 sorst/jst, saitama, japan glur␣2(glur2) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣2, we generated a conditional glur␣2 knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra2f using c57bl/6(b6) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra2f was used as a target of glur␣2 line. by crossing gra2f and tlcn-cre that expressed cre in germ line cells, glur␣2 null ko mice were produced, but most of them died within 3 days after birth. to overcome the lethality, the glur␣2 mutation was transferred onto b6/129 or b6/cd-1 genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣2 in the distribution of functional ampa receptors in vivo. ps1a-c033 gisp: a novel brain specific protein that binds to the gaba b1 subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific 130 kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b 1 subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b 1 and gaba b 2 from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b 1 protein and also promotes gaba b 1 surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b 1/gaba b 2 complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b 1-binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut1-3) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut1 and vglut2 seem to participate in this glutamate accumulation. in the present study, we found that vglut3 mrna is also expressed in the adult rat pinealocytes. vglut3 immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut1-ir or vglut2-ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito 1,2,3 , kenji nakamura 4 , toshikazu kakizaki 1,3 , satoe ebihara 5 , masakazu uematsu 6 , shigeo takamori 7 , minesuke yokoyama 4 , shiro konishi 4 , masayoshi mishina 3,8 , jun-ichi miyazaki 9 , kunihiko obata 10 , yuchio yanagawa 1,3 1 gunma univ., maebashi, japan; 2 sokendai, hayama, japan; 3 sorst, kawaguchi, japan; 4 mitsubishi kagaku inst. life sci., machida, japan; 5 kumamoto univ., kumamoto, japan; 6 toyohashi univ. tech., toyohashi, japan; 7 tokyo med. den. univ., tokyo, japan; 8 univ. tokyo, tokyo, japan; 9 osaka univ., suita, japan; 10 riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi (17052002) ps1a-c036 postnatal changes in the colocalization of vglut1 and vglut2 immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura 1,2 , akiya watakabe 3 , hiroyuki hioki 1 , fumino fujiyama 1 , yasuyo tanaka 1 , tetsuo yamamori 3 , takeshi kaneko 1,2 1 dept. morphol brain sci., grad. sch. med., kyoto univ., japan; 2 crest, jst, japan; 3 div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut)1 and vglut2 accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut1 and vglut2 in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p7. the cc was higher in area s1 than areas m1 and area v1 throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of 5 amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r1 and r2. the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin (5-hydroxytryptamine, 5-ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several 5-ht receptor subtypes, 5-ht transporter (5-htt) and vesicular monoamine transporter 1 (vmat1) in primary cultured mouse osteoblasts and mc3t3-e1 osteoblastic cells. sustained exposure to fluoxetine, a selective 5-ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc3t3-e1. these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦2 glutamate receptor (glur␦2). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦2 were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho15j, was caused by a new type of intragenic deletion of the grid2 gene, which was indeed translated as glur␦2 lacking 52-amino acids in the n-terminus. mutant glur␦2 proteins were retained in the soma of purkinje cells and degraded. as a result, ho15j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦2 signaling and cerebellar functions. research funds: kakenhi (16200024) ps1a-d040 role of the dry motif in melanin-concentrating hormone receptor 1 in signaling yumiko saito 1 , yoshimi aizaki 1 , mituse nakano 2 , kei maruyama 1 1 dept. pharamacol., saitama med. sch., saitama, japan; 2 international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp140, arg141 and tyr142 in the dry of rat melanin-concentrating hormone receptor 1 (mch1r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec50 value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch1r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a 1 r and p2y 1 r electrophysiologically using xenopus oocyte expression system. when a 1 r and p2y 1 r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/11 pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps1a-d043 dual signaling via metabotropic glutamate receptor1␣ is regulated by a cytoskeletal protein 4.1g michihiro tateyama 1,2 , yoshihiro kubo 1,2 1 department of biophysics and neurology, nips, aichi, japan; 2 sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor 1␣ (mglur1) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur1 are differentially regulated by different types of ligands, glutamate and gd 3+ . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur1. these proteins, such as homer and 4.1g, are also known to change the membrane expression of and modulate the function of mglur1. here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur1. interestingly, the functional couplings of mglur1 to gq and gs pathways were altered by co-expression of 4.1g, but not by homer. deletion of the c-terminal tail abolished the effect of 4.1g, indicating that the interaction of 4.1g with the c-terminal tail of mglur1 regulates the multi path signaling. ps1a-d044 modulation of the eaac1-mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto 1,2 , saori akiduki 1,2 1 age dimension research center, aist, ibaraki, japan; 2 graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein 3-18 (gtrap3-18), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier 1 (eaac1). it contains two potential pkc phosphorylation motifs at positions 18-20 and 138-140. however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac1-mediated glutamate transport activity in c6bu-1 cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine 18 into alanine (s18a) or at serine 138 into alanine (s138a). as compared with wt, s18a had no inhibitory effect on glutamate transport activity under exposure to 100 nm pma, and had increased glutamate transport activity under normal condition. by contrast, s138a had the same glutamate transport activity as that of wt. thus, the eaac1-mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine 18 in addicsin. kaori akashi 1 , manabu abe 1 , toshikazu kakizaki 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, japan; 2 sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤1-3(glur5-7) and glur␥1-2(ka1-2) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤2ko mice from c57bl/6 es cell line. the glur␤2ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥1 and glur␥2 proteins were significantly decreased in the crude fraction of ca3 region of glur␤2ko. furthermore, subcellular localizations of both subunits were also changed in glur␤2ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤2 subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps1a-d046 sema4d/plexin-b1 activates gsk-3␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin 4d (sema4d) receptor plexin-b1 induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b1-mediated r-ras gap activity, leading to growth cone collapse. sema4d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk-3␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk-3␤ antagonist suppressed the sema4d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b1-induced dephosphorylation of akt and gsk-3␤. plexin-a1 also induced dephosphorylation of akt and gsk-3␤ through its r-ras gap activity. thus, we conclude that plexin-b1 dephosphorylates akt and gsk-3␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos7 cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur2. hops was co-immunoprecipitated with grip1 (glutamate receptor interacting protein 1) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur2 via grip1 binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc-1. a neural amino acid transporter snat2/ata2 also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc-1 immunoreactivity (asc-1-ir) was detected in dendrites and somata of pyramidal neurons. the snat2-ir was widely detected in neurons, whose intracellular localization was similar to that of asc-1-ir. deferent from asc-1-ir, snat2-ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc-1 and snat2 to the neuronal uptake of l-serine. snat2 might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps1a-d049 neuronal glutamate transporter eaat4 controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake 1 , si-young song 2 , shiro konishi 3 , keiji imoto 1 1 natl. inst. physiol. sci. (nips), okazaki, japan; 2 mitsubishi kagaku inst life sci, tokyo, japan; 3 tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat4/glt-1 blocker threo-3-methylglutamate, but not the glt-1 blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat4 expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat4 was abundant. the results suggest that eaat4 plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat4 expression in postsynaptic pcs. chitoshi takayama 1 , yoshiro inoue 1 1 department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k-2cl co-transporter 1 (nkcc1) and k-cl co-transporter 2 (kcc2). in this study, we focused on kcc2, which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc2 with special reference to the neuronal development in the cerebellum. kcc2 was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc2. the kcc2-protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc2-expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima 1 , hisashi mori 1 1 molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d1-like and d2-like according to their pharmacological properties. the d1-like receptors consist of d1 and d5 receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d1 receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop (3i loop) domain of d1r fused with egfp effectively reduce camp production mediated by d1 and d5 receptors. interestingly, we also identified that the 3i loop domain of d1r interfere with gs coupled beta2 adrenergic receptor signaling. these results suggest that the third intracellular loop of the d1 receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d1-like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama 1 , yoichi ogawa 2 , hiroki yoshino 1 , masayuki yamashita 2 , toshifumi kishimto 1 1 dept. psychiatr.; 2 dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca1 pyramidal cells. da (100 m) profoundly decreased gaba a receptor-mediated postsynaptic currents to 32% in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to 29% in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d1-like receptor, since skf 83959, a selective agonist for pi-linked d1-like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d1-like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina 1 , jinko konno 1 , sachine yoshida 1 , hideki ohmomo 1 , hitoshi kawano 2 , fumihiro shutoh 1 , haruo nogami 1 , setsuji hisano 1 1 graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; 2 tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut1 or -2 mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day 13 (e13), vglut1 immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e15, vglut1-ir was seen as puncta close to l1-ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l1or tag1-ir in the imz, whereas vglut2-ir was first observed in the sp and upper imz where l1-ir existed. these results show that vglut1ir corticofugal fibers appose to elongating vglut2-ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps1a-d054 developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca1 regions michie kon 1 , yoichi avil 2 , hiroshi tsubokawa 1,2 1 dept. of information engineering, tohoku univ., sendai, japan; 2 grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca1 region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca1 region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under 15 days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca1 region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca1 region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine (2.5-10 mg/kg) produced a dose-dependent cpp. using a 3-day schedule of conditioning, it was found that intra-ca1 administration of nmda receptor antagonist, mk-801 (2 and 4 g/rat) significantly attenuated the morphine (7.5 mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda (0.1, 0.2 and 1 g/rat) significantly potentiated the morphine (2.5 mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk-801 receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura 1 , kouichi nakamura 2,3 , hiroyuki hioki 2 , masanori uemura 1 , takeshi kaneko 2,3 1 dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; 2 dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; 3 crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d1 receptor are expressed in striatonigral neurons and d2 receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d1, d2, d3, d4, d5). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p10-15) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since (1) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, (2) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, (3) depolarizing agent released tpa from isolated neurosecretosomes, (4) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u2st, galnac4-6st, d4st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda 1 , maki ishii 1 , isao nagata 2 , yumiko shimazaki 1 1 dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; 2 dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo-225 epitopes, glca(2s)␤1-3galnac(6s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa 1 , mineko kengaku 2 , nobuaki maeda 1 1 dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; 2 lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka 1 , tohru marunouchi 1 1 division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of 3 m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp1, has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp1 gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp1 or sulffp2 appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of 6-o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of 5-hydroxytryptoamine (5-ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day 16 or 18 rat cerebral cortex was cultured in the presence of 5-ht, na or da. after 4 days, we analyzed dendrite formation using anti-map2 antibody. after 14-21 days, we analyzed synaptogenesis with anti-psd-95, anti-synaptophysin, and anti-map2 antibodies. the addition of 5-ht (100-10000 nm), na (100-1000 nm) or da (10-1000 nm) increased dendritic length of pyramidal neurons. 5-ht (100-1000 nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by 5-ht 1a receptor, ␣ 2a receptor and d 2 receptor, while inhibited by 5-ht 2a and ␤receptors. in addition, synaptogenesis was promoted by 5-ht 2a and 5-ht 2c receptors, whereas inhibited by 5-ht 1a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in 293t cell, singar associated with p85 and p110, the subunits of pi3kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps1a-e068 lrfn2, a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd-95 naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn2 was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn2 protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd-95, a postsynaptic scaffold protein. we examined the physical interaction between lrfn2 and psd-95 by immunocrecipitation and pull-down assay, since lrfn2 contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn2 associated with psd-95/nmda receptor complex in the brain extracts and lrfn2 directly bound to psd-95 via its pdz domain-binding motif. in this study, we suggest that lrfn2 may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt1-8). among the prmt proteins, the prmt3 has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt3 in the rat brain. the prmt3 was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt3 expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt3 relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt3 in the brain, we tried to identify novel interacting proteins with the prmt3 in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps1a-e070 proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi 1,2 , michihiro igarashi 1,2 1 div mol cell biol, grad. sch. med dent sci; 2 trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than 150 identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi 17023019; project-promoting grant from niigata univ ps1a-e071 netrin-1 is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda 1 , keisuke watanabe 2 , kazuhiro ikenaka 2 , katsuhiko ono 2 , hiroyuki yaginuma 1 1 dept. anat., fukushima med univ. sch. of med., fukushima, japan; 2 div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin-1 is known to be a chemorepellent for a subset of axons, the role of netrin-1 for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin-1 in this repulsion. in the mouse embryo at e11, netrin-1 is expressed in the floor plate and the dermamyotome, and the netrin-1 receptor unc5c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin-1 repelled chick e5 drg axons. moreover, using function-blocking antibody against netrin-1, we revealed the fact that netrin-1 plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin-1 to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst 17590166 to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p6 neonates, but the ventral neurons became unresponsive after p9. the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe 1 , hirohide takebayashi 1,2 , kazuhiro ikenaka 1 , katsuhiko ono 1 1 div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; 2 dev stem cell biol. program, ucsf, usa netrin-1 is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin-1 also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin-1 −/− background, using olig3-lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig3 +/lacz ;netrin-1 −/− mice, ventral migration of olig3 cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig3-positive cells. these results suggest that netrin-1 exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g1 and netrin-g2 are vertebrate-specific membrane-anchored members of the unc-6/netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g1 and netrin-g2 proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g1 and netrin-g2 differentially bind to lrrcontaining proteins, ngl-1 and a related molecule nag14, in vitro. ngl-1 and nag14 in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g1 and netrin-g2, respectively. furthermore, in netrin-g1 and netrin-g2 deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang 1 , ryuichi sakai 2 , teiichi furuichi 1 1 lab. molecular neurogenesis, brain science institute of riken, saitama, japan; 2 division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p4-p8, stimulation of parallel fibers produces excitation of purkinje cells lasting for 600-800 ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p5-p7. since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p4 and that they then locate in purkinje cells, in cell body on p5 and in primary dendrites on p6-p7. the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya-1 and mmahya-2) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya-1 protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya-1 is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya-1 for the early cerebellum development. toshitaka morishima 1 , erina fukushi 1 , kazuto kobayashi 2 , naohiro hozumi 1 , sachiko yoshida 1 1 toyohashi university of technology, toyohashi, japan; 2 honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto 1 , hideyuki dekimoto 1 , tomiyoshi setsu 1 , masahiko watanabe 2 , mikio hoshino 3 , yo-ichi nabeshima 3 , toshio terashima 1 1 dept. of anat., kobe univ. grad. sch. of med., kobe, japan; 2 dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; 3 dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf1a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida 1 , tomoko nishiyama 1 , hitoshi tatsumi 1 , masahiro sokabe 1,2 1 department of physiology, nagoya university graduate school of medicine, nagoya, japan; 2 icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately 12 h and the elongation started at approximately 24 h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca3. once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca3 region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca3 toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca3, which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a 37 amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) 16 or e18 rat. dendritic outgrowth of pyramidal neurons was analyzed after 4 days using anti-map2 antibody. synapse formation was analyzed after 2-3 weeks, using anti-psd-95 and anti-synaptophysin antibodies. in the presence of cgrp (10-1000 nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of 5hydroxytryptoamine (5-ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day 18 rat hippocampus was cultured in the presence of 5-ht or na. after 5 days, we analyzed formation of dendrites using anti-map2 antibody. after 21 days, we analyzed formation of synapses using anti-psd-95, anti-synaptophysin, and anti-map2 antibodies. the addition of 5-ht (500 nm) or na (500 nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites 5-ht (100-1000 nm) and na (10-1000 nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ 2a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, 5-ht 2a receptor and ␣ 2a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within 24 h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c4 inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c4 inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di-2 anepeq) and the imaging system (micam02). in dscam-ko mice, the optical signal which precedes c4 inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps1a-f088 hippocampal cells cultured on 3d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after 2 weeks in vitro, neurons formed a dense layer, >0.1 mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, (1) collagenasespecific inhibitor did not affect cell depth, (2) limiting gliosis by antimitotics reduced the thickness of the tgl by 40%, (3), neither glial nor neuronal cell body were found in the tgl by gfap/map2 detection, (4) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after 2 weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca2+ by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama 1 , kazuto kujira 1 , tetsuya kawabe 2 , takao ito 1 , yoshihiro tsuruo 1 1 department of cell biology and anatomy, wakayama medical university, wakayama, japan; 2 department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) (17590600) ps1a-f091 metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama 1 , hitoo nishino 1 , takehiro yahata 2 1 nagoya city university graduate school of medical sciences, nagoya, japan; 2 nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit 1 , anand goswami 1 , nobuyuki nukina 2 , nihar ranjan jana 1 1 national brain research centre, india; 2 laboratory for structural neuropathology, riken brain science institute, 2-1 hirosawa, wakoshi, saitama 351-0198, japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin-3 for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc70. finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of 11,092 ests (5,442 non-redundant sequences). 2858 of them matched known genes of rodents in the genbank databases, but 2584 remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of 0, 2, or 4 weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps1a-f096 transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb-431542, an inhibitor of the type 1 tgf-␤ receptor (t␤r1). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r1 signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps (2 h restraint; 20 min forced-swimming; ether anesthesia) then they were put in untouchable situation for 7 days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming 15 min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c57bl/6j mice. pups were separated from mothers daily (3 h) on postnatal days 1 through 14. starting at 13 weeks of age they were tested for (1) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; (2) responses to social stimuli in social investigation (sit) and social preference tests; and (3) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of 90 db (so), or were forced to swim (sw). these manipulations were performed for 15 min once a day at 2 weeks after birth (from postnatal days 14 to 18) or 3 weeks after birth (from postnatal day 21 to 25). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of 6 weeks. it was found that the spatial learning activity of both so and sw mice manipulated 2 weeks after birth was impaired as compared to no mice. so mice manipulated 3 weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated 3 weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo 1 , yukiko yamada 2 , mitsuo iinuma 2 , yasuo tamura 2 , fumihiko iwaku 1 , kazuko watanabe 3 , minoru onozuka 4 1 dept. oral anat., asahi univ. sch. dent., japan; 2 dept. ped. dent., asahi univ. sch. dent.; 3 dept. physiol., gifu univ. sch. med., japan; 4 dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp8 mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca3 region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for 12 weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara 1 , yayoi kitamura 1 , junichi tanaka 2 , yukio hattori 1 , yasushi hayashi 1 1 department of human nutrition, notre dame seishin university, okayama, japan; 2 department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c57bl/6j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a 10-min interval, to measure the basal temperature (t 1 ) and stress-enhanced temperature (t 2 ), respectively. the difference ( t = t 2 − t 1 ) gives the sih. in control mice, t was approximately 1 • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t 1 . furthermore, spermidine and spermine also lowered t and t 1 at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox-1 and cox-2. it has been known that, under some stressful conditions, cox-2 is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for 6 h and subjected them to the elevated plus maze task 24 h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox-2 by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps1a-g104 imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded 15o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when 11 male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang 1 , takesi sakurai 2 , yasuitirou fukuda 3 , tomoyuki kuwaki 1,3 1 dept. molec. integ. physiol., chiba univ., japan; 2 dept. pharmacol., univ. tsukuba, japan; 3 dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of 3-8 weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of 5 weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between 3 and 5 weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of 3 weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between 3 and 8 weeks of age, and in the hippocampus at the age of 3 weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi #14760187 ps1a-g108 a systematic analysis of genetic factors associated with behavioral diversity between msm and c57bl/6 koide tsuyoshi 1,2 , aki takahashi 1,2 , toshihiko shiroishi 2,3 , akinori nishi 1 1 mgrl, national institute of genetics, mishima, japan; 2 sokendai, hayama, japan; 3 mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c57bl/6. in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b6-6cmsm, which carries chromosome 6 of msm, showed extreme behavioral differences from c57bl/6. the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga 1 , yu kajiyama 2 , shigenobu shibata 2 , hiroshi kunugi 1 1 department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; 2 department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for 4 days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e2) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e2) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e2 mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri 1 , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps1a-g113 synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi (15500238) ps1a-g114 involvement of nr2b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary 1 , takanobu nakazawa 1 , yuji kiyama 2 , toshiya manabe 2 , tadashi yamamoto 1 1 div. of oncology, ims, univ. of tokyo, tokyo, japan; 2 div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr2b is tyrosine-phosphorylated, with tyr-1472 being its major phosphorylation site. to investigate the role of tyr-1472 phosphorylation, we generated mice with a tyr1472phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr-1472 and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr2b is involved in the control of emotional responses by regulating crf content. ps1a-g115 increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca 2+ signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for 5 weeks, locomotor activity and longinterval (200, 1000 and 5000 ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for 5 weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui 1,3 , yosky kataoka 1,2 , yasuhisa tamura 3 , yasuyoshi watanabe 2,3 , hisao yamada 1 1 department of anatomy and cell science, kansai medical university, osaka, japan; 2 department of physiology, osaka city university graduate school of medicine, osaka, japan; 3 molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at 50-60 times/min), inhibition periods (less than 10 times/min) were often observed 2 h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns-398, a selective cox-2 inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps1a-g119 impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima 1 , naoya aoki 2 , andras csillag 3 1 biology, hokkaido univ., sapporo, japan; 2 agriculture, nagoya univ., nagoya, japan; 3 anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task 1, chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to 3 s. in task 2, chicks chose between small reward located at 20 cm and large reward at −140 cm, where cues signaled the distance of invisible food. task 3 was similar to the task 2, except that cues signaled the food quantity. in task 4, total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi (15370033, 17021018) ps1a-g120 analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe401, which had a missense mutation in the casy-1 gene. several casy-1 deletion mutants also showed learning defects. casy-1 has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x11l/mint2. however, after dissociation of x11l, alcadein is susceptible to cleavage by protease(s). we found that casy-1 was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy-1 can also be proteolytically cleaved. ps1a-g122 insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama 1 , atsushi kuhara 1 , akiko mohri 1,2 , kotaro kimura 1,3 , masatoshi okumura 1 , masahiro tomioka 4 , yuichi iino 4 , ikue mori 1,5 1 div. of biol. sci., nagoya univ., japan; 2 present address: univ. of texas, health sci. cent., usa; 3 present address: natl. inst. of genet., japan; 4 mol. genet. res. lab., univ. of tokyo, japan; 5 inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins-1 encoding insulin homologue caused defective associative learning, mutations in daf-2 and age-1 encoding the homologues of insulin receptor and pi 3-kinase, respectively, suppressed the defect of ins-1, and the mutation in daf-16 encoding forkhead transcriptional factor caused the learning defect. this suggests that ins-1 antagonizes daf-2 insulin-like signaling for associative learning. interestingly, age-1 animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age-1 in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins-1. we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps1a-g123 analysis of ttx-8: novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx-8(nj21) and ttx-8(nj34) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx-8 encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx-8 is similar to ric-3, identified in c. elegans at first and conserved among several species (halevi et al., 2002 (halevi et al., , 2003 . ric-3 is thought to be required for the maturation of acetylcoline receptor (halevi et al., 2002) , so ttx-8 may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps1a-g124 analysis of aho-3 mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio 1 , akiko mohri 1,2 , eiji kodama 1 , atsushi kuhara 1 , mizuho koike 1 , kotaro kimura 1,3 , ikue mori 1,4 1 div. of biol. sci., nagoya univ., japan; 2 present address: univ. of texas, health sci. cent., usa; 3 present address: natl. inst. of genet., japan; 4 inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho-3(nj15) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., 2000) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho-3 gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho-3 is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho-3 gene further. we have demonstrated aversive conditioning in lymnaea using 100 mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b3, located in the buccal ganglion. an associative memory, lasting 24 h, was produced with 20 pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b3 neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b3 neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura 1 , dai hatakeyama 1 , tetsuro horikoshi 1 , etsuro ito 2 , manabu sakakibara 1 1 lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; 2 cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts1 in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno 1 , minoru saitoe 1,2 , tim tully 3 1 tokyo metropolitan institute for neuroscience, tokyo, japan; 2 department of biology, tokyo metropolitan university, japan; 3 cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. 1 day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi 17005050 ke zhang 1 , jian z. guo 1 , ai k. guo 1,2 1 institute of neuroscience, chinese academy of sciences, china; 2 institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal4/uas-shi ts1 system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (<4 min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased 2 months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari 1,2 , kazuo okanoya 1,2,3 1 lab. for biolinguistics, riken-bsi, wako, japan; 2 grad. sch. of sci. and tech., chiba university, chiba, japan; 3 presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day 120, developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps1a-h137 facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi 17023034 ps1a-h138 the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment 1, rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of 0.15 m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment 2, rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi (17730431) ps1a-h139 transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps1a-h140 gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c57bl/6 mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over 80%, while those in the mscand ptx-injected mice decreased to 30%. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦2 that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps1a-h143 post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao 1,2 , takehito okamoto 1 , fumihiro shutoh 1,3 1 lab for motor learning control, riken bsi, saitama, japan; 2 sorst, jst, saitama, japan; 3 dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of 1 h daily training for 1 week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi (16500204) ps1a-h144 modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri 1,2 , takehito okamoto 1 , shin-ichi nishimura 1 , fumihiro shutoh 1,3 , soichi nagao 1,4 1 lab. for motor learning control, riken bsi, saitama, japan; 2 univ of the air, chiba, japan; 3 dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; 4 sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., 2006) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps1a-h145 influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho 1,2 , t kobayashi 1,2 , e hori 1,2 , k umeno 1,2 , t ono 1 , h nishijo 1,2 1 system emotional science, univ. of toyama, toyama, japan; 2 crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of 133 place cells recorded, activity of 40 was significantly modulated by the car speed and/or distance between the car and rat; 21, 12 and 7 cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, 16 place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a 2.0-3.0 mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about 40 ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas 5 and 7, contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area 5 to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca1 performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca1 neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca1 plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca 2+ but is blocked by mg 2+ in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg 2+ binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca1 cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l1 is a component of the ubiquitin system. uch-l1 is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l1 in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl1. gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca1. in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l1 is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps1a-i151 learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage 0). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band (64-120 hz) was strong at stage 0 and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps1a-i152 longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about 1.5 mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of 1 mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal1 ubiquitously expresses in brain. bmal1 functions by forming a heterodimer with either clock or npas2, which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal1 in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal1 function in a forebrain. using a dominant negative mutant of bmal1 (bmal1 r91a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., 2004), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal1 r91a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal1 in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn (0, 70, or 100 db; intermittent white noise). 70 db bgn induced significant decreases in number of error and time to goal in maze compared with 0 and 100 db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, 100 db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn 1,2 , anucha pongpanparadorn 2 , wasana pratchayasakul 2 , anchalee pongchaidacha 2 , nipon chattipakorn 2 1 fac. dent. cmu, chiang mai, thailand; 2 cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were 101 ± 14, 124 ± 19 and 108 ± 22 in the groups administered 250, 500 and 1000 mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was 17.4 ± 6.3, 22.7 ± 6.9 and 16.6 ± 5.0 for 250, 500 and 1000 mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a 2-h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a 2-h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses (10, 60 and 120 min). percentage inhibition of cache activity following a single tde injection at doses 500 and 1000 mg/kg significantly decreased at 10 and 60 min after tde injection, but not at 120 min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣4and ␣7-nachr and kinases involved in pi3k-akt pathway, and increase the levels of phosphorylated akt and bcl-2. these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi3k-akt pathway via ␣4and ␣7-nachrs. ps1a-i160 arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae 1 , yoshiyuki ishikura 2 , shigeaki fujikawa 2 , yoshinobu kiso 2 , manabu sakakibara 1 1 lab. neurobiol. engr., tokai univ., numazu, japan; 2 inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in 20-month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps1a-i161 thimet oligopeptidase co-exists in gfap-and cd11b-positive glia in rat pc/rsc treated with mk-801 takeshi kato 1 , mohammad arif 1 , michiyuki yamada 2 , toshiyuki chikuma 3 , md. mahiuddin ahmed 4 1 lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; 2 grad. sch. integr. sci., yokohama city univ., yokohama, japan; 3 dept. hygien. chem., showa pharmaceut. univ., machida, japan; 4 dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep24.15) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk-801 activated gfapand cd11b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) 3 day after the treatment. mk-801 also increased ep24.15 and prolyl oligopeptidase. immunohistochem data showed that ep24.15 co-localized with gfap and cd11b positive glial cells. since mk-801 causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep24.15 in the pc/rsc. these data suggest that ep24.15 in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps1a-i162 effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) 1 and 2 in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) 1 and 2, code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps1 shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps1 and ps2 with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps2 protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps1 level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps1a-i163 effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of 10-week-old adult mice. in this study, a␤42 peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤42 peptide. we found that treatment of nsc with oligomeric form of a␤42 peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤42 peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein-1 (hmgb1) is a non-histone chromosomal protein. here, hmgb1 was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb1 immunoreactivity was observed around dying neurons in the kainic acid-and a␤1-42 (a␤42)-injected rat hippocampi. hmgb1 was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb1 delayed the clearance of a␤ and accelerated neurodegeneration in a␤42-injected rats. these results suggest that hmgb1 released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps1a-i166 x11-like protein regulates metabolism of app in the mouse brain yoshitake sano 1,2 , tadashi nakaya 2 , shigeyoshi itohara 1 , toshiharu suzuki 2 1 riken bsi, saitama, japan; 2 hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x11-like protein (x11l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells (1999 . j. biol. chem. 274, 2243 2003. j. biol. chem. 278, 49448) . there is no evidence, however, that endogenous x11l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x11l in app metabolism in the brain, we generated x11l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x11l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin-1 protein by 2d-electrophoresis and tof-mass delineate that mutant at1 and htt proteins similarly reduce transcriptional factor x1 and x2. immunoprecipitation and pull-down assays support interaction between polyq and factor x1 and x2. immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x1 and x2 into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x1 and x2 ameliorates poly-q pathology in htt-/at1-expressing neurons and transgenic drosophila. these results suggest that factor x1 and x2 are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps1a-j170 ba1-42 was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, 10 rats/group (g). group 1 received e2 at 2.5 g/kg per day; groups 2 and 3 received ycj at 20 ml, and 100 ml/kg day, respectively, once everyday. group 4 received ycj 100 ml/kg plus e2 at 2.5 g/kg day twice a week, all for 5 weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e2 in group 3 was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid 1-42 antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer 3 and layer 5 of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group 3 was lesser than ovx and control groups. amyloid deposition was correlated with e2 serum at r = −0.4. ps1a-j171 correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki 1 , shigeo kinomura 1 , kazunori sato 1 , shinya uchida 1 , ryoi goto 1 , kentaro inoue 1 , ichiro tsuji 2 , hiroyuki arai 2 , ryuta kawashima 3 , hiroshi fukuda 1 1 department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; 2 tohoku univ. grad. school of med., sendai, japan; 3 niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of 111 community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h13-kenko-008), (h13-choju-007, h13-choju-023) ps1a-j172 effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the 8 weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook 1 , d.g. goodenowe 1 , y. yamazaki 1 , j. flax 2 1 phenomenome discoveries inc., saskatoon, canada; 2 precisionmed inc., san diego, ca, usa dementia affects about 10% of the population over the age of 65 and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an 8-metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the 8-metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin-2 (chr2). in this study, we quantitatively investigated the rapidness of the light-gated current of chr2 expressed in pc12 cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about 10-fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr2 is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori 1 , shigeki ohta 1 , kenji hamada 2 , naofumi yamada-okabe 2 , yonehiro kanemura 3 , hideyuki okano 4 , yutaka kawakami 5 , masahiro toda 1,6 1 neuroimmnology research group, keio univ., tokyo, japan; 2 chugai co. ltd., kanagawa, japan; 3 inst. cli. res., onh, japan; 4 physiology, keio univ., tokyo, japan; 5 cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; 6 neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa1110 gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa1110 homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa1110 protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day 15 were dissociated. after the cells were treated with 100 nm estradiol-17beta (e2) or ethanol for 8 days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis (2d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than 4000 spots were detected from 2d dige. compared with ethanol treatment, e2 increased the expression of 501 spots in the hypothalamic cells and 169 spots in the cortical cells (p < 0.2, difference >1.5). proteomics analysis showed different effects of e2 for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e14 days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp90 client protein family. 17-allylamino-17-demethoxygeldanamycin (17-aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. 17-aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of 17-aag in a mouse model of sbma. administration of 17-aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of 17-aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp90 function by 17-aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb9 promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately 5-20% of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps1a-k181 selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura 1 , yoshiyuki ishii 1 , kazuto kobayashi 2 , yasufumi sato 3 , keiichi itoi 1 1 lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; 2 dept. mol. genet., fukushima med. univ., japan; 3 inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a1, a2 and a6) and dopaminergic (a9, a10 and a12) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg4b has been shown to play an important role in the processing of lc3, a mammalian homologue of yeast atg8, but the tissue distribution of atg4b remains unknown. to understand the role of atg4b in rat tissue cells, we prepared an antibody to atg4b and pc12 cells in which atg4b expression was knocked down by rnai. in rnaitreated pc12 cells where atg4b expression was 10% of that in the wild-type pc12 cells, the expression of cytosolic lc3-i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc3 to lc3-i. moreover, the expression of atg4b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg4b was intense in neurons, especially in the cerebellum. these results suggest that atg4b plays a major role in the processing of lc3, while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps1a-k183 spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev 67n strain into the central nervous system was examined after its subcutaneous inoculation (10 5 pfu) in the rat hind footpad. on day 3 post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day 4 p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day 5 p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the 4th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki 1 , hiroshi kameda 1 , hisashi nakamura 1 , taro okunomiya 1 , koji ohira 1 , kouichi nakamura 1,2 , takahiro furuta 1 , takeshi kaneko 1,2 1 dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; 2 crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn1, rta1, mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap43 n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣1 and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has 10 armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca1 hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos7 cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr2 increased the surface expression of glur2 in cos7 cells compared with grip/glur2 or ␦-catenin/glur2 transfection. this study points to ␦-catenin as a regulator of glur2 receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca1 area in wild type (wt) and gad65 knockout (ko) mice. this parameter was twice lower in gad65ko mice (wt: 2.0 ± 0.5 pa/pf, n = 7; gad65ko: 0.9 ± 0.3 pa/pf, n = 8; p = 0.038). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba (5 m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad65ko animals (wt: 12.4 ± 0.7 ms, n = 14; gad65ko: 10.8 ± 0.6 ms, n = 12; p = 0.047). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad65ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that 5 hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p5 to p8) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent 5 hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur1 but not by mglur5 because it was antagonized by the mglur1 antagonist ly367385 but not by the mglur5 antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur1 is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps1p-a004 involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita 1 , michiko ohtomi 2 1 cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; 2 department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d1 receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d1 antagonist sch23390 into pfc by using a microdialysis method impaired md-pfc ltp but the d2 antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by 110-120%). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer 2/3 pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya 464-8601, japan we showed in previous study that somatic inhibitory synapses of neocortical layer 5 pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer 5 pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer 5 pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer 6 pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate (200 nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr1 in the regulatory mechanisms for ltp in the amygdala, we utilized ntr1-knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr1-ko mice than in wild-type mice. the bla-ltp in ntr1-ko mice was attenuated by sulpiride, a d2 receptor antagonist. these results suggest that d2-ntr1 interaction regulates the extent of ltp in the mouse la-bla synapses. ps1p-a010 facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over 28-day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca 2+ sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca 2+ at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin-1, and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps1p-a012 inhibitory modulation of the hippocampal ca3 transmission and plasticity by glucagon-like peptide-2 jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode-2 (glp-2) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp-2 (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp-2 effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp-2 on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices (400 m) were prepared from 7 to 35 days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca3 in the whole-cell mode using glass microelectrodes (resistance: 4-8 m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp-2 (10 nm-1 m) inhibited spontaneous excitatory postsynaptic current. glp-2 (10 nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp-2 may play an inhibitory role in the dg-ca3 transmission. ps1p-b013 quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp-2 and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a1, a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, 100 hz for 1 s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, 1 hz for 900 s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of 50 m apv, 3 m bicuculline, but not 500 m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when 20 mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at −10 mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine (5-100 m) or capsaicin (0.1-1 m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin (1 m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after 5 min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv1), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji 1 , akira komatu 2 , akinao nose 1 1 dept. phys., univ. tokyo, tokyo, japan; 2 dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila 3rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca 2+ -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t287d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t287d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr1* flies did not show mg 2+ blockade, and dnr1* flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf-1, and notch. we examined how mg 2+ blockade of dnr links to these transcription factors. research funds: kakenhi ps1p-b020 response properties of wind-sensitive giant interneurons in the 4th-instar nymphs of the cricket tetsuya matsuura 1 , masamichi kanou 2 1 dept. of welfare eng., iwate univ., morioka, japan; 2 dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) 8-1, 9-1, 9-2 and 9-3 in the 4th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from 12 different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi 8-1 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. the response magnitudes of gi 9-1 reached a plateau at a stimulus velocity of 30 mm/s in most stimulus directions. the response magnitudes of gis 9-2 and 9-3 increased with stimulus velocity up to 300 mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi 8-1, the ipsilateralfront and contralateral-rear in gi 9-1, the ipsilateral-rear in gi 9-2 and the ipsilateral-front in gi 9-3, designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca1. serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca 2+ , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca1 region. we found that ptp induced by the 100-hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca 2+ channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca 2+ channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma 1 , kunihiko kaneko 1,2 , makoto asashima 1,2 1 grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; 2 jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca 2+ concentration ([ca 2+ ] i ) may change because of nonsynaptic communication. we loaded pc12 cells with a ca 2+ indicator, indo-1 am, and the [ca 2+ ] i of more than 10,000 cells was measured using flowcytometry. the [ca 2+ ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca 2+ ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca 2+ -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss5 from human and mouse cns. in mouse cns, four isoforms (types 1-4) were expressed. subcellular localization analysis revealed that type 4 (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type1 variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc34 and nb2a derived from neuronal cell express only type 4, whereas os3 and kt-5 derived from astrocyte express both type 4 and type 1. interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os3 and kt-5. we analyzed promoter region of m-spinesin gene, and identified that 5 -flanking region from −224 to −188 bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme 3-phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca 2+ signals, such as intra-and intercellular ca 2+ waves; however, their physiological roles remain elusive. the overexpression of an ip 3 -hydrolyzing enzyme, ip 3 5-phosphatase, suppressed the spontaneous ca 2+ signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca 2+ -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca 2+ -signal-deficient astrocytes. these results indicate that spontaneous ca 2+ signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai 1 , noriko osumi 1,2 1 tohoku univ. sch. med., japan; 2 crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax6 transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax6 in postnatal mouse brain and found that pax6 was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax6 in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax6 heterozygote mutant (sey/+) mice at 8 weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, 6 and 24 h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax6 regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim21 to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., 2006) . we previously reported a unique expression of dim21 ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., 2005) . we show here that mab dim21 also recognizes adult neural stem cells and glial cells at postnatal period. dim21, brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim21 ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk2 (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l7 promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk2 cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad1/5/8 (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk2. lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in 1950s and 1960s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in 6-ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after 48 h (34 and 19%) and 72 h (56 and 33%) exposure to 10 −4 and 10 −5 m 6-ohda respectively. it was accompanied by morphological changes and induction of gfap, s-100 and ox42. 6-ohda (10 −6 m, 72 h) was found to cause a significant impairment in 3 h glutamic acid uptake (31%) and gsh levels (27%). further neurons (in coculture with 6-ohda pre exposed glial cells) on exposure to 6-ohda (10 −6 m), showed loss of th expression and significant neuronal cell death (34%). the results of the present study suggest that 6-ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at 1 day after injection, then started to repair at 3 days and returned to the normal levels at 6 weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at 3∼7 days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng2) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba1+ cells accumulated in stab wounds and ischemic lesions were found to express ng2, of which molecular weight of its core protein was higher by 10 kda than that of ng2 expressed in contralateral brain region. this was due to the lack of shedding of ng2 in the brain lesions. we found that iba1+ cells accumulated in stab wounds and ischemic core lesion, most of which were ng2+/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd163 and von willebrand factor. ng2+ mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng2+ mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy 1 all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from 20 cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from <1 to 40 m 2 . most of the fibers were myelinated and counts produced a mean of 16,891 (12,000-21,500). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from 0.29 to 3.16 m (from fetal age to 60 years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p5-10. costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca1, ca2 pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps1p-d042 impact of s100b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani 1 , akiko seto-ohshima 2 , shigeyoshi itohara 2 , hajime hirase 1 1 neuronal circuit mechanisms research group, japan; 2 lab. for behavioral genetics, bsi, riken, wako, japan s100b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s100b, we have recorded hippocampal spontaneous activities from urethane anesthetized s100b ko and wt mice. typical eeg patterns including theta (3-8 hz) and sharp wave associated fast ripple (120-180 hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca1 radiatum, where ictal event was characterized by hyper-synchronous gamma band (30-80 hz) activity. while both populations developed ictal event within 40 min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s100b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s100b related pathways could potentially be activated. yoshiko takagishi 1 , erina okabe 2 , xiaoyang sun 1 , sen-ichi oda 2 , yoshiharu murata 1 1 riem, nagoya univ, nagoya, japan; 2 grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps1p-d044 regulation of hippocampal neurocircuit activity by glutamate transporter glt-1 noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt-1, the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap5) and the glt-1 blocker (dhk) with the signal subtraction, we could visualize the activity of glt-1 as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt-1 by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt-1 function in the ca3 region was elicited by much lower concentration of dhk than that in the ca1 region. ps1p-d045 monoclonal antibody rip specifically recognizes 2 , 3 -cyclic nucleotide 3 -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg-4 cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was 2 ,3 -cyclic nucleotide 3phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg-4 cells. moreover, the same localization was also observed in rat cnp1 transfected hek293t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca 2+ signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca 2+ signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca 2+ signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki 1,2 , hisatsugu koshimizu 1 , megumi kashihara 1 , tomoko hara 1 , masami kojima 1,2 1 research institute for cell engineering; 2 sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that 3-day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p38) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p38 expressed in the neural progenitor cells at embryonic 14 days (e14) mice brain. to ascertain the function of p38 on these cells, we treated the cultured e14 cells with the selective chemical inhibitor for p38 (sb203580) for 7 days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p38 in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit 1 , takashi yamazaki 1,2 , takeshi oya 1 , yoko ishii 1 , masakiyo sasahara 1 1 pathology ii, university of toyama, toyama, japan; 2 oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on 3 to 28 days after injury, and activation of pdgfrs, mapks, akt and p38 were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from 3 to 21 days after injury. p-tyr was highly detected from 3 to 21 days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to 7 days after injury and then gradually decreased. activation of akt and p38 continuously increased from 3 to 28 day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln1 is a cerebellum-specific protein structurally related to the c1q and tumor necrosis factor families. recently, we have shown that cbln1 is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln1 was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln1 needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln1 proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln1 mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln1 released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to 3 -untranslated region (utr) of bdnf mrna to investigate the effect of the 3 utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf3 utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf3 utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer1a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps1p-e056 bmp4 expression in the adult rat brain bone morphogenetic protein-4 (bmp4) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp4 expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp4 expression in the adult rat cns using immunohistochemistry. bmp4 is intensely expressed in most neurons and their dendrites. in addition, intense bmp4 expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp4 protein. these data indicate that bmp4 is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp4 plays pivotal roles also in the adult brain. ps1p-e057 hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and 30 min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl-2 protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl-2 expression, decreasing the level of bax expression in c57bl/6 mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase 1. here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda 1 , yoko tohyama 1 , shinichi kohsaka 2 , tadashi kurihara 1 , kazuyuki nakajima 1,2 1 soka university, hachioji, tokyo, japan; 2 dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt-3, and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤2 among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba 1 , chiaki nakagawa 1 , masako tsuzuki 1 , motoko matsuda 1 , satoshi ichisaka 2 , yoshio hata 3 1 inst. dev. res., aichi human ser. ctr., kasugai, japan; 2 div. neurobiol., tottori univ., sch. med., yonago, japan; 3 div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats 12 h after beginning an 8-h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin-3 (nt-3) in selected brain regions from 14-day-old male rats and compared to those from adults. the changes in levels of bdnf and nt-3 were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci (200 kdyne, ih injury device) at t8/9 and were assigned to three groups; a (placebo), b (mp treated; 30 mg/kg i.v. immediately after injury), c (g-csf treated; 15 g/kg i.v. for 5 days after injury). animals were assessed with the bbb locomotor rating scale for 6 w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi (16390427) ps1p-e064 glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons (12 div) exposed to glutamate for 15 min at 100 m. glutamate exposure led to a decrease in mrna of nd1 and nd6, which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least 2-12 h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk-801. these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj-1 is enhanced by oxidative stresses. although exact functional significance of dj-1 has still unknown, it is thus proposed that dj-1 is protective against neural damage under oxidative stresses. in this study, we tested expression of dj-1 in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj-1 was markedly increased in the molecular layer of the dentate gyrus on days 4 and 14 post tmt injection. on day 14 post tmt injection, enhanced expression of dj-1 was observed in the stratum lucidum of the ca3. in glutathione-depleted mice, tmt was more effective in enhancing expression of dj-1, compared with that in untreated mice. furthermore, double staining of dj-1 and gfap demonstrated that most of cells highly immunoreactive to dj-1 were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj-1 is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt (2.8 mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus 2 days later, with visually apparent recovery of the granule cell layer 14 days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone (0.5-5 mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of 2.0 mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb (1 g/ml) on the pc12 cells was elicited through apoptotic cell death. activation of caspase-3/7 was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin (1 nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc12 cells. ps1p-e068 kinesin superfamily protein 4 (kif4) regulates activity-dependent neuronal survival by suppressing parp-1 enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein 4 (kif4), a microtubule-based molecular motor. the c-terminal domain of kif4 is a module that suppresses the activity of poly (adp-ribose) polymerase-1 (parp-1), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif4 from parp-1, resulting in upregulation of parp-1 activity, which supports neuron survival. after dissociation from parp-1, kif4 enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif4 controls the activity-dependent survival of postmitotic neurons by regulating parp-1 activity in brain development. research funds: 1684304 ps1p-e069 expression of hsp105, apg-1, and apg-2 in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt (2.8 mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp105, apg-1, and apg-2 in the granule cell layer of the dentate gyrus, but not in ca1-ca3 pyramidal cell layer, 16 h to 2 days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp105 highly expressed in survival neurons in the culture, apg-1 and apg-2 highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day 18. thirty gray of x-irradiations were performed on the cultured cells at 7 or 21 days in vitro (div). then the cells were fixed at 24 h after the irradiation with dapi. at 7-div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at 21-div. this indicates that the radiosensitivity of 7-div immature neurons is higher than that of 21-div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦2 glutamate receptor (glur␦2), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦2 is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca 2+ , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin-1 and found a novel gene down-regulated in response to mutant ataxin-1 in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s6 as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca1 molecular pathology. ps1p-f080 permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca 2+ -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca 2+ -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi3 kinase (pi3k) pathways. interestingly the up-regulation of bcl-2, a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat3 probe. scat3 is an indicator protein for caspase-3 activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase-3 cleavage sequence. using this probe, we could monitor the activation of caspase-3 at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt (2.8 mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex 2 days later. in cortical neurons, tmt exposure for 48 h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases 3 and 8, but not caspase 9, were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride (2.5 mg/kg, i.p.) in 3-(3w) and 7-week-old (7w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in 3w mice. by contrast, slight, diffuse neuronal damage was found in the ca1 and ca3 subfields and dentate gyrus of 7w mice. the hippocampal putrescine level was elevated markedly in 3w mice on tmt administration, whereas a minor putrescine increase was detected in 7w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps1p-f086 establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai 1 , toshinori hongo 2 , kimisato naito 2 , shigeto sasaki 2 1 department of physiology, kyorin university, school of medicine, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for 0.3-1.0 s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of 0.1 s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in 17 volunteers. experiment was performed by three tasks, control task is a forearm rotation, task1 is simulation of tools-manipulation, task2 is execution of tools-manipulation. we found different brain regions by this experiment. task2 to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at 1 khz, to record simultaneously and independently the 12 • horizontal saccades of each eye for 12 subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at 1 day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi (17022042) ps1p-f094 dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼20 hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from 35 healthy subjects, high and low scorers (n = 12 each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band (18-24 hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a 3-t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = 2.7 s) with online monitoring of meps. the intensity of tms pulses was varied from 30% to 95% at a 5% step (frequency at ∼0.15 hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above 80% stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = 0.46, p = 0.001). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps1p-f097 shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda 1,2 , yukihiro gomi 1 , itsuki imai 3 , nobuaki shimoda 1,2 , hiroyuki kato 1,2 1 international university of health and welfare, ohtawara, japan; 2 crest, jst, kawaguchi, japan; 3 nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels (22 channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of 15 s task and 30 s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky 1,2 , rieko osu 2 , hiroshi imamizu 2 , mitsuo kawato 1,2 1 computational brain project, icorp, jst, japan; 2 atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed 1 hz isometric force pulses to seven targets separated by 30 • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a 2d strain gauge. twenty repetitions of each condition were performed in 12 s blocks (tr = 2 s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of 661 voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only 31.3% (chance: 14.3%), errors were distributed normally about the target such that 80.4% (chance: 59.2%) of the data was classified correctly to within 60 • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from 28 • c, fish showed repetition of short time swimming in the range 32.5-40 • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in 3-5 days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in 3 days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps1p-f100 to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu 1 , ken-ich morishige 2 , jun nakanishi 1,3 , hiroyuki miyamoto 2 , mitsuo kawato 1 1 atr computational neuroscience labs, kyoto, japan; 2 kyushu institute of technology, japan; 3 jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p1 pups of the surviving cases increased greatly between p4 (30% of the control value) and p7 (52%), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p1, dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p4 and p7 pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p4 to p7. we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully (180 • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative 1, 4, and 7 days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps1p-g110 role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa 1 , tatsumi nagahama 2 1 dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; 2 fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps1p-g112 neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto 1 , sayaka hihara 1 , kazuo okanoya 2 , atsushi iriki 1 1 lab for symbolic cognitive development, bsi, riken, japan; 2 lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about 38.5 • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps1p-g115 prostaglandin e 2 -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, 1-23-1 toyama, shinjuku,162-8636, japan unilateral microinjection of pge 2 into the region around the rostroventral wall of the third ventricle (av3v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of 20-1000 pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide (0.5 mm, 100 nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge 2 injection site was examined. this treatment alone had no effect on the o 2 consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge 2 -induced responses were blocked 10 min after the pretreatment with bicuculline, and recovered at ∼70 min. pretreatment with vehicle saline had no effect on the pge 2 -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge 2 -induced thermogenesis. tetsufumi ito 1 , hiroyuki hioki 2 , kouichi nakamura 2 , takeshi kaneko 2 , yoshiaki nojyo 1 1 dept. anat., univ. of fukui, fukui, japan; 2 dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad67-ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad67-positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad67. we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad67-ir. after cutting t1-t4 vrs and drs, almost all gad67-ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama 1 , ning ma 1 , reiji semba 2 , satoru naruse 3 1 dept. of anat. ii, mie univ. school of med., tsu, japan; 2 inst. for developmental research, kasugai, japan; 3 dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin 1 (aqp1) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp1-immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp1-ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp1-ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp1-ir fibers were found both in the myenteric and submucosal plexi. many aqp1-ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp1-ir fibers penetrated into the muscle layers. these results suggest that aqp1-ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi (13137204) ps1p-g118 abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days 1-4. the increase in the volume and frequency setting of the artificial ventilator (fio2 = 50%, fico2 = 0%) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico2 was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad67-gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = 40) in medullary transverse slices. about 20% of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about 50% of gabaergic neurons recorded in the superficial rvm were co 2 inhibitory, and all of the remaining neurons were co 2 insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with 0 and 10 mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with 10 mv forehead spla, in comparison to subgroups 0 mv forehead spla. such law was common for both for sg and for cg. in two subgroups of 0 and 5 mv nasal zone spla in sg the greater rt gain after il was in 0 mv nasal spla subgroup, and for cg in 5 mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin (5ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via 5ht2 receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain 5ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c57bl/6n) were pretreated with pcpa (0.1 g/10 ml/kg body weight) or saline intraperitoneally for consecutive 4 days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o 2 inhalation was changed to stepwise 5, 7 and 9% co 2 in o 2 inhalation every 5 min. hypercapniainduced increases in tidal volume and minute ventilation during 9% co 2 inhalation were reduced by pcpa pretreatment; these results suggest that 5ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a 1.08% gosha-jinki-gan diet group. after 4 weeks, continuous cystometry with physiological saline or 0.1% acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with 0.1% acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps1p-g127 differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to 90 db white-noise for 10 min. 90 db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the 90 db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase 67 (gad67), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad67 mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were 2.0 on the small neurons, 3.1 on the medium-sized neurons, 1.2 on the large neurons, and 4.2 on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about 80% of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large (3.16 m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps1p-h131 neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c4 nerve and facial nerve activities. addition of 4 mm k + to the standard superfusate decreased burst rate of c4 activity. addition of 6 or 8 mm k + caused initial inhibition of c4 burst and subsequent high frequency c4 burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi (16500208) ps1p-h132 regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp (1 m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at −60 mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin 1 receptor (nk1r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk1r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps1p-h133 effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for 1 h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min-6 cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm-3000, shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda 2300, usa) at the right middle finger from 12 volunteers (22.8 ± 2.1 years). mild thermal stimuli (17 ± 1 • or 40 ± 1 • ) were administered every 2 min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm-3000. we examined the effects of color environments on cognitive function in 12 healthy subjects and 12 patients after traumatic head injury using p300 components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p300 latency in the red environment was significantly shorter in controls than in patients. the p300 amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area 10 in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about 800 ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps1p-h139 neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi 1,2 , mikinobu takeuchi 1,2 , takahiro omote 3 , naoyuki yosimura 3 , etsuro hori 1,2 , kazuo sasaki 3 , taketoshi ono 2,4 , hisao nishijo 1,2 1 system emotional science, univ. toyama, toyama, japan; 2 crest, japan science and technology agency, japan; 3 bio-information engineering, univ. toyama, toyama, japan; 4 molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems (103 channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate (1) human facial stimuli with different gaze directions and (2) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the 3d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi 1 , sachiko koyama 2 , ayumu matani 3 , andrzej cichocki 1 1 riken, wako, japan; 2 hokkaido university, sapporo, japan; 3 university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at 40 hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: (1) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. (2) the functions were averaged across subjects. (3) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. (4) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi (17022004) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps1p-h142 the reflection of category perception of sound in the auditory evoked n1m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for 2 different fundamental frequencies (f0) and 12 different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n1m source strength for nonlinear sounds was significantly larger than that for linear sounds. n1m peak latency only for vocal sounds was not affected by f0. these results suggest that perceptual categorization was reflected in n1m source strength and location (linear or nonlinear), and in n1m latency (vocal or nonvocal). sunao iwaki 1 , hiroko kou 1 , kouichi sutani 1 , mitsuo tonoike 2 1 national institute of advanced industrial science and technology, osaka, japan; 2 chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p3, related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p3 components, suggested the involvement of frontal and anterior cingulate cortex in the early p3 component (p3a) and the contribution of the parietal and temporal regions to the later component (p3b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l1 and l2, respectively). we compared the brain activity of 11 chinese learners of japanese as l2 with that of 28 japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm2. while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l1 and l2. neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around 100 ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around 200 ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around 500 ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a 1.5 t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for 6 times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya 1 , mie matsui 2,3 , tsuneyuki kobayashi 2,4 , hisao nishijo 2,4 , michio suzuki 2,5 , yasuhiro kawasaki 2,5 , masayoshi kurachi 2,5 , jun nakazawa 6 , kyo noguchi 7 , hikaru seto 7 1 neuropsychiatry, univ. toyama, toyama, japan; 2 crest, japan science and technology corporation, japan; 3 psychology, univ. toyama, toyama, japan; 4 system emotional science, univ. toyama, toyama, japan; 5 neuropsychiatry, univ. toyama, toyama, japan; 6 developmental psychology, univ. chiba, chiba, japan; 7 radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura 1 , atsushi sekiguchi 2 , keisuke wakusawa 2,3 , yuko sassa 2,4 , hyeonjeong jeong 2,5 , kaoru horie 5,6 , shigeru sato 5,6 , ryuta kawashima 2,6 1 miyagi university of education, sendai, japan; 2 niche, tohoku univ., japan; 3 dep. pediatrics, tohoku univ. school of medicine, japan; 4 ristex, jst, japan; 5 gsics, tohoku univ., japan; 6 lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain 10, 50, 100 points or lose 100, 50, 10 points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps1p-h152 brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara 1 , masato taira 2,3 , toshio iijima 1 , ken-ichiro tsutsui 1 1 div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; 2 arish, nihon university, tokyo, japan; 3 appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either 0 yen or a gain or loss of 200, 400, or 800 yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps1p-i153 neural activation during experience-based reasoning chisato suzuki 1,2 , takashi tsukiura 1 , hiroko mochizuki-kawai 1 , yayoi shigemune 1,2 , toshio iijima 2 1 neurosci. res. inst., aist, japan; 2 div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in 16 healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps1p-i156 the difference of brain activity elicited by different styles of art hiromi yamamura 1 , yasuyuki kowatari 1,2 , shigeru yamane 2 , miyuki yamamoto 1,2 1 comprehensive human sciences, university of tsukuba, tsukuba, japan; 2 system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm2. while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba9, ba46). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j-3) ps1p-i157 effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were 20-30 aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi 17,6577 ps1p-i158 the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo 1 , michio nomura 2 , jun kawaguchi 3 1 ntt commun. sci. labs., ntt corp., atsugi, japan; 2 dept. psychol., tokai women's univ., kakamigahara, japan; 3 dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo 1,2,3 , yulri nonaka 1 , katsumi mizuno 4 , kazuo okanoya 1,5 1 riken, bsi, biolinguistics, saitama, japan; 2 chiba university, chiba, japan; 3 jsps, japan; 4 department of pediatrics, showa university, tokyo, japan; 5 presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for 18 neonates within 3 days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three (3) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented 1 s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for 12 different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around 700-900 ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism (9:4-14:11) and 18 agematched typically developing children (9:5-15:2) were instructed to name 50 pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = 1.2 ± 0.2, loudness = 1.4 ± 0.3) than in the children with autism (duration = 1.1 ± 0.2, loudness = 1.2 ± 0.3) (p < 0.001). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at 0.5/s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser (532 nm, 180 mw) to the muscle surface (28.3 mm 2 ) significantly delayed its attenuation (p < 0.05). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for 2 min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < 0.05). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps1p-i165 nedl1, a novel e3 ubiquitin ligase for dishevelled-1, targets mutant superoxide dismutase-1 and interacts with p53 yuanyuan li 1,2,3 , kou miyazaki 1 , toshinori ozaki 1 , akira nakagawara 1 1 division of biochemistry, chiba cancer center research institute, chiba, japan; 2 production technology development center, the furukawa electric co., ltd., ichihara, japan; 3 hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e3 ubiquitin ligase gene termed nedl1. previous study has shown that nedl1 is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled-1, a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl1 for uniquitination and proteasome-mediated degradation. on the other hand, nedl1 bound and ubiquitinated mutant (but not wild-type) sod1 in a mutant sod1 type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl1 physically bound p53, and induced apoptosis in a p53-dpendent manner. taken together, our results suggest that nedl1 may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod1 and p53. spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc70interacting protein), u-box type e3 ubiquitin ligase, has been shown to interact with hsp90 or hsp70 and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc12 cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca 2+ current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox-2 inhibitor, rofecoxib in pentylenetetrazol (ptz, 40 mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily 45 min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the 16th day of ptz treatment. chronic treatment with rofecoxib (2.0 and 5.0 mg/kg, p.o.) for 15 days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced (80 mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered 45 min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin (10 and 20 mg/kg, p.o.), naproxen (7 and 14 mg/kg, p.o.), nimesulide (1-5 mg/kg, p.o.) or rofecoxib (1-4 mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib (1 mg/kg) or nimesulide (1 mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox-2 inhibitors. cox-2 inhibitors also antagonized the effect of flumazenil (4 mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps1p-j171 cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca3 subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps1p-j172 a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus (4 mg/4 min). after 20 min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that 8-oxoguanine (8-oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca3 subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that 8-oxog accumulated greatly in mtdna of the ca3 microglia. wild-type and mth1-null mice, the latter lacking an ability to hydrolyze 8-oxo-dgtp and 8-oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca3 neuron loss after kainate administration, however, levels of 8-oxog accumulated in mtdna and cellular rna in the ca3 microglia were significantly increased in mth1null mice in comparison to wild-type mice. we thus demonstrated that mth1 efficiently suppresses the accumulation of 8-oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps1p-j176 transcription factor nrf2 regulates brain response to kainate-induced excitotoxicity yukihiko dan 1 , kosuke kajitani 1 , noriko yutsudo 1 , ken itoh 2 , masayuki yamamoto 2 , yusaku nakabeppu 1 1 kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; 2 univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e2 related factor 2 (nrf2) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf2 has been reported to increase neuronal death under increased oxidative stress, nrf2 seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca3 subregion of hippocampus. to unveil the functional significance of nrf2 in the brain, we compared seizure responses between wild-type and nrf2-null mice after systemic kainate administration. we found that nrf2-null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps1p-j177 synaptic plasticity and 4-aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine (4-ap) at the concentration below 0.1 mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the 4-ap induced discharges in ca3 region was studied. ten m 4-ap induced the epileptic discharges with the frequency of 0.33 ± 0.04 hz (mean ± s.e.m.; n = 3) and the amplitude of 1.23 ± 0.86 mv. when ap-5, an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap-5 was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of 4-ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. 7ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps2a-a001 influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno 1 , hugh p.c. robinson 2 1 engineering science, osaka university, osaka, japan; 2 university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣1a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the 3-day-old rat pup spinal subarachnoid space at the rate of 1 l/2.5 min. the rats were sacrificed 48 h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣1a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps2a-a003 on the membrane potential profile of ca1 pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga 1,2 , yoko tominaga 1 1 dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; 2 lab. for dynamics of emergent intelligence, riken bsi, hirosawa 2-1, wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps2a-a004 overexpression of inwardly rectifying k + channel 2.1 in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) 2.1 have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca1 pyramidal neurons. then, egfp and kir2.1 expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer 5 pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer 5 pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer 2/3 neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer 5 pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types 1 and 2 of ryanodine receptors (ryr1 and ryr2) in the cerebellum of a ca 2+ channel ␣ 1a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr1 but not ryr2 in the rmn cerebellum: a less ryr1 mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr1 immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr2 immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr1 expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr1-mediated ca 2+ release, contributing altered ca 2+ homeostasis in the rmn purkinje cells. ps2a-a007 dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than 5 s. d1 receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi 1,2 , hiroyoshi miyakawa 3 , masashi inoue 3 , masato okada 2,4 1 tokyo institute of technology, japan; 2 brain science institute, riken, japan; 3 tokyo university of pharmacy and life science, japan; 4 the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai 1 , maya yamazaki 1,2 , mika tsujita 1 , manabu abe 1 , rie natsume 1,2 , kenji sakimura 1,2 1 department of cellular neurobiology, brain research institute, niigata university, niigata, japan; 2 sorst/jst, saitama, japan we previously reported that stargazin family (␥2, ␥3, ␥4, and ␥8) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., 2004) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥4 subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥4 subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day 11, while the reduction was not so significant in adult brain. these results suggested that ␥4 might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph 6.8) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop1). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop1-transfected cells. mvsop1induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn 2+ sensitivity. neutralization of a positive charge in the s4-like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca1 pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh414). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca1 region. in the pyramidal layer, the amplitudes of the responses to the 1-hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the 3-10-hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels 2 (asic2) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic2-expressing cells in rat central nervous system (e17-p14) using immunohistochemical techniques. asic2positive fiber first appeared in brain stem and spinal cord at e17-18 stage. asic2-expresseing cells appeared in white matter of brain stem and spinal cord at e20 stage. in early postnatal stages asic2expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p4 stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic2 mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval (1st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long 1st isi by whole-cell recordings in rat slices. application of 100 m apamin, an sk-type ca 2+ -activated k + (kca) channel blocker, shortened the 1st isi and decreased the amplitude of the slow ahp. the shortening of the 1st isi was observed when membrane potentials were depolarized. moreover, application of 3 m mibefradil, a t-type ca 2+ channel blocker, shortened the 1st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca 2+ influx through t-type ca 2+ channels. research funds: kakenhi (c) (17500270) jafar vatanparast 1,2 , mahyar janahmadi 1 , houri sepehri 2 , ali haeri-rohani 2 , ali reza asgari 1 1 neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; 2 dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px (0.6 m, within 5 min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within 20 min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca 2+ spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca 2+ dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca1 slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca1 pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm8). repeated menthol application induced trpm8 desensitization. this desensitization was depended on extracellular ca 2+ , indicating that involvement with ca 2+ -dependent kinase. pkc activator (pma) desensitized trpm8 and go6976 (pkc inhibitor) abolished pma-induced trpm8 desensitization. pma similarly desensitized wild type trpm8 and mutant trpm8, in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm8. as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm8 causes pkc to desensitize trpm8 itself. yosuke sawada 1 , hiroshi hosokawa 1 , kiyoshi matsumura 2 , shigeo kobayashi 1 1 dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; 2 dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below 17 • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa1 is activated by pungent compounds stimuli. if trpa1 responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa1 is activated by cooling or not is still controversial. here, we investigated that trpa1-expressing hek293 cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was 17.5 ± 2.7 • c. in inside-out singlechannel recording, cooling activated trpa1 directly. single channel conductance was 74.1 ± 18.8 ps. single channel currents showed inword rectification. in conclusion, trpa1 is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn1-4) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn1-4 demonstrated that samples from the nerve and the heart showed an hcn2 band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn1, hcn3 and hcn4 could not be found in those samples. when an hcn channel blocker, zd7288 was applied on the stimulus portion of sciatic nerve and the nerve was elicited at 0.5/s by a duration of 10 ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < 0.01). it is suggested that hcn2 channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ 1 adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir3) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir1.1 and kir2.1 channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps2a-b022 proliferation of rat c6 glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c6), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control (140 mm) to 190 or 240 mm. an increase in [na + ] i was revealed by an image processor in c6 cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr-1, which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr-1 level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than 1 khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma (20-50 m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma (5 m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi (17021021) il-sung jang 1 , in-sun choi 1 , eun-ju park 1 , jin-wha cho 1 , man-gee lee 2 , byung-ju choi 1 1 kyungpook national university, school of dentistry, south korea; 2 kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca3 pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (<10 m), but decreased those induced by gaba at higher concentrations (>100 m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav1.1-deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav1.1-deficient mice as negative controls to examine nav1.1 distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav1.1 expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav1.1 distribution, in the present study, higher magnification analysis revealed that nav1.1 is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav1.1 antibodies used in these previous studies, none of which utilized nav1.1-deficient mice. based on our findings, we propose that nav1.1 might be involved in propagating action potential to presynapses. keiji miura 1,2 , masato okada 2,3,4 , shun-ichi amari 4 1 department of physics, kyoto university, kyoto, japan; 2 "intelligent cooperation and control", presto, jst, japan; 3 department of complexity science and engineering, university of tokyo, chiba, japan; 4 brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. 14084212 and 16500093) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [1-13 c] glucose intravenously (1 g/kg) 24 or 48 h after the intraventricular injection of colchicine (75 g/10 l) and the amino acid fractions were extracted from the brains at 10, 20 or 40 min after the glucose injection. the amount of [1-13 c] glucose in the cerebra was increased, however, the 13 c incorporation into glutamine, glutamate, gaba and aspartate from [1-13 c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes1 or hes5 gene, phes1-and phes5-egfp transgenic (tg) mice were generated (ohtsuka et al., 2006) . in each transgenic mouse, a promoter of hes1 or hes5 gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes1 or hes5 regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day 10 (e10) to postnatal day 60 (p60). in the phes1-egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e10 to p60. in the phes5-egfp tg mouse inner ear, gfp-ir cells were observed from e16.5 to p15. gfp-ir cells in phes1-gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., 2006. mol. cell neurosci. ps2a-c033 expression of zfh-5 in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine 1 , kenji nakamura 2 , motoya katsuki 1 , tetsuo yamamori 1 1 national institute for basic biology, okazaki, japan; 2 mitsubishi kagaku institute of life science, machida, japan zfh-5 is a transcription factor containing three homeodomains and 18 zn fingers and expressed in differentiating neurons. we have reported that the level of zfh-5 mrna is negatively regulated by antisense transcripts of the zfh-5 gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh-5 antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh-5 mrna starts to be expressed. recently, we have raised an antibody against mouse zfh-5 and examined the expression profile of the zfh-5 protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh-5 protein was not detected even when the zfh-5 mrna was already expressed. this observation together with other data suggested that the zfh-5 protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue 1 , maya ota 1 , katsuhiko mikoshiba 2 , jun aruga 1 1 laboratory for comparative neurogenesis, riken bsi, saitama, japan; 2 laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic1-5) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic1/zic3 compound mutant mice. mice carrying homozygous zic1 mutant allele together with zic3 null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic1-or zic3-single mutant. these observations indicate that zic1, in cooperation with zic3, have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp58, a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp58 gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp58 has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp58 gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp58 transcripts are first detected at embryonic day 10 in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day 12-13 embryos, rp58 transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e15, rp58 transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp58 is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c2hc-type zinc finger motifs. using p19 cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf-2 and nzf-3 respectively. nzf-2 null mice are born alive, but die within 10 min after birth with cyanosis. on the other hand, nzf-3 null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf-2 and nzf-3 by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , 2006) . in the present study, to examine the role of runx3 in the development of drg in more detail, we examined the development of drg neurons in runx3-deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx3−/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days 14.5 and 15.5. shin hisahara 1,2 , susumu chiba 2 , hiroyuki matsumoto 2 , yoshiyuki horio 1 1 department of pharmacology, sapporo medical university, sapporo, japan; 2 department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt1 is still unclear. recent studies indicated that sirt1 interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes1 promoter. we performed overexpression of sirt1 and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt1 and n-cor in the sirt1 with n-cor. sirt1 and n-cor suppressed hes1 transcription by notch-icd in the luciferase assay. hes1 transcription was suppressed in overexpression of sirt1 and n-cor, suggesting that interaction between sirt1 and n-cor represses hes1 transcription. consistent with this, chip assays revealed that not only n-cor but also sirt1 bind to the promoter of hes1 gene. taken together, these results indicate that sirt1 and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes1 promoter site and repressing hes1 transcription. yasushi maruyama 1 , mitsuhiko kurusu 2 , masataka okabe 3 , katsuo furukubo-tokunaga 1 1 grad. school life and envir. sci., univ. tsukuba, japan; 2 natl. inst. genetics, mishima, japan; 3 inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx3 mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx3 regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., 2002) . furthermore, we announced previously that runx3 mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx3 on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx3 knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx3 knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi 1 , hirohide takebayashi 2 , takayuki manabe 1 , kazuhiro ikenaka 2 , akio wanaka 1 1 dept. anatomy, nara med. univ., kashihara, nara, japan; 2 division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng2proteoglycan and olig2 at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig2 promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig2 is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig2 lineage cells in the forebrain is largely unknown. here we examined fates of olig2 expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig2-creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) 12.5 or 14.5, when most of olig2+ cells are observed in the basal forebrain. the olig2+ cells at e12.5 gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig2 lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig2+ cells at e14.5 generated more glial cells than neurons. these results indicate that olig2 lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi 1,2 , masato yano 1,2 , satoru hayashi 1,2 , hirotaka j. okano 1,2 , hideyuki okano 1,2 1 dept. physiol., keio univ. sch. med, tokyo, japan; 2 sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf90 family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p21, p27 and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf90 and nf45, binding partner of nf90, respectively. in mouse embryonic brains, we found that nf45/90 expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf45/90 formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er81 and brn2) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi (16027202) ps2a-d047 protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes 2 days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for 1-4 weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng2 + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng2 is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng2 + cells in the neocortex are the opcs. we investigated about subtypes of ng2 + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng2 + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng2 + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math1 + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp4 and wnt3a, a significant proportion of these neural cells become math1 + . the induced math1 + cells mitotically active and express markers characteristic of granule cells precursors (pax6, zic1, and zipro 1). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math1 + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l7 + /calbindin-d28k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf8 rather than by wnt3a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p21 cip1 , p27 kip1 and p57 kip2 mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p27 kip1 mrna was shown. transfection of p27 kip1 into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e12.5 rat midbrain and hb1.f3 cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p27 kip1 elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g1/s phase did not activate mapk and pi3-k signaling. yuichi tanaka 1 , yusuke tozuka 1 , dai muramatsu 1 , kin-ichi nakashima 2 , tatsuhiro hisatsune 1 1 departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; 2 graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng2+ and olig2+ cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj-1+ or map-2+ neuronal cells by adding retinoic acid or bdnf. more than 90% of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod1 significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi 1 , michiko imanishi 2 , teiko kuroda 2 , masao tachibana 1 , masahiko takada 2 1 department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv3 subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv3 channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv3.1b and kv3.3 were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv3.3 in the frog retina and by kv3.1b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv3.1b. thus, the differential expression of kv3 channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou 1,2 , kazunori yamamoto 1 , masayuki hara 1 , akinori homma 1 , akimichi kaneko 3 , masahiro yamada 1 1 tokyo metropolitan univ., hino, tokyo, japan; 2 astellas pharma. inc., osaka, japan; 3 sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko (2003) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by 5-hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as 0.21 ± 0.05 ph unit by 100 mm k + . acidification of pho was suppressed by 0.4 m bafilomycin a1, a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps2a-e055 analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar 64-channel field potential recording system in slice preparations obtained from 16 to 24 days old mice. stimulation at ssc induced negative field potential with short latency and short duration (100-200 ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing 10 m bicuculline, the same stimulus induced a large negative field response with long duration (200-400 ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of 50 m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya 1 , ken tsutsui 2 , kimiko tsutsui 1 1 dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; 2 dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap-2 binding, and the c-terminal sh3 domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with 3 -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe-19, also expresses amph ir. arpe-19 thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe-19 cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura 1 , satoshi ichisaka 2 , chihiro hayashi 2 , hirotoshi maki 2 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v1) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v1 and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v1 receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi (176959) ps2a-e058 rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut)1 and vglut2, respectively. a complementary distribution of vglut1 and vglut2 was established by postnatal day (p)18 and both signal intensities increased further by p29-30 (peak cp). the immunoreactivity for vglut2 decreased around layer iv after brief monocular deprivation (4dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to 4dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area 17, with a small projection to layer vi of areas 17 and 18a. cells in layer vi of area 17 projected to dlgn. in addition, cells in layer v of area 17 projected to dlgn, which is not observed in normal rats. in area 18a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of 18a. cells in layers v and vi of area 18a projected to lp. smaller number of cells in layer v of area 17 also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps2a-e060 activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida 1 , takemasa satoh 2 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v1) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v1 to the lgn. we focused on the density of type 1 metabotropic glutamate receptor ␣ (mglur1␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur1␣ in the lgn decreased after elimination of v1, showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur1␣ signal in the lgn of young rats. yu morishima 1 , hiroshi sakamoto 2 , takahumi akasaki 1 , yoshio hata 1 1 div. integrative biosci., tottori univ. grad. sch. med. sci., japan; 2 div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, (1) how rapidly the reverse od shift proceeds and (2) whether the shift is induced only in young animals. we infused the cortex with muscimol in 4-week-old kittens and in adults. the reverse od shift was observed after 6 days md, but not significant after 3 days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after 6 days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps2a-e062 experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai 1 , nafiseh atapour 1 , john renger 1 , john roder 2 , peter seeburg 3 , takao hensch 1 1 neuronal circuit dev., riken, bsi, wako, japan; 2 mount sinai hospital, toronto, canada; 3 max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur1, 2) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation (1d md) rapidly alters both the phosphorylation state and surface expression of glur1 in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur1 (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after 4d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as 20d md eventually led to full od shifts. in glur2 ko mice, even 4d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur1 may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx2 promotor and dlx5/6 enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca1 and revealed a nonpyramidal neuron projecting to ca3 and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita 1 , takao oishi 2 , motoharu hayashi 2 1 div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; 2 dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than 60% of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area (5.3%) than in other areas, such as the prefrontal (10.8%), primary motor (9.8%), somatosensory (10.0%) and secondary visual areas (8.5%), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than 80% of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe 1 , guy n. elston 2,3 , tomofumi oga 1 , hiroshi tamura 1,3 , ichiro fujita 1,3 1 osaka univ., japan; 2 univ. queensland, australia; 3 crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v1). pyramidal cells in prefrontal cortex, such as area 12, have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v1 (n = 52), area te (n = 46), and area 12 (n = 43) of a 3-week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area 12, followed by area te, and v1. thus, considerable heterogeneity in pyramidal cell structure already exists 3-weeks after birth. hiroaki matsushita 1 , mahito ohkuma 1 , masami watanabe 2 , ei-ichi miyachi 1 1 department of physiology, fujita health university, aichi, japan; 2 department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura-2 based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the 50% of axotomized rgcs. bath application of 100 m carbachol, an ach receptor agonist, rose [ca 2+ ] i in 22% of normal rgcs. although the 85% of rgcs responded to carbachol at 3 days after axotomy, no responsive rgcs appeared during 7-15 days. ach responsiveness recovered in axonal regenerated rgcs (17%). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi (17500217 to em, 16591780 to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., 2004) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor (75 hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at 30 deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames (107 ms) or only one frame (13 ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps2a-e069 motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima 1,3 , tohru kiryu 2 , kazuhiko ukai 3 , takeshiko bando 1 1 div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; 2 div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; 3 dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. 20 subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura 1 , kenichiro miura 1 , masakatsu taki 1 , hiromitsu tabata 1 , naoko inaba 1 , kenji kawano 1 , frederick a. miles 2 1 grad. schl. med., kyoto univ., kyoto, japan; 2 lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn 2005) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency 3f and 5f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps (100 hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the 5f forwards and the 3f backwards. the contrast of the 5f was fixed at 4, 8, or 16%, while the contrast of the 3f was varied from one-fourth to four times the contrast of the 5f. when the contrast of the 3f (5f) was less than about half that of the 5f (3f), the 5f (3f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < 0.05). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito 1 , yaoxing chen 2 , yukiko tanada 1 1 dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; 2 china agricul. univ., beijing, china twenty white-leghorn chicks (p0-8) were perfused with 1% paraformaldehyde through the heart under deep anesthesia of nembutal (32 mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of 225 rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, 2004). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to 32 in 121 tec-rgcs, 28 in 77 tha-rgcs, and 16 in 38 hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers 1-3). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie 1 , kenji yuda 3 , eiichi okawa 4 , katsuyoshi maruyama 4 , hiroshi uozato 5 , hiroko horie 1 , satomi nakajima 1 , kenkichi tanioka 6 , yuji ohkawa 6 , tomoki matsubara 6 , wolfram tetzlaff 7 1 advanced res. centr. biological sci., waseda univ., tokyo, japan; 2 technomaster co. ltd., yokohama, japan; 3 kikuna yuda eye clinic, yokohama, japan; 4 healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; 5 dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; 6 nhk sci. technical res. labo., tokyo, japan; 7 icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between 25 and 70 cm toward and away in a high speed (top speed: 1000 mm/s) from the subjects. the device significantly improved visual performances in over 60% of the school-aged children with myopia and 80% of adults with presbyopia. hiroyuki miyamoto 1 , toral s. surti 2 , takao k hensch 1 1 laboratory for neuronal circuit development, riken brain science institute, wako, japan; 2 san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v1) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v1. here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad65 ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with 4 day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa 1 , miquel bosch 2 , shuzo sakata 3 , yoshio sakurai 4 , tetsuo yamamori 1 1 division of brain biology, national institute for basic biology, okazaki, japan; 2 mit, ma, usa; 3 rutgers university, nj, usa; 4 department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., 2004) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v2lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v2lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki 1 , youichi miyawaki 2 , tsunehiro takeda 1 1 department of complexity science and engineering, university of tokyo, chiba, japan; 2 mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly (10 ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = 0-990 ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa (10 and 20 ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p1, n170 and area under curve of a late positive potential expanding from 300 to 500 ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse 1 , ayumu matani 1,2 , tomoe hayakawa 2,3 , norio fujimaki 2 1 university of tokyo, kashiwa, japan; 2 nict, kobe, japan; 3 teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around 500 ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of 1000 flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p100 amplitude becomes. this suggested that a factor of the variability of the p100 amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. 16300083) hitoshi sasaki 1 , takuya ishida 1 , masayoshi todorokihara 2 , junichiro miyachi 1 , tahei kitamura 3 , ryozo aoki 1 1 dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; 2 dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; 3 dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama 1 , nobuo usui 1 , izuru nose 1,2 , masato taira 1,3 1 department of applied system neuroscience, nihon university school of medical science, tokyo; 2 faculty of human science, bunkyo university, saitama, japan; 3 arish, nihon university, tokyo, japan when an object is moving, perception of its 3d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps2a-f083 local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a1) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = 830 nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., 1998) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., 1993) . moreover, aaf has dense synaptic connections within the area (budinger et al., 2000) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were 50-ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima 1 , yasuo takeda 2 , yasushi shimoda 1 , kazutada watanabe 1 1 department of bioengineering, nagaoka university of technology, nagaoka, japan; 2 department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb-2 that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb-2 expression is prominent in the auditory system. nb-2 knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb-2 is indispensable for the function of auditory system. here we report the detailed analysis of the nb-2 localization using anti-nb-2 monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb-2 was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb-2 immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb-2 regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation (4 khz octave band, 125 db, 5 h), and then subjected to electrophoretic mobility shift assay to determine activator protein-1 (ap-1) dna binding. ap-1 binding was increased 2-12 h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea 2 h later. taken together, ap-1 may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki 1 department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c57bl/6 mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli (5-26 khz for 500 ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by 5 khz were different from those elicited by 20 or 25 khz. however, the areas activated by 5 khz were also activated by the mixture of 20 plus 25 khz but not by that of 19 plus 26 khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps2a-f092 functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh795 were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by 55-90%, whereas the inhibition of dc resulted in reduction only by 20-50%. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh-795. a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato 1,2 , kazuo okanoya 1 1 laboratory for biolinguistics, riken bsi, wako, saitama; 2 graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, 2000) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented 100-500 times at a fixed interval, through a speaker at 50-70 db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (<10 hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (>100 hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki 1,2,3,4,5,6 1 hokkaido university, sapporo, japan; 2 hokkaido university, sapporo, japan; 3 sakushin gakuin university, utsunomiya, japan; 4 kobe shoin women's university, kobe, japan; 5 riken, wako, japan; 6 sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children (8-12 years old, n = 8) and adults (19-23 years old, n = 12). there were six gap conditions with durations of 8, 16, 32, 64, 128 and 256 ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was 500 hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n1 and p2 complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps2a-f098 an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua 1 , tamaki miyamoto 2 , atsushi terao 2 , sachiko koyama 2 , takashi omori 2 , harumitsu murohashi 2 , shinya kuriki 2 1 jst, saitama, japan; 2 hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba9), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation (12 subjects, p < 0.05 corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi 1 , yuko toyosawa 2 , fumiya takeuchi 3 , michinao matsui 4 , shinya kuriki 1 1 research institute for elecronic science, hokkaido university, sappro, japan; 2 jst, saitama, japan; 3 hokkaido university, school of medicine, japan; 4 kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = 11). four synthesized vowels were used as stimuli (stimulus duration 600 ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used (1200/4800 ms). for the native vowels, an early sf (250-300 ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf (400-600 ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi (16500166) ps2a-f100 spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto 1,2 , junsei horikawa 1 1 department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; 2 riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh 795) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli (5-20 khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for 40 min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe 1 , go ashida 1,2 , kazuo funabiki 2 1 graduate school of informatics, kyoto university, kyoto, japan; 2 hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi 1 , shigenori takebayashi 2 , masahiko bundo 2 , masazumi fujii 3 , toshihiko wakabayashi 3 , jun yoshida 3 , hiroyuki fujisawa 1 , kazunori ando 1 , kazumasa hayasaka 1 1 department of radiology, national hospital for geriatric medicine, obu, japan; 2 department of neurosurgery, national hospital for geriatric medicine, obu, japan; 3 department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using 1.5 tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in 11 out of 12 patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = 8) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, 1987) . surprisingly, subjects tended to misidentify the second toe as the third (47.2%), and the third toe as the fourth (37.2%), while the reverse misidentification rarely occurred (third as second, 4.5%; fourth as third, 7.1%). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps2a-g105 effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi 1,2 , shunjiro moizumi 1 , ayami okuzumi 1 , humine saito 1 , shigeru kitazawa 1,2 1 department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; 2 crest, jst, saitama, japan morrone et al. (2005) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade (24 • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of 100 ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within 300 ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps2a-g106 function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda 1,3 , miki taoka 2,3 1 department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; 2 secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; 3 department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas 3b and 1 of four conscious macaque monkeys was studied by recording single-neuron activities. a total of 115 electrode penetrations were made in areas 3b and 1. in 44 penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining 71 penetrations, however, 14% of adjacent pairs (311/2153) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas 3b and 1 might be responsible for forming composite rfs of area 2 neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi (12771111, 14771029) miki taoka 1 , michio tanaka 1 , hisayuki ojima 1 , atsushi iriki 2 1 secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; 2 laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded 168 neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them (44% contra-, 55% bi-and 1% ipsilateral hand movements), we could determine receptive fields for only 43 neurons (25%). most of activities (138 neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta 1,2 , kouichi nakamura 1 , takeshi kaneko 1 1 department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; 2 crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata 1,2 , shanlin chen 2 , hiroki kitaura 1 , masaharu kudoh 1 , minoru shibata 2 , katsuei shibuki 1 1 department of neurophysiology, brain research institute, niigata university; 2 department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane (1.7 g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation (50 hz for 1 s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi 1 , nanae fukushima 1 , tetsuhiro fukuyama 2 , akira kakegawa 1 , tetsuji moriizumi 1 1 department of anatomy, shinshu university school of medicine, matsumoto, japan; 2 department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima 1 , takeshi kawano 1 , hidekuni takao 1,2,4 , kazuaki sawada 1,2,4 , hidekazu kaneko 3,4 , makoto ishida 1,2,4 1 department electrical & electronic engineering, toyohashi university of technology, aichi, japan; 2 issrc, toyohashi university of technology, aichi, japan; 3 aist, hsbe, ibaraki, japan; 4 jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, 2 m in diameter, 60 m in length; array: 40-m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = 0.91). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi (17206038), the 21st century coe program "intelligent human sensing" ps2a-g113 a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko 1 , hiroshi tamura 2 , shinya s. suzuki 1 , takahiro kawashima 3 1 aist, hsbe, ibaraki, japan; 2 laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; 3 department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over 2 h. seiji matsuda 1 , takehiro terashita 1 , tetsuya shimokawa 1 , kyoujy miyawaki 1 , yuji miguchi 1 , takuya doihara 1 , jie chen 1 , shuang-yan gao 1 , chun-yu li 1 , min wang 1 , zhong wang 1 , bing xue 1 , naoto kobayashi 1,2 , kazuhiro shigemoto 3 1 department anatomy, ehime university of medicine, ehime, japan; 2 medical education c, ehime university of medicine, ehime, japan; 3 department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than 1.2 m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat3, whereas other genes known to be regulated by stat3 were not detected. stat3 is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat3 in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat3 in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat3 and stat3phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat3 are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto 1 , susumu hyodo 2 , makoto kawasaki 1 , minori shibata 1 , takeshi saito 1 , hiroaki fujihara 1 , takashi higuchi 3 , yoshio takei 2 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; 3 department of integrative physiology, university of fukui, japan adrenomedullin 2 (am2) (identical to intermedin) belongs to the super family of am. centrally administered am and am2 activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am2 on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am2 was stronger than those of am and the effects of am2 on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am2 would have unknown receptor except cgrp and am receptor. arata oh-nishi 1 , makoto saji 1 , taku uchida 1 , sen-ichi furudate 2 , nobuyuki suzuki 1 1 division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; 2 division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr1 expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, 2004) . here we report that the density and morphology of spines of pyramidal neurons in ca1 region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for 2 h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca1, both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day 8-15, the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day 15. the staining of somatostain mrna in individual neurons was diminished when examined on day 35 or 70, albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps2a-g121 short chain sugar acid, 2-buten-4-olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki 1 , tatsushi onaka 2 , hirofumi hashimoto 1 , hiroaki fujihara 1 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 department of physiology, jichi medical school, tochigi, japan 2-buten-4-olide (2-b4o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of 2-b4o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at 15-60 min after intraperitoneal (i.p.) administration of 2-b4o (100 mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei 120 min after i.p. administration of 2-b4o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of 2-b4o. these results suggest that peripherally administered high dose of 2-b4o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps2a-g122 estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within 4 h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto 1,2 , shinpei higo 1,2 , yasushi hojo 1,2 , kouhei nakajima 1,2 , hironori nakanishi 1,2 , hirotaka ishii 1,2 , suguru kawato 1,2 1 department of biophysics & life science, university of tokyo, tokyo, japan; 2 crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps2a-g125 gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from −40 to -10 mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than -50 mv and most of them were ∼−80 mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr1, galr2 and galr3, in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr1 selective agonist m617 induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr1. hiroaki fujihara 1 , tomoki fujio 1 , david murphy 2 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking 2% saline for 5 days in 3, 6, 12 and 24 months old tg rats. in 3 and 6 months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats (12 and 24 months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps2a-h128 prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera 1 , hiroaki fujihara 2 , hirofumi hashimoto 2 , makoto kawasaki 2 , tatsushi onaka 3 , takakazu oka 1 , sadatoshi tsuji 1 , yoichi ueta 2 1 department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; 2 department of physiology, school of medicine, university of occupational and environmental health, japan; 3 department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin 1 , afsaneh eliassi 1 , nasser naghdi 2 , ali ghanbari 1 1 ghsemi; 2 pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d1 receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above (0.5 mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from 30 min up to the end of experiments (90 min). at the peak acid response, the vmh microinjection skf38393 (0.1,1) significantly reduced the amount of gas (p < 0.001). there was no any effect by microinjected sch23390 (0.1) into the vmh. injecting skf38393 into the vmh, 5 min after sch23390, had no effect on gas in compare with control. but, the acid suppressant effect of skf38393 was completely removed by peripheral injection of sch23390 (p < 0.01). our results show that the vmh d1 dopamine receptors have regulatory mechanisms of gas by interaction with h2 receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p 2x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p 2y purinoceptors (p 2y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p 2y r agonists increased [ca ++ ] i in son, with p 2y1 r agonist being the most effective. the possibility that p 2y1 r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p 2y1 r antagonist, mrs2179. atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs2179 (100 m) in 2 mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs2179. furthermore, combined treatment with mrs2179 and ppads (10 m, a p 2x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p 2y1 r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps2a-h131 analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii 1,2 , yasuhiro sonoki 1,2 , aizo furukawa 2,3 , yasushi hojo 2 , tetsuya kimoto 1,2 , suguru kawato 1,2 1 department of biophysics and life science, university of tokyo, tokyo, japan; 2 crest, jst, japan; 3 kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day 1(pd1) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd1 to pd14 with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai 1,2 , gen murakami 1 , shirou kominami 3 , john h morrison 4 , william g.m janssen 4 , tetsuya kimoto 1,2 , suguru kawato 1,2 1 department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo 153-8902, japan; 2 crest project, jst, japan; 3 faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima 739, japan; 4 kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc-19, mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca1, ca3, and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud 1 , song t. yao 1 , jing qiu 1 , julian fr paton 2 , david murphy 1 1 university of bristol, hw-line, uk; 2 department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic (2d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from 3 days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase 1, has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt1-7. delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt1-7 cells cultured in dmem supplemented with 10% fbs for 3 days. bk current was increased by addition of 17␤-estradiol (e2) in culture medium in a physiological concentration range. this action of e2 was blocked by ici-182,780, a potent estrogen receptor (er) antagonist. we further examined whether e2 acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e2 but ppt had no effect. e2 had no effect on the i k . these results indicate that e2 increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi 16590180, 16086210 ps2a-h136 myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi 1 , haruko kumanogoh 2 , shun nakamura 2 , seiji miyata 3 , shohei maekawa 1 1 department of biosystems science, kobe-university, kobe, japan; 2 division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; 3 department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, 25 kda protein, was identified as myelin protein zero (p0) from mass analysis and this result was confirmed by western blotting that a specific antibody to 25 kda band reacted to an authentic p0 prepared from rat sciatic nerve myelin. p0 is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p0 has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p0 expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan 17␤-estradiol (e2) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e2 (hojo et al., 2004) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e2 and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca1, ca3 and dg with a custom made multi-electrode measuring system (med64). ltd was enhanced by e2 dose-dependently in ca1, ca3 and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito 1 , takushi x. watanabe 2 , tomoko urabe 2 , hirofumi hashimoto 1 , hiroaki fujihara 1 , yukio hirata 3 , yoichi ueta 1 1 department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; 2 peptide institute, inc., osaka, japan; 3 department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for 5 days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto 1 , hao-gang xue 1 , yuji ishikawa 2 , yoshitaka oka 3 , hitoshi ozawa 1 1 department of anatomy and neurobiology, nippon medical school, tokyo, japan; 2 national institute of radiological sciences, chiba, japan; 3 department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki 1 , eiji munetsuna 1 , asuka kamogawa 1 , suguru kawato 2 , shiro kominami 1 1 graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; 2 graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p450arom in the hippocampus. method: the cultured hippocampus slices from 10 days male rat were treated with 100-1000 nm of tbt for 48 h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p450arom was determined by quantification of [3h]estradiol from [3h]testosterone. result: forty-eight hours treatment of hippocampal slices with 100 nm tbt induced increases in mrna contents of p450arom, and with 1000 nm tbt induced that of 3␤-hsd. estradiol content was increased by the treatment with 100 nm tbt, but not affected by 1000 nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p450arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for 4 h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of 30, 60, 90 and 120 ng in 200 nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent 4 h. the 30 ng dose of bicuculline methoiodide had minimum effect whereas 60 and 90 ng produced arousal. maximal wakefulness was observed at dose of 90 ng with no further increase in wakefulness at higher dose of 120 ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe 1 , hiroyuki tsuda 2 1 department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; 2 department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee1). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., 200 mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in 24 h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, (1 antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at 1 h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at 1 h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via 1-opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d 2 is a potent somnogenic substance, and isomerized from pgh 2 through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl 4 into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl 4 decreased rapid eye movement (rem) and non-rem sleep for 5 h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl 4 administrations. these findings indicate that pgd 2 plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd 2 involved in sleep-wake regulation. under baseline conditions, h 1 r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (<16 s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h 1 r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h 1 r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., 2006) . ps2a-h147 dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = 500 nm). the scn neurons responded to the light in intensities above 10 11 photons/cm 2 /s and were activated maximally at around 10 15 photons/cm 2 /s, they were about 1.6 log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca 2+ in scn neurons. since cytosolic ca 2+ regulates diverse cellular systems, we have hypothesized that the cytosolic ca 2+ rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca 2+ rhythms, mouse bmal1 and its dominant negative sequence (mbmf1r5) are transfected into the organotypic culture of scn with a yellow cameleon ca 2+ sensor by the gene gun. the results demonstrated that over-expression of bmal1 or mbmf1r5 significantly inhibited the circadian ca 2+ rhythms and thus we concluded that the native bmal1 rhythm is essential for cellular output processes of the murine clock system. ps2a-h149 the activation of ␣2 adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca3 region of rat hippocampal slices. carbachol (30 m) induced ␤ oscillation with the frequency and the amplitude of 14.8 ± 0.1 hz, and 0.7 ± 0.1 mv, respectively (mean ± s.e.m.; n = 3). epinephrine (50 m) significantly increased the frequency of 16.2 ± 0.1 hz (**p < 0.01), not change the amplitude. clonidine (50 m), an ␣2 receptor agonist, alone significantly increased the frequency at the concentration of 50 m (*p < 0.05). yohimbine, an ␣2 receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣2 receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart 0.4 mm from each other in the hippocampal ca3 region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency 100-300 ms), oscillatory (2-4 hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties (8/9 slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca3 region. ryozo aoki 1 , hiroshi wake 2 , hitoshi sasaki 1 , kiyokazu agata 3 1 dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; 2 dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; 3 dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as 1/f, ranging from 10 − 1 to 10 + 2 hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond 40 min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to 60 min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over 30 min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida 1 , hiroki sato 1,2 , naoki tanaka 2 , atsushi maki 1,2 1 japan science and technology agency, crest, saitama, japan; 2 advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from 8 to 12 hz) decreases and the sleep spindle power (from 12 to 16 hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from 1 to 8 s in (i), and from 8 to 15 s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim 1 , kazuki nakajima 1 , yutaka oomura 2 , kazuo sasaki 1 1 div. of bio-information eng., univ. of toyama, toyama, japan; 2 dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca 2+ exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng 1 , wei zhang 1 , akira nakamura 2 , masashi yanagisawa 3 , yasuichiro fukuda 2 , tomoyuki kuwaki 1,2 1 dept. molec. integ. physiol., chiba univ., japan; 2 dept. autonom. physiol., chiba univ., japan; 3 dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb-334867 (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb-334867. icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co2-sensitivity at least during waking periods. research funds: kakenhi 16590162,17590183 junko hara 1 , taizo matsuki 2 , katsutoshi goto 1 , masashi yanagisawa 3 , takeshi sakurai 1,2 1 department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; 2 yanagisawa orphan receptor project, erato, jst, tokyo, japan; 3 howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps2a-i157 prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto 1 , kazuhiko kubo 2 , shuji aou 1 1 dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; 2 dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. 2006) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. 16209006, s.a.) shuji aou 1 , tetsuya fujimoto 1 , yumi ichihara 1 , kimiya narikiyo 1 , toru ishidao 2 , hajime hori 2 , yukiko fueta 2 1 dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; 2 dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan 1-bromopropane (1-bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to 1-bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to 700 ppm of 1-bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. 1-bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that 1-bp is the potential candidate of endocrine disruptors which affect brain development. (16651027) ps2a-i159 changes in hippocampal excitability of rats prenatally exposed to 1-bromopropane yukiko fueta 1 , toru ishidao 1 , susumu ueno 2 , yasuhiro yoshida 3 , hajime hori 1 1 department of environmental management, school of health sciences; 2 department of pharmacology; 3 department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to 1-bromopropane (1-bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to 1-bp (700 ppm) during gestational day 1-20 (6 h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca1 at the pnd 12-15. conversely, s/r curves of fepsp as well as ps in the ca1 were inhibited at the age of 6-8 weeks. our results suggest that 1-bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca1 of the offspring during development. research funds: grant-in-aid for exploratory research (16651027) ps2a-i160 effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura 1,2 , kyoko itoh 1 , takeshi yaoi 1 , tohru sugimoto 2 , shinji fushiki 1 1 dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; 2 dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously 20 g/kg of bpa daily from embryonic day 0 (e0). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e14.5 and e16.5, whereas those cells increased in the cortical plate at e14.5, as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant 15390334 keiko ikemoto 1 , teruko uwano 2 , hisao nishijo 2 , taketoshi ono 2 , masayuki ito 3 , ikuko nagatsu 4 , katsuji nishi 5 , shin-ichi niwa 1 1 dept. neuropsychiat., fukushima med. univ. sch. med.; 2 toyama med. pharm. univ.; 3 faculty med., mie univ., mie, japan; 4 fujita health univ. sch. med., toyoake, japan; 5 dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day 9 and 19 after fertilization. pups were perfused at postnatal day 8. in the frontal cortex, the number of largesized (more than 7 m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c1/a1 catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps2a-i162 developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi 1,2,3 , kaori morohoshi 3,4 , rie yanagisawa 3 , erina saita 5 , gen watanabe 6,7 , masatoshi morita 3 , kazuyoshi taya 6,7 , hirohisa takano 3,4 , toshiyuki himi 1,2 , hideki imai 3,8 1 dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; 2 res. inst. pharmaceut. sci., musashino univ., tokyo, japan; 3 nation. inst. for environ. stud., ibaraki, japan; 4 grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; 5 wildlife rescue veterinarian associ., tokyo, japan; 6 facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; 7 the united grad. sch. veterinary sci., gifu univ., gifu, japan; 8 div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps2a-i163 the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic (5-ht) systems in stressinduced anxiety, we measured brain da and 5-ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of 5-ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for 60 min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and 5-ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although 5-ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl1-4) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl3 and nl4 in autistic disorders. nls have a relative identical structure (∼70%); nl1 and nl2 localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl3 seems express in the olfactory glia, but nl4 distribution is unknown. here we have generated antibody against human nl4, and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl4 was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl4 was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl4 is important for neuroendocrine function. nl4 cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl4 were rough and thicker than those of nl1 or nl3 transformants. it suggested the unique activity of nl4 for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca1 pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting 1-3% of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below 50. here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura 1 , daisuke horiuchi 1 , tomoyuki miyashita 2 , minoru saitoe 2 , hitoshi okazawa 1 1 department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; 2 tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein-1 (pqbp-1) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp-1 disease that pqbp-1 mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp-1 in xlmr pathology, we produced two kinds of flies, human pqbp-1 overexpression flies (hpqbp1 flies) and drosophila pqbp-1 knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp1 flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp-1 in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun 1 , sentaro takahashi 1 , yoshihisa kubota 1 , rui zhang 2 , chun cui 2 , yoshihiro fukui 2 1 natl. inst. radiol. sci., chiba, japan; 2 sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr1 subunits paired with two nr2 subunits. nr1 and nr2 (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr1/nr2a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr1/nr2b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca1 pyramidal neurons from mice (at 12 weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr2b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca1 region after ischemia. in blood, corticosterone levels increased biphasically at 30 min and 12 h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca1 region. by 3 days after ischemia, mr and gr were not significantly altered in the ca1 region. from 4 days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca1 region, and at 7 days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase-3 and midkine in the mca area. stroke was induced by a 90-min mca occlusion using an intraluminal filament. rats were exercised for 20 min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase-3 expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps2a-j174 reversed astrocytic glutamate transporter glt-1 crucial to the ca 2+ paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca 2+ paradox" is the phenomenon whereby the intracellular concentration of ca 2+ paradoxically increases during reperfusion with normal ca 2+ -containing media after brief exposure to a low ca 2+ solution. the present study aims to characterize the ca 2+ paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca 2+ solution for 60 min significantly injured only neurons after reperfusion with a normal ca 2+ medium for 24 h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt-1. the present findings suggested that ca 2+ paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt-1 during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch3 concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch3 were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c577t, which results in pro167ser. the second patient carried the missense mutation c302g, which results in arg75pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch3 receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps2a-j177 mci -186, a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3kinase madinyet niyaz 1 , tadahiro numakawa 2 , yoshinori matsuki 1 , emi kumamaru 2 , yuki yagazaki 2 , harumi kitazawa 1 , hiroshi kunugi 2 , motoshige kudo 1 1 pathology department of tokyo medical university, tokyo, japan; 2 department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci -186, a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci -186 prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci -186 exposure induced the activation of both the map kinase (mapk) and pi3 kinase (pi3k) pathways and that the mci -186-dependent survival effect was blocked by the inhibitors, u0126 (an mapk pathway inhibitor) or ly294002 (a pi3k pathway inhibitor). these results suggested that mci -186 exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru 1 , noriko yoshimura 1 , tetsuro sakamoto 1 , takaaki kitano 2 , naoko nisimaru 1 , isao yokoi 1 1 dept. physiol., sch. med., oita univ., japan; 2 med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by 31 p-nuclear magnetic resonance (25 • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with 2 mm ep (ep-1), acsf replaced by acsf with 2 mm ep after ischemia (ep-2), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for 1 h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep-1 than in control (p < 0.01). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) #17591639 from mext to t.k. hideaki tamai 1 , kuniko shimazaki 2 , norimasa seo 1 1 department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; 2 department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc6), yintang (ex-hn3) and sanyinjiao (sp6), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through 5-bromo-2,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc6, ex-hn3 and sp6. the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago 1 , keiko takahashi 1 , shigeo nakamura 1 , akemichi baba 2 , toshio matsuda 1 1 laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; 2 laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara 1 , tatsuya mishima 1 , takefumi kofuji 2 , kimio akagawa 1 1 department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; 2 radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc-1/syntaxin 1a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc-1/syntaxin1a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc-1/syntaxin1a knock-out mouse will be discussed. research funds: kakenhi (15700292) ps2a-k182 effects of local administration of the gaba agonists into the hippocampus ca1 area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka 1 , hiroko miyakubo 1 , junichi tanaka 1 , yasushi hayashi 2 , yukio hattori 2 , masahiko nomura 3 1 department of curriculum, teaching and memory, naruto university of education, tokushima, japan; 2 department of human nutrition, notre dame seishin university, okayama, japan; 3 department of physiology, saitama medical school, saitama, japan in fischer 344 male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca1 area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite 5-hydroxytryptamine (5-hiaa) concentration in the ca1 area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca1 area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad-67. fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka 1 , sho koyasu 1 , akinori nishi 2 , tomoyuki furuyashiki 1 , toshiyuki matsuoka 1 , shuh narumiya 1 1 department of pharmacology, university of kyoto, kyoto, japan; 2 department of physiology, university of kurume, kurume, japan prostaglandins e2 (pge 2 ) exert their actions in various organs through specific receptor, ep1 to 4. the previous study suggests that ep1 modulates da system. to investigate the roles of ep1 in da system, we examined ep1ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼300% in both wild-type and ep1ko mice. however, increase of locomotor activity in ep1ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d1 receptor (d1r) signaling, we tested the density of d1r and d1r signaling with phosphorylation of darpp-32. there were no differences in d1r binding. d1r signaling was significantly attenuated in the striatal slices from ep1ko mice. the effect of d1r agonist on locomotor activity was also attenuated in ep1ko mice. these results indicate that pge 2 has enhancing effects on locomotor activity via ep1 by potentiating the d1r signaling. central serotonin (5-ht) function has been implicated in impulsivity. the present study examined rats with 5-ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of 5-ht and its metabolites (5-hiaa) in the brain. for both tasks, significant negative correlations between learning performance and 5-ht and 5-hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and 5-ht and 5-hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of 5-ht roles on selective indices of impulsivity. yuki sato 1,2,3 , tatsushi onaka 2 , norimasa seo 1 , eiji kobayashi 3 1 dept. anesthesiol., jichi med. univ., tochigi, japan; 2 dept. physiol., jichi med. univ., tochigi, japan; 3 div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p1-21; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p4 in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p7; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p11. within the vestibular nucleus of p7-9 rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p21 and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p1 to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p3 in the vestibular nucleus as fuzziness about neuronal cell bodies. from p9 onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p12. during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p21. with ul, the pn network on the lesioned side remained compact until 5 days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by 11 days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by 14 days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca1 region of rat hippocampal slices, 100 hz/20 pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at 100 hz. ikuko yao 1 , hiroshi takagi 1 , hiroshi ageta 1 , tomoaki kahyo 1 , ken hatanaka 1 , kaoru inokuchi 1 , mitsutoshi setou 1,2 1 mitsubishi kagaku institute of life sciences, tokyo, japan; 2 university of tokyo, tokyo, japan; 3 okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps2p-a003 presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., 10 mv, 1 s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca 2+ currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca 2+ channel knockout mice. we conclude that the p/q-type specific ca 2+ channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi 1 , koji ikegami 1 , ken hatanaka 1,2,3 , yoko fujiwara-tsukamoto 1 , mineo matsumoto 1 , ikuko yao 1 , mitsutoshi setou 1,2,4 1 mitsubishi kagaku institute of life sciences, japan; 2 presto, japan; 3 school of pharmaceutical sciences, the university of tokyo, japan; 4 okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa22(−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa22 mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki 1 , shingo kimura 1 , reiko fujita 2 , shuji watanabe 1 , kazuhiko sasaki 1 1 dept. of physiol., sch. of med., iwate medical univ., morioka, japan; 2 dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c3, a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p50rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦2 glutamate receptor (␦2r) plays a crucial role in cerebellar functions. although ␦2r has a putative channel pore domain, and ␦2r displayed ca 2+ -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦2r functions as a channel. here we introduced a ␦2r transgene, which had a mutation (gln618arg) in the putative channel pore conserved in ca 2+ -permeable glutamate receptors, into ␦2 −/− mice. surprisingly, a mutant ␦2r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦2 −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca 2+ influx through ␦2r is not required for its function in the cerebellum in vivo, and that wild-type ␦2r may not function as a ca 2+ -permeable ion channel. research funds: kakenhi (17700316) and takeda science foundation ps2p-a007 distribution of tarp -8 on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya 1 , mika tsujita 2 , maya yamazaki 2 , etsuko kushiya 2 , manabu abe 2 , kaori akashi 2 , masanobu kano 3 , haruyuki kamiya 4 , kenji sakimura 2 , masahiko watanabe 1 1 department of anatomy, hokkaido university school of medicine, sapporo, japan; 2 department of cellular neurobiology, brain research institute, niigata, japan; 3 department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; 4 department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the -8 is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized -8 on excitatory synaptic and extrasynaptic membrane. in -8-ko mice, ampars were reduced in hippocampal homogenates (46% of control) and psd fraction (35%). immunogold labeling also exhibited reduction of extrasynpatic (47%) and synaptic (35%) ampars in ca1 pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites (36%) and spines (38%). ampar-mediated responses were reduced at ca1 synapses (52%). therefore, -8 is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa 1 , yugo fukazawa 1 , elek molnar 2 , masahiko watanabe 3 , ryuichi shigemoto 1,4 1 div. cerebral structure, nips, okazaki, japan; 2 mrc, univ. of bristol, bristol, uk; 3 hokkaido univ., sapporo, japan; 4 sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed 1.7 to 3 times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed 1.6 to 3 times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda 1,2,3 , hitoshi kita 1 1 dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; 2 japan society for promotion of science, tokyo, japan; 3 dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp55845 abolished the sipsp. an mglur1 antagonist, but not an mglur5 antagonist, partially blocked the sdepo. in addition, cgp55845 enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur1-mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps2p-a010 essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto 1 , yoshikazu isomura 1,2 , michiko imanishi 1 , tomoki fukai 2 , masahiko takada 1 1 system neurosci., tokyo met. inst. neurosci., tokyo, japan; 2 neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for >20 s after the induction. gaba amediated cl-influx lasted for 1.5 s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura 1 , yuko sekino 1,2 , toshiya manabe 1,2 1 division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; 2 crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < 30), using electrophysiological methods and optical imaging. in immature animals (p10), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p5) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p2-18). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p14-16 compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca1 area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣1, ␣2, and ␤2/3 subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣1, ␣2, and ␤2/3 subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ 1 -or ␣ 2 -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ 1 -adrenoceptor agonist phenylephrine, while reduced by the ␣ 2 -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ 1 -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ 2 -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ 1 -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ 2 -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi (16700344) ps2p-b015 involvement of basal pkc and erk 1/2 activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa 1 , takahiko chimura 2 , azumi matsumoto 2 , kazuhiko yamaguchi 2 1 lab. for motor learning control, riken, bsi, japan; 2 lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk1/2 activities at pf-pc synapses. taisuke miyazaki 1 , kohichi tanaka 2 , masahiko watanabe 1 1 department of anatomy, school of medicine, hokkaido university, sapporo, japan; 2 department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki 1,2 , kouichi hashimoto 2 , taisuke miyazaki 1 , masahiko watanabe 1 , masanobu kano 2 1 dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; 2 dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto 1,2 , takashi kodama 1,2 , ryuichi shigemoto 2,3,4 , yugo fukazawa 2,3 , yasuo mori 5 1 division of neural signaling, nips, okazaki, japan; 2 school of life science, graduate university for advanced studies (sokendai), okazaki, japan; 3 division of cerebral structure, nips, okazaki, japan; 4 sorst, japan science and technology agency, kawaguchi, japan; 5 department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v 2.1 (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v 2.1 mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v 2.1 exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n1) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n2) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n3 component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps2p-b020 target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from 0.4 ± 0.2 to 1.1 ± 0.2 (n = 6; mean ± s.d.) by reducing [ca 2+ ] o from 2 to 0.5 mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps2p-b021 response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at 1/15 s, and were depressed completely by cnqx. when stimulus rate was increased to 1/s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at 1/s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at 1/15 s, but not at 1/s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at 1/sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono 1 , keisuke shiba 2 , ken nakazawa 3 , ichiro shimoyama 4 1 department of otolaryngology, chiba university, chiba, japan; 2 department of otolaryngology, kimitsu central hospital, chiba, japan; 3 department of integrative neurophysiology, chiba university, chiba, japan; 4 section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps2p-b023 in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada 1,2 , eiji shigetomi 1,3 , fusao kato 1 1 lab. neurophysiol., jikei univ. sch. med., tokyo, japan; 2 dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; 3 jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi (17650116, 176865) ps2p-b024 laminar sources of synaptic input to layer 1 neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer 1 of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer 1 neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer 2/3. neurogliaform cells receive more excitatory inputs from layers 4 and 5. the dominant inhibitory synaptic inputs onto both types of cells originate from layer 2/3. these neurons also get inhibitory inputs from layers 1 and 4-6. the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer 1 are highly integrative. takako nishi 1 , tsukasa gotow 2 , shiro nakagawa 2 1 ins. nat. sci., senshu univ., kawasaki, japan; 2 dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p-1, es-1, ip-1 and ip-2 which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p-1/es-1 of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip-1/ip-2. the present study was conducted to examine the functional significance of the above ip-1/ip-2, having a peak sensitivity at 510 nm light. the body wall of the animal transmitted enough 510 nm to hyperpolarize ip-1/ip-2 in the ganglion covered by the body wall. ip-1/ip-2 were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip-1/ip-2 were not coupled to a-p-1/es-1 one another. finally, ip-1/ip-2, the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps2p-c026 cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the 7-day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e7. it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e7-e8), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada 1 , nyberg tobias 1 , nahoko kasai 1 , yuriko furukawa 2 , keiichi torimitsu 1,2 , kunihiko obata 3 , yuchio yanagawa 2,4 , tadaharu tsumoto 2,3 1 ntt basic research laboratories, ntt corporation, kanagawa, japan; 2 sorst, jst, saitama, japan; 3 neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; 4 department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase 67-green fluorescence protein (gad67-gfp) knock-in mice at postnatal day 0-1 were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first 5 days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least 14 div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps2p-c030 pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda 1 , toshiyuki ishii 2,4 , yasuhide shigematsu 3 , toshihiko hosoya 2 , yukio shimoda 3 1 dept. physiol., keio univ. sch. med., tokyo, japan; 2 bsi, riken, wako, japan; 3 mri, tokyo womens' medical univ., tokyo, japan; 4 dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p2x2-purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p2x2-purinoceptors. furthermore, a p2x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p2x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca 2+ . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca 2+ oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of 2mesatp was observed. the inhibition of 2mesatp response by p2y1 receptor specific antagonist mrs 2179 indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca 2+ oscillations. data will be presented on the p2 receptor mediated regulation of synchronized ca 2+ oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d1-like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf83959 was shown to selectively stimulate plc-linked d1-like dopamine receptors. darpp-32 is a phosphoprotein that regulates the efficacy of d1 receptor signaling. activation of d1 receptors that couple to golf/pka signaling leads to darpp-32 phosphorylation at thr34. we investigated the effect of skf83959 on darpp-32 phosphorylation at thr34 by using mouse neostriatal slices. treatment of neostriatal slices with skf83959 increased thr34 phosphorylation. the effect of skf83959 was enhanced by a plc inhibitor, u73122. in contrast, the effect of skf83959 was partially blocked by a d1 receptor antagonist, sch23390, and the sch23390-insensitive increase in thr34 phosphorylation was completely abolished by an adenosine a 2a receptor antagonist, zm241385. thus, analysis of darpp-32 phosphorylation revealed that skf83959 activates at least three signaling cascades in the neostriatum: (1) plc, (2) d1 receptor/golf/pka, (3) adenosine a 2a receptors. ps2p-c033 thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c57bl6 mouse were dyed with a voltage-sensitive dye rh414. in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal (200 mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca1 stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx (20 mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no.15390471 ps2p-c034 brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip3r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur1 localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur1 at psd within 20 min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca 2+ transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca 2+ rise, that is, ca 2+ release from ip3r and ca 2+ influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur1 at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca 2+ signaling regulation. research funds: health sciences research grant of nano-1 hiroyuki konishi 1 , kazuhiko namikawa 1,2 , keiji shikata 3 , yuji kobatake 1 , taro tachibana 3 , hiroshi kiyama 1 1 dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; 2 dept. anat., asahikawa med. col., hokkaido, japan; 3 dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc12 cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t1, is involved in the control of cell morphology via the regulation of rho proteins via rho gdi1. however, it is unclear whether t1 regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c6 rat glioma cells, which express t1 endogenously. rho gdi1 was dissociated from t1 in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t1-specific sequences reduced the effects of bdnf. these results suggest that t1 regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d1, bcl2. the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf-1 in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind1 in response to igf-1 both at protein & rna levels but no change was observed in c-myc, bcl2 levels. interestingly, mapk-erk, which is also known to activate cd1 in other systems, had no effect on the cd1 level in neuronal cells. on the other hand, pi3kinase not only activated jak/stat pathway but also affected on cyclind1 level. thus it appears that jak/stat is activated by pi3k, which in turn upregulates cyclind1 level. pi3k also activated erk without changing cd1 level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi3k. cd1 is a one of the major downstream targets of jak/stat. hiroko inoue 1 , haruhisa kawasaki 2 , ritsuko fujii 2 , tohru yoshioka 2 , kazunori sango 3 , toshihiko kadoya 4 , hidenori horie 2 1 sch. of sci. and eng., waseda univ., tokyo, japan; 2 advanced res. center for biol. sci., waseda univ., tokyo, japan; 3 tokyo metropolitan inst. for neurosci., tokyo, japan; 4 pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin-1 (gal-1/ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal-1/ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal-1/ox enhanced mrna expression of nerve regeneration-related factors such as il-6, il-1 ␤. however, those of gdnf, ngf, bdnf, nt-3, nt-4, igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal-1/ox. since il -6, il-1, and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal-1/ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca 2+ concentration, ␣camkii is activated by the interaction with ca 2+ /cam. prolonged increase in ca 2+ level leads to further activation of ␣camkii through autophosphorylation at t286. to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca 2+ level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy5y cells, the increase in ca 2+ level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦3 isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦1-4 antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦3 isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps2p-d041 the role of ca 2+ /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata 1,2 , hiroyuki sakagami 3 , keiji imoto 1,2 , kunihiko obata 4 , yuchio yanagawa 5 1 okazaki, japan; 2 sokendai, okazaki, japan; 3 tohoku univ., sendai, japan; 4 riken, wako, japan; 5 gunma univ., maebashi, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k42 with r42 to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , #17500218) ps2p-d042 characterization of ca 2+ -dependent membrane binding proteins of rat brain shohei maekawa 1 , katsutoshi taguchi 1 , haruko kumanogoh 2 , shun nakamura 2 1 division of biosystems science, grad. sch. of sci/tech, kobe-u kobe 657-8501, japan; 2 division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo 187-0031, japan ca 2+ -dependent membrane binding proteins were purified through a ca 2+ -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca 2+ -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps2p-d043 generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa251∼322) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa238∼402). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal4-dbd with mutants of crest; crest c ( 238∼402) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal4 one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps2p-d044 characterization of iq-arfgef, a novel exchange factor for arf6, in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor 6 (arf6) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf6. in this study, we determined the entire sequence of a partially identified cdna (mkiaa0522) encoding a novel sec 7 domain. the deduced protein contained coiled coil, iq, sec7, plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf6 more selectively than arf1. thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf6 and interaction with various pdz proteins. eriko miura 1 , masahiro fukaya 1 , tokiharu sato 2 , kazushi sugihara 3 , masahide asano 3 , katsuji yoshioka 2 , masahiko watanabe 1 1 dept. anat., hokkaido univ. sch. med., sapporo, japan; 2 dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; 3 adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap1 (or jip3) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap1 mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk1∼3 mrnas. in embryos, jsap1 immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap1 was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori 1 , shigeo uchino 2 , tomoko isosaka 1,3 , shinichi kohsaka 2 , takeshi yagi 3 , shigeki yuasa 1 1 dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; 2 dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; 3 kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr2b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr2b phosphorylation and the nmda-induced calcium responses increased after dopamine d 2 -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d 2 -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr2b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp1: dystrobrevin binding protein 1) at 6p22.3. however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi 1 , naoko oda 2 , ryuzo shingai 1,2 1 21st coe program, iwate university, morioka, japan; 2 department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at 10 • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa 1 , masaki nomura 2,3 , toshio aoyagi 2 , tomoki fukai 1 1 riken brain science institute, saitama, japan; 2 graduate school of informatics, kyoto university, kyoto, japan; 3 crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused 7 days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki 1,2 , tatsumi hirata 1 1 division of brain function, national institute of genetics, sok-endai, mishima, japan; 2 prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps2p-d053 an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc-12 cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama 1 , masahiro matsuura 2 , nobuhiko yamamoto 1 1 grad. sch. of frontier biosci., osaka univ., suita japan; 2 riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer 4. to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a5, sema7a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a5, sema7a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi 17023030, kakenhi 15300107 ps2p-e055 expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami 1 , katsuhiko ono 2 1 dept. anat., juntendo univ. sch. of med., tokyo, japan; 2 div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin-1 expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin-1 receptor neogenin. slit-1 was expressed in the forebrain, but its receptor robo-1 was not expressed in gnrh neurons. semaphorin (sema) 3a expression was detected in the olfactory-forebrain region. within the forebrain, sema3a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema3a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema3a receptor neuropilin-1, suggesting that sema3a may act as a repellent during the migration of gnrh neurons. kazuto fujishima 1,2 , qing-hua jin 1 , junko kurisu 1 , tomoo hirano 2 , mineko kengaku 1 1 riken, bsi, lab. for neural cell polarity, saitama, japan; 2 department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., 2005) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer2/vldlr induces dab1 phosphorylation, dab1 is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp2 suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab1. reelin treatment of the neurons derived from dab1 deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab1 signal pathways. hidenobu mizuno 1,2 , tomoo hirano 1,2 , yoshiaki tagawa 1,2 1 dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; 2 crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area 17/18 border of the other hemisphere, where they terminate in layers 2/3 and 5. we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the 17/18 border and started to invade the cortical plate around p5. they generated exuberant axonal arborizations by p13 then underwent remodeling to form the adult pattern by p15. neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir2.1 in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers 2/3. area-specific targeting to the 17/18 border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps2p-e060 identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp22like protein (nplp) that has a distant homology to pmp22/claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp22 resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc12 cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata 1 , shin-ichi sakakibara 2 , hirotaka j okano 1 , hideyuki okano 1,3 1 dept. physiol., keio univ., sch. med., japan; 2 dept. histol. neurobiol., dokkyo med. univ., japan ; 3 crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi1 and msi2, which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi1 and msi2 deficient mice. msi1−/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi2−/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi1−/− msi2−/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu 1 , satoshi yamagoe 2 , kazuo suzuki 2 , michiko ohtomi 1 1 department of biomolecular science, graduate school of science, toho university, chiba, japan; 2 department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect2 (leukocyte cell-derived chemotaxin 2) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect2 and tau or map2 in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect2 in neurons. at the early stages in cultured neurons, lect2 was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect2 was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect2 influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect2 may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda 1,2 , takahiro moriya 1 , hideyuki terazono 1 , suguru kudo 5 , masahiro yamaguchi 3 , kenji yasuda 4 , izumi nagata 2 , kazuyuki shinohara 1 1 div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; 2 dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; 3 dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; 4 dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; 5 special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e12.5 nestin-promoter gfp transgenic mice were placed into 32 pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng108 cells were co-cultured with vascular smooth muscle cells (sm-3). sm-3 cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin-1 mrna was specifically up regulated in ng108 cells. but the western blotting analysis did not show the significant increase of neuropilin-1. sema3a, a candidate of ligands for neuropilin-1, was found in ng108 cells rather than in sm-3 cells. the immunocytochemical analysis revealed that neuropilin-1 was increased in the surface of ng108 cells co-cultured with sm-3 cells or cultured with the conditioned medium of sm-3 cells. these results suggest that neuropilin-1 may be transferred from cytoplasmic pool to surface of ng108 cells by some soluble factors released by sm-3 cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa 1 , j. nakanishi 2 , h. hamada 1 , y. yokomachi 1 , k. yamaguchi 3 , n. takeda 1 1 inst. biomed. eng., waseda univ., tokyo, japan; 2 riken, japan; 3 kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc12 cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc12 cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca-1 was shown to be required for cdc42 to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca-1. toca-1 is strongly expressed in developing brain. knockdown of toca-1 in pc12 cells significantly enhances neurite elongation. consistently, overexpression of toca-1 suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc12 cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca-1 in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca-1 regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi (17079003) ps2p-e067 pragmin, a novel effector of rnd2 gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd2 is specifically expressed in brain. although rnd1 and rnd3 display an antagonistic action for rhoa, signaling pathways of rnd2 are not well known. here we have performed a yeast two-hybrid screen using rnd2 as bait and identified a novel rnd2-effector protein, pragmin (pragma of rnd2). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd2 among the rho family gtpases in a gtp-dependent manner. rnd2-bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc12 cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd2, and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd2 controls neurite outgrowth by regulating rhoa activity through pragmin. ps2p-e068 regulation of growth cone motility by collapsin response mediator protein-2 (crmp-2) masumi iketani 1 , yanai shigeki 1 , fumio nakamura 1 , yoshio goshima 1,2 , kohtaro takei 1,2 1 dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; 2 crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein-2 (crmp-2) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp-2 within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp-2 in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp-2 in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp-2 activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps2p-e069 eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani 1,2 , masaru ihara 1,2 , hiraki sakuta 1,2 , hiroo takahashi 1,2 , ikuko watakabe 1 , masaharu noda 1,2 1 division of molecular neurobiology, national institute for basic biology, okazaki, japan; 2 crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps2p-e070 neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa 1 and lpa 2 . here we examine how lpa 3 , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma 12 (pc12) cells. pc12 cells expressed low levels of endogenous lpa 3 mrna. when pc12 cells were infected with retrovirus expressing lpa 3 and treated with nerve growth factor under serum-free conditions for 3 days, neurite formation was observed in 55% of total infected cells, which was similar to that in control virus-infected pc12 cells. however, the mean length of neurites was increased in pc12 cells expressing lpa3, compared with that in control pc12 cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa 3 -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa 3 was involved in neuritogenesis and branch formation. masao sakurai 1 , tomoko aoki 1 , kyoko ishikawa 1 , shingo yoshikawa 2 , john b. thomas 2 , chihiro hama 1 1 cdb, riken, hyogo, japan; 2 salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt5 as a regulatory factor for glomerular patterning. in the wnt5 mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt5 expression in orns. previous studies in embryos have shown that wnt5 repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt5 mutant. this and other genetic data suggest that drl is an antagonist for wnt5 signaling. we propose that wnt5 signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps2p-e073 a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps2p-e074 differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino 1 , masatomo kobayasi 1 , taiki nakajima 1 , lydia michaut 2 , indrayani ghangrekar 1 , kuniaki takahashi 3 , ryu ueda 3 , kaoru saigo 4 , walter j. gehring 2 , katsuo furukubo-tokunaga 1 1 grad. sch. life envir. sci., univ. tsukuba, japan; 2 biozentrum, univ. of basel, switzerland; 3 genet. strains res. ctr., natl. inst. genet.; 4 dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified 1,465 genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected 102 genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki 1 , yoshie suzuki 1 , hiroyuki aburatani 2 , akinao nose 1 1 dept. phys., univ. tokyo, tokyo, japan; 2 rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, 12 and 13, which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼170 genes with more than two-fold difference in expression level. for 26 candidate genes out of 33 (78%), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles 12 or 13, implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet-2. to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet-2 signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet-2. loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc5 receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc5 homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps2p-e078 analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi 1 , takaaki hirotsu 2 , eriko kage 1 , hideaki takeuchi 1 , hirofumi kunitomo 3 , yuichi iino 3 , takeo kubo 1 1 dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; 2 dept. biol., fac. sci., kyushu univ. grad. sch., japan; 3 mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr-1 is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr-1 mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr-1 functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura 1 , ken-ichi ogura 1 , yoshio goshima 1,2 1 dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; 2 crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc-6 is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc-6 is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc-6 is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc-6 during the axonal guidance of the hsn neurons. silencing of netrin/unc-6 only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc-6 only at the vulval precursor cells in unc-6 null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc-6 expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn3a-hsp70:gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal4/uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb2a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere 2. these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) 16700296 masahiko taniguchi 1 , yoshinori mikami 2 , tomoyuki masuda 3 , tomoyuki yoshida 2 , naoto matsuda 2 , masayoshi mishina 2 , takao shimizu 4 1 department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; 2 department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; 3 department of anatomy, fukushima medical university, japan; 4 dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin 6d (sema6d). the orf of sema6d is 3054 bp (1018 aa). zebrafish sema6d protein showed a 70.8% identity with mouse sema6d protein. to clarify the expression pattern of sema6d, in situ hybridization was performed. in the embryos, sema6d is expressed in the lens and hindbrain. we also found that sema6d has the repulsive activity. these results indicate that sema6d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext (17023013) mayumi yamada 1 , shohei maekawa 2 , seiji miyata 1 1 department of applied biology, kyoto institute of technology, kyoto, japan; 2 division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps2p-f083 semaphorin 4d inhibits ␤1 integrin activity through the r-rasgap activity of plexin-b1 izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin 4d (sema4d) receptor, plexin-b1 functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema4d/plexin-b1 in cell migration. activation of plexin-b1 by sema4d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi3-k activation, and ␤1 integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤1 integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤1 integrin activation and cell migration, and that inactivation of r-ras by sema4d/plexin-b1-mediated r-ras gap activity control cell migration by modulating the activity of ␤1 integrins. shun hamada 1 , emi fukuda 2,3 , shota katori 2,3 , takeshi yagi 2,3 1 dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; 2 grad. sch. frontier biosci., osaka univ., osaka japan, 3 crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise >50 cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking 34 or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga1 is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga1 is expressed by layer 2/3 neurons throughout the neocortex at p7 mice, but is absent in adults. between e15.5 and late p0, stages that span the generation and radial migration of layer 2/3 neurons, mdga is expressed in patterns consistent with its expression by migrating layer 2/3 neurons, suggesting a role for mdga1 in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e15.5 to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga1 protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga1 acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca1 pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on 7 days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on 14 div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day 2, but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps2p-f088 analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono 1 , yoshiyuki kuroda 1 , fumiko matsui 1 , yoshihito tokita 1 , keiko nakanishi 1 , michiru ida 1 , masahito ikawa 2 , masaru okabe 2 , katsuhiko ono 3 , atsuhiko oohira 1 1 institute for developmental research, aichi human service ctr., kasugai, japan; 2 research institute for microbial diseases, osaka university, suita, japan; 3 national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi (17500246) ps2p-f089 phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata 1 , tatsuhisa watanabe 2 , ikuko suzuki 1 , junichi kitagawa 1 , akiko ogawa 3 , kenro kanda 4 , kazunao kuramoto 5 1 dept. of physiol., sch. of dent., nihon univ., tokyo, japan; 2 dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; 3 dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; 4 shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; 5 division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats (30-34 months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura 1 , koichi obata 1 , masafumi sakagami 2 , koichi noguchi 1 1 department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; 2 department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk5 (also known as big mapk 1 or bmk1) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk5, mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days 1 and 3 after cfa injection. furthermore, time course of phosphorylated-erk5 level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk5 in drg neurons may contribute to the development of inflammatory pain. ps2p-f091 activation of erk5 in drg neurons contributes to acute pain toshiyuki mizushima 1,2 , koichi obata 1 , takashi mashimo 2 , koichi noguchi 1 1 department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; 2 department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p38 in dorsal root ganglion (drg) neurons. however, there has been no study examining erk5 (also known as big mapk 1 or bmk1) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk5 phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk5 in small-to-medium diameter sensory neurons with a peak at 2 min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk5 in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) 1/2 and p38 mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk5 (also known as big mapk 1 or bmk1) is activated in the drg and spinal cord and participate in pain-related behaviors in the l5 spinal nerve ligation (snl) model. l5 snl induced an increase in the phosphorylation of erk5 not only in the injured l5 drg, but also in the spared l4 drg at day 7 after surgery. furthermore, l5 snl induced a striking increase in erk5 phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk5 in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai 1 , minoru asada 1 , naoki seno 2 , hidenori suzuki 1 1 department of pharmacology, nippon medical school, tokyo, japan; 2 pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣-1, which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps2p-f094 interleukin-1␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda 1 , jun kadoi 1 , msanori nasu 2 , masayuki takahashi 1 , shigeji matsumoto 1 1 dep. physiol and 2 res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il-1␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il-1␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il-1␤ induced depolarization in inflamed rats was larger than that in control rats. il-1␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il-1␤ paracrine mechanism following peripheral inflammation. junichi kitagawa 1 , mamoru takeda 2 , jun kadoi 2 , yoshiyuki tsuboi 1 , shigeji matsumoto 2 , koichi iwata 1 1 dept. of physiol., sch. of dent., nihon univ., tokyo, japan; 2 dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day 3 after ion treatment and the threshold decrement was lasting more than day 14. single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day 3-14 after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for 90 min. at 6, 12, 24, 48 and 120 h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at 6 h, peaked at 12 h and then decreased. intense gfap immunoreactivities were also detected at 6 and 12 h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for 90 min at 25 m/min. after the running, rats were applied the weight on the gastrocnemius muscles for 30 min, and then spinal cord and soleus muscles were removed from the rats. productions of il-1beta, il-6 and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at 6 h post-running, number of fos-positive neurons was increased, peaked at 12 h and then decreased to control level at 24 h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at 2 days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo 1 , yusuke sakai 2 , aya masawaki 3 , takashi shimoda 2 , masayuki moritani 1 , motohide takemura 1 1 dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; 2 dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; 3 dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip 2 weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for 90 min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep3 receptor (ep3r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca 2+ ] i ) in a ptx-sensitive manner in cho cells expressing canine ep3r and mouse bk b2 receptor (b2r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk (1 nm) was applied twice with a 6-min interval to the cells expressing b2r, the second [ca 2+ ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h-89 (1 m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk (10 pm) treatment for 1 h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca 2+ ] i was at least in part mediated by pka. ps2p-f100 contribution of peripheral 5ht2a or 5ht3 receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral 5ht2a or 5ht3/r in male rats with tmj inflammation. here we tested if these two 5ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c2. fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c2 transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for 7 days compared to naive rats. we tested if local 5ht2a or 5ht3/r antagonist affects fos expression in both groups. the number of fos-li in the vc/c2 but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral 5ht2a and 5ht3/rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of 5-ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼60% of sg neurons examined, 5-ht induced an outward current. the outward current was mimicked and suppressed by a 5-ht 1a agonist and 5-ht 1a antagonist, respectively. in ∼10% of sg neurons, 5-ht evoked an inward current which was mimicked by a 5-ht 3 agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that 5-ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of 5-ht 1a and 5-ht 3 receptors, respectively. the reciprocal postsynaptic actions of 5-ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by 5-ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic 50 of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic 50 of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps2p-g103 nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than 2 h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi (17700306) and km (16500262) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla 2 activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about 90% of neurons examined, melittin (1 m) superfused for 3 min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at 0 mv in the presence of bicuculline. this action was visible about 2 min after the beginning of its superfusion and subsided within 8 min after washout. these melittin actions were reduced in extent by a pla 2 inhibitor 4-bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla 2 activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi (17500275) ps2p-g105 presynaptic p2y 1 receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p2y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied 2-methylthio adp (2mesadp, 30 m), a p2y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about 20% of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by 2mesadp with no change in the amplitude. the 2mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs2179 (30 m), a selective p2y 1 receptor antagonist. these results indicate that the activation of presynaptic p2y 1 receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p2y 1 receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi (17700370), the japanese health sciences foundation (kh21006) ps2p-g106 presynaptic enhancement by proteinaseactivated receptor-1 agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)-1 but not par-2 agonist (each 1 m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par-1 mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par-1 agonist (sfllrn, 1 m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at -70 mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par-1 antagonist (yfllrnp, 1 m). these results indicate that the activation of par-1s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par-1 activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi (16700340) ps2p-g107 effect of tramadol metabolite m1 on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m1, which is one of its metabolites, at 1 mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m1 reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at −70 mv. this action was not seen in the presence of a -opioid receptor antagonist ctap (1 m). m1 also reduced the peak amplitudes of epscs which were monosynaptically evoked at −70 mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m1 inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m1 would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk-801 or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk-801 injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk-801. these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps2p-g109 studies on pain control system (rept. 57): changes in phosphorylation of nr2b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for 14 weeks. under these conditions, the immunoreactivities of phosphorylated-ser-1303 nr2b (p-ser-1303 nr2b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr-1472 nr2b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr2b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr2b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps2p-g110 prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, 100 hz, 20 a, 30 s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by 6-ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps2p-g111 the nav1.3 sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav1.3, a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav1.3. four weeks post-sci, nav1.3 protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav1.3 caused a significant reduction in nav1.3 expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda 1 , hiroyuki ichikawa 2 , ryuji terayama 2 , tomosada sugimoto 2 1 department of oral maxillofacial rehabilitation, okayama university, okayama, japan; 2 department of oral function and anatomy, okayama university, okayama, japan ib4-sap is a neurotoxin designed for targeting primary nociceptors with ib4 binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib4-sap (2.5 l of 0.067% solution for each ganglion) directly into the 4th and 5th lumbar (l4 and 5) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib4-binding. after counterstain, the cell body size of neurons were measured. ib4-sap reduced the total number of drg neurons in l4 and 5 ganglia combined by 21% (11652 ± 2213 on untreated side versus 9208 ± 1609 on treated side). small neurons (<1200 m 2 ) were reduced by 28% whereas large ones (≥1200 m 2 ) were not affected. ib4-binding neurons were mostly small (≥96%) and were reduced by 46%. the number of small neurons, that were not stained for ib4-binding, increased by 35% (1994 ± 286 versus 2700 ± 600). schuichi koizumi 1 , kaoru nasu-tada 1 , makoto tsuda 2 , emiko kunifusa 1,2 , kazuhide inoue 2 1 div. pharmacol., natl. inst. hlth. sci., tokyo, japan; 2 dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p2x4 receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p2x4 receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p2x4 receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p2x4 receptor in microglia, leading to neuropathic pain. research funds: mf16, kakenhi (170230570) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. 2004). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at 24 h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi 17109015 ps2p-g115 lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. 2004). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at 1 mm decreased the protein levels of myelin basic protein (mbp) at 24 h. this action was significantly inhibited by botulinum neurotoxin/c3 (bont/c3). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp22, mag, p0 in cultured fibers. the maximal decrease was observed all at as early as 3 h after the addition of lpa. bont/c3 and y27632 abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at 0 and 3, 7, 14, 21, 28 days after operation. pre-emptive co-administration of mk-801 and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk-801 decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama 1,2 , naomi akiyama 1 , mikie hirata 1 , takayuki nakagawa 2 , shuji kaneko 2 , masabumi minami 1 1 dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; 2 dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for 3 h per day between postnatal day 1 and 14. visceral sensitivities were assessed in stomach and colon at 10 weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research 16500216 ps2p-g119 change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered 1 to 3 days after exercise in rats (8 w old). c-fos expression in the superficial dorsal horn increased in l4 spinal segment when the edl muscle was compressed 2 days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats 8, 80 (81-84) and 130 (131-133) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in 80 w old rats, but not significant. after lec, the threshold started to decrease 1 day after lec in all three age groups. it returned to the pre-lec level 4 days after lec in 8 and 80 w old rats only. recovery of 130 w old rats delayed up to 7 days after lec. increased c-fos expression in the superficial dorsal horn was observed in l4 as well as in l5 in 130 w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal4 males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal4 males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama 1 , masaharu akita 2 1 kanagawa life-science res., japan; 2 kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day 11 to 13 of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days 25-39 of age, they did not show the first estrus before 52 days of age with mean age of 54.7 ± 2.0 day. in contrast, most of normal mouse igg-treated controls showed the first estrus by 43 days of age with mean age of 42.8 ± 2.2 day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss-1 gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days 29 of age (d29), and first estrus on around d34. brain tissues were obtained on d21, 26, 31, 36 and 41. kiss-1 mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d21 to 26 and was kept at a high level thereafter. gpr54 mrna expression in the medial preoptic area increased significantly from d21 to 31. metastin-immunoreactive cells were not found on d21 but were apparent in the arc-me on d26 onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito 1 , sei-etsu fujiwara 1 , kenjiro konno 2 , takashi yamaguchi 3 , tetsu nemoto 4 , etsuko kasuya 1 , ryosuke sakumoto 1 1 anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; 2 inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; 3 grad. sch. sci. & eng., yamagata univ., yonezawa, japan; 4 sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps2p-g126 hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi 1 , teruko uwano 1,3 , anh tran 2,3 , eturou hori 1,3 , taketoshi ono 2,3 , hisao nishijo 1 1 system emotional science, univ. of toyama, japan; 2 integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; 3 crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a 2 s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of 85 hf neurons, 26 responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue 1 , atsu aiba 2 , kaoru inokuchi 1 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 grad. sch. med., kobe univ., kobe, japan vesl-1s/homer-1a and vesl-1l/homer-1c are splicing isoforms encoded by the vesl-1 gene. vesl proteins bind and regulate mglur1/5, ip3 receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl-1s is upregulated by tetanic stimulation that elicits l-ltp. vesl-1s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl-1s gene-targeting mice (ko) and examined whether vesl-1s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl-1s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta 1 , r. migishima 1 , s. kida 2 , k. inokuchi 1,3 1 mitsubishi kagaku inst. life sci (mitils), japan, 2 tokyo univ. agricul., japan; 3 crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within 3 d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps2p-h129 sex differences in the effects of chronic estrogen treatment on fear conditioning in c57bl/6j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c57bl/6j mice were implanted with a silastic capsule containing 0, 5, 10 or 20 g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps2p-h130 analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested 48 h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for 2 weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps2p-h132 analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps2p-h133 analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor 1 (cb1) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb1 and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb1 or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb1 and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb1 and lvgccs, we also found that cb1 is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda 1 , takahiro furuta 1 , kouichi nakamura 1,2 , takeshi kaneko 1,2 ps2p-h136 difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama 1 , tomo unzai 1 , kouichi nakamura 1,3 , sakashi nomura 2 , takeshi kaneko 1,3 1 department of morphological brain science, kyoto university, kyoto, japan; 2 department of physical therapy, kyoto university, kyoto, japan; 3 crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut)2, thalamostriatal inputs, were less dense in patch region, whereas vglut1-positive corticostriatal inputs were evenly distributed. quantitative analysis revealed 84% of vglut2positive synapses in patch region were formed with spines, whereas 70% in matrix region were made with dendritic shafts. in contrast, the targets of vglut1-positive inputs were mainly spines in both regions. moreover, vglut2-positive axospinous synapses in patch region were larger than vglut1-positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi (16500217, 17022024,16200025, 17022020 (0131),17650100) ps2p-h137 single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai 1 , fumino fujiyama 1 , takeshi kaneko 1,2 1 department of morphological brain science, university of kyoto, kyoto, japan; 2 crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi (16500217, 17022024, 16200025, 17022020(0131) , 17650100) ps2p-h138 lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui 1 , kayo nishizawa 1 , nobuyuki kai 2 , kazuto kobayashi 2,3 1 dept. of psychology, fukushima univ., japan; 2 dept. mol. genet., fukushima medi. univ., japan; 3 crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of 6-ohda injections into the nucleus accumbens (acb) in c57bl/6j mice. all mice were trained to press the lever for a food pellet using a fixed ratio 5 (fr5) schedule. the mice were injected with ascorbate vehicle or 6-ohda into the acb, and then tested post-surgically using the fr5 schedule again. the 6-ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area 29 is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area 29, it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area 29 in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area 29d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area 29c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas 29a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area 29, suggesting different functional roles played by each subdivision of area 29 in spatial memory and behavior. eiichi jodo 1 , yoshiaki suzuki 2 , tadahiro katayama 1 , ken-yo hoshino 2 , yukihiko kayama 1 1 dep. of physiol., fukushima med. univ., fukushima, japan; 2 dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, 30%) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about 0.1 s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in 19% out of 438 vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only 6% out of 254 it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., 1994) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., 2005) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., 2005) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of 2d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of 75 sessions in phase 3, although they learned easily the four problems in phases 1 and 2. our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto 1 , noriyuki higo 1 , munetaka shidara 2 1 neuroscience research inst., aist, tsukuba, japan; 2 grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d2 receptor study using antisense cdna showed that d2 receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d2mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d2 receptor antipeptide antiserum. d2 receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area 36 of the perirhinal cortex than in area teav. the differential localization between area 36 and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano 1 , natsuko yoshida 2 , kiyohisa natsume 3 1 kyushu kyoritsu university, fukuoka, japan; 2 kyushu institute of technology, fukuoka, japan; 3 kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f3 and f4 was the highest and suggests that the theta rhythm at f3 and f4 may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after 6-month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi (1565081) ps2p-h146 neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe 1 , sho yagishita 1 , hideyuki kikyo 1,2 1 department of physiology, the university of tokyo school of medicine, tokyo, japan; 2 department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a 1.5 t scanner system was used (te = 50 ms; tr = 14 s; acquisition time = 3.0 s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong 1 , motoaki sugiura 3 , yuko sassa 2 , keisuke wakusawa 2,4 , kaoru horie 1,5 , shigeru sato 1,5 , ryuta kawashima 2,5 1 gsics, tohoku university, sendai, japan; 2 niche, tohoku university, japan; 3 miyagi university of education, sendai, japan; 4 department of pediatrics, school of medicine, tohoku university, japan; 5 the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz 1 , christie l. sahley 2 , kenneth j. muller 1 1 physio. & biophys., univ. of miami, miami, fl, usa; 2 biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps2p-h150 sensory/motor modules regulating the development of peer social relationship mamiko koshiba 1,2 , shun nakamura 2 1 jst, crest, japan; 2 ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata 1 , minoru hattori 2 1 department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; 2 graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) 30-s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi 30-s procedure over 30 days. the 3-s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the 30-s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi 30-s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps2p-i152 the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka 1,2 , ritz oshio 1,3 , norihiro sadato 1,4 , manabu honda 5,6 1 nips, okazaki, japan; 2 jsps, tokyo, japan; 3 nagoya univ., nagoya, japan; 4 ristex, jst, tokyo, japan; 5 sorst, jst, tokyo, japan; 6 ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps2p-i153 sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi3k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi3k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi (17500429) ps2p-i154 selective manipulation of working memory through d1 and d2 receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d1 and d2 receptors. the computer simulation with this model shows that d1 receptor activation mainly affect working memory activity itself, while d2 receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d1 and d2 receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or 25 mg/kg mam on the 15th day of gestation. the activity of male and female offspring was measured for each 1 h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every 1 min interval for 1 h under 5 min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca1 pyramidal layer and ectopic neuron mass). ps2p-i156 long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur1. hamid gholamipour 1 , shirin babri 2 , khameneh saied 3 1 department of physiology, university of tabriz, tabriz, iran; 2 university of tabriz, iran; 3 university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca1region of hippocampus on memory formation. fifty male rats were divided into five groups. (1) control (2) sham operation (3) test (4) diabetic/saline (5) diabetic/insulin. groups 4 and 5 were made diabetic by treatment with stz (50 mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca1 region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < 0.05). compared to sham group diabetic/insulin group showed increased latency (p < 0.05) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca 2+ /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of 19 per 100,000 in india. we studied the park 4 polymorphism in north indian population and parkin expression in early parkinson's disease (n = 30) and sporadic parkinson's disease (n = 30). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon-1, 3 and 6 in early pd and exon-1 and 12 in sporadic pd, heterozygous mutations in exon 4 and 9 in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position 167 of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin 4 mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul 1 , kittisak sripanichkulchai 2 , david finkelstein 3 1 faculty of medicine, srinakharinwirot university, thailand; 2 faculty of medicine, khon kaen university, thailand; 3 university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c57bl/6 mice were pretreated with 17beta-estradiol (e2), injected with mptp on the day 6 and brains were collected on day 11. e2 pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e2 was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e2 neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps2p-i163 effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki 1 , fumihiko yoshino 2 , masaichi chang-il lee 2 1 dept. of food sci. and nutrition, showa women's univ., tokyo, japan; 2 clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi (17700577) ps2p-i164 recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi 1 , kaori sawada 1 , haruo okado 1 , atsushi nambu 2 , masahiko takada 1 1 tokyo met. inst. neurosci., fuchu, tokyo, japan; 2 natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype-2 raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type-1) of raav (raav1) carrying αs cdna (raav1-␣s), and a cocktail of raav1-␣s and raav1 carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav1-␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser129 residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged 70-80s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of 0.5-1 (i), 1.5-2 (ii), 2.5-3 (iii), and 3.5-4 (iv). the ␤waves were rated with total periods (%) of 13-27 hz wavelets in 3-s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated 65-70% in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about 55% in group iv. the pvp ␤-waves were 60-80%, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana 1,2 , hirokazu iwamuro 1,3 , masahiko takada 4 , atsushi nambu 1,2 1 div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; 2 sokendai; 3 dept. neurosurg., univ. tokyo, tokyo, japan; 4 dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd44 expression on microglia and also enhanced the mt1-mmp expression to shed off cd44 at the cell surface and degrade surrounding ecm to open the migratory way. a53t mutant ␣-synuclein showed greater level of cd44 shedding and cell migration. extracellular treated ␣-synuclein also increased cd44 and mt1-mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant (2004 grant ( -2006 ps2p-j169 serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai 1 , hiromasa yamada 1 , yoshinari aimi 1 , ikuko nagatsu 2 1 department of anatomy, shiga university of medical science, otsu, japan; 2 fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of 6hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba 1-and tb4distribution in microglia were immunohistochemically investigated at 0 h and 7 days after twice mptp-treatments (one treatment comprised of 4 intraperitoneal injections of 20 mg/kg mptp at 2 h interval) to c57bl/6 and balb/c at 6 months (mo) interval. the recognizable change of iba 1-and tb4-distibution in microglia of both mice strains was observed even 6 mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps2p-j171 activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada 1 , ryohei hishida 2 , yoko hirata 3 , kenji ono 4 , hiromi suzuki 4 , shin-ichi muramatsu 2 , imaharu nakano 2 , kunihiro tsuchida 1 , toshiharu nagatsu 1,4 , makoto sawada 4 1 school of medicine, fujita health university, aichi, japan; 2 division of neurology, jichi medical university, japan; 3 department of biomolecular science, gifu university, japan; 4 research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the 60 week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps2p-j173 phosphorylation of erp57 in adult rat brain with neonatal 6-ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of 6-ohda at 3(p3) and 6 days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p56, tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the 6-ohda lesioned rats at p56, and it was identified as disulfide-isomerase a3 precursor (erp57) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp57 may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi (20 or 25 nm, 48 h) was applied to cultures at 7 days in vitro and succeeding changes of da neurons were investigated up to 35 days. more than 95% of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately 2 weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps2p-j175 specific expression of proapoptotic factor pag608 on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p53-activated gene 608 (pag608), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p53 expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag608 in the internal capsule of l-dopatreated and constitutive expression in the smi-32-immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag608 in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag608 expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag608 in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps2p-j176 an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang 1,2 , yuzuru imai 2 , haruhisa inoue 1,2 , ayane kataoka 2 , sachiko iita 2 , nobuyuki nukina 2 , ryosuke takahashi 1,2 1 neurology, university of kyoto, kyoto, japan; 2 bsi, riken, saitama, japan since loss of parkin e3 activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., 2004) have found that application of an antisense oligodeoxynucleotide specific for nr1 have successfully knockdown the expression of nr1 gene expression in the striatum of 6-hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr1 was re-addressed using a small interfering rna (sirna) specific for nr1. in pc12 cells, reductions of nr1 proteins after a single application of nr1 sirna were found by western blot experiments. and after one single application of nr1 sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr1 immunofluorescence were found in the striatum after the sirna treatments. lai et al., 2004. neurochem. int. 45, 11-22. research funds: faculty research grant, frg/04-05/ii-27, hong kong baptist university ps2p-j178 homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy (0.25-1 mol in 1 l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on 19th day. animals with 1 mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps2p-j179 ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca14 takahiro seki 1 , takayuki shimahara 1 , naoko adachi 2 , naoaki saito 2 , norio sakai 1 1 dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; 2 lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type 14 (sca14), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca14. however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca 2+ homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f9 cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately 300 compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki 1 , yo sasaki 2 1 department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; 2 washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase1 (nmnat1) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least 6 different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at 10 min, but next increased at 2 h and returned to basal at 6 h after cocaine challenge. actin showed a similar pattern but was maintained at high level at 6 h. both psd-95 and glur1 were increased between 2 h and 6 h like actin. by contrast, some proteins such as drebrin were decreased after the peak at 2 h. interestingly, the 20 s proteasome subunit demonstrated a dramatic upregulation at 2 h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps2p-k184 effects of mdma and 5-meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, 3,4methylenedioxymethamphetamine (mdma) and 5-methoxy-n,ndiisopropyltryptamine (5-meo-dipt), on sert and dat were examined. as previously reported, mdma (0.3-10 m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, 5-meo-dipt (0.3-300 nm) evoked an outward current response in sertexpressing cells similarly to that of selective 5-ht reuptake inhibitors. no current response was observed when 5-meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that 5-meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka 1 , nobue kitanaka 1 , tomohiro tatsuta 1,2 , yoshio morita 2 , motohiko takemura 1 1 department of pharmacology, hyogo college of medicine, nishinomiya, japan; 2 department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine (30 mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of 0.1 mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of 1 and 10 mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth (0.5 mg/kg, i.p.), pretreatment with subchronic clorgyline (0.1-10 mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura 1 , nobue kitanaka 1 , tomohiro tatsuta 1,2 , yoshio morita 2 , junichi kitanaka 1 1 department of pharmacology, hyogo college of medicine, nishinomiya, japan; 2 department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline (3.0-30 mg/kg, i.p.) 15 min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except 30 mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum 20 min after drug challenge. these results suggested that the inhibitory effect of lobeline (3.0-10 mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with 1 mg/kg meth and locomotor activity measured daily for 14 days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, 5 nmol ufp-101 (a nop receptor antagonist) and 1 mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp-101 strongly suppressed locomotor activity. thus, it was unclear if ufp-101 suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp-101 co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d2dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine (1-10 mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine (0.5-5 mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine (1, 2.5 and 5 mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine (5 mg/kg, i.p.) caused more than 80% mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine (20 mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps2p-d191 regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: (1) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis 3(ca3) were increased after fs. the rule of this kind of changing was of region-and time-specific. (2) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi 1,2 , tomoaki shirao 1 1 dept of neurobiol & behav.; 2 ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only 22.7 ± 3.0% of drebrin in the spine is stable, with a turnover time of 5.8 ± 0.4 min. blockade of ampar by 20 m cnqx reduces the population of stable drebrin (8.9 ± 3.6%), and has no effect on a turnover time. on the other hand, blockade of nmdar by 100 m ap5 has no effect on the population of stable drebrin, whereas shortens a turnover time (4.0 ± 0.3 min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu 1 , yoshito saitoh 1 , kaoru inokuchi 1,2 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at 1 h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki 1,2 , daisuke okada 1 , kaoru inokuchi 1,2,3 1 mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; 2 yokohama natl. univ. environment information sci., kanagawa, japan; 3 crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk2 cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto 1 , mitsutoshi setou 1,2 , kaoru inokuchi 1 1 laboratory for molecular gerontology, mitils, japan; 2 laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps3a-a005 an activity that traps vesl-1s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl-1s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at 1 m, but not 50 nm. because 50 nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps3a-a006 acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu 1 , ling chen 2 , masahiro sokabe 1,3,4 1 dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; 2 dept. physiol., nanjing med. univ., nanjing, china; 3 sorst cell mechanosening, jst, nagoya, japan; 4 dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca1 region in rat hippocampal slices. an application of 0.1 dheas for 10 min to the slice augmented instantly the epsp, which was terminated within 30 min. however, even 1 h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for 10 min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai 1 , ling chen 2 , masahiro sokabe 1,3,4 1 dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; 2 dept. physiol., nanjing med. univ., nanjing, china; 3 sorst cell mechanosensing, jst, nagoya, japan; 4 dept. mol. physiol., nips, okazaki, japan to know whether 17␤-estradiol (e2) can protect ca1 neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion (4vo) for 10 min, and the effect of e2 against this ischemic injury was examined. the electrophysiological properties of ca3-ca1 synapses were examined by a real-time optical recording method 7 days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e2 (1 mg/kg) 3 h before 4vo was able to protect ca1 neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt (2 mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn (8 mg/kg) did not. above results suggest that e2 can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps3a-a008 non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima 1 , mari ogiue-ikeda 1 , yuki oishi 2 , suguru kawato 1,2 1 department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; 2 department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca1, ca3, and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps3a-a010 morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki 1 , gen murakami 2,3 , tetsuya kimoto 2,3 , suguru kawato 2,3 1 college of humanities and sciences, nihon university, tokyo, japan; 2 department of biophysics and life sciences, university of tokyo, tokyo, japan; 3 crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of 100 nm dex induced rapid modulation of the density and morphology of dendritic spines in ca1 pyramidal neurons within 1 h. the total spine density increased from 0.88 spines/m to 1.36 spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of 10 m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura 1 , yuji ikegaya 2 , sadao shiosaka 1 1 division of structural cell biology, naist, nara, japan; 2 laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: 21st century coe research ps3a-a012 actin organizations within single dendritic spines in ca1 pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca1 pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of 1.2 min and 17 min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds (0.2-1.2 um/min) and lengths (0.2-0.7 um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori 1 , katsushige sato 2 , kohichi tanaka 1 , hiroko hamazaki 1 1 mol. neurosci., tmdu, tokyo, japan; 2 physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at 1 or 4 days post-hatching (p1 or p4), and at p7. this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p4, but they lose this ability at p7. in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps3a-b015 effect of estrogen on hippocampus in male and female mice takanori sugawara 1 , shinji hayashi 1 , victoria luine 2 1 graduated school of integrated sience, yokohama city university, yokohama, japan; 2 department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received 17 ␤-estradiol (e2) or oil-vehicle injections at adult before golgi impregnation. spine densities 10 m of apical dendrites of the pyramidal neurons in the hippocampus ca1 region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e2 treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto 1,2 , takafumi inoue 2,3 , akinori kuruma 1 , katsuhiko mikoshiba 1,2,3 1 lab. developmental neurobiology, brain science inst., riken, saitama, japan; 2 div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; 3 calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca 2+ reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p38 mapk and c-jun nh2-terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u0126, a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb203580 and sp600125, antagonists for p38 mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps3a-b018 regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey 1 , chou-chi li 2 , yumiko motoyama 1 , junko yamaoka 1 , masao ito 1 1 riken brain sci. inst., japan; 2 national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur2 co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur2 c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur2. glur2 phospho-ser880 promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep2m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur2-vcp interaction. yuichi kitagawa 1,2 , shin-ya kawaguchi 1,2 , tomoo hirano 1,2 1 dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp-1 and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps3a-b020 long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr2b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from 35 to 42 days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over 60 days old, when song learning is nearly complete, but ltd remained possible in birds over 60 days old who had been isolated from song tutors, and thus retained the capacity for learning. ps3a-b021 involvement of ca 2+ -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that 3 exposures to glu of cultured rat hippocampal slices at 24 h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur1 and glur2, showed that the number of glur1-positive puncta increased transiently after the repeated glu exposures, whereas the number of glur2-positive puncta increased gradually and persistently. jstx (a ca 2+ -permeable ampar blocker) suppressed fepsp amplitude recorded at ca3-ca1 synapses by 20-40% in the period corresponding to the transient increase of glur1-positive puncta. this transient increase should represent the delivery of ca 2+ -permeable (glur2-lacking/glur1-including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca 2+ -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka 560-0043, japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after 3 inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; 3 inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd 3 times (24 h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of 5-ht (10 m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the 5-ht 1b receptor agonist cp93129 mimicked the presynaptic inhibitory effect of 5-ht. 5-ht 1b receptor antagonist nas-181 reversed the 5-ht inhibitory effect, indicating that the 5-ht induced inhibitory effect occurs by mediating 5-ht 1b receptors. the presynaptic inhibitory effect of 5-ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, 5-ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this 5-ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that 5-ht, upon activating 5-ht 1b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku 1 , masato ano 1 , yuki hashimotodani 2 , tadasato nagano 3 , masanobu kano 3 1 dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; 2 dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; 3 dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, 5-ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a1, a2a, a2b and a3). adenosine a1 and a3 receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a1 and a3 receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a1 agonist (cpa) and a3 agonist (2-cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and 2-cl-ib-meca as reported previously. although ca currents were decreased by 2-cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of 2-cl-ib-meca. at administration of cpa and 2-cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a3 receptor may inhibit adenosine a1 receptor throughout a intracellular pathway in neurons. ps3a-c028 influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa 1 , j. yamada 2 , k. inoue 1 , y. yanagawa 3 , a. fukuda 1, 2 1 dept. physiol., hamamatsu univ. sch. med., japan; 2 dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; 3 dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad67-gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa 1 , hiroyuki hioki 1 , kouichi nakamura 1,2 , hisashi nakamura 1 , takeshi kaneko 1,2 1 dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter 3(vglut3)-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut3-positive cells showed immunoreactivity for 5-hydroxytryptamine (5ht), a significant proportion (12.3%) of vglut3-postive cells was 5ht-negative. in this study, triple immunofluorescence staining was performed for vglut3, 5ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad67) and tyrosine hydroxylase (th). our results showed that all of the vglut3-positive/5ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut3positive/5ht-negative neurons didn't show any immunoreactivities for gad67 nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut3-positive neurons in these nuclei. research funds: kakenhi 16200025, 17022020, 17650100 ps3a-c030 cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig 1 , mika ushimaru 1 , yoshiyuki kubota 1 , akiya watakabe 2 , tetsuo yamamori 2 , yuchio yanagawa 3 , yasuo kawaguchi 3 1 div. cerebral circuitry, nips, okazaki, japan; 2 div. brain biology, nibb, okazaki, japan; 3 dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin (5-ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, 60% of the cs were inhibited by 5-ht through 5-ht 1a r and 6% were excited through 5-ht 2a r. however, 44% of the fs cells were inhibited and 28% excited. these results show that cs cells are more inhibited by 5-ht than fs. the expression of 5-htr was confirmed by in situ hybridization. research funds: jsps pe04061 and 15300110 ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v4, teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer 3 but also in layer 5 and/or 6. in each of the 3 cases, about 50-100 gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad67, somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine (1) their other features, (2) possible species variability, (3) their functional significance. supported by riken bsi. withdrawn ps3a-c033 regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge 1,2 , susanna b. park 2 , greg j. stuart 2 , yasuo kawaguchi 1 1 national institute for physiological sciences, japan; 2 div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in 3 neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure (100 m) inhibited layer 5 pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers 2/3 were generally less responsive to ach, but ach inhibited layer 3 cells in visual cortex. prefrontal layer 5 pyramidal neurons were more responsive to ach than were layer 5 cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations (5 mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in 37% of non-fs interneurons (24 of 65 cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki 1,2 , kenzi saito 1,3 , yuchio yanagawa 1,2 1 department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; 2 sorst, jst, kawaguchi, japan; 3 sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad67-deficient mouse fetus. ps3a-c035 gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal 4 to 6 day in egl. its release, especially from premigratory zone, was inhibited by ni 2+ , but cd 2+ couldn't. we suggest that gaba excitation induces granule cell migration. ps3a-c036 gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi 1 , hiroaki wake 2 , junichi nabekura 2 , osamu takahashi 1 1 department of histology, kanagawa dental college, yokosuka, japan; 2 national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata 1 , kunihiko obata 2 , jonathan davies 3 , mark bellingham 3 , peter g. noakes 3 1 lab. for memory & learning, riken-bsi, wako, japan; 2 obata res. unit, riken-bsi, wako, japan; 3 sch. biomed. sci., univ. queensland, queensland, 4072, australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., 2005) . we investigated the involvement of gaba alone in the mn death using gad67-deficient mice, in which cerebral gaba is reduced to less than 10% of the wild-type. mn numbers at embryonic day (e) 18 were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e18 embryos and subjected to electrical recording from the c4 and c8 ventral roots and also gaba measurement. in gad67-deficient embryos, increase in number of brachial mns (139%) and decrease in both spontaneous discharges in the c4, c8 roots and gaba content (less than 20%) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek293 cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣1␤1␥2 and ␣2␤1␥2 combinations (75% reduction) but had no effect at ␣1␤1␥1 or ␣2␤1␥1. in slice recordings dmcm blocked ipscs by 70% in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥2 subunits whereas in the central amygdala some inputs contain ␥1 while others contain ␥2 subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ 1 -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps3a-d040 hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane 1 , takashi iwai 2 , yasuo nagai 1 , junichiro oka 2 , hideo kimura 1 1 dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; 2 lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h 2 s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h 2 s. we examined the effect of h 2 s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na 2 s), a donor of h 2 s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na 2 s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na 2 s. these observations suggest that h 2 s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after 5 days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by (1 l) lidocaein (2%) 5 min before training. animals were allowed to self administer morphine (1 mg/kg per inf.) ten consecutive daily 2-h session. during all morphine self administration session lever pressing was measured. our results show that: (1) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. (2) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca 2+ wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of 17␤ estradiol from glucocorticoid activity yuki oishi 1 , suguru kawato 2 1 department of physics, graduate school of science, university of tokyo, tokyo, japan; 2 graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, 17␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. 17␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that 17␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps3a-d044 the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex (0.2 mg/100 g b.w., sc) or sesame oil (vehicle control) everyday for 9-10 days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf-1 mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf-1, estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf-1 and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps3a-d046 mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of 6-hydroxydopamine. ps3a-d047 the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba 1 , kimi watanabe 1 , munekazu komada 2 1 institute for developmental research, aichi human service center, aichi, japan; 2 graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida 1,2 , asif k mustafa 2 , kenji hashimoto 3 , kumiko fujii 2,4 , kazuhisa maeda 2,5 , hiroshi ujike 6 , richard l. huganir 2 , solomon h. snyder 2 , akira sawa 2 1 dept. of systems biology, obi, suita, japan; 2 depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; 3 chiba univ. forensic mental health, chiba, japan; 4 dept. of psychiat, shiga univ. med. sci., shiga, japan; 5 div. of neuropsychiat, tottori univ., yonago, japan; 6 dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick1 as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick1 knockout mice were significantly lower than those of their wildtype littermates at age of p7, but not in adults, suggesting regulation of pick1 on sr at developing stage. in case-control association study, we observed an association of the pick1 gene with sz, which is more prominent in disorganized sz. our findings suggest that pick1 contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps3a-d050 epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe 1,2 , werner kilb 2 , ileana l. hanganu 2 , taizhe qian 2 , daiichiro nakahara 3 , atsuo fukuda 1 , heiko j. luhmann 2 1 dept. of physiol., hamamatsu, japan; 2 inst. of physiol., mainz, germany; 3 dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca3 region of an intact preparation. 5 mm taurine blocked epileptiform activity induced by mg 2+ free acsf and 20 m 4-ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps3a-d051 responses of pge 2 in undifferentiated and differentiated ng108-15 cells kayoko matsushima 1 , takashi imanishi 1 , akinori kawaguchi 1 , tetsuyuki wada 1 , shigeru yoshida 2 , seiji ichida 1 1 school of pharm. sci., kinki univ., osaka, japan; 2 school of pharm. sci., kinki univ., osaka, japan; 3 school of pharm. sci., kinki univ., osaka, japan; 4 school of pharm. sci., kinki univ., osaka, japan; 5 school of sci. & eng., kinki univ., osaka, japan; 6 school of pharm. sci., kinki univ., osaka, japan our previous findings showed that 5-ht-and bk-induced [ca 2+ ] i increases were enlarged in differentiated ng108-15 cells. for the next stage, we investigated the effect of pge 2 , an inflammatory mediator for 5-ht and bk, on the cells. ng108-15 cells were loaded with fura-2/am, and the change in [ca 2+ ] i was monitored by an image processor. the results showed: (1) pge 2 -induced response was decreased when ng108-15 cells were differentiated by bt 2 camp, (2) 10 −5 m ah6809 and sc19220 irreversibly inhibited pge 2 -induced response by about 50% and 26%, respectively, while 10 −5 m ah23848 and sulprostone had no effect, and (3) pge 2 -induced response was abolished under ca 2+free conditions in about 70% of both ng108-15 cells. these results indicate that the response to pge 2 , via ep1 and ep2 receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from 11-to 21-day-old rats. bath application of serotonin (5-ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during 5-ht application. under the current-clamp recording, 5-ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that 5-ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps3a-d053 searching for endogenous ligands of trace amine receptors in mammals (1) akira komatsu 1 , airi yamaguchi 2 , noriko makikusa 2 , osamu koizumi 2 1 dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; 2 neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by 2% dms, post-fixed by 4% formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps3a-d054 effects of mg 2+ on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa 1,2 , nahoko kasai 1 , akiyoshi shimada 1 , keiichi torimitsu 1,2 , kunihiko obata 3 , yuchio yanagawa 2,4 , tadaharu tsumoto 2,3 1 ntt basic research laboratories, kanagawa, japan; 2 sorst/jst, saitama, japan; 3 neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; 4 dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg 2+ plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca 2+ concentration are largely affected by mg 2+ removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg 2+ removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg 2+ concentration and their actions on neural activity using a mg 2+ indicator, kmg-20-am together with fluo4-am. we observed different effects of the mg 2+ removal on gabaergic and non-gabaergic neurons by using gad67-gfp knock-in mice. research funds: jst/sorst ps3a-e055 transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p7-p13. zn + cortical neurons were also labeled, intrinsic and extrinsic, from p5. unlike in the adult, these were in layer 5, instead of layers 2, 3, and 6. at p9, neurons occurred in layers 2, 3, and 5 and, in some areas, layer 4. at p15, zn + neurons first appeared in layer 6; and at p22, there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. 17024064 ps3a-e056 systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano 1,3 , ryohei kanzaki 2 , kei ito 1,3 1 center for bioinform., imcb, univ. of tokyo, tokyo, japan; 2 dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; 3 bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi 1,2 , taizo nakazato 2 , shigeru kitazawa 2 1 neurol, juntendo univ., tokyo, japan; 2 physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, 2005) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns2005). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse (100 to 250 mv for da; 300 to 450 mv for 5ht) was applied at 4 hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps3a-e059 time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui 1,4 , yuko sekino 2,3 , tomoaki sirao 1 1 dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; 2 div. of neural network, inst. med. sci. univ. of tokyo, tokyo; 3 crest, jst, kawaguchi, japan; 4 jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at 14 days in vitro (div), and analyzed the dynamic localization of gfp-da at 16 div. glutamate stimulation started gfp-da translocating within 10 s and completed in 2 min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in 10 min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps3a-e060 distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa 1 , jun shiota 1 , hiroyuki tsutsumishita 1 , hiroyuki sakagami 2 , masaaki tsuda 1 , akiko tabuchi 1 1 dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; 2 dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr-1, c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi (17790055) to a.t. shoko shimizu 1 , shinsuke matsuzaki 1 , tsuyoshi hattori 1 , ko miyoshi 2 , masaya tohyama 1 1 department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; 2 department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia 1 (disc1) was identified as a novel gene disrupted by a (1;11) (q42.1;q14.3) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc1 at centrosome and residues 446-533 of disc1 (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc1 downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc1 to target to the centrosome. furthermore, we have shown that inhibition of the disc1-kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc1 is important to the disc1-kendrin interaction and that a truncated form of disc1 lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca 2+ or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca 2+ and cl − imaging. we found that the relatively low density a172 glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca 2+ oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps3a-e063 involvement of ca 2+ influx in the unpolarized non-vesicular release of fgf-1 hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf-1 lacking a conventional signal peptide sequence. we found that fgf-1 is co-released with s100a13, a ca 2+ binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf-1 is co-released with s100a13 from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf-1 and s100a13 required ca 2+ . the overexpression of s100a13 88-98 mutant lacking an ability of interaction with fgf-1 inhibited the their release, suggesting that s100a13 is a cargo molecule. the release of fgf-1 upon either stimulation was abolished by voltage-dependent n-type ca 2+ channel blocker. these findings suggest that ca 2+ influx may be involved in the unpolarized non-vesicular release of fgf-1. in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc12 cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that 5-ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca 2+ ] i ) in pc12 cells. however, it is unclear how [ca 2+ ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca 2+ ] i on actin dynamics in pc12 cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a23187. treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to 5-ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc12 cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp36 is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp36 is endogenously expressed in pc12 cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp36 is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp36 in actin dynamics in growth cones and found that knockdown of clp36 expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp36 deletion mutants that both of pdz and lim domains are necessary for the proper function of clp36. ps3a-e066 screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura 1 , tsuyoshi kobayashi 1 , yuchio yanagawa 2 , kunihiko obata 3 , fujio murakami 1,4 1 grad. sch. of frontier biosci., osaka univ., osaka, japan; 2 grad. sch. of medicine, gunma univ., maebashi, japan; 3 bsi, riken, wako, japan; 4 sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the 2208 clones tested, two genes, neuronatin and seizure related gene 6 (sez-6) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu 1,2 , tomoko matsumoto 1,2 , sakae mikami 3 , takashi nagasawa 3 , fujio murakami 1,2 1 grad. sch. of frontier biosci., osaka univ., japan; 2 sorst, jst, japan; 3 inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf-1 and its receptor cxcr4 in the precerebellar migration. we show that cxcr4 is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr4 and sdf-1 knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps3a-e068 involvement of an immunoglobulin superfamily molecule, neph2/mkirre in the migration of precerebellar neurons kazuhiko nishida 1,2 , kazuhide nakayama 1 , saori yoshimura 1,2 , fujio murakami 1,2 1 grad. sch. of frontier biosci., osaka univ., osaka, japan; 2 sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph1, neph2/mkirre, and neph3, only neph2/mkirre was strongly expressed in pcns. expression of neph2/mkirre was detected from e13.5 when pcns migrate tangentially. the expression level became weaker at p1, when pcns stop the radial migration, raising the possibility that neph2/mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph2/mkirre in the migration of pcns. hiroki umeshima 1,2 , toshio ohshima 3 , tomoo hirano 2 , mineko kengaku 1 1 lab. for neural cell polarity, riken bsi, wako, japan; 2 department of biophysics, kyoto university, kyoto, japan; 3 lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk5 (cdk5-dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin2-gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk5-dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito 1,2 , takahiko kawasaki 1,2 , tatsumi hirata 1,2 1 division of brain function, national institute of genetics, mishima, shizuoka, japan; 2 department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k252a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k252a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k252a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k252a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc51 functions with unc-76 to regulate axonal transport in drosophila hiroaki mochizuki 1 , hirofumi toda 1,3 , emiko suzuki 2 , joseph gindhart 4 , toshifumi tomoda 3 , katsuo furukubo-tokunaga 1 1 grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; 2 gene net. lab., natl. inst. genet. mishima; 3 beckman res. inst., city of hope, ca, usa; 4 dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc51, which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc51 is essential for neural development in drosophila. loss of function of drosophila unc51 results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc51 genetically interacts with unc-76, an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc51 mutants, unc-76 was separated from synaptotagmin vesicles. these results suggest that unc51 coordinates kinesin-cargo interaction via unc-76 to regulate dynamic axonal transport. ps3a-e072 change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi 1 , daisuke takahashi 2 1 life science inst. sophia university, tokyo, japan; 2 waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after 24 h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi (16500193) and kakenhi on priority areas (16027207) ps3a-e073 generation and analysis of region-specific rac1-deficient mice hidetoshi kassai 1 , masahiro fukaya 2 , eriko miura 2 , mizuho sakahara 1 , masahiko watanabe 1,2 , atsu aiba 1 1 div. cell biol., kobe univ. grad. sch. med., kobe, japan; 2 div. physiol. sci., hokkaido univ. grad. sch. med., japan rac1 is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac1 in the cns is poorly understood because of the embryonic lethality of rac1 knockout mice. in this study, we generated and analyzed region-specific rac1-deficient mice (emx1-rac1 ko mice) by the cre-loxp system, in which a promoter for emx1 homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx1-rac1 ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx1-rac1 ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac1 regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map1b containing nterminal 126 amino acids promoted neuronal death. to reveal the mechanism of map1b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map1b by two-hybrid system. alpha-tubulin was found to interact with map1b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map1b n-terminal has not yet been reported. beta-tubulin was also found to interact with map1b n-terminal. when ␤ tubulin was divided in 2 fragment at between amino acid 213 and 214, there was no interaction between ␤ tubulin fragments and map1b n-terminal. interaction needs the continuous region over aa 213 and 214. there were much proportion of round formed cos7 cells in n-terminal containing map1b transfected cells than in n-terminal lacking map1b transfected cells. there might be some interference in interaction between map1b and tubulin in cells express map1b containing n-terminal. otone endo 1,2 , masaaki mizuno 3 , yasukazu kajita 2 , jun yoshida 2 1 department of neurosurgery, ja kainan hospital, aichi, japan; 2 department of neurosurgery, nagoya university, nagoya, japan; 3 department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta 1 , masae yaguchi 1 , yumi matsuzaki 2 , yoshiaki toyama 3 , yutaka kawakami 4 , hideyuki okano 2 , masahiro toda 1,5 1 neuroimmunology research group, keio univ., tokyo, japan; 2 physiology, keio univ., tokyo, japan; 3 orthopaedic surgery, keio univ., tokyo, japan; 4 institute for advanced medical research, keio univ., tokyo, japan; 5 neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd11c + dcs, cd8␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd8␣ + dcs compared to cd11c + dcs. these results suggest that cd8␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps3a-e077 von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo 1 , hiroshi kanno 1 , takaakira yokoyama 1 , shuichi nakano 2 , naoki sugimoto 2 , nahoko kobayashi 3 , tetsuhiko yoshida 3 , isao yamamoto 1 1 dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; 2 fiber and dept. of chemistry, konan university, kobe, japan; 3 toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly 100%, and peptide with the elongin c binding site (residues 157-171) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps3a-e078 the effect of pueraria mirifica on erk1/2 and s-100 following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e2) on the expression of erk1/2 and s-100 following sciatic nerve crush and transection in rats. protein levels of perk1/2 and s-100 in distal segments of nerve at day 7 were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e2, caused a significant decrease in the expression of perk1/2 levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk1/2 phosphorylation. following treatments, levels of s-100 were significantly decreased both in crushed and transected nerves with respect to control group at p < 0.05. this estrogenic effect was blocked by ici182,780. because of their structural similarity to e2, pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day 7. this study suggested that pm as well as g could enhance nerve regeneration like e2 by interfering with the injury-induced erk signaling pathway. yasuhiro kato 1 , takafumi suzuki 2 , kunihiko mabuchi 2 1 department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; 2 department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed 5 untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf-3 and stat3, which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p6-10 wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y27632, a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level 7 days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin (5ht) positive cells increased in number progressively from 1 day to 6 weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and 5ht cells surrounded the axons and glia. thus, 5ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda 1 , yukio someya 2 , ryo kadota 2 , chikato mannoji 2 , tomohiro miyashita 2 , atsushi murata 3 , masashi yamazaki 2 1 department of orthopaedic surgery, togane hospital, 2 department of ortopaedic surgery, graduate school of medicine, chiba university, japan; 3 division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected 24 h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt (2.8 mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), 5-bromo-2 -deoxyuridine-5 -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days 1 after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days 2 after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day 2 after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps3a-f087 early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski 1 , hanna kozlowska 1 , marcin jurga 1 , aleksandra habich 1 , elzbieta wanacka 1 , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed 24 h, 48 h, 72 h and 7 days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue 1, 2, 3 and 7 days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext2, one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext2 mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican-1 and syndecan-1 were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe 1 , hiroe sagawa 2 , masahiro ichikawa 3 , yoshihito tokita 1 1 dept. perinatol., int. dev. res., kasugai, japan; 2 dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; 3 dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y39983, can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for 14d. after fixation, the neurites were stained with anti-tuj1 antibody to obtain number and length of tuj1 neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day 12, wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of 15-days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of 15-days-old embryonic mice by culturing in dmem/f12 medium for 9 days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of 0-9 div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for 3-9 div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for 7 days. dha increased the number of tuj1(+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first 24 h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi (17590205) ps3a-f092 interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j19 kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± 10 • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps3a-f094 effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating 3d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the 3d structure of surrounding stimuli defined by binocular disparity. ps3a-f095 rotational motion aftereffect in positive direction for 3-dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for 3-dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known 3dimensional mae. this finding suggests that the visual system has a mechanism that detect 3-dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j19 takanori uka, ryo sasaki department of physiology 1, juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch (3 deg) of a center/surround display presented 10 degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always 0 percent. for each of 11 subjects, we calculated direction discrimination thresholds (at 82% correct) at each surround size. consistent with crowding, thresholds increased when surround size was 4.5 and 6 degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was 9 and 12 degrees, relative to 6 degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps3a-f097 generation of receptive fields in higher visual areas based on v1 columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v2 and v4, respond to stimuli with complex shapes. how do these neurons integrate signals from v1? in particular, does the well-known columnar organization of v1 play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v1 neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about 15% of model neurons exhibit responses similar to those of v2 and v4 neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a 3-d surface slant is perceived (blakemore, 1980) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa 2006) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext(13041033), jsps(13308048), coe21 ps3a-f099 consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi 1 , kota sasaki 2 , izumi ohzawa 1,2 1 grad. school of frontier biosci., osaka univ., japan; 2 grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and 2-d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by 2-d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext(13041033), jsps(13308048), coe21 ps3a-f100 firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate 1,2 , hiroshi tamura 1,3 , ichiro fujita 1,3 1 graduate school of frontier biosciences, osaka university, osaka, japan; 2 jsps, japan; 3 crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi 16-07757 ps3a-f101 spatial-frequency dependency of receptive field size and surround suppression in lgn and v1 hironobu osaki 1 , tomoyuki naito 2 , osamu sadakane 2 , masahiro okamoto 3 , hiromichi sato 2,3 1 med. sch., osaka univ.; 2 grad. sch. med., osaka univ.; 3 grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v1), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v1 (n = 93) and lateral geniculate nucleus (lgn) (n = 63) in anesthetized cats. first, we found that v1 neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v1 has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura 1 , akichika mikami 2 , kazuo itoh 1 1 department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; 2 department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v3a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v3a, we injected a bi-directional tracer biotinylated dextran amine into the v3a. our results indicated that the v3a has connections with the occipital, parietal and temporal cortices. the v3a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v3a has commissural connections with the v3, the v3a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v2. these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps3a-g103 activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera (100 fps), and were also monitored with a 2d or 3d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity 10-40 ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps3a-g104 timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki 1,2 , arman abrahamyan 3 , catherine stevens 3 , andreas a. ioannides 1,2 1 brain science institute, riken, saitama, japan; 2 graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; 3 school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately 170 ms (n170) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within 100 ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about 100 ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto 1 , shoutaro akaho 1 , kenji fujikumi 2 , yasuko sugase-miyamoto 1 , masato okada 3 1 aist, ibaraki, japan; 2 ism, tokyo, japan; 3 university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of 45 neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of 45 neurons for 38 visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps3a-g106 relationship between color and shape selectivity in area teo of the monkey masaharu yasuda 1,2 , hidehiko komatsu 1,2 1 national institute for physiological science, okazaki, japan; 2 sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps3a-g107 neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a 3-choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was 0.58, a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across 2-3 simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant (17022025) ps3a-g108 behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki 1 , atsuko komori 1 , tomonori manabe 2 , toshihiko hosoya 2 , hiroshi sagara 3 , emiko suzuki 3 , hitoshi okamoto 2 , ichiro masai 1 1 masai initiative research unit, riken, wako, japan; 2 riken bsi, wako, japan; 3 ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde6c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde6c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde6c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until 9 day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde6c activity is important for the sensitivity of vision. ps3a-g109 localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: 50-94 years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from 50 to 80 years of age, and showed a decline and lack of ir in older retinas (85-94 years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from 50 to 91 years of age, and absent at 94 years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps3a-g110 temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa 1 , norio fujimaki 1 , toshihide imaruoka 2 1 nict, kobe, japan; 2 kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak (117, 215 ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity (125, 237 ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around 196 ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano 1,2 , derek arnold 3 , alan johnston 4 , tsunehiro takeda 1 1 univ. tokyo, chiba, japan; 2 ntt cs lab., kanagawa, japan; 3 univ. sydney, sydney, australia; 4 ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, 2003) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around 10 hz and matched with the alpha frequency of meg. as 10 hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical 10 hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps3a-g112 the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya 1,2 , kei ito 1,2,3 1 center for bioinform., imcb, univ. of tokyo, tokyo, japan; 2 dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; 3 bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified 12 discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal4 enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps3a-g113 dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi 1,2 , toshiki tani 2 , shigeru tanaka 1,2 1 graduate school of life science & systems engineering, kyushu institute of technology, japan; 2 laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of 5 weeks. we also found that when the goggles were removed after 1 or 2 weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, 2003) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than 0.3 hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps3a-g116 retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., 2003. neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada 1 , yasuhiro enami 1 , hiroshi jouhou 2 , takehiko saito 3 , kaj djupsund 4 1 tokyo metropol. univ., hino, tokyo; 2 astellas pharma. inc., osaka, japan; 3 suny upstate med. univ., center for vision and ophthal., ny, usa; 4 univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph 0 ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph 0 only 0.2 units from the baseline of 7.60, we found a remarkable reduction of the conduction velocity by 20-30%, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps3a-g118 analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi (17500195) ps3a-g119 stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of 30% (17 of 57) of neuron pairs (inter-neuronal distance >500 m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the 57 pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to 28 visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in 71% (12/17) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii 1,2 , toshihiko hosoya 1 1 bsi, riken, japan; 2 dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over 200 ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v1). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v1. this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v1 neurons. masayoshi tsuruoka 1 , masako maeda 1 , bunsho hayashi 1 , ikuko nagasawa 2 , tomio inoue 1 1 dept. physiol. showa univ. sch. dent. tokyo, japan; 2 dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t13-l2 dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention (80 mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil (5%, 1 ml). the increased emg activity significantly reduced following bilateral l6-s3 ventral rhizotomies. a baseline emg did not significantly alter when the l6-s3 ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps3a-g123 hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba40) was significantly greater (p < 0.001, uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < 0.001, uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm8 in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm8 agonist (l-menthol: 0.06-6 mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = 7) or boo rat (n = 8). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm8 on the urothelium cell of the urinary bladder. ps3a-g125 caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito 1 , a.d. craig 2 1 dept. physiol, shimane univ. sch. med., izumo, japan; 2 atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol 2005) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns40413 ps3a-g126 ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps3a-h128 fasting induced long-chain fatty acid receptor gpr120 expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor 120 (gpr120) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of 48 h fasting on gpr120 expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr120 mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. 48 h fasting induced gpr120 mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr120-like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr120 at least fasting period. ps3a-h129 ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that 3hydroxybutylate (3hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls (4v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of 4v respond to the change in 3hb. change in the intracellular calcium concentration ([ca 2+ ] i ) in vitro was measured in dispersed ependymocytes taken from the 4v in rats. the present results showed that the [ca 2+ ] i increased in response to 3hb, but the increase was blocked by ␣-cyano-4-hydroxycinnamic acid, which is a monocarboxylate transporter 1 (mct1) inhibitor. immunohistochemistry showed that mct1-immunoreactivities were located on the 4v ependymocytes. these results indicate that the ependymocytes may sense 3hb through a mct1-dependent mechanism. research funds: kakenhi 14360177 ps3a-h130 comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin (1.3 mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with 6-hydroxydopamine (6-ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at 4 weeks after 6-ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at 0 mm; anteroposterior, +3 mm; lateral and −5 and −7 mm; dorsoventral from bregma at 2 weeks after 6-ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better 6-ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b 17791323 most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: (1) retrieving the food with a rake (a motortool), (2) retrieving the hidden food with a mirror-attached rake, (3) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, (4) moving a mirror hung along the rail by hand to find the food, (5) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada 1,2 , shigehiro miyachi 2 , michiko imanishi 2 , masato taira 1 , masahiko takada 2 1 div. applied system neurosci., nihon univ. sch. med., tokyo, japan; 2 dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area 9 (9m), dorsal area 46 (46d), and ventral area 46 (46v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex 3 days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from 9m, 46d, and 46v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that 9m, 46v, and 46d receive multisynaptic projections from the entorhinal cortex, area 36, and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps3a-h137 new neural activities of reward anticipation and task errors h. ogawa 1 , h. ifuku 2 , t. nakamura 3 , s. hirata 4 1 kumamoto kinoh hosp, kumamoto, japan; 2 fac educ, kumamoto univ., kumamoto, japan; 3 nat kikuchi hosp, kumamoto, japan; 4 dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, 3 & 1-2) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca 2 s after led offset at correct trials. relations between cues and responses were reversed. of 169 reward-related neurons found, 88.7% showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = 96) only at correct trial, i-type (n = 5) at around possible reward onset only at incorrect trials, and c-i type (n = 49) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all 3 types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai 1 , takayuki hosokawa 2 , masato inoue 1 , akichika mikami 1 1 section of brain sciences, primate research institute, kyoto university, inuyama, japan; 2 department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps3a-h140 neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue1), and centrally allocated one of the objects presented in cue1 (cue2). the frequency of which object was presented as cue2, i.e., task context, was manipulated across blocks. we focused on cue2 period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps3a-h141 anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki 1 , barry j. richmond 3 , munetaka shidara 1,2 1 grad. sch. of tsukuba univ., ibaraki, japan; 2 neurosci. ri., aist, tsukuba, japan; 3 lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in 2 conditions: (1) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], (2) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition 84/120 neurons responded in all trials. in the certain condition 50/84 neurons responded in the rewarded trials only. 48 of these 50 showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas17022052), aist masamichi sakagami 1,3 , kosuke sawa 2 , xiaochuan pan 1,3 1 bsrc, tamagawa university, tokyo, japan; 2 senshu university, kanagawa, japan; 3 presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a1-b1-c1 and a2-b2-c2. after monkeys could acquire the sequences, new pairs of stimuli (i.e., d1 and d2, e1 and e2, etc) were introduced to associated with b1 or b2 (d1-b1, d2-b2, etc). the asymmetric reward rule was instructed by pairing c (c1 or c2) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a1 and a2, which were associated with c1 and c2 in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi (17022037), hsfp, presto, jst ps3a-h143 reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan 1,2 , kosuke sawa 1,3 , masamichi sakagami 1,2 1 bsrc, research institute, tamagawa university, japan; 2 presto, jst, japan; 3 department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a1-b1-c1 and a2-b2-c2. the asymmetric reward rule was instructed by pairing c (c1 or c2) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of 202 neurons from the lateral pfc, 31% showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about 10% of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated 3 times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every 4 pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than 3 consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps3a-h146 neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima 1 , hajime mushiake 1 , jun tanji 2 1 department of physiology, tohoku university school of medicine, sendai, japan; 2 brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the 2-sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps3a-h147 neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. 2005) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = 73) preferentially when the instruction was associated with small reward. the instructions activated 3 groups of putamen neurons preferring small reward-(n = 19), large reward-action (n = 3) and both types of action (n = 26). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by 30-50 and 100-150 ms, respectively. putamen neuron activation lead that of cm neurons by 50-70 ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi (17022032) ps3a-h148 encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t1), second (t2) and third (t3) choices with reward probabilities of about 30, 50 and 80%, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r1, 95%). most da neurons (66/85) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t1 to t2 and to t3 trials, then decreased in r1 trial. in another task with two repetition trials (r1 and r2, 96%), magnitude of start-cue responses decreased gradually from t3 to r1 and to r2 trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi (17022032) ps3a-h149 striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada 1,2 , hitoshi inokawa 1 , minoru kimura 1 1 dept. of physiol. kyoto prefect univ. med., kyoto, japan; 2 jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded 146 striatal projection neurons from a monkey. after depressing a start button, the monkey chose 1 of 3 target buttons with correct rates at 1st, 2nd, 3rd and repetition trials of 33, 50, 89 and 96%, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice (11/55) rather than to upcoming choice of target (2/55). during the target choice, another subset of neurons showed firings selective to choosing particular target (29/57) rather than to expected reward probability (12/57). after the target choices, another group of neurons fired related to expected reward (41/56) rather than to chosen action (19/56). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi (17022032), jsps fellows ps3a-h150 encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of 169 tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose 1 of 3 target buttons at average correct rates of 33 (first), 50 (second), 89 (third) and 96% (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans (80/169) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi (17022032) ps3a-i151 representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda 1 , k. samejima 2 , k. doya 3 , m. kimura 1 1 dept. physiol., kyoto pref. univ. med.; 2 brain sci. res. center, tamagawa univ.; 3 irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of 236 striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a 1 s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of 10, 50 or 90%. a linear regression of neuronal discharge rates showed: 39 neurons encoded reward values of either action during delay period before go signal, with most (64%) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = 33), while other 37 neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi (17022032) ps3a-i152 delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c1), first delay (d1), second cue (c2), second delay (d2), and response periods. activity during the d1 and d2 periods was analyzed in this study. firing rates during the d1 period linearly depended on c1 durations. on the other hand, d2 period activity depended on trial types (ls and sl), but not on the variety of c2 durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi (17021039) ps3a-i153 neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku 1 , ryoi tamura 1 , teruko uwano 1 , taketoshi ono 2 1 dept. integrative neurosci., univ. toyama, toyama, japan; 2 dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize 4 paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi (16500260 17021016) ps3a-i154 reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori 1 , mikio inagaki 1 , wu lisa 2 , taijiro doi 1 , eishi hirasaki 1 , hiroo kumakura 1 , ichiro fujita 1 1 osaka univ., japan; 2 massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from 17022025 and takeda science foundation ps3a-i155 response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and 7a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, (1) performance of visually guided sex discrimination task and (2) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) (16209006) (sa) and coe program in kit from the mext ps3a-i157 activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa 1 , satoshi katai 2 , masanori saruwatari 1 , masato inoue 1 , akichika mikami 1 1 section of brain sciences, primate research institute, kyoto university, inuyama, japan; 2 third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro 2004) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto 1 , tetsuya iidaka 2 1 department of psychology, nagoya university, nagoya, japan; 2 department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: 30-80 hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the 400-600 ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the 600-700 ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included 16 images (4 objects × 4 views). 4 novel objects were generated by deforming a prototype in four directions. for 30 deg-interval object sets, 4 views were obtained by rotating each object with the interval of 30 deg, 60 deg-interval set and 90 deg-interval set were with 60 deg and 90 deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across 60 deg separated views was significantly higher in the trials with 30 deg-interval sets than those with 60 deg-interval sets. similarly, the difference was also found in the performances across 90 deg separated views between those with 30 deg-interval sets and 90 deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps3a-i160 brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps3a-i161 obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, 2005 ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: 1682 middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps3a-i162 sirna-induced nr1 knockdown causes hypofunction of nmda-r and cognitive deficit m. saji 1,2 , t. utida 2 , a. ohnishi 2 , k. noda 1 , m. ogata 1 , h. akita 1 , n. suzuki 1,2 1 physiol, health sci. sch. kitasato univ., sagamihara, japan; 2 brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr1 using hvj-envelope vectors. we confirmed that marked down-regulation (50%) of nr1 expression occurred only in the hippocampus among various brain regions 4-8 days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr1 knockdown, the nr1 down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr1 knockdown, the nr1 down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka 1,2 , hiroshi ageta 2 , ikuko yao 2 , kaoru inokuchi 2 , yutaka kirino 1 , mitsutoshi setou 2,3 1 graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; 2 mitsubishi kagaku institute for life sciences, tokyo, japan; 3 okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl3 mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl3 is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl3 mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl3 wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps3a-i164 decreased interneurons in the pax6 mutant mouse limbic system hasumi haba 1 , tadashi nomura 1 , yoshinobu hara 1,2 , noriko osumi 1,2 1 div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; 2 crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax6 heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax6 heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at 8 weeks, but not at 4 weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp1) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine (0.2 mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: 16790712, 17025055 ps3a-i166 gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba 1,2 , satoko hattori 1 , hiroaki hori 1 , tetsuo nakabayashi 3 , hiroshi kunugi 1 , ryota hashimoto 1 1 department of mental disorder research, national institute of neuroscience; 2 tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; 3 musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp1) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of 38 patients with schizophrenia and 38 age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: 16790712, 17025055 ps3a-i167 genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto 1 , hiroko noguchi 1 , hiroaki hori 1 , tetsuo nakabayashi 2 , satoko hattori 1 , sachie chiba 1 , seiichi harada 2 , osamu saito 2 , hiroshi kunugi 1 1 department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; 2 musashi hospital, ncnp, kodaira, japan; 3 tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp1) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in 165 healthy volunteers and 72 patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: 16790712, 17025055 ps3a-i168 detection of 18f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura 1 , nicola bright 3 , toshio yanagida 1 , masatoshi takeda 2 , paul grasby 3 1 department of physiology, osaka university, japan; 2 department of psychiatry, osaka university, japan; 3 cyclotron unit, imperial college, hammersmith hospital, uk we used 18 f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and 18 f-dopa scans were obtained from a database of scans published in mcgowan et al. 2004 , archives general psychiatry. the 14 schizophrenic patients all met dsm-iv criteria on medication and 10 healthy volunteers were compared. we attempted to improve the quality of the 18 f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in 18 f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > 0.02). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p300 component of the visual eventrelated potential in 14 patients with schizophrenia and 14 healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p300 amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p300 amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p300 amplitude between the 2 faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area 10 between the 2 faces in controls, while in patients, there was no significant activity difference between the 2 faces. stimulation with crying face induced higher activities in the 10 and right 21 areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps3a-j170 inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura 1 , shoji tanaka 2 , koki yamashita 2 , motoichiro kato 1 , haruo kashima 1 1 department of neuropsychiatry, school of medicine, keio university; 2 department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps3a-j171 pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc1, a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as 5-ht6 are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in 7 of the 10 sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps3a-j173 alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta 1 , ryo yamamoto 1 , shigeki sugiura 2 , kaoru inokuchi 3 , nobuo kato 1 1 dept. integrat. brain sci., grad. sch. med., kyoto univ.; 2 nara med. univ.; 3 mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer1a/vesl-1s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer 1a protein by diffusion from patch pipettes. homer 1a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer 1a may be a biological mediator of ecs effects. masanori kasai 1 , nozomi miyagi 1 , norio kawashiro 2 , daisuke torizuka 2 1 dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; 2 sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant (1.0 mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the 11th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the 2nd, 7th, 14th, 21st, 28th and 35th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc (75.1 ± 12.1 g/dl, n = 7) on the 35the day was significantly lower than that in intact rats (139.4 ± 11.9 g/dl, n = 7). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps3a-j175 molecular cloning of a novel candidate for ethanol-responsive genes, yy1ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy1ap (yy1-associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy1-binding and transactivation domains previously demonstrated in yy1ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy1 genes was demonstrated in the same neurons. analogous to yy1ap as a co-activator of transcription factor yy1, it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps3a-j176 excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr2b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr2b-selective nmda antagonists ifenprodil (3 and 10 m) and ro25-6981 (0.1 and 1 m) markedly inhibited degeneration of orexin neurons induced by 24 h application of nmda (45 m) or qa (1.5 mm). we also show that stimulation of gaba a receptors by muscimol (10 and 30 m) or isoguvacine (30 and 100 m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba (100 m) plus a gaba uptake blocker nipecotic acid (1 mm) was abolished by a gaba a antagonist picrotoxin (100 m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr2b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh795). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of 1 ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps3a-j178 a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi 1 , osamu fukayama 2 , takashi sato 2 , takafumi suzuki 2 , kunihiko mabuchi 1,2 1 dept. biomed. eng., univ. tokyo, tokyo, japan; 2 dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps3a-j179 on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki 1 , yasuhiko jimbo 2 , kenji yasuda 1 1 department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; 2 department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip 8 um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are 100 khz in 64ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita 1 , tetsu okumura 1 , kazuo okanoya 2 , jun tani 1 1 lab. for behavior & dynamic cognition, riken-bsi, japan; 2 lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps3a-k181 local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura 1 , yuichi yamashita 1 , kazuo okanoya 2 , jun tani 1 1 behav & dynamic cognition, riken-bsi, saitama, japan; 2 biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata 1 , shinya ishii 1 , munetaka shidara 2 1 dept. of e&e engineering, oita univ., oita, japan; 2 grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [1] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, 2006 behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than 3 consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki 1 , shinya yamamoto 2 , sunao uchida 3 , shigeru kitazawa 4,5 1 faculty of hum sci., waseda univ., tokorozawa, japan; 2 neurosci. res. inst, aist, tsukuba, japan; 3 faculty of sport sci., waseda univ., tokorozawa, japan; 4 dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; 5 crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ±80 ms, s.d. = 80 ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about 50 ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi (15200031) ps3a-k185 single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama 1 , fumiko sato 2 , ken nakazawa 3 , kenichi ono 3 1 chiba university, japan; 2 field of home economics, faculty of education, chiba univ., japan; 3 department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm-3000, shi-madzu) over the frontal area (45 channels) for 7 volunteers (19-23 y). thirty tasks were presented visually every 30 s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between 2 tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was −0.4+/−0.45 between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi 1,2 , kosuke hamaguchi 3 , masato okada 1,2,3 1 department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; 2 jst, japan; 3 riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated 2 different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi (14084212) and (16500093) ps3a-k187 auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa 1,2 , sachiko koyama 1,2 , takashi omori 3 , takashi morotomi 4 1 research institue for electronic science, hokkaido university, sapporo, japan; 2 japan science and technology agency, saitama, japan; 3 graduate school of information science and technology, hokkaido university, sapporo, japan; 4 sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = 13). the erps were elicited by tones (500 hz) with different durations (8, 16, 32, 64, 128, 256 ms) and gaps (8, 16, 32, 64, 128 , 256 ms) embedded in a continuous pure tone (500 hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for 32 and 64 ms were high both for the tones (32 ms, 38%; 64 ms, 61%) and the gaps (32 ms, 46%; 64 ms, 46%). ps3a-k188 determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps3a-k189 theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to 50% till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene, and 1-[4 (trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc42 in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp5 expression correlates with wnts/bmps in the cortical hem, sp8 with fgfs in the cop, and sp9 with shh in the ventral midline and mge. by using in utero electroporation, we show that sp8 regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp8 and fgf8 exhibit reciprocal induction, indicating that sp8 is a positive feedback regulator of fgf8. surprisingly, though, ectopic expression of both sp8 and its dominant active form shift cortical areas in the opposite manner to fgf8, suggesting that sp8 activates additional targets that overcome fgf8 function. our results indicate that fgf10 is an additional target of sp8, showing effect on patterning similar to sp8. these findings indicate that sp8 balance the proper cortical arealization through fgf8 and fgf10. research funds: nihr37ns31558 ps3p-c003 fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira 1 , kotaro hattori 1 , takeshi yagi 2 , shigeki yuasa 1 1 dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; 2 kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi 1 , kazuko keino-masu 1 , tatsuyuki ohto 1 , satoshi kunita 2 , satoru takahashi 2 , masayuki masu 1 1 dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; 2 laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp1, has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp1 gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext1 gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp1-deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp1/sulfatase-1 and sulffp2/sulfatase-2, which have hs 6-o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp1 and sulffp2. here we report the isolation and the characterization of the third homologue, sulffp3. sulffp3 has about 56% and 71% overall amino acid homology with sulffp1 and sulffp2, respectively. at 24 h postfertilization, sulffp3 is expressed in the ventral region of spinal cord, whereas sulffp1 is expressed only in the floor plate and sulffp2 is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp3 will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp2 was shown to mediate the repulsive effect of sema3a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp2, 3, 4, and 5 are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class 3 semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps3p-c007 developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa 1 , n. yamamoto 2 , m. yoshimoto 2 , t. yasuda 1 , k. maruyama 1 , t. kage 1 , h. takeda 3 , h. ito 2 1 nat. inst. rad. sci., chiba, japan; 2 nippon med. sch., japan; 3 tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax6-or dlx2-positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er81 is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er81 homologue (zfer81), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer81 was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er81 expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is 70% identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta 1,2 , hiroo takahashi 1,2 , takafumi shintani 1,2 , kazuma etani 1 , masaharu noda 1,2 1 div. of mol. neurobiol., nibb, okazaki, japan; 2 crest, jst, japan in the developing chick retina, the expression of bmp4 is relieved by that of bmp2 at around e5 with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina2, are expressed in a double-gradient fashion from e6 on like ventroptin and bmp2. when bmp2 expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp2 knockdown and ephrina2-misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp4 to bmp2 thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en2, wnt7b and pcp2/l7 gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and 4di-10asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and 4di-10asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or 4di-10asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru 1,2 , takahisa taguchi 1 1 aist, ikeda, japan; 2 presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a 2-dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps3p-d014 laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps3p-d015 synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki 1 , toshio aoyagi 2 1 department of physics, kyoto university, kyoto, japan; 2 graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf-1 mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into 4 weekold wistar strain rats in several doses followed by successive injections of brdu for the next 4 days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as 1 mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps3p-d017 the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato 1 , james e. goldman 2 1 division of pharmacology, national institute of health sciences, tokyo, japan; 2 department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p0 rat svz, sagital sections of forebrain were made at p3 and cultured for 6 days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣3 integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of 2nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps3p-d020 localization of neurotrophin receptors trka in pc12 cells: 3d reconstruction analysis of membrane proteins tomoki nishida 1 , hiroshi jinnai 2 , tatuo arii 3 , akio takaoka 4 , ryoichi yoshimura 1 , yasuhisa endo 1 1 department of applied biology, kyoto institute of technology, kyoto, japan; 2 department of polymer science and engineering, kyoto institute of technology, kyoto, japan; 3 national institute for physiological sciences, myodaiji, okazaki, japan; 4 osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin-1 in pc12 cells, analyzed by high-voltage electron microscopy, and reconstructed 3d structure of their subcellular distribution by imod. our results indicated that localization of caveolin-1, known as an integral membrane protein of caveolae, was never found in the invagination structure in pc12 cells, but trka and caveolin-1 immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd1, a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd1 mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd1 in the wnt-mediated neuronal development. there are three subtypes of mouse ccd1 gene products, ccd1a, ccd1b and ccd1c, which are generated by different promotor usage. mouse ccd1a as well as zebrafish ccd2a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi (15770137, 17300098) ps3p-d022 analysis of a role of r-spondin2 on proliferation of the cortical neuroepithelium yumiko hatanaka 1 , masahiro yamaguchi 2 , fujio murakami 3 , masayuki masu 1 1 grad. school of comprehensive human sci., univ. of tsukuba, japan; 2 grad. school of med., univ. of tokyo; 3 grad. school of frontier biosci., osaka univ r-spondin2 (rspo2) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. 2004) . rspo2 is expressed in the developing medial cerebral wall and transgenic mice expressing rspo2 in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. 2005) . since wnt3a has a role for expansion of caudomedial cortical progenitor cells (lee et al. 2000) , these findings lead us to the idea that rspo2 may synergistically promote proliferation of cortical neuroepithlial cells together with wnt3a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day 11.5 mouse. an application of wnt3a on these cells increased level of the reporter expression, and an addition of rspo2 further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt3a and rspo2 directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il-1␤, il-11, lif, gdnf) or lowered o2 (3.5%), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells (1.86-fold) as compared to normal o2. cytokine cocktail significantly increased th (+) cells (2.11-fold) compared to nontreated control. treatment of lif and il-1␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps3p-d024 structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui 1 , terukazu nogi 1 , mitsuharu hattori 2 , kenji iwasaki 1,3 , junichi takagi 1 1 research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; 2 dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; 3 core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer2 and vldlr), resulting in tyrosine phosphorylation of dab1. in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic 3d reconstruction of four-domain reelin fragment (i.e. r3-6) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r5-6) was capable of binding to the receptor (apoer2), and was also able to induce tyrosine phospholylation of dab1 in primary neuronal culture. ps3p-d025 effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax3 and pax7 compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps3p-d027 neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res 79:287, 2005) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi-3k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps3p-d028 fabp7 is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa 1 , miho matsumata 2,3 , yuji owada 2 , shigeki yuasa 1 , noriko osumi 2,3 1 natl. inst. of neurosci., ncnp, tokyo, japan; 2 tohoku univ. sch. of med., sendai, japan; 3 crest, jst pax6 transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax6 is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp7, a downstream of pax6, regulating maintenance of embryonic neural stem/progenitor cells (arai et al., 2005) . fabp7 was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). 56% of fabp7-expressing cells co-expressed gfap (a marker for early progenitors), and 33% of them co-expressed psa-ncam (a marker for late progenitors). fabp7 expression was also overlapped with pax6, and expression of fabp7 was down-regulated in the dg of pax6 deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp7 knockout mice. taking all together, it is concluded that fabp7 is required for maintenance of neural stem/progenitor cells in dg. ps3p-d029 involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day 4-7. sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani 1,6 , shiki okamoto 2 , masato imada 3 , kouichi itoh 4 , atsushi irie 5 , hitoshi sakuraba 6 , hideo kubo 7 1 department of molecular biologu, ohu univ., koriyama, japan; 2 dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; 3 dept. anatomy, nihon univ. shl. med., tokyo, japan; 4 dept. mol. pharma., univ. tokushima bunri, sanuki, japan; 5 dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; 6 department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; 7 dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam-2 shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam-2 is possible. in the present, we show that the isolated randam-2 high+ cells enrich nscs. the randam-2 high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam-2 low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam-2 is a usefule marker for the isolation of nscs by facs. yasuharu takamori 1 , yasuhisa tamura 1,3 , yosky kataoka 1,2 , yilong cui 1,3 , hisao yamada 1 1 department of anatomy and cell science, kansai medical university, osaka, japan; 2 department of physiology, osaka city university graduate school of medicine, osaka, japan; 3 morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b1 and b2 are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b1 (++), b2 (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b1 (+++), b2 (+), and mature neurons possess lamin a/c (++), b1 (+), b2 (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka 1 , yuichi tanaka 1 , tatsuhiro hisatsune 1 1 department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps3p-e033 new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba 1,2 , hideo namiki 2 , tatsunori seki 1 1 dept. of anat, juntendo univ. sch. of med., tokyo, japan; 2 integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p5. three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., 1987) . the dividing pattern is currently under investigation. akiya watakabe 1 , noritaka ichinohe 2 , sonoko ohsawa 1 , tsutomu hashikawa 3 , kathleen s. rockland 2 , tetsuo yamamori 1 1 div. of brain biol, nibb, okazaki, japan; 2 lab. for cortical organization and systematics, bsi, riken, wako, japan, 3 lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer 6 neurons in several areas of monkey neocortex. we previously reported that nurr1, ctgf and sema3e mrnas are specifically expressed in subsets of layer 6 neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer 6. by double ish, layer 6 neurons in monkeys are roughly divisible into cck(+) and sema3e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema3e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi 17024055 ps3p-e035 rbp-j regulates the cortical laminar formation kenji tanigaki 1 , kazue muraki 1 , norio yamamoto 2 , tasuku honjo 2 1 shiga medical center, research institute, shiga, japan; 2 department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps3p-e036 search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi 1 , tomoko ikawa 1 , yuya kasukawa 2 , hironori ueda 2,3 , kazuki kurimoto 4 , michinori saitou 4 , fumio matsuzaki 1,5 1 lab. for asymmetric cell division, cdb, riken, kobe, japan; 2 functional genomics subunit, cdb, riken, kobe, japan; 3 lab. for systems biology, cdb, riken, kobe, japan; 4 lab. for mammalian germ cell biology, cdb, riken, kobe, japan; 5 crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. 30 samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi (15700264) ryosuke tatsuno 1 , tomoaki sai 2,3 , masahiro otsu 2 , kuniko akama 1 , takashi nakayama 4 , tosifusa toda 5 1 grad. sch. of sci. and tech., chiba univ., chiba, japan; 2 lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; 3 dept. orthop. surg., jikei univ. sch. med., tokyo. japan; 4 dept. biochem., yokohama city univ. sch. med., yokohama, japan; 5 proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using 2-d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps3p-e038 identification of se90, a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified 150 genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se90 in developing and matured cns. teiichi furuichi 1 , akira sto 1,2 , yukiko sekine 1 , noriyuki morita 1 , tetsushi sadakata 1 , satoshi shoji 1 , jin-hong huang 1 , toshio kojima 2 1 laboratory for molecular neurogenesis, riken brain science institute, japan; 2 comparative systems biology team, riken genome sciences center, yokohama 230-0045, japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki 1 , makoto mizuno 2 , kazuhisa sakai 4 , yoshimoto kiyohara 4 , kazuhiko yamaguchi 3 , tsutomu hashikawa 4 , hiroyuki nawa 2 1 institute of biomedical engineering, waseda university, tokyo, japan; 2 department of molecular neurobiology, brain research institute, niigata university, niigata, japan; 3 laboratory for memory and learning, bsi, riken, saitama, japan; 4 laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg1 knock out mice (nrg1 type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps3p-e041 psd-95 protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama 1,2 , satoshi wakisaka 1 , shiho honma 1 , akira ito 1,2 , kei azuma 1,2 , mikihiko kogo 1 1 department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; 2 first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd-95 implicates in formation and maturation of excitatory synapses. psd-95 regulates the localization of the nmda receptor by means of binding with nr2. rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd-95 protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd-95 expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf97541. in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps3p-e043 ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps3p-e044 comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi 1 , kenji mizutani 2 , hiroto ogawa 3 , kohji hotta 1 , kotaro oka 1 1 ctr. for biosci. and info, keio univ., yokohama, japan; 2 neuro, karolinska inst, stockholm, sweden; 3 bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to 50 cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion (25% or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps3p-e045 three dimensional (3d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in 3d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that 3d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether 3d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in 3 monkeys. of a total of 77 p-cells that were examined during both frontal and vergence-pursuit, 46% discharged for both, 40% only for vergence, and 14% only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had 3d pursuit signals. majority (70%) of these p-cells discharged before onset of vergence eye movements with the typical lead time of 50 ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi (17022001) ps3p-e046 information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey13308, rr00165 aya takemura 1 , yumi murata 1,3 , kenji kawano 1,2 1 neurosci. res. insti, aist, tsukuba, japan; 2 dept. integ brain sci., grad. sch. med., kyoto univ., japan; 3 grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid (15 mg/ml, 36-37 l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at 20 • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of 58 ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps3p-e049 the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara 1 , kenji kunita 2 , kaoru maeda 1 , takeo kiyota 1 1 department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; 2 institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were 17 undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for 30 ms with 800-ms intervals. the oscillation trial (0.5-hz frequency and 2.5-cm amplitude) (65-75 s) was repeated 5 times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the 5th trial. a significant correlation was shown between 5th-1st trial differences in mean speed and vep amplitude (r = 0.79). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps3p-e051 primary motor cortex contributes to generating manual following response toshitaka kimura 1 , naoki saijo 1 , hiroaki gomi 1,2 1 ntt cs labs, kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m1), that (1) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and (2) intracortical inhibition within m1 assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m1. such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo 1 , hiroaki gomi 1,2 1 ntt cs labs., kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in 8 radial directions applied during reaching. the data show that the direction of the initial phase (150-200 ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase (250-300 ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps3p-e053 spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa 1 , hiroaki gomi 1,2 1 ntt cs labs., kanagawa, japan; 2 erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps3p-f055 influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka 1 , takashi obu 2 , kazuyuki kanosue 3 1 asmew, waseda university, saitama, japan; 2 graduate school of human sciences, waseda university, saitama, japan; 3 faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = 6) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at 1.5, 1.75, and 2.0 hz. over 99% success rate was observed in all tasks except oppo (78-97%). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps3p-f056 simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu 1 , tetsuro muraoka 3 , kazuyuki kanosue 1,2,3 1 faculty of human sciences, waseda university, saitama, japan; 2 faculty of sport sciences, waseda university, saitama, japan; 3 asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at 1, 1.33, and 1.66 hz. they were asked to perform 4 tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps3p-f057 activities of erector spinae muscles during jaw clenching in man kayoko yasunaga 1,2 , tadachika yabushita 1 , kazuo toda 2 , kunimichi soma 1 1 orthodontic science, tokyo med. & dent. univ., tokyo, japan; 2 div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada 1 , sachiko motoyama 1 , futoshi mori 1 , shigemi mori 2 1 department of veterinary physiology, yamaguchi university, yamaguchi, japan; 2 national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for 12-20 weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps3p-f059 neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima 1 , futoshi mori 2 , akira murata 1 , masahiko inase 1 1 dept. of physiol., kinki univ. schl. of med., osakasayama, japan; 2 dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m1 of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m1 hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m1 modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m1 output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi (16500271) ps3p-f060 activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara 1,2 , nobuhiko hatanaka 1,2 , masahiko takada 3 , atsushi nambu 1,2 1 school of life science, the graduate university for advanced studies, japan; 2 division of system neurophysiology, national institute for physiological sciences, japan; 3 tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu 1 , sei-ichi sasaki 2 , yuichiro cho 1 , kenji sato 1 1 anatomy and physiological science, tokyo medical and dental university, tokyo, japan; 2 department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s1 and s2 spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl7 -s3 spinal segments which contain afferent fibers of eas muscles. after 3 weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for 4 consecutive days. we compared adaptation in day1 and that in day4. the gain decrease for the first 700 saccades in day 4 (0.140 ± 0.036) was larger than that in day 1 (0.080 ± 0.021) (p = 0.026, n = 5, paired-t test). the rate of adaptation in day 4 (1.739 ± 0.393 × 10 −4 /sac) was higher than that in day 1 (1.120 ± 0.240 × 10 −4 /sac) (p = 0.011). the overall gain change (1800 saccades) in day 4 (0.219 ± 0.030) was larger than that in day 1 (0.112 ± 0.022) (p = 0.002). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi (16300129) ps3p-f064 asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v1. the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = 7) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by 4 s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in 60% of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida 1 , xiaofeng lu 1,2 , shogo ohmae 1 , toshimitsu takahashi 1,2 , shigeru kitazawa 1,2 1 dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; 2 crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of 16 targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi 17022033 ps3p-f066 frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa 1 , soichi nagao 1,2 1 lab. for motor learning control, riken bsi, saitama, japan; 2 sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps3p-f068 projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki 1,2 , kei ito 1,2,3 1 dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; 2 center for bioinform., imcb, univ. of tokyo, japan; 3 bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal4 enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening 4,000 strains, we identified about 180 candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps3p-f069 immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura 1 , yoshitaka ohtubo 1 , takashi kumazawa 2 , kiyonori yoshii 1 1 graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; 2 department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi (16300094) and the 21st coe program (center #19) granted by mext of japan ps3p-f070 inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa 1 , atsushi kuhara 1 , hitoshi inada 1 , eiji kodama 1 , takafumi mizuno 1 , ikue mori 1,2 1 lab. of mol. neurobiol., nagoya univ., japan; 2 institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx-7 gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx-7 mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx-7 mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps3p-f071 postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut1 and -2) during rat ob development from postnatal day 1 (p1) to p10 by in situ hybridization and immunohistochemistry. at p1 vglut1 immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut1 mrna signals were detectable in the mitral cell layer from p1 to p10. in contrast, vglut2 ir was prominent in the glomerulus at all days examined, and only at p1 and p3 in mitral cells. despite mitral vglut2 ir disappeared at p10, the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps3p-f072 v1r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito 1,2 , masumi ichikawa 3 , yuji mori 2 , kimiko hagino-yamagishi 1 1 tokyo metrop. inst. med. sci., tokyo, japan; 2 the univ. of tokyo, tokyo, japan, 3 tokyo metrop. inst. neurosci., tokyo, japan in rodent, v1r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v1r gene, which is expressed in the olfactory epithelium (oe). to examine when the v1rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified 23 v1r sequences. these v1rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v1rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps3p-f073 analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t1r2/t1r3 heterodimer plays a role as a sweet taste receptor. but, mice lacking t1r3 showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t1r3-independent sweetener binding site also exist in mice. in this study, to predict binding sites on t1r2/t1r3 and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [(1) sucrose, glcose, fructose, (2) saccharin, aspartame, acesulfame-k, glycine, (3) d-phenylalanine, (4) d-tryptophan, (5) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t1r2 and t1r3, respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu 1 , sachiko saito 2 , yuko murata 3 , ding ming 4 , tatsu kobayakawa 5 , robert f. margolskee 4 , yuzo ninomiya 1 1 sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; 2 saito sachiko taste and smell research institution, ibaraki, japan; 3 national res. institute of fisheries sci., kanagawa, japan; 4 dept. of physiol. & biophys., mount sinai sch. med., new york, usa; 5 national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t2r receptors which coupled with ggustducin. ps3p-f075 newborn infant body odor attenuates their mother's postpartum moods shota nishitani 1 , mayumi kokuryo 1 , tsunetake miyamura 2 , kazuyuki shinohara 1 1 div. neurobiol. & behav., nagasaki university, japan; 2 obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps3p-f076 human prefrontal activity in taste encoding: an fnirs study masako okamoto 1 , mari matsunami 2 , haruka dan 1 , tomoko kohata 2 , kaoru kohyama 1 , ippeita dan 1 1 national food research institute, tsukuba, japan; 2 nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = 10) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes (42 subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes (36 subfamilies) were expressed in the dorso-medial zone (zone 1) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone 1. these results indicate that there is a clear boundary between zone 1 and non-zone 1 areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone 1 tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi (16500196) ps3p-g079 taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to (1) best-taste profiles and (2) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi (11671860) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼100 members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi (16300105) ps3p-g081 identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka 1 , kazuhisa sakai 2 , tsutomu hashikawa 2 , kensaku mori 1 , masahiro yamaguchi 1 1 dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; 2 laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps3p-g083 nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi (15209061), kakenhi (17791325) ps3p-g084 integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than 25% of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi (17023012) ps3p-g085 odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to 8 classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than 2 classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi (13gs0007) ps3p-g086 odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa 1 , takaaki sato 2 , akira shimizu 1 , ken-ichiro tsutsui 1 , toshio iijima 1 1 div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; 2 res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range (20 hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps3p-g087 chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when 3 m lysine was spotted on ammonium chloride background, 0.003-0.01 m and 0.05 m background did not influence lysine chemotaxis, while 0.03 m background augmented and 0.07-0.1 m background suppressed the chemotaxis. in contrast, when 0.1 m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps3p-g088 glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu 1,2,3 , takeshi ishihara 1 , eisuke nishida 3 , yuichi iino 2 1 dept. biol., fac. sci., kyushu univ., japan; 2 mol. genet. res. lab., univ. of tokyo, japan; 3 grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for 5 min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr-1, a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr-1 appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr-1. in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr-1. these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr-1 localization in aiy neurons. kohei ueno 1 , yoshiaki kidokoro 2 1 dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; 2 inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼1%), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the 26th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal4/uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps3p-g090 spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki 1,2 1 graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; 2 department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps3p-g091 medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka 1 , yoshinori sahara 2 , shinichiro horie 2 , masumi ichikawa 3 , shun nakamura 2 1 st. marianna univ. schl. med., kawasaki, japan; 2 ntl. inst. neurosci., ncnp, tokyo, japan; 3 tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than 10 s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps3p-g093 synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about 1 hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi (17023012) ps3p-g094 rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii 1,2 , ken yoshikawa 2 , yuki takatori 1 , koichiro kawashima 2 1 dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; 2 dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca 2+ signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca 2+ signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m 1 , m 3 , m 4 and m 5 machr subtypes, and ␣3, ␣5, ␣6, ␣7, ␣9, ␣10 and ␤4 nachr subunits. transfection of anti-m 3 , anti-m 5 and anti-␣7 small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca 2+ signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m 3 or anti-m 5 sirna. ca 2+ signals evoked by nicotine were reduced by anti-␣7 sirna. these findings indicate that m 3 , m 5 machr and ␣7 nachr subtypes play major roles in ca 2+ signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi (16590060) ps3p-g101 is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto 1 , sachiho kubo 2 , seiji matsuda 3 , naoki maruyama 2 1 dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; 2 dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; 3 dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, 2006) research funds: kakenhi (16590831) ps3p-g102 dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day 50. electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day 45. analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around 25 hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c1 from kansai medical university ps3p-h103 unusually folded sod1 species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno 1 , yumiko simazaki 1 , fuminori saitoh 1 , ryosuke takahashi 2 , toshiyuki araki 1 1 national institute of neuroscience (ncnp), tokyo, japan; 2 dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod1 protein is thought to be responsible for the selective loss of motoneurons in sod1-related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod1 species are not yet clarified. since we have detected accumulation of unusual sod1 species in motoneuronal axons from g93a sod1-tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod1 species. immunoprecipitation analyses revealed specific interaction of unusual sod1 species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod1-tg compared with wt sod1-tg mice. these data strongly suggest that the toxic property of unusual sod1 proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c (17590907) ps3p-h104 relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately 30% of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day 24. the mice started to show ncl symptoms on p25, and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from 30% to 50% of that in the wild-type mice. research funds: kakenhi (10343253) ps3p-h105 neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro2a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura 1 , katsumi higaki 1 , junichiro matsuda 2 , yoshiyuki suzuki 3 , eiji nanba 1 1 division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; 2 national institute of biomedical innovation, osaka, japan; 3 clinical research center, international university of health and welfare, tochigi, japan g m1 -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm1 is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm1 is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm1 accumulation of lysosome and pm and the membrane fluidity was also reduced. gm1-bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m1 -gangliosidosis. katsuya inoue 1,2 , katsuaki endo 1 , takamitsu fujikawa 1 , seijyun fukuda 2 , tatsuo nakamura 2 1 department of physical therapy, university of aino, osaka, japan; 2 institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after 40 min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after 40 min occulusion. ps3p-h108 development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando 1 , yuya kawanaka 1 , minoru saito 2 , hiroaki mochizuki 1 , ken honjo 1 , hirofumi toda 1,3 , toshifumi tomoda 3 , akira sawa 4 , katsuo furukubo-tokunaga 1 1 grad. school of life & envir. sci., univ. tsukuba, japan; 2 molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; 3 beckman res. inst., city of hope. california, usa; 4 dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia-1 (disc1) gene, originally identified at the breakpoint of a chromosome (1;11) (q42.1; q14.3) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc1 functions in vivo, we expressed disc1 in drosophila and examined developmental and behavioral phenotypes. overexpression of disc1 resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps3p-h110 effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake 1 , ayumi kamo 1 , kenji minato 1 , hitoshi haruna 1 , hiritsugu fukuda 2 , yuji murata 2 , takayoshi hosono 1 1 department of bomedical engineering, osaka electro-communication university, japan; 2 graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of 22 of 25 7-day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing 8% oxygen at 40 • c for 15 min. after the insult, 11 of the 22 rats were put in a box at 35 • c for 12 h (hypothermia, h-group). the other 11 rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps3p-h112 tau hyperphosphorylation in ts1cje, a partial trisomy 16 mouse model for down syndrome ebrahim abdul 1 , a. shimohata 1 , w. yu 1 , m. yamaguchi 1 , m. murayama 3 , d. chui 3 , t. akagi 2 , t. takeuchi 1 , k. amano 1 , h.s. karthik 1 , t. hashikawa 2 , h. sago 4 , c.j. epstein 5 , a. takashima 3 , k. yamakawa 1 1 research scientist; 2 lab. for neural arch.; 3 lab. for alzheimers disease; 4 div. of fetal med. ncchd; 5 ucsf, usa although down syndrome (ds) or trisomy 21 is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts1cje, a ds mouse model partially trisomic for chromosome 16, shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome 21q22, has about 97 genes. importantly, sod1 and app, which may contribute to the ds phenotype, are excluded from the ts1cje trisomic segment. here we report that ts1cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk3␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts1cje segment other than app and sod1 can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: 17790737 ps3p-h113 increased oxidative stress and mitochondrial dysfunction in ts1cje, a down syndrome mouse model atsushi shimohata 1 , ebrahim a. s. 1 , m. yamaguchi 1 , w. yu 1 , h. sago 2 , c.j. epstein 3 , k. yamakawa 1 1 lab. for neurogenetics, riken-bsi, japan; 2 div. of fetal med. ncchd, japan; 3 dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome 21(hsa21) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts1cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome 16 extending from sod1 to znf295, containing 97 genes syntenic with hsa21. since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts1cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts1cje brains. collectively, our results implicate dosage imbalanced genes other than sod1 and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts1cje ds mental retardation-like phenotype. ps3p-h114 polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan 1,2 , kouko tatsumi 2 , takayuki manabe 2 , takahira yamauchi 1,2 , eri makinodan 2 , juro shimoda 1 , toshifumi kishimoto 1 , akio wanaka 2 1 department of psychiatry, nara medical university, kashihara, japan; 2 department of 2nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded (62q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. 7200 genes, encompassing around 50% of the drosophila genome, were screened, resulting in the identification of 370 candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps3p-h117 analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. (2005) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the 2 , 3 ,-cyclic nucleotide 3 -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps3p-h118 dysregulation of sodium channel ␤4 subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤4 (␤4) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤4 as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤4 started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤4 expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤4 gene in pc12 cells. forskolin, an activator of the camp/pka pathway, did not affect b4 promoter activity, indicating that ␤4 is not camp-responsive gene. these findings strongly suggest that sodium channel ␤4 subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps3p-h119 repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki 1 , yuji takahashi 1 , jun goto 1 , mutsuo oyake 2 , toshiya sato 3 , shoji tsuji 1 1 department of neurology, the university of tokyo, tokyo, japan; 2 department of neurology, brain research institute, niigata university, niigata, japan; 3 center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of 76 (q76), 96 (q96), 113 (q113), or 129 (q129). in the accelerating rotarod (9w), the latencies of q76, q96, q113 and q129 were 113%, 81%, 34% and 24%, respectively. in the open field, moving distances of q96, q113, and q129 were decreased to 98%, 50%, and 20%, respectively, while that of q76 was increased to 134%. home cage activity was decreased depending on the repeat length. the q113 mice, however, showed increased ratios of the activity during the light time to that during the total day at 8 weeks (115%) and 24 weeks (208%), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps3p-h120 the mice lacking schnurri-2 show multiple behavioral abnormalities related to psychiatric disorders keizo takao 1 , nobuyuki yamasaki 1 , keiko toyama 1 , tsuyoshi takagi 2 , shunsuke ishii 2 , tsuyoshi miyakawa 1 1 hmro, kyoto university graduate school of medicine, kyoto, japan; 2 riken, tsukuba, japan schnurri-2 (shn-2) is a zinc finger transcription factor, a mouse homologue of human hiv-ep2, that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn-2 is known to play important roles in the mammalian immune systems. however, the role of shn-2 in the central nervous system (cns) is still unknown. to investigate the functional significance of shn-2 in mammalian brain, we analyzed the shn-2 knockout (ko) mice using a comprehensive behavioral test battery. shn-2 ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn-2 mice. our results demonstrate a critical role of shn-2 in cns and suggest that shn-2 ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h121 comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca 2+ /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h122 effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c57bl/6j, c57bl/6n, c57bl/6c, 129svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than 1200 mice (, including wild type and mutant mice from 25 strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h124 comprehensive behaivoral analysis of ryanodine receptor type3 knockout mouse suzuko ohsako 1 , koichi tanda 1,2 , nobuyuki yamasaki 1 , keiko toyama 1 , hiroshi takeshima 3 , tsuyoshi miyakawa 1 1 kyoto university graduate school of medicine, kyoto, japan; 2 dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; 3 dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca 2+ signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr3 is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr3 deficiency, we subjected ryr3 knockoout mice to a battery of behavioral tests. ryr3 knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr3 in regulating general locomotor activity and social behavior. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h125 comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama 1 , koichi tanda 1,2 , nobuyuki yamasaki 1 , tsuyoshi miyakawa 1 1 hmro, kyoto university graduate school of medicine, kyoto, japan; 2 dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi (16680015, 16653065, 17017021, 17025023) , jst bird ps3p-h126 primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi 1 , keiko shimizu 2 1 grad. sch. human and environmental std, kyoto univ., kyoto, japan; 2 primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected 6-ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. 6-ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in 6-ohda injected monkeys. these results suggest that monkeys injected 6-ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi (17021022) ps3p-i127 training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata 1,2,3 , noriyuki higo 1,3 , takao oishi 1,3,4 , akiko yamashita 5 , keiji matsuda 1 , motoharu hayashi 4 1 neurosci. res. inst, aist, tsukuba, japan; 2 grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; 3 crest, jst, kawaguchi, japan; 4 dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; 5 div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext (17021055) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about 2,500 g1 animals (dba/2j × enu-treated c57bl/6j) for home-cage activity, open-field activity, and passive avoidance response, and obtained 13 lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in 5 of 13 mutant lines. hyperactivity was predominant phenotype, and 7 of 13 mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps3p-i129 ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., 2005) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg7-knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., 2005) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc3, and found that in our ncl model mice, positive signals for ubiquitin and lc3 were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)(17790141) and creative scientific research (16gs0315) ps3p-i130 activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama 1 , eiichi jodo 1 , yoshiaki suzuki 2 , ken-yo hoshino 2 , yukihiko kayama 1 1 dept. of physiology, fukushima medical university, fukushima, japan; 2 dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch23390, mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps3p-i131 increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara 1 , hiroyuki sakagami 2 , hisatake kondoh 2 , kohji fukunaga 1 1 department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; 2 department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps3p-i132 severity of audiogenic seizures is influenced by multiple factors in vlgr1-mutated mice hideshi yagi 1,2 , makoto sato 1,2 1 division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; 2 research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr1-mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c57bl/6 backcrossed vlgr1-mutated mice and 129/svs4 backcrossed vlgr1-mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr1-mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps3p-i133 reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota 1 , ken kadota 1,2 1 department of bioenvironmental medicine, university of chiba, chiba, japan; 2 chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of 5 mg/kg of map i.p. for 12 days (d 1 d 12) and then withdrawn from the drug for 7 28 days (wd 7 wd 28). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg 1) and mfc. the decrease in the density of pac was first observed in cg 1 and then in mfc. the reduction began on d 6 and advanced to higher levels on d 12 and subsequently wd 7. these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg 1 and mfc, particularly in layers ii and iii. ps3p-i134 up-regulation of ␤ 2 -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ 2 -adrenergic receptor (␤ 2 -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ 2 -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ 2 -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ 2 -ar-immunoreactive cells were labeled with ki-67, a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ 2 -ar-immunoreactive cells also exhibited fgf-2-ir. these findings suggest that ␤ 2 -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf-2. ps3p-i135 effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita 1 , norihiro muraoka 2 , hiroshi hasuo 1 , takashi akasu 1 , minoru shigemori 2 1 dept. of physiology, kurume univ. sch. of med., kurume, japan; 2 dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh482). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity (10-50 v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was 30-40 v. the amplitude of slow peak in fpi group was about 50% larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps3p-i136 comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca 2+ imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca 2+ imaging. the slices (300 m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for 1 h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of 1 ml/min. green autofluorescence (>520 nm) of the slices illuminated by blue light (460-490 nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca 2+ imaging was also performed in the slices. slices were incubated in acsf medium containing 10 m of fluo3/4 am for 1 h at 37 • c. the ca 2+ image was recorded with an excitation wavelength of 460-490 nmand an emission wave length of >520 nm. the autofluorescence and ca 2+ responses wereobserved in slices perfused with l-glutamate (10 mm). takuya hayashi 1 , hiroshi sato 1 , shinichi abe 2 , takashi hanakawa 2 , hiroshi watabe 1 , hidenao fukuyama 2 , babak aldekani 3 , hidehiro iida 1 1 department of investigative radiology, national cardiovascular center research institute, osaka, japan; 2 human brain research center, kyoto university, kyoto, japan; 3 nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area 9 corresponded to that revealed by the tracer (mncl 2 ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h17-kokoro-025 ps3p-i138 optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata 1,2 , yasutake mori 1,2 , tsuya taneda 1,2 , hiroaki okuda 1,2 , masaya tohyama 1,2 1 department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; 2 21st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps3p-i140 a model selection of glm applied to fmri data using aic jobu watanabe 1 , fumikazu miwakeichi 2 , andreas galka 3,4 , ryuta kawashima 4 , tohru ozaki 3 , sunao uchida 1,5 1 institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; 2 department of medical system engineering, faculty of engineering, chiba university, chiba, japan; 3 institute for statistical mathematics, tokyo, japan; 4 institute of experimental and applied physics, university of kiel, keil, germany; 5 new industry creation hatchery center, tohoku university, sendai, japan; 6 faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for 20-30 min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps3p-j148 flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata 951-8585, japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps3p-j149 effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune 1,2 , takashi tsukiura 1 , hiroko mochizuki-kawai 1 , chisato suzuki 1,2 , toshio iijima 2 1 neurosci. res. inst., aist, tsukuba, japan; 2 div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps3p-j150 different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno 1 , kenji kansaku 2 , yu aramaki 2 , mitsuo kawato 1 1 atr cns, kyoto, japan; 2 institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = 14) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, 50 or 10 yen is provided with a probability of 0.8, but only with 0.2 if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < 0.01) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps3p-j151 brain networks for communicative speech production: feeling inference and speech content production yuko sassa 1,2 , motoaki sugiura 3 , hyeonjeong jeong 1,4 , keisuke wakusawa 1,5 , kaoru horie 6 , shigeru sato 6 , ryuta kawashima 1 1 niche, tohoku university, sendai, japan; 2 ristex, jst, tokyo, japan; 3 miyagi university of education, sendai, japan; 4 gsics, tohoku university, sendai, japan; 5 department of pediatrics, tohoku university, sendai, japan; 6 the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the 21st coe program ps3p-j152 the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi 1 , yasuhisa fujibayashi 2 , yoshiharu yonekura 2 , tatsuya asai 2 1 department of human and intelligence systems, university of fukui, fukui, japan; 2 biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps3p-j153 imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai 1 , kuniyoshi shinya 1 , tetsuo araki 2 , masahiro kusakabe 2 , yasuhisa fujibayashi 3 , yoshiharu yonekura 3 1 department of nuclear power and energy safety engineering, university of fukui, fukui, japan; 2 department of human and intelligence systems, university of fukui, fukui, japan; 3 biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura 1,2 , winfried denk 3 , michael hausser 2 1 department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; 2 university college london, london, uk; 3 max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps3p-k161 analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka 1 , yasuhiro tanaka 1 , takeshi kaneko 1,2 1 dept. of morphological brain science, kyoto univ., kyoto, japan; 2 crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad67-gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after 3 h incubation in nmdg-based solution than in nacl-based solution. cutting at 0 • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at 20 • c. the viability after 3 h incubation was better kept at 20 • c than at 0 or 37 • c. we thus recommend that slices be cut at 20 • c in na-free solution, and incubated at 20 • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi (16200025,17022020,17650100) ps3p-k162 contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh 1 , yoshichika yoshioka 2 1 faculty of engineering, iwate university, morioka, japan; 2 iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal (2.95 ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals (3.03, 3.23, and 3.34 ppm). a phantom solution mimicking the normal condition (gsh:gssg = 100:1) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = 7:3:1:1), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was 1.9 mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area 9) and the right superior and middle frontal gyri (brodmann area 10) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps3p-k164 cortical commissural connection in macaque and human callosum using diffusion mri rishu piao 1 , takuya hayashi 1 , hiroshi sato 1 , shinichi abe 1,2 , takashi hanakawa 2 , hidenao fukuyama 2 , hidehiro iida 1 1 national cardiovascular center, osaka, japan; 2 human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in 9 subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps3p-k165 optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong 1,2 1 niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, 2 niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the 3-d neural cortical activity using the 2-d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps3a-c192 sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp8) nobuyuki karasawa 1 , kazuko watanabe 2 , keiki yamada 3 1 seijoh universitry, tokai, japan, 2 dept. physio., sch. med., gifu univ., gifu, japan, 3 dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, 2,4-diamino-6-hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp8) mice. time course of immunoreactive intensity for serotonergic (5-ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that 5-ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and 5-ht neurons. katsuya yamada 1,2,5 , yoshihiro matsumura 2 , takashi miki 3 , makoto wakui 1 ␣-smooth muscle actin + arterioles were fewer in kir6.1(−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir6.1(−/−) barrel cortex than in wild-type one for short (2 s, 5 hz) but not long (30 s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps3a-h135 learning to use sensory-tools by japanese monkeys yumiko yamazaki 1 , hiromi namba 1 support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps2p-h147 mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h1 receptor (h1r) in cognition in physiological and pathological conditions by using h1r mutant (h1−/−) mice. in normal condition, several behavioral studies indicated h1−/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h1−/− mice. these results indicate h1 receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h1−/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h1−/− mice. mutation of h1 receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h1r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h1r impairs cognition in normal conditions, whereas h1r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi (17390156; 14370027) ps3a-h133 5-ht2a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura 1,2 , hirohito-m. kondo 2 , makio kashino 2,3 1 department of psychology, tokai women's university, kakamigahara, japan; 2 ntt communication science laboratories, ntt corporation, atsugi, japan; 3 shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of 5-ht neurotransmission. we examined whether this 5-ht2a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the 5-ht2a receptor gene polymorphism in impulsive behavior (nomura et al., 2005) , together with the present findings, this observation indicates the involvement of 5-ht2a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps3a-h134 role of cortical thin arterioles in neurovascular coupling; analyses of kir6.1-containing atpsensitive potassium channel-deficient mice ps3p-g095 rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal 1 , chinmay k. mukhopadhyay 2 , birendra n. mallick 1 1 school of life sciences, jawaharlal nehru university, new delhi, india; 2 special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv-1 and the direct infection of hsv-1 are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv-1 jrfl-infected macrophages or with hsv-1. the generation of primary neurospheres did not suppressed by hiv-1-infection or by hsv-1 infection at no more that moi 10. in the secondary neurospheres, cd133 + neural stem cells were intact in these infections, although beta-3-tubulin + cells were decreased in hiv-1 infection and intact in hsv-1-infection. in the differentiation assay, neun + nfp + neurons in hiv-1-infection and gfap + s100 + astrocytes in hsv-1 infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv-1 infection and in hsv-1 infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv-1 and hsv-1 infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi 17790579 grant-in-aid for young scientists(b) ps3p-g097 effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga 1,2 , taeko yamauchi 2 , toshihiro konagaya 2 , hideki ohira 1 1 department of psychology, nagoya university, japan; 2 department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka 1 , shen jin-song 2 , saburo saito 1 , shigeki yuasa 3 1 dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; 2 dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; 3 dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori 1 , tomoko yamamoto 1 , kiyoshi matsumura 2 , hiroshi hosokawa 1 , shigeo kobayashi 1 1 dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; 2 dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e 2 (pge 2 ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge 2 but also induce an enzyme cyclooxygenase-2 (cox-2) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox-2, and whether cox-2 is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox-2 was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox-2 specific inhibitor, ns-398. these results indicate that aa itself or its metabolites induces cox-2 that accelerates the formation of pge 2 from aa, and, hence, enhances fever. mitsunari abe 1 , tatsuya mima 1 , shinichi urayama 1 , toshihiko aso 1 , nobukatsu sawamoto 2 , hidenao fukuyama 1 1 human brain research center, kyoto university graduate school of medicine, japan; 2 nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at 0.9 hz (10 min; 90% of the rest motor threshold) applied over the left primary motor area (m1). we scanned 4 sets of dwi (before, and 0 min, 10 min and 20 min after rtms) using 3-t mr scanner, and calculated apparent diffusion coefficient (adc). in 3 out of 4 subjects, the adc decreased (mean 1.72 × 10 −5 /mm 2 s −1 ) in the left m1 just after the rtms, which recovered at 20 min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v6 and v6a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v6a located more dorsal has a larger receptive field size then v6. both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v6 and v6a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v1 in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v6 and v6a.ps3p-i144 measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro 9-channel magnetometer consisting of a 3 × 3 matrix of superconducting quantum interference device (squid) with its integrated base of 2.5 × 2.5 mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with 60-80 ms peak by the white led flashing stimulation and the aef wave with 10-20 ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of 19 bits was used, along with a "blue dye" (rh 1691) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were 0.1-0.05% of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto 1 , yoko hoshi 1 , chie sato 1 , susumu terakawa 2 1 tokyo institute of psychiatry, tokyo, japan; 2 photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals (586 and 610 nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around 20-ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps3p-j147 multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh414, a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a 4t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a 4-s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba18), superior occipital gyrus around the parieto-occipital sulcus (ba19) and superior parietal lobule (ba7). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps3p-j155 influence of task difficulty during meter inspection: an fmri study naoki miura 1,2 , makoto takahashi 3 , jobu watanabe 2,4 , shinya uchida 2,5 , shigeru sato 6 , kaoru horie 6 , masaharu kitamura 2 , toshio wakabayashi 2 , katsuki nakamura 1,7 , ryuta kawashima 2 1 crest, jst, kawaguchi, japan; 2 niche, tohoku univ. sendai, japan; 3 graduate school of engineering, tohoku univ. sendai, japan; 4 bme institute, waseda univ., tokyo, japan; 5 idac, tohoku univ., sendai, japan; 6 lbc research center, tohoku univ., sendai, japan; 7 department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps3p-j156 neural connectivity among brain areas related to language function shinya uchida 1,2,3 , naoki miura 2,4 , jobu watanabe 5 , shigeo kinomura 1 , kazunori sato 1 , yasuyuki taki 1 , kentaro inoue 1 , ryoi goto 1 , ai fukushima 2 , kaoru horie 6 , shigeru sato 6 , katsuki nakamura 3 , hiroshi fukuda 1 , ryuta kawashima 2 1 department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; 2 niche, tohoku university, sendai, japan; 3 national institute of neuroscience, ncnp, kodaira, japan; 4 japan science and technology agency, kawaguchi, japan; 5 bme institute, asmew, waseda university, tokyo, japan; 6 graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than 80% of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa 1,2 , motoaki sugiura 3 , yuko sassa 1,4 , hyeonjeong jeong 1,5 , kaoru horie 5 , shigeru sato 5 , hiroyuki yokoyama 2 , kazuie inuma 2 , ryuta kawashima 1 1 niche, tohoku univ., sendai, japan; 2 department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; 3 miyagi univ. of education, sendai, japan; 4 ristex, jst, tohoku univ., sendai, japan; 5 gsics, tohoku univ., sendai, japan; 6 lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda 1 , yasumasa okamoto 1 , kazuhiro shishida 1 , akiko kinoshita 1 , shigeru toki 1 , kazutaka ueda 2 , hidehisa yamashita 1 , shigeto yamawaki 1 1 department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; 2 training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada 1,3,4 , kenji yoshimi 1,2 , noriyuki higo 4 , yong-ri ren 2 , hideki mochizuki 2 , yoshikuni mizuno 2 , shigeru kitazawa 1,3,4 1 dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; 2 dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; 3 crest, jst, tokyo, japan; 4 neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba-1 positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma −5.9 mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections. key: cord-009997-oecpqf1j authors: nan title: 2018 aspho abstracts date: 2018-03-31 journal: pediatr blood cancer doi: 10.1002/pbc.27057 sha: doc_id: 9997 cord_uid: oecpqf1j nan myelodysplastic syndrome (mds) and frequently arise in the context of inherited bone marrow failure (bmf) syndromes, such as shwachman diamond syndrome (sds). monosomy 7/del(7q) is associated with high grade mds and propensity to progress to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited bmf. development of non-transplant strategies to treat bone marrow failure without simultaneously stimulating outgrowth of malignant clones remains a major challenge. objectives: the aim of this study is to investigate the molecular consequences of del(7q) in the context of bmf with the goal of developing more effective treatments. design/method: to study the biological and molecular consequences of monosomy/del(7q) in bmf, induced pluripotent stem cells were generated from sds patients (sds-ipsc) . a deletion of the mds-associated region of the long arm of chromosome 7 was then introduced using a previously published modified cre-lox approach. results: the sds ipsc phenocopied bone marrow failure with slow proliferation and impaired hematopoietic differentiation. we next explored whether deletion of 7q conferred a relative fitness advantage within the context of bone marrow failure. proliferation of the sds-del(7q) ipscs was reduced below that of both the isogenic sds ipscs and normal controls without an increase in cell death. sds-del(7q) demonstrated reduced hematopoietic differentiation compared with isogenic sds cells. these data demonstrate that deletion of 7q fails to confer a relative growth advantage relative to isogenic sds ipscs and results in further impairment of hematopoiesis. to gain insight into the mechanisms of del7q-associated clonal evolution in sds, we performed rna sequencing (rnaseq) of sds+/-del(7q) ipsc. expression of tgf pathways and their downstream targets were reduced in sds-del(7q) ipscs compared to isogenic sds ipsc. single cell rnaseq analysis of primary sds bone marrow cells confirmed that the tgf pathway is hyperactivated in sds. western blot analysis showed increased phospho-smad2 levels in sds ipscs compared to sds-del(7q) and normal controls, while total levels of smad2 were unchanged. pharmacological targeting of tfg with small molecule inhibitors resulted in selective improvement of sds hematopoietic colony formation and myeloid differentiation without stimulating outgrowth of the isogenic sds-del(7q) cells or normal controls. these results demonstrate that del(7q) reverses the tgf pathway hyperactivation of sds. furthermore, inhibition of tgf selectively rescues hematopoiesis in sds but not in isogenic del7q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del7q clonal outgrowth. background: standard therapy of medulloblastoma consists of treatment with alkylating agents and radiation after surgical resection. although a statistically significant increase in survival is reported with this regimen, 1/3rd recur and become resistant this class of agents ultimately leading to mortality. large numbers of somatic mutations were observed in recurrent medulloblastoma (rm) after alkylating agent and radiation treatment. high mutation rates in tumors can have twofold effect; 1) a large number of non-synonymous mutations that have no role as drivers can still cause functional tumor antigens increasing the neoantigen burden and immunogenicity. moreover, 2) such tumors can gain mutations in canonical or non-canonical dna repair pathways leading to a gain in the number of mutations as seen in case of glioblastoma, this can lead to even higher accelerated mutational rate. evidences suggest that high mutational load can cause higher neoantigen burden thereby making the tumor more susceptible to immune checkpoint inhibition. we propose that post therapy recurrent medulloblastoma gain mutational signature and immunophenotype of malignancies demonstrating clinical response to immune checkpoint therapy. objectives: 1) rm has molecular signatures identical to tumors with high immunogenicity and clinical response to immune check point inhibition. 2) rm has the immune inflammatory phenotype; harboring high percentage of tumor infiltrating lymphocytes (tils), macrophages and monocytes. design/method: to test our hypothesis, we downloaded the raw bam files of previously published data from international cancer genome consortium (icgc) . this set of about 30 matched primaries and recurrent medulloblastoma cases forms our discovery cohort. we have called somatic variants using the gatk pipeline by the broad institute. to validate our key findings, we have procured human medulloblastoma specimens and are conducting whole exome sequencing. the primary assays utilized to assess immunogenicity are immunohistochemical (ihc) staining of formalin fixed and embedded recurrent medulloblastoma tissue to identify tils, tumor associated macrophages and other markers. 300 mg/m2 had dlts of dyspnea (grade 4)/hypoxia (grade 3) but no dlts were observed in any other cohort. adverse events were generally mild to moderate, consistent with the safety profile observed in adults. across the desc cohorts, plasma concentrations were dose-proportional and steady state concentrations were lower on day 15 vs. day 1. mean systemic exposure in the 1200 mg/m2 cohort was ∼ 4-fold greater compared with the adult rp2d of 800 mg bid. a pk:pd relationship between tazemetostat exposure and h3k27me3 levels in peripheral blood monocytes and granulocytes was observed in the desc phase. consistent and significant post-dose reductions in h3k27me3 occurred at doses ≥900 mg/m2. further analysis of twelve patients treated at the rp2d confirmed that h3k27me3 inhibition was maximally inhibited. doses 520-900 mg/m2 showed confirmed objective responses (cr/pr) per recist/rano in patients with es (n = 1), chordoma (n = 2), and atrt (n = 1). background: previous studies established that the platelet/ fibrin(ogen) axis promotes metastatic potential by impeding the clearance of newly formed micrometastases by natural killer (nk) cells. however, multiple important questions remain, including the potential of fibrin(ogen) to promote metastasis through interactions with cells other than platelets (e.g., inflammatory cells), and the fundamental question of whether fibrin polymerization is required for metastasis. objectives: determine the role of fibrin polymerization and fibrin(ogen) engagement of integrins iib 3 and m 2 in metastasis. design/method: we performed experimental and spontaneous metastasis assays in immunocompetent mice carrying specific fibrinogen structure/function alterations. results: expression of a mutant fibrinogen lacking the binding motif for the leukocyte integrin m 2 (fib 390-396a) significantly decreased metastatic potential relative to wildtype fibrinogen, suggesting a role for fibrin(ogen)inflammatory cell interactions mediated by m 2 in metastasis. to directly determine the importance of thrombinmediated fibrin polymerization in metastasis, we analyzed metastatic potential in fibaek mice, which carry a form of fibrinogen essentially "locked" in the soluble state due to a mutation in the a chain thrombin cleavage site. metastatic potential in fibaek mice was diminished relative to control mice, speaking to the importance of thrombin-mediated fibrin polymerization in the metastatic process. however, the fibaek mice retained significant metastatic potential relative to complete fibrinogen deficiency, indicating that fibrinogen monomer retains significant prometastatic properties. in order to better define the role of fibrin(ogen)-platelet interactions in metastasis, we compared metastatic potential in control and fib δ5 mice, carrying a form of fibrinogen lacking the chain binding motif for the platelet integrin iib 3. surprisingly, this mutation had no impact on metastatic potential. together, these studies suggest fibrinogen plays a multifaceted role in metastasis. fibrin(ogen)-leukocyte interactions mediated by m 2 appear to have a role in metastasis. previous studies showed that macrophages promote the metastatic potential of circulating tumor cells, which may represent at least one important m 2 expressing cell type whose prometastatic behavior is influenced by fibrin(ogen) interactions. these studies show that thrombin-mediated fibrin polymerization promotes metastasis, but soluble fibrinogen retains some significant prometastatic capacity. surprisingly, loss of the fibrinogen chain iib 3 binding motif had no impact on metastasis. given the established importance of platelets in metastasis, these findings suggest that fibrin (ogen) is capable of platelet stabilization through mechanism(s) independent of this iib 3 binding motif. platelets may bind polymerized fibrin at other sites, and/or fibrin interactions with other matrix proteins capable of binding iib 3 are sufficient to support platelet functions required for metastasis. the role of platelets in hemostasis and thrombosis is well defined, but it is becoming increasingly evident that platelets also assist in host defense and inflammation. platelets participate in the innate immune system through direct antimicrobial activity and interactions with effector cells (chapman2012, garlanda2013, kapur 2016). in the adaptive immune system, platelets recruit and costimulate t-cells, and promote b-cell differentiation and antibody class switching (kapur2016, morrell2017). the question remains: which mechanisms influence platelet immune function and are they developmentally regulated? preliminary studies in the palis lab have revealed significant dif-ferences in embryonic versus adult platelet gene expression, including regulators of immune and inflammatory responses such as beta2-microglobulin (b2m) and major histocompatibility complex class i (mhc1). mhc1 is expressed on all cell surfaces except red blood cells and its molecular chaperone b2m is a marker of inflammation highly expressed in platelet alpha granules (zufferey2014). preliminary data from the morrell lab reveals a mass release of b2m during platelet activation, which drives monocyte differentiation to an inflammatory phenotype through tgfb receptor signaling. we therefore sought to determine whether developmental changes in platelet b2m expression mediate differences in platelet-mediated monocyte activation. with trilineage hematopoiesis with a predominance of early myeloid precursors, with full maturation. microarray, elane and sbds sequencing and deletion/duplication analyses were negative. immunologic evaluation was significant for agammaglobulinemia and an absence of memory (cd19+cd27+) b cells. a 207 gene primary immunodeficiency panel revealed two variants of unknown significance-c.1957g>a and c.2292g>t in dnmt3b; one previously reported in association with icf1. parental testing demonstrated parental heterozygosity. centromeric instability was confirmed in mitogen stimulated lymphocytes showing characteristic, multibranched chromosomes containing at least 3 arms of chromosome 1 and 16 joined near the centromere. decondensation of the 1qh and 16qh regions and triradial configuration of chromosome1 was noted, and a diagnosis of icf1 syndrome was made. the patient was started on monthly intravenous immunoglobulin (ivig). prophylaxis for pneumocystis jiroveci pneumonia and respiratory syncytial virus was initiated. a 10/10 matched sibling hsct is being planned. demonstrated the diagnosis of high grade osteosarcoma. the patient was started on multi-agent chemotherapy with planned a whole femur prosthesis at time of local control. 7 cases of osteosarcoma have been described in the literature in patients with nf1 (median age; 25 years, range 17-37 years) with slightly male predominance (4 cases). the femur was the most common site of involvement (5 cases). four patients died of metastatic disease despite surgery and multi-agent chemotherapy. conclusion: nf1 represents a major risk factor for development of malignancy and uncommonly osteosarcoma in adolescents and adults. we report a rare case of an extensive involvement of osteosarcoma of the left femur in a child with known diagnosis nf1. this presentation should alert the pediatric oncologists to monitor for bone tumors in patients with nf1 by physical exam and detailed medical history. hasbro children's hospital, providence, rhode island, united states background: dysautonomia is a paraneoplastic syndrome most commonly described in adult malignancies. despite current therapies aimed at symptoms management, it is often debilitating. we present a case of a 16-year-old girl who initially presented with autonomic dysfunction and was subsequently found to have hodgkin lymphoma. objectives: describe hodgkin lymphoma presenting with dysautonomia and discuss symptom management with rituximab design/method: case report a 16 year-old-girl presented with severe symptoms of orthostatic hypotension necessitating prone positioning to prevent syncopal episodes. additionally, she reported anhidrosis, xerostomia, urinary retention, and constipation. she had unmanageable peripheral neuropathic pain despite multiple analgesia medications. initially, it was suspected that her symptoms were caused by an atypical presentation of guillain-barre syndrome. she was treated with intravenous immunoglobulin g, without response. due to a suspicion of a paraneoplastic syndrome a positron emission test/cat scan (pet/ct) was performed and revealed widespread fdg-avid nodal and splenic disease. pathology from a thoracoscopic biopsy of a mediastinal lymph node demonstrated classical hodgkin lymphoma. she was classified as stage ivb. a paraneoplastic panel obtained during the first cycle of chemotherapy revealed elevated anti-amphiphysin antibodies and glutamic acid decarboxylase (gad) antibodies. therapy was initiated with abe-pc (doxorubicin, bleomycin, etoposide, prednisone, cyclophosphamide) ; vincristine was held given her significant neuropathy. due to persistence of autonomic symptoms following her first cycle and presence of antiamphiphysin and gad antibodies, rituximab was incorporated into her treatment. following two cycles abe-pc, she had a rapid early response by fdg-pet/ct. she completed an additional three cycles of abd-pc. end of therapy imaging demonstrated complete response with a single persistent mildly fdg-pet avid lymph node (deauville 2) and her antibodies were negative. she continues treatment of maintenance rituximab with significant improvement, but not resolution, of her orthostatic hypotension. at this time, the patient can ambulate with assistance. constipation and urinary retention have fully resolved and, her peripheral neuropathy, xerostomia, anhidrosis have improved. conclusion: this is rare case of a pediatric hodgkin lymphoma patient developing dysautonomia associated with antiamphiphysin and glutamic acid decarboxylase antibodies and subsequently managed with chemotherapy and rituximab. clinicians should be suspicious of a paraneoplastic syndrome when a neurologic disorder fails to improve with standard treatment. results: labs obtained at an outside hospital one month prior to presentation showed absolute neutrophil count (anc) 78 and hemoglobin 10.2 g/dl. she presented to our institution with 11 days of fever, hepatomegaly 1 cm below costal margin, a white plaque on her tongue, and circumferential perianal ulceration. labs were significant for anc 0 and hemoglobin 7.8 g/dl. anti-granulocyte antibody testing was positive. bone marrow biopsy showed arrest of neutrophil maturation. after initiation of filgrastim (2.7 mcg/kg/day), her anc increased to >500 and repeat bone marrow biopsy demonstrated left shifted myelopoiesis. biopsy of her oral lesion demonstrated invasive actinomyces prompting a prolonged course of antibiotics. biopsies of her oral and anal lesions were reported as myeloid sarcoma without mll rearrangement. chemotherapy was not initiated due to complete resolution of both lesions within 6 weeks of initiating filgrastim and appropriate antibiotic coverage. she has not developed any further lesions concerning for malignancy. testing for common genes associated with severe congenital neutropenia and autoimmune lymphoproliferative syndrome was negative. her immunoglobulin levels and the measurement of age-appropriate vaccine responses were normal. after her lymphocyte subpopulation analysis indicated a selective deficiency in cd8 positive t-lymphocytes (absolute cd8 cell count 185), the severe combined immunodeficiency panel from genedx showed compound heterozygous mutations in results: a male infant was born with a large thigh mass. the child was clinically well aside from restricted movement of affected leg. mri showed mass expanding into pelvis without other lesions. an interventional-radiology guided core biopsy of the mass was reported as high-grade spindle cell sarcoma without etv6 rearrangement. surgery was deferred because of concern that it would result in excessive morbidity. the mass was treated with vincristine and dactinomycin per infantile fibrosarcoma protocols. after 3 months of therapy, no significant change in size of the mass was noted on physical exam or imaging. repeat biopsy was obtained to confirm diagnosis and allow for expanded tumor testing. this biopsy showed triphasic distribution of adipose, fibrous and mesenchymal tissue consistent with fhi with rare sarcomatous foci. additional chemotherapy was deferred and the child was followed clinically. his tumor has remained approximately the same size and still unresectable. next generation sequencing of tumor utilizing panel based technology revealed braf-erc1 fusion consistent with braf activating mutation. this mutation was confirmed by fluorescent in situ hybridization (fish) probe for braf. braf and mek inhibitors have been pursued as treatments to decrease size of tumor and allow for resection. conclusion: braf mutations have been characterized in a variety of malignancies. inhibition of braf and downstream signaling components has produced promising results in a variety of patients. this is the first case report of a braf mutation in a fhi. although management of fhi is typically surgical, this does suggest a potential therapeutic target and may allow for improved surgical outcomes especially in cases where up-front surgery would result in unacceptable morbidity. genetic sequencing of fhi and other rare tumors is an important tool and has the potential to identify mutations amenable to targeted therapies. background: icf is a rare autosomal recessive disorder characterized by hypo-or agammaglobulinemia and often opportunistic infections suggesting t-cell dysfunction. it is further categorized into subtypes 1-4 based on mutations in dna methylation. mutations in the helicase-lymphoid specific (hells) gene, which is required for t-cell proliferation and participates in de novo dna methylation, are characteristic of icf type 4 (icf4). of approximately 70 reported cases of icf, less than 10 percent are characterized as icf4. while malignancy has been reported in icf1 (angiosarcoma, acute lymphoblastic leukemia), and icf2 (hodgkin lymphoma), here we describe the diagnosis and management of an icf4 patient with neuroblastoma and neutropenia, which has not been previously described. objectives: describe a novel phenotype and mutation of icf4 and its management to further expand our understanding of this disease. results: a 6 month ex-31 week premature male with bronchopulmonary disease and failure to thrive presented with acute respiratory failure in the setting of recent viral bronchiolitis with associated chronic diarrhea. he was subsequently diagnosed with multiple infections including pjp pneumonia, norovirus, parainfluenza, rhinovirus, and pseudemonal cellulitis. he presented with profound neutropenia and agammaglobulinemia with presence of b and t cells on lymphocyte phenotyping. ct revealed a paraspinal mass that was mibg-avid on further study, strongly suggesting neuroblastoma. bone marrow was normocellular and negative for malignancy, however revealed marked granulocytic hypoplasia and maturation arrest concerning for severe congenital or, less likely, immune-mediated neutropenia. metastatic workup was negative. whole exome sequencing revealed a homozygous variant of unknown significance (c. 668t>c) in the hells gene, portending a working diagnosis of icf4 syndrome. immunoglobulin supplementation, pentamidine prophylaxis, and g-csf were initiated. he was able discontinue g-csf after 4 months of treatment. his neuroblastoma, initially categorized as l1, met criteria for observation. however, followup mri revealed interval growth nearing the spinal canal. he underwent tumor resection, confirming mycn non-amplified, favorable histology neuroblastoma. after infectious prophylaxis and immunologic support were initiated, he incurred two other hospitalizations, the first for g-tube cellulitis and the second for parainfluenza respiratory illness. he now has stable neutrophil counts off g-csf and remains in remission from neuroblastoma. current plan is to proceed with bone marrow transplantation for immunodeficiency. conclusion: icf4 has not previously been described with neutropenia or neuroblastoma. this report not only describes a novel mutation and phenotype of icf4 and the management thereof, but also reveals the potential curative role of bone marrow transplantation in such disease. staten island university hospital -northwell health, staten island, new york, united states background: desmoid tumors are rare tumors that arise from highly differentiated fibroblasts. they occur in isolation or as part of the disease spectrum of familial adenomatous polyposis (fap) . fap mutations between codons 1445-1578 typically correlate with increased extraintestinal disease such as desmoid tumors and upper gastrointestinal polyps. we describe a patient with a large intra-abdominal desmoid tumor who is heterozygous for a c.8161c>t (p.arg2721cys) apc gene mutation. we are not aware of any other patients reported with this germline apc mutation presenting with a desmoid tumor. objectives: to discuss a novel apc mutation and the presentation of a rare case. design/method: review of clinical presentation, genetic analysis and management of a rare tumor. a 17-year-old female with no significant medical history presented with abdominal asymmetry and intermittent pain. she reported urinary urgency, shortness of breath, early satiety, decreased appetite and a 20-pound weight loss over the course of 7 months. ct scan of the abdomen demonstrated a 24 × 15cm abdominal tumor abutting the local organs but no presence of bowel obstruction. a biopsy revealed a spindle cell neoplasm favoring fibromatosis. there was no known family history of fap, colon cancer, or desmoid tumors. apc gene mutation analysis demonstrated a c.8161c>t (p.arg2721cys) heterozygous gene variant. due to size and location of the tumor, it was initially deemed unresectable. the patient was started on a course of monthly liposomal doxorubicin. she tolerated the initial cycles well and interval ct after 3 cycles of chemotherapy revealed a 40% decrease in tumor volume. variability exists in phenotypic presentation with regards to the location of the afp mutation locus. while fap mutations associated with desmoid tumors typically have changes in the 1445-1645 codon region, our patient presented with a heterozygous mutation resulting in a missense mutation at codon 2721. due to the change in polarity and size, the mutation is not considered to be of conservative nature. we are only aware of one other report of this mutation, which occurred in an individual with a personal and family history of colon cancer. we are not aware of any patients with desmoid tumors who also have this germline apc gene mutation. our case report highlights an apc gene mutation that is not well-described; we are not aware of any other cases of this mutation reported in patients with desmoid tumors. future evaluation and tracking of this mutation may lead to the determination of further clinical significance. background: over time, advanced care planning for location of death has been associated with increased deaths at home rather than in the hospital. in some cases, however, complex management and symptom control can prevent families from achieving their goal of keeping their child out of the hospital and at home at the end of life. ascites is a sequelae of many conditions including malignancy that might lead to significant morbidity. increasingly, interventional procedures are being utilized. peritoneovenous "denver" shunts are placed internally with one end in the peritoneal space and the other buried within a major vessel such as the svc. a one-way valve and pump buried under the skin allows the patient to pump fluid from the peritoneal to the vascular space. the shunt is used frequently in adults, but has not seen much use in pediatric oncology patients. objectives: to describe a case of a terminally ill patient with refractory wilms tumor with ivc involvement who received symptomatic relief with denver shunt placement. results: an 8-year-old female was diagnosed with relapsed, refractory, metastatic wilms tumor with pulmonary and hepatic involvement, with tumor extension to the hepatic veins and ivc. multiple chemotherapeutic regimens and palliative radiation to the ivc were administered, but her disease continued to progress, leading to pressure on the portal vein and portal hypertension. the resulting ascites was causing the patient significant pain and was difficult to manage. the patient's code status was changed to dnr/dni after discussion with her mother, who identified a desire to have the child die at home as comfortably as possible. a peritoneovenous shunt was placed in order to control the patient's pain and avoid frequent medical procedures and therapies. despite initial anxiety, the patient was able to utilize the pump and achieve significant improvement in her ascites and pain. she was able to spend the remaining six weeks of her life at home. ascites is a common phenomenon of end stage disease. peritoneovenous shunts are a treatment modality that may be considered to allow for pain control at the end of life for pediatric oncology patients with ascites. the procedure is relatively low risk, allows for self-control of the pump to maintain comfort, and is easy enough to use by the patient or family. background: extraneural metastases (enm) from pediatric glioblastoma multiforme (gbm) are rare, with an estimated frequency of 0.3%. etiologic factors include multiple neurosurgical procedures and sarcomatous dedifferentiation. their occurrence can seriously affect the patient's quality of life and survival. while enms have been well documented in adults, pediatric cases have not been previously summarized. a 15 year old male with a cerebral gbm developed extension of disease outside of the neuraxis approximately 18 months post initial presentation and at the time of disease progression. metastases included exracranial temporal lesions, cervical and mediastinal lymph nodes and s29 of s301 bilateral lung nodules. a large pleural-based soft tissue metastatic focus was identified on imaging when the patient presented with respiratory distress secondary to a right tension pneumothorax, which was recognized and managed promptly. we summarize the main reported cases in literature to better define risk factors for and evaluate the proposed mechanisms underlying these systemic metastases. design/method: we performed a literature review on the pubmed database using the terms gbm and enm. patients under 21 years of age who met the weiss criteria for the diagnosis of enm from primary cns tumors were included. results: our patient fulfilled two of the three weiss criteria with confirmed gbm at the primary site with all enm in the temporal soft tissue and cervical lymph nodes displaying histopathologic features similar to the primary cns tumor. the intrathoracic adenopathy and lung nodules detected upon chest imaging during workup for respiratory distress were assumed to represent additional metastatic foci. our literature review identified 22 pediatric patients with enm from gbm with a median age of 12 years (range 3.5 -21 years) and a slight female predominance (55% females vs. 45% males). the most common sites of metastases reported were pleura/lungs, bones, lymph nodes and liver. in 9 of 22 patients, metastases were associated with csf shunting. conclusion: pediatric oncologists should have an increased index of suspicion when caring for patients with gbm, particularly those who have undergone shunting procedures and present with systemic symptoms including bony pain, respiratory changes, transaminitis or cytopenias which should prompt timely investigation for enm. although enm of cns tumors carry very poor prognosis, their diagnosis has potential therapeutic importance because treatment of metastatic lesions may alleviate symptoms and improve the quality of life. additional studies may be warranted to evaluate the incidence of enm that can provide valuable insight into the pathogenesis and biology of high-grade gliomas. nicklaus children's hospital, miami, florida, united states background: sinusoidal obstruction syndrome (sos) has been reported in patients undergoing intensive chemotherapy and as a complication post-hematopoietic stem cell transplan-tation. sos may be complicated by portal hypertension, hepatorenal disease or multi-organ failure. however, despite treatment, there may be further potential complications that can be anticipated in patients with history sos. we report two patients with history of sos presented with post-procedural bleeding after gastric tube placement. we believe that their presentations may be associated to their previous diagnosis of sos. design/method: pubmed search was done with search for terminology including "sinusoidal obstruction syndrome" "defibrotide", and "bleeding". papers relevant to our cases were selected for literature review. results: case 1: a 4 year-old female with history of desmosplastic medulloblastoma status-post resection and intensive chemotherapy was diagnosed with sos one month after her second part of planned tandem transplant. she was managed with paracentesis and defibrotide. due to malnourishment, patient had a gastric tube placement 5 months after she completed therapy and had an episode of upper gastrointestinal bleeding postoperatively from the g tube site. case 2: similarly, a 4 year-old male diagnosed with anaplastic medulloblastoma status post resection and adjuvant multiagent chemotherapy. his treatment course was complicated with sos after the second cycle of induction chemotherapy which responded to 21-day course of defibrotide. likewise, the patient had a major bleeding event from the g-tube site approximately two months after sos diagnosis. defibrotide was discontinued in both cases before g-tube placement. both patients had no previous history of bleeding disorders or relevant family history. in addition, comprehensive laboratory evaluations were within normal limits before both procedures. in sos, there is blockage of fluid out of the liver that leads to congestion, ascites, ischemia of the liver, and post-sinusoidal portal hypertension. two related causes of sos should be considered as an explanation for g-tube bleeding. similar patients should have close monitoring postoperatively or if possible surgical intervention should be delayed until the sos process has been evolved. nicklaus children's hospital, miami, florida, united states background: the development of treatment related acute myeloid leukemia (t-aml) and myelodysplastic syndromes (t-mds) is a potential complication after cytotoxic chemotherapy or radiation therapy. the incidence of development of t-aml/t-mds varies from 1-20% depending on the treatment regimen used. cutaneous myeloid sarcoma (ms) is a common presentation of extramedullary leukemia and usually occurs in the setting of aml. we report a rare case of cutaneous ms in an adolescent female after successful treatment for ovarian yolk sac tumor (yst) stage i with bep (bleomycin, etoposide and cisplatin) therapy. the ms was managed only with biopsy and close observation. design/method: a pubmed search was conducted for queries including t-aml/t-mds, cytotoxic agents, cutaneous myeloid sarcoma, regression. relevant papers were selected for literature review. a 13 year-old female was diagnosed with a left ovarian yolk sac tumor, for which she underwent left salpingooophorectomy and successfully completed 4 cycles of bep over 4 months. during routine follow-up 8 months after initiation of treatment for ovarian yst, she was noted to have a small, non-tender, indurated nodule on the left side of her upper back approximately 1cm in diameter. punch biopsy of the skin nodule was performed and pathology was positive for cutaneous myeloid sarcoma. at the time of next follow-up less than one month later, the skin lesion had resolved. two subsequent bone marrow aspirates were performed one month apart and were negative for leukemic involvement or mds. examinations and work-up including whole body pet with ct scan were negative for evidence of disease. although cutaneous ms can be regarded as the herald of systemic myeloid disease rather than a localized process, our patient was monitored periodically with physical exam and laboratory evaluations. she remains free of disease more than four years after the presentation of cutaneous ms without any further treatment. spontaneous regression ms has been previously reported. the authors would like to stress that a conservative approach with close observation could be an option in cutaneous ms even with history of chemotherapy exposure. nesreen ali, iman sidhom, sonia soliman, sherine salem national cancer institute, cairouniversity, egypt children cancer hospital egypt, egypt background: acute leukemia is the commonest malignancy in childhood. the coincidental occurrence of leukemia with hemophilia is extremely rare. hemophilia is a congenital rare x linked bleeding disorder. the main complication of the two diseases is bleeding diathesis which may be lifethreatening due to many factors, deficiency of coagulation factors in hemophilic patients, thrombocytopenia from disease and chemotherapy in leukemic patients, certain cytotoxic drugs such as asparaginase which may result in coagulation disorders and infection which may lead to disseminated intravascular coagulation. objectives: reporting such a case is imperative to set up treatment guidelines for prevention of bleeding and to optimize the therapeutic approach for these patients. design/method: seventeen years old boy, presented to children cancer hospital egypt in june 2015 with pallor and multiple ecchymoses.he was diagnosed with precursor b acute lymphoblastic leukemia, cerebrospinal fluid (csf) was free, the chromosomal analysis revealed hypodiploidy 36, xy. he had moderate type of hemophilia a since birth, factor viii level was 1.5 % at time of diagnosis, coagulation profile revealed prolonged partial thromboplastin time 89 (normal 26-45), factor viii was low 1%, prothrombin concentration and prothrombin time were normal100% and 13 seconds, virology screening for hepatitis b core igg/igm, hbs ag, hiv and hc igg /igm were negative.the patient started induction total xv sjcrh protocol, factor viii 40 unit/kg was given at presentation before doing bone marrow aspiration(bma), csf and as a prophylactic before intramuscular asparaginase injection, intrathecal and bma. it was given immediately within 2 hours before the procedures and platelets transfusion was given regularly to maintain platelets count about 50,000. the minimal residual disease by flow cytometry was 0.81% and 0.11% at d15 and d42 induction. results: our patient received his induction and reintensification chemotherapy without any major bleeding event which reveals the success of our guidelines for the prevention of bleeding. he developed very early relapse at w7 maintenance by the same clone. he received salvage chemotherapy but didn't achieve remission and died out of disease and resistant clone. the development of leukemia on top of hemophilia is a major problem. bleeding complication during chemotherapy can be prevented by regular prophylactic factor viii and platelets concentrate transfusion with good supportive care. life threating bleeding complication may be correlated with the severity of hemophilia. we need to collect data about the biology of leukemic cells, complications, and cause of death to optimize care for these patients. background: mucoepidermoid carcinoma (mec) is a rare malignancy that arises from exocrine glands in the upper aerodigestive tract and tracheobronchial tree. conventionally, mec diagnosis is based on histology, with prognosis based on the extent of resection and detection of metastases. mec is characterized by a translocation of chromosomes 11q and 19p resulting in a fusion between the mect1 and maml2 genes, that occurs in 38-81% of cases. this fusion transcript has been recognized to have a favorable impact on disease features and prognosis of mec. however, recent studies indicate that high grade mec can have mect1-maml2 fusion positivity and multiple other genomic imbalances that have not been studied in much detail. owing to the rarity of mec tumors, more definitive data related to the clinical and prognostic significance of these molecular markers are limited. objectives: 1. identify the presence or absence of mect1-maml2 fusion in the tissue of our patient. 2. analyze the incidence of the fusion in 25 mec cases in children and young adults retrieved from the iowa cancer registry. 3. determine if fusion status correlates with clinical, pathological and outcome data in our cohort. design/method: we describe the case of a 12 year-old caucasian male who presented with recurrent pneumonia, persistent cough and radiographic evidence of right lobar collapse. bronchoscopy revealed an endobronchial lesion and the patient underwent right upper lobe sleeve resection. pathology report was consistent with low grade muco-epidermoid carcinoma. we retrieved 25 archived formalin-fixed paraffinembedded (ffpe) specimens of pediatric and young adult mec cases (ages 0-29) reported in iowa from 1973-2016 using the iowa cancer registry. testing for the mect1-maml2 fusion in the index case and 25 ffpe specimens will be done using a custom-designed laboratory validated next generation sequencing (ngs) assay with the ability to detect novel fusion partners. clinical, pathological and outcome data (age, sex, tumor site, tumor size, nodal metastases, clinical stage, histologic grade, treatment and follow up) will be analyzed to correlate with fusion status. the mect1-maml2 fusion tested positive in our index patient. we will obtain irb approval to test for the fusion in the 25 archived ffpe specimens and correlate clinical, pathological and outcome data. conclusion: mect1-maml2 fusion is a frequent event in mec that has prognostic and potential therapeutic applications in adults. the results of this study may enlighten the clinical management of mec in children and young adults. children 's mercy hospital, kansas city, missouri, united states background: mutations in the samd9 gene are associated with a rare syndrome comprising of myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (mirage syndrome). diagnosis is made through exome sequencing. in the largest reported case series, of eleven patients diagnosed with mirage syndrome, two developed loss of chromosome 7. given the potent growth restricting activity of samd9 mutants, the loss of chromosome 7 is considered the first documentation of adaptation by aneuploidy mechanisms in humans and led to myelodysplastic syndrome (mds), with deaths occurring from related complications at 2 and 5 years of age. objectives: to report a case of mirage syndrome with congenital thrombocytopenia progressing to bone marrow failure, managed uniquely with bone marrow transplantation. results: male born at 29 weeks gestation with prenatal diagnosis of iugr, two vessel cord, oligohydramnios was found to have ambiguous genitalia, adrenal insufficiency, partial panhypopituitarism and congenital thrombocytopenia with bone marrow showing absence of megakaryocytic precursors. severe thrombocytopenia was present from birth. bone marrow evaluation demonstrated a hypocellular marrow with markedly reduced megakaryocytic and myeloid precursors and no evidence of myelodysplasia. he required gastric tube placement for failure to thrive, had a laryngeal cleft repaired and developed focal segmental glomerulosclerosis. mpl gene testing for congenital amegakaryocytic thrombocytopenia was negative. testing for fanconi anemia, shwachman-diamond syndrome and dyskeratosis congenita was also negative. approximately 30% of cells had loss of heterozygosity on chromosome 7q. exome sequencing showed that he is heterozygous for a de novo gain of function variant, c.2471g>a (p.arg824gln), identified in the samd9 gene, confirmed by sanger sequencing and consistent with a diagnosis of mirage syndrome. at 6 years of age, he developed pancytopenia requiring frequent transfusions with platelets and packed red blood cells. he underwent a successful bone marrow transplant at 7 years of age without significant complications, and remains transfusion independent without cytopenias greater than 18 months from bone marrow transplantation. conclusion: it is imperative to pursue work up for persistent congenital thrombocytopenia in a stepwise multidisciplinary manner. to the best of our knowledge, this is the first case of mirage syndrome associated bone marrow failure treated with bone marrow transplant. due to the individual rarity of mirage syndrome and pediatric myelodysplastic syndrome, it is important to maintain an index of suspicion given their association and explore bone marrow transplant as a therapeutic option. results: the patient demonstrated disease regression, initially, and continued without disease progression for 36 months. the regimen has been well tolerated with only minimal side effects of dry skin (ctcae grade 1) and a transient episode of brief erythrodysesthesia (ctacae grade 2) that resolved spontaneously. the combination of sorafenib and capecitabine was effective and well tolerated in this adolescent patient with fl-hcc. our observations, although in a single patient, lend support for further testing of this novel oral chemotherapy regimen in patients with fl-hcc, a disease for which there is no effective standard chemotherapy approach. background: epstein-barr virus (ebv) is a ubiquitous virus associated with a broad range of malignancies due to its oncogenic potential. history of organ or bone marrow transplantation, immunosuppressive therapy, and primary or acquired immunodeficiency syndromes increases the risk of ebvassociated tumors. epstein-barr virus associated smooth muscle tumors (ebv-smt) are unique and rare neoplasms typically discovered in immunocompromised patients. most information related to pathogenesis and therapeutic options is limited to case reports and case series of adult patients. there are several gene expression pathways that ebv utilizes, the most notable of which is the mammalian target of rapamycin (mtor) pathway. the mtor pathway performs a key role through integrating various cell growth signals and factors to regulate protein synthesis and metabolism related to smooth muscle proliferation. sirolimus is an immune modulating therapy that targets the mtor pathway to block activation of lymphocytes. objectives: several case reports have demonstrated shortterm clinical remission of ebv-smt in adult patients with the use of sirolimus. we report the first case of long-term background: bilateral neuroblastoma is characterized as neuroblastoma arising in both adrenal glands, a rare presentation with little data on its genetic make-up. a two-monthold patient was diagnosed with bilateral neuroblastoma in our clinic. her risk assignment was based on biopsy of the left adrenal lesion, which showed mycn amplification, an unfavorable genetic marker. treatment regimen was intensified accordingly and after 5 courses of chemotherapy tumors were excised. patient went on to receive a stem cell transplant and immunotherapy. with no knowledge of genetic similarity between the two tumors it is unclear whether biopsy of the right lesion would have yielded similar results or whether bilateral biopsies are needed for risk assessment of bilateral neuroblastoma. objectives: utilize whole exome sequencing (wes) to characterize the genomic signature of bilateral adrenal neuroblastomas excised following chemotherapy treatment. design/method: paraffin-embedded samples from left (l) and right (r) tumors underwent wes at the broad institute. we analyzed resulting data including somatic variant calls, indel mutations, and copy number variants (cnvs) using ingenuity software to evaluate and compare differences between the two tumor samples. preliminary analysis of the data shows important descriptive information on the two tumor samples. out of 29 somatic mutations in the r tumor cells and 25 mutations in the l tumor cells, only two common somatic mutations were present. out of 40 cnv calls in the r tumor and 60 in the l tumor, 31 cnvs were common between the two tumors, or 73% of each tumor's cnv calls. there was a 14 fold higher frequency in gains versus losses. the median size of the common cnvs was 56,683 (range 220 to 3,323,064 bp). cancerrelated genes with increased copy numbers included transcription factors, receptors for signal transduction pathways, and histone methylation proteins. conclusion: preliminary analysis of the wes results of the two adrenal tumors show some genomic divergence. because the tumor tissue was exposed to chemotherapy prior to excision it is difficult to determine whether genomic divergence is a result of independently originated tumors or subsequent adaptation to chemotherapy of a clonal cell population. the high number of common cnvs in the two tumors points to a common cell of origin, however the low number of common somatic mutations does not fit that picture. a future study to help elucidate the question will be wes of the original biopsy tissue to provide information on tumor mutations prior to the effects of chemotherapy. baylor college of medicine, houston, texas, united states background: although there has been significant improvement in the overall survival rates of children with cancer many children will still die from their illness or complications secondary to treatment. research surrounding the deaths of children who succumb to their disease is warranted to ensure we are providing the best care possible for these patients. objectives: this case series aims to explore pediatric cancer deaths by focusing on perhaps the most extreme cases of high intensity end of life care. we explore those patients whom we know are dying or our very likely to die as evidence by their do not resuscitate (dnr) orders. in all of these cases despite the patients very grim prognosis, their great likelihood of death and limitations placed of resuscitation methods all patients continued end of life care in the pediatric intensive care unit (picu). the primary medical records of all children with a cancer diagnosis who died between february 1, 2011 and january 31, 2017 in the picu with a dnr order seven days or earlier prior to death. each medical history included disease-directed treatment history and response with particular attention to the events surrounding the terminal admission. results: eight patients met criteria for this study representing 1.9% of all cancer patients who died during this time period and 7.4% of those who died in the icu. the average time between dnr and death is 19.6 days (7 days -32 days). the average length of terminal admission was 43.5 days (1 day -153 days). the average time between diagnosis and dnr is 10.75 months (0 months -22 months). the average time between diagnosis and death is 11.25 months (0 months -23 months). conclusion: these cases highlight the journey that patients, families and providers endure leading up to death. medical care is complex, there are very few absolutes that are encountered when caring for patients and decisions around limiting or withdrawing medical care are made in a context of the prior journey. . these cases help to understand the complexity of death and how two seemingly opposite ideals can be congruent in the event of an anticipated death. most of these cases show the need for improved anticipatory guidance surrounding death and greater consideration for de-escalation of care when death is expected. the hospital for sick children, toronto, ontario, canada background: rhabdomyosarcoma (rms) is the most common soft tissue sarcoma in children, with embryonal (erms) and alveolar (arms) representing the most common subtypes. arms tumors are associated with inferior outcome when compared to erms, and they are characterized in about 80% of the cases by a t (2;13) or t(1;13) chromosomal translocation with creation of a pax3-foxo1 or pax7-foxo1 fusion gene, respectively. it is increasingly clear that the pax-foxo1 fusion status is an important poor prognostic factor, thus the histological classification tends to be replaced by the fusion status, particularly in terms of risk stratifica-tion in contrast to arms, there are no recurrent chromosome alterations in erms; however, there are multiple numerical chromosome changes that are frequent in these tumours: gain of chromosome 2,8, 12 and 13 have been found in 25 to 50 % of emrs karyotypes. moreover, erms tumors show frequently allelic loss, the 11.p15.5 chromosomal region being the most frequently involved. recently, novel gene fusions have been described also in erms tumours. these fusions involved mainly the ncoa2 and or the vggl2 genes. the rearrangement partners are variable, and include, i.e. pax 3 (2q35), srf (8q11) and tead 1 (11p15). objectives: to present a patient who died as a consequence of brain metastases while on therapy in the setting of an foxo1negative rms and the identification of a new translocation t(2;15)(q21;q22). design/method: case report and retrospective review of the literature. we report a case of pelvic embryonal rhabdomyosarcoma in a 3-month old boy. he was treated as per cog arst 0531 intermediate risk group, but unfortunately was found to have a large cerebellar tumour during the course of his chemotherapy treatment and he subsequently passed away. a novel translocation between chromosomes 2 and 15 was observed in 11 of 20 metaphase cells by g-band analysis in the autopsy sample of the brain lesion. breakpoints of the translocation were estimated to be at 2q21 and 15q22. there were no additional clonal chromosome abnormalities in the tumour cells. conclusion: erms tumors with fusion genes involved have been exclusively described in patients less than 12 months of age; they seem to be associated with spindle cell histology and, a favorable outcome. in our patient, a novel (2;15) translocation was found and clinically, the patient had a dismal outcome. further studies are indicated to inquire whether this finding is of significance in term of prognosis for these patients. children 's national medical center, washington, district of columbia, united states background: iatrogenic immunodeficiency-associated lymphoproliferative disorders (lpds) are a group of lymphoid s35 of s301 proliferations or lymphomas that are well known to be associated with an immunosuppressed state. these disorders most commonly occur following hematopoietic or solid organ transplantation (called post-transplant lymphoproliferative disorders or ptld), but cases have also been described during the treatment of autoimmune and rheumatologic disorders by immunosuppressive and immunomodulatory medications. these disorders are strongly associated with infection by the epstein-barr virus (ebv) as a result of impaired immune function in the immunosuppressed state. while this phenomenon has been well documented in autoimmune conditions, cases affecting pediatric patients while on antileukemia chemotherapy are lacking. background: atypical teratoid/rhabdoid tumor (at/rt) of the central nervous system (cns) in children younger than 3 years old has a prevalence of 1% to 2% and accounts for 1.6% of all pediatric cns tumors. only 15-30% of patients have leptomeningeal dissemination. rhabdomyosarcoma is the most common soft tissue tumor in childhood, but represent only 3-4% of all pediatric cancers. rarely, it can metastasize or even directly extend into the cns, but typically, cases of cns involvement arise either from parameningeal areas or other primary sites. primary spinal or meningeal rhabdomyosarcoma is extremely rare. objectives: our objective is to describe two unique cns malignancies presenting as rare, primary leptomeningeal disease. design/method: case 1 a 19-month-old female presented with vomiting, fatigue and listlessness, despite a normal head ct and brain mri. csf showed hypoglycorrhachia and mild pleocytosis. ceftriaxone was started, but she developed nuchal rigidity and cranial nerve vii palsy. repeat brain mri showed evolving leptomeningeal enhancement concerning for meningitis. she gradually developed worsening opisthotonus and ultimately a brain biopsy of the temporal lobe was consistent with at/rt. case 2 a 3-year-old male presented with new generalized tonic-clonic seizure activity and intermittent headaches with photophobia, phonophobia, and vomiting. brain mri was significant for enhancement of interpenducular and suprasellar cisterns extending to the optic nerves and chiasm most consistent with meningitis. neurosurgery ultimately placed a lumbar drain for hydrocephalus, and a tissue biopsy demonstrated primary meningeal rhabdomyosacroma. results: in case 1, our patient's temporal lobe biopsy demonstrated grade iv malignant tumor cells consistent with atypical teratoid/rhabdoid tumor. fish demonstrated a homozygous deletion of smarcb1 (22q11.23). she was started on chemotherapy per the dana farber at/rt protocol but ultimately was discharged home on hospice. in case 2, our patient's lumbar arachnoid biopsy demonstrated cellular tumor consistent with group iiia embryonal rhabdomyosarcoma. immunostaining was positive for cd99, desmin, myogenin, and myo-d1 with neural markers ema and gfap highlighting the meninges but without a neural component to the tumor. he completed craniospinal radiation to 36gy total with lumbar boost to 50.4gy total. he is currently receiving chemotherapy per arst0431 protocol. conclusion: these two cases are particularly instructive because of their similar initial presentations and neuroimaging, but with very different and unique diagnoses. university of iowa, iowa city, iowa, united states background: ebf1-pdgfrb fusion causes ph-like b-cell acute lymphoblastic leukemia (b-all), which has a philadelphia positive phenotype without the bcr-abl translocation. this is one of several mutations associated with ph-like b-all and leads to downstream overexpression of tyrosine kinase. ebf1-pdgfrb fusion accounts for about 8% of children with ph-like b-all. patients with ph-like b-all previously had poorer outcomes with conventional chemotherapy. the addition of tyrosine kinase inhibitors (tki), like imatinib, has improved the outcome for many patients predicted to have tki sensitive mutations. objectives: to review clinical characteristics and outcomes of two cases of ph-like b-all at the university of iowa stead family children's hospital and to compare these outcomes to similar cases reported in the literature. design/method: a retrospective chart review was performed for two cases of ph-like b-all diagnosed and treated at the university of iowa stead family children's hospital. results: both patients were males diagnosed at 8 years of age with high wbc count (110,700 and 347,200) and positive for ebf1-pdgfrb gene fusion. patient 1 (pt1) was cns 2b at presentation while patient 2 (pt2) was cns negative; neither had testicular involvement. both started treatment according to cog protocol aall1131. peripheral blasts cleared by induction day 22 for pt1 and induction day 14 for pt2. at end of induction, pt1 had m3 bone marrow and pt2 had m1 bone marrow but mrd 8%. dasatinib was started induction day 13 for pt1 and induction day 31 for pt2. pt1 was still not in remission at end of consolidation; bone marrow cell culture for tki resistance showed best response to dasatinib. pt1 proceeded to anti-cd19 car t-cell therapy followed by tbi-based matched unrelated donor bone marrow transplant. pt2 had negative mrd at the end of consolidation and continues chemotherapy according to aall1131, dasatinib arm. both patients are currently clinically well. our patients had the same tyrosine kinase gene fusion and similar initial clinical courses. while both patients had persistent disease at end of induction, pt1 had almost 80% blasts while pt2 had significant reduction of disease burden before starting tki. pt2 showed good response with the addition of dasatinib while pt1 did not. these findings suggest that response to conventional chemotherapy may potentiate the effect of tki and may predict overall outcome. there are likely additional factors which must be taken into account when determining response to tki for patients with ph-like b-all which have not yet been identified. background: medulloblastoma is the most common malignant brain tumor of childhood. classically, medulloblastoma presents as a well-defined mass lesion in the cerebellum, with a high rate of metastatic dissemination. primary leptomeningeal medulloblastoma (plmb) is an exceedingly rare type of medulloblastoma presentation with a dismal prognosis in which patients present with isolated leptomeningeal disease without an associated mass. to our knowledge, only three pediatric and three adult cases of plmb (ages 5 -30 years) have been reported, all of which died within 6 months of diagnosis. this is the first case of plmb to report a molecular classification. objectives: to report the case of a pediatric patient with plmb in which histopathologic and molecular characterization was performed and to describe the patient's treatment and clinical course. design/method: retrospective review of the patient's electronic medical record and review of the literature. a 9-year-old boy presented with headache, vomiting, diplopia, and fatigue. physical examination revealed upward gaze palsy, left-sided extremity and facial weakness, and ataxia. magnetic resonance imaging (mri) of the brain revealed diffuse cerebellar leptomeningeal enhancement and edema without an identifiable mass and moderate hydrocephalus. mri of the spine and cerebral spinal fluid analysis were normal. a diagnosis of cerebellitis was rendered, and the patient underwent placement of a ventriculoperitoneal shunt. an extensive infectious, neurologic, rheumatologic, and oncologic workup did not identify an etiology. empiric antibiotics, high-dose steroids, and intravenous immunoglobulin therapy yielded minimal improvement. two months later, repeat mri of the brain performed for declining mental status demonstrated progressive thickening of cerebellar leptomeningeal disease. a suboccipital craniectomy with decompression and cerebellar biopsy were performed. pathologic examination revealed a diagnosis of plmb, classic histology, non-wnt/non-shh, without gain/amplification of myc/mycn, and p53 wild type pattern. craniospinal radiation to 4140 cgy with a 1440 cgy boost to the posterior fossa was delivered with concurrent carboplatin/vincristine over six weeks. two months following chemoradiation, mri of s37 of s301 the brain demonstrates significantly reduced pathological leptomeningeal enhancement of the cerebellum, and the patient is awaiting initiation of systemic chemotherapy while recovering from a surgical wound infection. conclusion: plmb is extremely rare but should be considered in patients with cerebellitis and diffuse leptomeningeal involvement who are refractory to medical management or in whom an etiology has not been identified. cerebellar biopsy is recommended early to enable timely treatment and improved outcomes. molecular classification should be performed in cases of plmb to further characterize this disease, inform treatment decisions, and improve clinical outcomes. background: primary intracerebral osteosarcoma is extremely rare and limited to case reports. ptpn11 gain of function is associated with noonan syndrome, which has increased risk of multiple cancer types including brain tumors, but osteosarcoma has never been described. ptpn11 mutations have been reported in many cancers as both oncogenes and tumor suppressors, however no ptpn11 mutations have been described in osteosarcoma. pdgfr-a is a growth factor receptor whose activation is implicated in several malignancies. pdgfr-a and ptpn11 concurrent mutations are described in glioblastoma. there is no known link between holoprosencephaly, noonan syndrome, and osteosarcoma. we report a case of multifocal intracerebral osteosarcoma in a child with lobar holoprosencephaly and chronic subdural hemorrhage and discuss the genetic changes found in the tumor. design/method: a seven-year-old caucasian female, with a known diagnosis of lobar holoprosencephaly, chronic subdural hemorrhage and well controlled seizure disorder presented with status epilepticus shortly after completing antibiotic therapy for infection of subdural hematoma. mri showed diffuse dural thickening with mass lesions in the frontal lobe, temporal lobe, and the parasagittal region, the largest of which was contiguous with the subdural space but none of the lesions were associated with bone on mri or by direct neurosurgical visualization. tissue obtained for concern for recurrent infec-tion resulted in a diagnosis of high grade osteosarcoma. dna analysis was performed to help guide treatment choice. results: standard metastatic work-up was negative for skeletal primary tumor or metastatic lesions outside of the brain. she was treated with high dose methotrexate for two cycles per modified aost1331. despite maximal supportive care, she quickly developed rapid tumor growth as well as intratumoral hemorrhage with resultant herniation and death from respiratory failure just three months after diagnosis. tumor gene sequencing discovered three mutations with described roles in cancer: pdgfra d842>vr, kdm6a loss of exons 1-4, and ptpn11 a72v. conclusion: to our knowledge, primary multifocal extraosseus intracerebral osteosarcoma has not been previously described. despite known cns penetration of high dose methotrexate, this tumor proved resistant and aggressive. holoprosencephaly is associated with a multitude of known genetic drivers, but none are found in this case. furthermore, the genetic changes in this tumor are not typical for osteosarcoma. pdgfr-a over-expression is described in osteosarcoma, but is not clearly correlated with worse overall survival. further research is required to determine the role of ptpn11 in osteosarcoma. background: anaplastic lymphoma kinase (alk) encodes a receptor tyrosine kinase whose activation induces pathways associated with cell proliferation, angiogenesis, and cell survival. alk rearrangements are rare in neuroblastoma, while alk mutations and gene amplification occur more frequently. alk mutations have been found to be associated with increased alk protein expression that is associated with a worse prognosis. alk is commonly mutated in neuroblastoma at three hotspots (f1174, r1275, and f1245). the eml4-alk rearrangement has mostly been associated with lung adenocarcinomas, with only a few cases of non-lung cancers found. it has never been reported in neuroblastoma. multimodal therapy and to report the successful management of treatment related iron overload. results: a 10-year old male presented with abdominal swelling and ct showed a right kidney mass and bilateral lung nodules. he underwent right radical nephrectomy with lymph node sampling. pathology was reviewed centrally and revealed wilms tumor with diffuse anaplasia with rhabdomyosarcoma arising within the stromal component and 3 of 13 nodes positive. he received adjuvant intensive chemotherapy and radiation to the hemiabdomen and whole lungs. the 49-week chemotherapy regimen was vincristine, doxorubicin, cyclophosphamide (per cog arst0431) alternating with carboplatin and etoposide (per cog aren0321 revised uh-1). treatment was complicated by multiple episodes of fever and neutropenia and anorexia requiring g-tube placement. post-therapy, he had persistent neutropenia and thrombocytopenia without related complications. every 6 months for 3 evaluations he underwent a bone marrow which revealed normocellular marrow with maturing trilineage hematopoiesis. evaluation for a bone marrow failure syndrome was unrevealing. starting at 6 months into therapy and all posttherapy imaging showed splenomegaly. he received 29 units of packed red blood cells through the duration of therapy. he was diagnosed with iron overload based on serum ferritin and imaging, including t2*mri. he received therapeutic phlebotomy for 2 years with normalization of serum iron studies, t2* of the heart, and liver iron concentration. he is more than 6 years from completing therapy with no evidence of recurrent disease. asymptomatic cytopenias persist and he has no evidence of iron overload. conclusion: though a rare development, clonal sarcomatous transformation can occur in wilms tumor. our patient's tumor was successfully treated with intensive multimodal therapy targeting the diffusely anaplastic wilms and the rhabdomyosarcomatous component. treatment-related iron overload in a pediatric patient with a solid tumor was successfully treated with phlebotomy. consideration should be given to screen patients with solid tumors who receive multiple packed red cell transfusions for iron overload at the completion of cancer therapy. primary children's hospital, university of utah, salt lake city, utah, united states background: malignant solid tumors are less frequently encountered in infants. primitive myxoid mesenchymal tumors of infancy (pmmti) are a myofibroblastic malignancy and cases are rarely reported in the literature. cure is achieved in the majority of cases with surgical resection, however treatment for unresectable cases remains an enigma. recently published literature postulates that the newly discovered bcor duplication found in pmmti is tumorigenic via an epigenetic pathway. this molecular signature resembles that of clear cell sarcoma of the kidney (ccsk) and the growing number of bcor mutated sarcomas. a similar chemotherapeutic backbone and local control used for ccsk, has been proposed for the unresectable subset of pmmti. utilizing this approach a 19 month-old with relapsed disease has remained disease free for 12 months. however, given the rarity of this disease and the lack of published literature, there is no known standard of care treatment for unresectable and/or recurrent ppmti. we report a case of unresectable recurrent pmmti, a rare infant tumor, with less than 20 cases reported. design/method: medical record, radiological studies, pathology and literature was reviewed. results: our patient is a now 12 month-old female who presented with constipation and lower extremity weakness in the first weeks of life. an mri demonstrated a large lumbar epidural mass with spinal cord impingement. given prolonged (>14 days) neurological symptoms and location, emergent chemotherapy was initiated. biopsy showed a bcor positive, primitive myxoid mesenchymal tumor of infancy (pmmti). she was treated with ifosfamide, carboplatin and etoposide, and demonstrated clinical and radiographic response. we gave two additional cycles of cyclophosphamide, carboplatin and etoposide until surgical resection was feasible followed by two post-surgical cycles of chemotherapy. unfortunately, four month post-therapy mri demonstrated two new lesions; an unresectable paraspinal soft tissue mass and a left iliopsoas groove mass. given bcor association and reported successful therapy with vincristine, doxorubicin, cyclophosphamide alternating with ifosfamide and etoposide, we elected to incorporate vinca-alkaloid and anthracycline into her regimen. she is being treated with vdc/ie with plan for radiation consolidation. conclusion: pmmti is a locally aggressive tumor, for which surgical resection is curative. for those not amendable to resection, best care practices are still being determined. we report a case of pmmti initially responsive to chemotherapy, but not curative. this is the second case to conclusively demonstrate chemo-responsiveness. bcor mutation seems to be a common feature of this cancer; its role in the pathogenesis and as a target is an area of investigation. medical college of wisconsin, milwaukee, wisconsin, united states background: atypical teratoid/rhabdoid tumors (atrt) are central nervous system (cns) tumors that most commonly occur in very young children. there is no widely accepted standard of care for atrt patients, and while survival rates are improving they are historically poor. patients with metastatic disease to the spine at diagnosis have a worse prognosis, and for patients >3 years old, the presence of metastatic disease often results in the use of craniospinal radiation. the importance of correctly identifying metastatic disease at diagnosis aids in decision making and can have both prognostic and therapeutic implications. mr imaging at diagnosis is used to identify metastatic disease; however, here we present a case of diffuse leptomeningeal enhancement that spontaneously resolved after resection of a primary supratentorial atrt. objectives: to describe the resolution of diffuse leptomeningeal enhancement after resection of a primary atrt tumor in a 12-month-old prior to any adjuvant therapy. results: a 12-month-old male presented with a 2 month history of vomiting and weight loss, regression of gross motor developmental milestones, and left hemiparesis. a brain mri demonstrated a 7 × 5.8 × 5.7 cm solid and cystic right atrial mass with diffusion restriction and post-contrast enhancement. smooth diffuse enhancement was noted along the surface of the brainstem and within the interpeduncular fossa. a spine mri demonstrated diffuse circumferential post-contrast enhancement along the surface of the entire spinal cord. the patient underwent a successful near total surgical resection of the primary mass. pathology confirmed the loss of ini-1 staining in tumor cells, consistent with a diagnosis of atrt. no immediate adjuvant radiation or chemotherapy was given. repeat imaging was completed 15 days after resection. brain mr demonstrated expected post-operative changes within the surgical cavity without definitive residual mass or leptomeningeal enhancement. spine mr demonstrated complete resolution of the previously seen circumferential enhance-ment along the entire spinal cord. csf evaluation at that time was negative for tumor cells. after recovery from surgery, chemotherapy treatment was initiated. conclusion: leptomeningeal enhancement at the time of diagnosis of atrt has historically been considered clear evidence of metastatic disease. this case raises questions about the previously accepted etiology of these imaging changes and suggests that widespread leptomeningeal enhancement should be carefully interpreted in future patients with similar imaging findings. in this setting, clinicians should consider repeat imaging following primary surgical resection in order to provide appropriate prognostic information and inform therapeutic decisions. poster # 047 primary ewings sarcoma of cervical cord mimicking cauda equina syndrome sucharita bhaumik, joshua chan nyu winthrop hospital, mineola, new york, united states background: ewing's sarcoma (es) is a malignant primary bone tumor usually involving long bones. primary es of spine is quite uncommon (0.9%) and its location in the cervical spine is even more rare. cauda equina syndrome (ces) is symptoms due to damage to the bundle of nerves below the end of the spinal cord known as the cauda equina (low back pain, radiating shooting pain down the legs, paraplegia, and loss of bowel or bladder control). it often occurs with lesions of lumbosacral spine. treatment with high-dose steroids may provide pain relief and improved neurologic function (by reducing edema) while awaiting diagnostic studies objectives: to demonstrate an unusual clinical presentation and emergent management of cervical es presenting with ces like symptoms. : 15 year old male presented with a left sided posterior neck mass. soon after, he developed weakness of left arm, urinary and stool retention and inability to walk or bear weight in both legs. on physical exam a left tempero-occipital 6 × 4cm fixed, non-tender, non-fluctuant mass was noted as well as motor and sensory impairment of left upper extremity, bilateral spastic paraplegia and loss of sphincter control. mri cervical spine showed a left cervical tumor with moth eaten appearance involving the vertebral bodies of c2-c3, adjacent muscles, displacing vital structures of the neck and compressing the cervical spinal cord. the thoracic and lumbosacral spine had no disease involvement. due to rapidly worsening spinal cord compression he was emergently treated with high dose steroids. he gained back all function in his extremities and regained bowel and bladder control. this eliminated need for urgent neurosurgical intervention. results: biopsy of the neck mass showed small blue round cells consistent with es with ewsr1 gene rearrangement. staging work up revealed no additional metastatic involvement. he then initiated treatment for localized es with systemic chemotherapy and radiotherapy and has had excellent response to treatment so far. conclusion: this is the first known case of non metastatic primary cervical es mimicking ces where an acutely enlarging mass presented with rapidly progressive neurologic deficits due to compression of anterior spinothalamic tract. in these unusual presentations of ces without lumbodorsal involvement it is important to consider cervical lesions. early rapid steroid initiation should be considered while awaiting biopsy results to prevent worsening cord compression followed by es focused treatments. this increases the chance of a successful outcome. the initial improvement with steroids may confuse the tumor with being a lymphoma children 's mercy hospital, kansas city, missouri, united states background: von willebrand disease (vwd) is a relatively common bleeding disorder with a high degree of genotypic and phenotypic variation. bleeding is usually mucocutaneous but can be severe and include muscle and joint bleeds especially in type 3 vwd patients. most common bleeding management consists of desmopressin, anti-fibrinolytics, and/or plasma-derived antihemophilic factor/von willebrand factor (ahf/vwf) complex. a recombinant vwf has become available in the last few years. anaphylaxis and inhibitor development in vwd are rare. objectives: to describe the rare clinical manifestation of anaphylaxis to factor concentrate in a patient with severe type 1 vwd. results: a 13-year-old female with severe type 1 vwd [baseline vwag 10%, activity < 10%, factor viii (fviii) 26%] originally presented with heavy menstrual bleeding (hmb) leading to anemia requiring blood transfusion. she underwent placement of a levonorgestrel-releasing intrauterine device (lngiud) and began norethindrone. her hmb continued despite the lngiud and an increase in norethindrone dosing. plasma derived ahf/vwf complex was administered, which she had previously received. following the infusion, the patient developed anaphylaxis with hives, wheezing, tachycardia, and itching requiring 2 doses of diphenhydramine and 1 dose of hydrocortisone with resolution of symptoms. subsequently, she received recombinant vwf without incident. however, due to her low fviii level, she also required treatment with a full length recombinant fviii product. she again developed hives and itching after this infusion. she has since received recombinant vwf with recombinant fviii/fc fusion protein without further allergic reaction. there was no evidence of an inhibitor with her most recent post-infusion vwf level was 101%, factor viii 199%. conclusion: anaphylaxis to plasma derived factor products has been documented far less frequently within the vwd population compared to those with hemophilia and is typically seen in those with large gene deletions, usually with type 3 disease. therefore, similar type 1 vwd patients with severe disease may benefit from gene sequencing. it is unclear in this patient's case to which aspect of her treatment she is allergic, as she reacted to plasma-derived ahf/vwf and full length recombinant fviii, but not recombinant vwf or recombinant fviii/fc fusion protein. we hypothesize that she may be allergic to an epitope in the fviii b domain, or that the presence of fc fusion may have had a protective effect. further investigation including genetic analysis is planned. nodules. biopsies were consistent with neuroendocrine carcinoma, large cell type (g3). next generation sequencing revealed a khdrbs2-braf fusion. he received conventional cytotoxic chemotherapy regimens both with cisplatin/doxorubicin, capecitabine/temozolomide, and doxorubicin/etoposide, but achieved a minimal response followed by rapid disease progression, massive ascites, and renal failure secondary to bilateral ureteral obstruction. results: based on his prior genomic testing, therapy with single agent mek inhibitor (trametinib) was initiated. this produced a rapid, dramatic response with greatly reduced disease burden at all sites, resolution of ascites and return to completely normal activity within 2 months. this response lasted for approximately 6 months before the tumor again progressed. further therapy with an erk inhibitor was ineffective, and the patient expired from progressive disease. located on the chromosome 7q34, the braf oncogene, as part of the ras/mapk pathway, is involved in cellular proliferation, differentiation, migration, and apoptosis. braf mutations are recognized in a wide range of adult malignancies: thyroid cancers, non-small cell lung cancer, cholangiocarcinoma, ovarian cancers, and multiple myeloma. braf mutations have also been described in adult neuroendocrine carcinoma of the colon. trametinib is a highly specific inhibitor of mek1/mek2, a downstream mediator in the braf pathway. it has demonstrated activity in a number of tumors including advanced melanoma and gliomas. trametinib was chosen for this patient based on his atypical braf fusion. we believe this is the first documented case of its successful use in neuroendocrine carcinoma in the pediatric population. conclusion: this case demonstrates the presence of braf fusion in a case of pediatric neuroendocrine carcinoma and significant response to single agent mek inhibition in this context. this cases raises the question as to whether the combination of a targeted inhibitor, in addition to either conventional chemotherapy or other braf inhibitors, might offer a better approach to therapy than current treatment options. albany medical center, albany, new york, united states background: warm autoimmune hemolytic anemia (waiha) is characterized by autoantibody, and occasional complement binding of protein antigens, on the surface of red blood cells at temperatures ≥37 oc resulting in targeted destruction. we describe the case of a 17 year old male with a history of evan's syndrome, poor immune response to vaccines and lymphoid hyperplasia, presenting with altered mental status and severe anemia, found to have a warm igg pan agglutinin with evidence of both intra and extravascular hemolysis. his course was complicated by respiratory failure requiring intubation, pulmonary emboli, enterococcus bacteremia and hypertension. he received multiple transfusions with only transient increases in hemoglobin. the aiha was refractory to multiple rounds of treatment with high dose steroids, ivig, rituximab, cyclophosphamide, bortezomib, plasma exchange and mycophenolate mofetil (mmf). objectives: given the refractory nature of our patient's aiha the decision was made to trial eculizumab, a monoclonal antibody targeting c5 complement, preventing its cleavage and activation, and shown to be effective in treatment of atypical hemolytic uremic syndrome and hemolysis due to an igm cold agglutinin. prior to eculizumab infusion, ch50 and sc5b-9 assays were significantly elevated. design/method: the patient was given two doses of eculizimab 6 days apart. results: his hemoglobin steadily rose independent of red cell transfusions with a corresponding decrease in reticulocyte count, ldh and ch50 levels. the patient has remained stable with a normal hemoglobin (12-14 g/dl) on maintenance steroids and mmf. although we cannot definitively conclude that eculizumab directly caused his recovery, the clinical course post-eculizumab suggests this may be an efficacious treatment for aiha. genetic testing showed monoallelic frameshift mutation of the nfkb1 gene and monoallelic missense mutation of the dock2 gene. given the role of nfkb1 in both immunodeficiency and autoimmunity, it is thought that the patient's phenotype is due to nfkb1 haploinsufficiency and he is currently considering hematopoietic stem cell transplant. st. joseph's regional medical center, paterson, new jersey, united states background: heterozygous -thalassemia typically manifests as thalassemia minor, characterized by mild microcytic hypochromic anemia with minimal clinical ramifications. coinheritance of -globin gene triplication has been reported to exacerbate the clinical and hematological phenotype ofthalassemia trait, due to increase in the alpha/non-alpha-chain imbalance. reported phenotypes range from asymptomatic thalassemia minor to moderate thalassemia intermedia, usually diagnosed in adulthood without transfusion dependence. this combination has been described in mediterranean, european and asian populations, but rarely reported in hispanics. objectives: to report two cases of unusually severethalassemia intermedia in hispanic patients with heterozygosity for triplicated -globin gene and a (0)-thalassemia allele. results: case 1: sixteen-month-old male of mexican descent presented with persistent microcytic anemia and jaundice. peripheral smear showed nucleated rbcs with basophilic stippling and target cells. hemoglobin electrophoresis revealed: hba-79%, hbf-17%, hba2-4.3%. -globin gene testing revealed heterozygosity for (0) mutation (ivsi-i, g→a). given the unusually severe anemia, -gene testing was performed which showed -globin gene(anti 3.7) triplication ( / ). at four years, he had splenomegaly and bilateral maxillary prominence. head ct showed irregular contour of the parieto-occipital region due to medullary expansion. due to significant persistent anemia (6-8g/dl) and progressive bony deformities of the skull, patient began chronic transfusions at age eight after family declined splenectomy.case 2: fifteen-year-old female, of peruvian and honduran descent, presented for evaluation prior to cholecystectomy for gallstones and recurrent ruq pain. father had known thalassemia trait. her hb was 9.2 g/dl with hypochromia, microcytosis, and target cells. electrophoresis indicated -thalassemia trait (hba-94%, hba2-4.7%, hbf-1.3%), confirmed by gene testing (heterozygous for a (0) mutation in codon 39 c>t). given jaundice and gallstones, -globin gene analysis was ordered showing triplication ( / ). ruq pain resolved post-cholecystectomy, but she developed persistent painful splenomegaly. she began hydroxyurea to increase gamma-globin production and decrease excess alpha chains, but it was discontinued due to hematological toxicity. due to recurrent luq pain and progressive splenomegaly, she underwent laparoscopic splenectomy at age 22 with resolution of symptoms and improved hemoglobin. conclusion: -globin gene testing should be considered in -thalassemia carriers with an atypical clinical presentation including hispanic patients. the wide variability in the phenotypic expression of (anti 3.7) mutation andthalassemia trait suggest interplay of other genetic factors which remain undefined. the clinically significant presentation amongst certain subjects, as in our two cases, makes it imperative to identify these factors to aid in phenotype prediction and genetic counseling. ashley bonheur, shivakumar subramaniyam, jogarao vedula, sucharita bhaumik nyu winthrop hospital, mineola, new york, united states background: wilms tumor (wt) is one of the most common solid malignant neoplasms in children. a diverse range of genes and mechanisms are implicated in wt pathogenesis. predisposing syndromes result from a disruption of wt1 gene, crucial for renal and gonadal embryogenesis. another gene is wt2 gene locus at 11p15, an area of imprinting. the p53 tumor suppressor gene on chromosome 17p13.1 is seen in patients with anaplastic histology. in addition to these genes, whole and partial chromosome gains of 1q, 2, 7q, 8, 12, & 13 and losses of 1p, 7p, 16q, 22q, as well as loss of heterozygosity (loh) are commonly seen. some genetic markers appear to be predictive of outcome and are now incorporated into the assigning of risk-directed therapy. patients with loh at chromosome 1p and 16q are treated with more intensive chemotherapy, as they have been associated with increased risk of relapse and mortality. objectives: to describe a new complex translocation involving chromosome 2, 7, and 12 in a case of pediatric wt. design/method: a four-year old female presented with abdominal pain and emesis. on exam, patient had a firm and large abdominal mass. radiologic studies revealed a complex lobulated right renal mass. right radical nephrectomy was performed. histopathologic studies showed wt with triphasic histologic features with blastema predominance, invasion of the lymphovascular and perinephric adipose tissues, perinephric lymph node involvement and no anaplasia. chest ct scan showed bilateral lung metastases. tumor cytogenetics showed an abnormal karyotype, a complex translocation of 2, 7, and 12. the rearrangement occurred due to translocation between chromosomal bands 7q22 and 12q15, with an insertion of 7q22-32 on the 2q21 region. pcr based genotyping using microsatellite markers additionally identified loh for chromosome 1p36 and 16q22. the patient was treated for high risk stage iv wilms tumor with favorable histology and received intensive chemotherapy and radiation therapy to the flank and the lungs. she is now in remission 8 months after, with no evidence of recurrence on surveillance scans. complex translocations associated with wt have not been rigorously studied. a question for further study is whether there is any relationship between recurrence potential with a complex translocation compared to common chromosomal abnormalities. further knowledge of the molecular pathology and genetic changes in wt will help the development of new targeted therapies, as well as new biomarkers to aid diagnosis, risk stratification, and monitoring of treatment and relapse. results: a 4 week-old girl was referred for evaluation of an abnormal newborn screen. mother was a known carrier of hb khartoum trait while father was a known carrier of thalassemia trait. patient's hemoglobin quantification performed by capillary zone electrophoresis showed hbf 92%, hb variant 8%, and no detectable hba. the hb variant ran in the d zone, a pattern consistent with mother's hb. alkaline agarose gel electrophoresis banding pattern showed f/s. acid agarose gel electrophoresis pattern showed v/f. later testing revealed abnormal isopropanol stability with 3+ precipitation at 20 minutes. this electrophoresis pattern is consistent with the pattern previously reported of hb khartoum. clinically, the patient is a healthy, active child whom we have followed for two years. she has not had any significant anemia outside of her physiologic nadir. she has not had any hemolytic episodes, and her bilirubin levels have always been within the normal range conclusion: to the best of our knowledge, this is the only reported case of hb khartoum/ thalassemia. the proline to arginine substitution of hb khartoum introduces a charged group on the chain at the site of 1 1 contact. the resulting unstable 1 1 chains can dissociate into monomers and favor the formation of methemoglobin, leading to hemoglobin instability. we had wondered if this unstable hemoglobin might result in clinical hemolysis when challenged with oxidative stress, such as in periods of infection. however, in the two years we have followed this patient, she has never had a hemolytic episode. at two years of age, she has hbf 7.8%, hb khartoum 85.5%, and hba2 6.7%. whether hbf elevation is protective from oxidative stress remains to be determined as we continue to follow this child. university of puerto rico -medical science campus, san juan, puerto rico, united states background: gm1 gangliosidosis is a lysosomal disorder caused by -galactosidase deficiency due to mutations in the glb1 gene. it is a rare autosomal recessive neurodegenerative disorder with an incidence of about 1:100,000-1:200,000 live births worldwide. this neurological disorder has three clinical forms. gm1 type 1, or infantile form is characterized by psychomotor regression by the age of 6 months, visceromegaly (hepatosplenomegaly), macular cherry red spot, facial and skeletal abnormalities, seizures, and profound intellectual disability. we present a 4-year-old female with gm1 type 1 and acute lymphocytic leukemia (all). design/method: she was diagnosed with gm 1 type 1 at the 1st months of age and family history was remarkable for an older sister with gm 1 type 1. diagnostic studies reveal homozygous exon 7 of the glb1 gene for a sequence variant defined as c.622c>t, predicted to an amino acid substitution p.aarg208cs. results: patient presented to our hospital with petechiae in lower extremities, pallor and intermittent tracheal bleeding. physical examination shows a hemodynamically stable girl that is chronically ill dependent of mechanical ventilation, severe mental retardation and scatter petechiae at upper and lower extremities. laboratory workup revealed severe normocytic anemia (hgb: 5.7g/dl) with immature peripheral cells and thrombocytopenia (81 × 109/l). serum chemistry revealed increase ldh (695u/l), increase hepatic enzymes (ast: 63u/l), normal uric acid level. there was no evidence coagulopathy. chest x ray was unremarkable except for evidence of chronic pulmonary illness. abdominal sonogram hepatosplenomegaly. during hospitalization, bone marrow aspirate and biopsy was performed which was diagnostic of b cell acute lymphoblastic leukemia (all) with 26.5% lymphoblast and orderly myeloid/erythroid maturation. flow cytometry: 26% b lymphoblast with aberrant phenotype c/w b-acute lymphoblastic leukemia. karyotype revealed hyperdiploid female of favorable prognosis. cytogenetic by fish: hyperdiploid all with extra copies of runx1 and igh (no bcr-abl translocation). family was oriented about the new diagnosis and the dismal prognosis in conjunction to her primary condition. parents agree on no chemotherapy treatment for all with only supportive treatment. to this date, there is no evidence in literature that has previously described association of gm1 and leukemia. life expectancy of patient's primary condition is null therefore, correlation with leukemia might not be a coincidental finding. this patient opens the possibility of malignancy as part of gm1 type 1 thus, malignancy diagnosis should be considered as part of their medical lifetime course. university of south florida, tampa, florida, united states background: hematological manifestations related to hiv infection are not uncommon, with thrombocytopenia having an estimated prevalence of 5-15%. the pathophysiology is likely multifactorial. studies suggest that the primary mechanism may be immunologic resulting in accelerated platelet destruction. additional theories suggest that infection of megakaryocytes may also play a role causing inadequate platelet production. treatment of hiv-related thrombocytopenia is challenging. first-line treatments include initiation and optimization of antiretroviral therapies, immunoglobulin (ivig), and glucocorticoids. however, this approach is not effective in all patients and second line treatment options are less well studied, particularly in the pediatric population. objectives: we aim to present and discuss the case of a 13 year old patient with perinatally acquired hiv-1 infection and persistent thrombocytopenia who, after failing first line therapies, showed normalization of platelet count on the novel thrombopoietin receptor agonist, eltrombopag. design/method: a retrospective chart review of the case patient's medical record was conducted. additionally, a thorough literature review was performed on this topic including the pathophysiology of hiv related thrombocytopenia and its treatment modalities. the patient required monthly ivig infusions for about 1 year, but did not show a sustained response, often with platelet count dropping to less than 10,000 in between infusions. after initiation of 50 mg eltrombopag daily the patient showed a sustained increase in platelet count (range 32,000-88,000). during a brief 2 week lapse in eltrombopag treatment his platelet count dropped to 17,000. upon re-initiation of therapy his count increased to 84,000. the patient has remained asymptomatic, off of ivig for over one year, with undetectable hiv viral load and greater than 500 cd4 t cell counts. no side effects or grade 2 laboratory abnormalities were reported. conclusion: treatment of hiv-related thrombocytopenia can be challenging. first line therapies, including ivig and glucocorticoids, are not effective in all patients. several other treatment modalities have been utilized, including anti-d immunoglobulin, dapsone, danazol, interferon alfa, vincristine, thrombopoetic growth factors including romiplostim and eltrombopag, or splenectomy, but these are less well studied. this represents the first reported case of a pediatric patient with hiv who showed a positive response to eltrombopag with a sustained improvement in platelet count and no adverse effects from treatment. eltrombopag may be a safe alternative to first line therapies in those patients with hiv and refractory thrombocytopenia, however additional studies are needed. university of illinois college of medicine at peoria, peoria, illinois, united states background: achromobacter xylosoxidans is a gram negative rod with peritrichous flagella which causes rare opportunistic infections most commonly encountered by immunocompromised patients. it is primarily associated with uncomplicated bacteremia, cather-associated infections, and pneumonia. most reports of bacteremia associated with a. xylosoxidans are nosocomial, associated with neoplasm, and occurring mainly in adults. most reported infections with a. xylosoxidans in children are associated with cystic fibrosis. there are very few reported cases of septic shock from a. xylosoxidans bacteremia and pneumonia in the pediatric oncology population. objectives: to describe a rare case of a. xylosoxidans septic shock in a pediatric patient with relapsed neuroblastoma results: a 4-year old boy with history of stage iv highrisk neuroblastoma underwent standard frontline therapy with chemotherapy, hematopoietic stem cell transplant, radiation therapy, and immunotherapy, followed by a dfmo trial for maintenance. his 3-month follow-up scans demonstrated relapse and he was subsequently treated with additional chemotherapy, surgical resection, and mibg therapy, crizotinib for an eml4-alk fusion and finally ifosfamide, carboplatin and etoposide (ice). he developed neutropenic fevers and was started on cefepime, vancomycin and fluconazole. blood cultures were initially negative. on the 4th day of fever, his previously scheduled pet scan was performed during hospitalization and showed new pulmonary opacities. he did not have respiratory symptoms, but therapy was escalated to meropenem, vancomycin and amphotericin. emergent bronchoscopy was performed the same day, with all bacterial and fungal cultures remaining negative. overnight, he developed tachypnea and saturations in the upper 80s, requiring nasal cannula. ir-guided lung biopsy was performed the next day, a flexible bronchoscopy was done to remove blood clots in the airway, the patient was placed on a ventilator, femoral lines were placed, granulocytes ordered and pressors were started for deterioration to presumed septic shock. arterial and femoral lines were placed but patient continued to have hemodynamic instability on multiple pressors. the following day, blood and respiratory cultures returned positive for results: at 33 days after the start of iti, the inhibitor was <0.1 bu and continued undetectable 6 months after initiation of iti therapy. in this patient, iti with high-dose plasma-derived factor viii and von willebrand factor (vwf) complex was well tolerated and effective. genetic analysis confirmed a large factor viii gene duplication of exons 7 to 22. we believe our patient developed inhibitor so quickly (14 exposure days) due to the possibility of this mutation causing a frameshift that introduces a premature termination codon. this might be functionally similar to a deletion in the factor viii gene which poses the highest risk for inhibitor development in patients with severe hemophilia a. this variant has only been identified previously in two unrelated patients diagnosed with severe hemophilia a. this duplication is not listed in dbsnp variant database, nor observed in the general population database. our case proves the effectiveness of this method for patients with severe hemophilia a and an inhibitor. it also shows that more research is needed to identify patients at risk for inhibitor development. background: mercaptopurine (6-mp) is a prodrug that is a core component of maintenance chemotherapy for patients with a diagnosis of acute lymphoblastic leukemia (all). suppression of the neutrophil count is used to demonstrate adequate dosing of 6-mp during this phase of therapy. bone marrow suppression is mediated by the active metabolite 6-thioguanine (6-tgn), whereas the metabolite 6-methylmercaptopurine nucleotides (6-mmpn) has been shown to cause hepatotoxicity. allopurinol has been used infrequently in all maintenance therapy in the setting of skewed metabolism when adequate myelosuppression is difficult to achieve due to excessive hepatic toxicity. when given in combination with allopurinol a reduced dose of 6-mp may result in both increased 6-tgn levels and decreased 6-mmpn levels. objectives: describe the characteristics and clinical course of patients treated with allopurinol and reduced dose 6-mp during maintenance chemotherapy for all. we performed a retrospective chart review of patients at aflac cancer and blood disorders center of children's healthcare of atlanta with new diagnoses of b or t-cell all who received allopurinol during maintenance chemotherapy. we identified eleven patients with b-cell or tcell all who received allopurinol adjunctive therapy during maintenance chemotherapy at a single institution between 2014-2017. these 11 patients received adjunctive allopurinol for 2-120 weeks (median 52 weeks) with reduced 6-mp (25-65% of full dose). all ten patients with genetic testing for thiopurine s-methyltransferase (tpmt) had wildtype genotype associated with normal enzyme levels. indications for allopurinol use were most commonly unfavorable 6-mp metabolite levels, transaminitis (n = 8), pancreatitis (n = 3) and hyperbilirubinemia (n = 3). favorable metabolite shift was achieved in all patients. liver enzymes improved in 6 of 10 patients with transaminitis after initiation of allopurinol/reduced 6-mp. three patients who experienced pancreatitis during maintenance did not have recurrence after initiation of allopurinol (2 of these patients previously reported). six patients developed pancytopenia while on allopurinol, and two of those patients developed pancytopenia severe enough to require allopurinol cessation. four patients developed isolated anemia (hgb <11.0 g/dl) without thrombocytopenia or severe neutropenia. no patient has experienced a recurrence of leukemia. overall, treatment with allopurinol and reduced dose 6-mp was successful in producing a favorable 6-mp metabolite distribution and reducing toxicity. therapy was generally tolerated; however a major and notable side effect was pancytopenia, in two cases severe enough to stop allopurinol treatment. anemia may be more prominent with allopurinol usage. allopurinol effect is variable among individual patients despite normal tpmt genotypes. baylor college of medicine, houston, texas, united states background: congenital sideroblastic anemia, b-cell immunodeficiency, periodic fevers and developmental delay syndrome (sifd) is a rare inherited sideroblastic anemia syndrome, first described in 2013 with 12 clinically similar cases. genetic variations of trnt1 were identified as causative. objectives: to present an unusual presentation of a patient with sifd complicated by diagnosis of concomitant alpha thalassemia trait. design/method: retrospective chart review. a five month old male infant was referred to our hematology center for evaluation of elevated hemoglobin barts identified on newborn screen. despite numerous attempts, blood work was unable to be collected. at seven months of age he had microcytic anemia (hemoglobin 7.5 g/dl, mean corpuscular volume 44 fl) more severe than what would be expected with alpha thalassemia trait. no variant hemoglobin was identified with isoelectric focusing or high performance liquid chromatography. by nine months of age he developed growth failure, intermittent emesis with fevers, developmental delays (predominantly gross motor), hearing loss, a disproportionally large head and coarse, thinning hair. over the next ten months, he was seen by numerous specialists for seemingly unconnected problems including sensorineural hearing loss, elevated liver enzymes and growth hormone deficiency. alpha globin analysis revealed deletion of two alpha globin genes. at 20 months of age, he was admitted with one week of fevers, jaundice, and emesis. peripheral blood smear showed microcytic hypochromic anemia with marked anisopoikilocytosis including target cells, elliptocytes, tear drops, spherocytes, poikilocytes, marked polychromasia, and coarse basophilic stippling. given the inconsistency of his laboratory findings with the diagnosis of alpha thalassemia trait and clinical syndromic findings, bone marrow biopsy was performed which revealed rare ringed sideroblasts. one month later whole exome sequencing revealed trnt1 splicing variant c.1057-7c>g and novel missense variant c.1092a>t consistent with sifd. hemoglobin barts on newborn screen with moderate to severe microcytic anemia directed initial diagnostic work-up towards variant alpha thalassemia. as additional medical conditions developed the focus shifted to a unifying syndrome. compared to previously described cases, our patient was diagnosed at an older age, presented with anemia rather than episodes of febrile illnesses, and had rare sideroblasts on bone marrow examination. diagnosis in this case led to identification of the novel c.1092a>t variant in his sister who had similar, but milder, features. sifd is a rare disease with variable phenotypic severity making diagnosis challenging without high index of suspicion which is crucial for appropriate management. wiseman, blood, 2013 . chakraborty, blood, 2014 background: cholelithiasis is uncommon in childhood. cholelithiasis is known to occur more frequently in children with predispositions, including female sex, obesity, parenteral nutrition, previous abdominal surgery, use of oral contraceptives, family history of gallstones, chronic hemolytic anemias, hepatobiliary disease, or exposure to specific drugs. although there have been occasional case reports linking cholelithiasis to childhood leukemia or leukemia therapy, the prevalence and risk factors of cholelithiasis in patients with childhood leukemia remain unclear. objectives: to estimate the prevalence of cholelithiasis in patients diagnosed with childhood acute lymphoblastic leukemia (all), and to evaluate possible risk factors for the development of cholelithiasis in patients with childhood all. we performed a computer-assisted review of the electronic medical records of 503 patients diagnosed for b or t-cell all at children's healthcare of atlanta in the period from 2010 to 2016. patients with diagnoses of cholelithiasis, cholecystitis or who had a cholecystectomy were identified. possible risk factors of age, sex, bmi, history of abdominal surgery and parenteral nutrition use were abstracted. patients with underlying chronic hemolytic anemia or pre-existing gallbladder disease were excluded. results: seventeen cases of cholelithiasis and 2 cases of cholecystitis without documented cholelithiasis were identified. among patients with cholelithiasis, 8 were female. median age at diagnosis of cholelithiasis was 14.0 (range 2.7 -21.6) years. seven patients had no symptoms referable to cholelithiasis at the time of diagnosis. the median age of leukemia diagnosis among these patients was 12.1 (range 0.9 -18.8) years. the median interval from diagnosis of leukemia to gallbladder disease was 1.0 years. four patients had bmi over the 95th percentile for age. two patients had a prior history of intraabdominal surgery. no patient received oral contraceptive pills. six patients received parenteral nutrition for more than 30 days. there was no documented family history of cholelithiasis. seven patients did not receive any cholelithiasis directed therapy. two patients were managed with medical management only, 1 with endoscopic retrograde cholangiopancreatogram with stone extraction, and 7 with cholecystectomy. our study estimates the prevalence of cholelithiasis in childhood lymphoblastic leukemia to be 3.3%, higher than the reported prevalence in the general pediatric population of 0.13-0.22%. although our cohort size is small, it appears that all therapy and supportive care modalities associated with all are likely to play a larger role in the development of cholelithiasis than known predisposing factors in the general population. further studies are warranted. background: an uncommon side effect of intravenous immunoglobulin (ivig) administration is clinically apparent, sometimes severe hemolysis. we describe a severe case of coombs-positive hemolytic anemia secondary to ivig administration. ivig is a blood derivative manufactured from pools of 5,000 to 10,000 individual plasma donations. ivig is not abo-type restricted, so anti-a, anti-b and anti-a,b isoagglutinins are detectable. objectives: to describe a rare but serious type of transfusion reaction leading to gross hemolysis after ivig administration. results: a 16-year-old male with a past medical history of obstructive sleep apnea and obesity was admitted to the pediatric intensive care unit for adenoviral pneumonia and subsequent respiratory failure requiring mechanical ventilation. he had a complex hospital course with many complications including acute respiratory distress syndrome (ards), septic-shock, and coombs-positive hemolytic anemia. the patient was treated with commercial ivig (baxter/baxalta) 400-mg/kg daily for five days. he had two isolated episodes of severe hemolysis in relation to ivig administration requiring multiple transfusions of packed red blood cells (prbc). examination of pre-transfusion peripheral blood smear showed spherocytosis with rouleaux formation and large clumped rbc aggregates. the patient's blood type was classified as blood group a, rh-negative and his initial prbc transfusions were of this type. subsequently, the patient's coombs test was found to be positive using polyspecific and anti-igg typing sera. the patient's antibody screen against reagent group o screening cells was negative ruling out autoimmune hemolytic anemia. however, type specific anti-a antibodies were detected in his plasma as well as the acid eludate prepared from the coombs-positive red blood cells. it was concluded that the patient's hemolysis was due to anti-a antibodies presumed to arise from ivig. the patient's rbc transfusions were changed to o-negative blood and the hemolytic process resolved. the patient ultimately died due to complications of ards. although hemolysis is a known side effect of ivig, it is rarely considered when deciding to administer ivig. in addition, it has rarely been described in the pediatric population. ivig is used in the treatment of a growing number of medical conditions. due to the critical nature of many of these patients, hemolysis secondary to ivig may not be considered and continued blood transfusions with the patient's specific blood type may be used. it is crucial to remember that severe hemolysis can occur from ivig, and the importance of transfusing with blood group o, rh-negative blood when applicable. university of maryland medical center, children's hospital, baltimore, maryland, united states background: coagulopathy is a well-described complication of acute promyelocytic leukemia (apml), and remains a leading cause in induction failure. with treatment, coagulopathy associated with apml has been shown to rapidly improve. multiple organ dysfunction syndrome (mods) in apml, including acute respiratory distress syndrome (ards), has been associated with infection, traumatic injury, malignant infiltration, and cytokine release syndrome. when mechanical ventilation is no longer sufficient, extracorporeal membrane oxygenation (ecmo) can be considered; however, coagulopathy, severe end-organ damage, and malignancy are all relative contraindications to initiation of treatment. we report the case of a 17-year-old female presenting in respiratory failure, disseminated intravascular coagulopathy (dic), with intracranial hemorrhage, and mods, diagnosed with apml, successfully treated with ecmo therapy. design/method: retrospective case analysis and literature review. our patient, a 17-year-old female was admitted in respiratory failure and altered mental status, following a fall shortly prior to presentation. initial laboratory values were notable for pancytopenia, dic, and acute renal failure. a non-contrast head ct showed left temporal lobe intraparenchymal hemorrhage. she was diagnosed with apml by peripheral smear, later confirmed by fish for t(15:17), and was started immediately on high-risk induction chemotherapy as per cog protocol aaml1331, including all-trans retinoic acid, arsenic trioxide, idarubicin, and dexamethasone. cvvhd was required for acute renal failure. despite maximal respiratory support, she remained hypoxemic, with oxygenation index of 46, pao2/fio2 ratio of 70. ecmo was initiated 24 hours after start of induction, 48 hours after admission. coagulopathy resolved on day 6 of induction, ecmo was discontinued after 9 days, mechanical ventilation and cvvhd were stopped after 15 days and she continued to improve, eventually achieving remission with few neurologic side effects. despite relative contraindications to ecmo, this patient was successfully treated with ecmo without significant neurologic side effects. the correction of her coagulopathy was multifactorial: 1) restoration of adequate oxygen delivery via ecmo improving endothelial function; 2) successful organ support to allow sufficient response to induction chemotherapy with atra leading to the terminal differentiation of leukemic blasts; 3) complement and contact system activation through contact with ecmo circuitry. this case illustrates that ecmo can still be considered in patients despite coagulopathy and end organ damage. sinai hospital of baltimore, baltimore, maryland, united states background: primary polycythemia vera is an extremely rare diagnosis in the pediatric patient and is defined by a marked elevation of red blood cells due to erythropoietin-independent mechanisms. presentations of this disorder range from the asymptomatic person to severe thrombotic events, such as budd-chiari syndrome or cerebrovascular stroke. mutations in the jak2 gene are found in adult and pediatric patients with polycythemia vera; however, the jak2 v617f mutation is less commonly identified in pediatric patients. we describe an otherwise healthy 16-year-old female who presented with a significantly elevated total erythrocyte count, hemoglobin, and platelets, incidentally discovered upon routine annual blood work obtained by her pediatrician. design/method: this is a report and discussion of a rare case. demonstrated cellular marrow with trilineage hematopoiesis and no dysplasia. cytogenetics were not assessed. his hemoglobin and platelet count recovered but leukopenia and neutropenia persisted. follow-up evaluation at three months revealed fevers, ongoing cytopenias, a one-month of a nodular skin rash on the trunk and extremities resembling erythema nodosum, and hepatitis (peak alt and ast of 1,176 and 1,008, respectively). following clinical evaluation, a skin biopsy was performed and was remarkable for atypical lymphocytes within the subcutis with t-cell markers, a high ki-67, and positive tia-1, perforin, and -f1 immunoperoxidase stains. negative stains for cd56, cd30, and ebv were noted. these results are consistent with sptcl. additional evaluation did not support a diagnosis of hlh. a staging evaluation was performed. pet-ct showed widespread hypermetabolic subcutaneous activity in the legs, trunk and skull and diffuse marrow hyperplasia. bone marrow demonstrated involvement with precursor b-cell acute lymphoblastic leukemia, with a mll gene rearranagement. his skin biopsy was retrospectively stained with tdt, cd34, pax-5, cd79a, and cd20 with negative results, and a blood smear taken at the time of the skin biopsy did not demonstrate leukemic cells. conclusion: this is the first report of a patient with sptcl having a synchronous malignancy. the patient is doing well, currently in the maintenance phase of treatment for his all, and his skin disease has resolved on pet-ct. while it is possible that his presentation was a function of chance, the possibility of an underlying immune dysfunction or cancer predisposition warrants further investigation. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hereditary xerocytosis (hx) is a rare red blood cell (rbc) dehydration disorder, characterized by variable hemolysis and propensity to iron overload. hx is often misdiagnosed as hereditary spherocytosis (hs). while splenectomy is curative for hs, it is relatively contraindicated in hx due to a substantial thromboembolism risk, signifying the importance of delineating these diseases. blood smear abnormalities are variable and often insufficient to make an accurate diagnosis. osmotic-gradient ektacytometry and genetic confirmation are critical in distinguishing these overlapping disorders. objectives: describe a family with hx, initially misdiagnosed as hs. discuss the importance of distinguishing these disorders and the utility of ektacytometry in making this distinction. design/method: a 13-year-old caucasian male was diagnosed with hs after presenting with prolonged neonatal jaundice starting on the first day of life. he described mild scleral icterus and history of intermittent jaundice and dark urine, without need for transfusions. his father, paternal uncle and paternal grandmother were all diagnosed with hs during childhood and underwent cholecystectomy. additionally, his father underwent splenectomy for abdominal pain. the child's blood counts revealed compensated anemia (hb 12.9 gm/dl) and reticulocytosis (arc 347 × 103/mcl) with increased mcv (96.6 fl) and mchc (38.2 gm/dl). blood smear showed increased polychromasia and poikilocytosis with rare spherocytes and few stomatocytes. while the child had normal ferritin, his father had iron overload (ferritin 800 ng/ml) despite no prior transfusions. osmotic-gradient ektacytometry profile of the child and father's rbcs showed a characteristic left-shifted, bell-shaped curve with decreased omin and ohyp, diagnostic of hx. the family is currently undergoing genetic studies. despite clinical similarities between hs and hx, distinguishing these diseases has significant management implications. hx is a disorder of rbc permeability, causing shortened rbc survival. stomatocytes on blood smear can raise suspicion for hx, but are insufficient to make an accurate diagnosis. identifying characteristic biomechanical membrane properties using osmotic-gradient ektacytometry is the gold standard for clinical diagnosis, which can then be confirmed by molecular studies. hs and hx can be easily and reliably distinguished using ektacytometry, as both disorders have very distinctive curves representing different rbc deformability patterns. after hx diagnosis was made, we counseled the family against splenectomy, as the risk of thromboembolism is significantly increased in hx compared to hs, and the father was diagnosed with iron overload. conclusion: hx is commonly misdiagnosed as hs. this case highlights the importance of making this distinction, and the utility of osmotic-gradient ektacytometry in reliably distinguishing these conditions. penn state health children's hospital, hershey, pennsylvania, united states background: relapsed acute myeloid leukemia (aml) presenting as an isolated central nervous system myeloid sarcoma (cns ms) is very rare and its treatment is not well-defined. thiotepa, vinorelbine, topotecan and clofarabine (tvtc) has been successful for re-induction therapy to induce remission prior to hematopoietic stem cell transplant (hsct). objectives: to describe our experience in utilizing tvtc therapy in two children with no extramedullary disease at initial diagnosis who presented with relapsed aml as intracranial myeloid sarcomas. results: case 1: 34 month-old female was diagnosed with flt3 negative aml and completed treatment per the children's oncology group (cog) aaml1031 study on the low risk arm without bortezomib. cerebral spinal fluid (csf) negative at diagnosis. fish testing positive for tcf3 gene deletion of unknown significance. mrd was undetectable after induction i and remained undetectable after each cycle. nine months off therapy, recurrent headaches prompted mri imaging which revealed two posterior fossa masses. csf and bone marrow testing were negative. stereotactic biopsy of the larger mass confirmed recurrence of aml. patient underwent two cycles of tvtc with a total of seven doses of intrathecal cytarabine with almost near resolution of the cns ms. completed cranial radiation and proceeded to allogeneic stem cell transplant with unrelated cord marrow donor and is disease free at approximately day +200.case 2: 5 year-old female diagnosed with flt3 and mll negative aml and completed treatment per cog aaml1031 study on the low risk arm without bortezomib. csf negative at diagnosis. mrd was undetectable after induction i and completed therapy without complications. two months off therapy, a retrospective analysis of her diagnostic bone marrow by the cytogenetic laboratory to test a new panel identifying novel 11q partners revealed a cryptic insertional 10:11(mllt10/mll(kmt2a) translocation. at four months off therapy, acute mental status changes prompted mri imaging which revealed two intracranial ms and lumbar spine involvement. resection of the larger lesion for symptomatic relief confirmed the mllt10/mll(kmt2a) fusion. csf positive for blasts and marrow negative for relapsed disease. patient completed two cycles of tvtc with a total of seven doses of it cytarabine with near resolution of cns disease (only 3 mm contrast enhancement in the medulla). she received craniospinal radiation and is awaiting improvement in her cardiac function before proceeding to hsct. conclusion: tvtc is a successful reinduction regimen for relapsed aml with cns ms prior to hsct. background: acute severe anemia can be a life-threatening medical condition. the differential is quite broad for possible etiologies of acute severe anemia, including autoimmune hemolytic anemia (aiha) and atypical hemolytic uremic syndrome (ahus). autoimmune hemolytic anemia is an antibody-mediated process that targets the protein antigens located on the surface of red blood cells. treatment options for aiha include corticosteroids, with up to 80% of patients being responsive, with some requiring splenectomy. atypical hemolytic uremic syndrome is a medical urgency, defined as the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. the etiology is usually due to genetic causes, or less commonly, due to autoantibodies or idiopathic reasons. prognosis is very poor. objectives: differentiating between autoimmune hemolytic anemia and atypical hemolytic uremic syndrome can be a time-sensitive diagnostic dilemma while the patient is in critical condition, but this important delineation can vastly alter therapeutic options. design/method: here we discuss two cases highlighting the diagnostic workup involved in differentiating between atypical hemolytic uremic syndrome and autoimmune hemolytic anemia. patient a is a 3-year-old male who presented in extremis with severe anemia, uremic encephalopathy, and severe acute renal injury requiring hemodialysis and multiple blood transfusions. patient b is a 10-month-old male, who also presented in extremis with respiratory failure secondary to adenovirus/rhinovirus/enterovirus, with acute progressive renal failure and microangiopathic hemolytic anemia, requiring hemodialysis and cardiorespiratory support. : patient a underwent a full hematologic and infectious disease workup. subsequent laboratory studies confirmed enteropathogenic e.coli (epec) in the patient's stool; blood cultures remained negative. renal biopsy results were consistent pigment nephropathy. bloodwork indicated positive direct coombs. patient a was ultimately treated with steroids 2mg/kg/day, with significant improvement. patient b also included a full hematologic work-up, including adamts13 activity and ahus genetic panel, as well as full infectious disease work-up. subsequent laboratory test-ing revealed blood cultures growing streptococcus pneumoniae, with adamts13 activity at 41% (adult ref range: >/ = 70%), and normal complement levels. imaging findings also supported diagnosis of ahus. the management of a critically ill patient with acute severe anemia requires a thorough hematologic and infectious disease work-up. while molecular and genetic are helpful in definitive diagnosis of ahus, the utility of such results is limited by time. overlapping clinical presentation of a patient in extremis due to acute severe hemolytic anemia with progressive renal failure presents a rather broad differential, with time-sensitive treatment and prognostic implications. the favorable response to steroids delineates aiha from hus. background: d-2-hydroxyglutaric aciduria (d-2-hga) is a rare metabolic disorder characterized by developmental delay, hypotonia, and bi-allelic mutations in d-2hydroxyglutarate dehydrogenase (d2hgdh) or isocitrate dehydrogenase 2 (idh2). metaphyseal chondromatosis with d-2-hydroxyglutaric aciduria (mc-hga) is a type of d-2-hga that has been previously reported in seven patients (omim 614875; pmid 24049096), three of whom had somatic mosaicism for r132 variants in isocitrate dehydrogenase 1 (idh1). we describe a 3-year-old boy with mc-hga who subsequently developed acute myeloid leukemia (aml) and was found to have a r132 variant in idh1 in a leukemic bone marrow sample. we report the first case of aml with this metabolic disorder. design/method: a 1-year-old hispanic boy presented with short stature, developmental delay, abnormal skin pigmentation, and unilateral congenital cataract. workup revealed multiple skeletal enchondromatosis and elevated urine d-2-hydroxyglutaric acid levels. he was diagnosed with mc-hga. no pathogenic variants in d2hgdh, idh1 and idh2 were identified in peripheral blood. germline testing with biopsies of skin lesions was declined by the family. two years later, he presented with streptococcal sepsis and pancytopenia. blasts were noted on peripheral smear. bone marrow morphology was consistent with acute myelomonocytic leukemia (∼23% blasts). chromosome analysis showed normal 46 xy, and molecular testing by pyrosequencing idh1 and idh2 revealed a r132c variant in idh1 (25% mosaicism). the patient is being treated as per the cog study aaml1031. end of induction i bone marrow aspirate was hemodiluted, but there was no obvious residual disease by flow cytometry (0.01-0.01% sensitivity) or morphology. the previously identified idh1 variant was no longer detectable (limit of detection <10%). although targeted therapy for aml with idh1 mutation is currently in phase i clinical trials in adults, there is no safety or efficacy data for using idh1 inhibitors in children. treatment with ivosidenib is therefore not currently an option for our patient. conclusion: this is the first case of aml reported with this rare metabolic disorder. somatic r132 variants in idh1 have been identified in three other mc-hga cases. this same mutation leads to the accumulation of d-2-hydroxyglutarate in gliomas and aml. without any confirmed germline mutation or somatic mosaicism testing of multiple specimen sources, we can only speculate that the patient has an underlying somatic idh1 mutation associated with mc-hga which subsequently led to leukemogenesis. we present the first case of this association, to increase index of suspicion for development of aml in children with metabolic disorders associated with variants in idh1. background: congenital combined deficiency of the vitamin k-dependent coagulating factors (vkcfd) is a rare heterogeneous autosomal recessive bleeding disorder. vkcfd is caused by mutations in the genes of either gamma-glutamyl carboxylase (ggcx) or vitamin k epoxide reductase complex (vkorc), which are responsible for the gammacarboxylation of vitamin k dependent proteins (vkdps) allowing for their activation. the clinical presentation ranges from no bleeding to intracranial hemorrhage. to date, vkcfd has been reported in few patients worldwide. objectives: we report a case of a girl with novel homozygous mutation of the ggcx gene, highlighting her clinical and biochemical characteristics with a review of the literature. a 3-month-old girl of consanguineous emirati parents, presented to our hospital with a history of bleeding from puncture site after receiving her second-month vaccine. that was associated with episodes of mild mucosal bleeding. review of systems was negative for jaundice, steatorrhea and failure to thrive and physical exam was unremarkable. investigations revealed markedly prolonged pt and aptt with high inr. fibrinogen, hemoglobin and platelets were always normal. activities of vitamin k-dependent factors including fii, fvii, fix, fx, protein c and s were all low. a measurement of proteins induced by vitamin k absence (pivka-ii) was done and came very high. this was associated with a mild elevation in liver enzymes but normal liver function test. the picture was supporting vitamin k deficiency, and as a result, she was started on oral vitamin k supplements of 1 mg/day. she responded partially to vitamin k and required higher doses to stabilize her inr. after excluding acquired causes and due to her requirement of high doses of vitamin k, a mutation in either ggcx or vkorc genes was suspected. genetic analysis was conducted for her which revealed a novel missense homozygous mutation in the ggcx gene (c.548a>t) confirming the diagnosis of combined deficiency of vitamin k-dependent clotting factors type 1. the asymptomatic parents were both heterozygous for the same mutation. results: she is currently stable on 10 mg/day of vitamin k supplements. conclusion: vkcfd is a rare bleeding disorder with an overall good prognosis due to the availability of several effective therapeutic options. the function of the mutated gene is unknown. our patient demonstrated a partial response to vitamin k supplements suggesting presence of a residual carboxylation capacity and a possible role of this gene in the enzymesubstrate interactions. university of alabama at birmingham, birmingham, alabama, united states s55 of s301 background: gata2 is a zinc finger transcription factor that plays a critical role in the regulation of hematopoiesis and lymphatic angiogenesis. mutations leading to gata2 deficiency (gd) have been linked to a variety of clinical conditions. patients with gd have a striking predisposition to develop myelodysplastic syndrome (mds), acute myeloid leukemia (aml), or chronic myelomonocytic leukemia (cmml). acute lymphoblastic leukemia (all) has not been associated with gd, although the association of bcell all and gd has been previously reported. objectives: to describe a unique association of gata2 deficiency and t-cell all in a young child. results: an 8-year-old female presented with a one-week history of fever and malaise. she had a significant past medical history of verruca plantaris and self-resolving leukopenia associated with febrile illnesses. significant family history included sister with neutropenia and human papilloma virus (hpv) infection, and mother with neutropenia, monocytopenia, atypical mycobacterial infections, and hpv infection. peripheral blood revealed hemoglobin 9.3 g/dl, hematocrit 26.7%, platelets 176,000/ul, and white blood cell 1,740/ul (neutrophils 122/ul, lymphocytes 1601/ul, monocytes 17/ul). patient underwent a bone marrow biopsy demonstrating lymphoblast infiltration. flow cytometry analysis demonstrated monoclonal lymphoid blast population that co-expressed cd117, cd34, cd13, nuclear tdt, cd2, however, lacked expression of cd33, cd10, cd3, cd19, hla-dr, or myeloperoxidase. findings were consistent with tcell all with aberrant myeloid markers. cytogenetics analysis revealed 45,xx,dic(21;22)(p11.2;p11.2). patient began treatment as per children's oncology group aall0434 and achieved remission at the end of induction. course of therapy was complicated by episodes of fever, reciprocating junctional tachycardia, asparaginase-associated thrombosis, viral meningitis, recurrent episodes of verruca plantaris, and resistant streptococcus pneumoniae or haemophilus parainfluenza infections causing chronic cough. later, she was also found to have low igm levels; after completion of therapy, she developed monocytopenia. lymphocyte subset panel revealed absent b cells, decreased number of natural killer (nk) cells, and cd4/cd8 inversion. further work-up included gata2 sequence analysis that showed heterozygous nonsense mutation (c.58c > t/c; reference nm_001145661) likely resulting in gata2 haploinsufficiency. patient continues to be in remission, is receiving monthly immunoglobulin replacement and is on azithromycin for atypical mycobacterial prophylaxis. surveillance bone marrow biopsies have shown no evidence of mds or leukemia, however, have demonstrated persistent hypocellularity. the possibility of undergoing an allogeneic bone marrow transplant is actively being discussed given its curative potential. clinicians should be aware that t-cell all may be associated with gata2 deficiency. cincinnati children's hospital medical center, cincinnati, ohio, united states background: treatment for severe hemophilia a is centered on factor viii (fviii) replacement therapy. development of an alloantibody (inhibitor) against fviii is a significant treatment complication occurring in as many as 25-30% of patients. high titer inhibitors render treatment with factor viii ineffective, necessitating the use of bypass agents that may not achieve hemostasis with the same efficacy. considering the substantial ramifications of inhibitor development on treatment, eradication of inhibitors is of great importance to achieve adequate hemostasis in this patient population. desensitization by immune tolerance induction (iti) is the primary method of inhibitor elimination. however, not all patients respond to iti. immunomodulation may be considered as the next line of therapy, although controversy remains in regards to agent selection and use. objectives: there is incomplete data on the use of immunomodulation therapy for inhibitor eradication in severe hemophilia a. we present a case of a pediatric patient with severe hemophilia a and high titer inhibitor who failed initial iti therapy to better illustrate potential treatment options for the future. design/method: a retrospective chart review was performed on a patient with severe hemophilia a at cincinnati children's hospital medical center. results: an 8-year-old caucasian male with severe hemophilia a secondary to intron 22 inversion, was initially diagnosed following extensive bleeding after circumcision at birth. he was identified as having an inhibitor (312 bethesda units (bu)) at 12 months of age after 15 exposure days of treatment. he failed multiple attempts of iti, with recombinant and plasma-derived (pd) fviii. he was advanced to immunomodulation therapy in combination with pdfviii, however demonstrated anaphylaxis to rituximab and ofatumumab. he underwent tolerization to rituximab, and received a six month course with a partial response (nadir of 0.56 bu). 12 months following last dose of rituximab, a rising inhibitor titer (7.44 bu) was found. mycophenolate mofetil (mmf) was initiated with subsequent inhibitor stabilization and a decreasing titer (1.48 bu) over the course of the following year. mmf has been well tolerated without major side effects or infection throughout therapy. conclusion: development of an inhibitor against fviii is a considerable complication in patients with severe hemophilia a. use of immunomodulatory therapies following iti failure remains controversial. mmf has not been well studied in this patient population. we report a case of a patient who is being successfully treated with mmf with minimal side effects. further prospective studies should be considered to further define the role of mmf immunomodulation therapy. background: down syndrome (ds) children with aml (ds aml) have higher cure rates than their non-ds counterparts. outcomes for refractory/relapsed cases, however, remain dismal. somatic mutations of the gene encoding the transcription factor gata1 in ds aml patients are responsible for the observed hypersensitivity of ds aml blasts to cytosine arabinoside (ara-c). in view of excellent survival rates (approaching 90%) of ds aml patients, the ongoing children's oncology group (cog) aaml1531 study seeks to determine the feasibility of treating standard risk (minimal residual disease/mrd negative) ds aml patients using a reduced dose (7-fold decrease) ara-c backbone. although results from japanese trials with this approach are promising, north american and european data are conflicting. although chromosome 7 rearrangements in ds aml do not appear to carry the same adverse prognostic significance as in non-ds aml, monosomy 7 in ds aml patients has been associated with a moderately worse outcome. isochromosome 7q, however, is rare and has only been reported in 3 previous cases of ds aml. objectives: to report our institutional experience of very early relapse involving 2 cases of ds aml patients treated per the reduced dose ara-c arm (3.8 g/m2) of the aaml1531 study. design/method: we hereby report the disease course and cytogenetics of the above 2 ds aml patients. : patient 1 is a 20 month old caucasian female who had gata1 mutation negative aml. patient 2 is a 3-year old caucasian male whose chromosomal analysis revealed isochromosome 7q (3 copies of the long arm of chromosome 7). both patients achieved negative mrd (<0.05%) after induction i chemotherapy with thioguanine, low-dose ara-c and daunorubicin and proceeded per the reduced dose ara-c arm of aaml1531. patient 1 relapsed immediately after completion of chemotherapy. salvage chemotherapy with mitoxantrone/high dose ara-c (hidac) failed to induce a second remission and the patient subsequently died of disease. patient 2 relapsed within 4 months from end of therapy. the patient underwent salvage chemotherapy utilizing a hidac backbone and remains in disease remission. the noted very early relapse following a reduced dose ara-c regimen in our 2 above ds aml children suggests that testing for gata 1 mutation and chromosome 7 rearrangements may play a useful role in the development of future risk-stratified treatment strategies for ds aml. university of rochester, rochester, new york, united states background: in developed countries in the 21st century, severe nutritional deficiency is not an often considered differential diagnosis of unexplained childhood anemia. aside from iron deficiency anemia, vitamin deficiency severe enough to impact hematopoiesis is uncommon in the general pediatric population. here we present the unique case of a 10-monthold infant who presented with intermittent emesis, failure to thrive (ftt), developmental delay, macrocytic anemia, and neutropenia which was initially concerning for a congenital bone marrow failure syndrome. instead, she was discovered to have an underlying, potentially familial deficiency of b12. objectives: 1. to describe the unique case of an infant with b12 deficiency. 2. to outline the importance of including b12 deficiency in the differential diagnosis of unexplained megaloblastic anemia in children. a 10-month-old exclusively breastfed infant presented for gastroenterology evaluation due to persistent emesis and poor weight gain over the course of 2 months. her history was notable for delayed developmental s57 of s301 milestones and hypoactivity. marked pallor prompted hematologic evaluation, which revealed concern for macrocytic anemia (hemoglobin 7.1 g/dl, mcv 107), reticulocytopenia (48.1 × 10^3/ l), and neutropenia (anc 0.4 × 10^9/l). an otherwise reassuring physical examination and laboratory evaluation was notable only for the discovery of an undetectable b12 level and marked hyperhomocysteinemia (162 mol/l). her hemoglobin (hgb) continued to decline (to 5.9 g/dl) over the first few days after presentation, and she required red blood cell (rbc) transfusion. within only a few days of initiation, daily cyanocobalamin injections resulted in a robust reticulocytosis response, improved hgb, immediate normalization in the neutrophil count, and resolution of hyperhomocysteinemia. additional history and laboratory evaluation from the patient's mother revealed a concurrent, asymptomatic maternal b12 deficiency as well as a history of a need for b12 supplementation in the maternal grandfather, raising concern for an inherited etiology. despite the rarity of vitamin-deficient hematologic abnormalities in the general pediatric population, b12 deficiency should be considered as a potential cause of an otherwise unexplained megaloblastic anemia, especially in the setting of concurrent ftt and neurodevelopmental delay. a detailed family history should be obtained in such cases and may have helped to prevent this patient's clinical sequelae had the deficiency been discovered sooner. our patient has experienced a favorable clinical response to b12 supplementation, attesting to the importance of vitamin b12 in early childhood growth and development. background: peg-asparaginase is universally utilized in the treatment of pediatric acute lymphoblastic leukemia (all). despite its high efficacy in this disease, it is associated with hypersensitivity and allergy in 10 -20% of patients. protracted anaphylaxis has been described in circumstances such as severe food allergy with ongoing allergen exposure; however, it has not yet been described in relation to peg-asparaginase. we describe the first reported case of protracted anaphylaxis after peg-asparaginase administration, provide guidance as to time course and management of protracted anaphylaxis, as well as evidence that erwinia asparaginase may be safely administered even in this high risk population. objectives: to provide guidance regarding the duration, course and management of protracted, severe anaphylaxis after peg-asparaginase therapy. a 15 year old male with very high risk all presented for consolidation therapy with peg-asparaginase (intramuscular) and vincristine. one hour after administration, he developed generalized hives and angioedema, for which he was given diphenhydramine. he then quickly developed progressive hives, angioedema, subjective throat and chest tightness, and wheezing. he was treated with diphenhydramine, epinephrine, albuterol, and methylprednisolone with resolution of symptoms. one hour later, symptoms recurred and the patient became hypotensive; he was retreated with methylprednisolone and epinephrine, and was transferred to the pediatric intensive care unit (picu). in the picu, he was placed on an epinephrine drip, and continued on methylprednisolone, diphenhydramine, cetirizine, albuterol, and ranitidine. the epinephrine drip was successfully discontinued after 48 hours, and his other medications were gradually weaned over the course of two weeks. of note, the patient did have st segment changes in his electrocardiogram during the first 48 hours of anaphylaxis. these were associated with normal ventricular function as per echocardiogram, and resolved within one week. this patient has subsequently tolerated multiple doses of erwinia asparaginase (intramuscular) without premedication. this patient was acutely managed in the pediatric intensive care unit with steroids, anti-histamines, and continuous infusion epinephrine. symptoms consistent with severe anaphylaxis including hives, angioedema, throat and chest tightness, wheezing, and hypotension persisted for a total of four days before finally resolving. he has thus far tolerated multiple doses of erwinia asparaginase without any symptoms of allergy, hypersensitivity, or anaphylaxis. protracted severe anaphylaxis after peg-asparaginase therapy can be successfully managed with multi-agent therapy, including antihistamines, steroids, and continuous infusion epinephrine. re-challenge with an alternate form of asparaginase may be tolerated, even in a patient with protracted anaphylaxis to peg-asparaginase. ucsf benioff children's hospital oakland, oakland, california, united states background: vincristine (vcr) is widely used in pediatric cancers. unlike most cytotoxic agents, hematopoietic toxicity is uncommon. vcr-induced anemia has been observed but its mechanism has not been well studied. vinca alkaloid-induced membrane changes were seen in early studies of hereditary spherocytosis (hs) and anecdotal cases suggest vcr may increase hemolysis in such patients. here we describe a case involving severe vcr-induced anemia in a patient with hs and an explanation as to the mechanism. objectives: to describe the mechanism of vcr-induced anemia in hs. design/method: case report. a 6 year-old female with hs was diagnosed with t-lymphoblastic lymphoma. she had required 2 packed red blood cell (prbc) transfusions as a neonate and thereafter had done well without episodes of acute hemolysis or aplasia. complete blood counts (cbc's) demonstrated a compensated hemolysis, and she did not require further transfusions until she commenced chemotherapy. by the start of maintenance she had received many more prbc transfusions than the average patient. intermittent drops in hemoglobin (hb) did not correlate with any particular agent, and she had stable, mild splenomegaly. a clear pattern emerged during maintenance. her hb was 8-9 g/dl at monthly clinic visits, when she received vcr, intermittent intrathecal methotrexate, and corticosteroids. within 3-4 days, her hb dropped to 5.8±0.6 g/dl, and reticulocyte count decreased from 13.4 to 4.3±0.7%. transfusion at day 4 corrected hb, and the reticulocytes and hb returned to baseline. white blood cell and platelet counts did not change after vcr. blood samples from pre, immediately post, and 4 days post vcr were analyzed and rbc characteristics and markers of hemolysis were not significantly different. ektacytometry showed identical curves, indicating no change in rbc deformability. in vitro incubation of patient blood samples with vcr also did not affect the osmotic deformability, confirming that a change in rbc rigidity was unlikely the reason for the drop in hb. these data indicate that a dysregulation of erythropoiesis was responsible for the anemia after vcr, rather than damage of peripheral rbc's. in most patients, maintenance therapy for lymphoblastic lymphoma does not cause severe anemia, likely because a temporary reduction in erythropoiesis in patients with a normal rbc survival and low reticulocyte count is not noticed. however, in a patient with decreased rbc survival and a brisk reticulocytosis, a disruption in rbc generation is more apparent. in conclusion, vcr administration to patients with an rbc disorder warrants close observation for potentially severe vcr-induced anemia. background: the addition of tyrosine kinase inhibitors (tki) to conventional chemotherapy has improved outcomes for pediatric patients with philadelphia chromosome-positive (ph+) acute lymphoblastic leukemia (all), however there remains an increased risk of relapse compared to other types of childhood all. typically, in relapsed disease the philadelphia chromosome persists and several mechanisms of resistance involving acquired mutations of the bcr-abl1 chimeric oncoprotein have been reported. objectives: describe a unique case of a pediatric patient with ph+ b-precursor all relapsing with b-precursor all without the philadelphia chromosome. results: an 8-year-old boy was diagnosed with ph+ bprecursor all with the presence of the t(9;22)/bcr-abl1 translocation by cytogenetics and fluorescence in situ hybridization (fish), respectively. additional abnormalities included gains of runx1 and loss of one copy of etv6. a remission bone marrow with negative minimal residual disease (mrd) was achieved at the end of induction with dasatinib and the esphall chemotherapy backbone. duration of tki therapy was two years post diagnosis. nearly one year after the completion of therapy, cytopenias prompted a bone marrow investigation. relapsed b-precursor all was established by immunophenotyping, however fish analysis did not identify the bcr-abl1 rearrangement. moreover, quantitative reverse transcriptase pcr was negative for the bcr-abl1 fusion transcript. again fish analysis of the bone marrow revealed multiple additional copies of runx1 and mono-allelic loss of etv6, similar to the initial diagnostic sample. the patient was re-induced per aall0232 anticipating a ph+ all relapse. however, with confirmation of the loss of the ph+ clone, tki therapy was not re-initiated. due to positive mrd of 3.5% at the end of re-induction therapy, the patient was salvaged with blinatumomab therapy and subsequently underwent an allogenic stem cell transplant with a sibling donor. conclusion: this is the first known report of a pediatric patient with ph+ b-precursor all who developed recurrent b-precursor all without the philadelphia chromosome. the persistent findings of gain of runx1 and loss of etv6 makes it unlikely that a second unrelated b-precursor all developed following successful treatment of the original disease. this case highlights the possibility of a genetically distinct subclone present at the onset of disease that shared abnormalities of runx1 and etv6 but did not contain the philadelphia chromosome. nevertheless, the subclone harbored leukemogenic potential in the absence bcr-abl1 expression. it is plausible that the predominant clone present at diagnosis was effectively treated with dasatinib and extinguished, but the bcr-abl1-negative clone persisted in the face of tki therapy. background: ligneous conjunctivitis is a rare form of pseudomembranous conjunctivitis that develops specifically in patients with type 1 plasminogen deficiency. lack of plasmin activity in those patients result in defective fibrinolysis and formation of fibrin-rich membranous material/ masses that develops on the palpebral conjunctiva as well as other sites in the body.current management involve surgical excision of the masses that is usually complicated by multiple recurrences. recently, use of topical plasminogen concentrates helped delaying recurrence, but currently, those concentrates are not commercially available. we report on a 7-year-old omani girl, with hypoplasminogenemia who required optimization of plasminogen level at the time of surgery to delay/ prevent recurrence. objectives: case report on the peri-operative use of ffp versus cryopricipitate transfusion as an alternative replacement of plasminogen during surgical excision of ligneous conjunctivitis. design/method: pharmacokinetic study was performed to assess plasminogen recovery after ffp (15 ml/kg) and precipitate (1 bag/5kg) transfusion results: plasminogen levels remained subnormal after either ffp or cryoprecipitate administration. with ffp, the maximum concentration reached was almost 50% of normal. although half-life of plasminogen is known to be 2-2.5 days, the patient seemed to have a high catabolic rate after receiv-ing cryoprecipitate, with plasminogen levels reaching basal levels within 4 hours. because of the better recovery profile with ffp, we opted to give ffp before and after surgery. peri-operative management included ffp transfusion at 20 ml/kg/12 hours one day before and for 3 days post operatively, followed by 10 ml/kg once daily from day 4-6, then 20 ml/kg on 7th post-operative day. topical treatment was initiated using antibiotic and steroids ed on the day of surgery, followed by heparin ed on the second day. on follow up, she used topical heparin, cyclosporine, prednisolone, and topical lubricant eye drops for variable duration. clinical picture remained stable for almost 1 year post operatively, when she started to develop recurrence of ligneous lesions again. background: ponatinib (inclusig®, ariad pharmaceutical) is a 3rd generation multi-targeted tyrosine kinase inhibitor (tki) approved for treatment of adults with chronic myeloid leukemia (cml) and philadelphia chromosomepositive acute lymphoblastic leukemia (ph+ all) resistant to or intolerant of other tkis. ponatinib has numerous drug-drug interactions and a black box warning for associated serious adverse vascular events and hepatotoxicity. for this reason, ponatinib use has been confined to specific high-risk populations. however, in patients who prove refractory to other therapies, the potential benefits of ponatinib may outweigh risks. to date, ponatinib has not been studied in the pediatric/adolescent and young adult (aya) population. furthermore, literature describing the use of ponatinib alone or in combination with other agents in pediatric oncology patients is scarce. objectives: to describe a single institutional experience using ponatinib in the pediatric patients with ph+ all. design/method: two cases of ponatinib use in pediatric ph+ patients resistant to other tkis were identified at our institution and are described. peripheral blood samples obtained from both patients identified bcr-abl1 p190 fusion transcripts and sanger sequencing was used to identify resistant mutations. results: our first case is a 15-year-old female who received upfront multi-agent chemotherapy plus dasatinib for ph+ all. relapse was confirmed on end-of-therapy bone marrow evaluation, thus bcr-abl mutation testing was performed and revealed a t315i mutation. ponatinib was initiated then discontinued after one week due to clinically significant fluid retention with peripheral edema and bilateral pleural/pericardial effusions. the second case is a lateadolescent female with ph+ all who relapsed 4-years after stem cell transplant (sct). following relapse, tki therapy included both imatinib and dasatinib. due to persistence of bcr-abl fusion transcript despite tki therapy she was switched to ponatinib. shortly following initiation of ponatinib she developed a diffuse, maculopapular rash, which persisted despite dose reduction, resulting in ultimate discontinuation of the drug. bcr-abl mutation testing identified f317l and f357v resistance-conferring mutations. to date, there is scant existing literature detailing the use of ponatinib in pediatric patients. appropriate dosing is undefined and side effect profile not well described, particularly when used concurrently with other chemotherapeutic agents. thus, this case series reporting the response to and toxicity of ponatinib in pediatric ph+ all patients has important clinical implications. additionally, this is the first report of a pediatric ph+ all patient with documented t315i mutation underscoring the importance of bcr-abl mutational testing, particularly at the time of relapse. cooper university hospital, camden, new jersey, united states background: myh9-related disorder is a rare autosomal dominant disease, encompassing several subtypes: may hegglin anomaly, epstein syndrome, fechtner's syndrome, and sebastian syndrome. heterozygous mutations are seen in the gene encoding non-muscle myosin heavy chain iia (nmmhc-iia) which is involved in cell motility as well as functions to maintain cellular shape and integrity. the presentation of myh9-rd is mainly characterized by macrothrombocytopenia, but various related expressions exist: nephritis often leading to renal failure, cataracts and sensorineural deafness (1). a 4-year-old girl with history of extensive dental caries, hyperactivity, and speech delay due to suspected hearing loss was incidentally found to have thrombocytopenia at the time of genetic evaluation. she did not have any bruising or excessive bleeding. she did not respond to observation, immunoglobulins, or steroid therapy. her platelet count remained persistently low (4-23 k/ul). she underwent extensive evaluation to rule out platelet disorder vs. coagulation defect. her peripheral smear showed enlarged platelets by giemsa stain but no inclusion bodies were noted in granulocytes. her platelet aggregation and platelet surface glycoprotein by flow cytometry were negative. her coagulation profile was also normal. objectives: this case report summarizes the complexity in diagnosing myh9-rd in a pediatric patient. design/method: since a unifying diagnosis for her clinical presentation was not apparent, whole exome sequencing (wes) was undertaken. results: wes revealed the r702c heterozygous pathogenic variant, located in exon 17 in the myh9 gene. myh9 gene alteration explained the patient's clinical features of macrothrombocytopenia and hearing loss. this mutation was paternally inherited, and her father demonstrates mosaicism. he was asymptomatic with normal platelet count but his morphology showed enlarged platelets with no inclusion bodies in granulocytes. when dealing with patients who have mild or no symptoms of bleeding diathesis but evidence of persistent macrothrombocytopenia, considering a platelet disorder belonging to myh9-rd can help delineate certain predisposing syndromes and guide clinical management. patients are likely to benefit from early genetic testing while receiving supportive therapy. wes can highlight syndromes and provide information on recurrence risk for families. the renal and hearing abnormalities are indistinguishable between epstein and fechtner's syndromes, but the pathogenic variants differ (2). the genotype-phenotype correlation implies that our patient may have either syndrome, although clinical features compatible with nephritis have yet to manifest. patients should be monitored closely for long-term progression of myh9 disease, and treatments should be initiated accordingly. we present an 11-year old female evaluated by genetics at birth due to prenatal microcephaly. chromosomes and microarray were normal. at age 3 she developed standard risk pre-b-cell acute lymphoblastic leukemia (all). she completed treatment in 2012 and has been doing well in the interim, remaining in complete clinical remission. during and after treatment she exhibited developmental delay and neurocognitive deficits. at age 11 her height and weight were at or below the 5th centile and head circumference was below the 2nd centile (approximately 6 standard deviations below the mean and corresponding to the 50th centile for a 9-month-old girl). bone age was appropriate. she had a distinctive triangular face with micrognathia and a pointed nose resembling a seckel-like syndrome. the patient also had clinodactyly of the 4th toes, zygodactylous triradius involving the 2nd and 3rd left toes, tendency to sydney line in the right palm and a radial loop in the left middle finger. the patient's unique clinical presentation prompted a more thorough genetic evaluation, which led to a novel finding we feel is clinically significant with regard to the development of malignancy. design/method: whole exome sequencing (wes) was performed on the patient as well as her biological parents (trio). a de novo heterozygous mutation in the gene pcdh17 with potential relation to the phenotype was discovered. this c.716dupa variant causes a frameshift starting with codon asparagine239, changing this amino acid to a lysine residue and creating a premature stop codon at position 34 of the new reading frame denoted p.asn239lysfsx34. this variant is predicted to cause loss of normal protein function via protein truncation or nonsense-mediated mrna decay. conclusion: pcdh17 is a member of the protocadherins family which is important in cell-to-cell adhesion and synaptic function in the central nervous system and is highly expressed in areas of the brain involved in higher cortical function and speech. aberrant expression of protocadherins has been associated with the development of malignancies in many organ systems. with regards to leukemia, the methylation status of this gene at diagnosis has been implicated in the prognosis of all and could be used as a biomarker to predict relapse. this patient's de novo mutation and clinical presentation are unique to what has been previously presented in the literature. we feel that this mutation is a clinically significant finding that may shed light on the role of this gene in the development of hematopoeitic malignancies. background: acquired hemophilia a (aha) is an uncommon and potentially life-threatening hemorrhagic disease characterized by sudden onset of bleeding in patients with neither personal nor family history of bleeding dyscrasia. it is usually seen in adults with autoimmune diseases, solid tumors, lymphoproliferative diseases, pregnancy or during the postpartum period; occurrence in the pediatric population has rarely been reported. we report a case of an otherwise healthy teenager who was found to have aha when he presented with acute onset of atraumatic soft tissue hematoma. results: a 13-year old male of middle eastern descent with history of congenital absence of the right external ear, but otherwise in good general health, presented to our emergency department with a three day history of progressive worsening of right lower leg pain, swelling, and paresthesia, without preceding history of trauma. evaluation by the pediatric orthopedics service documented significantly elevated compartment pressures, necessitating immediate four-compartment fasciotomy. pre-operative labs were significant for prolonged activated partial thromboplastin time (aptt) of 67.7 (23.4-38.9) seconds with normal prothrombin time (pt) and international normalized ratio (inr). ptt did not correct on mixing studies, suggesting the presence of a circulating anticoagulant. factors xii and xi were in the normal range; factor ix was elevated, 247 (60-150). factor viii level was 4% and fviii inhibitor level was 5.3 bethesda units (<0.8), confirming the diagnosis of aha. work up for autoimmune disease was negative. his bleeding and surgical hemostasis were managed with recombinant factor vii (novoseven) 90 mcg/kg every 3 hours for 24 hours post operatively, with gradual interval prolongation. factor viii antibody eradication was managed with prednisone 1 mg/kg/day. factor viii and inhibitor levels normalized by day 5 of hospitalization. recombinant factor vii was discontinued; steroids were gradually tapered and discontinued at discharge (hospital day 15). conclusion: acquired hemophilia is likely an underdiagnosed condition in pediatrics. while it is typically seen in adults with underlying autoimmune disease, solid tumors, lymphoproliferative disease, or during pregnancy or the postpartum period, pediatric cases may have no identifiable etiology. this case highlights the importance of considering this diagnosis in any patient with unexplained bleeding regardless of their age, so as to intervene early and prevent adverse consequences. university of oklahoma, oklahoma city, oklahoma, united states background: myeloid neoplasms associated with eosinophilia is a rare subtype of chronic leukemia characterized by clonal eosinophilia. the true incidence is unknown due to its rarity and possible classification as idiopathic hypereosinophilia syndrome. the most common chromosomal aberrations involve platelet-derived growth factor receptors (pdgfrs). we report one such rare case in a pediatric patient. most of the pediatric management of this entity is derived from adult case reports and case series. objectives: to describe a case of chronic leukemia presenting as eosinophilia results: a previously healthy 15 year old caucasian male presented with a several week history of migrating joint pain, splenomegaly, and abnormal blood counts with leukocytosis, thrombocytopenia and absolute eosinophilia. white blood cell differential showed myeloid precursors suggestive of chronic myeloid leukemia. bone marrow evaluation showed 10% blasts and 18% eosinophils. bcr-abl testing was negative, ruling out cml. fish analysis for eosinophilic clonality revealed deletion of chic2 gene, resulting in fip1l1/pdgfra fusion gene, diagnostic for myeloid neoplasm with eosinophilia associated with pdgfr abnormalities. treatment was started with tyrosine kinase inhibitor (tki), imatinib 100mg daily. within 3 months, fish analysis for fusion gene was negative. after approximately 18 months of daily imatinib, he was switched to maintenance dose of 200mg weekly. he is approximately 36 months since diagnosis and doing well on maintenance imatinib. in 2008, the who revised its classification of some chronic eosinophilic leukemias to myeloid and lymphoid neoplasms associated with eosinophilia and rearrangement of pdgfra, pdgfrb, fgfr1. the most common abnormality is the fip1l1/pdgfra fusion gene. other less common abnormalities include fusion genes kif5b-pdgfra and etv6-pdgfrb and point mutations in pdgfra22. some features of chronic eosinophilic leukemia include absolute eosinophilia, splenomegaly, elevated vitamin b12 and tryptase levels, and organ damage from eosinophil infiltrates and cytokine release. patients with rearrangements or mutations involving pdgfra are usually very responsive to imatinib. starting doses have not been well studied or established. experts recommend co-administration of corticosteroids during the first few days of imatinib therapy in patients with a history of cardiac involvement and/or elevated serum troponin levels to prevent myocardial necrosis, a rare complication of imatinib therapy in eosinophilic patients. fortunately our patient did not have cardiac involvement and to date has not exhibited signs of chronic tki toxicity. conclusion: myeloid neoplasms with eosinophilia constitute a rare form of chronic leukemias. they are often associated with pdgfr abnormalities and are usually very responsive to tyrosine kinase inhibitor therapy. walter reed national military medical center, bethesda, maryland, united states background: germline samd9l mutation is a rare cause of constitutional bone marrow failure with a unique propensity for clonal evolution to monosomy 7 and mds. objectives: previous case series have demonstrated diverse clinical outcomes in patients with a germline samd9l mutation. our case presents a novel samd9l mutation (p.val1551leu). additionally, the case highlights the challenges in clinical decision making for a patient with a gene mutation that is known for clonal evolution towards monosomy 7 with risk of progression to myeloid malignancy, but also known for self-correction through uniparental disomy or inactivating mutations which results in disease remission. design/method: a retrospective chart review and review of the literature was performed. dna was isolated from peripheral blood and used for whole exome sequencing. a peripheral blood sample from the patient's mother and father showed no samd9l mutation. skin biopsies of the patient and parents were evaluated for uniparental disomy or new mutations. to determine the pathogenicity of this novel mutation, the specific samd9l mutant dna was transfected into the 293human embryonic kidney cell line to assess its role in inhibiting cell proliferation. our patient presented at 8 months of age with pancytopenia and hypocellular bone marrow in the setting of s63 of s301 sepsis. he had evidence of dysfunctional immune activation with hemophagocytosis and elevated soluble il2 with simultaneous severe hypogammaglobulinemia. analysis of the peripheral blood showed no increase in chromosomal breakage, normal telomere length, and normal flow cytometry. gene testing for primary hemophagocytic lymphohistiocytosis and inherited bone marrow failure were negative. after the patient recovered from his presenting illness, a repeat bone marrow biopsy demonstrated improved cellularity with myelodysplasia and cytogenetics significant for monsomy 7.whole exome testing demonstrated a novel samd9l mutation. the patient continued to require intermittent ivig and failed to demonstrate appropriate leukocytosis with intermittent infections. on repeat bone marrow evaluation over the course of 9 months, the patient demonstrated no evidence of evolution towards self-correction and had a persistent monosomy 7 clone. the patient is scheduled to undergo a matched unrelated donor bone marrow transplant. our case highlights the unique clinical picture associated with constitutional marrow failure and clonal evolution secondary to a novel samd9l mutation which is thought to cause pancytopenia by inhibiting cellular proliferation and often results in the development of monosomy 7 which rescues hematopoiesis but with a risk for malignancy. background: notable labs developed a flow cytometricbased assay with a custom robotic platform to test fdaapproved drugs for anti-cancer activity against individual patient's tumor cells. this personalized assay is a potential method for identifying novel agents and drug combinations to treat aml patients who have failed standard therapies. objectives: to present the case of a teen who underwent successful treatment of relapsed aml post-sct with bortezomib, panobinostat, and dexamethasone-a regimen selected based upon results of notable lab testing. results: a 15-year-old male with m4-aml had an isolated bone marrow relapse 8 months after completion of scheduled therapy. at relapse, his aml was flt3-itd positive. he achieved a second remission with negative mrd and underwent matched sibling donor bmt after busulfan/cyclophosphamide conditioning. bma performed on day +180 was mrd positive (0.13%). repeat bma done on day +204 showed 5.7% mrd. he started sorafenib on day +212. he received donor lymphocyte infusion (dli) on day +246, then received 2 cycles of azacitadine (aza) followed by dli. marrow mrd by flow after sorafenib alone, sorafenib with dli, and sorafenib with aza/dli were 16%, 15.7%, and 0.16%, respectively. treatment was complicated by varicella meningitis, grade i skin agvhd, febrile neutropenia and c. difficile colitis, and metapneumovirus pneumonia. despite extremely low levels of leukemia (marrow mrd 0.16%), notable lab testing performed on the patient's leukemia cells from marrow collected after aza/dli/sorafenib revealed sensitivity of his leukemic blasts to a combination of bortezomib, panobinostat, and dexamethasone. because of prolonged cytopenias, multiple infectious complications, and persistently positive mrd, he discontinued aza/dli/sorafenib and on day +368 started bortezomib 1.3 mg/m2 iv on days 1, 4, 8, and 9; panobinostat 20 mg po on days 1, 3, 5, 8, 10, 12; and dexamethasone 20 mg po on days 1, 2, 4, 5, 8, 9, 11, and 12 . chemotherapy cycle 2 started 21 days later. he tolerated treatment without side effects and with resolution of rash and cytopenias. he achieved full donor chimerism, negative flt3-itd, and complete remission by morphology and flow after two cycles. notable lab testing is a powerful tool for evaluating the sensitivity of small populations of leukemic blasts to novel drug therapy. results from notable lab testing may serve as a useful guide for treatment selection after failure of standard aml therapy. this patient achieved morphologic and mrd remission post-sct with bortezomib, panobinostat, and dexamethasone-a regimen predicted to be efficacious based upon notable lab results. maria ahmad-nabi, christine knoll, sanjay shah, esteban gomez, lori wagner phoenix children's hospital, phoenix, arizona, united states background: development of inhibitors in patients with factor ix deficiency (fixd) is a well-recognized complication occurring in 1-3% of patients. within this subset a small percentage can develop anaphylaxis to factor. desensitization with cyclophosphamide, an alkylating agent used in the management of various oncologic malignancies, and reported for use in factor viii desensitization has been previously unreported for use in desensitization in patients with fixd. rituximab, an anti-cd 20 antibody, however has been used. objectives: to induce immune tolerance (it) in patients with inhibitors to factor ix with either novel or under reported methods using cyclophosphamide and/or rituximab. we report a case series of 2 patients at phoenix children's hospital with fixd who achieved it with cyclophosphamide and/or rituximab. results: patient one was a 14 year old male with severe fixd, who at the time of desensitization had inhibitor levels of 12 bu. he was desensitized with cyclophosphamide, then admitted for infusion of recombinant factor ix. he experienced a few minor symptoms of intolerance including an urticarial rash which was self-limited, and hemarthrosis of the right elbow on day 1 which responded to novo 7. he tolerated the remainder of his infusion without issues. he continued recombinant factor ix daily, and returned to clinic for monthly cyclophosphamide for 6 months. he did develop urticaria with hemarthrosis and spontaneous muscle bleeds which were tempered with zantac, zyrtec, solumedrol, and benadryl. he remained without a recurrence of inhibitors, however did have intermittent hemarthrosis of his ankles thereafter requiring prophylactic twice daily dosing recombinant factor ix. patient two was a 10 year old male with severe fixd and a family history of anaphylaxis to factor causing early death in all male relatives with the disease. he had never received factor ix and did not have a detectable inhibitor prior to desensitization. he successfully underwent desensitization to recombinant factor ix with rituximab in the icu, and returned to clinic for weekly infusions x 4. he experienced no adverse reactions concerning for anaphylaxis. he continued to tolerate factor ix products without evidence of intolerance, development of inhibitors, and continues on as prophylactic dosing of recombinant factor ix every other day. our experience at a single institution proves cyclophosphamide as a novel agent for inducing it in those with fixd and anaphylaxis. it also provides further evidence that rituximab can desensitize patients with severe fixd. differences include longer duration for cyclophosphamide therapy (6 months vs 1 month). background: cartilage-hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with defective cell-mediated immunity caused by mutations in the ribonuclease mitochondrial rna processing (rmrp) gene. cancer incidence is 7-fold higher in patients with chh than in the general population, especially non-hodgkin lymphoma. the use of rituximab, an anti cd20 antibody, results in decreased host b-cell number and impaired humoral function for 6-9 months. the safety of rituximab in pediatric patients with cancer and immunodeficiency is not well documented. a diagnosis of underlying immunodeficiency may discourage physicians from using rituximab due to the risk of severe bacterial infection or viral re-activation. objectives: to report a case of burkitt lymphoma in a young adult female with chh and defective cellular immunity successfully treated with rituximab. results: an 18-year old amish female with disproportionate short stature presented to our center for management of stage iv biopsy proven burkitt lymphoma with myc rearrangement. she had presented a week earlier with cervical, occipital, and submandibular lymphadenopathy, splenomegaly; fevers, night sweats, and weight loss for 2-4 weeks. on exam, her height was three feet associated with brachydactyly, mild bowing of the legs, normal size head without frontal bossing, fine and sparse hair. she had normal intelligence. her pattern of dysmorphisms was suggestive of chh (genetic testing not performed at time of diagnosis). pet-ct scan showed stage iv disease with involvement of cervical lymph nodes, spleen, iliac bone and bone marrow. treatment with standardintensity fab/lmb therapy (group c) with the addition of rituximab was initiated. she had an incomplete response to cop (∼80% reduction of tumoral masses) but achieved complete remission after copadam1. her course was complicated with severe varicella zoster but she completed therapy and remains in complete disease remission for 24 months after treatment completion. genetic testing subsequently performed proved homozygosity for chh with a n.71a>g variant. she had no other opportunistic infections during or after therapy. conclusion: the use of rituximab was both safe and beneficial in our patient despite defective cell mediated immunity secondary to chh suggesting that rituximab may be safe to use in patients with cellular immune deficiencies. background: hemophilia a and b are bleeding disorders characterized by deficiency in factor viii or ix, respectively. spontaneous or provoked hemarthrosis is a known complication of hemophilia. repetitive episodes of hemarthrosis can lead to debilitating hemophilic arthropathy. lyme disease is a tick-born infection which is endemic to increasing parts of the united states. chronic lyme disease, the phase in which lyme arthritis typically develops, occurs months to years after initial infection and is characterized by swelling of one or more large joints generally in the absence of systemic symptoms. objectives: review cases of hemophilia a and b patients with episodes of provoked hemarthrosis refractory to intensive recombinant factor replacement therapy found to have concurrent lyme arthritis. design/method: we report two clinical cases and review relevant literature. results: first, we report a 12 year-old male with moderate hemophilia a with a provoked knee hemarthrosis which failed to improve despite 3 months of intense factor replacement therapy requiring multiple hospitalizations. factor replacement regimens included twice daily standard half-life recombinant factor viii products or daily to every other day extended half-life recombinant factor viii products with trough levels aimed as high as 50-80%. factor viii pk studies were obtained for dosing, to confirm adherence, and to evaluate for subclinical inhibitors (inhibitor testing was negative). given protracted symptoms additional workup for hemarthrosis was pursed. lyme titers were positive for (8)igg, though negative for igm. he was treated with 28 days of doxycycline during which time hemarthrosis greatly improved on examination and imaging, and he was able to recover function through physical therapy. second, we report a 6 year-old male with moderate hemophilia b who required multiple hospital admissions for a provoked knee hemarthro-sis with no improvement in symptoms despite weeks of daily or twice daily factor replacement with standard halflife recombinant factor ix products aiming for 100% correction. we performed inhibitor testing (which was negative) and pk studies to assess for non-detectable inhibitors, dosing and adherence. lyme testing was positive for (6)igg, though negative for igm. he was treated with amoxicillin for 28 days during which time hemarthrosis significantly improved on examination and imaging. diagnosis and follow-up imaging studies for both patients included mri and serial bedside ultrasounds performed as per uc san diego school of medicine mskus guidelines. background: relapse/refractory aml following allogeneic hematopoietic stem cell transplant (hsct) holds a high mortality rate. current relapse/refractory therapy modalities for younger patients may include re-induction with a clofarabinebased regimen followed by second allogeneic hsct. even for patients who undergo second hsct, the five-year survival rate is dismal. new therapies, including small molecule inhibitors, are being studied in the post-hsct relapse setting or those unfit for hsct with promising results. venetoclax is a small molecule inhibitor that has received breakthrough designation for aml treatment in elderly patients objectives: to report a young adult aml patient with relapse post hsct who was successfully re-induced with topotecan, vinorelbine, thiotepa, clofarabine (tvtc) and has sustained remission with venetoclax maintenance therapy. this approach appears to be unique in terms of reported literature. results: our patient is now a 23-year-old female noted to have mll rearranged aml at initial diagnosis when she was 21 years old. she underwent chemotherapy consisting of cytarabine/daunorubicin according to standard 7+3. due to persistent disease, she was re-induced with g-csf, clofarabine, and high-dose cytarabine (gclac) which put her in cr. her course was complicated by sepsis, colitis, gastrointestinal bleed, deep venous thrombosis, and transfusionassociated circulatory overload. given her co-morbidities, she received another cycle of clofarabine/cytarabine, and then proceeded to reduced intensity allogeneic hsct, according to bmt ctn 1101. the patient tolerated hsct well and experienced no transplant-related complications, including no acute or chronic gvhd. unfortunately, she relapsed about 10 month's post-hsct. initial salvage therapy consisted of another course of g-clac, but due to persistent disease the decision was made to re-induce her with topotecan, vinorelbine, thiotepa, and clofarabine (tvtc). during this time however, she was found to have extensive infection with a fusarium species requiring a course of anti-fungal therapy. bone marrow evaluation showed no residual disease with an mrd of <0.1%. once the absolute neutrophil count recovered, the patient was started on single-agent venetoclax for maintenance therapy, which has been well-tolerated. she remains in morphologic remission for over 8 months. we describe herein a young adult with multiply relapsed aml wherein tvtc re-induction, followed by maintenance with venetoclax were safely used in the post-hsct setting. venetoclax therapy in the relapsed aml setting warrants further study. background: vitamin b12 deficiency is uncommon in children in developed countries, especially in the absence of risk factors like malabsorption or inadequate dietary intake. it often presents with non-specific symptoms and signs and can elude diagnosis. the recognition and treatment of vitamin b12 deficiency is critical as it can lead to bone marrow failure as well as severe neurological and developmental problems in children. to increase index of suspicion of vitamin b12 deficiency anemia in children. we report a rare case of vita-min b12 deficiency anemia in a child who presented with a severe macrocytic anemia, with signs of hemolysis and concern of malignancy. design/method: an almost three-year-old previously healthy girl presented with a few day history of fever, emesis, fatigue and pallor. she had no dysmorphic features, hepatosplenomegaly or lymphadenopathy on exam, growth and development were normal. laboratory findings showed severe macrocytic anemia (hemoglobin 4.4 grams/dl; mcv 104.1 fl) with reticulocytopenia. signs of intravascular hemolysis were present with elevated lactate dehydrogenase (3,842 units/l) and haptoglobin below assay limit. immune-mediated hemolysis was ruled out. initial picture of a hemolytic anemia was compounded by other findings of moderate neutropenia, mild thrombocytopenia and peripheral smear showing occasional blasts. further workup was done with a broad differential diagnosis that included leukemias, hemolytic anemias, bone marrow failure syndromes, and specific deficiencies. results: workup revealed abnormally low vitamin b12 levels along with significantly elevated homocysteine and methylmalonic acid levels indicating functional vitamin b12 deficiency. bone marrow evaluation showed megaloblastic anemia and dyserythropoiesis consistent with vitamin b12 deficiency, and ruled out leukemia. vitamin b12 deficiency can cause a hemolytic anemia like picture secondary to intramedullary hemolysis due to ineffective erythropoiesis. myeloid precursors are also affected which can lead to neutropenia, thrombocytopenia, and abnormal peripheral blood cells. in our patient, initial symptomatic anemia was treated with blood transfusion, followed by intramuscular vitamin b12 injections with normalizing lab values. so far, workup for an etiology for vitamin b12 deficiency is negative except for an equivocal range of anti-parietal cell antibodies raising concerns for pernicious anemia; however it is rare in this age group. another rare condition is an inborn error of the cobalamin transporter. she is currently on oral vitamin b12 supplementation and further workup will be planned based on response. conclusion: this case highlights the importance of early consideration and thorough evaluation of vitamin b12 deficiency in children with unclear etiology of anemia, so that prompt treatment can be initiated. memorial hospital/ university of miami, miami, florida, united states background: despite great success in the treatment of acute lymphoblastic leukemia (all), the outcomes for patients with relapsed all remain poor. prognostic indicators include timing and site of relapse. blinatumomab, is the first agent in its class that simultaneously binds cd3-positive cytotoxic t cells to cd19-positive b cells resulting in lysis of malignant cells. however, mechanisms of leukemia resistance to blinatumomab are unclear. objectives: to describe a case with multiple sites of extramedullary (em) relapse during blinatumomab therapy. results: a 9-year-old hispanic male with philadelphia positive, cd19-positive b-precursor cell all refractory to chemotherapy, had failed a bone marrow (bm) and was placed on blinatumomab and imatinib. he achieved minimal residual disease (mrd)-negative systemic remission, but during his fifth cycle developed bilateral periorbital masses. biopsies confirmed cd19-negative isolated em relapsed disease, which was treated with radiation therapy (rt). there was notable resolution of em disease and he continued systemic therapy. subsequently, he presented with a painful left scapular swelling. imaging showed muscle and lung parenchymal em relapse with cd19-positivity confirmed on histology. he continued on blinatumomab with localized rt while awaiting car-t cell therapy. his bm mrd remained negative until he developed systemic mrd-positivity with cd19-positive blasts following the sixth cycle. primary resistance to blinatumomab is poorly understood. it is proposed that expansion of cd19-negative clones or downregulation of cd19 following blinatumomab may play a role. this was observed in our patient's periorbital relapse; but subsequent em and systemic relapses were cd19-positive, consistent with the co-existence of multiple clones in relapsed all. it has also been postulated that em relapse could be linked to the failure of blinatumomab or t cells to migrate to em sites of disease or drug inactivation by the microenvironment. the second em relapse in our patient, with cd19-positive disease suggests this as a possible mechanism of relapse. this was reported in patients with cd19 positive non-hodgkin lymphoma (nhl), and higher doses of blinatumomab however, have shown promising results in this population. despite blinatumomab's effectiveness in inducing remissions in patients with refractory/relapsed all, it appears to have limitations in patients with em disease. these may arise either from the multiclonality associated with relapsed all or due to the emergence of resistance to blinatumomab, including failure to migrate to em sites. background: cyclic neutropenia is a rare hereditary disorder, characterized by recurrent neutropenia, cycling at about 3 week intervals, with variable associated symptoms including oral ulcers and fever. there are 4 reported cases of cyclic neutropenia associated with chronic inflammation leading to development of reactive aa amyloidosis. one patient also presented with amyloid goiter. we report a new case of cyclic neutropenia with associated renal and thyroid amyloid. design/method: a 12-year-old female presented with a 1 month history of thyromegaly, and recurrent aphthous ulcers associated with fevers. laboratory workup showed severe neutropenia, anemia, azotemia, and abnormal thyroid function, with an absolute neutrophil count -0/ l, hemoglobin -9.0 g/dl, serum creatinine -1.89 mg/dl, and uric acid -9.0 mg/dl. thyroid stimulating hormone was elevated -12.5 iu/ml, and normal free t4. urinalysis showed 2+ protein, 2+ blood, and 5-10 urine red blood cells/hpf. chest radiograph showed mild narrowing of the trachea from thyroid compression. bone marrow biopsy showed a hypocellular marrow, with tri-lineage hematopoiesis, left shifted myeloid maturation with very rare mature neutrophils. both renal biopsy and thyroid fine needle aspiration revealed abundant amyloid. of note, her father had aa amyloidosis, resulting in end-stage renal disease (esrd) requiring hemodialysis, and recurrent aphthous ulcers. the family history suggested a familial predisposition. genetic testing revealed a pathogenic elane c.358 a>t gene mutation with autosomal dominant inheritance confirming the diagnosis of cyclic neutropenia. we treated our patient with daily granulocyte colony stimulating factor to reduce the burden of chronic inflammation induced by cyclic neutropenia, and to preserve renal and other end organ function affected by further amyloid deposition. results: proband with elane gene mutation positive cyclic neutropenia, amyloidosis of thyroid and kidney, with a positive paternal history of aa amyloidosis resulting in esrd. cyclic neutropenia may result in chronic inflammatory states leading to secondary amyloidosis. university of kentucky, lexington, kentucky, united states background: overall survival of burkitt lymphoma (bl), regardless of stage, is greater than 85% in the pediatric population when treated with multi-agent chemotherapy. adenovirus is a common, usually self-limited infection within the pediatric population; however, findings can vary within an immunocompromised host. hepatitis is a rare complication, with very few reports of radiologic findings in this patient population. we discuss a three year old male with history of bl who presented with clinical and radiographic evidence of relapse but was found to have adenovirus hepatitis. design/method: a case report of a patient with bl in complete remission after completion of standard of care chemotherapy, who presented with return of high fever, elevated ldh, transaminitis and hepatic lesions. we describe the hepatic imaging and pathology consistent with adenovirus hepatitis in this immunocompromised host. our patient presented at three years old with a six week history of worsening abdominal pain and fevers. he was found to have a right sided pleural effusion, multiple lesions of the liver, and diffuse abdominal lymphadenopathy; biopsy of lymph tissue was consistent with bl. he completed therapy per anhl1131 arm b and was in a complete remission at the end of planned therapy. one month after completion of therapy, he returned with high fever, abdominal pain and transaminitis, similar to his initial presentation. ct scan showed multiple hypodense discrete lesions throughout the liver and re-accumulation of right sided pleural effusion. ldh peaked at 3580 u/l (uln 370 u/l). uric acid remained within normal limits. bilirubin peaked at 4.0 mg/dl, conjugated 3.2mg/dl. liver biopsy was performed, showing smudgy nuclei with immunohistochemical staining positive for adenovirus. there was no evidence of lymphomatous involvement. resolution of hepatic lesions and transaminitis, with normalization of ldh and fever, occurred with symptomatic treatment alone. adenovirus is known to cause systemic disease in immunocompromised patients and rarely hepatitis. no pediatric patients with discrete hepatic lesions secondary to adenovirus have been reported in the literature. three cases of discrete hepatic lesions have been reported in adult immunocompromised patients, two with fatal fulminant liver failure and one who required cidofovir. this case demonstrates that a common pediatric viral infection can present with lesions concerning for metastatic disease in a pediatric lymphoma patient. prompt diagnosis is vital in the management of these patients when recurrent lymphoma is in the differential. background: heparin induced thrombocytopenia (hit) is an immunologic process in which antibodies bind a heparin complex and cause a paradoxical hypercoagulable state. ramifications of this process may include a multitude of thrombotic events and bleeding complications secondary to platelet consumption. in our patient, hit manifested as increased bruising, an acute decrease in platelet count, and continual clotting of her crrt circuit. hit, although rare in pediatrics, should be included in the differential for children with thrombocytopenia who have received heparin products. to present a unique case report of a critically ill pediatric patient who developed hit in the presence of multiorgan system failure and to discuss the challenges encountered with identification of an alternative anti-coagulant. results: a 12yo obese, caucasian female child presented to our facility with bilateral pulmonary emboli (of unclear etiology). initially, she was started on a continuous heparin infusion, but was transitioned to enoxaparin within 3 days without issue. five days after enoxaparin was initiated, the patient developed acute kidney injury (evidenced by increasing creatinine) attributable to her biventricular heart failure. due to her need for continuous renal replacement therapy (crrt), she was transitioned back to a continuous heparin infusion. whereas her initial platelet count on transition was normal, she developed severe thrombocytopenia (15,000ul) within 48 hours. due to intermediate risk but low suspicion for hit, pf4 antibodies were sent which were positive. after much discussion, she was transitioned to an argatroban infusion which was titrated according to ptt levels. within 48 hours, her platelet count normalized. at discharge, she was prescribed apixaban for anti-coagulant management. conclusion: hit is an uncommon presentation in the pediatric population. given its rarity, there is often a delay in diagnosis which increases risk of complications such as bleeding, stroke, and limb ischemia. even if the diagnosis is suspected or proven, there may be challenges in initiating alternative agents as limited data exists on pediatric options. as argatroban remains the treatment of choice for patients with hit, experience in pediatric patients is limited, and dosing recommendations have been extrapolated from adult studies. anecdotal data exists for use of bivalirudin in children, although studies, primarily, focus on use in specific cardiac cases. in our patient's case, choice was further complicated by renal failure. this case study highlights the need for further research regarding the identification of a secondary anti-coagulant agent for use in pediatric patients with hit. background: subcutaneous panniculitis-like t-cell lymphoma (sptl) is a rare form of non-hodgkin's lymphoma characterized by infiltration of cytotoxic t-cells into subcutaneous tissue. sptl occurs in both adults and children and can present in both patient populations as either alpha/beta or gamma/delta subtypes. patients with the gamma-delta phenotype have an overall poorer survival, although the exact etiology is unclear. interestingly, both subtypes of sptl can present with secondary hemophagocytic lymphohistiocytosis (hlh), and this is associated with a worse prognosis. currently, there are no standardized treatment protocols for sptl, and clinical management includes watchful waiting, corticosteroids/immunosuppression, chemotherapy, and stem cell transplant. the primary objective was to compare how two patients with the same diagnosis responded acutely to therapy. we performed a retrospective chart review of two pediatric patients at our institution who were diagnosed with alpha/beta sptl and secondary hlh. we examined each presentation, treatment course, and outcome. we then completed a brief review of the current literature describing treatment of and outcomes for sptl with secondary hlh. results: these two patients presented in a similar manner with signs and symptoms of hlh. each was then subse-quently diagnosed with alpha/beta sptl after biopsy of cutaneous nodules and each had diffuse disease, as measured by pet. however, they demonstrated vastly different acute responses to therapy. one patient was pre-treated with systemic glucocorticoids before receiving definitive chemotherapy and tolerated therapy well as an outpatient. the other patient started systemic chemotherapy without steroid pretreatment and developed severe cytokine storm characterized by hypotension, cardiac dysfunction, multi-organ failure and cytokine elevation. both patients achieved complete remission (cr) after treatment with chop chemotherapy and remain disease-free 12-24 months off therapy. in patients presenting with sptl and secondary hlh, we propose that initial treatment with antiinflammatory or anti-cytokine therapy can decrease, or even prevent, the possibility of life threatening cytokine release as a result of cytotoxic chemotherapy. background: congenital dyserythropoietic anemia type ii (cda ii) is a rare autosomal recessive disorder, rarely presenting in the neonatal period. iron overload often occurs as a late sequela of ineffective erythropoiesis and intramedullary hemolysis. objectives: to report the novel use of iron chelation in an infant with cda ii associated with severe iron overload. the patient is a 3-month-old, former 27-week infant with prenatal non-immune hydrops and transfusion-dependent fetal anemia who presented with persistent anemia, reticulocytopenia, hyperbilirubinemia, liver dysfunction, and hyperferritinemia. his initial ferritin was 4822.3 ng/ml, tibc 185 ug/dl, and transferrin 116 mg/dl. his bone marrow biopsy showed trilineage hematopoiesis and erythroid dyspoiesis characterized by binucleation of late-stage precursors. genetic testing revealed a compound heterozygous missense mutation and splice site mutation in the sec23b gene, confirming the diagnosis of cda ii. initial liver biopsy revealed mild portal fibrous expansion, and abundant hepatic iron deposition. his ferritin continued to increase, peaking at 21,114 ng/ml, along with liver enzymes peaking at an alanine aminotransferase (alt) of 505 u/l and aspartate aminotransferase (ast) of 776 u/l. ferriscan showed an elevated estimated liver concentration of 2.8 mg/g dry tissue. repeat liver biopsy 3 months later showed giant cell hepatitis with worsening mild portal fibrosis and hemosiderosis. additionally, tissue liver iron concentration was 4755 mcg/g dry weight. cardiac t2* mri revealed mild cardiac iron deposition. given his significant degree of iron overload, deferoxamine was used to reduce hemosiderosis and liver morbidity in preparation for bone marrow transplantation. the patient received deferoxamine 15 mg/kg/day iv x 5 days/week for three months, without any clinically significant adverse events. blood counts and hepatic and renal function were monitored weekly without any abnormalities. growth parameters and liver enzymes significantly improved while receiving chelation therapy. as a noninvasive, cost-effective method, serum ferritin levels were monitored monthly to gauge response to treatment. despite receiving blood transfusions every 3-4 weeks, serum ferritin decreased to 344 ng/ml and liver enzymes decreased to alt 29 u/l and ast 26 u/l prior to bone marrow transplantation. we report the use of deferoxamine in a patient with cda ii less than 2 years of age, for treatment of iron overload. our patient tolerated deferoxamine well without significant adverse events or organ toxicity. deferoxamine may be a well-tolerated method of reducing iron burden in young patients with iron-loading pathologies. background: low grade gliomas with kiaa-1549-braf fusions typically have a favorable prognosis with infrequent rates of high grade transformation, low rates of metastasis and even lower rates of extra cns metastasis. while highgrade transformation has been reported for tumors with braf v600e mutations and cdkn2a deletions, it has not been pre-viously reported in gliomas with kiaa-1549-braf fusions. while there are case reports of high-grade cns malignancies metastasizing through a ventriculo-peritoneal (vp) shunt, low-grade gliomas metastasizing in this manner are extremely rare. objectives: to describe a unique case of peritoneal tumor dissemination of a braf fusion positive high grade neuroepithelial tumor in a child with a vp shunt placed for multifocal braf fusion positive low grade astrocytomas results: an eight-year-old male was initially diagnosed with multifocal low-grade astrocytomas of the hypothalamus and c2-c4 spinal cord. initial testing revealed the kiaa-1549-braf fusion, but no cdkn2a or braf v600e mutation. initial surgical management included a vp shunt and resection of the cervical spinal lesion. he received vincristine and carboplatin, followed by transition to vinblastine given new thoracic metastatic lesions after 10 months of therapy. at 15 months after diagnosis, scans were concerning for diffuse leptomeningeal progressive disease and new intracranial lesions, necessitating craniospinal radiation. following a near cr, he presented 13 months later with acute onset of abdominal pain. a ct scan revealed peri-renal and perirectal soft tissue masses, confirmed by exploratory laparotomy to be peritoneal tumor dissemination of high grade neuroepithelial tumor. a kiaa1549-braf fusion was noted and confirmed by rt-pcr, identical to that seen in the original cns tumors. additional findings included deletion of chromosome 1p (without 19q loss) and heterozygous and homozygous deletion of cdkn2a found by fish. brisk mitotic activity justified a high-grade designation. salvage chemotherapy consisted of 4 cycles of ice with subsequent resolution of pet-avid disease and only minimal peri-nephric tissue remaining. given the favorable response, surgical resection and multiple tissue biopsies were performed which documented no residual active disease. the shunt was revised and he started trametinib for maintenance. we present a unique case of peritoneal dissemination of high grade neuroepitheial tumors with the same kiaa-1549-braf fusion as multifocal low grade astrocytomas in a child with a vp shunt. this raises suspicion for tumor metastasis and transformation to a higher grade malignancy versus two distinct diseases, which may be indicative of an underlying cancer predisposition. texas children's hospital, houston, texas, united states background: polycythemia is a common referral to hematology. it is important to evaluate for a high oxygen affinity hemoglobinopathy, ensuring appropriate testing is performed for early diagnosis and avoidance of additional tests and procedures. a 17 year old mexican female presented with an elevated hemoglobin and hematocrit, symptoms of plethora of her hands and feet, chest pain, palpitations, and fatigue. further confounding the picture, she also had significant menorrhagia and iron deficiency. she was diagnosed with the rare high oxygen affinity hemoglobin new mexico variant, only previously described once in the literature in a 4 year old black boy. objectives: the patient initially presented at age 14 with a hemoglobin of 16.7g/dl and a hematocrit of 52.4%. initial work up consisted of a hemoglobin electrophoresis which diagnosed sickle cell trait, a co-oximetry panel which was normal, and erythropoietin level of 7mu/ml, also normal. she was then lost to follow up and re-referred at age 17. she is a competitive basketball athlete, and at that time, she presented with a hemoglobin of 17.1g/dl, and hematocrit of 50%. erythropoietin level continued to be normal at 13mu/ml. design/method: cardiology was consulted regarding chest pain and palpitations with a normal evaluation. chest x-ray was also normal. a bone marrow aspirate and biopsy was performed with results significant for mild erythroid hyperplasia and mild reticulin fibrosis. jak 2 mutation, von hippel lindau, bpgm, and hereditary erythrocytosis mutations including phd2, hif2a, and epor mutation analysis were sent, all of which were normal. testing to mayo clinic for p50 rbc oxygen dissociation returned low at 19mmhg (24-30mmhg normal range) and subsequently a hemoglobin electrophoresis identified a hemoglobin variant leading to beta globin gene sequencing. results: patient found to be heterozygous for hemoglobin new mexico, with 41.9% hb new mexico and 54.5% hba, and 3.6% hba2. there was no evidence of hbs. when evaluating patients with polycythemia, maintaining a high index of suspicion for high affinity hemoglobinopathies may eliminate further unnecessary and invasive testing for patients. caution should be used when using hemoglobin electrophoresis testing since hb new mexico is known to migrate similarly to hbs on hplc with minimal change that may not be detected in regular laboratories. most high affinity hemoglobinopathies are reported to not have significant symptoms. in this case, our patient complains of fatigue, occasional palpitations and plethora of hands and feet. we will need to further follow this patient for possible attributable symptomatology. divya keerthy, simone chang, warren alperstein, patricia delgado, claudia rojas, ofelia alvarez, matteo trucco university of miami jackson memorial hospital, miami, florida, united states background: improved technology is enabling detection of previously unidentified translocations and mutations in otherwise unclassified sarcomas. one such mutation is the bcl-6 co-repressor -internal tandem duplication (bcor-itd) allowing for the new classification of bcor positive undifferentiated round cell sarcomas (urcs). this sarcoma has a similar appearance to clear cell sarcoma of the kidney (ccsk), potentially representing an extra-renal manifestation of this tumor, but their clinical pathologic features are not identical. objectives: this case highlights how recombinant polymerase chain reaction (rt-pcr) and bcor immunohistochemical staining can ease the diagnosis of this rare sarcoma. results: a 5 month-old female presented for right sided pre-septal cellulitis and a temporal subcutaneous mass. the detection of multiple other subcutaneous nodules on exam raised the concern for malignancy and she was admitted for evaluation. she had two subcutaneous masses on her abdomen, with more cutaneous masses on her legs, back, shoulder, cheek and submandibular areas. she lacked spontaneous lower limb movement and had bilateral clonus. imaging confirmed multiple masses throughout the body including paravertebral area from t3 to l2, bilateral adrenal glands, left kidney and muscles of upper and lower extremities. initial differential included neuroblastoma, infantile myofibromatosis, rhabdomyosarcoma or atypical presentation of a renal tumor. however, synaptophysin and chromogranin stains were negative. with standard immunohistochemistry, the tumor could be only broadly classified as "undifferentiated sarcoma" maintaining the diagnostic challenge. using rt-pcr in the setting of a morphologically primitive round cell neoplasm with strong bcor expression, two external institutes simultaneously diagnosed the tumor as bcor-urcs. the primary lesion is unknown but potentially may have arose from the kidney. bcor-urcs has a heterogeneous histology with tumor cells appearing monomorphic in nests of 6-10 cells separated by septa with uniform nuclei. there is frequently an "orphan annie eye" appearance and sparse cytoplasm to the cells. diagnosis cannot be made solely on evaluation of this nonspecific histology. rt-pcr uses the genetic abnormality in undifferentiated sarcomas to narrow the differential and bcor immunohistochemical staining provides further context. bcor has significant diagnostic value given its sensitivity and specificity in urcs. another potential marker includes ywhae-nutm2b fusions, which occur in smaller subset of cases, but requires further study. rt-pcr has helped further classify tumors leading to the diagnosis of a rare undifferentiated sarcoma with bcor overexpression. while this technology is beneficial, its availability is limited. if accessibility improves, earlier identification and treatment may be possible maximizing the chance for a positive outcome. background: hematohidrosis is a rare condition that mimics bleeding disorders. cases present with oozing blood tinged fluid from various sites like eyes, ears, nose, skin, etc. reported causes of this condition were stress or fear, physical activity, psychological disorders. the condition is self-limited and don't affect the general condition of the patients, but it may contributes to psychosocial problems and may increases their stress and anxiety. so this condition needs to be promptly treated. to test the response of this disease and the associated headache to propranolol treatment. design/method: our case female patient 11 years old 1st offspring of non consanguineous marriage, was admitted with recurrent episodes of oozing blood tinged fluid from eyes, ears and nose 2 months before admission, about 0.5-1 ml from each orifice, lasted 5-10 minutes and subsided spontaneously. it could involve the 3 sites simultaneously or 1-2 sites. the number of attacks was 3-4 times per day then gradually increased to 15-20 times per day. later on the patient developed a bleeding attack from umbilicus. these attacks were aggravated by stress and physical activity and decreased with rest and sleep. the condition was associated with severe headache involving the whole head, throbbing in nature of gradual onset, increased by physical activity and relieved by analgesics. the condition was not associated with vomiting, blurring or diminution of vision, ocular pain, eye discoloration. no earache, tinnitus or diminution of hearing. there was no other form of discharge from eyes, ears or nose. no history of ecchymotic patches, bleeding from other orifices or blood product transfusion. no history of trauma, drug intake, fever or rash. no symptoms of other system affection. past history of recurrent attacks of epistaxis and two operations were done that passed without remarkable bleeding. no similar condition in the family physical examination was free, no evidence of psychological problems. complete blood count, coagulation profile, platelets function, factor 13 and c.t brain were normal. oozing fluid from the patient was analyzed showed the same components as blood. results: our case started oral propranolol 0.5mg/kg/day based on its use in similar cases in literature. the frequency of attacks and headache reduced then stopped after 2 months of treatment and didn't recur after stoppage of propranolol. propranolol can treat this condition successfully. further investigations are needed to determine the link between this condition and severe headache our case was suffering from. background: wilms tumor is the most common renal solid tumors of childhood and is derived from primitive metanephric cells located in the kidney. primary extra-renal wilms tumors (erwt) are extremely rare, estimated to comprise 0.5-1% of all wilms tumors. despite similar histologic appearance intrarenal and erwts differ in embryologic tissues of origin. erwts arise from the more primitive mesonephric or pronephric origin and, therefore, can develop anywhere along the craniocaudal migration pathway of these primitive tissues, most often retroperitoneal, inguinal/genital, lumbosacral/pelvic and mediastinal. these tumors are typically staged and treated per national wilms tumor study (nwts) guidelines, and, by definition, are stage ii or greater due to location beyond the kidney borders. based on the cases reported in the literature, outcomes for erwt are comparable to renal wilms tumors with an 11% local recurrence rate and an 85% two-year event-free survival. we report the first case of a stage iii testicular extrarenal wilms tumor in an 8-month-old male with an intrabdominal undescended testis who underwent complete surgical excision followed by chemotherapy and inguinal radiation. results: a full term 8-month old male underwent orchipexy for an undescended left testicle. the testicle was noted to be grossly abnormal with a pea-sized thickened tissue adherent to the upper pole and a separate mass outside of the scrotum on the superior epididymis. both masses were removed, and s73 of s301 pathology demonstrated wilms tumor with favorable histology and negative margins. ct imaging of the chest, abdomen and pelvis were negative for a primary renal tumor, local residual disease, pathologic lymph node enlargement or distant metastases. the tumor was classified per nwts as stage iii due to tumor removal in multiple pieces. the patient completed dd-4a treatment with vincristine, doxorubicin and dactinomycin per aren0534 with 10 cgy left inguinal radiation. he is currently 15 months off therapy without clinical or radiographic evidence of recurrent disease. primary erwt is an extremely rare malignant neoplasm associated with challenges in diagnosis, staging and treatment. based on the 80 cases reported in the literature, outcomes are similar to that of intrarenal wilms tumor. there are four pediatric paratesticular wilms tumors reported in the literature and, to the best of our knowledge, this is the first case of stage iii testicular wilms tumor successfully treated with dd-4a chemotherapy and radiation. in erwt, nwts guidelines for staging and treatment should be applied with evaluation of both kidneys to exclude an intrarenal primary tumor. background: patient is a 20 yo f, with esrd secondary to atypical hus versus ttp, who presented with thrombotic microangiopathy, aki, thrombocytopenia and anemia after a living unrelated donor kidney transplant. patient initially had downtrending creatinine. on post-op day 2, hematology was consulted for an increasing ldh and drop in platelets. peripheral smear was notable for an absence of schistocytes. yet, biopsy of the kidney revealed microthrombi. the patient was diagnosed with a thrombotic microangiopathy. plasmapharesis was initiated on day #3, at which time ms r was noted to have significantly elevated creatinine. plasmapharesis did not yield any correction in labs and significant bruising developed. patient was started on eculizimab; plasmapharesis was stopped. shortly after, creatinine, anemia and thrombocytopenia corrected to levels at which she was discharged. overall, patient was found to have progressive anemia, thrombocytopenia, an increasing creatinine and ldh (600s) concerning for atypical hus, despite absence of schistocytes on peripheral smear. she responded well to eculizimab, with correction of hematologic changes during induction. she was discharged on eculizimab and continued to respond with normalizing platelet counts and hemoglobin. the differential in light of patient's thrombotic microangiopathy and thrombocytope-nia also included ttp. yet, adamts13 remained normal. dic was unlikely given normal fibrinogen level and d-dimer. objectives: presentations of atypical hus vs ttp. discuss eculizumab as a treatment of atypical hus. highlight atypical presentations of illness in transplant patients. results: despite absence of schistocytes by smear, pt was diagnosed with atypical hus based on presentation and after failing plasmapharesis, she responded well to eculizumab. though her presentation was abnormal, her response to this antibody that blocks the complement cascade suggests that she was experiencing a complement-mediated process. there are rare documented cases in the literature of atypical hus without schistocytes. hemolytic uremic syndrome (hus) is characterized by hemolytic anemia, thrombocytopenia and acute kidney injury. atypical hus is a diagnosis of exclusion, not due common etiologies such as shiga toxin. among atypical causes are complement-mediated forms, caused by an antibody to complement factor. in addition to plasmapharesis, renal transplant and supportive care, the mainstay of treatment for atypical hus is eculizumab (an antibody that blocks the complement terminal cascade). this case describes a patient unique in that, she was diagnosed with atypical hus without any schistocytes by smear. secondly, she responded to eculizumab, with unremarkable gene studies. finally, this case highlights that transplant patients often have unique presentations. nicklaus children's hospital, miami, florida, united states background: synovial sarcoma is a spindle cell tumor categorized as a soft tissue sarcoma. the chromosomal translocation t(x;18) leading to the ss18-ssx fusion protein is unique to this sarcoma. it is a slow growing tumor with common recurrences and often, at presentation, with evidence of metastatic disease. if resection is not feasible, then neoadjuvant with adjuvant chemotherapy is recommended. metastasis carries an unfavorable prognosis given synovial sarcoma historically does not respond well to chemotherapy. trabectedin is a well-tolerated alkylating agent currently indicated for the treatment of liposarcoma and leiomyosarcoma. we present a 17-year-old male with metastatic synovial sarcoma to the lungs that progressed and was refractory to chemotherapy. he was administered trabectedin as a form of palliative chemotherapy, with significant clinical and radiographic response. design/method: pubmed search was done with search for terminology including "synovial sarcoma" and "trabectedin". papers relevant to our case were selected for literature review. a 17-year-old male patient presented with a large right axillary mass. initial imaging showed a heterogeneous multiseptated mass invading the subscapularis and teres major muscles along with innumerable lung nodules. biopsy confirmed diagnosis of monophasic synovial sarcoma. the patient was started on protocol arst 0332 with ifosfomide, mesna, doxorubicin. he completed 4 cycles followed by radical resection and 33 sessions of radiation. due to progression of disease multiple chemotherapy regimens were tried including topotecan and cyclophosphamide, protocol advl 1522 with lorvotuzumab, and pazopanib. imaging of the chest continued to show significant progression of metastasis. the patient's clinical status deteriorated with worsening respiratory status, requiring 10l of oxygen therapy, and inability to ambulate. he was started on trabectedin 1.2mg/m2 for palliative care. after 2 cycles of treatment patient was no longer requiring oxygen and was ambulating without assistance. radiological imaging showed significant reduction in number and size of lung nodules. trabectedin is a recently approved alkylating agent for the management of sarcomas resistant to first line treatment. response in synovial sarcoma is scarcely documented in the pediatric population. epidemiology places the most common age group in the young adults and children. our case opens the doors to further consideration of the use of trabectedin in the pediatric patient with metastatic synovial sarcoma. background: gata1 is an x-linked gene that plays critical role in hematopoiesis. mutations of gata1 gene can be associated to various blood disorders including diamond blackfan anemia, cytopenia, congenital dyserythropoietc anemia and acute megakaryoblastic leukemia. we report a patient with macrocytic anemia and platelet dysfunction who carries a novel gata1 mutation that has not been reported. results: a now 28-month-old male with complex medical history including prematurity at 33 weeks, dysmorphic features, global developmental delay, hyperinsulinism, hypogonadotropic hypogonadism, growth hormone deficiency, micropenis, failure to thrive, patent ductus arteriosus status post ligation, and severe hypotonia, was referred to hematology at 16 months old for resolved, transient thrombocytopenia and macrocytic anemia since 1 month of age. chromosomal microarray showed chromosome deletion of 14q21.3, which is the rps29 gene. he doesn't have a family history of diamond blackfan anemia (dba), despite mom having the same rps29 mutation. he was then diagnosed with dba. his lab workup showed mild macrocytic anemia (hgb 9.1 g/dl, mcv 95fl), normal to inappropriately low reticulocyte count, normal white blood cell and platelet counts, hgf 0%, erythroid ada 1.39 eu/gm hgb (elevated). he has abnormal pfa-100, with prolonged closure time of both adp and epinephrine. he had low von willebrand antigen and ristocetin cofactor activity. he has severe pancreatic insufficiency. bone marrow biopsy showed normocellular marrow with trilineage hematopoietic maturation, without ringed sideroblasts. since mother has the same rps29 gene mutation, maternal labs were done and showed no evidence of macroytosis or anemia. the diagnosis of dba was questioned. whole exome sequencing did not identify any pathogenic sequence changes in the coding regions of rps29 gene, but detected a gata1 mutation r140w, which was reported variant of uncertain significance. his mother shares the same mutation and is asymptomatic, but she may not be affected since gata1 iis xlinked. his father doesn't harbor the gata1 mutation. conclusion: gata1 gene encodes zinc finger dna binding hematopoietic transcription factor, which is important during erythroid differentiation. gata1 mutation r140w has not been reported in literature and is a novel variant of gata1 mutation, which might be contributing to this patient's clinical picture. further studies are warranted to confirm gata1 mutation r140w to be a pathogenic sequence change. alexander boucher, tomoyuki mizuno, alexander vinks, greg tiao, stuart goldstein, james geller cincinnati children's hospital medical center, cincinnati, ohio, united states background: hepatoblastoma (hb), the most common pediatric primary hepatic malignancy, can be associated with specific congenital syndromes. recently, chronic kidney disease and genitourinary anomalies have been linked to hb. cisplatin is a key chemotherapeutic agent in treating hb but its renal clearance and toxicity profile can limit its use for those with end-stage renal disease (esrd). objectives: using an institutional case series, we present data using cisplatin for hb in dialysis-dependent esrd and define recommended dosing for future use. design/method: a chart review of patients with concurrent hb and esrd on dialysis treated with cisplatin at our institution was undertaken. demographic data, diagnostic history, tumor pathology, alpha fetoprotein (afp), hearing assessments, dosing schema, treatment outcomes, and therapyrelated toxicities were reviewed. total cisplatin levels were collected at 5 time points within 10 days after each infusion. free cisplatin levels were also collected for 2 infusions, as were dialysate cisplatin levels. pk parameters were generated using bayesian estimation with a published population pk model as a priori information. results: three patients meeting these criteria were identified. each had "low risk" (non-metastatic resectable) disease at presentation and underwent upfront resections. all had congenital renal anomalies with esrd prior to their hb diagnosis. all cisplatin infusions were given over 3 hours, followed 3 hours later by hemodialysis. patients 1 and 3 received cisplatin at 50% of children's oncology group's ahep0731 weight-based dosing (1.67 mg/kg). patient 2 received 50% of ahep0731 body surface area-based dosing (50 mg/m2) during cycle 1 but required a second dose reduction (25 mg/m2) for cycle 2 due to prolonged cisplatin exposure (total area under the curve 342 mg⋅h/l; average for all seven evaluable cycles 238 mg⋅h/l) and early sensorineural hearing loss at 2000-4000 hz. no other hearing loss in any patient was identified; mild toxicities also included grade 1-2 emesis and grade 1 neutropenia and thrombocytopenia. the median (range) of clearance, volume of distribution at steady-state, and elimination half-life at terminal phase for total platinum were 0.19 (0.15-0.31) l/hour/70 kg, 69.1 (59.0-105.7) l/70 kg and 155 (102-185) hours, respectively. patients 1 and 2 received 2 cycles with rapid afp normalization. patient 3 required an additional 2 cycles, for a likely second primary hb 1 year after initial therapy. cisplatin can be used successfully in pediatric patients with esrd on hemodialysis to treat hb with minimal morbidity using 50% standard mg/kg-based dosing (1.67 mg/kg), achieving pharmacologically appropriate cisplatin exposures. background: treatment for immune thrombocytopenia (itp) has been grouped into rescue and maintenance therapy and often is reserved for patients with bleeding, severe thrombocytopenia, or for improvement in quality of life. splenectomy is considered one of the more invasive but definitive treatments with success rates of 70-80%. treatment of itp can be more difficult in the setting of previous treatment with immune modulation or when the patient is immunocompromised and not a candidate for splenectomy. objectives: present an interesting case of a patient with an autoimmune disease that presented with severe thrombocytopenia, un-responsive to rescue therapy, and requiring emergent splenectomy in the setting of acute intracranial hemorrhage (ich). a 13 year old female with a history of juvenile dermatomyositis presented with a fine purpuric rash on her extremities, wet purpura, and a platelet count of 1k/ l. bone marrow evaluation at that time was consistent with itp. she was on cyclosporine and plaquenil for dermatomyositis. platelets failed to increase after three doses of intravenous immunoglobulin and high dose steroids. following a two week course of oral prednisone and eltrombopag, she presented with persistent severe thrombocytopenia of 1k/ l, anemia of 6.4 g/dl, and a lower gi bleed. she was started on amicar, novo-seven, rituximab, and given platelet transfusions with no improvement in bleeding. subsequently, she developed a subdural hematoma with midline shift. surgery performed an emergent open splenectomy with concurrent continuous platelet transfusion. results: she was monitored closely post operatively and, due to ich, transfused to maintain platelets greater than 100k/ l. by 1 week post-op she had normal platelet counts off transfusions. all medications were stopped within three days of discharge. she represented eight days later with abdominal pain and thrombocytosis and was found to have a portal vein, splenic vein and mesenteric vein thrombosis. she was started on lovenox therapy and admitted for monitoring due to her history of ich. it is unknown whether our patient's underlying immune dysregulation and history of treatment with immunosuppressive medications may have contributed to her unresponsiveness to multiple therapeutic agents. in addition, her significant bleeding did not allow us to fully evaluate her response to second tier therapy. this adds to the scarcity of literature of itp response in pediatric patients with autoimmune disease, and may support more aggressive therapy upfront in these patients. background: multivisceral organ transplantation involves concurrent transplantation of the stomach, pancreas, liver, and intestine with splenectomy, and has been classically used in the pediatric population for infants with intestinal failure from disorders affecting foregut integrity. while there is some data demonstrating its efficacy in adults with low-grade abdominal malignancies, it has not been traditionally used for hepatocellular carcinoma treatment. to describe a unique pediatric case of multivisceral organ transplantation as definitive therapy for refractory fibrolamellar hepatocellular carcinoma in an adolescent male. a 16 year old male presents with a history of fibrolamellar hepatocellular carcinoma, tumor invasion of the portal vein, severe portal hypertension complicated by bleeding esophageal varices and hypersplenism. he had two treatments with yttrium-90 radioembolization, without significant response. he completed six cycles of traditional chemotherapy in combination with sorafenib with resolution of petavidity, but minimal decrease in tumor size and continued portal hypertension. since his disease remained relatively stable for over 2 years, he was evaluated and listed for multivisceral organ transplantation. at approximately 2 years and 7 months after diagnosis, he underwent en bloc liver, pancreas, stomach, small bowel, and colon transplant with splenectomy. a single lymph node was positive for malignancy at the time of resection. in addition to expected post-transplant complications, he also developed skin only acute graft versus host disease at 2 weeks after transplant, treated successfully with a thymoglobulin course. he clinically improved and was back to his baseline activity level, on full oral feedings within 3 months post-transplantation. at three and six month post-transplantation, there is no concern for relapsed hepatocellular carcinoma on comprehensive imaging and evaluation. he is maintained on protocol immunosuppression and posttransplant support. we present the first known case of successful multivisceral organ transplantation in the treatment of refractory pediatric fibrolamellar hepatocellular carcinoma. background: hematohidrosis is a rare disorder that presents with spontaneous excretion of whole blood from intact skin or mucosa. diagnosis is based on clinical observation of the occurrence with the proven presence of erythrocytes and other blood components, without other abnormalities to account for the phenomenon. the existing literature is scarce and consists of primarily case studies. most reports describe bleeding from facial sites around the eyes, ears, and nose. the available literature suggests anxiety and physical or emotional stress reactions as the most common inciting events. little evidence exists regarding the ideal therapeutic approach, however propranolol has been used successfully to reduce bleeding frequency and severity in multiple case reports. a specific genetic etiology has not been elucidated, and no familial cases have previously been reported. we present a pair of half-siblings, both of whom presented with spontaneous cutaneous and mucosal bleeding before two years of age, and report on preliminary results of propranolol therapy. tanzania. at 20 months of age, he became ill and developed spontaneous bleeding from his ears, nose, and scalp. he continued to have frequent bleeding episodes, usually related to illness or physical distress. a bleeding diathesis work-up was unremarkable, however some episodes were severe enough to require transfusions. the patient was subsequently diagnosed with hiv and hepatitis b, presumably acquired via unscreened blood product transfusions. patient b is an infant female born to the same mother as patient a, with a different father. she was healthy until two months of age when she developed spontaneous bleeding from the hairline, eyelids, ears and genital/rectal area. bleeding episodes were nearly always associated with irritability and crying. extensive coagulation workup was unremarkable. results: propranolol therapy was started in both patients, titrated to a goal of 2 mg/kg/day. in both patients, the frequency and duration of bleeding episodes significantly improved. patient b continues to have milder occasional bleeding episodes from her eyes, ears and scalp but has significantly less discomfort and irritability during the episodes. conclusion: to our knowledge, there are no prior reports involving two related patients with hematohidrosis. this case series suggests that there may be a genetic predisposition which has yet to be identified. propranolol has shown effectiveness in reducing symptom frequency and severity. background: gliomas are the most common central nervous system tumors in children. they are classified into different grades based on genotype (idh, braf, tsc, etc.). lowgrade gliomas such as oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas are classified as grades i and ii. of the molecular level alterations this case report focuses on the braf v600e mutation. braf is a member of the raf family of serine/threonine protein kinases and it plays an important role in cell survival, proliferation and terminal differentiation. objectives: here we discuss two cases where dabrafenib, a braf kinase inhibitor, was utilized in the management of gliomas. the cases focus on the use of dabrafenib late versus early in disease course. design/method: patient jl is a 20 year old female who was diagnosed with a low-grade glioneuronal tumor (c7-t1 with a metastatic lesion to the brain) in 2007. jl was treated with chemotherapy, radiation, and surgical resection. despite treatment, the patient's disease progressed. she developed lower extremity dysfunction, urinary incontinence, poor truncal control, and hydrocephalus. dabrafenib was started after the braf v600e mutation was confirmed. patient lg is a 12 year old female who presented in november 2016 with left facial and upper extremity weakness. ct and mri scans demonstrated a mixed solid and cystic lesion extending from the optic chiasm and hypothalamus to the right thalamus and posterior basal ganglia with additional involvement of the right cerebral peduncle. neurosurgical intervention was undertaken and dabrafenib was started after the braf v600e mutation was confirmed. results: patient jl's mri scans have demonstrated improvement of the spine with diminished areas of enhancement along thecal margins, decreased volume and enhancement within the trigeminal plate cistern and resolution of ependymal enhancement within the right ventricle. the patient's most recent mri exhibits no disease progression in head or spine. jl has shown improvement clinically since starting dabrafenib. patient lg has shown improvement in strength and recent mri of the brain has shown resolution of enhancement along surgical resection margins, decreased hyperintensity along the inferomedial aspect of the right basal ganglia and no new enhancements. conclusion: low grade gliomas can alter a person's quality of life and even lead to life threatening complications. often the standard chemotherapy, radiation and surgery don't prevent these complications. genetic analysis can help clinicians target therapy towards certain mutations such as braf v600e. dabrafenib has shown to decrease tumor burden, early utilization as therapy can help prevent morbidity and mortality. children's hospital of pittsburgh of upmc, pittsburgh, pennsylvania, united states background: copper is an essential cofactor in enzymatic reactions essential to proper hematologic, skeletal, neurologic and vascular function. copper requirements in children over the age of 4 are 15 mg/day, which is readily acquired in a typical diet. copper deficiency is known to occur in patients with the rare x-linked mutation and in older individuals with gastrointestinal bypass surgery; however, it is rarely reported in other conditions. objectives: to highlight individuals with autism spectrum disorders or developmental delay with a limited dietary repertoire are at risk for copper deficiency, thus a high index of suspicion must exist in order to diagnose the disorder. design/method: a 15 y/o boy with a prior diagnosis of global developmental delay and oral aversion presented with slowly progressive fatigue, weakness, gait instability, and weight loss. his longstanding feeding difficulties were refractory to intensive feeding programs. his daily diet consisted of 50-60oz of milk and 25-30 individual servings of butterscotch pudding (1680-1880calories/day, 0.7mg iron/day). initial complete blood count demonstrated white blood cell count of 3.3, absolute neutrophil count of 760, hemoglobin of 4.4, mean corpuscular volume of <50, reticulocyte count of 0.5, platelet count of 392. review of his peripheral blood smear revealed microcytic, hypochromic red cells without marked fragmentation, anisopoikilocytosis and ringed sideroblasts; there were no morphologic abnormalities of his leukocytes or platelets. iron studies demonstrated ferritin of 45, total iron binding capacity of 514, and 2% iron saturation. he had no evidence of b12, folate deficiency or blood loss. additional evaluation revealed a serum copper level of 6 (range 60-190), and cerulosplasmin of 2.1 (range 22-58). results: once a diagnosis of copper deficiency was made, the patient promptly began a course of parenteral copper repletion. he received iv copper 35mcg/kg/day x 3 days then weekly intravenous infusions. given his malnutrition, a gtube was placed to begin oral copper repletion and enteral nutrition. within 3 weeks his copper level improved as well as his blood counts. unfortunately, although his blood counts and copper levels normalized, his neurologic status remains below his old baseline although, he has made gains in his gross and fine motor abilities. conclusion: acquired copper deficiency in the pediatric population is a rare event but given the hematologic and neurologic consequences, prompt recognition and treatment is important. this patient's clinical course demonstrates the need to have a high index of suspicion of concomitant nutritional deficiencies other than those routinely evaluated such as iron, b12 and folate. background: lymphoepithelioma-like thymic carcinoma (lelc) is a rare, aggressive neoplasm with a high rate of invasion, metastasis and recurrence. there are no known curative therapies for metastatic lelc. we report the case of a 16-year-old male who presented with metastatic ebv positive lelc. sites of disease included a large primary anterior mediastinal mass and metastases to hilar lymph nodes, lungs and liver. he was initially treated with cisplatin and 5fluorouracil followed by mediastinal radiation. he had a partial response to therapy but his end of therapy scans showed disease progression in lungs, liver, and hilar, supraclavicular and axillary lymph nodes. objectives: molecularly targeted therapies tailored to the patient's genetic profile offer a novel approach to obtain improved survival outcomes. design/method: the patient enrolled on a precision medicine trial, nmtrc009: molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancer (nct02162732). in this study, tumor/normal whole exome sequencing and tumor rna sequencing were performed and a molecular report detailing the results of genomic and gene expression analysis was generated. a treatment plan was designed within a molecular tumor board comprising oncologists, pharmacists, genomicists, and molecular biologists with domain expertise. results: exome sequencing revealed 26 somatic coding point mutations and no structural mutations (focal copy number changes or translocations). candidate somatic driver mutations included tp53 s94x and r248w as well as kit n655k. both genes have been previously implicated in thymic carcinoma. rna expression analysis demonstrated aberrant activation of biological pathways, including overexpression of kit, hdac1, 2 and 9, tyms, and dhfr. the molecular tumor board selected the combination of pemetrexed (500 mg/m2) on day 1 of a 21 day cycle, imatinib (400 mg daily), and vorinostat (400 mg days 1-5, 8-12, and 15-19) . on day 8 of cycle 1, he was admitted with a herpes zoster infection and imatinib was discontinued in order to reduce risk of herpes zoster recurrence. imaging after 2 cycles showed a complete metabolic response on f-18 fdg pet and a partial response by ct size criteria. as of december 2017, the patient had received 15 cycles of pemetrexed and vorinostat. scans in december 2017 showed an increase in the size and metabolic activity of two right lower lobe pulmonary nodules. there were no new sites of disease and imatinib was re-started. background: systemic lupus erythematosus (sle) is a chronic autoimmune disease that affects multiple organ systems and is associated with many different autoantibodies. patients can present with vague constitutional symptoms including fever, rash, fatigue, and weight loss. some of the various hematologic manifestations of sle include anemia of chronic disease, leukopenia, autoimmune hemolytic anemia (aiha), and idiopathic thrombocytopenic purpura (itp). these can be the presenting signs of sle. evans syndrome (es), a disease characterized by itp and aiha, is a rare hematologic manifestation of sle. neurofibromatosis 1 (nf1) is a relatively common neurocutaneous disorder. these patients are at risk of developing benign and malignant tumors. its association with autoimmune disorders, including sle, remains rare. objectives: there are few cases in the literature that have patients with the combination of sle and nf1. this is the only case that has a patient with sle, nf1, and es. results: a 16-year-old caucasian female presents with two months of vaginal bleeding, weight loss and petechiae. her exam is remarkable for petechiae and café au lait macules. laboratory findings show severe anemia and thrombocytopenia. she receives blood and platelet transfusions during stabilization, and a bone marrow aspirate is performed to rule out a malignancy which is negative. based on the presence of thrombocytopenia and a positive coombs test, an autoimmune process such as es is considered. screening tests for sle reveal positive antinuclear and anti-double stranded dna antibodies as well as low complement. she receives intravenous immunoglobulin and methylprednisolone and eventually her vaginal bleeding slows and her counts recover. she begins sle therapy with hydroxychloroquine and azathioprine. due to the presence of café au lait macules on her exam, a genetics evaluation is performed and the patient is also diagnosed with nf1. to date, there are seven cases of sle with nf1 reported in the literature, only two of which are pediatric cases. there are no reports of the combination of sle, nf1, and es. conclusion: es is a rare hematologic manifestation of sle but can be the initial presentation of this disease. one large study estimates 2% of childhood-onset sle cases are observed to have es. screening for sle should be considered in all es patients even in the absence of typical clinical findings. association of nf1 and sle has been rarely described. whether this association reflects a causal relationship or is coincidental needs more investigation. (lube, ped blood & cancer, 2016) . university of california san diego, la jolla, california, united states background: high grade glioma (hgg) has poor outcomes in adults and children. extraneural metastases are very rare in hgg, and poorly characterized with only a few small case series in adults and only isolated case reports in pediatrics. no genomic data has previously been published for any children with hgg who develop extraneural metastases. objectives: our objective is to describe the natural history of two children with hgg and bony extraneural metastases, comparing their clinical characteristics as well as whole exome sequencing data for both tumors. this information would suggest similar patients should be monitored closely for extraneural metastasis and may benefit from more systemic therapy. design/method: we present a case series of two patients who presented with hgg and had development of bony metastases less than six months after initial diagnosis. both patients had molecular profiling with whole exome sequencing (wes). the first patient was an 11-year-old male with a tumor found in the left lateral ventricle invading into the fornices, hypothalamus, and left midbrain, who had subtotal resection. bony metastasis were found at 3.5 months after diagnosis, and he died 9 months after diagnosis. he initially received radiation, followed by nivolimuab. the second was a 12-year-old female with a tectal/pineal tumor and multiple spinal cord metastases, who had subtotal resection. she developed bony metastasis at 5.5 months after diagnosis and died 13 months after diagnosis. her histologic diagnosis was pineoblastoma, revised to hgg after whole exome sequencing. she received craniospinal radiation followed by chemotherapy per acns0332 (cisplatin, vincristine, and cyclophosphamide) for 2 cycles. when she failed to respond satisfactorily to this therapy, wes of tumor was performed and the findings were consistent with hgg. treatment was transitioned to temodar and lomustine after hgg diagnosis was given. she had ongoing progressive disease despite this therapy as well as trials of nivolumab, everolimus, and vorinostat. neither patient had extraneural metastasis at presentation. in both tumors, whole exome sequencing identified the h3f3a k27m mutation. both tumors also had additional known mutations associated with hgg but no other overlapping mutations. this case series represents the first description of the genetic alterations of pediatric hgg patients who developed extraneural metastases. while h3f3a k27m is a common mutation in pediatric midline hgg, especially dipg, and is associated with more aggressive disease, there has not been an association with extraneural metastasis prior to this series. background: deferiprone-induced agranulocytosis is a well -known albeit rare side effect of the drug. incidence of agranulocytosis varies from 0.5-3.6%, while milder neutropenia is reported in 8.5% of patients treated with deferiprone. deferasirox is unknown to cause such a complication. clinical trials and post marketing side effect monitoring studied possible correlations between different risk factors and development of agranulocytosis. unfortunately, no studies directly addressed a special risk in a community with background of ethnic neutropenia, like oman. objectives: to report on the incidence of neutropenia among omani children with b thalassemia using different iron chelators design/method: a retrospective study conducted on patients < 21 year-old with b thalassemia treated with different iron chelators. electronic patients records were reviewed to detect episodes of neutropenia either mild (anc 1.0-<1.5/cmm), moderate (anc 0.5-<1), severe < 0.5, or agranulocytosis anc = 0). data were collected including sex, age, personal or family history of ethnic neutropenia, iron chelating agent, infective complications, management and outcome. detailed clinical, laboratory ± radiological information were reported for patients who developed life-threatening agranulocytosis. among 179 young patients with b thalassemia, treated between 2007-2017 in squh, neutropenia, was reported in 78 patients (43.6%).severe neutropenia was encountered on 14 occasions in 11 patients (11/179: 6.1%) (8 on deferiprone including 5 episodes of agranulocytosis, 1 on defersirox, 1 on combined chelation, and 5 off chelation). moderate neutropenia was encountered in 29 patients (29/179: 16.2%), on 36 occasions: deferiprone (15), deferasirox (8), combined chelation (4), and 9 episodes off chelation. mild neutropenia was more prevalent, encountered in 59 patients (32.9%) on 124 occasions (30 on deferiprone, 44 on defersirox, 19 on combined chelation, and 31 off chelation) of 85 patients exposed to deferiprone, 35 patients had neutropenia (41%), higher than previously reported. deferiproneinduced agranulocytosis was encountered in 4 patients (4/85 = 4.7%). three of them had life threatening complications. one patient developed pneumonia complicated by rupture of pulmonary artery aneurysm-massive hemoptysis, who recovered fully after catheter embolization. the second had facial cellulitis and treatment with gcsf was complicated by frequent ventricular extrasystoles. the third had sepsis, disseminated herpes simplex and required admission to icu for inotropic support. in a community with background ethnic neutropenia, neutropenia is more common to be encountered among thalassemic patients, both on and off chelation therapy. careful monitoring of anc and rational choice/modification of chelating agents is required for optimal management of iron overload and to avoid life threatening complications. objectives: this case control study aimed to evaluate the systolic and diastolic cardiac function in 2 groups of children with ti: non transfused group and a group that received early regular blood transfusion comparing them to healthy controls. design/method: thirteen regularly transfused patients with ti with a mean age of 11.8+5.6 years were compared with eight patients who are non-transfused or minimally transfused (< 3 rbcs transfusion/year); mean age 11.8+9.4 years and 18 healthy controls with a mean age of 8.8 ± 3.9 years. clinical parameters and standard echocardiographic and tissue doppler imaging (tdi) were compared. results: young non-transfused ti patients had a statistically significant higher peak late diastolic velocity of the left ventricular inflow doppler, a mitral valve a wave duration over the pulmonary vein a wave duration ratio and the pulmonary s81 of s301 vein s/d velocities ratio compared to the transfused group with p values of 0.028, 0.01, 0.01 respectively. in addition, they have a lower e/a ratio of the mitral valve inflow and a larger left atrial to aortic diameter ratio compared to the control group with p values of 0.025 and 0.01 respectively. the diameters of the right and left outflow tract were significantly larger in the non transfused group with a trend to have a higher cardiac index compare to the transfused group. systolic function was similar in the 3 studied groups and none of the patients had evidence of pulmonary hypertension. young patients with ti who are receiving early regular blood transfusion have normal systolic function. diastolic function assessment revealed indicators of an abnormal relaxation of the left ventricle in the non transfused group which indicate diastolic dysfunction. the abnormalities affected multiple diastolic function parameters which give an indication that the changes are clinically significant. a statistically significant increase in the diameters of the outflow tracts are likely attributed to high cardiac output status in nontransfused ti patients as they had a trend to have a higher cardiac index. these findings support the early commencing of regular blood transfusion therapy for ti patients to prevent serious cardiac complications in adult life. background: in the 52-week sustain study, crizanlizumab 5.0 mg/kg significantly reduced the frequency of scpcs versus placebo (1.6 vs 3.0, p = 0.01) and increased the time to first on-treatment scpc (4.1 vs 1.4 months, p = 0.001) in patients with sickle cell disease (scd). to evaluate time to first scpc in sustain study subgroups and the likelihood of not experiencing scpc for the duration of the trial using post hoc analyses. design/method: sustain was a randomized, double-blind, placebo-controlled, phase 2 study (nct01895361). inclusion criteria were: scd patients aged 16-65 years; 2-10 scpcs in previous 12 months; concomitant hydroxyurea use permitted if ≥6 months and stable dose for ≥3 months. patients were randomized 1:1:1 to receive intravenous crizanlizumab 5.0 mg/kg, 2.5 mg/kg, or placebo. study treatments were administered on days 1 and 15, then every 4 weeks to week 50, with the final assessment at week 52. median time to first scpc after first dose was summarized for crizanlizumab 5.0 mg/kg or placebo in these subgroups: 2-4 or 5-10 scpcs in previous 12 months; scd genotype; and hydroxyurea use at baseline. hazard ratios (hrs) for crizanlizumab 5.0 mg/kg versus placebo were calculated based on cox regression analysis, with treatment as a covariate. descriptive statistics were used to summarize the frequency of patients who were scpc event-free for the duration of the study by prior scpc events, scd genotype, and hydroxyurea use at baseline. : 67 patients received crizanlizumab 5.0 mg/kg and 65 received placebo. there was a meaningful delay in time to first scpc with crizanlizumab 5.0 mg/kg versus placebo observed in the entire study population. the effect was present in both scpc subgroups, and the largest treatment difference was observed in hbss scd versus other genotypes (4.1 vs 1.1 months; hr: 0.50). in patients taking hydroxyurea who experienced 2-10 scpcs in the previous year, time to first onstudy scpc was longer with crizanlizumab 5.0 mg/kg versus placebo (2.4 vs 1.2 months; hr: 0.58). a greater proportion of patients treated with crizanlizumab 5.0 mg/kg were scpc event-free versus placebo in each of the analyzed subgroups. one third of patients who were taking hydroxyurea and treated with crizanlizumab 5.0 mg/kg were scpc event-free during the study versus 17.5% with placebo, possibly suggesting an additive effect. with crizanlizumab 5.0 mg/kg, there was a clinically meaningful delay in time to first scpc and an increased likelihood of being scpc-free versus placebo in all subgroups investigated. cincinnati children's hospital medical center, cincinnati, ohio, united states background: shwachman-diamond syndrome (sds) is an inherited marrow failure syndrome associated with increased risk of myelodysplasia (mds) and acute myeloid leukemia (aml). objectives: this multi-institutional retrospective study investigated clinical features, treatment, and outcomes of 38 sds patients who developed mds or aml by central pathology review. design/method: nine individuals presented with aml (4 male, 5 female), 5 mds-eb1/2 (3 males, 2 females, 23 with mds (11 male and 12 female), and one male with isolated persistent somatic tp53 mutation. one mds-eb1 and 1 mds patient progressed to aml. median age (years) at diagnosis of mds was 16 (range 0.5-30), mds-eb1/2 was 9 (range 0.7-20) and aml was 28.8 (range 5.5-47). complex cytogenetics were noted in 10/11 aml cases, with one having normal cytogenetics. complex clonal cytogenetic abnormalities were noted in 4 of 5 mds-eb1/eb2 patients and clonal abnormalities in 17 of 18 mds patients. follow up was available for 10 aml patients; 9 are deceased. 9 received chemotherapy with intent to proceed to hematopoietic stem cell transplant (hsct). four failed to achieve remission and died with disease without proceeding to transplant. one patient proceeded to hsct without prior chemotherapy. four of six transplanted subjects died with relapsed disease. treatment related mortality was largely infectious or gvhd. the sole surviving aml patient had normal cytogenetics, achieved remission with chemotherapy and underwent hscts with 3 separate stem cell infusions due to two primary graft failures. he remains alive in remission more than 4 years after diagnosis. of the 5 mds-eb1/2 patients, 4 underwent ric hsct, three of whom are alive, one died of infection. the fifth patient has stable disease on continued decitabine monotherapy for 4.75 years. of 19 mds patients with treatment data, 13 had upfront hsct therapy, 2 upfront chemotherapy and 4 had no therapy. three patients required ≥2 hscts all due to graft failure. follow up is available for 18, 11 of whom are deceased, 6 with relapsed disease. treatment related mortality was largely infectious or graft failure. one individual died of hepatic failure unrelated to mds. seven mds patients are alive in remission. in summary, prognosis is poor for patients with sds who develop aml due to resistant disease and treatment-related complications. better markers for risk stratification are needed to identify patients who would benefit from early transplant. novel therapeutic strategies are urgently needed to improve outcomes of sds patients with mds or aml. background: unlike primary myelofibrosis (pmf) in adults, which is associated with somatic mutations in jak2, mpl, or calr, myelofibrosis in children is rare and the underlying genetic mechanisms remain elusive. here we describe 3 families with autosomal recessive congenital macrothrombocytopenia with focal myelofibrosis (cmtfm) due to germline mutations in the megakaryocyte-specific immune receptor tyrosine-based inhibitory motif (itim) receptor g6b-b. objectives: to characterize the clinical phenotype, histological features and identify the causative gene for cmtfm. we performed affymetrix snp 6.0 genotyping on the index family to identify shared regions of homozygosity by descent. whole exome sequencing (ws) was performed on all three pedigrees to identify potentially causative mutations. we studied 6 affected children from 3 families, with macrothrombocytopenia, anemia, mild leukocytosis and a distinctive pattern of bone marrow (bm) fibrosis centered around clusters of atypical megakaryocytes. affected children had mild to moderate bleeding symptoms and required platelet and red cell transfusions. none showed evidence of extramedullary hematopoiesis, and all were negative for mutations in jak2, mpl, and calr. snp genotyping identified multiple statistically non-significant genomic loci, including the region of the major histocompatibility locus (mhc) on chromosome 6p (lod = 2.01). we focused on this region because affected individuals in two families shared a common homozygous human leukocyte antigen (hla) type and had congenital adrenal hyperplasia (cah) due to 21-hydroxylase (cyp21a2) mutation; the cyp21a2 and hla loci are located at 6p21.33 and 6p21.32-6p22.1. wes revealed homozygous frameshift mutations in the megakaryocyte and platelet inhibitory receptor g6b-b, encoded within the candidate linkage region. we identified two distinct g6b-b frameshift mutations (c.61_61+1dup; p.20fs and c.147inst; p.49fs) in 7 individuals within these three families. no other mutations that segregated with the phenotype were identified. to validate g6b-b as a potential disease-causing gene, we evaluated g6b-b expression in bm biopsy specimens from affected patient and control samples by immunohistochemical staining using a monoclonal antibody. g6b-b was strongly s83 of s301 and selectively expressed in megakaryocytes of control samples, but completely absent in clinically affected individuals. a murine knockout that lacks g6b-b has a strikingly similar phenotype with macrothrombocytopenia, myelofibrosis and aberrant platelet production and function, further affirming the causality of g6b-b mutations. we showed that autosomal recessive loss-offunction mutations in g6b-b cause cmtfm, uncovering the molecular basis of this rare disease. loss of g6b-b-dependent inhibition of megakaryocyte activation likely underlies the distinctive focal myelofibrotic phenotype and might be important in other forms of marrow fibrosis. cardinal glennon, saint louis, missouri, united states background: intrauterine transfusion is the method of choice for management of fetal anemia due to red blood cell alloimmunization. despite the decrease in prevalence of anemia due to rhesus d alloimmunization with prophylactic administration of anti-rhd immunoglobulin in rh d negative patients, maternal red red blood cell alloimmunization with other type of red blood cell antigens remains an important cause of fetal anemia. newborn who received intrauterine transfusion for hemolytic disease may have prolonged postnatal transfusion requirement. objectives: 1-to evaluate clinical outcome of fetuses and newborns who received intrauterine transfusions. 2-to determine the need of packed red blood cell transfusions until 6 months of age. we conducted a retrospective case series study of all intrauterine transfusions due to anemia secondary to red blood cell alloimmunization performed in our regional center ssm in st louis missouri, between april 2011 and january 2016. we evaluated the indications, diagnosis, gestational age, and frequency of intrauterine transfusions, along with the infant's gestational age at birth, duration of admission, timing of blood transfusion and monitoring of hemoglobin. results: 37 intrauterine transfusions were performed in 14 patients. the most common causes of alloimmunization were due to d antibodies (n = 7, 50%) and kell antibodies (n = 5, 35.7%). the median gestational age of the first intrauterine transfusion was 28.1 weeks, and the median pre-transfusion hemoglobin was 8.9 g/dl. the gestational age at the first intrauterine transfusions was found to be significantly correlated with the number of postnatal transfusions (r = 0.8. p = 0.001). the median gestational age at birth was found to be 35 weeks (28.6-36.9 weeks), with a hemoglobin of 13.1 (10.8-14.1). in our population, 6 patients (42%) received postnatal transfusions, of which 4 were during the first 3 weeks of life, and close monitoring follow up with a hematologist was established in 6 patients at their discharge from the nursery/nicu. one neonatal death occurred and severe morbidity due to severe anemia occurred in one infant. despite the continuing risk factor for persistent anemia, only 8 patients had follow up hemoglobin monitored by their primary care provider. conclusion: infants with anemia due to red blood cell alloimmunization treated with intrauterine transfusion should be monitored closely via regular complete blood count for persistent anemia due to suppression of fetal erythropoiesis. sebastian hesse, piotr grabowski, juri rappsilber, christoph klein dr. von hauner childen's hospital, lmu university hospital, munich, munich, germany background: neutrophil granulocytes are the most abundant leukocytes in the peripheral blood. validated diagnostic options for these cells are limited, leaving many patients with functional neutrophil defects without a defined diagnosis. objectives: here we evaluate proteomics as a new diagnostic tool to investigate defects of neutrophil granulocytes. we analyzed neutrophil granulocytes from 6 children with severe congenital neutropenia (scn) associated with elane mutations, 4 children with chronic granulomatous disease (cgd) with cyba (2) or cybb (2) mutations and 2 children with leukocyte adhesion deficiency (lad) due to itgb2 mutations. in addition we collected samples of children with genetically undetermined neutrophil defects. neutrophils from 68 healthy individuals served as controls. cells were isolated from fresh venous blood using negative selection (purity >99%). whole cell proteome analysis was done by data-independent acquisition. showed a correlation coefficient of ∼0.9. principal component analysis demonstrated unequivocal separation of the proteome of healthy and diseased cells. differential expression analysis showed minimal proteome aberrations in lad with deficiency in cell surface receptors and upregulation of alpl (total downregulated proteins: 7/ total upregulated proteins: 5). analysis of neutrophils from cgd patients also showed limited proteome aberration. cyba and cybb were both diminished independent of genotype, whereas protein clusters around a stat1/2 centered network were increased (total down: 11/ up: 23). neutrophils with elane mutations showed the gravest proteome disturbance (total down: 47/ up: 93) with an upregulated translational apparatus (srpdependent ribosomes and protein folding complexes) and increased mitochondrial proteins. proteins of each granule subset were dysregulated and metabolic pathways upregulated. a detailed analysis of the proteome from patients with genetically undefined diseases is currently ongoing. one patient with clinical phenotype of cgd was found to have no mutations of nadph oxidase members in whole exome sequencing but critically low levels of ncf1 on protein level. heterozygosity mapping showed autozytocity in the ncf1 region warranting current efforts to sequence promoters and intronic regions of the gene. mass spectrometry based proteomics promises exciting new insights into monogenic disease of neutrophil granulocytes and may offer new diagnostic options, in particular in synergy with genome sequencing. by virtue of our international care-for-rare alliance, open to new partners, we hope that our proteome focus may lead to better delineation of as yet unknown disease of neutrophil granulocytes. background: warm autoimmune hemolytic anemia (aiha) is an igg mediated disease. although it can be post-viral, it is often idiopathic and can also be a forme fruste for malignancy or an autoimmune disease. initial management includes steroids. it often relapses on steroid wean and can be refractory to the use of second line treatment such as rituximab. objectives: abatacept (ctla-4-ig fusion protein, ctla-4 mimetic) has been used to ameliorate autoimmune manifestation associated with ctla-4 haploinsufficiency. we used abatacept as a novel therapeutic agent to manage patients with refractory aiha. design/method: a retrospective case series of two patients at phoenix children's hospital with severe refractory aiha. results: patient 1, a previously healthy 12 year old female, presented with 8 weeks of icterus, fatigue, and hemoglobinuria. spleen was enlarged 8cm below the costal margin. laboratory evaluation demonstrated: hemoglobin 3.8 g/dl, mild leukopenia 3200/microliter, platelets 99,000/microliter, reticulocytosis 14.3%, positive direct coombs' test, mycoplasma igm and igg positive. bone marrow evaluation showed a hypercellular marrow. she continued to need packed red blood cell (prbc) transfusions despite receiving high dose steroids, ivig and rituximab from may-july 2017. in august, she started sirolimus decreasing her transfusion requirement. after starting abatacept (10mg/kg/dose bi-monthly for three doses and then monthly) in october, she maintained hemoglobin of 9-10 g/dl without transfusion. patient 2, a previously healthy 2 month old male, presented with one week of progressive fatigue, jaundice, and poor feeding. splenomegaly was absent. laboratory evaluation revealed hemoglobin 3.8g/dl, leukocytosis 20,200/microliter, platelets 324,000/microliter, reticulocytosis 16.1%, negative direct coombs' test, and non-specific reactivity on antibody screen. evaluation for inherited hemolytic anemia including a next generation sequencing panel was negative. further evaluation by blood bank showed 2+ positive coombs' for c3d due to a warm antibody. cold agglutinin disease was ruled out. bone marrow evaluation was normal. he received high dose ivig as a steroid sparing agent but continued to require prbc transfusions weekly. when prednisone did not seem to slow down hemolysis, treatment with abatacept was initiated and he has not required transfusions for two months. steroids are being weaned. we present successful treatment of two refractory aiha cases with abatacept. patient 1 is steroid and transfusion free and continues on monthly abatacept and sirolimus. patient 2 is also transfusion free and continues on a steroid taper. ctla-4 is crucial for suppressive function of treg cells. abatacept by binding to cd80/86 seems to enhance treg activity ameliorating autoimmune hemolysis. children's minnesota, minneapolis, minnesota, united states s85 of s301 background: transfusional iron overload is common in patients receiving chronic red cell transfusions. as a result, iron chelation is required to minimize toxicity from iron overload. chelation with a single agent can be inadequate at controlling or reducing iron burden. when combination therapy is required deferoxamine may be added to oral chelation. deferoxamine is generally given subcutaneous over 8-12 hours for 5-6 days a week at 25-60mg/kg/day. many patients struggle to remain compliant with this schedule which has prompted trials of intravenous high-dose (hd) deferoxamine. prior reports of short-term hd deferoxamine have shown minimal side effects however, prolonged use of hd deferoxamine has known toxicity. when compliance is a concern, our center has used hd deferoxamine infusions at 15mg/kg/hr x 48hours every 2 to 4 weeks. objectives: evaluate the safety and efficacy of hd deferoxamine at our institution to help guide future therapy. design/method: a retrospective review was completed of patients previously treated with hd deferoxamine between april 2011 and september 2017 at children's minnesota. final sample included 8 patients ages 3 to 14 years with underlying diagnosis of thalassemia (7) and diamond-blackfan anemia (1). deferoxamine infusions were given for 48 hours every 24-35 days with a mean length of treatment of 279 days. results: all patients were on combination therapy with deferasirox, however deferasirox was held during deferoxamine infusion. mean pre-deferoxamine liver iron concentration (lic) was 31.75mg/g and mean post lic was 12.11mg/g (p = 0.0008). ferritin mean pre-deferoxamine was 2677ng/ml compared with mean post 1594ng/ml (p = 0.0107). two patients had possible allergy, leading to deferoxamine discontinuation. one patient developed hives, eye swelling and cough while the other had emesis and cough. another patient experienced facial nerve palsy of unclear etiology, which did not recur with resumption of deferoxamine. no respiratory complications were seen. results showed significant decrease in iron burden following combination therapy with high dose deferoxamine and deferasirox. no significant pulmonary, liver, renal, vision, or hearing toxicities were observed. three patients reported reactions to deferoxamine infusions. however, one of these was able to successfully continue deferoxamine without further incident. short-term, hd deferoxamine was effective at reducing lic in combination with oral chelation but requires further evaluation to assess for potential increased risk of toxicity. short-term hd deferoxamine may be considered in the setting of poor compliance of subcutaneous administration or inadequate chelation with single agent therapy. further studies are needed to clarify ideal dosing, timing and risk of toxicity. background: immune thrombocytopenia (itp) is the most common cause of symptomatic thrombocytopenia in childhood but remains a diagnosis of exclusion warranting further evaluation if atypical findings are present. two male children (15 months and 3 years old) with newly diagnosed immune thrombocytopenia (itp) were found on initial evaluation to have persistent elevations of lactate dehydrogenase (ldh), alanine aminotransferase (alt), and aspartate aminotransferase (ast). these serum enzyme abnormalities cannot be attributed to itp. in the setting of thrombocytopenia, elevated transaminases and ldh create diagnostic complexity for the hematology/oncology provider as their elevation raises concern for malignancy, hemolytic disease, and other systemic diseases. to raise awareness about an unexpected pattern of duchenne muscular dystrophy in patients undergoing evaluation for itp. to expand the differential of a hematologist/oncologist when abnormal labs support a nonhematologic diagnosis design/method: this case-series of two patients with their clinical and laboratory findings were discovered with retrospective chart review. results: after a thorough evaluation for hemolytic anemias, liver disease and infectious etiologies was negative, bone marrow and liver biopsies were considered. eventually, both children were found to have severely elevated serum creatine kinase (ck). skeletal muscle has the highest concentration of ck of any tissue. thus, significant ck elevation is almost exclusively attributable to muscle injury and is the most sensitive and specific enzyme for diagnosis of muscle disease. referral to a neuromuscular specialist and further genetic testing confirmed the diagnosis of duchenne muscular dystrophy in both children allowing initiation of appropriate interventions. to date, there is no clear genetic predisposition to itp in patients with muscular dystrophy although further investigation may be needed. hematology/oncology providers should consider obtaining a serum ck to rule out muscle disease in any male child with unexplained elevations of serum ldh and/or aminotransferases, as it provides an easy and inexpensive, non-invasive approach to screening. additionally, clinical history and physical examination can aid in the diagnosis of muscular dystrophy, with gross motor delay, abnormal muscle bulk, gower's sign, and proximal muscle weakness all possible findings. objectives: to identify the range of cbcs in patients with ds without infections, hematologic or immune disorders and to create more accurate reference ranges for total white blood count; hemoglobin; hematocrit; mcv; platelet count and absolute neutrophils (anc), lymphocytes, monocytes, eosinophils, and basophils. design/method: a retrospective investigation of healthy pediatric patients with ds who received a cbc between 2011 and 2017 as part of their medical care at a single, large, pediatric teaching hospital. the study group consisted of 562 children with ds (male = 310, 55.2%; mean age = 2.10 years, sd = 3.17) at time of blood draw. initially 692 children were reviewed for possible participation in the study; however, 130 patients were excluded due to not meeting the study's inclusion criteria. descriptive statistics were performed on demographic and clinical characteristics. kruskal-wallis h tests, anova, and t-tests were run to determine the significant associations between independent means. results: a significant difference in absolute neutrophils between racial groups, f(2, 183.553) = 3.990, p = 0.020, was observed. there was an increase in anc from 2.5 +/-1.1 with african americans to 3.0 +/-1.6 in the other racial groups and to 2.9 +/-2.0 with caucasians. differences were also found in anc in hispanics/latinos versus non-hispanic/latinos. the results were higher in non-hispanics and latinos, a significant difference of -.584 (95% ci, -.791 to -.376), t(1255) = 2.572, p = 0.001. preliminary kruskal-wallis h tests run determined that there were significant differences between age groups for total white blood cell, hemoglobin, hematocrit, platelets, lymphocytes and anc. further studies are being run to evaluate in which age groups these differences lie and create reference ranges by age, race and sex. conclusion: among patients with ds, there are differences between racial groups and age groups. this data has been compared to previously established reference ranges for cbcs, but we are currently establishing healthy cbc controls which we will use to validate the reference ranges. these ranges will be published to help guide providers in workup and management of patients with ds. background: transfusion is a critical part of the care provided in the neonatal intensive care unit, but it is not without risks. low birth weight and premature infants can become anaemic from an immature haematopoietic system and frequent phlebotomy. these infants often receive multiple red blood cell transfusions. identifying infants more likely to require such intervention is important in ensuring the appropriate usage of this scarce resource. to determine whether birth weight, gestational age, gender, length of stay and mode of delivery can predict red cell concentrate (rcc) transfusion, units required, donor exposure and time to exposure. design/method: a retrospective chart review of all infants born below 32 weeks gestation and/or birth weight less than 1,500g who received a red blood cell transfusion between july 2009 and july 2012 in the cork university maternity hospital neonatal unit. results: 224 infants met the inclusion criteria, 105 (46.87%) received a rcc transfusion. our study showed lower gestational age (p<0.01) and lower birth weight (p<0.01) infants are more likely to be transfused. donor exposure increases with a lower birth weight (p = 0.016). multivariate analysis showed infants with a lower gestational age (or -0.019 per day; p<0.05); lower birth weight (or -0.002 per 1g; p<0.01) and a longer length of stay (or 0.016 per day; p<0.05) are more likely to receive a higher number of rcc transfusions. the time to first rcc transfusion is shorter in those with lower birth weight (or 0.013 per 1g; p<0.05) and lower gestational age (or 0.356 per day; p<0.01). gender and mode of delivery were not found to be predictors of red blood cell transfusion in this study. conclusion: low birth weight and premature infants are more likely to receive a rcc transfusion during admission to the neonatal unit. our study highlights predictors of rcc transfusion, donor exposure and time to transfusion. these can be used in identifying at risk infants, counselling parents and in anticipating transfusion requirements. emily southard, r. grant rowe, david williams, akiko shimamura, taizo nakano children's hospital colorado, aurora, colorado, united states background: the mecom locus encodes transcription factors that regulate hematopoietic stem cell self-renewal and maintenance. overexpression of mecom has been noted in 5-10% of acute myeloid leukemia, several solid tumors, and denotes a poor prognosis. mutations that reduce mecom expression or that disrupt protein function, however, have been implicated in the development of bone marrow failure (bmf) through undefined pathways. an association between mecom mutations and radioulnar synostosis with amegakaryocytic thrombocytopenia (rusat) syndrome has been reported, however further characterization of this phenotype has yet to be explored. to characterize the phenotypic spectrum of a cohort of pediatric patients with novel mecom mutations. we performed a retrospective review of five patients with mecom mutations who were referred to hematology at children's hospital colorado or boston children's hospital. clinical, laboratory, and genetic data was collected on subjects and available family members. results: four of 5 subjects were identified in infancy presenting with congenital cytopenias or physical dysmorphisms that prompted broad genetic screening. platforms for genetic detection included microarray, targeted genetic panels, and whole exome sequencing. three of 4 subjects with cytopenias presented with congenital thrombocytopenia, 1 of whom rapidly progressed to severe aplastic anemia. four of 5 subjects presented with congenital anomalies, 3 of whom demonstrated radioulnar synostosis. additional dysmorphic features identified include craniofacial (low set ears x1), cardiac (pda x1, vsd x1, aortic root dilation x1), pulmonary (pulmonary hypertension x1, arteriovenous malformations x1), and developmental delay. one subject presented at age 13 years with acute pancytopenia, hypocellular marrow, no dysmorphisms, and a mecom variant of unknown signif-icance. the identified mecom mutations include one 4.1 mb deletion involving several genes including mecom, one variant affecting a splice acceptor consensus sequence predicted to disrupt splicing, and three novel missense mutations, tyr949cys, arg950thr, and tyr1118cys, all of which were absent from public databases and were predicted in silico to be deleterious. we describe the phenotypic spectrum of 5 patients with novel mecom variants. a subset of patients lacked radio-ulnar synostosis and had presence of additional systemic anomalies, demonstrating a varied clinical phenotype that is not isolated to rusat syndrome. a centralized publically accessible database to share clinically annotated mecom variants, together with analysis by experts in mecom function would advance our understanding of the clinical interpretation of mecom variants. mecom should be considered in the differential diagnosis of bone marrow failure and we advocate for the inclusion of mecom in targeted sequencing panels. cairo university, cairo, egypt background: beta thalassemia is regarded as a serious public health problem in the mediterranean region, southeast asia, and the middle east. however, very few studies have been conducted to assess the quality of life (qol) among thalassemia major patients. objectives: to assess the quality of life among b-thalassemia major patients using short form (sf)-36 questionnaire and to determine the factors associated with their quality of life. design/method: a cross-sectional study was conducted among thalassemia major patients who were attending the hematology outpatient clinic at cairo university hospital, during the study period. data were collected between october 2016 and march 2017. the quality of life was assessed for patients aged ≥17 years. the mean age of the studied group was 18.32± 1.33 years. the majority (93.63%) had one monthly blood transfusion. the mean total score of sf-36 was 44.90±7.54. general health perception domain was the most affected domain with mean score, while vitality was the least affected one. there was no statistically significant difference between males and females regarding different quality domains except for vitality where the mean score was significantly higher in males than females (p = 0.05). age at onset of disease, and at first blood transfusion were the most documented factors positively correlated with the quality of life among the enrolled thalassemia patients. conclusion: the quality of life in thalassemia major patient was found to be compromised. all thalassemia patients should undergo assessment of the quality of life so that interventions focusing on the affected domains can be implemented. background: international adoption of children with special needs has become more prevalent in recent years leading to tremendous growth in the number of u.s. thalassemia patients adopted from foreign countries. currently 13% of the 1,119 thalassemia patients registered in the cooley's anemia foundation (caf) patient database have been adopted from foreign countries, primarily china. as this population continues to grow, further information is needed in order to provide these families with best supportive care. the primary goal of this study is to characterize the socio-demographics and health statuses of adopted children with thalassemia and their families. a secondary goal is to describe adoptive families' motivations, experiences, challenges, and support resources. design/method: a redcap survey was accessed by families of adopted children with thalassemia through the caf website and caf social media from january to august 2017. following a four-question screen, eligible subjects were directed to complete an adoption questionnaire. families who had at least one adopted child with thalassemia receiving care at a participating thalassemia treatment center or hematology office in the u.s. were considered eligible. descriptive statistics were analyzed using sas 9.4. respondents who were ineligible or who provided incomplete data were removed from the dataset prior to analysis. of 78 survey respondents, 67 qualified and completed the survey. these households had adopted a total of 74 children with thalassemia (33.8% male), most from china (93.2%), where they had been living in orphanages (73.0%). legal guardians identified primarily as christian (87.3%). the majority had completed post-secondary education (76.9%) with reported household incomes greater than $80,000 (76.1%). most adoptive families were connected to an adoption group or community including online groups, local support groups, and adoption networks (98.5%). commonly cited challenges were: 1) volume of frequent medical appointments, 2) insufficient support from their local care centers, and 3) financial burdens. the reality of care for the population of adopted patients with thalassemia in the u.s does not seem to match the expectations set by their providers. we are hopeful this data will be used to assist adoptive families navigating the complexities of thalassemia care. the findings suggest that this population would benefit from additional outreach, education, guidance, and advocacy resources -especially in the early stages of adoption and during initiation of post-adoption medical care. background: in many higher-income countries, thalassemia major has become a chronic disorder; many outcomes are different in emerging countries with more limited resources. most analyzes of health-related quality of life (qofl) in thalassemia have been conducted in high-income settings. objectives: to assess the impact of health status on qofl in thalassemia patients in an emerging country. we assessed qofl in 110 randomly-selected patients (72 thalassemia major; 33 with hemoglobin e thalassemia; five thalassemia intermedia) at the national thalassemia center in kurunegala, sri lanka where approximately 800 patients are managed. treatment is free, but compared to north america/europe, access to tertiary staff and other resources are limited. overall, control of body iron as estimated by serum ferritin concentration (mean± sem, 2906±267 g/l) was not optimal in many patients. to understand the impact of health status on qofl, we used the sf36v2 health survey, analyzing scores of physical function, pain, general health, social functioning, emotional and mental health, to generate overall physical and mental component scores. results: compared to reports from higher-income countries (american journal of hematology 2011; 86:92-5), physical function scores (mean±sd, 48.16±10.72) were similar in sri lankan patients; indeed, in three categories (physical role, social function, emotional role), sri lankan scores were slightly higher. by contrast, compared to scores from higherincome settings, those estimating bodily pain, general health, and mental health were significantly lower, resulting overall in a significantly lower physical component score in sri lankan patients. male sri lankan patients reported higher scores than females, and somewhat surprisingly, in four categories (physical function, physical role, social function and emotional role) reported higher scores than those obtained in higher-income settings. lower scores in physical functioning, leading to an overall lower physical component score, were recorded by females. patients with hemoglobin e thalassemia reported generally poorer qofl than those with thalassemia major. the lack of differences in qofl in patients with "high" and "low" hemoglobins was likely related to low pre-transfusion hbs (mean±sem, 8.31 ± 0.14 g/dl) in nearly all patients. these early data in a small cohort of thalassemia patients in an emerging setting suggest that in many patients bodily pain, reduced mental health, and poorer views of general health affect overall qofl. prospective studies in larger cohorts including evaluation of adequacy of transfusions and chelation therapy, complications, and overall accessibility of care may guide approaches to improve qofl in lower-income settings of thalassemia care. geetanjali bora, anand prakash dubey, tarun sekhri, mammen puliyel, aparna roy maulana azad medical college, new delhi, delhi, india background: in the last two decades, the presence of osteopenia has been described in optimally treated patients with transfusion dependent thalassemia, the pathogenesis of which seems to differ from osteopenia in non-transfused patients. the prevalence rate of low bone mineral density (bmd) in pediatric population is highly variable amongst studies done worldwide. furthermore, the role of metabolic and endocrine factors in determining bone mass in this population is not well understood. objectives: to assess bmd in subjects with transfusion dependent beta thalassemia by dual-energy-x rayabsorptiometry and find its co-relation with clinical, biochemical and hematological parameters. design/method: this is a comparative cross-sectional study and includes patients with transfusion dependent beta thalassemia between ages 6 to 16 years enrolled from a thalassemia day care center in the year 2012 -2013. at the time of enrollment age, sex, bmi z scores, pubertal staging, duration and type of chelation therapy were noted. enrolled subjects were scanned for bmd at lumbar spine l2-3 and left femoral neck using dexa scan. the bmd was expressed in mean values and z scores. age, bmi, ethnicity and gender matched historic controls were used to generate z scores. 5 ml of pre transfusion fasting venous blood samples were obtained to test for serum calcium, phosphate, alkaline phosphatase, pth, thyroid function panel, serum ferritin and serum igf-1 levels. mean values for pretransfusion hemoglobin and serum ferritin over last 12 months were calculated. results: total no of subjects 50, median age 11.6 years, male 32 (64%), female 18 (36%), ethnicity 100 % asian, bmi < 3rd centile 13 (26%), pre pubertal 50%, all receiving transfusion and chelation therapy. prevalence of low (z score < -1 sd) and very low (< -2.5 sd) bmd was 74%, 52 % at l1-l2 respectively and 66 %, 24% at left femoral neck respectively. there was trend of lower bmd z scores with advancing age. statistically significant co-relation (p value < 0.05) was found between low bmd and low mean pretransfusion hemoglobin, serum phosphate, igf -1 and vitamin d levels conclusion: a sizable proportion of children and adolescents with transfusion dependent thalassemia have suboptimal bone mineral density and this decline may start as early as 6-7 years of age despite being on transfusion regimen highlighting the importance of yearly dexa screening and optimization of pre-transfusion hemoglobin, vitamin d and igf 1 levels. vanderbilt university medical center, nashville, tennessee, united states background: it is well described that iron deficiency anemia (ida) can co-present with thrombocytosis or thrombocytopenia, though cases of thrombocytopenia are less frequent than thrombocytosis. prior reports of thrombocytopenia have included adult and pediatric patients with menorrhagia (1-2), menorrhagia due to uterine fibroids (3), or other gynecologic abnormalities (4). our cases highlight the pattern of ida, thrombocytopenia, and menorrhagia in the setting of significant menstrual clotting without observed gynecologic abnormalities in african-american adolescents. objectives: to describe the clinical course of three adolescent females with severe ida, menorrhagia, and thrombocytopenia. results: our cases included three female african-american patients ages 12-17 who presented with severe anemia and concurrent thrombocytopenia in the setting of menorrhagia. all three patients reported heavy and prolonged menstrual cycle bleeding with significant clots. two of the three were admitted for transfusions at presentation and noted to have significant menstrual bleeding with continued blood loss requiring additional transfusions until bleeding was controlled with estrogen therapy. these two patients were evaluated with pelvic ultrasounds revealing a prominent endometrium in both patients and hyperechoic material consistent with a clot in one patient. average hemoglobin on presentation was 4.3 gm/dl (2.8-6.5), average platelet count was 70,000/mcl (18,000-99,000), and average mcv was 63 (54-68). all had severe iron deficiency with an average ferritin of 4 ng/ml (2-7) subsequently treated with oral iron. one patient had a prior history of ida that required transfusion and had subsequent normalization of her complete blood count. two patients had subsequent thrombocytosis before normalization of their platelet counts. two patients received platelet transfusions: one due to recent neurosurgical intervention with a higher goal platelet count and the other to help control menstrual bleeding after a nadir platelet count of 9,000. a review of the clinical history and red cell indices pointed to ida and ongoing blood loss from menorrhagia as the reason for the bicytopenias. the thrombocytopenia in these cases may have been exacerbated by consumption of platelets in the significant clots all three patients reported. it is reasonable to treat with iron supplementation and supportive care which may include transfusions or management of menorrhagia with oral contraceptives or other hormonal methods. background: sickle cell disease is one of the most common inherited red blood cell disorders, yet many are not aware of their carrier status. the american college of obstetricians and gynecologists' guidelines recommend that pregnant women of african, mediterranean and southeast asian descent be screened for hemoglobinopathies with a cbc and hemoglobin electrophoresis1. however, adherence to this practice and frequency of improper screening with sickledex is unknown. proper screening and counseling can impact families' knowledge, allowing for establishing relationships with pediatric hematology providers earlier. objectives: we sought to assess prenatal hemoglobinopathy screening practice patterns and methods of obstetrics & gynecology (obgyn) and family medicine providers in the nyc regional area. design/method: a cross-sectional electronic survey was administered to obgyn and family medicine practitioners from four nyc institutions. questions focused on prenatal hemoglobinopathy screening practices using case scenarios with variations on parental trait status and ethnicities. chisquare analyses were used to compare the two provider groups on categorical variables. there were 98 total responses; 71 surveys were complete, of which 48 were obgyn and 23 family medicine providers. respondents were mainly from academic medical centers, with the majority being faculty (68% of the obgyns and 41% of family medicine). no significant difference was found in frequencies of screening patients with a positive family history of a hemoglobinopathy. when asked about screening practices for patients without a personal/family history of a hemoglobinopathy, 92% of obgyns versus 70% of family medicine providers "always" screened for hemoglobinpathies (p = 0.03). when analyzed by ethnic background, there were significant differences by group in screening patients of white (92% vs 70%), black (94% vs 74%), mediterranean (94% vs 74%), and asian descent (94% vs 70%) (p≤0.05 for all). however, in cases where the hemoglobinopathy carrier status of both parents was known, there was no difference in screening with a hemoglobin electrophoresis. furthermore, >20% of all respondents use sickledex for screening in the case scenarios. conclusion: this pilot survey highlights a difference in the methods and likelihood of prenatal hemoglobinopathy screening based on the type of prenatal care provider. screening differences can lead to variations in prenatal guidance, diagnostic procedures, informed decision-making and knowledge of families referred to pediatric hematology clinics. this is the first study analyzing prenatal screening for hemoglobinopathies in obgyn and family medicine. improving prenatal screening practices by collaborating with hematologists may decrease health care disparities and allow for earlier relationship building with pediatric hematology. 1. acog, opinion#691, 2017 poster # 220 hermansky-pudlak syndrome: spectrum in oman background: hermansky-pudlak syndrome (hps) is a rare autosomal recessive disorder, characterized by the triad of oculocutaneous albinism, a hemorrhagic diathesis resulting from storage pool-deficient platelets, and accumulation of ceroid/lipofuscin-like material in various tissues. before 2016, nine different types of hermansky-pudlak syndrome were identified, which can be distinguished by their signs and symptoms and underlying genetic cause. in 2016, a tenth type was defined based on mutations in the ap3d1 gene. hps type 2 is characterized in addition by severe neutropenia and recurrent sinopulmonary infection. the disease is more common in puerto rico, and this is the first report from oman. to describe the clinical, laboratory and genetic characteristics of hps sub-types in oman, including the first 2 cases of hps type 2. design/method: this is a retrospective study, including 7 cases with hps that had been suspected clinically and confirmed through genetic mutation analysis. clinical data included sex, age at presentation, initial clinical presentation (skin, eyes, development, neurological involvement, bleeding tendency, recurrent infections) and course of disease. laboratory data (complete blood counts, platelet and absolute neutrophil counts, coagulation screening, platelet function tests by platelet function analyzer, and platelet aggregation studies using different agonist had been recorded. pcr and next generation sequencing for genetic confirmation by testing mutations in hps1, ap3b1, hps3, hps4, hps5, hps6, dtnbp1,, bloc1s3, bloc1s6 genes had been done. results: seven omani cases with hps have been identified (4 males and 3 females). their age ranged between 0 (at birth) to 10 years. two patients had hps type 2, 1 patient had type 6, while the other 4 cases had hps type 3. no other sub-types were encountered in oman. all patients were products of consanguineous marriage. one patient had adrenal hge, while the others had mild hemorrhagic phenotype, characterized by recurrent bruising and mild epistaxis. laboratory testing confirmed variable platelet aggregation defects with different platelet agonists. all patients had characteristic hypopigmentation, iris transillumination, nystagmus, and foveal hypoplasia. both patients with hps type 2 had the same homozygous mutation in the ap3b1 gene (c.12_13delta), and presented with severe neutropenia. early diagnosis and initiation of gcsf on one of them improved outcome and prevented the development of complications. late diagnosis in the other patient resulted in the development of bronchiectasis as a result of recurrent sinopulmonary infections. background: sickle cell disease (scd), a genetic disorder characterized by defective sickle hemoglobin (hbs), triggers red blood cell sickling, hemolysis, vaso-occlusion, and inflammation. ischemic injury from scd starts in infancy and accumulates over a lifetime, causing pain, fatigue, and progressive end-organ damage that culminates in early mortality. voxelotor (gbt440) is an oral, once-daily therapy that modulates hemoglobin's oxygen affinity, thereby inhibiting hemoglobin polymerization. objectives: to assess the safety, pharmacokinetics, and efficacy of voxelotor in pediatric patients with scd. design/method: this ongoing study is being conducted in 2 parts: part a: a single dose of voxelotor 600 mg in pediatric and adolescent patients; part b: multiple doses of voxelotor 900 mg/d or 1500 mg/d for 24 weeks in adolescents. part b's primary objective is to assess the effect of voxelotor on modifying anemia. secondary objectives include measuring other markers of disease modification, such as hemolysis; daily scd symptoms, using a patient-reported outcome (pro) measure; and safety. results: as of november 6, 2017, 24 patients (10 females) had received voxelotor 900 mg and 12 patients (6 females) had received voxelotor for ≥16 weeks. the median age for the 12 patients was 13 years, 92% were receiving hydroxyurea (hu), and 41% had ≥1 painful crises in the past year. data for hemolysis measures are available for 11 patients who received voxelotor for 16 weeks. six of the 11 patients achieved a hemoglobin (hb) response of >1 g/dl increase. laboratory markers of hemolysis improved concordantly; the median reductions in reticulocytes and indirect bilirubin were 11% and 40%, respectively. ten of 12 patients showed reduction in total symptom scores (tss) at week 16, with a 94% median reduction in tss from baseline. there were no treatmentrelated serious adverse events (aes) or drug discontinuations due to aes. voxelotor 900 mg for 16 weeks in adolescents with scd, the majority receiving hu, demonstrated consistent, sustained efficacy on hb levels and measures of hemolysis; >50% of patients showed a >1 g/dl improvement in hb. improvement in tss in mildly symptomatic patients suggests that the pro is sensitive to treatment effect and supports use in the ongoing hope phase 3 study. voxelotor's reassuring safety profile is consistent with results in adults. these interim results support ongoing clinical evaluation of voxelotor as a potential disease-modifying therapy for adults and children with scd. supported by global blood therapeutics. background: acute kidney injury (aki) is a common complication in sickle cell disease (scd), and a potential risk factor for sickle nephropathy. aki is associated with acute decline in hemoglobin (hb) during vaso-occlusive pain crisis and acute chest syndrome (acs). it is unclear which pathologic factor plays a stronger role in aki development during hb drop: increase in free heme during vaso-occlusive events secondary to hemolysis or hb decline itself. objectives: to investigate if hb decline alone is associated with aki, we tested if the renal function of patients with scd worsened during parvovirus b19-induced transient aplastic crisis (tac), in the absence of accentuated hemolysis. design/method: with irb approval, a retrospective study of patients who had laboratory confirmed parvovirus-b19 was conducted. serum creatinine (scr), both during and within 12 months from the tac event, was collected. comparisons of the clinical and laboratory characteristics were analyzed using the wilcoxon test for continuous variables. aki was defined as an increase in scr by ≥0.3 mg/dl or a 50% increase in scr from baseline. to evaluate differences in change in hb on aki risk, changes in scr during tac were compared to those during pain crisis or acs admissions by fitting a generalized linear mixed model for binary outcome. a comparative sample of 149 acs events and 197 vaso-occlusive pain crisis were used to estimate rates of aki according to hb levels. results: three (9%) of the 34 patients with scd developed aki during tac. no association was identified between change in hb from baseline to tac event (p = 0.08). no cases of aki were identified until hb decreased <5.0 g/dl or the change in hb was ≥4.5 g/dl from baseline. next, we developed a model to evaluate the impact of change in hb from baseline for patients admitted with tac, pain crisis or acs on aki. with a 2 g/dl decrease in admission hb from baseline, patients with tac had a 3% probability of developing aki, while acute chest syndrome and pain crisis would have a 9% and 27% probability, respectively. our data suggest that aki is still prevalent during parvovirus b19-induced tac. however, the risk of aki during a tac event is 3 and 9 times lower than that from severe anemia induced by acute chest syndrome and vasoocclusive pain events, respectively. hemolysis-induced anemia during scd crisis appears to have a more significant role in the development of aki as compared to agenerative anemia. background: the natural history of hemoglobin e beta thalassemia (hbethal), the commonest form of severe beta thalassemia worldwide, has been examined in very few longterm studies. previously, we reported findings in 109 hbethal patients in sri lanka.1 objectives: to evaluate longterm requirements for transfusion and splenectomy, complications and death in hbethal patients. design/method: all available patients were reviewed 1-4 times annually over 12 years. results: 33 patients (30%) died, aged (mean ± sem) 29.3 ± 4.2 years; the (known) causes commonly included iron overload (9) and infection (12); 76 patients surviving patients are aged 33.4 ± 1.2 years. of 109 patients originally classified by severity (group 1 the mildest, and group 5 the most severe, phenotypes), 62 (57%) were assessed as mild (groups 1 and 2), of whom transfusions had been discontinued in 46. ultimately, 23/46 (50%) resumed transfusions, often following shifts to increasingly severe phenotypes including increasing intolerance to anemia. age at resumption of transfusions (following a transfusion-free interval of 14.9 ± 1.6 years) was 32.8 ± 2.7 years; in the more severe groups 4 and 5, regular transfusions were stopped in 27/33 patients and resumed in 22/27 (81%), at younger ages (19.9±1.8 years) and after shorter transfusion-free periods (10.1±1.4 years) than in "milder" patients. mid-parental height (mph) was ultimately achieved in 53%. 84 patients (77%) were splenectomized; updated analysis of responses to splenectomy (originally "group 3" patients), showed that splenectomy (at 9.0 ± 0.9 years) was followed by an extended, but impermanent, transfusion-free interval (11.1 ± 1.4 years); 50% patients resumed transfusions, usually related to exercise intolerance or poor growth. in groups 1 and 2, complications of anemia and ineffective erythropoiesis, including leg ulcers (in 38% and 50%) and gallstones (44% and 54%), were more frequent than in groups 4 and 5; fractures were observed (25-48%) across all groups, except for regularly-transfused group 5 patients (0%). pulmonary artery pressures >30 mm were recorded in 39% patients. evaluation of patients with hbethal requires observations over years, without which definition of patients as "mild" or "severe" may be misleading. while in many patients transfusions may be withheld or reduced in frequency, troublesome complications may surface with advancing age even in "milder" patients. although individual consideration of transfusion requirements is critical, the availability of effective chelation, where this can be provided without prohibitive cost, may alter the balance of risks and benefits of regular transfusions in hbethal. 1(premawardhena a. lancet 2005). background: social determinants of health (sdh) are environmental and socioeconomic factors, such as access to food and housing that affect health outcomes. pediatricians are increasingly screening for sdh as part of primary care visits, however less is known about screening for sdh in pediatric hematology. evidence suggests that sdh play a role in disease severity for children with scd, who face significant socio-economic and racial disparities. the goal of our quality improvement (qi) project was to increase the percentage of patients with scd who were connected to community resources for unmet social needs. design/method: we based our intervention on the successful implementation of wecare in our institution's pediatric primary care clinic. eligible patients were identified at the start of each clinic session. on arrival the parent was given a self-reported screening tool for six sdh (childcare, education, employment, food, utilities and housing). results were entered in the electronic health record by the physician or social worker who then printed a pre-existing resource list for patients with a positive screen. we used a series of plan-do-study-act (pdsa) cycles to study tests of change. we tracked process measures (percentage of patients screened, percentage of patients with an unmet social need who received a resource sheet), outcome measures (percentage of patients with an unmet social need who connected with a community resource) and balancing measures (staff, patient and provider satisfaction). run charts were reviewed weekly and then monthly to inform further tests of change. examples of pdsa cycles include who gave the paper survey to patients (social worker or physician versus medical assistant) and length of time between surveys (3 to 6 months). results: between august and december 2017 screening rates improved from 57% to 88%. of the patients screened, 67% report at least one unmet social need; of those 33% received a targeted list of community resources in the first month of the project, and 92% in the fifth month. finally, 44% of patients reached by phone had connected with a community resource within 2 weeks of the clinic visit. we have successfully implemented universal screening for sdh for patients with scd in our urban pediatric hematology clinic without requiring extra staff. next steps include further pdsa cycles to connect more patients to appropriate resources, and tracking improvement in health care utilization outcomes from addressing sdh in this vulnerable patient population. background: the clinical manifestations of sickle cell disease (scd), chronic hemolytic anemia, and vaso-occlusion occur as a direct result of sickle hemoglobin (hbs) polymerization. voxelotor (gbt440) is a first-in-class, oral, oncedaily investigational agent designed to modulate hemoglobin's oxygen affinity in a targeted approach to inhibit hbs polymerization. objectives: to examine the pharmacokinetics (pk), safety, and dosing of voxelotor in children (aged 6-11 years) and adolescents (aged 12-17 years) with scd from part a of the gbt440-007 study. design/method: gbt440-007 is an ongoing, open-label, phase 2a study in patients aged 6-17 years with scd (sickle cell anemia or sickle beta zero thalassemia). part a of this study (the focus of this abstract) is examining pk of singledose (600 mg) voxelotor. pk samples to measure whole blood and plasma voxelotor concentrations were collected up to 15 days following single-dose administration. separate population pk (ppk) models were developed to describe the concentration versus time profiles of voxelotor in whole blood and plasma using nonlinear mixed effects modeling (non-mem, version 7.3). ppk modeling and physiologically based pk (pbpk) modeling were used to simulate voxelotor pk parameters and support dose selection for future evaluation in younger children. : part a included 7 adolescents (4 females; median age 16 years [range 14-16]) and 6 children (3 females; median age 8.5 years [range 6-10]). mean weight was 52.8 kg (range 45-66 kg) and 21.1 kg (range 16-38 kg) in adolescents and children, respectively. voxelotor was well tolerated with no drugrelated grade ≥3 adverse events (ae) or serious aes. a 2compartment model with first-order absorption best described the pk of voxelotor (and was the same model structure used for adults with scd). voxelotor pk exposures in adolescents were comparable to those observed in adults, but higher exposures were observed in children. ppk and pbpk modeling support the use of a weight-based dosing strategy in younger children (aged <12 years) in future trials. adult voxelotor doses can be used in adolescents. however, based on higher pk exposures, a lower weight-based dosing strategy is recommended in children. ppk and pbpk modeling provides an innovative approach to minimize experimental dosing in children and accelerate dose selection of voxelotor in ongoing and future clinical studies. this abstract is supported by global blood therapeutics. background: hydroxyurea (hu) reduces rates of acute complications, and improves long term outcomes in patients with sickle cell disease (scd) and is now fda approved for children. through previous work we have increased the number of eligible patients on hu in our clinic, however accessing a compounding pharmacy remained a significant barrier to hu adherence for infants and children who cannot swallow capsules. objectives: the objective of our quality improvement project was to improve adherence to hu among pediatric patients with scd at our urban safety net hospital by addressing barriers to obtaining liquid hu. design/method: to begin we met with the leadership of our outpatient pharmacy which offers mail order delivery. however, like most retail pharmacies, they do not have the necessary protective equipment to compound liquid hu. through a series of discussions, we began a unique partnership with our institution's inpatient chemotherapy pharmacy who compounds the liquid hu and delivers it to the outpatient s95 of s301 pharmacy, who then dispenses liquid hu to families. using a series of plan-do-study-act (pdsa) cycles we tracked adherence by calculating the medication possession ratio (mpr), defined as the percentage of days in a given period of time that each patient had their medication on hand. the mpr for liquid hu mpr among enrolled patients was tracked by pharmacy staff and reviewed monthly. additional pdsa cycles included adding automatic refills and reminder calls by pharmacy staff and improving communication about delivery. we also tracked patient satisfaction. results: between march 2016 and december 2017, a total of thirty pediatric patients were enrolled in our program for on-site compounding and free mail order delivery of liquid hu. mpr for liquid hu is currently 93.8% among enrolled patients, significantly higher than the mpr of 60% reported in the literature, and has risen steadily since the beginning of the project. families are highly satisfied with the program, specifically appreciating the convenience of mail order delivery, saving on delivery fees, and reminder calls when refills were due. by compounding and dispensing liquid hu directly from our institution's outpatient pharmacy we have significantly improved adherence to this hu therapy in our high-risk population. next steps include analysis of change in clinical outcomes for patients enrolled in this program. as adherence to hydroxyurea is associated with decreased acute care utilization and cost, programs such as ours could play a crucial role in reducing the excessive costs and ed utilization among this patient population. background: experience with the iron-chelator deferasirox is reported widely in higher-income settings. by contrast, real-life experiences in emerging countries are infrequently reported. objectives: to evaluate, in a non-trial setting, the real-life response to deferasirox in an emerging country. design/method: in sri lanka's national thalassemia center which manages 800 patients without tertiary staff, quantitative evaluations of body iron or estimates of extra-hepatic iron, the records of 328 patients who began deferasirox in 2010/11 were retrospectively reviewed. results: baseline assessments (mean±sem) indicated substantial iron loading [serum ferritin (sf) 4,529±125 ug/l; serum alt 111±3.5 u/l (normal ≤ 40 u/l)]. deferasirox was introduced at low doses (21.2 ± 0.3 mg/kg/day); many patients started at <20 mg/kg and, after 12 months, doses remained ≤30 mg/kg/day in 60% patients. after 24 months, sf in 50% patients remained >2,500 ug/l; only by 48 months had (mean) sf declined to <2,500 ug/l (2475±344; p<0.001). similarly, mean alt normalized (to 35 ± 5 u/l) only by 60 months. death and complications were not systematically recorded by staff who had been charged, without provision of additional resources, with the introduction of this new drug in hundreds of patients. these results contrast to those in sri lanka's tertiary thalassemia center where, in 107 patients following the introduction of deferasirox 32 ± 0.1mg/kg/day, sf declined rapidly, even in relatively less ironloaded patients (from 3,231±278 to 2,153±218 g/l after 24 months; p = 0.0002). these findings underscore the importance, during the implementation of new drug regimens in lowerincome centers with marginal resources, for investments in methods to quantitate body iron burden, hands-on educational initiatives to guide day-to-day management by competent but non-expert staff, and data systems to record efficacy, effectiveness, toxicity and compliance. such investment is critical to optimising therapy and improving complications in thalassemia patients worldwide: even in sri lanka, where resources directed to thalassemia management are greater than in most of asia, results in the oldest living cohort (born 1980-1990) indicate under-treatment [elevated iron burdens (sf 3,565±323 ug/l) and high prevalences of diabetes (27%) and hypothyroidism (29%)]. even in a younger cohort (born 1990-2000) which has benefitted from improved treatments, the prevalence of many complications exceeds those reported from high-income settings. over the next decade, and two decades after the 2006 who declaration that the impact of thalassemia on global mortality and morbidity is underrecognized, increased investments by governmental and nongovernmental sources will be necessary to improve outcomes for asian patients with thalassemia. background: a major barrier to success in hydroxyurea (hu) treatment of patients with sickle cell disease (scd) is non-adherence. objectives: to optimize hu adherence in patients with scd. design/method: a care model was designed by the sickle cell (qi) team at children's hospital to improve hu adherence among scd patients. the original model included bimonthly family phone contact, monthly dispensing pharmacy phone contact and lab monitoring. adherence measures included obtaining hu from pharmacy monthly, completion of monthly labs, hb f percentage and mcv, and mtd achievement. from 6/2016 -6/2017, several pdsa cycles refined our care model. a one-year follow-up survey gathered feedback on the care model. the first-year data involved ∼ 30 patients. the biggest improvements resulted from making pharmacy calls before patient/family calls, shipping liquid hu to outlying patients, and tracking call time/content. the qi goal was 75 % hu adherence by 12/2017. the 33% baseline adherence rate increased to 80% by 12/2016, and has remained in that range. the completion rate of patient/parent phone calls increased from 33% the first month to 77% at six months. pharmacy prescription pick-up has increased from 50 % to 77% per month. lack of liquid hu availability was overcome by shipping the medication to the patient's home. parental hesitance to share information by phone, especially with qi team members with whom they had no established relationship, was overcome by having the longtime sickle cell nurse do many of the early calls. however, survey feedback showed families became comfortable with several clinic personnel calling. the calls gave families the opportunity to ask questions about their child and/or get additional information about scd. the calls also provided an opportunity for seasonal flu shot or tcd testing reminders. the surveys gave information on the optimal time of day to reach each family, providing individualization and further increasing the percentage of completed calls. two families surveyed said they no longer needed two calls a month because they were now able to remember to pick up hu, administer it, and get labs on their own. this qi project has not only improved hu adherence, but also fostered health education/counseling, increased patient/parent satisfaction, and enhanced service utilization. medical team member and patient/family comments demonstrate that it has helped build relationships and trust between families and the medical care system. based on survey feedback, we will further individualize care to increase adherence rate and sustain improvements. cincinnati children's hospital medical center, cincinnati, ohio, united states background: the thalassemias are a heterogeneous group of genetic blood disorders caused by mutations that decrease or eliminate the synthesis of the -and/or -globin subunits of hemoglobin. the phenotype of thalassemia depends on the interaction of the -and -globin gene clusters, because both loci determine the -/ -chain balance. for example, a -thalassemia phenotype can be more severe than expected when coinherited with -globin gene triplication (copy number gain), which exacerbates the -/ -globin imbalance. objectives: describe four individuals with an incorrect diagnosis of -thalassemia trait who were later properly diagnosed by comprehensive genetic testing to have -thalassemia intermedia caused by heterozygous -thalassemia mutations coinherited with triplicated -globin loci. design/method: sequence analysis of the -globin (hba1/hba2) and -globin (hbb) genes, and copy number variation analysis of the -and -globin gene clusters by multiplex ligand-dependent probe amplification. results: four unrelated individuals of northern european ancestry were evaluated for signs and symptoms not explained by a diagnosis of -thalassemia trait (previously made by a pediatric hematologist), including growth delay, splenomegaly, moderate anemia, marked elevation of hemoglobin f, thalassemic facies, reticulocytosis, and/or indirect hyperbilirubinemia. genetic testing revealed that all were heterozygous ( / 0) for the same, single -globin mutation [hbb.c.118c>t (p.q40*)] and also heterozygous for an -globin triplication ( / anti-3.7). their previous diagnoses of thalassemia trait had been made by complete blood counts, hemoglobin electrophoresis, and/or sequence analysis of the -globin genes only. these individuals' phenotypes ranged from moderate anemia only to multiple stigmata of thalassemia, demonstrating the phenotypic variation of a thalassemia genotype. correct diagnosis was made at an average age of 8.9 years. a trial of chronic transfusions was initiated for one patient for growth failure. all were educated about the potential for exacerbations of anemia, gallstones, osteoporosis, and iron overload (even without transfusions). parental genetic testing was recommended to assess reproductive risk, because inheritance of this complex genotype can be apparently autosomal dominant. conclusion: heterozygosity for a -thalassemia mutation does not necessarily indicate -thalassemia minor or "trait". when coinherited with -globin gene triplication, a symptomatic form of -thalassemia can occur. correct and timely diagnosis of thalassemia requires careful consideration of the degree of anemia and examination for organomegaly, bony changes, and jaundice. sequence analysis and copy number variation analysis of both the -and -globin gene clusters is key. hematologists need to be aware of this diagnostic possibility and how to test for it to prevent inaccurate or delayed diagnosis. background: the burden of healthcare costs for sickle cell disease (scd) is nationally estimated at over $488 billion. the major components of these costs are inpatient and emergency center (ec) visits, many of which are potentially avoidable. in several chronic conditions, a subset of patients account for most of the avoidable encounters. identifying these patients is the first step in targeted care delivery. objectives: to measure and analyze scd patient utilization patterns in the ec and inpatient at texas children's hospital (tch). we identified all individuals under 21 years old with any encounter at tch associated with an international classification of disease (icd)-9 or 10 code for scd, including hgb ss, hgb sc, and hgb s/beta thalassemia. for each patient, we identified all inpatient and ec encounters in the 365 days prior to their most recent encounter. finally, each encounter was classified as associated with pain, acute chest syndrome (acs), or "other" using an algorithm of discharge diagnosis codes and pharmaceutical delivery. the total number of scd-associated ec and inpatient encounters over the prior year was calculated for each patient. we stratified each patient according to their utilization patterns: low (0-1 encounters), intermediate (2-3 encounters), and high (≥4 encounters). we identified 952 unique patients with scd that had at least one encounter from july 2016 until june 2017. there were 1,100 scd-related encounters in the 365 days prior to their most recent encounter. most (74%, n = 701) patients exhibited low-utilization patterns and 18% (n = 174) were intermediate. finally, a small subset (8%, n = 77) demonstrated high-utilization patterns and accounted for 41% of all encounters. high-utilization was associated with older age and public payment mechanisms. pain encounters were predominantly in pre-adolescents and teenagers with high-and intermediate-utilization patterns. acs was most frequent in pre-teens and younger teens in the intermediate-utilization group. finally, the youngest-aged high and intermediate users presented for other reasons such as febrile episodes and splenic sequestration. our findings reflect national trends in that a significant portion of encounters are attributed to a small subset of patients exhibiting a high-or "super-" utilization pattern. at our institution, scd super-utilization is associated with older age and pain. we also identified a group of infants and toddlers with frequent encounters for fever. to comprehensively address this burden, it will be important to design interventions targeted toward age and specific medical needs. background: background: the rarity of diamond blackfan anemia (dba) has hindered describing the spectrum of disease, identifying predictive correlations, and guiding datadriven recommendations. long-term toxicities from steroid or transfusion therapy that start in childhood remain the major clinical problems in patients with dba who do not receive stem cell transplant. objectives: objective: to define the dba patient population at st. jude children's research hospital including treatment responses and toxicities to help inform recommendations on treatment and monitoring. design/method: method: medical records were reviewed for all patients with dba treated at st. jude between 1997 and 2017 for diagnostic testing, treatment types and regimens, and outcomes. two-sample t-test or wilcoxon rank sum test was used to compare continuous variables in two groups depending on the normality of the data tested by shapiro-wilk test. results: a total of 22 patients with dba were identified with a median age of 8.29 years (range 3 months -35 years) at last follow up. a ribosomal protein gene mutation was identified in 15/22 patients (68%) with an rps19 mutation 8/22 (36%). thirteen different congenital malformations were described in 9/22 patients (41%). fourteen of twenty (70%) patients treated with corticosteroids had an initial response and 3 of those achieved full remission. three patients became steroid-refractory and 2 were unable to wean to an acceptable dose. five of twenty patients continue on lower-dose steroids. five patients currently require no therapy. univariate analysis revealed no statistically significant genetic predictors of response or remission, however, 3/3 rpl11 patients responded to steroids with 2/3 (66%) in long-term remission. ten patients are maintained on chronic transfusions and 2 have undergone successful hematopoietic stem cell transplant. nineteen of 20 treated patients (95%) had a treatment-related toxicity. patients on steroids were more likely to have short stature than patients on transfusions or in remission (p = 0.005). severe bone mineral density deficit occurred in 4/20 (20%) patients, in 2 before age 7 years. eight patients had hepatic iron overload, in one documented by age 2 years. other severe toxicities included restrictive cardiomyopathy from iron overload, pathologic fracture, diabetes mellitus, and premature ovarian failure in one patient each. this genotypically and phenotypically heterogeneous dba cohort had a high rate of treatment-related toxicities, notably growth retardation, bone density loss, and hepatic iron overload even in very young children. these findings underscore the need for early standardized monitoring. background: patients with sickle cell disease (scd) face worsening morbidity and mortality between ages 18 and 30, when they must transition from pediatric to adult healthcare.(1) an effective curriculum addressing disease knowledge, educational and vocational skills, self-efficacy, and social supports is critical to a successful transition. traditional didactic approaches have not led to durable knowledge retention. (2) technology-based methods have been attempted, but the best educational approach remains unknown. objectives: 1. to understand how adolescent and young adult (aya) patients with scd view existing transition education. 2. to include patient preferences in improving our transition curriculum. we developed a qualitative survey to assess patient views of existing approaches for learning about scd and their opinions about preferred transition topics. thirty patients with scd aged 12 to 24 years old were recruited between january and december 2017. responses were managed using redcap electronic data tools hosted at the university of rochester.(3,4) qualitative and quantitative data analyses were performed, including independent t-testing to compare responses between age groups. results: approximately 68% of subjects were under 18 years of age, while 32% were 18 or older. seventy-one percent had a computer, and 93.5% had a cell phone, with most reporting daily use. subjects reported greatest satisfaction with learning from their doctor during clinic visits (83.9% agree or strongly agree) and websites on a cell phone (77.4% agree or strongly agree); the least popular methods were online chat rooms and microsoft® powerpoint presentations. satisfaction was similar across age groups. recommended transition topics were viewed positively, with subjects ranking highest understanding their bloodwork (87.1% agree or strongly agree) and understanding laws protecting students with chronic disease (93.6% agree or strongly agree). older subjects (18-24 years old) agreed more strongly with learning about opioid addiction and understanding differences between adult and pediatric doctors than did younger subjects (12-17 years old) (p < 0.05). this pilot study was successful in helping us to understand the educational needs of aya patients with scd. preliminary data underscore the importance of education provided by the pediatric hematologist. our results also suggest that the optimal use of technology-based methods requires further investigation and that tailoring transition education by age group may be useful. background: similar to patients with transfusion-dependent beta-thalassemias (tdt-beta), survivors of hemoglobin barts hydrops fetalis (homozygous alpha-0-thalassemia, tdtalpha) will require lifelong transfusions of erythrocytes. we have previously shown that a transfusion strategy that is based on the guidelines developed for tdt-beta (conventional transfusion) is suboptimal for these patients owing to the differences in the pathophysiology of anemia in the two conditions: in tdt-alpha, conventional transfusion strategy will lead to a gradual increase in non-functional hbh with subsequent tissue hypoxia and hemolysis. an aggressive transfusion strategy that was based on reduction of hbh and increase in "functional" hemoglobin level resulted in improvement of tissue oxygenation and reduction of hemolysis but was associated with significant increase in transfusional iron burden [amid et al, blood 2016] . objectives: to define the optimal chronic blood transfusion targets for hbh% and functional hemoglobin in patients with tdt-alpha. design/method: following research ethics board approval, longitudinal data of 6 patients with tdt-alpha (2 males, median age 11.5 (2.1-18.0) were retrospectively collected. variables of interest included total pre-transfusion hemoglobin, hbh%, and "functional" hemoglobin [measured as total hemoglobin x (1-hbh/100)]. outcome variables were lactate dehydrogenase (ldh, marker of hemolysis), and soluble transferrin receptor (str, marker of erythropoiesis). hemoglobin analysis was done using high-performance liquid chromatography and capillary zone electrophoresis. we examined the association of "functional" hemoglobin with str, and hbh% with ldh, using repeated-measures anova to adjust for the effect of multiple testing. we constructed receiver operating characteristic curve and calculated the area under the curve to define the best cut-off values for variables of interests. there was a strong association between functional hb and str, as well as hbh and ldh. the optimal cut-off for "functional" hemoglobin that was associated with str <2.0 mg/l was 98 g/l (auc = 0.94, sensitivity and specificity of 82.76% and 100% respectively). the optimal cut-off for hbh to supress ldh to <1000 u/l was 18% (auc = 0.94, sensitivity and specificity of 87.5% and 87% respectively). the optimal pre-transfusion hbh% for reduction of hemolysis was 18% and the optimal "functional" hemoglobin to adequately supress erythropoiesis was 98 g/l. to meet these hbh% and functional hb targets by simple blood transfusions, patients with tdt-alpha would require a hypertransfusion regimen with a minimum pre-transfusion total hb of 116 g/l and consequently high transfusional iron burden. an alternative approach using exchange transfusion to reduce hbh% and improve functional hemoglobin would be associated with less volume of transfusion and potentially better long-term outcome. hospital sacre coeur, milot, haiti background: initial results of work developing a pediatric sickle cell disease (scd) clinic at the hôpital sacré coeur (hsc) in milot, northern haiti were presented at aspho 2017. the purpose of this clinic is for a pediatrician with a special interest in scd to provide scd care, advising on trait and managing disease with penicillin prophylaxis (pcn) and hydroxyurea therapy (hu) for select patients. this clinic was started in collaboration with a us based hematologist and support from yale-new haven hospital. objectives: to describe the success and challenges of providing pcn and hu in the scd clinic at hsc through a review of patient records. design/method: since this clinic's inception, a database of patients, with basic clinical information has been kept and made accessible, through 'drop-box', to the us hematologist. the records of those that presented to the clinic were reviewed. the hemoglobin diagnosis was made either by clinical history and sickle cell prep or by hemoglobin electrophoresis through alpha laboratory, port-au-prince, haiti. results: ninety-nine individuals were seen in the first 2 years of the program. fifty-six underwent a hemoglobin electrophoresis. of these 99, 49 are ≤ 6 years old. thirty-two were started on pcn vk, of which 10/32 (31%) were ≤ 3 years old. eleven patients were started hu therapy. all patients on hu have shown progressive increases in hemoglobin. there have been no clinical complications of hu therapy. none of the patients taking hu have required hospitalization or transfusion in 2017. three patients (not on hu) were hospitalized in 2017 for complications of scd (osteomyelitis, pain). in 2016, with less than half the numbers in the program, there were 7 admissions for severe anemia, pain, stroke and splenic sequestration. with ongoing external support and a local reputation for excellence in sickle cell care, the clinic at hsc has been able to expand services and improve the health of a growing number of patients with scd. early data suggests that pcn and hu therapies are helping to reduce complications and improve quality of life. challenges to date have included lack of funding for transportation to clinics, for hospitalizations and to cover the cost of electrophoreses. at the same time as continuing providing excellent care and gathering data, it is crucial to explore opportunities for collaboration and cooperation in ways that will assure that the clinic can become independently sustainable while continuing to improve the quality of life for the individuals it serves. background: ykl-40 is an inflammatory glycoprotein expressed by infiltrating macrophages in various inflammatory conditions. it has been found to be elevated in patients with different pathological conditions like acute and chronic inflammations, increased remodeling of the extracellular matrix (ecm), development of fibrosis and cancer. several studies have found elevated ykl-40 concentrations in sera of patients with liver diseases such as hepatic fibrosis by hepatitis c virus. it has been suggested that ykl-40 concentrations reflect the degree of liver fibrosis. to evaluate serum ykl-40 levels in patients with -thalassemia and its relation to viral hepatitis, liver stiffness as assessed by transient elastography (fibroscan, fs) and hepatic iron concentration. design/method: a prospective study included 100 patients with -tm (43 males and 57 females) with mean age 13.8 ± 2.7 years (range: 5-18 years). serum ferritin level, liver enzymes (alt and ast), hbs ag, anti hcv ab and serum ykl-40 using elisa kit were evaluated. all patients were subjected to liver mri t2* to detect liver iron content by the sequence and transient elastography (fibroscan, fs) to assess degree of liver stiffness. results: mean fibroscan value was (10.99±11.5) kpa with a median 6.7 (range 1.3 to 47) kpa. 64 (64%) patients were categorized as f0-1 and 17 (17%) were stage f2-3, 19 (19%) patients had severe fibrosis. their median serum ferritin was 3100 ng∖ml, with 61 (61%) patients had values exceeding 2500 g/l. median cardiac t2* was 24.2 with 30 patients had values below 20 ms, and the median lic was 16.21 mg/g dw with 68 patients showed readings above 7 mg/g dw. nyl-40 was evaluated as a marker of inflammation and liver fibrosis and showed mean value 1505.1 (±960.9) pg/ml, and range from 500 to 3529 pg/ml. mean ykl-40 was significantly higher among males (p = 0.03), patients on chelation therapy (p = 0.002), patients on dfs (p≤0.001), in those with abnormal liver enzymes, splenectomised patients, patients with hbv sero-positivity, those with moderate elevation of t2* and patients with high grades of liver fibrosis (p<0.05). ykl-40 showed positive correlation with the rate of transfusion, lic, ferritin, alt and ast but negative correlation with weight, height and t2*. roc curve analysis revealed that the cutoff value of ykl-40 at 1500 pg/ml could differentiate -tm patients with and without viral hepatitis with 86.7% sensitivity and specificity of 91.4%, area under the curve (auc) 0.933, positive predictive value 81.2 and negative predictive value 94.1 (p<0.001). roc curve analysis revealed that the cutoff value of ykl-40 at 1600 pg/ml could detect -tm patients with liver cirrhosis with 93.4% sensitivity and specificity of 97.1%, area under the curve (auc) 0.972, positive predictive value 93.7 and negative predictive value 97.1 (p<0.001). conclusion: serum ykl-40 levels are elevated in patients with -thalassemia and can detect patients with active viral hepatitis and liver stiffness. background: the most common splenic complication in pediatric patients with sickle cell disease (scd) is acute splenic sequestration (ass), which has often been managed with splenectomy. although splenectomy has been a treatment of choice for years, long-term vascular complications have not been thoroughly evaluated. pulmonary hypertension (phtn) is a severe complication of scd. in adults with scd, phtn has been associated with a 40-month mortality rate of approximately 40%. it has been reported that splenectomized patients with hemolytic disorders are at even greater risk of phtn. several medications exist to treat phtn, but with few studies of their efficacy or toxicities in patients with scd. additionally, these patients are often treated with either chronic prbc transfusions or hydroxyurea (hu) to raise hemoglobin, reduce hemolysis, and prevent vaso-occlusive events. objectives: to evaluate effect of chronic prbc or hu vs. no intervention, on tricuspid regurgitant jet velocities (trv) in pediatric patients with scd and history of splenectomy. design/method: retrospective chart review of splenectomized patients with hbss followed at marian anderson center at st. christopher's hospital for children, philadelphia, between 1999 and 2017. we analyzed 73 trvs (16 hu, 40 prbc, and 17 from control group receiving neither treatment) from 35 patients (10 hu, 13 prbc, 12 neither). mean age at echo was 13.31 +/-5.1. data was analyzed with linear correlations and analysis of variance (anova), including the post hoc test of least significant difference (lsd) for all pairs of treatment groups. results: trv was not significantly correlated with age at time of assessment or with time between splenectomy and trv. univariate anova among groups yielded trv means of: 214.0 +/-36.0 cm/s (hu), 231.3 +/-28.1 (prbc), 231.3 +/-25.4 (neither). we found a notable difference as the mean of the hu group was almost 18 cm/s lower than the others, but no overall statistically significant association for any of the groups exists. however, when we performed post hoc tests to adjust for multiple comparisons and looked at all 3 pairings within the anova, we found that the lsd between the hu and the prbc groups was statistically significant (p = .051), and that a trend exists between the hu group and the neither treatment group (p = .095). our data suggests that treatment with hu is correlated with a reduction in trv in pediatric patients with scd who underwent splenectomy. given these promising results, we believe our data warrants further study with larger treatment groups. nancy olivieri, gaurav sharma, susmita nath, rajib de, tuphan kanti dolai, prakas kumar mandal, abhijit phukan, amir sabouhanian, robert yamashita, angela allen, david weatherall, prantar chakrabarti background: hemoglobin e thalassemia (hbethal), which accounts for 50% of all severe beta thalassemia worldwide, has an estimated prevalence of 1.4/10,000 in west bengal, from which little information about clinical findings has been reported. objectives: to document clinical and laboratory findings in patients with hbethal, ultimately to improve resources for clinical management. design/method: we reviewed records from: a database recording patient names; clinic charts; "special" charts containing additional details; and, in transfused patients, transfusion day-care records. additionally, because in india's public hospitals original lab/imaging reports are commonly retained at home, 20% of families were interviewed to provide additional information. we excluded records of patients aged <5 years and patients aged <30 years who had not been reviewed since 2014. results: while at least one visit had been recorded in 1,398 hbethal patients at nrs hospital, most patients are not regularly reviewed there. we examined 219 charts [84 (38%) aged ≥30 years; 135 (62%) aged 5-29 years; 61% male], representing approximately 70% of regularly-reviewed patients. most families (84.9%) reported monthly incomes (<5,000 indian rupees), below the monthly cost of living (70,000 rupees) in kolkata. mean (±sem) hemoglobin was 6.9±1.1 g/dl. 43% patients were receiving eight or more transfusions per year; from 2013, 40% had been treated with deferasirox, 26.5±8.5 mg/kg/day. iron control estimated by serum ferritin concentration (1357.2±1187 g/l) was highly variable. a total of 24% patients were splenectomized. a substantial obstacle to documenting complications was the lack of recording, in any of the five sources, of many relevant parameters: for example, the status of sexual maturation (normal, delayed, or absent) was documented in less than 60%, and measurements of fasting blood glucose in less than 50%, of records. where recorded, complication rates were high: delayed/abnormal sexual maturation was recorded in 15% patients aged >30 years; in the patients aged >30 years and those aged 5-29 years, respectively, hypothyroidism was recorded in 31% and 44%, and elevated serum alt in 30% and 35%. in most evaluable patients >30 years, height was measured between the 3rd-10th percentiles. cardiac findings, rarely documented, included pulmonary hypertension and reduced left ventricular ejection fractions in a few patients. despite dedicated attention to many aspects of thalassemia care, insufficient documentation limited a clear understanding of the current morbidity in hbethal patients. investment in personnel and technology will be critical to record relevant information, ultimately to improve clinical management, over the next decade. children's hospital of richmond at vcu health, richmond, virginia, united states background: sepsis is a common cause of death in children with sickle cell disease (scd). recommendations for care of fever in children with scd include immediate medical evaluation including blood culture and initiation of broad-spectrum antibiotic therapy. the increasing availability of pcr-based respiratory pathogen panels (rpp) provide the opportunity to rapidly identify viral causes of fever. the role for rpps in identifying the source of fever in children with scd and how it affects provider practice is not well studied. (1) to determine the epidemiology of respiratory virus-associated fever in children with scd and (2) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all children with scd seen in our emergency department (ed) with temperature ≥38.3oc at home or in the ed from january 1, 2016, through september 30, 2017, as well as, all febrile children for whom rpps were sent since the introduction of rpps april 2014. we reviewed the results of blood cultures, rpps, chest radiographs, and ed notes and discharge summaries to identify sources of infections. independent t test and chi-square analysis were used as appropriate to compare results using spss©. overall, the rate of bacteremia was 1%. there were no cases of bacteremia among children with positive rpps. 4% of children with negative rpps had true bacteremia. a positive rpp did not reduce the likelihood of bacteremia (p 0.11). patients with bacteremia had higher presenting temperatures than those without bacteremia (39.5oc vs 37.9oc, p 0.017). the most common rpp findings were rhinovirus/enterovirus (38%), human metapneumovirus (13%), and influenza a (10%). sending an rpp did not affect admission rate (29% and 26% respectively, p 0.70); however, likelihood of admission was lower in patients with positive rpps (21% vs 49%, or 0.27 [0.13-0.56], p 0.004). length of stay (los) was shorter in patients for whom an rpp was not sent (3.1 vs 4.5 days, p 0.036). as previously reported, bacteremia in febrile children with scd is very low, but remains a serious concern, particularly in the setting of high fever (>39oc). a positive rpp did not reduce the odds of bacteremia, but did have a sta-tistically significant impact on both admission rate and los. more work is needed to understand how rpp results impact provider decision-making and care for children with scd. cincinnati children's hospital medical center, cincinnati, ohio, united states background: diffuse myocardial fibrosis is a common, if not defining, feature of the heart in sickle cell anemia (sca) that is strongly associated with diastolic dysfunction. we found diffuse myocardial fibrosis in every patient in a sca cohort (n = 25) ranging in age from 6 to 61 years (niss 2017). the treatment and prevention of this complication of sca has not been studied before. objectives: because diffuse myocardial fibrosis must begin in early childhood, we hypothesized that early initiation and uninterrupted use of disease-modifying therapy for sca can prevent it. design/method: we use cardiac magnetic resonance imaging (cmr) to measure the myocardial extracellular volume fraction (ecv) to quantify diffuse myocardial fibrosis in individuals with sca who have been treated, uninterrupted, with hydroxyurea or chronic transfusion therapy since ≤4 years of age. two comparison groups were used: individuals with sca who have not been treated with disease-modifying therapy since ≤4 years of age (n = 25) and controls without sca (n = 16). results: we studied 7 individuals (3m/4f) with a mean age of 13.4 years (range 7 -24). mean age at the start of diseasemodifying therapy was 2.5 ± 0.4 years (range 1-4). only 1 had evidence of mild diffuse myocardial fibrosis (ecv 0.339); the other 6 had no detectable diffuse fibrosis (all had ecv <0.304, the upper limit of normal). mean ecv was 0.283 ± 0.012, which was significantly lower than the ecv of individuals with sca who have not received early uninterrupted therapy (0.441 ± 0.016; p = 0.009) and not statistically different from normal controls (0.257 ± 0.004; p = 0.898). none had macroscopic fibrosis by late gadolinium enhancement or evidence of myocardial hemosiderosis by t2* imaging. no patient had diastolic dysfunction by echocardiographic classification, right heart catheterization, or both. disease-modifying therapy for sca can prevent diffuse myocardial fibrosis, and possibly diastolic dysfunction, if started in early childhood. prospective trials of disease-modifying and anti-fibrotic therapy are planned to prevent diffuse myocardial fibrosis, which can be monitored noninvasively by cmr, and improve outcomes in sca. (niss, blood, 2017) . background: a statewide sickle cell surveillance system (sscss) was developed with the goal of determining the prevalence of sickle cell disease (scd) in indiana and the level of care that patients receive throughout the state. persons with scd are at high risk of infection, especially with encapsulated organisms, as well as at increased complications from influenza. utilizing sscss data, the relationship between vaccination status and mortality was explored. to determine if vaccination status is associated with mortality in persons with scd. the project was granted a waiver of consent by the st. vincent irb. death certificates were obtained to identify cause of death. deceased patients (cases) were matched by age, gender, and sickle genotype to living patients (controls). vaccination data were collected from the medical record and the children and hoosier immunization registry program (chirp) through the date of death for each case. cases and controls were assigned a point for completion of the pneumococcus, meningococcus and haemophilus influenza type b (hib) vaccine series and one point if the influenza vaccine was given within a year prior to death of the cases [max vaccine status score (vss): 4]. total points were compared between the cases and controls. two tailed t-tests to compare means of continuous data and wilcoxon signed-rank test to compare ordinal data. one thousand forty-eight individuals were included in the sscss. six hundred and seven (48.6%) were seen at one institution and included in this analysis (mean age = 21 years). thirty-three of the 607 (5.4%) were deceased at the time of analysis. six point one (6.1)% of controls and 12.1% of cases received a vss of 4. the mean vss for cases was 0.7±1.3 and 0.6±1.1 for controls. thirty point three (30.3) % of controls had a vss of one or more, compared to 27% of cases (p = 0.41). patients who died of infection [streptococ-cus (n = 1), pseudomonas (n = 1) and unidentified organisms (n = 4)] were not up to date on vaccination against encapsulated organisms, but two had received the influenza vaccine in the year prior to death. in this sample, mortality occurred exclusively among adult patients, which is consistent with current patterns in developed countries. among these adults, vss and mortality rates were not related. limitations to the study include small sample size and potential incompleteness of vaccine records. vaccination rates and other standard of care indicators should be explored in a larger cohort of patients to determine associations with mortality. background: sickle cell disease (scd) is a genetic disorder resulting in acute and chronic complications, including delayed puberty. delayed puberty can have adverse physical and psychosocial effects on affected children and families. there are no published reports from ghana on pubertal timing in children with scd. the aim of this cross-sectional study was to describe pubertal changes in children with scd at korle bu teaching hospital (kbth), accra, and compare these findings to those in a control group without scd. design/method: 178 children with scd and 174 children with hb aa, ages 8-19 years, were consecutively recruited and matched for age, sex and socioeconomic status. investigator-administered questionnaires were used to obtain demographic data for all participants and information on menarche (girls only). pubertal status was assessed by physical examination using tanner staging. testicular volumes were determined in boys using a prader orchidometer. body mass index (bmi) and socioeconomic status (ses) of participants were analyzed to determine if there were any associations with tanner stage. of the 178 with scd, 133 (74.7%) were hb ss and 45 (25.3%) hb sc. females comprised 51.1% (cases and controls). mean age at onset of breast development was significantly delayed in girls with scd (13.1 ± 1.9 years) compared to controls (10.8 ± 1.9 years) but there was no significant age difference at onset of pubic hair development. mean age at menarche was significantly delayed in girls with hb ss (14.0 ± 1.8 years) and hb sc (13.5 ± 1.5 years), compared to those with hb aa (12.5 ± 1.3 years). in boys, the mean ages at onset of puberty were significantly delayed in those with scd (13.6 ± 2.7 years, for genital development and 15.1 ± 2.2 years, for pubic hair development), compared to those without scd (11.3 ± 1.9 years and 11.4 ± 1.9 years, respectively). mean testicular volumes were significantly lower in cases compared to controls, across all age ranges (p<0.001). mean bmi in both cases and controls were similar at onset of breast development in girls. however, in boys with and without scd, mean bmi values were significantly different at pubertal onset. in univariate analysis, ses was not associated with tanner stage for both genital and breast development. mean ages at pubertal onset were significantly delayed in children with scd. longitudinal studies are needed to further characterize any associations with bmi and determine potentially modifiable risk factors affecting pubertal onset in scd. background: sickle-cell disease (scd) is a life-threatening genetic disorder associated with multiple chronic and acute complications. specific monitoring and treatment for children is a major part of the medical focus, but there remains a lack of real-world evidence of the disease burden and practice patterns among the pediatric scd population. objectives: to examine the clinical burden and management of scd among pediatric patients. design/method: a retrospective claims study was conducted using the medicaid analytic extracts database from 01jan2009-31dec2013. pediatric patients (aged <18 years) with scd were identified using icd-9-cm diagnosis codes (282.41-282.42, 282.60-282.69 ). the first observed scd diagnosis during the identification period was designated as the index date. patients were required to have continuous medical and pharmacy benefits for at least 6 months pre-and 12 months post-index period. patient data were assessed until the earliest occurrence of the following events: disenrollment, death, or the end of the study period. patient demographic and baseline clinical characteristics, clinical outcomes (mortality, incidence of pain crisis, complications), scd management, and healthcare utilization were examined. all variables were analyzed descriptively. results: a total of 12,388 patients met the study inclusion criteria, with a mean age of 7.7 years. most patients were black (59.9%) and had a charlson comorbidity index score of 0 (80.9%). mortality during follow-up was 0.1 in 100 personyears, and the event rate of pain crisis in the inpatient setting was 54.0 in 100 person-years. the three most common complications after pain crisis (highest rates in 100 person-years) were fever (31.9), infectious and parasitic diseases (27.7), and asthma (14.5). rates of life-threatening complications were also examined in 100 person-years, including acute chest syndrome (7.0), stroke (1.8), splenic sequestration (1.1), pulmonary hypertension (0.3), and pulmonary embolism (0.1). 83.9% of patients were prescribed antibiotics during the one-year post-index period. other frequent medications utilized among children were folic acid (39.2%), nonsteroidal anti-inflammatory drugs (37.4%), opioids (11.2%), and hydroxyurea (11.5%). 16.0% of patients had a blood transfusion within one year post-index date. patients had frequent health care utilizations in the inpatient (1 visit), emergency room (2 visits), office (7 visits), and pharmacy (11 visits) settings during the one-year follow-up period. pediatric scd patients are burdened with a high rate of complications including pain crisis. in addition, patients utilized a substantial amount of health care resources including outpatient office care and acute care visits. background: novel use of hydroxyurea in an african region with malaria (noharm, nct01976416) is a randomized controlled trial of hydroxyurea for very young children with sickle cell anemia living in uganda. during year 1, study participants received blinded study treatment of hydroxyurea or placebo; those receiving hydroxyurea had no increased risk of malaria, but had both laboratory and clinical benefits. during year 2, all study participants received openlabel hydroxyurea treatment. to assess the effects of open-label hydroxyurea treatment in a very young population of children with sickle s105 of s301 cell anemia living in uganda. study endpoints included the rates and severity of malaria infections, clinical sickle-related events, and laboratory effects. design/method: all children in the noharm trial were enrolled at mulago hospital sickle cell clinic in kampala uganda. during year 2, all children received open-label fixeddose hydroxyurea (20 mg/kg/day) for 12 months, after previously receiving either hydroxyurea or placebo for 12 months. results: a total of 198 children entered year 2 of the noharm trial and received fixed-dose hydroxyurea, including 107 males and 91 females, at an average age of 3.3 ± 0.9 years. among 99 children previously on placebo, there were 6 malaria events in 6 children, including 3 with severity grade ≥3, and three deaths (two acute chest syndrome, one sepsis). clinical adverse event rates dropped from 3.0 to 1.6 per patient year, and hospitalizations were reduced from 35 to 7. expected hematological benefits of increased hemoglobin, mcv, and fetal hemoglobin, along with decreased neutrophils and reticulocytes, were rapidly achieved. laboratory adverse events were infrequent at 0.2 events per patient-year, and only half of those were dose-limiting hematological toxicities. among 99 children previously on hydroxyurea, there were 7 malaria events in 5 children, including 2 with severity grade ≥3, and two deaths (one acute chest syndrome, one sepsis). clinical adverse event rates and hospitalizations were maintained at low rates, the hematological benefits of hydroxyurea continued throughout the extended treatment period, and dose-limiting toxicities remained infrequent. fixed-dose hydroxyurea treatment of young children with sickle cell anemia living in uganda is associated with no increased risk for malaria. clinical and laboratory benefits occur, including children previously on placebo who crossed-over to hydroxyurea treatment. future studies should focus on the optimal dosing and monitoring strategies, in an effort to determine the overall feasibility and safety of introducing hydroxyurea therapy across sub-saharan africa. background: acute chest syndrome (acs) is the second most common cause of hospitalization in patients with sickle cell disease and is a leading cause of morbidity and mortality. in mid-2009, an algorithm was implemented at cohen children's medical center to initiate transfusions within four hours of diagnosis of acs in order to improve patient outcomes. objectives: the aim of this project was to analyze the effect of early blood transfusion on the outcomes of patients with acs. we focused on the number of total transfusions, need for exchange transfusion, need for intensive care unit (icu) stay, and length of hospitalization. design/method: a retrospective chart review was completed on patients admitted to ccmc with a primary diagnosis of sickle cell disease and a secondary diagnosis of either acs or pneumonia during the years of 2006-2012. data from the three years directly prior to implementation of the algorithm was compared to data from the three years directly after implementation of the algorithm. a total of 118 patients were analyzed, of which 45 belonged to the pre-algorithm group and 73 to the postalgorithm group. patients from the post-algorithm group had a higher incidence of transfusions (78% with a mean transfusion number of 1.49 pre versus 86% with a mean of 1.83 post) as well as exchange transfusion (17% pre versus 27% post). the post-algorithm group had a shorter overall length of stay (mean of 6.0 days pre versus 5.0 days post). while the overall percentage of patients requiring an icu admission was similar in each group (27% pre versus 29% post), the post-protocol group had a lower likelihood of requiring an icu admission for reasons outside of line placement for exchange transfusion, most commonly for icu-level respiratory support (13% pre versus 4% post). despite a higher total number of transfusions, early recognition and transfusion for acs can lead to decreased lengths of hospitalization as well as decreased need for icu-level respiratory support. further studies comparing different center's clinical practice guidelines are necessary to improve the standard of care. background: novel use of hydroxyurea in an african region with malaria (noharm) was the first placebocontrolled randomized clinical trial of hydroxyurea in sub-saharan africa. in noharm, young children with sca received either hydroxyurea or placebo during year 1, followed by open-label hydroxyurea for all study participants during year 2. an ancillary noharm project was designed to determine if hydroxyurea treatment lowers transcranial doppler (tcd) velocities and possibly reduces stroke risk in this very young cohort. objectives: to perform tcd screening on the noharm cohort, measuring the time-averaged mean velocity (tamv) at the end of both year 1 and year 2. we hypothesized that the maximum tamv would be lower for noharm study participants receiving hydroxyurea compared to those receiving placebo, and that key clinical and laboratory parameters would also influence tcd velocities. design/method: all children enrolled in noharm were eligible to undergo tcd examination at two study time points: month 10-12 when they were completing the blinded treatment phase, and again at month 22-24 at the end of the open-label treatment phase. tcd measurements included tamv readings from the main intracranial arteries: middle cerebral artery, distal internal carotid artery, and bifurcation on tcd. all tcd examinations were scored and classified as normal (less than 170 cm/sec), conditional (170-199 cm/sec) or abnormal (greater than or equal to 200 cm/sec), with higher scores correlating to greater risk of stroke. results: at the end of year 1, 185 tcd exams were conducted of which 164 were suitable for analysis (81 hydroxyurea, 83 placebo). based on the maximum tamv, the median velocity was 138 cm/sec (iqr 120 -159) for children on hydroxyurea and 150 cm/sec (iqr 134 -168) on placebo, p = 0.0509. maximum tamv values had negative correlations with hemoglobin concentration (-0.47), fetal hemoglobin (-0.32), and oxygen saturation (-0.27); positive correlations were noted with age (0.27) and absolute neutrophil count (0.27). at the end of year 2, 187 tcd exams were conducted and all were suitable for analysis; the median velocity was 137 cm/sec on open-label hydroxyurea treatment, regardless of previous blinded treatment. all correlations with tamv were maintained except for age. conclusion: compared to placebo, hydroxyurea treatment for young children with sca living in uganda was associated with lower tcd velocities, which have been correlated in other studies with lower risk of primary stroke. tcd velocities were correlated with hematological and clinical parameters that can be improved by hydroxyurea therapy. children's hospital of richmond at virginia commonwealth university, richmond, virginia, united states background: acute chest syndrome (acs), defined by respiratory symptoms and a new pulmonary infiltrate, is a serious complication of sickle cell disease (scd). acs can occur during hospitalization for non-pulmonary conditions, such as a vaso-occlusive crisis or after surgery. nih clinical practice guidelines encourage incentive spirometry (is) which decreases the incidence of acs. it is additionally widely accepted that early, frequent ambulation in post-operative and pneumonia patients decreases the length of stay (los). to decrease acs events in children with scd at our children's hospital, we aimed for is use in 100% of ageappropriate pediatric sickle cell admissions. design/method: a multidisciplinary team examined inpatient acs prevention practices, including is, at children's hospital of richmond. key drivers were identified, including educational awareness of patients and healthcare staff, order placement, and documentation. we aimed for all scd patients ≥ 15 months of age hospitalized with any admission diagnosis to participate in is with the use of a traditional incentive spirometer or similar age-and ability-appropriate devices (e.g. positive expiratory pressure devices, bubbles, and pinwheels). we secondarily aimed to increase activity events, specifically ambulation and out of bed time. educational and outreach tools included patient informational brochure and incentive program, and staff informational sessions and reference materials at workstations. a disease-specific order set was implemented including desired is and activity orders. data were collected prospectively may through november 2017, during which 3 pdsa cycles were conducted. admissions during the corresponding months of the previous year were reviewed for comparison. independent t-test analysis was performed using graftpad prism 6 statistical analysis software. results: improvements reaching statistical significance included increase in is order placement from 44% to 89% of admissions (p < 0.01), and admissions with documented is use increased from 32% to 59% (p < 0.01). los decreased from a mean of 3.7 days to 2.7 days (p 0.02). post-admission development of acs also decreased from 12% to 4% of admissions, but did not reach statistical significance (p 0.18). there was an additional increase in appropriate activity order placement and documentation of activity events. conclusion: improving education and outreach to patients and staff, including implementation of a disease-specific order set, can improve is use and activity events. the decline seen in incidence of acs development during hospitalization, though not statistically significant, and the decreased los are encouraging, and efforts continue to improve on these trends. background: painful vaso-occlusive crises (voc) are a frequent and debilitating complication of sickle cell disease (scd) and are thought to occur due to progressive blockage of the microvasculature with rigid sickle shaped red blood cells. any trigger that decreases the microvascular blood flow (mbf) can promote entrapment of sickled cells in the microvasculature and progression to voc. exposure to cold wind and changes in weather are common triggers of voc and are associated with increased frequency of hospitalizations for pain in patients with scd. there is limited experimental data on the physiologic effects of these factors on peripheral perfusion in scd. to study the effect of graded thermal stimuli on the peripheral mbf in scd. design/method: 17 scd and 16 control (healthy or sickle trait) subjects aging 13 to 39 years were exposed to their individual threshold temperatures for heat and cold detection, heat and cold pain via tsa-ii thermode that was placed on the thenar eminence. mbf was measured on the contralateral thumb using photo-plethysmography (ppg). the vasoconstriction response within the complex ppg signal was detected using cross-correlation technique. mean mbf was derived from the ppg amplitude during each of these stimuli and compared to baseline mbf. cross correlation analysis showed that cold pain caused significant vasoconstriction response in 67% of the subjects, followed by heat pain (58%), cold detection (36%) and heat detection (18%).there was a significant drop in the mbf during cold pain (p <0.0001), heat pain (p <0.0001), heat detection (p = 0.0005) and cold detection (p = 0.02) when compared to baseline mbf, with cold pain causing the greatest drop in mbf. thermal sensitivity and mbf responses were comparable between scd and controls. conclusion: exposure to graded thermal stimuli causes a progressive drop in mbf with exposure to cold pain eliciting the strongest vasoconstriction response. vasoconstriction occurred in the contralateral hand at an average of 11 seconds after the stimuli, suggesting a neurally mediated mechanism. although there was no significant difference in vasoconstriction responses between scd and controls, the drop in mbf in patients with sickle cell disease can increase the likelihood of entrapment of the sickled red blood cells, leading to vaso-occlusion. these findings are consistent with extensive reports in literature that exposure to cold weather is associated with a higher frequency of voc. this suggests that neurally mediated vasoconstriction is likely an important factor in the pathophysiology behind cold exposure leading to voc in scd. background: vaso-occlusive crisis (voc) is a major cause of hospital admissions in children with sickle cell disease (scd). although the use of clinical biomarkers in voc has been studied, especially with regards to acute chest syndrome (acs), there is less data regarding overall voc severity prediction. in addition new biomarkers such as platelet to lymphocyte ratio (plr), neutrophil to lymphocyte ratio (nlr), and lymphocyte to monocyte ratio (lmr) have been little studied with regards to scd. objectives: to identify whether admission laboratory values, changes from well baseline laboratory values, and new biomarkers such as plr, nlr, and lmr could predict severity of vaso-occlusive crisis in children with sickle cell disease admitted with voc. design/method: this was a retrospective single center observational study of admissions of voc in children aged 1 -21 years with hbss or hbs-b0thal from september 2014 to november 2017 excluding those on hyper-transfusion protocol or having an admission diagnosis of acs. univariate analysis was done using student's t-test, mann-whitney non parametric test, or fischer's exact test as appropriate depending on the distribution between admission laboratory data of complete blood count (cbc), reticulocyte count, comprehensive metabolic panel, lactate dehydrogenase (ldh), change from well baseline cbc values within 6 months previously, plr, nlr, lmr, and the development of complicated voc. complicated voc was defined as the development of secondary acute chest syndrome, prolonged admission duration > 5 days (120 hours), requirement of blood transfusion, and readmission within 30 days. results: a total of 109 admissions were studied. fifty-nine (54.1%) were female. of the 109, 50 (45.9%) were complicated with no significant differences in sex (p 0.447) or age (p 0.435). univariate analysis revealed significant elevations in total bilirubin (p 0.017), ldh (p 0.010), and platelet count (p 0.019) in those with complicated voc. there is also significant difference in the percentage change of platelet count from baseline with greater decline in uncomplicated voc (p 0.014). there were no significant differences in plr (p 0.186), nlr (p 0.775), or lmr (p 0.445). conclusion: elevations in total bilirubin, ldh, and platelet count in admission laboratory values are associated with developing complicated voc. in addition, those with complicated voc present with significantly less decline in platelet count from baseline well cbc. plr, nlr, and lmr do not seem to be useful predictive biomarkers for severity of voc. background: sickle cell disease (scd) causes health problems of varying frequency and severity. the only validated biomarker for children with scd is transcranial doppler. if reliable predictors existed for scd severity, children with scd could be treated according to risk category. many patients with scd face psychosocial or economic hardships, but these factors have not been evaluated as risk markers for medical or functional severity of scd. objectives: the goal of this project was to develop and stratify a preliminary list of psychosocial risk factors for health outcomes that could be used as scd severity predictors. st. vincent institutional review board. a list of potential psychosocial risk factors for adverse health outcomes was compiled based on assessment materials utilized by the sickle safe program (indiana's hemoglobinopathy newborn screening follow-up program). this list of 39 items was distributed to child abuse prevention (12) and scd (17) experts, who ranked each item on a likert scale of 1 (least important) to 5 (most important). mean scores were calculated using spss version 24; 163 assessments were retrospectively analyzed to determine psychosocial risk factor frequency. risk factors occurring in ≥15% of homes were considered high frequency events. overall, there was high agreement among experts on the risk factors that were considered the most important predictors of severe scd outcomes. the risk factor with the highest frequency (92%) was eligibility for public assistance programs. fifteen risk factors were rated ≥4 by the experts. four (26.7%) were high frequency events occurring in ≥15% of homes: a child with hbss or hbs 0thalassemia not taking hydroxyurea (15%); parent report that they had treated a fever (>101®f) at home in the past 6 months (25%); tobacco use by someone in the household (23%); and the family reporting significant psychosocial stressors in the past year (30%). tobacco use in the home was significantly correlated with several other risk factors (smoking during pregnancy [r = 0.503], other health concerns in the child [r = 0.459], and child having health insurance [r = -0.459]), suggesting that it is part of a constellation of health risk. in general, the risk factors that were rated as most important for health outcomes occurred less frequently in the sample. this study represents important progress toward identifying a group of psychosocial risk factors for scd severity, which is a necessary first step for future investigation of empirical relationships between candidate risk factors and scd outcomes. unitversity of cartagena, cartagena, bolivar, colombia s109 of s301 background: sickle cell disease is an autosomal recessive disorder characterized by a mutation in the -globin chain, which produces hbs. acute and chronic complications as aplastic crisis, acute chest syndrome, priapism, stroke, leg ulcers and primary/secondary prevention of stroke can be treated with simple transfusion or exchange transfusion. the latter offers advantages as lower iron overload, post-treatment hbs goal control, lower viscosity and improved microvascular circulation. but it is not a widely-used option because is associated with technical difficulties. objectives: standardization of a new partial exchange transfusion protocol in a group of patients with sickle cell disease, within the framework of a chronic transfusion program. design/method: this is a prospective descriptive study, which included 25 patients under 18 years with sickle cell disease (20 hbss, 5 hbs-tal), with indication of partial exchange transfusion in a chronic transfusion program, according to the institutional protocol; patients who fulfilled the inclusion criteria were enrolled in the study between february 2016 and december 2017. a registry of the medical and technical complications was made in each of the procedures. a database was constructed in excel, and the graph-pad prism® version 6 oc software was used for statistical analysis. the sequence is as follows: isovolemic phlebotomy and transfusion of packed red cells. depending of the recent hemoglobin level (48 hrs), we do the phlebotomy there: hb:7-7.9: 10 cc/kg, hb: 8-8.9: 15 cc/kg, hb>9: 15 cc/kg; isovolemic solution (ns 0,9%) there: hb:7-7.9: 10 cc/kg, hb: 8-8.9: 15 cc/kg, hb>9: 15 cc/kg and packed red cell transfusion there: hb:7-7.9: 15 cc/kg, hb: 8-8.9: 15 cc/kg, hb>9: 10 cc/kg. the safety of this exchange transfusion protocol was analyzed in 25 patients with sickle cell disease (176 procedures). there were no differences in the sex distribution, and the median age was 8 years. 80% of the population was homozygous. the indication of transfusion was 52.27%(92/176) primary stroke prevention, 44.31%(78/176) secondary stroke prevention and 2.84%(5/176) was other reason. a low percentage of complications was found (7.3%); of which, those of medical origin (hypotension and nausea/vomiting) were only presented in 2.2% of the total procedures. the standardization of this protocol was safe and its use could be extended to other low-income centers that treat patients with sickle cell disease that need chronic transfusion program including patient with hemoglobin level until 7gr/dl. we suggest do studies for measure the security and efficacy of this protocol in patients with acute complications. background: clinical trials that aim to achieve pain reduction have challenges achieving clinical endpoints as pain has no quantifiable biomarkers and may be unrelated to scd. furthermore, the threshold of seeking medical care differs between patients and vocs that occur at home are missed. we present a non-interventional, longitudinal study to identify vocs in patients with scd. objectives: to examine the longitudinal relationship between pros and biomarkers in subjects with scd before, during, and after a self-reported voc event, in order to build a model of in-home and clinical voc and to collect longitudinal pros and biomarker data from subjects that span voc events in the home, clinic and the hospital. design/method: longitudinal measures of pain, fatigue, function, activity, and biomarkers from scd patients in steady state and voc were studied over a six month period. patients self-reported pain, fatigue, function, and medication use using a novel epro tool. voc was reported in real-time, triggering a mobile phlebotomy team. blood was collected sequentially after self-reported voc (at home or hospital). blood samples were drawn two days after resolution of voc, as reported by the patient. during non-voc periods, blood was drawn every 3 weeks to establish a baseline. biomarkers included leukocyte-platelet aggregates and circulating microparticles, cell and soluble adhesion molecules, cytokines, inflammatory mediators and coagulation factors. patients wore an actigraphy device to track sleep and activity and rest. results: twenty-seven of thirty-five patients experienced a total of 286 days with voc >4 hr, of which only 58 days resulted in healthcare utilization. voc days had significantly higher pain and fatigue scores. voc days were associated with significantly decreased functional scores, with significantly greater decreases during vocs requiring medical contact compared to at-home vocs. different activity profiles were identified for non-voc, at-home voc and medical contact voc days by actigraphy monitoring. at-home voc days exhibited increased daytime resting compared to non-voc days. medical contact vocs had decreased average and peak activity, and increased daytime resting compared to non-voc days. a sleep fragmentation index trended up for both at-home (16%) and medical contact voc days (18%). significant changes during voc days were observed in: c-reactive protein (54% increase), nucleated rbc (34% increase), monocyte-platelet aggregates (25% increase) and neutrophil-platelet aggregates (35% increase), interleukin-6 (112% increase), interleukin-10 (19% increase) and tnfalpha (14% increase). the identification and assessment of at-home vocs through use of epros, actigraphy and biomarkers is feasible as demonstrated by this innovative at-home study design. background: risk-stratifying sickle cell disease (scd) patients and demonstrating response to disease-modifying therapies is challenging due to the phenotypical heterogeneity of scd. a pathogenic role for procoagulant von willebrand factor (vwf) via excess vwf high molecular weight multimers (hmwm) has been proposed, with variable reports of increased vwf and hmwm in crisis vs. steady-state in adults, but less so for vwf in children with scd. moreover, vwf and multimers have not been studied in sickle trait. objectives: our pilot study evaluated the potential for vwf antigen (vwf:ag) and hmwm on densitometric tracings to serve as biomarkers for disease severity or treatment response in children and young adults with scd compared to sickle trait (hbas) siblings. design/method: we evaluated vwf:ag, vwf multimers and retrospective clinical data from 10 hbss, 3 hbsc and 5 hbas subjects at steady state. one hbsc subject also had a crisis sample. median scd age was 17 years (8.0-20.1 years). 46% were female. scd severity was judged by annual vasoocclusive and acute chest events, or stroke/elevated tcd. eight of 13 (6 hbss and 2 hbsc) took hydroxyurea. four hbss subjects had severe scd, all of whom were chronically transfused. results: mean vwf:ag (normal 50-160 iu/dl) was higher for hbss (175+/-17.4) and severe hbss (195+/-33.5) compared to hbsc (103+/-3.2, p = 0.049 and 0.044, respectively); however, lacked statistical significance when compared to hbas (152+/-34.5, p = 0.52 and 0.41, respectively). vwf:ag was elevated in 7/10 (70%) steady-state, including 3/4 (75%) with "severe" disease on chronic transfusion and 4/6 (67%) taking hydroxyurea, in 1 hbsc crisis but no hbsc 3/3 (100%) at baseline. vwf:ag was high in 2/5 (40%) hbas siblings. four (31%) had increased hmwm at baseline: 1 hbss/severe disease/chronic transfusion, 2 hbss/hydroxyurea and 1 hbsc untreated. hmwm were increased only during vaso-occlusive crisis in 1 hydroxyureatreated hbsc subject. no ultra-large hmwm were observed. in this preliminary study, in young scd subjects, vwf:ag trended higher in hbss vs. hbsc and in severe hbss participants at a single time-point, but serial evaluations at baseline, in crisis and with optimized diseasemodifying therapy are needed to determine the potential of vwf:ag and hmwm as biomarkers for severity or treatment response. surprisingly, vwf:ag was high in some sickle trait subjects. since hbas is associated with some health challenges such as increased thrombosis risk, further examination of vwf and endothelial dysfunction in sickle trait may provide novel insights into its role as a biomarker. background: the 2014 national heart lung & blood institute(nhlbi) guidelines for acute management of voe recommends rapid evaluation and treatment of pain, including administration of a parenteral opioid within 30-minutes of triage or 60-minutes from registration, pain reassessment & repeat opioid delivery within 15-30-minutes. inf use has been increasing in peds due to its rapid onset and ease of administration. objectives: to evaluate ped utilization of inf & its effect on intravenous (iv) opioid administration and pain control for the treatment of voe. design/method: a retrospective review of 250 emr was performed on children with scd±2years presenting to a ped with voe (pain scores 6 on a 0-10 scale) from jan-june 2017. variables studied were median time (iqr,95%ci) from ped arrival to first-parenteral-opioid-administration, time-to-first-iv-opioid, first & final pain score, disposition and readmission rate. time-to-first-iv-opioid was also compared to historical data (jan-dec2012,n = 231) prior to inf protocol initiation. . additionally, 15% patients received iv opioids within 60 minutes of ed arrival in the inf+iv opioid vs. 40% in the iv opioids alone group (p<0.01). no differences in 72-hour-returnrates were found in any of the groups, including inf alone group. conclusion: use of inf in the ped for voe is an excellent strategy to shorten time-to-first-parenteral-opioidadministration, improve pain scores & improve adherence to the nhlbi guidelines. however we had 2 distinct unexpected findings: (1) delays in iv opioid delivery after inf use & (2) inf alone appeared to provide sufficient pain control without iv opioids for disposition home in 17% of voe patients. whether the latter reflects insufficient pain management or that there is a milder subgroup for whom inf alone is sufficient, requires further investigation. this study illustrates our experience with a ped-based inf protocol in terms of unanticipated delays in iv opioids and also discharges after inf alone. efforts are underway to further improve use of inf in voe management. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: folate supplementation is commonly included as standard management in patients with sickle cell disease. however, clear evidence supporting the clinical benefits of this practice is lacking. a single study demonstrated improvement on the occurrence of repeat dactylitis at a higher dose of folic acid. to compare clinical outcomes in pediatric patients with sickle cell disease treated with folate supplementation versus those who were not. design/method: this study was a retrospective chart review that included patients 3 to 23 years old with sickle cell disease type ss and s 0 followed at st. christopher's hospital for children. data collected included information about folate supplementation, red cell indices and the presence or absence of clinical outcomes including vaso-occlusive crisis requiring hospitalization in the last six months, acute chest syndrome, infections, asthma, sleep apnea, nephropathy, cerebral vascular disease, stroke and avascular necrosis. analysis of variance (anova) was used to evaluate mean differences between age, number of infections, number of voc events, hemoglobin, reticulocyte count, and mean corpuscular volumes. additionally, chi square analysis was implemented to evaluate differences in folate and non-folate groups for left ventricular remodeling (lvr), sickle cell nephropathy, asthma, obstructive sleep apnea (osa), nocturnal hypoxia, and avascular necrosis (avn). mean differences between the folate and non-folate groups were compared for patients on and off hydroxyurea therapy. one hundred and seven patients met inclusion criteria following review of clinical data. of the patients included in the study, 45 patients were found to be taking folate (42%), while 62 patients were not (58%). statistical analysis showed that there were no significant differences in the incidence of clinical outcomes between patients on folate versus those who were not on folate. of the patients who were not on hydroxyurea, hemoglobin levels were significantly higher in patients on folate versus those who were not (p = 0.053), but not significantly different for the patients on hydroxyurea. this study suggests that folate supplementation makes no significant impact on the red blood cell indices of anemia nor on the incidence of adverse clinical outcomes in children with sickle cell disease. however, a larger prospective study is needed to guide future considerations for folate supplementation in sickle cell patients in the clinical setting. background: tanzania ranks 3rd globally for the number of infants born annually with sickle cell disease (scd) but lacks a national newborn screening program. the prevalence of sickle cell trait (sct) and scd is highest in the northwestern regions around lake victoria served by bugando medical centre (bmc) a teaching and consultancy hospital in mwanza. bmc also houses the hiv early infant diagnosis (eid) laboratory that tests dried blood spots (dbs) from hivexposed infants. dbs can be tested for hiv and then retested for sickle cell trait and disease. to determine the prevalence of sickle trait and disease by region and district in northwestern tanzania using existing public health infrastructure. secondary objectives explored associations between sct, scd, malaria and hiv. design/method: the tanzania sickle surveillance study (ts3) is a prospective year-long cross-sectional study of hivexposed infants born in northwestern tanzania, whose dbs collected by the eid program are tested at bmc and available for further testing of sct and scd. samples from children ≤24 months of age were tested by isoelectric focusing (ief) and scored independently by two tanzanian staff as normal, sct, scd, variant, or uninterpretable. dbs samples scored as disease or variant were repeated. over the course of 9 months, 157 ief gels have been run. a total of 10,019 dbs samples have been scored, including 9,567 from children less than 24-months old. the overall prevalence of sct is 20.65% and the prevalence of scd is 0.99%, along with 0.10% hemoglobin variants. quality of the laboratory results is extremely high, with only 0.15% dbs samples yielding an uninterpretable result. geospatial mapping of the first 5,900 samples revealed a regional scd prevalence ranging from 0.3% up to 2.0% among the 9 regions served by bmc. the prevalence of sct and scd is very high in northwestern tanzania. geospatial mapping will identify high prevalence areas where targeted newborn screening can be started using existing public health infrastructure with minimal start-up cost and training. further data will enhance the accuracy of the map to the district level. background: pediatric patients with sickle cell disease (scd) could develop obstructive, restrictive or mixed abnormalities of pulmonary function (pf). several publications report progressive worsening of pf over time, which could lead to severe morbidity in adult patients with sickle cell disease. in adults with sickle cell anemia up to 20-30 % of mortality is related to lung disease. early intervention aimed at improvement of lung function could significantly decrease morbidity and possibly improve life expectancy. among disease modifying approaches commonly used in scd are hydroxyurea (hu) and chronic prbc transfusions. both interventions lead to increase of hemoglobin, decrease of hbs fraction, leading to decreased hemolysis. reports of effect of hu on pulmonary function are conflicting with some suggesting no effect and others proposing a slower decline of pulmonary function. the goal of our study is to evaluate effect of disease modifying therapies, like hu and chronic prbc on change of pulmonary function in pediatric patients with sickle cell disease. design/method: this study utilized a retrospective chart review of children with scd who had multiple pfts. we analyzed 286 pfts from 80 patients done during clinic visits. scd patients were divided into three treatment groups: hydroxyurea, chronic transfusions or neither. data was analyzed with linear correlations and analysis of variance (anova). comparison were made between the three groups specifically observing the changes in absolute numbers on pfts over time using the first and last pft the patient had. results: there were a total of 80 patients with multiple pfts (ranging from 2-7); control (40), hydroxyurea (23) and chronic transfusion (10). the mean changes of the control, and hydroxyurea for the pft parameters fev1 (-5.53 the chronic transfusion group demonstrated a small improvement in pfts over time for fev1 (0.300), fvc (1.300), fef25-75 (0.400), however there was a decline in fev1/fvc (-0.013). however, there was no statistically significant (p-value <0.005) in the difference in any pfts parameters between any of the groups. in children with scd there is a decline of pf parameters over time. although no significant differences were seen between the three groups it appears chronic transfusion may improve or limit the decline in pfts. larger studies need to be done to evaluate difference in pf decline in patients with scd patients. background: the use of mobile technology in health care has been a growing trend. patients with chronic diseases such as sickle cell disease (scd) require close monitoring to provide appropriate treatment recommendations and avoid complications. we conducted a feasibility study for patients with scd hospitalized for pain using our self-developed mobile application (tru-pain: technology resources to better understand pain) and a wearable activity tracker. subjective symptoms such as pain and objective data such as heart rate (hr) were measured. we aimed to 1) correlate nursing recordings with mobile technology recordings; 2) get feedback from patients about usability. design/method: we enrolled patients with scd >8 years old and <36 hours from admission for uncomplicated vasoocclusive crisis, excluding patients admitted to icu. patients were given an ipad and a wearable device. they were instructed to record in the application at least once per day and to keep the wearable on, removing only to charge. prior to discharge, patients completed a feasibility questionnaire. we enrolled 20 patients, 40% females, median age 17.5 (range 13 to 54) who were admitted for a median 5 days (range 2 to 8) for uncomplicated pain crisis. patients used the application throughout hospitalization and made one entry/day (range 0 to 2). pain scores recorded via tru-pain correlated well (r = 0.74, p<0.005) with pain scores recorded in emr. there was an average of 10,930 data points recorded per day, by the wearable, with a maximum of 54,693 data points/day. the median amount of hours of wearable data per day was 4.21 (maximum of 18.05). the hr recorded via the wearable correlated significantly with the hr recorded in emr (r = 0.69, p-value < 0.005). as for usability, 70% of patients indicated never having a problem with the technology, 90% found tru-pain 'very easy' or 'somewhat easy' to use, and 60% were 'very satisfied' with their participation in the study, indicating that it helped them track their pain. our pilot study during hospitalization shows strong potential for using tru-pain for patients with scd. pain data from application and hr from wearable correlated well to the emr data. according to the feedback received, our application was easy to use and helped patients track their pain. despite limitations of battery life, the use of wearable technology is feasible, providing additional data such as activity. we are optimistic that we can continue to improve our tru-pain system to help improve care in patients with scd. background: hydroxyurea, chronic blood transfusion, and bone marrow transplantation can reduce complications, and improve survival in sickle cell disease (scd), but are associated with a significant decisional dilemma because of the inherent risk-benefit tradeoffs, and the lack of comparative studies. these treatments are underutilized leading to avoidable morbidity and premature mortality. there is a need for tools to provide patients high-quality information about their treatment options, the associated risks, and benefits, help them clarify their values, and allow them to share in the process of informed medical decision making. objectives: to develop a health literacy sensitive, web-based, decision aid (ptda) to help patients with scd make informed choices about treatments, and to estimate in a randomized clinical trial the acceptability and effectiveness of the ptda in improving patient knowledge, involvement in decisionmaking and decision-making quality. design/method: we conducted qualitative interviews of scd patients, caregivers, stakeholders, and healthcare providers for a decisional needs assessment to identify decisional conflict, knowledge, expectations, values, support, resources, decision types, timing, stages, and learning, and personal clinical characteristics, and to guide the development of a ptda. transcripts were coded using qsr nvivo 10. stakeholders completed alpha and beta testing of ptda. we conducted a randomized clinical trial of adults, and of caregivers of pediatric patients to evaluate the comparative efficacy of the ptda, vs. standard of care. results: ptda (www.sickleoptions.org) was developed per decisional needs described by 223 stakeholders and finalized following alpha testing, and beta testing by 68 and 87 stakeholders respectively. in a randomized trial of 120 subjects considering various treatment options, qualitative interviews revealed a high level of usability, acceptability, and utility in education, values clarification, and preparedness for decision making of the ptda. a median 68% rated the acceptability of ptda as good or excellent and provided narrative comments endorsing the acceptability, ease of use, and utility in preparation for decision making. the ptda met international standards for content, development process, and efficacy with the exception of having a full range of positive and negative experiences in patient stories. compared to baseline ptda group had statistically significant improvement in preparedness for decision making (p = 0.005) and informed subscale of decisional conflict (p = 0.02) but not for decisional self-efficacy, knowledge, choice predisposition, or stages of decision-making. a ptda for patients with scd developed following extensive engagement of key stakeholders was found to be acceptable, useful, easy to use, to improve preparedness for decision making, and decrease decisional conflict. background: painful vaso-occlusive crisis (voc) accounts for the majority of emergency department (ed) visits and hos-pitalizations in sickle cell disease (scd). we are interested in studying mental stress and associated autonomic nervous system (ans) imbalance that cause vaso-constriction as possible triggers of scd pain. to this end, we developed a mobile phone application (app) to record daily pain frequency and intensity as clinical endpoints that might be predicted by ans parameters measured in the laboratory. in particular, we think that the aura may represent ans instability that precedes or even triggers change in blood flow and voc. objectives: to assess the feasibility of using an app to evaluate frequency and severity of voc and its potential association with mental stress and presence of aura. design/method: an app was developed for both ios and android systems to allow patients to track pain, stress, and aura. the idea was to create an app that was easy to use with the intent to only capture pain episodes, rather than detailed description of the pain. all scd patients were eligible and a parent version was available for younger children. de-identified data was automatically transferred to a hipaa compliant database via a cloud-based server interfaced to the main research project database. a feedback questionnaire was implemented after at least a month of utilization to assess usability. of the 51 scd patients enrolled, 39 participants utilized the app and 21 of the 23 participants that provided feedback indicated the app was easy to navigate. the mean pain scale was 6 out of 10 (standard deviation 1.97) for those that entered they had pain that day. although the mean stress level was 3 out of 10, there was a statistically significant correlation between increasing stress levels and increasing pain scores (p < 0.05). aura was reported by 26 patients, with 5 patients reporting more than 10 episodes. moreover, on days aura was present there was greater incidence that pain was present as well (p < 0.05). however, there was no statistically significant association between pain intensity and presence of an aura (p = 0.14). conclusion: consistent with prior research, reported pain intensity is significantly associated with reported stress intensity. although there was an association between presence of aura and pain, it did not seem to correlate with pain intensity. this uniquely designed app can monitor scd pain clinically and help understand the role of sickle dysautonomia in the genesis of scd pain. university of florida college of medicine, gainesville, florida, united states background: evidenced-based guidelines recommend the emergent evaluation of fever in children with sickle cell disease (scd). as the prevalence of bacteremia has decreased, outpatient management has become more common. however, fever can sometimes herald other complications of scd, such as acute chest syndrome, vaso-occlusive pain crisis, splenic sequestration, or aplastic crisis. institutional practices regarding fever management in scd remain variable, and little is known about the clinical outcomes of children hospitalized for uncomplicated fever. objectives: the primary objective was to determine the rate of bacteremia or scd-related complications per febrile episode in children with scd admitted to a single institution between january 2014 and june 2017 for uncomplicated fever. this was a retrospective cohort study of febrile patients up to 21 years of age with scd, any genotype, admitted to the university of florida during the defined study period. eligible patients were identified by a database search using admitting diagnosis codes for scd and fever based on the international classification of diseases 9th and 10th revisions. encounters were manually reviewed to confirm eligibility. patients were excluded if they had other indications for hospitalization apparent at the time of admission, such as an acute vaso-occlusive episode requiring parental narcotics, asthma exacerbation, or additional complications of scd. the database search identified 211 encounters, of which 83 were excluded based on confounding indications for hospitalization. sixty-three eligible patients accounted for 128 hospitalizations. the median age was 2 years (range 5 weeks-17 years); 60.2% were male. mean duration of hospitalization was 2.6 days (range 1-13 days). eight positive blood cultures were identified; six of these were classified as contaminants. bacteremia or the development of a scd-related complication was identified in 18 (14.06%) admissions. these included acute chest syndrome (n = 4), bacteremia (n = 2), splenic sequestration (n = 1), and red cell transfusion (n = 11). exploratory analyses of potential predictors of bacteremia or scd-related complications showed no association with the presenting white blood cell count or degree of fever (p = 0.36). of the patients classified as having a scd-related complication, 94% had hemoglobin ss disease and 78% had at least one prior documented complication. 64% of the patients transfused had at least one prior transfusion. conclusion: while improvements in preventative care have substantially lowered rates of bacteremia in children with scd, fever warrants careful evaluation for other acute scdrelated complications. providers should consider inpatient observation in select cases. additional studies are warranted to define subsets of patients suitable for outpatient fever management. background: children with sickle cell disease (scd) exhibit lower neurocognitive functioning than healthy peers, even in the absence of stroke. among the domains commonly affected, working memory (wm) seems particularly affected by disease processes and wm deficits have significant implications for academic achievement and disease selfmanagement. few interventions to improve working memory in pediatric scd have been evaluated. to determine the effects of cogmed, a homebased computerized wm training intervention, in children with scd using a randomized controlled trial design. design/method: participants (ages 7-16) with scd completed a baseline neuropsychological assessment and those with wm deficits were randomized to either begin cogmed immediately or enter an 8-week waitlist. cogmed is a homebased intervention completed on an ipad that consists of 12 increasingly challenging exercises targeting visual-spatial and verbal wm, practiced over 25 sessions. at the end of training, participants completed a post-intervention neuropsychological assessment, including tests of visual-spatial and verbal wm from the wechsler intelligence scale for children-fifth edition (wisc-v). results: ninety-one participants (m age = 10.43, sd = 2.93; 59% female; 69% hbss) enrolled in the study; 52% (n = 47) exhibited wm deficits and were randomized to either begin cogmed immediately or wait 5-8 weeks before starting cogmed. among those that have received the intervention and reached the end of their training period (n = 42), 27 participants (59%) completed at least 5 cogmed sessions, 19 (41%) finished at least 10 sessions, and 7 finished at least 20 sessions (15%). the mean number of completed cogmed sessions was 9.10 (sd = 7.77). paired samples t-tests revealed significant improvements on the working memory index (t[38] = -2.44, p = 0.020) and on the digit span (t[40] = -3.02, p = 0.004), and spatial span-backward (t[39] = -2.83, p = 0.007) subtests. improvements were especially pronounced for participants completing at least 10 sessions. partial correlations controlling for respective baseline scores indicated that the number of cogmed sessions completed was positively correlated with post-test scores on digit span (r = .38, p = .017) and spatial span-backward (r = 0.45, p = 0.004) subtests. among participants who completed at least 10 cogmed sessions, 77% scored in the average range or higher on the working memory index at the post-intervention assessment, compared to 58% at baseline. results support the efficacy of cogmed in producing significant improvements in wm. a dose-effect was observed such that participants who completed more cogmed sessions had greater improvements in wm. home-based cognitive training programs may ameliorate scd-related wm deficits but methods for motivating and supporting patients as they complete home-based interventions are needed to enhance adherence and effectiveness. background: sickle cell disease is associated with myriad complications that lead to significant morbidity and early mortality. hydroxyurea has been used successfully to reduce the incidence of these complications and has led to significant improvements in quality and duration of life. at children's minnesota we recommend hydroxyurea in all patients with hb ss/s 0 thalassemia as early as 5 months of age with a goal of starting all patients before 12 months of age. objectives: the purpose of this study was to evaluate the use of hydroxyurea therapy in young patients with sickle cell disease, with particular attention to those children less than one year of age. design/method: a retrospective chart review was conducted on patients less than 5 years of age with sickle cell disease who began hydroxyurea therapy between january 1, 2008 and december 31, 2016. the study population was divided into three cohorts based upon age at hydroxyurea initiation: cohort 1 (0-1 year), cohort 2 (1-2 years), and cohort 3 (2-5 years). outcomes included laboratory data, clinical events (hospitalization, dactylitis, pain crisis, transfusion, splenic sequestration, acute chest syndrome), and toxicity occurring in the first 2 years of life. results: a total of 65 patients were included in cohorts 1 (n = 35, mean age 7.2 months), 2 (n = 13, mean age 19.5 months), and 3 (n = 17, mean age 35.5 months). patients in cohort 1 had higher hemoglobin (p = 0.0003) and mcv (p = 0.0199) and lower absolute reticulocyte count (p = 0.0304) when compared to cohort 3. the wbc (p = 0.0007, <0.0001) and anc (p = 0.0364, 0.0025) were significantly lower compared to both older cohorts. however, no patient had therapy held because of neutropenia. the mean baseline hemoglobin f in cohort 1 was 31.5% compared to 19.7% and 16.5% in cohorts 2 and 3 respectively (p = 0.002, p<0.0001). the mean duration of therapy in cohort 1 was 31.3 months, compared to 57.6 months in cohort 2 (p = 0.018) and 29.1 months in cohort 3 (p = 0.401). during this time, hb f levels remained higher in cohort 1 (mean 29.9%) compared to cohorts 2 and 3 (mean 20.4%, p = 0.007 and mean 20.6%, p = 0.003). patients in cohort 1 experienced fewer hospitalizations (p = 0.0025), pain crises (p = 0.0618), and transfusions (p = 0.0426). there was no difference in toxicity between groups. hydroxyurea was used safely in infants 5 to 12 months of age and resulted in more robust hematologic responses and a decrease in sickle-related complications when compared with patients starting hydroxyurea later in life. children's national health system, washington, district of columbia, united states background: children with sickle cell disease (scd) have a significantly greater risk of silent or overt cerebral infarction than the general population. infarcts are associated with declines in cognitive functioning and academic achievement. while infarcts are reliably identified using mri, scans are expensive and occasionally necessitate sedation. moreover, mri's are not recommended for routine monitoring of cerebral infarcts. additional tools are needed for discriminating the presence of a cerebral infarct that are brief, noninvasive, inexpensive, and repeatable. objectives: to evaluate differences in performance on cogstate, a computerized neurocognitive assessment, in patients with scd with and without history of cerebral infarct. design/method: participants included 112 children with scd ages 7-16 (m = 10.61, sd = 2.91; 58% female; 70% s117 of s301 hbss) enrolled in a cognitive intervention trial. participants completed the cogstate pediatric battery, which measures processing speed, sustained attention, verbal learning, working memory, and executive functioning. history of silent or overt infarct was determined via health record review. participants also completed measures of intelligence (iq) and math fluency. results: participants' standard scores across most neurocognitive measures were lower than expected compared to the standardization sample (mean iq = 91.03, sd = 13.34). thirty percent of participants (n = 33) had a documented history of cerebral infarct. participants with a history of cerebral infarct scored lower on cogstate tasks measuring sustained attention (t[108] = 2.93, p = 0.004) and executive functioning (t[98] = 2.46, p = 0.016), as well as on a measure of math fluency (t[88] = 2.16, p = 0.033). receiver operating characteristic (roc) analyses demonstrated that the cogstate task measuring sustained attention was a fair discriminant of patients with and without a history of infarct (auc = 0.75, ci95 = 0.65-0.84, p = 0.0001), whereas iq score was not (auc = 0.58, ci95 = 0.46-0.71, p = 0.19). cogstate processing speed and sustained attention tasks fairly discriminated between patients with at least average or below average intelligence (auc = 0.77, ci95 = 0.67-0.86, p = 0.00001 and auc = 0.74, ci95 = 0.64-0.84, p = 0.0001, respectively). finally, the cogstate processing speed task was good at discriminating between at least average or below average math fluency (auc = 0.80, ci95 = 0.70-0.90, p<0.00001). multiple tasks in the cogstate pediatric battery appear to adequately identify patients with a history of cerebral infarcts. in addition, cogstate tasks appear to be fair predictors of impairments in iq and academic achievement outcomes. cogstate is inexpensive and can be easily administered in a medical setting with minimal training in approximately 20 minutes. results support the potential for cogstate to be used as a screening tool for medical and neuropsychological abnormalities in children with scd. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: cardiovascular disease contributes to the morbidity and mortality of patients with sickle cell disease (scd). hydroxyurea therapy in scd has known clinical efficacy including improving anemia, decreasing episodes of vasoocclusive crisis and acute chest syndrome, and decreasing mortality. effect of hydroxyurea on cardiac function in children with scd is not well studied. an earlier study suggested the protective effect of hydroxyurea on left ventricular (lv) hypertrophy in scd. we hypothesized that hydroxyurea use would be associated with decreased lv remodeling and improved cardiac function. we aimed to evaluate the association between hydroxyurea use and lv remodeling and cardiac dysfunction in children with scd. design/method: we completed a retrospective study of patients with scd who were 10 to 22 years old, followed at st. christopher's hospital for children and had an echocardiogram completed in the past 18 months. data collected included gender, bmi, scd genotype, hydroxyurea use, chronic transfusion use, and 2d and doppler echocardiographic parameters. cardiac structure, geometry, systolic function, and diastolic function echocardiogram parameters were included. analysis of variance (anova) tests were performed to assess for statistical significance of differences in cardiac parameters between patients with and without hydroxyurea use. analysis of covariance (ancova) tests were performed to control for age. results: demographic and echocardiogram data was collected on all 93 patients who met inclusion criteria. of the 93 patients included, 31 (33%) were on hydroxyurea therapy. patients on hydroxyurea had significantly lower mean relative wall thickness (p = 0.026) and significantly higher mean peak early lv filling velocities (p = 0.032) and peak early lv filling/septal annuli early peak (e/ea) velocities (p = 0.002); however, only the e/ea velocities remained significant when controlling for age (p = 0.001). mean peak early lv filling velocities approached significance when controlling for age (p = 0.052). hydroxyurea therapy resulted in a significantly higher e/ea velocity, suggesting that these patients had worse diastolic function. it is possible that the patients initiated on hydroxyurea already had worse disease manifestations than those not on hydroxyurea, possibly accounting for the decreased diastolic function. when controlling for age, hydroxyurea use did not result in significant differences in cardiac structure parameters, systolic function parameters or cardiac geometry. prospective studies and larger sample size are needed to validate our findings, examine for additional statistically significant differences, and develop preventive strategies for cardiovascular disease in children with scd. background: acute chest syndrome (acs) is now the leading cause of death in children with sickle cell disease; mortality in the u.s. is reported to be 1-2% and is mostly due to respiratory failure. early transfusion improves clinical outcomes. although patients with concurrent asthma are considered at increased risk for poor outcomes, risk factors for respiratory failure in pediatric acs have not been well-defined. to determine whether specific epidemiological and clinical features of children hospitalized with acs are predictive of the need for mechanical ventilation. design/method: data from the kids' inpatient database were reviewed to identify patients age < 20 years with a discharge diagnosis of acs for the years 2003, 2006, 2009, and 2012 . outcomes were defined by the international classification of diseases, ninth revision, clinical modification code. data were weighted to estimate total annual hospitalizations according to hospital characteristics in the united states. trends in healthcare costs, length of hospital stay, transfusion, and mechanical ventilation use were analyzed using multivariable linear regression. in addition, multivariable logistic regression was used to ascertain specific clinical or epidemiologic factors associated with mechanical ventilation use after adjusting for patient and hospital characteristics. the total hospitalizations for acs were 5,018 in 2003; 6,058 in 2006; 6,072 in 2009; and 6,360 in 2012. reported use of mechanical ventilation ranged from 2.8% to 5.6% and was associated with non-black compared to black children (or, 1.53; 95%ci, 1.02 to 2.31) and the fall season (or, 1.36; 95%ci, 1.05 to 1.74), but not with age, preexisting asthma or hb-genotype. comorbidities of obesity (or, 3.35; 95%ci, 1.94 to 5.78), obstructive sleep apnea (or, 3.72; 95%ci, 2.23 to 6.20) and heart disease (or, 2.19; 95%ci, 1.47 to 3.27) were associated with mechanical ventilation use. the use of simple and exchange transfusion during all acs admissions ranged from 30.1% to 40.5% and 2.6% to 2.9%, respectively. among pediatric acs patients, those with obesity, obstructive sleep apnea or heart disease were at increased risk for respiratory failure and might benefit from early intervention (e.g., transfusion). surprisingly, asthma in children with acs does not appear to be a distinct risk factor for respiratory failure, and further studies are needed to clarify whether differences in treatment approach (e.g., addition of corticosteroids, bronchodilators) might impact on acs progression and/or severity even in high risk patients without asthma. objectives: to compare pulmonary functions between aa and k children with scd and to assess if a high hb f level contributes to better function. design/method: a cross sectional study was done on children with scd (hb ss disease) followed in comprehensive sickle cell programs. aa patients were followed at brookdale hospital, ny and k patients were followed in mubarak hospital, kuwait. children between the ages of 6 and 22 years who had pulmonary function tests (pft) done as a routine screening were enrolled. pft was done using spirometer and plethysmography. patients with congenital or anatomical lung abnormality, heart disease, pulmonary disease such as acute chest syndrome, acute asthma or pneumonia within 4 weeks were excluded. results: there were 74 children (37 in each group) with scd,. restrictive pattern on pft was seen in 18/37 (49%) of aa vs. 10/37 (27%) of k (p>0.05). obstructive pattern was seen in 6/37 (16%) of aa vs. 13/37 (35%) of the k group (p>0.05). in both groups, 13 children (35%) had normal pft. three/13 (15%) in the aa group had a hb f>20% as compared to 11/13 (85%) in the k group (p<0.01). abnormal pft was noted in 24/37 children (65%) in each group. hbf was >20% in 3/24 (13%) in the aa group vs. 15/24 (63%) in the s119 of s301 k group (p<0.01). in patients with abnormal pft, mean hbf was 10.4±8.4 in aa group, compared to 22.4±8 in k group (p<0.01). conclusion: abnormal pft is highly prevalent among children with scd in both groups. aa children are more likely to have restrictive disease and k to have an obstructive pattern. level of hbf did not seem to protect k patients from abnormalities on pft. this finding should emphasize the importance of performing pft as part of the initial evaluation of all children with scd. background: sickle cell disease (scd) is a life-threatening disease with varied clinical spectrum and severity leading to premature death. there is a lack of validated prognostic marker in scd. recent evidence suggests that inflammation and platelet adhesion plays a critical role in the pathophysiology of vaso-occlusion in scd. elevated mean platelet volume(mpv) values are associated with a higher degree of inflammation in many disease states but it's effect on sickle cell disease or it's severity is unknown. objectives: to analyze the role of mpv in predicting disease severity/mortality in pediatric patients with scd. design/method: this is a single center retrospective study and included patients with sickle cell disease between 6 months and 18 years of age during a 10-year period (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . demographic information, lab data and clinical information including acute chest syndrome (acs), priapism, transfusions, sepsis, pain crisis, avascular necrosis were collected. all laboratory data were collected in steady state with no crisis in the recent past 3 months. the disease severity score/probability of death was calculated using a validated model to predict risk of death in sickle cell disease (sebastiani et al. blood 2007) . pearson test was used to analyze correlation between mpv and probability of death. results: total no. of patients = 92; male 45 (48.9%); female 47(51.1%). median age is 6.0 years. all patients were of african-american origin. disease severity, hb ss -58(63%); hb sc -25(27.2%) and sickle-beta thalassemia 9(9.8%). patients on hydroxyurea has significantly lower mpv, p = 0.023 and this is independent of hb f levels. mpv has a significant positive correlation with the probability of death, p = 0.016 and correlation coefficient, r = 0.254. on subgroup analysis, the correlation is even more significant in the age group between 6 and 18 years, p = 0.004, r = 0.405. using linear regression model, with probability of death as a dependent variable and hydroxyurea, mpv as independent variables, mpv maintains a significant association with probability of death (p = 0.016). conclusion: mpv is an independent biomarker predicting disease severity and probability of death in pediatric patients with sickle cell disease. hydroxyurea a known disease ameliorating agent is associated with lower mpv values. this effect is independent of the levels of fetal hemoglobin and may be due to anti-inflammatory effect of hydroxyurea or effect on the platelets. background: major success with initial qi projects by the sickle cell care team at children's hospital has precipitated ongoing inclusion of the qi approach to many other aspects of patient care. objectives: to optimize scd patient care utilizing qi processes. design/method: success of the scd qi team's initial project on transcranial doppler studies (tcds) and a second more complex project on hydroxyurea (hu) adherence, led to additional projects on completion of key immunizations, rbc phenotyping, and vitamin d level testing. using similar processes and principles from the hu adherence project, plan-do-study-act (pdsa) cycles were used to conduct smallscale tests of change. patient chart prep sheets, created for bi-monthly pre-appointment chart prep meetings, were significantly modified to include these focused care qi objectives. because of difficulty with emr database capability, data collected from the emr was tracked in excel spreadsheets or other unique tracking vehicles for the various parameters. for example, due to the clinic's diffuse, geographically scattered population, many separate non-shared primary care emrs, and lack of a mandatory state immunization registry; immunization records needed to be retrieved from pcps, outlying hospitals, public health departments, and fqhcs, and added to the emr and excel database. starting in 12/2016, all such data was collected and updated monthly. in one year's time (2016 -2017) , the average immunization completion rate for seven key immunizations (pcv 13, pcv 23, hepatitis a, hepatitis b, meningococcal a, meningococcal b, and hpv) has increased by 20%. the biggest improvements were a 57% and 44% increase in completion for meningococcal a and meningococcal b, respectively. completion rate for rbc phenotyping rose from 34.8% to 74.4%. patients with at least one vitamin d lab test increased from 27.8% to 67.9%. since starting the tcd project in 2013, the percent of patients who have completed their annual tcd has gone from a baseline of 50% to a sustained value of > 80%. conclusion: these qi projects have not only increased adherence to national recommendations for care of scd patients, they have helped establish a scd clinic methodology to create and implement sustainable processes. having the focused care initiatives prominently displayed on the patients' chart prep sheet serve as a reminder to medical team members to check the status of that item. this methodology is currently being used to formulate additional qi projects on annual renal function parameters and specialty visits, such as annual eye and dental exams. background: dominican republic has a high burden of sickle cell disease, and 5-10% of children with homozygous hbss (sickle cell anemia, sca) will develop primary stroke. transcranial doppler (tcd) ultrasonography is an effective screening tool for primary stroke risk, but is not routinely available in dominican republic. hydroxyurea and blood transfusions are available, but no prospective screening and treatment program for stroke prevention has been implemented to date. (1) to screen a large cohort of children with sca living in dominican republic, using tcd to identify elevated stroke risk; (2) to determine the effects of treatments for stroke prevention (hydroxyurea for conditional velocities and transfusions for abnormal velocities). we hypothesized that both hydroxyurea and blood transfusions will decrease elevated tcd velocities and help prevent primary stroke. design/method: stroke avoidance for children with república dominicana (sacred, nct02769845) features a research partnership between cincinnati children's hospital and robert reid cabral children's hospital in dominican republic. the protocol, consent forms, and redcap database were prepared collaboratively and translated into spanish, and then irb approval was obtained at both institutions. in the initial prospective phase, children receive tcd screening over a 12-month period; those with conditional tcd velocities (maximum time-averaged velocity 170-199 cm/sec) receive fixed-dose hydroxyurea at 20 mg/kg/day, followed by dose escalation to maximum tolerated dose, while those with abnormal tcd velocities (≥200 cm/sec) receive monthly transfusions for stroke prevention. results: a total of 283 children were enrolled in sacred, with an average age of 8.7 ± 3.4 years. initial tcd screening revealed 200 (70.7%) normal, 63 (22.3%) conditional, 11 (3.9%) abnormal, and 9 (3.1%) inadequate velocities. among 48 children (25 males, 23 females, average age 6.8 ± 2.8 years) who initiated hydroxyurea at 20 mg/kg/day for conditional tcd velocities, 42 completed six months of treatment with expected hematological benefits including significant increases in hemoglobin concentration (7.5 to 8.5 g/dl) and fetal hemoglobin (15.8 to 28.4%). no clinical strokes have occurred in the treatment group. repeat tcd examination after 6-months of hydroxyurea treatment revealed 69% (29/42) with previous conditional velocities had normal tcd velocities. the prevalence of conditional tcd velocities in the dominican republic is high, indicating an elevated stroke risk among children with sca. hydroxyurea treatment is associated with improved hematological parameters, lower tcd velocities, and probable decreased stroke risk. sacred is an important prospective and collaborative research trial providing epidemiological data regarding tcd screening, stroke risk, and hydroxyurea effects among children with sca. background: red blood cell aggregation is a rheologic property that explains the shear-thinning behavior of blood. at lower shear rate blood flow, red cells tend to aggregate, s121 of s301 whereas in higher shear rate blood flow, these aggregates are dispersed. this property is especially important in the venous system, where low shear rate blood flow predominates. there is inconsistent data in the literature concerning aggregation and aggregability in sickle cell disease (scd). objectives: because the lorrca and myrenne instruments have been shown to be similarly effective methodologies in red cell aggregation measurements, we aimed to determine whether the measurement of aggregation indices in scd, by myrenne and by lorrca, is consistent in our lab. design/method: we measured aggregation in blood samples corrected to 40% hematocrit. aggregability was measured using 70kda dextran in the myrenne but not the lor-rca. aggregation index using lorrca was measured in 26 patients with scd and 22 healthy subjects enrolled in a study of blood flow between 2014 and 2017. aggregation and aggregability using the myrenne was measured in 67 patients with scd and 15 healthy subjects enrolled in a separate study of blood flow between 2008 and 2013. results: using lorrca, we found that aggregation index in patients with scd was less than that of healthy subjects (p<0.001). in the myrenne, aggregation at stasis was slightly higher in patients with scd compared to healthy subjects (p = 0.05) but aggregation at low shear rotation was not different. aggregability was higher in the patients with scd compared to healthy subjects at both stasis and low shear rotation (p<0.0001). red cell aggregation is an important determinant of low shear blood flow. deoxygenated venous blood is particularly important to low shear blood flow in patients with sickle cell disease. we found that two different aggregometers predict different aggregation results for scd. it is unclear why there is a systematic difference between the two methods, but there are some possibilities. first, the syllectogram in the lorrca is generated by the backscatter of light from the laser, while the myrenne measures transmitted light. second, the distance between the bob and cup in the lorrca is 300 microns, while the gap between plates in the myrenne is 50 microns, which might affect the disaggregation of red cells. further work is needed to understand the differences in red cell aggregation and aggregability when using these instruments, particularly when using aggregation as a predictor of blood flow and tissue perfusion. background: children with sickle cell disease (scd) are at risk of acute splenic sequestration crisis (assc). assc is a life-threatening complication characterized by splenomegaly, pain and severe anemia. assc most often occurs in young children with the most severe forms of scd and one-third of patients will have more than one episode. treatment is based primarily on expert opinion and includes blood transfusion and surgical splenectomy. objectives: we plan to assess the clinical practice patterns of physicians treating children with assc. design/method: a survey study was performed. the survey included six scenarios of severe scd with variation in age, hydroxyurea-use, and episode number of assc; questions focused on the acute and chronic management of assc. the survey was disseminated on three occasions over a six-month period, using an online survey tool, surveymonkey, to pediatric hematologist-oncologists participating in the american society of pediatric hematology-oncology hemoglobinopathy special interest group. the survey had a response rate of 43% (28/65). most respondents were recent graduates (61%; 17/28) practicing in academic urban centers with greater than 100 sickle-cell patients. seventy-nine percent (22/28) recommended hydroxyurea initiation in 9-12 m/o with severe scd. prophylactic penicillin after surgical splenectomy was continued by 93% (26/28) after 5 years. for the acute management of assc results did not vary despite patient age, hydroxyurea use, and the number of previous assc episodes. simple transfusion was preferred by 89% (25/28), with 54% (15/28) recommending slow transfusion and 36% (10/28) recommending routine simple transfusion. for the chronic management of assc, results varied based on patient age and the number of previous assc episodes. for a 12 m/o after the first episode, 36% (10/28) recommended observation and 32% (9/28) hydroxyurea initiation. for a 12 m/o with any prior episode of assc, 39% (11/28) recommended chronic transfusion therapy and 36% (10/28) surgical referral for splenectomy. for a 3 y/o after the first episode, 39% (11/28) recommended surgical splenectomy and 32% (9/28) increasing hydroxyurea dose. for a 3 y/o with any prior assc episode, 64% (18/28) recommended referral for surgical splenectomy. in this survey, we found most providers continue to recommend simple transfusions for assc and surgical splenectomy after two episodes. the majority of providers continue to delay referral for surgical splenectomy until age two, but earlier referral in children under two and use of chronic transfusion therapy were also reported. variability in chronic management highlights the need for further research of splenic sequestration. background: developing therapies for sickle cell disease (scd) is challenging in part because the accepted endpoint, vaso-occlusive crisis (voc), occurs infrequently, does not measure full disease burden, and is a measure of healthcare utilization. in phase 1/2 studies of patients with scd, voxelotor (gbt440) has demonstrated increased hemoglobin (hb) levels and reduced hemolysis and has been safe and welltolerated. voxelotor is being evaluated in the ongoing hope phase 3 trial. objectives: to report the innovative phase 2/3 hope trial design with novel primary and secondary outcomes to accelerate drug development. design/method: hope (nct03036813) is a phase 3, randomized, placebo-controlled, multicenter study of oral voxelotor in patients with scd (aged 12-65 years) with baseline hb 5.5-10.5 g/dl and 1-10 episodes of voc in the prior year. to accelerate clinical trials to support drug development, the study combines a phase 2 exploratory, dose-selection phase (group 1) with a pivotal phase (groups 2/3). patients in group 1 will be randomized 1:1:1 to voxelotor 900 or 1500 mg/day or placebo. analysis for dose selection will occur when the final patient has received 12 weeks of treatment. group 2 will continue enrollment with randomization 1:1:1 until dose selection based on analysis of the group 1 cohort. group 2 will allow for a seamless transition into group 3, which will randomize patients 1:1 to the selected dose or placebo. the final data analysis set will include group 2 patients who received placebo or the selected dose and all group 3 patients. the primary endpoint is an objective laboratory measure and surrogate of clinical benefit, increase in hb >1 g/dl, from baseline to 24 weeks based on voxelotor mechanism of action (inhibition of hb polymerization). this trial is the first to use a patient-reported outcome (pro), the 9-item sickle cell disease severity measure, as a secondary endpoint. this novel electronic pro, developed specifically for the hope study following fda guidance, will evaluate changes in scd symptom exacerbation and total symptom score from baseline to 24 weeks. additional secondary endpoints include measures of hemolysis, rates of voc, transfusions, and opioid use. the study was designed to enable selection of pro-defined symptom exacerbations or traditionally defined voc as the key secondary endpoint after the group 1 analysis. results: this study is ongoing. the hope trial, expected to complete enrollment by late 2018, will evaluate the efficacy and safety of voxelotor compared with placebo in patients with scd. supported by global blood therapeutics. background: inflammation, coagulation activation, oxidative stress and blood cell adhesion are elements of sickle cell disease (scd) pathophysiology. patients with scd have low levels of the omega-3 fatty docosahexaenoic acid (dha) and eicosatetraenoic acid (epa) in plasma and blood cell membranes. dha is a bioactive fatty acid with anti-inflammatory, anti-blood cell adhesion and anti-oxidant properties. altemi-atm is a novel dha ethyl ester formulation with a proprietary delivery platform (advanced lipid technology® (alt®)) that enhances oral dha bioavailability. the scot trial investigated the effects of altemiatm in children with scd. objectives: to demonstrate the effects of altemiatm on blood cell membrane omega-3 index and selected biomarkers of inflammation, coagulation, adhesion and haemolysis associated with scd. s123 of s301 design/method: children with scd, aged 5-17 years (n = 67), were enrolled. subjects were randomized to receive either placebo or one of three daily oral doses of altemiatm (12-26, 26-48 or 51-72 mg/kg/day dha) for two months. the effects of altemiatm on red blood cell (rbc), white blood cell and platelet membrane omega-3 fatty acids index (total dha + epa levels) were assessed after four weeks of treatment. the effects of altemiatm on markers of inflammation, adhesion, coagulation, and hemolysis were assessed after eight weeks of treatment. cell membrane dha and epa concentration was determined by using lc-ms/ms method. the percent changes from baseline on blood cell membrane omega-3 index and select scd biomarkers were compared between the three dose groups and placebo using a mixed-model repeatedmeasures (mmrm) analysis with baseline blood cell membrane omega-3 index, hydroxyurea use, and treatment as fixed effects and patient as a random effect. after four weeks of treatment, blood cell membrane dha and epa levels were significantly increased in all altemiatm doses (p<0.01). after eight weeks of treatment, significant reductions were observed in se-selectin (p = 0.0219), and d-dimer (p = 0.025) in patients exposed to altemiatm dose level 2 vs. placebo. hemoglobin was significantly increased at altemiatm dose level 1 versus placebo. plasma high-sensitivity c-reactive protein, lactate dehydrogenase, soluble vascular cell adhesion molecule-1 and white blood cell count showed improvement after 8 weeks of treatment in all three altemiatm doses levels but did not reach significance. conclusion: treatment with altemiatm enriches dha and epa in blood cell membranes of patients with scd and improves select sickle cell disease biomarkers of blood cell adhesion and thrombin generation. these findings provide insight into the mechanisms of action of altemiatm in sickle cell disease. brown university -hasbro children's hospital, providence, rhode island, united states background: despite clinical advances in the treatment of sickle cell disease (scd) in pediatric and young adult patients, pain remains a significant source of disease-related morbidity. physical therapy has been shown to be useful for the treatment of pain in children and young adults with various chronic illnesses of which pain is a significant component, however no data exists regarding potential benefits of physical therapy in pediatric and young adult patients with scd. objectives: to query healthcare providers and others involved in the care of pediatric and young adult scd patients regarding possible benefits of and barriers to physical therapy as a potential treatment modality. design/method: we conducted a web-based survey of healthcare providers within the new england pediatric sickle cell consortium (nepscc) in an attempt to identify potential benefits of and barriers to outpatient physical therapy in this patient population. results: nearly 92% of survey participants felt that physical therapy had the potential to be "somewhat beneficial" or "very beneficial" in pediatric and young adult patients with scd. a majority of physicians reported having referred patients with scd for physical therapy in the past. the most frequently identified perceived potential benefits included improved functional mobility, improvement of chronic pain symptoms, decreased use of opiates, improved mood symptoms, improved acute pain symptoms, and improved adherence with medications and clinic visits. significant perceived barriers identified included lack of transportation, time constraints, patient lack of understanding, and difficulty with insurance coverage. our study indicates that healthcare providers have an overwhelmingly positive view of the use of physical therapy in the management of pediatric and young adult patients with scd. significant barriers exist which need to be addressed. future research should focus on patient and parent perspectives regarding physical therapy, as well as a randomized controlled trial of a physical therapy intervention in this patient population. background: vitamin-d deficiency is fast becoming increasingly recognized in patients with sickle cell disease (scd). while it is estimated that these patients are five times more likely to develop vitamin-d deficiency, the exact clinical significance of this is largely unknown. given that this deficiency can be inexpensively and easily treated, our study sought to establish the prevalence of vitamin-d deficiency in our patient population and its relationship with disease severity. objectives: to estimate the prevalence of vitamin-d deficiency in patients with scd in our institution and to analyze their disease severity in relation to their vitamin-d level. design/method: through retrospective chart review we analyzed subjects that represent a cohort of patients followed at the adult and pediatric hematology services at university of miami with known diagnosis of scd that had a vitamin-d level drawn between january 01st, 2013 and august 31st, 2016. we conducted a cross-sectional study and recorded the first vitamin-d level during this period. patient demographics, medical and social history information were collected along with laboratory data. the number of admissions for vaso-occlusive crisis (voc) and acute chest syndrome within one year preceding the collection the vitamin-d level was also recorded. results: a total of 476 charts were reviewed, 279 adult charts and 207 pediatric charts. after exclusion, 119 patients were enrolled. subclinical vitamin-d deficiency is only evident on laboratory blood testing of vitamin-d (25-hydroxy) and according to this laboratory result patients were classified as sufficient (≥32 ng/ml), insufficient (<32 to 20 ng/ml) and deficient (<20 ng/ml). out of the 119 cases, 61.7% (74/119) were deficient, 21.7% (26/119) were insufficient and 15.8 % (19/119) were optimal. after statistical analysis two negative correlations were identified, increasing vitamin-d levels with decreasing white blood cell count (ci 95%-0.1931133 (-0.36057544, -0.01359017)and decreasing incidence voc (ci 95%-0.3149722 (-0.4684118, -0.1430889). conclusion: this study confirms that there is a significant prevalence of vitamin-d deficiency in patients with scd. furthermore, the results of this investigation proved that vitamin-d deficiency is associated with acute pain and leukocytosis in patients with scd. given the multitude of confounding factors that affect vitamin-d absorption and intake, multivariate analyses are required to truly further investigate this relationship. texas children's hospital, houston, texas, united states background: hemophagocytic lymphohistiocytosis (hlh) is a rare but life-threatening condition of hyper-inflammation that is characterized by splenomegaly, cytopenias, hyperferritinemia, hypertriglyceridemia, hemophagocytosis and coagulopathy. although timely diagnosis is imperative, it is often challenging as these individual signs and symptoms may occur in a variety of clinical conditions. to report a case of undiagnosed sickle cell anemia presenting with severe ebv viremia and associated hemophagocytic lymphohistiocytosis results: a 21-month-old previously healthy male presented with respiratory distress, increased fatigue, and a focal seizure following a two-week history of cough and lowgrade fevers. physical exam was consistent with hypovolemic shock and revealed significant splenomegaly. laboratory testing revealed severe hypoglycemia, acidosis and electrolyte disturbances including hyperkalemia, hyperphosphatemia, and hyperuricemia. labs showed a leukocytosis (wbc 53,000), severely low hemoglobin (1.9), and platelets of 56,000. coagulation testing revealed prolonged pt/inr and ptt, hypofibrinogenemia and a highly elevated d-dimer. additional workup was completed to determine etiology of acute presentation, given broad differential diagnosis. infectious studies were consistent with an acute ebv infection (plasma ebv pcr >550,000). elevated levels of soluble il-2 and ferritin completed 6/8 criteria for the diagnosis of hlh. bone marrow evaluation showed trilineage hematopoiesis with no abnormal blast population or hemophagocytosis. results from hemoglobin electrophoresis sent from the initial cbc sample were notable for hbs 72.5%, hbf 25.0%, and hba of 0%, confirming the diagnosis of sickle cell disease. the patient was started on hydroxyurea and penicillin and splenomegaly resolved. with supportive care, he demonstrated gradual improvement in symptoms and laboratory abnormalities, including normalization of soluble il-2, ferritin, cd163, il-18 levels, immunoglobulins, and declining ebv titers. nk cell function has remained abnormally low, not eliminating the possibility of acquired hlh despite spontaneous improvement. conclusion: splenic sequestration associated with sickle cell disease in combination with acute infectious mononucleosis could have explained many of the presenting symptoms including anemia, thrombocytopenia, and splenomegaly. however, it does not explain the unusually high ebv titer and degree of inflammation meeting diagnostic criteria for hlh, which raises concern for an underlying immunologic abnormality such as x-linked lymphoproliferative disorder (xlp). although testing for xlp was negative, he will require s125 of s301 continued monitoring in the future for signs of relapse. this case illustrates the complexity of diagnosing lymphohistiocytic disorders and the significant overlap in presentation between these disorders and other medical conditions. background: vaso-occlusive crisis (voc) is one of the most distressing occurrences in patients with sickle cell disease (scd). patient controlled analgesia (pca) is recommended by nih and expert opinions favor its early use. we aim to review the use of pca in patients with voc and to evaluate if its early use is associated with faster pain control and reduced length of stay (los). design/method: this retrospective single center study included all pediatric patients admitted and treated with pca for a severe voc from 2010 to 2016. "early" use was defined as start of pca within 48 hours of arrival in the emergency department (ed) and "late" use after 48 hours. time to reach adequate analgesia was defined as oucher, verbal scale or faces pain scale < 5/10 obtained twice consecutively in a 4-hours interval. time to reach adequate analgesia and los were compared between early-pca and late-pca groups. results: a total of 46 patients presented 87 episodes of voc treated with pca during the study. sixty-one episodes (70%) were treated with early-pca and 26 (30%) with late-pca. both groups were comparable in terms of age (13.2 vs 12.8 years old), gender (55.8% female vs 57.7%), hemoglobin phenotype (80.3% hbss vs 76.9%), but median pain score at admission was higher in early-pca than in late-pca (9/10 vs 7/10, median difference 1 (95% ci 0, 2). early-pca was associated with a median reduction in los of 3.15 days (95% ci 1.65, 4.82) (median early-pca los 6.4 vs late-pca 10.0 days). time to reach analgesia could be evaluated only in a subset of patients (20 in early-pca and 12 in late-pca group). although time to reach adequate analgesia tended to be shorter in the early-pca group, it was not statistically different: median102.9 hours vs 123.5 hours, difference of 30.4 (95% ci -4.0, 72.5). side effects were observed during 29 (33.3%) pca treatments (19/61 (31.2%) episodes in early-pca, 10/26 (38.5%) in late-pca group) among which 16 (18.6%) were significant adverse events. these were observed in 15 patients who required interventions: 2 desaturations requiring oxygen without intubation, 8 neurologic abnormalities (hallucinations, visual abnormalities, no stroke), 6 urinary retentions. conclusion: early use of pca for severe voc was associated with a reduced length of hospital stay despite that these patients had higher pain score on admission. prospective studies are needed to support these positive outcomes. background: acute chest syndrome is one of the leading causes of death in children with sickle cell disease1-2. while the cause of acute chest syndrome most commonly is not identified, fat embolism and infectious causes are believed to be most common. with an extremely high mortality rate, rapid identification and initiation of therapy is essential for survival. case presentation: we describe the case of an 18-year-old female with sickle cell sc disease who was admitted for vasoocclusive pain crisis and quickly progressed to multi-system organ failure due to fat embolism syndrome and parvovirus b19 infection objectives: the case highlights the presentation and diagnosis so other providers can optimize outcomes for those with this under-recognized syndrome design/method: her parvovirus studies returned after 7 days which showed: parvovirus b19 dna pcr detected; parvo igg 1.99 (positive > 1.1); and igm 12.98 (positive > 1.1). the patient experienced an approximately 2.5 g/dl drop in hemoglobin(8.7 to 6.0 g/dl/24 hrs) with progressive thrombocytopenia (from 269,000 to 54,000/ul) and a peripheral smear showed microcytic,normochromic red cells with nucleated rbcs and occasional nuclear budding, slight polychromasia, schistocytes, and polymorphic cells with toxic granules that suggested leukoerythroblastosis. she was emergently transferred to the regional quaternary care hospital for ongoing ecmo therapy where she experienced a change in her pupillary exam prompting a stat ct scan that showed severe, diffuse cerebral edema with transtentorial herniation. the decision was made to withdraw life-sustaining therapies and her family refused a post-mortem autopsy examination. fat embolism syndrome is a severe and uncommonly recognized complication of sickle cell disease, seen most commonly in those with a non-ss phenotype and previous mild disease course who present with severe, unrelenting vaso-occlusive pain episode and/or acute chest syndrome that progresses to respiratory distress with altered mental status and cutaneous changes. rapid identification and initiation of exchange transfusion therapy should be initiated with clinical suspicion because of the extremely high mortality rate. although previously considered rare, it needs to be considered in the differential diagnosis of more commonly encountered complications of sickle cell disease. background: patients with sickle cell disease (scd) experience vaso-occlusive crisis (voc), which results in extreme pain, often requiring opioids and admission. genetic and environmental factors affect the frequency and severity of these episodes. previous research has born conflicting evidence on whether environmental temperature is contributory. edmonton, alberta is the northern most city with a population over a million in north america. there is an increasing sickle cell population which is exposed to extreme winter conditions. this provides a suitable population and atmosphere to study the influence on cold external temperatures in scd. this study sought to identify if pediatric patients with scd, experience greater morbidity in cold external temperatures. board approved retrospective case control series. patients were identified through a clinical database, and emergency visit, phone call and admission data was collected over a fiveyear period. the average, minimum and change in temperature on day of presentation, 24 and 48 hours prior, was collected from the government of alberta, and was statistically analyzed using descriptive statistics, to determine the relation to vaso-occlusive events. results: one-hundred and eighteen patients were identified, and 258 voc events reviewed. the mean patient age was 6.6 years of age with a range from 0.3-17 years old. the female to male ratio was equivalent with 133 female (51.6%) and 125 male (48.4%) voc events. eight records (3%) had docu-mented cold exposures. the analysis between the temperature and the frequency of events did not yield significant correlation. average and minimum temperature on day of admission had the largest percentage of voc events occur at mild temperatures, from -4.99 to 20 • c and -4.99 to 5 respectively. change in temperature on day of admission, 24 and 48 hours had the largest percentage of voc events at a mild to moderate change in temperature of 10-15 degrees. data at 24 & 48 hours prior to admission showed similar results. secondary data analysis accounting for the lower proportion of extreme weather days in comparison to moderate temperate days showed no significant impact. there was no correlation of average, minimum or change in temperature on day of admission, 24 or 48 hours prior. multiple cofounding factors likely contribute to these results. as it was a retrospective study many confounding and precipitant factors may not be recorded or identified. a prospective study to better record specific cold exposure is warranted. children's national health system, washington, district of columbia, united states background: achieving optimal anticoagulation with unfractionated heparin (ufh) in pediatric patients receiving extracorporeal membrane oxygenation (ecmo) is often challenging due to antithrombin (at)-mediated heparin resistance (hr). intermittent at dosing during pediatric ecmo support does not maintain adequate at levels. continuous at infusion (cati) presents an alternative strategy to achieving consistent goal at levels and optimizing heparinization. however, cati during pediatric ecmo has not been adequately studied. objectives: to describe our center's experience with an ecmo cati protocol. design/method: in 2014, we modified our ecmo anticoagulation protocols to include ufh titration according to anti-factor xa (anti-fxa) levels and cati in patients with at-mediated hr. the cati rate was calculated using baseline and goal at levels while accounting for the circuit volume. cati was administered with ufh into the circuit via a s127 of s301 y-infusion set. at and anti-fxa levels were monitored every 6 hours. recombinant at (r-at) concentrate was used at our center until 2015 with subsequent transition to a plasmaderived at (pd-at) concentrate. due to the longer half-life of pd-at concentrate, the protocol was modified so cati is stopped once target at and anti-fxa levels are achieved. we conducted a retrospective study of all patients who received cati during ecmo support at our center. data are reported as median and interquartile range and compared using the mann-whitney u test. two-tailed p-value <0.05 was considered statistically significant. since 2014, 24 patients [13 males, age 1 month (0.03-8)] on ecmo support received 27 catis (12 rat, 15 pd-at) per our protocol (3 patients received 2 pd-at infusions during one ecmo run). the duration of cati was 48 hours (23-72). cati administration led to significant increases in at and anti-fxa levels from baseline of 43% (39-53) and 0.11 units/ml (0.08-0.22) to the first level within goal of 64% (55-83) and 0.39 units/ml (0.35-0.5), respectively (p<0.00001). the respective times to achieve goal at and anti-fxa levels were 9 hours (5-21) and 13 hours (6-23). the respective peak at and anti-fxa levels were 83% (70-99) and 0.53 units/ml (0.35-0.63). during cati, no patient required circuit change, 1 patient developed cannula thrombosis and 5 patients experienced non-fatal major bleeding. conclusion: cati in pediatric patients receiving ecmo support with close monitoring of at and anti-fxa levels was associated with significant rapid increase in at, optimization of heparin effect, and reduction in thrombotic complications without increase in major bleeding compared to prior reports. a prospective study of this at dosing strategy is warranted. children's hospital of orange county, orange, california, united states background: inherited factor xiii (f13) deficiency is a rare bleeding disorder with wide heterogeneity in clinical manifestations ranging from mild bruising, and mucosal and umbilical stump bleeding to spontaneous, severe intracranial bleeding. the bleeding phenotype is influenced not just by zygosity of the fxiii mutation alone, but also by co-inheritance of variants in other clotting protein genes that also play a major role in clot formation and stability. we present a series of three siblings found with f13a1 gene variant and platelet dysfunction linked to bleeding phenotype. design/method: retrospective chart review of the index case, coagulation studies and whole gene sequencing. the index patient presented at two years of age with a subdural hematoma after a fall, requiring emergent craniotomy. a week after initial evacuation, she re-bled, prompting an extensive work-up for potential bleeding disorders, including f13 activity, von willebrand profile, comprehensive fibrinolysis panel, pai-1 antigen level, platelet mapping thromboelastogram (plt-teg), and f13 genetic analysis. the patient's identical twin and older sibling, who had symptoms of bruising, underwent a similar evaluation. the index patient demonstrated consistently low f13 activity (31-49%), and platelet function testing revealed decreased response to adp agonists. the twin and older sibling had normal f13 levels, and only slightly decreased response to adp in platelet studies. whole gene analysis of f13 and 21 other genes on our next generation panel, revealed several intronic deletions in the index patient that were not shared by her siblings, which likely account for her decrease in circulating f13 levels. her symptoms have responded well to monthly treatment with factor 13 concentrate. all three children shared the f13 variant, pro564leu, previously described as a risk factor for intracranial hemorrhage. the f13 mutation, pro564leu, has been associated with intracranial hemorrhage in young women, but the presence of the variant alone may not be enough to cause a severe bleeding phenotype. family studies identified novel deletions in the index patient which may account for her decreased f13 levels, which would have been overlooked with standard sequencing. future studies, including evaluation of 'platelet' f13 levels, should be performed when platelet dysfunction is detected. further laboratory and clinical evaluation is required to delineate the long term implications of the interaction of even mild f13 deficiency if present with additional clotting disorders such as the platelet function defect in these siblings. background: acquired hemolytic anemia can occur due to mechanical shearing of red blood cells and is classically seen in patients with prosthetic heart valves. there are reports of this same traumatic effect with other repairs, including annuloplasty. following valvular procedures flow disturbances can exist across the valve that lead to shear stress and hemolysis. although von willebrand disease (vwd) is typically seen due to an inherited disorder in the pediatric population, flow disturbances in the setting of valve abnormalities can lead to acquired von willebrand syndrome (avws). von willebrand factor multimers become unfolded and elongated in the setting of shear stress resulting in increased susceptibility to cleavage by adamsts-13. specifically, loss of high molecular weight multimers (hmwms) can lead to a syndrome akin to type 2a vwd. objectives: to describe a case of mechanical hemolysis with acquired type 2a vwd design/method: a 3-month-old girl with history of hypoplastic left heart syndrome and severe tricuspid valve insufficiency underwent norwood procedure, blalok-taussig shunt placement and subsequently a bidirectional glenn and tricuspid valve annuloplasty. during the following month she requires weekly red blood cell (rbc) transfusions due to intermittent anemia. she also experienced bloody stools and dark urine. laboratory evaluation was notable for normocytic anemia, reticulocytosis, elevated lactate dehydrogenase, and low haptoglobin consistent with hemolytic process. immune-mediated hemolysis from transfusion reaction or presence of autoimmune or alloimmune antibodies testing was negative. to investigate gi bleeding, work up for vwd revealed normal vw activity and antigen but with loss of high molecular weight multimers consistent with acquired type 2a vwd. in consultation with cardiology, it was felt her tricuspid valve insufficiency jet could be leading to mechanical hemolysis and avws. a repeat echo showed persistent moderate tricuspid insufficiency but no other significant changes. due to the patient's continued need for weekly rbc transfusions she was subsequently trialed on pentoxifylline which is used in adult patients to decrease blood viscosity and increase erythrocyte flexibility in patients with mechanical hemolysis. her transfusion needs remained the same and the medication was discontinued after two weeks. she required one transfusion a week later but no transfusions since that time. although not commonly seen in pediatric patients, the diagnosis of mechanical hemolysis accompanied by avws should be pursued in a patient with congenital heart disease with significant anemia and/or bleeding. the work up in these patients is difficult as echocardiograms can be inconclusive thus an extensive hematologic evaluation is usually necessary. objectives: our aim was to assess incidence of and potential risk factors for central line-related dvt at our institution between 2011-2016. additionally, our goal was to analyze if that incidence differed between the three central line types and identification of line-specific risks. design/method: a retrospective chart review of 377 central line placements in pediatric patients at cleveland clinic between 2011-2016 was conducted. data included demographics, potential risk factors, line characteristics and any related thrombotic events. the study cohort consisted of 377 lines in 326 pediatric patients aged 1-18 years of age. there were 1.5 thrombi (95% ci 1.0-2.3) per 10,000 line days. statistically significant risk factors for thrombus include diagnosis group (liquid tumor highest rate of 16%, solid tumor lowest at 2%), type of line (picc 5%, broviac 29%, and mediport 4%), location of line, greater number of lines per patient, peg asparaginase (23% vs 4%), sepsis, and history of procoagulant state. line characteristics such as lumen size and number of lumens were not identified as a significant risk. there was a significantly higher rate of thrombus in 2016 than in the previous years when pooled (12% in 2016 vs 4.3% from 2011-2015, p = 0.020). the incidence of dvt in pediatric patients at our institution was highest with broviac lines, and significant risk factors in our patient population included liquid tumor, femoral vein location, peg asparaginase, sepsis, and history of a procoagulant state. the incidence of thrombi was highest in 2016, and therefore highlights the urgent need for improvement in nationwide hospital practices to minimize risk of thrombi formation and early detection in the higher-risk s129 of s301 populations. there is still much to be learned regarding the characteristics specific to different central lines, which would influence thrombi formation. nyu winthrop hospital, mineola, new york, united states background: pediatric immune thrombocytopenic purpura (itp) is an autoimmune disorder with platelet counts <100000 causing increased risk for significant hemorrhage. there is increased immunologic platelet destruction due to production of specific autoantibodies along with inhibition of platelet production. few randomized trials exist to guide management and ultimately each patient requires an individualized treatment plan. itp may be acute (diagnosis to 3 m) or chronic (> 12 months). one of the treatments of chronic itp is laparoscopic splenectomy (ls), which is very well tolerated. a rare complication of ls is splenosis, an autotransplantation or implantation of ectopic splenic tissue within the abdominal cavity or in any other unusual body compartment. splenosis is sometimes associated with relapsed itp due to preserved immune activity. the usual management of symptomatic splenosis is surgical resection. objectives: to describe medical management in a young patient with itp relapsed due to extensive unresectable splenosis following ls design/method: our patient was originally diagnosed at 2 years with itp and was treated with ls at 5 years of age for chronic severe thrombocytopenia and persistent bleeding not responding to first line therapies. she tolerated it well and had a complete response (cr) defined as a platelet count of >100000 measured on 2 occasions >7 days apart and absence of bleeding. she maintained a normal platelet count for twelve years after which she relapsed (loss of response after cr) with severe thrombocytopenia and hematuria necessitating high dose steroids. ct scans showed multiple wellcircumscribed soft tissue masses in the left lower quadrant adjacent to uterus and left ovary, involving left omentum and the anterior abdominal wall partly. findings were confirmed by damaged rbc nuclear scan to be splenosis. during laparoscopy the splenosis lesions were deemed too extensive and were not resected completely to avoid postoperative morbidity. she was started on sirolimus around the same time for treatment of her relapsed itp and steroids were weaned off. results: eight months since beginning sirolimus with therapeutic levels she remains in cr with no bleeding and has not required any steroids, immunoglobulins or anti d immunoglobulin. conclusion: sirolimus is a safe and effective steroid-sparing agent in treatment of chronic itp. this is the first instance of a patient with poorly resectable splenosis responding well to medications for itp. more data is needed regarding the longterm efficacy of such an intervention and whether it will eliminate the need for a second surgery in relapsed itp patients with extensive splenosis. background: storage pool disorders affecting platelets result in bleeding symptoms related to a deficiency or defect in alpha granules or delta granules. in delta-storage pool disorders (dspd,) there is a deficiency of the delta granules and their constituents, which results in the inability of platelets to properly activate as well as lack of proper constriction of blood vessels during bleeding episodes. amongst patients with dspd, females most commonly present with menorrhagia, while males tend to present with epistaxis and easy bruising. the international society on thrombosis and hemostasis (isth) developed a screening bleeding assessment tool (bat) for mild bleeding disorders, shown to be a validated tool in children. diagnosis of dspd is classically made with a platelet electron microscopy (pem) value <3.69 delta granules per platelet (dg/pl), but recently lower diagnostic thresholds of 2 dg/pl or even 1.2 dg/pl have been suggested. objectives: evaluate the correlation between pem and bleeding scores, and also examine various cut-off values used to diagnose and risk stratify patients with dspd. design/method: retrospective chart review of 96 pediatric patients followed by hematology with a diagnosis of dspd was performed. clinicians obtained bleeding scores for each patient as standard of care in the hemostasis clinic. quartile ranges were established to appropriate three stages of severity based upon bleeding scores. statistical analysis was performed using software r and exploratory data analysis to evaluate for a correlation. results: amongst all patients, the average bat score was 6.17 and pem was 2.37 dg/pl. the average bleeding score for pem between 3.69 dg/pl and 2 dg/pl was 6.17, while the average bleeding score for pem below 2 dg/pl was 4.65. the correlation coefficient between pem and bleeding scores is 0.30. using a threshold of 2 dg/pl, 31% of patients would have met diagnostic criteria. quartile ranges for the bleeding scores are as follows: 1st quartile was 2-4, 2nd quartile was 5-7, and 3rd quartile was >8. conclusion: patients with a more marked granule deficiency do not exhibit a more severe bleeding phenotype, suggesting proper platelet function is not solely determined by granule quantity in these patients. bleeding severity may be more appropriately assessed with bleeding scores rather than pem values, and using quartile ranges may aide in risk stratification and therapeutic interventions for dspd patients. further work remains to determine the optimal diagnostic threshold of pem dspd in pediatric populations. texas children's hospital, houston, texas, united states background: warfarin management has many challenging aspects including pharmacogenomics, food and drug interactions, lack of standardized dosing, patient compliance, tracking lab results from multiple lab locations, and the potential for significant bleeding or thrombotic complications. a literature review revealed limited data highlighting anticoagulation monitoring workflow and emr documentation and specifically, no data in the pediatric population. historically, the texas children's hospital cardiology and hematology centers were each documenting anticoagulation data within the epic tm system differently. epic's tm original design for anticoagulation documenting resulted in the necessity to duplicate documentation in order to see at-a-glance critical anticoagulation monitoring information. objectives: the objective of this project was to standardize inr documentation across departments to reduce the risk of patient safety events and improve workflow. design/method: a workgroup assembled consisting of nurses from the cardiology and hematology departments, along with staff members from the epic tm is support group. the workgroup identified current documentation practices, available epic tm tools, and brainstormed ideas to streamline and improve both documentation with the current epic tm tools. physician partners were identified in cardiology, hematology and coagulation laboratory to gain their input. a new anti-coag (ac) encounter was developed and first made available in an epic tm practice environment, then once approved, epic tm written education and training session were completed by both departments' staff. results: surveys were sent to 19 health care providers in the cardiology and hematology centers prior to the new ac encounter, and also to 27 health care providers six months after implementing the ac encounter. six responses were received for each survey. the pre-implementation survey showed the most problematic part of the documentation system for anticoagulation was no single place in the emr to find a complete anticoagulation picture. post ac encounter implementation survey results revealed more health care providers using the epic tm inr reminder pool, less time needed to compile a report of three months of anticoagulation information, less time needed to document individual encounters, less locations needed to document ac information and decreased amount of types of documentation used. standardized ac encounters improves workflow with less time needed to document and compile information, less types of documentation utilized and easier access to patients ac information. next steps include retrospective review of patients' inr time in therapeutic range to determine if there was an impact on patient compliance and continue to evaluate and modify the ac encounter to enhance user friendliness. caitlin tydings, jennifer meldau, christine guelcher, carole hennessey, eena kapoor, michael guerrera, yaser diab s131 of s301 children's national health system, washington, district of columbia, united states background: venous anatomic abnormalities (vaas) are considered a risk factor for developing deep vein thromboses (dvts) that occur as a result of significant alterations in venous blood flow. identification of predisposing vaas can be challenging. hence, diagnosis can be delayed or overlooked especially in pediatric patients. dvts in children or adolescents with predisposing vaas have been only described in sporadic case reports and small case series. objectives: to describe characteristics and outcomes of dvts in pediatric patients with underlying vaa treated at our center. design/method: we conducted a retrospective chart review of all pediatric patients with objectively confirmed extremity dvt treated at our institution over a 6-year period from 2011 to 2017 and identified all patients with underlying vaas. patients were managed according to standardized institutional protocols based on published guidelines. post-thrombotic syndrome (pts) was assessed at our center using the manco-johnson instrument. relevant data were collected and summarized using descriptive statistics. during the study period, 20 of 227 pediatric patients (9%) [14 females, median age 17 years (range 11-20)] diagnosed with extremity dvt at our center were found to have an underlying vaa. vaas included may-thurner anomaly (13 patients), venous thoracic outlet obstruction (5 patients) and inferior vena cava (ivc) atresia (2 patients). additional provoking factors were identified in 14 patients at time of presentation. dvt locations included upper extremity veins (5 patients), lower extremity veins (9 patients) and lower extremity veins and ivc (6 patients). the majority of dvts [17 patients, (85%)] were completely occlusive. high risk thrombophilia (defined as inherited deficiency of antithrombin, protein c, or protein s, or antiphospholipid antibody syndrome) was present in 6 patients (30%). all patients were treated with therapeutic anticoagulation with 6 patients continuing indefinite anticoagulation. endovascular interventions were performed in 18 patients and included percutaneous pharmacomechanical thrombectomy and/or catheter-directed thrombolysis (15 patients), balloon angioplasty (11 patients) and stent angioplasty (9 patients). surgical interventions included thoracic decompressive surgery (5 patients) and surgical thrombectomy (1 patient vvas represent an important risk factor for developing extensive extremity dvt in adolescents. this special population is at risk for short-term and long-term com-plications. early identification and correction of vaas may improve outcomes. however, multicenter, prospective studies are needed for developing optimal evidence-based treatment approaches. alexander glaros, roland chu, sureyya savasan, meera chitlur, madhvi rajpurkar, yaddanapudi ravindranath children's hospital of michigan, detroit, michigan, united states background: acute budd-chiari syndrome (bcs) is a rare thrombotic emergency in children, and etiologies/treatment are less well-defined than in adults. in adults, a systematic approach including anticoagulation, relief of venous obstruction, and treatment of the underlying cause has proven successful. more recently treatment has tilted towards aggressive surgical interventions, which carry significant risk and are often not feasible. objectives: review our experience with three different patients with bcs and suggest a mechanistic based approach to treatment. the records of three patients with bcs were reviewed and their presentations, etiologies, treatment, and outcomes were reported. results: patient a was a 17-year-old female with paroxysmal nocturnal hemoglobinuria who presented with recurrent worsening abdominal pain over several months. narrowing of inferior vena cava (ivc) and hepatic veins was noted on imaging. liver transplant was not considered surgically feasible. she was treated with eculizumab, steroids, and anticoagulation with restoration of hepatic venous flow in 4 weeks. patient b was a 14-year-old male with several weeks of right upper quadrant pain, fatigue, and pre-syncopal episodes, with a history of blunt abdominal trauma from football scrimmage 4 weeks earlier. he was found to have near complete occlusion of the ivc and hepatic veins. liver transplant was not considered feasible. he was successfully treated with anticoagulation alone. patient c was a 3-yearold male with acute myeloid leukemia in induction cycle 2 who developed severe pancytopenia; typhlitis was diagnosed and managed medically. days later he acutely decompensated, arrested, and was placed on extra corporeal membrane oxygenation, and imaging showed complete occlusion of the portal vein, hepatic veins, and ivc to the level of the atrium, with bilateral pulmonary emboli. emergency liver transplant or catheter based interventions was deemed not feasible. treatment with eculizumab was considered for presumed inflammation induced complement activation (c3 59mg/dl [normal 77-171]; ch50 was 12u/ml [normal 42-91]) as a trigger for thrombosis, but the patient progressed quickly and died before it could be initiated. our experience with bcs shows that invasive interventional options and liver transplant may not be feasible in most patients for multiple reasons. rapid diagnosis and aggressive etiology-based medical management are paramount to successful treatment of this rare complication. eculizumab may be considered in treating bcs with complement activation not only due to innate disorders, but also secondary to acute inflammation when proper laboratory evidence is present. background: platelet aggregation studies are the gold standard for the diagnosis of platelet function defects during the evaluation of a patient with bleeding problems. the platelet aggregation test measures how well platelets clot in response to different concentrations of epinephrine, adenosine diphosphate (adp), collagen, arachidonic acid and ristocetin. because platelet function defects are often under-recognized and under-diagnosed in the pediatric patient, the true incidence is unknown. we report our experience in the diagnosis of platelet defects at our institution over a 5-year period in order to add some clarity to the limited pediatric data available. objectives: our primary objective is to document correlations/trends between less well-known platelet function abnormalities and clinically significant bleeding at our institution over a 5-year period. design/method: after appropriate irb approval obtained, we performed a retrospective chart review of all children who had platelet aggregation testing done from 2011 to 2015. data collected included demographics (age, sex, race), personal and family history of bleeding, screening for coagulation defects and platelet aggregation test results. symptoms examined in our data were limited to epistaxis and heavy menstrual periods. for each of these symptoms, results were further analyzed to those with abnormal responses to adp and epinephrine. patients with existing bleeding diagnoses and those with incomplete medical records were excluded. we identified 159 patients. of the patients with epistaxis, 70% had abnormal platelet aggregation testing while only 36% of those with heavy menstrual periods had abnormal results. within our population, abnormal platelet function assay (pfa-100) results or race did not appear to correlate with abnormal platelet aggregation testing. in the cases of epistaxis, sex was also noncontributory. our preliminary results suggest that platelet aggregation testing was more useful in predicting platelet defects in those with a clinical bleeding history of epistaxis as opposed to heavy menstrual periods. for other presenting symptoms, platelet aggregation testing did not offer diagnostic benefit. abnormal response to adp in the platelet aggregation test was the most common finding in our population; the clinical significance of which is not well understood. going forward, we plan to document whether abnormal results correlated significantly with the subsequent final diagnoses of our patients. background: decision making for severe hemophilia a in previously untreated patients (pups) has recently become a significant ethical debate. recombinant factor viii (rfviii) products previously were recommended to avoid transmission of blood borne pathogens associated with plasma-derived fviii (pdfviii) products. however, the increased incidence of fviii alloantibody inhibitors with rfviii products compared to pdfviii products has challenged this former standard of care. despite the support of the medical and scientific advisory council, recommendations considering pdfviii products for a pup remains controversial. design/method: we used a modified utilitarian approach involving clinical, public health, and research ethics. shared decision making permeates the framework to maximize understanding, minimize bias, respect informed consent or dissent, and provide care that aligns with patient and family values when medically and practically feasible. the framework has three tiers. first, it evaluates whether resources are scarce or abundant for equitable resource allocation. if fviii products are scarce, we s133 of s301 recommend developing a central supply for emergency use and then evaluating the needs of the severe hemophilia a patients. prioritization of who receives the factor products would be decided by a designated team based on the availability of the factor products and clinical scenarios, with no preference given to those on research trials. however, if resources are abundant, treatment for acute bleeding and standard of care prophylaxis measures, including primary prophylaxis, could continue. the second tier accounts for whether there is a new infectious epidemic or concern where a pathogen cannot be eliminated. if there is, healthcare and public health workers may limit the use of pdfviii products. if not, pdfviii and rfviii products are to be equally considered. the third tier evaluates whether the clinical scenario is emergent or not. if there is acute, emergent bleeding, the immediately available resource should be used, along with bypassing and/or adjuvant resources as needed until the bleeding has resolved or improved. to align with patient and family preferences, attempts to have both pdfviii and rfviii products available at similar costs in institutions would be ideal. this ethical framework endeavors to balance autonomy, beneficence, nonmaleficence and justice in helping guide discussions among providers, pups with severe hemophilia a, and their families. disclaimer: findings and conclusions are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention, emory university, or children's healthcare of atlanta. background: von willebrand disease (vwd) is a common bleeding disorder which affects up to 1% of the population without gender predilection. bleeding associated with this condition results from a deficiency or abnormality in von willebrand factor interfering with formation of primary hemostasis. ehlers-danlos syndrome (eds) is a group of rare inherited connective tissue disorders which may have an associated bleeding manifestation without abnormalities in coagulation testing. bleeding symptoms reported in eds result from capillary and tissue fragility. joint hypermobility syndrome (jhs) is an inherited condition which is nearly indistinguishable from eds iii. reports of coinheritance of vwd and eds or jhs are infrequent. the objective of this retrospective study was to review patients with coexisting vwd and eds or jhs at the indiana hemophilia and thrombosis center in order to describe the type and severity of bleeding symptoms, physical examination findings, and pertinent laboratory data. design/method: the electronic medical record database of the indiana hemophilia and thrombosis center was queried for patients with a diagnosis of vwd and one of the following descriptors: hypermobility syndrome, hypermobility, hypermobile joints, or ehlers-danlos syndrome. the records of identified patients were reviewed for demographics, type and severity of bleeding symptoms, beighton scores (bs), vwd antigen, ristocetin cofactor, factor viii levels, vwd multimer pattern, vwd subtype, genetic testing for eds, and family history of eds. results: a total of 6 patients with dual diagnoses of vwd and eds and 21 patients with vwd and hypermobility were identified with this query. two patients had completed genetic testing for eds, and one had a col1a1 gene mutation identified. significant bleeding symptoms in the vwd and eds group included hematuria and postoperative hemorrhage. two of these patients had delayed wound healing postoperatively. seven of the 19 patients identified to have type i vwd and jhs had moderately severe and somewhat unusual bleeding episodes reported including hematuria, hematemesis, and hemoptysis; 4 of these patients had significant perioperative bleeding. females composed 83% of the vwd and eds group and 76% of the vwd and jhs group. conclusion: coinheritance of vwd and eds is an uncommon phenomenon. patients with vwd and eds or jhs may have atypical and moderately severe bleeding, especially with procedural intervention. incorporation of bs into the assessment of patients with bleeding disorders is useful to identify potential inherited collagen disorders, as diagnosis of these conditions may impact clinical management. in the year-long phase ii study (ro1fd003712), 11/12 khe patients responded. patients were followed for 5 years after study completion, collecting data on growth and development, complications of therapy, unexpected toxicities, and need for continuing sirolimus. objectives: after study therapy treatment of one year, objectives include: 1. assess long term toxicity over the 4-5 year period after study therapy completion 2. assess unexpected toxicity 3. assess overall condition of the patient 4. assess need for restart or continuation of sirolimus therapy design/method: prospective follow-up of patients with a diagnosis of khe from 2 institutions. inclusion criteria: follow-up for 4-5 years post-study. results: follow-up included data at 5 year (n = 5) and 4-4.5 year (n = 4) time points. average age at the start of treatment was 12 months. 9 of 12 patients were available for follow up. four patients are no longer on sirolimus: one patient completed study therapy and remains off treatment (ot) (7 years), 1 required 2 years of treatment and is now 2.5 years ot and 2 required an additional treatment course prior to successful discontinuation now 17 and 22 months ot. of the 5 patients still on sirolimus, all restarted medication for symptoms of pain, swelling and/or edema interfering with quality of life and have made an average of 2.5 attempts to discontinue sirolimus. no patient had reoccurrence of kmp. all patients had improvement in clinical and radiologic appearance of khe but all have residual lesions noted on imaging and/or clinical exam. no unexpected toxicity, growth delay, developmental issues or other long term toxicity of sirolimus was noted. conclusion: this is the first prospective data on long-term follow up of khe patients treated with sirolimus. although numbers are small, sirolimus is well tolerated; however, over half the patients were still on medication at 4-5 year follow up. this stresses the need for continued long term follow up in these young patients and investigation of the mechanism of sirolimus effect. nationwide children's hospital, columbus, ohio, united states background: recent studies have identified that adult persons with hemophilia (pwh) have a higher prevalence of hypertension and renal disease than the general population. while hematuria is a known complication of hemophilia a and b (ha, hb), its long-term impact on pwh is not currently known. by annually screening our patients with urinalysis, our pediatric center identified that just under half of our patients demonstrated hematuria over a four-year period. motivated by a desire to identify early markers of hypertension and renal disease, we sought to determine if this finding is reflected in the pediatric hemophilia population as a whole. objectives: establish the population-wide prevalence of hematuria in pediatric pwh. design/method: we used the pediatric health information system (phis) database, which contains clinical and resource utilization data for inpatients from 45 hospitals nationwide, to analyze the prevalence of hematuria, hypertension, renal disease and related diagnosis codes in pediatric pwh who were admitted from january 2010 to september 2015. results: during the five-year period, 2,197 unique pediatric pwh accounted for 4,802 admissions. while the majority of admissions were for bleeding or infectious concerns, 96 (4.4%) patients had an affiliated admission code for hematuria. for admissions as a whole, the median age was 7 years with 12% of those admitted being infants, 32% toddlers, 27% children, 28% adolescents, 2% older than 21. we identified 83% of admissions were for ha with the remaining 17% were for hb. there were 1254 (26%) admits in which a bypassing agent was administered. the median length of stay for persons with hematuria was 2 days compared to 3 days for nonhematuria/other bleeding. there were 120 (2.5%) admissions with hypertension reported; though, only 3 patients received an antihypertensive medication during that admission. additionally, only 31 (0.6%) admissions reported a diagnosis code of renal disease. our study demonstrated that pediatric pwh are experiencing hematuria. in general, only patients with persistent hematuria require hospital admission so we suspect this data underrepresents the numbers of pwh experiencing hematuria that is managed in the outpatient setting. we also suspect that hypertension is grossly underreported and undertreated in pediatric pwh. additionally, there are a low number of patients experiencing renal disease requiring hospital admission among this cohort. given that there is little research into the long-term impact of hematuria in hemophilia, we feel these findings support the need for further vigilance of our pediatric pwh. background: gla and gsd can aggressively destroy bone, with significant impact on morbidity and mortality. the mtor inhibitor, sirolimus has been shown to be effective in the treatment of these diseases. based on the addition of mtor inhibition to bisphosphonate therapy in metastatic cancer therapy, regimens have been used for refractory or high risk gla and gsd but there is heterogeneity of diagnosis, and variability of drug regimens and assessment of effectiveness. objectives: 1. assess the variability of clinical features of gla and gsd 2. assess the heterogeneity of diagnosis 3. assess drug regimens and response assessment across multiple institutions design/method: we conducted a retrospective review from 5 institutions of 19 cases of gla and gsd treated with sirolimus and a bisphosphonate for at least 2 months with assessment of clinical features, treatment protocols, response regimens and side effects. results: patients included gla (n = 8) and gsd (n = 11). the average age at diagnosis was 10 years. clinical features included effusions: gla (n = 4), soft tissue lymphatic malformations: gla (n = 3), gsd (n = 1), multiple splenic lesions: gla (n = 3), and soft tissue swelling at the site of bony lesion: gsd (n = 3). the presenting symptom in 17 patients was pain with 2 patients (gla) presenting with shortness of breath. fracture was noted in 5 patients: gla (1), gsd (4). diagnostic and/or response imaging included mri, ct, bone scan, skeletal survey and dexa scan. treatment consisted of: initial sirolimus use with the addition of bisphosphonate secondary to worsening disease (n = 4), initial therapy with other agents (interferon, chemotherapeutic agents, radiation) and change to sirolimus and bisphosphonate secondary to toxicity (n = 6), sirolimus and bisphosphonates (n = 7) and sirolimus, bisphosphonates and interferon (n = 2). seventeen patients had stable disease and 8 patients had improvement of disease. sirolimus protocol was standard; however, bisphosphonate protocol varied in dosing and frequency. side effects were tolerable and expected with no grade iii or iv toxicity. sirolimus and bisphosphonates are a safe and effective therapy for gsd and gla. a consistent medication regimen, redefined response and an improved radiologic classification will be important for the development of a prospective clinical trial. background: hemophilia a is a bleeding disorder from the deficiency of clotting factor viii. the most significant sequelae of hemophilia a is the tendency to develop hemarthrosis that incites joint destruction. the prevalence of overweight and obesity has been increasing in the general and hemophilia population and leads to several morbidities including arthropathy. this is a particular concern for hemophilia a as arthropathy is a consequence of joint bleeding. objectives: the purpose of this study was to detect the relation between body mass index (bmi) and joint health endpoints in a pediatric hemophilia population. design/method: participants in this study included 64 patients from the hemostasis and thrombosis center at children's hospital los angeles. participants were pre-screened and approached for this study during routine follow-up appointments. patients aged 4-18 years old who have been diagnosed with hemophilia a, including mild, moderate, and severe, qualified for the study. informed consent was obtained from the patients or parents before enrollment. joint health was objectively measured by physical therapists from children's hospital los angeles using the hemophilia joint health score (hjhs). an hjhs total score is calculated by assessing: swelling, duration of swelling, muscle atrophy, crepitus on motion, flexion loss, extension loss, joint pain, and muscle strength in 6 major joints. subjective data was also obtained by patients recording their annual bleed rate within the past year. of the 64 patients, 28 (44%) were normal weight, 12 (19%) overweight, and 24 (38%) obese. we used chi-square analysis to compare joint scores across bmi classifications (chi square = 2.87, df = 2, p-value = 0.24). although, this did not approach statistical significance, the average hjhs score in patients who had a hjhs >0 shows an increasing trend among bmi classifications: 6.19 in normal bmi patients, 6.75 in overweight bmi patients, and 7.00 in obese bmi patients. the average number of annual bleeds in those with positive values show: 8 in normal bmi patients, 5 in overweight bmi patients, and 12 in obese bmi patients. although a positive effect of adiposity was found in the joints of hemophilia a pediatric patients, the effect shows there was not enough evidence to conclude a difference. future studies are needed to address whether obesity has an effect on hemophilia and to determine whether overweight/obesity can lead to further complications in hemophilic joints. background: stagnant blood flow in slow-flow vascular malformations (vm), particularly in their venous components, can lead to localized intravascular coagulation (lic) that is characterized by elevated d-dimer levels, low fibrinogen and decreased platelet count this coagulation derangement can lead to localized thrombosis or bleeding which can result in pain, functional limitations, and possible progression to disseminated intravascular coagulopathy (dic). the treatment of vm and their associated coagulopathy has proven difficult. patients with complex vm are frequently managed with sirolimus, an mtor inhibitor, and have clinical benefits, including reduction of pain and improvement in functional impairment. it is possible that some of these improvements from sirolimus could be secondary to improvement in the coexisting lic. objectives: this study assessed the use of sirolimus to manage the coagulopathy seen in slow-flow vm. design/method: we reviewed charts of patients with vm who are followed in the vascular anomalies center at arkansas children's hospital and were started on sirolimus. efficacy was objectively assessed through improvement of ddimer, fibrinogen and platelet count. three sets of lab values (pre-sirolimus, 1-3 months post-sirolimus, and most recent) were obtained for each patient when available. we identified a total of 35 patients who had been prescribed sirolimus. eighteen were excluded based on underlying condition other than slow-flow vascular malformation and 1 for inadequate medical records. a total of 16 patients (13 combined vascular, 3 venous) were included in the study. all 16 had elevated d-dimer levels (mean 4.64 mcg/ml feu, median 2.99 mcg/ml feu, range (0.83-14.65)) prior to treatment. two patients had an associated low fibrinogen (below 175 mg/dl), indicating severe lic. with treatment, 14 (87.5%) patients showed an overall decrease in d-dimer levels with an average decrease of 1.52 mcg/ml feu between pre-and post-sirolimus labs, and an average decrease of 1.03 mcg/ml feu between pre-sirolimus and most recent values. the two patients with low fibrinogen prior to treatment showed a decrease in d-dimer levels (mean decrease of 7.845 mcg/ml feu) and an increase and normalization in fibrinogen (mean increase 83.95 mg/dl) after beginning sirolimus. no patient had thrombocytopenia. we report that treatment with sirolimus was effective in improving coagulopathy associated with slowflow vm as evidenced by decreased d-dimer levels and increased fibrinogen and/or platelets. long-term use of this medication in this population may decrease the bleeding and thrombotic complications that these patients experience, especially following invasive vascular procedures. background: safety and efficacy of bay 94-9027, a sitespecifically pegylated b-domain-deleted recombinant factor viii, in previously treated adolescents and adults aged 12-65 years with severe hemophilia a was demonstrated in the phase 2/3 protect viii study and ongoing extension. objectives: this subanalysis examines the efficacy and safety of bay 94-9027 in adolescents in protect viii and the ongoing extension study (data cutoff, january 2015). design/method: in protect viii, 134 patients (including 12 adolescents) received bay 94-9027 on demand or as prophylaxis for 36 weeks. prophylaxis regimens for weeks 10-36 were twice-weekly (30-40 iu/kg), every-5-days (45-60 iu/kg), or once-weekly (60 iu/kg) infusions based on bleeding during a 10-week run-in period of 25 iu/kg twice-weekly prophylaxis. patients continued their prophylaxis regimens in the extension or changed regimens at any time. results: twelve patients aged 12-17 years were included in the protect viii intent-to-treat population; 1 s137 of s301 additional patient discontinued after 1 dose (included in safety population). for 11 patients receiving prophylaxis before study enrollment, median (range) number of total and joint bleeds in the 12 months before study entry was 8.0 (0-15) and 6.0 (0-10), respectively. ten patients (83.3%) had target joints at baseline (median [range],1 [0-4] per patient). during weeks 10-36 of protect viii for the entire time patients remained on their designated prophylaxis dosing frequency, the median (quartile [q]1; q3) annualized bleeding rate (abr) for patients receiving twice-weekly (n = 3), every-5-days (n = 6), and once-weekly prophylaxis (n = 3) was 0 (0; 2.0), 1.1 (0; 8.2), and 18.4 (0; 19.3), respectively (overall prophylaxis [n = 12], 1.0 [0.0; 10.1]). two patients switched from once-weekly to twice-weekly (n = 1) or every-5-days prophylaxis (n = 1), and number of bleeds decreased from 2 to 1 in one patient and 6 to 4 in the other. all 12 patients from the main study continued in the extension; mean abr in the extension was 3.2 and varied by dosing regimen (twice weekly [n = 3], 3.2; every 5 days [n = 5], 5.2; once weekly [n = 2], 0.9). two patients changed from every-5-days to once-weekly prophylaxis during extension (mean abr, 0.6). one patient had a nonneutralizing antibody to bay 94-9027 at baseline; end-of-study titers were negative. no patient developed anti-peg antibodies or factor viii inhibitors or experienced a serious adverse event related to bay 94-9027 during the main study or extension. in previously treated adolescents with severe hemophilia a, bay 94-9027 prophylaxis was effective in prevention of bleeds, with less bleeding overall versus prestudy, and was generally well tolerated. funded by bayer. cincinnati children's hospital medical center, cincinnati, ohio, united states background: vascular malformations (vms) consist of a heterogeneous group of congenital disorders characterized by the abnormal development of blood and/or lymphatic vessels, which cause a broad spectrum of clinical manifestations. although considered benign, vms are frequently associated with cutaneous complications that can cause significant morbidity such as nodular overgrowth, skin thickening, pruritus, oozing or bleeding of lymphatic blebs and secondary infection. oral sirolimus has shown to be effective in the treatment of complicated vascular malformations but has known side effects and need for frequent laboratory monitoring. currently, there are limited studies on the use of topical sirolimus for the treatment of cutaneous manifestations of vascular malformations. objectives: to evaluate the efficacy and safety of topical sirolimus in vms with cutaneous complications and propose indications for use. design/method: this is a retrospective review of medical records of patients with vascular malformations treated with topical sirolimus from january 2012 to december 2017. response was determined by subjective and objective improvement. results: twenty-four patients, 16 (66%) females and 8 (33%) males, with vascular malformations and cutaneous manifestations were treated with topical sirolimus. age ranged from 4-27 years. indications for treatment were: blebs (79%, n = 19) causing either leaking, bleeding, pain, pruritus, swelling or recurrent infection; nodular overgrowth 8% (n = 2); pyogenic granuloma 4% (n = 1); bleeding 4% (n = 1) and cosmetic 4% (n = 1). treatment course ranged from 1-18 months. no major side effects were reported. one patient reported burning and itching sensation. regarding clinical response: 83% (n = 20) patients had improvement in cutaneous lesions; 12% (n = 3) had a stable lesions; and 4% (n = 1) stopped treatment due to side effects. for prior/concomitant treatment: 83% (n = 20) had prior surgery, laser or sclerotherapy; 37% (n = 9) had concomitant oral sirolimus. of the 15 patients not receiving concomitant systemic sirolimus, only 13% (n = 2/15) had been on oral sirolimus. of these patients, 80% (n = 12/15) had a very good response to topical treatment. : topical sirolimus appears to be beneficial and well-tolerated with a minimal side effect profile for the treatment of cutaneous manifestations of vascular malformations as a single agent or as adjuvant therapy with systemic sirolimus when symptoms are not adequately controlled. further studies are needed to prospectively analyze efficacy and safety of topical sirolimus in this patient population. objectives: to evaluate the safety and efficacy of long-term romiplostim in children with itp. design/method: all patients received weekly sc romiplostim from 1-10 g/kg to target platelet counts of 50-200 × 10(9)/l. median (min-max) treatment for the 65 patients was 135 (5-363) weeks for a total of 182 patient-years, or 2.8 years per patient. at baseline, median (min-max) age was 11 (3-18) years; 56% were female; 9.1% had prior splenectomy. median (min-max) average weekly dose was 4.8 (0.1-10.0) g/kg, including escalation to a stable dose; 20 patients started on 1 g/kg. reasons for discontinuing romiplostim (n = 28, 42%) included consent withdrawn (n = 10), required other therapy (n = 6), and ae (n = 2) (asthenia, headache, dehydration, and vomiting in one patient and anxiety in the other; none treatment related). fifty four serious aes occurred in 19 patients but were treatment related in one (concurrent grade 4 thrombocytopenia, grade 3 epistaxis, and grade 2 anemia). anti-romiplostim neutralizing antibodies were detected in one patient who discontinued to receive other therapy; antibodies were absent on retesting. from week 2 on, median platelet counts remained >50 × 10(9)/l; median platelet counts were >100 × 10(9)/l from weeks 24-260. nearly all (94%, 61/65) patients had ≥1 platelet response (platelet counts ≥50 × 10(9)/l, excluding ≤4 weeks after rescue medication). most (72%, 47/65) patients had a platelet response ≥75% of the time and 58% (38/65) did ≥90% of the time. sixty (92%) patients (or caregivers) self-administered romiplostim. fifteen (23%) patients had treatment-free periods of platelet counts ≥50 × 10(9)/l for ≥24 weeks (ie, remission); these patients (9 girls, 6 boys) had had itp for a median (min-max) of 3.5 (1.3-13) years, none had prior splenectomy, and had received romiplostim for 2.1 (0.7-6) years. all 15 had platelet counts >100 × 10(9)/l for ≥3 months and 12/15 for ≥6 months; the median (min-max) duration of being ≥100 × 10(9)/l was 42 (13-109) weeks. of baseline characteristics such as sex, platelet counts, itp duration, and number of past itp treatments (1, 2, 3, >3), only age <6 years was predictive of developing treatment-free periods ≥24 weeks (p = 0.0035). in this seven-year open-label extension, >90% of children with itp achieved a platelet response and romiplostim was well tolerated. importantly, 23% of patients were able to discontinue all itp medications for ≥6 months. funded by amgen inc. background: sirolimus is an immunosuppressive drug that is widely used in solid organ and bone marrow transplantation, and more recently for the treatment of vascular and lymphatic anomalies. sirolimus has been associated with decreased immunity in the transplant setting in patients that have received other immunosuppressive drugs or were immunosuppressed from previous chemotherapy. the effects of sirolimus on the immune system in chemotherapy naïve children who have not received other immunosuppressive agents are not well understood, and there is variability in the approach to fever and pcp prophylaxis. to understand the effects of sirolimus on the immune system of patients with non-complicated vascular or lymphatic anomalies by evaluating anc, alc prior to and after sirolimus therapy. design/method: multi-institutional retrospective review was done to include patients with non-complicated vascular or lymphatic anomalies. those with effusions/ascites, multiorgan involvement, or history of vascular-anomaly-related infections prior to treatment were excluded. results: twenty patients with kaposiform hemangioendothelioma (n = 6), generalized lymphatic anomaly (n = 2), cloves syndrome (1), and simple vascular malformation (n = 11) were included. age at initiation of sirolimus treatment ranged from 0.5 -20 years. male to female ratio was 9:11. sirolimus was initiated due to extensive disease, lack of response to steroids or bisphosphonates, pain, dment, lymphatic drainage, and prevention of ongoing overgrowth. prior to the start of sirolimus (sir-0) the mean anc was 3850 and alc was 2875. the target level of sirolimus varied by indication and patient, and ranged from 6 to 10. after the 1st steady state level, 1 month after sirolimus (sir-1) the mean anc decreased to 2951 and alc was 2793. at 3 months after sirolimus (sir-3) the mean anc was 3108 and alc was 2874. the first sirolimus levels (sir-1) mean was 11.3; and sir-3 level was 7.8. nine patients were placed on pcp prophylaxis at the start of sirolimus. none of these patients had an infectious complication while on sirolimus at a median f/u of 13 months. one patient had mild neutropenia (anc >500) which normalized after discontinuation of pjp prophylaxis. conclusion: in this small cohort of patients we found that the anc and alc level in patients with non-complicated vascular or lymphatic anomalies at sir-0 was not different from the sir-1 or sir-3. prospective studies that specifically track anc, alc, igg, and lymphocyte function should be conducted to better understand the effects of sirolimus in the immune system. this data will allow for uniform recommendations regarding prophylaxis and management of febrile episodes. background: acute infections and the associated systemic inflammation can increase the risk of venous thromboembolism (vte) and in certain well-defined clinical scenarios may be the primary trigger of vte in pediatric patients. pediatric data on vte in the setting of acute infection are sparse. objectives: to describe characteristics and outcomes of vte in pediatric patients with acute infections. we conducted a retrospective chart review of all pediatric patients with objectively confirmed vte treated at our institution since 2011 and identified all patients in whom an acute infection was identified as a vte trigger. patients were managed according to standardized institutional protocols based on published guidelines. relevant demographic, clinical and laboratory data were collected and summarized using descriptive statistics. since 2011, acute infection was identified as a trigger in 147 of 429 vtes (34%) diagnosed at our center. the median age at time of vte diagnosis in this group was 2.3 years (interquartile range 0.3-16). males were more commonly affected than females, representing 56% of cases. neonatal vte events accounted for 14% of cases. sepsis was the most common acute infection to be identified as a vte trigger [59/147 cases (40%)]. most vte events (80%) associated with acute infections were considered hospital-associated vtes. at time of vte diagnosis, 61% of patients were critically ill. extensive vte (defined as completely occlusive thrombosis involving >1 venous segment) occurred in 16% of patients. acute infection was deemed to be the primary trigger for vte in 30/147 patients (20%). infection-associated vtes in this cohort included cerebral sinus venous thrombosis due to sinus or cns infection (13 patients, 43%), septic throm-bophlebitis (11 patients, 37%), lemierre's or lemierre's-like syndrome (4 patients, 13%) and osteomyelitis-associated deep vein thrombosis (2 patients, 7%). systemic anticoagulation was prescribed in 124/147 patients (84%). anticoagulationrelated major bleeding occurred in 10/124 patients (8%). vte complications included vte recurrence (16 patients, 10%), vte progression (1 patient), acute pulmonary embolism (2 patients) and arterial ischemic stroke (2 patients). our study indicates that acute infection is a common risk factor for pediatric vte, especially in critically ill children, and can be the primary trigger in a significant proportion of vte cases associated with acute infections. anticoagulation appeared to be overall safe in this population and was associated with low rates of serious vte-related acute complications. however, our study also suggests that this population may be at increased risk for vte recurrence and anticoagulation-related major bleeding. background: epithelioid hemangiomas (eh) are rare benign vascular tumors that occur in soft tissues and bone and present between the third and sixth decades of life. a subset (29%) of eh harbor fos rearrangement. eh has been described in children, but little is known about the long-term outcomes of pediatric eh. the main objective is to obtain data to be used for improved understanding of this rare disease in order to provide standardization of care and development of future research studies. board-approved retrospective review of clinical, pathologic, and radiographic characteristics, and treatment outcomes in 11 patients diagnosed with eh between 1999 and 2017. results: eight patients were male; mean age at diagnosis was 14.8 years (range: 6-23). lesions involved the lower extremities (n = 5), cranium (n = 3), pelvis (n = 2), and spine (n = 1). multifocal disease was identified in five patients. the most common presentations involved significant localized pain and neurologic symptoms: headache, cranial nerve injury, loss of consciousness. radiographic studies identified variable features, such as multifocal lytic bony lesions with sclerotic margins, enhancing soft tissue component, and surrounding inflammatory edema. histologically, all specimens were composed of vascular channels lined by epithelioid endothelial cells without significant cytologic atypia; solid cellular areas (n = 2). endothelial cells were positive for cd31 and egr, and negative for camta1. fos rearrangement was assessed in only one specimen and detected. mean follow-up time was 545 days (range: 23-2642). patients were treated with surgical resection, intravascular embolization, bisphosphonates, propranolol, interferon, and sirolimus. one patient treated with interferon and one with sirolimus exhibited partial response for mean follow-up of 1566.5 days. although eh is a benign neoplasm, it is difficult to manage without standard protocols and portends considerable morbidity. our findings suggest medical management, particularly sirolimus, may benefit these patients; however, long-term follow-up is needed in treated children. novel fos inhibitors are in development and may benefit patients with fos rearrangement. penn state health children's hospital, hershey, pennsylvania, united states background: central venous catheters (cvc) are often required in critical care settings in order to provide a secure point of access for life sustaining care. clinical studies identify cvc presence as the single most important risk factor for deep vein thrombosis (dvt) in children. venous thromboembolic event (vte) incidence rates in critically ill children with a cvc range from 0.3-18% and 0.06-32.5 per 1000 catheter days depending on the population studied. per institutional protocol, the penn state health children's hospital picu (hershey, pa) utilizes a low dose continuous infusion of unfractionated heparin (ldufh) at 10 units/kg/hr as prophylaxis against cvc-related vte and to maintain line patency. the efficacy of this approach has never been evaluated. to determine if ldufh for prophylaxis results in lower incidence of cvc-related vte, catheter dysfunction and central line associated blood stream infection (clabsi) without increasing morbidities. to determine if the incidence of catheter related vte is lower than historical published data, a retrospective chart review was conducted utilizing the institutional electronic medical record for all patients in 2015, aged 0-17.99 years, who had a cvc during a picu admission. secondary objectives such as the incidence of catheter dysfunction, clabsi, and any associated bleeding complications are also being analyzed. results: interim data analysis revealed 478 cvcs (400 nontunneled cvc, 18 totally implantable devices, 19 tunneled lines, 41 peripherally inserted central catheters [picc] ) in 374 total patients with a median age of 1.9 years. overall vte incidence was 1.88% (9/478) with 7 vtes associated with non-tunneled cvc and 2 with piccs. sixty one percent of non-tunneled cvcs received ldufh and 85% (6/7) of the patients with vtes associated with non-tunneled cvcs did receive ldufh prophylaxis. vte incidence rate of nontunneled cvcs with ldufh was 2.5% (6/243) and 2.56 per 1000 picu catheter days. the only other vte events identified within our study cohort were in the picc group where two patients experienced vte, one of which was receiving ldufh. clabsi incidence was 1.2% (4 non-tunneled cvc, 1 tunnel cvc, 1 picc). no major bleeding complications were associated with ldufh. preliminary data demonstrates ldufh is efficacious in preventing cvc-related vte in comparison to published reports. further analysis will compare another similar sized and acuity level picu which does not practice the same method. background: fibroadipose vascular anomaly (fava) is a rare, challenging disorder associated with pik3ca mutations. fava often causes painful replacement of muscle and soft tissues with fibrotic and adipose tissue and is associated with ectatic draining veins. treatments for focal lesions are surgical excision, cryoablation or sclerotherapy and the role of medical therapy is unclear. some fava lesions are too extensive or directly involve neurovascular structure, resulting in refractory pain. objectives: to retrospectively evaluate the efficacy of sirolimus in patient with residual symptoms after procedural therapies for fava design/method: retrospective review of individual 7 cases from 6 institutions of fava refractory to other therapies treated with sirolimus for at least 3 months. cases were s141 of s301 identified by polling member of the aspho vascular anomalies special interest group. results: all seven patients report improvement on sirolimus therapy. all patients had received prior procedures, including sclerotherapy (6 patients), cryoablation (2 patients) and/or resection (3 patients). mean age at sirolimus initiation was 16y (range 6-29y). mean length of therapy is 18.4 months (range 3-29 months). six patients were treated with bid dosing and one adult received daily dosing. goals of sirolimus were improvement in pain or musculoskeletal dysfunction. pain and function improved in all patients, including discontinuation of narcotic use and resumption of participation in sports. time to symptom improvement ranged from 1-4 weeks. in four patients for whom dose was lowered, pain recurred in all four and responded to restarting or increasing sirolimus dose. while all patients do not have pre-and postsirolimus imaging, decrease in fava lesion size is seen in cases with available imaging. sirolimus side effects are similar to prior reports, most commonly mouth sores, elevated lipids and acne. we report the first known data supporting a role of sirolimus in refractory fava cases. sirolimus is welltolerated and initial improvement is rapid, within 4 weeks of initiation. whether sirolimus has a role in upfront therapy to reduce lesion size prior to procedures deserves further study. objectives: to assess platelet responses in children with itp receiving romiplostim. design/method: eligible children had itp for ≥6 months, ≥1 prior therapy, and screening platelet counts ≤30 × 10(9)/l or uncontrolled bleeding. weekly dosing was from 1-10 g/kg to target platelet counts of 50-200 × 10(9)/l. bone marrow biopsies were evaluated in europe at baseline and after 1 or 2 years (cohorts 1 and 2). as of mar 2017, 203 patients received ≥1 dose. at baseline, median (min-max) age was 10 (1-17) years, itp duration was 1.8 (0.5-13.8) years, and platelet count was 14 (2-265) × 10(9)/l; 10 patients (5%) had had prior splenectomy. the median (q1, q3) % time with a platelet response (platelet count ≥50 × 10(9)/l, no rescue medications past 4 weeks) in months 0-6 was 50% (17%, 83%) (primary endpoint). over the course of the study, 88% (179/203) of patients had a platelet response. four patients maintained platelet counts ≥50 × 10(9)/l with no itp medications for ≥24 weeks. median (min-max) treatment duration was 53 (8-119) weeks for 226 patient-years in total. median (min-max) average weekly romiplostim dose over the course of the study was 6.9 (0.2-9.5) g/kg; the median dose was 9 g/kg at 1 year (n = 106) and 10 g/kg at 2 years (n = 17). most (63%) patients initiated self-administration. sixty-four patients (31%) discontinued treatment, most frequently for lack of efficacy (n = 38), patient request (n = 7), and adverse event (ae) (n = 7). fortyone (20%) patients had serious aes (saes) including epistaxis (5%) and decreased platelet count (3%). five patients had treatment-related saes: 2 headaches, 2 abdominal pain, and 1 each of presyncope and neutralizing antibodies (ab). there were 6 cases of neutralizing ab to romiplostim (of 201 patients tested), but none to tpo; 5/6 had continued elevated platelet counts and in 2/6 cases ab were not found on retesting. for cohort 1, of 30 patients with baseline bone marrow biopsies, 27 had evaluable on-study biopsies scheduled for 1 year; 1 patient had an increase from grade 0 to 2. there were no findings of collagen or abnormalities. in this interim datacut of a romiplostim openlabel study in children with itp, 88% of children had a platelet response. overall, the median dose was 6.9 g/kg; the median romiplostim dose over time reached 10 g/kg. no new safety signals were observed over 226 patient-years. funded by amgen inc. background: hepatic hemangiomas are benign vascular tumors without a medical home, managed by multiple specialties. the diagnosis has been assigned historically to various vascular lesions affecting the liver with completely different clinical presentations, resulting in difficult standardized management. objectives: the consensus steering committee identified an acute need of clear definitions and evaluation guidelines using the updated international society for the study of vascular anomalies (issva) classification. the goal was to formulate recommendations that will be adopted by all specialties involved in the care of children with hepatic hemangiomas. design/method: we used a rigorous, transparent consensus protocol, with input from multiple pediatric experts in vascular anomalies from hematology-oncology, surgery, pathology, radiology and gastroenterology. in the first section, we precisely define the subtypes of hepatic hemangiomas seen in children (congenital and infantile) using clinical course, histology and radiologic characteristics. inclusion and exclusion limits to the diagnosis are noted. the following two sections describe these subtypes in further detail, including complications to be considered during monitoring and respectively recommended screening evaluations. conclusion: while institutional variations may exist for specific clinical details, a clear understanding of the diagnosis of hepatic hemangiomas affecting the pediatric population and the possible complications that require screening during the monitoring period should be standard. as patients with hepatic hemangiomas are managed by different medical and surgical specialties, a multidisciplinary consensus based on current literature, on the data extracted from the liver hemangioma registry and on expert opinion was required and was accomplished by this manuscript. objectives: to investigate the association between routine prophylaxis with bay 81-8973 and bleeding outcomes after adjusting for key patient and pharmacokinetic (pk) characteristics. design/method: the leopold kids study evaluated safety and efficacy of bay 81-8973 prophylaxis in 51 previously treated boys aged ≤12 years with severe hemophilia a. patients received bay 81-8973 25-50 iu/kg 2x/wk (n = 21) or >2x/wk (n = 30) and were followed up for 6-8 months. prophylaxis dose and frequency were assigned by investigators. pk parameters, including area under the curve (auc), half-life, and clearance, were derived from a population pk model and reflect predicted pk values with a 50-iu/kg dose. patient characteristics were compared between the 2x/wk and >2x/wk groups using wilcoxon rank sum or chi-square tests. negative binomial regression was used to model the association between prophylaxis frequency and annualized bleeding rate (abr) for total bleeds, first without adjustment and then adjusting for age, pk parameters, and bleed history. results: mean ± sd age for patients in this analysis was 6.4±3.0 years. patients receiving prophylaxis 2x/wk had more bleeding episodes in the 12 months before study entry (mean ± sd, 13.0±16.6 [median, 6.0] for 2x/wk vs 4.3±5.7 [1.0] for >2x/wk; p = 0.027) and were more likely to have been treated on demand (38% vs 10%; p = 0.035). pk parameters were similar between the 2x/wk and >2x/wk groups. without adjustments, abr during the study was 12% higher in the 2x/wk group compared with the >2x/wk group (rate ratio [rr], 1.12; 95% ci, 0.44-2.90; p = 0.81). abr was 36% lower in the 2x/wk group (rr, 0.64; 95% ci, 0.24-1.70; p = 0.37) after adjusting for age, auc, and number of bleeds in the prior 12 months. conclusion: abr was numerically lower but not significantly different between the 2x/wk and >2x/wk groups after adjusting for age and pk parameters. these findings suggest that even among patient groups that are homogeneous with respect to age, pk, and bleed history, further individualization of bay 81-8973 prophylaxis based on other characteristics may help reduce bleeding episodes even at a lower treatment frequency. larger real-world studies are needed to verify these findings. funded by bayer. stanford, palo alto, california, united states s143 of s301 background: vascular malformations may be of lymphatic, arterial, venous or capillary endothelial origin. they may be simple or complex, with complex malformations being a combination soft tissue and skeletal overgrowth. although likely present at birth, these malformations often become symptomatic with puberty or infection, and range from little or no clinical impact to life threatening symptoms. in malformations primarily of venous origin, pain may be significant and hypothesized to be caused by phlebolith development (intra-malformation thrombi), inflammation, consumptive coagulopathy, vascular engorgement, and endothelial proliferation. anti-angiogenic and anti-platelet therapies have been reported to relieve pain. however, the use of anticoagulation for pain is not well described. objectives: to report clinical features and outcomes of patients with vascular malformations of venous origin treated with anticoagulation for pain. we performed a retrospective review of patients with vascular malformations followed by the hematology service between january 2010 and december 2017 who were treated for pain with anticoagulation. pain relief was determined both by wong-baker pain scales and patient report. clinical data were extracted from electronic medical records. we identified five patients with venous malformations (vm) who had received anticoagulation for pain. four patients were female and median age was 8 years old (range 4 to 29 years old) at time of initiation of anticoagulation. all five patients had vm of the extremity, two with vm of the lower extremity, and three patients had vm of the upper extremity. two patients had concomitant coagulopathy and demonstrated decreased d-dimer after initiation of anticoagulation. four patients received enoxaparin, and one adult patient received rivaroxaban. all patients reported improvement in pain after administration of anticoagulation. one patient exhibited mild epistaxis and bruising at the injection site. there was no significant bleeding or other complications. pain is a significant complication in patients with venous malformations. our case series suggests that anticoagulation is a safe and effective therapy for pain relief in this population. further investigation is indicated to compare the effect of anticoagulation to other therapeutic interventions such sclerotherapy, surgery, and sirolimus in the treatment of pain associated with venous malformation. maria ahmad-nabi, christine knoll, sanjay shah, lucia mirea phoenix children's hospital, phoenix, arizona, united states background: estimates of the incidence of dvt in patients with osteomyelitis range widely from 5%-30%, however risk factors and outcomes of dvt in this cohort have not been thoroughly established. objectives: this study aims to estimate the incidence of dvt in patients with osteomyelitis, and to assess risk factors and outcomes of dvt in this cohort. design/method: after irb approval, a retrospective chart review was conducted for patients aged 0-18 years seen at phoenix children's hospital between 2012-2016 with icd 9/10 codes for osteomyelitis. exclusion criteria included chronic recurrent multifocal osteomyelitis, and chronic dvt. demographics, clinical factors and outcomes were compared between osteomyelitis patients with and without dvt using the fisher-exact and wilcoxon-rank sum tests, as appropriate for the data distribution. results: a total of 179 study subjects with osteomyelitis had a mean (standard deviation) age of 8.4 (5.7) years. dvt was present in 14 (8% of 179) patients, and 4 (28%), 5 (36%) and 5 (36%) patients received anticoagulation for < 6, 6-12 and ≥12 weeks, respectively. patients with vs without dvt were more likely to be male (86% vs 59%; p-value = 0.05), and had significantly higher rates of bacteremia (64% vs 24%; p-value = 0.003). rates of central lines were comparable between dvt and non-dvt patients (71% vs 68%; p-value = 1.00); however patients with dvt vs without dvt had significantly longer mean length of stay (18 vs 9 days; p-value <0.0001) and higher rates of icu admission (71% vs 16%; p-value <0.0001). the incidence of dvt among osteomyelitis pediatric patients was estimated at 8%, with risk increased by male sex and bacteremia. patients with dvt had significantly higher rates of icu admission and longer length of hospital stay. many of these patients had standard practice management of their dvt with 6-12 weeks of anticoagulation. our data highlights the need for recognition of high risk patients, and the need for future efforts targeting dvt prophylaxis. baylor college of medicine, houston, texas, united states background: lymphatic malformations (lm) frequently occur in the head and neck and can often be disfiguring and even life-threatening. management options include observation, surgery, sclerotherapy, and sirolimus. the optimal sequence of therapeutic interventions has not been determined due to the lack of comparative clinical trials or established guidelines. thus, prenatal planning with a multidisciplinary team is beneficial. we present a case series of ten children with head and neck lms evaluated in 2017 at our multidisciplinary vascular anomalies center. a chart review was performed to assess treatment modalities and recent trends. results: seven of 10 patients (70%) with head and neck lms were diagnosed prenatally. six patients required an ex utero intrapartum treatment procedure. all patients were started on sirolimus at a median age of 12.5 months (range 12 days -18 years). four patients most recently started on sirolimus were less than 3 months of age at the time of initiation. six patients underwent partial excision of lm during the first year of life; none of whom received sirolimus prior to surgery. sirolimus was discontinued in one patient given chronic clostridium difficile infections, and non-compliance in another patient. five patients received sclerotherapy. tracheostomy was necessary in six patients; one patient was de-cannulated after 7 months on sirolimus. all patients have had radiographic and clinical improvement of lm with varying treatment modalities. current clinical observations show improved response with sirolimus and demonstrate tolerability of sirolimus at a young age. conclusion: treatment of pediatric head and neck lms is challenging and a multidisciplinary approach is necessary. as the majority of patients are diagnosed prenatally, prenatal planning and discussion of potential use of sirolimus is beneficial. availability of vascular anomalies experts in the prenatal/neonatal period offers the best management results, and early initiation of sirolimus should be considered for complex lesions. long-term follow up is warranted to investigate the efficacy and timing of treatment options. yale school of medicine, new haven, connecticut, united states background: to mitigate transfusion of pathogencontaminated platelets, amotosalen, a synthetic psoralen compound, is added to sdp components. exposure to uv-a light activates amotosalen and crosslinks dna/rna base pairs, preventing replication of a broad spectrum of viral, bacterial, and other pathogens that may contaminate platelets. pr-sdps were fda approved for clinical use with no age restrictions in 2014. we initiated use of pr-sdps in november of 2016 for all patients. we retrospectively analyzed usage of pr-sdp vs conventional (non-pr) platelets (cp) in neonatal and pediatric patients with thrombocytopenia to compare hemostatic efficacy and the incidence of transfusion reactions (tr) for these products, after one year of a dual platelet inventory. design/method: since pr-sdp were fda-licensed, no irb approval was required; pr-sdp and cp were both considered standard of care. we evaluated transfusions for all pediatric patients age 0-18 years who received any platelet transfusion between november 2016 and november 2017. we determined the volume (mean ml ± 1sd) of each type of platelet component transfused, the number of platelet transfusion episodes, and reported trs based on cdc hemovigilance guidelines. a subgroup analysis was performed for thrombocytopenic neonates (0-4 months). results: patients 0-18 years who received only cps (n = 46) received a total of 8,030 ml of platelets (175 ± 151 ml/patient) over 62 transfusions (1.3 ± 0.6 episodes/patient). for comparison, in 38 patients who received only pr-sdp, a total of 4,350 ml of platelets (115 ± 107 ml/patient, p = 0.04) were infused over 61 transfusions (1.6 ± 0.9 episodes/patient, p = 0.12). for neonates (0-4 months, n = 26) who received only cps, 2,195 ml of cps (84 ± 105 ml/ patient) were transfused over 36 episodes (1.4 ± 0.6 episodes/patient). for comparison, those who received only pr-sdp (n = 27), received 1,613 ml of pr-sdp (60 ± 41 ml/patient, p = 0.27), transfused over 48 episodes (1.8 ± 0.9 episodes/patient, p = 0.08). for all recipients 0-18 years (n = 162), including additional patients who received both cp and pr-sdp, there were three reported allergic trs over 757 transfusion episodes, while no allergic reactions were reported with 537 pr-sdp transfusions. one febrile tr was reported to cp transfusion, while three were reported for pr-sdp. in conclusion, pr-sdps, in our pediatric population age 0-18 years, were comparable to cp products in regards to volume and episodes of platelet transfusions, and incidence/type of transfusion reactions. pr-sdp were safe and effective for use in this pediatric patient population. background: vascular anomalies are classified as either vascular tumors or vascular malformations. fibro-adipose vascular anomaly (fava) is a newly described entity which presents with distinct clinical, radiographic and histopathologic findings. we present a case in which the diagnosis of fava was complicated by a persistent low platelet count secondary to immune thrombocytopenia (itp). to describe a challenging diagnosis of a novel vascular anomaly (fava) complicated by severe thrombocytopenia. a 17 year old male presented to hospital with bruising and left thigh pain related to a remote sports injury. blood work revealed a platelet count of 9 × 109/l, but with an otherwise normal complete blood count. the following were also normal: aptt and fibrinogen; d dimer levels were slightly increased. he was treated with one dose of ivig (0.8 mg/kg) for presumed itp and responded well with his platelet count increasing to 118 × 109/l. he returned to hospital 3 weeks later with recurrent thrombocytopenia and worsening leg pain. an ultrasound of the left thigh revealed a 7.7cm x 4.0cm x 2.9cm lesion within the vastus medialis. the diagnosis of an intramuscular hematoma secondary to persistent thrombocytopenia was made. the patient presented with multiple episodes of thrombocytopenia over the next several months. his itp did not respond to oral prednisone (150 mg/day for 4 days). he continued to have short-lived responses to ivig requiring infusions every other week as his platelet count would fall below 10 × 109/l. his leg pain progressed, restricting him to a wheelchair. further imaging by mri brought into question the diagnosis of a hematoma and a biopsy of the thigh lesion was performed. the results were consistent with a diagnosis of fava; this was subsequently excised. conclusion: this is a unique case where a vascular anomaly was misdiagnosed as a hematoma due to a patient's persistent thrombocytopenia and history of an injury. fava is a newer entity which, unlike other vascular anomalies, has not been linked to thrombocytopenia or a localized consumptive coagulopathy. after excision of the fava, the patient's chronic pain, and mobility resolved, though his itp persisted. objectives: this preliminary, exploratory analysis of realworld administrative data was conducted to determine units dispensed and factor replacement product-related direct expenditures associated with a currently marketed shl or ehl rfix product. design/method: de-identified claims data from the commercially available truven health marketscan® research u.s. claims database were used to identify direct expenditures and number of international units (ius) dispensed for all patients aged 0-17 years with a diagnosis code of icd-9 286.0/icd-10 d66 who used nonacog alfa or eftrenonacog alfa during the study period (june 1, 2014 to july 31, 2017). reference weight measurements from the centers for disease control and prevention national center for health statistics' (cdc nchs) anthropometric data were used to estimate product dispensation on an iu per kg basis. the nonacog alfa and eftrenonacog groups comprised 37 and 11 patients, respectively. the median [iqr] age in the two groups was 8.0 [9.0] and 13.0 [4.0] years, respectively. while 10 of the 11 patients in the eftrenonacog alfa group had >1 calendar quarter of available data, only 23 of the 37 patients in the nonacog alfa group had >1 available quarter. the median rfix product dispensation per quarter was 29,074ius (iqr, 13, 967 ius) in the nonacog alfa group and 62,268ius (iqr, 18, 882 ius) in the eftrenonacog alfa group. incorporating attributed weight values, the median rfix product iu dispensation per kg per week was 97.39 iu/kg/wk (iqr, 31.92 iu/kg/wk-153.92 iu/kg/wk) in the nonacog alfa group, and 96.27 iu/kg/wk (iqr, 53.05-154.03 iu/kg/wk) in the eftrenonacog alfa group. applying 2016 wac prices (eftrenonacog alfa = $2.97/iu; nonacog alfa = $1.37/iu), the calculated estimates of $/kg/week were $133 and $286 in the nonacog alfa and eftranonacog alfa groups, respectively. conclusion: preliminary real-world data derived from a large u.s. claims database revealed differences in product dispensation and factor product-related expenditures among pediatric patients with any severity of hemophilia b to whom an shl or ehl rfix product was prescribed. refinements of these data, potentially to exclude instances of sporadic usage, may shed light on real-world dispensation of rfix products among pediatric hemophilia b patients. background: vascular malformations can be classified as simple (including capillary, venous, lymphatic, arteriovenous), combined, malformations of major named vessels or associated with other anomalies. multiple modalities including laser treatments, sclerotherapy, embolization, surgery and pharmacological intervention (with mtor inhibitors like sirolimus) have been used for treatment of vascular malformations. these interventions have been used alone or in combination with varied outcomes. we present our institution's experience with a multimodal approach to simple and combined vascular malformations. design/method: we performed a retrospective chart review of patients with vascular malformations who were referred to our center for an interventional radiology evaluation from june 2015 -july 2017. we included 22 patients (age at presentation:4 months -25 years), referred initially for interventional radiology procedures (irp) for vascular malformations. all patients had symptoms of pain and/or swelling/deformity. diagnosis of was based on vascular imaging (doppler ultrasound, mri/a/v). nine patients had venous malformations (vm), five had macrocystic lymphatic malformations (lm), six had lymphatic-venous malformations (lvm), and two arteriovenous malformations (avm). 19 patients initially underwent interventional radiology procedures. all the vm patients responded to sclerotherapy alone. three patients with lm responded to sclerotherapy alone, remainder required surgical intervention. one avm patient responded well to embolization, the other needed surgical resection after embolization. four lvm patients underwent irp with minimal improvement in symptoms (3-8 procedures attempted), surgical resection was attempted in 3 patients with poor response and 5 patients were started on sirolimus (0.8mg/m2/dose twice a day). all lvm patients started on sirolimus have responded well (decreased pain and swelling); time to initial symptom response ranged from 2 weeks -1 month from starting medication. in this case series, patients with simple vm responded well to sclerotherapy alone, avm and lm patients needed irp and/or surgery for complete response. complex lvm did not respond well to surgery or irp; 83.3% had improvement in clinical symptoms with addition of sirolimus to the treatment regimen. response to various modalities of treatment varied based on the type of vascular malformation. a multidisciplinary approach to management of vascular malformations is essential to provide multimodal therapeutic options for rapid symptom relief and improve the quality of life of these fragile patients, especially those with complex malformations. background: von willebrand disease (vwd) is the most common bleeding disorder in humans, affecting ∼1% of the united states' population. desmopressin (ddavp) is a longacting vasopressin analog that induces vasoconstriction and release of vwf. ddavp is used in patients with vwd and as a surgical prophylaxis, but carries anti-diuretic properties. to avoid electrolyte imbalance and hyponatremia, fluid restrictions are recommended in the 24 hours post-ddavp administration. objectives: this study sought to examine perioperative practices and outcomes following ddavp administration and a fluid restriction protocol in a population of pediatric patients with von willebrand disease. design/method: a retrospective chart review was conducted for patients with von willebrand disease who underwent surgical procedures at children's hospital of pittsburgh of upmc between january 1, 2015 and december 31, 2016. patient age, sex, weight, diagnosis, surgical procedure, total fluids administered, and post-operative sodium level were recorded. the primary outcomes noted were the proportion of patients exceeding 50% of the recommended fluid consumption for the 12-and 24-hour periods post-ddavp s147 of s301 administration, as defined by local guidelines. secondary outcomes were the presence of any bleeding requiring an er visit or readmission or hyponatremic seizures within 72 hours of ddavp administration. results: data was compiled for 42 patients (23 females, 19 males). the mean age was 11.19 years (sd 5.13 years), median age was 12 years (range 3 to 19 years). procedures included dental (13), otolaryngology (9), orthopedics (7), gastrointestinal (5), plastics (3), neurosurgery (1), ophthalmology (1), dermatology (1), general surgery (1) and gynecology (1). 30% of patients exceeded 50% of the fluid volume recommended for the first 12-hour period post-ddavp administration while still in the surgical setting. no patients exceeded 50% of the fluid volume recommended for the total 24-hour period post-ddavp administration. post-operative sodium levels were obtained in only 7 of 42 patients. no patients returned to the er or were admitted for bleeding in the 72 hours post-ddavp administration. no patients returned to the er or were admitted for hyponatremia or seizures in the 72 hours post-ddavp administration. maintenance of a fluid restriction protocol effectively deterred negative outcomes in this cohort. however, a significant fluid volume was administered in nearly a third of patients despite the restrictions. given the risk of hyponatremia, and limited compliance with fluid restrictions, postoperative sodium levels should be recorded in following ddavp administration to assess the possibility of a hyponatremia and to reinforce the importance of fluid restrictions and their communication. results: a male fetus required in utero insertion of a pleuroamniotic shunt for bilateral pleural effusions diagnosed antenatally by ultrasound. shortly after delivery at term, he developed respiratory distress and was found to have reaccumulation of the pleural effusions. blood work on day 1 of life showed a platelet count of 157,000/ l, which then decreased precipitously. he demonstrated schistocytes on blood-smear, signs of consumptive coagulopathy with hypofibrinogenemia and high d-dimers, and compensatory reticulocytosis. he required multiple transfusions and admissions to the intensive care unit for respiratory support. investigations ruled out congenital ttp, neonatal alloimmune thrombocytopenia, and noonan syndrome. given high clinical suspicion for an underlying vascular lesion causing kmp, a full body mri without contrast was undertaken. this showed a focal area of suspicious signal intensity in the upper paraspinal musculature. an ultrasound and mri with contrast demonstrated an extensive infiltrative vascular lesion involving the paraspinal musculature, prevertebral space, posterior extrapleural space, mediastinum, and neck. the child was commenced on prednisone (2mg/kg/day) and rapamycin (0.9 mg/m2 twice/day). there was no clinical or laboratory improvement after one month. a biopsy was performed which confirmed khe. in the second month of rapamycin therapy, the platelet count gradually normalized and the patient was discharged from hospital at 3.5-months of life. prednisone was weaned off at 4.5 months of life. a repeat mri at 7 months showed significant reduction in the khe. he is now almost 2 years into therapy and doing well. conclusion: this is a unique case of khe with kmp that initially presented with extensive and recurrent pleural and pericardial effusions. this case demonstrates the importance of suspecting an underlying vascular malformation in the presence of kmp. our patient had a delayed but overall good response to rapamycin. further studies investigating duration of rapamycin therapy is key for the optimal management of these patients. rosa diaz, donald mahoney, lakshmi srivaths, donald yee texas children's hospital, houston, texas, united states background: since von willebrand disease (vwd) is the most common inherited bleeding disorder, it must co-exist with other less common bleeding disorders in some dually affected patients. however, reports of combined deficiencies in factor viii (fviii) and von willebrand factor (vwf) are rare. objectives: to study the prevalence and bleeding phenotype of combined deficiencies of fviii and vwf in males with hemophilia a in a hemophilia treatment center. design/method: we retrospectively reviewed the electronic medical records of 99 males with hemophilia a followed at our institution during the past 10 years. the primary and secondary outcomes for the study were (1) the prevalence of combined fviii and vwf deficiencies and (2) the bleeding phenotype of these patients. we identified vwf deficiencies in 9% (n = 9) of the patients with hemophilia a. most (n = 6, 67%) patients were tested for vwf deficiency as part of the initial hemostatic evaluation, but one-third were tested due to clinical concern for inadequate response to fviii concentrate. the median duration of follow up was 9.5 years (range 3.4 to 17.2). patients were referred to our clinic at a median age of 12 months (range 0 to 6 years) for evaluation of easy bruising (n = 4, 45%), mucosal (n = 3, 33%) and surgical bleeding (n = 2, 22%). primary diagnoses included 4 with severe, 3 moderate and 2 mild discrepant hemophilia a. secondary diagnoses included 6 with low vwf activity, 2 type 1 vwd and 1 with type 2 unclassified. patients experienced episodes of musculoskeletal (n = 7, 78%), mucocutaneous (n = 6, 67%) and cns bleeding (n = 1, 11%). a total of 8 patients received factor prophylaxis. half of the patients were initially treated with fviii concentrates but subsequently changed to combined fviii/vwf products due to the frequency of breakthrough bleeding despite good compliance. all patients are on combined fviii/vwf products at the time of this review. a total of 7 (78%) of this cohort developed chronic joint disease manifest as decreased range of motion and/or abnormal mri findings. combined deficiencies of fviii and vwf were present in 9% of our center's hemophilia patients. these patients exhibited a severe bleeding phenotype as evidenced by the high frequency of hemarthrosis, need for prophylaxis and high prevalence of chronic joint disease. while the optimal treatment strategy remains to be elucidated, early recognition of a combined deficiency may have important clinical implications, particularly in patients who demonstrate a suboptimal response to fviii concentrate alone. background: childhood neutropenia is heterogeneous and may be congenital or acquired. cerebral cavernous malformation 3 (ccm3) is a neurovascular malformation disorder where lesions consist of low flow, dilated capillary endothelial channels with increased permeability, predisposing to hemorrhage and thrombosis. programmed cell death protein 10 (pdcd10) activity has been implicated in glia and neuron migration, and recently linked to the dysregulation of the actin and microtubule cytoskeleton, thereby affecting cellular morphology and migration. variants of pdcd10 encoding pdcd10 have been associated with ccm3. ccm3 causes a greater and earlier disease burden than other ccms, with 26% presenting younger than 10 years. some patients have associated extra-neuronal manifestations, suggesting that pdcd10 plays a role in other tissues. we describe a patient with significant blood cytopenias associated with ccm3. design/method: retrospective chart review to obtain patient data. results: an 8-month old female presented with seizure and was found to have multiple intracranial cystic lesions and abscesses due to s. pneumonia serotype 33f. during her treatment, she developed anemia (hemoglobin 7.6-8.7 g/dl), thrombocytopenia (platelets 73,000-128,000 cells/l), and profound neutropenia (absolute neutrophil counts of zero). initial bone marrow evaluation revealed a normocellular marrow but with marked granulocytic hypoplasia and 38% hematogones on flow cytometry. florescent in situ hybridization excluded cytogenetic changes characteristic of myelodysplastic syndrome. further evaluation included testing for neutrophil antibodies, chromosome breakage, and telomere length and results were normal. whole exome sequencing excluded mutations affecting congenital neutropenia genes, but detected a de novo pdcd10 variant (c.474+5g>a), thereby diagnosing ccm3. the neutropenia has responded well to granulocyte colony stimulation factor (gcsf), which is still needed at 26 months of age. moreover, the thrombocytopenia has progressed, requiring periodic platelet transfusions. over time, the bone marrow hematogone population has decreased to 8% at 20 months of age, though the granulocytic hypoplasia persists. conclusion: our case describes the first patient with neutropenia and thrombocytopenia associated with ccm3. we hypothesize the pdcd10 variant is the etiology of bone marrow dysfunction due to its role in actin and microtubule cytoskeleton formation, akin to the pathophysiology of xlinked neutropenia. supportive features of an underlying genetic cause of marrow dysfunction include the persistence of cytopenias beyond infection resolution as well as presence of hematogones. hematogones were previously reported to occur in patients with other congenital neutropenia disorders, indicating they could be a feature of congenital neutropenia and may be reactive to surrounding cell apoptosis. further testing of pdcd10 role in hematopoiesis should be explored. background: 10-15% of adult women will suffer from heavy menstrual bleeding (hmb) during their lifetime. 90% of women with inherited bleeding disorders suffer from hmb. there is a paucity of data about hmb among adolescents and young adults (aya), a population in which hmb may have large social and educational effects. objectives: to study the social and academic implications of hmb in an aya population. design/method: this is a questionnaire based survey conducted in a medium-sized city in california. we recruited females 14-24 years of age from one high school and from local university. the questionnaire was set up in research electronic data capture (redcap) at our institute which allowed us to obtain objective data about the respondents' menstrual cycles. a link was sent to the high school students via their online portal schoolloop and to the university students via social media and word of mouth. data was collected over 12 weeks from may 2017 to august 2017. we received 145 replies, some were not complete. using regression analysis, data was analyzed from 115 respondents in the age group of 18-24 (with a mean age of 19) years. we developed a composite score for hmb based on factors including saturation levels, number of pads, duration of bleeding, soaking of a pad within two hours, passage of clots, size and number of clots, and gushing sensation. we conducted statistical analysis of the drivers and implications of hmb based on the composite score. results indicate that having a relative with hmb, having other bleeding problems, and having anemia are drivers of higher hmb score. the results also indicate that hmb adversely affects quality of life as measured by participation in sports, social activities, after-school activities, tiredness, absenteeism, and gpa. hmb is also associated with increased rates of anemia and use of anti-depressants. hmb-driven anemia further adversely affects gpa. under-represented minorities are more likely to have a higher hmb score, as well as an increased adverse effect of hmb on gpa. the results suggest that the social costs of hmb are pervasive in the aya population, and especially pronounced among minorities. a relative with hmb is a significant driver of heavy menstrual bleeding. a hemostatic screen should be included when assessing the aya population with hmb. johns hopkins all children 's hospital, st. petersburg, florida, united states background: propranolol is a non-cardioselective beta blocker medication frequently prescribed for hemangiomas and hyperthyroidism. propranolol inhibits types i and ii iodothyronine deiodinases, enzymes that convert bioinactive thyroxine (t4) into bioactive triiodothyronine (t3). hypothyroidism is a well-recognized complication of diffuse hepatic hemangiomas that produce type iii deiodinase, an enzyme that converts t4 into bioinactive reverse t3 and t3 into diiodothyronine. thyroxine is typically selected for replacement in this population, even though doses up to 60% above physiologic may be necessary. we hypothesized that low dose, nearly physiologic t3 would be safer and equally effective because it bypasses propranolol's impact on the pituitarythyroid axis. we report an infant with diffuse hepatic hemangiomatosis and acquired hypothyroidism successfully treated with propranolol, prednisone, and triiodothyronine. design/method: a 7mo healthy female presented with abdominal distension, poor oral intake, and hepatomegaly. mri confirmed diffuse hepatic hemangiomatosis, the largest lesion measuring 4.4 cm by 3.9 cm. thyrotropin (tsh) was elevated at 47.9 (reference range* 0.5-6 mcgiu/ml), total t3# 165 (rr 60-300 ng/dl), and total t4^18.8 (rr 6-14 mcg/dl). treatment was started with prednisone (2 mg/kg/day) for three weeks, propranolol (3 mg/kg/day) and t3 (0.64mcg/kg/day). the t3 dose was slowly titrated to a maximum of 1.72 mcg/kg/day. thyroid hormone levels rapidly improved on t3 replacement. after two weeks, the tsh was 14.4, tt3 92, and tt4 16.5. after eight months, the tsh was 2.9, tt3 161, and tt4 10.1. at twelve months, the tsh dropped to 0.6, tt3 294, and tt4 3.7, suggesting decreased tumor production of type iii iodothyronine deiodinase. liver mri confirmed fewer hemangiomas, largest being 1.3 cm by 1.6 cm. the patient's t3 dose was reduced. both propranolol and t3 were discontinued after twenty-four months of treatment. one year off all therapy, this child has normal growth and development, only two <1.3cm hepatic hemangiomas and no evidence of hypothyroidism (tsh 2.2; tt3 153; tt4 6.4). conclusion: t3 at near physiologic doses corrects the consumptive hypothyroidism associated with diffuse hepatic hemangiomas. t3 replacement is preferable to thyroxine due to its lower risk of rebound hyperthyroidism as the hemangiomas involute and type iii deiodinase production declines. there are two prior case reports describing t3 use without t4, one employing propranolol and the other utilizing steroids for hemangioma management. this is the first case report with long term follow-up of a child treated with multimodal therapy including propranolol, prednisone, and triiodothyronine. *rr = reference range; #tt3 = total t3;^tt4 = total t4 background: multifocal lymphangioendotheliomatosis with thrombocytopenia (mlt) is a rare congenital disorder first described in 2004 that is characterized by multiple vascular abnormalities commonly involving the skin and gastrointestinal tract as well as consumptive coagulopathy often resulting in gi bleeding in infancy(1). to describe an unusual presentation and successful management of mlt in a neonate. design/method: baby h was born at full term after a pregnancy complicated by maternal sinus venous thrombosis requiring anticoagulation beginning at 28 weeks. at birth, she was diagnosed with multiple hemangiomas based on clinical exam. at two weeks of age, she developed melena and hematemesis. cbc revealed platelet count of 70 and she was referred to the ed. abdominal ultrasound was concerning for abnormal hepatic waveform; cxr showed multiple pulmonary nodules. workup revealed no other lesions and no further hematologic abnormalities. biopsy of presumed hemangioma ultimately revealed a smooth muscle-lined vascular proliferation without glut-1 immunoreactivity, consistent with mlt. her early course was complicated by an acute hemodynamically significant gi bleed; esophagogastroduodenoscopy identified six bleeding vascular malformations within the stomach that were injected with epinephrine and sclerosed with successful hemostasis. she received multiple prbc and platelet transfusions. central access was obtained and she was started on oral sirolimus based on previous reports of successful use in management of vascular malformations given its antiangiogenic and immunosuppressive effects (2). she has tolerated it well with no evidence of toxicity and has achieved a partial response with stable of hemoglobin >8 and platelet count >90. cutaneous lesions have diminished in intensity and she has had no further signs of gi bleeding. she receives pentamidine for pcp prophylaxis. she continues to have appropriate growth and development. we describe here an unusual presentation of an already rare disease. while cutaneous and gi lesions are typical of mlt, pulmonary involvement is not well-described in the literature. early identification of tissue-based diagnosis enabled timely stabilization and treatment of the patient. five months later, she continues to tolerate sirolimus and has shown significant response with diminished coloration of cutaneous lesions, stable blood counts, and no further bleeding. mlt is a relatively newly-recognized disorder with significant phenotypic variability. given that bleeding secondary to a kasabach-merritt-type consumptive thrombocytopenia is the major cause of morbidity and mortality in the first year of life in children with mlt, it is essential to recognize the diagnosis and initiate appropriate treatment as early as possible. 1north, arch background: patients with generalized joint hypermobility (jhm) may experience easy bruising or bleeding given the association between these symptoms and abnormalities in collagen, a required component of primary hemostasis. heavy menstrual bleeding (hmb) is a common initial presentation for females with underlying hemostatic defects and may be the sole manifestation of a bleeding disorder. however, limited reports describe jhm as a cause of hmb, leading to under recognition. objectives: to describe the clinical characteristics and management of young women presenting with hmb in the setting of jhm. design/method: this study utilized our hmb research registry. we included subjects 11-18 years, seen in the nationwide children's young women's hematology clinic between february 2014 and november 2017 with both hmb and jhm. medical records were retrospectively reviewed for history of presentation, menorrhagia impact questionnaire (miq): a validated quality-of-life tool for females with hmb, medication profiles and relevant laboratory studies. results: twenty-five patients met inclusion criteria (median age 15 years, range 11-18) with an average beighton score of 6.2 (range 4 to 9). participants presented an average of 3.2 years (range 5 months to 6 years) after menarche despite 76% of patients reporting heavy to very heavy menses since menarche. according to the miq responses, most participants expressed hmb-associated limitations in physical activities (84%), social activities (68%), and work or school activities (64%). of the participants, 92% reported bleeding symptoms in addition to hmb, most commonly easy bruising (56%), epistaxis (48%) and cutaneous bleeding (44%). forty percent of young women presented with anemia due to chronic blood loss. results of hemostatic testing were unremarkable, with the exception of one patient who was also found to have type 1 von willebrand disease. additionally, 44% of females reported arthralgia, with knees and ankles the most commonly affected joints. at time of presentation, 32% of participants reported failure of initial therapies and most patients (88%) were managed long-term with oral hormone therapy. in a small population of young women found to have jhm who initially presented with hmb, patients were likely to have prior bleeding symptoms as well as substantial delays from menarche to timing of presentation at our young women's hematology clinic despite limitations in activities of daily life. greater awareness of the associations between bleeding symptoms and jhm, despite typically normal hemostatic laboratory results, is necessary so that patients can more easily be identified and receive appropriate therapy. the objective is to determine the impact of cl care practices involving the home environment on ambulatory clabsi rates. design/method: information for the pi was collected through a comprehensive survey that was completed annually by the ccbdn member hospitals. responses to the questions about cl care practices involving the home environment were selected from the pi for 2015. ambulatory clabsi rates and ambulatory total bloodstream infection (bsi) rates were obtained from another ccbdn database. the proportion of hospitals that did or did not employ a particular cl care practice was tallied. the mean ambulatory clabsi rate and mean ambulatory total bsi rate of the hospitals that did or did not employ a particular cl care practice were compared using generalized linear model techniques assuming an underlying negative binomial distribution. results: twenty-five hospitals submitted responses to the 8 questions about cl care practices involving the home environment. one hospital was excluded for lack of bsi data. sixty-three percent of the hospitals programmatically educated parents about all aspects of the cl care bundle. the mean ambulatory clabsi rate for the hospitals that educated parents was significantly lower than that of the hospitals that did not (0.20 infections/1000 cl days vs. 0.30 infections/1000 cl days; p = 0.02). the mean ambulatory total bsi rate was also significantly lower (0.35 infections/1000 cl days vs. 0.53 infections/1000 cl days; p = 0.01). the mean ambulatory clabsi rates and mean ambulatory total bsi rates were not significantly different for the other 7 cl care practices. conclusion: an analysis of cl care practices involving the home environment reveals that parental education of all aspects of the cl care bundle is associated with a lower ambulatory clabsi rate and lower ambulatory total bsi rate. this finding highlights the importance of systematically teaching family members the proper method of handling cl. background: children undergoing chemotherapy are at a high risk for developing nausea. dr. amy baxter in collaboration with pediatric oncology patients and nurses, developed and validated a pictorial nausea rating scale for children aged 7-18 years, called the baxter retching faces (barf) nausea scale. staff nurses at a large, academic, pediatric hospital located within washington, d.c., have identified variability in nursing assessment and documentation of chemotherapy induced nausea and vomiting (cinv) in pediatric oncology patients. the purpose of this quality improvement project was to utilize the barf scale to standardize assessment and documentation of nausea in pediatric oncology patients receiving chemotherapy. the primary aims of this project were to: assess feasibility of the barf scale in clinical practice; increase nursing knowledge about cinv through education sessions; increase documentation of nausea assessments through the use of the scale. the secondary aim of this project was to: increase the recognition of nausea through the use of a standardized assessment tool. design/method: the pdsa model was used to guide the design and implementation plan. in the first phase of the project data was collected to identify the prevalence of nausea in patients admitted for chemotherapy in the prior three months. education sessions discussing cinv and the utilization of the barf scale were conducted. pre and post assessment of nurses' knowledge of cinv and documentation were assessed. in the second phase the barf scale was implemented into practice. nurses were asked to utilize the barf scale to assess and document nausea scores in patients, aged 7 to 18 years, receiving chemotherapy. at the end of the implementation period nurses were surveyed about the feasibility of the scale. post data was collected to identify the prevalence of nausea documented in the electronic health record. this project was undertaken as a quality improvement initiative at children's national and it does not constitute as human subjects research. as such it was not under the oversight of the institutional review board. results: all data has been collected; however complete data analysis will be conducted in the upcoming weeks. background: sickle cell disease (scd) is the most common inherited blood disorder in the united states (us); however, there are few quality measurements to evaluate scd practice. in 2014, the nhlbi published guidelines that include two key interventions for children with sickle cell anemia (sca): the use of transcranial doppler (tcd) screening for stroke prevention and hydroxyurea (hu) to prevent scd pain crisis. we conducted a national survey of scd management sent to providers in over 20 institutions in the us to better assess knowledge of the guidelines and barriers to hu counseling and tcd screening guideline implementation. it was hypothesized that the barriers to tcd screening are different than barriers to hu counseling and prescribing. a 33-question anonymous survey was sent to 49 providers by mail (follow-up by email). survey themes included nhlbi guidelines knowledge and comfort with understanding and implementing both tcd screening and hu use. the response rate was 59% (29/49) however one survey was incomplete. thus, 28 were analyzed in the final data set. all of the respondents are in active practice, 96% s153 of s301 in academics and all care for children with scd. the majority of providers (96%) felt "very" or "extremely" confident in their knowledge of tcd screening and interpretation. similarly, 100% of providers felt "very" or "extremely" familiar with hu dosing and management. for tcd screening, 36% of providers estimated their screening rates were >90% and 64% providers felt their annual screening rates were 75-90%. the two biggest barriers to tcd screening noted by providers (of moderate to extreme significance) included: lack of support staff (36%) and lack of time during a patient visit (26%). regarding hu prescribing practices, 71% of providers offered hu to at least 90% of children with sca over nine months of age. the biggest barrier to hu prescribing noted by 46% of providers was concerns about patient adherence or access to the medication. only 7% providers felt that lack of support staff was a moderately significant barrier to hu prescribing. the pediatric scd providers surveyed all have access to the nhlbi guidelines. despite widespread guideline knowledge, there are different barriers for tcd screening versus hu prescribing, which prevent optimal implementation. as a result, although both recommendations are from the same nhlbi guideline, they likely will require different implementation strategies (systems-based interventions for tcd screening; interventions to improve patient adherence for hu counseling) to improve outcomes. background: invasive fungal disease (ifd) is a major cause of mortality and morbidity among pediatric immunocompromised patients such as those who receive chemotherapy or hematopoietic stem cell transplantation. the current diagnostic 'gold standard' of ifd remains culture of infected tissue obtained by biopsy. noninvasive biomarker testing for galactomannan or 1,3-beta-d-glucan (bg) can have low sensitivity and does not provide species-level identification. nextgeneration sequencing (ngs) of cell-free plasma is a promis-ing noninvasive approach to providing species-level identification of ifd via a blood test and can further guide specific treatment. objectives: describe the incidence of positivity for fungal specific pathogens on ngs analysis in a high-risk immunocompromised pediatric population and correlate results with other 'standard' infectious studies if performed. design/method: immunocompromised pediatric patients with suspected ifd were enrolled and plasma was collected at time of enrollment. ngs was performed on extracted dna in cell-free plasma (karius, redwood city, ca). after removing human reads, remaining sequences were aligned to a curated database including 1251 pathogens. organisms present at a significance-level above a predefined threshold were reported. results: twenty-seven samples from 33 enrolled patients have been processed thus far. of these 27 subjects, 14 were enrolled for prolonged febrile neutropenia (≥96 hours) despite broad-spectrum antibiotics, 5 for recrudescent febrile neutropenia, 5 for abnormal imaging, and 3 with other findings. after evaluation of routine studies performed, 4 patients met criteria for proven ifd, 2 for probable ifd, and 9 for possible ifd using eortc/msg guidelines. the ngs plasma test identified the same pathogen as cultured from infected tissue or blood in 100% (4/4) of the proven cases. in the probable cases, pneumocystis jirovecii was identified in a patient with a positive bg (389 pg/ml) and pneumonia. among the possible cases, toxoplasma gondii was detected in a patient with prolonged febrile neutropenia and lung imaging suggestive of ifd. additionally, candida glabrata was isolated in a patient with prolonged febrile neutropenia but no other criteria for ifd. numerous pathogens were also identified that could explain the above clinical parameters, including hsv1, cmv, vzv, hhv6, ebv, bk polyoma virus, and ureaplasma parvum. the cell-free plasma ngs test can detect invasive fungal infections from blood. the test identified fungi from proven ifd, detected pathogens in both probable and possible ifd cases, and is a useful diagnostic tool in the evaluation of ifd. supplies and sample shipment and processing supported by karius, inc. baylor college of medicine, texas children's hospital, houston, texas, united states background: practicing medicine is a lifelong learning process. as noted in the institute of medicine's seminal report, 'to err is human,' adverse outcomes do not typically result from individual recklessness; rather, they result from faulty systems, processes, or conditions that provide an environment conducive to making a mistake, or failing to prevent one. learning to systematically review errors and translate lessons learned into quality improvement (qi) initiatives is a critical component of practice-based learning and improvement for practitioners at all career levels. objectives: to develop a methodical, self-reflective and nonthreatening approach to incident analysis and translation of lessons learned into qi initiatives. design/method: we used a validated, structured case audit approach, modified from szostek et al: 1) review all documentation relating to the case and identify all health care providers involved; 2) interview stakeholders, including those who directly provided and supported care; 3) use a qi tool to conduct a root-cause analysis; 4) identify a systems issue that contributed to the outcome; and 5) propose systems-level interventions and prioritize initiatives based on effort-yield projections. results: pdsa cycle 1: plan: establish a committee to 1) identify potential cases, 2) triage cases for conference presentation, 3) determine timing and frequency of conferences, 4) develop a training manual, 5) record identified qi initiatives. do: we established a quarterly section-wide meeting to which all members of the pediatric hematology/oncology service are invited, including administrative and nursing leadership. we developed a training manual and structured presentation template. prioritized cases were discussed in advance during multidisciplinary case review sessions, and presented by senior fellows who were instructed to focus discussion on potential opportunities for qi. study: we identified 23 cases, 10 meeting criteria for mmi presentation. qi initiatives identified from this conference resulted in a number of systemic practice changes; however, we encountered challenges to sustaining these changes over time. act: objectives for the next pdsa cycle are to 1) establish a method for tracking the adherence to recommended changes in practice, 2) maximize sustainability by integrating qi initiatives into institutional qi leadership and practice standardization committees. we have successfully implemented an mmi conference that meets 5 out of 6 institute of medicine quality domains: safety, effectiveness, patient-centeredness, timeliness, and efficiency. a standardized, consistent approach to mmi presentations that includes identification of contributing factors and specific qi implications has the potential for improving both provider education and patient care/safety. johns hopkins university, baltimore, maryland, united states background: receiving a cancer diagnosis is a life-changing event for patients and caregivers, although little is known about the experience. while some oncologists receive dedicated training in delivering this bad news, the initial conversation is often with a primary pediatrician, and these providers often feel they do not receive adequate training in the communication of a cancer diagnosis. objectives: our objectives were two-fold: first, to better define the experiences of caregivers/patients when told of a cancer diagnosis, and to query how caregivers/patients believe providers can improve the disclosure of this bad news. secondly, to assess what, if any, training primary pediatricians received in this skill, and to assess how comfortable providers in various settings and stages of training are with communicating cancer diagnoses. design/method: from november 2016-2017, semistructured, in-depth interviews were conducted with pediatric oncology patients and caregivers of patients (n = 6) diagnosed in the past year regarding their experiences receiving the diagnosis at our institution. in addition, pediatric residents (n = 6), outpatient pediatric primary care physicians and pediatric emergency medicine physicians (n = 6) were interviewed regarding their experiences delivering cancer diagnoses. interviews were analyzed following principles of thematic analysis. interviewers with patients and caregivers had two common themes: 1) all emphasized their wish for direct and thorough information; 2) both patients and caregivers emphasized the gratitude they felt for physicians who gave them hope by emphasizing the good prognosis of their child's cancer. lack of training in this area, as well as lack of comfort delivering this news was common will all providers. additionally, providers report variable approaches to giving bad news, including 1) whether to tell caregivers separately or tell the child and parents together, and 2) whether to give favorable prognostic information. additionally, attending physicians also differed significantly in their approaches to teaching residents. while some believed residents should give the news to gain experience, others felt that this is not appropriate if residents are inexperienced. only one resident reported ever receiving feedback on his communication skills in this type of discussion. conclusion: we plan to build on these interviews to develop a national survey of patients, caregivers, and providers to better understand the issues surrounding this discussion. we will use the findings to develop a communication curriculum for pediatric residents, focusing on the discussions that occur in the outpatient setting by primary pediatricians. background: human papilloma virus (hpv), common in both females and males, is responsible for pathologies ranging from benign genital warts to cervical and penile cancer. hpv strains 16 and 18 are responsible for 21,000 malignancies each year in the united states, and one third of them arise in men. pharmaceutical companies have now developed a vaccine that will help prevent the virus-associated malignancies. the cdc initially recommended that females ages 11-26 years receive the vaccine series, then starting in 2011 they expanded the eligibility to males ages 11-21 years. despite being widely available and highly publicized, only 40% of eligible females receive the full vaccine series. objectives: this study aims to assess the knowledge of hpv, the attitudes towards the hpv vaccine, and identify barriers preventing its full utilization. once identified, we aim to overcome the barrier(s) in order to improve vaccination rates in eligible adolescents. we distributed a standardized questionnaire to the parents of eligible female and male patients in our pediatric hematology-oncology clinic. it assessed the parents' knowledge of hpv and the vaccine, their views of the vaccine, and reasons why they may oppose it. results: approximately 80% of parents claim they have been educated about hpv, mostly by their primary care physician. however, 35% did not know what disorders hpv caused; 35% felt the vaccine should not be added to the typical vaccine schedule; 25% of parents do not intend to vaccinate their child. of those that opposed the vaccine, one-third were concerned about potential side effects and nearly 35% feel they do not have enough information. additionally, 25% of parents are not aware that the vaccine is available at their child's doctor and only 30% of parents have discussed the hpv vaccine with their child's doctor. the largest barrier to the utilization of the hpv vaccine that we have identified appears to be lack of educa-tion. as a result, we have begun distributing the cdc's hpv and vaccine patient guide to our patients' families as an intervention. we are currently in the process of re-administering our survey to these families after implementing the intervention to assess its success in increasing both knowledge and utilization of the hpv vaccine. cancer institute, chennai, chennai, tamilnadu, india background: rasburicase is a recombinant urate oxidase enzyme approved for use in tumor lysis syndrome (tls) and it acts by reducing serum uric acid levels. using rasburicase at the recommended dose of 0.2mg/kg/day for 5 days is expensive and it is not known whether this extended schedule is clinically beneficial compared to a single fixed dose of 1.5 mg. the aim of the present study was to evaluate the efficacy of single dose rasburicase 1.5 mg in prevention and management of tls. design/method: rasburicase is available as single use 1.5 mg vial. at our institution a single dose of rasburicase 1.5 mg irrespective of bodyweight has been used in adults and in children a dose of 0.15 mg/kg (maximum 1.5 mg) has been used since 2012 for prevention and management of tls and subsequent doses are given based on biochemical response and clinical condition. we retrospectively analysed the case records of patients who had received rasburicase from january 2012 to january 2017. the study included 186 patients with hematological malignancies who received rasburicase. children accounted for 56.4% (n = 105) patients and males comprised 73% (n = 135). rasburicase was used prophylactically in 59 (31.7%) patients, for laboratory tls in 76 patients (40.8%) and for clinical tls in 51 (27.4%) patients. single fixed dose rasburicase prevented laboratory/clinical tls in 87% of the prophylactic group and prevented clinical tls in 72% of the laboratory tls group. none of the patients in prophylactic and laboratory tls group developed clinical tls. however, majority of the patients with clinical tls required more than one dose rasburicase. single dose of 1.5 mg (1 vial) rasburicase is efficient in preventing and managing laboratory tls and is economically viable in resource constrained settings. nicole wood, lauren amos, nicholas clark, chris klockau, karen lewing, alan gamis children's mercy kansas city, kansas city, missouri, united states background: medication reconciliation for newly diagnosed oncology patients is complicated and cumbersome. these patients are often admitted on no medications, and leave on multiple. chemotherapy and supportive medications are crucial. despite numerous individuals overseeing this process, prescribing errors or omissions still occur. when reviewing the literature, improvement occurs when there is an interprofessional and standardized process to medication reconciliation. objectives: this project's aim was to improve the accuracy of the discharge medication reconciliation process from 74% to 90% from february 2017-august 2017. the process measure was the percentage of patients discharged with an accurate checklist. additional time for staff spent in completing the checklist and avoiding an increased error rate by changing the prescribing process were followed as balancing measures. we created a discharge medication checklist which included a list of required home medications prescribed by the resident, ideally 24 hours prior to discharge. it required fellow or attending review and pharmacy to review the list and educate the family. checklists were collected monthly and reviewed against the electronic medical record (emr) for accuracy. results: six pdsa cycles were completed. there were 2 errors during the data collection time frame. in pdsa cycle 1, a patient received acetaminophen for pain control which is avoided at home. in addition, this patient received diphenhydramine instead of ondansetron, which is preferred as an antiemetic. in pdsa cycle 4, a patient with a pending diagnosis was sent home with acetaminophen. of note, this patient did not have a checklist completed upon discharge. this project provides a novel and important method to standardize the discharge medication reconciliation process in a complex patient population. it clarifies which types of medications these patients need, provides pharmacy teaching to families which was not done previously, and prescribes discharge medications to families sooner. after the first medication reconciliation error, the checklist was revised. no further errors were made following revision, with the exception of one patient without a completed checklist at dis-charge. our accuracy rate increased from 74% at baseline to 92% following implementation. we are in the process of making the checklist electronic and accessible in the emr. in the interim between the end of data collection and implementation into the emr, a leukemia patient was sent home without an epinephrine pen, further demonstrating the importance of this standardized discharge process. for this reason, we have re-instituted the checklist until the electronic version is available. background: survivors of pediatric cancer are at risk of losing pre-existing protective antibodies to vaccine preventable diseases. in a prior study, 35% of children < 7 years lost humoral immunity to measles as a result of chemotherapy induced alterations in immune system. measles in recipients of immunosuppressive chemotherapy has mortality rates up to 50%. because of volitional vaccine refusal, there has been a dramatic increase in measles infection from 63 cases in 2010 to 677 in 2014, including several statewide outbreaks. small pediatric oncology practices frequently share floor/clinic space with the general pediatric patients putting them at risk for measles since virulence starts 48 hours prior to symptoms. there is no standard protocol for revaccinating post-chemotherapy patients. to assess measles risk based on serial humoral immune status in a cohort of pediatric oncology patients receiving intensive chemotherapy design/method: patients < 21 years age with known vaccination status receiving intensive chemotherapy between july 2015-june 2017 at our institution's pediatric oncology practice were included in this prospective study. serial measles igg antibodies were measured at diagnosis, 6 months and 12 months after initiation of chemotherapy using elisa. measles immunity was defined per lab standards. a comparison of pre-chemotherapy and serial post-chemotherapy immunization titers was made for all patients by diagnosis. the study population consisted of 31 children (17 male); 8 patients had all, 7 non-hodgkin lymphoma, 11 sarcoma and 5 other solid tumors. two patients (6.4%), both unvaccinated had non-protective measles antibody levels at s157 of s301 baseline. of the remaining 29 patients, 13.7% patients (2 leukemia, 1 lymphoma and 1 sarcoma) lost protective antibody titers at 6 months after initiation of chemotherapy and 27.5% (4 leukemia, 1 lymphoma and 3 sarcoma) at 12 months after initiation of therapy. 60% of the remaining 21 patients who retained measles antibody titers within protective range at 12 months also demonstrated a steady decline in antibody titers at 6 and 12 months from therapy initiation. the loss of protective measles humoral immunity occurred significantly more often in patients with leukemia compared to other malignancies. oncology patients in our practice undergoing intensive chemotherapy demonstrated progressive waning of protective measles igg titers. our data suggests that it should be standard practice to check all patients for measles humoral immunity prior to starting chemotherapy and at completion. larger studies need to be performed to establish guidelines for revaccinating post-chemotherapy pediatric patients, an intervention that is easily applicable and of low cost. background: the accurate determination of glomerular filtration rate (gfr) is important to screen for acute kidney injury, to dose chemo-therapy, and to identify risk for chronic kidney disease.being correlated with inulin clearance, measured gfr by iohexol plasma disappearance (igfr) is a new gold standard for measurement of gfr in pediatric cohort studies. igfr is based on the clearance of an exogenous marker and is unaffected by endogenous compounds or a patient's muscle mass. we compared igfr with 24-hour urine creatinine clearance (24crcl) and gfr estimating equations using serum creatinine (scr) and serum cystatin c (cystc) in pediatric patients with cancer. we recruited participants who were ages 6 to 16 yrs, continent of urine, and diagnosed with a malignancy in the past 5 years. eligible subjects had stable kidney function for at least two weeks prior to the assessment of igfr. consented subjects had baseline assessments including height, weight and vital signs. blood samples were obtained for serum chemistry, and time zero iohexol. igfr determined by 5ml iohexol solution infused over 1-2 minutes followed by 10ml of sterile saline. blood was drawn at 10, 30, 120 and 300 minutes.at the same time of igfr, the 24crcl was collected. igfr was calculated using a two-compartment model and area under the curve. we compared igfr to published gfr equations (schwartz et al, kidney int 2012). results: ten subjects (7 female/3male) agreed to participate. the distribution of diagnoses for the subjects: all = 6, lymphoma = 1, brain tumors = 2 and hepatocellular carcinoma = 1. six patients were off therapy. the lower gfrs are noted in patients who had malignancies other than leukemia, likely due to the use of cisplatin based therapy. the average igfr was 85ml/min/1.73m^2 whereas 24crcl was 155.8 ml/min/1.73m^2; demonstrating the 24crcl overestimates gfr compared to igfr. comparing igfr to univariate equations using scr, cystc, and the multivariate equation with both, the univariate cystc equation correlated well with igfr; the others overestimated igfr. we found that 24crcl overestimated igfr. the univariate cystc equation better correlated to igfr than equations with scr. the poor performance of scr based methods to assess gfr might be due to decreased muscle mass and inadequate nutritional status. creatinine-based determinations of gfr alone, may not be accurate in this population. further study is needed to determine if igfr should be a standard of care to assess gfr in children with cancer particularly who are receiving nephrotoxic medications and incontinent of urine. background: pediatric oncology patients undergoing chemotherapy through indwelling venous catheters are at increased risk for severe sepsis especially when neutropenic due to chemotherapy. rapid triage and early recognition are essential because delayed initiation of antibiotics and fluids in these patients or delayed transfer to higher level of care after initial stabilization is associated with poor clinical outcome. our pediatric oncology out-patient clinic is designated as an article 28 unit whereby the providers can initiate and give treatment such as intravenous fluid, antibiotics, chemotherapy and blood products. objectives: global aim-optimize management of early sepsis and decreased morbidity, mortality and hospital length of stay in the high risk pediatric oncology patients. smart aim-improve timely management with initiation of fluids and antibiotics and transfer of septic patients to higher levels of care by 10% in 6 months in above patients design/method: multidisciplinary team with physicians and nurses was created. retropective chart review of sepsis patients treated at the clinic from april 2016 to october 2016 was done using an audit sheet to identify the barriers in the delivery of care. three patients were identified and data analyzed prior to intervention; two were analyzed post interventions. a key driver diagram was created by the group to drive intervention. a process map was designed to identify the different steps in the care of these patients to pinpoint areas needing improvement. different timed data points were used starting from time of arrival to clinic, time to antibiotics and fluids and time to transfer to higher level of care. rapid pdsa cycles were done to improve the processes and delivery of care. run charts were created. there was an improvement close to the goal of 10 % for all data points used. pdsa cycles for improvement included conducting frequent mock codes with appropriate feedback real time coaching and process planning with nursing staff. we partnered with pharmacy for close loop communication with clinic staff and we improved communication between physicans at different levels. conclusion: sepsis in neutropenic pediatric oncology patients is deadly and can be reversed with timely management at different levels. given the promising results of the above project, we want re-inforcement of the processes to be a part of the daily practice of first line clinical staff. eventually we will extend the principles learnt in management and triage of sepsis to other outpatient emergencies chemotherapy related anaphylaxis background: chemotherapy-induced nausea and vomiting (cinv) is a common side effect in children receiving antineoplastic chemotherapy. recommended prophylactic antiemetic medications are based on the classification of chemotherapy emetogenicity. however, despite appropriate use of these antiemetic agents, some patients will still experience nausea and/or vomiting. children's oncology group clinical practice guidelines recommend the addition of olanzapine to prophylactic regimens for management of breakthrough cinv. objectives: our pediatric hematology oncology center implemented a quality improvement (qi) project aimed to increase the use of olanzapine in pediatric cancer patients 7 years of age and older receiving moderately or highly emetogenic chemotherapy and experiencing breakthrough cinv over a 3 month period. design/method: this qi project was conducted utilizing plan-do-study-act (pdsa) cycles. for the first pdsa cycle, baseline data was collected through chart review to determine the rate of olanzapine use for breakthrough cinv over a 6 month period from july 2016 to december 2016. breakthrough cinv was defined as use of 2 or more doses of antiemetic agents other than those given for cinv prophylaxis. guidelines for treatment of breakthrough cinv were reviewed with pediatric hematology/oncology attending physicians and fellows. flyers were created that listed chemotherapy regimens considered moderately and highly emetogenic. if a patient experienced breakthrough cinv, a flyer was to be placed in the patient's roadmap binder to signal olanzapine should be added to the next chemotherapy block. data was collected over a 1 month period in september 2017 following this first intervention. the second pdsa cycle consisted of didactic education and training of pediatric oncology nurses as well as pediatric residents regarding the addition of olanzapine for breakthrough cinv. rates of olanzapine use were then collected from october 2017 through november 2017. results: olanzapine use increased from 3.8% at baseline to 58.3% after the first pdsa cycle ( 2 = 14.666, p = 0.000). after the second pdsa cycle, olanzapine use increased another 14.1% to 72.4% ( 2 = 0.777, p = 0.378). the administration of olanzapine was successfully increased by modifying patients' roadmaps after patients experienced breakthrough cinv as well as with education and training of pediatric oncology staff, fellows, residents, and nurses. background: venous thromboembolism (vte) is increasingly affecting children. according to an administrative database study, there was a 70% increase in the incidence of vte among children admitted to free-standing children's hospitals in the united states from 2001 to 2007. risk factors for hospital-acquired vte are well-known and well-studied in adults, with evidence-based preventative measures available. similar guidelines are lacking for children. objectives: there is an ongoing national-initiative to develop and institute methods for screening and preventing hospitalacquired vte in children. in 01/2014, nationwide children's hospital instituted an electronic screening form required for all patients admitted ≥24 hours. patients were scored and riskstratified based on eight risk-categories. a summated score was used to determine the vte risk level, and used to make prophylaxis recommendations for patients ≥18 years; as well as patients ≥14 years who were admitted to an intensive care (icu), surgical, or trauma unit. the purpose of this irb exempt, quality improvement initiative was to retrospectively review our experience with this risk-stratification tool. results: 262 hospital-acquired vte events occurred in 232 unique subjects. median age at vte diagnosis was 2 years. only 69 (26%) vte occurred in children ≥14 years of age. 237 (91%) vte were deep vein thrombosis (dvt), and 16 (6.1%) involved pulmonary embolism. vte was most common in subspecialty units including the pediatric and cardiac icus 87 (33.2%); neonatal icu, 43 (16.4%); and hematologyoncology, 31 (11.8%). 184 (70%) vte were associated with central venous catheters (cvc) and 144 events (55%) were associated with altered mobility. congenital heart disease/heart failure was the most common chronic medical condition associated with vte (69 (26.3%) events); whereas infection and trauma/surgery were the most common acute medical conditions associated with vte (137 (52.3%) and 89 (34%) events, respectively). during 249 (95%) events, subjects scored a summated score ≥3. in summary, in this single institution, prospectively maintained database, cvc remains the most common risk factor for vte, followed by cardiac disease, infection and trauma/surgery. most subjects who developed vte scored high (score ≥ 3) on our screening tool. only a small proportion of vte occurred in patients older than 14 years and thus eligible for thromboprophylaxis. our results indicate that future vte prevention endeavors should include these age groups in addition to exploring more aggressive prophylactic modalities including pharmacological prophylaxis. background: pediatric fellows are required to have active engagement in quality improvement (qi) activities, and yet a national acgme review found most trainees had "limited knowledge of qi methods" and "limited participation in interprofessional qi teams". the twenty fellows in our pediatric hematology/oncology training program identified blood culture utilization as their qi priority. our institution recently introduced a hospital-wide decision algorithm to guide providers regarding when to obtain blood cultures. there is often a low threshold to obtain blood cultures in immunocompromised pediatric oncology patients, but these are often low-yield or result in falsepositives. our fellows spearheaded a project to implement the algorithm in the inpatient pediatric oncology population and improve the proportion of appropriately drawn blood cultures. we investigated how appropriately the algorithm was being utilized on the inpatient pediatric oncology floor prior to and after several educational steps aimed at disseminating the algorithm to members of the care team. our primary endpoint was to quantify the proportion of culture episodes drawn "inappropriately", with a goal of reducing inappropriate episodes to ≤10%. the algorithm was initially introduced to the nursing staff and residents covering the twenty-bed inpatient unit in september 2016. qi project planning took place with upper level fellows in january 2017. fellows and faculty received intensive training on the algorithm in july-august 2017. we then conducted a retrospective chart review of blood culture episodes drawn between august 2016 and november 2017. upper level fellows scored ∼500 culture episodes as to whether the decision to culture and number of cultures drawn were "appropriate" or "inappropriate", and catalogued the indications for culture episodes and if applicable, why the episode was found to be inappropriate. additionally, fellows discussed inappropriate culture episodes with the team onservice, to provide direct feedback on where the algorithm failed. results: between august -december 2016 on average 337 cultures/1000 patient-days were drawn. forty-nine percent of culture episodes were inappropriate. from january -october 2017, following targeted education on the algorithm, the rate of blood cultures drawn decreased to 263 cultures/1000 patient-days. the average proportion of inappropriate culture episodes fell to 16.7%, representing a 66% decrease in inappropriate culture utilization. correct application of a decision algorithm for blood culture utilization can reduce total cultures drawn on an inpatient pediatric oncology unit. fellow-led education of the multi-disciplinary team decreases the rate of inappropriate culture episodes as well as provides active engagement in qi. background: inadequate understanding of sickle cell disease (scd) is common and can affect patients' compliance and therefore their morbidity and mortality, especially after transition to adult care. optimal clinical care for scd includes disease education, which can be difficult given the breadth of possible topics and limited time in clinic. it is unclear how best to provide personalized, efficient education for adolescents with scd. this quality improvement (qi) study aimed to implement a questionnaire-based system to improve patients' knowledge of their scd and documentation of education by the nurse or physician. the study objective was to improve provider documentation and patient knowledge about their scd by identifying patients' gaps in comprehension. by january 2017, the study aimed to increase education documentation from 50% to 75%. by april 2017, the study aimed to increase use of a smart phrase for education documentation from 0% to 50%. by june 2018, the study aimed to increase patients' knowledge about their disease by 20%. design/method: twenty-one scd patients enrolled on an irb approved qi study, with twenty active patients. our comprehensive team generated a questionnaire with knowledgebased questions for two age groups: 12-14 and 15-18 years old. at each comprehensive visit, a questionnaire was distributed, with at least 3-month intervals. the provider scored questionnaires and reviewed two educational topics, with wrong answers taking priority. plan-do-study-act (pdsa) cycles included pdsa#1: patients completed questionnaire. pdsa#2: a smart phrase addressing questionnaire topics was created and shared with providers. pdsa#3: patients received education handouts during clinic education. documentation in clinic notes was the process measure and questionnaire scores was the outcome measure. results: pdsa#1 is complete, pdsa#2 has four patients remaining, and pdsa#3 is ongoing. due to variable visit frequency, there are multiple concurrent cycles. after pdsa#1, free text documentation was completed an average of 61% over the course of 3 months. after pdsa #2 documentation increased to 100% within 3 months and questionnaire scores increased from an average of 59% to 78%. of the questions that patients got wrong on their first visit, they were significantly more likely to improve on retesting if the topic was taught to them than if it was not addressed (72% vs. 33%, p = 0.04). we are currently completing pdsa#3 and collection of post pdsa#3 data. questionnaire-based scd education coupled with standardized smart phrases improves patients' scd knowledge and documentation by providers. further improvement in knowledge is expected with the addition of handouts. background: exposure to suffering can have a profound impact on the wellness of caregivers, often referred to as the "cost of caring". this cost is especially high in pediatric hematology/oncology. repeated exposure to suffering has the potential to negatively impact resilience and increases the risk of burnout, thus impacting quality of care and patient satisfaction. we have developed a peer support team utilizing the critical incident stress management (cism) model. this model has been successfully used in other professions that frequently face traumatic events such as fire fighters, police and emergency medical technicians. the h.o.p.e.s. team (helping our peers endure stress) consists of 18 volunteer multidisciplinary staff members who have received training to provide both group and peer support following any 'critical incident' that may impact one or more staff members. we hypothesize that implementation of the h.o.p.e.s. team will improve staff resilience, decrease overall rates of burnout and improve compassion satisfaction. s161 of s301 design/method: we are using both empiric metrics and anecdotal reports to assess the impact of the h.o.p.e.s. team. prior to the activation of the team, all pediatric hematology/oncology clinical staff members were surveyed using validated tools to assess their levels of resilience, burnout, secondary trauma and compassion satisfaction (proqolv5 and brief resilience scale). they were also asked to rate the number of times they had experienced critical incidents, as well as their perceived level of distress after dealing with traumatic events. after the h.o.p.e.s. team has been functional for 6 months, we will send the same survey to staff members to measure changes, paying special attention to resilience and rates of burnout and compassion satisfaction. results: enthusiasm for development of the team has been high. 18 of 19 people approached to volunteer their time to participate in the multidisciplinary team agreed, including attending physicians, fellows, nurses, nurse practitioners, child life specialists, social workers, clergy and psychologists. all volunteers participated in a 3-day training conducted by an instructor from the international critical incident stress foundation. engagement in the first staff survey has been high, with 91 of 150 responding to date. data collection is ongoing. clinical staff in pediatric hematology/oncology may be particularly vulnerable to burnout and decreased resilience by repeatedly witnessing suffering and trauma. peer support interventions following critical incidents may lead to increased resilience and compassion satisfaction while decreasing rates of burnout. enthusiasm for the development of a peer support team has been high. background: monthly blood transfusions are an indicated therapy for pediatric patients with sickle cell disease with certain complications. maximizing transfusion efficiency in a busy infusion clinic requires: ensuring that appropriate blood units are available in the hospital blood bank; laboratory specimens are obtained from patients in advance; and coordination of clinic appointment and nursing availability. we sought to improve clinic efficiency through identifying ways to better communicate with patients/families regarding upcoming laboratory and transfusion appointments, and to assess the efficacy of implementing a web-based personalized text reminder (pinger.com). we measured the baseline frequency with which transfusion appointments were missed by families, moved to later within the week, or delayed due to late labs. a convenience sample of patients receiving monthly transfusions received a questionnaire about patient/parent preferences for appointment reminders and barriers to keeping appointments. those patients/parents who did not opt-out of an additional text reminder received personalized texts from their care team reminding them of lab and transfusion appointments. rates of missed/moved/delayed appointments were compared between the group receiving the additional text messages and the group only receiving standard, hospitalgenerated appointment reminders (telephone call). results: forty-one families (45 patients) responded to the survey, capturing information on 63% of patients receiving chronic transfusion therapy. thirteen families (32%) declined the additional text reminders. families reported a preference for text reminders (66%), more often than email (49%) or telephone (37%), and 80% of families wanted to receive reminders for both transfusion and laboratory appointments. the majority (43%) of families reported competing work/life priorities as the reason for missed/late appointments. other families noted transportation/travel (29%), fear/illness/pain (21%), and lack of reminders (21%) as the reason for missed appointments. at baseline (twelve weeks), 3.7% of appointments were missed on a weekly basis (range 0-3 of 20 available per week), 10.4% were moved, and 5% of appointments were delayed. during our intervention period (twelve weeks), 7% were missed, 9.2% were moved, and 8.7% were delayed (combined, both groups). there was no difference in missed (7.0% texted vs 7.1% standard), moved (8.0% texted vs 10.0% standard) or delayed (8.0% text vs 9.3% standard) appointments. though families at our center reported a preference for a text-based reminder, personalized text reminders for appointments did not improve clinic efficiency as measured by missed, moved or delayed transfusion appointments. there was no improvement in appointment adherence in the group receiving personalized texts in addition to standard hospital reminders. university of utah, salt lake city, utah, united states background: childhood cancer outcomes have improved significantly, in large part due to multi-institution collaborative clinical trials run by the children's oncology group (cog). approximately half of eligible children with cancer will enroll on a therapeutic trial, but little is known about the factors affecting caregiver decision-making regarding enrollment or how well the required elements of informed consent are conveyed during the consent process. objectives: 1. assess coverage of ten of the required elements of informed consent for cog therapeutic trials. 2. describe factors affecting caregiver decision-making regarding therapeutic trial enrollment. we surveyed families of children who were offered enrollment onto a phase 3 cog therapeutic study for an initial cancer diagnosis in the previous 18 months. fisher's exact or wilcoxon rank-sum tests were utilized to compare demographic and other motivating factors related to enrollment decision-making. results: seventy participants were surveyed. regarding 10 of the basic required elements of informed consent, 96% knew the trial involved research, 99% knew consent was required, 76% knew the enrollment length for the trial, 97% knew they could continue care independent of enrollment, 73% knew who to contact with questions, 71% knew there were options besides enrollment, 83% knew they could withdraw at any time, 93% knew the information was confidential, 34% knew there were risks associated with the trial, and 46% knew there were benefits. of all participants, 84% (n = 59/70) enrolled onto a therapeutic study. among enrollees, 37% (n = 22/59) of the primary caregivers had completed college compared to 18% (n = 2/11) of those not enrolled (p = 0.3). when asked about factors impacting their decision, 69% (n = 41/59) of those enrolled said they felt there were no risks or did not know if there were risks associated with the study compared to 45% (n = 5/11) of those choosing not to enroll (p = 0.17). of those enrolled, 61% (n = 36/59) reported the physician recommendation "somewhat" or "strongly" affected their decision to enroll compared to 0% (n = 0/11) of those not enrolling (p = 0.0001). of those who enrolled, 17% (n = 10/59) reported feeling pressured to enroll while 45% (n = 5/11) of those not enrolled reported pressure (p = 0.05). of enrollees, 10% (n = 6/59) reported they did not have enough time to decide compared to 36% (n = 4/11) of those not enrolled (p = 0.03). failure to convey all 10 required elements of informed consent highlights possible deficiencies in the consent process for cog therapeutic trials. caregivers' perception of being pressured and lack of time to make an informed decision may impact clinical trial enrollment. background: abnormal uterine bleeding (aub) is a frequent adolescent gynecologic complaint. however, limited research exists to guide management, and acute care varies. we sought to improve emergency care for adolescents with aub by developing a clinical effectiveness guideline (ceg) and assessing its impact on quality of care. design/method: a stakeholder engagement group consisting of members from the departments of hematology/oncology, adolescent medicine, general pediatrics, and emergency medicine designed a ceg algorithm for emergency aub management. pediatric residents received ceg training and their knowledge and attitudes were assessed using pre and post intervention surveys. icd-9 and 10 codes identified electronic health record data for patients presenting to the pediatric emergency department (ed) for aub 6 months before and after ceg implementation. pre-pubertal patients and those with vaginal bleeding from trauma were excluded. a weighted, 20-point scoring system consisting of prioritized aspects of history, laboratory studies and management was developed to quantify the quality of care provided. t-test, chi square test, wilcoxon rank sum test, and a run chart were used for analysis. of the 91 patients identified, 62 met inclusion criteria. there were 37% of patients currently using some form of contraception, while 12.9% had bleeding related to a current or recent pregnancy. median aub quality care scores were 14 pre-and 16 post-intervention (p = 0.064). run chart data showed no shifts or trends (overall median score, 14-points). both pre and post-implementation, points were deducted most frequently for not assessing personal/family clotting disorder history and inappropriate use/dosing of oral contraceptives. we successfully designed and implemented a ceg and educational intervention for aub management in a pediatric ed. these data suggest our ceg may be an effective tool to improve emergency aub care for adolescents, though additional cycles are needed. background: high-dose methotrexate (hd-mtx) is a common chemotherapy administered inpatient at most centers. its administration is particularly susceptible to error due to the need for frequent drug levels with resulting changes in supportive care. errors can prolong patient stay and cause patient harm. objectives: global aim-to reduce the length of stay (los) of hd-mtx admissions. smart aims-to increase the percentage of patients whose pre-hydration fluids are started by 10am from 0% to 20% by 1/31/18, and to increase the percentage of patients who receive hd-mtx by 5pm from 43% to 100% by 6/30/18. we used rapid process improvement methods to target earlier methotrexate administration. a key driver of prolonged los was hypothesized to be drug levels returning overnight rather than in the day time due to delayed hd-mtx start. changes implemented have included scheduling hd-mtx patients as the first patients of the day for their exam in clinic and scheduling labs to pass for hd-mtx on the day prior to admission. there are ongoing pdsa cycles to change the location of pre-hydration start from the inpatient room to the clinic exam room in order to meet hd-mtx administration time goals. we are piloting two different education materials to improve patient experience. one explains hd-mtx levels in a red/yellow/green stoplight format and the other reminds patients how to prepare for the admission. other interventions regarding how we test urine ph and safety checks in the ordering process for history of delayed clearance are in the planning stage. the project is ongoing, but as of 12/12/17, we start methotrexate by 5pm 50% of the time which is improved from a baseline of 43%. when the project was started, pre-hydration was never started before 10am. now, fluids are started by 10am 40% of the time. pdsa cycles are ongoing and we have yet to sustain reductions in los, but some months have shown decreased los by as much as 19 hours from baseline measurements. rapid cycle improvement can be utilized to decrease los hd-mtx admissions. this has important financial implications as well as the potential to reduce secondary harm from unnecessary time in the hospital. pediatric cancer centers should schedule hd-mtx admissions first thing in the morning so that data regarding kidney injury and drug clearance can be interpreted by the day team and children are not cleared for discharge in the middle of the night. background: education and training for interdisciplinary pediatric oncology providers requires training in principles of palliative and end-of-life (eol) care. the experiences of bereaved parents can inform and enhance palliative care educational curricula in uniquely powerful and valuable ways. the objective of this study is to present an innovative palliative care educational program for oncology providers facilitated by trained bereaved parents who serve as volunteer educators in local and national palliative care educational forums and to describe how incorporation of bereaved parents in these educational forums affects participant comfort with communication and management of children at the eol. design/method: survey tools were adapted to determine how bereaved parent educators affected participant experiences in 3 different educational forums: institutional seminars on pediatric palliative and eol care, role-play based communication training sessions, and an international symposium on pediatric palliative oncology. pre-and post-session surveys with incorporation of retrospective pre-program assessment item to control for response shift were used in the evaluation of institutional seminars and communication training sessions. results from feedback surveys sent to all attendees were used to appraise the participants experience in the international oncology symposium. results: involvement of trained parent educators across diverse, interdisciplinary educational forums improved attendee comfort in communicating with, and caring for, patients and families with serious illness. importantly, parent educators also derive benefit from educational with interdisciplinary clinicians. integration of bereaved parents into palliative and eol care education is an innovative and effective model that benefits both interdisciplinary clinicians and bereaved parents. background: poorly controlled chemotherapy-induced nausea and vomiting (cinv) significantly impairs patients' quality of life and contributes to ongoing medical costs through increased length of stay in the hospital or readmissions and outpatient visits for control of nausea, vomiting or dehydration. lack of adherence to national evidenced-based guidelines that dictate antiemetic prescribing for variably emetogenic chemotherapy leaves patients vulnerable to increased cinv and its ensuing complications. objectives: to review our institution's antiemetic prescribing practices and their consistency with the antiemesis guidelines from the national comprehensive cancer network (nccn) and children's oncology group (cog)-endorsed supportive care guidelines and to further develop tools to increase adherence to these national-based guidelines to improve control of cinv. we performed a retrospective chart review of inpatient chemotherapy encounters. we evaluated emetogenicty of chemotherapy (high, medium, low), initial antiemetic regimen ordered, number of as needed medications required and adherence to national evidenced based guidelines tailored to each level of emetogenicity in the prescription of antiemetics. results: fifty-five total inpatient chemotherapy encounters were reviewed over 8 months. eighteen of these encounters were considered to have been highly emetogenic chemotherapy (hec) with the remaining 37 of these considered to be moderately emetogenic. only 9 out of 18 hec encounters completely included all guideline-recommended agents. there was a demonstrable lack of consistency across providers with dosing of aprepitant and most as needed medications. there was significant variation in order of first, second and third line anti-emetics ordered -with lorazepam and promethazine being used most frequently. with an aim of improving antiemetic prescribing practices for our patients, we are currently rebuilding chemotherapy treatment plans in our electronic medical record to incorporate antiemetic drug order sets that follow evidenced-based guidelines for variably emetogenic chemotherapy. this will be used in conjunction with an education initiative about best practices in supportive care for all prescribers of antiemetics. review of our department's recent inpatient chemotherapy encounters show we are falling short in following nationally recommended standards for appropriate antiemetic coverage during chemotherapy. identification of these deficiencies allows for implementation of quality initiatives to improve prescriber adherence to evidenced-based guidelines for better control of cinv. background: there are currently no consensus guidelines for the management of pediatric oncology patients presenting with fever without neutropenia. historically, these patients had been treated similarly to neutropenic patients with empiric antibiotics. while there has been a shift towards reducing unnecessary empiric treatment, there has been limited research into the outcomes associated with withholding empiric iv antibiotics in this patient population. we assessed the safety and efficacy of our institution's current protocol of observing well-appearing patients who present with fever without neutropenia and compared the outcomes of the patients who did and did not receive empiric iv antibiotics. design/method: this was a prospective, single-institution cohort study. patients were included if they were currently undergoing chemotherapy for an oncologic diagnosis and presented initially as an outpatient with fever and nonneutropenia (defined as anc ≥ 500 cells/mm3). for each episode we recorded lab and blood culture results, signs and symptoms of initial presentation, and clinical outcomes, including antibiotic administration and hospital admission. results: a total of 242 episodes of well-appearing patients with fever without neutropenia were identified. compliance with the institutional protocol was high; 81.8% of patients were observed without receiving empiric iv antibiotics. the majority of patients were discharged home and there were no serious complications or infectious deaths. the incidence of positive blood cultures was low (3.7% including several likely contaminants), despite the presence of central venous catheters in the majority (84.7%) of patients. there were no significant differences in age, oncologic diagnosis, central s165 of s301 line access, anc value, or incidence of bacteremia between patients who did and did not receive empiric iv antibiotics. patients who were admitted to the hospital were significantly more likely to have received iv antibiotics (p <0.001) despite documentation of a reassuring exam. however, admitted patients who initially received iv antibiotics were just as likely to discharge within 48 hours compared to patients who were observed. we propose that empiric iv antibiotic administration in febrile, non-neutropenic, otherwise well-appearing patients is unnecessary. our study demonstrated no adverse consequences of observation and no significant differences in clinical outcomes between patients who did and did not receive iv antibiotics aside from rate of hospitalization. this supports the practice of observation without empiric antibiotics for such patients. background: children with hepatoblastoma (hb) undergo repetitive computed tomography (ct) scans to determine response to treatment and assess for relapse. this imaging exposes children to radiation, anesthesia, and imposes financial and emotional burden. objectives: review our institutional experience to determine if afp measurements are sufficient to assess response to treatment and detect relapse. we conducted a retrospective chart review of all patients diagnosed with hb at our institution between 1978-2017. data collected included serum afp, total number and type of imaging studies during and post treatment, and how relapse or progressive disease was detected. results: thirty-one patients were diagnosed with afp positive hb. during therapy, 173 ct scans were performed: 118 to assess for response to therapy or surgical planning (average 4 scans/patient) and 55 due to concern for progression with rising afp. off therapy, 213 surveillance ct scans were performed (average of 5.3 scans/patient) and 72 (33%) included the chest in patients with no lung metastasis at diagnosis. relapsed patients averaged 12.5 surveillance scans, 6.5 of which were done before relapse was noted on imaging. there were no cases of radiographic evidence of relapse without a prior increase in afp. during treatment, response to therapy based on imaging correlated with a decline in afp in all patients, arguing that repetitive scans are not needed in this setting unless required for surgical planning. only 3 of 213 scans performed during off therapy surveillance displayed evidence of relapse, all of which were preceded by rise in afp. our study represents the largest cohort of hb patients. prior studies suggest similar results, but included fewer patients, lower stage of disease and less than 10 years of surveillance monitoring. at our institution, the cost of a ct c/a/p is $15,169 with reimbursement varying from 30-50%. in comparison, the cost of an afp measurement is $101.50. many scans also require anesthesia and result in emotional toil for families concerned about this procedure as well as the results. thus, afp demonstrates greater sensitivity, with significant cost savings and decreased emotional burden, and should be used for monitoring both during and off therapy, replacing routine serial imaging. background: we observed that our practice of drawing daily blood cultures in hospitalized patients with fever and neutropenia was wasteful; it resulted in excessive negative cultures that did not add to patient care. the smart aim of this quality improvement project was to reduce the number of negative blood cultures drawn on hospitalized patients with fever and neutropenia by 25% in 6 months. design/method: after reviewing published evidence suggesting drawing daily blood cultures in febrile neutropenic patients was unnecessary, a new blood culture guideline was implemented: cultures were drawn at presentation for fever with neutropenia and, if negative at 24 hours, repeat cultures were not drawn except for clinical change, new fever after being afebrile >24 hours, or antimicrobials were being changed/broadened. to impact key drivers, we educated staff and changed blood culture order sets to require providers to select a reason for ordering the culture and to eliminate a nursing order to draw daily cultures with fever. we compared the number of blood cultures drawn per 1000 central linedays (/1000-cld) and the proportion of positive versus negative cultures pre-guideline (july 2015-may 2016) and postguideline (june 2016-december 2016). we calculated the cost savings from reducing cultures. to assess patient safety, potential septic events without a corresponding positive blood culture were reviewed. data were analyzed by service (oncology and stem cell transplant). a chi-square test was used to compare rates. in stem cell transplant patients, pre vs. postguideline, there were 492 vs. 258 total cultures drawn/1000-cld; 25 vs. 21 positive (16% decrease, p = 0.404) and 467 vs. 237 negative cultures/1000-cld (51% decrease, p<0.0000001). in oncology patients, pre vs. post-guideline, there were 266 vs. 181 total cultures drawn/1000-cld; 17 vs. 13 positive (24% decrease, p = 0.024) and 249 vs. 168 negative cultures/1000-cld (32% decrease, p<0.0000001). the decreased positive culture rate among oncology patients may be due to decreased culture contaminants and/or the effect of a concurrent initiative to decrease clabsi in that group. there were 2 safety concerns; however, chart review concluded that the guideline did not lead to missed infections in these patients. for the first 6 months of the guideline, the total cost savings in blood cultures was $31,454.31. the implementation of our new blood culture guideline successfully led to a substantial reduction in the collection of negative cultures and a cost savings without compromising the detection of bacteremia in hospitalized pediatric patients with fever and neutropenia. background: there are various evidence-based guidelines for treatment of adult cancers, such as the nccn guidelines. previously, care was standardized for most new diagnosis pediatric cancer patients through enrollment on a clinical trial. with decreasing clinical trial availability and enrollment and few, if any, evidence-based guidelines for pediatric cancer, care standardization is challenging for pediatric cancers. objectives: to assess consistency of care, as determined by plan of treatment by diagnosis, for pediatric patients receiving chemotherapy for newly diagnosed cancer at a single center. design/method: patients with a new cancer diagnosis at a large, tertiary care pediatric oncology center in calendar year 2016 were identified through reports from the chemotherapy order entry (coe) system. reports included diagnosis (recorded through standardized options) and the plan of treatment. chart review was used to exclude patients who started treatment elsewhere and patients being treated for relapse, to clarify diagnosis if the standardized options in coe were unclear, and to clarify treatment plan if needed. data was entered and analyzed in a redcap database. specific diagnoses were clustered into higher level disease groups and the distribution of treatment plans for patients within each was determined. this project was deemed exempt from irb approval for human subject research as a qualifying quality improvement project. of the 324 patients with a first chemotherapy order in 2016, 142 were excluded due to one or more reasons: stem cell transplant (62), transfer of care (54), relapse (25), and other (9). an additional 61 patients were excluded because <5 patients/year/diagnosis. there was no cns tumor disease group with >5 patients. thus, 121 patients with hematologic malignancies or non-cns solid tumors are the focus of this analysis. for patients with intermediate risk rhabdomyosarcoma, the plan of treatment was the standard arm of a cog protocol, arst0531 for 3 patients and arst 1431 for 1 subsequent patient after protocol activation. for all other diseases including lymphoblastic leukemia/lymphoma (excluding infants), classical hodgkin lymphoma, aml (excluding trisomy 21 and apml), stage iii/iv burkitt lymphoma/diffuse large b-cell lymphoma, posttransplant lymphoproliferative disease, wilms tumor, rhabdomyosarcoma, ewings sarcoma, osteosarcoma, neuroblastoma, and retinoblastoma, only one treatment plan per risk category was used. conclusion: this analysis demonstrates highly consistent chemotherapy treatment at a single center for patients with hematologic malignancies and non-cns solid tumors. next steps include exploring strategies to group diagnoses for cns tumors and assessing the quality of evidence supporting the treatments given. background: rapid initiation of empiric antibiotics in patients with fever and neutropenia has been shown to reduce morbidity and mortality. current practice guidelines call for the initiation of antibiotics in these patients within sixty minutes and time-to-antibiotic (tta) has been suggested as a quality-of-care measure. many institutions, including our own, face barriers to meeting this time limit. objectives: utilizing a quality improvement model, determine barriers and implement an intervention to reduce the time-to-antibiotics for pediatric febrile patients with suspected neutropenia who present to the emergency department (ed) at our institution. we have identified and implemented an intervention utilizing the plan-do-study-act model for quality improvement. a twelve-month retrospective review was conducted to evaluate the efficacy of the current practice algorithm at our large, academic tertiary-care hospital. subjects identified were pediatric oncology patients undergoing active chemotherapy who presented to the ed with febrile neutropenia. we identified two specific barriers, triage level assignments and delay in ordering antibiotics. to address these barriers, we have created a wallet sized "fever card" that patients were instructed to show upon arrive to the ed. in collaboration with the ed staff, efforts were also made to educate all pediatric staff on the use of the fever card. post-intervention data collection is currently underway and pre-and post-intervention antibiotic delivery times will be compared. the pre-intervention cohort consisted of thirty-three encounters with a mean time-to-antibiotic delivery of 135 minutes, or seventy-five minutes greater than the accepted standard of care. only one patient received antibiotics within sixty minutes of arrival. post-intervention data collection is currently underway. since identifying two barriers to meeting the standard of care at our institution, we have implemented a quality improvement measure that empowers patient families to direct appropriate triage in the ed as well as simplifying the treatment protocol for ed providers. we expect to identify an improvement in time-to-antibiotics from the pre-intervention to the post-intervention period. background: sickle cell disease (scd) is a genetic disorder in which sickle hemoglobin (hbs) triggers multiple downstream effects, including red cell sickling, hemolysis, vaso-occlusion, and inflammation. scd, a lifelong disease initiated at birth with injury that accumulates over time, causes significant end-organ damage and clinical complications that are undertreated and associated with early death. homozygous mutation (hbss) causes the severe form of scd. individuals with scd are at increased risk of infection, stroke, and retinopathy. clinical guidelines for pediatric patients with scd recommend prophylactic penicillin use (ages 2-5), annual screening for stroke with transcranial doppler (tcd) imaging (ages 2-16), and annual ophthalmology exams to assess for retinopathy (ages ≥10). there are limited real-world data on implementation of these nhlbi-based recommendations. objectives: to describe utilization of penicillin, tcd screening, and ophthalmology care in children with hbss disease. medicaid administrative claims databases were used to identify us patients aged 2-16 years at first indication of hbss recorded in each calendar year from 2009 to 2014. patients were required to have medical and pharmacy benefits for the calendar year in which they were identified and for 12 months prior to their first recorded hbss indication. prior year utilization of penicillin, tcds, and ophthalmologist visits was measured for each annual cohort. annual cohorts included 347-438 commercial (mean age 9.5 years, 52% female) and 1024-1557 medicaid (mean age 8.3 years, 48% female) patients with hbss disease. fewer than half of all patients had received a tcd scan in the previous year, with similar rates seen across all age groups for both payers. ophthalmologist visits increased as patients aged, and while patients aged 12-16 years had the highest proportion with an ophthalmologist visit in both payer populations, the overall implementation remained low. in contrast to the low use of tcd and ophthalmology visits, penicillin use was highest in the 1-5 year age group: >80% use in any given year for both payers. conclusion: although our data demonstrated high penicillin use in the 1-5 year age group, consistent with guidelines there is an opportunity to improve implementation of other guidelines-based recommended screening. for example, tcd screening can identify children at risk of scd-related stroke in order to initiate preventive therapies. further research to understand potential barriers to proper screening and to evaluate strategies to improve awareness, adherence, and implementation of recommended screenings in children with scd is warranted. supported by global blood therapeutics. background: childhood cancer therapy has improved where there are many long-term survivors. while psychosocial difficulties in pediatric cancer survivors are recognized, the prevalence of these problems at initial survivorship presentation is unclear. objectives: to examine the prevalence of overall internalizing symptoms (e.g., depression/anxiety) in pediatric cancer survivors presenting to a survivorship clinic and to examine how this is mitigated by receiving psychological services and by evidence of parental depression/anxiety. design/method: pediatric cancer survivors attending their first visit at the reach for survivorship clinic at vanderbilt (ages 3-18) were included. survivors' parents (93% female) completed the child behavior checklist (cbcl), beck depression inventory-ii, and beck anxiety inventory. survivors >12 years completed a self-report. the wilcoxon rank-sum and pearson's 2 test were used for univariate analyses. the effect size and 95% confidence intervals (ci) estimated from the multivariable linear regressions were reported. results: 142 childhood cancer survivors a median of 12 years old and 3.3 years off therapy were included. thirty one survivors (22%) showed at least borderline clinical internalizing problems (t score >60) on the cbcl, but only 8 of these patients (26%) reported receiving psychological services. nine other survivors with normal t score ≤60 also reported receiving psychological services. parental depressive and anxiety symptoms were correlated to the parental report of survivor overall internalizing symptoms (spearman = 0.415, p = <0.001 and = 0.476, p = <0.001 respectively), however they were not correlated to survivor selfreports. furthermore, parents with mild to severe depressive symptoms or mild to severe anxiety symptoms were more likely to rate their child as having higher overall internalizing symptoms (p = 0.001; p = 0.008, respectively). multivariable linear regression showed that when adjusted for age, gender, cancer diagnosis and time off treatment, reported utilization of psychological services ( = 8.58, 95% ci [3.54, 13.62],p = 0.001), and parent depressive symptoms ( = 0.43, [.22, .65 ],p<0.001) were significantly associated with child overall internalizing symptoms. in an otherwise identical alternate model substituting parental anxiety for parental depression, parental anxiety was also a significant risk factor ( = 0.70, [.38, 1.03], p<0.001). alternatively, parent anxiety/depressive symptoms were not significantly associated with child self-report of internalizing symptoms. childhood cancer survivors have an elevated prevalence of experiencing internalizing symptoms but seldom report receiving psychological services. childhood cancer survivors' parents with anxious/depressed symptoms are more likely to rate their children as having more internalizing problems, compared to patient self-reports. ongoing longitudinal analyses will help clarify the best timing for potential interventions. background: life expectancy for adults with sickle cell disease (scd) has remained unchanged over the past 30 years despite improvements in pediatric scd survival. at greatest risk are the adolescents and young adults (ayas) transitioning from pediatric to adult care. allen county ranks 3rd in scd incidence among the 92 counties in indiana, and has 2 board certified pediatric hematologist-oncologists. when children "age out" of the pediatric system, there are few providers knowledgeable about managing adults with scd in the region. a novel partnership between hematologists and the family medicine residency program in allen county was initiated to educate family medicine residents (fps) about scd, hydroxyurea (hu), and management of scd-related complications with the goal to increase the number of knowledgeable providers to care for adults with scd. to determine the effectiveness of online learning modules in educating fps about hu, best practices for aya scd care and transition. three online learning modules about scd (comprehensive care of ayas with scd, hu, best practices in aya transition) were developed and cme-accredited. electronic pre-and post-tests were distributed to 32 fps with five questions for each module covering: contraception; screening tests; hu indications, dosing and monitoring; developmental milestones and scd knowledge assessments. the st vincent irb reviewed the protocol and granted a waiver of consent. results: twenty-six fps (81%) completed the pre-and posttests. over two-thirds correctly identified the clinical benefits of hu on both assessments. knowledge about the rationale for hu therapy increased after the completion of the hu module (30% correct on pre-test vs. 62% on post-test, p = 0.009). the proportion of correct responses increased for all comprehensive aya scd care post-test questions, but only the leading cause of death and the priapism-related questions reached statistical significance (15% vs. 35%, p = 0.048; 0% vs. 23%, p = 0.003, respectively). the proportion of correct responses for 2 of the transition-focused questions was unchanged (89% for both), while the proportion of correct post-test responses on the self-care assessment question significantly increased (0% vs. 12%, p = 0.04). after module completion, fps were able to correctly identify common scd complications and why hu is an effective treatment for individuals with scd. the best practices of transition clinic module may need modification to improve physician understanding of the intricacies in establishing and maintaining a scd transition clinic. overall, online training is effective at educating fps and could be used to increase the number of providers knowledgeable about scd care. background: survival rates for pediatric hodgkin lymphoma (hl) exceed 95% with contemporary therapy. studies of pediatric hl survivors treated in the 1970s-1990s have shown increased risk for treatment-related chronic health conditions. risk-adapted therapy, including tailored radiotherapy, has been developed to reduce long-term morbidity while maintaining excellent survival. little is known about chronic conditions associated with contemporary therapy presenting during the first 5 years from therapy completion (early outcomes). objectives: to analyze survival and early outcomes of pediatric hl patients treated with contemporary therapy. we conducted a retrospective review of hl patients diagnosed <21 years of age at our institution from 2010-2014. three-year overall (os) and event-free (efs) survival were calculated with kaplan meier statistics using sas 9.4. results of standardized screening for targeted toxicities that developed between 2 -5 years from therapy completion were identified and graded per ctcae criteria. censoring occurred at date of death, 5 years from therapy completion, or december 31, 2016. data from the last collection point were used for prevalence calculations in cases with multiple evaluations. we identified 83 patients (51% male; 43% non-hispanic white; mean age at diagnosis 13.6 ± 3.7 years) with a median time since therapy completion of 3.3 years (range 0.1-5.0). initial treatment included: 56 (67%) chemotherapy only and 27 (32%) multimodality treatment. all patients received anthracyclines (median dose 200mg/m2) and 96% received alkylating agents (median cyclophosphamide equivalent dose [ced] 3600mg/m2). the 3-year os was 99% with an efs of 90% (90% chemotherapy only, 91% multimodality treatment; p = 0.76). patients with relapsed/refractory disease received salvage treatment including chemotherapy only (n = 1), multimodality therapy (n = 1), or multimodality treatment including stem cell transplant (autologous n = 4; autologous+allogeneic n = 1). no patients developed thyroid dysfunction, cardiac dysfunction, subsequent neoplasm, or male gonadal dysfunction during the study period. pulmonary dysfunction was limited to ctcae grade 1. anti-mullerian hormone (amh) below the normal range was found in 10/11 pubertal females who received ced ≥7000mg/m2 compared to 0/12 females who received ced <7000mg/m2. two of the females with low amh also had follicle stimulating hormone >30iu/ml. this study is the first to evaluate early outcomes in pediatric hl survivors. the results indicate contemporary chemotherapy and a lower rate of radiotherapy utilization lead to excellent 3-year survival rates with minimal early toxicities. females exposed to ced ≥7000mg/m2 are at increased risk for gonadal dysfunction and should be prioritized for fertility preservation approaches prior to initiation of cancer therapy. background: cancer is one of the leading disease-related causes of death among individuals aged <20 years in the united states. recent evaluations of national trends of pediatric cancer used data from before 2010, or covered ≤28% of the us population. objectives: this study describes pediatric cancer incidence rates and trends by using the most recent and comprehensive cancer registry data available in the us. design/method: data from us cancer statistics were used to evaluate cancer incidence rates and trends among individuals aged <20 years during 2001-2014. data were from 48 states and covered 98% of the us population. we assessed trends by calculating average annual percent change (aapc) in rates using joinpoint regression. rates and trends were stratified by sex, age, race/ethnicity, us census region, county-based economic status, and county-based rural/urban classification, and cancer type, as grouped by the international classification of childhood cancer (iccc). we identified 196,200 cases of pediatric cancer during 2001-2014. the overall cancer incidence rate was 173.0 per 1 million; incidence rates were highest for leukemia (45.6), brain tumors (30.8), and lymphoma (26.0). rates were highest among males, aged 0-4 years, non-hispanic whites, the northeast us census region, the top 25% of counties by economic status, and metropolitan counties. the overall pediatric cancer incidence rate increased (aapc = 0.7, 95% ci, 0.5-0.8) during 2001-2014 and contained no joinpoints. rates increased in each stratum of sex, age, race/ethnicity (except non-hispanic american indian/alaska native), region, economic status, and rural/urban classification. rates were stable for most individual cancer types, but increased for non-hodgkin lymphomas except burkitt lymphoma (iccc group ii(b), aapc = 1.2, 95% ci, 0.4-2.0), central nervous system neoplasms (group iii, aapc = 0.4, 95% ci, 0.1-0.8), renal tumors (group vi, aapc = 0.6, 95% ci, 0.1-1.1), hepatic tumors (group vii, aapc = 2.5, 95% ci, 1.0-4.0), and thyroid carcinomas (group xi(b), aapc = 4.8, 95% ci, 4.2-5.5). rates of malignant melanoma decreased (group xi(d), aapc = -2.6, 95% ci, -4.7--0.4). this study documents increased rates of pediatric cancer during 2001-2014, in each of the demographic variables examined. increased overall rates of hepatic cancer and decreased rates of melanoma are novel findings using data since 2010. next steps in addressing changing rates could include investigation of diagnostic and reporting standards, host biologic factors, environmental exposures, or potential interventions for reducing cancer risk. increasing pediatric cancer incidence rates may necessitate changes related to treatment and survivorship care capacity. background: while childhood cancer treatment modalities have improved, the delayed effects of cancer treatment continue to compromise the quality of life in survivors. metabolic syndrome (ms) is diagnosed based on the presence of three of the following findings -obesity, dyslipidemia, hypertension and insulin resistance per the world health organization (who) criteria. the increased risk of ms among childhood cancer survivors was first reported in the 1990's and is known to increase the incidence of cardiovascular disease in these individuals. objectives: assess the frequency of ms in childhood cancer survivors at our institution. . we conducted a retrospective chart review on pediatric cancer survivors, 4 -18 years of age, who had been treated at sri ramachandra medical institute and research foundation between august 2015 and august 2017. patients who received at least one year of treatment with s171 of s301 chemotherapy and/or radiation and surgery were included. medical history, family history of diabetes, cardiovascular diseases, and hypercholesterolemia, tanner staging, weight for height (<5y per who criteria), bmi (>5y per indian academy of pediatrics iap), blood pressure (nhlbi criteria), fasting blood sugar levels and lipid profile were obtained from the charts. statistical analysis of the data was done using ibm spss statistical software (version 22). results: 97 patients were studied, 63.9% were male. 22.68% were under 5 years of age, 38.14% between 6-10 years and 39.18% above 11 years. leukemia survivors comprised 39.18% of the sample and non-leukemic's were 60.82%. 51.5% were treated with chemotherapy alone, 19.5% with radiotherapy and chemotherapy, and 28.8% underwent surgery with radiotherapy and chemotherapy. hypertension was found in 19.5% of the study group, dyslipidemia in 68%, impaired fasting blood glucose in 4.12% and 36.08 % were found to be obese. 10% of the study group was diagnosed with ms based on who criteria. conclusion: 10 % of our study population was found to have ms per who criteria. individual metabolic complications were detected in 68% of the population. acute lymphoblastic leukemia (all) survivors appeared to be at high risk in our population. ms has been known to increase cardiovascular complications in cancer survivors. a multidisciplinary team approach to management of these patients is important to closely monitor and manage the long-term complications related to ms such as type 2 diabetes and atherosclerosis. such an approach is essential to decrease long term morbidity and mortality from ms in this vulnerable population. background: the 5-year survival rate for childhood cancer exceeds 80%. however, up to 38% of these children require admission to the pediatric intensive care unit (picu) within three years of diagnosis. these children account for approximately 10% of all picu deaths, with mortality being higher for those post-hematopoietic stem cell transplant (hsct). national guidelines recommend that providers share informa-tion regarding prognosis and treatment options within the first 48 hours of icu admission. these prognostic goals of care conversations (pgocc) are critical to the care of children with malignancies, a subpopulation at risk for increased mortality. to determine the frequency of pgocc as well as describe differences in patient characteristics and critical care therapies by pgocc status. design/method: a retrospective cohort study was conducted using the university of michigan virtual picu system database. picu admissions lasting longer than 24 hours for patients ages 0 to 25 years between july 1, 2011 and june 30, 2016 with an oncologic diagnosis and/or hsct were identified. data on pgocc, patient demographics, diagnoses, picu interventions, and outcomes were recorded and compared between children with pgocc and those without using chi square test for categorical variables and kruskal-wallis test for continuous data. of 128 picu admissions, 53% were male; the mean age was 10.5 years. the leading diagnoses were acute lymphoblastic leukemia (46%), acute myeloid leukemia (14%), lymphoma (14%), neuroblastoma (5%), and brain tumors (5%), and 43% of patients were post-hsct. pgocc was documented in 35 (27%) patients. in comparison with patients who did not have a pgocc, children with a pgocc were more likely to be readmitted to the picu (45% vs. 15%, p <0.01) and more likely to have had relapse of disease (41% vs. 18%, p<0.01). patients with a pgocc had higher severity of illness scores (p = 0.02), higher use of non-invasive (22.9% vs. 8.6%, p = 0.04) and invasive conventional ventilation (68.6% vs. 23.7%, p<0.01), and high frequency ventilation (25.7% vs. 4.3%, p <0.01). also, patients with pgocc were more likely to receive continuous renal replacement therapy (37.1% vs. 5.4%, p<0.01), arterial catheterization (54.3% vs. 23.7%, p<0.01), and cardiopulmonary resuscitation (22.9% vs.1.1%, p<0.01). in only 1 in 3 critically ill children with hematologic-oncologic disease is pgocc held. children with pgocc were sicker and received more critical care interventions. future research is needed to evaluate the content of pgocc. background: central nervous system (cns) tumors and autism spectrum disorder (asd) represent significant disease cohorts in the pediatric population. asd diagnoses in children have a prevalence of 2%, 1 in every 88 children in the united states. additionally, more than 4,000 cns tumors are reported in children age 0 to 19 years in the united states with brain tumors being the most common solid tumor and the leading cause of death among all childhood cancers. the genetic etiology of autism and cns tumors is complex. specific gene alterations present in certain cancers have similarly been described and suspected to play a role in asd subtypes. targeted therapy panels, like foundation one (fo), have been beneficial in guiding treatment for some cancers based on distinct gene alterations. given the genetic overlap, the potential for therapeutic benefit and crossover from such actionable gene target panels merit further exploration in asd and cns tumors. we aim to identify and describe genetic alterations with known actionable targets in cancer therapy from fo as potential diagnostic, therapeutic and research targets for neurodevelopmental diseases. we plan to discuss the common genetic alterations between our cancers and neurodevelopmental diseases described in the literature. fo data was extracted and compared to the literature. each reported gene alteration from fo plus the keywords "autism", "psych" were used on pubmed to search for a suspected association if any with a neurodevelopmental disorder. results: twenty-one patients representing a cohort of six unique (astrocytoma-five, ependymoma-six, gbm-four, glioma-three, nerve sheath tumor-one, etmr-two) cns tumors were investigated. fo produced eighty total with sixty unique gene alterations. thirty-one (52%) of these yielded at least one published, suspected association to a neurodevelopmental disorder. the most common gene alterations were tp53-four, cdkn2a/b-five and braf-four. the main functional categories were cellular: proliferation, structure, differentiation and degradation; chromatin modeling; histone transcriptional modification; dna methylation and repair; strna; and neural signaling. sixty unique gene alterations were found in our cns tumor set using foundation one. thirty-one (52%) of these discrete alterations paired with at least one description in the literature as having been similarly altered in an asd subtype. many of these alterations have actionable targeted therapies presented through foundation one for our cns tumors and may be a relevant guide in the future of targeted therapy and research in asd subtypes. monoclonal antibody therapy usage is associated with significantly improved survival in b-cell nhl aya patients. although the usage has increased in the aya population from 2006 to 2013, the magnitude of the increase is low. factors that affect the use of mab include race and insurance s173 of s301 type. further research is warranted to identify why privately insured patients are less likely to receive these drugs. background: prevention of chemotherapy-induced nausea and vomiting (cinv) remains a challenge despite advances in pharmacotherapy and the development of cinv clinical practice guidelines by the pediatric oncology group of ontario (pogo) that have been endorsed by the children's oncology group. achieving control of cinv in pediatrics further is complicated by the difficulty young children have vocalizing their symptoms. use of a validated nausea-assessment tool in conjunction with improved adherence to evidence-based guidelines may result in better quantification of symptoms and reduction of both nausea severity and vomiting frequency for pediatric patients undergoing chemotherapy. the pediatric nausea assessment tool (penat) has been validated for children ages 4-18, and its integration into clinical practice may help optimize cinv control. objectives: this single-institution study sought to improve control of cinv in patients admitted for chemotherapy by standardizing the antiemetic regimens prescribed by all providers according to an institutional cinv algorithm developed from the pogo guidelines. we hypothesized that treatment using a standardized guideline would improve cinv control in patients admitted for chemotherapy. a baseline cohort of 70 admissions for chemotherapy completed penat assessments and cinv diaries prior to receiving chemotherapy, four times daily during each admission, and daily for 7 days following completion of chemotherapy from may 1, 2013 to january 31, 2014. providers then were provided an institutional cinv treatment algorithm based on the pogo guidelines and received education at departmental meetings on appropriate implementation of this algorithm. a second cohort of 78 admissions completed penat assessments and cinv diaries in a similar fashion from july 1, 2014 to december 31, 2016. results: complete control of vomiting markedly improved following cinv guideline implementation (72% vs 52%, p <.0001) with treatment failure also significantly reduced (6% vs 23%, p <.0001). after controlling for the degree of emetogenicity of chemotherapy received, a patient was 3.33 times more likely to vomit prior to guideline implementation (or 3.33, ci 1.96-5.56). there was no difference in nausea control, even after adjusting for the emetogenicity of chemotherapy. conclusion: control of chemotherapy-induced vomiting (civ) improved following widespread implementation of an institutional cinv treatment algorithm at a single institution. the severity of nausea reported remained unchanged which may reflect the difficulty of assessing nausea or an inadequate sample size. future research may focus on cinv treatment management through the use of guidelines specifically for breakthrough cinv and delayed cinv. background: aspho's professional development committee (pdc) recognized pediatric hematologists-oncologists (phos) serving in the united states (us) military have unique professional development needs that may not be addressed by aspho or a similar professional society. these individuals may also encounter challenges when transitioning to a civilian career. however, barriers to professional development have not been systematically characterized. the objectives were to characterize the number of phos with current or prior military service (mphos) and to identify any unmet professional development needs. design/method: a working group consisting of pdc members and both senior and early career mphos was formed. initial comments were solicited by email from known mphos regarding potential gaps in professional development and interest in working with aspho to improve support of mphos. a survey was developed and piloted with four members of the advisory group, questions were revised based on their feedback, and a final version was distributed via the aspho website and online community forum. targeted emails were sent to mphos identified through aspho and military databases. eligibility to complete the survey included 1) completion of a fellowship in pediatric hematologyoncology, and 2) current or prior service as an active duty military provider. quantitative and qualitative information were collected, including demographic data and perceived barriers to professional development. responses were summarized using descriptive statistics. results: sixty-five mphos were identified and 34 surveys were completed for a 52% response rate. respondents were engaged in a variety of professional activities; 64% were male, 52% were serving active duty commitments, and 32% felt there were professional development gaps. areas of concern were categorized into nine themes with the most concerning being 1) limited civilian knowledge of mpho practices (76% of participants), 2) inability to attend professional society meetings (60%), and 3 possibility of deployment (56%). participants expressed a desire for educational products to meet their specific needs and for networking opportunities with civilian colleagues. qualitative analyses identified concerns about low patient numbers and practice size. a subset of mphos perceive significant gaps in professional development. additional research is needed to better define areas for intervention, but many of the concerns align with those of similarly sized civilian programs and may be addressed through professional society networking opportunities, such as an aspho special interest group. background: infertility is an established cause of distress and has a negative impact on quality of life among childhood cancer survivors. the american society of clinical oncology has established guidelines on fertility counseling for individuals of reproductive age diagnosed with cancer, with the goal of improving reproductive and psychosocial outcomes. studies have shown that instituting a fertility team that can provide counseling and discuss fertility preservation (fp) options results in improved patient satisfaction in patients with cancer. objectives: the goal of this study was to examine predictors of referrals to the multidisciplinary fertility team, and documented fp interventions among these patients. design/method: an irb-approved retrospective medical record review was performed at a large pediatric academic center. all patients with new cancer diagnoses receiving chemotherapy were included from january 2015 (when the fertility team was established) to present. a standardized abstraction form was used to collect information about: age at diagnosis, gender, cancer type, whether a fertility consult was placed, and documented fp interventions. data were summarized descriptively and comparisons were made using nonparametric statistical methods. results: 265 patients met inclusion criteria, of which 152 (57%) were male. cancer types were as follows: 126 leukemia/lymphoma, 51 cns tumors, 44 sarcomas, 37 embryonal tumors, and 7 langerhan's cell histiocytosis (lch). the mean age was 8.2 years, (range <1-31 years). overall, 27% of all patients had a consultation with the fertility team. patients were significantly less likely to have a fertility consult if they were younger (p<0.001). further, there were differences in the consultation rate between diagnoses, with 53% of sarcoma patients completing a consult, compared to 31% of those with cns tumors, 41% of those with embryonal tumor, 44% of those with leukemia/lymphoma and none of the patients with lch. our findings show that many children, adolescents, and young adults newly diagnosed with cancer are still not receiving fertility counseling despite: 1) an expanding body of literature supporting the need to provide this counseling, 2) guidelines published by several organizations recommending discussions about infertility risk and fp options, and 3) presence of a multidisciplinary fertility team. specific strategies need to be developed to improve access for younger children, and for disease groups in whom fertility consults are underutilized, such as youth with cns tumors, embryonal tumors, and leukemia/lymphoma. background: socioeconomic status (ses) has on impact on overall survival in the pediatric oncology population. unfortunately, data are insufficiently detailed to explain the mechanism behind this phenomenon. how parents handle the health management demands placed on them at the time of a child's cancer diagnosis may represent a point of differentiation in health outcomes. objectives: determine the association between socioeconomic factors, cancer literacy, and parents' understanding of home emergency management and their responses to instances of pain, nausea, and fever. in a prospective observational study of parents whose children were newly diagnosed with cancer, we obtained demographic information and, using a validated instrument, (dumenci, 2014) we evaluated cancer literacy. we tested understanding of the education parents received about home emergency management with a 6-item multiple-choice vignette-based questionnaire focused on actions needed in home scenarios. we then followed parents' actual behavior through periodic phone calls assessing instances of nausea, pain, and fever and their responses to these episodes. results: preliminary analysis of 24 participants showed an average score of 4 on the 6-item parental understanding questionnaire (range 0-6). variables associated with increased score were college-level education by 1.2 points (95% ci [.3 to 2.1]), private insurance by 0.9 points [.13 to 1.7] and adequate cancer literacy by 1.2 points [.2 to 2.2]. actual behavior reported by families indicated that married parents and those with income above $75,000 were less likely to treat instances of pain by 51% (95% ci [28 to 72]) and 43% [6.6 to 79], respectively. white parents, those with college-level education, and those with adequate cancer literacy were less likely to treat instances of nausea by 31% [6 to 57], 29% [5 to 53] and 29% [5 to 53], respectively. no associations were found between socioeconomic markers and parental responses to instances of fever. our findings suggest an association between demographic and socioeconomic markers and improved parental understanding of home emergency management. paradoxically, the same markers show a decrease in treatment response to pain and nausea. larger prospective studies are needed to link this behavior pattern to health outcomes, and help inform the extent of ses impact on home emergency management. emory university/children's heathcare of atlanta, atlanta, georgia, united states background: cardiovascular disease is a leading cause of morbidity and mortality in childhood cancer survivors (ccs). previous research showed wide practice variation in referral patterns to cardiology from the survivor clinic and in recommendations from cardiologists about the need for further testing or exercise restrictions. to develop a cardio-oncology algorithm in order to standardize referrals to cardiology and provide guidelines for cardiologists evaluating pediatric ccs. design/method: survivorship and cardiology experts developed a weighted scoring system for pediatric ccs who received cardiotoxic therapy based on time since treatment and risk factors identified by the children's oncology group (cog) and american heart association (aha). the cardiooncology algorithm assigned a score of 1-21. the score range was categorized to guide cardiology referral: screening echo only (1-3), consider cardiology referral (4-6), recommend cardiology referral (7-9), and regular cardiology follow-up (≥10). the algorithm also provides recommendations to cardiologists for screening and exercise modifications based on the score. after establishment of the algorithm, a convenience sample of institutional survivor clinic patient charts were retrospectively reviewed from the first month of each quarter from april 2015-march 2016 to validate the algorithm, evaluate referral patterns to cardiology, and assess cardiology recommendations. the retrospective chart review evaluated 243 patients (51% male; 61% non-hispanic white; 46% leukemia survivors; median age at diagnosis 4.0 years [range 0-19.7]; median time off-therapy 7.2 years [range 2.3-18.2]). 230 patients (95%) received anthracyclines (median dose 154mg/m2, range 30-512) and 73 (30%) received cardiac radiation. assigned cardio-oncology scores resulted in: 35% echo only, 47% consider cardiology referral, 16% recommend cardiology referral, and 3% regular cardiology followup. when evaluating detection rates of late effects by cardiooncology score, 12 survivors (5%) had an abnormal echo: 1/83 echo only, 4/115 consider referral, 4/38 recommend referral, and 3/6 regular cardiology follow-up. assessing referral patterns prior to initiation of the algorithm revealed forty-two survivors (17%) referred to cardiology: 8/83 echo only, 16/115 consider referral, 14/38 recommend referral, and 4/6 regular cardiology follow-up. of the 37 patients seen by a cardiologist at our institution, 4 had further diagnostic testing ordered (i.e., stress test) and 7 received exercise restrictions. a cardio-oncology algorithm and guidelines will standardize cardiac care for survivors by assigning a score to guide referral and cardiology practice after referral. prospective clinical use has begun and review will occur in one year to determine changes in detection rates of cardiac late effects, referrals, and recommendations from cardiologists. oregon health and science university, portland, oregon, united states background: delirium affects 10-30% of patients (pts) in pediatric intensive care units (picu) and is associated with increased length of stay, decreased attention in school, and post-traumatic stress disorder. the diagnostic and statistical manual of mental disorders (dsm v) defines delirium as a "disturbance of consciousness […] with reduced ability to focus, sustain or shift attention" due to an underlying medical condition. despite the medical complexity of the hospitalized pho population, there are no published prospective studies looking at delirium in these pts. hypothesizing that delirium is under recognized in the pho population, we designed a year-long prospective study using a validated screening tool to determine the frequency of delirium in hospitalized pho pts and to identify associated clinical factors. design/method: baseline frequency of pts with symptoms suggestive of delirium was determined through retrospective chart review using a data mining program of electronic medical records (emr). for the prospective study, pho and picu nurses were trained to use the cornell assessment for pediatric delirium and to record scores within the emr on all pho pts once every 12-hour shift. predetermined demographic and clinical variables were entered daily into a red-cap database on all hospitalized pho pts. results: baseline frequency of delirium, without active screening, was determined to be 4.5% of hospitalized pho pts. in the first 6 months of the prospective study, 405 consecutive admissions occurred among 152 unique pho pts: 347 oncology, 30 hematology, and 23 stem cell transplant pts. 25 pts had at least 1 positive delirium screen, for a prevalence per admission of 6.2%. statistically significant variables associated with delirium, at p < 0.0002 by univariate logistical regression, included prolonged length of stay, pt location (picu vs pho unit), and fever. adjusting for length of stay, administration of benzodiazepines and opiates were also significantly associated with delirium, p = 0.048 and 0.044, respectively. on average, nurses completed delirium screening in 70% of each pts' 12-hour shifts. study accrual ends in jan 2018 and final data analyses will be reported in the abstract presentation. conclusion: delirium does occur in the pho hospitalized population and screening by trained nursing staff is feasible. pts at highest risk appear to be pts with prolonged hospital stays, picu admissions, or frequent use of benzodiazepines/opioids. routine screening should improve our recognition of delirium and allow us to promptly intervene, or prevent delirium in an effort to avoid potential acute and long term consequences. background: with high survival rates for children and adolescents with hodgkin lymphoma (hl), treatment regimens are now designed to maximize cure while decreasing risk of long-term health outcomes associated with chemotherapy and radiation therapy. within contemporary treatment regimens, the comparison of toxicities experienced by patients receiving chemotherapy plus radiotherapy (crt) versus only chemotherapy (co) has not been studied extensively. objectives: this study examines select self-reported adverse health outcomes in survivors of contemporarily-treated pediatric hl to better understand the balance between efficacy and toxicity associated with chemotherapy and radiation therapy. (cog) ahod0031 that evaluated a response-based treatment paradigm in pediatric hl. patient who received initial chemotherapy were randomized based on early response to continued chemotherapy, chemotherapy plus radiotherapy or augmented chemotherapy plus radiotherapy. patients completed self-report questionnaires on health problems at 0, 1, 3, and 5 years following therapy. we examined selected patient-reported pulmonary, gastrointestinal (gi), cardiac and endocrine outcomes. kaplan-meier survival curves were used to determine probability of survival without the selected adverse health outcome. log-rank tests were used to compare the co versus the crt group. results: a total of 1,051 enrolled patients, 251 patients in the co group and 800 patients in the crt group, completed 2,134 questionnaires at a median of 1.3 years after s177 of s301 completion of therapy (q1, q3: 0.6, 3.3) which were analyzed. the cumulative 10-year incidence of endocrine dysfunction was significantly greater in the crt group versus those in the co group (17% versus 6%; p<0.001), driven by the incidence of hypothyroidism (11% versus 2%; p<0.001). there were no significant differences in cardiac (7% versus 9%; p = 0.226), pulmonary (11% versus 10% p = 0.068), and gastrointestinal dysfunction (20% versus 17%; p = 0.317) between the co and crt patients. conclusion: this study demonstrates low cumulative incidence overall of organ dysfunction early post completion of contemporary therapy for hl. the addition of radiation therapy significantly increased risk for hypothyroidism, but with no higher risk noted for cardiac, pulmonary or gi dysfunction. limitations include self-report status, potential selection bias, and relatively short latency period following end of therapy. longer follow-up is needed to determine more delayed risks for organ dysfunction in order to best define the balance between therapeutic efficacy and long-term adverse health outcomes related to chemotherapy and/or radiation therapy. background: identification of an organism via bronchoalveolar lavage (bal) or respiratory tract biopsy (rtb) has historically been considered the gold standard for diagnosis of invasive fungal infection (ifi); however, data previously published by our group showed that these procedures infrequently lead to a change in management in children with an oncological diagnosis or undergoing hematopoietic stem cell transplant (hsct). there is also a paucity of data on the cost of ifi in this population. to compare the costs of work-up and management of pulmonary ifi diagnosed based on ct scan alone versus ct scan or chest x-ray prompting a bal or rtb. design/method: we collected cost data on patients at ann & robert h. lurie children's hospital of chicago undergoing chemotherapy or within 6 months of hsct who were suspected of having an ifi between 2007 and 2012. in order to include sufficient time to account for post-procedure compli-cations but avoid including costs unrelated to ifi, data were included for 14 days from the day of their diagnostic scan or procedure. cost data was available for 76 of the 101 patients previously studied. thirty-six of these patients were diagnosed with suspected ifi based on ct only and 40 patients underwent bal or rtb. when evaluating specific costs, inpatient beds costs were higher in the bal and rtb group (median $1,555 versus $1,255, p = 0.01), yet there was only a trend towards higher costs for antifungal agents (median $1,635 versus $1,089, p = 0.26) and respiratory support (median $257 versus $0, p = 0.14). many of the initial ct scans were not captured in the 14-day evaluation period for the bal or rtb group based on the study design; however, even when accounting for ct scans up to a week prior these procedures, the total cost of ct scans was higher in the ct only group (median $963 versus $635, p = 0.0002), as they had more scans. despite this, total costs were significantly higher for patients who underwent bal or rtb versus ct scan only (median $14,087 versus $5,900, p <0.0001). combined with our previous data that bal and rtb infrequently leads to a change in management in children with an oncological diagnosis or undergoing hsct suspected to have an ifi, the significantly higher costs associated with these procedures makes these invasive diagnostic techniques even less desirable. batra, pediatr blood cancer, 2015. background: while infants >12 months of age with acute lymphoblastic leukemia (all) have a poor prognosis, infants with acute myeloid leukemia (aml) fare better despite more intensive therapy. there are limited data on this difference, particularly differences in supportive care requirements during induction therapy for infants. objectives: to compare induction mortality and resource utilization in infants relative to non-infants aged <10 years, separately for all and aml. design/method: we used previously established cohorts of children treated for new onset all or aml at children's hospitals in the us contributing to the pediatric health information system. patients with down syndrome were excluded. follow-up started on the first day of induction chemotherapy and continued until the earliest of: 35 days after commencement of chemotherapy, start of the subsequent course, or death. high acuity of presentation, defined as icu requirements involving 2 or more organ systems within the first 72 hours following initial admission were compared using log binomial regression. 35-day inpatient mortality was compared using cox regression. resource utilization rates (days of use per 100 inpatient days) were compared using poisson regression. results: a total of 10359 all (405 infants, 9954 non-infants) and 871 aml (189 infants, 682 non-infants) were included in the analyses. infants were more likely to present with high acuity compared to non-infants for both all (12% and 1%, rr = 12.2, 95% ci:8.6, 17.5; p<0.0001) and aml (6% vs 3%; rr = 2.0, 95% ci: 0.96, 4.3; p = 0.06). infants with all had higher inpatient mortality compared to non-infants even after accounting for differences in acuity of presentation (2.7% vs 0.5%, adjusted hr = 2.7 95% ci: 1.2, 6.1; p = 0.015). in contrast, inpatient mortality was more similar for infants and noninfants with aml (3.2% vs 2.1%, adjusted hr = 1.2 95% ci: 0.3, 3.9; p = 0.73) and comparable to rates among infants with all. infants with all and aml had higher rates of utilization of fresh frozen plasma, cryoprecipitate, diuretics, supplemental oxygen, and ventilation relative to non-infants. infants with all also had higher rates of total parenteral nutrition, ecmo, and patient controlled analgesics compared to noninfants. infants with all experienced significantly higher induction mortality compared to noninfants, a difference not entirely explained by acuity at presentation. differences in ru among infants may reflect higher presentation acuity and greater treatment related toxicity. further work is needed to elucidate the contribution of treatment related toxicity to early mortality in infants with all. background: fever in a child with cancer is a medical emergency due to the significant risk of a serious bacterial infection. many attempts have been made to risk stratify these patients. the respiratory pathogen panel (rpp) is a panel of polymerase chain reaction tests that identify seventeen common respiratory viruses and three bacterial infections. samples are taken via nasopharyngeal swab. rpps are frequently sent, but we do not have data to determine whether a positive result can lead to stratification to a lower risk of bacterial infection. (1) to determine the epidemiology of respiratory virus-associated fever in pediatric oncology patients (2) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all pediatric oncology patients seen in our emergency department (ed) with fever from the introduction of the rpp in april 2014 to september 30, 2017. we reviewed the results of blood cultures, rpp, chest radiographs, and discharge summaries to identify sources of infection. we also identified the patients' cancer diagnosis, age, absolute neutrophil count (anc), and absolute lymphocyte count (alc). results: 107 positive rpps were found among pediatric oncology patients who presented to the ed with fever. the most common positive rpp findings were rhinovirus/enterovirus (rev) (45%), parainfluenza (14%), influenza (11%), coronavirus (11%), and polyviral (10%). among patients with a positive rpp, 4% had bacteremia compared to 12% bacteremia among all pediatric oncology patients with fever (or 0.42 [0.18-0.99], p 0.048). all cases of bacteremia were associated with rev. there was no bacteremia identified in patients with rpps positive for other viruses (or 0.0596 [0.0037-0.9715], p 0.048). rev positivity did not confer a lower risk of bacteremia than rpp negative patients ], p 0.97). anc (p = 0.87) and alc (p = 0.89) less than 500, and number of patients with severe neutropenia (p = 0.27) were not statistically different between the rev and non-rev positive rpp groups. rpps positive for viruses other than rev reduced the likelihood of bacteremia in febrile pediatric oncology patients in the ed setting. patients with bacteremia may have concurrent infection with rev. a larger study is warranted to determine if positive rpp results can inform clinical management of a child with febrile neutropenia. emily mueller, anneli cochrane, seethal jacob, aaron carroll s179 of s301 background: the usage of mobile health (mhealth), which refers to the application of mobile or wireless communication technologies to health and healthcare, has grown exponentially in recent years. mhealth tools have been used by caregivers of other vulnerable populations, but little has been focused on caregivers of children with cancer. objectives: to conduct a survey to understand the mobile technology usage, barriers, and desired mhealth tools by caregivers of children with cancer. we conducted a mailed cross-sectional paper survey of caregivers of all children who were diagnosed with cancer at riley hospital for children between june, 2015 and june, 2017. the survey contained 13 questions, both fixed and open-ended, in both english and spanish. up to three rounds of surveys were sent to those who did not respond. of the 121 respondents, they were primarily parents (93.2%), median age was 40.7 years (range 23-63), and most were white (78.5%) and non-hispanic/latino (87.1%). the top three annual household income brackets included $50,000 to $74,999 (21.2%), $25,000 to $49,999 (20.3%) and under $25,000 (17.8%). the majority had an education: 35.6% college graduates, 22% graduate degree, and 18.6% high school education or ged. nearly all respondents owned a smart phone (99.2%) and 61.2% owned a tablet. the majority used an ios operating system (62.8%), while 49.6% reported use of a device with an android operating system. all caregivers reported use of at least one mobile website/app regularly for their personal use. while 35.5% of respondents reported no barriers to mobile technology use, the top barrier selected was "data limitations" (21.5%). overall, 84.5% wanted at least one medical managementrelated website/app: medical knowledge (58.7%), healthcare symptom tracking/management (47.1%), and medication reminders (43%). healthcare system-related desires were high, as 59.5% wanted access to their child's medical record and 56.2% wanted a website/app to facilitate better communication with medical providers. there were no significant associations between socioeconomic status (income or education) with barriers or types of websites/apps desired by caregivers. since the vast majority of caregivers use mobile technology with minimal barriers, future research should focus on designing an mhealth tool to address the medical management needs by caregivers of children with cancer. by supporting caregivers through this type of mhealth tool, it could positively impact patient clinical outcomes through greater adherence to medications and treatment protocols. background: in children with fever and neutropenia, early initiation of targeted antibiotic therapy improves outcomes, yet there are no standards for choice of empiric antibiotics. in 2013 our institution implemented an early empiric ceftriaxone (eec) protocol to reduce time to antibiotic administration in febrile hematology-oncology patients who are potentially neutropenic when the absolute neutrophil count is not yet know. ceftriaxone is given immediately after obtaining blood for culture and lab studies. in patients found to be neutropenic, ceftriaxone is discontinued and cefepime is initiated. the purpose of this retrospective study was to evaluate our eec protocol in neutropenic patients by assessing ceftriaxone sensitivity of positive blood cultures and comparing rates of adverse outcomes with a cohort of patients treated prior to implementation of the protocol. we are now conducting a prospective study to more thoroughly investigate antibiotic sensitivities of organisms isolated from blood cultures of neutropenic patients. design/method: hematology-oncology patients with at least one positive blood culture between january 2011 and december 2013 were identified. patient demographics, neutrophil count, antibiotic treatment, isolated organisms and sensitivities, and adverse outcomes (increased respiratory support, hypotension requiring intervention, and icu admission) were obtained by retrospective chart review. fisher exact test was used to compare dichotomous variables between patient groups. we are now prospectively identifying febrile neutropenic patients with positive blood cultures and performing antibiotic sensitivity testing to several antibiotics commonly used as empiric therapy for febrile neutropenia. results: retrospectively, we identified 58 neutropenic patients with a total of 127 bacterial isolates from blood cultures. of organisms isolated, 47 were tested for sensitivity to ceftriaxone and 23 (49%) were not sensitive, 6/18 (33%) of gram-positive cultures and 18/29 (62%) of gram-negative cultures. ten of 16 (63%) eec patients had an adverse outcome versus 13/26 (50 %) of non-eec patients (p = 0.277). notably, 31% of eec patients required icu admission versus 4% of non-eec patients (p = 0.049). thus far our data obtained prospectively is revealing similar rates of ceftriaxone resistance with 9/19 cultures not sensitive to ceftriaxone (47%, ci 24.9%-71.1%). in our retrospective study, no statistically significant difference was seen in overall adverse outcome rate between the two cohorts, though icu admission rates were significantly higher in eec patients. ceftriaxone resistance rates were high in tested isolates, which is further supported by preliminary data from our ongoing prospective study. given these data, eec may not be effective at improving outcomes in febrile neutropenic pediatric hematology-oncology patients. background: approximately 1 in 5 children diagnosed with cancer will die of their disease, despite advances in treatment. results: two focus groups of six parents each met in june 2017. the parents were predominantly female (11 female, 1 male) and had lost their children an average of 2.8 years prior (range 1-5.3 years). two parents were in the same family. nearly all patients were offered palliative care (10/11), all were offered hospice and most died at home (9 at home, 2 in the icu). parent discussion uncovered six broad themes: beneficial provider qualities, optimal communication, helpful systematic supports, struggles to feel like a good parent, struggles with a loss of control and unmet needs. parents appreciated providers who were consistent, reliable and honest. parents desired communication that was sensitive to the needs of the patient and family with a balance of hope and realism. parents appreciated the tangible supports pro-vided by social work and the emotional support of child life both for the patient and their siblings. some parents struggled to define and advocate for their child's quality of life, especially when it led to disagreeing with the medical team. several parents expressed frustration with unfamiliar caregivers in the hospital, especially trainees. they expressed a strong desire for more anticipatory guidance about the end of life including how to discuss it with their children. they also wished for a cancer-specific support group for bereaved parents. conclusion: bereaved parents of pediatric oncology patients in our focus groups appreciated consistent, reliable providers who communicated with a balance of realism and hope. they appreciated the tangible and emotional support they received and wanted more anticipatory guidance at the end of their child's life. these results can help guide clinical care, especially in communities without strong palliative care support. further research is needed to develop interventions to improve end of life care. background: clinical trials involving human subjects depend on informed consent (ic) to ensure ethical protections for participants. parents of children with cancer often lack full understanding of the basic elements of ic for clinical trials. additionally, the stress of their child's cancer diagnosis may affect their decision-making capabilities. this is especially problematic as these children rely on parents to fully comprehend clinical trials and weigh their benefits and risks. physician communication is critical for effective family-centered care. the acgme mandates that training programs teach and assess trainees' communication skills. however, there are currently no published curricula aimed at training pediatric hematology/oncology fellows to deliver ic effectively for cancer clinical trials. to develop and pilot-test a simulation-based curriculum to enhance communication skills of pediatric s181 of s301 hematology/oncology fellows in the delivery of ic for cancer clinical trials. we developed, tested, and implemented the curriculum from 2016 to 2017 in two phases. in phase-1, we reviewed literature on simulation-based curricula and completed a needs assessment to create a clinical scenario and full curriculum using standardized patients. using miller's pyramid model, fellows' assessments included: immediate de-brief, surveys to assess pre/post confidence and knowledge of the basic ic elements ("knows" and "knows how"), and 360-degree summative assessments compiled from fellow self-assessments, faculty, and standardized patients ("shows how"). after initial testing and refinements done with 1 fellow, in phase-2, we implemented the curriculum with our 8 fellows. likert scale (1 strongly disagree-5 strongly agree) and basic p values are reported. results: fellows gave high mean ratings for training relevance (4.7) and standardized patients' preparedness (5). almost all (4.9) reported they have used the knowledge gained in their clinical practice. increase in self-reported confidence (pre/post) was noted in all domains: general -describing possible benefits of the clinical trial 3.5/5 vs.4.1/5 (p = 0.025), risks and potential side effects 3.5/5 vs.4.3/5 (p = 0.004), and explaining alternatives 3.1/5 vs.3.6/5 (p = 0.016); research -discussing purpose of the clinical trial 3.1/5 vs.3.7/5 (p = 0.006), and randomization 3.3/5 vs.4.0/5 (p = 0.046); and family-centered -addressing emotions during ic 3.6/5 vs.4.5/5 (p = 0.004), and delivering bad news 3.1/5 vs.3.6/5 (p = 0.016). summative evaluation mean ratings for all fellows were 4.5 (range 4.1-4.9). our novel simulated-based ic curriculum, significantly increased fellows' self-reported confidence and skills during ic delivery. importantly, our ic curriculum addressed not just research-related content but also management of parental emotional needs during the ic discussion. next phase includes kirkpatrick model program evaluation and dissemination across other training programs in our institution. national kaohsiung normal university, kaohsiung, taiwan, province of china background: taiwan's childhood cancer foundation reported in 2016 that the 5-year survival rate of childhood cancer was 75%. as a result, many childhood cancer survivors were back in school after treatment. however, childhood cancer survivors' educational outcomes suffered because of their long-term absence from school and late effects of cancer and cancer treatment. a few school reentry protocols have been developed by the nursing professionals in taiwan to facilitate students' return to school but remained experimental in nature and hardly accessible. parents, students, and teachers were left to their own devices to make individual school reentry plans. objectives: this study aimed to examine and uncover the commonalities among three middle school students' successful school reentry experiences from their teachers' perspectives and to analyze the factors contributing to their success. design/method: this is a qualitative interview study. indepth semi-structured interviews were conducted with three middle school teachers in december 2017 about their perceptions, observations, and experiences working with adolescent childhood cancer survivors. the students were two boys with leukemia and one girl with bone cancer. they were diagnosed in the first year of middle school when they were 12-13 years old and returned to school for the third and the final year. these students met the following criteria for successful school reentry: regular school attendance, average/above average academic performance, friendship maintenance, and high school diploma. the theme -bring the class to the hospital was found to be the key to the adolescents' successful return to school. without a prescribed school reentry protocol and in the face of limited bedside education services, the homeroom teachers, as links between school, home, and hospital, brought the class to their hospitalized students. they doubled as bedside teachers conducting lessons at the hospital or students' homes, became friends with the parents, witnessed firsthand the students' pain and triumph during treatment, brought the students back to school for visits and celebrations, delivered the classmates' wishes and news to the students, encouraged and welcomed classmates' visits to the hospital, and, together with parents and other teachers, developed flexible school reentry schedules for the students. this on-going study demonstrated the critical roles and functions of homeroom teachers in successfully bringing the students back to school during and/or after cancer treatment. further analysis will be focused on how and why these three homeroom teachers were able to carry out this unexpected task on top of their already full workload. jennifer kesselheim, shicheng weng, victoria allen, collaborative group fellowship program directors dana-farber/boston children's cancer and blood disorders center, boston, massachusetts, united states background: a novel, 4-module, case-based curriculum entitled "humanism and professionalism for pediatric hematology-oncology" (hp-pho) aims to foster pho fellows' reflection on grief and loss, competing demands of fellowship, difficult relationships with patients and families, and physician well-being and burnout. in small group facilitated sessions, fellows work to identify coping strategies and explore how the challenges of fellowship influence both their own doctoring and the patient experience. objectives: to administer the hp-pho curriculum in a prospective, cluster-randomized trial, measuring whether exposure to this educational intervention, compared to standard conditions, fosters humanism and professionalism and improves satisfaction with training. design/method: pho fellowship programs (n = 20) were cluster-randomized to deliver usual training in humanism and professionalism (control) or the novel curriculum (intervention) during the 2016-2017 academic year. the primary outcome measure was the pediatric hematology-oncology self-assessment in humanism (phosah). secondary measures included a 5-point satisfaction scale, the maslach burnout inventory (mbi), the patient-provider orientation scale, and the empowerment at work scale. participating fellows were pre-tested in summer 2016 and post-tested in spring 2017. a change score was calculated for each study instrument. we compared each outcome between arms using mixed effect models adjusted for pre-test score as a fixed effect and site as a random effect. results: randomization yielded 59 intervention and 41 control fellows. the two arms did not significantly differ in distribution of fellow age, gender, or post-graduate year. the 9 intervention sites successfully administered 33 of 36 (92%) modules. change scores on the phosah were not significantly different between the control and intervention arms (adjusted mean difference = 0.5; 95% confidence interval [ci] -1.0, 2.0; p = 0.5). compared to the control arm, fellows' exposed to the curriculum gave significantly higher ratings on several items within the satisfaction scale including satisfaction with their training on "physician burnout" (adjusted mean difference = 0.8; 95% ci 0.4, 1.2; p<0.001), "physician depression" (adjusted mean difference = 0.9; 95% ci 0.4, 1.4; p<0.001), "balancing professional duties and personal life" (adjusted mean difference = 0.7; 95% ci 0.3, 1.1; p = 0.002), and "humanism overall" (adjusted mean difference = 0.4; 95% ci 0.03, 0.9; p = 0.03). change scores on other secondary measures were not significantly different between study arms. conclusion: exposure to the hp-pho curriculum did not alter fellows' self-assessed humanism and professionalism. however, the curriculum proved feasible to administer and intervention fellows expressed higher levels of satisfaction in their humanism training, indicating the curriculum's positive impact both for fellows and their learning environment. background: recent work has documented significant levels of unmet needs among adolescents and young adults with cancer, particularly psychosocial challenges during the transition to adulthood, (e.g., abrupt disruption to school and social life, and social isolation). given that adolescents and young adults drive mobile app use, a mobile-phone may be an ideal way to deliver a psychosocial intervention to adolescents and young adults with cancer. to use a patient-centered approach to inform a mobile-based mindfulness and social support intervention for adolescent and young adult patients with cancer. design/method: participants were ten aya with sarcoma (50% female; 50% adolescents); parents of the five adolescents, and six healthcare providers (n = 21). formative research involved three steps: (1) in-depth interviews were conducted with ten aya with sarcoma; parents of the five adolescents, and six healthcare providers (n = 21). (2) adaptations were made to an existing mindfulness app which offers a program for youth. modifications included creating a 4-week "mindfulness for resilience in illness" program, with 28 relaxation exercises, and the addition of videos featuring two sarcoma survivors as program hosts. content was informed by the mindfulness curriculum for adolescents, learning to breathe. (3) a private facebook usability group was organized to (i) elicit beliefs about the mindfulness app and potential future enhancements, and (ii) promote social support. results of the in-depth interviews revealed themes around adolescents' functioning and coping, including body image concerns; recurrence-related anxiety; anger over loss; and being overwhelmed by medical information. themes from the interviews were incorporated into a demonstration version of the mobile app. a patient-centered approach is widely recommended in the development of mobile-based health behavior change interventions and may be a useful way to inform development of a mobile-based mindfulness and social support intervention for adolescents and young adults with cancer. background: medical trainees consistently report suboptimal instruction and poor self-confidence in communication skills. despite these deficits, few training programs provide comprehensive pediatric-specific communication education, particularly in the provision of "bad news." an in-depth survey to examine the historical experience and communication needs of pediatric fellows was conducted at a large academic pediatric center as the first step towards the development of a comprehensive communication curriculum. to determine the previous educational and clinical experiences of pediatric subspecialty fellows, assess their levels of comfort in the context of various communication topics, and query potential modalities and topics for future communication training. design/method: the needs assessment survey was developed using previously developed and validated questions and review of the literature. the survey was reviewed by internal and external pediatric oncology and palliative experts and pre-tested with a subset of trainees to enhance content validity. results: thirty-two out of a total of 38 fellows completed the survey (84% completion rate), of which 81% were pediatric hematology-oncology or subspecialty fellows. most fellows had participated in previous teaching sessions (97%), including those involving role play or simulation (75%). however, few fellows had received feedback from senior clinicians on their communication skills (31% of fellows had received feedback ≤ 3times). on a scale of 1-x, with 1 indicating "not well prepared," the mean score for 12 of 23 communication items was <3. fellows felt least prepared to lead discussions around informed consent for experimental therapies, end of life care, and autopsy. fellows indicated that didactic educational sessions and additional coursework were less useful strategies for improving their communication skills, whereas small group role play sessions with faculty and/or bereaved parent educators were most useful. fellows' overall communication preparedness score was not correlated with post-graduate year but was positively associated with the number of times they previously had delivered bad news to patients and families. fellows requested additional training on many topics, with greatest interest in learning skills to optimize communication with an angry patient or family. additional topic requests included placing limitations on resuscitation, withdrawing/withholding further therapy, and ageappropriate inclusion of patients in difficult discussions. despite self-report of prior communication skills training, pediatric subspecialty fellows felt underprepared to participate in difficult discussions with patients and families. learners identified role-playing and coaching with real-time feedback from other physicians and bereaved parents as more useful training strategies as compared to didactic sessions. background: when children die of cancer, parents must adjust to their child's absence amidst the lingering turmoil of what preceded their death: witnessing their child undergo painful treatments, making difficult decisions, and anticipating a devastating loss, all the while hoping for a recovery. adjustment to a child's death, as depicted by current bereavement literature, necessitates making meaning of one's loss. professional care staff can help parents make sense of their child's illness, and in turn, of their own parental experience during treatment. however, the extent to which relationships with professional care team members influence parents' ability to make sense of, and successfully cope with, their loss has not been examined. objectives: to examine how bereaved parents' interactions with their deceased child's pediatric oncology professional care team have impacted their grief symptoms design/method: to better understand how interactions with professional care staff relate to parents' grief outcomes, we conducted a mixed-methods study examining staff impact on parental grief. thirty participants whose children died of cancer one to three years ago completed an in-depth interview and psychometrically validated surveys measuring meaningmaking, depression, and grief symptoms. results: correlational analyses of the measures found that an increase in meaning making was associated with lower depressive and grief symptoms. a content analysis of the interviews found that many participants regarded staff "like family," had on-going relationships with staff after their child died, and described various ways staff interactions during treatment and after the child's death helped them make sense of their loss. in particular, participants described how interactions with staff have helped them find benefits in their loss and learn to create a new relationship with their child despite their physical absence. quantifying the interview data and statistically analyzing it along with the measures found that participants' increased frequency of describing staff's positive impact on their grief correlated with higher meaning-making scores and lower grief symptom scores. our study found that bereaved parents who lost their children to cancer were articulate in sharing their experiences of staff engagement and communication during treatment, offering numerous examples of how staff aided them in making meaning of their loss that were reliably associated with their subsequent grief. we hope the results of this mixed methods research encourage further study of the importance of staff interaction with families during the critical period of their children's care, and the lasting impact this can have regardless of the treatment outcome. memorial sloan kettering cancer center, new york, new york, united states background: although resiliency has been recognized as necessary for healthcare professionals, trainees feel unprepared for the emotional challenges inherent in caring for sick and dying patients. compounded by long hours, challenging work environments, and lack of formal training on handling emotionally difficult situations, many institutions are recognizing the need for interventions to reduce trainee distress. the goals of this fellow-led quality improvement initiative were: 1) to determine whether there is a need for emotional support amongst pediatric hematology and oncology fellows, 2) to provide formal resiliency and debriefing sessions, and 3) to measure feasibility, acceptability and effectiveness of implemented curriculum. design/method: an anonymous survey to determine need for resiliency and debriefing sessions following a traumatic event was distributed to 24 active pediatric hematology & oncology fellows at memorial sloan kettering cancer center in january 2017. once need was established, an intervention consisting of a formal curriculum was developed and initiated in june 2017, involving: 1) scheduled and ad hoc debriefing sessions in response to traumatic events (including patient death, codes, interpersonal conflicts, end-of-life care); led by a psychiatrist and social worker with fellows and a pediatric oncologist mentor in attendance, and 2) a resiliency didactic curriculum, led by a palliative medicine specialist, focused on skills such as contesting cognitive distortions and mindfulness. the effectiveness of these sessions will be measured using follow-up anonymous surveys at 6 months (currently underway) and 12 months post-initiation of intervention. the initial survey demonstrated most trainees (19/24) were present at 3 or more deaths during their training, while less than half of respondents had attended a post-event debriefing session. 85% of respondents felt there was not sufficient emotional support from the institution for physicians caring for dying patients. a separate pre-intervention survey found all respondents (14/14) expressed a need for regular debriefings, and nearly all anticipated that they would benefit from such debriefings. concerns identified by trainees that would preclude participation in the curriculum included preference to deal with emotional situations privately and time constraints. trainees identified a need for formal debriefings and resiliency skill development. the program was easily implemented, and is both feasible and acceptable with good attendance. feedback received at the 6-month mark will determine deficits and possible improvements to the curriculum. the 12-month survey will measure effectiveness of the program and whether it should be continued. background: acute kidney injury (aki) is a common but under-recognized complication among patients with leukemia. it is associated with prolonged hospital stays, increased mortality, progression to chronic kidney disease, and delays or changes in cancer therapy which may affect a patient's prognosis. however, data on aki in pediatric patients with cancer is still lacking overall. we investigated the incidence of aki in patients who were newly diagnosed with all at our center from january 2009 to september 2017. we performed a retrospective chart review of all patients who were newly diagnosed with all from neonate to 18 years in our facility. we determined the incidence of aki in our population using the kidney disease: improving global outcomes (kdigo) diagnostic criteria. we also assessed for nephrotoxic exposures, nci all risk stratification and risk of aki, and tumor lysis syndrome (tls). we identified 62 patients diagnosed during the study period who met inclusion criteria. median follow-up time was 29.5 months (range 3.6-60.3). the cohort was predominantly male (54.8%) and hispanic (93.5%). our analysis showed 51.6% had aki by kdigo criteria (29% grade 1, 14.5% grade 2, and 8% grade 3), 62.5% had aki on presentation, and 75% had multiple aki episodes during the study period. older age and longer length of hospitalization were associated with aki (p = 0.019 and p = 0.009, respectively). there was no association between aki and nci all risk classification, contrast exposure, hyponatremia, elevated white blood cell count, uric acid levels, antimicrobial therapy, or diuretic use in this study. conclusion: aki was a common finding in our study population. the majority had grade 1 aki by kdigo criteria. however, aki was associated with older age and a longer length of stay. further study is needed to determine the short-and long-term impact of aki on pediatric patients with all. st. jude children's research hospital, memphis, tennessee, united states background: in some regions, the availability of trained pediatric oncologists is a limiting barrier for the care of children with cancer. in 2003, the unidad nacional de oncología pediátrica (unop) and the universidad francisco marroquín school of medicine in guatemala established a pediatric hematology/oncology fellowship program sponsored by st jude children's research hospital to provide central america and the caribbean with well-trained specialists. a systematic analysis of the impact of fellowship programs in pediatric oncology has never been done, especially in the context of a regional education program. objectives: this study sought to analyze the impact of the unop fellowship program based on the regional number of providers, pediatric cancer centers and patient volume. in addition, it sought to characterize the jobs and scientific output of the graduates. the impact will be evaluated in the context of a cost analysis. to define the volume of providers, pediatric cancer centers and patients, the directors of pediatric cancer centers in central america were sent an online survey to obtain these data. all the centers contacted maintain an updated hospital-based patient registry. in addition, the 22 graduates of the fellowship program were also sent an online survey, asking about their job at graduation, current role and scientific productivity. the cost analysis will include assessment of direct costs including salaries and stipends for away rotations, as well as the indirect costs of faculty time spent teaching. since the establishment of the unop fellowship program, the region has more providers for pediatric cancer (p<0.05) and centers treat a larger volume of patients (p<0.05). two new centers have opened with graduates of the program. all but one graduate practice pediatric oncology (21/22) and the majority do it in their country of origin (19/21). no graduate practices outside of this region. almost half of the graduates (44%) hold a leadership role at their institution. the majority of their time is spent in the public sector (>95%). the majority of graduates participate in clinical research (61%) and have participated in the creation or implementation of therapeutic protocols (67%). on average, the graduates have published 2 peer-reviewed articles since completion of training. the unop fellowship program has had a favorable impact on pediatric cancer care in the region, contributing to the capacity to treat a larger volume of patients. graduates practice pediatric oncology in the region in the public sector, frequently hold leadership roles and are scientifically productive. background: abandonment of treatment is a major cause of treatment failure and poor survival in children with cancer in low-and middle-income countries. the incidence of abandonment in peru has not been reported. objectives: the aim of this study was to examine the prevalence and associated factors of treatment abandonment in pediatric patients with cancer of peru. we retrospectively reviewed the sociodemographic and clinical data of children referred between january 2012 and december 2014 to the two main tertiary centers for childhood cancer, located in lima, peru. definition of treatment abandonment was used from the siop (international society of paediatric oncology) podc (paediatric oncology in developing countries) abandonment of treatment working group recommendation. results: data of 1135 children diagnosed with malignant solid tumors and lymphomas were analyzed, of which 209 (18.4%) abandoned treatment. univariate logistic regression analysis showed significant higher abandonment rates in children living outside the capital city, lima (p<0.001); prolonged travel time to a tertiary center (> 5 hours; or 2.75, p = 0.002); living in a rural setting (or 3.44; p<0.001) and lack of parental formal job (or 4.39; p = 0.001). according to cancer diagnosis, children with retinoblastoma were more likely to abandon compared with other solid tumors. in multivariate regression analyses, rural origin and lack of formal parental employment were independently predictive of abandonment. conclusion: treatment abandonment prevalence in our country is high and closely related to socio-demographical factors. treatment outcomes could be substantially improved by strategies that help prevent abandonment of therapy based on these results. st. jude children's research hospital, memphis, tennessee, united states background: to improve the quality of a pediatric hematology/oncology fellowship program, a systematic assessment must be performed that can evaluate its current state and identify areas of opportunity, as well as modifications over time. unfortunately, widely agreed-upon metrics of quality for pediatric hematology/oncology fellowship programs currently do not exist. this is particularly important in this field due to the global shortage of specialists. for this reason, an assessment instrument that is applicable throughout the world must be created. objectives: the st. jude global education program assessment tool (epat) is a novel instrument that seeks to evaluate pediatric hematology/oncology fellowship programs around the world in systematic and objective way. epat will help determine key performance indexes that are relevant for quality education in pediatric hematology/oncology fellowship programs and establish the framework for improvement. design/method: firstly, key domains to be evaluated for program assessment were identified a priori based on the continuum of pediatric hematology/oncology fellowship programs in the context of geography and educational structure. subsequently, questions were formulated to evaluate these key domains, seeking to assess elements involved in ensuring competence in clinical practice, academic productivity and regional impact. due to the novelty of this tool and the lack of defined metrics of quality, epat relies on expert opinion in a two-step process: internally in the department of global pediatric medicine at st. jude children's research hospital and, subsequently, from a panel of experts in global pediatric oncology and medical education from around the world. ten key domains were identified to evaluate all aspects relevant to training programs around the world, regardless of educational and geographic context. questions have been created to assess these domains and, to make epat quantitative, these have assigned weights with a value reflective of their relative importance. this grading system allows for a score in each key domain, permitting monitoring of changes over time. epat is currently at the stage of external expert review, and subsequently will be piloted in five fellowship programs around the world to provide different geographical and patient care contexts for its validation. once epat is finalized, it will be distributed to pediatric hematology/oncology fellowship programs around the world to be applied. epat proposes a novel strategy to assess training programs in a systematic way that includes all aspects relevant for a training program in a global context. this tool will help guide improvements in pediatric hematology/oncology fellowship programs and assure a well-trained workforce. background: with the improvement in pediatric oncology patient survival and outcomes in the past several decades, monitoring for recurrence and long-term effects of therapy has become even more important. the utilization of personalized treatment summaries and survivorship care plans (scps) is one way to communicate this information with patients and families. the american college of surgeons commission on cancer (coc) created a standard regarding provision of scps to 50% of eligible patients by december 31, 2017 as a metric for accreditation of all cancer centers. the standard applies to all patients with stage i, ii, and iii cancer diagnoses and requires creation of the scp within one year of diagnosis or six months of completing treatment. during implementation at our pediatric cancer center, we identified barriers to use of the guidelines in the childhood cancer setting. objectives: define eligibility for an scp for pediatric oncology patients to include all patients with curative intent and to deliver scps within six months of finishing therapy. design/method: using chart review and a cancer center registry query, we identified childhood cancer patients potentially eligible for an scp by collecting stage, goal of therapy, and dates of treatment. all patients with curative intent were deemed eligible for an scp regardless of stage i-iv. patients being followed in the oncology clinic for posttreatment surveillance and care were included even if they had received an scp in the survivorship program or were greater than six months off therapy at time of implementation. as expected in the pediatric oncology population, acute lymphoblastic leukemia (all) was the most common diagnosis comprising 31.5% of patients. all is stratified into risk groups instead of surgical staging categories, and treatment duration is greater than one year, unlike many adult-onset malignancies. these differences required interpretation of the guidelines to apply to our pediatric population for all and other pediatric diagnoses with non-surgically based staging. our pediatric oncology clinic has to date provided scps to 141 of 277 eligible patients by adapting the guidelines to focus on patients with curative intent to receive an scp by six months off therapy. cancer staging guidelines and goals for curative intent as well as lengths of treatment vary between the pediatric and adult populations. the coc guidelines require adaptation for optimal applicability to the pediatric oncology population. background: education in communication for fellows in fields that require difficult discussions with families are few in nature. adult learning pedagogies such as role play are under-utilized in medical education, and have been shown to be as effective as traditional teaching methods such as lecture. an 8-module course for fellows in hematology/oncology, hospice and palliative medicine, radiation oncology, and pediatric hematology/oncology was implemented in january/february 2017. 12 fellows participated in the program. topics covered including fundamentals of communication, coping and spirituality, delivery of bad news, communicating with families, sexual dysfunction during treatment, palliative care/death and dying, and burnout. objectives: overall goal of this course is to foster holistic physicians who views their patients as people with cancer, not cancer patients, and physicians that can communicate effectively with their patients throughout the disease continuum. by the end of the course, learners should be able to practice the fundamental principles of good communication. design/method: fellows initially participated in a pre-course osce to establish baseline skills. osce was facilitated by the center for learning and innovation at northwell, and included actors portraying a pediatric patient and family member to whom the fellow had to break bad news. two months later, the course was carried out over the span of eight weeks and included didactic sessions followed by 45 minutes of role play scenarios. five of the eight modules included role play, with faculty members serving as simulated patients. after the course, a second breaking bad news osce was held. both osces were filmed, and feedback was given by the on-site actors. additionally, faculty members were given access to the videos in an on-line format and were given an evaluation tool to assess the fellows' performance pre-and post-intervention. fellows were given subjective surveys pre-and post-course as well. results: subjective data from participants showed a noticeable increase in comfort level in all areas on the pre-and post-course survey. data obtained from osce videos showed improvement in communication skills as assessed by sps and faculty members using a new evaluation tool developed by faculty. initial first-run data shows that this course is successful in improving communication skills as well as increasing fellows' comfort level across several domains of communication. future directions for our course include improving and validating our assessment tool, expanding our topic base to include more aya and pediatric scenarios, faculty development for improved role play, and investigating impact on practice after course completion. background: acute lymphoblastic leukemia (all) is the most common form of childhood cancer with approximately 2900 children diagnosed each year. survival rates have improved significantly over the past several years. children with all are at risk for developing musculoskeletal complications during and after completion of treatment, which can contribute to impaired activity, elevated body mass index (bmi), and risk for complications. interventions involving physical activity could improve musculoskeletal strength as well as overall health in these children. the aims of this study are to examine the feasibility of a directed physical activity program for children with newly diagnosed all during the initial intensive phase of therapy and to evaluate the overall health and quality of life of children participating in the directed physical activity program. design/method: all subjects will receive education materials about the importance and safety of physical activity and a nutrition handout. all subjects will also participate in the directed physical activity program under the supervision of a trained physical therapist for at least 40 minutes every week for 12 weeks. the program will entail four stations including a cardiovascular, balance/proprioception, strength and flexibility, and coordination and cardio. feasibility will be assessed by tracking the participation rate throughout the study period. other assessments will be made at study entry, at the end of 12 weeks of physical activity initiative and 3 months after completion of the intervention. assessments include overall strength and flexibility, weight, height, bmi, blood pressure and performance scores. descriptive statistics will be used for this study. results: a total of 10 patients, 3 male and 7 female, enrolled in the study over a 9.5 month period. patient ages ranged from 5-16 years. half of the patients enrolled have completed the 12 week program and all 5 patients had stability or improvement of their physical functioning scores. further data collection and analysis is ongoing. patients in the early intensive phase of all therapy are at risk for complications that can affect their physical functioning. a directed physical activity protocol may improve their overall physical functioning. patients may not need specific physical therapy; however a directed physical activity program appears to be beneficial for these patients. the main roadblocks to successful completion of the program were difficulty with scheduling, strain on the parents and patient from treatment, unplanned admissions for fever, as well as nausea and fatigue at time of visit. albany medical center, albany, new york, united states background: communication skills are a core competency highlighted by the acgme. increasing resident confidence in delivering difficult news has been shown to lead to more s189 of s301 effective communication. currently, the majority of residency programs lack formal training in communication skills. our objective was to demonstrate feasibility and efficacy of integrating a standardized-patient based training program for communication skills into the curriculum of pediatric residents design/method: to date, 10 pediatric and 4 medicine/pediatric residents have participated in the program during the intern year. the program consists of three, two-hour long sessions, in which each resident is given several opportunities to act out case scenarios with a standardized patient. scenarios included informing a parent of their child's new cancer diagnosis and disclosure of a positive hiv test to a teenager. residents received post hoc peer to peer, and preceptor to learner feedback. pre and post-program surveys were completed by residents. results: following course completion residents reported an increase in confidence in multiple areas of communication including giving a difficult diagnosis (p<0.05), discussing a poor prognosis (p<0.02), responding to different patient/family member emotional responses i.e. crying or anger (p<0.01), and organizing vital information to be relayed (p<0.01). in conclusion, communication skills training of pediatric residents is feasible and provides a platform for developing valuable skills not taught elsewhere within the curriculum. background: for children with cancer, transitioning back to school during or after treatment can be challenging. literature supports the need for school re-entry programs to ease this transition. however, these programs vary widely among pediatric cancer institutions with little data addressing their program components. data from this study provides information on current school re-entry programs across these institutions. objectives: one objective of this study was to assess for correlation between the presence of a school re-entry program and other factors, such as geographic location and institution size. a second objective was to establish a list of differences between institutions' school re-entry program components. finally, we aimed to describe current school reentry practices, as well as program benefits and perceived areas for improvement. states with membership in the children's oncology group were offered enrollment in this study. a member of each institution was invited to participate in a survey established by the research team. this person was closely associated with the institution's school re-entry practices. each interview queried institution demographics, as well as program components (e.g., participants, target audience, resources). comment was also collected on program benefits and potential for improvements. analysis of transcripts was performed using pearson's correlation to assess for relationships between institution size, geographic location, and program presence. grounded theory was used for analysis of benefits and improvements. results: thirty-nine of forty-one pediatric institutions who were offered enrollment participated in this study. twentynine institutions (76%) indicated the presence of a school reentry program, and ten (24%) stated they had none. no correlation was found between institution size and the presence of a school re-entry program (p = 0.627, ns). there was also no correlation found between institution location and the presence of a school re-entry program (p = 0.921, ns). a major theme surrounding the benefits of having a program included education for the returning student's peers. for those with programs, perceived improvements included increasing staffing and the ability to offer more services. the results do not support the hypothesis that the presence of a school re-entry program is influenced by the size and geographic location of the treating institution. however, data seem to suggest that available staffing may influence the presence of a program. future studies are needed to address other potential influences, as well as to take an evidence-based approach to determine the effectiveness of the interventions present in these programs. cohen children's medical center/ zucker school of medicine at hofstra-northwell, new hyde park, new york, united states background: genetics/genomics is evolving at an extremely rapid pace. current advances lead to individual algorithms toward disease treatment for each disease with multiple branch points. fellows learn only a fraction of the knowledge and there is no formal approach to teaching critical analysis of information and application algorithms toward disease. additionally, as knowledge evolves extremely rapidly, any approach must teach self-acquisition and application of evolving discoveries. objectives: to create, implement and evaluate a novel curriculum for genetics/genomics targeted toward pediatric hematology/oncology fellows design/method: the curriculum includes four components: 1) genetic and genomic medical knowledge, with one initial team-based learning session and weekly online multiple choice questions; 2) essential pathways, which will teach molecular pathways common in oncogenesis and relevant to targeted therapy in microteaching sessions with using auditory, visual and tactile learning; 3) knowledge acquisition and clinical judgment, to allow learners to gain experience into researching data available, then developing and prioritizing potential treatment plans using problem-based learning sessions in which they will stage a patient, research treatment options, prioritize and present findings; and 4) synthesis to demonstrate independent ability to research and recommend therapy through an independent project in which the learner, given a case, will present the case and research findings, genetics/genomics, molecular pathways and make recommendations for therapy in molecular tumor board for faculty and fellows. to evaluate, we plan to recruit 12 to 16 institutions, match for size of programs and implement in half and evaluate 2nd and 3rd year fellows in both groups by mcq exam and satisfaction surveys. the creation of a multi-module, adult-learning based curriculum for genetics and genomics in pediatric oncology is feasible. implementation and evaluation are necessary to demonstrate efficacy. background: neuroblastoma is the most common extracranial solid tumor in children. chimeric anti-gd2 antibody ch14.18 (dinutuximab) therapy has improved the survival of children with newly diagnosed high-risk, neuroblastoma patients as well at the time of first relapse/progression. acute neuropathic pain is a well-documented side effect of dinutuximab administration. however, additional adverse effects including sensorimotor neuropathy, ocular symptoms, and behavioral changes have been described. the incidence and severity of these effects are currently not well-documented in pediatric patients. with improved long term survival of patients receiving this modality, it is important to look for the potential late effects of dinutuximab. objectives: to determine the incidence and severity of neurologic, ophthalmologic, or behavioral changes after dinutuximab administration at our institution. we performed a retrospective chart review using our electronic medical record. we included all patients with high-risk neuroblastoma between the ages of 1 and 21 years at our institution diagnosed between 1997 and 2017 who received dinutuximab. patients with history of opsoclonus-myoclonus syndrome or gross sensorimotor neuropathy prior to receiving dinutuximab were excluded. we examined clinical documentation for subjective reports and objective exam findings of neurologic, ophthalmologic, or behavioral changes. we also looked for referrals made to neurology, ophthalmology, physical medicine & rehabilitation (pm&r), and psychology. : twenty-two patients met inclusion criteria. at the time of chart review, 15 patients were alive and 7 were deceased. eighteen patients received dinutuximab per anbl0032; 5 patients received dinutuximab per anbl1221. of these 22 patients, 11 patients reported symptoms of interest and 5 reported multiple symptoms. six patients reported symptoms that began at least 12 months after completing dinutuximab. nine patients had objective findings on exam, including decreased deep tendon reflexes, abnormal pupils, and nearsightedness. for 10 patients, 15 referrals were made to ophthalmology, pm&r for neuropsychologic testing, or neurology. two patients who reported symptoms of interest were not referred to a specialist. conclusion: neurologic, ophthalmologic, and behavioral symptoms were commonly reported and demonstrated on exam among pediatric patients with high-risk neuroblastoma who received dinutuximab. it is important to identify these effects so that appropriate specialist referrals can be placed for adequate management of these changes. we recognize that these symptoms may not be solely due to dinutuximab as these patients receive other agents including opioids, so a prospective trial is needed to further evaluate the long-term effects of dinutuximab and to determine how best to screen for these effects. akron children's hospital, akron, ohio, united states background: pediatric cancer is the leading cause of diseaserelated death in children in the united states (u.s.). in 2014, over fifteen thousand children were diagnosed with cancer in the u.s. this population is at high risk for malnutrition due to the multimodal therapies they receive: surgery, chemotherapy, radiation therapy, antibody therapy, and/or bone marrow transplant. adverse effects of these therapies include taste changes, loss of appetite, diarrhea, vomiting, and/or mucositis, making it difficult for the children to be able to consume adequate amounts of nutrition during therapy. there is no "gold standard" measurement tool for identifying patients at risk for malnutrition. nutritional status is not frequently evaluated as a component of clinical trials. assessment of anthropometric measurements (weight, height, z-scores) at diagnosis, as well as over the duration of treatment, can assist in the early identification of malnutrition. the incidence and prevalence of malnutrition in this population is unknown at akron children's hospital. the purpose of this study is to describe the nutritional status and provision of nutritional support therapies in pediatric patients during their first year post new oncologic diagnosis. objectives: identify the incidence and prevalence of malnutrition across oncologic diagnostic categories over the first twelve months post diagnosis. we performed a retrospective records review of all patients newly diagnosed with cancer in 2015 at akron children's hospital. demographic and anthropometric data was collected at time of diagnosis and nutritional status categorized by z score. anthropometric and nutrition support data was then collected every two months for the first year after diagnosis along with incidence of unplanned inpatient admissions. results: a total of 65 patients were included in the analysis, with 6.2% malnourished at time of diagnosis; 12.3% developed malnutrition the first year. patients with solid tumors represented 50% of patients with pre-existing or acquired malnutrition. overall, 47% of patients received at least one nutritional support modality. patients with pre-existing or acquired malnutrition had a non-significant increase in unplanned admissions (p = 0.1196). our study demonstrated that patients with solid tumors were found to be at increased risk of pre-existing and acquired malnutrition, followed by leukemias, and experienced higher incidence of unplanned admissions in the time period observed. prospective, multi-center replication of this study, including detailed collection of nutrition therapies is recommended to guide development of diagnosis specific nutrition support guidelines. background: pediatric and young adult oncology patients treated with intense chemotherapy have a high incidence of transfusional iron overload. iron deposition can lead to heart failure/arrhythmias, liver abnormalities, endocrine dysfunction, ineffective erythropoiesis, and increased cancer and mortality risk. however, there is a paucity of data regarding recommendations for management of transfusional iron overload in these cancer survivors. consequently, long-term complications of transfusional iron overload specific to these patients have not been assessed. objectives: to assess screening and phlebotomy-based treatment algorithms for this population. design/method: a retrospective chart review of pediatric and young adults who completed oncology management, had iron overload, and initiated phlebotomy treatment was conducted. tiered screening occurred in patients that received at least 5 packed red blood cell (prbc) transfusions. patients were recommended for evaluation and possible phlebotomy if: (1) liver iron concentration (lic) >5 mg of iron/gram dry weight liver tissue by ferriscan and/or (2) cardiac mri t2* < 20 ms. during phlebotomy, iron status was assessed quarterly and phlebotomy discontinued with lic <5 or normalization of ferritin/imaging lic verification. descriptive statistics were employed to report the characteristics of the study population. spearman correlations were utilized to describe associations between transfusions, lic, ferritin, iron saturation and number of phlebotomy sessions. results: twenty five survivors underwent phlebotomy. the mean age was 11.6 years (sd 6.1) and 10 (40%) were female. oncologic diagnoses: all (36%), aml (8%), nhl (12%), ewing sarcoma (16%), osteosarcoma (4%), neuroblastoma (12%) and cns (12%). patients received a median of 25.0 (iqr 17 -34) transfusions. median number of phlebotomy sessions was 6 (iqr 4-8) over 0.36 years (iqr 0.28 -0.59). prior to phlebotomy, median lic was 7.5 mg/g (iqr 5.6-9.0) and ferritin was 1110.0 ng/ml (iqr 700 -2030) . no patients demonstrated abnormal cardiac t2* mri (n = 18). 23 (92%) patients completed phlebotomy. one discontinued due to poor vascular access. no patients developed iron deficiency. lic was reduced by a median of 2.4 mg/g (iqr 1.1 -3.6) and ferritin by 586 ng/ml . correlation between number of transfusions and phlebotomy sessions was poor (r2 = 0.017). conclusion: management guidelines are lacking for transfusional iron overload in pediatric and young adult survivors of cancer. we demonstrate a phlebotomy algorithm that is effective and tolerated. correlation between number of transfusions received and phlebotomy treatments was poor, necessitating serial assessments. using this management algorithm, prospective studies can evaluate the effect of iron removal on iron overload complications in this patient population. penn state children's hospital, hershey, pennsylvania, united states background: cancer therapy leads to an impaired immune system that takes time to recover. it is important to ensure that these survivors have adequate immunity to prevent common yet potentially severe childhood illnesses. no validated guidelines currently exist for surveillance testing or re-immunization in this population. retrospective analysis involving a small cohort of pediatric cancer patients treated at penn state children's hospital showed 46% of patients screened for varicella immunity after therapy completion did not have adequate disease titers. to determine the proportion of pediatric cancer survivors who have lost humoral immunity to previously received vaccines; to determine the rate of response to single dose boosters or full vaccine series in seronegative subjects after one booster. design/method: pediatric cancer survivors treated at the children's hospital who are at least 12 months from completion of cancer therapy are prospectively tested for antibody levels to hepatitis b, tetanus, varicella, measles, and 6 strains of pneumococcus (4, 6b, 9v, 18c, 19f, and 23f). samples are analyzed by the cdc for measles and varicella avidity. seronegative subjects by commercial studies, are eligible to receive booster vaccines. titers are rechecked at least 6 weeks after boosters to re-evaluate immunity; if still seronegative, subjects will receive the entire vaccine series. titers are finally tested at least 6 weeks after the final dose of the vaccine series. immunity analyzed after therapy, after boosters, and after vaccine series. results: of 37 pediatric cancers survivors who completed therapy, 78% were non-immune to hepatitis b, 92% nonimmune to >50% of pneumococcal strains tested, 24% nonimmune to measles, 54% non-immune to varicella, and 2% non-immune to tetanus. 1 of 13 subjects who received mmr vaccine after therapy and prior to study enrollment did not have protective antibodies to measles. of the 15 subjects who received varicella vaccine after end of therapy and prior to study enrollment, 6 did not maintain protective antibody levels. cdc results for measles and varicella are pending, as well as repeat studies after vaccine boosters and series. conclusion: a significant percentage of pediatric cancer survivors do not retain immunity to hepatitis b, pneumococcus, measles, and varicella. after one booster, a high percentage of subjects did not develop protective immunity to varicella. only 1 subject did not have immunity to tetanus, which is consistent with the high immunogenicity of tetanus toxoid. formal guidelines are needed to protect this population from vaccine-preventable illness post-therapy. children's hospital of richmond at virginia commonwealth university health system, richmond, virginia, united states background: childhood cancer survivors are at risk for being overweight. diet and physical exercise are important in maintaining a healthy lifestyle and weight; however, it has been reported that cancer survivors are less active than their peers. one reason for this may be that there are no clearly established risk-based exercise recommendations for cancer survivors. another reason may be that providers tend to focus s193 of s301 recommendations for exercise more towards patients who are overweight. objectives: to describe changes in physical fitness of childhood cancer survivors who exercise. design/method: 'moving forward' is a wellness and physical fitness program that the center for care beyond the cure at chor offers in partnership with the ask childhood cancer foundation and the ymca. the program is available for any childhood cancer survivor between 8y and 18y age, being seen at our center. survivors define their fitness or wellness goals and then work with a trainer once a week (at least) for 30 min sessions throughout the year to achieve these goals. baseline and ongoing measurements for core strength, endurance, overall strength and balance were collected. the average of each of the parameters of all participants were compared from the beginning to the end of the program. over the year, there was a 30% increase in endurance as measured by the average of the miles walked in 6 minutes, 40% increase in core strength as measured by the average number of sit-ups in 30 secs, an 80% and 44% increase in overall strength as measured by the average weight lifted by leg press and the average weight lifted by chest press, and a 10% increase in balance as measured by the average number of seconds balancing on a single leg. in addition, each child had actually gained weight in the process with an approximately 10% increase in the average of the weights of all children. there are benefits to regular exercise beyond weight control, and improvements in physical fitness can be seen even without weight loss. regular physical exercise results in improved physical fitness and should be universally advocated to all patients. determining insulin resistance, measuring changes in fatigue and wellness perception following exercise are future directions that we intend to explore. dana-farber cancer institute, boston, massachusetts, united states background: improvements in adolescent and young adult cancer patient (aya) survival rates and quality of life outcomes have lagged behind those of children and older adults, highlighting a need for research targeting this unique population. current literature supports the value of strong ayaclinician communication, notably in facilitating therapeutic alliance, however little is known about aya communication priorities during cancer care and barriers to optimal ayaclinician communication. objectives: to explore aya and oncology clinician communication priorities and to identify barriers and facilitators to aya-oncology clinician communication. design/method: semi-structured interviews were held with 21 aya cancer patients and survivors (ages 15-25 years) from a single large academic institution and 22 oncology clinicians (physicians and nurse practitioners) from 7 academic institutions in the northeastern united states. interviews were conducted in english by phone or in person. all interviews were audio-recorded and transcribed verbatim. analyses were aided by nvivo11 software. ayas identified a wide range of topics as important to discuss with clinicians. the most frequently identified topics were 1) side effects of treatment (with an emphasis on physical appearance and function, n = 16), 2) social issues (including friendship, family, and school, n = 13), 3) looking ahead to the future (n = 12), and 4) sexual & reproductive health (including future fertility, contraception, and romantic relationships, n = 8). clinicians prioritized 1) cancer treatment and side effects (n = 17), 2) emotional and psychological health (n = 11), and 3) sexual and reproductive health with a focus on fertility risk and fertility preservation (n = 8). aya reported facilitators to good communication including an open and long-established relationship with the clinician (n = 16) and clinician engagement in age-appropriate and patient-directed conversations (n = 7). barriers included parental presence during visits (n = 7). clinicians reported barriers including 1) clinician discomfort (not feeling wellequipped to discuss psychosocial topics such as sexual health, spirituality, and relationships with peers, n = 13), 2) presence of parents/family (n = 12), and 3) perceived patient discomfort discussing specific topics (such as sexual health, n = 10). clinicians acknowledged the need for collaborative efforts with additional team members (i.e. nurses, psychosocial providers) to assist in meeting aya communication needs. conclusion: aya and clinician-reported communication priorities are largely aligned. however, ayas emphasize some topics, such as social function, appearance, and sexual health that are not highly prioritized by clinicians, which may result in gaps in care for ayas in treatment and in survivorship. these data identify opportunities for intervention, including clinician education, patient and family education, clinic-based intervention, and systems-based changes that can be developed and tested. background: primary care physicians (pcps) cite lack of knowledge and inadequate communication with the oncology team as major barriers to providing recommended surveillance for late effects of treatment to childhood cancer survivors. a standardized telephone handoff to pcps posttherapy is a potential strategy to increase survivorship care by pcps through interactive communication. to determine the feasibility of a structured telephone communication using the situation, background, assessment, and recommendation (sbar) communication tool delivered by a trained oncology nurse to increase pcp knowledge and willingness to provide survivorship care. design/method: from 12/12/16 to 1/23/17, a registered nurse expert in childhood cancer survivorship attempted to contact by telephone the pcps of the 30 most recent patients attending yale's childhood cancer survivorship clinic that were <18 years old, english-speaking, and ≥2 years posttreatment. all pcps had been previously sent an individualized survivorship care plan (scp) that listed the patient's previous treatment history and recommended surveillance tests. upon successful contact and after confirming receipt of the scp, the nurse explained the definition of late effects, description of patient's diagnosis and treatment history, and associated potential late complications and schedule of recommended surveillance tests. the pcp was also asked about his/her ability and willingness to provide needed surveillance for late effects in the future. overall, 26 of 30 pcps were successfully contacted with a median of 1 phone call (range: 1-3) that lasted a median of 6 minutes (range: 3-10) after a median of 1 business day (range: 0-18). no pcps ended the call mid-conversation. all 26 pcps were receptive and expressed appreciation for the call. twenty-five of 26 (96%) pcps expressed an understand-ing of the material discussed and endorsed belief in their ability and willingness to provide late effects surveillance for their patients. no pcps questioned discussing their patient's care with a nurse versus a physician. interactive, structured communications between nurses and pcps by telephone are feasible and are associated with high-levels of pcp confidence in providing survivorship care. background: childhood cancer (cc) admissions account for 5% of non-newborn pediatric hospitalizations. these hospitalizations are longer and more expensive than other hospitalizations. admission payer (medicaid or commercial) reflects both health policy and sociodemographic status. the objective of this study was to determine if length of stay (los) or cost of cc admissions differed by payer. we used the 2012 kids inpatient database, a sampling of all pediatric hospital discharges in the united states. analysis for this study was limited to admissions containing a cancer diagnosis in any discharge icd-9 codes. admissions were further subcategorized by discharge codes according to diagnosis (leukemia, lymphoma, solid tumor and brain tumor) and reason for admission (chemotherapy, procedure, infection, non-infectious toxicity or "other"). charges were converted to costs using cost-to-charge ratios. multivariable linear regression models were performed to control for age, gender, race, reason for admission, and diagnosis. results: there were 105,752 weighted admissions for children with a cancer diagnosis in 2012. of these admissions, 40.5% had medicaid, 50.6% had commercial insurance, and less than 1% had other payers. the mean los for medicaid admissions was 7.37 days (95% ci 7.2-7.5), compared with 6.33 days (95% ci 6.2-6.4) for commercial insurance. surgical admissions accounted for the largest difference in length of stay with medicaid admissions being 2.77 days longer than those covered by commercial insurance (11.19 days vs 8.42 days), however, the difference was significantly different for all reasons for admission. in multivariable analysis admissions associated with commercial insurance were 6% shorter s195 of s301 (p<0.001), accounting for approximately one hospital day, than admissions associated with medicaid after controlling for other variables including race. the mean overall cost for medicaid admissions was $23,464 (95% ci 22840-24088), compared with $21,849 (95% ci 21343-22356) for commercial insurance. in the multivariable model, cost was collinear with race. conclusion: los and cost of admissions associated with medicaid differed from those associate with commercial payers. medicaid admissions were 6% longer on average than commercial insurance, accounting for a difference in length of stay of approximately one day although the difference varied with the reason for hospitalization (chemotherapy, surgical procedure, infection, other toxicity, other). costs of admissions were not independent of race. further investigation into potential explanations for this difference including differential access to home care needs, outpatient reimbursement differences, social indications for prolonged hospitalization, and provider biases, is warranted. background: pediatric cancer is a major cause of morbidity and mortality among children surpassed only by accidents. despite improved outcomes in high income countries (hic) survival rates remain poor in the developing word. there are various diagnostic and therapeutic limitations contributing significantly for the survival gap. the main objective of the study is to to evaluate the outcomes of pediatric cancer in armenia and identify diagnostic and therapeutic limitations in the country. we conducted a retrospective study among 97 (≤18 years old) children with cancer (solid tumors and hematological malignancies), who were diagnosed and treated at the clinic of chemotherapy of muratsan hospital complex of yerevan state medical university between 2008 and 2016. those patients, who didn't receive chemotherapy for any reason were not included in the study cohort. epidemiological, social, medical information was collected through the patient charts review. this included patient age at diagnosis, sex, place of residence (city vs village), the educational level and employment status of parents, type of cancer, stage, presentation of symptoms, first medical specialty consulted and the time consulted, initial work-up, the type of treatment received, information on the diagnosis/treatment received abroad. results: at our clinic during the mentioned period of time the majority of patients presented with hematologic malignancies-71%. 77 (74.6%) patients had information on diagnosis delay. average delay in diagnosis was about 42 days. in 33% of cases the first contact with "healthcare system" was through pediatrician, and in 20% with surgeon. out of 19 relapsed patients 10 received salvage treatment in armenia and 4 abroad. from those who stayed for treatment in armenia 4 patients survived. majority of relapsed patients had acute lymphoblastic leukemia. from 35 leukemia patients immunophenotyping and cytogenetics were available for 26 (74.3%) patients; the majority of missing cases were between 2008 and 2012, when these diagnostic modalities were not available or affordable in the country. 43 (45%) patients received part of diagnosis and/or treatment abroad. the most frequent reason for going abroad was bone marrow transplantation, otherwise none available in armenia. out of 97 patients 22 were lost to follow-up, 15 patients had a fatal outcome. 60 patients were in remission at a median follow up of 3.57 years. conclusion: unavailability of cancer registry and several essential diagnostic/treatment modalities, luck of multidisciplinary care and palliative support, high rate of out-of-pocket expenses were among the main challenges of pediatric cancer care in armenia. background: adverse drug reactions (adrs) are increasingly recognized as important and sometimes irreversible complications of cancer treatment. anthracyclines and cisplatin are effective chemotherapeutic agents, but their use can be limited by cardiotoxicity (anthracyclines) and ototoxicity (cisplatin) in up to 60% of patients. genetic variants that can be used to predict who is most at risk of developing these adrs have been discovered and replicated. objectives: to create pharmacogenetic risk prediction models for anthracycline and cisplatin toxicities and discuss results with oncologists to facilitate incorporation into treatment decision-making when appropriate. design/method: risk prediction models were developed from the linear regression of strongly-predictive genomic variants (odds ratios ≥ 3) discovered and replicated in at least three patient populations. these models were used to assess an individual patient's genomic risk of developing cardiotoxicity from anthracyclines or hearing loss from cisplatin. risk results were returned to oncologists showing where the specific patient's genetic risk of toxicity lies on a continuum between the lowest and highest risk groups across all studied patients using a multi-gene model. interviews were conducted with patients, families, and oncologists to determine how results were valued and utilized. results: 227 patients have been genotyped and had their genetic risk results returned to their oncologists. the first 140 patients have been characterized to determine the impact these test results have had on their clinical care. results were described as being useful in decision-making by patients and/or oncologists in 100% of cases. additionally, for patients in the most extreme risk groups (highest and lowest risk), a change in treatment plan was ordered 30% of the time for cisplatin patients and 35% of the time for anthracycline patients. this included increased cardiac and audiological monitoring, the addition of a protective agent, or choosing an alternative treatment protocol if the risk outweighed the benefits of remaining on the current treatment plan. in interviews, patients indicated that they felt more involved in decision making, and felt reassured by understanding their genetic risk of toxicities. genetic risk prediction models for anthracycline cardiotoxicity and cisplatin ototoxicity were highly utilized by patients and oncologists in decision-making. results were found to be an important tool for informing patients of the risk of adrs during cancer treatment, and resulted in patients and their families feeling more involved in decision-making. background: childhood cancer survivors are at increased risk of developing executive dysfunction, and low socioe-conomic status (ses) has been identified as one of the mediators of executive functioning. previous studies have used traditional measures of ses, such as parents' education level, family annual income and occupation. but more recently, area based socioeconomic measures like block group poverty status are deemed to be more useful in monitoring of social inequalities in health in the united states. block groups are statistical divisions of census tracts and generally contain between 600 and 3,000 people. the current study aims to understand the association of block group poverty status (percentage of households in family's block group of residence living below the federal poverty level) with executive functioning among cancer survivor children. design/method: we used a retrospective cohort of 67 childhood cancer survivors. relevant information was collected from the medical record, administrative data sets and parent-filled surveys. address information was geocoded using arcgis 10.2 to obtain data on the block group poverty status. a priori cut-points were set to represent block groups with families living below poverty level at 0%, 0.1% to 9.9%, and ≥10.0%. executive functioning were assessed through a parent-rated instrument, the behavior rating inventory of executive functions (brief). multiple linear regressions were used to determine the relationship between block group poverty status and the brief scores. results: data was examined from 67 families of childhood cancer survivors, ranging in age from 6 to 18 years. in this sample, 32.8% families reported an annual income <$60,000, 32.8% reported income between $60,000 and $100,000 while 34.3% reported annual income ≥$100,000. primary care giver of 85.1% of cancer survivors had more than more high school education, and 31.3%, 41.8% and 26.9%, of families were living in a block groups with 0%, 0.1-9.9% and ≥10% poor households respectively. block group poverty level was not significantly associated with annual income levels (spearman's rho = 0.14, p = 0.25), or parental education level (spearman's rho = -0.02, p = 0.84). in a step-wise multiple linear regression, there was no statistically significant association seen between block group poverty status and executive functioning after adjusting for co-variables in the final model. future prospective study with a bigger sample size, longer follow up period and more robust measures of the executive functioning like a clinician administered test are needed to understand the effect of block group poverty status on executive functioning. to d100 completion was 45.5 days (range 21-63). all parents strongly agreed/agreed that d100 was helpful and would recommend d100 participation to another family. ten parents (100%) reported time spent on d100 was "just right." no parent felt more worried due to the intervention, though 1 parent found d100 participation stressful. this interim analysis suggests that parents have a favorable d100 experience and recommend the intervention. to date, <20% of enrolled parents fail to participate. d100 shows promise as an acceptable interdisciplinary communication intervention targeted to the early treatment period for childhood cancer. children 's hospital and research center oakland, oakland, california, united states background: screening echocardiograms are recommended by children's oncology group (cog) guidelines to assess for anthracycline-induced left ventricular (lv) systolic dysfunction. the yield of screening echocardiograms during chemotherapy and in the immediate post-therapy period is uncertain. objectives: to assess the incidence of lv dysfunction detected by screening echocardiograms during chemotherapy and in the immediate post-therapy period, defined as 0-24 months off-therapy. design/method: children diagnosed with cancer between january 2013-march 2016 who received anthracycline chemotherapy were identified. echocardiograms were performed as per protocol, institutional and cog guidelines, and were reviewed retrospectively. lv dysfunction was defined as fractional shortening (fs) <28% or ejection fraction (ef) <55% (1) results: in this cohort (n = 195, median age 6 years), the most common diagnosis was all (54.8%), followed by aml (9.7%). of 357 echocardiograms, 224 (62.7%) were performed during treatment and 133 in the immediate posttreatment period. thirty-eight (19.3%) patients had a >10% decrease in fs compared to their pre-treatment echocardiograms. none of these patients required any treatment modification or cardiac medications. only 1 patient (0.5%) had echocardiogram-proven lv dysfunction discovered on a screening echocardiogram during her treatment course. she eventually died due to multi-organ failure following septic shock. this patient was receiving treatment for aml and had received 300 mg/m2 of doxorubicin-equivalent anthracyclines at the time of the abnormal echocardiogram. one patient with metastatic ewing sarcoma had borderline lv dysfunction with a fs of 30% detected a month before completion of therapy. she had received 375mg/m2 of doxorubicin equivalent anthracyclines at the time of the abnormal echocardiogram. she did not require any therapy modification or additional cardiac medications. serial echocardiograms done on this patient have shown stable ventricular function. no off-therapy screening echocardiograms identified lv dysfunction. in our experience, the yield of echocardiograms to detect anthracycline-related cardiac dysfunction during treatment and in the immediate post-therapy period is very low. one patient developed lv dysfunction during treatment and one had borderline fs, while no lv dysfunction was identified within 24 months of completing chemotherapy. though fs decreased in 19% of patients, none required intervention. further study is needed to optimize the use of echocardiography screening in children treated with anthracyclines. references: 1. landier w et al. jco 2012. background: platinum-based chemotherapy increases the risk of sensorineural hearing loss in children with cancer. little is known about the impact of hearing loss on cognitive and emotional functioning in survivors. to determine the association of severe/profound hearing loss after platinum-based chemotherapy with 1) cognitive impairment and 2) emotional distress (i.e. anxiety and/or depression). cross-sectional study of all patients attending yale's childhood cancer survivorship clinic ≥2 years off therapy for cancer diagnosed at <21 years and treated with cisplatin and/or carboplatin, but with no history of cns tumor, cranial radiation, congenital hearing loss, or developmental delay. hearing loss severity and hearing aid data were abstracted from audiograms and detailed clinical history. cognitive impairment was defined as behavior rating inventory of executive function t score ≥65, assessment by neuropsychologist, and/or history of special education. emotional distress was determined by brief symptom inventory t score ≥63 (global or two subscales) or behavioral and emotional screening system t score ≥61, psychologist interview, and/or history of psychotropic medication/psychotherapy. the most recent available patient data were used. logistic regression with sas software, version 9.4 was performed. results: overall, 37 patients (57% female, 78% white) met eligibility criteria with a median age of 9.0 years (iqr = 12.1) at diagnosis and 22.3 years at evaluation (iqr = 10.4) after a diagnosis of sarcoma (36%), neuroblastoma (32%), or other (32%) for which 84% received cisplatin and 30% received carboplatin. fifteen patients (41%) had severe/profound hearing loss in at least one ear. patients with severe/profound hearing loss had a significantly increased risk of cognitive impairment (or = 5.14; 95% ci = 1.17-22.69), but not emotional distress, compared to patients without severe/profound hearing loss. there was no significant association between age at diagnosis, current age, time since diagnosis, sex, race, ethnicity, or diagnosis with either cognitive impairment or emotional distress. similarly, there was no significant interaction between 1) age at diagnosis and hearing loss or 2) sex and hearing loss with either cognitive impairment or emotional distress. ten of the 15 (67%) patients with severe/profound hearing loss in at least one ear were recommended hearing aids, of which 3 (30%) reported compliance most of the time. we conclude that severe/profound hearing loss is significantly associated with cognitive impairment, but not emotional distress, in childhood cancer survivors. our data supports the need for interventions to improve hearing in these patients, including compliance with hearing aids. background: who grade 3 anaplastic astrocytoma is a high grade glioma dependent on vascular endothelial s199 of s301 growth factor (vegf) mediated angiogenesis for its growth and infiltration. bevacizumab is a recombinant humanized monoclonal antibody which binds vegf-a and inhibits angiogenesis. common adverse effects of bevacizumab are hypertension, proteinuria, thrombosis and bleeding. while animal model based studies have shown that bevacizumab may impair ovarian function the effects of bevacizumab therapy on human fertility are not clear. since the physiology of pregnancy involves neovascularization/angiogenesis it is recommended that conception be avoided for at least 6 months following exposure to bevacizumab. to describe the course of a young adult who became pregnant after receiving bevacizumab and radiation therapy for treatment of an anaplastic astrocytoma. a 20 year old woman diagnosed with a localized hemispheric who 3 anaplastic astrocytoma was treated with chemotherapy and radiation (temozolomide/59.4 gy) followed by 12 cycles of bi-weekly bevacizumab/temozolomide. patient opted not to pursue fertility preservation prior to initiation treatment. she experienced bevacizumab-associated proteinuria and hypertension during treatment but received all protocol mandated doses (cumulative doses: bevacizumab = 240 mg/kg; temozolomide = 15.78 gm/m2). she had a spontaneous unassisted pregnancy 18 months after completing treatment. her pregnancy was uneventful and she was normotensive throughout. fetal ultrasonography at 16, 20, 27, 33 weeks revealed no abnormality of the brain, heart, great vessels, kidney, extremities, placenta and umbilical cord. at 39 weeks she delivered a female infant via cesarean section (birth weight: 3890 grams, apgars: 95 and 1010) excessive post-partum hemorrhage was not reported. placenta was bi-lobed and weighed 604 g. histological analysis revealed normal placental villous development and maturation and two small infarcts. conclusion: exposure to bevacizumab in our patient had no detrimental effect on fertility and on placental/fetal vascular development. we hope this report will add to the existing data on the effects of bevacizumab therapy on fertility. children's healthcare of atlanta, emory university school of medicine, atlanta, georgia, united states background: reports of malnutrition incidence and prevalence in young cancer patients are variable and not well established. previous research suggests children, especially less than 3 years old, treated with intensive cancer-directed therapy are at higher risk for malnutrition. however, no standardized assessment has been used to evaluate risk in this population. objectives: we aim to assess the trends of weight-for-age for patients following cancer diagnosis. this study will be the first to use a standardized measure of treatment intensity (intensity treatment rating scale, itr-2) and will assist in targeting interventions for identification and treatment of malnutrition. design/method: this observational, retrospective study obtained data through the center's pediatric cancer registry and electronic medical record. patients were classified by tumor type (brain or non-brain tumor) and treatment intensity (itr-2). itr-2 incorporates diagnosis, chemotherapy, radiation, and surgery, beginning with lowest intensity (1) to highest intensity (4). inclusion criteria included new cancer diagnosis 2007-2015 at less than 3 years old, with weight obtained and available within 2 days of therapy start date. incomplete data, alternate growth charts, or treatment intensity of 1, were excluded. weight was obtained at start of therapy and through 2 years after treatment initiation (approximately 750 days) and converted to z-scores adjusted for age and sex. weight trajectories were modeled using generalized linear mixed models with subject-specific random intercepts and spline functions. separate functions were constructed for subgroups of interest (tumor type and itr). results: there were 402 patients included: 53 patients with brain tumors (13.2%) and 349 with non-brain tumors (86.8%). of included patients, 165 had treatment intensity of 2 (41.0%), 192 of 3 (47.8%) and 45 of 4 (11.2%). over the observation period, 34,593 valid weights were recorded. at initiation of treatment, no difference existed between z-score by tumor type (p = 0.880) or by intensity (2 vs. 3, p = 0.879; 2 vs. 4, p = 0.665; 3 vs. 4, p = 0.558). tumor type did not affect z-score through the follow up period. z-scores were higher for intensity rating 2 vs. 3 and 2 vs. 4 (p = <0.001 and p = 0.015 respectively) at 240 days after the start of treatment and persisted through 720 days (p = 0.003 and p<0.001 respectively). higher treatment intensity is associated with decline in z-score and failure to return to baseline. future directions include further analysis on specific risk factors and timing of weight loss, longer-term follow-up of weight trends, and targeted interventions for identification, prevention, and treatment of malnutrition. objectives: asses the pt requirements for bleeding episodes in a prospective cohort of pcp using a <10 × 10e9 threshold compared to a <20 × 10e9/l threshold in a historical cohort. we collected pt data in all pcps treated at our center between january/2013 through december/2017. diagnosis, prescription for pt (prophylaxis vs bleeding disorder), plt count and transfused units were assessed for each pt. pcps treated from january/2013 through june 2015 received prophylactic pt with a <20 × 10e9 threshold (cohort a), and pts treated from july/2015 through december/2017 received prophylactic pt with a <10 × 10e9 threshold. pts done for procedures and pts with concomitant hemorrhagic pathology were excluded. we compared the number of pts prescribed as prophylaxis vs bleeding episode between cohorts. data analyzed: graphpad prims 6.0®. statistical analysis: percentages with confidence interval (ci); t-student test (parametric variables) and mann-whitney test (nonparametric variables). statistical significance: p<0.05. we reviewed 2093 pts (871 in cohort a, 1222 cohort b) in 209 patients. 62% had acute leukemia, 33% received and auto or allo hsct. diagnoses and the proportion of patients undergoing hsct was comparable in both cohorts. the average number of pts per patient was 8,54 in cohort a and 8,24 in cohort b (p = ns), but a significant difference was found when hsct patients were excluded from this comparison (7,34 pt per patient in cohort a vs 6,07 in cohort b, p = 0,005), which resulted in an estimated 16,4% reduction in pts prescription. furthermore 61 (7,1%) pts were prescribed for bleeding episodes in cohort a versus 99 (8,2%) in cohort b (p = ns). patients receiving hsct in the entire group ver-sus those not receiving hsct had similar pt requirements for bleeding episodes (10% vs 8,5% p = ns) conclusion: a <10 × 10e9 plt count threshold for prophylactic pts is safe in pcp in chemotherapy and hsct. it can result in a significant reduction in pt usage. key words: platelets, transfusions, prophylaxis, cancer, childhood. ucsf benioff children's hospital oakland, oakland, california, united states background: transition of care for adolescent and young adult (aya) survivors of childhood cancer from pediatric to adult-oriented long-term follow-up (ltfu) is complex. loss to follow-up is common, and little is known about the success rates among different models. the survivors of childhood cancer program (sccp) at ucsf benioff children's hospital oakland employs a community-based model for transitional care. our multidisciplinary team provides aya survivors a comprehensive treatment summary and recommendations, then facilitates transition to primary care or adult oncology ltfu programs. evaluate the success rate for transition of care among aya survivors of childhood cancer in our ltfu program, and identify barriers to successful transition. design/method: aya patients seen from november 2010 to august 2017 in the sccp with intent to transition were asked by email or telephone if they had followed up with their designated provider. the primary outcome was successful transition, defined as establishing care within 18 months of their visit. patients were also asked about barriers to transition and to rate the new provider's familiarity with their cancer history and ltfu needs. results: transition was intended for 88 patients. eightyseven were contacted and 43 responded. of these, 29 (67%) successfully transitioned, while 14 (33%) were lost to followup. ages ranged from 19 to 48 years, at 2 to 32 years since completion of therapy. ten (34%) transitioned to a primary care provider, 20 (69%) to an adult oncology ltfu program, and 1 (3%) to a pediatrician. patients rated their new provider's knowledge above average (3.76) on a 5-point scale from poor (1) to excellent (5). survivors lost to follow up indicated the following barriers to transition: loss/change of insurance (3), inability to find a provider (1), too busy/forgot (4), problems with transportation (1), concerns about cost/copay (2), and s201 of s301 other (4). twelve patients requested further assistance with transition. conclusion: two-thirds of responding patients successfully transitioned. more work is needed to overcome various barriers to transition for one third of aya survivors. albany medical center, albany, new york, united states background: the transition from active treatment, to offtherapy follow-up, is a stressful event for parents of children with cancer. the psychosocial needs of parents after therapy have received limited attention in the united states with only 3 published quantitative studies, the largest with 35 parents. we have secured funding for and recruited a transition care coordinator (tcc) to investigate this further. objectives: our objective is to assess and screen parents at the end of their child's treatment, and to develop interventions to support parents during this time and thereafter. design/method: after informed consent, a standardized questionnaire, the psychosocial assessment tool (pat 2.0), was administered to parents at end of therapy (t1), 6 months later (t2) and 1 year later (t3). the tcc provided "universal" intervention to all families with an end of therapy binder containing a treatment summary, follow-up roadmaps, information on late effects, and survivor scholarships. based on their pat2.0 scores, some parents were provided intervention specific to symptoms (targeted intervention for scores 1-1.99) or referred to a behavioral health specialist through the clinic social worker for counseling (for scores >2). results: analysis of pat1 data showed that 45% of parents (n = 45) scored in the targeted or clinical ranges; 19% of parents scored in those ranges at pat2. significant gender differences were revealed with the mean score for men of 0.7 and for women of 1.13. this was confirmed by showing statistical significance (p = 0.017) when analysis was conducted for only a subgroup of data composed of couples (n = 24). analysis of pat2 data by couples (n = 10) showed the mean score for men was 0.64 and for women was 0.93 (p = 0.12). gender differences were most apparent in caregiver stress reaction questions that focused on ptsd symptoms. when the subgroup of couples' scores (n = 24) for caregiver stress reaction at pat1 was analyzed, there was a significant difference (p = 0.005) in caregiver stress reaction with a mean of 0.08 for men versus 0.3 for women. [note: subcategory scores range from 0 to 1]. this study was initiated in october 2013 using a tcc and the pat2.0 screening tool. the results suggest greater stress on mothers after therapy, with a substantial proportion of parents having symptoms of ptsd after therapy. background: hodgkin lymphoma (hl) is a common childhood cancer characterized by an inflammatory microenvironment. chemotherapy and radiation may exacerbate this inflammation and contribute to the development of late effects (pneumonitis or pulmonary fibrosis). in a heterogeneous cohort of childhood cancer survivors exposed to pulmonarytoxic therapy, no association between pro-inflammatory cytokines and late pulmonary dysfunction was observed. our objective was to test this association in a relatively uniform cohort of survivors of hl, given the well-recognized proinflammatory background of this disease. objectives: to characterize off-therapy pulmonary function in survivors of hl treated with contemporary therapy, and to investigate its association with persistent systemic inflammation. design/method: blood samples, clinical data, and pulmonary function tests were obtained from survivors of hl ≥6 months off therapy. lung function score (lfs), a validated method for assessing degree of pulmonary dysfunction on a scale of i to iv, was determined from diffusion capacity and forced expiratory volume in one second (fev1). for a control group, blood samples from patients with benign, noninflammatory hematologic conditions were used. plasma concentrations of 50 inflammatory cytokines were measured on a luminex platform (emd millipore). associations between clinical features or cytokine levels and lfs i (normal) vs. ii-iv were evaluated using logistic regression or wilcoxon rank sum tests, respectively. results: of 77 survivors (mean age at diagnosis: 14 years, range: 3-18; mean time off therapy: 3.3 years, range: 0.5-24), 70% were categorized as lfs ii (mild dysfunction), 8% as lfs iii (moderate dysfunction), and no survivors as lfs iv (severe dysfunction). higher lfs was associated with female sex (p = 0.01) but not other demographic, disease, or treatment factors. forty-eight survivors had blood samples collected at a mean age of 18.5 years (range: 10-32) with a mean time since treatment completion of 3.8 years (range: 0.6-6.1). of 31 controls, the mean age at time of blood collection was 12 years (range: 4-17). survivors did not have significantly elevated cytokine levels compared to controls. female survivors of hl ≥6 months off therapy are at increased risk of pulmonary dysfunction. neither evidence for pulmonary dysfunction, as measured by lfs, nor duration of time off therapy were related to systemic inflammation in this study. pulmonary function deterioration and clinical pulmonary symptoms are rarely observed immediately following therapy but increase over time. future studies may consider exploring the contribution of systemic inflammation to pulmonary late effects in survivors farther off therapy, when risk for this late effect is greater. background: thyroid carcinoma is a very rare tumor in pediatrics, accounting for 1.5-3% of childhood carcinomas in the united states and europe. we aim to detect the risk of second malignancies among pediatric thyroid cancer survivors. the cohort analysis consisted of pediatric cancer patients aged less than 20 years diagnosed with a primary thyroid cancer and identified by site code icd-0-3: c739, reported to a seer 9 database between 1973 and 2013. they were followed up by death or the end of the study period (december 31, 2013) . out of 1769 patients diagnosed primarily with thyroid carcinoma, there were 42 patients who had 45 incidences of subsequent malignancies. the mean age of patients at initial diagnosis of thyroid cancer was 16 years. females (90.5%) had significantly higher incidence of second malignancies (sm) than males (9.5%). the overall standardized incidence ratio (sir) of sm in thyroid pediatric patients was higher than expected (sir = 1.48). some specific sites showed significantly higher incidences: salivary gland (sir = 33.95), gum and other mouth (sir = 24.53) and kidney (sir = 5.72). the overall risk of sm in patients received radioactive iodine was higher than expected (sir = 4.41). the cumulative inci-dence of sms from the initial diagnosis of thyroid cancer was calculated with the survival methodology of competing risk, death treated as a competing event. cumulative incidence of sm was 2.7% [95 % ci (1.62, 3.83 %)] at 25 years and substantially expanded after 15 years, reaching 11.92% [95 % ci (4.9, 18.8%)] at 40 years. the cumulative incidence of each tumor type at 40 years was 0.452% [95 % ci (0.139, 0.765 %)] for breast cancer, 0.28% [95 % ci (0.034, 0.53 %)] for salivary gland, 0.22% [95 % ci (0.00034, 0.448 %)] for each one of kidney and cervix uteri and 0.169% [95 % ci (0, 0.361 %)] for each one of ovary and melanoma of the skin. cumulative incidence of sm was stratified based on race, gender and radiotherapy exposure, but there was no statistical difference in each of them. conclusion: race, gender, histological subtypes, and radioactive iodine may play an important role as prognostic factors for developing sm among pediatric thyroid cancer survivors. identification of underlying mechanisms that raise the risk of sm is important for both treatment and follow-up strategy. background: the ethical practice of informed consent requires it be both voluntary and understood by the research participant. in pediatric oncology, parents must undergo informed consent to enroll their child with cancer into clinical trials, but often it can be difficult to understand especially for parents with low english proficiency. previous research has shown that parents of children with cancer have difficulty understanding voluntariness, and that parental satisfaction with informed consent does not always correlate with adequate comprehension. objectives: to examine socio-demographic and contextual correlates of comprehension of informed consent, voluntariness, and satisfaction in parents who consented to participation of their child in a cancer clinical trial. we focused on characterizing differences between non-hispanics and hispanics, the fastest growing ethnic group in the u.s. design/method: parents/guardians (n = 121) of children aged 0-17 years with newly diagnosed cancer, who had consented to participation of their child in a clinical trial for cancer treatment at rady children's hospital-san diego were s203 of s301 prospectively recruited. parents completed questionnaires assessing comprehension, voluntariness, satisfaction, health literacy, socio-demographics, and acculturation level, if hispanic. comprehension was surveyed at baseline and longitudinally at 3 months. comprehension, voluntariness and satisfaction outcomes were analyzed by socio-demographics, health literacy, and acculturation level using logistic regression. results: of the 121 participants surveyed, 61 (50.4%) were hispanic and 60 (49.6%) were non-hispanic. we found that higher health literacy was associated with greater objective comprehension (p<0.001), voluntariness (p<0.001), socioeconomic status (p<0.001), and acculturation (p<0.001). hispanics reported lower objective comprehension (p = 0.025), voluntariness (p = 0.029), health literacy (p<0.001) and ses (p = 0.015) compared to non-hispanics. spanish-speakers reported lower voluntariness (p = 0.016), health literacy (p<0.001), and acculturation (p<0.001) compared to englishspeakers. at the 3-month follow-up, comprehension in hispanics significantly improved (p = 0.012) compared to their baseline comprehension. satisfaction was moderately high across all subgroups and was not significantly impacted by socio-demographics, health literacy, or acculturation. in this study, with equivalent numbers of hispanic and non-hispanic participants, we found that hispanic and spanish-speaking parents of children with newly diagnosed cancer had inadequate informed consent comprehension, voluntariness and health literacy despite high satisfaction. our study suggests that hispanics and individuals with limited english proficiency are not making truly informed decisions for their child with cancer. to ensure the ethical practice of research in pediatric oncology, the informed consent and decision-making process must be improved with culturally and linguistically interventions for these underserved populations. memorial sloan kettering cancer center, new york, new york, united states background: pediatric oncology patients undergo repeated bone marrow aspirations and biopsies (bma/bx). these potentially painful procedures can exacerbate anxiety and distress. standard practice at memorial sloan kettering (msk) department of pediatrics is to use propofol, which has amnestic but no analgesic properties. we sought to evaluate whether the addition of local anesthetic would improve patient experience with bma/bx. the purpose of reppair: reducing procedural pain and improving recovery of quality of life (qol) (nct02924324) is to evaluate the efficacy of local anesthesia with ropivacaine in reducing procedural pain and improving post-procedure qol in pediatric neuroblastoma patients undergoing bma/bx with general anesthesia. reppair is a prospective, randomized, crossover clinical trial that opened for enrollment october 2016. eligible patients were 3 -18 years old with neuroblastoma. participants were observed on trial for two sequential bm procedures; one procedure with intervention a: propofol alone (pa), and the other with intervention b: propofol plus ropivacaine (p+r). participants were randomized to intervention sequence ab or ba and were blinded to the order of interventions. participants and recovery room (rr) nurses, who were also blinded, followed a standardized postprocedure pain management algorithm. the primary endpoint was percentage of participants requiring opioid analgesia in the 24 hours post-procedure. secondary endpoints included total opioid in 24 hours, non-opioid analgesia use, pain scores, time to first opioid, and short-term qol. qol was assessed by a parent-proxy metric that evaluated pain interference with sleep, physical, emotional, and social recovery. as of january 2018, 105 patients were assessed for eligibility and 56 patients were randomized (47 have completed both procedures). for the primary endpoint, a slightly higher proportion of participants required opioid for pa than p+r (24% versus 21%, p = 0.6). pain scores in the rr were significantly higher for pa than p+r (median [25th, 75th percentile]: 2 [0,4] versus 0 [0,2], p = 0.004). there were no statistically significant differences in total opioid or non-opioid analgesia, 6-and 24-hour pain scores, median time to first opioid, or pain interference scores. there were no adverse events. conclusion: preliminary findings of the reppair trial suggest that local anesthesia does not reduce the need for opioid analgesia or improve short-term qol in pediatric patients undergoing bma/bx with general anesthesia. local anesthesia did improve pain scores in the immediate recovery period. final results of this study will help establish evidence-based guidelines and optimize the experience of pediatric patients with bone marrow procedures at our center. background: children with advanced cancer experience a range of symptoms throughout treatment or at end of life, some of which are poorly controlled. minimizing suffering, including effective symptom management, in children with advanced cancer is a central value for pediatric oncology clinicians. patient-reported outcomes have been used in symptomrelated research in pediatric oncology patients; however the majority of literature specific to symptoms during palliative care and end of life for children and adolescents with advanced cancer is based primarily upon medical record reviews and to a lesser extent, patient self-report. the purpose of this study was to prospectively describe symptom frequency, severity, and level of distress in children/adolescents with advanced cancer using patient selfreport and parent proxy. design/method: a prospective cohort design was used for this study. five pediatric oncology institutions from across the united states participated. children and adolescents were eligible to participate if they were 7-18 years of age, englishspeaking, and had a diagnosis of advanced cancer, defined as a 2-week history of progressive, recurrent, or non-responsive disease or a decision not to pursue curative-focused therapy. a modified version of the memorial symptom assessment scale (msas) was used to measure symptom frequency, severity, and level of distress and was administered to child/parent dyads electronically via smartphones every two weeks. information regarding disease status and cancer treatment was collected concurrently. data was analyzed using descriptive statistics and univariate logistic regression analysis. results: a total of 47 children and adolescents and 47 parents participated in the study. the median age of child participants was 13 years, with half being male. the median age of parents was 46 years. the child participants had a variety of primary diagnosis, including: leukemia/lymphoma (n = 11, 23%), solid tumor (n = 21, 45%), and brain tumor (n = 15, 32%). the most frequently reported symptoms by children with advanced cancer and parents were pain (n = 195/562, 34.70%), lack of energy (n = 186/561, 33.16%), and nausea (n = 156/560, 27.86%). presence of disease (p = <0.0001), recent disease progression (p = 0.0002), and receiving cancer therapy (p = 0.0004) were significant factors on the presence of pain. high intensity cancer therapy was a significant factor on pain frequency (p = 0.0445) and level of distress (p = 0.0224). it is feasible to collect data prospectively in children with advanced cancer regarding symptom frequency, severity, and level distress. clinicians' increased understanding of the symptom experience may promote communication with children and adolescents and timely intervention. more research is needed to understand symptom clusters in children with advanced cancer. vanderbilt children's hospital, nashville, tennessee, united states background: febrile neutropenia (fn) is a frequent occurrence in children undergoing chemotherapy. though guidelines recommend adding a second antibiotic to broad-spectrum antipseudomonal coverage in specific scenarios, augmenting empiric therapy with a second antibiotic is common practice. additional empiric antibiotic (aea) use increases the risk of antibiotic toxicity and future antimicrobial resistance. data clarifying the indications for aea are limited in pediatric patients. objectives: to identify risk factors for gram-positive (gp) and gram-negative (gn) bacteremia in patients presenting with fn to determine situations in which aea use is warranted. design/method: a retrospective chart review was conducted of pediatric severe fn with absolute neutrophil count <500/ l occurring at a single institution between 2006 and 2013. potential a priori risk factors based on clinical reasons for antibiotic expansion were chills, hypotension, mucositis, skin or soft tissue infections (sstis), recent administration of highdose cytarabine (hdac), and a diagnosis of acute myeloid leukemia (aml). potential factors for gn bacteremia were chills, hypotension, mucositis, and abdominal pain. the association between each potential risk factor and gp or gn s205 of s301 bacteremia was identified. logistic regression was used for multi-variable analysis. the review yielded 701 episodes. gp bacteremia was isolated in 75 cases (10.7%) and gn bacteremia in 37 episodes (5.3%). in multivariable analysis, hypotension (or 3.5 (95% ci 1.7, 7.2), p = 0.001) and sstis (or 3.1 (1.1, 8.7) , p = 0.036) were independently associated with increased risk of gp bacteremia, while mucositis (p = 0.376), recent administration of hdac (p = 0.34) and chills (p = 0.161) were not. ten patients with aml didn't receive hdac, thus the association between aml and gp bacteremia could not be reliably estimated. hypotension (or 4.9 (2.2, 11.0), p<0.001) and chills (or 5.0 (2.5, 10.1), p<0.001) were independently associated with a higher risk of gn bacteremia, while mucositis (p = 0.196) and abdominal pain (p = 0.509) were not. of the 37 gn infections, 6 (16%) were resistant to cefepime, the empiric agent of choice at our institution. patients with fn with sstis, hypotension, or recent hdac had increased risk of gp bacteremia indicating potential benefit of empiric vancomycin in these settings, while mucositis and chills were not associated with gp bacteremia. hypotension and chills were associated with gn bacteremia, potentially warranting empiric antibiotic expansion, while mucositis and abdominal pain were not. identifying specific indications for aea use in pediatric severe fn use may improve antimicrobial utilization, decrease unnecessary antibiotic use, and improve patient outcomes. background: for children/young adults with incurable high grade gliomas (hggs), like diffuse intrinsic pontine glioma (dipg) or glioblastoma multiforme (gbm), oncologists endeavor to align therapy with patient/family goals of care, but may be influenced by providers' preferences or limited resources. ethical challenges can arise around the perceived purpose, risks and benefits of therapy options, provider conflicts of interest, access to care, deciding decisional priority between patients and families, and conflicts around end-oflife care. objectives: evaluate factors that play into longitudinal decision making for children and young adults with hggs, their families and oncologists using a qualitative approach with ethnographic elements. design/method: eligible patients were aged 0-21 with dipg, gbm, or secondary hgg. patient exclusions included: non-english speaking, in state custody, death prior to diagnosis, seen by oncology once, or an oncologist declined participation. key decision making visits (e.g. mri reviews) were serially audio-recorded, along with subsequent 1:1 semistructured interviews with patients and/or parents about the decision making process. field notes from clinician meetings, chart notes, and oncologist questionnaires were obtained. discussions and interviews were transcribed and independently coded by three investigators. inter-rater reliability was assessed during code book development. discrepancies were discussed until consensus met. constant comparison analysis with maxqda software continued until thematic saturation. results: twenty-two of 34 eligible patients were approached; 15 agreed to participate. one withdrew upon transferring care. mean age was 9.9 years (sd 5.9); 71% male, 50% caucasian, 29% african american, 14% hispanic, and 7% asian. four encounters, (2.5 hours), were recorded on average per patient. parent/patient interview themes included: 1) hope (for a cure, prolonged life, and quality of life), 2) importance of physician recommendations, 3) importance of support systems (family, community, social media), 4) food (as cancer etiology, intervention) 5) finances (personal, research funding), 6) communication (with medical providers, family, community), 7) death, and 8) god (beliefs, prayer, existential questions). oncologists desired prolonged quality of life, while patients/families transitioned to that hope from hope for a cure. decisions made in the setting of hggs are multi-factorial, ultimately reflecting the competing values of decision makers. optimism about treatment efficacy is held in tension with poor prognosis, allowing for functional hope. acknowledging patients' and families' shifting hopes allows for changes in goals of care and shared decision making. future work is needed to 1) develop preference tools for pediatric patients and families to inform medical providers and 2) provide training in communication and shared decision making with oncologists. emory university, atlanta, georgia, united states background: bone marrow transplantation (bmt) is a potentially curative but underutilized treatment for scd. our previous work has shown that there is variation in physician philosophy and practice in considering bmt as a treatment option for patients with scd, and physicians may not discuss this with patients and families as a potential treatment option. in a randomized clinical trial to test the effectiveness of a decision aid for disease modifying therapies for sickle cell disease, adult patients with scd as well as caregivers of adult/pediatric patients were interviewed about how they seek or have sought information related to scd, made decisions about treatments for scd, and identified a treatment option they were interested in learning more about using the decision aid tool. we performed a secondary analysis of these baseline data to understand patient information needs and attitudes regarding bmt as a treatment option for scd. the goals of this analyses was to understand patient and caregivers' attitudes and perceived information needs regarding bmt as a treatment option for scd. we performed an analysis of baseline interviews from caregivers of patients with scd or adult patients from a randomized control trial for a decision aid tool for scd. 13 of the 38 interviews belonged to caregivers of patients with scd. in addition to reviewing interviews for discussion of bmt, we interrogated for mention of terms such as 'bone marrow transplant' or 'cure' or 'stem cell transplant'. interviews were coded using nvivo10 and analyzed for emerging themes. results: of the 98 baseline interviews, 38 interviews met selection criteria. thirteen of the 38 interviews were with caregivers of pediatric patients, and the remainder were with adult patients, including young adult patients with scd. the majority of participants want to learn about bmt or curative options. in many participants, this was expressed despite knowledge that they were not a likely candidate for transplant. desired information about bmt included eligibility, benefits, risks, long-term effects, quality of life and financial aspects related to bmt. of the patients who discussed how they learnt about bmt, approximately half mentioned that their healthcare provider had not previously mentioned this to them. we then examined knowledge of bmt and attitudes with demographic and clinical variables. patients and caregivers of pediatric patients with scd want to learn about bmt as a treatment option. healthcare providers should consider discussing bmt with their patients with scd. natasha frederick, anna revette, alexis michaud, jennifer mack, sharon bober dana-farber cancer institute, boston, massachusetts, united states background: adolescents and young adults (ayas) consistently identify the need for improved patient-clinician communication on sexual and reproductive health (srh) issues. however, oncology clinicians do not routinely integrate srh conversations with ayas through disease treatment and survivorship. little is known about why these conversations do not take place. objectives: explore aya perceptions of and receptiveness to srh communication with oncology clinicians and to identify barriers and facilitators to these conversations. design/method: semi-structured interviews were held with 21 aya cancer patients and survivors (ages 15-25 years, 6 men, 15 women). twelve participants were on active treatment and 9 were within 2 years of treatment completion. interviews were conducted in english by phone or in person. the interview transcript underwent pre-testing with ayas. all interviews were audio-recorded and transcribed verbatim. transcripts were analyzed and summarized by two trained qualitative researchers according to standard comprehensive thematic qualitative analysis methods. analyses were aided by nvivo11 software. results: ayas perceived existing srh communication between ayas and oncology providers as inadequate. all ayas reported a need for improved srh communication with oncology providers, and three key areas of need emerged: 1) general education; 2) addressing specific srh issues experienced during treatment and survivorship; and 3) understanding the long-term impact of cancer and treatment on srh. ayas felt that current srh discussions are limited and too narrow in scope and scale. ayas reported that most srh conversations focus exclusively on fertility (n = 17), usually taking place at the start of treatment. other additional yet limited communication reported was about sexual activity (n = 7), contraception (n = 7), sexual function (n = 1). no ayas reported conversations about potential treatment complications related to sexuality other than infertility. key barriers to srh conversations include patient discomfort initiating conversation (n = 14) and presence of family members (n = 10), with additional reported barriers including perceived provider discomfort (n = 4), lack of rapport with provider (n = 4), and age/gender differences (n = 4). ayas felt that s207 of s301 communication tools such as handouts, brochures, and websites would be helpful facilitators to direct communication from the oncology clinician, and wanted conversations to start before treatment initiation and to continue through treatment and survivorship conclusion: ayas identify a key role for pediatric oncology providers in srh care from diagnosis through survivorship, however multiple barriers interfere with discussions about srh on a regular basis. identified barriers suggest that future efforts should focus on provider education and training in srh and srh-related communication in order to optimize care provided to this unique patient population. background: peripherally inserted central venous catheters (picc) provide secure vascular access in pediatric patients for the delivery of necessary therapies. the ease of placement in the inpatient and outpatient settings has expanded their utilization. however, recent data analyses show a significant increase in venous thromboembolism (vte) risk with the use of picc lines. with its rising use, modifiable risk factors need to be understood for preventative measures. objectives: in this study we aim to understand patient and catheter specific characteristics in relation to the development of vte. design/method: with irb approval, a retrospective interrogation of the electronic medical record and a picc database, at rainbow babies and children's hospital, was completed. the study cohort contained patients < 18 years of age who had a picc line placed between january of 2004 and december of 2016. data collected included indication for line placement, line dwell time, location of insertion including blood vessel and extremity, number of attempts at line placement, lumen size and indwelling line length. in addition, we collected number of days to vte formation, associated symptoms and location of vte. chi-squared analyses and fischer's exact test were used where appropriate for statistical analysis. we analyzed 3729(1098 neonatal) newly placed picc lines. fifty line-associated vte events were found, for an incidence of 1.3%. all vte occurred with the placement of the first picc line. intravenous therapies were the most common reason for line placement. no statistical significance was found between various indications for placement. the most common symptom of vte manifestation was extremity swelling, follow by extremity pain. right extremity picc was found to have a higher incidence of vte. larger catheter lumen sizes (> 4 french) had a higher incidence of vte. we found a mean time of 20.07 days to vte detection. we were unable to find any clinical, patient or line specific factors leading to increased vte formation after statistical analysis. special consideration should be given to the duration of picc line use as this may reduce the incidence and comorbities associated with vte. there is still much to be understood about catheter associated vte formation as our analyses indicates the need for prospective data collection on a larger scale in hopes to create guidelines related to catheter use in pediatrics. background: the decision to transfuse a patient is a complex one and is never based solely on a number; however, certain hemoglobin or platelet count thresholds have been proposed in aiding physicians make transfusion decisions. in our hospital, the thresholds for packed red blood cell (prbc) and platelet transfusion in pediatric oncology patients are hemoglobin levels below 8.0 g/dl and platelet counts below 20,000/mm3 (< 35,000 for brain tumors), respectively. recently, these thresholds have been questioned and we were asked whether we could safely lower the thresholds to < 7.0 g/dl of hemoglobin and < 10,000 /mm3 platelet count objectives: to investigate platelet and hemoglobin transfusion thresholds for oncology patients at children hospital of michigan design/method: retrospective chart review over a 6-month period, examining platelet and hemoglobin pretransfusion levels for each prbc and platelet transfusion given to oncology patients results: over the course of 6 months, 60 eligible oncology patients (median age 6 years) received 584 transfusions (233 prbc transfusions and 351 platelet transfusions). the mean pretransfusion hemoglobin level was 7.6 ± 1.0 g/dl (range 2.3-11.9) (n = 233) for total prbc transfusions and this was not different among disease categories (p = 0.146). patients who had anemia symptoms and signs (n = 190) had a slightly lower hemoglobin level compared to those who did not (n = 43): 7.5 ± 1.0 vs 7.8 ± 0.9 g/dl (p = 0.058). the mean pretransfusion platelet count was 24,570 ± 15,151 /mm3 (range 2,000 -104,000) for total platelet transfusions (n = 351); 32,800 ± 14,586 /mm3 in patients with brain tumors (n = 84); 20,610 ± 15,270 in patients with leukemia (n = 119); and 23,090 ± 13,628 in patients with solid tumors (n = 148). the mean pretransfusion platelet count was significantly higher in transfusions for brain tumors compared to that in the other disease groups (p<0.001 for both). the mean pretransfusion platelet count was not different among those patients who had bleeding/bruising symptoms (25,150 ± 17,898, n = 115) versus those who did not (24,290 ± 13,648, n = 236) (p = 0.620). the bleeding/bruising rate was slightly but insignificantly higher in those who had platelet counts <10,000 vs those who had ≥ 10,000 (38.2% vs 32.2%, p = 0.564). since most patients develop symptoms of anemia at hemoglobin above 7 g/dl and about 1/3 of patients develop bleeding/bruising symptoms at platelet counts above 20,000 /mm3, our current policy so far reflects a safe threshold for transfusion, and further lowering of the thresholds should be investigated in prospective studies. background: renal impairment is an important complication of childhood cancer and its treatment. serum creatinine level is frequently used as a screening test to monitor renal function; however, patients can have significantly decreased glomerular filtration rate (gfr) with normal serum creatinine. to determine the prevalence of chronic kidney disease (ckd) among children with cancer diagnosis, based on calculated gfr. to compare the difference between using serum creatinine value alone versus gfr in detecting ckd. design/method: retrospective review of medical records of 150 patients, age 1-18 years, diagnosed between 1/2011-12/2016 with solid tumors were analyzed. serum creatinine and calculated gfr using schwartz formula were recorded. ckd as classified by the foundation of kidney disease and outcome quality initiative was used: ckd stage 2: gfr (60 to 89 ml/min per 1.73 m2) ckd stage 3: gfr (30 to 59 ml/min per 1.73 m2) statistical analysis using spss software v.23. chi-squared test for proportions within group, and pearson chi-squared and fisher exact tests for statistical differences between groups. p-value <0.05 was considered to indicate significance results: out of the 150 records reviewed, 81 (54%) were males and 69 (46%) females, with mean age of 9.2±5.6 years. 37 (24.6%) patients received one or more of nephrotoxic chemotherapy drugs; cisplatinum, carboplatinum, or ifosphamide mainly in the non-wilms solid tumors group (94.5%) compared to (5.5%) in the wilms tumor (wt) group. based on calculated gfr (by schwartz formula) ckd stage 2/or 3 was diagnosed in 66 (44%) patients with overwhelming majority (98%) were in the mild stage 2 ckd, only 3 (4.5%) of those patients had abnormally high serum creatinine levels (p = 0.006). 56.7% of patients who received nephrotoxic chemotherapy developed ckd, compared to 39.4% in those who did not receive it, (p = 0.01). despite that only 2/18 (11%) of wt group patients received nephrotoxic chemotherapy, yet this group had higher percentage of ckd (83.3 %) compared to non-wt group (34.8%) p = 0.02. significantly lower mean gfr 73.8±10 was noticed in the wt group compared to 99.8±29 in non-wt group (p = 0.001) conclusion: high prevalence of mild ckd was found among solid tumor patients. using serum creatinine alone as measure of renal function significantly under estimates renal impairment in those patients. early identification of ckd is easily achieved by using calculated gfr, which can helps providers and care givers to avoid potential nephrotoxic antibiotics, contrast media, nsaids and dehydration that may further deteriorate renal function the university of texas southwestern medical center, dallas, texas, united states background: children with down syndrome (ds) have increased risk of developing leukemia. pediatric patients with ds-associated acute lymphoblastic leukemia (ds-all) are known to have significant toxicities with reinduction chemotherapy and historically poor outcomes with stem cell transplant (sct). anti-cd19 chimeric antigen receptor (car) t-cell therapy, tisagenlecleucel, demonstrated high rates of durable complete remission (cr) and a manageable safety profile in children with r/r b-cell acute lymphoblastic leukemia (b-all). objectives: characterize the efficacy and safety of tisagenlecleucel in pediatric/young adults with ds-all. design/method: pooled data from 2 single-arm, multicenter, phase 2 trials of tisagenlecleucel in pediatric/young-adult patients with r/r b-all (eliana, nct02435849; ensign, nct02228096) were analyzed. eight patients with ds-all were enrolled (data cutoff: eliana, 23 november 2016; ensign, 1 february 2016). seven were infused with tisagenlecleucel; 1 patient died from all progression and intracranial hemorrhage before infusion. no manufacturing issues occurred during production. 5/7 infused patients were male, 2/7 had prior sct (age range, 6-16 years). 6/7 patients achieved cr or cr with incomplete blood count recovery (cri) by day (d) 28 (cr+cri, 86%); 1 died before d28 and was not evaluable. analysis of minimal residual disease was negative in bone marrow in responding patients. two patients had cd19negative relapses at 5 and 8 months. ongoing remissions in 4 patients without relapse ranged from 2 to 11 months. the safety profile (n = 7) appears similar to that in patients without ds in the same trials (n = 90). grade (g) 3/4 cytokine release syndrome occurred in 43% (3/7) of patients with ds and in 44% without ds. rates of other g3/4 adverse events of special interest did not appear to favor a consistent trend between patients with/without ds (febrile neutropenia: 43% vs 36%; neurological events: 14% vs 11%; tumor lysis syndrome: 14% vs 2%). g3/4 infections were not observed in patients with ds (0% vs 23%). one patient died after infusion due to intracranial parenchymal hemorrhage on d15 associated with ongoing coagulopathy. time and extent of tisagenlecleucel expansion and long-term persistence were similar between groups. conclusion: this is the first analysis of car t-cell therapy in pediatric patients with r/r b-all and ds. these data suggest that toxicities appear similar to those in patients with b-all without ds, remission rates in ds-all are high, and longterm outcomes with sustained persistence appear promising. further exploration of tisagenlecleucel as an alternative to sct in children with r/r ds-all is warranted. sponsored by novartis. background: hispanic adolescence and young adults are twice as likely to develop acute lymphoblastic leukemia (all) with high risk features as non-hispanic whites. they also have poor prognosis and 39% higher death rate. b-all with crlf2 overexpression caused by genetic alteration of the cytokine receptor, crlf2 is five times more common in this subgroup. approximately 80% of crlf2 b-all cases also have ikzf1 genetic alterations. ikaros is involved in transcriptional regulation of several important genes involved in leukemogenesis. overexpressed casein kinase ii (ck2) impairs functions of ikaros. objectives: understand the molecular mechanisms that regulate crlf2 expression in crlf2 b-all. here we present evidence that ikaros-mediated repression of crlf2 transcription in b-all in hispanic children is regulated by ck2. design/method: primary b-all patient samples from hispanic children were used. ikaros retroviral transduction, ikaros shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: ikaros binding to promoter of crlf2 was confirmed using quantitative chip. functional experiments such as overexpression of ikaros in b-all primary cells results in transcriptional repression of crlf2 whereas ikaros silencing using shrna resulted in increased transcription. these results suggest that ikaros negatively regulates crlf2 expression. molecular inhibition of ck2 with shrna targeting the ck2 catalytic subunit, as well as pharmacological targeting of ck2 with cx4945 resulted in transcriptional repression of crlf2. ck2 inhibition was associated with increased ikaros dnabinding to the promoter of crlf2. however, the ability of cx4945 to repress crlf2 is lost or severely reduced, in cells with shrna silencing of ikaros, as compared to cells with intact ikaros. moreover, similar results were noted following treatment with cx4945 in leukemia cells obtained from high risk b-all patients with deletion of one ikzf1 allele. ikaros binds poorly to promoters of crlf2 gene in these cells. treatment with cx4945 restores ikaros dnabinding to the promoters of crlf2, which is associated with its strong repression. serial qchip analysis of the epigenetic signature at the crlf2 promoter showed that increased ikaros binding to the crlf2 promoter, following ck2 inhibition, is associated with enrichment for the h3k9me3 histone modification, which is a marker of repressive chromatin. results demonstrate that crlf2 expression is epigenetically regulated by the ck2-ikaros axis .cx4945 show antileukemic effect via restoration of ikaros tumor suppressor function, resulting in crlf2 repression suggesting advantage of using ck2 inhibitors as potential therapeutic approach in crlf2 altered b-all. results: hypodiploid all (modal chromosome number <44 and/or di <0.81) was identified in 131 patients (1.6% of all patients; 0.9% of nci standard risk (sr) and 2.9% of nci high risk (hr)), who were removed from frontline protocol therapy post-induction. overall 5-year efs and os were 52.1%±4.9% and 58.9%±4.8%. transplant status was retrospectively available for 113/131 (86%), 61 of whom underwent hsct in cr1. five-year efs with hsct was 57.4%±7.0% vs. 47.8%±7.5% without (p = 0.48). 5-year os with and without hsct was 66.2%±6.6% vs. 53.8%±7.6% (p = 0.34). when corrected for the median time to hsct (137 days), there were no significant differences in 5-year efs or os rates with and without hsct: 53.1%±7.3% and 64.2%±6.9% vs. 48.8%±7.8% and 53.8%±7.8%. no nci risk group or mrd subset benefitted significantly from cr1 hsct. sr patients (n = 42) had 5-year efs and os of 68.8±10.3% and 77.3%±9.2% with hsct (n = 27) vs. 57.1%±13.2% and 64.3%±12.8% without. hr patients (n = 71) had 5-year efs and os of 48.3%±9.0% and 57.6%±8.8% with hsct (n = 34) vs. 44.4%±9.2% and 49.9%±9.4% without. for those with end-induction mrd <0.01% (n = 74), 5-year efs and os were 66.3%±7.9% and 79.5%±6.7% with hsct (n = 39) vs. 60.3%±9.2% and 66.7%±8.8% without. end-induction mrd-positive patients (n = 30) fared poorly with both 5year efs and os of 29.4%±14.3% with hsct (n = 18) vs. 16.7%±10.8% and 22.2%±13.9% without. multivariate regression analysis including nci risk group, mrd, and cr1 hsct, showed only mrd negativity was significantly associated with efs (hr 0.256, p<0.0001) and os (hr 0.216, p<0.0001). patients with hypodiploid all fare poorly, particularly those with end-induction mrd ≥0.01%. while cr1 hsct is a standard treatment approach, it does not confer significant benefit. we were unable to assess bridging therapy prior to hsct, and comparator groups are small. taken together, however, new strategies are urgently needed for these patients. background: ras-pathway mutations are known to play a pivotal role in a significant proportion of myeloid malignancies, including upwards of 20% of pediatric aml cases. ras-pathway mutations in myeloid malignancy commonly co-occur with mutations of epigenetic regulators, suggesting cooperative leukemogenesis. among the epigenetic modifiers most frequently mutated in myeloid malignancy are regulators of dna methylation. this indicates that the alteration of dna methylation contributes to leukemogenesis. the ten-eleven translocation 2 (tet2) is an epigenetic regulator that plays an important role in regulation of dna methylation through its action of hydroxylation of 5-methylcytosine, which ultimately leads to passive de-methylation of dna cytosines. in myeloid malignancy, loss of function tet2 mutation is one of the most frequently co-occurring lesions in ras mutated malignancy. how specifically the altered methylation patterns in ras-pathway driven diseases promotes leukemogenesis is unclear. objectives: we hypothesize in mice with a ras-pathway mutation, that when an epigenetic modifier co-occurs, such as loss of function of tet2, this primes stem cells and/or early differentiating progenitors for transformation by preventing the repression of stem cell self-renewal genes, inhibiting differentiation, enhancing ras signaling and leading to leukemogenesis. we have generated a novel murine model with constitutive deletion of tet2 (tet2-/-) combined with an inducible activating krasg12d mutation (krasg12d/wt). mice have been tracked for evidence of hematologic malignancies and compared to mice with corresponding single genetic lesions. cooperative leukemogenesis will be demonstrated by decreased latency to disease onset, impact on malignancy lineage, in addition to investigating mechanistically through which pathways leukemogenesis may be promoted. results: krasg12d/wt/ tet2-/-mice demonstrate statistically significant differences in peripheral white blood cell count, hemoglobin, and platelet levels as early as 4-weeks post ras-pathway activation. peripheral cell lineage analysis demonstrates early skewing toward myeloid differentiation and marked splenomegaly in mice harboring both genetic lesions compared to wild type or mice with single genetic lesions. phospho-flow cytometric analysis reveals increased perk and ps6 activation in krasg12d/wt/ tet2-/-sca-1 enriched bone marrow cells compared to either genetic lesion alone. our study utilizing a murine model to examine how in ras-pathway mutations the addition of a co-occurring epigenetic lesion demonstrates that these lesions appear to cooperate to promote early myeloid differentiation with attendant changes in signaling pathways. this exploration to elucidate the mechanics of ras-pathway mediated disease lay the foundation for identification of patients who may benefit from existing therapies, such as dmtis, or identify new signaling targets for therapeutic exploration. background: the humoral immunogenicity of car19, a chimeric antigen receptor (car) with a murine scfv domain developed for treatment with tisagenlecleucel in relapsed/refractory (r/r) pediatric/young-adult acute lymphoblastic leukemia (all), was evaluated in 2 studies. little is known about the presence/impact of preexisting/treatmentinduced anti-murine car19 (mcar19) antibodies in patients treated with car therapy. objectives: patients from eliana (nct02435849; n = 68) and ensign (nct02228096; n = 29) were evaluated before and after tisagenlecleucel infusion to determine the impact of anti-mcar19 antibodies on cellular kinetics, efficacy, and safety. design/method: anti-mcar19 antibodies were determined by flow cytometry and reported as median fluorescence intensity. assay validation included evaluation of the interferences of intravenous immunoglobulin (ivig) treatment with the anti-mcar19 antibody assay. impact of preexisting and treatment-induced immunogenicity on cellular kinetics, efficacy, and safety was determined. treatment-induced immunogenicity was defined by a positive increase in anti-mcar19 antibody levels over baseline and was assessed by calculating the fold-change between preexisting (ie, baseline) and postinfusion levels. results: 90% of patients displayed preexisting anti-mcar19 antibodies; a similar incidence was detected in healthy volunteer samples during method validation. 35% of patients developed treatment-induced anti-mcar19 antibodies. no relationship was identified between tisagenlecleucel expansion (auc0-28d) and preexisting/treatment-induced anti-mcar19 antibodies (r2<0.001 and r2 = 0.006, respectively); similar results were seen for cmax. presence of treatment-induced anti-mcar19 antibodies did not appear to impact transgene persistence or response. kaplan-meier estimates showed that preexisting/treatment-induced anti-mcar19 antibodies did not appear to impact duration of response or event-free survival. strip plots showed consistent levels of preexisting/treatment-induced anti-mcar19 antibodies across patients with safety events, including cytokine release syndrome, neutropenia, thrombocytopenia, and neurological events. there was no apparent relationship between treatment-induced anti-mcar19 antibodies and b-cell recovery categories (≤3months, >3 and ≤6months, >6months, and ongoing sustained aplasia). no association existed between time of b-cell recovery and presence of treatment-induced anti-mcar19 antibodies. b-cell aplasia requiring ivig occurred following tisagenlecleucel in the majority of patients. the tisagenlecleucel concentration-time profiles in patients with treatment-induced anti-mcar19 antibodies were categorized by time following ivig administration. time of ivig administration had no impact on in vivo transgene expansion and persistence. we report the first comprehensive assessment of the impact of anti-mcar19 antibodies on clinical endpoints with car therapy. pediatric/young-adult patients with r/r all had a high frequency of baseline anti-mcar19 antibodies, and preexisting/treatment-induced anti-mcar19 antibodies did not impact the cellular kinetics, safety, and efficacy of tisagenlecleucel. cell-mediated immunity studies are ongoing. sponsored by novartis. background: adoptive immunotherapy, using cd123 engager (cd123-eng) t-cells, has shown success in preclinical studies, recognizing and killing acute myeloid leukemia (aml) blasts in vitro and in vivo. cd123-eng t-cells secrete bispecific molecules that recognize cd3 (t-cells) and cd123 (aml blasts), and are able to direct transduced t-cells and recruit bystander t-cells to kill cd123-positive blasts. however, cd123-engs do not provide costimulation and have not shown the capability for sequential killing of targets in vitro. we are seeking to improve the expansion, persistence and sequential killing capabilities of cd123-engs by genetically modifying these cells with an inducible costimulatory molecule, which can be activated by a chemical inducer of dimerization (cid). we generated a retroviral vector encoding cd123-eng and the inducible costimulatory molecule myd88.cd40 linked by a 2a sequence (cd123-eng.2a.imc). cd123-eng and cd123-eng.imc t-cells were generated by retroviral transduction, and their effector function was compared with and without cid. we used flow cytometric analysis to assess transduction efficiency, chromium release assays to evaluate cytolytic activity, and elisa to determine cytokine production. we successfully generated cd123-eng.imc tcells and achieved a mean initial transduction efficiency of 63% that was maintained above 50% throughout our study period. cd123-eng.imc t-cells +/-cid and cd123-eng t-cells readily killed cd123-positive aml blasts (molm13 and kg1a) in cytotoxicity assays when compared to the cd123-negative control (k562). in co-culture assays, cd123-eng.imc t-cells secreted increased il-2 and ifn-gamma in the presence of cid and cd123-positive targets (kg1a and molm13) when compared to co-culture with cd123-positive targets in the absence of cid. in addition, cd123-eng.imc t-cells displayed enhanced sequential killing capabilities and ifn-gamma secretion when stimulated weekly with cid and tumor cells at a 1:1 ratio when compared to cd123-eng t-cells. conclusion: cd123-eng.imc t-cells are able to recognize and kill cd123-positive aml blasts in an antigen dependent manner. cd123-eng.imc t-cells have improved effector function in the presence of cid as judged by cytokine production and their ability to sequentially kill cd123-positive target cells. thus, inducible myd88 and cd40 costimulation is a promising strategy to improve the effector function of cd123-eng t-cells, and warrants further active exploration in preclinical studies. background: eliana (nct02435849; n = 75) is a pivotal multicenter study testing the efficacy of tisagenlecleucel, anti-cd19 car-t, in children/young adults with r/r b-all. tocilizumab (toci) has been used for management of moderate/severe (grade 3/4) crs in ≈38% of patients treated with tisagenlecleucel at equivalent doses used in approved nononcological pediatric indications (<30kg received 12mg/kg; ≥30kg received 8mg/kg [800mg max dose]).(1) crs onset, as graded by the penn grading scale, generally occurred at a median of 3 days (range, 1-22) after infusion, requiring administration of 1-3 toci doses in some patients via a protocol-specific treatment algorithm. toci is a humanized monoclonal antibody that inhibits il-6 receptor (il-6r) signaling. the pharmacokinetics (pk) and pharmacodynamics (pd) of toci in pediatric patients with b-all with carassociated crs have not previously been described. objectives: characterize toci pk/pd for crs management following tisagenlecleucel infusion and describe its impact on cellular kinetics. design/method: toci pk and levels of soluble il-6r (sil-6r) were determined from serum and quantified using validated assays. maximum toci concentration (cmax) was derived using noncompartmental methods. sil-6r, proinflammatory cytokines, and crs resolution time were characterized to describe toci pd. summary statistics and graphical analyses of tisagenlecleucel exposure by number of doses were performed to describe the impact of toci on tisagenlecleucel kinetics in patients responding to tisagenlecleucel infusion. : 28/58 patients with crs received the first toci dose at a median of 5 days (range, 1-18) after crs onset. seventeen patients received 1 dose (range, 6.9-12 mg/kg); 8 received 2 doses (8-12 mg/kg); 3 received 3 doses (8-12 mg/kg), per the crs treatment algorithm. first-dose mean cmax (sd) was ≈111(30.6) g/ml; second dose, ≈265(376) g/ml. individual patient pd concentration-time profiles showed increased sil-6r levels after the first toci dose which remained elevated following the second dose. following toci administration, median time to crs resolution (including fever resolution) was 5 days (range, 2-29). crs onset coincided with tisagenlecleucel expansion, followed by a peak in serum cytokines, including il-6. the geometric mean auc0-28day and cmax of tisagenlecleucel transgene (by pcr) were 358% and 216% higher in tisagenlecleucel-responding toci-treated patients. conclusion: crs symptoms resolved within a median of 5 days after toci administration. toci levels achieved in patients with b-all were similar to reported pediatric nononcological indications (tocilizumab label) and resulted in concentration/time-dependent sil-6r increases. transgene continued to expand and persist following toci administration. these data support treatment with toci for crs management. (1) buechner, eha, 2017. sponsored by novartis. background: in acute myeloid leukemia (aml), mesenchymal stem and stromal cells (mscs) in the bone marrow microenvironment contribute to extrinsically mediated chemo-resistance and are therefore important potential therapeutic targets. the study of patient-derived mscs is at a competitive disadvantage, however, because traditional means of isolating mscs from a bone marrow aspirate interferes with isolating the more highly prioritized leukemic cells. many opportunities to study mscs are therefore missed. objectives: to develop a novel method of isolating mscs using the otherwise discarded portion of a bone marrow aspirate, thereby de-coupling the isolation of primary mscs from the isolation of leukemia cells. design/method: aml patient bone marrow aspirates were obtained prospectively from the children's oncology group. healthy patient marrow was purchased. experimental mscs were isolated from the bottom-most layer (rbc-layer) produced by density-gradient separation of a bone marrow aspirate, which is typically discarded. control mscs were isolated from the buffy coat (mnc layer). non-adherent cells were removed after 72 hours, and adherent cells were cultured at 5% co2 with mem-alpha containing 20% fbs. growth curves were obtained by seeding 6-well plates with 10,000 cells per well. cells were stained using oil red o to observe adipocyte differentiation. results: rbc-layer mscs grow successfully following overnight shipment of the aspirate. identical to mnc-layer mscs, rbc-layer mscs exhibit a fibroblastic morphology and are adherent to plastic. rbc-layer mscs persist in culture up to 14 passages before senescence. they exhibit a slower growth curve relative to mnc-layer mscs, but their overall doubling time is similar at approximately 120 hours. surprisingly, mscs from the rbc-layer exhibit adipocyte differentiation on stimulation, revealing their stem-cell like qualities. we present a method of isolating mscs from the discarded portion of a bone marrow aspirate that does not interfere with the isolation of leukemia cells from the same patient. this portion of the aspirate can be shipped, or can sit for at least 24 hours, without sacrificing its mscs. rbclayer mscs are nearly identical to mscs obtained conventionally. perhaps most importantly, rbc-layer mscs retain a stem-cell like capacity, showing them to be a highly valuable cell population in aml research. future plans include investigating potential selective enrichment of stem-cell mscs in the rbc-layer, which could explain the unexpected difference in growth kinetics. aml researchers now have the opportunity to study this exciting component of the bone marrow microenvironment without sacrificing valuable leukemic cells in the process. background: neutropenia is one of the most frequent side effect of chemotherapy associated with an increase in the risk of infection, especially in the cases when the depth and duration of neutropenia are extended. some genes, as variations of darc, gsdma and cxcl2 are known to influence white blood cell and neutrophil counts. our previous study conducted in children with acute lymphoblastic leukemia (all), showed that polymorphisms in these genes might play a role in the onset of chemotherapy complications during consolidation and maintenance treatment. objectives: in order to support our previous finding, we have expanded the study to the induction period in a cohort of 233 all children treated at the sainte-justine university health center between july 1995 and july 2005. design/method: previous associated single nucleotide polymorphisms (snps) in darc, gsdma and cxcl2 genes were analyzed for an association with the complications occurring during induction including the duration of low neutrophil count (pnn) and low absolute phagocyte count (apc), proven infections and delay between induction and consolidation phases. results: significant effect was found for all studied polymorphims. minor alleles of darc rs3027012, cxcl2 rs1680408 and gsdma rs3859192 were all associated with higher risk of complications during induction treatment, whereas that of darc rs12075 (particularly gg genotype) had a protective effect. the gg genotype of rs12075 was associated with a lower risk of post-induction delay (p = 0.02 or = 0.1, 95%ci 0.02-1.0), less frequent febrile episodes (p = 0.02) and lower number of days with apc/pnn count reduction (p = 0.008 for apc<0.5 and p = 0.02 for pnn<0.5). in contrast, the minor t allele of another darc polymorphism (rs 3027012), was associated with longer apc/pnn count reduction (p = 0.01 for apc<0.5 and p = 0.02 for pnn <0.5), as it was the tt genotype of gsdma rs3859192 (p = 0.02 for apc<0.5 and p = 0.04 for pnn<0.5). the patients with the gsdma rs3859192 had also a higher risk of documented febrile episodes (p = 0.04 or = 2.4 95%ci 1-5.5). the aa genotype of rs1680408 cxcl2 was associated with a higher risk of post-induction delay due to infection (p = 0.04, or = 3.4, 95% ci 1.1-11.5). conclusion: this complementary study confirmed our previous results, showing overall that variations in darc, gsdma and cxcl2 genes influence the onset of chemotherapy complications in pediatric all, regardless of treatment phases. these polymorphisms might be useful pharmacogenetics markers possibly guiding an adjustment of chemotherapy intensity. background: pediatric acute myeloid leukemia (aml) has a poor survival rate of about 70% and there is an urgent need for newer targeted therapies. car t-cell based therapies are effective against all but similar therapies against aml are still under development. recent clinical trials have highlighted the concerns about toxicity and therapy related deaths from car t-cells. antigen selection is the key factor determining the specificity, efficacy and toxicity of car t-cells. while contemporary adoptive t-cell therapies use monoclonal antibodies against tumor associated antigens we employed the naturally occurring flt3 ligand (fl) to target aml cells expressing flt3 receptors. flt3 receptor is expressed on multipotent and myelomonocytic progenitors as well as myeloid leukemia cells. to generate fl containing chimeric tlymphocytes designated flcar t-cells and to evaluate their efficacy against aml cells. design/method: flcar was constructed by fusing the coding sequences of the human fl, cd28 costimulatory domain, and cd3-zeta chain (intracellular region) in series. it was then cloned into the phiv-egfp lentiviral vector for expression in cell lines and primary t cells obtained from healthy donors. the empty phiv-egfp vector was used as a negative control. flcar was expressed on both cd4+ and cd8+ t-lymphocytes, confirmed by western blot. cell cytoxicity was evaluated by co-culturing flcar t-cells and aml cells followed by flow cytometric analyses. cytokine production was assessed by analyzing expression of interleukin-2 using quantitative rt-pcr. results: flcar t-cells were generated from cd4+ jurkat and cd8+ tk-1 cell lines with up to 70% lentiviral transduction efficiency. the efficiency for primary t cells was lower (5-10%). flcar was expressed as a ∼42 kda protein in cells and was partially phosphorylated on tyrosine. the expression of flcar on lymphocytes lead to increased basal il-2 expression in the cells. this was further augmented (by > 5 folds) upon co-incubating flcar t-cells with flt3expressing target cells. jurkat cells, tk-1 cells and primary human t cells expressing flcar suppressed the growth of flt3-expressing aml cell lines and primary aml cells in vitro. notably, flcar t-cells generated from healthy donors caused strong inhibition of aml cells even at a lower transduction efficiency. in vivo experiments using nsg-sgm mice xenografted with human aml cells are underway. our data demonstrate that flcar can be effectively expressed on t-lymphocytes and mediate potent cytotoxicity against flt3-expressing aml cells in vitro. being a completely human derived chimeric protein, it represents a promising candidate for further therapeutic development. holly pacenta, kelly sullivan, ahwan pandey, kelly maloney, joaquin espinosa children's hospital colorado, denver, colorado, united states background: individuals with down syndrome (ds) have a 20-fold higher risk of developing acute lymphoblastic leukemia (all) than the typical population. there are several important differences between all in individuals with ds (ds-all) and all in individuals without ds (nds-all): first, patients with ds-all have a lower percentage of favorable cytogenetic features compared to nds-all. second, patients with ds-all are more likely to have activating mutations in jak2, crlf2 overexpression, and ikzf1 deletions. despite these clear genotypic differences, this knowledge has not yet been exploited for therapeutic purposes in ds-all. when outcomes for ds-all are compared to nds-all with similar cytogenetic features, the survival rates are similar. however, individuals with ds-all have an increased risk of treatment-related mortality (trm). current therapy for ds-all is similar to that for nds-all, with the exception of small changes to decrease toxicities that are more prevalent in ds-all. it was recently identified that interferon signaling is constitutively activated in healthy individuals with t21. we hypothesize that aberrant interferon signaling could play a role in the unique leukemias observed in ds patients. objectives: to identify differences in gene expression and intracellular signaling cascades that are unique to individuals with ds-all, relative to both nds-all and healthy individuals with ds that can be exploited for therapeutic use. design/method: bone marrow samples were obtained from ds-all patients and matched nds-all patients based on clinical characteristics and genetic features. rna sequencing of these samples was performed and a total of 19 samples were used for the transcriptome analysis (6 ds-all vs. 13 nds-all). the differential expression data was generated by deseq2 and analyzed using ingenuity pathway analysis. the analysis revealed that the chromosome 21 genes that have been implicated in leukemogenesis are not differentially expressed in the ds-all samples, relative to nds-all. an inflammatory signature was identified, which included interferon gamma as an upstream regulator with predicted activation in ds-all. this finding is consistent with prior observations from healthy individuals with ds. other examples of results with potentially actionable targets include the upregulation of several genes in the ras pathway and genes involved in histone methylation. the increased interferon signaling seen in healthy individuals with ds was also identified in ds-all. this may contribute to the development of mutations in inflammatory pathways such as jak2 and crlf2 in ds-all. targeting these common pathways with small molecule inhibitors may have a therapeutic benefit in ds-all. cincinnati children's hospital medical center, cincinnati, ohio, united states background: next-generation sequencing (ngs) guides precision medicine approaches in oncology using therapies targeting molecular alterations found within an individual cancer. increased availability of ngs coupled with a proliferation of targeted drugs in development heightens the need for reliable pre-clinical animal models. here we report a patientderived xenograft (pdx) system with integrated molecular profiling for pre-clinical testing of conventional cytotoxic and novel targeted agents. objectives: to utilize ngs from patients with pediatric leukemia to guide rational pre-clinical trials in pdx leukemia avatars, and to determine pdx mice tolerance of and response to cytotoxic and targeted therapies. pediatric acute lymphoblastic leukemia (all) samples were obtained in adherence to an irb-approved protocol and xenografted into nod/rag/interleukin-2 (il-2)rg (nrg) mice. ngs was performed clinically using the foundationone® heme panel. a de novo all sample bearing mutations involving jak2, crlf2, ntrk3, cdkn2a/b, ptpn11 and wt1 was used for pre-clinical testing. thirty-seven nrg mice were transplanted with 2 million patient cells/mouse via iv injection. standard 4-drug induction chemotherapy was administered consisting of vincristine, dexamethasone, pegaspargase, and daunorubicin [vxpd, n = 6 mice], in comparison to vehicle control [n = 8]. parallel pdx cohorts were treated with single agent targeted therapies based on ngs findings, including ruxolitinib [n = 7], crizotinib [n = 8] and loxo-101 [n = 8]. the four-week treatment period began on day +30 from transplant after confirmation of engraftment. following completion of therapy, residual disease burden was analyzed by flow cytometry (hcd19+, mcd45-cells) in the bone marrow [bm] . to date, pdx models have been established using over thirty ngs-profiled pediatric all samples, including six samples bearing philadelphia (ph) chromosome or phlike mutations. pre-clinical testing was performed in a repreconclusion: ngs reveals concomitant mutations in ph-like all that may represent additional targets for therapy, or predict tyrosine kinase inhibitor (tki) resistance. we show that all xenograft nrg mice can tolerate a 4-week multi-agent cytotoxic chemotherapy induction regimen, as well as rational targeted agents, and serve as a robust pre-clinical model for precision medicine trials. background: osteonecrosis is a well-characterized all therapeutic toxicity attributed to glucocorticoids, asparaginase, and methotrexate that disproportionately affects adolescents. in ccg-1961, alternate-week dexamethasone during double delayed intensification (di) reduced osteonecrosis vs continuous dexamethasone with single di in rapid early responders (rer) ≥10y. to compare efs and os between hr-all patients with vs without osteonecrosis. design/method: hr-all patients 1-30y on aall0232 (2004-11) received cog augmented therapy with a 2 × 2 randomization to: (1) induction dexamethasone (10 mg/m2 d1-14) vs prednisone (60 mg/m2 d1-28), and (2) interim maintenance (im) high-dose methotrexate (hdm) vs escalating-dose methotrexate/pegaspargase (ema). rer received single, and slow early responders (ser) double, im/di. initially, all received monthly dexamethasone maintenance pulses, patients ≥13y received di alternate-week dexamethasone, and patients ≤12y received di continuous s217 of s301 dexamethasone. there were 2 osteonecrosis-related amendments: after 10/2006 all patients ≥10y received di alternateweek dexamethasone; after 6/2008 all patients ≥10y were assigned to induction prednisone, and all patients received di alternate-week dexamethasone and maintenance prednisone pulses. results: osteonecrosis was confirmed in 315/2817 patients. the 5y cumulative incidence (ci) was 12.7% overall and increased with age: 1-9y 2.6%, 10-12y 15.3% (alternateweek dexamethasone 10.2% vs continuous dexamethasone 23.5%; p<0.0001), ≥13y 19.7% (p<0.0001). among randomized rer patients ≥10y, ci differed by glucocorticoid (dexamethasone 25.0% vs prednisone 15.0%; p = 0.0003) but not methotrexate assignment (hdm 18.8% vs ema 21.5%; p = 0.7). among randomized ser patients ≥10y, ci was 18.9% with no difference by regimen. results were similar for patients ≥13y. in the entire study population, patients with osteonecrosis had superior 5y efs (89.0% vs 76.1%; p<0.0001) and os (95.8% vs 85.9%; p<0.0001) than those without osteonecrosis. 5y efs was significantly higher among randomized patients ≥10y with vs without osteonecrosis (88.5% vs 72.9%; p<0.0001); this finding was present in different age ranges (≥10y, ≥13y, ≥16y) and rer/ser subsets within each, especially in the ≥10y rer (92.6% vs 81.8%; p = 0.0004) and ser (74.0% vs 43.7%; p<0.0001) cohorts. across groups, asparaginase allergy was significantly associated with reduced osteonecrosis risk (≥10y: hr 0.43; p = 0.0013). patients who develop osteonecrosis have significantly increased efs and os, suggesting host differences that increase sensitivity to develop osteonecrosis and render all cells more chemo-responsive. pennsylvania state university, hershey, pennsylvania, united states background: cdc42 (cell division cycle protein 42) belongs to rho family of small gtpases in ras-oncogene superfamily. pro-oncogenic role of overexpressed cdc42 in ras driven solid tumors are well known. however, role of cdc42 in leukemia is yet to be established. ikzf1 encodes ikaros protein which has important role in regulation of lymphoid development and tumor suppression in leukemia. casein kinase ii (ck2) oncogene is overexpressed in leukemia. ck2 impairs ikaros function which can be restored by using ck2 inhibitors. objectives: to investigate role of cdc42 in leukemia and regulation of cdc42 by ikaros and ck2 in b-cell acute lymphoblastic leukemia (b-all). shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: cdc42 is identified as one of the ikaros target genes by analysis of genome-wide dna binding of ikaros using chip-seq and qchip in b-all primary cells. expression of cdc42 was also noted to be higher in all patient samples compared to normal bone marrow. functional experiments showed that ikaros overexpression via retroviral transduction results in transcriptional repression of cdc42. ikaros silencing using shrna resulted in increased expression of cdc42. these data suggest that ikaros negatively regulates transcription and expression of cdc42. ck2 directly phosphorylates ikaros and impairs its function as transcription factor. we noted that molecular inhibition of ck2 via sirna as well as treatment with specific ck2 inhibitor, cx4945 also decreases expression of cdc42. treatment with cx4945 of primary b-all with ikaros haploinsufficiency restores ikaros binding to cdc42 promoter and represses cdc42 expression. however, this effect is evident only in presence of ikaros. treatment with cx4945 in ikaros silenced (ikaros shrna) cells showed no change in expression of cdc42. these results emphasizes the importance of ikaros in regulating cdc42 expression. furthermore, we analyzed the changes in epigenetic signature at the cdc42 promoter following treatment with cx4945. results show that loss of histone marker of open chromatin (h3k9ac) and increased histone marker for repressive chromatin (h3k27me3), at the cdc42 promoter. these data suggest that ikaros transcriptionally represses cdc42 via chromatin remodeling. a specific cdc42 inhibitor, ml141 showed cytotoxic effects on primary b-all cells. conclusion: cdc42 may have important role in hematologic malignancies. expression of cdc42 in b cell all is regulated by ikaros and ck2. these results suggest that targeting cdc42 could be a potential therapeutic strategy in leukemia. caitlyn duffy, laura hall, justin godown, koyama tatsuki, scott borinstein monroe carell jr. children's hospital at vanderbilt, nashville, tennessee, united states background: systemic corticosteroids are widely used as treatment of acute lymphoblastic leukemia (all) and lymphoblastic lymphoma. there are anecdotal reports of bradycardia in pediatric patients receiving corticosteroids, but a more extensive analysis of this effect is needed. objectives: the aim of this study was to describe the incidence, severity, and timing of steroid-induced bradycardia and document any adverse events associated with bradycardia. design/method: we performed a retrospective review of all newly diagnosed patients at our center (2010-2016) with all/lymphoblastic lymphoma who received corticosteroids (dexamethasone 3-5mg/m 2 /dose or prednisone 30mg/m 2 /dose) during induction chemotherapy. patients were excluded if they had a pre-existing cardiac abnormality or if they received prior corticosteroids. the average 24 hour heart rate (hr) was assessed for the period prior to initiating steroid therapy and for the 24 hour period surrounding the nadir following steroid administration. the degree and time of steroid induced bradycardia was assessed. adverse patient events and concomitant medication use was documented to identify other contributing factors to bradycardia. a total of 153 children (80 females, 73 males, 16 months-27 years) were included in the analysis with 159 demonstrating a decrease in mean hr following steroid administration. median hr decrease was 22.9 beats per minute (quartiles 12.5-32) from prior to initiating steroids to surrounding nadir. sixty one percent developed bradycardia less than or equal to the 1st percentile for their age range. nadir occurred 7 doses (range 5-10) into treatment, which corresponded to 79 hours (55-109) after initiation of therapy. of 94 patients who experienced bradycardia, 78% were associated with dexamethasone rather than prednisone. hr nadir was not associated with other vital sign abnormalities. after completion of induction chemotherapy, 87% of patients had documented resolution of bradycardia with hr greater than the 5th percentile for age. it was observed that the children who continued to have relatively low hr were often younger (20 months-5 years old). examination of nadir hr during subsequent hospitalizations in which steroids were not being administered (excluding hr during procedural sedation) did not demonstrate a significant incidence of bradycardia. concomitant opioid, beta-blocker, or other medication exposure did not contribute to the incidence of bradycardia. corticosteroid-induced bradycardia is extremely common in children, teenagers, and young adults with all receiving induction chemotherapy. bradycardia was not associated with clinical adverse events and resolved after completion of corticosteroid treatment. therefore, further cardiac assessment may not be warranted in the presence of bradycardia suspected to be secondary to steroid administration. baylor college of medicine, houston, texas, united states background: survival in newly diagnosed pediatric acute myeloid leukemia (aml) is approximately 65%; however survival falls dramatically if a patient relapses. currently, approximately one-third of patients with pediatric aml relapse on standard chemotherapy regimens. aml cells are exposed to proteotoxic stress at baseline due to their rapid and inefficient metabolism; proteotoxic stress increases after chemotherapy due to accumulation of reactive oxygen species resulting in misfolded proteins. this leads to activation of cell stress pathways, such as the unfolded protein response (upr) in the endoplasmic reticulum. because an activated upr can make cells more sensitive to proteotoxic stress, we hypothesize that upr activation correlates with response to chemotherapy. objectives: determine the status of upr in pediatric aml and its correlation with chemosensitivity; design/method: peripheral blood samples from pediatric patients with aml were collected at the start of induction chemotherapy, 6-10 hours (h) and 24 h post initiation of systemic chemotherapy. tumor cells were sorted from peripheral blood mononuclear cells. expression of upr proteins was determined by chemiluminescence using an automated capillary electrophoresis system. clinical correlations were performed using an annotated database. we measured five upr proteins: grp78 (glucose regulated protein 78kda), phospho-eif2 , inositol-requiring enzyme 1 (ire1) and activating transcription factor (atf6). patients with aml had 2-5 times higher expression of upr proteins (except atf6) at baseline than normal controls. grp78-the key upr driver-had the highest level of protein expression in myeloid blasts. there was a wide variability in the level of baseline upr expression. eight out of 38 samples expressed >5 fold increase in grp78 above those with the lowest grp78 levels. similarly, 7 and 9 patients respectively, had a >5 fold increase in peif2 and ire1, compared to patients with low basal expression of these upr proteins. in our limited sample set, there was a trend towards lower overall survival (os) and event-free survival in patients with low baseline grp78 and ire1. conclusion: upr has a variable expression at baseline in pediatric aml, with a trend towards lower os in patients with a low basal grp78 and low ire1 expression, suggesting less chemosensitivity in this subgroup. conversely, it is possible that blasts with an upregulated upr prior to chemotherapy manage proteotoxic stress less effectively, having faster apoptosis and hence a better response to chemotherapy in patients with a high basal upr. we are currently expanding our findings in a larger cohort of patients enrolled in the children's oncology group aaml1031 protocol. background: children with newly diagnosed acute lymphoblastic leukemia (all) undergo chest x-ray (cxr) evaluation during initial diagnostic workup to ensure safe airway management. however, to our knowledge, no systematic assessment of cxr findings has been reported. objectives: to evaluate cxr findings at diagnosis of all and their associations with clinical characteristics. we reviewed the cxr findings at diagnosis of all in patients treated on the total xv and xvi protocols at st. jude children's research hospital. findings were evaluated for associations with clinical characteristics at presentation, and the clinical management of mediastinal masses was reviewed. mediastinal masses were seen in 107 (10.8%) of 990 patients evaluated and were more common in older patients (mean age, 8.51 years) than in younger patients (mean age, 7.14 years) (p = 0.005), in males than in females (p = 0.017), and in patients with t-all than in those with b-all (p<0.001). also associated with mediastinal masses were a higher white blood cell count (wbc) at diagnosis (mean, 107.02 × 109/l) (vs. a lower wbc; mean, 36.65 × 109/l) (p<0.001), cns involvement (vs. no involvement) (p = 0.028), and standard/high-risk disease (vs. low-risk disease) (p<0.001). other cxr findings included pulmonary opacity (160 patients [16.2%]), bronchial/perihilar thickening (187 patients [18.9%]), cardiomegaly (68 patients [6.9%]), and osteopenia/fracture/periosteal lesions (132 patients [13.3%]). pulmonary opacity was more common in younger patients (mean age, 6.51 years) than in older patients (mean age, 7.44 years) (p = 0.023) and in those with t-all (vs. b-all) (p = 0.010). bronchial/perihilar thickening, cardiomegaly, and osteopenia/fracture/periosteal lesions were also more common in younger patients than in older ones (p<0.001, p = 0.002, and p<0.001, respectively) and in those with low-risk disease (versus standard/high-risk disease) (p<0.001, p = 0.001, and p = 0.005, respectively). of the 107 patients with a mediastinal mass on cxr, 56 underwent a confirmatory chest ct scan, and 48 (85.7%) were confirmed to have a mediastinal mass. notably, 23 patients (41.1%) had airway compression, and compression of venous structures was identified in 18 of 48 patients (37.5%) who received iv contrast. the clinical course was evaluated for 107 patients with mediastinal masses detected by cxr. fifty patients (46.7%) required icu admission (mean stay, 3.0 days). general anesthesia was used for only 52 patients (48.6%), and 68 patients (63.6%) had a less invasive peripherally inserted central catheter. no deaths occurred in the acute phase. conclusion: cxr at the time of all diagnosis can detect various intrathoracic lesions and is helpful in planning initial diagnostic workup and management. background: mertk is a receptor tyrosine kinase that is aberrantly expressed in 80% of pediatric primary aml samples. mertk inhibition with the small molecule tyrosine kinase inhibitor (tki) mrx-2843 decreases tumor burden and prolongs survival in aml xenografts. while treatment with mrx-2843 reduces leukemia in the peripheral blood, it is less effective in the bone marrow, suggesting a role for the marrow microenvironment in therapeutic resistance. the jak/stat pathway has been implicated as a mediator of bone marrow derived resistance to tkis and inhibitors of this pathway are in clinical development for the treatment of aml. to determine the role of the bone marrow stromal niche in mediating resistance to mertk inhibition and to evaluate the efficacy of combined mertk and jak/stat inhibition. design/method: aml cell lines were cultured with or without the hs27 stromal cell line or hs27 conditioned medium, then treated with mrx-2843 +/-the jak/stat inhibitor ruxolitinib, or control. induction of apoptosis and cell cycle arrest in aml cells was measured by flow cytometry. expression of h2ax and total and phosphorylated stat5 were determined by immunoblot. results: co-culture with stromal cells significantly reduced aml cell death and g2/m phase arrest in response to treatment with 200nm mrx-2843 compared to no co-culture (cell death: 31.9% versus 61.2%, p<0.05; g2/m arrest: 8.9% versus 14.8%, p<0.01). g2/m arrest was accompanied by an increase in h2ax expression which was similarly abrogated in co-culture. conditioned medium did not provide protection from mrx-2843 induced apoptosis, g2/m arrest, or h2ax induction. mrx-2843 inhibited stat5 phosphorylation but direct co-culture and conditioned medium potently increased basal stat5 phosphorylation which was not inhibited by mrx-2843. to determine whether the observed induction of stat5 phosphorylation was functionally relevant, cocultures were treated with both mrx-2843 and ruxolitinib. while ruxolitinib potently inhibited the phosphorylation of stat5 in the presence of co-culture, combination treatment did not overcome stromal mediated protection from mrx-2843 induced apoptosis. similarly, the addition of exogenous gm-csf induced stat5 phosphorylation but did not yield protection from mrx-2843 functional effects in the absence of co-culture. together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from mrx-2843 induced apoptosis, cell cycle alterations, and dna damage. while co-culture potently induces phosphorylation of stat5 in leukemia cells, this is neither necessary nor sufficient for stromal-cell mediated protection from mertk inhibition and combined treatment with jak/stat inhibitors is unlikely to be therapeutically efficacious. background: mercaptopurine (6-mp) is an immunosuppressive thiopurine drug that is a key component of acute lymphoblastic leukemia (all) treatment. 6-mp is metabolized into 6-thioguanine (6-tgn), which is responsible for anti-leukemic effects, as well as 6-methylmercaptopurine nucleotides (6-mmpn/6-mmp), which are associated with hepatotoxicity. some patients preferentially metabolize 6-mp to 6-mmpn/6-mmp, increasing their risk for hepatotoxicity and potentially reducing anti-leukemic effects. hepatotoxicity can cause interruptions or delays in therapy that may jeopardize cure rates. allopurinol has been increasingly used in patients with inflammatory bowel disease (ibd) to shunt 6-mp metabolism toward 6-tgn and away from 6-mmpn to minimize hepatotoxicity and preserve therapeutic effects. objectives: this retrospective chart review expands upon our previously published case series of three patients with all in whom allopurinol was successfully used to redirect 6-mp metabolism. twelve additional patients have subsequently received allopurinol and 6-mp combination therapy at texas children's hospital. data from this larger patient sample, with longer follow up, is being analyzed to increase knowledge of the effectiveness and longitudinal effects of adding allopurinol to 6-mp to reduce risk of hepatotoxicity. design/method: data were abstracted from the electronic medical records of 15 patients with all treated at texas children's hospital from 2012 to present, who had been found to have evidence of altered 6-mp metabolism and in whom allopurinol was added to 6-mp therapy due to concern for risk or recurrence of hepatotoxicity. metabolite levels, 6-mp dose, and alanine transaminase (alt) prior to initiation of allopurinol and approximately 8 weeks later were compared. wilcoxon signed-rank test was applied for statistical analysis. : after the addition of allopurinol, patients experienced a significant decrease in mean levels of 6-mmpn (p = 0.0007), correlating with a significant decrease in mean alt (p = 0.004). with the initiation of allopurinol, the mean 6-mp dose was decreased from 84 to 32 mg/m2/day over an 8-week period. mean 6-tgn levels increased (p = 0.14). in follow up beyond 8 weeks, no patients had further holds in 6-mp due to hepatotoxicity. addition of allopurinol appears to shift metabolism from 6-mmpn toward 6-tgn, with increases in mean 6-tgn levels despite a decrease in mean 6-mp dose. this may limit negative side effects, thus resulting in fewer gaps in therapy and possible improved outcomes. further analysis of 6-mp dose titration and effects on anc over time as well as effects on overall survival is ongoing. prospective background: alterations in epigenetic patterning are a fundamental feature in acute myeloid leukemia (aml). treatment with dna methyltransferase inhibitors (dnmti) yields responses in aml, but the molecular mechanisms underlying this effect are poorly understood. in prior work, we demonstrated induction of genes involved in the pirna rna (piwi) silencing pathway as a common gene feature of 4 aml cell lines treated with decitabine. the piwi pathway is an rna silencing system, distinct from classical small rna transcriptional silencing, responsible for transposon-silencing in gametogenesis; emerging data suggest a role for this system in somatic cells. based on these data, we postulate that piwi induction plays a crucial role in aml recovery following demethylation and that disruption of this pathway would modulate response and/or recovery from decitabine treatment. to assess the effect contribution of the pirna pathway response following dnmti treatment in aml. design/method: to choose target genes in the pirna pathway for disruption, molm13 cells were first treated with escalating doses of decitabine. using quantitative rt-pcr, the dose-dependent expression of several pirna-associated genes were analyzed. two genes, mael and piwil2, were selected for disruption experiments based on preliminary data suggesting decitabine dose-dependent responses. molm13 cells were transduced with shrna targeting these genes using a lentivirus delivery system with selection in puromycin. knockdown efficiency was assessed by rt-qpcr. to determine how gene disruption affected cell growth, knockdown cells were treated with decitabine 20nm. proliferation was assessed by celltiter glo assay following decitabine treatment. clonogenic potential was assessed by colony forming assays of transduced cells after treatment with decitabine at 5nm and 10nm. results: following decitabine exposure in molm13, there was a markedly increased expression of mael and piwil2 compared to untreated cells (2363:1 and 41:1, respectively) . thus, these were the candidate genes chosen for disruption. of 4 mael shrna constructs, two resulted in a 25% relative expression of mael compared to controls. of the 3 piwil2 shrna constructs, the best knockdown showed 75% relative expression. there were no significant differences in proliferation or clonogenicity of stably selected mael or piwil2 knock-down molm13 cells following decitabine treatment. using gene knockdown procedures, mael and piwl2 do not appear to have a marked effect on growth and response to decitabine treatment in molm13. however, these results may be limited by inefficient knockdown using shrna targeting methods. further work using a cas9/crispr based inactivation of these genes is ongoing. children cancer hospital cairo, egypt background: hypodiploidy <44 chromosomes is very uncommon and have particularly poor outcomes in childhood acute lymphoblastic leukemia (all). it is subdivided into: near-haploid (24-31chromosomes), lowhypodiploid (32-39chromosomes) and high-hypodiploid (40-43chromosomes). to determine if minimal residual disease (mrd) can identify a group of patients with better prognosis in the hypodploid population who can be treated with intensive chemotherapy alone. design/method: a retrospective study that included all patients under age of 18 diagnosed as hypodiploid b-precursor all during the period between january 2008-december 2015 and treated at children's cancer hospital egypt on sjcrh total study-xv for ir/hr all. sixteen patients had <44 chromosomes (9 nearhaploid and 7 low-hypodiploid), constituting 1% of all pediatric patients with b-precursor all during the study period. patients with near-haploid all had a median age of 6 years (range 2-17), initial leukocyte count (wbc) median of 7.5 × 109/l (range 1.3-86.6), 4 (44.4%) were males and 5/9 (55.6%) had hr-nci criteria. four patients (44.4%) are alive in complete remission(cr) (range 25-48 months, median 30), one died in induction and 4 (44.4%) had hematological relapse (range 6.8-33 months, median 15). patients with low-hypodiploid all had significantly older age (median 15 years, range 13-17), median wbc 4.3 × 109/l (range 3.5-13.5), 5/7 (71.4%) were males. one patient (14.3%) is alive in cr, one died in induction, one failed to achieve cr post-induction and 4 patients(57%) had hematological relapse (range 3.9-5.6 months, median 4.8). mrd<0.01% by flow-cytometry on day-15 and end of induction was achieved in 5/9 (55.6%) and 6/8 patients(75%) with near-haploid, compared with 1/6 (16.7%) and 3/6 patients(50%) with low-hypodiploid; respectively (p = 0.287, p = 0.58;respectively). allogeneic transplantation was performed during initial remission only in 2 mrd negative patients (one relapsed and one is in cr) and in the patient with induction failure (relapsed post-transplant). five of the total six patients who had negative mrd on day-15 and end of induction are alive in cr (4/5 with chemotherapy alone). all 3 patients with negative mrd at end of induction but with mrd levels≥0.01% on day-15 (range 0.02-0.33%) relapsed as well as all 4 patients with detectable mrd at the end of induction. the difference in relapse was statistically significant in relation to negative-mrd on day-15 (p = 0.005), but not at end of induction(p = 0.105). conclusion: children with hypodiploid all and negative mrd on day-15 of induction are highly curable with intensive chemotherapy alone, while patients with negative mrd at the end of induction and detectable mrd on day-15 had dismal outcome. background: overall survival in pediatric acute myeloid leukemia (aml) has plateaued between 60-70%, with death during induction chemotherapy seen in 4-11% of patients. respiratory complications contribute to morbidity and mortality in pediatric aml induction, however the incidence, patterns, and predictors of respiratory adverse events (aes) during this period are unknown. to estimate the incidence of respiratory aes during induction therapy for de novo pediatric aml, to characterize and grade these respiratory aes, and to identify predictors of respiratory ae development. we conducted a retrospective longitudinal study from presentation to day 42 in institutional de novo pediatric aml patients (≤ 21 years) between march 2009 and december 2016. outcomes included any nci ctcae grade 2-5 respiratory ae or death from another cause. demographic, disease, and treatment-related data were abstracted. the most specific, best-fitting ctcae category and grade for each ae was determined. descriptive statistics, survival analysis, multivariable logistic regression analysis, and time-toevent distributions were performed (sas v9.4, cary, nc) . among 113 eligible patients, 54.0% (n = 61) experienced 69 discrete respiratory aes. incidence of grade 3-5 aes was 46.0% (n = 52). a bimodal time-to-event distribution demonstrated peaks at treatment days 0 and 14. induction death occurred in 4.4% (n = 5) including 3 deaths from respiratory failure associated with disseminated fungal disease. in univariate analysis, those experiencing aes differed significantly in regards to older age at diagnosis (p<0.0001), higher initial wbc (p = 0.012), higher initial peripheral blast percentage (p = 0.002), coagulopathy at diagnosis (pt (p = 0.004), d-dimer (p = 0.002)), fluid overload status (p<0.0001), occurrence of infection (p = 0.01), and occurrence of tumor lysis syndrome (tls) (p = 0.016). patients with hyperleukocytosis (p = 0.005), fluid overload (p<0.0001), and fab m3 morphology (p = 0.0016) each had a significantly decreased probability of completing the follow up period without experiencing a respiratory ae. on multivariable analysis, fluid overload (aor 45.2 [95% ci: 5.4-376.0) and older age (aor 1.10 [95% ci: 1.01-1.20) were significantly associated with ae occurrence when gender, hyperleukocytosis, tls, and infection status were held constant. we describe a high incidence of respiratory aes during pediatric aml induction. fluid overload and older age at diagnosis are independently associated with ae development when controlling for other proposed risk factors. interventions focused on conservative fluid management and offset of fluid overload should be explored in newly diagnosed pediatric aml in an effort to reduce respiratory complications during induction. overall, all survival rates are outstanding and have continued to improve with risk-adapted therapy. the most striking improvement occurred in t-all where 5-year os rates now exceed 90% and parallel b-all. survival improvements, however, have not been observed uniformly across all subgroups. while the gap in outcome differences narrowed among blacks, outcomes for hispanics have remained static. further, no improvements in survival were observed in infants or ayas and new treatment approaches have been implemented for these populations. background: acute myeloid leukemia (aml) accounts for approximately 18% of new childhood leukemia cases. chest x-ray (cxr) is performed in all newly diagnosed aml cases to evaluate the safety of airway management for anesthesia during diagnostic procedures; however, cxr results in pediatric patients with aml have not been described. objectives: the primary objective was to evaluate cxr findings at diagnosis in patients with aml. the secondary objectives included assessing associations between cxr findings and clinical characteristics, with the overall goal of aiding in the evaluation of the use of cxrs as an initial diagnostic study in pediatric patients with aml. design/method: cxr findings and clinical characteristics were evaluated in patients with newly diagnosed aml who were enrolled in one of three protocols at st. jude children's research hospital (aml97, aml02, and aml08). the findings were categorized based on radiologic reports. further, the associations of these findings and clinical characteristics were evaluated. we evaluated cxr findings in a total of 302 patients: 85 from aml97; 101 from aml02; and 116 from aml08. common cxr findings were pulmonary opacity (n = 51, 16.9%), bronchial/perihilar thickening (n = 38, 12.6%), splenomegaly (n = 38, 12.6%), mediastinal mass and lymph nodes (n = 27, 8.9%), pleural effusion/thickening (n = 17, 5.6%), demineralization/fracture/periosteal lesions (n = 10, 3.3%), scoliosis (n = 10, 3.3%), and granulomatous disease (n = 7, 2.3%). three cxr findings were associated with younger age at diagnosis: pulmonary opacity (median age, 4.5 years in patients with positive findings vs. 10.1 years in those with negative findings, p<0.01), bronchial/perihilar thickening (median age, 2.7 years vs. 10.3 years, p<0.01), and demineralization/fracture/periosteal lesions (median age; 2.7 years vs. 9.3 years, p = 0.04). two cxr findings were associated with older age at diagnosis: scoliosis (median age, 16.5 years vs. 9.0 years, p<0.01) and granulomatous disease (median age, 15.1 years vs. 9.1 years, p = 0.02). higher white blood cell counts (wbcs) at diagnosis were associated with cxrs showing pulmonary opacity (median wbc; 33.7 × 10^9/l vs. 14.8 × 10^9/l, p = 0.01) or splenomegaly (median wbc; 36.4 × 10^9/l vs. 15.2 × 10^9/l, p = 0.02). french-american-british (fab) m4/m5 subtypes were more frequently associated with pulmonary opacity compared with others (p<0.01). we did not find significant differences between female and male patients. conclusion: cxr in patients with newly diagnosed aml showed a variety of thoracic, abdominal, and bony lesions that are important for the initial evaluation and management. pulmonary opacity was the most common finding and was frequently seen in patients who were younger or had higher wbcs at diagnosis or fab m4/m5. background: children diagnosed with acute lymphoblastic leukemia (all) require a central venous catheter (cvc) to administer chemotherapy safely. both external and internal cvcs carry risks of complications including thrombosis, infection, and possible replacement. internal catheters, such as a port, are generally used for the majority of patients for the duration of treatment since therapy lasts for several years. many institutions place a port at the time of diagnosis. other institutions prefer to start induction therapy via placement of a peripherally inserted central catheter (picc) and defer port placement until the completion of induction therapy due to concerns of increased risk of infectious complications with port placement. objectives: to compare rates of common cvc associated complications by type of cvc placed at start of induction therapy in children treated for newly diagnosed all at the jimmy everest center (jec) at the university of oklahoma health sciences center. design/method: a retrospective chart review analyzed data from newly diagnosed all patients treated at the jec between 2010-2017. data was collected on complications including thrombosis, bacteremia, insertion site infection, cvc malfunction and need for removal. data collection began at the start of induction and was completed at the end of induction therapy. statistical analysis used a univariate and multivariate logistic regression model to compare complication rates between those who had a port versus those who had a picc placed at start of induction. results: data was collected on 128 patients. fifty-six patients had a port placed at start of therapy while 72 had a picc placed. fourteen percent of patients had a cvc associated complication. univariate analysis showed no statistically significant difference in rates of cvc associated complications between the groups (port 16%, picc 12.5% p = 0.564). the rates of hospitalization for cvc associated complications were similar between both groups (port 14%, picc 11% p = 0.590). rates of cvc removal were also similar between both groups (port 4%, picc 4% p = 0.863). multivariate model that included baseline patient characteristics including type of all, patient body surface area, gender, ethnicity and age continued to demonstrate no significant difference in cvc associated complications between both groups. conclusion: this single institution study showed that there was no significant difference in cvc associated complications between port and picc line placement at the start of childhood all induction therapy. port placement can be considered as a safe option at the start of induction therapy. complete remission [cr] or cr with incomplete blood count recovery [cri]) within treatment cycles 1-4. interim data are reported (nct02538965). results: seventeen patients were enrolled and received ≥1 dose of lenalidomide; median age was 12 years (range 5-18); 7 patients were female. patients received median 5 prior regimens (range 2-13). nine patients had previously undergone bone marrow transplantation (bmt). four patients had relapsed aml and 13 were refractory to immediate prior treatment. median duration of study treatment was 4 weeks (range 1-12); patients completed a median of 1 treatment cycle (range 0-3). all patients were evaluable for primary outcome; 1 achieved morphologic cri after 2 cycles (no patients achieved cr). the responder was a 13-year-old male with history of r/r aml after first-and second-line treatment, bmt, and salvage chemotherapy. at baseline, he had a complex cytogenetic karyotype (monoallelic −21q22.12, −3q, −4q13.3, −8p12) with no identifiable molecular mutation; he was also positive for del(5q) (−5q11, −5q23). his post-treatment karyotype showed no abnormalities. sixteen patients experienced treatment failure; 12 due to resistant disease, 3 of indeterminate cause, and 1 had treatment failure before a post-baseline assessment was performed. all patients experienced ≥1 grade 3-4 treatment-emergent adverse event (teae). the most commonly reported were thrombocytopenia (n = 10), anemia (n = 7), febrile neutropenia (n = 7), and hypokalemia (n = 7). fifteen patients experienced ≥1 teae related to lenalidomide. all patients discontinued treatment; 3 remain in follow-up. the study is now closed to enrollment. ten patients died on study: 3 during treatment, 7 during follow-up. all deaths were attributed to aml or complications due to aml. conclusion: third-line lenalidomide monotherapy was associated with clinical response in 1 of 17 pediatric patients with r/r aml; however, treatment exposure was limited. safety data are consistent with the known profile of lenalidomide. lenalidomide was not an efficacious treatment for r/r pediatric aml. funding: celgene corporation, summit, nj, usa. cook children's medical center, fort worth, texas, united states background: it is well documented that pediatric patients with acute lymphoblastic leukemia (all) often experience significant weight gain during induction therapy and later struggle with obesity. however, some patients experience unintended weight loss during induction therapy; since this issue is not well reported, it often goes undertreated. although malnutrition is reported to be associated with decreased survival, increased risk of infection, and loss of lean body mass, there remains a scarcity of in-depth analysis of prevalence and risk factors that contribute to this problem. our study attempts to address this critical yet unmet need. objectives: our aim was to identify the clinical risk factors and outcomes associated with weight loss during induction therapy for pediatric all. design/method: this was a retrospective chart review of patients between 2 and 20 years of age diagnosed with all at cook children's medical center from 4/1/14 to 3/31/17. for each patient, we collected height, weight, age, body mass index (bmi) z-scores at diagnosis and end of induction therapy, risk stratification, and whether consolidation was delayed. patients with a bmi >85th percentile at diagnosis were categorized as being overweight or obese. using logistic regression analyses, we examined which variables predicted whether the patient had an increase or decrease in bmi z-score throughout induction. a critical alpha level of 0.05 indicated statistical significance. results: ninety-six patients met our inclusion criteria. of these, 40% experienced a decrease in bmi during induction therapy. compared to patients whose bmi increased during induction, patients with a decrease in bmi were more likely to be overweight or obese at diagnosis (55% vs. 22%; p<0.001), to be ≥10 years of age (53% vs. 16%; p<0.0001), to have a high-or very-high-risk stratification (87% vs. 31%; p<0.0001), and to experience a delay in the start of consolidation therapy (47% vs. 21%; p<0.01). conclusion: this research highlights a risk not previously identified in the literature that may impact outcomes. patients treated on high-or very-high-risk protocols, who are overweight or obese at diagnosis, and who are ≥10 years of age at diagnosis should be monitored closely for weight loss during induction therapy. patients who experience weight loss should receive prompt intervention. it is our hope that this information can be used for future prospective studies and to help develop evidence-based guidelines. background: 17p abnormalities have been observed in some patients with hematologic malignancies. loss of p53 function as a tumor suppressor gene in the chromosome 17 plays an important role for development of leukemia. these patients usually have poor outcome due to the chemotherapy and are associated with poor prognosis. objectives: this study aimed to identify frequency of 17p abnormalities between iranian children and adult patients with aml (acute myeloid leukemia) malignancy. design/method: the 17p abnormalities were analyzed via bone marrow karyotyping and fish method in 669 acute myeloid leukemia patients. in this study, 17p abnormalities were observed in 52 (8%) patients out of 669 diagnosed cases. a significant strong correlation between 17p abnormalities and other high risk factors (poor risk cytogenetic) were observed. from 52 patients with aml malignancy (17p abnormalities), 45 (86%) patients have complex karyotype, 35 (67%) patients monosomal karyotype and 34 (65%) patients have monosomal karyotype accompanied with a complex karyotype. overall, 17p abnormalities are independent risk factor in acute myeloid leukemia and evaluation of these abnormalities by fish or other complementary techniques prior to treatment, might help for better risk stratification of high risk aml patients. background: hepatotoxicity in treatment of acute lymphoblastic leukemia (all) is well studied and transiently affects most patients receiving antimetabolite therapy. rarely, patients develop liver injury severe or prolonged enough to undergo a liver biopsy. little is known about how these patients differ from patients that develop transient hepatotoxicity. we sought to describe disease and treatment characteristics for all patients that developed hepatotoxicity severe enough to undergo liver biopsy. we also looked for pre-dictive factors for liver biopsy, including signs of early hepatic injury from the initial treatment protocol. design/method: pathology reports of all patients from the liver biopsy database at children's healthcare of atlanta were collected. controls were matched 2:1 for age, all subtype, and treatment protocol. demographics, treatment protocols, and overall outcomes were collected through the electronic health record. hepatic lab results for transaminases, coagulation, and albumin were collected for induction, consolidation, interim maintenance, delayed intensification, and maintenance. results: sixteen patients diagnosed between 2003-2013 (median age at diagnosis 10 years, range 1-16; 50% male; 75% pre-b all) were included in the case series. the median time from diagnosis to liver biopsy was 1.5 years (range 1-11). eight patients (50%) were in maintenance at the time of biopsy; none had active disease. eight (50%) were postbone marrow transplant. biopsy results included: steatosis (3), acute inflammatory/infectious (3), liver infiltration (1), fibrosis (6) and graft-vs-host disease (gvhd) (2). six patients were deceased; 5-year all-cause mortality from diagnosis was 31%. thiopurine methyltransferase (tpmt) status was known in 44% cases and 80% controls. all cases had intermediate or wildtype status, which did not differ from controls (p > 0.05). patients requiring liver biopsy did not have evidence of acute hepatotoxicity (ast/alt > 10× normal values) during their initial treatment protocol. hepatotoxicity requiring liver biopsy is a rare outcome of all treatment. these patients had elevated rates of relapse, bmt, and 5-year all-cause mortality, suggestive of a more severe disease process. however, it is difficult to sort out the temporality of relapse, bmt, and hepatoxicity requiring biopsy in this limited sample. additionally, patients with bmt preceding liver biopsy have other confounding factors that makes them difficult to include in the analysis. finally, our limited descriptive data show no notable correlation between early hepatotoxicity and later indication for liver biopsy. future cohort or case-control studies with larger sample sizes are required to further explore early predictive factors for severe hepatotoxicity requiring liver biopsy. nathan gossai, joanna perkins, michael richards, yoav messinger, bruce bostrom background: the majority of chemotherapeutic agents used to treat hodgkin lymphoma are teratogenic. pregnancy screening prior to the start of chemotherapy is supported by clinical guidelines and baseline testing is a standard component in therapeutic trials. there is limited data available on the incidence of pregnancy screening prior to the start of hodgkin therapy but previous studies suggest that pregnancy screening, especially at pediatric institutions, is not consistently completed. objectives: the objective of this study is to evaluate the incidence of pregnancy screening and contraceptive counseling prior to the start of therapy in females diagnosed with hodgkin lymphoma. design/method: a retrospective chart review was performed for all female patients newly diagnosed with hodgkin lymphoma from 2000 to 2015 at the hospital for sick children in toronto, ontario. all patients who were intended to receive multi-agent chemotherapy were included, regardless of age. data collected included demographic and disease information, chemotherapy regimen and enrollment on clinical trial. all pregnancy testing within two weeks prior to the start of therapy was captured, as well as type of pregnancy test performed, documentation of menstrual status, contraceptive counseling and contraceptive provision. univariate and multivariate analyses were used to describe factors influencing the incidence of pregnancy testing. results: a total of 122 female patients with newly diagnosed hodgkin lymphoma between the ages of 5 and 17 years were identified. sixty patients (49%) had pregnancy testing done prior to the start of therapy. testing modalities included serum and urine screens as well as quantitative beta-hcg measures. older age (p = 0.0016), documentation of menstrual status at diagnosis (p = 0.019) and diagnosis between 2008 and 2015 (p = 0.004) were associated with higher incidence of screening. enrollment on a therapeutic trial was not associated with a higher incidence of screening (p = 0.374). contraceptive counseling was documented for 19 patients (16%) and 11 patients (9%) were prescribed contraceptive medications during therapy. pre-chemotherapy pregnancy testing was completed on 49% of females with newly diagnosed hodgkin lymphoma. improvement is required and interventions, including clarification of institutional standards, modification of chemotherapy order sets and staff education, are planned. (rao et al., cancer, 2016) . university of louisville, louisville, kentucky, united states background: granulocytic sarcomas (also known as chloromas or leukemia cutis) were first described by a. burns in 1811. they are solid tumors comprised of immature granulocytic cells and represent extramedullary manifestations of underlying leukemia. chloromas are most commonly associated with acute myeloid leukemia. they may arise in other myeloproliferative disorders but are rarely seen in b or t cell acute lymphoblastic leukemia (all). objectives: although patients with all rarely have chloromas, it should remain on the differential for patients with unusual swelling or masses. design/method: we present a case series of two patients from our institution diagnosed with b cell all who had a chloroma as the presenting symptom. the first patient is a 4yo who presented to his primary provider with nasal congestion and a one-week history of bilateral eye swelling and was referred to an allergist when the symptoms did not resolve with anti-histamines. his review of systems was otherwise negative. he was referred urgently to ent two months later for a 3 × 3cm mass palpated along the medial border of the left eye. an mri showed a left facial mass surrounding the zygoma and extending into the anterior inferior left orbit. biopsy revealed b cell acute lymphoblastic lymphoma, and bone marrow aspirate and biopsy confirmed the diagnosis as b cell all. the second patient is a 10yo who presented to his primary doctor for rapid growth of a scalp nodule that had been present for about 2 months. he was referred to dermatology and treated for a supposed kerion from tinea capitis. the lesion continued to grow and became more irritated with this treatment. punch biopsy revealed a complicated phenotype of lymphoblastic lymphoma. however, after a lymph node biopsy and bone marrow aspirate and biopsy, the diagnosis was confirmed as b all. his only other positive point on review of systems was a questionably pathologic 20-pound weight loss and an area of matted cervical lymph nodes. for both of our patients, the chloromas completely disappeared during induction therapy. it is worth noting that both of these patients presented with the chloroma as the only symptom of the underlying leukemia. this led to initial misdiagnosis and delay in identifying their leukemia. therefore, while it is very rare for a patient with b all to present with a chloroma, our experience shows that all should be on the differential for patients presenting with unusual swelling or masses. background: hodgkin lymphoma (hl) is a lymphoproliferative neoplasm that commonly presents with history of adenopathy and a predictable pattern of disease involvement with or without systemic symptoms of fever and/or weight loss. in the hands of an experienced oncologist the diagnosis of hl is usually not a challenge. occasionally a diagnostic challenge is presented by a patient who has an atypical presentation which is suggestive of an alternative diagnosis. we describe a case series of patients diagnosed with hl whose initial clinical presentations lead to a diagnosis different form hl. honduras, nicaragua and the united states. results: six pediatric oncology centers from the american continent conducted a retrospective review of patients diagnosed with hl since 2010. patients that had an initial presentation not suggestive of hl or who were initially diagnosed with a disease other that hl were included for a total of 25 patients. argentina n = 8, guatemala n = 7, honduras n = 1, nicaragua n = 2, united states n = 7. five patients were female and 20 male. patient's ages ranged from 2 to 18 years. most patients (n = 19) were older than 11 years. three patients (15%) presented with non-immune cytopenias without overt lymphadenopathy, of those one had active hemophagocytic syndrome. five patients (20%) were suspected to have localized solid tumors: ewing sarcoma n = 2, rhabdomyosarcoma n = 1, hepatocellular carcinoma n = 1, and soft tissue tumor of the cheek n = 1. two (8%) metastatic solid malignancy as they presented with disseminated pulmonary nodules. five (20%) with autoimmune disorders: hashimoto thyroiditis n = 1, autoimmune hemolytic anemia n = 1, nephrotic syndrome n = 3. ten (40%) with chronic infectious processes: brucella n = 1, tonsillar abscess n = 1, splenic abscess n = 1, and tuberculosis (tb) n = 7. patients with suspected tuberculosis were diagnosed outside of the united states. six of 7 patients were ultimately diagnosed as having both tb and hl. seventeen patients had ann-arbor stage iii or iv, seven patient had stage ii with either b symptoms or bulky disease. patients were treated with various chemotherapy regimens according to the treating center: abvd, abve-pc oepa-copdac, avpc, beacopp. two patients had recurrent disease, one died of disease progression and one died from causes not related to hl conclusion: a small proportion of hl patients have atypical or unusual presentations. hl should be included in the differential diagnosis of solid tumors, autoimmune disorders, infections or cytopenias. the most common atypical presentation is an infectious process. background: acute lymphoblastic leukemia (all) represents the largest group of pediatric malignancies. the high cure rate of childhood all represents one of the most remarkable success stories in the war on cancer. in a lower middle income country (lmic) like the philippines, we reviewed the five year survival in a tertiary referral center. objectives: this retrospective cohort study aims to determine the survival of children 1-18 years old with all treated at a tertiary referral center for childhood cancer in the philippines from january 2012 to december 2016. design/method: this is a retrospective cohort study that reviewed medical charts of newly diagnosed all ages 1 to 18 years old from january 2012 to december 2016. a total of 435 subjects were included in the study. the 5 year overall survival (os) and event free survival (efs) were 65.3% and 62.8%, respectively. the 5 year os for standard risk all was 68.8% and for high risk patients was 50%. the 5 year os for the patients on remission was 83.7% and for those who relapsed was 21.1%. univariate and multivariate by cox proportional hazards regression revealed wbc count at diagnosis, risk classification, immunophenotyping, and development of relapse showed significant prognostic impact for mortality. age and gender were reported with no prognostic significance. the 5-year os and efs were lower compared to developed countries but are comparable with other lmics. the prognostic factors for relapse and mortality were compatible with the literature. overall, the adopted treatment protocols for childhood all in this institution showed acceptable results. relapse has a significant prognostic impact for mortality. development of accessibility to care, increase awareness, early detection and resources at hand should be achieved. improvement in the follow up protocol to prevent delays in the treatment, patient education to prevent non-compliance and psychosocial support, to developed better supportive care, and expand facilities should be given emphasis to further improve survival and prevent relapse. objectives: here, we seek to further characterize this entity by describing the pathologic and clinical features of 4 pediatric cases of burkitt-like lymphoma with 11q aberration. we collected pathologic and clinical data from the medical record on all pediatric high grade b-cell lymphoma (hgbcl) cases diagnosed at our institution over a 5-year period (2012-2017) . for those cases classified as neither burkitt lymphoma nor diffuse large b-cell lymphoma (dlbcl), fish for myc, bcl-2 and bcl-6, as well as array comparative genomic hybridization (acgh), were performed. we identified 38 cases of hgbcl, including 5 cases of burkitt lymphoma presenting as purely leukemic phase. of the hgbcl cases, 20 had burkitt lymphoma as defined by myc rearrangements, and 13 had dlbcl. collectively, the majority of these 33 patients had primary disease outside of the head/neck, and most patients presented with advanced stage (iii-iv) disease. of the 5 remaining cases, 11q aberration was identified in 4 cases using acgh. all 4 cases histologically and immunophenotypically resembled burkitt lymphoma but lacked myc rearrangement, instead showing proximal gains in 11q13-q23 and telomeric losses in 11q24.1qter. all 4 cases involved primary disease in the cervical lymph node and/or tonsil. three of these cases were localized (stage ii), and the fourth case involved a few metabolically active but non-enlarged lymph nodes in the chest and abdomen (stage iii). all 4 patients achieved complete remission with standard therapy for mature b-cell lymphoma, and were alive with no clinical evidence of disease at a median follow-up of 23 months. although the number is small, our results suggest that the majority of non-burkitt, non-dlbcl cases of pediatric hgbcl carry 11q aberrations. in addition, patients with 11q aberrations appear to be more likely to present with lower stage disease, thus requiring less intensive therapy, and also tend to have primary disease in the head/neck. these findings further support the classification of burkitt-like lymphoma with 11q aberration as a distinct pathologic and clinical entity, and we propose that all pediatric non-burkitt, non-dlbcl cases of hgbcl regularly undergo further workup for possible 11q aberrations. marie claire milady auguste, joseph bernard st damien hospital, port-au-prince, port-au-prince, haiti background: hodgkin lymphoma (hl) and non-hodgkin lymphoma (nhl) account for 7% of cancers in the united states pediatric population (1, 2). in central america and the caribbean, they are in second position among all types of pediatric cancers (3). a previous study on pediatric cancers in haiti showed that the lymphomas were in fifth place after the leukemias, wilms tumor, retinoblastoma and the sarcomas (4). the main objective of this study is to present the epidemiological profile of lymphomas managed at a haitian pediatric hospital. design/method: this is a retrospective study conducted on the cases of lymphoma diagnosed and managed at st damien hospital from january 2006 to december 2016. key variables such as age, gender, stage at diagnosis, histopathological types and outcome were collected to present the characteristics of this retrospective cohort. of the 407 cases of cancer diagnosed during the study period, 23 (5.7%) had the diagnosis of lymphoma. the sex ratio was 2.8 (17 males for 6 females) and the average age was 8.9 years [0-19 years]. there were 11 cases of hl (47.8%) and 12 cases of nhl (52.2%). 69.6% of the patients were diagnosed at stages iii and iv. among the hl cases, 6 (54.5%) were nodular sclerosis lymphoma, 3 (27.3%) with mixed cellularity and 2 (18.2%) with lymphocytic predominance. for the nhl cases, 4 (36.4%) were burkitt's lymphoma and 3 (27.3%) lymphoblastic t-cell lymphoma. among the 12 patients for who immunohistochemistry was found, the 4 cases of hl were cd30-positive and 6 out of 8 cases of nhl were cd20-negative. only 1 patient was hiv-positive, and 4 patients had a confirmed exposure to epstein-barr virus. 8 patients (34.8%) were lost to follow-up, 7 (30.4%) were in remission, 3 (13 %) relapsed, 2 (8.7%) were still in treatment and 3 (13%) were deceased. university of chicago, chicago, illinois, united states background: due to the adoption of risk-adapted therapy, pediatric and adolescent acute lymphoblastic leukemia (all) is associated with high cure rates. despite excellent outcomes in most children, patients with certain blast cytogenetic features do not fare as well. furthermore, african american, native american, and hispanic patients have worse outcomes than caucasian patients. while the outcome discrepancies are certainly multifactorial, and blast cytogenetics are related to age, it remains unclear whether ethnicity and blast cytogenetics correlate. the diverse patient population at the university of chicago provides an opportunity to evaluate for such a correlation. objectives: to describe cytogenetic findings in a racially and ethnically diverse population of patients of all age groups diagnosed with all at university of chicago from 2006 to 2016 and determine if there is a correlation between race/ethnicity and blast cytogenetics. results: a total of 191 newly diagnosed patients with all between the ages of 1-100 from 2006-2016 were included in this study. of those, 167 patients (87.4%) had b-all, 22 had t-all (11.5%), one had early t-cell precursor all and one had mixed phenotype all (b/t). caucasians accounted for 46% of patients, african americans (aa) 22%, hispanics 24.6%, asians 5.24%, and 2% were of other races. age distribution had a bimodal pattern, with a peak in incidence at 5 and another at 58 years of age, consistent with published data. cytogenetic categories included: t(12;21)(p13;q22), 11q23 rearrangements (kmt2a), iamp21, t(1;19)(q23;p13.1), t(9;22) (q34;q11), hypodiploidy, hyperdiploidy and double trisomy of chromosomes 4 and 10. aa and hispanic patients with b-all presented more frequently between the ages of 10-18 years compared to caucasians (p = 0.002 and 0.02, respectively). in aa patients, t(1;19) (q23;p13.3) was overrepresented (p = 0.04 when compared to caucasians), and was mainly observed in patients between 10-18 years. caucasian patients were more likely than non-caucasians to have hyperdiploidy (p = 0.04), especially in patients aged 1-9 years. the rate of t(1;19)(q23;p13.2) was significantly higher in aa patients in our cohort, in particular in patients between the ages of 10-18 years. hyperdiploidy was more likely in caucasians aged 1-9 years. these findings may suggest that varying blast cytogenetics could contribute to outcome differences between races. ahmed elgammal, yasser elborai, mohamed fawzy, asmaa salama, eman d el-desouky, lobna shalaby national cancer institute, cairo, cairo, egypt background: hodgkin lymphoma (hl) in children is one of the malignancies that have a high chance of cure. stage iv hl remains a challenge for getting good clinical outcome as in other stages. many treatment protocols used to give combination chemotherapy while combined modality treatment is the mainstay in other treatment protocols. objectives: we aimed in to assess the outcome using consolidation radiotherapy to chemotherapy (combined modality treatment) versus combination chemotherapy alone in treatment of stage iv hl. design/method: we included patients with stage iv hl and whose data were retrieved from the medical records of the pediatric oncology department, national cancer institute, cairo university, egypt from 2005 till june 2013 and were followed till august 2015. treatment was either to give 8 cycles of abvd (adriamycin, bleomycin, vinblastine, dacarbazine) only or to give 6 cycles of abvd followed by consolidation radiotherapy. the study included 22 cases; 17 were males and 5 were females. mean age was 10.66 years ranging from 4 to 17 years. the histopathology subtype was nodular sclerosis in the majority of cases (15 cases) followed by mixed cellularity (6 cases) then only one case of lymphocyte rich. nine cases were initially bulky while 13 cases were not. constitutional manifestations were present in 10 cases while it was absent in 12 cases. bone marrow was involved in only 4 cases. radiotherapy was given after completion of chemotherapy to 10 cases while 12 cases received chemotherapy only. the 5-year overall survival for patients who received radiotherapy was superior to those who received chemotherapy alone; 100% versus 45.8% respectively with statistical significance (p = 0.02). the 5-year progression free survival was also higher with radiotherapy than others; 90% versus 44.4% (p = 0.095). patients with stage iv hl who received consolidation radiotherapy apparently had a better outcome than those who received chemotherapy only. this suggests that radiotherapy contributes significantly with chemotherapy to the cure rate for those patients. the feinstein institute for medical research, manhasset, new york, united states background: microrna (mirnas) are short non-coding rnas that play a decisive role in cancer biology, including leukemia. exosomes are microvesicles (30-100 nm) produced by most cells in biological fluids. exosomes represent the fingerprint of the parental tumor and are loaded with bioactive markers such as mirnas, which may regulate tumor growth. exosomal cargo can be transferred into target cells changing their biological properties. our study investigates a functional role for exosomal mir-181a in pediatric acute lymphoid leukemia (p-all). objectives: 1/ to demonstrate that p-all exosomes induce cell proliferation2/ to confirm that exosome-induced cell proliferation is disease-stage specific 3/ to analyze exosomal mir-181a expression profiles in p-all4/ to authenticate that inhibition of exosomal mir-181a reduces leukemia proliferation design/method: exosomes were isolated by ultracentrifugation from healthy donors (hd) & p-all serum and conditioned medium (cm) of sup-b15, jm1, and cl-01 (control) human cell lines. cell lines were exposed to different sources of leukemia-derived exosomes in a paracrine or autocrine fashion for 24hrs in triplicates. proliferation was assessed by microscopic cell counting and confirmed by gene expression for proliferation, pro-survival and pro-apoptotic genes. mirna profiling was performed with the human cancer pathway finder microarray (qiagen). silencing of exosomal mir181a was carried out by a mir-181a inhibitor (qiagen), utilizing exo-fecttm exosome transfection reagent (sbi, system biosciences). further, exosomal mir-181a silencing was confirmed by q-pcr. cellular uptake of texred-sirna (sbi, system biosciences) was confirmed by flow cytometry. transfer of exosomal mir181a to the target cells was evaluated by q-pcr. we elucidated that cm-derived exosomes from sup-b15 and jm1 cell lines induce cell proliferation in sup-b15, jm1 (autocrine and paracrine) and cl-01 cells (paracrine) (p<0.01). serum p-all exosomes promote paracrine cell proliferation in all cell lines compared to hdderived exosomes (p<0.0001). heatmap analysis of mirna profiles of leukemia exosomes (all cell lines and p-all) identified mir-181a significantly upregulated in leukemia exosomes compared to controls. mir-181a was also upregulated in all cell lines after exposure to leukemia exosomes that induced proliferation. moreover, exosomal mir-181a inhibition reduces leukemic proliferation in pediatric all. our data suggest that all exosomes induce cell proliferation of leukemic cell lines in both paracrine and autocrine fashion. exosomes regulate these phenomena in a highly orchestrated way, by transfer of functional exosomal mirnas such as mir-181a. the results of this study suggest s233 of s301 that exosomal mir-181a inhibition can act as a novel way for growth-suppression of pediatric leukemia. results: a total of 199 disease sites were detected at pet/ct, while 172 sites were detected at contrast-enhanced ct and bone marrow biopsy (bmb). pet/ct showed improved detection of nodal lesions (p <0.0001) (kappa value = 0.633), extranodal lesions (p <0.0001) (kappa value = 0.632) and bone marrow (p <0.0001) (kappa value = 0.728) compared to contrast enhanced ct and bmb. pet/ct had upstaged 15 cases (16%) and down-staged 4 cases (4.3%) (p <0.001) (kappa value = 0.649). among the upstaged 15 cases, 10 patients (10.9%) were upstaged from stage ii to iii, based on residual in pet/ct not seen in contrast enhanced ct after abdominal mass excision. four patients (4.3%) were upstaged from stage iii to iv based on bone marrow uptake in fdg-pet without positivity in bma or bmb.regarding response assessment, sensitivity was 60% for pet and 80% for contrastenhanced ct (p = 0.56). specificity was 100% for pet and 65% for ct (p< 0.0001). positive predictive value for pet was 100%, while was 12% for ct scan (p< 0.0001). negative predictive value for both pet and ct was 98% (p = 0.82). five patients had 2nd biopsy to confirm viability of the residual lesions, 4 lesions were negative in pathological examination (all of them were metabolic negative in pet/ct; deauville score below 4). one lesion was positive in pathological examination (was positive in pet/ct; deauville score of 4). conclusion: pet/ct detected additional sites compared with contrast-enhanced ct and resulted in changing stage of disease. pet scan is significantly more specific than ct in the management of children with burkitt lymphoma. background: deep sequencing of the immunoglobulin heavy chain (igh) locus indicates that each b all is composed of innumerable subclones. in many cases, subclones exhibit differing phenotypic qualities. however, it remains unclear whether subclones demonstrate distinct tissue distribution within a patient. objectives: 1. to quantify the extent of clonal heterogeneity in diagnostic b all specimens; 2. to identify variability in clonal composition between bone marrow (bm) and peripheral blood (pb) disease sites. design/method: igh sequencing was performed on purified dna from 10 pairs of matched bm and pb patient specimens. multiplex pcr was used to globally amplify the igh locus; next generation sequencing (ngs) was performed using illu-mina® miseq. index clones (defined as ≥ 5% of all sequence reads in a specimen) and their subclone progeny (defined by 6 shared nucleotide bases immediately upstream of a common jh, or 6n_jx) were identified using igblast-determined vh and jh alignments (http://www.ncbi.nlm.nih.gov/igblast/) and an established in-house computational pipeline. results: up to 3 index clones per specimen were discovered in 16 of the 20 samples. in the remaining 4 (2 bm/pb pairs), 1 pair did not reveal a clonal igh and was eliminated from analysis; in the other, clone frequency did not reach the 5% index threshold, but predominant clonal precursors were inferred by the prevalence of their subclone progeny. subclone counts ranged from 2 to 2,619 per index clone. a combined 2,900 subclones derived from 11 pb index clones were observed; in contrast, 12 bm index clones gave rise to only 400 subclones. subclone heterogeneity was observed between all paired specimens. in 6 bm/pb pairs, index clones existed in equivalent proportions between disease sites. in contrast, 1 bm/pb pair demonstrated 2 high-frequency index clones in the bm (32.6% & 12.5%) with limited representation of these clones in the pb (0.6% & 1.4%, respectively); in this case, the most prevalent clone in the pb (10.8%) matched the least frequent index clone in the bm (5.0%). similarly, another pair showed a predominant index clone in the pb (5.8%) which was below index threshold (3.6%) in the bm. in 2 paired patient specimens, index clone predominance was discovered to be overtly distinct between bm and pb. among all pairs, the extent of subclone progeny derived from each index clone showed marked variability, with far higher subclone frequency in the pb than in the bm. our data indicate that b all clonal composition differs between disease sites. valley children's healthcare, madera, california, united states background: tuberculosis (tb) presenting with hodgkin lymphoma (hl) is rare. their coexistence could lead to delay in diagnosis of both tb and hodgkin lymphoma due to the similarities in signs and symptoms of presentation. most cases have been reported in the adult literature. we describe a case series of 11 children that were suspected to have tb and were found to have coexisting tb and hl. results: a retrospective review of hl patients in guatemala and argentina over 6 six years, uncovered 11 patients with simultaneous diagnosis of tb and hl. eight patients were from guatemala (incidence of 4.5%) and 3 from argentina (incidence of 2.5%). there were 4 females and 7 males. age ranged from 4 -17 years (mean 10.5 years, media 9 years). nine patients were suspected to have tb at presentation by the referring physician. two patients were found to have tb at the time of relapse through routine tissue culture. initial systemic symptoms included fever (n = 5), weight loss (n = 2), and night sweats (n = 2). six patients had a second systemic symptom in addition to fever. time for referral to oncology center ranged from 2 weeks to 5 months. nine patients were diagnosed with tb and hl through a tissue cultures and 1 with serum quantiferon. one patient was found to have hl without tb. two patients had no systemic symptoms and the diagnosis of tb came to light through routine tissue culture. five patients had stage iiib and ivb, two stage iia and one iib at diagnosis. hl treatment was given according to the insti-tutional standards depending on stage and risk with abvd, oepa/copdac +/-radiation therapy, and ice for relapse. five patients started anti tb treatment (isoniazid, rifampin, pyrazinamide +/-ethambutol for 2 months followed by isoniazid and rifampin for 30-52 weeks) simultaneously with chemotherapy, and three others after completing 2 cycles. the two relapsed patients started tb treatment after 2 cycles of chemotherapy. seven patients are alive and have been followed for 5 months -6 years. one patient died during therapy, another died for causes not related to tb or hl and one is currently receiving treatment. conclusion: tuberculosis can coexist with hl. in areas were the prevalence of tb is high, microbiology investigations of biopsy specimen should be strongly considered. therapy for tb can be given simultaneously with chemotherapy. coexistence of tb and hl does not appear to affect outcomes. the children's hospital affiliated to the capital institute of pediatrics, united states background: the pi3k/akt signaling pathway plays a central role in cell growth, proliferation and survival in physiological conditions. this signal pathway is considered to be an innovative targeted therapy of cancer, and its abnormal activation has been proved to be related to t-cell acute lymphoblastic leukemia (t-all). despite improved treatment strategies, such as multi-drug combination, high-dose chemotherapy and all kinds of application and popularization of hematopoietic stem cell transplantation, children with drug resistance or relapse t-all are still rather worse and its overall outcome and prognosis are much poorer than the more common b-lineage all. objectives: to explore the relationship between the pi3k/akt pathway and the pediatric t-all, so as to probe the exact molecular mechanisms of t-all and provide more directions for its treatment. design/method: 7 cases of new or recurrent acute t lymphocyte leukemia children with clinical information were collected in the children's hospital affiliated to the capital institute of pediatrics from dec.2015 to oct.2017, with 7 age and gender matched healty children as control (all was informed consent). the expressions of key genes in pi3k pathway were s235 of s301 analyzed by western blot rt-pcr analysis, the pi3k enzyme activities were detected by elisa,and the ccrf -cem's proliferation and its apoptosis were tested by mtt and flow cytometry technology on t-all cell lines ccrf-cem in different treatment group. the results of t-all children in clinical showed that pi3k protein and gene expression level were higher apparent than the control group (p<0.05), and pi3k enzyme activity increased as well (p<0.05); pi3k inhibitor ly294002 made a significant inhibition of cell proliferation and promoted cell apoptosis. ly294002 also enhanced the effectiveness of clinical commonly used chemotherapeutic drug dnr. in combination ly294002 and dnr treatment group cell viability dramatically declined, apoptosis and the apoptosis relation protein casepase3 expression in t-all patients was obviously higher than the control and the single drug group; pi3k/akt signaling pathway related proteins and gene expression level, pi3k, akt, gsk3 transcription in ccrf-cem were significantly higher than the control (p< 0.05), while pten transcription was significantly lower than the control (p<0.05). the abnormal activation of pi3k/akt signaling pathway might play an important role in pediatric t-all patients, especially in the cell proliferation or apoptosis. the results might provide new train of thought and direction in targeted suppress this signal pathway or in combination with other chemotherapy drugs therapy in looking for the more effective and less cytotoxic treatment of pediatric t-all. cleveland clinic children's hospital, cleveland, ohio, united states background: non-hodgkin lymphomas (nhls) are a heterogeneous group of lymphoproliferative diseases which comprise 7% of all childhood malignancies. nhls can be divided in to b cell lymphomas and t cell/natural killer (nk) cell lymphomas depending on immunophenotype, molecular biology, and clinical response to treatment. although nk/t cell lymphomas occurring in childhood and adolescence comprise a small portion of all lymphomas, they present many diagnostic and therapeutic challenges. the role of angiogenesis in lymphoma pathogenesis is becoming more evident. high molecular weight kininogen (hk) is a central compo-nent of the kallikrein-kinin system. it has been previously reported that cleaved hk (hka) induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in matrigel plug and corneal angiogenesis models. however, the role of endogenous kininogen in regulation of angiogenesis is in tumor microenvironment is unknown. objectives: to elaborate the role of hk in lymphoma angiogenesis, we used a murine t-cell lymphoma model and compared angiogenesis and tumor growth between wild-type and kininogen deficient (mkng1-/-) mice. we also evaluated the effect of hka on lymphoma cell proliferation. design/method: el-4 murine t-cell lymphoma cells (5 × 10^5) were implanted into wild-type and mkng1-/-mice. tumor size was measured using calipers and tumor volume was calculated using the formula volume = length × width^2 × 0.52. seventeen days after cell implantation, tumors were harvested and processed by immunoblotting and immunofluorescent staining. cell proliferation assays (mts) were performed to investigate any possible inhibitory effect of hka on el-4 cell growth, with human umbilical vein endothelial cells (huvec) were used as a positive control. results: el-4 lymphomas grew more rapidly and to larger sizes in mkng1-/-mice compared to wild-type mice, with significant differences apparent by day 11 after tumor implantation (p<0.01). by day 17, the volume of tumors in mkng1-/-mice was approximately 1.4-fold larger than in wild-type mice (mean volume ± standard deviation; 2120 ± 536 vs. 1485 ± 272 mm3, respectively, p<0.01). mts assays showed that hka does not directly inhibit the proliferation of el-4 cells in vitro, though it does significantly impair the viability of ecs studied simultaneously. conclusion: these findings suggest that hk is an important endogenous regulator of angiogenesis and tumor growth in this t-cell lymphoma model, and suggests that hka specifically modulates endothelial proliferation in tumor microenvironment. further work is needed to understand the mechanisms underlying these findings and provide future anti-angiogenic approaches to increase the therapeutic options for patients with nhl. bruce bostrom, jack knudson, nathan gossai, joanna perkins, michael richards, jawhar rawwas, susan sencer, julie chu, nancy mcallister, yoav messinger children's minnesota, minneapolis, minnesota, united states background: osteonecrosis causes significant pain and morbidity in older patients treated for acute lymphoblastic leukemia. besides altering the schedule of dexamethasone in delayed intensification there is no other intervention known to reduce the incidence of symptomatic osteonecrosis. pamidronate has been shown to reduce bone pain from osteonecrosis but not to prevent joint collapse when advanced. objectives: to compare the incidence of symptomatic osteonecrosis in patients who received prophylactic pamidronate compared with concurrent controls. to describe any increase in side effects from the use of pamidronate. design/method: patients age 10 to 28 years at time of all diagnosis were given intravenous pamidronate monthly for one year at the discretion of the primary oncologist starting in the first year of therapy. concurrent controls were patients age 10 to 28 who did not receive pamidronate. all patients were treated according to the concurrent cog protocols and received intermittent dexamethasone during delayed intensification. patients with bcr-abl all were excluded as the use of imatinib may increase the risk of osteonecrosis. imaging was only done if osteonecrosis was suspect based on clinical symptoms. patients were censored at the time of relapse. data were analyzed as of 1/1/2018. this retrospective study was approved by the children's minnesota irb. of the 62 patients evaluated 58% were male and 42% female, 74% had b-cell and 26% t-cell. the median followup is 2.4 years with a range of 0.3 to 7 years. pamidronate was given to 23 patients with 2 developing symptomatic osteonecrosis. there were 39 concurrent controls with 14 developing osteonecrosis. there was no significant difference in the leukemia lineage, gender distribution or body mass index (bmi) at diagnosis between groups. for all patients the median bmi was 22 with a range of 15 to 47. the age at diagnosis was significantly higher in the pamidronate group with a median of 18.6 years vs. 15.7 in the controls (p = 0.014). by kaplan-meier analyses the incidence of symptomatic osteonecrosis was significantly lower in the pamidronate group at 14% vs. 43% in controls. the log-rank p-value was 0.049 and the breslow p-value, which is more sensitive to early events, was 0.039. there were no untoward side-effects from pamidronate. pamidronate infusions significantly reduced the incidence of symptomatic osteonecrosis in patients over the age of 10 compared to concurrent controls who did not receive pamidronate. arahana awasthi, dina edani, janet ayello, christian klein, mitchell cairo new york medical college, valhalla, new york, united states background: mature b-nhl, including bl and pmbl express cd20+/cd79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. efs, cairo et al. blood. 2007; gerrard/cairo et al., blood, 2013 , goldman/cairo et al. leukemia, 2013 . pv has been demonstrated to possess significant preclinical activity against indolent cd79b+nhl (polson et al. can. res.2009 ). we previously observed that obinutuzumab (anti-cd20 mab) significantly enhanced cell death and increased overall survival against bl (awasthi/cairo et al., bjh 2015) in xenografted nsg mice. however, additive/synergistic effects of pv with obinutuzumab against mature pmbl/bl are unknown. to determine the efficacy of the pv or obinutuzumab/rtx alone or in combination against pmbl and rituximab (rtx) sensitive/resistant bl cell lines. design/method: raji4rh (provided by m. barth, md, roswell park cancer institute) and raji/ karpas1106p (atcc, usa) were cultured in rpmi. tumor cells were incubated with pv, and/or anti-cd79b, mmae (generously supplied by genentech inc.) with obinutuzumab /rituximab (100ug/ml) for 4 hr with nk cells at 10:1 e: t ratio and cytotoxicity was determined by delfia cytotoxicity assay. six to 8 week old female nsg (nod.cg-prkdcscid il2rgtm1wjl/szj), were divided into 5 groups: pbs, isotype control, pv, anticd79b mab and mmae (5mg/kg). mice were xenografted with intravenous injections of luc+ bl and pmbl cells and tumor burden was monitored by ivis spectrum system. results: os of mice receiving pv alone was significantly increased compared to anticd79b ab or isotype control in raji (35.5 vs.17 vs.19.5 our preliminary data indicates that pv significantly increased survival in bl and pmbl nsg xenografts compared to anti-cd79b ab alone. furthermore, pv in combination with obinutuzumab significantly enhances in-vitro cytotoxicity in bl and pmbl compared to obinutuzumab or pv alone. results: maximal grades (g)1/2, 3, and 4 crs occurred in 36, 19, and 24 patients, respectively. median lowest fibrinogen levels were 3.5, 3.3, and 1.2g/l in patients with maximal g1-2, 3, and 4 crs, respectively. 3%, 11%, and 25% of patients with maximal g1-2, 3, and 4 crs had lowest reported fibrinogen levels of ≥1 to <1.5g/l. eight patients (all with g4 crs) had very low fibrinogen levels (<1g/l), which occurred before (n = 1) or during (n = 6) maximal crs grade or at time of improvement (n = 1). no patients with maximal g1-3 crs had <1g/l fibrinogen levels. at the onset of <1g/l fibrinogen levels, 1 patient had concurrent g3, and 7 had g0-2 increased international normalized ratio and activated partial thromboplastin. cryoprecipitate was the primary treatment in the us, and fibrinogen concentrate (fc) guidelines for tisagenlecleucel-associated coagulopathy were developed for other countries because administration of fresh frozen plasma can be problematic. fc was available at 7/25 sites for 20 infused patients: 3/7 (g4 crs) and 0/8 (g1-3 crs). cryoprecipitate was available at 18/25 sites for 77 infused patients: 12/17 (g4 crs), 2/15 (g3 crs), and 0/32 (g1-2 crs). risk of bleeding increases in pediatric patients with comorbid thrombocytopenia and anticoagulant treatments. 5/8 patients had g3/4 decreased platelets within 1 day of <1g/l fibrinogen levels. 1 fatal case of intraparenchymal cranial hemorrhage occurred during resolving crs with g3 hypofibrinogenemia, ongoing thrombocytopenia, and continuous veno-venous hemofiltration with citrate. hypofibrinogenemia was observed more frequently in patients with higher crs grades during/when crs was improving or resolving. fc and cryoprecipitate treatment guidelines were developed. frequent monitoring and fibrinogen replacement are needed in patients with g3/4 crs. sponsored by novartis. its prolonged cns half-life, may allow a reduction in the number of intrathecal injections. objectives: to safely reduce the burden of therapy by reducing the number of it injections and reducing the total dose of doxorubicin with the addition of liposomal cytarabine and rituximab. design/method: patients (3-31 years) with cd20+ b-nhl with fab group b good risk (=stage i/ii and stage iii with ldh < 2xuln), fab group b intermediate risk (=stage iii ldh ≥2xuln and stage iv {bm blasts < 25%}) and fab group c high risk were eligible. patients received fab backbone therapy with the addition of six rituximab (375mg/m2) doses; two doses prior to each of two induction courses and one dose prior to each of two consolidation courses. cumulative doxorubicin was reduced from 120 to 50 mg/m2 in gr patients. after systemic methotrexate clearance, patients received age based dosing of it liposomal cytarabine. it injections were reduced from nine to five. the primary outcome is safety and toxic deaths among 40 evaluable patients with an estimated 3-year survival above 90%, monitored by an independent dsmb. results: to date, 32 evaluable patients, 25 fab group b and 7 group c (6 cns positive), median age 12 years (range 3-23), 20 males, 16 burkitt/16 dlbcl with 18 gr, 7 ir and 7 hr have enrolled. there has been one grade 3 anaphylactic reaction to rituximab and one grade 3 facial nerve palsy. no other serious adverse events were attributable to protocol therapy. there has been 1 death from progressive disease and 1 relapse at a median follow up of 30 months. efs and os are 94% and 97%, respectively. our initial results show excellent efs and os, consistent with published standard of care outcomes, with the addition of rituximab and intrathecal liposomal cytarabine despite the reductions in therapy. further enrollment is ongoing and continued long term outcomes are needed to confirm early results. future randomized studies are needed to examine both short term (mucositis, infections, hospitalization days) and long term (late cardiac toxicity) endpoints. 1. goldman etal, leukemia, 2013 2. cairo etal, jco 2012 st. jude children's research hospital, memphis, tennessee, united states background: bereaved parents identify significant spiritual needs around time of death and throughout their bereavement journeys. spirituality has been identified as a primary means by which bereaved parents can find meaning in their losses, and this ability to find meaning is associated with lower maladaptive grief symptoms. the use of spiritual coping strategies has been associated with improved coping and mental health outcomes among bereaved parents. objectives: to better understand how bereaved parents' experiences with spirituality throughout bereavement effects objective measures of grief, depression, and meaning-making. design/method: thirty participants whose children died of progressive cancer or related complications one to three years prior to participation completed an in-depth semi-structured telephone interview about their experiences with grief. participants were prompted to describe the impact of their spirituality on their bereavement processes. additionally, participants completed surveys related to grief (prolonged grief disorder questionnaire, pg-13), depression (beck depression inventory, bdi), and meaning-making (integration of stressful life experiences scale, isles). results were analyzed using a mixed methods approach including semantic content analysis of qualitative content and kruskal-wallis h test and post-hoc analyses of quantitative data. results: correlation analyses demonstrated significant differences between participants with positive and negative spiritual experiences of bereavement. participants with negative experiences of bereavement had a statistically significant increase in scores on the pg-13 compared to those with positive spiritual experiences signifying greater symptoms of prolonged grief. participants with negative spiritual experiences with grief had significantly lower scores on the isles, suggesting a lesser degree of adaptive integration of their losses. there were no significant differences in depression scores between groups. conclusion: bereaved parents that have a negative spiritual experience of bereavement are at increased risk for prolonged grief symptoms and are less likely to find meaning in their children's deaths than bereaved parents that describe a positive spiritual experience of bereavement. providers should consider exploration of spiritual beliefs and provision of spiritual care for parents of children facing life-limiting illnesses during treatment and bereavement. background: langerhans cell histiocytosis (lch) is an inflammatory myeloid neoplasia characterized by frequent relapse, with treatment failure associated with higher risk of death and neurodegenerative disease (lch-nd). activating somatic mutations in mapk pathway genes have been identified in almost all cases, with braf-v600e in approximately 60% of lesions. targeted therapies have been successful in treating other refractory cancers with braf v600e mutations (such as melanoma). given the central role of mapk pathway activation in lch, mapk pathway inhibition may be an effective therapeutic strategy for children with lch. objectives: the purpose of this study was to report the efficacy and toxicity profile of a retrospective cohort of patients with lch treated with mapk pathway inhibitors. design/method: medical records from 12 pediatric patients with lch (systemic and/or lch-associated neurodegeneration) who were treated with a mapk pathway inhibitor were retrospectively reviewed from five institutions. all patients had failed at least one prior systemic therapy and had a proven mapk pathway mutation. results: all patients in this series were less than 21 years old (median = 10.1 years; range: 2-20 years) with a median of three prior treatments (range: 1-9). at the time of initial mapk inhibitor use, nine of the 12 patients had lch-nd diagnosed clinically and/or by radiographic imaging; the remaining three patients had systemic disease. patients were treated for a median of 9 months (range: 1-20 months) with various reasons for discontinuation. three patients received combination mapk inhibitor therapies and three patients received other concurrent lch-directed therapies. four of the twelve patients had a grade 3 or 4 toxicity reported and three of these patients required dose reduction in order to be able to successfully resume therapy. overall survival was 92% with median 20 month follow-up (range: 1-42 months) with only one patient achieving transient complete response. the remaining ten patients had partial response or stable disease and four of these patients developed progressive disease while on therapy. conclusion: mapk pathway inhibitors may be a relatively safe salvage therapy for refractory systemic lch and lch-nd but the efficacy and durability of this strategy remains to be defined. combination with cytotoxic chemotherapies may be required in order to eradicate the disease-causing cell. future prospective trials of mapk pathway inhibitors for patients with refractory lch are needed in order to directly compare their efficacy and toxicity relative to other current salvage strategies. cincinnati children's hospital medical center, cincinnati, ohio, united states background: medication adherence during maintenance therapy has been shown to have a direct relationship with disease relapse in pediatric leukemia. previous research determined that patients who are ≤ 95% adherent to 6mercaptopurine (6mp) have a greater risk for relapse. the primary aim of the present study is to examine the relationship between metabolite profiles of 6mp with behavioral adherence rates obtained via electronic monitoring at 5, 10, and 30 days. it is hypothesized that patients demonstrating low levels of thioguanine (tgn) and methylated mercaptopurine (mmp) will have lower behavioral adherence rates prior to the blood draw. design/method: in a multisite, prospective study of 139 patients ages 7-19 years diagnosed with acute lymphoblastic leukemia (all) or lymphoblastic lymphoma (lbl), 6mp adherence was measured across 15 months of maintenance therapy using behavioral adherence (electronic monitoring) and pharmacological (metabolites) measures of 6mp. 6mp is metabolized into mmp and tgn. cluster analysis was used to generate three mutually-exclusive profiles of 6mp adherence. behavioral adherence rates were calculated for 5, 10, and 30 days prior to the blood draw. results: this study identified three metabolite profiles of 6mp across 15 months. previous research indicated that low levels of both metabolites suggest nonadherence to medication. low levels of one metabolite with high levels of another metabolite indicate adherence to 6mp. in this study, 51.2% of the low tgn-low mmp group had 5-day behavioral adherence rates ≥ 95% (mean = 100%); 48.8% had adherence rates < 84% (mean = 48.5%). in the high tgn-low mmp group, 77.6% had a mean 5-day adherence of 100%; 22.4% had adherence rates < 84% (mean = 32.9%). the low tgn-high mmp group had 74% of patients with a mean 5-day adherence level of 100%; 26% had adherence rates < 84% (m = 54.6%). at 10 and 30-days, 62 to 66% of patients in the low tgn-low mmp group had adherence rates < 95%. conclusion: these findings suggest that electronic monitoring and metabolite concentrations can be used to monitor 6mp medication adherence during maintenance therapy. it is notable that there is a sub-sample of pediatric patients who are identified as being nonadherent to 6mp based on electronic monitoring, however, metabolite levels indicate adherence to 6mp. similarly, a sub-sample of patients were identified as being adherent based on electronic monitoring, but metabolite profiles indicated sub-therapeutic levels of 6mp. our findings underscore the clinical significance of using both objective measures of medication adherence to inform clinical decision making. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hemophagocytic lymphohistiocytosis (hlh) is a life-threatening hyperinflammatory syndrome characterized by non-remitting fevers, rash, hepatosplenomegaly, cytopenias, liver dysfunction and coagulopathy, and can include central nervous system involvement. several genetic diseases cause hlh by impairing normal lymphocyte or macrophage function. the hlh panel at the cincinnati children's genetics laboratories includes 14 genes associated with hlh and other lymphoproliferative diseases, including the genes that cause primary hlh (prf1, unc13d, stxbp2, stx11, rab27a), x-linked lymphoproliferative diseases (sh2d1a, xiap), itk deficiency (itk), hermansky-pudlak syndrome types 2 and 9 (ap3b1 and bloc1s6), chediak-higashi syndrome (lyst), cd27 deficiency (cd27), xmen syndrome (magt1) and lysinuric protein intolerance (slc7a7). deletion/duplication analysis is available as a reflex test for all 14 genes, as copy number variations (cnvs) are not directly assessed by sequencing. objectives: the prevalence of cnvs among large groups of patients with hlh in north america is unknown. we assessed the frequency of cnvs in the genes on the hlh panel through a retrospective review of 522 orders for deletion/duplication analysis performed after next-generation or sanger sequencing: 397 orders for all 14 genes on the panel, and 125 orders of 1-5 genes from the panel. deletion/duplication analysis was performed on a custom 4 × 180k microarray annotated against ncbi build 37 (ucsc hg19, march 2006). deletion/duplication analysis resulted in a confirmatory diagnosis in 11 of 522 cases (2.1%). pathogenic or likely pathogenic cnvs were most common in the three x-linked genes: sh2d1a (3 deletions), xiap (3 deletions, 1 duplication), and magt1 (3 deletions). hemizygous deletions in xlinked genes in male patients were typically suspected after amplification failure during previous sequencing. of the autosomal recessive genes, pathogenic cnvs were observed once in each of three genes: rab27a (heterozygous), lyst (heterozygous), and stxbp2 (homozygous). in the two heterozygous cases, a second change was not identified by sequencing, so deletion/duplication analysis did not offer a confirmatory diagnosis. in 25 patients, deletion/duplication analysis was performed after a pathogenic or likely pathogenic variant was identified in an autosomal recessive gene during sequencing; however, in no case was a second mutation uncovered by cnv analysis. we recommend that deletion/duplication analysis be routinely performed in all male patients with hlh who lack a genetic diagnosis after sequencing of hlh-associated genes, especially if any regions failed to amplify. deletion/duplication analysis may be performed in female patients after sequencing if a genetic form of hlh is highly suspected, but the yield is expected to be low. cleveland clinic children's hospital, cleveland, ohio, united states background: the development of post-transplant neoplasia, typically from lymphoproliferative disease (ptld), is a severe complication in transplant recipients and affects approximately 12% of pediatric solid organ recipients. rates of lymphoma in adult heart transplantation patients are comparatively low, at less two percent at ten years. there are few published reports of the long-term outcomes of neoplasia after pediatric heart transplantation. we aimed to identify the subsequent malignancies that occurred in pediatric heart transplantation patients in a large single institution, and describe their treatment and subsequent clinical course. we performed a retrospective chart review of all pediatric heart transplant recipients followed at the cleveland clinic children's hospital from january 1985 to october 2012. we excluded patients who died within 30 days of heart transplantation. we reviewed in depth the history and clinical course of subjects who developed neoplasms. results: between 1985 and 2012, 101 patients underwent heart transplantation and survived at least 30 days post transplantation. nine patients (8.9%) developed a subsequent malignancy. in this case series, the median age at heart transplant was 3 years old and the median time to develop neoplasia was 88.6 months. primary neoplasia included monomorphic ptld (3), polymorphic ptld (1), burkitt lymphoma (2), hodgkin's lymphoma (1), plasmacytoma-like lymphoma (1) and epstein-barr virus-associated smooth muscle tumor (ebv-smt) (1). one patient with hodgkin lymphoma subsequently developed monomorphic ptld, one patient with polymorphic ptld subsequently developed ebv-smt and later, an undifferentiated gastric cancer. one patient with monomorphic ptld developed an ebv-smt. evidence of epstein-barr virus was present in six of nine patients at diagnosis of first malignancy. four of nine patients received reduction in immunosuppression as a primary intervention for the initial malignancy, with two complete responses (cr), one partial response, and one with progressive disease. five patients were treated with chemotherapy, with four cr and one with progressive disease. three patients died of malignancy (recurrent ebv-smt, undifferentiated gastric cancer, and monomorphic ptld post-hodgkin disease) and two patients died of other transplant related complications. conclusion: secondary malignancies represent a significant disease burden to survivors of cardiac transplantation. as expected, much of the malignancy burden is driven by ebv. despite aggressive histology, many malignancies can be successfully cured in this setting with a multidisciplinary approach. stanford university school of medicine, palo alto, california, united states background: current treatment of langerhans cell histiocytosis (lch) is based on extent of organ system involvement and if high risk systems are affected. gastrointestinal (gi) involvement is diagnosed in about 2% of lch patients, and classically presents in children under 2 years of age with malabsorption, failure to thrive, bloody diarrhea and anemia. although the gi system is considered standard risk, a mortality rate over 50% occurring within 2 years of diagnosis has been reported. this study was performed due to this discrepancy and the limited number of published cases. objectives: to review the clinical course and outcomes of patients diagnosed with gi lch. design/method: a retrospective chart review of patients with histologically confirmed gi lch diagnosed in the last 15 years identified from the bass center histiocytosis clinical database was performed. two other pediatric hematology/oncology centers (ucsf benioff children's hospital oakland and san francisco) were queried for additional cases. results: four patients with biopsy proven gi lch [3 subjects (2.9%) from 105 database records and l from center queries] were identified. failure to thrive, hypoalbuminemia, bloody diarrhea and rash were the most common presenting symptoms. lch of the skin was found in all patients. risk organ systems were involved in 2 patients. of note, 2 subjects were of african racial background. the median age at diagnosis was 3.5 months (1.5 months to 16 years), mean albumin 2.2 g/dl (1.2 -2.9 g/dl), mean esr of 56 mm/hr (37 -78 mm/hr). all patients initially received combination therapy per lchiii protocol (vinblastine, prednisone, and 6 mercaptopurine). two patients had recurrent disease and received second line therapy (cytarabine, 2cda, and local radiation therapy). all patients are alive without active disease at last follow-up (8 to 107 months after completion of therapy). a systematic approach to evaluate gi involvement should be performed in children diagnosed with lch. from our experience, combination chemotherapy for patients with lch involving the gi tract is an effective intervention for active disease. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bhatia indicated that rates of 6mp adherence ≥ 95% have better clinical outcomes. those with adherence rates ≤ 95% have an increased risk for disease relapse. the present study investigated patterns of 6mp medication adherence using group-based trajectory modeling in a large sample of pediatric patients. to describe patterns of behavioral adherence during the maintenance phase of therapy for a cohort of pediatric patients ages 7-19 years who were diagnosed with acute lymphoblastic leukemia or lymphoblastic lymphoma (n = 139). previous research has documented the relationship between optimal levels of medication adherence with positive health outcomes. it was hypothesized that three groups would be identified: optimal adherence, deteriorating adherence, and chronic nonadherence. it was hypothesized that patients in the optimal adherence group would have adherence rates ≥ 95%. those with poor adherence would have adherence rates ≤ 95%. design/method: the present study was a longitudinal, multisite study investigating adherence to 6-mercaptopurine in a pediatric cohort of patients using electronic monitoring devices. daily adherence rates (electronic monitoring of 6mp) were examined across 15-months. health outcomes were measured at quarterly intervals through medical chart reviews. results: unconditional growth curve modeling indicated that the mean percentage of behavioral adherence was 84.4% at baseline and declined to 75.2% at 15-months. three trajectories of 6mp behavioral adherence were identified: 1) optimal adherence (67% of patients): averaging 95% behavioral adherence across 15 months; 2) moderate adherence (20%): relatively stable nonadherence with rates of 67% across 15 months; and, 3) chronically nonadherent (13%): adherence decreased from 63% to 30%. with respect to patterns of medication adherence and relationship to clinically-relevant health outcomes, there were no significant differences in health outcomes between patients in the adherent versus nonadherent trajectories, including mean absolute neutrophil counts (anc), risk for infection as measured by anc, healthcare utilization, or risk for disease relapse. although longitudinal patterns of 6mp behavioral adherence were not related to health outcomes, it is notable that only 67% of the current sample had adherence rates ≥ 95%. in fact, 33% of the current sample demonstrated adherence rates ≤ 95%. our findings are important for development of future adherence promotion studies in pediatric cancer. our findings underscore the relative significance of tailoring adherence promotion interventions to subgroups of patients, including those with problematic patterns of adherence. patients who demonstrate adequate levels of adherence could still benefit from less intensive, preventative interventions to sustain and improve adherence. sophie gatineau-sailliant, pascale grimard, marie-claude miron, guy grimard, anne-sophie carret, jean-marie leclerc chu sainte-justine, montreal, quebec, canada background: vertebral involvement in langerhans cell histiocytosis (lch) is still a subject of interest, due to its low frequency and the absence of management's guidelines. objectives: to provide additional information on presentation, treatment and morbidity of pediatric lch vertebral lesions, we report cases of 11 children with vertebral lesion of biopsy-proven lch, between january 1st 2000 and december 31st 2015, at sainte-justine university health center (montreal, quebec, canada). we conducted a retrospective study by reviewing charts and imaging of vertebral lch in a population of 11 children (median age of 8.25 years at lch diagnosis), followed for a median duration of 34 months. symptoms at presentation, treatment modalities and morbidities were collected. results: vertebral lesions were present at lch diagnosis in 9 of 11 cases. they were usually diagnosed secondary to back pain in 10 of 11 cases and were asymptomatic in only one case. despite an epidural extension in 6 of 11 cases, no child developed neurological symptoms. lesions frequently involved vertebral body (10 of 11 cases) and were rarely unstable (2 of 11 cases). out of 29 vertebral lesions, most of them had a dorsal localization (15 of 29 lesions) and 8 of 11 patients had lch in multiple vertebrae. at diagnosis, median vertebral height loss was 37.5% compared to 25% at last imaging control. most used imaging modalities were pet-scan and plain x-rays. treatments were diverse and consisted in chemotherapy in all children but three and bisphosphonates in only 3 cases. radiation therapy was not used in any patient. six out 11 patients did benefit of an orthosis. a lch recurrence was observed in 6 patients and involved vertebrae in 4 cases. one patient with treatment-resistant lch disease had 5 relapses, and required multiple lines of treatment. all children were alive and disease-free at their last follow-up, 10 patients having radiological vertebral sequelae and only 3 had clinical sequelae. our study is consistent with the epidemiological data described in larger cohorts of children with vertebral lesions of lch and the favorable prognosis associated with such lesions. nevertheless, aggressive treatment and long term follow-up seemed to be essential as recurrences are s243 of s301 not rare and spontaneous bone regeneration often incomplete. plain x-rays appears to be a good follow-up tool for vertebral lesions as it allows reliable measures, less exposure to radiation at lower cost. national cancer institue, giza, giza, egypt background: acute lymphoblastic leukemia (all) is the most common type of childhood cancer and also the most complicated in the treatment, so it requires many interventions for both treatment and to alleviate suffer form side effects. pancreatitis is one of the toxicities, which is more common in all as it appears in about 16% of the patients. it occurs in many drug combinations which induce pre-pancreatitis and even direct destruction of pancreatic tissues. pancreatitis can be induced by many drugs used in the treatment such as chemotherapeutic agents or supportive treatment. lasparaginase is the backbone drug of the treatment of all in which 6 to 9 doses are required to achieve complete remission status in the induction phase of treatment and 12 to 19 doses in the maintenance phase.it is an enzyme that destructs the l-asparagine amino acid into aspartic acid and ammonia thus deplete the asparagine from the extracellular matrix . many drugs are investigated for their effect on treatment of induced pancreatitis such as interleukin-10, nsaid as antiinflammatory, glycerin tri nitrates as improvement of microcirculation, tnf-alpha antibody, paf inhibitor as specific anti-inflammatory and low molecular weight heparin .none of the drugs was investigated for their ability to prevent the occurrence of pancreatitis. objectives: this study was designed to evaluate the protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis design/method: acute pancreatitis was induced in rats by intra-muscular injection of l-asparaginase (1000 i.u/kg) given daily for five days. enoxaparin was given subcutaneous (100 i.u/kg) and diclofenac was given intra-peritoneal (2 mg/kg) daily for five days. then, markers of pancreatic injury, lipids, immune cell infiltration and oxidative stress were analyzed with histo-pathological examination of the pancreatic tissue results: during acute pancreatitis, oxidative stress markers were significantly changed as indicated by reduced tis-sue glutathione and increased malondialdehyde levels. this was accompanied with significant increase in immune cells infiltration as indicated by high levels of myeloperoxidase and pro-inflammatory cytokine tnf-alpha. triglyceride only showed increase level. treatment with enoxaparin and/or diclofenac restored levels of biochemical markers including serum alpha-amylase, reduced glutathione, malondialdehyde, pro-inflammatory cytokine tnf-alpha, myeloperoxidase and triglyceride. histological injuries of pancreatic tissues as vacuolation and necrosis of epithelial lining pancreatic acini, inflammatory cells infiltration and focal pancreatic hemorrhage were also reduced by treatment with enoxaparin and/or diclofenac. the present study emphasizes the potential protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis background: rosai dorfman disease (rdd), or sinus histiocytosis with massive lymphadenopathy (shml), is a rare condition of immune dysregulation of unknown etiology arising from the massive accumulation of non-langerhans type histiocytic cells inside lymph nodes. the disease classically presents as bulky, painless lymphadenopathy often associated with infection showing distension of lymph node sinuses by abundant histiocytic cells (cd1a(-), s-100(+)/cd68(+)). in some cases, the disease can be self-limiting, but in cases with a prolonged chronic course of exacerbations and remissions, those with extranodal involvement, or disease that threatens vitals structures, treatment may be necessary. there is no treatment consensus. to describe a case of life-threatening, unresectable, recurrent rdd successfully treated with langerhans cell histiocytosis (lch) 2009-inspired therapy. design/method: we compared this case to the current literature on chemotherapeutic treatments for rdd. we searched pubmed, ovid, and google scholar for similar cases. we believe this to be the first reported case of using lch therapy to successfully treat rdd. an 8-year-old male presented to an outside hospital with two years of massive neck swelling causing torticollis. biopsy confirmed rdd. he was intermittently treated with courses of antibiotics with partial response. surgical removal of the affected lymph nodes was unsuccessful due to proximity to the spinal cord. two years later, the patient presented to our institution. he was initially treated with prednisone with a fast tapering dose, but after a second relapse the decision was made to try chemotherapy following the lch-2009 protocol of weekly vinblastine (6 mg/m2), 6-mp (75 mg/m2), and high dose steroid bursts. he experienced two additional relapses off therapy at ages 12 and 14 years old, including cmv(+) associated septic shock and cytokine storm requiring rapid response, picu admission, and ionotropic support. this last episode was treated with a more prolonged induction and maintenance therapy. an extended and slowly tapered maintenance therapy regimen of 2.5 years of daily 6-mp, monthly vinblastine and steroids with a slowly tapered dose during his fourth remission has resulted in 38-months of continuous complete remission-the longest stretch of his life. no similar cases were found. literature search demonstrated no consensus regarding the most effective treatment of rdd, with no previous cases being successfully treated following lch chemotherapy protocols. we hypothesize that the multi-agent relatively mild lch-2009 therapy mitigates the immune dysregulation of rdd. this case suggests that lch-2009 therapy can be used to treat cases of rdd that is not amendable to surgery or observation. nicklaus children's hospital, miami, florida, united states background: central venous catheters (cvc) are necessary in the management of patients with malignancies, especially children. patients with acute leukemia (al) have higher rates of central line associated complications such as bloodstream infections compared with other malignancies. objectives: to examine the choice of placement of cvc and the differences in outcome between peripherally inserted central catheters (picc) and ports in patients with leukemia during induction. design/method: retrospective chart review of patients with newly diagnosed leukemia at nicklaus children's hospital between 2010 and 2016. results: ninety four patients with a new diagnosis of leukemia undergoing induction chemotherapy were identified. the average age was 6.9 years. overall, 51 (54.3%) patients had a port placed and 43 (45.7%) had a picc placed. the decision for picc or port was subjective and physician based. the main outcome measures were local inflammation/infection, bacteremia, thrombophlebitis, blocked catheter and premature removal. the most common complication was bacteremia (12. 8%). in a multiple logistic regression analysis for predicting whether patients had at least one complication, results showed that having at least one complication is 3.4 times the odds in patients with aml compared to patients with all (p = 0.032). when comparing picc vs. ports, patients with picc had more frequent episodes of blocked catheters (23.3%) and premature removal (20.9%) compared to the patients with ports (2.0% and 0.0%) (p = 0.002 and p = 0.001 respectively) during induction. local inflammation, bacteremia and thrombophlebitis were not statistically different (p = 1.0, p = 0.54 and p = 2.4 respectively). the most common place for port placement was the right subclavian vein (55%). there was no significant association between port location and having at least one complication (p = 0.112). acute lymphocytic leukemia subgroup analysis: fourteen patients (61%) in the picc group had at least one complication and 9 (39%) in the port group but that was not statistically significant (p = 0.128). our series showed a higher incidence of blocked catheters and premature removals with picc compared to ports in patients with leukemia during induction. the choice of placement of picc vs port was subjective and physician based. patients with all, despite receiving steroids and asparaginase during induction, did not show a statistically significant increase risk in thrombosis or infection but larger numbers may be needed in future studies. university of california, san francisco, san francisco, california, united states background: hemophagocytic lymphohistiocytosis (hlh) is classically a disorder of young children meeting systemic hyperinflammation criteria. presentation in late adolescence is uncommon. furthermore, though cns signs occur in 30-70% of cases, initial isolated neurologic presentation is rare, frequently resembling encephalitis or demyelinating disorders. these cns signs can be isolated or precede systemic disease, delaying hlh diagnosis. hlh declaring in adolescence with predominant psychiatric features has not been well documented. objectives: to describe a case of cns hlh presenting with neuropsychiatric features in absence of classic hlh criteria. design/method: retrospective review of clinical, radiologic, histologic, immunophenotypic, and molecular features of a patient with cns hlh. a 19-year-old female presented with acute-onset headaches following nine months of progressive anxiety, short-term memory loss, emotional lability, perceptual disturbances, and hypomania. brain mri demonstrated numerous enhancing t2 hyperintense supratentorial and infratentorial white matter lesions in the left thalamus and caudate head. brain biopsy showed histiocyte-rich inflammation and associated demyelination. extensive evaluation including universal microbial pcr failed to reveal underlying infection or malignancy. past medical history was notable for presumptive pulmonary sarcoidosis diagnosed 14 months prior with progressive respiratory failure with associated granulomatous pulmonary nodules which responded to systemic immunosuppression. at presentation of her neuropsychiatric symptoms, she had normal sil-2r, ferritin, fibrinogen, and triglycerides. there was no pancytopenia, coagulopathy, bone marrow hemophagocytosis, fevers, or splenomegaly. given the possibility of partial immune suppression of systemic symptoms and the prominent neurologic symptoms, hlh screening labs were sent and notable for decreased natural killer and cytotoxic t lymphocyte function, normal granzyme expression and cd107a mobilization, and absent perforin expression. genetic testing confirmed compound heterozygous mutations in prf1 (c.227g>a, c.626a>c) and familial hlh type 1. she was treated with low-dose dexamethasone and intrathecal chemotherapy per hlh-94. due to lack of evidence of systemic inflammation, vp-16 and high-dose steroids were held. within one week of initiating therapy, she had decreased anxiety and improved cognition, with sustained, incremental neuropsychiatric improvement with additional intrathecal treatments. she tolerated dexamethasone tapering without symptom flare. mri also demonstrated parenchymal lesion improvement. for definitive treatment, she underwent unrelated allogeneic hematopoietic cell transplantation and remains at neurologic baseline as of eight months post-transplant with ongoing imaging improvement. conclusion: this case of familial hlh with compound heterozygous perforin mutations in an adolescent with isolated neuropsychiatric symptoms illustrates that cns hlh may be an underrecognized phenomenon in absence of systemic signs. standard hlh therapy may effectively reverse these symptoms with associated radiologic responses. rush university children's hospital, chicago, illinois, united states background: posterior reversible encephalopathy syndrome (pres), a recognized complication of pediatric leukemia treatment has been reported in up to 5% patients in various series. hypertension, chemotherapy and cortical spreading depression have been implicated in the pathophysiology. due to the combinations used, it is difficult to identify the offending drug, several have been implicated. since delay of chemotherapeutic treatment in children with high risk leukemia is unfavorable, it is important to recognize the characteristic radiologic findings, manage appropriately and reintroduce the treatment as soon as possible. pharmacoethnicity is now recognized as an important factor for variation in neurotoxicity in children with all. ethnic differences in reported pres events in pediatric patients with all has not been well described in literature. to describe the factors associated with pres in a cohort of high risk pediatric all patients at a single institution. design/method: a total of 12 children with an average age of 9 years (1-20 years) diagnosed with all between 2013-2017 were retrospectively reviewed for the occurrence of pres. various demographic factors, therapy received, clinical features, radiology related findings and management were reviewed. a search for all published articles on pres in leukemia was conducted using pubmed databases. results: five (42%) children (average age 8.5 years) developed pres during days 10-29 of induction. 80% of the patients that developed and 45% of those that did not develop pres were hispanic. all the patients that developed pres and 43% of those that did not were diagnosed with high risk all. all patients received vincristine, 80% received daunomycin and intrathecal methotrexate and 20% received asparaginase in the 1 week prior to the event. mri findings confirmed pres in all 5 patients with no evidence of methotrexate related leukoencephalopathy or leukemia. at the time of pres all patients were in remission based on mrd and spinal fluid cytology. two-thirds of the patients had seizures and hypertension at the time of the event with no prior history of either. all patients had complete recovery of normal mental status after resolution of pres. a higher incidence of pres than previously reported was noted in our series. hispanic ethnicity, high-risk all and exposure to vincristine, daunomycin and intrathecal methotrexate in induction were associated with pres in our cohort. a new association that emerged was that of hispanic ethnicity with pres .larger studies to understand the importance of pharmacoethnicity in pres may help in individualization of chemotherapy based on ethnic differences. children's hospital of illinois, peoria, illinois, united states background: hyper ige syndrome is a primary immunodeficiency characterized by susceptibility to skin and lung infections as well as increased propensity for malignancy. hemophagocytic lymphohistiocytosis (hlh) is a syndrome characterized by overwhelming activation of t lymphocytes and macrophages occurring as either primary hlh caused by genetic abnormalities or secondary hlh associated with infectious, malignant, metabolic, or immunodeficiency causes. we describe the first case to our knowledge of hlh in a patient with hyper ige syndrome. to describe a case of hlh in a pediatric patient with hyper ige syndrome. results: a 7-year old caucasian male with known autosomal dominant hyper ige syndrome (stat3 mutation) was transferred to the pediatric intensive care unit secondary to concern for septic shock. the patient had persistent slow bleeding from oral lesions and central catheter sites despite the addition of aminocaproic acid and recombinant factor viia. he also required numerous blood product transfusions sec-ondary to anemia and thrombocytopenia. clinical suspicion was high for hlh and the patient met criteria for diagnosis of hlh with the following: ferritin > 40,000 ng/ml, triglycerides 264 mg/dl, decreased nk cell function with the sample only containing 1% nk cells, elevated soluble il-2 receptor at 4215 u/ml, splenomegaly, and fever. infectious workup was remarkable for a positive ebv qpcr with 80,700 copies/ml suggestive of ebv driven secondary hlh. familial hlh testing was unable to be completed. therapy was initiated based upon the hlh-94 study. the addition of ruxolitinib and anakinra were considered but the patient declined rapidly prior to treatment. ct of the head was concerning for a stroke with signs of edema and increased intracranial pressure likely leading to the development of symptoms consistent with brain stem herniation. the decision was then made to withdraw care. conclusion: to our knowledge, this is the first report of hlh in a patient with hyper ige syndrome. diagnosing hlh requires a high index of suspicion in critically ill patients, and prompt initiation of therapy is essential. this challenging case of hlh in a patient with hyper ige syndrome highlights the diagnostic challenge, variable presentation, and need for effective therapy in this vulnerable patient population. background: adolescents and young adults (ayas) with cancer are at risk for psycho-social as well as physical symptom burden during cancer therapy. the purpose of this study is to explore psychological and physical symptoms endorsed by aya while receiving therapy for cancer design/method: surveys were given in both inpatient and outpatient settings during cancer therapy. symptom screening in pediatrics tool (sspedi) and memorial symptom assessment scale (msas). symptoms severity was rated by teens on a 5 point likert scale. spss 22, used for statistical analysis. results: : a total of 39 aya on cancer therapy (age range 13-19.9 years) 35% female, 65% male, 43.6% acute leukemia, 48.7% solid tumors, and 7.7% diagnosis was not reported. 78% of aya on cancer therapy reported at least 1 or more symptoms, 45% reported >3 symptoms cluster. of the physical symptoms that were reported as most distressing to the teens, mouth sores and headaches were the top causes. of the physical symptoms that were most frequently endorsed; fatigue was on the top (58%), followed by change in appetite 45 %, vomiting 43%, and pain 40%., the least was bowel habit changes. aya rated sadness as the most frequent psychological symptom 38%, followed by feeling angry 32%, and scared 30%. statistically significant difference was noticed based on gender difference with more females reported symptoms (p = 0.01), while type of cancer (acute leukemia versus solid tumors) was not statistically different. conclusion: aya with cancer reported multiple physical and psychological symptoms with significant distress. females seem to report more symptoms compared to males. screening aya for cancer therapy related symptoms is feasible during routine visits and adds important information about the aya well-being. background: sinus histiocytosis with massive lymphadenopathy (shml), also known as rosai-dorfman disease, is a rare histiocytic proliferative disorder of unknown etiology. many treatment modalities have been employed; however, no uniform guidelines exist. objectives: literature review of treatment options for shml. design/method: chart review was performed on pediatric patients diagnosed with shml at the children's hospital at montefiore between 2010 and 2012 after irb approval. inclusion criteria included children between the ages of 0 and 21 years with shml. exclusion criteria included children with cutaneous shml. four cases of shml seen at montefiore are described. a comprehensive review of the literature identified 102 additional cases published between 1978 and 2018. manuscripts that did not include the treatment modality or outcome were excluded. results: many of the 106 patients with shml responded to observation alone. of 106 patients, 46 patients were observed, with 35 (76%) having resolution of disease, five having stable disease, and five being lost to follow-up. one patient received subsequent systemic therapy. surgical management was con-ducted upfront in 29 patients. of those, 18 (62%) had resolution of disease, one had stable disease, and one had recurrence with no further therapy noted. of the remaining nine patients, 77% were successfully treated with systemic therapy, consisting of either steroids (5) or steroids and chemotherapy (4). systemic therapy was used as first-line therapy in 31 patients. steroids alone or in conjunction with chemotherapy resulted in resolution of disease in 12/15 and 7/11 patients (19/26, 73%), respectively, with four patients having stable and three with progressive disease. chemotherapy without steroids resulted in resolution of or stable disease in 3/5 patients. radiation was ineffective. conclusion: shml is a rare disease with no published guidelines for treatment. from the results of the cases and a detailed review of the literature, it can be suggested that observation may be considered as first line management in patients providing there are no significant symptoms. for patients who are symptomatic or have significant progression, surgery may be considered. in patients with recurrence or refractory disease, steroids and/or chemotherapy may be used. the presence of nodal or extra-nodal disease did not seem to have a significant impact on the course of treatment. given the rarity of the disease, it is difficult to conduct a randomized control trial. further work, involving collaboration between centers and cooperation with the international rare histiocytic disorders registry would be helpful. boston children's hospital, boston, massechusettes, united states background: increasing census and intensified work compression on the inpatient oncology service at our institution was identified as leading to resident dissatisfaction, impaired resident learning and decreased perceived quality of patient care. objectives: to evaluate the impact of a redesign of a pediatric inpatient hematologic malignancy (ihm) service on resident perceptions of the educational value of the rotation and safety of patient care. design/method: during the 2016-2017 academic year, we initiated a bundled intervention on the ihm service. modifications included 1) decreased patient volume: the ihm service was divided into two teams, utilizing an extra attending -a teaching service consisting of residents and fellows and a team comprised of nurse practitioners. 2) intentional patient team assignment: patients were deliberately assigned to a care team based on educational opportunities and provider skill sets. 3) intentional attending faculty selection: attending faculty with deeper clinical and teaching experience were selected to supervise on the teaching team. 4) increased weekend staffing. after completing the service, junior residents completed an electronic survey to evaluate their perceptions of the educational value of the rotation, as well as their ability to deliver safe care while on the rotation. fisher's exact tests were used to compare responses from residents in 2017 who experienced the redesign to residents in 2016, whose experience results: survey completion rates were 70% (28/40) in 2016 and 57% (29/51) in 2017. intervention residents were significantly more likely than comparison group residents to choose the answers "very good" or "excellent" to describe both the overall quality of the rotation (76% intervention vs. 25% comparison, p<0.001) and the educational experience on rounds (52% intervention vs. 7% comparison, p<0.001). intervention residents also reported caring for fewer average primary patients daily on weekdays as compared to comparison residents (4.8 vs 8.7 patients, p<0.0002, 95% ci -5.14 to -2.64). furthermore, intervention residents were more likely than comparison residents to "agree" or "strongly agree" that they could provide safe patient care on weekend days (79% intervention vs. 14% comparison, p<0.001) and on nights (69% intervention vs. 25% comparison, p<0.01) while on the oncology service. a redesign initiative of an oncology service with the development of a new teaching service led to improved resident perceptions of the educational value of the rotation and ability to provide safe care to patients. this approach could be useful to other services and institutions to promote similar outcomes in resident education and patient care. background: alk-positive histiocytosis is a rare histiocytic proliferative disorder that has been reported in three infants presenting primarily with hepatosplenomegaly, anemia, and thrombocytopenia. given the rarity of this disease, there are no standard treatment algorithms for this diagnosis and the disease course and outcomes remain largely unknown. the published series describes treatment ranging from monitoring alone to multi-drug chemotherapy regimens. there was ulti-mately resolution of presenting symptoms in all three cases despite varying treatment strategies. objectives: to report a newly diagnosed case of alkpositive histiocytosis that was treated with a novel approach using cytarabine monotherapy. results: a full term male infant presented at birth with difficulty feeding and hyperbilirubinemia. over the first few weeks of his life, he subsequently developed thrombocytopenia, transaminitis, and profound hypoalbuminemia. by six weeks of life, he was experiencing significant abdominal ascites requiring repeat paracenteses, massive hepatosplenomegaly, respiratory distress secondary to abdominal distension, anemia, and coagulopathy. he underwent numerous diagnostic tests, including a liver biopsy followed by a bone marrow biopsy that showed alk-positive histiocytic infiltrates in both sites. treatment was initiated with cytarabine 170 mg/kg/day x 5 days, repeating every 4 weeks. throughout his course of five cycles of treatment, he experienced intermittent fevers and mild nausea with no other adverse events. by the end of five cycles, his hepatosplenomegaly resolved, his blood counts normalized, he demonstrated weight gain on oral feeds, and his liver enzymes normalized. he is currently 12 months post completion of therapy and remains well with a normal physical exam and laboratory values. conclusion: treatment of alk-positive histiocytosis with lose dose cytarabine resulted in complete resolution of our patient's symptoms with minimal treatment related adverse effects, and few long-term treatment related risks. given the rarity of the diagnosis, the reporting of effective novel treatment options is important for future patient care. background: adult patients with melanoma or lung cancer harboring braf v600e have benefitted from the development and subsequent approval of specific braf inhibitors. as such, delineating the subset of similarly targetable pediatric oncology patients may spur development and rational use of these inhibitors in children. importantly, other point mutations and fusions of braf may also be targetable in s249 of s301 children analogous to recent emerging data in adult cancer patients. objectives: to define the genomic landscape of known and novel braf alterations and raf1 fusions in pediatric malignancies and report index cases with clinical response to braf or mek inhibitors. design/method: dna was extracted from 40 microns of ffpe sections of 3,633 tumors from pediatric (<21 years of age) oncology patients, and cgp was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of 579x for up to 315 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. genomic alterations (ga) included base substitutions, indels, copy number alterations and fusions/rearrangements. a total of 221 (6.1%) braf-altered pediatric malignancies were identified. 172 (77.8%) harbored a single kinaseactivating braf short variant, indel, or fusion. an alteration resulting in reduced braf kinase activity was identified in 8 (3.6%) tumors while 7 (3.2%) tumors harbored multiple braf alterations, 3 of which contained at least a single activating short variant. the remaining 34 tumors (15.4%) contained functionally uncharacterized variants. kinaseactivating braf alterations were identified in diverse tumor spectra comprised of brain tumors (75.3%; 18 subtypes), carcinomas (10.6%; 6 subtypes, with melanoma constituting 50% of cases), hematological malignancies (8.8%; 5 subtypes), sarcomas (2.9%; 3 subtypes), and extracranial embryonal tumors (2.4%; 2 subtypes). seventy-two (32.6% of braf-altered cases) braf fusions were identified, 64 (88.9%) of which were kiaa1549-braf; 2 involved the novel fusion partners: stard3nl and khdrbs2. seven (0.2%) raf1 fusionpositive cases, predominantly brain tumors (5), were identified; 2 involved the novel fusion partners: tmf1 and sox6. index cases of response to therapy of intracranial tumors will be presented. we describe a population of pediatric patients with targetable braf alterations predominantly enriched in primary intracranial tumors, but spanning diverse solid tumor types and hematologic malignancies. we additionally report a cohort of raf1 fusion-positive patients. an index case and multiple previous reports suggest raf or mek inhibitors may benefit pediatric patients with either intracranial or extracranial disease, and development of such drugs in pediatric indications is strongly warranted. background: diffuse midline gliomas (dmg) with h3k27m mutation, including diffuse intrinsic pontine glioma (dipg), are the leading cause of brain tumor-related deaths in children. there are no effective therapeutic strategies and the median survival remains dismal. genomic studies have identified a recurrent mutation in the majority of dmgs involving a lysine to methionine substitution (k27m) in histones 3.1 and 3.3, resulting in changes in the epigenetic landscape that dysregulate gene expression and promote gliomagenesis. panobinostat, a multiple histone deacetylase (hdac) inhibitor, was found to be one of the most effective agents against dipg patient-derived cell cultures and xenograft models in previous studies and is presently in clinical trial for dipg. hdac inhibition with panobinostat may also exhibit activity against h3k27m+ diffuse midline gliomas of the thalamus and spinal cord. to evaluate the effect of panobinostat as a single agent against patient-derived thalamic and spinal cord h3k27m+ diffuse midline glioma cell cultures and in an orthotopic xenograft murine model of h3k27m+ spinal cord glioma. design/method: patient-derived thalamic and spinal cord h3k27m+ diffuse midline glioma cell cultures were treated with single agent panobinostat at a range of concentrations. cell viability was evaluated using the celltiter-glo assay. panobinostat was systemically administered to orthotopic xenograft murine models of luciferase-expressing spinal cord h3k27m+ diffuse midline glioma. response to panobinostat was evaluated with ivis in vivo imaging. results: hdac inhibition with panobinostat significantly decreases cell proliferation with an ic50 of 30 nm and 41 nm in the spinal cord and thalamic glioma patient-derived cell cultures respectively. panobinostat slowed tumor growth in murine models of spinal cord glioma by 1.5-fold in the brain (p = 0.0219, n = 5) and 2-fold in the spinal cord (p = 0.0176, n = 5) when compared to vehicle controls after 1 week of administration. panobinostat is in clinical trials for dipg. this study suggests that hdac inhibition with panobinostat may also be beneficial for patients with thalamic and spinal cord diffuse midline glioma h3k27m mutants. background: brain tumors are the most common solid tumor of childhood and the leading cause of childhood cancer deaths. while medulloblastoma is the most common malignant brain tumor of childhood with a 5-year survival 70-80%, children with high-grade gliomas (hggs) such as glioblastoma multiforme (gbm) fare much worse with a 5-year survival of 15-35%. implicated in this poor outcome is the presence of treatment resistant brain tumor stem-like cells. gbm stem-like cells (gscs) have been implicated in tumor growth, treatment resistance and patient relapse, making them a key therapeutic priority. antipsychotic drugs (apds) have been used for decades in various psychiatric clinical settings and are associated with a lower incidence of cancer, including malignant brain tumors. currently, atypical apds are being evaluated for their potential to alleviate cancer and treatment induced side effects. furthermore these drugs may have direct anti-tumor effects, potentially via inhibition of dopamine d2 receptors (drd2). objectives: determine the anti-cancer effects of atypical apds on gbm stem-like cells design/method: the anti-cancer effects of apds (quetiapine and risperidone) were evaluated on gbm stem-like cell lines developed in our laboratory (glio 3 and 38) and the group 3 medulloblastoma cell line hdmbo3. cell proliferation/viability was determined using trypan blue exclusion and mts assays. the effect of apds on cancer stem cell self-renewal was determined by neurosphere assay. receptor expression and apds effect on cell cycle proteins were examined by western blot analysis. results: western blot analysis of gscs and hdmbo3 demonstrated robust drd2 expression indicating a viable therapeutic target. both apds induced dose dependent cell death of all cell lines tested. treatment with only 2um of either apd for 10 days significantly reduced cell proliferation by 60% (hdmbo3) and 50-90% (gscs). consistent with these findings, we observed an increase in cell cycle inhibitors p21 and p27. furthermore at day 10 both apds induced a robust increase in gsc death, approximately 60% compared to only 10% in non-treated controls. lastly, 1um apds significantly reduced gsc neurosphere formation compared to untreated controls by up to 35% suggesting inhibition of gbm stem cell self-renewal. our data indicates that clinically relevant concentrations (low micromolar) of these apds induce anticancer effects in both gscs, which are enriched with tumor initiation/propagation properties, and in the group 3 (myc amplified) medulloblastoma cell line. these apds represent strong candidates as potential adjuvant therapies for the treatment of these brain tumors. background: while the poor prognosis for high risk neuroblastoma (hrnb) underscores the need for new treatment strategies, the elucidation of specific biologic subsets of neuroblastoma suggests a way to improve disease management. the identification of agents that target specific molecular pathways associated with the development or progression of diseases holds promise. dfmo, an inhibitor of odc, has been shown to decrease lin28 and mycn and target cancer stem cells in preclinical studies. currently 14% of patients undergoing immunotherapy relapse. dfmo is in studies to prevent relapse after immunotherapy and may be helpful during immunotherapy as well. the hypotheses for this study were that: 1) the incorporation of a targeted therapy, selected based upon upfront tumor genomic interrogation, into standard induction chemotherapy for hrnb is safe, feasible and may increase the pr/cr/vgpr response rate at the end of induction therapy; and 2) the addition of dfmo as maintenance during immunotherapy is safe and feasible and may decrease the relapse rate for hrnb. a multicenter feasibility pilot trial in subjects with newly diagnosed hrnb within the beat childhood cancer consortium. at diagnosis, patients' tumors underwent dna exome and rna sequencing which were analyzed within a molecular tumor board to identify the single best drug of 6 targeted agents to be added to cycles 3-6 of induction chemotherapy. after consolidation with asct and radiation, the patients received dfmo along with standard dinutuximab and retinoic acid and dfmo for 2 years after immunotherapy. patients were evaluated for additional toxicities with the addition of targeted agents and dfmo in addition to induction response. results: the pilot study of 20 eligible patients has shown this process to be feasible. all 20 patients have completed induction portions of the study. the combination of targeted agent with chemotherapy was shown to be safe without any unexpected toxicities. delays between induction cycles were < 2 weeks and related to surgery, infection, or thrombocytopenia. the induction response demonstrated 88% cr/vgpr/pr rate, which suggests improvement over historical 80%. in addition, 15 patients were eligible for the combination of dfmo with dinutuximab and retinoic acid was well tolerated and safe without additional toxicities due to dfmo. the pilot study of 20 patients has shown the process of genomic sequencing and addition of a targeted agent to upfront chemotherapy and addition of dfmo to dinutuximab and retinoic acid maintenance therapy in newly diagnosed hrnb patients and is feasible and safe without any unexpected toxicities. background: identifying sub-populations of medulloblastoma tumors with stem cell-like properties holds promise for reducing disease recurrence, but there is no known unifying marker of medulloblastoma cancer stem cells. the granulocyte stimulating factor receptor (gcsf-r or cd114) is well understood in the context of hematopoiesis, but its role in solid tumor pathogenesis is less clear. neuroblastoma and melanoma subpopulations expressing gcsf-r have cancer stem cell properties of chemoresistance and increased tumorigenicity, and are enriched in tumors after chemotherapy. gcsf-r activation leads to signaling through the jak-stat pathway, suggesting a potential therapeutic target. we hypothesized that a subpopulation of medulloblastoma cells would express the gcsf-r and that this subpopulation would demonstrate chemoresistance and response to inhibitors of the jak/stat pathway. objectives: our objective was to identify a subpopulation of medulloblastoma cells expressing the gcsf-r and determine their relative growth rates, tumorigenicity, and responses to chemotherapy and jak/stat inhibition. design/method: medulloblastoma cell lines were sorted via flow cytometry for gcsf-r surface expression. subpopulations of gcsf-r-positive and -negative medulloblastoma cells were then monitored for growth by continuous live cell imaging. responses to chemotherapy were measured in subpopulations of gcsf-r-positive and -negative medulloblastoma cells using continuous live cell imaging to measure percent cell confluence and cell viability assays. ic50 values were calculated for each cell line and each agent. parental medulloblastoma cell lines and isolated gcsf-r-positive and -negative subpopulations were also treated with the jak1/2 inhibitor ruxolitinib and growth rates, viability, and ic50 values were calculated. results: gcsf-r surface expression was identified on 0.2-1.3% of medulloblastoma cell lines. isolated gcsf-r positive cells demonstrate a slower growth rate compared to gcsf-rnegative or parental unsorted medulloblastoma cells. gcsf-r positive cells are more resistant in vitro to vincristine, etoposide, and carboplatin, when compared to the gcsf-r negative population and an unsorted population of the same cell line. ruxolitinib is cytotoxic to medulloblastoma cells in vitro, with higher ic50 values noted in gcsf-r positive cells compared to unsorted and gcsf-r negative cells. we show that a subpopulation of gcsf-r positive cells are present in multiple medulloblastoma cell lines via flow cytometry, and that isolated gcsf-r-positive cells have a slower growth rate than gcsf-r-negative or unsorted populations. we also show that ruxolitinib has in vitro activity against medulloblastoma cell lines. we propose that jak inhibition may represent an adjunct therapy targeting overall tumor burden and specifically targeting the gcsf-r-positive subpopulation of medulloblastoma cells that may drive tumor recurrence. we investigated the efficacy of intensified adjuvant chemotherapy in osteosarcoma patients. design/method: we retrospectively analyzed the medical records of 48 children with osteosarcoma treated at asan medical center between 2006 and 2015. all patients received a 3-drug induction consisting of 2 cycles of cisplatin and doxorubicin along with 4 cycles of methotrexate (map), and proceeded to surgical resection. adjuvant ct was map or map with the additional ifosfamide and etoposide (mapie), and mapie was mainly considered for poor responders (tumor necrosis below 90%) or patients with metastases. results: among 48 patients, 6 patients had metastases at diagnosis. surgery was conducted in 43 patients who responded to induction ct, and 17 showed over 90% tumor necrosis. among 43 patients who proceeded to adjuvant ct, 19 and 24 patients received to map and mapie protocols. with a median follow-up of 73 months, the 5-year overall survival (os) and event-free survival (efs) rates of all patients were 80% and 46.7%. of those 43 patients, 16 patients recurred, and 5 of them died of disease progression. relapsed patients received salvage ct and/or surgery, and 2 were rescued after autologous stem cell transplantation (sct). three patients developed treatment-related acute myeloid leukemia, and they are alive after allogeneic sct. according to the response to neoadjuvant ct, the os rates of good responders (n = 18) and poor responders (n = 30) were 100% and 68.1% (p = 0.011), and efs rates were 63.8% and 41.7% (p = 0.084). of the 30 poor responders, 9 patients received map as adjuvant ct, and the other 21 received mapie. the os rates of map and mapie group were 68.6% and 70.3% (p = 0.568), and efs rates were 30.0% and 48.3% (p = 0.247), respectively. when patients were classified into three groups: 1. localized disease & necrosis ≥ 90% (n = 17), 2. localized disease & necrosis < 90% (n = 24), 3. metastatic disease (n = 6), survival rates were in the order of group 1>2>3 (os = 100%:81.8%:16.7%, efs = 67.6%:43.6%:0%). in each group, intensified adjuvant ct by mapie did not improve survival outcomes. conclusion: initial metastatic disease and poor histological response to neoadjuvant ct were major risk factors for poor survival in osteosarcoma patients. we found that adding ifosfamide and etoposide to map did not improve survival outcomes of patients with adverse risk factors. more effective adjuvant therapy for these patients is needed. background: circulating cell-free dna (cfdna) that shed from tumors into circulation have been used for noninvasive molecular profiling in adult cancers but little is known about its utility in pediatric cancers. pediatric patients with metastatic and refractory solid tumors are known to have poor survival rates, and a key challenge in their management is obtaining biopsy samples especially at times when disease is widely spread or the patient is physically unfit for sampling. the development of a noninvasive profiling strategy is critical for optimizing molecularly guided therapy and assessing response to treatment. in this study, we want to determine the utility of cfdna to noninvasively analyze the molecular profiles of pediatric solid tumors such as neuroblastoma (nb), osteosarcoma (os), and wilms tumor (wt). design/method: tumor, plasma, and matched controls were collected from patients with nb, wt, and os, at diagnosis or time of disease progression. cfdna was extracted from the plasma and analyzed through multiple methodologies including a targeted next generation sequencing panels and shallow whole genome sequencing (swgs). results: fifteen nb patients, 10 os patients, and 2 wt patients had tumor molecular profiles known from different targeted next-generation sequencing platforms. in the cfdna of 7/15 nb patients, somatic mutations and copy number alterations previously reported in the tumors were detected, including recurrent nb drivers such as mycn amplification, alk, and atrx mutations. mutations not detected in the original tumor were also found in 6/15 nb patients including nras, mll2, arid1b, some of which are potentially actionable. in os, mutations known from the tumor were found in the cfdna of 5 of 10 patients, including atrx and notch3 mutations, as well as copy number alterations such as cdk4 amplification, which has targetable therapeutics available. of the two wt patients analyzed, cfdna revealed the same mutations as tumor in one patient, however in a cohort of patients where tumor was not available, cfdna revealed recurrent driver mutations such as amer1, dicer1. it is feasible to noninvasively identify somatic mutations and copy number alterations in cfdna of patients with pediatric solid tumors. establishing a platform using cfdna to identify molecular profiles of these tumors can serve as a powerful tool for guiding treatment and monitoring response to treatment. background: despite multi-modality therapy, the prognosis for patients with metastatic osteosarcoma remains poor necessitating development of novel targeted therapies. immunotherapy can be exploited to target osteosarcoma with exquisite specificity but remains limited by insufficient tumor specific targets. objectives: to overcome the dearth in tumor specific antigens, we have explored the use of tumor derived mrna (representing a tumor specific transcriptome) for development of personalized nanoparticle vaccines. design/method: rna-nanoparticles (rna-nps) can be amplified from limited amounts of biopsied tissue for induction of tumor specific t cells against osteosarcoma. since local vaccination strategies are mired by poor overall immunogenicity, we assessed the feasibility, immunogenicity and antitumor activity of intravenously administered rna-nps (tumor mrna complexed to dotap nanoliposomes) in pre-clinical murine and canine tumor models. we identified a clinically translatable np formulation for the delivery of rna to antigen presenting cells (apcs) that induces in vivo gene expression and preserves rna stability over time. tumor derived rna-nps induced antigen specific t cell immunity and mediated anti-tumor efficacy in several pre-clinical solid tumor models (i.e. b16f10, kr158b). when administered intravenously, rna-nps increased expression of co-stimulatory molecules (i.e. cd80, cd86, cd40, ccr7) and pd-l1 on cd11c+ cells throughout reticuloendothelial organs (i.e. spleen, liver, bone marrow) and within the tumor microenvironment; this phenotype was strictly dependent on type i interferon. targeted inhibition of type i interferon signaling (via infar1 mabs) abrogated anti-tumor efficacy mediated by rna-nps. we enhanced the immunogenicity of this platform by simply combining mrnas encoding for immunomodulatory molecules (i.e. hcv-pamps, gm-csf) or by combining rna-nps with immune checkpoint inhibitors. addition of checkpoint inhibitors (pd-l1 mabs) to rna-nps increased tumor infiltrating lymphocytes, and intratumoral mhc class i/ii expression, and mediated synergistic anti-tumor activity in settings where pd-1 or pd-l1 inhibition alone did not confer therapeutic benefit. we then explored the feasibility of rna-nps in a large animal osteosarcoma model. in ongoing studies for canines with osteosarcomas, we have shown that sufficient amounts of rna can be extracted, amplified, and manufactured into personalized rna-np vaccines. conclusion: rna-nps reprogram systemic immunity and mediate anti-tumor activity providing near immediate immune induction without the complexity of cellular immunotherapy. the immune correlate of preclinical response to rna-nps is hallmarked by interferon dependent pd-l1 expression on activated apcs (cd11c+ mhcii+ cd86+ cells). based on these findings, we are exploring the preclinical safety, efficacy and immunologic effects of rna-nps targeting canine osteosarcoma before first in-human evaluation. background: ewing sarcoma is an aggressive bone tumor affecting mainly adolescent and young adults. treatments are based on compressed schedule chemotherapy combined with local control (surgery and/or radiation). prognosis is poorer for patients with metastatic disease, older age and central primaries. survival when disease recurs within two years of diagnosis is <10%. the ews-fli1 fusion gene t(11; 22) (q24; q12) has been well characterized as a dominant ews driver-gene. the most common variation is ews exon 7 with fli1 exon 6 (60% of fusion positive patients). we designed a novel pbi-shrna tm ews/fli1 type 1 lpx which has demonstrated, safety and efficacy in animal model (rao et all). the pbi-shrna strategy silences target gene expression by concurrently inducing translational repression and p-body sequestration as well as post-transcriptional mrna cleavage. to determine the safety and maximum tolerated dose of intravenous administration of pbi-shrna tm ews/fli1 type 1 lipoplex in patients advanced ews. design/method: phase i study 3 × 3 escalation cohort. testing pbi-shrna tm ews/fli1 type 1 lpx (starting iv dose of 0.04 mg/kg) on patients (≥ age 8) with advanced ewing's sarcoma, all with a type 1 translocation. intravenous infusion was given twice a week for 4 weeks with the following escalation schema: 50% → 33% → 25% → 25% → 25%. required kps >80% and adequate organ function. cytokines induction pre and post-infusion was analyzed (il-12, il-6, tnf-alpha, il1ra). first cohort of patients has been enrolled (ages between 17-35 years). three relapsed patients had >3 lines of therapy and 1 patient had refractory disease, 3 patients received a complete cycle of pbi-shrna tm ews/fli1 type 1 lpx with twice a week infusions. a total of 69 doses were given. the most prominent related toxicity has been hematological, 1 patient developed transient g3 neutropenia, another patient developed g3 anemia that required prbc transfusion, and of note this patient had significant bone and bone marrow involvement. one patient only received two lpx infusions; she developed a fatal rsv pneumonia. other reported grade 2 toxicity includes fatigue and headache. evaluable patients (n3) had stable disease between 4 and 12 months before progression. one patient had sustained response for 12 month before progression, two patients are still alive. our preliminary experience supports the safety and potential efficacy of pbi-shrna tm ews/fli1 type 1 lpx as novel treatment for advanced ews with limited toxicity. il-6 increase correlates with higher bi-shrnai ews/fli1 lpx infusion rate and clinical symptoms. further clinical testing is indicated. background: as more children with cns malignancies (bt) are surviving, the late effects of the therapies they receive are better described. studies show that radiation therapy is particularly harmful to neurocognitive functioning, specifically processing speed, working memory, and attention span. these deficits have negative effects on quality of life, especially in academic and professional settings. a large proportion of s255 of s301 adult survivors of bt are unable to reach adult milestones such as living on their own, holding a steady job, and getting married. proton beam radiation therapy (pbrt), is touted for the potential to have fewer and less severe side effects than traditional photon radiation therapy (xrt). because of the properties of protons, the amount of damaging energy released in non-target healthy tissue is reduced when compared to xrt. although a study comparing iq testing between pbrt and xrt found no difference between the two therapies, no studies have compared the specific neurocognitive domains. it would be valuable to evaluate full neurocognitive testing scores (nct) since the specific domains, particularly processing speed (psi), appear to be most vulnerable to radiation therapy. objectives: our primary aim was to assess differences in psi for patients with bt who underwent pbrt versus xrt. a secondary aim was to assess differences in iq (fsiq) and working memory (wmi). we retrospectively evaluated all patients treated for bt at the jimmy everest cancer center within the past 20 years who received rt and had nct post radiation. we examined the full nct results for both subsets of participants to evaluate differences in the specific domains of processing speed, working memory, and iq by measuring percentiles scored in these domains. objectives: we report our experience on imaging children with mm treated uniformly on an institutional melanoma trial. we retrospectively reviewed the clinical and imaging findings of patients with ajcc stage iic-iv cutaneous mm treated on our institutional mel06 protocol. brain mri/ct, pet/ct, ct chest, abdomen, and pelvis (ctcap) were performed at diagnosis in all patients. on treatment, stratum a patients (peg-interferon; ajcc iic, iiia, iiib) (n = 16) had the same imaging repeated every 6 months; stratum b1 (peg-interferon and temozolomide; unresectable measurable disease metastatic, or recurrent) (n = 2) had pet scans every 2 months and brain imaging every 4 months; those in stratum b2 (peg-interferon and temozolomide; unresectable non-measurable, metastatic, or recurrent) (n = 3) had the same imaging performed every 4 months. off therapy all patients continued same imaging every 6 months for 3 years. results: there were 21 patients (11 female; median age 14 years). eleven had spitzoid and 10 conventional melanoma. primary sites included head/neck (n = 6), trunk (n = 7), and extremities (n = 8). patients with spitzoid melanoma had 236 imaging studies (86 pet, 81 ctcap, 11 ct chest, 10 ct brain, and 48 mri brain) with a median of 8, 7, 0, 4 and 0 studies/patient respectively. median cost per patient was $32,718. thirteen studies (5.8%) showed suspicious lesions with 28 additional scans and 2 diagnostic biopsies of which one only was positive stratum a with tert promoter mutation and died from disease). for conventional mm, 162 studies (61 pet, 57 ctcap, 8 ct chest, 7 ct brain, and 29 mri brain) were performed with a median of 7, 6.5, 0, 1, 3 studies/patient respectively. median cost per patient was $23,420. twenty (14%) showed suspicious lesions with 19 additional scans and 6 diagnostic biopsies; four were positive (two at diagnosis); both died of disease; the other two recurred locoregionally and were detected clinically; both are alive and disease free; one patient had diffuse metastases and died shortly after enrollment. after a median follow up of 6.3 years (range 0.4-9.2) 17 patients are alive and disease free. children with spitzoid melanoma should have minimal imaging at diagnosis and follow-up given the low risk of recurrence and low yield and high cost of aggressive imaging protocols. patients with conventional mm should be imaged according to the adult guidelines. nationwide children's hospital, columbus, ohio, united states background: the role of infections in the long term outcome of patients with bone tumors is controversial. two retrospective studies have shown increased survival in osteosarcoma patients who had a post-operative wound infection, while another showed no changes in overall survival. to determine the relationship between wound infections and/or bloodstream infection (bsi) on survival in pediatric and young adult patients with osteosarcoma and ewing sarcoma treated at a tertiary children's hospital. design/method: a retrospective chart review was performed for patients with diagnosis of osteosarcoma or ewing sarcoma from 2006-2016. patients received standard chemotherapy regimens for their disease type and stage. local control included surgical resection and/or radiation therapy. presence of infection was determined by bsi or wound cultures while receiving treatment for primary tumor. the median age of 85 patients was 14 (range 2-46 years) at diagnosis. 53% had a diagnosis of osteosarcoma and 47% had ewing sarcoma. of these, 46% of patients developed an infection during treatment; 25% had bsi, 26% had wound infections, and 5% had both. patients with bsi had a 5 year os of 63.3%, compared to 81% in those without bsi (p = 0.0015). those with both bsi and wound infections had the poorest overall survival of 50%, compared to 80.8% for patients without any infection. patients with wound infections alone had a 5 year os of 80.2%, compared to 75% of patients without a wound infection. our analysis revealed decreased os in patients with bsi; however, this could be due to other confounding factors in the presence of bsi. those with bsi or bsi and wound infections had the poorest survival. wound infections without bsi were associated with a slight increase in survival; however, this study was limited by the number of patients that had local wound infections. with the use of newer surgical techniques, availability of antimicrobials and routine use of prophylactic antibiotics, the incidence of infections while undergoing treatment is low. however, the importance of this clinical observation indicates a likely enhanced immune system associated with infection, supporting the role of immunotherapy for treatment of these aggressive tumors. background: hypoalbuminemia is a well-recognized effect of cancer and other chronic illnesses and is often regarded as a marker of malnutrition. in adults, hypoalbuminemia has been associated with adverse outcomes in patients with cancers of the lung, pelvis, head and neck, gastrointestinal tract, and bone marrow, as well as in some pediatric patients with ewing sarcoma and hodgkin lymphoma. hypoalbuminemia has not been well studied in children with cancer. to determine the incidence of hypoalbuminemia (using age-specific references) in children with cancer receiving chemotherapy at baseline (prior to starting chemotherapy) and to determine whether hypoalbuminemia is associated with inferior 5-year overall survival. design/method: we performed a single institution, irbapproved, retrospective review of pediatric oncology patients diagnosed between 1998 and 2012. five-year survival was estimated using the kaplan-meier method; groups were compared using cox regression. we identified 863 pediatric patients with a first diagnosis of cancer, brain tumor, or other condition possibly requiring chemotherapy. of these 863 patients, 204 were excluded for reasons including not receiving chemotherapy and missing data, leaving 659 patients who had a serum albumin level within 60 days prior to starting chemotherapy. the mean age was 8.1 years (sd 5.8 years); 62% were male; 92% were non-hispanic. the most common diagnosis was acute lymphoblastic leukemia (201 of 659; 31%). one hundred thirty nine of 659 (21%) had hypoalbuminemia prior to starting chemotherapy. there was no statistically significant difference in 5-year overall survival between those with and without hypoalbuminemia (78% vs. 82%, respectively; hazard ratio 1.27, 95% c.i. 0.85 -1.90). conclusion: hypoalbuminemia at baseline in pediatric oncology patients requiring chemotherapy is common (one in five), and was not associated with inferior 5-year overall survival in this cohort. leptomeningeal metastases at diagnosis. standard treatment for completely resected, non-anaplastic supratentorial ependymomas is close observation. treatment for anaplastic or incompletely resected non-anaplastic ependymomas is maximal safe surgical resection followed by focal radiation. however, up to 50% of localized ependymomas recur. the role of chemotherapy in treating ependymomas is under investigation. extraneural metastases of anaplastic ependymomas have rarely been reported and the outcome is dismal. objectives: to report extraneural cervical node metastases of a non-anaplastic ependymoma and successful treatment with surgical resection, radiation, and systemic chemotherapy. design/method: retrospective review of patient medical records, including radiographic imaging and tumor tissue pathology, and comprehensive literature review. results: a previously healthy 3-year-old girl underwent gross total resection (gtr) of an isolated right parietal lobe ependymoma (who grade ii). at age 4 years, magnetic resonance imaging (mri) revealed an isolated localized recurrence. she underwent gtr followed by observation. at age 6 years, she again experienced isolated localized recurrence and underwent gtr followed by 59.4 gy focal conformal photon radiation. at each recurrence, pathology revealed a non-anaplastic ependymoma, and cerebral spinal fluid (csf) cytopathology and spine mri were negative. at age 10 years, she developed an enlarged right posterior cervical chain lymph node. subsequent mri revealed a large rim-enhancing, t2 hyperintense lymph node and multiple abnormally enhancing regional nodes consistent with metastases. biopsy revealed a non-anaplastic ependymoma. mri of the brain and spine, computed tomography of the chest, abdomen, and pelvis, and csf and marrow evaluations were unremarkable. chemotherapy according to acns0831 was initiated. mri after course 3 demonstrated significant node size reduction. she underwent right neck node dissection. only one right level ii lymph node showed metastases. she was treated with 59.4 gy irradiation to the neck and 3 additional courses of chemotherapy. she remains in remission 24 months and 17 months after diagnosis of metastatic disease and end of therapy, respectively. literature review reveals rare reports of extraneural metastatic disease of anaplastic ependymomas to bone, lung, or liver, and only 2 involving lymph nodes, all associated with a poor outcome despite multimodal therapy. to our knowledge, this is the first report of extraneural metastases of a non-anaplastic ependymoma. extraneural metastases should be considered in children previously treated for non-anaplastic ependymomas who experience systemic symptoms, even in absence of cns relapse. multimodal treatment offers potential long-term disease control with acceptable toxicity. arun gurunathan, joel sorger, andrew trout, joseph pressey, rajaram nagarajan, brian turpin cincinnati children's hospital medical center, cincinnati, ohio, united states background: pigmented villonodular synovitis (pvns) is a benign neoplasm of the synovium. standard treatment is surgery, but post-operative recurrence rate is as high as 60%. radiation therapy can be used for local control, but is associated with late effects. while pvns is rarely fatal, aggressive disease and/or extensive surgery can result in substantial functional impairment. colony stimulating factor-1 (csf1) overexpression, often due to chromosomal translocation involving csf1, drives pvns through recruitment of synovial-like mononuclear cells expressing the csf1-receptor. tyrosine kinase inhibitors such as imatinib are active against the csf1-receptor, and have shown benefit in the post-surgical relapse setting. however, questions remain regarding the broader application of imatinib and regarding optimal response assessment. to present three patients with pvns, each with different clinical scenarios, who demonstrate clinical response to imatinib monitored by changes in metabolic activity (maximum suv) on pet/ct. results: three patients with pvns demonstrate pet/ct response to imatinib, guiding management of their challenging clinical scenarios. patient 1 is a 20 year-old female with left hip pvns and high grade articular cartilage loss, with decrease in metabolic activity (suvmax 8.3 to 4.7 in 3 months) on neoadjuvant imatinib, enabling total hip replacement surgery planning. patient 2 is a 16 year-old female with left knee pvns with recurrences after synovectomies, spared subsequent surgical control attempts after clinical improvement correlating with pet/ct response to imatinib (suvmax 10.2 to 4.3 in 2 months). patient 3 is a 27 year-old male with right knee pvns that recurred after total knee replacement, now with clinical improvement correlating with pet/ct response to imatinib (suvmax 11.2 to 5.2 in 3 months). all patients would have been characterized as stable disease by response evaluation criteria in solid tumors (recist). in each of these patients, imatinib has been tolerated well, with no therapy interruptions and absent or easily managed side effects (one patient takes dronabinol for decreased appetite, one patient takes prn immodium for diarrhea). all patients are currently still taking imatinib, with therapy length ranging from five to eleven months. in our series of three patients with pvns, imatinib shows promise for disease management in neoadjuvant and adjuvant settings with a tolerable side effect profile. imatinib should be considered in the treatment of pvns to spare surgical and radiotherapy related morbidity, and treatment effect can be monitored by pet/ct. background: metastatic rhabdomyosarcoma (rms) carries a poor prognosis with three-year event free survival rates ranging between 20%-69% (depending on oberlin risk factors) due to the lack of significantly effective breakthroughs in the recent past. there is an urgent and unmet need for new treatment strategies against this disease. metastatic rms cell lines exhibit increased expression of the erm family membrane-cytoskeleton linker protein ezrin. knockdown of ezrin expression using sirnas decreases the metastatic potential of these cells, whereas forced expression of ezrin results in increased degree of metastasis. the activity of ezrin is controlled by its phosphorylation at the threonine 567 (thr567) residue at the c-terminus of the protein, suggesting that alteration of ezrin phosphorylation may control rms growth and metastasis. our goal was to determine if pharmacological inhibition of thr567 phosphorylation in ezrin affects the growth, survival and metastasis in rms in vitro as well as in vivo. design/method: rms cell lines representative of the alveolar and embryonal histological subtypes were used. rms cells were treated with a small molecule inhibitor of ezrin, nsc668394, which specifically dephosphorylates ezrin at the thr567 residue. baseline expression of ezrin and perm levels as well as the effect of nsc668394 on perm levels in the rms cell lines was determined by western blotting of cell lysates. viability of cells was assessed by trypan blue exclusion, and morphology visualized by bright field microscopy. the extent of apoptosis was detected by imaging caspase 3/7 activation using fluorescent microscopy. motility of rms cells was examined by performing a wound-healing assay. subcutaneous and orthotopic xenografts were established in nsg mice using rd cells (embryonal rms). mice harbor-ing xenografts were treated with intraperitoneal injections of nsc668394 or dmso. results: ezrin is constitutively phosphorylated at the thr567 residue in a majority of the rms cell lines examined. nsc668394 dephosphorylates ezrin at the thr567 residue in these cell lines. treatment with nsc668394 inhibits growth, induces apoptosis and inhibits the migration of rms cell lines in vitro. further, treatment of nsg mice bearing subcutaneous or orthotopic embryonal rhabdomyosarcoma xenografts with nsc668394 significantly impedes tumor progression without any obvious adverse effects. our findings suggest that dephosphorylation of ezrin at the threonine 567 residue may have the potential to be a novel therapeutic strategy for rms patients. all india institute of medical sciences, new delhi, new delhi, delhi, india background: the role of laparoscopy in the management of pediatric intra-abdominal solid tumors is yet to be established. the safety of laparoscopic management of pediatric intra-abdominal tumors is still questionable. we study the results of the initial case series of pediatric intraabdominal tumors managed laparoscopically at our institute from july 2013 onwards. design/method: total 11 children (8-males, 3 females) who presented to us with pediatric intra-abdominal tumors were included. the tumors included wilms tumor (n = 7), neuroblastoma(n = 2), adrenal cortical tumor(n = 1), ovarian teratoma(n = 1).children were between 10months -7years and 4 received neo-adjuvant chemotherapy. a 4-port laparoscopic nephrectomy and lymph node sampling for wilms tumor and adrenalectomy for adrenal tumors was performed. the tumors were removed in-toto with no rupture (except in one). specimens were retrieved through a lumbar incision (n = 8) or an inguinal incision(n = 1). all the children are under regular follow up. two children with wilms tumor had recurrence. the neuroblastoma child underwent open surgery for recurrence later. conclusion: laparoscopy/laparoscopic assisted removal of pediatric intra abdominal tumor is a feasible and safe option. it has the advantage of less postoperative pain, shorter hospital stay and a better cosmetic result. proper patient selection, port placement and laparoscopic experience are contributory. background: targeting of proteins and cell surface antigens specific to cancer cells with monoclonal antibodies has proven to be an effective form of treatment in many forms of cancer. gd2 is a cell surface disialoganglioside that is expressed on the cell surface of some normal tissues including nerve cells, melanocytes, and mesenchymal stromal cells and is overexpressed in some pediatric cancers like neuroblastoma and osteosarcoma. dinutuiximab is a chimeric monoclonal antibody that is fda approved for the treatment of patients with high risk neuroblastoma and under investigation for the treatment of relapsed osteosarcoma. little is known about the patterns of gd2 expression in other pediatric malignancies. objectives: we sought to describe the patterns of gd2 expression in the following pediatric sarcomas: synovial sarcoma, rhabdomyosarcoma and ewing sarcoma. design/method: synovial sarcoma (n = 44), rhabdomyosarcoma (n = 35) and ewing's sarcomas (n = 12) formalin fixed, paraffin embedded cores were obtained from the seattle children's research institute tissue microarray (tma) biorepository. tma blocks consisting of melanoma cores stained with and without gd2 antibody were used as positive and negative controls, respectively. slides were incubated with anti-ganglioside gd2 antibody clone 2q594 (ab68456 from abcam) diluted 1:50 in 10% normal goat serum and 5% bsa in tbs overnight at 4˚c. the negative control of human melanoma section was incubated in 10% normal goat serum and 5% bsa in tbs without primary antibody. the expression of gd2 was indicated by characteristic brown diaminobenzidine staining. the intensity and location of tissue staining were assessed and compared to positive and negative controls. staining was considered positive (+++) if the intensity of the staining was consistent with that of the positive control with 67-100% of cells staining positive. classification of intermediate gd2 expression (++) was assigned to slides in which 34-66% of cells stained positive. slides were classified as sporadic staining (+) if 1-33% of cells stained positive. tissue was considered (-) if there was complete absence of staining, similar to the negative control. objectives: to evaluate the clinical presentation, management and treatment outcomes of children with malignant germ cell tumor at our institute design/method: a prospective study was conducted from june 1994 to dec 2016 in the department of pediatric surgery in a tertiary care institute in a developing country. all patients were evaluated for local disease and metastatic disease by imaging and tumor markers. risk stratified chemotherapy was used with low risk tumor receiving no chemotherapy, intermediate risk: 4 courses of peb chemotherapy and high risk: 4 courses of peb + 2 courses of pe. upfront resection of the primary or the residual disease after neoadjuvant chemotherapy if feasible was performed. follow up was done with monthly tumor markers for 6 months and imaging studies every 3-6 months for initial 3 years. five year overall survival and disease free survival was calculated. results: during the study we treated 152 children who formed the study group. of these 83 (55%) were gonadal (45;30% testicular and 30;25% ovarian) and the remaining 69 (45%) were extragonadal with sacrococcygeal (sct) being the most common site 48 (31%). one hundred and thirteen children (75%) presented to us primarily while the remaining 39 had received treatment elsewhere. stage 3 or stage 4 disease at presentation was present in 104 (68%) children. recurrence was noted in 50 (33%) patients. respectively. patients with testicular mgct and children with age 2-5 years and males had significantly poor rfs rates. conclusion: patients with mgct should be staged correctly and adjuvant chemotherapy is advisable to all patients except stage i endermal sinus tumor of testis. awareness regarding the same is still lacking in our country. meticulous follow up is needed as more than 30% of will recur. cure rates are dismal in children with recurrent mgct especially those who are not chemotherapy naïve. nemours children's specialty care, jacksonville, florida, united states background: radiotherapy for pediatric head and neck tumors often results in mucositis, limiting oral intake and compromising patients' nutritional status. this may be reduced through the improved conformality offered by proton therapy. despite widespread use of enteral tube feeding through a percutaneous gastrostomy (peg) or nasogastric tube (ngt), there is little data available regarding overall incidence of ngt/peg placement and perspectives of pediatric patients and caregivers. objectives: to (a) estimate the need for ngt/peg support and (b) characterize patient and caregiver perceptions surrounding enteral feeding in children with head and neck tumors undergoing proton therapy. design/method: dependent on development stage, patient (n = 16) or parents (n = 6) filled out a series of customized surveys according to a prospective irb approved study. seventythree percent of patients also received concurrent chemotherapy. questions addressed their current feeding route and perception, for example, "what aspect(s) of tube feedings are beneficial to you?" and "what aspect(s) of tube feeding worry or scare you?" fifty-five surveys were distributed before and after radiation, and with any change in feeding route. results: at the start of proton therapy, 1 patient had a ngt and 8 patients had peg. of these, 8 patients (36%) had a ngt/peg in place exclusively for the administration of medication; only 1 patient (4%) needed a ngt/peg for nutrition. in those patients without ngt/peg, 46% would "consider" enteral feeds. in patients without ngt/peg, the most commonly cited benefit was "maximizing my nutrition" (67%) and the most common negative aspect was "fear" of tube placement (100% of patients). all sub-populations (32% of patients) cited change in appearance as a negative aspect. in patients without ngt/peg at the start of proton therapy, 46% of patients/caregivers felt enteral feeding to be "unnecessary," and 83% of these patients would not "consider" ngt/peg even if their "physician advised it." over the course of proton therapy, the patients/caregivers who deemed enteral feeding "unnecessary" decreased from 46% to 18%. at completion of treatment, 7 patients (32%) were using a ngt/peg tube for nutritional support but only one (4%) patient relied exclusively on their enteral feeds. two patients (without ngt/peg) (9%) required parenteral support. our data does not support prophylactic placement of ngt/peg in of children with head and neck tumors undergoing proton therapy. ongoing research is needed to identify which patients will need ngt or peg to supplement their diet. in this cohort, anticipatory counseling should focus on pain, cosmesis, and utility. children's national medical center, washington, district of columbia, united states background: ovarian sex cord-stromal tumors (osct) are rare neoplasms that typically present with signs/symptoms of an adnexal mass and signs of hormonal production1 approximately 20% of ovarian sex cord-stromal tumors in children are sertoli-leydig cell tumors (slct) with median age of presentation 25 years overall.1 to our knowledge the youngest reported case in the literature describes a 9-month old female in china with a slct that was treated with oophorectomy alone.2 some studies have found an association in families between pleuopulmonary blastoma and osct with a germline mutation leading to dicer1 syndrome, which has been associated with a younger age at diagnosis.3,4 objectives: to describe an unusual case presentation of slct in an infant results: 3-month old, twin female, ex-32 week premature infant presented to the emergency department on multiple occasions for abdominal distention and feeding intolerance initially thought to be related to previous omphalocele repair and umbilical hernia. an ultrasound demonstrated an 8 × 6 cm mass arising from the right ovary with large volume ascites. she required admission to the intensive care unit due to s261 of s301 respiratory distress from her significant ascites. serum tumor marker including hcg, afp and ldh were negative. patient underwent right oophorectomy with tumor capsule noted to be open at time of surgery. further imaging post operatively demonstrated no other sites of disease. the patient was classified as figo stage ic due to the presence of her significant abdominal ascites that was presumed to be malignant pre-operative tumor rupture.5 the pathological diagnosis was challenging and eventually resulted as a mixed germ cell sex cord stromal tumor with pattern of sertoli cell tumor with neuroendocrine differentiation. based on the staging of figo ic with pre-operative rupture, the decision was made to treat with a standard platinum based regimen as there is a higher incidence of relapse in stage ic patients when compared to ia treated with observation alone.6 our patient tolerated four cycles of chemotherapy well and end of therapy scans showed no evidence of disease. interestingly, her dicer mutation genetics performed by ion torrent tm next generation sequencing was negative in germline and tumor studies. to our knowledge, our patient is the youngest described with slct. she will continue to be followed with serial imaging alone as she had no evidence of elevated tumor markers at diagnosis.6,7 due to young age and unusual diagnosis, she was referred to cancer genetics team. background: approximately 12% of patients with wilms tumor (wt) have metastatic disease at diagnosis and often have a grave prognosis. limited cell lines are available for the study of metastatic wt and long-term passaged cell lines do not always recapitulate the human condition. focal adhesion kinase (fak) is a non-receptor tyrosine kinase that controls cellular pathways involved in the tumorigenesis of pediatric renal tumors. using a novel patient-derived xenograft (pdx) model from a patient's primary wt (coa 25) and matched isogenic metastatic wt (coa 42), we previously demonstrated that fak is expressed and its inhibition led to decreased tumorigenicity of both the primary and metastatic pdxs. kinomic profiling is an innovative, high-throughput method used to investigate kinase signaling to identify potential therapeutic targets. to date, the kinomic profile of primary and metastatic wt has not been examined. objectives: investigate baseline kinomic differences between primary and metastatic wt and evaluate kinases upstream and downstream of fak as potential targetable therapies. design/method: cells from coa 25 and coa 42 were treated with pf-573,228 (pf), a small molecule fak inhibitor. protein from cell lysates of treated and untreated coa 25 and coa 42 were combined with kinase buffer, atp, and fluorescently labeled antibodies and loaded into a phosphotyrosine kinase or serine-threonine kinase pam-chip® per the uab kinome core protocol. phosphopeptide substrate analysis with the pamstation®12 kinomics workstation (pamgene® international), pamchip® protocol using evolve2 software, and bionavigator v. 6.0 were used to analyze kinases upstream and downstream of fak. the primary wt had increased epha8, ror1 sgk307 and decreased pdgfrb relative to the paired metastatic wt at baseline. treatment with pf increased ron, pdgfrb, p70s6kb, mak, camk2g, vacamkl, camk2d, ck1a1 and pskh1 in the primary wt. treatment with pf decreased tnk1, lmr1, cck4, epha5, pdk1, sgk196, lkb1 and increased pskh1 in the paired metastatic wt. primary wt displayed a different kinomic profile compared to metastatic wt in a matched isogenic pdx model. these data reveal that alternative therapies to specifically target metastases are needed. furthermore, fak inhibition resulted in diverse kinomic alterations between primary and metastatic wt. inhibitors targeting many of these pathways, such as pdgfrb inhibitors, are currently available and potentially could be combined with fak inhibitors in the treatment of wt. the results of the current study indicate that kinases upstream and downstream of fak in primary and metastatic wt warrant further investigation. background: use of high-dose methotrexate (hd-mtx, 12 g/m^2) is a mainstay of standard therapy for pediatric osteosarcoma (os) in north america. in pediatric os, there is a narrow therapeutic window for hd-mtx, with decreased tumor response rate with mtx concentrations <1000 m and decreased survival due to severe toxicity with concentrations >1500 m. risk factors for hd-mtx toxicity have been defined in adults, including body mass index (bmi) and male gender, but such studies have not been conducted in children. we sought to examine the relationship between mtx levels and toxicities during hd-mtx infusion for pedi-atric os, thereby identifying risk factors for increased toxicity and providing a framework for therapeutic drug monitoring. design/method: this retrospective chart review included patients treated at texas children's hospital with hd-mtx as first-line therapy for os from 2009-2015. data abstracted from electronic records included patient characteristics, bmi and body surface area (bsa), baseline and post-treatment laboratory values, mtx levels 4 and 24 hours after dose given (4h, 24h), hour mtx cleared (mtx <0.1 um), grade 3/4 mucositis, myleosuppression, persistent lft elevation (ctace v4.0), and % tumor necrosis. correlation between 4h mtx level and other covariates was summarized using descriptive statistics. we reviewed 128 hd-mtx infusions corresponding to 12 patients. bmi was found to significantly impact 4h mtx level (p<0.05). female gender was also significantly associated with higher 4h mtx level (p<0.001). percent necrosis (available in 9 patients) was associated with 4h mtx levels at near-statistical significance (p = 0.07). 4h mtx level was not found to contribute to toxicities or associate significantly with mtx clearance. analysis in a larger cohort is ongoing. we have identified at least one patient factor (bmi) that significantly impacts 4h mtx levels and is of potential use for future modeling, as current models incorporate bsa only. our findings concord with studies in adult os in that bmi significantly impacts 4h mtx level but diverge in that female gender is associated with higher 4h levels. importantly, these data support targeting 4h mtx levels to ensure that minimum concentration for adequate tumor necrosis is reached. these results do not suggest that monitoring 4h levels would prevent toxicities, thus necessitating further characterization of any intrinsic patient factors that associate with toxicity. overall, our definition of the clinical factors that associate with 4h mtx levels contributes to a framework for therapeutic drug monitoring in pediatric os. children 's mercy hospital kansas city, kansas city, missouri, united states background: post consolidation immunotherapy with dinutuximab, aldesleukin (il-2), granulocyte macrophage colony stimulating factor (gmcsf) and isotretinoin is standard of care for children with high risk neuroblastoma. dinutuximab is combined in 5 alternating cycles with s263 of s301 gmcsf or il2, followed by a 6th cycle with isotretinoin alone. il-2 is administered as a 96hour continuous infusion on days 0-4 at 3miu/m2/day followed by a higher infusion dose, 4.5miu/m2/day, in combination with dinutuximab on days 7-10 of cycles 2 and 4. the 3miu/m2/day dose may be administered inpatient or in the ambulatory setting. objectives: to retrospectively compare the incidence of inpatient and outpatient side effects and complications associated with low dose (3miu) il2 to provide the tolerability data necessary to evaluate these venues for future administration options. design/method: this study was a descriptive, singlecentered definitive study utilizing a retrospective convenience sample population of children with high risk neuroblastoma who received low dose il2 either as an inpatient or an outpatient without exclusion from may 2012 to june 2017. subjects were identified by a tumor registry query post irb approval. electronic and paper medical records were reviewed for the dates and location of the infusions, the home health company used if applicable and all documentation regarding clinical status, side effects and toxicity. demographics was limited to age and gender. results: infusion venue was chosen by provider preference. twenty-six infusions, 9 inpatient and 17 outpatient via 3 separate home health companies were all administered in entirety and without interruption. there were 10 males and 4 females ranging from 2-7 years of age. two children received a single outpatient infusion due to intolerance of il2 when combined with dinutuximab and 2 received therapy in both settings. fever, 3 inpatient and 1 outpatient was the only common side effect. no source of infection was ever identified. there was one incidence of diarrhea and one patient with pruritus in both the outpatient and inpatient settings respectively. no planned outpatient infusions required subsequent admission however the outpatient fever did necessitate an er evaluation. conclusion: low dose il 2 can successfully be administered outpatient. the medication has minimal side effects with fever occurring in 15%, none of which were associated with infection. no outpatient infusion required a subsequent admission. no patients who received cycle 2 infusions outpatient opted to receive the next cycle inpatient. baylor college of medicine, houston, texas, united states background: metastatic ewing sarcoma (es) has an extremely poor overall survival, necessitating investigations into molecular mechanisms to identify novel targets and develop new therapies. we previously performed an in vivo study, using our mouse model, designed to provide insights into transcriptomic and proteomic signatures for metastatic es to identify potential therapeutic targets. comparing profiles of primary tumors to corresponding metastatic lesions, we identified aberrant expression of integrin ß3 (itgb3) and downstream activation of integrin-linked kinase (ilk) in metastatic lesions compared to primary tumors, implicating this pathway as a key regulator in the ability of es to establish and enhance metastasis. our hypothesis is that upregulation of itgb3 and its downstream signaling events play a key role in es metastasis and are viable therapeutic targets. objectives: to investigate the role of itgb3 and its downstream signaling pathways in driving the establishment and enhancement of metastasis in es and to investigate this pathway as a potential therapeutic target. to investigate the role of itgb3 and ilk in es metastasis, we used sirna to knock down itgb3 and ilk expression in established es cell lines and then performed functional assays in vitro, including cell proliferation and invasion/migration assays. we also tested inhibition of this itgb3 signaling pathway using available small molecule inhibitors targeting itgb3, ilk and the downstream target ap-1, using cilengitide, compound 22 and sr11302, respectively. we are currently using these small molecule inhibitors as treatment in vivo and assessing rates of metastatic tumor formation. we generated stable itgb3 and ilk overexpression and knockdown cell lines, which we are using for similar in vitro and in vivo investigations. knockdown of itgb3 and ilk in our sirna cell lines resulted in decreased cell proliferation and decreased invasion and migration compared to controls. we also found significantly decreased cell proliferation using each of the small molecule inhibitors in vitro. our preliminary studies using compound 22 in vivo established a safety profile and dose escalation is underway to assess the effectiveness of inhibiting es metastasis. these results support our hypothesis that itgb3 and its downstream signaling events play a key role in the ability of es to establish metastatic foci and may serve as a potential therapeutic target. we continue to investigate this pathway in vitro. we are also using our small molecule inhibitors and itgb3 and ilk overexpression and knockdown approaches to study these effects on metastatic tumor development in vivo using our mouse model. background: neuroblastoma (nbl) is characterized by phenotypic heterogeneity. outcome is excellent for patients with low-(lr) and intermediate-risk (ir) disease, whereas only 50% of high-risk (hr) patients will survive. 5-hydroxymethylcytosine (5hmc) is an epigenetic marker of active gene transcription, and 5hmc profiles are prognostic in many types of adult cancers. we hypothesized that 5hmc profiles will serve as robust biomarkers in children with nbl tumors, refining current risk stratification. objectives: analyze genome-wide 5hmc in nbl tumors and correlate 5hmc deposition with chromosomal copy number and gene expression. design/method: 5hmc was quantified by nano-hmc-seal-seq from the dna extracted from 15 hr, 11 ir and 27 lr nbl tumors. read counts and clinical data were analyzed with deseq to identify genes with differential 5hmc patterns between risk groups. chromosomal copy number was assessed by chromosomal microarray analysis (cma) in a subset of samples (3lr and 9hr). expression of genes located on chromosome 1p was evaluated using publically available microarrays (e-mtab-1781) of 171 hr nbl tumors with known 1p loh status. results: globally, lr tumors had more 5hmc peaks (140,062) than ir (102,398, p = 0.36) tumors, or hr tumors (79,727, p = 0.01). 1,049 genes had different patterns of 5hmc deposition in hr versus lr tumors. 315 (30%) of these genes mapped to chromosome 1p and had decreased 5hmc in hr versus lr tumors (padj <0.05). in the cma analysis 1p deletion was detected in 5 of the 9 tumors tested. in the tumors with 1p loss, 322 genes that map to 1p showed decreased 5hmc deposition compared to the 4 hr tumors without 1p loss (p<0.05). further, compared to the tumors without 1p loss, the expression of 188 of the 322 1p genes was decreased (p<1 × 10-5), including chd5, camta1, and arid1a, known and proposed tumor suppressor genes in nbl. conclusion: different patterns of 5hmc accumulation are associated with neuroblastoma risk classification. nano-hmc-seal-seq is sensitive to copy number variations and has the potential to identify these changes in patient tumors. our results suggest that 5hmc deposition contributes to the silencing of tumor suppressor genes in 1p and may also regulate the transcription of other genes that drive tumor phenotype. background: metastatic osteosarcoma has a 5-year survival rate of 15-40%. pulmonary metastases remain a major treatment challenge in osteosarcoma. current treatment with conventional chemotherapy shows inadequate activity towards metastases and has toxic systemic side effects. chloroquine is a widely used anti-malarial drug and has been shown to have promising anti-cancer and anti-metastatic activity. polymeric drugs have been shown to have multiple advantages over their small molecular parent drugs, including enhancing the therapeutic efficacy, an improved pharmacokinetics profile and decreased systemic toxicity. we hypothesized that by developing chloroquine into a polymeric drug and combining it with conventional chemotherapy it will improve the treatment of metastatic osteosarcoma. objectives: to identify the optimal combination of polymeric chloroquine (pcq) with conventional chemotherapy active in osteosarcoma as a new means of treating metastatic disease in a murine osteosarcoma model. we synthesized and developed pcq and evaluated its anti-invasive activity using an osteosarcoma cell migration and invasion assay. we evaluated the efficacy of cell killing using combination drug therapies with pcq and a panel of conventional chemotherapy agents (doxorubicin, docetaxel, cisplatin and paclitaxel) using celltiter blue cell viability assay. to develop the murine osteosarcoma model, we intravenously injected luciferase-expressing human osteosarcoma cells 143b into nsg mice. we administered the drug combination that showed the strongest in vitro synergy to the mice and evaluated their anti-cancer and anti-metastatic effects in vivo. tumor growth and suppression were evaluated using whole body bioluminescence imaging. results: we successfully synthesized pcq that contains 16.7% chloroquine with a molecular weight of 18.9 kd. pcq was also found to decrease the toxicity of the parent chloroquine. pcq showed strong inhibition of osteosarcoma cell migration with 51% inhibition compared to 13% by chloroquine. we screened the combination drug therapies and found the combination of pcq and doxorubicin to show the strongest synergism. the pcq/doxorubicin combination is currently being evaluated in the murine model. combination drug therapy using pcq and doxorubicin showed synergistic cell killing and inhibition of cell migration in vitro. the combination represents a promising treatment strategy for pulmonary metastatic osteosarcoma. emory university/children's healthcare of atlanta, atlanta, georgia, united states background: survival for relapsed high-risk neuroblastoma (rnb) is < 5%, underscoring the critical need for novel therapies. rnbs have increased ras/raf/mapk mutations and increased yes-associated protein (yap) transcriptional activity. yap is a transcriptional co-activator that binds with tea-domain (tead) transcription factors to regulate cellular proliferation, self-renewal, and survival. we found that shrna inhibition of yap decreases nb cell proliferation and sensitizes ras-mutated nbs to mek inhibitors, supporting yap as a tractable therapeutic target. verteporfin (vp), a photodynamic drug used for macular degeneration, is the only drug found to inhibit yap expression or yap:tead binding to kill tumor-derived cells. peptide 17 is a 17mer yap peptidomimetic that also disrupts yap:tead interactions. we sought to determine whether these compounds are potent in nb via yap direct effects. design/method: yap expressing (nlf, sk-n-as) or yap null (ngp, lan5, sk-n-as-shyap) human-derived nbs were incubated with vp, with and without direct light exposure, or with peptide 17. celltiter-glo and immunoblots were used to assess for cell death and yap-downstream protein expression, respectively. results: without direct light exposure, vp inhibits yap expression at nm dosing, yet no nb cell death was observed at equal or higher concentrations. egfr and erk1/2 were inhibited along with yap, confirming yap/ras pathway coregulation. when vp was exposed to direct incandescent light for 30 minutes, > 80% nb cell death occurred in all nbs tested, even those lacking yap. peptide 17 caused no cell death or yap inhibition up to 75 um. neuroblastomas are resistant to vp at doses sufficient to inhibit yap expression. in macular degeneration, light-activated vp produces reactive oxygen species, which we hypothesize is the off target mechanism killing nbs independent of yap. given the off target effects and the need for light activation, vp is not an ideal preclinical or clinical yap inhibitor. accordingly, peptide 17 has poor cell permeability and low tead affinity, leading to its lack of efficacy. given the relevance of yap in rnb and other cancers, we are chemically optimizing a yap peptidomimetic with enhanced permeability, nuclear localization, and tead affinity to create a bonafide yap inhibitor for preclinical and clinical application. kayeleigh higgerson, aaron sugalski, rajiv rajani, josefine heim-hall, jaclyn hung, anne-marie langevin ut health san antonio, san antonio, texas, united states background: osteosarcoma is the most common bone malignancy in children, adolescents, and young adults. most study cohorts have 10 to 15% hispanic patients that encompass many different hispanic backgrounds. the university of texas health science center at san antonio (uthscsa) sarcoma team serves a latino population that is predominantly mexican american, thus providing a unique opportunity for evaluation this population. this study expands on previous data collected from january 2000 to december 2010 from the same institution, providing increased insight into outcomes of mexican american children, adolescents, and young adults with osteosarcoma. objectives: to further understanding of osteosarcoma in latino children, adolescents and young adults. design/method: a retrospective analysis of demographics, tumor characteristics, response to treatment, and survival outcome of all localized osteosarcoma of the extremity patients below 30 years of age diagnosed and treated by the uthscsa sarcoma team between january 2000 and june 2017 was performed. results: in our original cohort from january 2000 to december 2010, we observed a significantly decreased 5-year eventfree survival (efs) in patients diagnosed before age 12 (preadolescent) relative to patients diagnosed between ages 12 and 29 (11% vs. 57%, p<0.001). patients had a 5-year overall survival (os) and event-free survival of 65% and 48% respectively. in our expanded cohort from january 2000 to june 2017 we evaluated sixty-six patients with a median age of 14 (range, 2 to 28 y) with localized high-grade osteosarcoma of the extremity. the expanded cohort was 68% mexican american, with a median follow-up of 59 months (range, 5 to 192). the analysis of our expanded cohort is ongoing and we postulate that the findings will hold true, as we increase the cohort size and length of follow-up. conclusion: analysis of our previous cohort, predominantly of mexican american ethnicity, showed that preadolescent patients had an increased rate of relapse when compared with previous large studies. we also showed a trend towards decreased efs for the entire cohort. we hypothesize that we will further validate these findings with this expanded cohort and this will support further investigation into potential causes of poor outcome in this vulnerable latino population. background: neuroblastoma in infants has the potential to regress or mature spontaneously. growing literature showed that some cases subjected to initial observation didn't show inferior outcome compared to actively treated similar categories. objectives: we investigated whether early active treatment can be safely avoided/deferred in selected favorable cases at the children's cancer hospital-egypt (cche). design/method: patients enrolled on the watch and see strategy (w&s) at cche had small primary tumor; inss stage 1-2, uncomplicated stage 4s or stage 3 infants (< 365 days). tissue biopsy was not mandatory for infants below 6 months of age with localized adrenal mass (stage 1-2). on progression, immediate intervention took place according to stage and risk of disease after biological characterization. results: thirty four nbl patients were enrolled on w&s strategy; m/f:2.4/1. eighteen patients had stage 4s disease, 12 patients had stage 1-2 and 4 were stage 3. primary adrenal site was reported in 29 patients (85.3%), 21 patients (61.77%) had small mass measuring ≤5 cm in its largest diameter. the 5-year os & efs were 88.2±8.8% and 72.5±9%, respectively, with 43 months median follow-up (range: 1-106 months). spontaneous total/near total resolution of mass occurred in 16/34 patients (47%). median time to eliciting regression was 1.7 months (range: 0.4-14.7 months), and 20.7 months (range: 7-63 months) till complete resolution. only 8/34 patients (23.5%) witnessed progression (2 local, 2 distant and 4 combined local and distant progression); median time to progression was 8 months (range: 1-32 months) with 2/8 deaths after starting chemotherapy. watch and see strategy is a safe approach in localized and uncomplicated stage 4s neuroblastoma. progressive cases could be rescued. baylor college of medicine, houston, texas, united states background: ga-68 dotatate binds to somatostatin receptor 2 expressed in neuroendocrine tumors (nets). it was approved by fda in 2016 for use with pet/ct scan for localization of somatostatin receptor positive nets in adult and pediatric patients. pediatric approval was based mainly on extrapolation of data from adults. objectives: to describe the use of ga-68 dotatate pet/ct scan in children with neuroendocrine tumors and compare with other imaging modalities. design/method: patients with nets enrolled in texas children's rare tumor registry between february and october 2017 were reviewed and those patients who underwent ga-68 scan were included. results: four patients with nets underwent ga-68 scans without any adverse reactions. first patient was a 15-yearold female with small bowel net with multiple liver metastases. mri abdomen and fdg pet at diagnosis showed s267 of s301 multiple liver metastases but could not identify the primary lesion. ga-68 scan was able to accurately identify the enlarged lymph nodes in the small bowel and was better than fdg pet in delineating the liver metastases. second patient was a 13-year-old female with recurrent small bowel net with liver, lung and paraspinal metastases. the lesions were initially detected by ct scan. octreotide scan failed to show any uptake in the identified lesions while ga-68 was taken up by the liver lesions, lung lesions >1 cm in size and the paraspinal lesion. third patient is an 8year-old male with pancreatic net with peripancreatic lymphadenopathy, multiple liver metastases and cardiophrenic lymph node involvement. the primary lesion in the pancreas could not be identified by ct scan, ct angiogram, mibg scan, or octreotide scan. in addition, there was uncertainty about involvement of the enlarged cardiophrenic lymph node. in addition to clearly identifying the primary lesion, ga-68 scan was able to detect multiple peripancreatic lymph nodes not detected by other scans and revealed uptake in the cardiophrenic lymph node confirming its involvement by the tumor. fourth patient is a 15-year-old female with malignant abdominal paraganglioma with solitary lung metastasis. both mibg scan and ga-68 scan were able to identify the primary lesion. ga-68 scan was performed after the lung metastasis was removed and thus its ability to detect it could not be confirmed. background: neuroblastoma is the most common extracranial solid tumor of childhood, with overall survival for high-risk patients (hrnbl) near 50%. the outcomes of hrnbl have improved with high dose chemotherapy followed by autologous stem cell rescue (abmt). data about factors influencing the rate of hematopoietic recovery following abmt in hrnbl is lacking in the literature. our objective was to identify factors influencing the rate of hematopoietic recovery following abmt in hrnbl. design/method: this was a retrospective chart review of 55 patients with hrnbl treated at texas children's hospital from 2006 to 2016. neutrophil engraftment was considered the first of three consecutive days with post-transplant neutrophil count greater than 500 cells/ul. red blood cell and platelet engraftment were considered at a hemoglobin greater than 8g/dl and platelets greater than 20,000/ul three days after the last transfusion. race and conditioning regimen were analyzed using one-way anova; amount of infused cells was analyzed using pearson correlation coefficients; chemotherapy delay and bone marrow (bm) involvement after cycle 2 of induction chemotherapy were analyzed using independent sample t-tests. the study included 32 males and 23 females with a median age at diagnosis of 2.5 years. thirtyeight patients were caucasian, 6 african-american, 5 hispanic, 2 asian, and 4 did not have race documented. the mean dose of infused cd34+ cells was 3.36 × 10^8 cells/kg. forty-five patients received conditioning therapy with carboplatin/etoposide/melphalan (cem), 8 received busulfan/melphalan (bu/mel), and 2 received thiotepa/cyclophosphamide (thiotepa/cpm). the conditioning regimen administered was significant (p = 0.037) for time to engraftment of neutrophils, with bu/mel at 16.6 days, cem at 12.1 days, and thiotepa/cpm at 10 days. a delay of chemotherapy during induction (n = 25) was significant (p = 0.001) for time to platelet engraftment of greater than 75,000/ul and trended towards significance (p = 0.088) for time to neutrophil engraftment. bm involvement at diagnosis and after cycle 2 of induction was not significant for time to engraftment. dose of stem cells infused was the only variable significant for hemoglobin engraftment. background: osteosarcoma (os) is the most prevalent aggressive primary malignancy of the bone affecting children and young adults. approximately 10% to 20% of patients have metastatic disease at initial presentation, and 61% of those patients have isolated pulmonary metastases. although overall survival in patients with os has improved with advances in therapy, there have been no significant improvements in survival outcome in patients with metastatic disease. recent studies suggest that tumor-associated vascular cell adhesion molecule 1 (tvcam-1 or cd106) plays a critical role in the metastatic progression of various tumors. indirect evidence from these studies suggest that vcam-1/ 4 1 integrin signaling promotes tumor survival and metastatic progression by changing the tumor niche and associated immune response. to determine if interfering vcam-1/ 4 1 signaling between pulmonary metastatic osteosarcoma (pos) and macrophages (macs) by down-regulating vcam-1, depleting macs or blocking vcam-1/ 4 1 signaling will reduce pos and improve overall disease-free survival. design/method: we used a pair of spontaneous, high-grade murine os cell lines from balb/c mouse (h-2d), k7 and k7m2 (derived from in vivo k7 metastasis). we used lentiviral shrnas to knockdown vcam-1 mrna and protein expression in k7m2 (vcam-1kd). we introduced luciferase into k7, k7m2 and various k7m2 shrna cell lines to follow lung metastasis by bioluminescence (bli). we depleted macs by intranasal administration of liposomal clodronate formulation. we tested the ability of k7 and k7m2 supernatants to polarize m0 macs into m1 or m2 phenotype in vitro. we also administered anti-4 monoclonal antibody (anti-4 mab) intranasally to assess the outcome of functional blockade of vcam-1/ 4 1 signaling. results: k7m2 over-expressed vcam-1 compared to k7. mac depletion in k7m2-bearing animals exhibited reduced pos. weekly administration of anti-4 mab resulted in 80% tumor-free rescue among mice with established k7m2 pos. interestingly, supernatant from k7m2 but not k7 preferentially induced m2-like macs, suggesting a novel integrin-mediated mechanism of m2 differentiation. validation data with additional os cell lines will be presented. despite aggressive multimodal therapy, overall outcome for patients with pos remains dismal at 25-30%. for this reason, novel and directed therapy approaches are desperately needed. molecular targeted approaches for therapy are challenging, due to the complex genetic heterogeneity of os. immune-modifying therapy is a promising new alternative approach for pos. university of chicago, chicago, illinois, united states background: only half of all patients diagnosed with high-risk neuroblastoma achieve long-term survival. 123imetaiodobenzylguanidine (mibg) scans are routinely used to evaluate disease at diagnosis and following treatment, and the extent of disease is quantified using the curie scoring system. a previous study by yanik et al., has shown that for high-risk patients with mycn non-ampliified tumors, scores less than versus greater than 2 following 6 cycles chemotherapy are associated of superior survival, whereas scores less than versus greater than 0 were prognostic in patients with mycn-amplified tumors. however, the prognostic significance of specific sites of metastatic disease at diagnosis is not known. to determine if site of metastatic disease determined by 123i-metaiodobenzylguanidine (mibg) imaging in high-risk patients at the time of diagnosis was associated with outcome design/method: we performed a retrospective chart review of high-risk neuroblastoma patients treated at comer children's hospital and lurie children's hospital in chicago between 2006 and 2017 with positive mibg scans at the time of diagnosis. we collected imaging data as well as other clinical data including bone marrow status. sites of disease were defined as curie regions with any positive value. kaplan-meier analysis was performed to evaluate the association with disease sites and survival. pearson correlation coefficients were calculated to compare bone marrow disease to sites of positivity on mibg scan. the cohort consisted of 49 high-risk patients. 31 had skull disease, and 30 had pelvic disease. the presence of mibg positive disease in the skull and in the pelvis trended toward worse efs. efs at 3 years for patients with disease in the skull at diagnosis was 52 ± 10% and for patients without skull disease was 78 ± 10 % (p = 0.16). efs at 3 years for patients with and without pelvic disease was 49 ± 10% and 80 ± 9% (p = 0.10). consistent with prior data, we found that the presence of bone marrow disease was associated with worse survival with 3 year efs of 47 ± 10% and 88 ± 8% with and without marrow disease at diagnosis (p = 0.02). there is the highest correlation between pelvic disease on mibg scan and bone marrow disease with pearson coefficient 0.79. pelvic disease noted on mibg scan likely reflects underlying bone marrow disease. in patients with high-risk neuroblastoma, skull disease and pelvic disease on mibg scan at diagnosis may predict worse event free survival. background: osteosarcoma is one of the deadliest cancers in the pediatric population with little progress in morbidity and recurrence rates since the 1980's. oncolytic herpes simplex-1 virus (ohsv) is an attenuated virus that has shown encouraging results against certain solid tumors. programmed cell death protein (pd)-1-mediated t cell suppression via engagement of its ligand, pd-l1, is also of particular interest due to recent successes in selected cancers, especially those with high genetic mutational loads. most pediatric cancers do not have a wide variety of mutations; however, osteosarcoma has a chaotic genome, prone to genetic mutations. it has been shown through numerous other studies that pd-1 inhibition alone is not sufficient to result in statistically significant tumor growth delays in osteosarcoma models and patients. we hypothesize the addition of ohsv therapy as an immunologic stimulus to pd-1 inhibition is efficacious for osteosarcoma. (1) to determine whether ohsv therapy enhances response to pd-1 inhibition in immunocompetent murine models of osteosarcoma and (2) to quantify and characterize the anti-tumor t-cells infiltration after treatment with ohsv and pd-1 inhibition individually and in combination. we utilized an immunocompetent transplantable murine model using a cell line derived from a spontaneous metastatic osteosarcoma (k7m2, balb/c background). we transplanted established tumor wedges subcutaneously and monitored tumor volume by caliper measurement. once tumors reached 200-400mm3, we administered intratumoral injections of hsv1716 (1 × 108 plaque-forming units) every other day for a total of 3 injections. we then gave intraperitoneal injections of 250ug anti-pd-1 or control antibody twice weekly, up to 4 weeks, starting from the last dose of virus treatment. we monitored tumor growth via calipers twice weekly until tumors reached 2500mm3 or 2cm diameter. we quantified and characterized innate and adaptive immune cell infiltrates in tumors using flow analysis. we found significantly prolonged survival with our combination therapy group compared to all other groups. we found that anti-pd-1 by itself had little impact on t cell recruitment while the combination group had higher influx of cd8+ cells with a reduced amount of t-regulatory cells (cd4+foxp3+cd25+). we also found an increase in cd44+ effector memory cells. osteosarcoma is a deadly cancer with therapeutics remaining unchanged for the last 30 years. here, we describe prolonged murine survival after treatment with combination of pd-1 inhibition and ohsv injection. the combination treatment changed the microenvironment to be more inflammatory. our data support further preclinical and clinical studies. background: neuroblastoma is the second most common cause of cancer related death in children. treatment for high-risk neuroblastoma has improved significantly over the past twenty years, however cure rates remain below 50%. immunotherapy has emerged as an effective therapy for neuroblastoma, however new modalities and targets are needed to improve outcomes. objectives: our lab has developed a chimeric antigen receptor (car) that targets b7-h3 (cd276), an immune checkpoint molecule overexpressed on many cancers, including neuroblastoma. we hypothesized that b7-h3 would be a good target for car based immunotherapy for neuroblastoma. design/method: neuroblastoma tissue microarrays of primary patient samples were screened for b7-h3 expression by immunohistochemistry and cell lines were screened using flow cytometry. b7-h3 car t cells were tested in vitro by measuring tumor cell killing and cytokine production after coculture with tumor cell lines and in vivo in an orthotopic model of neuroblastoma. results: b7-h3 expression was detected by ihc on 82% of the 186 screened neuroblastoma patient samples. b7-h3 was expressed at high levels (2+ or 3+) in more than half of these samples (56%). almost all cell lines screened were homogeneously positive for b7-h3 by flow cytometry. retrovirally transduced b7-h3.4-1bb. car t cells were cocultured with three b7-h3 positive neuroblastoma cell lines (sk-n-be2, kcnr, and chla255) and robust tumor cell killing was demonstrated using an incucyte assay. supernatant from the co-cultures was harvested after 24 hours and both interferon gamma and il-2 production were detected by elisa.in an orthotopic subrenal capsule xenograft model of neuroblastoma, mice treated with b7-h3 car t cells show significant reductions in tumor growth and prolonged survival compared to those treated with untransduced control t cells. however, the treatment is not always curative.b7-h3 car t cells express high levels of exhaustion markers (pd1, tim3, and lag3) when compared to cd19 car controls. in order to overcome inhibition from exhaustion, b7-h3 car t cells were co-cultured with neuroblastoma cell lines and pd-1 blocking antibody. nivolumab significantly increased the production of il-2 and interferon-gamma by b7-h3 car t cells. further studies are underway to determine if b7-h3 car t cell activity is enhanced in vivo by treating animals with pd-1 blockade along with car t cells. conclusion: b7-h3 is expressed on a majority of neuroblastoma samples and appears to be a promising candidate for car t cell therapy. b7-h3 car t cells demonstrate activity against neuroblastoma xenografts that may be enhanced by the addition of pd1 inhibitors. helen devos children's hospital, michigan state university, grand rapids, michigan, united states background: osteosarcoma is the most common bone tumor in children. it is often metastatic at diagnosis and in this scenario less than 30% of children survive. polyamines, small molecules found in all cells, are involved in many cell processes including cell cycle regulation, immune modulation, cell signaling and apoptosis. they are also involved in tumor development, invasion and metastasis. in neuroblastoma, inhibition of the polyamine biosynthesis pathway with odc inhibitor alpha-difluoromethylornithine (dfmo) results in decreased cell proliferation and differentiation. these finding have led to multiple phase i and phase 2 ii multicenter clinical trials in pediatric neuroblastoma patients. dfmo is an attractive drug as it is oral, well-tolerated, can be given for prolonged periods and is already used in pediatric patients. the polyamine pathway has not been evaluated in osteosarcoma. objectives: evaluate effect of inhibition of polyamine biosynthesis with dfmo on osteosarcoma proliferation and cell differentiation. design/method: up to three osteosarcoma cell lines were used: mg-63, u-2 os and saos-2. cells were exposed to 5 mm dfmo for 6 days with replacement of media and dfmo on day 3. intracellular polyamine levels were measured by high performance liquid chromatography (hplc). cell numbers were obtained with a hemocytometer using trypan blue. flow cytometry cell cycle distribution (facs) and propidium iodide were used to evaluate for cell cycle arrest. the protein expression of several osteosarcoma differentiation markers was measured by sds-page and western blot using differentiation specific antibodies. a bioluminescent cell viability assay was used to measure cell recovery over several days after dfmo was removed and replaced with standard media. results: dfmo exposure resulted in significantly decreased cell proliferation in all cell lines. after treatment, intracellular spermidine levels were nearly eliminated in all cells. cell cycle arrest at g2 was observed in u-2 os. cell differentiation was most pronounced in mg-63 and u-2 os cells as determined by increased osteopontin levels. remarkably, cell proliferation continued to be suppressed for several days after removal of dfmo. conclusion: based on our findings dfmo is a promising new adjunct to the current osteosarcoma therapy for high risk patients. it is a well-tolerated oral drug that is currently in phase ii clinical trials in pediatric neuroblastoma patients as a maintenance therapy. the same type of regimen may also improve outcomes in metastatic or recurrent osteosarcoma patients for whom there have been essentially no medical advances in the last 30 years. background: recent studies demonstrate that lower levels of the ews-fli1 fusion oncoprotein are associated with enhanced metastatic capability in ewing sarcoma. the nf-kb transcription factor is a critical mediator of cxcr4 and cxcr7 -driven metastasis in multiple cancers, and increased cxcr4 and cxcr7 expression have each been associated with increased metastasis and poor prognosis in ewing sarcoma. we thus sought to investigate the impact of ews-fli1 on cxcr4/cxcr7-dependent nf-kb signaling in ewing sarcoma. objectives: the goals of this study are 1) to determine the impact of cxcr4/cxcr7 signaling on metastasis-associated nf-kb target gene expression in ewing sarcoma and then 2) to investigate how the ews-fli1 fusion oncoprotein modulates this response . design/method: we utilized multiple ewing sarcoma cells lines including a673, chla9, chla10, tc32 and tc71. cxcr4/cxcr7 cell surface expression was determined by flow cytometry. ews-fli1 level was modulated using sirna and expression levels were confirmed by western blot and rt-pcr. p65 dna binding was measured via elisa. nf-kb target gene expression was assessed via rt-pcr. results: consistent with ihc analysis of primary and metastatic patient tumor samples, the paired primary and metastatic ewing sarcoma cell lines chla9 and chla10 showed dramatic differences in cxcr4 and cxcr7 expression, with the metastatic chla10 line demonstrating much higher expression of both receptors. other cell lines (nonpaired) showed variable cxcr4/cxcr7 expression. genetic knock-out of cxcr4 lead to significant decrease in expression of both cxcl12/sdf-1 and il-6, two nf-kb transcriptional targets known to play a key role in tumor metastasis. knock-out of cxcr4 did not alter endogenous ews-fli1 mrna levels. conversely, lowering the level of ews-fli1 using sirna lead to enhanced nf-kb signaling, indicated by an increase in p65 dna binding. consistent with this observation, treating ewing cell lines with ews-fli1 sirna also resulted in significantly increased nf-kb target gene expression compared to control cells and target gene expression was then further enhanced upon cxcr4/cxcr7 receptor stimulation with the receptor ligand cxcl12/sdf-1. our findings indicate that the ews-fli1 oncoprotein negatively modulates cxcr4/cxcr7-dependent nf-kb signaling. this suggests that ews-fli1 low, cxcr4/cxcr7 high cells, which are associated with enhanced metastasis and poor prognosis, would be anticipated to exhibit enhanced expression of key nf-kb target genes. importantly, the nf-kb pathway is a druggable target that could potentially serve as an "achilles heel" in this subset of high risk tumors. current work is evaluating nf-kb inhibition as an approach to treating metastatic and refractory ewing sarcoma. background: acute graft versus host disease (agvhd) is a major cause of morbidity and mortality following allogeneic bone marrow transplant (bmt) in pediatric patients. gastrointestinal (gi) agvhd is the most serious manifestation. recently, decreased paneth cell (pc) in a predominantly adult cohort was shown to correlate with agvhd clinical grading and response to treatment. we aim to demonstrate the relationship between pc counts and gi agvhd stage and response to therapy. design/method: charts of patients who underwent endoscopy following bmt between 2004-2014 were reviewed. for repeated biopsies during the course of agvhd, only the first was included for analysis. one pathologist retrospectively reviewed the biopsies and counted pcs in 3 high powered fields; the average pc count was analyzed. twenty-six percent of biopsies were reviewed by a second blinded pathologist. statistical associations between pc counts and day 28 (d28) response, agvhd stage, and other study covariates of interest were gauged using general linear regression. agreement in pathologist pc counts was quantified by intraclass correlation (icc). the research was approved by the children's healthcare of atlanta irb. results: seventy-eight biopsies were included in the analysis. mean age at transplant was 10.5 years ± 5.8 (range: 2 months -20 years). most patients underwent transplant for hematologic malignancies (63, 74%). the majority of transplants used a matched unrelated donor graft -including cords (59, 69%) and myeloablative conditioning regimens (71, 82%) -52% received total body irradiation. of these, 64% were diagnosed clinically with gi agvhd (stage 1, 42%; stage 2, 14%; stage 3, 22%; stage 4, 22%). icc showed good agreement (0.833) between the pathologists. mean pc was 16.8 for patients with no gut agvhd, 21.3 for stage 1, 22.9 for stage 2, 9.4 for stage 3 and 5.9 for stage 4 (p = 0.001). on multivariate analysis pc was strongly associated with gi agvhd stage (p<0.001) after controlling for age, preparative regimen intensity, and diagnosis (malignant vs. non-malignant). mean pc counts were significantly lower in patients with no response to steroid therapy at d28 (complete response (mean 17.8) vs. persistent disease (4.2) vs. partial response (4.5) (p = 0.001)). patients diagnosed with gi agvhd with pc counts less than 10 had a higher risk of mortality (hr 3.1, 95% ci: 1.17, 8.09; p = 0.023). lower pc count correlated with stage 4 gi agvhd, refractory disease at d28, and mortality. incorporating pc count in pathology review during gi agvhd work-up may help in agvhd risk stratification. background: there have been increasing discussions pressuring health care teams and institutions for potentially bearing the cost of clostridium difficile infections (cdi) as a health care-associated infection in the recent years. the pediatric oncology patient population, though small, accounts for significant portion of all cdi with 10-15-fold increased risk. hematopoietic stem cell transplant (hsct) recipients constitute a unique subset with distinct risk factors, such as severe immune deficiency state and graft versus host disease (gvhd). although there is ample data on cdi in adult hsct recipients, reports on pediatric experience are limited. objectives: to evaluate the incidence and patterns of cdi among pediatric hematology, oncology and hsct inpatients at our institution. a retrospective review of all clostridium difficile (cd) stool tests performed using toxin enzyme immunoassay and later, polymerase chain reaction targeting toxin genes between 2007 and 2017 in a large, urban academic children's hospital was performed. the data were analyzed for hematology, oncology, hsct inpatient population and all the other cases separately and statistical comparisons were performed. results: a total of 5271 samples were submitted to the microbiology laboratory for cd testing during the study period. while hematology patients constituted 1.7%, oncology 5.9%, hsct 2.0% and others 90.4% of the cases on whom cd testing was done; per patient average test number was 2.0, 2.8, 6.2, and 1.5, respectively. of all the cd tests per-formed, 15.6% were positive. test positivity was higher in hsct (47.6%) and oncology (42.4%) cases tested compared with hematology (21.2%) and other cases (17.1%) with statistical significance (p<0.001). overall recurrence rate was 4.3%; hsct patients had the highest recurrence with a rate of 27% followed by oncology (13.6%), hematology (7.7%) and other (3.2%) cases, again reaching statistical significance (p<0.001). again, hsct patients had the highest average number of recurrences at 3.1 (2-6) followed by oncology 2.8 (2-10), general 2.52 (2-6) and hematology 2.25 (2-3) groups. there was no seasonal variability in the incidence of cdi among populations analyzed. prolonged hospital stay/antibiotic use and persistent diarrhea due to gvhd are the likely reasons for higher rate of cd testing in hsct as a result of increased monitoring and thus might have even caused underrepresentation of positive cd test frequency. higher incidence and frequencies of recurrence underscores the inevitable nature of cdi in hsct population as a consequence of the current therapies and may lead to future radical treatment approaches like fecal implantation. background: viral infections remain a challenge to treat post hct in children, and significantly contribute to morbidity and mortality. virus specific t cells (vsts) have shown tremendous clinical efficacy in treating viral infections post-hct, with minimal toxicity and long term efficacy. we have used donor-derived vsts in individual patients, however not all donors are agreeable to the process, and numerous patients may benefit from vsts who do not have an identified donor/have other disease indications objectives: we sought to actively build a third-party vst bank, for "off the shelf" use in eligible patients. design/method: vsts targeting cmv, adenovirus and ebv were manufactured using one of 2 techniques. initially ebv transformed b cells were genetically modified with an ad5f35pp65 vector and used as antigen presenting cells (apc) to stimulate and expand ebv, ad and cmvpp65 specific t cells. more recently, vsts were expanded using s273 of s301 apc pulsed with commercially available peptide pools (pep-mixes) to expand ebv/cmv/ad specific t cells. products were entered into the "bank" via two mechanisms: a) left over products from our "donor-derived" protocol when patients no longer required vsts or were not at risk of developing viral infections, or b) by targeting regular blood donors based on their hla typing to ensure an appropriate mix of high frequency hla types for optimal patient matching and antigen presentation based on current knowledge of antigen presentation. results: a total of 30 products are currently in the thirdparty vst bank ready for use. twenty seven of these are from our donor derived protocol, and three from targeted donors. all vst products met safety and in vitro efficacy testing. thirteen vst infusions have been given to 7 patients. eleven infusions have been given for cmv and two for adenovirus. five out of seven patients responded to thirdparty vst infusions, with a median of 2 vst infusions per patient (range 1-4). the median hla matching was 2 out of 10 per patient (range 1 to 4) no patients experienced adverse reactions, gvhd or other toxicity related to the vst infusion. a third-party vst bank is feasible and produces clinically appropriate vsts for use in patients with viral infections. hla typing and matching of vst products is essential to reduce toxicity and promote appropriate antigen presentation and expansion of vsts in vivo. further work is underway to further characterize the vsts using epitope mapping to better define the hla restriction and immunogenicity of each vst product. akron children's hospital, akron, ohio, united states background: acute graft-versus-host disease (agvhd) is a well-known complication of hematopoietic stem cell transplant (hsct) and a major cause of post-transplant related morbidity and mortality. first line therapy of agvhd involves corticosteroids and calcineurin inhibition. in patients with severe refractory gvhd, mortality can reach up to 90%. currently, there is no standard of care for the treatment of steroid refractory agvhd. many centers have looked at the use of antibody mediated control of agvhd to competitively inhibit the inflammatory cascade. basiliximab, a chimeric monoclonal antibody against the t-cell il-2 receptor, has been used in adults with steroid refractory agvhd. patients receiving this medication have demonstrated complete and partial responses to therapy with minimal toxicities. objectives: report the successful use of basiliximab in the treatment of agvhd in a 2-year-old following matched unrelated (mud) hsct. design/method: a 2-year-old male underwent mud transplant for high risk aml with monosomy 7. conditioning regimen included busulfan, fludarabine and equine atg. his clinical course was complicated by fever, mucositis and agvhd (stage 3 skin; stage 1 gi-biopsy proven). gvhd prophylaxis included tacrolimus and methotrexate, however with progressive skin rash, diarrhea, and early satiety, gvhd treatment with corticosteroids was initiated. as the patient continued to have worsening symptoms, basiliximab therapy was started. the patient received 2 doses (10mg) iv basiliximab on two consecutive days and then received weekly therapy for a total of 4 doses leading to initial improvement. the patient further developed acute on chronic gvhd on day +100, and subsequently received a second course of basiliximab. after initial administration of basiliximab, the patient had near complete resolution of symptoms. however, with a small wean in his tacrolimus dose, the patient experienced another skin gvhd flare prompting the second basiliximab course. the patient was subsequently weaned off all immunosuppression by day +376. the only acute complication the patient experienced while receiving basiliximab was right toe paronychia and asymptomatic low ebv titer. the patient is currently off all immunosuppression at the time of report without evidence of cgvhd. conclusion: this single case report, in a young pediatric patient, demonstrates the use of basiliximab may be a safe and efficacious treatment for pediatric patients with agvhd. university of california, san diego, la jolla, california, united states background: clinical outcomes after allogeneic hematopoietic stem cell transplantation (hsct) depend on restoration of t lymphocyte populations. association between recovery of cd4+foxp3+ regulatory t cells (tregs) and protection from chronic graft versus host disease (cgvhd) has been described in adult hsct. in adults, t cell recovery is driven by expansion of donor t cells and treg reconstitution is hypothesized to result from peripheral conversion. restoration of t cells in pediatric patients has a larger contribution from thymopoiesis, however, the relationship between thymopoiesis and treg recovery is undefined. objectives: we hypothesized that effective thymopoiesis is important for restoration of treg populations and protection from cgvhd in pediatric hsct patients. design/method: we performed longitudinal flow cytometry of peripheral blood t cells from 17 pediatric hsct patients and 9 age-matched healthy donors. laboratory data were correlated with clinical outcomes to evaluate impact. recovery of tregs occurred in 11/17 (64.7%) patients by post-transplant day 90. day 90 treg frequency in patients that developed cgvhd (1.7 ± 1.1% of cd4+ t cells) was reduced compared to cgvhd-free patients (3.2 ± 0.7%). failure to restore tregs to >2.0% of cd4+ cells by day 90 was associated with increased risk of cgvhd in the first year post-hsct (rr = 7.3, p = 0.05). a majority (60.8 ± 11.9%) of tregs from patients recovering the peripheral treg compartment expressed helios, a marker of thymic-derived tregs; only 21.5 ± 11.4% of tregs expressed helios in patients failing to restore adequate tregs. this prompted examining the relationship between defects in thymopoiesis and inability to restore tregs. we evaluated thymic function by flow cytometry quantification of cd45ra+cd31+ptk7+ recent thymic emigrant (rte) cd4+ cells (confirmed by qpcr for trec content). most (13/17, 76.5%) hsct patients had detectable rtes by day 30 post-hsct. thymic production of rtes was persistently absent in patients that developed cgvhd (<10/10^4 cd4+ cells in 5/5 patients), compared to cgvhd-free patients (7/12 patients >10 rte/10^4 cd4+ cells by day 30, average 22.3 ± 7.1/10^4 cd4+ cells). post-hsct thymic activity as measured by rte enumeration correlated with treg restoration; 6/8 (75%) rte+ patients restored tregs, compared to 3/9 (33%) of rte-patients. conclusion: failure to restore tregs after allogeneic hsct results in increased risk for cgvhd. in pediatric patients thymic generation of new t cells is an important contributor to restoration of the treg compartment. this data supports further investigation into mechanisms impairing post-hsct thymopoiesis and suggests peripheral blood tregs may be a prognostic biomarker for cgvhd. background: haploidentical stem cell transplantation (haplo sct) is riddled with unique challenges. objectives: we present our experience in the use of haplo sct with post-transplant cyclophosphamide (ptcy) and the adaptations required for each disorder for optimal outcome. design/method: we performed a retrospective study at the pediatric blood and marrow transplant unit, apollo cancer institutes, chennai, india. children up to 18 years of age, diagnosed to have benign disorders and underwent haplo sct with ptcy from 2002 to july 2017 were included. results: ptcy was used in 36 i.e. 73% haplo transplants for children with benign disorders. the underlying conditions included fanconi anemia 10, severe aplastic anemia 6, mds 1, jmml 1, hemoglobinopathy 3, prca 1, xld 1 and primary immunedeficiency disorders (pid) 13. source of stem cells was peripheral blood in 58%, bone marrow in 41%. conditioning included fludarabine with treosulphan or cyclophosphamide for pids and aplastic anemia respectively. neutrophil engraftment by day+16-21 with a durable graft was noted in 75% transplants with graft versus host disease in 20%, cmv reactivation in 35%. mortality rate was 45% with 2 infants less than 6 months of age developing severe fatal cytokine release syndrome. the median follow up is 1 year with 3 years being the longest. no significant late effects have been noted with chronic skin gvhd in 3 children. survival rate was superior among children with pids with survival of 70% in this group. haplo sct with ptcy is a feasible and costeffective option for cure in children with life-threatening benign disorders with no compatible family or matched unrelated donor. careful patient selection, reducing cyclophosphamide related free radical toxicity with the use of n acetylcysteine, limiting t cell numbers by capping cd34 at 5 × 106/kg, post-transplant viral monitoring protocols are required to reduce morbidity and mortality. we have been working on universal access to care for children from s275 of s301 all socioeconomic background and incorporating innovations to reduce the cost of hsct without compromising outcomes. haploidentical hsct using tcr / depletion costs 18000 usd as compared to ptcy priced at 25 usd. children with severe aplastic anemia and pids can be transplanted using reduced intensity conditioning and ptcy. in hemoglobinopathies, pretransplant immunosuppression is required to prevent graft rejection. graft versus host disease remains the main cause of mortality in children with fanconi anemia. mortality in infants less than 6 months after ptcy has been high, tcr / depletion would be superior in this cohort. cincinnati children's hospital medical center, cincinnati, ohio, united states background: fanconi anemia (fa) is a congenital bone marrow failure syndrome with hsct the only curative option for associated bone marrow failure. patients with fa undergoing hsct may experience increased toxicity related to either their underlying disease, or the effects of medications, resulting in the inability to tolerate prophylactic medications or sideeffects from anti-microbial therapy. objectives: we postulated that increased cd34 cell dose would be associated with a rapid immune reconstitution and therefore early withdrawal of anti-infective prophylactic medications. design/method: patients with fa transplanted at cchmc from an unrelated donor had peripheral blood stem cell grafts collected and cd34 selection performed. where possible, patients had serial measurements of their immune system performed at varying intervals post hsct. we defined immune reconstitution as normalization of lymphocyte subsets-cd3, cd4, cd8 and cd19 cells, as well as a normal response to mitogen stimulation including phytohemagglutinin, concanavalin a and pokeweed. the first measurement of either normal cell number or mitogen response was recorded for each patient. results: a total of 35 patients underwent hsct for fa at cchmc between 2012 and 2017. patient demographics included a median age of 8 years at hsct, the vast majority of patients having a fully matched or one anti-gen mismatched donor, and the majority of patients transplanted for bone marrow failure. there was a statistically significantly decreased time post-transplant to immune cell recovery in patients receiving >20 × 106/kg cd34 cells (median 25.7) compared to those receiving <20 × 106/kg cd34 cells (median 11.9). the median time to normalization of cd3 count was 224 days (cd34 count >20/kg) versus 371 days (cd34 count <20/kg), cd4 count 211 days (cd34 count >20/kg) versus 489 days (cd34 count <20/kg), cd8 count 193 days (cd34 count >20/kg) versus 344 days (cd34 count <20/kg) and cd19 count 93 days (cd34 count >20/kg) versus 109 days (cd34 count <20/kg). time to normalization of mitogen response was decreased posttransplant in those patients receiving increased cd34 cell dose at time of transplant, though this was not significant, reflecting low number of patients with evaluable responses. no patients in either group experienced gvhd or graft failure. patients with fa who are transplanted with higher cd34 cell doses have quicker immune reconstitution than those who receive lower cell doses. along with benefit to patients including less risk of infection and early termination of immune-prophylaxis medications, this supports the use of high dose cd34 selected grafts in this vulnerable population. background: parvovirus b19 (pvb19) infection after transplantation was first reported in 1986. since then, numerous cases of pvb19 infections after hematopoietic stem cell transplantation (hsct) and solid organ transplantation (sot) have been reported. most report anemia as the predominant clinical manifestation. however, pvb19 has been associated with pancytopenia, hepatitis, myocarditis, and allograft rejection. we present a patient with acute lymphoblastic leukemia who developed bone pain and pancytopenia following hsct in the setting of pvb19 infection. to describe an unusual presentation of pvb19 in a patient with acute lymphoblastic leukemia following hsct. design/method: a search of the english-language medical literature was performed using pubmed and medline databases. a review of the patient's medical history was performed. a 7 year old male with relapsed b-cell all and history of "fifth disease" in infancy presented four months after hsct with focal left arm pain and difficulties fully extending the arm. bone mri showed enhancement of the medullary space centered within incomplete transverse cortical fracture interpreted as pathologic fracture due to neoplastic involvement of the ulna with no history of inciting injury. subsequently, peripheral blood counts decreased from low normal values to wbc 1.9 k/microl, anc 310/microl, plt 57k/microl, and hemoglobin 8.6 g/dl. the patient's chimerism remained 100% donor. a bone marrow biopsy and aspirate were performed to assess for recurrent leukemia given persistence of bone pain and developing pancytopenia. marrow findings included morphologic cytopathic effects with erythroid precursors and strong parvovirus staining with no signs of red cell aplasia or recurrent b-cell disease by morphology or flow cytometry. pvb19 was detected in blood by pcr and immunoglobulins with resolution of cytopenia and bone pain. this case highlights an unusual constellation of symptoms following hsct in a child with all. unexplained bone pain and medullary infiltrates with pancytopenia suggestive of recurrent leukemia were likely triggered by pvb19 infection. the question remains if he had reactivation of pvb19, a primary infection by a new strain, or the virus was aquired through stem cells. bone biopsy could not be justified in light of clinical improvement. so far, bone lesions have only been described with congenital pvb19 infection. pvb19 appears to be uncommon after hsct, with a review of literature yielding 15 pediatric cases. however, it may be underestimated due to lack of routine screening. our patient's presentation supports that evaluating for pvb19 may be warranted in hsct patients presenting with symptoms suggestive of relapsed leukemia. background: cardiac injury may occur during hematopoietic stem cell transplant (hsct) in pediatric patients and can be asymptomatic for many years. recommendations for screening are available for patients who received anthracyclines or chest irradiation, but no guidelines exist for unexposed longterm survivors. we sought to define the prevalence of echocardiographic abnormalities in long-term survivors of pediatric hsct and determine the need for screening in asymptomatic patients. design/method: we analyzed echocardiograms performed on long-term survivors (≥ five years) who underwent hsct at cincinnati children's hospital between 1982 and 2006. we analyzed echocardiograms for left ventricular ejection fraction (ef), end-diastolic dimension (lvedd), septal thickness, posterior wall thickness, and global longitudinal strain (gls). we normalized linear measurements for age and patient body surface area. we included for further analysis patients who had echocardiogram obtained for routine surveillance. results: a total of 389 patients underwent hsct and were alive more than 5 years after transplant in 2017, with 114 having an echocardiogram obtained ≥ five years postinfusion. those with an echocardiogram were transplanted more recently (median 2003 vs. 1998 ). however, no difference between screened and unscreened individuals was noted for age at transplant, sex, transplant indication, anthracycline exposure, chest irradiation, or cyclophosphamide based preparative regimen. indications for echocardiograms included: cardiac symptoms 5 (4.4%), congenital cardiac anomalies 8 (7.0%), hypertension 2 (1.8%), known cardiac or pulmonary disease 2 (1.8%), routine post-hsct surveillance 95 (83.3%), and unknown 2 (1.8%). the mean time post-hsct was 11.7 years. among routine surveillance echocardiograms, the mean ef z-score was -0.97. mean lvedd zscore was -0.94, mean septal thickness z-score -1.00, mean posterior wall thickness z-score -0.98, and mean gls -21.96%. for patients that had echocardiogram performed for routine surveillance, 77/95 patients (82.1%) had ef measured, and 10/77 (13.0%) had ef z-scores ≤ -2.0 (abnormally low). patients exposed to anthracyclines had a mean z-score ef of -1.19 vs. unexposed patients -0.50 (p = 0.003). among individuals who received neither anthracyclines nor tbi only 1/31 (3.2%) was found to have an abnormal ef, 51.4% (z-score -2.73) or gls (-14.28%). only one patient who had a normal ejection fraction (z-score -0.39, ef 61.7%) had an abnormal gls, -15.9% (normal ≤ -16.0). long-term survivors of pediatric hsct who are asymptomatic and did not receive radiation or anthracyclines likely do not require surveillance echocardiograms, unless indicated by clinical symptoms. patients exposed to anthracyclines or tbi require close echocardiographic s277 of s301 screening and clinical monitoring for the development of cardiac complications. duke children's hospital, durham, north carolina, united states background: children undergoing pediatric blood and marrow transplants (pbmt) experience significant symptom distress. mobile health (mhealth) technologies can be leveraged to collect and monitor patient generated health data, and subsequently enhance our understanding of pbmt symptom clusters, patterns, and trajectories. better understanding of symptom complexity can foster development of precision health strategies to improve patient outcomes. however, limited research exists in integrating mhealth technology into pbmt management. we aimed to explore the feasibility, acceptability, and usability of using a pbmt specific mobile application to collect and monitor symptoms and wearable technology (apple watch) to measure objective data such as heart rate (hr) and activity. design/method: an exploratory mixed method design began in october 2017 to monitor pbmt symptoms for 20 patients using real-time data from: 1) a self-developed mhealth application (app) to collect subjective symptom data; and 2) apple watch to collect physiologic measures such as heart rate and number of daily steps. data is collected pre-transplant through 90 days. acceptability will be assessed through satisfaction surveys at study completion. we have enrolled 4 patients to date who are all currently using the app and watch. patients' average frequency of daily charting in the app 80%. the wearable average daily recorded measurements are 144 for hr and 29 for step count. most common symptoms recorded within the app include fatigue and pain. we have noted trends in data including a decrease in activity following transplant and gvhd and an increase following engraftment. patients have stated "the app is helpful to keep track of how my pain is doing day to day" and "i try to take more steps each day than the day before". patients often remove the watch for charging, then forget to put it back on, but consistently put it on upon reminder. finally, parents often were required to make app entries with patients too sick to record. we continue to enroll patients with enthusiasm from both patients and parents to use mhealth during pbmt. preliminary findings suggest feasibility of using the mhealth devices is strongly correlated to the patient's post-transplant stage and is facilitated by caregiver participation with device management (charging devices, reminders to wear watch and record in app). patients reported satisfaction and ease of use with devices, but found it difficult to keep up with charging and charting. these findings indicate using mobile devices may be useful methods to collect patient generated health data. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bacterial bloodstream infections (bsi) are a common complication following hematopoietic stem cell transplantation (hsct) in both pediatric and adult populations, and are associated with poor outcomes. there is limited data describing the outcomes and characteristics of patients who develop three or more bsi after hsct. objectives: to describe the characteristics and outcomes of pediatric patients who develop three or more blood stream infections in the first-year post hsct. design/method: we performed a retrospective chart review of 373 consecutive patients who underwent hsct at our institution from 2011 through 2016 to compile this case series. data were collected through the first year post-hsct including: patient demographics, underlying disease and therapy characteristics; and transplant complications such as thrombotic microangiopathy (tma), graft versus host disease (gvhd) and overall survival. bsis were classified according to current center of disease control guidelines. results: of 373 patients, 18 (5%) developed 3 or more bsi in the first-year post transplant (total bsi cases = 77 including all patients). of the 18 cases, the majority underwent allogeneic hsct (n = 17/18; 94%). most cases were from unrelated donor (n = 15/18, 83%). more than half of patients had grade 2-4 gvhd (n = 11/18, 61%). sixteen (89%) had tma. of these 16 cases, tma preceded the first bsi in n = 10/16 (62%). the majority of bsis were classified as central line-associated bloodstream infections (clabsis, n = 37/77, 48%), followed by mucosal barrier injury laboratory-confirmed bloodstream infections (n = 29/77, 38%) and secondary bsi (n = 11/77, 14%). the majority of isolated organisms (45%) were associated with mucosal barrier injury pathogens. one-year overall survival in the cohort was 44% (n = 8/18). pediatric patients undergoing hsct who develop 3 or more bsis in the first-year post transplant demonstrated an increased rate of tma compared to the overall institutional incidence of roughly 30%. tma diagnosis preceded the first bsi in over half of patients, suggesting that tma may predispose to recurrent bsi. improved strategies for early detection and treatment of tma as well as prevention of clabsis may help reduce the number of bsis ultimately leading to decreased morbidity and mortality in this patient population. background: in neutropenic pediatric patients, infection remains a significant cause of morbidity and mortality. while granulocyte transfusions have been utilized for decades to treat infections, including in the pediatric population, the efficacy of this intervention remains poorly described. previous guidelines have primarily utilized information from adult populations. furthermore, recruitment of donors typically involves friends or relatives of the patient with periodic involvement of community donors. the use of a readily available local donor population to improve availability has yet to be well described. as the immunocompromised population is particularly susceptible to worsening infection and clinical deterioration, the ability to rapidly harvest and deliver granulocytes warrants further investigation. to investigate the efficacy, safety, and outcomes of severely immunocompromised patients receiving granulocyte transfusions from a local altruistic granulocyte program in a pediatric tertiary care center. design/method: a retrospective review was performed to evaluate the context for receiving a transfusion as well as primary outcomes including infection clearance, survival to discharge, and overall mortality. the indiana blood bank assisted with timing the interval from initial order placement to onset of first granulocyte infusion. results: among the patient population reviewed, 22 patients received 23 separate granulocyte regimens. ages ranged from 0-18 years with a mean neutrophil count of 77 at time of first transfusion. indications for transfusions included bacteremia (n = 11), fungal pneumonia (n = 6), and fungemia (n = 5). primary outcomes included clearing infection (70%) and surviving to discharge (57%). the median time from initial order placement to infusion was 46 hours, although there was no significant difference between responders who cleared the infection and non-responders who did not. however, additional investigation found that ward patients had a 75% chance of surviving to discharge while patients in the icu at time of initial transfusion had a 36% chance of survival to discharge. the readily available granulocyte transfusion program allows patients to quickly receive therapy in neutropenic settings. this is beneficial for patients as transfusion prior to clinical decompensation correlates with increased likelihood of infection clearance, and subsequently improved mortality. further investigation is needed, likely as a prospective study, to better explore circumstances that are beneficial for granulocyte transfusions. background: donor lymphocyte infusions (dli) are composed of immune cells to treat relapse after hematopoietic cell transplantation (hct). to date, data regarding its efficacy is limited in pediatric populations. furthermore, while outcomes related to cd3 content have been characterized, to our knowledge, the relationship between outcomes and other cellular content in dli has never been reported. objectives: determine whether the primary hematological malignancy, presence/absence of graft-versus-host disease s279 of s301 (gvhd), and unique phenotypic content of each dli impact overall survival (os) in pediatric patients with hematological malignancies. design/method: irb-approved, retrospective study investigating all consecutive dlis given to patients at the children's hospital of wisconsin. analyses were conducted using mann-whitney, fisher's exact, and chi-square. from 1980 from -2016 patients ≤20 years old with hematologic malignancies [myeloid (aml/ mds/cml/jmml),n = 23; lymphoid (all),n = 7] underwent 55 dlis (72%% ≥2 dlis). the median time between hct and dli was 0.6 (range, 0.1-5.8) years. there were significant differences between the lymphoid and myeloid groups, respectively, in regard to median age at hct (14.7 vs 7.5 yrs, p = 0.022) and at first dli (20 vs 8 years, p = 0.006). ultimately, there were no statistically significant differences in gvhd or os in products with either higher or lower cd3, cd4, cd8, cd56, or cd19 cellular content. however, the median cd3/kg content was more than double in the patients who developed gvhd as compared to patients who exhibited no gvhd after dli (29.99 × 106 vs 10.03 × 106, p = 0.346). patients receiving one dli had a 6-year os of 21 ± 9% vs those receiving 2+ dli of 52 ± 16% (p = 0.012). with a median follow-up of 0.74 (range, 0.04-16.61) years, the 6 year estimated os of patients in the lymphoid group was higher at 71 ± 17% vs 22 ±9% in the myeloid group, although not significant (p = 0.11). our results indicate a survival benefit when using dli in a subset of patients who relapse after hct. unlike adult studies demonstrating little effect of dli in lymphoid diseases, many children with all achieved durable remission. while our analysis did not demonstrate that dli cellular content had a statistically significant effect on gvhd or os, it is possible that differences could be found if a larger population and more targeted cell doses were studied. more data will be needed to further define these relationships and identify patients who stand to benefit most. cincinnati children's hospital medical center, cincinnati, ohio, united states background: many arabic speaking muslim parents of children requiring bone marrow transplantation (bmt) receive medical care in the united states. providers may not understand the impact of islamic parents' religious beliefs and practices on their health care experience. objectives: to explore how islamic parents used religion in decision making and to understand the impact of their religious beliefs and practices on their overall health care experience. design/method: we used grounded theory, an inductive method gathering data from interviews and analyzing text, to identify core themes. ten caregivers of bmt children from middle eastern countries were interviewed by an arabicspeaking provider; interviews were coded by an interdisciplinary team. we identified 5 key themes: 1. patience is a core belief in islam. patience results from the acceptance of allah's will. behaviors showing patience include praying rather than questioning and crying. 2. al qur'an provides comfort, healing, and protection. families listen to recitations of al qur'an in the patient's room because they feel that this practice not only comforts them but promotes healing as well. for some, certain portions of the qur'an were especially meaningful such as surat al-baqara, which explains that while we may think something is bad for us, allah will know it is good for us. 3. religious care in the medical center helped families feel respected. religious care in the medical center included interactions with chaplains, who were understood to be "religion experts," and provision of space for prayer and religious resources. 4. seeking religious consultation. religious consultation from imams or religious scholars (muftis or sheikhs) provides interpretations of the qur'an applied to the family's specific situation helps families make difficult decisions and follow allah's plan. 5. muslim beliefs guided decision making; muslim practices brought comfort, strength, and peace. drawn from the parents' understanding of islam. parents who addressed this topic said they would only do what islam allowed. they did indicate that most aspects of healthcare were understood to be allowed within islam. additionally, muslim practices of prayer, reading/listening to qur'an, and giving alms all provided comfort, strength and peace. we identified several recurring themes through our interviews that allowed us to understand how families use their muslim faith to deal with their children's illnesses and how it influences their decision making. we believe this better understanding will allow for more informed conversations about patients' health care and decision making, and shows respect for religious beliefs and practices. nemours/dupont hospital for children, wilmington, delaware, united states background: virtually all children will be infected with human herpesvirus 6 (hhv-6) by the age of two. hhv-6 reactivation after stem cell transplantation causes multiorgan toxicities, including encephalitis, with inflammation and destruction of the temporal lobes and hippocampi, memory loss, and seizures. catatonia is characterized by posturing, immobility, mutism, and autonomic instability, and it's associated with various psychiatric and medical conditions. we describe a patient with hhv-6 encephalitis and unusual neurologic sequelae, including cognitive and neurobehavioral dysfunction and catatonia, which may impact our understanding of the pathophysiology of hhv-6 reactivation encephalitis. objectives: describe a case of hhv6 encephalitis with practice implications for stem cell transplantation. results: our patient was diagnosed with acute myeloid leukemia at age 14. within 2 years, he relapsed and received two stem cell transplants. on the 29th day after his second transplant, he developed hyponatremia and refractory seizures. brain mri showed edema in the medial right temporal lobe with linear ischemic change. eeg showed diffuse encephalopathy. cerebrospinal fluid (csf) demonstrated 5 white blood cells, 2 red blood cells, and hhv-6 by pcr. his prophylactic antiviral was switched to foscarnet and ganciclovir. repeat mri showed abnormal signals in bilateral medial temporal lobes and the right insula. three months later he developed episodes of diaphoresis, hypothermia, agitation, mutism, and unusual posturing, recurring almost daily, recognized as catatonia. mri showed improvement of the abnormalities in the bilateral medial temporal lobes and hippocampi. eegs showed diffuse slowing. after 4 months of antiviral therapy, csf was negative for hhv-6. over the ensuing 3 years, he had numerous episodes of diaphoresis, hypertension, hypothermia, pruritis, confusion, agitation, cogwheel rigidity, and bizarre posturing. dopamine blocking agents did not help. clonazepam helped reduce their frequency, and hot showers helped break acute episodes. further mris showed generalized cortical volume loss. he suffered from depression and severely impaired sleep and cognitive function. we describe a novel, debilitating outcome of hhv6 encephalitis which may provide diagnostic considerations as we continue to improve our understanding of the breadth of possible neurologic sequelae in transplant patients. hhv-6 is understood to infect and destroy the temporal lobes and hippocampi, but our patient's autonomic dysfunction indicate involvement of the hypothalamus and basal ganglia. antidopaminergic agents may worsen catatonia, and they were not effective for our patient. treatment of catatonia includes benzodiazepines; electroconvulsive therapy was not attempted in this case but may also be useful. background: epstein-barr virus (ebv)-related posttransplant lymphoproliferative disorder (ptld) is a lifethreatening complication in patients following hematopoietic stem cell transplantation, with a frequency estimated at 3.2% and a cumulative incidence of mortality estimated as high as 31%. studies of ebv have hypothesized that the tonsils are critical for propagating this infection, as tonsillar epithelial cells have been shown to be the site of primary viral infection and continued viral shedding; however, to date no studies have been performed assessing the role of tonsillectomy in patients with ebv ptld. objectives: identify patients with localized ebv ptld treated with tonsillectomy to identify prognostic factors that may be able to help guide future treatment decisions. design/method: patients treated at memorial sloan kettering cancer center who had received hematopoietic stem cell transplantation and had billing codes for both ebv and tonsillectomy were eligible for inclusion in this study. a retrospective chart review was performed, assessing patient demographics, transplant characteristics, laboratory values, tonsillar pathology, and clinical course. any patient who did not have unilateral or bilateral tonsillectomy performed or who had non-localized disease (defined as disease involvement outside of the oropharynx and neck) was subsequently immunodeficiency; 17% (n = 17/100) fanconi anemia (fa); 17% (n = 17/100) hemoglobinopathy; 12% (n = 12/100) non-fa marrow failure and 3% (n = 3/100) a metabolic disorder. seventy one percent (n = 71/100) had normal amh for age pre-transplant, 29% (n = 29/100) had low amh for age pre-transplant; of these, 37% (n = 11/29) had an oncologic diagnosis; 37% (n = 11/29) had fa; 10% (n = 3/29) had previously treated hlh; 6% (n = 2/29) had non-fa marrow failure; one had a metabolic disorder and one a hemoglobinopathy. of the 33 patients with post-transplant amh measurement 72% (n = 24/33) had low levels. of the 25 patients with previously normal pre-transplant amh 52% (n = 13/25) underwent myeloablative conditioning (mac) regimen with a 100% (n = 13/13) having low amh levels post-transplant compared to 48 %(n = 12/25) who underwent reduced intensity conditioning (ric) regimen with 25% (n = 3/12) having low amh levels post-transplant (p 0.0002). fifteen percent (n = 5/33) had low levels pre-transplant and underwent mac regimen with 100% (n = 5/5) remaining low; 80% of these patients (n = 4/5) had fa. nine percent (n = 3/33) had low levels and underwent a ric regimen with 100% (n = 3/3) of amh levels remaining low; 66% (n = 2/3) of these patients had hlh treated prior to transplant. conclusion: amh levels can be used for detection of premature ovarian failure and fertility counseling. there is a higher risk of premature ovarian failure with mac regimens and prior chemotherapy vs ric regimens. follow up of this cohort will provide more information to understand the effects of hsct in ovarian function and the usefulness of amh as a predictor of fertility potential. background: there are no proven strategies to prevent blood stream infections (bsi) secondary to oral mucosal barrier injury after hematopoietic stem cell transplant (hsct). additionally, we recently reported progressive gingivitis and dental plaque accumulation in hsct recipients despite our current oral standard of care (three times daily oral rinse). xylitol is a non-fermentable sugar alcohol that reduces dental caries, plaque accumulation, and oral disease progression by inhibiting bacterial growth. we hypothesized that the addition of xylitol to standard oral care will decrease dental plaque accumulation, gingivitis and bacteremia from oral flora. objectives: identify a clinically effective strategy to improve oral health and prevent bsi secondary to bacterial translocation through the oral mucosa in patients undergoing hsct. we are conducting a prospective randomized control study to test our hypothesis. those in the intervention arm receive our current standard of care (three times daily oral rinse) in addition to daily xylitol wipes; controls receive oral standard of care alone. oral exams are performed at baseline and weekly for the first 28 days post hsct. metagenomic shotgun sequencing (mss) of gingival samples is performed at all time points to evaluate microbiome diversity and pathogenic bacterial load. finally, we performed whole genome sequencing of pathogenic bacterial isolates causing bacteremia to assess for genetic relatedness to corresponding strains present within the patient's oral microbiome preceding the infection. : preliminary interim analysis of 21 patients demonstrates improved oral health in patients receiving xylitol (n = 10) over those receiving standard of care (n = 11), measured by the oral hygiene index (p = 0.03) and gingivitis index (p = 0.02). in the nine patients having complete oral mss analysis, xylitol appeared to be associated with decreased streptococcus mitis/oralis domination in the oral microbiome. finally, patients receiving xylitol had no incidence of streptococcus mitis/oralis bacteremia through the first 21 days compared to three patients (27%) in standard of care arm. interestingly, streptococcus mitis/oralis comprised 70% of the oral microbiome in one child who subsequently developed a streptococcus mitis/oralis bsi. we expect to complete this study in the next 4 months (n = 50). the addition of xylitol to oral standard care appears to decrease dental plaque and gingivitis in patients undergoing hsct. xylitol may also impede streptococcus mitis/oralis dominance in the oral microbiome with potential reduction in blood stream infections. (range:4-159 days). twenty-one mdli (49%) were administered because of lymphopenia, fourteen of them (33%) in patients with concomitant viral/opportunistic infections. mixed chimerism/graft failure was the motive of 37% of the mdli (n = 16) and six (14%) were administered to accelerate immune reconstitution. all infusions were well tolerated without appearance or worsening of gvhd. an increase in t-cell counts was observed following six mdli (28.57%), although it was a transitory response (3-8 weeks) in five cases. viral/opportunistic infections were controlled in five cases (35.71%), requiring a median of 2 mdli to achieve this response. none of the mdli administered in cases of mixed chimerism/graft failure were effective in reverting this situation. our preliminary data suggests that mdli, is a safe adoptive immunotherapy strategy even with high dose of t-cells without infusion side effects or gvhd complications. some efficacy has been observed in patients with lymphopenia and opportunistic infections, with no positive results in patients with mixed chimerism/graft failure, up to date. however, to determine the real efficacy of this strategy, prospective studies are required. jun zhao, kristen beebe, lucia mirea, alexandra walsh, shane lipskind, alexander, ngwube phoenix children's hospital, phoenix, arizona, united states background: male adolescents undergoing myeloablative hematopoietic stem cell transplantation (hsct) develop infertility with impaired spermatogenesis with reported rates ranging from 17% to 80%. in nonmalignant diseases, myeloablative regimens have been replaced with reduced intensity conditioning (ric) with the hopes of better survival rate, less organ toxicity and improved quality of life. despite the increased use of ric regimens for hsct, the effects of ric on fertility remain unknown. objectives: to assess fertility following ric hsct in young adult males. we assessed gonadal function and semen characteristics in adolescent males (>14 years) who received a single ric hsct at phoenix children's hospital for nonmalignant diseases during 2006-2016. male patients who were a minimum of 1 year from ric hsct and had postpubertal development at tanner stage iii or above were eligible for this study. gonadal status was assessed by measuring fsh, lh, testosterone, and inhibin b levels, and semen anal-yses assessed fertility indicators (semen volume, sperm concentration, motility, viability, forward progression, morphology, and total count). results: hormone levels and semen analysis have been obtained for 3 patients thus far. the median time between transplant and semen analysis was 4 years. post hsct, 2 (67%) patients showed abnormally elevated lh levels, but fsh, testosterone (total and free), and inhibin b levels were within normal range for all patients. sperm morphology and viability testing were not able to be performed due to low concentrations and volumes. as a result, the total motile sperm count, the most useful estimate for fertile potential, is essentially 0 for all 3 patients. conclusion: recruitment is ongoing, but so far our limited results suggest that ric hsct may have detrimental longterm effects on male fertility. a multi-institutional trial may be appropriate due to small patient numbers at each institution. we are currently exploring options to expand to other centers. further consideration is warranted regarding decisions made by providers, ways to improve anticipatory counseling provided to patients and their families prior to transplant, and how to augment the preventive care of these patients in longterm follow-up. currently all male patients being considered for ric transplant should be counseled to sperm bank prior to transplant. background: a previous systematic literature review identified all published studies of defibrotide treatment for patients of all ages with vod/sos. to assess day+100 survival for defibrotidetreated pediatric patients (≤18 or ≤16 years, per study) all patients exhibited infectious complications with at least 1 viral infection. four patients also had bacterial infections. of note, no patient developed evidence of fungal infections. conclusion: early institution of ecp in patients with high risk acute gvhd (grade 3-4) was very effective at treating agvhd, allowed for an aggressive steroid taper and contributed to excellent overall survival rates (83%). infectious complications were primarily viral and bacterial, with no fungal infections in this very high risk population. background: vod/sos is a life-threatening complication of hsct conditioning. vod/sos with multi-organ dysfunction (mod) may be associated with >80% mortality. defibrotide is approved to treat hepatic vod/sos with renal/pulmonary dysfunction post-hsct in the us and severe hepatic vod/sos post-hsct patients aged >1 month in the eu. there are few published data on survival of neuroblastoma patients with vod/sos post-hsct. objectives: to report day+100 survival and safety post hoc for patients with neuroblastoma and vod/sos post-hsct in the defibrotide t-ind trial. design/method: vod/sos was diagnosed by baltimore or modified seattle criteria or biopsy, with/without mod, after hsct or chemotherapy. defibrotide treatment (25 mg/kg/day) was recommended for ≥21 days. this post hoc analysis is based on 1154 adult and pediatric patients receiving ≥1 dose of defibrotide, including 571 with mod. results: among 111 patients with neuroblastoma, 106 developed vod/sos after hsct. for these post-hsct patients, 60.4% were male and 39.6% were female, median age was 3 years (range 1-17 years): 7.6% aged 0-23 months, 90.6% 2-11 years, 0.9% 12-16 years, and 1 patient >16 years. day+100 survival data were available for 105/106 of these neuroblastoma patients (43 with mod and 62 without mod); 103 had autologous and 2 had allogeneic transplants. kaplan-meier estimated day+100 survival for the neuroblastoma group was 87.2% (95% confidence interval [ci] , 79.0%-92.4%). for the mod and no mod subgroups, kaplan-meier estimated day+100 survival was 78.4% (95% ci, 62.6%-88.2%) and 93.5% (95% ci, 83.6%-97.5%), respectively. in the overall t-ind hsct population aged ≤16 years (n = 570) and pediatric autologous hsct subgroup (n = 127), kaplan-meier estimated day+100 survival was 67.9% and 87.1%, respectively. treatment emergent adverse events (teaes) occurred in 45.3% (n = 48/106), with serious teaes in 23.6% (25/106; most common: multi-organ failure, 4.7% [5/106]). teaes lead to treatment discontinuation in 17.0% (n = 18; most common: pulmonary hemorrhage, n = 3); death occurred in 10.4% (n = 11; >2%: multi-organ failure, 4.7%; vod/sos, 2.8%). treatment-related adverse events, as assessed by investigators, occurred in 17.0% (n = 18; most common: pulmonary hemorrhage, 2.8%). this post hoc analysis found kaplan-meier estimated day+100 survival of 87.2% in patients with neuroblastoma and vod/sos post-hsct, which was consistent with outcomes in pediatric patients after autologous hsct. the safety profile of defibrotide in neuroblastoma patients was consistent with the overall hsct population in this study and other defibrotide studies in pediatric patients. cincinnati children's hospital medical center, cincinnati, ohio, united states background: blood stream infections occur in nearly 30% of patients undergoing hematopoietic stem cell transplant (hsct) and fever is often the first symptom. timely administration of antibiotics is associated with improved outcomes, thus, early recognition of fever is paramount. current standard of care (soc) includes episodic monitoring of temperature in hospitalized patients, which may delay fever detection. therefore, continuous real-time body temperature measurement may detect fever prior to the current soc. temptraq is a food and drug administration cleared class ii medical device and consists of a soft, comfortable, disposable patch that results: of 100 patients, 98 were started on a pca in the 30 days post hct. 65% were male with median age of 11y. 46% had all, and 37% aml. matched related donors were used in 21% and 79% received tbi. pca was initiated median d+2. oral mucositis alone was the most common indication (80%). a majority of patients were started on hydromorphone (79%); 20% started on morphine and 1% started on fentanyl. 49% started on continuous infusion. pca was used for a median of 20 days (range 4-144 days). median pain score was highest d+3 of pca use, however, there was inconsistency in charting of numerical pain scores. on d+3, 12 patients had insufficient data to determine efficacy of pain control; of the remaining 86 patients, 24% had good pain control while 60% had moderate and 15% had poor pain control using our devised scale. the most common toxicity observed was respiratory depression (∼30%), however, etiology was often multifactorial and not due to opiates alone. analysis is ongoing to assess variables predicting pca use as well as efficacy of pain control and correlation between current reporting scales and patient perception. conclusion: pca use is common in pediatric hct yet pain control remains inadequate. there's a need for better evaluation of pca management, especially uniform assessment of pain, thereby improving quality of life post hct. children's national health system, washington, district of columbia, united states background: actinomycosis is a rare invasive anaerobic gram-positive bacterial disease caused by actinomyces spp. that may colonize the oropathynx, gastrointestinal tract and urogenitial tract and can lead to abscesses. respiratory tract actinomycosis is characterized by pulmonary cavities, nodules, consolidations and pleural effusions. although actinomyces are nearly always sensitive to penicillin they are frequently resistant to cephalosporins and variable sensitives to fluoroquinolones. although rare in children, immunosuppressed patients are at increased risk for actinomycosis. to describe a case of next-generation sequencing identification of actinomycosis. a 13-year-old male with a history of very high risk b-cell acute lymphoblastic leukemia who was 5 months status post a 7/8 matched unrelated donor bone marrow transplant complicated by prolonged fevers, persistent weight loss, and splenic lesions, treated with posaconazole and levofloxacin developed fever and cough in the setting of neutropenia. blood cultures demonstrated staphylococcus epidermidis. ct showed micronodules and effusion not consistent with s. epi, prompting bronchoscopy. all bacterial cultures were negative. patient was prescribed a three-week course of vancomycin with rapid improvement. design/method: 16s next generation sequencing (ngs) from bronchoalveolar levage sample was performed at the university of washington laboratory results: ngs assay from bronchoalveolar lavage showed major abundance of actinomyces most closely related to meyeri or oodontolyticus. demonstrated actinomyces. the patient was started on a six month course of amoxicillin with continued clinical improvement. in retrospect, the splenic nodules that were presumed fungal disease were likely actinomycosis, partially treated with levofloxacin. this case highlights the potential utility of ngs in the diagnosis of rare diseases in immunocompromised patients. actinomycosis was only demonstrated through ngs and led to a change in treatment regimen and durable clinical improvement. because actinomyces often mimics malignancy, tuberculosis or nocardiosis, the use of this novel test both targeted appropriate therapy and reduced the exposure to unnecessary medications to treat the differential diagnosis. finally, we highlight that actinomyces should be considered in patients who present with unexplained fevers, weight loss, and night sweats. haneen shalabi, cynthia delbrook, maryalice stetler-stevenson, constance yuan, bonnie yates, terry j. fry, nirali n. shah center for cancer research, national cancer institute, national institute of health, bethesda, maryland, united states background: car-t therapy, while effective, may not be durable for all, and antigen negative escape is a growing problem. hct, in relapsed/refractory all, can be curative, particularly for those in an mrd negative remission. we demonstrated that cd19 directed car-t therapy effectively rendered patients into mrd negative remissions (by flow cytometry) and the leukemia free survival post-hct was high 1 . in pastorek, jesssica bruce, michael a. pulsipher, chloe anthias, peter bader, andre willasch, jennifer sees, jennifer hoag, wendy pelletier, brent logan, pintip chitphakdithai, lori wiener university of pittsburgh, pittsburgh, pennsylvania, united states background: more than 4,500 pediatric hscts are performed in north american and europe each year. the ethics of exposing a healthy child to donation procedures which have some risks and no direct medical benefits continue to be a topic of debate. pediatric donors may experience psychological distress and poorer quality-of-life during and after donation compared to healthy controls. although there are fact/jacie requirements related to the management of pediatric donors, it is unclear what standardized practices exist for psychosocial assessment/management of this group. objectives: to describe transplant center practices for psychosocial evaluation/ management of pediatric donors (<18 years) and to examine differences in practices by location (cibmtr/ebmt) and number of harvests (volume). design/method: data were collected via a single crosssectional survey distributed electronically to cibmtr and ebmt centers between 9/18/17 and 11/20/17. : 55/98 (56%) of cibmtr and 85/147 (58%) of ebmt centers completed the survey. most centers had written eligibility guidelines for pediatric donors (91%). most also had a process for ensuring that donors were freely assenting to donate (78%), managed by a transplant physician (61%). a single physician often jointly managed donor/recipient care (44%). half of centers had a pediatric donor advocate (51%), who was most often a physician (38%) or social worker (16%). cost was the largest barrier to having a donor advocate (82%). most centers performed psychosocial screening of donors (69%) but rarely declined donors based on psychosocial concerns (13%). less than half of centers provided post-donation psychosocial follow-up (41%). comparisons by center location indicated that ebmt centers were more likely to have a physician doing joint donor/recipient care (60% vs. 23%; p = .001), less likely to have a psychosocial assessment policy (53% vs. 79%; p = .011), less likely to have a donor advocate (37% vs. 69%; p = .002), but marginally more likely to do post-donation psychosocial follow-up (49% vs. 31%; p = .063). large volume centers were more likely to have a psychosocial assessment policy than their medium/smaller counterparts (69% vs. 23%, 27%; p = .003) â€"there were no other differences on key psychosocial management variables by volume. although most centers have written guidelines for pediatric donor eligibility and mechanisms for ensuring assent, substantial numbers of donors do not undergo psychosocial assessment, are jointly managed with the recipient by a single physician without an assigned donor advocate, and do not receive psychosocial follow-up. the field would benefit from guideline development for the psychosocial management of pediatric donors. background: germline mutations in samd9 and samd9l genes cause mirage (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy) and ataxia-pancytopenia syndromes, respectively, and are associated with chromosome 7 deletions, mds and bone marrow failure (bmf). there are limited data on outcomes of hct in these patients. to describe outcomes of allogeneic hct in patients with hematologic disorders associated with samd9/samd9l mutations. results: seven patients underwent allogeneic hct for primary mds (n = 5), congenital amegakaryocytic thrombocytopenia (camt)(n = 1), and dyskeratosis congenita (n = 1). retrospective exome sequencing revealed gain-of-function mutations in samd9 (n = 4) or samd9l (n = 3) genes. constitutional mosaic monosomy 7 was present in 6 cases. two samd9 patients had features of mirage syndrome. unusual findings of panhypopituitarism, laryngeal cleft, and glomerulosclerosis were noted in one case. in another case with a samd9 mutation hypospadias & bifid scrotum were the only findings. the remaining patients had no phenotypic abnormalities. median age at hct was 5y (range: 1.4 -12.8). patients received transplants from bone marrow (matched unrelated (n = 3) & hla identical sibling (n = 2)), or unrelated cord blood (ucb) (n = 2). five mds patients received myeloablative s295 of s301 conditioning (busulfan-based (n = 3) or tbi-based (n = 2)); 2 patients (mds (n = 1); camt (n = 1)) received reducedintensity conditioning (ric) (fludarabine, cyclophosphamide, with ratg or alemtuzumab). syndrome-related comorbidities (diarrhea, infections, malnutrition, electrolyte imbalance, lung disease and hypoxia) were present in both patients with mirage syndrome. one patient with a familial samd9l mutation, mds and morbid obesity failed to engraft following ric double ucbt. she died one year later from refractory aml. all other patients achieved neutrophil and platelet engraftment, at a median (range) of 15 (12-19) and 16 (12-31) days, respectively. posttransplant complications included severe hypertension (n = 1), pericardial effusions (n = 2), veno-occlusive disease of liver (n = 1), and recurrent aspiration pneumonias (n = 1). one patient developed grade iii agvhd which resolved with treatment. one patient developed mild skin cgvhd and suffers from chronic lung disease. all surviving patients had resolution of hematological disorder and sustained peripheral blood donor chimerism (98-100%). overall survival was 86% with a median follow-up of 3 years (range: 1.1 -14.7 y). patients with hematological disorders associated with germline samd9 /samd9l mutations tolerated transplant conditioning without unusual, or unexpectedly severe toxicities. allogeneic hct led to successful resolution of mds or bmf, with excellent overall survival. more data is needed to refine transplant approaches in samd9/samd9l patients with significant comorbidities, and develop guidelines for their long-term follow-up. shyamli singla, tiffany simms-waldrip, andrew y. koh, victor m. aquino background: steroid-refractory acute graft versus host disease (agvhd) is a potentially fatal complication of allogeneic hematopoietic stem cell transplantation (hsct). basiliximab (anti-il2-r monoclonal antibody) as a single agent or in combination infliximab (anti-tnf-monoclonal antibody) has demonstrated efficacy in adult cohorts with steroid-refractory agvhd, but has not been well studied in the pediatric population. we adopted the use of basiliximab and infliximab as our institutional standard of care for steroid-refractory agvhd in pediatric hsct patients. to determine the response and survival of hsct children who received basiliximab and infliximab for the treatment of steroid-refractory agvhd. design/method: we retrospectively reviewed children who received basiliximab and infliximab for steroid-refractory agvhd refractory between september 2011 and december 2017. complete response (cr) was defined as resolution of all clinical signs of agvhd. partial response (pr) was defined as at least one grade reduction in one target organ (e.g. skin, gut or liver) without increased grade in another target organ. no response was defined as either no improvement or progressive worsening of agvhd in at least one organ. baseline demographics, transplant details, laboratory findings, and treatment outcomes were also evaluated. results: of the 214 evaluable hsct patients, 15 children (median age 11 yrs, range 9 mo-19 yrs) with steroid-refractory agvhd received combination monoclonal antibody (mab) therapy. the median time from the start of steroid therapy to initiation of mab was 13 days. the overall glucksberg grade of agvhd at the time of initiating mab therapy was grade i (n = 1, 6.7%) ii (n = 3; 20%), iii (n = 8; 53%) or iv (n = 3; 20%). the overall response rate was 53%, with 3 (20%) patients achieving cr, 5 (33.3%) patients achieving pr, and 7 (46.7 %) patients with no response at 30 days following the start of mab therapy. the median overall survival was 613, 292, and 84 days for patients who exhibited cr, pr, and no response, respectively. the overall survival at 1 year following start of mab therapy was 40%. background: the role of high dose chemotherapy (hdc) and autologous stem cell rescue (ascr) in patients with high risk (advanced metastatic or relapsed) soft tissue sarcomas is controversial. despite multimodal chemotherapy, radiotherapy, and local control measure advancements, prognosis of patients with advanced metastatic or unresectable and relapsed sarcomas remains poor, with less than 10% 5 years disease free survival. objectives: to determine if consolidation with myeloablative hdc and ascr improves relapse free (rfs) and overall survival (os) outcomes in a high risk patient subgroup. we performed retrospective review of all high risk soft tissue sarcoma patients who underwent hdc and ascr at the children's hospital at montefiore, bronx, ny between october 2014 and january 2018. the protocol was approved by albert einstein college of medicine institutional review board. results: 7 patients (4 primary metastatic high risk disease, 3 relapsed or recurrent disease) received hdc with ascr. primary diagnoses were rhabdomyosarcoma (rms) (n = 2, alveolar histology), primary site nasopharynx (n = 1) and lower extremity (n = 1). ewing's sarcoma (ews) (n = 5), axial site (pelvic) in 3 patients (60%). median age 12 years (range 6-35 years), 5 (71%) were male. all patients were in complete metabolic remission before transplant. median pre transplant comorbidity index was 3 (range 0-4). 5 patients (2 rms and 3 ews) received conditioning with carboplatin, etoposide and melphalan. remaining 2 patients with ews received conditioning with busulfan, melphalan and topotecan. all patients received peripheral blood mobilized hematopoietic stem cell transplantation. stem cell mobilization achieved with high dose filgrastim in all patients except one who required addition of plerixafor. median cd34+/kg s297 of s301 recipient body weight cell dose infused was 6.34 × 10^6 (range 2.72-12.03 × 10^6). median times to neutrophil and platelet (>20,000/â l) engraftment were 9 (range 8-13) and 49 (20-76) days respectively. 2 patients (28%) developed bk viuria (one with grade iii hemorrhagic cystitis); 3 (43%) developed cmv viremia; and one patient (14%) had asymptomatic ebv viremia. there was no graft failure, sinusoidal obstruction syndrome or transplant related mortality. median follow up post-transplant was 452 days (range 155-1141 days). 3 year probability of os and rfs were 80% and 50% respectively. hdc with ascr is a promising therapeutic strategy to consolidate remission and improve survival in select high risk soft tissue sarcoma patient subgroups. prospective clinical trials will inform the impact of disease status prior to hdc and ascr on outcome, optimal conditioning and long term relapse free and overall survival. background: absence of minimal residual disease is paramount for cure of pediatric acute lymphoblastic leukemia (all). the testis may harbor occult leukemia and this disease may result in treatment failure. objectives: the purpose of this study was to assess the longterm outcomes of boys with or without testicular leukemia pre-hematopoietic stem cell transplantation (hsct). design/method: retrospective analysis of 16 boys with high-risk de novo (2 with hypodiploidy all) or recurrent/refractory all was conducted. flow cytometry of bone marrow mononuclear cells was used to determine remission status. testicular evaluations were performed by physical examination and wedge biopsy pre-hsct. the median age at time of transplant was 12.3 years. all patients were in remission by flow cytometry of bone marrow mononuclear cells at the time of transplant and none had evidence of clinically apparent testicular disease. testicular leukemia was detected in 1 patient and he underwent bilateral orchiectomy. he developed acute graft versus host disease (gvhd) of the duodenum and sigmoid colon which resolved, and the leukemia remains in second complete remission and he is free of hsct-related morbidity 33.6 months post-hsct. of the 15 patients without testicular leukemia 4 died a median of 8.2 months (range, 2.5 to 12.5) post-hsct (2 with adenovirus infection and 1 each with thrombotic microangiopathy and aspergillus pneumonia); 6 experienced infection (staphylococcus species, corynebacterium, enterococcus, klebsiella, citrobacter, e. coli, epstein barr virus, adenovirus, bk virus, human herpesvirus-6, candida albicans, fusarium, aspergillus, yeast, and other fungus); 11 experienced gvhd (8 of the gi tract, 6 of the skin, 5 of the liver, 3 of the eyes, 2 of the mouth, and 1 of the lungs); and 1 developed a second neoplasia (right lower leg leiomyosarcoma). one patient developed bone marrow minimal residual disease (2.7% phenotypically abnormal cells detected 9 months after 6/6 matched sibling hsct). reinduction therapy comprised 5 weekly doses of rituxan, 2 courses of blinatumomab and 2 donor lymphocyte infusions with il-2. two subsequent bone marrow evaluations were minimal residual disease negative. thirteen months post-hsct residual disease recurred (0.012%) and he will receive inotumumab. overall median survival post-transplant of the 16 boys is 32.1 months (range, 2.5 to 75.3) and of the 12 surviving boys is 37.3 months (range, 11 to 75.3). conclusion: testicular biopsy can detect occult leukemia pre-hsct. testicular leukemia pre-hsct does not appear to increase the risk of subsequent relapse or other hsct-related adverse events compared to those without it. yaya chu, nang kham su, sarah alter, emily k. jeng, peter r. rhode, mathew barth, dean a. lee, hing c. wong, mitchell s. cairo new york medical college, valhalla, new york, united states background: rituximab has been widely used in frontline treatment of b-nhl including burkitt lymphoma (bl), however, some patients retreated with rituximab relapse, which limit patient treatment options. novel therapies are desperately needed for relapsed/refractory b-nhl patients. several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering immune cells with chimeric antigen receptors (car), as well as second-generation anti-cd20 antibodies. nature killer (nk) cells play important roles in the rejection of tumors. however, nk therapy is limited by small numbers of active nk cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of nk cell immunosurveillance. our group has successfully expanded functional and active peripheral blood nk cells (expbnk). 2b8t2m was generated by fusing alt-803, an il-15 superagonist, to four single-chains of rituximab. 2b8t2m displayed tri-specific binding activity through its recognition of the cd20 molecule on tumor cells, activated nk cells to enhance adcc, and induced apoptosis of b-lymphoma cells. objectives: to examine if 2b8t2m significantly enhances the cytotoxicity of expbnk against rituximab-sensitive and -resistant bl cells. design/method: expbnks were expanded with lethally irradiated k562-mbil21-41bbl and isolated using miltenyi nk cell isolation kit. alt-803 and 2b8t2m were generously provided by altor bioscience. nk receptors expression and cytotoxicity were examined as we previous described. ifng and granzyme b levels were examined by elisa assays. equal doses of rituximab, alt-803, rituximab+alt-803, obinutuzumab (obinu) were used for comparison. igg was used as controls. anti-cd20 car expbnk cells were generated as we previously described by mrna electroporation. rituximab-sensitive raji andresistant bl cells raji-2r and raji-4rh, were used as target cells. results: 2b8t2m significantly enhanced expbnk cytotoxicity against rituximab-sensitive raji cells, rituximab-resistant raji-2r cells and resistant raji-4rh cells compared to the controls igg, rituximab, alt-803, rituximab+alt-803, obinu (p<0.001, e:t = 1:1). furthermore, we confirmed the enhanced cytotoxicity by measuring ifn-g and granzyme b production. 2b8t2m significantly enhanced ifn-g and granzyme b production from expbnk against raji, raji-2r and raji-4rh compared to igg (p<0.001), rituximab (p<0.001), alt-803 (p<0.001), rituximab+alt-803 (p<0.001), and obinutuzumab (p<0.001). when compared to anti-cd20 car expbnk cells, 2b8t2m + expbnk had the similar cytotoxicity against raji, raji-2r and raji-4rh as anti-cd20 car expbnk cells did (p>0.05). conclusion: 2b8t2m significantly enhanced expbnk activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and -resistant bl cells. the in vivo functions of 2b8t2m with expbnk against rituximab-sensitive and -resistant bl cells using humanized nsg models are under investigation. background: cardiac dysfunction, including left ventricular systolic dysfunction (lvsd), is a known complication in stem cell transplant (sct) survivors. while detection of lvsd by echocardiography is important in this population, there has been minimal research to determine if subclinical cardiac dysfunction exists in sct patients. cardiopulmonary exercise testing (cpet) is a valuable tool to assess cardiac function, and to determine how the heart responds to the stress of exercise. no studies have been performed to determine if sct patients with normal lvsd on standard echocardiography may have abnormal cpet. to determine the feasibility of cpet, as well as additional echocardiographic parameters, to detect dysfunction in sct patients with a normal ejection fraction on echocardiogram. design/method: we performed a cross-sectional analysis of sct survivors who were at least 3 years post sct, 8 years of age or older and with an ejection fraction > 50% (low end of normal range) on echocardiogram. we assessed the exercise capacity of all patients with cpet, and sub-clinical cardiac dysfunction through tissue doppler and strain analysis from the echocardiogram. results: seven patients (6 male) have qualified and completed this study so far with an average age of 12.2±3.4 years. the median time from transplant is 4.4±0.8 years. all seven patients had a normal ejection fraction, however four patients had abnormalities on their cpet. these abnormalities included abnormal predicted peak oxygen consumption (vo2) (61%±9.8, normal > 70%) (the best predictor of functional capacity), predicted oxygen pulse (62%±10.1, normal > 70%) (measure of cardiac stroke volume) and ventilatory efficiency (ve/vco2 slope) (39±7.6, normal < 30). submaximal exercise data, used when patients are unable to complete a maximal effort test, demonstrated low-normal predicted vo2 at anaerobic threshold (45.8%±7.2%, normal >45% of was 19.4 days while patients who received autologous infusions had a mean number of days to engraftment of 13.3. engraftment after hsct needs to be prompt to minimize duration of neutropenia and maximize survival rates 6 . our data demonstrates that the infusion of hematopoietic stem cell products with a syringe or iv pump is an effective method of delivery for stem cell products and does not delay the time to engraftment. the median days to neutrophil engraftment was 14.5 days. this is comparable to data from the nmdp, which reports engraftment occurs within 14-20 days. the main limitation to this study was its small sample size due to the number of transplants done at our center. however, it does provide evidence to support that infusion of stem cell products via pump mechanism is a safe alternative to the infusion by gravity method in the process of the hematopoietic stem cell administration. johns hopkins all children 's hospital, st. petersburg, florida, united states background: leukemic relapse remains the most common cause of treatment failure after allogeneic hematopoietic cell transplant (allohct) for myeloid malignancies. most children who relapse post-allohct will die of their disease, making interventions to minimize this risk a high priority. objectives: to evaluate the safety and efficacy of posttransplant azacitidine for relapse prevention in children undergoing allohct for myeloid malignancy. design/method: we retrospectively reviewed the charts of children undergoing allohct for myeloid malignancies between february 2015 and november 2016 at johns hopkins all children's hospital. results: during the study period, 18 children (ages 2 to 20 years, median 12) underwent allohct for myeloid malignancies: de novo acute myeloid leukemia (aml), 11; mixed phenotype acute leukemia, 2; treatment-related aml, 2; juvenile myelomonocytic leukemia with aml transformation, 2; and myelodysplasia/aml, 1. thirteen were in first complete remission, 5 were in cr2 or greater. most patients (13/18) received fludarabine/melphalan/thiotepa conditioning; 11 received hla-identical related or unrelated donors, and 7 received haploidentical bone marrow grafts with post-transplant cyclophosphamide. three patients never received planned azacitidine (2 early relapse; 1 early trm), leaving 15 evaluable patients. azacitidine (32mg/m 2 /dose for 5 days, in 28-day cycles for up to 9 cycles) was started at a median of 66 days post-transplant (range 42-118). two-thirds (10/15) of patients received eight or more cycles. of five patients who stopped therapy early, only one was due to toxicity; other reasons included severe gvhd (1), parental preference (1), and relapse (2). cycle delays occurred in 9 patients, with a median 2 cycles delayed per patient, mostly for mild myelosuppression with early cycles. no patient required blood product transfusion during therapy, but g-csf was used in three patients to maintain anc>500/ l. dose-modifications were made in 3 patients (renal tubular acidosis, acute kidney injury, and myelosuppression). there were 3 relapses (20%), two of which occurred in patients in cr2, for a relapse incidence of 9% in patients in cr1, with a median follow-up of 20 months (range 12.5 to 28). no patients who received azacitidine died of transplant-related mortality. conclusion: administration of azacitidine in children undergoing allohct for myeloid malignancies is safe and feasible, with most patients successfully receiving all planned cycles. toxicity was acceptable and there was no trm or secondary graft failure. despite the limitations of a small cohort, relapse incidence-particularly in patients transplanted in cr1suggests a potential benefit in disease control that warrants investigation in follow-up studies. background: despite significant improvements in the success rate of hematopoietic cell transplantation (hct), graft failure remains an important complication in patients transplanted for severe aplastic anemia (saa). second allogeneic hct can salvage patients, but 5-year overall survival (os) rates have been reported as low as 60% 1 . objectives: identify patients who developed dropping donor chimerism, graft rejection, and/or graft failure after first hct for saa, necessitating additional hcts or cellular boosts (defined as stem cell products infused without preceding chemotherapy), and evaluate treatment-related complications and os. with vod/sos with and without multi-organ dysfunction (mod) pubmed and embase databases were searched for "defibrotide and retrospective chart reviews; excluded publication types were: case reports (<10 cases); meta-analyses; reviews; animal, modeling, pharmacokinetic, chromatography, and adult-only studies; guidelines; articles; and letters. resulting reports were screened for exclusion criteria. full-text articles were then reviewed for eligibility. study characteristics of selected publications were summarized, and publications were categorized by patients' mod status. when necessary, additional data tables were requested. a random effects model was used for pooling data for efficacy. interstudy heterogeneity was assessed with cochran's q-test. percentage of total variation across studies due to heterogeneity (i2) was evaluated we quantified ∼4300 proteins in each sample. reproducibility for one donor at 3 different time points children 's minnesota, minneapolis, minnesota, united states background: pediatric and young adult hodgkin lymphoma (hl) has five-year survival rates >90%. chemotherapy required to achieve this rate is associated with a lifetime risk of cardiac deaths, second malignancies, pulmonary disease and infertility. as effective salvage therapy exists, outcomes may be improved by de-intensifying initial therapy to lessen toxicity.objectives: we piloted a regimen in low and intermediate risk hl patients using agents without known association to significant late effects. this retrospective chart review was approved by children's minnesota irb.design/method: the bvg(p) regimen incorporated bortezomib (1.3 mg/m2 day 1,4,8,11); vinorelbine (25 mg/m2 day 1,8); gemcitabine (1000 mg/m2 day 1,8) every 21 days and prednisone (20 mg/m2/dose bid x 11 days). we treated 5 newly diagnosed patients, ages 10-18 years, with non-bulk stage iia (n = 4) or iib (n = 1) hl. two patients received bvg and 3 received bvgp with the addition of prednisone.results: newly diagnosed patients were all pet negative after the first or second cycle and remained pet negative at end of therapy, 4 cycles. nausea was well controlled with 5-ht3 antagonists and scopolamine. pegfilgrastim was not necessary due to the high absolute neutrophil count nadir [median 1.16 and minimum 0.56 × 109/l]. there were no episodes of febrile neutropenia, infection or transfusion need. no patients experienced alopecia. one patient developed sensory neuropathy after the eighth dose of bortezomib that was controlled with gabapentin and a switch to subcutaneous bortezomib administration. of the five newly diagnosed patients, four remain in remission at 227, 270, 557, 1191 days; 1 relapsed at previous disease sites at 861 days and subsequently achieved remission with bvgp with the addition of brentuximab. this series provides early evidence to stimulate expansion of this pilot experience and subsequent multiinstitutional study leading to a randomized trial of bvgp and current chemotherapy for low and intermediate hl. st jude affiliate clinic at st francis hospital, tulsa, oklahoma, united states background: symptoms suggestive of morning hypoglycemia has been noticed in children receiving all chemotherapy. only few small studies looked at this therapy related complication. factors increase risk of hypoglycemia in all patients include accelerated starvation, steroid induced adrenal suppression, mercaptopurine therapy and prolonged fasting for procedures.objectives: to study the prevalence and risk factors for hypoglycemia during all therapy design/method: medical records of of children (up to 18 years old) treated for all between 2011-2016 (86 patients) were studied for evidence of morning hypoglycemia defined as blood sugar (bs) < 60 mg/dl. statistical mean differences between the subgroups were analyzed with spss using a nonparametric mann-whitney u test.results: fifty two percent (52%) of patients developed hypoglycemia during all treatment, with an average of 2.2 episodes/patient. 59% were males and 41% females. almost 2/3 (65%) of patients with hypoglycemia were in maintenance phase of therapy. 34% of hypoglycemic episodes occurred in 10% of patients. majority of hypoglycemic episodes (78.2%) occurred on the day of procedure when patients were fasting overnight. 48.9% of hypoglycemic episodes occurred in children ≤3 years, with 75.8% in ≤6 years. patients who developed hypoglycemia were significantly younger (mean age at time of diagnosis of all was 4.93 ± 3.69 at the hypoglycemia group versus the non-hypoglycemia (7.27 ± 4.98) p<0.05. no statistically significant difference was found regarding sex, or tpmt genotype. 6% of hypoglycemic children-all <3years of age-presented with life threatening hypoglycemia symptoms including seizure and loss of consciousness. this study showed high prevalence of hypoglycemia during childhood all therapy. younger age, especially ≤ 3 years, is associated with higher risk of hypoglycemia as well as life-threatening episodes. to decrease fasting hypoglycemia during therapy for childhood all, we recommend that children under the age of 6 years receive bed time snack high in proteins and complex carbohydrates, and to get them up early the day of procedure to take clear sugary drink. hospital for sick children, toronto, ontario, canada ann & robert h. lurie children's hospital of chicago, chicago, illinois, united states background: childhood brain tumors are the most common solid malignancy and the leading cause of cancer-related mortality in children. the most aggressive type of pediatric central nervous system (cns) tumors is diffuse intrinsic pontine glioma (dipg). despite decades of clinical trials, there has been no substantial improvement with respect to therapeutic outcomes with most children eventually succumbing to the disease. research on adult high-grade gliomas has shown a targetable pathway through the inflammationinduced expression of indoleamine 2,3 dioxygenase 1 (ido1) and its recognized ability to suppress the anti-tumor immune response. a limited understanding into the role of ido1 in pediatric central nervous system tumors serves as the foundation of this research project. furthermore, the integration of nanotechnology is a fundamental step for the investigation and targeting of ido1. spherical nucleic acids (snas) composed of nanoparticles have been shown to transverse cellular membranes, exhibit stability in physiological environments, escape from degradation, and create precise targeting in brain tumors.objectives: the purpose of our project is to delineate the role of ido1 in pediatric dipg, and develop small inhibitory (si)rna oligonucleotides and snas aimed at therapeutically inhibiting the gene expression of immunosuppressive ido1. our specific aims are to: (1) confirm the gene expression ido1 in different human dipg cell lines; (2) generate and characterize sirna oligonucleotides targeting human ido1 in vitro; and (3) generate and characterize gold nanoparticles for targeted inhibition of ido1.design/method: unique patient-derived dipg cell lines were grown in culture, stimulated with increasing concentrations of the proinflammatory cytokine, ifn , and analyzed for mrna levels. sirna specific to ido1 was transfected into cells. sna generation is in progress.results: ido1 is expressed in multiple human pediatric dipg cell lines. sirna targeting ido1 among exons 9 and 10 results in a significant decrease in overall ido1 expression by dipg cells. sna generation for targeting ido1 with improved penetration & stability is ongoing, with preliminary results demonstrating a robust ability to inhibit ido1 expression. the grim prognosis of children with dipg, the lack of effective therapies, and the expression of ido1 by human dipg cells emphasize the importance of developing the treatment capability to inhibit ido1 gene expression, as a excluded from this study. the remaining patients were analyzed using descriptive statistics.results: a total of 17 patients meeting inclusion criteria were identified. of these, 4 patients (23.5%) received tonsillectomy alone, 1 (5.9%) underwent tonsillectomy and decreased immunosuppression, 6 (35.3%) received tonsillectomy and rituximab, and another 6 (35.3%) received tonsillectomy with additional therapy (including ebv-specific cytotoxic tlymphocytes, donor leukocyte infusion, and chemotherapy). of the 5 patients who received tonsillectomy with or without a decrease in immunosuppression, all were diagnosed with high-grade lymphoma and achieved clinical remission following tonsillectomy with no evidence of relapse to date. on further analysis looking at ptld risk factors, all patients were under 50 years of age, all received t-cell depleted grafts, and none had significant graft-versus-host disease (gvhd) at the time of ptld diagnosis. we have identified a population of patients with localized ebv ptld that achieved clinical remission with no evidence of recurrence following tonsillectomy, suggesting that tonsillectomy alone may be an adequate treatment for localized ebv ptld in a specific subgroup of patients. further analysis is needed to identify characteristics of this subgroup to determine which patients would be most likely to respond to this treatment. university of rochester, rochester, new york, united states background: malignant central nervous system (cns) tumors in young children have a poor prognosis and pose a significant therapeutic challenge. consolidation therapy with carboplatin and thiotepa was piloted in ccg-99703, cog acns0333, and cog acns0334 with the goals of intensifying therapy and omitting or delaying radiation.objectives: to document outcomes for patients undergoing carboplatin/thiotepa consolidation with autologous stem cell rescue (ascr) and to demonstrate the feasibility and toxicity of this regimen.design/method: patients up to 3 years old (median age: 12 months) with malignant cns tumors treated at the university of rochester from 2012-2017 with at least one cycle of carboplatin (17 mg/kg/day x 2 days) and thiotepa (10 mg/kg x 2 days) followed by peripheral blood ascr were included in retrospective analysis. data were recorded on time to engraftment (defined by absolute neutrophil count (anc) recovery to > 0.5 × 10^9/l), length of hospitalization, toxicity with each consolidation cycle, progression free survival (pfs) and overall survival (os). stem cell harvest data were also collected.results: eleven patients with malignant cns tumors (6 atypical teratoid/rhabdoid tumor, 3 primitive neuroectodermal tumor, 1 glioblastoma multiforme, and 1 pineoblastoma) received a total of 30 cycles of carboplatin/thiotepa. of these, 9 underwent stem cell harvest at our institution, with complications limited to procedure-related hypotension for 1 patient with known autonomic instability, and catheter-associated deep vein thrombosis (dvt) for 1 patient. four patients were in complete remission (cr) 1/status-post gross total resection, 1 was in cr2, and 6 had residual tumor at the time of consolidation. nine patients received 3 planned consolidation cycles, 1 patient 2 (of 2) planned cycles, and 1 patient 1 of an anticipated 3 cycles thus far. average time to engraftment for these 30 cycles was 10.2 (+/-1.4) days, with a mean hospital length of stay of 16 (+/-3.2) days. fever occurred in 17 of 30 cycles (57%); infectious toxicity included documented bacterial infection in 2 cases (enterococcus faecalis bacteremia in 1, klebsiella pneumoniae in 1). there were no regimenrelated deaths. with a mean follow-up of 23 months, 2 survivors have not yet completed all therapies, and 5 patients have relapsed (4 have died of disease). of the 7 survivors, 4 have been disease-free for >12 months. background: autologous hematopoietic stem cell transplantation (auto-hsct) has resulted in improved survival for patients with high-risk neuroblastoma. treatment intensification is however associated with greater complications. data on early infectious complications in low-and-middle income countries are limited.objectives: to review the early infectious complications following auto-hsct in patients with high-risk neuroblastoma.design/method: a retrospective chart review of pediatric patients with high-risk neuroblastoma who underwent auto-hsct at the american university of beirut medical center between 2003 and 2017 was conducted. infectious complications during the first 100 days post-transplant were reviewed.results: forty-three patients (27 males and 16 females) with a median age at diagnosis of 4.35 years [range: 0.5-13.9] years underwent auto-hsct during the above-mentioned period. conditioning regimen consisted of melphalan, etoposide and carboplatin. all patients received antiviral and antifungal prophylaxis. median time for neutrophil engraftment was 10 days [range: 8-19]. bacteremia and clostridium difficile infections occurred in 16 (37%) and 12 (28 %) patients respectively. seven (16%) patients developed enterocolitis diagnosed by imaging, 4 were adenovirus induced. cmv viremia was diagnosed in 7 (16%) patients, 5 of whom required treatment. varicella zoster reactivation, parvovirus viremia, toxoplasmosis encephalitis, bk virus cystitis (2 patients) and central nervous system ebv related post-transplant lymphoproliferative disorder were diagnosed in 6 different patients. there was no invasive fungal infection. sixteen (37%) patients have died, 6 of whom died in the early post-transplant period, 1 due to disease progression and 5 (11.6%) due to infectious complications. among the 5 patients who died due to infection, 1 developed toxoplasmosis encephalitis, 4 developed severe enterocolitis, 2 of which were adenovirus related. the mean igg level within one week post-transplant was lower in patients with clinically significant viral infection compared to others (4 vs 5.6 mg/dl, p: 0.08). the mean igg level at the time of clinically significant bacterial infection was lower in infected patients compared to others (4.4 vs 6.3 mg/dl, p: 0.03). neither absolute lymphocyte count nor absolute neutrophil count at day 20 post-transplant affected the incidence of clinically significant infections. our results show that the rate of infections during the early post auto-hsct period is higher than what has been described in developed countries and has a significant impact on mortality. prevention, early detection and improvement in the treatment is required to improve outcome. university of miami, miami, florida, united states background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for many malignant and non-malignant (bone marrow failure, immunodeficiency, or metabolic diseases) in pediatrics. despite advances in medicine, graft-versus-host-disease (gvhd) remains a significant cause of non-relapsed morbidity and mortality, specifically in those with malignant diseases.objectives: to highlight the complexity to acute gvhd management and seldom-described treatment approach. a 7 year male with a history of high risk acute myeloid leukemia (aml) due to failed induction therapy. he received a matched (10/10) unrelated donor hsctmarrow product-conditioned with busulfan, fludarabine, and anti-thymoglobulin (atg). his post-transplant course was complicated by hhv-6 viremia, pres (prompting a change from prograf to cyclosporine), mucositis, and grade iii acute gvhd (skin s3, gut s2, liver s0) around post transplant day 101, which later morphed to ocular involvement by d+120. he was started on 2 mg/kg steroids with good response but flared up with each attempt to taper steroid dose. a course of rituximab and later atg were tried without success in weaning off steroids. switching cyclosporine to sirolimus did not provide any additional benefit either. extracorporeal photopheresis (ecp) was started 3 times a week. he initially responded well, yet was not able to wean off steroids. in addition, he developed a flare when ecp session was reduced to 2 days per week. ecp was therefore increased to 5 days per week, which appeared to stabilize skin lesions. a trial of weekly methotrexate was attempted to wean off steroids and photopheresis, which provided no response. finally, a trial of bortezomib on days 1, 4, 8, and 11 of a 21 day cycle as published in a case series of multiple myeloma patients who developed post hsct gvhd. skin lesions improved remarkably however dose had to be reduced due to related pancytopenia. given the response to therapy, he was continued on a weekly dose of bortezomib, receiving a total 8 doses, which has permitted the slow taper of prednisone that has since been discontinued without a major flare. he however is currently maintained on ecp 3 times per week, which is now been slowly withdrawn.conclusion: management of acute gvhd in pediatric patients after hsct can be challenging with no definite options for those who fail steroids or become steroid dependent after initial response. in these situations, bortezomib could be a valid therapeutic option. background: neuroblastoma (nbl) is the second most common solid tumor in children and despite recent treatment advances, overall survival for high risk nbl remains <50%. the addition of immunotherapy has improved survival and includes anti-gd2 antibody therapy. the success of antibody therapy in neuroblastoma is primarily due to natural killer (nk) cell mediated antibody dependent cellular cytotoxicity. we previously demonstrated that nk cells from patients with high risk nbl can be successfully isolated and expanded to large numbers and exhibit potent anti-tumor effects against nbl (1). thus, infusions of autologous expanded nk cells in high risk nbl in combination with anti-gd2 antibody are being studied in clinical trials. toll-like receptors (tlr) present on the surface of leukocytes are responsible for pathogen recognition, and activation of these receptors stimulate the production of cytokines that critically link innate and adaptive immune responses. the tlr3 agonist, poly(ic) is a synthetic analog of dsrna that has previously been shown to directly stimulate cytokine production and improve cytotoxicity in primary nk cells through activation of genes regulated by interferon-response elements (ire) (2). we hypothesized that ex vivo activation of tlr pathways in nk cells during our normal 14-day expansion using k562 feeder cells expressing membrane bound il-21 would enhance their function.design/method: nk cells were isolated from peripheral blood mononuclear cells and expanded with our previously described expansion protocol in media containing il-2 and 50 ug/ml poly(ic) (3). at the end of the 14-day expansion, nk cells expanded with poly(ic) were compared to controls using a calcein cytotoxicity assay to measure cytotoxicity against high risk neuroblastoma and cytometric bead array to measure cytokine production. : surprisingly, the addition of poly(ic) during nk cell expansion did not improve proliferation, cytokine production or cytotoxicity compared to our standard expansion method. rnaseq demonstrated that our standard expansion method results in a modest decrease in tlr3 expression at the transcriptional level, but significant upregulation of several ireregulated genes. we conclude that either our standard approach interferes with tlr signaling or saturates the innate immune response pathway such that co-stimulation with poly ic does not produce an additive effect. we are performing expression analysis on nk cells receiving poly(ic) during expansion to further explore this hypothesis. background: gonadal dysfunction leading to infertility is a complication after hematopoietic stem cell transplant (hsct). anti-mã¼llerian hormone (amh) is a marker of ovarian reserve; it is not controlled by gonadotropins and has minimal inter-cycle variations, therefore, it can be used as a marker of ovarian reserve and aid in fertility counseling.objectives: assess ovarian reserve in hsct patients utilizing amh levels. background: tgf beta is an immune suppressive cytokine frequently elevated in the tumor microenvironment causing tumor immune evasion. acute tgf beta treatment potently inhibits nk cell cytotoxicity, cytokine secretion, and proliferation. however, tumor infiltrating nk cells receive chronic inhibitory tgf beta signals in conjunction with activating signals from tumor cells. objectives: to this end, we hypothesized that long-term tgf beta-cultured nk cells would induce functional and phenotypical changes on nk cells that differ from short-term tgf beta treatment.design/method: to explore this, primary human nk cells were cultured with the leukemia cell line, k562, alone or with exogenous tgf beta for 2 weeks. : surprisingly, nk cells cultured in tgf beta proliferated faster, and upon challenge with a variety of cell line targets they secreted much greater quantities of ifnî 3 (5-to 282-fold increase against 8/8 cell lines) and tnf (3-to 33-fold increase against 7/8 cell lines). further, the high cytokine secretion induced in these nk cells was no longer inhibited by adding additional tgf beta. degranulation was also increased (2/3 cell lines), however cytotoxicity was not enhanced in a 4-hour cytotoxicity assay. after resting in il-2, the cytokine hypersecretion of tgf betacultured nk cells was maintained for several weeks suggesting this functional change might involve cellular reprogramming. we investigated the mechanism behind these functional changes and profiled 92 genes involved in tgf beta signaling. we found significant reduction of smad3 transcription which corresponded to a striking decrease in smad3 chromatin accessibility. we also found significantly increased smad6 and decreased tgfbr3 expression. phenotypic analysis revealed that tgf beta also induced remodeling of the nk receptor repertoire with decreased nkp30, cd16, and klrg1 and upregulation of trail. the functional consequences of these tgf beta-induced changes on in vitro and in vivo nk cell function are currently under investigation. background: the use of t-cell depleted grafts in haploidentical stem cell transplantation (hsct) has been associated with a delay in early t-cell recovery which increases the risk of viral infections, relapse or graft rejection. conventional donor lymphocyte infusion (dli) after hsct transplantation is effective but conditioned because of a high prevalence of gvhd. the infusion of selected lymphocyte subpopulations with low aloreactivity is emerging as an effective strategy to rectify this issue. the depletion of cd45ra+ naive lymphocytes, preserving cd45ro+ memory t-cells, could provide a safe source of functional lymphocytes with anti-infection, antileukemic and anti-rejection properties, and lower rates of adverse effects. our objective is to present data of patients that have received cd45ro+ memory t-cells dli (mdli) and assess its safety and outcome. we present data of mdli performed after hsct in cases of mixed chimerism, persistent lymphopenia, viral/opportunistic infections or as a strategy to accelerate immune reconstitution.results: fifteen patients with diagnosis of all (n = 6), aml (n = 3), mds (n = 1), saa (n = 3), sideroblastic anemia (n = 1) and cgd (n = 1), received mdli after hsct. a total of forty-three mdli were infused. the median dose of cd45ro+ memory t-cells infused was 5.00 × 107/kg (range:4.8 × 104-4.25 × 108/kg), with a median dose of cd45ra+ naive t-cells of 3.90 × 102/kg (range:0-1.3 × 104/kg). the mdli were infused at a median of seventy-seven days after hsct (range:14-407 days), with a median interval between mdli of thirty-four days results: eight published studies reported survival outcomes for pediatric vod/sos patients (n = 1036), across all defibrotide doses. estimated day+100 survival (95% confidence interval) was 60% (53%-67%). for vod/sos with mod, 4 studies were identified (n = 402) with pooled estimated day+100 survival of 57% (52%-61%). only one openlabel expanded-access study, the treatment-ind, reported outcomes separately for pediatric vod/sos patients without mod (n = 289 patients aged ≤16 years). the day+100 kaplan-meier estimated survival for those patients was 78% (72%-82%). safety results were not pooled due to differences in reporting methodology; however, study results were consistent with the safety profile of the phase 3 historicallycontrolled trial in vod/sos patients with mod (43% pediatric), in which 101/102 defibrotide-treated patients and all 32 controls experienced ≥1 ae. hypotension was the most frequent ae (39%, defibrotide; 50%, controls); common hemorrhagic aes (ie, pulmonary alveolar and gastrointestinal hemorrhage) occurred in 64% of defibrotide-treated patients and 75% of controls. in this pooled analysis of studies with defibrotide-treated pediatric patients with vod/sos, estimated day +100 survival was 60% (without mod, 78%; with mod, 57%). safety results in individual studies were generally consistent with the known safety profile of defibrotide. taken together, these results show a largely consistent defibrotide treatment effect in pediatric patients treated with defibrotide for vod/sos, with or without mod. results: six patients met inclusion/exclusion criteria. all patients were started on ecp while concurrently receiving 1.5 to 2 mg/kg steroid therapy for agvhd plus a calcineurin inhibitor. patients had initiation of ecp within a maximum of 2 weeks from initial diagnosis of agvhd (range 3-12 days). patients had grade 2-4 agvhd (3/6 patients with grade 4) with skin, liver, and gi gvhd represented. patients received ei-ecp 2-3 times per week for the first 6 weeks and then had ei-ecp frequency tapered based on initial response.after 6 weeks of therapy 1 patient had a decrease in overall gvhd grade by 1 grade. all patients were able to have steroids tapered, with doses decreased by an average of 62% (33% -100% decrease).at 12 weeks of therapy, one patient with grade 4 agvhd died of mof associated with infections. three patients had complete resolution of agvhd and 2 patients decreased by 1 grade. steroid doses were decreased by an average of 91% (69% -100% decrease). continuously measures axillary temperature and wirelessly transmits real time-time data. the primary aim of the study was to evaluate the feasibility, safety and tolerability of continuous temperature monitoring in hsct patients using temptraq. we are performing a prospective observational study of pediatric patients (1-12 years of age) undergoing hsct at cincinnati children's hospital in cincinnati, ohio. enrolled patients wore a temptraq patch for 5 days. a 1-10 rating scale survey was completed by the parent/guardian at the end of the study to determine tolerability, ease of use, satisfaction and desire for future use in the inpatient and outpatient setting. temperature data from the temptraq patch was compared to the standard episodic temperature monitoring to determine detection of febrile episodes. seven of ten patients have completed screening. we anticipate completion of the study in early february. the temptraq patch was well tolerated by study subjects (mean tolerability rating of 8.7/10). one patient developed skin breakdown at the site of the temptraq patch attributed to recent thiotepa. the patch was easy to apply with an easy of application rating of 9.7/10. parents were overall satisfied (rating 8.4/10) and would like to use the temptraq patches in future hospitalizations (rating 8.4/10) and at home (rating 8.9/10). temptraq patch identified fever (≥ 100.4 • f) in 4 patients. the fever was never detected by episodic monitoring (soc) in 2 patients and significantly delayed in the other 2 patients (>12 hours). temptraq was well tolerated in pediatric hsct patients. timely fever detection was improved in temptraq over the current soc. background: serotherapy is commonly used in patients undergoing hematopoietic stem cell transplant (hsct) to reduce the incidences of engraftment failure and graft versus host disease. however, one well-known side effect is fever. as children undergoing hsct have compromised immune defenses, fever may also be an early indicator of bloodstream infection, which would warrant prompt use of broad-spectrum antibiotics. in a subset of patients with serotherapy-associated fever, antibiotics, which may induce antibiotic resistance and increase costs, may be unnecessary. we aimed to determine the incidence and characteristics of serotherapy-related fever, as well as the likelihood of concomitant bacteremia, in our institutional experience. a 5-year retrospective chart review was conducted of pediatric patients who received serotherapy as part of hsct conditioning at the university of minnesota. one-hundred sixty eight consecutive hsct patients who received serotherapy -either atg (n = 99) or alentuzumab (n = 69) -were identified. the median age at hsct was 6-years (range, 0.4-18 years). a total of 133 patients (79 %) developed fever while on serotherapy (atg = 79, alentuzumab = 54). one-hundred sixteen patients presented fever following the first infusion, and the median onset of fever was 7 hours after commencing infusion (range, 0.1-22 hours). fever resolved at a median 8 hours (range, 1-48 hours). one hundred and fourteen patients (98%) underwent blood cultures. only seven patient were not started on (6%) empiric antibiotics, while 14% (n = 17) were on antibiotic treatment prior to serotherapy for previously known or suspected infections. nine patients (7% of febrile patients, 4% of all patients) had positive blood cultures (atg = 6; alentuzumab = 3). no infection-associated deaths were observed.conclusion: while fever is common during serotherapy conditioning in children undergoing sct, episodes of concomitant bloodstream infection are rare. ongoing analysis identified potential risk factors for bacteremia as recent history of infection, first episode of fever following second or subsequent infusions, and previous central line placement. further analysis is being conducted to identify subgroups of patients for whom close monitoring alone may be safe. background: hsct is potentially curative for caya with high-risk leukemias; however, most lack an hla-matched 2018 aspho abstracts related donor. the risk of gvhd is increased with unrelated (urd) or partially matched related (pmrd) donors. selective t-cell depletion based on the elimination of t cells carrying and chains of the t-cell receptor may greatly reduce the gvhd risks, while allowing the maintenance of mature donor-derived alloreactive nk cells and / (+) t cells, which may augment the anti-leukemia effect.objectives: this is a prospective study of caya with acute leukemia who underwent hsct with mmrd or urds and tcr / /cd19 depletion. outcomes included engraftment, toxicities, viral reactivation, and relapse.design/method: this study included 36 caya with acute leukemia transplanted between october 2014 and may 2017. all received a myeloablative preparative regimen with targeted busulfan (n = 15) or tbi (1200 cgy/6 fractions) (n = 21), with thiotepa (10 mg/kg) and cyclophosphamide (120 mg/kg). atg (3 mg/kg x 3) was given to those receiving haploidentical grafts and to the first 17 who received urd grafts. immune suppression was not given post-hsct. the stem cell source was mobilized peripheral blood stem cells (pscs), which then underwent tcr / /cd19 depletion utilizing the clinimacs device under gmp conditions in the chop cellular immunotherapy lab.results: median age was 11 (range 1.3-21.7). diagnoses included all (9-b-cell, 3-t-cell) and aml (24; 3-secondary aml). urd were used for 28; 13 were 10/10 allele matched and 15 were 9/10 matched. haploidentical donors were used for 8. median cd34(+) dose -10.1 × 106, / (+) cd3(+) cells -5.63 × 106, and b cells -8.95 × 104. all patients achieved an anc at a median of d+13 (8-30), and 94% had platelet engraftment at median d+17 (12-40). nine patients (25%) developed acute gvhd (all skin, grades i-iv). five developed chronic gvhd (skin, gut, lung): limited in 4, extensive in 1. viral reactivations included: adenovirus (5, 14%), bk virus (8, 22%), cmv (10, 28%), and hhv6 (2, 6%). nine (25%) patients relapsed at a median of 147 days (range 57-625) post-hsct, including 7 aml patients (29.2%) and 2 all patients (16.7%). transplant-related mortality was 14%; causes included sepsis (6) and ards (2). os was 72%; efs was 58% (gvhd-free efs 39%, lfs 61%). hsct with tcr / /cd19 depletion demonstrates excellent engraftment kinetics with limited gvhd without immune suppression. elimination of post-hsct immunosuppression may offer an excellent platform to augment anti-leukemic immune therapy or to enhance immune reconstitution. background: hematopoietic cell transplantation (hct) is the only curative treatment available for patients with sickle cell disease (scd). low bone mineral density (bmd) has been described in scd, but little is known about the impact of curative hct on this outcome. to determine the prevalence of low bmd and variables associated with low bmd in scd patients after hct. we conducted a retrospective chart review of scd patients who underwent hct at children's healthcare of atlanta (choa) between 12/1993 and 12/2016 and survived ≥1 year post-hct. transplant characteristics, post-hct dual-energy x-ray absorptiometry (dexa) scan results, vitamin d levels, graft-versus-host-disease (gvhd) status, and fsh levels were reviewed. for patients 2-20 years of age, height corrected z-scores were calculated using a nihvalidated calculator, with t-scores used for older patients. bmd was categorized as low if between -1 and -2 sd below the mean and clinically significantly low if >-2 sd, in accordance with the children's oncology group long-term follow-up guidelines. vitamin d levels <20 ng/mol were considered deficient, and fsh levels >40miu/ml suggestive of premature ovarian failure. fisher's exact test was used to compare variables in those with normal versus abnormal dexa scan results, with p<0.05 considered significant.results: hct was performed on 71 patients with scd, with 67 surviving ≥1 year post-hct. dexa scans were obtained in 40 patients (55% female), with mean time from hct to dexa scan being 4 years (1.3-11.2 years) and mean age at time of dexa 13.1 years (6.1-22.3 years). patients with and without dexa scans did not differ by sex, donor source, age at transplant, or vitamin d status. low bmd was noted in 10 patients (25.0%), with these patients more likely to be >13 years (pubertal; 90.0 versus 40.0%, p = 0.009). acute gvhd was more common in patients with low bmd (50.0 versus 16.7%), but not statistically significant (p = 0.085). clinically significant low bmd was noted in 3 patients (7.5% of those with dexa scans). these patients were older (16.2 years at testing), were more likely to be male (66.6%), and all had acute and chronic gvhd, while none had evidence of gonadal failure.conclusion: clinically significant low bmd is uncommon after hsct for scd. patients at risk for low bmd include older patients and likely those with gvhd. this preliminary data suggests routine dexas may not be indicated for all patients who undergo hct for scd, but further data is needed. background: causes of renal dysfunction after hematopoietic cell transplantation (hct) include damage from radiation, nephrotoxic medications, graft vs. host disease (gvhd), hepatorenal syndrome, viral infections, or transplant associated microangiopathy. we sought to investigate the incidence of, and risk factors for, acute kidney injury in pediatric hct patients and associated risk with mortality.design/method: data from patients who underwent hct between 2013 and 2016 at a single institution were sequentially retrospectively captured on irb approved protocol. acute kidney injury (aki) was defined at multiple time points post-hct using the standardized criteria: kidney disease: improving global outcomes (kdigo). interval differences between values were analyzed using wilcoxon rank sum testing and categorical variables were analyzed using chi-square analysis.results: ninety-eight patients were included in the study: allogeneic (n = 96) and autologous (n = 2), mean age 8.7 years, of whom 50% were african american, 3% asian, 21% caucasian, 13% latino, and 13% mixed race. forty-seven percent of patients developed aki within the first 2 years of hct. increased risk for aki was associated with a lower pre-transplant creatinine level (p = 0.001), abnormal pretransplant bun (p = 0.019) and an unrelated donor (p = 0.022) while preparative regimen intensity, race, or primary disease were not. twenty-six percent of patients developed aki within 30 days of hct. of those with aki, 41% were exposed to either cidofovir, aminoglycosides, and/or ambisome for at least 5 days versus 18% without aki and 74% were exposed to vancomycin compared to 49% without aki. evaluating outcomes at 1 year after hct, of those with stage 1 aki: 10% had reduced gfr and 37% died, while 14% had reduced gfr and 43% had died for patients with aki stage 2 or 3. the absence of aki by day 30 was associated with 24% reduced gfr and 8% death at 1-year after hct. overall, those with aki at any time in the first year post-hct had a 3.7 fold increased risk of death compared to those without. for patients who required renal replacement therapy (rrt, n = 8), the risk of death was 19.5 fold greater compared to those who did not. in the 25% of patients who survived rrt, both recovered renal function within 2 years.conclusion: acute kidney injury is common after pediatric hct, and may be associated with low creatinine, abnormal bun, unrelated donor pre-hct, and renal toxic medications. early-onset aki post hct is associated with an increased risk of mortality. these data should be validated in a larger prospective study but may offer opportunities to intervene and enhance outcomes. background: myeloablative hematopoietic stem cell transplant (hct) for pediatric malignant disease is associated with significant morbidity with 90% patients experiencing mucositis. patient controlled analgesia (pca) utilizing opioids is an effective strategy for pain management. we sought to describe and analyze pca use in d+30 days post myeloablative hct for malignancies at lurie children's hospital of chicago from 2010-17.design/method: utilizing retrospective chart review, pca details were collected: indication, initiation day, pca duration, team managing pca (anesthesia or palliative), medication and dose in morphine equivalents, and pca toxicities. efficacy of pca was evaluated on pca day + 3, +7, +14, +21 using demands %, maximum pain score (rflacc, faces, vas) and subjective patient, parent and/or pain team perception of pain control. we devised a scale based on the above to designate pain control as good, moderate or poor. variables being analyzed include recipient age, sex, donor type, source, diagnosis, tbi use, gvhd/trm. this analysis, we analyze the depth of remission, car-t persistence, and post-transplant gvhd on our phase i anti-cd22 car-t protocol (nct02315612) to better understand the role of car-t in the peri-hct setting.design/method: children and young adults with relapsed/refractory cd22+ all treated on our phase i anti-cd22 car-t protocol were analyzed. mrd was assessed by flow cytometry (fc) in all, with pcr-based mrd analysis using igh or tcr testing assessed in select patients. hcts were performed at each patient's local institution based on standard of care and included varying conditioning regimens, donor types, stem cell source, and gvhd prophylaxis.results: on our cd22 car trial, 36 patients were treated, the majority of patients (n = 29) having relapsed following a prior hct. 23/36 patients (64%) attained a cr, 18 of whom were mrd negative by fc. concurrent pcr based mrd analysis available in 8 patients demonstrated that all patients achieved pcr based negativity. in 6, this was simultaneous with the 1 month mrd negative fc, and in 2, pcr negativity was achieved over time (fc remained negative). 4 patients proceeded to hct at a median time of 70 days (range: 54-117 days) post-car-t, which was a first hct in 2. these two patients remain in an mrd negative cr, 1 year post-car-t. no patients developed acute or chronic gvhd. car persistence was seen in 3 patients who had detectable car-t cells on the pre-hct marrow suggesting the possibility of ongoing anti-leukemia surveillance prior to initiation of the conditioning regimen.conclusion: by inducing pcr negativity, car-t therapy may have a synergistic role with hct to improve leukemia free survival, prior to emergence of antigen negative leukemia, without an increased risk of gvhd. while the sample size is small, car-t therapy may offer an effective bridge to hct, particularly for those who are pcr negative, and those who have not had a previous transplant. given the underlying risk of hct related trm, pre-hct car may potentially allow for hct conditioning de-intensification as it may not be needed to eradicate residual disease.1 lee dw, ash abstract 218, 2016 background: post-transplant lymphoproliferative disease (ptld) is a complication after solid organ transplantation (sot) that is frequently due to epstein -barr virus (ebv) as a decrease in ebv-specific t cell immunity due to immune suppression allows for uncontrolled proliferation of ebv-infected b cells. outcomes for ptld are suboptimal with relapse rates approaching 50%. however, ebv-infected b cells in ptld express the ebv antigens lmp1 and lmp2 that can be targeted with immune therapy.objectives: we hypothesize that third party "off the shelf" lmp-specific t cell products may improve outcomes and decrease associated co-morbidities for patients with ptld by not only target the lymphoproliferating ebv-infected b cells but also restoring ebv-specific immunity.design/method: lmp-specific t cells (lmp-tcs) are manufactured from eligible donors with a broad range of hla types in our gmp facility to be used in a children's oncology group (cog) trial (anhl1522) for patients with ptld after sot. lmp-tc products are manufactured from healthy donors using autologous monocytes and lymphoblastoid cell lines (lcl) transduced with an adenoviral vector expressing δlmp1 and lmp2 as antigen presenting cells. lmp-tc products undergo comprehensive characterization by ifn-elispot assay to determine lmpspecific epitopes, class i and/or ii response, and hla restriction to guide selection of lmp-tc product for each patient.results: thus far, 27 lmp-tc products have been manufactured. lmp-tcs were active against lmp2 (mean: 158 sfu/1 × 10^5 cells; range: 1-800), lmp1 (31; 0-355), and lcl (105; 0-424) as determined by ifn-elispot assay. at the time of cryopreservation, the lmp-tc products comprised a mean of 36% cd8+ t-cells, 45% cd4+ t-cells, and 8% nk cells. no b cells or monocytes were detected in the final products. thus far, we have identified 3 novel lmp epitopes (lmp1 specific: n = 1; lmp2 specific: n = 2). approximately 80% of the lmp-tc products have lmp-specific activity through multiple hla alleles, and 67% have a mixed class i and class ii response. conclusion: thus, lmp-specific t cell products can be expanded from healthy donors to creat a third party bank, and identifying epitopes and hla alleles with lmp activity will facilitate selecting the most appropriate product for patients. while lmp-specific t cells have previously demonstrated safety and efficacy in phase i studies, anhl1522 is the first trial using cellular therapy within a cooperative group setting. children's cancer hospital at the university of texas md anderson cancer center, houston, texas, united states background: in 2017, the united states food and drug administration (fda) approved the first chimeric antigen receptor t cell (car-t) therapy; tisagenlecleucel. this cd19-directed genetically modified autologous t cell immunotherapy has shown response rates of almost 90% among children and young adults with b-cell precursor acute lymphoblastic leukemia (all) that are refractory or in second or later relapse. cytokine release syndrome (crs) and car-t cell related encephalopathy syndrome (cres) are well described toxicities associated with car-t therapy. crs is a systemic inflammatory response and is typically characterized by fever, hypoxia, tachycardia, hypotension and multi-organ toxicity. cres may occur concurrently or following crs, or without any associated crs symptoms and is characterized by encephalopathy, delirium, seizures and rarely cerebral edema. almost half of patients who receive tisagenlecleucel may require pediatric intensive care unit (picu) support. crs and cres are generally reversible but may be associated with fatal outcomes. pediatric specific management guidelines, comprehensive training of multidisciplinary staff, effective communication and phased infrastructure ensure that adequate resources are available to facilitate early diagnosis and appropriate management of pediatric patients with crs and cres and allow for optimal patient outcomes and accreditation by the foundation for accreditation of cellular therapy (fact).objectives: develop a comprehensive program to ensure safe administration of immune effector cell (iec) therapy to pediatric patients.design/method: an inter-disciplinary pediatric cartox (car t cell therapy associated toxicity) committee consisting of cell therapy and picu physicians, neurologists, fellows, nursing leadership, advanced practice practitioners, pharmacists, registered nurses and social workers was created to monitor patient toxicity and establish specific clinical guidelines and diagnostic and treatments algorithms for pediatric patients receiving iec therapy. educational modules were developed as (i) live in-services and (ii) an online module with a competency based assessment. electronic medical record (emr) order sets and documentation and warning systems were also developed by the committee. the pediatric cartox committee developed a diagnostic and treatment algorithm for patients receiving iec therapy. emr orders and flowsheets were developed to support adherence to the algorithm. inter-disciplinary staff training and competency assessments were closely tracked. almost 97% of identified staff have completed training and achieved competency including, pediatric cell therapy staff, emergency center, picu, outpatient clinic/triage, neurology and sub-specialty staff and nocturnalists.conclusion: an inter-disciplinary approach can assist in institutional readiness for an iec program, promote quality assurance and perhaps fact iec accreditation. future directions include a program for ongoing staff competency assessments. predicted peak vo2) and abnormal oxygen uptake efficiency slope at the anaerobic threshold (1359.3±297.8.9, normal 1790±310). additionally, on echocardiogram three patients had evidence of diastolic dysfunction as evidenced by an elevated e/a ratio (1.9±0.4) on tissue doppler. three patients demonstrated depressed longitudinal peak systolic strain (-17.7±2.8), indicating dysfunction not captured by ejection fraction. in this feasibility study, sct patients without evidence of lvsd on standard measures by resting echocardiogram can demonstrate abnormal exercise capacity. additionally, they can demonstrate systolic and diastolic dysfunction by measures not always included in standard echocardiography. these data suggest the need for a more thorough screening of survivors, and will be further validated as additional patients are recruited for this study. background: in hematopoietic transplantation, the t lymphocytes of the inoculum play a determining role in promoting hematopoiesis, transferring immunity to pathogens and acting as mediators of the graft-versus-leukemia effect (gvl). however, they are also responsible for graft-versus-host disease (gvhd), the main cause of post-transplant morbidity and mortality. the depletion of cd45 ra lymphocytes, by eliminating naive t lymphocytes from the inoculum, aims to conserve the gvl without producing gvhd.design/method: since april 2016, 14 patients (8 boys and 6 girls), with a median age of 8 years, have undergone an allogeneic hematopoietic transplant from an hla donor identical with cd45 ra/cd19 depletion. the indication for transplant was: acute lymphoblastic leukemia (4), acute myeloblastic leukemia (4), myelodysplasia (5) and medullary aplasia (1). the donor was familiar in 4 cases and unrelated in 10. the conditioning regimen was with fludarabine, busulfan and thiotepa. the median of cd34 + cells infused was 6.82 × 10 6 / kg. on the day 0, +15 and +30 a programmed infusion of 1 × 106 / kg lymphocytes cd45ra-was performed.results: all the patients grafted with a median leukocyte (> 0.5 × 109 / l) and platelet (> 20 × 109 / l) engraftment time of 15 and 10 days, respectively. only one patient has developed acute gvhd grade i and no patient has developed chronic gvhd. immune reconstitution was early and rapid in all t cell subsets no patient has relapsed so far and only 1 patient with myelodysplasia has developed an aml. she has received a 2nd transplant and has died of relapse. there was no case of toxic mortality. the event-free survival (sle) was 90 â± 10% with a median follow-up of 10 months. at present, 13 patients are alive, out of immunosuppressive treatment and doing well. allogeneic transplantation with cd45 lymphocytes ra depletion resulted on very encouraging results, with a very low incidence of acute and chronic gvhd, but preserving the gvl effect by infusing cd45 ra-donor lymphocytes. miami children's health system, miami, florida, united states background: hematopoietic stem cell transplantation (hsct) using autologous or allogeneic progenitor cells is a potentially curative treatment for patients with high-risk malignancies and nonmalignant conditions. the american society for blood and marrow transplantation developed a task force to establish consensus guidelines for defining patient care in hsct and advocated for further studies to delineate safe procedural steps as an increasing amount of hsct are being offered to patients. there is limited evidence to support engraftment in recipients who receive their infusions via iv or syringe pump. we present novel data from patients who achieved neutrophil engraftment following hsct by a pump mechanism.objectives: to provide evidence supporting the use of pump (intravenous or syringe) infusion method in hematopoietic stem cell transplantations.design/method: a retrospective review was completed for 114 patients who underwent hsct between 2003 and 2012. inclusion criteria included patients who had received hematopoietic stem cell transplants between 2003 and 2012 and who were ages 6 months to 21 years old. the main outcome measure was days to neutrophil engraftment (defined as the first of three consecutive days with an anc > 5 × 10 9 /l).results: among 114 patients who received infusion of hematopoietic stem cell products via pump mechanism, 63 patients (55.3%) received autologous products and 51 (44.7%) received stem cells from allogeneic donors. neutrophil engraftment (anc > 5 × 10 9 /l) occurred in a median of 14.5 days after stem cell infusion. the mean number of days to engraftment for patients who received allogeneic infusions s301 of s301 design/method: a retrospective chart review was performed at the children's hospital of wisconsin. statistical analyses included kaplan-meier estimate for os, mann-whitney test for comparing outcomes between subjects, and descriptive analyses.results: from 2005-2016, 14 patients with a median age of 7.5 (4.3-23.0) years at 1 st hct were identified. patients were conditioned with cy/atg (n = 3), cy/flu/atg (n = 6), or cy/flu/atg/tbi (n = 5) and received marrow (n = 13) or cord blood (n = 1) with median cd34/kg dose of 6.82 (1.88-10.90) ×10 6 . two patients developed grade i acute graftversus-host disease (gvhd); none developed chronic gvhd. due to dropping chimerism, graft rejection, or graft failure, 2 nd hct (n = 9) or boost (n = 5) was offered. the median cd3 chimerism prior to hct/boost was 53 (0-100)%. median time between 1 st hct and 2 nd hct or boost was 134 days (42 days-5.1 years). in 9 patients receiving 2 nd hct, 5 used the same donor, of which 3 used the same stem cell source (marrow) and 2 switched to peripheral blood stem cells (pbsc). in 4 patients who switched donors, 3 used pbsc and 1 used cord blood. most patients receiving 2nd hct underwent a uniform conditioning regimen of cy200/flu150/equine atg/8 gy tli (n = 5) or cy120/flu120/rabbit atg/2 gy tbi (n = 3); one received cy/atg. acute and chronic gvhd (limited seen in 83%) developed in 50% and 43% of patients, respectively. four patients required 18 additional boosts and 1 additional hct. after final intervention, cd3 and whole-blood chimerism at last follow-up was between 95-100% (n = 9) and 90-100% (n = 5), respectively. with a median follow-up of 8.6 (3.8-12.8) years, 13 of 14 patients are alive with an estimated 10-year os of 92.3 ± 0.07%, having performance status ≥90% (n = 12) or 80% (n = 1). one patient developed chronic extensive gvhd and died of fungal infection 2.1 years after 2 nd hct. our single-center experience demonstrates excellent ability to salvage patients who develop graft failure after initial hct. transplant-related complications such as gvhd and infections remain significant concerns.